Construction And Analysis Of Bioactive Injectable Hybrid Gel For Bone Tissue Engineering

Objective: Bone defect is an important issue for clinical work today.In 2011,there were more than 1 million surgery involving the repair of bone defect in the United States.At present,the commonly method are autologous bone grafts and allografts,which are limited by limited sources,donor damage,immune rejection and other problems.Therefore,it has become an important direction of bone defects repair by constructing bone biological engineering materials.Hybrid gel has become an important candidate in bioengineering because of its good biocompatibility and safety.Injectable hydrogels are an ideal material for minimally invasive repair of bone defects due to their responsiveness to temperature,pH or pressure.Injectable hydrogels can reach the bone defect area through injection and reshaped under control to fill the defect area.In order to obtain biomaterials with biological activity and inductive osteogenesis potential,the ideal material for bone defect repair should be obtained by hydrogel composite seed cells and osteogenic factors.The human body has a relatively stable PH environment and temperature,so hydrogels with pH sensitivity andtemperature sensitivity have innate advantages.The combination of seed cells,induction factors and smart injectable hydrogel is expected to be an ideal bioengineered material.The effect of two kinds of smart injectable gels as bone tissue engneering materials were tested by in vitro and in vivo.The experiment is mainly divided into two parts:1.Composite graphene oxide and PPCN-g obtained temperature sensitive hydrogel GOP.First,increased elastic modulus and temperature responsiveness ability of GOP were observed by using rheological experiments.Fluorescence microscopy and scanning electron microscopy confirmed that cells can adhere well in GOP and generated collagen and mineralization sections under BMP9 induction.Gluc detection confirmed that iMADs cells could be stable in the long term and express target protein continuously.Alkaline phosphatase detection found that GOP can assist in the increase of alkaline phosphatase expression in iMADs.The expression of osteogenic factors in all stages was significantly increased by qPCR.The subcutaneous ectopic bone formation in nude mice further showed that the application of GOP gel could enhance the effect of BMP9 in inducing osteogenesis of iMADs cells in vivo.Immunohistochemical staining suggested that the enhancement of osteogenesis was related to vascular induction ability of GOP.2.CMC-ACP nanoparticles were formed through the organiccombination of amorphous calcium phosphate and chitosan.CMC-ACP has good stability.And CMC-ACP shown pH responsiveness to complete self-assembly.CMC-ACP hybrid gel is injectable and available for 3D printing within the PH range of the human body.After iMADs cells were loaded into CMC-ACP hybrid gel for culture,SEM and fluorescence microscopy showed that iMADs cells could adhere to the gel and significant mineralization were observed.Gluc assy suggested that cells could survive in the gel for up to 14 days and continuously express the target protein.CMC-ACP hybrid gel provides a favorable environment for the osteogenic differentiation of MSCs: alkaline phosphatase staining and calcium salt deposition staining suggest that CMC-ACP hybrid gel can effectively coordinate BMP9 to enhance the osteogenic differentiation of mesenchymal stem cells.qPCR assay showed that CMC-ACP hybrid gel induced osteogenic differentiation of iMADs and inhibited adipogenic differentiation.The results were also confirmed in experiments of ectopic subcutaneous osteogenesis in nude mice: although CMC-ACP hybrid gel was not sufficient to mediate ectopic osteogenesis solely,it shown synergistic effect of BMP9 mediated ectopic osteogenesis: MicroCT showed higher and more uniform bone formation in the BMP9-CMC-ACP groups compared with the BMP9-NA groups.The CMC-ACP hybrid gel also promte the vascularization.Histological examination showed that CMC-ACP hybrid gel,as a biological scaffold,effectively improvedBMP9's osteogenic capacity,obtained more orderly bone trabecula formation,more abundant blood vessel growth and less production of adipose tissue.According to the above studies,we believe the combination of injectable gel and BMP9 induced mesenchymal stem cells is an effective model to construct biologically active bone repair materials.The intelligent hydrogel-based bone repair materials provide a feasible direction for the repair of bone defects.