Self-activating anti-infection implant

Implanted devices often fail due to early bacterial infections and insufficient osseointegration, which prolong recovery and increase morbidity. Conventional strategies co-immobilize antibacterial agents (organic bactericides, antibiotics, or inorganic ions such as Ag, Zn, Sr, Cu, Mg) with osteogenic peptides/growth factors, but sustained release can cause cytotoxicity and antibiotic resistance, limiting clinical translation. Targeting fundamental metabolic differences between mammalian cells and bacteria offers an alternative. Bacteria export electrons during respiration via outer membrane cytochromes, nanowires, or flavins, whereas mammalian cells generate ATP through mitochondrial inner membrane electron transfer. Hydroxyapatite (HA) is osteoconductive and widely used to promote osseointegration, while molybdenum sulfide (MoS2) is a biocompatible 2D material with tunable semiconducting/metallic properties and reported broad-spectrum antibacterial activity. The authors hypothesize that an HA/MoS2 coating on Ti6Al4V implants can self-activate upon bacterial contact to extract bacterial electrons, disrupt redox homeostasis and respiration, thereby killing bacteria, while concurrently modulating mesenchymal stem cell (MSC) bioelectric states to enhance osteogenic differentiation. The work develops a laser-cladded and CVD-sulfurized HA/MoS2 heterojunction coating and evaluates its antibacterial and osteogenic performance in vitro and in vivo.

Prior implant surface strategies combine antibacterial agents (organic bactericides, antibiotics, or inorganic ions such as Ag/Zn, Ag/Sr, Cu/Mg) with osteogenic cues to prevent infection and promote osseointegration, but can elicit tissue toxicity and drive bacterial resistance due to sustained release. HA provides proven osteoconductivity and is widely used on dental/orthopedic implants, yet inert surfaces are prone to biofilm formation. MoS2, a 2D transition metal dichalcogenide, has adjustable semiconducting/metallic behavior, high surface activity, and reported antibacterial, anticancer, and diagnostic applications. Its layered structure and tunable energy states can facilitate charge separation and surface reactions, and Mo and S are biologically relevant elements. Bacterial extracellular electron transfer via cytochromes, nanowires, and flavins is well established, as is the central role of mitochondrial electron transfer for ATP production in eukaryotic cells, suggesting a bioelectronic platform that exploits these differences. However, the biological role of an HA/MoS2 coating in mediating electron transfer at the bacteria-material interface had not been elucidated.

Materials preparation: Ti6Al4V (Ti6) plates were polished (SiC grits 240/400/800), cleaned, and pre-coated by drop-casting HA, MoS2, or HA/MoS2 (w/w 50:50) suspensions (10 mg mL−1 each) with ultrasonication. Laser cladding (CW 2 kW Nd:YAG, 1.06 μm; current 90 A; pulse width 2 ms; 20 Hz; spot 0.6 mm; 5 mm s−1) produced HA-Ti6. For MoS2-Ti6 and HA/MoS2-Ti6, sulfurization by CVD was performed with sulfur powder in N2 (1 atm), heating to 750 °C (10 °C min−1) for 1 h, then cooling. Characterization: Surface/microstructure characterized by SEM (JSM-6510LV, JEM-2100F), TEM (Talos F200x), HRTEM, HAADF-STEM with EDS mapping; Raman (532 nm) to confirm MoS2 (E1 2g and A1 g modes); XPS survey and core levels (Mo 3d, S 2p); XRD for crystal phases (MoS2 (002), (110); HA (002), (211)); EPR to assess vacancies; water contact angle; UV-Vis-NIR to estimate bandgaps via Tauc plots; UPS (He I 21.22 eV) to determine work function, valence band maximum (VB), and compute conduction band (CB) minima. Antibacterial assays in vitro: Bacteria (S. aureus ATCC 25923, MRSA 43300, E. coli ATCC 8099, P. aeruginosa ATCC 15692) cultured in LB. Spread-plate quantification after incubation on samples (e.g., 2.5×10^7 CFU mL−1; 37 °C; 6 h S. aureus/MRSA; 12 h E. coli; 6–12 h P. aeruginosa), serial dilutions, and CFU counting. MIC and MBC of HA/MoS2 determined by dose series (0–40 mg mL−1) using OD and colony counting. Live/dead staining (BacLight) after 12 h. SEM imaging of bacterial morphology after glutaraldehyde fixation, ethanol dehydration, and freeze-drying. Mechanistic assays: RNA-seq of S. aureus after 6 h on Ti6 vs HA/MoS2-Ti6 (TRIzol extraction; HiSeq 4000; analysis with FastqStat, RSeQC, edgeR; GO/KEGG). Intracellular ROS by DCFH-DA fluorescence (6 h S. aureus; 12 h E. coli). Linear sweep voltammetry (LSV) of bacteria-material systems. Membrane potential with DiBAC4(3) fluorescence for S. aureus. Zeta potential of bacteria. ATP levels (luminescence) in S. aureus after 12 h. Dissolved oxygen measurements of media (indicative of O2 consumption). MSC assays: Rat MSCs cultured in alpha-MEM + 10% FBS + pen/strep. Cell membrane potential by DiBAC4(3) (24 h). Mitochondrial membrane potential by JC-1 assay. Intracellular Ca2+ by Fluo-3 AM imaging. Cell morphology by DAPI/TRITC-phalloidin. Proliferation by MTT at days 1/3/7. Osteogenic differentiation in induction medium (β-glycerophosphate, dexamethasone, ascorbic acid): ALP activity (normalized to protein) at days 3/7/14; osteogenic gene expression (RT-qPCR for RUNX2, ALP, COL-I) at day 14; mineralization by Alizarin Red S staining and quantification at day 14. In vivo rat model: Sprague Dawley male rats (300–350 g), ethics approved. Ti6 or HA/MoS2-Ti6 rods implanted in tibia near knee. For infected groups, rods pre-coated with 20 μL S. aureus (10^7 CFU mL−1). Animals grouped for 2- and 4-week endpoints (n=8/group). At 2 weeks: explant rolling on agar and culture for residual CFU; H&E and Giemsa staining of peri-implant tissues. At 4 weeks: micro-CT (BV/TV in defined cylindrical ROI), histology with methylene blue–acid fuchsin (bone-implant contact), and Safranin-O/Fast Green (osteogenesis vs cartilage; histomorphometry in 20 μm peri-implant region). Major organ histology (heart, liver, spleen, lung, kidney). Statistics: t-test, one-way or two-way ANOVA with Dunnett’s multiple comparisons; significance at p<0.05.

- HA/MoS2 heterojunction coating fully covered Ti6 and showed homogeneous Ca, P, Mo, S distribution; HRTEM revealed MoS2 (002) lattice spacing 0.60 nm and HA (002) 0.34 nm indicating compact interface contact. - Optical/electronic properties: Bandgaps (Tauc) were 1.73 eV (MoS2-Ti6) and 1.38 eV (HA/MoS2-Ti6). UPS-derived work functions were 4.77 eV and 4.70 eV, respectively. Conduction band minima were approximately −4.19 eV (MoS2-Ti6) and −4.20 eV (HA/MoS2-Ti6); valence band minima −5.92 eV and −5.58 eV. HA/MoS2-Ti6 potential lay below the bacterial biological redox potential (−4.12 to −4.84 eV), enabling spontaneous electron extraction from bacteria. - Antibacterial efficacy in vitro: HA/MoS2-Ti6 reduced bacterial counts by 1.81-log (S. aureus) and 1.88-log (E. coli) vs Ti6; MRSA reduction was 2.36-log. With initial 10^5 CFU mL−1, counts decreased from 7.32-log to 5.22-log after 6 h; with 10^6 CFU mL−1, from 8.66-log to 6.26-log; Ti6 showed negligible change. MIC ≈ 5 mg mL−1; MBC ≈ 10 mg mL−1 (MBC/MIC = 2). Live/dead staining and SEM revealed extensive membrane disruption only on HA/MoS2-Ti6. - Mechanism: RNA-seq showed 54 genes upregulated and 47 downregulated on HA/MoS2-Ti6 vs Ti6. Downregulated GO terms: ATP metabolic process, cytochrome-c oxidase activity, ABC transporter complex; Upregulated: plasma membrane respiratory chain complex II, anaerobic electron transport chain, anaerobic respiration. LSV indicated higher saturated current when S. aureus contacted HA/MoS2-Ti6, consistent with electron flow to the coating. DiBAC4(3) fluorescence increased (membrane depolarization), zeta potential became more negative, intracellular ROS increased markedly, ATP levels were lowest on HA/MoS2-Ti6, and oxygen consumption decreased—together indicating a shift from aerobic to anaerobic respiration and redox imbalance leading to death. HA/MoS2-Ti6 had minimal activity against strictly aerobic P. aeruginosa, supporting a metabolism-switch mechanism. - MSC responses: HA/MoS2-Ti6 increased cell membrane potential signal (DiBAC4(3)), altered mitochondrial membrane potential (decreased JC-1 J-monomer/J-aggregate ratio indicating increased Δψm), elevated intracellular Ca2+, enhanced proliferation (MTT) at days 1/3/7, and showed high biocompatibility. Osteogenesis markers increased: ALP activity (days 3/7/14), RUNX2/ALP/COL-I mRNA (day 14), and mineralized matrix (Alizarin Red S) vs Ti6; HA contributed osteoconductivity and HA/MoS2 potential likely activated Wnt/β-catenin and Wnt/Ca2+ pathways. - In vivo outcomes: At 14 days, fewer neutrophils and bacteria around HA/MoS2-Ti6 implants; 2.65-log reduction in CFU vs Ti6 in infected rats. At 4 weeks, BV/TV: HA/MoS2-Ti6 + S. aureus 44.62%, HA/MoS2-Ti6 (no bacteria) 51.67% vs Ti6 + S. aureus 25.40% and Ti6 (no bacteria) 35.13%. Bone-implant contact ratios: HA/MoS2-Ti6 + S. aureus 80.15%, HA/MoS2-Ti6 (no bacteria) 89.58% vs Ti6 (no bacteria) 44.18% and Ti6 + S. aureus 12.12%. Safranin-O/Fast Green indicated robust osteogenesis with minimal cartilage on HA/MoS2-Ti6 groups. No histological toxicity observed in major organs.

The HA/MoS2 heterojunction coating establishes an energetically favorable pathway for bacterial electrons to flow to the material due to its conduction band position below the bacterial biological redox potential. This spontaneous, contact-activated electron extraction perturbs bacterial electron transport chains, downregulates aerobic respiration (e.g., cytochrome c oxidase activity), upregulates anaerobic pathways, elevates intracellular ROS, depletes ATP, and reduces O2 consumption, culminating in membrane damage and cell death without external energy input or antibiotic release. The lack of efficacy against strictly aerobic P. aeruginosa underscores the dependence on respiration pathway modulation. Concurrently, the coating’s potential modulates MSC bioelectric states—altering plasma membrane and mitochondrial membrane potentials—and increases intracellular Ca2+, which together promote mitochondrial metabolism and activate osteogenic signaling (e.g., Wnt/β-catenin and Wnt/Ca2+), yielding enhanced proliferation, ALP activity, osteogenic gene expression, and mineralization. In vivo, these dual actions translate into significant infection control and superior osseointegration (higher BV/TV and bone-implant contact), with no detectable systemic toxicity. The strategy exploits fundamental metabolism/electron-transfer differences between bacteria and host cells to simultaneously solve infection and osseointegration challenges.

A laser-cladded and CVD-sulfurized HA/MoS2 coating on Ti6 implants provides a self-activating antibacterial mechanism by extracting electrons from adherent bacteria, shifting their metabolism from aerobic to anaerobic respiration, disrupting redox balance, and inducing death. Simultaneously, the coating enhances MSC proliferation and osteogenic differentiation by modulating membrane potentials and elevating intracellular Ca2+, resulting in strong in vitro osteogenesis and improved in vivo bone integration. This antibiotic-free, contact-activated approach offers a promising, multifunctional implant surface strategy to address peri-implant infection and osseointegration. Future work should evaluate long-term durability and stability of the coating under physiological loading, broaden pathogen spectrum assessments (including biofilm-forming and strictly aerobic species), and explore clinical translation.

- Limited antibacterial activity against strictly aerobic Pseudomonas aeruginosa, indicating mechanism specificity tied to respiration pathway modulation. - In vivo evaluations were performed in a rat tibia model with 2–4 week endpoints; long-term performance, durability, and mechanical stability of the coating under load were not assessed. - While broad antibacterial efficacy was shown for S. aureus, MRSA, and E. coli, activity against diverse clinical pathogens and mature biofilms requires further study. - Potential effects of wear, corrosion, and ion release over extended implantation periods were not addressed.