Histosec

Subsequently, the pieces were washed in tap water for 24 h and placed in 10% ethylenediaminetetraacetic acid (EDTA) solution containing 4.13% Titriplex^TM III (Merck KGaA, Darmstadt, Germany) and 0.44% sodium hydroxide (Labsynth^TM, São Paulo, Brazil) for a period of 7 weeks. Specimens were submitted to standard histological procedures for embedding in Histosec^TM (Merck KGaA, Darmstadt, Germany).
Semi-serial sections with a 5 μm thick coronal plane were used to reach the center of the 8 mm defect using the scale free size tool available in DP controller software (3.2.1.276—2001-2006, Olympus Corporation^TM, Tokyo, Japan). Subsequently, the sections were stained by Masson’s trichrome, hematoxylin, and eosin (for analysis of osteocytes), picrosirius red solution and against Harris hematoxylin stain (for evaluation of total collagen fibers, type 1 and 2), and immunostaining for markers of bone formation and resorption (vascular endothelial growth factor, VEGF; bone morphogenetic protein; BMP—2/4; osteocalcin, OCN; tartrate-resistant acid phosphatase, TRAP).

The specimens were washed in tap water for 24 h and immersed in 10% ethylenediaminetetraacetic acid (EDTA—a solution containing 4.13% Titriplex™ III Merck KGaA, Darmstadt, Germany and 0.44% sodium hydroxide Labsynth, São Paulo, Brazil), for a period of approximately 60 days [52 (link)]. Then, the collected bone fragments underwent successive standard histological staging and were finally included in Histosec^TM (Merck KGaA, Darmstadt, Germany). Semi-serial coronal cuts of 5 µm thickness were performed, prioritising the centre of the circular defect and stained with haematoxylin-eosin.

The pieces were then washed in running water for 24 h and subjected to demineralization in 10% EDTA (4.13% tritiplex^® III, Merck KGaA, Hessen, Germany and 0.44% sodium hydroxide^®, Labsynth, São Paulo, Brazil), with weekly changes of the solution for a period of approximately six weeks, carried out at the Anatomy Laboratory of Bauru School of Dentistry (University of São Paulo).
The bone fragments collected were submitted to standard histological processing and included in a HistosecTM (Merck KGaA^®, Darmstadt, Germany) at the Histology Laboratory of the University of Marília (Unimar). Semi-serial coronal slices with 5 µm thicknesses were made, prioritizing the center of the defect, and stained with hematoxylin-eosin, Masson’s trichrome, and picrosirius-red.
The defect images were obtained using the Leica DFC 310FX high resolution digital camera (Leica^®, Microsystems, Wetzlar, Germany) connected to the Leica DM IRBE inverted laser microscope and LAS 4.0.0 capture system (Leica^®, Microsystems, Heerbrugg, Switzerland).
Each type of fiber by color was analyzed using the Axio Vision Rel. 4.8 Ink image software (Carl Zeiss^® MicroImaging GmbH, Jena, Germany). The interlaced bone was recognized for its random and unorganized fibrillar pattern, usually with polarization colors ranging from red/orange to light green/yellow, depending on the width of the fiber.