Summary

PPP and non-neutrophil containing PRP preparations subjected to a second-spin (ss) to remove the platelets lead to induction of myoblast cells into the muscle differentiation pathway, while unmodified PRP lead to myoblast proliferation.

Abstract

I. Introduction
Autologous platelet-rich plasma, PRP, is broadly used to promote healing of soft tissues. However, the lack of consistent clinical effect of PRP suggests tailoring the current formulation may be necessary, depending on the type of tissue, to achieve a reliable clinical effect. We created modified indication-specific PRP and altered its composition by subtracting some PRP components. Myostatin (MSTN) and transforming growth factor beta (TGFß1) are known to be detrimental for muscle regeneration, so their depletion may promote myoblast differentiation.
The purpose of this study is to compare the effects of non-neutrophil-containing PRP, of modified PRP (Mod PRP= TGF-ß1 and MSTN were immuno-depleted), and of platelet poor plasma (PPP) on human skeletal muscle myoblast (HSMM) differentiation.

II. Methods

Blood was obtained from seven healthy donors. A complete blood count was performed, and five plasma formulations were created using PRP kit: 1) PRP, 2) Mod PRP, 3) PPP, 4) PRP subjected to a second-stage spin to remove platelets (PRP(ss)), and, similarly, 5) Mod PRP(ss).
Myoblast cultures were treated with 2% blood plasma formulations in myogenic media, in triplicate per condition. The positive control (PC) media was 2% horse serum, and the negative control (NC) media was 10% FBS. Cells were counted and viability was determined. Cells were collected after four days for Western blot (WB) and RT-PCR analyses, and six days for myotubule formation/muscle cell fusion.
Plasma formulations and cell extracts were analyzed for TGF-ß1, MSTN, and myosin heavy chain (MYH2) using SDS PAGE and Western blot analysis.
Total RNA was collected and expression levels of MYH2, paired box gene 7 (PAX7), and MSTN were determined via RT-PCR. Immunofluorescent staining was also performed using MYH2 antibody.


III. Results
Cells in the positive control group had high levels of MYH2 in immunofluorescent staining and formed well-organized polynucleated myotubes, while the negative controls exhibited none. PPP and additionally spun fractions (PRPss) induced a significant increase in MYH2 staining, cell differentiation, and myotubule formation, but the standard PRP formulation showed no significant effects of myocyte differentiation.
Western blot analysis of myoblast cell extracts confirmed no increase of MYH in the PRP group, but increased expression of MYH in the PPP, Mod PRP and spun groups. RT-PCR also showed no increase in MYH in the PRP group (1.06 ± 0.65) compared to the negative control. PPP and spun groups (PRPss) had a significant increase in MYH (12.4 ± 5.8; 9.7 ± 6.1) compared to PRP (p=0.03). MSTN and PAX7 showed no significant changes. Immunoblotting exhibited upregulation of MYH expression in the PPP group with an increase in concentration from 1 to 4%.

IV. Discussion and Conclusion
PPP and non-neutrophil containing PRP preparations subjected to a second-spin (ss) to remove the platelets lead to induction of myoblast cells into the muscle differentiation pathway, while unmodified PRP lead to myoblast proliferation. Clinical studies are required to confirm the effect of these biologics on muscle regeneration.