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This study explored the feasibility of constructing a tissue engineered muscle layer in the oesophagus using oesophageal acellular matrix (OAM) scaffolds and human aortic smooth muscle cells (hASMCs) or human adipose-derived stem cells (hASCs). The second objective was to investigate the effect of hypoxic preconditioning of seeding cells on cell viability and migration depth. Our results demonstrated that hASMCs and hASCs could attach and adhere to the decellularized OAM scaffold and survive and proliferate for at least 7 days depending on the growth conditions. This indicates adipose-derived stem cells (ASCs) have the potential to substitute for smooth muscle cells (SMCs) in the construction of tissue engineered oesophageal muscle layers.

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.

Objectives/hypothesis Despite surgical advances, childhood tracheal stenosis is associated with high morbidity and mortality. Various tracheal scaffold strategies have been developed as the basis for bioengineered substitutes, but there is no consensus on which may be superior in vivo. We hypothesized that there would be no difference in morbidity and mortality between three competing scaffold strategies in rabbits. Study design Pilot preclinical study. Methods Tracheal scaffolds were prepared by three methods that have been applied clinically and reported: preserved cadaveric ("Herberhold") allografts, detergent-enzymatically decellularized allografts, and synthetic scaffolds (nanocomposite polymer [polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU)]). Scaffolds were implanted into cervical trachea of New Zealand White rabbits (n = 4 per group) without cell seeding. Control animals (n = 4) received autotransplanted tracheal segments using the same technique. Animals underwent bronchoscopic monitoring of the grafts for 30 days. Macroscopic evaluation of tissue integration, graft stenosis, and collapsibility and histological examinations were performed on explants at termination. Results All surgical controls survived to termination without airway compromise. Mild to moderate anastomotic stenosis from granulation tissue was detected, but there was evidence suggestive of vascular reconnection with minimal fibrous encapsulation. In contrast, three of the four animals in the Herberhold and POSS-PCU groups, and all animals receiving decellularized allografts, required early termination due to respiratory distress. Herberhold grafts showed intense inflammatory reactions, anastomotic stenoses, and mucus plugging. Synthetic graft integration and vascularization were poor, whereas decellularized grafts demonstrated malacia and collapse but had features suggestive of vascular connection or revascularization. Conclusions There are mirror-image benefits and drawbacks to nonrecellularized, decellularized, and synthetic grafts, such that none emerged as the preferred option. Results from prevascularized and/or cell-seeded grafts (as applied clinically) may elucidate clearer advantages of one scaffold type over another. Level of evidence NA. Laryngoscope, 127:E449-E457, 2017.

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.

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.

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.

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.

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.

Tissue engineered tracheae have been successfully implanted to treat a small number of patients on compassionate grounds. The treatment has not become mainstream due to the time taken to produce the scaffold and the resultant financial costs. We have developed a method for decellularization (DC) based on vacuum technology, which when combined with an enzyme/detergent protocol significantly reduces the time required to create clinically suitable scaffolds. We have applied this technology to prepare porcine tracheal scaffolds and compared the results to scaffolds produced under normal atmospheric pressures. The principal outcome measures were the reduction in time (9 days to prepare the scaffold) followed by a reduction in residual DNA levels (DC no-vac: 137.8±48.82 ng/mg vs. DC vac 36.83±18.45 ng/mg, p

As biocompatible matrices, porcine dermal scaffolds have limited application in tissue engineering due to rapid degradation following implantation. This study compared the physical, chemical and biomechanical changes that occurred when genipin and quercetin were used to crosslink dermal scaffolds and to determine whether quercetin could be used as an alternative to genipin. Physicochemical changes in the collagen were assessed using spectroscopic methods [X-ray diffraction analysis (XRD) and nuclear magnetic resonance (NMR) analysis]. The crosslinking reaction was evaluated by quantification of amino acids and the degree of this reaction by ninhydrin assay. Because the mechanical behaviour of the collagen matrices is highly influenced by crosslinking, the tensile strength of both sets of scaffolds was evaluated. The highest mechanical strength, stiffness, degree of crosslinking and changes in the packing features of collagen (measured by XRD) were achieved using genipin. Some of the results found in the quercetin-crosslinked scaffolds were possibly due to hydration and dehydration effects elicited by the solvents (phosphate-buffered saline or ethanol), as seen in the NMR results. In the quercetin-ethanol-crosslinked scaffolds, possible reorientation of the amino groups of the collagen molecule may have taken place. Therefore, depending on their proximity to the crosslinking reagent, different types and numbers of interactions may have occurred, inducing a higher crosslinking degree (as evidenced by the ninhydrin assay) and reduction in the free amino acids after reaction. Both crosslinking agents and solvents interfere in the physicochemical properties of collagen thereby inducing variations in the matrix structure. Quercetin-crosslinked scaffolds may have broader clinical application where a lower degree of crosslinking and stiffness is required. Copyright © 2016 John Wiley & Sons, Ltd.

Background Laryngeal functional impairment relating to swallowing, vocalisation and respiration can be life changing and devastating for patients. A tissue engineering approach to regenerating vocal folds would represent a significant advantage over current clinical practice. Method Porcine hemi-larynx were de-cellularised under negative pressure. The resultant acellular scaffold was seeded with human bone marrow derived mesenchymal stem cells and primary human epithelial cells. Seeded scaffolds were implanted orthotopically into a defect created in the thyroid cartilage in 8 pigs and monitored in vivo for 2 months. In vivo assessments consisted of mucosal brushing and bronchoscopy at 1, 2, 4 and 8 weeks post implantation followed by histological evaluation post termination. Results The implanted graft had no adverse effect on respiratory function in 6 of the 8 pigs, none of the pigs had problems with swallowing or vocalization. Six out of the 8 animals survived to the planned termination date, 2 animals were terminated due to mild stenosis and deep tissue abscess formation respectively. Human epithelial cells from mucosal brushings could only be identified at weeks 1 and 4. The explanted tissue showed complete epithelialization of the mucosal surface and the development of rudimentary vocal folds. However, there was no evidence of cartilage remodelling at the relatively early censor point. Conclusion Single stage partial laryngeal replacement is a safe surgical procedure. Replacement with a tissue engineered laryngeal graft as a single procedure is surgically feasible and results in appropriate mucosal coverage and rudimentary vocal fold development.

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.

Poly(3-hydroxybutyrate) (P(3HB)) foams exhibiting highly interconnected porosity (85% porosity) were prepared using a unique combination of solvent casting and particulate leaching techniques by employing commercially available sugar cubes as porogen. Bioactive glass (BG) particles of 45S5 Bioglass((R)) grade were introduced in the scaffold microstructure, both in micrometer ((m-BG)

Biologically derived scaffolds are becoming viable treatment options for tissue/organ repair and regeneration. A continuing hurdle is the need for a functional blood supply to and from the implanted scaffold. We have addressed this problem by constructing an acellular ileal scaffold with an attached vascular network suitable for implantation and immediate reperfusion with the host's blood. Using a vascular perfusion approach, a segment of porcine ileum up to 30 cm long, together with its attached vasculature, was decellularized as a single entity. The quality of the decellularized scaffold was assessed histologically and using molecular tools. To establish vascular perfusion potentials of the scaffold, a right-sided nephrectomy and end-to-end anastomosis of the decellularized scaffold's vasculature to a renal artery and vein were performed in a pig of similar size to the donor animal. Lengths of ileal scaffold, together with its attached vasculature, were successfully decellularized, with no evidence of intact cells/nuclear material or collagen degradation. The scaffold's decellularized vascular network demonstrated optimum perfusion at 1, 2 and 24 h post-implantation and the mesenteric arcade remained patent throughout the assessment. The 1, 2 and 24 h explanted scaffolds demonstrated signs of cellular attachment, with cells positive for CD68 and CD133 on the vascular luminal aspect. It is possible to decellularize clinically relevant lengths of small intestine, together with the associated vasculature, as a single segment. The functional vascular network may represent a route for recellularization for future regeneration of bowel tissue for patients with short bowel syndrome.

This work investigated the effect of adding nanoparticulate (29 nm) bioactive glass particles on the bioactivity, degradation and in vitro cytocompatibility of poly(3-hydroxybutyrate) (P(3HB)) composites/nano-sized bioactive glass (n-BG). Two different concentrations (10 and 20 wt %) of nanoscale bioactive glass particles of 45S5 Bioglass composition were used to prepare composite films. Several techniques (Raman spectroscopy, scanning electron microscopy, atomic force microscopy, energy dispersive X-ray) were used to monitor their surface and bioreactivity over a 45-day period of immersion in simulated body fluid (SBF). All results suggested the P(3HB)/n-BG composites to be highly bioactive, confirmed by the formation of hydroxyapatite on material surfaces upon immersion in SBF. The weight loss and water uptake were found to increase on increasing bioactive glass content. Cytocompatibility study (cell proliferation, cell attachment, alkaline phosphatase activity and osteocalcin production) using human MG-63 osteoblast-like cells in osteogenic and non-osteogenic medium showed that the composite substrates are suitable for cell attachment, proliferation and differentiation.

Background Successful replacement of airways in patients has been done with tissue engineered constructs. However, replacing the larynx where active movement is crucial, requires functional muscle tissue. Methods Decellularised scaffolds were characterised with histological, immunohistochemical, and molecular techniques and xenogenically implanted to determine the effect of implantation on scaffold biodegradation time and immunogenicity in vivo. The cellular host immune response to the scaffold was quantified by stereology and by fluorescence activated cell sorting in vitro. Findings Decellularisation results in total DNA clearance and downregulation of MHC classes I/II and myosin heavy chain expression, with relative preservation of the scaffold's structural integrity (collagen, elastin, s-glycosaminoglycans content) and biomechanical properties. Decellularisation altered the host response to the scaffold in vivo, resulting in a prolonged degradation time and increased neoangiogenic potential relative to fresh tissue. In addition, we proved for the first time that decellularised scaffolds trigger a lower cellular mediated immune response, resulting in a reduced T-cell proliferative response in vitro, and alter the cellular immune profile towards the scaffolds in vivo, with a reduction in CD3+ cells and a shift towards the M2-macrophage phenotype. Interpretation Decellularised laryngeal muscles are non-immunogenic and may provide the optimum scaffold source for a tissue-engineered larynx. Funding UK Medical Research Council, Sparks Children's Charity, and Royal College of Surgeons of England.

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 sug- gest 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.