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Shockwave treatment promotes bone healing of nonunion fractures. In this study, we investigated whether this effect could be due to adenosine 5′‐triphosphate (ATP) release‐induced differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells. Cultured bone marrow‐derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase activity, osteocalcin production, and calcium nodule formation. Expression of P2X7 receptors and c‐fos and c‐jun mRNA was determined with real‐time reverse transcription polymerase chain reaction and Western blotting. P2X7‐siRNA, apyrase, P2 receptor antagonists, and p38 MAPK inhibitors were used to evaluate the roles of ATP release, P2X7 receptors, and p38 MAPK signaling in shockwave‐induced osteogenic hMSCs differentiation. Shockwave treatment released significant amounts (∼7 μM) of ATP from hMSCs. Shockwaves and exogenous ATP induced c‐fos and c‐jun mRNA transcription, p38 MAPK activation, and hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7‐siRNA or selective antagonists, or blockade of p38 MAPK with SB203580 prevented osteogenic differentiation of hMSCs. Our findings indicate that shockwaves release cellular ATP that activates P2X7 receptors and downstream signaling events that caused osteogenic differentiation of hMSCs. We conclude that shockwave therapy promotes bone healing through P2X7 receptor signaling, which contributes to hMSC differentiation. S TEM  C ells   2013;31:1170–1180

Shockwaves Induce Osteogenic Differentiation of Human Mesenchymal Stem Cells Through ATP Release and Activation of P2X7 Receptors