|Year : 2019 | Volume
| Issue : 5 | Page : 755-762
|Role of angiogenesis in oral submucous fibrosis using vascular endothelial growth factor and CD34: An immunohistochemical study
Ettishree Sharma1, Nutan Tyagi1, Vineeta Gupta1, Anjali Narwal2, Hitesh Vij3, Dheeraj Lakhnotra4
1 Department of Oral Pathology and Microbiology, Institute of Dental Studies and Technologies College, Modinagar, Uttar Pradesh, India
2 Department of Oral Pathology, Post Graduate Institute of Dental Sciences, Rohtak, Haryana, India
3 Department of Diagnostic Sciences and Oral Biology, Division of Oral Pathology, King Khalid University, Abha, Kingdom of Saudi Arabia
4 Dental Surgeon, Institute of Dental Sciences, Jammu, Jammu and Kashmir, India
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|Date of Submission||14-Aug-2018|
|Date of Decision||20-Jul-2017|
|Date of Acceptance||17-Oct-2017|
|Date of Web Publication||18-Dec-2019|
| Abstract|| |
Background: Oral submucous fibrosis (OSF) is an insidious, chronic, disabling disease, in which there is lack of perfusion due to reduced level of the vasculature and this is said to be responsible for the epithelial atrophy seen in OSF. The degree of vasculature of the affected mucosa and its effects on the epithelial thickness remains controversial till date. Aims: This study attempts to analyze the role of angiogenesis in OSF and its progression using vascular endothelial growth factor (VEGF) and CD34 markers. Materials and Methods: The study samples for the present study comprised of 10 cases each of early OSF, moderately advanced, advanced OSF, and 10 cases of normal oral mucosa were used as controls. All the cases were subjected to immunohistochemical staining with VEGF and CD34 markers. Results: Among the different grades of OSF, we did not find any noticeable difference in VEGF expression although we found a upregulation in microvessel density (CD34) in early and moderately advanced OSF followed by a downregulation in advanced OSF. Conclusions: As the disease progresses, there is an increased production of the extracellular matrix component (collagen I and II and fibronectin) and results in fibrosis. Subsequently, it leads to the reduction in the level of corium vascularity and results in hypoxia which ultimately causes reduction and constriction of the vascular channels. This sequence of events alerts us to the relevance of early disease diagnosis and management in an irreversible pathology such as OSF.
Keywords: Angiogenesis, angiogenic markers, CD34, immunohistochemistry, oral submucous fibrosis, vascularity, vascular endothelial growth factor
|How to cite this article:|
Sharma E, Tyagi N, Gupta V, Narwal A, Vij H, Lakhnotra D. Role of angiogenesis in oral submucous fibrosis using vascular endothelial growth factor and CD34: An immunohistochemical study. Indian J Dent Res 2019;30:755-62
|How to cite this URL:|
Sharma E, Tyagi N, Gupta V, Narwal A, Vij H, Lakhnotra D. Role of angiogenesis in oral submucous fibrosis using vascular endothelial growth factor and CD34: An immunohistochemical study. Indian J Dent Res [serial online] 2019 [cited 2020 Apr 2];30:755-62. Available from: http://www.ijdr.in/text.asp?2019/30/5/755/273416
| Introduction|| |
Oral submucous fibrosis (OSF) is a chronic, insidious disease characterized by juxta-epithelial inflammatory reaction and progressive fibrosis of the submucosal tissue which primarily involves the oral cavity, oropharynx, and upper third of the esophagus., It is considered as an “Oral Potentially Malignant Disorder” which is seen predominantly in the Asian population. In ancient Indian medical literature, a condition similar to OSF was described by “Sushruta” as Vidari showing features such as reduction in mouth opening, depigmentation of oral mucosa, and pain on eating food. Later, in 1952, Schwartz reported the same condition in five Indian women who emigrated to East Africa and entitled it as “Atrophia Idiopathica (tropica) mucosae oris.” In 1953, another case was described by Joshi which he termed as “submucous fibrosis of the palate and pillars.” Various other terms such as “diffuse oral submucous fibrosis,” “idiopathic scleroderma of the mouth,” “idiopathic palatal fibrosis,” and “sclerosing stomatitis” were suggested, but the most widely accepted term is “submucous fibrosis.”
Results from various studies done in 2002 concluded that more than 5 million people in India alone have OSF (0.5% of the Indian population). The disease is most frequent in southern parts of India and with the highest prevalence in Kerala. OSF cases have also been reported among people of Indian origin across the world mostly in Taiwan, Nepal, Sri Lanka, Malaysia, Thailand, Vietnam, Uganda, and South Africa.
Epidemiological studies reveal that the malignant transformation rate of OSF is 4.5%–7.6%. Paymaster in 1956, first mentioned about the precancerous nature of OSF after observing the development of squamous cell carcinoma in one-third of his OSF patients. It is generally believed that malignant changes are mostly attributed to an atrophic epithelium more than an epithelium with normal thickness, and thus, there is more tendency to develop oral cancer from potent carcinogens in any lesion with atrophic epithelium. The presence of altered cytokine activity accompanied with dense fibrosis and reduced vascularity generate a favorable environment for the carcinogenic products to produce changes in the epithelium. It has been observed that there is a higher incidence of leukoplakia among submucous fibrosis patients than those without the disease.
Histological features of this disease include juxta-epithelial fibrosis, hyalinized collagen accumulation beneath the basement membrane and progressive reduction of vascularity. The degree of vascularity of the diseased mucosa and its effect on the epithelial thickness in OSF has always been a matter of debate. The measurement of vascularity cannot be done directly, but quantification can be done indirectly by means of angiogenic markers such as CD34, CD105, and CD35.
Angiogenesis is the formation of new capillary blood vessels, it is one of the most universal and fundamentally important biological phenomenon occurring in mammalian organisms. Angiogenesis is a significant process that occurs in a number of physiological events, such as embryonic growth, chronic inflammation, wound healing, and in pathological conditions such as progression of a tumor.
The quantification of microvasculature can be done by the assessment of microvessel density (MVD) using endothelial markers such as CD34, CD31, CD105, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (FGF)., CD34 (human hematopoietic progenitor cell antigen) is a surface glycophosphoprotein of unknown functions. It is regarded as a significant marker for vascularization demonstrating MVD in the tissues. The expression of CD34 is seen in the lymphohematopoietic stem and progenitor cells, the endothelium, embryonic fibroblasts, the interstitial cells of Cajal and in the dendritic cells present in the dermis, around blood vessels and in the nerve sheath., Immunohistochemically, CD34 is mainly expressed on small or newly formed vessels; however, the endothelial cells of small and large blood vessels in both normal and tumor tissue have been reported to be stained with equal intensity.
VEGFs and its receptors VEGFR-1, VEGFR-2 are the essential molecules in the development of vasculature. It helps not only in the regulation of blood and lymphatic vessel development and homeostasis but also has intense effects on neural cells. Hypoxia plays a major role in the production of VEGF. Under hypoxic conditions, endothelial, hematopoietic, and stromal cells get stimulated by growth factors such as transforming growth factors (TGF), interleukins, or platelet-derived growth factor and results in secretion of VEGF. VEGF receptor-2 is a major pro-angiogenic receptor and mediates most of the downstream effects of VEGF-A. This study attempts to analyze angiogenesis in OSF and to evaluate its possible role in the progression of the disease using VEGF and CD34.
| Materials and Methods|| |
The material for present study comprised 40 formalin-fixed paraffin-embedded tissue blocks comprising of previously diagnosed cases of normal mucosa (Group I; n = 10), of early OSF (Group II; n = 10), moderately advanced OSF (Group III; n = 10), and advanced OSF (Group IV; n = 10), which were retrieved from the archives of the Department of Oral Pathology and Microbiology, IDST, Modinagar and a private laboratory in Jaipur. Histological diagnosis was made based on examination of H and E-stained slides and was assessed based on Sirsat and Pindborg classification given in 1966. Cases of Renal glomerulus and fibrosis were taken as positive control for VEGF and CD34, respectively. Omitting the use of the primary antibody and carrying out the successive steps of immunohistochemistry as usual, gave negative control stained CD34 and VEGF slides. From each paraffin-embedded block, 2 sections were cut maintaining the thickness of 3–4 μm.
All glasswares used in the staining process were gently cleaned with running distilled water before usage. From each paraffin-embedded block, 2 sections were cut using a semiautomatic rotary microtome maintaining the thickness at 3–4 μm. The sections were then lifted on to the precoated (3-Aminopropyltriethoxysilane) slides.
The slides were placed on the slide racks and then into the hot air oven at a temperature of 55°C to 65°C for ½ h. The sections were cleared by passing them through 3 changes of xylene for 5 min each and the sections were hydrated by passing them through 3 changes of acetone for 5 min each, then the slides were immersed in distilled water for 5 min. The sections were covered with peroxidase block followed by washing in Tris buffer solution. The slides, after buffer treatment were immersed in a larger glass microwave oven container filled with antigen retrieval solution (20 ml Diva in 400 ml of distilled water). The microwave oven was run for 2 cycles of 10 min and 5 min at a temperature of 95°C and 99°C, respectively, with an interval of 5 min between the two cycles.
Following this, the slides were transferred to Coplin jars filled with wash buffer for 5 min. The excess buffer on the slides was tapped off and covered with protein block (normal horse serum) for 10 min. The protein block solution was gently tapped off the slides. The sections were then covered with primary anti-human rabbit monoclonal antibody VEGF (Biocare Medical Manufacturers) and mouse monoclonal anti-human CD34 antibody (Biocare Medical Manufacturers), and the slides were incubated for one h at room temperature in a humidifying chamber. The slides were then washed gently with TBS thoroughly and kept in the TBS buffer bath for five min. After tapping off the excess buffer, the sections were incubated with the secondary conjugated polymer (Biocare Medical Manufacturers) for 25 min at room temperature in a humidifying chamber.
Then, the slides were washed gently with TBS and kept in the TBS buffer bath for five min. Chromogen 3,3'-diaminobenzidine (DAB) was freshly prepared by adding one drop of DAB to 1 ml of stable DAB substrate buffer. The prepared DAB was kept in a dark place till usage. Excess buffer was tapped off, and the tissue sections were covered with freshly prepared substrate chromogen (DAB) solution for five min. Subsequently, the slides were gently rinsed in distilled water and counterstained with Mayer's hematoxylin for 4 min, and then washed gently under running water for about 10 min, they were dehydrated by passing through two changes of acetone and then cleared by dipping in xylene and later mounted in DPX. The slides were interpreted using a light microscope (Olympus CX41).
All the slides were viewed under the (Olympus CX41) light microscope at 400X magnification by three observers. The positively stained CD34 and VEGF areas showed uptake of brown color. For the assessment of VEGF immunostaining, semi-quantitative scoring was done in the epithelium in five consecutive nonoverlapping fields to evaluate the overall percentage area that stained positive and also to judge the level of intensity of immunostaining. The categorization of grading for an area based on percentage was assigned as: No stain = 0; <25% = 1; 25%–50% =2; 50%–75% = 3; >75% = 4. For scoring the VEGF immunostaining intensity, the criteria considered was: None = 0; Mild = 1; Moderate = 2; and Intense = 3. The final score was attained by multiplying percentage area score (0–4) with intensity score (0–3). Thus, the final score ranging between 0 and 4 was considered as negative staining, whereas a score ranging 5–12 was regarded as positive staining.
All brown-stained structures containing lumen were regarded as CD34 positive microvessels. The vessel count was recorded from areas showing high vascular density adjacent to the basement membrane in five consecutive nonoverlapping high power fields (hotspots) in each slide. These areas were assessed independently by two other observers also (O2 and O3). The mean of the vessel count from the five fields was considered as the final score for evaluating the expression of CD34.
The statistical software, namely, SPSS version 19.0 (SPSS, IBM, Armonk, NY) was used to analyze the data, and Microsoft Excel was used to generate graphs, tables, etc. Results on continuous measurements are presented on mean ± standard deviation and results on categorical measurements are presented in numbers (%). The significance is assessed at 5% level of significance. Chi-square, Independent t-test, ANOVA with post hoc, Bonferroni for multiple comparison, and Pearson's correlation tests have been used to find the significance of study parameters on the categorical scale and ordinal scale between two or greater than two groups, Cronbach's alpha reliability test for inter-observer agreement. A probability value of ≤0.05 was considered to be statistically significant.
| Results|| |
In normal mucosa cases, there were 8 (80%) male and 2 (20%) female, whereas in OSF cases, there were 28 (93.3%) male and 2 (6.6%) female. The mean age for normal mucosa cases was 23.7 ± 4.05 and for OSF it was 29.47 ± 6.18.
Inter-observer agreement was assessed statistically by applying Cronbach's alpha reliability test and the results were found to be in agreement. Therefore, for further statistical analysis, only the findings of observer 1 were considered.
Evaluation of vascular endothelial growth factor immunostaining
The VEGF immunostaining was found to be heterogeneous, diffuse, brownish, and exhibited a granular staining of the cytoplasm of the epithelial cells along with a few stromal components such as fibroblasts, endothelial cells, inflammatory cells, salivary glands, and muscle fiber bundles. Immunolocalization of VEGF was found in the basal layer of normal oral mucosa with a negative-to -mild staining intensity. All scores of staining intensity were observed in early OSF and moderately advanced OSF while only mild and moderate staining intensities were seen in advanced OSF [Figure 1].
|Figure 1: Photomicrograph showing (a) Negative vascular endothelial growth factor immunostaining in normal mucosa. (b) Mild vascular endothelial growth factor immunostaining in early oral submucous fibrosis. (c) Moderate vascular endothelial growth factor immunostaining in early oral submucous fibrosis. (d) Intense vascular endothelial growth factor Immunostaining in early oral submucous fibrosis. (e) Mild vascular endothelial growth factor immunostaining in moderately advanced oral submucous fibrosis. (f) Moderate vascular endothelial growth factor immunostaining in moderately advanced oral submucous fibrosis. (g) Intense vascular endothelial growth factor immunostaining in moderately advanced oral submucous fibrosis. (h) Mild vascular endothelial growth factor immunostaining in advanced oral submucous fibrosis. (i) Moderate vascular endothelial growth factor immunostaining in advanced oral submucous fibrosis (IHC, ×400)|
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Percentage area distribution of vascular endothelial growth factor immunoreactivity
The mean of the overall distribution of percentage area using VEGF in normal mucosa was found to be 0.90 ± 0.59, whereas in OSF, the mean value was observed as 2.01 ± 0.80 which was considered to be highly significant (P < 0.001). The group-wise mean of the overall distribution of percentage area in Group I, II, III, and IV were 0.90 ± 0.59, 2.42 ± 0.69, 2.02 ± 0.64 and 1.6 ± 0.88, respectively. All the readings were highly significant (P < 0.001). By comparing the percentage area distribution of VEGF among normal mucosa and different grades of OSF, significant results were seen when Group 1 was compared with Group II (P < 0.001) and Group III (P = 0.008) as listed in [Table 1]a.
Intensity of vascular endothelial growth factor immunostaining
The overall VEGF staining intensity was evaluated, and the mean value in normal mucosa was observed as 0.56 ± 0.37and 1.63 ± 0.7 in OSF. A statistically significant correlation (P < 0.001) was observed between the mean VEGF staining intensity in normal oral mucosa and OSF. The group-wise analysis of staining intensity by VEGF was assessed and the mean value for Group I was found as 0.56 ± 0.37. The staining intensity in Group II, Group III, and Group IV was observed as 1.68 ± 0.55; 1.7 ± 0.7; 1.42 ± 0.80, respectively. All the values were statistically significant (P = 0.001). On assessment between all the groups, statistically significant results were found between Group I when compared individually with Group II, III, and IV as shown in [Table 1]b.
Evaluation of vascular endothelial growth factor final score
The mean of the final score of VEGF expression in normal mucosa (Group I) was 0.70 ± 0.62, whereas in OSF it was 3.65 ± 2.22. A statistically significant difference (P < 0.001) was found between VEGF final score in normal oral mucosa and OSF. The group-wise mean of the final score of VEGF expression in Group II was 4.32 ± 2.19, in Group III was 3.75 ± 2.21, and Group IV was 2.88 ± 2.24 and all the values were found to be statistically significant (P = 0.001). While comparing all the groups, significant results were seen only when Group I was compared with Group II and with Group III [Table 1]c.
The overall VEGF-positive immunostaining was observed in 40% (12) cases of OSF. A statistically significant correlation (P = 0.017) was observed between VEGF positivity in normal oral mucosa and OSF. The group-wise VEGF immunoreactivity observed in Group II, III, IV was 50% (5), 30% (3), and 40% (3), respectively. We observed all the scores of staining intensity in Group I and II while only mild and moderate staining intensities were seen in advanced OSF.
Evaluation of CD34 immunostaining
In the present study, expression of CD34 was detected in many microvessels. A sharp and crisp brown color indicating CD34-positive immunostaining was seen in the endothelial cells of the blood vessels in normal oral mucosa, all grades of OSF as illustrated in [Figure 2] and also in the positive control. The overall MVD observed in normal mucosa was found as 3.84 ± 0.56, whereas in OSF, the mean value was found to be 12.52 ± 4.51. All the findings were found to be significant (P < 0.001). The group-wise quantitative analysis for MVD in Group I, II, III, and IV was found as 3.84 ± 0.56; 15.44 ± 4.12; 11.92 ± 2.77; 10.22 ± 5.02, respectively. A statistically significant correlation (P < 0.001) was found between the mean MVD in normal oral mucosa and mean MVD in OSF. On comparison among all the groups, statistically significant results were found in all the groups except between Group II and III, and between Group III and IV [Table 2].
|Figure 2: Photomicrograph showing CD34-positive endothelial lined blood vessels. (a) normal mucosa, (b) early oral submucous fibrosis, (c) moderately advanced oral submucous fibrosis, and (d) advanced oral submucous fibrosis (IHC, ×400)|
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|Table 2: Immunohistochemical analysis of microvessel density in normal mucosa and oral submucous fibrosis using CD34|
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Correlation between vascular endothelial growth factor and CD34 expression
The correlation between CD34 and VEGF expressions was found to be statistically significant with a P < 0.001 as shown in [Table 3].
|Table 3: Correlation between CD34 and vascular endothelial growth factor expression|
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| Discussion|| |
OSF has a relentless course of progression and is not amenable to reversal at any stage of the disease process even after termination of the alleged causative factor, that as areca nut chewing. Different studies have shown it to be a consequence of disturbances in the homeostatic equilibrium between synthesis and degradation of the extracellular matrix (ECM), wherein collagen forms a major component, thus OSF can be considered as a collagen-metabolic disorder.,,,
As the disease progresses, there is lack of perfusion due to reduced level of the vasculature and this is said to be responsible for the epithelial atrophy seen in OSF. The degree of vasculature of the ailing mucosa and its effects on the epithelial thickness remains controversial till date. The direct measurement of vasculature is not possible; however, quantification can be done indirectly by means of various angiogenic markers. Angiogenic properties are known to be associated with the aggressiveness of the disease.
Literature studies have stated that MVD can be considered as an indirect indicator of neoangiogenesis. To stain microvessels, CD34 antibody has been used as a marker for the early hematopoietic stem/progenitor cells and has been established as a potent hematopoietic cell-surface antigen.
A limited literature pool exists on studies which have been conducted focusing on the role of angiogenesis in OSF. We attempt here to analyze the role of angiogenesis in OSF using both VEGF and CD34 markers and to evaluate their role in the progression of this disease.
Our study demonstrates a very diffuse, brownish granular expression in the cytoplasm of epithelial cells and of the stromal components such as fibroblasts, inflammatory cells, and muscles. In case of normal mucosa, negative–to-mild expression of VEGF was observed, whereas in case of OSF, all the staining intensities were seen in early and moderately advanced OSF. In case of advanced OSF only mild-to-moderate staining intensities were observed [Figure 1].
A significant increase in VEGF expression (percentage area distribution and staining intensity) (P < 0.001) were observed as the disease progressed from normal oral mucosa to OSF. Similar results were noted by Nayak et al. in 2013, where they compared the expression of VEGF with decorin and reported positive VEGF-A immunostaining in the lining epithelium in OSF. Another study done by Anura et al. 2014 showed contradictory results as they found a nonsignificant correlation between normal oral mucosa and OSF as there was a decrease in the VEGF expression in case of OSF with dysplasia when compared to OSF without dysplasia and also in cases of OSF without dysplasia as compared to normal oral mucosa. They mentioned that the reduced expression of VEGF in OSF without dysplasia could be due to increased fibrosis which leads to reduction and constriction of vascular channels in the corium along with flattened rete pegs.
There was a significant increase (P = 0.001) in VEGF expression from normal-to-early OSF and a downregulated expression in moderately advanced and advanced OSF. The overall VEGF expression was compared and significant results were largely noticed between normal oral mucosa and all groups of OSF (P = 0.001 and 0.007) but for advanced OSF (P = 0.099). Till date, there is no literature that explains the expression of VEGF amid different grades of OSF.
In the present study, expression of CD34 was detected in many microvessels. A sharp and crisp brown color indicating CD34-positive immunostaining was seen in the endothelial cells of the blood vessels in normal oral mucosa, all grades of OSF and also in the positive control. There was a significant upregulation (P < 0.001) in the vascularity as the disease progressed from normal oral mucosa to OSF [Figure 2]. Desai et al. in 2010 reported insignificant results regarding MVD in OSF and normal mucosa. They did not find any noticeable reduction in the vasculature in OSF as compared to that seen in normal mucosa. According to them, the stroma becomes more hyalinized by continuous deposition and cross-linking of mature collagen bundles which created tissue hypoxia followed by the development of new blood vessels to compensate the hypoxia. Pandiar and Shameena in 2014 also reported a contradictory finding as they observed more vascularity in normal oral mucosa than OSF.
There was a significant increase (P < 0.001) in vascularity as the disease progressed from normal to early OSF. Then, a downregulated expression was noticed as the grade proceeded from early to advanced OSF. We found the expression of CD34 to be statistically significant (P < 0.001) between normal oral mucosa and the total of all the cases of OSF. However, among the OSF grades, statistically significant (P = 0.013) results were found between the early and advanced OSF cases only, whereas nonsignificant results were observed when early OSF was compared with moderate OSF (P = 0.197) and advanced OSF (P = 1.000).
These findings were in agreement with the outcomes of Ekanayaka and Tilakaratne in 2013 who reviewed the literature and suggested that with advanced fibrosis, the vascularity of the mucosa decreases. Singh et al. in 2010 reported a marked increase in MVD in Grade II (early OSF) and a noticeable reduction in Grade III (moderately advanced OSF) and Grade IV (advanced OSF). They also correlated collagen thickness with advancing grades and epithelial cells density. As the grade increased, the thickness of collagen was also found to increase but the epithelial cell density decreased. They also suggested that muscle fiber bundles in the stroma get invaded by collagen as the disease progresses, and this may cause a persistent insult which leads to constriction and obliteration of blood vessels along with reduction in their number. Debnath et al. in 2013, reported an inversely proportional relation of blood vessel density to advancing grades of OSF and supported the hypothesis given by Singh et al. in 2010.
Contrarily, the findings of Rajendran et al. in 2005 did not correlate with our findings as they did not find any significant changes in MVD among the different grades of OSF in comparison with normal mucosa. They hypothesized that to compensate for hypoxia caused by collagen predominance; there was an increased vasculature as an adaptive response.
Till date, there is no definitive data in the literature that elucidates the correlation between CD34 and VEGF in OSF. In the present study, we, however, noticed significant correlation between CD34 and VEGF expressions. As the disease progresses, there is an increased production of ECM component (collagen I, collagen II, and fibronectin) which is responsible for the fibrosis seen. This in turn leads to reduction in the level of corium vascularity which in-turn results in hypoxia and stimulates an overexpression of HIF1-α. These hypoxic conditions affect the connective tissue fibroblasts and also the surface epithelium. Consequently, apoptosis is induced leading to epithelial atrophy and ulcerations. There is a decrease in VEGF expression in advanced cases owing to increased fibrosis which ultimately causes reduction and constriction of the vascular channels, that is, decrease in vessel diameter. Further, with progressive fibrosis, the chances of development of dysplasia also increase. HIF 1-α is also associated with both upregulation and downregulation of different growth factors such as VEGF, TGF-β, platelet-derived growth factor, FGF, and epidermal growth factor receptor which are responsible for the progression and eventual malignant transformation of OSF.
| Conclusion|| |
As the disease progresses, there is an increased production of the extracellular matrix component (collagen I and II and fibronectin) and results in fibrosis. Subsequently, it leads to the reduction in the level of corium vascularity and results in hypoxia which ultimately causes reduction and constriction of the vascular channels. This sequence of events alerts us to the relevance of early disease diagnosis and management in an irreversible pathology such as OSF. This can be aided by incorporating more immunomarker studies and molecular researches with a larger sample size.
We would like to thank Mr. Mudit Sharma for his technical assistance with immunohistochemistry.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Ettishree Sharma
Department of Oral Pathology and Microbiology, Institute of Dental Studies and Technologies College, Kadrabad, Modinagar - 201 201, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]
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