A Preliminary Study On The Related Signal Pathway Of Chondrogenesis In The Early Response Of ATDC5 Cells Under Compressive Stress

BackgroundsTemporomandibular joint is one of the finest and most frequent load-bearing joints in human body.Accordingly,the condylar cartilage,which plays an important role in the process of dispersed masticatory pressure,has become one of the structures most closely related to mechanical stimulation in the joint system.Pressure is an essential physiological factor to maintain joint stability.Appropriate pressure in the articular cavity helps to maintain the health of articular cartilage.However,when the pressure load is too high,it is very likely to cause irreversible damage to articular cartilage,and even lead to osteoarthritis(OA).It has been confirmed that stress stimulation can activate the signal pathway in prechondrocytes and regulate the process of cartilage formation,but few studies have focused on the activation of the early signal pathway of chondrogenic differentiation under compressive stress.As a kind of chondrocyte line,the differentiation process of ATDC5 cell line is similar to that of chondrocytes,and can be used to simulate the differentiation process of chondrogenesis in vitro.ObjectivesThis experiment attempts to determine the optimal compressive stress conditions for the chondrogenesis of ATDC5,to explore the activation of signal pathways involved in early response in ATDC5,and to screen out the key signal factors involved in the regulation of chondrogenic function in the early response of compressive stress stimulation.Methods1.The chondrogenesis of ATDC5 cells was induced by agarose gel modified micromass method for 7 days.The formation of glycosaminoglycan was detected by Alcian blue staining test,and the expression of SOX9,collagen Ⅱ and Aggrecan was detected by immunofluorescence test,RT-qPCR and Western Blot.2.The three-dimensional compressive stress loading model of chondrocytes was established in transwell chamber.0,1,2,3,4,5 g/cm2 compressive stress was applied to ATDC5 cells(setting 0 g/cm2 as control group)for 24 hours.After the collagen block was pressurized and stained with FITC-labeled phalloidin,the three-dimensional culture morphology of ATDC5 cells was observed by fluorescence confocal microscope,the cell activity was detected by CCK8 method,and the expression of SOX9,collagen Ⅱ and Aggrecan was observed by RT-qPCR and Western Blot,so as to select the best compressive stress value in early response.3.Using the established three-dimensional compressive stress loading model of chondroblasts,ATDC5 cells were subjected to 0 and 4 g/cm2 compressive stress respectively(setting 0 g/cm2 as the control group)for 6 hours.The phosphorylated protein expression of MAPK and PI3K-Akt signal pathways in early biological response was detected by phosphorylated protein chip technology and GO enrichment analysis.Results1.ATDC5 cells were successfully induced to form mature cartilage nodules within 7 days by agarose modified micromass method;the extracellular matrix was sky blue in Alcian blue staining;SOX9,CollagenⅡ and Aggrecan were highly expressed in immunofluorescence assay;SOX9,CollagenⅡ and Aggrecan showed high expression synchronization in RT-qPCR and Western Blot at the same time point after chondrogenesis induction.2.After the compressive stress of 0,1,2,3,4,5 g/cm2 were applied to ATDC5 cells for 24 hours,the actin of ATDC5 cells was fluorescent green in FITC-labeled phalloidin staining,and it was found under fluorescence confocal microscope that ATDC5 cells maintained the interconnected three-dimensional growth pattern under compressive stress.And there was no significant difference in OD value among the six groups in CCK8 assay.When the compressive stress of 4g/cm2 was applied to ATDC5 cells for 24 hours,the expression of SOX9,collagen Ⅱ and Aggrecan in RT-qPCR and Western Blot was significantly higher than that in the control group.3.When ATDC5 cells were subjected to 4 g/cm2 compressive stress for 6 h,the expression levels of p-MKK6,p-ERK1/2,p-AKT,p-MSK2,p-P70S6k,p-CREB,pGSK3β,p-HSP27,p-RSK1,p-mTOR,p-p53,p-MEK1,p-p38 phosphorylated proteins were higher than those in the control group(foldchange>1.2).Conclusions1.The chondrogenic induction model of ATDC5 cells in vitro could be established within 7 days by agarose modified micromass method,and the expression of SOX9,collagen Ⅱ and Aggrecan could be detected at both mRNA and protein levels.2.The compressive stress loading model of chondroblasts in this experiment can maintain the three-dimensional culture conditions of chondroblasts,and does not affect the activity of chondroblasts.3.4 g/cm2 is the key force for the early response of cartilage-related signaling pathway under compressive stress.4.p-ERK1/2,p-AKT,p-CREB,p-RSK1,p-mTOR,p-p38 are probably the important phosphorylated proteins involved in the early biological response to appropriate compressive stress in cartilage formation in this experimental model.