Bioactive glass (BAG) S53P4 is a clinically approved bone substitute with antibacterial, osteoconductive and osteostimulatory properties. Its antibacterial effect is associated with ion release, local pH elevation and osmolality, but the precise biochemical and biophysical mode-of-action is unclear. This study investigates the antibacterial mechanism of BAG S53P4 eluates. BAG eluates, collected at 2, 4, 8, and 24 h, eradicated Staphylococcus aureus. Elemental analysis revealed an early increase in concentrations of Si and Na, a later rise in Ca, depletion of P over time and rapid loss of Mg. Membrane disturbances occurred within 5 min, evident by permeability for SYTOX, aligning with time-kill kinetics for S. aureus and Bacillus subtilis. In B. subtilis, 2h-BAG-eluate induced rapid delocalization of marker proteins for cell division and DNA repair, signaling membrane potential collapse and nucleoid condensation. Transcriptomics revealed early transcription remodeling reflecting ionic and energetic imbalance, including disruption of central metabolism, redox homeostasis, and translational stability. Scanning electron microscopy revealed severe cell surface damage and particulate deposits on S. aureus. Transmission electron microscopy showed cell envelop disruptions and cytoplasmic leakage. Energy dispersive X-ray analysis identified Si on bacterial cell surface at 4 h and intracellular accumulation in punctured, empty cells at 24 h. Overall, BAG ionic dissolution products kill bacteria through a stepwise mechanism involving membrane damage, protein delocalization and metabolic impairment, accompanied by Si deposition on bacterial surfaces and loss of Mg. This finally leads to cell wall degradation, cytoplasmic content leakage and further Si deposition on the cells and inside cell ghosts.

This study reports the development and characterization of thermoresponsive injectable hydrogels based on S53P4 bioactive glass (BG) extracts and poloxamer 407. S53P4 BG was selected for its established release of therapeutic ions, including silicon species, calcium, sodium, and phosphate. Hydrogels were prepared using ion‐rich S53P4 BG extracts, and their pH, ion release, rheological behavior, syringeability, biocompatibility, and antibacterial activity were evaluated. The hydrogels exhibited gelation temperatures between 17.2°C and 19.7°C, enabling application in a sol state and rapid gelation upon warming. Syringeability tests demonstrated high mass‐delivery fidelity, with 97%–100% recovery for sol‐state formulations and 91%–95% recovery for gel‐state formulations. Frequency‐sweep confirmed shear‐thinning behavior, supporting practical injectability. Inductively coupled plasma‐optical emission spectrometer (ICP‐OES) analysis verified stable ionic composition before and after thermal cycling. Biocompatibility assays with L929 fibroblasts and human umbilical vein endothelial cells (HUVECs) exhibited ≥70% cell viability for all formulations, meeting ISO 10 993–5 criteria. Antibacterial testing demonstrated up to a 5‐log reduction in Staphylococcus aureus ATCC 25 923 and Escherichia coli ATCC 25 922 compared to the initial inoculum, confirming strong bactericidal effects.Overall, the S53P4 BG extract–poloxamer 407 hydrogels display injectability, thermoreversible gelation, stability, biocompatibility, and potent antibacterial properties, supporting their suitability for future in vivo applications.