P1 - Calcium Silicate Loaded-Porous Chitosan Scaffold for Pulp-Dentin Complex Regeneration

Abstract

Innovative pulp therapy strategies based on the use of biomaterials capable of stimulating dental pulp cells to participate effectively of the complete pulp-dentin complex regeneration is a challenge in Dentistry. Therefore, the aim of this study was to develop and characterize several formulations of calcium silicate (SiCa)-loaded porous chitosan (CH) scaffolds, and evaluate their bioactive and chemotactic potential on human dental pulp cells (hDPCs). Different concentrations of SiCa suspensions (0.5, 1.0 and 2.0%, w/v) were either incorporated (1:5; v/v) or not to 2% CH solution, establishing the following groups: CH (control); CH+0.5SiCa; CH+1.0SiCa; CH+2.0SiCa. The resulting solutions were submitted to thermally induced phase separation followed by freeze drying to obtain porous scaffolds. The topography and chemical characterization (SEM/EDS) of the scaffolds, as well as their pH and calcium release kinetics were assessed. Next, hDPCs were seeded onto the scaffolds to assess their bioactivity potential according with the parameters of cell viability (Live/Dead assay; 1, 7 and 14-days), proliferation (AlamarBlue assay; 1, 7 and 14-days), adhesion and spreading (F-actin assay; 1, 7 and 14-days). The pulp cells were also evaluated for total protein synthesis (Lowry Protein assay; 14-days), alkaline phosphatase activity (ALP Assay Kit; 14-days), mineralized matrix deposition (Alizarin Red assay; 21-days), and gene expression of odontogenic differentiation markers (ALP, DSPP, and DMP-1; RT-qPCR assay, 21-days). The chemotactic stimulus of the scaffolds on pulp cells (Transwell assay; 24 h) was also assessed. Data were analyzed by ANOVA and Tukey post-hoc (?=5%). The incorporation of SiCa in the chitosan scaffold formulations increased the pore diameter in comparison to control (p<0.05). High amounts of SiCa changed the surface nano-topography of the chitosan scaffolds (Image 1) and increased the pH and Ca release (p<0.05). Greater cell viability, proliferation, adhesion and spreading, as well as expression of odontogenic markers and mineralized matrix deposition was observed in the CH+1.0SiCa and CH+2.0SiCa groups (p<0.05). However, increased synthesis of total protein and mineralized matrix, as well as expression of DMP-1 and DSPP occurred only in group CH+2.0SiCa (p<0.05). Based on the fact that porous chitosan scaffolds enriched with 2% of calcium silicate showed improved bioactive and chemotactic potential on hDPCs, it should be considered as a potential biomaterial for cell-free tissue engineering strategy to drive pulp-dentin complex regeneration.