2. Skeletal analyses of the archeological individuals

A total of 100 individuals affected by pathological illness will be selected from human remains unearthed at the necropolis of Isola Sacra and Velia (1st-3rd century AD), the catacomb of Santa Mustiola (4th-6th century AD), the catacomb of Santa Sofia di Canosa (4th-6th century AD), and from the necropolises of Amiternum (4th-6th century AD) and Santa Giusta (13-14th AD). Skeletal lesions will be described in detail through imaging techniques (MICRO CT scans, 3D photogrammetry) to better clarify the nature of the pathology and the need for specific treatment to ease the pain. A screening of oral health will also be conducted, that will include, caries, abscess, antemortem tooth loss. A few apparently unaffected individuals will be also selected as control sample for the calculus analysis.

3. Ancient dental calculus analysis

The analysis of ancient dental calculus will follow the accepted protocols, involving optical and polarized microscopy analysis. At Sapienza, the ancient dental calculus will be examined to identify residues of food or therapeutic substances at the DANTE—Diet and Ancient Technology Laboratory, using a Zeiss Imager2 cross-polarized microscope. Plant structures such as phytoliths and starch granules will be identified and interpreted through comparison with reference collection store in the laboratory.

4. Clinical and Forensic analyses

Chemical analysis will be conducted to detect biomolecules, assessing dietary habits and phytotherapeutic practices of ancient individuals. This will take place at the Forensic Toxicology Research Laboratory (FT-LAB) at the University of Florence, using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The project will also include comprehensive clinical and forensic analyses involving two distinct modern populations: non-drug users and individuals who use substances such as cannabis, cocaine, opiates, alcohol, amphetamines, and benzodiazepines. Non-drug users will provide "blank" dental calculus for method validation, while substance users will provide "positive" samples of dental calculus, hair, and saliva for forensic investigation. An experimental protocol will be developed to test the environmental stability of dental calculus. 

5. Statistical analysis                                                                       All results obtained on ancient and modern samples will be the object of computational, statistical, and interpretative analyses using the software Stata/SE 17.0. Validation parameters for the different analytical methods will be calculated following the most recent international guidelines. Moreover, sensitivity and specificity of dental calculus as matrix for past/chronic consumption will be statistically evaluated by the comparison with hair test and self-reported questionnaires.