Characterization of novel calcium hydroxide-mediated highly porous chitosan-calcium scaffolds for potential application in dentin tissue engineering

The aim of this study was to develop a highly porous calcium-containing chitosan scaffold suitable for dentin regeneration. A calcium hydroxide (Ca[OH](2)) suspension was used to modulate the degree of porosity and chemical composition of chitosan scaffolds. The chitosan solution concentration and freezing protocol were adjusted to optimize the porous architecture using the phase-separation technique. Scanning electron microscopy/energy-dispersive spectroscopy demonstrated the fabrication of a highly porous calcium-linked chitosan scaffold (CH-Ca), with a well-organized and interconnected porous network. Scaffolds were cross-linked on glutaraldehyde (GA) vapor. Following a 28-day incubation in water, cross-linked CH scaffold had no changes on humid mass, and CH-Ca featured a controlled degradability profile since the significant humid mass loss was observed only after 21 (26.0%) and 28 days (42.2%). Fourier-transform infrared spectroscopy indicated the establishment of Schiff base on cross-linked scaffolds, along with calcium complexation for CH-Ca. Cross-linked CH-Ca scaffold featured a sustained Ca2+ release up to 21 days in a humid environment. This porous and stable architecture allowed for human dental pulp cells (HDPCs) to spread throughout the scaffold, with cells exhibiting a widely stretched cytoplasm; whereas, the cells seeded onto CH scaffold were organized in clusters. HDPCs seeded onto CH-Ca featured significantly higher ALP activity, and gene expressions for ALP, Col1, DMP-1, and DSPP in comparison to CH, leading to a significant 3.5 times increase in calcium-rich matrix deposition. In sum, our findings suggest that CH-Ca scaffolds are attractive candidates for creating a highly porous and bioactive substrate for dentin tissue engineering.