Silk membrane
B. mori cocoons were obtained from the Rural Development Administration (Jeonju, Korea). The raw cocoons were cut to 1 cm × 1-cm size pieces and used for the total group. The inner and outermost layers of the cocoons were removed by mechanical peeling (Fig. 1). The thickness of the removed portion was approximately 0.2 mm for each. Then, the remaining middle layer was cut as 1 cm × 1 cm and used for the middle group. Each membrane was then washed with ethanol. Subsequently, the silk membranes were sterilized by autoclave at 121 °C and 1.5 atmospheric pressure for 30 min. The thickness was measured by caliper.
SEM imaging
All materials were prepared for scanning electron microscope (SEM) examination. The prepared specimens underwent SEM analysis at Gangneung Center in Korea Basic Science Institute (KBSI). After immobilization of the samples on the plate, each sample was coated with gold. Specimens were imaged using a SU-70 microscope (Hitachi, Japan) operating at 5 keV.
Cell culture
In vitro tests were performed using MG-63 osteoblast-like cells (ATCC, Manassas, VA, USA). The cells were grown to 80 % confluence in Dulbecco’s modified Eagle’s medium-high glucose (PAA Laboratories, Linz, Austria) containing 1 % penicillin/streptomycin (100×), supplemented with 10 % fetal bovine serum (PAA Laboratories, Etobicoke, ON, Canada) at 37 °C in an atmosphere of 5 % CO2 and 99 % relative humidity. Media were changed every 3 days.
The mat from the total group and middle group was cut as 1 cm2-sized rectangular shape. The samples were sterilized by autoclave at 121 °C and 1.5 atmospheric pressure for 30 min and passivated in DMEM culture medium for 12 h, prior to seeding cells. A seeding density of 20,000 cells/cm2 was used, and the specified amounts of cells were seeded using 1 ml of DMEM medium. The plates were incubated in a humidified environment for a period of up to 14 days, and the medium was changed every second day. At specific time intervals, samples were analyzed for tetrazolium salt 3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and alkaline phosphatase (ALP) assay.
MTT assay and alkaline phosphatase assay
Cell viability was quantified after 24, 48, and 96 h of culture growth using the tetrazolium salt MTT assay. Briefly, cells were incubated with MTT solution (Cell proliferation kit I; Roche Molecular Biochemicals, Mannheim, Germany) in six-well plates for 4 h at 37 °C in an atmosphere of 5 % CO2 and 99 % relative humidity. Formazan crystals were solubilized with DMSO at room temperature overnight, and the product concentration was determined by measuring the absorbance at 540 nm with a Victor Multilabel counter (Perkin-Elmer-Wallac, Freiburg, Germany).
ALP was evaluated by the transformation of p-nitrophenylphosphate into p-nitrophenol at 37 °C and pH 10.2 using appropriate reactive chemicals (all from Sigma, St. Louis, MO, USA), while the specific activity of ALP was calculated based on the protein concentration of lysates determined using a commercially available colorimetric assay (#71230, AnaSpec, Freemont, CA, USA). The product concentration was determined by measuring the absorbance at 415 nm with an iMark microplate absorbance reader (BioRad, Hercules, CA, USA). ALP standard was prepared in a serial dilution of 1:50 and detected using an absorbance of 405 nm.
Animals and surgical procedure
Ten 12-week-old New Zealand white rabbits with an average weight of 2.3 kg (range 2.0–2.5 kg) were used for this experiment. This experiment was approved by the Institutional Animal Care and Use Committee of the Gangneung-Wonju National University, Gangneung, Korea (GWNU-2014-5).
General anesthesia was administered by intramuscular injection of a combination of Zoletil 50 (15 mg/kg; Vibac, Carros, France) and Rumpun (0.2 mL/kg; Bayer Korea, Seoul, Korea). The cranial area was shaved and disinfected with povidone-iodine. Two percent lidocaine with epinephrine (1:100,000) was applied to the cranial area. A midline incision was made in the sagittal plane of the skull, and subperiosteal dissection was performed to expose the calvaria. A trephine bur was used under saline irrigation to create a calvarial defect. Two 8-mm-diameter defects were created, one on each side of the midline. The calvarial defects were covered with the total or middle membranes.
Following treatment, the pericranium and skin were closed in layers with 3–0 black silk (AILEE, Busan, Korea). After surgery, the rabbits received 1 mg/kg gentamicin (Kookje, Seoul, Korea) and 0.5 mL/kg pyrin (Green Cross Veterinary Products, Seoul, Korea) intramuscularly, three times daily for 3 days.
Each rabbit was individually caged and received food and water. All animals were killed at 4 or 8 weeks after surgery. Specimens were separated and fixed in 10 % formalin. After μCT analysis, histological analysis was performed.
μCT analysis
The prepared specimens were analyzed by μCT using an animal PET/CT/SPECT system (Inveon, Siemens, Erlangen, Germany) at the Ochang Center of the Korea Basic Science Institute. The μCT scanner was set to 80 kV for the X-ray tube, with a 500-μA current for the X-ray source and a 210-ms exposure time. The detector and X-ray source were rotated through 360° in 360 steps, with 30 calibration exposures. The system magnification was set to produce an axial field of view (FOV) of 30.74 mm and a trans-axial FOV of 30.74 mm. The scanned images were reconstructed using the Inveon Research Workplace software (Siemens). Gross profiles of the specimens were obtained from reconstructed three-dimensional images. Because the initial defect was round in shape with an 8.0-mm diameter, the region of interest (ROI) was set based on the initial defect size and shape. The ROI of each specimen was analyzed for bone volume (BV).
Histomorphometric evaluation
The calvarial samples were harvested, decalcified in 5 % nitric acid for 5 days, and dehydrated in ethyl alcohol and xylene. After separation of the parietal bones through the midline sagittal suture, the calvarial samples were embedded in paraffin blocks. The paraffin blocks were sliced into sections that were then stained with hematoxylin and eosin. The section with the largest defect area was selected, along with sections 50 μm proximal and distal to the largest defect section.
The staining procedure for hematoxylin and eosin staining was as follows. First, de-wax and hydrate paraffin sections. The slide was stained in hematoxylin for 5 min. Overstained sections can easily be differentiated by agitating for a second in acid-alcohol, then washing in tap water for 5 min. The slides were immersed in eosin for 30 s and then wash them in running tap water for 1 min. The slides were dehydrated and clear in xylene.
Digital images of the selected sections were captured with a digital camera (DP-73; Olympus, Tokyo, Japan). The images were analyzed by Sigma Scan pro (SPSS, Chicago, IL). The new bone formation was calculated as the percentage of newly formed bone in the calvarial defect area.
Statistical analysis
SPSS for window ver. 19 (IBM Co., Armonk, NY, USA) were used for statistical analysis. The differences between the mean values of the total group and the middle group in the MTT and alkaline phosphatase (ALP) assays were evaluated by independent sample t tests. Paired t test was used for comparison the samples within the same animal. The level of significance was set as P < 0.05.