P8 - Polycaprolactone/Nano-Hydroxyapatite Nanofibrous Scaffold as a Cell Homing-Based Therapy

Abstract

This study investigated the behavior of human dental pulp cells (HDPCs) seeded on the surface of polycaprolactone/nano-hydroxyapatite nanofibrous scaffolds, aiming a dentin tissue engineering-based vital pulp therapy (VPT). Polycaprolactone (PCL)-based solutions (10% w/v in 8:2 chloroform and dimethylformamide) containing nano-hydroxyapatite (nHA) particles (0.5; 1.0; or 2.0% w/v) were electrospun into nanofibrous scaffolds. PCL scaffolds without nHA were used as control. The scaffolds were blindly characterized for morphology and composition (SEM/EDS; n=12), solubility (n=6), the release of calcium/phosphate (n=6), and change of medium pH (n=6). A culture of HDPCs was obtained from sound third molars and characterized for the presence of a subpopulation of mesenchymal stem cells. Then, HDPCs were seeded on the surface of the scaffolds and evaluated for cell viability (alamarBlue; n=8), cell proliferation (Live/Dead assay; n=4), adhesion and spreading (F-actin; n=4), total protein content (TP; Lowry method; n=8), alkaline phosphatase activity (ALP; thymolphthalein assay; n=8), expression of odontogenic genes (RT-qPCR; DSPP, DMP1, COL1A1, and ALPL; n=6), and formation of a mineralized matrix (Alizarin Red staining; n=8). All experiments were repeated at least twice. Sample sizes were calculated using G-Power. Data were analyzed with confidence intervals and one- or two-way ANOVA followed by Tukey, Sidak, or Games-Howell post-hocs (?=5%). All formulations generated randomly arranged fibers ranging from 600 to 900 nm in diameter. Higher nHA concentrations roughened the surface of the nanofibers, while PCL+2%nHA increased the interfibrillar spaces. PCL+1%nHA and PCL+2%nHA significantly released calcium/phosphate over a period of 28 days in comparison with PCL+0.5%nHA. Nonetheless, the medium pH was maintained below 8.0. HDPCs viability and proliferation were not affected by the incorporation of nHA, while cell adhesion/spreading was stimulated, especially for PCL+2%nHA. Higher total protein content and increased ALP activity were seen in the presence of nHA. PCL+1%nHA and PCL+2%nHA upregulated the expression of DSPP and DMP1 in 14 days; and COL1A1, ALPL, and DMP1 in 21 days. The formation of a mineralized matrix after 21 days was concentration-dependent, and it was about 9× higher for PCL+2%nHA formulation compared to control.