Infected bone defects present a major challenge in orthopedics, characterized by a high treatment failure rate. Bacterial infection severely impedes bone regeneration, particularly in oral cavities where open defects facilitate colonization and biofilm formation. Biofilms lead to osteoinflammatory hyperplasia and avascular necrosis, hindering healing. Current clinical strategies often combine bone grafting with antibiotics; however, antibiotic resistance and the disruption of the body's microbial ecosystem limit this approach. This necessitates novel strategies to balance antibacterial and osteoinductive properties. Native bone's piezoelectricity is crucial for its physiological function. Bone generates endogenous electrical signals from collagen fibril polarization under shear stress. These signals influence stem cell differentiation and tissue regeneration by increasing Ca²⁺ influx via voltage-gated Ca²⁺ channels (VGCCs), activating Ca²⁺ signaling pathways, promoting cell metabolism, and accelerating ATP depletion to achieve cytoskeleton reorganization. Furthermore, electrical stimulation enhances osteoblast attachment and proliferation while inhibiting bacterial activity. Therefore, effective bone tissue engineering scaffolds should mimic natural bone's chemical properties, micro-nanotopology, and electrical microenvironment. This study introduces a self-promoted electroactive mineralized scaffold (sp-EMS) designed to address these challenges. The sp-EMS is constructed through biomimetic self-assembly of mineralized collagen fibrils and silver ultrathin nanowires (Ag uNWs). This approach ensures a biomimetic bone-like interface with excellent bone regeneration potential, while the Ag uNWs provide antibacterial properties and enhanced osteoinductive capabilities. The sp-EMS continuously generates a weak current via mild electrochemical reactions, promoting BMSC recruitment, osteogenic differentiation, and angiogenesis. Simultaneously, electrical stimulation synergizes with silver ions to combat bacteria.

The literature extensively highlights the challenges of treating infected bone defects, emphasizing the limitations of antibiotic-based approaches in the face of drug-resistant bacteria and the disruption of the microbiome. Studies demonstrate the critical role of native bone's piezoelectricity in stem cell differentiation and tissue regeneration. Research on electrical stimulation has shown its positive effects on osteoblast activity and bacterial inhibition. Previous work has explored biomimetic self-assembly strategies for creating bone-like scaffolds with excellent biocompatibility and biodegradability. However, the integration of intrinsic electroactivity into these scaffolds for enhanced bone regeneration and infection control remains largely unexplored, forming the gap this study aims to fill.

The study involved the synthesis of silver ultrathin nanowires (Ag uNWs) via thermal decomposition of silver nitrate in ethylene glycol, using polyvinylpyrrolidone (PVP) as a reducing agent and sodium bromide as a shape-directing agent. The average diameter of Ag uNWs was approximately 19.20 ± 4.00 nm. The self-promoted electroactive mineralized scaffolds (sp-EMS) were fabricated using a biomimetic self-assembly approach, combining Ag uNWs with type I collagen and inducing mineralization. Control scaffolds (MS) lacking Ag uNWs were also prepared. To investigate the role of electrical stimulation, passivated silver nanowires (Ag@AgCl and Ag@Ag2S) were synthesized to inhibit the electrochemical reactions. Various characterization techniques, including transmission electron microscopy (TEM), atomic force microscopy (AFM), X-ray diffraction (XRD), thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), and inductively coupled plasma optical emission spectroscopy (ICP-OES), were employed to analyze the physical and chemical properties of the scaffolds. Electrochemical measurements, including cyclic voltammetry (CV) and chronoamperometry, were performed to assess the self-promoted electrical stimulation generated by the sp-EMS. In vitro studies utilized human bone marrow mesenchymal stem cells (BMSCs) cultured on different scaffolds. Cell viability, proliferation, intracellular calcium levels, actin remodeling, and osteogenic differentiation were evaluated using CCK-8 assay, Fluo-3 calcium assay, western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR). Antibacterial effects were assessed using colony-forming unit (CFU) assays, live/dead staining, scanning electron microscopy (SEM), and TEM. The release of silver ions (Ag⁺) and reactive oxygen species (ROS) was also quantified. In vivo studies employed rat calvarial defects (both infected and non-infected), rabbit open alveolar bone defects, and beagle dog vertical bone defects. Bone regeneration was assessed using micro-computed tomography (µCT) and histological analysis. Immunofluorescence and immunohistochemistry were used to evaluate the expression of relevant markers.

The sp-EMS exhibited a biomimetic hierarchical structure similar to native bone, with Ag uNWs distributed within the collagen fibrils. The sp-EMS generated a stable, weak current (around -4 µA in PBS) due to the electrochemical corrosion of Ag uNWs. In vitro studies showed that the sp-EMS significantly promoted BMSC proliferation and osteogenic differentiation. This effect was mediated by increased Ca²⁺ influx via the activation of voltage-gated calcium channels (CACNA2D1), enhanced ATP synthesis (evidenced by upregulation of P2X7), and subsequent actin remodeling, ultimately activating the BMP2/Smad5 pathway. Passivation of the Ag uNWs, eliminating the self-promoted electrical stimulation, significantly reduced the osteogenic effects, confirming the crucial role of electrical stimulation. The sp-EMS also demonstrated excellent antibacterial activity against S. aureus, effectively inhibiting bacterial growth and disrupting bacterial cell membranes. This antibacterial effect was attributed to the release of Ag⁺ and ROS, with ROS generation being significantly higher in the sp-EMS group compared to controls. In vivo studies showed that the sp-EMS achieved near-complete bone regeneration in rat calvarial defects (both infected and non-infected), significantly outperforming the control groups. In the infected model, the sp-EMS promoted M2 macrophage polarization, contributing to reduced inflammation. Furthermore, the sp-EMS effectively promoted bone regeneration in rabbit open alveolar bone defects and beagle dog vertical bone defects, demonstrating its potential for treating complex clinical scenarios. The generated electrical stimulation persisted in vivo for at least 21 days post-implantation.

The findings demonstrate the successful development of a novel self-promoted electroactive scaffold for effective bone regeneration and infection control. The sp-EMS's biomimetic design, combined with its intrinsic electroactivity, overcomes the limitations of traditional bone graft materials and antibiotic therapies. The observed enhancement of osteogenic differentiation is directly linked to the self-generated electrical stimulation, activating key intracellular pathways involved in bone formation. The significant reduction in inflammation in the infected bone models underscores the scaffold's potential in mitigating the detrimental effects of bacterial infection. The successful translation of the sp-EMS's efficacy across different animal models, including those with complex bacterial microenvironments, strongly suggests its clinical translatability.

This study successfully demonstrates the efficacy of the self-promoted electroactive mineralized scaffold (sp-EMS) in promoting bone regeneration and inhibiting bacterial activity in infected bone defects. The sp-EMS provides a unique approach to bone tissue engineering by leveraging the intrinsic electroactivity of a biocompatible scaffold to stimulate bone formation and combat infection. Future research could focus on optimizing the scaffold's design and exploring its potential in other clinical applications, such as treating large bone defects and complex fractures. Clinical trials are warranted to further evaluate the safety and efficacy of the sp-EMS in humans.

The study primarily focused on S. aureus as the infecting bacteria. Further investigations are needed to assess the sp-EMS’s efficacy against a broader range of bacterial species and biofilms. The animal models used, while representing a significant step towards clinical translation, may not perfectly capture the complexity of human bone defects. Long-term in vivo studies are required to evaluate the durability and potential long-term effects of the sp-EMS on bone remodeling and overall health. Further research is needed to optimize the Ag uNW concentration and assess potential systemic toxicity associated with prolonged silver release, albeit current findings suggest excellent biosafety.