Effect Of Rolling Compression Bioreactor And Different State Of Chondrocytes On Chondrogenesis Of Bone Mesenchymal Stem Cells

Cartilage is a tissue with characterized semitransparent and elasticity which relies on a crucial balance of structure and function. When this balance is disturbed, whether from disease or injury, the body is unable to adequately repair the damage. The self repairing capability of injuried cartilage is limited owing to avascular,no innervation and no lymphatic return. Along with the development of engineering,hylology and cytobiology, the methods of tissue engineering will be most promising on cartilage repairing. Due to easily available and fast growthing, bone mesenchymal stem cells were considered as best seed cells in cartilage tissue engineering. Mechanical load and biochemical factors play the pivotal role on chondrogenesis of bone mesenchymal stem cells. As a result, the experiment was divided into two parts to study of the effect of rolling compression loading bioreactor on chondrogenesis of rabbit bone marrow mesenchymal stem cells and Chondrogenesis of bone mesenchymal stem cells with different states of chondrocyte in co-culture system1 The effect of rolling compression loading bioreactor on chondrogenesis of rabbit bone marrow mesenchymal stem cellsObjective To explore the effect of rolling compression loading bioreactor on chondrogenesis of rabbit bone marrow mesenchymal stem cells.Methods BMSCs were isolated from New zealand rabbits, , at approximately 2.5 months old. Passage3 BMSCs were used to make BMSCs-agarose constructs(diameter=4mm;height=4mm). Samples were divided into four groups: TGF-β1 + loading, TGF-β1, loading, and control groups. Gene expression and GAG synthesis were tested at three time points.Results Type II collagen gene expression of group TGF-β1 + loading was upregulated at three time points, and obviously increased at days 21. type Ⅱand aggrecan gene expression of TGF-β1 + loading, TGF-β1 and loading groups upregulated (2.5±0.24(P<0.05),1.8±0.12和1.9±0.20 folds) , (2.7±0.34(P<0.05),2.6±0.06和2.1±0.07) at days 7, (5.7±0.61(P<0.05),3.4±0.54(P<0.05),3.2±0.36 folds), (8.6±1.61 ( P<0.05),7.7±1.42(P<0.05),6.7±0.93 folds) at days 14, (7.1±1.21(P<0.05),4.9±0.89(P<0.05),5.6±0.92(P<0.05)folds ), (14.3±1.81(P<0.05),9.7±1.12(P<0.05),10.5±0.90 folds) at days 21.The aggrecan gene expression of TGF-β1 + loading group was also significantly increaseed at days 21. The aggrecan and typeⅡcollagen gene expression of control group was not obviously upregulated. The GAG content of TGF-β1 + loading, TGF-β1 , loading and control group was 2.10±0.33,1.69±0.12,1.58±0.16,0.13±0.05μg/(mg wet weight) at days 7, 3.56±0.32,2.33±0.25,2.64±0.085,0.20±0.04μg/(mg wet weight) at days 14, 4.63±0.28,2.68±0.35,3.35±0.34,0.27±0.0118μg/(mg wet weight) at days 21. The GAG content of TGF-β1 + loading group was greater than other groups at three time points(P<0.05). Toluidine blue O stain result showed that TGF-β1 + loading group had obviously blue-stain, and even appeared cartilage lacunae.Conclusion The rolling compression loading bioreactor had positive effect on chondrogenesis of rabbit bone marrow mesenchymal stem cells, And this effect was further enhanced by combined with TGF-β1.2 Chondrogenesis of bone mesenchymal stem cells with different states of chondrocyte in co-culture systemObjective To explore the effect osteoarthritis chondrocytes and normal chondrocyte on chondrogenesis of bone mesenchymal stem cells(BMSCs) in self designed co-culture system.Methods BMSCs and chondrocytes were isolated from New zealand rabbits. Six-month old rabbit was used to establish osteoarthritis model, and osteoarthritis chondrocytes was isolated for further experiments. BMSCs-low melting agarose constructs were put onto the self-made 6 well plates lattice assembly. Real-time PCR, 1,9-dimethylmethylene blue(DMMB) test were performed at 3, 7, 14 day in all groups, respectively. Results Rabbit osteoarthritis model was successfully established. Articular surface was gray and rough. TypeⅡcollagen gene expression of Normal P0-BMSCs group was 5.11±0.3,7.2±0.39,11.2±0.58 folds ,typeⅠcollagen gene expression was 0.25±0.2,0.75±0.35,1.05±0.48 folds, aggrecan gene expression was 2.1±0.22,3.8±0.41,22.5±0.39 folds comparing with control group of same time point at days 3,7,14. TypeⅡcollagen gene expression of Normal P3-BMSCs group was 0.71±0.3,1.5±0.57,1.8±0.38 folds ,typeⅠcollagen gene expression was 0.92±0.13,1.2±0.33,1.8±0.30 folds, aggrecan gene expression was 0.7±0.22,1.0±0.28,1.58±0.40 folds comparing with control group of same time point at days 3,7,14. TypeⅡcollagen gene expression of OA P0-BMSCs group was 1.0±0.37,1.73±0.74,3.6±0.36 folds ,typeⅠcollagen gene expression was 0.98±0.22,0.65±0.14,1.21±0.22 folds, aggrecan gene expression was 0.95±0.15,6.2±0.68,7.8±0.44 folds comparing with control group of same time point at days 3,7,14. TypeⅡcollagen gene expression of OA P3-BMSCs group was 1.1±0.35,0.67±0.39,1.51±0.22 folds ,typeⅠcollagen gene expression was 0.4±0.12,0.6±0.4,0.8±0.13 folds, aggrecan gene expression was 1.28±0.28,0.95±0.39,1.12±0.41 folds comparing with control group of same time point at days 3,7,14. TypeⅡcollagen gene expression was up-regulated in group Normal P0-BMSCs. Type I,Ⅱcollagen and aggrecan gene expression was not obviously up-regulated. TypeI collagen gene expression level of group OA P3-BMSCs is lower than control group in 3,7,14 day. GAG content of group Normal P0-BMSCs,Normal P3-BMSCs,OA P0-BMSCs,OA P3-BMSCs was 5.7±0.49,2.3±0.41,2.5±0.31,1.8±0.59 (mg/wet weight). GAG content of group Normal P0-BMSCs was 5.7±0.49μg/(mg/wet weight) more than that of other groups. GAG content of group OA P0-BMSCs and control group were of no statistical difference (P>0.05). There were significant differences between experimental groups and control group(P<0.05).Conclusion Rabbit normal P0 chondrocytes and rabbit OA P0 chondrocytes secreted morphogens enhanced chondrogenic differentiation of rabbit BMSCs. Rabbit normal P3 chondrocytes and rabbit OA P3 chondrocytes secreted morphogens do not have a significant effect on chondrogenic differentiation of rabbit BMSCs.