## 5 Discussion  
The anatomy of teeth has been extensively studied and is globally well known. However, some aspects warrant re-analysis considering recent advancements in dental technology. One such aspect is the pulp chamber, as precisely understanding its anatomy and dimensions (particularly its volume) is crucial for accurate diagnosis, treatment planning, and monitoring [18]. This study was designed to validate the use of CB-CT for evaluating human pulp chamber volume using molars as a model. We compared in vivo CB-CT images--both raw and after manipulation by two trained experts and an algorithm--with ex vivo micro-CT images. Molars were chosen due to their large pulp chamber size [19]. The anatomy and dimensions of the pulp chamber are of interest in several dental fields, including endodontics [20; 21], age estimation [22; 23], dental pulp tissue engineering [24], and preclinical teaching and training of endodontic postgraduates [25; 26]. An algorithm was developed to automatically calculate pulp chamber volume based on DICOM CB-CT images commonly used in dental clinics. The results demonstrate that the volumes obtained from in vivo CB-CT images differ from those obtained from ex vivo micro-CT images of the same teeth. However, the volumes were very similar when using the viewer-set CB-CT threshold and almost identical when processed with the algorithm, with relative errors not exceeding 5.7%. Therefore, the results suggest that in vivo CB-CT achieves high precision and reproducibility for evaluating pulp chamber volume when using the viewer-set threshold. The discrepancies between in vivo CB-CT and ex vivo micro-CT measurements may be partly due to the imprecision of CB-CT volumetric measurements caused by superimposed structures [2]. It should be noted that the measurements were obtained using only the Promax 3D Max CB-CT unit, and results could vary with other brands. Additionally, the Romexis Planmeca software offers a tool for pulp volume measurement, and the data obtained were included in the study. The automatic volumes generated by the viewer with an automatic threshold differed significantly from those obtained by trained experts adjusting the threshold. Data obtained by threshold adjustment were much closer to the micro-CT data, indicating that the Romexis Planmeca viewer's automatic tool is not initially reliable for calculating dental pulp volume. For samples 1 to 4, a voltage of 120 kV and a current of 5 mA were used, while for sample 5, 90 kV and 10 mA were used due to different scanner specifications. The voxel size also varied from 0.25 mm in the first three samples to 0.2 mm in the fourth sample and 0.15 mm in the fifth sample. These variations could explain significant differences between the volume results obtained by micro-CT and other methods [27; 28]. The volumes obtained by adjusting the CB-CT image threshold closely matched those of the micro-CT, showing that dental pulp volume measurements are operator dependent. Despite keeping all parameters unchanged, notable discrepancies were found between the algorithm and micro-CT results in sample 5, possibly due to differing kV and mA settings and multiple pulp calcifications observed in the micro-CT images. When calculating pulp chamber volume for sample 5 with the proposed algorithm and a voxel size of 0.2 mm, a volume of 11.06 \(\pm\) 0.52 mm\({}^{3}\) was obtained. However, using a voxel size of 0.25 mm resulted in a volume of 21.61 \(\pm\) 1.01 mm\({}^{3}\), highlighting the substantial influence of voxel size on measurements. While sample 5 showed discrepancies, similar calcifications in sample 3 did not affect alignment between micro-CT and algorithm results, indicating an unclear cause for these differences. To our knowledge, this is the first study applying an algorithm with in vivo CB-CT to calculate dental pulp chamber volume and comparing it with ex vivo micro-CT results. However, the results should be interpreted cautiously due to the small sample size and differences between maxillary and mandibular molars [19].