Experimental Study On Articular Cartilage Defects Repair Of Diannan Small-ear Pigs With Bone Marrow Stromal Cells Transfected By Adenovirus Vector Mediated TGF-β3 And BMP-2 Seeded On Demineralized Bone Matrix

[Objective] To observe the possibility of using Demineralized bone matrix as a scaffold for cartilage tissue engineering, determine the optimal co-culture time for implantation, and the feasibility of articular cartilage repair of Diannan small-ear pigs with bone marrow stromal cells transfected by adenovirus vector mediated TGF-β3 and BMP-2 seeded on demineralized bone matrix.[Methods] 1.Demineralized bone matrix was made according to Urist's method. The Ultra structure of DBM was observed with scanning electron microscope (SEM). Bone marrow stromal cells transfected TGF-β3 and BMP-2 were cultured with DBM, and MTT assay was used to analyze the growth curve of Bone marrow stromal cells on DBM. The growth of Bone marrow stromal cells on DBM was also observed with SEM.2. Prepared full thickness defects both knee joints of Diannan small-ear pig. The DBM/BMSCs transfected TGF-β3 and BMP-2 was co-cultured and transplanted into full thickness defects on femoral condyle of both knee joints. Eight adult Diannan small-ear pigs were used and divided into four groups.Group A is the weight-bearing articular surface of lateral femoral condyle of the right knee (DBM/BMSCs transfected by two genes). Group B is the weight-bearing articular surface of medial femoral condyle of the right knee (DBM/BMSCs). Group C is the weight-bearing articular surface of medial femoral condyle of the left knee (only DBM).Group D is the weight-bearing articular surface of lateral femoral condyle of the left knee(empty defects). Two Diannan small-ear pigs were killed at 2th,4th,8th and 12th week after the operation, and the reparative tissue samples were evaluated grossly, histologically, immunohistochemically and evaluate the repairing effect by O'driscoll's score system. SPSS 11.5 software was applied to proceed the statistic analysis.[Results] 1.The DBM scaffold was white three-dimensional cancellous feature which had great biocompatibility, could promote cell adhesion and proliferation. So it should be a good scaffold for tissue-engineered cartilage. SEM showed the holes were connected each other, BMSCs transfected by two genes co-cultured with the DBM ampilified,which had the peak until the sixth day.2. Defective regions of group A were repaired by hyaline cartilage at 4 weeks after transplantation and were barely recognizable with natural articular cartilage at 12 weeks.TypeⅡcollagenase immunohistochemistry stain expression was positive after 12 weeks.The positive reaction was showed in TGF-β3,BMP-2 immunohistochemistry stain after 8,12 weeks.[Conclusion] 1. The DBM scaffold had a three-dimensional structure of tissue engineering scaffold. DBM could promote the proliferation of target cells. So it should be a good scaffold for tissue-engineered cartilage.2. DBM/BMSCs transfected by two genes could be an efficient alternative for articular cartilage defects repair.