PF127 hydrogel-assisted in-situ Staphylococcus aureus biofilm fabrication endowing pathological animal model for chronic osteomyelitis

Chronic osteomyelitis driven by biofilms remains difficult to treat and thus the exploration of its clinical therapy and pathological mechanism largely depend on effective animal models. However, standardized and efficient methods for establishing stable and reproducible animal models for chronic osteomyelitis are still lacking. This study developed a Hydrogel Assisted In-situ Biofilm Fabrication (HAIBF) method by coupling thermosensitive Pluronic F-127 (PF127) hydrogel with biofilm fragments, which stably mimic the pathological process of biofilm-infected chronic osteomyelitis. Forty-eight rats were randomly allocated into four groups: a planktonic bacteria injection group, a direct biofilm injection group, a PF127-loaded biofilm injection group, and a blank control group. Rats were euthanized, and tibial samples were harvested once a week for the following month after infection. Macroscopic examination, SEM, CLSM, micro-CT with three-dimensional reconstruction and quantitative analysis, and H&E staining were combined to evaluate biofilm localization, bone destruction, and tissue pathology. PF127 facilitated the stable localization of biofilm within bone tissue. Collectively, these results validated the successful establishment of a chronic osteomyelitis model characterized by persistent biofilm infection, pathological bone remodeling, and impaired bone repair. HAIBF integrates low-metabolism biofilm fragments with an injectable, thermoresponsive PF127 hydrogel to establish an implant-free bone infection model that undergoes in situ gelation and promotes the local retention of biofilm-derived material. Within a relatively short infection progression period, this model reliably reproduces persistent biofilm-associated infection and selected pathological features of chronic osteomyelitis, thereby providing a more stable and consistent experimental platform for mechanistic studies and therapeutic evaluation.