Development and Characterization of a Porcine Liver Scaffold.

The growing number of patients requiring liver transplantation for chronic liver disease cannot be currently met due to a shortage in donor tissue. As such, alternative tissue engineering approaches combining the use of acellular biological scaffolds and different cell populations (hepatic or progenitor) are being explored to augment the demand for functional organs. Our goal was to produce a clinically relevant sized scaffold from a sustainable source within 24 h, while preserving the extracellular matrix (ECM) to facilitate cell repopulation at a later stage. Whole porcine livers underwent perfusion decellularization via the hepatic artery and hepatic portal vein using a combination of saponin, sodium deoxycholate, and deionized water washes resulting in an acellular scaffold with an intact vasculature and preserved ECM. Molecular and immunohistochemical analysis (collagen I and IV and laminin) showed complete removal of any DNA material, together with excellent retention of glycosaminoglycans and collagen. Fourier-transform infrared spectroscopy (FTIR) analysis showed both absence of nuclear material and removal of any detergent residue, which was successfully achieved after additional ethanol gradient washes. Samples of the decellularized scaffold were assessed for cytotoxicity by seeding with porcine adipose-derived mesenchymal stem cells in vitro, these cells over a 10-day period showed attachment and proliferation. Perfusion of the vascular tree with contrast media followed by computed tomography (CT) imaging showed an intact vascular network. In vivo implantation of whole intact nonseeded livers, into a porcine model (as auxiliary graft) showed uniform perfusion macroscopically and histologically. Using this method, it is possible to create an acellular, clinically sized, liver scaffold with intact vasculature in less than 24 h.

Application of porcine gastrointestinal organoid units as a potential in vitro tool for drug discovery and development.

The gastrointestinal tract (GI) is a crucial part of the body for growth and development and its dysregulation can lead to several diseases with detrimental effects. Most of these diseases lack effective treatment, occurring as a result of inappropriate models to develop safe and potent therapies. Organoids are three-dimensional self-organizing and self-renewing structures that are composed of a cluster of different cells in vitro that resemble their organ of origin in architecture and function. Over recent years, organoids have been increasingly used to study developmental biology, disease progression, i.e., cancer, tissue engineering and regenerative medicine and other biological processes. Owing to their complex nature and ability to retain the morphological and molecular patterns of their tissue-of-origin, they have great potential as alternative tools/models for drug screening, development and biomarker discovery. Using a species with similar genetic homology to humans as a source of organoids, such as the porcine model may offer huge translational relevance. This review focuses on the culture and establishment of porcine organoid units and their potential use and application as in vitro models to further the science of drug discovery, by overcoming current limitations of established two- and three-dimensional models. It also highlights the translational application of using porcine organoids as a model of different disease contexts.

Development of a novel hybrid bioactive hydrogel for future clinical applications.

Three-dimensional hydrogels are ideal for tissue engineering applications due to their structural integrity and similarity to native soft tissues; however, they can lack mechanical stability. Our objective was to develop a bioactive and mechanically stable hydrogel for clinical application. Auricular cartilage was decellularised using a combination of hypertonic and hypotonic solutions with and without enzymes to produce acellular tissue. Methacryloyl groups were crosslinked with alginate and PVA main chains via 2-aminoethylmathacrylate and the entire macromonomer further crosslinked with the acellular tissue. The resultant hydrogels were characterised for its physicochemical properties (using NMR), in vitro degradation (via GPC analysis), mechanical stability (compression tests) and in vitro biocompatibility (co-culture with bone marrow-derived mesenchymal stem cells). Following decellularisation, the cartilage tissue showed to be acellular at a significant level (DNA content 25.33 ng/mg vs. 351.46 ng/mg control tissue), with good structural and molecular integrity of the retained extra cellular matrix (s-GAG= 0.19 µg/mg vs. 0.65 µg/mg ±0.001 control tissue). Proteomic analysis showed that collagen subtypes and proteoglycans were retained, and SEM and TEM showed preserved matrix ultra-structure. The hybrid hydrogel was successfully cross-linked with biological and polymer components, and it was stable for 30 days in simulated body fluid (poly dispersal index for alginate with tissue was stable at 1.08 and for PVA with tissue was stable at 1.16). It was also mechanically stable (Young's modulus of 0.46 ± 0.31 KPa) and biocompatible, as it was able to support the development of a multi-cellular feature with active cellular proliferation in vitro. We have shown that it is possible to successfully combine biological tissue with both a synthetic and natural polymer and create a hybrid bioactive hydrogel for clinical application.

Creation of an Acellular Vaginal Matrix for Potential Vaginal Augmentation and Cloacal Repair.

STUDY OBJECTIVE: Our aim was to use porcine vagina to create a vaginal matrix and test its cellular biocompatibility. DESIGN, SETTING, AND PARTICIPANTS: Vagina was harvested from pigs and decellularized (DC) using a combination of detergents (Triton X-100 and sodium deoxycholate) and enzymes (DNAse/RNAse). INTERVENTIONS: The presence of cellular material, collagen structural integrity, and basement membrane proteins were assessed histologically. To address cytocompatibility, porcine adipose-derived mesenchymal stem cells were harvested from abdominal fat together with vaginal epithelial cells and seeded onto the mucosal aspect of the vaginal scaffold. Both cell populations were seeded individually and assessed histologically at days 3 and 10. MAIN OUTCOME MEASURES AND RESULTS: The combination of enzymes and detergents resulted in a totally acellular matrix with very low DNA amount (control = 97.5 ng/µL ± 10.8 vs DC = 40.1 ng/µL ± 0.33; P = .02). The extracellular matrix showed retention of collagen fibers and elastin and a 50% retention in glycosaminoglycan content (control = 1.18 µg/mg ± 0.28; DC = 1.35 µg/mg ± 0.1; P = .03) and an intact basement membrane (positive for laminin and collagen IV). Seeded scaffolds showed cell attachment with adipose-derived mesenchymal stem cells and vaginal epithelial cells at days 3 and 10. CONCLUSION: It is possible to generate an acellular porcine vaginal matrix capable of supporting cells to reconstruct the vagina for future preclinical testing, and holds promise for creating clinically relevant-sized tissue for human application.

Tracheal Replacement Therapy with a Stem Cell-Seeded Graft: Lessons from Compassionate Use Application of a GMP-Compliant Tissue-Engineered Medicine.

Tracheal replacement for the treatment of end-stage airway disease remains an elusive goal. The use of tissue-engineered tracheae in compassionate use cases suggests that such an approach is a viable option. Here, a stem cell-seeded, decellularized tissue-engineered tracheal graft was used on a compassionate basis for a girl with critical tracheal stenosis after conventional reconstructive techniques failed. The graft represents the first cell-seeded tracheal graft manufactured to full good manufacturing practice (GMP) standards. We report important preclinical and clinical data from the case, which ended in the death of the recipient. Early results were encouraging, but an acute event, hypothesized to be an intrathoracic bleed, caused sudden airway obstruction 3 weeks post-transplantation, resulting in her death. We detail the clinical events and identify areas of priority to improve future grafts. In particular, we advocate the use of stents during the first few months post-implantation. The negative outcome of this case highlights the inherent difficulties in clinical translation where preclinical in vivo models cannot replicate complex clinical scenarios that are encountered. The practical difficulties in delivering GMP grafts underscore the need to refine protocols for phase I clinical trials. Stem Cells Translational Medicine 2017;6:1458-1464.

Growth factors and their use in short bowel.

PURPOSE OF REVIEW: To examine the most recent literature on the clinical trials associated with the relevant growth factors that have been of interest in the treatment of short bowel. RECENT FINDINGS: Short bowel is a rare but devastating condition that condemns patients to lifelong parenteral support. Historically, treatment options negating the need for parenteral support were limited. Therapeutic growth factor use is of interest, but the clinical trial data are inconclusive. The STEPS-2 trial was the first trial that showed a sustained positive effect of the growth factor glucagon-like peptide-2 (GLP-2). This led to a phase shift in the management of short bowel, with the US Food and Drug Administration approval of the GLP-2 analogue teduglutide in 2012. This review summarizes all the relevant clinical trials of growth factors in the treatment of short bowel. SUMMARY: GLP-2 has shown that growth factors can revolutionize the treatment of short bowel. Data however are lacking with regards to the solitary use of other factors. This review highlights the need for further work using the factors in combination as well as considering their use in novel methods for example in the field of regenerative medicine.

Stem Cell-Based Tissue-Engineered Laryngeal Replacement.

Patients with laryngeal disorders may have severe morbidity relating to swallowing, vocalization, and respiratory function, for which conventional therapies are suboptimal. A tissue-engineered approach would aim to restore the vocal folds and maintain respiratory function while limiting the extent of scarring in the regenerated tissue. Under Good Laboratory Practice conditions, we decellularized porcine larynges, using detergents and enzymes under negative pressure to produce an acellular scaffold comprising cartilage, muscle, and mucosa. To assess safety and functionality before clinical trials, a decellularized hemilarynx seeded with human bone marrow-derived mesenchymal stem cells and a tissue-engineered oral mucosal sheet was implanted orthotopically into six pigs. The seeded grafts were left in situ for 6 months and assessed using computed tomography imaging, bronchoscopy, and mucosal brushings, together with vocal recording and histological analysis on explantation. The graft caused no adverse respiratory function, nor did it impact swallowing or vocalization. Rudimentary vocal folds covered by contiguous epithelium were easily identifiable. In conclusion, the proposed tissue-engineered approach represents a viable alternative treatment for laryngeal defects. Stem Cells Translational Medicine 2017;6:677-687.

Mechanisms underlying heterologous skin scaffold-mediated tissue remodeling.

Biocompatibility of two newly developed porcine skin scaffolds was assessed after 3, 14, 21 and 90 days of implantation in rats. Both scaffolds showed absence of cells, preservation of ECM and mechanical properties comparable to non-decellularised skin before implantation. Host cell infiltration was much prominent on both scaffolds when compared to Permacol (surgical control). At day 3, the grafts were surrounded by polymorphonuclear cells, which were replaced by a notable number of IL-6-positive cells at day 14. Simultaneously, the number of pro-inflammatory M1-macrophage was enhanced. Interestingly, a predominant pro-remodeling M2 response, with newly formed vessels, myofibroblasts activation and a shift on the type of collagen expression was sequentially delayed (around 21 days). The gene expression of some trophic factors involved in tissue remodeling was congruent with the cellular events. Our findings suggested that the responsiveness of macrophages after non-crosslinked skin scaffolds implantation seemed to intimately affect various cell responses and molecular events; and this range of mutually reinforcing actions was predictive of a positive tissue remodeling that was essential for the long-standing success of the implants. Furthermore, our study indicates that non-crosslinked biologic scaffold implantation is biocompatible to the host tissue and somehow underlying molecular events involved in tissue repair.

Biocompatibility and potential of decellularized porcine small intestine to support cellular attachment and growth.

The aim of this study was to decellularize a 30 cm long segment of porcine small intestine, determine its in vivo behaviour and assess the type of immunological reaction it induces in a quantitative manner. A segment of porcine ileum up to 30 cm long, together with its attached vasculature, was decellularized via its mesenteric arcade as a single entity. The quality of the acellular scaffold was assessed histologically and using molecular tools. The host response to the scaffold was evaluated in a rodent model. Stereological techniques were incorporated into quantitative analysis of the phenotype of the macrophages infiltrating the scaffold in vivo. Lengths of ileal scaffold, together with its attached vasculature, were successfully decellularized, with no evidence of intact cells and DNA or collagen and GAGs overdegradation. Analysis of explants harvested over 2 months postimplantation revealed full-thickness recellularization and no signs of foreign body or immune reactions. Macrophage profiling proved that between weeks 4 and 8 in vivo there was a switch from an M1 (pro-inflammatory) to an M2 (pro-remodelling) type of response. We show here that the decellularization process results in a biocompatible and non-toxic matrix that upon implantation triggers cellular infiltration and angiogenesis, primarily characterized by a pro-remodelling type of mononuclear response, without inducing foreign body reaction or fibrosis.

Immunomodulatory effect of a decellularized skeletal muscle scaffold in a discordant xenotransplantation model.

Decellularized (acellular) scaffolds, composed of natural extracellular matrix, form the basis of an emerging generation of tissue-engineered organ and tissue replacements capable of transforming healthcare. Prime requirements for allogeneic, or xenogeneic, decellularized scaffolds are biocompatibility and absence of rejection. The humoral immune response to decellularized scaffolds has been well documented, but there is a lack of data on the cell-mediated immune response toward them in vitro and in vivo. Skeletal muscle scaffolds were decellularized, characterized in vitro, and xenotransplanted. The cellular immune response toward scaffolds was evaluated by immunohistochemistry and quantified stereologically. T-cell proliferation and cytokines, as assessed by flow cytometry using carboxy-fluorescein diacetate succinimidyl ester dye and cytometric bead array, formed an in vitro surrogate marker and correlate of the in vivo host immune response toward the scaffold. Decellularized scaffolds were free of major histocompatibility complex class I and II antigens and were found to exert anti-inflammatory and immunosuppressive effects, as evidenced by delayed biodegradation time in vivo; reduced sensitized T-cell proliferative activity in vitro; reduced IL-2, IFN-γ, and raised IL-10 levels in cell-culture supernatants; polarization of the macrophage response in vivo toward an M2 phenotype; and improved survival of donor-derived xenogeneic cells at 2 and 4 wk in vivo. Decellularized scaffolds polarize host responses away from a classical TH1-proinflammatory profile and appear to down-regulate T-cell xeno responses and TH1 effector function by inducing a state of peripheral T-cell hyporesponsiveness. These results have substantial implications for the future clinical application of tissue-engineered therapies.

Decellularized rabbit cricoarytenoid dorsalis muscle for laryngeal regeneration.

OBJECTIVES: Although considerable progress has been made in regenerative medicine, a quantum step would be the replacement and/or regeneration of functional muscle tissue. For example, although patients' airways can now be successfully replaced with stem cell-based techniques, a much greater patient need would be addressed by regeneration of the muscles required for engineering a functional larynx, in which active movement is critical. The rabbit cricoarytenoid dorsalis muscle was chosen for the present study because it is equivalent to the posterior cricoarytenoid muscle, the only significant abductor muscle in human larynges. METHODS: Rabbit cricoarytenoid dorsalis muscles were harvested, and different decellularization methods were compared by use of a combination of histologic, immunohistochemical, and molecular techniques. Decellularized scaffolds were implanted into Sprague-Dawley rats as part of a 2-week biocompatibility study to assess immunogenicity. RESULTS: Decellularization with a combination of latrunculin B, potassium iodide, potassium chloride, and deoxyribonuclease resulted in total DNA clearance and reduced levels of major histocompatibility complex class II expression, with relative preservation of the scaffold's structural integrity (collagen, elastin, and glycosaminoglycan content). The scaffolds showed minimal signs of rejection at 2 weeks in a cross-species (xenotransplantation) study. CONCLUSIONS: Decellularized laryngeal muscles, which are nonimmunogenic, may provide the optimal scaffold source for the generation of a fully functional tissue-engineered larynx.

Esophageal tissue engineering: a new approach for esophageal replacement.

A number of congenital and acquired disorders require esophageal tissue replacement. Various surgical techniques, such as gastric and colonic interposition, are standards of treatment, but frequently complicated by stenosis and other problems. Regenerative medicine approaches facilitate the use of biological constructs to replace or regenerate normal tissue function. We review the literature of esophageal tissue engineering, discuss its implications, compare the methodologies that have been employed and suggest possible directions for the future. Medline, Embase, the Cochrane Library, National Research Register and ClinicalTrials.gov databases were searched with the following search terms: stem cell and esophagus, esophageal replacement, esophageal tissue engineering, esophageal substitution. Reference lists of papers identified were also examined and experts in this field contacted for further information. All full-text articles in English of all potentially relevant abstracts were reviewed. Tissue engineering has involved acellular scaffolds that were either transplanted with the aim of being repopulated by host cells or seeded prior to transplantation. When acellular scaffolds were used to replace patch and short tubular defects they allowed epithelial and partial muscular migration whereas when employed for long tubular defects the results were poor leading to an increased rate of stenosis and mortality. Stenting has been shown as an effective means to reduce stenotic changes and promote cell migration, whilst omental wrapping to induce vascularization of the construct has an uncertain benefit. Decellularized matrices have been recently suggested as the optimal choice for scaffolds, but smart polymers that will incorporate signalling to promote cell-scaffold interaction may provide a more reproducible and available solution. Results in animal models that have used seeded scaffolds strongly suggest that seeding of both muscle and epithelial cells on scaffolds prior to implantation is a prerequisite for complete esophageal replacement. Novel approaches need to be designed to allow for peristalsis and vascularization in the engineered esophagus. Although esophageal tissue engineering potentially offers a real alternative to conventional treatments for severe esophageal disease, important barriers remain that need to be addressed.

The pro-angiogenic properties of multi-functional bioactive glass composite scaffolds.

The angiogenic properties of micron-sized (m-BG) and nano-sized (n-BG) bioactive glass (BG) filled poly(D,L lactide) (PDLLA) composites were investigated. On the basis of cell culture work investigating the secretion of vascular endothelial growth factor (VEGF) by human fibroblasts in contact with composite films (0, 5, 10, 20 wt %), porous 3D composite scaffolds, optimised with respect to the BG filler content capable of inducing angiogenic response, were produced. The in vivo vascularisation of the scaffolds was studied in a rat animal model and quantified using stereological analyses. The prepared scaffolds had high porosities (81-93%), permeability (k = 5.4-8.6 x 10⁻9 m²) and compressive strength values (0.4-1.6 MPa) all in the range of trabecular bone. On composite films containing 20 wt % m-BG or n-BG, human fibroblasts produced 5 times higher VEGF than on pure PDLLA films. After 8 weeks of implantation, m-BG and n-BG containing scaffolds were well-infiltrated with newly formed tissue and demonstrated higher vascularisation and percentage blood vessel to tissue (11.6-15.1%) than PDLLA scaffolds (8.5%). This work thus shows potential for the regeneration of hard-soft tissue defects and increased bone formation arising from enhanced vascularisation of the construct.

Smooth muscle cells in porcine vein graft intimal hyperplasia are derived from the local vessel wall.

BACKGROUND: Accelerated intimal hyperplasia (IH) is an important cause of morbidity and mortality in patients with atherosclerotic vascular disease treated with bypass vein grafts. We used an interposition vein graft model to determine the source of neointimal cells in a clinically relevant large animal model. METHODS: Jugular vein segments from sex-mismatched, MHC-in-bred pigs were implanted into common carotid arteries bilaterally and harvested up to 8 weeks postsurgery for stereological, histological, and immunofluorescence analyses. RESULTS: Progressive IH lesions contained macrophages and smooth muscle cells (SMC). Fluorescent in situ hybridization following grafting of female veins into male arteries revealed that only ∼10% of the SMC were male, confirming that the majority of intimal SMC derived from the local vessel wall. CONCLUSIONS: The majority of neointimal SMC in the IH seen after interposition vein grafting derive from the engrafted local vessel wall. These are the first results from a clinically relevant large animal model that confirm data from rodent models. They have implications for the utility of therapeutic stem cells in this type of intimal hyperplasia.

Pilot study: fibrin sealant in anal fistula model.

PURPOSE: The aim of this study was to investigate the failure of fibrin sealant treatment for fistula-in-ano in an experimental porcine model and to determine histologic changes associated with the sealant and setons. METHODS: Three surgically created fistulas were treated by seton drainage in each of eight male pigs. After 26 days, magnetic resonance imaging was performed and setons were removed. Two pigs were killed as controls for stereologic histologic fistula track assessment. In six, fistulas were curetted, and in four the fistulas were treated with fibrin sealant. In these four sealant and two seton pigs, magnetic resonance imaging was repeated a median of 47.5 days after fistula formation. The pigs were killed and stereologic histologic fistula track examination was performed to determine granulation tissue and fistula lumen volumes. These values were compared among control, seton, and sealant groups over time, and related to fistula volumes derived from magnetic resonance imaging. RESULTS: Sealant was not visible microscopically within tracks, although some sections revealed a foreign body-type reaction. On stereologic assessment, granulation tissue volumes were smaller in sealant and seton groups than in controls (median, 88 vs. 187 vs. 453 mm3, respectively; P = 0.002) and decreased over time (median, 408 and 152 mm3 (Day 42) vs. 88 and 75 (Day 53), respectively; P = 0.002). Fistula lumen (P < 0.001), and granulation tissue combined with fistula lumen volumes (P = 0.002) were similarly smaller. Magnetic resonance imaging of fistula intensity was less in the sealant group than in the seton group and controls (mean, 777 vs. 978 vs. 1214 units/mm2, P = 0.003). Magnetic resonance imaging fistula volumes were least in sealant and seton groups vs. controls (P = 0.024), decreasing significantly in the sealant group over time (P = 0.018). No direct relationship was found between imaging and histologic volumes. CONCLUSIONS: In an experimental porcine model of anal fistula, granulation tissue was still present, albeit diminished, following track curettage combined with seton or sealant therapy, and was minimal in the sealant group, confirming some benefit from this procedure. Eradication of all longstanding granulation tissue may ensure complete success of fibrin sealant therapy.