Scanning Electron Microscopy of Implants

Rabbits were euthanized on post-operative days 28 and 56 for SEM, as previously described (Pribaz et al., 2012 (link)). Briefly, the ex vivo implants were carefully extracted as not to disturb the surface structures and were immediately fixed in buffered formaldehyde (4%) and glutaraldehyde (2.5%) for 48 h and rinsed with buffer thrice (10 min each). Secondary fixation was performed for 1 h with osmium tetroxide (1%) in PBS. Dehydration was carried out by incubating the implants (15 min each) in increasing concentrations of ethanol (30%, 50%, 67%, 80%, 90%, 100% and 100%). Subsequently, the implants were immersed in decreasing mixtures of ethanol and hexamethyldisilazane (HMDS) (ethanol:HMDS=3:1, 1:1 and 1:3) followed by 100% HMDS dehydration twice (15 min each). Samples were air-dried overnight to ensure evaporation of HMDS. All implants were mounted on aluminum stub mounts, sputter-coated with gold-palladium prior to imaging under a field-emission scanning electron microscope (JSM-6700F FE-SEM; JEOL, Tokyo, Japan) at 35×, 70×, 350× and 1500× magnification.