Redifferentiation Of Genetically Modified Dedifferentiated Chondrocytes In A Three-dimensional Microcavitary Hydrogel

ObjectiveDedifferentiated chondrocytes were transfected by lentiviral vector and adenovirus vector carrying TGF-β3 gene and cultured in a three-dimensional microcavitary hydrogel.Redifferentiation effect of each viral vector was evaluated,thus providing experimental basis for cartilage injury repair.Method1.Isolation,cultivation and expansion of porcine articular chondrocytes（AC）Chondrocytes were isolated from fresh porcine femoral condyle,which were digested with type Ⅱ collagenase,and then collected and cultured in a flask with chondrogenic medium.Cells were subcultured and frozen for the following experiment at about 80%confluency.2.Transfection of articular chondrocytes with lentiviral vector carrying TGF-β3geneChondrocytes were transfected with lentiviral vector overexpressing TGF-β3 gene purchased from Land M Biological Company according to the instructions.Lv-P4 and Lv-P6 were obtained by screening and passage.They were resuscitated with 1.2%alginate solution at 6×10⁶/ml cell density and dropped into 102m M calcium chloride solution（CaCl₂）with a special gun to form beads by crosslinking.They were named as3D-Lv-P4 and 3D-Lv-P6 respectively.P4 and P6 were encapsulated in the same method,named as 3D-P4 and 3D-P6.At the same time,P4 and P6 in monolayer culture were used as control,named 2D-P4 and 2D-P6,respectively.These beads were washed by PBS and placed in a 48-well plate for three-dimensional culture in a cell incubator.The samples at each time point were used to detect the cell viability and the expression of cartilage-related genes.3.Transfection of articular chondrocytes with adenovirus vector carrying TGF-β3geneChondrocytes were transfected with adenovirus vector overexpressing TGF-β3gene,which has been constructed in a previous study.3D-Ad-P4,3D-Ad-P6,3D-P4and 3D-P6 were obtained follow the same method as above.Similarly,2D-P4 and 2D-P6 were used as control.The detection of the cell viability and cartilage-specific genes were conducted at the corresponding time points.4.Gelatin microspheres were prepared by double emulsification,and gelatin microspheres with a pore size of 80-120μm were screened,which were mixed with 1.6%alginate solution according to 0.3g:1ml ratio.And P6,Ad-P6,Lv-P6 were resuspend at the density of 6×10⁶/ml cells with the mix solution,named Gel-P6,Gel-Ad-P6,Gel-Lv-P6,respectively.The above beads were washed by PBS and placed in a 48-well plate and cultured in chondrogenic induction to establish a three-dimensional culture system.During this period,the culture medium of Gel-Lv-P6 and Gel-Ad-P6 at each time point was collected to detect the release of TGF-β3.The cell viability,gens and protein level of specific marker were detected.Result1.During subculture of chondrocytes,the cell morphology gradually changed from round or polygonal to fibrous or fusiform.In terms of gene expression,the expression of Col Ⅰ gene of P1,P4,and P6 gradually increased,while the expression of cartilage-specific gene Col Ⅱ and ACAN decreased gradually.2.The proliferation activity of 3D-Lv-P4 and 3D-Lv-P6 was higher than that of the control group and the expression of Col Ⅰ and Col X gene was significantly lower than that of the control group.However,the ACAN gene expression of 3D-Lv-P4 and Col Ⅱ,ACAN genes in 3D-Lv-P6 group were significantly increased.3.The cell viability of 3D-Ad-P4 and 3D-Ad-P6 was also higher than that of the control group.The Col Ⅰ gene expression in 3D-Ad-P4 group was higher than that of2D-P4,while the Col Ⅰ gene expression of 3D-Ad-P6 was lower than 2D-P6.In addition,the expressions of Col Ⅱ and ACAN genes in 3D-Ad-P4 group and 3D-Ad-P6 group were significantly higher than their control group.4.The cell viability of Gel-P6,Gel-Lv-P6 and Gel-Ad-P6 was higher than that day1 respectively.After 14 days of culture,the Col Ⅰ gene expression of Gel-Ad-P6 and Gel-Lv-P6 was lower than Gel-P6,while the expression of Col Ⅱ,ACAN and Col X genes of Gel-Ad-P6 was the highest in all groups.On day 28,the m RNA level of Col Ⅰ in Gel-Lv-P6 group was the highest in all groups,while the expression of Col Ⅱ and Col X genes in group Gel-Ad-P6 and Gel-Lv-P6 were higher than Gel-P6.Similarly,the expression of Col Ⅱ,ACAN genes in group Gel-Ad-P6 was the highest.Western blotting showed that the expression of COL Ⅰ protein in Gel-Lv-P6 was the highest,while the COL Ⅱ protein level in Gel-Ad-P6 and Gel-Lv-P6 was higher than that in Gel-P6.On the other hand,the expression levels of TGF-β3 in Gel-Ad-P6 and Gel-Lv-P6 group were different.The TGF-β3 expression of Gel-Lv-P6 was low and maintained at about 600pg/ml throughout the culture time,while the level of TGF-β3 in Gel-Ad-P6 group remained about 45-55ng/ml in the first 18 days,then declined to a low level,about 4-4.5ng/ml.Conclusion1.More chondrocytes with better cellular activity can be obtained by modified multiple digestion.During the process of subculture,chondrocytes gradually appear the phenomenon of dedifferentiation.2.In the three-dimensional culture of Lv-TGF-β3/Ad-TGF-β3 and sodium alginate,the proliferative activity of chondrocytes P4 and P6 was significantly increased,and the expression of specific genes of chondrocytes was promoted,which achieved the redifferentiation to a certain extent.3.The target gene of lentiviral vector can be expressed continuously and stably,but the expression abundance is relatively low.Rather,the expression abundance of adenovirus vector is high,but the expression is relatively unstable.4.Cells have high proliferative activity in microporous hydrogels.Dedifferentiated chondrocytes P6 transfected by virus vector carrying TGF-β3 in a three-dimensional microcavitary hydrogel can significantly promoted the expression of cartilage-specific related genes or proteins and realized the redifferentiation,but Ad-TGF-β3 increased chondrocyte hypertrophy gene expression.