Despite the emergence of various monoculture and multispecies in vitro systems, current protocols inadequately mimic the bacterial diversity of oral biofilms which may contain up to 700 bacterial taxa, with many species yet to be identified.
The primary aim of this study was to establish an in vitro biofilm model of the supra-gingival oral microbiome which reflects biofilm diversity. The establishment of a successful model will allow the development of anti-microbial/biofilm agents that selectively target bacteria associated with oral diseases including caries and periodontal disease.
Supra-gingival plaque samples (n=6) were individually inoculated into polypropylene 3D printed flow cells and cultured on hydroxyapatite (HA) discs for 14 days in Artificial Saliva Medium (ASM) and a new-modified medium (SHI). New generation sequencing (NGS) was used to analyse the microbiome at plaque collection and after 14 days growth.
Confocal microscope and scanning electron microscope imaging demonstrated the successful development of plaque biofilms over 14 days in ASM and SHI. Biofilm thickness, bio-volume and viability showed no significant difference at 14 days for either culture media (p>0.05). NGS demonstrated a significant decrease in bacterial composition and diversity between the original inoculum and 14 day biofilms when using ASM (p<0.001). However, this alteration was limited to a common phylum, Actinobacteria (PERMANOVA test: 25.34; p<0.01). Five domain phyla and over 26 genera were maintained in 14-day biofilms grown in ASM, representing 65% of the original inoculum. LIVE/DEAD™ BacLight™ staining revealed that the established biofilm consisted of an average of 73% live and 27% dead bacteria across experiments regardless of which growth media was used.
The study demonstrated the capability to grow oral biofilms from supragingival plaque with a highly complex and diverse range of bacterial species in 3D-printed flow cells over 14 days using ASM or SHI.