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首页> 外文期刊>Orthopaedic Journal of Sports Medicine >The Use of Platelet-Rich and Platelet-Poor Plasma to Enhance Differentiation of Skeletal Myoblasts
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The Use of Platelet-Rich and Platelet-Poor Plasma to Enhance Differentiation of Skeletal Myoblasts

机译：使用富含血小板和贫血小板的血浆增强骨骼肌成肌细胞的分化

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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.
机译：目的：富血小板血浆（PRP）已被用于增强肌肉骨骼损伤后的组织修复和再生。但是，越来越多的临床证据表明PRP和相关血液制品未显示出一致的临床效果。这项研究的目的是比较含有非中性粒细胞的PRP（LP-PRP），修饰的LP-PRP（Mod LP-PRP）（其中TGF-β1和Myostatin（MSTN）耗尽）和血小板贫血（PPP）的影响）对人骨骼肌成肌细胞（HSMM）的分化。我们的假设是，LP-PRP会导致成肌细胞增殖，而不是分化，而PRP制剂的修饰会增加成肌细胞的分化，这是骨骼肌再生所必需的。方法：从8位健康人类供体中同时采集血液，以创建LP-PRP，Mod-LP-PRP，PPP和第二次自旋（ss）PRP和Mod-PRP组。使用连接至无菌珠的抗体去除TGF-β1和MSTN来创建Mod-PRP。然后将生物制剂分别添加到人体骨骼肌成肌细胞（HSMM）中，并进行四天的分析。对成肌细胞增殖和分化途径的诱导分析包括蛋白质印迹和RT-PCR，以及共聚焦显微镜以评估多核肌小管的形成。结果：与PPP相比，LP-PRP处理导致成肌细胞增殖增加（1.01 x 106 vs 5.1 x 105细胞），但与阴性相比，没有证据显示通过成肌管形成或通过诱导肌球蛋白重链（MHC）RNA分化成肌肉细胞对照（0.1倍变化; p> 0.05）。 TGF-β1和MSTN已成功耗尽Mod-PRP，但这种修饰几乎没有改善成肌细胞的分化（MHC RNA与对照相比变化了0.2倍; p> 0.05）。与阴性对照相比，将PPP应用于培养物诱导的成肌细胞分化，包括可见的多核肌小管形成和MHC诱导（9.8倍变化; p <0.05）。第二次离心旋转（去除血小板）导致PRP和Mod-PRP制剂中的成肌细胞分化显着增加，与PPP和2％马血清阳性对照的水平相似（8.0x vs 6.7x vs 9.8x vs 6.0x分别增加MHC RNA的3倍;与LP-PRP，Mod-LP-PRP和阴性对照相比，所有p <0.05）。 Western印迹和RT-PCR分析证实MSTN和TGF-β1在包括Mod-LP-PRP在内的所有第二次旋转的组中都进一步耗尽。结论：PPP和PRP制剂经过第二次旋转以去除血小板，导致诱导成肌细胞进入肌肉分化途径，而未修饰的PRP导致诱导进入增殖途径。这些结果表明，传统配方的PRP不应用于诱导肌肉再生。实验室证据表明，应进行第二次旋转以去除血小板的贫血小板血浆（PPP）或LP-PRP可以刺激成肌细胞分化，这是骨骼肌再生所必需的。需要进行临床研究以确认这些生物制剂对肌肉再生的作用。

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