Characterization of the molecular composition and in vitro regenerative capacity of platelet-based bioproducts and related subfractions.

The use and demand of platelet-based bioproducts in regenerative medicine is steadily increasing. However, it is very difficult to establish the real clinical benefits of these therapies, as the lack of characterization and detailed production methods of platelet-based bioproducts persists in the literature and precludes cross-study comparisons. We characterized the molecular composition and in vitro regenerative capacity of platelet-rich plasma (PRP) produced in a closed-system. Furthermore, we performed a parallel characterization on different PRP subfractions (plasma and plasma-free platelet lysate), identifying that the fractions containing platelet-derived cargo exert the most potent regenerative capacity. This observation led us to develop a method to obtain a platelet secretome highly enriched in growth factors, free of plasma and cellular components (PCT/IB2022/057936), with the aim of establishing a superior bioproduct. The molecular characterization of secretomes revealed agonist-dependent differences, which correlates with beneficial grades of regenerative capacity. Importantly, secretomes showed general superiority to PRP in vitro. We discuss the variables influencing the bioproduct quality (inter-donor variation, platelet source and processing methods). Finally, we propose that the characteristics of secretomes circumvents certain limitations of PRP (autologous vs allogeneic), and envision that optimizing post-processing protocols (nanoencapsulation, lyophilization), would allow their clinical application even beyond regenerative medicine. STATEMENT OF SIGNIFICANCE: The use and demand of platelet-based bioproducts in regenerative medicine is steadily increasing. However, it is very difficult to establish the real clinical benefits of these therapies, or to improve/personalize them, as the lack of characterization of the bioproducts and their production methods is a constant in the literature, reason that precludes cross-study comparisons. In the present manuscript, we provide a comprehensive molecular and functional characterization of platelet-based bioproducts and subfractions, including platelet rich plasma, plasma fractions and platelet secretomes produced with a methodology developed by our group. Our results show that the molecular composition of each fraction correlates with its regenerative capacity in vitro. Thus, a rigorous characterization of platelet-derived bioproducts will potentially allow universal use, customizing and new applications.

Platelet-derived bio-products: Classification update, applications, concerns and new perspectives.

Platelet derived bio-products in the form of platelet rich plasma, plasma rich in growth factors, or plasma-free platelet releasates, are being studied worldwide with the aim of proving their efficacy in tissue regeneration within many different clinical areas, such as traumatology, maxillofacial surgery, ophthalmology, dermatology and otorhinolaryngology, amongst others. The current lack of consensus in the preparation method and application form, or in the quality assessment of each bio-product, precludes adequate interpretation of the relevance of reported clinical outcomes, and, while many in clinicians are very positive about them, many are sceptic. Relevant aspects of these products are considered to propose a classification nomenclature which would aid a comprehensive comparison of clinical outcomes of bio-products of the same characteristics. Finally, the uses of platelet-derived bio-products in in vitro culture (for cell therapy purposes) as a substitute of animal-origin sera, and other future perspectives of applications of platelet-derived bio-products are discussed.

Optimising platelet secretomes to deliver robust tissue-specific regeneration.

Promoting cell proliferation is the cornerstone of most tissue regeneration therapies. As platelet-based applications promote cell division and can be customised for tissue-specific 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.

Implementation of a closed platelet-rich-plasma preparation method using the local blood bank infrastructure at the Principality of Asturias (Spain): Back to basic methodology and a demographics perspective after 1 year.

Platelet-rich plasma (PRP) is used with increasing demand as autologous adjuvant therapy in many areas of regenerative medicine, thanks to the platelet rich content of growth factors and bio-active molecules. However, to date there is a lack of consensus on PRP preparation methods, on processing and application forms, on clinical application guidelines and on knowledge-based composition at the cellular and molecular level, making difficult the assessment of clinical results from different groups in different clinical areas. Here we describe the implementation of PRP production on a closed-system using the infrastructure of a certified blood bank, detailing methodology, and validation and production results 1 year after its implementation. Our methodology provides a reproducible, safe, practical and yet affordable PRP bio-product that will allow further studies to better define PRP applications in regenerative medicine and personalized therapeutic regimes.

Platelet releasate promotes skeletal myogenesis by increasing muscle stem cell commitment to differentiation and accelerates muscle regeneration following acute injury.

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.