Abstract
Platelet-Rich Plasma (PRP) is enriched in molecular messengers with restorative effects on altered tissue environments. Upon activation, platelets release a plethora of growth factors and cytokines, either in free form or encapsulated in exosomes, which have been proven to promote tissue repair and regeneration. Translational research on the potential of exosomes as a safe nanosystem for therapeutic cargo delivery requires standardizing exosome isolation methods along with their molecular and morphological characterization. With this aim, we isolated and characterized the exosomes released by human PRP platelets. Western blot analysis revealed that CaCl2-activated platelets (PLT-Exos-Ca2+) released more exosomes than non-activated ones (PLT-Exos). Moreover, PLT-Exos-Ca2+ exhibited a molecular signature that meets the most up-to-date biochemical criteria for platelet-derived exosomes and possessed morphological features typical of exosomes as assessed by transmission electron microscopy. Array analysis of 105 analytes including growth factors and cytokines showed that PLT-Exos-Ca2+ exhibited lower levels of most analytes compared to PLT-Exos, but relatively higher levels of those consistently validated as components of the protein cargo of platelet exosomes. In summary, the present study provides new insights into the molecular composition of human platelet-derived exosomes and validates a method for isolating highly pure platelet exosomes as a basis for future preclinical studies in regenerative medicine and drug delivery.
Highlights
This article is an open access articleFor more than a decade, intra-articular delivery of autologous Platelet-Rich Plasma (PRP) has emerged as a safe and promising biotechnological alternative for the treatment of different pathologies [1]
The PRP used in this study presents a concentration of platelets 2-fold higher than in blood, with no leukocytes or erythrocytes (Table 1)
The results revealed that PLT-Exos exhibited some degree of contamination with all the non-exosome cell components, whereas immunoreactivity was undetectable or barely above detection threshold for all the antigens analyzed in PLTExos-Ca2+, indicating that exosomes isolated from calcium-activated platelets were of high purity
Summary
This article is an open access articleFor more than a decade, intra-articular delivery of autologous Platelet-Rich Plasma (PRP) has emerged as a safe and promising biotechnological alternative for the treatment of different pathologies [1]. PRP is a plasma product from patients’ own blood, which aims to increase the number of platelets and the concentration of molecular mediators that exert therapeutic effects, while eliminating unwanted elements such as red blood cells [2]. The selective enrichment in growth factors and anti-inflammatory cytokines is considered to be responsible for the effects of PRP in improving clinical conditions in a variety of disease situations [3–6]. Exosomes isolated directly from PRP (PRP-derived exosomes, PRP-Exos), which are highly enriched in platelet-derived exosomes and include exosomes from other different cellular sources, or exosomes obtained from washed platelets (platelet-derived exosomes, PLT-Exos) have attracted increasing attention as potential mediators of the effects of PRP and platelet lysates in tissue regeneration [9]. There is evidence that PLT-Exos are rich in the molecular mediators responsible for the healing effect of PRP [10]. Homologous PRP-Exos have shown to promote proliferation and inhibit apoptosis of rabbit chondrocytes [13] and to promote the formation of vessel-like structures from cultured human umbilical vein endothelial cells while increasing their proliferation and migration rates [14]
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