## 4 Discussion  
Since the development of micro-CT systems, their usefulness has been demonstrated in a wide variety of applications in dental research [11]. The widespread use of micro-CT in dental studies has led to the development of standardised image acquisition protocols that can be applied to images taken with different technologies. One of the main aims of these protocols is reproducibility; thus, many authors, including us, have focused on demonstrating high degrees of agreement between measurements by different observers. [PERSON] et al. [39] developed a reliable and reproducible protocol to measure implant bone thickness based on CBCT images. They found a high level of agreement between observers. Likewise, [PERSON] et al. [40] tested a protocol to enable the comparison of anemoterm per-riapical radiographs with images extracted from postmortem CBCT exams through one angle, linear measurements, and the proportion between these distances. They reported a high level of reproducibility. [PERSON] et al. [41] calculated the volume of the cleft region using the Osirix Dicom Viewer and reported a nonsignificant difference in bone volumetric measurements between observers. This protocol uses HUs, and it is important to acknowledge an ongoing debate: Obtaining HUs from grey levels has not been sufficiently studied [42], and researchers must assume that there is a constant correlation between both [43]. Of note, [PERSON] et al. [44] analysed mineral density that was estimated based on the HUs of CBCT images and determined from measurements in parts of the jaw and tooth. Even recognising these aforementioned limitations, the authors verified the repeatability of the method, denoted by a high degree of agreement. Hence, we can deduce that HUs are comparable and allow us to evaluate the reproducibility of our proposed protocol to establish sectors to estimate density. Although the calculated CCCs demonstrated the high concordance of the proposed measurement method, there were important differences between the measurement points. For enamel, given its thickness in the teeth we assessed, this proposal allows the movement of the position of the generated prisms to assess external or internal layers of the tissue. For dentine, the lowest CCCs on the lingual surface can be related to more pronounced differences between its layers compared with the buccal surface. There are more dentinal tubules in the crown, and their number is reduced toward the apex [45]. Moreover, the layers closest to the pulp chamber are denser than the middle and outer ones, particularly in the crown, where there is a significant increase from the middle to the internal layer near the pulp [46; 47]. Given these differences in density, the choice of the exact frame from which to begin working to establish the reference points can affect the density values obtained, which may explain the lower concordance in the crown area compared with the cervical area. The root results were the least concordant. A possible explanation is the narrowness of the sampling area, which forced us to increase the flexibility of the cube position and could have increased the differences between the observers. Despite these limitations, this proposal produced results with high levels of concordance. This outcome can be explained by the ability of micro-CT to provide structural information of objects and to allow morphometric reconstructions, which are advantageous for analysis of volumes and densities compared with other instruments such as radiographs [48]. In other words, because the differences in density in dental tissues seem to be due to the formation and arrangement of the layers that comprise them, a 2D method that allows an estimation of the density of a single layer would be less effective than one that covers a set of layers. Although the agreement results can be attributed to the characteristics of bovine teeth, there do not seem to be morphological differences in the composition of enamel, dentine, and cementum between bovine and human teeth [49; 50; 51].