Control and Monitoring of Scaffold Architecture for Tissue Engineering - Biomaterials Fabrication and Processing - page 75

FIGURE 3.1 Light microscopy images of MG63 bone cells growth in a PLLA scaffold for 4 weeks (H&E
staining) with 200 × magnifi cation. The arrow points to the porous wall of the PLLA scaffold.
time consuming, but may also introduce structural artifacts or lead to loss of some components
because all biological specimens must be subjected to a dehydration and rehydration cycle. Changes
in the hydration of the matrix may lead to the separation of the cells and matrix and the formation
of lacunar spaces, thus impairing the factual examination of the constructs. 128 Most importantly,
observation by microscopy is an end-point evaluation and a destructive analysis.
3.4.1.3 Confocal Microscopy
The emergence of confocal microscopy (CM) was a revolution in the fi eld of microscopy. By label-
ing with fl uorophores, optically nontransparent specimens can be visualized at high resolution in
3-D. CM enables the acquisition of sample images up to a few hundred micrometers thickness by
using a focused laser beam and collecting the emitted fl uorescent signal through a pinhole aperture
that spatially rejects light from out of focus areas. Changing the plane of focus enables the rapid
sample sectioning along the xz - or yz -plane to obtain sample cross-sections. 129 Optical sectioning
eliminates the requirements of physical sectioning as in histological analysis, enabling the observa-
tion of viable cells in the scaffold and the online measurement of cell activities and tissue turnover.
A confocal microscope image of a chitosan scaffold is presented in Figure 3.2.
The main drawbacks of CM are the limited penetration depth, the lack of temporal resolution,
which renders it unsuitable to detect transient cell shape changes, and the requirement of fl uoro-
phores. Except for a few engineered tissues, for instance, skin and cornea, the engineered constructs
are relatively thick, around a few millimeters to centimeter range. The penetration depth in CM
limits the observation to few hundred micrometers from the surface of the constructs. Furthermore
the fl uorescent labeling may affect the long-term viability of cells in the constructs.
The use of CM in refl ectance mode overcomes the requirement of fl uorophores. In this case,
the contrast is achieved by utilizing the inherent refractive index properties of the various cellu-
lar microstructures. The radiated laser beam is scattered irregularly by the heterogeneous tissue
components. Backscattered in-focus signals are captured, transmitted to a spectrometer, and sub-
mitted for visualization. Usually, a laser light with near-infrared (near-IR) wavelengths is used for
in vivo refl ectance mode measurements. Even though this mode is more appropriate for in vivo