Experimental Study About The Effect Of Controlled-release TGF-?3 Based On Coculture System On Chondrogenic Differentiation Of ATDC5

Object:Different ratios of ATDC5 cells to transfected chondrocytes by adenovirus were used to construct a coculture system to determine the release level of TGF-?3,and to evaluate the effect of the coculture system on promoting the chondrogenic differentiation of ATDC5 cells.Method:1.Cells harvest and culturePrimary chondroctyes were isolated from poker from local butchery,and expanded in a 150cm² flask,then were passaged to the third generation and frozen for further usage.ATDC5 cell line was purchased from Abgent?USA?,and also expanded in a150cm² flask with its specific culture medium.HEK-293 cells were purchased from American type culture collection and cultured with HEK-293 medium in a 75cm² flask.2.The amplification,purification and titer determination of adenovirusAdenovirus encoding TGF-?3 constructed in previous studies,was transfected into HEK-293 cells to amplified to a sufficient quantity,then was purified and titered.3.The construction of alginate hydrogel coculture system1:1 and 3:1 ratio of ATDC5 to chondrocytes?named as 1A1C and 3A1C?were mixed respectively.Two groups of pure ATDC5 were constructed as control groups?A group?,and exogenous TGF-?3?10ng/ml?was added to one of them in the culture medium?ArT group?.These 4 groups of cells were mixed in an alginate solution respectively,and then the cell/alginate mixture was droped into CaCl₂ sulotion to form beads.All beads were collected to 24-well plate and cultured in chondrogenesis induction medium.4.The release of TGF-?3 in two experimental groups,the cell viability and the chondrogenesis of each group.The culture media of the 1A1C and 3A1C were collected to test the release of TGF-?3 by Elisa.After the cultivation,the cell viability and chondrogenesis of each group were observed and analyzed by cell counting Kit-8?CCK-8?,quantitative real-time PCR?Q-PCR?,western blot?WB?and immunohistochemistry?IHC?.Result:1.Chondrocytes were isolated from fresh pork bones successfully,and expanded to the third generation.ATDC5 and HEK-293 were expanded to a sufficient number for further usage.In this process,the primary chondrocyte was observed,and found that it was polygonal and grow slowly.After expansion to the third passage,the chondrocytes became larger and their growth rate accelerated.ATDC5 cells was small,round,and grew rapidly;HEK-293 with the shape of round or oval,slightly adhered to the botton of the flask,and grows fast.2.Recombinant adenovirus carrying TGF-?3 gene was successfully transfected into HEK-293 cells,and the virus was amplified,purified,and titered,and its titer was4.38×10⁹ ifu/ml.3.TGF-?3 could be detected in the culture medium of both experimental groups,and both reached a peak on the third day,and fell rapidly on the sixth day,and thereafter maintained a low level.The cell proliferation activity of the 3A1C group was the highest,which was 3.5 times that of the beginning of the experiment,while the cell proliferation activity of the two control groups did not change much.The expression levels of cartilage marker genes?type II collagen and ACAN?in the two experimental groups were higher than those of P4 chondrocytes,which was comparable to that of porcine cartilage.In histological examination,HE staining showed that the cells of the two experimental groups were densely grown,and the cells of the two control groups were sparse,especially group A.In the safranine O staining,the sections of the two experimental groups showed a large number of red-stained areas around the cells,while the control group had fewer red-stained areas.Type?collagen immunohistochemical staining showed a large number of tan areas in the sections of the two experimental groups,while the two control groups were rarely?ArT?or even missing?A?.These datas confirm that the coculture system constructed in this study could promote ATDC5chondrogenic differentiation.Conclusion:The TGF-?3 controlled-release system constructed in this study can effectively promote the chondrogenic differentiation of ATDC5,moreover,in the case of reducing chondrocytes,the role of promoting cartilage differentiation is not affected.Base on this study,future efforts should be focus on improving this coculture system,such as using lentivirus as the vector,and further optimizing the proportion of cells in the coculture system,in order to control the amount and duration of TGF-?3 production more effetively,which can provide a better microenvironment for the cartilage differentiation of seed cells,and provide a new strategy for cartilage repair.