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

Abstract

Shockwave fractures 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 sig naling 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.

Shock Wave Treatment Enhances Cell Proliferation and Improves Wound Healing by ATP Release-coupled Extracellular Signal-regulated Kinase (ERK) Activation

“…Mechanical stimulation has been shown to mediate the release of cellular ATP of various cells such as astrocytes, osteoblasts, chondrocytes, and epithelial cells (46 -49). Studies by Sun et al (23) and Yu et al (22) support our finding that ESWT at our chosen settings releases ATP without negatively affecting cell viability as they showed that subjection of human mesenchymal stem cells to Ͻ200 shock wave pulses at 0.18 mJ/mm 2 resulted in Ͼ95% viability directly or 24 h after ESWT.…”

“…We found that the released ATP activated P2 receptors and increased Erk1/2 and p38 MAPK signaling via mechanisms similar to those reported in osteoblasts, mesenchymal stem cells, and T cells (20,(22)(23)(24). In addition to Erk1/2 and p38 MAPK, the downstream signaling mechanisms triggered by shock wave-induced P2 receptor activation may also involve additional signaling intermediates such as Ras and hypoxiainducible factor 1a transactivation (20,23).…”

“…In addition to Erk1/2 and p38 MAPK, the downstream signaling mechanisms triggered by shock wave-induced P2 receptor activation may also involve additional signaling intermediates such as Ras and hypoxiainducible factor 1a transactivation (20,23). A study dealing with stretch-induced injury reported the activation of Erk1/2 to be dependent on the rate and amplitude of mechanical impact, involving released ATP and activated P2 receptors (46).…”

“…Recent evidence suggests that shock waves trigger adenosine triphosphate (ATP) release by activating purinergic signaling and p38 MAPK activation (22,23). The crucial role of purinergic signaling in the regulation of numerous physiological processes has only recently been appreciated (29,30).…”

Early Characterization of Blast-related Heterotopic Ossification in a Rat Model

“…Moreover, they report that these orthopaedic disease processes can be attenuated/modulated by targeting adenosine triphosphate (ATP) hydrolysis and SMAD1/5/8 phosphorylation at the burn site using apyrase [19]. Interestingly, it has been reported that focused extracorporeal shockwave therapy (ESWT; low-density shockwaves administered orders of magnitude below blast overpressure conditions used in the study) has been shown to induce osteogenic differentiation of marrow-derived mesenchymal stem cells through ATP release and downstream transcriptional signaling events resulting in activation of P2X7 receptors [28]. Consistent with the finding of Peterson et al, removal of ATP using apyrase inhibited ESWT-induced osteogenic differentiation.…”