Matrix-Bound Nanovesicles: The Extracellular Matrix Vesicle

1. Introduction

The extracellular matrix (ECM) serves as both the structural framework and signaling hub for cells, creating the microenvironment necessary for cellular function. This critical role of the ECM stems from a highly complex and finely tuned composition of proteins, lipids, glycans, cytokines, chemokines and growth factors [1, 2, 3]. These ECM components and their interactions have been extensively studied [4, 5, 6], leading to the development of regenerative medicine tools, usually in the form of biomaterials. These biomaterials offer natural biocompatibility and low immunogenicity and can be tailored for specific applications. Examples include isolated proteins (i.e., collagen [7, 8, 9], fibronectin [9], elastin [10, 11]), glycosaminoglycans (i.e., hyaluronic acid [12, 13], chondroitin sulfate [14]), and decellularized biologic scaffolds (small intestinal submucosa [15, 16], urinary bladder [17, 18], dermis [19, 20, 21], peritoneum [22, 23]), which have been extensively utilized in clinical applications [4]. Despite decades of ECM research, a previously unidentified component was discovered less than ten years ago: the matrix-bound nanovesicle (MBV, Figure 1). First described in 2016 [24], MBV are a distinct extracellular vesicle (EV) subtype (100–200 nm) found covalently attached to decellularized biologic scaffolds and present in virtually all mammalian tissues. While much about MBV remains unknown, their existence introduces a new layer of complexity to the composition and function of ECM and expands the ECM regenerative medicine toolbox. In this chapter, we compile current knowledge on MBV composition, biogenesis, biological significance and therapeutic potential.

2. The extracellular matrix bioactivity

The ECM was initially thought to be a simple scaffolding that provided structural support for cell processes such as migration and proliferation [25]. However, this view evolved when it became clear that the ECM influences cell behavior, with different tissues and phenotypes being shaped by the specific tissue microenvironment. For example, basement membranes, which are rich in ECM components like collagen type IV, collagen type VIII, laminin and nidogen, play a critical role in driving the proliferation and phenotype of epithelial and endothelial cells [26, 27]. Additionally, in vitro studies have shown that isolated ECM components or solubilized ECM extracts [28] can modulate the cell phenotype.

It is now evident that the ECM provides biological signaling to resident cells while supporting their growth. This signaling occurs through receptors on the cell membrane, which activate various signaling pathways and cascades. For instance, integrins can recognize specific motifs from different ECM components [29] and modulate their phenotype through kinase cascades. Another key ECM mechanism that modulates cell behavior is mechanotransduction, where the mechanical properties of ECM, such as stiffness, are recognized by transmembrane receptors, including integrins [30]. These receptors activate pathways like YAP/TAZ and Hippo [31, 32], which govern multiple cellular responses. Growth factors also interact with ECM components, such as fibronectin [33] or glycosaminoglycans [34], providing additional bioactivity in the ECM that modulates cell behavior. Finally, peptides derived from ECM degradation during processes like inflammation, ECM remodeling and angiogenesis are known to influence cell phenotype [35, 36]. In fact, the release of these peptides, growth factors, and cytokines is a key factor in the beneficial effects of ECM bioscaffolds after implantation [37]. Overall, the ECM is a complex, bioactive substrate that modulates cell behavior. Moreover, many pathways modulated by the ECM through transmembrane receptors are tightly linked to the deposition and alteration of ECM components [25, 38, 39]. These pathways can also activate the expression of enzymes that degrade the ECM, such as matrix metalloproteinases [40] (MMP-1, MMP-3, MMP-9). This bi-directional crosstalk between the cell and its environment, often referred to as cell-ECM dynamic reciprocity [2, 41], underscores the of ECM composition and structure on cell development, wound healing and tissue homeostasis.

The discovery of MBV embedded within the ECM introduces new possibilities for understanding how cell-ECM interactions are modulated. Although MBV share morphologic features similar to all EV, their compartmentalization within the matrix suggests a unique biogenesis pathway. Additionally, the attachment of MBV to the ECM suggests that cells responsible for ECM production may also be involved in attaching these vesicles to the matrix, potentially altering its properties. Moreover, the covalent attachment of MBV to the ECM suggests that cells may have specialized machinery for this task, distinct from the EV released into fluids. While these mechanisms are only partially understood, recent research in EV biology has aided in advancing our knowledge of vesicle formation and function. Herein, we will discuss the composition and functions of MBV integrated into the ECM, highlight how they differ from fluid phase-vesicles, and explore potential biogenesis pathways for EV that interface with the ECM. We will also review the known roles of EV in these contexts, focusing on how they affect cell phenotype and how the matrix may influence these effects.

3. Extracellular vesicles and the extracellular matrix

To date, research on EV has largely focused on their role as potent mediators of cell signaling, as they are capable of moving freely between cells and distant sites, using biological fluids as a mobile medium. Indeed, the identification of EV was documented for the first time in 1967 when transmission electron microscopy revealed lipid-rich nano particles extruding from platelets, called “platelet dust” [42]. Shortly after, vesicles were also found in the synaptic fluid bordering nerve endings in the mouse atrium [43] and in fetal bovine serum [44]. However, it wasn’t until 1981 that the term “exosome” was proposed to describe vesicles ranging from 40 to 1000 nm secreted in the cell culture supernatant from both normal and neoplastic cell lines [45]. While these early studies documented the presence of EV in body fluids or cell culture supernatants, it wasn’t until pioneering studies in the 1980s that exosome formation, derived from multivesicular endosomes fusing with the plasma membrane and releasing their contents through exocytosis, was shown [46, 47, 48]. Breakthrough studies in the 1990s and early 2000s demonstrated for the first time that EV play a role in cell-to-cell communication—specifically, exosomes enriched with MHC class II released from B cells were shown to present antigens to T cells [49]. This was followed by the demonstration of exosome-mediated exchange of RNA and protein between cells in 2006 [50, 51], a phenomenon which would eventually be exploited in vitro to produce a “recombinant exosome” that incorporates recognition or homing molecules for tissue-specific targeting [52]. Over the past two decades, the EV biology field has seen significant acceleration of investigations concerning the role of EV as mediators of cell-to-cell communication within a liquid modality. However, the presence of EV within solid substrates (e.g. ECM of soft tissue, and cartilaginous structures of bone) and their role in tissue homeostasis and repair, matrix remodeling, and immunomodulation is an emerging paradigm in the field of EV biology with significant implications toward novel therapeutic and diagnostic uses of nanovesicles.

Numerous studies have shown that EV contain surface proteins and ECM molecules that can mediate binding with cells and ECM molecules. However, the concept of a unique subpopulation of EV embedded within the fibrillar network of the ECM has only recently become an area of interest to the field. In the context of EV biology, the dense, fibrillar nature of the ECM naturally impedes not only intercellular communication, but also EV biogenesis and trafficking across cell membranes. As a result, the widely accepted model for transport of EV between cells—which relies upon a liquid phase—has largely ignored the possibility of EV embedded into the ECM. Although studies have shown that cells secrete heterogeneous subpopulations of EV into the cell culture supernatant and ECM [53], these studies often rely on traditional in vitro cell culture models that fail to distinguish between the EV secreted into the culture supernatant and those trapped within the ECM produced by the cells. Recently, it was shown using a 3T3 fibroblast model that specific populations of EV could be harvested based on their compartmentalization into either the liquid-phase cell culture medium or the solid-phase ECM substrate [53]. Under long-term culture, 3T3 fibroblasts deposit an underlying layer of ECM that can be harvested using established decellularization protocols to carefully remove the cells and cellular debris while preserving the composition and ultrastructure of the ECM [4, 53, 54, 55]. This decellularized ECM, along with the MBV it contains, can be isolated and analyzed separately from exosomes harvested from the cell culture supernatant. Comprehensive lipidomics and RNA sequencing of these MBV isolated from the ECM of 3T3 fibroblasts revealed a distinct miRNA and lipid profile compared to exosomes in the cell culture supernatant and the parent cell (Figure 2A). These findings suggest that molecular sorting occurs during vesicle biogenesis, distributing specific miRNA and lipids to vesicles destined for different extracellular locations.

In addition to in vitro produced vesicles, EV have also been isolated directly from tissues and organs. Over the past decade, there has been a growing body of literature highlighting methods of isolating and characterizing a subpopulation of EV from brain and central nervous system tissue [56, 57, 58, 59, 60, 61, 62, 63, 64]. While these studies have shown that EV can be isolated from whole tissue, these approaches are heavily limited by the inability to differentiate between vesicles embedded into the ECM of the tissue parenchyma from exosomes secreted into tissue fluid, and the inability to differentiate between cellular contaminants introduced during the tissue homogenization process. Furthermore, a separate class of lipid nanovesicles (20–200 nm) have been identified as a unique subset of small EV that play vital roles in the mineralization of cartilage, bone and dentin [65, 66]. Unlike other EV, these mineralization vesicles release their bioactive cargo directly into the ECM, rather than to neighboring cells [67]. Given these unique attributes, mineralization vesicles are often reported as a class of vesicle completely independent from exosomes whose function focuses on the transmission of their bioactive cargo to recipient cells. The first clear evidence that EV are stably and functionally integrated into the ECM of tissues and organs came from studies isolating lipid-bound nanovesicles from decellularized mammalian tissue [24]. Decellularization of the native tissue carefully removes the cellular component while maintaining the underlying ultrastructure and composition of the ECM [53]. Transmission electron microscopy of decellularized urinary bladder showed the presence of nanometer-sized lipid vesicles tightly associated within the fibrillar matrix, and subsequent enzymatic degradation of the matrix was required to release these vesicles from the ECM. Given their intimate association with the ECM, these vesicles were termed matrix-bound nanovesicles (MBV) [24]. The fact that these nanovesicles remain tightly integrated within the ECM, even after decellularization with detergents and chaotropic agents and extensive washing, underscores their stable integration within the matrix. This also addresses concerns regarding passive inclusion of fluid-phase exosomes that may bind to the superficial surface of the ECM, or cellular organelles which may contaminate EV preparations from whole tissue. However, isolating MBV from ECM remains challenging due to the complex ultrastructure of ECM molecules. Different isolation methods, such as papain [56, 59, 60, 62, 64], collagenase [58, 61], proteinase K, pepsin [24], and mechanical agitation with a chaotropic salt [57] have been employed to disrupt the ECM and release EV. Unfortunately, these methods can affect the integrity of vesicle surface markers, potentially altering their biochemical composition and biological function. In fact, a recent study showed that the choice of enzyme used to digest the matrix has a significant impact on MBV yield, purity, cellular uptake, and biological activity [68].

MBV have demonstrated the ability to reproduce the functional and phenotypical characteristics of the ECM from which they are derived. This includes influencing stem cell fate and inducing an anti-inflammatory, wound-healing macrophage phenotype—both distinct processes of constructive tissue remodeling [24, 53, 69, 70, 71, 72]. Unlike EV secreted into body fluids, which are readily available for cell-cell communication, MBV remain tightly associated with the tissue ECM and require matrix degradation to be released. This requirement for ECM breakdown may be integral to their mechanism of action. In addition to their role as potent mediators of immunomodulation, MBV may also serve as delivery vehicles for ECM components. Numerous studies have reported various ECM components as EV cargo such as ECM remodeling enzymes (aggrecanase, heparanase, neutrophil elastase, transglutaminase, lysyl oxidase, and matrix metalloproteinases) [73, 74, 75, 76, 77, 78, 79] and other ECM molecules, like fibronectin [80, 81, 82, 83], hyaluronic acid [84, 85], syndecan [86], glypican [87, 88, 89], tenascin C and nidogen [90].

4. MBV biogenesis and composition

There is increasing evidence that cells can selectively release unique populations of EV into either liquid substrates or into dense fibrillar ECM. The ability of cells to distinguish between liquid and solid environments and to selectively deposit distinct vesicle subpopulations—with unique lipid compositions—into these different substrates, suggests that MBV may follow a unique and independent biogenesis pathway from EV secreted into a liquid phase [53, 91]. In epithelial cells, experimental evidence for independent mechanisms of apical or basolateral EV release has been shown using Madin-Darby canine kidney (MDCK) cells grown on cells culture inserts [92]. For instance, basolateral nanovesicle release was shown to rely on the sphingomyelinase-dependent ceramide production machinery, while apical vesicle release was shown to rely on an ESCRT (endosomal sorting complexes required for transport)-related protein, ALIX. Thus, polarized vesicle release from MDCK epithelial cells is governed by two distinct mechanisms: the ALIX–Syntenin1–Syndecan1 complex on the apical side and the sphingomyelinase-dependent ceramide production pathway on the basolateral side [92]. Moreover, enrichment of Wnt proteins within the different EV populations have demonstrated dependence on cell polarity [93]. Similar results have been observed in intestinal epithelial cells, where EV were shown to be differentially secreted from the apical and basolateral sides (Figure 2C) [94]. Interrogation of the molecular cargo of these two populations revealed that, although there were commonalities in the proteomic signature, EV released from the apical side of cells were enriched in molecules involved in apical shedding of endosomes. In contrast, basolateral EV were enriched with cell adhesion molecules [94]. In human cholangiocytes, epithelial cells which line bile ducts, polarized cultures have shown significant differences in the composition and yield of apically and basolaterally released EV [95]. Importantly, polarized cholangiocyte apical EV were notably enriched in cholesterol compared with basolateral EV, further supporting the idea that the membrane sources for apical and basolateral EV are maintained separately [95].

While these 2D cell culture studies, and others [92, 96], show that differential populations of EV are secreted from apical or basolateral sides of cells into the surrounding environment, 3D cell culture offers a more dynamic perspective on how vesicles are secreted into solid or fluid phase environments. For example, human colon carcinoma LIM1863 cells, grown as free-floating multicellular organoids with a central lumen, were shown to release two distinct EV populations. These populations were differentiated based on their enrichment of apical or basolateral sorting proteins [97]. Furthermore, MDCK cells cultured in a 3D matrix from a basement membrane extract, which more closely recapitulated the physical restrains of the ECM, showed the secretion of hyaluronan-coated EV from the basal side of epithelial cysts into the surrounding matrix [85]. These in vitro cell culture studies clearly show that cells can deposit a unique population of EV into the ECM with differing molecular cargo and functions compared to EV secreted into cell culture supernatant.

While these studies highlight independent biogenesis mechanisms for apical and basolateral EV released from epithelial cells, more research is needed to determine if similar mechanisms exist for cells that reside in the interstitial matrix like fibroblasts, which do not experience the liquid interphase that is typical for epithelial cells. Furthermore, while published in vitro studies of apical or basolateral release of EV have been conducted using cell culture inserts, these methodologies are lacking a true ECM component. Recent studies have shown that under long-term culture, fibroblasts will deposit a layer of ECM that is rich in MBV [53]. Importantly, lipidomic analysis revealed that MBV deposited into the ECM have a significantly different lipid composition compared to EV secreted into the cell culture supernatant, again suggesting that these two populations follow independent biogenesis pathways. Although the deposition of EV directly into the ECM has been observed [53, 85], the mechanisms by which the fibrous and dense network of the ECM regulates and restricts MBV deposition and release, are still largely unexplored.

Beyond their distinct biogenesis pathways, MBV and liquid-phase EV exhibit unique molecular signatures, including differences in lipid, protein, and RNA cargo (Table 1). Lipidomic analysis revealed that MBV possess a highly distinct lipid profile compared to liquid-phase EV, with notable enrichment of phospholipids in MBV [53]. Specifically, MBV contain significantly higher levels of phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), bis-monoacylglycerophosphate (BMP), polyunsaturated fatty acids (PUFAs), particularly the oxygenated species PUFA species. Additionally, cardiolipin (CL), arachidonic acid (AA), docosahexaenoic acid (DHA), and docosapentaenoic fatty acids (DPA) are strongly enriched in MBV relative to the EV collected from cell culture media [53]. The presence of CL in MBV is particularly interesting, as CL is primarily localized to mitochondrial membranes, suggesting a potential link between MBV biogenesis and the mitochondria. In addition to lipid differences, MBV exhibit a distinct protein profile (Table 1). Silver staining of proteins separated via electrophoresis demonstrated clear differences between liquid-phase EV and MBV protein cargo. Immunoblot analysis further confirmed these differences, showing a marked decrease in exosomal markers, like CD63, CD81, and CD9 in MBV [24, 53]. Another study that analyzed MBV surface marker expression using an exosome antibody array also demonstrated a decrease in expression of CD81 and CD63, as well as other surface markers commonly expressed by exosomes, like ICAM-1, ALIX, and ANXA5 compared to exosomes [98]. Among all surface markers tested, CD81 had the highest expression in MBV [98]. While further studies are needed to comprehensively characterize MBV-associated surface and luminal proteins compared to liquid-phase EV proteins, these findings reinforce the concept that MBV represent a distinct vesicle population within the ECM. Beyond lipid and protein composition, MBV also demonstrate significant differences in RNA cargo compared to liquid-phase EV. Next-generation RNA sequencing revealed that RNA molecules between 100 and 200 nt significantly decreased in MBV, while 28 miRNAs are differentially expressed in MBV compared to the fluid-phase EV. Among these, miR-163-5p, miR-27a-5p, and miR-92a-1-5p are notably enriched in MBV, whereas miR-451a, miR-93b-5p, miR-99b-5p are significantly reduced relative to liquid-phase EV [53]. Ingenuity Pathway Analysis further demonstrated that the miRNAs enriched in MBV are predominantly associated with organ and system development and function whereas miRNAs enriched in liquid-phase EV are linked to cellular growth, development, proliferation, and morphology [53]. These molecular differences further corroborate the hypothesis that MBV follow a distinct biogenesis pathway from EV and highlight their specialized nature and potential role in ECM- associated signaling.

While MBV are distinct from liquid-phase EV in their biogenesis and molecular cargo, MBV isolated from different tissue sources also exhibit unique compositional profiles (Figure 2B). Comparative analysis of MBV harvested from urinary bladder matrix (UBM), small intestinal submucosa (SIS), dermis, liver, esophagus, and cardiac tissue revealed substantial variation in lipid, protein, and RNA cargo, and size [98]. Quantitative evaluation by nanoparticle tracking analysis demonstrated a range of MBV size depending on the source tissue, with cardiac MBV being the largest (375.6 ± 19.4 nm), while SIS-derived MBV were the smallest (111.7 ± 3.3) [98]. Unique cytokine profiles between MBV isolated from different sources further highlights these tissue-specific differences. Overall, MBV harvested from liver ECM exhibited the highest cytokine expression, while UBM MBV showed the lowest [98]. Despite this variation, certain cytokines, such as ANG-1, IL-28B, RANTES, and TIMP-1, were consistently abundant across all MBV sources, whereas PECAM-1, IFN-α, TGF-α, and IL-13 remained low. However, distinct cytokine signatures were still apparent in MBV from different sources. Liver MBV were enriched in in IL-15, IL-17A, PIGF-2, TGF-β1, and TweakR compared to exosomes, while cardiac, esophageal, and dermal MBVs exhibited elevated levels of VEGF and IL-22 [98]. Additionally, IFN- c, IL-17F, and IL-1α were more abundant in liver MBV than in dermis MBV, and IFN-beta expression was highest in liver MBV compared to those from cardiac, dermis, and SIS⁹⁶. Among the most differentially expressed cytokines, RANTES, Eotaxin-1, and IL-8 displayed the greatest variation across tissue sources [98]. Overall, the cytokine profiling demonstrates that though MBV from different sources have common cytokines, each profile is unique and specific to the tissue from which they were derived [98]. Total RNA composition also differs across MBV sources. SIS-derived MBV contained the lowest concentration of RNA per vesicle, whereas dermis-derived MBV had the highest. Principal component analysis and sequencing further demonstrated that MBV carry tissue-specific miRNA cargo [24, 98]. Notably, MBV from cardiac and liver tissue clustered separately from those derived from soft tissues, like SIS and dermis, suggesting fundamental differences in their miRNA signatures [98]. Collectively, these findings demonstrate that MBV retain signatures reflective of their tissue of origin, offering additional insight into their biogenesis and functional properties.

Given that MBV integrate within the ECM’s dense, fibrous network, it is plausible that they are secreted concurrently with ECM components during processes like matrix deposition, tissue development, homeostasis maintenance, and dynamic remodeling in response to injury. For example, studies examining the formation of collagen fibrils have identified the presence of lipid nanovesicles. During collagen synthesis, collagen fibrils are shuttled to the ECM via plasma membrane protrusions, called “fibripositors” [100, 101]. The tip of this “fibripositor” is the site of fibril deposition to the ECM. Transmission electron microscopy has shown that EV are deposited into the ECM during fibril deposition, though they are not immunoreactive for collagen, suggesting that they are not collagen-specific secretory vesicles. Interestingly, invadopodia—actin-rich subcellular structures formed by migratory cells that protrude into and degrade ECM—have been identified as sites of polarized secretion of exosomes [102]. The release of hyaluronan-coated EV from 3D organoids also points to concerted EV release during matrix synthesis. It was found that cells with high levels of hyaluronan synthesis release large amounts of HA-coated EV from their basal surface into the ECM [85]. In cancer cells, EV secretion containing the ECM molecule fibronectin, was found to be essential for directional and efficient cell migration by reinforcing transient polarization states and promoting adhesion formation [81]. More recently, it was shown that Cav1 modulates EV cargo sorting and segregation of EV subpopulations, specifically facilitating the incorporation of ECM components, including Tenascin-C (TnC), through a cholesterol-dependent mechanism [90]. This suggests that exosome secretion is necessary for the deposition of ECM components.

Overall, identifying and characterizing the MBV subpopulation presents exciting possibilities for EV-based therapeutics, particularly in the design of innovative biomaterials and the engineering of synthetic EV for clinical translation. Given the enrichment of specific lipid and molecular cargo within MBV, additional investigation into their biogenesis could be fundamental in enhancing EV cargo-loading strategies or in integrating EV into ECM-based biomaterials for use as inductive scaffolds in tissue repair.

5. Therapeutic uses of MBV

Given their nanometer size, potent immunomodulatory properties, and ability to mediate constructive remodeling outcomes independent of the parent ECM [103], MBV can be utilized in clinical applications previously unfeasible for ECM sheets or hydrogels, such as intravenous injections or microinjections into the CNS (Figure 3A). MBV can be isolated from FDA-approved ECM bioscaffolds on the market that have been safely used in millions of patients, thus presenting a low clinical risk of adverse side effects [24]. Simply stated, millions of patients implanted with an ECM scaffold over the last twenty years have received a therapeutic dose of MBV. Recognizing the presence of MBV within ECM bioscaffolds and understanding their biologic activity provides opportunities for new therapeutic approaches. Thus, MBV are a strong candidate for a clinically translatable, regenerative medicine intervention.

In this regard, preclinical studies have already demonstrated MBV cytocompatibility in vitro and biocompatibility in vivo across several routes of administration [104], similar to other EV [105]. Cellular uptake of MBV has also been confirmed in various cell types, including osteoblasts, fibroblasts, mesenchymal stem cells and perivascular stem cells [104, 106]. However, macrophages are undoubtedly the most extensively studied cell type in MBV interactions (Figure 3B) [68, 69, 70, 98, 107]. The ability of MBV—and consequently ECM bioscaffolds—to shift macrophages and other myeloid cells toward a pro-remodeling phenotype is thought to be a key driver of their therapeutic potential. For example, in a mouse model of influenza infection, systemic MBV administration reduced mononuclear cell infiltration, dampened inflammatory phenotypes and decreased systemic production of cytokines [108], resulting in reduced pulmonary inflammation. Similarly, in an optic nerve regeneration model, MBV (in combination with fluvastatin) promoted axon growth and neuroprotection by modulating neutrophils and mononuclear cells [109, 110]. MBV from different decellularized tissues have also been shown to support CNS regeneration [111]. In another model, MBV administration—both locally and systemically—mitigated rheumatoid arthritis-related inflammation, shifting macrophages toward an M2-like phenotype via CD43hi/His48lo/CD206+ monocytes [112]. Across these studies, MBVs have also been shown to influence T-cells, fibroblasts, and neurons at genetic and marker expression levels. However, it remains unclear whether MBV directly target differentiated tissue cells (e.g., synoviocytes, lung fibroblasts, or neurons) or if their effects are primarily mediated through myeloid cells. Alternatively, the robust immune response triggered by MBVs could be overshadowing any direct effects on these cells. A recent study also reported the interaction of MBV with osteoclasts in vitro and an amelioration of osteolysis in a mouse calvarial particulate-induced osteolysis model [106], showing a decrease in inflammation but not a direct change in myeloid cells populations. However, the effect on tissue resident macrophages was also hypothesized by the authors.

Notably, in several studies, MBV-driven shifts in myeloid cell phenotypes persisted long after administration. For instance, Crum et al. observed MBV-induced changes in myeloid populations more than 50 days after the final MBV dose, in a rheumatoid arthritis model [112]. This suggests that MBVs may induce a form “memory,” in myeloid cells, commonly referred to as “trained immunity” [113, 114]. Trained immunity is driven by epigenetic modifications—heritable but reversible changes to DNA or chromatin structure that alter gene expression without modifying the genetic sequence [114]. What’s more, this trained immunity phenomena has been observed in a recent study assessing the effect of MBV on adaptative immunity [115]. In this study, it was demonstrated that MBVs not only modulate immune responses without inducing immunosuppression but may also imprint long-term memory in macrophages [115]. While the epigenetic effects of other EV on immune cells has been reported [116, 117], direct evidence for MBV-driven epigenetic remodeling remains to be seen. However, if confirmed, this would provide valuable insight into the regenerative mechanisms of ECM bioscaffolds and expand MBV applications as a new immunomodulatory therapy.

6. Conclusion and future directions

Conceptually, the investigation of an EV population embedded within the ECM is unique among studies addressing EV biology. MBV were originally identified as potent bioactive factors within FDA-approved ECM bioscaffolds used clinically for regenerative medicine applications [24]. Over the past decade, molecular and cellular-based studies have established MBV as a unique class of vesicle bound within the collagen network of the ECM within mammalian soft tissues and organs. While substantial progress has been made in (i) developing MBV isolation techniques for various tissues and cell culture sources, (ii) characterizing recipient cell responses to MBV, and (iii) demonstrating their regenerative potential in preclinical studies, major gaps in our understanding of MBV biology remain. These include understanding their biogenesis pathway, core-proteome and co-secretome, intra-tissue heterogeneity, and how their composition and function change with aging and disease. Given the enormous potential for MBV in regenerative medicine applications, a deeper understanding of their basic biology is warranted and essential to guide the development of the next generation of biomaterials and engineered EV-based therapeutics. For example, given the selective loading of specific molecular cargo within MBV, Understanding MBV biogenesis could guide new strategies for cargo loading or for integrating EV into solid-state biomaterials used as an inductive substrate for tissue repair. Furthermore, defining MBV’s role in tissue homeostasis, disease, and regeneration will provide a springboard for future research focusing on targeting key signaling pathways involved in these processes. Although MBV are not FDA approved as a stand-alone therapy, they are naturally present in all commercially available ECM bioscaffolds. Simply put, every patient who has received an ECM scaffold over the last twenty years [4] has received a therapeutic dose of MBV. Recognizing the presence of MBV within ECM bioscaffolds and understanding their biologic activity provides opportunities for new therapeutic approaches. Maximizing the full clinical potential of MBV, and EV in general, requires a comprehensive understanding of their intricate biology, which will directly guide the design and application of vesicle-based therapeutics. These findings could lead to the development of MBV-driven therapeutic strategies that direct endogenous immune cells to orchestrate immunomodulation and constructive remodeling after injury. Separately, ECM-based therapies can now be re-evaluated through a new lens, offering insights that will drive the design and development of next-generation diagnostic and therapeutic technologies.

Conflict of interest

PL declares no conflict of interest. HC is employed by Viscus Biologics LLC, GSH is VP at ECM Therapeutics.