Multifunctional, enzyme/pH-responsive gelatin microspheres with aptamer-targeted antibacterial and ionic-mediated dual therapy for infected bone defects.

The treatment of infectious bone defects requires simultaneous resolution of bacteria-associated antibiotic resistance, inflammatory microenvironment dysregulation, and impaired bone regeneration. Here, we developed an injectable, self-assembling designed gelatin micro-/nano-sphere system (GHMs@G1-N-A/T) that addresses the tripartite challenges of infectious bone defects through innovative material engineering: Antibacterial module featuring aptamer-conjugated gelatin nanospheres (AGN-Apt/Te) for MRSA-specific targeting, coupled with dual enzyme/pH-responsive release mechanisms (gelatinase-triggered nanosphere detachment and MgO2-derived ROS generation); A self-assembling microsphere scaffold (GHMs) constructed through vanillin-mediated crosslinking and nano-hydroxyapatite (n-HA)/MgO2 incorporation, enabling sequential release of Mg2+/Ca2+; and A gelatinase-sensitive peptide (G-1) interface that dynamically links these components, ensuring microenvironment-responsive functionality. Results demonstrated that gelatinase-triggered AGN-Apt/Te nanospheres detachment enabled bacteria-specific antibiotic delivery, achieving greater than 95 % eradication of S. aureus and MRSA through synergistic biofilm disruption (via MgO2-derived ROS bursts) and Te-mediated membrane damage. In vitro, self-assembling GHMs scaffold ensured sustained release of Mg2+/Ca2+, thereby promoting HUVEC tube formation (1.9-fold) and osteogenic differentiation of BMSCs. In a rat osteomyelitis model, GHMs@G1-N-A/T demonstrated sequential therapeutic efficacy: rapid infection control (greater than 95 % reduction within 7 days) followed by functional bone regeneration (46.32 % BV/TV at day 28). This work offers a new multifunctional biomaterial design that integrates hierarchical modular assembly, infection microenvironment-responsive logic and sequential transition from antibacterial to regenerative for the repair of complex infectious bone defects.