Influence of the Processes Parameters on the Properties of The ...

Chapter 4.
Experimental Procedures and Protocols for Analyses
During <strong>the</strong> commissioning <strong>of</strong> <strong>the</strong> metalizing high voltage, low current passes through a gold foil,
ionize <strong>the</strong> atoms and <strong>the</strong>y are deposited on <strong>the</strong> surface <strong>of</strong> <strong>the</strong> sample. The thickness <strong>of</strong> <strong>the</strong> gold layer
deposited depends on <strong>the</strong> time <strong>of</strong> filing but does not exceed 10 Å. The gas pressure was less than 50 mTorr
and <strong>the</strong> current was about 40 mA. The coating time was 120 s. The metalizing chamber is <strong>the</strong>n reduced to
atmospheric pressure and <strong>the</strong>n <strong>the</strong> samples are introduced into <strong>the</strong> microscope chamber. Complete schematic
procedure is shown in Figure 4.22. A working distance between <strong>the</strong> bottom <strong>of</strong> <strong>the</strong> barrel and <strong>the</strong> sample
between 30 and 37 mm is recommended. An acceleration voltage <strong>of</strong> electrons between 10 and 15 kV is
generally accepted, and a probe current <strong>of</strong> between 50 and 150 Å.
4.2.2 SCION Image Analysis
Image analysis <strong>of</strong> SEM micrographs was used for <strong>the</strong> observation <strong>of</strong> <strong>the</strong> internal pore morphology
<strong>of</strong> <strong>the</strong> freeze-dried foams. Polymeric and composite foams images are taken on <strong>the</strong> surface, at <strong>the</strong> cross
section and inside <strong>the</strong> pores. Cross sectional images are taken at five different points (<strong>the</strong> centre, top left,
bottom left, top right and bottom right) to verify homogeneity <strong>of</strong> pores and <strong>the</strong>ir distribution (cf. Figure
4.23). Normally magnifications <strong>of</strong> image are (25, 40, 100, 200, 300, 400, 500) depending on <strong>the</strong> pore size.
Higher magnification up to 1K, 2K and 3K is recorded, if internal surface <strong>of</strong> <strong>the</strong> pores walls is to be
observed.
The SEM micrographs were treated and statistically analyzed using <strong>the</strong> s<strong>of</strong>tware SCION ® Image
analysis. The images were digitized on a matrix <strong>of</strong> 1024 1024 pixels with 256 gray levels. The foams were
duplicated and five images <strong>of</strong> different areas <strong>of</strong> <strong>the</strong> same foam were analyzed. Effect <strong>of</strong> <strong>the</strong> successive
image transformations can be seen in Figure 4.24.
Scaffold
surface
Top left-50×
Top Right-100×
Scaffold cross- section-20×
Bottom left-100×
Bottom right-100×
Centre-100×
Figure 4.23: SEM Images <strong>of</strong> cross sectional foam.
An example <strong>of</strong> <strong>the</strong> data obtained after SCION ® image analysis is presented in Table 4.3. From <strong>the</strong>
data, it is possible to define three pores categories (cf. Table 4.4): Micro, meso and macro pores are pores
with dimension <strong>of</strong> equivalent diameter less than 25 m, 25−150 m and above 150 m respectively. The
micro pores are necessary for movement <strong>of</strong> <strong>the</strong> liquids and nutrients in <strong>the</strong> scaffold. Meso pores are for <strong>the</strong>
accommodation <strong>of</strong> <strong>the</strong> human mesenchymal stem cells, as <strong>the</strong>ir size varies from 100−150 m. Macro pores
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