The Effects Of Indirectly Co-cultured Chondron In Vitro On Chondrocytes Biological Characteristics

Objective:The purpose of this study is using Transwell indirect co-culture technique to discover the effects of chondron on chondrocytes biological characteristics and provide the theoretical basis for the construction of seed cells of tissue engineering technology?Methods:Two month old New Zealand rabbit knee cartilage were digested into chondrocytes and chondrons with different digestive enzymes.The rabbit chondrocytes and chondrons will be seeded in Transwell Permeable Supports(in 2*105 cells/well).All cells were divided into the experiment group which chondrocytes were seeded in the lower layer while chondrons were seeded in the upper layer and the control group which chondrocytes were seeded in the lower layer while upper layer were seeded nothing.Abstract the chondrocytes cultured in the lower layers in each group at day 2,4,6,8.For detecting all indicators.The chondrocytes proliferation rate was detected by Flow Cytometer,each groups 's chondrocytes will be labelled by PCNA.The chondrocytes early apoptosis rate was detected by Flow Cytometer,each groups 's chondrocytes will be labelled by Annexin V ? 7-AAD.Besides fluorescence microscopy was used to detect the apoptosis rate by TUNEL staining and proliferation rate by EdU staining.The genes expression was detected Through PCR technology including AGG?Col-II ?MMP-13.Results:The detection of chondrocytes early apoptosis rate labelled by Annexin V ?7-AAD by using the flow cytometry has find out that,at day 2 and day 4,the chondrocytes early apoptosis rate between the experiment group and control group are considered no significant difference,the result has no statical significances.But the chondrocytes early apoptosis rate in the control group in day 6 and day 8 are obvious higher than the experiment group,the result has statistical significances.The results of TUNEL staining show that there were no significant difference between the experiment group and control group in chondrocytes apoptosis rate,but there were obvious trend that chondrocytes apoptosis rate in the control group are higher than the experiment group.The detection of chondrocytes proliferation rate by using the flow cytometry has find out that,at day 2 and day 4,the chondrocytes proliferation rate between the experiment group and control group are considered no significant difference,the result has no statical significances(p>0.05).But the chondrocytes proliferation rate in the experiment group in day 6 and day 8 are obvious higher than the control group(p<0.05),the result has statistical significances.The results of EdU staining show that there were significant difference between the experiment group and control group in chondrocytes proliferation rate at day 6(p<0.05).Rt-PCR showed that chondrocytes genes expression between the experiment group and control group are considered no significant difference at day 2 and day 4.But AGG expression and Col-II expression in the experiment group are obvious higher than the control group(p<0.05)at day 6 and day 8.MMP-13 expression in the experiment group obvious decreased than the control group(p<0.05)at day 8.Conclusion:From all these findings we conclude,first,that Chondron acted as a basic functional unit of articular cartilage can delay the apoptosis of chondrocytes for a longer period of time and enhance the proliferation of chondrocytes,and actively regulate the gene expression of cartilage matrix related substances.secondly,Chondron can be used as seed cells for the repairment of cartilage in the tissue engineering technology.