Measurement of the variation in salivary mucosal epithelial cell numbers

PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.Thesis (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2015 (Department of Molecular and Cell Biology).Includes bibliography: leaves 55-64.Oral epithelial cells are one of the major cellular components in human whole saliva. They primarily represent desquamated oral mucosal epithelial cells. Oral mucosa coats the soft tissues of the oral cavity and protects the underlying structures. Like other epithelial tissues, oral mucosal epithelium maintains its function and stability by cell renewal, which in most tissues is a process where cell mitosis balances cell loss to finally reach an equilibrium status. In the oral mucosal epithelium, cells are produced from the basal layer, then mature and move to the surface, to subsequently shed from the top layer. Epithelial cells derived from the oral cavities have been shown to contain biomarkers for oral disease such as oral cancer. In order to reliably compare epithelial cell-derived biomarker levels in human saliva among subjects, it is important to normalize for inter-individual differences in salivary epithelial cell numbers. The aim of our study was to explore inter-subject and intra-subject daily variations in salivary epithelial cell counts and to develop cell counting-independent approaches for the epithelial cell quantification, with the ultimate aim for their application in salivary diagnostics devices. In this project salivary epithelial cell numbers were first quantitated by traditional cell counting using a hemocytometer, by optical density measurement using a spectrophotometer, and with a keratin 10 ELISA. Inter-individual differences in cell counts were studied (n=8 subjects), as well as daily variations measured over a week time interval (n=4 subjects), and hourly variations (n=1 subject, performed in duplicate). Correlations between cell counts, whole saliva OD[620] values and keratin 10 levels were studied in samples collected at the Living Laboratory in the Museum of Science (n=1 6 subjects). Lastly, age-dependent variations in these parameters were investigated. Results indicated that there was a 7.4 fold variation in oral epithelial cell counts among individuals. The values found ranged from 12.8x10[4] to 94.5x10[4] cells/ml. The counts in 4 subjects monitored for a week showed that the salivary epithelial numbers are not stable and varied 2.2 to 13.3 fold within the four subjects during the week. We observed that cell numbers showed a remarkable reduction in counts when samples were collected immediately after a meal. This result was highly reproducible. A strong positive linear relationship was observed between salivary OD[620] value and oral epithelial cell counts in saliva (R[2]=0.8867, P[less than]0.001) where it was established that an OD of 1.0 corresponded to an epithelial cell density of approximately 155.8x10[4] cells/ml. Keratin 10 was highly expressed in oral epithelial cells but showed a weak correlation with epithelial cell counts (R[2]=0.21 82, P=0.067) which may be related to the variable expression of this marker in keratinized and non-keratinized epithelium. Proper quantitation of epithelial cells in saliva is important for salivary diagnostic investigations relying on cell-associated biomarkers. The studies conducted show inter- and intra-subject variations in such cell numbers, emphasizing the need for normalization. The whole saliva OD[620] showed the best correlation with epithelial cell numbers and appears to be a suitable alternative to labor-intensive cell counting. Furthermore, epithelial cell-specific markers other than keratin 10 are still being pursued