Perpetuation of Subgingival Biofilms in an In Vitro Model

Objective: The purpose of this study was to evaluate the reproducibility of in vitro grown biofilms intiated with subgingival plaque from periodontally diseased patients and then continued by re-inoculating new biofilms from previously growing biofilms. Methods: Subgingival plaque samples from a 5mm bleeding pocket along with 5ml of saliva were collected from chronic periodontitis patients. Calcium hydroxyapatite (HA) discs were coated with 10% filtered sterilized saliva for 2hrs, placed in 5ml of Trypticase Soy Broth (TSB) and innoculated with 50ul of dispersed subgingival plaque. The biofilms were grown anaerobically at 37ºC for 10 days with transfer to fresh medium at 48 h intervals. At specific intervals, the biofilms were enumerated for total viable counts, and the species present were semi-quantitated by DNA-DNA ‘checkerboard' hybridization. The initial biofilms were used as the inoculum for additional sets of saliva-coated HA disks and the same procedures were repeated. Results: All generations of biofilms demonstrated similar rates of growth and reached steady-state at day 7 (ca. 10^8 CFUs). No differences were found for viable counts among the different generations at the same age (p=0.132). Likewise, the proportions of species present demonstrated a high similarity among the different sets of the same age. Conclusions: This study demonstrated that the growth of subgingival periodontal species in this biofilm model is reproducible and can be perpetuated in vitro through several generations. This model could be useful in future studies to characterize different periodontopathogenic properties and biofilm interactions. Supported by NIDCR grant R01-DE014714.