Validation of In Vitro Model of Oral Fibrosis Via Genetic and Protein Characterization

Background: Oral submucous fibrosis (OSMF) poses a significant health challenge due to its association with restricted mouth opening and an elevated risk of oral cancer. Despite its impact, a lack of well-characterized and validated in vitro models has impeded a thorough understanding of the molecular mechanisms driving OSMF progression. Objectives: The primary objective of the present study was to establish and characterize in vitro primary cell line, consisting of fibroblasts, from OSMF biopsy sample. Materials and Methods: The study involved the isolation of fibroblasts from tissue affected by OSMF. Following isolation, the fibroblasts were cultured and passaged, with the process continued up to passage 2. Cell viability and Population Doubling Time calculated cell proliferation, while morphological changes were reported. Immunocytochemistry assessed fibroblast markers (vimentin, β actin, and CD90), and qPCR measured gene expressions (TGFBR-1, COMP, TGM-2, TIMP-1, MMP-7, MMP-9) across passages, comparing with OSMF tissue and control fibroblasts. Results: The in vitro OSMF cell line demonstrated successful isolation, subculturing, and sustained fibroblast proliferation. Population Doubling Time calculations and morphological observations from passages 0 to 2 provided insights into growth patterns. Immunocytochemistry confirmed fibroblast identity through positive expression of vimentin, beta-actin, and CD90. QPCR revealed varied expressions of pathological genes across passages, closely resembling OSMF tissue, while distinct differences were noted as compared to control fibroblasts. Conclusions: The in vitro OSMF primary cell line was developed directly from the patient sample. Validation of this model of oral fibrosis was achieved successfully through genetic and protein characterization therefore, presenting it as a reliable experimental platform for future investigations into OSMF pathogenesis and potential therapeutic interventions. Clinical application: This study provides a validated in vitro model for OSMF, offering a foundation to explore disease mechanisms, identify biomarkers, and develop targeted therapies. By closely mimicking the molecular and cellular characteristics of OSMF, the model facilitates testing of potential treatments, understanding fibrosis-to-cancer transitions, and tailoring personalized interventions. It holds the potential for advancing precision medicine, improving early detection, and guiding regenerative therapies to restore oral function in affected patients.