κ-Carrageenan-Based Hydrogels Support Viability and Odontogenic Differentiation of Stem Cells From the Apical Papilla

Immature teeth with pulp necrosis remain challenging because conventional root canal treatment does not restore pulp-dentin vitality. Hydrogel scaffolds for regenerative endodontics should support stem cell viability and provide a microenvironment favourable for dentin-pulp regeneration. This study evaluated κ-Carrageenan (κC)-based hydrogels, with and without gelatin, as scaffolds for stem cells from the apical papilla (SCAP), and examined how formulation-dependent properties influenced SCAP viability and odontogenic differentiation.

Hydrogels were prepared as 1% gelatin, 1.5% κC, 2.5% κC, 1.5% κC+gelatin (κCG) and 2.5% κCG, with cells cultured without hydrogel as a control. Physicochemical characterisation included scanning electron microscopy, Fourier-transform infrared spectroscopy, rheology, degradation and swelling at days 1, 3, 7 and 14. SCAP were isolated and characterised using flow cytometry. Cell viability, metabolic activity, spreading, migration, alkaline phosphatase activity, Alizarin Red S staining and qPCR of differentiation-related markers were also assessed.

The hydrogels showed formulation-dependent microstructure, degradation, swelling and viscoelastic properties. κC-containing formulations behaved as viscoelastic solids, and 2.5% κCG exhibited the highest storage modulus and lowest Tan (δ). SCAP remained predominantly viable in all groups. Gelatin-containing hydrogels improved long-term cytocompatibility and greater cell spreading compared to lower-concentration κC alone. Migration was comparable across groups. During differentiation, κC-containing formulations showed higher alkaline phosphatase activity and mineral deposition, with increased late-stage mineralisation observed in 2.5% κCG. Gene expression analysis indicated early upregulation of RUNX2 and BSP and increased late-stage expression of OPN, OCN, DMP1 and DSPP, with a more evident maturation-associated response in 2.5% κCG.

κC-based hydrogels supported SCAP viability and promoted odontogenic differentiation in vitro. Gelatin incorporation improved late-stage mineralisation and odontogenic marker expression, with 2.5% κCG demonstrating the most favourable overall response.

κC-gelatin hydrogels may serve as promising scaffold candidates for dentin-pulp regenerative strategies by supporting SCAP survival and odontogenic maturation within a tunable hydrogel microenvironment.