The ex-vivo study presented in this chapter succeeded in-vitro work by Mulhall and colleagues [55], in which the dielectric characteristics of normal oral primary cell-line kératinocytes, pre-cancerous cell-line kératinocytes and cancerous cell-line kératinocytes were examined using the 3DEP DEP-Well testing system used in this study. The work by Mulhall and colleagues concluded that oral cancer cells achieved greater membrane capacitance values than oral dysplastic cells, which achieved achieved greater membrane capacitance values than normal oral cells, but found the opposite trend for cytoplasmic conductivity.
In the present study however, the OBB sample group which achieved the greatest mean membrane capacitance was the Healthy group (6.5 + 2.6 mF /m^), and the lowest mean membrane capacitance was achieved by samples from the Dysplastic and the Normal from OSCC/ Dysplastic groups (3.1 + 0.5 mF/m^ and 3.1 + 0.3 m F/ m^ respectively). OBB samples comprising the OSCC group achieved the greatest mean cytoplasmic conductivity (55 + 5 mS/ m), with OBB samples comprising the Healthy group achieving the second greatest mean cytoplasmic conductivity (52 + 3 mS/ m).
The disparity between the ex vivo results presented in this study and the in vitro results presented in the study by Mulhall and colleagues could be partly due to the heterogeneity of OBB samples, be they from healthy, dysplastic or cancerous tissue. Rovers Medical Devices B.V., the manufacturers of the 0 reel lex brushes used in this study, state that their brushes collect cells from all layers o f oral
epithelial tissue [68]. This implies that oral epithelial cells from four different layers in keratinized tissue and five different layers in non-keratinzed tissue [69], each possessing a different phenotype according to the layer they constitute [69], would theoretically be found in samples from healthy, normal oral epithelial tissue. Additionally, after performing the recommended 10 rotations of the brush head during the brush biopsy procedure [68], erythrocytes (red blood cells, RBCs) were also regularly found, in small concentrations, in Healthy OBB samples. If epithelial cells from each layer were to be considered as distinct cell types, for the purposes of modelling (as they possessed different radii and phenotypes), and if RBCs were also present, that would result in five or six different cell types being present in OBB samples from healthy oral tissue. This number does not included a variety of other cell types such as melanocytes and Langerhans cells also found in normal oral epithelium [69]. The number of different cell types contained in dysplastic and OSCC samples would theoretically be even greater, due to the presence of immune cells (such as macrophages, leukocytes and lymphocytes) and cancer-associated fibroblasts [69].
Therefore, the DEP spectra produced by OBB samples from all four tissue groups examined in this study, were in fact mean spectra produced by contributions from the multiple cell types present in each sample. Whilst significant differences between OSCC and Healthy OBB samples were found when examining membrane capacitance (p = 0.007 when duplicate samples were included and p = 0.045 when duplicate samples were excluded), it remains to be seen which of the multiple cell types contained within each OBB sample contributed to this significant difference. M em brane capacitance reflects the ability of the plasma membrane to store electrical charge, and increased surface area results in an increase in membrane capacitance. The surface area of the membrane can be increased due to the presence of microvilli, membrane folds or any form of membrane protrusion. However, it is not possible, in the present form of this study, to attribute this increased membrane capacitance of cells from healthy tissue when compared to cells from OSCC tissue, to one particular cell type.
One other significant difference in dielectric properties was found. Upon consulting the results shown in Table 2.3, the mean cytoplasmic conductivity of cells from OSCC samples was significantly different to that of cells from samples of oral dysplasia (p = 0.048). However, this difference was not considered significant when the previously excluded samples were included (Table 2.3 of the Results section). Cytoplasmic conductivity, the ability of the cell cytoplasm to conduct electric charge, is indicative of cytoplasmic ionic content but, as was the case with membrane conductance, it is not possible to attribute the increased value for cells from OSCC samples compared with cells from samples of oral dysplasia, to the ionic content of one cell type. No significant differences w ere found, for any of the OBB sample classification groups examined, in terms of membrane conductance when
duplicate samples were included, though a significant difference was found for OSCC vs. All Normal (Healthy and Normal from OSCC/ Dysplasia) samples when duplicates were excluded (p = 0.034, Table 2.3).
Of note, other than for mean cell radius (a variable not determined by DEP testing, rather required for calculation of tw o dielectric properties), there were no significant differences in dielectric properties found when comparing cells from Dysplastic OBB samples with cells from Healthy OBB samples. This result suggested that the dielectric properties cytoplasmic conductivity, membrane conductance and membrane capacitance could not be used to differentiate between dysplastic and healthy oral tissue.
Considering the highly heterogeneous nature of OBB samples, it is possible that the disparity between the results in the present study and those in the study by Mulhall and colleagues is also partly due to the application of a model (the single-shell model) which assumes all cells to be spherical, and of similar radii (as only one mean radius value is considered). It could therefore be argued that the application of this model to OBB samples, was inappropriate, hence the reason behind the development and examination of two, novel variables in this study: the frequency span of the membrane transition region of DEP spectra and the median membrane midpoint frequency.