Dotslide 2

Undecalcified histologic thin ground sections oriented in the middle of the tooth in its longitudinal axis were prepared of the second incisors of ind1, ind2 and ind3 (ref. ^{86 (link)}). For ind3 the section was carried out through the maxillary fragment hereby cutting the second incisor. Specimens were ground stepwise to a thickness of first 400 µm, then 250 µm and finally 100 µm and scanned at every step with an Olympus BX61VS digital virtual microscopy system (dotSlide 2.4; Olympus Tokyo, Japan), with a resolution of 0.32 µm/pixel. The final 100-µm-thick slides remain as a permanent record and were also used to determine the spatial distribution of Barium.
Poor enamel preservation did not allow direct daily increment count in histologic ground sections and were therefore measured in a similar way as µCT images, using the method described by Birch and Dean^{45 (link)}. Histologic images were analysed and measured independently by a different observer than µCT images.

The tissue specimens underwent a progressive dehydration process using increasing alcohol concentrations and were subsequently embedded in a light-curing resin (Technovit 7200 VLC + BPO; Kulzer & Co., Wehrheim, Germany). Precise thin-ground sections were then meticulously prepared, following the sagittal suture and centered on the defect, employing visualization software (Amira-Avizo 3D 2021.2, Thermo Fisher Scientific, Waltham, MA, USA). Further processing of the resin blocks involved cutting and grinding equipment (Exakt Apparatebau, Norderstedt, Germany). The prepared sections were stained using Levai-Laczko dye, combining azure II, methylene blue and pararosaniline. Systematic scanning and evaluation of the stained slides were conducted using an Olympus BX61VS digital virtual microscopy system (DotSlide 2.4; Olympus, Japan, Tokyo), equipped with a 20X objective providing a resolution of 0.32 µm per pixel. A comprehensive descriptive analysis was performed on the microscopy data to capture additional intricacies and details meticulously.

Ten samples were dehydrated with ascending alcohol grades and embedded in light-curing resin (Technovit 7200 VLC + BPO; Kulzer & Co., Wehrheim, Germany). Blocks were further processed using Exakt cutting and grinding equipment (Exakt Apparatebau, Norderstedt, Germany). Thin-ground sections from all samples were prepared (31 (link)), in a plane parallel to the sagittal suture and through the middle of the alveolar socket and stained with Levai–Laczko dye (Figure 1B). The slices of around 100 µm were scanned using an Olympus BX61VS digital virtual microscopy system (DotSlide 2.4, Olympus, Tokyo, Japan) with a 20x objective resulting in a resolution of 0.32 µm per pixel and then evaluated.

Calvarial samples were dehydrated with ascending alcohol grades and embedded in light-curing resin (Technovit 7200 VLC + BPO, Kulzer & Co., Wehrheim, Germany). Blocks were further processed using Exakt cutting and grinding equipment (Exakt Apparatebau, Norderstedt, Germany). Thin-ground sections, from all samples, were prepared and stained with Levai–Laczko dye in a plane parallel to the sagittal suture and through the center of the augmented area. The slices were scanned using an Olympus BX61VS digital virtual microscopy system (DotSlide 2.4, Olympus, Japan, Tokyo) with a 20× objective resulting in a resolution of 0.32 µm per pixel, and then evaluated.

After 7 or 15 days, tibiae were harvested and dissected to analyze each implant individually. Formalin-fixed (10%) samples were dehydrated in ascending grades of ethanol and embedded in LR white resin (Sigma-Aldrich, Saint Louis, MO, USA). Blocks were further processed using the EXAKT cutting and grinding equipment (EXAKT Apparatebau, Norderstedt, Germany). Undecalcified thin-ground sections were prepared through the center of the implant and stained with Levai–Laczko dye, a variant of the Giemsa dye that allows distinguishing reliably between old bone, newly formed bone, and bone debris resulting from the surgical procedure and implant placement. In this staining, woven bone appears dark pink, mature bone light pink, and soft tissue blue. The slices were scanned using an Olympus BX61VS digital virtual microscopy system (DotSlide 2.4, Olympus, Tokyo, Japan) with 20× magnification at a resolution of 0.32 μm per pixel.

All samples underwent dehydration using increasing grades of alcohol and embeded in light-curing resin (Technovit 7200 VLC + BPO, Kulzer and Co., Wehrheim, Germany). Samples were further handled using a cutting and grinding equipment (Exakt Apparatebau, Norderstedt, Germany). Thin- ground sections of around 100 µm were prepared in a plane with the sagittal suture and through the center of the defect, before being stained with Levai-Laczko dye, a variant of the Giemsa dye that allows to distinguish reliably between old bone and new bone [26 (link)].
The slices were scanned using an Olympus BX61VS digital virtual microscopy system (DotSlide 2.4, Olympus, Tokyo, Japan) with a 20× objective resulting in a resolution of 0.32 µm per pixel and then were segmented manually using Photoshop CS 4 (Adobe Systems Inc., San Jose, CA, USA). The segmented images were then measured in Fiji using the Bone J plugin based on the color-coded thresholding that was obtained from the correspondent histograms [27 (link)]. The histomorphometric analysis was performed at three regions of interest (ROIs) representing the central compartment within the defect margins, the adjacent ectocranial compartments, and the outer compartment on the surface of the host’s cortical bone.

The 16 samples were dehydrated with ascending alcohol grades and embedded in light‐curing resin (Technovit 7200 VLC + BPO; Kulzer & Co., Wehrheim, Germany). Blocks were further processed using EXAKT cutting and grinding equipment (Exakt Apparatebau, Norderstedt, Germany). Thin‐ground sections from all samples were prepared in a plane parallel to the sagittal suture and through the center of the defect and stained with Levai–Laczko dye. The slices were scanned using an Olympus BX61VS digital virtual microscopy system (DotSlide 2.4, Olympus, Japan, Tokyo) with a 20× objective resulting in a resolution of 0.32 μm per pixel and then quantified using Adobe Photoshop® software (Adobe, San Jose, CA). Histomorphometric analysis was performed at three ROIs representing (a) the central compartment within the defect margins, (b) the adjacent ectocranial compartments, and (c) the outer compartment on the surface of the host's cortical bone.