## 2 Materials and Methods
### Microstructural Analysis  
The dental samples were processed and analyzed at the National Center for Electron Microscopy in collaboration with the UCM. The entire methodological process of sample preparation comes from the internal protocol for analysis of biological mineralized structures, and according to dental tissues analysis with SEM [26]. The microstructural analysis was carried out using a JEOL-JSM 6400 SEM scanning microscope (JEOL Ltd., Tokyo, Japan), and was carried out within a maximum period of three months from the collection of each sample. Teeth were longitudinally sectioned in a vestibular-lingual direction [19; 23] at the point of maximum dental contour to be able to analyze all points of the dentin of different depths. The longitudinal section allows us, in a single dental section, to visualize all the dentin from the pulp tissue to the dentin-enamel junction (DEJ). It was decided to carry out this section method due to dental fragility and the difficulty of obtaining teeth from subjects with OI. Furthermore, knowledge of the S-shape direction of the tubules would allow the view of the tubules in two different modes: longitudinal (cut parallel to the tubular direction) or transverse (cut perpendicular to the tubular direction). The surface was polished with silicon carbide abrasive discs (metalograph polishing machine 50-8435 MICRODUO-I AUTO, METALOGRAT, no data), in a decreasing sequence of the abrasion degree (disc 600, disc 800/2400 and disc 1200/400). Subsequently, the teeth were treated with 37% orthophosphoric acid gel (Scotchbond(tm) etchant, 3M(tm) ESPET(tm), St. Paul, MN, USA) for 20s, evaporated (Quorum Q150T STM, Quorum Technologies, Lewes, UK) and plated with gold (Quorum Q150R STM, Quorum Technologies, Lewes, UK), to avoid surface charges. All the samples were collected, processed, and analyzed by the same operator (A.M.-V.), and previously calibrated.  
The systematic analysis of the dentin was based on taking images at 2000 magnifications at four points of the dentin. In order to standardize the regions analyzed, the total thickness of the dentin was divided into four areas of equal thickness, and the analysis was conducted in four portions, from the most external to the most internal. These were referred to as external or outer, middle, deep and pulpal dentin (adjacent to pulpal tissue), respectively. Because a longitudinal section of the tooth is not the best way to visualize the tubules--although, due to its serpentine shape, it allows, in many cases, a transverse view of them--an approximation to the tubular density and diameter was made. At each point of the dentin, the dentinal tubule count was recorded (initially in mm\({}^{2}\) and converted to mm\({}^{2}\)), and the diameters (um) of the tubules present in the image were measured with the ImageJ/Fiji software for Windows ([[https://fiji.sc/](https://fiji.sc/)]([https://fiji.sc/](https://fiji.sc/)), No copyright, accessed on 8 March 2017), obtaining the average in each microscopic section. It was anticipated that due to the meandering path of the dentinal tubules, some cuts would obtain a cross section and others a longitudinal section, affecting the variables of count and tubular diameter, so the cutting pattern was included as a variable, eliminating its action as analysis bias.