Measures of dentin GLGs

Counts of dentin GLGs followed previous protocols (Adulyanukosol, Amorena & Miyazaki, 1998; Mitchell, 1978), incomplete or worn tusks were excluded from this study. The tusks were sectioned longitudinally in the mid-sagittal plane using a Buehler Isomet low speed saw (Struers Minitom, Denmark), polished using progressively finer grades of sandpaper (150–1,200), and then etched in 10% formic acid for 1–3 h. Tusks were washed in tap water for 10 min to remove the etching reagent, followed by immersion in 100% acetone for 2–3 min to increase the clarity of the GLGs. Samples were rewashed in tap water for 12 h to remove the formic acid. The etched tusk was dried at room temperature for 1–2 days and then rubbed with laser toner powder (Canon, Thailand). The number of GLGs in each tusk was counted in triplicate (Fig. 2).