4.4. Quantitative Analysis of Histological Bone Biopsy Samples
Quantitative histological analysis of bone biopsies taken after six months of healing was performed on the same samples previously prepared for pathohistological analysis. The digital photomicrographs were stored in an uncompressed format used to store high-resolution images until analysis. The digital photomicrographs were loaded into the free ImageJ computer program (Wayne Rasband, National Institute of Health, Bethesda, MD, USA). All the photomicrographs were taken under the same conditions (magnification of 100, 200, and 400 times, PNG format). Before starting the analysis, parameters such as setting the scale on the basis of the known distance and converting it to the unit of length (μm) were set. Also, the possibility of manual correction was set to exclude from the analysis any edge regions that were not completely clear or that represented an artifact. Each sample was analyzed individually such that the digital micrograph was adjusted using the threshold option, with manual manipulation allowing areas of interest (ROI) to be marked in different colors. Accordingly, the areas of newly formed bone, residual BSB, and soft tissue structures were determined in relation to the total area of the histological sample in the field of view, and after all the results were processed, the obtained areas were converted into volume percentages (%). All the preparations were additionally examined by two independent researchers.