Dental dimorphism can be used for discriminating sex in forensic contexts. Geometric morphometric analysis (GMA) allows the evaluation of the shape and size, separately, of uneven 3D objects. This study presents experiments using a novel combination of GMA and an artificial neural network (ANN) for sex classification, applied to premolars of Caucasian Italian adults (50 females and 50 males). General Procrustes superimposition (GPS) and the partial least square (PLS) method were performed, respectively, to study the shape variance between sexes and to eliminate landmark variations. The “set-aside” approach was used to assess the accuracy of the proposed neural networks. As the main findings of the pilot study, the proposed method applied to the first upper premolar correctly classified 90% of females and 73% of males of the test sample. The accuracy was 0.84 and 0.80 for the training and test samples, respectively. The sexual dimorphism resulting from GMA was low, although statistically significant. GMA combined with the ANN demonstrated better sex classification ability than previous odontometric or dental morphometric methods. Future research could overcome some limitations by considering a larger sample of subjects and other kinds of teeth and experimenting with the use of computer vision for automatic landmark positioning.

Teeth have proven to be a reliable source of DNA for forensic analysis as the pulp is rich in cells and protected from damaging factors and contamination by dental hard tissues. The pilot study aims to evaluate the feasibility of Next-Generation sequencing analysis on dental pulp to detect genetic mutations in DNA caused by post-mortem cell necrosis. We used a 56-gene oncopanel kit on a sample of 17 teeth extracted from living patients. Time of the tooth avulsion was assumed as death of the individual and Post-mortem Interval (PMI) was the time elapse since the DNA extraction and analysis. Days and Accumulated Degree Days (ADD) were assumed as measures of PMI that ranged between 0 to 34 days. Only 38 of the 56 considered genes proved to be affected by mutations (101), thus being of forensic interest. More specifically, 14 mutations occurred only in a specific range of PMIs/ADD; 67 were detected (alone or as clusters of the same gene) at specific PMI/ADD; 22 occurred at every PMI/ADD, except for some specific intervals. Since dental pulp was not targeted by any oncological diseases and all teeth were intact, vital, and from patients with unremarkable medical history, it could be assumed that mutations were due to post-mortem DNA changes induced by pulp death and the increasing time elapse since death. This pilot study found encouraging results in the application of NGS analysis on dental DNA, especially for PMIs of several days for which the traditional tools for PMI estimation have limitations. Further research on a larger sample of PMI and validation research on a larger sample of PMI and validation of the results are indeed necessary.

Teeth are known to be reliable substrates for human identification and are endowed with significant sexual dimorphism not only in the size but also in the shape of the crowns. In the preliminary phase of our study (already published in 2021), a novel sex estimation method based on dental morphometric geometric (GMA) analysis combined with the artificial neural network (ANN) was developed and validated on a single dental element (first upper premolar) with an accuracy rate of 80%. This study aims to experiment and validate the combination of GMA–ANN on the upper first and second left premolars and the upper left first molar to obtain a reliable classification model based on the sexual dimorphic traits of multiple maxillary teeth of Caucasian Italian adults (115 males and 115 females). A general procrustes superimposition (GPS) and principal component analysis (PCA) were performed to study the shape variance between the sexes and to reduce the data variations. The “set-aside” approach was used to validate the accuracy of the proposed ANN. As the main findings, the proposed method correctly classified 94% of females and 68% of males from the test sample and the overall accuracy gained was 82%, higher than the odontometric methods that similarly consider multiple teeth. The shape variation between male and female premolars represents the best dimorphic feature compared with the first upper molar. Future research could overcome some limitations by considering a larger sample of subjects and experimenting with the use of computer vision for automatic landmark positioning and should verify the present evidence in samples with different ancestry.

Recent studies show that Langerhans cell density increases in vital lesions and ligature marks when compared with post-mortal wounds. The enzyme, iNOS, has been established as a marker for estimating time of agony and serves in the regulation of dendritic cell behaviour. It is the aim of this paper, therefore, to evaluate the expression of this enzyme by Langerhans cells and the possible consequences that may be related to the production of nitric oxide by these cells in other types of lesions, including hanging furrows. The results show a greater expression of iNOS by Langerhans cells at the level of the hanging furrow when compared with other examined groups. Apart from an increase in the expression of iNOS, a large fraction of the mast cell population in the class II MHC molecules was observed in the hanging furrow. This corroborates that interactions between mast cells and dendritic cells are critical for the differentiation of these latter cellular types. Hence, the results suggest that iNOS plays a crucial role in the forensic practice of establishing the time interval in defining the vitality in hanging marks.

Post-mortem interval (PMI) estimation remains one of the major challenges in forensic practice, especially for late PMIs beyond 7–10 days after the death of the subject. In 2022, an innovative method to investigate the occurrence of mutations induced by the death of a subject in the DNA of post-mortem dental pulps at different PMIs was developed, applying a next-generation sequencing (NGS) analysis. The present study aims to apply the same method of analysis to a small sample of teeth belonging to the same subject and analyzed at different PMIs/accumulated degree days (ADDs), and of teeth extracted from different subjects but analyzed at the same PMI/ADD to verify the repeatability of the results obtained in relation to the time elapsed since death. A total of 10 teeth were collected from 6 patients (3 males and 3 females) with PMI varying from 8 to 35 days, and ADD from 157.4 to 753.8. We found 1754 mutations in 56 genes, with more than 700 mutations having a prevalence > 5% and more than 300 variants considered of interest for the purposes of the study. Mutations that were not present at lower PMIs but manifested in later PMIs in pulps belonging to the same subject demonstrate that they can only have been acquired by the subject after death and according to the time elapsed since death. In total, 67 somatic mutations in 29 out of the 56 genes of the used panel occurred in a fashion that allows an association with specific PMI/ADD ranges (within 8 days, between 17 and 28, and beyond 30 days after death). The results suggest that temperature and humidity could influence the rate of DNA degeneration in dental pulps, thus PMI should be estimated in ADD more than days. The preliminary validation supports the hypothesis that the innovative method could be a useful tool for estimating the post-mortem interval even beyond the first week after death, but further analyses are needed to customize a specific genetic panel for forensic investigations and verify the influence of degenerative processes of soft tissues surrounding dental elements on DNA degeneration of pulps.

Post-mortem toxicology constantly deals with the research of reliable alternative matrices to be applied in case of highly damaged corpses (such us carbonized, skeletonized, human remains, etc.). Teeth represent a promising alternative matrix since dental tissues are endowed by different features, resistance and stability after death. Since scant literature reported on the pharmacokinetics and mechanism of incorporation of xenobiotics into dental tissues, this pilot research aims to investigate whether in the pulp can be detected the same substances found in blood in drug related death cases. Secondly, the study is addressed to disclose the possible deposit of drugs in dental hard tissues (dentine and/or enamel), thus contributing to reconstruct the drug abuse history (timing, e.g.). The study experimented with a novel method to separately analyse dental enamel, dentin, and pulp, applied to 10 teeth collected during autopsies of drug-related deaths along with blood and hair samples for classic toxicological analyses. Each tooth was prepared by "pulverization technique" and then analysed by gas chromatography paired with mass spectrometry (GC-MS) and ultra high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC/HR-MS) for searching cocaine, opiates, and metabolites. The results were then compared with those obtained from blood and hair samples. Preliminary results demonstrated that teeth differ from any other classic matrix (blood and hairs) since the qualitative correspondence of the detected substances between pulp and blood as well as dental hard tissues and hair suggests that they can be useful in post-mortem evaluation as a unique matrix for both acute and chronic assumptions of drugs. The mechanism of accumulation of substances in mineralized dental tissues emerged the most significant result, being influenced by the type of molecule and the method of assumption. The main limitation of this study is the limited availability of the sample and the absence of anamnestic information of the time, rates and method of drug assumption during life. Further research is necessary to systematically investigate the distribution of different substances within the different tissues of the tooth.