1 Volume 23 2024 e243996 Short Communication Braz J Oral Sci. 2024;23:e243996http://dx.doi.org/10.20396/bjos.v23i00.8673996 1 Department of Pathology and Oral Diagnosis, Dental School of the Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 2 Forensic Odontology Service, Afrânio Peixoto Legal Medicine Institute, Rio de Janeiro, Brazil. 3 Department of Nuclear Instrumentation Laboratory, Technology Center of Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 4 Department of Pediatric Dentistry and Orthodontics, Dental School of the Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 5 Department of Forensic Dentistry and Public Health, Dental School of the Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil. Corresponding author: Maria Augusta Visconti Rua Professor Rodolpho Paulo Rocco, 325, Cidade Universitária, Rio de Janeiro, RJ – Brazil. Zip Code: 21941-617. Telephone number: + (55) 21 98889-9383. E-mail: gutavisconti@odonto.ufrj.br Editor: Dr. Altair A. Del Bel Cury Received: July 14, 2023 Accepted: September 22, 2023 Forensic significance of dental cementum: pilot study on new micro-CT energy parameters for age estimation in human teeth Pedro Américo Felizardo dos Santos1 , Vanessa Moreira Andrade2 , Luan Ferreira Bastos3 , Luciana Pereira da Silva4 , Ricardo Tadeu Lopes3 , Aline de Almeida Neves4 , Andreia Cristina Breda de Souza2,5 , Maria Augusta Visconti1* Aim: To determine new energy parameters in micro-CT to allow appropriate segmentation of dental cementum from the surrounding structures. Methods: For this pilot study, 11 single-rooted premolar teeth of young subjects aged 12 to 21 were selected. The teeth were scanned using high-energy microtomography (SkyScan 1173, Bruker) with varying acquisition energies (60, 70, and 80 kV). Each tooth was scanned three times. The images were reconstructed and analyzed by calculating the volume (mm³) of dental cementum in the cervical, middle, and apical root thirds among the different scanned stacks. The mean cementum volume in the root thirds was obtained, and correlated with the patient’s age, using Pearson’s correlation. Results: The mean age of the patients in the sample was 16,73 years. For the 60kV energy, the cementum volumes were 1.01 mm³, 1.00 mm³, and 0.58 mm³, for the cervical, middle, and apical thirds, respectively. For 70kV, it was 1.08 mm3, 1.20 mm3, and 0.71 mm3 for the respective thirds. And for 80kV, 0.55 mm3, 0.91 mm3, and 0.67 mm3 for the respective thirds. Conclusion: An acquisition energy of 70kV resulted in the best segmentation and reproducibility of the results. Micro-CT can be a useful tool to non-destructively evaluate the dental cementum. Keywords: Forensic dentistry. X-ray microtomography. Dental cementum. https://orcid.org/0000-0001-8798-9998 https://orcid.org/0000-0002-1291-9642 https://orcid.org/0000-0001-9948-5500 https://orcid.org/0000-0002-0719-5002 https://orcid.org/0000-0001-7250-824X https://orcid.org/0000-0002-6049-0588 https://orcid.org/0000-0001-9820-4279 https://orcid.org/0000-0002-8837-8387 2 Santos et al. Braz J Oral Sci. 2024;23:e243996 Introduction Dental cementum is a vital tissue with a continuous apposition throughout life. How- ever, it is distributed unevenly in layers on the root surface, which are larger in the apical areas and smaller in the areas near the cementoenamel junction1. This physi- ological characteristic makes it an excellent source of information for age estimation in cases of human identification1. The adjacent position and mineral content of cementum-like dentin make it difficult to differentiate from conventional radiographs2. And the micro-computed tomogra- phy (micro-CT), a microscopic version of cone-beam computed tomography (CBCT), which also uses X-radiation to obtain images3, can contribute to dental tissue analysis. Currently, the main applications for micro-CT involve its use in qualitative hard tissue analysis, such as three-dimensionally visualizing specific characteristics of bone tra- beculae and their quality4, or quantitative, such as measuring the area, volume, poros- ity, density, and concentration of minerals5,6. Other than bone tissues, micro-CT has also been used for quantitative analysis of dental tissues, such as enamel, dentin, and root canal, often associated with age estimation methods1,7. Only one study has been conducted to analyze dental cementum using micro-CT, albeit with dental samples from rodents2. Thus, this pilot study aimed to establish acquisition parameters for images obtained by micro-CT that allow the qualitative and quantitative analysis of human dental cementum and its volumetric quantification correlating with age. Materials and Methods After approval by the Research Ethics Committee (CAAE 92868518.0.0000.5257), patients with scheduled extraction of premolars due to various clinical indications were approached as potential participants of the study. Upon thorough review and comprehension of the informed consent document, patients who willingly consented to contribute their extracted teeth became integral participants in this study. Eligibility criteria and sample selection We included single-rooted premolar teeth of individuals aged between 12 and 21, with intact root portions. Tooth elements that had been damaged during the extraction procedure or presented any alteration in the root portion were excluded. After selec- tion, the teeth (n=11) were stored individually, in containers containing formaldehyde 10%, identified by codes and patient age. Micro-CT scanning and reconstruction procedures All specimens were scanned using a high-energy microtomographic device (SkyScan, 1173; Bruker-micro-CT, Kontich, Belgium), with each tooth individually fixed with utility wax on its base. Each specimen was scanned at three different energy inputs (60, 70, and 80 kV) to generate reconstructions with different contrasts. The other parameters were fixed and consisted of a current of 100 µA; aluminum filter of 1.0 mm in thick- ness; rotation step of 0.5°; average rotation of 5 and 360; exposure time of 1100 ms; pixel size of 8.98 μm per image in a matrix of 2240 by 2240 pixels each image8-11. 3 Santos et al. Braz J Oral Sci. 2024;23:e243996 After the acquisition, all images were reconstructed using InstaReconTM (v.1.7.3.0) software (Skyscan, Kontich, Belgium). Some reconstruction parameters were adjusted to ensure the best image quality, such as smoothing, misalignment compensation, beam hardening, and ring artifacts. Image processing was performed using CTAnTM (v.1.18.4.0) software (Skyscan, Kontich, Belgium). Cementum evaluation Using the CTAnTM (v.1.18.4.0) software (Skyscan, Kontich, Belgium), the length of the roots of each tooth was measured and divided into three-thirds of equal lengths: cervical, middle, and apical thirds. From this, regions of interest (ROIs) were established manually throughout the length of the tooth where the dental cementum is normally located (Figure 1). 16.750 15.075 13.400 11.725 10.050 8.375 6.700 5.025 3.350 1.675 16.750 15.075 13.400 11.725 10.050 8.375 6.700 5.025 3.350 1.675 16.750 15.075 13.400 11.725 10.050 8.375 6.700 5.025 3.350 1.675 A B C Figure 1. Axial and coronal reconstructions of a scanned tooth with three different energies using image correction factors. The acquisition parameters were: 8.98 μm pixel size, 100 ms integration time, 0.50 thick pitch, and 1.0 mm thick aluminum filter. (a). 60 kV; (b). 70 kV; (c). 80kV. A gray value histogram was obtained from each ROI, allowing segmentation of the cementum (Figures 2 and 3). Furthermore, a global threshold technique was employed to obtain the total volume of the cementum for each third. These steps were performed in each of the respective thirds three times for each tooth, all by a single experienced and trained examiner, for all acquisition parameters evaluated (60kV, 70kV, and 80kV). A B C Figure 2. Axial reconstructions of the same tooth show the region of interest (ROI). (a). 60 kV; (b). 70 kV; (c). 80kV. 4 Santos et al. Braz J Oral Sci. 2024;23:e243996 Figure 3. Illustrative representation of the Histogram. Is a software tool capable of allowing the selection of the gray levels of the image. The representation shows the selection of all gray levels between 165 and 255. Statistical analysis The variables were represented by the mean and standard deviation for all three regions of the tooth and discriminated against for the different energy levels. The mean of the values measured in the three regions was calculated and correlated with the patient’s age using Pearson’s Correlation test. The software IBM-SPSS v.25 was used for the analyses, and ExcelTM was used for the graphs. The adopted significance level was 5%. Results The volume of dental cementum (mm³), obtained in each root third of the same tooth, and their respective means, for the three energies tested, are shown in Table 1. Table 1. Values of dental cementum volume (mm³) and average between the thirds obtained in each tooth, in the three energies tested, about age. Energy Tooth Age Cervical Middle Apical Mean Pearson’s Correlation 60 kV 1 14 4.14 1.99 1.22 2.45 r = -0.227 (p=0.504) 2 21 1.09 1.51 1.36 1.32 3 17 0.93 0.63 0.37 0.64 4 16 0.93 0.89 0.38 0.73 5 14 1.97 2.55 1.64 2.05 6 16 0.09 0.16 0.25 0.17 7 17 0.87 0.41 0.33 0.54 8 21 0.83 1.4 0.23 0.82 9 16 0.15 0.86 0.29 0.43 10 16 0.01 0.35 0.09 0.15 11 16 0.13 0.3 0.23 0.22 Continue 5 Santos et al. Braz J Oral Sci. 2024;23:e243996 Continuation 70 kV 1 14 2.75 1.22 1.08 1.68 r = -0.539 (p=0.088) 2 21 0.34 0.85 0.97 0.72 3 17 0.9 0.57 0.28 0.58 4 16 1.52 1.07 0.41 1.00 5 14 2.4 2.77 1.69 2.29 6 16 2.4 2.77 1.69 2.29 7 17 0.69 0.54 0.3 0.51 8 21 0.34 0.71 0.15 0.40 9 16 0.1 0.92 0.27 0.43 10 16 0.27 1.32 0.45 0.68 11 16 0.19 0.5 0.5 0.40 80 kV 1 14 1.71 0.79 0.62 1.04 r = -0.045 (p=0.899) 2 21 1.11 1.59 1.4 1.37 3 17 0.59 0.53 0.22 0.45 4 16 0.54 0.69 0.2 0.48 5 14 0.57 1.75 1.65 1.32 6 16 0.57 1.75 1.65 1.32 7 17 0.23 0.46 0.32 0.34 8 21 0.41 0.82 0.15 0.46 9 16 0.02 0.51 0.27 0.27 10 16 0 0.45 0.2 0.22 11 16 0.25 0.68 0.68 0.54 The mean age of the participants was 16.73, with a standard deviation (SD) of 2.3 years and a variance of 5.42. For the 60-kV energy, the means (SD) and variance obtained among all cementum volumes were 1.01 (1.19) mm³ and 1.42 variance, 1.00 (0.77) mm³ and 0.59, and 0.58 (0.54) mm³ and 0.30 for the cervical, middle, and apical thirds, respectively. For the 70kV energy, they were 1.08 (1.01) mm³ and 1.02 variance, 1.20 (0.82) mm³ and 0.67 and 0.71 (0.56) mm³ and 0.32 for the respective thirds. At 80kV, the values were 0.55 (0.50) mm³ and 0.25 variance, 0.91 (0.52) mm³ and 0.27 and 0.67 (0.60) mm³ and 0.37 for the respective thirds. The gray levels used to visualize the dental cementum in each root third were also established to measure the volume, employing the histogram (Table 2). These data were subsequently correlated with age as shown in Figure 4. The mean (SD) and vari- ance of the gray level values for the 60 kV energy were 147.6 (11.7) and 136.85 vari- ances, for the 70 kV energy was 118.6 (10.0) and 100.1 variances, and for the 80 kV, energy was 105.5 (28.0) and 784.5 variances. 6 Santos et al. Braz J Oral Sci. 2024;23:e243996 Table 2. Gray levels were adopted to visualize the dental cementum in each root third to measure the volume. Energy Tooth Age Cervical Middle Apical Mean Pearson’s Correlation 60 kV 1 14 165 165 165 165 r = 0.033 (p=0.924) 2 21 150 150 150 150 3 17 147 147 147 147 4 16 135 135 135 135 5 14 147 147 147 147 6 16 145 145 145 145 7 17 162 162 162 162 8 21 151 151 151 151 9 16 152 152 152 152 10 16 122 122 122 122 11 16 148 148 148 148 70 kV 1 14 132 132 132 132 r = 0.055 (p=0.871) 2 21 122 122 122 122 3 17 114 114 114 114 4 16 100 100 100 100 5 14 116 116 116 116 6 16 126 126 126 126 7 17 134 134 134 134 8 21 120 120 120 120 9 16 118 118 118 118 10 16 107 107 107 107 11 16 116 116 116 116 80 kV 1 14 138 138 138 138 r = -0.289 (P=0.388) 2 21 90 90 90 90 3 17 93 93 93 93 4 16 82 82 82 82 5 14 96 96 96 96 6 16 176 176 176 176 7 17 115 115 115 115 8 21 93 93 93 93 9 16 95 95 95 95 10 16 90 90 90 90 11 16 92 92 92 92 7 Santos et al. Braz J Oral Sci. 2024;23:e243996 A D B E C F r = -0,227 r = 0,033 r = 0,055r = -0,539 r = -0,045 r = -0,289 3.0 2.5 2.0 1.5 1.0 0.5 0.0 170.0 150.0 130.0 110.0 90.0 70.0 12 14 16 18 20 22 Age 60kV 70 kV 80 kV Ce m en tu m V ol um e G ra y Le ve ls 12 14 16 18 20 22 Age 60kV 70 kV 80 kV 3.0 2.5 2.0 1.5 1.0 0.5 0.0 170.0 150.0 130.0 110.0 90.0 70.0 12 14 16 18 20 22 Age 12 14 16 18 20 22 Age 3.0 2.5 2.0 1.5 1.0 0.5 0.0 170.0 150.0 130.0 110.0 90.0 70.0 12 14 16 18 20 22 Age 12 14 16 18 20 22 Age Ce m en tu m V ol um e G ra y Le ve ls Ce m en tu m V ol um e G ra y Le ve ls Figure 4. Graphs of correlations between age and cementum volume in mm3 (A. B. and C) and between age and gray levels (D, E, and F). for each of the three energies tested (60, 70, and 80 kV). None of the obtained dental cementum volumes means, or gray levels were correlated with age (p>0.05) (Figure 4). Nevertheless, the highest correlation (in absolute value) between dental cementum volume and age was found when using 70 kV energy (r = - 0.539; p=0.088). Discussion Many studies have been conducted to analyze and measure the volume and thickness of cementum, but most of them used destructive methods, such as histologic anal- ysis1,12,13. In contrast, micro-CT allows the scanning of samples in a non-destructive 8 Santos et al. Braz J Oral Sci. 2024;23:e243996 manner, maintaining the physical integrity of the analyzed structure3,8, which could be advantageous, especially in keeping counter-evidence in forensic cases. As this was a pilot study, and due to the scarcity of previous similar studies, the sam- ple size used was 11 teeth. This number was supported in preliminary studies based on microtomographic analysis of other biological tissues or microscopic analysis of the dental cementum itself1,8,11. Earlier studies have outlined dental tissue analysis parameters encompassing energy levels (70-100kV), amperage (100-114µA), isotropic resolution (5-20µm), and integra- tion time (300-900ms). Image reconstruction employed NRecon software (V1.6.10.4; Bruker, Belgium), quantitative analysis utilized CTAn software (V.1.14.4, Bruker, Belgium)9-11, with some studies also incorporating filters2. On the other hand, none of them presented values adapted for the evaluation of the dental cementum over the entire root length. According to the results of this study, the mean volume of cementum in all root thirds was measured and correlated with the patients’ age, showing better results when the images were obtained with 70kV (p=0.088). Despite this, the values were not statis- tically significant (p<0.05), suggesting that a study with a larger number of samples may provide better results. In summary, existing studies differ regarding the selection of micro-CT acquisition parameters for dental tissue analysis. Specifically regarding the energy, where the values found vary from 60 to 100kV3,9,11, there is, in fact, no literal consensus on the subject. Based on the analysis of the different gray levels used in the histogram to delimit the region of the dental cementum, it is believed that the large variation in these numbers was due to the different thicknesses of cementum among the root thirds of each sample, making this value unique for each one. Most studies conducted to analyze dental tissues such as enamel and dentin do not have the habit of disclosing the val- ues used in the histogram14,15. However, in order to make the research method repro- ducible, we have chosen to disclose the gray levels that were manually determined by the operator. Due to the density similarity between dentin and cementum, it is challenging to seg- ment and differentiate them. But, based on the results of the present study, and in comparison with data from a previous study1, it is possible to suggest that the combi- nation of an exposure time greater than 1000ms and a voxel less than 5µm along with adequate power and filter should be used in future studies. An important consideration to acknowledge in this study pertains to the exclusivity of the tooth type selected for analysis, i.e., premolars, which are commonly subjected to orthodontic extraction. While this choice was made for specific research reasons, it is crucial to recognize its potential impact on the generalizability of findings to a broader population of tooth types. In light of this limitation, future investigations could substantially enhance the depth and breadth of knowledge in this field. It is recommended that forthcoming studies encompass a broader spectrum of tooth types to represent dental tissues compre- hensively. Moreover, expanding the scope to include larger sample sizes and more 9 Santos et al. Braz J Oral Sci. 2024;23:e243996 diverse age groups will undoubtedly facilitate a more nuanced understanding of the correlation between cementum thickness and age. This approach has the potential to yield invaluable insights into the intricate dynamics of dental tissue aging. By broaden- ing the study’s focus and incorporating a wider range of tooth specimens and patient demographics, future research endeavors can effectively address the limitations mentioned and foster a more comprehensive comprehension of the subject matter. Therefore, we conclude that the micro-CT allows a non-destructive analysis of the dental cementum, possibly becoming a tool for human age estimation, and that the acquisition parameters to ensure a better quality of the quantitative analysis are: 8.98 μm pixel size, 2240 x 2240 pixels matrix per image, 100 μA, 1100 ms integration time, 0.5º rotation step, 1.0 mm thick aluminum filter, and 70kV energy. Additionally, for a subjective visual analysis of the cementum, the 60kV energy guaranteed greater sharpness in the teeth analyzed. Financial Support This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Conflict of interest The authors declare that they have no conflict of interest. Author Contribution Pedro Américo Felizardo dos Santos: study concepts and study design, data acquisi- tion, data analysis and interpretation, statistical analysis and manuscript editing, man- uscript preparation. 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