Nitric oxide-generating metallic wires for enhanced metal implants

The global orthopedic market is booming, with metal-based implants (titanium, stainless steel, cobalt alloys) preferred due to biocompatibility and mechanical properties. However, implant-associated infections (0.5-30% of cases) are a significant complication. Biofilms on implant surfaces protect bacteria from the immune system and antibiotics, leading to implant failure, prolonged treatment, increased antibiotic use, and economic burdens. Localized therapy is a promising approach. Drug-eluting implants have limitations due to finite drug reservoirs. Localized drug synthesis, such as enzyme-prodrug therapies, is another option, but challenges include enzyme biodegradability, membrane permeability, and ensuring target-site specificity. This study proposes a simpler alternative: utilizing the inherent qualities of implant materials to catalyze prodrug conversion, specifically focusing on nitric oxide (NO) generation from S-nitrosothiols, given NO's antibacterial and antibiofilm properties. Previous research has shown iron-containing implants mediating antibiotic prodrug hydrolysis. This study explores the feasibility and efficacy of a simpler, direct approach using a thermal treatment to enhance the catalytic activity of common implant materials for NO generation.

Extensive research exists on drug-eluting implants using various agents (anti-inflammatory drugs, antibiotics, growth factors). However, these approaches are limited by the finite amount of drug that can be loaded onto the implant. Localized drug synthesis using enzyme-prodrug therapies is another area of research. Enzymes can convert prodrugs into active therapeutic agents, but challenges remain related to enzyme stability, delivery, and off-target effects. Studies have also investigated the use of transition metal ions (Cu²⁺, Co²⁺, Fe²⁺) and metal-based complexes for NO prodrug decomposition, highlighting the potential of material-driven NO generation. However, there is a need for approaches that enhance the efficiency and practicality of NO generation in biomedical applications. This study builds upon this existing research by focusing on a simpler and more direct method for local NO generation using thermal treatment of common implant materials.

Stainless steel, stellite, titanium, and silver wires were used. A thermal treatment (calcination) was applied to modify the wires' catalytic properties. The effect of wire length on NO generation from S-nitrosoglutathione (GSNO) was evaluated using a Griess assay. The NO generation kinetics of stainless steel and stellite wires were determined at various time points. The impact of GSNO concentration on NO generation was investigated. Different NO donors (DPTA NONOate, DETA NONOate, NOC-5, β-gal NONOate, SNAP) were tested. The effect of calcination temperature and time on NO generation was explored. The recyclability of the wires was assessed over multiple cycles. X-ray Photoelectron Spectroscopy (XPS), Energy-Dispersive X-ray Spectroscopy (EDS), Raman Spectroscopy, and Atomic Force Microscopy (AFM) were used to characterize the surface chemistry and morphology of the wires before and after calcination. Tensile testing was conducted to evaluate changes in mechanical properties. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was used to quantify metal ion leaching. The cytotoxicity and effect on cell proliferation were assessed using HUVEC and HCASMC cells and an alamarBlue assay. Biofilm inhibition properties were evaluated using Pseudomonas aeruginosa (PAO1) and crystal violet staining. Statistical analyses (one-way ANOVA with Tukey post hoc test) were performed.

Calcination significantly enhanced NO generation from GSNO for stainless steel and stellite wires. Titanium and silver wires showed little to no NO generation. Stellite wires exhibited higher NO generation efficiency than stainless steel wires. A linear increase in NO generation was observed with increasing wire length (number of wires) for both materials. XPS analysis revealed a greater coexistence of different oxidation states of transition metals after calcination. EDS and Raman spectroscopy confirmed the formation of an oxide layer after calcination. AFM showed increased surface roughness after calcination, particularly for stellite. The optimal calcination temperature was found to be 600 °C. Stellite wires maintained their NO generation capacity over five cycles, while stainless steel wires showed a significant decrease. The calcination process did not significantly affect the mechanical properties of the wires. Metal ion leaching increased after calcination, but the leached ions did not significantly contribute to NO generation. The calcinated wires showed no cytotoxicity, did not affect cell proliferation rates, and effectively inhibited Pseudomonas aeruginosa biofilm formation. The biofilm inhibition increased with repeated GSNO/GSH additions.

The findings address the research question by demonstrating that a simple thermal treatment can transform commonly used biocompatible metallic implant materials into NO-generating catalysts. The significantly improved antibacterial properties and lack of cytotoxicity make this approach promising for preventing implant-associated infections. The ability to tune NO generation by adjusting parameters (wire length, GSNO concentration, calcination conditions) offers versatility and control over the therapeutic effect. The superior performance of stellite compared to stainless steel highlights the importance of material selection. While minor changes in mechanical properties and increased ion release are noted, the overall benefits of the improved antibacterial action outweigh these drawbacks. This study presents a novel, material-driven approach to combat a major clinical challenge in implant medicine. Further studies are needed to comprehensively assess the long-term in vivo performance and potential clinical translation of these findings.

This research successfully demonstrated a simple thermal treatment to modify metallic implant materials (stainless steel and stellite) for localized NO generation, thus preventing implant infections. Stellite showed superior sustained NO release. The method's biocompatibility and biofilm inhibition properties make it a promising, simplified strategy to address implant-related infections. Future work should focus on in vivo studies to confirm efficacy and investigate long-term biocompatibility and corrosion resistance.

The study primarily focused on in vitro assessments. Further in vivo studies are necessary to fully evaluate the efficacy and long-term effects of the calcinated wires. The limited number of bacterial species tested (Pseudomonas aeruginosa) warrants investigation with a broader range of pathogens. Although mechanical properties were investigated, additional testing to confirm these treated wires meet medical implant standards is necessary.