Les cellules de comptage (Malassez, Thoma …) sont conçues pour permettre le dénombrement de cellules sous microscope et la détermination de leur concentration grâce au volume calibré de la grille apparaissant dans l’image microscopique. Le comptage manuel présente des inconvénients majeurs : subjectivité, non-répétabilité… Il existe des solutions commerciales de comptage automatique dont l’inconvénient est de nécessiter un environnement bien contrôlé qu’il n’est pas possible d’obtenir dans le cadre de certaines études (ex. : le glycérol influe grandement sur la qualité des images). L’objectif du projet est donc double : un comptage des cellules automatisé et suffisamment robuste pour être réalisable, quelles que soient les conditions d’acquisition.Dans un premier temps, une méthode basée sur la transformée de Fourier a été développée pour détecter, caractériser et effacer la grille de la cellule de comptage. Les caractéristiques de la grille extraites par cette méthode servent à déterminer une zone d’intérêt et son effacement permet de faciliter la détection des cellules à compter.Pour réaliser le comptage, la problématique principale est d’obtenir une méthode de détection des cellules suffisamment robuste pour s’adapter aux conditions d’acquisition variables. Les méthodes basées sur les accumulations de gradients ont été améliorées par l’adjonction de structures permettant une détection plus fine des pics d’accumulation. La méthode proposée permet une détection précise des cellules et limite l’apparition de faux positifs.Les résultats obtenus montrent que la combinaison de ces 2 méthodes permet d’obtenir un comptage répétable et représentatif d’un consensus des comptages manuels réalisés par des opérateurs. / The counting cells (Malassez, Thoma ...) are designed to allow the enumeration of cells under a microscope and the determination of their concentration thanks to the calibrated volume of the grid appearing in the microscopic image. Manual counting has major disadvantages: subjectivity, non-repeatability ... There are commercial automatic counting solutions, the disadvantage of which is that a well-controlled environment is required which can’t be obtained in certain studies ( eg glycerol greatly affects the quality of the images ). The objective of the project is therefore twofold: an automated cell count and sufficiently robust to be feasible regardless of the acquisition conditions.In a first step, a method based on the Fourier transform has been developed to detect, characterize and erase the grid of the counting cell. The characteristics of the grid extracted by this method serve to determine an area of interest and its erasure makes it easier to detect the cells to count.To perform the count, the main problem is to obtain a cell detection method robust enough to adapt to the variable acquisition conditions. The methods based on gradient accumulations have been improved by the addition of structures allowing a finer detection of accumulation peaks. The proposed method allows accurate detection of cells and limits the appearance of false positives.The results obtained show that the combination of these two methods makes it possible to obtain a repeatable and representative count of a consensus of manual counts made by operators.

The current study investigated the distribution of periodontal epithelial cells and nerve fibres within the furcations of rat maxillary molar teeth subjected to hypothermic injury. The upper right first molars of 30 Sprague-Dawley rats were subjected to a single 20 minute application of dry ice in order to produce aseptic necrosis within the periodontal ligament, while the contralateral first molar served as an untreated control. Five animals were each sacrificed via cardiac perfusion after 7, 10, 14, 18, 21 and 28 days respectively and the maxillae were dissected out. After fixation in paraformaldehyde and processing, the tissues were embedded in paraffin wax and cut into 7µm serial coronal sections through the furcation region. Consecutive sections were then stained with H&E, cytokeratin AE1/AE3 and PGP 9.5 immunostains. Light microscopic examination of the H&E stained sections revealed that ankylosis had not developed in all of the experimental teeth, and in some of the observation groups fewer teeth were ankylosed than unaffected. The morphology of the ankylotic areas appeared to change with time, initially consisting of fine bony trabeculae, then progressing to solid bone occupying the entire furcation before becoming less solid again by the latest observation periods. Root resorption was often seen adjacent to areas of ankylosis, but the cementum of the tooth root at the point of ankylotic union was usually intact and free of resorption. Changes within the pulp chambers of the experimental teeth were also noted, with reduction in cellularity and tissue disorganisation initially, then increasing cellularity and formation of a cementum-like material on the chamber walls later. Cytokeratin AE1/AE3 immunostaining successfully identified epithelial cells within the periodontal ligament and their distribution around control teeth was similar to previous reports. Counting of these cells revealed lower numbers around experimental teeth, with the lowest counts around experimental teeth which had developed ankylosis. No change in the epithelial cell counts was detected over time, and these cells did not appear to regenerate after necrosis regardless of whether or not ankylosis developed. Statistical analysis indicated that the probability of ankylosis decreased as the number of epithelial cells increased. The PGP 9.5 immunostain identified periodontal nerve fibres, but the use of this stain was quite technique sensitive. The furcations of the molar teeth were noted to have relatively sparse innervation, with most of the visible nerve fibres being closely associated with blood vessels and located in the outer two-thirds of the ligament. Counting of the nerve fibres revealed fewer fibres around experimental teeth compared to control teeth, especially in the part of the ligament closest to the tooth root. There was no relationship detected between nerve count and time or between nerve and epithelial cell counts. Resorption was found to be more prevalent in experimental teeth, and the probability of resorption in a given tooth decreased as the epithelial cell count increased. The findings of this study suggest that the epithelial cells within the periodontal ligament have a protective function in the prevention of dentoalveolar ankylosis and resorption. Evidence of an intimate interrelationship between periodontal nerve fibre and epithelial cell numbers could not be confirmed. The null hypothesis that epithelial cell rests of Malassez do not provide a protective function against ankylosis and external root resorption was rejected, and the null hypothesis that nerve fibres and epithelial cells are not inter-dependent was retained. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297409 / Thesis (D.Clin.Dent.) -- School of Dentistry, 2007

Epithelial rest cells of Malassez (ERM) are small groups of epithelial cells within the periodontal ligament closely approximated to the radicular cementum surface. The cells have a high nuclear/cytoplasm ratio. In oblique sections of the periodontal ligament, the cell rests can be seen, not as isolated groups of cells but as a network, similar to a fish-net, surrounding the root. The function of the ERM is unknown and their participation in some dental pathological conditions is still controversial. Some new publications have described the isolation of ERM from human periodontal ligaments. To date no publications have described the expression of bone-related proteins by ERM. ERM were cultured and isolated from porcine periodontal ligaments (chapter 2). An immunohistochemical study was carried out in rat porcine and human periodontal sections using AE1/AE3 antibody. The expression of cytokeratins by ERM was demonstrated in all species (chapter 3). Characterization and identification of ERM was achieved by Transmission Electron Microscopy (TEM), immunohistochemistry and Western blot. The results demonstrated the epithelial nature of these cells obtained from the mid radicular third of porcine first deciduous molars (chapter 4). An in vitro study using a semi quantitated RT-PCR technique was carried out in four different types of porcine periodontal cells (GF, PDLF, ERM and alveolar bone cells). These cell types were compared for the expression of the bone related proteins osteopontin and bone sialoprotein. The strongest expression of osteopontin was for the ERM compared to alveolar bone cells, PDLF and GF. These results demonstrated for the first time the expression of osteopontin from cultured porcine ERM suggesting a possible role of these cells in cementogenesis (chapter 5). Finally, emdogain (EMD), an enamel matrix derivative protein, was utilized at different concentrations to stimulate periodontal ligament cells and determine its role in proliferation, attachment and by RT-PCR expression of osteopontin or bone sialoprotein in vitro (chapter6). EMD demonstrated proliferative and attachment responses in a dose dependent manner. EMD stimulated the expression of OPN m RNA by porcine ERM and alveolar bone cells. The results contribute to explaining the different regenerative events associated with EMD in periodontal regenerative therapy. The findings of this study contribute to a broader understanding of possible functions of the ERM and suggests a role for these cells in cementogenesis by their strong OPN expression.

The current study investigated the distribution of periodontal epithelial cells and nerve fibres within the furcations of rat maxillary molar teeth subjected to hypothermic injury. The upper right first molars of 30 Sprague-Dawley rats were subjected to a single 20 minute application of dry ice in order to produce aseptic necrosis within the periodontal ligament, while the contralateral first molar served as an untreated control. Five animals were each sacrificed via cardiac perfusion after 7, 10, 14, 18, 21 and 28 days respectively and the maxillae were dissected out. After fixation in paraformaldehyde and processing, the tissues were embedded in paraffin wax and cut into 7µm serial coronal sections through the furcation region. Consecutive sections were then stained with H&E, cytokeratin AE1/AE3 and PGP 9.5 immunostains. Light microscopic examination of the H&E stained sections revealed that ankylosis had not developed in all of the experimental teeth, and in some of the observation groups fewer teeth were ankylosed than unaffected. The morphology of the ankylotic areas appeared to change with time, initially consisting of fine bony trabeculae, then progressing to solid bone occupying the entire furcation before becoming less solid again by the latest observation periods. Root resorption was often seen adjacent to areas of ankylosis, but the cementum of the tooth root at the point of ankylotic union was usually intact and free of resorption. Changes within the pulp chambers of the experimental teeth were also noted, with reduction in cellularity and tissue disorganisation initially, then increasing cellularity and formation of a cementum-like material on the chamber walls later. Cytokeratin AE1/AE3 immunostaining successfully identified epithelial cells within the periodontal ligament and their distribution around control teeth was similar to previous reports. Counting of these cells revealed lower numbers around experimental teeth, with the lowest counts around experimental teeth which had developed ankylosis. No change in the epithelial cell counts was detected over time, and these cells did not appear to regenerate after necrosis regardless of whether or not ankylosis developed. Statistical analysis indicated that the probability of ankylosis decreased as the number of epithelial cells increased. The PGP 9.5 immunostain identified periodontal nerve fibres, but the use of this stain was quite technique sensitive. The furcations of the molar teeth were noted to have relatively sparse innervation, with most of the visible nerve fibres being closely associated with blood vessels and located in the outer two-thirds of the ligament. Counting of the nerve fibres revealed fewer fibres around experimental teeth compared to control teeth, especially in the part of the ligament closest to the tooth root. There was no relationship detected between nerve count and time or between nerve and epithelial cell counts. Resorption was found to be more prevalent in experimental teeth, and the probability of resorption in a given tooth decreased as the epithelial cell count increased. The findings of this study suggest that the epithelial cells within the periodontal ligament have a protective function in the prevention of dentoalveolar ankylosis and resorption. Evidence of an intimate interrelationship between periodontal nerve fibre and epithelial cell numbers could not be confirmed. The null hypothesis that epithelial cell rests of Malassez do not provide a protective function against ankylosis and external root resorption was rejected, and the null hypothesis that nerve fibres and epithelial cells are not inter-dependent was retained. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297409 / Thesis (D.Clin.Dent.) -- School of Dentistry, 2007

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