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Fracture Healing and the Underexposed Role of Extracellular Vesicle-Based Cross Talk

Qiao, Zhi∗; Greven, Johannes∗; Horst, Klemens∗; Pfeifer, Roman†; Kobbe, Philipp∗; Pape, Hans-Christoph†; Hildebrand, Frank∗

Author Information

∗Department of Trauma and Reconstructive Surgery, RWTH Aachen University Hospital, Aachen, Germany

†Department of Orthopaedic Trauma and Harald-Tscherne Laboratory, University Hospital Zurich, University of Zurich; Zurich, Switzerland

Address reprint requests to Zhi Qiao, MSc, Department of Trauma and Reconstructive Surgery, RWTH Aachen University Hospital, Pauwelsstreet 30, 52074 Aachen, Germany. E-mail: [email protected]

Received 27 June, 2017

Revised 23 July, 2017

Accepted 20 September, 2017

ZQ is supported by the China Scholarship council (No. 201508080049).

The authors report no conflicts of interest.

doi: 10.1097/SHK.0000000000001002
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Abstract

Fracture healing is a complex process, involving the interaction of immunity, bone, and vessel systems. During bone healing, cells from different systems have to communicate with each other. There are three cell–cell signal transduction modes that are commonly known and accepted (1):

  • (1) secretion molecules,
  • (2) cell–cell contact, and
  • (3) tunneling nanotubules.

Recent studies have introduced the potential for communication via extracellular vesicles (EVs) (2, 3). It is known that EVs from osteoblasts, chondroblasts, and osteoclasts are important for tissue calcification and bone remodeling. Furthermore, studies have shown a crucial role of EV-based cross talk in the physiological and pathophysiological processes of coagulation, inflammation, and osteogenesis (3, 4). These processes are involved in fracture healing, and thus, EV-based cross talk communication occurs throughout the whole process of fracture healing. As different types of EVs can be released under different conditions (5, 6), this review focuses on studies dealing with the role of EVs in the process of fracture healing.

WHAT ARE EVS

EVs are membranous vesicles generated from a variety of cells, with diameters ranging from 30 to 5000 nm. Three kinds of extracellular EVs have been observed: exosomes, shedding microvesicles, and apoptotic blebs (7). Exosomes are cup-shaped membranous EVs, with a diameter of 30 to 100 nm. They generated by inward budding of endosomal membranes into large multivesicular bodies (MVBs), and secreted by exocytosis (8), with surface markers like Alix, HCS70, CD63, CD81, and CD9 (7). Shedding microvesicles are generated by blebbing and shedding of the plasma membrane from almost all cell types, with a diameter of 100 to 1000 nm, and have cell markers such as selectins and integrins (9). In some publications, shedding microvesicles were also called microparticles (10), whereas in some others, microparticles referred as those isolated from platelets or endothelial cells (9). Apoptotic blebs are membrane vesicles generated by apoptotic or dying cells (7), with a diameter of 50 to 5000 nm. Inside of these EVs, proteins, RNAs, small molecules and mitochondria DNA can be found. In this article, we focus on exosomes and shedding microvesicles.

EV-based cross talk

EVs are competent information mediators. The membranous structure of EVs enables them to transport molecules within their inner cytosol, and membrane-dependent molecules at the surface. The specialties of EV-based molecular transport are:

  • (1) transport of RNAs between cells,
  • (2) transport of components of specific pathways, and
  • (3) Transport of surface molecules and membranous molecules.

The transportation of these molecules enables them to coordinate cellular reactions; this process can control gene expression by regulating mRNA turnover, ultimately contributing to the epigenetic and proteomic properties of the target cells (11), as well as changing the maturation and differentiation of target cells (12). At the same time, cells can activate surface molecular pathways of remote cells.

Organelles have also been found to be transferred between different cells by EVs (13). Studies have found mitochondrial DNAs in EVs from platelets, astrocytes, glioblastoma, and bone marrow-derived stromal cells (14, 15). According to the results from recent studies, the release of mitochondria is able to elevate inflammatory reactions (14, 16).

In addition to other mechanisms, the interaction of EVs with cells includes surface molecules, EV-cell fusion, and cargo releasing.

EVs carry surface molecules and transmembrane proteins on their surfaces and, therefore, include characteristics of the cells they were released from. These surface proteins help to interact with specific target cells, increase the specificity of cargo delivery, and trigger signaling cascades via receptor interactions (7).

Through EV-cell fusion, proteins and RNAs can be transported into recipient cells. The EV-cell interactions and fusions are mediated by receptor binding (17). Using membrane-bound structures, EVs can receive signals and fuse with their target cells (3). Due to the presence of surface receptors, EVs only interact with cells they recognize (18).

EVs also have the potential to reach the extracellular spaces and blood, releasing their content and membrane components (18, 19). EVs contain proteinase, cytokines, growth factors, and organelles (1, 20) that function in the extracellular matrix.

EVS FROM FRACTURE HEALING-RELATED CELLS

Osteoblastic EVs (OBEVs)

Osteoblasts play a central role in the fracture healing process. It has already been reported that osteoblasts directly communicate with osteoclasts (4, 21), mesenchymal stem cell (MSC) (22, 23), and others (24), via EVs. Proteomic analysis of OBEVs/exosomes revealed (25) proteins that modulate osteogenesis, which are involved in pathways such as the eukaryotic initiation factor 2 (EIF2) signaling pathway, integrin signaling pathway, Rho-guanosine triphosphate (Rho-GTP) signaling pathway, and mammalian target of rapamycin (mTOR) signaling pathway (25).

OBEVs were observed to induce differentiation of multipotential stem cells into osteoblasts (24, 26, 27). OBEVs/exosomes from mineralizing osteoblasts have the ability to promote the differentiation of bone marrow stromal cells into osteoblasts, a process that is mediated by the Wnt signaling pathway (27). Several studies have also found evidence to show that osteoblasts communicate with osteoclasts through OBEVs. Receptor activator of nuclear factor-κB ligand (RANKL) proteins, which are able to induce generation of osteoclasts, was found in EVs/microvesicles originated from osteoblasts (4). In rats, hypoxia stimulates the release of EVs/microvesicles from osteoblasts, containing vesicular adenosine triphosphate (ATP), which was proved to influence functions of osteoclasts (28).

Mesenchymal stem cell (MSC)-derived EVS (MSCEV)

MSCs play an active role in the processes of immune modulation and tissue regeneration. Cytokines, micro-RNAs (miRNAs), surface molecules, and signaling pathway components from MSCEVs have been shown to be involved in cell–cell communications.

Ischemia-activated MSCs can release EVs with high concentrations of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), and other angiogenesis-related molecules (29). The release of these factors into the extracellular matrix promotes angiogenesis and osteogenesis.

Important fracture healing-related pathways have been found in EVs/microvesicles from activated MSCs, including in the Wnt signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, transforming growth factor (TGF) signaling pathway, peroxisome proliferator-activated receptors (PPAR) signaling pathway, and the bone morphogenetic protein (BMP) signaling pathway (30). A variety of surface molecules from MSCEVs/microvesicles were found to be involved in fracture healing processes, including PDGF receptors, EGF receptors, ezrin (EZR), integrins, and IQ motif containing GTPase activating protein 1 (IQGAP-1) (30). These pathways and molecules are involved in the recruitment and differentiation of MSCs (31), the regulation of endothelial cell proliferation, angiogenesis (32), and epithelial proliferation (33).

A variety of miRNAs have been detected in MSCEVs/microvesicles (34), some of which were also shown to have functions related to fracture healing. Nakamura et al. (35) reported that MSC-derived miRNA promotes myogenesis and angiogenesis. MiR-196a (22) was observed to promote osteogenesis, whereas miR-378 (36), miR-223 (37), and miR-100 (38) were found to inhibit osteogenesis. MiR-378 (39) and miR-223 (40) were also found to be involved in angiogenetic remodeling. It is also reported that, at different time points during fracture healing, MSCs release EVs with different profiles of miRNAs, which are involved in osteoblastogenesis or leukocyte migration (23).

Endothelial EVS (EEVS)

The generation of endothelial EVs was found to be stimulated by inflammatory cytokines, bacterial lipopolysaccharides, reactive oxygen species, thrombin, C-reactive protein, and uremic toxins (41). The generation of EEVs after TNF-α and thrombin stimulation involves several signaling pathways, including the caspase, rho-kinase, nuclear factor kappa B (NF-κB) pathways, and MAPK (41, 42). Tumor necrosis factor α (TNF-α), interleukin-1 (IL-1), and intergrin-1a may amplify the release of EEVs through the NF-kβ and MAPK pathways (43).

EEVs have been found to be important elements in the processes of coagulation, inflammation, angiogenesis, and osteogenesis. In the early phase of fracture healing, phospholipids and proteins at the surface of EEVs were found to control the coagulation equilibrium, among them are endothelial protein C receptor (EPCR), thrombomodulin (TM), tissue factor (TF), von Willebrand factor (vWf), tissue plasminogen activator (tPA), and phosphatidylserines (PS) (43–46). Inflammation-activated EEVs bear inflammation modulating molecules such as platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), and endothelial-selectin (E-selectin) (42, 47). These molecules are known to have diverse roles in vascular biology, including angiogenesis, thrombosis, and regulation of multiple stages of leukocyte migration. At the same time, studies have reported that EEVs modulate proliferation, angiogenesis, and apoptosis of endothelial cells through RNA transportation. Horizontal transportation of miRNAs between endothelial cells by EEVs/microvesicles promotes angiogenesis after Akt activation and endothelial nitric oxide synthase (eNOS) expression (48), and promotes vascular endothelial reparation (49). Furthermore, EEVs contain matrix metalloproteases, which are involved in the extracellular matrix degradation, as well as the release of growth factors that play crucial roles in bone remodeling and angiogenesis (50, 51). The role of EEVs in osteogenesis has been reported in several studies. Inflammation-induced EEVs express molecules, such as PECAM-1 and ICAM-1, which were found to promote osteoclastogenesis (52, 53). EEVs/microvesicles from TNF-α-stimulated endothelial cells were also found to contain a significant amount of BMP-2, and were able to enhance vascular smooth muscle cell osteogenesis and calcification (54).

Osteoclastic EVS (OCEVS)

Osteoclasts are an important element in fracture healing and bone remodeling. A study tested the profile of OCEV-miRNAs from TNF-α and RANKL-stimulated osteoclasts, showing a variety of miRNAs (miR-378, miR-223, mir-210, miR-21, miR-155, miR-146a, and miR-199a) that may be involved in angiogenesis, osteogenesis, and chondrogenesis (55). RANK was also found to be present on the surface of osteoclastic EVs, and is capable of regulating osteoclastogenesis (56). More studies are needed to investigate the communication functions of OCEVs.

Macrophage and monocyte-derived EVS

Macrophages and monocytes are important for both initiation of inflammatory reactions and tissue repair. Granulocyte-macrophage colony-stimulating factor (GM-CSF) (57), lipopolysaccharides (LPS) (58), and Fas ligand (59) were found to be able to stimulate generation of EVs by macrophages.

EVs/microvesicles from cultured macrophages have been shown to transport functional RNA molecules into target cells, including monocytes, endothelial cells, epithelial cells, and fibroblasts (57). EVs/microvesicles from monocytes and macrophages were found to be rich of tissue factor (TF), which can bind platelets through P-selectin glycoprotein ligand-1 (PSGL-1) on the EVs, and P-selectin on the platelets, thereby enhancing coagulation (60). EVs/microvesicles from monocytes were observed to interact with endothelial cells, inducing procoagulatory and proinflammatory endothelial phenotypes (59). In vitro capillary tube formation and in vivo angiogenesis assays have shown that monocyte-derived EVs/microvesicles have strong proangiogenic activities (61). MSCs/exosomes incubated by monocyte-derived EVs showed increased runt-related transcription factor 2 (RUNX2) and BMP-2, indicating the osteogenic differentiation of MSCs (58).

Neutrophil-derived EVS (NEVS)

The initiation of an inflammatory and coagulatory response has the potential to stimulate the generation of NEVs. TNF-α, the complement system, IL-8, platelet activating factor protein kinase C activator, and nitric oxide synthase inhibitor have been found to increase the production of NEVs (62). It has been reported that, under inflammatory conditions, the amount of NEVs will increase significantly, compared with normal conditions (63, 64).

Although there are no studies referring to the functions of NEVs in fracture healing, several studies have indicated a role of NEVs in fracture healing-related reactions. During coagulation, platelets were found to be activated by NEVs in an Akt phosphorylation-dependent manner (65). In addition, NEVs seem to be important inflammation modulators, as they may have both anti-inflammatory (10, 66) and proinflammatory functions (10, 67, 68), depending on the background of the microenvironment (10, 62). Studies on proteomes of NEVs/microvesicles have revealed over 400 proteins, covering mainly integrin signaling, EGF, and FGF signaling pathways (10). Although it has not been proven yet, the EGF and FGF signaling pathways found in NEVs/microvesicles may stimulate the differentiation of MSCs and angiogenesis (10).

Platelet-derived EVS (PEVS)

Platelets are activated under coagulatory and inflammatory conditions, with the subsequent release of large amounts of functional PEVs (69). A study demonstrated that 87% of the release of platelet-activating factor (PAF), a key factor for triggering inflammatory and thrombotic cascades, was associated with EVs (70, 71). There is also evidence that PEVs may transfer platelet-specific immunoreactive antigens to the surface of other cells, modulating cellular functions (72).

In the early phase of fracture healing, expansion of coagulation platelet–platelet cross talk is one of the most important features of PEVs. PEVs express anionic phospholipids (AP) and TF on their surfaces; arachidonic acids (AA) from platelets were reported to have the potential to activate the clotting cascades in platelets (73, 74). PEVs have also been found to be able to bind to the subendothelial matrix in vitro and in vivo, acting as substrates for further platelet binding (75).

Posttraumatic modulation of inflammation by PEV-based cross talk was shown in several publications (76). In a clinical study, high posttraumatic levels of PEVs were associated with the development of acute respiratory distress syndrome (ARDS) and multiple organ failure (MOF) (73). Through PEV AA and bioactive prostanoids, PEVs were found to promote inflammatory reactions by activating MAPK pathways (77). Through the MAPK pathway, PEVs can induce the production of important inflammation mediators like Cyclooxygenase-2 (COX-2) and prostacyclin (PGI2), in human endothelial cells (77). PEVs were shown to activate monocytes and macrophages, and reprogram their functions toward a phagocytic phenotype, by regulating their RNA expression (78) and activating the NF-kβ pathway (79). Neutrophils (80) and leukocytes (16, 81) are also the targets of PEVs. One study found that the binding of PEVs to neutrophils can induce a significant increase in phagocytic activity in a concentration-dependent manner (80). Mitochondria found in PEVs can perform as inflammation mediators, activating leukocytes and neutrophils, causing inflammatory responses (16).

Studies have shown that PEVs can modulate the proliferation, survival, chemotaxis, and function of endothelial cells through the VEGF pathway (82) and by argonaute 2 (Ago2)-microRNA complex-delivering (83). It has also been demonstrated that PEVs are able to stimulate mitogenic activity of bone cells, contributing to bone regeneration (84). In addition, a study showed that surface molecules on PEVs can active hematopoietic cells by chemotaxis, increasing adhesion, proliferation, and survival, and activating signaling cascades including MAPK p42/44, phosphatidylinositol-3 kinase (PI-3K), AKT, and signal transducer and activator of transcription (STAT) proteins (85).

OTHER CELLULAR EVS INVOLVED IN FRACTURE HEALING PROCESSES

Other cell types have also been observed to release EVs under inflammatory conditions. Even though there is limited evidence in the literature, the existing data may provide the first evidence for the role of EV-based cross talk in fracture healing. Erythrocytes activated by lysophosphatidic acid can secrete procoagulatory EVs, which contribute to coagulation processes (86). Vascular smooth muscle cells incubated by LDL are found to produce TF-bearing EVs, promoting coagulation cascades (87).

EVs from chondrocytes have been detected in the growth plate. EVs are released in a polarized fashion from the lateral edges of growth plate chondrocytes. They have been found to be involved in the mineralization of bone matrix (88). Later, BMP, VEGF, bone sialoprotein (BSP), osteonectin, and osteocalcin were also found in these EVs, highlighting the communicative role of chondroblastic EVs (88–91). VEGF, if released into the matrix, regulates angiogenesis and osteogenesis, and stimulates endothelial cells, chondroblasts, and osteoblasts (92). BSP in EVs was shown to promote differentiation of osteoblasts and repair of bone defects (91), as well as to promote differentiation of chondrocytes and osteoblasts (in growth periods) (90). Furthermore, osteopontin, osteonectin, and osteocalcin were found to be able to inhibit mineralization in vitro(88).

EV-BASED CROSS TALK NETWORK DURING FRACTURE HEALING

Fracture healing involves very complex systemic reactions. It involves sophisticated cooperation between a variety of cells, cytokines, and inflammatory factors (Fig. 1). In this article, we will briefly overview the network of EV-based cross talk during fracture healing, highlighting the networks of cytokines and inflammatory factors.

F1
Fig. 1:
Known MV-based cross talk mechanisms during fracture healing.

Coagulation and inflammatory reactions are the first steps for fracture healing. In this phase, the ruptured blood vessels, tissue necrosis, and other ongoing damage stimulate the inflammatory cascades. The activation of platelets, neutrophils, and macrophages by hypoxia, coagulation, and inflammation is observed to produce not only a variety of cytokines and inflammatory factors, but also large amounts of active EVs (1, 93). The appearance of EVs may indicate that they have important functions.

A short time after blood vessel and tissue injury, procoagulatory EVs can be released from platelets and neutrophils. The positive feedback model of PEVs seems to further stimulate PEV productions (74), as well as activate and attract neutrophils (16, 80) and macrophages (78, 79). EEVs from activated endothelial cells also promote coagulation via EPCR, TM, TF, vWf, tPA, PS, and plasminogen (43, 94, 95).

Inflammatory background is important for fracture healing. EVs derived from a variety of cells participate in the inflammatory regulation of the fracture site. PEVs are able to stimulate the differentiation of monocytes and macrophages, inducing the production of proinflammatory factors (78, 79). The activation of neutrophils results in secretion of NEVs that also regulate inflammatory reactions. In addition, NEVs can increase the inflammatory response by the activation and aggregation of macrophages (67), inducing production of IL-6, IL-8, MAC-1, and oxygen active species (10, 96). However, NEVs can also inhibit the aggregation of neutrophils (97), induce the production of anti-inflammatory factors from macrophages such as IL-10 and TGF-β1 (66, 98), and inhibit proinflammatory actions of endothelial cells (10). Although EVs from MSCs have been shown to have the potential to modulate the inflammatory response, further studies are needed to clarify their role in the process of fracture healing.

A variety of EVs take part in angiogenesis modulation during fracture healing. EVs from platelets, neutrophils, macrophages, MSCs, and endothelial cells have been shown to have the ability to stimulate differentiation of stem cells or endothelial cells. EVs from MSCs (29, 31) and chondrocytes (92) have been shown to release multiple growth factors, providing a microenvironment for angiogenesis and tissue regeneration. Surface molecules from MSCEVs (32, 33) and EEVs (99) have also been shown to modulate angiogenesis. Pathway molecules and miRNAs from PEVs (83), MSCEVs (35), EEVs (48, 49, 100), and osteoclast-derived (101) and monocyte-derived EVs (61) also participate in the modulation of angiogenesis.

During tissue regeneration and bone remodeling, multiple EVs take part in the process of activation of fibroblasts, chondroblasts, and osteoblasts. EVs from MSCs have a significant role in wound healing. They were found to enhance wound healing via promotion of collagen synthesis and angiogenesis (102). One study also found that MSC-derived exosomes caused an increase in proliferation and migration of fibroblasts (103). In the microenvironment of osteogenesis, a mixture of EVs has been found to modulate the osteogenesis reactions. Matrix vesicles found in the growth plate serve as carriers of morphogenetic information to nearby chondrocytes and osteoblasts. These matrix vesicles were observed to be released from the lateral edges of growth plate chondrocytes, the osteoid-facing surfaces of osteoblasts, and the apical surfaces of odontoblasts (88). Cultured chondrocytes treated with these matrix vesicles showed a 2- to 3-fold increase in alkaline phosphatase activity (88). OBEVs contain miRNAs and pathway molecules, which have the potential to stimulate osteogenesis of MSCs (26, 27), and communicate with osteoclasts for bone remodeling (4, 28). Molecules from EEVs, such as BMP-2 (54) and PECAM-1 (104), have been shown to involve in the modulation of angiogenesis, osteogenesis, and bone remodeling.

In addition to their communicative functions, EVs from osteoblasts, chondroblasts, and osteoclasts are also involved in osteogenesis and bone remodeling. They are the practical workers for bone matrix generation and calcification. Aspects of matrix generation, calcification, and bone remodeling are reviewed elsewhere (105).

A summary of EV-based miRNAs related to osteogenesis, obtained from ExoCarta (12/2016) (106), can be found in Figure 2, Figure 3, and Table 1.

F2
Fig. 2:
MV-originated miRNAs related to osteoblastic functions.
F3
Fig. 3:
MV-originated miRNAs related to endothelial cell functions.
T1
Table 1:
Summary of EV-based miRNAs related to osteogenesis

THE POTENTIAL APPLICATION VALUE OF EVS DURING FRACTURE HEALING

Molecular diagnosis

Multiple components of EVs, especially miRNAs, are suggested as biomarkers for molecular diagnosis. Several studies have discussed the diagnostic value of EVs in neurological disorders (107), cardiovascular diseases (108), osteoporosis (48), and sepsis (109).

With regard to the skeleton, the diagnostic potentials have also been investigated. One study tested the proportion of EVs in rheumatoid arthritis (RA) patients. The authors found an increase of plasmatic CD3(+), CD14(+), CD19(+), CD41(+), CD62E(+) MPs, and urinary CD14(+), CD3(+), and CD19(+) MPs in RA patients with high disease activity (110). More interestingly, compared with a healthy population, the level of miR-92a was found to decrease 24 h after a bone fracture, whereas it was decreased 14 days after a lumbar compression fracture (111). These studies showed the potential of EVs as a diagnostic tool. However, more research is needed to bring this marker into clinical practice as a routine parameter.

EV-based therapies

Compared with proteins and RNAs, EV structures may be more tolerant to the microenvironment in vivo(112). Thus, the use of EEV-mediated delivery is expected to be more efficient than using soluble proteins or RNA molecules. One study also indicated that EV structures may be at lower risk of immunogenicity and toxicity (112).

The therapeutic potential of EVs has been studied in the treatment of several diseases, such as acute lung injury (113), myocardial ischemia/reperfusion injury (114), kidney injury (115), peri-implant bone defect (116), and others; the results seem to be promising. Moreover, some researchers have developed manufactured EVs to improve treatment of fracture healing. In this context, researchers aimed to remodel cell-derived EVs, thereby making them more effective for treatment. These small molecules, miRNAs, and exogenous proteins have been successfully delivered by EVs (117, 118). In addition, specifically generated gelatin EVs have been used as vehicles to transport molecules. With this technique, bone defects have been treated successfully by transporting BMPs, VEGFs, TGF-β, and other molecules (119, 120), to target cells. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were also shown to be a potential vehicle in guiding bone regeneration (121).

PROSPECT FOR RESEARCH AND CLINICAL USE

To date, the mechanism of EVs involvement in bone matrix generation and mineralization has been thoroughly investigated. However, there is still little information about the role of information transfer during the process of fracture healing. Therefore, further studies should focus on this area of research. To date, knowledge gained from studies has only shown the important role of EV-based communication in fracture healing, whereas the more detailed mechanisms remain unknown.

Many potential advantages of EVs make them worthy for future studies. EVs can be easily collected from blood or body fluids, with comparable characteristics of their original solid cells; this is advantageous for the diagnostic application of EVs. MiRNAs and surface molecules in EVs are more stable. However, little is known about the specificity of EVs in fracture healing-related states. Further studies are needed to look into the components and surface markers of EVs that may be able to predict the fracture healing states and the potential threats.

The application of EVs in tissue engineering offers many possibilities for treatment options. Researchers have tried to use the advantages of EVs in improving fracture healing and treating bone defects, with optimistic results. However, some restrictions limit their large-scale clinical use. For example, the production of EVs should be generated with more specific components, and needs to be produced in large scale. Thus, specific tissue-engineering cells maybe a good option for the next research step. Tissue-engineering cells or bacteria can produce highly concentrated and purified EVs. At the same time, highly specified surface molecules can accurately aim at targeted cells. In addition, the clearance of EVs in vivo also needs to be considered; more stable EVs may need to be developed for effective treatments. Furthermore, man-made EVs represent a very promising field for research and treatment.

CONCLUSIONS

EVs play an important role during fracture healing, with their information transportation and bone matrix generation. Although the mechanism of EVs involvement in bone matrix generation is clear, the mechanism of EVs role in information transportation remains underexposed and is a field of research that requires further study. It is likely that, in the near future, EVs will be useful in clinical diagnosis and treatment.

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      Keywords:

      Cross talk; endothelial cell; extracellular vesicle; intercellular communication; miRNA; osteoblast; osteoclast; platelet

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