Polaron e500

Unstained polished sections of the resin-embedded implants were imaged using backscattered scanning electron microscopy (SEM-EVO MA10, Zeiss, White Plains, NY, USA) equipped with Oxford Aztec (Zeiss, USA). To increase the conductivity of the materials and, consequently, image quality, copper SEM tape was attached to each sample and to the stub prior to the imaging and samples were sputter coated with a layer of gold palladium (Polaron e500) (Quorum Technologies, Laughton, UK). SEM parameters chosen for imaging were electron high tension (EHT) 20.00 kV, magnification 26–30× and, I probe 600 pas. Images were analyzed for new bone formation (%) using ImageJ v. 1.8.0 (NIH, Madison, WI, USA). A circular region (ROI), corresponding to the defect area, was initially cropped. Then, both the total area of the defect and the area of the new bone were binarized using an interactive threshold method (default). Thresholding enabled the isolation and quantification of bone formation based on greyscale. The percentage of new bone formation was determined according to the following formula: $$\left(\frac{A bone}{A tot}\right)\times 100$$
where, A bone represents the area (in pixels) of the new bone ingrowth and A tot is the total area of interest.

On days 7 and 21, samples were rinsed with 0.1 M cacodylate buffer and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate (Merck Life Science, Gillingham, UK) overnight at 4 °C. Following fixation, the samples were washed 3 times with deionised water and dehydrated in an ethanol series (20 min each at 50%, 70%, 90%, and 100%) followed by treatment with 1:2 and 2:1 HMDS (hexamethyldisilane): EtOH (20 min) and 100% HMDS (overnight) (Merck Life Science, Gillingham, UK). The samples were then mounted on stubs and gold- sputtered coated using a Polaron e500 (Quorum Technologies, Lewes, UK). Imaging was performed with an SEM with an accelerating voltage of 20 kV (EVO ma10, Zeiss, White Plains, NY, USA).