Enhancing osteoporosis treatment: emerging roles of engineered exosomes in bone regeneration and repair

Abstract

Background

Osteoporosis, a prevalent metabolic bone disease, is characterized by reduced bone density and an increased risk of fractures. The primary challenge in treating osteoporosis lies in correcting the imbalance between bone resorption and formation while minimizing the associated risks of treatment. Current drugs, such as bisphosphonates, teriparatide, and romosozumab, help reduce fracture risk but have limitations, including long-term safety concerns, incomplete protection, and poor patient adherence.

Main body

Exosomes, small extracellular vesicles with natural biocompatibility and low immunogenicity, have emerged as promising therapeutic candidates for osteoporosis. These vesicles carry bioactive molecules that regulate bone remodeling, promote osteogenesis, and inhibit osteoclastogenesis. Advances in bioengineering have improved the targeting efficiency and drug-loading capacity of exosomes, while their combination with biomaterials supports localized and sustained bone regeneration. The review explores the signaling mechanisms that contribute to osteoporosis and highlights the biological functions of exosomes. Furthermore, it addresses the challenges in translating exosome-based therapies, such as variability in exosome content, production standardization, scalability, and unresolved safety concerns. Preclinical models play a key role in assessing therapeutic efficacy. The review also considers interdisciplinary innovations, including nanotechnology, biomaterials, advanced imaging, and artificial intelligence, and their potential to enhance the translation of exosome therapies.

Conclusions

Exosome therapies represent a promising next-generation strategy for the treatment of osteoporosis. With continued advancements in engineering, clinical evaluation, and interdisciplinary innovations, exosomes could offer safer, more effective, and personalized treatment options for osteoporosis. However, challenges remain in ensuring the consistency, safety, and scalability of exosome production, which must be addressed before widespread clinical adoption.

Introduction

Bone integrity relies on the precise coupling of bone formation and bone resorption. When this balance is disrupted, osteoporosis develops, characterized by reduced bone mineral density (BMD), deterioration of trabecular and cortical microarchitecture, and consequently, increased bone fragility and fracture risk [1, 2]. With the accelerating pace of global population aging, both the prevalence and the societal burden of osteoporosis are rising [3]. Osteoporotic fractures are associated with substantial morbidity and mortality, loss of mobility and independence, and considerable direct and indirect healthcare costs [4]. These outcomes underline the urgent need for more durable and individualized strategies to preserve skeletal strength and prevent fractures across diverse at risk populations.

Clinically, available pharmacologic therapies can be broadly divided into two categories [5]. Antiresorptive agents-including bisphosphonates, denosumab, selective estrogen receptor modulators, and hormone replacement therapy, reduce bone turnover and lower fracture risk, but their utility is constrained by adverse effects, adherence challenges, rare complications such as osteonecrosis of the jaw (ONJ), and rebound bone loss following discontinuation in some regimens [6]. Anabolic therapies-such as teriparatide, abaloparatide, and romosozumab-stimulate bone formation and rapidly increase BMD. However, their duration of use is restricted, their optimal sequencing with antiresorptives remains complex, and their efficacy may be diminished in patients with chronic inflammation, glucocorticoid exposure, or multimorbidity [7]. Despite notable progress, residual fracture risk and concerns regarding long-term safety and adherence persist, underscoring the need for innovative, targeted, and durable therapeutic approaches capable of restoring bone remodeling while minimizing systemic toxicity [8, 9].

Against this backdrop, exosomes, small extracellular vesicles (EVs) secreted by a variety of cells, have emerged as promising candidates for both mechanistic insights and therapeutic innovation [10, 11]. With inherent biocompatibility, low immunogenicity, and the ability to transport proteins, lipids, and nucleic acids, exosomes mediate communication among osteoblasts, osteoclasts, immune cells, and the bone vasculature [12, 13]. Accumulating evidence shows that exosomes can transmit pro-osteogenic or pro-resorptive signals, serve as biomarkers, and act as therapeutic carriers that modulate bone remodeling dynamics [14, 15]. Recent studies have further suggested that dietary sources, such as milk-derived exosomes, may support musculoskeletal health and provide a practical avenue for osteoporosis management [16]. Their multifunctional properties provide new opportunities to understand the pathophysiology of osteoporosis and to design novel interventions.

Building on these considerations, this review emphasizes the emerging concept of engineered exosomes as innovative tools for osteoporosis therapy. Beyond summarizing the physiological and pathological foundations of bone remodeling and the limitations of current pharmacologic treatments, it emphasizes how advances in exosome engineering, including surface modification, gene editing, nanotechnology, and integration with biomaterials, can address unmet clinical needs by enhancing targeting, cargo delivery, and functional efficacy. The roles of exosomes in osteogenesis, bone resorption, and immune modulation are also categorized to provide mechanistic context. By prioritizing the translational potential of engineered exosomes, this review offers a forward-looking framework that complements existing therapies and opens new avenues for durable and personalized management of osteoporosis.

Current strategies for osteoporosis therapy

Antiresorptive therapies

Bisphosphonates (alendronate, risedronate, zoledronate) inhibit farnesyl pyrophosphate synthase, reduce bone turnover, and lower vertebral and non-vertebral fracture risk [17]. Their long skeletal retention supports durable effects yet complicates management of rare adverse events such as atypical femoral fractures (AFF) and ONJ [18]. Oral regimens face adherence barriers-including gastrointestinal intolerance and posture/fasting requirements-while annual intravenous zoledronate improves persistence but necessitates renal monitoring [19, 20]. Denosumab, a monoclonal antibody to receptor activator of nuclear factor κB ligand (RANKL), provides robust gains in spine and hip BMD with significant fracture risk reduction across diverse risk strata. However, abrupt discontinuation may trigger rebound bone turnover and multiple vertebral fractures, so most patients ultimately choose to transition to a potent bisphosphonate [21]. Selective estrogen receptor modulators (such as raloxifene) protect against vertebral fractures and confer favorable effects on breast cancer risk, though they do not reduce hip fractures and may increase venous thromboembolism risk [22]. Hormone replacement therapy reduces fractures and relieves vasomotor symptoms in early postmenopause but requires individualized risk-benefit assessment given breast and vascular risks [23]. Across these antiresorptive classes, safety surveillance for AFF/ONJ, vigilance for denosumab rebound, and strategies to sustain adherence remain crucial [24]. Shared decision-making, simplified dosing regimens, infusion services, and reminder systems are increasingly recognized as essential to maintain long-term benefit. Despite these measures, residual fracture risk and concerns regarding cumulative exposure underscore the need for innovative and more durable therapeutic approaches.

Anabolic therapies

Teriparatide and abaloparatide—intermittent analogs of parathyroid hormone (PTH) and PTH-related peptide-stimulate bone formation and rapidly increase spine BMD, with proven reductions in vertebral and some non-vertebral fractures [25]. Their use is label-restricted to typically 18–24 months, after which consolidation with an antiresorptive agent is required to preserve gains [7]. Romosozumab, a sclerostin inhibitor, exerts dual actions by transiently stimulating bone formation and concurrently suppressing resorption, leading to rapid BMD increases and early fracture risk reduction; however, its duration is limited to 12 months, and subsequent antiresorptive therapy is advised [26]. Cardiovascular risk assessment is recommended before use [27]. Sequencing strategies are clinically important: an “anabolic-first then antiresorptive” sequence maximizes BMD accrual and accelerates fracture risk reduction in very-high-risk patients, whereas prior long-term potent antiresorptive exposure may blunt subsequent anabolic response [28]. Drug holidays can be considered in selected patients on bisphosphonates but are not appropriate for denosumab, where continuity is essential [29]. Even with these strategies, adherence challenges, the complexity of sequencing, and concerns regarding long-term safety leave substantial unmet needs.

Current unmet needs in osteoporosis management

Despite advances in antiresorptive and anabolic therapies, substantial unmet needs remain in osteoporosis management, as many patients still experience fragility fractures, highlighting the persistence of fracture risk. Long-term safety concerns, such as AFF and ONJ associated with bisphosphonates, rebound bone turnover with abrupt discontinuation of denosumab, and potential cardiovascular risks linked to romosozumab, further complicate management decisions [21, 27]. These issues, along with suboptimal adherence and persistence due to complex dosing schedules, injection burdens, and the asymptomatic nature of the disease, contribute to reduced real-world effectiveness [30]. Furthermore, treatment algorithms still lack precision. Although current biomarkers and imaging technologies have yielded encouraging results, they are still insufficient to predict individualized responses or guide optimal sequencing of anabolic and anti-resorptive agents [31]. There is also a clear need for therapies that not only offer effective short-term fracture risk reduction but also provide durable, long-term skeletal protection, especially in patients with comorbidities such as chronic inflammation or glucocorticoid use. To address these challenges, innovative, personalized approaches are needed to enhance adherence, tailor treatment, and prevent fractures while minimizing risks. This highlights the need for novel modalities like engineered exosomes, offering cell-type specificity, durable effects, and reduced toxicity.

Physiological and pathological mechanisms of bone homeostasis

Mechanisms of osteoblasts in osteoporosis

In normal bone metabolism, bone formation and resorption are balanced to maintain bone homeostasis [32, 33]. Osteoblasts form new bone by secreting and mineralizing matrix proteins, a process regulated by key signaling pathways, particularly wingless-related integration site (Wnt)/β-catenin and bone morphogenetic protein (BMP)/Smad [34, 35]. The Wnt/β-catenin pathway is central to bone formation [36]. When Wnt ligands, such as Wnt3a and Wnt10b, bind to the Frizzled receptor and low-density lipoprotein receptor-related protein (LRP)5/6 co-receptor on osteoblasts, glycogen synthase kinase (GSK)-3β activity is inhibited, preventing β-catenin degradation [37, 38]. This allows β-catenin to accumulate, cross the membrane, and enter the nucleus, where it binds to T-cell factor (TCF)/Lymphoid enhancer-binding factor (LEF) transcription factors. These complexes activate genes like runt-related transcription factor (RUNX) 2, Osterix, and osteocalcin, promoting osteoblast proliferation, differentiation, and matrix protein production, such as type I collagen [39, 40]. These matrix proteins mineralize to form solid bone (Fig. 1). Mutations in LRP5 can disrupt this signaling, leading to conditions like osteoporosis or osteosclerosis [41].

Fig. 1.

The Wnt pathway and BMP pathway in normal osteoblasts. This diagram spotlights the key components and interactions within each pathway that contribute to osteoblast differentiation and bone formation. The Wnt pathway is depicted on the left, showing its role in promoting osteoblast proliferation and maturation, while the BMP pathway on the right emphasizes its influence on osteoblast differentiation and activity

The Wnt pathway also interacts with other pathways, like BMP and Notch, to regulate bone growth and repair [42]. The Jagged1/Notch1 pathway affects osteoclastogenesis by modifying the RANKL/osteoprotegerin (OPG) ratio in stromal cells and reducing osteoclast progenitors [43]. Jagged1, a type I transmembrane glycoprotein, interacts with Notch receptors, and this signaling is crucial for regulating cell proliferation, differentiation, and fate. Direct cell-cell contact is necessary to activate Notch signaling [44]. However, there are no current treatments targeting Jagged1/Notch1, requiring further research into its potential for osteoporosis therapy.

The BMP/Smad pathway plays a critical role in bone formation and fracture healing. BMPs, especially BMP2 and BMP4, bind to type I (BMPR1A) and type II (BMPR2) receptors on osteoblasts, activating Smad1/5/8 [45]. Phosphorylated Smad1/5/8 proteins bind Smad4, forming a complex that translocates to the nucleus. In the nucleus, this complex activates osteogenesis-related genes, such as RUNX2 and Osterix, promoting osteoblast differentiation [46]. RUNX2 and Osterix enable osteoblasts to mature and secrete bone matrix proteins, like type I collagen and osteocalcin [47]. These proteins mineralize to form bone tissue. BMP/Smad signaling also supports angiogenesis, supplying osteoblasts with nutrients [46]. Disruption of BMP signaling impairs bone formation and fracture healing, deciding its essential role in bone regeneration.

Osteoblast signaling pathways are central to osteoporosis pathogenesis. By contrast, BMP and Notch signaling remain important yet lack effective clinical translation, leaving opportunities for safer, targeted therapies in the future.

Mechanisms of osteoclasts in osteoporosis

The RANK/RANKL signaling pathway plays a critical role in bone resorption, regulating continuous bone remodeling and repair [48]. This pathway is centered around RANKL, which controls the formation and activity of osteoclasts [49]. RANKL is primarily secreted by osteoblasts, bone marrow stromal cells, and other cell types, and it binds to the RANK receptor on osteoclast precursors. This binding triggers the differentiation, maturation, and activation of osteoclasts. Upon RANKL binding to RANK, intracellular signaling pathways, including nuclear factor κB (NF-κB), c-Jun n-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), are activated in osteoclast precursors [50]. These pathways promote the expression of genes essential for osteoclast development, enhance osteoclast attachment to the bone surface, and increase their resorptive activity. Mature osteoclasts secrete acidic substances and lysosomal enzymes like cathepsin K to degrade the mineralized bone matrix, releasing minerals such as calcium and phosphorus, which completes bone resorption (Fig. 2) [51].

Fig. 2.

The RANK/RANKL signaling pathway in normal osteoclasts. RANKL, secreted by osteoblasts, fibroblasts, and T cells, binds to the RANK receptor on the surface of osteoclasts and their precursors. Upon binding, RANKL activates a series of signaling pathways, including NF-κB, MAPK, and NFATc1. NF-κB activation is mediated through TNF receptor-associated factor (TRAF) 6, which translocates into the nucleus to promote the expression of genes related to osteoclastogenesis. The MAPK pathway activates downstream molecules such as extracellular signal-regulated kinase (ERK), JNK, and p38, regulating the transcription factor activator protein (AP) −1. Nuclear factor of activated T cells, cytoplasmic (NFATc) 1 serves as the key regulatory factor for osteoclast differentiation

OPG, a “decoy receptor” for RANKL. OPG binds to RANKL, preventing its interaction with RANK, thus inhibiting osteoclast differentiation and activity. This creates a balance between bone resorption and formation. Under normal conditions, the ratio of RANKL to OPG maintains bone homeostasis. When RANKL levels exceed OPG, osteoclast activity increases, leading to excessive bone resorption and conditions like osteoporosis [52–54]. Conversely, an excess of OPG reduces osteoclast activity, resulting in abnormally high bone density [55, 56]. The RANK/RANKL/OPG pathway is a key regulator of osteoclast generation, maturation, and function in bone metabolism.

In osteoporosis, especially postmenopausal osteoporosis, estrogen deficiency shifts the RANKL/OPG balance by upregulating RANKL and downregulating OPG, thereby promoting excessive osteoclast activity and bone loss. This central role of the RANK/RANKL/OPG axis has also led to successful therapeutic translation. The anti-RANKL monoclonal antibody denosumab effectively suppresses osteoclastogenesis, reduces bone resorption, and lowers fracture risk [28]. However, rapid bone loss and rebound fractures after discontinuation remain significant clinical challenges, underscoring the need for safer and more durable therapies targeting this pathway [57].

Immune microenvironment in osteoporosis

The immune system plays a critical role in osteoporosis, with immune cells deeply intertwined in the process of bone remodeling. Chronic low-grade inflammation, driven by cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6, promotes osteoclastogenesis while inhibiting osteoblast function, leading to bone loss [56, 58]. In innate immunity, M1 macrophages release pro-inflammatory cytokines that enhance osteoclast activity, while M2 macrophages and bone-resident osteomacs support osteogenesis [59]. Neutrophil extracellular traps and dendritic cell-derived cytokines further amplify RANKL-driven bone resorption during inflammation [60]. In adaptive immunity, Th17 cells secrete IL-17 to stimulate RANKL expression, whereas Treg cells suppress osteoclast differentiation through cytotoxic T-lymphocyte antigen (CTLA)-4 and IL-10 [61, 62]. B cells have dual effects by producing both RANKL and its decoy receptor OPG, but aging and estrogen deficiency shift this balance toward bone loss [63, 64].

These immune effects converge on the RANKL–RANK–OPG axis, where pro-inflammatory cytokines, including TNF-α, IL-6, IL-17, and interferon (IFN)-γ, increase osteoclast activity and alter osteoblast lineage commitment. The gut-bone axis and microbiome also play a key role in bone health by affecting calcium absorption and modulating immune responses that influence bone metabolism [65–67]. Oxidative stress, particularly in aging, exacerbates osteoporosis by promoting osteoclast activity and inducing osteoblast apoptosis [68]. Additionally, endocrine factors like PTH and glucocorticoids promote bone resorption by inducing osteoblast apoptosis and suppressing osteogenesis [69]. Collectively, these immune and endocrine mechanisms contribute to accelerated bone loss and increased fracture risk in osteoporosis.

From a translational perspective, these mechanisms offer potential therapeutic avenues. Denosumab exemplifies the successful targeting of RANKL, while emerging approaches aim to modulate inflammatory cytokines (e.g., IL-17 or TNF-α blockade), restore gut microbiota balance through probiotics, or reduce oxidative stress with antioxidant strategies [70, 71]. Although still in exploratory stages, such interventions demonstrate how immune and endocrine insights may translate into novel osteoporosis therapies.

Exosomes and osteoporosis

Formation and biological properties of exosomes

EVs are a type of membrane-bound vesicles actively secreted by cells, which mainly include exosomes (30–150 nm) and microvesicles (100–1000 nm) [10]. They are produced by nearly all cell types and serve as critical tools for intercellular communication, extensively involved in various physiological and pathological processes within the body. Exosomes can be derived from various cell types, including osteoblasts, osteoclasts, mesenchymal stem cells (MSCs), and immune cells, each contributing differently to bone metabolism [11, 72, 73]. Therefore, exosomes, a subset of EVs, have a more specialized formation process in bone metabolism.

The formation of exosomes begins with endocytosis at the plasma membrane, leading to the creation of early endosomes. These early endosomes mature into late endosomes, during which they accumulate intraluminal vesicles (ILVs) through endosomal sorting complex required for transport (ESCRT)-dependent or independent mechanisms, becoming multivesicular bodies (MVBs) [74]. During this process, small vesicles within MVBs gradually accumulate various biomolecules. These molecules are not randomly incorporated into exosomes but are selectively packaged through molecular sorting mechanisms within the cell [75, 76]. This cargo is selectively loaded into exosomes based on the cell of origin and its physiological state, allowing exosomes to convey specific signals to recipient cells. Some MVBs fuse with lysosomes for degradation, while others fuse with the plasma membrane, releasing the ILVs into the extracellular environment as exosomes (Fig. 3). Exosomes act as mediators for cellular communication by transporting bioactive molecules such as proteins, RNA, and microRNA (miRNA), and play essential roles in immune regulation and the process of disease [74, 77]. Therefore, exosomes are not merely waste products expelled by cells; they are finely regulated carriers of biological information, capable of transmitting crucial biological signals between cells. Moreover, the lipid bilayer of exosomes provides stability and protects their cargo from degradation during transit in bodily fluids such as blood, lymph, or synovial fluid [78]. This stability, combined with their small size, enables exosomes to traverse biological barriers and reach distant target cells [79]. The lipid bilayer of exosomes also plays a role in interactions with recipient cells. Specific proteins and lipids on the surface of exosomes play a crucial role in their ability to interact with recipient cells. These surface molecules can bind to membrane proteins of recipient cells, thereby initiating a series of cell signaling pathways that influence various cellular functions, including cell proliferation, differentiation, and immune responses [77].

Fig. 3.

Mechanism of exosome formation,biological characteristics and main functions. Exosome formation begins with the endocytosis of the plasma membrane, leading to the maturation of early endosomes into MVBs. Within MVBs, ILVs are formed, which selectively package biomolecules like proteins, RNA, and miRNA. Some MVBs fuse with the plasma membrane to release ILVs as exosomes. These exosomes carry bioactive molecules and play a critical role in cellular communication, immune regulation, and disease processes

However, changes in the cargo composition of exosomes under different physiological or pathological conditions can alter their function in target cells [78]. For example, tetraspanins, a family of proteins commonly found on the surface of exosomes, are known to facilitate the binding of exosomes to target cells [80, 81]. This interaction can activate intracellular signaling pathways, such as the MAPK/ERK pathway, which is involved in cell proliferation and differentiation [82, 83]. In cancer biology, exosomes carrying tetraspanins have been shown to promote tumor growth by transferring oncogenic factors to surrounding cells, thereby creating a more favorable environment for tumor expansion [84, 85]. Additionally, integrins on the exosome surface play a significant role in determining the tissue tropism of exosomes [86, 87]. For example, integrin αvβ6 on exosomes can specifically bind to fibronectin and laminin on the extracellular matrix of certain tissues, directing the exosomes to those tissues [88]. This binding can activate the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, which is critical for cell survival and proliferation [89]. Therefore, the diversity of EVs reflects their broad physiological functions, while exosomes, a subset of EVs, rely on finely-tuned intracellular membrane systems and exhibit unique biological activities [90]. Together, EVs and exosomes contribute to complex intercellular signaling networks, maintaining cellular function and systemic homeostasis.

Exosomes in osteoporosis

Promotion of osteogenesis

Exosomes contribute to osteoblast differentiation and bone mineralization by transferring bioactive molecules, including miRNAs and proteins. Key osteogenic miRNAs, such as miR-196a and miR-217, activate the Wnt/β-catenin pathway, enhancing osteogenic markers like Runx2 and alkaline phosphatase (ALP) [91, 92]. Other miRNAs, including miR-29b, miR-23a, and miR-140, regulate both osteogenic and adipogenic differentiation, though their off-target effects complicate therapeutic application [93–95].

Proteins within exosomes, such as osteocalcin and ALP, directly support bone mineralization, while exosomal vascular endothelial growth factor (VEGF) facilitates angiogenesis essential for bone health [10]. MSC-derived exosomes enriched with Wnt proteins or BMPs enhance bone density and osteogenesis through BMP/Smad and PI3K/Akt pathways [96–98]. Additionally, modulation of sirtuin (SIRT) 1 activity by exosomes influences bone metabolism, making it a promising therapeutic target for osteoporosis (Table 1) [107, 113–115].

Table 1.

Exosome promotion of osteoblast differentiation and mineralization

Exosome-derived	Models	Signaling pathway	Main effect	References
MSC-derived exosomes	Osteoarthritis rat model	TGFβ1, miR-135b, Sp1	Chondrocyte proliferation	[99]
Bone marrow-derived macrophage	T2DM rat model	miR-144-5p	Osteoblast growth	[100]
Bone marrow MSCs	OVX rat model/MG-63 model	ERK-estrogen receptor α	Osteoblast mineralization	[101]
Human MSCs	Osteoblast precursor model	MAPK, ROS	Osteoblast apoptosis	[102]
Adipose tissues derived MSCs	Diabetic osteoporosis model	miR-146a, TNF-α, IL-1β	Bone loss	[103]
M2 macrophages	Stem cell model/ Nude mice model	VEGF	Bone regeneration	[104]
Vascular smooth muscle cell	Osteoblast model	WNT/β-catenin	Osteoblast mineralization	[105]
Plum-derived nano-vesicles	Osteoblast precursor model	BMP-2, p38, JNK, SMAD1	Osteoblast differentiation	[106]
Mechanically strained osteocytes	Osteoblast precursor model	Mapk14, Sf1	Osteoclast differentiation	[107]
Non-small cell lung cancer cell	Osteoclast precursor model	miR-17-5p, PTEN, PI3K/Akt	Osteoclast differentiation	[108]
Endothelial progenitor cell	Mouse bone fracture model	miRNA-124	Osteoclastogenesis	[109]
P2X7 receptor-activated Exosomes	Osteoclast precursor model /OVX rat model	PI3K-Akt-GSK3β	Bone resorption	[110]
Bone metastatic mammary tumor cell	Osteoblast precursor model	JNK	Osteoblast differentiation	[111]
Myoblast-derived exosome	Mouse osteoporosis model	lncRNA-MIR22HG-Hippo	Osteogenic differentiation	[112]

Activation of osteoclastogenesis

Exosomes also regulate osteoclast activity and contribute to osteoporosis progression. Osteoclast-derived exosomes, enriched with miR-214, TRAF6, and c-Fos, promote osteoclastogenesis and bone resorption while inhibiting osteoblast function, thereby disrupting bone homeostasis [116–118]. These exosomes further amplify inflammation, upregulating RANKL expression, which stimulates osteoclast activity.

In contrast, MSC- and osteoblast-derived exosomes containing OPG and miR-503 counteract RANKL signaling, reducing osteoclast activity and promoting bone formation. These opposing properties highlight exosomes as dual modulators of bone remodeling and potential therapeutic tools for osteoporosis, with approaches such as exosomal OPG or miR-503 delivery showing promise in mitigating excessive bone resorption (Table 1) [119, 120].

Immunomodulation and remodeling balance

Exosomal communication integrates immune, vascular, and stromal cues to tune remodeling dynamics [121, 122]. M1-polarized macrophages release vesicles containing pro-inflammatory mediators (e.g., TNF-α, IL-6) and miRNAs (e.g., miR-155/miR-98) that potentiate osteoclastogenesis and inhibit osteoblast activity, whereas M2-derived exosomes convey IL-10/miR-21 and support repair [123–126]. T-cell derived vesicles exemplify bidirectionality: cargos such as Wnt3a stimulate osteoblastogenesis via Wnt/β-catenin, while RANKL-containing vesicles enhance osteoclastogenesis [41, 127]. Treg-derived exosomes carrying IL-10/transforming growth factor (TGF)-β suppress resorption [128]. Endothelial and osteoblast exosomes add pro-angiogenic signals (e.g., VEGF), coordinating type-H vessel formation with bone formation [129]. These modulatory functions explain how dysregulated exosomal signaling in osteoporosis increases resorption and reduces formation, and they motivate engineering strategies (targeting, cargo loading) to bias the exosomal milieu toward net anabolism and durable fracture-risk reduction (Table 1).

Engineered exosomes for osteoporosis therapy

Natural exosomes may have some limitations in therapeutic efficiency and targeting specificity [130]. In addition, there are many current exosome isolation methods (Fig. 4). But each method has its advantages and disadvantages (Table 2) [131–133]. Recent years, engineering technologies are employed to modify exosomes, significantly enhancing their therapeutic performance. We summarize deeply the applications of surface modification technologies, gene editing technologies, and nanotechnology in exosome engineering.

Fig. 4.

Schematic representation of common exosomal separation techniques. (a) Ultracentrifugation. (b) Density gradient centrifugation. (c) Dead-end filtration. (d) Tangential flow filtration. (e) Size-exclusion chromatography. (f) lmmunoafinity [131]. Copyright, 2021 Chen Jiaci et al.

Table 2.

Overview of several exosome isolation and purification methods

Method	Principle	Advantages	Disadvantages	Translational relevance
Differential ultracentrifugation	Separation by size/density	Large-scale processing; Widely used in labs	Time-consuming; Contamination; Sample loss	Limited GMP feasibility; Labor-intensive
Density gradient centrifugation	Separation by density	High precision; Removes contaminants	Complex operation; Expensive	Difficult to scale up; Mainly research use
Ultrafiltration	Size-based membrane filtration	Simple operation; Concentrating exosomes	Membrane clogging; Sample loss	Potentially automatable; Moderate scalability
Immunoaffinity capture	Antibody–antigen binding	High purity; Targeted studies	High cost; Limited yield	Suitable for biomarker; Not routine GMP
Size exclusion chromatography	Column-based size separation	Maintains structure; Sensitive samples	Limited yield; Small-scale analysis	GMP-adaptable; For clinical translation
PEG precipitation	Polymer-induced aggregation	Easy to perform; Low cost	Easy contamination	Widely used in preclinical work; Not GMP-standard
Microfluidics technology	Chip-based separation	High precision; Small sample analysis	Expensive equipment; Technical expertise	Personalized medicine Early-stage

Surface modification technology

Surface modification technology involves altering the surface properties of exosomes through chemical or biological methods to enhance their targeting specificity and stability [134]. This process typically includes binding specific targeting molecules, antibodies, peptides, or other biomolecules to the surface of exosomes, enabling them to specifically recognize and bind to target tissues or cells (Table 3) [145, 146].

Table 3.

Surface modification technology of exosomes

Technique	Principle	Application and advantages	References
PEGylation	Attaching PEG chains to exosome surface	Reduces immune clearance; Improves in vivo stability	[135, 136]
Antibody decoration	Using antibodies to target specific cell antigens	Enhances targeting; Improves delivery efficiency	[137]
Click chemistry	Bio-orthogonal reactions for mild modifications	Maintains exosome structure; Improves functionality	[138]
Lipid modification	Modifying membrane with lipid molecules	Enhances stability; Improves cellular compatibility	[139]
Magnetic nanocoating	Decorating exosomes with magnetic nanoparticles	Enables magnetic field-guided delivery; Improves targeting precision	[140, 141]
Aptamer modification	Using aptamers to recognize specific cell targets	Increases specificity; Reduces non-specific uptake	[142, 143]
Hydrogel loading	Encapsulating exosomes in hydrogels	Provides sustained release, Enhances therapeutic efficacy	[144]

There is extensive research showing that arginine-glycine-aspartic acid (RGD) peptides have a strong affinity for integrin receptors, particularly integrin αvβ3, which is relevant in tissue targeting and regeneration, including applications in cancer therapy and bone repair [147, 148]. Research on RGD peptides has demonstrated that these sequences bind effectively to integrin αvβ3, which plays a role in cellular adhesion, angiogenesis, and other physiological processes [139]. These interactions are also being explored in fields like tumor imaging and targeted drug delivery, where RGD-conjugated nanocarriers have shown promising results in enhancing the precision of targeted therapies (e.g., in osteosarcoma models) by accumulating more efficiently at target tissues expressing integrin αvβ3 receptors. However, there is no direct report indicating that RGD peptide-modified exosomes have been used to enhance exosome accumulation specifically in bone tissue through integrin receptor targeting in preclinical studies. Although current research primarily focuses on RGD-modified nanocarriers, their potential has already been explored in regenerative medicine [149, 150]. This suggests that applying the same strategy to bone tissue targeting is feasible, given that integrin αvβ3 is also expressed during bone remodeling and angiogenesis. However, there is still a lack of direct experimental evidence supporting the accumulation of RGD-modified exosomes in bone tissue, and further research is needed to validate this concept.

Additionally, by modifying exosome surfaces with anti-osteocalcin antibodies, researchers have successfully achieved efficient delivery of exosomes to bone formation regions, significantly enhancing osteoblast activity and bone formation rates [151]. The application of these modification techniques lays the foundation for developing more efficient exosome-based therapies for osteoporosis. Another successful surface modification technique involves coating exosome surfaces with polyethylene glycol (PEG) [152]. PEG is a highly hydrophilic polymer that forms a hydration barrier, preventing exosomes from being rapidly cleared by the immune system [153, 154]. By PEGylating exosomes, their circulation half-life in the bloodstream can be significantly extended, thereby increasing their accumulation in target tissues [155, 156]. For example, in animal models, PEGylated exosomes have demonstrated higher bone-targeting specificity, reduced nonspecific distribution, and better therapeutic outcomes in the treatment of osteoporosis (Fig. 5) [134].

Fig. 5.

Effects of PL, PL-exo and PL-exo-ALN on MPS-induced bone mass loss in vivo. (a) 2D and 3D reconstructive μ-CT images in the distal femurs of five groups. (b) Quantification of the BMD, trabecular bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) by CTAn of five groups. (c) Images of dynamic bone formation with different treatments were monitored the fluorochrome labeling of five groups (scale bar: 300 μm). (d) Bone minerals of different groups were examined by Von Kossa staining of five groups [134]. Copyright, 2021 Gang Zhang et al.

Moreover, the acidic and hypoxic tumor microenvironment has been found to remodel the lipid composition of exosomes, thereby affecting the uptake of exosomes by tumor cells. These lipid composition changes may alter the biophysical properties of exosomes, making them either easier or harder for target cells to internalize [157]. There is great interest in targeting exosomes and engineering them to carry specific cargo to modulate pathological responses. These advances have the potential to revolutionize current therapies by directly delivering therapeutic payloads to target cells in tumors or other bone diseases, while avoiding the toxicity associated with traditional systemic treatments. With ongoing research efforts, we look forward to significant progress in the field of exosome engineering. These surface-modified exosomes hold significant promise in precision medicine, especially in the treatment of cancers, neurological diseases, and bone regeneration. Ongoing research is exploring their use as vehicles for delivering small interfering RNA (siRNA), clustered regularly interspaced short palindromic repeats (CRISPR) components, and chemotherapeutic agents with high specificity and minimal off-target effects. These approaches not only expand the therapeutic repertoire of exosome engineering but also align with the principles of precision medicine, enabling interventions tailored to specific molecular pathways and patient subgroups in osteoporosis.

Gene editing technology

The convergence of exosome biology with CRISPR-Cas9 gene editing technology offers an innovative platform for precision therapy in osteoporosis [158]. The CRISPR-Cas9 system, composed of the Cas9 nuclease and a single-guide RNA (sgRNA), enables sequence-specific double-strand DNA cleavage and has revolutionized the field of genome editing [159]. However, clinical translation is limited by safe and efficient delivery of CRISPR components to target tissues [160]. Traditional viral vectors are effective but raise concerns regarding immunogenicity, random genome integration, and long-term safety [161]. Synthetic nanoparticles also face limitations in stability and targeting. In this context, exosomes are increasingly recognized as promising delivery vehicles due to their natural biocompatibility, ability to cross physiological barriers, and intrinsic cell–cell communication functions [162].

Most proof-of-concept studies have emerged from oncology and infectious diseases, where exosome-mediated CRISPR delivery has been used to silence oncogenes such as Kirsten rat sarcoma viral oncogene homolog (KRAS) or modulate immune checkpoints such as Programmed death-ligand (PD-L) 1, and to suppress viral replication in hepatitis B infection [158]. These advances underscore the feasibility of packaging and transporting CRISPR machinery using EVs. Importantly, such lessons can be leveraged to develop bone-specific applications. In skeletal research, hybrid exosomes fused with cartilage-affinity peptides and loaded with CRISPR constructs targeting fibroblast growth factor (FGF) 18 achieved selective uptake by chondrocytes, significantly improving osteoarthritis pathology (Fig. 6) [163]. Similarly, nanoscale hybrid carriers that combine engineered exosomes with liposomes have been employed to encapsulate CRISPR/Cas9 plasmids, delivering them into chondrocytes and alleviating cartilage damage [164]. Although primarily applied in osteoarthritis, these strategies demonstrate the potential of exosome-based CRISPR delivery to target joint and bone tissues with high specificity.

Fig. 6.

The development of an injectable CAP/FGF18-hyExo@HMs system that combines with chondrocyte-targeted in vivo FGF18 gene-editing and self-renewable lubrication towards a cell-free OA treatment [163]. Copyright, 2021 Manyu Chen et al.

A more direct osteoporosis-related advance involves bacterial extracellular vesicles (BEVs) engineered for targeted delivery. Researchers modified probiotic Escherichia coli Nissle 1917 to overexpress human C-X-C chemokine receptor type (hCXCR) 4 on vesicle membranes, generating BEVs capable of homing to bone marrow MSCs. These vesicles were then loaded with sclerostin siRNA, a potent inhibitor of bone formation. The engineered BEV-CS particles not only displayed excellent targeting and compatibility but also promoted osteogenic differentiation in vitro and reversed osteoporotic phenotypes in ovariectomized (OVX) mouse models, without long-term toxicity [165]. This work demonstrates how gene editing approaches can regulate exosome cargo composition to enhance their therapeutic efficacy in skeletal disease.

Taken together, these findings suggest two complementary strategies for osteoporosis: (i) employing exosomes as carriers for CRISPR/Cas9 systems to edit genes regulating osteogenesis and osteoclastogenesis, and (ii) applying CRISPR-based engineering to modify exosome composition, thereby improving their targeting and function. Both approaches hold promise to promote bone formation, inhibit excessive bone resorption, and overcome the limitations of existing pharmacotherapies. Future work should prioritize bone-specific targeting ligands, optimization of loading efficiency, and rigorous preclinical evaluation of safety and durability. If successful, exosome-mediated CRISPR delivery could represent a transformative step toward precision gene therapy in osteoporosis.

Application of nanotechnology

As naturally occurring nanoscale EVs, exosomes have unique physical and biological properties, giving them broad application potential in the field of nanotechnology. Recent years, the application of nanotechnology has brought new breakthroughs in the engineering of exosomes, particularly in enhancing the stability, loading capacity, and targeted delivery efficiency of exosomes (Fig. 7) [166, 167]. By combining exosomes with nano materials, exosomes not only retain their natural biocompatibility but also acquire additional functional properties, further improving their potential for use in the treatment of osteoporosis [168].

Fig. 7.

Exosome engineering process. (a) Surface modification: targeting molecules (e.g., antibodies or peptides) are added to exosomes for cell or tissue binding. (b) Gene delivery: exosomes carry the Cas9 gene delivery system for genetic modifications. (c) CRISPR-Cas9 editing: exosomes are engineered using CRISPR-Cas9 for gene editing. (d) Nanotechnology applications: nanotechnology enhances exosomes through nanoparticle loading and surface modifications to improve targeting and therapy

Nanotechnology can be used to combine exosomes with functional nano particles, forming exosome-nanoparticle composites, thereby enhancing their targeting and therapeutic effects. For example, researchers have integrated magnetic nano particles into exosomes to create magnetic exosomes that can respond to external magnetic fields [169]. In osteoporosis models, these magnetic exosomes can theoretically be precisely directed to areas of bone loss under the guidance of an external magnetic field, significantly improving therapeutic efficiency. More importantly, exosomes derived from MSCs derived from human induced pluripotent stem cells combined with tricalcium phosphate have also been shown to activate the PI3K/Akt signaling pathway and enhance the osteoinductive ability of β-tricalcium phosphate (β-TCP), thereby potentially being used to repair bone defects. This technology not only enhances the targeting ability of exosomes but also provides a non-invasive method of external control, further expanding the potential applications of exosomes.

Exosomes can load a variety of biologically active substances, such as proteins, nucleic acids (DNA, mRNA, miRNA, siRNA) and small molecule drugs, and deliver them to target cells or tissues. However, the natural drug-carrying capacity of exosomes is limited, but nanotechnology can significantly improve its drug-carrying capacity by combining exosomes with nanocarriers [170]. For example, by fusing nano liposomes with exosomes, multiple drug molecules can be loaded simultaneously, enabling multi-targeted combination therapy [171]. This multifunctional exosome-nanoparticle composite can theoretically show significant advantages under complex pathological conditions, especially in the treatment of osteoporosis, where multiple signaling pathways need to be regulated simultaneously. For instance, researchers have successfully co-loaded anti-resorptive drugs and pro-osteogenic drugs into exosome-nanoparticle composites, significantly improving bone density and quality in mouse models [172, 173]. The same time, the researchers developed an exosome delivery system based on exosomes secreted by human induced pluripotent stem cells MSCs, engineered exosomes BT-Exo-siShn3, to take advantage of the intrinsic resistance of these special exosomes. Osteoporosis function, and cooperates with Shn3 gene-loaded siRNA to enhance osteogenesis and inhibit osteoclasts [174].

Nanotechnology can also enhance the in vivo stability of exosomes by coating or integrating them with nano materials [175, 176]. Studies have shown that encapsulating exosomes in nano capsules can prevent their rapid degradation in the bloodstream, prolonging their half-life [177]. These stabilized exosomes have shown more sustained therapeutic effects in the treatment of disease, reducing the need for frequent dosing and significantly improving patient compliance [178]. Nanotechnology also provides new tools for the detection and tracking of exosomes. By loading fluorescent or radioactive nano particle probes into exosomes, researchers can achieve real-time monitoring of exosomes in vivo [179, 180]. This imaging technology not only helps scientists better understand the bio-distribution and delivery efficiency of exosomes but also provides valuable tools for evaluating therapeutic outcomes in clinical applications [181]. For example, in animal models, exosomes labeled with nano particle probes have shown clear in vivo distribution patterns, helping to optimize exosome-based treatment plans [180, 182].

Collectively. by incorporating surface modification technology, gene editing technology, and nanotechnology, exosomes have shown great potential in the treatment of osteoporosis. As these technologies continue to advance, future exosome-based therapies will become more precise and efficient, offering more effective treatment options for patients with osteoporosis.

Discussion and future directions

Exosome-based therapy shows great potential in the treatment of osteoporosis, particularly in the context of personalized medicine. Osteoporosis is a multifactorial disease influenced by age, sex, genetics, and lifestyle, underscoring the need for tailored interventions [3]. Exosomes, with their cell-specific molecular cargo, could enable precision delivery to distinct patient subgroups. However, this personalized approach introduces significant challenges for clinical application, as heterogeneity in exosome content based on donor age, health status, or cell passage can significantly affect therapeutic efficacy. Addressing these sources of variability is critical for translation into routine care [183].

Cell-based therapies, particularly MSCs, have shown significant potential in the treatment of osteoporosis in recent years, as they can promote bone repair and reverse bone loss through their regenerative capabilities [184]. However, stem cell therapies face numerous challenges, including issues of homogeneity, safety, and the long-term survival and integration of transplanted cells, which limit their widespread clinical application and efficacy [185]. Given these limitations, researchers have increasingly turned to exosome therapy as an alternative. However, despite its promise, exosome therapy also faces its own set of challenges in clinical translation. Exosomes, as an alternative to cell-based therapies, can carry beneficial bioactive molecules, reduce immunogenic reactions, and are easier to standardize for production [10, 11]. Although exosome therapy has shown potential in preclinical studies, it still faces challenges in clinical translation, including immunogenicity, long-term safety, and scalability, some of which are also present in engineered exosome technologies [133].

A major translational barrier lies in the standardization and scale-up of exosome production [186]. Current isolation methods, such as ultracentrifugation, size-exclusion chromatography, and immunoaffinity capture, are labor-intensive, costly, and difficult to adapt for large-scale good manufacturing practice (GMP)-grade production without compromising yield or purity [187]. Exosome secretion is regulated by pathways such as Rab27- and Rab31-mediated vesicle trafficking and ceramide synthesis, yet harnessing these regulators for clinical manufacturing remains challenging [188]. Furthermore, reproducibility is compromised by factors such as fluctuations in culture conditions, nutrient availability, and variability in donor cell characteristics [189]. Establishing standardized protocols for production, purification, and characterization is essential to ensure consistent quality and therapeutic performance. Emerging tools such as microfluidic devices and bioreactor-based culture systems may improve efficiency, but clinical readiness will require rigorous validation and harmonization of manufacturing pipelines [190].

Safety and immunogenicity are also key challenges [191]. Although exosomes are naturally biocompatible, engineered exosomes modified to carry therapeutic cargos or surface ligands may provoke immune responses, raising concerns about patient-specific safety profiles in osteoporosis therapy [192]. While exosomes are naturally biocompatible and less likely to provoke immune responses compared to stem cells, engineered modifications may alter this response. Pharmacokinetic and biodistribution data remain limited, and the long-term clearance mechanisms of engineered exosomes in vivo are not fully understood. Rigorous preclinical testing is therefore essential prior to clinical trials. In this context, osteoporosis animal models provide valuable platforms. OVX rodents remain the most widely used to mimic postmenopausal bone loss, while glucocorticoid-induced and aging models capture additional clinical scenarios [193]. Transgenic models targeting Wnt or RANKL pathways have further advanced mechanistic understanding. Importantly, several studies have demonstrated that mesenchymal stem cell-derived exosomes can restore bone mass in OVX models, demonstrating their therapeutic promise [194, 195]. Nonetheless, differences in bone remodeling dynamics, immune milieu, and metabolism between animals and humans must be acknowledged, as they may limit translational fidelity.

Beyond overcoming barriers, technological innovations continue to expand the therapeutic horizon. Advances in nanotechnology have enabled the design of exosome-mimicking nanoparticles with improved stability and controlled drug release, while incorporation into biomaterials such as hydrogels or scaffolds enhances localized bone regeneration, particularly in fracture repair [196]. Imaging modalities, including PET and MRI, now allow real-time tracking of exosome biodistribution, facilitating optimization of therapeutic strategies [197]. In parallel, artificial intelligence and machine learning tools are emerging as powerful adjuncts to exosome research [198]. By integrating complex molecular signatures with imaging and clinical parameters, AI-driven approaches could improve diagnostic accuracy, risk prediction, and treatment monitoring in osteoporosis, although these applications remain at an exploratory stage [199, 200].

In summary, exosome-based therapies for osteoporosis face several translational barriers, including variability in production, challenges in large-scale standardization, unresolved safety concerns, and gaps in bridging preclinical models with human disease. Nevertheless, with advances in bioengineering, nanotechnology, AI, and interdisciplinary collaboration, exosomes hold significant promise as next-generation therapeutics. In the future, engineered exosomes may evolve into a cornerstone of osteoporosis treatment, offering more precise, durable, and effective solutions for diverse patient populations.

Conclusion

The engineering of exosomes is emerging as a transformative strategy for osteoporosis therapy, with the capacity to enhance osteoblast differentiation, suppress osteoclast activity, and rebalance bone remodeling. Advances in bioengineering, including surface modification, gene editing, nanotechnology, and integration with biomaterials, have improved the stability, targeting, and therapeutic efficacy of exosomes. Nonetheless, key challenges remain, particularly in large-scale production, safety validation, and the development of personalized delivery strategies. Integrating engineered exosomes with conventional pharmacologic regimens and leveraging emerging tools such as multi-omics profiling may further refine their clinical utility. By aligning mechanistic insights with translational innovation, engineered exosomes are positioned to complement and extend current therapies, offering a forward-looking framework for durable and individualized management of osteoporosis.

Abbreviations

Akt

Protein kinase B

ALP

Alkaline phosphatase

AP

Activator protein

BEVs

Bacterial extracellular vesicles

BMP

Bone morphogenetic protein

BMD

Bone mineral density

CRISPR

Clustered regularly interspaced short palindromic repeats

CTLA

Cytotoxic T-lymphocyte antigen

ERK

Extracellular signal-regulated kinase

EVs

Extracellular vesicles

FGF

Fibroblast growth factor

GMP

Good manufacturing practice

GSK

Glycogen synthase kinase

IFN

Interferon

IL

Interleukin

ILVs

Intraluminal vesicles

JNK

c-Jun n-terminal kinase

KRAS

Kirsten rat sarcoma viral oncogene homolog

LRP

Low-density lipoprotein receptor-related protein

MAPK

Mitogen-activated protein kinase

MVBs

Multivesicular bodies

NF-κB

Nuclear factor kappa-light-chain-enhancer of activated B cells

NFATc

Nuclear factor of activated T cells, cytoplasmic

OPG

Osteoprotegerin

OVX

Ovariectomized

PTH

Parathyroid hormone

PD-L

Programmed death-ligand

PI3K

Phosphoinositide 3-kinase

RANKL

Receptor activator of nuclear factor κB ligand

RGD

Arginine-glycine-aspartic acid

RUNX

Runt-related transcription factor

sgRNA

single-guide

siRNA

Small interfering RNA

SIRT

Sirtuin

TCF

T-cell factor

TRAF

TNF receptor-associated factor

VEGF

Vascular endothelial growth factor

Wnt

Wingless-related integration site

β-TCP

β-tricalcium phosphate

hCXCR

Human C-X-C chemokine receptor type

Author contributions

FMS and LHT designed and completed this initial draft, PHY optimized the images in the text, and LHT optimized the tables.

Funding

This study was funding by the Beijing Traditional Chinese Medicine Science and Technology Development Fund Project (BJZYYB-2023-01), Beijing Natural Science Foundation (L258078), and the China Academy of Chinese Medical Sciences Science and Technology Innovation Project (CI2021B010).

Data availability

This review is based on previously published studies and publicly available resources. No new data or materials were generated or analyzed in this study

Declarations

Ethical approval

Not applicable.

Conflict of interest

The authors declare that they have no competing interests.