Superoxide mediates shock wave induction of ERK-dependent osteogenic transcription factor (CBFA1) and mesenchymal cell differentiation toward osteoprogenitors

Extracorporeal shock wave (ESW) is an alternative non-invasive method for the promotion of bone growth and tendon repair. In an animal model, we have reported that ESW promoted bone marrow osteoprogenitor growth through transforming growth factor-β1 induction. We have further explored the mechanism for the ESW promotion of osteogenesis. Results showed that an optimal ESW treatment at 0.16 mJ/mm ² for 500 impulses rapidly induced a higher O ₂ ^- and ONOO ^- production associated with a decrease of nitric oxide level in 1 h, and induced a higher transforming growth factor-β1 production in 24 h, and a higher colony-forming units-osteoprogenitor formation in 12 days. The colony-forming units-osteoprogenitor colonies revealed positive staining of bone alkaline phosphatase and turned into bone nodules in 21 days. Early scavenging of O ₂ ^- but not Ca ²⁺, H ₂O ₂, or prostaglandin E ₂ suppressed osteoprogenitor cell growth and maturation. Scavenging of O ₂ ^- by superoxide dismutase raised the nitric oxide level back to the basal level and suppressed ESW-promoted osteoprogenitor cell growth, whereas inhibition of ONOO ^- by urate or NO by N-nitro-L-arginine methyl ester did not affect ESW promotion of osteogenesis, indicating that O ₂ ^- acted as an early signal for ESW-induced cell growth. Further studies demonstrated that ESW induced ERK activation, and blockage of O ₂ ^- production or inhibition of tyrosine kinase, but not protein kinase A and C inhibitors, suppressed ESW-induced ERK activation. In support that O ₂ ^- mediated the ESW-induced ERK activation and osteogenic differentiation, we further demonstrated that scavenging of O ₂ ^- by superoxide dismutase and inhibition of ERK activation by PD98059 decreased specific osteogenic transcription factor, core binding factor A1 activation, and decreased osteocalcin expression. Taken together, we showed that ESW-induced O ₂ ^- production followed by tyrosine kinase-mediated ERK activation and core binding factor A1 activation resulted in osteogenic cell growth and maturation. Thus, an appropriate modulation of redox reaction by ESW may have some positive effect on the bone regeneration.