Shockwaves Inhibit Chondrogenic Differentiation Of Human Mesenchymal Stem Cells In Association With Adenosine And A2B Receptors

Cartilage is a highly organized,avascular connective tissue that provides low friction,shock absorbance,load bearing and distribution for effective joint movement.Cartilage defects cause joint pain,stiffness and loss of mobility due to osteoarthritis,trauma,aging,and developmental disorders.Due to the limited regenerative capacity of chondrocyte,the cartilage defects cannot fully repair itself.Mesenchymal stem cells(MSCs)are able to self-replicate and differentiate into a variety of cell types such as osteoblasts,chondrocytes,adipocytes,and smooth muscle cells.Differentiated MSC producing hyaline cartilage tissue have shown promising early results in the repair of localized cartilage defects and may provide further treatment approaches for more extended cartilage damages in the future.In the past decades,extracorporeal shockwave therapy(ESW)had emerged as the important choice in the treatment of many orthopedic disorders including shoulder pathology,chronic diabetic foot ulcers,osteoarthritis of the knee,chronic patellar tendinopathy,bone nonunion and avascular necrosis of the femoral head.However,the exact mechanisms of shockwave therapy in musculoskeletal healing are still unknown.Our recent study suggested that the mechanism of shockwave promoting osteogenesis may be related to adenosine 5'-triphosphate(ATP).However,ATP cannot survive outside the cell,and is hydrolyzed into adenosine in a short time.Therefore,adenosine may be an important factor in the biological effects of shockwaves.Adenosine in the extracellular compartment functions as a signaling molecule through the activation of G-protein-coupled receptors.Four distinct adenosine receptors(ARs)have been described,A1,-2A,-2B,and-3,which differ in their affinity for adenosine and the biological effects of their associated signaling pathways.The inhibition of A1 and A3 ARs and the activation of A2 A and A2 B ARs ad enylyl cyclase causing decreases and increases in the intracellular concentration of Cyclic Adenosine monophosphate(c AMP),respectively.The four ARs are widely involved in neurodegenerative,immune,cardiac,inflammatory disorders,cancer and bone homeostasis.A2 B receptor(A2BAR)is the most insensitive AR of the four.Its engagement stimulates osteoblast differentiation of MSC precursors.In addition,A2 BAR occupancy suppresses osteoclast differentiation and function as well.A2 BAR signaling pathway can stimulate the production of IL-6,which may have an important role in the differentiation of MSCs.Objectives:To investigate the effect of shockwave treatment on h MSCs chondrogenesis and explored the potential mechanisms.Methods:1.Flow cytometry detects surface markers of BMSCs.2.Calcium nodules Alizarin red staining was used to identify the osteogenic differentiation ability of the isolated cultured cells.3.Toluidine blue staining,type II collagen immunohistochemical identification the chondrogenic differentiation ability of the isolated cultured cells.4.Oil red staining identifies the chondrogenic differentiation ability of the isolated cultured cells.5.CCK8 assay cell proliferation rate;Trypan blue staining to detect the effect of ESW on MSC activity.6.ELISA measurement of extracellular ATP,ADP,AMP and adenosine content after ESW exposure to MSCs.7.Western blotting detection of shock waves increased the expression of A2 BAR in h MSCs under three-dimensional culture conditions.8.Real-time PCR was used to detect the expression of SOX6,Sox9,COL1A2,COL2A1 and ACAN m RNA levels.Results:1.Shockwave treatment increases the release of ATP,ADP,AMP,and adenosine from h MSCs.The concentration of adenosine as well as ATP,ADP,AMP in shockwave treated group dramatically increased from 0 to 150 impulses in a dose-dependent way.For the treatment group of impulses more than 150,the results were more confused.Differential trend of ATP,ADP,AMP,and adenosine was apparent.There were no significant differences for ATP,ADP and AMP compared with the 200 impulses group,while the release of adenosine increased significantly(p<0.01).2.Shockwave exerts differential influence on the viability of h MSCs.The viability of MSCs was evaluated by via an CCK-8 assay.a.Shockwave reduced cell viability in the short term: Viability of cells subjected to <200 shockwave impulses remained at >95% when examined immediately after shockwave treatment.However,cells exposed to 200 impulses showed significantly decreased viability after one hour.b.Shockwave promoted cell viability in the long term: Cell viability was enhanced in h MSCs dose-dependently.There was only slight effect at 50 impulses,but a significant increase in viability was observed in the cells treated with 150 and above impulses compared to 0 impulses.In groups with 200 and above impulses,no significant difference was found compared with cells treated with 150 impulses shockwave(p >0.05).3.Shockwave treatment increased the levels of A2 BAR in h MSCs under 3-D culture conditions.The expression of the four known adenosine receptors(A1AR,A2 AAR,A2BAR,A3AR)in MSCs were evaluated by Western blotting.A2 BAR was significantly increased in MSCs following 100,150,200,250 impulses treatment wit h shockwave.After mechanical stimulation of MSCs,A2 BAR was up-regulated by a mean factor of 0.41.No consistent change was noticed between different groups in the levels of A1 AR,A2AAR and A3 AR.The concentration of HIF-1? in shockwave treated group dramatically increased from 0 to 150 impulses in a dose-dependent way.4.Shockwave treatment inhibits chondrogenic differentiation of h MSCs under 3D culture conditions.Upon treatment of shockwave,a suppressed accumulation of the cartilage matrix was detected by toluidine blue staining and immunostaining with anti–type II collagen antibody.Gene expression levels of the differentiated cells were assessed for hyaline cartilage markers COL2A1,Sox9,SOX6 and ACAN,fibrocartilage marker(collagen type I)and the internal control marker of glyceraldehyde-3-phosphate dehydrogenase(GAPDH).There was a suppressed chondrogenic response with changes in gene expression in cells treated by 0,50,150 and 250 impulses of shockwave.The expressions of SOX6,collagen types II and I,and ACAN were significantly decreased by 19-,12-,7-,and 6-fold,respectively(p<0.05).5.Shockwave treatment inhibits chondrogenic differentiation of h MSCs through or partially through the adenosine receptor A2 B.Sox9,COL1A2,COL2A1 and ACAN were determined by real-time PCR.While pellets were treated by adenosine or A2 BAR specific agonist BAY60-6583,there was no significant difference in the gene expression of SOX6,COL1A2,COL2A1 and ACAN compared with the shockwave group(p > 0.05).The antagonist of A2 BAR can inhibit the effect of shockwave.While pellets were treated by A2 BAR specific antagonist PSB115,there was significant increase in the gene expressions of SOX6,COL1A2,COL2A1 and ACAN compared with control group(p < 0.01).While the combination of shockwave treatment and PSB115 resulted significant counteracted effect in the gene expression of SOX6,COL1A2,COL2A1 and ACAN compared with shockwave group(p < 0.01).7.Shockwave treatment inhibits chondrogenic differentiation of h MSCs possibly related with IL-6.And the extracellular concentration of IL-6 was detected by enzyme-linked immunosorbent assay(ELISA).The shockwave treatment increased the secretion of IL-6,while the agonist and antagonist of A2 BAR promoted and inhibited the secretion of IL-6(p < 0.05).Conclusions:1.Shock waves inhibit the chondrogenic differentiation of h MSCs pellets.2.The shock wave can stimulate the concentration of extracellular adenosine in h MSCs and increase the expression of A2 BAR.3.Activation of A2 BAR can inhibit the chondrogenic differentiation of h MSCs.4.The shock wave inhibits the chondrogenic differentiation of h MSCs by activating the A2 B adenosine receptor.