Zainab F.doc


J Bagh College Dentistry                               Vol. 25(3), September 2013                                X-ray diffraction  
 

Restorative Dentistry  62 
 

X-ray diffraction and biocompatibility of glass ionomer 
cement reinforced by different ratios of synthetic 

hydroxyapatite 
 
Mohammed R. Al.Jabouri, B.D.S., M.Sc., Ph.D. (1)  
Zainab M. Abdul-Ameer, B.D.S., M.Sc. (1)  
 
ABSTRACT 
Background: This study was done to assist X-ray diffraction and biocompatability of glass ionomer cement reinforced 
by different ratios of Hydroxyapatite. 
Materials and Methods: The powder of glass ionomer cement reinforced by different ratios of Hydroxyapatite were 
used to get X-ray diffraction pattern by X-ray diffraction machine, While for biocompatibility test, A polyethylene 
tubes containing glass ionomer cement reinforced by different ratios of Hydroxyapatite were implanted on the 
dorsal submucosal site of Rabbit's tissues and histological slide were prepared for histopathological study. 
Results: X-ray diffraction test showed that all elements of glass ionomer cement reinforced by different ratios of 
Hydroxyapatite were react with each other and all the final products none crystalline in nature with small amount of 
Hydroxyapatite present unreacted may be act as cores for final reacted elements. The histological test showed mild 
irritation to Rabbit's tissues by glass ionomer cement reinforced by different ratios of Hydroxyapatite, this irritation 
subsided with time. 
Conclusions: there is chemical reaction of all elements of glass ionomer cement reinforced by different ratios of 
Hydroxyapatite and new final products were results .Also glass ionomer cements reinforced by different ratios of 
Hydroxyapatite were biocompatible with Rabbit’s tissues. 
Key words: X -ray diffraction, Biocompatibility, glass ionomer cement. (J Bagh Coll Dentistry 2013; 25(3):62-68). 
 
INTRODUCTION 

Glass ionomer cement was first introduced to 
the dental profession by Wilson and Kent in 1972. 
Their main characteristics are an ability to 
chemically bond to enamel and dentine with 
insignificant heat formation or shrinkage; 
Biocompatibility with the pulp and periodental 
tissues, fluoride release producing a cariostatic 
and antimicrobial action, less volumetric setting 
contraction and a similar coefficient of thermal 
expansion to tooth structure. These advantages 
have made them successful as luting cement and 
lining materials. However, as a restorative 
material, their sensitivity to moisture and low 
mechanical strength and wear resistance make 
them the least durable. This may be adequate for 
primary teeth because they will exfoliate in a 
number of years.1,2  

When the powder and liquid in conventional 
glass ionomer are mixed together, an acid-base 
reaction occurs between the polyalkenoate acid 
and ion leachable glass, resulting in a plastic paste 
which then hardens to a sold mass. The final set 
structure is a complex composite of the original 
glass particles sheathed by a siliceous hydrogel 
and bonded together by a matrix phase of 
hydrated fluoridated calcium and aluminum 
polyacrylate .3, 4 While GICs have been used 
successfully for over 30 years in dentistry, there 
are still concerns regarding GIC biocompatibility 
in non-dental applications.  
(1)Assistant professor, Department of Conservative Dentistry, 
College of Dentistry, University of Baghdad. 

 
In particular, Al3+ release has been associated 

with poor local bone mineralization and local 
neurotoxicity .5  

Numerous cell culture studies of cytotoxicity 
have reported cell inhibition by specific GIC 
compositions, and Brook and Hatton reviewed 
this work in 1998 .6 It appeared that the in vitro 
toxicity of GICs was due to a complex mechanism 
based on both ion release (in particular, Al3+ and 
F-) and pH effects .7 Early animal studies of GIC 
bone cements provided evidence of good 
biocompatibility. 8   

In this study we assist the X-ray diffraction of 
glass ionomer cement reinforced by different 
ratios of Hydroxyapatite and its biocompatability 
with Rabbit’s tissues.  
    
MATERIALS AND METHODS 
Preparation of experimental cement: 

 A powder of glass ionomer cement (medicem; 
Promedica; neumonster; Germany) was mixed 
with different ratios (10%, 15%, 20%, 25%, 30% 
by weight) of Hydroxyapatite then the mixed 
powder was applied in agate machine for half an 
hour and hour for grinding and to get homogenous 
mixture (better distribution of  Hydroxyapatite 
particles throughout the glass ionomer powder) 
while the liquid of glass ionomer cement 
remained unchanged. 
X-ray diffraction test: 
  A sample of experimental materials were 
prepared to undergo this test by mixing the 



J Bagh College Dentistry                               Vol. 25(3), September 2013                                X-ray diffraction  
 

Restorative Dentistry  63 
 

experimental powder and liquid materials and 
preparation of a cylindrical specimen by using a 
stainless steel mould constructed according to 
ADA specification No.6 then the sample were 
pulverized and exposed to CuKα radiation by 
using Mn filter, the values of diffraction will be 
measured and spacing will be calculated 
according to Bragg`s equation: 
2d=λ /sinθ 
Where: d: spacing (distance), λ : is the wavelength 
of CuKα radiation  
θ: is the diffraction angle 
 Using the value 1.54Aº for the wavelength of 
CuKα radiation, the figures of spacing (d) will be 
calculated and compared with ASTM standard 
table to detect the crystal components in the set 
materials. 
Histopathological study:  

Biocompatibility test was carried out on the 
rabbits by submucosal implantation of the control 
group (glass ionomer cement) and experimental 
groups {glass ionomer cement reinforced by 
different rations of Hydroxyapatite only (10%, 
20% and 30% was used for this test)} by using 
polyethylene tube as a carrier.   Females rabbits 
weight 2-2.5Kg was used in this study. The 
rabbits were divided into three groups according 
to period of implantation of the polyethyline tubes 
(3, 10 and 21 days).  Each rabbit has received two 
implants submucosal on the dorsal position (in 
order not to get trauma on the implanted site 
during movement of the rabbits).   
Anesthesia:  

General anesthesia was performed by 
intramuscular injection of a mixture of 88mg/Kg 
of body weight of Ketamine chloride (50gm/ml) 
and 10 mg/Kg body weight of Xylazin (2%). 
Sterilization:  

The sterilization of the polyethylene tube was 
performed as follows  

a- The tubes were immersed in pure ethyl 
alcohol for 15 minutes. 

b- Then the tubes were washed with normal 
saline solution. 

c- Autoclaving the tubes for half an hour at 
1000C was the last step of sterilization the 
polyethylene tube.  
All the instruments that were in contact with 

the rabbits were pre-sterilized by autoclaving. 
Preparation of the implants: 

The polyethylene tubes of 0.5mm internal 
diameter and 1mm external diameter were loaded 
with cement materials after mixing , and cut to 
pieces of 5mm in length(a negative pressure were 
used to easy the loading the base materials by 
sucking the other end of the tube by manual 
sucker).  

Preparation of the implanted site:                                      
The rabbit was anesthetized by intramuscular 

injections then the anesthetized rabbit was placed 
on its abdominal side on a sterilized surgical 
board. The dorsal skin of the intended 
implantation area was shaved and disinfected by 
povidone iodine solution, the area was located 5-6 
cm from the center of the dorsal side toward the 
tail, where it was observed to be the most difficult 
place to be scratched by the animal itself.  
Implantation of the base materials: 

Under the aseptic conditions, two incisions of 
approximately 10mm in length were made 
through the skin, one on each side of the dorsal 
side. The submucosal tissue was opened by blunt 
dissection, then the Polyethelene tubes filled with 
cement materials were held from the middle by a 
straight tweezers and inserted at the implanted site 
at least 2cm from the line of the incision after the 
implantation of each tube the wounds were 
sutured and the skin was scrubbed again with 
povidon iodion disinfectant. 
Animals grouping: 

 The rabbits were grouped for three time 
intervals of three days, ten days and three weeks 
(21 days).                                                                             
Euthanasia: 
  The rabbits of a time period were sacrified, 
each rabbit was injected intra-muscular with large 
dose of anesthetic Ketamine chloride and Xylesin. 
Preparation of histological section:                         

 The tubes were removed with the tissue, 
which was cut out in rectangular pieces to 
facilitate directional embedding and correct 
sectioning, then  were immersed in 10% buffered 
formalin. The tissue was fixed and processed for 
Parafin wax embedding, serial sections were cut 
to a thickness of 8μm by a microtome. One of 
every 10 slices was taken and placed in a water 
bath and then placed on a slide, which was taken 
to an oven at 40oC to adhere the slice to the slide.  
The slide was placed in Xylol to remove the 
paraffin surrounding the tissue. The slide was 
placed in a bath containing Haematoxelin and 
Eosin stain and was left for 10 minutes to stain the 
tissue. The slide was removed from staining bath, 
rinsed with distilled water and a glass cover was 
luted on the stained tissue with Canada balsam. 
Slides examination: 

Each slide was examined under light 
microscope at magnification of 12.5X and 20X to 
evaluate the intensity and degree of inflammatory 
reactions around each tube end, and the 
subsequent tissue healing at the sites of 
implantation. Slides were examined with aid of 
specialist in oral pathology, using double blind 
technique. 



J Bagh College Dentistry                               Vol. 25(3), September 2013                                X-ray diffraction  
 

Restorative Dentistry  64 
 

RESULTS 
A- X-ray Diffraction test: 
 X-ray diffraction test was performed on the 
following 
1- For the Hydroxyapatite, powder of glass 
ionomer cement, liquid of glass ionomer: The 
x-ray patterns showed in figure(1, 2, 3).The 
results showed that  both Hydroxyapatite and 
glass ionomer powder have crystalline structure 
while the liquid of glass ionomer cement has no 
crystalline structure because it composed of 
inorganic acids. 

Figure 1. x-ray diffraction pattern of 
synthetic Hydroxyapatite. 

 
Figure 2.  x-ray diffraction pattern of  

powder of glass ionomer cement. 
 

 
Figure 3. x-ray diffraction pattern of  liquid  

of glass ionomer cement. 
 
2-For the powder of set  glass ionomer cement 
material figure (4): The  results of this test 
showed that the final set material of glass ionomer 
cement has crystal of unreacted ZnO, may be act 
as cores for the set material. 

 
Figure 4. x-ray diffraction pattern of  set 

glass ionomer cement. 
 

3-For the powder of set  glass ionomer 
reinforced by (10%, 15%, 20 %, 25%  and 
30%) cement materials figure (5 ,6 ,7, 8 ,9):                                                   

The  results of this test showed that the final 
set material of glass ionomer cement reinforced 
by different ratios of  Hydroxyapatite has crystals 
of unreacted ZnO and Hydroxyapatite may be act 
as cores for the set material while other products 
of setting reaction non crystalline in nature also 
the results showed that most of Hydroxyapatite 
particles shear in the setting reactions giving new 
products non crystalline in nature. 
 

 
Figure 5. x-ray diffraction pattern of  set 
glass ionomer cement reinforced by 10% 

Hydroxyapatite. 

 
Figure 6. x-ray diffraction pattern of  set 
glass ionomer cement reinforced by 15% 

Hydroxyapatite. 



J Bagh College Dentistry                               Vol. 25(3), September 2013                                X-ray diffraction  
 

Restorative Dentistry  65 
 

 
Figure 7. x-ray diffraction pattern of  set 
glass ionomer cement reinforced by 20% 

Hydroxyapatite. 
 

 
Figure 8. x-ray diffraction pattern of  set 
glass ionomer cement reinforced by 25% 

Hydroxyapatite. 

 
Figure 9. x-ray diffraction pattern of  set 
glass ionomer cement reinforced by 30% 

Hydroxyapatite. 
 
B-Histopathological test: 
The histopathological pictures were qualitatively 
examined under light microscope regarding the 
intensity of the inflammatory response of the 
rabbit’s submucosal tissues to the   implanted 
controls and experimental groups and the degree 
of the subsequent tissue healing at different time 
periods. 
1-For the glass ionomer cement: 
At three days:Sever inflammatory response 
extend to the lateral side of the tube with necrosis 
near the implanted material figure(10).   

  
Figure 10. Polyethylene tube filled glass 

ionomer cement after 3 days Hematoxylin 
and Eosin.X 20. 

 
At ten days: There is large mass of 

granulation tissue figure (11). 
                   

 
Figure 11. Polyethylene tube filled glass 

ionomer cement after 10 days Hematoxylin 
and Eosin.X 20. 

At three weeks: there was connective tissue, 
hyalinization, fibrous tissue appeared in large area 
as hallow around the end of the implanted tube. 
There was coagulation degeneration adjacent to 
the base material figure (12). 
                   

 
Figure 12. Polyethylene tube filled glass 

ionomer cement after 3 weeks Hematoxylin 
and Eosin.X 20. 

 
2-For the glass ionomer cement reinforced 
by 10% Hydroxyapatite: 
At three days: 
  Sever inflammatory response extend to the 
lateral side of the tube with necrosis near the 
implanted material figure (13). 



J Bagh College Dentistry                               Vol. 25(3), September 2013                                X-ray diffraction  
 

Restorative Dentistry  66 
 

                

 
Figure13. Polyethylene tube filled glass 

ionomer cement reinforced by 10% 
Hydroxyapatite after 3 days Hematoxylin 

and Eosin.X 20. 
 

At ten days: There is large mass of 
granulation tissue figure (14).  
  

 
Figure 14. Polyethylene tube filled glass 

ionomer cement reinforced by 10% 
Hydroxyapatite after 10 days Hematoxylin 

and Eosin.X 20 
 

At three weeks: there was connective tissue, 
hyalinization, fibrous tissue appeared in large area 
as hallow around the end of the implanted tube. 
There was coagulation degeneration adjacent to 
the base material figure (15). 
                      

 
Figure 15. Polyethylene tube filled glass 

ionomer cement reinforced by 10% 
Hydroxyapatite after 3 weeks Hematoxylin 

and Eosin.X 20. 
 
 3-For the glass ionomer cement reinforced by 
20% Hydroxyapatite: 
At three days:  A cute inflammatory cells mainly 
neutrophiles and there was fibrous tissue 

figure(16).                

 
Figure 16. Polyethylene tube filled glass 

ionomer cement reinforced by 20% 
Hydroxyapatite after 3 days Hematoxylin 

and Eosin.X 20. 
 
At ten days: Thick dense fibrous tissue with 
mature fibroblast cells figure (17). 
                    

 
Figure 17. Polyethylene tube filled glass 

ionomer cement reinforced by 20% 
Hydroxyapatite after 10 days Hematoxylin 

and Eosin.X 20. 
 
At three weeks: A small mass of loose fibrous 

tissue with large active fibroblast cells (no 
inflammation) figure 

(18). 

Figure18. Polyethylene tube filled glass 
ionomer cement reinforced by 20% 

Hydroxyapatite after 3 weeks Hematoxylin 
and Eosin.X 20. 

 
4-For the glass ionomer cement reinforced by 
30% Hydroxyapatite: 
At three days:  A cute inflammatory cells mainly 
neutrophiles and there was fibrous tissue 



J Bagh College Dentistry                               Vol. 25(3), September 2013                                X-ray diffraction  
 

Restorative Dentistry  67 
 

figure(19).                       

 
Figure 19. Polyethylene tube filled glass 

ionomer cement reinforced by 30% 
Hydroxyapatite after 3 days Hematoxylin 

and Eosin.X 20. 
 
At ten days: Thick dense fibrous tissue with 

mature fibroblast cells figure (20). 
                           

 
Figure 20. Polyethylene tube filled glass 

ionomer cement reinforced by 30% 
Hydroxyapatite after 10 days Hematoxylin 

and Eosin.X 20. 
 

At three weeks: Thin loose fibrous tissue 
without inflammation figure (21). 
                       

 
Figure 21. Polyethylene tube filled glass 

ionomer cement reinforced by 30% 
Hydroxyapatite after 3 weeks Hematoxylin 

and Eosin.X 20. 
 
DISCASSION 

Hydroxyaptite (HA) is the main biomineral 
component of  human hard tissues (tooth and 
bone) and it's chemistry is represented by the 
formula ( Ca10(PO4)6OH) .

9,10It is biocompatible 
material when synthesized artificially as 
biomaterial and if  fluoride ions substitute 
hydroxyl ions on the hexagonal unit cell they give 
rise to fluorapatite ( Ca10(PO4)6F2). 

11 Associated 

with the biometric process, crystal growth can be 
used for dentistry applications on the enamel. 
Crystal growth conventionally involves the 
application of a sulphated acid solution to the 
enamel .12   X-ray diffraction instruments are used 
to measure crystal structure, grain size, texture 
and/or residual stress of materials and compounds 
through interaction of X-ray beam with a sample.  

Before introducing a new material to the 
market, its properties and biocompatibility must 
be previously studied. From a biological point of 
view, its irritant potential must be evaluated 
because eventual toxic components may cause 
irritation, degeneration or even necrosis of the 
tissues adjacent to the material. 13 This restorative 
cement is produced via an acid-base reaction 
between the glass (Ca-FAlSi) and an organic 
polymer acid (e.g., polyacrylic acid), which 
results in very interesting physical and chemical 
properties, such as biocompatibility, high 
adhesiveness compared to other restorative 
materials, and cariostatic properties owing to the 
sustained release of fluorides. 14, 15 

According to the methodology used in this 
study, implantation of standard polyethylene tubes 
containing the material for analysis, which only 
comes into contact with the subcutaneous 
connective tissue through the tubular opening on 
one side, as the other side is sealed, allows a 
comparative analysis among the experimental 
groups in a standardized manner, without 
interferences determined by variables of volume 
and areas of juxtaposition. In turn, the external 
walls of the polyethylene tube serve as control 
due to their low irritating potential, as a basic 
parameter of the ideal model of reactionary 
development. Thus, a comparative analysis of the 
experimental groups could be safely done 
between the test groups and between them and the 
control 16, 17-19 

In order to increase bonding to bone, 
hydroxyapatite reinforced glass ionomer cements 
(HA-GIC) have been developed. 20 A number of 
researchers have attempted to evaluate the effect 
of the addition of HA powders to restorative 
dental materials.17 In this study, the powder of 
glass ionomer cement reinforced by different 
ratios of Hydroxyapatite (HA) has excellent 
biological behavior, and this is agree with other 
studies.20, 21 The results of histopathological test 
shows that increase the percentage of 
hydroxyapatite may improve the biocompatibility 
of glass ionomer cement especially after three 
weeks which may related to that hydroxyapatite 
(HA) has excellent biological behavior, and its 
composition and crystal structure are similar to 



J Bagh College Dentistry                               Vol. 25(3), September 2013                                X-ray diffraction  
 

Restorative Dentistry  68 
 

the apatite in the human dental structure and 
skeletal system. 21  

In spite of the positive results found for the 
experimental GIC in this study, it is worth 
emphasizing that further research is needed before 
this material can be indicated for clinical use. 
 
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