Myofibroblasts in odontogenic cysts and tumors: An immunohistochemical study

Soujanya Pinisetti; Durgaprasad Tadi; Ravikanth Manyam

doi:10.4103/jmau.jmau_64_21

ORIGINAL ARTICLE

Year : 2023 | Volume : 11 | Issue : 1 | Page : 68-73

Myofibroblasts in odontogenic cysts and tumors: An immunohistochemical study

Soujanya Pinisetti¹, Durgaprasad Tadi², Ravikanth Manyam³
¹ Department of Oral Pathology, Government Dental College and Hospital, Vijayawada, Andhra Pradesh, India
² Department of Prosthodontics, Drs. Sudha and Nageswara Rao Siddhartha Institute of Dental Sciences, Krishna, Andhra Pradesh, India
³ Department of Oral Pathology, Vishnu Dental College, Bhimavaram, Andhra Pradesh, India

Date of Submission	02-Jul-2021
Date of Decision	25-Aug-2021
Date of Acceptance	13-Feb-2022
Date of Web Publication	06-Jun-2022

Correspondence Address:
Dr. Soujanya Pinisetti
Department of Oral Pathology, Government Dental College and Hospital, Vijayawada, Krishna - 520 008, Andhra Pradesh
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jmau.jmau_64_21

Abstract

Objective: The objective of this study was to assess immunohistochemically the presence of myofibroblasts both qualitatively and quantitatively in odontogenic cysts and tumors and to compare with the control cases of squamous cell carcinoma and to correlate the results with biologic behavior of these lesions. Materials and Methodology: Formalin-fixed, paraffin-embedded blocks of odontogenic cysts and tumors were retrieved from institutional archives. The sample size is 40; these include ten cases of odontogenic keratocyst (OKC) (n = 10), five cases of dentigerous cyst (n = 5), ten cases of solid ameloblastoma (n = 10), and five cases of unicystic ameloblastoma (n = 5). Ten cases of squamous cell carcinoma (n = 10) served as control. Sections were taken and stained immunohistochemically using alpha-smooth muscle actin for evaluation of myofibroblasts. The number of positive stromal cells was evaluated both for quantitative and qualitative analyses. Results: The present study showed that the mean number of myofibroblasts among the odontogenic cysts and tumors was higher in locally aggressive lesions such as OKC (23.79 ± 19.95), solid ameloblastoma (26.38 ± 17.00), and unicystic ameloblastoma (20.74 ± 14.86) which were comparable to squamous cell carcinoma (21.49 ± 9.76) when compared to benign lesions like dentigerous cyst which showed the least number of myofibroblasts (13.1 ± 7.71). Qualitatively, the staining intensity of myofibroblasts showed a significant variation within the same lesion and among different lesions. There was a distinct difference in the morphology, pattern of arrangement, and distribution of myofibroblasts among the studied lesions. Conclusion: We conclude that the increase in the number of myofibroblasts could be one of the contributory factors for the locally aggressive behavior of benign lesions such as ameloblastomas and OKCs. Further studies are suggested to understand the mechanism by which these important cellular elements exert their effects on stromal and epithelial tissue compartments.

Keywords: Biologic behavior, myofibroblasts, odontogenic cysts, odontogenic tumors

How to cite this article:
Pinisetti S, Tadi D, Manyam R. Myofibroblasts in odontogenic cysts and tumors: An immunohistochemical study. J Microsc Ultrastruct 2023;11:68-73

How to cite this URL:
Pinisetti S, Tadi D, Manyam R. Myofibroblasts in odontogenic cysts and tumors: An immunohistochemical study. J Microsc Ultrastruct [serial online] 2023 [cited 2023 Mar 20];11:68-73. Available from: https://www.jmau.org/text.asp?2023/11/1/68/346625

Introduction

Odontogenic cysts and tumors comprise a group of lesions derived from the tooth-forming apparatus and odontogenic epithelial remnants. Majority of these lesions are usually benign, but some lesions such as odontogenic keratocyst (OKC) and ameloblastoma exhibit a locally aggressive behavior.^[1] Understanding the biologic behavior of these lesions is of fundamental importance in final diagnosis and treatment planning.

The difference in the degree of aggressiveness in the biologic behavior of these lesions is attributed not only to the epithelial component but also to connective tissue components. Stroma is essential for the maintenance of epithelial tissues. The cooperation between the parenchymal and stromal cells plays an important role in pathologic phenomena such as tumor invasion and progression.^[2] It is well known that many epithelial tumors are characterized by local accumulation of connective tissue cells which is called as stromal reaction. Initially, these cells were called as the peritumoral fibroblasts or tumor-associated fibroblasts, but these cells were different from fibroblasts and were found to express alpha-smooth muscle actin and were called as myofibroblasts.^[3]

Myofibroblasts are a modified form of fibroblasts with contractile property similar to smooth muscle cells. The presence of myofibroblasts has been reported in various normal and pathologic tissues. They play an important role in extracellular matrix synthesis, reorganization, and tissue contraction during wound healing and pathologic process like tumorigenesis.^[4]

With this background, we have planned our study to evaluate the presence of myofibroblasts immunohistochemically in odontogenic cysts and tumors and to compare the biologic behavior of these lesions by taking squamous cell carcinoma as control.

Materials and Methodology

This is a retrospective study. Formalin-fixed, paraffin-embedded blocks of odontogenic cysts and tumors were retrieved from the institutional archives. Institutional ethical comitee approval was obtained with reference number :VDC/IEC/2017/56. The sample size is 40; these include ten cases of OKC (n = 10), five cases of dentigerous cyst (n = 5), ten cases of solid ameloblastoma (n = 10), and five cases of unicystic ameloblastoma (n = 5). Ten cases of squamous cell carcinoma (n = 10) served as control. Diagnosis for the selected cases was established on hematoxylin- and eosin-stained sections according to the WHO criteria for odontogenic cysts and tumors. Tissue sections with sufficient representation of stromal connective tissue are included and sections with considerable inflammatory infiltrate in the stroma and cystic walls were excluded.

Staining procedure and evaluation of myofibroblasts

The histologic sections were immunohistochemically stained with alpha-smooth muscle actin (α-SMA) mouse anti-human antibody (BioGenex, clone IA4). Human colon was used as the external positive control. Whereas, α-SMA-positive cells within the blood vessels served as the positive control for the specificity of the stain. The number of positive stromal cells was counted for quantitative analysis. Counts were performed on Olympus microscope using Image-Pro Plus image analysis software. Representative fields were randomly selected and ten fields were chosen for each section at 40× magnification. Each α-SMA-positive cell, excluding those surrounding blood vessels, was counted and the total number of positive cells for all ten examined fields per case was calculated. This allowed calculation of the mean number of α-SMA-positive cells per field. Results were presented as the mean number of α-SMA-positive cells per field. Differences in the mean number of α-SMA-positive cells per field among all types of lesions and the differences among the group of odontogenic cysts and among the group of odontogenic tumors were analyzed using one-way analysis of variance test.

Results

In our study, the mean number of myofibroblasts among the odontogenic cysts and tumors was higher in OKC (23.79 ± 19.95), solid ameloblastoma (26.38 ± 17.00), and unicystic ameloblastoma (20.74 ± 14.86) which were comparable to squamous cell carcinoma. Dentigerous cyst showed the least number of myofibroblasts [Table 1].

Table 1: Mean number of alpha-smooth muscle actin-positive cells per field in odontogenic lesions and squamous cell carcinoma

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Among the odontogenic cysts, the mean number of myofibroblasts was higher in OKC when compared to dentigerous cyst, and this difference was statistically significant (P < 0.05). Among the odontogenic tumors, there was a significant difference in the mean number of myofibroblasts between solid and unicystic ameloblastomas. Solid ameloblastomas showed more number of myofibroblasts when compared to unicystic ameloblastomas, and this difference was statistically significant. When odontogenic cysts were compared with squamous cell carcinomas, the mean number of myofibroblasts per field in squamous cell carcinoma was not statistically significant from that of OKC with P value of 0.174. When odontogenic tumors have been compared with squamous cell carcinomas, the mean number of myofibroblasts was higher in solid ameloblastomas, and this difference was statistically significant.

According to Chi-square test, the staining intensity of myofibroblasts showed a significant variation within the same lesion and among different lesions. The morphology of myofibroblasts is spindle shaped in all cases, but we observed difference in the arrangement, few appear to be isolated and others in network interconnected with one another [Table 2].

Table 2: Morphology and arrangement of myofibroblasts in odontogenic cysts and tumors and squamous cell carcinoma

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For the analysis of distribution of myofibroblasts and their significance to the epithelium, the lesions have been broadly classified into cystic and solid lesions. Cystic lesions included all odontogenic cysts and unicystic ameloblastoma, whereas the solid lesions included solid ameloblastoma and control cases of squamous cell carcinomas.

Distribution of myofibroblasts in the connective tissue capsule of the cystic lesions has broadly been classified into a subepithelial zone and midzone and periphery of the lesion [Table 3].

Table 3: Pattern of distribution or arrangement of myofibroblasts in odontogenic cysts

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The distribution of myofibroblasts in odontogenic tumors has been seen as associated with connective tissue stroma adjacent to the epithelial island and in the connective tissue stroma at the advancing front. Whereas, in squamous cell carcinomas, these cells are present predominately at the invasion front.

Discussion

Myofibroblasts are specialized fibroblasts that exhibit features of both fibroblasts and smooth muscle cells. These cells are present both in physiologic situations and in pathologic situations such as wound healing and tumor progression. Tumor-derived cytokines, mainly transforming growth factor-beta (TGF-β) and platelet-derived growth factor, are mainly responsible for the emergence of myofibroblasts. These myofibroblasts were in turn thought to produce numerous growth factors and extracellular proteases that help in tumor growth and invasion.^[5]

The role of myofibroblasts in squamous cell carcinoma has been the topic of interest, and numerous studies carried out indicate that the myofibroblasts contributed to the neoplastic growth and invasion of these lesions.^{[6],[7],[8],[9]} Few studies have been done depicting the role of myofibroblasts in odontogenic cysts and tumors^{[5],[10],[11],[12],[13]} revealed that odontogenic lesions with an aggressive biologic behavior such as OKC and SAM contained high frequency of stromal myofibroblasts which were comparable to cases of squamous cell carcinomas. These studies implied that myofibroblasts could contribute to the biologic behavior of these lesions and suggested the use of therapeutic agents which can act as antimyofibroblast drugs to reduce the extent of lesion prior to surgery.^{[5],[10],[14],[15]}

In our study, we observed that among the odontogenic cysts and tumors, the mean number of myofibroblasts was significantly higher in locally aggressive lesions like in OKC solid ameloblastomas which were comparable to cases of squamous cell carcinoma [Table 1], which was similar to the study reported by Vered et al.^[5] and Gabhane et al.^[16]

Among the odontogenic cysts, OKC, a well-known entity for its locally aggressive behavior characterized by rapid rate of growth and tendency to invade adjacent structures, showed a high mean number of myofibroblasts than dentigerous cyst [Table 1] and [Figure 1] and [Figure 2]. This could be due to increased expression of TGF-β by the epithelium of the OKC when compared to other cysts^[17] that helps in transdifferentiation of more number of fibroblasts to myofibroblasts. These myofibroblasts were in turn thought to produce numerous growth molecules and extracellular proteases that help in further cyst growth and expansion. The mean number of myofibroblasts in OKC was not statistically different from squamous cell carcinoma which is a malignant neoplasm, suggesting that they could contribute to the locally aggressive behavior of OKC.

Figure 1: Alpha-smooth muscle actin-positive myofibroblasts in odontogenic keratocyst (×200)

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Figure 2: Dentigerous cyst showing alpha-smooth muscle actin-positive myofibroblasts (×200)

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Another interesting observation in this study was that in odontogenic cysts, we identified three distinct zones of distribution of these myofibroblasts: a subepithelial zone, midzone, and periphery of the lesion [Table 2] and [Figure 3] and [Figure 4]. A similar finding of distribution of myofibroblasts in three zones was reported by Lombardi and Morgan in their study of α-SMA expression in odontogenic cysts.^[11]

Figure 3: Subepithelial parallelly arranged myofibroblasts beneath epithelial crypt formation in odontogenic keratocyst (×400)

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Figure 4: Parallelly arranged myofibroblasts at the periphery of lesion adjacent to bone in odontogenic keratocyst (×400)

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In our study, 4 out of the 5 cases of dentigerous cyst and 7 out of the 10 cases of OKC predominately showed myofibroblasts in the midzone and in the periphery of the lesion. As already mentioned, TGF-β along with the mechanical tension within the tissues is mainly responsible for transdifferentiation of fibroblasts to myofibroblasts. This increased number of myofibroblasts in the periphery of the lesion adjacent to the bone could be explained by the fact that expanding cyst exerts pressure on the surrounding bone which is a rigid structure and this pressure is maximum at bone connective tissue interface, thereby leading to transdifferentiation of more number of myofibroblasts.

These transdifferentiated myofibroblasts with their inherent contractile property might exert pressure on the underlying bone inducing the osteoclastic differentiation adjacent to the bone surface. These activated osteoclasts demineralize the bone releasing the organic components which were further degraded by the extracellular proteinases, mainly matrix metalloproteinases secreted by these cells.

The presence of myofibroblasts in odontogenic tumors has been reported in few studies. Earlier studies done using electron microscopy revealed that the predominant cell form in the stromal component of these odontogenic neoplasm is myofibroblast and is responsible for the stromal support and its locally aggressive behavior.^[18],[19] One study reported a significant correlation between presence of myofibroblasts and MMP-2 expression in ameloblastomas and suggested that the co-expression of myofibroblasts and MMP-2 may be associated with a more aggressive infiltrative behavior.^[20]

In the present study, all the cases of solid ameloblastomas contained myofibroblasts [Figure 5] and their mean number in solid ameloblastomas was higher compared to unicystic ameloblastomas and this difference was statistically significant [Table 1]; a similar finding was in a previous study done by Vered et al.^[5] who demonstrated that the number of myofibroblasts in solid ameloblastomas is significantly higher than in unicystic variants, suggesting that myofibroblasts can contribute to the biological behavior of aggressive lesions. This difference can contribute to the more locally aggressive behavior of solid ameloblastomas when compared to unicystic ameloblastomas which shows biologically low-grade course, with a limited recurrence potential even when treated with simple curettage.^[21],[22]

Figure 5: Myofibroblasts around odontogenic epithelial islands in follicular ameloblastoma (×200)

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Qualitatively, there was a lot of variation in staining pattern of myofibroblasts in comparison with the blood vessels within the same lesion and in between the lesions. This difference in staining intensity might be due to the variable amount of cytoplasmic actin present in the cell.

“Cellular form follows function.” The shape and structure of a cell reflects the function it needs to accomplish, for example a plump cell usually indicate an actively synthesizing cell whereas a thin and elongated morphology reflects a resting state. The morphology of myofibroblasts is spindle shaped in all cases, but we observed difference in the pattern of arrangement. Few α-SMA positive were spindle shaped and had no contact with the adjacent cells [Table 3] and [Figure 6] and others were plump, spindle with elongated cytoplasmic extensions which were interconnected with the neighboring myofibroblasts in a “network” like fashion [Figure 7]. A similar finding was reported by Vered M et al. in their study on myofibroblasts in tongue dysplastic lesions and squamous cell carcinomas.^[6] Benign lesions like dentigerous cysts showed the spindle isolated pattern, while the locally aggressive lesions predominately showed network pattern whereas squamous cell carcinomas showed only network pattern [Figure 8]. This distinct arrangement pattern between benign, locally agressive and malignant neoplastic lesions might represent different subsets of population of myofibroblasts and should be answered further studies.

Figure 6: Spindle and isolated pattern of arrangement of myofibroblasts (×400)

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Figure 7: Spindle and network pattern of arrangement of myofibroblasts (×400)

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Figure 8: Myofibroblasts in well-differentiated squamous cell carcinoma (×400)

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The distribution of myofibroblasts in solid ameloblastomas was both in the connective tissue surrounding the islands and also in the stroma away from the islands. Similar finding was reported by Mashhadiabbas et al.^[10] Myofibroblasts surrounding the tumoral islands may be actively involved in secreting growth molecules which might help in the growth of the tumor islands. Whereas, the cells in the connective tissue stroma at the advancing front may be actively involved in secreting extracellular proteinases that help in tumor invasion.

Conclusion

Thus, the increase in the number of myofibroblasts could be one of the contributory factors for the locally aggressive behavior of benign lesions such as ameloblastomas and OKCs. Further studies are suggested to understand the mechanism by which these important cellular elements exert their effects on stromal and epithelial tissue compartments. In addition, future strategies like therapeutic targeting of myofibroblasts, their by products, or factors responsible for the transdifferentiation may be used to restrict the extent of lesions prior to surgery and prevent recurrence thereafter.

Acknowledgment

We would like to thank Dr. T.R. Saraswathi.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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