Stem cell-derived extracellular vesicles and artificial nanovesicles: a translational framework for cell-free wound repair

Abstract

Chronic nonhealing wounds remain a major clinical challenge, driven by persistent inflammation, impaired angiogenesis, defective extracellular matrix remodeling, and incomplete functional restoration. While stem cell therapies can modulate these processes, their clinical use is limited by low engraftment, variability, and safety concerns. Stem cell-derived extracellular vesicles (SC-EVs) and artificial nanovesicles (SC-ANVs) have emerged as cell-free alternatives that aim to capture key paracrine functions of stem cells. In this review, we evaluate these vesicle-based approaches within a problem-oriented framework linking molecular and cellular effects to clinically meaningful outcomes. SC-EVs and SC-ANVs influence inflammation, angiogenesis, and tissue regeneration in preclinical wound models; however, current evidence is largely limited to improvements in wound closure and histological parameters. We highlight that proof of concept for clinically relevant endpoints, such as durable function, reduced scarring, and recurrence prevention, remains insufficient. Key translational challenges, including delivery, dosing, and endpoint selection, are discussed, along with a framework for future studies required to establish therapeutic efficacy. Collectively, SC-EVs and SC-ANVs represent emerging platforms whose clinical potential depends on rigorous validation against defined clinical benchmarks.

Introduction

Chronic nonhealing wounds, including diabetic foot ulcers, venous leg ulcers, and pressure injuries, represent a major global health challenge, affecting more than 100 million individuals worldwide¹, and imposing annual healthcare costs exceeding $25 billion in the United States alone². Beyond delayed closure, the central clinical challenge lies in the failure to restore durable function and tissue integrity, often resulting in impaired mobility, recurrent breakdown, infection, and, in severe cases, amputation (Level 1)^3–5. These outcomes are compounded by intermediate clinical complications such as persistent exudation, wound instability, and high recurrence rates (Level 2)^6–9. To contextualize these challenges, a hierarchical framework is adopted, linking clinically meaningful outcomes to underlying biological processes. At the tissue level (Level 3), successful healing requires formation of a stable epithelial barrier and organized dermal architecture^6,10. At the vascular level (Level 4), adequate and functional angiogenesis is essential, particularly in ischemic and diabetic wounds^7,8. At the cellular and molecular levels (Level 5 and below), coordinated regulation of inflammation, macrophage polarization, fibroblast and keratinocyte activity, protease balance, and extracellular matrix (ECM) remodeling is required^9,11. Disruption across these levels creates a self-sustaining pathological cycle that prevents resolution and undermines higher-level clinical outcomes.

Current therapies, including advanced dressings and cell-based approaches, have shown limited success in consistently addressing this multiscale complexity⁹. Consequently, next-generation wound therapies must meet defined criteria that include resolution of chronic inflammation, restoration of functional angiogenesis, support of fibroblast and keratinocyte activity, reduction of pathological scarring, and feasibility in terms of safety, scalability, and clinical integration^6,12–21. Stem cell-based strategies, particularly those using mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), have demonstrated the ability to modulate multiple components of the wound environment. However, their clinical translation has been constrained by low engraftment, phenotypic variability, and safety concerns²².

Increasing evidence suggests that many of the therapeutic effects of stem cells are mediated through paracrine signaling rather than long-term cellular integration²². In this context, stem cell-derived extracellular vesicles (SC-EVs) and stem cell-engineered artificial nanovesicles (SC-ANVs) have emerged as cell-free approaches that aim to reproduce key aspects of stem cell signaling. These nanoscale vesicles deliver bioactive cargo, including proteins, lipids, and nucleic acids, that can influence inflammation, angiogenesis, and tissue regeneration^6,7,11. While this positions them as potential alternatives to cell-based therapies, it remains unclear whether these effects are sufficient to achieve clinically meaningful outcomes in complex wounds. Whether SC-EVs and SC-ANVs can fully recapitulate these effects without dynamic cellular feedback remains unclear.

In this review, we evaluate SC-EVs and SC-ANVs within a problem-oriented framework that connects biological activity at lower levels (Levels 3–5) to clinical endpoints (Levels 1–2). Rather than cataloging molecular effects in isolation, we assess the extent to which vesicle-mediated modulation of key processes such as inflammation resolution, vascularization, and tissue remodeling addresses defined barriers to healing. We further compare these approaches to stem cell therapies, highlighting both shared mechanisms and limitations associated with the absence of dynamic cellular feedback. Finally, we examine evidence from preclinical and early clinical studies, focusing on what constitutes proof of concept for therapeutic efficacy. We argue that improvements in wound closure or histological parameters alone are insufficient, and that future studies must demonstrate meaningful impact on functional outcomes, scar quality, and recurrence. By integrating mechanistic insights with translational considerations, this review aims to define both the potential and the current limitations of SC-EVs and SC-ANVs as cell-free strategies for complex wound repair.

Biology of wound healing

Wound healing is a highly coordinated and dynamic process comprising overlapping phases of hemostasis, inflammation, proliferation, and remodeling^23,24. These phases are regulated through tightly controlled cellular and molecular interactions that collectively determine whether tissue repair proceeds toward regeneration or fibrosis.

During hemostasis, vasoconstriction and platelet aggregation prevent blood loss while initiating repair signaling. Activated platelets release growth factors such as PDGF, TGF-β, and VEGF, which promote fibrin clot formation and recruit inflammatory and stromal cells. These early signals establish the transition into the inflammatory phase by promoting leukocyte chemotaxis and activating resident fibroblasts and endothelial cells²⁵. The inflammatory phase is characterized by sequential infiltration of neutrophils and monocyte-derived macrophages. Macrophages play a central regulatory role by transitioning from pro-inflammatory (M1) to pro-repair (M2) phenotypes, thereby coordinating pathogen clearance and tissue repair. However, in chronic wounds, this transition is frequently impaired, leading to persistent cytokine production (TNF-α, IL-1β, and IL-6) and sustained inflammatory signaling^16,26. Although macrophage polarization is widely recognized as a key regulatory axis, the in vivo temporal and spatial cues governing this process in human chronic wounds remain incompletely defined.

The proliferative phase involves granulation tissue formation, angiogenesis, and re-epithelialization. Fibroblasts deposit provisional ECM, keratinocytes restore epidermal integrity, and endothelial cells form new vascular networks under VEGF and FGF signaling²⁴, and are regulated by pathways including Wnt/β-catenin and Notch, which control cell proliferation, migration, and fibroblast-to-myofibroblast differentiation^16,17,25,26. Macrophages further contribute by secreting growth factors such as TGF-β and EGF, linking inflammatory resolution to tissue regeneration²⁷. The remodeling phase involves ECM maturation, wound contraction, and restoration of tensile strength. Myofibroblasts drive contraction and collagen deposition, while matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) regulate ECM turnover. Dysregulated TGF-β signaling during this phase contributes to excessive fibrosis and pathological scarring^{16–19,25–38}. Notably, most mechanistic insights are derived from animal models, and the coordination of these pathways in human chronic wounds remains insufficiently understood, particularly in the context of dysregulated rather than absent signaling^18,19,25.

In contrast to acute healing, chronic wounds are characterized by persistent arrest in the inflammatory or early proliferative phase. They exhibit sustained neutrophil and M1 macrophage dominance, elevated protease activity that degrades growth factors and ECM components, and reduced responsiveness of fibroblasts and keratinocytes^{16–19,23–26,28}. Persistent NF-κB activation reinforces inflammation, while impaired Wnt/β-catenin signaling contributes to defective tissue formation²⁹. Additionally, ischemia and impaired perfusion, particularly in diabetic wounds, limit oxygen and nutrient delivery, further impairing repair.

Collectively, chronic wounds represent a state of multilevel dysregulation spanning immune, vascular, cellular, and ECM components (Levels 3–5), ultimately preventing progression toward stable tissue restoration (Levels 1–2). Conventional therapies, including debridement and dressings, fail to correct these underlying dysfunctions. While emerging approaches such as growth factor delivery, gene therapy, and cell- or vesicle-based strategies show promise, their clinical translation remains limited by incomplete mechanistic understanding, variability in response, and challenges in scalability and standardization^30,31.

Nanovesicle biology and function

Nanovesicles are lipid bilayer-bound particles that mediate intercellular communication through the transfer of bioactive cargo. They include naturally occurring extracellular vesicles (EVs) as well as engineered artificial nanovesicles (ANVs), both of which are increasingly being explored as cell-free therapeutic platforms in regenerative medicine.

Natural EVs are broadly classified into exosomes (30–150 nm, endosomal origin), microvesicles (100–1000 nm, plasma membrane derived), and apoptotic bodies (1000–5000 nm), with distinct biogenesis pathways illustrated in Fig. 1. Standard characterization typically relies on nanoparticle tracking analysis or dynamic light scattering for size distribution, electron or atomic force microscopy for morphology, and molecular profiling of canonical markers such as CD9, CD63, and CD81 to ensure purity and identity³². In parallel, ANVs are engineered using methods such as extrusion, sonication, or microfluidics to mimic the structural and functional properties of natural EVs. Importantly, while EVs reflect the native cellular state, ANVs are decoupled from endogenous regulation and therefore allow controlled engineering of composition and dose but lack physiological adaptive feedback. A key advantage of ANVs is their scalability and higher production yield compared to endogenous EV isolation, making them more suitable for large-scale manufacturing under controlled conditions³³.

Fig. 1. Biogenesis and classification of extracellular vesicles.

The figure illustrates the three main types of extracellular vesicles: exosomes (30–150 nm), formed via endosomal pathways; microvesicles (100–1000 nm), generated by plasma membrane budding; and apoptotic bodies (1000–5000 nm), released during cell apoptosis. Each type differs in origin, size, and molecular cargo, including proteins, lipids, and nucleic acids. Created in BioRender. Gangadaran, P. (2026) https://BioRender.com/8wyiqj0.

Functionally, both natural and engineered nanovesicles act as mediators of intercellular signaling within the wound microenvironment. Their biological activity primarily maps onto intermediate regulatory processes (Levels 3–5), including modulation of inflammatory signaling, angiogenic activation, and regulation of ECM remodeling. These vesicles influence recipient cell behavior through transfer of proteins, lipids, and nucleic acids that alter gene expression and signaling pathways^34–36. In the inflammatory compartment, vesicles can influence macrophage polarization states and cytokine profiles through bioactive cargo, including miRNAs and immunomodulatory proteins⁶. In the vascular niche, they modulate endothelial cell behavior and angiogenic signaling pathways such as PI3K/Akt and MAPK, thereby contributing to neovascular responses²³. In the stromal compartment, vesicle-mediated signaling affects fibroblast activation, collagen deposition, and ECM remodeling through regulation of protease activity and growth factor signaling^35,36.

Despite these mechanistic effects being consistently observed in preclinical wound models, including diabetic, burn, and aged skin systems, translation to clinically meaningful outcomes remains limited. Despite consistent preclinical effects, clinical translation remains limited by lack of standardized potency assays, batch variability, and challenges in Good Manufacturing Practice (GMP)-compliant manufacturing³⁴. Although Minimal Information for Studies of Extracellular Vesicles (MISEV) 2023 and International Society for Extracellular Vesicles (ISEV) guidelines have improved EV characterization frameworks, they primarily define identity criteria rather than functional benchmarks required for clinical translation^27,32. To address these limitations, current efforts focus on defining robust characterization frameworks, identifying functional potency signatures (e.g., miRNA or protein cargo profiles), and developing optimized delivery systems such as hydrogel-based sustained release platforms. Early-phase clinical investigations in chronic wounds, including diabetic ulcers and burn injuries, represent initial steps toward clinical translation³⁷.

Taken together, nanovesicles represent a versatile platform for cell-free therapeutic delivery. Their biological effects arise from modulation of cellular signaling networks; however, the extent to which these effects can be translated into consistent and clinically meaningful outcomes remains unresolved. The following section focuses on SC-EVs and SC-ANVs in details and highlights their role in modulating the wound microenvironment.

SC-EVs in wound healing

As mentioned above, successful wound healing requires a tightly regulated sequence of overlapping phases. SC-EVs have emerged as key mediators of stem cell paracrine signaling. Rather than acting as structural replacements, they function as nanoscale information carriers that modulate recipient cell behavior by transferring proteins, lipids, and nucleic acids³⁸. Their therapeutic relevance lies in their ability to influence intermediate regulatory processes (Levels 3–5), which collectively govern higher-order tissue outcomes (Levels 1–2). The following sections examine the origin and characteristics of SC-EVs, their mechanistic roles within the wound microenvironment, and the extent to which these effects address key barriers to effective wound repair.

SC-EV origin and characterization

SC-EVs are derived from a range of stem and progenitor cell sources, including bone marrow, adipose tissue, umbilical cord, iPSCs, and fetal dermis. While these vesicles share conserved structural features (e.g., nanoscale size and expression of canonical exosomal markers such as CD9, CD63, and CD81), their cargo composition and functional properties vary depending on the parent cell type.

Major SC-EV sources and defining features

• Bone marrow-derived macrophage exosomes (M-Exos): A subset of these vesicles, termed M2-Exos, originates from alternatively activated macrophages and typically ranges from 30 to 150 nm. These vesicles were notable for their ability to reprogram pro-inflammatory M1 macrophages toward an pro-repair M2 macrophages, with reported conversion efficiencies approaching 100%. This effect was mediated by delivery of regulatory molecules such as CCL22, CCL24, and MFG-E8, along with additional factors including IL-4, CXCL12, and bFGF that supported tissue repair and wound healing³⁹.

• Fetal dermal MSC-derived exosomes (FDMSC-Exos): These vesicles (40–100 nm) are isolated from fetal skin and contain diverse nucleic acid, lipid, and protein cargo. Their morphology is typically confirmed by transmission electron microscopy (TEM)³⁸.

• Adipose-derived stem cell exosomes (ADSC-Exos/ASC-Exos): Typically, ~90–100 nm and isolated from conditioned media, these vesicles express canonical exosomal markers such as CD63. They may contain regulatory long noncoding RNAs such as MALAT1 and can be further engineered with targeted miRNAs (e.g., miR-146a) to enhance therapeutic potency⁴⁰.

• iPSC-derived MSC exosomes (iPSC-MSC-Exos): These vesicles (30–100 nm) exhibit classical exosomal marker expression (CD9, CD63, CD81) and are associated with strong regenerative and angiogenic signaling potential⁴¹.

• Umbilical cord MSC–derived EVs

  • ucMSC-Exos/uMSC-Exos: ~100 nm vesicles with phospholipid bilayer structure and canonical marker expression⁴².
  • UCB-Exos: 30–150 nm vesicles isolated via ultracentrifugation, also expressing CD9, CD63, CD81⁴².

• Amniotic MSC-derived exosomes (AMSC-Exos): These vesicles (30–150 nm) similarly express CD9, CD63, and CD81 and carry cargo associated with regenerative and angiogenic functions⁴³.

Table 1 summarizes recent preclinical and emerging clinical studies investigating mesenchymal stem cell (MSC)-derived EVs for wound healing. Collectively, these studies demonstrate consistent modulation of key biological processes, including inflammation resolution, angiogenesis, and ECM remodeling. However, most evidence is derived from small animal models, predominantly murine systems in which wound closure is driven largely by contraction, thereby limiting direct translational relevance. In addition, mechanistic attribution to specific exosomal cargo remains incomplete, and challenges related to scalable manufacturing, standardization, and safety continue to constrain clinical application.

Table 1.

MSC-derived EVs for wound healing

EV Source (Abbreviation)	Original Source	Size (nm)	Key Markers & Contents	Key Features
M-Exos (M2-Exos)	Alternatively activated M2 macrophages	30–150	CCL22, CCL24, MFG-E8, IL-4, CXCL-12, bFGF	Induces M1 to M2 macrophage reprogramming.
FDMSC-Exos	Fetal dermal MSCs (fetal skin)	40–100	Nucleic acids, lipids, proteins	Structure commonly visualized with TEM.
hADSC-Exos/ASC-Exos	Human adipose-derived stem cells (hADSCs)	90–100	CD63, lncRNA MALAT1	Isolated from conditioned media (CM); can be fortified with miRNAs (e.g., miR-146a).
hiPSC-MSC-Exos	Human iPSC-derived MSCs	30–100	CD9, CD63, CD81	-
hucMSC-Exos/uMSC-Exos	Human umbilical cord MSCs	~100	CD63, CD81, CD9	Spherical vesicles with a phospholipid bilayer structure.
UCB-Exos	Human umbilical cord blood plasma	30–150	CD9, CD63, CD81	Can be isolated by ultracentrifugation.
hAMSC-Exos	Human amniotic MSCs	30–150	CD9, CD63, CD81	-

EVs extracellular vesicles, Exos exosomes, MSCs mesenchymal stem/stromal cells, M-Exos macrophage-derived exosomes, M2-Exos alternatively activated (M2) macrophage-derived exosomes, FDMSC-Exos fetal dermal mesenchymal stem cell-derived exosomes, hADSC-Exos/ASC-Exos human adipose-derived stem cell-derived exosomes, hiPSC-MSC-Exos human induced pluripotent stem cell-derived mesenchymal stem cell exosomes, hucMSC-Exos/uMSC-Exos human umbilical cord mesenchymal stem cell-derived exosomes, UCB-Exos umbilical cord blood-derived exosomes, hAMSC-Exos human amniotic mesenchymal stem cell-derived exosomes, TEM transmission electron microscopy, CM conditioned medium, bFGF basic fibroblast growth factor.

Modes of action in tissue repair and regulation of wound microenvironment

Despite heterogeneity in cellular origin, SC-EVs exhibit conserved structural features and carry overlapping regenerative cargo profiles that enable modulation of key cellular components within the wound microenvironment. Their primary biological effects occur at intermediate regulatory levels (Levels 3–5), where they influence immune responses, vascular dynamics, and stromal cell function.

Immune regulation and inflammatory resolution

As mentioned previously, a critical barrier to effective wound healing is the failure to transition from a pro-inflammatory to a reparative state. Chronic wounds are characterized by sustained dominance of M1 macrophages, which perpetuate inflammation and impair progression to tissue repair⁴⁴. SC-EVs, particularly the M2-Exos, promote a shift toward M2 polarization through delivery of regulatory mediators, including CCL22, CCL24, and MFG-E8⁴⁵. This phenotypic transition facilitates resolution of inflammation and supports progression into the proliferative phase. However, the extent to which this macrophage reprogramming is sustained in vivo, particularly in chronic human wounds, remains unclear.

Stromal regulation and extracellular matrix remodeling

SC-EVs regulate fibroblast behavior, a key determinant of ECM deposition and tissue remodeling. FDMSC-Exos enhance proliferation, migration, and ECM production in adult dermal fibroblasts through activation of Notch signaling, particularly via Jagged1⁴⁶. Similarly, adipose-derived EVs containing the long noncoding RNA MALAT1, promote fibroblast migration, with loss-of-function studies demonstrating reduced activity upon MALAT1 depletion⁴⁷. Additional EV populations, including iPSC-MSC-Exos and UCB-derived EVs, have been shown to increase collagen synthesis (types I and III), elastin production, and fibronectin expression, while promoting fibroblast proliferation and migration in a dose-dependent manner^37,48. Collectively, these effects contribute to matrix deposition and wound closure; however, excessive fibroblast activation may also contribute to fibrosis if not properly regulated.

Vascular regulation and angiogenesis

Neovascularization is essential for oxygen delivery, nutrient supply, and support of proliferative processes during wound repair. SC-EVs promote angiogenesis through both direct effects on endothelial cells and indirect effects mediated by macrophage signaling. M2-Exos enhance VEGF production and endothelial tube formation, potentially through activation of mTOR-dependent pathways⁴⁹. Other EV populations, including iPSC-MSC-Exos and AMSC-Exos, stimulate endothelial cell proliferation, migration, and tube formation, driven in part by pro-angiogenic cargo such as lncRNAs (e.g., PANTR1, H19, OIP5-AS1) and growth factor signaling^50,51. UCB-derived EVs further enhance angiogenesis through miR-21-3p–mediated inhibition of PTEN and SPRY1, promoting endothelial activation. The application of UCB-Exos significantly accelerated overall wound closure compared to controls. However, analysis of wound contour and histopathology indicates that much of this closure in the treated group was attributable to increased contraction rather than enhanced re-epithelialization as shown in Fig. 2 (Fig. 2A, B for macroscopic closure, Fig. 2C for histology). Thus, while these data support an effect on healing kinetics (Level 3), they do not provide evidence for improved regenerative healing or reduced scarring⁵². In another study, miR-146a-enriched hASC secretome significantly enhanced HUVEC proliferation, migration, and tube formation, consistent with increased pro-angiogenic activity. This secretome was enriched in pro-angiogenic growth factors, including HGF, FGF-1, FGF-2, and uPA, and contained relatively lower levels of anti-angiogenic miRNAs⁴⁴.

Fig. 2. UCB-Exos promote cutaneous wound healing in mice.

A Representative macroscopic images of wounds treated with UCB-Exos or PBS on days 2, 5, and 8 post-injury. B Wound closure rates following different treatments (n = 10 per group). C H&E-stained sections of wounds treated with UCB-Exos or PBS at day 8 post-operation, with black arrows indicating scar edges (scale bar: 500 μm). D Quantification of scar width and re-epithelialization (n = 3 per group). E Masson’s trichrome staining of wound sections from both treatment groups. *P < 0.05 versus PBS (control). The figure has been reproduced/adapted from an open-access publication distributed under the Creative Commons Attribution (CC BY-NC) license from Hu et al.⁵².

Collectively, across immune, stromal, and vascular compartments, SC-EVs consistently modulate signaling pathways associated with wound repair, including PI3K/Akt, MAPK, Notch, and TGF-β signaling. These effects collectively contribute to improved wound closure and histological organization in preclinical models. However, the majority of evidence reflects modulation of intermediate biological processes (Levels 3–5), and direct linkage to clinically meaningful outcomes, such as durable tissue function, scar reduction, or recurrence prevention (Levels 1–2) remains insufficiently established. A key limitation in interpreting these findings is the absence of clearly defined thresholds that link improvements at intermediate biological levels to clinically acceptable outcomes. Establishing such criteria will be essential for determining whether modulation of Levels 3–5 is sufficient to achieve meaningful therapeutic benefit at Levels 1–2.

Inflammation modulation and scar reduction

While section “Modes of action in tissue repair and regulation of wound microenvironment” describes the underlying cellular and molecular mechanisms, the functional relevance of these effects lies in their ability to influence higher-order outcomes such as inflammation resolution and scar quality (Level 2). Effective wound healing requires not only timely resolution of inflammation but also controlled tissue remodeling to minimize fibrosis and scar formation. While SC-EVs modulate immune and stromal pathways at intermediate levels, their functional relevance is best evaluated through their impact on inflammatory resolution and scar quality.

SC-EVs attenuate inflammation by reducing the persistence of pro-inflammatory mediators⁴⁵. In parallel, EV-mediated effects on endothelial activation, such as reduced expression of adhesion molecules (VCAM-1, ICAM-1) and inflammatory cytokines (e.g., MCP-1) in activated endothelial cells suggest a broader role in limiting leukocyte recruitment and sustaining inflammatory cycles⁴⁴. In the context of tissue remodeling, multiple EV populations have been shown to influence fibroblast-to-myofibroblast transition and ECM organization. For example, uMSC-Exos reduce myofibroblast accumulation and scar formation in preclinical models through downregulation of the TGF-β2/SMAD-2 signaling axis, mediated in part by exosomal miRNAs such as miR-21, miR-23a, miR-125b, and miR-145⁴². Similarly, MSC-derived EVs regulate collagen dynamics by promoting early deposition while limiting excessive accumulation at later stages, thereby influencing scar architecture⁵³. Additional studies demonstrate that MSC-EVs and UCB-derived EVs modulate expression of key fibrosis-associated genes, including Acta2, Pdgfra, and Engrailed-1, while shifting collagen balance toward a more regenerative profile (increased collagen III and TGF-β3, reduced collagen I and TGF-β1)⁴⁸. Spatial transcriptomic analyses further support these findings, indicating coordinated suppression of profibrotic TGF-β signaling alongside enhancement of Wnt-associated pathways that favor tissue repair over fibrosis⁵⁴.

Despite these promising findings, most evidence is derived from small animal models, where reduced scar width and improved histological organization do not necessarily correspond to restoration of mechanical strength, functional integrity, or long-term outcomes. As such, while SC-EVs clearly influence pathways associated with inflammation resolution and scar modulation (Level 2), their ability to achieve clinically meaningful improvements in tissue function and long-term remodeling (Level 1) remains to be established. Addressing this gap will require studies designed to meet predefined clinical endpoints, including restoration of durable tissue function, reduction in long-term scar burden, and prevention of recurrence. This will necessitate longitudinal in vivo models and early-phase clinical trials that go beyond wound closure metrics and incorporate functional and structural outcome measures. Thus SC-EV-mediated modulation of wound biology predominantly operates at Levels 3–5 and partially at Level 2 but does not yet constitute sufficient evidence for consistent Level 1 clinical benefit.

SC-ANVs

Building on the biological effects described for SC-EVs (section “SC-EVs in wound healing”), SC-ANVs have been developed as engineered systems designed to overcome key translational limitations while attempting to preserve therapeutic functionality.

Definition and fabrication methods

SC-ANVs are synthetically generated nanoscale vesicles (50–400 nm) designed to replicate key structural and functional properties of natural SC-EVs⁵⁵ while addressing limitations related to scalability, yield, and compositional control. In contrast to biologically secreted EVs, SC-ANVs are produced through controlled physical or mechanical processes, enabling substantially higher production yields (approximately 70–250-fold greater than natural exosomes) and tunable cargo loading. This design flexibility is particularly relevant for translation, where reproducibility, dose standardization, and manufacturing scalability remain major barriers for EV-based therapies. Functionally, SC-ANVs are engineered to encapsulate bioactive cargo including growth factors (e.g., VEGF, FGF-2), nucleic acids (miRNAs, mRNAs), and enzymes within a lipid bilayer that may retain selected membrane proteins derived from parent stem cells (e.g., CD9, CD63, CD81)⁵⁶. Through this, they aim to reproduce key paracrine signaling functions of stem cells while enabling greater control over composition and dosing. These engineered systems have emerged in part to address intrinsic limitations of natural EVs, including low yield, heterogeneity in cargo composition, and challenges in isolation and purification, which constrain reproducibility and large-scale clinical translation⁵⁷. However, unlike living cells, SC-ANVs lack adaptive biofeedback mechanisms, raising important questions about whether static cargo delivery is sufficient to regulate complex and dynamic wound environments.

Several fabrication strategies have been developed to generate SC-ANVs. Serial extrusion remains the most widely used method, involving mechanical disruption of cells through polycarbonate membranes with progressively smaller pore sizes (e.g., 10 μm to 400 nm), resulting in reassembly of membrane fragments into vesicles. This approach produces high particle yields (e.g., ~71-fold increase for iPSC-derived SC-ANVs) and is typically followed by density gradient ultracentrifugation for purification⁵⁸. Microfluidic approaches generate vesicles by subjecting cells to controlled shear stress within microchannels (5000–20,000 s⁻¹), producing more uniform vesicles (~120 nm) and offering potential for scalable, GMP-compatible production. Alternative methods such as freeze-thaw cycling and sonication can also generate vesicles but are associated with limitations including protein denaturation and heterogeneity in vesicle size and cargo integrity.

Importantly, SC-ANVs generally exhibit favorable physicochemical properties, including colloidal stability (zeta potential < −15 mV), and can retain functional biomolecules derived from parent cells⁵⁸. Despite these advantages, the extent to which engineered vesicles can reproducibly match the functional complexity and therapeutic efficacy of natural SC-EVs or stem cells in vivo remains to be established. As such, their development should be evaluated not only in terms of production efficiency but also in their ability to meet defined biological and clinical performance thresholds. Establishing whether these engineered advantages translate into reproducible improvements in clinically meaningful outcomes (Levels 1–2), rather than simply enhanced production efficiency or intermediate biological effects (Levels 3–5), remains a key unmet requirement.

Cargo loading and surface engineering

Cargo loading and surface engineering are central to the design of SC-ANVs, as they directly determine the ability of these vesicles to modulate specific biological processes within the wound microenvironment (Levels 3–5) and, ultimately, influence therapeutic outcomes. In contrast to natural EVs, SC-ANVs offer the advantage of tunable composition, enabling targeted delivery of defined bioactive molecules. This controllability is particularly relevant for addressing key barriers in chronic wounds. Cargo encapsulation strategies can be broadly classified as endogenous or exogenous⁵⁹. Endogenous loading involves genetic modification of parent stem cells to overexpress specific therapeutic molecules such as miRNAs (e.g., miR-126-3p) or proteins (e.g., IL-10), which are subsequently incorporated into SC-ANVs during vesicle formation. This approach enables efficient and biologically relevant cargo packaging; however, it requires extended cell culture and may introduce cellular stress or toxicity⁶⁰. In contrast, exogenous loading introduces therapeutic cargo into preformed vesicles, offering greater flexibility in cargo selection and dosing. Techniques such as electroporation generate transient membrane pores (100–500 V) to facilitate entry of nucleic acids (siRNA/mRNA), although vesicle aggregation can occur. Sonication transiently disrupts vesicle membranes to allow incorporation of small-molecule drugs (e.g., doxorubicin), achieving loading efficiencies of 60–80%. Saponin-assisted permeabilization enables incorporation of hydrophobic compounds (e.g., paclitaxel) but requires careful removal of residual detergent to maintain vesicle integrity.

Beyond cargo loading, surface engineering strategies are employed to enhance targeting specificity and retention at the wound site. Similar to endogenous loading, surface functionalization can be achieved through genetic modification of parent cells to express targeting ligands on vesicle membranes⁶¹. Post-fabrication approaches further expand this capability. For example, click chemistry enables conjugation of targeting peptides (e.g., RGD motifs for integrin binding), improving wound localization by approximately 3.5-fold. Aptamer functionalization allows selective targeting of cell-surface receptors (e.g., EGFR on keratinocytes), while membrane hybridization with liposomes incorporating collagen-binding peptides enhances dermal retention. Additional approaches include lactadherin fusion (C1C2 domain–mediated anchoring, >95% efficiency) and palmitoylation-based conjugation of cytokines (e.g., IL-4), enabling stable presentation of bioactive signals on the vesicle surface⁶². Collectively, these strategies enable spatially controlled delivery and improved targeting of therapeutic cargo, with the potential to enhance efficacy while reducing off-target effects. Defining optimal combinations of cargo, targeting strategy, and dosing will be critical for demonstrating proof of concept in vivo. Importantly, these design strategies must be evaluated within application-specific frameworks, where defined biological effects at intermediate levels are quantitatively linked to clinically acceptable outcomes. Whether increased precision in cargo delivery and targeting translates into consistent improvements in higher-order clinical outcomes (Levels 1–2) remains to be established.

Functional evidence in wound models

SC-ANVs have demonstrated therapeutic activity across multiple preclinical wound models, including diabetic, burn, aged, and infected wounds. These studies consistently show modulation of key biological processes at intermediate levels (Levels 3–5), particularly inflammation, angiogenesis, and cellular function. However, as with SC-EVs, the critical question remains whether these effects are sufficient to achieve clinically meaningful outcomes (Levels 1–2), such as durable tissue restoration and reduced scarring.

Modulation of angiogenesis and inflammation in diabetic wounds

In diabetic wound models, SC-ANVs have been engineered to address impaired vascularization and persistent inflammation, the two central barriers to healing. For example, MSC-derived SC-ANVs loaded with miR-126-3p promoted angiogenesis by inhibiting SPRED-1 and activating ERK/VEGF signaling, resulting in increased capillary density (CD31⁺ cells) and accelerated wound closure compared to controls^63,64. In parallel, delivery of miR-146a reduced pro-inflammatory cytokines (TNF-α and IL-6) through inhibition of the TLR4/NF-κB pathway. While these findings demonstrate effective modulation of vascular and inflammatory pathways (Levels 4–5), the primary endpoint remains accelerated wound closure. Evidence linking these changes to improved tissue quality, long-term function, or recurrence prevention (Levels 1–2) is not established.

Effects on epithelial repair and scar modulation in burn models

In burn wound models, umbilical cord MSC-derived SC-ANVs enhanced keratinocyte migration and increased expression of growth factors such as FGF-7 and VEGF, leading to reduced healing time. In addition, miR-29b-loaded SC-ANVs downregulated collagen-associated genes (COL1A1, COL3A1) in fibroblasts, resulting in reduced scar formation⁶⁵. While their ability to meaningfully improve scar quality at the clinical level remains uncertain, these studies suggest that SC-ANVs can influence both re-epithelialization and matrix remodeling. Moreover, scar reduction is typically assessed through histological or molecular endpoints, without corresponding evaluation of mechanical strength, elasticity, or long-term tissue function.

Reversal of cellular dysfunction in aged and chronic wounds

In aged or chronic wound models, SC-ANVs have been used to target cellular senescence, a key contributor to impaired repair. iPSC-derived SC-ANVs reduced expression of senescence markers (e.g., p16ᴵᴺᴷ⁴ᵃ) and increased SIRT1 activity, leading to decreased senescent fibroblast populations and restoration of collagen synthesis⁶⁶. Although these findings indicate reversal of cellular dysfunction (Level 5), it remains unclear whether such effects translate into sustained improvements in tissue organization, functional integrity, or resistance to re-injury in vivo.

Antimicrobial activity and biofilm disruption

SC-ANVs have also been engineered for antimicrobial applications. However, whether such antimicrobial effects translate into improved healing trajectories or reduced recurrence rates in complex wound settings remains to be determined. For example, vancomycin-loaded SC-ANVs functionalized with lysostaphin demonstrated effective eradication of MRSA biofilms, reducing bacterial load by several orders of magnitude. These findings highlight the potential of SC-ANVs to address infection-related barriers to healing, which are particularly relevant in chronic wounds⁶⁵. In addition to engineered antimicrobial loading strategies, neutrophil-derived ANVs have been explored as innate immune–mimetic systems that deliver bactericidal proteins such as lysozymes, thereby directly targeting wound-associated infections⁶⁷.

Pharmacokinetics, targeting, and in vivo retention

Pharmacokinetic analyses indicate that engineered SC-ANVs can achieve improved retention at wound sites. For instance, RGD-modified SC-ANVs exhibited prolonged wound localization compared to unmodified vesicles, with detectable distribution across the liver, spleen, and wound tissue following systemic administration⁶⁸. While these findings support enhanced delivery efficiency, the relationship between biodistribution profiles and therapeutic efficacy has not been clearly established.

LncRNA-based therapeutic delivery and limitations of current models

Targeted delivery of long noncoding RNAs represents another emerging application. In diabetic wounds, LncRNA-H19–loaded EV-mimetic nanovesicles (H19-EMNVs) restored angiogenic signaling and improved wound closure under hyperglycemic conditions (Fig. 3)⁶⁹. However, detailed histological analysis (Fig. 3) revealed that wound closure was largely driven by contraction, with incomplete re-epithelialization and ongoing dermal remodeling at the study endpoint. These findings highlight a recurring limitation across preclinical studies: while vesicle-based therapies accelerate closure kinetics (Level 3), they do not consistently demonstrate full-thickness skin regeneration or restoration of normal tissue architecture.

Fig. 3. ^H19EMNVs accelerate diabetic wound healing.

a Representative photographs of full-thickness skin defects in diabetic rats, either untreated (control) or treated with SAH, SAH-²⁹³EMNVs, SAH- ^H19EMNVs, or SAH- ^H19EMNVs combined with RI (BMS-754807), captured at 0-, 3-, 7-, and 14-days post-surgery. Scale bar: 10 mm. b Quantification of wound closure (%) for the corresponding groups at 3, 7, and 14 days. *p < 0.05 versus control; #p < 0.05 between SAH- ^H19EMNVs and SAH-²⁹³EMNVs. c H&E-stained sections of wounds from each group at day 14. The full image width represents the initial defect size (1.8 cm), and white arrows indicate the length of complete full-thickness wound healing. Scale bar: 2 mm. d Measurement of the total length of newly regenerated full-thickness skin in each group at 14 days. *p < 0.05 versus control; #p < 0.05 between SAH- ^H19EMNVs and SAH-²⁹³EMNVs. e Immunofluorescence staining for p-Akt with DAPI nuclear counterstain. Scale bar: 100 μm. f Quantification of p-Akt–positive cells (%) corresponding to (e). *p < 0.05 versus control; #p < 0.05 between SAH- ^H19EMNVs and SAH-²⁹³EMNVs. The figure has been reproduced/adapted from an open-access publication distributed under the Creative Commons Attribution (CC BY-NC) license from Tao et al.⁶⁹.

Safety-by-design for SC-ANVs

Although most studies on SC-ANVs remain at the preclinical stage³⁷, their clinical translation will require a dedicated safety-by-design framework that systematically defines pharmacokinetics (PK), biodistribution, clearance, and long-term persistence^70,71. These parameters are essential for understanding the in vivo behavior and for establishing dosing regimens that are both effective and safe. Current evidence from EV studies suggests that nanovesicle-based systems generally exhibit low to moderate immunogenicity in preclinical models^72–74. However, it remains inappropriate to directly extrapolate these findings to SC-ANVs due to differences in fabrication methods, cargo loading strategies, and surface engineering approaches. Accordingly, immunogenicity and off-target effects must be evaluated independently rather than inferred from EV data. A major limitation in the current literature is the absence of comprehensive PK and toxicology datasets specific to SC-ANVs. This includes incomplete characterization of tissue distribution, degradation kinetics, and long-term retention in key organs such as the liver and spleen. These data gaps significantly hinder regulatory assessment and complicate the design of first-in-human studies. Therefore, bridging studies that integrate standardized biodistribution profiling, immunological assessment, and dose–response relationships will be essential before SC-ANVs can progress toward clinical translation.

Therapeutic delivery strategies for SC-ANVs

A major barrier to the clinical translation of SC-ANV-based therapies is the limited efficiency of in vivo delivery, particularly in chronic wounds where impaired vascularization, biofilm formation, and protease-rich environments reduce vesicle retention and bioavailability. Following systemic administration, nanovesicles are rapidly sequestered by the mononuclear phagocyte system, resulting in poor accumulation at the wound site and suboptimal therapeutic exposure⁷⁵. These limitations highlight that therapeutic efficacy depends not only on vesicle design but also on effective delivery and retention within the target tissue. To address these challenges, multiple complementary delivery strategies have been developed that can be broadly categorized into targeted delivery and biomaterial-assisted retention systems.

Targeted delivery approaches aim to improve SC-ANV accumulation at injury sites by enhancing molecular recognition within the wound microenvironment. Surface modification strategies, including conjugation of peptides, antibodies, or aptamers, enable vesicles to bind selectively to receptors that are upregulated during tissue injury and repair⁷⁶. This receptor-mediated targeting increases local concentration at the wound site and reduces off-target biodistribution, thereby improving the efficiency of intermediate biological modulation (Levels 3–5). However, while targeting strategies improve spatial specificity, their effectiveness is still constrained by rapid systemic clearance and the dynamic nature of the wound microenvironment, particularly in chronic wounds with heterogeneous receptor expression. Given these constraints, biomaterial-assisted delivery has emerged as a more robust strategy for improving vesicle retention and sustained activity.

Biomaterial-based delivery systems

Biomaterial-assisted delivery has emerged as a more robust strategy to overcome limitations of rapid clearance and short vesicle half-life. Incorporation of SC-ANVs into hydrogels, electrospun nanofiber scaffolds, or microneedle patches enables the formation of bioactive “living dressings” that localize vesicles at the wound site⁷⁷. For example, hyaluronic acid–based hydrogels incorporating MSC-derived EVs have been shown to enhance epithelial closure and improve wound healing outcomes in diabetic models⁷⁸. In addition to passive retention, biomaterial systems can actively modulate vesicle-matrix interactions. By mimicking ECM components or incorporating binding motifs, these platforms enhance vesicle retention and facilitate controlled interactions with the pathological ECM⁷⁹. This not only improves local bioavailability but may also contribute to the restoration of a more regenerative microenvironment by modulating ECM structure and signaling.

Collectively, these delivery strategies emphasize that therapeutic efficacy of SC-ANVs is not determined solely by cargo composition or intrinsic biological activity, but also by their ability to reach and persist within the target tissue. Despite encouraging preclinical advances, most delivery systems remain at the proof-of-concept stage, and their impact on clinically relevant endpoints such as durable tissue regeneration, scar quality, and recurrence prevention (Levels 1–2) has yet to be systematically demonstrated.

Translational considerations and comparative positioning of SC-ANVs

From a translational perspective, SC-ANVs are positioned as engineered alternatives to SC-EVs, designed to address key limitations including low yield, heterogeneity, and challenges in standardization. They have been developed as engineered alternatives to natural EVs⁵⁷, to mimic EV structure and function while offering improved scalability, tunability, and reproducibility. Unlike EVs, ANVs can be engineered to encapsulate defined therapeutic cargo, target specific tissues, and exhibit enhanced stability and biodistribution profiles^15,80. As such, they represent a complementary platform that may overcome key bottlenecks associated with natural vesicle systems.

Preclinical evidence and translational limitations of SC-EVs and SC-ANVs

A critical evaluation of the current literature indicates that the majority of evidence supporting SC-EVs and SC-ANVs in wound healing is derived from preclinical studies, with effects primarily observed at intermediate regulatory levels (Levels 3–5)^35,81. These studies consistently demonstrate modulation of key biological processes, however, direct evidence linking these mechanistic and tissue-level changes to clinically meaningful outcomes (Level 1), such as restoration of durable tissue function, reduction in amputation rates, or long-term scar quality remains limited⁸². As such, current findings should be interpreted primarily as proof of biological activity rather than definitive therapeutic efficacy. This distinction is essential, as improvements in intermediate processes do not necessarily translate into meaningful clinical benefit^82,83.

Evidence from wound healing models

Preclinical wound studies have shown that SC-EVs can enhance multiple aspects of repair, including inflammation resolution, angiogenesis, re-epithelialization, and ECM remodeling. These effects are most consistently demonstrated in diabetic, burn, and ischemic wound models, where treatment is associated with faster wound closure and improved histological organization^84,85. Similarly, SC-ANVs have shown promising results in early studies. For example, nanovesicle-based systems embedded within biomaterial scaffolds, such as methacrylated hyaluronic acid hydrogels, have been shown to enhance endothelial and fibroblast activity, promote angiogenesis, and improve matrix deposition⁸⁶. In burn models, engineered nanovesicle-containing hydrogels have demonstrated combined antibacterial and regenerative effects through modulation of reactive oxygen species and tissue repair pathways⁸⁷. Despite these encouraging findings, the number of studies specifically evaluating SC-ANVs remains limited compared to SC-EVs, and systematic comparisons between the two platforms are largely lacking.

Cross-disease evidence and implications for regenerative potential

Beyond wound healing, SC-EVs have demonstrated regenerative and immunomodulatory effects across multiple preclinical disease models. These include reduced infarct size and improved functional recovery in ischemic stroke⁸⁸, attenuation of neuroinflammation and demyelination in multiple sclerosis models⁸⁹, antifibrotic and anti-inflammatory effects in pulmonary hypertension⁹⁰, and enhanced tissue regeneration in bone and periodontal disease models^91,92. In reproductive models, EVs have been shown to restore ovarian function through modulation of PI3K/Akt and TGF-β/SMAD signaling pathways⁹³. Early clinical observations, such as the use of umbilical cord MSC-derived EVs in osteoarthritis, further suggest potential translational relevance⁹⁴. While these findings support the broader regenerative capacity of EV-based therapies, their applicability to wound healing should be interpreted cautiously. The biological requirements for tissue repair differ across organ systems, and cross-disease efficacy does not directly establish clinical benefit in cutaneous wound healing.

Limitations of preclinical endpoints

A major limitation in the current literature lies in how wound healing outcomes are quantified. Most studies rely on gross measures such as percentage wound closure or reduction in wound area over time, which reflect overall healing rate but do not distinguish between underlying mechanisms⁹⁵. Specifically, wound closure can occur through two distinct processes: re-epithelialization and wound contraction. Increased contraction may accelerate apparent closure but is often associated with fibrosis and poor functional outcomes⁹⁶. This distinction is particularly relevant in small animal models, where contraction is the dominant mechanism of wound closure⁹⁷. As a result, interventions that improve closure kinetics in preclinical models may not necessarily promote regenerative healing or improved scar quality. To address this limitation, future studies should:

• Quantify re-epithelialization and contraction separately

• Incorporate functional and biomechanical outcome measures

• Evaluate long-term remodeling and recurrence

Such approaches will be essential for establishing whether vesicle-based therapies can meaningfully impact higher-level clinical outcomes (Levels 1–2). Taken together, preclinical evidence supports a consistent role for SC-EVs and SC-ANVs in modulating biological processes relevant to wound repair. However, the current body of literature remains heavily weighted toward mechanistic and histological endpoints. Bridging the gap between these intermediate effects and clinically meaningful outcomes will require: 1. Standardized experimental designs, 2. Improved endpoint selection, 3. Longitudinal and large-animal studies, and 4. Direct comparison between EV and ANV platforms. Until such data are available, SC-EVs and SC-ANVs should be considered promising but still investigational approaches, with their ultimate clinical value dependent on rigorous validation against defined therapeutic benchmarks. This reinforces the central premise of this review that therapeutic success in wound healing must be evaluated not only at the level of biological modulation (Levels 3–5), but through demonstrable improvements in durable functional outcomes (Levels 1–2).

Clinical trials and translational status

SC-EVs

Clinical evaluation of SC-EVs is steadily expanding, with a particular focus on MSC-EVs, owing to their immunomodulatory and regenerative properties. Researchers are particularly focusing on MSC-EVs because of their robust immunomodulatory and regenerative capabilities. Early-phase clinical trials have primarily focused on safety and feasibility rather than definitive efficacy. For example, in a randomized phase I trial (NCT04389385), inhaled MSC-EVs administered to patients with acute respiratory distress syndrome were well tolerated and associated with trends toward reduced inflammatory cytokines and improved oxygenation⁹⁸. Similarly, in a separate trial (NCT04276987) involving patients with severe SARS-CoV-2 pneumonia, aerosolized MSC-EVs demonstrated a favorable safety profile without severe adverse events⁹⁹. While these studies support the clinical safety of EV-based therapies, their relevance to wound healing remains indirect. A pilot study evaluating human umbilical cord MSC-derived EVs (hucMSC-Exos) for chronic cutaneous wounds reported improvements in re-epithelialization and local inflammation^100,101. However, these findings are based on small cohorts and early-stage designs, and do not yet establish efficacy in terms of durable tissue function, scar quality, or recurrence prevention. Across studies, most clinical endpoints remain focused on surrogate or intermediate outcomes, such as wound area reduction or histological improvement, rather than higher-order clinical outcomes (Levels 1–2)^27,102.

From a translational perspective, SC-EVs offer several advantages over cell-based therapies, including reduced risks of tumorigenicity, immune rejection, and complications associated with cell engraftment. In addition, EVs can be engineered to enhance targeting and therapeutic payload delivery¹⁰³. However, substantial challenges remain. These include the need for standardized isolation and purification methods (e.g., ultracentrifugation, size-exclusion chromatography, precipitation), establishment of reproducible potency assays, and control of inter-batch variability arising from donor differences, culture conditions, and manufacturing processes. Biological heterogeneity remains a central barrier, complicating dose-response relationships and mechanistic attribution of therapeutic effects¹⁰⁴. As emphasized by the ISEV, rigorous characterization standards are essential but insufficient on their own; future progress will depend on linking vesicle composition to functional potency and clinical outcomes¹⁰⁵. Regulatory classification further complicates clinical translation. Agencies such as the U.S. Food and Drug Administration and European Medicines Agency are still determining whether EV-based therapies should be categorized as biologics, cell-derived products, or advanced therapy medicinal products (ATMPs). As a result, approval pathways remain heterogeneous and often require case-specific regulatory strategies. Current efforts are focused on developing GMP-compliant production systems, improving vesicle tracking and characterization through multiomics approaches, and integrating engineered or synthetic EV mimetics to enhance consistency and scalability. While these advances position SC-EVs as a promising therapeutic platform, clinical evidence remains limited to early-phase trials. Robust demonstration of efficacy, particularly with respect to functional tissue restoration, scar reduction, and long-term outcomes remains an unmet requirement for widespread clinical adoption^{27,102,106,107}. Collectively, current clinical evidence supports safety and biological activity but falls short of demonstrating that SC-EVs can reproducibly achieve clinically meaningful endpoints in complex wound healing. Table 2 presents the current clinical trial status of some SC-EVs.

Table 2.

Clinical trials involving extracellular vesicle-based products for skin repair and wound healing

Trial ID	Product	Phase/Status	Indication	Reference
NCT02565264	Plasma-derived exosomes	Phase 1/Unknown	Intractable cutaneous wounds	132
NCT04134676	Wharton’s jelly MSC-conditioned medium (containing EVs)	Phase 1/Completed	Chronic wounds	133
NCT04235296	MSC-conditioned medium	Phase 1/Completed	Skin wounds	134
NCT04173650	Commercial MSC-EVs	Phase 1/2 A/Not yet recruiting	Dystrophic epidermolysis bullosa	135
NCT05508191	Adipose MSC-conditioned medium	N.A./Completed	Skin aging	136
NCT05475418	Adipose MSC-exosomes (purified EVs)	Not yet recruiting	Wound healing	137
NCT05004779	MSC-conditioned medium	Not yet recruiting	Hypertrophic scar	138
NCT04326959	Umbilical cord MSC-conditioned medium	Phase 1/2/Unknown	Keloid formation	139

NCT National Clinical Trial, MSC mesenchymal stem cell, EVs extracellular vesicles, MSC-EVs mesenchymal stem cell-derived extracellular vesicles, NA not available.

SC-ANVs

Unlike naturally secreted EVs, which are heterogeneous and yield-limited, SC-ANVs can be generated through controlled fabrication methods, enabling improved scalability, compositional uniformity, and tuneable therapeutic loading. These vesicles are typically produced by mechanically disrupting parent stem cells (e.g., MSCs or iPSCs) using techniques such as extrusion or sonication, resulting in membrane-bound structures that retain key surface proteins and bioactive cargo from the source cells¹⁰⁸. To date, most of the evidence supporting SC-ANVs remains preclinical. In wound models, particularly in diabetic and ischemic settings, MSC-derived ANVs have been shown to enhance angiogenesis, promote collagen remodeling, and accelerate re-epithelialization¹⁰⁹. In addition, engineered ANVs loaded with pro-regenerative miRNAs or cytokines have demonstrated the capacity to modulate inflammation and improve healing kinetics in experimental systems^110,111. However, like SC-EVs, these outcomes are largely confined to intermediate biological and histological endpoints (Levels 3–5), and do not yet establish improvements in clinically meaningful outcomes such as functional tissue restoration, scar quality, or recurrence prevention. At present, clinical translation of SC-ANVs is at an early stage. Registered clinical trials are limited, and most efforts are focused on preclinical optimization and regulatory preparation. Academic and industry groups are actively developing GMP-compliant production pipelines and conducting Investigational New Drug (IND)-enabling studies, including evaluation of PK, biodistribution, and immunogenicity in relevant models. These efforts represent important steps toward clinical application but do not yet provide evidence of therapeutic efficacy in humans.

From a translational standpoint, SC-ANVs offer several theoretical advantages over natural EVs, including higher production yield, reduced batch-to-batch variability, and greater flexibility for cargo loading and surface functionalization. Fabrication approaches such as mechanical extrusion and microfluidic processing enable reproducible large-scale production, supporting their potential for industrial translation¹¹². In addition, ANVs can be engineered with targeting ligands or incorporated into biomaterial-based delivery systems (e.g., hydrogels or scaffolds) to enhance retention and localized release at wound sites. Their cargo profiles, which may include growth factors (e.g., VEGF, FGF-2), anti-inflammatory mediators (e.g., IL-10), and regulatory RNAs, contribute to modulation of angiogenesis, inflammation, and matrix remodeling¹¹³. However, these functional attributes remain largely inferred from preclinical models and require validation in clinical contexts. Regulatory classification of SC-ANVs presents additional complexity. Because these vesicles are derived from biological materials but are artificially engineered, they occupy an intermediate position between biologics and nanomedicine. Regulatory agencies such as the U.S. Food and Drug Administration and European Medicines Agency may classify them as biologics, combination products, or ATMPs, depending on their composition and intended use. This ambiguity necessitates case-specific regulatory strategies, comprehensive safety assessment, and early engagement with regulatory authorities. Key challenges for clinical translation also include ensuring consistency in vesicle size and cargo composition, minimizing inter-batch variability, establishing standardized potency assays, and defining long-term safety and immunogenicity profiles. Emerging approaches, including integration with 3D bioprinting platforms, scaffold-based delivery systems, and bio responsive release technologies, may enhance therapeutic precision and retention at wound sites¹¹⁴. Overall, SC-ANVs represent a highly adaptable and scalable platform with significant engineering advantages over natural EVs. However, their clinical development remains in an early phase, and robust evidence demonstrating efficacy in terms of functional tissue repair and long-term outcomes in wound healing is currently lacking. Nonetheless, assessment of PK, biodistribution, and immune response, for the SC-ANVs is ongoing in translational programs by research groups in Korea, the U.S., and Europe. As such, their clinical value will ultimately depend on demonstrating consistent and clinically meaningful benefit beyond accelerated wound closure.

Emerging strategies to enhance vesicle-based therapeutics

Recent advances in SC-EVs and SC-ANVs have focused on overcoming key translational limitations, including low yield, limited retention, and variable therapeutic potency. These strategies aim not only to enhance biological activity at the cellular level but also to improve delivery efficiency and reproducibility, thereby bridging the gap between mechanistic effects (Levels 3–5) and clinically meaningful outcomes (Levels 1–2).

Preconditioning and cargo optimization

Pre-isolation conditioning of parent cells has emerged as an effective strategy to enhance EV yield and functional cargo composition. For instance, hypoxic culture conditions or exposure to inflammatory cytokines (e.g., TNF-α) can enrich EVs with pro-angiogenic and immunomodulatory factors, including specific miRNAs and growth factors^115,116. In parallel, genetic or biochemical approaches enable selective enrichment of therapeutic cargo, such as miR-21-5p or miR-126-3p, which enhance angiogenic and reparative signaling in recipient cells. Exogenous cargo loading strategies including electroporation and transfection allow incorporation of defined molecules such as growth factors (e.g., FGF-2, EGF) or regulatory RNAs to augment specific regenerative pathways. While these approaches improve targeting individual mechanisms, their impact on integrated tissue-level outcomes remains to be established.

Surface engineering and targeted delivery

Surface functionalization of vesicles has been explored to improve localization and retention within the wound microenvironment. For example, collagen-binding peptides such as SILY enhances ECM affinity and prolongs vesicle residence time at injury sites. Similarly, incorporation of targeting ligands or membrane modifications enables selective interaction with specific cell populations, including endothelial cells and fibroblasts. These approaches are often combined with biomaterial-based delivery systems. Hydrogel–vesicle composites, particularly those based on chitosan or hyaluronic acid, provide sustained release and protect vesicles from degradation, thereby extending their therapeutic window^116,117. Integration with decellularized ECM scaffolds further supports vascularization and modulates matrix remodeling¹¹⁶.

Scalable manufacturing and vesicle mimetics

A major limitation of natural EVs is their low yield and batch variability. To address this, engineered vesicle mimetics including extruded nanovesicles have been developed through mechanical fragmentation of cell membranes. These vesicles can achieve substantially higher production yields while retaining key functional properties of natural EVs^33,118,119. Similarly, synthetic vesicles incorporating stem cell–derived membranes or exosomal proteins provide an alternative platform for controlled cargo delivery^116,120. Advances in 3D culture systems, such as MSC spheroids, further enhance EV production efficiency and consistency compared to traditional monolayer cultures^116,121. Improved purification techniques also contribute to reduced batch variability and enhanced reproducibility.

Combination therapies and multifunctional platforms

Emerging strategies increasingly focus on combining vesicle-based therapies with additional functional components to address the multifactorial nature of chronic wounds. For example, vesicles loaded with antimicrobial peptides or metallic nanoparticles (e.g., silver) can simultaneously target infection and promote tissue repair. Similarly, co-delivery of small-molecule drugs such as metformin enables concurrent modulation of systemic factors (e.g., hyperglycemia) and local wound processes⁶⁹. These multifunctional platforms highlight the potential of vesicles as integrated therapeutic systems capable of addressing multiple pathological axes, including inflammation, infection, and impaired angiogenesis.

Despite these advances, most emerging strategies remain focused on enhancing mechanistic endpoints, such as angiogenesis, cell proliferation, or inflammatory modulation. Evidence demonstrates that these innovations translate into improved functional outcomes such as durable tissue integrity, reduced scarring, or prevention of recurrence remains limited. In addition, increased engineering complexity may introduce new challenges related to scalability, regulatory approval, and safety.

Discussion

SC-EVs and SC-ANVs represent promising cell-free therapeutic platforms for regenerative wound healing by recapitulating key aspects of stem cell paracrine signaling. These vesicles carry bioactive cargo, including proteins, nucleic acids, and lipids, that modulate angiogenesis, immune responses, and ECM remodeling. In preclinical models, they consistently enhance intermediate biological processes (Levels 3–5), including inflammation resolution, endothelial activation, and fibroblast function, ultimately accelerating wound closure and improving histological organization. Despite these encouraging findings, several translational gaps remain before vesicle-based therapies can be considered clinically definitive.

Heterogeneity of wound biology limits one-size-fits-all strategies

A key limitation is the biological heterogeneity of chronic wounds. Diabetic foot ulcers, venous leg ulcers, pressure injuries, and burn wounds share overlapping dysregulated pathways but differ significantly in dominant pathological drivers across Levels 3–5. As a result, a universal vesicle formulation is unlikely to be effective across all indications. More plausible is the development of indication-specific vesicle designs, such as pro-angiogenic and anti-inflammatory formulations for ischemic diabetic ulcers, and antifibrotic, keratinocyte-supportive vesicles for burn wounds¹²².

Limitations of preclinical wound models

A substantial portion of reported efficacy is derived from rodent models, particularly diabetic mouse and rat systems. These models rely heavily on the panniculus carnosus, leading to wound closure predominantly via contraction rather than true tissue regeneration. Consequently, improvements in wound area reduction may overestimate regenerative efficacy. Historically, therapies that performed well in these models have often failed to reproduce similar outcomes in human chronic wounds³⁵. This highlights the need for standardized evaluation metrics that distinguish contraction-driven closure from true re-epithelialization and tissue restoration.

Lack of dynamic biological feedback in vesicle-based therapies

Unlike living stem cells, SC-EVs and SC-ANVs lack intrinsic biological adaptability. Stem cells can sense inflammatory, hypoxic, and mechanical cues and dynamically adjust their secretome accordingly, whereas vesicles represent a fixed snapshot of cellular output. This absence of feedback regulation imposes a significant design constraint, requiring external engineering of cargo composition, dosing, and release kinetics to approximate dynamic biological responses¹⁵.

Role of SC-EVs and SC-ANVs within regenerative architectures

Clinical experience in wound care suggests that structural scaffolds, particularly decellularized matrices and biomimetic dressings, are currently among the most effective strategies for addressing Level 1 clinical deficits such as loss of tissue architecture and vascular support¹²³. In contrast, bioactive factors delivered in poorly structured or ischemic wound beds often demonstrate limited efficacy. Therefore, SC-EVs and SC-ANVs are more likely to achieve clinical success when used as adjuncts within scaffold-based or cell-based systems, rather than as standalone therapies¹²³.

Positioning relative to cell-based therapies

A key translational advantage of SC-EVs and SC-ANVs is their “off-the-shelf” nature, enabling immediate availability and reduced risk of tumorigenicity, immune rejection, and engraftment failure compared to stem cell transplantation. However, autologous stem cell approaches remain biologically closer to native regenerative processes, albeit with higher cost, complexity, and processing time. Determining the optimal positioning of vesicle-based versus cell-based therapies will require direct comparative clinical trials^124,125.

Conceptual insight from fetal wound healing

Fetal skin regeneration provides an important conceptual benchmark for scarless healing. Unlike adult wounds, fetal wounds heal with minimal inflammation, reduced fibrosis, and, in some cases, regenerative restoration of complex structures. This difference is attributed to higher cellular plasticity and distinct inflammatory signaling environments¹²⁶. SC-EVs and SC-ANVs may partially recapitulate aspects of this regenerative signaling; however, as acellular systems, they lack the full feedback-driven coordination of living tissues. As a result, they are unlikely to fully reproduce fetal-like regenerative orchestration on their own¹²⁷.

Intriguingly, beyond stem cell–derived systems, plant-derived exosome-like nanovesicles have emerged as alternative therapeutic platforms. These vesicles offer advantages including low immunogenicity, high stability, and accessibility, while modulating inflammatory pathways and promoting tissue repair through naturally derived bioactive compounds¹²⁸. However, their integration into wound healing paradigms remains at an early stage, and comparative efficacy relative to mammalian vesicle systems is not yet established. Taken together, SC-EVs and SC-ANVs represent a powerful but biologically constrained class of regenerative therapeutics. Their strengths lie in targeted modulation of intermediate wound-healing processes (Levels 3–5), scalability, and safety. Their limitations stem from lack of biological adaptability, model-system overestimation of efficacy, and insufficient demonstration of robust Level 1 clinical outcomes such as durable functional restoration and scar-free healing. Future progress will depend on integrating vesicle technologies with biomaterial scaffolds, improving indication-specific engineering, and critically designing clinical studies that directly measure functional outcomes rather than surrogate wound closure metrics.

Conclusion

SC-EVs and SC-ANVs have proven to be multifunctional agents that modulate key biological processes involved in wound healing. However, current evidence remains insufficient to establish their effectiveness in addressing definitive clinical outcomes (Level 1), such as prevention of amputation, durable tissue restoration, or long-term reduction in scarring in complex human wounds. The majority of available data derive from rodent models in which wound closure is predominantly driven by contraction rather than true regeneration⁹⁵, and clinical studies remain limited in size, heterogeneity, and focus on hard functional endpoints³⁵. While vesicle-based approaches offer clear advantages over cell-based therapies including reduced risks of uncontrolled proliferation, immune rejection, and ectopic engraftment, they introduce new translational challenges related to delivery efficiency, dosing standardization, biological variability, and loss of intrinsic cellular feedback mechanisms¹²⁹. These limitations underscore that SC-EVs and SC-ANVs are not yet standalone solutions but rather emerging components of a broader regenerative toolkit. Future progress will depend on moving beyond proof-of-mechanism toward clinically grounded validation. This includes the development of indication-specific vesicle designs, rigorous linkage between molecular changes (Levels 3–5) and functional outcomes (Level 1), and well-controlled early-phase clinical trials that prioritize tissue function, scar quality, and recurrence rather than wound closure alone³⁵. Within this framework, SC-EVs and SC-ANVs are most likely to achieve clinical impact as part of combination strategies integrating biomaterial scaffolds and, where appropriate, cell-based therapies¹²⁹. Ultimately, whether these vesicle-based systems can transition from promising biologics to clinically transformative therapies will depend on their successful integration into standardized, mechanism-driven, and outcome-focused regenerative medicine frameworks^68,130,131.

Author contributions

R.L.R., A.O., and R.B. contributed equally to this work. R.L.R., A.O., and R.B. conceptualized and designed the study, performed the literature review, and co-wrote the original draft. S.K.J., A.M., M.K., and S.B. contributed to the literature survey, manuscript editing, figure preparation, and data interpretation. A.M., A.P., M.S., S.G., A.D., and K.N.A assisted in manuscript editing and critical review of the content. E.J.O. and K.Y.C. contributed clinical & translational insights acquired the funding and assisted in manuscript revision. P.G. supervised the study design, reviewed the manuscript, acquired the funding, and provided intellectual input. H.Y.C. and B.-C.A. provided overall supervision, final manuscript editing, and project administration.

Data availability

No new data were generated or analyzed in this review. Data sharing is not applicable to this article.

Competing interests

The authors declare no competing interests.

Declaration of Generative AI and AI-Assisted Technologies in the Writing Process

An AI-based tools (Wordvice AI or ChatGPT by OpenAI, GPT-5.2, accessed April 2026) was used under author supervision in a limited manner to improve English expression, clarify structure, and adjust word count. The authors reviewed and edited the content independently and took full responsibility for the final manuscript.