Bilayer Heterogeneous Methacrylated-Silk Fibroin Scaffold with Antibacterial and Osteoinductive Functions for Treating Infectious Bone Defects

Infectious bone defects (IBDs) pose a significant clinical challenge due to the simultaneous requirements for effective infection control and robust bone regeneration, and guided bone regeneration (GBR) using multifunctional scaffolds offers a promising therapeutic strategy. Herein, we developed a bilayer hydrogel scaffold (DSHM) based on methacrylated silk fibroin (SilMA) integrated with hydroxyapatite (HA) and minocycline hydrochloride (MH) to address these demands. By precisely optimizing the concentrations of SilMA, photoinitiator, HA, and MH, we engineered a bilayer structure featuring a porous HA-loaded layer (PSH, ~432.1 µm) to enhance mechanical strength and support osteogenic differentiation, and a dense MH-loaded layer (DSM, ~34.6 µm) to act as a barrier against fibrous tissue ingrowth and provide sustained antibacterial effects. The interface-crosslinked DSHM scaffold exhibited a well-defined heterogeneous architecture, improved mechanical properties, tunable degradation, and excellent biocompatibility. Notably, DSHM demonstrated synergistic multifunctionality, including inhibition of fibroblast infiltration, promotion of bone tissue regeneration, and effective infection control. Overall, the DSHM scaffold presents a robust approach for localized and targeted treatment of IBDs, with strong potential for clinical translation. Overall design: To explore the potential antibacterial and osteogenic mechanisms of the materials, whole-transcriptome RNA sequencing (RNA-seq) was performed on tissue samples using high-throughput transcriptomic analysis. Six SD rats (aged 6–8 weeks) were used to establish infectious cranial defect models as previously described and were randomly assigned to two groups: one receiving DSHM implantation and the other serving as a blank control. Two weeks post-implantation, the rats were euthanized, and tissues from the defect regions were carefully dissected, rinsed with pre-chilled sterile PBS, blotted dry, and immediately transferred into cryovials containing RNA-later (5?mL, Beyotime). Samples were snap-frozen in liquid nitrogen and sent to Suzhou Genewiz Biological Technology Co., Ltd. (China) for high-throughput RNA sequencing and subsequent bioinformatics analysis.