Development and Analysis of Calcium-Containing Simvastatin-Releasing Scaffolds for Pulp-Dentin Regeneration

Objectives: The objective of this study was to develop a porous chitosan-calcium scaffold capable of releasing simvastatin (SV) at a bioactive level, stimulating the migration and odontoblastic phenotype of dental pulp cells (DPCs), aiming to obtain an innovative cell-free tissue-engineering system for pulp-dentin complex regeneration.
Methods: A calcium-containing chitosan scaffold (CH-Ca) was developed by phase-separation. Cell spread, viability and odontoblastic markers expression (ALP, DSPP, DMP-1, Col1 and Ca deposition) was evaluated on DPCs seeded on the biomaterial. Then, bioactive dosage of simvastatin (SV) was selected and the synergistic effect with CH-Ca scaffold on markers expression and chemotaxis was determined. After that, CH-Ca capable of releasing bioactive SV dosage (CH-Ca-SV) was obtained and evaluated on DPCs 3D-culture, using an artificial pulp chamber with simulated pulp pressure. The presence of viable cells, cell spread and odontoblastic markers expression (ALP, Col1A1, DSPP/DSP, DMP-1, and Ca deposition) was assessed on the 3D matrix and scaffold surfaces. Scaffolds with no Ca or SV were used as controls (CT) (ANOVA/Tukey a=5%).
Results: The CH-Ca scaffold featured interconnected pore network, allowing viable cells to exhibit stretched actin fibers, whereas cells of CT were organized in clusters. The markers expression was increased in CH-Ca scaffold compared to CT (p<0.05). DPC/CH-Ca construct cultured in contact with 0.1 uM SV exhibited viable cells with strong stained stress fibers and intense markers expression (p<0.05). The DPC 3D-culture in contact with CH-Ca-SV scaffold adapted to dentin disc featured enhanced ALP, DSPP, DMP-1 and Col1 expression, with viable DSP-positive migrating cells being observed on scaffold periphery (p<0.05).
Conclusions: Highly porous calcium-containing chitosan scaffold allows DPCs adhesion, spread and proliferation throughout its structure, strongly inducing an odontoblastic phenotype. Incorporation of low-dosage simvastatin on this innovative biomaterial increases its bioactivity by inducing the chemotaxis and stimulating the regenerative potential of resident stem cells.