Nitric oxide-generating metallic wires for enhanced metal implants

The study addresses the challenge of implant-associated infections, a common complication in orthopaedic and other implant surgeries due to antibiotic-resistant biofilms forming on implant surfaces. While metals such as titanium, stainless steel, and cobalt-based alloys are widely used for their biocompatibility and mechanical properties, localized therapeutic strategies are needed to prevent infections. Two prevailing localized therapy approaches are drug-eluting implants (limited by finite reservoirs) and localized drug synthesis via enzyme-prodrug therapy (limited by enzyme stability, delivery, and specificity). The authors propose leveraging intrinsic properties of implant materials to catalyze prodrug activation, thereby enabling sustained, localized generation of nitric oxide (NO)—a broad-spectrum antibacterial and antibiofilm agent—without added enzymes or complex coatings. They hypothesize that simple thermal (calcination) treatment of metallic implants can endow catalytic activity to decompose endogenous S-nitrosothiols (e.g., GSNO) into NO, providing an implant-intrinsic anti-infective function.

Prior work has shown enzyme–prodrug systems can locally synthesize therapeutics (including NO and antibiotics) on implant surfaces but face challenges of enzyme biodegradability, membrane impermeability, delivery carriers, and off-target activation. Transition metal ions and complexes (Cu2+, Co2+, Fe2+; copper-based MOFs) catalyze NO release from S-nitrosothiol prodrugs, and Zelikin’s group observed metal implants mediating prodrug hydrolysis to antibiotics. Material surface activation by calcination or etching can enhance catalytic properties by creating oxide layers and mixed oxidation states, facilitating electron transfer. Alternative NO-generation platforms include polymeric amines, ceria, zinc oxide particles, and MOFs that catalyze GSNO decomposition. However, a practical, manufacturing-simple approach for clinically relevant metals to generate NO in situ remains needed.

Materials: Stainless steel (AISI 316L), stellite (Co–Cr–Fe–Ni–Mo–Mn–C–Be), titanium, and silver wires (250 µm diameter) were cut to 5 mm length. Wires were cleaned by sonication (acetone, then water). Calcination: Unless varied, wires were heat-treated at 600 °C for 30 min (7.5 °C/min) in a muffle furnace to yield calcinated wires. Variables tested included calcination temperature (300, 600, 900 °C for 0.5 h) and time (0.5, 1, 2 h at 600 °C). NO generation assays: NO release was quantified by Griess assay (546 nm) of supernatants after incubations in PBS at 37 °C, 200 rpm, protected from light. Standard protocol: 0–20 wires (5 mm, 0.25 mm diameter) incubated with 200 µL of GSNO in PBS for 24 h (unless noted). Kinetics were obtained with 5 wires sampled at 0.5–24 h. GSNO concentration dependence tested 0–200 µM (200 µL, 24 h). Alternative donors (50 µM each; 0.5 h incubation) included DPTA NONOate, DETA NONOate, NOC-5 (spontaneous donors), β-gal NONOate (enzyme-activated donor), and SNAP (S-nitrosothiol). EDTA (0.1 mM) was added in some experiments to chelate leached metal ions and assess their contribution to NO generation. Surface modification controls: Surface roughness was modified by HCl etching or mechanical abrasion (120-grit sandpaper) to compare effects of roughness versus calcination-induced surface chemistry on NO generation. Recyclability and storage: Five-use cycles were performed with washing between cycles; long-term storage tested with calcinated wires stored 10 months at room temperature before NO tests. Characterization: XPS (ESCALAB 250Xi) to analyze oxidation states; FE-SEM/EDS for morphology and elemental composition; Raman spectroscopy (532 nm) to assess oxide phases; AFM (peak force tapping) to quantify surface roughness. Mechanical testing: tensile tests (Instron 5565) to measure tensile strength, ductility (strain at max force), and Young’s modulus pre/post calcination. ICP-MS (Perkin Elmer Nexion 5000) quantified ion leaching (24 h to 4 weeks) from non-calcinated and calcinated wires. Biocompatibility: HUVEC and HCASMC cytotoxicity assessed via alamarBlue after 24 h exposure to 2 or 5 wires; cell proliferation over 1–3 days with 5 wires. Antibiofilm assays: Pseudomonas aeruginosa PAO1 biofilm formation on wires in M9 minimal medium with glucose at 37 °C, 180 rpm, 6–6.5 h. GSNO (50 µM) and GSH (1 mM) were added once or repeatedly (up to 4 additions at 2 h intervals) to assess NO-mediated biofilm inhibition. Biofilm biomass quantified by crystal violet staining (OD595).

• Calcination-enabled NO generation: Among stainless steel, stellite, titanium, and silver wires, only stainless steel and stellite generated NO from GSNO, with calcination enhancing activity versus non-calcinated counterparts. Titanium and silver showed little-to-no effect and were not pursued further.
• Length/surface dependence: Increasing number of stainless steel or stellite wires (5 mm length each) increased cumulative NO generation; stellite reached a plateau with as few as 5 wires within 24 h, indicating higher catalytic efficiency than stainless steel.
• Kinetics: Linear NO generation profiles over 24 h for both materials at 50 µM GSNO; stellite exhibited an average rate 57% higher than stainless steel for the same wire count.
• GSNO concentration: NO output increased with GSNO concentration (0–200 µM) for both materials, enabling tunable dosing by prodrug availability.
• Alternative donors: No enhancement with calcinated wires for spontaneous NONOates (DPTA, DETA, NOC-5) or enzyme-activated β-gal NONOate. SNAP generated substantial NO with both materials over 0.5 h; stainless steel produced more NO from SNAP (41.3 µM) than stellite (27.8 µM), whereas stellite was superior for GSNO over longer times.
• Surface chemistry and morphology: XPS revealed increased coexistence of multiple oxidation states after calcination (Fe/Cr/Ni for stainless steel; Co/Cr/Fe for stellite). EDS and Raman confirmed increased surface oxides after calcination. AFM showed roughness increases: stainless steel Rq ×2.6; stellite Rq ×19.7. Roughness changes via abrasion/etching indicated: in stainless steel, surface chemistry (from calcination) dominated NO generation; in stellite, roughness significantly contributed (mechanical abrasion approximated calcination performance).
• Optimal calcination: Stainless steel showed optimal NO generation at 600 °C for 1 h; 300 °C and 900 °C (0.5 h) or times 0.5 h and 2 h at 600 °C yielded negligible-to-low activity. Stellite performed similarly at 600–900 °C (0.5 h) and at 1–2 h at 600 °C; 300 °C or 0.5 h yielded low activity. Raman suggested oxide phases consistent with Fe2O3 (stainless, 600 °C) and Cr2O3/Co3O4 (stellite).
• Recyclability and storage: Stainless steel lost ~64% NO generation after one GSNO cycle and none after two; stellite maintained performance over 5 cycles. After 10 months storage, stainless steel showed a small decrease; stellite showed no decrease. XPS changes suggested stainless steel oxide layer diminished after GSNO exposure, correlating with activity loss; stellite retained oxide characteristics.
• Mechanical properties: Calcination caused small changes. Stainless steel: tensile strength 1336.8→1318.4 MPa (slight decrease), ductility 6.3→3.9%, Young’s modulus 32.4→43.2 GPa (increase). Stellite: tensile strength 1934.3→2436.5 MPa (increase), ductility 8.2→6.5%, Young’s modulus 33.1→48.7 GPa (increase). Overall, stiffness increased with modest ductility reduction.
• Ion leaching: ICP-MS showed increased ppb-level ion release from calcinated wires over time. EDTA (0.1 mM) modestly reduced NO output, indicating leached ions did not significantly drive NO generation.
• Biocompatibility: No cytotoxicity to HUVECs or HCASMCs after 24 h exposure to non-calcinated or calcinated wires; proliferation over 3 days was unaffected.
• Antibiofilm efficacy: With GSNO (50 µM) and GSH (1 mM), calcinated stellite reduced biofilm biomass by 28% versus non-calcinated wires under the same conditions and by 40% versus non-calcinated wires without GSNO/GSH. For stainless steel, reductions were 13% and 27%, respectively. Repeated GSNO+GSH additions further enhanced inhibition for both materials.

The findings demonstrate that simple calcination of clinically relevant metal alloys (stainless steel, stellite) imparts catalytic activity to decompose endogenous S-nitrosothiols (GSNO) into NO, addressing the need for localized, sustained antibacterial action on implant surfaces without enzyme coatings or drug reservoirs. The linear kinetics and dependence on GSNO concentration enable tunable NO delivery by adjusting material (stellite vs stainless steel), available surface (wire number/length), and prodrug level. Mechanistically, enhanced catalytic performance correlates with calcination-induced surface oxide layers exhibiting mixed metal oxidation states (facilitating electron transfer) and, for stellite particularly, increased surface roughness that exposes active sites. Stellite outperforms stainless steel in sustained activity due to better retention of its oxide layer and roughness over multiple cycles and long-term storage. The biocompatibility data (no cytotoxicity, no effect on proliferation) and antibiofilm efficacy support the therapeutic potential of this materials-intrinsic approach. Mechanical testing indicates only modest changes post-calcination, suggesting feasibility for implant applications, though application-specific standards must be verified. Ion leaching remains low (ppb) and does not significantly contribute to NO generation, but long-term corrosion behavior requires evaluation. Overall, the study validates a practical pathway to integrate NO therapy into metal implants by leveraging intrinsic material transformations rather than complex surface chemistries or enzymes.

Thermal treatment (calcination) renders stainless steel and stellite wires catalytically active for generating nitric oxide from endogenous GSNO, providing a simple, materials-driven method to endow metallic implants with antibiofilm functionality. NO output is tunable via material selection (stellite superior for sustained use), available surface area, GSNO concentration, and calcination parameters (optimal ~600 °C; 1 h for stainless steel; 1–2 h for stellite). The approach is biocompatible (no cytotoxicity; no proliferation impact), inhibits Pseudomonas aeruginosa biofilm formation, and induces only modest changes in mechanical properties. Stellite exhibits excellent recyclability and storage stability, making it a promising candidate for sustained NO delivery. Future work should assess long-term corrosion resistance and ion release in physiological environments, expand biofilm studies to additional pathogens and in vivo models, evaluate complex implant geometries and surfaces, and investigate clinical manufacturing integration while preserving device classifications.

• Corrosion behavior and long-term stability of calcinated alloys in physiological environments were not assessed and may affect implant viability.
• Ion leaching increased (ppb range) after calcination; biological impacts beyond in vitro assays require further study.
• Recyclability was limited for stainless steel (rapid loss of activity after one to two cycles), potentially restricting sustained use for this alloy.
• Experiments were conducted in vitro (PBS, cell lines, short-term biofilm assays) and may not fully reflect in vivo conditions.
• Biofilm studies focused on Pseudomonas aeruginosa over short durations (6–6.5 h); broader pathogen panels and longer timelines are needed.
• Only wire geometries were tested; translation to full implant forms and complex surfaces was not demonstrated.
• Optimal calcination conditions were identified empirically; comprehensive process windows and manufacturing scalability were not established.