Primary Experimental Study On In Vivo Construction Of Tissue-engineered Osteochondral Composite Using A Combination Of Biomimetic Scaffold Col-CS/NHAC-PLA And Gene-transducted Bone Mesenchymal Stem Cells

[Objective]To discuss the possibility of TGF-β1and BMP-2gene-modified bone Mesenchymal Stem Cells (BMSCs) composite with Col-CS/nHAC-PLA biomimetic scaffold to repair the cartilage and subchondral bone complex injury, in order to provide experimental basis for the clinical application of tissue-engineered bone and cartilage.[Methods]Porcine BMSCs were isolated, cultured and purified by combining density gradient centrifugation with suitable culture, BMSCs were transfected by TGF-β1and BMP-2gene recombinant lentivirus, the transfect efficiency was observed by fluorescence microscopy;The transfected cells were composited with Col-CS/nHAC-PLA scaffold, The porosity and pore size of Col-CS/nHAC-PLA and the adhesion growth of cells with BMSCs scaffold were detected by the scanning electron microscope (SEM). The model of femoral condyle Osteochondral injury in pigs was established and repaired by TGF-β1and BMP-2gene-modified BMSCs composite with Col-CS/nHAC-PLA biomimetic scaffold. The cartilage part of the injury knee was took out at12and24weeks post-operation and was observed by HE staining and evaluated of the repair of cartilage and subchondral bone injury by AKP staining、ABPAS staining and type2collagen immunohistochemical staining.[Results]1、Porcine BMSCs are successfully isolated, cultured and purified by combining density gradient centrifugation with suitable culture.BMSCs were successfully transfected by recombinant lentivirus vectors of TGF-β1and BMP-2gene. Fluorescent protein was successfully observed by fluorescence microscopy and indirectly reflected the expression of TGF-01and BMP-2protein.2、The porosity and pore size of Col-CS/nHAC-PLA was detected by SEM and could provide adhesion growth of BMSCs in the bracket on the micro-environment, and the porosity was about80percent, the CS pore size was about30-100u m, HA pore size was between50-120μm.3、The model of femoral condyle Osteochondral injury in pigs was successfully established and repaired by the tissue-engineered bone and cartilage.12weeks, Compared with in the simple material group, gross and histological observation showed that new cartilage was more thick and surface was more smooth, the continuity of new cartilage and subchondral bone was better, the brown alkaline phosphatase particles in the cytoplasm of osteoblasts was more, the continuity of new cartilage matrix was better and the dyed brown type Ⅱ collagen was more obviously in the cell composite group.24weeks, Compared with in the simple material group, gross and histological observation showed that new cartilage was complete and more transparent, the continuity of new cartilage and subchondral bone was better and was regular arrangement and the dyed brown type Ⅱ collagen was more obviously in the cell composite group.4、The repair of the organization as a whole is evaluated by0’Driscoll, Keeley and Salter organization morphological score. Statistics showed that The score after the repair of osteochondral defects of experimental group was superior to that of the simple material group at week12and24(p<0.05).[Conclusions]The Combination of Col-CS/nHAC-PLA biomimetic scaffold and Gene-transducted bone Mesenchymal Stem Cells can effectively repair c a r ti l a ge a n d s u bc h o nd r a l b o n e c o mp l e x i n j ur y, pr o v id e s a n e w ap p r oa c hf or t he t r e at m en t o f b o ne a nd ca r ti la g e d am a g e.