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The limited supply of autologous small-diameter vascular grafts remains a significant challenge in vascular surgery. Synthetic polymers are used for the production of larger prostheses but lack long-term hemocompatibility for application in small-diameter replacements. By tissue engineering of vascular implants from autologous fibrin we may be able to eliminate this bottleneck. Therefore, the aim of this in vitro study was to assess the hemocompatibility and the thrombotic response of fibrin-based small-caliber vascular grafts compared to commercially available synthetic grafts in accordance with ISO 10993-4.

Fibrin-based vascular prostheses with a diameter of 5 mm were generated using a previously established approach. For hemocompatibility testing, fibrin grafts with and without heparin coating and commercially available same-sized synthetic heparin coated prostheses (VascuGraft® FLOW, B.Braun, Germany) were exposed to whole blood from healthy human donors (n=8) in a dynamic circulation system for 4 h. Subsequently, the thrombotic response was analyzed via platelet count, while quantification of ß-Thromboglobulin, SC5b9 and TAT via ELISA was performed to assess the activation state of thrombocytes, the complement- and coagulation system in the Chandler loop setting. Furthermore, adhesion of thrombocytes was examined using Scanning Electron Microscopy (SEM).

Irrespective of the specimen, no thrombotic occlusions were detected during the experiments. No significant difference in the mean deviation from the initial thrombocyte concentration (233 ± 49.73 x103/µl) was observed between the fibrin and synthetic grafts. Preliminary examinations of thrombocyte and complement activation further suggest no substantial differences. Accordingly, SEM imaging revealed a uniformly dispersed adhesion of activated thrombocytes on all sample surfaces.

Heparin-free fibrin-based small-diameter vascular grafts exhibited similar humoral hemocompatibility compared to heparin-coated synthetic grafts. These findings mark an important step towards an increased availability of regenerative small-diameter vascular replacements for critical patients and unlock various opportunities in the production and clinical availability of further fibrin-based grafts for the use in vascular surgery in the future.

Synthetic vascular prostheses are associated with severe complications, including bacterial infections with biofilm formation and lack of patency due to insufficient hemo- and biocompatibility. Biohybrid prostheses could constitute a possibility to avoid these limitations by using off-the-shelf grafts with reduced immunogenic and inflammatory reactions and improved biocompatibility. Unprocessed fibrin matrices have insufficient biomechanical stability to serve as vascular substitutes for vessel replacement. To address this issue, we developed a method to coat 3D printed tubular scaffolds out of the biodegradable polymer polycaprolactone (PCL) with fibrin.

A meshed 3D printed PCL scaffold was placed into a custom made cylindrical vascular mold. Cryoprecipitated human fibrinogen was filled into the mold and cross-linked by deliberate coagulation using thrombin and Calcium. By inflation of a luminally inserted percutaneous angioplasty (PTA) balloon (Mustang™, Boston Scientific, USA) for 60 minutes, compaction of the graft wall was facilitated. The biomechanical analysis was performed using uniaxial longitudinal and circumferential tensile testing, three-dimensional burst- and compliance testing, and suture retention tests. All tests were conducted in accordance with DIN EN ISO 7198:2017-07.

With the procedure described above, vascular prostheses with a smooth luminal surface were manufactured with dimensions of 60 mm in length, 6 mm in luminal diameter and 2 mm wall thickness.
Compared to compacted and uncompacted fibrin vessel controls without inner wall scafold, biomechanical analyses confirmed that grafts with incorporated PCL scafolds exhibit significant improved biomechanical properties, matching those of native vessels.

Transluminal balloon compression of the fibrin coated 3D printed PCL scaffolds proved to be an efficient and reproducible method for significantly increasing the mechanical stability of biohybrid prosthesis. Biomechanical tests confirmed that the developed prosthesis could withstand even supraphysiological forces, making them applicable for translational and potentially clinical applications in vascular reconstructive surgery.

As the human cardiovascular system is built up by a hierarchical vascular network, regenerative approaches for the vascular system have to account for a wide range of different vessel sizes as well as various biomechanical and biochemical conditions. Emulation of these highly complex conditions in vitro requires sophisticated dynamic culture techniques. We here present an overview of the bioreactor systems and perfusion techniques used in our working group to emulate physiological as well as pathological conditions throughout the vascular tree.

A modular hemodynamic emulation system was established which could be adapted to simulate the physiological biomechanical conditions specific for the large- and small-diameter arteries as well as for the capillary microvascular network and peripheral and central venous vessels. For this, different pump modules, compliance modules, bioreactors and resistors were fabricated and arranged as required to emulate the desired physiological or pathological mechanical environment. With this system, both native and tissue engineered bioartificial vessels and tissues were exposed to the respective biomechanical forces present in the human body. Monitoring of flow, pressure, and oxygen saturation was facilitated by a multisensor monitoring system.

The bioreactor perfusion system facilitated accurate simulation of both, arterial and venous pressure and flow curves of different sites of the cardiovascular system. With that, the characteristic biomechanical environment of the aorta, large-diameter and small-diameter arteries were emulated. Further, peripheral and central venous pressure- and flow curves and the arteriovenous oxygen gradient of the microvascular and capillary circulation were simulated. Stimulation of cellularized bioartificial vessels and microvascular constructs resulted in physiological cell alignment in larger vessels and facilitated directed capillary network formation in the microvascular tissue.

The modular bioreactor perfusion system can accurately emulate a specific biomechanical environment of a target component of the cardiovascular system. Alternatively, by combining different components, it facilitates biomechanical simulation of the whole heirarchical vascular system on a desktop. With that, it can be used for regenerative approaches as well as for cardiovascular disease modelling or drug testing.

Tissue engineered vascular prostheses (TEVPs) are expected to improve clinical outcomes in patients requiring vascular replacement. Their successful realization demands sufficient in vitro testing that accurately mimics the physiological biomechanical environment of the human circulation system. The Organ Care System Heart (OCS™, Transmedics, Andover, USA), designed for ex vivo perfusion of donor organs, enables the standardized cultivation of biological three-dimensional constructs under defined biomechanical stimuli. In this proof-of-concept study, we assessed the suitability of the OCS for perfusion of bioartificial fibrin-based aortic TEVPs.

Bioartificial aortic grafts were produced using blood-derived compacted fibrin as a scaffold material and cellularized with human umbilical vein endothelial cells, adipogenic stem cells, and fibroblasts replicating the physiological dimensions and three-layered wall structure of the human aorta. The TEVP was mounted in a custom-built bioreactor and integrated into the OCS. Flow and pressure for dynamic perfusion were set to match physiological parameters, while oxygen and carbon dioxide saturation were maintained through an external gas supply during the five-day incubation period. Immunofluorescence imaging was performed to examine the wall structure as well as cell morphology and viability.

The perfusion of three-layered bioartificial vessels (n=3) in the OCS facilitated stable cultivation conditions throughout the perfusion period. Continuous monitoring confirmed a corporal systemic pressure (120/60 mmHg) with a physiological pressure waveform at a steady pulse frequency of 60 bpm under physiological pH, gas concentrations and temperature. Perfusion in the OCS facilitated the generation of a three-layered aortic prostheses with physiological cell morphology and alignment.

The OCS Heart is suitable for standardized in vitro perfusion of TEVPs, precisely imitating the intended mechanical and biochemical circulation conditions. Thus, it offers a promising approach for long-term maturation and evaluation of three-dimensional biomimetic tissue constructs by mimicking the physiological stimuli to enhance cellular activity and induce physiological aortic wall morphology. Thus, ex-vivo maturation in the OCS is a viable option to optimize the biomimetic properties of tissue engineered bioartificial aortic grafts.

Die in vitro Endothelialisierung von Gefässgrafts hat in zahlreichen Studien gezeigt, dass die Offenheitsrate von solchen Grafts signifikant erhöht werden kann. Tissue Engineering ist in der EU gesetzlich als Arzneimittelherstellung geregelt. Das bedeutet, dass solche Produkte nach dem pharmazeutischen Industriestandard, der als GMP bezeichnet wird, hergestellt werden müssen. Unsere Abteilung führte diese Methode schon die letzten 25 Jahre erfolgreich durch, musste aber ein neues Labor errichten um die neuen strengen gestzlichen Auflagen zu erfüllen. Seit September 2020 ist dieses Labor voll einsatzfähig und konnte, neben anderen Produkten, in vitro endothelialisierte Gefässbypässe herstellen.

Patienten mit OP Indikation aber ohne brauchbare Vena saphena magna kamen für die Methode in Frage. In einer ersten Operation wurde den Patienten ein kurzes Segment einer oberflächlichen Armvene entnommen. Daraus wurden die Endothelzellen isoliert und zu Massenkulturen hochgezüchtet. Damit wurden die PTFE Grafts konfluent beschichtet und anschliessend standardmässig implantiert.

82 endothelialisierte Grafts wurden implantiert. 27 Grafts wurden in fem-pop I, 5 Grafts in fem-pop II und 36 Grafts in fem pop III Postion implantiert. Weiters wurden 13 Grafts in kruraler Position implantiert. 1 Graft wurde als transversaler Bypass verwendet. 8 Grafts zeigten einen primären Verschluss im Beobachtungszeitraum. Es gab keine Graftbedingte Infektion. Die Offenheitsrate betrug 92% nach einem Jahr, 87% nach zwei und 84% nach 3 Jahren.

Die In vitro Endothelialisierung von Gefässgrafts erhöht die Offenenheitsrate von PTFE Grafts substantiell. Die Implantation kann standardmässig ohne weiteren Aufwand durchgeführt werden. Die Produktion in einem GMP Labor ist schnell und kontrolliert und bietet ein Maß an höchstmöglicher Sicherheit für ein qualitativ hochwertiges Produkt.