| Background:The damage of articular cartilage is very difficult to repair itself due to without vasculars, nerves and lymphs. It has been comfirmed that the tissue engineering technology promote repair of cartilage injury study. the chondrocytes as seed cells in vitro culture were easy to dedifferentiate in vitro; in vivo, the tissue repair and mechanical properties of phenotypes were lower than that of the normal articular cartilage, and with the time of repair in vivo, tissue repair appeared the characteristics of rapid degeneration, which restricted its clinical application prospects. Chondron is used as the basic functional structure of articular cartilage, which is mainly composed of chondrocytes and pericellular matrix(PCM), and PCM plays an important role in the metabolism and mechanical transmission of cartilage cells. In vitro experiments confirmed that co-culture of sodium alginate gel beads are composed of chondrocytes and cartilage in a ratio of 1:1, and its biological characteristics are better than the other ratios; however, Chondron group and the ratio of 1:1 co-culture group to repair cartilage defects in vivo is unknown.Objective:To explore the use of alginate three-dimensional culture of Chondrons, or three-dimensional co-culture of Chondron and chondrocyte(1:1) transplantation on the repair of defects in articular cartilage of rabbit knee, and comparative analysis of both the repair effect.Methods:1.Chondrocytes and chondrons from the both knee joints of 2-month-old New Zealand white rabbits were digested using different enzyme, and chondrocytes or chondrons were mixed with 1.2% sodium alginate in a cell density of 5.8×106/mL respectively. At last, the three alginate gel balls(about 25?L) were prepared by chondrocytes, chondrons and co-culture of chondrocytes and chondrons cells(1:1);2.Forty five 4-month-old New Zealand white rabbits were chosen, and full-thickness articular defect with a diameter 4 mm were produced in the femoral condyle fossa of both knee joints. Alginate beads combined chondrocytes?chondrons or co-cultured(1:1)group cultured for two days in vitro was implanted into the articular defects; 3. After the implantation to 6, 12 and 24 weeks, we assessed by MRI, gross observation, HE staining,safranin O staining, immunohistochemical staining; RT-PCR detection about mRNA expression and TUNEL detection of apoptosis, in order to comprehensive judgment defect repairing.Results:1. MRI score Roberts shows that: from 6 weeks to 24 weeks. Scores of three groups showed increasing trend; at 12 weeks, the score of co-cultured group were significantly higher than the chondrocytes and chondrons group(P < 0.05); the score of co-cultured group is higher than the chondron group and chondrocyte group significantly at 24 weeks(P < 0.05). 2.The immunohistochemistry of collagen-II: from 6 weeks to 24 weeks, the expression of chondron group and co-cultured group showed an increasing trend; at 6weeks and 12 weeks, the expression of chondron group were higher than that the co-cultured group, and the co-cultured group were significantly higher than those in the chondron group at 24 weeks. 3.Wakitani tissue repair and score showed that over time,chondron group and co-cultured group score showed decreasing, and compared with 6weeks, 12 weeks was statistically significant(P < 0.05); each co-culture groups were significantly lower than those of thechondron groups(P < 0.05). 4.Rt-PCR showed that 6weeks to 24 weeks, co-cultured group in COL-II, Sox-9 mRNA expression was gradually increased, and the COL-II mRNA expression increased in chondron group; from 6 weeks to 12 weeks, COL-X and MMP-13 mRNA expression decreased at the chondron groupand co-cultured group, but both increased at 24 weeks;COL-II and Sox-9 of chondron group were higher than those in the co-cultured group at 6 weeks and 12 weeks, and at 24 weeks two index of co-cultured group were higher than chondron group; at three times,COL-X and MMP-13 expression in co-cultured group were lower than chondron group.5.TUNEL detection of cell apoptosis showed that the cells apoptosis rate of chondrocytes showed an increasing trend from 6 weeks to 24 weeks, and the increasing was statistically significant compared with 24 weeks and 12 weeks(P < 0.05); cell apoptosis rate in chondron group increased from 6 weeks to 24 weeks; apoptosis rate of the co-cultured group was significantly lower from 6 weeks and 12 weeks(P < 0.05),however the apoptosis rate of co-cultured group chondron at 24 weeks were more increased significantly than that at 12 weeks(P < 0.05). what's more, the apoptosis rate of co-cultured group were lower than chondron group and chondrocytes group at three points.Conclusion:1.chondrons and chondrocytes(1:1) were cultured as seed cells, have good effect on early repair of tissue defect, and it could delay the repair of tissue degradation.2.The chondrons has the function of delaying chondrocyte apotosis. |
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