The major functions of platelets are preventing acute blood loss and repairing vascular walls and adjacent tissues after injury. During wound healing, platelets are activated by contact with collagen, exposed to the bloodstream after endothelial injury. Platelets secrete stored intercellular mediators and cytokines from the cytoplasmic pool and release their α-granule content after aggregation. This secretion is intense in the first hour and platelets continue synthesizing more cytokines and growth factors from their mRNA reserves for at least another 7 days . More than 800 different proteins are secreted into the surrounding media [1, 2], having a paracrine effect on different cell types: myocytes , tendon cells [3–7], mesenchymal stem cells from different origins [8–11], chondrocytes [12–14], osteoblasts [3, 15, 16], fibroblasts [17–19] and endothelial cells . Cell proliferation, angiogenesis and cell migration are stimulated, resulting in tissue regeneration. There are also reports confirming that platelets secrete antimicrobial peptides, suggesting an antibiotic effect .
Other properties were already proven for platelets related to their anti-inflammatory and analgesic effects [22–24]. A clinical trial showed that platelet concentrates had an analgesic effect  and Asfaha and colleagues showed PAR4-mediated analgesic effects in vitro. El-Sharkawy and colleagues studied platelet secretions and their effect on macrophage cultures, concluding that platelet concentrates function as an anti-inflammatory agent, because of the high RANTES and LXA4 concentrations .
Platelet-derived products include platelet-rich plasma (PRP), which can be used with or without previous platelet activation. Such preparations have been used since the 1970s and they have been increasingly popular since the 1990s . Since then, different ways of preparing PRP have emerged: from conventional blood centrifugation to commercial systems; activated by adding collagen, calcium and/or thrombin, by glass contact or by freezing cycles; applied as platelet suspension or as a gel; and the methodology continues to broaden [29–31].
The application of PRP in different tissues has given promising results in different pathologies such as acute and chronic injuries of bone and cartilage. Kon and colleagues reported observation of 91 patients (115 knees) treated with PRP, which showed that PRP treatment is safe, reduces pain and improves knee function, especially in younger patients at 12 months . This was superior to hyaluronic acid viscosupplementation . Subsequent analysis at 24 months, however, showed a progressive loss of the improvement and opened up the question of possible repeated therapies . This example indicates the necessity for further studies even in series with an extensive number of patients and provided with controls. General opinion in recent reviews is that the majority of reported clinical studies do not have sufficient statistical power to give conclusive results. In view of multiple potential PRP applications in orthopedics, sports medicine and reparative surgery, comparative analyses of different clinical scenarios would be useful. These comparisons are not feasible, mainly because PRP is a biological product, prepared using different protocols, sometimes without even controlling whether platelets were effectively concentrated and purified or whether an early activation occurred, discarding all of the secreted growth factors within the platelet-poor plasma (PPP). Another issue that is not even mentioned in clinical reports is whether there is a correlation between the platelet concentration or the PRP volume applied per injured area or volume. Studies have already demonstrated that low platelet concentration is inefficient and that high concentrations have an inhibitory effect on cell growth, but results are still contradictory [34–36]. Although still not deeply characterized, the leukocyte content was also shown to be an important factor, increasing inflammation and reducing tissue regeneration in tendinopathies . Preparation procedures are also relevant, as shown by studies of the chondro-inductive and osteo-inductive potential of PRP, which is reported to be lost by thrombin activation  and retained after freeze–thaw activation . Consistent nomenclatures, standardized protocols to produce PRP as well as a full characterization of the final product are still missing and would highly improve the comparability of studies [40, 41].
Combinations of PRP and mesenchymal stem cells have been widely studied in vitro[8–11]. All of the authors concluded that PRP increased cell proliferation but divergences were found regarding the stem cell differentiation capacity, some concluding that PRP favored the osteogenic differentiation  and others demonstrating a chondrogenic compromise [10, 12]. These variations could be due to different PRP preparations, including or not the leukocyte fraction, which can modify its growth factor content. In both cases, PRP seemed to be a promising additive for stem cell transplantation for orthopedic applications, by increasing the number of transplanted stem cells and guiding their differentiation to a defined cell type.
The present study was proposed to establish an optimized and reproducible method for PRP preparation, and to characterize the content in growth factors and cytokines of the obtained fractions before and after platelet activation. Our final goal is to develop and characterize a PRP preparation for therapeutic purposes, focusing on high platelet yield, purity and recovery without growth factor secretion throughout sample manipulation.