Metabolic assessment of therapies for the development of drug-eluting stents

Metabolic assessment of therapies for the development of drug-eluting stent

Biocompatibility and immunogenicity of decellularised allogeneic aorta in the orthotopic rat model

Background and aim of the study: Peripheral arterial disease causes blood vessel dysfunction that requires surgical intervention. Current surgical interventions employ synthetic or allogeneic vascular grafts, which offer biocompatible materials solutions that are not able to regenerate or grow with the patient. Decellularised scaffolds have gained significant momentum in the past few years, since they have the potential to regenerate in the patient. The aim of this study was to investigate the effects of modified decellularisation protocol on the biocompatibility and immunogenicity of allogeneic rat abdominal aorta in an orthotopic rat model. Methods: Native syngeneic Wistar (W) and allogeneic Dark Agouti (DA) aortas, together with decellularised allogeneic DA aortas, were assessed histologically, immunohistochemically and biomechanically. The immunogenicity of the untreated and decellularized syngeneic and allogeneic grafts was assessed in W rats, implanted orthotopically. Following implantation for 6 weeks, the grafts were explanted and assessed for the presence of T cells and macrophages by immunohistochemistry, and for their biomechanical integrity and histoarchitecture. Results: No obvious histoarchitectural differences were observed between the native W and DA aortas, with both presenting similar three-layered structures. Histological analysis of decellularized DA aortas did not reveal any remaining cells. Explanted native DA allografts showed media necrosis, partial elastic fibre degradation and adventitia thickening, as well as infiltration by lymphocytes (CD3+, CD4+) and macrophages (CD68+) in the adventitia. The explanted decellularized DA allografts indicated reduced immune injury compared to the explanted native DA allografts. The explanted native W syngeneic grafts showed a mild immune response, with an intact media and no lymphocyte infiltration. The explanted native DA allografts showed significantly lower collagen phase slope than the decellularized DA allografts prior implantation, and significantly higher thickness than the explanted decellularized DA allografts. Conclusions: The results indicated that the modified decellularization protocol did not affect significantly the mechanical and histological properties of the native DA rat aorta. Overall, the immune response was improved by decellularization. Native DA allografts induced an adverse immune response in W rats, whereas syngeneic W grafts showed good tissue integration

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

© 2018, The Author(s). 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

Identifying an optimal seeding protocol and endothelial cell substrate for biohybrid lung development

© 2018 John Wiley & Sons, Ltd. Several key prerequisites need to be fulfilled for the development of a biohybrid lung, which can offer an actual alternative to lung transplantation. A major aspect is an optimized haemocompatibility of the device's artificial surfaces via endothelial cell seeding. In this study, four different types of polymeric gas exchange hollow fibre membranes (HFMs) were analysed utilizing four different seeding protocols in order to identify the ideal combination for sufficient long-term endothelialization. Human cord blood-derived endothelial cells (HCBECs) were used for the endothelialization of polypropylene HFMs with two different pore sizes and poly-4-methyl-1-pentene HFMs, both with and without heparin/albumin coating. The qualitative and quantitative impact of four different rotational seeding protocols regarding long-term HFM endothelialization and the impact of inflammatory stimulation on the seeded HCBECs were examined by fluorescence microscopy, cell counting, and analysis of relative expression levels of activation, shear stress, and thrombogenic state markers. Optimized endothelial cell seeding and long-term cultivation were only achieved using heparin/albumin-coated poly-4-methyl-1-pentene HFMs, applying 24 hr of rotational speed at 1 rpm followed by 120 hr of static culture. Neither cell-to-HFM contact nor the rotational cultivation procedure showed an impact on the physiological anti-thrombogenic and anti-inflammatory HCBEC activation status. Additionally, the cells maintained their physiological responsiveness towards inflammatory stimulation. Rotational seeding strategies and a seamless heparin/albumin coating of the HFMs are crucial requirements for a sufficient and long-lasting endothelialization and thus a key element in the future development and in vivo application of the biohybrid lung

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. Endothelialisation 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 modelled using computational fluid dynamics (CFD), with a view to assessing the magnitude and distribution of the shear stress on the wall of 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 wall shear stress and regions of stagnant flow, implying both regions of increased flow-induced blood damage and a variable flow-induced stimulation on seeded ECs in a biohybrid setting

Consequences of ventricular tachyarrhythmia in patients with a left ventricular assist device: Live recording in the ICU

Consequences of ventricular tachyarrhythmia in patients with a left ventricular assist device: Live recording in the IC

Immunoprecipitation of immunogenic proteins.

<p>1 mg of dEAC_ord and dEAC_intens extracts were incubated with plasma from mice immunized with dEAC_ord (plasma_ord) and dEAC_intens (plasma_intens) overnight, immune complexes were precipitated with protein A agarose, separated by SDS-PAGE and coomassie-stained. Bands form the gel were excised as indicated by the brackets and submitted as samples S2–S9 to mass spectrometry.</p

Correlative volumetric visualization of the carotid wall for native EAC, dEAC_ord and dEAC_intens by Scanning Laser Optical Tomography (SLOT).

<p>SLOT (A–C) was performed in transmission mode displaying autofluorescence at 532 nm on tissue pieces of 1.5 cm in length from the indicated tissues and by Multi Photon Microscopy (D–F) with maximum intensity projections of axial cross sections of the indicated tissues representing the autofluorescence at 800 nm excitation wavelength. TA: tunica adventitia; TM: tunica media; TI: tunica intima.</p

Prevention of rejection of allogeneic endothelial cells in a biohybrid lung by silencing HLA-class I expression

Variability in Human Leukocyte Antigens (HLA) remains a hurdle to the application of allogeneic cellular products. Due to insufficient autologous endothelial cell harvesting for the biohybrid lung, allogeneic human cord blood derived endothelial cells (HCBEC) were used for the endothelialization of poly-4-methyl-1-pentene (PMP) gas exchange membranes. Therefore, HLA class I expression was silenced stably in HCBECs to prevent rejection. The capacity of HLA class I-silenced HCBEC to abrogate allogeneic immune responses, their functional properties and suitability for endothelialization of PMP membranes were investigated. Delivery of β2-microglobulin (β2m)-specific shRNAs reduced β2m mRNA levels by up to 90% and caused a knockdown of HLA class I expression by up to 85%. HLA-silenced HCBEC abrogated T-cell responses and escaped antibody-mediated complement-dependent cytotoxicity. The EC phenotype and cytokine secretion profiles between HLA-expressing or -silenced HCBEC remained unaltered. EC specific activation (e.g. ICAM) and thrombogenic markers (e.g. thrombomodulin) remained unaffected by HLA-silencing, but their expression was upregulated by TNFα-stimulation. Furthermore, HLA-silenced HCBECs showed high proliferation rates and built an EC monolayer onto PMP membranes. This study represents a new therapeutic concept in the field of cell and organ transplantation and may bring the bioartificial lung as an alternative to lung transplantation closer to reality

Effects of combined cryopreservation and decellularization on the biomechanical, structural and biochemical properties of porcine pulmonary heart valves

Non-fixed, decellularized allogeneic heart valve scaffolds seem to be the best choice for heart valve replacement, their availability, however, is quite limited. Cryopreservation could prolong their shelf-life, allowing for their ideal match to a recipient. In this study, porcine pulmonary valves were decellularized using detergents, either prior or after cryopreservation, and analyzed. Mechanical integrity was analyzed by uniaxial tensile testing, histoarchitecture by histological staining, and composition by DNA, collagen (hydroxyproline) and GAG (chondroitin sulfate) quantification. Residual sodium dodecyl sulfate (SDS) in the scaffold was quantified by applying a methylene blue activation assay (MBAS). Cryopreserved decellularized scaffolds (DC) and scaffolds that were decellularized after cryopreservation (CD) were compared to fresh valves (F), cryopreserved native valves (C), and decellularized only scaffolds (D). The E-modulus and tensile strength of decellularized (D) tissue showed no significant difference compared to DC and CD. The decellularization resulted in an overall reduction of DNA and GAG, with DC containing the lowest amount of GAGs. The DNA content in the valvular wall of the CD group was higher than in the D and DC groups. CD valves showed slightly more residual SDS than DC valves, which might be harmful to recipient cells. In conclusion, cryopreservation after decellularization was shown to be preferable over cryopreservation before decellularization. However, in vivo testing would be necessary to determine whether these differences are significant in biocompatibility or immunogenicity of the scaffold