Kahina Abbas

Micael Hardy

Florent Poulhès

Hakim Karoui

Paul Tordo

Olivier Ouari

Fabienne Peyrot
email: fabienne.peyrot@parisdescartes.fr

Medium-throughput ESR

Keywords: EPR spectroscopy, Spin trapping, RAW macrophages, Free radical, CD-DIPPMPO

come

Introduction

ROS are important by-products of aerobic metabolism, involved in various diseases, but they also play a major part in cell signaling [START_REF] Bartosz | Reactive oxygen species: destroyers or messengers?[END_REF]. Spin trapping with cyclic nitrones combined to ESR is the gold standard for the detection of oxygen radical species, such as superoxide (O2 .-) or hydroxyl radical (HO . ), in cellular systems [START_REF] Janzen | Spin Trapping[END_REF][START_REF] Green | The production of hydroxyl and superoxide radicals by stimulated human neutrophils -measurements by EPR spectroscopy[END_REF][START_REF] Rosen | Hydroxyl radical generation by polymorphonuclear leukocytes measured by electron spin resonance spectroscopy[END_REF][START_REF] Hume | The production of oxygen-centered radicals by bacillus-Calmette-Guerin-activated macrophages. An electron paramagnetic resonance study of the response to phorbol myristate acetate[END_REF][START_REF] Rosen | Detection of superoxide generated by endothelial cells[END_REF][START_REF] Bannister | Production of oxygen-centered radicals by neutrophils and macrophages as studied by electron spin resonance (ESR)[END_REF][START_REF] Britigan | Do human neutrophils make hydroxyl radical -Determination of free radicals generated by human neutrophils activated with a soluble or particulate stimulus using electron paramagnetic resonance spectrometry[END_REF][START_REF] Britigan | Stimulated human neutrophils limit iron-catalyzed hydroxyl radical formation as detected by spin-trapping techniques[END_REF][START_REF] Hassett | Bacteria form intracellular free radicals in response to paraquat and streptonigrin. Demonstration of the potency of hydroxyl radical[END_REF][START_REF] Pou | Spin-trapping and human neutrophils. Limits of detection of hydroxyl radical[END_REF][START_REF] Ramos | Spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes[END_REF][START_REF] Bačić | Spin-trapping of oxygen free radicals in chemical and biological systems: New traps, radicals and possibilities[END_REF]. However interpretation of spin trapping results in cells may be difficult and lots of controls are required. DMPO (5,5-dimethyl-1pyrroline N-oxide), the historical spin trap, reacts with hydroxyl radical to form the adduct DMPO-OH. Detection of DMPO-OH following incubation with stimulated neutrophils was initially considered a proof of hydroxyl radical production by these cells [START_REF] Green | The production of hydroxyl and superoxide radicals by stimulated human neutrophils -measurements by EPR spectroscopy[END_REF][START_REF] Rosen | Hydroxyl radical generation by polymorphonuclear leukocytes measured by electron spin resonance spectroscopy[END_REF]. However DMPO-OOH, the adduct obtained by reaction with O2 .-, spontaneously decomposes into several species, including DMPO-OH [START_REF] Finkelstein | Production of hydroxyl radical by decomposition of superoxide spin-trapped adducts[END_REF][START_REF] Locigno | Nitric oxide release from the unimolecular decomposition of the superoxide radical anion adduct of cyclic nitrones in aqueous medium[END_REF]. In addition DMPO-OOH can be rapidly reduced to DMPO-OH by glutathione-peroxidase [START_REF] Rosen | Detection of superoxide generated by endothelial cells[END_REF], an enzyme present in cells, such as endothelial cells, neutrophils, or macrophages [START_REF] Kim | PEP-1-SIRT2 inhibits inflammatory response and oxidative stress-induced cell death via expression of antioxidant enzymes in murine macrophages[END_REF]. Thus detection of DMPO-OH does not unequivocally prove the formation of hydroxyl radical (Figure 1) and stimulated neutrophils have then been shown not to produce hydroxyl radical [START_REF] Britigan | Do human neutrophils make hydroxyl radical -Determination of free radicals generated by human neutrophils activated with a soluble or particulate stimulus using electron paramagnetic resonance spectrometry[END_REF]. Amelioration of the technique has occurred over the years with spin traps that form superoxide adducts with longer life time in buffer and that do not spontaneously decompose to superoxide adducts [START_REF] Ouari | Recent developments and applications of the coupled EPR/Spin trapping technique (EPR/ST)[END_REF]. Today, DEPMPO [START_REF] Frejaville | 5-Diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) -a new phosphorylated nitrone for the efficient in-vitro and in-vivo spin-trapping of oxygencentered radicals[END_REF][START_REF] Liu | Evaluation of DEPMPO as a spin trapping agent in biological systems[END_REF][START_REF] Timmins | Trapping of free radicals with direct in vivo EPR detection: a comparison of 5,5-dimethyl-1-pyrroline-N-oxide and 5diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide as spin traps for HO • and SO4[END_REF][START_REF] Khan | Spin traps: in vitro toxicity and stability of radical adducts[END_REF] is frequently used as a spin trap but novel spin traps, derived from the diisopropyloxyphosphoryl analogue DIPPMPO [START_REF] Chalier | 5-Diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO, a crystalline analog of the nitrone DEPMPO: synthesis and spin trapping properties[END_REF] and bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated -cyclodextrin moiety (CD-DIPPMPO), were recently demonstrated to form adducts of increased stability in buffer (Figure 1) [START_REF] Bézière | Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps[END_REF]. CD-DIPPMPO is especially sensitive and specific of extracellular superoxide, as demonstrated with living macrophage suspensions studied in gas-permeable Teflon tubing [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF]. These properties were attributed to the -cyclodextrin moiety that prevents the spin trap to enter cells and ensures hydroxyl radical does not react with the nitrone function [START_REF] Bézière | Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps[END_REF][START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF]. The penetration of cells by spin traps is an important parameter to understand spin trapping results. DMPO and DEPMPO, and their hydroxyl adducts, have been shown to rapidly penetrate lipid bilayers [START_REF] Anzai | ESR measurement of rapid penetration of DMPO and DEPMPO spin traps through lipid bilayer membranes[END_REF]. DIPPMPO, being more lipophilic, is supposed to be more suitable for trapping in subcellular compartments [START_REF] Chalier | 5-Diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO, a crystalline analog of the nitrone DEPMPO: synthesis and spin trapping properties[END_REF] and Mito-DIPPMPO, being conjugated to a large lipophilic triphenylphophonium cation, is assumed to be accumulated 5-10 fold in the cytoplasm, driven by the plasma membrane potential, and further accumulated in the mitochondria [START_REF] Ross | Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology[END_REF]. Inside cells, superoxide adducts of all these spin traps are subject to conversion to the corresponding hydroxyl adducts by action of thiols with or without catalysis by glutathione-peroxidase as shown with purified enzymatic systems or subcellular fractions [START_REF] Bézière | Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps[END_REF][START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF][START_REF] Culcasi | The line asymmetry of electron spin resonance spectra as a tool to determine the cis:trans ratio for spin-trapping adducts of chiral pyrrolines N-oxides: the mechanism of formation of hydroxyl radical adducts of EMPO, DEPMPO, and DIPPMPO in the ischemic-reperfused rat liver[END_REF][START_REF] Hardy | Improving the trapping of superoxide radical with a betacyclodextrin-5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) conjugate[END_REF].

Results obtained with suspensions of adherent cells may occasionally differ from physiological responses. To overcome this issue, a number of solutions have been proposed, such as allowing cells to adhere to quartz flat ESR cuvettes [START_REF] Tanigawa | Spin trapping of superoxide from glass adherent polymorphonuclear leukocytes induced by N-formylmethionyl-leucyl-phenylalanine[END_REF], using a custom made flowtype quartz cuvette [START_REF] Nakagawa | Induction of superoxide in glioma cell line U87 stimulated with lipopolysaccharide and interferon-gamma: ESR using a new flow-type quartz cell[END_REF] or ESR cavity [START_REF] Kotake | Determination of the rate of superoxide generation from biological systems by spin trapping: use of rapid oxygen depletion to measure the decay rate of spin adducts[END_REF], or using microcarrier bead cultures [START_REF] Britigan | Insight into the nature and site of oxygencentered free radical generation by endothelial cell monolayers using a novel spin trapping technique[END_REF][START_REF] Danhier | Influence of cell detachment on the respiration rate of tumor and endothelial cells[END_REF]. Oxygen supply can however be an issue with some of these setups. Since experimentation on biological material requires the comparison of numerous conditions and repetition of experiments for statistical analysis, it is desired to find easily accessible and convenient systems to analyze large numbers of samples in a single day under near-physiological conditions. In this study, we show that ESR detection can be performed directly on coverslip glasses initially designed for microscope studies.

Methods

Reagents

DIPPMPO was synthesized according to previously published protocols [START_REF] Chalier | 5-Diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO, a crystalline analog of the nitrone DEPMPO: synthesis and spin trapping properties[END_REF]. Purity was determined by NMR and HPLC coupled with mass spectrometry and was above 98%. Mito-DIPPMPO and CD-DIPPMPO were prepared using the procedures described for diethoxyphosphoryl analogues [START_REF] Hardy | Improving the trapping of superoxide radical with a betacyclodextrin-5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) conjugate[END_REF][START_REF] Hardy | Mito-DEPMPO synthesized from a novel NH2-reactive DEPMPO spin trap: a new and improved trap for the detection of superoxide[END_REF][START_REF] Hardy | Detection, characterization, and decay kinetics of ROS and thiyl adducts of Mito-DEPMPO spin trap[END_REF]. Their NMR and electrospray ionization-highresolution mass spectrometry (ESI-HRMS) characterizations were previously published [START_REF] Bézière | Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps[END_REF]. DEPMPO was obtained from Radical Vision (Marseille, France). Diethylene triamine pentaacetic acid (DTPA), xanthine (X), xanthine oxidase (XO), superoxide dismutase conjugated with polyethylene glycol (SOD-PEG), phosphate-buffered saline (PBS, pH 7.1-7.5) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France).

Culture of RAW macrophages

Macrophages (RAW 264.7 cells), originally purchased from American Type Culture Collection (ATCC, CRL-9609™), were a kind gift from Dr Jean-Claude Drapier (CNRS UPR 2301, Gif-sur-Yvette, France). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM AQmedia, Sigma-Aldrich) supplemented with 5% fetal bovine serum (Lonza, Levallois-Perret, France) and 1% penicillin/streptomycin (Invitrogen) in a water-jacketed incubator under 5% CO2 at 37°C.

Spin trapping of superoxide radical produced by macrophages

On the day prior to experiment, RAW cells were placed on round microscope coverslip glasses (diameter 12 mm, thickness 0.13-0.16 mm, Thermo Scientific Menzel, Braunschweig, Germany) inserted in a 24-well culture plate and covered with 500 µL DMEM. When indicated, confluent cells on two coverslips (250 000 cells/coverslip) were stimulated with PMA (5 µM, 20 min, 37°C), then washed twice with 500 µL of PBS, and incubated for 20 min at 21°C with the desired spin trap (5-10 mM), DTPA (1 mM), and PBS (final volume 150 µL). The back of the coverslips were slightly dabbed with paper tissue, secured (cell-layer facing outwards) on a quartz slide (130x12x1.5 mm) with a minimum of ESR-silent silicon vacuum grease (Bluestar silicones, Caronno Pertusella, Italy), and introduced in the ESR cavity equipped with a flat quartz protection sleeve (Figure 2). A 24-well culture plate represents thus the opportunity of screening 12 conditions on the same batch of culture cells within half a day.

For comparison purposes, cell suspensions of RAW macrophages (3.6±0.3x10 6 cells/100 µL) prepared as described in a previous work [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF] were inserted in gas-permeable PTFE tubing (Extruded Sub-Lite-Wall®, inside diameter: 0.635 mm, wall thickness: 0.051 mm, Zeus Industrial Products Ltd., Ireland) or glass microcapillary pipettes (50 µL, Hirschmann).

ESR spectroscopy

ESR measurements were performed using an Elexsys 500 ESR spectrometer (Bruker, Wissembourg, France), operating at X-band (9.8 GHz) and equipped with a TM cavity. Typical settings used were: microwave power, 10 mW; modulation frequency, 100 kHz; modulation amplitude, 0. were recorded sequentially at 21°C. Data acquisition and processing were performed using Bruker Xepr software. Baseline correction of ESR spectra was performed by subtracting the signal obtained when the spin trap was omitted.

Computer simulations

Computer simulations of the ESR spectra were performed using the program of Rockenbauer and Korecz [START_REF] Rockenbauer | Automatic computer simulations of ESR spectra[END_REF]. In addition to the g-factor and hyperfine coupling constants, the relaxation and exchange parameters were optimized from the best resolved spectra, and when necessary, two or three superimposed components were taken into account as described in previous works [START_REF] Bézière | Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps[END_REF][START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF]. These parameters are listed in Table 1. Normalized line shapes were used, giving concentration data for all components in arbitrary units.

Results

Detection of superoxide on adherent cells with DEPMPO

Superoxide radical production by RAW macrophages attached to microscope coverslip glasses was induced by 20 min incubation with 5 µM PMA at 37°C. Subsequent trapping of the radical with 10 mM DEPMPO enabled the detection of a significant ESR signal, characteristic of a mixture of superoxide and hydroxyl radical spin adducts in a 1:9 ratio (Figure 3, upper-left panel). Addition of 200 U of SOD-PEG, a membrane-permeative superoxide dismutase but that is incorporated very slowly within the cells [START_REF] Beckman | Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance[END_REF], strongly inhibited the detection of both superoxide and hydroxyl radical adducts, whereas no signal was observed in the absence of PMA (Supplementary Figure S1).

Optimization of the conditions of ESR recordings was performed. It was observed that best results were obtained after 20 min incubation with the spin trap, post PMA-stimulation. Earlier recording start times did not allow optimal accumulation of the spin adducts. Besides, spin adduct decay could not be ignored when measurement start was delayed by more than 20 min. Use of two coverslip glasses as indicated in Figure 2 increased the signal compared to the use of a single coverslip because the microwave excitation spreads over a sample height of ~ 2 cm in the resonator. In our first attempts, the cell-layer on the coverslips was arranged facing the quartz slide but we observed 50% more signal when the cell-layer faced outwards, with the coverslips mounted onto the quartz slide using vacuum grease, probably due to better diffusion of oxygen to the cell layer. The ESR measurement was then restricted to 10 min to prevent the cell-layer from drying out; the signal remained stable during this period.

Comparison of the spin traps

Representative ESR spectra obtained for four cyclic nitrone spin traps with PMA-stimulated RAW macrophages attached to two coverslip glasses are shown in Figure 3. A concentration of 10 mM was used for each spin trap except for CD-DIPPMPO (5 mM), because aggregation tends to reduce the spin trapping rate of the latter at higher concentrations. In most cases, the PMA-dependent signal resulted from overlapping components and computer simulation was required to extract the respective contributions of superoxide and hydroxyl radical adducts displayed in Figure 4. DIPPMPO behaved in a similar way to DEPMPO but with half the sensitivity; DIPPMPO-OH was responsible for 90% of the signal. On the

Discussion

The production of superoxide by PMA-stimulated RAW macrophages was measured by ESR spectroscopy without detachment of the cells from microscope coverslip glasses. PMA is a specific activator of protein kinase C, which in turn performs the phosphorylation of a critical residue in NADPH oxidase (NOX2) in the macrophage membrane [START_REF] Forman | Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling[END_REF]. According to the principles of spin trapping, superoxide radical reacts with the ESR-silent cyclic nitrone, e. g. DEPMPO, to form a persistent radical, referred to as the superoxide adduct (Figure 1), characterized by a distinctive ESR spectrum. The stability of this adduct within cells can be decreased by various pathways (involving reductases, ascorbate, thiols, or heme proteins) as shown in previous works [START_REF] Rosen | Detection of superoxide generated by endothelial cells[END_REF][START_REF] Bézière | Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps[END_REF][START_REF] Reinke | Degradation of DMPO adducts from hydroxyl and 1-hydroxyethyl radicals by rat liver microsomes[END_REF][START_REF] Khramtsov | NMR spin trapping: Detection of free radical reactions using a phosphorus-containing nitrone spin trap[END_REF][START_REF] Bézière | Metabolic stability of superoxide and hydroxyl radical adducts of a cyclic nitrone toward rat liver microsomes and cytosol: A stopped-flow ESR spectroscopy study[END_REF]. Especially, DEPMPO-OOH is converted to DEPMPO-OH by thiols and glutathione-peroxidase [START_REF] Culcasi | The line asymmetry of electron spin resonance spectra as a tool to determine the cis:trans ratio for spin-trapping adducts of chiral pyrrolines N-oxides: the mechanism of formation of hydroxyl radical adducts of EMPO, DEPMPO, and DIPPMPO in the ischemic-reperfused rat liver[END_REF], an enzyme present in RAW macrophages [START_REF] Kim | PEP-1-SIRT2 inhibits inflammatory response and oxidative stress-induced cell death via expression of antioxidant enzymes in murine macrophages[END_REF]. In the present study, a high proportion of hydroxyl radical adduct was observed in the experiment with DEPMPO (Figure 3 and Figure 4), but its level was reduced upon incubation with SOD-PEG (Supplementary Figure S1). Under our conditions with rather short incubation times, most of SOD-PEG remains in the extracellular space where it reacts with superoxide at diffusion-controlled rates [START_REF] Beckman | Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance[END_REF] and efficiently competes with the spin trap (which reacts with superoxide at rates that do not approach 100 M -1 s -1 ) outside cells but not within cells. The hydroxyl radical adduct arose thus from the reduction of DEPMPO-OOH in cells and not from trapping of hydroxyl radical resulting from superoxide disproportionation (Figure 1) [START_REF] Bézière | Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps[END_REF].

Oxygen supply is critical in spin trapping experiments [START_REF] Kotake | Determination of the rate of superoxide generation from biological systems by spin trapping: use of rapid oxygen depletion to measure the decay rate of spin adducts[END_REF]. Previously we observed, using PMA-stimulated RAW macrophage suspensions [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF], that spin trapping was enhanced in gaspermeable PTFE tubing, compared to glass microcapillary pipettes. Here, using adherent cells, different levels of detection were obtained depending on the way the coverslip glasses were mounted onto the quartz slide. When the cell-layer faces outwards, molecular oxygen is constantly replenished through equilibration with air, whereas it is expected to be rapidly consumed when the macrophages are sandwiched between glass and quartz walls or enclosed in ESR flat cells. The increase in ESR signal when the cell-layer faced outwards could be linked to a better diffusion of oxygen to the cell layer in two ways. First, increase in formation of DEPMPO-OOH (and subsequently of DEPMPO-OH) could be the result of an increased superoxide production by NOX2 when availability of oxygen, the substrate of the enzyme, increases. Second, reductases are known to be activated under anaerobic conditions and could reduce the spin adducts to diamagnetic compounds faster upon oxygen deprivation, a process that has been studied with sterically hindered nitroxides [START_REF] Swartz | Use of nitroxides to measure redox metabolism in cells and tissues[END_REF][START_REF] Rosen | Formation and reduction of a nitroxide radical by liver microsomes[END_REF].

Our protocol using cells attached to microscope coverslip glasses has many advantages over previous methods. ESR measurement is performed on adherent cells and preserves cellular integrity. Cell harvesting generally involves enzymatic digestion, such as trypsinization, and mechanical detachment methods such as scraping. However these harvesting methods are known to cause damage to the cells. Enzymatic treatment damages cell membranes by partial hydrolysis of various membrane-expressed proteins and thus impairs cell function [START_REF] Revel | Adhesion of culture cells to their substratum[END_REF][START_REF] Wu | Versican protects cells from oxidative stress-induced apoptosis[END_REF][START_REF] Piercy | Effect of harvesting and sorting on β-1 integrin in canine microvascular cells[END_REF][START_REF] Mutin | Reevaluation of trypsin-EDTA for endothelial cell detachment before flow cytometry analysis[END_REF][START_REF] Hirai | Characterization of cellular motions through direct observation of individual cells at early stage in anchorage-dependent culture[END_REF]. Scraping inflicts physical damage on cells and reduces their viability [START_REF] Heng | Mechanical dissociation of human embryonic stem cell colonies by manual scraping after collagenase treatment is much more detrimental to cellular viability than is trypsinization with gentle pipetting[END_REF][START_REF] Mcneil | A method for incorporating macromolecules into adherent cells[END_REF]. Scraping also alters the metabolic rates of cells (measured as nitroxide reduction kinetics) [START_REF] Batista | Effects of different detachment procedures on viability, nitroxide reduction kinetics and plasma membrane heterogeneity of V-79 cells[END_REF]. Moreover it was shown in several studies that cell detachment induces an increase in cellular ROS [START_REF] Lee | Activation of the leukotriene B4 receptor 2-reactive oxygen species (BLT2-ROS) cascade following detachment confers anoikis resistance in prostate cancer cells[END_REF][START_REF] Chanvorachote | Caveolin-1 regulates endothelial adhesion of lung cancer cells via reactive oxygen species-dependent mechanism[END_REF][START_REF] Kerkweg | Disruption of skeletal myocytes initiates superoxide release: contribution of NADPH oxidase[END_REF]. The procedure described in this work is thus considerably less liable to artifacts linked with non-physiological production of ROS and to misinterpretation of spin adduct accumulation in cells with impaired metabolism as implying higher ROS production. Moreover, commercial disposable coverslip glasses are easy to handle and enable studies on large numbers of samples and controls over a short period of time, which is not the case when the expensive and fragile ESR flat-cells are used. In addition, superoxide detection is performed with only 500 000 cells, which is almost one-tenth as many cells as were used for our previous study using gas-permeable PTFE tubing [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF].

On the whole, the relative amounts of each adduct obtained on adherent cells with different spin traps correlate those previously obtained on cell suspensions with larger cell numbers (1-5x10 6 cells/100 µL) [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF], suggesting that like for polymorphonuclear leucocytes PMAinduced superoxide release by macrophages was independent of cell adhesion [START_REF] Tanigawa | Spin trapping of superoxide from glass adherent polymorphonuclear leukocytes induced by N-formylmethionyl-leucyl-phenylalanine[END_REF]. DIPPMPO, like DEPMPO, is characterized by a higher detection of the hydroxyl adduct as a product of reduction of the superoxide adduct inside cell, since these spin traps are known to enter cells [START_REF] Chalier | 5-Diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO, a crystalline analog of the nitrone DEPMPO: synthesis and spin trapping properties[END_REF][START_REF] Anzai | ESR measurement of rapid penetration of DMPO and DEPMPO spin traps through lipid bilayer membranes[END_REF]. Mito-DIPPMPO and CD-DIPPMPO, which are known not to form ESRdetectable adducts from low fluxes of hydroxyl radical [START_REF] Bézière | Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps[END_REF], appear as more specific of superoxide. SOD-controls are then largely unnecessary with these spin traps to discriminate between superoxide or hydroxyl radical as the origin of the adduct formation. The extracellular localization of CD-DIPPMPO explains why the adducts are protected from degradation by intracellular components, whereas partial conversion of the superoxide to the hydroxyl adduct is observed with Mito-DIPPMPO, which was designed to enter the cell and target the mitochondria [START_REF] Hardy | Mito-DEPMPO synthesized from a novel NH2-reactive DEPMPO spin trap: a new and improved trap for the detection of superoxide[END_REF][START_REF] Hardy | Detection, characterization, and decay kinetics of ROS and thiyl adducts of Mito-DEPMPO spin trap[END_REF].

Conclusion

In conclusion, we confirmed that spin trapping with CD-DIPPMPO coupled to ESR detection enable specific detection of superoxide produced by PMA-stimulated macrophages, as previously observed with cell suspensions [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF], but here the detection was performed on adherent cells without detachment from microscope coverslip glasses. The sample preparation proposed in this work is straightforward, requires small numbers of adherent cells, and can be used when stress involved in trypsinization or mechanical detachment has to be avoided. In addition, we expect the oxygen supply to be constant in our system, as opposed to closed ESR flat cells that promote hypoxia. ESR detection performed on undetached cells using this setup paves the way for a large number of applications. For example, it would enable convenient medium-throughput screening of antioxidants and ROSmodulating agents in various cell lines. Alternatively, it could be used to screen the stability of ESR imaging contrast agents, such as aminoxyl or triarylmethyl radicals [START_REF] Bézière | First combined in vivo X-ray tomography and high-resolution molecular electron paramagnetic resonance (EPR) imaging of the mouse knee joint taking into account the disappearance kinetics of the EPR probe[END_REF], in the presence of cells, prior to in vivo studies. Table 1: Parameters used for the simulation of the spectra in Figure 1 1 are shown as grey lines. We observe on the lower-right panel that the linewidth of CD-DIPPMPO-OOH spectrum is larger than that of DEPMPO-OOH and DEPMPO-OH adducts (upper-left panel) due to a higher molecular tumbling correlation time. As a consequence, the signal-to-noise ratio obtained with CD-DIPPMPO is not as high as with DEPMPO even if the amount of spin adducts is similar.

Tables

Figure 3 :

 3 Figure 3: Comparison of ESR spectra obtained in the presence of PMA-stimulated RAW macrophages attached to two microscope coverslip glasses. Activation of the macrophages (250 000 cells/coverslip) was performed for 20 min with 5 µM PMA at 37°C and followed by incubation with DEPMPO (10 mM), DIPPMPO (10 mM), Mito-DIPPMPO (10 mM), or CD-DIPPMPO (5 mM) for 20 min in PBS (pH 7.1-7.4) containing 1 mM DTPA at 21°C. ESR settings are described under Methods. The experimental spectra (black lines) are the sum of ten scans and are representative of three independent experiments. Calculated signals using parameters from Table1are shown as grey lines. We observe on the lower-right panel that the linewidth of CD-DIPPMPO-OOH spectrum is larger than that of DEPMPO-OOH and DEPMPO-OH adducts (upper-left panel) due to a higher molecular tumbling correlation time. As a consequence, the signal-to-noise ratio obtained with CD-DIPPMPO is not as high as with DEPMPO even if the amount of spin adducts is similar.

Figure 4 :FiguresFigure 1

 41 Figure 4: Concentration of superoxide and hydroxyl radical adducts of the studied spin traps obtained with PMA-stimulated RAW macrophages attached to two microscope coverslip glasses.Activation of the macrophages (250 000 cells/coverslip) was performed for 20 min with 5 µM PMA at 37°C and followed by incubation with DEPMPO (10 mM), DIPPMPO (10 mM), Mito-DIPPMPO (10 mM), or CD-DIPPMPO (5 mM) for 20 min in PBS (pH 7.1-7.4) and 1 mM DTPA at 21°C. The concentration of each adduct expressed in arbitrary unit was derived from computer simulation of the ESR spectra by normalized lineshapes as described under Methods. The data are the average of three independent experiments and the error bars reflect the standard deviation. The hydroxyl adduct of CD-DIPPMPO was under the limit of detection of our method.

Figure

  Figure 4

4

 4 Figure 4

Legends for figures Figure 1: Structure of the spin traps under study and general principles of spin adduct formation and decay. Figure 2: Experimental procedure for superoxide detection on adherent cells.

  . On the day prior to experiment, RAW macrophages in 500 µL DMEM were placed on round microscope coverslip glasses inserted in a 24-well culture plate. When indicated, confluent cells on two coverslips (250 000 cells/coverslip) were stimulated with PMA (5 µM, 20 min, 37°C), then washed twice with PBS, and incubated for 20 min at 21°C with the desired spin trap (5-10 mM) in 150 µL PBS containing DTPA (1 mM). Both coverslips were then mounted on a quartz slide using vacuum grease and introduced in the ESR cavity equipped with a flat quartz protection sleeve. Sequential ESR spectra were recorded for 10 min as indicated under Methods.

	This is an Accepted Manuscript of an article published by Taylor & Francis in Free Radical Research on 5
	June 2015, available online: http://www.tandfonline.com/0.3109/10715762.2015.1045504
	Spin adduct (% in the simulation)	Conformer	AP (mT)	AN (mT)	AH (mT)	AHγ (mT)
	DEPMPO-OOH trans (10%)	T1 (53%)	5.19	1.31	1.19	0.08
		T2 (47%)	4.85	1.32	1.01	0.09
	DEPMPO-OH (90%)	-	4.71	1.40	1.32	-
	DIPPMPO-OOH trans (10%)	T1 (48%)	5.17	1.30	1.20	0.06
		T2 (52%)	4.79	1.32	1.02	0.09
	DIPPMPO-OH (90%)		4.68	1.40	1.33	-
		-
	Mito-DIPPMPO-OOH trans (80%)	T1 (52%)	5.33	1.28	1.48	-
		T2 (48%)	5.29	1.29	0.87	-
	Mito-DIPPMPO-OH (20%)	-	5.29	1.36	1.05	-
	CD-DIPPMPO-OOH trans (100%)	T1 (54%)	5.34	1.26	1.25	-
		T2 (46%)	5.26	1.30	1.12	-

© 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 13/17

© 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

Acknowledgments

The authors wish to thank Dr. J.-C. Drapier (CNRS UPR 2301, Gif-sur-Yvette, France) for the generous gift of RAW 264.7 macrophages. They are also deeply grateful to Dr. A. Rockenbauer (Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Budapest, Hungary) for the use of his simulation program and for his precious advice

Figure 2 This is an Accepted Manuscript of an article published by Taylor & Francis in Free Radical Research on 5 June 2015, available online: http://www.tandfonline.com/0.3109/10715762.2015.1045504 Figure 3 This is an Accepted Manuscript of an article published by Taylor & Francis in Free Radical Research on 5 June 2015, available online: http://www.tandfonline.com/0.3109/10715762.2015.1045504

Declaration of interest

The authors are indebted to CNRS and the Agence Nationale de la Recherche (ANR-09-BLAN-0193-02, SPIN BioRad) for their financial support.

Abbreviations

Supplementary material available online

Supplementary Figure 1 to be found online at http://informahealthcare.com/doi/abs/10.3109/10715762.2015.1045504


Emmanuel Richaud

Ludmila Audouin

Xavier Colin

Bruno Fayolle

Jacques Verdu

New Developments in the Field of Radiochemical Ageing of Aromatic Polymers

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Polymers having an aromatic backbone polymers have a high mechanical strength and a high modulus. Their aromaticity increases their resistance for use in relatively severe conditions especially in aerospace and nuclear industry for which lifetime prediction is a key issue. For example, a challenge for nuclear plants is to extend lifetime from the initially planned 40 years duration to 50 or 60 years, which makes necessary to determine lifetime by a non-empirical method. Since polymers mechanical failure originates from chain scission or crosslinking of the backbone, the ideal method of lifetime prediction would first involve the elaboration of a kinetic model for chain scission and crosslinking. Then, the changes of molecular mass would be related to the changes of mechanical properties using the available laws of polymers physics. Lifetime would be then determined using a pertinent lifetime criterion. A noticeable difficulty comes here from the fact that oxidation, which plays a key major role in chain scission, is diffusion controlled and thus heterogeneously distributed in sample thickness. It is crucial, indeed, to determine experimentally and to predict this depth distribution of chain scission and crosslinking because it will play a key role on fracture properties. This chapter will be henceforward devoted to the effect of aromaticity on radiostability, the effect on temperature on the chain scission/crosslinking competition, the diffusion limited oxidation (which will be illustrated by the effect of dose rate, atmosphere and sample thickness), then some concluding remarks on oxidative stability of aromatic polymers and the possible link with the absence of macromolecular mobility below T g . We will start by some basics of radiochemistry which are necessary for the good understanding of this paper, and especially the quantitative treatment for crosslinking and chain scission.

Basics of radiochemical degradation

Let us first consider the reaction:

A + h  B The radiochemical yield is defined as the number of B molecules that are generated per absorbed joule: where: -G' is the radiochemical yield expressed in mol J -1 , -n is the number of moles of B which is formed by the radiochemical reaction, -E' is the amount of absorbed energy in J, -E is the amount of absorbed energy in 100 eV, -N is the number of B molecules, -N Av is the Avogadro's number, -G is the yield in molecules per 100 eV absorbed. Since the absorbed dose (denoted by  and expressed in Gy) is defined as the amount of received energy (in J) per kilogram of polymer, the following equation can be derived: (2)

So that: 7 dc r1 0 . G . I dt

  (3)
r being the rate of a radiochemical event (in mol l -1 s -1 ), I the dose rate in Gy s -1 and c the concentration of reacted or generated species (in mol kg -1 even if one often considers that it is the same than in mol l -1 ).

Mathematical treatment for chain scission and crosslinking

Irradiation can provoke both chain scission and crosslinking, the relative proportion of these phenomena depending on many factors which will be presented in the following. The radical coupling may lead to trifunctionnal or tetrafunctionnal crosslinking noduli called respectively Y-and H-crosslinkings (Fig. 1):

°°H-crosslinking °°Y-crosslinking The radiochemical yields for chain scissions G(s) and crosslinking G(x) in thick samples can be tentatively assessed using -The Charlesby-Pinner's [START_REF] Charlesby | Atomic Radiation and Polymer[END_REF] from an analysis of the residual soluble fraction after beginning of sol-gel transition, -Saito's equations [START_REF] Saito | On the Effect of High Energy Radiation to Polymers I. Cross-linking and Degradation[END_REF] describing the average molar mass changes for soluble polymers before the sol-gel transition. The corresponding mathematical treatment of these theories differs for Y-and Hcrosslinking: 1. In the case of H-crosslinking mode:

  7 HH NN 0 11 s x 10 . G (s) G (x) .δ MM      (4) 7 H H WW 0 11 s G ( s ) 2x 10 . 2G (x) .δ 22 MM            (5) 7 1/2 H SS W0 H H G( s ) 1 0 ww 2G (x) M. G ( x ) . δ   (6)
2. In the case of Y-crosslinking mode:

  7 YY NN 0 11 s x 10 . G (s) G (x) .δ MM      (7) 7 Y Y WW 0 11 s G ( s ) x1 0. G ( x ) . δ 22 MM            (8) 7 1/2 Y S N0 Y Y 2G (s) 1.93 10 13 w G( x ) M. G ( x ) . δ   (9)
where:

w S = 1 -w I is the soluble fraction, -G H (s), G H (x), G Y (s), and G Y (x) are the radiochemical yields expressed for 100 eV respectively for chain scissions and crosslinking for an H-and a Y-crosslinking mechanism, - is the dose (Gy) -M N0 and M W0 are respectively the initial number and weight average molar mass (kg mol -1 ).

Effect of aromaticity on radiostability

The degradation of aromatic polymers was studied by many authors: 1. By monitoring mechanical properties [START_REF] Sasuga | Degradation in tensile properties of aromatic polymers by electron beam irradiation[END_REF], see Table 1):

These results give a first indication on the relative radiostability of aromatic polymers. Let us first mention that these lethal doses are considerably greater than those compiled by Wilski for polymer having an aliphatic backbone [START_REF] Wilski | The radiation induced degradation of polymers[END_REF]. It can be attributed to the well-known stabilizing effect of aromatic groups which was first observed on methyl methacrylate-styrene copolymers [START_REF] Charlesby | Energy Transfer in Macromolecules Exposed to Ionizing Radiations[END_REF], Kellman et al., 1990[START_REF] Thominette | Radiochemical ageing of poly(vinyl chloride) plasticized by didecylphthalate[END_REF]. However, the results of these studies cannot be directly used in kinetic models for aromatic polymers. Table 1. Dose for reducing strain at break to 50% and 20% of initial value for various aromatic polymers submitted to 5.10 3 Gy s -1 in air (sample thickness ~ 0.1-0.2 mm) [START_REF] Sasuga | Degradation in tensile properties of aromatic polymers by electron beam irradiation[END_REF].

2. By radical appearance measured by Electron Spin Resonance spectroscopy [START_REF] Heiland | Measurement of Radical Yields To Assess Radiation Resistance in Engineering Thermoplastics[END_REF], see Fig. ) dose (kGy) Fig. 2. Concentration in radicals versus dose measured in PPO (), Kevlar (), PSU (), PEPO (), Ultem (), Kapton (), PEEK () in aromatic polymers under 1 -5 kGy h -1 dose rate at 77 K [START_REF] Heiland | Measurement of Radical Yields To Assess Radiation Resistance in Engineering Thermoplastics[END_REF].

Data presented in Fig. 2 permit the yield in radical build-up to be calculated according to the simplified scheme for radiochemical degradation in the absence of oxygen:

Polymer (PH) + h  P° r i P° + PH  PH + P° k P P°  P° + s k R P° + P°  P-P + x k C P° + P°  F + PH k D Initiation: Propagation: -scission:
Termination by coupling: Termination by disproportionnation:

Where F is a double bond. The rate of radical formation is:

    2 it dP r2 k P dt    (10)
with:

k t = k C + k D (11)
Fig. 2 indicates the existence of an asymptote characterizing a steady-state at which the concentration in radicals is:

  i t r P 2k   (12)
Eq. 10 gives:

            i dP
G(P°) = G i =   t0 dP dt      (16)
The corresponding yields for radical build-up are given in -By gas yield emission: Concerning the yield for total gas emission, it can be checked that there is no great difference between gamma, proton and electron-beam irradiation [START_REF] Hegazy | Irradiation effects on aromatic polymers: 2. Gas evolution during electron-beam irradiation[END_REF], Hill & Hopewell, 1996) and that these values are considerably lower than those measured for aliphatic polymers such as PE or PP [START_REF] Schnabel | Degradation by High Energy Irradiation[END_REF]. An example is worth to be mentioned [START_REF] Schnabel | Degradation by High Energy Irradiation[END_REF]: aliphatic polysulfones have a gas emission yield equal to 39 (poly butene-1sulfone) or 71 (poly hexene-1-sulfone) meanwhile PES or PSU gas emission yields are lower than 1, illustrating here the protective effect by aromatic rings. However, the relative proportion of each emitted gas can vary due to some differences in temperature rising under irradiation. As it will be seen below, this difference due to irradiation nature has a lower influence on the radio induced degradation of polymer than temperature, nature of environment atmosphere and oxygen gradient in sample thickness.

I (kGy h -1 ) Gaz Kapton Upilex-R Upilex-S PEEK-c PEEK-a PES U-PS U-
SO 2 ----- 2 3 . 0 4 3 . 0 - 10 6120
Table 3. 103.G(gas) for irradiation at 30°C 100 m films vacuum (Hegazy et al., 1992a) 3. Let us turn to the radiation-induced crosslinking observed in PEEK and PSU. Fig. 3 depicts the changes in thermal behavior which are induced by radio-ageing for a thick PEEK sheath under air (Richaud et al., 2010a). As it will be seen later, the observed behavior is comparable to the one obtained for an irradiation under vacuum. Virgin and degraded samples were characterized by DSC heating-cooling-heating cycle. -In the case of virgin sample, melting endotherms for the first and the second heating ramps are very comparable (peak temperature and enthalpy). The crystallization from molten state is characterized by a sharp exotherm at c.a. 300°C. -

In the case of 30.7 MGy irradiated sample, melting endotherm for the second heating ramp and crystallization exotherm are shifted to the lower temperatures with a lower transition enthalpy. During irradiation of semi-crystalline polymers, chain scission and crosslinking occur only in amorphous phase, the crystalline phase undergoes only small changes so that melting characteristics remain almost unchanged at the first heating scan. After a first melting however, the sample is homogenized, and the changes of molar mass, branching ratio or crosslink density resulting from irradiation affect melting and crystallization. Irradiation effects become then observable by DSC. Here, the decrease of crystallization temperature and enthalpy is explained by the occurrence of radiation-induced crosslinking [START_REF] Sasuga | Electron irradiation effects on dynamic viscoelastic properties and crystallization behaviour of aromatic polyimides[END_REF]. This latter disfavors crystallization because it lowers the transport rate of chain segments from the melt to growing crystals. As a consequence, the degree of crystallinity and the lamellae thickness are decreased that explains the observed decrease of melting point and melting enthalpy. Comparable data were obtained by Hegazy (Hegazy et al., 1992c). T g values from this study are reported (Fig. 4): It is well known that for a linear polymer, T g is an increasing function of molar mass.

According to Fox and Flory (Fox & Flory, 1950):

FF gg 0 N k TT M  (17)
Where: -T g0 is the T g of a virtual infinite polymer (K). -M N is the number average molar mass (kg mol -1 ). -k FF is a constant characteristic of the chain chemical structure (K kg mol -1 ). Where: -T gl is the T g of a virtual linear polymer containing all the structural units of polymer except crosslinks (K). -F is a flex parameter linked to chain stiffness (kg mol -1 ). - A is the concentration in elastically active chains, which is calculated from crosslinks concentration (mol kg -1 ). Under vacuum, chain scission is generally negligible. Above gel point, it seems to us that T g changes are given by combining Fox-Flory relationship and Saito's equation (assuming first that s << x): [START_REF] Murakami | Gamma-rays irradiation effects on polysulfone at high temperature[END_REF][START_REF] Richaud | Diffusioncontrolled radiochemical oxidation of bisphenol A polysulfone[END_REF], and PEEK (: 24 kGy h -1 , ,,: 60 MGy h -1 for 3 different initial crystalline microstructures) samples irradiated at several temperatures [START_REF] Vaughan | On crystallization, morphology and radiation effects in poly(ether ether ketone)[END_REF], Richaud, 2010a).

From the above given mechanistic scheme where chain scission results from radical rearrangement and crosslinking results from radical coupling scheme, the following stationary rate expressions can be derived: -Rate of chain scission:

ds dt = 1/2 Ri CD kr 2kk      (20)
-Rate of crosslinking:

C i CD k dx r dt 2 k k   (21)
Assuming that disproporationation is negligible compared to coupling for polymers under study, it comes:

i dx r dt 2  ( 22
)
So that : G i = 2G(x) in the absence of oxygen. It seems that this relationship disagrees with experimental results in the case of aromatic polymers which will be discussed next.

As a conclusion of this short review of experimental results, it is clear that aromatic polymers belong the family of relatively radiostable polymers because primary radiochemical events, presumably radiolytic chain scissions of C-H bonds, have a radiochemical yield less than 0.5, i.e. 5 to 50 times lower than for aliphatic polymers. Aromatic groups are actually able to dissipate a great part of the absorbed energy into reversible processes (fluorescence, phosphorescence...). Data militate in favour of the following ranking:

N C C C N C O O O O O C O N H C O C CH 3 CH 3 S O O > > > >
A comparison of the rate of gel content increase (Fig. 4) for samples irradiated at various temperatures and dose rates shows that gelation is more sudden in the case of PSU and more progressive in PEEK which means that radiation generated radicals would be more reactive for PSU than PEEK. Data also show unambiguously: -The protective effect of aromaticity: Kapton®, which is the most aromatic polymer, presents a very limited degradation, or even no significant degradation [START_REF] Kang | Effect of electron beam irradiation on polyimide film[END_REF], Richaud et al, 2010b). -

The role of SO 2 moiety: experimental results for radiochemical yields values [START_REF] Horie | On the kinetics of polymer degradation in solution: Part XI-Radiolysis of poly(olefin sulfones)[END_REF] shows that aliphatic polysulfones are among the less stable polymers with G(s) values on the order of 10. High yields of SO 2 emission are also observed indicating thus the probable existence of the weak [START_REF] Li | Thermal degradation of bisphenol A polysulfone by highresolution thermogravimetry[END_REF][START_REF] Molnár | Thermal degradation of chemically modified polysulfones[END_REF] carbon-sulfone bond cleavage. It seems reasonable to suppose that such events occur also in PSU even though the yield is considerably lower owing to the well-known protective effect of aromatic nuclei illustrated for instance in studies on isobutylenestyrene.

-

The role of isopopylidene group: gelation is undoubtedly due to the coupling of alkyl radicals. The difference in gelation rate would be explained by the nature of radicals: in PEEK, only aryl radicals can react meanwhile both aryl and primary methyl radical can react in PSU.

°°C

CH 3 CH 2 °C CH 2 °CH 3 C CH 3 CH 2 CH 2 C CH 3
Theoretically, it seems that yields in gas emission G(gas) is half of yield radicals G(P°) as illustrated by proposal of degradation mechanisms below: Analogous mechanisms could lead to carbon monoxide formation from PEEK. Data for Kapton®, PEEK and PSU (Table 1 andTable 2) seem also to show that: G(P°) >> 2G(x) and 2G(gas)

PEEK and PSU values gathered in this work are compared with those for polyethylene [START_REF] Khelidj | Absolute Rate Constants for Hydrocarbon Autoxidation. XXI. Activation Energies for Propagation and the Correlation of Propagation Rate Constants with Carbon-Hydrogen Bond Strengths[END_REF], and those compiled for cyclohexane and benzene [START_REF] Ferry | Comportement des verres cyclohexane/benzène et des copolymères éthylène/styrène sous rayonnements ionisants: Transferts d'énergie et d'espèces entre les groupements aliphatiques et aromatiques[END_REF] 4. Yields for radicals, gas emission, crosslinking and double bond formation (corresponding to disproportionnation process). *: G(H 2 ) is expected to be close to G(gas), **: G(F) is expected to be negligible in aromatic polymers.

These results call for the following comments: 1. The effect of aromaticity on radiostability is confirmed. 2. The ratio G(P°)/G(gas) is surprisingly lower than 1 for cyclohexane, is fairly close to 2 for benzene and PE, but increases for PSU and PEEK. A possible explanation is the existence of very stable radicals that would be detected by ESR but would contribute neither to gas emission nor to crosslinking (see later) as for example:

O O °Let us also note that the absence of contribution to gas emission seems easily explainable because the probability for generating a short and volatile segments from such macroradicals is very low. 3. The ratio G(P°)/(G(x)+G(F)) is also clearly greater than 2 for PSU and particularly for PEEK meanwhile it is close to 1 in PE and in cyclohexane. The comparison with benzene suggests that this behaviour is due to the macromolecular structure of radicals preventing them to react by coupling to give crosslinking. In fact, even if a negative concavity is observed in Fig. 1 and indicates the existence of a termination process even at 77 K, the relatively low value of G(x) indicates that segmental mobility would be reduced below T g , which will be discussed in a first section dedicated to temperature effect and another one dealing with oxidizability. (Sasuga &[START_REF] Sasuga | Radiation deterioration of several aromatic polymers under oxidative conditions[END_REF][START_REF] Murakami | Gamma-rays irradiation effects on polysulfone at high temperature[END_REF][START_REF] Murakami | Gamma-rays irradiation effects on polysulfone at high temperature[END_REF].

Effect of temperature

Data in

As previously suggested, the only knowledge of changes in macroscopic (engineering) properties does not permit to ascribe the right cause of failure. Yields in chain scission and crosslinking permit to elucidate the degradation mechanism. Let us first comment the T g changes in PES [START_REF] Li | Preparation of the crosslinked polyethersulfone films by high-temperature electron-beam irradiation[END_REF]

Tg (°C)

Fig. 6. Change in T g for PES submitted to electron beam irradiation at room temperature () or 230°C () [START_REF] Li | Preparation of the crosslinked polyethersulfone films by high-temperature electron-beam irradiation[END_REF].

Before sol-gel transition, T g changes are given by combining Fox-Flory law (Fox & Flory, 1950) with Saito's equation [START_REF] Saito | On the Effect of High Energy Radiation to Polymers I. Cross-linking and Degradation[END_REF] irrespectively of H-or Y-crosslinking mode:

  7 gg 0 F F F F NN 0 11 TT k . 1 0 . k . G ( s ) G ( x ) . MM           (23)
It turns into:

for a pure crosslinking mechanism:

g 7 FF dT 10 .k .G(x) d    (24)
for a pure chain scission mechanism:

g 7 FF dT 10 .k .G(s) d    (25)
Results presented in Fig. 5 indicate that crosslinking predominates above T g , whereas chain scission predominates below T g . Comparable exploitation can be done from other published results [START_REF] Murakami | Gamma-rays irradiation effects on polysulfone at high temperature[END_REF][START_REF] Brown | Effect of gamma radiation on two aromatic polysulfones II. A comparison of irradiation at various temperatures in air-vacuum environments[END_REF]: All data converge towards the fact that G(x) increases with temperature. Despite some scattering, it also suggests that G(s)/G(x) decreases with temperature. According to [START_REF] Brown | Effect of gamma radiation on two aromatic polysulfones II. A comparison of irradiation at various temperatures in air-vacuum environments[END_REF], it falls to 0 above T g .

The results in Table 6 can be explained as follows: irradiation creates macroradicals. At low temperature, they react by an unimolecular process which generates a chain scission (possibly accompanied by gas emission). At high temperature, macromolecular mobility is sufficient to permit macroradicals to react by coupling to give a crosslinking. According to Zhen [START_REF] Zhen | The effect of chain flexibility and chain mobility on radiation crosslinking of polymers[END_REF] [START_REF] Murakami | Gamma-rays irradiation effects on polysulfone at high temperature[END_REF][START_REF] Brown | Effect of gamma radiation on two aromatic polysulfones II. A comparison of irradiation at various temperatures in air-vacuum environments[END_REF].

Let us now discuss of the possibility of Y-and H-crosslinking.

-At 150°C, Hill [START_REF] Hill | The crosslinking mechanism in gamma irradiation of polyarylsulfone: evidence for Y-links[END_REF] unambiguously showed that PSU crosslinks in Y-mode.

A comparable analysis was performed by Li [START_REF] Li | Preparation of the crosslinked polyethersulfone films by high-temperature electron-beam irradiation[END_REF]. A proposal of mechanism is shown below:

C CH 3 CH 2 °C CH 3 CH 2 °-
Kudo [START_REF] Murakami | Gamma-rays irradiation effects on polysulfone at high temperature[END_REF] proposed a Y-mechanism also. However, by reexaminating his results using the above equations, his conclusion is questioned. -Richaud [START_REF] Richaud | Diffusioncontrolled radiochemical oxidation of bisphenol A polysulfone[END_REF] proposed a H-crosslinking mechanism at 60°C, basing on the observation that if isopropopylidene groups are generated, the C-CH 2 ° group is considerably more reactive than aryl one and should react by coupling to give C-CH 2 -CH 2 -C crosslinking bridges corresponding more to a H-crosslinking mechanism. As a conclusion, it seems that elevating the temperature promotes the crosslinking. However, the nature of this latter and presumably the role of temperature remain unclear.

Effect of atmosphere and sample thickness

Results in Fig. 7 [START_REF] Sasuga | Radiation deterioration of several aromatic polymers under oxidative conditions[END_REF] clearly show that oxygen accelerates the degradation of aromatic polymers. The nature of the responsible process is suggested in a study on the degradation of PET films monitored by T g measurements [START_REF] Burillo | Electron beam irradiation effects on poly(ethylene terephthalate)[END_REF]: a strong T g decrease is observed in air at the beginning and is certainly due to chain scission whereas a moderate T g decrease is observed for irradiation under vacuum. As proposed by Sasuga [START_REF] Sasuga | Oxidative irradiation effects on several aromatic polyimides[END_REF], polymer radiodegraded under anaerobic conditions would undergo mainly crosslinking meanwhile they would undergo chain scissions when they are degraded under aerobic (oxidative) conditions. The effect of atmosphere nature and the effect of thickness have the same origin linked to oxygen diffusion: thick samples present a diffusion limited oxidation (DLO), i.e. that their surface undergoes an oxidative degradation leading to chain scission meanwhile bulk undergoes an anaerobic radio-ageing generating radicals reacting only by coupling. This effect was quantified by comparing the degradation of thin and thick samples by means of GPC (for molar mass assessment and subsequently chain scission and crosslinking yields assessment) and T g [START_REF] Richaud | Diffusioncontrolled radiochemical oxidation of bisphenol A polysulfone[END_REF]. T g decreases for both 2 mm and 200 µm thick samples, but more significantly for thin films (Fig. 8) which is not surprising since T g decreases with chain scissions and increases with crosslinking. 1. Case of thin films Thin films undergo certainly a "pure" random chain scissions process. A graphical estimation (Fig. 5) gave for the thin samples (using k FF ~ 220 K kg mol -1 for PSU): from which one obtains G(s) ~ 0.15 2. Case of thick samples Here, the simultaneous crosslinking compensates partially chain scissions consequences on T g changes. For ideal networks having no dangling chains, the effect of crosslinking on T g obeys the DiMarzio's law (DiMarzio,1964 -equation ( 18)) with: -T gl ~ 473 K -F PSU = 0.1105 kg mol -1 -k DM is the DiMarzio's constant: k DM is close to 1 - is the elastically active chains concentration. For ideal tetrafunctional networks:

 = 2.x (26)
x being the concentration of crosslinks events. Supposing that DiMarzio's equation can be applied for non-ideal networks, Eq. 23 turns into the Fox-Loshaek [START_REF] Fox | Influence of molecular weight and degree of crosslinking on the specific volume and glass temperature of polymers[END_REF] equation for low conversion of the crosslinking process:

T g = T gl + k FL .x (27)
with the Fox-Loshaek constant k FL being equal to: k FL = 2.T gl .F.k DM (28) so that: k FL ~ 104 K mol kg -1 Finally, the global changes of T g would be given by: In other words, effect of sample thickness is explained by the effect of oxygen diffusion (which will be developed in the next section): irradiation generates radicals. In the absence of oxygen (i.e. for the core a bulk sample), these later react together by coupling which gives a crosslinking phenomenon. When oxygen is present (which is the case for thin samples or the superficial layer of a bulk sample), it orientates the degradation towards an oxidative mechanism with the formation of peroxy radicals POO°, and POOH of which decomposition induces chain scissions.

Effect of dose rate

This effect is illustrated by some results by Tavlet [START_REF] Tavlet | Aging of organic materials around high-energy particle accelerators[END_REF] or deformation (%) Fig. 9. Radiation degradation in an epoxy resin on strain (,), stress (,). Open symbols correspond to long term (low dose rate) ageing [START_REF] Tavlet | Aging of organic materials around high-energy particle accelerators[END_REF].

There are two effects to be distinguished: -An increase in temperature involved by enhancing the dose rate, -A physical and chemical effect linked to Diffusion Limited Oxidation, which will be described below. From a simplified theory of diffusion controlled oxidation [START_REF] Audouin | Role of oxygen diffusion in polymer ageing: kinetic and mechanical aspects[END_REF], a rough estimation of oxygen consumption rate r OX in films or superficial layers of thick samples can be done:

O2 2 S OX 2 D. [ O ] r TOL  (30)
where:

-TOL is the thickness of oxidized layer (m), -D O2 is the oxygen diffusion coefficient in polymer amorphous phase (m 2 s -1 ), -[O 2 ] S is the equilibrium concentration under atmospheric pressure given by Henry's law:

[O 2 ] = s O2 P O2 (31) s O2 being the solubility coefficient of oxygen in polymer amorphous phase. r OX can be expressed from a simplified mechanistic scheme for radio-thermal oxidation:

PH + h  P° + ½ H 2 r i P° + O 2  P° k 2 POO° + PH  POOH + P° k 3 POO° + POO°  inact. prod. + O 2
k 6 k i being the rate constants (expressed in l mol -1 s -1 ). The oxidation rate for oxygen consumption can be expressed as:

  2 OX dO r dt  = -k 2 .[P°][O 2 ] + k 6 .[POO°] 2 (32)
Using the classical steady-state assumption:

k 2 .[P°][O 2 ] = r i + k 3 .[POO°].[PH] (33)
 TOL  I -1/4 (40)
It is thus demonstrated that radiothermal oxidation is characterized by a skin-core structure: superficial layers are oxidized meanwhile bulk not. Results by Tavlet [START_REF] Tavlet | Aging of organic materials around high-energy particle accelerators[END_REF] are thus explained as follows: for irradiation performed under a relatively elevated dose rate, only a very thin polymer layer is degraded and time for drop of elongation at break is longer than for irradiation performed under a moderate dose rate. This reasoning is confirmed by the comparison of a bulk sample (4 mm) for which no dose rate effect is observed undoubtedly because the thickness of degraded superficial layer is negligible compared to the total thickness of bulk material. Examples of Oxidized layers measured for radiochemical degradation of aromatic polymers (Richaud et al., 2010a) are presented in Fig. 10 and confirm the existence of a Diffusion Limited Oxidation.

On the radio-induced oxidizability of aromatic polymers

It seemed to us interesting to compare the k 3 2 /k 6 ratio estimated from radiodegradation of aromatic polymers with polypropylene or polyethylene ones. Let us recall that k 3 2 /k 6 describes the oxidizability of a polymer irrespectively of the oxidation mode (i.e. photo, thermo-or radio-induced). k 3 2 /k 6 can be calculated from Eq. (32) using: -

[PH] = /M,  being the polymer density and M the molar mass of repetitive unit, r i = 10 -7 .G(P°).I from Heiland's results (Table 2), -r OX from TOL measured for example in FTIR mapping mode (Eq (30)). One can use with a good approximation the values of oxygen permeation of BisPhenol A Polycarbonate values for in PEEK and PSU (Van Krevelen & Te Nijenhuis, 2009) at 60°C : -D O2 = 8.110 -8 m² s -1 -s O2 = 410 -8 mol l -1 Pa -1 For samples exposed under 1.5 bar oxygen, TOL were found close to: -100 µm in PSU [START_REF] Richaud | Diffusioncontrolled radiochemical oxidation of bisphenol A polysulfone[END_REF], -70 µm in PEEK (Fig. 10). so that one can calculate (Table 6): -k 3 2 /k 6 = 1.310 -5 l mol -1 s -1 for PSU k 3 2 /k 6 = 1.810 -6 l mol -1 s -1 for PEEK These orders of magnitude are surprisingly on comparable or higher than values reported in PP [START_REF] Richaud | Effect of oxygen pressure on the oxidation kinetics of unstabilised polypropylene[END_REF] and PE [START_REF] Khelidj | Absolute Rate Constants for Hydrocarbon Autoxidation. XXI. Activation Energies for Propagation and the Correlation of Propagation Rate Constants with Carbon-Hydrogen Bond Strengths[END_REF]. k 3 can be conveniently estimated from the paper by [START_REF] Khelidj | Absolute Rate Constants for Hydrocarbon Autoxidation. XXI. Activation Energies for Propagation and the Correlation of Propagation Rate Constants with Carbon-Hydrogen Bond Strengths[END_REF] using the relations: log k 3 s-POO° = 16.4 -0.2BDE(C-H) at 30°C log k 3 t-POO° = 15.4 -0.2BDE(C-H) at 30°C E 3 s-POO° = 0.55.(BDE(C-H) -65) E 3 t-POO° = 0.55.(BDE(C-H) -62.5) s-POO° and t-POO° representing respectively secondary and tertiary peroxy radicals. Given the difference in Bond Dissociation Energies for C-H (denoted by BDE(C-H)) in benzene [START_REF] Davico | The C-H Bond Energy of Benzene[END_REF] or in -CH 3 [START_REF] Khelidj | Absolute Rate Constants for Hydrocarbon Autoxidation. XXI. Activation Energies for Propagation and the Correlation of Propagation Rate Constants with Carbon-Hydrogen Bond Strengths[END_REF], it is clear that: k 3 (PEEK) < k 3 (PSU) < k 3 (PE) < k 3 (PP) Thus, it suggests that (Table 7): k 6 (PEEK, PSU) << k 6 (PE, PP) From a chemical point of view, the reactivity of aryl or primary peroxy radicals is not low enough to justify such values (Table 6). A possible explanation is that the termination between two POO° radicals is a process involving two rare species. The mobility of segments favouring the coupling could thus control the kinetics of the reaction. The low order of magnitude below T g could be hence justified. Aromatic polymers could be interesting substrate for studying the Waite's theory:

TOL r OX

G i r i [PH] BDE k 3 2 /k 6 k 3 k 6 (m) (mol l -1 s -1 ) (mol/100 eV) (mol l -1 s -1 )( m o l l -1 )( k J m o l -1 )( l m o l -1 s -1 )( l m o l -1 s -1 )( l m o l -1 s -1 ) PEEK 1.00E-
2 0 A0 r dC 4N rD1 C dt πDt       (41)
where: -r 0 is the cage radius, which corresponds to the maximal distance permitting the reaction: It would be on the order of r 0 ~ 1-10 nm (Emanuel & Buchachenko, 1990), -D is the diffusion coefficient of radical species. On the order of D ~ 10 -22 -10 -20 m 2 s -1 (Emanuel & Buchachenko, 1990), -N A is Avogadro's number. According to this theory, kinetic constant depends on the diffusivity of a radical and in other words of macromolecular mobility.

Conclusions

A comparison of yields for gas emission, gel d o s e o r c r i t i c a l d o s e f o r r e a c h i n g a n unacceptable level of mechanical properties shows that aromatic polymers radiochemical behaviour can be first explained by their aromatic character which is doubly favourable: -Yield for radical formation is very low compared with aliphatic polymers, because aromatic groups are able to dissipate a great part of the absorbed energy into reversible processes (fluorescence, phosphorescence...). -

In the case of radio-oxidation, the propagation rate constant of hydrogen abstraction by peroxy radicals (k 3 ) is a decreasing function of the C-H bond dissociation energy, that takes a high value for aromatic hydrogen. These positive effects are partially counterbalanced by the relatively low termination rate linked to the low segmental mobility in glassy state. It is noteworthy that P° + P° termination (which leads to crosslink when oxygen is lacking) is more efficient than expected because P° radicals can diffuse independently of segmental mobility by the valence migration process (P 1 ° + P 2 H  P 1 H + P 2 °). This chapter also illustrates the effect of dose rate, thickness and oxygen concentration in polymers layers, which are linked by the theory of Diffusion Limited Oxidation: chain scissions predominate in aerobic degradation (i.e. thin sample, and low dose rate) and crosslinking in case of anaerobic degradation (thick sample, high dose rate). The effect of temperature can be considered as not partially understood.

Fig. 1 .

 1 Fig. 1. Reaction of H-and Y-crosslinking.

Fig. 3 .

 3 Fig. 3. Heating-cooling-heating cycle by DSC for non-irradiated (a) and 30.7 MGy irradiated PEEK sample.

Fig

  Fig. 4. T g changes for PEEK amorphous () and semi-crystalline () samples irradiated under vacuum.For a network, T g is an increasing function of crosslink density. According to Di Marzio (DiMarzio, 1964):

  ) ~ 0.012 for amorphous PEEK, -G(x) ~ 0.023 for semi-crystalline one. It seemed to us interesting to compare radio-induced gelation for PEEK, PSU and PES (Fig.5).

  Fig.5. Gel formation for PSU at 210°C (), 180°C (), 100°C (), 60°C (), room temperature () from[START_REF] Murakami | Gamma-rays irradiation effects on polysulfone at high temperature[END_REF][START_REF] Richaud | Diffusioncontrolled radiochemical oxidation of bisphenol A polysulfone[END_REF], and PEEK (: 24 kGy h -1 , ,,: 60 MGy h -1 for 3 different initial crystalline microstructures) samples irradiated at several temperatures[START_REF] Vaughan | On crystallization, morphology and radiation effects in poly(ether ether ketone)[END_REF] , Richaud, 2010a).

Fig. 7 .

 7 Fig. 7. Residual elongation as a function of dose for PSU (a) and PES (b) submitted to g-rays under air () or 0.7 MPa oxygen ().

Fig. 8 .

 8 Fig. 8. T g changes with dose for thin films () or thick samples ().

Fig. 10 .

 10 Fig.10. Oxidation profiles in PEEK sheaths submitted to 10 MGy (), 20 MGy (), 30.7 MGy () -rays exposure(Richaud et al., 2010a).

Table 2

 2

	.

Table 4 : thickness T (K) dose rate atmosphere  50% (MGy)  gel (MGy) G (gas)

 4 in Table

		298			> 0.3
	100 µm	423 463	0.1 kGy s -1	He	> 0.15 0.1
		503			0.1
		298			3	0.025
	50 µm	373 453	1.7 Gy s -1	vacuum	1.25 0.5	0.07 0.17
		483			< 0.25	2

) dose rate atmosphere  50 % (MGy)  gel (MGy) G(gas)

  Table5confirm that lethal dose (here. the necessary dose to half the initial elongation at break) and gel dose vary oppositely with temperature meanwhile gas emission yield would increase.

	thickness T (K100 µm 298			> 0.3	-	-
	100 µm 100 µm	423 463	0.1 kGy s -1	He	> 0.15 0.1	--	--
	100 µm	503			0.1	-	-
	50 µm	298			-	3	0.025
	50 µm 50 µm	373 453	1.7 Gy s -1	vacuum	--	1.25 0.5	0.07 0.17
	50 µm	483			-	< 0.25	2

Table 5 .

 5 Effect of temperature on lethal dose for PES

Table 6 .

 6 most polymers can only crosslink above their melting point. Effect of temperature on radiochemical yields in chain scissions and crosslinking (envisaging the possibility of H-or Y-crosslinking mode)

	dose rate method T (K)	G H (s)	G Y (s)	G H (x)	G Y (x) G(s)/G(x)thickness
			308	0.03		0.04		0.75	3 mm
	8 Gy s -1 Sol gel	353 398	0.05 0.3		0.05 0.2		1.00 1.50	3 mm 3 mm
			493	0		0.67		0.00	3 mm
			298	0.100	0.373	0.100	0.301	1.00	50 µm
	1.7 Gy s -1	GPC	373 423	0.104 0.147	0.318 0.455	0.104 0.147	0.313 0.442	1.00 1.00	50 µm 50 µm
			453	0.216	0.601	0.216	0.647	1.00	50 µm
			298	0.067	0.065	0.104	0.147	0.64	50 µm
	1.7 Gy s -1 Sol gel	373	0.170	0.138	0.245	0.312	0.69	50 µm
			453	0.075	0.268	0.170	0.575	0.44	50 µm

Table 7 .

 7 Kinetic parameters of PEEK and PSU estimated from DLO theory.


Patrice Peyre
email: patrice.peyre@ensam.eu

Additive Layer Manufacturing using Metal Deposition

Introduction and Scope

Among the additive layer manufacturing techniques for metals, those involving metal deposition, including laser cladding/Direct Energy Deposition (DED, with powder feeding) or Wire and Arc Additive Manufacturing (WAAM, with wire feeding), exhibit several attractive features. For instance, one can mention high mass efficiency (50-80% for LMD, 100% for WAAM), large build rates (more than 100 cm 3 /h), sound microstructures with a limited amount of porosities, and the ability to build graded or multimaterials. Even though corresponding processes have been developed a rather long time ago, there is still an important demand for research work in various topics such as deposition of novel or graded materials, postprocessing, and wear behavior of deposited materials.

The current Special Issue, including six contributions, aims at presenting recent and original work dedicated to all these aspects, with a more specific focus on coatings than on 3D structures.

Contributions

Six papers have been published in this Special Issue of Metals on ALM using metal deposition. They can be roughly separated in three distinct topics, presented here below.

In-Situ Manufacturing of Innovative Materials

Novel materials can easily be manufactured in-situ by metal deposition techniques, either from alloying parent materials or from in-situ chemical reactions.

An interesting example is the DED manufacturing of γ-TiAl coatings on Ti-6Al-4V substrates, starting from pure Al and Ti elemental powders [START_REF] Hoosain | Heat Treatment of In Situ Laser-Fabricated Titanium Aluminide[END_REF] instead of prealloyed powders. The paper demonstrates the feasibility of obtaining satisfactory and low-cost Ti-Al deposits under normal atmospheric conditions, versus more usual EBM conditions involving a stronger limitation of O 2 content. This was made possible by an optimization of the Ti and Al powder feeding, through a control of two separate powder hoppers. One major conclusion is the dominant role played by Al at % versus heat-treatment conditions in determining hardness and the mechanical properties. Metallurgically, twinning was also considered as an important contributor to elevated hardness values.

The WAAM process was investigated as a promising technique to generate Fe/Al walls by fusing simulateously an AISI304 stainless steel wire and a A4043 Al-Si alloy wire on a low carbon steel substrate. The optimization of the process, and especially of the inter-layer dwell time to limit cold cracking, was carried out through a thermal camera control of the surface temperature (less than 400 • C) between subsequent deposits. An interesting point to notice is that a +50% hardness increase can be obtained with an optimization of the continuous current regime (at higher scan speed), attributed by the authors to a grain refinement effect. Future investigations, focused on the influence of Al, Cr, and Ni contents in the welds, and especially solid-solution strengthening of the steel matrix by increased Al fraction, or an estimation of the Al 1.1 Ni 0.9 intermetallic volume fraction, should add a more precise understanding of observed results.

Wear Resistance of Deposited Materials

Laser metal deposition is a well known efficient method for improving wear resistance, and more widely the lifetime, of metalic materials. The main novelty of Liu et al.'s work [START_REF] Liu | Formation of SUS304/Aluminum Alloys Using Wire and Arc Additive Manufacturing[END_REF][START_REF] Liu | Fe-Si-Al Coatings with Stable Wear Resistance Prepared by Laser Cladding Industrial Wastes[END_REF] is to use nonspherical powders coming from industrial Fe-Si-Al ribbons leftovers, thus reducing the cost of raw materials and of the global process. Through the deposition of a 2 mm thick coating on a 1045 carbon steel, significant improvements of the wear resistance (factor 10 reduction of mass loss) are demonstrated, coming from the high hardness deposit, and surprisingly with a rather low influence of (P,V) process parameters.

In nearly the same topic, high-wear resistance steel (HWS) coatings are obtained by Baek et al. [START_REF] Baek | Mechanical Properties of Tool Steels with High Wear Resistance via Directed Energy Deposition[END_REF] on carbon steel substrates. In this work, a specific focus is put on the influence of a postdeposition quenching/tempering heat treatment. Through a detailed microstructural and mechanical investigation of laser coatings, a satisfactory compromise between toughness and tensile resistance was demonstrated for heat-treated HWS, mostly attributed to an increase of martensite volume fraction and the formation of fine and uniformly distributed carbides.

The preservation of the amorphous nature during laser deposition is partly confirmed in the innovative paper of Hou et al. [START_REF] Hou | Microstructure and Wear Resistance of Fe-Cr-Mo-Co-C-B Amorphous Composite Coatings Synthesized by Laser Cladding[END_REF]. Considering a Fe-Cr-Mo-Co-C-B alloy powder, and rather classical DED conditions, sound laser deposits were obtained, which were subsequently analyzed by X-ray Diffraction (XRD). XRD indicated a 53% volume fraction of amorphous phase, compared with 85% volume fraction in the starting powder, with the difference beeing attributed to dilution effects. Due to a six-times-higher hardness in the clad, significant improvements of the wear resistance were then obtained.

Hybrided Processes

Postprocessing heat or mechanical treatments applied on as-built ALM materials are mandatory to obtain desired metallurgical-mechanical properties. However, hybrid processes, involving sequences of manufacturing/post-treatments all along the building process, are not that common. On a similar topic, one can mention Kalentics's work involving SLM and laser shock peening [START_REF] Kalentics | Tailoring residual stress profile of Selective laser Melted parts by Laser Shock Peening[END_REF]. In the paper by Yang et al. [START_REF] Yang | Residual Stress, Mechanical Properties, and Grain Morphology of Ti-6Al-4V Alloy Produced by Ultrasonic Impact Treatment Assisted Wire and Arc Additive Manufacturing[END_REF], a combined ultrasonic impact treatment (UIT) + WAAM process was used to modify microstructures and mechanical properties of a Ti-6Al-4V alloy. The beneficial effect of this hybrid process was as follows: a reduction of Von Mises equivalent stresses, the formation of a refined and more equiaxed grain structure, and an improvement of yield and ultimate tensile strengths. The only detrimental effect was found to be a severe reduction of elongation to failures.

Conclusions

In the six original research papers presented in this Special Issue on ALM using metal deposition, the reader will benefit from an overview of current research works, which mainly highlight the wide spectrum of possible materials' developments.

Conflicts of Interest:

The author declares no conflict of interest.