Esw Promotes The Differentiation Of Hmscs Into Osteoblast By ATP Influencing On The P2X₇Receptor

There are a lot of biological mechanisms in using the suitable quantity of ExtracorporealShock Waves（ESW）to stimulate the human Mesenchymal Stem cell’s （hMSCs） differentiationinto osteoblast.Following the indepth investigation,the shock wave can be used for thetreatment of Bone Fracture Disunion,Chronic Tendinitis,Aseptic Necrosis of Femoral Head andhave achieved good results.In vitro experiments, ESW could enhances the proteinphosphorylation of c-Fos,c-Jun in MAPK pathway within the hMSCs to mediate transforminginto osteoblast and promoting the osteogenesis of hMSCs.The recent research shows that theP2X₇receptors （nucleotide receptor） on the cell membrane surface are closely related with thebone formation,such as the osteogenic ability of the mouse which the P2X₇receptor’s gene hasbeen knockout obviously depressed.ATP is a natural ligands of the P2X receptor,and themembrane channel opening leading to the influx of Na⁺,Ca²⁺and the efflux of K+,whenextracellular ATP acting on the P2X receptors,According to these findings we speculate that theESWs could precipitate the hMSCs cells to release ATP,and the ATP could induced the hMSCsdifferentiating into osteoblast,this research was deployed based on thses study.Objective: To study the human bone arrow stromal cells （hMSCs） expresses the P2X₇receptors,and the Extracorporeal Shock Wave （ESW） can promote the intracellular ATP of thehMSCs be secreted out of the cell,that the high concentration of ATP in extracellularenvironment activating with the P2X₇receptor on hMSCs membrane to promote the hMSCsdifferentiation into osteoblast.Method: Obtaining the primary hMSCs by using the density gradient centrifugation todeal with the human bone marrow blood,culturing and subculturing,and observing themorphological changes,identifying the potential of multiple differentiation and useing the Flow cytometry to identificate the receptor.Adding different P2receptors’ inhibitors/associatedblockers （KN-62,8-SPT, APPS, PPADS, APYRASE） in cell nutrient medium When usingextracorporeal shock wave induced human bone arrow stromal cells differentiating intoosteoblasts,and treating the human bone arrow stromal cells with different concentrations ofATP to induced human bone arrow stromal cells into osteoblasts differentiation.By observingthe cell’s morphological,micro-plate reader and calcium nodules （Alizarin red staining）detectionto compare the inhibition rate of various inhibitors and the differentiate effect of differentconcentration of ATP in order to determine which P2subtype receptor and whether the ATPcould mediate the differentiation of osteoblast, and useing the immunofluorescence technique tomark the P2subtype receptor.Result: The cultured hMSCs had uniform appearance,fibroblast-like and whorlslygrowed,the subculture hMSCs growed slowly,and about15-20days passaged once.The cellsexpressed multiple membrane phenotypes,using the flow cytometry to detect phenotypes,wefound that the hMSCs weakly expressed the CD34and CD45,strongly expressed the CD73andCD105,denoting the cultured hMSCs were homogeneous.The ALP activity of every group cellwas significantly higher then the ALP activity of control group cell （P>0.05）,and calciumnodules had been deposited by alizarin red staining,but not the control group cell,when usingthe ESW to stimulatie the hMSCs differentiating into osteoblast.The ALP activity of cell withKN-62and APYRASE was significantly lower than the ALP activity of other highly selectivereceptor inhibitor groups （P>0.05）,suggesting that the P2X₇receptor inhibited by KN-62andATP could mediate the hMSCs’ differentiation into osteoblast.The ALP activity of groups cellinduced by ATP （1μm/L） was significantly higher than the control group’s and the ALP activityof cells induced by ATP（1μm/L） was higher than other ATP concentrationgroups’（P>0.05）,suggesting that the ATP could mediated the hMSCs differentiating intoosteoblast,and the suitable concentration is1μm/L.We found that about95%of cells appearingred staining by immunofluorescence microscopy,useing the antibody of P2X₇receptor labellingthe hMSCs and incubating with the second antibody,but not the control group cells（P>0.05）,certifying the hMSCs can express the P2X₇receptor.Conclusion: Human Mesenchymal Stem cell release intracellular ATP out of thecell,enhancing the extracellular ATP concentrations,and the ATP could activate the P2X₇ receptor which located on the cell membrane to mediate human Mesenchymal Stem celldifferentiating into osteoblast,is an important regulatory pathway in Extracorporeal ShockWaves inducing the cultured human Mesenchymal Stem cell differentiating into osteoblasts.