Self-activating anti-infection implant

Bacterial infections and insufficient osseointegration significantly hinder post-implantation recovery, increasing morbidity. Successful implantation hinges on rapid osteoblast differentiation and simultaneous infection prevention. Current strategies often involve integrating antibacterial agents (organic bactericides, antibiotics, inorganic agents like silver/zinc, silver/strontium, or copper/magnesium) with osteogenic peptides or growth factors. However, these methods can cause tissue toxicity or bacterial resistance due to sustained release of agents, limiting clinical application. Therefore, antibiotic-free strategies are crucial for preventing infection and enhancing osseointegration. Electron transfer is fundamental to cellular energy metabolism. Disrupting this process generates intracellular reactive oxygen species (ROS). Bacteria transfer electrons to extracellular acceptors via outer membrane cytochromes, nanowires, and flavins. In contrast, cells generate ATP through electron transfer within mitochondrial inner membranes. This metabolic difference provides a platform to modulate cell fate and bacterial metabolism, potentially inducing osteogenic differentiation in mesenchymal stem cells (MSCs) and killing bacteria by altering metabolic pathways. Hydroxyapatite (HA) is widely used in osseointegration due to its osteoconductivity, but bacteria adhering to inert implants form biofilms, causing implant failure. Molybdenum sulfide (MoS2) exhibits metallic or semiconducting properties depending on its structure, making it suitable for biomedical applications including anti-cancer and antibacterial therapies and diagnostics. Its unique properties, particularly in its 2D nanosheet form, offer tunable electronic energy states and enhanced bactericidal ability against both Gram-positive and Gram-negative bacteria. Mo and S are biological elements, further enhancing MoS2's potential. This study hypothesizes that an HA/MoS2 coating on titanium implants (Ti6Al4V) can simultaneously enhance osteogenic ability and antibacterial activity.

The paper reviews existing strategies for enhancing osseointegration and preventing infection in implants, highlighting the limitations of approaches involving sustained release of antibacterial agents. It discusses the importance of electron transfer in bacterial and cellular metabolism and how this difference can be exploited. The biocompatibility and antibacterial properties of hydroxyapatite (HA) and molybdenum sulfide (MoS2) are reviewed, setting the stage for the proposed HA/MoS2 composite coating. The review notes existing literature on the use of MoS2 in various biomedical applications, such as anti-cancer therapy and antibacterial therapy, but highlights a gap in understanding its role in electron transfer between bacteria and implant materials.

The study used laser cladding and chemical vapor deposition (CVD) to create an HA/MoS2 coating on Ti6Al4V implants. First, HA, MoS2, or a mixture of both were applied to the Ti6 surface, followed by laser cladding. The CVD process involved sulfuration at 750°C. Various characterization techniques were employed, including SEM, TEM, XRD, XPS, UPS, Raman spectroscopy, and contact angle measurements to analyze the morphology, structure, and chemical composition of the coating. The antibacterial properties of Ti6, HA-Ti6, MoS2-Ti6, and HA/MoS2-Ti6 were assessed using the spread plate method against S. aureus, E. coli, and MRSA. Live/dead staining and SEM were used to examine bacterial morphology and membrane integrity. RNA sequencing analyzed the gene expression changes in S. aureus cultured on HA/MoS2-Ti6 compared to Ti6. ROS production was measured using DCFH-DA. Linear sweep voltammetry (LSV) was used to study electron transfer between bacteria and the materials. The effect of the HA/MoS2 coating on MSCs was investigated by assessing cell membrane and mitochondrial membrane potential, intracellular Ca2+ levels, cell viability (MTT assay), ALP activity, osteogenic gene expression (RT-qPCR), and matrix mineralization (Alizarin Red S staining). In vivo studies involved implanting the materials in rat tibiae with and without S. aureus infection. Post-implantation assessment included H&E and Giemsa staining for inflammation and bacterial presence, micro-CT for bone regeneration, and histopathological staining (methylene blue-acid magenta and Safranin-O/Fast Green) to evaluate bone-implant contact and osteogenesis. Organ histology was examined to assess toxicity.

The HA/MoS2 coating effectively eradicated both S. aureus and E. coli, achieving 1.81-log and 1.88-log reductions in bacterial counts, respectively, and even higher reduction against MRSA (2.36-log). RNA sequencing revealed that the expression of anaerobic respiration genes increased, while aerobic respiration genes decreased in S. aureus when cultured on HA/MoS2-Ti6, suggesting a shift in bacterial metabolism. This was further evidenced by increased ROS production in bacteria on the HA/MoS2-Ti6 surface, indicating a disruption of redox equilibrium. LSV analysis confirmed electron transfer from S. aureus to the HA/MoS2-Ti6 surface, and a decrease in ATP levels confirmed the disruption of bacterial respiration. The HA/MoS2 coating also improved MSC viability and promoted osteogenic differentiation, evidenced by increased ALP activity, RUNX2 and COL-I gene expression, and matrix mineralization. In vivo studies showed that the HA/MoS2 coating significantly reduced inflammation and bacterial counts compared to the control Ti6 group. Micro-CT analysis revealed significantly increased bone formation in the HA/MoS2-Ti6 group, both with and without initial S. aureus infection. Histological analysis confirmed enhanced bone-implant contact and osteogenesis in the HA/MoS2-Ti6 group, with a minimal presence of chondrocytes. No significant organ toxicity was observed.

The findings demonstrate that the HA/MoS2 coating effectively addresses both the challenges of bacterial infection and osseointegration in implants. The self-activating antibacterial mechanism, based on electron transfer and metabolic disruption in bacteria, is highly efficient and eliminates the need for sustained release of antibiotics. The concomitant promotion of osteogenic differentiation by the coating significantly improves bone regeneration. The in vivo results validate the effectiveness of this strategy. The superior performance of HA/MoS2-Ti6 compared to HA-Ti6 and MoS2-Ti6 alone highlights the synergistic effect of the composite coating.

This study successfully demonstrated a novel self-activating anti-infection implant using an HA/MoS2 coating. The coating effectively kills bacteria by altering their metabolic pathways through electron transfer, while also promoting bone regeneration by modulating cell membrane potentials. This dual functionality addresses critical challenges in orthopedic implant technology. Future research could focus on optimizing the coating parameters, exploring other bacterial species, and conducting longer-term in vivo studies to further assess its clinical potential.

The in vivo study used a rat model, which may not fully replicate human bone tissue and immune responses. The study focused on two specific bacterial strains, and the effectiveness of the coating against a broader range of pathogens should be investigated. Longer-term in vivo studies are needed to evaluate the long-term stability and biocompatibility of the coating. The mechanism of action might be more nuanced and may involve other factors beyond electron transfer.