Introduction

Bone defects caused by trauma, infection and tumor resection are a common clinic scenario in orthopaedic practice.1 The bone is a rigid organ that not only protects various internal organs, but also participates in hematopoiesis, storing minerals, supporting our body’s physical structure and maintaining mobility.2 Given the important roles of bone, it is crucial to develop effective methods to repair and reconstruct bone defects. Currently, the gold standard for treating large bone defects is autologous bone grafting. However, this strategy faces many problems, including potential donor site complications, limited autografts, and risk of potential graft failures.3 Over the past few decades, bone tissue engineering has emerged as a promising strategy for treating large bone defects. This approach generally involves the integration of a three-dimensional (3D) biocompatible scaffold that provides shape and mechanical strength, seed cells with osteogenic potential, and molecular signals that can induce osteogenic differentiation and vascularization.4 An ideal cellular source for bone tissue engineering approaches should be non-immunogenic, non-tumorigenic, and posses potent proliferative and osteogenic potentials. Among the various cell types evaluated for bone tissue engineering applications, mesenchymal stem cells (MSCs) are considered one of the most promising cellular sources due to their easy acquisition, potent proliferative and osteogenic potentials, low immunogenicity, and well-defined osteogenic differentiation pathway.5 However, there are still many concerns about the potential risks associated with MSC transplantation, including ectopic tissue formation, malignant transformation, cell embolism, and immune rejection.6 Additionally, even when MSCs are successfully administered, variables such as donor age, the number of in vitro expansion passages, culture conditions, and transplantation procedures may adversely affect their therapeutic efficacy.7

In recent years, numerous studies have demonstrated that the therapeutic effects of MSCs in tissue repair are primarily mediated by paracrine factors, rather than through direct differentiation into parenchymal cells to restore or replace the damaged tissue. Among these paracrine factors, extracellular vesicles (EVs) play a pivotal role.8 According to the most recent consensus from the International Society for Extracellular Vesicles (ISEV2023), EVs are lipid bilayer-enclosed particles released by cells that lack the ability to self-replicate.9 Once considered cellular debris, EVs are now recognized as critical mediators of intercellular communication, capable of delivering bioactive cargo to recipient cells or interacting with cellular receptors via surface proteins. Traditionally, EVs have been classified into three main subtypes based on their size, origin, and biogenesis: exosomes, microvesicles, and apoptotic bodies. Exosomes are formed through the inward budding of endosomal membranes, microvesicles (also referred to as ectosomes) arise from the outward budding of the plasma membrane, and apoptotic bodies are generated during cellular fragmentation in apoptosis (Table 1).10 However, current isolation methods are unable to reliably distinguish EV subtypes based on their biogenesis, and universal molecular markers for definitive classification remain lacking. Consequently, ISEV discourages the use of biogenesis-based terminology unless specific EV subpopulations have been rigorously isolated and characterized. Despite the continued prevalence of the term “exosomes” in the literature, ISEV recommends the use of the broader, more inclusive term “EVs”.9

Table 1 The Three Classical Types of EVs

During EV formation, they selectively package proteins, nucleic acids, and lipids from their parent cells, functioning as ‘signaling complexes’. They transmit biological information through direct membrane fusion, endocytosis or ligand‒receptor interactions, thereby influencing the behaviors of the recipient cells.11 The precise biological function of EVs is a reflection of their parent cells and the local microenvironment.12 Stem cell-derived EVs have gained prominence in regenerative medicine research, as numerous studies have demonstrated that they are the key mediators of the biological functions of their parent cells.13 EVs offer several compelling advantages, including eminent bioactivity, stability, low immune-rejection, desirable biocompatibility, and high feasibility for modularized customized modification.14 Although stem cells have long been considered a major component of bone tissue engineering strategies, EVs are emerging as an advanced substitute, capable of recapitulating the therapeutic potentials of stem cells while avoiding the potential risks associated with in vivo stem cell administration.15 In recent years, considerable research has focused on the use of EVs in bone tissue engineering, though their development remains in the early stages. One major challenge is that free EVs do not achieve durable retention and controlled release at defect sites, leading to the combination of scaffolds as carriers for EVs.16 An increasing number of studies have explored the use of various bioactive scaffolds integrated with EVs for bone tissue engineering applications. The purpose of this review is to discuss the roles of EVs in bone regeneration and their application in bone tissue engineering. We specifically focus on current strategies for integrating EVs with various bioactive scaffolds and the latest advances in achieving controlled and sustained release of EVs from the scaffolds at bone defect sites.

The Roles of EVs in Bone Regeneration

Bone regeneration is a complex and highly orchestrated biological process involving dynamic intercellular communication among MSCs, bone-forming osteoblasts, bone-resorbing osteoclasts, osteocytes, immune cells, and other cell types.17 It is now well established that EVs play a crucial role in mediating these cellular interactions within the bone microenvironment. Several comprehensive reviews have thoroughly discussed the roles of EVs and their interactions with recipient cells during bone regeneration.18,19 In this section, we elaborate on the sources of parent cells for EVs used in bone tissue engineering and elucidate the roles and potential molecular mechanisms of EVs in bone regeneration.

Parent Cells of EVs

Numerous studies have investigated the use of both natural and engineered EVs derived from various parent cells in bone tissue engineering. Among theses parent cells, MSCs stand out as the predominant cellular source of EVs. Additionally, EVs derived from bone cells, immune cells, and endothelial cells are also employed in bone tissue engineering applications. A summary of studies on the application of EVs derived from different parent cell sources in bone regeneration is provided in Table S1 (supplementary data).

MSCs

Researchers have utilized EVs secreted by MSCs derived from diverse tissues, such as bone marrow (BMSCs),20–36 adipose tissue (ASCs),37–44 umbilical cord (UCMSCs),45–47 induced pluripotent stem cells (iPS-MSCs),48,49 dental tissues (DMSCs),50–57 and synovial membranes (SMSCs),58 in combination with biomaterials to promote bone regeneration in preclinical animal models.

MSCs derived from different tissues offer distinct advantages. Among them, BMSCs are one of the most thoroughly researched and utilized MSC types in both academic and practical settings. As a vital cellular component within the bone microenvironment, their ability to proliferate, migrate, and differentiate into osteoblasts is crucial for successful bone regeneration.59 Studies have shown that BMSC-derived EVs (BMSC-EVs) enhance osteogenic differentiation of both MSCs and osteoblasts in vitro.20,25,28,60–62 Furthermore, when integrated with biomaterials, BMSC-EVs can significantly accelerate bone regeneration at bone defect sites in vivo.20,25,26,28,60,62

ASCs are abundantly distributed throughout the body and are easily accessible, making them a highly practical source of parent cells for EVs.37 Research has shown that ASCs produce a greater quantity of EVs compared to BMSCs. Although a considerable body of studies have demonstrated that ASC-derived EVs (ASC-EVs) can accelerate bone regeneration when integrated with biomaterials in vivo,37–42 there is still some controversy regarding their osteogenic potential. For instance, studies by Li et al and Liu et al found that only EVs from osteogenically induced ASCs were capable of promoting osteogenic differentiation of BMSCs in vitro. Conversely, EVs from non-osteogenically induced ASCs did not exhibit the ability to enhance osteogenic differentiation of BMSCs.37,63 However, research by Gandolfi et al, Kim et al and Xing et al reported that EVs from non-osteogenically induced ASCs enhanced the osteogenic potential of ASCs, BMSCs and MC3T3-E1 cells (murine calvariae preosteoblast cell line) in vitro.39,41,42 One possible explanation for this disparity is that Li et al and Liu et al evaluated the osteogenic potential of ASC-EVs under 2D cell culture conditions, whereas Gandolfi et al, Kim et al and Xing et al assessed it using MSCs seeded on scaffolds in a 3D culture environment.

UCMSCs are derived from postnatal waste tissues. Compared to MSCs derived from other tissue origins such as BMSCs and ASCs, UCMSCs possess higher self-renewal capacity, lower immunogenicity and fewer ethical concerns.64 Studies have shown that EVs secreted by UCMSCs (UCMSC-EVs) can promote osteogenic differentiation of BMSCs, mouse osteoblast progenitor cells and MC3T3-E1 cells in vitro.45–47 Additionally, when combined with biomaterials, UCMSC-EVs have been shown to enhance bone regeneration at bone defect sites in vivo.45–47

iPS-MSCs combine the advantages of both iPSs and MSCs. Even after 40 passages in vitro, iPS-MSCs retain their self-renewal capacity and do not exhibit tumorigenic risk.65 Researchers have demonstrated that EVs secreted by iPS-MSCs (iPS-MSC-EVs) can promote osteogenic differentiation of BMSCs in vitro and enhance bone regeneration when combined with scaffolds in vivo.48,49

Human DMSCs derived mainly from dental pulp (DPSCs), gingiva (GMSCs), apical papilla (SCAPs) and periodontal ligament (PDLSCs) are promising candidates for bone regeneration due to their minimally invasive tissue collection methods and strong osteogenic differentiation potential.66 Studies have demonstrated that EVs secreted by DPSCs (DPSC-EVs),51,52 GMSCs (GMSC-EVs),53–55 SCAPs (SCAP-EVs)50 and PDLSCs (PDLSC-EVs)56,57 can promote osteogenic differentiation in vitro and accelerate bone regeneration at bone defect sites in vivo.

Currently, there is no consensus on the optimal parent cells for EVs in bone tissue engineering applications. Li et al conducted a comparative analysis of the osteogenic potential of BMSC-EVs, ASC-EVs, and EVs secreted by SMSCs (SMSC-EVs). Their findings indicated that ASC-EVs exhibited superior effects in enhancing migration, proliferation, and osteogenesis of BMSCs in vitro, as well as promoting bone regeneration in a mouse model in vivo, compared to BMSC-EVs and SMSC-EVs.58 However, a study by Liu et al reported that BMSC-EVs showed stronger osteogenic potential compared to ASC-EVs.63 The inconsistency between studies may be attributed to differences in EV concentrations and pretreatment conditions of the parent cells.

Bone Cells

Osteoblasts are the primary functional cells responsible for bone formation, playing a key role in the secretion, synthesis, and mineralization of the bone matrix.67 Mizukami et al demonstrated that EVs secreted by mature primary osteoblasts promoted osteogenic differentiation of mouse mesenchymal stromal cells in vitro. Furthermore, local administration of EVs encapsulated in a gelatin hydrogel at bone defect sites significantly enhanced bone healing in a mouse femoral bone defect model.68 MC3T3-E1 is an osteoblast precursor cell line derived from mouse calvaria. Previous studies have demonstrated the osteo-inductive potential of EVs derived from osteogenically differentiated MC3T3-E1 cells. These EVs have also been utilized in combination with scaffolds or hydrogels to improve bone regeneration in vivo.69,70

Osteoclasts play a crucial role in maintaining bone homeostasis, primarily through their function in resorbing the bone matrix.67 Studies have shown that EVs derived from osteoclasts significantly upregulate expression of osteogenic markers and promote mineralization in MSCs in vitro. When integrated with biomaterials, osteoclasts-derived EVs have been demonstrated to enhance bone regeneration in vivo.71,72

Immune Cells

Immune cells play a pivotal role in bone regeneration. Numerous studies have highlighted the influence of immune cells, such as T cells, B cells, and macrophages, on MSC-mediated bone regeneration. Optimizing the host immune microenvironment has been demonstrated to improve stem cell-based bone regeneration.73,74 Within bone defect sites, MSCs can modulate immune cell functions through EVs, while immune cells can, in turn, impact MSC differentiation by releasing EVs.14,15

Macrophages are immune-regulating cells that play pivotal roles in both initiating and resolving inflammation through polarization into various phenotypes. M1 macrophages are characterized by their pro-inflammatory phenotype, while M2 macrophages exhibit an anti-inflammatory phenotype. Promoting the polarization of macrophages toward the M2 phenotype has been shown to effectively enhance bone angiogenesis and accelerate bone healing.75 Peng et al demonstrated that EVs secreted by M2 macrophages not only promoted polarization of macrophages toward the M2 phenotype but also enhanced osteogenic differentiation and mineral deposition in BMSCs. When encapsulated in a multifunctional DNA-based hydrogel, these M2 macrophage-derived EVs accelerated the healing of alveolar bone defects in vivo.76 In a separate study, Wei et al used EVs derived from BMP2-pretreated RAW 264.7 cells (a murine macrophage cell line) to modify titanium nanotube implants, thereby promoting osteogenesis in BMSCs.77

In addition to macrophage-derived EVs, those secreted by polymorphonuclear leukocytes (PMNs) and dendritic cells have also been integrated with biomaterials to enhance bone regeneration. Wang et al demonstrated that PMN-derived EVs (PMN-EVs) promoted the proliferation and osteogenic differentiation of BMSCs in vitro. BMSC-based cell sheets integrated with PMN-EVs significantly accelerated bone regeneration in a rat calvarial defect model.78 In another study, Cao et al demonstrated that EVs from mature dendritic cells promoted the proliferation and osteogenic differentiation of BMSCs in vitro and enhanced BMSC-mediated bone regeneration in a rat femoral defect model in vivo.79

Other Cells

In addition to MSCs, bone cells and immune cells, EVs secreted by other cell types, such as chondrogenic progenitor cells, endothelial cells and Schwann cells, have also been utilized in combination with biomaterials to enhance bone regeneration. ATDC5 is a chondrogenic progenitor cell line known for its significant capacity for osteogenic differentiation.80 Zha et al introduced vascular endothelial growth factor (VEGF) plasmid into EVs secreted by ATDC5 cells (ATDC5-EVs) through electroporation. This approach achieved dual functions: promoting osteogenic differentiation of BMSCs and enabling controlled delivery of the VEGF gene. These engineered EVs were subsequently integrated with 3D-printed porous bone scaffolds, which effectively improved vascularized bone regeneration in vivo.81

Evidence has suggested that EVs secreted by vascular endothelial cells have the potential to induce bone remodeling.82 Lin et al successfully enriched programmed cell death ligand 1 (PD-L1) in EVs derived from genetically modified human umbilical vein endothelial cells (HUVECs). These PD-L1-overexpressing EVs were demonstrated to suppress T cell activation and induce osteogenic differentiation of BMSCs when pre-cultured with T cells in vitro. Furthermore, incorporating HUVEC-derived EVs into an injectable hydrogel significantly accelerated fracture healing in a murine model.83

Schwann cells are typically observed in peripheral nerves, and EVs derived from Schwann cells (SC-EVs) have been proved to effectively promote nerve regeneration.84 A study by Wu et al discovered that SC-EVs also play a direct role in bone regeneration. Their research demonstrated that SC-EVs promoted the migration, proliferation, and osteogenic differentiation of BMSCs in vitro. Additionally, when combined with titanium alloy scaffolds, SC-EVs improved bone regeneration in vivo.85

Function and Potential Mechanisms of EVs in Bone Regeneration

In the previous section, we introduced the sources of parent cells of EVs applied in bone tissue engineering applications. It is evident that EVs, particularly those derived from MSCs, play pivotal roles in promoting bone regeneration. Extensive research has shown that the beneficial effects of EVs in bone regeneration are primarily due to their ability to induce osteogenic differentiation, facilitate angiogenesis, and modulate immune responses.15 It is well established that the biological characteristics and functions of EVs are determined by their cargoes, which encompass a diverse array of bioactive molecules inherited from their parent cells, including proteins, nucleic acids, and lipids.11,12 The lipid bilayer membrane of EVs acts as a protective barrier, shielding their cargoes from degradation by extracellular proteases, nucleases, and other enzymes. By transferring their cargoes between cells, EVs mediate genetic alteration in target cells, leading to cell fate change. Several previous reviews have already examined the mechanisms through which EVs contribute to bone regeneration.19,86 In this section, we will elaborate on the roles and potential mechanisms of EVs in bone regeneration, focusing on their ability to induce osteogenic differentiation, promote angiogenesis and regulate immune responses.

Promoting Osteogenesis

As previously mentioned, numerous studies have demonstrated that EVs released by MSCs and some other cells can directly enhance the osteogenic differentiation of MSCs, osteoblasts, and osteoprogenitor cells.20,25,45,46,50,60–62,82,85 The process of bone regeneration, involving the osteogenic differentiation of MSCs into mature osteoblasts and their subsequent mineralization, is intricately regulated by various miRNAs.87 MiRNAs are small non-coding RNAs (containing about 18–22 nucleotides) that regulate gene expression at the post-transcriptional level by binding to target mRNAs and inducing their degradation and/or translational inhibition.88 Among the various molecules contained in EVs, miRNAs have garnered significant attention due to their regulatory roles in gene expression.89 EVs have been shown to be enriched with osteogenesis-related miRNAs. For instance, Qin et al extracted small RNAs from BMSC-EVs and subjected them to miRNA/small RNA-sequencing analysis, finding that three miRNAs critical for osteogenesis (miR-196a, miR-206 and miR-27a) were highly enriched in BMSC-EVs. Functional tests demonstrated that all these three miRNA mimics exhibited osteogenic effects, with miR-196a exhibiting the highest potency.25 Guo et al demonstrated that BMSC-EVs promoted osteogenic differentiation of BMSCs by suppressing the expression of WWP1, an inhibitor of osteoblast differentiation, through the delivery of miR-19b-3p.33 Chen et al analyzed miRNAs contained in ASC-EVs and found that among the top 30 most highly enriched miRNAs in the EVs, five miRNAs (miR-21, miR-199b, miR-10a, miR-10b and miR-let-7f) were reported to be involved in maintaining bone homeostasis or promoting stem cell osteogenic differentiation.38 Jing et al demonstrated that the enhanced osteogenesis induced by SCAP-EVs was mediated through the highly expressed miRNA-150-5p. Further bioinformatic analysis predicted that miRNA-150-5p might facilitate osteogenesis by regulating the PI3K-Akt, Wnt, and MAPK signaling pathways.50 Hu et al found that UCMSC-EVs enhanced calcium deposition and endothelial network formation, promoting both osteogenic differentiation and angiogenesis by delivering miR-23a-3p, which activated the PTEN/AKT signaling pathway.47 Wang et al conducted small RNA sequencing analysis of DPSC-EVs and identified the highly expressed miR-1246 as a potential key regulator of DPSC-EVs in promoting bone tissue regeneration.90 In another study by Cao et al, the promoting effect of EVs derived from mature dendritic cells on proliferation and osteogenesis of BMSCs was demonstrated to be mediated by highly expressed miR-335. Further investigation revealed that miR-335 was transferred to BMSCs by EVs and inhibited the Hippo signaling pathway by targeting large tongue suppressor kinase 1 (LATS1).79

Furthermore, researchers have discovered that miRNA profiles within EVs change depending on the stage of osteogenic differentiation. Zhai et al demonstrated that EV released from MSCs exposed to longer osteogenic differentiation time showed enhanced osteogenic potential compared to that of MSCs exposed to shorter osteogenic differentiation time. Specifically, osteogenic miRNAs (miR-503-5p, miR-146a-5p, miR-129-5p, and miR-483-3p) were upregulated and anti-osteogenic miRNAs (miR-133a-3p, miR-32-5p, and miR-204-5p) were downregulated in EVs derived from late-stage osteogenic cultures (day 10 and day 15), compared to those from early-stage osteogenic cultures (day 0 and day 4). Further bioinformatic analysis suggested that these differentially expressed miRNAs might activate the PI3K/Akt and MAPK signaling pathways.91 In line with this finding, Pishavar et al observed a similar trend in EVs derived from placental stem cells (PSCs). They found that miR-10, miR-27a, and miR-192, which have been reported as late markers of osteogenesis, were upregulated in EVs derived from late-stage osteogenic cultures.61

Other researchers compared miRNA profiles within EVs secreted by MSCs derived from different tissue origins. Liu et al found that only three miRNAs were significantly different (1 up-regulated: miR-23b-3p; 2 down-regulated: miR-199a-3p and miR-214-3p) between osteogenically induced BMSC-EVs and ASC-EVs. All three miRNAs were reported to be involved in regulating osteogenic and/or adipogenic differentiation. They further concluded that the osteo-inductive potential of BMSC-EVs was attributed to multiple miRNAs (let-7a-5p, let-7c-5p, miR-31a-5p, and miR-328a-5p), which targeted Acvr2b/Acvr1, regulating the competitive balance of Bmpr2/Acvr2b towards Bmpr-elicited Smad1/5/9 phosphorylation.63

Recent research has revealed that long non-coding RNAs (lncRNAs) can be transferred to target cells via EVs. These lncRNAs act as competing endogenous RNAs, binding and sequestering miRNAs to prevent them from interacting with their target mRNAs.92 For instance, Yang et al demonstrated that BMSC-EVs carrying lncRNA MALAT1 significantly enhanced osteogenic activity and mitigated osteoporosis symptoms in ovariectomized mice by acting as a miR-34c sponge, leading to the upregulation of SATB2 expression.93 Behera et al identified that lnc-H19 was enriched in BMSC-EVs and functioned as an activator of the ANGPT-Tie2 axis. By serving as a miRNA-106a sponge, lnc-H19 promoted both BMSC osteogenesis and endothelial angiogenesis.94 Additionally, Qi et al reported that EVs derived from osteogenically-induced BMSCs transferred lncRNA-ENSRNOG00000056625, which acted as a sponge to sequester miR-1843a-5p, preventing its binding to Mob3a. This interaction promoted YAP dephosphorylation and nuclear translocation, ultimately alleviating senescence-related phenotypes, and enhancing proliferation and osteogenic differentiation of senescent BMSCs.30

In addition to nucleic acids, the abundant proteins encapsulated in EVs also play a vital role in cell-to-cell communication. Li et al performed quantitative proteomics to compare the protein profiles of BMSC-EVs, ASC-EVs, and SMSC-EVs. They discovered that proteins associated with regulation of actin cytoskeleton, focal adhesion, extracellular matrix (ECM)-receptor interaction, PI3K-Akt signaling pathway, and cAMP signaling pathway were more abundant in ASC-EVs compared to BMSC-EVs and SMSC-EVs, which could account for the enhanced osteogenic potential of ASC-EVs.58 Al‑Sharabi et al compared the protein profiles of EVs derived from osteogenically induced MSCs with those from non-osteogenically induced MSCs. The findings revealed that the upregulated differentially expressed proteins in EVs derived from osteogenically induced MSCs were mainly involved in pathways related to wound and bone healing. In contrast, the upregulated proteins in EVs derived from non-osteogenically induced MSCs appeared to be involved in pathways related to EV formation and biogenesis.28 Ge et al performed proteomic analysis of EVs derived from osteoblast cell line MC3T3. Their findings indicated that proteins encapsulated in EVs from osteoblasts were highly enriched in osteogenesis-associated signal pathways, including integrin signaling, eukaryotic initiation factor 2 signaling, and mTOR signaling pathways.95

In conclusion, EVs promote osteogenesis in MSCs and osteoblasts by delivering their bioactive cargoes, particularly nucleic acids and proteins. This effect is likely mediated through the modulation of key signaling pathways, including PI3K/Akt, BMP/Smad, Wnt/β-Catenin, AMPK, and Hippo. However, the precise mechanisms underlying EVs’ osteogenic activity remain incompletely understood. Further research is needed to elucidate the detailed mechanisms and related downstream signaling cascades.

Enhancing Angiogenesis

Improving vascularization of the implants remains a significant challenge in bone tissue engineering. In cases of large bone defects, the implanted seed cells are typically positioned several hundred microns away from the nearest capillary supply, leading to hypoxia and subsequent apoptosis of the seed cells, which compromises the efficacy of the implants.96 Therefore, promoting angiogenesis has consistently proven to be an effective strategy to facilitate bone regeneration.97 Numerous studies have shown that MSCs derived from various tissues (such as bone marrow, adipose tissue, placenta, and umbilical cord) secrete EVs that not only enhance osteogenesis but also exhibit potent pro-angiogenic effects. In vitro experiments have demonstrated that MSC-EVs can significantly enhance the proliferation, migration, and tube formation of endothelial cells.98–101 Additionally, in animal models, biomaterials integrated with MSC-EVs have been shown to effectively promote bone regeneration by enhancing vascularization at bone defect sites.50,60,102 Furthermore, direct injection of MSC-EVs into bone fracture sites has been reported to accelerate fracture healing through improved vascularization.103

The pro-angiogenic effects MSC-EVs are closely associated with their encapsulated bioactive cargoes. MSC-EVs are enriched with angiogenesis-promoting biomolecules, including VEGF, angiogenin, basic fibroblast growth factors (bFGF), and angiopoietin-1 (ANG-1). Notably, levels of VEGF, angiogenin, monocyte chemotactic protein-1 (MCP-1) and the receptor-2 for VEGF (VEGF-R2) are even higher in EVs than in their parent MSCs.99 In addition to these angiogenic factors, EV-encapsulated miRNAs also play an important role in their pro-angiogenic effects. For instance, Gong et al demonstrated that EVs secreted by the MSC line C3H10T1/2 enhanced the proliferation, migration and angiogenesis of HUVECs by delivering miR-30b, which downregulated the expression of DLL4. DLL4 is a membrane-bound ligand from the Notch signaling family that plays a negative regulatory role in vascular sprouting and vessel branching.104 Wang et al reported that miR-210, which targeted the angiogenesis-related gene Efna3, was enriched in BMSC-EVs. EVs collected from BMSCs with silenced miR-210 exhibited significantly reduced pro-angiogenic effects both in vitro and in vivo.105 Other studies reported that ASC- EVs exerted pro-angiogenic effects via the transfer of miR-125a and miR-31, which targeted DLL4 and hypoxia-inducible factor-1α (HIF-1α), respectively, in recipient endothelial cells.106 Additionally, Jing et al found that miR-126-5p, highly expressed in SCAP-EVs, was transferred to HUVECs to enhance expression of angiogenic genes such as VEGF and ANG-1.50

The pro-angiogenic effects of EVs can be further enhanced by hypoxia pretreatment of the parent cells. Liu et al found that EVs released by hypoxia-treated UCMSCs promoted angiogenesis of HUVECs in vitro and bone fracture healing in vivo through miR-126 and the SPRED1/Ras/Erk signaling pathway. Hypoxia preconditioning resulted in elevated miR-126 levels in UCMSC-EVs through HIF-1α activation.103 Additionally, certain biomaterials can amplify the angiogenic potential of EVs. For instance, Liu et al demonstrated that lithium-containing biomaterials upregulated the expression of miR-130a in BMSC-EVs, which resulted in the downregulation of the PTEN protein and activation of the AKT pathway, thereby enhancing the proliferation, migration, and tube formation of HUVECs.107 In summary, EVs can enhance bone regeneration by delivering their encapsulated pro-angiogenic biomolecules to endothelial cells and promoting vascularization.

Regulating Immune Responses

As mentioned above, immune cells play a pivotal role in bone regeneration. Typically, a large number of immune and inflammatory cells are located in the microenvironment of bone defect sites. The implanted biomaterials are often recognized as foreign substances by the host immune system, triggering a cascade of immune responses.20 Studies have shown that prolonged exposure to pro-inflammatory cytokines can lead to chronic inflammation, resulting in fibrous encapsulation around the bone graft and osseointegration failure.108 Therefore, focusing solely on direct osteogenesis while neglecting the immune reactions caused by biomaterials and seed cells is insufficient for constructing an ideal tissue-engineered bone graft. Only mild inflammatory responses are beneficial for bone regeneration.109

EVs can modulate immune responses by interacting with immune effector cells, including T cell, B cells, macrophages, and other immune cells, through mechanisms such as membrane fusion, ligand‒receptor interactions and delivery of bioactive cargoes.110 Growing evidence indicates that MSC-EVs possess immunosuppressive functions. They have been shown to inhibit immune cell activation, promote the expression of anti-inflammatory factors and reduce inflammatory responses.111 For instance, MSC-EVs can suppress the proliferation of T cells and B cells, promote the conversion of T cells into regulatory T cells, inhibit dendritic cell maturation, and attenuate the function of natural killer cells.112 Additionally, MSC-EVs have been found to facilitate tissue damage repair by modulating the M1/M2 polarization of macrophages localized at the site of tissue injury.113,114 In a study performed by Fan et al, both in vitro and in vivo results demonstrated that BMSC-EVs promoted macrophage M2 polarization via the NF-κB pathway. Moreover, EV-functionalized scaffolds created a more favorable immune microenvironment for bone regeneration compared to scaffolds without EV modification.20 Consistent with this finding, Li et al observed that ASC-EVs inhibited inflammation and promoted the polarization of M1 macrophages to M2 macrophages. Their data further indicated that ASC-EVs exerted their immunomodulatory effects through miR-451a, which targeted the macrophage migration inhibitory factor (MIF).115

In summary, the roles of EVs in bone regeneration encompass three main aspects: firstly, they directly promote osteogenic differentiation of MSCs and osteoblasts; secondly, they stimulate angiogenesis, thereby accelerating vascularized bone regeneration; and thirdly, they modulate the inflammatory response at the site of injury, creating a favorable immune environment for bone regeneration. As previously discussed, the beneficial effects of EVs are primarily attributed to their encapsulated bioactive molecules, particularly miRNAs and proteins. Table 2 summarizes the miRNAs enclosed in EVs that are implicated in bone regeneration.

Table 2 MiRNAs Enclosed in EVs That are Involved in Bone Regeneration

EV-Biomaterial Delivery System in Bone Tissue Engineering

In The Roles of EVs in Bone Regeneration, we introduced the sources of parent cells for EVs applied in bone tissue engineering and elucidated the roles and potential molecular mechanisms of EVs in bone regeneration. EVs are emerging as an ideal candidate for developing cell-free therapies for bone regeneration due to their inherent advantages, such as low immune-rejection, stability, biocompatibility, and high feasibility for modularized customized modification.14 However, one of the major challenges in applying EVs to bone tissue engineering is that free EVs do not allow for durable retention at the defect site.16 To address this issue, researchers have proposed an excellent solution: combining EVs with biomaterial scaffolds to achieve sustained aggregation and controlled release of EVs at the defect site. To date, numerous studies have successfully combined EVs with biomaterial scaffolds, demonstrating the strong potential of these scaffolds for efficient EV loading and release modulation.15 Traditionally, biomaterial scaffolds, stem cells, and growth factors have been considered the three essential elements for bone tissue engineering.116 With the development of EV-integrated scaffolds, this three-basic-element model has the potential to be simplified to two-basic-element model: biomaterial scaffolds and EVs.117 However, the use of EVs alone is often insufficient for complete tissue regeneration, especially in challenging scenarios like large bone defects.118 Therefore, various techniques have been applied to engineer EVs to enhance their therapeutic potential in bone regeneration. These engineering methods can generally be divided into two main categories: endogenous engineering strategies and exogenous engineering strategies.15,119 Endogenous engineering strategies involve modifying the parent cells of EVs, while exogenous engineering strategies involve modifying EVs after they have been isolated from their parent cells. Additionally, the way in which EVs are integrated with biomaterial scaffolds plays a critical role in determining the bone regenerative potency of these EV-loaded scaffolds.119 In this section, we elaborate on the methods for engineering EVs and strategies for integrating EVs with biomaterial scaffolds.

Endogenous Engineering of EVs

In Parent Cells of EVs, we have introduced the diverse sources of parent cells for EVs applied in bone tissue engineering. It is well established that EVs exert their therapeutic effects in a content-dependent manner, with proteins, mRNAs, and/or microRNAs playing pivotal roles in their biological functions. As miniature versions of cells, the contents of EVs are determined not only by the type of parent cells but also by their culture conditions.120 Furthermore, studies have shown that modifying parent cells before EV isolation can alter the contents and biological functions of EVs.121 In this section, we discuss methods for engineering EVs at the cellular level through physical manipulation, pre-conditioning treatment and genetic engineering of parent cells, as illustrated in Figure 1.

Figure 1 Schematic summary of strategies for endogenous engineering of EVs at the cellular level, including physical manipulation, pre-conditioning treatment (including osteogenic induction, hypoxic exposure and biochemical pretreatment) and genetic engineering of parent cells (such as lentivirus transduction and the EXPLOR technique). Adapted from Biomaterials, Volume 283, Tao SC, Li XR, Wei WJ, et al, Polymeric coating on beta-TCP scaffolds provides immobilization of small extracellular vesicles with surface-functionalization and ZEB1-Loading for bone defect repair in diabetes mellitus, Page 121465, Copyright 2022, with permission from Elsevier.117

Physical Manipulation of Parent Cells

Mechanical loading of the skeleton system is crucial for bone development, growth, and maintenance. Piezo1 is a mechanotransducer that confers mechanosensitivity to bone-forming osteoblasts, thereby regulating mechanical load-induced bone formation and remodeling.122,123 He et al demonstrated that EVs derived from BMSCs pretreated with Yoda1, a Piezo1 agonist, exhibited enhanced osteogenic capabilities compared to control EVs. Moreover, these Yoda1-pretreated BMSC-derived EVs, when incorporated into hydrogels, displayed significantly improved bone regenerative capacity in both subcutaneous ectopic bone formation model in nude mice and rat calvarial bone defect model.32

Extruding physical forces directly onto cells is a commonly used methods for the physical modification of parent cells. The traditional method of isolating EVs via differential centrifugation is widely recognized as time-consuming, labor-intensive, and producing EVs with low purity and yield, which somewhat limits their potential for large-scale clinical application.124 To address these deficiencies in the production of natural EVs, researchers have utilized cell-derived plasma membrane fragments to produce EV mimetics (EMs) via lipid self-assembly. EMs, which closely resemble EVs in shape and content, can be fabricated by serially extruding cells through polycarbonate membrane filters with decreasing pore sizes using a microextruder.125 This extrusion technique yields 100–200 times more EMs than the conventional differential centrifugation method, significantly improving production efficiency and substance loading.126 Compared to natural EVs, EMs exhibit similar size, structure, surface markers, and can be modified using both endogenous and exogenous engineering strategies. To date, EMs derived from various parent cells, including MSCs, iPSC-ECs, ATDC5 cells, and others, have been effectively utilized in bone tissue engineering.118,124,127,128 Although EMs are not naturally secreted by parent cells, they are considered more promising vesicles than EVs for gene and drug delivery due to their higher yields, less time-consuming isolation process, and relatively simpler cargoes.124 Therefore, in this review, we also include EMs when discussing the application of EVs in bone regeneration.

In addition to exerting physical forces to parent cells, researchers have discovered that magnetic scaffolds can influence the contents of EVs. Zhu et al found that modification of hydroxyapatite scaffolds with magnetic nanoparticles reduced the levels of certain proteins, such as reactive oxygen species, ubiquitin, and ATP in osteoclast-derived EVs, while increasing Rho kinase levels. This modification weakened the negative impact of EVs on osteogenic cells and promoted bone regeneration.129

Pre-Conditioning of Parent Cells

Pre-conditioning of parent cells can enhance the biologic activity of EVs derived from them. Researchers have utilized various methods including osteogenic induction, hypoxia pretreatment, and stimulation with growth factors, cytokines and other biochemical reagents to optimize the pro-osteogenic efficacy of EVs. Although the relationship between cell state and EV production remains incompletely understood, pre-conditioning of parent cells offers advantages such as operational simplicity, low cost, preservation of EV structure, and elimination of additional purification steps, highlighting its potential for scalable manufacturing.

Osteogenic Induction

Pre-culturing parent cells in osteogenic induction medium is an effective strategy to enhance the osteogenic potential of EVs. Studies have shown that EVs derived from osteogenically-induced MSCs are more effective in promoting osteogenic differentiation of MSCs in vitro and bone regeneration in vivo compared to EVs derived from undifferentiated MSCs.28 Additionally, several studies have reported that EVs secreted by non-osteogenically induced ASCs lack osteogenic potential, whereas EVs secreted by osteogenically induced ASCs significantly promote osteogenic differentiation of BMSCs.37,63 Furthermore, researchers have demonstrated that EVs induce osteogenic differentiation of MSCs in a stage-dependent manner. Specifically, EV released from MSCs exposed to longer osteogenic differentiation time induce osteogenic differentiation more efficiently than those released from MSCs exposed to shorter osteogenic differentiation time.61,91 Taken together, these findings suggest that osteogenic induction is one of the most cost-effective and practical methods for enhancing the osteogenic potential of EVs.

Hypoxia Treatment

Preconditioning parent cells in a hypoxic environment is another widely applied pretreatment strategy to enhance the osteogenic potential of EVs. It is well established that MSCs naturally reside in hypoxic conditions.130 Cells respond to hypoxic environment by expressing HIF-1α, an important regulator of bone repair and regeneration that mediates key biological processes such as angiogenesis and osteogenesis.131,132 Hypoxic pretreatment not only stimulates MSCs to secrete more EVs, but also enhances the angiogenic potential of EVs both in vitro and in vivo.100,103 In a study by Liu et al, hypoxic preconditioning of UCMSCs was shown to enhance the pro-angiogenic potential of EVs by transferring functional miR-126 to HUVECs, resulting in the down-regulation of SPRED1. Furthermore, they demonstrated that hypoxia preconditioning amplified the therapeutic efficacy of EVs, thereby accelerating bone fracture healing in a murine femoral fracture model.103 Zhuang et al also discovered that EVs derived from hypoxia-pretreated BMSCs promoted the proliferation, migration, and angiogenesis of HUVECs and ultimately enhanced vascularized bone regeneration in a rat calvarial bone defect model. Mechanistically, hypoxia induced overexpression of miR-210-3p in EVs, which inhibited EFNA3 expression and subsequently activated the PI3K/AKT pathway.133 In another study by Cui et al, EMs were generated from hypoxia-preconditioned endothelial cells derived from human induced pluripotent stem cells (hiPSCs) through an extrusion approach. Although these EMs did not directly induce osteogenic differentiation of BMSCs, they exhibited potent pro-angiogenic activities and re-educated bone marrow endothelial cells to secret cytokines that promoted osteogenic differentiation of BMSCs and alleviated the pro-inflammatory microenvironment dominated by M1 macrophages in osteoporotic bones.127 In addition to hypoxic pretreatment of parent cells, researchers also use chemicals that stabilize the expression of HIF-1α to mimic hypoxia in cells under normal oxygen levels. As mentioned earlier, cells respond to hypoxic treatment by expressing HIF-1α, which regulates several hypoxia-responsive genes. Under normoxic conditions, HIF-1α is proteasomally degraded by the iron-containing prolyl hydroxylase (PHD) enzyme. Dimethyloxaloylglycine (DMOG) is a small angiogenic molecule that inhibits PHD enzyme, thereby stabilizing HIF-1α.134 Liang et al found that EVs derived from BMSCs preconditioned with a low dose of DMOG promoted neovascularization via the AKT/mTOR pathway and enhanced bone regeneration in critical-sized bone defects in rats.22

Biochemical Pretreatment

Preconditioning parent cells with various growth factors and cytokines can further enhance the osteogenic potential of EVs. BMP-2 is a well-established osteogenic factor and the only osteo-inductive growth factor approved by the Food and Drug Administration.52 Wei et al demonstrated that EVs from BMP2-stimulated macrophages induced osteogenic differentiation of BMSCs more efficiently than control EVs and enhanced the bio-functionality of titanium nanotubes.77 Previous studies indicated that inflammatory mediators could induce the regenerative capacity of MSC-EVs.135 In a study by Lu et al, tumor necrosis factor-α (TNF-α), a major inflammatory factor, was used as a preconditioning agent to mimic the acute inflammatory phase upon bone injury. Their study showed that TNF-a preconditioning enhanced the osteogenic potential of ASC-EVs by increasing their Wnt-3a content.136 Sun et al found that pretreatment of BMSCs with lipopolysaccharide (LPS), a potent inducer of inflammatory cytokine release, significantly enhanced the immunoregulatory potential of EVs by increasing EV secretion and promoting the polarization of macrophages from M1 to M2 phenotype.137

Beyond the biochemical agents mentioned above, researchers have employed other agents, such as dexamethasone,31 tauroursodeoxycholic acid,44 hydrogen peroxide,138 and strontium-substituted calcium silicate ceramics,139 to pretreat MSCs and enhance the therapeutic potential of EVs in bone regeneration. Furthermore, rather than using a single biochemical agent, researchers have explored the synergistic effects of multiple agents on parent cells to further enhance the therapeutic potential of EVs. For instance, Man et al investigated the synergistic effects of the hypoxia mimetic agent deferoxamine (DFO) and the DNA methyltransferase inhibitor 5-azacytidine (AZT) on the therapeutic efficacy of BMSC-EVs for bone repair.132 DFO is an iron chelating agent that induces cellular hypoxia by inhibiting the activity of PHD, thereby stabilizing HIF-1α expression.140 AZT is a DNA methyltransferase inhibitor that promotes osteogenic differentiation of MSCs.141 Interestingly, the demethylation of HIF-1α is essential for its stabilization during hypoxia, enhancing its transcriptional activity.132 Their study demonstrated that inducing hypomethylation during hypoxia synergistically enhanced osteogenesis in BMSCs, thereby increasing the pro-angiogenic and osteogenic potentials of their secreted EVs.

Genetic Engineering of Parent Cells

The strategy of loading EVs with therapeutic molecules via genetic modification of the parent cells can improve the therapeutic efficacy of EVs. By utilizing biological tools, such as viral vectors or plasmids, parent cells can be genetically engineered to increase the expression of endogenous molecules, which are subsequently encapsulated within their derived EVs through the cell’s biomolecular synthesis mechanisms. Endogenous cargoes are usually miRNAs and proteins with therapeutic benefits.

Lentivirus and adenovirus transduction are commonly used by researchers to genetically modify parent cells in bone tissue engineering applications. For instance, Huang et al established a stable BMSC line that constitutively overexpresses BMP2 via lentivirus transduction.27 They discovered that EVs derived from these BMP2-overexpressing BMSCs retained the general physical and biochemical characteristics of naïve BMSC-EVs in terms of size distribution, surface marker expression and endocytic properties. However, these EVs showed significantly enhanced bone regenerative potential compared to naïve BMSC-EVs in a rat calvarial bone defect model in vivo. Interestingly, despite BMP2 being constitutively expressed in the parental cells, BMP2 protein was absent as a constituent of the secreted EVs. Further investigations revealed that the enhanced pro-osteogenic potential of EVs was partly attributed to altered EV cargoes, including miRNAs that potentiate the BMP2 signaling cascade. Subsequently, the same research group utilized EVs derived from BMP2-overexpressing BMSCs in combination with hydrogels to repair rat calvarial bone defects.142 In another study by Fan et al, EMs were obtained from MSCs in which the expression of noggin, a natural BMP antagonist, was downregulated by transduction of lentivirus particles encoding noggin shRNA.118 They demonstrated that EMs from noggin-suppressed MSCs significantly accelerated bone healing compared to naïve EMs in a rat calvarial bone defect model. Mechanistic studies revealed that the enhanced osteogenic potential of EMs from noggin-suppressed MSCs was mediated via inhibition of miR-29a. Meanwhile, Chen et al generated a stable ASC line in which miR-375, a positive regulator in the osteogenic differentiation of MSCs, was upregulated by lentivirus transduction.40 Administration of EVs from miR-375-overexpressing ASCs improved osteogenic differentiation of BMSCs in vitro and enhanced bone regeneration in a rat model of calvarial bone defect in vivo. In another study, Li et al transfected BMSCs with adenovirus carrying triple point-mutations (amino acids 402, 564, and 803) in the HIF-1α coding sequence.24 The mutant HIF-1α effectively maintained cellular expression under normoxic conditions. They discovered that EVs derived from these mutant HIF-1α-modified BMSCs exhibited enhanced pro-osteogenic and pro-angiogenic potential in vitro. Furthermore, injecting these EVs into the necrotic region significantly accelerated bone regeneration and angiogenesis in a rabbit model of steroid-induced avascular necrosis of the femoral head. Another group demonstrated that EVs derived from mutant HIF-1α-modified BMSCs, in combination with β-tricalcium phosphate (β-TCP) scaffolds, effectively repaired critical-sized bone defects by promoting bone regeneration and neovascularization.21

Besides viral transduction, chemical transfection methods are also utilized by researches to genetically modify parent cells. As mentioned earlier, excessive infiltration or persistence of inflammatory cells contributes to chronic inflammation and hinder bone regeneration. Studies have shown that TIM3, a membrane protein encoded by Havcr2, promotes the polarization of M2 macrophages, which in turn secrete cytokines that suppress inflammation. Lu et al transfected BMSCs with Havcr2 overexpression plasmid with a commercial DNA transfection reagent and obtained engineered EVs that highly expressed TIM3. Their results demonstrated that these TIM3-overexpressing EVs, when encapsulated in hydrogel, accelerated bone regeneration in a mouse calvarial bone defect model by modulating the immune microenvironment and mitigating the adverse effects of excessive inflammation.29 In a separate study, Liu et al generated BMSC-EVs overexpressing miR-20a, a microRNA known for its potent pro-osteogenic potential, by transfecting BMSCs with miR-20a mimics using a commercial transfection reagent. Their study showed that these BMSC-EVs overexpressing miR-20a significantly promoted the migration and osteogenesis in BMSCs in vitro and improved titanium alloy scaffold osteointegration in osteoporotic rats.143

Recently, researchers have developed an innovative technique for loading proteins into exosomes using optogenetics, known as EXPLOR (exosomes for protein loading via optically reversible protein-protein interactions) technology.144 This technique exploits the natural process of exosome biogenesis and optogenetically controlled reversible protein-protein interactions, enabling precise, reversible and efficient delivery of the target proteins into exosomes. To load target proteins into exosomes, two vectors expressing fusion proteins are introduced into parent cells: CIBN (a truncated version of CIB1) conjugated with an exosome-associated tetraspanin protein CD9, and CRY2-conjugated cargo protein. A single pulse of 488-nm laser irradiation triggers the rapid translocation of CRY2-conjugated cargo proteins from the cytosol to the plasma membrane and the membrane of multivesicular bodies (MVBs), where CIBN-conjugated CD9 proteins are co-localized. During the process of endogenous biogenesis, the cargo proteins are incorporated into exosomes. Upon removal of the illumination source, the cargo proteins are detached from the CD9-fused CIBN, resulting in their release into the exosomes’ intraluminal space.145 Zinc Finger E-Box Binding Homeobox 1 (ZEB1), which is highly expressed in CD31hi endomucinhi endothelial cells, promotes angiogenesis-dependent bone formation and is considered a very promising therapeutic molecule.146 To efficiently load ZEB1 into exosomes, Tao et al transfected SMSCs with two vectors expressing fusion proteins: a CIBN-CD9 expression vector and a CRY2-ZEB1 expression vect