Development of a dual-component infection-resistant arterial replacement for small-caliber reconstructions: A proof-of-concept study.

Introduction: Synthetic vascular grafts perform poorly in small-caliber (<6mm) anastomoses, due to intimal hyperplasia and thrombosis, whereas homografts are associated with limited availability and immunogenicity, and bioprostheses are prone to aneurysmal degeneration and calcification. Infection is another important limitation with vascular grafting. This study developed a dual-component graft for small-caliber reconstructions, comprising a decellularized tibial artery scaffold and an antibiotic-releasing, electrospun polycaprolactone (PCL)/polyethylene glycol (PEG) blend sleeve. Methods: The study investigated the effect of nucleases, as part of the decellularization technique, and two sterilization methods (peracetic acid and γ-irradiation), on the scaffold's biological and biomechanical integrity. It also investigated the effect of different PCL/PEG ratios on the antimicrobial, biological and biomechanical properties of the sleeves. Tibial arteries were decellularized using Triton X-100 and sodium-dodecyl-sulfate. Results: The scaffolds retained the general native histoarchitecture and biomechanics but were depleted of glycosaminoglycans. Sterilization with peracetic acid depleted collagen IV and produced ultrastructural changes in the collagen and elastic fibers. The two PCL/PEG ratios used (150:50 and 100:50) demonstrated differences in the structural, biomechanical and antimicrobial properties of the sleeves. Differences in the antimicrobial activity were also found between sleeves fabricated with antibiotics supplemented in the electrospinning solution, and sleeves soaked in antibiotics. Discussion: The study demonstrated the feasibility of fabricating a dual-component small-caliber graft, comprising a scaffold with sufficient biological and biomechanical functionality, and an electrospun PCL/PEG sleeve with tailored biomechanics and antibiotic release.

Hemodynamic Assessment of Hollow-Fiber Membrane Oxygenators Using Computational Fluid Dynamics in Heterogeneous Membrane Models.

Extracorporeal membrane oxygenation (ECMO) has been used clinically for more than 40 years as a bridge to transplantation, with hollow-fiber membrane (HFM) oxygenators gaining in popularity due to their high gas transfer and low flow resistance. In spite of the technological advances in ECMO devices, the inevitable contact of the perfused blood with the polymer hollow-fiber gas-exchange membrane, and the subsequent thrombus formation, limits their clinical usage to only 2-4 weeks. In addition, the inhomogeneous flow in the device can further enhance thrombus formation and limit gas-transport efficiency. Endothelialization of the blood contacting surfaces of ECMO devices offers a potential solution to their inherent thrombogenicity. However, abnormal shear stresses and inhomogeneous blood flow might affect the function and activation status of the seeded endothelial cells (ECs). In this study, the blood flow through two HFM oxygenators, including the commercially available iLA® MiniLung Petite Novalung (Xenios AG, Germany) and an experimental one for the rat animal model, was modeled using computational fluid dynamics (CFD), with a view to assessing the magnitude and distribution of the wall shear stress (WSS) on the hollow fibers and flow fields in the oxygenators. This work demonstrated significant inhomogeneity in the flow dynamics of both oxygenators, with regions of high hollow-fiber WSS and regions of stagnant flow, implying a variable flow-induced stimulation on seeded ECs and possible EC activation and damage in a biohybrid oxygenator setting.

Hypothermic preservation of endothelialized gas-exchange membranes.

Endothelialization of the blood contacting surfaces of blood-contacting medical devices, such as cardiovascular prostheses or biohybrid oxygenators, represents a plausible strategy for increasing their hemocompatibility. Nevertheless, isolation and expansion of autologous endothelial cells (ECs) usually requires multiple processing steps and time to obtain sufficient cell numbers. This excludes endothelialization from application in acute situations. Off-the-shelf availability of cell-seeded biohybrid devices could be potentially facilitated by hypothermic storage. In this study, the survival of cord-blood-derived endothelial colony forming cells (ECFCs) that were seeded onto polymethylpentene (PMP) gas-exchange membranes and stored for up to 2 weeks in different commercially available and commonly used preservation media was measured. While storage at 4°C in normal growth medium (EGM-2) for 3 days resulted in massive disruption of the ECFC monolayer and a significant decline in viability, ECFC monolayers preserved in Chillprotec could recover after up to 14 days with negligible effects on their integrity and viability. ECFC monolayers preserved in Celsior, HTS-FRS, or Rokepie medium showed a significant decrease in viability after 7 days or longer periods. These results demonstrated the feasibility of hypothermic preservation of ECFC monolayers on gas-exchange membranes for up to 2 weeks, with potential application on the preservation of pre-endothelialized oxygenators and further biohybrid cardiovascular devices.

Vitrified Human Umbilical Arteries as Potential Grafts for Vascular Tissue Engineering.

BACKGROUND: The development of a biological based small diameter vascular graft (d < 6 mm), that can be properly stored over a long time period at - 196 °C, in order to directly be used to the patients, still remains a challenge. In this study the decellularized umbilical arteries (UAs) where vitrified, evaluated their composition and implanted to a porcine model, thus serving as vascular graft. METHODS: Human UAs were decellularized using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) detergents. Then, vitrified with vitrification solution 55 (VS55) solution, remained for 6 months in liquid nitrogen and their extracellular matrix composition was compared to conventionally cryopreserved UAs. Additionally, total hydroxyproline, sulphated glycosaminoglycan and DNA content were quantified in all samples. Finally, the vitrified umbilical arteries implanted as common carotid artery interposition graft to a porcine animal model. RESULTS: Decellularized and vitrified UAs characterized by proper preservation of extracellular matrix proteins and tissue architecture, whereas conventionally cryopreserved samples exhibited a disorganized structure. Total hydroxyproline content was preserved, although sulphated glycosaminoglycan and DNA contents presented significantly alterations in all samples. Implanted UAs successfully recellularized and remodeled as indicated by the histological analysis. CONCLUSION: Decellularized and vitrified UAs retained their structure function properties and can be possible used as an alternative source for readily accessible small diameter vascular grafts.

Effect of 'in air' freezing on post-thaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds.

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me2SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs. Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me2SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen 'in air' and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me2SO alone (60%). No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of 'ready-to-use' TECs.

Biocompatibility and Immunogenicity of Decellularized Allogeneic Aorta in the Orthotopic Rat Model.

IMPACT STATEMENT: The generation of a small-caliber arterial graft, utilizing a large vessel of a small animal, such as the aorta of the rat or rabbit, for clinical use in the peripheral arterial tree, can widen the options for arterial prostheses. This in vivo study demonstrated the ability of the decellularization protocol that was used to produce a noncytotoxic acellular small-caliber arterial graft, with sufficient biomechanical and biological integrity to withstand the demanding flow and pressure environment of the rat aorta. This work also demonstrated the superiority of the decellularized homograft over its intact counterpart, in terms of lower immunogenicity.

Use of sucrose to diminish pore formation in freeze-dried heart valves.

Freeze-dried storage of decellularized heart valves provides easy storage and transport for clinical use. Freeze-drying without protectants, however, results in a disrupted histoarchitecture after rehydration. In this study, heart valves were incubated in solutions of various sucrose concentrations and subsequently freeze-dried. Porosity of rehydrated valves was determined from histological images. In the absence of sucrose, freeze-dried valves were shown to have pores after rehydration in the cusp, artery and muscle sections. Use of sucrose reduced pore formation in a dose-dependent manner, and pretreatment of the valves in a 40% (w/v) sucrose solution prior to freeze-drying was found to be sufficient to completely diminish pore formation. The presence of pores in freeze-dried valves was found to coincide with altered biomechanical characteristics, whereas biomechanical parameters of valves freeze-dried with enough sucrose were not significantly different from those of valves not exposed to freeze-drying. Multiphoton imaging, Fourier transform infrared spectroscopy, and differential scanning calorimetry studies revealed that matrix proteins (i.e. collagen and elastin) were not affected by freeze-drying.

Endothelialization and characterization of titanium dioxide-coated gas-exchange membranes for application in the bioartificial lung.

Fouling on the gas-exchange hollow-fiber membrane (HFM) of extracorporeal membrane oxygenation (ECMO) devices by blood components and pathogens represents the major hurdle to their long-term application in patients with lung deficiency or unstable hemodynamics. Although patients are treated with anticoagulants, deposition of blood proteins onto the membrane surface may still occur after few days, leading to insufficient gas transfer and, consequently, to device failure. The aim of this study was to establish an endothelial cell (EC) monolayer onto the gas-exchange membrane of an ECMO device with a view to developing a hemocompatible bioartificial lung. Poly(4-methyl-1-pentene) (PMP) gas-exchange membranes were coated with titanium dioxide (TiO2), using the pulsed vacuum cathodic arc plasma deposition (PVCAPD) technique, in order to generate a stable interlayer, enabling cell adhesion onto the strongly hydrophobic PMP membrane. The TiO2 coating reduced the oxygen transfer rate (OTR) of the membrane by 22%, and it successfully mediated EC attachment. The adhered ECs formed a confluent monolayer, which retained a non-thrombogenic state and showed cell-to-cell, as well as cell-to-substrate contacts. The established monolayer was able to withstand physiological shear stress and possessed a "self-healing" capacity at areas of induced monolayer disruption. The study demonstrated that the TiO2 coating mediated EC attachment and the establishment of a functional EC monolayer. STATEMENT OF SIGNIFICANCE: Surface endothelialization is considered an effective approach to achieve complete hamocompatibility of blood-contacting devices. Several strategies to enable endothelial cell adhesion onto stents and vascular prostheses have already been described in the literature. However, only few studies investigated the feasibility of establishing an endothelial monolayer onto the gas exchange membrane of ECMO devices, using peptides or proteins that were weakly adsorbed via dip coating techniques. This study demonstrated the effectiveness of an alternative and stable titanium dioxide coating for gas-exchange membranes, which enabled the establishment of a confluent, functional and non-activated endothelial monolayer, while maintaining oxygen permeability.

Developing a biohybrid lung - sufficient endothelialization of poly-4-methly-1-pentene gas exchange hollow-fiber membranes.

Working towards establishing a biohybrid lung with optimized hemocompatibility, this study analyzed the feasibility of establishing flow-resistant endothelium on heparin/albumin coated poly-4-methly-1-pentene hollow fiber gas exchange membranes (PMP-HFs). The seeding efficiency and proliferation of human cord blood derived endothelial cells (HCBEC) on PMP-HFs were analyzed under static conditions by WST-8 cell proliferation assay and fluorescence microscopy. The HCBEC monolayer integrity under different flow conditions was also assessed. Endothelial-specific phenotype verification, expression activation levels and thrombogenic state markers were quantified by real-time RT-PCR for cell-to-PMP-HF contact under static and dynamic conditions. The results demonstrated the feasibility of establishing a viable, confluent, and flow-resistant endothelial monolayer on the blood-contact surface of PMP-HFs, which maintained a physiological response to TNFα-stimulation and flow conditions. The endothelial phenotype, expression levels of adhesion molecules and thrombogenic state markers were unaffected by cell-to-PMP-HFs contact. These results represent a significant step towards establishing a biohybrid lung.