Research On The Construction Of Co-cultured System In Microcavitary Hydrogel And Its Application In Chondrogenesis

ObjectiveIn this study,we aimed to optimize the microenvironment in order to improve the effect of chondrogenesis of stem cells.We introduced a co-culture method between ATDC5 cells and primary chondrocytes into microcavitary hydrogel,and explored the idealcondition for this culture system so as to eventually obtain the ideal engineered cartilage,for clinical cartilage repair.Method?.Co-culture cells harvest and cultureThe primary chondrocytes were isolated from poker from local butchery,and expanded in a 175cm2 flask with cartilage culture medium.The mice ATDC5 cell line was bought from Abgent(San Diego,USA),and also expanded in a 175cm2 flask with ATDC5 culture medium.?.Gelatin microspheres preparation and the culture of primary chondrocytes in the microcavitary hydrogelThe gelatin microspheres of 80-120?m size were made by double emulsion method.After stained with Rhodamine,the degradation of gelatin microspheres was observed through fluorescence microscope.Then gelatin microspheres were encapsulated into alginate hydrogel with primary chondrocytes.The situation of cells in microcavitary alginate hydrogel was determined by live/dead staining and Cell Counting kit-8(CCK-8)testing.?.The construction of co-culture system in the microcavitary alginate hydrogelMix the chondrocytes and ATDC5 cells with ratio of 1:3,1:1 and 3:1(group 1C3 A,AC and 3C1A).The single primary chondrocytes(C)and ATDC5 cells(A)were set as control groups.These cells were mixed with gelatin microspheres in the sodium alginate solution and made the microcavitary alginate hydrogel and cultured inchondrogenesis inducing medium.?.Cell viability and cartilage phenotypes analysisAfter the culture period,observe and analyze the cell viability and chondrocyte phenotypes by cell counting Kit-8(CCK-8),quantitative real-time PCR(qRt-PCR),immunohistochemistry(IHC)and western blot(WB).Result?.Succeed to extract the primary chondrocytes from fresh porcine cartilage and thaw the P7ATDC5 cells.And succeed to expand these cells in vitro,observe their growthform,cell viability and draw the curve of cell vitality.?.The gelatin microspheres with different diameters were obtained,and the faster the stirring speed was,the smaller the microspheres were.The gelatin microspheres could degrade spontaneously at 37? and thus form microcavities inside the alginate hydrogel.The live/dead staining result showed the primary chondrocytes could distribute uniformly and grow flourishly.The CCK-8 data indicated cells in the microcavitary hydrogel group had superior ability of growth to those in the control group.?.The co-culture system of primary chondrocytes and ATDC5 cells was successfully constructed.In this co-culture system,the best cell ratio of chondrocyte and ATDC5 cell was 1:3 by the analysis of cell viability and chondrocyte phenotypes analysis.P44/42 of the mitogen-activated protein kinase(MAPK)also involved the cell proliferation.ConclusionThe microcavitary alginate hydrogel co-culture system could provide an ideal environment for chondrocytic differentiation of ATDC5 cells,especially in the cell ratio of 1:3(chondrocytes toATDC5 cells).This could provide a more effective way to build artificial cartilage in tissue engineering and a ideal strategy for repairing damaged cartilage in clinic.