Leica sp1600

Bone morphogenesis around the implants was examined to assess potential bone formation following PTH[1–34] treatment. The specimens containing implants were embedded in resin for sectioning (n = 6 per group from different rats). Sections (50 μm thickness) were cut perpendicularly to the longitudinal axis of the implants using a saw microtome (Leica-SP1600, Leica Biosystems, Nussloch, Germany). Scanning electron microscope (SEM, TM–1000, Japan) images of the sections were used to assess bone–implant contact, thickness of lamellar bone around the implant, and trabecular area in the SEM visual field (Fig 4A–4C). The labeled bone surface and interlabeled thickness were detected by fluorescence microscopy (Leica DM5 500B, Leica Microsystems, Bensheim, Germany) and using Image-Pro Plus 6.0 software (IPP 6.0, Media Cybernetics Inc., Rockville, MD, USA). The data were used to calculate mineral apposition rate as follows: mineral apposition rate = interlabeled thickness per 10 days (expressed as μm/day). Then the sections were stained with a tartrate-resistant acid phosphatase (TRAP) staining kit following the manufacturer’s protocol (Sangon Biotech, Shanghai, CHN) to investigate changes at the cellular level.

The specimens euthanized after 7 days were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 45 days and embedded in paraffin. The implants were unscrewed and 5‑μm‑thick sections were prepared and stained with hematoxylin and eosin (H&E). For immunohistochemistry, sections were incubated with rabbit anti‑CD63 antibody (1:100; Abclonal), rabbit anti‑periostin antibody (1:1000; Abcam) and rabbit anti‑CD11b antibody (1:4000; Abcam) overnight at 4 ℃ followed by incubation with a horseradish peroxidase (HRP)‑conjugated secondary antibody. Sections were examined with a digital pathology scanner (Aperio AT2; Leica Biosystems, USA) and evaluated by calculating the mean optical density (MOD) of CD63, periostin and CD11b expression using Image-Pro Plus 6.0 (Media Cybernetics, USA).
The specimens euthanized after 28 days were dehydrated in a gradient series of ethanol and embedded in polymethylmethacrylate. Sections with a final thickness of 50 μm were prepared using a saw (Leica SP1600; Leica Biosystems) and stained with methylene blue acid solution. Digital images at 25× magnification were acquired with an Axio Imager.Z2 (Zeiss) and analyzed for the bone-implant contact (BIC) ratio with Image-Pro Plus.

Each RHG with the attached abutment was rinsed in saline and then chemically fixed in buffered 10% formaldehyde solution (Merck KGaA, Darmstadt, Germany) for 1 day at 4°C, followed by rinsing in tap water, dehydrating in ethanol, and embedding in methylmethacrylate.29 Using a microtome (Leica SP1600, Leica Biosystems, Wetzlar, Germany), the tissue blocks were cut through the longitudinal axis of the implants into 250‐μm‐thick slices (3‐4 total, 500 μm apart) according to a systematic random sampling protocol.30 All slices were then glued to Plexiglas specimen holders and ground down to a final thickness of 80 to 100 μm. The slices were then surface‐polished and surface‐stained with McNeal's Tetrachrome, basic Fuchsine, and Toluidine blue.31 The microscopic sections were visualized and recorded with a Nikon Eclipse 80i microscope. Epithelial down‐growth along the abutment surface was determined from photographs using NIS‐Elements AR 2.10 imaging software (Nikon Instruments Europe B.V., Amsterdam, The Netherlands).