Evaluation of long-term biocompatibility and osteogenic differentiation of graphene nanosheet doped calcium phosphate-chitosan AZ91D composites

Abstract To improve the biodegradable performance and osteoinductivity of magnesium (Mg) alloy, a novel graphene nanosheet (GNS) doped micro-arc oxidized-AZ91D (MAO-AZ91D) based calcium phosphate (CaP)-chitosan (CS) (GNS-CaP-CS/AZ91D) composite was fabricated. Their long-term in vitro biocompatibility, osteoinductivity and the related signaling pathway were evaluated. The electrochemical test, Mg concentration and SEM results showed that the corrosion rate of GNS-CaP-CS/AZ91D became much slower, whose corrosion degree with immersion time being 90 d was similar to that of AZ91D being 16 d. Good biocompatibility and non-cytotoxicity were observed during the whole immersion process of GNS-CaP-CS/AZ91D. Extracts from GNS-CaP-CS/AZ91D especially in the early stage (16 days and before) could significantly enhance alkaline phosphatase (ALP) activity, calcium mineral deposition and osteoblast-related genes expression of human bone marrow mesenchymal stem cells (hBMSCs). GNS released from GNS-CaP-CS/AZ91D composites might serve a critical role to induce osteogenic differentiation through ERK/MAPK pathway in the long-term immersion process, while, PI3K/Akt signaling only significantly in the early stage. Moreover, Mg element could promote osteogenic differentiation through ERK/MAPK pathway and the maximum concentration was about the range between 200 and 500 ppm.