title: The Use of Platelet-Rich and Platelet-Poor Plasma to Enhance Differentiation of Skeletal Myoblasts: Implications for the Use of Autologous Blood Products for Muscle Regeneration.
abstract: BACKGROUND
  Platelet-rich plasma (PRP) has been used to augment tissue repair and regeneration after musculoskeletal injury. However, there is increasing clinical evidence that PRP does not show a consistent clinical effect. Purpose/Hypothesis: This study aimed to compare the effects of the following non-neutrophil-containing (leukocyte-poor) plasma fractions on human skeletal muscle myoblast (HSMM) differentiation: (1) PRP, (2) modified PRP (Mod-PRP), in which transforming growth factor β1 (TGF-β1) and myostatin (MSTN) were depleted, and (3) platelet-poor plasma (PPP). The hypothesis was that leukocyte-poor PRP would lead to myoblast proliferation (not differentiation), whereas certain modifications of PRP preparations would increase myoblast differentiation, which is necessary for skeletal muscle regeneration.
  
  
  METHODS
  Controlled laboratory study.
  
  
  METHODS
  Blood from 7 human donors was individually processed to simultaneously create leukocyte-poor fractions: PRP, Mod-PRP, PPP, and secondarily spun PRP and Mod-PRP (PRPssand Mod-PRPss, respectively). Mod-PRP was produced by removing TGF-β1 and MSTN from PRP using antibodies attached to sterile beads, while a second-stage centrifugal spin of PRP was performed to remove platelets. The biologics were individually added to cell culture groups. Analysis for induction into myoblast differentiation pathways included Western blot analysis, reverse-transcription polymerase chain reaction, and immunohistochemistry, as well as confocal microscopy to assess polynucleated myotubule formation.
  
  
  RESULTS
  HSMMs cultured with PRP showed an increase in proliferation but no evidence of differentiation. Western blot analysis confirmed that MSTN and TGF-β1 could be decreased in Mod-PRP using antibody-coated beads, but this modification mildly improved myoblast differentiation. However, cell culture with PPP, PRPss, and Mod-PRPssled to a decreased proliferation rate but a significant induction of myoblast differentiation verified by increased multinucleated myotubule formation and myosin heavy chain expression (mean 8-fold change in mRNA level; P < .05), which was comparable with 2% horse serum, the positive control.
  
  
  CONCLUSIONS
  PPP and leukocyte-poor PRP preparations subjected to a second spin to remove the platelets led to induction of myoblast cells into the muscle differentiation pathway, whereas unmodified leukocyte-poor PRP led to myoblast proliferation.
  
  
  CONCLUSIONS
  These results indicate that traditionally formulated PRP may not be appropriate to induce muscle regeneration. Laboratory evidence suggests that PPP or non-neutrophil-containing PRPss, subjected to an additional 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.
authors.affiliation: Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Olga
authors.initials: O
authors.lastname: Miroshnychenko
authors.affiliation: Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Wen-Teh
authors.initials: WT
authors.lastname: Chang
authors.affiliation: Sports Medicine Center, Stanford University Medical Center, Redwood City, California, USA.
authors.forename: Jason L
authors.initials: JL
authors.lastname: Dragoo
mesh_terms: D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D006801:Humans; D007962:Leukocytes; D032448:Myoblasts, Skeletal / Q000166:cytology*; D055435:Myostatin; D010949:Plasma / Q000502:physiology*; D053657:Platelet-Rich Plasma / Q000502:physiology*; D012038:Regeneration; D053773:Transforming Growth Factor beta1; D014945:Wound Healing
chemical_list: C525911:MSTN protein, human; D055435:Myostatin; D053773:Transforming Growth Factor beta1
keywords: PPP; PRP; autologous blood product; growth factor; muscle; myoblast; myoblast differentiation; myoblast proliferation; myostatin; platelet-poor plasma; platelet-rich plasma; transforming growth factor beta