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.

Matrix-Bound Nanovesicles: The Effects of Isolation Method upon Yield, Purity, and Function.

Identification of matrix-bound nanovesicles (MBV) as ubiquitous components of the extracellular matrix (ECM) raises questions regarding their biologic functions and their potential theranostic application. Unlike liquid-phase extracellular vesicles (e.g., exosomes), MBV are tightly bound to the ECM, which makes their isolation and harvesting more challenging. The indiscriminate use of different methods to harvest MBV can alter or disrupt their structural and/or functional integrity. The objective of the present study was to compare the effect of various MBV harvesting methods upon yield, purity, and biologic activity. Combinations of four methods to solubilize the ECM (collagenase [COL], liberase [LIB], or proteinase K [PK] and nonenzymatic elution with potassium chloride) and four isolation methods (ultracentrifugation, ultrafiltration [UF], density barrier, and size exclusion chromatography [SEC]) were used to isolate MBV from urinary bladder-derived ECM. All combinations of solubilization and isolation methods allowed for the harvesting of MBV, however, distinct differences were noted. The highest yield, purity, cellular uptake, and biologic activity were seen with MBV isolated by a combination of liberase or collagenase followed by SEC. The combination of proteinase K and UF was shown to have detrimental effects on bioactivity. The results show the importance of selecting appropriate MBV harvesting methods for the characterization and evaluation of MBV and for analysis of their potential theranostic application. Impact statement Identification of matrix-bound nanovesicles (MBV) as ubiquitous components of the extracellular matrix (ECM) has raised questions regarding their biologic functions and their potential theranostic application. This study demonstrates that the harvesting methods used can result in samples with physical and biochemical properties that are unique to the isolation and solubilization methods used. Consequently, developing harvesting methods that minimize sample contamination with ECM remnants and/or solubilization agents will be essential in determining the theranostic potential of MBV in future studies.

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.

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.

Sutureless nerve repair with ECM bioscaffolds and laser-activated chitosan adhesive.

The outcome of peripheral nerve repair following transection is influenced by a number of factors but almost all approaches require anastomosis of the nerve using technically demanding microsurgical procedures. However, the use of sutures presents a number of unavoidable challenges including additional nerve trauma, stimulation of an inflammatory response, and endoneural fibrosis. The objective of the present study was to determine the efficacy of a sutureless approach to nerve repair. A rat sciatic nerve transection model was used with a laser-activated, chitosan-based adhesive (SurgiLux), combined with different forms of extracellular matrix (ECM), known to promote Schwann cell proliferation and nerve growth both in peripheral nerve applications. Following a 5 mm transection of the sciatic nerve, nerve guide wraps were prepared using: (1) laser-activated adhesive (SurgiLux) alone, (2) SurgiLux incorporating ECM (SurgiLux ECM), (3) ECM secured using SurgiLux, and (4) ECM secured using 8-0 Prolene sutures. A no treatment groups was used as a negative control. Evaluation of tissue remodeling was conducted with histolomorphometric assessment of neuroma, integrity of repair, nerve immunolabeling, ratio of myelinated to non-myelinated fibers, and amount of connective tissue. Quantitative and semi-quantitative analysis of the repaired nerve transections at 6 and 12 weeks showed that that SurgiLux incorporating powdered ECM (SurgiLux ECM), SurgiLux alone and ECM alone all improved the healing response compared to no-treatment controls, with less fibrotic tissue and more nerve staining. Histologic scoring showed that the SurgiLux ECM group showed the greatest increase in histologic score between the two time points tested. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1698-1711, 2018.

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.

Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide.

Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold's regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cell-removing detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology. STATEMENT OF SIGNIFICANCE: Preservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules (e.g., collagen) during decellularization can strongly influence the cellular response. Unfortunately, conventional techniques (SEM, SHG) are not conducive to identifying denatured collagen molecules in tissues. We demonstrate the first investigation into the molecular denaturation of collagen in decellularized ECM enabled by a novel Collagen Hybridizing Peptide (CHP) that specifically binds to unfolded collagen chains. We show that SDS and Triton X-100 denature collagen molecules while CHAPS and SD cannot. Such detection has been nearly impossible with other existing techniques. The CHP technique will advance our understanding about the effect of the cell-removing process on ECM, and lead to development of the decellularization technology.

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.

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.

A panel data set on harvest and perfusion decellularization of porcine rectus abdominis.

In this dataset, we particularly depicted the harvest and perfusion decellularization of porcine rectus abdominis (RA), accompanied with displaying of the retained vascular trees within the perfusion-decellularized skeletal muscle matrix (pM-ECM) using vascular corrosion casting. In addition, several important tips for successful pM-ECM preparation were emphasized, which including using anatomically isolated skeletal muscle as tissue source with all main feeding and draining vessels perfused, preserving the internal microcirculation availability, aseptic technique and pyrogen free in all steps, sequential perfusion via artery or vein, and longtime washing after decellularization. The data are supplemental to our original research article describing detailed associations of pM-ECM as a clinically relevant scale, three-dimensional scaffold with a vascular network template for tissue-specific regeneration, "Perfusion-decellularized skeletal muscle as a three-dimensional scaffold with a vascular network template" Zhang et al. (2016) [1].

Perfusion-decellularized skeletal muscle as a three-dimensional scaffold with a vascular network template.

There exists a great need for repair grafts with similar volume to human skeletal muscle that can promote the innate ability of muscle to regenerate following volumetric muscle loss. Perfusion decellularization is an attractive technique for extracellular matrix (ECM) scaffold from intact mammalian organ or tissue which has been successfully used in tissue reconstruction. The perfusion-decellularization of skeletal muscle has been poorly assessed and characterized, but the bioactivity and functional capacity of the obtained perfusion skeletal muscle ECM (pM-ECM) to remodel in vivo is unknown. In the present study, pM-ECM was prepared from porcine rectus abdominis (RA). Perfusion-decellularization of porcine RA effectively removed cellular and nuclear material while retaining the intricate three-dimensional microarchitecture and vasculature networks of the native RA, and many of the bioactive ECM components and mechanical properties. In vivo, partial-thickness abdominal wall defects in rats repaired with pM-ECM showed improved neovascularization, myogenesis and functional recellularization compared to porcine-derived small intestinal submucosa (SIS). These findings show the biologic potential of RA pM-ECM as a scaffold for supporting site appropriate, tissue reconstruction, and provide a better understanding of the importance maintaining the tissue-specific complex three-dimensional architecture of ECM during decellularization and regeneration.

The Use of Biologic Scaffolds in the Treatment of Chronic Nonhealing Wounds.

Significance: Injuries to the skin as a result of illness or injury, particularly chronic nonhealing wounds, present a major healthcare problem. Traditional wound care approaches attempt to control the underlying causes, such as infection and ischemia, while the application of wound dressings aims to modify a poorly healing wound environment into a microenvironment more closely resembling an acute wound allowing the body to heal the wound naturally. Recent Advances: Regenerative medicine approaches, such as the use of biologic scaffold materials comprising an intact extracellular matrix (ECM) or individual components of the ECM, are providing new therapeutic options that focus upon the provision of biochemical cues that alter the wound microenvironment to facilitate rapid restoration of normal skin architecture. Critical Issues: The incidence of chronic nonhealing wounds continues to increase. For example, between 15% and 20% of diabetics are likely to develop chronic, nonhealing foot wounds creating an increasing burden on healthcare systems worldwide. Future Directions: Developing a thorough understanding of wound microenvironment and the mechanisms by which biologic scaffolds work in vivo has the potential to markedly improve outcomes in the clinical translation for the treatment of chronic wounds.

Regional variations in the histology of porcine skin.

Porcine skin is commonly used as a model for human skin injury and as a source material for biologic scaffold materials. Although remarkable similarities between porcine and human skin exist, regional anatomic variations present in human skin are also present in porcine skin. The objective of this study was to evaluate the structure of porcine skin from 11 different anatomic regions in the American Yorkshire crossbreed. Both qualitative and quantitative methods were used, with emphasis on epidermal and dermal thickness, hair follicle density, and collagen and elastin composition and distribution. The results showed that significant regional differences in skin histology exist, particularly with regard to the thickness of the dermis and epidermis and the amount of collagen and elastin within each tissue. Differences were also seen in the distribution of type I and type III collagen within the dermis. Therefore, while porcine skin shares many similarities with human skin, distinct regional differences in composition and morphology exist. This study highlights the importance of appreciating these regional differences to avoid misinterpretation of experimental results when using porcine skin as a human analogue.

An acellular biologic scaffold promotes skeletal muscle formation in mice and humans with volumetric muscle loss.

Biologic scaffolds composed of naturally occurring extracellular matrix (ECM) can provide a microenvironmental niche that alters the default healing response toward a constructive and functional outcome. The present study showed similarities in the remodeling characteristics of xenogeneic ECM scaffolds when used as a surgical treatment for volumetric muscle loss in both a preclinical rodent model and five male patients. Porcine urinary bladder ECM scaffold implantation was associated with perivascular stem cell mobilization and accumulation within the site of injury, and de novo formation of skeletal muscle cells. The ECM-mediated constructive remodeling was associated with stimulus-responsive skeletal muscle in rodents and functional improvement in three of the five human patients.

Human NELL1 protein augments constructive tissue remodeling with biologic scaffolds.

Biologic scaffolds composed of extracellular matrix (ECM) derived from decellularized tissues effectively reprogram key stages of the mammalian response to injury, altering the wound microenvironment from one that promotes scar tissue formation to one that stimulates constructive and functional tissue remodeling. In contrast, engineered scaffolds, composed of purified ECM components such as collagen, lack the complex ultrastructure and composition of intact ECM and may promote wound healing but lack factors that facilitate constructive and functional tissue remodeling. The objective of the present study was to test the hypothesis that addition of NELL1, a signaling protein that controls cell growth and differentiation, enhances the constructive tissue remodeling of a purified collagen scaffold. An abdominal wall defect model in the rat of 1.5-cm(2) partial thickness was used to compare the constructive remodeling of a bovine type I collagen scaffold to a biologic scaffold derived from small intestinal submucosa (SIS)-ECM with and without augmentation with 17 µg NELL1 protein. Samples were evaluated histologically at 14 days and 4 months. The contractile response of the defect site was also evaluated at 4 months. Addition of NELL1 protein improved the constructive remodeling of collagen scaffolds but not SIS-ECM scaffolds. Results showed an increase in the contractile force of the remodeled skeletal muscle and a fast:slow muscle composition similar to native tissue in the collagen-treated group. The already robust remodeling response to SIS-ECM was not enhanced by NELL1 at the dose tested. These findings suggest that NELL1 protein does contribute to the enhanced constructive remodeling of skeletal muscle.

Lessons from developmental biology for regenerative medicine.

The ultimate goal of regenerative medicine is the functional restoration of lost or damaged tissues and organs. Since most tissues in man lack true regenerative properties and instead respond to injury with an inflammatory response and scar tissue formation, regenerative medicine strategies that include combinations of cells, scaffolds, and bioactive molecules to replace injured or missing tissues have been developed. The physical, chemical, and electrical cues that define the microenvironmental niche and the effect of these influences upon cell behavior during development are of interest to developmental biologists, with obvious overlap to the interest of the regenerative medicine field. This manuscript provides an overview of current approaches for tissue restoration being investigated in the field of regenerative medicine and attempts to identify areas of mutual beneficial interest with the field of developmental biology.

Biologic scaffolds for musculotendinous tissue repair.

Traumatic injuries to the musculoskeletal system are common events and volumetric muscle loss (VML) is no longer a rare occurrence. Surgical intervention is typically the only option for restoration of partial function. Surgical intervention for VML however does not regenerate the lost tissue and typically results in alterations of both the anatomy and biomechanics at the site of injury. Non-traditional approaches to the restoration of functional musculoskeletal tissue, including those provided by tissue engineering and regenerative medicine strategies, become viable alternative therapies when the expected outcome is bleak. One such strategy involves the delivery of constructive cues and modulation of the micro-environmental niche via biologic scaffold materials. These materials ideally retain the native structure and composition of the extracellular matrix of the tissue from which they are derived. Some of the recent advances in the use of biologic scaffolds to target key stages of the musculotendinous repair process and promote the restoration of functional tissue are described herein.

Quantitative multispectral imaging of Herovici's polychrome for the assessment of collagen content and tissue remodelling.

Bioprosthetic devices, constructed from a variety of materials, are routinely implanted in a variety of anatomical locations. Essential to their success is the formation of a non-destructive interface with the host tissue and appropriate tissue remodelling. Traditionally, the main method of assessing the host-material interface has been qualitative histological evaluation, using pattern recognition and comparative assessment to identify changes in the normal tissue architecture that are characteristic of scar tissue. In the present study, the recently developed technique of multispectral imaging was used to revisit a little-described histological stain, Herovici's polychrome, which is capable of distinguishing between types I and III collagen. Combined, these techniques allowed quantification of collagen content and distribution of collagen types within a tissue sample. Samples of rat tail and human scar tissue were used to optimize the staining, while comparison with immunolabelled samples was used to develop a reproducible quantification system, based on the specific colour profiles for types I and III collagen. Finally the remodelling of rat abdominal wall defects repaired with crosslinked or non-crosslinked extracellular matrix scaffolds derived from porcine urinary bladder was assessed with this technique. Compared to standard histological assessment, the combination of multispectral imaging and Herovici's polychrome staining presents a quick, simple, reliable technique that can provide accurate quantification of tissue remodelling and specifically identify the expression and distribution of types I and III collagen.

Bone marrow-derived cells participate in the long-term remodeling in a mouse model of esophageal reconstruction.

BACKGROUND: The default response of the esophagus to injury includes inflammation and scar tissue formation often leading to stricture. Biologic scaffolds composed of extracellular matrix (ECM) have been associated with the reconstitution of functional esophageal tissue in preclinical studies and clinical case reports of esophageal mucosal resection, anastomotic reinforcement, and full circumferential replacement. However, the mechanisms responsible for this change in the default response to esophageal injury are not fully understood. METHODS: The objective of the present study was to determine whether bone marrow-derived cells (BMCs) participate in the long-term remodeling of ECM scaffolds in the esophageal location in a mouse model. RESULTS: BMCs were present in low numbers in remodeling ECM scaffolds. Compared with the untreated control mice, the ECM-implanted animals showed better remodeling of the epithelial layer. CONCLUSIONS: BMCs are involved in ECM remodeling process during tissue repair after esophageal injury, but the low numbers argue against any significant involvement in the constructive remodeling process.

A murine model of volumetric muscle loss and a regenerative medicine approach for tissue replacement.

Volumetric muscle loss (VML) resulting from traumatic accidents, tumor ablation, or degenerative disease is associated with limited treatment options and high morbidity. The lack of a reliable and reproducible animal model of VML has hindered the development of effective therapeutic strategies. The present study describes a critical-sized excisional defect within the mouse quadriceps muscle that results in an irrecoverable volumetric defect. This model of VML was used to evaluate the efficacy of a surgically placed inductive biologic scaffold material composed of porcine small intestinal submucosa-extracellular matrix (SIS-ECM). The targeted placement of an SIS-ECM scaffold within the defect was associated with constructive tissue remodeling including the formation of site-appropriate skeletal muscle tissue. The present study provides a reproducible animal model with which to study VML and shows the therapeutic potential of a bioscaffold-based regenerative medicine approach to VML.