Study Of Construction Of Tissue Engineering Intervertebral Disc Based On Bone Scaffold Compounding With SD Rat’s BMSCs Transfected With Sox-9

Objective: To explore the feasibility of construction of tissue engineered intervertebral disc based on the rat bone marrow mesenchymal stem cells(BMSCs) transfected with Sox-9 gene and the homologous scaffold. The cell-scaffold hybrids impanted subcutaneously into Balb/c nude mice. Evaluation of the hybrids were carrid out with in physicochemical propert and biocompatibility, providing the evidence of it become the intervertebral disc tissue engineering intervertebral disc.Methods:(1) The rat bone marrow mesenchymal stem cells transfected with the Sox-9 gene were resuscitate, culture and observed for cell morphology under inverted microscope. Putting the homologous scaffold which had already disinfected into the 24 pot. The gene transfected cells were seeded onto the scaffold. The cell-scaffold hybrids cultured in vitro for 48 hours were evaluated through inverted microscope, scanning electron microscopy and histological staining.(2) In aseptic operation platform of the SPF Laboratory, the cell-scaffold hybrids which had cultured in vitro for 48 hours were implanted subcutaneously into nude mice. After 2 months culture in vivo, evaluation of the hybrids were carried out through scanning electron microscopy, histological staining and type I and II collagen immunohistochemical staining.Detecting the gene expression of type I, II collagen and aggrecan in disc cells of native disc and cell-scaffold hybrids by q RT-PCR technique.Results:(1)Gene transfected BMSCs in vitro were long spindle shaped.Scanning electron microscopy displayed good cell adhesion on the scaffold.(2)After 2 months in vivo culture, hybrids were positive in type I collagen immunohistochemistry but negative in type II collagen immunohistochemistry staining. I, II collagen and aggrecan of cells in hybrids were higher than that in native disc.Conclusion:(1) After removal cells, blending, freeze drying method and operation steps of processing get off cell decalcified bone tissue engineering porous composite scaffold of acellular completely, retained the removal of cells from the bone matrix main component, have the appropriate aperture and porosity.(2) Sox-9 gene transfected BMSCs co-culture with scaffold stay in a proper status. After implanting subcutaneously into nude mice, it shows well cell compatibility and the cartilage performance, as it can be one of the test methods of building tissue engineering intervertebral disc.