Platelet‐rich plasma‐derived extracellular vesicles: A superior alternative in regenerative medicine?

Abstract Platelet‐rich plasma (PRP), due to its promising therapeutic properties, has been used in regenerative medicine for more than 30 years and numerous encouraging outcomes have been obtained. Currently, by benefiting from new insights into PRP mechanisms and the excellent performance of extracellular vesicles (EVs) in the field of tissue repair and regeneration, studies have found that a large number of EVs released from activated platelets also participate in the regulation of tissue repair. A growing number of preclinical studies are exploring the functions of PRP‐derived EVs (PRP‐EVs), especially in tissue regeneration. Here, we summarize the latest progress in PRP‐EVs as a superior alternative cell‐free therapeutic strategy in regenerative medicine, clarify their underlying molecular mechanisms, and discuss the advantages and limitations of the upcoming clinical applications. This review highlights the potential of PRP‐EVs to replace the application of PRP or even become a superior alternative in regenerative medicine.


| INTRODUC TI ON
As the human population continues to age and the incidence of degenerative and traumatic diseases continues to increase, developing therapeutic strategies to repair and regenerate damaged tissues and restore their normal functions is the most important goal of regenerative medicine. 1 To date, the strategies of regenerative medicine have focused on using materials science and engineering techniques to develop novel biomaterials that can regulate cellular functions and activate their innate regenerative potential. [2][3][4] However, in recent decades, the potential of platelet-rich plasma (PRP) therapies has attracted considerable interest in regenerative medicine. Many high-quality systematic reviews and meta-analyses have summarized the encouraging results of PRP therapies in a wide range of clinical fields, including orthopaedic surgery, 5 plastic surgery, 6 dermatology, 7 trichology, 8,9 cardiac surgery, 10 maxillofacial surgery, 11 pain management, 12 spinal disorders, 13 sports medicine 14 and others. 15,16 Emerging clinical evidence suggests that PRP therapies are promising, but there remain some disadvantages and limitations that need to be considered.
In the past couple of decades, the discovery of extracellular vesicles (EVs) has been one of the most revolutionary contributions to cell biology. 17 The ability of EVs to transport proteins, nucleic acids and lipids to target specific tissues and maintain the stability of therapeutic cargo makes EVs interesting as part of new strategies for the treatment of various diseases. 18 Similarly, EV-based subcellular therapies are expected to pave the way for the clinical application of regenerative medicine by overcoming the challenges of cell-based therapies. However, most recent preclinical studies in regenerative medicine focus on mesenchymal stem cells (MSCs) as a source of EVs 19,20 ; relatively little attention is paid to exploring EVs derived from other cells or tissues.
Platelet-derived EVs, as the largest part of blood EVs, have a long history of discovery. 21,22 Traditionally, due to a lack of in-depth understanding of the functions of EVs, platelet-derived EVs have been described as procoagulant materials released from activated platelets. 23 Over time, this definition has been gradually accepted as they are multipurpose. Due to the great potential of PRP to promote tissue repair and regeneration, PRP-derived EVs (PRP-EVs) have also attracted great interest in regenerative medicine. However, as a novel promising therapy in regenerative medicine, the possible mechanisms behind PRP-EVs and the advantages and limitations of their application still need to be further understood. (Since PRP extraction is an indispensable step in the process of extracting plateletderived EVs and in order to compare the role of PRP in regenerative medicine, this review uses the abbreviation "PRP-EVs" to refer to both platelet-derived EVs and PRP-derived EVs.) In this review, we systematically summarize the latest reported progress of PRP-EVs as a superior alternative cell-free therapeutic strategy in regenerative medicine, clarifying their underlying molecular mechanisms and discussing the advantages and limitations of the upcoming clinical applications.

| DEFINITION , R ATIONALE S AND LIMITATI ON S OF PRP IN REG ENER ATIVE MEDICINE
PRP is a kind of blood-derived product that is produced via centrifugation or the apheresis process for the platelet enrichment of plasma from autologous or allogenic blood. It is characterized by a higher proportion of platelets than that of normal blood.
First, once released from platelets, many PRP-derived biomolecules fail to be protected from the phospholipid membrane, which may be damaged by lytic enzymes from the extracellular environment and lose their biological activity quickly. Additionally, recent systematic reviews have revealed that the absence of unified standards for PRP preparations, classifications and clinical applications makes it more difficult to evaluate the biological functions and clinical effectiveness of PRP between different studies. 15,16 Failures in product standardization limit the scope of PRP clinical applications and the development of commercial PRP-related products. Moreover, as one of the most comprehensively investigated blood-derived products, the components of PRP are highly influenced by individual characteristics, including intrinsic, versatile and adaptive characteristics. The individual states of the donor, including gender, age and health status, may be present in the PRP products. The individualized features of PRP make it difficult to compare PRP therapy outcomes and may result in many studies being unable to be validated repeatedly. 15,36 Similarly, the components of PRP are as complex as those of blood and are likely more complex than many traditional pharmaceutical drugs. 15 It is extremely difficult to explain the individual functions of different components. Thus, although no serious complications have been reported, some possible teratogenic and carcinogenic risks still need to be a focus of attention during PRP therapy. More importantly, it is well-known that platelets lack an integral cellular structure, but cases of slight immunological rejection between allogenic individuals nonetheless exist, which may limit the clinical application of PRP to some extent.
However, current strategies face difficulties in breaking these bottlenecks. Therefore, PRP-derived products, such as PRP-EVs or PRP hydrogel, are expected to replace PRP in the future as more efficient and safer clinical candidates in the field of tissue repair and regeneration.

| E X TR ACELLUL AR VE S I CLE S: A NE W PAR ADIG M FOR SUBCELLUL AR THER APY
Due to the excellent proliferation and differentiation potentials, the role of progenitor/stem cells is becoming increasingly prominent and of central importance in regenerative medicine. 37,38 In recent decades, progenitor/stem cell-based regenerative medicine strategies have developed rapidly and with many encouraging results. While promising, there have been several challenges in transitioning this strategy from bench to bedside, including immune compatibility, tumourigenicity and transmission of infections. 39,40 More importantly, diverging from early studies suggesting that the therapeutic effects of progenitor/stem cells are derived from their engraftment and differentiation at damaged tissue sites, recent studies have demonstrated that the tissue repair and regeneration by progenitor/ stem cells may be driven by the paracrine activity of their secreted factors, including EVs and soluble factors. [41][42][43] In the past decade, EV-based subcellular therapies have emerged as more promising strategies to overcome these challenges associated with progenitor/ stem cell-based therapies for tissue and organ regeneration. 20 EVs are natural nano-sized membrane vesicles encapsulated by phospholipid bilayers, which can be secreted by various types of cells in normal or stress conditions. 44,45 According to the differences in their triggering mechanisms and biophysiological properties, EVs can be subdivided into three major types: exosomes (30-150 nm in diameter), microvesicles (50 nm-1 μm in diameter) and apoptotic bodies (100 nm-5 μm in diameter). 17,46,47 In 1976, EVs were first found in the secretomes derived from platelets and defined as "platelet dust", which are involved in bone mineralization. 48 In the early 1980s, EVs were regarded to act as "garbage bags" with the major function of removing cellular waste. 49 However, in the past decade, EVs have been thought of as important mediators for intercellular communication that are linked to both physiological and pathological functions. Owing to their natural properties as excellent vectors for biological messengers and trophic factors, as well as their ability to surmount biological barriers, EVs are increasingly being studied as promising therapeutic agents. 45 Studies are exploring EVs for potential cell-free biotherapies for regenerative medicine and as delivery vehicles for therapeutic agents to treat cancer and inflammatory and immune diseases. 45

| HIS TORIC AL BACKG ROUND, B I OG ENE S IS , ISOL ATI ON AND FE ATURE S OF PRP-E VS
As mentioned above, due to its convenient, safe and efficient properties, PRP is widely used in various clinical fields to promote tissue repair and regeneration. However, the mechanisms of PRP in regenerative medicine are not yet completely understood. Previous prevailing views suggested that the powerful repair ability of PRP is derived mainly from the abundant amounts of secreted growth factors. However, recent studies have revealed that in addition to growth factors, a large number of EVs are released after PRP activation to participate in the regulation of tissue repair. [64][65][66] PRP-EVs are described as a kind of subcellular vesicle released from platelets under conditions of activation, shear stress, apoptosis and injury ( Figure 1). 21 In 1967, using electron microscopic techniques, Wolf first observed these shed membrane fragments from activated platelets and described them as "platelet dust". 48 In 1972, Warren et al. illustrated this release process in more detail. 67 Early studies believed that these membrane fragments shared many functional features with platelets. For instance, Sinauridze et al. demonstrated that the surfaces of these membrane fragments have a 50-to 100-fold higher specific procoagulant activity than that of activated platelets. 68 However, due to the lack of knowledge of EVs, in-depth studies regarding their features and functions are rare. Recently, with the increased understanding of EVs, PRP-EVs have attracted increasing attention, not only because of their excellent procoagulant activity but also because of their great potential to promote tissue repair and regeneration. 69 It is well known that EVs are present in a wide range of body fluids including blood, urine, saliva, CSF, amniotic fluid, breast milk and ascites. 51 In healthy individuals, blood is one of the richest and most F I G U R E 1 The origins and comparison of contents in PRP and PRP-EVs. Some studies have confirmed the higher concentration of growth factors in PRP-EVs as compared to PRP. Besides, PRP and PRP-EVs were demonstrated with great potential in regenerative medicine easily accessible sources of EVs, whereas platelet-derived EVs contribute to the majority of blood EVs (up to 70%-90%). 22 Recently, more accurate detection methods have also demonstrated that nearly 50% of blood EVs are derived from platelets or megakaryocytes. 70,71 Additionally, Aatonen et al. summarized the isolation procedure for PRP-EVs, which includes three key steps: step one, PRP is extracted from whole blood; step two, PRP is activated to promote the release of EVs; and step three, PRP-EVs are isolated by the differential centrifugation method ( Figure 2). 72 There are two subtypes of PRP-EVs released from activated platelets: exosomes (40-100 nm in diameter), which are generated by exocytosis from the multivesicular body (MVB) and alphagranules, and microvesicles or microparticles (100-1000 nm in diameter), which are generated by the surface budding of the cytoplasmic membrane. 23,72,73 There are many significant differences between these two types of PRP-EVs in terms of formation mechanisms and their features (Table 1) inhibited. This leads to the reorganization of phospholipids, the breakage of bonds between the cytoskeleton and the partial degradation of actin filaments, which promote the formation and release of microvesicles. 23,48,75 Moreover, some other molecular mechanisms are involved in the formation of microvesicles, including membrane curvature proteins, rho-associated protein kinase 1, adenosine diphosphate-ribosylation factor 6 and the contraction of actin-myosin. 76,77 In general, the cargo and biological properties of EVs are defined by the types and characteristics in parental cells. According to the different methods of activating platelets, platelet-derived microvesicles are characterized by a high expression of CD41, CD42 and phosphatidylserine (PS). 78 Similarly, platelet-derived exosomes are characterized by a high expression of marker proteins of exosomes, such as CD9, CD63, TSG101 and ALIX ( Figure 3). 79 However, it is difficult for the current isolation protocols to distinguish these two types of PRP-EVs. Unless the experimental conditions can identify that the capturing vesicles are from cell membrane budding or intracellular vesicles, the current consensus is to use the umbrella term "extracellular vesicles". 47 Many important features are used for the unified identification of PRP-EVs, including platelet endothelium adhesion molecule (CD31), CD41, CD42a, CD61, CD62p, CD63 and PS. [80][81][82] In addition, many critical biomolecules are rich in PRP-EVs, including growth factors, cytokines, chemokines, lipids and nucleic acids, as well as procoagulant and anticoagulant, pro-inflammatory and antiinflammatory, and proangiogenic and antiangiogenic factors. 23,83,84 These components and features of PRP-EVs support their prospective therapeutic application in regenerative medicine.

| PRP-E VS IN REG ENER ATIVE MEDICINE
When PRP-EVs were first discovered, their main functions were considered to be the transport of procoagulant materials, performing most of the same functions as platelets. 85 With the continued increase in interest in PRP-EVs, an increasing number of other functions have been confirmed. They were also demonstrated to be involved in haemostasis, vascular integrity, immunoregulation and inflammatory regulation. [86][87][88] Additionally, platelet-derived EVs have been reported to be associated with the pathological processes of some diseases, such as rheumatoid arthritis, 89,90 cancer 91 and cardiovascular diseases. 92 More importantly, a recent study found that PRP-EVs have great potential in the field of tissue repair and regeneration ( Table 2). 83  Despite these encouraging preclinical results, the underlying tissueregeneration mechanisms still need to be confirmed for future clinical applications of PRP-EV-based therapies, as briefly discussed here (Figures 1 and 3).

| PRP-EVs for procoagulant activity and haemostasis
It is well known that platelets play an important physiological role in haemostasis and coagulation. 93 For trauma patients with severe bleeding, haemostasis is the first as well as a very critical physiological process necessary to prevent and treat the acute injury.
However, compared with platelets, PRP-EVs seem to be a superior alternative for local coagulation and haemostasis following injury. For example, PRP-EVs derived from resting platelets showed milder pro-coagulant and haemostatic properties than that derived from thrombin-activated platelets. 99 Therefore, to meet the purpose of local administration, PRP-EVs derived from specific trigger is necessary to reduce the pro-coagulant activity.

| PRP-EVs in angiogenesis
Angiogenesis is an integral process in regenerative medicine. Newly   105 Overall, the excellent capability in angiogenesis is an important mechanism of PRP-EVs in promoting tissue repair and regeneration.

| The pro-inflammatory and anti-inflammatory properties of PRP-EVs
As well as their important roles in haemostasis and angiogenesis,

PRP-EVs have prominent pro-inflammatory and anti-inflammatory
properties. In fact, PRP-EVs have been traditionally regarded as powerful pro-inflammatory mediators. For example, some scholars believe that PRP-EVs may mediate the inflammatory reaction following some cases of platelet transfusion. 106 On the contrary, some scholars also suggest that PRP-EVs may contribute to anti-inflammatory effects. For example, PRP-EVs may reduce the inflammatory reaction via inhibiting the production of pro-inflammatory factors, such as TNFα from macrophages 88 or TNFα and IL-8 from plasmacytoid dendritic cells. 112 Moreover, EVs from stored PRP can polarize macrophages and transfer them to an anti-inflammatory phenotype. 88 PRP-EVs can also improve the production of lipoxin A4 to resolve inflammation through providing 12-lipoxygenase to mast cells. 113

| PRP-EVs as delivery vehicles
With the development of precision medicine, EVs, as delivery vec-  122 Other studies have also verified the high loading and delivery efficiency for antiviral and antitumor drugs. 123,124 Overall, the excellent loading capacity of PRP-EVs opens up more possibilities in regenerative medicine. Moreover, compared with EVs derived from stem cells, a large amount of PRP-EVs can be directly produced from platelet concentrates, which seem to be a more convenient and feasible candidate as a drug carrier.

| ADVANTAG E S AND LIMITATI ON S OF PRP-E VS IN REG ENER ATIVE MED I CINE
To date, studies have shown the great potential of PRP-EVs in the field of tissue repair and regeneration and, to some extent, revealed their related mechanisms. Recent findings suggest that PRP-EVs may be a superior alternative in regenerative medicine, compared to the well-studied PRP. [64][65][66]83 Although it remains to be further to their participation in intercellular communication. 106,125 Furthermore, as compared with EVs from other sources, especially stem cells, PRP-EVs show some advantages in several aspects.
First, owing to the direct extraction from platelet concentrates, the isolation of PRP-EVs lowers the requirements of upstream expansion and avoids the critical procedure and quality control issues associated with cell amplification, which is indispensable for EVs from other cells. 50 Additionally, the platelet concentrate is a kind of essential medicine authorized by the World Health Organization.
Blood donation is legal and encouraged in the majority of countries.
However, most blood donation is administrated to meet the clinical need for red blood cells; only 20% is collected for the preparation of platelet concentrates, which implies a rich allogeneic platelet source for the isolation of PRP-EVs. 83 In some emergency situations, autologous blood is also a very convenient option. However, although PRP-EVs are derived from platelet concentrates, current regulatory authorities classify EVs as biological medicinal products, thus, their regulatory rules are significantly different from those of blood products. 83 In future, as other kinds of blood products and EVs, the man-

CO N FLI C T S O F I NTE R E S T
All authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Conceptualization, J. Wu, Q. Liu and X. Yang; writing original draft preparation, J. Wu; writing-review and editing, Y. Piao, Q. Liu and X.
Yang. All authors have read and agreed to the published version of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article, as no new data were created or analysed in this paper.