INTRODUCTION
Osteoradionecrosis (ORN) represents one of the most devastating late complications of ionizing radiation therapy, typically emerging months to years after treatment for malignancies in anatomical regions adjacent to bone. While historically associated with head and neck cancers - including oral, pharyngeal, and laryngeal carcinomas - ORN also occurs following radiotherapy for breast, pelvic, and spinal tumors, reflecting the systemic vulnerability of skeletal tissue to radiation-induced damage[1]. Clinically, ORN is defined by the presence of exposed, non-healing necrotic bone persisting for more than three months in the absence of tumor recurrence[2]. Despite significant advances in radiation delivery technologies - such as intensity-modulated radiotherapy, volumetric modulated arc therapy, and proton beam therapy - the incidence of ORN remains alarmingly high, ranging from 2% to over 20%, depending on total radiation dose, fractionation schedule, field volume, and concomitant surgical or dental interventions[3-5].
Current therapeutic paradigms, including hyperbaric oxygen, prolonged antibiotic regimens, and surgical debridement or microvascular reconstruction, are largely palliative; they address secondary infection or remove devitalized tissue but fail to reverse the underlying pathophysiological milieu that prevents endogenous regeneration[6]. Consequently, patients often endure recurrent episodes, progressive functional impairment, and severely diminished quality of life, underscoring an urgent unmet clinical need for biologically rational, regenerative strategies.
In this context, extracellular vesicles (EVs) - particularly small EVs (sEVs), commonly termed exosomes - have emerged as a transformative therapeutic modality in regenerative medicine[7,8]. The sEVs are naturally occurring, nanoscale (30-150 nm) lipid bilayer nanoparticles secreted by nearly all cell types, functioning as sophisticated intercellular messengers[9,10]. They encapsulate complex and bioactive cargo comprising proteins, lipids, mRNAs, microRNAs (miRNAs), long non-coding RNAs, and even mitochondrial components such as mitochondrial DNA and respiratory chain proteins[11,12]. Upon uptake by recipient cells, this cargo can reprogram cellular metabolism, suppress inflammatory signaling, enhance survival pathways, and stimulate tissue repair[8]. Critically, as acellular entities, sEVs circumvent the safety concerns associated with whole-cell therapies - including tumorigenicity, ectopic differentiation, and immune rejection - while retaining the multifaceted regenerative capacity of their parent cells[7]. Their intrinsic biocompatibility, low immunogenicity, and potential for targeted delivery further position them as ideal candidates for precision regenerative interventions in complex pathological settings like ORN.
Recently, a growing number of studies have demonstrated that human amniotic mesenchymal stem cell-derived sEVs (hAMSC-sEVs) can play a substantial role in facilitating angiogenesis and bone regeneration[13,14]. To further explore the potential of hAMSC-sEVs in restoring bone vascular homeostasis, this review conducts a comprehensive analysis of the underlying therapeutic mechanisms, integrating recent advances in mitochondrial biology, epigenetics, signal transduction, and immunomodulation (Figure 1). Furthermore, we critically evaluate the current limitations impeding clinical translation and outline future directions necessary to realize the full potential of this innovative nanotherapeutic platform.
Figure 1 Human amniotic mesenchymal stem cell-derived small extracellular vesicles promote bone regeneration in irradiated tissue by delivering mitochondria, microRNAs, and signaling molecules to restore energy metabolism, reprogram progenitor cells, and shift macrophages from pro-inflammatory to anti-inflammatory phenotypes.
hAMSC-sEVs: Human amniotic mesenchymal stem cell-derived small extracellular vesicles; miRNA: MicroRNA; RUNX2: Runt-related transcription factor 2.
PATHOPHYSIOLOGY OF ORN: SYSTEMIC COLLAPSE OF THE BONE-VASCULAR COUPLING SYSTEM
The understanding of ORN pathogenesis has evolved from the classical “3H” hypothesis - hypocellularity, hypovascularity, and hypoxia - to a dynamic, self-amplifying cascade centered on the breakdown of the osteo-angiogenic unit[15]. Ionizing radiation inflicts direct DNA damage and sustained oxidative stress, triggering senescence or apoptosis in both osteolineage cells and vascular endothelial cells[16-18]. These initial injuries establish a maladaptive feedback loop where irradiated cells adopt a senescence-associated secretory phenotype (SASP), characterized by the persistent release of pro-inflammatory and pro-fibrotic mediators like interleukin-6 (IL-6) and transforming growth factor-β (TGF-β)[19]. These factors concurrently suppress osteogenic differentiation via nuclear factor-kappa B signaling and impair microvascular integrity, leading to a systemic collapse of the bone-vascular coupling system[20,21]. Notably, while the ORN microenvironment generally suppresses the activity of most resident bone cells, accumulating evidence confirms that aberrant osteoclast hyperactivation is a pivotal driver in the progression of the disease[22-24]. This process ultimately creates a fibrotic, metabolically inert tissue state that actively resists endogenous regeneration and conventional clinical interventions.
MECHANISMS AND STRATEGIES OF SEVS IN TARGETING ORN
The hAMSC-sEVs represent a paradigm-shifting, cell-free nanotherapeutic platform uniquely equipped to dismantle the interconnected pathological axes of ORN[13,25]. Unlike conventional monotherapies that target isolated symptoms or single molecular pathways, hAMSC-sEVs’ therapeutic efficacy arises from four interdependent mechanistic pillars - mitochondrial rescue, epigenetic reprogramming, multi-pathway activation, and immune microenvironment remodeling - that collectively restore the metabolic, transcriptional, signaling, and biomechanical foundations necessary for coupled osteo-angiogenic regeneration.
Mitochondrial transfer and metabolic reprogramming: Rescuing the bioenergetic collapse
Ionizing radiation induces profound mitochondrial dysfunction in osteoblasts and endothelial cells, characterized by mtDNA fragmentation, impaired electron transport chain activity, and excessive reactive oxygen species production[26,27]. This bioenergetic crisis leads to ATP depletion, compromising essential processes such as collagen synthesis, mineralization, and angiogenic sprouting[28]. sEVs directly counteract this metabolic failure through intercellular transfer of mitochondrial cargo. This process is mediated by surface tetraspanins (CD63/CD9) on sEVs interacting with adhesion receptors (such as CXC receptor-4 and intercellular adhesion molecule-1) on recipient cells, leading to internalization via endocytosis or membrane fusion. While parent mesenchymal stem cells (MSCs) often employ tunneling nanotubes for direct organelle transfer, sEVs provide a distinct, cell-free mechanism for restoring mitochondrial function. Once internalized, these exogenous mitochondria integrate into the host mitochondrial network, restoring oxidative phosphorylation, boosting ATP generation, and normalizing redox homeostasis[29,30]. In parallel, sEVs drive adaptive metabolic reprogramming by upregulating peroxisome proliferator-activated receptor gamma coactivator 1-alpha - a master regulator of mitochondrial biogenesis[31] - and stabilizing hypoxia-inducible factor-1α under residual hypoxia[32,33]. This dual strategy enhances both mitochondrial respiration and glycolytic flux, enabling irradiated cells to dynamically switch energy substrates according to local oxygen availability - a critical adaptation for survival and function in the compromised bone marrow microenvironment (Figure 2).
Figure 2 Human amniotic mesenchymal stem cell-derived small extracellular vesicles mediate mitochondrial transfer to damaged osteoblasts and endothelial cells via tunneling nanotubes, restoring oxidative phosphorylation and ATP production, while promoting glycolytic adaptation in endothelial cells to support vascular repair.
ROS: Reactive oxygen species; hAMSC-sEVs: Human amniotic mesenchymal stem cell-derived small extracellular vesicles; CXCR4: CXC receptor-4; ICAM-1: Intercellular adhesion molecule-1; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; OXPHOS: Oxidative phosphorylation; HIF-1α: Hypoxia-inducible factor-1α.
Epigenetic rejuvenation: Reversing radiation-induced transcriptional silencing
Beyond metabolic rescue, sEVs address the persistent epigenetic dysregulation imposed by ionizing radiation[34,35]. Exposure to radiation triggers durable epigenetic modifications, particularly CpG island hypermethylation at promoter regions of key osteogenic genes - including Runt-related transcription factor 2, SP7/Osterix, and BGLAP - effectively locking mesenchymal progenitors into a non-regenerative state[36]. It is hypothesized that sEVs may mitigate this “epigenetic scar” through the delivery of epigenetically active cargo. Notably, they are highly enriched in miR, which directly suppresses DNA methyltransferases DNMT3A and DNMT3B, leading to global DNA demethylation and reactivation of silenced osteogenic programs[37]. Additionally, sEVs carry histone-modifying complexes, including histone deacetylase and histone acetyltransferase activators, which promote an open chromatin conformation at regenerative gene loci[38,39]. Such proposed epigenetic remodeling could potentially restore differentiation competence and confer long-term transcriptional resilience, distinguishing sEVs from transient signaling agonists. While further in vivo validation in ORN models is required, this theoretical capacity for rewriting the epigenetic landscape suggests a potent strategy to rejuvenate the regenerative potential of irradiated progenitor cells.
Multi-pathway synergistic activation: Orchestrating coupled regeneration
The sEVs do not rely on a single signaling axis but instead co-activate a synergistic and evolutionarily conserved network of pro-regenerative pathways that collectively restore the osteo-angiogenic unit compromised in ORN[40,41].
Central to this response is the concurrent activation of the phosphatidylinositol 3-kinase/protein kinase B and Wnt/β-catenin cascades - key regulators of cell survival, osteoblast differentiation, vascular endothelial growth factor-mediated angiogenesis, glucose metabolism, and mammalian target of rapamycin-dependent protein synthesis[42]. Critically, emerging studies demonstrate that sEVs also modulate the Hippo-YAP/TAZ pathway by downregulating LATS1/2 kinase activity, thereby promoting YAP/TAZ nuclear translocation and TEAD-dependent transcription of CTGF, CYR61, and Runt-related transcription factor 2. This mechanism functionally links extracellular matrix mechanics - such as the increased stiffness of fibrotic bone - to regenerative gene expression[43]. Simultaneously, sEVs robustly engage the nuclear factor-erythroid-related factor 2/antioxidant response element antioxidant axis, inducing cytoprotective enzymes including heme oxygenase-1, NQO1, and superoxide dismutase 2 to neutralize chronic reactive oxygen species and disrupt the oxidative stress - inflammation feedback loop. Far from operating in isolation, these pathways exhibit extensive crosstalk: YAP/TAZ physically interacts with β-catenin to potentiate Wnt signaling[44], nuclear factor-erythroid-related factor 2 stabilizes hypoxia-inducible factor-1α under residual hypoxia to support metabolic adaptation[45], and protein kinase B-mediated phosphorylation of glycogen synthase kinase 3beta further enhances β-catenin stability. This integrated, self-reinforcing signaling circuitry enables sEVs to orchestrate a holistic regenerative program that markedly surpasses the therapeutic potential of single-pathway interventions.
Immune microenvironment remodeling: Disrupting the SASP-fibrosis axis
sEVs fundamentally reshape the irradiated bone microenvironment by dismantling the self-sustaining SASP-driven inflammatory-fibrotic axis that underlies tissue dysfunction in ORN[46,47]. Irradiated osteocytes and endothelial cells secrete a pathological cocktail of IL-6, tumor necrosis factor-α, matrix metalloproteinases, and TGF-β, which perpetuates chronic inflammation, stromal fibrosis, and impaired regenerative capacity[48]. sEVs counteract this cascade through integrated immunomodulatory and anti-fibrotic actions: They deliver anti-inflammatory miRNAs - particularly miR-21-5p and miR-146a-5p - that suppress nuclear factor-kappa B and signal transducer and activator of transcription 3 signaling in myeloid cells, thereby reprogramming macrophage polarization from a pro-inflammatory M1 state toward an M2-like, pro-reparative phenotype marked by elevated CD206 and IL-10 expression[49]. Concurrently, they directly mitigate cellular senescence in irradiated stromal populations by downregulating key cell cycle inhibitors[50], while also inhibiting cGAS-STING pathway activation triggered by cytosolic mitochondrial DNA leakage - a major instigator of radiation-induced sterile inflammation[51]. In parallel, sEVs attenuate TGF-β/Smad2/3 signaling in fibroblasts, rebalance the matrix metalloproteinases/tissue inhibitor of metalloproteinases equilibrium, and reduce excessive collagen deposition, effectively softening the fibrotic extracellular matrix[52]. This restoration of biomechanical permissiveness is essential for enabling vascular ingrowth and osteoblast migration. Collectively, these coordinated mechanisms transform a hostile, non-regenerative niche into a pro-healing microenvironment that actively supports coupled osteo-angiogenic regeneration. However, it must be noted that reshaping the irradiated niche is complex endeavor, as the signaling pathways involved often exhibit pleiotropic effects on oncogenesis. For instance, while suppressing tumor necrosis factor-α is vital for neutralizing the SASP and reducing fibrosis, this cytokine embodies a “double-edged sword” in the tumor microenvironment - possessing the capacity to both exert anti-tumor cytotoxicity and facilitate metastatic spread.
Together, these four mechanistic pillars position sEVs not merely as a drug delivery vehicle, but as a dynamic biological system capable of simultaneously rescuing cellular energetics, rewriting epigenetic memory, activating synergistic regenerative circuits, and reprogramming the immune landscape - thereby achieving true restoration of the bone-vascular coupling essential for healing in ORN.
MSC-DERIVED SEVS IN ORN THERAPY AND ADVANTAGES OF HAMSC- SEVS
Although exosomes derived from traditional MSCs (e.g., bone marrow and adipose tissue) have likewise demonstrated considerable potential in bone tissue regeneration, their clinical translation is frequently hindered by invasive harvesting procedures, limited cell yields, and an age-dependent decline in therapeutic efficacy[53,54]. Nevertheless, these limitations do not diminish the tremendous potential of MSC-sEVs in the treatment of ORN. Compared to their parent cells, MSC-sEVs exhibit superior biocompatibility, translating to a minimized risk of immune rejection. Concurrently, MSC-sEVs can enhance the radioresistance of healthy resident bone cells via multiple signaling pathways, exerting potent pro-angiogenic, anti-inflammatory, and ferroptosis-regulating effects[55-57]. Another notable advantage is that MSC-sEVs can be integrated with diverse biomaterials to facilitate bone regeneration[58,59].
To overcome these translational bottlenecks, hAMSC-sEVs have emerged as a highly promising alternative. Sourced from discarded term placental tissues, hAMSCs fundamentally circumvent ethical controversies and invasive extractions. Moreover, residing at an early developmental stage, these cells exhibit robust proliferative capacity and a notably higher yield of sEVs[60,61]. Beyond their logistical and manufacturing advantages, the feasibility of applying hAMSC-sEVs in ORN therapy is fundamentally rooted in their molecular mechanisms that drive coupled osteo-angiogenesis. As delineated earlier in this review, hAMSC-sEVs may offer a biologically superior, cell-free therapeutic strategy for the management of ORN.
LIMITATIONS AND FUTURE PERSPECTIVES
Although hAMSC-sEVs show exceptional promise for ORN therapy, their clinical translation faces key challenges - amplified by the need to justify their use over other MSC-sEV sources. Compared to sEVs from adult tissues like bone marrow or adipose tissue, hAMSC-sEVs originate from an ethically non-controversial, fetal-derived source with inherent immune privilege and exhibit robust proliferative capacity. Their regenerative cargo is enriched in pro-angiogenic factors, osteoinductive miRNAs, and - critically - functional mitochondria, providing a strong biological rationale for their application in ORN. This developmental advantage enables multi-targeted repair of ORN’s metabolic, epigenetic, and inflammatory axes.
Nevertheless, three barriers remain. First, sEV heterogeneity - driven by donor variability, culture conditions, and isolation methods - compromises batch consistency and regulatory approval. Second, scalable GMP-compliant manufacturing is limited by low-yield purification techniques. Third, targeted delivery to avascular, fibrotic ORN lesions is inefficient; systemic administration yields poor retention. Future strategies must therefore integrate biomaterial carriers (e.g., hyaluronic acid hydrogels) for local sustained release or engineer sEV surfaces with bone-homing ligands (e.g., RGD peptides). Addressing these through interdisciplinary collaboration will be essential to harness hAMSC-sEVs’ full therapeutic potential.
CONCLUSION
The hAMSC-sEVs represent a paradigm-shifting approach to ORN, simultaneously rescuing mitochondrial bioenergetics, reversing epigenetic silencing, and reprogramming the fibro-inflammatory niche to restore osteovascular coupling. Their ability to deliver functional mitochondria and developmentally informed signals offers a regenerative - not merely palliative - solution to a historically untreatable condition. While standardization, scalable production, and targeted delivery remain hurdles, advances in EV engineering and biomaterials provide a clear translational pathway. In the future, it is imperative to concentrate on the construction of efficient drug delivery systems using biomaterials that meet the requirements, and to expand clinical trials to validate the effectiveness and biological safety of sEVs therapy.
Peer review: Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Cell and tissue engineering
Country of origin: China
Peer-review report’s classification
Scientific quality: Grade B, Grade B, Grade C
Novelty: Grade B, Grade B, Grade C
Creativity or innovation: Grade A, Grade B, Grade B
Scientific significance: Grade A, Grade C, Grade C
P-Reviewer: Chen GY, Assistant Professor, MD, Germany; Sukocheva OA, Assistant Professor, PhD, Senior Researcher, Australia S-Editor: Wang JJ L-Editor: A P-Editor: Zhao YQ
