Effect Of Mechanical Microenvironment On Mechanosensitive Channels In Knee Joint Chondrocyte

Chondrocytes sense changes in the mechanical microenvironment via mechanosensitive channels,thereby regulating cell functions.This mechanism plays an important role in the cartilage health and injury.However,the role of different mechanosensitive channels in mechanotransduction are not yet fully understood.Transient receptor potential vanilloid 4(TRPV4)and piezo-type mechanosensitive ion channel component 1,-2(Piezo1 and Piezo2)are particularly important in the process of chondrocyte mechanotransduction.Therefore,we have studied the role of TRPV4,Piezo1,and Piezo2 in mechanotransduction of chondrocytes during sensing and responding to mechanical microenvironment,and further explored the potential impact of the mechanism on knee articular cartilage health and injury.Firstly,the effect of cyclic tensile strain(CTS)on the expression of mechanosensitive channel proteins(TRPV4,Piezo1,and Piezo2)were evaluated.The expression of TRPV4,Piezo1,and Piezo2 differently response to CTS with different amplitudes.Specifically,the TRPV4,Piezo1,and Piezo2 expression reached its highest level in 3%,13%,and 18% CTS,respectively.The resultsindicated that the role of TRPV4,Piezo1,and Piezo2 in chondrocyte sensing CTS with different amplitudes are different.In addition,the effect of CTS on the synthesis of collagen II and aggrecan which are the major components of cartilage matrix were studied.The results showed that expression levels of collagen II and aggrecan increase in 3%,8%,and 13% CTS,whereas decreases were observed in 18% CTS.The results indicate that normal loading enhances cartilage matrix synthesis,whereas excessive loading induces cartilage matrix degradation.Secondly,the effect of CTS on calcium signaling in chondrocytes using calcium imaging were investigated.The results indicated that calcium oscillation in chondrocyte was regulated by CTS in a magnitude-dependent manner.Further analysis of the role of TRPV4,Piezo1,and Piezo2 in CTS-induced calcium oscillation,the TRPV4,Piezo1,and Piezo2 knock-down(TRPV4-KD,Piezo1-KD,Piezo2-KD)chondrocytes using siRNA technology were established.Our results exhibited that the calcium oscillation induced by 3% and 8% CTS showed a significant decrease in only TRPV4-KD group relative to the Control group,whereas both Piezo1-KD and Piezo2-KD group in 13% CTS and only Piezo2-KD group in 18% CTS.The results indicate that TRPV4,Piezo,and Piezo2 mediated mechanotransduction in chondrocyte sensing 3% and 8%,13%,and 18% CTS,respectively.Thirdly,the effect of matrix stiffness on the expression of TRPV4,Piezo1,and Piezo2 with PDMS substrate matched with PCM stiffness underphysiological and pathological conditions were studied.The results showed that TRPV4 expression continued to increase with increased stiffness,whereas opposite expression were observed in Piezo1 and Piezo2.The results indicate that the role of TRPV4,Piezo1,and Piezo2 in mechanotransduction in response to different substrate stiffnesses in chondrocyte are different.In addition,the results showed that the synthesis of collagen II and aggrecan reached its highest level at the matrix with the stiffness(~78 kPa,~54 kPa),and then decreased in stiffer(~197 k Pa)or softer(~2 kPa)matrix.The results suggest that it will help maintain the cartilage phenotype on intermediate substrate,whereas be beneficial to the degradation of cartilage matrix on stiffer or softer substrate.Lastly,the role of TRPV4,Piezo1,and Piezo2 in stiffness-induced calcium oscillation were investigated.The results showed that calcium oscillation in chondrocyte were regulated by matrix stiffness in a stiffness-dependent manner.We found that the intensity and frequency of calcium oscillation induced by medium-and high-stiffness(~78 kPa,~197 kPa)showed a significant decrease in only TRPV4-KD group compared with the control group,whereas both Piezo1-KD and Piezo2-KD group in medium-and low-stiffness(~54 k Pa,~2kPa).The results indicate that TRPV4 and Piezo mediated mechanotransduction in chondrocyte responding to medium-and high-stiffness(~78 kPa,~197 kPa)and medium-and low-stiffness(~54 kPa,~2 kPa),respectively.In summary,TRPV4-mediated mechanotransduction regulates the anabolic response of chondrocytes to CTS with medium-and low-strain;Piezo2-mediated mechanotransduction regulates the catabolic response of chondrocytes to CTS with high-strain;Stiff matrix is mediated by TRPV4 channel while soft matrix is mediated by Piezo1 and Piezo channels.Our study may provide a potential novel drug target in chondrocyte function repair,and it may also provide a new strategy to optimize chondrocyte microenvironment for functional tissue engineering.