Objectives: Platelet-rich plasma (PRP) has been has been used to augment tissue repair and regeneration after musculoskeletal injury. However, there is increasing clinical evidence that PRP, and related blood products, do not show a consistent clinical effect. The purpose of this study is to compare the effects of non-neutrophil containing PRP (LP-PRP), modified LP-PRP (Mod LP-PRP) where TGF-β1 and myostatin (MSTN) were depleted, and platelet poor plasma (PPP) on human skeletal muscle myoblast (HSMM) differentiation. Our hypothesis was that LP-PRP would lead to myoblast proliferation, not differentiation, while modifications of PRP preparations will increase myoblast differentiation, which is necessary for skeletal muscle regeneration.
Methods: Blood was simultaneously processed from eight healthy human donors to create LP-PRP, Mod-LP-PRP, PPP and second spin (ss) PRP and Mod-PRP groups. Mod-PRP was created using antibodies attached to sterile beads to remove TGF- β1 and MSTN. The biologics were then individually added to human skeletal muscle myoblasts (HSMM) and were analyzed over four days. Analysis for induction into myoblast proliferation and differentiation pathways included Western blot and RT-PCR, as well as confocal microscopy to assess for polynucleated myotubule formation.
Results:LP-PRP treatment lead to increased myoblast proliferation compared to PPP (1.01 x 106 vs 5.1 x 105 cells), but showed no evidence differentiation into muscle cells either by myotubule formation or via inducing myosin heavy chain (MHC) RNA compared to negative controls (0.1x fold change; p>0.05). TGF- β1 and MSTN were successfully depleted in Mod-PRP, but this modification did little to improve myoblast differentiation (0.2x fold change MHC RNA vs control; p>0.05). Application of PPP to cultures induced myoblast differentiation that included visible multinucleated myotubule formation and MHC induction compared to negative controls (9.8x fold change; p<0.05). A second centrifugal spin (removes platelets) lead to a significant increase in myoblast differentiation in PRP and Mod-PRP preparations, similar to the level of PPP and the 2% horse serum positive control (8.0x vs 6.7x vs 9.8x vs 6.0x fold increase in MHC RNA, respectively; all p<0.05 compared to LP-PRP, Mod-LP-PRP and negative controls). Western blot and RT-PCR analyses confirmed that MSTN and TGF-β1 were further depleted in all groups, including Mod-LP-PRP, that were subjected to a second spin.
Conclusion: PPP, and PRP preparations subjected to a second spin to remove platelets, lead to induction of myoblast cells into the muscle differentiation pathway, while unmodified PRP lead to induction into the proliferation pathway. These results indicate that traditionally formulated PRP should not be used to induce muscle regeneration. Laboratory evidence suggests that platelet poor plasma (PPP) or LP-PRP subjected to a second spin to remove platelets should be used to stimulate myoblast differentiation, which is necessary for skeletal muscle regeneration. Clinical studies will be required to confirm the effect of these biologics on muscle regeneration.