Advances, challenges, and future directions in the clinical translation of ECM biomaterials for regenerative medicine applications.

Extracellular Matrix (ECM) scaffolds and biomaterials have been widely used for decades across a variety of diverse clinical applications and have been implanted in millions of patients worldwide. ECM-based biomaterials have been especially successful in soft tissue repair applications but their utility in other clinical applications such as for regeneration of bone or neural tissue is less well understood. The beneficial healing outcome with the use of ECM biomaterials is the result of their biocompatibility, their biophysical properties and their ability to modify cell behavior after injury. As a consequence of successful clinical outcomes, there has been motivation for the development of next-generation formulations of ECM materials ranging from hydrogels, bioinks, powders, to whole organ or tissue scaffolds. The continued development of novel ECM formulations as well as active research interest in these materials ensures a wealth of possibilities for future clinical translation and innovation in regenerative medicine. The clinical translation of next generation formulations ECM scaffolds faces predictable challenges such as manufacturing, manageable regulatory pathways, surgical implantation, and the cost required to address these challenges. The current status of ECM-based biomaterials, including clinical translation, novel formulations and therapies currently under development, and the challenges that limit clinical translation of ECM biomaterials are reviewed herein.

Mitigation of influenza-mediated inflammation by immunomodulatory matrix-bound nanovesicles.

Cytokine storm describes a life-threatening, systemic inflammatory syndrome characterized by elevated levels of proinflammatory cytokines and immune cell hyperactivation associated with multi-organ dysfunction. Matrix-bound nanovesicles (MBV) are a subclass of extracellular vesicle shown to down-regulate proinflammatory immune responses. The objective of this study was to assess the efficacy of MBV in mediating influenza-induced acute respiratory distress syndrome and cytokine storm in a murine model. Intravenous administration of MBV decreased influenza-mediated total lung inflammatory cell density, proinflammatory macrophage frequencies, and proinflammatory cytokines at 7 and 21 days following viral inoculation. MBV decreased long-lasting alveolitis and the proportion of lung undergoing inflammatory tissue repair at day 21. MBV increased the proportion of activated anti-viral CD4+ and CD8+ T cells at day 7 and memory-like CD62L+ CD44+, CD4+, and CD8+ T cells at day 21. These results show immunomodulatory properties of MBV that may benefit the treatment of viral-mediated pulmonary inflammation with applicability to other viral diseases such as SARS-CoV-2.

Biocompatibility and biodistribution of matrix-bound nanovesicles in vitro and in vivo.

Matrix-bound nanovesicles (MBV) are a distinct subtype of extracellular vesicles that are firmly embedded within biomaterials composed of extracellular matrix (ECM). MBV both store and transport a diverse, tissue specific portfolio of signaling molecules including proteins, miRNAs, and bioactive lipids. MBV function as a key mediator in ECM-mediated control of the local tissue microenvironment. One of the most important mechanisms by which MBV in ECM bioscaffolds support constructive tissue remodeling following injury is immunomodulation and, specifically, the promotion of an anti-inflammatory, pro-remodeling immune cell activation state. Recent in vivo studies have shown that isolated MBV have therapeutic efficacy in rodent models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV administered independent of the rest of the ECM, the in vitro and in vivo safety and biodistribution profile of MBV remain uncharacterized. The purpose of the present study was to thoroughly characterize the pre-clinical safety profile of MBV through a combination of in vitro cytotoxicity and MBV uptake studies and in vivo toxicity, immunotoxicity, and imaging studies. The results showed that MBV isolated from porcine urinary bladder are well-tolerated and are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive compared to the potent immunosuppressive drug cyclophosphamide. Furthermore, this safety profile was sustained across a wide range of MBV doses. STATEMENT OF SIGNIFICANCE: Matrix-bound nanovesicles (MBV) are a distinct subtype of bioactive extracellular vesicles that are embedded within biomaterials composed of extracellular matrix (ECM). Recent studies have shown therapeutic efficacy of MBV in models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV, the in vitro and in vivo biocompatibility and biodistribution profile of MBV remain uncharacterized. The results of the present study showed that MBV are a well-tolerated ECM-derived therapy that are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive. Collectively, these data highlight the translational feasibility of MBV therapeutics across a wide variety of clinical applications.

3D Bioprinted Patient-Specific Extracellular Matrix Scaffolds for Soft Tissue Defects.

Soft tissue injuries such as volumetric muscle loss (VML) are often too large to heal normally on their own, resulting in scar formation and functional deficits. Decellularized extracellular matrix (dECM) scaffolds placed into these wounds have shown the ability to modulate the immune response and drive constructive healing. This provides a potential solution for functional tissue regeneration, however, these acellular dECM scaffolds are challenging to fabricate into complex geometries. 3D bioprinting is uniquely positioned to address this, being able to create patient-specific scaffolds based on clinical 3D imaging data. Here, a process to use freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting and computed tomography (CT) imaging to build large volume, patient-specific dECM patches (≈12 × 8 × 2 cm) for implantation into canine VML wound models is developed. Quantitative analysis shows that these dECM patches are dimensionally accurate and conformally adapt to the surface of complex wounds. Finally, this approach is extended to a human VML injury to demonstrate the fabrication of clinically relevant dECM scaffolds with precise control over fiber alignment and micro-architecture. Together these advancements represent a step towards an improved, clinically translatable, patient-specific treatment for soft tissue defects from trauma, tumor resection, and other surgical procedures.

Matrix Bound Nanovesicles Have Tissue-Specific Characteristics That Suggest a Regulatory Role.

Recent studies have identified an extracellular vesicle population that is tightly anchored within the extracellular matrix (ECM) of tissues and organs until released by matrix turnover events. Evidence suggests that these matrix-bound nanovesicles (MBVs) are a ubiquitous component of the ECM, raising questions regarding their tissue-specific identity and their biologic function(s). The primary objective of this study was to examine MBVs isolated from six different tissues and compare their physical and compositional characteristics to determine the common and differentially expressed features. Accordingly, the results of this characterization show that while MBVs are a ubiquitous component of the ECM, they contain a protein and microRNA cargo that is tissue specific. The results furthermore suggest that MBVs have an important role in regulating tissue homeostasis.

Immunomodulatory matrix-bound nanovesicles mitigate acute and chronic pristane-induced rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and destruction of synovial joints affecting ~7.5 million people worldwide. Disease pathology is driven by an imbalance in the ratio of pro-inflammatory vs. anti-inflammatory immune cells, especially macrophages. Modulation of macrophage phenotype, specifically an M1 to M2, pro- to anti-inflammatory transition, can be induced by biologic scaffold materials composed of extracellular matrix (ECM). The ECM-based immunomodulatory effect is thought to be mediated in part through recently identified matrix-bound nanovesicles (MBV) embedded within ECM. Isolated MBV was delivered via intravenous (i.v.) or peri-articular (p.a.) injection to rats with pristane-induced arthritis (PIA). The results of MBV administration were compared to intraperitoneal (i.p.) administration of methotrexate (MTX), the clinical standard of care. Relative to the diseased animals, i.p. MTX, i.v. MBV, and p.a. MBV reduced arthritis scores in both acute and chronic pristane-induced arthritis, decreased synovial inflammation, decreased adverse joint remodeling, and reduced the ratio of synovial and splenic M1 to M2 macrophages (p < 0.05). Both p.a. and i.v. MBV reduced the serum concentration of RA and PIA biomarkers CXCL10 and MCP-3 in the acute and chronic phases of disease (p < 0.05). Flow-cytometry revealed the presence of a systemic CD43hi/His48lo/CD206+, immunoregulatory monocyte population unique to p.a. and i.v. MBV treatment associated with disease resolution. The results show that the therapeutic efficacy of MBV is equal to that of MTX for the management of acute and chronic pristane-induced arthritis and, further, this effect is associated with modulation of local synovial macrophages and systemic myeloid populations.

A liquid fraction of extracellular matrix inhibits glioma cell viability in vitro and in vivo.

Suppressive effects of extracellular matrix (ECM) upon various cancers have been reported. Glioblastoma multiforme has poor prognosis and new therapies are desired. This work investigated the effects of a saline-soluble fraction of urinary bladder ECM (ECM-SF) upon glioma cells. Viability at 24 hours in 1, 5, or 10 mg/mL ECM-SF-spiked media was evaluated in primary glioma cells (0319, 1015, 1119), glioma cell lines (A172, T98G, U87MG, C6), and brain cell lines (HCN-2, HMC3). Viability universally decreased at 5 and 10 mg/mL with U87MG, HCN-2, and HCM3 being least sensitive. Apoptosis in 0319 and 1119 cells was confirmed via NucView 488. Bi-weekly intravenous injection of ECM-SF (120 mg/kg) for 10 weeks in Sprague-Dawley rats did not affect weight, temperature, complete blood count, or multi-organ histology (N = 5). Intratumoral injection of ECM-SF (10 uL of 30 mg/mL) at weeks 2-4 post C6 inoculation in Wistar rats increased median survival from 24.5 to 51 days (hazard ratio for death 0.22) and decreased average tumor volume at time of death from 349 mm3 to 90 mm3 over 10 weeks (N = 6). Mass spectrometry identified 2,562 protein species in ECM-SF, parent ECM, and originating tissue. These results demonstrate the suppressive effects of ECM on glioma and warrant further study.

Role of 4-hydroxybutyrate in increased resistance to surgical site infections associated with surgical meshes.

An increased resistance to surgical site infections has been associated with surgical meshes composed of naturally occurring materials, including poly-4-hydroxybutrate (4HB). 4HB is a naturally occurring short-chain fatty acid that has been shown to promote endogenous expression of the Cramp gene coding for the antimicrobial peptide (AMP) cathelicidin LL-37 in murine bone marrow-derived macrophages. The molecular pathways involved in the 4HB-induced cathelicidin LL-37 expression have not yet been identified. The present study showed that transcriptional activation of the Cramp gene by 4HB is independent of inhibition of histone deacetylase (HDAC) activity, and that upregulation of Cramp is modulated by the G-protein coupled receptor GPR109A. Furthermore, an intracellular signaling cascade that promotes the activation of the MAP kinases, p38 and JNK, and a subsequent NF-κB phosphorylation downstream from p38 is essential for the AMP transcriptional response in 4HB-stimulated macrophages. The findings provide a solid scientific basis and rationale for the decreased incidence of surgical site infections with the use of this type of surgical meshes. Further clinical significance is found in the fact that the 4HB activated molecular pathway includes common targets of frequently used nonsteroidal anti-inflammatory drugs (NSAIDs) and other FDA approved drugs recognizing G-protein coupled receptors.

Graft IL-33 regulates infiltrating macrophages to protect against chronic rejection.

Alarmins, sequestered self-molecules containing damage-associated molecular patterns, are released during tissue injury to drive innate immune cell proinflammatory responses. Whether endogenous negative regulators controlling early immune responses are also released at the site of injury is poorly understood. Herein, we establish that the stromal cell-derived alarmin interleukin 33 (IL-33) is a local factor that directly restricts the proinflammatory capacity of graft-infiltrating macrophages early after transplantation. By assessing heart transplant recipient samples and using a mouse heart transplant model, we establish that IL-33 is upregulated in allografts to limit chronic rejection. Mouse cardiac transplants lacking IL-33 displayed dramatically accelerated vascular occlusion and subsequent fibrosis, which was not due to altered systemic immune responses. Instead, a lack of graft IL-33 caused local augmentation of proinflammatory iNOS+ macrophages that accelerated graft loss. IL-33 facilitated a metabolic program in macrophages associated with reparative and regulatory functions, and local delivery of IL-33 prevented the chronic rejection of IL-33-deficient cardiac transplants. Therefore, IL-33 represents what we believe is a novel regulatory alarmin in transplantation that limits chronic rejection by restraining the local activation of proinflammatory macrophages. The local delivery of IL-33 in extracellular matrix-based materials may be a promising biologic for chronic rejection prophylaxis.

Ultrasonic cavitation to prepare ECM hydrogels.

Hydrogels composed of extracellular matrix (ECM) have been used as a substrate for 3D organoid culture, and in numerous preclinical and clinical applications to facilitate repair and reconstruction of a variety of tissues. However, these ECM hydrogel materials are fabricated using lengthy methods that have focused on enzymatic digestion of the ECM with an acid protease in an acidic solution; or the use of chaotropic extraction buffers and dialysis procedures which can affect native protein structure and function. Herein we report a method to prepare hydrogels from ECM bioscaffolds using ultrasonic cavitation. The solubilized ECM can be induced to rapidly self-assemble into a gel by adjusting temperature, and the material properties of the gel can be tailored by adjusting ECM concentration and sonication parameters. The present study shows that ECM bioscaffolds can be successfully solubilized without enzymatic digestion and induced to repolymerize into a gel form capable of supporting cell growth. STATEMENT OF SIGNIFICANCE: ECM hydrogels have been used in numerous preclinical studies to facilitate repair of tissue following injury. However, there has been relatively little advancement in manufacturing techniques, thereby impeding progress in advancing this technology toward the clinic. Laboratory techniques for producing ECM hydrogels have focused on protease digestion methods, which require lengthy incubation times. The significance of this work lies in the development of a fundamentally different approach whereby an ECM hydrogel is rapidly formed without the need for acidic solutions or protease digestion. The ultrasonic cavitation method described herein represents a marked improvement in rheological properties and processing time over traditional enzymatic methods, and may lend itself as a platform for large-scale manufacturing of ECM hydrogels.

Lipidomics and RNA sequencing reveal a novel subpopulation of nanovesicle within extracellular matrix biomaterials.

Biomaterials composed of extracellular matrix (ECM) provide both mechanical support and a reservoir of constructive signaling molecules that promote functional tissue repair. Recently, matrix-bound nanovesicles (MBVs) have been reported as an integral component of ECM bioscaffolds. Although liquid-phase extracellular vesicles (EVs) have been the subject of intense investigation, their similarity to MBV is limited to size and shape. Liquid chromatography-mass spectrometry (LC-MS)-based lipidomics and redox lipidomics were used to conduct a detailed comparison of liquid-phase EV and MBV phospholipids. Combined with comprehensive RNA sequencing and bioinformatic analysis of the intravesicular cargo, we show that MBVs are a distinct and unique subpopulation of EV and a distinguishing feature of ECM-based biomaterials. The results begin to identify the differential biologic activities mediated by EV that are secreted by tissue-resident cells and deposited within the ECM.

Matrix bound nanovesicle-associated IL-33 activates a pro-remodeling macrophage phenotype via a non-canonical, ST2-independent pathway.

The regenerative healing response of injured skeletal muscle is dependent upon an appropriately timed switch from a local type-I to a type-II immune response. Biologic scaffolds derived from extracellular matrix (ECM) have been shown to facilitate a macrophage phenotype transition that leads to downstream site-appropriate functional tissue deposition and myogenesis. However, the mechanisms by which ECM directs the switching of immune cell phenotype are only partially understood. Herein, we provide the first evidence that matrix bound nanovesicles (MBV) embedded within ECM-scaffolds are a rich and stable source of interleukin-33 (IL-33), an alarmin/cytokine with emerging reparative properties. We show that IL-33 encapsulated within MBV bypass the classical IL33/ST2 receptor signaling pathway to direct macrophage differentiation into the reparative, pro-remodeling M2 phenotype, which in turn facilitates myogenesis of skeletal muscle progenitor cells. Our results suggest the potential of IL-33+ MBV as a clinical therapy to augment the restorative efficacy of existing ECM-based and non-ECM based approaches.

4-Hydroxybutyrate Promotes Endogenous Antimicrobial Peptide Expression in Macrophages.

IMPACT STATEMENT: This study evaluated the biological activity of hydroxylated derivatives of butyrate as inductors of antimicrobial peptides (AMPs) in murine bone marrow-derived macrophages in vitro. A differential modulation of AMP expression by the hydroxylated derivatives of butyrate is shown. The ability of sodium 4-hydroxybutyrate to upregulate AMP expression through a histone deacetylase inhibitory-independent mechanism, and to promote increased resistance to bacterial contamination in vivo are also shown. The findings provide an alternative for prevention of bacterial contamination of implanted biomaterials. Functionalization of biomaterials with hydroxylated derivatives of butyrate can enhance the endogenous antimicrobial activity of the immune system through increased production of AMPs by host cells, thus providing protection against bacterial contamination.

TGFß promotes breast cancer stem cell self-renewal through an ILEI/LIFR signaling axis.

FAM3C/Interleukin-like EMT Inducer (ILEI) is an oncogenic member of the FAM3 cytokine family and serves essential roles in both epithelial-mesenchymal transition (EMT) and breast cancer metastasis. ILEI expression levels are regulated through a non-canonical TGFß signaling pathway by 3'-UTR-mediated translational silencing at the mRNA level by hnRNP E1. TGFß stimulation or silencing of hnRNP E1 increases ILEI translation and induces an EMT program that correlates with enhanced invasion and migration. Recently, EMT has been linked to the formation of breast cancer stem cells (BCSCs) that confer both tumor cell heterogeneity as well as chemoresistant properties. Herein, we demonstrate that hnRNP E1 knockdown significantly shifts normal mammary epithelial cells to mesenchymal BCSCs in vitro and in vivo. We further validate that modulating ILEI protein levels results in the abrogation of these phenotypes, promoting further investigation into the unknown mechanism of ILEI signaling that drives tumor progression. We identify LIFR as the receptor for ILEI, which mediates signaling through STAT3 to drive both EMT and BCSC formation. Reduction of either ILEI or LIFR protein levels results in reduced tumor growth, fewer tumor initiating cells and reduced metastasis within the hnRNP E1 knock-down cell populations in vivo. These results reveal a novel ligand-receptor complex that drives the formation of BCSCs and represents a unique target for the development of metastatic breast cancer therapies.

Cytocompatibility and mechanical properties of surgical sealants for cardiovascular applications.

OBJECTIVES: The present study compared physical, mechanical, and biologic characteristics of 4 clinically available surgical sealants for cardiovascular repair. METHODS: BioGlue (Cryolife Inc, Kennesaw, Ga), PreveLeak (Mallinckrodt Pharmaceuticals, St Louis, Mo), Tridyne VS (BD, Franklin Lakes, NJ), and Coseal (Baxter Healthcare Corporation, Westlake Village, Calif) were compared for the following properties: hydrated swelling, cytocompatibility, burst strength, biaxial stretching (elasticity), and in vitro degradation. RESULTS: Sealants showed a wide range of swelling upon hydration. By gravimetric and volumetric measurement, swelling was greatest for Coseal followed by Tridyne VS, BioGlue, and PreveLeak. Tridyne VS was the most cytocompatible based on Alamar Blue assay results, supporting 85% cell survival compared with 36% to 39% survival with the other sealants. All sealants withstood pressure above mean arterial pressure (70-110 mm Hg) and physiologic systolic blood pressure (90-140 mm Hg) in an ex vivo arterial flow burst model; lowest peak pressure at failure was PreveLeak at 235 ± 48 mm Hg, and highest peak pressure at failure was BioGlue at 596 ± 72 mm Hg. Biaxial tensile testing showed no differences in elasticity between ex vivo porcine aorta and carotid arteries and Tridyne VS or Coseal, and BioGlue and PreveLeak were significantly stiffer. In vitro degradation time for Coseal was 6 days and 21 days for Tridyne VS. No degradation was observed in BioGlue or PreveLeak for 30 days. CONCLUSIONS: Although all sealants withstood supraphysiologic arterial pressure, there were differences in characteristics that may be important in clinical outcome. Coseal degradation time was short compared with other sealants, whereas BioGlue and PreveLeak showed a significant compliance mismatch with native porcine carotid artery. Tridyne VS was significantly more cytocompatible than the other 3 sealants.

Extracellular Matrix Degradation Products Downregulate Neoplastic Esophageal Cell Phenotype.

IMPACT STATEMENT: Extracellular matrix (ECM) biomaterials were used to treat esophageal cancer patients after cancer resection and promoted regrowth of normal mucosa without recurrence of cancer. The present study investigates the mechanisms by which these materials were successful to prevent the cancerous phenotype. ECM downregulated neoplastic esophageal cell function (proliferation, metabolism), but normal esophageal epithelial cells were unaffected in vitro, and suggests a molecular basis (downregulation of PI3K-Akt, cell cycle) for the promising clinical results. The therapeutic effect appeared to be enhanced using homologous esophageal ECM. This study suggests that ECM can be further investigated to treat cancer patients after resection or in combination with targeted therapy.

Extracellular Matrix Bioscaffolds for Building Gastrointestinal Tissue.

Regenerative medicine is a rapidly advancing field that uses principles of tissue engineering, developmental biology, stem cell biology, immunology, and bioengineering to reconstruct diseased or damaged tissues. Biologic scaffolds composed of extracellular matrix have shown great promise as an inductive substrate to facilitate the constructive remodeling of gastrointestinal (GI) tissue damaged by neoplasia, inflammatory bowel disease, and congenital or acquired defects. The present review summarizes the preparation and use of extracellular matrix scaffolds for bioengineering of the GI tract, identifies significant advances made in regenerative medicine for the reconstruction of functional GI tissue, and describes an emerging therapeutic approach.

The extracellular matrix of the gastrointestinal tract: a regenerative medicine platform.

The synthesis and secretion of components that constitute the extracellular matrix (ECM) by resident cell types occur at the earliest stages of embryonic development, and continue throughout life in both healthy and diseased physiological states. The ECM consists of a complex mixture of insoluble and soluble functional components that are arranged in a tissue-specific 3D ultrastructure, and it regulates numerous biological processes, including angiogenesis, innervation and stem cell differentiation. Owing to its composition and influence on embryonic development, as well as cellular and organ homeostasis, the ECM is an ideal therapeutic substrate for the repair of damaged or diseased tissues. Biologic scaffold materials that are composed of ECM have been used in various surgical and tissue-engineering applications. The gastrointestinal (GI) tract presents distinct challenges, such as diverse pH conditions and the requirement for motility and nutrient absorption. Despite these challenges, the use of homologous and heterologous ECM bioscaffolds for the focal or segmental reconstruction and regeneration of GI tissue has shown promise in early preclinical and clinical studies. This Review discusses the importance of tissue-specific ECM bioscaffolds and highlights the major advances that have been made in regenerative medicine strategies for the reconstruction of functional GI tissues.

Membrane-organizing protein moesin controls Treg differentiation and antitumor immunity via TGF-ß signaling.

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that are important for organizing membrane domains and receptor signaling and regulating the migration of effector T cells. Whether moesin plays any role during the generation of TGF-ß-induced Tregs (iTregs) is unknown. Here, we have discovered that moesin is translationally regulated by TGF-ß and is also required for optimal TGF-ß signaling that promotes efficient development of iTregs. Loss of moesin impaired the development and function of both peripherally derived iTregs and in vitro-induced Tregs. Mechanistically, we identified an interaction between moesin and TGF-ß receptor II (TßRII) that allows moesin to control the surface abundance and stability of TßRI and TßRII. We also found that moesin is required for iTreg conversion in the tumor microenvironment, and the deletion of moesin from recipient mice supported the rapid expansion of adoptively transferred CD8+ T cells against melanoma. Our study establishes moesin as an important regulator of the surface abundance and stability of TßRII and identifies moesin's role in facilitating the efficient generation of iTregs. It also provides an advancement to our understanding about the role of the ERM proteins in regulating signal transduction pathways and suggests that modulation of moesin is a potential therapeutic target for Treg-related immune disorders.

Matrix-bound nanovesicles within ECM bioscaffolds.

Biologic scaffold materials composed of extracellular matrix (ECM) have been used in a variety of surgical and tissue engineering/regenerative medicine applications and are associated with favorable constructive remodeling properties including angiogenesis, stem cell recruitment, and modulation of macrophage phenotype toward an anti-inflammatory effector cell type. However, the mechanisms by which these events are mediated are largely unknown. Matrix-bound nanovesicles (MBVs) are identified as an integral and functional component of ECM bioscaffolds. Extracellular vesicles (EVs) are potent vehicles of intercellular communication due to their ability to transfer RNA, proteins, enzymes, and lipids, thereby affecting physiologic and pathologic processes. Formerly identified exclusively in biologic fluids, the presence of EVs within the ECM of connective tissue has not been reported. In both laboratory-produced and commercially available biologic scaffolds, MBVs can be separated from the matrix only after enzymatic digestion of the ECM scaffold material, a temporal sequence similar to the functional activity attributed to implanted bioscaffolds during and following their degradation when used in clinical applications. The present study shows that MBVs contain microRNA capable of exerting phenotypical and functional effects on macrophage activation and neuroblastoma cell differentiation. The identification of MBVs embedded within the ECM of biologic scaffolds provides mechanistic insights not only into the inductive properties of ECM bioscaffolds but also into the regulation of tissue homeostasis.