SARS-CoV-2 infection and replication in human gastric organoids

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.Several clinical reports have described gastrointestinal symptoms for COVID-19, though whether the virus can replicate within the stomach remains unclear. Here the authors generate gastric organoids from human biopsies and show that the virus can efficiently infect gastric epithelium, suggesting that the stomach might have an active role in fecal-oral transmission

A new strategy for the decellularisation of large equine tendons as biocompatible tendon substitutes

Tendon ruptures and/or large losses remain to be a great clinical challenge and often require full replacement of the damaged tissue. The use of auto- and allografts or engineered scaffolds is an established approach to restore severe tendon injuries. However, these grafts are commonly related to scarce biocompatibility, site morbidity, chronic inflammation and poor biomechanical properties. Recently, the decellularisation techniques of allo- or xenografts using specific detergents have been studied and have been found to generate biocompatible substitutes that resemble the native tissue. This study aims to identify a novel decellularisation protocol for large equine tendons that would produce an extracellular matrix scaffold suitable for the regeneration of injured tendons in humans. Specifically, equine tendons were treated either with tri (n-butyl) phosphate alone, or associated to multiple concentrations of peracetic acid (1, 3 and 5 %), which has never before been tested in vitro. Samples were then analysed by histology and with biochemical, biomechanical, and cytotoxicity tests. The best decellularisation protocol, resulting from these examinations, was selected and the chosen scaffold was re-seeded with murine fibroblasts. Resulting grafts were tested for cell viability, histologic analysis, DNA and collagen content. The results identified 1 % tri (n-butyl) phosphate combined with 3 % peracetic acid as the most suitable decellularised matrix in terms of biochemical and biomechanical properties. Moreover, the non-cytotoxic nature of the decellularised matrix allowed for good fibroblast reseeding, thus demonstrating a biocompatible matrix that will be suitable for tendon tissue engineering and hopefully as substitutes in severe tendon damages

Alternating air-medium exposure in rotating bioreactors optimizes cell metabolism in 3D novel tubular scaffold polyurethane foams

Background: In vitro dynamic culture conditions play a pivotal role in developing engineered tissue grafts, where the supply of oxygen and nutrients, and waste removal must be permitted within construct thickness. For tubular scaffolds, mass transfer is enhanced by introducing a convective flow through rotating bioreactors with positive effects on cell proliferation, scaffold colonization and extracellular matrix deposition. We characterized a novel polyurethane-based tubular scaffold and investigated the impact of 3 different culture configurations over cell behavior: dynamic (i) single-phase (medium) rotation and (ii) double-phase exposure (medium-air) rotation; static (iii) single-phase static culture as control. Methods: A new mixture of polyol was tested to create polyurethane foams (PUFs) as 3D scaffold for tissue engineering. The structure obtained was morphologically and mechanically analyzed tested. Murine fibroblasts were externally seeded on the novel porous PUF scaffold, and cultured under different dynamic conditions. Viability assay, DNA quantification, SEM and histological analyses were performed at different time points. Results: The PUF scaffold presented interesting mechanical properties and morphology adequate to promote cell adhesion, highlighting its potential for tissue engineering purposes. Results showed that constructs under dynamic conditions contain enhanced viability and cell number, exponentially increased for double-phase rotation; under this last configuration, cells uniformly covered both the external surface and the lumen. Conclusions: The developed 3D structure combined with the alternated exposure to air and medium provided the optimal in vitro biochemical conditioning with adequate nutrient supply for cells. The results highlight a valuable combination of material and dynamic culture for tissue engineering applications

Estimation of the physiological mechanical conditioning in vascular tissue engineering by a predictive fluid-structure interaction approach

<p>The <i>in vitro</i> replication of physiological mechanical conditioning through bioreactors plays a crucial role in the development of functional Small-Caliber Tissue-Engineered Blood Vessels. An <i>in silico</i> scaffold-specific model under pulsatile perfusion provided by a bioreactor was implemented using a fluid-structure interaction (FSI) approach for viscoelastic tubular scaffolds (e.g. decellularized swine arteries, DSA). Results of working pressures, circumferential deformations, and wall shear stress on DSA fell within the desired physiological range and indicated the ability of this model to correctly predict the mechanical conditioning acting on the cells-scaffold system. Consequently, the FSI model allowed us to <i>a priori</i> define the stimulation pattern, driving <i>in vitro</i> physiological maturation of scaffolds, especially with viscoelastic properties.</p

Arterial Decellularized Scaffolds Produced Using an Innovative Automatic System

There is still an unmet clinical need for small-caliber artery substitution. Decellularized scaffolds in tissue engineering represent a promising solution. We have developed an innovative system for the automatic decellularization of blood vessels, used to process pig arteries. The system is able to automatically drive a decellularization process in a safe and reliable environment, with complex time patterns, using up to three different decellularization solutions, and providing at the same time a physical stress to improve the decellularization. The decellularization of pig arteries was evaluated by means of histology, DNA quantification and mechanical testing. Outcomes showed scaffolds with no cellular or nuclear remnants and a well-preserved tissue structure, corroborated by mechanical properties similar to native tissue. Decellularized scaffolds were seeded on the inner layer with human endothelial cells and implanted as iliac artery replacement in 4 pharmacologically immune-compromised pigs. This chimeric model was performed as a very preliminary evaluation to investigate the performances of these scaffolds in vivo, and to investigate the fate of seeded cells. Recipients were sacrificed on day 14 and day 70 after surgery, and vessels were found to be patent and with no evidence of thrombi formation. The inner layer was covered by endothelial cells, and the migration of cells positive for alpha-smooth-muscle actin was observed from the outer layer towards the tunica media. Intriguingly, the endothelial cells on explanted vessels were entirely derived from the host while the seeded cells were lost. In conclusion, this work presents a novel tool for a safe and controlled production of arterial scaffolds, with good decellularization outcomes and a good performance in a short-term, large-animal implantation. (C) 2015 S. Karger AG, Base