S 4700 2 fe sem

PCL nanofibers and spuncoat films were imaged by scanning electron microscopy (SEM). Samples were sputter-coated with thin layer of gold (Denton Vacuum Desk II, 15 kV, 80 s) prior to imaging (Hitachi S-4700-II FE-SEM, 5 kV, WD=12 cm). Nanofiber diameters were determined by analyzing electron micrographs using ImageJ software (NIH). All nanofiber and film samples for the study were made in one batch. For determining nanofiber diameter, two nanofiber samples were randomly chosen and three evenly spaced SEM images were collected on each sample. SEM Images were analyzed and the mean nanofiber diameter was found to be 325 nm (standard deviation (S.D.) = 256 nm, n = 60 fibers).
Surface roughness of the spuncoat films was measured using atomic force microscopy (AFM, Dimension Icon, Bruker, Billerica, MA). Three film samples were analyzed with three spots per sample for a total of nine spots measured. The spot size was 50 μm × 50 μm, the scans were acquired with 256 samples per line, and images were analyzed with Nanoscope Analysis (Bruker). The root mean square (RMS) roughness was determined for each analyzed spot and the mean RMS roughness was 92.8 nm (S.D. = 10.7 nm, n = 9).

Sterile porous gelatin sponges (GelfoamTM, GS) were obtained from Pfizer (New York, NY). For imaging the pore structure, GS were sputtercoated with gold for 60 s and imaged by scanning electron microscopy (SEM, Hitachi S-4700-II FE-SEM, 3 kV, image resolution 1280 pixels by 960 pixels, 8-bit pixel depth). Pores of approximate diameter of 100 μm were visible in SEMs of the GS (Suppl. Fig. 2a).

HA/TCP particles (65:35 by mass hydroxyapatite/β-tricalcium phosphate particles, 0.5 mm to 1.0 mm nominal particle size) were obtained from Zimmer, Inc. To confirm the composition of HA/TCP particles, X-ray diffraction (XRD, DMAX 220, Rigaku Denki) was performed using copper Kα radiation (wavelength 0.1541874 nm) in the 2θ range 10 ° to 60 ° (Suppl. Fig. 2b). Scanning electron microscopy (SEM, Hitachi S-4700-II FE-SEM, 10 kV, image resolution 1280 pixels by 960 pixels, 8-bit pixel depth) verified the HA/TCP particle nominal size range of 0.5 mm to 1 mm diameter.
HA/TCP particles were heat-sterilized at 200 °C for 2 h. For regular constructs and 7-hole combi-cassettes, 40 mg of HA/TCP particles were placed in round-bottomed 2 mL polypropylene cryotubes (Nunc) and vortexed with 1 mL of culture medium. The particles were allowed to settle by gravity and the supernatant was aspirated to remove ceramic dust. For 19-hole combicassettes, 120 mg of HA/TCP particles were washed by the same procedure with 2 mL of medium.