Surface & Coatings Technology 471 (2023) 129886 Contents lists available at ScienceDirect Surface & Coatings Technology journal homepage: www.elsevier.com/locate/surfcoat Accelerated biodegradation of FeMn porous alloy coated with ZnO: Effect on cytocompatibility and antibiofilm properties Aleksandra Bartkowska a, 1, Adam Benedict Turner b, c, 1, Andreu Blanquer d, *, Aliona Nicolenco e, Margarita Trobos b, c, *, Carme Nogues d, Eva Pellicer a, **, Jordi Sort a, f a Departament de Física, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès, Spain b Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden c Centre for Antibiotic Resistance Research in Gothenburg (CARe), University of Gothenburg, Gothenburg, Sweden d Departament de Biologia Cel⋅lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès, Spain e CIDETEC Surface Engineering, Basque Research and Technology Alliance (BRTA), Paseo Miramon 196, 20014 Donostia-San Sebastian, Spain f Institució Catalana de Recerca i Estudis Avançats (ICREA), Pg. Lluís Companys 23, E-08010 Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: Fe-based alloys are being studied as potential candidates for biodegradable implants; however, their degradation Zinc oxide coating rates remain too slow. To accelerate biodegradation while simultaneously hindering biofilm formation, a ZnO iron‑manganese alloys coating was deposited onto porous equiatomic FeMn alloy discs by sol-gel method using dip coating. The effect of Biofilm the ZnO coating on the microstructure, biodegradability, cytocompatibility, and antibacterial properties were Cytocompatibility Staphylococcus aureus investigated. Biodegradability experiments were performed by immersing the specimens in Hank's balanced salt Saos-2 solution and measuring ion release after up to 28 days of immersion. The experiments showed an increased degradation of the FeMn/ZnO sample due to Fe segregation towards the grain boundaries, formation of iron- manganese oxide, and limited formation of degradation products on ZnO. Further, indirect Saos-2 cell cyto­ toxicity testing in 24 h sample-conditioned media showed no significant cytotoxicity in concentrations equal to or below 50 %. In addition, the total biofilm biovolume formed by Staphylococcus aureus on the FeMn/ZnO surface was significantly reduced compared to the uncoated FeMn. Taken together, these results show that the ZnO coating on FeMn improves the degradation rate, maintains cytocompatibility, and reduces biofilm accu­ mulation when compared to an uncoated FeMn alloy. 1. Introduction is rather slow, limiting their clinical applications [2,3,10,11]. Therefore, the scientific community is currently exploring various solutions to In recent years, metallic bioresorbable or biodegradable metals have enhance the degradation rate of Fe-based alloys. The common ap­ emerged as potential candidates for stents and orthopaedic fixation proaches include alloying Fe with other elements (e.g., C, Mn, Si), devices such as bone pins, screws, wires, rods, or scaffolds [1–7]. The introducing porosity in the material, or macro-/nano-pattering main idea behind biodegradable metals is to provide mechanical support [3,4,12–17]. In the past decade, surface modification approaches have while gradually degrading over a desired period of time and becoming emerged as an alternative and more efficient way to improve the overall completely dissolved when their function is fulfilled. Owing to the use of performance of biodegradable metallic materials. These methods often biodegradable materials, implant removal surgery can be avoided and involve ion implantation [18], laser ablation [19] or deposition of thus the costs of medical treatment and risk of infection can be decreased coatings [5,20–22]. For instance, zinc ion implantation on the surface of [8,9]. Fe-based alloys are considered especially promising candidates for pure iron was found to accelerate the degradation rate by creating biodegradable implant applications due to their excellent mechanical galvanic pairs between Fe and Zn clusters [18]. Modification of the Fe- properties and good biocompatibility. However, their degradation rate Mn alloys surface by laser ablation increased their biodegradability * Corresponding authors. ** Correspondence to: E. Pellicer, Departament de Física, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès, Spain. E-mail addresses: andreu.blanquer@uab.cat (A. Blanquer), margarita.trobos@biomaterials.gu.se (M. Trobos), eva.pellicer@uab.cat (E. Pellicer). 1 A. Bartkowska and A.B. Turner contributed equally to this work as first authors. https://doi.org/10.1016/j.surfcoat.2023.129886 Received 8 June 2023; Received in revised form 1 August 2023; Accepted 2 August 2023 Available online 4 August 2023 0257-8972/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). A. Bartkowska et al. Surface & Coatings Technology 471 (2023) 129886 thanks to the formation of nanostructured metal oxides [19]. The pressing and sintering under vacuum at 900 ◦ C. Details regarding the deposition of coatings offers greater opportunities to adjust the corro­ synthesis of the FeMn discs can be found elsewhere [15]. The ZnO sion and other characteristics of the system because the ultimate prop­ precursor solution was prepared by mixing zinc acetate dihydrate (Zn erties will arise from the interplay between the two components (bulk (CH3COO)2⋅2H2O > 99.0 % purity, Merck, Germany) as a starting ma­ material and coating). terial, ethanol as a solvent, and monoethanolamine (C2H7NO, MEA, Recently, several authors have published reports on the improved >99 % purity, Merck, Germany) as a stabilizer. Zinc acetate and MEA properties of Fe-based alloys achieved by the deposition of second-phase were purchased from Merck and used without further purification. To a coatings. For instance, Qi et al. [22] reported the mechanism of accel­ solution of 0.5 M zinc acetate, MEA was added, keeping the zinc acetate eration of Fe corrosion by polylactic acid (PLA) coating. A decrease in to MEA ratio of 1:1. The solution was stirred for 2 h at 60 ◦ C using a the local pH due to PLA hydrolysis and the fact that the PLA coating magnetic stirrer working at 500 rpm to prepare a clear, homogenous hindered the formation of a passivation layer were behind the observed solution, and then aged for 24 h at room temperature. Before deposition, enhanced degradation. In another report, polyethylene glycol (PEG) the FeMn samples in the form of a flat disc with a diameter of 9 mm and coatings were deposited on Fe alloy foam which helped improve its thickness of 2 mm were polished up to #800 grit, ultrasonically cleaned corrosion rate [20,21]. In a study by Huang et al. [23], a collagen in ethanol and dried using a nitrogen gun. coating was applied to the Fe-Mn alloy, and although it did not increase The ZnO precursor solution was deposited by dip-coating onto FeMn the corrosion rate of the alloy, it helped reduce cytotoxicity and improve discs at a withdrawal rate of 300 mm/min using a Coater 5 AC (id Lab) osseointegration. operating at room temperature and 50 % relative humidity. To increase As biomaterial-associated infections are of concern, several ap­ the thickness of the ZnO layers and improve surface coverage, the dip­ proaches have been investigated to decrease the risk of infection ping process was repeated two times followed by mild annealing of the [24,25]. One possible solution is the deposition of antibacterial and deposited layers at 120 ◦ C for 20 min after each cycle. Finally, the coated antibiofilm coatings on the biomaterial surface [26–28]. ZnO has been FeMn substrates were annealed at 500 ◦ C for 3 h in air using a Carbolite studied as an antibacterial material, demonstrating its capability to furnace. Uncoated FeMn discs were annealed under the same conditions inhibit the growth of Escherichia coli [27] and Staphylococcus aureus and used as a reference for the ion release study. [29,30]. The mechanism of antibacterial activity of ZnO is not yet well The FeMn/ZnO samples were characterized using a scanning elec­ understood, but the production of reactive oxygen species on the surface tron microscope (SEM, Zeiss Merlin, Zeiss, Germany) coupled with of ZnO that cause oxidative stress to bacterial cells, ultimately causing energy-dispersive X-ray spectroscopy (EDS) detector, operating at 5–20 their death is one of the plausible explanations [31]. Thin ZnO films can kV. The thickness of the ZnO coating was assessed from SEM images of be deposited using a wide range of techniques such as sol-gel, spin- and the discs tilted 60◦ towards the detector. The phase composition was dip-coating, metal-organic chemical vapour deposition, molecular beam investigated using grazing incidence X-ray diffractometer (GIXRD) and epitaxy, pulsed laser deposition, or atomic layer deposition, amongst standard X-ray diffraction (XRD) analyses. The GIXRD patterns were others [27,32,33]. Sol-gel approaches are especially attractive for their acquired at room temperature using a Malvern Panalytical diffractom­ simplicity and ease of tailoring the properties of the synthesised films eter operating at an angular 2Θ range of 25◦ –75◦ , with a step size of [33–35]. Several parameters of the sol-gel process can be controlled, 0.02◦ and a counting time of 8 s. Standard XRD patterns were performed such as the type of precursor and its concentration, the type of solvent on a Panalytical X'Pert diffractometer (Malvern Panalytical, UK) with Cu and additives, the aging time of the mixture, the method of coating the Kα radiation, using an angular range of 20◦ –100◦ , with a step size of substrate and post-heat treatment of the materials. Sol-gel-derived ZnO 0.026◦ . coatings have been successfully deposited on degradable Mg alloys [36] to increase cytocompatibility and osseointegration and inhibit bacterial adhesion and growth. 2.2. Biodegradability test An implanted biodegradable material must be biocompatible (i.e., it must show an appropriate host response upon implantation), it cannot The biodegradability of FeMn and FeMn/ZnO was studied under cause a toxic or allergic inflammatory response or be thrombogenic. The static immersion of the samples in Hank's balanced salt solution (HBSS, biocompatibility of implant materials can be determined by two key H8264, Merck, Germany) according to the ASTM-G31–72 standard. factors – the host reaction (caused by implanted material) and the Prior to the tests, the samples were cleaned and sterilised in ethanol. degradation of the material in vivo [37]. When studying degradable Each disc, with a surface area of 1.25 cm2, was immersed in plastic metals, the second factor is crucial. The abundant degradation products containers filled with 30 mL of HBSS solution. The containers were then should not cause any inflammatory response. Enhanced biodegrad­ placed in a water bath and kept at a constant temperature of 37.5 ◦ C. The ability can influence cell viability and proliferation since the released discs were kept in HBSS for up to 28 days. After 1, 3, 7, 14 and 28 days, corrosion products can lead to histological changes in local tissues, due the solution was withdrawn, and the containers were refilled with fresh to toxic solutes and also to hypersensitivity to allergens [38]. media. The concentration of ions released from the material (Fe, Mn and The objective of this work was to study the effect of ZnO sol-gel- Zn ions) to the media was measured by inductively coupled plasma- derived coatings deposited on degradable porous FeMn alloys in terms optical emission spectroscopy (ICP-OES, Agilent 7900, Agilent, USA). of material biodegradability, cytocompatibility and microbial in­ Prior to analysis, the media was treated with HNO3 to dissolve the solid teractions. The purpose of the ZnO layer deposited on the FeMn alloy is corrosion products suspended in the solution. Five replicas of each two-fold: to favour the degradation of the alloy, and to confer anti­ sample were analysed, and the results are presented as an average ± biofilm properties to the material, without compromising its standard error (SE). cytocompatibility. The corrosion rate was estimated according to the release of Fe and Mn after 1 day of sample's immersion [39] (Eq. (1)): 2. Materials and methods C*V CR = (1) S*T 2.1. Synthesis and characterization of ZnO coating deposited on FeMn alloy where CR is the corrosion rate given in g/m2/day; C is the released ion concentration (g/L), V is the solution volume (L), S is the sample surface ZnO was deposited on FeMn porous alloy discs by dip-coating. The area (m2), and T is the incubation time (days). porous equiatomic FeMn alloys were fabricated by powder metallurgy, To evaluate the morphological and compositional changes of the namely by ball milling of initial Fe and Mn powders, followed by samples undergoing biodegradation, additional discs were immersed in 2 A. Bartkowska et al. Surface & Coatings Technology 471 (2023) 129886 the HBSS. After 3, 7, 14, and 28 days, the specimens were removed from Collection, Manassas, USA) was streaked from a − 80 ◦ C glycerol stock HBSS, rinsed with distilled water, dried using compressed nitrogen, and onto 5 % Horse Blood Columbia Agar (HBA; Media Department, Clinical kept in a desiccator until further examination. Analyses were performed Microbiology Laboratory, Sahlgrenska University Hospital, Sweden) both on the surface and the cross-section of the discs. Once removed followed by aerobic incubation at 37 ◦ C overnight. After incubation, from the HBSS, the specimens were not immersed again. The cross isolated single colonies were picked with a flocked nylon swab and sections were prepared by embedding the samples in resin, grinding, and inoculated into 4 mL Tryptic Soy Broth (TSB; Scharlau, Barcelona, polishing with diamond paste up to 1 μm. Top-down and cross-section Spain) to achieve an optical density (OD546) of 0.13, equivalent to 108 SEM observations and EDS determinations were carried out at 10 kV CFU/mL. This suspension was then diluted 1:1000 in TSB to achieve a and 20 kV, respectively. final working bacterial suspension (inoculum) of 105 CFU/mL. Prior to FeMn and FeMn/ZnO samples immersed for 14 and 28 days were inoculation with bacterial suspension, discs were sterilised by dry heat also characterized using X-ray diffraction to identify the products sterilisation at 180 ◦ C for 2 h. formed on the surface. The experiments were carried out on a Bruker diffractometer using Cu Kα radiation. The 2Θ region was between 200 2.4.2. Viability of S. aureus after 4 h or 24 h biofilm formation and 1000 with a step size of 0.0260. The peaks were identified using Dry-heat sterilised FeMn/ZnO and control FeMn discs (9 mm diam­ X'Pert Panalytical software. eter, 2 mm thickness) were added to wells of a 48-well plate, inoculated with 1 mL of 105 CFU/mL of the previously mentioned S. aureus ATCC 2.3. Cell proliferation assays and cytotoxicity analyses 25923 working bacterial suspension, and incubated aerobically and statically at 37 ◦ C for either 4 or 24 h. In vitro cell proliferation and cytotoxicity of FeMn and FeMn/ZnO After each time point, discs were carefully removed from the well samples were assessed using human osteosarcoma cells (Saos-2 cell line; plate, rinsed three times in sterile saline (0.9 %), and transferred to a 15 ATCC HTB-85). The cells were maintained in Dulbecco's Modified Ea­ mL Falcon Tube containing 1 mL sterile saline (0.9 %). Discs were then gle's medium (DMEM; Gibco, Thermo Fisher Scientific, USA) supple­ sonicated (42 kHz) in a Branson 3510MT Ultrasonic Cleaner (Branson, mented with 10 % fetal bovine serum (FBS; Gibco) at a controlled Brookfield CT, USA) for 30 s, followed by vortexing for 1 min at 10000 temperature of 37 ◦ C and 5 % CO2. rpm. Disaggregated biofilm cells were then ten-fold serially diluted in a Samples were ultrasonically cleaned with distilled water and ethanol combination of sterile saline (0.9 %) and Triton-X (0.1 %). These di­ and then sterilised with pure ethanol. To prepare the conditioned media, lutions were plated on 5 % HBA plates and incubated at 37 ◦ C overnight the samples were immersed in supplemented DMEM for 24 h under and the viable colony-forming units (CFU) were subsequently enumer­ standard conditions. A ratio of 1.25 cm2/mL was used, as recommended ated. Three independent biological experiments with duplicate samples by the ISO 10993-5:2009 standard. After 24 h, the samples were were performed. removed, and the conditioned media were kept at 4 ◦ C protected from light until use. Next, the conditioned media were diluted to obtain the 2.4.3. Confocal laser-scanning microscopy and quantitative image analysis extract at the following concentrations: 100 % (non-diluted), 50 %, 25 of S. aureus biofilms %, and 12.5 % for further analysis. In parallel, control media were Dry-heat sterilised FeMn/ZnO and control FeMn discs were added to prepared by incubating media for 24 h, without any sample to obtain wells of a 48-well plate, inoculated with 1 mL of 105 CFU/mL of S. aureus aged media. ICP-OES was used to identify the released ion concentra­ ATCC 25923 inoculum, and incubated aerobically and statically at 37 ◦ C tions of Fe, Mn, and Zn ions in the concentrated conditioned media (100 for either 4 or 24 h. Upon completion of each time-point, discs were % extract). carefully removed from the well plate and rinsed 3 times in sterile saline The metabolic activity of osteoblasts was determined using the (0.9 %). After which, S. aureus adhered to the surface were stained using Alamar Blue reagent (Thermo Fisher Scientific, USA) after 1 and 3 days FilmTracer LIVE/DEAD Biofilm Viability Kit (Invitrogen, Waltham, in order to evaluate the proliferation of cells exposed to the conditioned United States) in the dark at room temperature for 20 min. After stain­ media. A total amount of 5 × 104 osteoblasts were seeded in a 24-well ing, discs were rinsed in sterile saline (0.9 %) to remove unbound stain plate using fresh media. After 24 h, the media was replaced with and transferred to a 35 mm petri-dish containing 3 mL sterile saline for conditioned media at different concentrations (100 %, 50 %, 25 %, and imaging on a Nikon C2+ confocal laser-scanning microscope (CLSM; 12.5 %), and the cells were incubated for another 24 h under standard Nikon, Tokyo, Japan) with a 100× water-dipping objective (CFI Plan conditions. The conditioned media was then replaced with fresh media 100XC W). Images were taken with Z-slices of 3 μm through the S. aureus containing 10 % Alamar Blue and incubated for 4 h. Afterwards, the biofilms. Biofilm thickness and biovolume were measured using Bio­ supernatant was collected (day 1), and fluorescence was analysed at a filmQ [40]. Sections imaged were taken from five randomly selected wavelength of 585 nm after excitation at 560 nm using a Spark multi­ fields of view on each material sample. Three independent biological mode microplate reader (Tecan, Mannedorf, Switzerland). The cells experiments were performed with technical duplicates. were then incubated with the same conditioned media previously used and kept in the incubator during the Alamar Blue assay. Cells were 2.4.4. SEM imaging of S. aureus ATCC 25923 cultured for 72 h in this conditioned media and the Alamar Blue assay Dry-heat sterilised FeMn/ZnO and FeMn discs were added to wells of was repeated as previously described (day 3). Aged and fresh media a 48-well plate, inoculated with 1 mL of 105 CFU/mL of S. aureus ATCC were used as the controls. The experiments were performed in 25923, and incubated aerobically and statically at 37 ◦ C for 24 h. After triplicates. incubation, both the control FeMn and FeMn/ZnO samples were care­ In parallel, the cytotoxicity of conditioned media was assessed in fully removed from the well plate and rinsed 3 times in sterile saline (0.9 cells grown for 3 days using the live/dead viability/cytotoxicity kit for %). Following this, S. aureus adhered to the surface were fixed in 4 % mammalian cells (Invitrogen, United States) according to the manu­ formaldehyde (HistoLab AB, Sweden) for 1 h at room temperature facturer's protocol. Images of randomly selected regions were obtained before dehydration in a graded ethanol series (50-, 70-, 80-, 90-, 95-, using an Olympus IX71 inverted microscope equipped with 100 %), for 5 min at each concentration, with the final 100 % step being epifluorescence. repeated once. The discs and S. aureus biofilms adhered to the surface were gold 2.4. Assessment of S. aureus adhesion and viability sputtered (10 nm) using a Leica EM ACE600 Sputter Coater (Leica, Stockholm, Sweden) and imaged in secondary electron mode using an 2.4.1. Staphylococcus aureus ATCC 25923 culture conditions LEO 55 ULTRA FEG SEM (Zeiss, Germany) operating at 5 KV. Staphylococcus aureus ATCC 25923 (American Type Culture 3 A. Bartkowska et al. Surface & Coatings Technology 471 (2023) 129886 2.5. Statistical analysis possible to obtain a material using a sol or a gel as an intermediate step with the process being conducted at lower temperatures than traditional All quantitative data were analysed using GraphPad Prism 9 and methods [33,35]. The sol-gel processes can be divided into the (i) biodegradability and cytocompatibility data is presented as the mean ± preparation of precursor solution, (ii) sol deposition on the substrate by standard error of the mean. Statistical differences for biodegradability spin- or dip-coating and (iii) heat treatment of the xerogel film. During and cytocompatibility studies were obtained by one-way analysis of the drying process of sol-gel-derived films, when the solvent is removed, variance (ANOVA) with a Tukey correction. Microbiological data is compressive stress is generated in the system owing to the difference in presented as the mean ± standard deviation. Biofilm viability (CFU/mL) the thermal expansion coefficients between the film and the substrate. was analysed using an unpaired student's t-test. Differences in biofilm This likely led to the formation of a wrinkled structure in the gelated biovolume and thickness were analysed by paired Students t-test to ZnO film. In the samples described here, apart from the wrinkled ZnO compare between live and dead groups, and unpaired Students t-test for coating, a layer of Fe-Mn oxide was also present, which was formed the comparisons between the two biomaterial groups. A value of p < during the annealing process. 0.05 was considered significant. The GIXRD pattern of the dip-coated ZnO layer on the FeMn sub­ strate is shown in Fig. 2. This pattern confirms the formation of poly­ 3. Results crystalline ZnO in the hexagonal wurtzite phase (space group P63mc), as 3.1. Morphology of the ZnO coating deposited on FeMn substrate Fig. 1 displays the topography and morphology of the ZnO coating deposited on the FeMn substrate, together with the elemental distribu­ tion of Fe, Mn, Zn and O obtained by SEM + EDS. The porous FeMn substrate contains large pores, with a diameter of around 10 μm and nanopores, with a size around 100 nm, as presented in Fig. S1. The coating has a compact structure and is evenly distributed over the entire surface. However, Fig. 1A, B shows that the regions around the large pores have a distinct morphology. This was attributed to the difference in the wettability between the dense and porous areas of the sample. The regions around the pores were enriched in Mn, O, and Fe, with a very low Zn content, indicating the formation of iron‑manganese mixed ox­ ides. The ZnO coating formed a wrinkled network structure consisting of regions of higher thickness (i.e., ganglia-like hills) with a thickness of approximately 2 μm, as well as flat regions with a thickness of approx­ imately 600 nm, as shown in Fig. 1C, D. The flat areas of the ZnO coating possessed a mesoporous structure, as shown in Fig. 1E. Similar morphologies of ZnO thin films have been previously described in the literature [32,34]. Several explanations have been put forward to explain the morphology of ZnO films, such as stress relaxa­ tion and slow cooling conditions. In our case, we attributed the wrinkled morphology to stress relaxation due to the difference in the thermal Fig. 2. GIXRD pattern of FeMn/ZnO sample. Triangles correspond to the ZnO expansion coefficient between the metallic disc underneath and the thin hexagonal phase, while squares belong to the iron‑manganese oxide phase film, as suggested by Kwon et al. [41]. The sol-gel methods make it formed during annealing. Fig. 1. Morphology of the ZnO film deposited on the FeMn substrate: (a) low-magnification view of the coating, (b) SEM image and EDX map of the area, (c) tilted view of coating’ wrinkles, (d) magnified view of a wrinkle and (e) morphology of the coating at the flat areas, showing a detail of the mesoporosity in the inset. 4 A. Bartkowska et al. Surface & Coatings Technology 471 (2023) 129886 evidenced by the presence of characteristics peaks for this phase at the of immersion, new peaks matching the MnCO3 phase appear. Indeed, the 2Θ values of 31.9◦ , 34.4◦ and 36.3◦ , corresponding to (100), (002) and formation of corrosion products such as carbonates and phosphates (101) planes, respectively. No preferred orientation was observed; resulting from the reaction between samples and the surrounding HBSS therefore, we can conclude that the ZnO crystallites possess random environment is common [9]. The HBSS contains HCO−3 and HPO−4 ions orientation. Besides the ZnO phase, an additional peak was identified at which may react with the released metallic ions, thus forming insoluble 40.60, which is consistent with the (200) plane of iron manganese oxide metal carbonates or phosphates. The latter deposit on the metallic sur­ phase with space group Fm-3m. The presence of this oxide is in accor­ face, slowing down further degradation. In the case of the FeMn/ZnO dance with the EDS mapping presented in Fig. 1. sample (Fig. 4B), three characteristic peaks of ZnO, the γ-austenite phase, as well as the iron‑manganese oxide phase (identified as well in Fig. 2), are present in all samples. Moreover, one can observe the pres­ 3.2. Biodegradability of FeMn/ZnO ence of the α-Fe in the FeMn/ZnO samples, most probably due to the segregation of Fe on the grain boundaries after the annealing step A first biodegradability test was conducted to assess the changes in required to convert the ZnO precursor into ZnO. No peaks ascribed to the surface morphology and composition of the FeMn/ZnO samples and corrosion products are observed, which would agree with our previous to measure ion release upon immersion in HBSS. The in-plane views observation that the ZnO might hinder the formation of corrosion after 3, 7, 14, and 28 days of incubation are presented in Fig. 3. The products. Hence, the main difference between the uncoated and ZnO- results of the EDS analysis obtained from the areas highlighted in Fig. 3 coated discs resides in the phase composition of the FeMn substrate: (black letters A–D, corresponding to FeMn and numbers 1–4, corre­ austenite and ferrite phases coexist in the FeMn/ZnO sample, while the sponding to FeMn/ZnO) are shown in Table 1. naked substrate has a purely austenitic structure. It is anticipated that The corrosion products formed on the uncoated FeMn alloy after 3, 7, the occurrence of the austenite and ferrite phases in the FeMn/ZnO and 14 days of immersion had a size of a few micrometres and mostly samples might contribute to accelerating their degradation in bodily consisted of globular precipitates that were not uniformly distributed on fluids [3,43]. the surface. These were identified as Ca, P, and Cl-containing pre­ In addition, having analysed Figs. 3 and 4, it is likely that the ZnO cipitates by EDS. After immersion for 14 and 28 days, the amount of Ca coating acts as a physical barrier in the initial stages of degradation, and P precipitates formed on the surface of the FeMn alloys appeared protecting the surface of FeMn alloy from direct contact with the cor­ much higher than for the FeMn/ZnO samples (Table 1). After 28 days of rosive environment, therefore, limiting the deposition of insoluble immersion, a compact corrosion layer was formed, covering the entire corrosion products from HBSS. In fact, the amounts of Ca- and P-con­ surface. This compact corrosion layer can act as a protective barrier, taining products after 7 days of incubation are lower (Table 1). How­ hindering the progression of corrosion. ever, over time, the penetration of HBSS through the pores might allow The morphologies of the corrosion products formed on the surface of for the initiation of localized corrosion. Moreover, as the ZnO coating is the FeMn/ZnO samples showed different features. Following immersion not fully dense, media can easily permeate through the defects and pores for a maximum of 14 days, the distinctive morphology of the ZnO and cause micro galvanic corrosion between the semiconductor coating coating can be still observed, indicating that it had not been entirely and the metallic substrate. engulfed by corrosion products. The products primarily consist of Fig. 5 shows the cumulative ion release of Fe (A) and Mn (B) in loosely packed globular precipitates, identified as Ca, P and Cl pre­ (uncoated) FeMn, annealed FeMn and FeMn/ZnO samples after 1, 3, 7, cipitates. However, following a 28-day immersion period, a denser layer 14 and 28 days of immersion for both Fe and Mn release after 1 day (C) of corrosion products accumulated on the surface of the ZnO coating. and 28 days (D) of immersion in HBSS. It can be observed that the This layer included some platelet-like structures that have been recog­ release of both Fe and Mn increases over time, with significant differ­ nized as Ca-P apatite-like precipitates [4,14]. The formation of these ences between FeMn and FeMn/ZnO samples. The release of Fe and Mn precipitates is a priori positive, since it increases the bioactivity of the ions in FeMn/ZnO samples after 1 day was 5.50 ± 0.94 ppm and 9.00 ± surface, eventually leading to a better bone-bonding ability of the ma­ 0.86 ppm, respectively. In uncoated FeMn the release after 1 day in terial [42]. HBSS was much lower and reached 0.06 ± 0.02 ppm for Fe ions and 0.33 The XRD patterns of FeMn and FeMn/ZnO samples in the as- ± 0.04 ppm for Mn ions (Fig. 5A). Note that the release of Fe and Mn ions prepared state and after 14 and 28 days of degradation in HBSS are after 1 day is significantly different for FeMn/ZnO sample, while both displayed in Fig. 4A, B. FeMn specimens (Fig. 4A) in the as-prepared ions are released in similar amounts from the uncoated FeMn sample. state and after 14 days of immersion in HBSS show the same phase The release after 28 days in HBSS for FeMn/ZnO reached the value of composition (γ-austenite and Fe-Mn oxide phases), while after 28 days Fig. 3. Top-down SEM micrographs of the surface of FeMn and FeMn/ZnO samples after 3, 7, 14 and 28 days of immersion in HBSS at 37.5 ◦ C. Black letters A–D correspond to FeMn, and numbers 1–4 correspond to FeMn/ZnO immersed for 3, 7, 14, and 28 days, respectively. 5 A. Bartkowska et al. Surface & Coatings Technology 471 (2023) 129886 Table 1 EDS analysis of the areas presented in Fig. 3. A, B, C, and D correspond to areas of FeMn samples immersed for 3, 7, 14, and 28 days, respectively. Meanwhile, 1, 2, 3, and 4 correspond to areas of FeMn/ZnO samples immersed for 3, 7, 14, and 28 days. Sample EDS analysis Element (wt.%) Fe Mn O Zn Ca P Cl Mg FeMn 3d A 42.9 ± 5.0 35.4 ± 5.4 18.4 ± 2.0 X X X 3.3 ± 0.7 X FeMn/ZnO 3d 1 4.2 ± 0.1 32.1 ± 0.2 19.8 ± 0.2 38.4 ± 0.2 2.6 ± 0.1 1.3 ± 0.1 1.2 ± 0.1 0.5 ± 0.1 FeMn 7d B 41.9 ± 0.8 36.4 ± 0.8 16.4 ± 0.3 X 1.3 ± 0.1 X 4.1 ± 0.1 X FeMn/ZnO 7d 2 12.6 ± 0.6 26.5 ± 0.8 25.6 ± 0.4 30.6 ± 0.4 1.4 ± 0.1 2.3 ± 0.1 0.9 ± 0.1 X FeMn 14d C 33.6 ± 0.7 31.2 ± 0.9 23.8 ± 0.4 X 2.9 ± 0.1 4.0 ± 0.1 4.6 ± 0.1 X FeMn/ZnO 14d 3 1.8 ± 0.5 30.7 ± 0.6 22.3 ± 0.3 38.6 ± 0.4 0.7 ± 0.1 0.5 ± 0.1 0.8 ± 0.1 X FeMn 28d D 26.3 ± 0.7 29.7 ± 0.9 24.3 ± 0.4 X 9.7 ± 0.2 8.1 ± 0.2 0.9 ± 0.1 1.1 ± 0.1 FeMn/ZnO 28d 4 4.8 ± 0.1 36.1 ± 0.2 27.6 ± 0.2 17.5 ± 0.2 2.0 ± 0.1 3.1 ± 0.1 8.5 ± 0.1 0.3 ± 0.1 Fig. 4. X-ray diffraction patterns of the as-prepared and immersed for 14 and 28 days in HBSS samples of (A) FeMn and (B) FeMn/ZnO. 14.70 ± 1.55 ppm for Fe and 21.69 ± 1.38 ppm for Mn ions (again, sections of FeMn/ZnO discs in the as-prepared state and after 7, 14 significantly higher), while for FeMn it reached the value of 0.34 ± 0.05 and 28 days of immersion in HBSS. Linear EDS analysis was performed ppm for Fe and 2.9 ± 0.29 ppm for Mn (Fig. 5B). In general, the release on these samples to identify the distribution of Fe, Mn, O, Zn and Ca on of Fe ions is lower than that of Mn. The concentration of Zn2+ ions in the the cross-section of the samples. We can observe that already in the as- FeMn/ZnO samples was very low, reaching only 0.15 ppm after 28 days prepared state, there is a Mn- and O-enriched region, just below the ZnO of immersion (not shown). coating, that can be recognized as the iron-manganese-rich oxide. Just As a reference, ion release from annealed (uncoated) FeMn discs was below this layer we can observe a region enriched in Fe with reduced tested. An elevated ion release after 1 to 28 days of immersion was content of Mn, that, likewise, forms due to Mn diffusion towards the observed when compared to the uncoated, non-annealed FeMn disc surface to form the aforementioned oxide layer. After 28 days we can (Fig. 5C, D). Yet, the release of Fe and Mn ions was lower than for the observe that the Mn-rich region is becoming thicker upon degradation. FeMn/ZnO sample. The release of Mn was also lower when compared to The outermost layer contains Ca, coming from HBSS-derived the FeMn/ZnO sample, but it reached the same concentration after 28 precipitates. days of immersion. Fig. 8 shows cross-section areas of the FeMn and FeMn/ZnO samples Overall, the release of Fe after 28 days of immersion is 43 times in the as-prepared state and after 7, 14 and 28 days of immersion in higher (and the release of Mn 7.5 times higher) in FeMn/ZnO sample HBSS. Additional supplementary Fig. S2 presents the EDS mapping than in FeMn. The significant differences between FeMn, annealed FeMn performed on the samples immersed for 28 days, where annealed FeMn and FeMn/ZnO samples indicate that the ZnO coating and the formation sample was added as a reference as it has shown an ion release close to of secondary phases stemming from its synthesis would trigger the that of FeMn/ZnO. Uncoated FeMn and FeMn/ZnO samples present degradation of FeMn alloys in HBSS. similar microstructural features after 7 days of immersion, with a The concentration of metallic ions after 1 day of immersion in HBSS degradation layer at the upper region. However, after 14 and 28 days of was used to make a rough estimation of the corrosion rates according to immersion, samples show distinct microstructures. The corrosion layer Eq. (1), and the results are shown in Fig. 6. A significant increase in the formed on the FeMn disc becomes thicker and more compact and the corrosion rate was observed for the FeMn/ZnO sample when compared macropores are infilled with degradation products. Conversely, the to the uncoated FeMn alloy and the annealed (uncoated) FeMn samples. FeMn/ZnO sample does not show a thick corrosion layer on the top The corrosion rates of FeMn/ZnO were 36 times higher than that of surface. It does, however, show segregation of Fe around the pores, as FeMn and 3 times higher than that of annealed FeMn. This indicates that highlighted by yellow arrows, and confirmed by EDS mapping in the the enhanced degradability can be attributed to both Fe segregation due supplementary information. Moreover, an increase in the size of the to annealing and the presence of the ZnO coating. nanopores is observed in the denser region, highlighted by red arrows, Fig. 7 presents the SEM images and linear EDS analysis of cross- which would indicate the dissolution of the FeMn matrix. 6 A. Bartkowska et al. Surface & Coatings Technology 471 (2023) 129886 Fig. 5. Released ion concentration of Fe (A) and Mn (B) after 1, 3, 7, 14, and 28 days of immersion in HBSS for FeMn, annealed FeMn, and FeMn/ZnO samples. Analysis of significance for ion release after 1 (C) and 28 (D) days, comparing the release of Fe and Mn for the different samples. Bars marked with different alphabetical superscripts are significantly different from each other, while bars with the same superscript are not significantly different from each other. conditioned media used for the experiments were prepared at 100 %, 50 %, 25 % and 12.5 % concentrations. Similarly, the control media, conditioned for 1 day without any material, was also diluted with fresh media at previously mentioned concentrations. Fig. 9A shows the ion release concentration after 1 day of incubation in DMEM for 100 % extract. The release of Fe ions was not significantly different for the two samples, whereas the release of Mn was signifi­ cantly higher for the FeMn/ZnO sample. These results are in line with those previously observed for the ions leaching in HBSS (cf. Figs. 5C and 9A). The release of Zn ions (not shown in the plot) was 6.2 ± 0.7 ppm. Regarding cell viability (Fig. 9B and C), the percentage of live cells was approximately 75 % for FeMn and 51 % for FeMn/ZnO using the conditioned medium at a concentration of 100 %, which was signifi­ cantly lower than the control, FeMn and FeMn/ZnO conditioned media. However, the percentage of live cells was 90 % for both samples when the conditioned media was diluted to 50 and 12.5 %, without significant differences between both the samples and the concentrations. Results showed that with an increase in the dilution of the conditioned media, cell viability increased. The fluorescence microscopy images of the live/ Fig. 6. Calculated corrosion rates of FeMn and FeMn/ZnO samples based on dead assay (Fig. 9B) for 12.5 % and 50 % conditioned medium showed a the released ion concentration after 1 day of immersion in HBSS calculated for similar amount of live Saos-2 cells (green cells), with very few dead cells both Fe and Mn ion release. The total corrosion rate is the sum of Fe and Mn (red) for both samples. For the concentrated conditioned media (100 %), corrosion rates for each sample. ‘DR’ stands for degradation rate. fewer cells were found for both samples compared to the control and diluted conditioned media. In addition, the conditioned media derived 3.3. In vitro cytocompatibility and cell proliferation from FeMn/ZnO led to a higher ratio of dead cells than the FeMn sample. The metabolic activity results after 1 and 3 days of incubation are Cytocompatibility of the materials was assessed by cell proliferation presented in Fig. 9D–E and Supplementary Fig. S3. The fluorescence and cytotoxicity using an indirect in vitro model on Saos-2 cells. The intensity of Alamar Blue (which corresponds to metabolic activity of the 7 A. Bartkowska et al. Surface & Coatings Technology 471 (2023) 129886 Fig. 7. EDS line scans for O, Mn, Fe, Ca and Zn elements along the cross-section of FeMn/ZnO samples in the as-prepared state (A) and after 7 (B), 14 (C), and 28 (D) days of immersion in HBSS. The yellow arrow indicates the direction of the EDS line scan. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 8. Cross sections of FeMn and FeMn/ZnO samples immersed for 7, 14 and 28 days in HBSS. ‘DL’ stands for degradation layer formed on the surface. Yellow arrows indicate the Fe-enriched regions, while red arrows show increased pore size. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) cells) increased with increasing dilution of the conditioned media, presented. We observed that the metabolic activity in the 100 % although no significant differences were observed amongst the 50 %, 25 conditioned media increased over time for the 24 h-control media, % and 12.5 % dilutions. The 100 % concentrated conditioned media led whereas it remained low and without significant differences between the to significant differences between the control, FeMn and FeMn/ZnO FeMn and FeMn/ZnO samples. On the contrary, no significant differ­ samples. A drastic change was observed when comparing the 100 % ences were observed in cell proliferation between samples on day 1 of conditioned media with the diluted conditioned media for both mate­ culture in the 50 % conditioned media, whereas significant differences rials in the sense that all materials (FeMn, FeMn/ZnO and control) arose after 3 days of culture between the 24 h-control media and FeMn showed similar metabolic activity. In supplementary Fig. S3, the dif­ conditioned media compared to day 1. The proliferation of the FeMn/ ferences in cell proliferation between days 1 and 3 of culture are ZnO sample after day 3 was not significantly different from that on day 8 A. Bartkowska et al. Surface & Coatings Technology 471 (2023) 129886 Fig. 9. Cytocompatibility assessment of FeMn and FeMn/ZnO samples obtained through indirect assay. The concentration of Fe and Mn ions released to the culture media after 24 h by FeMn and FeMn/ZnO samples (A), Live/Dead images of Saos-2 cells cultured indirectly in conditioned media from FeMn and FeMn/ZnO (B), the viability of Saos-2 cells cultured indirectly in conditioned media from FeMn and FeMn/ZnO (C). Metabolic activity of Saos-2 cells cultured in diluted conditioned media from FeMn and FeMn/ZnO samples after 1 day (D) and 3 days (E). Media conditioned for 1 day without any material was used as a control and diluted with fresh media. Significant differences are marked with (*). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.) 1. The morphology of S. aureus biofilms adhered to either the control FeMn or the FeMn/ZnO surface after 24 h incubation was visualised by 3.4. Biofilm formation SEM (Fig. 10F–K). In agreement with the obtained data from CLSM, the total biomass adhered to the FeMn control surface (Fig. 10F–H) was In this study, the uncoated FeMn substrate was used as a control. noticeably greater than that adhered to the FeMn/ZnO surface S. aureus ATCC 25923 was incubated with and directly exposed to the (Fig. 10I–K). S. aureus biofilm formed on the FeMn control was thicker control FeMn and the FeMn/ZnO samples for 4- and 24 h. After both and more homogenously distributed across the FeMn surface compared time points, no significant change (p > 0.05) in bacterial viability was to the FeMn/ZnO surface, where smaller aggregates were common, and observed between the FeMn/ZnO specimens and the control FeMn the underlying material surface remained visible in many areas. Further, (Fig. 10A). less extracellular polymeric substances (EPS) between bacterial cells The live and dead biovolume of S. aureus biofilms adhered to the were observed on the FeMn/ZnO surface in comparison to the FeMn surface were measured by CLSM and quantified using BiofilmQ. After 4 group (Fig. 10H - red arrows) indicating a less matured and developed h no differences between the FeMn control and the FeMn/ZnO groups biofilm. In combination with this visible reduction in biofilm biomass, were identified regarding total biovolume or biofilm thickness of both more damaged, ruptured and/or dead S. aureus cells were observed on live and dead S. aureus cells (Fig. 10B, C). Similarly, after 24 h no sig­ the FeMn/ZnO surface (Fig. 10K - blue arrows) when compared to the nificant differences between the two materials were observed regarding S. aureus cells adhered to the FeMn control. the thickness of biofilms composed of live or dead bacterial cells, or the biovolume of dead cells (Fig. 10B, C). However, after 24 h a significant 4. Discussion reduction in live biofilm biovolume was observed on the FeMn/ZnO surface when compared to the FeMn surface (Fig. 10B). Further, while 4.1. Effect of the ZnO coating on the degradation of FeMn alloys an increase in live S. aureus biovolume over time was found in both groups, the fold increase in biovolume of live bacteria between 4- and Several approaches have been tested to increase the corrosion rate of 24 h was noticeably greater on the control FeMn (9.5-fold increase) than biodegradable iron-based materials, namely, (i) alloying Fe with other on the FeMn/ZnO coated specimen (4.3-fold increase). Moreover, on the elements, such as Mn to shift the corrosion potential towards more FeMn control, an increase in the dead biovolume of biofilms was also negative values and hence, make it more prone to corrosion (ii) intro­ observed over time. In addition, after 24 h of growth, significantly more ducing porosity to increase the total area susceptible to corrosion attack live S. aureus cells than dead were found on the FeMn surface, whereas and, (iii) introducing noble elements that could act as a cathode and no difference was found between the live/dead cell proportions on the cause the formation of micro-galvanic corrosion cells. FeMn/ZnO surface (Fig. 10B). Representative CLSM images of S. aureus In short, the phenomena taking place when a biodegradable metal is adhered to the FeMn (Fig. 10D) and the FeMn/ZnO surfaces (Fig. 10E) in contact with physiological fluids can be described as follows [4,9]: after 24 h reflect these observations. Once the degradable metal is immersed in the fluid, it undergoes an 9 A. Bartkowska et al. Surface & Coatings Technology 471 (2023) 129886 Fig. 10. A) Viable Staphylococcus aureus ATCC 25923 adhered to the control FeMn and the ZnO-coated material surfaces after 4 h and 24 h. Quantitative confocal laser-scanning microscopy (CLSM) of S. aureus biofilms grown on FeMn/ZnO and FeMn surfaces showing the total B) biofilm biovolume and C) biofilm thickness of live and dead bacteria after 4 h and 24 h. Representative CLSM images of live and dead stained S. aureus biofilms grown on the D) FeMn control and the E) FeMn/ZnO coated surfaces for 24 h in which live S. aureus cells stain green, and dead stain red. Scanning electron micrographs of S. aureus adhered to the surface of the control FeMn (F-H) and FeMn/ZnO (I-K) after 24 h of growth, obtained at increasing magnifications (200×, 1000×, 5000×). Red arrows indicate extracellular polymeric substances (EPS) in the biofilm, whereas blue arrows indicate damaged or dead S. aureus cells. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.) 10 A. Bartkowska et al. Surface & Coatings Technology 471 (2023) 129886 oxidation reaction, where metallic ions are released (Eq. (2)). The others have reported higher degradation rates when coatings are electrons generated by anodic reaction are consumed during the deposited on Fe-based alloys. The observed increased corrosion was cathodic reaction, which reduces the amount of dissolved oxygen (Eq. attributed either to the formation of micro-galvanic cells [45] or (3)). These reactions occur on the entire metallic surface, and the increased solubility of Fe corrosion products due to polymer hydrolysis dissolution of the metal can be accelerated by galvanic coupling be­ when polymeric coatings were deposited [22]. In our case, we attribute tween metallic matrix and grain boundaries or secondary phases the increased corrosion rate of the FeMn/ZnO composite to the segre­ because of their distinct potentials. Moreover, M(OH)n corrosion prod­ gation of Fe and the formation of micro-galvanic cells between Fe-rich ucts are deposited on the metal surface (Eq. (4)). regions and the FeMn matrix, as well as between the ZnO coating and the FeMn underlying material. M→M n+ + ne− (anodic reaction) (2) 2H2 O + O2 + 4e− →4OH − (cathodic reaction) (3) 4.2. Effect of the accelerated corrosion on the proliferation and cytotoxicity of Saos-2 cells M n+ + nOH − →M(OH)n (product formation) (4) Indirect cytocompatibility studies using biodegradable Fe-based al­ Physiological solutions such as HBSS contain abundant inorganic loys have been conducted on various types of cells such as fibroblasts ions, which can influence the degradation process by either (i) breaking [46], smooth muscle cells (SMC) [47] and osteoblasts [46], depending down the passive film and causing pitting corrosion or (ii) passivating on the target application. Metallic ions released during corrosion may the Fe and slowing down corrosion due to the formation of phosphates have harmful effects on cellular functions [9]. Hence, it is important to and carbonates. Ions such as Cl− and SO2−4 can break the passive film and demonstrate that the degradation products being formed during mate­ accelerate corrosion, while other ions like HPO2− 3− 4 /PO4 , HCO3 /CO3 − 2− rial dissolution are non-toxic to cells. Here, osteoblastic Saos-2 cells 2+ and Ca might form a protective layer which further delays degrada­ were used to investigate the cytocompatibility of the FeMn-based tion of the material [9]. materials. Apart from the reaction between the metal and physiological solu­ Experiments were conducted using conditioned media at concen­ tion, the structural properties of metals themselves can favour or hinder trations of 100 %, 50 %, 25 % and 12.5 %, showing that only the 100 % degradation. The segregation of elements is known for enhancing the extract had a cytotoxic effect. The ionic concentrations of Fe and Mn corrosion rate of alloys [44]. Two grains of different chemical compo­ correlate with cell viability. The maximum concentration of released Fe sition and crystal structure can cause corrosion, for example in the case in 100 % extract was 45.3 ± 6.67 mg/L (811 μmol/L), Mn was 57.3 ± of duplex steels, which consist of austenite and ferrite grains [43]. This 3.2 mg/L (1043 μmol/L), while Zn concentration reached 6.2 ± 0.7 mg/ can lead to the formation of galvanic couples between them, where one L. Our results obtained are in agreement with previous reports. For grain acts as anodic site and the other as cathodic site. This galvanic instance, it has been reported that Fe concentrations above 50 mg/L can coupling can lead to the selective dissolution of the less corrosion- result in decreased metabolic activity [38]. Mueller et al. [47] showed resistant grain. Similarly, Donik et al. [19] described that the that the SMC growth rate was reduced by the addition of soluble ferrous patterning of FeMn surface by laser ablation leads to the formation of ions to the cell culture medium. The minimum Mn concentration high-temperature oxides, that in turn leads to a shift in the ratio of Fe/ causing cytotoxic effects depends strongly on the type of cells used [48]; Mn to Mn, which ultimately enhances biodegradability of the material. for human lung epithelial cells, a concentration above 20 μmol/L was In our study, we observe a similar shift, namely, a difference in the Fe to cytotoxic [49], however, for mouse brain cells, the minimum Mn con­ Fe/Mn ratio around the pores, as well as higher Mn content at the sur­ centration causing cytotoxicity increased to 200 μmol/L [50] and for face due to the oxide formation. The layer just below the surface is HUVEC cells it was even higher, reaching 500 μmol/L [51]. For Saos-2 enriched in Fe that can also influence corrosion. cells cultured with the 50 % extract, containing Mn concentration of The standard corrosion potential of Fe and Mn is − 0.44 V and − 1.05 approximately 500 μmol/L, cytotoxicity was not observed. The rela­ V, respectively [39]. When mixed, the potential falls in between (around tively low release of Zn ions should not cause cytotoxicity, as it is below − 0.7 V), with the exact value depending on the alloy composition. In the threshold value. this case, apart from the austenitic FeMn phase, the material consists of While discussing the results of in vitro cytocompatibility, we should ZnO coating, iron‑manganese oxide, and the α-Fe phase. This α-Fe phase keep in mind that the tests were performed under static conditions, formed as a side product of iron‑manganese oxide formation during whereas real in vivo conditions are more dynamic [52]. As the cell annealing, as the particle boundaries adjacent to the oxide layer were viability presented in this study clearly depends on the level of dilution, depleted in Mn (Figs. 7–8), thus forming an Fe-rich phase. The α-Fe zone it could be presumed that the concentration of ions and degradation at the boundaries causes a difference in potential between the inner products in a dynamic environment is lower than all the dilutions pre­ regions of the grains and the boundary, thus making the material more sented here and, therefore, the material should not cause a cytotoxic prone to corrosion. Moreover, the ZnO and iron-manganese oxide phases effect in vivo. For instance, in a study by Loffredo et al. [52], the viability can form micro-galvanic cells, leading to an increased corrosion rate, of ECs cells treated with a 1 % eluate concentration of media condi­ when compared to the non-annealed FeMn. Based on the cross-section tioned with Ag-containing TWIP steels was significantly reduced images presented in Figs. 7 and 8, we suspect that the FeMn austenite compared to 316 L stainless steel, while the same materials did not show phase (with more negative standard corrosion potential) acts as the significant differences in the viability towards SMCs. Therefore, the type anode, while the α-Fe, iron-manganese oxide, and ZnO phases act as the of cells used also influences the results of in vitro cytocompatibility tests. cathode. The pits formed on the cross-sectional area in Fig. 8 indicate In vitro tests of Fe-based degradable alloys are directly affected by the that indeed, the austenite matrix may dissolve during the immersion in concentration of ions in the culture media [53]. Manganese, iron, and HBSS. It is important to point out that the pores are not clogged by the zinc are essential metals found in tissues. The role of Mn is essential for ZnO coating and hence the underlying alloy is still exposed to HBSS. The the immune system, bone growth, adjustment of blood sugar, cofactors corrosion rate of FeMn/ZnO, presented in Fig. 6, is one order of in enzymes, and others [54]. However, overexposure to Mn can be magnitude higher than corrosion rates given for Fe-based alloys hazardous in humans, such as Mn-induced neurotoxicity, which occurs immersed in HBSS under static conditions in the literature while the in workers exposed to dust-containing Mn. The toxic level of Mn in our corrosion rate of FeMn is in accordance with the values found elsewhere case was above 50 ppm. As the concentration of Fe was not significantly [39]. different between the samples, the cytotoxicity of 100 % extract could be Although previous studies have demonstrated that the deposition of attributed to the elevated concentration of Mn or to the release of Zn coatings on degradable materials leads to enhanced corrosion resistance, ions. Nevertheless, as the conditions in vivo are more dynamic, the 11 A. Bartkowska et al. Surface & Coatings Technology 471 (2023) 129886 toxicity of 100 % extract should not be of concern when considering the and Mn ions. Moreover, a significant reduction in the total biofilm cytocompatibility of FeMn/ZnO, as aforementioned. biovolume of S. aureus was observed at 24 h, indicating antibiofilm properties of the FeMn/ZnO sample. In conclusion, FeMn/ZnO possesses 4.3. Effect of the accelerated corrosion on S. aureus biofilm formation better degradability, and is more resistant to biofilm formation than uncoated FeMn, while maintaining good cytocompatibility. Implant-associated infections are an important and debilitating complication of medical implants and have been attributed to the for­ CRediT authorship contribution statement mation of biofilm on the surface of an implant material [24]. A biofilm is a community of bacterial cells irreversibly attached to a substrate and/ Aleksandra Bartkowska: Visualization, Investigation, Formal or each other that are embedded in a self-made matrix of extracellular analysis, Writing – original draft. Adam Benedict Turner: Visualiza­ polymeric substances (polysaccharides, protein, and eDNA) [55]. By tion, Investigation, Formal analysis. Andreu Blanquer: Methodology, adopting a biofilm phenotype, bacterial cells become significantly more Validation, Writing – review & editing, Supervision. Aliona Nicolenco: resistant to external environmental stresses and challenges, including Investigation. Margarita Trobos: Methodology, Resources, Writing – those from the host-immune system and antibiotic chemotherapy [56]. review & editing. Carme Nogues: Conceptualization, Resources, Su­ The stages of bacterial adhesion and biofilm formation are the following: pervision. Eva Pellicer: Conceptualization, Writing – review & editing, (a) bacterial adhesion, (b) formation of microcolonies, (c) biofilm Supervision. Jordi Sort: Conceptualization, Resources, Writing – review maturation and (d) biofilm dispersal [26,57]. Many strategies are & editing, Supervision, Funding acquisition. currently under investigation to avoid or limit the formation of biofilm on the surface of metallic implants such as the incorporation of anti­ Declaration of competing interest bacterial agents such as silver, zinc oxide, etc. Previous research has established that degradable alloys possess There is nothing to disclose. better infection resistance than non-degradable ones [25]. One of the There were no financial or personal relationships with other people potential reasons for their antibacterial/antibiofilm properties can be or organizations that could inappropriately influence our work. associated with the effect of degradation on bacterial adhesion [26,58]. AI and AI-assisted technologies were not used in the writing process. This degradation reduces the area available for initial bacterial attach­ ment and colonization and could therefore limit the progression of Data availability biofilm formation. Hence, degradable materials with higher corrosion rates should be more resistant to biofilm formation than those with Data will be made available on request. lower corrosion rates. In previous studies on degradable Mg-based al­ loys, the antibiofilm properties have been associated with an increase in Acknowledgments pH and a high release of Mg ions upon degradation [58]. Local pH changes at the material surface were found to limit bacterial attachment. This project has received funding from the European Union's Horizon Moreover, bacteria attach preferably to corrosion products and avoid 2020 research and innovation programme under the Marie Skłodowska- active corrosion sites. In our previous study, Ag-containing Fe-Mn alloys Curie grant agreement No. 861046 (‘Bioremia’ European Training showed an antibiofilm effect higher than in Ag-free Fe-Mn alloys, which Network), from the Spanish Government (grants PID2020-116844RB- could be associated with enhanced corrosion of the former [15]. In this C21 and PID2020 116844RB-C22), from the Generalitat de Catalunya study, a similar effect was observed, as the increased corrosion rate of (2021-SGR-00651 and 2021-SGR-00122), from the Swedish Research FeMn/ZnO reduced or delayed the development of biofilm formation Council (2022-00853), the Swedish state under the agreement between more effectively than the slowly corroding uncoated FeMn. the Swedish government and the country councils, the ALF-agreement Our results provide more evidence supporting the correlation be­ (ALFGBG-978896), and the Hjalmar Svensson Foundation. tween enhanced degradation and antibiofilm behaviour. The FeMn/ZnO composite, possessing a higher corrosion rate, has a more pronounced Appendix A. Supplementary data antibiofilm effect than the FeMn sample. As the FeMn/ZnO ion release did not show a bactericidal effect per se, the observed antibiofilm Supplementary data to this article can be found online at https://doi. properties of FeMn/ZnO may be attributed to the enhanced degradation. org/10.1016/j.surfcoat.2023.129886. It is possible, that the enhanced release of Fe and Mn ions, thus enhanced degradation, limit bacterial attachment and biofilm formation. References 5. Conclusions [1] H. Hermawan, Updates on the research and development of absorbable metals for biomedical applications, Prog. Biomater. 7 (2018) 93–110, https://doi.org/ The main goal of the current study was to determine the effect of sol- 10.1007/s40204-018-0091-4. [2] H. Hermawan, D. Dubé, D. 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