Cutting and grinding system

Rats were injected with alizarin-3-methyliminodiacetic acid (30 mg/kg body weight, i.p.) on the second day and calcein (20 mg/kg body weight, i.p.) on the 11th day. Rats were euthanized on the 14th day. Cross sections were cut parallel to the occlusal surface and polished to 60–80 μm with an EXAKT cutting and grinding system (EXAKT Apparatebau, Germany). The distal alveolar bone of the mesial palatal root was used for analysis. The bone mineral apposition rate (MAR) was measured using Bioquant software (BioQuant, San Diego, CA, USA). The maximum distance between the red fluorescent line and the green fluorescent line on the photo was measured.

Eight rabbits were euthanized for histomorphometric analysis at 4 weeks after implantation. Mini-implant specimens with the surrounding tissue were embedded in light-curing resin (Technovit 7200VLC; Heraeus Kulzer, Dormagen, Germany), sliced, and ground into 40–50 μm using the Exakt cutting and grinding system (Exakt Apparatebau, Norstedt, Germany) [23 (link)]. Specimens were stained with hematoxylin and eosin (HE).

After micro CT scanning, tibial bone specimens were trimmed to five cm length (3 cm defect zone with 1 cm adjacent cortical bone proximally and distally) and fixed in 4% paraformaldehyde for one week before being transferred to 70% ethanol. For histological analysis, the mid-defect regions were sectioned in the transverse and sagittal plane. The sagittally sectioned samples were used for paraffin embedding. For processing decalcified samples into paraffin, bone samples were decalcified in 15% EDTA for 6–8 weeks at 4 °C. The samples were then dehydrated in increasing ethanol concentrations in a tissue processor (Excelsior ES, Thermo Scientific, Waltham, MA, USA), and embedded in paraffin. Five-micrometer sections were obtained using a rotary microtome (RM 2265, Leica, Wetzlar, Germany).
The mineralised bone samples to be cut on the sagittal plane were embedded in methylmethacrylate resin (Technovit 9100, Heraeus Kulzer, Hanau, Germany). Six-micrometer sections of the full length of the defect (5 cm long) were cut using a sledge microtome (Polycut S, Reichert technologies, Depew, NY, USA) and 40–50 µm sections were cut using an EXAKT cutting and grinding system (EXAKT, Norderstedt, Germany). Sections were cut at two thicknesses because the thicker ones preserve gross morphology more accurately and the thin sections reveal more cellular detail.

For histological analysis, the extracted tissues from the PMMA/HA composite and PMMA containing barium sulfate were embedded in resin (Technovit 7200 VLC, Kulzer, Hanau, Germany). Thin sections of blocks at a thickness of 40 µm were prepared using an Exakt cutting and grinding system (Exakt cutting and grinding system, Exakt, Oklahoma City, OK, USA). They were then stained with Goldner’s trichrome. Optical microscopic images of samples were prepared using Axioskop microscopy (Olympus BX51, Olympus, Tokyo, Japan). In the optical images, the mature bone matrix was stained green [21 (link)]. The volumes of bone tissues newly formed around and within the PMMA and PMMA/HA composite were calculated on the basis of micro-CT analyses.