Effects of platelet-rich plasma derived growth factors and TGF-beta antagonists on skeletel muscle cells proliferation and differentiation

Uvod Regeneracija skeletne mišice po poškodbi je omejena s tvorbo brazgotinskega tkiva, počasnim celjenjem in relativno visoko verjetnostjo ponovitve poškodbe. Terapija, ki temelji na pripravkih avtolognih trombocitnih preparatov (ang. platelet-rich plasmaPRP) je v zadnjem času postala izjemno popularna, predvsem v primeru poškodb sklepnih vezi in mišičnih tetiv. Za zdravljenje mišičnih poškodb se PRP še ne uporablja, poglavitni pomislek je, da med ostalim vsebuje tudi rastni faktor TGF-β, ki je pomemben dejavnik brazgotinjenja v skeletno-mišičnem tkivu. V raziskavi smo želeli prirediti učinek PRP-ja za uporabo na skeletnem mišičju. Učinek rastnega faktorja TGF-β smo zavirali z njegovim zaviralcem s ciljem izločitve potencialno neželenega učinka v procesu mišične regeneracije. Materiali in metode Zaradi pomanjkanja znanstvenih temeljev o terapiji z avtologno trombocitno plazmo smo zasnovali predklinično raziskavo, v kateri smo uporabili humano, CD56-pozitivno mioblastno celično linijo. Po dodajanju PRP-ja, dekorina (zaviralca TGF-β) in njune kombinacije v gojilni medij smo preučevali stopnjo proliferacije mioblastov, njihove metabolne aktivnosti, profil izražanja fibrotičnih citokinov in ekspresijo miogenih regulatornih faktorjev. Z uporabo slikovne pretočne citometrije smo analizirali razmerje med hibernirajočimi, aktiviranimi in diferencirajočimi mioblasti ter rezultate primerjali med posameznimi skupinami. Dodatno smo opravili še vizualno analizo izražanja dezmina z uporabo visoko ločljivega konfokalnega mikroskopa ter ugotavljali stopnjo formacije več jedrnih miotubulov. Rezultati Ugotovili smo, da pride v mioblastnih celičnih kulturah pod vplivom PRP-ja do značilnega znižanja izražanja fibrotičnih citokinov, povečanja stopnje proliferacije in viabilnosti celic. Obenem pride prav tako do povečane ekspresije miogenih regulatornih faktorjev, kar pripomore k pomembnemu pomiku v miogeni diferenciaciji mioblastov. V kombinaciji PRP-ja z dekorinom smo zaznali pomembne dodatne sinergistične učinke. Razprava Izsledki raziskave dokazujejo, da PRP ne samo potencialno znižuje brazgotinjenje, temveč tudi pripomore k učinkovitejši mišični regeneraciji, še posebej v kombinaciji z zaviralcem TGF-β.Background: Muscle reinjury is often in sports medicine and as such an important target of reinjury prevention. Growth factors from platelet-rich plasma (PRP) with proven effects in tendinous and ligamentous healing have not yet been studied in skeletal muscles mainly due to the concern that exogenous TGF-β application could lead to even greater fibrosis development in an injured muscle. Therefore, only some TGF-β antagonists like decorin have shown their positive role in muscle repair so far. In our study, we investigated the effects of PRP in combination with TGF-β antagonist decorin in skeletal muscle regeneration. Objective: This study proves so far unknown positive effects of platelet-rich plasma in muscle healing, especially when combined with a TGF-β antagonist. Methodology: A novel human myoblast cell culture, defined as CD56 (NCAM)+ developed in our laboratory, was used for evaluation of potential bioactivity of PRP and decorin. The influence on the cells mitochondrial activity, expression of TGF-β was studied in parallel with cell proliferation. Further we have studied the ability of the therapeutic agents to influence the differential cascade of dormant myoblasts towards fully differentiated myotubes by monitoring step wise activation of single nuclear factors like MyoD and Myogenin via multicolor flow cytometry. Results: Our results clearly showed that PrP and decorin treated myoblasts have a significant increase in the mitochondrial activity and in the cell proliferation rate as compared to non-treated control cells. At the same time lower expression of TGF-β and MSTN was evident in PrP treated myoblasts, although PRP itself contains some amount of TGF-β. Conclusion: PrP can be a highly potential therapeutic agent for skeletal muscle regeneration and repair, especially if in combination with a TGF-β antagonist. In such way not only better healing but also lower reinjury rate could potentially be achieved