Icariin had been reported as a potential agent for osteogenesis, but the dose-effect relationship needed further research to realize the clinical application of icariin. We isolated and purified human bone mesenchymal stem cells (hBMSCs) and stimulated them with different concentrations of icariin. The cytotoxicity of icariin was evaluated by the methylthiazolytetrazolium (MTT) assay method. The proliferation and osteogenic differentiation of such hBMSCs were investigated for different concentrations of icariin. We found that icariin had a dose-dependent effect on the proliferation and osteogenic differentiation of hBMSCs in a suitable concentration range from 10−9 M to 10−6 M, but at concentrations above 10−5 M, the cytotoxicity limited its use. The extremely low cost of icariin and its high abundance make it appealing for bone regeneration.

Purpose: Poly(lactic-co-glycolic acid) (PLGA) is excellent as a scaffolding matrix due to feasibility of processing and tunable biodegradability, yet the virgin scaffolds lack osteoconduction and osteoinduction. In this study, nano-hydroxyapatite (nHA) was coated on the interior surfaces of PLGA scaffolds in order to facilitate in vivo bone defect restoration using biomimetic ceramics while keeping the polyester skeleton of the scaffolds. Methods: PLGA porous scaffolds were prepared and surface modification was carried out by incubation in modified simulated body fluids. The nHA coated PLGA scaffolds were compared to the virgin PLGA scaffolds both in vitro and in vivo. Viability and proliferation rate of bone marrow stromal cells of rabbits were examined. The constructs of scaffolds and autogenous bone marrow stromal cells were implanted into the segmental bone defect in the rabbit model, and the bone regeneration effects were observed. Results: In contrast to the relative smooth pore surface of the virgin PLGA scaffold, a biomimetic hierarchical nanostructure was found on the surface of the interior pores of the nHA coated PLGA scaffolds by scanning electron microscopy. Both the viability and proliferation rate of the cells seeded in nHA coated PLGA scaffolds were higher than those in PLGA scaffolds. For bone defect repairing, the radius defects had, after 12 weeks implantation of nHA coated PLGA scaffolds, completely recuperated with significantly better bone formation than in the group of virgin PLGA scaffolds, as shown by X-ray, Micro-computerized tomography and histological examinations. Conclusion: nHA coating on the interior pore surfaces can significantly improve the bioactivity of PLGA porous scaffolds.

Icariin, a plant-derived flavonol glycoside, has been proved as an osteoinductive agent for bone regeneration. For this reason, we developed an icariin-loaded chitosan/nano-sized hydroxyapatite (IC-CS/HA) system which controls the release kinetics of icariin to enhance bone repairing. First, by Fourier transform infrared spectroscopy, we found that icariin was stable in the system developed without undergoing any chemical changes. On the other hand, X-ray diffraction, scanning electron microscopy and mechanical test revealed that the introduction of icariin did not remarkably change the phase, morphology, porosity and mechanical strength of the CS/HA composite. Then the hydrolytic degradation and drug release kinetics in vitro were investigated by incubation in phosphate buffered saline solution. The results indicated that the icariin was released in a temporally controlled manner and the release kinetics could be governed by degradation of both chitosan and hydroxyapatite matrix. Finally the in vitro bioactivity assay revealed that the loaded icariin was biologically active as evidenced by stimulation of bone marrow derived stroma cell alkaline phosphatase activity and formation of mineralized nodules. This successful IC-CS/HA system offers a new delivery method of osteoinductive agents and a useful scaffold design for bone regeneration.

In recent years, graphene (G) and graphene oxide (GO) nanoparticles have begun to be applied in surgical implant surface modification. However, biosafety and antibacterial ability of G and GO are still unclear. In this study, the biosafety of G and GO in vitro was evaluated by co-culture with bone marrow mesenchymal stem cells (BMSCs) and biosafety in vivo was observed by implanting materials into mice muscle tissue. Biosafety results showed that 10 μg/ml was the safety critical concentration for G and GO. When the concentration was more than 10 μg/ml, the cytotoxicity of G and GO showed a dose-dependent manner.Antibacterial results showed that G presented the antibacterial ability with the concentration equal to and more than 100 μg/ml; GO presented the antibacterial ability with the concentration equal to and more than 50 μg/ml. The antibacterial effect of G and GO were in a dose-dependent manner in vitro.The GO or G concentration between 50 and 100 μg/ml may be the better range to keep the balance of cytotoxicity and antibacterial ability. Our study reveals that G and GO have potential to be used in clinic with good biosafety and antibacterial properties in a certain concentration range.