Rodent Model for Orthotopic Implantation of Engineered Liver Devices.

This study presents a novel surgical model developed to provide hematological support for implanted cellularized devices augmenting or replacing liver tissue function. Advances in bioengineering provide tools and materials to create living tissue replacements designed to restore that lost to disease, trauma, or congenital deformity. Such substitutes are often assembled and matured in vitro and need an immediate blood supply upon implantation, necessitating the development of supporting protocols. Animal translational models are required for continued development of engineered structures before clinical implementation, with rodent models often playing an essential early role. Our long-term goal has been generation of living tissue to provide liver function, utilizing advances in additive manufacturing technology to create 3D structures with intrinsic micron to millimeter scale channels modeled on natural vasculature. The surgical protocol developed enables testing various design iterations in vivo by anastomosis to the host rat vasculature. Lobation of rodent liver facilitates partial hepatectomy and repurposing the remaining vasculature to support implanted engineered tissue. Removal of the left lateral lobe exposes the underlying hepatic vasculature and can create space for a device. A shunt is created from the left portal vein to the left hepatic vein by cannulating each with separate silicone tubing. The device is then integrated into the shunt by connecting its inflow and outflow ports to the tubing and reestablishing blood flow. Sustained anticoagulation is maintained with an implanted osmotic pump. In our studies, animals were freely mobile after implantation; devices remained patent while maintaining blood flow through their millifluidic channels. This vascular anastomosis model has been greatly refined during the process of performing over 200 implantation procedures. We anticipate that the model described herein will find utility in developing preclinical translational protocols for evaluation of engineered liver tissue. Impact statement Tissue and organ transplantation are often the best clinically effective treatments for a variety of human ailments. However, the availability of suitable donor organs remains a critical problem. Advances in biotechnology hold potential in alleviating shortages, yet further work is required to surgically integrate large engineered tissues to host vasculature. Improved animal models such as the one described are valuable tools to support continued development and evaluation of novel therapies.

The Effect of Hematocrit on Platelet Adhesion: Experiments and Simulations.

The volume fraction of red blood cells (RBCs) in a capillary affects the degree to which platelets are promoted to marginate to near a vessel wall and form blood clots. In this work we investigate the relationship between RBC hematocrit and platelet adhesion activity. We perform experiments flowing blood samples through a microfluidic channel coated with type 1 collagen and observe the rate at which platelets adhere to the wall. We compare these results with three-dimensional boundary integral simulations of a suspension of RBCs and platelets in a periodic channel where platelets can adhere to the wall. In both cases, we find that the rate of platelet adhesion varies greatly with the RBC hematocrit. We observe that the relative decrease in platelet activity as hematocrit falls shows a similar profile for simulation and experiment.

In vitro measurement of particle margination in the microchannel flow: effect of varying hematocrit.

It has long been known that platelets undergo margination when flowing in blood vessels, such that there is an excess concentration near the vessel wall. We conduct experiments and three-dimensional boundary integral simulations of platelet-sized spherical particles in a microchannel 30 µm in height to measure the particle-concentration distribution profile and observe its margination at 10%, 20%, and 30% red blood cell hematocrit. The experiments involved adding 2.15-µm-diameter spheres into a solution of red blood cells, plasma, and water and flowing this mixture down a microfluidic channel at a wall shear rate of 1000 s(-1). Fluorescence imaging was used to determine the height and velocity of particles in the channel. Experimental results indicate that margination has largely occurred before particles travel 1 cm downstream and that hematocrit plays a role in the degree of margination. With simulations, we can track the trajectories of the particles with higher resolution. These simulations also confirm that margination from an initially uniform distribution of spheres and red blood cells occurs over the length scale of O(1 cm), with higher hematocrit showing faster margination. The results presented here, from both experiments and 3D simulations, may help explain the relationship between bleeding time in vessel trauma and red blood cell hematocrit as platelets move to a vessel wall.

Photodecomposition of Pigment Yellow 74, a pigment used in tattoo inks.

Tattooing has become a popular recreational practice among younger adults over the past decade. Although some of the pigments used in tattooing have been described, very little is known concerning the toxicology, phototoxicology or photochemistry of these pigments. Seven yellow tattoo inks were obtained from commercial sources and their pigments extracted, identified and quantitatively analyzed. The monoazo compound Pigment Yellow 74 (PY74; CI 11741) was found to be the major pigment in several of the tattoo inks. Solutions of commercial PY74 in tetrahydrofuran (THF) were deoxygenated using argon gas, and the photochemical reaction products were determined after exposure to simulated solar light generated by a filtered 6.5 kW xenon arc lamp. Spectrophotometric and high-pressure liquid chromatography (HPLC) analyses indicated that PY74 photodecomposed to multiple products that were isolated using a combination of silica chromatography and reversed-phase HPLC. Three of the major photodecomposition products were identified by nuclear magnetic resonance and mass spectrometry as N-(2-methoxyphenyl)-3-oxobutanamide (o-acetoacetanisidide), 2-(hydroxyimine)-N-(2-methoxyphenyl)-3-oxobutanamide and N,N''-bis(2-methoxyphenyl)urea. These results demonstrate that PY74 is not photostable in THF and that photochemical lysis occurs at several sites in PY74 including the hydrazone and amide groups. The data also suggest that the use of PY74 in tattoo inks could potentially result in the formation of photolysis products, resulting in toxicity at the tattoo site after irradiation with sunlight or more intense light sources.