Plank rychlo s solution

After the micro-CT measurements, tissue samples were prepared for histological analysis. The samples were decalcified in Plank-Rychlo’s solution (MUTO Pure Chemicals Co., Tokyo, Japan) for 5 days, dehydrated in graded ethanol baths, and then embedded in paraffin. The embedded samples were cut into 4-μm-thick sections using a microtome and stained with hematoxylin-eosin (HE; Sigma, St. Louis, MO) following standard protocols. Stained slide images were acquired using a standard light microscope (Leica DM500, Leica Microsystems, Wetzlar, Germany) equipped with a SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). In performing the histomorphometric analysis, four sites were randomly selected for each slide and the new bone formation area of each region was calculated using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MI, USA).

After the micro-CT measurements, the harvested samples were prepared for the histological and histomorphometric analyses. The samples were decalcified in Plank-Rychlo’s solution (MUTO Pure Chemicals Co., Tokyo, Japan) for 5 days, dehydrated in graded ethanol concentrations, and then embedded in paraffin. The embedded samples were longitudinally cut into 4-µm-thick sections and stained with hematoxylin and eosin (H&E; Sigma, St. Louis, MO, USA). For the qualitative analysis of the bone regenerative process, the stained samples were evaluated, especially in the border and center areas, using a standard light microscope (Leica DM500, Leica Microsystems, Wetzlar, Germany) connected to a SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Four sites in each sample were randomly selected to calculate the new bone formation percentage by using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).

After micro-CT analysis, samples were decalcified in Plank-Rychlo's solution (MUTO Pure Chemicals, Tokyo, Japan) for 7 days, dehydrated in graded ethanol baths (80–100%), and embedded in paraffin. Using a rotary microtome, 4-μm-thick sections were cut from the samples and stained with hematoxylin–eosin (HE; Sigma-Aldrich) and Masson’s trichrome. Histologic observations and images were acquired using a TissueFAXS plus system (TissueGnostics GmbH, Vienna, Austria). For histomorphometric analysis, four sites were randomly selected for each slide, and the new bone formation area of each region was calculated using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MI, USA).