Nano-hydroxy apatite/chitosan/gelatin scaffolds enriched by a combination of platelet-rich plasma and fibrin glue enhance proliferation and differentiation of seeded human dental pulp stem cells

Abstract

The purpose of this study was to investigate the effects of Fibrin Glue (FG) and activated platelet-rich plasma (a-PRP) on the proliferation and osteogenic differentiation of human dental pulp stem cells (h-DPSCs). Therefore, we planned to investigate in vitro behavior of porous composite scaffolds based on chitosan-gelatin/nanohydroxyapatite (CS-G/nHA) treated with FG and a-PRP. The porous structure of CS-G/nHA was prepared using combination of particle leaching and freeze-drying methods. The a-PRP was prepared from the centrifugation of whole blood activated with calcium chloride. Four groups of composite scaffolds were fabricated to seed h-DPSCs: (1) a-PRP-FG/CS-G/nHA; (2) FG/CS-G/nHA; (3) a-PRP/CS-G/nHA; (4) CS-G/nHA. The 14 days SEM image reveled organized fibrin network on scaffolds surface. All groups treated with FG and a-PRP, showed improved adhesion of seeded h-DPSCs compared to CS-G/nHA. Cytotoxicity of the composite scaffolds was assessed by MTT. Alizarin red staining confirmed the formation of bone minerals by h-DPSCs after 21 days of cell seeding. In addition, the a-PRP-FG treated scaffolds exhibited significantly elevated bone gamma-carboxyglutamic acid-containing protein (BGLAP), bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (RUNX2) gene expression. The present result the composite scaffolds treated with FG and a-PRP showed a fibrin network, preferentially on the surface of composite scaffold increasing the mineralization and osteoblastic differentiation of harvested cells. In addition, a-PRP-FG/ CS-G/nHA scaffold increased bone marker gene expressions from day 7 to day 21.

Keywords: Bone tissue engineering; Differentiation; Fibrin glue; Nano-hydroxy apatite; Platelet-rich plasma; Proliferation.