Background Restoration of dysfunctional tissue using autologous methods such as platelet-rich plasma (PRP) has become an attractive field of research as of late. Experimental evidence suggests that there is a positive effect of platelets on regenerating tissue. Both due to the cost-effective and safety-orientated nature of PRP applications to deliver growth factors in injured skeletal muscle, many studies have investigated the effect of autologous PRP in regenerative medicine. However, the molecular mechanisms involved in platelet-mediated muscle regeneration are currently poorly understood. The aim of this study was to determine the effect of platelets on myoblast proliferation and differentiation in vitro and establish the role of platelets in skeletal myogenesis using murine muscle fibres ex vivo.
Methods Due to largely controversial evidence among different groups, we firstly studied technical aspects of platelet preparation dissecting the role of platelet concentrations, platelet agonists, storage conditions and other platelet preparation variabilities. Secondly, we analysed the effect of platelet releasate on proliferation and differentiation of C2C12 myoblasts into myotubes by means of cell proliferation assays, immunohistochemistry and gene expression. Most importantly, we expanded in vitro cell culture findings on single muscle fibres by establishing the effect of platelet releasate on murine skeletal muscle stem cells using protein expression profiles for key myogenic regulatory factors. We also sought to determine whether the observed findings were mediated, by key growth factors such as VEGF and PDGF, by employing VEGF- and PDGF-receptor pharmacological inhibition.
Results We discuss technical findings regarding the role of different experimental variables of platelet preparation on skeletal myogenesis, that may shed light onto the discrepancy of the published evidence. Findings for three different platelet agonists (i.e. PAR-1, collagen and thrombin), platelet concentrations and centrifugation speeds are compared. We provide evidence that platelet releasate increases myoblast and muscle stem cell proliferation in a dose-dependent manner, which is mitigated by VEGFR and PDGFR inhibition. In particular, VEGFR and PDGFR inhibition, independently, ablated MyoD expression on proliferating muscle stem cells, compromising their commitment to differentiation in muscle fibres. Application of platelet releasate affected differentiation of myoblasts into myotubes in a temporal manner.
Conclusion Taken together, these data provide supporting evidence on a positive effect of platelet releasate on myoblast proliferation, with a temporal effect on cell differentiation. Therefore, platelet releasate may have a powerful role in skeletal myogenesis, that is mediated by growth factors such as VEGF and PDGF. Platelet-based methodologies aiming to provide autologous biomaterials have implications for regenerative medicine and muscle healing.