Internalization of Streptococci and Staphylococci by Cultured Osteoblasts

Oral implants can fail in both early and late stages of implant integration as a result of bacterial infections. Osteoblast cells are needed to produce new bone matrix material for an implant to fully integrate within the oral cavity. Objectives: A number of bacteria have been shown to invade osteoblasts and to influence cellular functions. We have investigated the interactions of commensal and pathogenic species of gram-positive bacteria with osteoblasts. Methods: Human MG63 osteoblast like cells were infected with bacteria, and levels of adherence and internalization determined by antibiotic exclusion assay and microscopy. Osteoblast responses were assessed morphologically and by alkaline phosphatase production subsequent to bacterial exposure. To investigate a role for fibronectin recognition in internalization, the fnbpA gene (encoding fibronectin-binding protein A) from Staphylococcus aureus Col was cloned and expressed in Streptococcus gordonii DL1. Surface expression of FnBPA was confirmed by Western immunoblot analysis and immunofluorescence microscopy. Results: S. aureus Col and P1, Streptococcus pyogenes A40, and S. gordonii DL1 were internalized by osteoblasts at levels up to 5% input bacterial cells. MG63 cell internalization levels of S. gordonii DL1 expressing FnBPA were increased >100-fold compared with S. gordonii DL1. MG63 cell morphology was relatively unaffected by intracellular S. aureus or S. gordonii, but monolayer integrity was lost following infection with S. pyogenes. S. aureus Col internalization led to reduced levels of metabolically active cells, and abrogated alkaline phosphatase production. Conversely S. gordonii did not have inhibitory effects on metabolising cell numbers, or alkaline phosphatase production. Similar effects were observed using primary bone cells. Conclusion: Commensal and pathogenic bacteria are internalized by osteoblasts, promoted by fibronectin, but have differential effects on osteoblast maturation. Supported by a P&G research award from the Oral & Dental Research Trust.