In the present study, calcium phosphate–coated bovine bone powder (Biocera®; Oscotec, Chunan, Korea) was used as the bone powder material, and fibrin glue from a Greenplast kit was used (Greenplast kit; Green Cross Corp., Seoul, Korea). To construct the scaffolds, 30 mg of bone powder was placed in each hexahedron-shaped hole of a mold (8 mm × 8 mm × 1.5 mm). Next, 0.2 mL of fibrinogen solution (40 mg/mL in PBS) was prepared. Recombinant human ANG (rhANG; MybioSource, San Diego, CA) (0.5 μg or 2.0 μg) was then added into the fibrinogen solution to prepare the ANG-containing scaffold. Then, the fibrinogen or fibrinogen/ANG solution was added to the bone powder and mixed well. Thrombin (Greenplast Kit; 5 U/mL) solution (0.1 mL) was added, and the composites were rapidly blended. The resulting polymerized mixture was freeze-dried for 3 days to obtain a fibrin/bone powder (FB) scaffold.
Micro-computerized tomography analysis
To evaluate the entire scaffold structure, samples were scanned with an aluminum filter using micro-computerized tomography (Micro-CT; Sky-Scan 1172TM; Skyscan, Kontich, Belgium). Three-dimensional and trans-sectional images were obtained from reconstructed scanned data set using CT-analyzer software (Skyscan).
To observe the structure of the scaffold, fabricated scaffolds were sputter-coated with gold for 120 s under vacuum. Then, the scaffolds were observed using a scanning electron microscope (SEM; EM-30; Coxem, Daejeon, Korea).
Porosity and compressive strength analysis
Porosity was measured using a mercury intrusion porosimeter (AutoPore IV9500, Oak Ridge, TN). Briefly, scaffolds were sealed in a penetrometer, weighed, and subjected to analysis . To evaluate the mechanical properties of the scaffold, compressive strength was analyzed. The fabricated scaffolds were subjected to a compression test using an Instron model 4505 universal test machine (Instron, Canton, MA) by applying a load via a 1 N load cell at a crosshead speed of 0.5 mm/min under ambient conditions.
Determination of ANG release from scaffolds in vitro
The kinetics of ANG release from the FB/ANG scaffold were determined. The fabricated scaffolds were immersed in 2 mL of phosphate-buffered saline (PBS, pH 7.4). The samples were incubated at 37 °C under continuous agitation. At various time points, the supernatant was collected and fresh buffer was replenished. The quantitative determination of ANG was performed using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). Then, the cumulative release of ANG was calculated and represented as a percentage of the total amount of ANG in the scaffold.
To evaluate the biocompatibility of the scaffold, immortalized HUVECs (EA.Hy926) were purchased from American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured in DMEM (Gibco-BRL, Gaithersburg, MD) containing 10 % fetal bovine serum (FBS) and 1 % antibiotics at 37 °C under 5 % CO2 and 100 % humidity.
Cell proliferation assay and evaluation of cytotoxicity
CellTiter96® Aqueous One solution (Promega, Madison, Wi) was used to measure cell proliferation. The EA.Hy926 cells were seeded and cultured on the FB and FB/ANG scaffolds. At predetermined time points (1, 5, 10, or 15 days), 200 μL of MTS reagent was mixed with 500 μL of culture media and added to each well. After incubation for 2 h, the supernatant was taken, and its absorbance was measured at 490 nm using an ELISA reader (SpectraMAX M3; Molecular Devices, Sunnyvale, CA). In addition, the cytotoxicity of the fabricated scaffold was evaluated using a Live/Dead® Viability/Cytotoxicity staining kit (Molecular Probe, Eugene, OR). After 3 days of cultivation, the specimen was rinsed with PBS to remove the phenol red, and reagent solution was added. After incubation for 30 min in a CO2 incubator, the samples were observed using an inverted fluorescence microscope (DM IL LED Fluo; Leica Microsystems, Wetzlar, Germany).
Cell adhesion observation
SEM was used to observe cell adhesion to the scaffolds. After 5 days of culture, the samples were fixed with 2.5 % glutaraldehyde, and post-fixation was performed with 0.1 % osmium tetroxide (OsO4; Sigma). The sample was then dehydrated with a graded ethanol series (50 %, 75 %, 95 %, 100 %, and 100 %). The samples were then sputter-coated with gold and observed by SEM (EM-30; Coxem).
All animal experiments were performed according to the guidelines of the Wonkwang University Institutional Animal Care and Use Committee (Wonkwang University IACUC; WKU11-31). In this study, 3-month-old New Zealand white rabbits weighing 2.5–3.0 kg were used. To generate calvarial defects, the rabbits were anesthetized, the calvarium was exposed through a skin incision, and circular calvarial defects were made using a trephine bur (8 mm diameter) . The FB and FB/ANG scaffolds were then implanted in the induced calvarial defects. After 1, 2, 4, and 8 weeks, the animals were sacrificed, and the bone defect regions were dissected out from the host bone. The extracted bone tissue was then fixed with 4 % paraformaldehyde buffered with 0.1 M phosphate (pH 7.2) for 5 days before further experiments.
To analyze the newly formed bone, the bone specimens were scanned using a micro-CT system (Sky-Scan 1172TM; Skyscan, Kontich, Belgium) at 60 kV and 167 μA. Then, the image data were reconstructed using CT-analyzer software (Skyscan), and a three-dimensional image was reconstructed. A cylindrical region of interest (ROI) was positioned over the defect site. The volume of the newly formed bone was measured by assigning a threshold, and the percentage of the bone volume (% BV) was calculated by dividing the volume of the newly formed bone by the total volume within the ROI.
After micro-CT scanning, the samples were dehydrated in a graded alcohol series (80–100 %), decalcified in 8 % formic acid/8 % HCl, and embedded in paraffin. Sections with a thickness of 5 μm were prepared from the samples and mounted on slides, and the samples were then stained with hematoxylin-eosin and Goldner’s Masson trichrome (MT) stain.
All experiments were performed in triplicate, and statistical analyses were performed using GraphPad Prism statistical analysis software (GraphPad Software, San Diego, CA). Significant differences among groups were identified by ANOVA followed by t-test. Values in the text are expressed as the means ± standard deviation (SD), and P < 0.05 was considered statistically significant.