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.

Regeneration of the oesophageal muscle layer from oesophagus acellular matrix scaffold using adipose-derived stem cells.

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.

Innovative organotypic in vitro models for safety assessment: aligning with regulatory requirements and understanding models of the heart, skin, and liver as paradigms.

The development of improved, innovative models for the detection of toxicity of drugs, chemicals, or chemicals in cosmetics is crucial to efficiently bring new products safely to market in a cost-effective and timely manner. In addition, improvement in models to detect toxicity may reduce the incidence of unexpected post-marketing toxicity and reduce or eliminate the need for animal testing. The safety of novel products of the pharmaceutical, chemical, or cosmetics industry must be assured; therefore, toxicological properties need to be assessed. Accepted methods for gathering the information required by law for approval of substances are often animal methods. To reduce, refine, and replace animal testing, innovative organotypic in vitro models have emerged. Such models appear at different levels of complexity ranging from simpler, self-organized three-dimensional (3D) cell cultures up to more advanced scaffold-based co-cultures consisting of multiple cell types. This review provides an overview of recent developments in the field of toxicity testing with in vitro models for three major organ types: heart, skin, and liver. This review also examines regulatory aspects of such models in Europe and the UK, and summarizes best practices to facilitate the acceptance and appropriate use of advanced in vitro models.

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.

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.

Novel lactoferrin-conjugated gallium complex to treat Pseudomonas aeruginosa wound infection.

Pseudomonas aeruginosa is one of the leading causes of opportunistic infections such as chronic wound infection that could lead to multiple organ failure and death. Gallium (Ga3+) ions are known to inhibit P. aeruginosa growth and biofilm formation but require carrier for localized controlled delivery. Lactoferrin (LTf), a two-lobed protein, can deliver Ga3+ at sites of infection. This study aimed to develop a Ga-LTf complex for the treatment of wound infection. The characterisation of the Ga-LTf complex was conducted using differential scanning calorimetry (DSC), Infra-Red (FTIR) and Inductive Coupled Plasma Optical Emission Spectrometry (ICP-OES). The antibacterial activity was assessed by agar disc diffusion, liquid broth and biofilm inhibition assays using the colony forming units (CFUs). The healing capacity and biocompatibility were evaluated using a P.aeruginosa infected wound in a rat model. DSC analyses showed thermal transition consistent with apo-lactoferrin; FTIR confirmed the complexation of gallium to lactoferrin. ICP-OES confirmed the controlled local delivery of Ga3+. Ga-LTf showed a 0.57 log10 CFUs reduction at 24 h compared with untreated control in planktonic liquid broth assay. Ga-LTf showed the highest antibiofilm activity with a 2.24 log10 CFUs reduction at 24 h. Furthermore, Ga-LTf complex is biocompatible without any adverse effect on brain, kidney, liver and spleen of rats tested in this study. Ga-LTf can be potentially promising novel therapeutic agent to treat pathogenic bacterial infections.

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.

Antibacterial, remineralising and matrix metalloproteinase inhibiting scandium-doped phosphate glasses for treatment of dental caries.

OBJECTIVES: Antibiotic resistance is increasingly a growing global threat. This study aimed to investigate the potential use of newly developed scandium-doped phosphate-based glasses (Sc-PBGs) as an antibacterial and anticariogenic agent through controlled release of Sc3+ ions. METHODS: Sc-PBGs with various calcium and sodium oxide contents were produced and characterised using thermal and spectroscopic analysis. Degradation behaviour, ion release, antibacterial action against Streptococcus mutans, anti-matrix metalloproteinase-2 (MMP-2) activity, remineralisation potential and in vivo biocompatibility were also investigated. RESULTS: The developed glass system showed linear Sc3+ ions release over time. The released Sc3+ shows statistically significant inhibition of S. mutans biofilm (1.2 log10 CFU reduction at 6 h) and matrix metalloproteinase-2 (MMP-2) activity, compared with Sc-free glass and positive control. When Sc-PBGs were mounted alongside enamel sections, subjected to acidic challenges, alternating hyper- and hypomineralisation layers consistent with periods of re- and demineralisation were observed demonstrating their potential remineralising action. Furthermore, Sc-PBGs produced a non-toxic response when implanted subcutaneously for 2 weeks in Sprague Dawley rats. SIGNIFICANCE: Since Sc3+ ions might act on various enzymes essential to the biological mechanisms underlying caries, Sc-PBGs could be a promising therapeutic agent against cariogenic bacteria.

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.

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.

Is quercetin an alternative natural crosslinking agent to genipin for long-term dermal scaffolds implantation?

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.

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.

Annexin A12-26 Treatment Improves Skin Heterologous Transplantation by Modulating Inflammation and Angiogenesis Processes.

Skin graft successful depends on reduction of local inflammation evoked by the surgical lesion and efficient neovascularization to nutrition the graft. It has been shown that N-terminal portion of the Annexin A1 protein (AnxA1) with its anti-inflammatory properties induces epithelial mucosa repair and presents potential therapeutic approaches. The role of AnxA1 on wound healing has not been explored and we investigated in this study the effect of the peptide Ac2-26 (N-terminal AnxA1 peptide Ac2-26; AnxA12-26) on heterologous skin scaffolds transplantation in BALB/c mice, focusing on inflammation and angiogenesis. Treatment with AnxA12-26, once a day, from day 3-60 after scaffold implantation improved the take of the implant, induced vessels formation, enhanced gene and protein levels of the vascular growth factor-A (VEGF-A) and fibroblast influx into allograft tissue. It also decreased pro- while increasing anti-inflammatory cytokines. The pro-angiogenic activity of AnxA12-26 was corroborated by topical application of AnxA12-26 on the subcutaneous tissue of mice. Moreover, treatment of human umbilical endothelial cells (HUVECs) with AnxA12-26 improved proliferation, shortened cycle, increased migration and actin polymerization similarly to those evoked by VEGF-A. The peptide treatment instead only potentiated the tube formation induced by VEGF-A. Collectively, our data showed that AnxA12-26 treatment favors the tissue regeneration after skin grafting by avoiding exacerbated inflammation and improving the angiogenesis process.

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.

A comparison of tracheal scaffold strategies for pediatric transplantation in a rabbit model.

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 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.

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.

The development and implantation of a biologically derived allograft scaffold.

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.

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.

Potential use of gallium-doped phosphate-based glass material for periodontitis treatment.

This study aimed at evaluating the potential effect of gallium-incorporated phosphate-based glasses towards periodontitis-associated bacteria, Porphyromonas gingivalis, and matrix metalloproteinase-13. Periodontitis describes a group of inflammatory diseases of the gingiva and supporting structures of the periodontium. They are initiated by the accumulation of plaque bacteria, such as the putative periodontal pathogen Porphyromonas gingivalis, but the host immune response such as elevated matrix metalloproteinases are the major contributing factor for destruction of periodontal tissues. Antibacterial assays of gallium-incorporated phosphate-based glasses were conducted on Porphyromonas gingivalis ATCC 33277 using disc diffusion assay on fastidious anaerobe agar and liquid broth assay in a modified tryptic soy broth. In vitro study investigated the effect of gallium on purified recombinant human matrix metalloproteinase-13 activity using matrix metalloproteinase assay kit. In vivo biocompatibility of gallium-incorporated phosphate-based glass was evaluated in rats as subcutaneous implants. Antibacterial assay of gallium displayed activity against Porphyromonas gingivalis (inhibition zone of 22 ± 0.5 mm compared with 0 mm for control glass, c-PBG). Gallium in the glass contributed to growth inhibitory effect on Porphyromonas gingivalis (up to 1.30 reductions in log 10 values of the viable counts compared with control) in a modified tryptic soy broth. In vitro study showed gallium-incorporated phosphate-based glasses inhibited matrix metalloproteinase activity significantly (p ≤ 0.01) compared with c-PBG. Evaluation of in vivo biocompatibility of gallium-incorporated phosphate-based glasses in rats showed a non-toxic and foreign body response after 2 weeks of implantation. The results indicate that gallium ions might act on multiple targets of biological mechanisms underlying periodontal disease. Moreover, gallium-incorporated phosphate-based glasses are biocompatible in a rat model. The findings warrant further investigation and will have important clinical implications in the future treatment and management of periodontitis.