Three-dimensional culture of rat BMMSCs in a porous chitosan-gelatin scaffold: A promising association for bone tissue engineering in oral reconstruction.

Abstract Objective This study investigated the in vitro effects of a chitosan-gelatin scaffold on growth and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMMSCs) in three-dimensional (3D) cultures and evaluated the biomaterial biocompatibility and degradability after its grafting into tooth sockets of rats. Design A porous chitosan-gelatin scaffold cross-linked by glutaraldehyde was synthesised and characterised by light (LM), scanning electronic microscopy (SEM), energy dispersion spectroscopy (EDS) and X-ray diffraction (XRD). Rat BMMSCs were isolated, expanded and seeded onto scaffold using Dulbecco's Modified Eagle's Medium (DMEM) with or without an osteogenic supplement. Cell viability by MTT assay, alkaline phosphatase (ALP) activity and morphological LM and SEM analysis were performed after 1, 3, 8 and 14 days in culture. Free-cell scaffolds were implanted into tooth sockets of Lewis rats after upper first molars extraction. Fifteen male recipient rats were sacrificed after 5, 21 and 35 days for histological analysis. Results Scaffold characterisation revealed the porous structure, organic and amorphous content. This biomaterial promoted the adhesion, spreading and in vitro viability of the BMMSCs. Osteogenic-supplemented media did not improve the cellular response compared to DMEM. The biomaterial presented high biocompatibility and slow biodegradation in vivo. Remains of biomaterial were still observed at 21 and 35 days after implantation. However, on the 21st day, alveolar bone and epithelial healing were completely established. Conclusions These results indicate that chitosan-gelatin support the adhesion and osteogenic differentiation of rat BMMSCs and offer adequate physico-chemical and biological properties for use as scaffolds in bone tissue engineering-related strategies.