Myofibroblasts in Oral submucous fibrosis, Oral squamous cell carcinoma and Normal mucosa- an Immunohistochemical Analysis

Myofibroblasts in Oral submucous fibrosis, Oral squamous cell carcinoma and Normal mucosa- an Immunohistochemical Analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Myofibroblasts in Oral submucous fibrosis, Oral squamous cell carcinoma and Normal mucosa- an Immunohistochemical Analysis Sivachandran Annamalai, Shaik Mohamed Shamsudeen, Rooban thavarajah, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.17936/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Myofibroblasts are spindle shaped smooth muscle like fibroblast involved in the physiological repair of tissue structure can also cause pathological remodeling of tissue by forming fibrosis. They express smooth muscle actin which can be evaluated for their biological behavior in the physiological and pathological conditions. Aim: To evaluate the expression of Myofibroblasts in oral submucous fibrosis, oral squamous cell carcinoma and normal mucosa by Immunohistochemical analysis. Methods: It is a cross-sectional study done to evaluate the expression of Myofibroblasts in oral submucous fibrosis (Group-1), oral squamous cell carcinoma (Group-2) and normal buccal mucosa (Group-3) using immunohistochemistry using alpha smooth muscle actin in formalin fixed, paraffin embedded tissue specimens. The localization of stain, nature of stain, intensity of stain and the percentage of cells stained among the three groups were studied and compared. Results: The gender distributions in the study were in favor of males. It was observed that Staining Intensity was seen more in group I (30%) than in group II (20%) and progressively increased from early (16.5%) to advance stage(50%) of oral submucous fibrosis. Higher staining intensity was observed in well differentiated oral squamous cell carcinoma. Conclusion: Myofibroblasts are important in normal healing process, but can induce fibrosis. In oral submucous fibrosis, they can be used to assess the severity of the lesion by their increased expression of alpha smooth muscle actin. In oral carcinogenesis increase in the number of alpha smooth muscle actin can change the distribution pattern results in tumor invasive characteristics. Key Words: Myofibroblasts, Alpha smooth muscle actin, Staining intensity, Oral Submucous Fibrosis, Oral Squamous Cell Carcinoma. Head & Neck Surgery Myofibroblasts Alpha smooth muscle actin Staining intensity Oral Submucous Fibrosis Oral Squamous Cell Carcinoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 background Myofibroblasts are spindle shaped cells which produce collagen and having contractile properties like smooth-muscle, hence known as smooth muscle like fibroblast. They express alpha smooth muscle actin (α SMA)[1].They primarily involved in wound healing by forming fibrous tissue and secrete extracellular matrixes helps in the repair process by reducing the physical size of the damage by their contractility properties. Once the tissue is repaired they disappear by apoptosis. Apart from the physiological repair process, they are involved in the pathological remodeling of the tissue in which they persist and form tissue deformation seen as hypertrophic scars [2]. Fibrosis is a progressive disease characterized by accumulation of scarring extracellular matrix proteins, which disrupt normal tissue architecture [2]. Oral Submucous Fibrosis (OSF) is one such disorder characterized by exuberant deposition of sub epithelial collagen in response to chronic areca nut chewing resulting in mucosal rigidity that leads to limitation in mouth opening. In various fibrotic disorders, the key cellular mediator of fibrosis is myofibroblasts, which when activated serves as primary collagen-producing cell [3, 4]. Myofibroblasts are also involved in the cancer progression by stimulating the microenvironment stromal cells. The activated Myofibroblasts express α-smooth muscle actin which represents the majority of tumor stromal cells [5].Thus, it is necessary to understand the expression of myofibroblast in the molecular mechanism of oral submucous fibrosis and oral cancer progression. Methods The study material comprised of 50 formalin fixed, paraffin embedded tissue specimens from archival blocks. The samples are divided into 3 groups namely: Group I- Oral submucous fibrosis (20 samples), Group II-Oral squamous cell carcinoma (20 samples) and Group III – normal buccal mucosa (10 samples) taken as control group. Tissue samples were taken from archival blocks. The tissue processing was done with routine haematoxylin and eosin staining (H & E). Immunohistochemical staining was done with α-smooth muscle actin as primary antibody (Biogenex –synthetic NH2 terminal decapeptide of α-smooth muscle actin, Mouse monoclonal category and IgG2a immunoglobulin) and Secondary antibody used were Biogenex-super sensitive IHC detection system kit (Poly Horse Radish Peroxidase –pretitrated anti-species immunoglobulin labeled with enzyme polymer, super enhancer reagent, anti-mouse monoclonal negative control serum, and liquid DAB- Diamino-benzidine-chromogen ) The technique for Immunohistochemical analysis was done as per the standardized method [Fig-1]. STAINING CRITERIA AND STAINING INDEX CALCULATION METHODS Calculation of staining intensity (SI) The calculation of staining intensity was considered as no stain visible as 0 (SI 0), if the staining visible only under 40X is calculated as 1 (SI 1), if staining visible under 10X as 2(SI 2) and staining visible even at 4X considered as 3 (SI 3). Calculation of percentage of cells (Labeling Index-LI) The calculation of percentage of cells labeling index was done as if there is no positive cells it was considered as 0 (LI 0), if 1-25% positive cells was seen it was calculated as 1 (LI 1), if 25-50% positive cells was seen calculated as 2 (LI 2)and 50-100% positive cells was seen as 3 (LI 3). Calculation of staining index It is derived from multiplication of staining intensity and percentage of cells. (SI x LI) The final score is grouped into no stain if the score is 0, mild if the score is 1-2, moderate if the score is 3-4 and intense categories if the score is 6-9. Statistical analysis: Statistical analysis was done using SPSS TM software (version 11.5). p ≤ 0.05 was considered to be statistically significant. Kruskal Wallis and Mann-Whitney test was done to compare tissue localization of stain, cellular location, and nature of stain, intensity of stain and the percentage of cells stained among the three study groups. The inter-observer variability for the intensity of stain and percentage of cells stained was assessed using kappa statistics. Results Twenty cases of OSF (Group I), 20 cases of OSCC (Group II) and 10 cases of clinically appearing normal mucosa (Group III) were analyzed for immune reactivity of myofibroblasts. All the samples in groups were taken from the buccal mucosa. DISTRIBUTION OF GENDER AMONG GROUPS The males were predominant in the study compromising 95% in group I, 75% in group II and 80%in group III. The age groups were divided into 20- 40 years, 41 - 60 years and 61+ years. In Group I, 65% belonged to age Group 20-40 years and 35% belonged to age Group 41-60 years. In Group II, 10% belonged to age Group 20-40 years and 65% belonged to age Group 61+years. In Group III 90% belonged to age Group 20-40 years and10% belonged to age Group 41-60 years DISTRIBUTION OF HABITS AMONG GROUP I (OSF) AND GROUP II (OSCC) In group I & II, 31 had habits of tobacco and rest of them had no habits. Within those who had habits 9.7 % had no stain, 51.6 % had mild staining, 22.6 % had moderate staining and 16.1% had intense staining. Those who are taking tobacco had no staining 55.6% had mild staining 33.3% and 11.1% had moderate staining. I.STAINING INTENSITY Distribution of Staining Intensity (SI) Of α-SMA (Alpha Smooth Muscle Actin) Among 3 Groups: α-SMA revealed positivity in group I, II and III. In Group I and Group II cases showed 85% staining for α-SMA , whereas in Group III, positive staining was observed 30%. In Group I out of 20 OSF cases 30 % had a score of 3 (SI3), 20 % had a score of 2 (SI2) ( Fig-2), 35 % had a score of 1 (SI1) (Fig-3) and 15% had a score of 0 (SI0). Group II out of 20 cases 20 % had a score of 3 (SI3), 30 % had a score of 2 (SI2) (Fig-4), 35 % had a score of 1 (SI1) (Fig-5) and 15% had a score of 0 (SI0). In Group III out of 10 normal cases 30 % had a score of 0 (SI0) and ( Fig -6) 70 % had a score of 1 (SI1) ( Fig-7A) Distribution of Staining Intensity among Different OSF Grading: In 20 cases of OSF, 6 cases belonged to Grade I, 10 cases belonged to Grade II and 4 cases belonged to Grade III. In Grade I 16.7% had a score of 0 (SI0), 50% had a score of 1 (SI1), 16.7 % had a score of 2 (SI2) and 16.7 % had a score of 3 (SI3). In Grade II 40 % had a score of1 (SI1), 30 % had a score of 2 (SI2), and 30% had a score of 3 (SI3), In Grade III 50 % had a score of 0 (SI0) and 50 % had a score of 3 (SI3) ( Fig-7B) Distribution of Staining Intensity among Different OSCC Grading: In 20 cases of OSCC, 13 cases belonged to well differentiated group, 3 cases belonged to moderately differentiated and 4 cases belonged to poorly differentiated. In well differentiated group, 23.1% had a score of 0 (SI0), 23.1% had a score of 1 (SI1), 23.1 % had a score of 2 (SI2) and 30.8 % had a score of 3 (SI3). In moderately differentiated group 66.7 % had a score of 1 (SI1) and 33.3 %had a score of 2 (SI2). In poorly differentiated group 50 % had a score of 1 (SI1) and 50 % had a score of 2 (SI2) ( Fig-7C) KAPPA STASTISTIC VALUE The inter-observer agreement for the staining intensity of stain for all the 3 groups was arrived using kappa statistics and kappa value is 0.689 . PERCENTAGE OF IMMUNOPOSITIVE CELLS- LABELING INDEX (LI) Distribution of Labeling Index (Li) Of α-SMA (Alpha Smooth Muscle Actin) Among 3 Groups: α-SMA revealed positivity in group I, II and III. In Group I and Group II cases showed 85% staining for α-SMA , whereas in Group III, positive staining was observed 40%. In Group I out of 20 OSF cases 5 % had a score of 3 (LI3), 40 % had a score of 2 (LI2), 40 % had a score of 1 (LI1) and 15% had a score of 0 (LI0). Group II out of 20 cases 15 % had a score of 0 (LI0) and 85 % had a score of 1 (LI1). In Group III out of 10 normal cases 60 % had a score of 0 (LI0) and 40 % had a score of 1 (LI1) ( Fig-8A) Distribution of Labeling Index (Li) Among Different OSF Grading: Total 20 cases of OSF, 6 cases belonged to Grade I, 10 cases belonged to Grade II and 4 cases belonged to Grade III. In Grade I 16.7% have a score of 0 (LI0), 50% had a score of 1 (LI1) and 33.3 % had a score of 2 (LI2). In Grade II 50 %had a score of1 (LI1), 40 % had a score of 2 (LI2) and 10% had a score of 3 (SI3). In Grade III 50 % had a score of 0 (LI0) and 50 % had a score of 2 (LI2) (Fig-8B) Distribution of Labeling Index (Li) Among Different OSCC Grading: Total 20 cases of OSCC, 13 cases belonged to well differentiated group, 3 cases belonged to moderately differentiated and 4 cases belonged to poorly differentiated. In well differentiated group, 23.1% had a score of 0 (LI0) and 76.9 % had a score of 1 (LI1). In moderately and poorly differentiated group all had a score of 1 (LI1) (Fig-8C). III. STAINING INDEX Distribution of Staining Index among the Groups. Using Kruskal-Wallis test the comparison among groups derived, there is a significant difference in the expression of myofibroblasts. ( Fig-9A) Comparison Of Staining Index Between Group I And Group III In OSF, 15% of the cases showed no staining while 40% mild staining, 20% moderate staining and 25% of the cases had intense staining in connective tissue. In normal mucosa, 40% exhibited mild staining and no staining was seen in 60% of the cases. The result is statistically significant. Comparison Of Staining Index Between Group II And Group III In OSCC, 15% of the cases showed no staining while 65% mild staining and 20% moderate staining in connective tissue. In normal mucosa, 40% exhibited mild staining and no staining was seen in 60% of the cases. The result is statistically significant. Comparison Of Staining Intensity Between Group I And Group II In OSF group, 15% of the cases showed no staining while 40% mild staining, 20% moderate staining, and 25% of the cases had intense staining in connective tissue. In OSCC group, 15% of the cases showed no staining while 65% mild staining and 20% moderate staining in connective tissue. As both the lesions expressing myofibroblasts, the result is statistically not significant. Comparison of Staining In Different OSF Grading In Grade I, 16.7% showed no stain, 50% showed mild stain 16.7% moderate and 16.7 % showed intense staining. In Grade II 50 % showed mild staining 20 % showed moderate and 30% showed intense staining. In Grade III 50 % showed no stain, 25% showed moderate and 25 % showed intense staining. (Fig-9B) Comparison of Staining In Different OSCC Grading In well differentiated group 23.1% showed no stain, 46.2% showed mild stain and 30.8% showed moderate staining. In OSCC moderately differentiated 100% showed mild staining. In OSCC poorly differentiated 100% showed mild staining (Fig-9C). discussion Myofibroblasts are smooth muscle like fibroblasts and one of the phenotypic heterogeneity among the fibroblasts. They can alter the inflammatory response by secreting the soluble mediators of inflammation. Furthermore myofibroblasts is suggested to be induced by TGFβ, a potent pro-inflammatory and pro-fibrotic cytokine related to imbalance between collagen deposition and degradation in Oral Submucous Fibrosis [6].Hence the role of myofibroblasts in the repair process has varying functions in the physiological and pathological conditions The distribution of genders in the study groups ranged from a ratio 4:1 in favor of Males: Females in the control group and 19:1 in the OSF group. The male preponderance among OSF has been documented in several studies and can be due to the habit of using tobacco [7]. The age distribution of OSF is higher between 20-40 years as similar to other studies reported in the literature [7]. Similarly the occurrence of OSCC commonly occurs among older age groups as reflected in our study. As normal controls were obtained during impaction procedures 90% of they were obtained from 20-40 age groups. Staining intensity: Staining intensity between the three study groups was not statistically significant and the expression was seen more in group I than in group II (20%). The staining intensity progressively increased from early OSF (16.5%) to advance OSF (grade III 50%). This indicates that OSF represents a failed wound healing process of the oral mucosa after chronic sustained injury resulting in scarring and fibrosis. This could be also in response to the hypersensitivity caused by arecoline and the resultant persistent (juxtra epithelial inflammatory response) in OSF, which acts as an initiating factor leading to a defective inflammatory response and activation of fibroblasts culminating in fibrosis [8]. The well differentiated OSCC showed higher staining intensity indicating that myofibroblasts are stimulated during the repair of extra cellular matrix and are capable of augmenting and down-regulating the inflammatory response by secretion of these soluble mediators of inflammation [9]. Labeling index: The labeling index indicates the more percentage of cells stain with α SMA in group I than in group II or III and can be due to chronic inflammation and continuous tissue remodeling in OSF as well as chronic sustaining injury to factors such as arecoline, micro trauma could cause the transient or continuous differentiation of fibroblast from undifferentiated stem cell or a differentiated fibroblast pool [10]. The labeling index among the various grades of OSF shows an increase from grade 1 to III. This indicates with increase of stimulus and disease grade, more amount of myofibroblasts differentiation occurs [11]. However the difference between the grades were not statistically significant in OSCC with varying grades of differentiation, no significant difference could be found in the labeling index. Hence the expression of α SMA indicates that differentiation of tumor does not appear to influence the differentiation of myofibroblasts [11]. Staining index: There was statistical significant difference in the pattern of staining index among the study group. Intense staining of α SMA was observed only in group I while moderate staining was seen in group I and II only. Group II had only the effect of inflammation and hence had a mild staining index in 40% of cases while no staining was seen in 60% of cases. The intense staining in group I was similar to the findings in the literature [13].Though 65% cases of group II showed a mild SI and 20% cases had moderate SI, none had intense SI. The absence of intense SI in group II needs to further analyzed. On comparing the Staining Index between OSF and normal tissue, it is observed that there was a wide difference between the expressions of SI using αSMA. The uniform mild expression in both cases probably is a result of the mild continuous inflammation in both the condition [14, 15]. On comparing the group II and group III, it is observed that there is only a mild difference between the grades of Staining Index. However it was statistically significant. The uniformity in expression probably relates to the chronic inflammation that exists between both the conditions [15]. There is no significant difference between group I and II (p=0.157) However 24% of cases exhibited intense staining in group I while no cases of group II had intense SI. This indicates that OSF had more myofibroblasts expression than OSCC [16, 17]. The staining index did not considerably vary within the grades of the OSF .This indicates that the staining intensity as well as labeling index is crucial factor for the staining index. In this cross sectional study the expression of SI 3 increased with increasing grades of OSF. While no such observation was seen in labeling index. Low number of cells expressed SMA positivity while the staining intensity varied. This indicates that with grades of OSF increasing only a small subset of fibroblast population expressed intensive staining while the number of cells expressing α SMA never altered[18,19]. Conclusions Persistent, chronic inflammation leads to formation of a subset of fibroblastic population-myofibroblasts. The progressive increase of myofibroblasts in the Oral submucous fibrosis can be used as a marker in evaluating their severity. Myofibroblasts stimulate tumor development and constitute the tumor stroma and their presence in the invasion front of the tumor suggests they are associated with tumorgenesis. Clinical evidence suggests that the presence of myofibroblasts is associated with a poor prognosis have to be evaluated. abbreviations α SMA- alpha smooth muscle actin OSF- Oral submucous fibrosis OSCC- Oral squamous cell carcinoma SI- staining intensity LI – labeling index TGFβ -transforming growth factor beta Declarations Ethics approval and Consent to participate: The study protocol was evaluated and approved by the Institutional Ethics Committee of the Ragas Dental College and Hospital, Chennai. Consent for Publication: Not applicable Availability of data and material: The data and material can be available on request. Competing interests: The authors declare that they have no competing interests. Funding: The research did not receive any financial support from funding agencies. Affiliations: Department of Oral pathology, SRM University, College Dentistry,kattankulathur,Chennai,India Dr. Sivachandran Annamalai Department of dental diagnostic Science & Oral Biology, College Of Dentistry, King Khalid University ,Abha,Saudi Arabia Dr. Shaik Mohamed Shamsudeen Department of Oral Pathology, Ragas Dental college and Hospital, Chennai, India Dr. Rooban T, Dr. Elizabeth Joshua, Dr. Umadevi K Rao Dr. Ranganathan Kannan Author’s contribution: Conception , Design the study (SA, EJ, UR, RK),Acquisition of data and biological samples(SA,SM), Data analysis and interpretation (EJ, UR, RK, SA,RT,SM )Manuscript drafting and editing (SM,SA),Final approval of manuscript (EJ, UR, RK, SA,RT,SM). Acknowledgment: NIL references Punnya V. Angadi, Alka D. Kale, Seema Hallikerimath. Evaluation of myofibroblasts in oral submucous fibrosis: correlation with disease severity. Journal of Oral Pathology & Medicine 2011; 40(3) : 208–213. Steven E. Mutsaers, Jill E. Bishop, Gus Mcgrouther, Geoffrey J. Laurent. Mechanisms of Tissue Repair: from Wound Healing to Fibrosis. J. Biochem. Cell Bid.1997; 29: 5-17. Kundendu AryaBishen, Raghu Radhakrishnan, Kapaettu Satyamoorthy. The role of basic fibroblast growth factor in oral submucous fibrosis pathogenesis. J Oral Pathol Med 2008; 37: 402–411. Tilakaratne WM, Klinikowski M.F, Takashi Saku, Peters TJ, SamanWarnakulasuriya. Oral submucous fibrosis: Review on aetiology and pathogenesis. Oral Oncology 2006; 42:561– 568. Hu Yanjia, JianXinchun.The role of epithelial–mesenchymal transition in oral squamous cell carcinoma and oral submucous fibrosis.Clinica Chimica Acta 2007; 383: 51–566. Mithani SK, Mydlarz WK, Grumbine FL, Smith IM, Califano JA. Molecular genetics of premalignant oral lesions. Oral Dis 2007; 13:126-33. Ranganathan K, Devi UM , Joshua E, Kirankumar K, SaraswathiR.Oral submucous fibrosis: a case-control study in Chennai, South India.. J Oral Pathol Med 2004; 33:274–277 Rajalalitha, S. Vali. Molecular pathogenesis of oral submucous fibrosis – a collagen metabolic disorder. J Oral Pathol Med. 2005; 34:321-8. Beacham DA, Cukierman E. Stromagenesis: the changing face of fibroblastic microenvironments during tumor progression. Semin Cancer Biol. 2005; 15:329–41. Xouri G, Christian S. Origin and function of tumor stromafibroblasts. Semin Cell Dev Biol 2010; 21:40-6. Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol 2008; 214:199-210. Lúcio PS, Cavalcanti AL, Alves PM, Godoy GP, Nonaka CF.Myofibroblasts and their relationship with oral squamous cellcarcinoma. Braz J Otorhinolaryngol 2013; 79:112‑ Philip T, Kumar TD, Rajkumar K, Karthik KR, Priyadharsini N,Kumar AR. Immunohistochemical evaluation of myofibroblasts using alpha-smooth muscle actin in oral submucous fibrosis. SRM J Res Dent Sci 2014; 4:243-7. Utsunomiya H, Tilakaratne WM, Oshiro K, Maruyama S, Suzuki M, Ida-Yonemochi H. Extracellular matrix remodeling in oral submucous fibrosis: Its stage-specific modes revealed byimmunohistochemistry and in situ J Oral PatholMed 2005;34:498-507. Darby I, Skalli O, Gabbiani G. Alpha‑smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab Invest 1990; 63:21‑9. Chaudhary M, Gadbail AR, Vidhale G, MankarGadbailMP,Gondivkar SM, Gawande M. Comparison of myofibroblasts expression in oral squamous cell carcinoma, verrucous carcinoma, high risk epithelial dysplasia, low risk epithelial dysplasia and normal oral mucosa. Head Neck Pathol 2012; 6:305‑ Kellermann MG, Sobral LM, Da Silva SD, et al. Myofibroblasts in the stroma of oral squamous cell carcinoma is associated with poor prognosis. Histopathology 2007; 51: 849–53. Desmoulière A, Guyot C, Gabbiani G. The stroma reaction myofibroblast: A key player in the control of tumor cell behavior. Int J Dev Biol 2004; 48:509-17. Gupta MK, Mhaske S, Ragavendra R, Imtiyaz. Oral submucous fibrosis — Current Concepts in etiopathogenesis. Peoples J Sci Res 2008; 1:39-44. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8565","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":231692,"identity":"b095b28b-9f4b-47d1-bbdf-9c7a28bc2855","order_by":1,"name":"Sivachandran Annamalai","email":"","orcid":"","institution":"SRM University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sivachandran","middleName":"","lastName":"Annamalai","suffix":""},{"id":231693,"identity":"27cf8e82-ff5d-4526-b431-b257e9f1b30e","order_by":2,"name":"Shaik Mohamed 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18:39:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115000,"visible":true,"origin":"","legend":"Immunohistochemical stained section showed myofibroblast in OSF-10x","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/3305fc6d-e91d-4349-a88e-d47d160d659d/v1/Fig-2.jpg"},{"id":194286,"identity":"36b8196c-11a1-4a95-9934-c6242f72fcf7","added_by":"auto","created_at":"2019-12-02 18:39:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90217,"visible":true,"origin":"","legend":"Immunohistochemical stained section showed myofibroblast in OSF -40x","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/3305fc6d-e91d-4349-a88e-d47d160d659d/v1/Fig-3.jpg"},{"id":194287,"identity":"59e7b3a9-004b-4901-abb0-325a7057dcd8","added_by":"auto","created_at":"2019-12-02 18:39:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81792,"visible":true,"origin":"","legend":"Immunohistochemical stained section showed myofibroblast in OSCC-10x","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/3305fc6d-e91d-4349-a88e-d47d160d659d/v1/Fig-4.jpg"},{"id":194288,"identity":"aa51f986-ea12-4a97-bf9e-94bf37349960","added_by":"auto","created_at":"2019-12-02 18:39:24","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":73025,"visible":true,"origin":"","legend":"Immunohistochemical stained section showed myofibroblast in OSCC -40x","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/3305fc6d-e91d-4349-a88e-d47d160d659d/v1/Fig-5.jpg"},{"id":194289,"identity":"4b495966-21ae-4059-9a12-cc3688620e81","added_by":"auto","created_at":"2019-12-02 18:39:24","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":78605,"visible":true,"origin":"","legend":"Immunohistochemical stained section showed myofibroblast in normal tissue-10x","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/3305fc6d-e91d-4349-a88e-d47d160d659d/v1/Fig-6.jpg"},{"id":194290,"identity":"a1a4e806-2b19-4709-97d8-fa535864a5f4","added_by":"auto","created_at":"2019-12-02 18:39:24","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":74090,"visible":true,"origin":"","legend":"A: Distribution of staining intensity among the groups \nB: Distribution of staining intensity among different OSF gradings \nC: Distribution of staining intensity among different OSCC gradings","description":"","filename":"Fig7.StainingIntensity.jpg","url":"https://assets-eu.researchsquare.com/files/3305fc6d-e91d-4349-a88e-d47d160d659d/v1/Fig-7. Staining Intensity.jpg"},{"id":194291,"identity":"97160664-009f-46fa-b50f-3612f527c4df","added_by":"auto","created_at":"2019-12-02 18:39:24","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":72100,"visible":true,"origin":"","legend":"A: Distribution of percentage of cells among the groups\nB: Distribution of percentage of cells among different OSF gradings \nC: Distribution of percentage of cells among different OSCC gradings","description":"","filename":"Fig8.Labellingindex.jpg","url":"https://assets-eu.researchsquare.com/files/3305fc6d-e91d-4349-a88e-d47d160d659d/v1/Fig-8.Labelling index.jpg"},{"id":194292,"identity":"ce860acf-c947-4ccf-9006-03622e0ec2fb","added_by":"auto","created_at":"2019-12-02 18:39:24","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":68450,"visible":true,"origin":"","legend":"A: Distribution of staining index among the groups\nB: distribution of staining index among different OSF gradings \nC: distribution of staining index among different OSCC gradings","description":"","filename":"Fig9.Stainingindex.jpg","url":"https://assets-eu.researchsquare.com/files/3305fc6d-e91d-4349-a88e-d47d160d659d/v1/Fig-9.Staining index.jpg"},{"id":13480260,"identity":"72fc8426-8da8-436a-a099-84c8e73baeff","added_by":"auto","created_at":"2021-09-16 21:42:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1356504,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8565/v1/9c12b5de-ebe8-43a4-81fb-2a9c1aac3084.pdf"}],"financialInterests":"","formattedTitle":"Myofibroblasts in Oral submucous fibrosis, Oral squamous cell carcinoma and Normal mucosa- an Immunohistochemical Analysis","fulltext":[{"header":"background ","content":"\u003cp\u003eMyofibroblasts are spindle shaped cells which produce collagen and having contractile properties like smooth-muscle, hence known as smooth muscle like fibroblast. They express alpha smooth muscle actin (\u0026alpha; SMA)[1].They primarily involved in wound healing by forming fibrous tissue and secrete extracellular matrixes helps in the repair process by reducing the physical size of the damage by their contractility properties. Once the tissue is repaired they disappear by apoptosis. Apart from the physiological repair process, they are involved in the pathological remodeling of the tissue in which they persist and form tissue deformation seen as hypertrophic scars [2].\u003c/p\u003e\n\u003cp\u003eFibrosis is a progressive disease characterized by accumulation of scarring extracellular matrix proteins, which disrupt normal tissue architecture [2]. Oral Submucous Fibrosis (OSF) is one such disorder characterized by exuberant deposition of sub epithelial collagen in response to chronic areca nut chewing resulting in mucosal rigidity that leads to limitation in mouth opening. In various fibrotic disorders, the key cellular mediator of fibrosis is myofibroblasts, which when activated serves as primary collagen-producing cell [3, 4].\u003c/p\u003e\n\u003cp\u003eMyofibroblasts are also involved in the cancer progression by stimulating the microenvironment stromal cells. The activated Myofibroblasts express \u0026alpha;-smooth muscle actin which represents the majority of tumor stromal cells [5].Thus, it is necessary to understand the expression of myofibroblast in the molecular mechanism of oral submucous fibrosis and oral cancer progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe study material comprised of 50 formalin fixed, paraffin embedded tissue specimens from archival blocks. The samples are divided into 3 groups namely: Group I- Oral submucous fibrosis (20 samples), Group II-Oral squamous cell carcinoma (20 samples) and Group III \u0026ndash; normal buccal mucosa (10 samples) taken as control group.\u003c/p\u003e\n\u003cp\u003eTissue samples were taken from archival blocks. The tissue processing was done with routine haematoxylin and eosin staining (H \u0026amp; E). \u0026nbsp;Immunohistochemical staining was done with \u0026alpha;-smooth muscle actin as primary antibody (Biogenex \u0026ndash;synthetic NH2 terminal decapeptide of \u0026alpha;-smooth muscle actin, Mouse monoclonal category and IgG2a immunoglobulin) and Secondary antibody used were Biogenex-super sensitive IHC detection system kit (Poly Horse Radish Peroxidase \u0026ndash;pretitrated anti-species immunoglobulin labeled with enzyme polymer, super enhancer reagent, anti-mouse monoclonal negative control serum, and liquid DAB- Diamino-benzidine-chromogen ) The technique for Immunohistochemical analysis was done as per the standardized method [Fig-1].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSTAINING CRITERIA AND STAINING INDEX CALCULATION METHODS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCalculation of staining intensity (SI)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe calculation of staining intensity was considered as no stain visible as 0 (SI 0), if the staining visible only under 40X is calculated as 1 (SI 1), if staining visible under 10X as 2(SI 2) and staining visible even at 4X considered as 3 (SI 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCalculation of percentage of cells (Labeling Index-LI) \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe calculation of percentage of cells labeling index was done as if there is no positive cells it was considered as 0 (LI 0), if 1-25% positive cells was seen it was calculated as 1 (LI 1), if\u0026nbsp; 25-50% positive cells was seen\u0026nbsp; calculated as 2 (LI 2)and 50-100% positive cells was seen\u0026nbsp; as 3 (LI 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCalculation of staining index\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt is derived from multiplication of staining intensity and percentage of cells. (SI x LI)\u003c/p\u003e\n\u003cp\u003eThe final score is grouped into no stain if the score is 0, mild if the score is 1-2, moderate if the score is 3-4 and intense categories if the score is 6-9.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was done using SPSS \u003csup\u003eTM \u003c/sup\u003esoftware (version 11.5).\u0026nbsp; p \u0026le; 0.05 was considered to be statistically significant.\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003eKruskal Wallis and Mann-Whitney test was done to compare tissue localization of stain, cellular location, and nature of stain, intensity of stain and the percentage of cells stained among the three study groups.\u003c/li\u003e\n\u003cli\u003eThe inter-observer variability for the intensity of stain and percentage of cells stained was assessed using kappa statistics.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTwenty cases of OSF (Group I), 20 cases of OSCC (Group II) and 10 cases of clinically appearing normal mucosa (Group III) were analyzed for immune reactivity of myofibroblasts. All the samples in groups were taken from the buccal mucosa.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDISTRIBUTION OF GENDER AMONG GROUPS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe males were predominant in the study compromising 95% in group I, 75% in group II and 80%in group III.\u003c/p\u003e\n\u003cp\u003eThe age groups were divided into 20- 40 years, 41 - 60 years and 61+ years. In Group I, 65% belonged to age Group 20-40 years and 35% belonged to age Group 41-60 years. In Group II, 10% belonged to age Group 20-40 years and 65% belonged to age Group 61+years. \u0026nbsp;In Group III 90% belonged to age Group 20-40 years and10% belonged to age Group 41-60 years\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDISTRIBUTION OF HABITS AMONG GROUP I (OSF) AND GROUP II (OSCC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn group I \u0026amp; II, 31 had habits of tobacco and rest of them had no habits. Within those who had habits 9.7 % had no stain, 51.6 % had mild staining, 22.6 % had moderate staining and 16.1% had intense staining. Those who are taking tobacco had no staining 55.6% had mild staining 33.3% and 11.1% had moderate staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI.STAINING INTENSITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of Staining Intensity (SI) Of \u0026alpha;-SMA (Alpha Smooth Muscle Actin) Among 3 Groups:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026alpha;-SMA\u003c/strong\u003e revealed positivity in group I, II and III. In Group I and Group II cases showed 85% staining for \u003cstrong\u003e\u0026alpha;-SMA\u003c/strong\u003e, whereas in Group III, positive staining was observed 30%.\u003c/p\u003e\n\u003cp\u003eIn Group I out of 20 OSF cases 30 % had a score of 3 (SI3), 20 % had a score of 2 (SI2) (\u003cstrong\u003eFig-2),\u003c/strong\u003e 35 % had a score of 1 (SI1) \u003cstrong\u003e(Fig-3) \u003c/strong\u003eand 15% had a score of 0 (SI0). Group II out of 20 cases 20 % had a score of 3 (SI3), 30 % had a score of 2 (SI2) \u003cstrong\u003e(Fig-4), \u003c/strong\u003e35 % had a score of 1 (SI1) \u003cstrong\u003e(Fig-5)\u003c/strong\u003eand 15% \u0026nbsp;had a score of 0 (SI0). In Group III out of 10 normal cases 30 % had a score of 0 (SI0) and (\u003cstrong\u003eFig -6)\u003c/strong\u003e 70 % had a score of 1 (SI1) (\u003cstrong\u003eFig-7A)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of Staining Intensity among Different OSF Grading:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn 20 cases of OSF, 6 cases belonged to Grade I, 10 cases belonged to Grade II and 4 cases belonged to Grade III. In Grade I 16.7% had a score of 0 (SI0), 50% had a score of 1 (SI1), 16.7 % had a score of 2 (SI2) and 16.7 % had a score of 3 (SI3). In Grade II 40 % had a score of1 (SI1), 30 % had a score of 2 (SI2), and 30% had a score of 3 (SI3), In Grade III 50 % had a score of 0 (SI0) and 50 % had a score of 3 (SI3) (\u003cstrong\u003eFig-7B)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of Staining Intensity among Different OSCC Grading:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn 20 cases of OSCC, 13 cases belonged to well differentiated group, 3 cases belonged to moderately differentiated and 4 cases belonged to poorly differentiated. In well differentiated group, 23.1% had a score of 0 (SI0), 23.1% had a score of 1 (SI1), 23.1 % had a score of 2 (SI2) and 30.8 % had a score of 3 (SI3). In moderately differentiated group 66.7 % had a score of 1 (SI1) and 33.3 %had a score of 2 (SI2). In poorly differentiated group 50 % had a score of 1 (SI1) and 50 % had a score of 2 (SI2) (\u003cstrong\u003eFig-7C)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKAPPA STASTISTIC VALUE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inter-observer agreement for the staining intensity of stain for all the 3 groups was arrived using kappa statistics and kappa value is \u003cstrong\u003e0.689\u003c/strong\u003e.\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003e\u003cstrong\u003e PERCENTAGE OF IMMUNOPOSITIVE CELLS- LABELING INDEX (LI)\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of Labeling Index (Li) Of \u0026alpha;-SMA (Alpha Smooth Muscle Actin) Among 3 Groups:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026alpha;-SMA\u003c/strong\u003e revealed positivity in group I, II and III. In Group I and Group II cases showed 85% staining for \u003cstrong\u003e\u0026alpha;-SMA\u003c/strong\u003e, whereas in Group III, positive staining was observed 40%.\u003c/p\u003e\n\u003cp\u003eIn Group I out of 20 OSF cases 5 % had a score of 3 (LI3), 40 % had a score of 2 (LI2), 40 % had a score of 1 (LI1) and 15% had a score of 0 (LI0). Group II out of 20 cases 15 % had a score of 0 (LI0) and 85 % had a score of 1 (LI1). In Group III out of 10 normal cases 60 % had a score of 0 (LI0) and 40 % had a score of 1 (LI1) (\u003cstrong\u003eFig-8A)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of Labeling Index (Li) Among Different OSF Grading:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal 20 cases of OSF, 6 cases belonged to Grade I, 10 cases belonged to Grade II and 4 cases belonged to Grade III. In Grade I 16.7% have a score of 0 (LI0), 50% had a score of 1 (LI1) and 33.3 % had a score of 2 (LI2). In Grade II 50 %had a score of1 (LI1), 40 % had a score of 2 (LI2) and 10% had a score of 3 (SI3). In Grade III 50 % had a score of 0 (LI0) and 50 % had a score of 2 (LI2) \u003cstrong\u003e(Fig-8B)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of Labeling Index (Li) Among Different OSCC Grading:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal 20 cases of OSCC, 13 cases belonged to well differentiated group, 3 cases belonged to moderately differentiated and 4 cases belonged to poorly differentiated. In well differentiated group, 23.1% had a score of 0 (LI0) and 76.9 % had a score of 1 (LI1). In moderately and poorly differentiated group all had a score of 1 (LI1)\u003cstrong\u003e(Fig-8C).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIII. STAINING INDEX\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of Staining Index among the Groups.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing Kruskal-Wallis test the comparison among groups derived, there is a significant difference in the expression of myofibroblasts. (\u003cstrong\u003eFig-9A)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison Of Staining Index\u0026nbsp; Between Group I And Group III\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eIn OSF, 15% of the cases showed no staining while 40% mild staining, 20% moderate staining and 25% of the cases had intense staining in connective tissue. In normal mucosa, 40% exhibited mild staining and no staining was seen in 60% of the cases. The result is statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison Of Staining Index Between Group II And Group III\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eIn OSCC, 15% of the cases showed no staining while 65% mild staining and 20% moderate staining in connective tissue. In normal mucosa, 40% exhibited mild staining and no staining was seen in 60% of the cases. The result is statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison Of Staining Intensity Between Group I And Group II\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eIn OSF group, 15% of the cases showed no staining while 40% mild staining, 20% moderate staining, and 25% of the cases had intense staining in connective tissue. In OSCC group, 15% of the cases showed no staining while 65% mild staining and 20% moderate staining in connective tissue. As both the lesions expressing myofibroblasts, the result is statistically not significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison of Staining In Different OSF Grading\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn Grade I, 16.7% showed no stain, 50% showed mild stain 16.7% moderate and 16.7 % showed intense staining. In Grade II 50 % showed mild staining 20 % showed moderate and 30% showed intense staining. In Grade III 50 % showed no stain, 25% showed moderate and 25 % showed intense staining. \u003cstrong\u003e(Fig-9B)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison of Staining In Different OSCC Grading\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In well differentiated group 23.1% showed no stain, 46.2% showed mild stain and 30.8% showed moderate staining. In OSCC moderately differentiated 100% showed mild staining. In OSCC poorly differentiated 100% showed mild staining \u003cstrong\u003e(Fig-9C).\u003c/strong\u003e\u003c/p\u003e"},{"header":"discussion","content":"\u003cp\u003eMyofibroblasts are smooth muscle like fibroblasts and one of the phenotypic heterogeneity among the fibroblasts. They can alter the inflammatory response by secreting the soluble mediators of inflammation. Furthermore myofibroblasts is suggested to be induced by TGF\u0026beta;, a potent pro-inflammatory and pro-fibrotic cytokine related to imbalance between collagen deposition and degradation in Oral Submucous Fibrosis [6].Hence the role of myofibroblasts in the repair process has varying functions in the physiological and pathological conditions\u003c/p\u003e\n\u003cp\u003eThe distribution of genders in the study groups ranged from a ratio 4:1 in favor of Males: Females in the control group and 19:1 in the OSF group. The male preponderance among OSF has been documented in several studies and can be due to the habit of using tobacco [7].\u003c/p\u003e\n\u003cp\u003eThe age distribution of OSF is higher between 20-40 years as similar to other studies reported in the literature [7]. Similarly the occurrence of OSCC commonly occurs among older age groups as reflected in our study. As normal controls were obtained during impaction procedures 90% of they were obtained from 20-40 age groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStaining intensity:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStaining intensity between the three study groups was not statistically significant and the expression was seen more in group I than in group II (20%). The staining intensity progressively increased from early OSF (16.5%) to advance OSF (grade III 50%). This indicates that OSF represents a failed wound healing process of the oral mucosa after chronic sustained injury resulting in scarring and fibrosis. This could be also in response to the hypersensitivity caused by arecoline and the resultant persistent (juxtra epithelial inflammatory response) in OSF, which acts as an initiating factor leading to a defective inflammatory response and activation of fibroblasts culminating in fibrosis [8].\u003c/p\u003e\n\u003cp\u003eThe well differentiated OSCC showed higher staining intensity indicating that myofibroblasts are stimulated during the repair of extra cellular matrix and are capable of augmenting and down-regulating the inflammatory response by secretion of these soluble mediators of inflammation [9].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLabeling index:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe labeling index indicates the more percentage of cells stain with \u0026alpha; SMA in group I than in group II or III and can be due to chronic inflammation and continuous tissue remodeling in OSF as well as chronic sustaining injury to factors such as arecoline, micro trauma could cause the transient or continuous differentiation of fibroblast from undifferentiated stem cell or a differentiated fibroblast pool [10].\u003c/p\u003e\n\u003cp\u003eThe labeling index among the various grades of OSF shows an increase from grade 1 to III. This indicates with increase of stimulus and disease grade, more amount of myofibroblasts differentiation occurs [11]. \u0026nbsp;However the difference between the grades were not statistically significant in OSCC with varying grades of differentiation, no significant difference could be found in the labeling index. Hence the expression of \u0026alpha; SMA indicates that differentiation of tumor does not appear to influence the differentiation of myofibroblasts [11].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStaining index:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was statistical significant difference in the pattern of staining index among the study group. Intense staining of \u0026alpha; SMA was observed only in group I while moderate staining was seen in group I and II only. Group II had only the effect of inflammation and hence had a mild staining index in 40% of cases while no staining was seen in 60% of cases. The intense staining in group I was similar to the findings in the literature [13].Though 65% cases of group II showed a mild SI and 20% cases had moderate SI, none had intense SI. The absence of intense SI in group II needs to further analyzed.\u003c/p\u003e\n\u003cp\u003eOn comparing the Staining Index between OSF and normal tissue, it is observed that there was a wide difference between the expressions of SI using \u0026alpha;SMA. The uniform mild expression in both cases probably is a result of the mild continuous inflammation in both the condition [14, 15].\u003c/p\u003e\n\u003cp\u003eOn comparing the group II and group III, it is observed that there is only a mild difference between the grades of Staining Index. However it was statistically significant. The uniformity in expression probably relates to the chronic inflammation that exists between both the conditions [15].\u003c/p\u003e\n\u003cp\u003eThere is no significant difference between group I and II (p=0.157) However 24% of cases exhibited intense staining in group I while no cases of group II had intense SI. This indicates that OSF had more myofibroblasts expression than OSCC [16, 17].\u003c/p\u003e\n\u003cp\u003eThe staining index did not considerably vary within the grades of the OSF .This indicates that the staining intensity as well as labeling index is crucial factor for the staining index. In this cross sectional study the expression of SI 3 increased with increasing grades of OSF. While no such observation was seen in labeling index. Low number of cells expressed SMA positivity while the staining intensity varied. This indicates that with grades of OSF increasing only a small subset of fibroblast population expressed intensive staining while the number of cells expressing \u0026alpha; SMA never altered[18,19].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003ePersistent, chronic inflammation leads to formation of a subset of fibroblastic population-myofibroblasts. The progressive increase of myofibroblasts in the Oral submucous fibrosis can be used as a marker in evaluating their severity. Myofibroblasts stimulate tumor development and constitute the tumor stroma and their presence in the invasion front of the tumor suggests they are associated with tumorgenesis. Clinical evidence suggests that the presence of myofibroblasts is associated with a poor prognosis have to be evaluated.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"abbreviations ","content":"\u003cp\u003e\u0026alpha; SMA- alpha smooth muscle actin\u003c/p\u003e\n\u003cp\u003eOSF- Oral submucous fibrosis\u003c/p\u003e\n\u003cp\u003eOSCC- Oral squamous cell carcinoma\u003c/p\u003e\n\u003cp\u003eSI- staining intensity\u003c/p\u003e\n\u003cp\u003eLI \u0026ndash; labeling index\u003c/p\u003e\n\u003cp\u003eTGF\u0026beta; -transforming growth factor beta\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and Consent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was evaluated and approved by the Institutional Ethics Committee of the Ragas Dental College and Hospital, Chennai.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication:\u003c/strong\u003e\u0026nbsp; Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e\u0026nbsp; The data and material can be available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The research did not receive any financial support from funding agencies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAffiliations:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDepartment of Oral pathology, SRM University, College Dentistry,kattankulathur,Chennai,India\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDr. Sivachandran Annamalai\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDepartment of dental diagnostic Science \u0026amp; Oral Biology, College Of Dentistry, King Khalid University ,Abha,Saudi Arabia\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDr. Shaik Mohamed Shamsudeen\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDepartment of Oral Pathology, Ragas Dental college and Hospital, Chennai, India\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDr. Rooban T, Dr. Elizabeth Joshua, Dr. Umadevi K Rao Dr. Ranganathan Kannan\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception\u0026nbsp; , Design the study (SA, EJ, UR, RK),Acquisition of data and biological samples(SA,SM), Data analysis and interpretation (EJ, UR, RK, SA,RT,SM )Manuscript\u0026nbsp; drafting and editing (SM,SA),Final approval of manuscript (EJ, UR, RK, SA,RT,SM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u003c/strong\u003e\u0026nbsp; NIL\u003c/p\u003e"},{"header":"references","content":"\u003col\u003e\n\u003cli\u003ePunnya V. Angadi, Alka D. Kale, Seema Hallikerimath. Evaluation of myofibroblasts in oral submucous fibrosis: correlation with disease severity. Journal of Oral Pathology \u0026amp; Medicine 2011; 40(3) : 208\u0026ndash;213.\u003c/li\u003e\n\u003cli\u003eSteven E. Mutsaers, Jill E. Bishop, Gus Mcgrouther, Geoffrey J. Laurent. Mechanisms of Tissue Repair: from Wound Healing to Fibrosis. J. Biochem. Cell Bid.1997; 29: 5-17.\u003c/li\u003e\n\u003cli\u003eKundendu AryaBishen, Raghu Radhakrishnan, Kapaettu Satyamoorthy. The role of basic fibroblast growth factor in oral submucous fibrosis pathogenesis. J Oral Pathol Med 2008; 37: 402\u0026ndash;411.\u003c/li\u003e\n\u003cli\u003eTilakaratne WM, Klinikowski M.F, Takashi Saku, Peters TJ, SamanWarnakulasuriya. Oral submucous fibrosis: Review on aetiology and pathogenesis. Oral Oncology 2006; 42:561\u0026ndash; 568.\u003c/li\u003e\n\u003cli\u003eHu Yanjia, JianXinchun.The role of epithelial\u0026ndash;mesenchymal transition in oral squamous cell carcinoma and oral submucous fibrosis.Clinica Chimica Acta 2007; 383: 51\u0026ndash;566.\u003c/li\u003e\n\u003cli\u003eMithani SK, Mydlarz WK, Grumbine FL, Smith IM, Califano JA. Molecular genetics of premalignant oral lesions. Oral Dis 2007; 13:126-33.\u003c/li\u003e\n\u003cli\u003eRanganathan K, Devi UM , Joshua E, Kirankumar K, SaraswathiR.Oral submucous fibrosis: a case-control study in Chennai, South India.. J Oral Pathol Med 2004; 33:274\u0026ndash;277\u003c/li\u003e\n\u003cli\u003eRajalalitha, S. Vali. Molecular pathogenesis of oral submucous fibrosis \u0026ndash; a collagen metabolic disorder. \u003cem\u003eJ Oral Pathol Med. \u003c/em\u003e2005; 34:321-8.\u003c/li\u003e\n\u003cli\u003eBeacham DA, Cukierman E. Stromagenesis: the changing face of fibroblastic microenvironments during tumor progression. Semin Cancer Biol. 2005; 15:329\u0026ndash;41.\u003c/li\u003e\n\u003cli\u003eXouri G, Christian S. Origin and function of tumor stromafibroblasts. Semin Cell Dev Biol 2010; 21:40-6.\u003c/li\u003e\n\u003cli\u003eWynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol 2008; 214:199-210.\u003c/li\u003e\n\u003cli\u003eL\u0026uacute;cio PS, Cavalcanti AL, Alves PM, Godoy GP, Nonaka CF.Myofibroblasts and their relationship with oral squamous cellcarcinoma. Braz J Otorhinolaryngol 2013; 79:112‑\u003c/li\u003e\n\u003cli\u003ePhilip T, Kumar TD, Rajkumar K, Karthik KR, Priyadharsini N,Kumar AR. Immunohistochemical evaluation of myofibroblasts using alpha-smooth muscle actin in oral submucous fibrosis. SRM J Res Dent Sci 2014; 4:243-7.\u003c/li\u003e\n\u003cli\u003eUtsunomiya H, Tilakaratne WM, Oshiro K, Maruyama S, Suzuki M, Ida-Yonemochi H. Extracellular matrix remodeling in oral submucous fibrosis: Its stage-specific modes revealed byimmunohistochemistry and in situ J Oral PatholMed 2005;34:498-507.\u003c/li\u003e\n\u003cli\u003eDarby I, Skalli O, Gabbiani G. Alpha‑smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab Invest 1990; 63:21‑9.\u003c/li\u003e\n\u003cli\u003eChaudhary M, Gadbail AR, Vidhale G, MankarGadbailMP,Gondivkar SM, Gawande M. Comparison of myofibroblasts expression in oral squamous cell carcinoma, verrucous carcinoma, high risk epithelial dysplasia, low risk epithelial dysplasia and normal oral mucosa. Head Neck Pathol 2012; 6:305‑\u003c/li\u003e\n\u003cli\u003eKellermann MG, Sobral LM, Da Silva SD, et al. Myofibroblasts in the stroma of oral squamous cell carcinoma is associated with poor prognosis. Histopathology 2007; 51: 849\u0026ndash;53.\u003c/li\u003e\n\u003cli\u003eDesmouli\u0026egrave;re A, Guyot C, Gabbiani G. The stroma reaction myofibroblast: A key player in the control of tumor cell behavior. Int J Dev Biol 2004; 48:509-17.\u003c/li\u003e\n\u003cli\u003eGupta MK, Mhaske S, Ragavendra R, Imtiyaz. Oral submucous fibrosis \u0026mdash; Current Concepts in etiopathogenesis. Peoples J Sci Res 2008; 1:39-44.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Myofibroblasts, Alpha smooth muscle actin, Staining intensity, Oral Submucous Fibrosis, Oral Squamous Cell Carcinoma","lastPublishedDoi":"10.21203/rs.2.17936/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.17936/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Myofibroblasts are spindle shaped smooth muscle like fibroblast involved in the physiological repair of tissue structure can also cause pathological remodeling of tissue by forming fibrosis. They express smooth muscle actin which can be evaluated\u0026nbsp;\u0026nbsp;for their biological behavior in the physiological and pathological conditions. \u003c/p\u003e\u003cp\u003eAim: To evaluate the expression of Myofibroblasts in oral submucous fibrosis, oral squamous cell carcinoma and normal mucosa by Immunohistochemical analysis. Methods: It is a cross-sectional study done to evaluate the expression of Myofibroblasts in oral submucous fibrosis (Group-1), oral squamous cell carcinoma (Group-2) and normal buccal mucosa (Group-3) using immunohistochemistry using alpha smooth muscle actin in formalin fixed, paraffin embedded tissue specimens. The localization of stain, nature of stain, intensity of stain and the percentage of cells stained among the three groups were studied and compared. \u003c/p\u003e\u003cp\u003eResults: The gender distributions in the study were in favor of males. It was observed that Staining Intensity was seen more in group I (30%) than in group II (20%) and progressively increased from early (16.5%) to advance stage(50%) of oral submucous fibrosis. Higher staining intensity was observed in well differentiated oral squamous cell carcinoma. \u003c/p\u003e\u003cp\u003eConclusion: Myofibroblasts are important in normal healing process, but can induce fibrosis. In oral submucous fibrosis, they can be used to assess the severity of the lesion by their increased expression of alpha smooth muscle actin. In oral carcinogenesis increase in the number of alpha smooth muscle actin can change the distribution pattern results in tumor invasive characteristics. Key Words: Myofibroblasts, Alpha smooth muscle actin, Staining intensity, Oral Submucous Fibrosis, Oral Squamous Cell Carcinoma.\u003c/p\u003e","manuscriptTitle":"Myofibroblasts in Oral submucous fibrosis, Oral squamous cell carcinoma and Normal mucosa- an Immunohistochemical Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2019-12-02 18:39:23","doi":"10.21203/rs.2.17936/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8881745c-967f-4721-9a62-380ea548da53","owner":[],"postedDate":"December 2nd, 2019","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":38831,"name":"Head \u0026 Neck Surgery"}],"tags":[],"updatedAt":"","versionOfRecord":[],"versionCreatedAt":"2019-12-02 18:39:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8565","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-8565","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}