Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect.

Background: HA modified by bisphosphonate (BP) (HA-BP) was synthesized by chemical reaction and possessed promising properties such as self-healing, injection ability, and strong adhesion. The main aim of this study was to confirm its role in promoting osteogenic differentiation in vitro and bone regeneration in vivo.

Methods: The cell biocompatibility of this material was determined using the CCK-8 assay. Alkaline phosphatase (ALP), osteocalcin (OT), vascular endothelial growth factor (VEGF), and collagen I were assessed by quantitative real-time polymerase chain reaction (Q-PCR) in the treated group. The number and density of calcium nodules and ALP were evaluated by Alizarin Red staining and ALP staining. We have successfully developed an animal model simulating osteonecrosis of the femoral head (ONFH). Utilizing this animal model, the impact of HA-BP/CaP on bone formation was assessed. The amount of bone regeneration at 1 and 2 months after HA-BP/CaP injection was estimated by micro-computed tomography (micro-CT) analysis and H&E, collagen I, and periostin staining.

Results: The number of cells gradually increased in the experimental group over time and was close to that of the blank control group. ALP, collagen I, and VEGF expression was significantly higher in the experimental group than in the blank group (VEGF, ALP, both **p < 0.01; collagen I, ***p<0.001). In addition, the number and density of calcium nodules and ALP was clearly greater in the material group than in the control group. The quantification analysis showed that the mineral contents of regenerated bone at 1 and 2 months after HA-BP/CaP injection were significantly greater than those in the control group, according to micro-CT evaluation (**p<0.01). The amount of organic components in the HA-BP/CaP group was greater than that in the control group after decalcification and H&E staining. In addition, collagen I and periostin staining further confirmed the results of H&E staining.

Conclusion: This material can boost proliferation and osteogenic differentiation of MC3T3-E1 cells in vitro. It can intensely accelerate bone regeneration in vivo, which is a promising strategy for tissue engineering.