| BackgroundIn clinic,articular cartilage injury caused by exercise causes local defects and degeneration of articular cartilage,leading to pain and limited joint activity,and then progresses to osteoarthritis.Due to the limited healing ability of cartilage tissue,autologous cell transplantation is the first choice in the treatment of local cartilage defects.At present,the problem of "dedifferentiation" of chondrocytes caused by passage cannot be avoided.How to make chondrocyte proliferate in a large number in a short time and maintain its characteristics and meet the clinical needs has become an urgent problem to be solved.ObjectivesTo investigate the isolation,culture and identification of chondrocytes from hyaline cartilage of knee joint of New Zealand white rabbits by collagenase digestion and sequential culture of cartilage tissue blocks.To observe the effects of combined intervention of fibroblast growth factor 18(FGF18)and small molecule drug Kartogenin(KGN)on chondrocyte proliferation and cell characteristics.Methods1.Bilateral knee joints of New Zealand rabbits were obtained and hyaline cartilage tissue was separated.Primary chondrocyte was isolated and cultured by simple 0.2% collagenase type II digestion method and tissue block culture method.Chondrocyte was identified by toluidine blue staining and collagen type II immunofluorescence.The growth curve of P1-P6 chondrocyte was detected and drawn by CCK8 test.Enzyme-linked immunosorbent assay(ELISA)was used to detect the content of collagen type I and II in P1 and P5 media and to calculate the ratio between them..2.Chondrocytes of the P1 and P5 were selected for experiment.After screening the concentration of FGF18 and KGN,FGF18 + KGN(concentration: 100 ng/ml,10 ?M),FGF18(100 ng/ml),KGN(concentration: 10 ?M)and control group were set up.CCK8 test,Toluidine blue staining,Type II collagen immunofluorescence,Cell scratch test,Enzyme-linked immunosorbent assay(ELISA)and Western blot were used to detect the proliferation,migration of chondrocytes and the secretion of extracellular matrix.Results1.The primary chondrocytes of New Zealand rabbits were successfully isolated and cultured by using 0.2% collagenase type II digestion and cartilage tissue culture sequentially.Under inverted microscope,the chondrocytes were round and polygonal,uniform in size and active in proliferation and division.2.Toluidine blue staining showed that the cells separated by sequential method were all stained,the nucleus was purple blue,the extracellular matrix was light blue,the intracellular and extracellular granules were purple blue,Aggrecan secretion existed,and type II collagen fluorescence could be seen in the cell membrane and cytoplasm by immunofluorescence,suggesting that the cells had the characteristics of chondrocyte,and the identification of chondrocyte was completed.3.The growth curve of P1-P6 cells was successfully plotted by CCK8 test.The results showed that the proliferation activity of each generation decreased after P5(P < 0.05).The collagen I and II secreted by chondrocytes were quantitatively detected by ELISA.The ratio of collagen I and II secreted by P1 and P5 chondrocytes in standard medium within 72 hours was 3.12 + 0.37,7.01 + 0.56(P < 0.05),respectively.4.Combination of FGF 18 and KGN interfered with chondrocyte proliferation.CCK8 experiment showed that FGF 18 + KGN promoted the proliferation of chondrocyte in logarithmic growth phase(P < 0.05)..5.Toluidine blue staining experiment showed that fibroblast growth factor 18 + KGN group stained more deeply in chondrocyte and extracellular,which confirmed that fibroblast growth factor 18 + KGN could promote the secretion of Aggrecan.Type II collagen immunofluorescence assay showed that the fluorescence of chondrocyte was enhanced in fibroblast growth factor 18 + KGN group,which confirmed that the expression of type II collagen protein was increased.6.Cell scratch test showed that the healing rate of fibroblast growth factor 18 + KGN group was significantly increased(P < 0.01),which could promote chondrocyte migration.7.ELISA showed that the content of type II collagen increased significantly(P < 0.01),type I collagen decreased significantly(P < 0.01)and the ratio of type I and type II collagen changed(P < 0.01)in FGF 18 + KGN group.8.Western blot results showed that type II collagen,SOX9 and Aggrecan,which represent the chondrocyte characteristics of hyaline cartilage tissue,increased significantly in the FGF 18 + KGN group(P < 0.01),while type I collagen and type VII collagen with dedifferentiation characteristics decreased(P < 0.01)Conclusions1.Sequential application of 0.2% collagenase digestion and chondrocyte culture can successfully establish a system of chondrocyte isolation,expansion and culture.2.FGF18 and KGN intervention in chondrocyte can promote chondrocyte proliferation and maintain hyaline chondrocyte characteristics... |
PDF Full Text Request