Naohiro Nakasone

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.

Altered gene expression in leukocyte transendothelial migration and cell communication pathways in periodontitis-affected gingival tissues

BACKGROUND AND OBJECTIVE Gene expression is related to the pathogenesis of periodontitis and plays a crucial role in local tissue destruction and disease susceptibility. The aims of the present study were to identify the expression of specific genes and biological pathways in periodontitis-affected gingival tissue using microarray and quantitative real-time RT-PCR analyses. MATERIAL AND METHODS Healthy and periodontitis-affected gingival tissues were taken from three patients with severe chronic periodontitis. Total RNAs from six gingival tissue samples were used for microarray analyses. Data-mining analyses, such as comparisons, gene ontology and pathway analyses, were performed and biological pathways with a significant role in periodontitis were identified. In addition, quantitative real-time RT-PCR analysis was performed on samples obtained from 14 patients with chronic periodontitis and from 14 healthy individuals in order to confirm the results of the pathway analysis. RESULTS Comparison analyses found 15 up-regulated and 13 down-regulated genes (all of which showed a change of more than twofold in expression levels) in periodontitis-affected gingival tissues. Pathway analysis identified 15 up-regulated biological pathways, including leukocyte transendothelial migration, and five down-regulated pathways, including cell communication. Quantitative real-time RT-PCR verified that five genes in the leukocyte transendothelial migration pathway were significantly up-regulated, and four genes in the cell communication pathway were significantly down-regulated, which was consistent with pathway analysis. CONCLUSION We identified up-regulated genes (ITGB-2, MMP-2, CXCL-12, CXCR-4 and Rac-2) and down-regulated genes (connexin, DSG-1, DSC-1 and nestin) in periodontitis-affected gingival tissues; these genes may be related to the stimulation of leukocyte transendothelial migration and to the the impairment of cell-to-cell communication in periodontitis.

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 Sharpeys fibers, and hypogenesis of cementum. Proliferating cells localized in Hertwigs 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.

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.

Amyloid beta (A4) precursor protein expression in human periodontitis-affected gingival tissues

OBJECTIVE Periodontitis involves periodontal tissue destruction and is associated with chronic inflammation and ageing. Periodontitis has recently been recognised as a risk factor for Alzheimers disease (AD). We showed upregulation of molecules in the AD pathway including amyloid beta (A4) precursor protein (APP), a key gene in AD, interleukin-1 beta (IL-1β), and complement component 1 (q subcomponent, A chain) (C1QA) in periodontitis compared to healthy tissues. Here, we quantitatively analysed the expression levels of APP, IL-1β, and C1QA and determined the localisation of APP in gingival tissues. DESIGN Fourteen chronic periodontitis patients and 14 healthy participants were enrolled. Six samples of total RNA from two distinct sites of healthy and periodontitis-affected gingival tissues from three randomly selected patients were used for microarray analyses, and significant biological pathways in periodontitis were identified. Differential gene expression of APP, IL-1β, and C1QA, which belong to the AD pathway, were analysed with quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) using samples from these 14 chronic periodontitis patients and 14 healthy controls. APP localisation was analysed with immunohistochemistry. RESULTS APP, IL-1β, and C1QA mRNA levels were significantly upregulated in periodontitis-affected gingival tissues. APP was mainly localised in macrophages in gingival connective tissues underneath the epithelial layers. CONCLUSIONS An association between AD and periodontitis was detected with microarray and computer-aided data mining analyses. qRT-PCR identified differential gene expression in periodontitis-affected gingival tissue that may be related to AD pathogenesis. Elevated APP, IL-1β, and C1QA transcripts and APP-expressing macrophages in periodontitis-affected gingival tissues were observed, suggesting a relationship between periodontitis and AD pathogenesis.

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-β1 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.

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.