Repair Of Cartilage Defect In The Rabbit With Mesenchymal Stem Cells Introduced To Chondrocytic Differentiation

Objective Bone mesenchymal stem cells(BMSCs) were isolated,purificated, introduced, and transplated into cartilage defect with technology of tissue engineering, by which the machnism and feasibility of BMSCs were investigated initially in cartilage resurfacing.Methods Bone marrow was aspirated from two-month-old New Zealandwhite rabbit. Then BMSCs were isolated and purificated from bone marrow by density gradient centrifuge and adherence culture. When cells grew to 80% confluence, they were harvested and designated as passage 1. These cells were further expanded with 1 : 3 splitting. BMSCs at passage 3 were selected to obtain their proliferation curve and adherence rate. Exposed to transforming growth factor-pi (TGF-pl), vitamin C, and dexamethasone in monolayer and in a three-dimensional matrix, chondrocytic differentiation of mesenchymal stem cells was existed. By the immunohistochemical staining of type Ⅱ collagen and the toluidine blue metachromasia, the effect of differentiation was demonstrated. At the same time, the effects of TGF-pl and IGF-1 in the process of separate or unite introducement could be shown. BMSCs, introduced in medium containing 5ng/ml TGF-β 1 for 4 days, were suspended in 1.2% w/v alginate solution in a density of 2.107 cells per milliliter and dropped into a solution of CaCl2 . The alginate/cell suspension gelled immediately and formed spherical beads. They were divided into two groups in random, one exposed to TGF-pl in a three-dimensional matrix, the other to IGF-1 and TGF-pl. By the same way, alginate gel whithout or with BMSCs can be made. Based on the difference of composition, alginate beads were divided into 4 groups. After that, the posterior weight -bearing aspect of the medial femoral condyle was exposed and a 3.5mm 8.0mm full -thickness defect was created with a low-speed burr to a depth of 3.0mm. Then the defects were filled with different alginate beads. At 2, 4, 8, 12, and 20 weeks postoperatively, the rabbits were killed and the distal femora were removed. After decalcified, the specimens were embedded in paraffin2and Sum sections were cut . Healing of the defect was investigated histologically using Safranin'O -Fast green staining and toluidine blue staining.Results From this experience, such results can be obtained: First, rabbitBMSCs may be isolated and proliferation by density gradient centrifuge and adherence culture. Exposed to TGF-pl, vitamin C, and dexamethasone, BMSCs can express markers of chondrogenesis. Second, it is the combination of TGF-pl and IGF-l that facilitates the proliferation of BMSCs and enhances the synthesis of extracellular cartilage matrix. Third, BMSCs in alginate gel grow well and have a low-rate proliferation. If in inductive culture medium, they can differentiate to type of chondrocyte. Fourth, after transplanted into cartilage defect, alginate/cell gel can transform to tissue like hyaline cartilage.Conclusion After introduced, BMSCs can express markers of chondrogenesis and facilitate the repair of cartilage defect in rabbit.