Experimental Study On The Effect Of Simulated Microgravity On Dedifferentiation Of Condylar Chondrocytes In SD Rats

Objectives:By establishing a simulated microgravity environment in vitro,three-dimensional culture of the condylar chondrocytes of SD rats was developed.To explore the effects of simulated microgravity environment on the biological characteristics of chondrocytes.Methods:Select 1-3 d condylar cartilage tissue blocks of SD rats in vitro,get the condylar chondrocytes,continuous subculture,selection of third-generation cells toluidine blue stain,type ii collagen immunohistochemical staining for identification.The 6th generation condyle cartilage cells were selected to be inoculated on The microcarrier cyrtodex-3 and placed in The microgravity environment simulated by The Rotary Cell Culture SystemTM rotating Cell Culture system(RCCS).In the same batch,the microcarriers of the cells were inoculated with the static conditions of the ground for cultivation as a control group.Training after 7 ds,were observed under inverted phase contrast microscope and scanning electron microscopy,and extraction of total RNA,two groups of cells for real-time Quantitative-PCR detection,compared two groups of cells into cartilage related genes of Sox9,col-Ⅱ alpha 1,Aggrecan and cartilage cells to differentiation tendency related col-Ⅰ alpha 1 expression difference.Results:1.The condylar chondrocytes obtained by tissue block culture method are presented with polygonal appearance,the nuclei are large and round,and they are closely aligned,presenting the appearance of "paving stone".P3 generation of generation of condylar cartilage cells,primitive cells with similar form,growth in a s-shaped curve type,has a good proliferation,toluidine blue staining was carried out on the cell and collagen type II immunohistochemical staining were positive.In the P6 generation,the proportion of elongated spindle cells increased significantly.presenting a "fish group" arrangement,presenting a dedifferentiated form.2.Using the inverted phase contrast microscope and scanning electron microscope to observe the condylar cartilage cells of the microcarrier cytodex-3,the cells were attached to the microcarriers and effectively grew and proliferated.3.Under simulated micro gravity environment condylar chondrocytes after 7 days,condylar chondrocytes col-Ⅰ alpha 1,col-Ⅱ alpha 1 mRNA expression of Aggrecan amount is lower than that of the ground under the stationary state of developing,however,Sox9 mRNA expression quantity is relatively higher.Conclusions:1.In vitro culture model of condyle chondrocytes was established by tissue block culture method.2.The condyle cartilage cells can be adhered to the surface of microcarrier cytodex-3 to grow and proliferate,and cytodex-3 can effectively increase the culture space and facilitate the large-scale expansion of the condylar cartilage cells.3.In the microgravity bioreaction,the condylar cartilage cells were cultured,the chondrocytes dedifferentiated and the related gene expression was decreased,and the rate of differentiation was delayed.The increased expression of the upstream gene expression of chondrocytes indicates that microgravity has definite significance for the maintenance of chondrocyte phenotype.