Bone Matrix

Serial sections (8 semi-serial sections of each maxilla, with a 5 μm thickness for each section) were obtained using a microtome (Leica RM2255, Germany) and stained with H.E. (hematoxylin and eosin). Morphometric measurements were performed by a single calibrated investigator with a binocular light microscope (Olympus Optical Co., Tokyo, Japan) using a 100x immersion objective and a Zeiss kpl 8X eyepiece containing a Zeiss II integration grid (Carl Zeiss Jena GmbH, Jena, Germany) with 10 parallel lines and 100 points in a quadrangular area. The grid image was successively superimposed on approximately 13 histological fields per histological section, comprised of all tooth sockets from the coronal limit adjacent to the gingival epithelium until the lower apical limit. For each animal/socket, sections from the coronal, medial and apical thirds were evaluated. In the morphometric analysis, points were counted coinciding with the images of the following components of the alveolar socket: clot, inflammatory cells, blood vessels, fibroblasts, collagen fibers, bone matrix, osteoblasts, osteoclasts and other components (empty space left by the inflammatory exudate or intercellular liquid and bone marrow); similar to previous descriptions [5 (link), 36 (link)–38 (link)]. The results are presented as the volume density (mean) for each evaluated structure.

Live staining of mineralized bone matrix was done by immersing medaka hatchlings (12 to 18 dpf) in either Alizarin Complexone solution (ALC; 0.1% in fish medium, Sigma A3882) for two hours or in a Calcein solution (0.01% in fish medium; Sigma C0875) for one hour in the dark at room temperature (RT). Stained hatchlings were rinsed with fish medium three times (15 mins per rinse, at RT) and mounted in 1.5% low melting agarose on a glass bottom petri dish for live fluorescence imaging (488 nm laser/GFP filter for Calcein; 568 nm laser/mCherry filter for ALC).

The above-mentioned remains were inside chamber O of the Ca’ Granda crypt. The access to the chamber was only possible from an opening in the ceiling and the manhole had remained sealed until the first opening. At that time, the archeologists accessed the chamber equipped with personal protective equipment to perform the first site inspection. After the preliminary inspection, the first excavation campaign begun. The remains were retrieved by the archaeologists, equipped with personal protective gear and gloves, under the supervision of the toxicologists. The crania, together with their preserved brain tissue, were placed in sterilized and sealed jars and left inside the Ca’ Granda crypt to maintain the same environmental conditions of the sepulcher chamber until the time of analysis. The post-cranial bones associated to the cranium C2 were also collected together with the cranium and placed in a separate sterilized box, in order to permit the anthropologists to study the entire skeleton. The samplings performed for each investigation (toxicological, radiocarbon, and histological investigations) were performed with sterilized scalpel and handsaw.
Well-preserved brain tissue samples were collected with a scalpel. The sample site was determined based on the consistency of the samples, choosing the best preserved area, as no specific zone of sampling is suggested for forensic toxicology specimen collection in such cases^{3 (link)}. For histological investigation, the section of sampling was selected based on the presence of well-preserved and well-visible encephalic convolutions, hypothesizing more preserved structures. For histological investigations three samples collected from the frontal, parietal and occipital brain area were selected.
The bone sample for the radiocarbon investigation was collected based on its weight and preservation, indeed the sample should weight about 5 g and be well-preserved with the cortical bone not affected by diagenesis or taphonomic processes. For toxicological investigations, the cranial sample was collected considering that the cranium was the only bone detected in the individuals under investigation apart from C2 that was associated with post-cranial bone samples. However, to perform a standardized sampling, we decided to collect only cranial samples, even if other bones were present (see post-cranial bone of C2). Moreover, the cranial sample can be considered a good bone matrix in forensic toxicology considering the excellent results obtained from previous studies that compared them with other bone sample sites^{5 (link),20} . The crania were cut with a sterilized handsaw on the occipital bone adjacent to the foramen magnum, and in cases where the foramen magnum was not preserved the parietal bone, in accordance with previous papers^{5 (link),20} .