Oral submucous fibrosis (OSMF) a chronic and an insidious disease that affects mainly the buccal mucosa is characterized by localized or diffuse localized or diffuse ulceration, burning sensation, fibrosis of oral tissues and trismus.1 This oral lesion may sometime extend to soft plate and pharynx. OSMF shows a strong confinement to Indian and South East Asian population. It has a strong predisposition to malignant transformation.2

In OSMF, the disturbance in equilibrium between matrix metalloproteinases (MMPs) and tissue inhibitor matrix metalloproteinases (TIMP) results in elevated deposition of extracellular matrix (ECM).3 MMPs play a crucial role in fibrosis of the oral cavity.

(MMPs) are a group of structurally related zinc-dependent endopeptidases and have the capability to degrade all components of extracellular Matrix. To date, at least 25 different vertebrate MMPs have been identified, 24 of which are present in humans, including two recently duplicated genes encoding MMP-23. These MMPs are classified into five main classes (collagenases, gelatinases, stromelysins, membrane-type and others, including the matrilysin) on the basis of their putative substrate specificity and internal homologies.4

MMP-3 also known as stromelysins I is a key member of this MMP family which is responsible for degradation of collagen type II, IV, V, IX and X, proteoglycans, gelatins, fibronectin, laminin and elastin. It plays an important role in activation of pro MMP-1 into the active form of MMP-1 in malignant tissues. MMP-3 expression is low in normal tissues but it is altered during tumour formation, where remodeling of ECM is required.5

As OSMF is considered as a high-risk pre-cancerous condition, knowledge of genetic susceptibility to malignant transformation is of immense importance, especially in developing cancer predictive markers for OSMF patients. The present study was conducted with an aim to evaluate MMP-3 as a possible biomarker for OSMF

This hospital-based study with case-control design was carried out in the Department of Biochemistry, Byramjee Jeejeebhoy Government Medical College and Sassoon General Hospital, Pune, Maharashtra for a period of 2 years (January 2023-December 2024). The protocol of the study was approved by Institutional Ethics Committee (Reference number 6/2018, dated 22/2/2018).

The study included a total of 118 clinically diagnosed OSMF patients. Informed consent was obtained from each participant Additionally total of 118 healthy individuals were recruited as controls. The diagnosis of OSMF was made by clinician. The operational definition used “OSMF is a chronic, complex, premalignant condition of the oral cavity, characterized by juxta-epithelial inflammatory reaction and progressive fibrosis of the sub mucosal tissues (the lamina propria and deeper connective tissues)”.

Inclusion Criteria.

1. Individuals with positive history of areca nut and tobacco chewing intake > 1
2. Presence of disease made by clinician on the basis of clinical inspection, a restricted mouth opening, and the presence of fibrous band and the complaint of burning sensation

Exclusion criteria.

1. individuals with a history of the consumption of tobacco in any other form such as cigar, bidi, mawa, etc.
2. patients with any history of local application, intra lesional injection or a systemic administration of drugs
3. not willing to be a part of the study

From these participants 5 ml of blood sample was aseptically with single use disposal syringe and needle from antecubital vein by venipuncture. The blood sample was collected in plain vacutainer. After collection the blood sample was allowed to stand for clotting at room temperature for 15 minutes. Blood sample was centrifuged at 3000 rpm for 10 minutes. The serum sample obtained was collected in vial and stored at -20◦C for estimation of MMP3,

MMP-3 was estimated using the TIMP ELISA method involves quantifying MMP-3 levels in a sample by utilizing an enzyme-linked immunosorbent assay (ELISA) that specifically detects MMP-3, often in conjunction with its inhibitor, TIMP (tissue inhibitor of metalloproteinases). Standards and samples were pipetted into the wells and any MMP-3 present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for MMP-3 was added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and color develops in proportion to the concentrations of total MMP-3 (pro-and/or active) bound in the initial step. After the color development was stopped then the intensity of the color was measured.

Among OSMF patients, 76 (64.4%) were males and 42 (35.6%) were females whereas in control group, 74 participants were males and 44 (37.3%) were females. There was no significant difference between gender wise distribution of recruits in patients and control groups (Chi square test, P>0.05). The age wise distribution of participants is shown in table 1. Maximum number of patients belonged to age group < 60 years (24.6%) followed by age groups 51-60 years (22.1%) and 41-50 years (20.3%). There was no significant difference between age wise distribution of recruits in patients and control groups (Chi square test, P>0.05).

Table 1. Age wise distribution of OSMF patients and controls.

The lesions of OSMF of 118 OSMF patients classified into four grades (grade 1 to grade 4). This classification was per the clinical grades of OSMF lesions diagnosed by clinician. When Z test was performed to know the difference between the proportions of the OSMF patients from different clinical grades of OSMF, there was no statistically significant difference noted.

Figure 1. Grade wise distribution of OSMF patients.

Poor oral hygiene, chronic alcoholism and consumption of spicy food were common confounders other than tobacco chewing and arecanut consumption found to be associated with OSMF. As shown in table 2, the mean serum levels of MMP-3 were higher in OSMF patients as compared to individuals belonging to control group and the difference was highly statistically significant (P value <0.0001**).

Table 2. Matrix metallo-proteinase -3 (MMP-3) in OSMF patients and controls.

As shown in figure 2, the mean MMP-3 of OSMF patients increased with severity of the disease (One-way ANOVA test, P value <0.0001).

Figure 2. Grade wise mean ± serum of MMP-3 OSMF patients

As per definition OSMF is a chronic condition that is featured by progressive stiffing of buccal mucosa that ultimate result in inability to open mouth.6 It is a chronic and latent malignant condition that is characterized by both hyalinization and fibrosis of oral sub- epithelium tissue. In addition, negatively affecting the individual’s quality of life and physical health, OSMF also7 poses as a regional and global serious public health issue. 7 In case of OSMF, the rate of ‘malignant transformation’ to ‘oral squamous cell carcinoma (OSCC)’ is reported between 7 to 13%.8The disease, OSMF is characteristic by appearance of sign of fibrosis that is progressive and featured appearance of myofibroblasts and expression of consistent “α-smooth muscle actin (α-SMA)”. The combination of these pathological features leads to alternation of oral microenvironment that finally progress to tumor.9

There are various mechanisms considered for overall pathophysiology of OSMF. The combination of various pathological events mediates the OSMF. These include avitaminosis, iron and other nutritional deficiencies, dietary habits, mutations, substance abuse (smokeless tobacco, alcohol) and infections.10Among the various etio-pathogenic mechanisms described till recent, alteration in the extracellular matrix (ECM), mediated by about by numerous proteolytic enzymes and their inhibitors, is suggested to play an important role. Numerous proteolytic enzymes and their inhibitors, is suggested to play an important role. Among the various proteolytic enzymes released by the stromal cells, matrix metalloproteinases (MMPs) play an important role in the modulation of ECM.11,12

MMPs are produced by a variety of cells during both physiologic conditions like normal development and wound healing and a wide variety of pathological processes. Currently, 28 human MMPs have been identified, and these enzymes have been classified according to their substrate specificity and structural similarities. The major subgroups are interstitial collagenases, gelatinases, stromelysins, and membrane- bound MMPs. They are produced by several cell types including fibroblasts, macrophages, neutrophils, and some epithelial cells. Their secretion is induced by certain stimuli including growth factors, cytokines, and physical stress. Most MMPs are synthesized as pro- peptides that require proteolytic cleavage for activation. 13, 14

In this study at estimation of serum MMP-3 levels in OSMF was done. The mean serum MMP-3 levels were higher in subjects with OSMF as compared to normal subjects and the difference was highly statistically significant (p <0.0001). The mean± matrix metallo- proteinase -3 (MMP-3) of OSMF patients increased with severity of the disease.

Although, national and international studies are conducted for various aspects of OSMF including epidemiology, pathophysiology, evaluation of diagnostic and prognostic biomarkers, and therapeutic modalities, the scope for still exist for evaluating the known concepts and unknown mechanisms responsible for this oral ailment. In this study, when the MMP-3 levels were assessed in OSMF patients, it was noted that elevated serum MMP-3 levels can be used as screening tool in the early detection of OSMF. Additionally, it can be ideal tool for monitoring the prognosis of the patients

1. Rani P, Singh RNP, Sharma S, Shahi AK, Chandra S, Singh B. Prevalence of Oral Submucous Fibrosis, Its Correlation of Clinical Grading to Various Habit Factors among Patients of Bihar: A Cross-Sectional Study. J Pharm Bioallied Sci. 2023 15(Suppl 1):S554-7.
2. Srivastava R, Jyoti B, Pradhan D, Siddiqui Z. Prevalence of oral submucous fibrosis in patients visiting dental OPD of a dental college in Kanpur:A demographic study. J Family Mad Prim Care. 2019;8: 2612–7.
3. Poonja P, Sattur A, Burde K, Hiremath S. A study on genetic polymorphism in Matrix Metalloproteinases-3 in oral submucous fibrosis patients and in healthy individuals. Appl Cancer Res 2017; 37: 4.
4. Venugopal A, Maheswari TU. Expression of matrix metalloproteinase-9 in oral potentially malignant disorders: A systematic review. J. Oral Maxillofac. Pathol. 2016;20(3):474-9.
5. Shrestha A, Carnelio S. Evaluation of matrix metalloproteinases-2 (MMP-2) and tissue inhibitors of metalloproteinases-2 (TIMP-2) in oral submucous fibrosis and their correlation with disease severity. Kathmandu University Me J. 2015;11(4):274- 81.
6. Xu H, Lyu FY, Song JY, Xu YM, Jiang EH, Shang ZJ, et al. Research Achievements of Oral Submucous Fibrosis: Progress and Prospect. Biomed Res Int. 2021; 2021:6631856.
7. Phulari R, Dave E. A systematic review on the mechanisms of malignant transformation of oral submucous fibrosis. Eur J Cancer Prev 2020;29(5):470–473.
8. Singh I, Juneja S, Tandon A, Jain A, Shetty DC, Sethi A. Immunoexpression of alpha smooth muscle actin correlates with serum transforming growth factor‐β1 levels in oral submucous fibrosis. Journal of Investigative and Clinical Dentistry. 2019;10(4): e12473.
9. Soumya S, Sanghvi R. Prevalence of Oral Submucous Fibrosis with Other Oral Potentially Malignant Disorders: A Clinical Retrospective Study. Cureus. 2023 Nov 29; 15(11): e49642.
10. Srivastava R, Jyoti B, Pradhan D, Siddiqui Z. Prevalence of oral submucous fibrosis in patients visiting dental OPD of a dental college in Kanpur: A demographic study. J Family Med Prim Care. 2019;8(8):2612-1
11. Satpute PS, Hazarey VK, Ganvir SM. Estimation of Matrix Metalloproteinases-2 Promoter Polymorphism as a Risk Factor for Oral Carcinogenesis in Indian Population. Sci Rep 2012;1(7):370.
12. Chaudhary AK, Singh M, Bharti AC, Asotra K, Sundaram S, Mehrotra R. Genetic polymorphisms of matrix metalloproteinases and their inhibitors in potentially malignant and malignant lesions of the head and neck. J Biomed Sci. 2010; 17:10.
13. Mishra G, Ranganathan K. Matrix metalloproteinase-1 expression in oral submucous fibrosis: An immunohistochemical study. Ind J Dent Res. 2010;21(3):320-5.
14. Bazarsad S, Zhang X, Kim K, Illeperuma R., Jayasinghe R., Tilakaratne W et al. Identification of a combined biomarker for malignant transformation in oral submucous fibrosis. J. Oral Pathol. Med. 2017; 46:431-8.