Establishment of a human osteocyte model to reveal mechanisms of chronic Staphylococcus epidermidis bone and joint infections

Aims

Chronic bone and joint infections caused by Staphylococcus epidermidis can be difficult to treat and are prone to recurrence. In this study, established human osteoblast- and osteocyte-like cell line models were used in vitro to investigate the intracellular persistence of S. epidermidis as a potential mechanism for infection persistence.

Methods

The SaOS2 human osteosarcoma cell line was differentiated into osteoblast-like (SaOS2-OB) and osteocyte-like (SaOS2-OY) stages and infected with four S. epidermidis strains at varying multiplicities of infection. Susceptibilities to lysostaphin, a known bacteriolytic enzyme, and a panel of antibiotics were determined to identify an antimicrobial capable of clearing extracellular bacteria while preserving intracellular bacterial viability. Bacterial survival and number were quantified by colony-forming unit and droplet digital polymerase chain reaction (PCR), while host gene expression was analyzed by real-time PCR.

Results

Lysostaphin was ineffective against all four S. epidermidis strains, while 10 × minimal bactericidal concentration levofloxacin consistently eliminated extracellular but not intracellular bacteria. Intracellular S. epidermidis at multiplicities of infection of 1,000 persisted in SaOS2-OY for 14 days at 10 × minimal bactericidal concentration levofloxacin, while it was cleared within five days in SaOS2-OB cells. Droplet digital PCR genome copy analysis identified a higher number of bacteria than conventional culture, indicating more accurate counting and a transition of the intracellular bacteria to a non-culturable state. Proinflammatory and bone remodelling responses by both bone cell types were evident upon infection, with increased messenger RNA levels of CCL5 , CXCL6 , CXCL10 , SOST , receptor activator of nuclear factor kappa-B ligand ( RANKL ), and matrix metalloproteinase 13 ( MMP13 ).

Conclusion

We report the first model of intracellular S. epidermidis persistence in osteocyte-like cells, which provides a robust platform for mechanistic studies of chronic bone and joint infections and the preclinical screening of novel therapeutics targeting intracellular bacteria.

Cite this article: Bone Joint Res  2026;15(8):987–997.