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.

Mechanical and microstructural analysis of a radially expandable vascular conduit for neonatal and pediatric cardiovascular surgery.

In pediatric cardiovascular surgery, there is a significant need for vascular prostheses that have the potential to grow with the patient following implantation. Current clinical options consist of nonexpanding conduits, requiring repeat surgeries as the patient outgrows the device. To address this issue, PECA Labs has developed a novel ePTFE vascular conduit with the capability of being radially expanded via balloon catheterization. In the described study, a systematic characterization and comparison of two proprietary ePTFE expandable conduits was conducted. Conduit sizes of 8 and 16 mm inner diameters for both conduits were evaluated before and after expansion with a 26 mm balloon. Comprehensive mechanical testing was completed, including quantification of circumferential, and longitudinal tensile strength, suture retention strength, burst strength, water entry pressure, dynamic compliance, and kink radius. Scanning electron microscopy was used to investigate the microstructural properties. Automated extraction of the fiber architectural features for each scanning electron micrograph was achieved with an algorithm for each conduit before and after expansion. Results showed that both conduits were able to expand significantly, to as much as 2.5× their original inner diameter. All mechanical properties were within clinically acceptable values following expansion. Analysis of the microstructure properties of the conduits revealed that the circumferential main angle of orientation, orientation index, and spatial periodicity did not significantly change following expansion, whereas the node area fraction decreased post expansion. Successful proof-of-concept of this novel product represents a critical step toward clinical translation and provides hope for newborns and growing children with congenital heart disease. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 659-671, 2018.

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.

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.

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.

Solubilized liver extracellular matrix maintains primary rat hepatocyte phenotype in-vitro.

Whole organ engineering and cell-based regenerative medicine approaches are being investigated as potential therapeutic options for end-stage liver failure. However, a major challenge of these strategies is the loss of hepatic specific function after hepatocytes are removed from their native microenvironment. The objective of the present study was to determine if solubilized liver extracellular matrix (ECM), when used as a media supplement, can better maintain hepatocyte phenotype compared to type I collagen alone or solubilized ECM harvested from a non-liver tissue source. Liver extracellular matrix (LECM) from four different species was isolated via liver tissue decellularization, solubilized, and then used as a media supplement for primary rat hepatocytes (PRH). The four species of LECM investigated were human, porcine, canine and rat. Cell morphology, albumin secretion, and ammonia metabolism were used to assess maintenance of hepatocyte phenotype. Biochemical and mechanical characterization of each LECM were also conducted. Results showed that PRH's supplemented with canine and porcine LECM maintained their phenotype to a greater extent compared to all other groups. PRH's supplemented with canine and porcine LECM showed increased bile production, increased albumin production, and the formation of multinucleate cells. The findings of the present study suggest that solubilized liver ECM can support in-vitro hepatocyte culture and should be considered for therapeutic and diagnostic techniques that utilize hepatocytes.

Decellularization and cell seeding of whole liver biologic scaffolds composed of extracellular matrix.

The definitive treatment for patients with end-stage liver disease is orthotropic transplantation. However, this option is limited by the disparity between the number of patients needing transplantation and the number of available livers. This issue is becoming more severe as the population ages and as the number of new cases of end-stage liver failure increases. Patients fortunate enough to receive a transplant are required to receive immunosuppressive therapy and must live with the associated morbidity. Whole organ engineering of the liver may offer a solution to this liver donor shortfall. It has been shown that perfusion decellularization of a whole allogeneic or xenogeneic liver generates a three-dimensional ECM scaffold with intact macro and micro architecture of the native liver. A decellularized liver provides an ideal transplantable scaffold with all the necessary ultrastructure and signaling cues for cell attachment, differentiation, vascularization, and function. In this review, an overview of complementary strategies for creating functional liver grafts suitable for transplantation is provided. Early milestones have been met by combining stem and progenitor cells with increasingly complex scaffold materials and culture conditions.

ECM hydrogel coating mitigates the chronic inflammatory response to polypropylene mesh.

Polypropylene has been used as a surgical mesh material for several decades. This non-degradable synthetic polymer provides mechanical strength, a predictable host response, and its use has resulted in reduced recurrence rates for ventral hernia and pelvic organ prolapse. However, polypropylene and similar synthetic materials are associated with a chronic local tissue inflammatory response and dense fibrous tissue deposition. These outcomes have prompted variations in mesh design to minimize the surface area interface and increase integration with host tissue. In contrast, biologic scaffold materials composed of extracellular matrix (ECM) are rapidly degraded in-vivo and are associated with constructive tissue remodeling and minimal fibrosis. The objective of the present study was to assess the effects of an ECM hydrogel coating on the long-term host tissue response to polypropylene mesh in a rodent model of abdominal muscle injury. At 14 days post implantation, the ECM coated polypropylene mesh devices showed a decreased inflammatory response as characterized by the number and distribution of M1 macrophages (CD86+/CD68+) around mesh fibers when compared to the uncoated mesh devices. At 180 days the ECM coated polypropylene showed decreased density of collagen and amount of mature type I collagen deposited between mesh fibers when compared to the uncoated mesh devices. This study confirms and extends previous findings that an ECM coating mitigates the chronic inflammatory response and associated scar tissue deposition characteristic of polypropylene.

Role of the extracellular matrix in whole organ engineering.

End-stage organ failure is a devastating problem with limited therapeutic options. The definitive treatment is orthotropic transplantation, however, there exists a severe shortage of viable donor organs, and this shortage is worsening with an aging demographic and as the number of new cases of organ failure increases. Patients fortunate enough to receive a transplant are required to receive immunosuppressive therapies and can face transplant rejection. The emerging concept of organ engineering may offer a new hope for these patients. Researchers in the field of regenerative medicine and tissue engineering are using three-dimensional whole organ scaffolds composed of allogeneic or xenogeneic extracellular matrix (ECM) for engineering functional tissue suitable for transplantation. Perfusion decellularization is an approach that generates native ECM scaffolds with intact 3D anatomical architecture and vasculature. Decellularized organs provide the ideal transplantable scaffold with all the necessary microstructure and extracellular cues for cell attachment, differentiation, vascularization, and function. The present manuscript will review the role of the ECM in normal development, the concept of ECM tissue specificity, and the effect of processing methods on eventual clinical outcomes. An overview of existing challenges and future directions will also be discussed.

Intestinal stem cells remain viable after prolonged tissue storage.

Intestinal stem cells (ISCs) are responsible for renewal of the epithelium both during normal homeostasis and following injury. As such, they have significant therapeutic potential. However, whether ISCs can survive tissue storage is unknown. We hypothesize that, although the majority of epithelial cells might die, ISCs would remain viable for at least 24 h at 4 °C. To explore this hypothesis, jejuna of C57Bl6/J or Lgr5-LacZ mice were removed and either processed immediately or placed in phosphate-buffered saline at 4 °C. Delayed isolation of epithelium was performed after 24, 30, or 48 h storage. At the light microscope level, despite extensive apoptosis of villus epithelial cells, small intestinal crypts remained morphologically intact for 30 h and ISCs were identifiable via Lgr5-LacZ positivity. Electron microscopy showed that ISCs retained high integrity for 24 h. When assessed by flow cytometry, ISCs were more resistant to degeneration than the rest of the epithelium, including neighboring Paneth cells, with higher viability across all time points. Cultured isolated crypts showed no loss of capacity to form complex enteroids after 24 h tissue storage, with efficiencies after 7 days of culture remaining above 80 %. By 30 h storage, efficiencies declined but budding capability was retained. We conclude that, with delay in isolation, ISCs remain viable and retain their proliferative capacity. In contrast, the remainder of the epithelium, including the Paneth cells, exhibits degeneration and programmed cell death. If these findings are recapitulated in human tissue, storage at 4 °C might offer a valuable temporal window for the harvesting of crypts or ISCs for therapeutic application.

Intestinal crypts reproducibly expand in culture.

BACKGROUND: In vitro growth techniques for intestinal crypts and single intestinal stem cells have been recently described, but several questions of translational importance remain unaddressed. The purpose of this study was to first, evaluate if intestinal crypts reproducibly expand in vitro; second, determine the impact of age and region of intestine on crypt growth in vitro; and third, determine the effects of cryopreservation on crypt growth in vitro. METHODS AND MATERIALS: Crypts were harvested from 5 cm of proximal, middle, and distal small intestine of C57BL/6J mice aged 4 wk, 6-8 wk, 12-14 wk, and 18-20 wk (n = 4-6 animals) and cultured. For each region, we determined the efficiency of crypts forming enterospheres (day 1) and progressing to enteroids (day 7). Subsequently, enteroids were passaged and cryopreserved to determine if growth was changed by these manipulations. RESULTS: Forty-three to 99% of intestinal crypts formed enterospheres, with higher efficiency in proximal small intestine and in younger mice. Twenty-five to 64% of enterospheres progressed to budding enteroids within 7 d. In vitro expansion was greater in proximal enteroids. This expansion continued in a logarithmic fashion, with ≈ 97% replating efficiency of isolated enteroid crypt buds. Following cryopreservation, ≈ 90% of enteroids recovered normal proliferative capacity. CONCLUSIONS: Intestinal crypt culture is efficient and significantly expands intestinal tissue in a reproducible manner. Regional and age growth differences may reflect distinct stem cell characteristics or differences in support cells. The ability to culture and expand intestinal tissue in vitro provides a potential translational approach toward understanding and treating patients with short bowel syndrome.

Catheter-deliverable hydrogel derived from decellularized ventricular extracellular matrix increases endogenous cardiomyocytes and preserves cardiac function post-myocardial infarction.

OBJECTIVES: This study evaluated the use of an injectable hydrogel derived from ventricular extracellular matrix (ECM) for treating myocardial infarction (MI) and its ability to be delivered percutaneously. BACKGROUND: Injectable materials offer promising alternatives to treat MI. Although most of the examined materials have shown preserved or improved cardiac function in small animal models, none have been specifically designed for the heart, and few have translated to catheter delivery in large animal models. METHODS: We have developed a myocardial-specific hydrogel, derived from decellularized ventricular ECM, which self-assembles when injected in vivo. Female Sprague-Dawley rats underwent ischemia reperfusion followed by injection of the hydrogel or saline 2 weeks later. The implantation response was assessed via histology and immunohistochemistry, and the potential for arrhythmogenesis was examined using programmed electrical stimulation 1 week post-injection. Cardiac function was analyzed with magnetic resonance imaging 1 week pre-injection and 4 weeks post-MI. In a porcine model, we delivered the hydrogel using the NOGA-guided MyoStar catheter (Biologics Delivery Systems, Irwindale, California), and utilized histology to assess retention of the material. RESULTS: We demonstrate that injection of the material in the rat MI model increases endogenous cardiomyocytes in the infarct area and maintains cardiac function without inducing arrhythmias. Furthermore, we demonstrate feasibility of transendocardial catheter injection in a porcine model. CONCLUSIONS: To our knowledge, this is the first in situ gelling material to be delivered via transendocardial injection in a large animal model, a critical step towards the translation of injectable materials for treating MI in humans. Our results warrant further study of this material in a large animal model of MI and suggest this may be a promising new therapy for treating MI.

Maintenance of human hepatocyte function in vitro by liver-derived extracellular matrix gels.

Tissue engineering and regenerative medicine (TE&RM) approaches to treating liver disease have the potential to provide temporary support with biohybrid-liver-assist devices or long-term therapy by replacing the diseased liver with functional constructs. A rate-limiting step for TE&RM strategies has been the loss of hepatocyte-specific functions after hepatocytes are isolated from their highly specialized in vivo microenvironment and placed in in vitro culture systems. The identification of a biologic substrate that can maintain a functional hepatocyte differentiation profile during in vitro culture would advance potential TE&RM therapeutic strategies. The present study compared two different biologic substrates for their ability to support human hepatocyte function in vitro: porcine-liver-derived extracellular matrix (PLECM) or Matrigel. Because Matrigel has been shown to be the most useful matrix for static, traditional hepatocyte culture, we directly compared PLECM with Matrigel in each experiment. Albumin secretion, hepatic transport activity, and ammonia metabolism were used to determine hepatocyte function. Hepatocytes cultured between two layers of PLECM or Matrigel showed equally high levels of albumin expression and secretion, ammonia metabolism, and hepatic transporter expression and function. We conclude that like Matrigel, PLECM represents a favorable substrate for in vitro culture of human hepatocytes.