Preparation of animal
Thirty-six Sprague-Dawley male rats (15 weeks old, Koatech, INC. Korea) weighing between 250 g and 300 g comprised the animal experimental model used. The animals were housed individually in standard rat cages maintained under an ambient temperature of 24 °C to 26 °C and a 12/12 h light/dark cycle. The animals had free access to drinking water and standard laboratory pellets. This study was conducted at the Pusan National University Institutional Animal Care and Use Committee (PNU-2011-000254 ).
Experimental materials and surgical procedures
Each bony defect was stuffed by Bio-Oss®, inorganic bovine bone, as xenograft material. rhBMP-2 was produced in E. coli using genetic engineering (Cowellmedi Co, Busan, Korea). As for rhBMP-2, 100 μg/1 mL concentrations were used. Alendronate (Sigma, St. Louis, MO, USA) was used as bisphosphonate. 1 mM (low concentration) and 10 mM (high concentration) alendronate were conducted this study.
The animals were anesthetized with a mixture of 10 mg/kg of xylazine hydrochloride (Rumpun® Bayer, Korea) and 100 mg/kg of ketamine chloride (Ketalar®, Yuhan Corporation, Korea). The dorsal area of the rat cranium was shaved before surgery, and the surgical field was prepared with an iodine solution. A midline skin incision was performed on the skull, and the periosteum with the temporalis muscle was reflected laterally. Two symmetrical round 5 mm diameter bony defects were then formed in the calvaria using 5 mm diameter trephine (Hee Sung Corp., Seoul, Korea). The bony defect were grafted with Bio-Oss® only (group 1, n = 9), Bio-Oss® wetted with rhBMP-2 (group 2, n = 9), Bio-Oss® wetted with rhBMP-2 and 1 mM alendronate (group 3, n = 9) and Bio-Oss® wetted with rhBMP-2 and 10 mM alendronate (group 4, n = 9). Then, the muscle layer was closed with 4–0 Vicryl® sutures in a continuous fashion, and the skin with 3-0 VicrylⓇ sutures. Gentamycin at 5 mg/kg was injected for prevention of infection after surgery. The animals from each group were sacrificed at 2, 4 and 8 weeks after surgery. The skin was dissected, the calvaria harvested and immediately immersed in a 10 % tempered solution of formaldehyde.
Histology
Each specimen was fixed in 10 % formaldehyde solution, decalcified in formic acid for 48 h, and embedded in paraffin. Serial cross-sections (5 μm) were cut through the larger diameter of the defect and stained with hematoxylin-eosin (H-E). The H-E stains reveal the cellular reactions indicating bone formation. The slides were photographed with the use of a virtual slide system (Scanscope CS system, Aperio Technologies, Vista,CA).
Histomorphometric analysis
The Aperio Technologies Scanscope CS system is useful for calculating new bone formation areas on H-E stained slides. The calculation, involving just the drawing of the newly formed bone outlines, is easily done. Slides in each group were scanned by virtual slide system microscopy (X100), and then 2 slides from each group were selected in the 2, 4, 8 weeks. To calculate the new bone formation area, 4 sites were randomly selected for each slide, the photographs of which were 0.600 mm × 0.500 mm. In this study, we applied 2 statistical methods to the significance testing of each group. The dependent variables of the control and experimental groups were averages and standard deviations. The difference of the dependent variables in each group for the 2, 4, 8 weeks was analyzed by Kruskal-Wallis test and Tukey’s post hoc test. The collected data were analyzed with the use of SPSS 18.0 software (SPSS Inc., Chicago, IL, USA).
Immunohistochemical analysis
The 5 μm thick slice maintained at 60 °C in an oven for an hour. After that, the sample was washed with distilled water after being hydrated using several steps of alcohol after the paraffin was removed. This treatment was repeated four times for five minutes with xylene. And then the sample was washed three times with the buffer solution for three minutes. In order to remove the intrinsic peroxide within the tissue, the sample was reacted with 0.3 % H2O2 solution. The sample was washed four times with buffer solution in order to inhibit the unusual combination within the tissue, after reacting with blocking serum (goat ImmunoCruz staining system, Santa Cruz Biotechnology, Inc., USA) for an hour, and reacting for a night with a dilution of each primary antibody, OPG, RANKL, Collagen type I (Santa Cruz biotechnology, OPG 1:120, RANKL 1:40, collagen type I 1:100). After being washed with the buffer solution, the sample was reacted with a secondary antibody labeled Biotin for an hour, washed four times again with the buffer solution, and reacted with the enzyme conjugate streptavidin (goat immunocruz staining system, Santa Cruz Biotechnology) for an hour at room temperature.
The tissue section was again washed in PBS three times for 10 min and was colored for 3 min in a solution mixed with diaminobenzidine chromogen and hydrogen peroxidase. The section was then washed in Tris buffer, PBS and distilled water for 10 min each. After that, it was counterstained with Harris hematoxylin and then mounted.
After each staining we evaluated the slides by immunohistochemical reactivity for RANKL using a scoring system of −, +, ++, and +++, which corresponded to absent staining, weak staining (<25 % of cells), moderate staining (<50 % of cells), and strong staining (>50 %), respectively. In the evaulation of OPG, collagen Type I expression, the boundary of cell counting was using 50 % and 75 % instead of 25 % and 50 %.