Bioresorption and Degradation of Biomaterials - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 326

Fig. 1 Cell-based resorption assay with an osteoclast (marked in pink) on the surface of a
calcium-orthophosphate (scanning electron microscopy 92300)
is critically dependent on the detection of resorption pits, which typically have to
be stained to be visible. The same is true for CLSM. If resorption is not detectable
in LM and CLSM due to lack of staining and low contrast of the material, scanning
electron microscopy can be used [ 97 ]. For this, the specimen has to be specially
prepared to be suitable for the necessary vacuum environment during the analysis.
Typically, cells are first removed from the material surface; the specimen is dried
in an increasing ethanol series and then sputtered with gold to enhance the surface
contrast. Several parameters such as pit number and pit area can be analyzed using
2D imaging in combination with image analysis software [ 88 ]. For 3D parameters
like pit depth and pit volume, other methodologies are necessary. For this purpose,
3D-SEM based on stereophotogrammetry methods has been tested [ 97 ]. This
technology uses images obtained from different angles of the pit to calculate the
volume of the pit [ 98 ], but is relatively labor-intensive. Our group has recently
introduced infinite focus microscopy as a possible tool for measuring bone sub-
stitute resorption [ 52 , 53 ].The IFM system makes use of the relatively small focal
plane of light microscopes and is based on algorithms that can automatically detect
focused areas in a series of images. The algorithm then combines only the focused
parts of each image with the position information of the image in the image stack.
From these data the 3D surface can be reconstructed with a high resolution (\1 lm).
No sample pre-processing step is necessary and the size of the detection area is
limited only by the computing power.