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In order to provide an alternative treatment option to lung transplantation for patients with end-stage lung disease, we aim for the development of an implantable biohybrid lung (BHL), based on hollow fiber membrane (HFM) technology used in extracorporeal membrane oxygenators. Complete hemocompatibility of all blood contacting surfaces is crucial for long-lasting BHL durability and can be achieved by their endothelialization. Autologous endothelial cells (ECs) would be the ideal cell source, but their limited proliferation potential excludes them for this purpose. As induced pluripotent stem cell-derived ECs enable the generation of a large number of ECs, we assessed and compared their capacity to form a viable and confluent monolayer on HFM, while indicating physiologic EC-specific anti-thrombogenic and anti-inflammatory properties. ECs were generated from three different human iPSC lines, and seeded onto fibronectin-coated poly-4-methyl-1-pentene (PMP) HFM. Following phenotypical characterization, ECs were analyzed for their thrombogenic and inflammatory behavior with or without TNFα induction, using FACS and qRT-PCR. Complementary, leukocyte- and platelet adhesion assays were carried out. The capacity of the iPSC-ECs to reendothelialize cell-free monolayer areas was assessed in a scratch assay. ECs sourced from umbilical cord blood (hCBECs) were used as control. iPSC-derived ECs formed confluent monolayers on the HFM and showed the typical EC-phenotype by expression of VE-cadherin and collagen-IV. A low protein and gene expression level of E-selectin and tissue factor was detected for all iPSC-ECs and the hCBECs, while a strong upregulation of these markers was noted upon stimulation with TNFα. This was in line with the physiological and strong induction of leukocyte adhesion detected after treatment with TNFα, iPSC-EC and hCBEC monolayers were capable of reducing thrombocyte adhesion and repopulating scratched areas. iPSCs offer the possibility to provide patient-specific ECs in abundant numbers needed to cover all blood contacting surfaces of the BHL with a viable, non-thrombogenic and non-inflammatory monolayer. iPSC-EC clones can differ in terms of their reendothelialization rate, and pro-inflammatory response. However, a less profound inflammatory response may even be advantageous for BHL application. With the proven ability of the seeded iPSC-ECs to reduce thrombocyte adhesion, we expect that thrombotic events that could lead to BHL occlusion can be avoided, and thus, justifies further studies on enabling BHL long-term application.

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.

© 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.

Decellularized scaffolds represent a promising alternative for mitral valve (MV) replacement. This work developed and characterized a protocol for the decellularization of whole MVs. Porcine MVs were decellularized with 0.5% (w/v) SDS and 0.5% (w/v) SD and sterilized with 0.1% (v/v) PAA. Decellularized samples were seeded with human foreskin fibroblasts and human adipose-derived stem cells to investigate cellular repopulation and infiltration, and with human colony-forming endothelial cells to investigate collagen IV formation. Histology revealed an acellular scaffold with a generally conserved histoarchitecture, but collagen IV loss. Following decellularization, no significant changes were observed in the hydroxyproline content, but there was a significant reduction in the glycosaminoglycan content. SEM/TEM analysis confirmed cellular removal and loss of some extracellular matrix components. Collagen and elastin were generally preserved. The endothelial cells produced newly formed collagen IV on the non-cytotoxic scaffold. The protocol produced acellular scaffolds with generally preserved histoarchitecture, biochemistry, and biomechanics.

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.

Purpose Lung transplantation is the only curative therapy option for patients suffering from end stage lung diseases. However, it is associated with high risks and only available for selected patients. In order to provide an alternative treatment option, this project aims for the development of an implantable biohybrid lung (BL), based on hollow fibre membrane (HFM) technology used in extracorporeal membrane oxygenators. A crucial requirement to achieve long-lasting durability of the BL is the optimized bio- and haemocompatibility of all blood contacting surfaces, which can be achieved by endothelialisation. Methods Albumin/Heparin (A/H) and Fibronectin (FN) coated HFM were compared for their eligibility to mediate the establishment of a viable, confluent and non-thrombogenic endothelial cell (EC) monolayer. Activation status of the seeded ECs was analyzed and verified by leukocyte- and thrombocyte adhesion assays. The behavior of HFM-seeded ECs under workload conditions, i.e. fluid flow and oxidative stress exposure, was assessed in a customized flow chamber setup. To identify a clinically relevant EC source, comparative studies including human cord blood derived endothelial cells (hCBECs) induced pluripotent stem cell derived endothelial cells and immunotolerable MHC-silenced hCBECs were conducted. Results A physiologic, viable and confluent EC monolayer could be sustained under both, static and flow dynamic culture conditions. All tested EC types preserved their non-thrombogenic and non-inflammatory status on both tested HMF. Exposure to relevant levels of hyperoxia or hypoxia did not affect the physiological function of the ECs. However, FN coated HFMs demonstrated an improved EC-monolayer resistance towards flow conditions. Conclusion Endothelialisation of HFM improves their hemocompatibility. Moreover, the endothelial monolayer on HFM can resist clinically relevant settings, like flow exposure and hyperoxia/hypoxia. MHC-silenced ECs could be considered as suitable cell source, escaping the recipient's immune response. These results may bring BL as alternative of lung transplantation for patients with end-stage lung diseases closer to reality.

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.

Low haemocompatibility of left ventricular assist devices (LVAD) surfaces necessitates anticoagulative therapy. Endothelial cell (EC) seeding can support haemocompatibility, however, the availability of autologous ECs is limited. In contrast, allogeneic ECs are readily available in sufficient quantity, but HLA disparities induce harmful immune responses causing EC loss. In this study, we investigated the feasibility of using allogeneic low immunogenic ECs to endothelialize LVAD sintered inflow cannulas (SIC). To reduce the immunogenicity of ECs, we applied an inducible lentiviral vector to deliver short-hairpins RNA to silence HLA class I expression. HLA class I expression on ECs was conditionally silenced by up to 70%. Sufficient and comparable endothelialization rates were achieved with HLA-expressing or HLA-silenced ECs. Cell proliferation was not impaired by cell-to-Sintered Inflow Cannulas (SIC) contact or by silencing HLA expression. The levels of endothelial phenotypic and thrombogenic markers or cytokine secretion profiles remained unaffected. HLA-silenced ECs-coated SIC exhibited reduced thrombogenicity. In contrast to native ECs, HLA-silenced ECs showed lower cell lysis rates when exposed to allogeneic T cells or specific anti-HLA antibodies. Allogeneic HLA-silenced ECs could potentially become a valuable source for LVAD endothelialization to reduce immunogenicity and correspondingly the need for anticoagulative therapy which can entail severe side effects.

Purpose Left ventricular assist devices (LVADs) are established implantable devices in patients with end-stage heart failure. Due to their artificial materials, anticoagulative therapy is needed, though leading to partly lethal complications and limited clinical applications. Improved LVAD hemocompatibility of all blood contacting surfaces can be achieved by their endothelialisation. Additionally, the endothelial monolayer need to resist clinically relevant flow-conditions, for which we established a new approach using magnetic forces and intracellular superparamagnetic nanoparticles (NP). Methods Endothelial cells (EC) were incubated with nanoparticles of different concentrations and coatings (sodium citrate, polyvinylpyrrolidone) for various time periods. Nanoparticulate absorption behaviour was visualized by Prussian blue staining. Batches with high intracellular nanoparticle quantities were analysed for cell viability by immunofluorescence staining and proliferation assay, possible cytotoxicity by flow cytometry. Following, cellular alignment induced by different magnetic forces was examined and transferred to LVAD impeller endothelialisation. Results Concentration-, coating- and time-dependent changes in cell viability, proliferation and cytotoxicity could be shown, indicating best results for low concentrated sodium citrate nanoparticles, which were used for subsequent cell migration experiments. Magnetic field sufficiently affected endothelialisation as significantly more NP loaded ECs adhered to the impeller surface compared to the unloaded control group. Conclusion These results are promising steps towards the effective biologisation of the LVAD, as the LVAD itself may provide the needed magnetic field for sufficient and flow-resistant endothelialisation. This hypothesis needs to be analysed within the following experiments.

In regenerative medicine, autologous endothelial colony forming cells (ECFCs) bear the greatest potential to be used for surface endothelialization of tissue engineered constructs, as they are easily attainable and possess a high proliferation rate. The aim of this study was to develop a standardized pre-conditioning protocol under dynamic conditions simulating the physiology of human circulation to improve the formation of a flow resistant monolayer of ECFCs and to enhance the antithrombogenicity of the endothelial cells. The main focus of the study was to consequently compare the cellular behavior under a steady laminar flow against a pulsatile flow. Mononuclear cells were isolated out of peripheral blood (PB) buffy coats and plated on uncoated tissue culture flasks in anticipation of guidelines for Advanced Therapy Medicinal Products. ECFCs were identified by typical surface markers such as CD31, CD146 and VE-Cadherin. To explore the effects of dynamic cultivation, ECFCs and human umbilical vein endothelial cells were comparatively cultured under either laminar or pulsatile (1 Hz) flow conditions with different grades of shear stress (5 dyn/cm2 versus 20 dyn/cm2). High shear stress of 20 dyn/cm2 led to a significant upregulation of the antithrombotic gene marker thrombomodulin in both cell types, but only ECFCs orientated and elongated significantly after shear stress application forming a confluent endothelial cell layer. The work therefore documents a suitable protocol to pre-condition PB-derived ECFCs for sustainable endothelialization of blood contacting surfaces and provides essential knowledge for future cultivations in bioreactor systems.

The correlation between extracellular matrix (ECM) components, cell shape, and stem cell guidance can shed light in understanding and mimicking the functionality of stem cell niches for various applications. This interplay on osteogenic guidance of human adipose-derived stem cells (hASCs) was focus of this study. Proliferation and osteogenic markers like alkaline phosphatase activity and calcium mineralization were slightly increased by the ECM components laminin (LA), collagen I (COL), and fibronectin (FIB); with control medium no differentiation occurred. ECM guided differentiation was rather dependent on osterix than on Runx2 pathway. FIB significantly enhanced cell elongation even in presence of actin polymerization blockers cytochalasin D (CytoD) and ROCK inhibitor Y-27632, which generally caused more rounded cells. Except for the COL surface, both inhibitors increased the extent of osterix, while the Runx2 pathway was more sensitive to the culture condition. Both inhibitors did not affect hASC proliferation. CytoD enabled osteogenic differentiation independently from the ECM, while it was rather blocked via Y-27632 treatment; on FIB the general highest extent of differentiation occurred. Taken together, the ECM effect on hASCs occurs indirectly and selectively via a dominant role of FIB: it sustains osteogenic differentiation in case of a tension-dependent control of actin polymerization.

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.

Purpose Lung transplantation remains the only treatment option for patients with end-stage lung disease. Although surgical techniques and organ preservation significantly improved over the last decades, ischemia and reperfusion injuries, which inter alia manifest in edema formation, still represent major limitations affecting clinical outcome. Here we investigated the hypothesis that different lung preservation solutions contribute to solution-specific degrees of transient leaks within the endothelial cell (EC) monolayer, therefore allowing liquid influx into the interstitial space leading to edema formation. Methods Intercellular connectivity within confluent EC monolayers was assessed after 1h, respectively 5.5h storage in the respective preservation solutions OCS™, Steen™, Perfadex™, PerfadexPlus™ or Celsior™ ± 10µM or 100µM 17β-Estradiol (17s), which was used to deliberate disintegration of the monolayer. Furthermore, 5.5h and 24h of regeneration in the preservation solution, respectively EC medium without 17s was analyzed. Monolayer permeability was evaluated using FITC-Dextran transwell-assays, cell viability (Calcein AM) and monolayer integrity (VE-Cadherin) were examined by fluorescence microscopy. Realtime qRT-PCR analyses were carried out to investigate responses on gene expression levels. Results FITC-Dextran assay indicated solution-specific monolayer permeability, after 1h incubation highest was identified for Celsior™, followed by Perfadex™ and OCS™, Steen™ and PerfadexPlus™ showed up with the lowest permeability. While addition of 10µM 17s did not affect solution-specific permeability, all groups treated with 100µM 17s become durable leaky. Comparable results were seen after 5.5h and 24h of regeneration, while previous incubation with 100µM 17s induced irreversible effects. These results could be confirmed by immunofluorescence staining. By contrast, independent of the treatment qRT-PCR analyses indicated negligible effects on gene expressions of activation and thrombogenic state markers. Conclusion The application of different preservation solutions results in solution-specific, partly irreversible endothelial leakage, which may partly explain edema and weight increase during organ preservation. Subsequent analysis will correlate these results with clinical parameters.

Surface pre-endothelialization is a promising approach to improve the hemocompatibility of implants, medical devices, and artificial organs. To promote the adhesive property of thermoplastic polyurethane (TPU) for endothelial cells (ECs), up to 1 wt % of gold (Au) or platinum (Pt) nanoparticles, fabricated by pulsed laser ablation in polymer solution, were embedded into the polymer matrix. The analysis of these nanocomposites showed a homogenous dispersion of the nanoparticles, with average diameters of 7 nm for Au or 9 nm for Pt. A dose-dependent effect was found when ECs were seeded onto nanocomposites comprising different nanoparticle concentrations, resulting in a fivefold improvement of proliferation at 0.1 wt % nanoparticle load. This effect was associated with a nanoparticle concentration-dependent hydrophilicity and negative charge of the nanocomposite. In dynamic flow tests, nanocomposites containing 0.1 wt % Au or Pt nanoparticles allowed for the generation of a confluent and resistant EC layer. Real-time polymerase chain reaction quantification of specific markers for EC activation indicated that ECs cultivated on nanocomposites remain in an inactivated, nonthrombogenic and noninflammatory state; however, maintain the ability to trigger an inflammatory response upon stimulation. These findings were confirmed by a platelet and leukocyte adhesion assay. The results of this study suggest the possible applicability of TPU nanocomposites, containing 0.1 wt % Au or Pt nanoparticles, for the generation of pre-endothelialized surfaces of medical devices.

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.

The extent of the indirect DNA damage generated in mammalian cells by visible light because of the presence of endogenous photosensitizers was studied by means of repair endonucleases. In immortalized human keratinocytes (HaCaT cells) exposed to low doses of natural sunlight, the yield of oxidative DNA base modifications sensitive to the repair endonuclease formamidopyrimidine-DNA glycosylase (Fpg protein) generated by this indirect mechanism was 10% of that of pyrimidine dimers (generated by direct DNA excitation). A similar yield of Fpg-sensitive modifications, which include 8-hydroxyguanine, was observed in primary keratinocytes. The relative yield of oxidative base modifications decreased at higher light doses, probably as a result of photodecomposition of the endogenous chromophore involved. For the three cell lines tested, viz. HaCaT cells, L1210 mouse leukemia cells and AS52 Chinese hamster cells, the yield of oxidative base modifications generated by a low dose of visible light appeared to be correlated with the basal concentrations of porphyrins in the cells. Induction of cellular porphyrin synthesis by pretreatment with 5-aminolaevulinic acid increased the light-induced oxidative damage in L1210 cells several-fold. In both induced and uninduced cells, the damage was inhibited by more than 50% in the presence of ascorbic acid (100 microM), while alpha-tocopherol and the iron chelator alpha-phenanthroline had no effect and beta-carotene even increased the damage. Even high doses of visible light did not significantly increase the numbers of micronuclei in L1210 cells or of gpt mutations in AS52 cells. The negative outcome can be fully explained by the photobleaching of the endogenous photosensitizers, which prevents the generation of sufficiently high levels of oxidative DNA damage. Therefore, the mutagenic risk arising from the indirectly generated oxidative DNA modifications induced by sunlight may be underestimated when results obtained at high doses are extrapolated to low doses or low dose rates.

Purified repair endonucleases such as Fpg protein, endonuclease III and IV allow a very sensitive quantification of various types of oxidative DNA modifications in mammalian cells. By means of these assays, the numbers of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine (8-oxoG), were determined to be less than 0.3 per 10(6) bp in several types of untreated cultured mammalian cells and human lymphocytes and less than 10 per 10(6) bp in mitochondrial DNA from rat and porcine liver. Oxidative 5,6-dihydropyrimidine derivatives sensitive to endonuclease III and sites of base loss sensitive to endonuclease IV or exonuclease III were much less frequent than Fpg-sensitive modifications. Here, we summarize our indications that all Fpg-sensitive modifications are recognized under the assay conditions and that on the other hand there is no artifactual generation of oxidative damage during the analysis. In addition, we show that the steady-state levels of Fpg-sensitive modifications in human lymphocytes and in two mammalian cell lines were higher in proliferating than in resting (confluent) cells. Only some of the Fpg-sensitive base modifications induced by various oxidants are 8-oxoG residues, as demonstrated for the damage under cell-free conditions. The percentage was dependent on the species ultimately responsible for the DNA damage and was approx. 40% in the case of hydroxyl radicals and peroxynitrite, 75% for type II photosensitizers (reacting via singlet oxygen) and only 20-30% in the case of type I photosensitizers such as riboflavin and acridine orange, which are assumed to react directly with the DNA.

The alkaline elution technique in combination with various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) was used to quantify steady-state (background) levels of oxidative base modifications in various types of mammalian cells. In human lymphocytes the number of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine, was 0.25 +/- 0.05 per 10(6) base pairs. Even lower levels (0.07 +/- 0.02 per 10(6) bp) were observed in HeLa cells. The numbers of sites sensitive to the other repair endonucleases were below the detection limit (0.05 per 10(6) bp). In a direct comparison, the background level of Fpg-sensitive modifications determined by alkaline elution was much lower than the background level of 8-hydroxydesoxyguanosine (8-oxodG) determined after enzymatic DNA hydrolysis by HPLC and electrochemical detection. However, the number of additional Fpg-sensitive modifications induced by a photosensitizer plus light was similar to the additional number of 8-oxodG residues determined by HPLC with electrochemical detection. This indicates that the enzyme assay does not systematically underestimate the number of lesions and points to an artefactual generation of 8-oxodG during DNA isolation and hydrolysis.

Surface pre-endothelialization is a promising approach to improve the hemocompatibility of implants, medical devices, and artificial organs. To promote the adhesive property of thermoplastic polyurethane (TPU) for endothelial cells (ECs), up to 1 wt % of gold (Au) or platinum (Pt) nanoparticles, fabricated by pulsed laser ablation in polymer solution, were embedded into the polymer matrix. The analysis of these nanocomposites showed a homogenous dispersion of the nanoparticles, with average diameters of 7 nm for Au or 9 nm for Pt. A dose-dependent effect was found when ECs were seeded onto nanocomposites comprising different nanoparticle concentrations, resulting in a fivefold improvement of proliferation at 0.1 wt % nanoparticle load. This effect was associated with a nanoparticle concentration-dependent hydrophilicity and negative charge of the nanocomposite. In dynamic flow tests, nanocomposites containing 0.1 wt % Au or Pt nanoparticles allowed for the generation of a confluent and resistant EC layer. Real-time polymerase chain reaction quantification of specific markers for EC activation indicated that ECs cultivated on nanocomposites remain in an inactivated, nonthrombogenic and noninflammatory state; however, maintain the ability to trigger an inflammatory response upon stimulation. These findings were confirmed by a platelet and leukocyte adhesion assay. The results of this study suggest the possible applicability of TPU nanocomposites, containing 0.1 wt % Au or Pt nanoparticles, for the generation of pre-endothelialized surfaces of medical devices.

Utilization of living cells for therapies in regenerative medicine requires a fundamental understanding of the interactions between different cells and their environment. Moreover, common models based on adherent two-dimensional cultures are not appropriate to simulate the complex interactions that occur in a three-dimensional (3D) cell-microenvironment in vivo. In this study, we present a computer-aided method for the printing of multiple cell types in a 3D array using laser-assisted bioprinting. By printing spots of human adipose-derived stem cells (ASCs) and endothelial colony-forming cells (ECFCs), we demonstrate that (i) these cell spots can be arranged layer-by-layer in a 3D array; (ii) any cell-cell ratio, cell quantity, cell-type combination, and spot spacing can be realized within this array; and (iii) the height of the 3D array is freely scalable. As a proof of concept, we printed separate spots of ASCs and ECFCs within a 3D array and observed cell-cell interactions in vascular endothelial growth factor-free medium. It has been demonstrated that direct cell-cell contacts trigger the development of stable vascular-like networks. This method can be applied to study complex and dynamic relationships between cells and their local environment.

In the present work, the two-photon polymerization (2PP) technique was applied to develop precisely defined biodegradable 3D tissue engineering scaffolds. The scaffolds were fabricated via photopolymerization of gelatin modified with methacrylamide moieties. The results indicate that the gelatin derivative (GelMod) preserves its enzymatic degradation capability after photopolymerization. In addition, the developed scaffolds using 2PP support primary adipose-derived stem cell (ASC) adhesion, proliferation and differentiation into the anticipated lineage.

Objectives To evaluate bioartificial haemodialysis access grafts in a sheep model with respect to patency and morphology. Material and methods Bovine internal thoracic arteries (n = 28) were decellularised. Fourteen grafts (DC grafts) were directly implanted as cervical AV shunts, the remaining were re-seeded with endothelial cells (ECs) derived from blood samples of the later ovine recipient (EC grafts) first. Following simulated punctures and duplex ultrasound scans to determine patency, grafts were explanted for immunohistochemical characterisation after 3 and 6 months, respectively. DC grafts underwent biomechanical testing for compliance (C), suture retention strength (SRT), and burst pressure (BP) before (n = 6) and after (n = 6) implantation. Results Following 3 and 6 months, the majority of EC (n = 6/6; n = 6/7) and DC grafts (n = 5/6; n = 5/7) were patent and not relevantly stenosed (peak systolic velocity: EC grafts = 76 cm s−1 ± 4; DC grafts = 77 cm s−1 ± 5). Simulated haemodialysis punctures revealed significantly shorter bleeding times in all bioartificial grafts than in native jugular veins (P > 0.001). Comparing native carotid arteries with DC grafts prior to and post-implantation, the latter differed significantly with respect to C (P > 0.001; P = 0.005), whereas only pre-implant DC grafts differed regarding BP (P = 0.002); no differences were observed for SRT. Histology revealed complete endothelial surface coverage of EC, but not DC grafts. Furthermore, DC grafts exhibited areas of pronounced tissue calcification. Conclusion The preclinical development of a bioartificial haemodialysis access graft with promising mechanical and morphological properties in a sheep model is feasible.

The extract obtained by solid-liquid extraction with pentane-dichloromethane (2+1, v/v) from dry mugwort (Artemisia vulgaris L.) herb was fractionated by means of gel permeation chromatography (GPC) and subsequent middle-pressure liquid chromatography (MPLC). Capillary gas chromatography (HRGC) and combined HRGC techniques, i.e. HRGC-mass spectrometry (HRGC-MS) and HRGC-Fourier transform infrared spectroscopy (HRGC-FTIR) of the separated MPLC fractions revealed the occurrence of 54 additional volatiles not described as yet in A. vulgaris herb. Among them, chiral evaluation of y-nonalactone carried out by on-line coupled multidimensional gas chromatography-mass spectrometry (MDGC-MS) revealed a distribution of (R): (S) = 34:66%.

The oxidative DNA damage induced by the polar photosensitizer Ro19-8022 in the presence of light was studied and correlated with the associated mutagenicity. Both in isolated DNA and AS52 Chinese hamster ovary cells, photoexcited Ro19-8022 gave rise to a DNA damage profile that was similar to that caused by singlet oxygen: base modifications sensitive to the repair endonuclease Fpg protein, which according to high-performance liquid chromatography (HPLC) analysis were predominantly 8-hydroxyguanine (8-oxoG) residues, were generated in much higher yield than single-strand breaks, sites of base loss (AP sites) and oxidative pyrimidine modifications sensitive to endonuclease III. Fifty percent of the Fpg-sensitive modifications were repaired within 2 h. Under conditions that induced 10 Fpg-sensitive modifications per 10(6) bp (six 8-oxoG residues per 10(6) bp), approximately 60 mutations per 10(6) cells were induced in the gpt locus of the AS52 cells. A rather similar mutation frequency was observed when a plasmid carrying the gpt gene was exposed to Ro19-8022 plus light under cell-free conditions and subsequently replicated in bacteria. Sequence analysis revealed that GC-->TA and GC-->CG transversions accounted for 90% of the base substitutions. A significant generation of micronuclei was detectable in AS52 cells exposed to the photosensitizer plus light as well.

Reactive oxygen species (ROS) are formed inside cells not only under the influence of exogenous agents (visible light, ionizing radiation, and many oxidants such as peroxides or quinones), but also under normal (physiological) conditions as byproducts of oxygen metabolism and other cellular redox reactions (Pryor 1986; Halliwell and Gutteridge 1986; Sies 1986; Clayson et al. 1994). ROS such as hydroxyl radicals and singlet oxygen are a serious threat to the integrity of the cellular genome, since they efficiently react with DNA to generate many types of DNA modifications, at least some of which are pre- mutagenic (Breimer 1990; Halliwell and Aruoma 1991; Epe 1991; Feig et al. 1994). Steady-state levels of 8-hydroxyguanine (8-oxoG) and other oxidative DNA base modifications observed in untreated cells indicate that the various cellular defense and DNA repair systems (Demple and Harrison 1994) do not completely eliminate the mutagenic risk associated with ROS formation even under normal growth conditions. This led to the assumption that oxidative DNA damage is a causal or ancillary risk factor for the development of cancer and several age-correlated degenerative diseases (Ames 1983; Wallace 1992; Gutteridge 1993). A strategy to verify this hypothesis and to quantify the mutagenic risk associated with oxidative DNA damage could be to determine (a) what type of oxidative DNA damage profile (pattern of DNA modifications) is generated in the cells under the conditions of interest and (b) the mutagenicity associated with this damage profile. Then, the quantification of any suitable marker modification of this damage profile should allow an estimation of the mutagenicity to be expected.

Fanconi anemia (FA) is an autosomal recessive disorder involving progressive pancytopenia, skeletal malformations, and a predisposition to leukemia. The in vitro growth of FA fibroblasts is impaired, due to a defective G2 phase traverse of the cell cycle. Analyzing the cell cycle of lymphoid cell lines (LCLs) obtained from peripheral blood of FA patients by transformation with Epstein-Barr virus, we found a similar G2 phase defect, which was dependent upon the oxygen concentration. In addition, FA cells exhibited hypersensitivity toward cis-dichlorodiammineplatinum and mitomycin C, and moderate sensitivity toward trans-dichlorodiammineplatinum. FA cells, however, showed no elevated sensitivity toward paraquat, an intracellular generator of superoxide radicals, or cumene hydroperoxide, a model organic peroxide. Chelating iron with low concentrations of o-phenanthrolin improved cell proliferation and G2 phase transit of FA cells at 20% oxygen, but little at 5% oxygen. LCL cultures from healthy subjects were inhibited in their proliferation rate at all concentrations of o-phenanthrolin. Exposure to excess iron, on the other hand, was very toxic to FA cells at 20%, but less toxic at 5% oxygen. In conclusion, the FA mutation leads to a cell cycle defect, which is expressed in cultures of lymphoid cells from FA patients, and involves hypersensitivity toward bifunctional alkylating agents, oxygen, and iron.

Reactive oxygen intermediates like hydrogen peroxide (H2O2) have been shown to serve as messengers in the induction of NF-kappa B and, then, in the activation and replication of human immunodeficiency virus (HIV)-1 in human cells. Because H2O2 can be converted into the highly reactive OH. at various locations inside the cells, we started to investigate the generation of Reactive oxygen intermediates by photosensitization. This technique is based on the use of a photosensitizer which is a molecule absorbing visible light and which can be located at various sites inside the cell depending on its physicochemical properties. In this work, we used proflavine (PF), a cationic molecule having a high affinity for DNA, capable of intercalating between DNA base pairs. Upon visible light irradiation, intercalated PF molecules oxidize guanine residues and generate DNA single-strand breaks. In lymphocytes or monocytes latently infected with HIV-1 (ACH-2 or U1, respectively), this photosensitizing treatment induced a cytotoxicity, an induction of NF-kappa B, and a reactivation of HIV-1 in cells surviving the treatment. NF-kappa B induction by PF-mediated photosensitization was not affected by the presence of N-acetyl-L-cysteine while strong inhibition was recorded when the induction was triggered by H2O2 or by phorbol 12-myristate 13-acetate. Another transcription factor like AP-1 is less activated by this photosensitizing treatment. In comparison with other inducing treatments, such as phorbol 12-myristate 13-acetate or tumor necrosis factor alpha, the activation of NF-kappa B is slow, being optimal 120 min after treatment. These kinetic data were obtained by following, on the same samples, both the appearance of NF-kappa B in the nucleus and the disappearance of I kappa B-alpha in cytoplasmic extracts. These data allow us to postulate that signaling events, initiated by DNA oxidative damages, are transmitted into the cytoplasm where the inactive NF-kappa B factor is resident and allow the translocation of p50/p65 subunits of NF-kappa B to the nucleus leading to HIV-1 gene expression.

The DNA damage induced by visible light in L1210 mouse leukaemia cells was analysed by an alkaline elution assay with specific repair endonucleases. DNA single-strand breaks and DNA modifications sensitive to FPG protein (formamidopyrimidine-DNA glycosylase), endonuclease III and exonuclease III were quantified in parallel. The light-induced cellular DNA damage was found to consist of many base modifications sensitive to FPG protein, which most probably are predominantly 7,8-dihydro-8-oxoguanine (8-hydroxyguanine) residues. Base modifications sensitive to endonuclease III are virtually absent. The yield of the FPG-sensitive base modifications is 10-fold higher than that of single-strand breaks plus AP sites (sites of base loss). The described ratios of the various modifications indicate that the damage most probably results from a reaction of DNA with singlet oxygen (type II reaction) or directly with an excited endogenous photosensitizer (type I reaction) and is not mediated by hydroxyl radicals. Experiments with cut-off filters indicate that wavelengths between 400 and 500 nm are responsible for most of the modifications. The FPG-sensitive base modifications are repaired efficiently (t1/2 approximately 1 h at 37 degrees C). This is perhaps why the light-induced DNA damage is apparently associated with only low mutagenicity.

The specific recognition of DNA modifications by repair endonucleases was used to characterize the DNA damage induced by photosensitizers in the presence of visible light. Under cell-free conditions, chemically unrelated photosensitizers (methylene blue, acridine orange, proflavin, riboflavin, hematoporphyrin) induce the same type of DNA damage. It is characterized by a high number of base modifications sensitive to the repair endonuclease FPG protein (formamidopyrimidine-DNA glycosylase), while both the number of DNA strand breaks and the number of sites of base loss (sensitive to exonuclease III or endonuclease IV) is low. Therefore the damage is markedly different from that induced by hydroxyl radicals. Mechanistically, the generation of the base modifications sensitive to FPG protein involves singlet oxygen in some, but possibly not all cases, as substituting D2O for H2O increases the reaction yield six-fold in the case of methylene blue, but only 1.4-fold in the case of acridine orange. In plasmids from Salmonella typhimurium strains treated with methylene blue or acridine orange plus light and from Escherichia coli strains treated with acridine orange or proflavin plus light, the same type of damage was observed as under cell-free conditions. In L1210 mouse leukemia cells exposed to acridine orange plus light, the numbers of modifications sensitive to FPG protein and exonuclease III were quantified, in addition to strand breaks, by a modified alkaline elution assay. Again, the number of base modifications sensitive to FPG protein was found to be several-fold higher than the number of strand breaks and sites of base loss. It has to be concluded that the DNA damage in the intact cells is not mediated by hydroxyl radicals or cellular nucleases, but by the same mechanism as operates under cell-free conditions with these agents.

A number of repair endonucleases, viz. endonuclease III, formamidopyrimidine-DNA glycosylase (FPG protein), endonuclease IV, exonuclease III and UV endonuclease, is used to simultaneously quantify various types of DNA modifications, which were induced by agents that generate reactive oxygen species. Under cell-free conditions, two types of DNA damage profiles are obtained. The profiles induced by chemically generated singlet oxygen and by various photosensitizers (acridine orange, methylene blue, riboflavin, hematoporphyrin) plus light are dominated by base modifications sensitive to FPG protein, while 5,6-dihydropyrimidines (recognized by endonuclease III), sites of base loss (AP sites, recognized by endonuclease IV and exonuclease III) and strand breaks are minor lesions. In contrast, the DNA damage profile induced by hydroxyl radicals (γ-rays) consists of approx. equal levels of base modifications, AP sites and strand breaks. The damage profiles induced by Fe(III)-EDTA in the presence of Superoxide and by Fe(III)-nitrilotriacetate in the presence of H2O2 do not differ from that by hydroxyl radicals. The damage profile induced by Cu(II)-phenanthroline deviates by high levels of AP sites that are recognized by endonuclease IV and exonuclease III — but not by those AP endonucleases which cleave at the 3' site — and probably represent AP sites oxidized at C-1'. The damage induced by Fe(III)-bleomycin plus H2O2 deviates by an increased level of double strand breaks and the absence of endonuclease-sensitive base modifications. Cellular DNA damage profiles are obtained from bacteria, cultured mammalian cells and mammalian mitochondria after exposure to acridine orange plus visible light. A comparison with the cell-free profiles reveals that the damage in all three systems is not induced indirectly by hydroxyl radicals or an activation of cellular nueleases, but by the same mechanism that is responsible for the cell-free DNA damage