## 2 Materials and Methods  
Five permanent molars, corresponding to 5 subjects (3 females and 2 males), with ages ranging between 59 and 66 years, were analyzed. The teeth used in the study were programmed for exodontia due to different odontological treatments and were free of pulp chamber damage. This is the reason why the pulp chamber was intact and free of damage despite the age of the patients. It should be considered that teeth with caries suffer retraction of the pulp chamber, decreasing the volume of the pulp chamber. Likewise, restorations of decayed teeth produce artifacts in the images that prevent the volume from being correctly assessed. Additionally, molars were chosen due to their relatively large pulp chambers compared to other teeth. This characteristic made them ideal candidates for testing the algorithm's effectiveness.  
Pieces were collected with written informed consent and obtained in accordance with Spanish legislation (RD 1301/2006; Law 14/2007; RD 1716/2011; Order ECC/1404/2013). Before exodontia, CB-CT images were obtained using tomographic equipment (ProMax3 DMid ProFace, Planmeca, Helsinki, Finland), with a voltage of 120 kV and a tube current of 5 mA, except for the tooth identified as 5, for which the settings were 90 kV and 10 mA. For the analysis of DICOM (Digital Imaging and Communication in Medicine) images, the software used was Planmeca Romexis 6.2.1.25. Briefly, measurements were made as it follows: The axial and transverse axes were oriented according to the direction of the tooth. The tool \"Measure a cube\" was used to trace the area that covered the pulp chamber in the 3 views: axial, panoramic, and sagittal/orthoradial. A point inside the cube belonging to the pulp chamber was selected as the reference density for the \"3D Growth Region\" tool with the preset \"Root Cavity\", adjusting the threshold favorably (Figure 1a-d). To accurately determine the volume using CB-CT software ([[https://anatomage.com/invivo/](https://anatomage.com/invivo/)]([https://anatomage.com/invivo/](https://anatomage.com/invivo/)), accessed on 1 March 2024), an expert observer's eye is essential for adjusting the threshold. The results obtained with the automatic threshold are less precise.  
After exodontia, teeth were preserved in physiological saline solution at 4 C until use for micro-CT study, which was carried out at the Centro Nacional de Investigacion sobre la Evolucion Humana (CENIEH; Burgos, Spain). The images were obtained with V I Tome I X s 240 from GE Sensing Inspection Technologies (Phoenix X-ray, Manufacturer: Baker Hughes, Celle, Germany), and for the analysis of the images, the software Dragonfly 2022.2 was used. The pulp canal was deleted from the rest of the tooth by adjusting the histogram. Once the pulp canals were isolated, a box was defined that corresponded to the region of the pulp chamber (Figure 1e-i). The software itself has a function that directly provides the volume value of the selected region (connected components; Figure 1i).  
To calculate the pulp chamber volume, 300 DICOM images for each of the 5 samples, corresponding to different axial planes, were available. Nevertheless, only the imagescorresponding to the pulp chamber were selected, resulting in a reduction of 14/300 for sample 1, 16/300 for sample 2, 9/300 for sample 3, 16/300 for sample 4, and 12/300 for sample 5. The selection was based on a meticulous visual analysis of each image by two experts, who established the initial and final planes containing parts of the pulp chamber. The image chosen as the initial one was the closest to the pulp canals among those that contained the pulp chamber, and the rest were selected in ascending order. On the other hand, the voxel size varied for each sample, with a side length of 0.25 mm for the first three samples, 0.2 mm for the fourth sample, and 0.15 mm for the fifth sample.