Abstract

Regenerative dentistry has advanced considerably in recent years. Among the emerging approaches, biopolymer hydrogel have shown great potential as therapeutic agents for preserving the vitality of dentin-pulp tissues. These hydrogels integrated with cells and bioactive materials to stimulate functional tissue regeneration. Our study evaluated whether three-dimensional alginate–gelatin hydrogels (AGHs) crosslinked with calcium chloride (CaCl2) enhance the osteo-odontogenic differentiation of odontoblast-like cells in vitro. Two seeding configurations were compared: inter-hydrogel (INT) surface seeding and intra-hydrogel (INTR) encapsulation. Here, the MDPC-23 cells were cultured in AGHs crosslinked with 70 or 100 mM CaCl2 and assessed for proliferation, cytoskeletal morphology, alkaline phosphatase (ALP) activity, osteo-odontogenic gene expression, and mineralized nodule formation. After 7 days, cell proliferation was significantly greater in the alginate–gelatin hydrogel (AGH) groups than in the control group. Cells in the intra alginate–gelatin hydrogel 100 (INTR-AGH100) remained predominantly rounded, whereas those in the inter alginate–gelatin hydrogel 100 (INT-AGH100) formed irregular clusters on the hydrogel surface. ALPase activity was highest in INTR-AGH100 at the early stage of culture. Both INT-AGH100 and INTR-AGH100 showed significantly increased expression of DSPP, DMP-1, BSP, OCN, OPN, and Runx-2, together with enhanced mineralized nodule formation. Although no significant differences were detected between the two seeding strategies in all assays, distinct morphological patterns were observed, and the INTR configuration showed relatively greater early differentiation-related activity. These findings suggest that 100 mM calcium chloride-crosslinked AGHs provide a favorable three-dimensional microenvironment under the present experimental conditions and represent a promising in vitro scaffold platform to support future studies of matrix-guided dentin regeneration.