Effect Of Chondrocytes With Different Passage In The Co-culture System On The Chondrocytic Differentiation Of ATDC5 Cells

PurposesBased on the three-dimensional co-culture system,porcine ACs with different passage were transfected by recombinant adenovirus and encapsulated with ATDC5 in hydrogel to establish a co-culture system that can effectively release growth factors,to explore the influence of different generations of chondrocytes on growth factor releasing and on the effect of chondrogenic differentiation of ATDC5 cells in the co-culture system.Methods1.Cell harvesting and expansionATDC5 cells were purchased from ABGENT in San Diego,USA,and cultured in a150cm²culture flask containing ATDC5 medium.Porcine chondrocytes were obtained from fresh porcine articular cartilage by enzymolysis and cultured in cartilage culture medium to the P1,P3,and P5 for the following experiment2.Morphological characteristics of chondrocytes and expression of cartilage related genesThe morphological changes of chondrocytes of different generations were observed by inverted microscope during the expansion and culture of chondrocytes in vitro,and the expression level of Col II,Col I and ACAN genes in chondrocytes were detected by quantitative real-time quantitative q-PCR.3.Adenovirus transfection of chondrocytes and construction of three-dimensional hydrogel co-culture systemThe P1、P3、P5 chondrocytes were transfected with recombinant adenovirus carrying TGF-β3 transfection gene,and the transfection effect was observed by Fluorescence microscope on the second day after transfection.Then a certain number of transfected chondrocytes were mixed with ATDC5 cells cultured in advance according to the ratio of1:3.After the supernatant was discarded by centrifugation,sodium alginate solution was used to resuspend the mixture of cells.And then the sodium alginate/cell mixed solution was cross-linked with CaCl₂ solution to form gelatin microspheres,and then transferred to a 24-well plate containing cartilage-inducing solution for 28 days of culture,and lastly,the three experimental groups were named P1,P3,and P5,respectively.4.Detection of TGF-β3 and evaluation of cartilage differentiation in vitroThe culture medium in the co-culture system was collected at the specified time point,and the concentration of TGF-β3 in the co-culture medium was measured by Elisa,and the controlled-release effect of TGF-β3 in different experimental groups was evaluated.On the 28th day of chondrogenic induction in the hydrogel,experimental samples were collected and the expression of the relevant chondrogenic specific genes was evaluated by q-PCR.Meanwhile,the secretion of cartilage marker proteins was observed by HE staining and immunohistochemistry.According to the data obtained,the chondrogenic effect in vitro was evaluated.Results1.The fresh primary chondrocytes extracted in this experiment were cultured in a monolayer in vitro and subcultured in vitro to P1,P3 and P5 after 80%fusion.In this process,the primary chondrocytes showed a multi-angle shape.After passage,the morphology of chondrocytes changed from polygonal to shuttle-shaped or oval-like shape.The results of q-PCR detection of chondrocyte-related marker genes showed that the expressions of ACAN and ColⅡwere relatively high in P1.With the increase of generation,the expression of ACAN and ColⅡgenes was decreased,and the expression of Col I gene was up-regulated,indicating that chondrocytes were dedifferentiated.2.The adenovirus carrying the TGF-β3 gene was infected with P1,P3,and P5chondrocytes.On the second day after transfection,the transfection efficiency was observed with a fluorescence microscope.It can be observed that the three groups of cells all have an obvious fluorescent expression with the transfection efficiency of 95%or better,of which the P5 group had the highest fluorescence effect,and the cell growth was observed in good condition.3.The results of the Elisa experiment showed that the Secretion trend of TGF-β3 in the three experimental groups of P1,P3,and P5 was the same.The secretion level of TGF-β3 reached the highest point on the third day.The highest peak was in the P3 group the lowest in the P1 Group.Then there was a significant decrease in expression on the 6th day.In the subsequent co-culture cycle（after the 12th day）,the TGF-β3 secreted by chondrocytes remains in a low range.The q-PCR results showed that the expression levels of the chondrogenic marker genes（ACAN and ColⅡ）of ATDC5 cells in the P5 group were higher than those in the P1 and P3 groups,while the ColⅠgene had the lowest expression level in the P3 group and the highest in the P1 group.In the co-culture system,the expression level of ACAN and ColⅡgene of chondrocytes in the P5 group was the lowest,while the expression level of ColⅠwas the highest.The results of HE staining showed that there were a large number of cells in each group of histological section samples,which grew in clumps,with the characteristics of Lacuna of chondrocytes.Safranin O staining shows the deposition of GAG.The figure shows that the three groups have obvious red staining areas,and the staining of the P3 group is relatively light.The staining results of typeⅡcollagen showed that the staining of P1 and P3 groups was weak only showing a small part of the area of tan area,while the staining of group P5 was relatively obvious,and the amount of cells and tan area were relatively large.ConclusionsThe results of this study show that chondrocytes of different generations have different effects on the chondrogenic differentiation effect of ATDC5 in the three-dimensional co-culture system.Among them,the chondrocytes in the P1 group had a poor chondrogenic induction effect,which may be due to the low release of TGF-β3from the P1 group of transgenic chondrocytes,which was not sufficient to induce ATDC5to differentiate into cartilage.Compared with the P1 and P3 groups,the P5 group showed a better effect of promoting cartilage differentiation.However,the degree of dedifferentiation of chondrocytes in the P5 group was the highest during the co-culture period.But these experimental data also further verified the feasibility of constructing a co-culture system for controlled release of TGF-β3.The longer culture time it takes,the more cells can be obtained,on the one hand,which can meet the requirement of the number of seed cells for cartilage tissue engineering.On the other hand,the higher the degree of dedifferentiation chondrocytes underwent,the closer to the state of apoptosis they got,which can stop the release of TGF-β3 by controlling the apoptosis of the transfected chondrocytes,thereby controlling the amount of TGF-β3 released and enabling mesenchymal stem cells to carry out cartilage differentiation at an optimal TGF-β3concentration and time to avoid exceeding the concentration of TGF-β3 which will lead to hypertrophy,calcification and even apoptosis of newly differentiated chondrocytes.