Bone Regeneration In A Critical Sized Defect Using P BMP4-loaded TACS@EG-HBC Particles

Objective Bone defects are often caused by trauma,infection,tumors.The traditional treatment methods,such as autologous bone graft,allograft bone transplantation or nonbone materiasl,have many complications.Therefore,the researchers combined the bone tissue engineering and gene therapy technology to explore the carrier for the safe and effective delivery and expression of the target gene in vitro.Recent studies show that chitosan(CS)can also be used as a gene vector.It has great biosafety and biocompatibility,which can form stable nanoparticles and degradate slowly in vivo so as to keep gene release sustainable.Bone morphogenetic protein(BMP)plays an important role in bone repair and bone regeneration.BMP4 protein is one of the most important proteins in the BMP family,which can induce the differentiation of bone cells and promote the formation of new bone.The aim of this study was to investigate the transfection efficiency of pBMP4-loaded chitosan nanoparticles in vitro and bone regeneration in a critical sized defect in vivo.Methods Alkylation of sulfhydryl chitosan(TACS)and polyethylene glycol modified hydroxyl chitosan(EG-HBC)were taken to interact with BMP4 plasmid to form TACS@EG-HBC/pBMP4.HEK293 T cells is transfected with TACS@EGHBC/pBMP4.Western blot was used to test its sustainable gene release and immunofluorescence to test transfection efficiency.Prepared 18 mm complete bone defect model at the bilateral radius,the experimental groups were implanted with TACS@EG-HBC/pBMP4/G and TACS/pBMP4/G,While control groups implanted only gelatin sponge.The radius bone defect were observed by bone mineral density and bone mineral content,anatomy,x ray,HE staining,immunohistochemistry and biomechanical testing at 2,4,8,12 weeks.Results Western blot showed the highest protein expressing level in TACS@EGHBC/pBMP4 group,the transfection rate of core-shell structure was gradually increased.The results showed that the transfection efficiency of TACS@EGHBC/pBMP4 was higher than that of TACS/pBMP4 group.At 12 weeks after operation,new bone formation were significantly increased by the TACS@EG-HBC/pBMP4/G treatment.X-ray showed in the above group the reunion of bone marrow cavity at 12 weeks.It demonstrated significant increase in BMC and BMD values compared to the other two at 2,4,8 and 12 weeks.It also showed the bending stiffness of regenerated new bones at 12 weeks have no statistical differences(p < 0.05)compared with the normal radius.Conclusion TACS@EG-HBC/pBMP4 owns good ability of sustained release and repairing bone defect.