Mesenchymal Stem Cell-Derived Exosomes: Applications in Regenerative Medicine

Exosomes are a type of extracellular vesicles, produced within multivesicular bodies, that are then released into the extracellular space through a merging of the multivesicular body with the plasma membrane. These vesicles are secreted by almost all cell types to aid in a vast array of cellular functions, including intercellular communication, cell differentiation and proliferation, angiogenesis, stress response, and immune signaling. This ability to contribute to several distinct processes is due to the complexity of exosomes, as they carry a multitude of signaling moieties, including proteins, lipids, cell surface receptors, enzymes, cytokines, transcription factors, and nucleic acids. The favorable biological properties of exosomes including biocompatibility, stability, low toxicity, and proficient exchange of molecular cargos make exosomes prime candidates for tissue engineering and regenerative medicine. Exploring the functions and molecular payloads of exosomes can facilitate tissue regeneration therapies and provide mechanistic insight into paracrine modulation of cellular activities. In this review, we summarize the current knowledge of exosome biogenesis, composition, and isolation methods. We also discuss emerging healing properties of exosomes and exosomal cargos, such as microRNAs, in brain injuries, cardiovascular disease, and COVID-19 amongst others. Overall, this review highlights the burgeoning roles and potential applications of exosomes in regenerative medicine.


Introduction
Exosomes are membranous extracellular vesicles that range from 30-200 nm in diameter. Exosomes have been found to be secreted by most cell types including immune cells (B cells, T cells, mast cells, dendritic cells), neuronal cells, epithelial cells, endothelial cells, embryonic cells, cancer cells, and mesenchymal stem cells (MSCs). The term "extracellular vesicle" broadly encompasses several types of vesicles including exosomes, microvesicles, and apoptotic bodies. However, the word "exosome" specifically denotes vesicles that are formed inside multivesicular bodies (MVBs) within cells [1][2][3]. Exosomes carry vital information and macromolecules from their source of origin and thus have a significant role in cell-cell communication. These macromolecules consist of a variety of proteins, enzymes, transcription factors, lipids, extracellular matrix proteins, receptors, and nucleic acids, and can be found both on the inside and outside of the exosomal surface ( Figure 1). Exosomes have been detected in almost all body fluids in both heathy and disease conditions, including fluids such as urine, blood, serum, breast milk, amniotic fluid, cerebrospinal fluid, malignant ascites, saliva, bile, and lymph ( Figure 2) [4][5][6][7][8][9][10][11][12]. Exosomes were first discovered by Pan and Johnstone while investigating the maturation mechanisms of sheep reticulocytes into erythrocytes [13][14][15]. The researchers discovered a type of vesicle (which they later named: "exosome") that was released from reticulocytes and contained lipids, proteins, and enzymes of reticulocyte origin [16]. Exosomes were initially assumed to be cellular debris or garbage disposal and considered signs of cell death [17][18][19]. Since their discovery, extensive research has been carried out to determine the biology, function, and potential clinical uses of exosomes. It is now established that exosomes are released by donor cells into the extracellular environment to perform diverse biological functions, including intracellular communication and the exchange of genetic material and proteins between a parent cell and surrounding cells (Figure 1) [20,21]. The clinical importance of exosomes has been established in their use as alternatives to liposome-mediated drug delivery in cancer immunotherapy. Exosomes are also a promising biological gene delivery system due to their microRNA and mRNA content [22][23][24][25]. However, there are still many aspects of exosomes that are not fully understood or characterized. For example, as many potential targets for cancer therapy are tumor-specific biomarkers, it is crucial to study the biomarkers present on the surface of exosomes in order to develop tumor-targeting therapies [26,27]. The great potential of these small wonder vesicles to aid in gene delivery, disease diagnostics, intracellular communication, drug delivery, and biomarker-driven therapies has progressively drawn the attention of researchers. Exosomes were first discovered by Pan and Johnstone while investigating the maturation mechanisms of sheep reticulocytes into erythrocytes [13][14][15]. The researchers discovered a type of vesicle (which they later named: "exosome") that was released from reticulocytes and contained lipids, proteins, and enzymes of reticulocyte origin [16]. Exosomes were initially assumed to be cellular debris or garbage disposal and considered signs of cell death [17][18][19]. Since their discovery, extensive research has been carried out to determine the biology, function, and potential clinical uses of exosomes. It is now established that exosomes are released by donor cells into the extracellular environment to perform diverse biological functions, including intracellular communication and the exchange of genetic material and proteins between a parent cell and surrounding cells ( Figure 1) [20,21]. The clinical importance of exosomes has been established in their use as alternatives to liposome-mediated drug delivery in cancer immunotherapy. Exosomes are also a promising biological gene delivery system due to their microRNA and mRNA content [22][23][24][25]. However, there are still many aspects of exosomes that are not fully understood or characterized. For example, as many potential targets for cancer therapy are tumor-specific biomarkers, it is crucial to study the biomarkers present on the surface of exosomes in order to develop tumor-targeting therapies [26,27]. The great potential of these small wonder vesicles to aid in gene delivery, disease diagnostics, intracellular communication, drug delivery, and biomarker-driven therapies has progressively drawn the attention of researchers. [28,29]. For example, MSC-derived exosomes (MSC-Exos) have been shown to induce repair in mouse models of wound healing and myocardial infarction ( Figure 3) [47][48][49][50][51]. In particular, investigations have revealed that exosomes secreted by placental umbilical cord MSCs play a significant role in wound healing and tissue regeneration [52]. Similarly, in the past few decades, studies have demonstrated that MSC-Exos can have advantageous effects in various contexts including neurological, respiratory, cartilage, kidney, cardiac, and liver diseases, bone repair, and cancer ( Figure 3) [28,29,48,[53][54][55][56][57][58][59]. In short, MSC-Exos can serve as a smart drug delivery approach through the transportation of exogenous chemicals and biomolecules for stem cell-free regenerative medicine. MSC-Exos have many potential therapeutic advantages when compared to synthetic nanoparticles, liposomes, single molecules, and cells. This stems from their novel beneficial characteristics such as smaller size, lower complexity, lack of nuclei (thus preventing neoplastic transformation), increased stability, easier production, longer preservation, and potential for loading proteins, small molecules, or RNAs for delivery of biomolecules [60]. MSC-Exos can also be modified to display distinct antibodies or surface receptors to transfer therapeutic payloads to specific organs, tissues, and cells. Additionally, MSC-Exos host numerous types of biological molecules, enabling them to participate in various therapeutic approaches simultaneously, which cannot be accomplished with conventional small molecules. Therefore, here, we review the recent advancements in the field of molecular mechanisms of exosomes in regenerative medicine and exosome research, as well as address the potential therapeutic approaches of exosomes in tissue regeneration due to disease and injury recovery.

Exosome Biogenesis, Secretion and Uptake
The structure and composition of exosomes depends on several factors including the donor cell, microenvironment, and physiological conditions. Exosomes are formed from endosomal vesicles via the exocytosis process ( Figure 4). As cargo transporters, exosomes can carry proteins, peptides, nucleic acids, and lipids. Studies investigating the protein composition of exosomes have shown that, while some proteins specifically arise from parental tissue, some were unique to exosomes [1,2,9]. Specific proteins contained within exosomes include those present in the endosome, plasma membrane, and cytoplasm, implying differential selection [20]. Additionally, several studies have shown that exosomes carry nucleic acids including different RNA types, e.g., microRNA, messenger RNA, and non-coding RNA [61][62][63][64][65][66][67]. Interestingly, the composition of proteins, peptides, and nucleic In short, MSC-Exos can serve as a smart drug delivery approach through the transportation of exogenous chemicals and biomolecules for stem cell-free regenerative medicine. MSC-Exos have many potential therapeutic advantages when compared to synthetic nanoparticles, liposomes, single molecules, and cells. This stems from their novel beneficial characteristics such as smaller size, lower complexity, lack of nuclei (thus preventing neoplastic transformation), increased stability, easier production, longer preservation, and potential for loading proteins, small molecules, or RNAs for delivery of biomolecules [60]. MSC-Exos can also be modified to display distinct antibodies or surface receptors to transfer therapeutic payloads to specific organs, tissues, and cells. Additionally, MSC-Exos host numerous types of biological molecules, enabling them to participate in various therapeutic approaches simultaneously, which cannot be accomplished with conventional small molecules. Therefore, here, we review the recent advancements in the field of molecular mechanisms of exosomes in regenerative medicine and exosome research, as well as address the potential therapeutic approaches of exosomes in tissue regeneration due to disease and injury recovery. exosomes have pleiotropic functions through paracrine signaling [20]. Currently, little is known about the underlying mechanism behind the sorting of exosomes into the different populations. Studies have revealed that MVBs specifically contain various lysosomeassociated molecules such as lysosomal-associated membrane protein 1, 2, and 3 (LAMP-1, -2, -3), tetraspanins, and a cluster of differentiation factors (CD-107a, CD-107b, CD-208 or CD-63) (Figure 1), whereas late endosomes possess major histocompatibility complex (MHC) class II [20,74]. Hanson and Cashikar explored the morphogenesis mechanism of MVBs and found that the endosomal sorting complex required for transport (ESCRT) plays a crucial role in driving both exosomal and ectosomal biogenesis [75]. ESCRT comprises approximately 30 different proteins that are organized into four machinery complexes, namely ESCRT-0, -I, -II, and -III in association with vacuolar protein sorting associated protein 4 (VPS4), vesicle trafficking 1, and apoptosis-linked gene 2-interacting protein X (Alix) which is also called programmed cell death six interacting protein [75]. The initial ESCRT-0 complex assists in recognizing and sorting ubiquitinated intracellular cargos that are prescribed for lysosomal degradation. ESCRT-I and -II contribute to deforming the membrane into buds with sequestered vesicles, whereas ESCRT-III plays a role in vesicle scission [76]. ESCRT-independent biogenesis mechanisms are proposed to involve tetraspanins (CD63, CD9, CD37, CD82 or CD81), which have been identified as exosomal markers. These proteins are vital in extracellular vesicle biogenesis and essential for extracellular vesicle secretion and uptake by receptor cells. Hydrolysis of sphingomyelin into ceramide is also known to contribute to the biogenesis of exosomes [20,77]. As a summary, Table 1 includes the composition and functions of proteins, e.g., ESCRT, AAA ATPases, ESCRT-associated proteins, SNAREs, Rabs, and other enzymes, that are actively involved in exosome biogenesis, sorting, transport, and secretion [78][79][80][81][82][83][84][85][86][87][88][89].

Protein Class Name
Metabolic

Proteins
Exosomal proteins perform various functions such as targeting/adhesion, anti-apoptosis, membrane fusion, signal transduction, metabolism, and structural dynamics [2,9]. There are similarities in protein content between species, as the available proteomic data for exosomes isolated from mouse and human dendritic cells (DCs) suggest that about 80% of the proteins are conserved in the two species [2,9,121]. However, based on proteomic studies, exosomes isolated from different cell types contain specific groups of proteins depending on the secreting cell types ( Table 2). Western blot and fluorescence-activated cell sorting analysis can identify known cellular proteins in exosomes prepared from different cell types [74,107,108,135,136]. For unknown cellular proteins, mass spectrometry associated with trypsin digestions can analyze exosomes derived from cells like mast cells, DCs, and enterocytes [2,105,112,121,[137][138][139][140]. These methods have been used to identify cytosolic proteins present in exosomes including actin, tubulin, cytoskeletal components, and actin-binding proteins, as well as Rab and annexins, which are important in intracellular membrane fusions and transport function (Tables 2 and 3). Exosomes also contain different types of 14-3-3, heterotrimeric G proteins, and protein kinases, which are critical in signal transduction during critical physiological processes. Further, exosomes derived from human DCs and enterocytes contain various types of metabolic enzymes such as enolases, peroxidases, and lipid kinases. The two constitutive forms of heat shock proteins (i.e., HSP70 and HSP90) are found in exosomes which perform the function of antigen presentation and aid in loading antigenic peptides onto MHC class I molecules (Table 3). Notably, the MHC class I molecules are present in most isolated exosomes [141][142][143][144]. Exosomes also consist of proteins that are involved in specific cellular functions. For example, MHC class II molecules are present in large amounts in exosomes. Exosomes derived from DCs contain CD86, which acts as costimulatory molecule for T cells, and can contain cell-specific transmembrane proteins such as αM, β2 on DCs, and T cells, α4B1. Other exosome proteins include immunoglobulin-family members, intercellular adhesion molecule 1 (ICAM1)/CD54 on B cells and cell-surface peptidases such as dipeptidylpeptidase IV/CD26 on enterocytes and aminopeptidase N/CD13 on mastocytes [145]. Additionally, exosomes harbor a vast variety of glycosylphosphatidylinositol (GPI) anchored proteins, nuclear proteins, and proteasome related proteins. Finally, the proteome of exosomes of various cell types are also divided into several signaling molecules and enzymes complexes (Tables 2 and 3) [122][123][124][125]146].

Lipids
Lipids are the least studied but most crucial components within exosomal membranes. Exosomes contain an abundance of lipids including glycosphingolipids, sphingomyelins (SM), cholesterol (CHOL), and phosphatidylserine (PS). Lipids not only play an important role in the structure of exosomal membranes, but also facilitate the formation of exosomes and their release into extracellular milieu [73,147,148]. Exosomes mainly contain monounsaturated fatty acids, polyunsaturated fatty acids, and saturated fatty acids, though the lipid composition of an exosome depends on the parent cells from which the exosome is derived [69][70][71][72]. Interestingly, exosomes can transport several bioactive lipids and lipid metabolism-related enzymes [126,149]. Fatty acids such as arachidonic acid (AA), leukotrienes prostaglandins, phosphatidic acid, docosahexaenoic acid (DHA), and lysophosphatidylcholine (LPC) have been found in MSC-Exos. The lipid metabolism enzymes of exosomes can modulate the cell homeostasis of a beneficiary [71,150].
The lipid classes found in different exosome types based on the published investigations are shown in Table 4. Methods such as thin-layer chromatography, gas-liquid chromatography, and mass spectrometry have been used to identify the lipids in exosomes [71]. Studies have shown that the content of CHOL, SM, glycosphingolipids, and PS is 2-3 times higher in exosomes compared to their parent cell. In addition to this, the majority of exosomes show lower content of PC and phosphatidylinositol (PI) than in parent cells. Additionally, lipid content can significantly vary by cell type. The lipid composition of exosomes isolated from hepatocyte cells (HepG2/C3a Oli-neu cells), prostate cancer cells (PC-3 cells), and PC-3 cells+ hexadecylglycerol (HG: a precursor of ether phospholipids) exhibit some similarities [66]. Furthermore, the exosome preparation from Oli-neu cells has lower enrichment of SM and higher enrichment of CHOL, as compared to exosomes isolated from PC-3 cells [66]. Reticulocyte-derived exosomes present high levels of CHOL as compared to other cells [56], while the enrichment of exosomes from adipocytes was found to contain a higher level of SM and lower level of PS than other various preparations in Table 4 [134].
Though lipidomic studies conducted on exosomes isolated from different cell types have been published, results are typically given only in the form of the number of different lipid classes. For example, the lipidomic study of exosomes isolated from colorectal cancer cells (LIM1215) identified a total of 500 lipid species [151], whereas Brzozowski et al. reported total of 187 lipid species identified in exosomes enriched from prostate cancer cell lines, i.e., PC-3, RWPE1, and NB26 [71,72,128]. Further studies have shown that exosomes secreted from epithelial cells (RWPE1) contain high amounts of fatty acids, prenol, and glycerolipids lipids. Exosomes from prostate cancer cell lines (PC-3 and NB26) show an abundant amount of sterol lipids, glycerophospholipids, and sphingolipids. In a lipidomic analysis of exosomes isolated from U87 (glioblastoma cells), Huh7 (hepatocellular carcinoma cells), and human bone marrow derived MSCs, Haraszti et al. showed that the lipid composition of MSCs and Huh7 exosomes were similar to each other but distinct from the U87 exosomes [152].

RNAs
Another important cargo of exosomes is RNAs, leading to the emergence of exosomes as a mediator of intracellular communication and a component of various signaling pathways [153][154][155]. MSC-Exos include RNA, which was found to be enclosed within cholesterol-rich phospholipid. This was demonstrated by the RNA cargo's vulnerability to RNase degradation only in the presence of sodium dodecyl sulfate (SDS)-based lysis buffer, a chelator of cholesterol, cyclodextrin, and phospholipase A2 [154,155].
Ethidium bromide staining, a technique routinely used for the detection of RNA, of MSC exosomal RNAs found that they consist of mainly short RNAs (<300 nt), whereas 28S and 18S RNAs were not visible [67,154,156,157]. A primary exosomal mechanism of action is thought to be post-transcriptional gene regulation via microRNA content (miRNAs, miRs), which are small, endogenous RNA molecules around 22 nucleotides in length. miRNAs have been shown to play pivotal roles in health and disease, including cancer, cardiovascular diseases, and wound healing [158]. Microarray hybridization of MSC-derived exosomal RNA against probes for 151 miRNAs revealed the existence of 60 miRNAs and ribosomal RNA degradative products [154,155]. Comparative analysis of the composition of MSC exosomal miRNAs with their cellular miRNA revealed that 106 miRNAs from the MSCs were not secreted in the MSC exosomes. These results suggested that MSCs secrete a select population of miRNA through a regulated process. Furthermore, an ample amount of passenger miRNA has been found in MSC-Exos [159].
There have been several studies conducted on exosomal miRNAs involved in intracellular communications and disease [61,62,[64][65][66]. The plasma-derived exosomal miR-92a showed an anti-apoptotic effect on fibroblast-like synoviocytes, which ultimately leads into the destruction of bone in rheumatoid arthritis patients [66]. Researchers have found that MSC-derived exosomal miRNAs can both promote [160] and reduce tumor growth [161]. For example, Lee et al. found that miR-16, a microRNA known to target vascular endothelial growth factor (VEGF), was abundantly present in MSC exosomes, leading to an antiangiogenic effect on tumor cells [161]. MSC-derived exosomal miRNAs also play an essential role in cardiovascular protection and repair by regeneration, as well as inhibition of cardiac apoptosis and fibrosis [162]. Shao et al. discovered that MSC-Exos enclosed a higher amount of cardioprotective miRNA such as miR-29 and miR-24, and lower amount of cardiac-offensive miR-21 and miR-15 as compared to MSCs [163]. Further, it was found that human amniotic epithelial cell-derived exosomal miRNAs play a crucial role in wound healing by promoting cell migration and proliferation of fibroblasts [164]. Human amnion MSC-derived exosomal miRNA, miR-135a, promotes wound healing and fibroblast migration by downregulating large tumor suppressor kinase 2 expression [165]. Wu el al. further found that MSC-derived exosomal miR-100 provides protection to the articular cartilage and helps in regulation of cartilage homeostasis in the OA mice model via inhibition of mTOR-autophagy pathway [149]. Further, it has been identified that plasma-derived exosomal miRNAs are involved in 'extracellular matrix-receptor interaction' and contribute to Hirschsprung's disease through interfering in cell junctions [166]. Human adipose stem cell-derived exosomes loaded with miR-21 mimics play a critical role in cell proliferation and migration of keratinocytes, and treatment of diabetic chronic wounds with miR-21 mimics results in accelerated healing by collagen remodeling, increasing re-epithelization, vessel maturation, and angiogenesis in vivo [167]. Further descriptions of the role of exosomal miRNA are discussed in later sections.

Emerging Technologies for Exosome Isolation
The majority of the cells in the body secrete exosomes in the extracellular milieu; however, exosomes are also found with other body fluids ( Figure 2). Since their discovery, several methods have been developed to isolate the exosomes from body fluids. In the past decades, there have been many advances in exosome detection and separation techniques, resulting in higher recovery, purity, sensitivity, and specificity of isolated exosomes  (Table 5). Still, due to overlapping size range, small sizes, and similar morphologies to other extracellular vesicles challenges persist in isolation methods. The most common and traditional method of exosome isolation is ultracentrifugation/differential ultracentrifugation, which separates exosomes based on size and density [170,171]. This technique is comparatively cost-effective and secure, though time consuming. It is generally used for the isolation of exosomes from large volumes of biological cultures. The main disadvantage of this method is a lack of specificity, in that the separated exosomes could contain other extracellular vesicles of similar sizes. To overcome this problem, it is recommended to use iodixanol or sucrose cushions in addition to differential ultracentrifugation [172][173][174].
Ultrafiltration is another conventional method used for the isolation of exosomes. In this method, exosomes can be isolated based on their molecular weight or size. For example, exosomes can be separated using defined molecular weight cut-off membrane filters. The filtration method is much faster than differential ultracentrifugation and does not require any kind of unique instrument [168,171,[175][176][177]. However, a major drawback is a lack of purity in the isolated fraction. Similar to ultracentrifugation, it is hard to omit compounds of other molecules with similar sizes to exosomes [178,179].
In addition to ultrafiltration, size exclusion chromatography (SEC) also separates exosomes on the basis of size or molecular weight [180,181]. SEC isolates exosomes with high purity and high yield, and acts as an essential tool in the process of exosome purification. In SEC, a column made with a solid-phase matrix of beads, with pores of different sizes, is used to separate macromolecules and other particulate matter [47,182]. SEC can be used in combination with ultracentrifugation or other techniques for higher yield of exosomes [183,184].
An immunoaffinity chromatography method can be used to enhance the purity of separated exosomes. Exosomal membrane proteins and receptor molecules are used to develop this highly specific method. In this technique, the exosomes can be captured on the column by immunoaffinitive proteins and their specific antibodies [185][186][187]. The immunoaffinity chromatography technique is appropriate for smaller-scale production of exosomes from fewer sample volumes. A microplate-based enzyme-linked immunosorbent assay (ELISA) is the best example of immunoaffinity-based chromatography approach used for quantifying the captured exosomes from biological samples such as a serum, plasma, and urine [188].
Exosome purification can also be carried out by precipitation [189,190]. Precipitationbased exosome isolation is used to concentrate the exosomes from biological fluids. Exosomes can be precipitated from cell culture media by altering their dispersibility and solubility. This can be achieved by commercially available precipitation reagents such as polyethylene glycol (PEG) [177,191]. Currently, numerous precipitation-based exosome isolation kits are available in the market. These are compatible with biological fluids such as urine, plasma, serum, cerebrospinal fluid, and cell culture medium [192,193].
Recently, microfluidic-based methods were developed for the rapid and efficient isolation of exosomes from biological samples. The main advantages of these techniques are remarkable reductions in reagent consumption, sample volume, analysis cost, and isolation time [194][195][196]. As scalability is enhanced through technique modification and available technologies, the needs of health care applications such as reproducibility, reliability, low cost, and speed can eventually be fulfilled. A full summary of advantages and disadvantages of the exosome isolation methods is given in Table 5.

Applications of Exosomes in Injury and Disease
Exosomes, and specifically exosomes derived from mesenchymal stem cells, have been found to have enormous benefits in a variety of diseases and injuries through the proteins and RNAs that they contain. Additionally, because exosomes are representations of their parent cells, as the cellular environment changes, so exosomes change. As such, the number and content of exosomes can be used as a biomarker for changing conditions in disease. In the following sections we summarize recent findings in problems such as wound healing, neurological damage, and hepatic diseases.

Wound Healing
Skin damage can commonly arise due to factors such as the sun, parasites, or a fall, often leading to open abrasions with potential for infection. Injuries to the skin are healed in an intricate process that takes place in four overlapping stages: (1) hemostasis, (2) inflammation, (3) proliferation; and (4) maturation/remodeling ( Figure 5) [197][198][199]. In the first stage, hemostasis, platelets form a blood clot to prevent blood loss. Simultaneously, the platelets secrete hormones, cytokines, and chemokines, including tumor growth factor-β (TGF-β), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF), to attract inflammatory cells (growth factors important in wound healing are summarized in Table 6) [9,40,[200][201][202][203][204]. Inflammation, the second step of wound healing, begins within 24 h of injury as neutrophils infiltrate the wound and secrete products, such as toll-like receptors (TLRs) and nuclear factor к-lightchain-enhancer of activated B cells (NF-кB), to attract and activate pro-inflammatory (M1) macrophages [9,40,201,202,204]. M1 macrophages phagocytose pathogens, produce an oxidative burst, and remove apoptotic cells before products including signal transducer and activator of transcription 3 (STAT-3) promote the polarization of M1 macrophages into anti-inflammatory M2 macrophages, thus stimulating inflammatory resolution [205][206][207][208]. Proliferation then begins as keratinocytes and fibroblasts proliferate at the edge of the wound. Increased levels of VEGF and FGF stimulate angiogenesis, the process by which new blood vessels are formed to transport necessary nutrients, oxygen, and growth factors to the damaged tissues. Fibroblasts secrete immature type III collagen to form a new extracellular matrix (ECM), and then differentiate into myofibroblasts. These cells have contractile abilities, pulling together the edges of the wound [197,198,203,206,209]. Finally, during the maturation phase, the former ECM gets degraded by a variety of enzymes, including matrix metalloproteinases and plasminogen activators, as the type III collagen is substituted by mature type I collagen. The remodeling of the scar is a longer process than other stages of wound healing: over months or years, the scar tissue reaches its final appearance [198,209]. The proper sequence, timing, and regulation of these stages are critical during wound healing; any delinquency in this progression can result in the formation of chronic ulcers or hypertrophic scarring [197,199,210]. The major risk factors in this are underlying conditions such as aging, diabetes, and recalcitrant infections. Intemperate fibroblast activity results in hypertrophic scarring and may degenerate into keloids [210][211][212].
One of the promising approaches for this is an exploitation of cellular therapies using MSCs and the possible role of MSC-Exos in wound healing and regeneration [39]. Though studies using MSCs have revealed that both autologous and allogenous MSCs give promising results [263], several studies have demonstrated that the effectiveness and the regenerative capacity of the conditioned media from MSCs is similar or greater than MSCs when applied to chronic wounds [37,[39][40][41]48,264]. As such, it has been found that the MSC-derived secretome, in the form of exosomes, carries soluble factors and metabolites that play an important part during wound healing [40,41,265]. Recent research has highlighted the potential of MSC-Exos, and in particular the growth factor and microRNA content of exosomes, as a therapeutic treatment of chronic skin ulcers and hypertrophic scarring, as well as the possible role of exosomes in the modulation of different stages of wound healing (see Table 7 for an expansive list of microRNAs in wound healing) [48,198,200,217,266,267]. Exosomes can target several pathways including phosphoinositide 3-kinase (PI3K)/AKT, ERK, and STAT-3 which are vital in facilitating and accelerating wound healing through downstream targets such as hepatocyte growth factor (HGF), insulin-like growth factor-1, nerve growth factor (NGF), and stromal cell-derived factor [48,50,198,[268][269][270][271][272][273]. Further, a study by Shabbir and team demonstrated a trending increase in VEGF induced by MSC-Exo administration [48]. In addition to activation of growth factors through downstream processes, growth factors such as VEGF, HGF, and PDGF have been found in exosomes isolated from MSCs of different sources [274,275]. TGF-β has been found in low levels in umbilical cord MSC-Exos [274,276], and when exosomes are further loaded with TGF-β cargo, can stimulate vascularization and matrix remodeling [277]. Exosomes, by proxy of their effect on and containment of growth factor, possess cellular proliferation and differentiation modulating properties along with high immunomodulating, immunosuppressing, and angiogenic activities, which have been demonstrated both in cell culture and animal models [50, 266,269,278]. M2 polarization through regulation of TLRs [285][286][287]. Studies have found that miR-132 located within MSC-Exos can elevate IL-10 expression and decrease levels of NF-кB, IL-6 and IL-1β in favor of inflammation resolution [288][289][290]. Broadly, exosomes have shown significant beneficial effects on proliferation, collagen deposition, and angiogenesis, even in states of chronic wounds and comorbidities such as diabetes [272,273,291]. Several microR-NAs participate in this step including miR-132, miR-126, and miR-21. miR-132 plays a role in proliferation, as it can increase the activity of the STAT-3 and ERK pathways, thereby promoting keratinocyte growth. Exosomes loaded with miR-132 improved angiogenesis by increasing the tube formation of endothelial cells [211,289]. In a hypoxia-like environment, exosomes have been found to be abundant with miR-126, which aids in angiogenesis through downstream activation of PI3K/AKT signaling [292,293]. Though many miRNAs can be found in exosomes, some are highly expressed, including miR-21 [280]. This important microRNA has been shown to be impactful in several diseases and injuries. miR-21 promotes migration of keratinocytes and fibroblasts, stimulating re-epithelialization, and promotes collagen synthesis [280,285,[294][295][296]. Further, miR-21 can resolve inflammation, as miR-21 is increased in macrophages after they envelop apoptotic neutrophils, a key process in the transition from inflammation into proliferation stages [39,263]. Additionally, miR-21 can downregulate TLR-4-mediated inflammation through the inhibition of the expression of programmed cell death protein 4 [39,281]. miR29a and miR29b are also shown to be have elevated levels in MSC-Exos and play pivotal roles in regulation of TGF-β and cell growth; in addition to the highly expressed miR-23a, the 29 family also has a large impact on angiogenesis [280,284]. Exosomes, and MSC-Exos specifically, show great potential for the promotion of rapid and efficient wound healing. Exosomes can be applied directly to an injury, which in animal models has been shown to promote collagen synthesis and the proliferation and migration of fibroblasts and keratinocytes (Table 8) [268,297,298]. These effects are shown to be due in part to exosomal regulations of microRNA levels and protease activities [297,299]. However, progressively more creative ways to utilize these versatile particles in novel technologies are being developed, such as the incorporation of exosomes into a gel, which is then applied to an injury. This treatment method has been shown to be more beneficial than a single, direct administration of exosomes due to a slow, steady rate of delivery, with some gels delivering exosomes for up to a week [300][301][302]. These scaffolds offer structure, hydration, and increased flexibility in treatment options, as hydrogels can been used for wounds from basic skin injuries to deep nerve damage [302,303]. In the continued development of wound healing strategies continue, the broad applicability and therapeutic benefits of exosomes will only expand the prospects and efficiency of wound healing technologies.

Brain Injury
Injuries to the brain, including traumatic brain injury (TBI) or stroke, can lead to longterm disability and decreased life expectancy, making them major health and economic issues [46, 308,309]. These traumas can prompt rapid acute and long-term damage to neuronal tissue and function. Successful treatment of brain injuries is limited due to the need for swift diagnosis and difficulties in delivering therapeutics past the blood-brain barrier (BBB). Additional complications arise due to the myriad of changes that take place following brain damages. MSC-Exos are not only capable of crossing the BBB through intravenous or intranasal delivery, but also have beneficial effects in treating chronic inflammation in and promoting healthy healing, making them a potential therapeutic for complex brain injuries ( Figure 6) [310][311][312][313].

Brain Injury
Injuries to the brain, including traumatic brain injury (TBI) or stroke, can lead to longterm disability and decreased life expectancy, making them major health and economic issues [46, 308,309]. These traumas can prompt rapid acute and long-term damage to neuronal tissue and function. Successful treatment of brain injuries is limited due to the need for swift diagnosis and difficulties in delivering therapeutics past the blood-brain barrier (BBB). Additional complications arise due to the myriad of changes that take place following brain damages. MSC-Exos are not only capable of crossing the BBB through intravenous or intranasal delivery, but also have beneficial effects in treating chronic inflammation in and promoting healthy healing, making them a potential therapeutic for complex brain injuries ( Figure 6) [310][311][312][313]. Exosomes released from different types of MSCs such as human umbilical cord, adipose tissue, bone marrow, and neural stem cells perform several investigated and suggested biological functions. Exosomes delivered via intranasal, intravenous, or other routes can migrate to the brain and penetrate the blood brain barrier. From there, the exosomes can enter general circulation and arrive at far off targets. Conversely, exosomes can travel across the blood brain barrier from inside the blood vessel into the central nervous system and be taken up by neurons and glial cells. Exosomes contain diverse contents (as depicted in Figure 1), that can influence inflammation, misfolded proteins, damage, and disease. In injury states such as TBI or stroke, exosomes interact with synaptic activity and neural survival, facilitating neurite outgrowth, and can promoting myelination and blood brain barrier repair. Exosomes delivered via intranasal, intravenous, or other routes can migrate to the brain and penetrate the blood brain barrier. From there, the exosomes can enter general circulation and arrive at far off targets. Conversely, exosomes can travel across the blood brain barrier from inside the blood vessel into the central nervous system and be taken up by neurons and glial cells. Exosomes contain diverse contents (as depicted in Figure 1), that can influence inflammation, misfolded proteins, damage, and disease. In injury states such as TBI or stroke, exosomes interact with synaptic activity and neural survival, facilitating neurite outgrowth, and can promoting myelination and blood brain barrier repair. Impacts from sports, car crashes, military experiences, or falls can lead to damage to the brain called a concussion, or traumatic brain injury (TBI). Originally thought to be an acute event, research has now shown that TBIs can lead to long-lasting effects on brain function, reducing life expectancy [308,309,314]. TBIs produce immediate trauma to the brain in which neurons, glia, and blood vessels stretch or tear, inducing apoptosis and damaging the BBB. This is followed by a pro-inflammatory immune response that recruits glial cells to the injury site; upon arrival to the injury, immune cells become activated, phagocytose dead and damaged cells, and secrete pro-inflammatory signals. In moderate to severe injuries, this inflammatory immune response does not properly resolve, causing chronic inflammation: complications of chronic inflammation can last weeks to years following injury [46,308,314]. MSC-Exos from various sources including bone marrow, umbilical cord, and adipose tissue have shown great potential in modulating the inflammatory response that follows a TBI [46,51,315]. When MSC-Exos were delivered 24 hours after injury in a model of TBI, controlled cortical impact, researchers found a decreased inflammatory response, which led to improved recovery via enhanced neurogenesis and angiogenesis [51, 309,315]. This effect is due in part to microRNAs contained within exosomes, such as miR-9, miR-124, and miR-125b. These microRNAs regulate important cytokines such as IL-1β, and thus promote neurogenesis. Studies have found that treatment with exosomes can decrease inflammatory markers in a dose-dependent fashion [46,308,316,317]. Additionally, exosomes can regulate TLR-4 and macrophage polarization, thereby promoting recovery following a TBI [46,136,314,317,318]. Importantly, administration of MSC-Exos following a concussion can not only help the molecular changes that take place, but also lead to improvements in motor and cognitive deficits that commonly occur after brain damage [51, 309,315,[317][318][319][320].
Another common injury to the brain is a stroke, which is a major cause of morbidity globally, with 15 million estimated strokes every year worldwide [310,313,321,322]. There are different types of strokes which can be difficult to differentiate between creating complications in diagnoses, including ischemic (development of a clot that blocks blood flow to a part of the brain) and hemorrhagic (a blood vessel ruptures) [74,323,324]. Following a stroke, there is a loss of oxygen to the brain, cell death, and excess inflammation. Interestingly, exosomes are being considered as a potential biomarker for stroke severity, and importantly for diagnosis, stroke type [8,322,325]. Kalani et al. found that the microRNA content in secreted exosomes is contingent on the type of stroke a patient suffered from, with miRs such as miR-21-3p, miR-27b-3p, and miR-132-3p elevated in patients with an ischemic stroke [8]. In addition to exosomes serving as a stroke biomarker, MSC-Exos have highly beneficial properties in the treatment of stroke. The primary standard of ischemic stroke care is the delivery of tissue plasminogen activator (tPA); interestingly, the addition of exosomes to tPA treatment significantly improved functional outcome following stroke compared to tPA treatment alone [313,317]. Delivery of MSC-Exos in stroke models leads to long-term neuroprotection, improved neurogenesis and neurovascular remodeling, as well as enhanced behavioral and neurological performances in motor function, coordination, sensorimotor, and spatial learning [136,317,324,[326][327][328][329][330]. Varied contents of exosomes have been shown to aid recuperation, from growth factors such as VEGF to microRNAs [46,331]. Zheng et al. identified that miR-25 in MSC-Exos improved cell viability following stroke through modulation of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3, while in a model of middle cerebral artery occlusion, miR-133b secreted from MSCs led to improved neurogenesis and stroke recovery [46,310,311,330]. Similar to studies in TBI, MSC-Exos can improve recovery through modulation of inflammation [43,136,310,313,328]. A study by Zhao et al. found that delivery of MSC exosomes significantly decreased inflammatory signaling and promoted the polarization of microglia from M1 to M2 activation [136]. miR-21, miR-199a, miR-124a, and miR-17 are a few of many exosomal microRNAs that have been shown to play beneficial roles in neuroprotection, immune regulation, and rejuvenation after injury [324,332]. Together, these studies demonstrate that exosomes are highly beneficial for neurological injuries, not only due to the vast therapeutic value provided by exosomal contents, but also because these vesicles can bypass the BBB through both intravenous and intranasal administration. See Figure 6 for depiction of exosomal role in brain healing.

Hepatic Diseases
Liver diseases include illnesses such as cirrhosis (the scarring of the liver) and hepatocellular carcinoma, and worldwide account for approximately 2 million deaths annually [333]. Treatments for liver diseases vary, but several studies related to acute liver injury and other hepatic diseases have identified that exosomes may have dual function of therapeutic agents as well as specific biomarkers for liver disease diagnosis [334][335][336][337][338]. Recently, a report from Momen-Heravi and group has demonstrated that in alcoholic hepatitis patients, the number of exosomes was found to be elevated compared to the healthy population [338]. Certain RNAs are differentially affected in exosomes derived from patients with liver diseases, with RNAs such as miR-21 elevated in exosomes of patients with hepatocellular carcinoma [334,335,338,339]. These findings set a stage for exosomes as biomarkers for noninvasive detection of hepatocellular carcinoma and other acute liver diseases [340][341][342].
The regenerative capacities of exosomes in liver have been explored as therapeutic agents, as exosomes carry cargo over large distances for cellular communication. Several studies have found that treatment with exosomes benefits liver repair and regeneration following hepatic failure, an effect thought to be due to the promotion of angiogenesis via the Wnt signaling pathway [343]. Further, hepatocyte-derived exosomes can deliver the synthetic machinery to form sphingosine-1-phosphate in target hepatocytes, enabling cell proliferation and liver regeneration after ischemia, reperfusion injuries, or after partial hepatectomy [344]. It has also demonstrated that hepatocyte-derived exosomes stimulate hepatocyte proliferation in vitro and promote liver regeneration in vivo during acute liver injury; the underlying mechanism is thought to involve exosome-mediated transfer of neutral ceramidase and sphingosine kinase 2 at the site of regeneration. Additionally, it was revealed that after liver injury, the enhanced levels of circulating exosomes have proliferative effects [344]. The regenerative potential of MSC-Exos in carbon tetrachloride (CCl4)-induced liver injury has also been investigated. It was found that these exosomes effectively attenuated the CCl4-induced liver injury by promoting proliferative and regenerative responses [345,346]. The potential effect of exosomes derived from human-induced pluripotent stem cell-derived mesenchymal stromal cells was studied in hepatic ischemiareperfusion injury. The administration of such exosomes showed promising effects in the recovery of hepatic ischemia-reperfusion injury, with suppressed inflammatory responses, attenuated oxidative stress responses, and inhibited cellular apoptosis, pointing to exosomes as a viable therapeutic option for liver diseases [347].

Cardiovascular Diseases
Cardiovascular diseases (CVDs), such as heart failure and coronary artery disease, are some of the prime causes of morbidities and mortalities in the United States, accounting for a total of around 655,000 attributable fatalities per year and are associated with immense health and financial expenditure [348,349]. Conventional CVD therapies primarily consist of transplantations and therapeutics; however, receiving a transplant can be a very drawnout process, and therapeutics have limited clinical efficacy. Therefore, focus has shifted to the development and validation of new therapeutics, with numerous cell-based therapeutic interventions initiated for the treatment of CVDs. Though cell-based treatment methods are promising, they face challenges such as low engraftment, poor survival rate of transplant cells, tumorigenesis potential, and immune rejection. Intriguingly, recent experimental data have suggested that myocardial protective functions through autocrine and/or paracrine actions of cell-based therapies may be achieved via through exosomes (Figure 7) [350,351]. Exosomes have major roles physiological and pathological cardiovascular processes including regulation of angiogenesis, cardiomyocyte hypertrophy, cardiac fibrosis, blood pressure control, and anti-apoptotic effect (survival) have been broadly acknowledged. Additionally, in the heart, cells including cardiomyocytes, cardiac fibroblasts, endothelial, vascular, cardiac progenitors, and stem cells release exosomes [352][353][354][355][356][357][358][359][360]. Moreover, exosomes lack some of the issues of cell-based therapies due to their low immunogenicity, minimal embolism risk, and biocompatibility, and can be delivered to the heart in a variety of ways, including engineered exosomes, endogenous exosomes, targeted exosomes, or exosomes contained in a patch [361,362]. The heart undergoes extensive cardiac remodeling following cardiac stressors such as myocardial infarction (MI), to restore contractile function. Numerous studies have indicated that after cardiac stressors, endogenous exosomes can ameliorate heart function (Figure 7) [363][364][365]. This demonstrates that exosomes can have effective therapeutic utility in the treatment of CVDs.
The therapeutic potential of exosome-based cell-free therapy for CVDs applies to several diseases including MI, atherosclerosis, and dilated cardiomyopathy. The cardioprotective effect of exosomes can be augmented by pretreatments like hypoxia preconditioning, gene programming, or drug intervention [294,366,367]. In MI increasing evidence has shown that the administration of exosomes can enhance cardiac repair. For example, a study has revealed that MSC-Exos improve cardiac function by down-regulating the expression of CD68 [163]. Exosomes obtained from miR-146a-modified adipose-derived stem cells (ADSCExos) have been shown to attenuates acute MI-induced myocardial damage by suppressing the local inflammatory response through inhibition of the release of proinflammatory cytokines (IL-6, IL-1β, and TNF-α). The same report further demonstrated that ADSCExos improves cardiac functions by arresting cardiomyocyte apoptosis via early growth response factor 1 downregulation [368]. Huang and coworkers have discovered that exosomes from atorvastatin (ATV)-pretreated MSC (MSCATV-Exos) ameliorated cardiac dysfunction and reduced infarct area by diminishing IL-6 and TNF-α levels, promoting angiogenesis, and preventing apoptosis following MI. MSCATV-Exos are abundant in lncRNA H19 that regulates miR-675 expression and activation of pro-angiogenic factors [366]. Adipose-derived stromal cells (ADSC)-derived miR-93-5p-containing exosomes are also beneficial in the treatment of MI by conferring protection against autophagy, apoptosis, and inflammation [369]. Separately, in a mouse model of MI, hypoxia-derived exosomal miR125b-5p exerts cardioprotective function and enhances cardiac repair by suppressing the expression of pro-apoptotic genes p53 and BAK1, thus inhibiting cardiomyocyte apoptosis [370].
The therapeutic promise of exosomes has also been investigated in the treatment of atherosclerosis. MSC-Exos serve a protective role in hindering the progression of atherosclerosis by inducing M1→M2 macrophage polarization through up-regulation of miR-let7 [371], an effect also displayed in different animal models of ischemia-reperfusion (I/R) injury [372,373]. Post myocardial I/R injury, exosomes aid in cardiac repair and contract myocardial infarct size by limiting cardiac fibroblast proliferation, creating an anti-inflammation microenvironment, and improving cardiac function mainly via the shuttling of miRNAs (miR-182, miR-146a, miR-181b, and miR-126) [372,373]. In addition, Lankford and colleagues have shown that the MSC-Exos are effective in dilated cardiomyopathy, as exosomes reduce ventricular dilation by hampering inflammatory cytokines expression and enhancing the production of anti-inflammatory M2 macrophages over M1 macrophages [374]. In chronic heart failure, a study has shown significantly elevated exosomal miR-146 levels, which can inhibit the inflammatory response [375]. low immunogenicity, minimal embolism risk, and biocompatibility, and can be delivered to the heart in a variety of ways, including engineered exosomes, endogenous exosomes, targeted exosomes, or exosomes contained in a patch [361,362]. The heart undergoes extensive cardiac remodeling following cardiac stressors such as myocardial infarction (MI), to restore contractile function. Numerous studies have indicated that after cardiac stressors, endogenous exosomes can ameliorate heart function (Figure 7) [363][364][365]. This demonstrates that exosomes can have effective therapeutic utility in the treatment of CVDs. MSCs or exosomes can be genetically modified with tRNA, miRNA or mRNA to express the desired gene using gene delivery methods or CRISPR/Cas9. MSC-derived exosomes can act as therapeutic vehicles to deliver biological molecules or drug molecules and further immersed in scaffolds and delivered as patch, injectable scaffold, or 3D tissue construct to increase the functions of exosomes. Similarly, exosomes can be chemically conjugated with targeted peptides to further enhance efficacy and retention when delivered intravenously.
The therapeutic potential of exosome-based cell-free therapy for CVDs applies to several diseases including MI, atherosclerosis, and dilated cardiomyopathy. The cardioprotective effect of exosomes can be augmented by pretreatments like hypoxia preconditioning, gene programming, or drug intervention [294,366,367]. In MI increasing evidence has shown that the administration of exosomes can enhance cardiac repair. For example, a study has revealed that MSC-Exos improve cardiac function by down-regulating the ex- Figure 7. Stem cell-derived exosomes for cardiac repair therapies. (A) Exosomes isolated from different types of mesenchymal stem cells carry and deliver proteins, nucleic acids (DNA, miRNAs, mRNAs, and other RNAs) and metabolites to the damage heart tissue, consequently promoting cardioprotective effects. (B) Schematic representation of tissue engineering approaches in exosomebased cardiac repair therapies. MSCs or exosomes can be genetically modified with tRNA, miRNA or mRNA to express the desired gene using gene delivery methods or CRISPR/Cas9. MSC-derived exosomes can act as therapeutic vehicles to deliver biological molecules or drug molecules and further immersed in scaffolds and delivered as patch, injectable scaffold, or 3D tissue construct to increase the functions of exosomes. Similarly, exosomes can be chemically conjugated with targeted peptides to further enhance efficacy and retention when delivered intravenously.
Though exosomes are heavily investigated for their therapeutic potential, they are also capable of propagating detrimental pathology in heart disease. In MI, miR-155enriched exosomes secreted by activated macrophages were found to negatively regulates fibroblast differentiation and promote inflammation, exacerbating cardiac rupture [376]. In atherosclerosis, Gao et al. reported that dendritic cell-derived exosomes induce the progression of atherosclerosis by triggering inflammatory responses [377]. Taken together, although these preliminary studies on CVDs have promising results, there are still many questions that must be answered before exosomal treatment can fully achieve a useful clinical outcome (Figure 7).

Bone Regeneration
Bone health is a rising issue and public health concern, as in the United States, approximately 850,000 people suffer from bone fractures per year [378,379], while approximately 25 million peoples are at high risk for injury due to low bone density [380,381]. In the bone remodeling and fracture healing process, osteoclasts reabsorb old or damaged bone, while new bone is synthesized by osteoblasts [381,382]. A healing cascade is initiated, leading to the recruitment of inflammatory cells, formation of new vessels, and establishment of hematoma at the fracture site [383]. The management of these bone fractures includes autologous and allogeneic transplantation; however, these procedures have a prolonged recovery time and thus increased likelihood of complications. In recent years, tissue engineering-based biomaterials and cell therapies have emerged as major players in the treatment of bone fractures. Research has identified that the biological effect of cell-based therapies is executed largely via exosomes, and as such, exosomal regulation of bone repair has become a viable therapeutic strategy. Exosomes are secreted by cell types including osteoblasts, osteoclasts, osteocytes, and bone marrow MSCs, which are known to mediate cellular communication and participate in the regulation of bone microenvironment [384][385][386][387][388][389][390]. Furthermore, numerous studies have corroborated the potential regulatory roles of exosomes in bone remodeling and fracture healing [381,382], suggesting exosomes can be utilized as individualized therapeutic strategies to promote bone tissue repair.
Exosomes derived from various sources have been demonstrated to be beneficial in bone repair. Chief amongst them are MSC-Exos, which play a promising role in the induction of osteo-differentiational activity through microRNA regulations [391]. Exosomes obtained from adipose tissue MSCs, preconditioned with TNF-α, promote primary osteoblast differentiation by regulating the Wnt pathway [392]. Separately, exosomes derived from mineralizing osteoblast cells promote new bone growth through directly regulating osteoblast proliferation and stimulating the differentiation of osteoblast precursors into mature osteoblasts via mediating miRNA profiles. This successively activates downstream signaling pathways for bone formation and matrix mineralization [384]. Another group has reported that exosomes isolated from human-induced pluripotent stem cell-derived MSCs potently enhance osteo-inductivity of β-TCP through activation of PI3K/AKT pathway [393]. Further, this study demonstrated that in an immunocompetent rat osteochondral defect model, the MSC-Exos promote cartilage regeneration by enhancing early cellular infiltration and proliferation, inducing synovial macrophage polarization, and exhibiting anti-apoptotic activity [393].
Bone remodeling is a long-lasting process in which can exosomes play crucial regulatory roles. Exosomes derived from osteoblasts stimulate the differentiation of osteoclasts in vivo, and as such exosome treatment can be used to enhance the removal of damaged tissue [394,395]. It has also been revealed that in a femur fracture model of CD9 − / − mice, which lacks exosome production, there is a delay of callus formation resulting in remission of a bone union. Providing further support for the beneficial role of exosomes in bone repair, the administration of MSC-Exos rescued this delayed repair [396]. The same study further explained that, when compared to exosomes isolated from MSCs cultured in preconditioned media, control exosomes were deprived of bone repair-related cytokines such as monocyte chemotactic protein-1(MCP-1), MCP-3, and stromal cell-derived factor-1 [396].
In a rat osteochondral defect model, the intra-articular administration of human embryonic MSC-Exos induces an enhanced gross appearance and improved histological scores; by 12 weeks of treatment, the cartilage and subchondral bone was fully restored [397]. In addition, studies have shown that overactivated inflammatory response or inflammation attenuation results in either bone tissue damage or accumulation of necrotic tissue, respectively [398][399][400]. MSC-Exos are shown to diminish inflammation-based delay of fracture healing by effectively suppressing levels of proinflammation factors IL-1β and TNF-α, whereas increasing levels of anti-inflammatory factor TGF-β [401].
Vascularization is an important event in bone formation as it improves diffusion of oxygen and nutritional components that are crucial for new bone formation [402]. Various reports have demonstrated the involvement exosomes in the activities of endothelial cells (ECs) including migration, proliferation, and tube formation. Exosomes derived from endothelial progenitor cells induce the formation of vessel-like structures both in vitro and in vivo by activating eNOS and PI3K/AKT pathways in human umbilical vein endothelial cells and human mammalian ECs [403]. In addition, the contribution of MSC-Exos in angiogenesis is well established [404,405]. Researchers have shown that MSC-Exos are internalized into tissue engineering-based matrix, which results in pro-angiogenic and pro-osteogenic activities, suggesting exosomes may regulate synergistic bone regeneration and enhance angiogenesis [406]. Moreover, in an in vivo study, MSC-Exos delivered to osteoporotic rats had elevated angiogenesis and osteogenesis eight weeks post-administration [407]. However, the coupling underlying mechanisms of angiogenesis and osteogenesis is still unclear.
A primary mechanism of action is osteopathic repair is through microRNA activity and regulation, many of which have been identified within exosomes. For example, researchers have identified that miR-199b is involved in the regulation of Runx 2, a master transcription factor for osteoblast and osteocyte differentiation [391]. Let-7 positively regulates osteogenesis and new bone formation by downregulating high-mobility group AT-hook 2 expression [408], while upregulated levels of miR-135b significantly inhibit osteogenic differentiation of MSCs [409]. In both human MSCs and human unrestricted somatic stem cells, miR-221 downregulation showed to stimulate osteogenic differentiation [410]. The crucial role of miR-21 in enhancing the rate of fracture healing has also been illustrated in a rat closed femur fracture model [411]. miR-21 rich exosomes possess anti-apoptotic properties and can contribute to osteoblastogenesis through the regulation of small mothers against decapentaplegic 7 (Smad7) and modulating PI3K/β-catenin pathways [412,413]. Exosomes can also serve as a biomarker, as altered miRNA profiles in MSC-Exos have been discovered during osteogenesis [391]. Therefore, the involvement of specialized cargo within exosomes enriched with specific factors such as miRNA, cytokines, or growth factors can play vital roles during bone repair and diagnostics [396]. This collective data and positive outcomes provided new insight for exosome-based synergetic therapy for bone fracture healing.

COVID-19
In late 2019, Chinese health authorities reported an outbreak of pneumonia of unknown origin in the city of Wuhan [414][415][416]. This led to the occurrence of a global pandemic, now known as COVID-19, resulting in thousands of deaths and infecting millions of people across the world (https://coronavirus.jhu.edu/map.html accessed 25 June 2021). COVID-19 has had an enormous destabilizing impact on society, including the healthcare sector and economies of even the most developed nations. A novel coronavirus named 2019-nCoV (later renamed as SARS-CoV-2) is known to be the causative agent and was first isolated from the airway epithelial cells of a patient [414][415][416]. SARS-CoV-2 was first sequenced in China and found to be different from previously identified viruses including MERS-CoV and SARS-CoV. It is now considered as the seventh member of the family of coronaviruses that are known to infect humans. SARS-CoV-2 is mainly transmitted via droplets within individuals of a susceptible population. It has also been shown to survive on glass and banknotes surfaces for 24-72 h [417]. However, the exact molecular mechanism of infection from SARS-CoV-2 is currently unknown.
MSCs have been substantially investigated as useful tools for stem cell-based therapy of degenerative diseases, cardiovascular disease, and lung diseases due to properties such as high proliferation rate, high differentiation, and vast source potential. Recently, several studies have posited that COVID-19 can hijack the immune system of the body and trigger an overreaction, ultimately leading to extreme inflammation and organ damage. In COVID-19, following its infection of the lungs, SARS CoV-2 virus triggers an immune response of cytokine secretion along with other immune cells, resulting in a hyper inflammation condition called cytokine storm. Numerous studies have shown that MSCs can interact with and regulate the function of immune cells such as B cells, DCs, natural killer cells, macrophages, T cells, and neutrophils [418][419][420]. In COVID-19, the SARS-CoV-2 enters the body by binding to angiotensin-converting enzyme 2 (ACE2), an enzyme located on the membrane of many cell types, including those in the lung [421,422]. MSCs do not contain any ACE2 receptors, making them immune to SARS-CoV-2. Recent research has found that transplanting MSCs in COVID-19 patients has immunomodulatory effects that can prevent the cytokine storm and decrease the damage to tissues through the regenerative potential of MSCs [423,424].
Interestingly, several studies have demonstrated that exosomes derived from MSCs suppress cytokine release and reduce the level of inflammation [418,425,426]. In addition to this, Dinh et al. showed that exosomes can help in lung regeneration in pulmonary fibrosis conditions [250]. Therefore, exosomes could possibly be used in the treatment of critically ill COVID-19 patients in combination with other therapies (Figure 8). To date, more than 90 MSCs based studies for respiratory diseases have been registered for clinical trials (https://clinicaltrials.gov, accessed 25 June 2021). In addition, Ruijin hospital in China registered a pilot clinical study to explore the inhalation of exosomes derived from allogenic adipose MSCs in patients critically ill with COVID-19 (ClinicalTrials.gov Identifier: NCT04276987). Similarly, several other, similar clinical trial studies have been registered worldwide (Table 9). Therefore, there is growing evidence that the use of MSC-Exos in COVID-19 therapy may limit the respiratory complications in patients.

Conclusions and Perceptive
In recent years, it has been established that exosomes have modulatory potential and play a critical role in diverse biological processes. Exosomes show tremendous therapeutic potential for disorders including chronic wound healing, neurological damages, and cardiovascular dysfunction. Exosomes have also gained widespread attention in the field of biomarker research and are now even being seen as an alternative strategy to stem cell-based regenerative therapies. Exosomes can be genetically engineered to deliver distinct therapeutic moieties to a desired target. These cargos include recombinant proteins, antagomirs, short interfering RNAs, antisense oligonucleotides, and immune modulators [427].
Although exosomes have attained significant achievements in several therapies, challenges remain. While numerous proteins, RNAs, lipids, and metabolic enzymes (Tables 2-4) have been identified in exosomes, little is known about their functions and sorting mechanisms. Exosomal cargo is also highly dependent on surrounding milieu and metabolic status of host cells. It remains ambiguous whether natural, physiological levels of small vesicles exert any pathological or regulatory roles in vivo. Despite several exosomal studies in chronic wound healing and skin regeneration, the exact molecular mechanism and the role of exosomes in these processes require further investigations. Additionally, for better exploitation of exosomes, an extensive study is needed in the areas of biogenesis, cellular uptake, and trafficking of exosomes. Moreover, other critical challenges involving the use of exosomes in regenerative medicine require further investigations, such as isolation, purification, optimization, standardization, quality control, and further exploration of molecular mechanisms of exosome communication with target cells. Overall, the potential of exosomes derived from various sources in regenerative medicine and tissue engineering is highly promising.  [CrossRef] [PubMed]