| Bone loss caused by congenital defects, traumatic injury, cancer, reconstructivesurgery or periodontal disease is always the most difficult challenge both at home andabroad. Autologous bone graft is the so-called gold standard in transplantation surgery.But the use of autologous bone may be restricted because the amount of donor bone islimited and the complication in donor site. The strategies of allogenic or xenogenicare associated with problems such as the risk of disease transmission and graftrejection with a concomitant necessity for secondary surgery, and cost much.Synthetic grafts are important substitutes, but they fail to provide similar mechanicalproperties of human bone tissue, often causing failure in bone healing. Bone tissueengineering attempts to solve the referred problems mentioned above. Recently, gene-and stem cell-based therapy have been applied in bone tissue engineering andachieved some good clinical results.Bone marrow mesenchymal stem cells (BMSCs) have been widely used for bonetissue engineering. They potentially can be used to develop bone，cartilage，muscle oradipose. Bone morphogenetic proteins (BMPs) are well known as osteoinductivegrowth factors which play an important role in bone regeneration process. Theseproteins are capable of inducing the osteogenic differentiation of mesenchymal cells.So BMP-2gene has been frequently used to induce the osteogenic differentiation ofBMSCs.Gene vectors are required for the transfer of exogenous gene into cells, the mainapproach to gene delivery in gene therapy is viral vector systems due to theirrelatively high transfection efficiency. But viral-based gene vectors are a kind ofengineered viruses, so they have the drawbacks of high cost, immune response againstrepeated administration, oncogenicity and difficulty in large-scale production.Therefore, many scientists have shifted their interest to develop non-viral gene vectors.Different types of polyethyleneimine (PEI) are regarded as a kind of the most effective non-viral gene vectors. However, PEI has the disadvantage of no specifictissues and cells targeting, relative lower transgene efficiency compared with viralvectors and high cytotoxicity. In order to overcome the drawbacks, it is necessary tomodify PEI.Scaffold is also a main factor of bone tissue engineering, including naturepolymer, ceramics, synthesis polymer and composite material. However, the scaffoldsstill face the problem of causing inflammation, the degradation rate of biomaterial notmatch the regeneration rate of bone and the lack of biological activity. So the newertechnology of cell sheet without scaffold has emerged. The cells are collected as asheet, which maintains the connections between cells and ECM, and the relativeproteins and biological activity factors. It also can provide a suitablemicroenvironment for bone regeneration.Preparation and characterization of PEI-alginate/plasmid complexesPEI-al gene carriers were prepared by electrostatic interation between the cationicPEI and polyanionic alginate, and physicochemical properties were characterized. Theresults showed that the average particle size of PEI-al nanocomposites were73.8nm,and the zeta potential distribution was+19.9mV; SEM images showed that PEI-alnanocomposites were spherical and monodispersed particles while some aggregationoccured when PEI-al nanocomposites were blended with pBMP-2; DLS resultsshowed that the average particle size of PEI-al/pBMP-2complexes were increased to149.0nm, and the zeta potential distribution was decreased to+18.8mV; Agarose gelelectrophoresis result suggested the formation of neutral or positive complexesbetween PEI-al and pBMP-2, when the weight ratio reaches1.5.The effects of PEI-al/pBMP-2on bone formation in vitro and in vivoPEI-al/pBMP-2complexes were transfected into MC3T3-E1cell line in vitro,and the capability of inducing osteogenic differentiation was evaluated byrealtime-PCR and alizarin red stain. PEI-al/pBMP-2complexes and PEI-alnanocomposites were absorbed into gelatin sponges, respectively, and the gelatinsponges were implanted into the rat calvarial defects in vivo. The effect of boneregeneration was observed by Micro-CT and histologic analysis. The results showedthat the expression of osteogenesis-related gene was increased post-transfected withPEI-al/pBMP-2complexes, and the calcium nodules formation was much more inPEI-al/pBMP-2group than PEI-al/pEGFP group; Micro-CT anlysis indicated that in the PEI-al/pBMP-2group, there are irregular high density thing in the central of thedefect site and the edge of defect is irregular; And more new bone formation wasobserved in PEI-al/pBMP-2group than in the PEI-al group. HE staining showed thatthere are many bone cores in the PEI-al/pBMP-2group, while in the PEI-al groups alldefects were repaired with fibrous connective tissue.The effects of PEI-al/pBMP-2engineered BMSCs cell sheet on bone formation invitro and in vivoPEI-al nanocomposites carrying BMP-2gene could efficiently transfect BMSCs,and the BMP-2protein producing cell sheet was made by culturing the cells invitamin C containing medium for10days. To quantify the secretion of BMP-2protein,ELISA assay was performed; the capability of inducing osteogenic differentiation wasevaluated by realtime-PCR and ALP activity. EGFP producing cellsheet (EGFP/CS)and BMP-2producing cellsheet (BMP/CS) were implanted into the rat calvarialdefects in vivo, then the effect of bone regeneration was observed by Micro-CT andhistologic analysis. The results showed that the genetically engineered cells releasedthe BMP-2for at least14days; The expression of osteogenesis-related gene wasincreased, which demonstrated the released BMP-2could effectively induce the cellsheet osteogenic differentiation in vitro. Micro-CT and histologic analysis indicatedthat BMP/CS group was more efficient than other groups on promoting boneformation in the defect area.Our study demonstrated that PEI-al nanocomposites could efficiently transfectBMSCs and the BMP-2gene engineered cell sheet could effectively enhance boneformation. |
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