Untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652649 Volume 17 2018 e18134 Original Article 1 D.D.S., M.S., Ph.D., Postgraduate Program in Dentistry, Federal University of Maranhão, São Luís, Maranhão, Brazil. 2 D.D.S., M.S., Ph.D., Department of Dentistry I, Federal University of Maranhão, São Luís, Maranhão, Brazil. 3 D.D.S., M.S., Ph.D., Department of Dentistry II, Federal University of Maranhão, São Luís, Maranhão, Brazil. 4 School of Medicine, Federal University of Maranhão, São Luís, Maranhão, Brazil. Corresponding author: Maria Carmen Fontoura Nogueira da Cruz Department of Dentistry II, Av. dos Portugueses, 1966, Bacanga, São Luís – MA, Brazil. CEP 65080-805 E-mail address: ma.carmen@uol. com.br Phone number: +55 98 3272-8572 Received: November 20, 2017 Accepted: March 28, 2018 Histopathological evaluation of dental follicles associated with impacted third molars Rogério Vera Cruz Ferro Marques1, Daniele Meira Conde Marques2, Fernanda Ferreira Lopes3, Leonardo Victor Galvão-Moreira4, Maria Carmen Fontoura Nogueira da Cruz3 Aim: To histologically evaluate dental follicles of impacted third molars with no radiographic evidence of pathology. Methods: We carried out both a quantitative and qualitative analysis of pericoronal follicles removed from impacted third molars and investigated the association with clinical data. The sample included 36 extracted dental follicles of impacted third molars, obtained from 28 patients, which presented with no radiographic evidence of pathologies. Results: None of the follicles analyzed showed any pathological entity. The epithelial lining was observed in 61.1% of samples, being identified as reduced enamel epithelium. We found a significant relation between the the presence of inflammatory infiltrate and the group aged over 21 years (64.3%; p<0.05). Conclusions: Considering the absence of pathological lesions, we suggest that the removal of impacted third molars, particularly in young-aged individuals, should be carefully indicated. Keywords: impacted tooth, third molar, dental follicle, pathology 2 Marques et al. Introduction The therapeutic removal of third molars is a common maxillofacial surgery that takes into account clinical diagnostic criteria, including the presence of symp- toms or pathologies, preventive measures or orthodontic indications in asymp- tomatic patients1,2. Several issues have been linked to the maintenance of these teeth, such as root resorption or carious lesions on the second molar, trismus, pericoronaritis, localized pain and development of cysts or tumors. In the pres- ence of such complications, the surgical removal has been widely indicated; however, this decision becomes more difficult when there is no symptom or associated pathology3,4. Even though prophylactic surgery has been justified on the basis that third molars might have a greater potential to develop associated diseases, this practice remains largely controversial5,6. Some authors have argued against surgery in asymptomatic impacted teeth based on the low incidence of pathologies related to third molars. Most studies have used data obtained only through radiographic analysis; nevertheless, the prevalence of pathological conditions may be higher than usually is expected on radiographies7,8. Thus, only radiographic aspects are inconclusive for the proper diagnosis of lesions associated with impacted third molars9. Moreover, although histological changes may exist in the surrounding tissues of impacted teeth, there is no accurate information regarding the prevalence of dis- eases in the pericoronal tissue, which is thought to range from 10% to 58.5%2,10. Hence, further research is necessary to identify a prevalence of alterations that can justify the prophylactic removal of impacted teeth. In this way, we aimed to his- tologically evaluate dental follicles of impacted third molars with no radiographic evidence of associated pathologies. Material and Methods The present study was approved by the Research Ethics Committee of the Federal University of Maranhão (protocol #23115.016756/2011-40). We conducted clini- cal, morphological and radiographic evaluations of dental follicles of impacted third molars with no radiographic evidence of associated pathologies. The sample included 36 dental follicles obtained from 28 patients seeking orthodontic treatment in a pri- vate clinic in São Luís, Brazil. Inclusion criteria were the presence of impacted third molars with an indication for extraction and a dental follicle radiographic space of up to 2.5mm10. Patients with any local or systemic inflammatory and/or infectious disease and those who did not agree to participate in the study were not included. Data obtained from each patient included age, gender, dental unit extracted, and indication for surgery. The radiographic evaluation was carried out by a specialist in a blind fashion, using scanned panoramic radiographies at the ratio of 1:1 that were processed by the Radiocef Studio 2 software (Radio Memory, Belo Horizonte, MG, Brazil). The width 3 Marques et al. of the pericoronal space was used as the largest distance from the crown to the sur- rounding bone. The Winter’s classification of impaction was also registered. For morphological analysis, dental follicles obtained during the extraction of third molars were fixed in 10% formaldehyde solution and embedded in paraffin. Histo- logical cuts (5µm) were performed, stained using the hematoxylin-eosin technique and observed on a microscope. The histopathological analysis of dental follicles was carried out by two independent oral pathologists that were blind to clinical and radio- graphic data. The Cohen’s kappa coefficient (κ) was calculated using SPSS 18.0 (IBM Corp, Armonk, NY, USA). The following characteristics were taken into account for the analysis of dental folli- cle specimens. Connective tissue: dense, loose, or myxoid, which may present occa- sional mineralization; epithelial odontogenic residues; and unspecific inflammatory infiltrate. Limiting epithelium: reduced enamel epithelium or absence of the epithe- lial lining. The presence of a stratified squamous epithelium was considered normal, with up to 3 cell layers, fragmented or separated from the surrounding connective tissue11 (Figure 1). Data were analyzed using SPSS 18.0. To verify statistically significant associations among variables, the Pearson’s chi-squared test (χ2) was utilized at a 5% significance level. Results A total of 36 pericoronal follicles of impacted third molars were collected from 28 patients, aged 14-37 years (mean age: 21.1 years). A total of 55.6% of follicles were obtained from females (n = 20) and 44.4% were from males (n = 16). Mean age for Figure 1. Histological analysis of a dental follicle from a patient under 21 years old: a) limiting enamel epithelium exhibiting discontinuity. b) connective tissue fragment. (original magnification, ×40; HE stain). 4 Marques et al. females was 19.75 years (95% CI: 18.58–20.92) and 22.88 (95% CI: 19.48–26.27) for males. The sample was then divided into two age groups: under 21 years old and over 21 years old. Most teeth included in the present study were lower left third molars (38.9%), followed by lower right third molars (36.1%). In relation to the third molars impaction type, according to the Winter’s classification, 47.2% were mesioangular, 30.6% vertical, 11.1% distoangular, and 11.1% horizontal. In regard to their location, 75% of all impacted teeth were in the mandible (Table 1). We found no association between classification and location with histological features. Regarding the histopathological analysis, inter-rater agreement was satisfactory (κ = 0.84). Microscopically, 38.9% of the dental follicles did not present epithelial lining, while 61.1% presented with a fragmented reduced enamel epithelium or separated from the surrounding connective tissue. The connective tissue exhibited altered myx- omatosis in 72.2% of cases, while epithelial odontogenic residues and calcification were found in 88.9% and 36.1%, respectively, but there was no association between these variables and the age group (p>0.05; Table 2). We observed the presence of nonspecific chronic inflammation in 41.7% of samples to be significantly associated with the age range of patients. The presence of inflam- matory infiltrate was positive in both groups, but was higher in patients aged over 21 years old (p=0.028) as shown in Table 2. Table 1. Distribution of groups according to the Winter’s classification and anatomical location. Winter’s Classification n % Mesial 17 47.2 Vertical 11 30.6 Horizontal 4 11.1 Distal 4 11.1 Location n % Mandible 27 75 Maxilla 9 25 Table 2. Association between the histological characteristics of samples and the age group. Age (years) Myxomatous áreas p Epitelial odontogenic residues p Calcifications p Inflammatory Infiltrate p Yes No Yes No Yes No Yes No n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%) < 21 14 (53.8) 8 (80.0) 0.14 19 (59.4) 3 (75.0) 0.54 8 (61.5) 14 (60.9) 0.96 6 (27.3) 16 (72.7) 0.02* > 21 12 (46.2) 2 (20.0) 13 (40.6) 1 (25.0) 5 (38.5) 9 (39.1) 9 (64.3) 5 (35.7) *p<0.05, according to the Pearson’s chi-squared test. 5 Marques et al. Discussion From data obtained, we observed an increased frequency of surgical removal of impacted third molars in female patients aged over 21 years old, corroborating with pre- vious findings10,11. Still, there was a higher incidence in the mandible as 75% of impacted teeth were lower third molars, similarly to the findings of Stathopoulos et al.5 (2011). Regarding the radiographic evaluation, we considered as a normal pericoronal space a width of up to 2.5mm. According to the literature, the normal space varies from 2mm to 4mm; however, it is important to correlate the radiographic findings with clinical and histopathological aspects3. Small cystic histological alterations may be present in dental follicles tissue and not be featured on radiographies, and the reverse may also occur if the enlargement of the pericoronal space represents a normal tissue on the histopathological exam10. Regarding the dental follicle, Stathopoulos et al.5 (2011) described a high transforma- tion potential in cysts or neoplasms. On the other hand, Khorasani and Samiezadeh12 (2008) reported that this potential may be overrated as dental follicles can be misdiag- nosed as pathological lesions, considering that the distinction between a small den- tigerous cyst and a large dental follicle may be difficult, requiring thereby histological, clinical and radiographic correlation. Immunohistochemical and molecular tests may be useful tools for differential diagnosis among these follicular alterations13. In this study, all dental follicles analyzed presented without any pathological entity. Histologically, the dental follicles analyzed were identified as elements of fibrous connec- tive tissue with different amounts of altered myxomatosis, epithelial calcification, epithelial odontogenic residues and epithelial lining. The incidence of epithelial lining on dental folli- cles has been reported in 69-87.8% of cases12,13. Here, we observed the presence of epithe- lium (reduced enamel epithelium) in 61.1% of samples, most probably due to the average age of patients (21.1 years). The reduced enamel epithelium is more common in young indi- viduals, whereas the incidence of squamous epithelium is regularly found in older subjects. A significant relation between the increasing age of patients and the presence of inflammatory infiltrate in the connective tissue was observed. Prior studies that inves- tigated the connection between the inflammatory infiltrate and oral tissues prolifer- ation found inflammation to cause chronic irritation, stimulate epithelial cells prolif- eration and modify a normal epithelium into a squamous epithelium, which is more resistant to external factors14. Metaplasia can be considered an adaptive reaction in cells vulnerable to stress, improving their tolerance to inadequate environmental con- ditions15, but this epithelial type was not found in the present study. Furthermore, 88.9% of samples presented odontogenic epithelium inside the connective tissue, more than the 79% reported by Kim and Ellis16 (1993). A similar reduction of epithelial islands in relation to increasing age was also reported in this study; however, this relation was not significant. Our data shows that only 27.8% of dental follicles presented with myx- omatous areas as compared to 40% reported by Khorasani and Samiezadeh12 (2008). A significant relationship between altered myxomatosis, age and gender was not found. In conclusion, although our histopathological evaluation of dental follicles did not show pathological alterations, the participants aged over 21 years presented with unspecific chronic inflammation. Therefore, we suggest that impacted third molars should be 6 Marques et al. extracted only due to pathological processes that may influence the patient’s well-be- ing or by specific indications, especially in young-aged patients. We also encourage further studies that include immunohistochemical and molecular analysis. References 1. Fardi A, Kondylidou-Sidira A, Bachour Z, Parisis N, Tsirlis A. Incidence of impacted and supernumerary teeth- a radiographic study in a North Greek population. Med Oral Patol Oral Cir Bucal. 2011;16:e56-61. 2. Kotrashetti VS, Kale AD, Bhalaerao SS, Hallikeremath SR. Histopathologic changes in soft tissue associated with radiographically normal impacted third molars. Indian J Dent Res. 2010 Jul-Sep;21(3):385-90. doi: 10.4103/0970-9290.70809. 3. Adelsperger J, Campbell JH, Coates DB, Summerlin DJ, Tomich CE. Early soft tissue pathosis associated with impacted third molars without pericoronal radiolucency. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000 Apr;89(4):402-6. 4. Marciani RD. Is there pathology associated with asymptomatic third molars? J Oral Maxillofac Surg. 2012 Sep;70(9 Suppl 1):S15-9. doi: 10.1016/j.joms.2012.04.025. 5. Stathopoulos P, Mezitis M, Kappatos C, Titsinides S, Stylogianni E. Cysts and tumors associated with impacted third molars: is prophylactic removal justified? J Oral Maxillofac Surg. 2011 Feb;69(2):405-8. doi: 10.1016/j.joms.2010.05.025. 6. Brkić A, Mutlu S, Koçak-Berberoğlu H, Olgaç V. Pathological changes and immunoexpression of p63 gene in dental follicles of asymptomatic impacted lower third molars: an immunohistochemical study. J Craniofac Surg. 2010 May;21(3):854-7. doi: 10.1097/SCS.0b013e3181d809ab. 7. Curran AE, Damm DD, Drummond JF. Pathologically significant pericoronal lesions in adults: Histopathologic evaluation. J Oral Maxillofac Surg. 2002 Jun;60(6):613-7; discussion 618. 8. van der Linden W, Cleaton-Jones P, Lownie M. Diseases and lesions associated with third molars. Review of 1001 cases. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1995 Feb;79(2):142-5. 9. Dudhia R, Monsour PA, Savage NW, Wilson RJ. Accuracy of angular measurements and assessment of distortion in the mandibular third molar region on panoramic radiographs. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011 Apr;111(4):508-16. doi: 10.1016/j.tripleo.2010.12.005. 10. Simşek-Kaya G, Ozbek E, Kalkan Y, Yapici G, Dayi E, Demirci T. Soft tissue pathosis associated with asymptomatic impacted lower third molars. Med Oral Patol Oral Cir Bucal. 2011 Nov;16:e929-36. 11. Costa FW, Viana TS, Cavalcante GM, de Barros Silva PG, Cavalcante RB, Nogueira AS et al. A clinicoradiographic and pathological study of pericoronal follicles associated to mandibular third molars. J Craniofac Surg. 2014 May;25(3):e283-7. doi: 10.1097/SCS.0000000000000712. 12. Khorasani M, Samiezadeh F. Histopathologic Evaluation of Follicular Tissues Associated with Impacted Third Molars. J Dent Med Tehran Univ Med Sci. 2008;5:65-70. 13. Villalba L, Stolbizer F, Blasco F, Mauriño NR, Piloni MJ, Keszler A. Pericoronal follicles of asymptomatic impacted teeth: a radiographic, histomorphologic, and immunohistochemical study. Int J Dent. 2012;2012:935310. doi: 10.1155/2012/935310. 14. de Paula AM, Carvalhais JN, Domingues MG, Barreto DC, Mesquita RA. Cell proliferation markers in the odontogenic keratocysts: Effect of inflammation. J Oral Pathol Med. 2000 Nov;29:477-82. 15. Kumar V, Abbas AK, Fausto N. Robbins and Cotran. Pathologic Basis of Disease. 7th ed. Philadelphia: Saunders; 2005. 16. Kim J, Ellis GL. Dental folliculartissue: misinterpretation as odontogenictumors. J Oral Maxillofac Surg. 1993 Jul;51(7):762-7.