# Evaluation of age, sex, and ancestry-related variation in cortical bone and dentine volumes in modern humans, and a preliminary assessment of cortical bone-dentine covariation in later Homo

Mathilde Augoyard, Clément Zanolli, Frédéric Santos, Anna C Oettlé, Ericka l'Abbé, Mona Le Luyer, Marine Cazenave, Thomas Colard, Jakobus W Hoffman, Antonio Profico, et al.

► To cite this version:

Mathilde Augoyard, Clément Zanolli, Frédéric Santos, Anna C Oettlé, Ericka l'Abbé, et al. Evaluation of age, sex, and ancestry-related variation in cortical bone and dentine volumes in modern humans, and a preliminary assessment of cortical bone-dentine covariation in later Homo. JASs. Journal of anthropological sciences, 2023, 101, 10.4436/JASS.10019. hal-04243158

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# Evaluation of age, sex, and ancestry-related variation in cortical bone and dentine volumes in modern humans, and a preliminary assessment of cortical bone-dentine covariation in later *Homo*

Mathilde Augoyard¹, Clément Zanolli¹,², Frédéric Santos¹, Anna C. Oettlé³, Ericka

N. L'Abbé⁴, Mona Le Luyer¹,⁵,⁶, Marine Cazenave⁷,⁸,⁹, Thomas Colard¹,¹⁰, Jakobus

Hoffman¹¹, Antonio Profico¹² & Priscilla Bayle¹

1) Univ. Bordeaux, CNRS, MCC, PACEA, UMR 5199, F-33600 Pessac, France

email: mathilde.augoyard@u-bordeaux.fr, priscilla.bayle@u-bordeaux.fr

2) Department of Maxillofacial and Oral Surgery, Sefako Makgatho Health Sciences University, Ga-Rankuwa, Pretoria, South Africa

3) Department of Anatomy and Histology, Sefako Makgatho Health Sciences University, Ga-Rankuwa, Pretoria, South Africa

4) Forensic Anthropology Research Centre, Department of Anatomy, Faculty of Health Sciences, University of Pretoria, Pretoria 0031, South Africa

5) Center for Genomic Medicine, Massachusetts General Hospital, Boston, USA

6) Department of Psychiatry, Harvard Medical School, Boston, USA

7) Division of Anthropology, American Museum of Natural History, New York, NY, USA

8) Skeletal Biology Research Centre, School of Anthropology and Conservation, University of Kent, Canterbury, UK

9) Department of Anatomy, Faculty of Health Sciences, University of Pretoria, South Africa

10) Department of Oral Radiology, University of Lille, Lille University Hospital, Lille, France

11) South African Nuclear Energy Corporation SOC Ltd., Pelindaba, South Africa

12) Department of Biology, University of Pisa, Pisa, Italy

**Summary** - Cortical bone and dentine share similarities in their embryological origin, development, and genetic background. Few analyses have combined the study of cortical bone and dentine to quantify their covariation relative to endogenous and exogenous factors. However, knowing how these tissues relate in individuals is of great importance to decipher the factors acting on their evolution, and ultimately to understand the mechanisms responsible for the different patterns of tissue proportions shown in hominins. The aims of this study are to examine age-, sex-, and ancestry-related variation in cortical bone and dentine volumes, and to preliminary assess the possible covariation between these tissues in modern humans and in five composite Neandertals. The modern analytical sample includes 12 immature individuals from France and 49 adults from France and South Africa. Three-dimensional tissue proportions were assessed from microtomographic records of radii and permanent maxillary canines. Results suggest ontogenic differences and a strong sexual dimorphism in cortical bone and dentine developments. The developmental pattern of dentine also seems to vary according to individual's ancestry. We measure a stronger covariation signal between cortical bone and dentine volumes than with any other dental tissue. A more complex covariation pattern is shown when splitting the modern sample by age, sex, and ancestry, as no signal is found in some subsamples while others show a covariation between cortical bone and either crown or radicular dentine. Finally, no difference in cortical bone volume is noticed between the modern young adults and the five young adult composite Neandertals from Marine Isotopic Stages (MIS) 5 and 3. Greater dentine
volumes are measured in the MIS 5 chimeric Neandertals whereas a strong interpopulation variation in dentine thickness is noticed in the MIS 3 chimeric Neandertals. Further research on the cortical bone-dentine covariation will increase understanding of the impact of endogenous and exogenous factors on the development of the mineralized tissues.

**Keywords** – Canine, Radius, Development, Sexual dimorphism, Bone/dentine covariation, Neandertal.

**Introduction**

In recent years, the development of three-
dimensional (3D) virtual imaging has enabled
access to internal bony and tooth structures
for the quantitative assessment of their endo-
structural organization (e.g., Bayle et al. 2010,
2011; Cazenave et al. 2017; Colombo et al.
2019; García-Campos et al. 2020; Genochio
et al. 2019; Le Cabec et al. 2013; Puymerail
2017; Puymerail et al. 2012; Swan et al. 2020;
Zanolli and Mazurier 2013; Zanolli et al. 2020).
Functional adaptation is often put forward to
explain differences in cortical bone and den-
tal volumes, as well as in internal geometry of
long bones and roots in hominins (Bondioli et
al. 2010; Churchill 1998; Kupczik and Hublin
2010; Trinkaus and Ruff 2012). The mechanical-
related adaptation of bone structure in response
to different loadings has been widely described
for decades (Pearson and Lieberman 2004; Ruff
2019; Shaw and Stock 2009; Wolff 1892) and the
internal bone structure of hominin postcranial
remains has been investigated for reconstructing
joint position, joint loading, and ultimately past
behaviors (see references in Cazenave and Kivell
2023; Kivell 2016). Regarding hominin roots,
several studies have also highlighted a change of
their volume to better sustain forces related to
(para)masticatory activities (Kupczik and Hublin
2010; Le Cabec et al. 2013; Zanolli et al. 2018).
However, phenotypic variation is multifactorial
and must be seen as "a compromise between
mechanical and other influences" (Ruff 2019,
p.191). For example, many studies on extant
and extinct humans have found consistent varia-
tion in stature, weight, and bone robustness due
to different selection pressures depending on

the ecogeographic regions (Bernal et al. 2006;
Cowgill et al. 2012; Foster and Collard 2013;
Ruff 1994; Savell et al. 2016; Weisensee 2014),
following Bergmann's and Allen's "rules" (Allen
1877; Bergmann 1847). Similarly, geographic
variation in dental morphology among human
groups has been the subject of numerous pub-
lications (Bernal et al. 2010; Irish 2016; rev.
in Pilloud et al. 2016; rev. in Scott and Turner
1997), but none of them questioned the differ-
ence in dentine thickness depending on ances-
try. Another factor that could lead to phenotypic
variation in hominins is the morphological inte-
gration between anatomical structures linked
together by functional and/or developmental
similarities (Hallgrímsson et al. 2009; Rolian
2014). For example, previous studies have
highlighted strong integration between dental
elements that have an important role in occlu-
sion and mastication (Gómez-Robles and Polly
2012). Many researchers have focused on devel-
opmental integration between mandible and
lower dentition, two anatomical units that influ-
ence each other during development (Boughner
and Hallgrímsson 2008; Coquerelle et al. 2012;
Workman et al. 2002). In their study, Coquerelle
and colleagues described interactions between
mandibular form and dental mineralization in
early development, highlighting a major influ-
ence of genetic background on both tooth and
mandible structures during pre- and postnatal
ontogeny. This mandible-dentition relationship
would then decline over time, under the action of
several exogenous factors, particularly functional
ones, leading to an independence between man-
dible form and dental development (Coquerelle
et al. 2010). Biological tissues whose formation
is induced by the same signaling pathways and
development is controlled by the same genetic
activation/inhibition ratio are therefore likely
to covary (Boughner and Hallgrímsson 2008;
Churchill 1998; Evans et al. 2016; Hallgrímsson
et al. 2007).

Cortical bone and dentine tissues of modern
humans share similarities in their composition,
embryonic origin, and responsiveness to specific
developmental signaling molecules, that can
lead to coordinated variation of these two tis-
sues. The mineralized fraction of cortical bone
and dentine is composed of non-collagenous
proteins that promote and control the deposi-
tion of hydroxyapatite on collagen fibers, and
of an organic matrix that is mainly composed
of type I collagen (Burr and Allen 2019; Dean
2017; Qin et al. 2004). Osteogenesis and den-
tinogenesis are driven by the same network of
genes encoding signaling pathways that are regu-
lated by epithelial-mesenchymal interactions
(Brown et al. 2018; Jernvall and Thesleff 2012).
Cortical bone amount is regulated by the activ-
ity of osteoblasts and osteoclasts responsible for
the formation and resorption of cortical bone
through the entire individual life, in response to
biomechanical constraints (Burr and Allen 2019;
Pearson and Lieberman 2004; Ruff et al. 2006).
To a lesser extent, dentine may also be sensitive
to functional loads induced by (para)masticatory
activities: at the end of tooth formation, regular
secondary dentine continues to form around the
pulp chamber walls (Dean 2017) and irregular
secondary dentine can be secreted by the odonto-
blasts in response to abrasion, attrition, erosion,
or tooth pathologies (Dean 2017; Teaford et
al. 2000). Some studies also highlighted direct
correlation between tensile stress, occlusal wear,
and dentine distribution pattern, suggesting that
mechanical forces may have an impact on sec-
ondary dentine formation (Benazzi et al. 2014;
Coon 1962; Kupczik and Hublin 2010; Oyama
et al. 2007).

Cortical bone and dentine also share similar
patterns of sexual dimorphism since their devel-
opment responds to hormonal and chromosomal
signals (Alvesalo 1997; Callewaert et al. 2010).
Previous studies have shown a sexual dimorphism

in the infra-cranial skeleton of modern humans
with males having higher bone mineral density,
periosteal circumference, and cortical bone area
than females (Burr and Allen 2019; Leonard et al.
2010; Stuck et al. 2020). Differences in absolute
and relative volumes of crown dentine (i.e., the
proportion of the total crown volume constituted
by dentine) were also assessed between males
and females, with the former showing a greater
volume of crown dentine (García-Campos et al.
2018; Saunders et al. 2007; Sorenti et al. 2019).

Despite these structural, biochemical,
genetic, and developmental similarities, no
study has investigated a possible correlation
between the volumes of cortical bone and den-
tine among humans. Indeed, variations in dental
and bone tissue proportions have been assessed
separately among fossil hominins, but not
together. For example, an extensive literature
has shown greater overall cortical robustness in
Neandertals than in modern humans (Churchill
1998; Genochio et al. 2019; Trinkaus and Ruff
2012; Trinkaus et al. 1999), reminiscent of that
described for the crown and radicular dentine
that is larger and much thicker in Neandertals
(Buti et al. 2017; Kupczik and Hublin 2010; Le
Cabec et al. 2013). The biomechanical history of
the skeleton may explain a significant part of the
differences in cortical bone thickness and inter-
nal diaphyseal geometry of long bones between
adult Neandertals and modern humans (De
Groote 2011; Puymerail et al. 2012; Trinkaus
and Ruff 1989; Volpato et al. 2011). However,
this pattern of postcranial and dental robust-
ness may already be present in the early stages
of development (Bayle 2008; Bayle et al. 2011;
Maureille 2002; Weaver et al. 2016).

Bone and dentine tissue proportions act as
a complex interaction between a host of factors
including endogenous (e.g., genetic, physiologic),
environmental (e.g., mechanical loadings), and
evolutionary ones. Hence, there is a clear need
for understanding how cortical bone and den-
tine thickness vary according to an individual's
age, sex, and ancestry, and whether a systematic
effect exists that affects both tissues, and that
could lead to their coordinated variation during
development. Results from modern samples may
provide an explanatory model for the more robust
bone/dentine pattern observed in Neandertals.

Within this exploratory study carried out on
a modern human ontogenic sample, we evalu-
ated the variation in cortical bone and dentine
volumes according to age, sex, and ancestry, and
documented the degree of possible covariation
between the volumes of these two tissues. More
specifically, we tested the hypotheses that (i) the
volumes of tissues vary according to individuals'
age, (ii) sex, and (iii) ancestry, and that (iv) due
to their structural, genetic, and developmen-
tal affinities, a coordinated variation in cortical
bone and dentine volumes should be observed
in immature individuals and should decline in
adults due to the increasing impact of exogenous
factors on tissue development. We used a sam-
ple of distal radii and permanent canine teeth
to quantify the absolute and scaled volumes of
cortical bone and dentine and to assess their
degree of covariation in two skeletal samples of
European- and African-derived immature and
adult modern humans. The first investigation of
five young adult composite Neandertals was also
conducted to quantitatively compare the volume
of cortical bone and dentine tissues and their
potential covariation, and to give novel insights
on the evolutionary mechanisms affecting both
bone and dentine developments.

Materials and Methods

To quantitatively assess cortical bone and
dentine (co)variation, we investigated bony and
dental elements for which development is less
dependent on the function. Finding bone por-
tions for which mechanical factors have a negligi-
gible impact on tissue development is difficult.
Minimal biomechanical variation of the midtho-
racic region has been observed among individu-
als, the ribs at levels 4-7 being less susceptible
to mechanical loadings than appendicular skel-
eton (Crowder and Rosella 2007; Roberts and
Chen 1972; Tommerup et al. 1993). However,
identification of rib number can be challenging,

especially in the archaeological/palaeoanthropo-
logical record where the ribs are often fragmented
or absent. To be able to further investigate this
topic in the fossil record in the future, we thus
decided to measure cortical bone volume from
the distal radius, an element that is relatively
frequent in the palaeoanthropological record
(Pérez-Criado and Rosas 2017; Rodríguez et al.
2016; Zipfel et al. 2020). Moreover, although
affected by mechanical constraints (Auerbach
and Ruff 2006; Haapasalo et al. 2000) that lead
to an asymmetry of the radius shaft (Hong et
al. 2021), several authors highlighted a negligi-
ble side difference in anatomical measurements,
cortical thickness, and mineral density in the
distal portion of the radius compared to other
long bones (Gray et al. 2019; Hildebrandt et al.
2016; Min et al. 2007). It should be noted that,
because this study partly relies on archaeologi-
cal specimens and/or on individuals where only
the right or left arm was available, we could not
test for their right vs. left handedness to select
the non-dominant arm for analysis. Dentine
volumes were measured from permanent upper
canine teeth (UC). All the selected UCs show
low wear degree (stages 1 to 3; Molnar 1971), so
the apposition of secondary dentine in the pulp
cavity, mainly induced by functional constraints
(Dean 2017), would be negligible.

The studied modern sample consists of micro-
computed tomography (microCT) scans of radii
and UCs from a European sample composed
of 12 immature and 17 adult modern humans.
The sample comes from the French medieval
cemeteries of Sains-en-Gohelle (Pas-de-Calais,
France), and Jau-Dignac-et-Loirac (Gironde,
France), dated from the High to the Low Middle
Ages (Beauval et al. 2012; Cartron and Castex
2006). This European assemblage is supple-
mented with a South African sample composed
of 32 adults of African and European ancestries
from the contemporary identified osteological
collection of Sefako Makgatho Health Sciences
University (SMU, Gauteng Province, South
Africa) and the Pretoria Bone Collection (PBC;
Tab. S1). Individuals from these osteological col-
lections are mostly migrant laborers from South
Africa or neighboring countries (i.e., Botswana, Mozambique, Zimbabwe, and Zambia), and reflect people from low socio-economic status (L'Abbé et al. 2021).

All individuals were selected under the condition that they each had one radius and UC well-preserved and free from observable pathology. The maturation stages of the UCs ranged from “root canal with parallel walls and apical end still partially open” to “root canal with apical end completely closed” (i.e., scores G to H of Demirjian et al. 1973). When necessary, reconstruction of the missing apical portion was done following Le Cabec et al. (2013) method. The age at death of the archaeological individuals has been assessed following Black and Scheuer (1996), Moorrees et al. (1963), Scheuer and Black (2000), and Schmitt (2005) methods. The sex of the adults from the archaeological samples has been determined following the DSP method based on hip-bone measurements (Murail et al. 2005). The immature individuals have been distributed between the age classes “Juvenile” 9–14 years (n = 5) and “Adolescence” 15–19 years (n = 7), according to Bogin (2003). The European adult sample is composed of 11 females and 7 males aged 20 to 49 years, whereas the South African sample consists of 14 females and 18 males aged 21 to 70 years (for a complete age distribution of the modern human sample, see Fig. S1).

Due to the scarcity of individuals having both their radius and canine preserved in association in the palaeoanthropological record, we created five young adult composite Neandertals from different archaeological sites (Tab. S1). To minimize phenotypic variation induced by evolutionary process, we combined bone and dental remains dated to either Marine Isotopic Stage (MIS) 5 or 3. To ensure optimum comparison with data obtained from modern humans, the selected radii were fully developed, and the canines showed low wear degree (stages 1 to 4; Molnar 1971). The three chimeric individuals from MIS 5 were created by combining the right radius from the Regourdou 1 individual (Regourdou, France) who has been described as a young adult (Volpato et al. 2012), with three UCs from

Krapina (Kr 36; Kr37, Kr76; Croatia). The first chimeric Neandertal from MIS 3 was represented by the association of the radius from the young adult Spy II (Shackelford 2014; Spy, Belgium) and the UC Vi 12.5 from Vindija (Croatia), whereas the second chimeric individual from MIS 3 was created by using the same radius in association with the UC Palomas 35 (La Sima de las Palomas del Cabezo Gordo, Spain). All the bone and dental elements, except for Palomas 35 (Bayle et al. 2017), were downloaded from the Digital Archive of the Neanderthal Museum (www.archiv.neanderthal.de).

Acquisitions parameters of the modern human and Neandertal samples are reported in Table S2. A semi-automatic threshold-based segmentation of bone and dental tissues was carried out with Watershed segmentation tool and manual corrections, using Avizo 7.0 software (FEI Visualization Sciences Groupe, VSG), and ImageJ 1.53. Surface rendering was performed using triangulation and constrained smoothing from the volumetric data (marching cube algorithm; Lorensen and Cline 1987).

Absolute and scaled volumes describing 3D tissue proportions were computed for one portion of the radius diaphysis, one portion of the UC root, and for the UC crown. Although epiphyses are under compressive articular loadings (Eschweiler et al. 2022; Rose 1993), some studies have found less bilateral asymmetry in surface area and size of upper limb bone articulations than in the midshaft (Lieberman et al. 2001; Ruff 2019; Trinkaus et al. 1994). A distal portion was therefore virtually cut between 20% and 30% of the total radius length (0% being at the distal end, and 100% at the proximal end), at the boundary between cortical and trabecular bone forming the radius distal end. Furthermore, a bigger portion between 20% and 80% of the total radius length, representing the entire cortical bone portion, was virtually extracted (Fig. 1A). As primary dentinogenesis begins from the future enamel-dentine junction towards the direction of the future pulp cavity (Dean 2017), and that crown formation of UC is completed around 5.5 years (AlQahtani et al. 2010), the
<figure><img src="image_2.png" /><figcaption>**Fig. 1** - Portions (highlighted) used for the measurements of absolute and scaled volumes: A) portions of radius (anterior view) representing 10% to 60% of its total length; B) portions of upper canine (buccal view) representing 40% of the root length, the crown dentine, and the total dentine.</figcaption></figure>

development of crown dentine may be strongly genetically controlled. Conversely, irregular secondary dentine can be secreted around the pulp cavity, and more specifically below the cementoenamel junction (Nudel et al. 2021), in response to environmental stimuli (Dean 2017; Teaford et al. 2000). Moreover, sex difference in the location of secondary dentine formation has been previously described (Zilberman and Smith 2001). It would appear that, within a single tooth, there are local variations in the degree of sensitivity of dental tissues to endogenous and exogenous factors. We therefore decided to select different portions of the UC to conduct this study: the radicular dentine portion between 50% and 90% of the total root length (0% being at the root apex, and 100% at the cervix), as well as the crown dentine portion, virtually extracted following the protocol described in Le Cabec et al. (2013) (Fig. 1B). Furthermore, the total dentine volume (i.e.,

crown and radicular dentine) was extracted (Fig. 1B). We hypothesize that, if there is a signal of cortical bone-dentine quantitative covariation, it should vary according to the dentine portion.

To avoid taking into account for an allometric consequence of change in individual body size in the measured tissue thickness variation, it was necessary to find the appropriate proxy to scale the volumes of cortical bone and dentine. The femoral head breadth, usually defined as the best proxy for body size estimation (McHenry 1992; Ruff et al. 1991), could not be measured in this study due to a lack of available femurs. OLS regression of base-10 logged variables and Spearman correlation tests were used to determine the relationship between tissue volumes and the total radius length (i.e., the maximum length parallel to the longitudinal axis of the radius, following Ruff 2002), and the total dentine length (i.e., the maximum length of the
dentine between the root and crown apices). The length measurement with the highest correlation with the cortical bone and dentine volumes was considered the best proxy for bone/dental size: the cortical bone volumes were therefore scaled to the total length of the radius (r = 0.78), and the dentine volumes were scaled to the total dentine length (r [0.73; 0.82]; Fig. S2).

The following variables were digitally measured or calculated using Avizo 7.0 software (Tab. S3, see also publicly available version on Zenodo; Augoyard et al. 2023): RL, the total radius length (mm); Vcor, the absolute volume of cortical bone for a specific bone portion (mm³); sVcor [= Vcor/RL], the scaled volume of cortical bone for a specific bone portion (mm²); DL, the total dentine length (mm); Ve, the volume of enamel (mm³); sVe [= Ve/DL], the scaled volume of enamel (mm²); Vp tot, the total volume of pulp (mm³); sVp tot [= Vp tot/DL], the scaled total volume of pulp (mm²); Vd tot, the total volume of dentine (mm³); sVd tot [= Vd tot/DL], the scaled total volume of dentine (mm²); Vd, the absolute volume of dentine for a specific dentine portion (mm³); sVd [= Vd/DL], the scaled volume of dentine for a specific dentine portion (mm²). The first author performed the analyses and Wilcoxon rank test for measurement accuracy revealed intra-observer differences less than 1% (see Tab. S4).

Due to small sample sizes, only nonparametric tests were performed. All plots and statistical analyses were computed in R 4.2.2 (R Core Team 2022; the R codes used in this study are available in Supporting Information). In this exploratory study, we interpreted *p*-values lesser than 0.05 as a substantial evidence against the null hypothesis, and *p*-values lesser than 0.10 as a weak to moderate evidence against the null hypothesis.

To compare cortical bone and dentine volumes between age group, sex, and ancestry, pairwise Wilcoxon rank sum tests were performed. These tests were supplemented by 95% confidence intervals (95% CIs) providing ranges of plausible values for the true difference between group means and obtained using a Bca bootstrap method (Zieffler et al. 2011). Boxplots

were computed to provide a visual representation of the median, standard deviation, minimum and maximum values of the variables for each subsample.

OLS regressions and Spearman correlation tests were first performed to get an overview of the relationship between the total volumes of cortical bone and the different dental tissues (i.e., enamel, dentine, pulp). Then, we focused our analyses on the distal part of the radius and on the crown and radicular dentine portions to provide a more thorough evaluation of the local variations in tissue thickness. To test the degree of cortical bone-dentine quantitative covariation through life in the modern sample, Spearman correlation tests were conducted for all immature individuals (n = 12) and for all adult regardless of sex and origin (n = 49). Then, to assess whether the degree of covariation can vary according to the sex, Spearman correlation tests were performed for female (n = 25) and male (n = 25) samples, separately. Finally, to evaluate if the ancestry can influence the degree of cortical bone-dentine covariation, we measured the Spearman correlation coefficients separately in the adults of African (n = 30) and European (n = 19) ancestry. The relationship between two variables was considered strong when their r value was larger than 0.7, moderate for r values between 0.5 and 0.7, and weak for r values between 0.3 and 0.5.

Cortical bone and dentine volumes of the five young adult composite Neandertals were first compared to the mean and to the limits of variation estimated in the entire sample of modern humans, using adjusted Z-score analysis. This test allows the comparison of parameters from small and unbalanced samples to a reference sample, using the inverse Student's t-test (Scolan et al. 2012). To ensure that the results obtained were not biased by the presence of modern individuals with different developmental stages (i.e., juvenile individuals, older adults etc.), the young adult composite Neandertals were secondly compared to chimeras created from modern adults aged 20-29 years. Results of the Wilcoxon rank sum test suggest that the
dentine/cortical bone ratios measured in modern young adult individuals (n = 12) are strictly comparable to those created by the random association of dentine and bone variables in the corresponding modern chimeras (n = 132; Fig. S3). We therefore assumed that the creation of composite individuals is a reliable way to preliminarily assess the bone-dentine relationship in a sparse palaeoanthropological record. The detailed composition of the modern young adult and Neandertal chimeric individuals and their associated cortical bone and dentine variables are presented in Table S5.

## Results

*Comparison of cortical bone and dentine volumes between age, sex, and ancestry*

Descriptive statistics of all absolute volumes of cortical bone and dentine are presented in Table S6 and Figure S4, and compared between subsamples in Table S7. All absolute variables should be considered as indicative only. Descriptive statistics of scaled volumes of cortical bone and dentine are presented in Table 1. The results of the Wilcoxon rank test and the 95% CIs are provided in Table 2.

Statistically reliable differences in scaled volumes of cortical bone were measured between the 9–14 years and the 15–19 years subsamples (*p*-values = 0.01), while no significant difference in the scaled volumes of dentine is noticed between the two immature subsamples (Tab. 2, Fig. 2). The scaled volumes of cortical bone and dentine are statistically different between the 9–14 years individuals and the total adult sample (all *p*-values <0.01; Tab. 2). When splitting the adult sample by ancestry, only the scaled volumes of crown dentine are similar between individuals of age class [9–14y] and the European adults (Fig. 2). Except for the scaled radicular dentine, no statistical differences are noticed in the scaled volumes of cortical bone and crown dentine and their ratios between the 15–19 years and adult samples, when looking at the Wilcoxon *p*-value (Tab. 2, Figs. 2, 3). This difference can

be extended to the volume of crown dentine, according to the 95% CIs (Tab. 2). The slightly skewed distribution in the total adult sample and the presence of several outliers in the 15–19 years and adult samples may explain the H1 acceptance for the 95% CIs that are sensitive to skewness and extreme values, unlike Wilcoxon test. Crown dentine volumes were lower in 15–19y individuals compared to African adults (*p*-value = 0.05), whereas similar volumes are measured between age class [15–19y] and European adults (Fig. 2). All males and females statistically differ in their scaled volumes of cortical bone and dentine (Tab. 2, Fig. 2). To try to better discriminate the impact of sex relative to that of ancestry, we compare the volumes of tissues between males and females of the same ancestry. The results also indicate significant sexual dimorphism in all scaled volumes of cortical bone and dentine both between African males and females, and between European males and females (Tab. 2, Fig. 2). Compared to European adults, the adults of African ancestry have similar cortical bone volumes but higher dentine volumes, mostly due to African males having significantly higher volumes of radicular dentine than any other male and female individuals (Tab. 2, Fig. 2).

*Correlation test performed on scaled volumes of cortical bone and dentine in the modern sample*

The OLS regressions and Spearman correlation tests reveal a moderate but significant relationship between the volume of the entire cortical portion (i.e., sVcor 20-80%) and the total dentine volume in African adults (r = 0.68; r² = 0.49; Fig. 3A). For both the enamel and pulp volumes, correlations with cortical bone were significantly weaker, with values for r coefficients of 0.33 and 0.47 respectively, and r² coefficients of 0.12 (Figs. 3B, 3C).

The Spearman correlation coefficients computed between the cortical bone volume of the distal radius and the crown and radicular dentine volumes in modern human subsamples are provided in Table 3. Differences are observed in the signal of cortical bone-dentine quantitative covariation between immature and adult
<table><caption>Tab. 1 - Mean, range, and standard deviation of scaled volumes of cortical bone and dentine and their ratios, measured in modern humans of different age groups, sexes, and ancestries.</caption><thead><tr><th>AGE GROUP</th><th>N</th><th>SEX</th><th>ANCESTRY</th><th>sVcor 20-30% (mm²)</th><th>sVd 50-90% (mm²)</th><th>sVd crown (mm²)</th><th>sVd 50-90% / sVcor 20-30% / sVd crown / sVcor 20-30%</th></tr></thead><tbody><tr><td rowspan="3">9-14y</td><td rowspan="3">5</td><td rowspan="3">u</td><td>mean</td><td>5.18</td><td>5.53</td><td>5.16</td><td>1.09</td><td>1.01</td></tr><tr><td>range</td><td>4.43-6.22</td><td>4.94-6.16</td><td>4.56-5.63</td><td>0.79-1.33</td><td>0.78-1.27</td></tr><tr><td>(s.d.)</td><td>(0.74)</td><td>(0.54)</td><td>(0.44)</td><td>(0.18)</td><td>(0.20)</td></tr><tr><td rowspan="3">15-19y</td><td rowspan="3">7</td><td rowspan="3">6 u + 1 F</td><td>mean</td><td>7.83</td><td>5.59</td><td>5.86</td><td>0.74</td><td>0.77</td></tr><tr><td>range</td><td>6-9.91</td><td>4.75-6.03</td><td>5.14-6.83</td><td>0.48-0.95</td><td>0.59-1.04</td></tr><tr><td>(s.d.)</td><td>(1.44)</td><td>(0.46)</td><td>(0.64)</td><td>(0.15)</td><td>(0.15)</td></tr><tr><td rowspan="12">Adult</td><td rowspan="3">7</td><td rowspan="3">M</td><td>European</td><td>mean</td><td>10.33</td><td>6.99</td><td>7.19</td><td>0.68</td><td>0.70</td></tr><tr><td>range</td><td>9.15-11.28</td><td>5.45-8.59</td><td>5.79-8.27</td><td>0.48-0.91</td><td>0.51-0.90</td></tr><tr><td>(s.d.)</td><td>(0.88)</td><td>(1.16)</td><td>(0.84)</td><td>(0.14)</td><td>(0.12)</td></tr><tr><td rowspan="3">12</td><td rowspan="3">F</td><td>mean</td><td>7.60</td><td>6.07</td><td>5.54</td><td>0.81</td><td>0.74</td></tr><tr><td>range</td><td>6.34-9.10</td><td>5.18-7.66</td><td>4.81-6.46</td><td>0.59-1.01</td><td>0.56-0.93</td></tr><tr><td>(s.d.)</td><td>(0.92)</td><td>(0.75)</td><td>(0.58)</td><td>(0.12)</td><td>(0.11)</td></tr><tr><td rowspan="3">18</td><td rowspan="3">M</td><td>African</td><td>mean</td><td>9.55</td><td>8.62</td><td>7.90</td><td>0.91</td><td>0.84</td></tr><tr><td>range</td><td>7.56-12.82</td><td>5.51-11.72</td><td>4.60-10.03</td><td>0.71-1.26</td><td>0.57-1.20</td></tr><tr><td>(s.d.)</td><td>(1.31)</td><td>(1.26)</td><td>(1.37)</td><td>(0.17)</td><td>(0.20)</td></tr><tr><td rowspan="3">12</td><td rowspan="3">F</td><td>African</td><td>mean</td><td>6.98</td><td>6.16</td><td>5.80</td><td>0.89</td><td>0.84</td></tr><tr><td>range</td><td>6.05-8.66</td><td>5.27-7.16</td><td>4.94-6.56</td><td>0.74-0.98</td><td>0.65-1.04</td></tr><tr><td>(s.d.)</td><td>(0.73)</td><td>(0.61)</td><td>(0.60)</td><td>(0.08)</td><td>(0.12)</td></tr></tbody></table>

Abbreviations: u, unknown; F, female; M, male; sVcor, the scaled volume of cortical bone for a specific bone portion (mm²); sVd, the scaled volume of dentine for a specific dentine portion (mm²); s.d., standard deviation.

individuals. The first ones present a weak positive correlation between the scaled volume of cortical bone and the scaled volume of crown dentine (r = 0.35, *p*-value = 0.27), whereas moderate positive correlations are observed in the total adult sample (r [0.56; 0.57], *p*-values < 0.01; Tab. 3).

When measuring the volumes of crown and radicular dentine relative to cortical bone volume (i.e., sVd 50-90% / sVcor 20-30% and sVd crown / sVcor 20-30% ratios), the values are significantly higher for the 9-14 years than for the 15-19 years and adult subsamples (Fig. 4).
<table><caption>**Tab. 2** - P-values of the Wilcoxon rank test and 95% CIs, allowing the comparison of scaled volumes of cortical bone and dentine and their ratios between modern samples of different age groups, sexes, and ancestries. A single asterisk indicates a moderately significant difference (0.01< p-values <0.05) and two asterisks indicate highly significant difference (p-values <0.01), according to the Wilcoxon test.</caption><thead><tr><th>SAMPLES PAIR<br>COMPARISON</th><th>N</th><th>p</th><th>sVcor 20-30%<br>(mm²)</th><th>sVd 50-90%<br>(mm²)</th><th>sVd crown<br>(mm²)</th><th>sVd 50-90% /<br>sVcor 20-30%</th><th>sVd crown /<br>sVcor 20-30%</th></tr></thead><tbody><tr><td rowspan="2">9-14y vs.<br>15-19y</td><td rowspan="2">5/7</td><td>p</td><td>0.01*</td><td>1</td><td>0.20</td><td>0.01*</td><td>0.03*</td></tr><tr><td>95% CI</td><td>[-3.75, -1.46]</td><td>[-0.55, 0.52]</td><td>[-1.27, -0.19]</td><td>[0.16, 0.54]</td><td>[0.07, 0.41]</td></tr><tr><td rowspan="2">9-14y vs.<br>Adult</td><td rowspan="2">5/49</td><td>p</td><td>&lt;0.01**</td><td>&lt;0.01**</td><td>&lt;0.01**</td><td>&lt;0.01**</td><td>&lt;0.01**</td></tr><tr><td>95% CI</td><td>[-4.10, -2.65]</td><td>[-2.21, -1.03]</td><td>[-2.10, -1.06]</td><td>[0.07, 0.39]</td><td>[0.08, 0.36]</td></tr><tr><td rowspan="2">15-19y vs.<br>Adult</td><td rowspan="2">7/49</td><td>p</td><td>0.31</td><td>&lt;0.01**</td><td>0.15</td><td>0.11</td><td>0.63</td></tr><tr><td>95% CI</td><td>[-1.84, 0.29]</td><td>[-2.13, -1.08]</td><td>[-1.41, -0.25]</td><td>[-0.23, -0.01]</td><td>[-0.13, 0.08]</td></tr><tr><td rowspan="2">Females vs.<br>Males</td><td rowspan="2">25/25</td><td>p</td><td>&lt;0.01**</td><td>&lt;0.01**</td><td>&lt;0.01**</td><td>0.70</td><td>0.73</td></tr><tr><td>95% CI</td><td>[-3.12, -1.96]</td><td>[-2.66, -1.44]</td><td>[-2.54, -1.45]</td><td>[-0.09, 0.08]</td><td>[-0.10, 0.08]</td></tr><tr><td rowspan="2">Adults AF vs.<br>Adults EU</td><td rowspan="2">30/19</td><td>p</td><td>0.85</td><td>0.01*</td><td>0.04*</td><td>&lt;0.01**</td><td>0.02*</td></tr><tr><td>95% CI</td><td>[-0.98, 0.87]</td><td>[0.51, 1.96]</td><td>[0.19, 1.64]</td><td>[0.07, 0.22]</td><td>[0.04, 0.20]</td></tr><tr><td rowspan="2">Males AF vs.<br>Males EU</td><td rowspan="2">18/7</td><td>p</td><td>0.11</td><td>&lt;0.01**</td><td>0.16</td><td>0.01*</td><td>0.16</td></tr><tr><td>95% CI</td><td>[-0.17, 1.56]</td><td>[-2.55, -0.63]</td><td>[-1.54, 0.15]</td><td>[-0.37, -0.11]</td><td>[-0.26, -0.02]</td></tr><tr><td rowspan="2">Females AF vs.<br>Females EU</td><td rowspan="2">12/13</td><td>p</td><td>0.23</td><td>0.41</td><td>0.31</td><td>0.16</td><td>0.13</td></tr><tr><td>95% CI</td><td>[-1.12, 0.16]</td><td>[-0.43, 0.57]</td><td>[-0.22, 0.67]</td><td>[-0.003, 0.15]</td><td>[-0.03, 0.17]</td></tr><tr><td rowspan="2">Males AF vs.<br>Females AF</td><td rowspan="2">18/12</td><td>p</td><td>&lt;0.01**</td><td>&lt;0.01**</td><td>&lt;0.01**</td><td>0.95</td><td>0.82</td></tr><tr><td>95% CI</td><td>[-3.32, -1.89]</td><td>[-3.12, -1.80]</td><td>[-2.78, -1.37]</td><td>[-0.12, 0.05]</td><td>[-0.12, 0.09]</td></tr><tr><td rowspan="2">Males EU vs.<br>Females EU</td><td rowspan="2">7/13</td><td>p</td><td>&lt;0.01**</td><td>0.05*</td><td>&lt;0.01**</td><td>0.06</td><td>0.38</td></tr><tr><td>95% CI</td><td>[-3.61, -2.06]</td><td>[-1.82, -0.05]</td><td>[-2.22, -0.92]</td><td>[0.01, 0.24]</td><td>[-0.05, 0.18]</td></tr></tbody></table>

Abbreviations: AF, African ancestry; EU, European ancestry; 95% CI, 95% bias-corrected and accelerated bootstrap confidence interval; sVcor, the scaled volume of cortical bone for a specific bone portion (mm²); sVd, the scaled volume of dentine for a specific dentine portion (mm²).

The results obtained from the correlation tests performed for male and female samples, show no clear signal of correlation (Tab. 3). Only a weak positive correlation between cortical bone

and radicular dentine volumes and a weak negative correlation between cortical bone and crown dentine volumes are measured for females and males respectively, although none of the *p*-values
<figure><img src="image_3.png" /><figcaption>**Fig. 2** - Boxplots of the scaled volumes of cortical bone (1st row), radicular, and crown dentine (2nd and 3rd rows, respectively), measured in modern subsamples and compared between age groups (A, B, C), ancestries (D, E, F), and sexes (G, H, I). A single asterisk indicates a moderately significant difference (0.01< p-values <0.05) and two or more asterisks indicate highly significant difference (p-values <0.01), according to the Wilcoxon test. Abbreviations: AF, African ancestry; EU, European ancestry; sVcor, the scaled volume of cortical bone for a specific bone portion (mm²); sVd, the scaled volume of dentine for a specific dentine portion (mm²); ns, non-significant Wilcoxon test p-value.</figcaption></figure>
are significant. These results are different when comparing the cortical bone-dentine covariation signal between the African and European adult samples (Tab. 3). For the first one, there are significant strong and moderate positive correlations between cortical bone volume and radicular and crown dentine volumes, respectively (r [0.70; 0.56]). For the European sample, there are weak to moderate significant positive correlations between the three volumes of tissues (r = 0.44, between cortical bone and radicular dentine, and r = 0.60, between cortical bone and crown dentine; Tab. 3). When looking at the dentine-bone ratios, the adults of African ancestry have higher volumes of crown and radicular dentine relative to cortical bone compared with the European adults. These differences in ratio values are particularly significant between the males of the two ancestries (Fig. 4).

*Comparison of absolute and scaled volumes of cortical bone and dentine and their ratios between the five composite Neandertals and modern humans*

Adjusted Z-score analyses were first performed to provide an overall comparison of cortical bone and dentine volumes between the five Neandertal chimeric individuals and the modern variability (Tab. S8). The results show that the five Neandertal chimeric individuals are within the 95% of variation of the modern human sample for both the absolute and scaled volumes of cortical bone. Regarding the volumes of crown and radicular dentine and the dentine/bone ratios, almost all Neandertal chimeric individuals fall well beyond modern variability. Only the MIS 3 chimeric individual created by the combination of the Spy II radius and the Palomas35 UC fall entirely within the 95% of modern human variation (Tab. S8). Z-scores of cortical bone and dentine variables of the composite Neandertals compared to the modern young adult chimeras are similar to those obtained in comparison with the global modern variability, except for the same MIS 3 chimera for whom only the scaled volume of radicular dentine (sVd 50-90%) is slightly above the 95% upper limit of variation (Fig. 5).

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

**Fig. 3** - Regressions of the total scaled dentine (A), enamel (B), and total pulp (C) volumes against the scaled volume of cortical bone measured between 20% and 80% of the total radius length, among African adults (n = 30). Abbreviations: sVcor, the scaled volume of cortical bone for a specific bone portion (mm²); sVd tot, the scaled total volume of dentine (mm³); sVe, the scaled volume of enamel (mm²); sVp tot, the scaled total volume of pulp (mm³).
<figure><img src="image_7.png" /><figcaption>**Fig. 4** - Boxplots of the radicular dentine-cortical bone and crown dentine-cortical bone ratios (1st and 2nd columns, respectively), measured in modern sub-samples and compared between age groups (A, B), ancestries (C, D), and sexes (E, F). A single asterisk indicates a moderately significant difference (0.01< p-values <0.05) and two or more asterisks indicate highly significant difference (p-values <0.01), according to the Wilcoxon test. Abbreviations: sVcor, the scaled volume of cortical bone for a specific bone portion (mm²); sVd, the scaled volume of dentine for a specific dentine portion (mm²); ns, non-significant Wilcoxon test p-value.</figcaption></figure>
<table><caption>Tab. 3 - Spearman correlation coefficients (r) and their associated p-values, performed on scaled volumes of cortical bone and dentine within modern human samples, according to the age group, sex, and ancestry.</caption><thead><tr><th rowspan="2">SAMPLES</th><th rowspan="2">3D variables</th><th colspan="3">IMMATURES (N = 12)</th><th colspan="3">ALL ADULTS (N = 49)</th></tr><tr><th>sVcor 20-30%</th><th>sVd 50-90%</th><th>sVd crown</th><th>sVcor 20-30%</th><th>sVd 50-90%</th><th>sVd crown</th></tr></thead><tbody><tr><td rowspan="2"></td><td>sVcor 20-30%</td><td>r</td><td>1</td><td>-0.10</td><td>0.35</td><td>1</td><td>0.56</td><td>0.57</td></tr><tr><td>p-value</td><td></td><td></td><td>0.77</td><td>0.27</td><td></td><td>&lt;0.01*</td><td>&lt;0.01*</td></tr><tr><td rowspan="2"></td><td>sVd 50-90%</td><td></td><td>1</td><td>-0.11</td><td></td><td>1</td><td>0.83</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>0.73</td><td></td><td></td><td>&lt;0.01*</td></tr><tr><td></td><td>sVd crown</td><td></td><td></td><td></td><td>1</td><td></td><td></td><td>1</td></tr></tbody></table><table><thead><tr><th rowspan="2">SAMPLES</th><th rowspan="2">3D variables</th><th colspan="3">ALL FEMALES (N = 25)</th><th colspan="3">ALL MALES (N = 25)</th></tr><tr><th>sVcor 20-30%</th><th>sVd 50-90%</th><th>sVd crown</th><th>sVcor 20-30%</th><th>sVd 50-90%</th><th>sVd crown</th></tr></thead><tbody><tr><td rowspan="2"></td><td>sVcor 20-30%</td><td>r</td><td>1</td><td>0.36</td><td>-0.09</td><td>1</td><td>-0.22</td><td>-0.32</td></tr><tr><td>p-value</td><td></td><td></td><td>0.07</td><td>0.66</td><td></td><td>0.28</td><td>0.11</td></tr><tr><td rowspan="2"></td><td>sVd 50-90%</td><td></td><td>1</td><td>0.42</td><td></td><td>1</td><td>0.69</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>0.03*</td><td></td><td></td><td>&lt;0.01*</td></tr><tr><td></td><td>sVd crown</td><td></td><td></td><td></td><td>1</td><td></td><td></td><td>1</td></tr></tbody></table><table><thead><tr><th rowspan="2">SAMPLES</th><th rowspan="2">3D variables</th><th colspan="3">ADULTS AF (N = 30)</th><th colspan="3">ADULTS EU (N = 19)</th></tr><tr><th>sVcor 20-30%</th><th>sVd 50-90%</th><th>sVd crown</th><th>sVcor 20-30%</th><th>sVd 50-90%</th><th>sVd crown</th></tr></thead><tbody><tr><td rowspan="2"></td><td>sVcor 20-30%</td><td>r</td><td>1</td><td>0.70</td><td>0.56</td><td>1</td><td>0.44</td><td>0.60</td></tr><tr><td>p-value</td><td></td><td></td><td>&lt;0.01*</td><td>&lt;0.01*</td><td></td><td>0.06</td><td>&lt;0.01*</td></tr><tr><td rowspan="2"></td><td>sVd 50-90%</td><td></td><td>1</td><td>0.85</td><td></td><td>1</td><td>0.70</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>&lt;0.01*</td><td></td><td></td><td>&lt;0.01*</td></tr><tr><td></td><td>sVd crown</td><td></td><td></td><td></td><td>1</td><td></td><td></td><td>1</td></tr></tbody></table>

Abbreviations: AF, African ancestry; EU, European ancestry; sVcor, the scaled volume of cortical bone for a specific bone portion (mm²); sVd, the scaled volume of dentine for a specific dentine portion (mm²).

Discussion

(Co)variation of cortical bone and dentine volumes over the lifetime

The results obtained from the comparison of cortical bone volumes between age groups highlight an age-related pattern in bone development.

The appositional bone growth in juveniles is characterized by a cortical bone deposition on the periosteal surface, simultaneously with its resorption in the endosteal cavity (Burr and Allen 2019). From adolescence to adult stages, cortical bone deposit takes place at both the endosteal and periosteal levels due to the drastic
<figure><img src="image_8.png" /><figcaption>**Fig. 5** - Adjusted z-scores of the absolute and scaled volumes of cortical bone and dentine and their ratios calculated for the five chimeric Neandertals and compared with the variability of the chimeric modern young adults. Zone defined between dotted lines encompass 95% of modern variability, the zero line represents the mean. Abbreviations: Vcor, the volume of cortical bone for a specific bone portion (mm³); sVcor, the scaled volume of cortical bone for a specific bone portion (mm²); Vd, the volume of dentine for a specific dentine portion (mm³); sVd, the scaled volume of dentine for a specific dentine portion (mm²).</figcaption></figure>

increase in sex steroids (Frisancho et al. 1970). Smaller cortical bone areas were indeed measured in juveniles compared with adolescents and adults (Cambra-Moo et al. 2014; Frisancho et al. 1970). The results of our study support these 2D observations as significant differences in absolute and scaled cortical bone volumes are observed in the age group 9–14 years, compared with adolescents and adults, with values increasing throughout life. The difference in absolute bone volumes between age group 15–19 years and adults, and the similarities in their scaled counterparts may reflect a growth allometry, where differences in absolute bone growth can cover up similarities in relative growth (Buschang 1982).

Surprisingly, even if the upper canines selected for this study have comparable maturational stages (i.e., scores G to H of Demirjian et al. 1973) and low occlusal wear, variation in dentine volumes can be observed between age classes. The slow and regular rate of secondary dentine formation that proceeds at the end of tooth formation (Dean 2017) could be an explanation of this age-related pattern in dentine development: the dentine portions that first reach their dental maturation, as this is the case for crown dentine, will be first subjected to this secondary dentine deposition.

Therefore, we observe a gradual increase in crown dentine volumes between juveniles, adolescents, and adults. Radicular dentine volumes remain significantly lower in immature individuals compared to adults, and no dentine thickening is noted in the 15–19 years compared with 9–14 years age groups. This could suggest that secondary dentine preferentially forms first closer to the crown and does not affect much root dentine volumes between adolescence and early adulthood.

Cortical bone and dentine tissues share similarities in mineral composition, in genetic and hormonal affinities, and respond to the same signaling molecular pathways (Alvesalo 1997; Brown et al. 2018; Burr and Allen 2019; Callewaert et al. 2010; Dean 2017; Jernvall and Thesleff 2012), that are not shared with other dental tissues (i.e., enamel, pulp). We therefore hypothesized that endogenous factors could affect both cortical bone and dentine volumes and could lead to their coordinated variation during development. Especially, we expected to detect a stronger covariation signal in adolescents, for whom the distal epiphysis of the radius is no longer used for alternative locomotion, such as crawling (Colombo et al. 2019). Moreover, these individuals exhibit a shorter
history of exposure to biomechanical factors compared with adults since their radii and upper canines are still under development or have just reached their skeletal and dental maturity (ages for fusion of the distal radius epiphysis range from 14 to 20 years, rev. in Scheuer and Black 2000, and upper canine formation is completed around 15.5 years, AlQahtani et al. 2010). Thus, deviation from the genetic trajectory of tissue development, mainly induced by bone remodeling and secondary dentine deposition, would be negligible. However, the secretion of sex hormones leading to an acceleration in bone growth inherent to the adolescence stage (Bogin 2003) may confound the covariation signal.

The preliminary correlation test performed on an adult subsample confirms the existence of a significantly stronger covariation signal between cortical bone and dentine than between cortical bone and any other dental tissue. However, and contrary to our assumptions, Spearman correlation tests show weak covariation signals between scaled volumes of cortical bone and dentine in immature individuals. When splitting the immature sample by developmental stages, the dentine-bone ratios indicate that, relative to cortical bone, the volumes of crown and radicular dentine are significantly higher in juvenile individuals than in adolescents and adults. Previous studies have also shown a loss of synchronization between bone and dental developments in individuals younger than twelve years of age, resulting in differences in age estimation from bone or dental elements (Conceição and Cardoso 2011; Lewis 1991). Similar statements have been made from hominin juvenile remains (Cazenave et al. 2020; Dean and Smith 2009). This developmental disjunction could be the result of a great sensitivity of skeletal development to environmental insults, and notably to the socioeconomic condition of individuals (Cardoso 2007; Conceição and Cardoso 2011). It has also been shown that individuals may experience accelerated dental eruption in relation to early life stress (McDermott et al. 2021). In this study, adolescents and adults do not show significant differences in dentine-bone ratios, although

comparison tests highlighted significant differences in dentine volumes between the two subsamples, and especially between the adolescents and the African adults. This could be evidence of comparable dentine-bone relationship from the adolescent stage through adulthood. While the small size of the immature subsamples prevents any firm conclusions regarding covariation, the forty-nine adults show significant moderate correlations between the volumes of cortical bone at distal-radius level and crown and radicular dente, suggesting a low impact of mechanical loading on the development of these portions, even in adulthood.

### Influence of sex and ancestry on the volumes of cortical bone and dentine and their covariation

Significantly greater absolute and scaled volumes of cortical bone are measured in males compared with females. To what extent this difference is the result of sexual dimorphism solely and/or is linked to the impact of various functional loads on the distal arm due to sex-related activities remains to be understood. The distal part of the radius is sensitive to age-related osteopenia resulting in the increment of cortical porosity in response to estrogen deficiency at menopause (Milovanovic et al. 2015; Nishiyama et al. 2010). However, this physiological disturbance likely has a limited influence on our measurements since the female sample is mainly composed of premenopausal women. As previously measured (Feeney et al. 2010; García-Campos et al. 2018; Sorenti et al. 2019; Zilberman and Smith 2001), males have significantly greater area and absolute volumes of crown dentine than females. Our study confirms these observations and, at least for the upper permanent canine, extends them to the radicular dente. Hormonal and chromosomal differences between sexes may explain the dimorphism in odontoblast activity, both at crown level (Alvesalo 1997; Zilberman and Smith 2001), but also at radicular level as supported by our results. These differences in both absolute and scaled volumes of dente are particularly significant between females and males of African ancestry, where the distribution
of the former fell below the 25th percentile of
the African male variation. These results high-
light the importance of considering crown and
radicular dentine volumes in sex determination
(García-Campos et al. 2018; García-Campos
et al. 2020; Sorenti et al. 2019), which may be
particularly useful in forensic or archaeological
context where human remains are fragmentary.
Splitting the adult sample according to sex, no
clear covariation signal is measured between
scaled volumes of cortical bone and dentine.
However, the similarity in dentine-bone ratios
between females and males may therefore suggest
a comparable relationship between these two tis-
sues between sexes.

Unlike other research showing a correla-
tion between variation in bone robustness and
the ecogeographic regions (Bernal et al. 2006;
Ruff 1994), we found no significant difference
in absolute and relative volumes of cortical bone
between the African and European samples.
Direct comparison of activity level between the
two adult subsamples is difficult as little infor-
mation is available for the French sample. Even
if the African males selected in this study were
mostly migrant manual laborers (L'Abbé et al.
2021), we expected higher bone thickness in
pre-industrial compared to post-industrial sam-
ples (French medieval vs. contemporary South
African populations, respectively). The similarity
in cortical bone volumes between samples could
also highlight the lesser effect of mechanical
loading on the distal portion of the radius.

Geographic variation in dental morphology
has been widely described (Bernal et al. 2010;
Irish 2016; rev. in Pilloud et al. 2016; rev. in Scott
and Turner 1997). Especially, Esan and Schepartz
(2018) showed advanced timing of tooth for-
mation and dental emergence in children of
African ancestry compared with European ones,
which could lead to bigger tooth dimensions in
African adults (Harris and Lease 2005; Pilloud
et al. 2014). However, in the present study, the
African adults have higher dentine volumes than
the European ones only because of African males
having much thicker dentine than any other sub-
sample. This result is difficult to interpret since

it may reflect between-groups environmental dif-
ferences (such as masticatory behavior), and/or
genetic diversity. Unlike what we observed in the
male and female subsamples, moderate to strong
correlation coefficients were measured for adults
of European and African ancestry, respectively.
The latter also have higher values of dentine-bone
ratios, due to statistically higher dentine volumes
for comparable cortical bone volumes between
the two adult subsamples. Hence, it seems that
discriminating samples by ancestry rather than by
sex is a better way to access the covariation signal
observed in the overall adult sample.

Discussion on the first application of this study to
evolutionary anthropology

Previous studies that have measured the dentine volume in Neandertals highlighted an overall thickening of this tissue compared to modern humans (Buti et al. 2017; Genochio et al. 2019; Kupczik and Hublin 2010; Le Cabec et al. 2013), variously interpreted as the influence of a host of endogenous and exogenous factors. This study partially confirmed these observations as greater absolute and scaled dentine volumes were measured in the three MIS 5 chimeric Neandertals and in the MIS 3 chimeric individual created by the combination of the Spy II radius and the Vi. 12.5 upper canine. For the SpyII/Palomas35 MIS 3 chimeric individual, however, dentine variables entirely fall within the modern variability. Although the permanent dentition of individuals from Palomas falls within Neandertal variability (Bayle et al. 2017), some of their mandible and dental characteristics extend the previously known ranges of variation observed in this taxon (Bayle et al. 2017; Walker et al. 2008). According to Walker et al. (2008), the morphological variability of the individuals from this site is much higher than what has been observed in other Neandertal populations. Previous studies support ecogeographical and chronological variability across Neandertal populations (Caramelli et al. 2006; Rosas et al. 2006; Walker et al. 2018). A strong interpopulation variation in dentine thickness between MIS 5 and MIS 3 Neandertals also emerges from our results.
Numerous studies showed greater cortical robustness in the long bones of Neandertals compared with modern humans (Churchill 1998; Trinkaus and Ruff 2012; Trinkaus et al. 1999), variously interpreted as a direct adaptation to cold climates (De Groote 2011; Trinkaus 1981), and/or the consequences of mechanical loading imposed by activity (Churchill 1998; Pearson 2000). In the present study, the absolute and scaled volumes of cortical bone measured on the radius were statistically identical between the modern young adult and the Neandertal chimeras. Pearson (2000) demonstrated higher robusticity indices of the lower limb in MIS 4 and MIS 3 Neandertals compared with recent humans, but significantly lower indices for the forelimb. Other studies confirmed the relative gracility of the radius in Neandertal that falls within the range of variation of recent human samples (Hambücken 1995; Vandermeersch and Trinkaus 1995), as supported by our results.

Analysis of the ratios of tissue volumes showed a similar bone-dentine relationship pattern between MIS 3 SpyII/Palomas35 Neandertal and modern humans, while MIS 5 chimeric individuals have higher dentine relative to cortical bone volumes. Comparing these tissue ratios between immature Neandertals where tissue development is more ontogenetically constrained and older adult specimens, would allow us to have a more precise information on the variation of this bone-dentine relationship through ontogeny in Neandertals. Indeed, the loss of synchronization of bone and tooth development observed in modern humans (Conceição and Cardoso 2011; Lewis 1991) and supported by our results, may also affect other hominin taxa (Cazenave et al. 2020; Dean and Smith 2009). The increasing number of discoveries associating hominin cranio-dental and postcranial remains, notably radii (e.g., Berger et al. 2010; Clarke 1998; Détroit et al. 2019; Morwood et al. 2005), will be of great relevance to question the timing of bone and dental developments in other hominin taxa than modern humans.

## Conclusion

Through this exploratory study, we investigated the age-, sex-, and ancestry-related variation in cortical bone and dentine volumes in modern humans. Moreover, we questioned the relationship between these two tissues, both in modern humans and in Neandertals.

By quantifying absolute and scaled volumes of biological hard tissues measured on distal radius portions, likely to be less sensitive to mechanical loadings than other long bones, and on upper canines with a low wear degree, the objective was to better assess the genetic basis of tissue development. Our results first indicate ontogenic differences in cortical bone and dentine developments, as well as strong sexual dimorphism in tissue formation. This last result confirms the reliability of using dental and bony elements for sex determination in archaeological populations. The developmental pattern of dentine also seems to vary according to individual's ancestry. To what extent this population difference is the result of between-groups genetic diversity solely and/or can reflect differences in mechanical behaviors remains to be further investigated.

The preliminary results have outlined a covariation trend between volumes of cortical bone and dentine that does not exist with enamel nor pulp. However, this covariation pattern is more complex than anticipated as no covariation signal is measured in the immature sample and, only when splitting the adult sample by ancestry a strong covariation signal is found. The difference in covariation strength measured in the adult sample according to dentine portion suggested that, along the upper permanent canine, crown and radicular dentine tissues may not have the same degree of sensibility to environmental stimuli. Thus, accessing the genetic basis of morphological variation may be compromised by the impact of environmental factors, particularly mechanical ones, which affect tissue developments throughout life.

It is challenging to understand the phenotypic variation in humans. Another complementary approach to document the local variation
of cortical bone and dentine volumes along the bone shaft and the dental remains would be to investigate their local topographic distribution using morphometric maps (for previous works that studied bone and/or dentine distribution independently based on this approach, see Bayle et al. 2010; Bondioli et al. 2010; Cazenave et al. 2017; Profico et al. 2021; Puymerail et al. 2012; Zanolli et al. 2018).

The study of cortical bone-dentine covariation from modern humans of known biological (e.g., age, sex, pathology, etc.) and cultural (e.g., profession, sport practice, etc.) parameters, could shed light on the pattern of joint tissue thickening observed in the palaeoanthropological record, and especially in the majority of Neandertal specimens. Additional analyses must be conducted on a larger modern sample covering a wide range of developmental stages, eco-geographic origins, activity level, and composed of bones and teeth with different degrees of sensitivity to biomechanical forces, to be able to better decipher the (onto)genetic and functional factors acting on tissue development. Ultimately, it could allow the better understanding of the factors that rule the evolution of bone and dental tissues through the evolution of hominins.

## Data sharing

The data that support the findings of this study are openly available in Zenodo at https://doi.org/10.5281/zenodo.6341546

## Acknowledgements

Research supported by the "Ministère de l'Enseignement supérieur et de la Recherche" (2021-24; doctoral grant received by M. Augoyard, University of Bordeaux), the CNRS, the "Investments for the future" program IdEx Bordeaux (3Dent'in project, ANR-10-IDEX-03-02, coord.: P. Bayle; STEP ITAP project, OPE-2018-0181, coord.: P. Bayle and J.-G. Bordes), the Région Nouvelle-Aquitaine (NATCH project, 2016-1R40204,

coord.: J.-Ph. Faivre), the Département des Sciences Archéologiques de Bordeaux, and the Bakeng se Afrika project 597924-EPP-1-2018-1-ZA-EPPKA2-CBHE-JP, an Erasmus plus project of the EU (coord.: E. L'Abbé). This research benefited from the scientific framework of the University of Bordeaux's IdEx "Investments for the Future" program / GPR "Human Past". For the South African sample, ethical approval was obtained from the Sefako Makgatho University Research Ethics Committee (SMUREC) and from the Faculty of Health Sciences Research Ethics Committee of the University of Pretoria (ref. no. 685/2021). MicroCT data used in this study were produced through the microCT facilities of the ISIS-4D, PLACAMAT, and MRI, LabEx CeMEB platforms for the French specimens, and Necsa platform for the South African specimens. For access to the specimens and technical support, we thank C. Beauval, D. Castex, NL Japhta, F. de Beer, E. de Jager, A. Masse, P. Semal, N. Vanderesse, and R. Ledevin. We are very grateful to R. Macchiarelli, A. Colombo, C. Ruff, and C. Dean for helpful discussions. We want to thank the editor of the JASs and the two anonymous reviewers for their comments and suggestions which greatly improved this manuscript.

## Authors contribution

MA, CZ, and PB designed the research and wrote the original draft. MA, CZ, FS, PB performed the formal analyses. FS, ACO, ENL, MLL, MC, TC, JH, AP provided microCT resources and methodological support. All authors read, provided feedback, and approved the manuscript.

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