Esophageal extracellular matrix hydrogel mitigates metaplastic change in a dog model of Barrett's esophagus.

Chronic inflammatory gastric reflux alters the esophageal microenvironment and induces metaplastic transformation of the epithelium, a precancerous condition termed Barrett's esophagus (BE). The microenvironmental niche, which includes the extracellular matrix (ECM), substantially influences cell phenotype. ECM harvested from normal porcine esophageal mucosa (eECM) was formulated as a mucoadhesive hydrogel, and shown to largely retain basement membrane and matrix-cell adhesion proteins. Dogs with BE were treated orally with eECM hydrogel and omeprazole (n = 6) or omeprazole alone (n = 2) for 30 days. eECM treatment resolved esophagitis, reverted metaplasia to a normal, squamous epithelium in four of six animals, and downregulated the pro-inflammatory tumor necrosis factor-α+ cell infiltrate compared to control animals. The metaplastic tissue in control animals (n = 2) did not regress. The results suggest that in vivo alteration of the microenvironment with a site-appropriate, mucoadhesive ECM hydrogel can mitigate the inflammatory and metaplastic response in a dog model of BE.

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.

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.

Extracellular Matrix for Myocardial Repair.

Multiple strategies have been investigated to restore functional myocardium following injury or disease including the local administration of cytokines or chemokines, stem/progenitor cell therapy, mechanical circulatory support, pharmacologic use, and the use of inductive biomaterials. The use of xenogeneic biologic scaffolds composed of extracellular matrix (ECM) has been shown to facilitate functional restoration of several tissues and organs including the esophagus, skeletal muscle, skin, and myocardium, among others. The present chapter describes the current understanding of specific components of biologic scaffolds composed of ECM, the mechanisms by which ECM bioscaffolds promote constructive cardiac remodeling after injury, determinants of remodeling outcome, and the versatility of ECM as a potential cardiac therapeutic.

Alarmins of the extracellular space.

The ability of the immune system to discriminate between healthy-self, abnormal-self, and non-self has been attributed mainly to alarmins signaling as "danger signals". It is now evident, however, that alarmins are much more complex and can perform specialized functions that can regulate a wide spectrum of processes ranging from propagation of disease to tissue homeostasis. As such, alarmins and their signaling mechanisms are now actively pursued as therapeutic targets. The clinical utility of alarmins requires an understanding of their specific localization. Specifically, many alarmins can function paradoxically depending upon their localization, intra or extracellular. The present review focuses upon alarmin presence and differential expression in the extracellular space versus within the cell and how variation of the localization of alarmins can reveal important mechanistic insights into alarmin functions and their efficacy as biomarkers of disease and therapeutic targets.

Injectable, porous, biohybrid hydrogels incorporating decellularized tissue components for soft tissue applications.

Biodegradable injectable hydrogels have been extensively studied and evaluated in various medical applications such as for bulking agents, drug delivery reservoirs, temporary barriers, adhesives, and cell delivery matrices. Where injectable hydrogels are intended to facilitate a healing response, it may be desirable to encourage rapid cellular infiltration into the hydrogel volume from the tissue surrounding the injection site. In this study, we developed a platform technique to rapidly form pores in a thermally responsive injectable hydrogel, poly(NIPAAm-co-VP-co-MAPLA) by using mannitol particles as porogens. In a rat hindlimb muscle injection model, hydrogels incorporating porosity had significantly accelerated cellular infiltration. To influence the inflammatory response to the injected hydrogel, enzymatically digested urinary bladder matrix (UBM) was mixed with the solubilized hydrogel. The presence of UBM was associated with greater polarization of the recruited macrophage population to the M2 phenotype, indicating a more constructive foreign body response. The hybrid hydrogel positively affected the wound healing outcomes of defects in rabbit adipose tissue with negligible inflammation and fibrosis, whereas scar formation and chronic inflammation were observed with autotransplantation and in saline injected groups. These results demonstrate the value of combining the effects of promoting cell infiltration and mediating the foreign body response for improved biomaterials options soft tissue defect filling applications. STATEMENT OF SIGNIFICANCE: Our objective was to develop a fabrication process to create porous injectable hydrogels incorporating decellularized tissue digest material. This new hydrogel material was expected to exhibit faster cellular infiltration and a greater extent of pro-M2 macrophage polarization compared to control groups not incorporating each of the functional components. Poly(NIPAAm-co-VP-co-MAPLA) was chosen as the representative thermoresponsive hydrogel, and mannitol particles and digested urinary bladder matrix (UBM) were selected as the porogen and the bioactive decellularized material components respectively. In rat hindlimb intramuscular injection models, this new hydrogel material induced more rapid cellular infiltration and a greater extent of M2 macrophage polarization compared to control groups not incorporating all of the functional components. The hybrid hydrogel positively affected the wound healing outcomes of defects in rabbit adipose tissue with negligible inflammation and fibrosis, whereas scar formation and chronic inflammation were observed with autotransplantation and in saline injected groups. The methodology of this report provides a straightforward and convenient mechanism to promote cell infiltration and mediate foreign body response in injectable hydrogels for soft tissue applications. We believe that the readership of Acta Biomaterialia will find the work of interest both for its specific results and general translatability of the findings.

The Effect of Mechanical Loading Upon Extracellular Matrix Bioscaffold-Mediated Skeletal Muscle Remodeling.

Mounting evidence suggests that site-appropriate loading of implanted extracellular matrix (ECM) bioscaffolds and the surrounding microenvironment is an important tissue remodeling determinant, although the role at the cellular level in ECM-mediated skeletal muscle remodeling remains unknown. This study evaluates crosstalk between progenitor cells and macrophages during mechanical loading in ECM-mediated skeletal muscle repair. Myoblasts were exposed to solubilized ECM bioscaffolds and were mechanically loaded at 10% strain, 1 Hz for 5 h. Conditioned media was collected and applied to bone marrow-derived macrophages followed by immunolabeling for proinflammatory M1-like markers and proremodeling M2-like markers. Macrophages were subjected to the same loading protocol and their secreted products were collected for myoblast migration, proliferation, and differentiation analysis. A mouse hind limb unloading volumetric muscle loss model was used to evaluate the effect of loading upon the skeletal muscle microenvironment after ECM implantation. Animals were sacrificed at 14 or 180 days. Isometric torque production was tested and tissue sections were immunolabeled for macrophage phenotype and muscle fiber content. Results show that loading augments the ability of myoblasts to promote an M2-like macrophage phenotype following exposure to ECM bioscaffolds. Mechanically loaded macrophages promote myoblast chemotaxis and differentiation. Lack of weight bearing impaired muscle remodeling as indicated by Masson's Trichrome stain. Isometric torque was significantly increased following ECM implantation when compared to controls, a response not present in the hind limb-unloaded group. This work provides an important mechanistic insight of the effects of rehabilitation upon ECM-mediated remodeling and could have broader implications in clinical practice, advocating multidisciplinary approaches to regenerative medicine, emphasizing rehabilitation.

Bioscaffold-mediated mucosal remodeling following short-segment colonic mucosal resection.

Precancerous or cancerous lesions of the gastrointestinal tract often require surgical resection via endomucosal resection. Although excision of the colonic mucosa is an effective cancer treatment, removal of large lesions is associated with high morbidity and complications including bleeding, perforation, fistula formation, and/or stricture, contributing to high clinical and economic costs and negatively impacting patient quality of life. The present study investigates the use of a biologic scaffold derived from extracellular matrix (ECM) to promote restoration of the colonic mucosa following short segment mucosal resection. Six healthy dogs were assigned to ECM-treated (tubular ECM scaffold) and mucosectomy only control groups following transanal full circumferential mucosal resection (4 cm in length). The temporal remodeling response was monitored using colonoscopy and biopsy collection. Animals were sacrificed at 6 and 10 wk, and explants were stained with hematoxylin and eosin (H&E), Alcian blue, and proliferating cell nuclear antigen (PCNA) to determine the temporal remodeling response. Both control animals developed stricture and bowel obstruction with no signs of neomucosal coverage after resection. ECM-treated animals showed an early mononuclear cell infiltrate (2 weeks post-surgery) which progressed to columnar epithelium and complex crypt structures nearly indistinguishable from normal colonic architecture by 6 weeks after surgery. ECM scaffold treatment restored colonic mucosa with appropriately located PCNA+ cells and goblet cells. The study shows that ECM scaffolds may represent a viable clinical option to prevent complications associated with endomucosal resection of cancerous lesions in the colon.

Regenerative Medicine Approaches for Age-Related Muscle Loss and Sarcopenia: A Mini-Review.

Sarcopenia is a complex and multifactorial disease that includes a decrease in the number, structure and physiology of muscle fibers, and age-related muscle mass loss, and is associated with loss of strength, increased frailty, and increased risk for fractures and falls. Treatment options are suboptimal and consist of exercise and nutrition as the cornerstone of therapy. Current treatment principles involve identification and modification of risk factors to prevent the disease, but these efforts are of limited value to the elderly individuals currently affected by sarcopenia. The development of new and effective therapies for sarcopenia is challenging. Potential therapies can target one or more of the proposed multiple etiologies such as the loss of regenerative capacity of muscle, age-related changes in the expression of signaling molecules such as growth hormone, IGF-1, myostatin, and other endocrine signaling molecules, and age-related changes in muscle physiology like denervation and mitochondrial dysfunction. The present paper reviews regenerative medicine strategies that seek to restore adequate skeletal muscle structure and function including exogenous delivery of cells and pharmacological therapies to induce myogenesis or reverse the physiologic changes that result in the disease. Approaches that modify the microenvironment to provide an environment conducive to reversal and mitigation of the disease represent a potential regenerative medicine approach that is discussed herein.

Macrophage phenotype in response to ECM bioscaffolds.

Macrophage presence and phenotype are critical determinants of the healing response following injury. Downregulation of the pro-inflammatory macrophage phenotype has been associated with the therapeutic use of bioscaffolds composed of extracellular matrix (ECM), but phenotypic characterization of macrophages has typically been limited to small number of non-specific cell surface markers or expressed proteins. The present study determined the response of both primary murine bone marrow derived macrophages (BMDM) and a transformed human mononuclear cell line (THP-1 cells) to degradation products of two different, commonly used ECM bioscaffolds; urinary bladder matrix (UBM-ECM) and small intestinal submucosa (SIS-ECM). Quantified cell responses included gene expression, protein expression, commonly used cell surface markers, and functional assays. Results showed that the phenotype elicited by ECM exposure (MECM) is distinct from both the classically activated IFNγ+LPS phenotype and the alternatively activated IL-4 phenotype. Furthermore, the BMDM and THP-1 macrophages responded differently to identical stimuli, and UBM-ECM and SIS-ECM bioscaffolds induced similar, yet distinct phenotypic profiles. The results of this study not only characterized an MECM phenotype that has anti-inflammatory traits but also showed the risks and challenges of making conclusions about the role of macrophage mediated events without consideration of the source of macrophages and the limitations of individual cell markers.

Matrix-Bound Nanovesicles Recapitulate Extracellular Matrix Effects on Macrophage Phenotype.

The early macrophage response to biomaterials has been shown to be a critical and predictive determinant of downstream outcomes. When properly prepared, bioscaffolds composed of mammalian extracellular matrix (ECM) have been shown to promote a transition in macrophage behavior from a proinflammatory to a regulatory/anti-inflammatory phenotype, which in turn has been associated with constructive and functional tissue repair. The mechanism by which ECM bioscaffolds promote this phenotypic transition, however, is poorly understood. The present study shows that matrix-bound nanovesicles (MBV), a component of ECM bioscaffolds, are capable of recapitulating the macrophage activation effects of the ECM bioscaffold from which they are derived. MBV isolated from two different source tissues, porcine urinary bladder and small intestinal submucosa, were found to be enriched in miRNA125b-5p, 143-3p, and 145-5p. Inhibition of these miRNAs within macrophages was associated with a gene and protein expression profile more consistent with a proinflammatory rather than an anti-inflammatory/regulatory phenotype. MBV and their associated miRNA cargo appear to play a significant role in mediating the effects of ECM bioscaffolds on macrophage phenotype.

Extracellular Matrix Bioscaffolds as Immunomodulatory Biomaterials.

Suppression of the recipient immune response is a common component of tissue and organ transplantation strategies and has also been used as a method of mitigating the inflammatory and scar tissue response to many biomaterials. It is now recognized, however, that long-term functional tissue replacement not only benefits from an intact host immune response but also depends upon such a response. The present article reviews the limitations associated with the traditionally held view of avoiding the immune response, the ability of acellular biologic scaffold materials to modulate the host immune response and promote a functional tissue replacement outcome, and current strategies within the fields of tissue engineering and biomaterials to develop immune-responsive and immunoregulatory biomaterials.

The effect of cell debris within biologic scaffolds upon the macrophage response.

All biomaterials, including biologic scaffolds composed of extracellular matrix (ECM), elicit a host immune response when implanted. The type and intensity of this response depends in part upon the thoroughness of decellularization and removal of cell debris from the source tissue. Proinflammatory responses have been associated with negative downstream remodeling events including scar tissue formation, encapsulation, and seroma formation. The relative effects of specific cellular components upon the inflammatory response are not known. The objective of the present study was to determine the effect of different cell remnants that may be present in ECM scaffold materials upon the host innate immune response, both in vitro and in vivo. Collagen scaffolds were supplemented with one of three different concentrations of DNA, mitochondria, or cell membranes. Murine macrophages were exposed to the various supplemented scaffolds and the effect upon macrophage phenotype was evaluated. In vivo studies were performed using an abdominal wall defect model in the rat to evaluate the effect of the scaffolds upon the macrophage response. Murine macrophages exposed in vitro to scaffolds supplemented with DNA, mitochondria, and cell membranes showed increased expression of proinflammatory M1 marker iNOS and no expression of the proremodeling M2 marker Fizz1 regardless of supplementation concentration. A dose-dependent response was observed in the rat model for collagen scaffolds supplemented with cell remnants. DNA, mitochondria, and cell membrane remnants in collagen scaffolds promote a proinflammatory M1 macrophage phenotype in vivo and in vitro. These results reinforce the importance of a thorough decellularization process for ECM biologic scaffold materials. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2109-2118, 2017.

Solubilized extracellular matrix bioscaffolds derived from diverse source tissues differentially influence macrophage phenotype.

The host response to biomaterials is a critical determinant of their success or failure in tissue-repair applications. Macrophages are among the first responders in the host response to biomaterials and have been shown to be predictors of downstream tissue remodeling events. Biomaterials composed of mammalian extracellular matrix (ECM) in particular have been shown to promote distinctive and constructive remodeling outcomes when compared to their synthetic counterparts, a property that has been largely attributed to their ability to modulate the host macrophage response. ECM bioscaffolds are prepared by decellularizing source tissues such as dermis and small intestinal submucosa. The differential ability of such scaffolds to influence macrophage behavior has not been determined. The present study determines the effects of ECM bioscaffolds derived from eight different source tissues upon macrophage surface marker expression, protein content, phagocytic capability, metabolism, and antimicrobial activity. The results show that macrophages exposed to small intestinal submucosa (SIS), urinary bladder matrix (UBM), brain ECM (bECM), esophageal ECM (eECM), and colonic ECM (coECM) express a predominant M2-like macrophage phenotype, which is pro-remodeling and anti-inflammatory (iNOS-/Fizz1+/CD206+). In contrast, macrophage exposure to dermal ECM resulted in a predominant M1-like, pro-inflammatory phenotype (iNOS+/Fizz1-/CD206-), whereas liver ECM (LECM) and skeletal muscle ECM (mECM) did not significantly change the expression of these markers. All solubilized ECM bioscaffold treatments resulted in an increased macrophage antimicrobial activity, but no differences were evident in macrophage phagocytic capabilities, and macrophage metabolism was decreased following exposure to UBM, bECM, mECM, coECM, and dECM. The present work could have important implications when considering the macrophage response following ECM implantation for site-appropriate tissue remodeling. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 138-147, 2017.

Immunomodulation and Mobilization of Progenitor Cells by Extracellular Matrix Bioscaffolds for Volumetric Muscle Loss Treatment.

Acellular bioscaffolds composed of extracellular matrix (ECM) have been effectively used to promote functional tissue remodeling in both preclinical and clinical studies of volumetric muscle loss, but the mechanisms that contribute to such outcomes are not fully understood. Thirty-two C57bl/6 mice were divided into eight groups of four animals each. A critical-sized defect was created in the quadriceps muscle and was repaired with a small intestinal submucosa ECM bioscaffold or left untreated. Animals were sacrificed at 3, 7, 14, or 56 days after surgery. The spatiotemporal cellular response in both treated and untreated groups was characterized by immunolabeling methods. Early time points showed a robust M2-like macrophage phenotype following ECM treatment in contrast to the predominant M1-like macrophage phenotype present in the untreated group. ECM implantation promoted perivascular stem cell mobilization, increased presence of neurogenic progenitor cells, and was associated with myotube formation. These cell types were present not only at the periphery of the defect near uninjured muscle, but also in the center of the ECM-filled defect. ECM bioscaffolds modify the default response to skeletal muscle injury, and provide a microenvironment conducive to a constructive healing response.

Mechanisms by which acellular biologic scaffolds promote functional skeletal muscle restoration.

Acellular biologic scaffolds derived from extracellular matrix have been investigated in preclinical and clinical studies as a regenerative medicine approach for volumetric muscle loss treatment. The present manuscript provides a review of previous studies supporting the use of extracellular matrix derived biologic scaffolds for the promotion of functional skeletal muscle tissue formation that is contractile and innervated. The manuscript also identifies key mechanisms that have been associated with ECM-mediated skeletal muscle repair, and provides hypotheses as to why there have been variable outcomes, ranging from successful to unsatisfactory, associated with ECM bioscaffold implantation in the skeletal muscle injury microenvironment.

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.

Strategies for functional bioscaffold-based skeletal muscle reconstruction.

Tissue engineering and regenerative medicine-based strategies for the reconstruction of functional skeletal muscle tissue have included cellular and acellular approaches. The use of acellular biologic scaffold material as a treatment for volumetric muscle loss (VML) in five patients has recently been reported with a generally favorable outcome. Further studies are necessary for a better understanding of the mechanism(s) behind acellular bioscaffold-mediated skeletal muscle repair, and for combination cell-based/bioscaffold based approaches. The present overview highlights the current thinking on bioscaffold-based remodeling including the associated mechanisms and the future of scaffold-based skeletal muscle reconstruction.

The promotion of a constructive macrophage phenotype by solubilized extracellular matrix.

The regenerative healing response of injured skeletal muscle is dependent upon a heterogeneous population of responding macrophages, which show a phenotypic transition from the pro-inflammatory M1 to the alternatively activated and constructive M2 phenotype. Biologic scaffolds derived from mammalian extracellular matrix (ECM) have been used for the repair and reconstruction of a variety of tissues, including skeletal muscle, and have been associated with an M2 phenotype and a constructive and functional tissue response. The mechanism(s) behind in-vivo macrophage phenotype transition in skeletal muscle and the enhanced M2:M1 ratio associated with ECM bioscaffold use in-vivo are only partially understood. The present study shows that degradation products from ECM bioscaffolds promote alternatively activated and constructive M2 macrophage polarization in-vitro, which in turn facilitates migration and myogenesis of skeletal muscle progenitor cells.