Graft preparation
The scapula of a cow was purchased from a local grocery. Using trephine bur (diameter 8.0 mm), round bone grafts were cut at a thickness of 1.0 mm. The control bone was placed into a conical tube containing a 10 % ethanol solution, and the experimental bone was placed in a 10 % ethanol and 3 % 4HR solution. These tubes were placed on a rotating machine for 24 h. Then, the grafts were placed in the drying oven for 8 h. They were sterilized with ethylene oxide gas and stored at room temperature before usage. The weight of dried grafts was measured and the amount of 4HR incorporation into the experimental bone was between 10 to 15 % by weight.
X-ray diffraction, Fourier transform infrared absorbance spectra, and endotoxin test
X-ray diffraction (XRD) patterns of the samples were collected in the range of 10 to 60° (2θ) using a diffractometer (PANalytical, X’Pert Pro MPD) with a Cu-Kα (λ = 1.5418 Å) radiation source. Fourier transform infrared (FT-IR) spectrum measurements were carried out using a Vertex 80 (Bruker Optics, Germany) spectrometer coupled with a Hyperion 3000 (Bruker Optics, Germany) microscope equipped with a germanium attenuated total reflectance objective lens (ATR ×20) and a liquid nitrogen-cooled mercury cadmium telluride detector.
An endotoxin test was performed using a commercial kit, and the subsequent procedure was in accord with the manufacturer’s protocol.
Antibacterial test
Two oral pathogens (Streptococcus sanguinis, ATCC 10556, and Aggregatibacter actinomycetemcomitans, ATCC 33384) and Staphylococcus aureus (ATCC 502A) were used for antibacterial tests. The bacterial strains were cultured with brain heart infusion (BHI) (Becton, Dickinson and Company, Sparks, MD, USA) broth under aerobic conditions supplemented with 5 % CO2. S. aureus was also cultured under aerobic conditions.
The control disc and experimental disc were placed on the surface of blood agar plates (Hangang, Gunpo-si, Korea) for S. sanguinis and S. aureus and BHI plates for A. actinomycetemcomitans. For comparison, two different types of antibiotic discs (vancomycin and penicillin) were also used. The plates of S. sanguinis and A. actinomycetemcomitans were incubated at 37 °C aerobically supplemented with 5 % CO2 for 2 days. For S. aureus, the plates were incubated at 37 °C aerobically for 1 day. The maximum diameter of the inhibition zone was observed.
Scanning electron microscopic examination with EDX microanalysis and cellular attachment assay
The specimens were coated with 0.7 nm of OsO4 (HPC-1SW, Japan). Each specimen was observed using a scanning electron microscope (Hitachi, SU-70) and underwent EDX microanalysis (EDAX Genesis; Pv 77, EDAX, USA) to analyze the elements of the area of interest. The compositions of the elements were compared.
Murine macrophages from the Cell Bank (RAW264.7; Korean Cell Line Bank No. 40071) were grown on the control and experimental discs. The growth of the cell culture was stopped at 1 h after seeding by fixing the samples. All materials including raw materials were prepared for scanning electron microscopic examination. After immobilization of the samples on the plate, each sample was coated with gold and examined using a scanning electron microscope (H-800, Hitachi, Japan).
Animal experiment
This animal study was approved by IACUC (GWNU-2014-14). Seventeen 12-week-old Sprague-Dawley rats with an average weight of 300 g were used for this experiment. A dental-trephine bur was used under copious saline solution irrigation to form a full-thickness calvarial defect. An 8-mm-diameter defect was created on the rat parietal bone, and the control graft or experimental graft was placed in the defect. The control group consisted of eight animals, and the experimental group included nine animals. After the grafting procedure, the pericranium and skin were sutured with 3-0 black silk. The animals were sacrificed 6 weeks after the operation.
Histological analysis
The calvarial samples were subjected to dehydration and embedding. The segments were embedded to show the sagittal sections in paraffin blocks. The paraffin blocks were sliced (5 μm) and stained with hematoxylin and eosin. The detailed staining procedure followed the standard method in the manufacturer’s manual. The selected sections were photographed with a digital camera (DP-73; Olympus, Tokyo, Japan). The images were analyzed with Sigma Scan Pro (SPSS, Chicago, IL). The ratio of the remaining graft was calculated based on the ratio of the remaining graft to the original size of the graft.
Statistical analysis
An independent sample t test was used to compare the control and experimental groups in the animal study. The significance level was set at P < 0.05.