SEM sample preparation for cells on 3D scaffolds by freeze-drying and HMDS

Common dehydration methods of cells on biomaterials for scanning electron microscopy (SEM) include air drying, hexamethyldisilazane (HMDS) or tetramethysilane (TMS) treatment and critical point drying (CPD). On the other side, freeze-drying has been widely employed in dehydrating biological samples and also in preparing porous biomaterial scaffolds but not in preparing cells on three-dimensional (3D) biomaterials for SEM examination. In this study, we compare cells on porous hydroxyapatite (HA) prepared by air drying, HMDS and freeze-drying. The effects of fixation and using phosphate buffered saline (PBS) in the fixation were also assessed on three porous calcium phosphate (CaP) materials, namely, HA, α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP) samples. There is no significant difference in samples prepared by HMDS treatment and freeze-drying viewed at low magnification. Besides, it is better not to use phosphate buffer in the fixation step for CaP materials to avoid undesirable spontaneous precipitation of CaPs. On the other hand, fewer exchanges of liquids are required for freeze-drying and hence chemical fixation may not be absolutely required for samples prepared by freeze-drying. Other technical details of the preparation were also investigated and discussed. This study suggests both HMDS and freeze-drying can be employed to dehydrate cells on 3D scaffolds for SEM examination.