Yasunobu Enokiya

Cell proliferation and death of Hertwig's epithelial root sheath in the rat

Abstract. Hertwigs epithelial root sheath (HERS) degenerates immediately after root dentin is formed. However, odontogenic tumors or cysts may originate from residual cells, although little is known about how HERS proliferates and disappears. This study investigated whether cell death is provoked in the tissues surrounding the root during eruption of the rat upper molar. We employed the TdT-mediated-dUTP nick end labeling (TUNEL) method and transmission electron microscopy (TEM) to observe the morphological features of cell death. We examined the activity of cell proliferation immunohistochemically using proliferative cell nuclear antigen (PCNA) and the continuity of HERS using polyclonal keratin antibody (PK). Cell death resembling apoptosis and apoptotic bodies phagocytosed by neighboring mesenchymal cells were detected in only a few cells by both TUNEL and TEM. We also found cells with electron-lucent cytoplasm which contained dilated or ruptured mitochondria and remarkably dilated rough endoplasmic reticulum (rER) which lay sparsely along the root. These cells seemed to be dead HERS cells based on their ultrastructural features, location, and stage. PCNA-positive cells were found in the apical end of the HERS cells, fibroblasts of the periodontal ligament, and odontoblasts. PK reacted with HERS; however, PK-positive cells partially disappeared after the 15th postnatal day when the root dentin had formed slightly. These results may indicate that HERS cells migrate into the periodontal ligament or die immediately after root dentin is formed and that various types of cell death such as apoptosis and cytoplasmic type occur in the tissues surrounding the root during tooth development.

Expression and function of laminin and integrins on adhesion/migration of primary culture cells derived from rat oral epithelium.

BACKGROUND AND OBJECTIVE It remains controversial whether or not the junctional epithelium cells that are directly attached to teeth migrate on the enamel surface, as those cells are able to adhere firmly to the enamel. The aim of this study was to investigate the expression patterns of laminin gamma(2), integrin beta(4) and integrin alpha(3), and to examine their potential function in cell migration. MATERIAL AND METHODS Oral epithelium cells obtained from Sprague-Dawley rats were established in primary culture. We employed a wound-healing assay to characterize the direction of cell extension at the start of cell migration, and observed different localizations of laminin and integrins using immunofluorescence. For functional analyses of integrins, we employed a phosphatidylinositol-3-kinase (PI3K) activator to promote integrin beta(4) function and used P1B5 to inhibit integrin alpha(3) function, and we analyzed the percentage of re-epithelialization as the migration function. RESULTS Marked accumulation of laminin gamma(2) was detected in the peripheral cytoplasm of cells adjacent to the wound area, as shown by the results of the migration assay. Integrin beta(4) was detected in the distal cell processes of actively migrating cells, while integrin alpha(3) was found in cell membranes of cells adjacent to the wound area. In the functional analyses, the percentage of re-epithelialization was significantly lower in the PI3K-activator group and in the P1B5-treated group (2.5% and 7.2%, respectively) than in the control group (39.0%) (p < 0.01). CONCLUSION The results suggest that laminin gamma(2) is secreted as a foothold for cell migration, that integrin beta(4) participates in cell adhesion and that integrin alpha(3) is involved in cell migration in the primary culture cells.

Proliferation, migration and apoptosis of periodontal ligament cells after tooth replantation.

OBJECTIVE The aim of this study was to investigate the proliferation, migration and death of periodontal ligament (PDL) cells after tooth replantation. MATERIALS AND METHODS Maxillary first molars were extracted from 4-week-old male (n = 28) Sprague-Dawley rats and immediately replanted, after which, proliferation, migration and death of PDL cells were investigated. RESULTS At 3 days after tooth replantation, many proliferative cell nuclear antigen (PCNA)-positive PDL cells were observed on the alveolar bone side, but fewer on the root side. However, while a gradual decrease was observed in number of PCNA-positive PDL cells on the alveolar bone side until 7 days, an increase was seen on the root side. At 3 weeks, cells labeled with PKH26 (fluorescent dye into plasma membrane) were located in the middle of the PDL space. However, these PKH26-labeled cells did not spread to the surface of the cementum or the alveolar bone. TUNEL-positive cells were observed on both the bone and root sides at 3 days. Number of apoptotic cells increased until 7 days on the bone sides, but decreased on root sides. CONCLUSION These results suggest that both cell proliferation and apoptosis occur in different patterns and at different times to maintain regular spacing of the PDL after tooth replantation.