2.6. Histomorphometric Analysis

After 7, 14, and 21 days post-surgery, histological analysis was carried out. In detail, fractured bones were excised with surrounding soft tissue, fixed overnight in 4% PFA and incubated in an accenting sugar gradient (10%, 20% and 30% each for 24 h). The dehydrated bones were placed in molds, immerged with SCEM embedding medium (Section Lab Co Ltd., Hiroshima, Japan) and frozen over cooled hexane (Carl Roth GmbH&CoKG, Karlsruhe, Germany). Following hardening, the bones were longitudinally cut in 5 µm transversal plane sections using a cryotome (Leica CM3050S, Leica Microsystems, Wetzlar, Germany). The sections were mounted on microscope slides using cryofilm (Cryofilm type II C, Section Lab Co Ltd., Hiroshima, Japan). Movat Pentachrome staining was performed for histomorphometric analysis. For that purpose, sections were stained in Alcian Blue (8GS, Chroma), Weigert’s hematoxylin (Merck, Darmstadt, Germany), Brilliant Crocein/Acid Fuchsin (Brilliant Crocein R, Chroma, and Acid Fuchsin, Merck, Darmstadt, Germany), 5% Phosphotungstic acid PTA (Chroma), and Saffron du Gâtinais (Chroma). The stained sections were mounted in Vitro-Clud (Langenbrinck, Emmendingen, Germany). For semi-quantitative assessment of callus bridging, mosaic images of the same samples were taken using the Axioscope 40 (Zeiss, Jena, Germany) and Axiovsion Rel.4.8 software. Histomorphometry was performed through the quantification of different tissue areas in the fracture gap defined as region of interest (ROI) using ImageJ software. Specifically, mineralized tissue and cartilage area were measured in mm² as well as the ratio in percent of these tissues to the total tissue areas comprised of total cortical area, total mineralized bone area, total cartilage area as described previously [15]. Callus bridging of the same samples at day 21 post osteotomy was evaluated with the following scoring: a = complete bridging (all four cortices bridged by callus), b = partial bridging (two to three cortices bridged by callus), c = incomplete bridging (callus present, but no bridging visible), and d = nonunion (rounded cortices, minimal presence of callus). Scoring was performed independently by two blinded reviewers and averages are depicted as stacked bar charts.