Rational Design of Artificial Cellular Niches for Tissue Engineering - Tissue Engineering: Computer Modeling, Biofabrication and Cell Behavior - page 130

Fig. 3 Left : Detailed image of an Adipose-Derived Stem Cell (ADSC) cultured on a collagen
hydrogel, where the alignment of fibrils is clearly visible around the cell-matrix adhesion sites.
Micrograph obtained using a FIB-FESEM instrument (FEI Quanta 3D™ DualBeam™). Right :
FIB-FESEM micrograph of an ADSC cultured on a collagen-agarose gel; alignment of collagen
fibrils is also remarkable at the main adhesion sites. Images courtesy of Laboratory of Tissue
Engineering and Biomaterials, CEIT, San Sebastian, Spain
the last two above-mentioned variations of microscopy have been particularly rele-
vant for the nanostructural and mechanical characterization of the natural tissue in
Tissue Engineering.
SEM allows for the acquisition of very high magnification images; and even
more, Field Emission Gun (FE)-SEM instruments, which are widely used for the
nanostructural characterization of surfaces [ 17 ]. Figure 3 shows two examples of
cells cultured on fibrous scaffolds. Moreover, these electron microscopes can be
combined with a Focused Ion Beam (FIB) module, which allows for high precision
milling of samples. As a result, 3D nanostructural analysis can be performed. The
main drawback of this technique is the need of working with dehydrated samples,
which enhances the risk of altering the microstructure during sample preparation.
To overcome this limitation, cryogenic modules have been developed which do not
require dehydration of samples.
Atomic Force Microscopy (AFM) and other local contact methods, such as
nanoindentation, are recently drawing attention for the mechanical characterization
of biological tissues [ 18 ]. In these tests, both the load and displacement of a small
probe, the indenter tip, are continuously monitored as the probe is loaded onto the
surface of interest. Local contact methods are ideal for probing small samples, local
gradients and heterogeneities in biological materials; they are also powerful tools for
examining hierarchical and multiscale organization of cells and tissues. In addition,
no extensive sample preparation is required prior to mechanical testing, in contrast
to other techniques. Finally, most nanoindentation instruments allow purposeful ex-
ploration of a variety of different deformation modes by changing experimental time
scale, indenter tip geometry and loading conditions. Although earliest applications
of these techniques had been designed for the analysis of metallic and ceramic ma-