Stem Cell Secretome Reverse Areca Nut Extract Induced Fibrosis in Human Fibroblast and Patient-Derived Biopsies

Oral submucous fibrosis is a progressive, oral potentially malignant condition that can affect the oral cavity and is often linked to areca nut consumption. This study aimed to investigate the potential of dental pulp mesenchymal stem cells secretome (DPMSCs-S) in the reversal of areca nut-induced fibrosis in human fibroblast cells and patient-derived buccal mucosa tissues.

The cytokine and growth factor levels in DPMSCs-S were analyzed by flow cytometry. Cytotoxicity and apoptotic features were analyzed using a cell viability assay and microscopy, respectively. Cellular senescence and reactive oxygen species generation were assessed in human fibroblast cells using a β-galactosidase assay and 2',7'-dichlorodihydrofluorescein diacetate probe, respectively. Fibrosis-related gene expression (COL15A1, COL1, COL3, α-SMA, TGFβ1, and TGFβ2) and protein expression (COL1 and Ki67) were assessed by RT-PCR and fluorescence microscopy, respectively.

Areca nut extract and arecoline resulted in a dose-dependent reduction in cell viability. Secretome treatment reduced cell death, apoptotic and senescent features in fibroblast cells exposed to areca nut extract and arecoline, decreased ROS levels, and significantly downregulated areca nut-induced fibrotic gene expressions (COL15A1, COL3, α-SMA, TGFβ2), and protein expression of COL1. Heat-inactivated secretome retained the activity, implying the involvement of heat-stable biomolecules in the reversal of fibrosis. Ex vivo results corroborated these findings, with reduced expression of COL15A1, COL3, and α-SMA in buccal tissues from oral submucous fibrosis patients treated with DPMSCs-S.

Secretome exhibited promising cytoprotective and anti-fibrotic properties, reduction in cell death, senescence, collagen expression, and oxidative stress. These findings support the potential of secretome as a novel therapeutic candidate for oral submucous fibrosis, meriting further investigation in preclinical and clinical trials.