Silk fabric membrane
The silk fabric membrane (SFM) was kindly provided by Sanju Myungju Co (Sangju, Korea). The silk fabric is the plain weave with a waft density of 45 yarns/inch and a weft density of 47 yarns/inch. The waft yarn is a 140-denier silk filament, and the weft yarn is a twisted yarn from 21d to 25d silk filaments. This silk fabric membrane was degummed to remove sericin before the experiment. The crystallinity of silk fabric is 55.7 % calculated from Fourier transform infrared spectroscopy measurement result. The average pore size of silk fabric membrane is 12,792 μm2 determined by digital optical microscope (Toolis, Daegu, Korea).
The SFM was prepared with 10 mm in length and 10 mm in width for animal experiment. The thickness of SFM was approximately 0.3 mm in dry condition and 0.5 mm in wet condition.
Animals and surgical procedures
Nine 10-week-old New Zealand white rabbits were used in this experiment, which was approved by the Institutional Animal Care and Use Committee of Gangneung-Wonju National University, Gangneung, Korea (IACUC GWNU- 2014–15). General anesthesia was induced by intramuscular injection of a combination of 0.5 mL of tiletamine and zolazepam (125 mg/mL; Zoletil; Bayer Korea, Seoul, Korea) and 0.5 mL of xylazine hydrochloride (10 mg/kg body weight; Rompun; Bayer Korea). The cranium area was shaved and disinfected with povidine-iodine. A longitudinal incision was made on the midline of the cranium area. Sharp subperiosteal dissection was performed to expose the parietal bones. A dental trephine bur was used under saline irrigation to create a bilateral full-thickness calvarial defect. Two defects 8 mm in diameter were created, one on each side of the midline. Either the 4HR-incorporated SFM or the conventional SFM was placed on the calvarial defects. Some defects remained uncovered and served as the control (Fig. 1). Assignment to each group for the corresponding defect was performed randomly, and each group was composed of six animals (six defects for each group). None of the animals received the same membrane in both calvarial defects. Then, the pericranium and skin were closed in layers with 3-0 black silk. Each rabbit was individually caged and received food and water. Nine animals were sacrificed at 8 weeks after the operation.
Hematoxylin and eosin staining
The bone samples were decalcified using 5 % nitric acid for 48 h. The right and left parietal bones were separated through the midline sagittal suture. Both segments were embedded to show the sagittal sections in the paraffin blocks. Then, the sections were sliced and stained with hematoxylin and eosin (H&E).
Paraffin-embedded tissue blocks were sliced to a thickness of 5 μm. The sections of each tissue were carefully placed on silane-coated slides. These slides were incubated at 60 °C for 24 h. After cooling at room temperature, the tissue slides were soaked in 100 % xylene for 5 min in triplicate. The tissue sections were then hydrated by the consecutive application of high- to low-grade ethyl alcohol. Fully hydrated tissue sections were washed with distilled water. After that, tissue sections were stained with Harris modified hematoxylin solution (Sigma Aldrich, St. Louis, MO, USA) for 10 min at room temperature. Then, de-staining was performed with 1 % acid alcohol for 1 s. De-stained tissue sections were washed in running tap water for 10 min. Next, Eosin Y solution (Sigma Aldrich, St. Louis, MO, USA) was applied on the tissue sections for 1 min. Then, after gradational hydration with ethyl alcohol and clearing with xylene, the tissue sections were fixed by paramount solutions.
Histomorphometric evaluation
The sagittal section showing the widest defect area was selected. Digital images of the selected sections were taken using a digital camera (DP-20; Olympus, Tokyo, Japan). The images were analyzed by SigmaScan Pro 5.0; SPSS Science, Chicago, IL, USA). The total amount of new bone was calculated as a percentage of the total region of the defect. The residual membrane was also calculated as a percentage of the residual membrane area 8 weeks postoperatively compared to the original area of membrane.
Statistical analysis
An ANOVA test was used for comparison of new bone formation of the three groups, and the LSD method was used as a post hoc test. An independent-samples t test was used for the comparison of the residual membrane of the two groups. Statistical significance was set at P < 0.05.