The Role Of Hypoxia In TGF-?1-induced Chondrocyte Hypertrophy

Objective:To study the effect of hypoxia on TGF-?1-induced chondrocyte hypertrophy,it is clear that the optimal oxygen concentration can maintain the chondrocyte phenotype,and lay a foundation for the construction of a normal functional tissue-engineered chondrocyte culture system.Methods:The articular chondrocytes from patients with osteoarthritis,femoral neck fracture and femoral head necrosis were isolated and the m RNA expression of COL1A1,COL2A1,COL10A1,MMP-13,IHH and PTHr P genes in chondrocytes from patients with different diseases was detected by RT-q PCR.The levels of COLI,COLII and COLX proteins in chondrocytes were detected by immunofluorescence staining.Toluidine blue staining was used to identify the cultured P0 chondrocyte phenotype.RT-q PCR was used to compare the m RNA expression levels of COL1A1,COL2A1,COL10A1,MMP-13,IHH and PTHr P genes in P0-P3 chondrocytes.Cartilage cells were treated with different TGF-?1concentrations(5 ng/m L,15 ng/m L)and oxygen concentrations(2%,20%)to detect m RNA expression of COL1A1,COL2A1,COL10A1,MMP-13,IHH and PTHr P and cartilage cell mitochondrial complexes ?activity.Results:Compared with chondrocytes of femoral neck fractures,the expression of COL1A1 in chondrocytes of osteoarthritis and femoral head necrosis increased by(12.78±2.24)folds and(7.58±2.24)folds respectively;the expression of COL2A1 decreased by(0.594±0.05)folds,(0.316±0.01)folds;COL10A1 expression levels increased by(2.47±0.25)folds and(1.22±0.12)folds respectively;MMP-13 expression levels increased by(3.287±1.12)folds and(5.517±1.05)folds respectively;IHH expression levels increased by(3.287±1.12)folds and(5.517±1.05)folds respectively;the expression levels of PTHr P increased by(2.532±0.25)folds and(2.378±0.20)folds respectively,and the difference between the two groups was statistically significant.Compared with cartilage cells from OA patients,femoral neck fractures express high levels of COLII protein and low expression of COLI and COLX proteins.Compared with P0 chondrocytes,the expression levels of COL1A1 in P1-P3 chondrocytes were(1.34±0.114)folds,(0.434±0.103)folds and(0.371±0.112)folds respectively;the change of COL2A1 expression levels was(3.813±)2.51)folds(101.136±10.51)folds and(12.13±4.21)folds;COL10A1 expression levels were(0.884±0.125)folds,(16.16±6.13)folds,and(3.574±1.51)folds;IHH expression levels were(81.39±8.02)folds,(145.63±13.95)folds and(10.40±4.15)folds;the changes of PTHr P expression levels were(0.895±0.15)folds,(7.31±3.11)folds and(3.61±2.51)folds respectively.Differences between groups were statistically significant.The morphological appearance of chondrocytes showed that the chondrocytes of P0 and P1 were mostly triangular,while the chondrocytes of P2 and P3 generations were mostly polygonal and had numerous pseudopodia.Compared with P2 chondrocytes,P1 chondrocytes express high levels of COLII protein and low expression of COLI and COLX proteins.Compared to the control group(normoxia),the expression levels of COL10A1,IHH and PTHr P in the normoxia + TGF-?1(5 ng/m L)group were(2.75±0.220)folds,(1.57±0.202)folds and(3.304±0.36)folds respectively;The expression levels of COL10A1,IHH and PTHr P in the normoxic + TGF-?1(15 ng/m L)group were(2.88±0.214)folds,(1.49±0.215)folds,and(4.98±0.65)folds respectively;the difference between the groups was statistically significant.Compared with the normoxic group,the expression levels of COL10A1,IHH and PTHr P in the normoxic + TGF-?1(5 ng/m L)group were(2.75±0.220)folds,(1.57±0.202)folds,and(3.304±0.36)folds;The expression levels of COL10A1,IHH and PTHr P in the hypoxia + TGF-?1(5ng/m L)group were(1.45±0.071)folds,(0.85±0.11)folds and(2.14±0.23)folds respectively;the difference between the groups was statistically significant.Compared with the hypoxia 0h group,the expression levels of COL10A1,IHHand PTHr P in the hypoxia + TGF-?1(5ng/m L)6h group were(3.61±0.302)folds,(3.43±0.341)folds and(1.81±0.17)folds respectively.The expression levels of COL10A1,IHHand PTHr P in the hypoxia + TGF-?1(5ng/m L)12h group were(1.60±0.21)folds,(1.20±0.12)folds and(2.34±0.22)folds respectively;differences between the groups were statistically significant.The rate of NADH consumption by mitochondria was(3.35±0.65),(4.69±0.57)and(1.77±0.48)in normoxic group,normoxia + TGF-?1(5ng/m L)group,and normoxia + TGF-? 1(15ng/m L)group,respectively.± 0.48);differences between groups were statistically significant.The rates of NADH consumption by mitochondria in the normoxic,hypoxic,normoxic + TGF-?1(5 ng/m L)and hypoxic +TGF-?1(5 ng/m L)groups were(3.35±0.65),(3.70±0.71),(4.69±0.57)and(8.01±0.88)respectively;differences between groups were statistically significant.The rate of NADH consumption by mitochondria in hypoxia + TGF-?1(5ng/m L)0h group,hypoxia + TGF-?1(5ng/m L)6h group and hypoxia+ TGF-?1(5ng/m L)12h group was(3.35)± 0.65),(5.59 ± 0.45)and(8.11 ± 0.98);differences between groups were statistically significant.Conclusion:1.Chondrocytes(knee OA,femoral head necrosis)undergo hypertrophy and dedifferentiation.2.P2 generation of chondrocytes hypertrophied in vitro monolayer culture.3.Hypoxia inhibits the induction of TGF-?1 on cartilage hypertrophy.4.Hypoxia inhibits the hypertrophy of chondrocytes by increasing the activity of mitochondrial complex enzyme I.