Developer xd2

Images were acquired with a Leica SP5 upright confocal microscope with ×10, ×20, ×40 and ×63 objectives and analyzed with Fiji software 1.0 and Definiens developer XD 2.5. Immunohistochemistry images were captured with Zen Blue Software v3 (Zeiss), and whole slides were acquired with a slide scanner (AxioScan Z1, Zeiss). Oncospheres were imaged with a Leica DMi1 inverted microscope.

Histomorphometric analysis was blinded and performed at two regions of interest, representing the cortical and medullary compartments. The respective areas were manually segmented, and we classified newly formed bone and old bone using Adobe Photoshop (Adobe Inc., Mountain View, CA, USA). New bone volume per tissue volume (nBV/TV) within 200 µm adjacent to the implant surface and new bone-to-implant contact (nBIC) were determined and are presented as percentages (Figure 2). All measurements were taken using Definiens Developer XD 2.7 (Definiens AG, Munich, Germany).

Healing was monitored by two independent observers. Anteroposterior and lateral radiological views were acquired under general anesthesia immediately post-operatively, after 1 and 2 weeks and then at biweekly intervals for all animals.
After fixation in 4% PFA in PBS, osteosynthesis plates were carefully removed, ensuring no fracture occurred. One sample of the AH-sEV group was excluded, as the femur broke during preparation (see Fig. 1). During µCT scanning, explanted femora were stored in polymer sample tubes filled with formalin to prevent dehydration. Of each group, nine to ten (n = 9–10) samples were scanned in a SCANCO µCT 50 (SCANCO Medical AG, Brüttisellen, Switzerland) at 70 kVp, 114 µA filtered with 0.5 mm Al. Over a field of view of 20.4 mm, 850 Projections/180° were integrated for 475 ms with averaging 1, HW-binning 2 and reconstructed to an isotropic resolution of 12 μm. Scans were exported as DICOM slices calibrated to mgHA/cm³. Image preprocessing and measurements were performed using Using Fiji [20 (link)] and Definiens Developer XD 2.7 (Definiens AG, Munich, Germany). Bone Volume (BV) over total Tissue Volume (TV) was determined for the defined, encompassing the newly formed bone in the medullary canal, within the osteotomy region, and the periosteal callus within a defined ROI (for further details see supplementary information). BV/TV are expressed in %.

The explanted femora were examined by μCT using a SCANCO micro-CT 50 system (SCANCO Medical, Brüttisellen, Switzerland). All samples were scanned nominally to the diaphyseal axis of the femur at 90 kVp and 200 μA with a 0.5 mm Al filter. In total, 500 projections/180° were integrated for 500 ms and reconstructed to an isotropic voxel size of 17.2 μm. Volume of interest (VOI) was selected using ImageJ/Fiji [46 (link)]. Image stacks were rotated so the femur was aligned with the Z axis and the fixation pins were horizontally aligned with the X axis. The bone up to 1.8 mm adjacent to the fixation pins was excluded from measurement, resulting in a VOI between 4.6 and 5.5 mm in height. The outer border of residual old bone was marked using the polygonal selection tool and interpolated over the osteotomy gap. The VOI was thresholded and exported as a mask. Measurements were performed with Definiens Developer XD 2.7 (Definiens AG, Munich, Germany). The measurement was split into a defect region, limited to a height of 4 mm centered on the VOI and a marginal region of the remaining VOI. Both regions were split into the central volume inside the interpolation of the old bone over the osteotomy gap and the surrounding volume. In all regions, Bone Volume (BV) and Tissue Volume (TV) were measured. BV/TV was calculated and is expressed as mean ± SD (%).

Digital images were semi-automatically segmented, and tissue types were classified: (1) pre-existing/native bone of the SF, (2) newly formed bone, (3) bone substitute material, and (4) soft tissue/marrow area within the augmented area (AA). Particles smaller than 0.008 mm² were considered to be debris and ascribed to void space. Inaccurately classified areas were manually corrected under microscopic control (Adobe Photoshop, Adobe, San Jose, CA, USA). Segmentation and histomorphometric measurements were performed using Definiens Developer XD 2.7 (Definiens, Munich, Germany).
The following histomorphometric parameters were assessed in serial 100 µm zones starting from the native bone of the SF towards the apical end of the biopsies [20 (link)].

The angiogenic response of the aortic rings was analyzed by quantifying the microvessel outgrowth. A ruleset was developed using Definiens Developer XD2 (Definiens, Munich, Germany) to analyze the 3D confocal microscopy images. A series of segmentation and classification operations was performed on the DAPI channel to exclude the ring from the volume measurements, and the total volume of the objects determined to be “outgrowth” was summed for each image stack and reported. Volume data were averaged for the same ring, and then was further averaged over the multiple rings for the same animal. A t-test was used to compare the volume data between the groups.

The angiogenic response of the aortic rings was analyzed by quantifying the microvessel outgrowth. A ruleset was developed using Definiens Developer XD2 (Definiens, Munich, Germany) to analyze the transmitted light images. A new image layer (or channel) with enhanced local contrast was produced using “contrast to neighbor pixels” to distinguish the newly formed microvessels sprouting from the aortic ring. Next, a series of segmentation and classification operations was performed on the channel to exclude the ring from the area measurements, and the total area of the objects determined to be “outgrowth” was reported. Data were averaged on the picture level for the same ring, then further averaged on the ring level for the same animal, followed by a Student's t-test for group comparisons.