Biologic scaffolds are extensively used in various clinical applications such as musculotendinous reconstruction, hernia repair or wound healing. Biologic scaffolds used in these applications vary in species, breed and tissue of origin, and other variables that affect their properties. Decellularization and sterilization processes also determine the characteristics of these scaffolds. The goal of the present study is to compare the composition and mechanical properties of decellularized porcine placental scaffolds from three different porcine breeds: Landrace, York and Duroc. Placental extracellular matrix (ECM) scaffolds from the three porcine breeds preserved the amnion/chorion ECM structure and the basement membrane markers laminin and collagen type IV. ECM placental scaffolds showed similar contents of collagen, elastin and lipids, and minimal differences in glycosaminoglycans content. Mechanical properties from the three breeds ECM placental scaffolds were also similar and stable for 24 months. While this study serves as preliminary characterization of porcine ECM scaffolds, future studies will determine their compatibility and suitability for tissue engineering applications.

Background: Extracellular matrix (ECM) bioscaffolds produced by decellularization of source tissue have been effectively used for numerous clinical applications. However, decellu-larized tracheal constructs have been unsuccessful due to the immediate requirement of a functional airway epithelium on surgical implantation. ECM can be solubilized to form hydrogels that have been shown to support growth of many different cell types. The purpose of the present study is to compare the ability of airway epithelial cells to attach, form a confluent monolayer, and differentiate on homologous (trachea) and heterologous (urinary bladder) ECM substrates for potential application in full tracheal replacement. Materials and methods: Porcine tracheas and urinary bladders were decellularized. Human bronchial epithelial cells (HBECs) were cultured under differentiation conditions on acel-lular tracheal ECM and urinary bladder matrix (UBM) bioscaffolds and hydrogels and were assessed by histology and immunolabeling for markers of ciliation, goblet cell formation, and basement membrane deposition. Results: Both trachea and urinary bladder tissues were successfully decellularized. HBEC formed a confluent layer on both trachea and UBM scaffolds and on hydrogels created from these bioscaffolds. Cells grown on tracheal and UBM hydrogels, but not on bioscaffolds, showed positive-acetylated tubulin staining and the presence of mucus-producing goblet cells. Collagen IV immunolabeling showed basement membrane deposition by these cells on the surface of the hydrogels. Conclusions: ECM hydrogels supported growth and differentiation of HBEC better than decellularized ECM bioscaffolds and show potential utility as substrates for promotion of a mature respiratory epithelium for regenerative medicine applications in the trachea. (c) 2021 Elsevier Inc. All rights reserved.

IMPACT STATEMENT This study developed and characterized human testis extracellular matrix (htECM) and porcine testis ECM (ptECM) for testing in human spermatogonial stem cell (hSSC) culture. Results confirmed the hypothesis that ECM from the homologous species (human) and homologous tissue (testis) is optimal for maintaining hSSCs. We describe a simplified feeder-free, serum-free condition for future iterative testing to achieve the long-term goal of stable hSSC cultures. To facilitate analysis and understand the fate of hSSCs in culture, we describe a multiparameter, high-throughput, quantitative flow cytometry approach to rapidly count undifferentiated spermatogonia, differentiated spermatogonia, apoptotic spermatogonia, and proliferative spermatogonia in hSSC cultures.

Extracellular matrix (ECM) hydrogels represent a growing subset of injectable biomaterials for tissue engineering and regenerative medicine applications. Whilst two-dimensional scaffolds composed of ECM have been in routine clinical use for decades, the clinical translation of ECM hydrogels has been limited to date. Evaluation of the stability and function of ECM hydrogels after sterilization and storage is necessary to advance therapeutic use of these materials. In the present study, a new form of ECM material, specifically, ‘lyophilized digest’ was introduced to mitigate potential storage issues and investigate the effects of sterilization. Both ECM powders and lyophilized digests were subjected to sterilization by gamma irradiation, electron beam irradiation, ethylene oxide and supercritical carbon dioxide prior to hydrogel formation. The efficacy and effect of sterilization upon mechanical properties, macrophage response and stem cell chemotaxis was determined. Independent of the form of ECM exposed to sterilization, irradiation at 30 kGy altered ECM properties and inhibited subsequent gelation. Interestingly, the form of the ECM exposed to sterilization had a dramatic impact upon hydrogel rheological properties. Hydrogels formed from sterilized powders had reduced mechanical properties whereas hydrogels formed from sterilized lyophilized digest had mechanical properties equivalent to the non-sterilized control. Sterilization did not affect the bioactivity of ECM hydrogels as measured by stem cell chemotaxis and macrophage response. Together these data provide practical insight required for the translation of ECM based hydrogels.

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 SignificanceWe 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. (C) 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Torres, Crisanto M. MD; Reing, Janet E.; Molina, Catalina Pineda; Hussey, George S. PhD; Badylak, Stephen F. MD, PhD Author Information

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 sitespecific 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. (C) 2015 Wiley Periodicals, Inc.

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 SignificanceThe 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. (C) 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The use of synthetic surgical mesh materials has been shown to decrease the incidence of hernia recurrence, but can be associated with undesirable effects such as infection, chronic discomfort, and adhesion to viscera. Surgical meshes composed of extracellular matrix (i.e., biologically-derived mesh) are an alternative to synthetic meshes and can reduce some of these undesirable effects but are less frequently used due to greater cost and perceived inadequate strength as the mesh material degrades and is replaced by host tissue. The present study assessed the temporal association between mechanical properties and degradation of biologic mesh composed of urinary bladder matrix (UBM) in a rodent model of full thickness abdominal wall defect. Mesh degradation was evaluated for non-chemically crosslinked scaffolds with the use of (14)C-radiolabeled UBM. UBM biologic mesh was 50% degraded by 26 days and was completely degraded by 90 days. The mechanical properties of the UBM biologic mesh showed a rapid initial decrease in strength and modulus that was not proportionately associated with its degradation as measured by (14)C. The loss of strength and modulus was followed by a gradual increase in these values that was associated with the deposition of new, host derived connective tissue. The strength and modulus values were comparable to or greater than those of the native abdominal wall at all time points.

Extracellular matrix (ECM) derived from mammalian tissues has been utilized to repair damaged or missing tissue and improve healing outcomes. More recently, processing of ECM into hydrogels has expanded the use of these materials to include platforms for 3-dimensional cell culture as well as injectable therapeutics that can be delivered by minimally invasive techniques and fill irregularly shaped cavities. At the cellular level, ECM hydrogels initiate a multifaceted host response that includes recruitment of endogenous stem/progenitor cells, regional angiogenesis, and modulation of the innate immune response. Unfortunately, little is known about the components of the hydrogel that drive these responses. We hypothesized that different components of ECM hydrogels could play distinctive roles in stem cell and macrophage behavior. Utilizing a well-characterized ECM hydrogel derived from urinary bladder matrix (UBM), we separated the soluble and structural components of UBM hydrogel and characterized their biological activity. Perivascular stem cells migrated toward and reduced their proliferation in response to both structural and soluble components of UBM hydrogel. Both components also altered macrophage behavior but with different fingerprints. Soluble components increased phagocytosis with an IL-1RA(high), TNFα(low), IL-1β(low), uPA(low) secretion profile. Structural components decreased phagocytosis with a PGE2(high), PGF2α(high), TNFα(low), IL-1β(low), uPA(low), MMP2(low), MMP9(low), secretion profile. The biologic activity of the soluble components was mediated by Notch and PI3K/Akt signaling, while the biologic activity of the structural components was mediated by integrins and MEK/ERK signaling. Collectively, these findings demonstrate that soluble and structural components of ECM hydrogels contribute to the host response but through different mechanisms.

BACKGROUND Acellular dermal matrices (ADMs) have been commonly used in expander-based breast reconstruction to provide inferolateral prosthesis coverage. Although the clinical performance of these biologic scaffold materials varies depending on a number of factors, an in-depth systematic characterization of the host response is yet to be performed. The present study evaluates the biochemical composition and structure of two ADMs, AlloDerm(®) Regenerative Tissue Matrix and AlloMax™ Surgical Graft, and provides a comprehensive spatiotemporal characterization in a porcine model of tissue expander breast reconstruction. METHODS Each ADM was characterized with regard to thickness, permeability, donor nucleic acid content, (residual double-stranded DNA [dsDNA]), and growth factors (basic fibroblast growth factor [bFGF], vascular endothelial growth factor [VEGF], and transforming growth factor-beta 1 [TGF-β1]). Cytocompatibility was evaluated by in vitro cell culture on the ADMs. The host response was evaluated at 4 and 12 weeks at various locations within the ADMs using established metrics of the inflammatory and tissue remodeling response: cell infiltration, multinucleate giant cell formation, extent of ADM remodeling, and neovascularization. RESULTS AlloMax incorporated more readily with surrounding host tissue as measured by earlier and greater cell infiltration, fewer foreign body giant cells, and faster remodeling of ADM. These findings correlated with the in vitro composition and cytocompatibility analysis, which showed AlloMax to more readily support in vitro cell growth. CONCLUSIONS AlloMax and AlloDerm demonstrated distinct remodeling characteristics in a porcine model of tissue expander breast reconstruction.

The basement membrane complex (BMC) is a critical component of the extracellular matrix (ECM) that supports and facilitates the growth of cells. This study investigates four detergents commonly used in the process of tissue decellularization and their effect upon the BMC. The BMC of porcine urinary bladder was subjected to 3% Triton-X 100, 8mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 4% sodium deoxycholate or 1% sodium dodecyl sulfate (SDS) for 24h. The BMC structure for each treatment group was assessed by immunolabeling, scanning electron microscopy (SEM) and second harmonic generation (SHG) imaging of the fiber network. The composition was assessed by quantification of dsDNA, glycosaminoglycans (GAG) and collagen content. The results showed that collagen fibers within samples treated with 1% SDS and 8mM CHAPS were denatured, and the ECM contained fewer GAG compared with samples treated with 3% Triton X-100 or 4% sodium deoxycholate. Human microvascular endothelial cells (HMEC) were seeded onto each BMC and cultured for 7 days. Cell-ECM interactions were investigated by immunolabeling for integrin β-1, SEM imaging and semi-quantitative assessment of cellular infiltration, phenotype and confluence. HMEC cultured on a BMC treated with 3% Triton X-100 were more confluent and had a normal phenotype compared with HMEC cultured on a BMC treated with 4% sodium deoxycholate, 8mM CHAPS and 1% SDS. Both 8mM CHAPS and 1% SDS damaged the BMC to the extent that seeded HMEC were able to infiltrate the damaged sub-basement membrane tissue, showed decreased confluence and an atypical phenotype. The choice of detergents used for tissue decellularization can have a marked effect upon the integrity of the BMC of the resultant bioscaffold.

Biologic scaffolds composed of extracellular matrix (ECM) are commonly used to facilitate a constructive remodeling response in several types of tissue, including the esophagus. Surgical manipulation of the esophagus is often complicated by stricture, but preclinical and clinical studies have shown that the use of an ECM scaffold can mitigate stricture and promote a constructive outcome after resection of full circumference esophageal mucosa. Recognizing the potential benefits of ECM derived from homologous tissue (i.e., site-specific ECM), the objective of the present study was to prepare, characterize, and assess the in-vivo remodeling properties of ECM from porcine esophageal mucosa. The developed protocol for esophageal ECM preparation is compliant with previously established criteria of decellularization and results in a scaffold that maintains important biologic components and an ultrastructure consistent with a basement membrane complex. Perivascular stem cells remained viable when seeded upon the esophageal ECM scaffold in-vitro, and the in-vivo host response showed a pattern of constructive remodeling when implanted in soft tissue.

Acellular biologic scaffolds are commonly used to facilitate the constructive remodeling of three of the four traditional tissue types: connective, epithelial, and muscle tissues. However, the application of extracellular matrix (ECM) scaffolds to neural tissue has been limited, particularly in the central nervous system (CNS) where intrinsic regenerative potential is low. The ability of decellularized liver, lung, muscle, and other tissues to support tissue-specific cell phenotype and function suggests that CNS-derived biologic scaffolds may help to overcome barriers to mammalian CNS repair. A method was developed to create CNS ECM scaffolds from porcine optic nerve, spinal cord, and brain, with decellularization verified against established criteria. CNS ECM scaffolds retained neurosupportive proteins and growth factors and, when tested with the PC12 cell line in vitro, were cytocompatible and stimulated proliferation, migration, and differentiation. Urinary bladder ECM (a non-CNS ECM scaffold) was also cytocompatible and stimulated PC12 proliferation but inhibited migration rather than acting as a chemoattractant over the same concentration range while inducing greater rates of PC12 differentiation compared to CNS ECM. These results suggest that CNS ECM may provide tissue-specific advantages in CNS regenerative medicine applications and that ECM scaffolds in general may aid functional recovery after CNS injury.

Biologic scaffold materials composed of extracellular matrix (ECM) have been shown to promote the formation of site-specific, functional, host tissue following placement in a number of preclinical and clinical studies. Endotoxin contamination of biomaterials is thought to result in deleterious immune responses that may affect the remodeling outcome when present in significant quantities. However, the exact amount of endotoxin contamination within or upon an ECM-based biologic scaffold that is required to elicit adverse effects in recipients is currently unknown. The present study examined the in vitro and in vivo effects of endotoxin contamination within an ECM scaffold derived from porcine dermis upon the host immune response and the downstream ability of the scaffold material to promote constructive tissue remodeling. Test articles with endotoxin values that exceed the current U. S. Food and Drug Administration (FDA) limit had similar or decreased immune responses both in vitro and in vivo when compared with devices that were below the current FDA limit. Dermal matrices spiked with large doses of endotoxin (100 ng/mL), equivalent to 10-20 times the FDA limit, elicited a robust immune response in vitro. However, by 35 days postimplantation, no difference in tissue remodeling was detected, regardless of the amount of endotoxin present within the material. These results suggest that current endotoxin standards may fall well below levels that induce an adverse acute proinflammatory response and associated long-term deleterious effects upon tissue remodeling outcomes.

Biologic scaffolds prepared from the extracellular matrix (ECM) of decellularized mammalian tissues have been shown to facilitate constructive remodeling in injured tissues such as skeletal muscle, the esophagus, and lower urinary tract, among others. The ECM of every tissue has a unique composition and structure that likely has direct effects on the host response and it is plausible that ECM harvested from a given tissue would provide distinct advantages over ECM harvested from nonhomologous tissues. For example, a tissue specific muscle ECM scaffold may be more suitable for constructive remodeling of skeletal muscle than non-homologous ECM tissue sources. The present study describes an enzymatic and chemical decellularization process for isolating skeletal muscle ECM scaffolds using established decellularization criteria and characterized the structure and chemical composition of the resulting ECM. The results were compared to those from a non-muscle ECM derived from small intestine (SIS). Muscle ECM was shown to contain growth factors, glycosaminoglycans, and basement membrane structural proteins which differed from those present in SIS. Myogenic cells survived and proliferated on muscle ECM scaffolds in vitro, and when implanted in a rat abdominal wall injury model in vivo was shown to induce a constructive remodeling response associated with scaffold degradation and myogenesis in the implant area; however, the remodeling outcome did not differ from that induced by SIS by 35 days post surgery. These results suggest that superior tissue remodeling outcomes are not universally dependent upon homologous tissue derived ECM scaffold materials.

Macrophages have been classified as having plastic phenotypes which exist along a spectrum between M1 (classically activated; pro-inflammatory) and M2 (alternatively activated; regulatory, homeostatic). To date, the effects of polarization towards an M1 or M2 phenotype have been studied largely in the context of response to pathogen or cancer. Recently, M1 and M2 macrophages have been shown to play distinct roles in tissue remodeling following injury. In the present study, the M1/M2 paradigm was utilized to examine the role of macrophages in the remodeling process following implantation of 14 biologically derived surgical mesh materials in the rat abdominal wall. In situ polarization of macrophages responding to the materials was examined and correlated to a quantitative measure of the observed tissue remodeling response to determine whether macrophage polarization is an accurate predictor of the ability of a biologic scaffold to promote constructive tissue remodeling. Additionally the ability of M1 and M2 macrophages to differentially recruit progenitor-like cells in vitro, which are commonly observed to participate in the remodeling of those ECM scaffolds which have a positive clinical outcome, was examined as a possible mechanism underlying the differences in the observed remodeling responses. The results of the present study show that there is a strong correlation between the early macrophage response to implanted materials and the outcome of tissue remodeling. Increased numbers of M2 macrophages and higher ratios of M2:M1 macrophages within the site of remodeling at 14 days were associated with more positive remodeling outcomes (r2 = 0.525–0.686, p < 0.05). Further, the results of the present study suggest that the constructive remodeling outcome may be due to the recruitment and survival of different cell populations to the sites of remodeling associated with materials that elicit an M1 vs. M2 response. Both M2 and M0 macrophage conditioned media were shown to have higher chemotactic activities than media conditioned by M1 macrophages (p < 0.05). A more thorough understanding of these issues will logically influence the design of next generation biomaterials and the development of regenerative medicine strategies for the formation of functional host tissues.

BACKGROUND & AIMS:The therapy of choice for end-stage liver disease is whole-organ liver transplantation, but this option is limited by a shortage of donor organs. Cell-based therapies and hepatic tissue engineering have been considered as alternatives to liver transplantation, but neither has proven effective to date. A regenerative medicine approach for liver replacement has recently been described that includes the use of a three-dimensional organ scaffold prepared by decellularization of xenogeneic liver. The present study investigates a new, minimally disruptive method for whole-organ liver decellularization and three different cell reseeding strategies to engineer functional liver tissue. METHODS:A combination of enzymatic, detergent, and mechanical methods are used to remove all cells from isolated rat livers. Whole-organ perfusion is used in a customized organ chamber and the decellularized livers are examined by morphologic, biochemical, and immunolabeling techniques for preservation of the native matrix architecture and composition. Three different methods for hepatocyte seeding of the resultant three-dimensional liver scaffolds are evaluated to maximize cell survival and function: (1) direct parenchymal injection, (2) multistep infusion, or (3) continuous perfusion. RESULTS:The decellularization process preserves the three-dimensional macrostructure, the ultrastructure, the composition of the extracellular matrix components, the native microvascular network of the liver, and the bile drainage system, and up to 50% of growth factor content. The three-dimensional liver matrix reseeded with the multistep infusion of hepatocytes generated ∼90% of cell engraftment and supported liver-specific functional capacities of the engrafted cells, including albumin production, urea metabolism, and cytochrome P450 induction. CONCLUSIONS:Whole-organ liver decellularization is possible with maintenance of structure and composition suitable to support functional hepatocytes.