The effect of glutathione on 2-hydroxyethylmethacrylate cytotoxicity and on resin-dentine bond strength.

AIM: To evaluate the influence of reduced glutathione (GSH) application on 2-hydroxyethylmethacrylate (HEMA) cytotoxicity on rat pulpal cells and evaluate the effect of etched-dentine treatment with GSH on the immediate microtensile bond strength (µTBS) of etch-and-rinse adhesive. METHODOLOGY: The cytotoxicity of 10 mmol L(-1) HEMA, 10 mmol L(-1) HEMA + 1 mmol L(-1) GSH, 10 mmol L(-1) HEMA + 5 mmol L(-1) GSH and 10 mmol L(-1) HEMA + 10 mmol L(-1) GSH was compared (6 h and 24 h). Cells viability was measured by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, followed by morphological observation of cells. Etched-dentine surfaces were rinsed and treated with one of the following solutions: 2% GSH, 5% GSH or 10% GSH, bonded with Adper Single Bond Plus (3M, ESPE, St. Paul, MN, USA) and restored with resin composite. The control group received no GSH treatment. After 1 day of water-storage at 37 °C, the specimens were subjected to µTBS testing. Cytotoxicity and µTBS data were analysed by one-way anova and Tukey post hoc tests (P < 0.05). RESULTS: There were significant differences between the groups. HEMA elicited a remarkable toxic effect. 10 mmol L(-1) GSH prevented HEMA-induced damage at both exposure times. Whilst 5 mmol L(-1) GSH lost its protective effect at 24-h exposure time and 1 mmol L(-1) GSH showed no protective effect at both exposure times, GSH had no significant effect on the immediate µTBS; however, 5% GSH had higher bond strength value when compared to 10% GSH (P = 0.003). CONCLUSION: Controlled concentrations of GSH had a protective effect against HEMA cytotoxicity. GSH had neither positive nor negative influence on µTBS.

Restricted expression of chromatin remodeling associated factor Chd3 during tooth root development.

BACKGROUND AND OBJECTIVE: The tooth root is one of the critical parts to maintain tooth function; however, the molecular mechanisms of root development remain unknown. We aimed to identify specific factors for root morphogenesis using a newly developed experimental system. MATERIAL AND METHODS: Tentative cementoblasts and periodontal ligament cells from mouse mandibular molars were isolated using laser capture microdissection. More than 500 cementoblasts and periodontal ligament cells were separately captured. After RNA extraction and amplification, mRNA expression in isolated cementoblasts was compared with that of periodontal ligament cells by cDNA microarray analysis. Then, putative cementoblast-specific genes were subjected to in situ hybridization analysis to confirm the results in mouse mandible. RESULTS: Approximately 2000 genes were differentially expressed between these tissues. Among those genes, zinc finger helicase (ZFH), also termed chromodomain-helicase-DNA-binding protein 3 (Chd3), was one of the highly expressed transcripts in tentative cementoblasts. In situ hybridization revealed that ZFH/Chd3 was strongly expressed in Hertwig's epithelial root sheath rather than in cementum. Moreover, its expression disappeared when root formation was advanced in the first molar. In contrast, Chd3 was continuously expressed in dental epithelial cells of the cervical loop, in which root extension is never terminated. CONCLUSION: These results suggest that ZFH/Chd3 might play an important role in tooth root development and subsequent cementogenesis.

Enhanced osteoblast and osteoclast responses to a thin film sputtered hydroxyapatite coating.

A sputtering technique followed by a low temperature hydrothermal treatment has been demonstrated to produce a dense-and-bioactive hydroxyapatite thin film coating. The purpose of the present study was to investigate osteoblast and osteoclast responses to the hydroxyapatite coated plates and titanium plates with similar roughness. Rat bone marrow stromal cells were cultured on these plates to induce osteoblasts. The cells showed a significantly enhanced proliferation on the hydroxyapatite surface, accompanied by increase of osteoblastic phenotypes. The co-cultured osteoclasts exhibited the significantly different cell number and morphology between the hydroxyapatite and the titanium surfaces. A series of osteoclast marker genes were more stimulated on the hydroxyapatite and thirty two percent of the hydroxyapatite surface area could be resorbed by osteoclasts. The thin film sputtered hydroxyapatite could provide a favorable surface for both osteoblast and osteoclast formation and their function, indicating its good osteoconductivity and biodegradability.

Insulin-like growth factor I regulates apoptosis in condylar cartilage.

Endogenous insulin-like growth factor-I (IGF-I) is known to affect the growth and development of condylar cartilage. However, the critical effect of IGF-I on cell survival is still unknown. We hypothesized that endogenous IGF-I could regulate the survival of cells of the mandibular condylar cartilage. Mandibular condyles dissected from 12-day-old rats were cultured for 1, 3, and 5 days in medium containing antisense oligodeoxynucleotide (AS-ODN) for IGF-I. Real-time RT-PCR analysis showed that the levels of IGF-I and IGF binding protein (IGFBP)3 mRNAs in the AS-ODN group were significantly decreased. After 3 days' culture, the number of necrotic cells was observed in the undifferentiated mesenchymal cell layer. These cells were TUNEL-positive and confirmed to be apoptotic by electron microscopic observation. Immunoblotting revealed that expression of cleaved caspase3 was increased with AS-ODN. These results may suggest that the cells in the undifferentiated mesenchymal cell layer of the mandibular condyle require IGF-I for survival.

Selective drug delivery system to bone: small peptide (Asp)6 conjugation.

Targeting a drug on hydroxyapatite (HA) could be a promising way for selective drug delivery to bone, because HA, an inorganic component in hard tissues (bone and teeth), does not exist in soft tissues. Several bone noncollagenous proteins, which bind to HA, have repeating sequences of acidic amino acids in their structures as possible HA-binding sites. Thus, we think that a small peptide of repetitive acidic amino acid could work as a carrier for selective drug delivery to the bone. To test this hypothesis, we conjugated (Asp)6 to fluorescein isothiocyanate (FITC), evaluated its affinity to HA in vitro, and examined its tissue distribution after injection into rats. Although fluorescein itself did not bind to HA, (Asp)6-FITC bound to HA as well as calceine and tetracycline. Twenty-four hours after intravenous injection of (Asp)6-FITC to rats, animals were killed, and ground sections of hard tissues and cryosections of soft tissues were made. Under a confocal laser scanning microscope, clear labeling lines were observed in bones and teeth, whereas no labeling was detected in soft tissues. In the rats administered with fluorescein alone, the fluorescent labeling was detected in neither hard nor soft tissues. Fluorescent analysis of blood, urine, and bones after (Asp)6-FITC administration revealed that biological half-life of FITC in blood was short (60 minutes) and that within 24 h, 95% of the administered FITC was excreted as urine whereas 2% of the FITC accumulated in bones. After subcutaneous administration of (Asp)6-FITC to mice, fluorescent intensity remaining in the femurs was measured periodically. In these mice the biological half-life of FITC in the femur was 14 days. Present results indicate that (Asp)6 is effective as a carrier for selective drug delivery to bone.

Expression of matrix metalloproteinase-9 mRNA and protein during deciduous tooth resorption in bovine odontoclasts.

Matrix metalloproteinase-9 (MMP-9, or gelatinase B) is an extracellular proteinase that is highly expressed in osteoclasts and has been postulated to play an important role in their resorptive activity. Although MMP-9 has been reported to play a role in bone resorption, the association of this enzyme during deciduous tooth resorption has not yet been clarified. The purpose of the present study was to increase our understanding of the role of MMP-9 during deciduous tooth resorption. Reverse transcription-polymerase chain reaction (RT-PCR) and northern blot analysis of total RNAs extracted from bovine root-resorbing tissues, which lie between the root of a deciduous tooth and its permanent successor, revealed the expression of mRNA for MMP-9 in the tissue. These results indicate that MMP-9 may be involved in the process of deciduous tooth resorption. In addition, in situ hybridization and immunohistochemistry were also performed to identify the cells that produced MMP-9 in bovine root-resorbing tissue. MMP-9 mRNA was highly expressed in odontoclasts that were aligned along the surface of the tissue. Immunohistochemistry confirmed the predominant localization of MMP-9 in odontoclasts. The present data demonstrate that odontoclasts in deciduous root resorption express MMP-9, which may participate in proteolysis during root resorption of deciduous tooth.

A new mutation of the Po gene in patients with Charcot-Marie-Tooth disease type 1B: screening of the Po gene by heteroduplex analysis.

Most of Charcot-Marie-Tooth (CMT) 1 families are associated with a duplication in chromosome 17p11.2-p12, which includes the gene encoding peripheral myelin protein-22 (PMP-22). Point mutations of the Po gene have been identified in a few of the CMT 1 families in whom no duplication was found. We investigated a new mutation of the Po gene in one of those families. A to G substitution of nucleotide 389 in exon 3 resulted in Lys 131 Arg substitution. This structural change of extracellular domain of Po would alter the function of Po and result in an impairment of peripheral myelin compaction.

cDNA cloning of S100 calcium-binding proteins from bovine periodontal ligament and their expression in oral tissues.

The periodontal ligament (PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. To characterize PDL cells at the molecular level, we constructed a cDNA library from bovine PDL tissue. We then focused on the isolation of S100 calcium-binding proteins (CaBPs), because they mediate Ca2+ signaling and control important cellular processes such as differentiation and metabolism. We screened the PDL cDNA library with a mouse S100A4 cDNA, and cloned the bovine cDNAs of two S100 CaBPs (S100A4 and S100A2). In northern blotting analysis, the highest expression of S100A4 was detected in PDL from erupted teeth (PDLE). PDL from teeth under eruption (PDLU) showed a lower expression of S100A4, and its expression in gingiva was faintly detectable. S100A4 expression was also high in the pulp tissue followed by the dental papilla of the tooth germ. S100A2 expression was high in PDLE and gingiva. Interestingly, only PDLE exhibited a high expression of both S100A4 and S100A2. PDLE also expressed the highest level of beta-actin, a target cytoskeletal protein for S100A4. It is conceivable that the high expression of S100A4 in PDLE is a result of the maturation of the PDL and/or a response to mechanical stress generated by mastication. Since there was a marked difference of S100A4 expression between PDL and gingiva, we propose that S100A4 could be a useful marker for distinguishing cells from these two tissues.

Tomaculous neuropathy in Charcot-Marie-Tooth disease with myelin protein zero gene mutation.

Mutation of the myelin protein zero (MPZ) gene is associated with a small number of Charcot-Marie-Tooth (CMT) patients. We present a patient with Lys 130 Arg substitution in the extracellular domain who showed tomacula formation in biopsied sural nerve. CMT patients with mutations Ly 96 Glu, Lys 130 Arg and Ile 135 Leu showed tomaculous neuropathy. Present and previously reported investigations suggest that the pathological phenotypes of peripheral nerve are probably related to the mutations of the MPZ gene.

Molecular and pathological studies in Charcot-Marie-Tooth disease 1A.

We analyzed a 1.5-Mb duplication of the p11.2-12 region of chromosome 17, including the PMP-22 gene (CMT1A duplication), seven families with Charcot-Marie-Tooth disease type I (CMT I) and six sporadic patients with suspected CMT I by Southern blot analysis. In order to detect the CMT 1A duplication, probe pVAW409R3a, probe PMP-22 cDNA and reference probe SF85 were used for Southern hybridization. In six out of seven families with CMT I, CMT1A duplication was identified. One of six sporadic CMT patients had CMT1A duplication. The probe pVAW4O9R3a was more informative than PMP-22 cDNA and SF85 for detecting CMT1A duplication. In pathological study of biopsied sural nerve, thickened myelin sheath was observed in some myelinated fibers in patients with CMT1A duplication.

De novo mutation of Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT 1A) is an autosomal dominant demyelinating polyneuropathy associated with a 1.5-Mb duplication of the p11.2-p12 region of chromosome 17, including the peripheral myelin protein-22 (PMP-22) gene (CMT 1A duplication). We report a male patient with a de novo CMT 1A diagnosed on clinical, electrophysiologic, and molecular grounds. Motor nerve conduction velocity (MCV) of the patient was 10.9 m/s in the ulnar nerve. The MCV of both his parents was within the normal range. Southern blot analysis of BamHI digestion showed reduced intensity rate of SF85/PMP-22, indicating CMT 1A duplication. Haplotype analysis with pVAW4093a, demonstrated that the de novo CMT 1A duplication was of paternal origin.

Charcot-Marie-Tooth disease type 1A duplication by PCR analysis.

By generating new junctional fragments from the recombinant Charcot-Marie-Tooth (CMT) 1A-REPs in CMT1A patients, a 3.2-kb recombination hot spot is observed in three quarters of CMT1A patients. By a polymerase chain reaction (PCR) method the authors analyzed eight patients CMT1A duplication, confirmed by Southern blot, to detect a recombination hot spot. Four patients had a novel 3.2-kb junctional fragment by PCR analysis. These four patients with a novel 3.2-kb junctional fragment had an abnormal 1789-bp fragment in addition to 1986-bp fragment after NsiI digestion (type 1). One patient who demonstrated no novel 3.2-kb junctional fragment had an abnormal 336-bp fragment in addition to 265 bp (type 2). Three patients with CMT1A duplication were not diagnosed as having CMT1A on the basis of PCR analysis. The PCR-based DNA test is valuable for screening to detect CMT1A duplication.

Expression of peripheral myelin protein zero in sural nerve of patients with Charcot-Marie-Tooth disease 1B.

A Charcot-Marie-Tooth disease 1B (CMT1B) family with a mutation of the Po gene is presented. A to G substitution of nucleotide 389 in exon 3 resulted in Lys 131 Arg substitution. Immunostaining for Po in biopsied sural nerve from one family member with CMT1B was expressed in a small number of myelinated fibers. Immunoblot analysis for Po revealed that it was of normal molecular weight (29 kDa) although significantly reduced in amount. This heterozygous mutation could lead to a reduction in the total amount of normal protein in peripheral nerves through a mechanism of loss of function.

Toxicity of camphorated phenol and camphorated parachlorophenol in dental pulp cell culture.

The toxicity of phenol, parachlorophenol, camphorated phenol, camphorated parachlorophenol, and camphor was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay on established rat dental pulp cells (RPC-C2A). RPC-C2A cells at the confluent stage were incubated for 24 h in an experimental medium containing each compound at different concentrations. All tested drugs showed cytotoxicity in the MTT assay in a concentration-dependent manner. It is believed that camphor is a vehicle, and it reduces the toxicity of phenol and parachlorophenol. However, camphor itself showed cytotoxicity, and the addition of camphor increased the toxicity of phenol and parachlorophenol, reconfirming the cytotoxicity of these classical antiseptics.

Autoradiographic investigation of the effect of 1-hydroxyethylidene-1, 1-bisphosphonate on matrix protein synthesis and secretion by secretory ameloblasts in rat incisors.

Seven daily subcutaneous injections of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) can induce enamel hypoplasia. Several enamel-free zones were observed along the crown-analogue side of rat incisors during the secretory stage of amelogenesis. Ameloblasts related to the enamel-free zones lay directly on the abnormally non-mineralized mantle dentine, whereas the adjacent ameloblasts, which were forming the enamel matrix layer, were associated with the region where mineralization of dentine was proceeding. The further purpose of this study was to investigate the synthetic and secretory activity of these two groups of ameloblasts and to trace the fate of the radioactively labelled proteins. [(3)H]-proline was administered to Wistar rats 12 h after the last injection of HEBP. Light-microscopic autoradiography was performed. Quantitative analysis indicated that the ameloblasts of the enamel-forming zones in the drug-treated group showed a distribution pattern of silver grains similar to that of the controls. The ameloblasts of the enamel-free zones also demonstrated incorporation of [(3)H]-proline at the same level. There was some labelling over the non-mineralized mantle dentine, which was supposed to indicate the penetration of ameloblast products. From these results, it is concluded that HEBP does not affect the ameloblast activity in protein synthesis. The complete failure of enamel-layer formation in some specific regions is probably due to the failure in protein secretion and protein deposition. This study provides additional evidence that the mineralization of dentine is an essential factor in successful enamel matrix secretion and deposition.

Confocal laser scanning-microscopic observations on the three-dimensional distribution of oxytalan fibres in mouse periodontal ligament.

The purpose was to improve confocal laser scanning-microscopic (CLSM) techniques to observe the three-dimensional (3-D) distribution of oxytalan fibres in mouse periodontal ligament and to clarify the 3-D relation between those fibres and blood vessels. As aldehyde fuchsin is a contrast agent and the specific wavelength affects the depth of penetration, CLSM reflectance imaging of oxytalan stained with aldehyde fuchsin after oxidization provides strong contrast. Oxytalan fibres, whose precise roles have not yet been clarified, are connective tissue fibres present in human periodontal ligament in addition to collagen fibres. Despite many studies on their arrangement and biomechanical characteristics, their 3-D distribution in relation to other structures has never been reported. Mandibular first molars of mice were sectioned mesiodistally, pre-oxidized by monopersulphate compound (Oxone), stained with aldehyde fuchsin and examined by CLSM. CLSM images clearly revealed the 3-D distribution, and relation of oxytalan fibres to blood vessels and other structures, as well as their branching patterns in the periodontal ligament. The marked anatomical correlations between the direction, distribution and branching patterns of oxytalan fibres and blood vessels suggest that they interact to perform a specialized physiological role in the periodontal ligament.

Gene expression and immunolocalization of amelogenin in enamel hypoplasia induced by successive injections of bisphosphonate in rat incisors.

Successive injections of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) in rats induce enamel hypoplasia. To elucidate the pathogenesis of this hypoplasia, male Wistar rats were daily injected with HEBP or physiological saline for 7 days. After the last injection, they were killed under anaesthesia and their maxillary incisors were examined using an in situ hybridization technique and immunohistochemical staining to detect the gene expression and localization of amelogenin protein, respectively. In the HEBP-injected rats, several islets of partially mineralized enamel were present along crown-analogous surface of the incisor in the secretory stage of amelogenesis and enamel-free zones existed between these islets. In situ hybridization demonstrated amelogenin gene expression over the ameloblasts facing the islets of the matrix enamel as well as over those of the enamel-free zones. Immunohistochemical studies using rabbit antiamelogenin antibody revealed positive reaction both in the enamel matrix of the control group and in the islets of enamel matrix of the HEBP-injected group. Some small granules immunoreactive to amelogenin antibody were found in the distal portions of the ameloblasts in the HEBP-injected rats. The results indicate that HEBP does not alter amelogenin gene expression over ameloblasts, or the protein's existence in enamel matrix. There appeared to be some accumulation of amelogenin in the HEBP-treated ameloblasts. It is therefore suggested that the enamel hypoplasia in this experiment may not be due to a disturbance in amelogenin synthesis but to a disturbance in a later process, presumably of protein secretion.

The effect of high salt intake on the mandibular bone loss in Dahl-Iwai salt-sensitive rat.

The purpose of this study was to evaluate the effect of high salt intake on the mandibular bone in Dahl-Iwai salt-sensitive (DS) rats. Twenty-eight 11-week-old male DS rats were divided into four groups (n=7). The control groups received a normal (0.2% NaCl) diet while the experimental groups received a diet supplemented with 8.0% NaCl. The systolic blood pressure was significantly increased in the experimental groups compared to the control groups. The animals were sacrificed under ether anesthesia at the 8th week or the 22nd week of the experiment. The biochemical data in plasma and urine suggested negative calcium balance in the experimental groups compared to the control groups. The bone mineral density was significantly reduced at the 22nd week of high salt loading. The histomorphometric analysis suggested that the reduction of the mandibular bone volume had already started by the 8th week of high salt loading along with the increased bone resorption and the decreased bone formation, and that the improper bone remodeling balance became normalized by the 22nd week of high salt loading. In conclusion, these results indicate that a high salt intake causes not only severe hypertension but also a mandibular bone reduction in the DS rats.

Effects of mechanical stress on the mRNA expression of S100A4 and cytoskeletal components by periodontal ligament cells.

The periodontal ligament (PDL) functions under constant mechanical stress, and PDL cells obviously control PDL functions under such conditions. We have previously found that the mRNA expression of the Ca2+-binding protein S100A4 and beta-actin is higher in the PDL from erupted teeth than in the PDL from teeth under eruption. This suggested a role for S100A4 in the response of PDL cells to mechanical stress, possibly by coupling Ca2+ and the cytoskeletal system. In the present study, we investigated the direct effects of cyclical stretching on the mRNA expression of S100A4 and two cytoskeletal components (beta-actin and alpha-tubulin) by PDL cells. In Northern blotting analysis, the expression of S100A4, beta-actin, and alpha-tubulin mRNAs was higher in the PDL from fully erupted and functional bovine teeth than in partially erupted ones. Similarly, when bovine PDL cells were mechanically stimulated by means of the Flexercell Strain Unit, the expression of S100A4, beta-actin, and alpha-tubulin mRNAs increased over the control levels. The results of our present study indicate that S100A4 is involved in the responses of PDL cells to mechanical stress possibly by coupling Ca2+ to the cytoskeletal system in these cells.

[Alveolar bone resorption process in molar tooth region in calcium-deficient rats].

The present study was carried out to investigate the alveolar bone resorption in the molar tooth region in the rats fed a low calcium diet. Male Wistar rats (70-85g in body weight) were either fed a low calcium diet (0.05% Ca, 0.35% P) or a control diet (0.5% Ca, 0.35% P) by using a pair feeding technique. The rats were sacrificed at intervals of 3, 6, 9 and 20 days during the experimental period. Contact microradiograph of the second molar region of the mandible showed that the cancellus bone as well as the endosteal surface of the cortical bone were progressively resorbed soon after the start of low calcium feeding. At day 9, the amount of bone was reduced to about half of the control rats and 80% of the bone was diminished at day 20. In spite of the severe bone resorption, the alveolar bone proper that surrounds the molar sockets and a few cancellus bone were seen remaining and the occlusal function of the molar tooth seemed to be maintained. These results suggest that the alveolar bone quickly responded to the low calcium diet resulting in bone resorption and that the bone in the supporting tissues of the molar tooth seems to be not affected by the calcium deficiency.