Materials Advances View Article Online PAPER View Journal | View Issue Biodegradable porous FeMn(–xAg) alloys: This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. assessment of cytocompatibility, mechanical, Open Access Article. Published on 06 December 2022. Downloaded on 3/2/2023 4:50:01 PM. Cite this: Mater. Adv., 2023, 4, 616 magnetic and antibiofilm properties† Aleksandra Bartkowska, *a Oriol Careta, b Adam Benedict Turner, c Andreu Blanquer, b Elena Ibañez, *b Margarita Trobos, c Carme Nogués, b Eva Pellicer *a and Jordi Sort ad In this work, porous FeMn(–xAg) alloys are fabricated through powder metallurgy methods. The effects of porosity and Ag addition on the microstructure, biodegradability, magnetic and mechanical properties of the alloys are investigated. Studies on the cytocompatibility, inflammatory cytokine response and anti- bacterial effect are also conducted. The fabricated alloys exhibit a macro- and nanoporous structure, with uniformly distributed silver particles. The biodegradability tests reveal that the release of Mn to the Hank’s solution is higher than that of Fe, without significant differences between the alloys. The degra- dation products consist mainly of Fe, Mn, O and compounds enriched in Ca, P and Cl. As-sintered alloys show a low saturation magnetization value (below 1 emu g1), which does not increase significantly with immersion time. The results on biocompatibility indicate that all tested alloys are non-cytotoxic, but the Received 16th August 2022, addition of Ag might interfere with cell proliferation. However, the ions released by the FeMn(–xAg) Accepted 1st December 2022 alloys do not induce an inflammatory response in macrophages. The obtained results on microbiological DOI: 10.1039/d2ma00867j interactions reveal that although no significant bactericidal effect is observed at 4 h between FeMn con- trol and FeMn–5Ag, a significant reduction in the total biofilm biomass of both live and dead bacteria is rsc.li/materials-advances observed after 24 h in Ag containing FeMn–5Ag surfaces. 1 Introduction increased during the revision surgery.8 It has been shown that biodegradable materials are in general less prone to biomaterial- In recent years, the application of biodegradable metals (BMs) associated infections than non-degradable materials, most and polymers as potential candidates for temporary medical probably due to the degradation processes that reduce the area implants has been of growing interest.1–6 This class of materials available for bacterial colonization and, therefore, the formation is expected to corrode gradually in vivo once they have fulfilled of biofilm.9 Among various tested alloys, Mg-based and their role.2,7 Their main advantage is the possibility of avoiding Fe-based alloys are especially attractive due to their very good the implant removal surgery, hence limiting the cost of medical biocompatibility.2,7 Mg-based alloys show very low toxicity and treatment and decreasing the risk of infections, which is their Young’s modulus is close to that of the human bone. However, their degradation rate is too rapid for some applications, a Departament de Fı́sica, Universitat Autònoma de Barcelona, E-08193, Bellaterra resulting in H2 gas evolution and bubbling upon immersion. In (Cerdanyola del Vallès), Spain. E-mail: aleksandra.bartkowska@uab.cat, addition, they are hard to process, and their strength and ductility eva.pellicer@uab.cat are not satisfactory. b Departament de Biologia Cellular, Fisiologia i Immunologia, Universitat Over the past few years, increasing attention has been paid Autònoma de Barcelona, E-08193, Bellaterra (Cerdanyola del Vallès), Spain. to Fe-based alloys as they show good processability, mechanical E-mail: elena.ibanez@uab.cat c Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, strength and low toxicity,10–13 however, the degradation rate is University of Gothenburg, Gothenburg, Sweden too slow to be applied as biodegradable implants.7,14 Moreover, d Institució Catalana de Recerca i Estudis Avançats (ICREA), Pg. Lluı́s Companys 23, Fe-based alloys tend to be ferromagnetic, which is undesirable E-08010, Barcelona, Spain for magnetic resonance imaging (MRI) purposes. Thus, several † Electronic supplementary information (ESI) available: SEM images of powders approaches to improve the degradation rate of Fe-based alloys and cold-pressed pellets, EDS mappings taken before and after incubation in HBSS solution, XRD patterns after incubation in HBSS, and number of live/dead are being explored, such as alloying with different elements cells grown on wells cultured with media conditioned. See DOI: https://doi.org/ (e.g., Mn),11,13,15 using new fabrication methods to tune the 10.1039/d2ma00867j microstructure such as selective laser melting (SLM),16,17 616 | Mater. Adv., 2023, 4, 616–630 © 2023 The Author(s). Published by the Royal Society of Chemistry View Article Online Paper Materials Advances introducing porosity,17–21 adding small amounts of noble cylindrical green pellets (discs) of 10 mm diameter and 2–3 mm in elements,22–25 or adding C to promote twinning-induced plas- thickness. The green pellets were then loaded in a vacuum ticity (TWIP),7,26–28 amongst others. The addition of elements furnace and sintered at 900 1C at 5  105 mbar for 4 h at a such as Cu and Ag, which are more noble than Fe or Mn, is heating rate of 1 1C min1. As Mn has a high vapor pressure, being investigated as a way of not only improving corrosion higher sintering temperatures and longer dwelling times might rates (through galvanic pairs) but also because they bring lead to significant losses of Mn content due to sublimation,18,31 antimicrobial properties to the resulting alloys.22,25 Powder therefore the temperature was kept at 900 1C. Moreover, it has metallurgy is especially appealing to introduce insoluble ele- been shown that the losses of Mn in ball-milled powders are lower ments like Ag to the Fe–Mn matrix in a homogenous manner. than in powders prepared using other methods.31 This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. However, the effect of adding Ag in terms of its role in the Open Access Article. Published on 06 December 2022. Downloaded on 3/2/2023 4:50:01 PM. degradation rate remains still controversial: while most authors 2.2 Structural characterization agree that Ag causes increased degradation rates of Fe-based The structural and compositional characterization of the fabri- alloys,22–24,29 some studies report that Ag additions promote cated materials was conducted using an SEM microscope (Zeiss galvanic coupling only in the first stages of degradation and, Merlin, Zeiss, Germany) operated at 15 kV equipped with an after formation of the degradation layer, the Ag role is not being energy-dispersive X-ray spectroscopy (EDS) detector. The phase fulfilled anymore.27 Similarly, one can find dissimilar conclu- compositions of ball-milled powders, sintered alloys and alloys sions on the effect of silver on antibacterial activity. In the after immersion in Hank’s balanced salt solution (HBSS) were report by Liu30 no antimicrobial properties were confirmed in examined by X-ray diffraction (XRD, Panalytical X’Pert powder Fe70.3–Mn28.9–Ag0.8 alloy, while in the research published by diffractometer, Malvern Panalytical, UK) using Cu Ka radiation. Sotoudehbagha22 antibacterial activity in the alloys containing The measurements were conducted in an angular range from 201 1 and 3 wt% of Ag was observed. to 1001 with a step size of 0.0261 in the Bragg-Brentano geometry. The main aim of this study is to fabricate and characterize The XRD results were analyzed using Rietveld refinement (MAUD porous Fe–Mn alloys with different additions of Ag in terms of software),32 from which crystallite size and lattice parameters were their microstructure, mechanical and magnetic properties, determined. ImageJ software was used to assess the macroporosity degradation, biocompatibility and antibacterial activity. For this of sintered alloys by analyzing the SEM images. Archimedes’ purpose, four different compositions have been investigated: method was used to determine the density of the specimens. equiatomic FeMn, and FeMn with 1, 3 and 5 wt% of silver. The Then, the porosity values were estimated by comparing the relatively high content of Mn is proposed to ensure the non- theoretical (i.e., for the fully dense materials) and experimental magnetic character of the investigated alloys. Up to date, neither densities, according to the formula (eqn (1)): a long-term biodegradability test nor a study of the evolution of    densityexperimental magnetic properties upon immersion has been performed in % porosity ¼ 1  (1) densitytheoretical terms of ion release and degradation surface analysis for porous, Ag-containing equiatomic Fe–Mn alloys. 2.3 Biodegradability tests Biodegradability tests were conducted by immersing samples of 2 Materials and methods as-fabricated alloy pellets for various periods (7, 14, 28, 56, and 84 days) in HBSS at 37.5  1 1C. Each sample, with a surface 2.1 Fabrication of alloys area of approximately 100 mm2, was immersed in a container Commercial powders of iron (97% purity, Merck, Germany), containing approximately 30 mL HBSS. The volume of the manganese (Z99% purity, Merck, Germany) and silver (Z99.9% solution adhered to the ASTM G31-72(2004) standard, using a purity, Merck, Germany) were used to prepare four types of volume/surface area ratio of 0.3 mL mm2. At least three samples: FeMn, FeMn–1Ag, FeMn–3Ag and FeMn–5Ag, where replicas were prepared at each time point. The HBSS (H8264, the ratio of Fe : Mn was adjusted to 50 : 50 (at%) and the additions Merck, Germany) is a popular physiological solution used to of silver are given in wt% (1, 3 and 5 wt%, respectively). Mixtures reproduce the conditions in the human body, which contains of powders were alloyed using a planetary ball milling device CaCl2, MgSO4, KCl, KH2PO4, NaHCO3, NaCl, Na2HPO4 and (Pulverisette 5, Fritsch, Germany) under an Ar atmosphere at a glucose. After each period, 2 mL of solution from at least three rotation speed of 300 rpm, for 10 h and with a 10 : 1 ball-to-powder replicas was taken for the ion release concentration analysis, weight ratio. The procedure of weighing powders and sealing the which was conducted using inductively coupled plasma mass container was conducted in a glove box under Ar atmosphere spectrometry (ICP-MS, Agilent 7900, Agilent, USA). Before the (H2O o 0.1 ppm, O2 o 0.1 ppm) to prevent oxidation and ICP-MS analysis, the aliquots were diluted with HNO3. Samples atmospheric contamination. The particle size of as-milled pow- immersed for 14, 28, 56, and 84 days were withdrawn from the ders was measured using the ImageJ software (National Institutes containers for structural analysis of corrosion products formed of Health, USA) from scanning electron microscopy (SEM) images. both on the surface and the cross-section of the pellets. XRD After ball-milling, the powders were further refined in an agate analysis was performed to identify the corrosion products mortar, to avoid leaving large agglomerates. Subsequently, as- formed on the surface of FeMn and FeMn–5Ag alloys after 7 prepared powders were uniaxially cold-pressed at 100 MPa to form and 28 days of immersion. © 2023 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2023, 4, 616–630 | 617 View Article Online Materials Advances Paper 2.4 Characterization of mechanical and magnetic properties Alamar Blue assay was repeated according to the previously The mechanical properties were studied using nanoindentation described protocol (day 3). Aged and fresh media were used as (Anton Paar NHT3 nanoindenter, Anton Paar, Austria). Before controls. Experiments were performed in triplicate. indentation, the discs were polished to a mirror-like surface, Immediately after proliferation assays, cytotoxicity was with the final polishing step using 1 mm diamond particles assessed in cells incubated with media conditioned for 7 and paste. Maximum applied loads of 100 and 500 mN were used 56 days using the live/dead viability/cytotoxicity kit for mam- to assess the behaviour of the material during deformation. malian cells (Invitrogen), according to the manufacturer’s pro- A series of at least 50 measurements were performed on each tocol. Images from different regions of the well were captured using an Olympus IX71 inverted microscope equipped with This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. sample to obtain statistically significant data. The load- displacement curves were used to calculate the hardness (HB) epifluorescence. Open Access Article. Published on 06 December 2022. Downloaded on 3/2/2023 4:50:01 PM. and reduced Young’s modulus (Er), using the method of Oliver 2.6 Quantification of inflammatory cytokines secretion and Pharr.33 Apart from that, the elastic (Welast) and plastic (Wplast) energies of deformation were obtained from the load- THP-1 monocyte cells were used to analyze the potential displacement curves by assessing the areas between the unloading immunological response induced by the FeMn(–xAg) alloys. curve and displacement axis (Welast) and between the loading and Monocytes were grown in RPMI 1640 medium (Gibco) supple- unloading curves (Wplast). Finally, the elastic recovery was calculated mented with 25% FBS (Gibco) under standard conditions. as the ratio between elastic (Welast) and total (Welast + Wplast) energies To differentiate monocytes into macrophages, 4  105 THP-1 during nanoindentation. cells were seeded into 24-well plates and treated with 0.16 mM A vibrating sample magnetometer (VSM, MicroSense, USA) phorbol-12-myristate-13-acetate (Sigma-Aldrich, Saint Louis, was used to analyze the magnetic properties of the samples MO, USA) for 72 h. Then, cells were washed and incubated in before (as-fabricated) and after incubation in HBSS, at room a fresh medium for 24 h before carrying out the experiments. temperature. The maximum applied magnetic field was 20 kOe. The fresh medium was replaced by a conditioned RPMI The results were normalized over sample weight to obtain the medium (prepared as explained above) and macrophages were value of saturation magnetization (MS). incubated for 5 h and 24 h to allow the secretion of inflamma- tory cytokines. As a positive control, 1 mg mL1 of lipopolysac- charide (LPS) (Sigma-Aldrich) was added to the macrophages 2.5 Cell proliferation assays and cytotoxicity tests culture. As a negative control, macrophages were cultured in a For cell proliferation and cytotoxicity experiments, human fresh medium. After 5 h and 24 h, supernatants were collected osteosarcoma Saos-2 cells (ATCC HTB-85) were used. Cells were and used to quantify cytokine secretion. Inflammatory cyto- maintained in Dulbecco’s Modified Eagle Medium (DMEM) kines TNF-a, IL-1b and IL-6 were evaluated by flow cytometry (Gibco, ThermoFisher Scientific, Waltham, MA, USA) supple- using cytometric bead array (CBA) (Becton–Dickinson, East mented with 10% fetal bovine serum (FBS; Gibco), under Rutherford, NJ, USA). Cytokine concentrations in the super- standard conditions (37 1C, 5% CO2). natant were analyzed according to the manufacturer’s protocol. Experiments were conducted in a medium conditioned in Negative control was considered as basal secretion level and the presence of the different FeMn(–xAg) alloys. To prepare the used for statistical comparisons. Experiments were performed conditioned media, the samples were sterilized with UV light in triplicate. for 3 min and incubated in 15 mL of supplemented DMEM under standard conditions for 7, 14, 28, and 56 days. Then, the 2.7 Assessment of S. aureus adhesion and viability alloys were removed, and the conditioned media were kept at From a frozen stock, S. aureus ATCC 25923 was streaked onto a 4 1C protected from light until used. In parallel, control media 5% horse blood agar (HBA) plate (Media Department, Clinical were also prepared following the same incubation times and Microbiology Laboratory, Sahlgrenska University Hospital, Sweden) conditions but without the presence of any alloy (aged media). and incubated at 37 1C overnight. After incubation, colonies were Osteoblasts (Saos-2 cells) proliferation was determined using taken and inoculated into 4 mL tryptic soy broth (TSB) (Scharlau, Alamar Blue cell viability Reagent (Thermo Fisher Scientific) Barcelona, Spain) to achieve an OD546 of 0.13, equivalent to before (day 0) and after (day 3) cells’ exposure to the conditioned 108 CFU mL1, which was diluted 1 : 1000 into 40 mL of TSB to media. Briefly, 5  104 cells were seeded into each well of a 24- achieve a suspension of 105 CFU mL1. One mL of this suspension well plate with fresh medium. Cells were cultured for 24 h to was then added to each well of a 48-well plate (Nunclon Delta- allow cell attachment, and then the medium was replaced with surface; Thermo Fisher Scientific, Waltham, USA) containing 9 mm fresh medium with 10% Alamar Blue, and cells were incubated diameter discs with the test surface (FeMn–5Ag) and controls of for 4 h in standard conditions in the dark. Then, the supernatant FeMn or Ti (Christers Finmekaniska AB, Skövde, Sweden). The was collected (day 0), and the fluorescence was measured at discs were then incubated statically at 37 1C for 4 or 24 h to assess 585 nm wavelength after excitation at 560 nm on a Spark bacterial adhesion and biofilm formation as well as bacterial multimode microplate reader (Tecan, Männedorf, Switzerland). viability. After supernatant collection, FeMn(–xAg) conditioned media for After each incubation period, each disc was rinsed three different incubation times were added to each well. Cells were times in sterile 0.9% saline to remove non-adhered bacteria. cultured for 3 more days in a conditioned medium and the Discs were then stained using LIVE/DEADt BacLightt Bacterial 618 | Mater. Adv., 2023, 4, 616–630 © 2023 The Author(s). Published by the Royal Society of Chemistry View Article Online Paper Materials Advances Viability Kit, for microscopy & quantitative assays (Invitrogen, decreases with increasing the Ag content. Crystallite sizes are Waltham, USA) following the manufacturer’s instructions. The 14, 14, 13 and 10 nm for the FeMn, FeMn–1Ag, FeMn–3Ag, discs were rinsed three times to remove any unbound stain and FeMn–5Ag, respectively. The lattice constant increases upon the fluorescence was assessed in a plate reader (FluostarOmega, addition of Ag, showing values of 3.625 Å for FeMn, 3.630 Å BMG LABTECH, Ortenberg, Germany) and confocal laser- FeMn–1Ag, 3.633 Å for FeMn–3Ag and 3.637 Å for FeMn–5Ag. scanning microscope (Nikon C2+, Nikon, Tokyo, Japan) with a The FeMn and FeMn–1Ag sintered alloys maintain the 100 water dipping objective (CFI Plan 100XC W). Five z-stacks austenitic structure, while FeMn–3Ag and FeMn–5Ag alloys were taken at randomly chosen fields of view on each sample and consist of a duplex structure, comprising g-austenite (fcc struc- images were taken at 3 mM intervals through the biofilm layers. ture) and e-martensite phase (hcp structure). The occurrence of This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Biofilm thickness and biomass were analyzed in BiofilmQ.34 From the martensite phase was not expected as alloys containing Open Access Article. Published on 06 December 2022. Downloaded on 3/2/2023 4:50:01 PM. the plate reader, the average fluorescence intensity of each well above 27.3 at% of Mn are known to be mainly austenitic.13 This scan was taken as the representative live/dead distribution for phase transition could be caused by the occurrence of discrete each disc. Experiments were performed in triplicate with dupli- inhomogeneities in the element distribution, forming regions cate material samples. of lower Mn content and thus promoting the martensitic transformation. This phenomenon was already observed before 2.8 Statistical analysis by Zhang,21 but the explanation of martensitic transformation All quantitative data were analyzed with GraphPad Prism 9 under furnace cooling conditions was not given. In this case, (GraphPad Software Inc., San Diego, CA, USA) and presented as the martensitic transformation could be further enhanced by the the mean  standard error of the mean. Statistical differences presence of Ag. Nevertheless, both g-austenite and e-martensite were assessed by one-way analysis of variance (ANOVA) with a phases are antiferromagnetic, therefore the occurrence of the Bonferroni correction for cytocompatibility and inflammatory martensitic phase should not interfere with MRI. A shift in the response analyses and with Fisher’s least significant difference diffraction peaks towards higher 2y angles is observed in post-test for microbiological analyses. A value of p o 0.05 was the sintered alloys with higher Ag contents. The crystallite size considered significant. of the alloys subjected to sintering increases when compared to as-milled powders, and values of 38, 39, 18 and 13 nm for FeMn, FeMn–1Ag, FeMn–3Ag and FeMn–5Ag, respectively, are obtained. 3 Results and discussion Therefore, as in the case of powders, the addition of silver is leading to a decrease in the crystallite size. A peak of low intensity 3.1 Microstructure and compositional analysis can be observed at 2y = 351, which was attributed to Fe2O3. The 3.1.1 Morphology and phase composition of as-sintered occurrence of the Fe2O3 phase was observed in other studies35 and alloys. The SEM images of the initial and ball-milled powders it originates from the sintering method, which is always con- are presented in Fig. S1 (ESI†). The initial particle size was less nected with some level of oxidation. Nevertheless, Fe2O3 is also than 50 mm for the initial Fe, Mn, and Ag powders. The sizes of biocompatible,37,38 therefore its presence should not compromise the ball-milled powders were in the range of 1–40 mm. Fig. 1 the biocompatibility of the tested alloys. shows the XRD patterns of the powders ball-milled for 10 h Fig. 2 shows SEM micrographs of the morphology of sin- (Fig. 1a) and the as-sintered FeMn, FeMn–1Ag, FeMn–3Ag and tered (a–c) FeMn, (d–f) FeMn–1Ag, (g–i) FeMn–3Ag and (j–l) FeMn–5Ag alloys (Fig. 1b). The ball-milled powders have an FeMn–5Ag alloys. The SEM images under various magnifications austenitic structure, which is in accordance with the Fe–Mn were analyzed using ImageJ software and the % area of macro- phase diagram for this Mn content.35 XRD patterns of ball- porosity and the size of pores were calculated. Manganese is milled powders show wide peaks and a crystallite size which foreseen to diffuse into the iron matrix during sintering and Fig. 1 XRD patterns of (a) ball-milled powders and (b) sintered FeMn(–xAg) alloys. © 2023 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2023, 4, 616–630 | 619 View Article Online Materials Advances Paper This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 06 December 2022. Downloaded on 3/2/2023 4:50:01 PM. Fig. 2 SEM micrographs of sintered and surface-polished samples of FeMn (a–c), FeMn–1Ag (d–f), FeMn–3Ag (g–i) and FeMn–5Ag (j–l) samples. The % area macroporosity was determined using ImageJ by analyzing SEM images of the regions of macroporosity. The unlabeled red arrows provide reference for the Ag particles. create a solid solution, while silver may form non-soluble decreased compressibility due to the presence of non-alloyed silver precipitates in the FeMn matrix, as the solubility of Ag in Fe is particles in the ball-milled powder.42 very low.39,40 The morphologies of the green pellets, i.e. pressed Silver particles are uniformly distributed within the Fe–Mn powders before sintering, are shown in Fig. S2 (ESI†). All tested matrix, as indicated by red arrows in Fig. 2. To further analyze alloys show a similar initial porosity in the range of 19–26%, as the distribution of Ag precipitates in the Fe–Mn matrix, EDS calculated using ImageJ based on SEM images. The process of compositional maps, presented in Fig. S3 and S4 (ESI†), show sintering typically creates some additional level of porosity that Ag precipitates exhibit a polydisperse distribution within a resulting from the rearranging of Fe and Mn, i.e., voids are left size range of 0.5–3 mm. Larger agglomerates of Ag were behind by the metals upon diffusion. Another factor causing observed, particularly in the FeMn–5Ag sintered alloy, as shown porosity is the change in the crystalline structure from bcc-Fe in Fig. S4b (ESI†). The presence of Ag-rich precipitates could in and bcc-Mn to fcc-g (FeMn) and hcp-e (FeMn). This porosity is principle enhance the micro galvanic corrosion, hence indu- expected to have a beneficial impact on the proliferation of cells cing faster degradation rate of tested alloys.22–24 within the pores and, what is more, it can also improve the EDS compositional analysis of the samples furnished the degradation rate due to an increased surface area exposed to the weight percentage of individual elements, as listed in Table 1. bodily environment.18,41 In the tested materials, two different The amounts of Ag are close to the nominal values (1, 3 and 5 wt%), levels of porosity can be distinguished – namely macroporosity with while the weight percentages of Fe and Mn are slightly different an average pore size of around 10 mm – and nanoporosity with a from the nominal equiatomic composition (50.5 wt% Fe and pore size of around 100 nm, as presented in Fig. 2(c, f, i and l). The 49.6 wt% Mn), with a lower amount of Mn than the initial calculated levels of total porosity are presented in Table 1, which value. Manganese is an element very prone to sublimation shows that porosity increases with Ag content. While the addition under high temperatures;39 therefore it may be assumed that of only 1 wt% of Ag does not cause a significant decrease in the partial sublimation of Mn occurred, lowering its final amount. density, the addition of 3 and 5 wt% of Ag leads to a higher total 3.1.2 Surface morphology and chemical composition upon porosity (above 30%). This phenomenon is attributed mainly to the immersion. The changes in surface morphology of FeMn and Table 1 Actual chemical composition, macroporosity, density, and total porosity of sintered alloys. Total porosity accounts for both macro- and nanopores. Macroporosity was estimated from ImageJ software processing of SEM images, whereas the total porosity was determined by the Archimedes method Sample wt% Fe wt% Mn wt% Ag Macro-porosity (% area) Density (g cm3) Total porosity (%) FeMn 54.3 45.7 0 18.39  3.00 5.50  0.16 28.2  2.1 FeMn–1Ag 56.7 42.0 1.3 17.80  1.54 5.38  0.11 30.1  1.4 FeMn–3Ag 54.9 42.5 2.6 20.61  1.34 4.94  0.04 36.3  0.5 FeMn–5Ag 54.5 41.1 4.4 22.02  0.30 4.87  0.12 37.7  0.6 620 | Mater. Adv., 2023, 4, 616–630 © 2023 The Author(s). Published by the Royal Society of Chemistry View Article Online Paper Materials Advances This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 06 December 2022. Downloaded on 3/2/2023 4:50:01 PM. Fig. 3 Top-view SEM images of the degraded surface of FeMn (a–d) and FeMn–5Ag (e–h) incubated for 14 (a and e), 28 (b and f), 56 (c and g) and 84 (d and h) days in HBSS. (EDS analysis on the highlighted areas and locations is presented in Table 2). FeMn–5Ag alloy upon immersion are presented in Fig. 3. The the alloy and they can break the passive oxidation layer, there- degradation process of the alloys led to the formation of a fore leading to the formation of the pits.2,23,46 As can be seen in degradation layer. The EDS analyses, performed on the areas Fig. 3d, Cl-rich precipitates have formed on the exposed surface highlighted in Fig. 3 and summarized in Table 2, reveal that the of FeMn alloys, most probably after Cl ions have broken the degradation layer consists not only of Fe, Mn, and O, but also oxide layer. degradation products enriched in Cl, P and Ca, which are To gain further insight into the degradation products elements present in the HBSS, show up. Silver was not detected formed on the surfaces upon immersion, XRD analysis was on the degradation surface, which suggests that it is not prone performed on the FeMn and FeMn–5Ag samples after 7 and 28 days to be released during incubation in HBSS. In the first stages of of immersion, as presented in Fig. S5 (ESI†). The XRD patterns degradation, namely after 14 days of immersion the corrosion obtained after 7 days did not demonstrate any additional products formed on the surface are enriched in Ca–P clusters, features compared with the as-prepared samples (Fig. 1b). This forming a flaky structure that could be identified as calcium can be explained by the thin nature of the oxide layer as well phosphate-based apatite. These clusters tend to disappear at as the less abundant presence of other corrosion products. longer immersion periods, being replaced by different corro- However, after 28 days of immersion, an increase in the intensity sion products. of some of the peaks was observed. Corrosion products such as The globular precipitates, shown in Fig. 3f and h mainly MnPO4, MnCO3, Fe2O3 and MnO were identified. It is worth consist of O, Mn, Ca and P. Increasing immersion period leads noting that some of the oxide peaks can overlap, therefore we to the formation of a relatively thick oxidation layer, as shown might expect more corrosion products on the surface. in Fig. 3c and h. Even though the layer has the appearance of To unveil the degradation mechanism, cross-sections of all compact and homogenous, more detailed analysis (Fig. 3c) tested alloys immersed for 14, 28, 56 and 84 days in HBSS were reveals a cracked structure caused most probably by dehydration examined (Fig. 4). One can observe the formation of a degrada- due to air-drying after removal from HBSS.44,45 The cracked tion layer at the utmost sample surface, which is particularly oxide layer is prone to detachment, as shown in Fig. 3h. After visible in the samples immersed for 14 and 28 days, with a detachment, the exposed area allowed HBSS to penetrate further thickness of around 30 mm. This layer was presumably cracked into the pores. As shown in Table 2, the degradation layer of due to dehydration upon air drying or during the curing of the samples incubated for 84 days (Fig. 3d and h) contains high mounting resin, as mentioned before.11,27 Remarkably, this levels of Cl, namely 12.89 and 15.4 wt% for FeMn and FeMn– layer becomes thinner or even disappear for longer immersion 5Ag, respectively. The Cl ions are aggressive to the surface of periods. At the same time, an increase of the incubation period favors higher penetration of the HBSS towards the bulk of the material Table 2 Chemical composition of areas and points indicated in Fig. 3 as since the HBSS is able to permeate through the open-pore measured by EDS structure of the pellets. As a result, corrosion products formed Elements (wt%) inside the pores, mainly consisting of iron and manganese oxides, Area/point Fe Mn O Cl P Ca as shown in Fig. S6 (ESI†). Brighter contrast (due to charging) is associated with low-conducting corrosion products made of O, Ca, FeMn 1 34.6 22.5 38.3 0.5 3.1 1.0 2 19.3 20.1 30.9 12.9 8.4 8.4 P and Cl. When comparing individual alloys, the degradation FeMn–5Ag 3 23.4 — 68.9 0.5 3.8 3.4 layer of Ag-containing alloys seems to be thinner after 56 and 4 12.5 22.3 38.3 — 8.8 18.2 84 days of immersion than that of FeMn. Nevertheless, at the 5 40.5 27.1 31.1 1.4 — — 6 27.9 21.1 42.2 0.8 5.8 2.2 same time, the corrosion products consist mainly of metal 7 6.3 42.9 25.1 15.4 — 10.4 hydroxides and carbonates, accompanied by calcium, phosphorus © 2023 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2023, 4, 616–630 | 621 View Article Online Materials Advances Paper This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 06 December 2022. Downloaded on 3/2/2023 4:50:01 PM. Fig. 4 Cross-section SEM images of FeMn, FeMn–1Ag, FeMn–3Ag and FeMn–5Ag specimens after 14, 28, 56 and 84 days of immersion in HBSS. ‘S’ stands for sample and ‘DL’ to the degradation layer formed on top of the sample. and chlorine elements, as mentioned before.11,20,47 The amount Fe–Mn matrix can lead to the formation of micro galvanic cells of Cl is notably higher in the Ag-containing alloys which can be that can cause an accelerated corrosion rate. Additions of Ag attributed to localized pitting. Since porosity increases with Ag particles into the Fe-based alloys have also been tested, but so content, the Cl-rich corrosion products are accumulated inside far mixed results have been reported in the literature. Most of the pores to a larger extent in samples with higher Ag wt%. the studies have shown an enhanced corrosion rate22–24 but a The EDS compositional maps of the cross-sections of the neutral effect has also been observed,26,27 as well as a decrease alloys immersed for 84 days, presented in Fig. S6 (ESI†), show in corrosion rate in Fe–Ag alloys.48 Silver has a more positive the distribution of Fe and Mn alloying elements as well as of standard corrosion potential compared to Fe and Mn, hence it foreign elements that have accumulated inside the pores. Fe can be assumed that during corrosion the Fe–Mn matrix acts as and Mn are uniformly distributed in the matrix, but the upper an anode, and it is therefore oxidized, while the Ag particles act degradation layer shows a lower concentration of those ele- as a cathode and thus accept electrons.23,30 Another possible ments than the bulk. The abundant presence of oxygen was mechanism to explain the role of Ag in the acceleration of the observed, both in the degradation layer and the sample, mostly corrosion rate is that during corrosion of the Fe–Mn matrix, the inside the pores. Other elements, such as Ca, Cl and P, were Ag particles will form a separate phase, which has the ability to also present, mostly in the degradation layer. The EDS analyses break the Fe oxide layers.23,43 performed in the degradation layer show that its Mn content is The concentration of Fe and Mn ions released from the lower than that of Fe. For example, in the case of FeMn–5Ag, materials into the HBSS after 7, 14, 28, 56 and 84 days of the content of Fe was around 4–5 times higher than the content immersion is presented in Fig. 5a and b. It can be observed that of Mn (33.3 wt% of Fe and 8.5 wt% of Mn after 14 days of Mn release is higher than Fe release for all tested alloys and immersion, and 51.7 wt% of Fe and 11.1 wt% of Mn after that the ion concentration increases with the immersion time, 84 days of immersion). This suggests that the release of Mn which is in agreement with the previously shown EDS results. ions to the HBSS occurs easier than the release of Fe ions. Mn is more prone to be released due to a lower standard 3.1.3 Released ion concentration. It has been shown before corrosion potential when compared with Fe (i.e., Mn2+ ions are that addition of noble elements, such as Pt and Pd,15,28,42 to the more susceptible to be released to the HBSS than Fe2+ or Fe3+). 622 | Mater. Adv., 2023, 4, 616–630 © 2023 The Author(s). Published by the Royal Society of Chemistry View Article Online Paper Materials Advances and resintering cycles, released around 1.5 ppm and 2 ppm for Mn and Fe ions after 84 days of immersion, respectively.11 In another report, FeMnSiPd alloys obtained through arc melting,15 released around 1.5 ppm for both elements up to 120 days of immersion. The release of ions in our tested alloys was higher, presumably due to the higher level of porosity, which enhances the degradation. 3.2 Magnetic properties This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 5 Concentration of (a) Mn and (b) Fe ions released into the HBSS at different immersion periods as measured with ICP-MS. Please note the The magnetic behavior of as-sintered and immersed alloys is Open Access Article. Published on 06 December 2022. Downloaded on 3/2/2023 4:50:01 PM. different Y-axis scale in panels of Mn (a) and Fe (b) release. presented in Fig. 6a–d. The as-sintered alloys (Fig. 6a) show very low saturation magnetization, with values of 0.019, 0.199, 0.626 and 1.077 emu g1 for FeMn, FeMn–1Ag, FeMn–3Ag and FeMn– In the first stage, up to 28 days of immersion, a sharp increase in 5Ag, respectively. For comparison, the value of saturation the release is observed, especially in the case of Mn for all tested magnetization (MS) of the 304 austenitic stainless steel SS304, specimens and it tends to slow down upon further immersion. commonly used as implant material is 1.25 emu g1.36,49 Nevertheless, an increase in both Mn and Fe release after 84 days On the other hand, the MS value of pure iron is as high as of immersion is observed in the case of FeMn and FeMn–5Ag, as 221.7 emu g1.50 This means that the FeMn–5Ag alloy, which well as an increase of Fe release of the FeMn–1Ag sample. has the highest value of MS among the tested alloys, has only However, no significant differences in the amounts of released 0.49% the magnetization of Fe. In fact, the Fe-based alloys with ions were observed between the tested alloys. The silver- manganese content above 30 wt% have been reported to be containing alloys did not show an increase in the ion release of antiferromagnetic as they consist mainly of antiferromagnetic either Fe or Mn when compared to the alloy without Ag. Similar g-austenite and e-martensite phases.51,52 therefore enhancing behavior has already been observed in literature with Fe–Mn–C– the materials’ compatibility with MRI imaging. In our case, the xAg alloy.26,27 Apparently, Ag had a negligible influence on the MS value increased with higher Ag content, which might be biodegradability of the material. Although Ag could in principle attributed to slightly higher porosity of those alloys (which in induce microgalvanic corrosion in contact with the less noble turn could lead to higher amount of Fe2O3 after sintering).53,54 FeMn environment, the amounts of leached Fe and Mn ions in Following immersion in HBSS, the magnetization of FeMn FeMn and FeMn–5Ag, as determined by ICP, were similar. This progressively increased with the immersion time, as shown in suggests that other effects, likely the porosity, play a major role. Fig. 6c. The alloys incubated for 84 days (Fig. 6b) showed a MS The trend in the curve’s shape of the ion concentration value of 2.301, 1.249, 0.0367 and 0.396 emu g1 for FeMn, indicated a linear dependence up to 28 days and then a more FeMn–1Ag, FeMn–3Ag and FeMn–5Ag, respectively. When com- stable increase during a longer immersion period. It can be paring the gradual differences in the magnetic properties upon mainly attributed to the degradation products formed on the increasing immersion time, a similar trend was noticed in the surface that are partially blocking the dissolution of Fe and FeMn alloy – the lowest value of magnetization is shown by Mn at longer immersion periods. Due to the same reason, the the as-sintered material and it increased gradually – after 14 corrosion rate does not increase with the immersion time for the and 28 days the value was similar, around 0.690 emu g1, with Ag-containing alloys, as the release of silver is being blocked by an increase up to 2.290 emu g1 after 3 months of immersion. corrosion products formed on the surface and inside the pores. This observation can be ascribed to a higher release of Mn than Nevertheless, the release of silver was also measured, and it was 3 Fe ions, which leads to a higher level of Fe-enriched regions in orders-of-magnitude lower than the release of Fe and Mn. Namely, the material than before immersion. On the contrary, in the the highest Ag release was observed for the FeMn–5Ag and was case of the FeMn–5Ag, an increase of the incubation time led to 1.23  0.21 mg L1 after 14 days of immersion, then it decreased to a small decrease in the MS values, starting from 1.077 emu g1 0.88  0.22 mg L1 after 56 days of immersion and again increased for as-sintered alloy and then decreasing, firstly to around up to 6.35  3.65 mg L1 after 84 days in HBSS. In the FeMn–1Ag 0.550 emu g1 after 14 days of immersion and then to a and FeMn–3Ag, the highest concentration of Ag was also observed minimum value of 0.393 emu g1 after 3 months of immersion. after 84 days of immersion and was 1.65  0.55 mg L1 and 1.3  In any case, even though the MS values change upon degrada- 0.34 mg L1, respectively. Thus, as could be suspected, the release tion, the level of MS is sufficiently low to assure good compat- of Ag ions increases with the initial Ag content. The increased ibility of the alloys with MRI. amount of Ag released after 84 days of immersion, when com- pared with the amount released after 56 days, can be connected 3.3 Mechanical properties with the increased amounts of Cl-rich precipitates on the degra- Nanoindentation was used to analyze the micro/nano-mechanical dation surface that can contribute to breaking the degradation properties of the tested alloys. The tests were conducted under layer and, hence, enabling further degradation. two different maximum loads, namely 100 and 500 mN. The lower A previous study reported that Fe–Mn alloys (Fe35Mn and load was used to extract information from the nanoporous regions Fe25Mn), obtained through powder sintering and cold rolling of the material, while the higher loads may be able to assess the © 2023 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2023, 4, 616–630 | 623 View Article Online Materials Advances Paper This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 06 December 2022. Downloaded on 3/2/2023 4:50:01 PM. Fig. 6 Magnetization curves (a–d) for (a) as-sintered samples, (b) alloys after immersion in HBSS for 84 days, and detailed changes of magnetization upon immersion for different periods for (c) FeMn and (d) FeMn–5Ag. Load-displacement curves (e) of the FeMn(–xAg) alloys under 100 and 500 mN (insert) maximum applied loads, and (f) corresponding reduced Young’s modulus (Er) under 100 mN (blue) and 500 mN (green) of maximum applied loads, together with hardness (HB) (red). Please note the different Y-axis scales between the panels. influence of both levels of porosity (macro and nano) on the hand, the curves obtained under 100 mN of maximum load reduced Young’s modulus (Er) and hardness (HB). Representative almost overlap and the resulting values of Er are more similar load-displacement curves of tested alloys, together with calculated among the various samples, as shown in Fig. 6f. The nanoinden- values of Er and HB are presented in Fig. 6e, f and Table 3, tation tests conducted under a maximum applied load of 500 mN respectively. The load-displacement curves (Fig. 6e) show higher show that the alloys have a comparable value of Er, around variability under higher load, which is mainly due to different 50 GPa, which is relatively close to that of a human bone (cortical levels of macroporosity among the tested alloys. On the other bone has Young’s modulus value of around 20 GPa).55 The Table 3 Values of Young’s modulus (Er), hardness (HB), HB/Er ratio and energies of deformation (Welast/Wtotal and Wplast/Wtotal) for FeMn, FeMn–1Ag, FeMn–3Ag and FeMn–5Ag alloys Sample Er (500 mN) GPa Er (100 mN) GPa HB (GPa) HB/Er (wear resistance) Welast/Wtotal Wplast/Wtotal FeMn 50.39  14.16 121.24  12.84 4.66  0.17 0.038  0.003 0.22  0.03 0.78  0.03 FeMn–1Ag 47.24  16.86 106.60  16.82 4.86  0.27 0.046  0.008 0.26  0.04 0.74  0.03 FeMn–3Ag 56.09  15.36 124.65  15.13 4.40  0.53 0.035  0.006 0.20  0.03 0.80  0.10 FeMn–5Ag 51.81  17.13 137.67  14.58 4.16  0.19 0.030  0.003 0.18  0.02 0.82  0.04 624 | Mater. Adv., 2023, 4, 616–630 © 2023 The Author(s). Published by the Royal Society of Chemistry View Article Online Paper Materials Advances average value of hardness performed under 500 mN was 1.48 GPa In these experiments, two different control media were used: for FeMn, 1.2 GPa for FeMn–3Ag and 1.14 GPa for FeMn–5Ag. fresh media prepared just prior the experiment and media that To understand the mechanical properties of the alloys, the were maintained in the incubator for the same time periods as results obtained under lower load (100 mN) were used to the conditioned ones (aged medium; see Section 2.5). further calculate HB, Er, HB/Er ratio and energies of deforma- We decided to include this aged medium as a control tion, as presented in Table 3. The FeMn–1Ag alloy had the because we reasoned that the medium and serum components highest hardness and the lowest Young’s modulus among all could degrade over time during the conditioning of the media tested alloys, while the FeMn–5Ag had the lowest HB and the with the alloys. Indeed, when comparing cell proliferation in highest Er. The hardness values of all tested alloys are above both types of control media, we observed a significant decrease This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 4 GPa, which is similar to that of fully dense Fe30Mn6Si1Pd.15 in Saos-2 cells proliferation when the cells were cultured in the Open Access Article. Published on 06 December 2022. Downloaded on 3/2/2023 4:50:01 PM. The values of hardness increase upon small additions of Ag and 28 and 56 days aged media, the difference being higher in the decrease at higher Ag amounts. As silver does not form an alloy last timepoint (Fig. 7). It is known that growth factors and other with the Fe–Mn system and stays in a form of precipitates proteins degrade when stored at room temperature or 37 1C. whose hardness is lower than the Fe–Mn matrix, the overall Each protein has a different degradation rate but, to our hardness of the material might be lower. The other factor knowledge, no research studies on serum degradation over influencing hardness values is the level of porosity (which time and its effect on cell proliferation have been published. increases with the Ag content), tending to reduce HB. However, the FBS information for users recommends to store The HB/Er ratio is an indirect estimation of wear resistance. the product at 10 to 40 1C, and not to use FBS stored at 2 to It has been shown that wear resistance is dependent on the 8 1C for more than 4 weeks (Freezing and Thawing FBS and hardness (HB) and the reciprocal value of elastic modulus Other Sera). Because of the differences found in cell prolifera- (Er),56,57 thus the highest HB/Er ratio of FeMn–1Ag alloys tion when using the two types of control media, the effect of the indicates the highest wear resistance for this composition. In different conditioned media on cell proliferation was only Table 3, the ratios of the elastic (Welast) and plastic (Wplast) compared with their corresponding aged medium. energies versus the total (Wtotal) energy of deformation during At day 0 (after one day in culture in fresh medium), the nanoindentation are presented. All tested alloys demonstrate a intensity of the signal obtained was similar among all samples of higher contribution of plastic energy in the total energy of the same conditioned time group, with no significant differences deformation. It is shown that the contribution of elastic energy (Fig. 7). After 3 days in culture in the conditioned media, cell is higher for the FeMn–1Ag alloy than for the other alloys, viability significantly increased in 7 and 14 days conditioned meaning that it possesses a slightly higher elastic recovery, media, regardless of the alloy composition (Fig. 7a and b), mean- which indicates the ability of the material to recover the initial ing that cells were able to proliferate. Nevertheless, cells cultured shape after deformation. in the aged control medium showed a higher proliferation rate Contrarily, the FeMn–5Ag alloy shows higher contribution of than in any of the alloys conditioned media tested. Some signifi- Wplast to the total energy of deformation. The so-called ‘plasti- cant differences were found when comparing all conditioned city index’ indicates higher plasticity of this alloy when com- media: cells cultured in FeMn–3Ag conditioned medium showed pared to FeMn, FeMn–1Ag and FeMn–3Ag counterparts. the highest proliferation rate among the 7 days conditioned media, and cells cultured in FeMn–5Ag conditioned medium showed the lowest proliferation rate among the 14 days condi- 3.4 Cytocompatibility of FeMn(–xAg) alloys tioned media (Fig. 7a and b). To assess the alloys’ cytocompatibility, two different analyses After 28 and 56 days of medium conditioning or aging, cell were performed in vitro: cell proliferation and cytotoxicity. proliferation was similar between conditioned and aged media Whilst cell proliferation analysis allows to assess whether cells and among conditioned media, except for a significant incubated with conditioned media can proliferate (i.e., increase decrease in the FeMn–5Ag conditioned medium compared with their number over time), cytotoxicity analysis can determine FeMn–3Ag at 28 days and with FeMn at 56 days (Fig. 7c and d). whether the ions released by the alloys produce a toxic effect Next, the live/dead assay was used to determine the cyto- (i.e., a decrease in the number of live cells over time). toxicity of the alloy-conditioned media. As shown in Fig. S7 First, we tried to perform direct cytocompatibility analyses, (ESI†), the number of cells grown on wells cultured with media by growing the cells directly on the surface of the alloys. We conditioned for 7 days with Fe–Mn, FeMn–1Ag, FeMn–3Ag and observed that the cells did not adhere properly to the alloys FeMn–5Ag was similar (A–D), but lower than the number of (data not shown), probably due to morphological changes cells on wells cultured with the aged and the fresh control that the surface of the alloys experienced upon immersion media (E and F). However, the number of cells grown on wells (see Fig. 3 and 4). It has been described that Fe-based alloys cultured with medium conditioned for 56 days was much lower. show a relatively high corrosion and degradation rate that Specifically, cell numbers seemed to decrease as the silver preclude cell adhesion.18 Hence, in order to analyze the effect percentage in the alloy increased (G–J). The number of cells of the alloys and of the ions released into the medium, we growing in culture with the 56 days aged medium was also low proceeded with the indirect cytocompatibility analyses, by (K), whereas cells cultured with fresh media appeared to be in culturing the cells in alloy-conditioned media. monolayer and in a higher number (L). The number of live cells © 2023 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2023, 4, 616–630 | 625 View Article Online Materials Advances Paper This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 06 December 2022. Downloaded on 3/2/2023 4:50:01 PM. Fig. 7 Cell proliferation of Saos-2 cells before (day 0) and after (day 3) culture with media conditioned for 7 (A), 14 (B), 28 (C) and 56 (D) days with FeMn, FeMn–1Ag, FeMn–3Ag and FeMn–5Ag alloys, measured by Alamar Blue fluorescence. Aged medium, consisting of DMEM medium incubated for the same incubation times and conditions but without any alloy, and fresh medium were used as controls. Bars with different alphabetical superscripts are significantly different from each other (p o 0.05), whereas bars with the same alphabetical superscripts are not significantly different from each other. (E) Logarithmic representation of cytokine release by macrophages exposed to 56 days FeMn(–Ag) conditioned media. Secretion was analyzed by the CBA test at 5 and 24 h of culture. LPS: Lipopolysaccharide, positive control. Asterisks above the columns indicate significant differences compared with the negative control. was higher than 90% in all cases. These results are in agree- a salt, as nanoparticles, or as part of the alloy. In our case, Ag ment with Alamar Blue results, and suggest that, while FeMn- was incorporated within the FeMn matrix in the form of (–xAg) materials might interfere with cell proliferation, they are particles during the fabrication step. not cytotoxic. It has been shown that the metal salt AgNO3 produces a high The cytotoxicity of biodegradable metallic alloys is mainly cytotoxic effect in mammalian cell cultures,59,60 with an IC50 of attributed to the released degradation particles58 and metallic AgNO3 in the range of 2–11 mM (300–1900 mg L1) depending on ions13 which can promote or inhibit both cell proliferation and the cell type.61 On the other hand, studies using nanoparticles metabolic activities. In addition, the corrosion of porous alloys concluded that 6 mM silver nanoparticles do not have a sig- can produce a general change in pH and osmolarity that can nificant impact on cytotoxicity of epithelial cells.62 Similar contribute to a decrease in the cell viability. The results obtained results were observed specifically using osteoblasts, as the showed that media conditioned with the FeMn–5Ag alloy reduced presence of silver nanoparticles coating-alloys did not induce cell proliferation compared with the other alloys containing less any cytotoxic effect on MG63 osteoblasts.63,64 amount of Ag, indicating that certain amount of silver can alter Our results showed values at the 0.88–6.35 mg L1 range of cell proliferation. In fact, the FeMn–5Ag alloy was the one with the Ag released from the alloys. The highest values observed for highest Ag release after 84 days in Hank’s solution (6.35 mg L1). FeMn–5Ag after 84 days were far from the concentrations The effect of Ag on cell viability is controversial and depends considered cytotoxic. In this regard, our cytotoxic results are on the state of the Ag, i.e. whether Ag is present in the culture as in agreement with those of previous studies (viability higher 626 | Mater. Adv., 2023, 4, 616–630 © 2023 The Author(s). Published by the Royal Society of Chemistry View Article Online Paper Materials Advances than 90%). However, the cell proliferation rate was reduced no secretion of IL-1b, IL-6 and TNF.68 It has been reported that when osteoblasts were cultured in the presence of the alloy- the presence of metal debris and even low concentrations of conditioned media. After culture with 7 and 14 days condi- metal ions can induce an inflammatory response.69 In this tioned medium, a decrease in cell proliferation was observed regard, it has been observed that the release of Fe, Mn and Ag regardless of the alloy, which indicates that not only Ag can could induce the secretion of inflammatory cytokines; Fe and interfere with the cell cycle. The presence of Fe and Mn ions, or Mn are considered no allergenic elements, although Ag is.70 the relatively high corrosion rate could be responsible for the However, the results obtained for cytokine release indicated reduced proliferation rate. The high corrosion rate could that concentrations lower than 0.5 mg L1 of Fe, 2.3 mg L1 of increase the pH and osmolality, as previously observed.65 The Mn and 0.9 mg L1 of Ag did not induce the inflammatory This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. release of Ag from the FeMn–5Ag after 28 and 56 of media response in macrophages. Open Access Article. Published on 06 December 2022. Downloaded on 3/2/2023 4:50:01 PM. conditioning could also contribute to the lowest cell prolifera- tion observed. 3.6 Microbial interactions with FeMn(–xAg) In this investigation, the effect of FeMn and FeMn–5Ag 3.5 Inflammatory cytokine response alloys on S. aureus viability and biofilm formation was tested. To find out whether the ions released by alloys activate the The addition of silver was tested due to its widely known secretion of inflammatory cytokines, 56 days conditioned medium antimicrobial capacity.71,72 Biofilm biomass and thickness were was added to macrophage cultures. We analyzed the pro- assessed using confocal laser-scanning microscopy (CLSM) inflammatory cytokines IL-1b, IL-6 and TNF, which are commonly (Fig. 8A–D) and fluorescence plate reading (Fig. 8E–H) after produced by macrophages in response to bacterial endotoxin or 4 and 24 h of growth. Moreover, representative images of the LPS.66 According to Chanput et al., stimulating THP-1 macro- biofilm formed on FeMn and FeMn–5Ag, obtained using CLSM phages with LPS results in a dramatic increase in the secretion of are shown (Fig. 8I and J). IL-1b, IL-6, and TNF, which reaches a plateau after 18 h of When comparing the FeMn–5Ag to the FeMn stimulation.67 material, whilst no significant difference in biofilm viability Results of the CBA showed that ions released by FeMn(–xAg) was observed at 4 h, a significant reduction in the total live and alloys did not activate the secretion of IL-1b, IL-6 or TNF in dead biomass was observed at the 24 h time point (Fig. 8A and B). macrophages when compared with a non-stimulated control. Importantly, the total biofilm thickness on FeMn–5Ag was directly However, when macrophages were exposed to LPS (positive comparable to that of the titanium control (Fig. 8C and D), known control), an increase in the concentration of the three cytokines for its biocompatibility in bone-implant applications, showing was detected in the culture medium, even though IL-1b analogous performance of the FeMn–5Ag materials. increase was not statistically significant (Fig. 7e). Ubanako According to the fluorescence plate reading data, at 4 h the et al. also obtained the same results when using low amounts total biofilm viability was unaffected by the alloying of 5% silver of LPS to stimulate THP-1 derived macrophages, that is, little or compared to FeMn (Fig. 8E and F). A reduction in biofilm Fig. 8 Staphylococcus aureus ATCC 25923 biofilm morphology on FeMn–5Ag compared to FeMn and machined titanium (Ti) controls after 4 and 24 h incubation, analysed using quantitative confocal laser-scanning microscopy (CLSM) to assess biofilm biomass (A and B) and thickness (C and D). Fluorescence intensity of total live and dead cells in biofilms grown for 4 h (E and F) and 24 h (G and H) on FeMn–5Ag, FeMn and Ti using fluorescence plate reading. Bars with different alphabetical superscripts are significantly different from each other (p o 0.05), whereas values with the same alphabetical superscripts are not significantly different from each other. CLSM images of the biofilm formed on the surface of FeMn (I) and FeMn–5Ag (J) alloys (NIS-Elements Viewer, Nikon). Green: Live S. aureus cells; Red: Dead S. aureus cells. © 2023 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2023, 4, 616–630 | 627 View Article Online Materials Advances Paper biomass on FeMn–5Ag compared to FeMn was also observed (f) The cytocompatibility experiments performed using through fluorescent plate reading at 24 h for live (Fig. 8G) but conditioned media revealed that the investigated alloys are not for dead (Fig. 8H) bacteria. non-cytotoxic. Although Ag-containing alloys have shown to The current state of the literature regarding the addition of be cytocompatible, their cell proliferation is lower than that silver into FeMn alloys as a potential antimicrobial agent is of FeMn. disputed and dissimilar conclusions have been reported.21,29 (g) The inflammatory cell response experiment, conducted The unchanged bacterial viability observed here by the addition using media conditioned for 56 days, shows that the ions of silver to FeMn is in agreement with certain literature,29 in released by FeMn(–xAg) alloys do not induce an inflammatory which no reduction in S. aureus viability was observed after response in macrophages in vitro. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. incubation with similar silver containing materials. With the (h) The viability and biofilm formation of Staphylococcus Open Access Article. Published on 06 December 2022. Downloaded on 3/2/2023 4:50:01 PM. concentrations of silver observed in the release assays under- aureus with FeMn(–xAg) alloys were tested. The obtained results taken in this study, it is likely that the total level of released reveal that whilst no significant increase in bacterial killing is silver into the culture medium was below the threshold for observed at 4 h between the FeMn and the FeMn–5Ag samples, achieving a significant bactericidal effect. a significant reduction in biofilm biomass of both live and dead Therefore, rather than a direct bactericidal activity of Ag, cell populations is observed at 24 h. these results show that the FeMn–5Ag material may influence (i) Overall, these results indicate a potential for a novel biofilm formation in S. aureus. The observed reduction in total material that achieves identical performance compared to the biofilm biomass adhered to the FeMn–5Ag surface after 24 h is, current state of the art, whilst reducing early bacterial adhesion to our knowledge, a novel finding (Fig. 8A and B). The potential and biofilm formation. mechanism for this is unknown but it could be related to the degradability of the bulk material, as seen in Fig. 3 and 4. By limiting the solid surface to which bacteria can adhere to, the Author contributions ability for biofilm to form may be reduced, a concept also A. Bartkowska: conceptualization; investigation; data curation; proposed by Daghighi et al.9 formal analysis; validation, visualization; writing – original draft. O. Careta: investigation; data curation; formal analysis; validation; visualization; writing – original draft. A. Turner: investigation; 4 Conclusions data curation; formal analysis; validation, visualization; writing – original draft. A. Blanquer: conceptualization; investigation; writ- In this study, porous FeMn alloys with additions of Ag were ing – review & editing; E. Ibáñez: conceptualization; methodology fabricated through a powder metallurgy route. An in-depth resources; supervision; writing – review & editing. M. Trobos: study of the influence of porosity and Ag additions on the conceptualization; methodology; resources; supervision; writing microstructure, biodegradability, degradation surface, magnetic – review & editing; funding acquisition C. Nogués: conceptualiza- and mechanical properties, has been undertaken. Furthermore, tion; methodology; resources; supervision; writing – review & studies on cytocompatibility, inflammatory cell response and bac- editing; project administration. E. Pellicer: conceptualization; terial interactions have been conducted. The results revealed that: methodology; resources; supervision; writing – review & editing; (a) FeMn and FeMn–1Ag sintered alloys consist mainly of a project administration. J. Sort: conceptualization; methodology; g-austenite phase, while FeMn–3Ag and FeMn–5Ag alloys con- resources; supervision; writing – review & editing; project admin- sist of a duplex structure, comprising g-austenite (fcc structure) istration; funding acquisition. and e-martensite phase (hcp structure). (b) The alloys have a porous structure, consisting both of macro- and nanoporosity, with the total level of porosity varying Conflicts of interest from 28% for FeMn alloy up to 37% for FeMn–5Ag alloy. (c) The biodegradability experiments, performed in HBSS up There are no conflicts to declare. to 84 days of immersion, reveal that the release of Mn is higher than the release of Fe, which was attributed to a lower standard Acknowledgements corrosion potential of Mn when compared to Fe. The degrada- tion surface changes over time, with corrosion products con- This project has received funding from the European Union’s sisting of Fe, Mn, O and enriched in Ca, P and Cl are present. Horizon 2020 research and innovation programme under the (d) The saturation magnetization value of FeMn alloys Marie Skłodowska-Curie grant agreement No. 861046 (‘Bioremia’ slightly increases with immersion time, with the highest Ms value European Training Network), the Spanish Ministry of Economy, after 84 days of immersion. Nevertheless, the Ms value even after Industry and Competitivity (Grant PID2020-116844RB-C21), the 84 days is sufficiently low not to interfere with MRI imaging. Generalitat de Catalunya (2017-SGR-0292 and 2017-SGR-503), the (e) No significant differences in the mechanical properties of Swedish Research Council (2022-00853), the Swedish state under the different alloys are observed, although the addition of 3% the agreement between the Swedish Government and the county and 5% Ag tends to increase the Young’s modulus and the councils, the ALF-agreement (ALFGBG-978896), the IngaBritt plasticity index. and Arne Lundberg Foundation (LU2021-0048); the Hjalmar 628 | Mater. Adv., 2023, 4, 616–630 © 2023 The Author(s). Published by the Royal Society of Chemistry View Article Online Paper Materials Advances Svensson Foundation, the Doctor Felix Neuberghs Foundation, 21 Q. Zhang and P. Cao, Mater. Chem. Phys., 2015, 163, 394–401. and the Adlerbertska Foundation. The authors would like to 22 P. Sotoudehbagha, S. Sheibani, M. Khakbiz, S. Ebrahimi- thank the staff from the Servei de Microscòpia of Universitat Barough and H. Hermawan, Mater. Sci. 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