Development of CaCO3 microsphere-based composite hydrogel for dual delivery of growth factor and Ca to enhance bone regeneration

Abstract

Injectable scaffolds have attracted much attention because of their minimum surgical invasiveness. However, limited osteogenic induction property and low mechanical properties hampered their application in bone tissue engineering. CaCO₃ microspheres, which possess osteoinductivity, rough surfaces and specific binding sites for BMP-2, were first fabricated; after BMP-2 uploading, microspheres were further entrapped in fibrin-glue hydrogel. CaCO₃ microspheres were co-functionalized with casein and heparin. To obtain a high encapsulation of heparin and thus BMP-2 uploading, along with controlled release and simultaneous maintenance of the presence of vaterite which had osteogenic induction property, fabrication parameters were optimized and microspheres were characterized using XRD, FITR and SEM. The formed CaCO₃ had a microsphere morphology of ∼1 μm. Both vaterite and calcite phases were present and the relative amount of calcite phase increased with the amount of heparin. Sample 25 mM_4-1Hep with the highest loading amount of heparin was selected as carrier for BMP-2 and BMP-2 loaded CaCO₃ microspheres were further entrapped in fibrin-glue hydrogel (FC-B). For the as-prepared composite hydrogel, mechanical properties were characterized and the presence of CaCO₃ significantly elevated the tensile strength; controlled release of BMP-2 was sustained until day 21. Based on ALP activity, alizarin red staining and RT-PCR, in vitro osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was found to be significantly enhanced under induction of FC-B. Rabbit tibia bone defect model was applied to evaluate its in vivo performance. After implantation for 4 weeks, presence of composite hydrogel was observed in defects. After 8 weeks, bone defects of FC-B group were nearly completely healed. Using the fact that autologous scaffolds can be derived based on fibrin-glue hydrogel, the well-designed BMP-2 loaded fibrin-glue composite hydrogel demonstrated good potential in bone tissue engineering.