Research On Healing Full-thickness Cartilage Defect Of Rabbit Using Bone Marrow-derived Mesenchymal Stem Cells

Objective:Isolating and culturing high-grade mesenchymal stem cells from Rabbits bone marrow and then differentiate into chondroblast in vitro. To evaluate the effect of bone marrow mesenchymal stromal cells (BM-MSCs) with Sodium hyaluronate healing full-thickness cartilage defects of rabbit knee.Methods:1. Bone marrow of rabbits was obtained from proximal tibia by sterile sternum puncture needle and Reforming ensity gradient centrifugation cultivation was utilized to culture BM-MSCs.it cultured in the DMEM with low glucose containing15%fetal bovine serum. Analyze the cell surface markers by flow cytometry in passage3.2. BM-MSCs from the second passage were divided into two groups:the induction group were, adding the chondrogenic medium; the control group were adding the common medium. The growth of the cultured cells was observed. Alcian blue staining were used to detect the secretion and expression of glycosaminoglycan in extracellular matrix.3.Eighteen rabbits were divided in to three groups randomly.the defects of the experimental group implanted BM-of MSCs/sodium hyaluronate complex; the control group were implanted Sodium hyaluronate; the blank group without special treatment. The animals were killed in6th and12th week later. Restored tissue was evaluated using naked eyes and histological section,score was given according tomodified Pineda grade standard-The total score was analyzed with statistic software SPSS13·0.Results:1.These cell populations are comprised of morphologically homogenous fibroblast-like cells and present plastic-adherent property. Flow cytometry analysis shows that these cells are homogenously positive for the mesenchymal markers CD29and CD44, but negative for the pan-leukocyte marker CD45.2. The induction group cells aggregation growth as flake-shaped or polygonal, which was positive for Alcian blue staining; control cells still showed the typical long fusiform adherent growth, Alcian blue staining was negative.3. Cartilage defect of experimental group were repaired with a thin-layer hyaline-like cartilage, which was positive for type Ⅱ collagen and Alcian blue staining, control group and blank group were repaired with fibrous tissues,which was weakly positive for type Ⅱ collagen and Alcian blue staining. Experimental group had statistical difference compared with control and blank group (P<0·05) in each period which showed that Experimental group got a better indication than other two groups.Conclusion:1.Reforming ensity gradient centrifugation cultivation method is an effective and convenient procedure to isolate and culture high-purity MSCs from Rabbits bone marrow.2. BM-MSCs can be induced to chondrogenic differentiation by TGF-of β3, insulin, sodium pyruvate, vitamin C and dexamethasone in vitro.3. Bone marrow mesenchymal stem cells/Sodium hyaluronate complexes is contribute to the repair of cartilage defects.