4.5. Specimen processing

Study II: The tissue specimens with implants were fixed in 10% PBS formalin for 7 to 14 days. After fixation, the soft tissue and fibrous capsule at one end of the implant were cut and the implant was extremely gently removed, leaving a tidy hole. The samples were processed and mounted in paraffin. The block was cut precisely perpendicular to the longitudinal axis of the implant hole. Several 4 µm sections were cut from each sample specimen at the midsagittal site of the implant hole. At least 2 sections of each sample were inspected.

Study III: The tissue specimens with implants were fixed in 10% PBS formalin for 7 days. After the fixation, one femur from each group was placed in a hard resin embedding process without removing the implant. These samples were dehydrated in a graded alcohol series and embedded in metacrylate (Technovit®, Kulzer Gmbh, Germany) using the standard method (Donath et al. 1982). The specimens were further cured at 50°C overnight, and then cut with a low-speed diamond saw along a perpendicular plane at the middle of the implant. One half was cut into thin ground sections (30 µm) using a sandwich method and the Exact Cutting-Grinding and Micro-Grinding system (EXAKT Apparatebau, Germany). The sections were mounted and stained with Goldner-Trichrome and Haematoxylin-Eosin stains for histology and a morphometric light-microscopic computer-aided examination. The other half of each sample embedded in hard resin was used for field emission scanning electron microscopy (FESEM). The metacrylate-embedded thick sections were treated for 10 min. in saturated NaOH in alcohol solution to remove the plastic from the tissues on the surface of the block before coating with carbon. The other four femurs in each group were decalcified and routinely processed and mounted in paraffin. Before embedding, the bone, the soft tissue and the fibrous capsule were cut at one end of the implant bed and the implant was gently removed, leaving a clear-cut hole. The block was cut perpendicular to the longitudinal axis of the bone and the implant hole. Sections of 4 µm were cut from each specimen at different midsagittal sites of the implant hole and stained with Haematoxylin-Eosin.

Study IV: The sample fixation and hard-resin embedding process were as described above. After that, the specimens were cut longitudinally with a diamond saw at the middle of the implant. One half was then cut transversely at the osteotomy site and at a site outside the osteotomy, but in the nail area. Thin ground sections (30 µm) were made, as in study III. The sections were mounted and stained with Goldner-Trichrome stain for histology. Two femurs in the 26- and 60-week groups and one in each of the earlier groups were taken for pQCT measurements after removal of the nail. The nail was taken for a FESEM analysis of corrosion marks. After pQCT, the bones were decalcified and then routinely processed and mounted in paraffin. Several 4 µm vertical sections were cut and stained with Haematoxylin-Eosin. Organs for metal ion examination were dissected using a plastic spoon, quick-frozen, stored in polyethylene casks and kept at -20 ºC until analysis.