The Construction Of Dual Gene-activated Tissue Engineering Osteochondral Complex

The destruction and lost of articular cartilage and subchondral bone caused by inflammation, trauma and tumor often leads to dysfunction of articular. Besides, no matter the destruction origin from the articular cartilage or the subchondral, the defect will deep into subchondral bone at last. Previous researches mainly focus on cartilage tissue engineering, and pay less attention to subchondral bone with important physiological function.The in situ gene delivery of gene-activited matrix is that the seeding cell capture or encounters the DNA condensed by biomaterials, different with the transfection of gene seeking target cell randomly; so it's a good strategy for localized delivery.Now we exploit the multilineage differentiation of Bone-marrow stem cells(MSC),construct osteochondral complex by in situ inducing MSC to differentiate to chondrocyte and osteoblast at different layer of a whole scaffold. We make the cartilage-like scaffold from chitosan and gelatin,which was activited by TGF-β1 plasmid;and we make the bone-like scaffold from chitosan,gelatin and hydroxyapatite,which was activited by BMP-2 plasmid.Then MSC was transfected in situ to differentiate to chondrocyte and osteoblast respectly.The two scaffolds were cultured separatly for the first week and combined into a osteochondral complex by fibrin glue.Chondrogenesis and osteogenesis potential of MSC in vitro was assessed using scanning electron micrograph, cell proliferation test, Q-PCR assay, ELISA assay, histological and immunohistochemical analysis.Furthermore, the complex was used at a rabbit full-thickness cartilage defect model for in vivo osteochondral reparation and the results were evaluated by histological and immunohistochemical staining.The SEM result shows that the scaffold was good porosity and the two layer was adhered to each other without fissure; mesenchymal stem cells seeded in each layer of the gene-activated osteochondral scaffold showed significant cell proliferation, high expression of TGF-β1 protein and BMP-2 protein respectively.The Q-PCR results showed that mRNA expressions of aggrecan and collagenⅡwere increased in pTGF-β1-activited layer and mRNA levels of OCN, OPN and collagenⅠwere also increased in pBMP-2-activited layer tested, indicating that bilayered scaffolds could make osteochondral composite by MSC simultaneously differentiating into osteoblast and chondrocyte induced by different cytokines, and this result was further confirmed by immunohistochemical staining.The in vivo results showed that the bilayer scffold promoted the articular cartilage and subchondral bone regeneration in the full-thickness rabbit knee ostochondral defect model.This study gives the evidence that bilayer gene-activited scaffold can induce MSC multi-lineage differentiation at different layer, and this complex can promote the repair of ostochondral defect.