Micro-CT scan

High-resolution micro-CT images of the Villabruna upper and lower dentition were obtained with a BIR Actis5 microtomographic system (Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany) using the following scan parameters: 130 kV, 100 μA, with 0.50 mm Brass filter. Volume data were reconstructed using isometric voxel length of 30 μm. The micro-CT images of the teeth were virtually segmented using a semiautomatic threshold-based approach in Avizo 7 (Visualization Sciences Group Inc.) both to reconstruct a complete 3D virtual model of the Villabruna dentition and to evaluate the extension of the carious lesion in the RM 3 (Fig. 1C).

Reconstruction of physiological occlusal relationship

The functional reconstruction of the Villabruna dentition follows indications provided by Benazzi et al.36 and Kullmer et al.37. In detail, the upper and lower dentition of the Villabruna specimen was reproduced with high-resolution epoxy and dental stone casts38. Moreover, digital surface data of the dentition was acquired with a white light 3D digitisation system (smartSCAN3D, Breuckmann GmbH, Germany), with an average resolutions of ~65 μm.

The casts of the upper and lower third molars were used to draw two-dimensional maps of all complementary wear facet pairs on the occlusal surface after their identification with a binocular (Leitz MZ12). In addition, the facets were interactively marked on the virtual models using PolyWorks® 12.0 software (InnovMetric Software Inc., Canada)39.

The facets were labelled applying the numbering system of Maier and Schneck39 and colour-coding in the facet maps follows39,40,41. The application of the dental occlusal compass determines relative occlusal movements for each individual wear facet pair. The point of maximum intercuspation (centric occlusion) marks the start point of directions of movements for the standardised colour-coding in OFA39. Blue coloured facets indicate occlusal contacts during latero- and lateroretrusive movements, yellow specifies lateroprotrusion facets, green shows medioretrusive movements, red and black indicate retrusion and protrusion, respectively (Fig. 2). The facet maps are used to identify directions of occlusal movements41,42 and support setup of the condyle boxes of the dental articulator.

For physiological repositioning of the teeth we aligned casts of each tooth crown in a dental articulator system (PROTAR, KaVo Dental GmbH, Germany). With a dental articulator system it is possible to reproduce natural occlusal movements, while macroscopically observe the contact situations36,37. The epoxy cast specimens were positioned in the articulator after taking its lower jaw dimensions (general geometry, condylar axis position, occlusal and the mid-sagittal plane) from a 3D-print of the complete lower jaw (data from micro-CT).

After positioning of the mandibular resin dentition in the articulator, the maxilla epoxy cast was positioned with the best-fit occlusal relationship possible. Once the initial position is setup, the epoxy casts are replaced with dental stone copies on a wax basis. A slight distortion in the original specimen prevents a proper occlusion. Therefore we used dental stone copies, which can be easily cut at the interproximal planes that each crown can be repositioned independently. Both arches were mounted with dental gypsum between duett-plates and montage-plates (Baumann Dental GmbH) in the articulator. All crowns were then removed from the arches bases.

The upper and lower right M3s of the right and left sides were repositioned first. Based on their occlusal fingerprint (wear facet pattern) they provide important occlusal precision for matching the antagonistic pairs. When the third molar pairs are positioned in maximum intercuspation, we set up the articulator condyle boxes to constrain possible articulator movements for the individual occlusal simulation. Subsequently the antagonistic occlusal pairs can be restored in their dental arches in the same way. The articulator allows testing of the occlusal position of each repositioned tooth pair to ensure consistency of functional movements throughout the tooth rows in accordance with the colour-coded occlusal compass. The anterior dentition of Villabruna was reconstructed last, because it does not show any contact in maximum intercuspation of the dental arches.

Virtual Occlusal Fingerprint Analysis (OFA)

Virtual Occlusal Fingerprint Analysis was applied to evaluate the physiological occlusal movements and crown contacts. The upper and lower jaw digital models were aligned with a virtual model generated from a surface scan from the physical reconstruction of the dental arches in maximum intercuspation (Supplementary Fig. S2), using a best-fit algorithm in IMInspect module of PolyWorks® 12.0.

We verified the kinematics of the occlusal movements (i.e., the pathway of incursive and excursive movements) applying the “Occlusal Fingerprint Analyser” (OFA) software. The OFA software is a virtual tool developed at the Senckenberg Research Institute in Frankfurt (Germany) to detect relief guided dental collisions of antagonistic tooth crowns43,44,45. The OFA software records the occlusal pathways and sequential surface contacts derived from collision detection, deflection and breakfree algorithms (Supplementary Fig. S3; Supplementary Videos 1,2).

Experimental replication of striations

Test 1

Experimental scratching/levering activities were carried out on the enamel surface of three recently extracted lower M 3 s to test three kinds of point tools (Supplementary Table S1): wood point (Supplementary Fig. S6C), bone point (Supplementary Fig. S7C) and microlithic point (Supplementary Fig. S8C). These tools were produced by Matteo Romandini e Rossella Duches (University of Ferrara). The wood point was produced on Larix decidua, as coniferous trees dominated the landscape during the period of the burial. The bone point was obtained from the diaphysis of a large size ungulate and is comparable to bone points found in the burial kit (Supplementary Fig. S1). The microlithic point was made by direct retouching of bladelets extracted from red and grey flint cores, comparable to those exploited by the Epigravettian settlers at Riparo Villabruna.

The same force was applied during the tests. The breakage of the point defined the end of the experiment (Supplementary Table S1).

Test 2

Experimental tests were carried out using microgravette Epigravettian points on six medieval carious human molars (Supplementary Table S2) collected from the Department of Cultural Heritage (University of Bologna, Italy). Different forces were applied in relation to dentine exposure and several parameters were evaluated, such as the type of tool used (tool shape efficacy), actions, directions, inclinations and duration of treatment (Supplementary Table S2).

Analysis of the cross-sectional geometry

The Villabruna RM 3 and the six archaeological human molars used for the experimental tests were analysed using a Hirox Digital Microscope KH-7700 with an MXG-10C body, OL-140II and OL-700II lenses and an AD-10S Directional Lighting Adapter. This portable instrument, housed at the University of Siena, provides a 3D composite image through the overlapping of a series of pictures taken at different focus levels. It enables us to observe the cross section of grooves and to collect metrical parameters46,47, as recently shown for the study of archaeological cutmarks and interproximal grooves on human teeth13,47,48. The following metrical parameters were collected: DC (depth of cut), BT (breadth at the top), BF (breadth at the floor) and RTF (ratio between the breadth at the top and the breadth at the floor of cut).

Three striations within the Villabruna RM 3 cavity were analysed. Two are located in the region 2 and one in the region 5 (Fig. 3). The striations are shallow (DC is less than 4 μm) and narrow (BT between 2.5 and 17.5 μm) and cross sections are V-shaped (Supplementary Fig. S4A,B,C). This characteristic is quantified by the high value of ratio between breadth at the top and breadth at the floor of grooves (RTF comprised between 6.3 and 8.3).

Experimental grooves inflicted by the use of Epigravettian points on exposed dentine are V-shaped, with RTF ranging from 5.2 and 13.1 (n = 6) (Supplementary Fig. S9), resembling those observed in the Villabruna RM 3 .

Gas Chromatography-Mass Spectrometry (GC-MS)

Beeswax has already been identified in ancient therapeutic dental practices11 and could potentially have been used in the case of the Villabruna individual. Another related possibility which has antibacterial and antifungal properties is propolis, a sticky material that honeybees collect from living plants, mix with wax and use to construct and repair their hives49. The chemical composition of both beeswax and propolis are known49,50 and characterisation can be carried out using organic residue analysis (ORA).

Sampling was carried out by CDS at the Max Planck Institute for Evolutionary Anthropology, Leipzig. Scrapings of the residue adhering to the outer and inner surfaces of the ilium were obtained for testing using a sterilised scalpel. A sample was taken from the organic material integrated within a carbonate concretion buried in close proximity to the left iliac crest and the surrounding soil was tested as a control. The material adhered to the cavity in the molar proved difficult to sample. To avoid damaging the inner surface of the tooth, repeated washings with small quantities of an organic solvent (dichloromethane:methanol, 2:1, v:v) were taken using a sterilised glass Pasteur pipette to directly dissolve organic compounds present in the residue. Supplementary Table S3 reports the sampling details.

Solvent Extraction

All solvents used were HPLC grade solvents (Roth) and the standard purity was ≥99% (Sigma-Aldrich). Glassware was sterilised before use and a method blank was included to monitor laboratory contamination. Isotopically labelled C 18:0 was used as an internal standard for quantification purposes.

Prior to extraction, 10 μg of isotopically labelled C 18:0 internal standard were added to all the samples. To each sample, 2 mL of dichloromethane:methanol (2:1; v:v) solution were added. The samples were shaken and sonicated for 15 minutes and then centrifuged (3500 rpm, 10 minutes, room temperature). The solvent containing the extracted lipid was pipetted into screw capped test tubes and the extraction was repeated twice more, combining the lipid extracts. The solvent was then evaporated to dryness under a gentle stream of nitrogen and mild heating (30 °C) to obtain the total lipid extract (TLE). Each sample was rehydrated using 120 μL of hexane and then partitioned [1:1]. The solvent was evaporated and both parts of the samples were stored at −20 °C pending further analysis. One part of each sample was derivatised (silylated) and analysed, the other stored.

Saponification

Potential ‘unbound’ lipid fractions in samples VIL01, VIL02, VIL03 and VIL04 were targeted by saponification. To each sample, 1 mL of 0.5 M methanolic sodium hydroxide solution made up in methanol:water (9:1, v:v) was added. The samples were shaken and vortexed and then heated (90 minutes, 70 °C). The samples were allowed to cool and centrifuged (4000 rpm, 10 minutes, room temperature). The supernatant was pipetted into screw-capped test tubes. The neutral fraction was extracted three times using 1 mL of hexane into small glass vials. The aqueous fraction was acidified to a pH 3 using c.0.4 mL of 6M hydrochloric acid. The acid fraction was extracted three times into small vials, using 1 mL of hexane. Both the acid and neutral fractions were evaporated to dryness using mild heating (30 °C) and a gentle stream of nitrogen. The neutral fraction was silylated prior to GC-MS analysis, while the acid fraction was methylated before silylation and GC-MS analysis.

Methylation

200 μL of Boron Triflouride (14% Methanol) were added to each of the samples, which were then heated for 1 hour at 70 °C. The reaction was quenched with 2 drops of double distilled water and allowed to cool. Methylated lipids were extracted three times using 2 mL hexane. Samples were evaporated to dryness using mild heating (30 °C) and a gentle stream of nitrogen, then rehydrate using 120 μL of hexane and partitioned [1:1]. The solvent was evaporated and samples store at −20 °C pending further analysis. One part of each sample was silylated prior to GC-MS analysis.

Silylation

30 μL of pyridine were added to the dried samples at room temperature, followed by 55 μL of MSTFA (N-Methyl-N-trifluoroacetamide). The samples were agitated for 30 minutes at 37 °C, then centrifuged to remove any remaining drops on the snap caps and transferred to autosampler vials containing micro inserts.

Gas-Chromatography Mass-Spectrometry (GC-MS) Analysis

GC-MS analysis was carried out on an Agilent 6890 Gas Chromatograph coupled with a Quadrupole Mass Spectrometer (MS) (Agilent, Germany), equipped with an Agilent 7683 series auto sampler (Agilent, Germany). A Hewlett Packard 5973 Mass Selective Detector (MSD) was used for GC-MS analysis. The GC was fitted with a 30 m DB-5MS (5% phenyl methyl siloxane) Agilent column, with a 0.25 mm internal diameter and a film thickness of 0.25 μm. The samples were injected in splitless mode at 300 °C. Helium was used as the carrier gas, with a flow rate of 1 mL min−1. The oven was programmed at 50 °C for 2 minutes, then ramped at 10 °C per minute to 325 °C and held for 15 minutes. The MS was operated in Electron Impact mode (EI; 70 eV), at a full scan range of m/z 50 to 550, with a scan time of 3s per scan. Data acquisition was carried out using Data Analysis Version 3.3 (Bruker Daltonics) data system. Data analysis was performed using MSD ChemStation Version D.00.01.

Plant microremain analysis

The Villabruna remains were brought to the archaeological material laboratory in the Max Planck Institute for Evolutionary Anthropology, where they were sampled by AGH. The caries on the lower molar was sampled for possible plant microremains by adding a small volume of double distilled water (~50 μl) to the cavity using an adjustable volume pipet with a plastic disposable tip, agitating the surface by pumping the water in and out of the pipet and finally transferring all of the water to a microcentrifuge tube. Later 1 ml of ddH2O was added and the tube vortexed for 15 sec and centrifuged for 5 min at 3 krpm. 950 μl of supernatant was removed and the pellet resuspended in the remaining 50 μl, 10 μl of which was mounted on a slide, with 10 μl 25% glycerin.

We also collected several kinds of control samples, including samples of the containers in which the fossil material was stored, to look for contamination from the post-excavation curation. We took samples from the bubble wrap and stuffing in the box in which the mandible was stored, as well as the stuffing from the skull box and small fragments from the bottom of the skull box. These controls were sampled by holding them with forceps over a 15 ml tube and washing them with a stream of ddH2O which was collected in the tube. The tube then centrifuged, then all but ~50 μl removed and 10 μl of this remainder mounted and examined. Finally, when a batch of samples was mounted for a day’s worth of microscopy, we prepared a blank slide, which contained only 10 μl dH2O and 10 μl 25% glycerin.

We performed regular cleaning and testing procedures to assess possible lab contamination. The laminar flow hood and the surrounding bench areas were cleaned once a week with hot tap water and starch-free soap, followed by a wipe with 5% bleach and a final tap water wipe rinse. Since the results of Crowther and colleagues51, we now recommend using NaOH instead of bleach. Every two weeks, the laminar flow hood and the bench work area were tested for contaminants by wiping the entire surface with a wet towel, rinsing the towel into a 50 ml tube, centrifuging this tube, pipetting off the supernatant and mounting the remainder on a slide. Records were kept of the contaminant load before and after cleaning, with photographs and written descriptions to allow comparison to the archaeological material. All of our reagents and mounting material were changed once a month and the water and glycerin containers were tested once every two months for contaminants. In addition to the weekly cleaning, the work areas were cleaned immediately prior to sampling with soap and a water rinse.