Ex Situ Left Ventricular Pressure-Volume Loop Analyses for Donor Hearts: Proof of Concept in an Ovine Experimental Model.

Ex situ heart perfusion (ESHP) has emerged as an important strategy to preserve donation after brain death (DBD) and donation after circulatory death (DCD) donor hearts. Clinically, both DBD and DCD hearts are successfully preserved using ESHP. Viability assessment is currently based on biochemical values, while a reliable method for graft function assessment in a physiologic working mode is unavailable. As functional assessment during ESHP has demonstrated the highest predictive value of outcome post-transplantation, this is an important area for improvement. In this study, a novel method for ex situ assessment of left ventricular function with pressure-volume loop analyses is evaluated. Ovine hearts were functionally evaluated during normothermic ESHP with the novel pressure-volume loop system. This system provides an afterload and adjustable preload to the left ventricle. By increasing the preload and measuring end-systolic elastance, the system could successfully assess the left ventricular function. End-systolic elastance at 60 min and 120 min was 2.8 ± 1.8 mmHg/mL and 2.7 ± 0.7 mmHg/mL, respectively. In this study we show a novel method for functional graft assessment with ex situ pressure-loop analyses during ESHP. When further validated, this method for pressure-volume assessments, could be used for better graft selection in both DBD and DCD donor hearts.

Liver preservation with machine perfusion and a newly developed cell-free oxygen carrier solution under subnormothermic conditions.

We describe a new preservation modality combining machine perfusion (MP) at subnormothermic conditions(21 °C) with a new hemoglobin-based oxygen carrier (HBOC) solution. MP (n=6) was compared to cold static preservation (CSP; n=6) in porcine orthotopic liver transplants after 9 h of cold ischemia and 5-day follow-up. Recipients' peripheral blood, serial liver biopsies, preservation solutions and bile specimens were collected before, during and after liver preservation. Clinical laboratorial and histological analyses were performed in addition to mitochondrial functional assays, transcriptomic, metabolomic and inflammatory inflammatory mediator analyses. Compared with CSP, MP animals had: (1) significantly higher survival (100%vs. 33%; p<0.05); (2) superior graft function (p<0.05);(3) eight times higher hepatic O2 delivery than O2 consumption (0.78 mL O2/g/h vs. 0.096 mL O2/g/h) during MP; and (4) significantly greater bile production (MP=378.5 ± 179.7; CS=151.6 ± 116.85). MP downregulated interferon (IFN)-α and IFN-γ in liver tissue. MP allografts cleared lactate, produced urea, sustained gluconeogenesis and produced hydrophilic bile after reperfusion. Enhanced oxygenation under subnormothermic conditions triggers regenerative and cell protective responses resulting in improved allograft function. MP at 21 °C with the HBOC solution significantly improves liver preservation compared to CSP.

Numerical simulation of asymmetrically altered growth as initiation mechanism of scoliosis.

The causes of idiopathic scoliosis are still uncertain; buckling is mentioned often, but never proven. The authors hypothesize another option: unilateral postponement of growth of MM Rotatores or of ligamentum flavum and intertransverse ligament. In this paper, both buckling and the two new theories of scoliotic initiation are studied using a new finite element model that simulates the mechanical behavior of the human spine. This model was validated by the stiffness data of Panjabi et al. (J. Biomech. 9:185-192, 1976). After a small correction of the prestrain of some ligaments and the MM Rotatores the model appeared to be valid. The postponement in growth was translated in the numerical model in an asymmetrical stiffness. The spine was loaded axially and the resulting deformation was analyzed for the presence of the coupling of lateral deviation and axial rotation that is characteristic for scoliosis. Only unilateral postponement of growth of ligamentum flavum and intertransverse ligament appeared to initiate scoliosis. Buckling did not initiate scoliosis.

The Groningen hypothermic liver perfusion pump: functional evaluation of a new machine perfusion system.

To improve preservation of donor livers, we have developed a portable hypothermic machine perfusion (HMP) system as an alternative for static cold storage. A prototype of the system was built and evaluated on functionality. Evaluation criteria included 24 h of adequate pressure controlled perfusion, sufficient oxygenation, a maintained 0-4 degrees C temperature and sterile conditions. Porcine livers were perfused with pump pressures that were set at 4 mmHg (continuous, portal vein) and 30/20 mmHg, at 60 BPM (pulsatile, hepatic artery). Control livers were preserved using the clinical golden standard: static cold storage. In the HMP group, pressure, flow and temperature were continuously monitored for 24 h. At time-points t = 0, 2, 4, 8, 12, and 24 h samples of University of Wisconsin machine preservation solution were taken for measurement of partial oxygen pressure (pO(2)) and lacto-dehydrogenase. Biopsies in every lobe were taken for histology and electron microscopy; samples of ice, preservation solution, liver surface, and bile were taken and cultured to determine sterility. Results showed that temperature was maintained at 0-4 degrees C; perfusion pressure was maintained at 4 mmHg and 30/20 mmHg for portal vein and hepatic artery, respectively. Flow was approximately 350 and 80 ml/min, respectively, but decreased in the portal vein, probably due to edema formation. Arterial pO(2) was kept at 100 kPa. Histology showed complete perfusion of the liver with no major damage to hepatocytes, bile ducts, and non-parenchymal cells compared to control livers. The machine perfusion system complied to the design criteria and will have to demonstrate the superiority of machine perfusion over cold storage in transplant experiments.

Development of the isolated dual perfused rat liver model as an improved reperfusion model for transplantation research.

The Isolated Perfused Liver (IPL) model is a widely used and appreciated in vitro method to demonstrate liver viability and metabolism. Reperfusion is performed in a controlled setting, however, via the portal vein only. To study transplant related questions concerning bile and transport of bile, the in vitro Isolated dual Perfused Liver model is revisited. The IdPL is an in vitro reperfusion model, using both portal vein and hepatic artery. Livers from 12 Wistar rats were flushed with University of Wisconsin-organ preservation solution, procured and reperfused in either the conventional IPL-model (n = 6) or the new IdPL-model (n = 6). Liver injury, assessed by the release of aspartate amino transferase and lactate dehydrogenase, showed similar levels during both IPL and I dPL reperfusion, only alanine amino transferase showed an improvement. Cumulative bile production showed an improvement: 176.3 +/- 8.4 in the IdPL compared to 126.1 +/- 12.2 microg/g-liver in the IPL (p < 0.05). Clearance of phenol red (PR) and taurocholic acid (TC) remained similar. At 90 minutes reperfusion the PR clearance showed 0.11 +/- 0.01 and 0.11 +/- 0.02 mg/30min/g-liver and the TC clearance 1.01 +/- 0.10 and 1.01 +/- 0.07 micromol/ml/30min/g-liver in the IPL and IdPL, respectively. Increasing the reperfusion time beyond the normally used 90 minutes resulted in a significant increase in transaminases and LDH and a decrease in bile production, liver morphology remained intact and glycogen content was appropriate. In conclusion, the IdPL-model showed similar or better results than the IPL-model, but the liver could not endure an extended reperfusion time using the IdPL.

Hypothermic machine perfusion of the liver and the critical balance between perfusion pressures and endothelial injury.

Hypothermic machine perfusion (HMP) provides better protection against cold ischemic injury than cold storage in marginal donor kidneys. Also, in liver transplantation a switch from static cold storage to HMP could be beneficial as it would allow longer preservation times and the use of marginal donors. A critical question concerning application of HMP in liver preservation is the crucial balance between perfusion pressure and occurrence of endothelial injury. Rat livers were cold-perfused for 24 hours to study perfusion pressures for both hepatic artery and portal vein. Cold storage served as control and was compared to HMP-preserved livers using a mean arterial perfusion pressure of 25 mm Hg and a portal perfusion pressure of 4 mm Hg (25% of normothermic liver circulation) and to HMP at 50 mm Hg and 8 mm Hg perfusion, respectively (50% of normothermic liver circulation). UW solution was enriched with 14.9 micromol/L propidium iodide (PI) to stain for dead cells and with an additional 13.5 micromol/L acridine orange to stain for viable hepatocytes. A low PI-positive cell count was found using HMP at 25% of normal circulation compared to cold storage. The PI count was high for the HMP group perfused at just 50% of normal circulation compared to HMP at 25% and compared to cold storage. In summary, for liver HMP, perfusion at 25% showed complete perfusion with minimal cellular injury. HMP using perfusion pressures of 25 mm Hg for the hepatic artery and 4 mm Hg for the portal vein is feasible without induction of endothelial injury.

Numerical simulation of the hepatic circulation.

Availability of donor livers and the relatively short preservation time limit the success of liver transplantation. The use of hypothermic machine perfusion could pave the way for expansion of the donor pool. To better define optimal settings of such a device, the feasibility of using a numerical simulation model of the hepatic circulation is determined. Hemodynamics in the hepatic arterial, portal venous and hepatic venous compartments of the hepatic vascular tree was modelled using an electrical analogue. Calculated pressure and flow profiles throughout the liver were in accordance with physiologic profiles in the total circulatory system. Comparison of calculated flow values with normal control values showed a discrepancy that was explained by inaccurate diameter input data. Until more precise methods for determining vascular dimensions become available, redefining vessel diameter makes the simulation model perfectly suitable for predicting influences of temperature and/or viscosity on hepatic hemodynamics and is thereby an excellent tool in defining optimal settings for our hypothermic liver perfusion system.

Comparison of velocity profiles for different flow chamber designs used in studies of microbial adhesion to surfaces.

Flow chambers are commonly used to study microbial adhesion to surfaces under environmentally relevant hydrodynamic conditions. The parallel plate flow chamber (PPFC) is the most common design, and mass transport occurs through slow convective diffusion. In this study, we analyzed four different PPFCs to determine whether the expected hydrodynamic conditions, which control both mass transport and detachment forces, are actually achieved. Furthermore, the different PPFCs were critically evaluated based on the size of the area where the velocity profile was established and constant with a range of flow rates, indicating that valid observations could be made. Velocity profiles in the different chambers were calculated by using a numerical simulation model based on the finite element method and were found to coincide with the profiles measured by particle image velocimetry. Environmentally relevant shear rates between 0 and 10,000 s(-1) could be measured over a sizeable proportion of the substratum surface for only two of the four PPFCs. Two models appeared to be flawed in the design of their inlets and outlets and allowed development of a stable velocity profile only for shear rates up to 0.5 and 500 s(-1). For these PPFCs the inlet and outlet were curved, and the modeled shear rates deviated from the calculated shear rates by up to 75%. We concluded that PPFCs used for studies of microbial adhesion to surfaces should be designed so that their inlets and outlets are in line with the flow channel. Alternatively, the channel length should be increased to allow a greater length for the establishment of the desired hydrodynamic conditions.

In vivo and in vitro experience with the PUCA-II, a single-valved pulsatile catheter-pump.

The Pulsatile Catheter (PUCA) pump is a trans-arterial pulsatile ventricular assist device that can be used for short-term left ventricular support. The separate inflow and outflow valves in the first version of the device (PUCA-I) were replaced by a single inflow/outflow valve in the latest PUCA pump version (PUCA-II). The new combined valve was tested during in vitro (mock circulation) and in vivo experiments for valve leakage, flow resistance, and thrombus formation. During the in vitro experiments a maximum valve leakage of 6% during ejection and 21% during aspiration was found. The maximum flow resistance coefficient (K) was 4. The animal experiments demonstrated that the PUCA-II could be positioned within a few minutes into the left ventricle without X-ray guidance and without using a vascular graft. Thrombi were not found in the combined valve after total pump time of 3 hours, which proved the good washout of the valve. Initial experiments to position the pump in the right ventricle through the pulmonary artery were successful and contributed to the development of a new application for the device.

Design and in vitro testing of a voice-producing element for laryngectomized patients.

A voice-producing element has been developed to improve speech quality after laryngectomy. The design process started with the formulation of a list of requirements. The lip principle has the best potential for fulfilling the requirements. A numerical model was made to find the optimal geometry of an element based on the lip principle. Extensive in vitro tests were performed to check all requirements. For this a test set-up with realistic acoustic and aerodynamic properties was developed. Results show that the protruding lip length dominates fundamental frequency, cross-sectional area dominates flow resistance and relation between flow and fundamental frequency. Most requirements have been fulfilled; both for males and females a potentially good functioning prototype could be selected. Clinical experiments will be performed to confirm the quality of the voice-producing prosthesis.