Platelet-based applications such as platelet-rich plasma (PRP) and platelet releasate have gained unprecedented attention in regenerative medicine across a variety of tissues as of late. The rationale behind utilizing PRP originates in the delivery of key cytokines and growth factors from α-granules to the targeted area, which in turn act as cell cycle regulators and promote the healing process across a variety of tissues. The aim of the present review is to assimilate current experimental evidence on the role of platelets as biomaterials in tissue regeneration, particularly in skeletal muscle, by integrating findings from human, animal and cell studies. This review is composed of 3 parts: firstly, we review key aspects of platelet biology that precede the preparation and use of platelet-related applications for tissue regeneration. Secondly, we critically discuss relevant evidence on platelet-mediated regeneration in skeletal muscle focusing on findings from (i) clinical trials, (ii) experimental animal studies and (iii) cell culture studies; and thirdly, we discuss the application of platelets in the regeneration of several other tissues including tendon, bone, liver, vessels and nerve. Finally, we review key technical variations in platelet preparation that may account for the large discrepancy in outcomes from different studies. This review provides an up-to-date reference tool for biomedical and clinical scientists involved in platelet-mediated tissue regenerative applications.

Aim:The use of platelets as biomaterials has gained intense research interest.However, the mechanisms regarding platelet-mediated skeletal myogenesis remain to be established. The aim of this study was to determine the role of platelet releasate in skeletal myogenesis and muscle stem cell fate in vitro and ex vivo respectively. Methods: We analysed the effect of platelet releasate on proliferation and differentiation of C2C12 myoblasts by means of cell proliferation assays, immunohistochemistry, gene expression and cell bioenergetics. We expanded in vitro findings on single muscle fibres by determining the effect of platelet releasate on murine skeletal muscle stem cells using protein expression profiles for key myogenic regulatory factors. Results: TRAP6 and collagen used for releasate preparation had a more pronounced effect on myoblast proliferation vs thrombin and sonicated platelets (P < 0.05). In addition, platelet concentration positively correlated with myoblast proliferation. Platelet releasate increased myoblast and muscle stem cell proliferation in a dose-dependent manner, which was mitigated by VEGFR and PDGFR inhibition. Inhibition of VEGFR and PDGFR ablated MyoD expression on proliferating muscle stem cells, compromising their commitment to differentiation in muscle fibres (P < 0.001). Platelet releasate was detrimental to myoblast fusion and affected differentiation of myoblasts in a temporal manner. Most importantly, we show that platelet releasate promotes skeletal myogenesis through the PDGF/ VEGF-Cyclin D1-MyoD-Scrib-Myogenin axis and accelerates skeletal muscle regeneration after acute injury. Conclusion: This study provides novel mechanistic insights on the role of platelet releasate in skeletal myogenesis and set the physiological basis for exploiting platelets as biomaterials in regenerative medicine. K E Y W O R D Sgrowth factors, platelet releasate, platelet-rich plasma, regeneration, satellite cells, skeletal muscle

Aim The prevalence of obesity is a major risk factor for cardiovascular and metabolic diseases including impaired skeletal muscle regeneration. Since skeletal muscle regenerative capacity is regulated by satellite cells, we aimed to investigate whether a high‐fat diet impairs satellite cell function and whether this is linked to fatty acid uptake via CD36. We also aimed to determine whether loss of CD36 impacts on muscle redox homeostasis and skeletal muscle regenerative capacity. Methods We studied the impact of a high‐fat diet and CD36 deficiency on murine skeletal muscle morphology, redox homeostasis, satellite cell function, bioenergetics and lipid accumulation in the liver. We also determined the effect of CD36 deficiency on skeletal muscle regeneration. Results High‐fat diet increased body weight, intramuscular lipid accumulation and oxidative stress in wild‐type mice that were significantly mitigated in CD36‐deficient mice. High‐fat diet and CD36 deficiency independently attenuated satellite cell function on single fibres and myogenic capacity on primary satellite cells. CD36 deficiency resulted in delayed skeletal muscle regeneration following acute injury with cardiotoxin. CD36‐deficient and wild‐type primary satellite cells had distinct bioenergetic profiles in response to palmitate. High‐fat diet induced hepatic steatosis in both genotypes that was more pronounced in the CD36‐deficient mice. Conclusion This study demonstrates that CD36 deficiency protects against diet‐induced obesity, intramuscular lipid deposition and oxidative stress but results in impaired muscle satellite cell function, delayed muscle regeneration and hepatic steatosis. CD36 is a key mediator of fatty acid uptake in skeletal muscle, linking obesity with satellite cell function and muscle regeneration.

Promoting cell proliferation is the cornerstone of most tissue regeneration therapies.As platelet-based applications promote cell division and can be customised for tissuespecific efficacy, this makes them strong candidates for developing novel regenerative therapies. Therefore, the aim of this study was to determine if platelet releasate could be optimised to promote cellular proliferation and differentiation of specific tissues. Growth factors in platelet releasate were profiled for physiological and supraphysiological platelet concentrations. We analysed the effect of physiological and supraphysiological releasate on C2C12 skeletal myoblasts, H9C2 rat cardiomyocytes, human dermal fibroblasts (HDF), HaCaT keratinocytes, and chondrocytes. Cellular proliferation and differentiation were assessed through proliferation assays, mRNA, and protein expression. We show that supraphysiological releasate is not simply a concentrated version of physiological releasate. Physiological releasate promoted C2C12, HDF, and chondrocyte proliferation with no effect on H9C2 or HaCaT cells. Supraphysiological releasate induced stronger proliferation in C2C12 and HDF cells compared with physiological releasate. Importantly, supraphysiological releasate induced proliferation of H9C2 cells. The proliferative effects of skeletal and cardiac muscle cells were in part driven by vascular endothelial growth factor alpha. Furthermore, supraphysiological releasate induced differentiation of H9C2 and C2C12, HDF, and keratinocytes. This study provides insights into the ability of releasate to promote muscle, heart, skin, and cartilage cell proliferation and differentiation and highlights the importance of optimising releasate composition for tissue-specific regeneration.

Platelet-based applications are currently used for the delivery of growth factors and other biomolecules as autologous biomaterials in regenerative medicine and cosmetic therapies. Many studies have revealed that platelet-based applications such as platelet-rich plasma and platelet releasate exhibit beneficial biological effects after a sports injury or trauma when administered locally by intramuscular injections. At present, treatment of the public, patients and athletes with platelet-based applications is permitted and regulated by the Food and Drug Administration and the World Anti-Doping Agency. Since 2011 the use of autologous platelet-rich plasma is permitted in competitive sports by the World Anti-Doping Agency, due to the lack of evidence in performance enhancement and anabolic effects. However, accumulating research has recently shed light on the role of platelet-derived growth factors in wound healing, skeletal myogenesis, muscle stem cell function and tissue regeneration. Although any ergogenic potential of platelet-rich plasma and platelet releasate on intact skeletal muscle and human sports performance remain to be established, novel evidence suggests that platelet-derived growth factors can modulate muscle, tendon, ligament, protein synthesis/degradation, vascularization, energy utilization and regenerative capacity in various experimental settings. Since platelet-based applications are currently not prohibited, they constitute a tool for potential abuse and doping in sports. The aim of this review is to critically discuss and assimilate current insights and biological evidence that set the ground for exploitation and misuse in competitive sports, and develop strategies to combat these activities.

New Findings What is the central question of this study?What is the impact of obesity‐independent hyperlipidaemia on skeletal muscle stem cell function of ApoE‐deficient (ApoE–/–) mice? What is the main finding and its importance?Compromised muscle stem cell function accounts for the impaired muscle regeneration in hyperlipidaemic ApoE–/– mice. Importantly, impaired muscle regeneration is normalised by administration of platelet releasate. Abstract Muscle satellite cells are important stem cells for skeletal muscle regeneration and repair after injury. ApoE‐deficient mice, an established mouse model of hyperlipidaemia and atherosclerosis, show evidence of oxidative stress‐induced lesions and fat infiltration in skeletal muscle followed by impaired repair after injury. However, the mechanisms underpinning attenuated muscle regeneration remain to be fully defined. Key to addressing the latter is to understand the properties of muscle stem cells from ApoE‐deficient mice and their myogenic potential. Muscle stem cells from ApoE‐deficient mice were cultured both ex vivo (on single fibres) and in vitro (primary myoblasts) and their myogenic capacity was determined. Skeletal muscle regeneration was studied on days 5 and 10 after cardiotoxin injury. ApoE‐deficient muscle stem cells showed delayed activation and differentiation on single muscle fibres ex vivo. Impaired proliferation and differentiation profiles were also evident on isolated primary muscle stem cells in culture. ApoE‐deficient mice displayed impaired skeletal muscle regeneration after acute injury in vivo. Administration of platelet releasate in ApoE‐deficient mice reversed the deficits of muscle regeneration after acute injury to wild‐type levels. These findings indicate that muscle stem cell myogenic potential is perturbed in skeletal muscle of a mouse model of hyperlipidaemia. We propose that platelet releasate could be a therapeutic intervention for conditions with associated myopathy such as peripheral arterial disease.

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