Cytotoxicity and endothelial dysfunction induced by iron-containing bioresorbable materials

The present thesis is part of an integrated and interdisciplinary project that aims to develop new Fe-based bioresorbable alloys for cardiovascular implants. The main purpose was to scrutinize and highlight the biological interactions between Fe-based materials and the surrounding tissue. During the last decade, biodegradable materials have been developed as alternatives to permanent coronary stents. Metals such as pure Fe and Fe-Mn alloys were investigated in this context for their good mechanical properties, adequate degradability and assumed biocompatibility. The main objectives were (1) to look at the impact of the released corrosion products, ions or ROS (Reactive Oxygen Species), as well as the material itself on the vascular tissue and (2) to develop new ex-vivo and valid in vitro models to address these questions. We demonstrated that Fe corrosion releases hydroxyl radicals (HO•), which contribute to induce cytotoxicity, oxidative stress, inhibition of NO production through a decreased eNOS enzymatic activity and endothelial dysfunction. Indirect contact tests (Fe ions only) did not reveal any effect. The implication of HO• on arterial wall was confirmed by the protective role of catalase, which totally prevented deleterious outcomes. Our data contribute to raise concerns about the biocompatibility of these new degradable alloys for vascular implants and the deleterious impact of HO• has to be considered when developing new Fe-based materials.Ce travail fait partie d'un projet interdisciplinaire qui vise à développer de nouveaux alliages biorésorbables à base de fer comme implants cardiovasculaires. L'objectif principal était d'analyser les interactions biologiques entre les matériaux à base de fer et le tissus artériel. Il s’agissait (1) d’examiner l’impact des produits de corrosion libérés, ions ou ROS (espèces réactives de l’oxygène), ainsi que du matériau même sur le tissu vasculaire et (2) de développer de nouveaux modèles ex-vivo et in vitro pour répondre à ces questions. Nous avons montré que la corrosion du fer libère des radicaux hydroxyle (HO•), qui contribuent à induire de la cytotoxicité, un stress oxydant, l’inhibition de la production de NO, par une activité enzymatique eNOS réduite, et un dysfonctionnement endothélial. L'implication de HO• dans la toxicité endothéliale a été confirmée par le rôle protecteur de la catalase, qui a totalement empêché les effets délétères. Les ions de fer libérés par la corrosion n'ont pas révélé d’effet. Nos données contribuent à susciter des inquiétudes quant à la biocompatibilité de ces nouveaux alliages dégradables comme implants cardiaques.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 202

Are Fe-Based Stenting Materials Biocompatible? A Critical Review of In Vitro and In Vivo Studies

e-based materials have increasingly been considered for the development of biodegradable cardiovascular stents. A wide range of in vitro and in vivo studies should be done to fully evaluate their biocompatibility. In this review, we summarized and analyzed the findings and the methodologies used to assess the biocompatibility of Fe materials. The majority of investigators drew conclusions about in vitro Fe toxicity based on indirect contact results. The setup applied in these tests seems to overlook the possible effects of Fe corrosion and does not allow for understanding of the complexity of released chemical forms and their possible impact on tissue. It is in particular important to ensure that test setups or interpretations of in vitro results do not hide some important mechanisms, leading to inappropriate subsequent in vivo experiments. On the other hand, the sample size of existing in vivo implantations is often limited, and effects such as local toxicity or endothelial function are not deeply scrutinized. The main advantages and limitations of in vitro design strategies applied in the development of Fe-based alloys and the correlation with in vivo studies are discussed. It is evident from this literature review that we are not yet ready to define an Fe-based material as safe or biocompatible

Mind your assays: Misleading cytotoxicity with the WST-1 assay in the presence of manganese

The WST-1 assay is the most common test to assess the in vitro cytotoxicity of chemicals. Tetrazolium-based assays can, however, be affected by the interference of tested chemicals,including carbon nanotubes or Mg particles. Here, we report a new interference of Mn materials with the WST-1 assay. Endothelial cells exposed to Mn particles (Mn alone or Fe-Mn alloy from 50 to 1600 μg/ml) were severely damaged according to the WST-1 assay, but not the ATP content assay. Subsequent experiments revealed that Mn particles interfere with the reduction of the tetrazolium salt to formazan. Therefore, the WST-1 assay is not suitable to evaluate the in vitro cytotoxicity of Mn-containing materials, and luminescencebased assays such as CellTiter-Glo® appear more appropriate

Neurotoxicity of Engineered Nanomaterials: Testing Considerations

As with toxicology in general, major challenges have emerged in its subfield neurotoxicology regarding the testing of engineered nanomaterials (ENM). This is on the one hand due to their complex physicochemical properties, like size, specific surface area, chemical composition as well as agglomeration and dissolution behavior in biological environments. On the other hand, toxicological risk assessment has faced an increasing demand for the development and implementation of non-animal alternative approaches. Regarding the investigation and interpretation of the potential adverse effects of ENM on the brain, toxicokinetic data are relatively scarce and thus hampers dose selection for in vitro neurotoxicity testing. Moreover, recent in vivo studies indicate that ENM can induce neurotoxic and behavioral effects in an indirect manner, depending on their physicochemical properties and route of exposure. Such indirect effects on the brain may proceed through the activation and spill-over of inflammatory mediators by ENM in the respiratory tract and other peripheral organs as well via ENM induced disturbance of the gut microbiome and intestinal mucus barrier. These ENM specific aspects should be incorporated into the ongoing developments of advanced in vitro neurotoxicity testing methods and strategies

Endothelial dysfunction induced by hydroxyl radicals – the hidden face of biodegradable Fe-based materials for coronary stents

Fe-based materials are currently considered for manufacturing biodegradable coronary stents. Here we show that Fe has a strong potential to generate hydroxyl radicals (HO·) during corrosion. This HO· generation, but not corrosion, can be inhibited by catalase. Oxidative stress was observed (increased HO-1 expression) in aortic rings after direct exposure to Fe, but not in the presence of catalase or after indirect exposure. This oxidative stress response induced an uncoupling of eNOS in, and a consequent reduced NO production by endothelial cells exposed to Fe. In isolated rat aortic rings NO production was also reduced by HO· generated during Fe corrosion, as indicated by the protective role of catalase. Finally, all these mechanisms contributed to impaired endothelium- dependent relaxation in aortic rings caused by HO· generated during the direct contact with Fe. This deleterious impact of Fe corrosion on the endothelial function should be integrated when considering the use of biodegradable Fe-based alloys for vascular implants

Hydroxyl radicals and oxidative stress: the dark side of Fe corrosion

Fe-based materials are considered for the manufacture of temporary implants that degrade through the corrosion of Fe by oxygen. Here we document the generation of hydroxyl radicals (HO˙) during this corrosion process, and their deleterious impacts on human endothelial (ECs) and smooth muscle cells (SMCs) in vitro. The generation of HO˙ was documented by two independent acellular assays, terephtalic acid hydroxylation (fluorescence) and spin trapping technique coupled with electron paramagnetic resonance spectroscopy. All Fe-based materials tested exhibited a strong potential to generate HO˙. The addition of catalase prevented the formation of HO˙. Cellular responses were assessed in two ECs and SMCs lines using different cytotoxicity assays (WST-1 and CellTiter-Glo). Cells were exposed directly to Fe powder in the presence/absence of catalase, or to extracts obtained from the corrosion of Fe. Cell viability was dose-dependently affected by the direct contact with Fe materials, but not in the presence of catalase or after indirect exposure to cell extracts. The deleterious effect of HO˙ on ECs and SMCs was confirmed by the dose-dependent increase of the transcripts of the oxidative stress gene heme oxygenase-1 4 h or 6 h after direct exposure to the particles, but not in presence of catalase or after indirect exposure. The demonstration of HO˙production during corrosion and consequent oxidative stress on human ECs and SMCs newly reveals a deleterious consequence of Fe-corrosion that should be integrated in the assessment of the biocompatibility of Fe-based alloys

Iron-based biodegradable alloys: importance of the testing parameters for corrosion evaluation via immersion tests.

n/

Iron-based biodegradable alloys: importance of the testing parameters for corrosion evaluation via immersion tests

Cardiovascular diseases such as arteriosclerosis are widespread nowadays. Implanting a stent in the injured blood vessel is a common solution. Currently, permanent stents are used and could lead to long-term complications, such as restenosis. To avoid these issues, biodegradable stents are developed. They have to offer the required mechanical support to the blood vessel during the healing period (about one year), be biocompatible, and corrode at a suitable rate, i.e. in 1-2 years [1]. This work deals with the evaluation of the corrosion properties of iron-based alloys by immersion tests. It highlights the importance to follow a well established protocol to obtain reliable and reproducible results

Iron-based biodegradable alloys: importance of the testing parameters for corrosion evaluation via immersion tests

Cardiovascular diseases such as arteriosclerosis are widespread nowadays. Implanting a stent in the injured blood vessel is a common solution. Currently, permanent stents are used and could lead to long-term complications, such as restenosis. To avoid these issues, biodegradable stents are developed. They have to offer the required mechanical support to the blood vessel during the healing period (about one year), be biocompatible, and corrode at a suitable rate, i.e. in 1-2 years [1]. This work deals with the evaluation of the corrosion properties of iron-based alloys by immersion tests. It highlights the importance to follow a well established protocol to obtain reliable and reproducible results

Iron-based biodegradable alloys: effect of composition and experimental parameters on the corrosion rate

This work investigates the degradation behaviour of iron-based alloys in pseudo physiological conditions. The goal is to identify the best composition leading to a corrosion rate adapted to biodegradable stent applications. In this context, TWIP steels (Fe-Mn-C alloys) with excellent mechanical properties are compared to pure iron, known for its too slow degradation rate. To assess the corrosion properties, two main kinds of tests were conducted, immersion and electrochemical tests. To reach reproducible and relevant results, the suitable experimental parameters were identified. The immersion tests in a pseudo physiological solution had two purposes, to estimate the corrosion rate by measuring the mass loss or concentration of ions released in the solution, and to scrutinise the corrosion mechanism owing to the characterisation of the corroded samples. SEM-EDS, XPS, ToF-SIMS and in situ AFM were used to look at the corroded surfaces. In this last case, the samples were immersed in a cell in the AFM while scans were conducted after different times, giving an insight into the formation of the different layers. Indeed, these analyses showed that different layers form on the surface. The bare metal is covered by oxides and hydroxides and then by calcium phosphates precipitating from the pseudo physiological solution. These layers actually protect the metal from corrosion. The results also show that many factors influence the corrosion rate such as the roughness or pre-oxidation of the surface, the composition of the solution, the solution stirring, the sample positioning, and the solution volume to sample surface ratio. This means that the test protocol has to be carefully chosen to reach reproducible results and to mimic the actual physiological conditions. Electrochemical tests such as potentiodynamic polarisation tests in a pseudo physiological medium aimed at comparing different materials. They also gave information about the tendency of the materials to passivate and the degree of protection offered by the passive layers. It is shown that TWIP steels corrode faster than commercial iron and that the degree of deformation of the TWIP samples does not seem to change noticeably their degradation rate