Fogorvosi szemle, 2005 (98. évfolyam, 1-6. szám)
2005-04-01 / 2. szám
76 FOGORVOSI SZEMLE ■ 98. évf. 2. sz. 2005. an advantageous environment for investigating events that regulate the formation of both collagen- and noncollagen-based calcified tissues. In order to study the role(s) and mechanism of action of matrix molecules, as well as the differentiation and physiology of the cells manufacturing them, we have developed an experimental system to access them in an environment that respects the local physiology as well as the whole-animal biology. The system consists of an osmotic minipump hooked to a “window” created in the bone on the buccal aspect of the rat hemimandible, near the apical end of the incisor. Three applications of this local targeting system will be reviewed: (a) tracer studies with noncollagenous bone matrix proteins (b) functional charaterization of mineral binding domains, in BSP, and c) local gene transfer using a lentivirai vector. This rodent hemimandible window model offers the possibility to study various aspects of calcified tissue formation within the context of the whole-animal biology. It represents a restricted environment that can be exploited for administering locally high concentrations of products that would be toxic systemically, or proteins that are available in limited amounts. The bony window can be used to create local transgenic conditions, and its use in mice opens the door to experimental manipulations in animals with well-defined physiological and phenotypic alterations. Acknowledgement: Supported by the Canadian Institutes of Health research. N. OBARA, H. LESOT* Department of Oral Anatomy, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan *INSERM U595, Faculté de Médecine, Strasbourg, France EXPRESSION AND LOCALIZATION OF ß-CATENIN IN DEVELOPING TOOTH GERMS Both cell adhesion and signaling are significant regulatory mechanisms of morphogenesis. Since ß-catenin is a component of cadherin-mediated cell adhesion and also plays an essential role in the canonical Wnt signaling pathway, this molecule might be a key player linking cell-cell adhesion and signaling in development. During tooth morphogenesis, two classical cadherins, E- and P- cadherins, are expressed in the enamel organ showing complementary patterning, while Wnt and related genes are differentially expressed both in the epithelial and the mesenchymal components of the tooth germ. We analyzed subcellular localization of ß-catenin in the molar tooth germs at the cap stage by using confocal microscopy, and detected specific nuclear localization of this molecule in the enamel knot, suggesting the presence of Wnt signal in this area. We also confirmed the coincident up-regulation of P-cadherin in the same region. These results imply the existence of a linkage between cell adhesion and signaling. Furthermore, in situ hybridization analyses showed obvious up-regulation of ß-catenin gene expression in the enamel knot at the cap stage and in the inner dental epithelium at later stages. This up-regulation may be related to the multiple roles for ß-catenin in tooth development. Acknowledgements : This work was supported by the COST action B23. A. PALMON, D. AFRAMIAN, E. SHAI, R. DAVID. Institute of Dental Sciences, Faculty of Dental Medicine, The Hebrew University, Jerusalem, Israel. ISOLATION, CULTIVATION AND PRIMARY CHARACTERIZATION OF RAT SUBMANDIBULAR SALIVARY GLAND PROGENITOR CELL CANDIDATES The major cause for salivary dysfunction is radiation therapy. Unfortunately, there is no satisfactory treatment for this condition. Affected patients mainly suffer from rampant dental caries, frequent mucosal infections, and difficulties while swallowing and chewing dry food, all of which considerably decrease their quality of life. New therapeutic strategies were suggested to handle postradiation impairment including; gene therapy, tissue engineering and recently stem cell biology. Previous reports suggested that cells derived from tissue damaged (duct ligation) collagen type I cultivated might be considered as salivary gland progenitor cells. We hypothesize that salivary gland basal epithelial cells (Integrin «6ß1 positive immunostained) from animals might be candidates for being adult salivary gland stem cell. Autologous graft of these cells might replace damaged salivary acinar cells and regain tissue functionality, after radiation therapy. In the present study we examined the possibility of isolating and cultivating those cells. Adult rats were subjected to short heat acclimation stress condition followed by isolation of salivary gland parenchymal cells using the Percoli gradient technique. Thereafter, a6ß1 positive cells were secluded by MACS methodology and analyzed by FACS before and after cultivation. We found by FACS analysis that stress conditions increased cx6ß1 positive isolated cells from 6% to 29% in the whole epithelial gland fraction population (These numbers correspond to 0.5% and -2.5% from the whole gland respectively). Cultivation of these cells for 17 days lead to 98% «6ß1 positive cell enriched fraction. These cells are currently being characterized with additional stem cell markers such as P63 and TRAF-4, and examined for their ability to differentiate to adult salivary gland cells. We conclude that adult cx6ß1 salivary gland positive cells can be isolated and cultivated and might be used for future new therapeutic regiments.
