The Effect Of Low-intensity Pulsed Ultrasound On Human Osteoarthritis Chondrocytes In Vitro And Related Transduction Mechanism

Part 1: the effect of low-intensity pulsed ultrasound on human osteoarthritis chondrocytes in vitroObjective: To investigate the effect of low-intensity pulsed ultrasound(LIPUS)on the apoptosis and extracellular matrix of human osteoarthritis chondrocytes primarily cultured in vitro.Methods: Human osteoarthritis chondrocytes acquired from abandoned articular cartilage were isolated and cultured.The cells were identified by Toluidine Blue staining and Type II collagen cells immunochemical staining.The cells(P2)were stimulated by LIPUS at different intensities(0、30、60、90 m W/cm2)for one week(20 min/d).The rate of cell apoptosis was evaluated by FCM(flow cytometer).The m RNA and protein expression levels of collagenⅡ、aggrecan and MMP13 were detected by qPCR and Western blot.Results: The rates of cell apoptosis(%)in group of 30、60、90 m W/cm2(7.75±0.79;8.52±0.91;11.38±0.85)were lower than those in control group(18.13±0.83)(P < 0.05).There were significant difference between 30、60、90 m W/cm2 groups and control group both in m RNA and protein expression level of collagen II 、 aggrecan and MMP13 in an intensity independent manner(P<0.05).Conclusions: LIPUS can reduce the rate of apoptosis of human osteoarthritis chondrocytes primarily cultured in vitro.Further LIPUS can promote the synthesis and inhibit the decomposition of ECM.It might be an useful treatment for OA and delay the progression of OA.Part 2: related transduction mechanism of low-intensity pulsed ultrasound on human osteoarthritis chondrocytes in vitroObjective: To investigate the effect of low-intensity pulsed ultrasound(LIPUS)on human osteoarthritis(OA)chondrocytes and explore the underlying mechanism.Methods: Human osteoarthritis chondrocytes were collected from abandoned articular cartilage.The P2 cells were randomly divided into three groups: an OA control group,an OA plus LIPUS group,an OA plus LIPUS and LY294002-treating group.The m RNA levels of COL2α1,aggrecan and MMP13 m RNA were determined by q PCR.The protein levels of COL2α1,aggrecan,Akt,p-Akt and MMP13 were evaluated by Western blotting.Results: Compared with the OA control group,the expressions of COL2α1 and aggrecan at both m RNA and protein levels were significantly increased,and that of MMP13 were significantly reduced in the OA plus LIPUS group.The p-Akt protein level was significantly increased after LIPUS stimulation,but there was no significant difference in the Akt protein levels between the two groups.Moreover,LY294002,an inhibitor of PI3K/Akt,significantly suppressed the biological effect activated by LIPUS.Conclusion: LIPUS could reduce the rate of apoptosis,enhance the ECM synthesis and inhibit the ECM decomposition of human osteoarthritis chondrocytes,which might be regulated through PI3K/Akt pathway.