Dental calculus is increasingly used by researchers to study dietary patterns in past populations. The
benefits of using dental calculus for this purpose have been clearly demonstrated in previous studies,
with dental calculus harbouring a wealth of microremains and biomarkers for health and diet within
its mineral matrix. Previous studies have demonstrated some of the limitations and biases of how
methods of processing may overlook, or even remove, some of the important information contained
within the mineralised matrix. However, there are many factors that are impossible to account for
in vivo and in archaeological material, such as exact dietary intake, and individual factors such as
pH and enzyme activity, leaving some limitations that may not be addressed through these types of
studies and will require a different approach.
We present a protocol for creating a calcifying oral biofilm model that can be used to explore the
biases and limitations of dental calculus as a medium for paleodietary reconstructions. We report the
microbial and mineral composition of our model in an effort to validate the model calculus as an ap-
propriate proxy to natural dental calculus. The microbial profile and species diversity of our model
was determined using metagenomic classification with the nf-core/eager pipeline and Kraken2, and
compared to various reference samples from oral sites, including saliva, plaque, and dental calculus.
We then assessed whether our model calculus mineralises in a manner similar to natural dental calcu-
lus using Fourier transform infrared (FTIR) spectroscopy. The metagenomic classification showed a
microbial profile predominantly made up of (facultative) anaerobes, with a community structure that
was somewhat distinct from other oral reference samples. The core genera of the model consisted
of oral species, but clustered separately from oral reference samples, with a higher abundance of
anaerobes.
Mineral and organic components of our model mimic that of the modern and archaeological ref-
erence calculus that was used as a comparison. There was an overall increase in the inorganic
component relative to organic over the course of the experiment, with carbonated hydroxyapatite as
the principal compound, consistent with natural human-derived calculus.
We conclude that oral biofilm models, such as the one presented in this study, have great potential to
validate current methods used in the analysis of archaeological dental calculus, and should be used
to complement, rather than replace current in vivo studies.