To Co-culture Chondroytes And Chondrons In Alginate And Research The Biological-mechanical Properties Of Cultured Cells

Objective:To co-culture chondroytes and chondrons in alginate, then research the biological-mechanical properties of cultured cells, in order to provide suitable seed cells for tissue engineering. Methods:chondrocytes and chondrons were isolated from three months old New Zealand white rabbits, then cultured in 2% alginate pellets(50ul) with densities of 2×105/ml, and divided into three groups: ①chondroytes;②co-cultures(chondrocytes and chondrons at1:1);③chondrons. The histomorphological were observed by hematoxylin-eosin(HE), cell proliferation were detected by CCK-8, GAG、Col-II and MMP-13 m RNA expression were analysed by RT-PCR. The micropipette aspiration technique was used to quantify changes in mechanical properties of chondrocytes. Results:1. Cell proliferation:training at 7th day, the rate of chondrocytes group ’s propagation was the fastest,co-culture group’s between the other groups and chondrons group’s was the slowest(P<0.05); at 14 th day, co-culture group’s was the fastest(P<0.05); at 21 th day, chondrocytes group ’s was the slowest(P<0.05); at 28 th day, chondrons group’s was the fastest(P<0.05). 2. RT-PCR: ①Collagen Ⅱ: at 7th day, Col-Ⅱ m RNA relative expression of chondrocytes group was higher than other groups(P<0.05); at 14 th day, co-culture group’s was the highest(P<0.05); at 21 th day, chondrocytes group ’s was the lowest(P<0.05), no significant difference between the other groups; at 28 th day, chondrocytes group ’s was the lowest(P<0.05), no significant difference between the other groups. ②AGG: at 7th day, GAG m RNA relative expression of chondrocytes group was higher than chondrons group’s(P<0.05); at 14 th day, there were no significant difference between the three groups; at 21 th day, chondrocytes group ’s was the lowest(P<0.05), no significant difference between the other groups; at 28 th day, chondrocytes group ’s was the lowest(P<0.05), no significant difference between the other groups. ③MMP-13: at 7th、14th、28th day, MMP-13 m RNA relative expression of three groups were no significant difference between the three groups; at 21 th day, chondrons group’s was the lowest(P<0.05), no significant difference between the other groups. 3. Visco-elastic properties of cells:from the statistical analysis of cell equilibrium modulus(E∞)、instantaneous modulus(E0) and apparent viscosity(μ),we can find, training at 7、14th day, chondrocytes group ’s E∞、E0、μ were the lowest,co-culture group’s between the other groups and chondrons group’s was the highest(P<0.05);at 21、28th day,chondrons group’s was the highest(P<0.05),no significant difference between the other groups. Conclusion:To co-culture chondrocytes and cartilage can maintain cell’s proliferation and related gene expression in a long period of time, even able to maintain good mechanical properties, but further research is needed to confirm that,it is will get the same results in vivo.