Exosomes promise better bone regeneration

Abstract

Fractures primarily result from high-energy trauma, leading to structural discontinuity of bone tissue. Contemporary therapeutic approaches continue to face persistent challenges including nonunion, infection, and inflammatory complications that pose significant clinical management difficulties. Emerging evidence demonstrates that extracellular vesicles (EVs), particularly exosomes, serve as critical mediators in diverse pathophysiological processes. Accumulating studies reveal that exosomal cargos enhance osteogenesis and angiogenesis through dynamic regulation of cellular components and molecular networks within the bone remodeling microenvironment, thereby potentiating fracture healing cascades. This comprehensive review systematically examines the mechanistic contributions of exosomes in coordinating osteoblastic differentiation, osteoclastic activity modulation, and neovascularization processes. In addition, we describe the role of exosomes from different cellular sources (e.g., mesenchymal stem cells, endothelial progenitor cells, and osteoblasts) in fracture repair. Finally, this paper elaborates on the potential challenges and future directions for the development of novel exosome-based therapeutic strategies for clinical fracture repair.

Graphical abstract

Role of exosomes in bone remodeling.

1. Introduction

A bone fracture is defined as the disruption of bone integrity or continuity and commonly occurs due to direct or indirect traumatic injuries, such as muscle strain or skeletal diseases. Following a fracture, haematoma rapidly develops, inducing the recruitment, proliferation, migration, and differentiation of progenitor cells and bone marrow stromal cells into osteoblasts and chondrocytes. This process facilitates the formation of healing tissue to bridge the fracture site and stimulates vascularization to provide nutrition and support to the regeneration of the skeleton. The ultimate outcome of this process is the reconstruction and restoration of the damaged bone tissue to its original structure and function, facilitated by mechanisms involving bone formation and resorption [1].

The reparative process of fracture healing is subject to regulation by various factors, including the nature and extent of the injury sustained, the stability of fracture fixation, and biological processes, such as immunological and developmental processes associated with bone ontogeny. The process is characterised by an increase in tissue volume, which is related to the de novo recruitment and differentiation of stem cells involved in the formation of bone and vascular tissue. While the majority of fractures do indeed result in the restoration of the cellular composition, structure, and biomechanical function of the injured skeletal organ to its pre-injury state, it is important to note that approximately 10 % of fractures fail to achieve normal healing standards or remain nonunion [2]. At present, the standard treatment for nonunion fractures is surgical intervention, frequently necessitating multiple revision surgeries. This results in protracted treatment times, heightened complication risks, considerable physical and mental health implications for patients, and a significant socioeconomic burden [3]. It is imperative, therefore, to develop novel therapeutic approaches that focus on promoting bone tissue healing and regeneration while simultaneously minimising the potential adverse effects associated with surgery.

EVs are membrane vesicles that are secreted by almost all cell types [108]. The classification of EVs is typically divided into three distinct categories, which are distinguished by their unique production pathways. These categories include exosome, microvesicle and apoptotic body. Of these, EVs with a diameter of 30–150 nm are termed 'exosomes' [106]. Exosomes are derived from the endocytic pathway and are released from the multivesicular bodies (MVBs), which contain multiple vesicles generated by the fusion of the exosome with the cell membrane. The generation of these particles is a process that occurs in four distinct stages, namely budding, invagination, MVB formation and secretion [99]. Extracellular vesicles (EVs) are cell-derived nanoscale vesicles that facilitate intercellular transport. The loading of therapeutic drugs, including small molecules and nucleic acids, into EVs allows for targeted delivery to specific cell types or tissues, thus facilitating precision medicine [[4], [5], [6], [7], [8], [9], [10], [11], [12], [13]]. It has been demonstrated that EVs have a pivotal function in the process of fracture repair. The advantages of EVs include high stability, low immunogenicity and strong targeting ability, which serve to overcome the limitations of traditional treatment methods [14]. This article provides a review of the role of exosomes in regulating osteoblastogenesis, osteoclastogenesis, and angiogenesis, as well as their therapeutic applications in bone fracture healing. It provides a comprehensive overview of the recent advancements in research concerning the promotion of fracture repair by exosomes derived from diverse sources. The objective of this review is to offer novel insights that will inform future treatment strategies for fracture repair.

2. Overview of exosomes

Exosomes are formed as a consequence of the endosomal maturation system, and their biogenesis involves early endosomes, late endosomes, and multivesicular bodies. The intraluminal vesicles are secreted in the form of exosome, with a diameter of approximately 30–150 nm, through fusion with the plasma membrane and exocytosis of multivesicular bodies (Fig. 1). These intraluminal vesicles released into the extracellular space are referred to as exosome [15]. As these particles emerge from endosomes, they are characterised by a substantial presence of diverse biomolecular components originating from the originating cells. These components encompass metabolites, lipids, proteins, and nucleic acids [16]. The membranes of classical exosomes contain tetraspanins, such as CD63, CD81, and CD9, as well as other marker proteins including heat shock proteins, adhesion molecules, integrins, and endosomal sorting complex required for transport (ESCRT)-associated proteins such as Alix and TSG101. The high abundance of tetraspanins, Alix, TSG101, and HSP70 has led to their widespread utilisation as exosomal marker proteins. Furthermore, exosome composition has been shown to include various genetic materials, including small and long non-coding and coding RNAs, as well as microRNAs (miRNAs) [17,18].

Fig. 1.

The biological journey of exosome: from biogenesis to uptake and its structural composition.

Exosomes are responsible for the transmission of signals between cells via the active biomolecules they carry, which include proteins, nucleic acids and metabolites. Upon uptake by recipient cells through mechanisms such as phagocytosis, micropinocytosis, endocytosis mediated by caveolin-dependent internalization, clathrin-dependent endocytosis, and plasma membrane fusion, these molecules trigger intracellular signaling in target cells [19]. The biological activities of these complexes are attributed to the unique properties they possess in regard to intercellular communication. It is widely acknowledged that they play a crucial role in the regulation of fundamental physiological processes, as well as in the diagnostic and therapeutic management of disease [20]. Exosomes have been shown to be important mediators of intercellular communication and to play unique biological roles in the regulation of normal physiological processes, as well as in the diagnosis and treatment of disease. Upon delivery of miRNAs to specific target cells via exosome, the resulting regulatory influence on diverse aspects of cell phenotype and behaviour becomes apparent. These aspects include, but are not limited to, the cell cycle, apoptosis, migration, inflammation, and angiogenesis [21]. It is evident that, in consideration of the aforementioned characteristics, the engineering of exosome-based delivery systems can be utilised to facilitate the delivery of specific therapeutic molecules. These molecules include short interfering RNAs, antisense oligonucleotides, chemotherapeutic agents and immunomodulators, which can be directed towards target cells with the intention of achieving therapeutic outcomes [22]. In comparison to alternative synthetic drug delivery systems, such as liposomes, nanoparticles, microspheres, and micelles, exosome-based natural nanocarriers demonstrate high biocompatibility, low immunogenicity, and low toxicity. Moreover, they possess the capacity to traverse the blood-brain barrier, rendering them optimal for utilisation as drug carriers [20,23].

Recent studies have demonstrated the significant role of exosome in orthopaedic diseases. These membrane-bound vesicles act as mediators of intercellular communication and material exchange in physiological and pathological processes. In addition to this, they facilitate the safe delivery of various biologically active substances to target cells through various pathways and sites. This process enables the participation of exosome in physiological regulation, including repair of bone and cartilage tissue, and the regulation of bone metabolism [24,25]. This provides a novel perspective for the treatment of various orthopaedic diseases, including osteoarthritis, rheumatoid arthritis, osteoporosis, femoral head necrosis, bone defects, and fractures. In the context of fracture healing, researchers have identified that exosome therapy from diverse sources can play a pivotal role in stimulating bone regeneration or repair processes through the activation of various signalling pathways and the regulation of gene expression mechanisms [26]. This article systematically reviews the sources of exosomes and their biological effects in fracture repair, including the generation mechanism and characteristics of these vesicles, as well as their positive roles in fracture repair. The review provides guidance for future research and clinical treatment of fractures.

3. The molecular mechanisms of exosomes in bone fracture repair

3.1. Exosomes and miRNAs

miRNAs, a class of non-coding RNAs, have been shown to play a crucial role in the regulation of gene expression. This process involves the targeting of mRNAs for degradation or the inhibition of their translation [27]. Primary microRNA (pri-miRNA) is first transcribed by RNA polymerase II, and then processed by ribonucleases into hairpin precursor microRNA (pre-miRNA). Subsequent to this process, pre-miRNAs are transported to the cytoplasm, where they generate double-stranded mature miRNAs. One strand is incorporated into the miRNA-induced silencing complex (miRISC), which contains the Argonaute protein, and interacts with target mRNA transcripts, resulting in gene expression inhibition. It has been demonstrated that miRNAs significantly regulate cell growth and metabolism by post-transcriptionally suppressing gene expression [28]. Despite the capacity of bones to undergo self-repair, intervention is frequently necessary to stimulate regeneration in damaged bones when effective regeneration at the site of fracture is not possible.

In recent years, extensive research has confirmed that miRNAs are key regulatory molecules in fracture healing. Their simple modulation with agonists and antagonists renders them ideal targets for future fracture treatment strategies, especially for pathophysiological fractures with non-union or delayed union of fractures [29,30]. It has been established that mesenchymal stem cells derived exosome (MSC-exos,derived from Rat) and endothelial progenitor cells derived exosome (EPC-exos,derived from human) carry microRNA-126, which has been shown to further activate the vascular endothelial growth factor receptor 2 (VEGFR2) signal transduction pathway [109,111]. It has been reported that miR-22 is one of the key regulators in the differentiation switch from stem cells to bone cells. For example, loading miRNA-22 and miRNA-126 mimics into PCL-NFs (polycaprolactone nanofibres) can promote the cellular viability and osteogenic differentiation of human iPSCs (induced pluripotent stem cells) and increase the expression of osteogenic cell markers, including RUNX2, BGLAP, ALP and SPARK. The loading of miRNAs, such as miR-22 and miR-126, onto polycaprolactone (PCL) nanofibres (NFs) as bone implants has been demonstrated to enhance the osteogenic differentiation [31]. A study of microarray data from a rat femoral fracture revealed that 317 miRNAs were found to be upregulated in normal healing fractures, with eight displaying the highest levels of expression. These include rno-miR-140-3p, rno-miR-140-5p, rno-miR-181a-5p, rno-miR181d-5p, rno-miR-208b-3p, rno-miR-451a, rno-miR-743b-5p, and rno-miR-879-3p [32]. It has been established that microRNAs (miRs) such as miR-181a-5p, miR-140-3p and miR-140-5p play a regulatory role in the initial stages of inflammation during fracture healing. This is achieved by targeting the NCOA1, NRIP1 and IL1A genes. Treatment with a mimic of miR-181d-5p in human mesenchymal stem cell experiments demonstrated that the suppression of miR-181d-5p expression can promote osteogenic differentiation through the MAPK pathway targeting RUNX2, and also through TGF-β secretion to facilitate osteoblast differentiation. Furthermore, both miR-181a-5p and miR-181d-5p have been shown to induce apoptosis in bone cells by upregulating BCL2. MiR-208b-3p, another highly expressed microRNA in fracture healing, has been shown to inhibit osteoblastogenesis in MC3T3-E1 cells(derived from mouse) by reducing Acvr1b translation, thereby suppressing bone formation and decreasing Bmp2 as well as downstream targets Smad1/4/5 and Runx2 [29]. Moreover, research findings have indicated a correlation between the expression of microRNA-92a-3p and the reduced expression of integrin-binding sialoprotein (IBSP) during tissue formation in response to injury. In vitro, the over-expression of miRNA-92a-3p has been demonstrated to inhibit IBSP expression and accelerate osteoblast differentiation, while the silencing of miRNA-92a-3p has been shown to suppress osteoblast activity. The reduction of IBSP has been demonstrated to promote osteoblast differentiation through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signalling pathway, and the expression of miRNAs-92a-3p has been shown to promote the differentiation of MC3T3-E1 cells into osteoblasts. Consequently, these findings imply that microRNA-92a-3p may be a pivotal factor in enhancing fracture healing in trauma patients [33].

Exosomes have the capacity to transport miRNAs through both paracrine and endocrine mechanisms. The composition of miRNAs loaded into exosome is primarily determined by the surface molecules of the exosomal membranes, the intracellular sorting complexes required for transport, and the specific binding motifs of the miRNAs themselves. The secretion of exosomal miRNAs is determined by the type and severity of tissue damage, and is part of maintaining the balance in the body [28]. In their study, Lijuan Yu et al. sought to ascertain the influence that exosome derivatives from osteoblasts (MDA PCa 2b,derived from human), osteoclasts (PC3,derived from human), and mixed prostate cancer cell lines(C4-2,derived from human) exert on the differentiation of osteoblasts and osteoclasts. The study revealed that all three types of exosomes promoted osteoclast formation in vitro and induced bone resorption in vivo. The results indicated that those exosomes derived from osteogenic tumors promoted osteoclast differentiation and inhibited osteoblastogenesis through the transfer of specific miRNAs. Among the delivered miRNAs, miR-92a-1-5p was found to be the most abundant, which downregulates the expression of type I collagen by directly targeting COL1A1. This disruption of bone homeostasis is known to promote osteoclast differentiation while inhibiting osteoblast differentiation. Consequently, it can be concluded that prostate cancer-derived exosome and metastatic miRNAs play a crucial role in disrupting bone homeostasis, degrading the bone matrix, inducing pathological bone remodeling, and ultimately creating a pre-metastatic niche for tumour growth [34].

There is evidence to suggest that certain miRNAs within the bone microenvironment regulate the proliferation, migration, and differentiation of bone marrow mesenchymal stem cells (BMSCs). These cells are involved in the process of bone repair and regeneration during fracture healing, and this regulation is achieved through exosomal delivery [35]. Recent studies have identified a role for miRNAs derived from macrophage-derived exosome in promoting the differentiation of bone marrow stromal cells (BMSCs) and influencing the process of fracture healing. Research has demonstrated that miR-5106 is highly expressed in macrophage-derived exosome (M2D-Exos,derived from mouse). These exosome have been found to induce osteogenic differentiation of BMSCs(derived from mouse) by directly targeting the SIK2 and SIK3 pathways [36]. It is noteworthy that in diabetic mice, the level of miR-144-5p was found to be considerably elevated in the exosome(dBMDM-exos,derived from rat) derived from bone marrow macrophages. These exosome have been shown to deliver miR-144-5p to BMSCs(derived from rat) and to regulate bone regeneration by targeting Smad1. This finding provides a novel and effective intervention target for the treatment of impaired fracture healing in diabetes [37]. Yun B. et al. used RNA sequencing to analyse the expression profiles of miRNAs in exosomes originating from the colostrum and mature milk of humans, cows and goats. They found that these exosome-derived miRNAs from the diet are highly conserved in human, bovine and caprine milk. Furthermore, the most abundant miRNAs in human milk are also conserved across species [114]. Zhou Y et al. isolated exosomes from rat PC12 cells and assessed their internalization by human cervical cancer (HeLa) cells. The expression of PTEN in HeLa cells was significantly deregulated due to the delivery of miR-21 by rat cell exosomes. The results show that exosomes can transfer between cells of different species and regulate protein expression in recipient cells by delivering enclosed miRNAs. This study suggests that we can utilise exosome vehicles containing miRNAs, regardless of species, to combat various diseases and regulate abnormal proteins [115].

In consideration of the research findings, it can be concluded that miRNAs function as pivotal regulatory molecules in the process of fracture healing, exerting a substantial influence on the promotion of bone formation and fracture repair through the of exosome. This provides a reliable theoretical basis for the clinical treatment of fracture healing.

3.2. Exosomes and osteoclasts

Osteoclasts, which are derived from the hematopoietic stem cell lineage, represent the primary cells involved in the process of bone resorption. On one hand, they secrete and synthesize metabolic cytokines, thereby mediating cell-to-cell crosstalk with endothelial progenitor cells, and promoting angiogenesis by releasing PDGF-BB15. On the other hand, they promote bone formation through RANKL reverse signalling. These primary mechanisms are of pivotal significance in the processes of bone remodeling and metabolism [38].

In a recent study, Raw264.7 cells(derived from mouse) were utilised as target cells to evaluate osteoclast differentiation through Trap staining and Trap activity detection after six days of treatment with recombinant sRANKL (Soluble Receptor Activator of Nuclear factor-kB Ligand) or non-recombinant mouse sRANKL combined with exosome or microRNA. The results of this study demonstrated that besides its role in osteoclast regulation, exosome also promotes osteoclast differentiation in a manner that is independent of RANKL. This finding suggests that exosome has the capacity to induce osteoclast generation [39]. In another animal experiment, the role of Circ_0008542 in osteoclasts was validated through the process of m6A(N6-methyladenosine) methylation, which occurs on the RNA molecule. The results demonstrated that RNA-m6A methylation plays a pivotal role in the post-transcriptional regulation of Circ_0008542 following exosome injection. The molecular sponge effect of Circ_0008542 in osteoclasts is facilitated by the interaction with the miR-185-5p/RANK axis, thereby promoting osteoclast differentiation and inducing bone resorption. Furthermore, the study demonstrated that the effects of osteoclast differentiation and bone resorption induced by exosome injection with Circ_0008542 overexpressed in MC3T3-E1 cells(derived from mouse) could be reversed by inhibiting METTL3 or overexpressing ALKBH5 [39]. However, studies have also found that exosome from PC-3 cells(derived from human) can inhibit osteoclast(derived from human) differentiation by downregulating microRNA-148a and blocking the PI3K/AKT/mTOR pathway [40]. Moreover, it has been demonstrated that exosome(PC-3-Derived Exosomes) can inhibit osteoclast(derived from human) differentiation by downregulating microRNA-214 and blocking the NF-κB signalling pathway [41]. TRAP-binding peptide-functionalized exosome(derived from mouse RBCs) that carries miR-214 can inhibit osteoclast differentiation and reverse bone loss [42]. Exosomes are responsible for the transmission of membrane components and the cytoplasmic content between donor and recipient cells, including proteins, lipids and genomic materials, i.e. messenger RNAs (mRNAs) and miRNAs, thereby facilitating intercellular communication [97,107].

These findings suggest that exosome regulation of osteoclast differentiation is achieved through cell-to-cell communication in the microenvironment, which may have implications for bone remodeling and fracture repair.

3.3. Exosomes and osteoblasts

Osteoblasts are primarily derived from mesenchymal stem cells located in the periosteum and matrix of bone marrow. These cells have the capacity to secrete a variety of bioactive substances, which are specifically to provide a cellular source for the repair of bone defects. In addition, they secrete bone-related extracellular matrix, thereby accelerating the process of bone repair.

Researchers have extracted exosome-rich fractions from mesenchymal stem cells(derived from rat) in bone marrow and added them to target cells in osteoporotic rats. The results obtained demonstrated that the levels of p-p38 and p-JNK in recipient cells were significantly increased in the exosome-treated group, thus indicating that exosome derived from mesenchymal stem cells activated the MAPK pathway to promote hFOB 1.19 cell(derived from human) proliferation and subsequently facilitate osteoblast differentiation, thereby inhibiting the progression of osteoporosis [43]. Liu et al. found that exosome derived from macrophages(Raw 264.7,derived from mouse) stimulated by different concentrations of zinc ions significantly increased alkaline phosphatase (ALP) activity in osteoblasts(MC3T3-E1 Subclone 14 pre-osteoblasts,derived from mouse) and promoted endothelial cell migration after downregulating the gene expression levels of M1 and M2 markers. However, further research is needed to investigate the in vitro and in vivo osteogenic and angiogenic abilities of these particles and their underlying mechanisms [44]. Furthermore, Jia et al. investigated the impact of neutrophil-derived exosomes(HL-60,derived from human), stimulated by sodium urate, on bone erosion development in gout. Results indicated that these exosomes could hinder osteoblast(hFOB, derived from human) activity through miRNA-1246, this leads to a decrease in ALP and OPG expression levels, coupled with an elevation in RANKL expression. Through its impact on the bone morphogenetic protein pathway, this particular miRNA assumes a crucial function in overseeing the activities of osteoblasts during the process of bone formation [45].

In conclusion, exosomes from various sources can regulate the role of osteoblasts in bone formation through different mechanisms, providing new ideas and methods for clinical treatment of fracture repair.

3.4. Exosomes and endothelial cells

The function of endothelial cells is generally understood to be the protection and transportation of substances, the control of vascular permeability, and the regulation of vascular tone. However, it is important to note that endothelial cells can also mediate immune reactions at sites of injury or infection, thereby serving as a central and active component of both the immune and vascular systems. The initial vascular plexus is established by merging vascular cells, and the final blood vessels are generated by budding of endothelial cells. This process is known as angiogenesis and facilitates the delivery of nutrients and a plentiful blood supply, thereby enabling tissue regeneration and repair [46].

Previous studies have shown that stimulation with rWNT5A in melanoma cells with low endogenous WNT5A expression can induce rapid release of exosomes containing immune regulatory cytokines IL-6 and pro-angiogenic factors IL-8, VEGF, and MMP2 [46]. In their study, Lin et al. investigated the effects of HeLa cell-derived exosomes(derived from human) on endothelial tight junctions and the underlying mechanisms responsible for these effects. Following the administration of engineered HeLa cell-derived exosomes to human umbilical vein endothelial cells, a significant reduction in the TJ protein-occludin and Claudin-5 was observed, resulting in increased permeability of the endothelial monolayer. In addition, the injection of HeLa cell-derived exosomes into mice resulted in an increase in vascular permeability in vivo, and under specific conditions, HeLa cells were found to be capable of disrupting vascular integrity [47]. A study using biochemical methods and animal experiments has determined the biological activity and potential mechanisms of exosomes released from WERI-Rb1 retinoblastoma cells(derived from human) in tumor angiogenesis. The experimental data showed that exosomes can be engulfed by human umbilical vein endothelial cells, significantly promoting cell viability and inducing an inflammatory response in endothelial cells by increasing the expression of a series of related genes such as IL-1, IL-6, IL-8, MCP-1, VCAM1, and ICAM1. Remarkable increases in migration and tube formation were also observed in human umbilical vein endothelial cells incubated with exosomes. Furthermore, experiments using a xenograft model in nude mice showed a significant increase in the number of endothelial cells and blood vessels in tissues treated with exosomes compared to control tissues [48]. The Dll4-Notch pathway has been reported to play an important role in the coupling of bone formation and angiogenesis. Disruption of this pathway has been demonstrated to affect both angiogenesis and bone formation [102].

Angiogenesis in the bone is coupled to ossification. In long bones, endothelial cells interact with osteoprogenitors during bone formation and healing. During the process of bone development, the formation of intraosseous blood vessels is of critical importance, and exosome is pivotal in this process [100].

4. Role of exosomes in bone remodeling

4.1. Mesenchymal stem cells derived exosomes (MSCs-exos)

Mesenchymal stem cells (MSCs) are a population of stromal cells found in bone marrow and most connective tissues. These cells possess the capacity to differentiate into mesenchymal tissues, such as bone and cartilage, by regulating the Wnt and transforming growth factor-beta/bone morphogenetic protein signalling pathways [49].

The implantation of mesenchymal stem cells (MSCs) has been the subject of extensive research in preclinical studies on the regeneration of bone tissue. mounting evidence suggests that transplanted MSCs play a pivotal role in promoting bone regeneration through paracrine signalling pathways [50,51]. Therefore, exosomes, as the main paracrine effectors, have attracted growing attention in promoting fracture healing [52]. Liu et al. directly promoted the angiogenic capacity of endothelial cells in vitro and the ability of new blood vessel ingrowth in vivo by stimulating exosomes derived from bone marrow stromal cells(derived from rat) using a lithium-containing bioactive scaffold. They induced the expression of exosomal miR-130a, which promoted endothelial cell(HUVECs, derived from human) proliferation, migration, tube formation, and upregulated the expression of angiogenic genes [53]. A recent study confirmed that exosome derived from bone marrow MSCs(derived from rat) can accelerate endothelial cell and osteoblast differentiation, proliferation, and migration by activating the HIF-1α/VEGF and BMP-2/Smad1/RUNX2 signalling pathways and regulating various miRNAs. This further promotes angiogenesis and osteogenesis to enhance fracture healing [54]. Furthermore, it has been demonstrated that exosome derived from bone marrow MSCs(HucMSC, derived from human) with low oxygen preconditioning can enhance the effects of exosomal miR-126 by activating HIF-1α, leading to increased angiogenesis, proliferation, and migration. This optimises the therapeutic effect of MSC-derived exosomes in promoting fracture repair [55]. In addition, there are studies that have implanted exosomes secreted by human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSC-Exos) into bone defect wounds in castrated rats. The results showed that hiPSC-MSC-Exos enhanced cell proliferation and alkaline phosphatase activity, upregulated the mRNA and protein expression of osteogenic markers in castrated rat bone marrow MSCs. In vivo experiments demonstrated that hiPSC-MSC-Exos significantly stimulated bone regeneration and angiogenesis in the bone defect of castrated rats [56]. The combination of hiPS-MSC-Exos with tricalcium phosphate (TCP) for bone defect repair has been shown to enhance the osteoinductivity of TCP by activating the PI3K/Akt signalling pathway in hBMSCs(derived from human), thereby improving the osteogenic activity of the scaffold [57]. Li et al. immobilised exosome-derived from human adipose-derived MSCs (Adi-MSC-Exos) on polydopamine-coated PLGA (PLGA/pDA) scaffolds. The results of this study demonstrated that this approach enhanced the migration, proliferation, and osteogenic differentiation of hBMSCs in vitro, and the combination of PLGA/pDA scaffolds with Exos enhanced bone regeneration in a mouse bone defect model [58]. In the study by Zhang et al., umbilical cord-derived MSC-derived Exosomes (uMSC-Exos) were transplanted into a rat femoral fracture model. The results demonstrated that uMSC-Exos expressed CD9, CD63, and CD81, and significantly enhanced both angiogenesis and the process of bone healing. In vitro, uMSC-Exos(derived from human) enhanced osteogenic differentiation, increased the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α, and were taken up by human umbilical vein endothelial cells (hUVECs), enhancing their proliferation, migration, and blood vessel formation. Finally, using specific RNA inhibitors or siRNAs, demonstrated that HIF-1α plays an important role in uMSC-Exos-induced VEGF expression, promoting angiogenesis and enhancing fracture healing [59]. Additionally, there are research findings that indicate the potential of uMSC-Exos to enhance COL1, OPN, and RUNX2 expression, thereby promoting fracture healing through the Wnt signalling pathway [60].

Collectively, these studies unveil the intricate communication patterns within the bone cell microenvironment mediated by BMSCs-exos, shedding light on the mechanisms underlying bone reconstruction. BMSCs-exos are involved in regulating bone-related cell proliferation and differentiation, promoting vascular regeneration, and immune modulation. These findings underscore the significance of BMSCs-exos as a promising source for promoting fracture repair.

4.2. Macrophage-derived exosomes (M-exos)

Macrophage-derived exosome are considered to be one of the most abundant sources of extracellular vesicles in peripheral blood. As an important component of innate and adaptive immunity, activated macrophages play a crucial role in inflammation, host defence, and tissue regeneration [61].

Xiong et al. found that the expression of microRNA-5106 was significantly increased in exosome-derived M2 macrophages(derived from mouse). Furthermore, the study demonstrated that exosomal miRNAs could induce osteogenic differentiation of bone marrow stromal cells (BMSCs, derived from mouse) by directly targeting the SIK2 and SIK3 genes. The local injection of macrophage-derived exosome has been demonstrated to accelerate fracture healing [36]. Recent studies have demonstrated that activated and non-activated macrophages exhibit divergent profiles of microRNA (miRNA), with miR-530, chr9_22532, and chr16_34840 being particularly abundant in activated macrophages [62]. Therefore, Wei et al. modified titanium nanotube implants with macrophage-derived exosome(RAW 264.7,derived from mouse) to promote osteogenesis. The results demonstrated a substantial increase in early osteogenic differentiation markers, ALP and BMP2 expression, thereby substantiating the osteogenic effect of titanium nanotubes co-incorporated with BMP2/macrophage-derived exosomes and their potential regulatory role in MSC (hBMSC, derived from human)osteogenic differentiation. Furthermore, it has been demonstrated that BMP2/M-exos-functionalized titanium nanotubes activated autophagy during osteogenic differentiation [63]. The role of autophagy in osteogenesis and bone remodeling is far more important than reported [110]. The present studies demonstrate the significant potential of macrophage-derived exosome for bone regeneration. Nevertheless, the precise mechanisms through which these effects are produced remain to be elucidated. Elucidation of the pivotal signalling mechanisms and regulatory factors in these exosome populations is poised to furnish a novel paradigm for the promotion of fracture repair.

4.3. Endothelial progenitor cell-derived exosomes (EPC-exos)

Angiogenesis represents a vital component of the fracture healing process, with defects in angiogenesis at the fracture site frequently resulting in suboptimal bone healing. Endothelial cells represent a pivotal cell type in the context of angiogenesis during fracture repair. These cells have been shown to stimulate neovascularization and osteogenesis, thereby promoting fracture healing [64].

A growing body of evidence indicates the beneficial role of exosome secretion by EPCs in promoting angiogenesis [65]. Exosomes derived from EPCs (derived from mouse) promote the mRNA expression levels of MMP9, CTSK, TRAP, and CAR2, which are associated with osteoclast(derived from mouse) differentiation. EPC-exosomes can also mediate the regulation of long non-coding RNA MALAT1 on mir-124 to enhance the recruitment and differentiation of osteoclast precursors, thereby promoting in vivo bone repair [66]. Jia et al. demonstrated that treatment with EPC-derived Exosome(derived from rat) significantly accelerated bone regeneration in rats. In vitro analysis demonstrated that EPC-exosomes enhanced the proliferation, migration, and angiogenic capacity of endothelial cells(derived from human) in a miR-126-dependent manner. Furthermore, EPC-exosomes have been shown to downregulate SPRED1 and activate the Raf/ERK signalling pathway, thereby stimulating angiogenesis and accelerating bone regeneration [67]. Lin et al. engineered exosomes derived from human umbilical vein endothelial cells to enrich for PD-L1 and demonstrated that overexpressed exosomal PD-L1 specifically bound to PD-1 on the surface of T cells, inhibiting T cell activation. Furthermore, when co-cultured with T cells, exosomal PD-L1 induced MSCs to undergo osteogenic differentiation and significantly promoted granulation tissue formation and fracture healing in a mouse model during the early hyperactive inflammatory stage [68]. Mi et al. developed an exosome-material composite scaffold by combining a naturally derived hyaluronic acid hydrogel, endothelial cell-derived exosomes(derived from mouse) overexpressing microRNA (miRNA) 26a-5p, and loaded with APY29 (an IRE-1α inhibitor). The composite scaffold demonstrated the following effects in a mouse model of femoral fracture: anti-inflammatory, osteogenic differentiation, osteoclast activation, angiogenesis, and immune modulation. Furthermore, the composite scaffold was found to regulate the balance between osteoblasts/osteoclasts and M1/M2 macrophages, thereby promoting fracture healing [69].

4.4. Chondrocyte-derived exosomes (CC-exos)

Tissue engineering based on chondrogenic progenitor cells has attracted attention due to their strong chondrogenic differentiation ability.

Chen et al. implanted chondrogenic progenitor cell-alginate constructs into nude mice and provided chondrogenic signals to the constructs in a subcutaneous environment using CC-Exos (derived from rabbit), effectively promoting ectopic cartilage regeneration. The experimental results showed that CC-Exos stimulated chondrogenic progenitor cell(derived from rabbit) proliferation and increased the expression of cartilage formation markers. They also increased collagen deposition in the engineered constructs and minimized vascular ingrowth, leading to effective and reproducible development of cartilage and promoting bone regeneration [70].

In conclusion, the use of CC-Exos in the field of fractures may represent a novel cell-free therapeutic approach for promoting cartilage regeneration in a subcutaneous environment, thereby facilitating bone regeneration.

4.5. Osteoblast-derived exosomes (OC-exos)

Osteoblasts are bone cells derived from mesenchymal cells, and they enhance the strength of bone healing through the generation and mineralization of bone matrix. During the process of fracture healing, exosomes derived from osteoblasts play a crucial role in information exchange between osteoblasts and other bone-related cells.

GE et al. isolated exosomes derived from osteoblasts(Mc3t3 cells, derived from mouse) and identified through multi-omics analysis that the EIF2 pathway mediates the important role of miR-21 in osteogenesis [71]. Other studies have found that miR-677-3p, miR-3084-3p, miR-680, or miR-378 enriched in exosomes derived from osteoblasts(MC3T3-E1,derived from mouse) promote osteogenesis. They found that exosomal delivery of miR-378 targets CASP3 to activate the PI3K/Akt pathway, thereby enhancing ALP activity and promoting the expression of osteogenic differentiation markers Runx2, osteocalcin, and osteopontin. On the other hand, miRNAs (miR-667-3p, miR-6769b-5p, miR-7044-5p, miR-7668-3p, and miR-874-3p) released by osteoblasts collectively target and inhibit Axin1, an important negative regulator of the Wnt signaling pathway, leading to increased expression of β-catenin and promoting osteogenic differentiation of ST2 cells(derived from mouse). However, exosomal RANKL, miR-30d-5p, miR-133-3p, and miR-140-5p derived from osteoblasts inhibit bone formation [72]. However, a recent study by Niedermair et al. found that exosomes derived from osteoblasts of patients with osteoporosis also impede osteogenic differentiation of BM-MSCs(derived from CA、OP、and CA/OP human patients) [73]. Therefore, the specific functions of exosomes derived from osteoblasts in osteoporosis are still uncertain.

In summary, Exosomes derived from diverse cellular sources play synergistic roles in bone remodeling (Table 1): Mesenchymal Stem Cell (MSC)-derived exosomes facilitate angiogenesis and osteogenic differentiation via bioactive factor transfer; M2 macrophage-derived exosomes regulate bone formation-related genes; EPC-derived exosomes drive vascularization and influence osteoclast activity; Chondrocyte-derived exosomes stimulate cartilage regeneration. Osteoblast-derived exosomes bidirectionally regulate bone metabolism through specific miRNAs, with exosomes from osteoporotic sources potentially inhibiting repair. Collectively, these exosomes mediate the "angiogenic-osteogenic coupling" mechanism, presenting a novel therapeutic approach for cell-free bone regeneration.

Table 1.

The role of exosomes in fracture repair.

Source of exosomes	vitro or vivo experiments	Substances rich in exosomes	recipient cell	Regulatory signaling pathways	function	Ref.
Bone marrow stromal cells derived from rat	Vivo	miR-130a	Endothelial cells	Unknown	Increased proliferation, migration, and vascularization of endothelial cells, up regulation of the expression of pro angiogenic genes	[53]
Bone marrow mesenchymal stem cells derived from rat	Vivo and vitro	Unknown	Endothelial cells, osteoblasts	HIF-1α/VEGF, SMURF1, BMP-2/Smad1/RUNX2	Promote angiogenesis and osteogenesis, promote osteogenic differentiation, proliferation, and migration	[54]
Hypoxic preconditioning of bone marrow mesenchymal stem cells derived from human	Vivo and vitro	miR-126	Mesenchymal stem cell	SPRED1/Ras/Erk	Promote angiogenesis, proliferation and migration, accelerate fracture healing	[55]
Mesenchymal stem cells derived from human induced pluripotent stem cells	Vivo	Unknown	Osteoblasts	PI3K/Akt	Up regulation of osteoblast mRNA and protein expression, enhanced cell proliferation and alkaline phosphatase activity, promote bone regeneration and angiogenesis	[56,57]
Adipose derived stem cells from human	Vivo and vitro	Unknown	Bone marrow mesenchymal stem cells	Unknown	Enhance the migration, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells	[58]
Umbilical cord mesenchymal stem cells from human	Vivo and vitro	CD9, CD63和CD81	Endothelial cells, osteoblasts	HIF-1α	Angiogenesis and promoting fracture repair	[59]
Umbilical cord mesenchymal stem cells from human	Vivo	Unknown	Osteoblasts	Wnt	Enhance the expression of COL-1, OPN and Runx2 and promote fracture healing	[60]
M2 macrophage from mouse	Vivo and vitro	MiR-5106	Osteoblasts, bone marrow mesenchymal stem cells	SIK2, SIK3	Promote osteoblast differentiation	[36]
Bmp2/macrophage from human	Vivo and vitro	Unknown	Mesenchymal stem cell	Unknown	Increase the expression of osteoblast differentiation markers, ALP and BMP2, regulation of osteogenic differentiation	[63]
Endothelial cells from mouse	Vivo and vitro	miR-124	Osteoclast	LncRNA-MALAT1	Increased neovascularization and enhanced fracture healing	[66]
Endothelial cells from rat	Vivo and vitro	miR-126	Endothelial cells	SPRED1, Raf/ERK	Enhance the proliferation, migration and angiogenesis of endothelial cells	[67]
Human umbilical vein endothelial cells	Vivo and vitro	PD-L1	Mesenchymal stem cell, T cells	Unknown	Promote callus formation and fracture healing	[68]
Endothelial cells from mouse	Vivo and vitro	Natural polymer hyaluronic-acid-based hydrogel, APY29, miR-26a-5p	Osteoblasts, osteoclast, macrophage	Unknown	Anti inflammatory, osteogenic differentiation, osteoclast activation, angiogenesis and immune regulation, promote fracture repair	[69]
Chondrocyte feom rabbit	Vivo and vitro	Unknown	Chondrocyte	Unknown	Increases the expression of chondrogenic markers while inhibiting angiogenesis	[70]
Osteoblasts from mouse	Vitro	miR-21	Osteoblasts	EIF2	Promote osteoblast differentiation	[71]
MC3T3-E1 cells from mouse	Vitro	miR-677-3p, miR-3084-3p, miR-680, miR-378, miR-6769b-5p, miR-7044-5p, miR-7668-3p, miR-874-3p	Osteoblasts	PI3K/Akt, Wnt	Acquire alkaline ALP activity and promote the expression of osteoblast differentiation markers, promote osteogenic differentiation	[72,73]
Osteoblasts from CA、OP、and CA/OP human patients	Vivo and vitro	miR-30d-5p, miR-133-3p, miR-140-5p	Osteoblasts	RANKL, BMP	Inhibit bone formation	[72,73]

5. Application of exosome in fractures

The process of fracture healing is subject to regulation by various factors, including the nature and severity of the injury sustained, the stability of fracture fixation, and biological processes such as immunological and developmental processes associated with bone formation. A salient feature of fracture healing is the augmentation of tissue volume, which is concomitant with the mobilisation and differentiation of stem cells that are instrumental in the formation of bone and vascular tissues [2].

Evidence indicates that exosomes play a pivotal role in intercellular communication through the transfer of bioactive molecules, including miRNAs, mRNAs, and proteins, to recipient cells. These vesicles exert multifaceted therapeutic effects on fracture healing through several interconnected mechanisms: (1) suppression of inflammatory responses; (2) activation of anti-inflammatory pathways; (3) facilitation of callus formation; (4) promotion of angiogenesis; and (5) modulation of bone cell activity to enhance osteogenesis. Collectively, these mechanisms contribute to the accelerated fracture repair process (Fig. 2) [74]. Further research on the application of exosome therapy in the context of bone fracture healing has demonstrated encouraging results in animal models.

Fig. 2.

The process of human fracture repair and the mechanism of Exosomes in fracture repair. (The "arrow" indicates the next stage of fracture healing process and the indicative role of extracellular vesicles from different sources on fracture healing).

Studies have explored the use of exosome therapy in various types of fractures, including traumatic, fragility, pathological, and delayed fracture healing, with positive outcomes observed in some cases. Violent fractures are a severe category of fractures resulting from mechanical trauma. Delayed fracture healing can be defined as a fracture that has not progressed for three consecutive months following surgery or a fracture that has not healed by nine months after surgery [105]. Fractures were considered to be cases of delayed healing if radiological signs of callus formation at the fracture location were not evident within the standard healing time period, and if the patient continued to experience pain and exhibited rocking movements [113]. The term 'fracture nonunion' was defined as the absence of radiographic healing over a period of 9 months, with no significant progression of healing observed within the last 3 months. Numerous factors have been posited as contributing to the intricate mechanisms underpinning bone nonunion, with these factors including biomechanics and immunology [98,103]. According to the World Health Organization (WHO), a fragility fracture is defined as "a fracture caused by trauma that would be insufficient to fracture a normal bone". It is evident that fragility fractures are the most obvious clinical outcomes of osteoporosis, and are characterised by increased bone fragility [95,104]. A pathologic fracture is one that occurs without adequate trauma and is caused by a benign or malignant bone lesion [96]. Pathologic fractures occur through these lesions due to altered biomechanics [94].

5.1. The use of exosomes in violent fractures

Violent fractures are mainly caused by sudden mechanical impact or traumatic injury to certain critical parts of the body and are the most common type of musculoskeletal injury. Recently, Huang et al. demonstrated that MSC-Exos-miR-19b(derived from human) promotes the healing of violent fractures by inhibiting the expression of WWP1 or Smurf2 and increasing the expression of KLF5 through the Wnt/β-catenin signaling pathway [75]. In order to explore the methods for repairing elderly hip fractures, Hai et al. isolated exosomes from human umbilical cord mesenchymal stem cells(hUMSCs)and added them to autologous bone marrow mesenchymal stem cells (ABMSCs). The experimental results showed that hUMSCs significantly promoted the metabolism and migration of ABMSCs and inhibited apoptosis. With an increasing concentration of exosomes derived from hUMSCs in ABMSCs, osteogenic differentiation and the expression of Collagen II in ABMSCs were significantly enhanced, thereby promoting the healing of violent fractures [76]. Furthermore, a study revealed that PD-L1, which is enriched in HUVECs-Exos, binds to PD-1 on the surface of T cells. This was then applied to a mouse model of fractures. The experimental results demonstrated that local administration of exosome-rich PD-L1 at the fracture site inhibited the activation of T cells in peripheral lymphoid tissues. Furthermore, exosomal PD-L1 has been observed to induce MSCs(derived from human) to differentiate into osteoblasts, thereby significantly promoting scar tissue formation and fracture healing during the early inflammatory stage [68].

Fracture in combination with traumatic brain injury (TBI) is among the most prevalent and severe types of polytrauma in clinical practice. It has been observed that patients with fractures accompanied by TBI exhibit significantly reduced fracture healing times in comparison to those with isolated fractures. A growing body of research has suggested a possible association between TBI and accelerated fracture healing [77]. In order to investigate this phenomenon, Lin et al. applied exosome-rich fluid derived from patients with traumatic brain injury (TBI) to a mouse fracture model. The results demonstrated that the active substances released by TBI-Exos enhanced the function of endothelial cells, thereby promoting angiogenesis at the fracture site. Furthermore, TBI-Exos have been shown to directly mediate the cellular functions of both hMSCs and vascular endothelial cells [78].

5.2. The use of exosomes in delayed fracture healing and nonunion

The physiological process of bone healing is multifaceted, involving numerous factors such as microstability, fracture morphology, and tissue perfusion. A minor imbalance in any of these factors can act as a critical bottleneck in the bone healing process, impeding the regenerative capacity of fractured bones and resulting in delayed or non-union fractures [79].

The ratio of osteoblasts/osteoclasts and M1/M2 macrophages plays a crucial role in delayed fracture healing. Mi et al. developed a cocktail therapy to regulate the balance of osteoblasts/osteoclasts and M1/M2 macrophages(derived from mouse) in a delayed fracture healing model. Engineered endothelial cell-derived exosomes (EC-ExosmiR-26a-5p,derived from mouse) and APY29 were specifically delivered to regulate osteoblasts/osteoclasts and macrophages, respectively, achieving repair and promotion of delayed fracture healing [[69], [80]]. Chen et al. synthesised an injectable, self-healing, adhesive HA@SDF-1α/M2D-Exos hydrogel by combining stromal cell-derived factor-1α and M2 macrophage-derived exosomes (M2D-Exos,derived from mouse) with hyaluronic acid hydrogel precursor solution. This novel hydrogel was then applied to a non-union fracture model. The experimental results demonstrated that the HA@SDF-1α/M2D-Exos hydrogel enhanced the proliferation and migration of human mesenchymal stem cells (HMSCs) and human umbilical vein endothelial cells (HUVECs), promoted osteogenesis and angiogenesis both in vitro and in vivo, and was compatible with the natural healing process of fractures. By integrating osteogenesis, angiogenesis, and resistance to infection at various stages, it offers a novel approach for accelerating bone repair [81].

Diabetes is a chronic metabolic disease characterised by elevated blood glucose levels. The elevated levels of blood glucose, advanced glycation end products, reactive oxygen species, and inflammation frequently result in increased osteoclast activity, reduced osteoblast activity, and diminished expression of factors that stimulate osteoblasts. Consequently, this results in delayed fracture healing or non-union in diabetic patients [82]. Researchers have found that exosomes derived from diabetic bone marrow-derived macrophages (dBMDM-exos,derived from rat) have lower osteogenic potential and impaired fracture repair ability compared to non-diabetic bone marrow-derived macrophages. The level of miR-144-5p in dBMDM-exos is significantly increased, and it can regulate bone regeneration by transferring into bone marrow mesenchymal stem cells and targeting Smad1. Therefore, inhibition of miR-144-5p can regulate Smad1 expression and reverse the negative effects of dBMDM-exos on bone repair and regeneration in vitro and in vivo in rat models [37]. Wang et al. conducted an animal experiment in which M2D-Exos (derived from mouse) were extracted and used for intervention in the bone repair of diabetic mouse fractures. The results demonstrated that M2D-Exos significantly regulated the bone immune microenvironment by reducing the proportion of M1 macrophages. The study demonstrated that M2D-Exos could induce the transformation of M1 macrophages into M2 macrophages by stimulating the PI3K/AKT pathway, thereby accelerating the healing of diabetic fractures [83]. Zhang et al. isolated and identified exosome-derived from adipose-derived stem cells (ASCs-exos,derived from rat) and evaluated their effects on osteogenic differentiation of bone marrow stromal cells (BMSCs), bone repair and regeneration in a diabetic rat model both in vitro and in vivo. In comparison with the control group, ASCs-exos enhanced the osteogenic potential of BMSCs by activating the Wnt/β-catenin signaling pathway. Furthermore, the study demonstrated that ASCs-exos promoted bone repair and regeneration in vivo, thus providing a novel direction for the treatment of non-union fractures in diabetic patients [84].

5.3. The use of exosomes in fragility fractures

Fragility fractures, also known as osteoporotic fractures, are typically caused by low-energy trauma, such as falls from standing height or lower [85]. Patients with this type of fracture often have poor bone quality, advanced age, and usually have underlying comorbidities, which pose many challenges in the treatment of fractures.

Recent research has provided insights into the differential expression profiles of serum exosomal miRNAs between postmenopausal women with severe osteoporosis and those with normal bone density. The results showed differential expression of serum exosomal miRNAs in postmenopausal women with osteoporosis and fragility fractures. MiR-324-3p, miR-766-3p, miR-1247-5p, miR-330-5p, and miR-3124-5p were found to be associated with bone density and may serve as potential diagnostic biomarkers and potentially involved in the pathophysiology of fragility fractures [86]. Xun et al. treated BMSCs from aged osteoporotic rats with serum exosomes derived from young rats under osteogenic induction conditions after fatigue loading-induced fragility fractures. They quantified the expression of osteogenesis-related miRNAs. The study found that serum exosomes from young rats with high expression of miRNA-19b-3p improved the impaired osteogenic differentiation ability of BMSCs from aged osteoporotic rats. Transfection with a miRNA-19b-3p mimic promoted osteogenic differentiation of BMSCs and reduced PTEN expression. Serum exosomes from young rats improved the impaired osteogenic differentiation ability of BMSCs from aged osteoporotic rats after fatigue loading, providing a new therapeutic strategy for repairing and preventing fragility fractures [87].

5.4. The use of exosomes in pathologic fractures

The term 'pathological fractures' refers to fractures that occur with minimal or no external force when the normal structure and strength of the bone are compromised by invasive diseases or destructive lesions [88]. Due to the presence of the pathology, bone cells, bone microenvironment, and blood supply around the fracture site are often affected, which poses many challenges in the clinical treatment of pathological fractures.

Li et al. found that exosome-mediated cell-cell communication between liver cancer and bone may be crucial for osteolytic bone destruction. They extracted exosomes from liver cancer cells and conducted cell experiments. The results showed that Huh-7-Exos promoted osteoclast differentiation in RAW 264.7 cells. The tumor necrosis factor-alpha (TNF-α) enriched in Huh-7-Exos regulated osteoclast differentiation and bone resorption activity through NF-κB/CTSK/TRAP expression. Therefore, TNF-α in exosomes may be an important therapeutic target for preventing and treating liver cancer-mediated pathological fractures and bone diseases [89]. Gupta et al. utilised a functionalized exosome-loaded biodegradable nano-cementitious carrier called amphotericin B (AmB) to simultaneously manage periosteum and bone formation in an osteosarcoma tumor resection model. The experimental results showed that the released AmB from the carrier successfully killed tumor cells, and importantly, the incorporation of exosomes facilitated bone formation. Compared to the blank group, the bone volume increased by approximately 2.6 times. Additionally, histological and immunofluorescence analysis confirmed that the carrier also contributed to periosteal development and mineralization of the callus. This indicates that this approach can be used for bone regeneration in bone tumors and prevent pathological fractures caused by incomplete development of periosteum and loss of bone [90].

6. Prospects of exosomes in bone fracture repair

Exosomes, defined as nanoscale membrane-bound vesicles secreted by cells, can be released into body fluids or other tissues through the process of cell extrusion. This process facilitates communication between cells and participates in various stages of bone regeneration, thereby achieving fracture treatment,we summarize the application of exosomes in the repair of different types of fractures in Table 2.

Table 2.

Application of exosomes in fracture repair.

Source of exosomes	vitro or vivo experiments	Substances rich in exosomes	Fracture type	mechanism	function	Ref.
Human umbilical cord mesenchymal stem cells	Vivo and vitro	Unknow	Violent fractures	Promote the metabolism and migration of ABMSCs and inhibit apoptosis	Promote the osteogenic differentiation of abmscs and significantly enhance the expression of collagen II Promote fracture healing	[76]
Human umbilical vein endothelial cells	Vivo and vitro	PD-L1	Violent fractures	Activation of T cells in peripheral lymphoid tissues is inhibited	Induce osteogenic differentiation of MSCs Promote soft tissue recovery and fracture healing	[68]
Traumatic brain injury derived from human	Vivo and vitro	Unknow	Violent fractures	Paracrine effect	Promote angiogenesis and osteogenic differentiation at the fracture site	[78]
Endothelial cells derived from mouse	Vivo and vitro	miR-26a-5p	Delayed healing and non healing	Regulation of the ratio of osteoblasts/osteoclasts and macrophages	Promote fracture repair	[80]
M2 macrophages derived from mouse	Vivo and vitro	Unknow	Delayed healing and non healing	Enhanced proliferation and migration of hMSCs and HUVECs	Promote osteogenesis and angiogenesis in vitro and in vivo	[81]
Diabetic bone marrow-derived macrophages derived from rat	Vivo and vitro	miR-144-5p	Delayed healing and non healing	Inhibit mir-144-5p 2. regulate Smad1 expression	Improve osteogenic potential and promote fracture repair	[37]
M2 macrophages derived from mouse	Vivo and vitro	Unknow	Delayed healing and non healing	Reduce the proportion of M1 macrophages, stimulate pi3k/akt pathway	Regulate bone immune microenvironment and accelerate fracture healing	[83]
ADSCs derived from rat	Vivo and vitro	Unknow	Delayed healing and non healing	Activating Wnt/β- catenin signaling pathway	Enhance the osteogenic potential of BMSCs, promote bone repair and regeneration ability	[84]
Serum derived from rat	Vivo and vitro	miRNA-19b-3p	Fragility fractures	Unknow	Promote osteogenic differentiation of BMSCs, reduce PTEN expression	[87]
Hepatoma cell derived from human	Vitro	TNF-α	Pathological fracture	Regulation of NF- κB/ctsk/trap expression	Regulation of osteoclast differentiation and bone resorption activity	[89]
Bone marrow stem cells derived from rat and mouse	Vivo and vitro	Adriamycin	Pathological fracture	Unknow	Promote bone formation, promote the development of periosteum and mineralization of callus	[90]

Exosomes possess a number of advantageous properties, including their capacity to carry hydrophilic and lipophilic substances, their ability to cross biological barriers, and their directional delivery properties. These characteristics make them particularly useful in the treatment of bone injuries, where they can facilitate the healing process by delivering therapeutic agents to target tissues [101]. In comparison with alternative synthetic drug delivery systems, the natural characteristics of exosome-based drug delivery systems render them highly biocompatible, low immunogenic and low cytotoxic. Moreover, their capacity to traverse various biological barriers renders them an optimal candidate for the development of clinical drug carriers.

The mechanisms of action of exosome derivatives from diverse cell sources in fracture healing principally involve the activation of various signalling pathways and the regulation of gene expression. This, in turn, influences the functions of endothelial cells, osteoblasts and osteoclasts involved in bone regeneration. However, further in-depth research is required to determine which cell source of exosome is more suitable as a novel biological agent for bone fracture repair. Concurrently, the pharmacokinetics of exosome remain to be fully elucidated, and there is as yet no consensus on the optimal dosage and frequency of exosome administration [91]. Consequently, it is challenging to attain optimal therapeutic outcomes.

In the majority of animal experiments, exosomal drugs are injected into the animal's blood circulation, resulting in their accumulation in the lungs and liver, as opposed to other organs [101]. Moreover, it has been documented that exosome elimination within the liver occurs rapidly [92]. This observation suggests the possibility that the therapeutic efficacy of exosome administration may be rapidly eliminated before it can exert its intended effect. It is imperative that the functionality, appropriate dosage, distribution, and clearance of exosome are thoroughly investigated to ensure their safety and efficacy [112]. However, it should be noted that the technology behind exosome extraction is not yet sufficiently advanced, particularly in cases where a large volume of extraction is required [101]. The prevailing methodologies for the isolation of exosome samples, predominantly encompassing ultracentrifugation and ultrafiltration, have been observed to demonstrate suboptimal efficiency [93]. This underscores the imperative for large-scale manufacturing of exosome samples prior to the initiation of clinical trials.

In conclusion, it is evident that exosome function as effective mediators of intercellular communication in the bone microenvironment and have significant advantages in fracture treatment. Nevertheless, the efficient separation and purification of extracellular vesicles in large quantities, as well as their transportation and storage, remains a significant challenge. Furthermore, the functions of many substances within extracellular vesicles are still unclear. These challenges hinder the translation of extracellular vesicle-based drug delivery systems into clinical practice. Consequently, further exploration is necessary to fully elucidate the potential of extracellular vesicles in clinical applications.

Author contributions

SWH drafted the main part of the manuscript and reviewed the current literature. YJL contributed the table and figure design. XHP provided oversight of the drafting of the manuscript and provided substantive improvements. SGW and BL provided substantive improvements. All authors read and approved the fnal manuscript.

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Funding sources

This work was funded by Sanming Project of medicine in Shenzhen (SZSM202106019 and 202208), the Science and Technology Innovation Committee of Shenzhen (No. KCXFZ20240903094059020 and JCYJ20190809113815103).

Declaration of competing interest

The authors declare no conflicts of interests that could have appeared to influence the work reported in this paper.