Apoptotic extracellular vesicles act as master regulators of the bone healing niche

Abstract

Apoptotic extracellular vesicles (ApoEVs), natural bilayer nanoparticles released during programmed cell death, have emerged as pivotal regulators and promising therapeutic agents for bone regeneration. They function as innate multimodal signaling entities, expertly coordinating the complex cellular interplay between osteogenesis, angiogenesis, innervation, and immunomodulation required for successful bone healing. This review systematically outlines the biogenesis, isolation, and fundamental mechanisms by which ApoEVs orchestrate the bone healing niche. We further explore their dual role in bone homeostasis and pathology, highlighting applications in treating osteoporosis and osteoarthritis, while acknowledging potential negative effects from specific cellular origins. The significant promise of ApoEVs is being unlocked through innovative engineering strategies to enhance their therapeutic efficacy and specificity. Critically, we discuss how emerging artificial intelligence (AI) tools are poised to overcome major translational hurdles. Despite the challenges in standardization and scalable production, the convergence of ApoEV biology and AI-driven design positions them as a transformative next-generation, cell-free platform for treating intractable bone and joint disorders.

Graphical abstract

Introduction

In multicellular organisms, the maintenance of tissue homeostasis relies on the continuous and programmed turnover of cells, a process in which apoptosis serves as a fundamental mechanism for the controlled removal of unwanted cells [1–3]. Far from a silent demise, the execution of apoptosis is a highly active event that generates apoptotic extracellular vesicles (ApoEVs). These vesicles are laden with a diverse cargo of bioactive molecules, including proteins, nucleic acids, and lipids, from their parent cells, functioning as key messengers in intercellular communication [4–7]. By transferring their molecular contents to recipient cells, ApoEVs can profoundly influence cellular behavior, a capability that positions them as critical regulators of tissue repair and regeneration. This is particularly evident in the context of bone healing, where emerging evidence highlights the role of ApoEVs in modulating the immune and osteogenic microenvironment to direct skeletal repair [8–10]. Consequently, the inherent bioactivity and delivery potential of ApoEVs not only underscore their physiological significance but also establish them as a promising and versatile platform for the development of engineered therapeutics for bone diseases [11–13].

The recognition of ApoEVs as central coordinators of tissue repair has brought their regenerative potential, particularly in the challenging context of bone healing, into sharp focus. Bone regeneration is a complex, multi-stage process requiring the precise spatiotemporal coordination of inflammation, angiogenesis, and osteogenesis. While traditional bone grafts remain the clinical mainstay, they are hampered by limitations such as donor-site morbidity and limited availability [14–16]. ApoEVs present a promising alternative, as they are increasingly shown to orchestrate this healing cascade by modulating key signaling pathways (e.g., Wnt/β-catenin, MAPK/ERK), directing immune cell polarization, and activating both endothelial cells and mesenchymal stem cells (MSCs) [17–20]; unlike canonical bone healing regulators including BMPs and Wnt ligands that typically act on single pathways to drive lineage commitment or matrix deposition, ApoEVs function as injury-responsive, multimodal signaling vehicles that deliver integrated packages of proteins, lipids, and nucleic acids, enabling them to coordinate parallel cellular programs within a single vesicle and creating synergistic crosstalk that requires the sequential action of multiple growth factors to replicate. To harness this potential, sophisticated engineering strategies, including surface modification for targeted delivery and incorporation into biomaterial scaffolds, are being developed to enhance their therapeutic efficacy and practicality. However, the path to clinical translation is not without challenges; significant hurdles remain in standardizing ApoEV function, optimizing their in vivo stability, and establishing long-term safety profiles. This review, therefore, aims to systematically synthesize the current understanding of ApoEVs as multimodal regulators of the bone-healing microenvironment, uniquely positioned as master orchestrators of the niche’s complex, multifaceted dynamics. We will explore their fundamental roles in bone homeostasis and repair, critically assess the burgeoning field of ApoEV-based therapeutics, and discuss the future perspectives and challenges for their successful clinical application in treating bone diseases.

ApoEVs and their characteristics

ApoEVs are a distinct class of extracellular vesicles generated during the programmed cell death process of apoptosis [21, 22]. They are primarily formed through the outward blebbing of the apoptotic cell membrane and are heterogeneous in size, ranging from large microparticles (1–5 μm) to smaller apoptotic bodies (100–1000 nm) [23–26]. Their molecular cargo is a complex and representative snapshot of the parent cell, packaged with proteins, lipids, and nucleic acids (including genomic DNA, mRNA, and microRNA). This unique composition, derived directly from the controlled process of apoptosis, differentiates ApoEVs from vesicles released from viable cells (e.g., exosomes) and underlies their potent and multifaceted bioactivity (Table 1).

Table 1.

Distinguishing ApoEVs from exosomes and microvesicles (simplified)

Feature	Exosomes	Microvesicles (ectosomes)	ApoEVs (apoptosis-derived EVs)	Ref.
Biogenesis/origin	Endosomal (MVB) release	Plasma-membrane budding	Apoptotic disassembly (blebbing/apoptopodia; fragmentation)	[27]
Typical size (diameter)	~ 30–150 nm	~ 100–1000 nm	Broad: ~100 nm–1 μm; apoptotic bodies ~ 1–5 μm	[28]
Signature surface feature (example)	CD63 (tetraspanin)	Annexin A1 (enrichment reported)	PS externalization (Annexin V/Lactadherin+)	[28]
Cargo tendency (example)	miRNAs (regulatory RNAs)	Cytosolic proteins	Genomic DNA / nuclear fragments (especially larger subtypes)	[29]
Dominant uptake & intracellular processing	Endocytosis → endo-lysosomal trafficking	Endocytosis/phagocytosis → lysosomal processing	Efferocytosis → phagolysosomal processing (PS-recognition)	[30]
In vivo fate/turnover	Clearance by RES (e.g., liver/spleen)	Clearance by RES (context-dependent)	Often rapid phagocyte clearance (efferocytosis-biased)	[27]

Biogenesis and subtype of ApoEVs

The formation of ApoEVs is a direct and active consequence of the apoptotic cascade, primarily orchestrated by the caspase family of proteases. This process gives rise to a spectrum of vesicular subtypes, historically classified by their size and biogenetic mechanism: large apoptotic bodies (1–5 μm), mid-sized apoptotic microvesicles (100 nm–1 μm), and small apoptotic exosomes (30–150 nm) [23–26]. The biogenesis of these subtypes is morphologically distinct; apoptotic bodies are generated through extensive membrane blebbing and the dynamic formation of apoptopodia, while apoptotic microvesicles bud directly from the plasma membrane [28, 31–33]. A defining event in this process is the caspase-mediated exposure of phosphatidylserine on the outer membrane of these vesicles. This surface phosphatidylserine acts as a universal “eat-me” signal, dictating their subsequent clearance by phagocytes and facilitating the targeted delivery of their cargo to recipient cells [34, 35]. Thus, the very mechanisms that create ApoEVs are intrinsically linked to their function, enabling them to transmit complex signals that orchestrate tissue remodeling and immune communication.

Cargo and functional programming of ApoEVs

The biological impact of ApoEVs is fundamentally dictated by their molecular cargo, a complex and heterogeneous mixture of proteins, nucleic acids, and lipids that reflects the identity and physiological state of the parent cell (Fig. 1). This cargo is not a random assortment but a selectively packaged “molecular legacy” that programs ApoEVs to regulate critical processes in recipient cells, including immune modulation, DNA repair, and tissue regeneration [36–41].

Fig. 1.

The multimodal cargo of apoptotic extracellular vesicles (ApoEVs). ApoEVs are loaded with a diverse repertoire of bioactive molecules, including proteins, nucleic acids, lipids, and organelles, from their parent cell. This cargo is selectively packaged and delivered to recipient cells via surface receptors, such as phosphatidylserine (PS), enabling ApoEVs to orchestrate complex cellular responses in both physiological and pathological contexts. Panels created with Figdraw.com under academic license

Protein/lipid-mediated direct signaling and recognition

The protein cargo is highly diverse, encompassing signaling molecules, enzymes, and structural components. For example, ApoEVs can deliver Fas ligand to induce apoptosis in target cells or carry enzymes like USP5 to promote DNA repair [42–48]. Crucially for bone healing, ApoEVs derived from mesenchymal stem cells (MSCs) are enriched with specific signaling proteins such as Ras and integrin α-5, which activate pathways like RAF1/MEK/ERK to directly drive osteogenic differentiation [42–49]. The lipid composition is equally functional, which can also be metabolized into pro-resolving mediators like resolvin D5, actively modulating macrophage polarization to resolve inflammation and promote a regenerative microenvironment [50–52].

Nucleic acid-mediated genomic and post-transcriptional regulation

Nucleic acids constitute a key regulatory component, including fragmented genomic DNA, mitochondrial DNA, and a rich profile of non-coding RNAs. The distribution of this cargo is size-dependent; larger apoptotic bodies may contain entire organelles and nuclear fragments, while smaller vesicles are enriched with regulatory microRNAs (miRNAs) that can silence target genes in recipient cells [26, 28, 45, 53–56]. A prime example is hsa-miR-4485-3p, a highly stable and abundant miRNA in MSC-derived ApoEVs that is strongly associated with enhanced bone regeneration [57]. This efficient transfer mechanism, however, can be subverted in disease, as ApoEVs are also capable of packaging and disseminating viral components, underscoring their dual role in physiology and pathology [58–62]. By integrating these diverse molecular signals, ApoEVs emerge as sophisticated multimodal regulators, capable of precisely coordinating the cellular responses necessary for complex processes like bone healing.

Induction methods for ApoEV generation

Reliable ApoEV production is essential for research and therapy. Current methods, chemical, physical, and physiological, each balance efficiency, practicality, and biological relevance. Chemical induction using agents such as staurosporine is efficient and reproducible. Success requires precise optimization of concentration and exposure time to prevent secondary necrosis while inducing apoptosis. Rigorous purification is also necessary to remove residual inducers. Physical induction, such as UV irradiation, provides a non-chemical alternative but risks DNA and protein damage, potentially altering ApoEV cargo and function. Physiological induction via serum starvation mimics natural apoptosis, yielding biologically relevant ApoEVs. However, this method is slower, produces lower yields, and requires careful timing optimization. A comparative summary is provided in Table 2.

Table 2.

Summary of common in vitro induction methods for ApoEVs

Method type	Specific inducer/ stimulus	Typical concentration/ duration	Origin cells (examples)	Ref.
Chemical	Staurosporine (STS)	0.5 µM for 20 h	Mesenchymal stem cells	[63]
		0.5 µM for 12 h	RAW264.7 macrophages in vitro	[64]
		0.5 µM for 3 h	Bone marrow macrophages, preosteoclasts, and mature osteoclasts in vitro	[65]
		0.5 µM for 8 h	Human ESCs, iPSCs, and umbilical cord MSCs	[66]
		0.5 µM for 12 h	Human deciduous pulp stem cells	[67]
		0.5 µM for 3 h	Neutrophils	[68]
	Alendronate (ALN)	500 µM for 24 h	Bone marrow macrophages, preosteoclasts, mature osteoclasts	[69]
	Etoposide	10 µM for 24 h	Human T lymphocytes	[70]
	Hydrogen peroxide (H₂O₂)	1 mmol/L for 12 h	Endplate chondrocyte	[71]
	Actinomycin-D	5 µg/mL for 12 h	Human T lymphocytes	[70]
	Tumor necrosis factor-α (TNF-α)	100 µM for 12 h	Human T lymphocytes	[70]
	Doxorubicin	25 µM for 48 h	Murine B16-F1 or B16-OVA cells in vitro	[27]
	Bortezomib	72 h	Human glycan-modified melanoma cell line Mel-JuSo	[72]
	Temozolomide/cisplatin	25–100 µM/20–50 µM for 24 h	Human Glioblastoma cells / LUAD cell	[73, 74]
	LPS	1 µg/mL for 24 h	RAW264.7 macrophages in vitro	[75]
	Anisomycin	5 µg/mL	HeLa cell	[76]
	Jurkat-derived microparticles (Membrane lipids)	3 × 106	RAW macrophages Human U937 cells	[77]
	CoCl2	50–100 µM for 2 d	PC12 cells	[78]
Physical	UV-B	100 mJ/cm2	Mutu I BL cells	[76]
		150 mJ/cm2	Jurkat cells	[76]
		90 mJ/cm2	Human lymphoblasts in vitro	[79]
	γ irradiation	6–12 Gy for 3–6 d	Human Glioblastoma cells in vitro	[73]
	Heat shock	56 °C for 30 min in a water bath	Jurkat cells	[70]
Physiological	Serum-free culture (Starvation)	Serum starvation for 4 h (time can vary significantly by cell type)	Monocytic leukemia cells, T cell lymphoma cells, neuronal cells, epithelial cells (human), Jurkat cells	[80, 81]

Isolation of ApoEVs

The isolation of pure and functional ApoEV populations is a critical prerequisite for their characterization and application. The most common techniques exploit differences in physical properties or surface markers, each offering a distinct balance of yield, purity, and specificity.

Differential Centrifugation remains the most widely used method, separating vesicles through a series of increasing centrifugal forces. Its key advantage is speed, minimizing processing time and potential vesicle degradation [82]. However, it yields heterogeneous pellets often contaminated with protein aggregates and non-vesicular material [83, 84]. Purity can be improved by coupling low-speed spins with size-based filtration, though this introduces risks of vesicle loss or membrane damage due to shear stress [85, 86].

Density Gradient Centrifugation significantly enhances purity by separating particles based on their buoyant density in a medium like iodixanol [87]. This allows for the collection of specific, high-purity fractions. The major drawbacks are the inconsistent density profiles of heterogeneous ApoEVs and substantial yield losses during fraction collection, limiting its scalability [88–91].

Fluorescence-Activated Cell Sorting (FACS) enables the high-purity isolation of specific ApoEV subpopulations by using fluorescent labels for surface markers, such as phosphatidylserine [92, 93]. A significant limitation is its detection threshold (~200–500 nm), which biases collection towards larger apoptotic bodies and fails to capture smaller ApoEVs, resulting in low overall yield. Furthermore, it is also difficult to distinguish the types of vesicles solely based on their diameter [94]. The technique is also complex and can compromise vesicle integrity. Emerging microfluidic platforms are being developed to overcome these limitations, offering promising avenues for high-throughput, gentle sorting in the future [95–98].

ApoEVs as versatile regulators: from systemic homeostasis to coordinated bone regeneration

ApoEVs are fundamental, dualistic mediators of intercellular communication. Their biological impact, spanning tissue homeostasis, disease propagation, and orchestrated repair, is determined by their cellular origin, molecular cargo, and the recipient microenvironment. This inherent versatility positions them as critical players in both systemic pathophysiology and, as detailed here, the precise coordination of complex regenerative processes such as bone healing.

Dualistic roles in physiology and pathology

ApoEVs are indispensable for maintaining tissue integrity and function as systemic conveyors of maintenance signals. For example, in the liver, they are internalized via specific receptors to promote hepatocyte regeneration [99–101]. Systemically, they are enriched with DNA repair enzymes that can rescue adjacent cells from genomic instability, counteracting aging phenotypes [102–106].

This homeostatic function can be subverted in disease. In cancer, tumor-derived ApoEVs transfer oncogenic cargo to promote chemotherapy resistance and metastasis [73, 74, 107]. In inflammatory conditions, they can exacerbate pathology by activating immune cells. Conversely, their inherent bioactivity can be therapeutically harnessed; for instance, MSC-derived ApoEVs exhibit potent anti-inflammatory and pro-regenerative capacities, accelerating diabetic wound healing and demonstrating anti-tumor effects [72, 108, 109]. This duality underscores that ApoEV actions are not inherently beneficial or detrimental but are a product of their context, making them both critical pathophysiological agents and promising therapeutic tools.

Central coordination of the bone regeneration cascade

Bone regeneration requires the precise spatiotemporal coordination of osteogenesis, angiogenesis, innervation, and immunomodulation [110]. For instance, in the early inflammatory phase, ApoEVs derived from immune cells rapidly modulate macrophage polarization toward an M2 phenotype, resolving inflammation and initiating tissue cleanup. During the repair phase, ApoEVs from vascular niche cells promote angiogenesis, while osteoclast-derived ApoEVs couple bone resorption and formation. In the later remodeling phase, MSC-derived ApoEVs drive osteogenic differentiation and matrix maturation. This staged delivery ensures that ApoEVs coordinate osteogenesis, immunomodulation, and angiogenesis according to the dynamic needs of the healing niche, rather than acting simultaneously on a single day. As masterful intercellular communicators, ApoEVs released following injury are now recognized as central coordinators of this entire cascade (Fig. 2) [31, 111–115]. Their roles can be systematically dissected across the key phases of repair.

Fig. 2.

Multimodal Regulation of Osteogenesis by Apoptotic Extracellular Vesicles (ApoEVs) from Diverse Cellular Origins. (A) MSC-derived ApoEVs (MSC-ApoEVs), released from transplanted exogenous bone marrow mesenchymal stem cells (BMSCs), enhance osteogenic differentiation by activating Ras/Raf1/MEK/ERK and ROS/JNK signaling pathways. (B) Osteoclast-derived ApoEVs (OC-ApoEVs) play a critical role in bone homeostasis; their deficiency contributes to osteoporosis, while exogenous administration rescues bone loss by activating mTOR signaling via RANKL reverse signaling. Multinucleated mature osteoclast-derived ApoEVs (mOC-ApoEVs) further promote osteoblast activity through surface RANK-mediated signaling. (C) Non-bone marrow-derived ApoEVs demonstrate potent osteogenic capacity: Dental pulp stem cell-derived ApoEVs (DPSCs-ApoEVs) promote bone formation via ERK1/2 activation and accelerate periodontal repair, while periodontal ligament stem cell-derived ApoEVs (PDLSCs-ApoEVs) coordinate regeneration through VDAC1-enriched mitophagy activation. (D) In cartilage regeneration, human umbilical cord MSC-derived ApoEVs (HUMSCs-ApoEVs) deliver miR-100-5p and let-7i-5p to synergistically drive M2 macrophage polarization and chondrogenesis via ERK/MAPK and eEF2K/p38 MAPK pathways, while hypoxia-preconditioned adipose-derived stem cell ApoEVs (ADSC-H-ApoEVs) precisely regulate chondrogenic differentiation through miR-1246 upregulation. Panels A-D created with Figdraw.com under academic license

Osteogenesis

The osteogenic potential of ApoEVs is source-dependent. MSC-derived ApoEVs activate the RAF1/MEK/ERK pathway to promote endogenous stem cell recruitment and differentiation [42, 116]. Osteoclast-derived ApoEVs (OC-ApoEVs) are crucial couplers of bone remodeling, promoting osteogenesis via RANKL reverse signaling and mTOR activation [69, 117, 118]. Dental and periodontal stem cell ApoEVs also enhance osteogenesis through ERK1/2 signaling and by modulating mitophagy. Notably, while the existing literature indicates that the specific molecular effectors of DPSC-ApoEVs remain incompletely defined, potential candidate molecules include encapsulated miRNAs and functional proteins, whose roles in mediating DPSC-ApoEVs’ osteogenic effects warrant further investigation [119–122]. Relatedly, ApoEVs from sources like umbilical cord MSCs contribute to cartilage regeneration by driving chondrogenesis and macrophage polarization [123–126].

Immunomodulation

ApoEVs masterfully regulate the bone immune landscape (Fig. 3). They shift macrophages from a pro-inflammatory (M1) to a pro-regenerative (M2) phenotype via mechanisms like the AMPK/SIRT1/NF-κB pathway, thereby attenuating osteoclastogenesis [127, 128]. T cell-derived ApoEVs modulate the microenvironment via ectonucleotidases (CD39/CD73) that generate immunomodulatory adenosine, and help rectify Treg/Th17 imbalances [129, 130]. They also promote the timely clearance of neutrophils, resolving the initial inflammatory phase [131].

Fig. 3.

Apoptotic extracellular vesicles (ApoEVs) mediate immunomodulation in bone healing niche. (A) BMSC-derived ApoEVs (BMSC-ApoEVs) mitigate inflammation by downregulating COX2 and the pro-inflammatory cytokines TNF-α and IL-6, while upregulating the anti-inflammatory cytokine IL-10 in macrophages. This immunomodulation is driven by the activation of the AMPK/SIRT1/NF-κB signaling pathway. The subsequent inhibition of TNF-α release suppresses pro-inflammatory macrophage polarization, ultimately attenuating osteoclastogenesis and bone resorption. Created with Figdraw.com under academic license. (B) A targeted nanoparticle system (TDNs) for site-specific immunomodulation. The TDNs, featuring a mesoporous silica core loaded with MCP-1 and a FasL corona, recruit T cells and induce their apoptosis. The resulting T cell-derived ApoEVs (T cell-ApoEVs) rebalance the Treg/Th17 axis by suppressing the secretion of pro-inflammatory cytokines from Th17 cells and promoting the production of anti-inflammatory cytokines from Treg cells, thereby ameliorating osteoporosis in ovariectomized (OVX) mice. Reproduced with permission [129] (C) Mechanistic insight into T cell-ApoEV function: Surface-enriched CD39 and CD73 on T cell-ApoEVs catalyze the hydrolysis of extracellular ATP to adenosine. Adenosine then activates the A2BR receptor and the downstream PKA signaling pathway, which concurrently enhances osteogenic differentiation and inhibits the secretion of inflammatory cytokines. Reproduced with permission [130]

Angiogenesis and vascular-osteogenic coupling

ApoEVs are pivotal for revascularization. Vesicles from vascular niche cells like red blood cells and platelets deliver cargos (e.g., carbonic anhydrase 1) that activate osteogenic pathways in MSCs [18, 20]. They execute sophisticated coupling programs; for example, PDLSC-ApoEVs upregulate STC1 in endothelial cells to concurrently enhance angiogenesis and osteogenesis [19]. Furthermore, pre-osteoclast ApoEVs function as targeted delivery vehicles for PDGF-BB to endothelial progenitor cells, directly promoting the formation of osteogenic type H vessels (Fig. 4) [118, 132].

Fig. 4.

ApoEVs orchestrate angiogenesis and osteogenic coupling for bone regeneration. (A) RBC- and platelet-derived ApoEVs directly stimulate osteogenesis. Following vascular injury, apoptotic red blood cells release RBC-ApoEVs that deliver carbonic anhydrase 1 (CA1) to human bone marrow stromal cells (hBMSCs), activating the P38 MAPK pathway to enhance osteogenic differentiation. Similarly, platelet-derived ApoEVs (PLT-ApoEVs) upregulate GOLPH2 to induce AKT phosphorylation in MSCs, facilitating bone repair. (B) PDLSC-derived ApoEVs promote angiogenic-osteogenic coupling. PDLSCs-ApoEVs activate the MAPK1/3 pathway in human umbilical vein endothelial cells, inducing the transcription factor FOS and subsequent upregulation of stanniocalcin 1 (STC1). This STC1-mediated signaling cascade concurrently enhances both angiogenesis and osteogenesis, accelerating bone repair. (C) Mononuclear preosteoclasts (pOC-ApoEVs) drive type H vessel formation. pOC-ApoEVs, inheriting pro-angiogenic cargo, deliver platelet-derived growth factor BB (PDGF-BB) to endothelial progenitor cells (EPCs). This targeted delivery promotes the formation of type H vessels, a specialized capillary subtype critical for coupling angiogenesis to bone formation. Panels A-C created with Figdraw.com under academic license

Homeostatic coupling

Fundamentally, ApoEVs are essential couplers of bone remodeling. Osteoclast-derived ApoEVs, including those from mononuclear preosteoclasts (pOCs) and multinuclear mature osteoclasts (mOCs), stimulate bone formation and angiogenesis at resorption sites [65], while MSC-derived ApoEVs can attenuate osteoclastogenesis via miRNA transfer [133]. Efficient clearance (efferocytosis) of ApoEVs is paramount; impaired clearance, as seen with aging, contributes to bone loss [30], whereas systemic ApoEV administration can deliver pro-osteogenic signals like RNF146 to restore stem cell function and bone mass [29]. Thus, ApoEVs integrate cell death, efferocytosis, and intercellular signaling to maintain skeletal integrity (Fig. 5).

Fig. 5.

ApoEVs regulate bone homeostasis through targeted molecular delivery. (A) Mononuclear preosteoclasts (pOCs) undergo induced fusion and differentiation to form multinuclear mature osteoclasts (mOCs). pOCs exhibit pro-angiogenic activity, while mOCs possess pro-osteogenic activity. Reproduced with permission [65] (B) BMSC-ApoEVs shift the bone remodeling balance toward anabolism by enhancing osteogenic differentiation and suppressing osteoclastogenesis. A key mechanism involves the delivery of miR-1324, which targets and inhibits sorting nexin 14 (SNX14), thereby activating the pro-osteogenic SMAD1/5 signaling pathway. Created with Figdraw.com under academic license. (C) Systemic administration of exogenous apoptotic bodies rescues the osteopenic phenotype in MRL/lpr and Casp3^−/− mouse models with inherent MSC dysfunction. (D, E) Functional validation of the underlying mechanism: (B) The pro-osteogenic effect of apoptotic bodies is abolished by β-catenin siRNA, confirming the necessity of Wnt/β-catenin signaling. (E) The Wnt pathway inhibitor XAV939 blocks the apoptotic body-induced upregulation of RNF146, downregulation of AXIN1, and subsequent increase in mineralized nodule formation, demonstrating that apoptotic bodies activate osteogenesis through the RNF146/AXIN1/β-catenin axis. Panels C-E reproduced with permission [29]

In summary, ApoEVs function as versatile signaling hubs that coordinate the multicellular program of bone regeneration. Their ability to simultaneously engage osteogenic, immunomodulatory, and angiogenic processes makes them indispensable, endogenous orchestrators of skeletal repair and promising blueprints for next-generation therapeutics.

ApoEVs as multifunctional therapeutics for bone diseases

Bone diseases such as osteoporosis, osteoarthritis, and rheumatoid arthritis are driven by a fundamental breakdown in skeletal homeostasis. Apoptotic extracellular vesicles (ApoEVs) represent a novel class of versatile, cell-free therapeutics with the unique capacity to restore this balance. Unlike canonical osteogenic proteins, which typically act as single, pathway-specific, soluble signals, ApoEVs function as integrated, multimodal “information packages.” As highlighted in Table 3, their membrane-encapsulated structure enables them to co-deliver a complex cargo of proteins, lipids, and nucleic acids.

Table 3.

Comparative analysis of ApoEVs vs. canonical osteogenic proteins/growth factors in bone healing

Comparison dimension	Apoptotic extracellular vesicles (ApoEVs)	Canonical osteogenic proteins/growth factors	Ref.
Signal structure & cargo	Membrane-encapsulated, multimodal carriers that act as integrated “information packages,” capable of co-delivering proteins, lipids, and nucleic acids simultaneously	Primarily soluble proteins (e.g., BMPs, TGF-β, IGFs, FGFs, PDGFs, VEGF) or pathway ligands (e.g., Wnts) that signal through their cognate cell-surface receptors	[86, 110, 115]
Multiplexing & pathway coverage	High intrinsic multiplexing potential: a single vesicle can carry multiple bioactive cues to coordinate diverse cellular programs, including immune modulation, matrix remodeling, osteogenesis, and angiogenesis-osteogenesis coupling	Typically act in a module- or pathway-specific manner. Broader niche coordination requires the sequential action of multiple factors within a network
Spatiotemporal availability	Injury-coupled production: generated locally in damaged tissues during repair/remodeling, as part of apoptosis and subsequent cellular clearance processes	Availability is governed by endogenous expression, release, diffusion, and degradation kinetics. Exogenous supplementation requires controlled delivery to achieve therapeutic timing and localization
Delivery & engineering	The bilayer membrane structure protects cargo and supports direct intercellular transfer. Conceptually well-suited for engineering strategies to enable targeted delivery or depot-based release	Soluble proteins often require additional carriers or depot systems to enhance local retention and dose control. Diffusion and rapid clearance can limit effective therapeutic exposure
Standardization & quality control	Demands rigorous standardization across the entire workflow: from defining the source material, inducing apoptosis, isolating vesicles, and characterizing them (including distinguishing them from EVs released by viable cells)	Molecular identity is well-defined and easily quantifiable. However, biological outcomes are highly dependent on dose, timing, and local presentation, often requiring specialized delivery systems for spatiotemporal control in repair settings

This intrinsic multiplexing enables ApoEVs to coordinately target the core pathological triad of bone diseases, stem cell dysfunction, chronic inflammation, and aberrant tissue remodeling, by engaging diverse cellular programs such as immune modulation, angiogenesis, and osteogenesis. Furthermore, their therapeutic potential is enhanced by injury-coupled production, as they are generated locally during tissue damage and repair, providing natural spatiotemporal relevance. Their bilayer structure also offers inherent advantages for delivery and engineering, protecting bioactive cargo and facilitating targeted intercellular transfer without the rapid diffusion and clearance challenges associated with traditional growth factors.

Thus, ApoEVs emerge as a sophisticated therapeutic platform, capable of restoring skeletal homeostasis by simultaneously addressing multiple disease mechanisms through a coordinated, biomimetic approach that mirrors and enhances the body’s own repair signals.

Rejuvenating aged and osteoporotic bone

Age-related osteoporosis is characterized by MSC dysfunction, leading to impaired osteogenesis and increased marrow adiposity. ApoEVs directly counteract this decline. For instance, ApoEVs from young MSCs deliver RAB7 to aged MSCs, enhancing lysosomal biogenesis and autophagic flux to restore their self-renewal and osteogenic capacity, thereby increasing bone mass and reducing marrow fat in aged models [134]. Similarly, embryonic stem cell-derived ApoEVs (ESC-ApoEVs) upregulate TCOF1 and activate FLVCR1 to stabilize mitochondrial function, reduce senescence, and rejuvenate aged MSCs, improving osteopenia [135]. In postmenopausal osteoporosis, the functional decline of MSCs is linked to a suppressed TGF-β/SMAD2/3–WNT/β-catenin axis. Systemic infusion of wild-type MSC-derived ApoEVs (WT-MSC-ApoEVs) delivers miR-145a-5p to MSCs in ovariectomized (OVX) mice, reactivating this critical osteogenic pathway and rescuing bone loss [136].

Modulating inflammation in arthritis

The therapeutic efficacy of MSC-ApoEVs is demonstrated in osteoarthritis by their dose-dependent amelioration of arthritis in MRL/lpr mice. Mechanistically, these vesicles use surface phosphatidylserine to engage T cell membranes, disrupting CD3 phosphorylation and TCR signaling to suppress IL-2 secretion and T cell-driven inflammation [137]. This immunomodulatory potential is being harnessed in advanced delivery systems, such as injectable hydrogels co-loaded with engineered T cell-derived apoptotic bodies to reduce inflammation and lubricating microspheres to address joint friction [138]. Further, ApoEVs-inspired strategies using BRD4 inhibitor-loaded nanoliposomes (JQ1@PSLs) achieve targeted M1 macrophage repression, effectively alleviating pain and cartilage destruction [139].

For the more complex autoimmune landscape of rheumatoid arthritis, sophisticated ApoEV engineering has been employed. MSCs are pretreated with dexamethasone and engineered to produce FasL-overexpressing ApoEVs, which are then cloaked with a ROS-responsive heparin shell (D@ApoEVFasL∩L). This construct effectively regulates neutrophils, macrophages, and ROS levels in the synovial microenvironment, triggering a restorative immune cascade and demonstrating potent therapeutic effects [140].

Targeting malignant bone disease

Beyond degenerative and inflammatory conditions, MSC-ApoEVs exhibit direct anti-tumor efficacy. In multiple myeloma (MM), these vesicles surface-express Fas ligand (Fasl), which engages Fas receptors on MM cells. This interaction triggers a critical intracellular calcium influx, promotes Fas translocation, and initiates caspase-dependent apoptosis, highlighting a promising targeted therapy for malignant bone disease [141].

In summary, ApoEVs represent a powerful, multifaceted therapeutic platform. Their inherent ability to deliver complex biological cargo, including proteins, miRNAs, and chemical inhibitors, enables precise targeting of dysregulated pathways in stem cell function, immune response, and tumor survival, offering a highly promising avenue for the next generation of bone disease treatments (Fig. 6).

Fig. 6.

Therapeutic mechanisms of ApoEVs in bone diseases. (A) Rejuvenation of aged MSCs: Young MSC-derived ApoEVs enrich RAB7 to enhance lysosomal biogenesis and autophagic flux, restoring self-renewal and osteogenic-adipogenic differentiation balance, thereby increasing bone mass and reducing marrow adiposity. (B) Mitochondrial stabilization: Embryonic stem cell-derived ApoEVs (ESC-ApoEVs) upregulate TCOF1 to enhance mitochondrial protein transcription and activate FLVCR1, stabilizing mitochondrial function, reducing senescence, and improving osteopenia. (C) Inhibition of marrow adiposity: BMSC-ApoEVs activate Wnt/β-catenin signaling to suppress adipogenic differentiation, indirectly improving bone metabolism in aging. (D) Immunomodulation in autoimmunity: ApoEVs expose surface phosphatidylserine (PtdSer) to engage T cell membranes, disrupting proximal TCR signaling and CD3 phosphorylation, thereby inhibiting pathogenic T cell activation, cytokine production, and lymphoproliferation. (E) Rescue of osteoporotic MSCs: In ovariectomized (OVX) models, WT-MSC-ApoEVs deliver miR-145a-5p to recipient MSCs, reactivating the impaired TGF-β/Smad2/3–Wnt/β-catenin signaling axis and restoring osteogenic differentiation. (F) Joint protection in rheumatoid arthritis: Osteoclast-derived ApoEVs (OC-ApoEVs) promote macrophage repolarization to an M2 phenotype, enhance chondrocyte function, suppress osteoclastogenesis, and augment BMSC differentiation, collectively resolving synovitis and preserving joint architecture. (G) Antitumor therapy: MSC-ApoEVs surface-express FasL to engage Fas receptors on multiple myeloma cells, triggering intracellular Ca²⁺ influx, Fas translocation, and caspase-dependent apoptosis. Panels A-G created with Figdraw.com under academic license

Adverse effects and pathological roles of ApoEVs

Despite the broad therapeutic potential of ApoEVs in bone regeneration, a nuanced understanding reveals that their biological impact is highly context-dependent, with certain subsets actively contributing to disease pathogenesis. The cellular origin and specific cargo of ApoEVs are critical determinants, as vesicles from specific sources can negatively regulate bone homeostasis and even drive pathological processes.

Inhibition of bone formation and promotion of resorption

Contrary to the osteogenic capacity of their parent cells, osteoblast-derived ApoEVs exhibit surprisingly limited pro-osteogenic effects, a paradox warranting further mechanistic investigation. More strikingly, ApoEVs from other lineages directly impair bone repair. For instance, osteocyte-derived ApoEVs localize to the area of bone resorption and potently stimulate the proliferation and differentiation of osteoclast precursors. This pro-osteoclastic effect is mediated not by RANKL, but through the induction of TNF-α, leading to localized bone loss [142]. Similarly, macrophage-derived ApoEVs influence MSC fate, shifting it toward adipogenesis and away from osteogenesis. This adverse outcome is driven by miR-155 enrichment, which modulates SMAD2/SMAD5 signaling, and by the delivery of miR-143-3p, which targets IGFBP5 to disrupt the osteoblast-osteoclast balance and contribute to periodontal bone destruction [64, 143, 144].

Drivers of pathological calcification and ectopic bone formation

The adverse effects of ApoEVs extend beyond bone loss to include aberrant bone formation. Ectopic calcification in soft tissues, a hallmark of conditions like osteoarthritis and heterotopic ossification, can be initiated by ApoEVs. During early heterotopic ossification, PROCR⁺ fibroblasts release calcified ApoEVs, resulting in calcium accumulation and microcrystal formation. Mechanical stress-induced rupture of these vesicles releases crystals that nucleate calcified nodules within the tendon extracellular matrix, initiating a pathogenic cascade reinforced by M2 macrophage polarization [145]. Furthermore, chondrocyte-derived ApoEVs can precipitate calcium via their enzymatic activities, contributing to pathologic cartilage calcification in aging and osteoarthritis [146, 147].

In summary, despite the tremendous potential of ApoEVs, their adverse effects necessitate a cautious and sophisticated strategy during therapeutic development. ApoEVs derived from cells under inflammatory or pathological conditions may contribute to disease progression; for example, tumor-derived ApoEVs can accelerate tumor growth and metastasis [74]. The duality of ApoEVs’ function, governed by origin, cargo, microenvironment, inherent heterogeneity, and standardization gap, underscores the imperative to thoroughly evaluate these factors. Future translation will depend on standardized characterization and targeted engineering strategies to precisely harness their regenerative benefits while mitigating their pathological potential.

Engineering ApoEVs for enhanced bone tissue engineering

The clinical translation of native ApoEVs is hindered by inherent limitations, including heterogeneity, a short circulatory half-life, and potential off-target effects. To harness their full therapeutic potential for bone regeneration, sophisticated engineering strategies are being developed to enhance their targeting, stability, and functional payload. This section reviews the leading engineering approaches, surface modification, composite carrier construction, functionalized loading, and lyophilization that are paving the way for the next generation of ApoEV-based bone therapeutics.

Surface-targeting modifications for precision homing

Surface engineering equips ApoEVs with molecular zip codes for precise delivery to bone tissue. A prominent strategy involves conjugating bone-homing peptides, such as SDSSD or (Asp-Ser-Ser)6 ((DSS)6), to the vesicle surface using crosslinkers like EDC/NHS. For instance, engineering placental MSC-derived ApoEVs (PMSC-apoEVs) with the SDSSD peptide enabled selective accumulation at pathological bone sites, where they modulate bone homeostasis via the Hippo and Notch signaling pathways [148]. Similarly, decorating BMSC-ApoEVs with (DSS)6 and co-loading the ubiquitin ligase RNF146 significantly enhanced bone-targeting and potentiated the osteogenic capacity of endogenous MSCs [149]. Beyond peptides, microenvironment-responsive systems are also employed, such as matrix metalloproteinase-activated cell-penetrating peptides (MAP) for targeted delivery to inflammatory sites [150–152].

Composite biomaterial carriers for sustained release

Integrating ApoEVs into biocompatible matrices addresses challenges of rapid clearance and provides sustained, localized release. Hydrogels and gelatin-based scaffolds are ideal for this purpose due to their tunable porosity and excellent biocompatibility [153, 154]. A notable example is the use of 3D-printed extracellular mesenchymal scaffolds to deliver ApoEVs derived from hypoxia-preconditioned adipose-derived MSCs (ADMSCs). This combination significantly enhanced chondral repair, with hypoxia-induced ApoEVs offering superior efficacy due to their increased yield and enriched regenerative cargo (e.g., miRNAs and functional proteins) [125]. This synergy between advanced biomaterials and engineered ApoEVs creates a protective niche for enhanced bone and cartilage regeneration.

Functionalized loading for enhanced therapeutic payload

ApoEVs can be functionally loaded with therapeutic agents to augment their innate capabilities. Techniques like electroporation or liposome-based encapsulation are used to load drugs (e.g., Bortezomib for multiple myeloma) or genetic materials (e.g., siRNAs, miRNAs) [28]. For example, MSCs undergoing apoptosis can efficiently incorporate exogenously added Bortezomib into their ApoEVs, achieving higher drug-loading efficiency than conventional sonication. The resulting Bortezomib-loaded MSC-ApoEVs demonstrated reduced off-target toxicity and enhanced anti-myeloma efficacy, highlighting their potential as targeted nanotherapies for bone-related cancers [155, 156].

Lyophilization for improved storage and clinical translation

Lyophilization (freeze-drying) is a critical step for overcoming the storage and stability bottlenecks that impede the clinical use of ApoEVs. Studies confirm that lyophilized ApoEVs maintain their structural integrity and bioactivity after long-term storage [157–159]. Clinically, lyophilized MSC-ApoEVs have proven effective, as they accelerate alveolar bone regeneration by upregulating TRIM71 and activating the ERK signaling pathway in MSCs. Trials have also demonstrated their ability to reduce hemostasis time in tooth extraction wounds [160]. Their utility can be further expanded by integrating them into composite materials, such as hydrogel sponges, to manage bleeding and promote healing in complex wounds [161].

Engineering strategies are progressively transforming ApoEVs from natural biological entities into sophisticated therapeutic platforms (Fig. 7). Peptide-modified ApoEVs enable precise bone homing, composite carriers ensure localized and sustained delivery, and lyophilization ensures a practical shelf life. However, the path to clinical adoption requires addressing key challenges: standardizing the preparation of heterogeneous ApoEVs, comprehensively evaluating their long-term immunogenicity and metabolic fate, and scaling up manufacturing under Good Manufacturing Practice (GMP) standards. Overcoming these hurdles will unlock the full potential of engineered ApoEVs as off-the-shelf therapeutics for bone tissue engineering.

Fig. 7.

Engineering strategies to augment ApoEV therapeutic potential for bone regeneration. (A) Surface-Targeting Modification: ApoEVs are functionalized with targeting ligands to achieve site-specific delivery. Examples include modification with a matrix metalloproteinase (MMP)-activated cell-penetrating peptide (MAP) for responsive targeting to inflamed tissues, or decoration with the bone-homing peptide (DSS)66 for enhanced affinity to bone matrix. (B) Composite Biomaterial Carriers: ApoEVs are embedded within a hybrid hydrogel scaffold, creating a protective depot for the sustained and localized release of vesicles, such as those derived from hypoxia-preconditioned MSCs, to enhance osteochondral repair. (C) Functionalized Loading: ApoEVs are engineered as targeted drug carriers. Therapeutic agents (e.g., Bortezomib, BTZ) are efficiently loaded during apoptosis, leveraging the cell’s innate machinery to achieve high encapsulation efficiency and subsequent targeted delivery to disease sites, such as multiple myeloma lesions. (D) Lyophilization for Storage: The lyophilization process enables the long-term preservation of ApoEVs, maintaining their structural integrity and bioactivity for ready-to-use clinical applications. Panels A-D created with Figdraw.com under academic license

Comparative summary of EVs engineering strategies

Engineering strategies can differentially optimize EVs across four key dimensions, targeting efficiency, payload capacity, release kinetics, and stability (Table 4). Surface-targeting modification prioritizes active homing and specificity, making it suitable for systemic targeted therapy. Composite biomaterial carriers (e.g., hydrogels) stand out for their high payload capacity and tunable sustained release, making them the preferred choice for local, long-term therapy and tissue repair. Functionalized loading enhances drug-loading compatibility and enables synergistic or stimulus-responsive delivery, but often requires combination with targeting modifications to improve precision. Lyophilization primarily addresses the shelf-life bottleneck for storage, transportation, and large-scale applications; however, it does not provide targeting or controlled release functions on its own and thus needs to be combined with other strategies. Given that a single method is difficult to meet the multi-index requirements of complex clinical scenarios, the integrated strategy of “targeting modification + composite carrier delivery + lyophilization storage” is more likely to achieve performance complementarity and clinical translatability.

Table 4.

Simplified core performance comparison of EVs’ engineering strategies

Engineering strategy	Targeting efficiency	Payload capacity	Release characteristics	Stability	Core advantages	Key limitations	Ref.
Surface-targeting modifications (e.g., peptide modification)	High (active targeting; 5–20x efficiency boost vs. unmodified EVs)	Moderate (additional drug-loading step required)	Rapid release (> 70% released in 1–6 h)	Moderate (in vivo half-life extended by 2–3x)	Precise targeting; suitable for systemic therapy	High modification difficulty; potential membrane damage	[162, 163]
Composite biomaterial carriers (e.g., hydrogel loading)	Moderate (strong local retention; poor systemic targeting)	High (3–10x payload increase; compatible with multiple drugs)	Controlled sustained release (> 7 days, two-phase release)	High (in vivo retention extended by 5–7x)	High payload; tunable release; safe for local long-term therapy	Unsuitable for systemic delivery; variable degradation rates	[164, 165]
Functionalized loading	Moderate (requires combination with targeting modifications)	High (supports synergistic multi-drug loading)	Controlled release (stimulus-responsive; leakage < 5%)	Moderate (enhanced membrane stability; potential immunogenicity)	High loading efficiency; synergistic/responsive delivery	Complex synthesis; higher cost	[166, 167]
Lyophilization	Moderate (> 90% retention with proper protectants)	Moderate (protects pre-loaded drugs; no direct loading function)	Consistent with fresh EVs (no inherent controlled release)	High (stable for 6–12 months at 4 °C/−20 °C)	Improves storage stability; facilitates clinical translation	Needs protectant optimization; high energy cost; requires combination with other strategies	[159, 161]

Challenges and prospects in ApoEV clinical translation

ApoEVs represent a promising cell-free therapeutic platform for bone regeneration, as they inherit bioactive cargo from parent cells and demonstrate high biocompatibility. While their mechanistic roles in bone homeostasis are being unraveled and engineering strategies are expanding their capabilities, several formidable challenges must be overcome to realize their clinical potential.

Primary challenges

The profound therapeutic potential of ApoEVs for bone regeneration is increasingly evident from compelling pre-clinical studies. However, the journey from laboratory bench to clinical bedside is fraught with significant and multifaceted hurdles. The clinical translation of ApoEVs is primarily hampered by unresolved challenges in their manufacturing, characterization, and safety profiling. This section will critically examine these key bottlenecks, including the standardization of scalable production, functional potency assessment, and comprehensive biodynamic understanding, which currently impede their progression into clinical applications (Fig. 8). Addressing these barriers is a critical prerequisite for harnessing the full regenerative power of ApoEVs in medicine.

Fig. 8.

Key challenges in the clinical translation of ApoEVs. (A) Heterogeneity and Safety Concerns: ApoEV populations are intrinsically heterogeneous, varying in size, cargo, and function. This diversity leads to inconsistent therapeutic outcomes and potential safety risks, including the carriage of infectious agents (e.g., viral particles) or oncogenic molecules from pathological cells. Furthermore, allogeneic ApoEVs face rapid immune clearance and may trigger adaptive immune responses upon repeated administration. (B) Targeting and Delivery Limitations: Current bioengineering strategies for enhancing ApoEV targeting lack standardization, resulting in unreliable delivery efficiency. A significant challenge is the inherent off-target accumulation of ApoEVs in organs of the mononuclear phagocyte system (e.g., liver and spleen), which drastically reduces their bioavailability at the intended site of bone injury. (C) Standardization and Pharmacokinetic Gaps: The field suffers from a lack of robust, scalable production methods. Conventional isolation techniques compromise vesicle purity and integrity, whereas chemical apoptosis inducers can introduce cytotoxic residues and alter the native ApoEV cargo. Critically, there is a profound lack of long-term pharmacokinetic data, with most studies tracking ApoEVs for only up to 72 h, leaving their long-term fate and clearance kinetics unknown. Panels A-C created with Figdraw.com under academic license

Inherent heterogeneity and safety

ApoEV preparations are highly heterogeneous in size, cargo, and function, even from identical sources, leading to inconsistent therapeutic outcomes [168]. Critically, ApoEVs derived from pathological microenvironments may carry infectious agents (e.g., HIV-1) [58–62] or oncogenic molecules, posing significant safety risks. The immunogenicity of allogeneic ApoEVs also remains a concern, as donor antigens presented on MHC molecules can trigger adverse immune responses upon repeated administration [169].

Targeting and bioengineering limitations

Current targeting strategies, whether through surface functionalization or parental cell engineering, lack standardization, resulting in variable efficiency. ApoEVs also exhibit natural tropism for the mononuclear phagocyte system, leading to off-target accumulation in organs like the liver and spleen, which reduces their bioavailability at bone defect sites [170]. Furthermore, bioengineering processes themselves may introduce unforeseen immunogenicity or toxicity.

Standardization and pharmacokinetic gaps

The transition to clinical use is hampered by the lack of scalable, reproducible production methods. Conventional ultracentrifugation often compromises vesicle integrity, while apoptosis inducers like STS can alter ApoEV cargo and introduce cytotoxic effects [9]. A critical knowledge gap exists in long-term pharmacokinetics; current studies have only tracked ApoEVs in vivo for up to 72 h, with no data on their clearance kinetics or long-term fate beyond 28 days, which is essential for regulatory approval.

Among these challenges, standardization of scalable production and comprehensive long-term safety and pharmacokinetic assessment stand out as the most critical and urgent bottlenecks. Their primacy stems from the fact that scalable, reproducible production is the foundational prerequisite for any clinical translation; without standardized manufacturing processes, consistent therapeutic potency cannot be guaranteed, and large-scale clinical trials (a core requirement for regulatory approval) are infeasible. Meanwhile, long-term safety and pharmacokinetic data are non-negotiable for regulatory clearance: without clarifying the long-term fate, clearance kinetics, and potential long-term toxic/immunogenic effects of ApoEVs in vivo, their clinical application cannot be justified, regardless of preclinical efficacy. Focusing on these two bottlenecks is therefore essential to prioritize future research efforts and accelerate the clinical translation of ApoEVs for bone regeneration.

Those significant translational barrier stems from the lack of clear regulatory pathways for ApoEVs-based therapies. The absence of standardized FDA guidelines for ApoEVs products exacerbates investor caution, complicates clinical trial design, and delays large-scale validation. For instance, ApoEV-based therapies have struggled with requirements for rigorous characterization and manufacturing consistency under current Good Manufacturing Practice (GMP), which directly impacts ApoEV development. Without established benchmarks for quality control and safety, ApoEVs risk being stalled in pre-clinical stages, mirroring the delays seen in ApoEVs drug applications. This regulatory uncertainty heightens the need for interdisciplinary collaboration to address standardization and pharmacokinetic gaps.

Innovative strategies and artificial intelligence-based future directions

To address these challenges, innovative engineering approaches are being explored. A promising strategy involves using disease-modifying drugs themselves to induce apoptosis, thereby generating drug-loaded ApoEVs in situ. Looking ahead, a more radical “hollowing and reloading” strategy can be envisioned: preserving the native ApoEV membrane for its targeting and stability properties while replacing the heterogeneous internal cargo with a defined, potent therapeutic payload (Fig. 9). This would maximize efficacy and safety by eliminating potentially adverse native components.

Fig. 9.

Advanced engineering paradigms for therapeutic ApoEVs: drug-induced biogenesis and membrane reconstitution. (A) Drug-Induced Biogenesis: A strategic method where mesenchymal stem cells (MSCs) are incubated with a specific therapeutic drug (e.g., Bortezomib, BTZ) at an optimized concentration, triggering apoptosis and the subsequent generation of drug-loaded ApoEVs (Drug-MSCs-ApoEVs) [155, 156]. This approach leverages the cell’s natural biogenesis machinery to encapsulate the therapeutic agent, combining the drug’s efficacy with the inherent bioactivity and targeting capabilities of the MSC-derived vesicle membrane. This method enhances delivery efficiency, reduces off-target toxicity associated with high-dose systemic administration, and avoids the chemical residues from non-therapeutic apoptosis inducers. (B) Membrane Reconstitution for Precision Therapy: A more sophisticated approach involving the isolation of ApoEV membranes from patient-derived cells (e.g., peripheral blood cells). The native internal cargo is removed to create a hollow, biocompatible shell. This shell is then reconstituted and reloaded with a defined payload of purified therapeutic molecules (e.g., drugs, nucleic acids). This “hollowing and reloading” strategy maximizes therapeutic predictability and safety by eliminating the heterogeneous and potentially adverse cargo of natural ApoEVs, while the autologous membrane minimizes immunogenic risks. Panels A-B were created with Figdraw.com under an academic license

The advancement of ApoEV-based bone regeneration is increasingly driven by the integration of artificial intelligence (AI) with multi-omics approaches. While the complex biological cargo of ApoEVs presents a significant analytical challenge, AI models are now being employed to decode this information. By processing integrated genomic, proteomic, and metabolomic data, these models can identify subtle, clinically relevant biomarker signatures essential for bone healing that were previously obscured by technical noise (Fig. 10) [171]. This not only maps pathological communication networks in bone diseases for unprecedented mechanistic insight but also enables predictive modeling. For instance, deep learning has been used to predict the osteogenic activity of peptides and generate optimized sequences, as demonstrated in the evaluation of camel milk-derived peptides [172].

Fig. 10.

Artificial Intelligence in EVs Research: Applications and Prospects (A) AI methodologies are revolutionizing EV studies by enabling intelligent isolation and characterization, enhancing targeted recognition and delivery, optimizing engineered EV design, mapping intercellular communication networks, integrating multi-omics data, and advancing disease diagnosis and therapy. Each domain leverages machine learning and deep learning algorithms to overcome challenges in EV heterogeneity, functional analysis, and clinical translation. Panels were created with Figdraw.com under an academic license

A key innovation involves leveraging machine learning to overcome these analytical bottlenecks. Algorithms can process large, high-dimensional ApoEV datasets to systematically evaluate their structural and functional properties [173, 174], paving the way for rational engineering. AI-driven platforms can predict how specific modifications will influence a vesicle’s bone-targeting efficiency and drug-loading capacity, significantly accelerating the development of bespoke therapeutic vesicles [175]. This approach is exemplified by a study where unsupervised deep learning mined protein intrinsic disorder regions (IDRs) to design the pentapeptide AIB5P, which was subsequently verified to promote osteogenesis and bone defect repair by binding integrin α5 and activating the FAK pathway [176].

A particularly compelling strategic direction is the use of AI for early diagnosis and patient stratification in bone-related disorders. By identifying optimal biomarker combinations from patient-derived EVs, AI can enhance the sensitivity of diagnosing conditions like osteoporosis, ensuring that future ApoEV therapies are applied to the right patients at the right time [177].

Looking forward, the future of ApoEV therapeutics hinges on the seamless convergence of multi-omics biology and AI-driven design. This powerful combination will enable the precise optimization of engineering parameters for bone regeneration. When coupled with the establishment of robust, scalable manufacturing protocols, these efforts will finally unlock the potential of precision ApoEV-based therapies, offering powerful new solutions for regenerating bone and treating complex metabolic bone diseases.

Conclusion

Compared to traditional strategies, ApoEVs have emerged as a potent, cell-free platform for bone regeneration, masterfully orchestrating repair by regulating osteogenesis, angiogenesis, and immunomodulation. While their innate bioactivity and scalability are promising, engineering strategies, such as surface modification and biomaterial integration, are being employed to overcome the limitations of native vesicles. The integration of artificial intelligence can significantly accelerate their clinical translation, leveraging predictive modeling to optimize ApoEV engineering and multi-omics analysis to decipher complex cargo-function relationships. To fully realize this potential, future work must focus on establishing standardized manufacturing, employing these AI-driven insights for mechanistic understanding, and validating long-term safety and efficacy. By systematically addressing these challenges, engineered ApoEV therapies are poised to transition from a compelling concept to a clinical reality, revolutionizing the treatment of complex bone disorders.

Author contributions

† Yadong Guo, Wenbo Du, and Shuguang Cheng contributed equally as cofirst authors.

Funding

This study was funded by the National Natural Science Foundation of China (32301129 and 82150410451), the Guangzhou Science and Technology Plan Project (2023B03J1240), and the Science and Technology Projects in Guangzhou (202201020396).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.