A Novel Gene-activated Matrix Composed Of PEI/plasmid-BMP2 Complex And Hydroxyapatite/Chitosan-Microspheres Promotes Bone Regeneration

Objective: The incorporation of pro-osteogenic growth factors into bone graft materials to enhance bone regeneration is a key research area within the field of bone tissue engineering and regenerative medicine.However,direct incorporation of growth factors in protein form is easily degraded,and has limited active half-life,which cannot exert long-term and stable osteoinductive and oteoconductive effects.The combination of gene therapy and tissue engineering through gene-activated matrix(GAM)may provide a good alternative solution to overcome such limitations.Scaffold materials can be combined together with plasmid DNA and chemical-based transfection agent to form GAM,through which transfected cells could secrete growth factors in a sustained manner over a longer time duration;thereby enabling bone graft materials to act as a repository of therapeutic genes,while providing structural support and a scaffold matrix for new bone tissue ingrowth.In this study,we prepared hydroxyapatite/chitosanmicrospheres(HA/CS-MS)with microfabrication technology and emulsification method,and loaded the Polyethylene imine/bone morphogenetic protein 2 plasmid(PEI/p BMP2)complex with high transfection capacity,thus forming a novel GAM system with superior bone regeneration capacity-PEI/p BMP2-HA/CS-MS.In the previous in vitro study,we selected MC3T3-E1 cells to optimize a series of conditions,and finally implanted GAM into animals to verify its osteogenic effect mediated by BMP2 overexpression.Methods: In vitro,hydroxyapatite/chitosan-microspheres with ECM-like nanofiber structure were prepared by physical hydrogel method.Followed by scanning electron microscopy(SEM),X-ray diffraction(XRD),Fourier transform infrared spectroscopy(FITR),the physical and chemical properties of the microsphere materials were tested.The complexes were formed with different doses of PEI and plasmid DNA,and optimized to the optimal ratio,which have strong transfection ability and less cytotoxicity.Next,the PEI/p DNA complex was loaded on the microsphere material,and the biocompatibility of the complex material was detected using CCK-8 reagent and live-dead dye reagent.Using inverted fluorescence microscopy,reporter genes and qPCR,the transfection of cells adhered to the composite was tested,and the ability of the composite to release PEI/p BMP2 was further tested.q-PCR was used to detect the expression of related osteogenic genes(RUNX2,SP7,OCN and COLI)in cells induced by the composite material.The effects of the composites on osteogenic differentiation were detected using alkaline phosphatase(ALP)and alizarin red(ARS)staining kits.In vivo,this study used 9 male SD rats(8 weeks old,weight 200-220g).Surgery was performed under anesthesia(60 mg/kg ketamine and 8 mg/kg xylazine).A hollow ring drill with a diameter of 5 mm was used to create a model of the skull defect.The defect area was randomly divided into the following study groups:(1)Vacant defect;(2)MS;(3)p BMP2-MS.Each rat was implanted with 0.5 mg of material,and the incision was sutured in layers with sterile silk thread.Each group consists of 3 rats.The animals were sacrificed 8 weeks after the operation,and the changes of bone mass in the implanted area were analyzed by micro CT and tissue sections.Results: In vitro: Firstly,hydroxyapatite/chitosan-microspheres(MS)with an ECM-like nanofiber structure were successfully prepared.PEI/p DNA complexes were prepared in the range of N/P ratios,and their optimal transfection parameters(N/P=10,2 ?g dose)and transfection efficiency(54.79±4.95%)were determined in monolayer cell culture.Using SEM to characterize MS,it can be seen that the entire microsphere is composed of a uniform nanofiber network,which has great similarity with the nanofiber structure of bone natural ECM.From the results of FTIR spectra and XRD images,it can be seen that the prepared microspheres contain two species of n HA and CS.The results of CCK-8 and live/dead staining of the cells demonstrated the excellent biocompatibility of the p BMP2-MS material.In vitro transfection assays showed that cells adhered to the material could be efficiently transfected,and the PEI/p BMP2 complex released from p BMP2-MS could also efficiently target cells to secrete BMP2 protein,which can increase calcium deposition and Alkaline phosphatase,while increasing the gene expression of RUNX2,SP7,OCN and COLI,finally induced the differentiation of MC3T3-E1 cells.In vitro: In vivo data from microcomputed tomography(micro-CT)and histological staining demonstrated that the use of composite activated scaffolds effectively promoted bone formation in bone defects compared to control animals.These findings suggest that the non-viral gene activation matrix p BMP2-MS is effective for bone regeneration and is an attractive gene delivery system with significant potential for clinical application.Conclusion: The new GAM can slowly release the plasmid to the surrounding area,effectively transfecting local target cells,and the cells adhering to the material can also endocytose the plasmid,thereby secreting the target protein and jointly promoting the osteogenesis effect.