Effect of wire length on NO generationStainless steel, stellite, titanium, and silver wires were calcinated and screened for their capacity to generate NO when exposed to an NO donor, specifically GSNO. Briefly, non-calcinated or calcinated wires for each material were placed in glass vials, followed by the addition of 200 μL of 50 μM GSNO in PBS, and incubated at 37 °C for 24 h. After incubation, a Griess assay was performed on the supernatant to quantify the NO generated (Fig. 1). This procedure was carried out using 0, 5, 10, 15, or 20 wires (5 mm length, 0.25 mm diameter), where an increasing number of wires correspond to a greater wire length. The choice to increase the number of wires rather than the wire length facilitates practical lab-scale procedures. To illustrate, it is more practical to use 20 wires that are 5 mm in length rather than 1 wire that is 100 mm in length. However, the exposed surface area is greater compared to the latter single-wire approach. Nevertheless, the difference in surface area, even with 20 wires, is less than 2.4%, which can be considered negligible. This extra surface area was calculated assuming the wires are perfect cylinders. As an example, the surface area of 1 wire 100 mm in length is 78.64 mm2. On the other hand, the surface area of 20 wires 5 mm in length is 80.5 mm2 (4.03 mm2 × 20 wires) which accounts for a 2.37% increase when using our “length” approach. The percentage increase difference is lower when using fewer wires.Fig. 1: NO generation from increasing amounts of non-calcinated or calcinated metal wires.Cumulative NO generation after incubating 0, 5, 10, 15, or 20 non-calcinated or calcinated a stainless steel, b stellite, c titanium, or d silver wires in 200 μL of 50 μM GSNO in PBS for 24 hours at 37 °C. *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey post-hoc test). n ≥ 3; error bars represent standard deviation.As shown in Fig. 1, when comparing calcinated wires with their non-calcinated counterpart, a difference in NO generation was only observed for stainless steel and stellite wires, while little-to-no difference was observed when using titanium or silver wires. For stainless steel, a linear increase in NO generation with an increasing number of both non-calcinated or calcinated wires can be observed. This means that non-calcinated stainless steel wires have inherent catalytic properties to decompose GSNO and generate NO, however the calcination process further improves the catalytic activity. The inherent catalytic activity of non-calcinated wires will be discussed later. Stellite demonstrated a greater efficiency in generating NO from GSNO compared to stainless steel. This is because only 5 wires were required to reach the NO generation plateau within the same time frame. Nevertheless, as with stainless steel, a linear increase in NO generation was observed for an increasing number of non-calcinated wires. This catalytic activity inherent in stainless steel and stellite wires can be attributed to the presence of transition metal elements (Fe, Ni, Mn, Cr, Co) within these alloys. Transition metals are known to act as effective catalysts due to their capacity to either donate or accept electrons from a reagent, depending on the nature of the reaction. Studies have shown that multimetallic catalysts abundant in these metal elements exhibit catalytic properties30,31. Thus, materials like stainless steel and stellite have a great potential to exhibit such properties. However, they are commonly used after being surface modified (e.g. acid leaching or coatings32), as has been shown in the fields of pollutant abatement33, organic synthesis34, and clean energy35. This is because they exhibit insufficient active sites for catalytic reactions and require significant energy (high overpotential) to initiate the reaction36,37, with many cases showing no activity unless surface modified38. These results show that GSNO can be catalytically degraded by unmodified stainless steel or stellite wires, whilst calcination further improves this degradation. To the best of our knowledge, this is the first reported case documenting this finding.To investigate the contributing factors of this enhanced catalytic activity, we performed X-ray Photoelectron Spectroscopy (XPS). The spectra obtained for both materials before and after calcination showed the presence of the respective transition metal elements, but after calcination the high-resolution spectra show a greater coexistence of different oxidation states for all species (Fig. 2 for most abundant elements, Supplementary Figs. 1, 2 for all others, Supplementary Table 1 for binding energies of each species for both materials). The ability of transition metals to adopt various oxidations has also been associated with enhanced catalytic activity39. In other words, their ability to easily change oxidation states allows them to act as electron transfer catalysts in many reactions.Fig. 2: XPS spectra of the three most abundant elements before and after calcination of stainless steel and stellite wires.XPS spectra for a non-calcinated and b calcinated stainless steel wires. i) Fe 2p, ii) Cr 2p, iii) Ni 2p. XPS spectra for c non-calcinated and d calcinated stellite wires. i) Co 2p, ii) Cr 2p, iii) Fe 2p.Furthermore, it has been shown that treatment via calcination or surface etching of these metals can activate their surface, endowing greater catalytic properties40. High-temperature conditions can cause these components to migrate from the bulk phase to the surface of the material, generating an oxide layer that significantly alters their physicochemical properties. To confirm the formation of this oxide layer after calcination, Energy-Dispersive X-ray Spectroscopy (EDS), Raman Spectroscopy, and XPS were carried out. For the former, an increase in the relative weight percentage of oxygen from 1.5 to 4.5% for stainless steel (Fig. 3a, b) and 1.2 to 5.1% for stellite (Fig. 4a, b) was observed. The Raman spectra were also shown to significantly change for both materials after calcination, indicating the presence of the oxide layer (Figs. 3c, 4c), as previously demonstrated41,42. Finally, for XPS, an increase in the ratio between M-O and oxygen defects is indicative of the presence of more metal oxides on the surface of the materials (Figs. 3d, 4d).Fig. 3: EDS elemental mapping and spectra as well as Raman and XPS (O 1 s) spectra before and after calcination of stainless steel wires.EDS a elemental mapping and b spectra of i) non-calcinated and ii) calcinated stainless steel wires. c Raman spectra of stainless steel wires before and after calcination. d XPS spectra (O 1 s) of i) non-calcinated and ii) calcinated stainless steel wires.Fig. 4: EDS elemental mapping and spectra as well as Raman and XPS (O 1s) spectra before and after calcination of stellite wires.EDS a elemental mapping and b spectra of i) non-calcinated and ii) calcinated stellite wires. c Raman spectra of stellite wires before and after calcination. d XPS spectra (O 1 s) of i) non-calcinated and ii) calcinated stellite wires.Moreover, this oxide layer formation can also lead to an increase in the surface roughness and thereby exposure of the active metal species43,44,45. To quantify differences in surface roughness, atomic force microscopy (AFM) was carried out and showed that the calcination process increased the root-mean-square roughness (Rq) of stainless steel and stellite wires by 2.6 and 19.7 times, respectively (Figs. 5, 6). In addition to the difference in metal alloy composition, this large difference in surface roughness can explain the higher catalytic activity of stellite compared to stainless steel. It should be noted that titanium wires also exhibited a change in Raman spectra upon calcination (Supplementary Fig. 3), indicative of the formation of an oxide layer46, however a negligible change in surface roughness was observed (Supplementary Fig. 4). This could explain the trend observed in Fig. 1 which depicts statistically significant NO generation, albeit small, when using >15 wires. On the other hand, silver wires did not show any significant changes in Raman spectra (Supplementary Fig. 5) when compared to the other three materials, as well as a negligible change in surface roughness (Supplementary Fig. 6). Since titanium and silver did not generate NO, they are not considered viable materials and will not be investigated further. Considering that both surface roughness and surface elements were altered following the heat treatment process, it was essential to determine which change had a more significant impact on NO generation. To investigate this, the surface roughness of stainless steel and stellite wires was modified using chemical etching (HCl) or mechanical abrasion (sandpaper, 120-grit), and an increase in surface roughness was observed (Supplementary Figs. 7, 8). Notably, neither surface roughness treatment affected NO generation in stainless steel wires compared to calcination, indicating that surface elements play a more critical role in NO generation. Conversely, mechanically abraded stellite wires generated NO to a similar extent as calcinated wires, suggesting that surface roughness is a significant factor for stellite (Supplementary Fig. 9). Although chemical etching led to a small increase in NO generation compared to untreated samples, it was not as effective as mechanical abrasion. Further optimization of treatment variables for chemical etching could potentially enhance NO generation. Nevertheless, calcination remains the ideal treatment to simply and consistently lead to NO generation for both materials and will be utilized henceforth.Fig. 5: FE-SEM and AFM before and after calcination of stainless steel wires.a FE-SEM images of i) non-calcinated and ii) calcinated stainless steel wires. b AFM images of i) non-calcinated and ii) calcinated stainless steel wires.Fig. 6: FE-SEM and AFM before and after calcination of stellite wires.a FE-SEM images of i) non-calcinated and ii) calcinated stellite wires. b AFM images of i) non-calcinated and ii) calcinated stellite wires.Kinetic NO generationUpon confirming only stainless steel and stellite wires could generate NO from GSNO, their generation profile was determined. Understanding the NO generation kinetics of the material is crucial in evaluating its effectiveness. Maintaining a constant generation of NO over time is preferred over a burst generation when considering antibacterial applications. While the latter case may initially eliminate bacteria, there is a potential risk of bacterial regrowth later. Therefore, it is essential to assess whether the generation rate of the wires exhibits a burst or constant NO generation over time. To obtain this profile, a Griess assay was performed at various time points on the supernatant of 5 non-calcinated or calcinated wires that had been incubated for 24 h at 37 oC with 200 μL of 50 μM GSNO in PBS. A linear profile was observed for both cases, suggesting a stable generation profile can be achieved (Fig. 7a, b). Consistent with the previous section, stellite showed an averaged generation rate 57% higher compared to stainless steel for the same number of wires. These results in conjunction with the previous section highlight the tunability of NO generation via material choice and number (or length) of wires.Fig. 7: NO release from non-calcinated and calcinated stainless steel and steillite wires.Cumulative NO generation after incubating 5 non-calcinated or calcinated a stainless steel or b stellite wires in 200 μL of 50 μM GSNO in PBS over a 24 h time period at 37 °C. Cumulative NO generation after incubating 5 non-calcinated or calcinated c stainless steel or d stellite wires in 200 μL of 0, 12.5, 25, 50, 100, or 200 μM GSNO in PBS over a 24 h time period at 37 °C. Cumulative NO generation after incubating 5 non-calcinated or calcinated e stainless steel or f stellite wires in 200 μL of 50 μM DPTA NONOate, DETA NONOate, NOC-5, β-gal NONOate, or SNAP in PBS over a 0.5 h time period at 37 °C. n ≥ 3; error bars represent standard deviation.Effect of GSNO concentration on NO generationIn addition to evaluating the kinetic generation profile of both materials, the effect of GSNO concentration on NO generation was also investigated. This is because NO has concentration-dependent effects47, therefore achieving tunable generation profiles that are biologically and therapeutically relevant is a desired feature. To achieve this, 5 non-calcinated or calcinated wires were incubated with 200 μL of 0, 12.5, 25, 50, 100, or 200 μM GSNO in PBS for 24 h at 37 oC. A Griess assay was carried out and, as expected for both stainless steel and stellite, NO generation increased with increasing GSNO concentration (Fig. 7c, d). The same trend was also observed for other systems which generate NO from GSNO using polymeric amines48, ceria nanoparticles49, zinc oxide particles50, or metal-organic frameworks51. Therefore, in addition to material choice and number of wires, the GSNO concentration can also be used to tune NO generation.Alternative donors for NO generationThus far, GSNO has been used as the NO donor as it is a natural NO prodrug present in the body and is therefore suitable to enable localized and sustained NO generation52. However, various other NO donors can be considered to expand potential applications for these heat-treated wires. Structural variations between NO donors can lead to significantly different NO-generation mechanisms. There are three main mechanisms by which NO can be generated from NO donors: (i) spontaneous generation through thermal or photochemical self-decomposition, (ii) enzymatic oxidation/hydrolysis through metabolic activation, or (iii) chemical reactions with acids, alkalis, metals, or thiols53. GSNO falls within the latter mechanism. NO donors from each category were chosen and incubated with non-calcinated and calcinated wires for both materials to determine whether NO could be generated. For the spontaneous generation donors, DPTA NONOate, DETA NONOate, and NOC-5 were chosen. β-gal NONOate was chosen as a donor of the enzymatic hydrolysis mechanism, while SNAP was included as an exogenous (rather than endogenous, as is the case with GSNO) donor, which generates NO via chemical reactions. Briefly, non-calcinated or calcinated wires were incubated with 200 μL of 50 μM DPTA NONOate, DETA NONOate, NOC-5, β-gal NONOate, SNAP, or GSNO (Fig. 7e, f). In contrast to previous sections, the incubation time was reduced from 24 to 0.5 h since the former was too long to show any difference between non-calcinated and calcinated wires. As expected, there was no difference in NO generation between non-calcinated or calcinated wires for NO donors which generate via the spontaneous mechanism. In other words, the spontaneous generation rate of these donors is more rapid than the benefit that could have been provided by the wires, if any. Similarly, the NO donor that relies on enzymatic hydrolysis, β-gal NONOate, did not show any difference in NO generation for either stainless steel or stellite wires. This is to be expected given that β-Gal-NONOate generates NO following activation by β-galactosidase54. Nevertheless, β-gal NONOate was still evaluated to determine whether calcinated wires could potentially demonstrate enzyme-mimicking properties. Finally, SNAP was able to generate NO for both wire materials. Interestingly, stainless steel wires were able to generate more NO from SNAP compared to stellite wires, 41.3 vs. 27.8 μM NO, respectively. This is in contrast to GSNO, where stellite wires proved to be more efficient than stainless steel (Fig. 1). Finally, despite both SNAP and GSNO falling under the same generation mechanism category, SNAP shows significantly greater NO generation rates compared to GSNO. To clarify, a greater amount of NO is generated over a shorter time period. This corresponds with the known stabilities of each compound, with SNAP being less stable than GSNO at these reaction conditions55.Effect of calcination conditions on NO generationAs previously discussed, heat treatment of both stainless steel and stellite wires can cause transition metal components within these alloys to migrate towards the surface, generating an oxide layer that alters their physicochemical properties. Differences in calcination temperature and time can result in changes in the abundance of major elements in the oxide layer56. Therefore, the effect of temperature and time were evaluated to determine whether differences in these variables could enhance the catalytic properties of the wires. First, the effect of calcination temperature was investigated by heat treating stainless steel or stellite wires for 0.5 h at 300, 600, or 900 °C. Then, 5 non-calcinated and calcinated wires were incubated with 200 μL of 50 μM GSNO in PBS for 8 h (Fig. 8). It should be noted that 8 h incubation was used instead of 24 h to ensure the NO generation plateau was not reached and each condition could be compared. Notably, stainless steel showed negligible-to-no NO generation for both 300 and 900 °C. These results correspond with the Raman spectra obtained for each condition, where only 600 °C calcination led to a different spectrum corresponding to Fe2O3 (hematite)41, while the other temperatures are similar to the control, corresponding to Fe3O4 (magnetite)57. On the other hand, stellite showed similar NO generation when using 600 and 900 °C calcination temperatures, while 300 °C resulted in no generation of NO. Interestingly, the Raman spectra peaks for both 300 and 600 oC are similar, corresponding to Cr2O3 and Co3O4 (first and second major peak, respectively)42. Since the spectra are similar, it suggests that a likely reason for the lack of NO generation could be due to the difference in the amount of oxide layer formed and surface roughness. On the other hand, the spectra for 900 oC also show the peaks corresponding to Cr2O3 and Co3O4, with additional peaks around 300 and 1350 cm−1. Since the difference in NO generation between 600 and 900 oC is negligible, a lower calcination temperature is preferable from an economic point of view. Therefore, these results show that the optimal calcination temperature for both stainless steel and stellite wires is 600 °C.Fig. 8: Effect of calcination temperature on NO release from stainless steel and stellite wires.i) Cumulative NO generation and ii) corresponding Raman spectra after incubating 5 non-calcinated or calcinated a stainless steel or b stellite wires in 200 μL of 50 μM GSNO in PBS for 8 hours at 37 °C. Wires were calcinated at 300, 600, or 900 °C for 0.5 h. *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey post-hoc test). The dashed line represents the average concentration of NO generated by non-calcinated wires. n ≥ 3; error bars and grey shaded area represent standard deviation.Having confirmed the optimal calcination temperature of 600 oC, the calcination time was then investigated. Briefly, the wires were calcinated for 0.5, 1, or 2 h, and evaluated for their NO generating capacity (Fig. 9). Similar trends can be observed for calcination times as were observed with calcination temperatures. Briefly, for stainless steel negligible NO generation was shown when calcinating for 0.5 and 2 h when compared to the non-calcinated counterpart, while calcinating for 1 h resulted in NO generation. On the other hand, stellite showed similar NO generation when calcinating for 1 and 2 h while a 0.5 h calcination led to a reduction of approximately 50% in NO generation. Interestingly, the Raman spectra were similar for all cases, regardless of calcination time, suggesting that differences in NO generation arise from differences in the quantity of oxide layer formed and surface roughness.Fig. 9: Effect of calcination time on NO release from stainless steel and stellite wires.i) Cumulative NO generation and ii) corresponding Raman spectra after incubating 5 non-calcinated or calcinated a stainless steel or b stellite wires in 200 μL of 50 μM GSNO in PBS for 8 hours at 37 °C. Wires were calcinated at 600 °C for 0.5, 1, and 2 h. *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey post-hoc test). The dashed line represents the average concentration of NO generated by non-calcinated wires. n ≥ 3; error bars and grey shaded area represent standard deviation.Recyclability of wiresA crucial aspect to consider when evaluating long-term sustained delivery is the ability to generate NO even after multiple uses. As such, this variable was examined for both stainless steel and stellite wires. As before, 5 non-calcinated or calcinated wires were incubated with 200 μL of 50 μM GSNO in PBS for 24 h at 37 oC, followed by a Griess assay on the supernatant to quantify the cumulative NO generated. The wires were then washed by dipping with ultrapure water and the process was repeated a total of 5 times (Fig. 10a, b). Interestingly, stainless steel showed a significant decrease in NO generation of approximately 64% after 1 use, with no NO being generated after 2 uses. On the other hand, stellite wires showed no decrease in NO generation even after 5 uses, suggesting greater stability over multiple uses. These results indicate that stellite is a more attractive material for sustained NO generation. Additionally, calcinated stainless steel and stellite wires were stored for 10 months in a glass vial at room temperature to evaluate the effect of long-term storage. A small decrease in NO generation was observed for stainless steel, while no decrease was observed for stellite wires (Fig. 10c). It is also worth noting the wire samples after GSNO incubation showed negligible-to-small changes in XPS spectra for all elements except for oxygen (Supplementary Figs. 10, 11). For stainless steel, the ratio of oxygen defects to M-O increased after one cycle of GSNO incubation, similar to before the calcination process (Fig. 3di), suggesting a decrease in the oxide layer. This implies the oxide layer had a significant effect on the catalytic performance of the calcinated wire given the decrease in NO generation after one GSNO cycle. On the other hand, this was not the case with stellite, where a low ratio of oxygen defects to M-O was still observed, similar to after the calcination process (Fig. 4dii),. However, additional C=O and C-O peaks were observed upon GSNO incubation.Fig. 10: Recyclability and long-term storage of non-calcinated and calcinated stainless steel and stelite wires.Cumulative NO generation after incubating 5 non-calcinated or calcinated a stainless steel or b stellite wires in 200 μL of 50 μM GSNO in PBS for 24 hours for 5 cycles at 37 oC. c Cumulative NO generation after incubating 5 non-calcinated or calcinated 10-month stored stainless steel or stellite wires in 200 μL of 50 μM GSNO in PBS for 24 hours at 37 °C. n ≥ 3; error bars represent standard deviation.Material integrity and performance evaluationThus far, we have successfully demonstrated that our heat treatment process enables the catalytic generation of NO from stainless steel and stellite wires. We investigated various parameters affecting NO generation, including wire length, kinetic generation rate, GSNO concentration, alternative NO prodrugs, and wire recyclability. However, recognizing that the heat treatment process could potentially alter mechanical properties, it is essential to evaluate these differences, as any changes could compromise the structural integrity of implants and increase the risk of failure. This section aims to highlight the differences in mechanical properties, rather than assert that the treated materials can still be used as implant materials. Further testing is required to ensure that they meet the necessary standards for medical implants, depending on their final application. To this end, tensile testing was carried out on both non-calcinated and calcinated stainless steel and stellite wires. The untreated and treated metal wires were subjected to controlled loading conditions until failure, allowing for the measurement of tensile strength, ductility, and Young’s modulus (Fig. 11). The mechanical properties of both stainless steel and stellite wires showed small changes after calcination (Supplementary Table 2). Specifically, for stainless steel it was observed that after the heat treatment process, the tensile strength decreased from 1336.8 to 1318.4 MPa, the ductility as measured by tensile strain at maximum force decreased from 6.3 to 3.9%, and the Young’s modulus increased from 32.4 to 43.2 GPa. On the other hand, for stellite the tensile strength increased from 1934.3 to 2436.5 MPa, while the ductility decreased from 8.2 to 6.5%, and the Young’s modulus increased from 33.1 to 48.7 GPa. These results confirm that the calcination process does not adversely affect the critical mechanical properties of the metal wires, in both cases leading to a small increase in stiffness and decrease in ductility.Fig. 11: Tensile test of non-calcinated and calcinated stainless steel and stellite wires.a Tensile testing of i) non-calcinated and ii) calcinated stainless steel wires. b Tensile testing of i) non-calcinated and ii) calcinated stellite wires. Each test was conducted n = 3.In addition to changes in mechanical properties, it is also important to evaluate leaching of metal ions into the surrounding environment. ICP-MS was used to quantify the elemental composition of the supernatant of solutions incubating untreated and treated wires at various time points (24 h, 1 week, 2 weeks, and 4 weeks). For both materials, an increase in elemental concentration was observed, suggesting the heat treatment process indeed enhances leaching of certain elements (Supplementary Tables 3, 4), albeit in the ppb range. It is therefore necessary to evaluate two additional variables: (i) the biological impact of increased ion release (reviewed in Section Cytotoxicity, rate of proliferation, and biofilm inhibition properties), as well as (ii) the effect these transition metal ions have on NO production. For the latter case, EDTA was added as a chelating agent to ensure NO generation in previous sections was not significantly affected by the metal ions leached from the samples. Briefly, 5 non-calcinated or calcinated wires for both materials were incubated with GSNO in the presence of EDTA for 24 h and the NO generated was quantified (Supplementary Fig. 12). A small decrease in overall NO generation was observed for all cases, suggesting the leached metal ions did not significantly contribute to NO generation. It is important to acknowledge that implant materials must exhibit high corrosion resistance to withstand the harsh physiological environment within the human body. The calcination process, by altering the microstructure or surface characteristics of the alloy, may impact its corrosion resistance. While this study primarily focuses on the catalytic generation of NO, we recognize the importance of corrosion resistance in medical implants58. Addressing the corrosion behavior of calcined alloys is outside the scope of this study. However, future investigations will be necessary to comprehensively assess their long-term stability in physiological conditions.Cytotoxicity, rate of proliferation, and biofilm inhibition propertiesNext, the biocompatibility of the heat-treated wires was evaluated in order to ensure the heat treatment process does not result in cell toxicity. Specifically, non-calcinated or calcinated wires were exposed to HUVEC or HCASMC for 24 h and an alamarBlue assay was used to confirm their viability after exposure. As shown in Fig. 12a, b, no decrease in cell viability was observed for either material regardless of being heat treated or not. Although the wires do not affect either cell viability, it is also important to determine whether they affect the rate at which the cells proliferate. To evaluate this, HUVEC or HCASMC were seeded with wires and their cell viability over a 3-day period was monitored. No difference in cell viability was observed for all materials, indicating the calcination process does not affect the rate at which cells proliferate (Fig. 12c, d). This was further supported by optical microscope images which showed the morphology and number of the cells did not change between samples for the same time points (Supplementary Figs. 13–16). Upon confirming the calcination process does not affect cell viability, the performance of the wires to inhibit biofilm formation was evaluated. As previously mentioned, biofilm formation in implantable medical devices (e.g., stents, catheters, vascular grafts, etc.) are a key limitation towards their application59, and NO has been reported to disperse biofilms by inducing bacterial death48. Therefore, the gram-negative Pseudomonas aeruginosa (PAO1) was chosen to evaluate the antibiofilm properties of both wire materials. Pseudomonas aeruginosa was chosen as it is one of the most common pathogens causing nosocomial infections in hospitals60. Briefly, bacterial suspensions of PAO1 were incubated with 5 stainless steel or stellite wires as well as GSNO (50 µM) and L-glutathione reduced (GSH) (1 mM) for 6 h. Then crystal violet staining was performed on the washed wires to quantify the biofilm (Fig. 12e). It should be noted that GSH was also included in this study as it is a biologically abundant biothiol61 and has been shown to act as a reducing agent62. Firstly, although the heat treatment process itself (wires non-cal. vs. wires cal.) resulted in an averaged 13% decrease in biofilm biomass prior to GSNO and GSH exposure, statistical analysis showed this difference was not significant. This was also the case between non-calcinated wires and non-calcinated wires exposed to GSNO. On the other hand, when exposed to GSNO and GSH, a statistically significant averaged decrease of 16% for both materials was observed. Consistent with previous results, calcinated stellite wires showed the highest reduction in biofilm formation when exposed to both GSNO and GSH, leading to 28% less biofilm formation compared to non-calcinated wires exposed to the same conditions, i.e. GSNO and GSH. When compared to non-calcinated wires which were not exposed to GSNO and GSH, a 40% inhibition in biofilm formation was observed. For stainless steel, the percentage decreases were 13 and 27%, respectively. These results demonstrate that the heat treatment process of the wires leads to an inhibition of bacterial biofilm formation via the generation of NO from GSNO. As demonstrated in the previous sections, the extent of biofilm inhibition can then be controlled by the amount of NO generated, which can be tuned via material choice, number of wires, GSNO concentration, and heat-treatment conditions. In practical applications, metallic implants will be subjected to a continuous exposure of GSNO and GSH rather than a single dose. Therefore, to assess the sustained antibiofilm efficacy under such conditions, repeated additions of GSNO and GSH were performed to simulate a biological environment. Specifically, GSNO and GSH were administered up to four times to determine if this would enhance the inhibitory effect. Consistent with expectations, both materials exhibited increased biofilm inhibition with additional GSNO and GSH doses (Fig. 12f). Comparatively, this approach aligns with recent studies on biofilm inhibition against Pseudomonas aeruginosa and other bacteria. Similar efficacy has been observed with other methods, including antibiotic-loaded block copolymers (58% or 74%)63, enzyme-loaded silver-doped silica nanoparticles (58.8% or 72.4%)64, or hydrogels (82%-98% or 64%)65, with inhibition ranges varying based on the bacterial species.Fig. 12: Cytotoxicity and biofilm inhibition properties of non-calcinated and calcinated stainless steel and stellite wires.Cytotoxicity of stainless steel or stellite wires before and after calcination towards a HUVEC and b HCASMC. Rate of proliferation of c HUVEC and d HCASMC in the presence of stainless steel or stellite wires before and after calcination. e Biofilm inhibition of non-calcinated and calcinated wires when exposed to 50 µM GSNO and 1 mM GSH. f Biofilm inhibition of calcinated wires when exposed to 50 µM GSNO and 1 mM GSH up to 4 times. *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey post-hoc test). n ≥ 3; error bars represent standard deviation. SS stainless steel, St stellite, uncal. before calcination, cal. after calcination.
