Figure 7

Changes in dentin composition over time. (a) Images of maxillary and mandibular first, second and third molars of 6- and 12-month-old pigs. 6 m: 6-month-old pig, 12 m: 12-month-old pig, 1st: first molar, 2nd: second molar, 3rd: third molar, Max: maxillary, Man: mandibular. Mandibular first (6^m−1, 12^m−1), second (6^m−2, 12^m−2) and third (12^m−3) molars, as indicated by a red square, were used for this study. (b) Schematic representation of the root furcation preparation. The apical roots of 6^m−1, 12^m−1 and 12^m−2 teeth were first separated by cutting with a jewellery saw (blue dotted line), and then, each of the root furcations (approximately 5 mm thick) were prepared by cutting at the cementum-enamel junction (CEJ) (red dotted line). The root furcations from 6^m−2 and 12^m−3 teeth were obtained by cutting at the CEJ with dissecting scissors (purple dotted line). (c) The densities of the porcine molars were measured using a pycnometer (n = 3). (d) Volumes of the porcine molars were calculated from the measured densities (n = 3). (e) Changes in the amount of DPP and DSP in the porcine molars. SDS-PAGE stained with Stains-all for the detection of DPP bands (top left) and western blots using a specific antibody against DSP (top right), showing AN extracts from each root furcation. Each protein sample amount for SDS-PAGE was normalized (see Supplementary Table S2). The areas of the DPP bands on the SDS-PAGE gel (bottom, left) and the DSP bands (bottom, right) on the western blots were determined using ImageJ software and normalized to the density of the 6^m−2 and 12^m−3 samples in order to compare the protein amount at each age. The areas of the DSP bands at each age were calculated as the total area of the band in each lane shown with asterisks. The full-length gel and blot are presented in Supplementary Fig. S4. (f) ALP-inducing activity of HPDL cells exposed to each of the AN extracts (n = 6). TGF-β1: recombinant human TGF-β1 with a carrier (0.3 ng mL⁻¹).