A combination of modern physics tools reveals the oldest dental filling, dating back to the Stone Age

Previous archaeological finds suggest that Neolithic humans could have practiced therapeutic dentistry. In 2006, Coppa and his group found several holes (probably made using a bow drill) in human molars from a 7500 to 9000-year-old graveyard in Pakistan, but there was no evidence of dental filling (Coppa et al., Early Neolithic tradition of dentistry, Nature 440, 775 (2006)).
In the present study, several research groups with different expertize have adopted a multi-disciplinary approach providing the earliest direct evidence of a therapeutic dental filling.
The discovery is based on the finding of extraneous material on the occlusal surface of a canine from a Neolithic human mandible. The jawbone fragment, bearing a canine, two premolars, and the first two molars, was found in 1911 near the village of Lonche in northern Istria (Slovenia) and, from that time, kept in the Natural History Museum of Trieste (Italy).

Figure 1:  Volume renderings of part of the Lonche jaw. In b) a longitudinal virtually cut section of the object is shown. The dotted yellow rectangles in a) and b) show the position of the longitudinal crack partially filled with beeswax. Images obtained at Elettra by conventional micro-CT (cubic voxel side: 18 μm).

The data for the study were obtained using conventional and synchrotron radiation computed X-ray microtomography (micro-CT), radiocarbon dating with accelerator mass spectrometry (AMS), infrared (IR) spectroscopy and scanning electron microscopy (SEM). These advanced analytical techniques provided crucial information on morphology and age of the mandible, age and composition of the dental filling and tooth microstructure. The experiment was conducted in several steps.
Conventional X-ray micro-CT (performed at the Tomolab station of Elettra) provided a high-resolution 3D reconstruction of the mandible (Figure 1). In particular, we could observe that the canine revealed a long vertical crack, and an area of enamel that had worn away to create a large cavity, which exposed the dentine (Figure 2-a). To increase the spatial resolution and obtain more detailed images, the tooth crown was then investigated using synchrotron X-ray micro-CT at the SYRMEP beamline of Elettra (Figures 2-b, 2-c). Thanks to the high spatial coherence properties of synchrotron radiation, we could use phase-contrast imaging to visualize an unusual light material forming a thin cap that perfectly filled the cavity and the upper part of the canine crack.

Figure 2: a) Conventional micro-CT virtual section of the Lonche canine (voxel side: 18 μm). b) Synchrotron X-ray micro-CT slice showing details of thecrown: the thickness of the filling material, afterward identified as beeswax, is visible. Beeswax exactly fills the shallow cavity in the exposed dentine and the upper part of the crack (voxel side: 9 μm). c) Synchrotron X-ray micro-CT axial slice of the tooth showing the enamel crack (beeswax colored in light green). d) Microphotograph of the tooth crown with indication of the surface covered by beeswax (within the yellow dotted line). e) Comparison between the IR spectrum of the material filling the Lonche canine (average of 10 spectra, 128 scans per spectrum, 4 cm-1 spectral resolution) and a modern sample of natural beeswax.

A minute amount (volume < 2 mm3) of the filling material was extracted from the canine, placed in a diamond anvil cell, and layered to allow an analysis in transmission in the mid-infrared spectral range (4000-500 cm-1) (Figure 2-e)). A set of standards, including modern natural beeswax, was run in a second step to fingerprint the unknown substance. Thanks to the high molecular sensitivity of the IR technique, the analysis revealed that the chemical markers (spectral features of wax ester compounds) of the tooth filling corresponded to beeswax, with some additional bands that reveal the presence of salts and oxidation processes, as expected after such ageing period. In conclusion, IR analysis clearly identified the unknown filling material as beeswax. 
Radiocarbon dating was performed with AMS in two independent laboratories. A bone sample of about 1 g was collected from the mandible using a conventional hand drill, its collagen was then extracted and subsequently measured by means of the CIRCE AMS system at the 2nd University of Naples (Caserta, Italy). A beeswax sample of 1.4 mg was finally extracted from the tooth and AMS 14C measurements were performed using the STAR facility at ANSTO. The AMS measurements determined that both mandible and beeswax filling were about 6,500 years old.
SEM images showed in detail the morphology of the tooth occlusal surface, demonstrating that beeswax not only filled the exposed area of the dentine, but also penetrated the longitudinal crack. The observation of the edges of the crack, before and after beeswax cleaning, gives information to assess whether the fracture originated antemortem, perimortem or postmortem.
The evidence obtained in this study, based on the use of advanced analytical methods, was finally interpreted with the involvement of medical dentists and palaeoanthropologists. In conclusion, the data strongly support the hypothesis of therapeutic dental treatment, likely used to alleviate the pain of chewing on a cracked tooth, although alternative postmortem practices cannot be completely ruled out. However beeswax may have been used to plug the cracked and worn tooth while its owner was still alive, which would make it the earliest known direct evidence of a therapeutic dental filling.
This teamwork shows how combining careful observation and advanced physics methods could help unlocking unknown aspects of the human way of life during the Stone Age.

This research was conducted by the research team of the 'Abdus Salam' International Center for Theoretical Physics, the University of Roma ‘La Sapienza’, Elettra - Sincrotrone Trieste, the University of Trieste, the 2ndUniversity of Napoli andthe Australian Nuclear Science and Technology Organization (ANSTO).

  • Federico Bernardini, "Abdus Salam” International Centre for Theoretical Physics, Trieste, Italy.
  • ClaudioTuniz, “Abdus Salam” International Centre for Theoretical Physics, Trieste, Italy and University of Wollongong, New South Wales, Australia.
  • Alfredo Coppa, Department of Environmental Biology, University ‘‘La Sapienza’’, Rome, Italy.
  • Lucia Mancini, Diego Dreossi (micro-CT), Diane Eichert (FTIR), Elettra - Sincrotrone Trieste S.C.p.A., Basovizza, Italy.
  • Gianluca Turco, Matteo Biasotto, Department of Medical Sciences, University of Trieste, Italy.
  • Filippo Terrasi, CIRCE, INNOVA and Department of Environmental Sciences, 2nd University of Naples, Caserta, Italy.
  • Nicola De Cesare,CIRCE, INNOVA and Department of Life Sciences, 2nd University of Naples, Caserta, Italy
  • Quan Hua, Vladimir Levchenko, Australian Nuclear Science and Technology Organization Lucas Heights, New South Wales, Australia.


Federico Bernardini, ClaudioTuniz, Alfredo Coppa, Lucia Mancini, Diego Dreossi, Diane Eichert, Gianluca Turco, Matteo Biasotto, Filippo Terrasi, Nicola De Cesare, Quan Hua, Vladimir Levchenko, “Beeswax as Dental Filling on a Neolithic Human Tooth", PLoS ONE 7, e44904 (2012); doi:10.1371/journal.pone.0044904.

Last Updated on Friday, 19 October 2012 15:45