5) (Media Cybernetics, Inc , MD, USA) The mean of all the measur

5) (Media Cybernetics, Inc., MD, USA). The mean of all the measurements (measured by one

blinded, previously calibrated examiner) was expressed as the total apposition for each animal, and then Tacrolimus divided by the time between administrations of the fluorescent markers to give the dentine apposition rate per day. It is important to comment here, that this study was reproduced twice under the same conditions. ALP plasma levels from C6 and T6 groups were measured as the release of thymolphthalein from thymolphthalein monophosphate using a commercial kit (Labtest Diagnostica S/A, MG, Brazil). Briefly, 50 μl of thymolphthalein monophosphate was mixed with 0.5 ml of diethanolamine buffer, 0.3 mmol/ml (pH 10.1), and left for 2 min at 37 °C. Afterwards, 50 μl of the plasma sample was added. This stood for 10 min at 37 °C, then 2 ml of a solution of Na2CO3 (0.09 mmol/ml) and NaOH (0.25 mmol/ml) was added to allow colour development. The absorbance was measured at 590 nm by ELISA (Molecular Devices, CA, USA), and ALP levels were calculated from a standard solution and data are expressed as U/L of ALP. The left hemimandibles from C10 and T10 groups were sectioned transversely at the first molar

region (Fig. 1a). The fragments obtained were embedded in epoxy resin (Buehler, Lake Bluff, IL, USA) and wet-polished for microhardness testing (Future Tech-FM-1E, Tokyo, Japan). Five indentations were performed INNO-406 cost at dentine mesial face of incisor, each separated 200 μm from another. Indentations were done with a 25 g load during 5 s. The mean of the values obtained from indentations was expressed as Knoop Number Hardness (KNH). Initially, it was observed in a pilot study that after polishing the incisor mesial face at a 200 μm depth into the tooth (from the outer surface), the dentine tubules were arranged transversely, exposing the peritubular and intertubular dentine features (Fig. 2). For the EDX microanalysis, the right hemimandibles GNAT2 of C10 and T10 were cross-sectioned at the first molar region (Fig.

2a). The incisors were extracted and the mesial face was wet-polished at a 200 μm depth from the outer surface, using 1200 and 2000-grit silicon carbide papers (Norton S/A, SP, Brazil) (Fig. 2b). After ultrasonic cleaning in deionized water (B-1210-MTH, Branson Ultrasonic Corporation, CT, USA), the specimens were dehydrated in alcohol until 100% and then dried in a recipient containing silica gel. The specimens were carbon-coated (Desk II Sputtering, Denton Vacuum, NJ, USA) and the elemental content of dentine was analyzed using EDX microanalysis by images obtained in SEM (JXA-840A; JEOL, Tokyo, Japan) under 25 kV and 3000× magnification. For each specimen, six images were obtained (1200 μm2 each) (Fig. 2c and d) and the element content in at.% of calcium (Ca), phosphorus (P), oxygen (O) and magnesium (Mg) was measured in the peritubular and intertubular dentine; later, the Ca/P ratio was calculated.

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