Biocompatible reduced graphene oxide (rGO) could deliver drugs for synergistically stimulating stem cells directed differentiation with influences on specific cellular activities. Here, we prepared a biodegradable gelatin reduced graphene oxide (rGO@Ge) to evaluate its functions in promoting rat adipose derived mesenchymal stem cells (ADSCs) chondrogenic differentiation through delivering kartogenin (KGN) into the stem cell efficiently. The optimum KGN concentration (approximately 1 μM) that promoted the proliferation and chondrogenic differentiation of ADSCs was clarified by a series of experiments, including immunofluorescent (IF) staining (Sox-9, Col II), alcian blue (Ab) staining, toluidine blue (Tb) staining and real-time quantitative PCR analysis of the chondrogenic markers. Meanwhile, the biocompatibility of rGO@Ge was evaluated to clearly define the nonhazardous concentration range, and the drug loading and releasing properties of rGO@Ge were tested with KGN for its further application in inducing ADSCs chondrogenic differentiation. Furthermore, the mechanism of rGO@Ge entering ADSCs was investigated by the different inhibitors that are involved in the endocytosis of the nanocarrier, and the degradation of the rGO@Ge in ADSCs was observed by transmission electron microscopy (TEM). The synergistic promoting effect of rGO@Ge nanocarrier on ADSCs chondrogenesis with KGN was also studied by the IF, Ab, Tb stainings and PCR analysis of the chondrogenic markers. Finally, the intracellular Reactive Oxygen Species (ROS) and autophagy induced by KGN/rGO@Ge complex composites were tested in details for clarification on the correlation between the autophagy and chondrogenesis in ADSCs induced by rGO@Ge. All the results show that rGO@Ge as a biocompatible nanocarrier can deliver KGN into ADSCs for exerting a pro-chondrogenic effect and assist the drug to promote ADSCs chondrogenesis synergistically through modification of the autophagy in vitro, which promised its further application in repairing cartilage defect in vivo.

Objectives. The objective of this study is to develop a load-bearing prevascularized construct by combining calcium phosphate cement (CPC) with cells within a three-dimensional (3D) hydrogel culture system, to accelerate the regeneration of alveolar bone defects. Methods. A 3D co-culture system was established by encapsulating human periodontal ligament stem cells (hPDLSCs) and human umbilical vein endothelial cells (hUVECs) within a gelatin methacryloyl (GelMA) hydrogel on 3D-printed porous CPC scaffolds. The mechanical properties, pore structure and angiogenic potency were determined in vitro. In vivo performance was evaluated using a nude rat subcutaneous implantation model and a rat alveolar bone defect model. Four groups were tested: (1) Blank group (surgery-only group); (2) CPC+GelMA group (non-prevascularized group)；(3) CPC+GelMA-cell group (prevascularized group)；(4) Natural Periodontiumgroup. Results. The novel construct had good mechanical properties and biocompatibility. The 3D co-culture in GelMA successfully induced microvascular formation in vitro. Subcutaneous implantation in nude rats showed that the CPC+GelMA-cell group exhibited markedly greater angiogenic capacity than the CPC+GelMA group after 6 weeks, with a neovascular density 1.93-fold higher than that of the non-prevascularized group. Among all groups, the CPC+GelMA-cell group exhibited the strongest capacity for repairing rat alveolar bone defects. Compared to CPC+GelMA group, CPC+GelMA-cell group significant enhanced bone regeneration in rats by 1.23-1.37 folds, and increased vascularization by 2.65 folds (p＜0.05). Conclusions: The novel three-dimensional prevascularized CPC construct combined appropriate mechanical properties with great efficacy for alveolar bone regeneration and vascularization in vivo in an animal model.