Contact-free monitoring of vessel graft stiffness - proof of concept as a tool for vascular tissue engineering

Tissue-engineered vessel grafts have to mimic the biomechanical properties of native blood vessels. Manufacturing processes often condition grafts to adapt them to the target flow conditions. Graft stiffness is influenced by material properties and dimensions and determines graft compliance. This proof-of-concept study evaluated a contact-free method to monitor biomechanical properties without compromising sterility. Forced vibration response analysis was performed on human umbilical vein (HUV) segments mounted in a buffer-filled tubing system. A linear motor and a dynamic signal analyser were used to excite the fluid by white noise (0-200 Hz). Vein responses were read out by laser triangulation and analysed by fast Fourier transformation. Modal analysis was performed by monitoring multiple positions of the vessel surface. As an inverse model of graft stiffening during conditioning, HUV were digested proteolytically, and the course of natural frequencies (NFs) was monitored over 120 min. Human umbilical vein showed up to five modes with NFs in the range of 5-100 Hz. The first natural frequencies of HUV did not alter over time while incubated in buffer (p = 0.555), whereas both collagenase (-35%, p = 0.0061) and elastase (-45%, p < 0.001) treatments caused significant decreases of NF within 120 min. Decellularized HUV showed similar results, indicating that changes of the extracellular matrix were responsible for the observed shift in NF. Performing vibration response analysis on vessel grafts is feasible without compromising sterility or integrity of the samples. This technique allows direct measurement of stiffness as an important biomechanical property, obviating the need to monitor surrogate parameters. Copyright (C) 2016 John Wiley & Sons, Ltd

Metabolic Requirements of Blood Vessels in a Perfusion Bioreactor

Small caliber vessel grafts are one of the major challenges of vascular tissue engineering. A variety of processes have been developed to create vascular grafts from scaffolds and donor cells in bioreactors. In order to optimize such processes, this study focused on monitoring vessel metabolism under conditions typically used in perfusion protocols. Bovine veins were perfused in a bioreactor for four days. Group 1 vessels served as controls and were perfused with standard medium. Medium of group 2 was adjusted to the viscosity of blood. Group 3 vessels were additionally challenged with elevated luminal pressure. Contractile function was assessed in an organ bath. Tissue viability was determined by tetrazolium dye reduction. Oxygen gradients, dextrose consumption, and lactate production were monitored using a blood gas analyzer. KCl induced contractions did not differ between days 0 and 4. Norepinephrine dose-response curves of group 3 vessels achieved a higher maximum contraction on day 4, with no changes of EC50. Tissue viability was not altered by any of the perfusion conditions. Oxygen gradients across the vessels did not change with time but were elevated in group 2, with no signs of oxygen depletion. Dextrose consumption and lactate formation of group 1 and 2 vessels appeared to be stoichiometric. In contrast, group 3 vessels produced more lactate than dextrose could supply. These results indicate that conventional oxygenation of culture media is sufficient to meet the oxygen consumption of a functional vessel. Elevated shear forces increased the oxygen demand without affecting dextrose consumption. Elevated shear forces and luminal pressure caused the utilization of alternative energy sources. Thus online monitoring of key metabolic parameters appears to be a desirable feature of perfusion bioreactors for vascular tissue engineering

Tissue Engineering of Small Caliber Vessel Grafts from Human Umbilical Veins

Human umbilical veins (HUV) have recently been suggested as a starting material for vascular tissue engineering. HUV possess a functional smooth muscle layer and could be turned into an immunologically inert graft with contractile properties by creating a neoendothelium from the recipient's own cells. This study investigated methods to remove the native endothelium without impairing the contractile function of the smooth muscle layer. These denuded HUV were then seeded with endothelial cells in a perfusion bioreactor, demonstrating the creation of a confluent, shear-resistant neoendothelium