01-intro.qmd

# Introduction {#chap-intro}

<!-- Putting the study of dental calculus in context -->

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## Summary {-}

What is dental calculus? Why does it grow on our teeth? Is it bad for us?
Why have archaeologists become endlessly fascinated by it? What is the point
of this dissertation? Am I going to answer any of these questions?

:::
:::

```{r}
#| label: setup
#| include: false

library(here)
library(patchwork)
source(here("scripts/wos_papers.R"))
```

<!-- The dissertation will be made widely available, so I want it to be as accessible as possible.
This is why the writing won't be particularly "academic" [**highly exagerated airquotes**]. That 
boring stuff is reserved for the articles -->

Dental calculus is becoming a popular substance in research on the behaviour and
biology of people in the past. You
may also know it as tartar or mineralised plaque. In other languages the word is
often related to "tooth stones". In fact, calculus is itself latin for 'pebble'.
This was originally used as a term for mathematical calculations using counting
stones, and only later used to describe various calcifications in the human body
(<https://www.etymonline.com/word/calculus>). This can be the cause of some confusion,
as calculus is also a branch of mathematics. If you see the term 'calculus' in
this dissertation, you can safely assume that I'm referring to stuff that grows
on your teeth and for which you receive lectures from your dentist, and not the
topic you dreaded in high school.

I will briefly describe the formation of dental calculus here, but for a more
thorough review of the entire process I refer you to [Chapter 2](#chap-background).
Dental calculus is formed from dental plaque, a substance that grows on your teeth
and consists mainly of bacteria and
a surrounding structure called the extracellular matrix.
When the local environment within and around the plaque reaches a favourable alkaline
pH, both the extracellular matrix and bacteria within will calcify
[@whiteDentalCalculus1997; @jinSupragingivalCalculus2002].
The alkaline pH causes minerals (especially calcium and phosphate)
from saliva to enter the plaque, causing the extracellular matrix
and eventually also the bacteria to harden, resulting in a concrete-like
deposit on the surface of the teeth.
The process repeats itself when new bacteria colonise the surface of the newly
formed dental calculus, creating a layered structure, though somewhat disorganised
[@jepsenCalculusRemoval2011; @akcaliDentalCalculus2018].
Dental plaque can accumulate more easily on teeth
(and dental calculus) because they are a hard, non-shedding surface.
Most of the surfaces in our mouth are covered by a layer of cells called the oral
epithelium. These cells are continuously renewed as
new cells are formed and dead cells fall off [@squierOralMucosa1998].
This constant turnover means that it is difficult for
bacteria to build the communities they require for producing biofilms. Enamel, the
white substance that covers the crown of your teeth, behaves differently. It stops
growing when the tooth has fully formed. After that, there is no renewal. This allows
bacteria to continue to grow and develop communities if there is no intervention from
you (or your dentist)<!--cite-->. Dental plaque can trap a variety of different
microparticles, including bacteria, human proteins, and small debris from the food
we eat
[@hendyProteomicCalculus2018; @henryCalculusSyria2008; @delafuenteDNAHuman2013].
When the plaque mineralises, it can preserve these microparticles over long periods
of time, even after
the person whose teeth provided a home for the calculus has died.
Also, the main crystal structures in calculus strongly bind DNA, making
calculus a fantastic source of ancient DNA (aDNA) from the mouth [@warinnerNewEra2015].
Another advantage of dental calculus is that it represents a more recent and direct
source of diet than teeth or other bones. While bones and teeth can take years to
remodel and incorporate a dietary signal, calculus forms on a much smaller
timescale and is in direct contact with the dietary material.
Calculus can form within weeks at any point during an individual's life
and may, therefore,
indicate a recent and direct consumption of food, while bone can take years to
show a (indirect) dietary signal, following food molecules entering the
bloodstream, and finally entering the bone from there.
Further, enamel stops forming after the crown of the
last tooth has developed---third molars, or 'wisdom teeth---at around 16 years of age,
and the turnover of dentin is very limited [@hillsonDentalAnthropology1996].
These properties are probably why archaeologists have become increasingly
interested in dental calculus.

## Dental calculus in archaeology {#intro-arch}

The main archaeological interest in dental calculus is to explore research questions
involving diet and the evolution of the oral biome and oral health. To this end,
it can contribute a surprising amount for such a small, seemingly insignificant
material. This relates to its ability to retain and preserve a wide variety
of different materials, from the food we eat to the bacteria that make their home
in our mouths
[@yatesOralMicrobiome2021; @henryCalculusSyria2008; @warinnerPathogensHost2014; @warinnerEvidenceMilk2014; @adlerSequencingAncient2013].
The goal of current studies targeting archaeological dental calculus have not changed much
since the early uses of dental calculus in archaeological research, but the methods
certainly have, allowing us to unearth<!--pun always intended--> information
that was previously not considered possible.
By my count, archaeological dental calculus has now been subject to various forms
of microscopy
[@middletonOpalPhytoliths1994; @charlierSEMCalculus2010; @powerSynchrotronRadiationbased2022];
extractions of biomolecules including DNA, proteins, and metabolites [@adlerSequencingAncient2013; @warinnerEvidenceMilk2014];
and stable isotope analyses.

```{r}
#| label: fig-plot-and-wordclouds
#| fig-cap: "Plot of the number of articles per year in bioarchaeology and clinical dentistry with the term 'dental calculus' in the title."
#| fig-width: 6
#| fig-height: 5

titles_plot + bioarch_title_wordcloud / dental_title_wordcloud + theme(legend.position = "top", axis.title.x = element_blank()) +
  plot_annotation(tag_levels = "A") + plot_layout(widths = c(5,4))
```

Perhaps the most common use of dental calculus is to recreate the diet
of past people and populations ([@fig-plot-and-wordclouds]B).
One of the ways to do this is by dissolving the calculus in a weak acid or
decalcifant, or mechanically breaking it up. This process releases any fragments
of plants that were trapped within the calculus and can be identified, for example
with a microscope. The tricky part is not destroying the plant fragments when
releasing them from the calculus.
As far as I can tell, the first attempt at this was the extraction of phytoliths
(silicified plant remains) from the teeth of cows, sheep, and horses
[@armitageExtractionIdentification1975].
This was a somewhat isolated use-case, and the method didn't really catch on until
the 1990s
[@ciochonOpalPhytoliths1990; Middleton 1990, in @middletonOpalPhytoliths1994].
The first extractions from human teeth followed shortly [@foxPhytolithCalculus1996],
and there are now studies using plant microremains
(especially starch granules and phytoliths)
from dental calculus to infer diet in past peoples from across the world,
including Pacific Islands [@dudgeonDietGeography2014], China [@chenStarchGrains2021],
Europe [@fiorinCombiningDental2021], and more
[@buckleyDentalCalculus2014; @henryCalculusSyria2008; @mickleburghNewInsights2012].
The durable nature of dental calculus also means that microremains within it can
survive for millennia, allowing us to look at the diets of early humans and other
hominins
[@pipernoStarchGrains2008; @henryCalculusSyria2008; @chenStarchGrains2021; @henryNeanderthalCalculus2014; @henryDietAustralopithecus2012; @buckleyDentalCalculus2014; @hardyNeanderthalMedics2012; @hardyStarchGranules2009].


That bacteria can become trapped within calculus has been known to archaeologists
for a while [@brothwellDiggingBones1981<!--check-->; @vandermeerschMiddlePaleolithic1994], but it wasn't
used in archaeological research until DNA extraction started to become more accessible
[@delafuenteDNAHuman2013].
Dental calculus then became part of the third scientific revolution in archaeology.
The early studies focused on oral health in the past
[@adlerSequencingAncient2013; @delafuenteDNAHuman2013; @warinnerPathogensHost2014].
Bacteria have shorter lifespans than humans which makes them useful when studying
the evolution of bacteria in the human mouth
[@delafuenteDNAHuman2013; @yatesOralMicrobiome2021].
Diet has also been a focus of paleogenetic research. This has mainly been
addressed by considering how long-term changes in the patterns of bacteria within
the mouths of our ancestors have changed that could be related to changes in diet.
Just like we adapt to deal with various diseases, climates, etc., we also adapt to
changes in our diet
[@adlerSequencingAncient2013; @yatesOralMicrobiome2021].
Directly identifying genetic
markers of plants and animals within dental calculus is difficult, but not impossible
(see @warinnerEvidenceMilk2014). Most of the
DNA within dental calculus will be oral bacteria, and this will overwhelm the
small signal from plant DNA, which makes species identifications problematic
[@fagernasMicrobialBiogeography2022].
A newer field of biomolecular archaeology, paleoproteomics, may be able to
address this issue by targeting plant proteins, along with a range of other
dietary protein sources.
Hendy and coauthors were able to identify a number of these in dental calculus,
as well as proteins from cereals, and milk proteins from
different sources [@hendyProteomicCalculus2018].
Dental calculus has also become a target for extracting other biomolecules that
may be related to diet, such as alkaloids, fatty acids, and carbohydrates
[@velskoDentalCalculus2017; @gismondiMultidisciplinaryApproach2020].
The methods used for this
have also proven to be useful in detecting compounds that are related to other
activities and ceremonies, such as nicotine [@eerkensDentalCalculus2018], and may
provide some evidence of medicinal practices [@gismondiMultidisciplinaryApproach2020].

To a lesser extent, the presence and amount of dental calculus on teeth has been used
as an indicator of dental health
[@drewettExcavationOval1975; @sagneStudiesPeriodontal1977; @zhangDentalDisease1982; @lieverseDentalHealth2007].
@pilloudOutliningDefinition2019
explored the terms associated with a number publications on dental or oral health,
dental calculus came up as one of them; albeit not the most common, which was
(unsurprisingly) dental caries (@fig-dental-terms).

To a lesser, lesser extent, it has also provided some interesting insights on
non-dietary activities, such as occupations and smoking habits. In a rare find,
blue particles were detected in the dental calculus of a Medieval German woman.
These blue particles originated from lapis lazuli, an exotic stone
often ground into pigments and used
to illuminate manuscripts [@radiniMedievalWomen2019]. Nicotine was detected in dental
calculus of pre-colonisation individuals from California using Ultra-Performance Liquid
Chromatography Mass Spectrometry (UPLC-MS), showing direct consumption of tobacco and
providing more detailed insights on the demographics of consumption in a way that no other
human-adjacent archaeological materials can.

![Word cloud of most common dental terms in articles. Figure is from @pilloudOutliningDefinition2019, Figure 1.](figures/wordcloud.png){#fig-dental-terms}

It wasn't always appreciated for the wealth of information hidden within its
hardened shell.<!-- can't really blame them, most dental research is focused on its prevention and removal...-->
Until roughly 20 years ago, archaeologists who encountered calculus had limited
use for this material.
Some researchers quantified it using a simple four-stage scoring method that was
developed for recording deposits
on archaeological dental calculus [@brothwellDiggingBones1981], similar
to a common clinical scoring system [@greeneSimplifiedOral1964]. 
The four-stage
system is probably still the most widely used among archaeologists. More detailed
methods are also available [@dobneyMethodEvaluating1987; @greeneQuantifyingCalculus2005],
but the original method is generally preferred for its simplicity.
Unfortunately,
knowing the size of a calculus deposit is not as valuable as being able to
analyse the deposit itself, and the deposits were often
removed because they obscured tooth and root morphology [@scottBriefHistory2015].
This had made a lot of people very angry and been widely regarded as a bad move
[@adamsRestaurantEnd2002, p.1]. Hindsight being what it is, it's hard to blame
anyone. A lot of dental research mainly focuses on the prevention and removal
of dental calculus.

The wide range of applications for dental calculus that we know about today,
and the fact that it's pretty much ubiquitous in the
past thanks to poor oral hygiene, makes it a really exciting target for future
(and current) paleodietary research.
That being said, the study of dental calculus doesn't seem to fit into any predefined
areas of study within (and beyond) archaeology. Most researchers seem to
see it as a means to the information contained within, rather than being worth studying
in its own right. This can be problematic.
Other than what we can see with our current methods, what do
we really know about dental calculus and how its growth and structure affect the
reliability of these methods and potentially distort our interpretations of the past?

## What is dental calculus? {#intro-what}

To answer these questions, we must first answer a single, surprisingly difficult
question: What is dental
calculus? I'm not referring to its formation or composition, which I briefly
described [above](#chap-intro). How do we categorise it? Is it a dental disease? An oral
health condition? A byproduct of oral conditions? We start by exploring various
definitions of oral health. Definitions in an introduction
are a little cliché and tedious, but often necessary<!--insert shrug emoji-->. Since oral health is a
complex topic, definitions of oral health are often purposefully (and confusingly)
broad, and they extend beyond physical well-being and into the realms of emotional
and social comfort.
The World Dental Federation (FDI) defines oral health as the ability to perform
mouth- and face-related functions with confidence and without pain
(including smiling, speaking, eating, etc.) [@fdiOralHealth]
(<https://www.fdiworlddental.org/fdis-definition-oral-health>).
Both the World Health Organisation (WHO) and FDI take a similar approach to
defining oral conditions, giving a list of conditions that cause
discomfort, pain, disfigurement, or death.
The list includes the dental conditions
tooth decay (caries), gum disease (periodontal disease), and dental trauma, but
not dental calculus [@whoOralHealth]
(<https://www.who.int/news-room/fact-sheets/detail/oral-health>).
While these are not likely to cause death, they are often the source of physical
and emotional discomfort, and may cause further health complications if they are
not dealt with in a timely fashion.

Dental calculus and dental plaque are not
considered oral conditions according to WHO. In fact, dental plaque is part of the normal
functioning of our oral biome [@marshDentalPlaque2006]. When plaque reaches a certain
level of acidity over a prolonged period of time, the normal functioning of the bacteria
within the plaque may shift towards a disease-causing function.
The biofilm will cause the surface of the enamel to demineralise, eventually resulting
in a cavity (or caries). Dental caries are unequivocally considered a dental disease.
If, instead, the biofilm calcifies, dental calculus is the result. Its status in oral
health is questionable.

Dental calculus is not known to be painful, nor does it affect the ability to perform
the functions listed above. However, with continued accumulation, it may affect the
confidence of the person
performing these tasks [@collinsHomelessDental2007], and in extreme cases it can affect
function [@balajiUnusualPresentation2019]. Most of the virulence and disease-causing
potential is lost when the bacteria within dental plaque calcify [@akcaliDentalCalculus2018].
It has been shown to contain pockets of living bacteria that can be detrimental
to oral and dental health [@tanCalculusUltrastructure2004; @tanBacterialViability2004].
The rough, porous surface of dental calculus is also
a great place for bacteria to attach more easily and develop a new layer of plaque on
the surface of the calculus. This is likely
why there is often a correlation (NOT causation) between dental calculus and
periodontitis, especially subgingival calculus
[@jepsenCalculusRemoval2011; @whiteDentalCalculus1997].
Since it seems to fulfill some of the criteria of an oral condition, it should be
considered as such, at least under the definitions provided by WHO and FDI.
Whether or not dental calculus can be considered an oral disease is more
questionable.
While it does grow on the surface of teeth, it doesn't seem to affect the underlying
enamel. And while there is a relationship with periodontal disease
(which has been defined as a dental disease), the nature of this
relationship is still under debate, with calculus likely being a secondary contributor
[@jepsenCalculusRemoval2011]. As such, we can probably limit the definition to an oral
condition and not necessarily a dental disease [@pilloudOutliningDefinition2019].
In fact, dental calculus is quite hard, so a layer of dental calculus on a tooth can
actually protect it from wearing down (although there are better options).

## The study of dental calculus {#intro-study}

It seems that the researchers who are studying dental calculus approach it
from a wide range of different fields and backgrounds, including genetics,
proteomics, botany, and (bio)archaeology. The paleogeneticists mine it for the
wealth of information it contains on oral health and disease in the past
[@yatesOralMicrobiome2021; @warinnerPathogensHost2014].
Paleodiet researchers extract microremains and residues from food
[@henryCalculusSyria2008; @mickleburghNewInsights2012]
to infer dietary practices. Bioarchaeologists use its presence and amount
to broadly infer diet, and dental and overall health
in a given population [@yaussyCalculusSurvivorship2019; @lieverseDentalHealth2007; @belcastroContinuityDiscontinuity2007; @novakDentalHealth2015; @slausDentalHealth2011]. <!-- need more examples. dental anthropologists? paleopathologists?-->
This leaves research output from studies of calculus scattered across
multiple venues, with no clear gathering point.
I think it's fair to say that dental calculus should be included in
discussions of pathological oral conditions, even if its role is secondary. But who
is currently studying dental calculus as a substance in its own right? And why do we
need to learn more about it if we're just interested in what's inside?
Related discussions have started to take place in recent years
[@radiniDirtyTeeth2022; @bucchiComparisonsMethods2019; @wrightAdvancingRefining2021].

The lack of a specific field of study for dental calculus to belong may be
related to how it's taught to students (and if it's taught at all).
Textbooks from the more established fields in bioarchaeology are probably
a good indicator of the teaching curricula, which also impacts research
focus.
The most popular osteoarchaeology textbooks only briefly mention dental
calculus as more of a footnote than anything else. A couple of lines describing
what it is (usually 'mineralised plaque') and that it can contain food
debris and bacteria [@whiteBoneManual2005, @whiteHumanOsteology2011].
They're not wrong.
Diseases that manifest themselves in the skeleton as lesions on the bones
have a very clear home in paleopathology. No one questions whether
or not the degeneration of vertebrae from tuberculosis should
be included in the paleopathology textbooks (at least not as far as I'm aware).

These textbooks often include chapters on dental disease, where more detailed
descriptions of dental calculus are usually found
[e.g. @robertsDentalDisease2007; @waldronPalaeopathology2020].
Dental caries, calculus' more famous sibling, will
often get a few pages.
In some cases, dental calculus may even be hidden within a section on periodontal
disease or plaque
[e.g. @ortnerIdentificationPathological2003; @aufderheidePaleopathology1998].
The focus of these (sub)sections is varied, with some simply describing what it is,
and others giving
brief discussion on the relationship between calculus and periodontal disease.
A more detailed section was dedicated to dental calculus in *Ortner's Identification
of Pathological Conditions in Human Skeletal Remains*, with a detailed description
of formation, structure, and application in (biomolecular) archaeology
[@kinastonOrtnerDentition2019]. The description extends well beyond any
(paleo)pathological significance of dental calculus.
Can we fault the authors/editors for not
giving it more attention? After all, it's not a dental disease, and its relationship
with other dental diseases is unclear. What is clear, is that it has implications for
oral health, and, for that very reason, could be addressed more extensively in
paleopathology; certainly in the textbooks that include dental disease.

<!--Dental anthropology. -->
On the surface, dental anthropology seems like a more suitable home for
the study of dental calculus. However, it's not included in
*A Companion to Dental Anthropology*, an otherwise great resource on
studying archaeological teeth.
The editors briefly acknowledge the valuable information
gained from calculus and that it holds a lot of potential; but that's it
[@scottBriefHistory2015].
Other notable absences include textbooks such as
*Technique and Application in Dental Anthropology* and
*New Direction in Dental Anthropology* [@townsendDentalAnthropology2012],
both of which dedicate considerable attention to dental caries.
<!-- Hillson, Dental Anthropology -->
Hillson's *Dental Anthropology*, a book that I consider to be
the 'bible' for dental anthropology, has a section on dental
calculus in the Dental Disease chapter.
It covers a basic description, the composition, microscopic structure,
methods used for recording archaeological calculus, and
the distribution in the dentition (i.e. which teeth are more prone to calculus buildup)
[@hillsonDentalAnthropology1996].
Considering these are entire books devoted to the dentition, it seems odd that
there is often only a few paragraphs (if that) on dental calculus. Granted, the
only function teeth serve in the
growth of dental calculus is as a suitable surface on which to attach;
though the role of substratum is an important role, as dental calculus is
seemingly unable to form on other surfaces in the oral cavity.

<!-- Sorry to do this to you again, but we need more definitions. -->
<!-- The *Medical Dictionary for the Dental Professions (2012)* defines dental anthropology as: -->

<!-- "a branch of physical anthropology concerned with the origin, evolution, and development -->
<!-- of dentition of primates, especially humans, and to the relationship between primates' -->
<!-- dentition and their physical and social relationships." [in @irishIntroductionDental2015]. -->

<!-- It could certainly be considered part of the development of dentition in primates -->
<!-- (if you consider both 'development' and 'dentition' more broadly). Perhaps the -->
<!-- description on -->
<!-- the Dental Anthropology Association's (DAA) website gives more room for interpretation: -->

<!-- "Dental anthropology utilizes the dentitions of humans and other non-human primates--both past and present--to answer questions of anthropological interest. These questions can include (but are by no means limited to): How are individuals and populations related? What did their diet look like? How healthy were they?" -->
<!-- [@daaDentalAnthropology] -->
<!-- (<http://www.dentalanthropology.org/>, accessed 30-Nov-2021). -->

<!-- This description more directly addresses a dietary and health perspective, which -->
<!-- certainly applies to dental calculus, as long as you consider it part of the dentition. -->
<!-- It's possible we'll see dental calculus included in more detail in future textbooks, -->
<!-- given how it has increased in popularity over the last decade or so. -->

Since the use of dental calculus in biomolecular archaeology is relatively new,
there are fewer available textbooks, and it rarely has a dedicated course.
The most common place to find descriptions of dental calculus is, therefore,
journal articles. There will be a short paragraph on dental calculus formation (and sometimes
composition) in the introduction section. These are quite variable and are often limited
by the word count of the journal. Despite this, the descriptions will often be as long,
if not longer, than the sections in textbooks devoted to dental calculus
[@velskoMicrobialDifferences2019]. The focus of these paragraphs are generally the same.
They describe the formation and mineral composition of dental calculus, and provide some
examples of how dental calculus has been used in related studies (not unlike the beginning
of this chapter).
The contribution of dental calculus to archaeology has been significant, so it
is likely to receive more and more attention going forward. In fact, an entire
chapter was recently devoted to dental calculus in the second edition of
*Handbook of Archaeological Sciences* [@fagernasDentalCalculus2023]. Take that,
dental caries!

## The challenges of studying dental calculus

What we know about dental calculus and the influence of diet was reviewed in an
article aimed at (bio)archaeologists. The overall conclusion reached in the article:
it's still pretty unclear [@lieverseDietAetiology1999]. Now, 20-some years later,
there has been limited progress on this point.
High-protein diets are linked to an increase of urea, which is linked to an increase
in oral pH, which is linked to mineral deposition
[@wongCalciumPhosphate2002; @dibdinOralUrea1998]. BUT, protein may also inhibit
crystalisation [@hidakaDietCalculus2007].
Starch consumption has been linked to increased rates of caries in early farming
populations [@storeyPaleopathologyOrigins1986]. This is
consistent with *in vitro* testing, at least for starches high in amylose content<!--examples-->.
So a high-starch diet causes caries, not calculus, right? Well, starches with a high
amylopectin content are linked to increased calcification [@hidakaDietCalculus2007].
It likely depends on what is consumed along with the starch [@hidakaStarchRole2008].
There is also some (*in vitro*) evidence to suggest that silica may promote dental calculus
formation by promoting mineral precipitation, i.e. the transfer of minerals from
saliva to the biofilm [@damenSilicicAcid1989]. Overall, this is an understudied
area in both clinical and archaeological contexts.

<!-- the mechanism of starch incorporation into dental calculus -->
Another aspect of diet and dental calculus where we are still looking for answers,
is the process that causes fragments of food and other environmental materials
to become entrapped in the dental calculus.
We know that it happens. Decades of research has shown dental calculus to be a
seemingly unlimited resource for dietary substances. We don't know exactly how
this happens, and herein lies the potential for bias.
Efforts have been made to understand
how much of the consumed food makes it into the calculus. These include studies
on modern humans [@leonardPlantMicroremains2015] and non-human primates
[@powerChimpCalculus2015; @powerRepresentativenessDental2021],
where food intake is meticulously documented, and calculus subsequently analysed.
These studies have common findings; the amount of the diet that becomes trapped
in the dental calculus of any one person has no clear relationship to the amount
of food that was consumed. The most likely reason is that the formation of dental
calculus differs between people [@powerChimpCalculus2015].
So, it's not a great way to study the diet of a single person, but generally suitable
to study patterns in the diet of a population. The more people you study, the more
likely you are to gain a complete picture of the diet in a population.
<!--kinda like the
more monkeys with typewriters you have, the more likely they are to reproduce
Shakespeare. This is not meant to be discouraging.-->
The fact that we can still see
(in some cases, literally) remains that were consumed thousands of years ago is
pretty cool. We just need a better understanding of why the record of diet from
dental calculus differs from the actual intake of food. This will allow us to make
more robust interpretations about past dietary practices.
Something that may influence the dietary record that we get from calculus is the
method we use to extract the dietary remains from calculus.
Our understanding of dental calculus extraction methods is improving, with studies looking
at the effect of various acids used to dissolve calculus (commonly EDTA or HCl)
[@trompEDTACalculus2017; @bucchiComparisonsMethods2019; @sotoCharacterizationDecontamination2019; @palmerComparingUse2021];
as is our understanding of how the choice of tooth may affect our results
[@fagernasMicrobialBiogeography2021], and that not all we see is related to
deliberate consumption [@delaneyMoreWhat2023].

These studies provide valuable insights into potential biases of our sampling methods
and the representation of diet within dental calculus, with a minor caveat.
Most of these studies have been conducted on living primates or archaeological
remains. An issue with using living (or once living) organisms is the inability
to control factors
related to the variability between subjects. Basically, studying humans is messy
and complicated because we're all unique. It's a lovely sentiment but it
can make for some messy science. Not bad science (not at all!). Just messy.
A method of study that offers more control is the growth of
plaque and calculus in a lab. This allows us to control many of the things that
are difficult to control in humans, such as the bacteria that colonise our mouth,
where each person has a pretty unique makeup of bacteria. We also have a very unique
genome (with the exception of identical twins) that plays a role in how quickly we form
calculus in our mouth (if at all). Certain enzymes start digesting our food as
soon as it enters our mouth, and the activity of these
enzymes fluctuates throughout the day, causing a lot of variability both within and
between individuals.<!--in response to meals, stress, etc.-->
Finally, the number of microremains that enter our mouth over days, weeks, and
months,
can be very different between people, even with the same diet.
All these things can muddy the results of research on living subjects, where a
lab-grown approach can help tease out <!--the pesky--> confounding factors.
I don't believe research conducted on lab-grown biofilms can in any way replace
studies with modern or archaeological individuals, nor should they. But it can
complement these studies by zooming in on certain aspects that are too difficult to
isolate in (once-)living people<!--expand justification-->.

Often we can draw from clinical studies as there are common goals, e.g. discovering
the aetiology and/or presentation of a disease. However, the motivation driving the
studies in archaeology and dental research are inherently different; although,
there is certainly overlap in some areas ([@fig-plot-and-wordclouds]B and C).
There is more interest in preventing dental calculus from forming in the first
place, so most studies focus on short-duration models to explore anti-microbial
treatments and inhibition of biofilm formation and plaque buildup [@extercateAAA2010].
As shown in a previous study, calculus and plaque have distinct microbial profiles
[@velskoMicrobialDifferences2019], so the applicability of short-term models
to explore archaeological questions on dental calculus are limited, since
plaque is rarely (if ever) preserved. Archaeologists are more interested in
questions related to how diet influences the growth of biofilms, and how fragments
become embedded inside, and what we can say about diet.
Further, the interest in dental calculus as a field of clinical research has been
declining since the 2000s, which, as far as I'm aware, is when the last studies
growing dental calculus in a lab were conducted. We can see this by the number of
clinical articles with the term dental calculus in the title
([@fig-plot-and-wordclouds]A). And they certainly aren't interested in how food
debris becomes trapped inside our calculus.
Dental calculus has also become less of a problem with the use of modern dental hygiene
practices and regular visits to the dentist [@velskoMicrobialDifferences2019].

To summarise: Bioarchaeologists are interested in how dental calculus relates
to dental and general health; paleodietary researchers are interested in the food
remains that are trapped inside; paleogeneticists are interested in accessing the
oral bacteria that have been fossilised within; clinial dentistry views it as a
nuisance to be removed and, ideally, prevented from forming in the first place.
This lack of systematic research specifically devoted to dental calculus as a substance,
rather than a means to an end, leaves a lot of questions regarding the expected
behaviour of dental calculus and how information from the past becomes trapped inside.
To summarise the summary: we need to ask more basic questions about dental calculus.

## Aims {#intro-aims}

This dissertation is a contribution to a dental-calculus-centric body of knowledge,
and addresses a gap in the fundamental research on dental calculus to further our
understanding of how we can use dental calculus to reconstruct the diets of people
in the past. The main aim is the development, validation, and application of
a calcifying oral biofilm model to improve interpretations on archaeological dental
calculus. By developing a model system we can isolate the effects of confounding
factors in dental calculus and diet, and explore new uses for dental calculus
in paleodietary reconstructions through fundamental experimentation. I also aim
to assess the potential and limitations of dental calculus to explore dietary
activities of past populations.

Every decision we make, from sampling to statistical analysis, leads us down a
unique path towards a different interpretation from the other possible paths in
the multiverse of analyses. It's important we fully understand the path we take,
to ensure that it is the right path given the limitations,
and one that maximises the validity and detail of our interpretations.

<!-- In short, can I grow calculus in the lab? Is what I'm growing actually a substance -->
<!-- that resembles calculus? And can the model be used to inform archaeological research; -->
<!-- specifically, how should we interpret the food debris extracted from dental calculus? -->

With these aims, I hope to address the following research questions:
<!-- objectives instead of questions? -->

*How can we improve the resolution of our interpretations using dental calculus on individuals and populations?* <!--(overarching theme - problem statement needs to lead up to this)-->
We are stuck in the identification of compounds, and unable to speak to the
quantity, since we know that it's not very representative of a single individual.

*Can we trust the system? (i.e., using dental calculus to reconstruct diet)*
Since we don't know the mechanism of incorporation, there are likely hidden biases
and limitations of our methods as a result. We don't know the starting point, i.e.,
exactly what and how much was originally trapped inside, so we have difficulty
validating our methods.

*How can a model improve our understanding of dietary reconstructions using dental calculus?*
How can it address current challenges in paleodietary reconstructions, and can it
help us produce a better understanding of how dietary intake relates to the
record of diet we extract from archaeological dental calculus?

<!--This means sometimes asking the apparently obvious questions, like how does food become trapped in dental calculus.
And occasionally you'll find that no one really has a good answer to these questions. (PREFACE?) -->


## Thesis outline and structure

If you have made it to this point, you have probably read most of [**Chapter 1**](#chap-intro),
in which I provide some context to the study of dental calculus in archaeology
and identify some areas that could benefit from further investigation.
[**Chapter 2**](#chap-background) provides some background information on oral biofilms
and oral biofilm models in more detail than I can do in the research articles
included in Chapters 3 and 4. So if you're already well-versed in oral
microbiology, feel free to skip to Chapter 3. If not, I recommend picking
up a textbook written by actual experts in the field of oral microbiology.
If, for some reason, you can't access one of these, feel free to read
[**Chapter 2**](#chap-background). I suppose there are worse
options than something written by a PhD student in archaeology.
The chapter reflects the current knowledge of biofilms and the oral microbiome
(as best I could summarise) at the time of writing, and no warranty is given for
the inevitable new developments that will change what we now believe to be true.

To address the aims of the dissertation outlined [above](#intro-aims),
I developed a protocol to grow dental calculus in a lab on plastic tubes instead
of looking at the real stuff you normally find inside your mouth. The reason
for using lab-grown biofilms instead of humans is that the *in vitro* lab model offers more
control over all the factors that go into the growth of dental calculus, at least
in theory. The real world is messy, and sometimes you need to remove things from
the real world to break it down and really get into the nitty gritty of how it works.
There are many different kinds of biofilm models, including single species of bacteria,
select species
determined by the researchers (defined consortium), and multiple species from some
natural source (the human mouth, for example). I will cover the different types of models
in more detail in [**Chapter 2**](#background).
Since there are many biofilm models to choose from, developing a new protocol may
seem counter-productive; however, few are developed for long-term
growth and even fewer with the purpose of mineralising the biofilm to form dental calculus.
One of the exceptions involves a highly complex
setup that is unlikely to be supported by budgets and facilities available to most
archaeological laboratories [@sissonsMultistationPlaque1991].

After developing a working protocol, the next step was to determine if the stuff
I grew in the lab is actually
dental calculus. Or at least something close enough that we can use
it to explore our research questions.
To do this, we (myself and coauthors) determined the mineral
and bacterial composition of our model using Fourier Transform Infrared (FTIR)
spectroscopy and metagenomic classification [**Chapter 3**](#byoc-valid).
We then compared the results of these analyses to naturally grown dental calculus,
both modern and archaeological.

Being confident that our model looks and behaves like human dental calculus,
we then set out to test some very basic behaviours of starch
grains within dental calculus.
[**Chapter 4**](#byoc-starch) is a research article where we 'fed' the biofilm with
a known quantity of starch granules during the growth period to see if the input
quantity/ratio matched the extracted quantity (or output). Those who are
familiar with dental calculus research will not be surprised that it did not.
The more interesting outcome of the study is the more detailed explanation of how
the input and output starch quantities were mismatched.

[**Chapter 5**](#mb11CalculusPilot) is a separate article, in the sense that it doesn't
involve the biofilm model in any way. Rather, it addresses the theme of the overall
utility of dental calculus in archaeological research.
We look at possible medicinal compounds in the dental
calculus of a Post-medieval Dutch population. We employed Ultra High Performance
Liquid Chromatography coupled with tandem Mass Spectrometry (UHPLC-MS/MS) to identify
various compounds in dental calculus, including alkaloids and other compounds.
It shows the potential of dental calculus to inform about past practices, but also
highlights some of the limitations we are currently experiencing in the field.
[**Chapter 6**](#chap-discussion) is a discussion on the limitations and future potential of
dental calculus in the field of archaeology, and what biofilm models can contribute to our
understanding of past diet.

## References cited {.unnumbered}