Technovit 7500

Histomorphometric assessment was performed to evaluate the intracortical remodeling. Nondecalcified mandibles were dehydrated in graded ethanol (70%, 95%, and 100%) and embedded in methyl methacrylate (MMA, Technovit 7500, Kulzer, Hamburg, Germany). Mandibles were sectioned (~100 μm thick) throughout the molar region in a mesial-distal direction using a diamond wire saw. The samples were mounted on slides and ground to a thickness of 80 μm.
To ensure the measurement was performed at a similar region in all the animals, sections containing the deepest root of first molar in each animal were selected. The sections were examined under fluorescence microscope (Nikon, Tokyo, Japan).

Three kinds of fluorochrome labeling, including alizarin red (25 mg/kg), calcin green (30 mg/kg), and oxytetracycline (50 mg/kg), were injected in chronological order (Table 1). For the rabbits sacrificed at week 4, the fluorochrome labeling was injected at week 1, week 2, and week 3, respectively. For the rabbits sacrificed at week 12, the fluorochrome labeling was injected at week 3, week 6, and week 9, respectively. After sacrifice, samples were embedded with methyl methacrylate (MMA, Technovit 7500, Kulzer, Hamburg, Germany). The embedded sample was sawed along the long axis of implant into a section with 200–500 μm thick, which was then polished to about 100 μm. The prepared slides were examined under fluorescent microscopy (FluoView FV 1000; Olympus, Tokyo, Japan). The bone growth rate was calculated as the average distance between every two fluorochrome-labeled lines over the known time interval of two corresponding injections.

Alizarin red (30 mg/kg) and calcein green (50 mg/kg) were injected sequentially at week 1 and week 2 after the implant surgery. A total dose volume of 3 ml of each labeling solution was used in each animal. The samples were dehydrated in gradient ethanol and embedded in methyl methacrylate (MMA, Technovit 7500, Kulzer, Hamburg, Germany). The sections were made by using a microtome saw (EXAKT E300CP, Germany). Different kinds of sandpapers were used to get the final thickness of 100 um slides. Fluorescent microscopy (Leica, TCS SP2, Germany) was used to analyze the slides. The average distance between the fluorochromes labeling each day was defined as the bone growth rate.