Prokaryotic/eukaryotic co-culture as a model for in vitro functional 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 aureus co-culture for assessment of a previously developed dual and sequential drug delivery system designed to release gentamicin followed by alendronate. MG-63 cells were initially exposed to 10^2 "-" 10^5CFU/mL of S. aureus for different periods. An inoculum of 10^2CFU/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 MSCs. The DDS-containing co-culture exhibited an increasing resazurin signal over 16 days and maintained adherent cells with an organised F-actin cytoskeleton. On day 25, protein-normalised ALP activity was 40 mol/µg protein in the DDS-containing co-culture, compared with 5 mol/µg protein in the basal-medium control and 63 mol/µg protein 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.