Chitosan/gelatin/PVA-based hydrogel as a 2D culture substrate for the differentiation of hDPSCs into insulin-producing cells

Abstract

Chitosan/poly(vinyl alcohol)/gelatin (CHS/PVA/GEL) hydrogels represent attractive two-dimensional (2D) biomaterial interfaces for directing stem cell behaviour in vitro. In this study, a CHS/PVA/GEL hydrogel was fabricated via solvent casting and rendered suitable for biological application through steam sterilisation (autoclaving). Physicochemical properties were evaluated by assessing uniformity, swelling capacity, and 21-day gravimetric stability in differentiation-relevant media. Biological performance was examined using human dental pulp stromal cells (hDPSCs), including verification of mesenchymal cell identity, cytocompatibility testing using extract-based MTT and LIVE/DEAD assays, together with direct-contact LIVE/DEAD assessment, and adhesion analysis. hDPSCs were subsequently differentiated into insulin-producing cells using a three-stage induction protocol over a 15-day differentiation workflow. Morphological changes, dithizone staining, and glucose-stimulated insulin secretion were used to assess differentiation. The hydrogel demonstrated cytocompatibility and supported cell adhesion. At the same time, differentiation yielded robust glucose responsiveness, with insulin secretion increasing from 627.4 μUI/mL per 1·105 cells (3.3 mM glucose) to 4121.3 μUI/mL per 1·10⁵ cells (16.7 mM glucose), corresponding to an approximately 6.6-fold increase. These findings highlight the CHS/PVA/GEL hydrogel as a stable planar substrate that enables strong functional outcomes.