A novel mouse calvarial defect model of osteomyelitis induced by Staphylococcus aureus (ATCC 6538)

Abstract Background Osteomyelitis is a severe bone infection that causes chronic inflammation, tissue necrosis, and vascular compromise. The infection often requires surgical debridement and prolonged high-dose antibiotic therapy. Despite numerous osteomyelitis models in different species and skeletal sites, a standardized mouse calvarial defect model that reflects cranial intramembranous ossification and enables research on craniofacial infection is unavailable. Therefore, this study aimed to establish a reproducible mouse calvarial defect osteomyelitis model and to define an inoculum threshold that reliably induces infection while preserving experimental feasibility for downstream mechanistic and therapeutic studies. Results Bilateral calvarial defects (diameter = 4 mm) were created in C57BL/6J mice, and the defects were inoculated with graded doses of Staphylococcus aureus (ATCC 6538). Four weeks postoperatively, micro-computed tomography (micro-CT), bacteriological culture, and histopathological scoring for inflammation, necrosis, and bacterial presence were performed. Mice receiving 5 µL of 10 8 colony-forming units (CFU)/mL showed clear osteomyelitis, with significantly lower bone volume/total volume and defect union scores, together with higher CFU counts and histologic scores, compared with the controls (all p < 0.05). Bone mineral density showed no significant pairwise difference between groups, but a significant linear decreasing trend was detected across the ordered groups ( p for trend = 0.0031). The lower inoculum groups exhibited only mild or no significant changes upon micro-CT imaging and histological evaluation, indicating incomplete or absent infection. Consequently, 10 8 CFU/mL was identified as a practical cutoff for consistently inducing destructive cranial osteomyelitis in this model. Conclusions This study established a mouse model of calvarial defect osteomyelitis that recapitulates the key features of cranial bone infection, including bone loss, impaired union, and robust inflammatory and bacterial burdens. This model provides a platform for studying the mechanisms of cranial osteomyelitis by incorporating genetically modified mice and testing local and systemic therapeutic strategies. Although only a single S. aureus strain and limited ancillary staining were used, the study data lay the groundwork for future investigations that incorporate diverse clinical isolates, comorbid conditions, and advanced regenerative or anti-infective interventions targeting craniofacial bone infections.