Development of a Coculture Model for Assessing Competing Host Mammalian Cell and Bacterial Attachment on Zirconia versus Titanium

<i><u>Objectives:</u></i> Coculture models are limited by bacteria rapidly outcompeting host mammalian cells for nutrients <i>in vitro</i>, resulting in mammalian cell death. The goal of this study was to develop a coculture model enabling survival of mammalian cells and oral bacterial species to assess their competition for growth on dental implant materials. <i><u>Methods:</u></i> Two early colonizing oral bacterial species, <i>Streptococcus mutans</i> or <i>Actinomyces naeslundii</i>, were grown in coculture with primary human macrophages or human gingival fibroblasts for up to 7 days on tissue-culture treated polystyrene or polished titanium and zirconia disks. Chloramphenicol was supplemented in cell culture medium at bacteriostatic concentrations to maintain stable bacterial inoculum size. Planktonic and adherent bacterial growth was assessed via spot plating while mammalian cell growth and attachment were evaluated using colorimetric metabolic assay and confocal fluorescence microscopy, respectively. <i><u>Results:</u></i> Macrophages and fibroblasts proliferated in the presence of <i>S. mutans</i> and maintained viability above 70% during coculture for up to 7 days on tissue-culture treated polystyrene and polished titanium and zirconia. In contrast, both mammalian cell types exhibited decreasing proliferation and surface coverage on titanium and zirconia over time in coculture with <i>A. naeslundii</i> versus control. <i>S. mutans</i> and <i>A. naeslundii</i> were maintained within an order of magnitude of seeding inoculum sizes throughout coculture. <i><u>Significance:</u></i> Cell culture medium supplemented with antibiotics at bacteriostatic concentrations can suppress bacterial overgrowth and facilitate mammalian cell viability in coculture model systems. Within the study's limitations, oral bacteria and mammalian cell growth in coculture are comparable on polished titanium and zirconia surfaces.