Negative controls were incubated in medium

lacking TdT enzyme. The specimens were examined and photographed in an OLYMPUS BX-60 microscope. No quantification has Enzalutamide chemical structure been carried out in TUNEL-labelled sections because it has been concluded that quantification produces uneven results due to the variable number of apoptotic bodies present in a given tissue. 21 Upper alveolar processes from 4 alendronate-treated and 4 control rats from each time point were fixed and decalcified as described. Then, they were post-fixed in 0.1 M cacodylate-buffered 1% osmium tetroxide for 2 h at room temperature, dehydrated in graded concentrations of ethanol, and embedded in Spurr epoxy resin (EMS). Toluidine blue-stained 1-μm thick sections were examined in a light microscope, and cervical regions of the tooth germ/alveolar bony crypt were selected for ultrathin sectioning. Sections 80-nm thick were obtained with a Thiazovivin molecular weight diamond knife on a Leica Ultracut R ultramicrotome (Leica, Buffalo, NY, USA), collected

onto 200-mesh copper grids, stained with uranyl acetate and lead citrate, and examined in a Jeol 1010 transmission electron microscope operated at 80 kV. The upper first molar germs of CON rats at 9 days presented normal morphology; the enamel matrix was almost completely secreted. They did not present immunolabelling to Smad-4 antibody (Fig. 1a, b). The first molar germs of ALN specimens at day 9 presented contacting bone trabeculae adjacent to ameloblasts and cells of the HERS. Weak immunolabelling was observed in some dental follicle cells (Fig. 1c, ADP ribosylation factor d). At day 12, CON specimens presented elongation of the root dentine that was still being formed, and the epithelial diaphragm was intact. They presented positive immunolabelling in the inner enamel organ epithelium. The fibroblasts and cementoblasts adjacent to the forming root were strongly immunopositive to Smad-4 (Fig. 1e, f). At the same time point, ALN-treated specimens presented ankylosis sites between the maturing enamel matrix and bone trabeculae from

the crypt. The bone trabeculae also contacted the cells of the cervical portion of the tooth germ. Immunopositive cells were detected at the inner enamel organ. Some epithelial cells of the cervical portion of the tooth germ were also immunopositive (Fig. 1g, h). At day 30, the CON specimens at day 30 presented normal root formation and eruption. Immunopositive cementoblasts were detected over the entire root surface of CON specimens (Fig. 1i, j). No tooth eruption and root elongation occurred until the thirtieth day in ALN specimens, and several ankylosis sites were observed over the first molar germ. They presented some immunopositive cells adjacent to the enamel organ (Fig. 1k, l). TUNEL labelling was carried out in the ALN specimens at 30 days (Fig. 2). The CON specimens at the same time point were not shown because their root development occurred normally.