Biomineralization on enzymatically cross-linked gelatin hydrogels in the absence of dexamethasone

A mechanical stimulus and chemical induction by dexamethasone have been important factors in dental pulp stem cell (DPSC) differentiation and biomineralization. We have demonstrated that the enzymatically crosslinked gelatin hydrogels are extremely effective substrates for DPSC differentiation towards odontoblasts. DPSCs were seeded on the crosslinked hard (similar to 8 kPa) and soft (similar to 0.15 kPa) gelatin hydrogels for 35 days with and without dexamethasone. Odontogenic differentiation markers such as OCN, ALP and DSPP were upregulated after 35 days of culture on crosslinked hydrogels with and without dexamethasone. SEM and Alizarin red staining of the crosslinked hydrogels showed a biomineralized sheet of hydroxyapatite deposits laid by the DPSCs on the top surface and inside the hydrogel. We found that the DPSC differentiation and biomineralization were independent of the hydrogel stiffness and dexamethasone. We hypothesize that this biomineralization was indeed triggered by the surface chemistry of the crosslinked gelatin hydrogels since we did not observe any biomineralization on the uncrosslinked gelatin or mTG. We also showed that the DPSCs, when removed from hard hydrogel surfaces and re-seeded on a TCPS, retained their odontogenic lineage and showed a permanent mineralization effect. Our results show the potential of enzymatically crosslinked gelatin hydrogels as scaffolds for dentin regeneration.