Stem cell-derived exosomes in wound healing: mechanistic insights and delivery strategies

ABSTRACT

Mesenchymal stem cells (MSCs) contribute significantly to wound healing due to their ability to self-renew, modulate immune responses, and differentiate into various cell types. However, challenges such as unpredictable growth, limited vascular transport efficiency, stringent storage and maintenance requirements that limit the widespread clinical use of MSC-based therapy, highlighting the need for developing effective cell-free alternatives. The regenerative effects of MSCs are mediated through paracrine signaling, primarily via their secretome, which includes extracellular vesicles and soluble factors, especially exosomes. Compared to MSC therapy, exosomes provide superior benefits in terms of storage, safety, and efficiency in targeting the wound sites due to their enhanced tissue penetration capabilities. However, a specific aspect that remains underexplored in exosome-based therapy for wound healing is the development of optimized delivery systems, to ensure controlled, sustained release and precise localization of the exosomes at the wound sites. This review uniquely focuses on this critical and emerging area, providing a detailed overview of the current advancements and limitations in exosomes-based wound healing therapies, with a focus on their delivery strategies. The insights presented in this review are expected to accelerate the development of innovative, effective treatments, revolutionizing wound care management and advancing regenerative medicine in clinical practice.

GRAPHICAL ABSTRACT

1. Introduction

Skin, being the outermost layer of the human body, is one of the largest organs serving as a barrier against external pathogens while regulating immunity, metabolism, and sensory perception [1]. Despite its major role in protection, skin remains highly susceptible to damage from intrinsic pathological conditions and external mechanical forces, particularly incision, pressure, and burn injuries, skin ulcers and chronic wounds. Wound healing is a multistage process involving coagulation, inflammation, proliferation and remodeling [2], regulated by various cell types, cytokines and growth factors. Disruption in these stages can lead to excessive scar formation and impaired tissue regeneration. Chronic wounds make this process more difficult and present treatment problems, especially in individuals with diabetes, ulcers, and burns [2,3]. According to new statistics from the 2022 Wound compendium, 10.5 million Americans with Medicare were impacted by chronic non-healing wounds in 2019—a rise of 2.3 million from 2014. The paper also states that the burden is expected to increase because of aging populations and rising rates of infection, diabetes, and obesity [4].

Conventional wound treatment includes debridement, infection control, vascular support, and offloading strategies. Even though these treatments provide temporary relief, ongoing infection and inflammation, particularly in chronic wounds, necessitate repeated treatments. Treatment is made more difficult by the emergence of microorganisms that are resistant to drugs and by poor angiogenesis [2].

Skin grafts and artificial replacements have been shown to be promising advancements [5–7], however, issues like graft rejection, high cost, fragility, and limited applicability reduce clinical utility [8–10]. Although there is promise in emerging technologies like 3D bioprinting [11], vascularity and scalability issues still exist.

Regenerative medicine offers new possibilities, with stem cells playing a crucial role because of their differentiation capacity and immunomodulatory activities [2]. Adipose tissue, bone marrow, and umbilical cord mesenchymal stem cells (MSCs) have been investigated extensively for their potential to repair wounds [12]. They secrete growth factors such as IGF-1, VEGF, TGF-β, MMP-1, and KGF that promote macrophage polarization, ECM remodeling, and angiogenesis [13]. Additionally, MSCs promote vascularization by means of proteins like EGFL6 via ERK signaling and growth factors like VEGF, HGF, PDGF, and bFGF [5].

Fibroblast growth factor (FGF), epidermal growth factor (EGF), and the newly discovered peptide RL-QN15 are examples of bioactive peptides that have long been known to be effective in promoting wound healing because of their roles in angiogenesis, migration, and cell proliferation [14]. These peptides belong to a well-known class of regenerative medicine. A promising cell-free method in this larger therapeutic landscape is stem cell-derived exosome treatment, which offers benefits including improved stability, immunomodulation, and targeted intercellular communication in addition to delivering bioactive compounds like peptides. There are a lot of promises for improving wound healing results by combining exosome therapy with peptide-based therapies.

MSCs also secrete indoleamine-2, 3-dioxygenase (IDO), prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α)-stimulated gene 6 (TSG-6), modulating immune responses and promoting scar-free healing [12]. Several studies have demonstrated the therapeutic application of MSCs in wound healing, wherein the local injection of MSCs into wounds and surrounding tissues has shown promising results in promoting tissue repair [15–18].

However, their clinical application is limited by issues like tumorigenesis, immune risks, in vitro expansion, and lack of standardization [19–21]. Culture and storage conditions can affect MSC survival.

Given these challenges, MSC-derived extracellular vesicles (EVs), particularly exosomes, have emerged as a safe and promising alternative. These cell-free nanovesicles retain the regenerative and anti-inflammatory characteristics of MSCs with no associated risks of live cell transplantations [22]. They serve as the primary mediators of MSC paracrine signaling and carry a wide range of bioactive molecules, including proteins, lipids, and nucleic acids that contribute to tissue repair. They offer several advantages including enhanced scalability, reduced immunogenicity and lower chances of tumor formation. Additionally, they can be tailored for specific therapeutic applications, further enhancing their clinical relevance. Moreover, MSC-derived exosomes do not require any custom culturing, making them practical, cost-effective, and easier to store and transport. While MSCs show promise in wound healing, their clinical use is restricted due to delivery and maintenance problems. Exosomes, as cell-free derivatives, provide a safer and more efficient alternative, controlling repair processes via tailored signaling. This review emphasizes the therapeutic potential of exosomes and the need for optimized delivery mechanisms to improve their clinical applicability in wound care. The delivery techniques for MSC-derived exosomes in wound healing are the particular focus of this study to address a major gap in the field. Despite its essential significance in therapeutic effectiveness, this issue is still little understood. Although the regeneration properties of MSCs and their exosomes have been extensively studied, only a few studies have thoroughly investigated the ways in which delivery techniques affect therapy results. Through an analysis of current developments in exosome-based treatments, with an emphasis on delivery methods including hydrogels, bioprinting, and scaffold coatings, this study offers fresh perspectives that can direct the development of more efficient, focused, and clinically applicable wound healing techniques. These insights have the potential to improve patient outcomes and advance regenerative medicine practices.

2. Exosomes

2.1. Structure

All prokaryotic and eukaryotic cells, in both normal physiology and disease states, release EVs. EVs, which have been isolated from body fluids, including serum, and conditioned cell media, consist of closed membrane vesicles that vary in size, shape, composition, and biogenesis. According to the guidelines of the International Society for Extracellular Vesicles (ISEV) 2018, extracellular vesicles (EVs) are naturally secreted, lipid bilayer-enclosed particles that lack the capacity for replication and do not contain a functional nucleus [23].

EVs comprise various subtypes, including exosomes, microvesicles, and apoptotic bodies, with diameters typically ranging from ~30 nm to 5000 nm. The production and classification of EVs are illustrated in Figure 1 [2,23]. Exosomes (30-160 nm) are small vesicles formed when multivesicular structures comprising intraluminal vesicles fuse with the plasma membrane. Microvesicles (0.1–1.0 μm) are shed by the outward blebbing of the plasma membrane. The largest EVs, apoptotic bodies (1–5 μm), are produced during the late phases of apoptosis [25,26]. Among extracellular vesicles, exosomes – small vesicles of endosomal origin – represent a key subtype involved in intercellular communication and the regulation of various cellular processes [23,27].

Figure 1.

The figure illustrates the production and assembly process of extracellular vesicles (EVs), highlighting different types of EVs, including apoptotic bodies, microvesicles, and exosomes, along with their distinct origins and sizes. The major stages of EV formation are depicted, including the budding of vesicles from the plasma membrane and the release of intraluminal vesicles (ILVs) from endosomal compartments. This illustration underscores the complexity and functional diversity of EVs in cellular communication and their potential roles in disease. Adapted from Gyorgy et al., 2011, Cell Mol Life Sci [24]. Created with biorender.

2.2. Molecular and biochemical composition of exosomes

Mesenchymal stem cell (MSC)-derived exosomes are nanoscale extracellular vesicles enriched with bioactive molecules that orchestrate wound healing through precise regulation of inflammation, angiogenesis, and tissue remodeling. The molecular composition of MSC-exosomes is diverse and includes proteins, lipids, and nucleic acids. Proteins are a major component, with exosomes typically expressing surface markers like CD9, CD63, and CD81, which are involved in exosome formation, secretion, and uptake by target cells [15]. Additional proteins present include adhesion molecules, antigen presentation proteins (such as MHC class I and II), membrane transport and fusion proteins (e.g., annexins, SNARE complex), and MSC-specific markers like CD29, CD73, CD90, CD44, and CD105 [28]. These proteins not only serve as biomarkers but also facilitate exosome maturation, immune modulation, and targeted delivery of cargo. Their composition includes nucleic acids, proteins, and lipids, which synergistically activate regenerative pathways in target cells. Exosomes derived from bone marrow, umbilical cord, or adipose tissue MSCs exhibit source-specific therapeutic properties, such as enhanced angiogenesis with adipose-derived exosomes (ADSC-exosomes) or superior collagen modulation with umbilical cord-derived exosomes (UCMSC-exosomes) [29,30].

The nucleic acid cargo of MSC-exosomes is equally important. Exosomes carry different forms of RNA e.g., messenger RNA (mRNA), microRNA (miRNA), and long non-coding RNA (lncRNA) [31]. An example of an enriched miRNA in MSC exosomes is miR-16, which targets vascular endothelial growth factor (VEGF). miRNA is known to modulate gene expression of recipient cells, which can lead to certain cellular processes, depending on the receptor cell (i.e., angiogenesis, inflammation, repair). Exosomes do not carry nuclear, mitochondria, endoplasmic reticulum (ER), or Golgi complex proteins further differentiating their cargo from outer forms of cellular debris. Nucleic acids form a critical component, with microRNAs (miRNAs) like miR-21-5p and miR-146a-3p driving angiogenesis by activating VEGFR and PI3K/AKT pathways in endothelial cells. Long non-coding RNAs (lncRNAs), such as H19 and MALAT1, regulate collagen synthesis and reduce scarring by modulating TGF-β/Smad and Hippo/YAP signaling [30]. Exosomal mRNA and tRNA further support protein synthesis in fibroblasts, enabling efficient extracellular matrix (ECM) deposition. Proteins, including VEGF, FGF-2, and TSG-6, directly stimulate endothelial cell proliferation and macrophage polarization toward anti-inflammatory M2 phenotypes, while Wnt4 and IL-10 enhance re-epithelialization and suppress excessive inflammation [2]. Lipids in exosomal membranes not only protect cargo but also facilitate fusion with target cells, ensuring efficient delivery of bioactive molecules. Additionally, exosomes regulate lipid metabolism by enhancing fatty acid utilization for energy during cell migration and modulating prostaglandin pathways to control inflammation [32]. During the inflammatory phase, exosomal miR-223 silences pknox1 to promote M2 macrophage polarization, reducing pro-inflammatory cytokines like TNF-α [33]. In the proliferative phase, ADSC-exosomes accelerate fibroblast and keratinocyte migration via AKT/HIF-1α and Wnt/β-catenin pathways, while VEGF and miR-126-3p stimulate angiogenesis [34,35]. For diabetic wounds, melatonin-primed exosomes improve healing by targeting PTEN/AKT pathways to enhance endothelial function [36]. During remodeling, exosomes balance collagen I/III ratios by suppressing TGF-β1 and upregulating TGF-β3, minimizing scar formation [37]. UCMSC-exosomes further inhibit myofibroblast differentiation via TGF-β2/Smad2 signaling, ensuring organized ECM deposition [38]. The composition and secretion of MSC-exosomes are influenced by various factors, including cell type, confluency, serum conditions, cytokines, and environmental stimuli such as hypoxia or stress. This dynamic regulation allows MSC-exosomes to adapt their cargo to specific physiological or pathological contexts, enhancing their therapeutic potential. MSC-exosomes are internalized by recipient cells through mechanisms such as endocytosis, ligand-receptor interactions, and direct membrane fusion, with integrins and tetraspanins (CD9, CD63, CD81) playing crucial roles in targeting and uptake [39]. Table 1 summarizes the molecular and biochemical composition of MSC-exosomes involved in wound healing.

Table 1.

Molecular and biochemical composition of MSCs-exosomes involved in wound healing.

Component Type	Key Molecules/Markers	Function in Wound Healing	Relevant Pathways/Effects	References
Proteins	CD9, CD63, CD81, TSG101, ALIX, CD73, VEGF, FGF-2, IL-10, TGF-Î2 3, Wnt4, MMP3	Promote angiogenesis, fibroblast proliferation, immunomodulation, ECM remodeling, and anti-scarring effects	Enhance endothelial cell migration, stimulate fibroblast proliferation, regulate immune response, balance collagen deposition, and inhibit myofibroblast differentiation	[40]
Lipids	Ceramides, sphingomyelin, cholesterol, phosphatidylserine, gangliosides	Structural stability, membrane fusion, regulation of lipid metabolism, energy supply for cell proliferation and migration	Modulate lipid mediators (prostaglandins, leukotrienes), enhance membrane biosynthesis and signaling, reduce abnormal scar formation	[41]
Nucleic Acids (miRNA, lncRNA, mRNA)	miR-21, miR-23a, miR-125b, miR-145, miR-223, miR-126-3p, miR-181c, lncRNA H19, MALAT1	Regulate inflammation, angiogenesis, fibroblast migration, collagen synthesis, and scar reduction	miR-223 promotes M2 macrophage polarization; miR-126-3p enhances angiogenesis; lncRNA H19 regulates PI3K/AKT pathway; miR-181c inhibits TLR4 signaling; miR-21/miR-23a/miR-125b/miR-145 inhibit myofibroblast differentiation	[40]
Enzymes/Signaling Molecules	Wnt4, TSG-6, MMP3, PI3K/AKT, ERK/MAPK	Stimulate cell migration, ECM remodeling, and re-epithelialization	Activate Î2 -catenin, regulate MMP3/TIMP1, modulate TGF-Î2/Smad and Hippo/YAP pathways for balanced tissue regeneration	[40]
Other Cargo	tRNA, aminoacyl-tRNA synthetases, mitochondrial mRNA	Support protein synthesis, cellular energy metabolism, and tissue rebuilding	Modulate amino acid metabolism, autophagy, and fibroblast function	[32]

2.3. Sources of therapeutic exosomes for wound healing applications

Exosomes can be secreted by a wide range of tissues and cells, each contributing distinct components and characteristics. Among these, mesenchymal stem cells (MSCs) have emerged as prominent and valuable source of exosomes due to their potent regenerative and immunomodulatory properties. Exosomes derived from various MSC sources, including human umbilical cord MSCs (hUCMSCs), adipose-derived MSCs (ADMSCs), and bone marrow MSCs (BMMSCs), have been extensively studied for their therapeutic potential in wound healing. Additionally, Wharton’s Jelly MSCs (WJ-MSCs), epidermal stem/progenitor cells (EPSCs), and others such as oral mucosal lamina propria-progenitor cells (OMLP-PCs) and amniotic fluid stem cells (hAFSCs) have demonstrated promising wound-healing benefits due to their diverse biological activities. Moreover, skin stem cells have also gained attention as a promising source of exosomes. Beyond stem cells, other contributors include endothelial cells and various immune cells, which can also produce exosomes with unique functional profiles and potential medical applications in wound care [42].

2.3.1. Human umbilical cord mesenchymal stem cells (hUCMSCs)

The human Umbilical cord (hUCMSCs) is a promising source of mesenchymal stem cells. They have rapid self-renewal qualities and are painless to extract and collect, which increases their applicability in both therapeutic and research settings [43]. These MSCs can be isolated from various compartments of the umbilical cord, such as Wharton’s jelly, veins, UC linings, sub amnion and arteries [44]. Compared with other sources of stem cells, hUCMSCs have stronger proliferation, high differentiation potential, enhanced angiogenesis capacity and improved immune-regulatory functions [45]. Due to their numerous advantages, hUCMSCs have emerged as a promising platform for stem cell-based drug delivery systems. Their therapeutic effectiveness is assessed in numerous trials for a variety of diseases [46]. However, exosomes derived from hucMSCs, (hUCMSC-exos) have demonstrated superior effectiveness in wound healing attributed to their anti-fibrotic, anti-inflammatory, anti-apoptotic properties, as well as their ability to promote tissue integration and regeneration [47]. Due to their low tumorigenicity and immunogenicity, hUCMSC-exos are increasingly being recognized as a viable substitute for whole-cell treatment. According to recent research, hUCMSC-exos may improve the healing of chronic wounds by promoting the shift from the inflammatory to the proliferative phase of tissue repair [48].

2.3.2. Adipose tissue-derived mesenchymal stem cells (ADMSCs)

Adipose tissue-derived MSCs (ADMSCs) are unique multipotent cells that can develop into osteocytes, adipocytes, neutral cells, vascular endothelial cells, cardiomyocytes, and pancreatic β-cells, among others, demonstrating multidirectional osteogenic and chondrogenic differentiation [49,50]. For these reasons, ADMSCs have received a lot of attention in regenerative medicine based on their possible therapeutic influence and potential unique biological characteristics, such as self-renewal capacity, high yield, which can be harvested fairly easily, and the propensity to differentiate into a variety of tissue-specific progenitor cells. Similarly, ADMSCs have autochthonous migratory abilities, which allow them to localize to a site of injury, and facilitate repair during both the autocrine and paracrine mechanisms of signaling [51]. ADMSCs can also secrete a large variety of paracrine factors, and extracellular vesicles that promote repair and tissue repair and regeneration [52]. ADMSCs have produced positive results in treating a wide range of conditions such as tissue atrophy, fibrosis, or even radiation-induced ulcers. They have also proven to be effective therapeutics for management of abnormal pathological wound healing, including scar formation by modulating macrophages, immune response, and promoting angiogenesis and epithelialization [53]. In addition to wound healing, ADMSCs have been used for systemic illness, including acute graft-versus-host disease, hematological, and immunologic disorders, including idiopathic thrombocytopenic purpura and refractory pure red cell aplasia [54].

In clinical application, ADMSCs are mainly used in the forms as “ADMSCs-scaffold composite” or in ‘cell-free therapies’ using ADMSC-exosomes or ADMSCs alone [55]. Among these, the cell-free extracts such as ADMSCs-derived exosomes have emerged as superior alternatives to traditional ADMSCs cell-based therapies due to their comparable regenerative effects and reduced immunogenicity [56,57]. Novel approaches such as encapsulating ADMSCs- derived exosomes in pluronic F127 hydrogel, have been shown to accelerate wound healing and tissue regeneration [58], particularly in diabetic and chronic wound models [59,60]. They are increasingly being explored in plastic and cosmetic surgeries, and tissue engineering, opening new avenues in science and research [61,62].

2.3.3. Bone marrow-derived mesenchymal stem cells (BMMSCs)

Bone marrow mesenchymal stem cells are one of the most widely used stem cells in clinical settings. They are multipotent cells isolated from the bone marrow of a donor [63]. They have been widely applied in regenerative medicine, cell therapy and tissue engineering, demonstrating unique therapeutic effects across various indications [64]. Importantly, BMMSCs possess distinct biological characteristics that make them irreplaceable in certain contexts [64]. However, compared to other mesenchymal stem cells (MSCs), such as human umbilical cord-derived MSCs (hUCMSCs) and adipose-derived MSCs (ADMSCs), the extraction of BMMSCs is more complex. They are difficult to isolate, and the collection process involves invasive procedures that pose certain risks. Moreover, BMMSCs represent only about 0.001–0.01% of the total bone marrow population, necessitating extensive in vitro expansion and culturing to obtain sufficient cell numbers for therapeutic applications [65].

Despite these challenges, BMMSCs have demonstrated a strong safety profile in numerous clinical trials and are emerging as promising candidates for cell free therapy. BMMSCs-derived exosomes (BMMSC-exos), which are small extracellular vesicles play a critical role in intercellular communication and tissue repair by delivering a range of bioactive molecules, including proteins and mRNAs, to recipient cells, which in turn help in tissue regeneration and the attenuation of inflammatory responses [66]. Therapeutically, BMMSC-exos have shown efficacy in promoting the repair of skin, bone, and cartilage defects, and in enhancing overall tissue healing [67]. Several studies have highlighted their potential to reduce tissue fibrosis, and aid in tissue regeneration through mechanisms such as anti-inflammation, anti-apoptosis and antioxidant activity. While they were initially recognized for their role in bone regeneration and internal organ repair, recent advancements have confirmed their effectiveness in wound healing as well [68–70].

2.3.4. Wharton’s jelly-derived MSCs (WJ- MSCs)

Wharton’s Jelly is a gelatin-like tissue within the umbilical cord which contains micro fibroblasts-like stromal cells [71]. Wharton’s Jelly-derived MSCs (WJ- MSCs) exhibit a significant advantage over conventionally derived exosomes from adipose tissue and bone marrow tissue. WJ-MSCs have higher expansion and differentiation capacity, superior immune-evasion capabilities and greater ethical advantages, making them a promising treatment option in conditions like graft-versus-host disease, tissue injuries and degenerative disorders [72–74]. Also, WJ-MSCs’ secretome containing exosomes, i.e., WJ-MSC-Exosomes are highly potent in facilitating immunomodulation and tissue repair [75,76].

2.3.5. Epidermal stem cells (EPSCs)

Epidermal stem cells are present in the basal layer of the epidermis and are responsible for skin homeostasis, including differentiation and self-renewal [77]. EPSCs are a multipotent cell type which largely contributes to wound healing and tissue regeneration, garnering considerable interest in the field of regenerative medicine [78]. EPSCs have been shown to promote angiogenesis and accelerate the healing of full-thickness wounds. The key contributor to their therapeutic activity lies in the exosomes they release (EPSCs-derived exosomes) which are responsible for exerting regenerative effects

In the diabetic mouse model, it was found that EpSC-exosomes enriched with miR-200b-3p alleviated the apoptosis of endothelial cells, thereby enhancing angiogenesis and promoting the healing of diabetic wounds [79]. In a study by Ma et al. [80], a bilayer cell patch composed of epidermal stem cells and angiogenesis-enhanced ADMSCs significantly improved diabetic wound healing [80]. Furthermore, EPSC-Exosomes have demonstrated the ability to enhance wound repair, by reducing scar formation and promoting the regeneration of skin appendages, nerve fibers, blood vessels, and deposition of collagen. This effect was achieved through the inhibition of growth factor induced by fibrotic responses and improved cellular communication. This is achieved through the inhibition of growth factor-induced fibrotic responses and improved cellular communication in the wound environment [81].

2.3.6. Fetal dermal MSCs (FD-MSCs)

Fetal dermal MSCs (FDMSCs) isolated from fetal skin, serve as a novel treatment approach in regenerative medicine. FDMSCs secrete exosomes, (FDMSCs-Exos) which are therapeutically potent in promoting wound healing. One of their key mechanisms involves activating the motility and secretory function of adult dermal fibroblasts (ADF) through the Notch signaling pathway, thereby promoting tissue regeneration [82]. In a study by Xia et al. [83] it was demonstrated that FDMSCs induce macrophage polarization from M1 to M2. This shift results in the downregulation of pro-inflammatory markers and upregulation of anti-inflammatory markers, ultimately leading to reduced inflammation and improved tissue repair [83].

2.3.7. Oral mucosa lamina propria-progenitor cells (OMLP-PCs)

OMLP-PCs are present beneath the oral mucosa, also known as lamina propria. They show potent immunomodulatory and antibacterial properties. These cells inhibit the growth of Gram-positive and Gram-negative bacteria, through the secretion of soluble factors like osteoprotegerin (OPG) and haptoglobin (Hp) [84]. The findings suggest that OMLP-PCs are crucial in maintaining bacterial homeostasis and can be used in the treatment of infectious diseases such as bacterial pneumonia and chronic non-healing wounds. Furthermore, OMLP-PCs exhibit low immunogenicity and potent immunosuppressive properties, functioning in both a dose dependent and HLA class II-independent manner. These characteristics make them highly suitable for allogeneic tissue engineering and immunomodulatory therapies [85]. Extracellular vesicles derived from OMLP-PCs (OMLP-PC-EVs) have shown enhanced efficacy in promoting wound healing processes, including cell proliferation and tissue repopulation, while inhibiting myofibroblast differentiation [86,87].

2.3.8. Human amnion MSCs (hAMSCs)

These cells are novel among all other sources of MSCs due to their unique tissue-specific characteristics, high yield and the absence of human leukocyte antigen (HLA) and co-stimulatory molecules. These features contribute to their low immunogenicity. Human amniotic mesenchymal stem cells (hAMSCs) also possess potent immunomodulatory and anti-inflammatory properties, pluripotent potential and strong paracrine activity, including the secretion of various growth factors that support tissue repair [88,89]. hAMSC-Exosomes have also shown significant therapeutic effects in the treatment of diabetic wounds and hypoxia/ischemia-induced cerebral palsy via regulation of cellular apoptosis [90,91]. Moreover, hAMSC-Exo offer protection to retinal pigment epithelium cells against oxidative stress by modulating the PI3K/Akt/FoxO3 signaling pathway, suggesting their potential for the treatment of age-related macular degeneration [89].

2.3.9. Human amniotic fluid stem cells (HAFSCs)

These multipotent cells exhibit features intermediate between embryonic and adult stem cells and can develop into all three germ layers. They are easy to obtain and have no ethical and legal limitations for their use in experiments [92,93]. Injections of dissociated hAFSCs have been found to accelerate cutaneous wound healing and reduce scarring. Additionally, hAFSC sheets were also found to exert anti-fibrotic properties without delaying wound closure [94]. In vivo studies showed that hAFSCs not only regulate innate immunity but also support acquired immunity by promoting T cell production, particularly in inflammation-related diseases [95]. According to the research findings, hAFSC-exosomes significantly accelerate wound healing, promote the natural distribution of collagen throughout the healing process, and support the regeneration of hair follicles, nerves, and blood vessels. Furthermore, hAFSC-exos reduce the excessive accumulation of myofibroblasts and the extracellular matrix [96].

Compared to exosomes from other cell sources, such as platelet-derived or epithelial cell-derived exosomes, MSC-derived exosomes exhibit superior regenerative and immunomodulatory capabilities. Platelet-derived exosomes are rich in growth factors like PDGF and VEGF and support early-stage wound healing, particularly hemostasis and angiogenesis [97,98]. However, they lack the broader anti-inflammatory and tissue remodeling function observed in MSC-derived exosomes. Epithelial cell-derived exosomes contribute to re-epithelialization and barrier repair but are generally more limited in their paracrine signaling range and immunosuppressive effects [99]. In contrast, MSC-derived exosomes provide a multifaceted therapeutic profile, offering anti-inflammatory, pro-angiogenic, anti-fibrotic, and immune-modulating effects that support all phases of wound healing – from inflammation resolution to tissue remodeling [45,48,52]. Furthermore, MSC exosomes are generally less immunogenic and more versatile across wound types, making them particularly promising for clinical translation [100]. These exosomes are essential for tissue regeneration, immune modulation, blood vessel development, and inflammation reduction. Depending on their cellular source, which affects characteristics like immunogenicity, ease of isolation, and particular wound healing functions, their therapeutic effects differ. The distinctions among different MSC-derived exosomes are compiled in Table 2.

Table 2.

Comparative functional characteristics of MSC-derived exosomes in wound healing.

MSC Source	Isolation Ease	Immuno-genicity	Key Functional Effects	Wound Healing Roles	Ref
hUCMSCs	Easy (noninvasive)	Low	Anti-inflammatory, pro-angiogenic	Promotes tissue regeneration, transition to proliferative phase	[45,48]
ADMSCs	Easy (liposuction)	Low – Moderate	Paracrine signaling, scar modulation	Enhances epithelialization, reduces fibrosis	[52,53]
BMMSCs	Invasive (bone marrow)	Low	Osteogenic, anti-apoptotic	Enhances bone/cartilage repair, reduces inflammation	[67–69]
WJ-MSCs	Easy	Very Low	Immunomodulation, high differentiation	Graft vs host, degenerative wound repair	[72–74]
EPSCs	Moderate	Low	Angiogenesis, anti-fibrotic	Promotes scarless healing, skin appendage regeneration	[79–81]
FD-MSCs	Limited availability	Low	M2 macrophage polarization	Enhances regeneration, reduces inflammation	[82,83]
OMLP-PCs	Moderate	Very Low	Antibacterial, immunosuppressive	Controls infection, promotes repopulation	[84,86,87]
hAMSCs	Easy	Very Low	Anti-inflammatory, pluripotent	Diabetic wound healing, CNS repair	[88–91]
hAFSCs	Moderate	Very Low	Multilineage differentiation	Enhances collagen alignment, vascularization	[94–96]

The biogenesis of exosomes is a highly intricate process involving endocytosis, cargo sorting, and multivesicular body (MVB) maturation as illustrated in Figure 2. Exosome formation begins with the process of endocytosis, which is initiated within the endocytic trafficking pathway through the inward budding of the plasma membrane, resulting in the formation of early endosomes. These endosomes travel toward the interior of the cells, wherein they undergo structural modifications and cargo selection. They function as primary sorting stations, and direct internalized molecules to distinct cellular fates. During this stage the endosomal sorting complex required for transport (ESCRT) has an important role in recognizing and selecting proteins, lipids, nucleic acids, and other biomolecules. ESCRT incorporates these biomolecules into the inward budding intraluminal vesicles (ILVs) within the endosomal lumen. As these early endosomes undergo maturation, they are transformed into late endosomes, characterized by vacuolar subdomains and further reorganization of the endosomal membrane. During this process of transition from early endosomes to late endosomes, repeated inward invagination of the endosomal limiting membrane results in the continuous biogenesis of ILVs. The accumulation of ILVs within the endosomal lumen results in the formation of multivesicular bodies (MVBs), which contain hundreds of vesicles. During invagination transmembrane and membrane-associated proteins are selectively sequestered into budding domains, while cytosolic components are encapsulated within the vesicle interior. This regulated sorting mechanism establishes the molecular diversity of ILVs and consequently of the exosomes which are released upon the fusion of MVBs with the plasma membrane. Most MVBs fuse with the plasma membrane, releasing the ILVs into the extracellular space, referred to as “exosomes.” This release is regulated by Rab GTPases (e.g., Rab27a, Rab27b) and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), which facilitate membrane fusion. These vesicles perform a variety of complex functions in molecular exchange and intracellular communication [103]. Each exosome has surface molecules that enable it to bind to cell receptors. After binding to targeted cells, exosomes can be internalized via phagocytosis or endocytosis, triggering signaling through receptor-ligand interaction. Exosomes can also fuse with the target cell’s membrane, releasing their contents into the cytoplasm. This process can alter the physiological state of the cell [104]. Exosomes generated by a specific parent cell can significantly impact the activity of neighboring cells, the microenvironment of the cell, and the physical characteristics of distant cells and tissues. Alternatively, MVBs are directed to the lysosome for degradation [104–106], where ILVs and their cargo are degraded and recycled. The balance between secretion and degradation determines the functional impact of exosomes in physiological and pathological conditions.

Figure 2.

A figure illustrating the production of exosomes. Exosomes are generated during endosomal maturation. As the early endosomes mature, they are either ESCRT dependent or ESCRT independent. The resulting structure known as multivesicular body (MVB) contains numerous intraluminal vesicles (ILVs). MVBs have two primary fates, they may fuse with lysosomes of ILVs called the multivesicular body (MVB), and they may fuse with lysosomes for degradation or merge with plasma membrane releasing exosomes. Adapted from Vaiaki and Falasca, 2024, Semin Cancer Biol.; Pircalabioru et al., 2024, Int J Mol Sci. [101,102]. Created with biorender.

2.4. Isolation of exosomes

Size exclusion liquid chromatography and ultracentrifugation are the commonly used techniques for isolating exosomes. Density gradient ultracentrifugation is a widely used and effective technique for isolating exosomes. Iodixanol (OptiPrep™) density gradient ultracentrifugation is a high-resolution method to isolate exosomes based on their buoyant density. After initial centrifugation is done to remove the debris, exosomes are pelleted and resuspended in iodixanol solution, followed by layering at the bottom of a density gradient. During the process of ultracentrifugation, exosomes migrate to their specific density zones, thereby allowing separation from protein aggregates and other contaminants (Figure 3). This technique specifically yields a highly purified exosomes fraction suitable for downstream analyses. Exosome purification is influenced by their physicochemical properties.

Figure 3.

1) Extracellular vesicles (EV) are released by cells during their normal activity. 2) For EV isolation, conditioned culture medium is harvested and major contaminants removed by consecutive low speed centrifugation. 3) The cleared supernatant is concentrated by ultracentrifugation, and the resulting 100K pellet is loaded on the bottom of an iodixanol gradient. EVs float upwards and EV-enriched fractions are collected and pelleted. The final sample is rich in EVs and free from contaminants. Adapted from Roszkowski, 2024, Clin Exp Med [67]. Created with biorender.

Differential centrifugation is also commonly used to separate exosomes from microvesicles, with exosomes requiring ultracentrifugation at 70,000–100,000 × g for 90–120 minutes, while microvesicles are pelleted at 10,000 × g for 30 minutes. However, exosome pellets may also contain lipoproteins, chylomicrons, protein clumps, ribonucleoprotein complexes, and microsomes, particularly when isolated from plasma or other body fluids following food intake [107,108]. Centrifugation mechanics and tube type can influence exosome recovery. Other techniques for identifying and characterizing exosomes include western blotting, flow cytometric analysis, enzyme-linked immunosorbent assay (ELISA), nanoparticle tracking analysis, atomic force microscopy, and scanning electron microscopy. Exosomes exhibit diverse cargo profiles, consisting of lipids, proteins, deoxyribonucleic acids (DNAs) and ribonucleic acids (RNAs). Their specific roles, such as immune response regulation and cell-to-cell communication, are influenced by their biological origin [107,108].

Additional purification methods, such as immunoaffinity purification, ultrafiltration, polymer coprecipitation, and microfluidics and field flow fractionation are also used which can introduce specific biases to some extent demonstrated in the supplementary Figure S1. These may include the unintended exclusion of certain exosome subpopulations, such as those with higher density or lacking specific surface markers. Therefore, the selection of a purification method plays a critical role, as it significantly influences the composition of isolated exosomes, thereby impacting the study outcomes [109].

3. MSC-derived exosomes and their therapeutic effects on wound healing

MSCs-derived exosomes can be delivered at the wound site via intravenous or subcutaneous injection [3]. Once administered they can actively participate in all the phases of the wound healing process through a variety of mechanisms. In the inflammatory phase, MSC exosomes can modulate the immune response by influencing the polarization of macrophages and regulating cytokine secretion, thus contributing to the reduction of inflammation. During the proliferative phase, they enhance the proliferation of fibroblasts, migration of keratinocytes and angiogenesis, thus ensuring rapid wound closure and tissue regeneration. In the remodeling phase, they contribute toward regulating collagen synthesis and deposition, ultimately leading to reduced scar formation. This section mainly discusses the mechanism of exosomes in the wound healing process

Mesenchymal stem cell-derived exosomes (MSC-exosomes) in wound healing and cutaneous tissue regeneration:

The process of skin regeneration can be summarized into four overlapping phases: the hemostatic phase, the inflammation phase, the proliferation phase, and the remodeling phase [110]. Research on wound healing and skin regeneration consistently highlights the role of MSC-exosomes in these critical phases.

3.1. MSC exosome in hemostasis

The MSCs-derived exosomes have been shown to affect the dynamics of blood coagulation. Following injury to the skin, mesenchymal stem cells (MSCs) located in the dermis release exosomes (MSC-exosomes), which have been shown to promote platelet activation and enhance blood clot formation. The exogenous administration of MSC-exosomes (e.g., via injection in a mouse model) supports the early hemostatic response by facilitating rapid clotting, thereby contributing to the initial phase of wound healing (Figure 4(a)). In a study reported by Silachev et al. [113] the exosomes derived from umbilical cord significantly reduced the time for formation of clot and lag phase of spontaneous clotting in human blood as compared to controls [113]. Also, the samples treated with exosomes demonstrated increased clot firmness and a larger clot area indicating enhanced hemostatic properties. The results of the proteomic analysis revealed that both MSCs and their EVs carry key coagulation-associated proteins, including CD9, phosphatidylserine (PS), myosin-9, talin-1, histones, and cytoplasmic actin. Remarkably, CD9 plays a crucial role in initiating platelet activation, stabilizing platelet aggregates, and enhancing fibrinogen binding. MSC-EVs also contain annexin V, a protein with known anticoagulant properties [113]. The presence of phosphatidylserine and tissue factor, in the umbilical MSCs and their extracellular vesicles contributed toward the procoagulant properties, demonstrating the role of MSCs derived exosomes in hemostasis.

Figure 4.

(a) Role of MSC-derived exosomes in the hemostasis phase of wound healing. following injury to the skin, mesenchymal stem cells (MSCs) located in the dermis release exosomes (MSC-exosomes), which are shown to promote platelet activation and enhance blood clot formation. The exogenous administration of MSC-exosomes (e.g., via injection in a mouse model) supports the early hemostatic response by facilitating rapid clotting, thereby contributing to the initial phase of wound healing. b) Role of MSC-derived exosomes in the inflammation phase of wound healing. during the inflammatory phase, MSC-exosomes contribute to immune modulation by promoting macrophage M2 polarization and releasing anti-inflammatory factors. These exosomes also influence various immune cells including neutrophils, T cells, and B cells, while reducing the levels of pro-inflammatory factors. This coordinated action fosters a controlled inflammatory response, facilitating the transition to subsequent healing phases and supporting tissue regeneration under the protective cover of the forming scab. (c) Role of MSC-derived exosomes in the proliferation phase of wound healing. MSC-exosomes enhance wound repair during the proliferative phase by promoting the proliferation and migration of keratinocytes and fibroblasts, while reducing apoptosis. They facilitate angiogenesis, collagen deposition, and the formation of granulation tissue by stimulating the activity of proliferating fibroblasts and myofibroblasts. These coordinated effects contribute to tissue regeneration and re-epithelialization of the wound bed. (d) role of MSC-derived exosomes in the remodeling phase of wound healing. during the remodeling phase, MSC-derived exosomes promote re-epithelialization, regulate myofibroblast activity, and modulate extracellular matrix remodeling by balancing collagen I and III deposition. Importantly, they help reduce excessive scar formation by limiting fibrosis and promoting organized tissue regeneration, ultimately supporting restoration of tissue structure, integration, and function. Adapted from Wong et al., 2025, Explore Immunol.; Huelsboemer et al., 2024, Mil Med Res. [111,112]. Created with biorender.

In a study by Chance et al. [94] the procoagulant activity of extracellular vesicles (EVs)-derived from monolayer and spheroid cultures of adipose-derived MSCs abbreviated as MAd-EVs and SAd-EVs respectively. Similarly, bone marrow MSCs (BMSCs) were also explored derived from monolayer and spheroid cultures abbreviated as MBM-EVs and SBM-EVs respectively. All EV types exhibited thrombogenic properties, significantly increasing thrombin generation and reducing the time to thrombin peak (p <0.0001) [94]. The total thrombin production was markedly elevated across all EV groups. This procoagulant activity was associated due to the presence of tissue factor (TF) and phosphatidylserine (PS) on the EV surfaces. AdMSC-EVs showed higher PS expression, resulting in greater thrombin output. EVs from AdMSC spheroids produced the strongest clots, while BMSC-EVs enabled faster clot initiation. Overall, the results of the study confirmed both AdMSC-EVs and BMSC-EVs exhibited significant procoagulant potential, AdMSC-EVs demonstrated more thrombogenic activity and exhibited higher amounts of phosphatidylserine (PS) as compared to BMSC-EVs. The study highlighted the variability in the source of EVs is an important parameter contributing to the specific therapeutic effect of the exosomes [114].

Fiedler et al. [115] investigated the hemostatic potential of exosomes derived from adipose-derived mesenchymal stem cells (AdMSCs) under both unstimulated and stimulated conditions induced by lipopolysaccharide (LPS) and tumor necrosis factor (TNF). The results of the study revealed that stimulation with LPS or TNF did not alter the quantity of EVs produced by AdMSCs. Regardless of inflammatory priming, all the exosomes exhibited activity procoagulant activity acting through both the intrinsic and extrinsic coagulation pathways.

Clotting assay performed using both the human reference plasma and plasma deficient in coagulation factors VII and XII. It was observed that in the reference plasma, all EVs induced similar clotting times, but no clot formation was observed in factor VII deficient plasma, suggesting the presence of tissue factor on the surface of EVs, which in turn activated the extrinsic clotting pathway. In factor XII-deficient plasma, prolonged clotting times were observed, particularly in unstimulated and TNF-stimulated EV groups, indicating the presence of phosphatidylserine (PS) that supports activation of factor XII via the intrinsic pathway. Importantly, the procoagulant activity of AdMSC-EVs was observed independent of inflammatory stimulation, highlighting their dual potential in initiating hemostasis through both intrinsic and extrinsic coagulation cascades [115]. Taken together, the role of MSCs exosomes in promoting hemostasis is mainly achieved due to the presence of phosphatidylserine (PS) and tissue factors.

3.2. MSC-exosome activity mechanism during the inflammatory phase

Since inflammation is the body’s protective response to harmful stimuli, it is beneficial for proper wound healing to have an early and well-regulated inflammatory response [116]. In contrast, a persistent and disrupted inflammatory response can promote fibrosis, interfere with wound healing, and inhibit re-epithelialization [117]. Macrophages play a significant role in skin regeneration, exhibiting pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. Dysregulated macrophages may promote fibrosis or severe inflammation [118]. MSC-derived exosomes play an important role in the inflammation phase of wound healing. During the inflammatory phase, MSC-exosomes contribute to immune modulation by promoting macrophage M2 polarization and releasing anti-inflammatory factors. These exosomes also influence various immune cells including neutrophils, T cells, and B cells, and reduce the levels of pro-inflammatory factors. This coordinated action fosters a controlled inflammatory response, facilitating the transition to subsequent healing phases and supporting tissue regeneration under the protective cover of the forming scab (Figure 4(b)).

A study reported by He X. revealed that the depletion of macrophages delayed wound healing despite MSC injection, indicating that MSC-mediated wound healing is dependent on macrophages. Moreover, the results of the study demonstrated that systematically infused bone marrow MSCs and jaw marrow MSCs migrated to the wound site, facilitating macrophage polarization toward the M2 phenotype promoting wound healing. Also, the M2 polarization was found to increase by in vitro co-culturing of MSCs with the macrophages. Mechanistically, it was proved that the exosomes derived from the MSCs had an important role to play in macrophage polarization. The study revealed that the depletion of MSC derived exosomes reduced the M2 phenotype of the macrophages, and infusing MSCs without exosomes resulted in fewer M2 macrophages at the wound site, causing delayed healing [119].

The findings support the role of MSCs in eliciting M2 polarization of macrophages and thereby accelerate wound healing by transferring exosome derived microRNAs, small, non-coding RNA molecules with a critical role in regulation of gene expression at the post transcriptional level. Thus, MSC-exosomes strongly influence recipient macrophages, promoting the acquisition of the anti-inflammatory M2 profile and facilitating the polarization of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype [120]. Exosomes derived from mesenchymal stem cells can regulate the differentiation, activation, and proliferation-like processes of B lymphocytes, while simultaneously reducing T-lymphocyte proliferation. Additionally, MSC-exosomes attenuate the immune response by converting activated T cells into T-regulatory phenotype. Furthermore, excessive cytokine production can harm tissue, and the regulation of inflammatory factors is essential for the regeneration of skin tissue. Exosomes from various MSC sources can reduce the inflammatory response triggered by multiple stimuli. This is achieved through the reduction of the action of various pro-inflammatory enzymes such as cyclooxygenase (COX)-2 and inducible nitric oxide synthases (iNOS) along with cytokines, TNF-α, IL-1β, and MCP-1. In addition, MSC-exosomes can increase the expression of IL-10, which is an anti-inflammatory cytokine that plays an important role in mitigating inflammation and scarring in the skin’s wounds in different pathologies [121,122].

Various studies have shown that MSC-exosomes contain certain miRNAs to regulate the immune system, contribute to macrophage polarization and intercellular communication. Three miRNAs, miRNA-21, miRNA-146a, and miRNA-181c, are particularly helpful in the regulation of inflammatory and immune responses and were shown to be most abundant in hUC-dMSCs through the analysis of miRNA expression patterns [123,124]. After further investigation, it was found that exosomes from hUC-dMSCs containing miRNA-181c helped reduce the excessive inflammation caused by burns by suppressing the Toll-like receptor 4 (TLR4) signaling pathway [125]. Additionally, exosomes containing anti-inflammatory microRNAs (miRNA-124a, miRNA-125b) inhibit the post-transcriptional expression of cytokines and chemokines, such as TNF-α and MCP-1, which helps maintain the reduction in the infiltration of the cells that cause inflammation throughout the process of wound healing [126]. In another study Szebeni et al. and Gou et al. demonstrated that the exosomes secreted by MSCs contain miR-223, which plays a crucial role in macrophage polarization and involved in regulating the balance between the proinflammatory (M1) and anti-inflammatory (M2) macrophages by causing the suppression of classic proinflammatory pathways, while enhancing alternative anti-inflammatory response [127,128].

It was observed that when miR-223 levels were high, the expression of pknox1 was downregulated, thereby promoting the polarization of M2. On the other hand when miR-223 was knocked down, pknox1 expression increased, reducing the M2 polarization and causing a shift of macrophages toward the M1 phenotype. The overall findings suggest the role of MSC-derived exosomes delivering miR-223 to macrophages, where it binds to pknox1 mRNA, suppressing its expression and facilitating M2 polarization required for wound healing. Previous studies have shown the role of exosomes from LPS preconditioned MSCs in nerve regeneration. A rat sciatic nerve injury model and in vitro inflammatory model were established using the RAW264.7 cells and LPS pre-Exos were injected locally at the injury site. The results of the study demonstrated accelerated functional recovery, axon regeneration, and remyelination while enhancing M2 macrophage polarization. Mechanistically, the LPS pre-Exos were enriched with TNF-stimulated gene-6 (TSG-6), which inhibited the NF-ΚΒ/NLRP3 signaling pathway, thereby promoting M2 macrophage polarization, highlighting the role of exosomes in addressing the immune response and enhance the peripheral nerve repair [129].

Cumulatively, the evidence supports the anti-inflammatory potential of MSCs exosomes in modulating the immune response to prevent inflammation.

3.3. MSC-exosome activity mechanism during the angiogenesis and proliferation phase

MSC-derived exosomes play a crucial role in angiogenesis by activating numerous signaling pathways and up-regulating pro-angiogenic growth factors. MSC-exosomes enhance wound repair during the proliferative phase by promoting the proliferation and migration of keratinocytes and fibroblasts, while reducing apoptosis. They facilitate angiogenesis, collagen deposition, and the formation of granulation tissue by stimulating the activity of proliferating fibroblasts and myofibroblasts. These coordinated effects contribute to tissue regeneration and re-epithelialization of the wound bed (Figure 4(c)). In endothelial and stromal cells, these exosomes activate the ERK, AKT, and STAT3 pathways, which results in elevated expression of hepatocyte growth factor (HGF), vascular endothelial growth factor A (VEGFA), stromal-derived factor 1 (SDF1), insulin-like growth factor 1 (IGF1), nerve growth factor (NGF), and interleukin-6 (IL-6). This is especially beneficial in chronic wounds, where these signaling pathways and growth factor levels are typically suppressed [130]. There are several stages of healing of which the development of new blood vessels, contraction of the wound, granulation tissue formation, deposition of collagen, and re-epithelialization are hallmark features [131]. Neoangiogenesis is one of the key factors in many physiological or pathological conditions such as healing of the wound and other tissues that need repair [110,132]. These exosomes are enriched with MSCs-derived angiogenesis-related proteins and RNAs (miRNAs) that activate the endothelial signal pathway. Additionally, exosomal miRNAs are essential for fostering angiogenesis. For instance, miR-146a, which is abundant in MSC exosomes, inhibits NF-κB signaling and downregulates phosphorylated IκB-α to increase endothelial cell stability and angiogenic activity [133].

The therapeutic potential of human umbilical cord mesenchymal stem cells (HUCMSCs) and their exosomes (HUC-Exos) in promoting diabetic wound healing was explored. In this study Human umbilical vein endothelial cells (HUVECs) were co-incubated with HUCMSCs, and HUC-Exos and their effect was evaluated in both in vitro and in vivo models to assess their effects on wound repair. The results of the study revealed that HUCMSCs regulated oxidative stress induced injuries in endothelial cells through exosome mediated signaling and displayed accelerated diabetic wound healing [134].

According to Shen et al. [68], bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos), enhance HaCaT cell migration and proliferation while reducing apoptosis, hence promoting skin wound healing. The pro-apoptotic gene APAF1 is targeted and downregulated by miR-93-3p, which regulates angiogenesis and cell survival. The miR-93-3p/APAF1 pathway is identified in the study as a crucial mechanism in skin regeneration triggered by BMSC exosomes [68].

Choi et al. [135] examined adipose-derived stem cell (ASC) exosomes and their relevant roles in skin regeneration. Their results showed that in adipose-derived stem cells (ASC) exosomes contain microRNA that inhibited the gene products NPM1, PDCD4, CCL5, and NUP62, all related to apoptosis, inflammation, and cell regulation. Inhibiting these genes showed a marked increase in proliferation of dermal fibroblasts, thus improving tissue regeneration and repair. The authors concluded how ASC exosomes represent a potential therapeutic target for tissue regeneration [135].

These exosomes also promote a range of trophic factors that allow for tissue regeneration [136]. Tissue regeneration of skin is dependent on cell proliferation and re-epithelization. Skin fibroblast play a major role in wound contraction, matrix deposition, tissue remodeling and skin healing and regeneration [137]. Recipient cells can uptake MSC-exosomes that encase proteins and RNAs, which impact a recipient cell’s proliferation and migration. MSC-exosomes can also impact the behavior of fibroblast and control their proliferation and migration through changing the expression of certain growth factors and signaling genes [138,139]. This indicates a process that enhances collagen formation to help complete structure healing of wounds and facilitates the process of granulation tissue formation [140]. Similarly, KLF3-AS1, a long non-coding RNA identified in BMSC-derived exosomes, has been demonstrated to upregulate VEGFA expression and increase angiogenesis in high-glucose treated HUVECs by increasing endothelial proliferation, tube formation, and decreasing apoptosis. In diabetic wound models, KLF3-AS1-expressing exosomes promoted wound healing and neovascularisation [141]. Together, these findings demonstrate that MSC-exosomes function as efficient angiogenesis mediators by delivering bioactive molecules that block inhibitory signals and upregulate growth factors, hence promoting wound healing.

3.4. MSC-exosome activity mechanism during the remodeling phase

The extracellular matrix (ECM) is made up of four principal types of biomolecules: collagen, non-collagen proteins, glycosaminoglycans, and proteoglycans. The ratio of collagen synthesis to its degradation is one of the most important parameters that determines ECM regeneration, as insufficient or excessive ECM formation can lead to delayed wound healing or pathological scarring. In addition to their primary effects on cells, exosomes from MSCs have been shown to regulate ECM re-synthesis, playing a vital role in the remodeling phase of wound healing as shown in Figure 4(d) [127]. During the remodeling phase, MSC-derived exosomes promote re-epithelialization, regulate myofibroblast activity, and modulate extracellular matrix remodeling by balancing collagen I and III deposition. Importantly, they help reduce excessive scar formation by limiting fibrosis and promoting organized tissue regeneration, ultimately supporting restoration of tissue structure, integration, and function.

These data indicate that wound healing is assisted by MSC-exosomes that impact the restoration of the ECM. Additionally, exosomes from stem cells residing in the adipose tissue (ADSC exosomes) have effects on collagen synthesis in certain aspects of wound healing. These have the potential to promote healing in early stages, by enhancing the initial phase of collagen synthesis, and then reducing the process of collagen synthesis in the later stages of healing, leading to less scar tissue [142]. This work demonstrates that there are MSC-exosomes that play a role in remodeling of the ECM and enhance the healing response while limiting scarring in the recovery phase. In a current study by Dalirfardouei, R. et al. [143], the therapeutic effects of exosomes derived from the mesenchymal stem cells (MSCs) on wound healing were explained [143].

Studies also showed that the treatment of full-thickness excision wound models with MenSC-derived exosomes demonstrated a reduction in inflammation and increased neoangiogenesis through the upregulation of VEGFA. Also, the expression of the Rela gene was increased in MenSC-derived exosomes. The Rela gene encodes the p65 subunit of the NF-kB family, an important transcription factor in activating the NF-kB signaling pathway. Elevated levels of this gene suggest these exosomes induce epithelial cell proliferation and migration by invoking NF-kB activation that supports re-epithelialization. Also, MenSC-derived exosomes have a crucial role to play during the remodeling stage of wound healing. In the early phases of tissue repair, they promote the expression of collagen I mRNA, involved in faster wound closure. As the process of healing progresses into the later phases, there was marked increase in collagen III mRNA expression causing a decrease in the Col1:Col3 ratio, which is associated with reduced scar formation, thereby highlighting the potential of MenSC-Exos in not only promoting enhanced wound healing but also improving the quality of tissue regeneration during the remodeling phase of wound healing. Taken together, MSCs-exosomes regulate the formation of collagen and reduce scar formation thereby promoting accelerated wound healing [143].

4. Engineered exosomes

It had been established that exosomes that underwent certain pretreatments have superior biological and therapeutic efficacy and represent a significant advancement in exosome-based therapies as compared to those which are solely derived from MSCs. Hence, engineered exosomes from mesenchymal stem cells (MSCs) are being developed as prospective transformative agents in regenerative medicine, as bioengineering approaches can enhance their therapeutic potential, which is a strategy to achieve standardization and reproducibility for their clinical use [2,144].

The development of engineered exosomes represents a significant advancement over their natural counterparts, by offering several advantages including enhanced loading efficiency of therapeutic molecules, improved therapeutic specificity and greater stability as mentioned in Table 3. This approach enables the incorporation of functional molecules in the exosomes, thereby amplifying the therapeutic potential of exosomes while at the same time preserving the inherent advantages of the natural exosomes. Additionally, the engineered exosomes effectively overcome the limitations of lower yield and enable the development of versatile and synergistic therapeutic strategies [2].

Table 3.

Summary of studies utilizing bioengineered exosomes.

Study	Model	Source of Exosomes	Type of Modification	Wound type	Outcome	References
MiR146a-loaded engineered exosomes from PMSCs with silk fibroin patch	in vivo/in vitro (Mice)	placental mesenchymal stem cells (PMSCs)	miR146a loading and integration into silk fibroin patch (SFP)	diabetic wound	Modulated 281 genes linked to inflammation; downregulated synthesis of inflammatory genes; upregulated insulin and (CAM) signaling pathway	[145]
miR-132-overexpressing exosomes from adipose derived stem cells	in vivo/in vitro C57BL/6 mice)	mouse adipose mesenchymal stem cells	Lentiviral transfection into ADSCs for miR-132 (miR-132-ADSCs) overexpression	diabetic wound	Promoted collagen I/III synthesis increased (CD31) and vascular smooth muscle cells α-smooth muscle actin (α-SMA) levels.	[146]
M2-polarized macrophages derived exosomes in 3D printed hydrogel	in vivo/in vitro	human bone marrow-derived mesenchymal stem cells and RAW 264.7 macrophages	3D printed AGP hydrogel with COL@d-ECM/M2-Exo	Full thickness wound	Activated JAK/STAT, IL-4 R, and RTK signaling pathways; promoted anti-inflammatory factors (CD163 and CD206)	[147]
Engineered Human Adipose Stem-Cell-Derived Exosomes Loaded with miR-21-5p	in vivo/in vitro (ICR mice)	human adipose stem cell	electroporation with miRNA and miR-21 mimics into hASC-exos	diabetic wound and scratch wound	Stimulated proliferation and migration of keratinocytes via Wnt/β-catenin signaling pathway; improved angiogenesis	[148]
HMOX1-overexpressing mesenchymal stem cell-derived exosomes	in vitro diabetic mice)	mesenchymal stem cells (MSC)	Heme oxygenase-1 (HO-1) encoded by the gene HMOX1 incorporated in exosomes	diabetic wound	Enhanced proliferation and migration of fibroblasts, keratinocytes and angiogenesisExo-HMOX1, promoted re-epithelialization, and improved collagen deposition.	[149]
Biomimetic small exosome enriched with TGF-β1	in vivo (mice)	human umbilical cord mesenchymal stem cells (HUMSCs) isoproterenol-induced)	TGF-β1 factor-enriched small exosome biomimetics exosomes	large scale wound	Facilitated keratinocyte migration, plasticity, and sweat gland regulation	[150]
Engineered Exosomes Containing Cathelicidin/LL-37	in vitro	U937 (monocytic) Cells-Derived Exosomes	Engineered Exosomes Containing Cathelicidin/LL-37	N/A	Promoted cell proliferation and migration, angiogenesis, antimicrobial activity,	[151]

Bioengineered exosomes are developed using various methods, including genetic modification, chemical modification or membrane fusion [152]. Chemical and physical modification of exosomes can also be considered as direct engineering of exosomes. Genetic modification of exosomes is considered indirect engineering of exosomes [153]. There are mainly two ways for the modification of exosomes: 1) pre-isolation modification or endogenous methods and 2) post-isolation modification or exogenous methods [154,155]. Pre-isolation modification method includes co-incubation and gene editing, while the post-incubation modification method includes passive incorporation (co-incubation) and active incorporation methods including sonication, chemical transfection, freeze-thaw method, extrusion and electroporation) [155,156]. These methods enable to directly load the therapeutic molecules in it and achieve optimal therapeutic cargo delivery with specific therapeutic properties and targeted delivery [2].

Electroporation is a simple technique to temporarily increase the permeability of exosomes using electrical pulses of high voltage allowing the creation of pores in the membrane enabling the loading of molecules into the endosomal cavity [2].

Xiong et al. [50], in their study highlighted the potential application of electroporation as an efficient method for loading therapeutic microRNA into the exosomes [157]. The miR-542-3p-loaded exosomes (miR-542-3p-Exos) were tested in vitro using human skin fibroblasts (HSFs) and human dermal microvascular endothelial cells, which effectively internalized the modified exosomes, resulting in increased cellular proliferation, migration and angiogenesis. In the in vivo study using the full thickness skin wound model in mice, the local injection of miR-542-3p-Exos significantly improved wound closure, enhanced collagen deposition, promoted neovascularization resulting in accelerated wound healing. The study highlighted electroporation as an effective method for developing engineered exosomes for enhanced wound healing therapies [157].

In a recent study, Hade, M.D., Suire, C.N. and Suo, Z. (2023) reported a novel peptide assisted strategy to enhance the cargo loading of exosomes, using a well-characterized cell-penetrating peptide YARA, derived from the HIV-1 trans-activator of transcription protein [158]. In this approach, YARA was covalently conjugated to miR-21-5p, and the resulting YARA-miR-21-5p conjugate was co-incubated with exosomes isolated from MSCs. This study demonstrated a significant increase in the therapeutic loading efficiency when compared to incubation of exosomes with free miR-21-5p. Once taken up by the mammalian cells, the YARA-miR-21-5p-loaded exosomes promoted fibroblast proliferation, migration and invasion essential for effective wound healing. The study highlighted the efficacy of engineered exosomes in achieving enhanced therapeutic load and delivery capacity for miR-21-5p based regenerative therapies by chemical transfection followed by co incubation method [158].

In a recent study by Jiang et al. [159], viral transfection was used as one of the methods to engineer the exosomes [159]. Using lentiviral transduction, human bone marrow derived MSCs (hBMSCs) were genetically modified to either overexpress or silence tSG-6. The in vivo study indicated that genetically modified TSG-6-enriched MSC-derived exosomes significantly reduced scarring in a murine wound model by lowering inflammation and collagen deposition as confirmed by histological and molecular analysis. Additionally, the study highlighted the loss of therapeutic effect when the expression of TSG-6 was silenced or neutralized with TSG-6-neutralizing antibody, thereby highlighting the role of TSG-6-modified MSC-derived exosomes in attenuating scar formation during wound healing, highlighting viral transduction as one of the methods for formulating engineered exosomes [159].

Overall, engineered exosomes hold immense potential, enabling the creation of tailored exosomes with distinct exosomal and biological effects. However, not all the MSC-derived exosomes are equally amenable to engineering, thereby highlighting the need for selective sourcing and optimization. With the increasing understanding of exosome biogenesis and the complex mechanism of wound healing, the development of precisely engineered exosomes with enhanced regenerative capabilities and targeted potential is highly anticipated, representing a significant step forward in personalized and effective wound care solutions.

5. Exosomes integrated with biomaterials

5.1. Enhancing wound healing through exosome-loaded biomaterials

Another challenge in treating wounds with exosomes is determining the optimal mode of administration, which could be addressed by using hydrogel-based biomaterials and strategies that provide localized, sustained delivery of exosomes to the damaged site. The various modes of exosome administration are depicted in Figure 5. They can be administered via a variety of delivery methods, each with unique benefits and drawbacks. Although intravenous, intra-articular, or direct injection at the defect site is a straightforward and widely used technique, it frequently leads to their poor retention at the damage site, requiring higher dosages and more frequent administration to maintain therapeutic effectiveness [160,161]. Hydrogels have been a successful delivery system to address this, improving their stability and retention [162]. They are ideal for controlled and prolonged release of exosomes, especially in injectable or thermosensitive forms, due to high-water content, biocompatibility, tunable degradation, and capacity to promote native cell proliferation and vascularization. Another potential technique is bio-printing, which allows the fabrication of precisely designed 3D scaffolds that enables the localized and sustained delivery of exosomes within a physiologically appropriate environment [161,163]. Furthermore, surface-coating scaffolds with exosomes (via linker molecules, extracellular matrix components, or antibodies) provides a tailored strategy that leverages exosomes surface markers to increase delivery specificity. Collectively, these delivery methods aim to enhance the therapeutic potential of exosomes by improving their localization, stability, and long-term release at the wound site.

Figure 5.

Various modes of exosome delivery.

In spite of its profound therapeutic potential, the direct delivery of MSCs-derived exosomes for clinical translation is challenging due to rapid systemic clearance, short half-life, and susceptibility to degradation. Additionally, the need for frequent administrations to maintain the therapeutic efficacy, significantly increases the cost of the treatment, primarily due to the high expenses coupled with their production and purification costs. Wound healing is a complex, multi-phase process, and administering a single high dose of exosomes may result in rapid clearance, degradation, or burst release, leading to only transient therapeutic effects. Hence using multiple small doses or controlled release systems will ensure a continuous supply of bioactive agents, to support tissue regeneration. This underscores the need for advanced delivery systems such as scaffolds, hydrogels which could prolong the release of the exosomes at the site of the wound, thereby enhancing the therapeutic efficacy and improving the wound healing properties. Biomaterials consists of both natural and synthetic components. The inherent biodegradable and biocompatible properties of the natural biomaterials make them ideal for mimicking the ECM. They have an important role in supporting cellular activities and facilitating the exchange of exosomes and cells for promoting synergistic tissue repair [2]. Conversely, synthetic biomaterials are more flexible and can effectively modulate the inflammatory microenvironment after injury and create a favorable environment for exosome-cell communication and targeting. Integrating exosomes into the biomaterials can enhance stability and amplify the biological effects of exosomes.

Xinrong Geng and colleagues first time demonstrated the therapeutic potential of a composite hydrogel composed of carboxyethyl chitosan (CEC) and dialdehyde carboxymethyl cellulose (DCMC) loaded with BMSC-derived exosomes (BMSC-Exos) for diabetic wound healing in rats [164]. This hydrogel displayed a multifaceted wound healing effect. The exosomes promoted angiogenesis and facilitated the polarization of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type, thereby reducing inflammation and accelerated wound healing in in type 1 diabetic rats, highlighting its strong potential for clinical application in chronic wound management. This composite hydrogel overcame the disadvantages of the conventional treatment strategies failing to effectively address the complex pathophysiology of diabetic wounds and highlighted the urgent need for advanced regenerative therapies like exosomes [164].

In another study by Xiao et al. [165], adipose-derived stem cell exosomes (ADSC-Exos) were delivered via a human acellular amniotic membrane (hAAM) scaffold for diabetic wound healing. ADSC-Exos significantly promoted the proliferation and migration of human dermal fibroblasts (HDFs) and enhanced tube formation and proliferation of HUVECs, in vitro thereby indicating its strong pro-regenerative and pro-angiogenic effects. On application to the diabetic mouse model, the hAAM-Exos composite accelerated wound healing by modulating inflammation, stimulating vascularization, and enhancing collagen deposition and extracellular matrix production [165].

OxOBand, an oxygen-releasing antioxidant cryogel based wound dressing loaded with ADSC-derived exosomes, was reported by Shiekh Singh, and Kumar [166]. The exosomes demonstrated enhanced migration of keratinocytes and fibroblasts and improved the survival of neuroblastoma cells under hyperglycemic conditions. In diabetic wound models, it significantly accelerated wound closure, enhanced collagen deposition, promoted re-epithelialization, and stimulated neovascularization, while effectively reducing oxidative stress. Additionally, Oxoband bound exosomes when tested in infected diabetic wounds, effectively prevented ulceration, controlled infection, and promoted wound healing [166].

Xiong et al. [148], highlighted the therapeutic potential of HUCMSC-derived exosomes encapsulated within a bioactive polyvinyl alcohol/alginate nanohydrogel scaffold (exo@H) for treating diabetic wound [148]. The composite hydrogel significantly enhanced the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. The in vivo studies in full-thickness wounds in diabetic rats, revealed that (exo@H) accelerated the wound closure and elevated the VEGF expression, thereby promoting neovascularization. The composite allowed sustained release and uniform distribution of the exosomes at the wound site [148].

A novel strategy using the gelatin microspheres fabricated by microfluidic technique for the local delivery of rat adipose-derived stem cells was developed by Shi et al. [167], to enhance diabetic wound healing [167]. The developed microspheres supported cell adhesion, proliferation, and provided a favorable environment for rADSC survival. In vivo, these microspheres promoted M2 macrophage polarization, collagen deposition, angiogenesis, and hair follicle formation. Notably, the fluorescence intensity around hair follicles was 17-fold higher than controls, indicating active participation of rADSCs through exosome-mediated signaling and paracrine effects of the exosomes which demonstrated an active role in wound repair [167].

In a recent study by Ashrafi et al. [168], hybrid scaffold combining nanofibers and hydrogel was formulated for the controlled delivery of exosomes isolated from human placenta-derived stem cells in full-thickness wound in rats [168]. The loaded exosomes into the alginate-based hydrogel layered with polycaprolactone nanofibers effectively mimicked the native bilayer structure of the dermis and epidermis. The results of the in vivo studies showed that exosomes loaded scaffold significantly accelerated wound closure and improved collagen synthesis by 22% compared to unloaded scaffold and by 33% relative to the untreated control [168].

A cell-free therapeutic strategy using adipose-derived stem cell (ADSC) exosomes encapsulated in an alginate-based hydrogel was developed by Shafei et al. [169], to enhance wound healing in full-thickness skin wounds [169]. Isolated exosomes from ADSC were incorporated into a biocompatible, biodegradable alginate based hydrogel scaffold. The exosome-loaded hydrogel scaffold preserved the activity of exosomes at the wound site and significantly improved wound closure, collagen synthesis, and angiogenesis in vivo. The study highlighted the paracrine role of ADSC-derived exosomes in tissue regeneration and supported their use in cell-free wound healing therapies through the use of biomaterials for encapsulating the exosomes [169].

5.2. Functional advantages of biomaterial-exosome integration

Encapsulating exosomes in hydrogels, cryogels, or microspheres shields them against enzymatic breakdown and environmental stress, preserving their bioactivity for long durations [164,169]. Many biomaterials (e.g., alginate, PVA, gelatin) allow for the prolonged and localized release of exosomes, reducing burst release and providing a consistent therapeutic concentration all over time. For example, the polyvinyl alcohol/alginate nanohydrogel scaffold (exo@H) provided prolonged release and uniform dispersion, greatly improving angiogenesis and wound healing in diabetic rats [148]. The porosity of the scaffold, charge interactions, and microstructure all have an impact on exosome loading and retention at the injury site. Microfluidic-engineered gelatin microspheres exhibited significant exosome retention, while also promoting M2 macrophage polarization and hair follicle regeneration [167]. Biomaterial platforms can help exosomes perform immunomodulatory tasks by influencing the local immunological microenvironment. For example, CEC/DCMC-based hydrogels loaded with BMSC-exosomes improved macrophage M2 polarization, decreased inflammation, and increased vascularization [164]. When integrated with exosomes, biomaterials can boost regeneration processes such as collagen deposition, angiogenesis, and re-epithelialization. OxOBand, a cryogel-based oxygen-releasing scaffold, demonstrated potent antioxidant and wound healing properties, particularly under diabetic and infected conditions [166]. Preclinical studies show that exosome-loaded biomaterials significantly improve wound healing in diabetic and full-thickness wound models, indicating a high clinical potential [164–169]. However, progress toward clinical use necessitates standardized methodologies for exosome separation and scaffold development.

Thus, the ntegrating of exosomes with tailored biomaterials presents a promising strategy to enhance exosome stability, retention, sustained release, and their biological efficacy in tissue repair. Studies related to these effects are briefly described in Table 4. The emerging evidence strongly supports that the application of exosomes in conjugation with biomaterials significantly amplifies the therapeutic outcomes. This combined strategy not only advances the field of wound healing but also opens up new avenues for developing biomedical materials for clinical applications in wound healing.

Table 4.

Overview of studies on exosome-incorporated biomaterials.

Study	Model	Exosome Source	Delivery System/Modification	Wound type	Outcome	References
hUCMSCs Exosomes combined with Pluronic F127 Hydrogel	in vivo/in vitro Sprague-Dawley (SD) rats	human umbilical cord mesenchymal stem cells (hUCMSCs)	hUCMSCs combined with Pluronic F127 Hydrogel	Scratch wound and diabetic wound	Upregulated VEGF, TGFβ-1; promoted skin regeneration	[170]
Platelet-rich Plasma Exosomes Assembled on Chitosan/Silk + Curcuma zedoaria Polysaccharide	in vivo SD) rats	Platelet-rich Plasma Exosomes	chitosan/silk hydrogel sponge loaded with polysaccharides from curcuma zedoria incorporated within Platelet-rich Plasma Exosomes	diabetic wound	Enhanced collagen; accelerated wound closure	[171]
ADSC-exosomes + Q5 protein hydrogel	in vivo/in vitro Db/db mice)	adipose-derived mesenchymal stromal cell (ADSC) generated by transduction with human telomerase lentivirus Htert	Exosomes with E. coli derived Q5 protein	diabetic wound	Effective topical application; similar to IV administered exosomes	[172]
MicroRNA-126-exosomes +chitosan hydrogel	in vivo/in vitro	synovial mesenchymal stem cells (SMSCs)	Chitosan hydrogel for sustained miR-126-3p exosome release	diabetic wound	Improved granulation, collagen deposition and angiogenesis	[173]
Silk fibroin – collagen hydrogel loaded with IGF1-CESCs exosomes	in vitro	cartilage endplate stem cell exosomes	The IGF1 gene from lentivirus incorporated into CESCs exosomes were encapsulated in an injectable silk fibroin – collagen hydrogel	Annulus fibrosus damage	Accelerated AF repair, reduced degeneration	[174]
Silk-hyaluronic acid self-healing hydrogel laden macrophage-derived exosomes	in vitro (Wistar rats)	M2 macrophage derived exosomes	M2Exo were conjugated with oxidized hyaluronic acid and mixed with PEGylated silk fibroin to develop self-healing Exo-gel	diabetic wound	75% wound closure was observed within 7 days enhanced regeneration and angiogenesis; promoted collagen deposition.	[175]
Multifunctional ADM hydrogel containing endothelial cell-exosomes for diabetic wound healing	in vivo/in vitro	human umbilical vein endothelial cells (HUVEC)	Dual network hydrogel (ADMFe3+@PA-Exos/GelMA)	diabetic wound	Inhibited bacterial growth; promoted collagen deposition; enhanced angiogenesis; reduced oxidative stress and inflammation	[176]
Wharton Jelly Derived Mesenchymal Stem Cells + Aloe-Emodin:	in vitro	Wharton Jelly derived mesenchymal stem cells (WJ-MSCs)	Wharton Jelly Derived Mesenchymal Stem Cell’s Exosomes Combined with Aloe-Emodin	leishmania and wound	Antimicrobial effect and rapid wound closure	[177]
collagen-based three-dimensional scaffold encapsulated with adipose mesenchymal stem cells-derived exosomes	in vivo (Wistar rats)	adipose mesenchymal stem cells	micro-porous collagen-based three dimensional scaffold (CTS) encapsulated with adipose mesenchymal stem cells (ASCs)-derived exosomes	diabetic wound	Enhanced wound closure; epidermal and dermal regeneration; angiogenesis collagen deposition	[178]
HUMSC MSC exosomes + AgNP microneedle patch	in vivo/in vitro	human umbilical cord mesenchymal stem cell (HUMSC)	GelMA/SilMA microneedle (MN) patch encapsulated with mesenchymal stem cell derived exosomes (MSC-exos) and (AgNPs) nanoparticles	diabetic wound	Antibacterial; stimulated angiogenesis; reduced inflammatory response; and localized delivery	[179]
HUCMSC-exosomes + PVA/Alg nanohydrogel	in vivo/in vitro	human umbilical cord mesenchymal stem cells (HUCMSCs)	HUCMSCs-derived exosomes encapsulated in a bioactive scaffold composed of polyvinyl alcohol (PVA)/alginate (Alg) nanohydrogel (exo@H)	diabetic wound	Facilitated proliferation, migration and angiogenesis of HUVECs; enhanced SMA, SR-B1, CD31 and VEGF via activation of ERK1/2 pathway.	[180]
Hyaluronic acid-liposomes fused with HucMSC exosomes	in vivo/in vitro	human umbilical cord mesenchymal stem cells (HUCMSCs)	Fusion of Exo with Hyaluronic Acid Liposomes (HL). HL@Exo.	photodamaged skin	Enhanced cell proliferation; repair and angiogenesis; reduced inflammation, and increased collagen regeneration	[181]
BMMSC-exosomes+ magnetic microneedle patch	in vivo/in vitro	bone marrow mesenchymal stem cell (BMMSC)	Microneedle patch (MMP) loaded with bone marrow mesenchymal stem cell-derived exosomes (Exos) and folic acid-magnetic nanoparticles (Fmns) within a methacrylated carboxymethyl chitosan (CMCSMA) microneedle	oral mucosal lesions	Improved re-epithelialization, and angiogenesis; reduced inflammatory cytokine levels and promoted M2 macrophage polarization	[182]
ADSC-exosomes+ MEW-PCL/GelMA scaffold	in vivo/in vitro	Adipose mesenchymal stem cells (ADSCs)	Controllable GelMA hydrogel-combined Melt Electrowriting (MEW)-PCL scaffold for in situ release of exosomes	wound	Stimulated cell proliferation, migration, accelerated re-epithelialization; improved collagen maturation, and enhanced angiogenesis	[183]
Milk exosome-infused fibrous matrix	in vivo/in vitro	Pasteurized bovine milk	Exosomes immobilized on polydopamine (PDA)-coated hyaluronic acid-based electrospun nanofibrous mesh (mEXO@PMAT)	wound	Sustained release of exosomes; enhanced keratinocyte and fibroblast activity	[184]
ADSC-exosomes + porcine ECM patch	in vivo/in vitro	Adipose mesenchymal stem cells (ADSCs)	ADSC-exosomes loaded onto decellularized porcine pericardial extracellular matrix bilayer patches	diabetic wound	Accelerated granulation; tissue formation, re-epithelialization, vascularization, and collagen production.	[185]
Exosome/metformin-loaded self-healing conductive hydrogel	in vivo/in vitro	Adipose mesenchymal stem cells (ADSCs)	Dual-loaded hydrogel (PEG/Ag/CNT) incorporating metformin and exosomes	chronic diabetic wound	Preserved F-actin., reduced ROS and improved microvessel integrity	[186]
ADSC-exosomes + porcine ECM patch	in vivo/in vitro	Human Gingival mesenchymal stem cells (HGMSCs)	Exosomes loaded onto porous PHE microspheres composed of PLGA, PLLA-PEG-PLLA, nano-hydroxyapatite (nHAP), and poly-ε-l-lysine (EPL)	diabetic wound	Promoted angiogenesis via Wnt/β-catenin signaling	[187]
Fiber-reinforced gelatin/β-cyclodextrin hydrogels loaded with platelet-rich plasma (PRP)-derived exosomes	in vivo/in vitro	Platelet-rich plasma (PRP)	PRP-exosomes covalently crosslinked with genipin and encapsulated in fiber-reinforced gelatin/β-cyclodextrin (GEL/β-CD) hydrogels	diabetic wound	Enhanced autophagy; inhibited apoptosis and improved regeneration	[188]
BMSCs Exosome-loaded tannic acid – thioctic acid hydrogel	in vivo/in vitro	Bone marrow mesenchymal stem cells (BMSCs)	Exosomes incorporated into a tannic acid-thioctic acid (TATA) hydrogel	coagulation disorders	Accelerated wound closure, enhanced healing; collagen deposition increased collagen deposition, hemostasis and antibacterial activity	[189]
Hypoxic USC-exosomes + SISMA hydrogel	in vivo/in vitro	Urine-derived stem cells (USCs)	SIS-modified hydrogel (SISMA) delivering miR-486-5p exosomes	diabetic wound	Regulated SERPINE1 activity, promoted angiogenesis via the HIF-1α signaling pathway	[190]
Injectable exosome-loaded quaternized chitosan/oxidized sodium alginate hydrogel	in vivo/in vitro	Induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs)	Exosomes encapsulated within an injectable hydrogel (HACC/OSA) composed of quaternized chitosan (HACC) and oxidized sodium alginate (OSA)	combined radiation-wound injury (CRWI)	Antibacterial, accelerated re-epithelialization, enhanced collagen deposition, and angiogenesis.	[191]

Taken together, the study highlights the importance of integrating the engineered exosomes with the biomaterials to maximize the therapeutic efficacy. This combination offers synergistic advantages including the enhanced stability of exosomes, targeted delivery with sustained release at the wound site, thereby paving the way for the development of novel biomaterials tailored for regenerative therapies.

6. Opportunities of exosomes in clinical setting

The application of exosome therapies in burns and wounds treatment and regenerative medicine promise new advancements. Exosomes provide a safer method of treatment when compared to stem cell therapy. Due to their nanoscale size, exosomes offer better tissue penetration and targeted delivery of the therapeutic cargo. They are effective because of their capability to carry specific bioactive molecules designed for wound healing, and their innate ability to modulate immune responses, promote angiogenesis, and improve tissue repair. Preferentially, miRNAs transferred by exosomes improve delivery when using hydrogels, topical nanoparticles, and other topical formulations, thus allowing customization for different types of burns. At the same time, synergistic integration with other existing therapies within the multi-modal approaches, such as pharmacological agents, skin grafts, and others can be more efficient in reducing the healing time, scarring, and functional outcomes. Exosome-based approaches are capable of filling the existing gaps in treatment, utilizing state-of-the-art advancements, targeted engineering, and support from legislations providing safe, highly effective, and scalable technology for effective management of wounds. Though the data lacks stating the efficacy of exosomes in clinical settings, it is observed that the exosomes can cure wounds efficiently in vivo and in vitro.

A systematic review and meta-analysis were conducted in 2022 to determine the therapeutic efficacy of cell-derived exosomes in diabetic wounds. Twenty-one studies, including 323 animals, were taken and analyzed. The results indicated that exosome therapy was superior to the control group in terms of wound repair rate, neovascular density, re-epithelialization, collagen deposition, scar width, and inflammatory down-regulation [192]. Similar results were discussed in another meta-analysis conducted by Gunjan et al. [193], including 18 preclinical and 4 clinical studies where they used MSC-derived exosomes from different sources [193]. Jing Wu et al. [29], in their review, mentioned 27 studies in which the exosomes combined with different APIs are used to treat diabetic wounds. The results showed a positive response in the animals with rapid improvement in wound closure [29]. Also, to assess the therapeutic potential of microRNA-engineered exosomes on diabetic wounds, a meta-analysis was conducted in 2023. Six studies, including 72 animals, were analyzed. The result showed that microRNA exosome treatment was superior compared to control therapy. These exosomes significantly accelerated the rates of wound healing, re-epithelialization, the number of neovascular formations, and also increased the deposition of collagen while down-regulating the inflammatory markers [194].

As per the systematic review conducted by Prasai et al. [195], which comprised 51 studies (32 mice and 19 rats) for identifying the efficacy of exosomes in treating dermal wounds, found that the exosomes improve wound healing by stabilizing and stimulating a diverse set of mediators activated during each phase [195]. These findings were consistent in all the studies, irrespective of the model used, route of administration, source, and concentration of exosomes [195]. This literature is also supported by a systematic review conducted from 2018 to 2023. The review, which was focused on exosome-based therapies for wound healing, revealed that exosome-based treatments improve wound healing by increasing angiogenesis, re-epithelialization, collagen deposition, and decreasing scar formation [196,197]. Additionally, the modified stem cell exosomes provide greater benefits than the existing exosomes therapy, providing a future perspective for bio-engineered and genetically modified exosomes [198].

Despite promising findings from various studies, the development of exosome-based therapies faces several challenges [199]. In clinical settings, the potential drawbacks of exosome-based treatments may, in some cases, outweigh their benefits. Difficulties in achieving a consistent therapeutic effect persist throughout the MSC-exosome development process, posing significant hurdles to their translation into effective medical applications.

Numerous studies have been carried out to assess the biodistribution of exosomes derived from various sources and their therapeutic applications across different disease conditions. However, further research is needed to elucidate the precise mechanism of action of MSC-derived exosomes at each stage of the healing process. The data regarding ongoing as well as completed clinical trials is discussed in Table 5. MSC-exosomes must reach their target location in a precise spatiotemporal manner to exert specific therapeutic effects. Currently, there are no definitive markers available to detect and analyze the biodistribution of MSC-derived exosomes. Additionally, genetic manipulation of donor MSCs is often used to down-regulate specific RNAs or proteins in MSC-exosomes, which may lead to significant alterations in their composition and therapeutic properties [23].

Table 5.

Clinical trials investigating the therapeutic potential of exosome-based treatments in wound healing.

Sr.NO	Clinical Study Name	ClinicalTrials.gov ID	Type of Study	Phase	Source of Exosomes	Disease condition	References
1	Pilot Study of Human Adipose Tissue Derived Exosomes Promoting Wound Healing	NCT05475418	one-arm pilot study	notapplicable	Adipose tissue	Wounds & Injuries	https://clinicaltrials.gov/search?cond=exosomes&term=Wound%20Healing
2	Effect of Plasma Derived Exosomes on Cutaneous Wound Healing	NCT02565264	an open prospective clinical trial	Early phase 1	plasma-derived exosomes	Ulcers	https://clinicaltrials.gov/study/NCT02565264?cond=exosomes&term=Wound%20Healing&rank=2
3	Efficacy and Safety of Wharton’s Jelly-Derived Mesenchymal Stem Cell Exosomes in the Treatment of Diabetic Foot Ulcers: a Double-blinded Randomized Controlled Clinical Trial (WJ-MSC)	NCT06812637	Double-blinded Randomized Controlled Clinical Trial	Phase 1	Wharton’s Jelly-Derived Mesenchymal Stem Cell Exosomes	Diabetic Foot ulcers	https://clinicaltrials.gov/study/NCT06812637?cond=exosomes&term=Wound%20HealingLimit=100&page=1&rank=3
4	Evaluation of Personalized Nutritional Intervention on Wound Healing of Cutaneous Ulcers in Diabetics	NCT05243368	triple-blind randomized controlled trial	not applicable	MSC-derived Exosomes	cutaneous ulcers in diabetes	https://clinicaltrials.gov/study/NCT05243368?cond=exosomes&term=Wound%20Healing&limit=100&page=1&rank=4
5	The Role of Mesenchymal Stem Cell and Exosome in Treating Pilonidal Sinus Disease in Children	NCT06391307	Prospective Randomized Controlled Trial	not applicable	MSC-derived Exosomes	Pilonidal Sinus Disease	https://clinicaltrials.gov/study/NCT06391307?cond=exosomes&term=Wound%20Healing&limit=100&page=1&rank=5

The various primary MSC sources, methodologies of isolating, purifying, and detecting exosomes, as well as the need for a consistent definition or description of MSC exosomes, must all be carefully considered [200]. Furthermore, factors such as local variables, distribution route, and the timing of intervention can influence the therapeutic efficacy of MSC-exosomes. The culture medium used may also yield non-MSC-derived exosomes [201]. After the administration of MSCs into the body, their biological effect should only occur after absorption by the targeted cells. Otherwise, immune cells may deplete MSC-exosomes in the bloodstream. MSCs can be administered through several routes, including intravenous (IV), subcutaneous (SC), and intraperitoneal (IP) injection. There are also limitations to administering exosomes. Exosomes administered systemically may trigger an immediate inflammatory response and rapid clearance from the bloodstream, leading to inadequate retention time and diminished homing ability. Locally injected exosomes may have a significantly shorter half-life and lower bioavailability due to flushing by body fluid, which results in poor exosome retention [202].

7. Conclusion

The captivating features of exosomes in promoting healing, controlling inflammation, stimulating new blood vessel growth (angiogenesis), and aiding tissue remodeling render them highly promising for regenerative medicine and MSC therapy. These attributes stand in stark contrast to conventional treatment methods, which face barriers such as immune rejection, a lack of donor tissues, and prolonged recovery times. However, several obstacles remain for translating exosome therapies into clinical practices, including variability in isolation methods, rapid clearance from circulation, challenges in large-scale manufacturing, targeted delivery, standardization, and potential immunogenicity. These issues must be addressed to ensure successful clinical application of exosome-based therapies. Future research should focus on bioengineering strategies such as hydrogel-exosome encapsulation and genetic modification to enhance stability and targeting. Despite existing regulatory, technical, and translational challenges, the expanding research, with evolving policy frameworks and ongoing advancements in MSC-derived exosome therapy hold strong potential to become a scalable and effective therapy for wound healing and tissue regeneration, helping bridge the gap from preclinical to clinical application and accelerate the translational progress.

8. Future perspective

Exosome-based therapies for burn wounds hold significant promise for the future. Overcoming challenges such as low effective concentration, instability, variable composition, and poor therapeutic benefit through various bioengineering strategies could lead to more potent therapies. Highly specialized mesenchymal stem cell (MSC) exosomes, capable of delivering proteins, drugs, molecules, or genetic material, represent a powerful approach to tissue regeneration. These exosomes can be engineered to specifically target cells or tissues, enhancing their therapeutic effects by enriching endogenous chemicals [203]. An innovative technique in controlled drug delivery systems is the hydrogel-exosome encapsulation technique [204]. This technology has the potential to enhance tissue regeneration, reduce inflammation, accelerate healing, and provide a sustained release of exosomal cargo, particularly in the treatment of burn wounds. As research progresses, integrating controlled-release 3D adipose-derived MSC (ADMSC)-derived exosome-loaded hyaluronan hydrogels and similar bioengineering techniques could revolutionize burn treatment, offering more effective, targeted, and regenerative solutions for patients [205]. Advancement in bioreactors and microfluidics will facilitate scalable and cost-effective methods for producing exosomes with consistent therapeutic potency and purity, addressing the limitations of current manufacturing processes. The incorporation of personalized therapies, through engineering exosomes to carry the bioactive molecules tailored to individual patient needs, based on severity of wounds, wound characteristics, and comorbidities, will enhance treatment outcomes. Also, the integration of exosomes with modern biomaterials, including 3D printed scaffolds, nanofibers, and hydrogels, has great promise in the creation of advanced smart dressings that allow for real-time monitoring of the wound, as well as controlled and gradual exosome release.

The therapeutic application of exosomes in the wound-based therapies have shown remarkable promise in recent years; however, significant challenges still exist that need to be addressed before these cell-free therapies can transition from experimental models to clinical practice for treatment of wounds. Even though the exosomes are considered to have low immunogenicity as compared to their parent MSCs, the wound environment especially in the case of chronic or infected wounds may alter their immune compatibility. The repeated administration of exosomes derived from allogeneic sources can still carry donor antigens that may provoke immune responses. Furthermore, there are chances that exosomes may interact unpredictably with the inflammatory milieu of chronic wounds, thereby exacerbating or dysregulating the immune response. The use of exosomes also demands long-term safety concerns that persist due to the off-target effects and unintended interactions of the exosomes within the skin tissue. One of the major challenges in clinical translation is the lack of standardized protocols for the isolation, purification, characterization and quality control of the exosomes. For example, the commonly used isolation methods for exosomes such as ultracentrifugation and polymer precipitation can result in the formation of exosomes but with different exosomal compositions and bioactivities. The ethical implications in using the MSCs derived from perinatal tissues, although offer the source of exosomes with rich exosomal content, issues exist around the donor consent, cultural acceptability, traceability which may influence ethical and regulatory scrutiny. Moreover, if genetically modified MSCs are used to enhance the potency of exosomes another layer of ethical scrutiny arises, particularly in terms of gene editing and long-term genetic effects. Although numerous preclinical studies have shown encouraging results of exosomes in wound healing, only a limited number of studies have progressed to early phase clinical trials thereby reflecting a wide translational gap driven by long term safety, undefined therapeutic window, and variable outcomes across the animal models. Additionally the complexity in the production of exosomes and their quality assurance contributes to high development costs, limiting their accessibility. Overall, advancement in this next generation therapy from bench to bedside could revolutionize the area of wound care by offering safer, cell-free and targeted regenerative solutions.

This study highlights the therapeutic potential of MSC-derived exosomes in wound healing and stresses how important delivery methods are crucial for increasing their clinical usefulness. By investigating bioengineered systems such as hydrogels and 3D-printed scaffolds, it demonstrates how better distribution might increase the efficacy of exosomes. To get these therapies closer to clinical translation and better patient care, standardized manufacturing and thorough clinical validation are crucial next steps.

Funding Statement

No funding is received for the work.

Article highlights

• A comprehensive review of exosomes derived from mesenchymal stem cells (MSCs) as potentially effective cell-free therapeutics for wound healing.

• Emphasizes the significance of delivery strategies in maximizing exosome therapeutic efficacy, which is often overlooked.

• Explores bioengineering techniques such as hydrogels, 3D-printed scaffolds, and controlled-release systems to improve exosome stability, localization, and sustained release.

• Issues addressed include inconsistencies in exosome composition, immunological compatibility difficulties, a lack of standardized isolation and quality control techniques, and high manufacturing costs.

• Recognizes the slow progression from promising preclinical data to clinical trials, emphasizing safety, reproducibility, and regulatory hurdles.

• Discusses emerging technologies like scalable manufacturing, personalized exosome modification, and integration with smart biomaterials to revolutionize wound care.

• Calls for standardized production methods, robust clinical evaluations, and clear regulatory frameworks to accelerate clinical adoption of exosome-based therapies.

• Envisions improved, targeted, and safer regenerative treatments for acute and chronic wounds, advancing the field of regenerative medicine.

Author contribution

Conceptualization: Vinita Patole; Investigation: Vinita Patole, Divya Kakade, Shantanu Date; Writing Original draft: Divya Kakade, Shantanu Date, Vinita Patole; Writing Review and Editing: Divya Kakade, Shantanu Date, Vinita Patole, Prabhanjan Giram, Avinash Sanap, Ajinkya Aher, Surekha K. Satpute; Revision: Vinita Patole, Ganesh Ingavle, Pawan Karwa; Supervision: Ganesh Ingavle, Prabhanjan Giram, Vinita Patole, Avinash Sanap; Project administration: Ganesh Ingavle, Vinita Patole, Prabhanjan Giram.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17460751.2025.2561449