The combined effect of PDX1, epidermal growth factor and poly-L-ornithine on human amnion epithelial cells’ differentiation

Comparison of transduction efficiency of various adenoviral titres. (a) hAECs were transduced with adenovirus harbouring an mPdx1 vector at various MOIs (multiplicity of infection). Cells were stained with an mPdx1-specific antibody (Texas-Red conjugate) at 24 h and 48 h post infection to determine the transduction efficiency. Nuclei were counter stained with DAPI (blue). (b) Calculation of transduction efficiency from two different microscopic fields of cells 24 h after transduction with 50 MOI of the mPdx1-harbouring adenovirus. Cells were viewed using the 10X objective of an Olympus inverted fluorescence microscope. Purple nuclei are those that are stained by both DAPI and Texas-Red conjugated secondary antibody. (ZIP 1201 kb

The fate of an endothelium layer after preconditioning

BackgroundA strategy in minimizing thrombotic events of vascular constructs is to seed the luminal surface with autologous endothelial cells (ECs). The task of seeding ECs can be achieved via bioreactors, which induce mechanical forces (shear stress, strain, pressure) onto the ECs. Although bioreactors can achieve a confluent layer of ECs in vitro, their acute response to blood remains unclear. Moreover, the necessary mechanical conditions that will increase EC adhesion and function remain unclear. We hypothesize that preconditioning seeded endothelium under physiological flow will enhance their retention and function.ObjectiveTo determine the role of varying preconditioning protocols on seeded ECs in vitro and in vivo.MethodsScaffolds derived from decelluarized arteries seeded with autologous ECs were preconditioned for 9 days. Three specific protocols, low steady shear stress (SS), high SS, and cyclic SS were investigated. After preconditioning, the seeded grafts were exposed to 15 minutes of blood via an ex vivo arteriovenous shunt model or alternately an in vivo arteriovenous bypass graft model.ResultsThe shunt model demonstrated ECs remained intact for all conditions. In the arteriovenous bypass model, only the cyclic preconditioned grafts remained intact, maintained morphology, and resisted the attachment of circulating blood elements such as platelets, red blood cells, and leukocytes. Western blotting analysis demonstrated an increase in the protein expression of eNOS and prostaglandin I synthase for the cyclic high shear stress-conditioned cells relative to cells conditioned with high shear stress alone.ConclusionCyclic preconditioning has been shown here to increase the ECs ability to resist blood flow-induced shear stress and the attachment of circulating blood elements, key attributes in minimizing thrombotic events. These studies may ultimately establish protocols for the formation of a more durable endothelial monolayer that may be useful in the context of small vessel arterial reconstruction.Clinical RelevanceThe importance of ECs toward patency has been demonstrated by the superior performance of endothelialized vein compared with prosthetic vascular graft materials. This article evaluates conditioning protocols for bioengineered vascular conduits to improve endothelial retention. This study describes approaches to improve bioengineered vessels as a potential alternative to conventional prosthetic vascular grafts

Developing repair materials for stress urinary incontinence to withstand dynamic distension

Polypropylene mesh used as a mid-urethral sling is associated with severe clinical complications in a significant minority of patients. Current in vitro mechanical testing shows that polypropylene responds inadequately to mechanical distension and is also poor at supporting cell proliferation.Our objective therefore is to produce materials with more appropriate mechanical properties for use as a sling material but which can also support cell integration.Scaffolds of two polyurethanes (PU), poly-L-lactic acid (PLA) and co-polymers of the two were produced by electrospinning. Mechanical properties of materials were assessed and compared to polypropylene. The interaction of adipose derived stem cells (ADSC) with the scaffolds was also assessed. Uniaxial tensiometry of scaffolds was performed before and after seven days of cyclical distension. Cell penetration (using DAPI and a fluorescent red cell tracker dye), viability (AlamarBlue assay) and total collagen production (Sirius red assay) were measured for ADSC cultured on scaffolds.Polypropylene was stronger than polyurethanes and PLA. However, polypropylene mesh deformed plastically after 7 days of sustained cyclical distention, while polyurethanes maintained their elasticity. Scaffolds of PU containing PLA were weaker and stiffer than PU or polypropylene but were significantly better than PU scaffolds alone at supporting ADSC.Therefore, prolonged mechanical distension in vitro causes polypropylene to fail. Materials with more appropriate mechanical properties for use as sling materials can be produced using PU. Combining PLA with PU greatly improves interaction of cells with this material

Environmental Toxin Screening Using Human-Derived 3D Bioengineered Liver and Cardiac Organoids

IntroductionEnvironmental toxins, such as lead and other heavy metals, pesticides, and other compounds, represent a significant health concern within the USA and around the world. Even in the twenty-first century, a plethora of cities and towns in the U.S. have suffered from exposures to lead in drinking water or other heavy metals in food or the earth, while there is a high possibility of further places to suffer such exposures in the near future.MethodsWe employed bioengineered 3D human liver and cardiac organoids to screen a panel of environmental toxins (lead, mercury, thallium, and glyphosate), and charted the response of the organoids to these compounds. Liver and cardiac organoids were exposed to lead (10 µM–10 mM), mercury (200 nM–200 µM), thallium (10 nM–10 µM), or glyphosate (25 µM–25 mM) for a duration of 48 h. The impacts of toxin exposure were then assessed by LIVE/DEAD viability and cytotoxicity staining, measuring ATP activity and determining IC50 values, and determining changes in cardiac organoid beating activity.ResultsAs expected, all of the toxins induced toxicity in the organoids. Both ATP and LIVE/DEAD assays showed toxicity in both liver and cardiac organoids. In particular, thallium was the most toxic, with IC50 values of 13.5 and 1.35 µM in liver and cardiac organoids, respectively. Conversely, glyphosate was the least toxic of the four compounds, with IC50 values of 10.53 and 10.85 mM in liver and cardiac organoids, respectively. Additionally, toxins had a negative influence on cardiac organoid beating activity as well. Thallium resulting in the most significant decreases in beating rate, followed by mercury, then glyphosate, and finally, lead. These results suggest that the 3D organoids have significant utility to be deployed in additional toxicity screening applications, and future development of treatments to mitigate exposures.Conclusion3D organoids have significant utility to be deployed in additional toxicity screening applications, such as future development of treatments to mitigate exposures, drug screening, and environmental toxin detection