Biomedical Engineering Reference
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(
7-9 days following the injury) when the wound bed is covered by a layer of
keratinocytes. This process is followed by the re-establishment of the stratified
epidermal layer.
To date, these major organizational changes during healing have been
substantially studied with molecular biology and immunofluorescence-based
approaches [42, 43]. Although the spatial distribution of gene products may
be mapped with the latter technique, destruction of the sample is usually re-
quired, and separate (microtomed) sections are needed for each protein whose
spatial distribution is of interest. The development of vibrational microscopy
approaches, which permit the simultaneous determination of the spatial dis-
tribution of several protein species, as well as non-protein species, would be
beneficial. The current experiments [6] demonstrate the feasibility of tracking
changes in the spatial distribution of the major skin proteins within the first
several days after wounding in an organ culture wound healing model and
correlate these changes with data obtained using microarray analysis.
∼
Human Organ Culture Wound Healing Model
Human skin specimens were obtained from reduction abdominoplasty (tummy
tuck) and used to generate acute wounds as described elsewhere [42]. A 3 mm
biopsy punch was used to create an acute wound. Skin specimens were main-
tained at the air-liquid interface at 37
◦
C, 5% CO
2
, and 95% relative humidity
for 6-7 days.
Validation of Vibrational Imaging Protocols with IR Microscopic
Imaging
The generic procedure for collecting either confocal Raman micrographs or
IR images from a healing wound is shown in Fig. 15.10a. The wound is gener-
ated with a punch biopsy and the residual tissue is retained for spectroscopic
examination.
For IR imaging, flash frozen samples were microtomed (5
m thick sec-
tions) perpendicular to the SC. The sections were placed on BaF
2
windows
and mapped with a Perkin-Elmer “Spotlight” system. Typical image sizes
are 300
μ
6000 spectra. The visible image of a
skin section acquired 4 days following wounding (Fig. 15.10b) reveals a clear
MET, indicating the persistence of biological events in the explants. Factor
loadings (Fig. 15.10c) in the 1475-1750 cm
−
1
region clearly identify collagen
(factor 1) and keratin (factor 2). The score images of these factors, as shown
in Fig. 15.10d and e, depict the collagen-rich epidermis and the keratin-rich
dermis, respectively.
×
750
μ
m and are sampled with
∼
Confocal Raman Microspectroscopy of a Healing Wound
Raman spectra were collected from an image plane encompassing both wounded
and non-wounded areas. In a typical experiment, a confocal Raman image was
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