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Objectives and questions: Bone infections, including osteomyelitis and post-surgical complications, remain a major challenge in orthopaedic and trauma surgery and can lead to implant failure, chronic inflammation, and impaired bone repair. Systemic antibiotics and surgical debridement are frequently insufficient due to recurrence and antibiotic resistance, motivating local sustained-release approaches. This study evaluated the antibacterial, antibiofilm, biocompatibility, and physicochemical performance of a novel injectable bone paste for infection-resistant bone repair applications.

Material and methods: This in vitro experimental study evaluated antibacterial activity using agar diffusion (inhibition-zone) assays on standardized bacterial lawns (10⁶ CFU/mL) and quantitative killing assays against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and methicillin-resistant S. aureus (MRSA). Antibiofilm efficacy was assessed by disrupting 72 h mature biofilms and quantifying viable bacteria by CFU enumeration after 16–18 h exposure (detection limit: 10 CFU). Biocompatibility was examined using Cell Counting Kit-8 (CCK-8) on human bone marrow stem cells exposed to paste-conditioned medium (5–20 mg/mL) for 24–72 h, complemented by fluorescence imaging of F-actin and nuclei. Flowability/injectability was quantified by measuring injection force through a syringe at a constant rate over 21 days. Degradation and structural stability were assessed in PBS at 37 °C for up to 4 weeks. All experiments were performed with n = 5 independent replicates. Statistics: in Prism; t-tests or ANOVA/Tukey, α=0.05; data shown as mean±SD.

Results: The bone paste produced inhibition zones of 23 ± 1.4 mm, 21 ± 1.2 mm, and 20 ± 1.1 mm against E. coli, S. aureus, and MRSA, respectively (n = 5). Conditioned medium (5–20 mg/mL) showed no cytotoxicity at 24, 48, or 72 h, with CCK-8 viability comparable to untreated controls (n = 5). Exposure to 10 mg/mL bone paste reduced viable counts by approximately 20,000-fold for E. coli, 60,000-fold for S. aureus, and 25,000-fold for MRSA (p ≤ 0.0001, n = 5). Biofilm disruption assays showed bacterial counts below the detection limit (<10 CFU) after overnight treatment with 10 mg/mL bone paste. Fluorescence imaging demonstrated normal cell morphology, intact nuclei, and well-organised F-actin across all tested concentrations. Injectability testing showed stable flow characteristics, with an injection force of 1.8 ± 0.2 N maintained over 21 days. Degradation testing showed preserved structural integrity for up to 4 weeks under physiological conditions.

Discussion and conclusions: The injectable bone paste combined broad-spectrum antibacterial and antibiofilm activity with in vitro biocompatibility, sustained injectability, and structural stability under simulated physiological conditions. These properties support further evaluation as a local infection-resistant biomaterial for bone repair; confirmation of regenerative capacity requires dedicated osteogenic and/or in vivo bone-formation testing.