Study On Bone Regeneration In A Critical Sized Defect Using HBC@TACS-pBMP4 Particles And HBC@TACS-pFGF2 Particles

Object Chronic infection of severe car accident trauma and tumor is a difficult problem in the repair of bone defect.Autologous bone graft,allograft,synthetic materials and other repair methods have many disadvantages,such as large trauma in the donor bone area,new defects in the donor area,limited donor bone,immune rej ection,spread of infectious diseases and high cost.Therefore,researchers have turned their attention to tissue engineering technology,exploring the combination of tissue engineering technology and gene therapy technology,and using safe and effective gene carriers to effectively express the target gene in vivo and in vitro to repair bone defects A large number of studies have shown that chitosan(CS)because of its good biocompatibility and low cytotoxicity,can be used as a carrier and formed after chemical graft modification of chitosan modification of chitosan,such as hydroxyl butyl(HBC)and thiol alkylated chitosan(TACS)after processing nano structures can be used as a slow-release carrier for gene therapy.Bone morphogenetic protein 4(BMP4)can induce osteogenic differentiation of bone marrow mesenchymal stem cells and promote the formation of new bone,while basic fibroblast growth factor(FGF2)can promote the formation of new blood vessels and play an important role in the repair of bone defects Therefore,this study intends to investigate the biological effects of modified chitosan coated with BMP4 gene and FGF2 gene respectively in vitro and its application in the repair of critical radial bone defect in rabbitsMethods Mercaptoalkyl chitosan(TACS)respectively with pFGF2 gene plasmid pBMP4 plasmid in accordance with the proportion of mixture through positive and negative electric role in gene surface carrier adsorption nuclear structure(TACS-pFGF2 and TACS-pBMP4),the quality of HBC with TACS mixed with nuclear structure,form the gene as the core of the double layer structure of surgery,and HBC@TACS pFGF2 and HBC@TACS pBMP4 slow-release microspheres.The sustained-release microspheres were co-cultured with bone marrow mesenchymal stem cells in vitro.CCK8 detects cytotoxicity of different doses of gene vectors.PCR was used to detect the mRNA expression of related osteogenic genes after co-culture of bone marrow mesenchymal stem cells with different gene vectors.Bilateral radial surgery in rabbits resulted in a radius defect of about 18mm in length,which was randomly divided into 6 groups.Will contain different doses pBMP4 gene plasmid and pFGF2 plasmid gene carrier solution to join gelatin sponge(18 mm*20 m)after lyophilization in volume growth of about 18 mm cylindrical filling to bone defect site(experimental group A:100 ug pBMP4,group B:100 ug pBMP4+10 ug pFGF2,group C:100 ug pBMP4+20 ug pFGF2 group D:100 ug pBMP4+40 ug pFGF2,control group E gelatin sponge column,blank control group F:At week 8,week 12 and week 16,X-ray examination was performed to observe the repair of bone defect,and then gross specimen was taken to observe the repair of bone defect,Masson staining and biomechanics test to evaluate the biological characteristics of the new boneResults Western blot showed that BMSCS transfected with HBC@tacs-pfgf2 and HBC@tacs-pfgf2 and HBC@tacs-pbmp4 could successfully express FGF2 protein and BMP4 protein,and the protein expression level was positively correlated with time Cytotoxicity assay showed that the proliferation rate of pBMP4 plasmid was(149.26±0.76)%.The proliferation rate of pFGF2 was(133.9±2.38)when the final concentration of pFGF2 plasmid was 100ug/ml.Bone marrow mesenchymal stem cells(BMSCS)co-cultured with pBMP4 plasmid whose final concentration was 100ug/ml with 40ug/ml tacs@tacs-pbmp4 and FGF2 plasmids showed higher expression of osteocalcin(OCG)mRNA in osteoblasts than other groups,and the difference was statistically significant.X-ray examination and gross specimen observation in the animal experimental group showed that good repair could be achieved at the radius bone defect site at 16 weeks after the final concentration of pBMP4 plasmid tacs@tacs-pbmp4 and FGF2 plasmids with the final concentration of pBMP4 plasmids with the final concentration of pBMP4 plasmids with the final concentration of pBMP4 plasmids with the final concentration of 40ug/ml tacs@hfc-pfgf2 during the bone defect repair process,and the bone marrow cavity recanalization.Biomechanical experiments showed that the maximum lateral stress of the repaired radius in group A at week 16 was 158.3 1n,reaching 93.73 of the lateral stress of the normal radius.There was no statistical difference.Masson staining:at all time points,the degree of mineralization of new bone in group A was higher than that in other groupsConclusion HBC and TACS can encapsulate pBMP4 plasmid and pFGF2 plasmid to form gene vector microspheres and successfully transfect cells to express BMP4 protein and FGF2 protein.Adding appropriate microspheres to the environment in vitro can promote the proliferation of BMSCs and express genes related to success.Adding appropriate dose of microspheres in vivo can promote the repair of critical radial bone defect in rabbits.