Prokaryotic/eukaryotic co-culture as a model forin vitrofunctional validation of a dual and sequential drug delivery system with antimicrobial and bone regenerative features

Bacteria-mammalian cell co-cultures can provide information on host-cell responses to biomaterials under bacterial challenge that is not available from separate monocultures. This study established a two-stage osteogenic cell-Staphylococcus aureusco-culture for assessment of a previously developed dual and sequential drug delivery system (DDS) designed to release gentamicin followed by alendronate. MG-63 cells were initially exposed toCFU/ml ofS. aureusfor different periods. An inoculum ofCFU/ml and a 2 h exposure were selected because MG-63 metabolic activity did not differ significantly from the monoculture control at this time point (p> 0.05). These conditions were subsequently transferred to primary human bone-marrow mesenchymal stromal cells. The DDS-containing co-culture exhibited an increasing resazurin signal over 16 d and maintained adherent cells with an organised F-actin cytoskeleton. On day 25, protein-normalised alkaline phosphatase activity was 40 mol µg^-1protein in the DDS-containing co-culture, compared with 5 mol µg^-1protein in the basal-medium control and 63 mol µg^-1protein in the osteogenic-medium control. The model supports preliminary assessment of mammalian-cell compatibility and osteogenic-associated activity during sequential drug release under an initial bacterial challenge.