Biomedical Engineering Reference
In-Depth Information
5.3.1.1 Scanning Electron Microscopy, Atomic Force
Microscopy, and Confocal Laser Microscopy
It is important to have a measure of the interfi ber distance and fi ber diameter of the electrospun
scaffolds as these properties infl uence cell adhesion, migration, differentiation, and proliferation.
There are two main methods that are commonly utilized: (1) laser scanning confocal microscopy
and (2) SEM. These techniques also have the ability to preserve the structure of any biological mate-
rial in relation to the fi bers.
The characterization of the fi bers using laser scanning confocal microscopy requires the usage
of some form of fl uorescence labeling, for example, doping the polymer solutions with 0.05% w/v
of rhodamine or fl uorescein. This method may also be employed to characterize colocalization of
fi bers during multifi ber spinning. Furthermore, this technique is particularly useful when immu-
nochemical method is employed to detect specifi c entities, that is, proteins on the fi bers by using
dye-labeled antibody. Confocal microscopy can also be used as an
in vitro
tool to assess viability
and distribution of cells on the nanofi ber membranes. Figure 5.11 shows images of a double-sided
seeding of PCL/Col nanofi brous membranes with SMCs. Cells situated on the top and bottom faces
of the membrane were stained prior to seeding with cell tracker red and green, respectively. SEM is
arguably the simplest method for fi ber dimensions analysis. Most SEM systems are equipped with
applications that allow point-to-point distance measurements. Immunogold labeling technique can
be used in SEM for more specifi c applications such as detection of a particular matrix protein in
the samples.
Jaeger et al.
[35] were the fi rst to report that when using AFM to measure the diameters of indi-
vidual electrospun fi bers, values were likely to be overestimated due to the geometry of the AFM
tip. Specifi c surface areas of porous and fi brous polymers can be measured by the BET gas absorp-
tion method. The theory behind this method can be found in many classic physical chemistry topics
[36]. BET gas adsorption has been used to measure specifi c surface areas of electrospun fi brous
membranes [37,38].
Steel rings
Nanofiber sheet
FIGURE 5.11
Schematic drawing showing the double-sided seeding of SMCs on PCL/Col nanofi ber sheets
using steel rings as an
in vitro
suspension culture method. Viability assays using cell tracker red and green and
laser confocal scanning microscopy revealed live cells at the top and bottom surfaces, respectively.
