Occlusion regulates tooth-root elongation during root development in rat molars.

Occlusion is commenced by contact of a tooth with an opposing tooth and is the mechanical force working against the periodontal ligament (PDL). However, the influences of occlusion during root development remain uncertain. By extracting the unerupted counterpart molars of rats, we established a non-occlusal model that directly examined the effects of the absence of occlusion in developing molars using micro-computed tomography (µ-CT) and histological procedures. The µ-CT data for experimental molars confirmed no attrition and hypogenesis of the alveolar bone. Root lengths in experimental groups increased more than in control groups. Histological findings of experimental molars showed a wide crown pulp, a long and narrow root, immature Sharpey's fibers, and hypogenesis of cementum. Proliferating cells localized in Hertwig's epithelial root sheath (HERS), the apical pulp, and the PDL of experimental teeth. Furthermore, cell-proliferative activity in experimental roots exceeded that in normal roots. These data indicate that cell proliferation is decreased by occlusion during root formation. Thus, occlusion is one factor that regulates root elongation.

An immunohistochemical study of the expression of heat-shock protein-25 and cell proliferation in the dental pulp and enamel organ during odontogenesis in rat molars.

OBJECTIVES: The aim of this study is to clarify the functional significance of heat-shock protein (HSP)-25 during tooth development. DESIGN: We compared the expression of HSP-25 in the dental epithelial and mesenchymal cells with their proliferative activity during odontogenesis in rat molars on postnatal days 1-100 by immunohistochemistry using anti-HSP-25 and anti-5-bromo-2'-deoxyuridine (BrdU) for cell proliferation assay. RESULTS: On day 1, BrdU-immunoreactive cells were densely located in the inner enamel epithelium in the cervical loop and intercusped areas and the dental pulp adjacent to them, whereas HSP-25-immunoractivity (IR) was restricted to the cusped area where odontoblasts and ameloblasts had already differentiated. Subsequently, BrdU-IR shifted in the apical direction to be localized around Hertwig's epithelial root sheath during days 5-30, never overlapping with concomitantly apically-shifted HSP-25-IR. On days 60-100, BrdU-immunoreactive cells were hardly recognizable in the dental pulp, where HSP-25-IR was exclusively localized in the odontoblast layer. Furthermore, the odontoblast- and ameloblast-lineage cells exhibited two steps in the expression of HSP-25 throughout the postnatal stages: first, dental epithelial and pulpal mesenchymal cells showed a weak IR for HSP-25 after the cessation of their proliferative activity, and subsequently odontoblasts and ameloblasts consistently expressed an intense HSP-25-IR. CONCLUSION: Odontoblast- and ameloblast-lineage cells acquire HSP-25-IR after they complete their cell division, suggesting that this protein acts as a switch between cell proliferation and differentiation during tooth development. The consistent expression of HSP-25-IR in the formative cells may be involved in the maintenance of their functional integrity.

The eternal tooth germ is formed at the apical end of continuously growing teeth.

Rodent incisors are known to be continuously growing teeth that are maintained by both the cell-proliferation at the apical end and the attrition of the incisal edge. This type of tooth had a special epithelial structure for the maintenance of stem cells, showing the bulbous epithelial protrusion at the apical end. The morphological transition of the epithelial-mesenchymal compartment by serial transverse sections of the apical end toward the incisal direction is likely to reflect the development of the tooth germ in the prenatal stage. Based on the present histological and previous molecular biological studies, the special structure at the apical end is obviously different from the cervical loop giving rise to Hertwig's epithelial root sheath (HERS), in human, mouse and rat molar tooth germs. Hence, we propose a new concept that the eternal tooth bud producing various dental progeny is formed at the apical end of continuously growing teeth, and a new term "apical bud" for indicating this specialized epithelial structure. Furthermore, BrdU labelling analysis suggested that the guinea-pig molars, which were continuously growing teeth, also possessed plural specific proliferative regions and "apical bud" at the apical end.

Gene and protein localisation of tumour necrosis factor (TNF)-α converting enzyme in gingival tissues from periodontitis patients with drug-induced gingival overgrowth.

OBJECTIVE: It is known that tumour necrosis factor (TNF)-α converting enzyme (TACE) plays a crucial role in fibrotic inflammatory diseases, and is specifically inhibited by tissue inhibitor of metalloproteinase (TIMP)-3. Fibrotic drug-induced gingival overgrowth (GO) is often combined with periodontitis. However, neither TACE nor TIMP-3 has been thoroughly examined in periodontal tissues to date. The aim of the present study was to analyse mRNA expression of TACE and TIMP-3, and protein localisation of TACE in gingival tissues removed from drug-(calcium-channel blocker) induced GO and periodontitis. METHODS: A total of 30 gingival tissue samples were taken from 15 GO and 15 periodontitis patients. The mRNA expression levels were analysed by quantitative reverse transcription polymerase chain-reaction (qRT-PCR) and the protein localisation was investigated by immunohistochemistry. Statistical analysis was performed using the Mann-Whitney U-test. RESULTS: TACE and TIMP-3 mRNA levels were significantly higher in GO compared to the periodontitis groups, as revealed by qRT-PCR (p<0.05). TACE-producing cells were immunohistochemically detected among monocytes/macrophages, plasma cells and some epithelial cells. TACE immunoreactivity was shown to be more intense in GO than in periodontitis-gingival tissue. CONCLUSIONS: We have demonstrated TACE expression in cells such as macrophages, plasma cells and epithelial cells, and its predominant expression in GO tissues. This data suggests that TACE expression in GO-gingiva could be involved in the pathogenesis of disease.

Microarray and quantitative RT-PCR analyses in calcium-channel blockers induced gingival overgrowth tissues of periodontitis patients.

OBJECTIVES: The purpose of the present study was to analyse transcriptomes and mRNA expression levels for specific genes in calcium-channel blocker-induced gingival overgrowth (GO) tissues. DESIGN: Eight gingival tissues samples (from both GO negative and positive sites) were harvested from four GO patients for microarray analyses. Twelve candidate genes were selected for further quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR) analyses. Ten GO tissues from periodontitis patients and ten control gingival tissues from healthy subjects were compared by qRT-PCR. Mann-Whitney U-test was used for statistical evaluation. RESULTS: In GO positive tissues, 163-1631 up-regulated and 100-695 down-regulated genes were identified with more than two-fold changes compared with GO negative tissues amongst patients by microarray experiments. No commonly expressed genes amongst the eight sets of microarray data were found. The clustering analysis confirmed that the entire transcriptome patterns showed similarities in individuals, but differences amongst the four patients. The qRT-PCR and statistical analyses for the candidate genes, though, revealed differential gene expressions between GO-positive and negative tissues. We found that matrix metalloproteinase (MMP)-1 and MMP-12 as well as cathepsin-L were significantly up-regulated whilst keratin-10 and transforming growth factor-ß1 were significantly down-regulated in GO tissues of periodontitis patients compared with the control gingival tissues of healthy subjects. CONCLUSION: The microarray analyses revealed that GO pathogenesis was complex and individually varied, though GO-affected gingival tissues were controlled at least by genes related to collagen metabolisms including regulated MMPs, cathepsin-L, growth factors, and keratins to maintain tissue homeostasis in vivo.

Differential gene and protein expression of tissue inhibitors of metalloproteinases (TIMP)-3 and TIMP-4 in gingival tissues from drug induced gingival overgrowth.

OBJECTIVES: The purpose of this study was to analyse mRNA expression and protein localization of tissue inhibitors of metalloproteinases (TIMP)-3 and TIMP-4 in gingival tissues removed from drug (calcium-channel blocker) induced gingival overgrowth and periodontitis patients. DESIGN: Employing RT-PCR, we evaluated TIMP-3 and TIMP-4 mRNA levels of 20 human gingival tissue samples taken from patients suffering gingival overgrowth (GO) and periodontitis (P). Then, using immunohistochemistry we investigated the TIMP-3 and TIMP-4 protein localization of five sample tissues from each group. RESULTS: TIMP-4 mRNA levels in GO-gingiva tended to be lower than in P-gingiva but the results differed little (p = 0.22). Varying degrees of inflammation in the protein localization of TIMP-3 and TIMP-4 were found. TIMP-4 immunoreactivity (IR) was weak in the endothelial cells, fibroblasts, epithelial basal and parabasal cells while the degree of inflammation differed as well. TIMP-3 and TIMP-4 IR in inflammatory cells, including lymphocytes, plasma cells, and macrophages, were faint and intense respectively. For P-gingiva, both TIMP-3 and TIMP-4 IR expression was weak in the endothelial cells, fibroblasts, basal and parabasal epithelial layers. Expression of TIMP-3 was faint in the inflammatory cells, whereas TIMP-4 IR was strong. CONCLUSION: Our findings suggest that TIMP-3 and TIMP-4 expression differs in GO and P-gingival tissues, both of which are potentially involved in pathogenesis.

Cell dynamics in the pulpal healing process following cavity preparation in rat molars.

Odontoblast-lineage cells acquire heat-shock protein (HSP)-25-immunoreactivity (IR) after they complete their cell division, suggesting that this protein acts as a switch between cell proliferation and differentiation during tooth development. However, there are few available data concerning the relationship between cell proliferation and differentiation following cavity preparation. The present study aims to clarify the expression of HSP-25 in the odontoblast-lineage cells with their proliferative activity after cavity preparation by immunocytochemistry for HSP-25 and cell proliferation assay using 5-bromo-2'-deoxyuridine (BrdU) labeling. In untreated control teeth, intense HSP-25-IR was found in odontoblasts and some subodontoblastic mesenchymal cells. Cavity preparation caused the destruction of odontoblasts and the disappearance of HSP-25-IR was conspicuous at the affected site, although some cells retained HSP-25-IR and subsequently most of them disappeared from the pulp-dentin border by postoperative day 1. Contrary, some subodontoblastic mesenchymal cells with weak HSP-25-IR began to take the place of degenerated cells, although no proliferative activity was recognizable in the dental pulp. Interestingly, proliferative cells in the dental pulp significantly increased in number on day 2 when the newly differentiating cells already arranged along the pulp-dentin border, and continued their proliferative activity in the wide range of the pulp tissue until day 5. These findings indicate that progenitor cells equipped in the subodontoblastic layer firstly migrate and differentiate into new odontoblast-like cells to compensate for the loss of the odontoblast layer, and subsequently the reorganization of dental pulp was completed by active proliferation of the mesenchymal cells occurring in a wide range of pulp tissue.

The relationship between the cusp pattern and plural stem cell compartments in Guinea pig cheek teeth by chasing BrdU-labeling.

Continuously growing rodent incisors have a special epithelial structure for maintaining adult stem cells that shows a bulbous epithelial protrusion at the apical end and is referred to as an "apical bud". Guinea pig cheek teeth (premolars and molars), also continuously growing teeth, have a complex crown shape consisting of plural cusps. The present study clarifies the existence of apical buds in guinea pig premolars/molars as it examines the relationship between the crown shape and the orientation of the apical buds by micro-computed tomography (micro-CT) and immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU). One premolar and three molar teeth in each side of the maxillae and mandibles assumed characteristic features: each horizontally-sectioned tooth showing a complex zigzag shape was composed of a core of dentin covered by a layer of enamel on all axial surfaces except the buccal of the uppers and the lingual of the lowers. Furthermore, four bulbous epithelial protrusions--including the stellate reticulum--were recognized in the apical end of each tooth, where slow-cycling long-term label-retaining cells resided 20 days after a peritoneal injection of BrdU. These data indicate that guinea pig premolars/molars have four apical buds where the epithelial adult stem cells reside. In contrast, rodent incisors, which show a single cone appearance, are covered by enamel on the labial side and possess only one apical bud. The results of this study suggest that plural apical buds, being arranged bucco-lingually and mesio-distally, produce the crown mold in a zigzag fashion.

The relationship between the termination of cell proliferation and expression of heat-shock protein-25 in the rat developing tooth germ.

Odontoblast- and ameloblast-lineage cells acquire heat-shock protein (HSP)-25 immunoreactivity after they complete cell division during postnatal odontogenesis in rat molars. However, there are no data available concerning the relationship between the termination of cell proliferation and HSP-25 immunoreactivity during tooth morphogenesis. We compared the expression of HSP-25 in tooth germs with their proliferative activity in the rat prenatal to perinatal molar and postnatal incisor to clarify the functional significance of HSP-25 during tooth morphogenesis by immunohistochemistry using anti-HSP-25 and anti-Ki67/5-bromo-2'-deoxyuridine (BrdU). Numerous proliferating cells in developing molars were distributed throughout the tooth germ and HSP-25 immunoreactivity was recognizable in the dental epithelial and mesenchymal cells after they completed cell division. However, both cell proliferation and immunoreaction for HSP-25 are absent in the enamel knots. The distribution pattern of the proliferating cells in the incisors was basically identical to that in the prenatal molars except for the lack of non-proliferating secondary enamel knots and the sparse distribution of proliferating cells in the apical bud. Thus, HSP-25 protein is suggested to act as a switch between cell proliferation and terminal cyto-differentiation during odontogenesis.