Biomedical Engineering Reference
In-Depth Information
of internal uneven surface structures of the scaffold under normal optical instruments, such as the
traditional light microscope. The inability to easily monitor the inner structure of scaffolds poses a
major challenge for TE as it impedes the precise control and adjustment of the parameters affecting
cell growth and ECM deposition in response to various mimicked culture conditions. In this section,
rather than providing an extensive description of the principles of operation of the most commonly
used imaging techniques, we outline their potential and limitations as monitoring tools for TE.
3.4.1 M
ICROSCOPY
3.4.1.1
Scanning Electron Microscopy
A common way for assessing general structural features of the scaffolds is by scanning electron
microscopy (SEM). In this technique, the samples are irradiated with a high-energy-focused elec-
tron beam. The excited molecules from the sample then emit secondary electrons that are detected
in a scintillator photomultiplier, and the ensuing signal is turned into two-dimensional (2-D) inten-
sity distribution, which can be viewed as a digital image. Because the brightness of the signal
depends on the angle of the electron bean in relation to the surface, the obtained digital images
refl ect some features of 3-D structure. Most of the polymeric and biological samples are not good
electron conductors. So, in order to achieve contrast, sample preparation involves coating with elec-
tronically dense molecules such as gold.
SEM can provide high-resolution images (up to 5 nm) evidencing general morphology, surface
topography, pore geometry, cell distribution, and morphology. SEM is also useful for monitoring the
morphological changes that occur along the
in vitro
culture. Nevertheless, this technique is limited
to the surface of the scaffolds and is not suitable for assessing the 3-D structure of the scaffold, nei-
ther the cell distribution throughout the construct. The combination of SEM with physical methods
such as mercury intrusion porosimetry, which determines porosity and pore interconnectivity, pro-
vides a more detailed description of the scaffolds' architecture.
122-124
However, besides the inability
to provide 3-D information, SEM presents another severe limitation: the processing requirements.
Not every sample can be easily processed for SEM analysis, and this is particularly verifi able for
samples containing cells and ECM. Furthermore, this method is destructive and requires the sacri-
fi ce of the analyzed samples, which sometimes are so diffi cult to obtain.
3.4.1.2
Light and Fluorescence Microscopy
Light microscopy is a traditional but very powerful tool in the biological fi eld. One of the most
important areas relying on the use of microscopy is Histology. Histology is the study of animal
and plant Tissue at a microscopic level. The use of conventional microscopy in combination with
various biochemical stains that selectively enhance contrast, enables the distinction of individual
components within a complex biological system. Traditional histological techniques have shown to
be useful in the assessment of the evolution of engineered tissue constructs, since they have enabled
the localization of cells, matrix proteins, and matrix calcifi cation throughout the constructs, as well
as the visualization of the morphological aspects of the scaffolds.
125,126
Figure 3.1 shows a light
microscope image of the grown in a PLLA scaffold for 4 weeks. Another variant of histology is
fl uorescent immunohistochemistry. The principles are quite the same, but in this case the molecules
of interest are marked with fl uorescent dyes, usually linked to very specifi c antibodies
127
, and the
tissue sections are analysed in a fl uorescent microscope. The high resolution and great contrast
associated with a relatively low cost of operation make histology an essential technique in every
biological laboratory. However, the penetration depth of visible and fl uorescent light is very limited.
Only thin and transparent samples can be visualized. Inevitably, all tissue or organ samples have
to be cut into thin sections about 7 µm through conventional paraffi n embedding and microtome
sectioning before being examined by bright light or fl uorescence microscopy. Alternatively cryo-
sectioning can be used. These sample preparation methods and histological analysis are not only
