Hodaka Sasaki

Involvement of laminin and integrins in adhesion and migration of junctional epithelium cells.

BACKGROUND AND OBJECTIVE The junctional epithelium attaches to the enamel surface with hemidesmosomes (of which laminin-5 and integrin-alpha(6)beta(4) are the main components) in the internal basal lamina. Laminin-5 is also involved in cell motility with integrin-alpha(3)beta(1), although their functions have not yet been clarified.The purpose of this study was to determine the functions of those adhesive components between the tooth and the junctional epithelium during cell migration.Because an idea has been proposed that directly attached to tooth cells (DAT cells) may not contribute to cell migration, 5-bromo-2-deoxyuridine staining was performed to confirm cell migration. MATERIAL AND METHODS We investigated laminin-gamma(2) (contained only in laminin-5), integrin-beta(4) (involved in cell-extracellular matrix contact) and integrin-alpha(3) (inducing cell migration) in the junctional epithelium, oral gingival epithelium and gingival sulcus epithelium of 6-wk-old ICR mice using laser microdissection, quantitative real-time reverse transcription-polymerase chain reaction, immunofluorescence and 5-bromo-2-deoxyuridine staining. RESULTS Laminin and integrins were clearly immuno-localized in the basal lamina of all epithelium. Quantitative analysis of laminin and integrin mRNAs by laser microdissection showed that they were more highly expressed in DAT cells than in basal cells in the oral gingival epithelium. In particular, a 12-fold higher expression of laminin-5 was observed in the junctional epithelium compared with the oral gingival epithelium. 5-Bromo-2-deoxyuridine staining showed rapid coronal migration of DAT cells. CONCLUSION These results suggest that the abundant expression of laminin-5 and integrin-alpha(6)beta(4) is involved in the attachment of DAT cells to teeth by hemidesmosomes. Abundant expression of laminin-5 and integrin-alpha(3)beta(1) might assist in DAT cell migration, confirmed by 5-bromo-2-deoxyuridine staining during the turnover of junctional epithelium.

Association of Shh and Ptc with keratin localization in the initiation of the formation of circumvallate papilla and von Ebner's gland

The development of gustatory papillae in mammalian embryos requires the coordination of a series of morphological events, such as proliferation, differentiation and innervation. In mice, the circumvallate papilla (CVP) is a specialized structure that develops in a characteristic spatial and temporal pattern in the posterior region of the tongue dorsal surface. The distinct expression patterns of Shh and Ptc, which play important roles in the development of other epithelial appendages, have been localized in the trench wall that gives rise to von Ebner’s gland (VEG). To define the cellular mechanisms responsible for morphogenesis and differentiation during early development of CVP and VEG, the localization patterns of keratins (cytokeratins) K7, K8, K18, K19, K14 and connexin-43, which are dependent on Shh expression in other developmental systems, have been examined in detail. The distinct localization of keratins K7, K8, K18, K19, K14 and connexin-43 in the epithelium giving rise to the CVP and VEG suggests that cytodifferentiation is established prior to morphological changes. Interestingly, the localization of proliferating cell nuclear antigen, a marker for cell proliferation, is similar to that of Shh. An understanding of the regulatory roles of cell-cell interactions and signalling molecules in orchestrating a mutual network will bring us nearer to defining the molecular and cellular mechanisms underlying morphogenesis in mammalian taste bud development.

Clinical evaluation of salivary periodontal pathogen levels by real-time polymerase chain reaction in patients before dental implant treatment.

Objective Periodontal pathogens in dental plaque are the main causative agents of periodontitis and peri-implantitis. Detection of the presence of such periodontal pathogens early would serve as a useful tool in the diagnosis and treatment of this disease. Therefore, the purpose of this study was to investigate whether the periodontal pathogen levels in saliva were correlated with the periodontal status of patients receiving implant treatment. Materials and Methods A total of 291 patients visiting Tokyo Dental College Chiba Hospital were divided into four groups: a no-periodontitis (np) group, a mild-periodontitis (mip) group, a moderate-periodontitis (mop) group, and a severe-periodontitis (sp) group. The levels of the following five periodontal pathogens in saliva were evaluated using real-time polymerase chain reaction: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Prevotella intermedia. Results The levels of P. gingivalis and T. forsythia were significantly higher in mop group than in np group (P < 0.05). The levels of all periodontal pathogens tested except A. actinomycetemcomitans were significantly higher in sp group than in np group (P < 0.05). Conclusion The detection levels of the periodontal pathogens targeted in saliva samples were correlated with the periodontal status. This suggests that using saliva to screen for periodontopathic bacteria offers an easier-to-use clinical tool than the paper point method in the diagnosis and treatment of periodontitis and peri-implantitis.

Down-regulated Genes in Mouse Dental Papillae and Pulp

Important factors involved in odontogenesis in mouse dental papillae disappear between the pre- and post-natal stages of development. Therefore, we hypothesized that certain genes involved in odontogenesis in dental papillae were subject to pre-/post-natal down-regulation. Our goal was to identify, by microarray analysis, which genes were down-regulated. Dental papillae were isolated from embryonic 16-day-, 18-day- (E16, E18), and post-natal 3-day-old (P3) murine first mandibular molar germs and analyzed by microarray. The number of down-regulated genes was 2269 between E16 and E18, and 3130 between E18 and P3. Drastic down-regulation (fold change > 10.0) of Adamts4, Aldha1a2, and Lef1 was observed at both E16 and E18, and quantitative RT-PCR revealed a post-natal reduction in their expression (Adamts4, 1/3; Aldh1a2, 1/13; and Lef1, 1/37). These results suggest that down-regulation of these three genes is an important factor in normal odontogenesis in dental papillae.

Proliferation and osteogenic differentiation of human mesenchymal stem cells on zirconia and titanium with different surface topography.

The purpose of this study was to elucidate behavior of human mesenchymal stem cells (hMSCs) on yttria stabilized tetragonal zirconia polycrystals (TZP) and commercial pure titanium (CpTi) with different surface topography. Mirror-polished (MS), sandblasted with 150-μm alumina (SB150) and SB150 acid-etched (SB150E) were prepared on TZP and CpTi. Proliferation, osteogenic differentiation of hMSCs was evaluated. The scanning electron microscopy showed that micro- and nano-topographies were created on both TZP and CpTi SB150E surfaces. The proliferation ability, ALP activity, expression of Runx2 on the both SB150E specimens was significantly higher than those on the other specimens. These results suggested that creation of micro- and nano-topographies on TZP and CpTi by blast and acid-etching may offer a promising method for enhancing the proliferation and differentiation of hMSCs in clinical application.

Release properties of atelocollagen-gelatin complexes as carriers for local administration of fluvastatin

The aim of this study was to investigate properties of atelocollagen/gelatin complexes (AC/Gel) and their characteristics of sustained statin release, to assess the utility of AC/Gel. AC/Gel were prepared by changing the mixing ratio of AC (0 to 40% of AC). Analysis of spectra of fluvastatin (Flu), gelatin (Gel), and Flu with Gel complex using a Fourier transform-infrared spectrometer indicates that Flu was bound to Gel through a bond involving the carboxyl and amino groups. Evaluation of characteristics of sustained release of Flu from the AC/Gel using an ultraviolet-visible spectrophotometer showed that the release rate of Flu decreased with increasing the AC content. The histological evaluation using of Sprague-Dawley rats suggest that, unlike the pure Gel sponge, the AC/Gel was not absorbed in an early stage. Therefore, the present study showed that sustained Flu release can be controlled by using an AC/Gel, suggesting the utility of this composite material.

Dental Implant Treatment with Computer-assisted Surgery for Bilateral Agenesis of Maxillary Lateral Incisors: A Case Report

Here, we report a case of dental implant treatment involving computer-assisted surgery for bilateral agenesis of the maxillary lateral incisors. The patient was a 39-year-old woman with the chief complaint of functional and esthetic disturbance due to maxillary and mandibular malocclusion. The treatment plan comprised non-extraction comprehensive orthodontic treatment and prosthodontic treatment for space due to the absence of bilateral maxillary lateral incisors. A preliminary examination revealed that the mesiodistal spaces left by the absent bilateral maxillary lateral incisors were too narrow for implant placement (right, 5.49 mm; left, 5.51 mm). Additional orthodontic treatment increased these spaces to approximately 6 mm, the minimum required for implant placement if risk of damage to the adjacent teeth due to inaccuracies in directionality of drilling is to be avoided. For dental implant treatment with computer-assisted surgery, preoperative planning/simulation was performed using Simplant® ver.12 software and a toothsupported surgical template fabricated using stereolithography. Two narrow-diameter implants were placed in a two-stage procedure. It was confirmed that there was sufficient distance between the implant fixtures and the roots of the adjacent teeth, together with no exposure of alveolar bone. Following a 4-month non-loading period, second-stage surgery and provisional restoration with a temporary screw-retained implant crown were performed. Cement-retained superstructures made of customized zirconia abutment and a zirconia-bonded ceramic crown were fitted as the final restoration. At 5 years after implant surgery, there were no complications, including inflammation of the peri-implant soft tissue and resorption of peri-implant bone. Computer-assisted implant surgery is useful in avoiding complications in bilateral agenesis of the maxillary lateral incisors when only a narrow mesiodistal space is available for implant placement.

Bone marrow stromal cells from low-turnover osteoporotic mouse model are less sensitive to the osteogenic effects of fluvastatin

This study aimed to investigate the effects of fluvastatin on the differentiation of bone marrow stromal cells (BMSCs) into osteoblasts in senescence-accelerated mouse prone 6 (SAMP6) compared with that in the normal senescence-accelerated-resistant mouse (SAMR1) model. SAMP strains arose spontaneously from the AKR/J background and display shortened life span and an array of signs of accelerated aging, compared with control SAMR strains. The dose effects of fluvastatin were also evaluated. BMSCs were cultured with/without fluvastatin (0 μM, 0.1 μM, 0.5 μM, and 1.0 μM). WST-1-based colorimetry was performed to evaluate cell proliferation. To evaluate cell differentiation, gene expression levels of bmp2 and runx2 were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and protein expression levels were determined using enzyme-linked immunosorbent assay (BMP2) and immunofluorescence staining (BMP2 and Runx2). Alkaline phosphatase (ALP) activity assay and histochemical detection were determined; the effect of noggin, a BMP-specific antagonist, was examined using ALP histochemical detection. To assess for mature osteogenic marker, gene expression levels of bglap2 were determined by qRT-PCR and mineralization was determined by alizarin red staining. RhoA activity was also examined by Western blotting. In SAMP6, BMP2, Runx2 and Bglap2 mRNA and protein expressions were significantly increased by fluvastatin, and ALP activity was increased by BMP2 action. RhoA activity was also inhibited by fluvastatin. The concentration of fluvastatin sufficient to increase BMP2 and Runx2 expression and ALP activity was 0.5 μM in SAMP6 and 0.1 μM in SAMR1. In conclusion, the present study revealed that fluvastatin promoted BMSC differentiation into osteoblasts by RhoA-BMP2 pathway in SAMP6. BMSCs of SAMP6 are less sensitive to the osteogenic effects of fluvastatin than SAMR1.

Comparison of Gene Expression in Peri-implant Soft Tissue and Oral Mucosal Tissue by Microarray Analysis.

PURPOSE Implant placement entails disruption of the epithelial continuity, which can lead to various complications. Therefore, the area of mucosal penetration is of particular interest clinically. The goal of the present study was to compare gene expression in peri-implant soft tissue (PIST) with that in oral mucosal tissue (OMT) using microarray analysis, and to investigate which genes were specifically expressed in PIST. MATERIALS AND METHODS The bilateral upper first molars were extracted from 4-week-old rats and titanium alloy implants placed only in the left-side extraction sockets. Four weeks after surgery, samples were harvested from the left-side PIST and right-side OMT and total RNA samples isolated. Microarray analysis was used to compare gene expression in PIST and OMT, which was then confirmed using quantitative real-time polymerase chain reaction. Immunohistochemical staining was also performed to confirm protein level expression. RESULTS The number of genes expressed with more than a twofold change in PIST compared with OMT was 1,102, of which 750 genes were upregulated and 352 genes were downregulated. The messenger RNA (mRNA) expression of three selected genes-Ceacam1, Ifitm1, and MUC4-were more significantly expressed in PIST than in OMT(P < .01). Immunohistochemical localization of CEACAM1, IFITM1, and MUC4 was observed in PIST, but no immunoreaction was recognized in OMT. CONCLUSION The result of microarray analysis showed that, because of implant placement, 750 genes were upregulated in PIST compared with OMT. CEACAM1, IFITM1, and MUC4 were specifically upregulated in PIST.