Following the methodology proposed in Cambra-Moo et al., 2012 and Cambra-Moo et al., 2014, in which we analysed sections of tibia and humeri midshafts at different ontogenetic stages, we selected three complete human crania from subjects of different ages (child, adolescent and young adult; Fig. 1), and we analysed the histology of selected areas of the frontal, parietal and occipital bones. The skeletal remains are part of the ossuary excavated in the Santa María de la Soledad mediaeval church that located in Almansa (Castilla-La Mancha, Spain), which was utilised as a cemetery from the 12th to 18th centuries. From each individual, we extracted three different samples from the vault bones, avoiding areas of muscle attachments (Fig. 1A). Before extracting the blocks, we proceeded to photograph and measure the bones of each cranium to preserve the maximum information from each individual. Due to the nature of the deposit (i.e., an ossuary in which the crania were recovered disarticulated), it was not possible to recognize the mandible of the postcranial skeletal elements. Therefore, each individual was aged based on teeth (i.e., the maxilla) development recommendations (AlQahtani et al., 2010, Buikstra and Ubelaker, 1994 and Ubelaker, 1978) and Brothwell's methodology (Brothwell, 1981) for surface macrowear of the molars in the case of the young adult individual. Thus, the three crania were categorised as follow: child (5.5 to 6.5 years old), adolescent (16.5 to 17.5 years old) and young adult (25 to 35 years old).

Using a manual trimmer, a 2 × 1.5 cm² bone sample was extracted from the middle area of the cranial bones (Fig. 1B). Then, each block was embedded in a solution composed of two components, a transparent resin (EpoFix Resin) and a catalyst (EpoFix hardener) (Fig. 1C). Using a vacuum pump to prevent the formation of bubbles, all bone cavities were completely filled with the mixture. Then, a total of 9 thin sections were obtained from the three individuals, which were cut using a circular diamond ISOMET low-speed saw (Buehler, Lake Bluff. Illinois, USA). After a fine polish with silicon carbide (SiC) sandpaper (4000 grit) using a manual polisher (Metaserv 3000, Buehler, Düsseldorf, Germany), thin sections were fixed on microscope slides with a mounting adhesive (Microtec Epoxy Adhesive, Resin Technology Group, Massachusetts). The resulting slides were later progressively polished using different grits to reach a final thickness of 100 μm. Finally, each thin section was observed and photographed using a linearly polarised light microscope (BX61 microscope equipped with a DP70 camera, both from Olympus, Hamburg, Germany), and a complete high-resolution photomontage was assembled using Photoshop CS5 software (Adobe Systems, San José, USA) (Fig. 1D).

Lastly, we mapped each section using geographical information systems (GIS) software (ArGIS 9.3, Esri, Redlands, USA) (Fig. 1E). The GIS software allowed us to finely draw each structure in the 9 thin sections to automatically calculate its size and spatial distribution and to provide qualitative descriptions regarding its nature (see details in Cambra-Moo et al., 2012 and Cambra-Moo et al., 2014). In this study, we drew the mineralised area (MA) of the section and the non-mineralised spaces found in each section (diploe cavities, primary osteons-PO, resorption spaces-RS and secondary osteons-SO), which were used to study the vascularisation (VASC) during the compartmentalisation analysis. In addition, we measured the thickness of the thin sections of all the individuals (five measurements were made for each bone section, see the thickness mean and standard deviation values plotted in Fig. 3).

After mapping the thin sections, histological and compartmentalisation analyses were carried out for the three bones of the three individuals.

Enlow (1963) was one of the first to investigate the histology of cranial bones. His work not only described the most important aspects of skull histology, but he also postulated a complete model for the growth of the skull bones (Enlow, 1968 and de Ricqlès, 2007). Despite this revolutionary contribution and many other notable histological studies that have been carried out on the cranial bones (mostly focused in the facial growth; Martínez-Maza, 2007), there is limited standardised data regarding the histology of cranium cross-sections that include tissue types descriptions (Cho and Hwang, 2012 and Trammell, 2012), and many histological variations that occur during cranial ontogeny and evolution remain poorly understood (Bartsiokas, 2002).

This study, at this phase, represents an introductory approximation of the histomorphological variations of vault bones, and despite the study explores a small number of individuals (which are not representative of the different age classes), from a late mediaeval ossuary (skeletal remains appeared disarticulated) in which intra- or inter-population differences were not under control (sex and/or dietary differences of the individuals were not possible to establish), the histological descriptions and the analysis of the tissue compartmentalisation of the three individuals viewed, made on an ontogenetic perspective, has revealed interesting preliminary results. First, it is important to highlight that, as has been largely described before (Scheuer and Black, 2000), bone section thickness increases with age (see the mean thickness variations in the lines plots of Fig. 3), and the occipital bone exhibits the large disparity in thickness. Second, the enlargement of the vault bones is critical until the end of the childhood (vault bones reach their final extension). Afterwards (during youth), the increasing bones thickness becomes more relevant (see the proposal to describe the main changes in cranium growth, Fig. 4). Third, these changes that describe the size increase are accompanied by important modifications in the histology and compartmentalisation of vault bone tissues (Fig. 3).

Regarding the child, the presence of an important mineralised area occupied by avascular lamellar bone (LB, mean extension: 32.37% of the total mineralised area [MA]; Fig. 3) is a priori unexpected. In most mammals, this bone tissue type is usually related to low rates of bone deposition (in adult individuals), which does not match the rapid growth rates described during the human infancy and childhood (Currey, 2002 and Scheuer and Black, 2000). However, the presence of an avascular LB in an individual approximately 6 years old could be easily assumable because, at this age, the complete cranium size is almost reached (90% of the brain size is reached by the 6th year, Aguila and Enlow, 1998 and Scheuer and Black, 2000). In addition, the vault bones have almost reached their final extension and may begin to increase in thickness (to reach adult values), which could imply a change in the growth rate while the growth rate on other bones of the cranium remains high (Lieberman, 2011). From the foetus phase to early infancy, when the cranium bones are not yet fused, the vault bones are highly porous in their macroscopic appearance (Scheuer and Black, 2000), and it is possible that they are configured by cancellous trabecular tissue in which the diploe cavities contact the ectocranial and endocranial membranes (highly vascularised bone that is considered embryonic-like, de Ricqlès et al., 1999). The presence of the avascular LB in the child could indicate that vault bones become “sealed”, minimizing their porosity, while simultaneously increasing their size. The data of the histological and GIS analysis are summarised in the upper row in Fig. 4, and a schematic of our proposal for the main growth changes in the cranium development is shown in the bottom row of Fig. 4. We speculate if this “sealing” process could be implicated in improving the fine control of the brain-head thermoregulation until it reaches its final maturation, which could be associated with decreasing the “temperature shielding length” from childhood to adulthood, as described by Zhu et al. (2006). However, we observed a large area of LB in the child frontal bone, and we also observed that the same LB began to remodel in the parietal and occipital bones (we identified SO and RS, Fig. 2A2–A4). Moreover, all of the individuals presented a similar pattern in which the VASC increased from the frontal to occipital bones (see Fig. 2A5–A7, B5–B7, C5–C7 and Fig. 3). Both characteristics are in accordance with the idea that the bone microstructure reproduces the preferential sense of growth in the vault bones, as described in the literature (occipital comes from the Latin occipio that means to begin; Scheuer and Black, 2000), which seems to be initiated from the posterior side of the head (see the black curved arrow in the bottom row of Fig. 4).

In contrast, the adolescent and young adult presented a different pattern. In the adolescent, the MA occupied a larger area of the bone section (mean value: 85.39%) and the diploe appeared markedly reduced (the VASC mean value was under 20%, Fig. 3). The MA was occupied by a highly remodelled fibrolamellar bone (FLB), which presented a convoluted-like structure in the external table (Fig. 2B4), and is partially constituted by LB (also highly remodelled) in the internal tables. The high values of the MA in the adolescent are in accordance with the maximum of mineralisation ascribed to cortical bone during youth (the largest bone robustness in the long bones) when the final cortex size is reached (Cambra-Moo et al., 2014, Ruff and Jones, 1981 and Ruff et al., 1994) (Fig. 3). In addition, as has been previously described in other studies, the number of secondary osteons (SO) increased during youth compared to infancy and childhood, and the SOs were small in size (Curtis and Nawrocki, 2010). Finally, the young adult presented the largest VASC of the three crania (mean value: 38.87%), which was highest in the occipital bone (similar to the child). The LB appeared extremely reduced and was only clearly visible in the external table of the frontal bone and in the internal table of the parietal bone (Fig. 2C1 and C2). The most striking result regarding the young adult was the existence of several fine canals by which capillaries could contact the ectocranial membrane (black arrows in Fig. 2C1–C4 and the upper row in Fig. 4). As previously proposed in the literature, cerebral blood flow increases during ontogeny, and therefore, the irregular appearance of LB and the presence of these fine canals in several areas of the vault bones could be related to local changes in heat exchange through the scalp (Zhu et al., 2006 and Zhu et al., 2009). Future investigations should analyse the possible relationships between these changes in the adult cortex remodelling process and the local variations in the endocranial and ectocranial surface remodelling areas that have been described by many authors (e.g., Kranioti et al., 2009, Martínez-Maza et al., 2006 and Mowbray, 2005). Our proposal establishes that during maturity, the microstructure of vault bones continues to change locally throughout the remodelling process. It is of interest to assess if these changes are related with heat dissipation through the vault bones during head thermoregulation (Bruner et al., 2011, Hershkovitz et al., 1999 and Jivraj et al., 2009).

The tissue type characterisation via the histological analysis using thin sections could be crucial in achieving not only a better understanding of the morphological variations of cranial bones but also in understanding the variations in the physiological regime during human ontogeny and human evolution. Through the histological analyses, we can describe both the changes in the size and shape of bones structures during ontogeny and the maturation of the different tissues. Additional individuals must be analysed, and the data of the biological characteristics of each individual must be improved to infer the biological aspects involved in ontogenetic variations. Notwithstanding, this study represents the first attempt to approximate the histomorphological variations in the cranium vault based on an integrative perspective. Further studies are necessary to explore more sections from each bone and additional bones from other locations in the cranium.

Analyses of the morphological variations in the cranial bones are important in anthropological studies. Histological analyses of cranium bones could contribute to a better understanding of these variations, not only from a developmental perspective but also regarding cranium evolution. The present preliminary approach analysed the histology of the frontal, parietal and occipital bones of three individuals with different ages (child, adolescent and young adult). The presence of different types of bone and the differential spatial distribution of the bone tissues during growth revealed that cranium development is a complex process. We found that child vault bones become larger during childhood and that at the same time they are sealed by an avascular lamellar bone (decreasing their porosity); adolescent bones increase in thickness (reaching almost adult size) and largely reduce in their vascularity; and adult bones become completely remodelled and interconnected with the ectocranial surface via fine vascularisation.

A deep understanding of these transformations during human ontogeny will allow us to analyse the complex processes of bone diseases and better understand the histological variations among different human groups or variations due to sex-age differentiation. Further studies are necessary to unveil important aspects of the histology of cranial vault bones that remains unresolved.