Animals
Eighteen adult male Sprague-Dawley rats (each weighing approximately 0.40 kg) were used in this study. The rats were housed in standard cages and were fed under standard laboratory diet. All animal handling and surgical procedures were performed according to the animal care guideline and use of laboratory animals. This experiment was approved by the Chosun University Institutional Animal Care and Use Committee, Gwangju, South Korea (CIACUC2014-A0027).
Surgical procedure
General anesthesia was induced by intramuscular injection with 0.5 mg/kg of a combination of Zoletil®50 (tiletamine + zolazepam 1:1; Virbac S.A., Carros, France) and Rompun (xylazine; Bayer Healthcare Korea, Korea) in a ratio of 1:1. After shaving and painting with povidone-iodine, local anesthesia was treated with 2 % lidocaine (Yuhan Co., Seoul, Korea) with 1/100,000 epinephrine. A mid-sagittal incision was performed for exposure of parietal bones. Using a diamond bur, two critical-sized defects (each diameter ≥8 mm) were created in both sides of the parietal bone under normal saline irrigation (Fig. 1). The defects were filled with the following: (1) group 1: no graft as control group (n = 10 defects); (2) group 2: Bio-Oss® (Geistlich Pharma Ag., Swiss, n = 11 defects); and (3) group 3: Bongros® (Bio@ Inc., Seongnam, Korea, n = 15 defects) were used by mixing with saline solution to fill one defect each. The wounds were sutured with Vicryl 4-0 (Ethicon Inc., GA, USA). All the animals received a single intramuscular injection with 0.1 ml/kg of antibiotics (gentamicin, Daesung Microbiological Labs. Co. Ltd., Seoul, Korea) for 3 days.
Histologic and histomorphometric analysis
Histologic analysis
The animals were sacrificed at 4 and 8 weeks after surgery. Histologic samples were harvested including graft sites. The samples were fixed in 10 % neutral-buffered formalin for 3 days and then decalcified in 12.5 % EDTA (Sigma-Aldrich) pH 7.0 for an additional 15 ± 3 days. The samples were serially dehydrated in ethanol in a tissue processor (Shandon Citadel 1000, Thermo Scientific, Franklin, MA, USA) and embedded with paraffin (Leica EG 160). Sections of 5-μm thickness were taken using a microtome (Leica RM 2135). The slides were deparaffinized with xylene and rehydrated with serial concentrations of ethanol.
The slides were stained with hematoxylin-eosin (Sigma-Aldrich) for the use of optical microscope. Using optical microscope, the sections were examined for evaluation of bone formation and integration of the reconstructed areas into the neighboring bone tissue.
Histomorphometric analysis
Using optical microscope (Primo Star, Carl Zeiss Co., Ltd., Germany) and imaging software (AxioVision 4.7.2), images were obtained for morphology and for analyzing fraction of bone healing. All images were transferred from AxioVision to Adobe Photoshop Elements 7.0 software. Histomorphometric assessment was performed by an individual who was blind to the treatments. The area of the defect and the newly formed bone was measured using Adobe Photoshop Elements 7.0 software. These measurements were used in the following formula to determine the fraction of bone regeneration. The average fraction of bone regeneration from each group was determined to be the average fraction of bone regeneration:
$$ \mathrm{Fraction}\ \mathrm{of}\ \mathrm{bone}\ \mathrm{regeneration} = \left({A}_{\mathrm{n}}/{A}_{\mathrm{d}}\right)\times 100\left(\%\right) $$
where A
d is the area of the original defect and A
n is the area of the newly formed bone within the defect site (Fig. 2).
Because bone regeneration was processed from the edge of the defect and towards the center of the defect, two portions (lateral and central portions) of the defect were used. First, the assessment of bone healing was performed for each portion, and then, the two portions were summarized by the following formula to evaluate the fraction of bone healing at the whole defect site:
$$ \mathrm{The}\ \mathrm{fraction}\ \mathrm{of}\ \mathrm{bone}\ \mathrm{regeneration} = \left({A}_{\ln }+{A}_{\mathrm{cn}}\right)/\left({A}_{\mathrm{ld}}+{A}_{\mathrm{cd}}\right) \times 100\ \left(\%\right) $$
where A
ld is the area of the original defect in the lateral portion, A
cd is the area of the original defect in the central portion, A
ln is the area of the newly formed bone within the lateral portion of the defect, and A
cn is the area of the newly formed bone within the central portion of the defect.
Statistical analysis
All data were expressed as mean and standard deviation. The new bone formation rate was analyzed via one-way ANOVA and post hoc test.