Poster Session 1 - Cardiopulmonary
P1
CONCEPTUALIZATION
, PROTOTYPING AND FIRST IN VITRO INVESTIGATIONS OF A STENT-BASED, ENDOVASCULAR APPROACH TO GENERATE LARGE-BORE VASCULAR ACCESS TO CENTRAL THORACIC VESSELS
J. Emunds1, F. Vorwold1, V. Gesche2, R. Zayat1, S. Kalverkamp1, J. Spillner 1, F. Hima1
1Division of Thoracic Surgery and Thoracic Organ Support, University Hospital Medical Faculty, RWTH Aachen University, Aachen, Germany. 2PerAGraft GmbH, Aachen, Germany.
Objectives: Generation of large-bore central vascular anastomosis for different purposes, especially organ support is still a surgical domain. We investigated an endovascular “inside-out” approach to generate such an anastomosis.
Methods: Two approaches were conceptualized. Both consisted of an endovascular stent graft prosthesis with an end-to-side connected side-graft. In the “elephant trunk technique” the side-arm was located inside the main graft and subsequently developed to the outside in analogy of the “elephant trunk technique”. In the “simultaneous approach” the side-arm was located on the outside of the main graft. With the deployment of the device the side-arm was developed to the outside. Prototypes were handcrafted from commercially available endoprosthesis, vascular prosthesis, nitinol wires and self-developed introducer sheathes. Both approaches were investigated in cadaveric specimen and a mock circulation loop.
Results: The elephant trunk technique used a thread connected to the distant end of the side-arm to retrieve it from the inside to the outside of the main graft. Thus, the side-arm reached out of the punctured vessel.
The simultaneous approach used a tight-fitting sheath around the side-arm. This sheath carried the side-arm through the vascular wall simultaneously during deployment. For both approaches a perforation through the vascular wall was necessary and feasible by endovascular puncture and electrosurgical perforation via an 8 mm introducer sheath. The subsequent deployment proved successful regarding expansion of the whole combined stent graft. Both approaches allowed continuous blood flow during deployment process.
In both approaches the side-arm could be retrieved using a minimally invasive approach.
Conclusions: A principal feasibility of our new minimally invasive inside-out approach could be approved in vitro. This endovascular approach to generate a large bore vascular anastomosis may allow future application in thoracic assist device surgery.
This work is supported by the German Research Foundation (Deutsche Forschungsgesellschaft, DFG): project-number: 347325614.
P2
IN SILICO INVESTIGATION OF FLOW AND GAS TRANSFER CHARACTERISTICS OF THREE-DIMENSIONAL MEMBRANE SHAPES FOR NOVEL ARTIFICIAL LUNG DESIGNS BASED ON 3D PRINTING
K.P. Barbian1, C. Certa2, J. Linkhorst2,3, M. Neidlin1, U. Steinseifer1, M. Wessling2,3, B. Wiegmann4,5,6, S.V. Jansen1
1Department of Cardiovascular Engineering, Institute of Applied Medical Engineering, RWTH Aachen University, Aachen, Germany. 2DWI - Leibniz-Institute for Interactive Materials, Aachen, Germany. 3Chemical Process Engineering, RWTH Aachen University, Aachen, Germany. 4German Center for Lung Research (DZL), BREATH, Hannover, Germany. 5Department of Cardiothoracic, Transplantation, and Vascular Surgery, Hannover Medical School, Hannover, Germany. 6Lower Saxony Centre for Biomedical Engineering, Implant Research and Development (NIFE), Hannover, Germany.
Objectives: The great progress of modern additive manufacturing techniques makes it possible to pursue a completely new approach for the design of artificial lungs. Three-dimensional membrane structures, based on the shapes of triply periodic minimal surfaces (TPMS), provide the advantages of intrinsic separation of two volumes, improved gas transfer rates through passive blood-mixing effects and the possibility to fulfill arbitrary design constraints of the outer housing geometry without the need of additional flow guidance parts. Our objective in this study is to evaluate different TPMS membrane configurations and their impact on gas transfer and flow resistance. Based on these results, design criteria for the use of three-dimensional membrane structures inside artificial lungs will be derived.
Methods: In our study, a computational fluid dynamics (CFD) model was set up to simulate blood flow, as well as O2 and CO2 gas transfer rates, inside TPMS structures. The model was validated on experimental data and used for a simulation series of different TPMS membrane geometries.
Results: The validation of the numerical model shows good correspondence with the experimental data. Within the results, three major effects are observed. An increased TPMS element size (larger membrane scale) results in A: a higher area-specific gas transfer, B: a smaller volume-specific membrane area that is available for gas transfer, and C: a higher permeability (lower flow resistance).
Conclusions: Concerning the desired application for the design of an artificial lung, the effect of exponentially growing gas exchange area with reduced membrane element size outweighs the other effects. Therefore, the use of a specific TPMS type (Schwartz’s Diamond) with the smallest resolution that can be currently achieved via additive manufacturing is recommended to maximize the ratio of gas transfer rate to pressure drop.
P3
DESIGN, REALIZATION AND PRELIMINARY VALIDATION OF A HIGH-FIDELITY BIOINSPIRED LUNGS SIMULATOR
S. Maglio, S. Tognarelli, A. Menciassi
The BioRobotics Institute, Scuola Superiore Sant’Anna, Pisa, Italy.
Objectives: Commercial respiratory simulators are typically low fidelity and are not effective training solutions [1]. Our project aims to realize an active high-fidelity airways neonatal simulator able to replicate physiology and biomechanical properties of human respiratory system. Herein, innovative fabrication process for high-fidelity lung parenchyma and bronchial tree realization are presented.
Methods: 3D models of neonatal lungs and bronchial tree were extracted from the CT scan of 1-month baby girl. The bronchial tree was made in Silicone TG material through fused deposition modeling technique by using a 3D-Bioplotter printer (Envisiontech, Germany). The lung parenchyma was replicated creating a porous silicone through sacrificial material technique. Ecoflex 0030 silicone (Smooth-On, USA) was mixed with fine salt at 20 %, 15 % and 12 % of silicone concentrations. After silicone curing the sacrificial material was melted in water obtaining an open cells porous silicone. On bench characterization of the obtained prototypes were performed with Instron 6800 machine (Instron, USA) by comparing the porous silicone samples biomechanical properties with porcine tissues data. The artificial bronchial tree was then integrated in the porous silicone and a faithful lungs shape was replicated through molding technique. To assure the airtightness, the simulated parenchyma was covered with a thin layer of silicone.
Results: Compression test highlighted the 15 % and 12 % concentrations have same mechanical properties of the porcine lung (i.e., 1.24 mPa elastic modulus and same trend), while 20 % concentration resulted stiffer (i.e., 4.07 mPa elastic modulus). The simulated lungs have been inflated and they are able to expand more than 200 % [2].
Conclusions: High-fidelity neonatal lungs and bronchial tree were realized mixing innovative rapid prototyping technologies with standard techniques. The simulator well replicates the human lungs properties. Future efforts will be dedicated to a well-structured validation process and to the design and realization of the remaining anatomical structures.
[1] A.K. Lefor et al. Int J Med Educ. 2020; 11:97-106.
[2] Y.K. Mariappan et al. J Magn Reson Imaging. 2011; 33(6):1351-61.
P4
INVESTIGATION OF FLOW DIRECTION DEPENDENT GAS TRANSFER IN ARTIFICIAL LUNGS
J.M. Focke1, K.P. Barbian1, N. Gendron1, U. Steinseifer1, J. Arens1,2, M. Neidlin1
1Department of Cardiovascular Engineering, Institute of Applied Medical Engineering, Medical Faculty, RWTH Aachen University, Aachen, Germany. 2University of Twente, Engineering Organ Support Technologies group, Department of Biomechanical Engineering, Enschede, Overijssel, Netherlands.
Objectives: The ability to predict local gas transfer inside artificial lungs is essential to improve their design and efficiency. This allows for smaller devices and therefore lower priming volume for pediatric applications or better mobility for ECMO patients. Gas transfer is influenced by blood flow conditions inside different membrane fiber configurations. Depending on these parameters, we derived directional mass transfer coefficients. The objective of our study was to investigate gas transfer on the fiber level with the ultimate aim of deriving directional mass transfer coefficients for new oxygenator designs.
Methods: Gas transfer was modeled at common blood flow rates in periodic elementary cells of fiber bundle configurations using CFD. The configurations represent different devices that either use stacked or wound fiber mats, differing in the angle (90° or 24°, respectively) the mats are arranged, as well as their main directions (X, Y, Z). To validate the model, gas transfer experiments were performed at small fiber bundle modules with the same fiber configurations using porcine blood.
Results: The transfer coefficients differ by a factor of up to 2.5 between fiber configurations. Flow rates influence the coefficients by a factor of 2.4. The simulation overestimates the experiment by a factor of 1.4 but shows the same tendencies as the simulation.
Conclusions: In this study, we were able to present the dependency of gas exchange within artificial lungs on the fiber bundle configuration and derived transfer coefficients. Differences between CFD and experiments still need to be investigated in the ongoing work. The coefficients will serve as input to numerically investigate the gas exchange efficiency of whole artificial lungs. These investigations have the potential to identify less efficient areas within the oxygenators and support the improved design of new generation devices.
P5
STENT-BASED CENTRAL ACCESS FOR THORACIC ORGAN SUPPORT
C. Hensen1, F. Hima1, N. Gendron2, L. Strudthoff2, R. Zayat1, S. Kalverkamp1, J. Spillner1, N. Pütz1
1Division of Thoracic Surgery and Thoracic Organ Support, University Hospital Medical Faculty, RWTH Aachen University, Aachen, Germany. 2Department of Cardiovascular Engineering, Institute of Applied Medical Engineering, Helmholtz Institute, Medical Faculty, RWTH Aachen University, Aachen, Germany.
Objectives: For future artificial lungs and current cardiac support techniques a central access is required. While a big surgery can be strenuous for critically ill patients, a minimally invasive approach would be favorable. Therefore, we investigated a stent-based approach to central structures.
Methods: We developed a “Lumen-apposing Stent”, which is a self-expanding, woven nitinol stent. Four prototypes with different wire diameters were conceptualized, built (Admedes GmbH, Pforzheim, Germany) and tested regarding their mechanical properties (radial force and crimping diameter). In vitro investigations were performed on porcine hearts to evaluate the functionality and fit of the stent. Coming from the outside, the stent was inserted in different positions of the heart (aorta, pulmonary artery, atria, ventricles) using the Seldinger-Technique and common implantation tools. To measure the leakage, a water-filled circulation system driven by a pump was connected to the stent and the vessels. Potential stent-coverings and the connection to vascular prosthesis were investigated.
Results: The mechanical evaluations showed that the 10 mm stent was crimpable to a minimum diameter of 3 mm. The average radial force of the different prototypes ranked between 1.1 N and 2.28 N and the maximum radial force between 3.74 N and 7.24 N. During in vitro experiments, the stent`s implantation in all positions was successful and a tight fit was achieved. The self-expanding stent provided a sufficient and leakproof lumen inside the vascular/heart wall with a water loss of 20 mL/min on average at 20 mmHg/27 cmH2O pressure.
Conclusions: A “Lumen-apposing” stent-based outside-in access to all relevant central structures seems feasible and may allow further minimalized central thoracic organ support for different purposes. Ongoing improvement of the device and additional in vivo and cadaveric trials are necessary and in progress.
This work is supported by the German Research Foundation (Deutsche Forschungsgesellschaft, DFG): project-number: 347325614.
P6
FUNDAMENTAL STUDY OF RATE OF WATER LOSS AND GAS INLET PRESSURE IN ARTIFICIAL LUNGS DURING PEDIATRIC ECMO
T. Ebine1,2, S. Kohira1,2, R. Komiyama1, K. Fujii1,2, K. Kokubo1,2
1Kitasato University School of Allied Health Sciences, Sagamihara, Japan. 2Kitasato University Graduate School of Medical Sciences, Sagamihara, Japan.
Objectives: Water condensation in artificial lungs (oxygenators) during extracorporeal membrane oxygenation (ECMO) can cause wet-lung. However, the influence of the operating conditions on the onset of condensation is unclear. The present study investigated the effect of the sweep gas flow rate on the water loss rate of the oxygenator and the onset of condensation in the oxygenator.
Methods: A closed circuit consisting of an oxygenator (porous polypropylene membrane; membrane area, 0.5 m2) and a roller pump was used. The circuit was primed with physiological saline solution. Saline solution was circulated in the blood side at flow rate of 1.0 L/min and the oxygenator inlet pressure of 110 mmHg. The oxygenator was supplied with 100 % oxygen gas, and the sweep gas flow rate was varied from 0.5 to 1.5 L/min. The system was circulated for 8 hours, and both the amount of water loss from the circuit and the gas inlet pressure were measured every hour.
Results: The water loss rate ranged from 1.94 to 4.03 mL/h. The higher the sweep gas flow rate, the higher the water loss rate and the gas inlet pressure. The gas inlet pressure initially increased slightly, then increased significantly from 4 to 7 hours, and then plateaued. The water loss rate remained constant despite the rapid increase in the gas inlet pressure. The cumulative amount of water loss at the time of the rapid increase in the gas inlet pressure ranged from 12.6 to 15.8 mL, with minimal difference depending on the sweep gas flow rate.
Conclusions: The higher the sweep gas flow rate, the higher the water loss rate. However, the cumulative amount of water loss at the onset of condensation in the oxygenator ranged from 12.6 to 15.8 mL, and the amount was minimally affected by the sweep gas flow rate.
P7
PERMEABILITY AND MASS TRANSFER FOR A BUNDLE OF TRIANGULAR MEMBRANES
J. Bornoff, K.H. Fraser
Department of Mechanical Engineering, University of Bath, Bath, UK.
Objectives: Extracorporeal membrane oxygenator (ECMO) devices use tubular hollow fibre membranes that are circular in cross section to oxygenate blood in cases of cardiovascular or respiratory related failures. Fibres with a triangular cross section potentially produce anisotropic pressure drops and reduce backflow. This study investigated this change to the fibre geometry by using computational fluid dynamics to assess the permeability and oxygen mass transfer of triangular fibre membrane bundle.
Methods: A single equilateral triangular fibre was considered in 2D with cyclic boundary conditions, efficiently approximating the entire fibre bundle as a square array. MATLAB v2019b was used to parameterise the unit cell, where the rotation of the triangular fibre was varied between 0 and 60 o, and porosity (ε, ratio of empty to fibre area) was varied between 0.6 and 0.9. OpenFOAM v7 was used to solve the fluid-flow and scalar transport equations for oxygen transfer. An average velocity was applied across the cell and was varied to vary the Reynolds number between 1 and 10. The fluid flow field was solved first, then the oxygen transfer on the solved flow field. The Darcy permeability was calculated using the pressure drop across the unit cell. The oxygen transfer rate was measured on the outlet plane.
Results: Permeability increased 100 x between ε = 0.3 and 0.9, and varied sinusoidally with fibre rotation, peaking at 30 o with a maximum 15x the minimum. Permeability decreased by approximately 13 %, and oxygen transfer increased 10 x, between Re = 1 and 10. When compared to a circular fibre array, permeability was approximately 1.5 - 5 x smaller, but mass transfer increased approximately 5 %.
Conclusion: The variation in properties of the triangular fibre opens the possibility for spatially varying properties within an ECMO device, whereby a combination of maximising/minimising permeability or oxygen transfer could be achieved for different regions.
P8
A RAT V-V ECMO MODEL TO INVESTIGATE BIOINCOMPATIBLE REACTIONS DURING EXTRACORPOREAL CIRCULATION
T. Furihata1,2, K. Kobayashi1,3 K. Kokubo1,3, B. Tsuchiya1,3, M. Ogata1,3, H. Nakajima2, M. Kubota1,3
1Kitasato University Graduate School of Medicine, Japan. 2University of Yamanashi Hospital, Japan. 3Kitasato University School of Allied Health Sciences, Japan.
Objectives: Brain injury has been reported as a complication of cardiac surgery requiring artificial heart and lung machines. Hypoxia in local organs, accelerated apoptosis caused by ischemia, and bioincompatible reactions have been reported as possible causes of brain injury. The objective of the present study was to construct a rat V-V ECMO model for investigating the effects of bioincompatible reactions caused by extracorporeal circulation on brain injury.
Methods: Male Sprague Dawley rats were used for the experiments. Four experimental groups were compared: a sham group, which underwent 8 hours of anesthesia; a cerebral infarction group, in which bilateral common carotid occlusion was performed for 2 hours as a model of brain injury (positive control); a low-flow group, in which the blood flow was 1 to 2 mL/min; and a high-flow group, in which the blood flow was 6 to 8 mL/min. For extracorporeal circulation, blood was removed from the right external jugular vein and returned via the right femoral vein. Extracorporeal circulation with an oxygenator were performed for 2 hours, followed by 6 hours of anesthesia. After the experiment, serum neuron specific enolase (NSE), an indicator of brain injury, and serum IL-6 (Interleukin-6) were measured.
Results: The NSE level of the sham group did not change during the experiment. In the cerebral infarction group, an increasing trend after arterial occlusion was observed, reaching the level after reperfusion for 4 hours that was about twice the starting level. The NSE levels in the low-flow and high-flow groups also increased with time and reached values of about twice the starting level. The IL-6 levels were higher in the low-flow and high-flow groups, compared with that in the sham group.
Conclusions: We developed a rat V-V ECMO model in which inflammatory reactions occurred after extracorporeal circulation, leading to brain injury.
P9
DEVICE FOR DYNAMIC FILTRATION OF MICROBUBBLES
A.P. Kuleshov, G.P. Itkin, A.S. Buchnev, A.A. Drobyshev
Federal State Budgetary Institution “Academician V.I. Shumakov Federal Research Center of Transplantology and Artificial Organs”, Ministry of Health of the Russian Federation, Russia.
Objectives: The device for dynamic filtration of air microbubbles has been developed to remove microbubbles from the arterial line of the heart-lung machine (HLM) and the extracorporeal membrane oxygenation (ECMO) to ensure effective filtration of the gas fraction in hemodynamic conditions of adult and pediatric patients. Reducing the number of microbubbles in the circulatory system circuit remains an important factor in patient safety.
Methods: In the course of the research, an alternative method based on the physical principles of centrifugal and centripetal effects on air microbubbles for their effective removal from the arterial line of the artificial circulation apparatus is proposed. The devices ensure the removal of microbubbles at the outlet of the arterial line with a size of more than 10 microns and minimize the number of microbubbles with a size of less than 10 microns.
Results: The main design indicators: the developed device has ergonomic dimensions and operating parameters acceptable for installation in the HLM output line. The device for dynamic filtration provides sufficient separation of bubbles in the blood flow range of 4.0 - 6.0 L/min for an adult and 0.5 - 2.0 L/min for a pediatric patient. The mock circulation loop studies of models have shown the decrease in the total number of microbubbles with a size of less than 10 microns by 70 %, microbubbles with a size from 10 to 50 microns by 87 %, microbubbles with a size of more than 50 microns by 99 %.
P10
A COMPARISON OF INDEPENDENT LUNG VENTILATION AND EXTRACORPOREAL MEMBRANE OXYGENATION IN A RESPIRATORY FAILURE - AN “IN SILICO” STUDY
K. Zieliński, P. Okrzeja, A. Stecka, R. Pasledni, M. Kozarski, M. Darowski
Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland.
Objectives: An Independent Lung Ventilation (ILV) can be used as an alternative treatment to the Extracorporeal Membrane Oxygenation (ECMO) in case of the refractory respiratory failure and asymmetrical lungs pathology. The aim of this study was to compare the effectiveness of ILV and EMCO in terms of oxygenation and hemodynamic stabilities by means of the previously elaborated Virtual Patient (VP).
Methods: Our VP is set of numerical respiratory and cardiovascular models, connecting into one cardiopulmonary system in the form of the computer application. VP parameters were adjusted to simulate a patient suffering from the respiratory failure. Numerical models of ECMO in veno-venous cannulation mode and ILV were developed and integrated with VP. Several simulations for different ILV (minute ventilation, flow division, positive end-expiratory pressure) as well as ECMO (flow rate, oxygen fraction) parameters were performed. The results were analyzed in terms of oxygenation as well as hemodynamic variables, especially cardiac output, and ventilation to perfusion ratio.
Results: Several case reports in the literature show the potential of ILV in patients with unilateral lung diseases. The preliminary numerical simulation results are generally agreed with them. ILV as ECMO is able to improve oxygenation thanks to correcting ventilation to perfusion mismatch.
Conclusions: Numerical simulation study shows that ILV seems to be a good alternative option in treatment of patients with refractory respiratory failure and asymmetrical lungs pathology because is less invasive than ECMO.
P11
A HYBRID (PHYSICAL-COMPUTATIONAL) CARDIOPULMONARY SIMULATOR FOR INDEPENDENT LUNGS VENTILATION
K. Zieliński, A. Stecka, M. Kozarski, P. Okrzeja, R. Pasledni, M. Darowski
Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland.
Objectives: An Independent Lung Ventilation (ILV) is a medical procedure to ventilate each lung separately by means of a double lumen tube. It is commonly used in asymmetrical lungs diseases. It can be an alternative option to the Extracorporeal Membrane Oxygenation in patients with the respiratory failure when the conventional mechanical ventilation by a single lumen tube gives no improvements in these patients. ILV can by applied by two ventilators or one ventilator with a special divider. The aim of this study was to provide a hybrid (physical-computational) cardiopulmonary simulator (HCPS) as an Artificial Patient to simulate ILV clinical scenarios with real ILV systems.
Methods: Our HCPS consist of two functional modules. The first is a numerical cardiopulmonary model executing in the real time. The second is a special interface connecting real medical devices like ventilator to the numerical model. It based on impedance conversion. This interface contains two piston-based airflow generators and is feedbacked with the left and right bronchi of the numerical model The clinical ventilator and ILV divider called Ventil were connected to the HCPS. The simulations of ILV for different ventilatory settings and for various asymmetrical lung diseases were performed. The simulation results were analyzed in terms of respiratory parameters and hemodynamic variables as well.
Results: The simulation results show valid collaboration of ventilator, Ventil and HCPS together. They evidence that in case of asymmetrical respiratory pathology ILV gives lower airway pressures in affected lung preventing barotrauma and lungs injury, reduces dead space and improves hemodynamic stability.
Conclusions: The presented HCPS can be useful as a test bench for ILV systems as well as a tool for training and education of medical stuff, medical and biomedical students.
P12
HEMODYNAMIC ASSESSMENT OF SIX COMMERCIALLY AVAILABLE OXYGENATORS AS PARACORPOREAL ARTIFICIAL LUNG BY A HYBRID SIMULATOR
M. Bézy1*, T. Vydt1*, L. Fresiello2, M. Rocchi2, K. Zieliński3, B. Meyns2, M. Vanierschot1, T. Verbelen2
1Department of Applied Mechanics and Energy conversion (TME), Group T, KU Leuven, Belgium. 2Department of Cardiovascular Sciences, KU Leuven, Belgium. 3Nalecz Institute of Biocybernetics and Biomedical Engineering (IBIB), Polish academy of Sciences, Poland. *Equally contributing authors.
Objectives: To compare the hemodynamic effects of six commercially available oxygenators as paracorporeal artificial lung (PAL).
Methods: A hybrid cardiovascular simulator was used, including a model of atrial, ventricular, pulmonary and systemic circulation. The simulator reproduced 4 pulmonary hypertension (PH) profiles: mild, moderate, severe and cardiogenic shock. Six different oxygenators: 1) A.L.ONE (Eurosets S.r.l, Milano, Italy), 2) Bionique (Hemovent GmbH, Aachen, Germany), 3) EOS ECMO (LivaNova PLC, London, UK), 4) Hilite 7000 LT (Medos, XENIOS AG, Heilbronn, Germany), 5) ILA (Novalung, XENIOS AG, Heilbronn, Germany) and 6) PLS-i (Maquet GmbH, Gettinge Group, Gothenburg, Sweden) were connected between the simulated pulmonary artery and left atrium. For each profile, the hemodynamic effects of each oxygenator were assessed.
Results: Improved organ perfusion, reflected by increases in arterial blood pressure (ABP) and cardiac output (CO) and right ventricular (RV) unloading, reflected by decreases in central venous pressure (CVP), RV end diastolic volume (RVEDV), RV peak pressure (RVPmax) and pressure-volume area (PVA) were achieved by every oxygenator in moderate and severe PH and in cardiogenic shock. RV unloading was most successful in severe PH and most pronounced when using a Bionique, a PLS-i or an ILA oxygenator. For the latter, e.g., mean ABP increased from 70 to 84 mmHg and CO from 3.5 to 4.6 L/min while CVP decreased from 22 to 21 mmHg, RVEDV from 265 to 258 mL, RVPmax from 93.0 to 86.1 mmHg and PVA from 12,601 to 11,552 mmHg.mL.
Conclusions: Currently, the Bionique, PLS-I and ILA oxygenators seem to be the best hemodynamically suited devices on the market to act as short-term PAL (e.g. during lung transplantation). For long-term use, gas exchange and hemocompatibility should be assessed and optimized. The most optimal moment for implantation seems to be in the phase of severe PH, not awaiting cardiogenic shock to occur.
P13
LIMITS AND CHALLENGES OF ADDITIVE MANUFACTURING TECHNIQUES IN THE PRODUCTION OF IMPLANTABLE METAL PROSTHESES
M. Sanguedolce1, E.M. Zanetti2, G. Fragomeni3, G. Pascoletti4, L. De Napoli1, G. Catapano1, L. Filice1
1Department of Mechanical, Energy and Management Engineering, University of Calabria, Rende, Italy. 2Department of Engineering, University of Perugia, Perugia, Italy. 3Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy. 4PolitoBIOMedLab, DIMEAS, Politecnico di Torino, Torino, Italy.
Objectives: There is growing interest in the adoption of additive manufacturing (AM) techniques for, but not limited to, patient-specific prostheses. This study aims to critically analyze advantages and limits to their use to produce metal implantable prostheses as compared to conventional manufacturing techniques.
Methods: This study was based on a non-systematic analysis of the scientific literature addressing theoretical and practical features of AM techniques for the production of metal implantable prostheses. Information was gathered from subjectively selected relevant peer-reviewed papers found with scientific and general-purpose search motors with pertinent keywords. Information was critically analyzed with respect to geometrical accuracy, mechanical and tribological properties, and functional outcomes of implanted prostheses. Challenges were also discussed in terms of production and quality monitoring that AM is to meet to successfully replace conventional manufacturing techniques.
Results: The search yielded a limited number of comprehensive quantitative studies reporting on characterization and short and long-term functional performance of implanted prostheses produced by AM. Metallic prostheses produced by AM exhibit peculiar features as a result of their characteristic manufacturing conditions. The thermal cycles they are subjected to yield microstructures that result in improved mechanical strength but at the expense of ductility and fatigue performance. They often present difficult to eliminate flaws, discontinuities and geometrical errors and frequently require secondary treatments. This often leads to controversial prostheses behavior in severe bodily environments and may hinder the rapid production and implantation of personalized prostheses, the most relevant advantage of additive manufacturing techniques.
Conclusions: Metal prostheses produced with traditional manufacturing techniques still feature more robust features and better performance than those produced by AM. The added complexities due to the relatively new technology still need to be thoroughly investigated to properly develop novel device evaluation procedures, likely followed by the reinvention of the overall production practice towards patient-specific prostheses.
P14
METHOD OF REDUCING EDEMA OF ORGANS BY REDUCING PRESSURE IN THE THORACIC LYMPHATIC DUCT
A.P. Kuleshov, G.P. Itkin, A.S. Buchnev, A.A. Drobyshev
Federal State Budgetary Institution “Academician V.I. Shumakov Federal Research Center of Transplantology and Artificial Organs”, Ministry of Health of the Russian Federation, Russia.
Objectives: Heart failure is often accompanied by edema of the organs, in which capillary pressure increases and fluid stagnation forms. This leads to an increase in the load on the lymphatic system. An increase in the flow in the thoracic lymphatic duct is incommensurable with the required outflow of interstitial fluid and leads to an impairment in lymph drainage in the liver, kidneys, intestines and partially in the lungs.
Methods: The method based on the introduction through the left internal jugular vein into the left brachiocephalic vein of the specialized catheter. The catheter takes blood cyclically from the area where the lymph enters the venous system and pushes it into the brachiocephalic vein. For this purpose, the catheter has an outlet valve at the distal tip with an inflatable balloon-limiter, and an inlet valve in the area of the lymph outlet. The catheter connects to a hydraulic pulsation system that regulates the vacuum and pressure parameters for blood taking and pushing.
In the phase of blood taking, the inlet valve is open, and the outlet valve is closed. Blood moves from the catheter to the pulsation system from the area confluence of the left internal jugular and left subclavian veins. In the phase of blood pushing, the inlet valve is closed, and the outlet valve is open. There is a necessary balance of venous blood flow which supported by parameters of system.
Results: The technical result achieved in mock circulation loop studies is a local cyclic decrease in venous pressure from values of 20 - 30 mmHg to values of 0 - 5 mmHg for a wide range of blood flow in the venous bed. This enables controlled regulation of the lymph drainage process from the main organs providing lymph drainage.
P15
LEFT VENTRICULAR ASSIST DEVICE - SINGLE CENTER EXPERIENCE
M. Gjerakaroska-Radovikj1, G. Severova2, S. Jovev1
1University Clinic for State Cardiac Surgery, Skopje, Republic of North Macedonia. 2University Clinic for Nephrology, Skopje, Republic of North Macedonia.
Objectives: We evaluated our initial single-center experience and outcomes of left ventricular assist device (LVAD) implantation in end-stage heart failure patients.
Methods: We evaluated demographic, echocardiography, laboratory parameters and adverse events of 12 consecutive patients in whom Heartware and HeartMate 3 pumps were implanted as bridge to heart transplant.
Results: All patients were male with average age of 56.0 ± 7.13 years. Pre-implant diagnosis was dilative cardiomyopathy in 9 (75 %). Five (41 %) patients received Heartware (Medtronic), and 7 patients (58 %) the HeartMate 3 (Abbott). 4 (33.3 %) patients had pre-implant INTERMACS (Interagency Registry for Mechanically Assisted Circulatory Support) profile 1 or 2, the remaining 8 (66.6 %) had INTERMACS profile 4. Mean duration support was 8.71 ± 8.64 months (range 0.06 - 33.3 months). Echo follow-up revealed significant improvement in terms of left ventricular size, function and degree of mitral regurgitation. Lab results confirmed improved tissue perfusion and absence of low cardiac output syndrome. In terms of complications there was 1 case of pump exchange and 1 case of pump malfunction. Bleeding requiring surgery occurred in 3 (25 %) patients, gastrointestinal bleeding in 1 (8 %) patients, LVAD-specific infections in 2 (16 %) patients, ischemic stroke in 2 (12 %) patients. During follow-up, 8 (66.6 %) patients remained in stable condition awaiting heart transplantation and 4 (33.3 %) patients died while on support.
Conclusion: LVADs are important long-term management option in end-stage heart failure. Further improvement of their design would reduce the rate of serious adverse events.
P16
IN VITRO STUDY OF NEWLY DESIGNED BICUSPID PEDIATRIC PULMONARY HEART VALVE
K. Suzuki1, H. Sumikura2, S.I. Sakamoto1
1Department of Cardiovascular Surgery, Nippon Medical School Musashikosugi Hospital, Japan. 2Department of Science and Engineering, Tokyo Denki University, Japan.
Objectives: A simulation study to assess the hemodynamics of a newly designed bicuspid valve conduit under hydrodynamic conditions of the pediatric pulmonary circulation system.
Methods: The designed bicuspid valves are constructed from a 0.1 mm expanded polytetrafluoroethylene (ePTFE) sheet leaflet. The valve leaflets are sutured to both the anterior and posterior walls of the conduit to offer a wide coaptation area consisting of the cusp and graft and to preserve the strength of the valve leaflets. Furthermore, the valve consists of two bulging sinuses on the anterior and posterior walls to reduce regurgitant flow.
The hemodynamic properties, under hydrodynamic conditions of the pediatric pulmonary mock circulation system of the ePTFE conduit along with those of the ePTFE tricuspid-valved conduit use at our institute were evaluated (14 mm dia.; T-conduit, n = 5). The newly developed conduit with a bicuspid valve (14 mm dia.; S-conduit, n = 5) and an ePTFE graft (22 mm dia.) with Magna EASE (19 mm dia.; M-conduit, n = 3) were also assessed. The transvalvular pressure gradient and regurgitant flow ratio were measured at heart rates (HR) of 100, 120, and 140 beats per min.
Results: The transvalvular pressure gradients of the T-, S-, and M-conduits were 13.4, 11.1, and 5.0 at HR100, 15.0, 13.6, and 8.1 at HR120, and 17.3, 17.7, and 9.2 at HR140, respectively. The S-conduit had a lower transvalvular pressure gradient than the T-conduit. Regurgitant flow ratios of the T-, S-, and M-conduits were 21.7, 14.7, and 14.7 at HR100, 19.4, 13.6, and 14.7 at HR120, and 17.7, 14.2, and 14.8 at HR140, respectively. The S-conduit regurgitant flow ratio tended to be lower than that of the T-conduit and equal to that of the M-conduit.
Conclusions: The newly designed conduit with a bicuspid valve provides good competence, lower pressure gradient, and lower regurgitant ratio.
P17
IN VITRO HAEMOCOMPATIBILITY EVALUATION OF AN INTRA-VENTRICULAR BALLOON PUMP AS SHORT-TERM MECHANICAL circulatory support
T. Sing1,2,3, M. Simmonds3,4, C. Semenzin1,2,3, A. McNamee3,4, J. Pauls1,2,3, G. Tansley1,2,3
1School of Engineering and Built Environment, Griffith University, Gold Coast, Australia. 2Innovative Cardiovascular Engineering and Technology Laboratory, Critical Care Research Group, The Prince Charles Hospital, Brisbane, Australia. 3Griffith University Mechanobiology Research Laboratory, Griffith University, Gold Coast, Australia. 4School of Health Sciences and Social Work, Griffith University, Gold Coast, Australia.
Objectives: As part of the pre-clinical evaluation process for mechanical circulatory support, blood compatibility is required to be assessed. A previously described Intra-Ventricular Balloon Pump (IVBP) - a low-cost, short-term mechanical circulatory support device - was evaluated within a physiological testing rig for blood compatibility as part of an ongoing pre-clinical evaluation process.
Methods: A low-volume (one unit of blood) human blood-compatible mock circulatory loop was designed and manufactured using biocompatible materials, such as addition-cured silicones, polycarbonate and 3D-printed resins. The mock circulatory loop comprised of a patient-specific decompensated dilated cardiomyopathic ventricle and atrium to represent a failing left heart. An IVBP was moulded from biocompatible silicone and inserted into the mock circulatory loop and inflated simultaneously with the model ventricle contraction. For measuring haemolysis, samples were taken every hour and assessed for plasma-free haemoglobin. For thrombosis, samples were taken after progressively titrating out anticoagulant and measuring the activated clotting time. Once thrombosis tests were completed, the loop was drained and inspected for presence of thrombi.
Results: A preliminary evaluation into the mock circulatory loop haemodynamics was able to induce representative failing left ventricular (peak systolic pressure 140.8 ± 15.2 mmHg), mean left atrial (21.2 ± 2.8 mmHg), and mean aortic pressures (94.8 ± 10.4 mmHg) at a flow rate of 1.49 ± 0.15 L/min. An IVBP implanted in the loop will increase the systolic pressure and flow rates seen in the loop.
Conclusions: A haemocompatible mock circulatory loop was designed and constructed, which was able to accurately represent the left heart and systemic circulation haemodynamics for severe left heart failure. An IVBP was designed to fit within the mock ventricle. Studies will be conducted and reported utilising human blood to assess the baseline haemolysis and thrombogenicity of the mock circulatory loop, and from the IVBP.
P18
A MODIFIED HYBRID MOCK CIRCULATION FOR THE EXPERIMENTAL EVALUATION OF A RIGHT VENTRICULAR ASSIST DEVICE
F. De Gaetano1, S. Vandenberghe2, S. Demertzis2, M.L. Costantino1
1Department of Chemistry, Material and Chemical Engineering “G.Natta”, Politecnico di Milano, Milan, Italy. 2Department of Cardiac Surgery, Cardiocentro Ticino, Lugano, Switzerland.
Objectives: Heart Failure affects mainly the left ventricle since it has to work at the highest-pressure level. Ventricular Assist Devices (VADs) can support the function of the failing left ventricle, but in 20 % of patients, they also cause secondary right heart failure. Currently no dedicated Right VAD exists and simply adapting a Left VAD is not advisable. In this project, we previously prototyped a specific RVAD and here we present a modified, validated Hybrid mock circulation to shift focus from systemic to pulmonary simulation with «hardware-in-the-loop».
Methods: A hybrid mock circulatory loop (HMCL) combines a detailed numerical model of the cardiovascular system with a hydraulic interface such that certain portions of the circulation are represented by a physical environment with fluid flow and pressure. To validate the HMCL’s performance and physiological responsiveness, experimental data were compared to a well-known commercial numerical model of the whole circulation: Harvi-Professor. Parameter study tests aimed at validating the right ventricular model have been carried out by analyzing the effects of preload, afterload, contractility, and heart rate (HR) variations in the numerical model.
Results: The hydraulic interface was controlled at lower pressure levels, prescribed by the numerical model. Changes in the numerical code included refinement of the time-varying elastance description of the RV and Right Atrium. The parameter variations all responded as expected, within the physiological ranges described in literature. The RV PV-loops with the same input parameters of the Harvi and HMCL models, are highly agreeing, both in physiological and in pathological conditions. The stroke work of the right ventricle (SWRV) was also calculated, and the results show a high correlation to the Harvi results (differences < 3.5 %).
Conclusions: The study demonstrates that the hybrid simulator developed in this work is a research tool for hardware-in-the-loop investigations of medical devices supporting right ventricular function.
P19
ENGINEERING OF BACTERIAL NANO CELLULOSE-BASED SMALL DIAMETER (CSD) VASCULAR GRAFT FOR CORONARY ARTERY BYPASS GRAFTING
D. Fusco1,2, F.G. Meissner1,2, A. Pisanu1,2, N. Christiaens1,2, B. Podesser3, B. Winkler4, A. Marsano1,2, F. Eckstein2
1Department of Biomedicine, University of Basel, Basel, Switzerland. 2Department of Surgery, University Hospital of Basel, Basel, Switzerland. 3Department of Biomedical Research, Medical University of Vienna, Vienna, Austria 4Medical University of Vienna, Vienna, Austria.
Objectives: Coronary artery disease, is one of the leading causes of death worldwide (Naghavi et al., 2015). In the context of coronary artery bypass grafting (CABG), internal mammary arteries, radial arteries and saphenous veins cannot be always used as autologous arterial substitutes (Ishii et al., 2016) e.g., in patients with multivessel disease (Wipperman et al., 2009). Synthetic vascular grafts (e.g., Dacron (PET), Teflon (ePTFE)) are an alternative option only for substitution of vessels with an inner diameter > 6 mm (Wippermann et al., 2009; Weber et al., 2017) and not suitable for CABG. The aim of the project is to develop a Cellulose-based Small Diameter (CSD, 3 - 4 mm) vascular graft for CABG made of Bacterial nano-Cellulose (BC) reinforced with a Cobalt Chrome mesh. The hypothesis of the study is that CSD vascular grafts are biomechanically suitable to substitute autologous small diameter vessels.
Methods: The CSD graft is generated in a customized bioreactor providing a temperature- and oxygen- controlled culture environment. BC is synthetized and deposited by Gluconacetobacter.
Results: We manufactured up to 20 cm long CSD vascular grafts with a 3 - 4 mm inner diameter (inner wall thickness 0.5 mm), a circumferential tensile strength of 0.34 ± 0.09 MPa (Force at the breaking point of 1.29 ± 0.28 N, n = 6), and a burst pressure up to 515.5 ± 94.7 mmHg (n = 6). Based on an in vitro cytotoxicity test CSD graft resulted not toxic (> 70 % cell viability, n = 3). In a proof-of-concept study predecessors of these CSD grafts were already used for CABG on pigs. After 4 weeks, angiography showed fully open grafts with no dissection and no narrowing at distal anastomoses. Histology of the explants showed endothelialization from autologous endothelial cells and formation of neointima.
Conclusions: These data indicate the engineered CSD graft as a promising alternative to autologous grafting for coronary artery bypass surgical procedure.
P20
EYE TRACKING SUPPORTED HUMAN FACTORS EVALUATION OF LEFT VENTRICULAR ASSIST DEVICE PERIPHERALS IN SIMULATED EVERYDAY AND EMERGENCY SITUATIONS
G. Widhalm1, T. Abart1, M. Noeske1, L. Kumer2, L. Rössler2, S. Ecker1, S. Kitzweger1, A. Berger2, G. Laufer1, D. Wiedemann1, D. Zimpfer1, H. Schima1,3,4, M. Wagner2, T. Schlöglhofer1,3,4
1Department of Cardiac Surgery, Medical University of Vienna, Vienna, Austria. 2Division of Neonatology, Pediatric Intensive Care and Neuropediatrics, Department of Pediatrics, Comprehensive Center for Pediatrics, Medical University of Vienna, Vienna, Austria. 3Ludwig Boltzmann Institute for Cardiovascular Research, Vienna, Austria. 4Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Vienna, Austria.
Objectives: Left ventricular assist devices (LVADs) are an established therapeutic option for end-stage heart failure, but limited usability of wearable peripherals affect patients’ quality of life. The aim of this study was to evaluate user experiences with HeartMate 3 LVAD peripherals in simulated everyday and emergency scenarios.
Methods: This prospective single-center study included untrained former LVAD patients who underwent heart transplantation between 2014 - 2022. Simulated scenarios included battery exchanges (in reaction to alarm, darkened surrounding, within consolidated bag), AC-power connection, driveline disconnection, and controller exchange. All scenarios were video recorded using four perspectives (SIMStation Pro) and eye-tracking glasses (Tobii Pro Glasses 2/3). Outcome measures were successful completion, duration to success, number of errors, need for a second attempt, pump-off-time, and an 18-item post-scenario-survey (4-point Likert-scale).
Results: Seven patients (Age: 60 ± 19 yrs, female: 28.6 %, median 965 (IQR: 1,417) days on HVAD support) completed the scenarios 1,938 ± 955 days after heart transplantation. 81.6 % (40/49) of all scenarios were solved on first attempt. Following a demonstration, all second attempts were successful. 85.7 % successfully exchanged batteries in a mean duration to success (MDTS) of 63.6 ± 51.3 s. The battery exchange as alarm reaction (MDTS = 33.8 ± 15.5 s), in the darkened room (MDTS = 22.0 ± 8.8 s), within the consolidated bag (MDTS = 108.7 ± 50.9 s), and the controller exchange (MDTS = 118.3 ± 64.4 s) were solved initially by all patients. Major problems occurred during AC-power connection, where only 28.6 % were successful, while three patients (42.9 %) erroneously disconnected the pump, resulting in a median pump-off-time of 13 s (range: 9 - 120 s). Only four patients (57 %) were able to correctly dis- and reconnect the driveline. Overall, 43 % of patients reported improvements in usability compared to their previously implanted device. Eye-tracking will enhance the understanding of patients gaze behavior.
Conclusions: The preliminary results of this ongoing study indicate limited intuitive handling and potential for user-centered design optimization of HeartMate 3 peripherals.
P21
DESIGN OF FORCE TEST RIG FOR THE REALHEART TAH LEFT PUMP
S. Andreou
Research and Development Department, Scandinavian Real Heart, Västerås, Sweden.
Objectives: This study showcases the design of a test rig with aim to capture the forces on of the left pump drive unit. The results will be used to verify motor choice and mechanical loads on the TAH components. Furthermore, another outcome of the planned tests is to determine the worst-case condition concerning the loads on the drive train, as FDA requires testing under these conditions.
Method: The test rig contains a left pump blood unit as mechanical and hydraulic path and a drive unit as actuator. A load cell is placed in between to record the force. It was critical that the test rig resembles the actual left pump of the TAH as close as possible. This adapted left side of the Realheart TAH is then connected to a circulatory mock loop.
The force profile is measured while the pump is meeting the respective hydraulic requirements which are representative of physiological parameters in a clinical setting for the left side of the RealHeart TAH. The hydraulic requirements include scenarios of maximum cardiac output and maximum afterload pressure. The experiment is to be repeated, n = 3, for each test scenario.
Results: The developed force test rig was evaluated, and results were gathered in various test. Relationships between different parameters and maximum mean force was established. The worst case of maximum mean force was measured to be 78.8 N in test scenario 1. The force profile in the latter, was plotted along with AV-plane movement.
Conclusion: The rig developed is representative of the left pump RealHeart TAH and can be used to draw conclusions on the forces acting on the drive unit. The rig is reliable and robust. Its design allows for interchangeability of the critical components, and ease of assembly.
P22
COMPUTATIONAL BLOOD FLOW ANALYSIS OF A SUTURELESS INFLOW CANNULA FOR VENTRICULAR ASSIST DEVICES
M. Azimi1,2, S. Liao1,2, D. McGiffin3, S.D. Gregory1,2
1Cardio-Respiratory Engineering and Technology Laboratory (CREATELab), Baker Heart and Diabetes Institute, Melbourne, Australia. 2Department of Mechanical and Aerospace Engineering, Monash University, Clayton, Australia. 3Department of Cardio-thoracic Surgery and Transplantation, The Alfred Hospital, Melbourne, Australia.
Objectives: A sutureless inflow cannula (SLIC) for ventricular assist devices (VADs) could provide rapid off-bypass implantation. However, intraventricular blood flow dynamics associated with SLIC have not been characterized. This study numerically investigated intraventricular blood flow dynamics associated with various SLIC insertion lengths and the subsequent thrombosis risk.
Methods: An idealized 3D-model of the left ventricle (LV) in three end-diastolic volumes (110, 170 and 240 mL) were developed using literature-based geometrical data. Nine SLICs of various length (4 - 40 mm) were generated. A 1,060 kg/m3 blood density and non-Newtonian viscosity (Carreau model) were defined. A total heart-failure scenario with no wall movement was implemented. A k-ω SST turbulent model was solved using PISO algorithm. The effects of (A) LV size and (B) VAD flow (4, 5 and 6 L/min) on flow dynamics were studied. Wall shear stress (WSS) on the inflow cannula, developed stasis in LV and the rate of LV blood clearance (washout) were compared as predictive measures of thrombosis.
Results: For all LV sizes, shorter insertion lengths (10 - 20 mm) resulted in 8.5 % lower WSS compared to longer lengths (> 30 mm). A shorter SLIC (12 mm) provided the lowest stasis in a 170 mL LV whereas the minimum stasis for the 240 mL LV occurred for a 22 mm SLIC. Similarly, the generated stasis for the 4 and 5 L/min VAD flow was minimized by 4 and 20 mm lengths, respectively. All models obtained complete washout in three seconds, lower than that reported in the literature (> 15 seconds). Fast washout was attributed to the constant VAD flow and the idealized LVs.
Conclusions: Shorter SLICs (10 - 22 mm) reduced the cannula WSS, LV stasis region and thus, thrombosis risk compared to longer lengths. However, the recommended length was different for various LV size and VAD flow. Further investigation is required to identify the relationship between cannula length and thrombosis risk in patient-specific LVs.
P23
DESIGN ENHANCEMENT OF A BI-VENTRICULAR BLOOD PUMP FOR ECMO/ECLS
S. Deininger1, C. Franzen1, E. Cuenca-Navalon1, O. Marseille1 L. Robers2, D. Eggert2, R. Malcher3, F. Eggert3, J. Hutzenlaub3
1Hemovent GmbH, Aachen, Germany. 2Tribecraft AG, Zürich, Switzerland 3Mecora Medizintechnik GmbH, Aachen, Germany.
Objectives: Extra Corporeal Membrane Oxygenation (ECMO) is used to treat acute respiratory and circulatory failure. Risk of thrombus formation and hemolysis are aspects to consider when developing dedicated blood pumps for ECMO. A novel blood pump introduced for clinical applications has been discussed in a recently published study indicating that the current design of the blood pump is already gentle on platelets. However, Hemovent sees potential to further develop the design in terms of cell damage and potential for thrombus formation. The focus of this development iteration was to enhance the bi-ventricular blood pump in areas of the membrane and the pump housing.
Methods: A thorough analysis of the current design is conducted via FE-modelling and -simulation, intended to identify areas prone to improvement. Results of detailed non-stationary CFD simulations are compared to ex vivo pumps and clinical data. Redesigning of the pump housing and membrane contour is followed by corresponding FE- and CFD-simulations with non-continuous flow aspects.
Results: The comparison of results between non-stationary CFD-simulations of the original design and post-clinical systems validate the model and boundary conditions defined in the calculations. Simulations of the bi-ventricular blood pump with implemented design enhancements show improved results on behalf of the considered factors. The pump cycles needed to washout a single filling of the pump was decreased by 30 %. Volume-fraction CFD-simulations indicate improved washout and less residual blood in all areas.
Conclusions: The enhancements propose potential advantages in terms of reduced thrombus formation and reduced haemolytic effects. The dwell time of the blood in the pump is reduced, resulting in less time the blood is prone to mechanical stress and in reduced absence of the metabolism. The enhanced design will be introduced in the next product iteration.
P24
DETERMINATION OF THE HEMATOCRIT IN THE MICROGAP OF A BLOOD PUMP MODEL
S. Krakowski, T. Bierewirtz, U. Kertzscher
Biofluid Mechanics Laboratory, Charité - Universitätsmedizin Berlin, Berlin, Germany.
Objectives: The aim is to investigate the radial gap of a piston pump with regard to the hematocrit in the hydrodynamic bearing. In the following, conclusions are to be drawn about the risk of hemolysis and the capacity of the bearing.
Methods: The hematocrit in the gap is measured using a pump model with a rotation motor, a transparent glass cylinder and a transparent piston. The light source is located in the piston so that the light transmits through the gap. The hematocrit is determined by a colour-value-analysis. An image of the gap radially to the piston is taken with a digital microscope and assigned to the measured gap width. The RGB image is converted into a grey scale image using a MATLAB script and the mean grey value (0 - 255) is output. Using a calibration curve created at idle state, the measured value can be compared with the expected value. If the values differ, the occurrence of plasma skimming is suspected. Both the load capacity of the bearing and the risk of hemolysis in the gap would be affected. The gap width is recorded with the help of eddy current sensors, the bearing force is detected by force sensors. Human blood is used as the test fluid. The hematocrit is known. The free hemoglobin is measured before and after the tests.
Results: Preliminary tests in flow channels were able to demonstrate a correlation between colour value and hematocrits with a known gap width. The experiments on the pump model were able to confirm these results.
Conclusions: The presented colour-value-analysis is suitable to investigate the microgap of the pump model with regard to the hematocrit. The transferability of the method to other microgaps requires a translucent construction as well as accessibility for a lens and is thus limited in its applicability.
P25
AUTOMATED TEST LOOP TO ASSESS PUMP-INDUCED HEMOLYSIS UNDER PULSATILE OPERATING CONDITIONS
P. Borchers, S. Leonhardt, M. Walter
RWTH Aachen University, Chair for Medical Information Technology, Aachen, Germany.
Objectives: Hemolysis is a crucial parameter to assess the hemocompatibility of left ventricular assist devices (LVADs). The standard ASTM-F1841 describes a basic test loop for the analysis of hemolysis induced by continuous flow blood pumps. To increase the accuracy and reproducibility of the operating conditions inside such loops, this work proposes the automatic control of the average pump flow and head pressure. Furthermore, the loop should allow the investigation of pulsatile speed profiles of rotary LVADs and remaining heart activity on pump-induced hemolysis.
Methods: The automated test loop contains a dSpace MicroLabBox, which synchronously collects all sensor readings and controls the actuators. The LVAD speed can be freely driven by a generic control unit for brushless DC motors and the head pressure across the pump can be adjusted by a controllable tube clamp. The average pump flow and the average pump head pressure levels are controlled using two averaging iterative learning controllers alternately. The pump speed profile can then be varied on top of the resulting average pump speed. Moreover, the test loop can be extended by a pressure chamber to generate remaining heart activity. To achieve this, a voice coil actuator pushes onto the membrane of the corresponding pressure chamber. Due to the cyclic nature of heart activity, an iterative learning approach is used to generate desired pressure curves.
Results: The automated test loop holds the average pump flow and head pressure closer to their intended operating levels. Furthermore, the pressure chamber allows the simulation of remaining heart activity.
Conclusions: An automated test loop for the assessment of pump-induced hemolysis under various pulsatile operating conditions has been designed. In the future, two of these automated test loops will be utilized in parallel to directly investigate the influence of pulsatile pump speed profiles and remaining heart activity on pump-induced hemolysis.
P26
HIGH-CONDUCTIVITY COATINGS ON METAL COMPONENTS IN A TRANSCUTANEOUS ENERGY TRANSFER SYSTEM (TETS)
J. F. Hansen
Abbott Laboratories, Pleasanton, California, USA.
Objectives: A Left Ventricular Assist Device (LVAD) requires substantially more sustained power (several watts) than almost any other implanted medical device (other devices typically need at most milliwatts and often only microwatts). This demand for power leads to many unique challenges when designing a wireless power transfer system, often referred to as a Transcutaneous Energy Transfer System (TETS), for an LVAD. We will discuss one of these challenges: the importance of reducing inductive heating of the TETS receiver module’s titanium housing.
Methods: We used extensive finite element computer simulations and then confirmed select results by calorimeter measurements of prototypes.
Results: Induction heating of the metal in TETS receivers of different designs was found to be from 270 mW to 501 mW. By partially coating the titanium receiver housing at strategic locations with high-conductivity metals, such as silver or gold, this inductive heating was reduced to a range of approximately 10 mW to 28 mW.
Conclusions: As the thermal budget of an implanted TETS receiver module is typically no more than 500 mW, without mitigation the inductive heating will consume at least half of that budget (and possibly all of it), severely limiting module functionality. We conclude that reducing induction heating by applying high-conductivity coatings may be a crucial step toward a successful TETS design.
Poster Session 1 - Hemo-/Biocompatibility
P27
MECHANICAL AND USABILITY TEST OF A LOW-COST DEVICE (BAMBI) DESIGNED TO MANAGE POSTPARTUM HEMORRHAGE IN LOW-RESOURCE SETTINGS
K. Osouli1, S. Candidori2, P. Russo2, S. Graziosi2, A. Zanini3, M.L. Costantino1, F. De Gaetano1
1Department of Chemistry, Materials and Chemical Engineering “G. Natta”, Politecnico di Milano, Milan, Italy. 2Department of Mechanical Engineering, Politecnico di Milano, Milan, Italy. 3Obstetrician gynecologist.
Objectives: Postpartum hemorrhage (PPH) is the leading cause of maternal mortality in low and middle-income countries (LMICs). Some devices are available in developed countries to tamponade uterine walls, but not in LMICs due to their prohibitive cost. This work aims to test the mechanical properties and usability of a low-cost device (BAMBI) designed by our group to manage PPH in low-resource settings.
Methods: The BAMBI device is inserted in the uterus and filled with saline solution until the balloon fits the uterine cavity exerting pressure on the proximal uterine wall to stop the bleeding. In vitro tests were performed to evaluate the intraluminal pressure (ILP) of the balloon at different filling stages both in the open air and in constrained conditions using a commercial uterus simulator. A qualitative assessment of the shape of the balloon and measuring the burst volume of the balloon were performed. To examine the pull-out force that the BAMBI device can sustain, tensile tests were conducted varying several design parameters. To perform the usability tests of BAMBI, a transparent and flexible uterus phantom was used. The phantom, obtained by SLA technique, was designed to fit the cavity of a commercial delivery simulator, used as a reference.
Results: In the open air, the ILP was 20 mmHg at a filling volume of 150 mL, reaching a plateau around 13 mmHg, increasing the volume up to 1500 mL. In the constrained condition, the ILP increased from 20 mmHg to 25 mmHg, varying the filling from 150 mL to 500 mL. The Average burst volume measured was 15 liters. The device connection sustained a maximum force of 29.4 N during the tensile tests.
Conclusions: The integrity and mechanical properties of the BAMBI device were tested and showed promising results compared to the existing commercial devices.
P28
FABRICATION OF MEDICAL DEVICES WITH NANOPRINTING: HAZARDS IDENTIFICATION FOR RISK ANALYSIS
G. D’Avenio, C. Daniele, M. Grigioni
National Center for Innovative Technologies in Public Health, Istituto Superiore di Sanità, Rome, Italy.
Objectives: Additive manufacturing (AM) is gaining diffusion in the field of medical devices (MDs). Recent advances in nanoprinting make use of additive multiphoton polymerization (MPP), which enables to use light at wavelenghts at which a polymer is transparent, in order to finely control polymerization at designed locations, with sub-diffraction limit resolution (100 nm feature size has been obtained).
The Regulation (EU) 2017/745 on MDs does not consider specifically AM-based MDs. Such MDs must be assessed carrying out a risk analysis, thus identifying each hazardous situation, and estimating the associated risks. The objective of the study is to provide useful elements for the risk analysis of this type of innovative devices.
Methods: We referred to EN ISO 14971, which is the harmonized standard for risk analysis and management in support of the European MD directive. An analysis of the recent literature was carried out in order to identify process-specific hazards associated to nanoprinting.
Results: Additive MPP has hitherto leveraged on general-purpose consumables (polymers, photoinitiators), typically adapting materials from stereolithography, not designed for biological applications. Polymerization in MPP generally involves the photoinitiator-enabled creation of free radical species, which are typically toxic. As already observed with traditional AM techniques (FDM, Fusion Deposition Modeling), the quite high temperature of the polymer during printing could create hazards. It has been demonstrated that extracts of a consumable material after FDM printing may have different physico-chemical signatures with respect to the same material before printing. Hence, possible contact of body tissues with undesirable substances leached from a nanoprinted MD may arise, depending on the fabrication process parameters.
Conclusions: Appropriate qualification of biological hazards should always be carried out for AM-based MDs. It is essential to evaluate all these hazards, in order to obtain safe MDs obtained with nanoprinting, and to exploit the capabilities offered by this technology.
P29
DESIGN AND REALIZATION OF A HIGH-FIDELITY UMBILICAL CORD SIMULATOR FOR EMERGENCY PROCEDURES TRAINING
M. Polizzotto, S. Maglio, S. Tognarelli, A. Menciassi
The BioRobotics Institute, Scuola Superiore Sant’Anna, Pisa, Italy.
Objectives: The objective of this study was the design and development of an affordable and high-fidelity umbilical cord simulator for training of neonatal catheterization. This is an emergency procedure that can lead to important consequences, e.g., vessel perforation, cardiac arrhythmias, endocarditis. Only few of the current commercial simulators replicate faithfully the neonatal umbilical cord; they are expensive and disposable, thus unsuitable for training programs in low-income countries. The simulator peculiar features were achieved combining soft materials and employing rapid prototyping techniques.
Methods: Using literature data, the umbilical cord 3D model was designed reproducing the real anatomy with the two arteries that coil around the central vein. To closely replicate the real catheterization procedure, a dedicated reservoir was incorporated at the base of the umbilical cord to store a blood-like fluid that will be used during the simulation.
The first high-fidelity prototype was realized through moulding technique. The simulator UV-curable resin mould was 3D printed using SGC technology. The mould pieces were firstly covered with two layers of Ecoflex 50 silicone (Smooth-On, USA) to confer the proper rigidity to the simulator walls and then the Ecoflex gel silicone was poured into the mould to replicate the gel consistency of the Wharton’s jelly, where vessels are embedded. Finally, the reservoir volume was filled with DragonSkin 10 Slow silicone to obtain a stiff and stable base structure.
Results and conclusions: The realized simulator was tested and evaluated by neonatologists from Azienda Ospedaliero Universitaria Pisana (Italy) who highlighted the high-fidelity of the system both in terms of dimensions, materials and tactile consistency. Future efforts will be dedicated to move laterally the central vein and replicate its curvature of about 90 degrees at the entrance of the neonatal body that represents the most difficult part of the catheterization procedure.
P30
INFLUENCE OF THE FLUID RHEOLOGY ON THE BLOOD FLOW HEMODYNAMICS WITHIN NUMEROUS PATIENT-SPECIFIC ARTERIAL NETWORKS OF VARIED COMPLEXITY
Z. Tyfa, P. Reorowicz, K. Jóźwik
Institute of Turbomachinery, Lodz University of Technology, Lodz, Poland.
Objectives: One can find hypotheses that blood can be treated as a Newtonian fluid in large arteries and small vessels characterized by a high blood flux. However, there is no clear answer concerning these issues - despite extensive studies, numerous research groups outline contradictory results. Thus, the main objective of the following study was to assess whether blood can be simplified to a Newtonian fluid during CFD (computational fluid dynamics) investigations for numerous patient-specific arterial models of varied complexity.
Methods: A sensitivity analysis of blood rheology comprised investigations within 3 types of geometries: large arterial system (entire aorta), cerebral vasculature and simple bifurcation of common carotid artery (CCA). All patient-specific models were reconstructed by means of a developed in-house software named AMR (Anatomical Model Reconstructor). After conducting a vast meta-analysis related to a mathematical description of shear-thinning behaviour of blood, 8 rheological models were selected and used in stationary and pulsatile simulations of blood flows.
Results: The following parameters were analysed during qualitative and quantitative assessment: flow intensity, wall shear stress (WSS), WSS spatial gradient (WSSG), oscillatory shear index (OSI), local and global non-Newtonian importance factor (IL and IG) and non-Newtonian effect factor (NNEF). High relative variation of all parameters, high values of IL and IG as well as non-negligible differences in maximal values of WSS/WSSG were observed for CCA bifurcation and entire aorta geometries. Hardly any changes in these parameters were noted for intracranial arterial network, both during stationary and transient analyses.
Conclusions: All hemodynamic parameters and their variation are strictly related to the model geometry and imposed boundary conditions. All results indicate that blood can be simplified to a Newtonian fluid for cerebral vasculature (small vessels with high flow intensity), while for CCA bifurcation and large vessels networks it should be modelled as a shear-thinning fluid.
P31
INFLUENCE OF PATIENT-SPECIFIC ARTERIAL MODEL RECONSTRUCTION TECHNIQUE ON GEOMETRY TOPOLOGY AND FLOW HEMODYNAMICS - CFD INVESTIGATIONS
Z. Tyfa, P. Reorowicz, K. Jóźwik
Institute of Turbomachinery, Lodz University of Technology, Lodz, Poland.
Objectives: A vast majority of researchers base their blood flow CFD (computational fluid dynamics) studies on either idealized or patient-specific geometries that are usually transformed from surface objects to volumetric ones, while corresponding CFD results and their correctness are taken for granted. Thus, the main objective of this research was to thoroughly assess an influence of the blood vessels model reconstruction technique on the numerical flow solution.
Methods: All patient-specific geometries were reconstructed in a developed software named AMR (Anatomical Model Reconstructor) and were exported as surface bodies stored in STL format. Afterwards, volumetric objects were generated with three different techniques: profiles-lofting, Scan-to-3D algorithm available in SolidWorks program and Skin methods available in ANSYS SpaceClaim module. Finally, models were categorized into 3 separate groups depending on their complexity and served as a basis for the volumetric meshes generation in ANSYS ICEM program. Stationary and pulsatile simulations were conducted in ANSYS CFX software.
Results: An entire analysis was divided into two parts: topological and hemodynamic. In both of them, the authors performed a thorough qualitative and quantitative comparison of the results. In the first one, geometrical parameters were analyzed, e.g. volume of the model as well as area of the chosen cross sections and neuralgic regions. In the second sub-investigation, the authors focused on a comparison of the chosen hemodynamic parameters, including flow intensity, shear stress distribution and blood wash-out efficiency.
Conclusions: All surface-to-volume conversion methods result in topology distortions that affect the blood flow solution, hence, a reliability of obtained geometries cannot be taken for granted. All investigated case studies indicated that ANSYS SpaceClaim module produces geometries of the highest similarity towards reference STL object, while profiles-lofting method is burdened with the highest topological and hemodynamic discrepancies.
P32
INVESTIGATION OF IMPEDANCE BASED AORTIC FLOW MEASUREMENTS
D. Voss, S. Leonhardt, M. Walter
Medical Information Technology, Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, Aachen, Germany.
Objectives: Setting the appropriate support level for LVADs in clinical use is a challenging task. Among other parameters cardiac output is a key parameter for patient assessment. We propose using an impedance-based flow measurement directly combined with a percutaneous LVAD to measure cardiac output continuously. In this work, a preliminary investigation is conducted to determine the flow dependent conductivity of the blood in the aorta.
Methods: The impedance of blood has been shown to be primarily dependent on hematocrit, temperature, and flow. The flow dependency is a consequence of the orientation and deformation of red blood cells. The analysis of the flow dependency of blood is performed experimentally in an aorta phantom representing the descending aorta. A blood compatible mock loop has been built, consisting of tubes, a LVAD as a pump, a flow sensor, and an aorta phantom. In this setup, the pump enables blood flows of up to 10 L/min. Multiple rigid aorta phantoms with inner diameters of 25 mm and 31 mm have been employed. The temperature of the mock loop is regulated to 37 °C. Fresh whole porcine blood from a slaughterhouse with added heparin is used. The impedance measurement is performed with a 4-pole diagnostic catheter, thus enabling 4-electrode measurements. The influence of the measurement line is compensated via standard open, closed and load compensation.
Results: The impedance shows the expected decrease with an increase of the flow. The derived specific conductivity increases by 16.7 % from 0.66 S/m up to 0.77 S/m.
Conclusions: This demonstrates that the flow dependent variation of blood impedance can be measured using a four-pole electrode catheter. Consequently, this method is therefore a promising approach for continuously estimating cardiac output in percutaneous VADs.
P33
MORPHOLOGICAL CHARACTERISATION OF NOVEL LAYERED HYDROGEL-LIPOSOME BEADS FOR HEMODYNAMIC FLOW STUDIES
T. Bode1*, G. Hentschel1*, T. Rittinghaus1, T. Hildebrand1, M. Müller1, B. Glasmacher1
1Institute for Multiphase Processes, Leibniz University Hannover, Garbsen, Germany. *Equally contributing authors.
Objectives: Particle image velocimetry (PIV) is used to gain data for haemodynamic flow models. PIV seeding particles, often made out of glass, suspended into the blood substitute are used for this purpose. In this study, a layered bead system made up of liposomes in an alginate matrix is presented, in which liposomes could act as flexible, biocompatible seeding particles. To assess the systems properties, liposome distribution, size and intra-bead morphology have to be analysed.
Methods: DSPC/cholesterol-liposomes (1:1 w/w) were produced in bi-distilled water by thin-film hydration and extrusion (12.5 mg/mL and 25 mg/mL) using a 1 µm PC-membrane. Electro-spraying (flow rate 7 mL/h) was used to produce alginate (2 wt.%) beads with (BL) and without (BW) embedded liposomes. The morphology was assessed using a cryo-SEM with a cryopreparation-station by freezing the samples in liquid nitrogen and abrading top layers. A lyophilisation-cycle of 4 min at -90 °C was then followed by sputtering for 40 sec. EDX was used to characterise and map the beads atomic composition.
Results: SEM-images showed a porous structure inside BL with large cavities (mean diameter ~3 ± 0.9 µm) evenly distributed throughout the samples. BW did not show these cavities but a similar porous structure. EDX maps showed large quantities of C, Na and Ca around the circumference of the inclusions and throughout the porous structure but significantly less to no signals inside the inclusions.
Conclusions: Liposomes were successfully embedded into alginate beads as shown by SEM and EDX, however, their diameter increased by a factor of 3. Cryo-SEM enabled more detailed information about the size and distribution of liposomes inside the matrix as well as liposome and bead morphology compared to a standard SEM or similar methods. Further studies need to determine causes for the liposome enlargement as well as their reflective properties as seeding particles.
P34
EFFICIENCY IN INTRACELLULAR DELIVERY INTO RBCS FROM ANIMALS AND HUMAN WITH A MICROFLUIDIC APPROACH
C. Bernardelli1,2, M. Piergiovanni3, E. Bianchi1,3, C. Carlo-Stella4, M.L. Costantino1, G. Casagrande1,3
1Laboratory of Biological Structure Mechanics LaBS, Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, Milan, Italy. 2Laboratory of Pharmacology, Department of Health Sciences, Università degli Studi di Milano, Milan, Italy. 3MERYLO’ srl, Varedo (MB), Italy 4Humanitas University, Milan, Italy.
Objectives: Red Blood Cells (RBCs) are promising drug delivery system for many drugs. By using microfluidics, it is possible to accurately control the intracellular delivery of molecules through fluidic shear stress acting on RBCs membrane. Before to get to pre-clinical studies others aspects has to be verified mainly related to the different RBCs physical characteristics in human or in other animals.
Methods: The same intracellular delivery procedures were repeated on blood from human, mouse, and rat. A PMMA chip with a nominal square section of 50 μm and a length of 58.5 mm was used. A syringe pump pushed the RBCs suspension at 5-30-50 µL/min. All the tests were performed at 1 % of haematocrit considering FD40S (0.1 mM) as probe molecule. Samples were washed in PBS to remove excess dextran, then RBCs were analysed with a flow cytometer. Two parameters, the number of molecules that are delivered (ΔF with respect to not-treated cells - %) and the cells that receive at least one molecule (LC - %) influence the loading efficiency.
Results: Probe molecule was successfully delivered either to human or animals RBCs (LC always > 50 %). The encapsulation is more effective in human (ΔF > 100 %), followed by mice (ΔF up to 80 %), while rats showed a ΔF < 50 %. This difference might be due to the RBCs rats and mice properties with respect to human; they are slightly smaller (-11 % and -17 % in diameter, -32 % and -45 % in volume respectively) and less deformable (Elongation index: -20 % and -8.6 % respectively).
Conclusions: To identify a proper animal model for future in vivo studies, the number of totally loaded cells should not have significant variation, even if the delivery efficacy is not as high as in human RBCs. Thus, the mouse appears to be the most suitable model for the next preclinical studies.
P35
MICROFLUIDIC STUDY ON A TRANSPARENT TWO-PHASE BLOOD MODEL FLUID
V. Froese, G. Gabel, M. Lommel, U. Kertzscher
Biofluid Mechanics Laboratory, Institute of Computer-assisted Cardiovascular Medicine, Charité - Universitätsmedizin Berlin, Berlin, Germany.
Objectives: Due to the intransparency of blood, flow phenomena can only be observed to a limited extent. During the last years several transparent models have been examined. An ideal blood model should be consistent with the rheologic behavior of whole blood. This is determined by the flow behavior of the red blood cells, as they make up 99 % of the blood cells. The objective of this work is the comparison of blood with a two-phase blood model in which alginate microspheres are modeling the red blood cells.
Methods: Transparent alginate microspheres in a size of 8 µm ± 2 µm were used as a model of red blood cells. A water - calcium - chloride solution was used as a model fluid of plasma. Different flow properties like the viscosity and the cell free layer as well as the deformation of cells and microspheres have been compared in straight and hyperbolic contracted microfluidic channels.
Results: A good agreement with the flow properties of blood could be observed. The viscosity of the blood model is in the same order of magnitude as blood and showed shear thinning behavior. At 37°C, the blood model reached 3.7 mPas from 1000 1/s onwards. The microspheres are less deformable than red blood cells: the deformation index in areas with high shear rates was 15 % and in areas with low shear rates 50 % lower. It was possible to visualize the flow field at several layers in the fluid.
Conclusions: The two-phase blood model fluid has similar flow properties as whole blood. The alginate microspheres are in the same order of magnitude as red blood cells and are slightly less deformable. In the long term, the model can be used for experiments on flow analysis and optimization in blood pumps or as a validation model for CFD to calculate multiphase flows.
P36
CHARACTERIZATION OF TISSUE FACTOR-BEARING EXTRACELLULAR VESICLES IN COVID-19
R. Weiss1, T. Eichhorn1, S. Huber2, M. Mostageer1, R. Würzner2, V. Weber1
1Center for Biomedical Technology, Department for Biomedical Research, University for Continuing Education Krems, Krems, Austria. 2Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria.
Objectives: The severe acute respiratory syndrome (SARS) coronavirus disease 2019 (COVID-19) has rapidly developed into a pandemic. Severe COVID-19 is frequently associated with thrombotic complications which can result in multiple organ dysfunction. There is evidence that increased platelet activation and platelet-monocyte aggregate formation in severe COVID-19 patients induce expression of tissue factor (TF) on monocytes. In this study we characterized TF-bearing extracellular vesicles (EVs) and their cellular origin in patient samples.
Methods: A total of 12 patients with SARS-CoV-2 infection admitted to the intensive care unit (ICU) requiring mechanical ventilation (time course, 134 samples in total) and 25 healthy controls were recruited for this study. Plasma was obtained by centrifugation of whole blood anticoagulated with EDTA at 2,000 x g for 15 min. EVs were characterized by flow cytometry using a CytoFLEX LX device (Beckman Coulter) with a combination of PC7-conjugated anti-CD41 as platelet marker, PB-conjugated anti-CD45 as leukocyte marker and FITC-conjugated anti-TF (VD8, BioMedica Diagnostics). APC-conjugated Annexin V was used as marker for EVs exposing phosphatidylserine. Calibration of the flow cytometer was performed with fluorescent silica beads and the EV gate was set below the 1 µm bead cloud.
Results: EV counts, as well as TF-positive EVs, were significantly increased in COVID-19 patients as compared to healthy controls (360,901 ± 29,877 vs. 94,620 ± 30,874 EVs/µl; 26,345 ± 2,211 vs. 4,487 ± 1,662 TF+ EVs/µl). 30 % of all TF+ EVs in patient samples co-expressed CD41 and CD45 on the surface whereas only 3 % of co-expression was detected on TF+ EVs in healthy controls (7,367 ± 991 vs. 128 ± 46 TF+CD41+CD45+ EVs/µl).
Conclusions: To our knowledge, this is the first report on TF+ EVs carrying both, platelet and monocyte markers, providing evidence for the interaction of platelets and monocytes in COVID-19 patients.
P37
BLOOD QUALITY INFLUENCES HEMOLYTIC CHARACTERIZATION OF BLOOD PUMPS
A.P. McNamee, L. Kuck, M.J. Simmonds
Biorheology Research Laboratory, Menzies Health Institute Queensland, Griffith University, Gold Coast, Australia.
Objectives: Ex vivo hemocompatibility testing largely relies on the assessment of plasma free-hemoglobin (i.e., hemolysis) in a mock circulatory blood loop. Current approaches ignore that the hemolysis in these tests does not account for baseline blood quality, overlooking that hemolysis is sensitive to erythrocyte health, age, and shear history, thus reducing the signal-to-noise of these tests. The present study aimed to investigate the influence of blood quality (“noise”) on the sensitivity of ex vivo blood pump hemolysis (“signal”).
Methods: Blood obtained from healthy individuals was treated with interventions known to decrease blood quality; i.e., i) incubation with phenazine methosulfate to oxidize blood cells, ii) incubation with a protein fixative (glutaraldehyde), or iii) aging blood via ex vivo storage for 24 h at 4 °C. Following treatment, each condition was compared with healthy control blood for erythrocyte deformability, mechanical fragility, and ex vivo hemolytic response to an explanted HeartMate 3 in a miniaturized blood loop (under standard ASTM F1841 conditions; Q = 5 L/min, ∆P = 100 mmHg).
Results: Decreased blood quality (characterized by decreases in cell deformability) changed the hemolytic signature of the ex vivo blood loop tests. Increased cell rigidification (from chemical treatment and ex vivo ageing) was positively correlated with larger values of hemolysis generated by the HeartMate 3 under identical pumping conditions. The Normalized Index of Hemolysis (NIH) for a given test sample was identified to be sensitive to baseline blood quality.
Conclusions: Blood quality should be characterized at baseline for each ex vivo hemocompatibility test in a mock circulatory blood loop. The type of blood used (animal vs. healthy vs. clinical) should thus be considered and reported against a standardized measure when performing evaluation of new/existing blood pumps.
P38
CONTROLLING AND EVALUATING THROMBUS FORMATION WITHIN CARDIOVASCULAR MEDICAL DEVICES
A.P. McNamee, L. Kuck, M.J. Simmonds
Biorheology Research Laboratory, Menzies Health Institute Queensland, Griffith University, Gold Coast, Australia.
Objectives: Suboptimal blood interactions with cardiovascular medical devices may lead to thrombus formation and thus severe secondary complications that substantially increase clinical burden. We sought to develop an assay capable of mimicking clinically-relevant thrombus formation within various types of cardiovascular devices (i.e., ventricular assist devices, heart valves, and implanted stents) in order to identify and evaluate thrombogenic regions within each system and the impact of thrombus formation on device performance and/or hemodynamics.
Methods: Extending methods proposed by Maruyama et. al. (2009), we developed a small benchtop assay that allows for any desired level of thrombogenic potential to be achieved within blood. Donor specific targets for thrombogenicity can then be scaled up to a large-volume mock circulatory loop, within which discrete medical devices can be implanted for evaluation. To evaluate the clinical relevance of our method, we designed bespoke test systems for various cardiovascular implants that facilitates controlled thrombus formation and subsequent evaluation, in tandem with traditional hemocompatibility assessment.
Results: Baseline blood thrombogenicity of a given participant was identified to be highly important for subsequent thrombogenic control in the mock circulatory rig. By evaluating variations in hemodynamics and evaluation of specific coagulation indices, we could successfully predict the onset of early thrombus development. Following initial thrombus identification, we could also successfully control growth/reversal of thrombi. Physical inspection of the medical devices identified that locations of formed clots mimicked those of clinically-reported case studies.
Conclusions: Our protocol successfully and reliably controls thrombus development within various cardiovascular medical devices to aid identification of formation sites that align with clinically-informed observations. Our method could be applied during early prototyping for reduced thrombogenicity, shed light on unresolved thrombi related complications of existing medical devices, or evaluate the success of possible interventions (e. g., pharmacological, material surface interaction, or mechanical wash out) to minimize thrombus formation.
P39
MODIFICATION OF THE VACUUM SUCTION CANNULA TO REDUCE BLOOD TRAUMA DURING ASPIRATION WITH USING THE HLM
A.P. Kuleshov, G.P. Itkin, A.S. Buchnev, A.A. Drobyshev, O.Y. Esipova
Federal State Budgetary Institution “Academician V.I. Shumakov Federal Research Center of Transplantology and Artificial Organs”, Ministry of Health of the Russian Federation, Russia.
Objectives: An improved blood aspiration system has been developed to improve the quality of surgical operations with the use of the heart-lung machine (HLM) devices.
Method: Currently, in many surgical operations with the use of HLM, blood is extracted from the wound cavities using a suction cannula connected to a blood suction system (roller or vacuum pump). In turn, the blood is coming in the recirculation system, which represented by a collecting tank and connecting tubes. According to experts, aspiration is the main factor of blood trauma in these operations and, along with microbubbles, increases the rehabilitation time of the patient after surgery.
An inexpensive, user-friendly, and low-traumatic dynamic blood aspiration system has been created, which is different:
- maximum separation of blood and air already at the stage of blood collection from the wound;
- reduction of the vacuum required for blood aspiration;
- minimal costs for the modification of the blood collection cannula.
The principle of operation is to separate the liquid and gas fractions of the blood-air mixture. The effect is achieved due to the centrifugal forces of the rotating blood-air flow in combination with the lifting Archimedean forces. For ergonomic and practical use, the handle of a conventional blood suction tube is modified as much as possible.
Result: more than 95 % separation of the gas fraction from the liquid fraction with low hemolysis was obtained.
Conclusions: Blood aspiration systems are applicable for surgical operations where the patient’s aspirated blood is reused (operations with artificial circulation, liver transplantation, some abdominal operations, etc.).
P40
ENDOTHELIUM-PROTECTIVE EFFECT OF HEPARIN-FUNCTIONALIZED ADSORBENTS THAT DEPLETE PRODUCTS OF PLATELET AND NEUTROPHIL ACTIVATION
M. Ebeyer-Masotta, T. Eichhorn, V. Weber
Center for Biomedical Technology, Department for Biomedical Research, University for Continuing Education Krems, Krems, Austria.
Objectives: Endothelial activation is a hallmark in sepsis and COVID-19. The concomitant activation of coagulation and the immune response is mainly driven by platelet and neutrophil activation. We and other groups have previously shown that heparin-functionalized matrices can deplete mediators of immunothrombosis, responsible for endothelial activation, as well as platelets and extracellular vesicles. Using a cell culture flow model, we aim to investigate the endothelium-protective role of heparin-functionalized surfaces.
Methods: Whole blood from healthy donors anticoagulated with heparin was stimulated using lipopolysaccharide from Escherichia coli (100 ng/mL). Stimulated whole blood was incubated with Heparin Sepharose, unmodified Sepharose or was left untreated for 60 min. Treated blood was centrifuged at 2,500 g for 15 min and plasma was diluted 1:10 in culture medium to prepare conditioned medium. Human umbilical vein endothelial cells (HUVECs) were seeded into microfluidic channel slides to allow cultivation under flow. After reaching confluence, the culture medium was changed to conditioned medium and stimulation of the HUVECs was carried out for 4 h. Adhesion of platelets and monocytes, adhesion molecule expression (ICAM-1 and E-selectin) and endothelial barrier function (VE-cadherin and zonula occludens-1) were assessed using brightfield and/or confocal microscopy. Mediators (platelet factor 4, histones, neutrophil elastase, tumor necrosis factor alpha) were quantified by ELISA before and after treatment with adsorbent.
Results: Treatment with Heparin Sepharose reduced the adhesion of platelets and monocytes to the endothelial layer as well as the expression of ICAM-1 and E-selectin. Endothelial barrier function was also preserved after treatment with Heparin Sepharose.
Conclusions: Heparin-functionalized matrices exert protective effects on the endothelium by reducing the adhesion of immune cells and preserving the endothelial barrier function. Our study adds knowledge to the mechanism of action of clinically used heparin-functionalized matrices, such as Seraph-100 (Exthera Medical), and provides evidence of the multimodal effect of these adsorption technologies.
P41
ALTERING THE FIBRIN ADSORPTION TOPOLOGY ON TITANIUM BY APPLYING A HIGH FREQUENCY ALTERNATING CURRENT
T. Bierewirtz, A. Hujeirat, U. Kertzscher
Biofluid Mechanics Laboratory, Institute of Computer-assisted Cardiovascular Medicine, Charité-Universitätsmedizin Berlin, Berlin, Germany.
Objectives: Thrombus formation remains one of the most critical adverse events on implants with blood contact, e.g. ventricular assist devices, which are mostly made of titanium. The initial mechanism of thrombus formation is mainly attributed to the formation of fibrin fibers onto the surface. The topology of this formation is, among other material characteristics, dependent on the surface charge at the material-blood interface. In this study, the fibrin topology is aimed to be altered towards less thrombogenic implants by adjusting the surface charge. We hypothesize that a high frequency alternating current (HFAC) causes a decrease of surface charge at the titanium-fluid interface due to the skin effect, leading to a less thrombogenic adsorption topology.
Methods: Phosphate-buffered saline with human fibrinogen and thrombin was flowed over titanium grade 5 probes in a closed circuit at 37 °C for 30 minutes with and without HFAC at 1 MHz and 100 uA. The probes were evaluated by scanning electron microscope imaging. Finite element methods (FEM) were used to simulate the surface charge distribution at the titanium-fluid interface.
Results: SEM images of probes with HFAC revealed a predominantly monolayered fibrin topology, whereas the reference probes exhibited thick fibrin strands. FEM simulations revealed a skin effect in the titanium, leading to a decrease of surface charge at the titanium-fluid interface.
Conclusions: We conclude that HFAC might decrease the surface charge at the titanium-fluid interface due to the skin effect, leading to less thick fibrin strands that are associated with high thrombogenicity. In future, this method could be used to find an ideal antithrombogenetic surface potential for artificial materials in blood contact.
P42
VISUALIZATION OF SHEAR-INDUCED PLATELET DEPOSITION IN A FLOW CHAMBER
I. Esslinger, I. Schulz, T. Bierewirtz, U. Kertzscher
Biofluid Mechanics Laboratory, Institute of Computer-assisted Cardiovascular Medicine (ICM), Charité - Universitätsmedizin Berlin, Berlin, Germany.
Objectives: The formation of thrombi is still a challenge when it comes to ventricular assist devices (VADs). This can be caused by the subsequent deposition of proteins and platelets on foreign surfaces. Especially regions of scratches offer high potential for the deposition of blood components. In VADs, the blood contacting components are mainly made of titanium alloys due to hemocompatible properties and corrosion resistance. However, surfaces where mechanical contact of the rotor and the stator can occur require high wear resistance due to the greater potential of scratches. This study aims to investigate the potential for platelet deposition of five different inorganic hard material coatings on titanium.
Methods: An in vitro test bench to optically investigate the deposition of fluorescent labelled platelets on the protein layer of different surfaces is hereby presented. The coating materials included Titanium Nitride (TiN), Titanium Niobium Nitride (TiNbN), Diamond-Like Carbon (Ionbond-Medthin-43 (DLCI) and Oerlikon Balimed A (DLCO) and Wolfram Carbid Oerlikon Balimed C (WC). Uncoated titanium (Ti) was investigated as reference. Heparinized human whole blood was incubated with Mepacrine (fluorescent dye) and then pumped with a defined shear rate condition through a flow chamber over the samples. The adhered platelets were visualized via inverted fluorescence microscopy. The analyzation of the green values and the binary image of the generated fluorescent picture offers conclusions about the platelet accumulation and the percentage of the covered surface area, respectively.
Results: Statistical analysis showed a significant lower potential for platelet deposition for TiN compared to Ti and no significant differences for the rest.
Conclusions: It can be concluded that none of these coatings have a higher potential for platelet deposition than Ti and therefore point towards suitability for blood contacting components.
P43
OPTIMIZATION OF COVALENT HEPARIN IMMOBILIZATION ON TITANIUM
J. Kuchinka1, T. Groth1,2
1Department Biomedical Materials, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany. 2Interdisciplinary Center of Material Science, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Objectives: Here, we optimize the covalent immobilization of heparin onto titanium to generate a primary heparin layer to improve the hemocompatibility of titanium surfaces and as basis for a polyelectrolyte multilayer system of physically adsorbed heparin.
Methods: Titanium surfaces, produced by vapor-depositing titanium onto glass, were treated with different amino-terminated silanes including 11-aminoundecyltriethoxy silane (AUDTES) and phosphonates providing functional groups for the covalent heparin immobilization. The covalent immobilization of heparin was carried out either using ethyl(dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) or by reductive amination. Reaction parameters were optimized regarding pH, concentration, time or temperature. Moreover, native heparin and low molecular weight heparin (LMWH) were used. Physicochemical properties of the surfaces were studied measuring thickness, wettability, surface potential and chemical composition. Amino group content of surfaces was determined by colorimetric assay (orange II). Studies on the direct anticoagulant activity of the heparin surfaces were studied with anti-factor Xa assay.
Results: Characterization of the amino-terminated surfaces showed that AUDTES is the most effective with respect to surface amino group density. Phosphonate coatings seemed to be less stable in acidic and alkaline environment than silanes. Measurements of water contact angle and zeta potentials reveal that end-on heparinized surfaces possess a more negative surface potential and are more wettable than side-on heparinized surfaces indicating higher yield of heparin binding. Heparin immobilization is influenced by the pH of the reaction mixture with best results at a slight acidic pH. The effectiveness, regarding its density, is also affected by the molecular weight of the heparin, where LMWH show better results. Anti-FXa assay confirmed heparin activity on the surfaces for side-on and end-on immobilized heparin and for native as well as for LMWH.
Conclusions: Optimized reaction conditions lead to a stable and dense first heparin layer on titanium and will be an optimal basis for further modification with multilayers.
Poster Session 2 - Kidney, Liver, Apheresis
P44
BIODERIVED THERAPEUTICS IN CHRONIC LIVER DISEASES
A. Ganguin, C. Zivko, P. Luciani
Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland.
Objectives: Hepatic stellate cells (HSCs) play a key role in liver fibrosis. Following liver injury, HSCs may transdifferentiate from a quiescent (anti-fibrotic) into a myofibroblast-like pro-fibrotic (activated) state, which, if unresolved, leads to fibrosis [1]. C. Zivko et. al. showed that extracellular vesicles (EVs) shed by quiescent LX-2 (human HSC cell line) cells can trigger an anti-fibrotic effect on naïve LX-2 cells [2]. Therefore, we hypothesized that LX-2 produced cell- derived nanovesicles (cdNVs) will preserve the fibrosis-resolving features of EVs and additionally increase the therapeutic applicability due to higher yield and better scaling potential compared to standard EVs.
Methods: CdNVs were produced by serial extrusion [3] of confluent LX-2 and purified by an iodixanol gradient, followed either by size exclusion chromatography (SEC) and ultracentrifugation (UC), or by UC only. LX-2 cells were either treated with polyenylphosphatidylcholines-rich lipid S80 (anti-fibrotic) or with transforming growth factor β1 (TGF-β1, pro-fibrotic) prior to cdNV production. The formed particles were characterized by concentration and size using nanoparticle tracking analysis, as well as by their protein content using a micro bicinchoninic acid (BCA) assay. The effect of cdNVs from differently treated LX-2 cells on seeded LX-2 (untreated) cells was investigated with fluorescent microscopy [4] and qPCR.
Results: CdNVs from differently treated LX-2 yielded similar yields, protein amounts, size distribution, and zeta potential values. Fluorescent microscopy results revealed an increase in cytosolic lipid droplets in LX-2 upon cdNV administration, which is directly correlated with fibrosis resolution. The anti-fibrotic effect of cdNVs was confirmed by qPCR on 3 different fibrosis markers.
Conclusions: Our results suggest an anti-fibrotic potential in cdNVs which could lead to the development of cell-derived therapeutics against liver fibrosis.
[1] S.L.F. Takuma Tsuchida Nat Rev Gastroenterol Hepatol 2017 14, 397-411.
[2] C. Zivko et al. 2021, Research Square https://doi.org/10.21203/rs.3.rs-1124359/v1].
[3] H. Lee et al. Int J Mol Sci 2020, 21.
[4] G. Valentino et al. Pharmaceutics 2019, 11.
P45
AN IN VITRO STUDY ON IMPROVING THE ENDOTOXIN RECOVERY RATE IN BLOOD SAMPLES USING THE KINETIC CHROMOGENIC LIMULUS AMOEBOCYTE LYSATE ASSAY
S. Harm, C. Schildböck, J. Hartmann
Department for Biomedical Research, University for Continuing Education, Krems, Austria.
Objectives: Endotoxin neutralization caused by plasma components makes it challenging to quantify endotoxins in human whole blood. In this study, we investigated which factors influence the recovery of endotoxins using Limulus Amoebocyte Lysate (LAL)-based assays. The aim of this study was to optimize the preparation of endotoxin-containing whole blood samples to achieve high endotoxin recovery in the LAL assay.
Methods: Endotoxin recovery in blood samples was optimized with respect to the anticoagulant, calcium and magnesium ion concentration of sample diluent, incubation time and incubation temperature. In total, 20 serum samples from healthy volunteer donors spiked with 50 ng/ml endotoxin were compared for endotoxin recovery using conventional sample preparation and our adapted sample preparation.
Results: Our data demonstrate that heparin increases the endotoxin recovery in whole blood, while citrate and EDTA anticoagulation decrease endotoxin recovery by removing divalent cations from the outer layer of the endotoxin micelles. Furthermore, we could show that the endotoxin activity in human plasma and serum decreases strongly over time. For the maximum recovery of endotoxins in human blood, samples were diluted tenfold with 10 IU/ml heparin solution containing 5 mM magnesium chloride. After incubation at room temperature for 3 h, samples were treated at 70 °C for 15 min and the endotoxin content was measured by the kinetic chromogenic based LAL assay. This adapted sample preparation resulted in 830 ± 200 % higher endotoxin recovery for plasma and serum samples compared to conventional sample preparations for the LAL assay.
Conclusions: By adapting the sample preparation, the recovery of endotoxin in whole blood using the LAL assay was significantly improved. Further efforts still need to be made to establish a standardized and reproducible method for the reliable quantification of endotoxin in whole blood samples.
P46
BIOARTIFICIAL LIVER SUPPORT TECHNOLOGIES FOR THE TREATMENT OF CHRONIC LIVER FAILURE - MORPHOFUNCTIONAL CHARACTERISTICS
M. Shagidulin1,2, A. Grechina2, N. Onishchenko1, M. Krasheninnikov4, A. Nikolskaya1, E. Volkova1, G. Piavchenko2, I. Kozlov5, I. Agapov1, L. Davydova3, V. Bogush3, S. Gautier1,2
1Federal State Budgetary Institution, Shumakov National Medical Research Center of Transplantology and Artificial Organs of the Ministry of Health of the Russian Federation, Moscow, Russia. 2Federal State Autonomous Educational Institution of Higher Education, I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation, Sechenov University, Moscow, Russia. 3National Research Center, Kurchatov Institute, Moscow, Russia. 4Federal State Autonomous Educational Institution of Higher Education, Pirogov Russian National Research Medical University of the Ministry of Health of the Russian Federation, Moscow, Russia. 5Orel State University, Orel, Russia.
Objectives: Treatment of chronic liver failure (CLF) and selection efficiency morphofunctional criteria of supporting and stimulating therapy at CLF is an actual problem.
Methods: CLF was modeled on Wistar rats by means of ССl4. Isolated liver cells (LC) and mesenchymal stromal cells (MSC) were obtained by standard procedure. Suspension of LC (2.5 - 4.0 х 106 cells/cm3) and MSC(0.5 - 0.8 х 106 cells/cm3) was immobilized on recombinant spidroin rS1/9 based microgel (gr.2, n = 40). Formed cellular constructs (CCs) were transplanted (TX) into damaged rat liver. The control (gr.1, n = 20) was without treatment (injected saline). Animal Survival, dynamics reduction of СLF indices; recovery morphology of liver; CCs morphology and cell viability were investigated within 90 days after TX. The transformation of histological sections images in the form of a 3D surface was performed in the MATLAB software with k-means clusterization algorithm. After that specific markers for the automated artificial intelligence analysis were identified and 3D surface image was built.
Results: In gr.2 all biochemical indices returned to normal levels within 30 days. CCs was fully integrated into structures of the rat liver. In CCs within 90 days were detected newly formed vessels and viable hepatocytes. In the liver parenchyma hepatocyte proliferation, restoration of a hepatic lobe structure were detected. Dystrophia, fatty vacuoles, connective tissue level and etc. parameters in gr.2 were less expressed in contrast to gr.1, where formation of liver cirrhosis took place without recovery of biochemical indices.
Conclusions: The selected informative morphofunctional criteria characterize the effectiveness of the correction and treatment of CLF with CCs. We consider that this technique bioartificial liver support technologies for correction and treatment CLF are effective, prospective and can be used in clinical practice for treatment of CLF, especially for recipients on a waiting list for the liver transplantation.
P47
MATHEMATICAL SIMULATION OF THE ACID-BASE STATUS IN PATIENTS DURING A WEEK-LONG HEMODIALYSIS CYCLE
M. Pietribiasi, J.K. Leypoldt, M. Debowska, J. Waniewski
Nalecz Institute of Biocybernetics and Biomedical Engineering PAS, Warsaw, Poland.
Objectives: Chronic acidosis in hemodialysis (HD) patients is corrected with bicarbonate added to the dialysis fluid. Due to the intermittent nature of the treatment, blood pH and bicarbonate concentration rise during HD sessions and decrease in the interdialytic periods. Because of the complexity of acid-base regulation it is often difficult to estimate the optimal dialysate bicarbonate concentration for each patient and its effects after several consecutive sessions. We used a mathematical model to predict the acid-base status during and after a week-long cycle of HD comprising 3 sessions (HD1, HD2, HD3).
Methods: The model describes acid-base chemistry of body fluids via the interactions of CO2 with bicarbonate and non-bicarbonate buffers. Bicarbonate and dissolved CO2 are transported from the dialysis fluid to the patient. Their dialysances, and the rate of endogenous acid generation (all constant during the week), were estimated from clinical data collected in a recent study in which a profiled bicarbonate dialysis fluid concentration (CD,Bic) was adopted. During the cycle described in this abstract, CD,Bic was increased from 30 to 35 mEq/L after 2 hours of treatment. Measurements of serum bicarbonate concentration (CBic), pH, and partial pressure of CO2 (PCO2) were available only immediately before HD1, every hour during HD2 and HD3, and before the start of the next weekly cycle.
Results: On average, the root-mean-square errors during HD itself were 1.53 ± 0.72 %, 0.13 ± 0.01 %, 2.15 ± 0.17 % of the measured values for CBic, pH and PCO2, respectively. The errors on the prediction of the values reached after each post-dialytic interval were 2.04 ± 2.64 %, 0.06 ± 0.22 %, 1.96 ± 1.14 % for CBic, pH and PCO2, respectively.
Conclusions: The reported model was able to describe clinical data with good accuracy throughout the whole week using only constant parameters. The small errors made on the pre-dialysis measurements of the following week highlight the potential of the model as a clinical predictive tool.
P48
DOES MEDICINES INTAKE INFLUENCE THE ACCURACY OF THE OPTICAL MONITORING OF DIALYSIS? THE CASE OF UREMIC TOXIN URIC ACID AND PARACETAMOL
J. Holmar1, J. Paats1, A. Adoberg1,2, L. Leis1,2, M. Luman1,2, J. Arund1, K. Pilt1, R. Tanner1, F. Uhlin1,3, I. Fridolin1
1Department of Health Technologies, Tallinn University of Technology, Tallinn, Estonia. 2Centre of Nephrology, North Estonia Medical Centre, Tallinn, Estonia. 3Department of Nephrology and Department of Medicine and Health Science, Linköping University, Linköping, Sweden
Objectives: Uric acid (UA) is one of the uremic toxins and the main UV-absorbing solute in spent dialysate at 280 nm. Optical monitoring of the concentration and removal of the uremic toxins during dialysis has been proposed. It has been shown that paracetamol and its metabolites notably affect the UV-absorbance signal of spent dialysate. Still, there is a poor understanding of the effect on the accuracy of the optical method. This study aimed to evaluate the effect of paracetamol intake on the accuracy of optical monitoring of UA concentration in the spent dialysate.
Methods: 43 chronic dialysis patients (33 M and 10 F, 63 ± 17 years old) from Tallinn, Estonia (22) and Linköping, Sweden (21) were studied. Each patient was followed during four midweek dialysis sessions. Spent dialysate samples were collected before dialysis, at 7, 60, 120, 180, and 240 min after the start of the session, and from a tank of total dialysate collection. The samples UA concentrations were determined by HPLC analysis and UV-absorbance spectra with the spectrophotometer. Linear regression analysis was used to study relationship between UV absorbance (220 - 400 nm) and uric acid concentration in spent dialysate samples of patients who received paracetamol (n = 200; daily dosage = 1.0 - 4.0 g) and who did not receive paracetamol (n = 736; dosage = 0 g).
Results: There was a strong linear correlation between UA concentration in dialysate and UV-absorbance of spent dialysate at 220 - 400 nm. Paracetamol intake decreased the correlation between UV-absorbance of spent dialysate and UA concentration in the region of UV-absorbance of paracetamol remarkably.
Conclusions: Intake of paracetamol in the relatively large daily dosage (1.0 - 4.0 g), decreases the accuracy of the UV-absorbance method for UA concentration determination in the spent dialysate. It should be considered while developing optical monitoring methods and using the region of UV-absorbance of paracetamol.
P49
BRACHIAL BASILIC ARTERIOVENOUS FISTULA (BBAVF) WITH SUPERFICIALIZATION AND TRANSPOSITION OF THE BRACHIAL VEIN IN ONE SURGICAL ACT
N. Gjorgjievski1,2, L. Trajceski1,2, P. Dzekova-Vidimsliki1,2, V. Gerasimovska1,2, V. Pushevski1,2, Z. Janesvski1,2, I.G. Nikolov1,2, G.J. Selim1,2, A. Canevska-Tanevska1,2, G. Spasovski1,2, I. Rambabova-Bushljetikj1,2, P. Dejanov1,2
1University Hospital of Nephrology, Skopje, N. Macedonia. 2Faculty of Medicine, University “Ss Cyril and Methodius”, Skopje, N. Macedonia.
Objectives: The basilic vein on the upper arm could be used for the creation of arteriovenous fistula for hemodialysis. The aim of our study was to determine the risk factors associated with the survival of brachial basilic arteriovenous fistula (BBAVF) with superficialization and transposition of the basilic vein in one surgical act.
Methods: Forty-two patients with chronic kidney disease stage 5 on dialysis with created BBAVF were analyzed. This prospective study was performed from 2019 to 2020. The BBAVF with superficialization and transposition of the basilic vein in one surgical act was created in all patients. Evaluation of blood vessels by Doppler ultrasound (DUS) was done before BBAVF creation. The follow-up period of BBAVF survival was 3, 6, and 12 months after the creation.
Results: The mean age of patients was 59.66 ± 14.24, of which 26 % (11/42) were males. Diabetes mellitus was present in 23.8 % (10/42) of patients, and in 26 % (11/42) of patients have previous tunneled jugular or subclavian catheters. The mean hemodialysis vintage of patients was 4.69 ± 4.43. During the follow-up period, the percentage of BBAVF survival at 3 months was 80.1 % (34/42), at 6 months was 78.6 % (33/42), and at 12 months was 69 % (29/42). The age, gender, hemodialysis vintage, diabetes, and previous tunneled catheters were not significantly associated with the survival of BBAVF in the follow-up period (p > 0.05). The comparative analysis of BBAVF survival at 3, 6, and 12 months in respect of previous tunneled catheters had no significant association (p > 0.05).
Conclusions: The creation of BBAVF with superficialization and transposition of the basilic vein in one surgical act was associated with good survival of the vascular access. The potential benefit of this technique is better optimization of health resources and a shorter duration of the use of tunneled catheters.
P50
SAFETY AND CLINICAL EFFICACY OF PLASMA-SAVING MEMBRANE-BASED THERAPEUTIC PLASMA EXCHANGE (MTPE) IN THE CRITICALLY ILL PATIENTS UNDERGOING CRRT
J.S. Ahn1, T.H. Kim2, A.R. Kim 2, S.Y. Kim2, J.H. Han2, J.W. Chang1
1Department of Internal Medicine. 2Department of Nursing, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea.
Objectives: The mTPE eliminating endotoxin, cytokine and abnormal antibody could be beneficial. To minimize adverse reaction caused by fresh frozen plasma (FFP) and bleeding risk, we used 5 - 20 % albumin, crystalloid fluid and 8 units of FFP as replacement solution. We investigated bleeding event, the changes of platelet count, prothrombin time (PT), activated partial thromboplastin time (aPTT) and 28-Day mortality rate as primary outcome.
Methods: In this retrospective study, 118 mTPE sessions were performed in 60 patients (age 59.6 ± 14.4 years, M:F = 34:26) undergoing CRRT and receiving antibiotics and vasopressors for severe sepsis/shock (n = 43), inflammatory disease (n = 12) and others (n = 5). Our subjects were divided into four groups according to the SOFA score: I (0-9, predicted mortality rate < 10 %), II (10 - 12, 10 - 30 %), III (13 - 14, 40 - 60 %), and IV (15 - 24, > 75 %). mTPE was repeated with the interval of 24 - 48 hours up to 10 times, if needed.
Results: There was no significant difference in SOFA scores, mean arterial pressure, vasopressor index score and PT. The hemoglobin (8.8 ± 2.0 vs. 8.4 ± 2.0 g/dL, p = 0.021) and platelet count (96K ± 101K vs. 80K ± 86K /mm3, p = 0.001) were significantly decreased after 1st mTPE. aPTT was significantly increased from 62.7 ± 38.6 to 74.5 ± 41.2 seconds (p = 0.037). The similar changes persisted after 2nd mTPE. However, there was no bleeding event within 24 hours from the end of mTPE sessions. The 28-Day mortality rates were 22.2 %, 12.5 %, 0 % and 67.6 % in the 4 groups, respectively. Analyzing survival according to the number of mTPE application, 14 patients survived among 29 who received only one time of mTPE and 4 among 20, two times of mTPE. Only 2 patients survived among 11 who received three time or more of mTPE.
Conclusions: Despite aPTT prolongation and decreased hemoglobin and platelet count, no bleeding events and lower-than-expected mortality suggest that mTPE could be safe and beneficial in these patients.
P51
EFFECTS OF EXTRACORPOREAL PERFUSION INVOLVING CONTACT BETWEEN BLOOD AND A HEMODIALYSIS MEMBRANE ON KIDNEY INJURY IN A RAT MODEL
Y. Kikugawa1, K. Kobayashi1,2, B. Tsuchiya1,2, M. Ogata1,2, K. Kokubo1,2, M. Kubota1,2
1Kitasato University Graduate School of Medical Sciences, Kanagawa, Japan. 2Kitasato University School of Allied Health Sciences, Kanagawa, Japan.
Objectives: Preserving residual renal function (RRF) is important for patients during the induction phase of hemodialysis. However, RRF is likely to decline rapidly after the start of hemodialysis treatment. In the present study, we investigated the effects of hemodialysis treatment on kidneys using a rat dialysis model, focusing on blood pressure fluctuations during dialysis and contact between blood and the dialysis membrane as possible factors for the decrease in RRF.
Methods: Male Sprague Dawley rats were used for the experiments. Three experimental groups were used: a sham group, an extracorporeal perfusion alone group (in which extracorporeal perfusion was performed without a dialyzer), and a dialyzer group (in which extracorporeal perfusion was performed with a dialyzer). The perfusion time was 4 hours. Arterial pressure and arterial blood oxygen saturation (SpO2) were measured during the perfusion experiment, and the urinary kidney injury molecule-1 (KIM-1) concentration was determined as a marker of renal injury after the perfusion was completed. After the perfusion experiment, the kidneys were harvested, and glomerular congestion was evaluated using HE staining.
Results: In the extracorporeal perfusion alone group, a significant upward trend in arterial pressure and a significant downward trend in SpO2 were observed during the first 2 hours of perfusion. In the dialyzer group, a significant increasing trend in arterial pressure during the first 4 hours of perfusion and a significant decreasing trend in SpO2 during the first 2 hours of perfusion were observed. The urinary KIM-1 levels were significantly higher in the dialyzer group than in the sham group. HE staining showed that the percentage of congested glomeruli to total glomeruli was significantly higher in the dialyzer group than in the sham group.
Conclusions: Contact between blood and the dialysis membrane during extracorporeal perfusion may cause kidney injury and congestion.
P52
ESTIMATING PARAMETERS OF BICARBONATE KINETICS DURING HEMODIALYSIS USING THE HYDROGEN ION MOBILIZATION MODEL
J.K. Leypoldt, M. Pietribiasi, M. Debowska, J. Waniewski
Nalecz Institute of Biocybernetics and Biomedical Engineering PAS, Warsaw, Poland.
Objectives: Previous studies describing bicarbonate (HCO3) kinetics using the hydrogen ion (H+) mobilization model (Sargent et al, Semin Dial 2018) allowed estimation of the mobilization parameter (mH+) by assuming a fixed HCO3 dialysance (D) for the hemodialyzer. Because D is difficult to determine empirically, we evaluated whether mH+ and D could be simultaneously estimated from the time dependence of blood HCO3 concentration ([HCO3]) when using a constant or time varying dialysate [HCO3].
Methods: We estimated mH+ when assuming a fixed D (0.198 L / min) and also estimated mH+ and D simultaneously by comparing measured blood [HCO3] during hemodialysis (HD) with H+ mobilization model predictions using nonlinear regression. Estimates were calculated from clinical study data where blood [HCO3] was measured in 11 HD patients during 4-hr treatments with A) constant dialysate [HCO3] of 35 mEq/L; B) dialysate [HCO3] decreasing from 35 to 30 mEq/L after 2 hrs; C) dialysate [HCO3] increasing from 30 to 35 mEq/L after 2 hrs. Study interventions were fixed during a given week; blood samples were collected only during the second and third sessions of the week. Acid-base composition was determined using a blood gas analyzer. Parameters were estimated individually for each patient and session and analyzed together.
Results: Values of mH+ (n = 62) were not significantly different when a fixed D was assumed (0.155 ± 0.095 mL/min, mean ± SD) or when mH+ and D were simultaneously estimated (mH+ = 0.137 ± 0.106 mL/min; D = 0.187 ± 0.092 mL/min). In the latter case, however, mH+ and D values were correlated (r = 0.724), indicating model overparameterization.
Conclusions: Robust parameter estimation using the H+ mobilization model during HD is limited to a single parameter (mH+).
P53
PLATELET FUNCTION DURING PLATELET-RICH PLASMA SEQUESTRATION IN COMPLEX CARDIAC SURGICAL PROCEDURES
R. Hajek1, O. Zuscich1, I. Fluger1, P. Caletka1, V. Lonsky1, P. Santavy1, L. Slavík2, J. Ulehlova2
1Department of Cardiac Surgery, University Hospital, Olomouc, Czech Republic. 2Department of Hemato - Oncology, University Hospital, Olomouc, Czech Republic.
Objectives: Contemporary cell-salvage technology can preserve platelets in platelet-rich plasma (PRP) sequestration. We tested the preservation of platelet functions during and after PRP sequestration.
Methods: Written informed consent, local ethic commitee approval. Elective surgery estimated CPB > 2 hours. Antiplatelet medication withdrawn according to ESA/ESC guidelines. Initial hematokrit > 0.35 (800 ml of whole blood processed) RBC+PRP sequestration simultaneously, PRP retransfused immediately after the CPB. CPB: X- coating, centrifugal pump, UFH: ACT > 400 s, TXA 30 mg/kg + 15 mg/kg in CPB. Cell saver: CPD-A bags, EDTA vacutainer, manufacturer protocol. Platelet count flow cytometry. Optical aggregometry: collagen, ADP, ristocetin and epinephrin induced aggregation. Whole blood sample before surgery, from processed PRP, after retransfusion, after surgery. Thromboelastography before/after surgery.
Results: Sequestration group: 21 patients (94 % male), control 12 patients (80 % M). Both group comparable by age (67 both), EuroSCORE (3.37 vs. 3.62 %), aortic (61 vs. 49 %), redo surgery (22 vs. 14 %). No difference in perioperative (605 vs. 564 mL) postoperative (711 vs. 652 mL) blood loss, transfusion of RBC (1.6 vs. 2.4 TU), FFP (1.7 vs. 1.7 TU), PLT (1 vs. 0 TU ), fibrinogen, PCC, additional TXA dose (18 vs. 14 %), reexploration because of bleeding (16 vs. 14 %), ICULOS (4 vs. 3.5 days), HLOS (11.5 vs. 10.5 days), 30 day mortality (1 vs. 0) and major adverese clinical events (myocardial ischemia, stroke, AKI, ALI, new onset of atrial fibrillation). Platelet count decreased (185 to 136 vs. 230 to 136). Platelet count after CPB was lower in PRP (105 vs. 196, p = 0.001) but not at the end (136 both). Significant decrease of collagen, ADP and epinephrin mediated aggregation but preserved values of ristocetin mediated aggregation recorded.
Conclusions: The using og PRP sequetrastion have no impact on bleeding, transfusion therapy and clinical outcome in complex cardiac surgery. It preserves platelet count and ristocetin, but no other receptor mediated aggregation.
Acknowledgment: Supported by institutional grant FN Olomouc 87-72.
P54
CHANGES IN RESPONSIVENESS OF LEUKOCYTES IN CONTACT WITH A HEMOFILTER DURING CONTINUOUS BLOOD PURIFICATION THERAPY
M. Sekiguchi1, A. Yamamoto2, Y. Kurihara1, K. Kokubo1,2, K. Kobayashi1,2, K. Masaru1,2
1Kitasato University Graduate School of Medical Sciences, Kanagawa, Japan. 2Kitasato University School of Allied Health Sciences, Kanagawa, Japan.
Objectives: Continuous blood purification therapy is widely used in patients with acute renal failure during intensive care. In this therapy, treatment is continuous for 24 hours or longer, so the blood is continuously in contact with a hemofilter. The present study aimed to clarify the changes in the responsiveness of leukocytes in contact with a hemofilter using an in vitro hemofiltration model using porcine whole blood.
Methods: Porcine whole blood adjusted to an ACT of 300 - 400 s was divided into three portions, and perfusion experiments were performed for 12 hours using a polysulfone (PS) membrane, an NV polymer-embedded PS membrane (NV-PS), or a polymethyl methacrylate (PMMA) membrane under the same conditions. To measure leukocyte responsiveness to stimulation, inflammatory cytokines (interleukin (IL) -6 and IL-8) were measured and evaluated using an enzyme-linked immunosorbent assay after stimulation with lipopolysaccharide (LPS).
Results: The transmembrane pressure for the PMMA (n = 6) membrane increased with time, while those for the PS (n = 6) and NV-PS (n = 7) membrane remained almost constant. IL-6 production of leukocytes was significantly lower in all the membrane contact groups than that without contact with the membrane, while IL-8 production increased after LPS stimulation in all the membrane contact groups. The 12 hours of circulation may have a significant effect on T cells and B cells, which are mainly involved in IL-6 production, while the effect on monocytes and natural killer cells, which are mainly involved in IL-8 production, considered to be less in all the membrane contact groups.
Conclusions: The change in responsiveness of leukocytes in contact with the hemofilter upon LPS stimulation suggests that contact with the membrane decreased the responsiveness of T and B cells, but not of monocytes or natural killer cells.
P55
REMOVAL OF PROTEIN-BOUND TOXINS BY HEMOPERFUSION: AN IN VITRO STUDY
C. Schildböck, S. Harm, J. Hartmann
Center for Biomedical Technology, University for Continuing Education Krems, Krems, Austria.
Objectives: The aim of this in vitro study was to determine the performance of the hemoperfusion adsorbents Biosky MG 350 (Biosun Med. Tech., Foshan, China) and Jafron HA-130 (Jafron Biomed., Zuhai, China) for the adsorption of uremic toxins and for liver toxins as well as to compare the adsorptive removal to the clearance by hemodialysis.
Methods: Human heparinized plasma (10 IU/mL) was spiked with the uremic toxins homocysteine, hippuric acid, indoxyl sulphate and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) and with the liver toxins bilirubin, cholic acid, tryptophane and phenol. The albumin-bound ratio of each toxin was determined by centrifugation of the spiked plasma through 30 kD membranes followed by the measurement of albumin and toxin concentrations in the filtrate. Hemoperfusion and dialysis experiments were carried out using 330 mL plasma at a flow rate of 30 mL/min over six hours. The hemoperfusion experiments were carried out with 30 mL adsorbent columns. For the hemodialysis experiments, pediatric high flux filters (FX paed, Fresenius Medical Care, Bad Homburg, Germany) were used with a dialysate flow rate of 1500 mL/h.
Results: Dialysis resulted in higher removal rates for hippuric acid, homocysteine, indoxyl sulphate and tryptophane than hemoperfusion, while hemoperfusion was more effective for the elimination of bilirubin, cholic acid and phenol. Jafron HA-130 and Biosky MG 350 showed similar results for the toxins tested in this study. CMPF, a strongly albumin-bound uremic toxin, could not be removed with any of the treatment procedures.
Conclusions: The use of porous and hydrophobic adsorbers in combination with conventional dialysis treatment could be promising for the removal of strongly albumin-bound toxins that can only be insufficiently removed by hemodialysis alone.
Poster Session 2 - Tissue Engineering
P56
FABRICATION OF MICROSTRUCTURED SURFACES AND ITS INFLUENCE ON BACTERIAL ADHESION AND GROWTH
S. Nilsson Zagiczek1, C. Grasl1,2, M. Kussmann3, M. Weiss-Tessbach3, D. Moser4, F. Moscato1,2,5, H. Schima1,2,6
1Centre for Medical Physics and Biomedical Engineering, Medical University of Vienna, Vienna, Austria. 2Ludwig Boltzmann Institute for Cardiovascular Research, Vienna, Austria. 3Department of Medicine I, Division of Infectious Diseases and Tropical Medicine, Medical University of Vienna, Vienna, Austria. 4Department of Cranio-Maxillofacial and Oral Surgery, Medical University of Vienna, Vienna. 5Austria Austrian Cluster for Tissue Regeneration, Vienna, Austria. 6Division of Cardiac Surgery, Medical University of Vienna, Vienna, Austria.
Objectives: Implantation of a foreign body is often associated with restoration of natural body functions to prolong life expectancy and improve quality of life. However, the procedure involves considerable risk for bacterial infections. Device-related infections account for a large proportion of hospital-acquired infections, and the ability of bacteria to form a biofilm as a protective shield usually makes treatment impossible without removal of the implant. Recently, there have been several approaches to prevent bacterial attachment and biofilm formation. In particular, topographic surfaces have attracted considerable attention in studies seeking antibacterial properties without additional antimicrobial substances.
Methods: Several surfaces with microcylinders in three different dimensions (1, 3 and 9 µm) were fabricated with a nanoprinter using two-photon lithography and evaluated for their antibacterial activity in terms of biofilm formers. The microstructured surfaces were cultured for 24 hours with different strains of Pseudomonas aeruginosa and Staphylococcus aureus to study bacterial attachment to the patterned surfaces. In addition, surface wettability was measured by a contact angle measurement.
Results: The contact angles increased with the size of the cylinders, indicating that the hydrophobic properties of the surface were favored. Since previous studies have proven that bacterial attachment is usually affected by surface wettability, a difference in bacterial adhesion should be observed. However, the results demonstrated that Staphylococcus aureus was not affected by the microstructures, while for Pseudomonas aeruginosa the bacterial amount increased with the size of the cylinders, and compared to a flat surface, a reduction of bacteria was observed only for one strain on the smallest cylinders.
Conclusions: This study indicates that microstructures from 1 to 9 µm have little to no antibacterial properties and that a change in wettability due to surface structures has no effect on bacterial adhesion.
P57
YAP/TAZ FACILITATES EXTRACELLULAR VESICLE RELEASE UPON MECHANICAL STIMULATION
R. Weiss2, F. Nägele1, T. Eichhorn2, V. Weber2, J. Holfeld1
1Department of Cardiac Surgery, Medical University of Innsbruck, Innsbruck, Austria. 2Center for Biomedical Technology, Department for Biomedical Research, Danube University Krems, Krems, Austria.
Objectives: Chronic heart failure, most commonly caused by ischemic heart disease, is the leading cause of death worldwide. Currently, there is no curative treatment available.
The physical stimulus of shockwave therapy (SWT) has a pro-angiogenic impact on ischemic tissue, representing a promising regenerative approach. The Hippo signaling pathway YAP/TAZ plays a key role in angiogenesis and can be regulated by mechanical signals. Both SWT stimulation and YAP/TAZ activation cause a release of extracellular vesicles (EVs). We aim to substantiate the mechanotransduction of SWT via YAP/TAZ facilitated EV release and subsequent angiogenic response.
Methods: Human umbilical vein endothelial cells (HUVECs) were stimulated with 300 impulses at a frequency of 3 Hz and an energy flux density of 0.1 mJ/mm2. 4 h thereafter, mRNA expression of YAP/TAZ target genes (ANKRD1, CYR61) was measured and the nuclear localization of YAP/TAZ was examined by immunofluorescence. The culture supernatant was collected. EV release was characterized by flow cytometry (Cytoflex LX calibrated with fluorescent silica beads) using the membrane dye Cell Mask Orange (CMO) and tetraspanin markers CD63 and CD81. Furthermore, EVs were analyzed by a bead-based flow cytometry assay with CD63-coupled magnetic beads.
Results: SWT of HUVECs resulted in a higher concentration of CMO+ EVs (9,953 ± 2,039 vs. 6,092 ± 1,503 EVs/µL) in the culture supernatant as compared to the untreated control. This was confirmed by a higher percentage of EV-decorated beads after SWT, accompanied by higher mRNA expression of YAP/TAZ target genes ANKRD1 (p = 0.0005, respectively) and CYR61 (p = 0.0006, respectively). Immunofluorescence staining showed nuclear translocation of YAP/TAZ upon SWT compared to untreated controls. These effects were abolished and the EV concentration was decreased upon pharmacological inhibition of YAP/TAZ nuclear translocation.
Conclusions: The mechanical stimulus of SWT activates the Hippo-Pathway YAP/TAZ and causes EV release. Our study provides substantial insight into cellular mechanotransduction and serves as the basis for translation into clinical practice.
P58
IDENTIFICATION AND CHARACTERIZATION OF ADRENAL GLAND DERIVED CALCIFICATION PARADOX MEDIATORS
S. Bhargava1,2, N. Gayrard3, V. Jankowski1, À. Argilés3, J. Jankowski1,2
1Institute of Molecular Cardiovascular Research, Medical Faculty, RWTH Aachen University, Aachen, Germany. 2Experimental Vascular Pathology, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands. 3RD-Néphrologie and EA7288 University of Montpellier, Montpellier, France.
Objectives: In the current study, we investigated a previously unknown systemic regulation of vascular calcification processes by adrenal glands.
Methods: Bovine adrenal gland homogenate was separated using chromatographic fractionation. The fractions were assessed in cells, aortic rings, and vitamin D3 plus nicotine renal failure rat model for effects on vascular calcification processes. Potential mediators were distinguished by mass spectrometry and comparison with pertinent databases.
Results: We identified a novel peptide from adrenal glands that inhibits the transdifferentiation of aortic smooth muscle cells into osteoblast like cells, which are associated with vascular calcification. This peptide is released from chromogranin A by enzymatic cleavage by calpain 1 and kallikrein and named Calcification Blocking Factor (CBF) owing to its protective effects against vascular calcification. CBF reduced the calcium content of aortic smooth muscle cells and aortic rings under calcifying culture conditions and in aortas of vitamin D and nicotine treated (VDN) animals. CBF treated VDN animals showed significantly reduced pulse pressure, a marker of arterial stiffness. CBF prevented aortic smooth muscle cell transdifferentiation into osteoblast-like cells within the vessel wall via the sodium-dependent phosphate transporter PIT-1, inhibiting NF-κB activation and subsequently inhibiting BMP2/p-SMAD signaling. Consistent with our preclinical results, concentration of CBF was found to be significantly reduced in diseases characterized by increased calcification shown in patients with chronic kidney disease. Smaller fragments of the 19 aa peptide were analyzed to identify the active site of CBF. A smaller 8 aa peptide reduced vascular calcification while promoting bone density in VDN animals.
Conclusions: In conclusion, we identified a novel 19 aa peptide which modulates vascular calcification and is derived from the adrenal glands. Furthermore, we identified a smaller 8 aa peptide that promotes bone density and reduces vascular calcification. These findings suggest a novel function of adrenal glands in calcium metabolism.
P59
SURFACE TOPOGRAPHY ENHANCE STEM CELL-DERIVED CARDIOMYOCYTE MATURATION AND CHANGE Β-MYOSIN HEAVY CHAIN EXPRESSION
L.R.X. Cortella1, I.A. Cestari1, M. Soldera2,3, A. Lasagni2,3, I.N. Cestari1
1Bioengineering Department, Heart Institute (InCor), University of São Paulo Medical School, São Paulo, Brazil. 2Institut for Manufacturing Technology, Technische Universität Dresden, Dresden, Germany. 3Fraunhofer-Institut für Werkstoff und Strahltechnik IWS, Dresden, Germany.
Objectives: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are possible substitutes for cardiac tissue but have an immature phenotype. We investigated the functionalization in the low micrometer range of polyethylene terephthalate (PET) sheets by laser ablation and hot embossing. We tested the ability of a line-like 3 µm spatial period pattern to induce changes in maturation and gene expression of hiPSC-CM.
Methods: We studied Ca2+ cycling and sarcomeric gene expression by seeding hiPSC-CM onto structured or onto unstructured PET scaffolds. Quantitative PCR analysis of alpha (MYH6) or beta myosin heavy chain (MYH7) were performed. Spontaneous contractile activities of hiPSC-CMs were assessed by measuring the Ca2+ fluorescence signal. The kinetic parameters analyzed were time-to-peak (tpeak), decay time (dct), peak width at 50 % (pw50), and decay rate (τ).
Results: After 4 weeks in culture, Ca2+ cycling and gene expression of iPSC-CM were compared between groups. Surface topography induced cellular alignment and increased sarcomere organization. Ca2+ cycling of hiPSC-CM cultured on structured substrates showed reduction of all measured parameters, except for tpeak, with the decay rate approximately 50 % faster (p < 0.05). Changes in MYH6 expression did not reach significance but MYH7 showed an increase expression when compared to controls with a ΔΔCt of 0.35 (p = 0.01).
Conclusion: Structured scaffolds can induce phenotypic changes in hiPSC-CM, with improve alignment and maturation. The kinetic parameters obtained from cells grown onto structured PET surfaces may be interpreted as reflecting faster Ca2+ removal mechanisms, closer to the adult myocardium. These changes were associated with modifications in sarcomeric gene expression. MYH7 encodes β-myosin heavy chain, which is the major left ventricular myosin heavy chain subtype in the human adult myocardium, suggesting that this change in expression may be part of the underlying mechanism for the phenotypic variation brought about by the patterned substrate. Support: Finep 1253/13.
P60
WHAT IS HISTORICAL EFFECT OF TANGENTIAL MECHANICAL FORCE FIELD BY CENTRIFUGE ON CELLS IN VITRO?
S. Hashimoto1, H. Yonezawa2, K. Kishimoto1, S. Saito1
1Mechanical Engineering, Kogakuin University, Tokyo, Japan. 2Systems Design, Kogakuin University, Tokyo, Japan.
Objectives: The myotube thickens in the normal mechanical force (hyper-gravitational) field. In this study, a tangential mechanical force field with respect to the scaffold plane was set in order to visualize the historical effect of the force field direction on the cell behavior. In order to align the longitudinal direction of the cells in advance, a stripe-pattern of low ridges was created on the surface of the scaffold. Historical effects after cultivation for 5 hours while exposed to the force field by centrifugal force were tracked by the behavior (deformation, migration) of cells on the scaffold surface.
Methods: Striped patterns of low ridges (height 0.7 μm, width 3 μm, spacing 3 μm) were created on the surface of the scaffold by photolithography technology. Variation was made on the angle between the longitudinal direction of the ridges and the direction of the tangential force of the centrifuge: 0 degrees, 45 degrees, and 90 degrees. Myoblasts (C2C12: mouse myoblast cell line) were used in the experiment. After centrifugation at 100 G for 5 hours, the 2D projection shape of each cell was tracked by time-lapse microscopic images for 24 hours.
Results: The experimental results showed the following. When exposed to a tangential force field perpendicular to the longitudinal direction of the cell, the movement and deformation of the cell becomes larger than when exposed to a tangential force field parallel to the longitudinal direction of the cell. This historical effect remains for 24 hours after the tangential field stimulus is stopped.
Conclusions: Stripe patterns of low ridges on the surface of the scaffold are effective for aligning the longitudinal direction of the cells with respect to the mechanical force field. The tangential force field on the cell is effective to visualize the historical effect in the direction of the force field.
P61
IN SITU GELLING HYDROGELS BASED ON OXIDIZED POLYSACCHARIDES FOR TISSUE REGENERATION
C. Willems1, T. Groth1,2
1Department of Biomedical Materials, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany. 2Interdisciplinary Center of Materials Science and Interdisciplinary Center of Applied Science, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany.
Objectives: Our objective was to synthesize in-situ gelling hydrogels based on oxidized polysaccharides and amine or hydrazide containing biopolymers to prepare biomimetic, biodegradable scaffolds for cartilage and bone replacement.
Methods: Oxidized polysaccharides and carboxymethylchitosan or hydrazide-modified gelatin were mixed at 37 °C, where an imine or hydrazone bond was formed to crosslink the gel. Different gels were investigated by using rheological and gravimetric measurements. The ratio of both components was optimized and different filler materials were used to increase the stability and rheological properties of the hydrogels. Different assays were applied to investigate the cytotoxicity and bioactivity of the hydrogels.
Results: The mechanical and bioactive properties could be varied depending on the type, molecular weight and degree of oxidation of the used polysaccharide. Developed hydrogels were stable for up to 14 days, while embedded cells were found to be viable.
Conclusions: It can be shown, that stable hydrogels can be synthesized using oxidized polysaccharides without the use of a potentially toxic additional crosslinking agent. These gels show a great potential to be used in tissue engineering and 3D bioprinting.
P62
AN OPTOGENETIC APPROACH TO CHARACTERISE HUMAN TLR2 HOMO- AND HETERODIMERS IN THP-1 AND THP-1 DERIVED MACROPHAGES
K. Colleselli, C. Pollhammer, A. Stierschneider, H. Hundsberger, C. Wiesner
Department of Medical and Pharmaceutical Biotechnology, IMC University of Applied Sciences, Krems, Austria.
Objectives: To date, little is known about the functional mechanisms of the Toll-like receptor 2 (TLR2) homodimer. Mainly the heterodimers with Toll-like receptor 1 (TLR1) and Toll-like receptor 6 (TLR6) which show pro-inflammatory properties are discussed. The scientific body is not in agreement about the role and function of TLR2 homodimers, especially since specific ligands for activation are unknown. However, a profound understanding can be particularly valuable for immunological dysfunctions as in sepsis.
Methods: We have established a novel optogenetic cell line using THP-1 TLR2 knock-out (KO) cells. TLR2 was fused with a light-oxygen-voltage-sensing (LOV) domain and the plasmid was then stably integrated into KO cells generating TLR2LOV knock-in cells by lentiviral transfection. This optogenetic cell line allows precise dimerisation and activation of TLR2 by blue light. We performed RT-qPCR to examine mRNA expression levels of common pro- and anti-inflammatory cytokines and chemokines (IL-6, IL-8, IL-10, TGFβ) and immunoblots for detection of p65, ERK1/2 and p38 expression. Furthermore, we established attachment and migration assays and differentiated THP-1, KO and TLR2LOV knock-in cells into macrophages.
Results: By using an integrated cell-reporter we were able to show the functionality and effective activation of our optogenetic TLR2LOV knock-in cell line. Preliminary data demonstrate significant differences in mRNA and protein expression levels between KO and TLR2LOV knock-in cells after light induction and a difference between homo- and heterodimers. Moreover, KO cells showed stronger attachment which could be reset by the knock-in cells. Macrophage differentiation was more prominent in KO cells than in THP-1 and TLR2LOV knock-in cells.
Conclusions: Our optogenetic cell lines are a convenient tool to further investigate principal mechanisms of TLR2 homo- and heterodimerisation and may provide new insights for therapeutical strategies for sepsis and other immunological disorders.
P63
DEVELOPMENT OF A TISSUE EQUIVALENT FROM DECELLULARIZED PORCINE ARTICULAR CARTILAGE
Y.B. Basok, A.D. Kirillova, E.A. Nemets, A.M. Grigoriev, L.A. Kirsanova, V.I. Sevastianov
Shumakov National Medical Research Center of Transplantology and Artificial Organs, Ministry of Health of the Russian Federation, Moscow, Russia.
Objectives: Development and comparison of protocols for decellularization of micronized porcine articular cartilage.
Methods: Decellularization was carried out according to three protocols: (1) -3 cycles of freezing/thawing +treatment with surfactants +DNase; (2) -surfactant +DNase + supercritical CO2; (3) -surfactant +ultrasound +DNase. The biochemical analysis of the composition of the obtained decellularized micronized porcine articular cartilage (DCp) included the determination of the content of DNA, glycosaminoglycans (GAG) and collagen. The morphology of the samples was evaluated using histological staining methods. The biological safety of DCp was investigated in vitro and in vivo. Functional activity in vitro was evaluated with respect to the ability of DCp to maintain adhesion, viability, and specific activity of human adipose tissue mesenchymal stromal cells (ADSC).
Results: Decellularization according to protocol 3 allowed to achieve a 95 % reduction in the amount of DNA with the greatest preservation of GAG (37 %) and collagen (82 %), compared with other protocols. The biological safety of DCp has been proven in in vitro and in vivo experiments. The ability of DCp to maintain a high level of proliferation and chondrogenic differentiation, manifested in the ability of cells to produce GAG and type II collagen, has been proven on the culture of ADSC.
Conclusions: An optimal protocol for obtaining tissue specific DCp with preservation of morphofunctional properties of the extracellular matrix has been developed. The high ability of DCp to maintain adhesion, proliferation and chondrogenic differentiation of ADSC in vitro makes it promising for the creation of a tissue equivalent of cartilage.
The research was carried out at the expense of the Russian Science Foundation Grant No. 21-15-00251, https://rscf.ru/en/project/21-15-00251/).
P64
CREATING AN IDEAL DIAPHRAGMATIC BIOSCAFFOLD: DEVELOPMENT AND OPTIMIZATION OF DECELLULARIZATION PROTOCOLS FOR DIAPHRAGMATIC TISSUE ENGINEERING IN MURINE MODEL
M.N. Andreas1*, A.K. Boehm1*, P. Tang1, S. Moosburner1, J.M. Gaßner1,2, D. Wulsten3, J. Neudecker1, S. Spuler4, J. Pratschke1,5, I.M. Sauer1,5,§, K.H. Hillebrandt1,2
1Department of Surgery, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany. 2Berlin Institute of Health, Charité - Universitätsmedizin Berlin, Berlin, Germany. 3Julius-Wolff-Institut für Biomechanik und Muskuloskeletale Regeneration, Berlin, Germany. 4Max-Delbrück-Centrum für Molekulare Medizin in der Helmholtz-Gemeinschaft, Berlin, Germany. 5Cluster of Excellence Matters of Activity, Charité - Universitäts-medizin Berlin, corporate me.mber of Freie Universität Berlin and Humboldt Universität zu Berlin. *Equally contributing authors.
Objectives: Tissue engineered bioscaffolds based on decellularized composites have gained increasing interest for treatment of various diaphragmatic impairments, including patch repair of diaphragmatic hernias, and defects due to trauma or oncologic resections (e. g. lung cancer, pleural metastasis).The in vivo performance of decellularized diaphragmatic tissues depends on bioengineering parameters such as applied agents and processing techniques, which vary in their efficacy to maximize cellular removal and minimize extracellular matrix (ECM) damage. We herein conduct the first study to systematically compare detergent-enzymatic treatments regarding several agents and application models for diaphragmatic decellularization.
Methods: Rat diaphragms (n = 84) were decellularized according to several detergent-enzymatic protocols with the main detergents 1 % and 0.1 % Sodium dodecyl sulfate (SDS) and 4 % Sodium deoxycholate (SDC) by orbital shaking or retrograde perfusion through the vena cava. Systematic comparison and evaluation were conducted by (1) qualitative assessment with macroscopic and microscopic evaluation by histological staining, immunohistochemistry, and scanning electron microscopy (SEM), (2) quantitative analysis including DNA quantification and biomechanical testing, and (3) semi-quantitative analysis by proteomics.
Results: All DET protocols generated histologically and functionally decellularized diaphragmatic specimens and allowed for preservation of extracellular matrix structures confirmed by ultrastructural SEM analysis. SDS 0.1 % treated samples achieved best performing outcomes in both DNA quantification and biomechanical testing compared to SDC 4 % and SDS 1 %. The application modality of retrograde perfusion seemed to benefit biomechanical performance only in SDS treatment. Differences in proteomic composition of the native and decellularized diaphragmatic matrisome were compared for the first time.
Conclusions: While all DET protocols proved efficient in removing cell nuclei whilst preserving ECM structures, our study indicates that SDS 0.1 % is best suitable for diaphragmatic decellularization. Finding an optimized protocol lays the foundation for further developmental experiments and clinical trials of bioengineered diaphragmatic surrogate tissues.
Image Space Material funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy - EXC 2025.
P65
IMPROVEMENT OF THE SIMPLER AND SHORTER DECELLULARIZATION PROCEDURE OF IN VIVO TISSUE ENGINEERED VASCULAR GRAFT USED FOR ALLOGENEIC TRANSPLANTATION
T. Gondai1, M. Yamanami1, K. Kanda1, T. Inoue1, H. Kawajiri1, T. Watanabe1, D. Kami2, S. Gojo2, H. Yaku1
1Department of Cardiovascular Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan. 2Department of Regenerative Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Objectives: We have been clinically applying autologous in vivo tissue engineered vascular grafts formed in the subcutaneous tissues of pediatric patients since 2015. However, there is concern about the mechanical strength of the graft in patients in severe condition because the formation of the graft depends on the patient’s ability to regenerate tissues. Therefore, we devised to apply decellularized allogeneic grafts formed in healthy adult humans. In order to complete graft harvest from the recipient and transplantation into the patient on the same day, it is necessary to develop a simpler and shorter decellularization procedure. In this study, we evaluated the different treatment from the previous circulation method using the beagle dog model.
Methods: A cylindrical silicone rods (diameter: 5 mm, length: 30 mm) were implanted in the subcutaneous tissue of beagle dogs for several months to prepare tubular tissue grafts. The grafts were decellularized by shaking at 37 °C in 1 % sodium lauryl ether sulfate (SLES). The shaking was performed by dividing into two groups. In one group, the conical tubes containing the grafts were placed vertically on the shaker (group V). And in another group, the conical tubes were placed horizontally to shake more vigorously (group H). After each treatment, residual DNA from the graft was quantified.
Results: In group H, the amount of residual DNA in the graft was less than 50 ng/mg dry weight after one hour of treatment, which indicated sufficient decellularization. Previously. at least 3 hours of treatment was required to meet this criterion.
Conclusions: The decellularization process could be significantly shortened by simple vigorous shaking in the horizontal direction. Further studies are needed to evaluate the effect of this method on the mechanical properties of the graft.
P66
INFLUENCE OF DIFFERENT APPROACHES FOR COATING OF BONE SUBSTITUTE MATERIAL WITH HEPARIN ON TISSUE REACTION IN VIVO
S. Stojanović1,2, H. AlKhoury3, M. Radenković1, S. Najman1,2, T. Groth3
1Department for Cell and Tissue Engineering, Scientific Research Center for Biomedicine, Faculty of Medicine, University of Niš, Niš, Serbia. 2Department of Biology and Human Genetics, Faculty of Medicine, University of Niš, Niš, Serbia. 3Department Biomedical Materials, Institute of Pharmacy, Martin Luther University Halle Wittenberg, Halle (Saale), Germany.
Objectives: Coating the bone substitute materials with anti-inflammatory agents is of great potential in reducing the inflammatory response resulted after biomaterial implantation and preserving biocompatibility in vivo but also modulating the course of tissue reaction, biomaterial degradation rate and fate of the implants. Our aim was to modify biphasic calcium phosphate (BCP) particles, as a model substrate, by coating with heparin, glycosaminoglycan with proven anti-inflammatory activity since it is well known that biodegradable and bioresorbable biomaterials such as BCP induce inflammatory tissue reaction after implantation.
Methods: We applied two approaches for coating of BCP particles with heparin: 1) covalent immobilization and 2) multilayered coating using layer-by-layer (LbL) technique. Tissue reaction, potential anti-inflammatory activity, and behavior of coated BCP particles in vivo were analyzed using mice subcutaneous implantation model. Gene expression, histological and histomorphometrical analysis were performed in implants explanted 15 and 30 days after implantation.
Results: The course of subcutaneous tissue reaction was shown to be dependent on the coating method used. Some differences in gene expression pattern and infiltrated tissue composition were observed between covalent and LbL coated BCP particles and also between coated compared to plain BCP particles, at both examined time points.
Conclusions: Obtained results showed that both approaches were successful in coating of BCP particles with heparin. Applied coating approaches led to the different tissue response to BCP particles after implantation which suggested that it may be used as an approach for modulating and guiding the biomaterial-induced tissue inflammatory response and initiation of regenerative processes.
P67
PANCREAS MORPHOLOGICAL FEATURES AS A DETERMINING FACTOR OF THE EFFECTIVE DECELLULARIZATION PROTOCOL
A. Ponomareva, N. Baranova, L. Kirsanova, G. Bubentsova, E. Nemets, I. Miloserdov, V. Sevastianov
The Shumakov National Medical Research Center of Transplantology and Artificial Organs, Moscow, Russia.
Objective: The scaffold from the decellularized human pancreas (DP scaffold) seems to be the most suitable for creating a tissue-engineered pancreatic construct (TEPC). The efficiency of the decellularization is influenced by the physical and mechanical properties, structural characteristics, and composition of the extracellular matrix of native tissue. The aim of the study is to develop an optimal protocol for decellularization of the post-mortem donors pancreas with different morphological characteristics of the original tissue.
Methods: A tissue-specific DP scaffold was obtained as a result of a physicochemical methods combination of decellularization under various modes of processing of pancreatic tissue fragments, including cyclic repetition of freezing and thawing and the method of osmotic shock in two versions. The first option included incubation in a 0.1 % solution of sodium dodecyl sulfate (SDS) of low and high ionic strength; in the second protocol, the sequence of processing steps was changed - a 0.1 % solution of SDS of high and low ionic strength were used. The pancreas and the DP scaffold samples were subjected to histological analysis.
Results: Three types of pancreatic tissue samples were identified: pancreas with evident lipomatosis signs, pancreas with diffuse fibrosis, and pancreas without morphological signs of pathology. Histological analysis showed that the protocol with using freeze and thawing cycling is suitable only for the human pancreas with lipomatosis; the protocol with using osmotic shock is suitable for the pancreas with diffuse fibrosis and pancreas without pronounced signs of fibrosis and lipomatosis, but with a different sequence of processing steps.
Conclusions: To achieve complete decellularization of human pancreatic fragments and obtain a tissue-specific DP scaffold, the protocol for processing pancreatic tissue must be correlated with the morphological features of the native tissue.
P68
ULTRASOUND STIMULATION OF PIEZOELECTRIC HYDROGELS BOOSTS CHONDROGENIC DIFFERENTIATION OF HUMAN ADIPOSE TISSUE-DERIVED STROMAL CELLS
L. Ricotti1,2, D. Trucco1,2,3, L. Vannozzi1,2, A. Cafarelli1,2, C. Manferdini3, E. Gabusi3, P. Dolzani3, Y. Saleh3, M. Columbaro4, G. Lisignoli3
1The BioRobotics Institute, Scuola Superiore Sant’Anna, Pisa, Italy. 2Department of Excellence in Robotics and AI, Scuola Superiore Sant’Anna, Pisa, Italy. 3SC Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, Istituto Ortopedico Rizzoli, Bologna, Italy. 4Piattaforma di Microscopia Elettronica, Istituto Ortopedico Rizzoli, Bologna, Italy.
Objectives: In this study we investigated the chondrogenic differentiation of adipose tissue-derived stromal cells (ASCs) embedded in a novel hydrogel doped with graphene oxide (GO) nanoflakes and piezoelectric barium titanate nanoparticles (BTNPs), periodically stimulated with low-intensity pulsed ultrasound, using a dedicated set-up enabling a precise control of the energy dose at the target.
Methods: ASCs at 2x106 cells/mL were embedded in 3D VitroGel RGD® hydrogels (TheWell Bioscience), without nanomaterials (controls) or doped with 25 µg/mL of GO nanoflakes and 50 µg/mL of BTNPs.
Hydrogel properties were tested in terms of: (1) rheological properties and printability; (2) injectability; (3) adhesion force to the cartilage tissue, performed on ex vivo bovine cartilage samples; (4) degradation time in PBS, PBS + lysozyme (120 µg/mL to simulate an inflamed environment) and in synthetic synovial fluid.
The hydrogels were exposed to ultrasound stimulation (1 MHz, 250 mW/cm2, duty cycle: 20 %, pulse repetition frequency: 1 kHz, stimulation time: 5 min) every 2 days, until day 10 of culture. Samples were chondrogenically differentiated for 2, 10 and 28 days. At each time point cell viability (Live and Dead), cytotoxicity (LDH), gene expression of collagen type 2 (COL2), aggrecan (ACAN), SOX9, and collagen type 1 (COL1), electron microscopy, histology, and immunohistochemistry (COL2, aggrecan, SOX9, and COL1) were evaluated.
Results: Rheometric measurements highlighted a shear-thinning behavior. However, the hydrogel resulted poorly printable. Injectability was good, with force values compatible with ISO 7886-1:2018. Interestingly, the nanomaterials increased considerably the hydrogel adhesion to the cartilage tissue. The hydrogel completely degraded in 3 months. Ultrasound stimulation considerably boosted the chondrogenic differentiation of ASCs laden in 3D piezoelectric hydrogel: COL2, aggrecan and SOX9 were considerably overexpressed, while the fibrotic marker COL1 decreased, compared to control samples (non piezoelectric hydrogels and piezoelectric hydrogels not stimulated with LIPUS).
Conclusions: These results suggest that the ultrasound stimulation of ASCs encapsulated in piezoelectric hydrogels represents an exciting paradigm for cartilage regeneration.
P69
THE EFFECT OF SMOOTH MUSCLE CELLS ON THE VIABILITY OF ENDOTHELIAL CELLS CULTURED IN THE MEDIUM WITH HIGH AND VARIABLE GLUCOSE
A. Ciechanowska, I.M. Gora, S. Sabalinska, P. Ladyzynski
Nalecz Institute of Biocybernetics and Biomedical Engineering PAS, Warsaw, Poland.
Objectives: The aim of the study was to investigate the effect of human umbilical artery smooth muscle cells (HUASMCs) on the viability of co-cultured human umbilical vein endothelial cells (HUVECs) depending on the concentration of glucose in the culture medium.
Methods: The cultures were conducted on 2 cm2 semi-permeable flat polysulfone membranes coated with fibronectin, immobilized in self-made inserts. The insert contained either HUVECs on a single membrane or HUASMCs and HUVECs on two parallel membranes located close to each other. Cultures were conducted for 7 or 14 days in the culture medium containing glucose in normal (5 mmol/L), high (20 mmol/L) or variable (alternately 5 or 20 mmol/L) concentration. The culture medium was changed daily. The flow cytometry analysis was used to investigate the cell apoptosis (Annexin V and 7-AAD staining), mitochondrial membrane potential (JC-1 staining), and the production of reactive oxygen species (ROS) (DCFDA staining).
Results: After 7 and 14 days of culturing in the medium with H glucose concentration the percentage of apoptotic HUVECs co-cultured with HUASMCs was lower than in cultures in medium with H/N glucose concentration (after 7 days: 14.3 % ± 0.8 % vs. 21.8 % ± 1.0 %, p = 0.001, and after 14 days: 9.1 % ± 0.8 %, vs. 16.4 % ± 0.8 %, p < 0.0001). This result was in agreement with the results of the mitochondrial membrane potential and ROS analyses, although the differences of these parameters were not significant.
Conclusions: In co-cultures of HUVECs and HUASMCs variable glucose concentration has a more negative effect on HUVEC viability than constant high glucose concentration. This effect of glucose concentration is qualitatively similar to the one observed in cultures of HUVECs without HUASMCs
P70
DEVELOPMENT OF A DYNAMIC BIOREACTOR TO CULTURE OVARIAN CORTICAL TISSUE UNDER SUITABLE OXYGEN CONCENTRATIONS AND MECHANICAL CHALLENGES
G. Fragomeni1, L. De Napoli2, M. Di Nardo3, V. Barbato3, V. Genovese3, V. De Gregorio3, A. Travaglione3, R. Gualtieri3, R. Talevi3, G. Catapano2
1Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy. 2Department of Mechanical, Energy and Management Engineering, University of Calabria, Rende, Italy. 3Department of Biology, University of Napoli Federico II, Napoli, Italy.
Objectives: Cryopreservation of ovarian tissue strips followed by thawing, in vitro culture until the antral stage, in vitro oocyte maturation, fertilization and embryo transfer is a promising strategy to exploit fertility potential of young women with ovarian malignancies or premature ovarian failure. In in vitro batch culture of ovarian tissue, it is difficult to keep follicles viable and guide their progression to the secondary stage. Recent evidence suggests that optimal perifollicular dissolved oxygen concentration and tissue mechanical deformation may foster follicle viability and progression. Herein, we report on the development of dynamic bioreactors to culture ovarian tissue under suitable O2 concentrations and mechanical challenges.
Methods: Digital representations of feasible dynamic bioreactor designs varying by conformation and geometry were prepared with state-of-the-art CAD software. CFD 3D models of medium flow were used to optimize geometry and operation of such bioreactors. Prototypes of promising bioreactors were manufactured by 3D SLA additive manufacturing. Bovine ovaries harvested from animals aged 8 - 24 months were cut in strips 1 x 1 mm2 large, and 0.5 mm thick. 10 strips were loaded in bioreactor prototypes and were cultured for 7 d feeding 4 mL/min medium equilibrated with air under 10 % compressive deformation. Controls were static dish cultures. Follicle staging and health were assessed histologically. Follicle viability was assessed by labeling with live-dead far red and propidium iodide, and confocal laser scanning microscopy.
Results: In preliminary dynamic bioreactor cultures, the combined effect of enhanced oxygen supply to follicles, due to disruption of the stagnant medium layer around the strips, and mechanical cues exerted on strips, such as compressive deformation and shear stresses, preserved follicle viability and promoted their progression to the secondary stage better than static culture.
Conclusions: Ovarian tissue culture in dynamic bioreactors is promising for multistep in vitro folliculogenesis provided that results are confirmed in larger trials and human tissue.
P71
NEW HYDROGELS FOR LIVER ORGANOIDS
S. Leal Marin1,2, M. Fernandez3, O. Gryshkov1,2, B. Glasmacher1,2, P. Baptista3,4
1Institute for Multiphase Processes, Leibniz University Hannover, Garbsen, Germany. 2Lower Saxony Center for Biomedical Engineering, Implant Research and Development, Hannover, Germany. 3Instituto de Investigación Sanitaria Aragón (IIS Aragón), Zaragoza, Spain. 4Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Zaragoza, Spain.
Objectives: Extracellular matrix (ECM) and different polymers have been explored as hydrogels to replace the use of Matrigel in 3D culture of Organoids. In this study, ECM components obtained from the human amniotic membrane (hAM) were mixed with fibrinogen and thrombin to obtain a suitable hydrogel for liver organoids.
Methods: hAM was digested to obtain an extract of the ECM components. The extract was mixed in different concentrations with hyaluronic acid, fibrinogen, and thrombin to obtain a hydrogel. Liver organoids from progenitor cells isolated from liver tissues were previously grown in Matrigel drops. The drops were digested, and the resulting pellet was mixed with the hydrogel and seeded in droplets on cell culture wells. After crosslinking, the droplets were cultured for 7 days. The expansion was tracked by optical microscopy. Past 7 days the organoid composition was mapped by Raman spectroscopy.
Results: The hydrogel was stable for cell culture and expansion of liver organoids. Raman spectroscopy allowed the identification of different components like lipids and DNA present in the hydrogel drops.
Conclusions: Future work will be performed to characterize the mechanical properties and degradation kinetics of the hydrogel as well as the differentiation of the organoids in these hydrogels as compared to Matrigel.
P72
QUICK AND RELIABLE METHOD FOR ESTIMATION OF THE QUALITY OF THE LIVER ISOLATE INTENDED FOR CULTURING IN THE HOLLOW FIBER FLOW BIOREACTOR
M.J. Wiśniewska, M. Jakubowska, A. Wencel, D.G. Pijanowska, K.D. Pluta
Hybrid and Analytical Microbiosystems Department, Nałęcz Institute of Biocybernetics and Biomedical Engineering Polish Academy of Sciences, Warsaw, Poland.
Objectives: Liver performs very important functions, but in liver failure its capabilities are limited. Currently, the only treatment is organ transplantation, but donor shortage is strong limitation of this therapy. One of the alternatives, most promising therapies for liver failure is the use of Bioartificial Liver devices, BALs. For these hybrid systems the best source of cells seems to be human isolated hepatocytes.
Methods: The two-step collagenase perfusion procedure is considered a golden standard for hepatocytes isolation. However, we have elaborated the isolation method based on mechanical defragmentation and enzymatic digestion of a liver tissue. This method is faster and less stressful for cells. We can isolate whole spectrum of liver cells, which is important to ensure good co-culture conditions for hepatocytes. The method for estimation of the preparation quality is based on microscopic evaluation of the isolates and their ability to form islands of the polygonal-shaped cells. Successfully evaluated preparations can be then thawed and used for seeding our self-manufactured hollow-fiber bioreactors.
Results: Results obtained from immunostaining, flow cytometry, and ELISA test showed that some of the isolated cells could produce albumin, a characteristic protein for hepatocytes. Also, we observed that 8 days following the cells isolation hepatocyte-like cells appear in the standard culture. Our hepatocyte isolation and evaluation method allow us to fast check if biological material is valuable for us and suitable for further use in a flow bioreactor.
Conclusions: Our hepatocytes isolation and evaluation method is a quick technique to estimate the quality of the liver isolate. Positively evaluated preparations can be subsequently used for culturing in flow bioreactors which can be used in drug testing in personalized medicine or as the function block of BAL.
P73
GENETICALLY MODIFIED C3A CELLS PERFORMED BETTER IN DYNAMIC CULTURE THEN THEIR UNMODIFIED COUNTERPARTS
M. Jakubowska, M. Wiśniewska, A. Wencel, D.G. Pijanowska, K.D. Pluta
Hybrid and Analytical Microbiosystems Department, Nalecz Institute of Biocybernetics and Biomedical Engineering Polish Academy of Sciences, Warsaw, Poland.
Objectives: Liver diseases that lead to its failure are one of the most frequent causes of death worldwide. Currently, transplantation is the only cure for liver insufficiency or failure. Unfortunately, donor shortage is a main problem of this therapy. One of the most promising alternatives for liver transplantation are bioartificial livers (BALs). These hybrid devices might constitute a bridging therapy for patients waiting for the transplantation. The most advanced one is ELAD, which came to the third phase of clinical trials. However, ELAD did not live up to expectations. The device utilizes the C3A cell line, which has non-functional urea cycle (lack of ARG1 and OTC genes expression) and this could be a reason for its clinical trials failure.
Methods: In order to restore urea cycle we conducted genetic modification of C3A cells using lentiviral vectors. We proposed two different approaches toward genetic modification - missing genes were carried on by the same lentiviral vector or on two different vectors. After establishment of the new cell lines we characterized them in stationary and flow culture and compared to unmodified C3A cells. Dynamic conditions were ensured by flow system constructed in our laboratory, based on polysulfone hollow fiber bioreactors. During the experiment, the most crucial parameters such as cell viability, glucose consumption, albumin and urea production were measured and then compared.
Results: Obtained results indicated that modified cell line produced more albumin and urea. What is more, genetically modified cells had higher proliferation rate and are more metabolically active (increased glucose consumption). Importantly, comparison of stationary and dynamic culture conditions showed that flow culture ensured better environment for hepatoma cells growth.
Conclusions: Summarizing, genetically modified cells outperformed their unmodified counterparts. Therefore, they could be used as a biologically active block of improved BAL devices.
P74
IN VITRO INVESTIGATION OF 3D ELECTROSPUN AND PRINTED SCAFFOLDS FOR BIOMIMETIC PROGRAMING OF BONE REPLACING ARTIFICIAL TISSUES
D.V. Portan1, D. Polyzos2, D.D. Deligianni1
1Laboratory of Biomechanics and Biomedical Engineering, Dept. of Mechanical Engineering and Aeronautics, University of Patras, Patras, Greece. 2Laboratory of Applied Mechanics and Vibrations, Dept. of Mechanical Engineering and Aeronautics, University of Patras, Patras, Greece.
Objectives: The synthesis of multi-functional biomaterials with enhanced mechanical properties for the replacement of damaged bone or skin is still a challenge due to the mismatch between the properties of the natural tissues vs. the artificial ones. While many biologic solutions are proposed to boost up cells’ response, such as coatings with growth factors, the replacement of metallic orthopedic implants is still not feasible. This investigation allowed to better understand the impact of bone micro-environment on materials with the purpose to achieve a biomimetic design of novel 3D bone replacing scaffolds.
Methods: Semi-empirical modeling has been used to evaluate the type of interaction between several synthetic biomaterials and osseous tissues. Several categories of 3D scaffolds were manufactured from PLA and PCL thermoplastic matrices reinforced with electrically conductive phases, by electrospinning and 3D printing technologies. The mechanical performance of the 3D scaffolds, the biodegradability mode and their electrical conductivity have been evaluated. Processes and key markers indicating primary bone cells appropriate development, morphology, and viability when these were in contact with flat surface and with 3D scaffolds, were investigated.
Results: Modeling results allowed the biomimetic design of 3D structures with optimum properties such as: appropriate mechanical performance, adjusted electrical conductivity and improved geometry. Primary cells have proven to be extremely sensitive and selective in relation to the substrate. The geometry and the nano-level of the electrospun substrates enabled a rapid migration of cells between the fibers. However, the 3D printed scaffolds are considerably superior with respect to their mechanical properties.
Conclusions: Biomimetic programming of biomaterials features consists in constructing multi-layered materials with gradient properties similar to the natural tissues. For artificial bone tissues, 3D printed gradient scaffolds show promising features from all perspectives.
Acknowledgements: To the ‘Hellenic Foundation for Research and Innovation (HFRI), Project Acronym: COMPACT, Project no. 2060.
P75
DESIGN OF A 3D SYSTEM FOR THE MIXED CULTURE OF CANCER-ASSOCIATED FIBROBLASTS (CAFS) AND TUMOR CELLS
L. Milian1,2, I. Monleón1,2, P. Martinez-Vallejo1,2, Z. Oguir1,2, M. Sancho-Tello1,2, J.J. Martín de Llano1,2, C. Carda1,2,3, M. Mata1,2,4
1Department of Pathology, Faculty of Medicine and Odontology, University of Valencia, Valencia, Spain. 2INCLIVA, Valencia, Spain. 3Biomedical Research Networking Center on Bioengineering, Biomaterials and Nanomedicina (CIBER-BBN), Valencia, Spain. 4Biomedical Research Networking Center of Respiratory Diseases (CIBERES), Madrid, Spain.
Objectives: CAFs are a key component of the tumor microenvironment in lung cancer. We propose to generate organoids composed of CAFs and tumor cells and culture them in a 3D-system generated from collagen hydrogels, which allows us to evaluate the growth, dissemination and phenotypic changes that occur because of the interaction of these cell types.
Methods: CAFs and A549 cells were cultured, and suspensions of 150,000 cells/mL were generated, according to the following ratios: 100 % A549, 25 % A549 and 75 % CAFs and 100 % CAFs. Cell suspensions were cultured according to the drop slope method for 72 h. The organoids generated were collected and cultured in type I collagen (4 mg/mL) hydrogels drops for 72 h. The size and morphology of the organoids were evaluated every 24 h, using a phase contrast microscope. Organoid and cells morphology was evaluated by fluorescent F-actin staining using phalloidin-rhodamine. Vimentin and pankeratin were evaluated by immunofluorescence. The relative gene expression of CDH1, CDH2 and VIM was evaluated by real-time RT-PCR.
Results: In organoids generated with 100 % CAFs, we observed a rapid dispersion of cells with a migratory phenotype that escaped from the organoid and colonized the entire collagen scaffold. In contrast, those organoids generated with 100 % A549 cells, this migratory profile was not observed. We also did not observe notable variations in the size of the organoid. In the organoids generated with both cell types, we observed, on the one hand, a notable increase in size, and on the other, the appearance of a population of migratory cells located on the periphery of the organoid, appearing cells with fibroblastic morphology and others with a more epithelioid morphology.
Conclusions: This model represents a suitable system to evaluate the relationship and interaction between different cell types that make up organoids, valuable for the study of lung cancer.
P76
VASCULARIZATION OF 3D-PCL SCAFFOLDS FOR CARTILAGE REGENERATION
L. Milian1,2, I. Peregrín1,2, M. Sancho-Tello1,2, M. Monleón3, M. Oliver-Ferrándiz1,2, J.J. Martín de Llano1,2, Z. Oguir1,2, C. Carda1,2,4, M. Mata1,2,5
1Department of Pathology, Faculty of Medicine and Odontology, University of Valencia, Valencia, Spain. 2INCLIVA, Valencia, Spain. 3Centro de Biomateriales e Ingeniería Tisular (CBIT), University Politechnic of Valencia, Valencia, Spain. 4Biomedical Research Networking Center on Bioengineering, Biomaterials and Nanomedicina (CIBER-BBN). Valencia, Spain. 5Biomedical Research Networking Center of Respiratory Diseases (CIBERES), Madrid, Spain.
Objectives: As occurs in the regeneration of the airway (e.g.), it is often necessary to replace large segments of organs that have been damaged by different causes, including cancer. In these cases, the vascularization of the substitutes to be implanted determines their success or failure. We have recently reported the potential use of 3D PCL scaffolds in combination with alginate and chondrocytes, for cartilage regeneration. We have observed that electromagnetic stimulation improved the chondrogenic properties of these scaffolds in vitro. To study vascularization, 3D-PCL scaffolds were manufactured and implanted in athymic nude mice.
Methods: PCL scaffolds were prepared by a mixed particle leaching/freeze extraction process. The following experimental groups were considered: PCL (control group), PCL + alginate, PCL + chondrocytes precultured in proliferation or differentiation medium, and PCL + alginate + chondrocytes precultured in proliferation or differentiation medium. Ferrite microparticles were also included in the scaffold to study the effect of electromagnetic stimulation. These constructs were implanted subcutaneously, on the back of nude mice. Three weeks after the implantation, a standard histopathological evaluation of the scaffolds was performed, and angiogenesis was assessed by CD31 immunohistochemistry. Vascularization was evaluated based on the total number of vessels detected in the scaffold and their size, considering large (> 20 µm), medium (10 - 20 µm), and small (< 10 µm) vessels.
Results: We did not detect any difference in relation to the content of alginate, nor that of chondrocytes. Those scaffolds precultured with differentiation medium had a greater number of vessels that were smaller in size than those precultured with proliferation medium. Regarding irradiation, we observed that the electromagnetically stimulated scaffolds had a higher percentage of medium and small caliber vessels than the non-stimulated ones.
Conclusions: Electromagnetic stimulation in combination with preculture in chondrogenic differentiation medium could improve vascularization of PCL scaffolds.
P77
MICROFLUIDIC DEVICE USING RAPID DRUG GRADIENT FORMATION FOR BACTERIAL RESISTANCE PREDICTION
S. Becke1, G. Gabel1, M. Lommel1, A. Hujeirat1,2, D. Roizman2, U. Kertzscher1, J. Rolff2
1Biofluid Mechanics Laboratory, Institute of Computer-assisted Cardiovascular Medicine, Charité - Universitätsmedizin Berlin, Berlin, Germany. 2Evolutionary Biology, Freie Universität Berlin, Berlin, Germany.
Objectives: Antibiotic resistance is one of the leading global health issues, predicted to kill more people in 2050 than cancer. Current measures to tackle antibiotic resistance include prudent use, new drug development, and rapid diagnostics. While these are all necessary, they are not sufficient. In contrast, the proposed microfluidic device uses rapid antibiotic gradient formation to predict the dynamics of resistance evolution before antibiotics are used.
Methods: Bacterial resistance evolves much faster when the bacterial population is exposed to a concentration gradient, and resistance can evolve even at very low doses due to high selection pressure. The microfluidic system therefore utilizes convective and diffusive mass transfer mechanisms for the rapid formation of antibiotic gradients. Within a convection unit, antibiotics, nutrient media and diluents are mixed in a porous medium, and thus steady-state gradients can be formed within minutes. The diffusion unit consists of an agar culture medium. Antibiotic gradients diffuse into the culture medium through a contact surface. This causes exposure of the bacterial population to all possible antibiotic concentration gradations or even combinations of two antibiotics. Evolutionary adaptation is continuously observed and allows immediate assessment of the sustainability of an antibiotic strategy.
Results: The microfluidic approach allows the generation of stable antibiotic gradients in a culture medium within hours. Gradient formation is numerically simulated with computational fluid dynamics and visualized and validated by fluorescent tracer fluids. Experiments demonstrated resistance evolution of E. coli MG1655 against Ampicillin within the microfluidic device.
Conclusions: While the proposed method showed promising results in preliminary experiments using model organisms, further validation experiments using different bacterial strains and antibiotics need to be performed. In addition, it is planned to dynamically change the gradients within the microfluidics, to allow in vitro simulation of pharmacokinetics within the human body.
P78
MODELING NAFLD USING HUMAN-INDUCED PLURIPOTENT STEM CELL AND ORGAN-ON-CHIP TECHNOLOGY
L. Morisseau1, R. Jellali1, A. Abderrahmani2, V. Pawlowski2, C. Legallais1, Y. Sakai3, E. Leclerc1,4
1Centre de Royallieu, CNRS, BMBI, Université de Technologie de Compiègne, Compiègne, France. 2Université de Lille, CNRS, Centrale Lille, Univ. Polytech. Hauts-de-France, Lille, France. 3Department of Chemical System Engineering, University of Tokyo, Japan. 4IRL 2820 LIMMS/CNRS, Institute of Industrial Science, University of Tokyo, Japan.
Objectives: Nonalcoholic fatty liver disease (NAFLD) is a metabolic and progressive disease whose incidence has dramatically increased in recent years with the rise of obesity. It is characterized by the process ranging from simple steatosis to fibrosis. However, a deep understanding of NAFLD progression remains challenging due to the lack of relevant in vitro human disease models. In this work, we investigated the development of a NAFLD model using hepatocytes-like cells (HLC) derived from human-induced pluripotent stem cells (hiPSCs) and organ-on-chip technology.
Methods: Our protocol consists in a step-by-step differentiation process from definitive endoderm (S1), to hepatic specification (S2), followed by a hepatic progenitor step (S3) and finally reaching HLC (S4). hiPSCs were cultivated on static condition in standard Petri dishes until S3, then detached and transferred in a microsystem for S4-maturation. We used a PDMS-based liver-on-chip model designed to ensure the dynamic culture of the hepatic cells with an optimal shear stress. Steatosis was induced by free fatty acid exposures (oleic acid). HLC differentiation and functionalities was assessed by staining or/and measuring the levels of albumin and urea. In addition, mRNA levels of hepatic differentiation genes and steatosis markers were also investigated. Finally, the steatosis state of HLC was evaluated by staining and measuring cytoplasmic lipid droplets.
Results: Our results showed cells with typical cuboidal hepatic phenotypes at differentiation end point. Albumin and urea detection demonstrated hepatic profile and basal functionality of HLC. Intracellular lipid droplets were observed in cells, which is a relevant symptom in fatty liver disease. Furthermore, NAFLD biomarkers kinetics are currently undergoing using metabolomic profiling and mass spectrometry.
Conclusions: The present study successfully proposes a liver organ-on-chip model based on hiPSCs. The hepatic differentiation toward HLC was confirmed and the steatosis assays revealed the potential of our protocol to generate a liver-like disease model.
P79
ELECTROSPUN BEADS-ON-STRING IN THICK HONEYCOMB SCAFFOLD FOR BONE TISSUE ENGINEERING
N. Rivoallan1,2, T. Baudequin1, M. Müller2, P. Vigneron1, A. Hébraud3, R. Jellali1, G. Schlatter3, C. Legallais1, B. Glasmacher2
1CNRS, UMR 7338 Biomechanics and Bioengineering, Sorbonne Universités, Université de Technologie de Compiégne, Compiégne, France. 2Institute for Multiphase Processes, Leibniz University Hannover, Hannover, Germany. 3ICPEES UMR 7515, Institut de Chimie et Procédés pour l’Energie, l’Environnement et la Santé, CNRS, Université de Strasbourg, Strasbourg, France.
Objectives: Thick honeycomb-like scaffolds with nanoparticles of hydroxyapatite (NP-HA) are known to promote proliferation and differentiation of MSCs C3H10T1/2 cells as osteoblasts [1]. In addition, the presence of NP-HA is necessary to produce thicker structures without losing the pattern. An alternative to achieve correct thickness is electrospinning of beads-on-string fibers to ensure space between the different layers [2]. However, the effects of the beads on the mechanical and biological properties are not well characterized.
Methods: Solutions of 10 and 12 % wt/v polycaprolactone (PCL) are used to produce fibers with/without beads, respectively. To microstructure the scaffolds, a honeycomb pattern is placed on the collector of the electrospinning device. The NP-HA can be electrosprayed between different layers of the scaffolds. Elastic moduli are determined thanks to uniaxial tensile test. C3H10T1/2 cells are then seeded on the scaffolds without any differentiation factor during one week. The viability and proliferation are evaluated by Live/Dead kit, MTT assay and the early differentiation by ALP Activity. Moreover, the scaffolds and the cells are observed on SEM.
Results: The production of beads-on-string during electrospinning permitted the scaffold to reach well-defined honeycomb wells with a 100 µm equivalent diameter spaced by walls of 40 µm. The overall thickness of the constructs was larger than 50 µm without NP-HA. The elastic modulus was in the range of 5 - 10 MPa. Beads-on-string fibers did not alter viability but the presence of NP-HA promotes cell differentiation towards bone lineage.
Conclusions: Beads-on-string fibers permitted to increase the thickness of structure without using NP-HA. The use of those beads in the scaffold did not alter the cell behavior. It will now be possible to evaluate the role of the structure on the differentiation in bone lineage compared to the effect of the NP-HA themselves.
[1] A. Garcia Garcia et al. ACS Biomater. Sci. Eng. 2018, 4(9):3317-3326.
[2] S. Nedjari et al. Materials Letters, 2015, 142:180-183.
