The effect of normal, metaplastic, and neoplastic esophageal extracellular matrix upon macrophage activation.

INTRODUCTION: Macrophages are capable of extreme plasticity and their activation state has been strongly associated with solid tumor growth progression and regression. Although the macrophage response to extracellular matrix (ECM) isolated from normal tissue is reasonably well understood, there is a relative dearth of information regarding their response to ECM isolated from chronically inflamed tissues, pre-neoplastic tissues, and neoplastic tissues. Esophageal adenocarcinoma (EAC) is a type of neoplasia driven by chronic inflammation in the distal esophagus, and the length of the esophagus provides the opportunity to investigate macrophage behavior in the presence of ECM isolated from a range of disease states within the same organ. METHODS: Normal, metaplastic, and neoplastic ECM hydrogels were prepared from decellularized EAC tissue. The hydrogels were evaluated for their nanofibrous structure (SEM), biochemical profile (targeted and global proteomics), and direct effect upon macrophage (THP-1 cell) activation state (qPCR, ELISA, immunolabeling) and indirect effect upon epithelial cell (Het-1A) migration (Boyden chamber). RESULTS: Nanofibrous ECM hydrogels from the three tissue types could be formed, and normal and neoplastic ECM showed distinctive protein profiles by targeted and global mass spectroscopy. ECM proteins functionally related to cancer and tumorigenesis were identified in the neoplastic esophageal ECM including collagen alpha-1(VIII) chain (COL8A1), lumican, and elastin. Metaplastic and neoplastic esophageal ECM induce distinctive effects upon THP-1 macrophage signaling compared to normal esophageal ECM. These effects include activation of pro-inflammatory IFNγ and TNFα gene expression and anti-inflammatory IL1RN gene expression. Most notably, neoplastic ECM robustly increased macrophage TNFα protein expression. The secretome of macrophages pre-treated with metaplastic and neoplastic ECM increases the migration of normal esophageal epithelial cells, similar behavior to that shown by tumor cells. Metaplastic ECM shows similar but less pronounced effects than neoplastic ECM suggesting the abnormal signals also exist within the pre-cancerous state. CONCLUSION: A progressively diseased ECM, as exists within the esophagus exposed to chronic gastric reflux, can provide insights into novel biomarkers of early disease and identify potential therapeutic targets.

In vivo biomolecular imaging of zebrafish embryos using confocal Raman spectroscopy.

Zebrafish embryos provide a unique opportunity to visualize complex biological processes, yet conventional imaging modalities are unable to access intricate biomolecular information without compromising the integrity of the embryos. Here, we report the use of confocal Raman spectroscopic imaging for the visualization and multivariate analysis of biomolecular information extracted from unlabeled zebrafish embryos. We outline broad applications of this method in: (i) visualizing the biomolecular distribution of whole embryos in three dimensions, (ii) resolving anatomical features at subcellular spatial resolution, (iii) biomolecular profiling and discrimination of wild type and ΔRD1 mutant Mycobacterium marinum strains in a zebrafish embryo model of tuberculosis and (iv) in vivo temporal monitoring of the wound response in living zebrafish embryos. Overall, this study demonstrates the application of confocal Raman spectroscopic imaging for the comparative bimolecular analysis of fully intact and living zebrafish embryos.

A blueprint for translational regenerative medicine.

The past few decades have produced a large number of proof-of-concept studies in regenerative medicine. However, the route to clinical adoption is fraught with technical and translational obstacles that frequently consign promising academic solutions to the so-called "valley of death." Here, we present a proposed blueprint for translational regenerative medicine. We offer principles to help guide the selection of cells and materials, present key in vivo imaging modalities, and argue that the host immune response should be considered throughout design and development. Last, we suggest a pathway to navigate the often complex regulatory and manufacturing landscape of translational regenerative medicine.

Neutrophils Enable Local and Non-Invasive Liposome Delivery to Inflamed Skeletal Muscle and Ischemic Heart.

Uncontrolled inflammation is a major pathological factor underlying a range of diseases including autoimmune conditions, cardiovascular disease, and cancer. Improving localized delivery of immunosuppressive drugs to inflamed tissue in a non-invasive manner offers significant promise to reduce severe side effects caused by systemic administration. Here, a neutrophil-mediated delivery system able to transport drug-loaded nanocarriers to inflamed tissue by exploiting the inherent ability of neutrophils to migrate to inflammatory tissue is reported. This hybrid system (neutrophils loaded with liposomes ex vivo) efficiently migrates in vitro following an inflammatory chemokine gradient. Furthermore, the triggered release of loaded liposomes and reuptake by target macrophages is studied. The migratory behavior of liposome-loaded neutrophils is confirmed in vivo by demonstrating the delivery of drug-loaded liposomes to an inflamed skeletal muscle in mice. A single low-dose injection of the hybrid system locally reduces inflammatory cytokine levels. Biodistribution of liposome-loaded neutrophils in a human-disease-relevant myocardial ischemia reperfusion injury mouse model after i.v. injection confirms the ability of injected neutrophils to carry loaded liposomes to inflammation sites. This strategy shows the potential of nanocarrier-loaded neutrophils as a universal platform to deliver anti-inflammatory drugs to promote tissue regeneration in inflammatory diseases.

Engineering Extracellular Vesicles with the Tools of Enzyme Prodrug Therapy.

Extracellular vesicles (EVs) have recently gained significant attention as important mediators of intercellular communication, potential drug carriers, and disease biomarkers. These natural cell-derived nanoparticles are postulated to be biocompatible, stable under physiological conditions, and to show reduced immunogenicity as compared to other synthetic nanoparticles. Although initial clinical trials are ongoing, the use of EVs for therapeutic applications may be limited due to undesired off-target activity and potential "dilution effects" upon systemic administration which may affect their ability to reach their target tissues. To fully exploit their therapeutic potential, EVs are embedded into implantable biomaterials designed to achieve local delivery of therapeutics taking advantage of enzyme prodrug therapy (EPT). In this first application of EVs for an EPT approach, EVs are used as smart carriers for stabilizing enzymes in a hydrogel for local controlled conversion of benign prodrugs to active antiinflammatory compounds. It is shown that the natural EVs' antiinflammatory potential is comparable or superior to synthetic carriers, in particular upon repeated long-term incubations and in different macrophage models of inflammation. Moreover, density-dependent color scanning electron microscopy imaging of EVs in a hydrogel is presented herein, an impactful tool for further understanding EVs in biological settings.

Scarring vs. functional healing: Matrix-based strategies to regulate tissue repair.

All vertebrates possess mechanisms to restore damaged tissues with outcomes ranging from regeneration to scarring. Unfortunately, the mammalian response to tissue injury most often culminates in scar formation. Accounting for nearly 45% of deaths in the developed world, fibrosis is a process that stands diametrically opposed to functional tissue regeneration. Strategies to improve wound healing outcomes therefore require methods to limit fibrosis. Wound healing is guided by precise spatiotemporal deposition and remodelling of the extracellular matrix (ECM). The ECM, comprising the non-cellular component of tissues, is a signalling depot that is differentially regulated in scarring and regenerative healing. This Review focuses on the importance of the native matrix components during mammalian wound healing alongside a comparison to scar-free healing and then presents an overview of matrix-based strategies that attempt to exploit the role of the ECM to improve wound healing outcomes.

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.

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.

Preparation and characterization of a biologic scaffold and hydrogel derived from colonic mucosa.

Gastrointestinal pathologies, injuries, and defects affect millions of individuals each year. While there are diverse treatment options for these individuals, no ideal solution exists. The repair or replacement of gastrointestinal tissue, therefore, represents a large unmet clinical need. Biomaterials derived from extracellular matrix (ECM) scaffolds have been effectively used to repair or replace numerous tissues throughout the body in both preclinical and clinical studies. Such scaffolds are prepared from decellularized tissues, and the biochemical, structural, and biologic properties vary depending upon the source tissue from which the ECM is derived. Given the potential benefit of a site-specific ECM scaffold for some applications, the objective of this study was to prepare, characterize, and determine the in vitro and in vivo cell response to ECM derived from porcine colon. Results of this study show that porcine colon can be effectively decellularized while retaining biochemical and structural constituents of the source tissue. Two forms of colonic ECM, scaffold and hydrogel, were shown to be cell friendly and facilitate the polarization of macrophages toward an M2 phenotype both in vitro and in vivo. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 291-306, 2017.

Restoring Mucosal Barrier Function and Modifying Macrophage Phenotype with an Extracellular Matrix Hydrogel: Potential Therapy for Ulcerative Colitis.

BACKGROUND AND AIMS: Despite advances in therapeutic options, more than half of all patients with ulcerative colitis [UC] do not achieve long-term remission, many require colectomy, and the disease still has a marked negative impact on quality of life. Extracellular matrix [ECM] bioscaffolds facilitate the functional repair of many soft tissues by mechanisms that include mitigation of pro-inflammatory macrophage phenotype and mobilization of endogenous stem/progenitor cells. The aim of the present study was to determine if an ECM hydrogel therapy could influence outcomes in an inducible rodent model of UC. METHODS: The dextran sodium sulphate [DSS]-colitis model was used in male Sprague Dawley rats. Animals were treated via enema with an ECM hydrogel and the severity of colitis was determined by clinical and histological criteria. Lamina propria cells were isolated and the production of inflammatory mediators was quantified. Mucosal permeability was assessed in vivo by administering TRITC-dextran and in vitro using transepithelial electrical resistance [TEER]. RESULTS: ECM hydrogel therapy accelerated healing and improved outcome. The hydrogel was adhesive to colonic tissue, which allowed for targeted delivery of the therapy, and resulted in a reduction in clinical and histological signs of disease. ECM hydrogel facilitated functional improvement of colonic epithelial barrier function and the resolution of the pro-inflammatory state of tissue macrophages. CONCLUSIONS: The present study shows that a non-surgical and non-pharmacological ECM-based therapy can abate DSS-colitis not by immunosuppression but by promoting phenotypic change in local macrophage phenotype and rapid replacement of the colonic mucosal barrier.

The impact of detergents on the tissue decellularization process: A ToF-SIMS study.

Biologic scaffolds are derived from mammalian tissues, which must be decellularized to remove cellular antigens that would otherwise incite an adverse immune response. Although widely used clinically, the optimum balance between cell removal and the disruption of matrix architecture and surface ligand landscape remains a considerable challenge. Here we describe the use of time of flight secondary ion mass spectroscopy (ToF-SIMS) to provide sensitive, molecular specific, localized analysis of detergent decellularized biologic scaffolds. We detected residual detergent fragments, specifically from Triton X-100, sodium deoxycholate and sodium dodecyl sulphate (SDS) in decellularized scaffolds; increased SDS concentrations from 0.1% to 1.0% increased both the intensity of SDS fragments and adverse cell outcomes. We also identified cellular remnants, by detecting phosphate and phosphocholine ions in PAA and CHAPS decellularized scaffolds. The present study demonstrates ToF-SIMS is not only a powerful tool for characterization of biologic scaffold surface molecular functionality, but also enables sensitive assessment of decellularization efficacy. STATEMENT OF SIGNIFICANCE: We report here on the use of a highly sensitive analytical technique, time of flight secondary ion mass spectroscopy (ToF-SIMS) to characterize detergent decellularized scaffolds. ToF-SIMS detected cellular remnants and residual detergent fragments; increased intensity of the detergent fragments correlated with adverse cell matrix interactions. This study demonstrates the importance of maintaining a balance between cell removal and detergent disruption of matrix architecture and matrix surface ligand landscape. This study also demonstrates the power of ToF-SIMS for the characterization of decellularized scaffolds and capability for assessment of decellularization efficacy. Future use of biologic scaffolds in clinical tissue reconstruction will benefit from the fundamental results described in this work.

The effect of terminal sterilization on the material properties and in vivo remodeling of a porcine dermal biologic scaffold.

Biologic scaffolds composed of extracellular matrix are commonly used in a variety of surgical procedures. The Food and Drug Administration typically regulates biologic scaffolds as medical devices, thus requiring terminal sterilization prior to clinical use. However, to date, no consensus exists for the most effective yet minimally destructive sterilization protocol for biologic scaffold materials. The objective of the present study was to characterize the effect of ethylene oxide, gamma irradiation and electron beam (e-beam) irradiation on the material properties and the elicited in vivo remodeling response of a porcine dermal biologic scaffold. Outcome measures included biochemical, structural, and mechanical properties as well as cytocompatibility in vitro. In vivo evaluation utilized a rodent model to examine the host response to the materials following 7, 14, and 35 days. The host response to each experimental group was determined by quantitative histologic methods and by immunolabeling for macrophage polarization (M1/M2). In vitro results show that increasing irradiation dosage resulted in a dose dependent decrease in mechanical properties compared to untreated controls. Ethylene oxide-treated porcine dermal ECM resulted in decreased DNA content, extractable total protein, and bFGF content compared to untreated controls. All ETO treated, gamma irradiated, and e-beam irradiated samples had similar cytocompatibility scores in vitro. However, in vivo results showed that increasing dosages of e-beam and gamma irradiation elicited an increased rate of degradation of the biologic scaffold material following 35 days. STATEMENT OF SIGNIFICANCE: The FDA typically regulates biologic scaffolds derived from mammalian tissues as medical devices, thus requiring terminal sterilization prior to clinical use. However, there is little data and no consensus for the most effective yet minimally destructive sterilization protocol for such materials. The present study characterized the effect of common sterilization methods: ethylene oxide, gamma irradiation and electron beam irradiation on the material properties and the elicited in vivo remodeling response of a porcine dermal biologic scaffold. The results of the study will aid in the meaningful selection of sterilization methods for biologic scaffold materials.

Intestinal stem cell growth and differentiation on a tubular scaffold with evaluation in small and large animals.

AIMS: To investigate the growth and differentiation of intestinal stem cells on a novel tubular scaffold in vitro and in vivo. MATERIALS & METHODS: Intestinal progenitor cells from mice or humans were cultured with myofibroblasts, macrophages and/or bacteria, and evaluated in mice via omental implantation. Mucosal regeneration was evaluated in dogs after rectal mucosectomy followed by scaffold implantation. RESULTS: Intestinal progenitor cells differentiated into crypt-villi structures on the scaffold. Differentiation and scaffold coverage was enhanced by coculture with myofibroblasts, macrophages and probiotic bacteria, while the implanted scaffolds enhanced mucosal regeneration in the dog rectum. CONCLUSION: Intestinal stem cell growth and differentiation on a novel tubular scaffold is enhanced through addition of cellular and microbial components, as validated in mice and dogs.

Inhibition of COX1/2 alters the host response and reduces ECM scaffold mediated constructive tissue remodeling in a rodent model of skeletal muscle injury.

Extracellular matrix (ECM) has been used as a biologic scaffold material to both reinforce the surgical repair of soft tissue and serve as an inductive template to promote a constructive tissue remodeling response. Success of such an approach is dependent on macrophage-mediated degradation and remodeling of the biologic scaffold. Macrophage phenotype during these processes is a predictive factor of the eventual remodeling outcome. ECM scaffolds have been shown to promote an anti-inflammatory or M2-like macrophage phenotype in vitro that includes secretion of downstream products of cycolooxygenases 1 and 2 (COX1/2). The present study investigated the effect of a common COX1/2 inhibitor (Aspirin) on macrophage phenotype and tissue remodeling in a rodent model of ECM scaffold treated skeletal muscle injury. Inhibition of COX1/2 reduced the constructive remodeling response by hindering myogenesis and collagen deposition in the defect area. The inhibited response was correlated with a reduction in M2-like macrophages in the defect area. The effects of Aspirin on macrophage phenotype were corroborated using an established in vitro macrophage model which showed a reduction in both ECM induced prostaglandin secretion and expression of a marker of M2-like macrophages (CD206). These results raise questions regarding the common peri-surgical administration of COX1/2 inhibitors when biologic scaffold materials are used to facilitate muscle repair/regeneration. STATEMENT OF SIGNIFICANCE: COX1/2 inhibitors such as nonsteroidal anti-inflammatory drugs (NSAIDs) are routinely administered post-surgically for analgesic purposes. While COX1/2 inhibitors are important in pain management, they have also been shown to delay or diminish the healing process, which calls to question their clinical use for treating musculotendinous injuries. The present study aimed to investigate the influence of a common NSAID, Aspirin, on the constructive remodeling response mediated by an ECM scaffold (UBM) in a rat skeletal muscle injury model. The COX1/2 inhibitor, Aspirin, was found to mitigate the ECM scaffold-mediated constructive remodeling response both in an in vitro co-culture system and an in vivo rat model of skeletal muscle injury. The results presented herein provide data showing that NSAIDs may significantly alter tissue remodeling outcomes when a biomaterial is used in a regenerative medicine/tissue engineering application. Thus, the decision to prescribe NSAIDs to manage the symptoms of inflammation post-ECM scaffold implantation should be carefully considered.

Concentration-dependent rheological properties of ECM hydrogel for intracerebral delivery to a stroke cavity.

Biomaterials composed of mammalian extracellular matrix (ECM) promote constructive tissue remodeling with minimal scar tissue formation in many anatomical sites. However, the optimal shape and form of ECM scaffold for each clinical application can vary markedly. ECM hydrogels have been shown to promote chemotaxis and differentiation of neuronal stem cells, but minimally invasive delivery of such scaffold materials to the central nervous system (CNS) would require an injectable form. These ECM materials can be manufactured to exist in fluid phase at room temperature, while forming hydrogels at body temperature in a concentration-dependent fashion. Implantation into the lesion cavity after a stroke could hence provide a means to support endogenous repair mechanisms. Herein, we characterize the rheological properties of an ECM hydrogel composed of urinary bladder matrix (UBM) that influence its delivery and in vivo interaction with host tissue. There was a notable concentration-dependence in viscosity, stiffness, and elasticity; all characteristics important for minimally invasive intracerebral delivery. An efficient MRI-guided injection with drainage of fluid from the cavity is described to assess in situ hydrogel formation and ECM retention at different concentrations (0, 1, 2, 3, 4, and 8mg/mL). Only ECM concentrations >3mg/mL gelled within the stroke cavity. Lower concentrations were not retained within the cavity, but extensive permeation of the liquid phase ECM into the peri-infarct area was evident. The concentration of ECM hydrogel is hence an important factor affecting gelation, host-biomaterial interface, as well intra-lesion distribution. STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM) hydrogel promotes constructive tissue remodeling in many tissues. Minimally invasive delivery of such scaffold materials to the central nervous system (CNS) would require an injectable form that exists in fluid phase at room temperature, while forming hydrogels at body temperature in a concentration-dependent fashion. We here report the rheological characterization of an injectable ECM hydrogel and its concentration-dependent delivery into a lesion cavity formed after a stroke based on MRI-guidance. The concentration of ECM determined its retention within the cavity or permeation into tissue and hence influenced its interaction with the host brain. This study demonstrates the importance of understanding the structure-function relationship of biomaterials to guide particular clinical applications.

Composite ECM-alginate microfibers produced by microfluidics as scaffolds with biomineralization potential.

A novel approach to produce artificial bone composites (microfibers) with distinctive features mimicking natural tissue was investigated. Currently proposed inorganic materials (e.g. apatite matrixes) lack self-assembly and thereby limit interactions between cells and the material. The present work investigates the feasibility of creating "bio-inspired materials" specifically designed to overcome certain limitations inherent to current biomaterials. We examined the dimensions, morphology, and constitutive features of a composite hydrogel which combined an alginate based microfiber with a gelatin solution or a particulate form of urinary bladder matrix (UBM). The effectiveness of the composite microfibers to induce and modulate osteoblastic differentiation in three-dimensional (3D) scaffolds without altering the viability and morphological characteristics of the cells was investigated. The present study describes a novel alginate microfiber production method with the use of microfluidics. The microfluidic procedure allowed for precise tuning of microfibers which resulted in enhanced viability and function of embedded cells.

Tissue-Specific Effects of Esophageal Extracellular Matrix.

Biologic scaffolds composed of extracellular matrix (ECM) have been used to facilitate repair or remodeling of numerous tissues, including the esophagus. The theoretically ideal scaffold for tissue repair is the ECM derived from the particular tissue to be treated, that is, site-specific or homologous ECM. The preference or potential advantage for the use of site-specific ECM remains unknown in the esophageal location. The objective of the present study was to characterize the in vitro cellular response and in vivo host response to a homologous esophageal ECM (eECM) versus nonhomologous ECMs derived from small intestinal submucosa and urinary bladder. The in vitro response of esophageal stem cells was characterized by migration, proliferation, and three-dimensional (3D) organoid formation assays. The in vivo remodeling response was evaluated in a rat model of esophageal mucosal resection. Results of the study showed that the eECM retains favorable tissue-specific characteristics that enhance the migration of esophageal stem cells and supports the formation of 3D organoids to a greater extent than heterologous ECMs. Implantation of eECM facilitates the remodeling of esophageal mucosa following mucosal resection, but no distinct advantage versus heterologous ECM could be identified.

A Rodent Model to Evaluate the Tissue Response to a Biological Scaffold When Adjacent to a Synthetic Material.

The use of biologic scaffold materials adjacent to synthetic meshes is commonplace. A prevalent clinical example is two-staged breast reconstruction, where biologic scaffolds are used to provide support and coverage for the inferior aspect of the synthetic expander. However, limited data exist regarding either the kinetics of biologic scaffold integration or the host tissue response to the biologic scaffold materials used for this application or other applications in which such scaffold materials are used. The present study evaluated the temporal host response to a biological scaffold when placed adjacent to a synthetic material. Evaluation criteria included quantification of material contracture and characterization of the host cell response and tissue remodeling events. Results show a decreased thickness of the collagenous tissue layer at biologic scaffold/silicone interface compared to the abdominal wall/silicone interface during the 12-week experimental time course. All test materials were readily incorporated into surrounding host tissue.