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Nitric oxide (NO) is associated with many pathophysiology of the central nervous system including brain ischemia, neurodegeneration and inflammation. Epigallocatechin gallate (EGCG) is a major compound of green tea polyphenol that has shown the protective activity against neuronal diseases. This study examined the effect of EGCG on NO-induced cell death in PC12 cells. The administration of sodium nitroprusside (SNP), a NO donor, decreased the cell viability and induced apoptosis showing characterization such as cell shrinkage and chromatin condensation as well as subG1 fraction of cell cycles. EGCG inhibited the cytotoxicity and apoptotic morphogenic changes induced by SNP. EGCG attenuated the production of reactive oxygen species (ROS) by SNP, and ameliorated the SNP-induced Bax to Bcl-2 expression ratio leading to apoptosis. In addition, EGCG prevented the release of cytochrome c from the mitochondria into the cytosol as well as the upregulation of the voltage-dependent anion channel (VDAC), a cytochrome c releasing channel, in the mitochondria of SNP-treated cells. EGCG abrogated the activation of caspase-9, caspase-8 and caspase-3 induced by SNP. These results demonstrate that EGCG has a protective effect against SNP-induced apoptosis in PC12 cells by scavenging ROS and modulating the signal molecules associated with cytochrome c, caspases, VDAC and the Bcl-2 family. These findings suggest that EGCG might be a natural neuroprotective substance.

Objective To determine the diameter of the crown, total crown area, individual cusp area, and occlusal table area in Korean maxillary permanent molars, as well as dental characteristics relevant to the hypocone reduction trait. Materials and methods Subjects included 121 dental school students in Korea (81 men and 40 women). A digital image analysis system was used for measurements and we relied on visual scoring to assign categories of hypocone expression. Results The mean crown dimension was larger in the first molar (M1) than in the second molar (M2). Regarding differences according to gender, the crown diameter, total crown area, and individual cusp area were significantly larger in the men than in the women. The mean occlusal table area ratios were 61% for M1 and 57% for M2, and these ratios increased in proportion to the total crown area. With respect to the hypocone and other features of the maxillary molars, differences between the men and women were more prominent for M1 than for M2. The M2 hypocone tended to be smaller than the M1 hypocone, and hypocone reduction was inversely related to the cusp area of the protocone. The protocone area may be considered a reliable parameter for comparing race and/or gender differences in maxillary molars. Conclusion This study concerning characteristics of the maxillary molar occlusal surface in Koreans provides useful information for comparative studies among human races.

Clinically, acupuncture therapy is useful for the control of acute or chronic pain. This study was designed to elucidate the antinociceptive mechanism of acupuncture and the mechanisms underlying cardiovascular reflex elicited by toothache. Expression of c-Fos, a neuronal activation marker, and the phenylethanalamine-N-methyltransferase (PNMT) were examined 1.5 hours after noxious intrapulpal tooth stimulation. Manual acupuncture was performed 20 min before noxious intrapulpal stimulation by 2 M KCl injection into upper or lower anterior tooth pulp. The acupuncture points were Li4 (Hegu) between the 1st and 2nd metacarpal bones or St36 (Zusanli) between the anterior crest of the tibial tuberosity and the fibula head below the patella. After noxious intrapulpal tooth stimulation, Fos-immunoreactive (IR) neurons were identified in the trigeminal subnucleus caudalis (Vc) and the transitional region between the subnucleus caudalis and the subnucleus interpolaris (Vi), in the inferior olivory nucleus (IO) connecting the cerebellum and other brain regions, and also the thalamic ventral posteromedial (VPM) nucleus and centrolateral (CL) nucleus, respectively. In addition, Fos-IR neurons were found in the central cardiovasuclar regulation centers, such as the hypothalamus supraoptic nucleus (SON) and paraventricular nucleus (PVN), and nucleus tractus solitarius (NTS) and rostral ventromedulla (RVLM). All acupuncture at St36 or Li4 significantly suppressed Fos-IR neurons in all Fos-expressed brain areas except the IO nucleus and attenuated the increases in arterial blood pressure (BP) and heart rate (HR) after noxious intrapulpal stimulation. Its Fos-suppressive effects were mostly blocked by naloxone, an opioid antagonist. In addition, acupuncture at St36 or Li4 significantly decreased Fos-containing PNMT, and this effect was also reversed by naloxone. These results suggest that: 1) tooth pulpal noxious signals transmit to the Vc and Vc/Vi transitional region and the 2nd afferent neuron synapse in the thalamic VPM and CL, 2) tooth pulpal pain elicits cardiovascular reflex mediated by NTS, VLM, hypothalamic SON and PVN, and 3) acupuncture reduces cardiovascular reflex elicited by toothache, is associated with the adrenergic system.

Photoluminescence (PL) and cathodoluminescence (CL) of light emitting diode samples with various InGaN/GaN multi-quantum-well (MQW) active structures and a range of dislocation density (∼7×108/cm2 to ∼9×109/cm2) were investigated. Results indicate that threading dislocations have impact on the luminescence of GaN thin films and act as a nonradiative recombination center. It was also observed that the optical emission is affected by MQW structure such as the number of quantum wells.

Objectives: Cyclosporin A (CsA) is known to elicit fibrous gingival overgrowth with changes of blood vessel profiles. In this study, we examined the expression of several angiogenic and angiostatic genes during the development of CsA-induced gingival overgrowth. Methods: For the development of gingival overgrowth, Sprague-Dawley rats received subcutaneous injections of CsA in daily doses of 5, 10, 15 mg/kg body weight for 6 weeks, and another group received 10 mg/kg of CsA for 3, 6, and 12 weeks. Human gingival tissues were obtained from three CsA-treated patients following the gingivectomy procedure and from three healthy patients following the crown-lengthening procedure as a control. Gingival fibroblasts were isolated from the healthy gingival tissues of the rat or the human, and cultured with 250–1000 ng/ml of CsA. Results: Reverse transcription–polymerase chain reaction (RT–PCR) analyses showed that expressions of some angiogenic genes such as angiopoietin 1, basic fibroblast growth factor, and vascular endothelial growth factor, and angiostatic genes such as angiopoietin 2, brain-specific angiogenesis inhibitor 1 and 2, and thrombospondin 1 were not changed significantly in both gingival tissues and cultured fibroblast cells under the CsA treatments. However, expression of thrombospondin 2 (TSP2) decreased dose- and time-dependently in rat and human gingival tissues. Western blot analyses showed that the expression of TSP2 protein was dose-dependently reduced by the CsA treatments in human cultured gingival fibroblasts. Conclusions: These results indicate that the decrease in angiostatic TSP2 expression may be attributed to the CsA-induced gingival vascularization rather than to the increased expression of angiogenic genes. It suggests that TSP2 is involved in the development of CsA-induced gingival overgrowth with the gingival vascularization.

Periodontal disease is one of the most prevalent chronic disorders worldwide. It is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss. Here, we focused on the role of adipokines, which are locally expressed by periodontal tissues, in the regulation of catabolic gene expression leading to periodontal inflammation. The expression of the nicotinamide phosphoribosyltransferase (NAMPT) adipokine was dramatically increased in inflamed human and mouse gingival tissues. NAMPT expression was also increased in lipopolysaccharide- and proinflammatory cytokine-stimulated primary cultured human gingival fibroblasts (GF). Adenovirus-mediated NAMPT (Ad-Nampt) overexpression upregulated the expression and activity of COX-2, MMP1 and MMP3 in human GF. The upregulation of IL-1β- or Ad-Nampt-induced catabolic factors was significantly abrogated by the intracellular NAMPT (iNAMPT) inhibitor, FK866 or by the sirtuin (SIRT) inhibitor, nicotinamide (NIC). Recombinant NAMPT protein or extracellular NAMPT (eNAMPT) inhibition using a blocking antibody did not alter NAMPT target gene expression levels. Moreover, intragingival Ad-Nampt injection mediated periodontitis-like phenotypes including alveolar bone loss in mice. SIRT2, a part of the SIRT family, was positively associated with NAMPT actions in human GF. Furthermore, in vivo inhibition of the NAMPT-NAD+-SIRT axis by NIC injection in mice ameliorated the periodontal inflammation and alveolar bone erosion caused by intragingival injection of Ad-Nampt. Our findings indicate that NAMPT is highly upregulated in human GF, while its enzymatic activity acts as a crucial mediator of periodontal inflammation and alveolar bone destruction via regulation of COX-2, MMP1, and MMP3 levels.

Summary: Compared with HIV-negative individuals, HIV-positive individuals have a higher prevalence of anogenital human papillomavirus (HPV) infection, as well as a higher incidence of HPV-associated anal cancer. Little is currently known of chromosomal changes occurring in anal intraepithelial neoplasia (AIN), the probable precursor to anal cancer. Genetic changes in AIN were characterized by comparative genomic hybridization (CGH) in a study of samples obtained from 19 HIV-positive and 11 HIV-negative men. The proportion with genetic changes significantly increased with the severity of the histopathologic grade with none diagnosed as (0%) AIN 1; 5 of 17 (29%) as AIN 2; and 5 of 9 (56%) AIN 3 showing genetic changes (p = .02). This correlation was also found in study subjects who had multiple biopsies with different grades of pathology concurrently or serially over time. The most common regional DNA copy number change was gain mapped to chromosome arm 3q (12% of AIN 2 and 33% of AIN 3). This alteration was previously reported to be commonest alteration in cervical cancer, which suggests a common molecular pathway for these two HPV-associated anogenital neoplasias.

Purpose: This study was performed to investigate the interobserver agreement on the detection of carotid artery calcifications on panoramic radiographs. Materials and Methods: This study consisted of panoramic radiographs acquired from 634 male patients of the age of 50 years or older. Having excluded carotids of no diagnostic quality, 1008 carotids from the panoramic radiographs of the patients were interpreted by two oral and maxillofacial radiologists independently for the presence of carotid artery calcifications. Statistical analysis was used to calculate the interobserver agreement. Results: Interobserver agreement was obtained for 932 carotids (92.4%). Inconsistent interpretation of 76 carotids (7.5%) between the two observers was found. Cohen’s kappa value was 0.688 (p⁄0.001). Conclusion: The probability of a match between the two observers was substantially high. (Imaging Sci Dent 2014; 44: 137-41)

Analysis of gene expression and correlation with clinical parameters has the potential to become an important factor in therapeutic decision making. The ability to analyze gene expression in archived tissues, for which clinical followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accurately reflect expression before fixation. In the present study we have optimized the RNA isolation and reverse transcription steps for quantitative reverse transcription-polymerase chain reaction (RT-PCR) on archival material. Using tissue taken directly from the operating room, mRNAs with half-lives from 10 minutes to >8 hours were isolated and reverse transcribed. Subsequent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fixation times (time from excision to fixation) of up to 12 hours. Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.

Purpose: Often tissues obtained from prostate adenocarcinoma tumors embedded in paraffin are heterogeneous in cell type and must be carefully microdissected to acquire tissue fragments that provide homogeneous aliquots of tumor clones. Such tissue fragments rarely contain sufficient DNA to perform genomic characterization needed as an early step in localizing relevant oncogenes or tumor suppressor genes. We report that PCR using a degenerate oligonucleotide primer (DOP-PCR) can be applied to DNA samples from microdissected paraffin-embedded prostate adenocarcinomas, and this provides sufficient product for fluorescent allelic imbalance measurements or comparative genomic hybridization (CGH).Materials and Methods: Samples were selected to be representative of those routinely obtained during prostatectomies, based on typical tumor stages (T2 and T3) and Gleason grades (range 3 + 3 to 4 +5). For DNA analysis without prior DOP-PCR, only large tumors were selected to be sectioned. More than 50 specimen...

Relaxin (Rln), a polypeptide hormone of the insulin superfamily, is an ovarian peptide hormone that is involved in a diverse range of physiological and pathological reactions. In this study, we investigated the effect of Rln on bone morphogenetic protein 2 (BMP-2)-induced osteoblast differentiation and bone formation. Expression of Rln receptors was examined in the primary mouse bone marrow stem cells (BMSCs) and mouse embryonic fibroblast cell line C3H/10T1/2 cells by RT-PCR and Western blot during BMP-2-induced osteoblast differentiation. The effect of Rln on osteoblast differentiation and mineralization was evaluated by measuring the alkaline phosphatase activity, osteocalcin production, and Alizarin red S staining. For the in vivo evaluation, BMP-2 and/or Rln were administered with type I collagen into the back of mice, and after 3 weeks, bone formation was analyzed by micro-computed tomography (µCT). Western blot was performed to determine the effect of Rln on osteoblast differentiation-related signaling pathway. Expression of Rxfp 1 in BMSCs and C3H/10T1/2 cells was significantly increased by BMP-2. In vitro, Rln augmented BMP-2-induced alkaline phosphatase expression, osteocalcin production, and matrix mineralization in BMSCs and C3H/10T1/2 cells. In addition, in vivo administration of Rln enhanced BMP-2-induced bone formation in a dose-dependent manner. Interestingly, Rln synergistically increased and sustained BMP-2-induced Smad, p38, and transforming growth factor-β activated kinase (TAK) 1 phosphorylation. BMP-2-induced Runx 2 expression and activity were also significantly augmented by Rln. These results show that Rln enhanced synergistically BMP-2-induced osteoblast differentiation and bone formation through its receptor, Rxfp 1, by augmenting and sustaining BMP-2-induced Smad and p38 phosphorylation, which upregulate Runx 2 expression and activity. These results suggest that Rln might be useful for therapeutic application in destructive bone diseases.

This article reports the experiences of women aged 55 to 75 with mobility impairments who attributed aspects of their limitations to workplace injuries and provides insight into worker’s compensation policies. The study sample includes Mexican American (MA) and non-Hispanic White (NHW) women aged 55 to 75 who participated in a 4-year ethnographic study of disablement. Ninety-two of the 122 participants in the study attributed aspects of their functional limitations to employment, and their experiences were analyzed using data from 354 meetings. Using Lipscomb and colleagues’ conceptual model of work and health disparities, the women’s experiences were grouped into three categories according to type of injury, assistance gained, and the consequences of a workplace injury; the results have broad implications for policies that influence aging outcomes. Workplace injuries causing permanent functional limitations compound the effects of age and gender on employment outcomes. Policies addressing health disparities should consider work related influences.

Background and Objective Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-oxidizing enzyme with immune-inhibitory effects. The aim of this study was to investigate the expression of IDO by lipopolysaccharide (LPS), a component of gram-negative bacteria, in human periodontal ligament (PDL) cells. Material and Methods Human PDL cells and gingival fibroblasts (GFs) were prepared from explants of human PDLs and from gingival tissues of clinically healthy donors, respectively. Real-time RT-PCR, western blotting and the IDO enzyme assay were performed to determine the expression of IDO following LPS treatment of cells. LPS was injected into mice tail veins to evaluate the effects of LPS in vivo in the maxillary first molar. Immunofluorescence staining and histological analysis were followed to localize IDO in mouse PDL. Results The level of expression of IDO mRNA in primary human PDL cells after LPS treatment was increased in a dose-dependent manner, reaching a peak 8 h after LPS treatment. The expression and activities of IDO protein were significantly increased in comparison with those of the control. In addition, the increased production of kynurenine in culture medium was observed 72 h after LPS treatment. In the immunofluorescence findings, stronger immunoreactivities were shown in PDL than in gingival tissues in the maxillae. In accordance with the immunofluorescence findings, LPS treatment induced a strong up-regulation of IDO mRNA in human PDL cells, whereas human GFs showed only a weak response to LPS. Conclusion These results clearly show that IDO was induced by LPS in primary human PDL cells, suggesting that PDL might be involved in the regulation of oral inflammatory disease.

Embryonic hypoxia/ischemia is a major cause of a poor fetal outcome and future neonatal and adult handicaps. However, biochemical cellular events in mouse embryonic stem (mES) cells during hypoxia remains unclear. This study investigated the underlying mechanism of apoptosis in mES cells under CoCl2-induced hypoxic/ischemic conditions. CoCl2 enhanced the expression of hypoxia-inducible factor-1α (HIF-1α) and the accumulation of reactive oxygen species in mES cells. The CoCl2-treated mES cells showed a decrease in cell viability as well as typical apoptotic changes, cell shrinkage, chromatin condensation, and nuclear fragmentation and an extended G2/M phase of the cell cycle. CoCl2 augmented the release of cytochrome c into the cytosol from the mitochondria with a concomitant loss of the mitochondrial transmembrane potential (ΔΨm) and upregulated the voltage-dependent anion channel. In addition, CoCl2-induced caspase-3, -8, and -9 activation and upregulation of p53 level, whereas downregulated Bcl-2 and Bcl-xL, a member of the anti-apoptotic Bcl-2 family in mES cells. Furthermore, CoCl2 led to the upregulation of Fas and Fas-ligand, which are the death receptor assemblies, as well as the cleavage of Bid in mES cells. These results suggest that CoCl2 induces apoptosis through both mitochondria- and death receptor-mediated pathways that are regulated by the Bcl-2 family in mES cells.

The purpose of this study is to investigate the antinociceptive effects of ginsenosides on toothache. c-Fos immunoreactive (IR) neurons were examined after noxious intrapulpal stimulation (NS) by intrapulpal injection of 2 M KCl into upper and lower incisor pulps exposed by bone cutter in Sprague Dawley rats. The number of Fos-IR neurons was increased in the trigeminal subnucleus caudalis (Vc) and the transitional region between Vc and subnucleus interpolaris (Vi) by NS to tooth. The intradental NS raised arterial blood pressure (BP) and heart rate (HR). The number of Fos-IR neurons was also enhanced in thalamic ventral posteromedial nucleus (VPMN) and centrolateral nucleus (CLN) by NS to tooth. The intradental NS increased the number of Fos-IR neurons in the nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM), hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN), central cardiovascular regulation centers. Ginsenosides reduced the number of c-Fos-IR increased by NS to tooth in the trigeminal Vc and thalamic VPMN and CLN. Naloxone, an opioid antagonist, did not block the effect of ginsenoside on the number of Fos-IR neurons enhanced by NS to tooth in the trigeminal Vc and thalamic VPMN and CLN. Ginsenosides ameliorated arterial BP and HR raised by NS to tooth and reduced the number of Fos-IR neurons increased by NS to tooth in the NTS, RVLM, hypothalamic SON, and PVN. These results suggest that ginsenosides have an antinociceptive effect on toothache through non-opioid system and attenuates BP and HR increased by NS to tooth.

Objectives The purpose of this study was to assess the ability of two new calcium silicate-based pulp-capping materials (Biodentine and BioAggregate) to induce healing in a rat pulp injury model and to compare them with mineral trioxide aggregate (MTA). Materials and Methods Eighteen rats were anesthetized, cavities were prepared and the pulp was capped with either of ProRoot MTA, Biodentine, or BioAggregate. The specimens were scanned using a high-resolution micro-computed tomography (micro-CT) system and were prepared and evaluated histologically and immunohistochemically using dentin sialoprotein (DSP). Results On micro-CT analysis, the ProRoot MTA and Biodentine groups showed significantly thicker hard tissue formation (p < 0.05). On H&E staining, ProRoot MTA showed complete dentin bridge formation with normal pulpal histology. In the Biodentine and BioAggregate groups, a thick, homogeneous hard tissue barrier was observed. The ProRoot MTA specimens showed strong immunopositive reaction for DSP. Conclusions Our results suggest that calcium silicate-based pulp-capping materials induce favorable effects on reparative processes during vital pulp therapy and that both Biodentine and BioAggregate could be considered as alternatives to ProRoot MTA.

The formation of dentin and enamel is initiated by the differentiation of odontogenic precursor cells into odontoblasts and ameloblasts, respectively. This study was performed to identify new molecules involved in the differentiation of odontogenic cells. The genes expressed differentially between the root stage (after the differentiation of odontogenic cells and dental hard-tissue formation) and the cap stage (before the differentiation of odontogenic cells and dental hard-tissue formation) were searched using differential display PCR. For the first time, synaptic vesicle protein (SV) 2b, an important transmembrane transporter of Ca(2+) -stimulated vesicle exocytosis, was identified as a differentially expressed molecule. Real-time PCR and western blotting revealed an increase in the transcriptional and translational levels of SV2b during or after the differentiation of odontogenic cells. Immunofluorescence revealed this molecule to be localized in not only fully differentiated odontoblasts but also in pre-odontoblasts before dentin matrix secretion. The expression pattern of the SV2a isoform was similar to that of the SV2b isoform, whereas the SV2c isoform showed a contrasting pattern of expression. After treatment with alendronate, an inhibitor of protein isoprenylation for the transport of secretory vesicles, the expression of SV2a and SV2b decreased, whereas that of SV2c increased. These results suggest that the SV2 isoforms are functional molecules of (pre)odontoblasts which may be involved in vesicle transport.

Bone morphogenetic protein 2 (BMP2) activates unfolded protein response (UPR) transducers, such as PERK and OASIS, in osteoblast cells. ATF6, a bZIP transcription factor, is also a UPR transducer. However, the involvement of ATF6 in BMP2-induced osteoblast differentiation has not yet been elucidated. In the present study, BMP2 treatment was shown to markedly induce the expression and activation of ATF6 with an increase in alkaline phosphatase (ALP) and OC expression in MC3T3E1 cells. In contrast, ATF6 activation by BMP2 was not observed in the Runx2(-/-) primary calvarial osteoblasts, and Runx2 overexpression recovered BMP2 action. BMP2 stimulated ATF6 transcription by enhancing the direct binding of Runx2 to the osteoblast-specific cis-acting element 2 (OSE2, ACCACA, -205 to -200 bp) motif of the Atf6 promoter region. In addition, the overexpression of ATF6 increased the Oc promoter activity by enhancing the direct binding to a putative ATF6 binding motif (TGACGT, -1126 to -1121 bp). The inhibition of ATF6 function with the dominant negative form of ATF6 (DN-ATF6) blocked BMP2- or Runx2-induced OC expression. Interestingly, OASIS, which is structurally similar to ATF6, did not induce Oc expression. ALP and Alizarin red staining results confirmed that BMP2-induced matrix mineralization was also dependent on ATF6 in vitro. Overall, these results suggest that BMP2 induces osteoblast differentiation through Runx2-dependent ATF6 expression, which directly regulates Oc transcription.

Relaxin (Rln) is an ovarian hormone that stimulates osteoclastic and osteoblastic activities and connective tissue turnover. To investigate the expression of Rln during orthodontic tooth movement, rats were implanted with orthodontic appliances that connected a spring from the upper incisors to the first molar with a 70 cN force. Rats in each group were killed 6, 48, and 144 h after activating the appliance, and the levels of Rln1 and Rln3 expression in the ovary were determined by real-time RT-PCR, northern blots, western blots, and immunofluorescence analyses. The amount of tooth movement induced by the orthodontic force increased in a time-dependent manner. The levels of Rln1 mRNA increased by 12-, 41-, and 263-fold at 6, 48, and 144 h, respectively, after orthodontic tooth movement. The time-dependent increase in the concentration of Rln 1 protein in the ovary was also confirmed by western blotting. Rln 1 was localized in the granulosa cells of the ovarian follicles, and the immunoreactivity against Rln 1 was increased by the movement. In contrast, the concentration of Rln 3 was below the level of detection. The results of this study suggest that local changes in periodontal tissues induced by orthodontic tooth movement may affect Rln1 expression in the ovary. However, further studies are needed to decipher the mechanisms involved and the possible contribution of the increased level of expression of Rln 1 to the tooth movement.

A complex and intricate cascade of gene expression is essential for late stage tooth development. This study was performed to detect molecules involved in dental hard tissue formation and tooth eruption by comparing gene expression in cap stage molar germs (before eruptive movement and dental hard tissue formation) with that in root formation stage molar germs (after eruptive movement and dental hard tissue formation). DD-PCR revealed that cyclophilin A (Cyp-A), a potent chemoattractant for monocytes as well as a ligand for extracellular matrix metalloproteinase inducer (EMMPRIN) was expressed differentially in the two stages molar germs. The levels of Cyp-A and EMMPRIN mRNA were significantly higher at the root formation stage than at the cap and crown stages of the molar germs. Immunofluorescence showed that Cyp-A and EMMPRIN were expressed strongly in the follicular cells overlaying the occlusal region of the molar germs at the root formation stage. In contrast, their immunoreactivity was weak in the follicular tissues and was not region-specific in molar germs at the cap stage. In addition, the MCP-1 and CSF-1 mRNA levels increased in parallel to that of Cyp-A mRNA and the increased number of osteoclasts at the occlusal region. Immunoreactivity against Cyp-A and EMMPRIN was also observed in the fully differentiated ameloblasts and odontoblasts. This study suggests that Cyp-A and EMMPRIN play roles in the maturation of dental hard tissue and the formation of an eruption pathway.

Metformin is an oral anti-diabetic drug of the biguanide class that is commonly used to treat type 2 diabetes mellitus. This study examined the molecular mechanism for the action of metformin on osteoblast differentiation. Metformin-induced mRNA expression of the osteogenic genes and small heterodimer partner (SHP) in MC3T3E1 cells were determined by RT-PCR and real-time PCR. Metformin increased significantly the expression of the key osteogenic genes, such as alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP) as well as SHP. Transient transfection assays were performed in MC3T3E1 cells to confirm the effects of metformin on SHP, OC and Runx2 promoter activities. Metformin increased the transcription of the SHP and OC genes, and the metformin effect was inhibited by dominant negative form of AMPK (DN-AMPK) or compound C (an inhibitor of AMPK). The adenoviral overexpression of SHP increased significantly the level of ALP staining and OC production. However, metformin did not have any significant effect on osteogenic gene expression, ALP staining and activity, and OC production in SHP null (SHP-/-) primary calvarial cells. Moreover, upstream stimulatory factor-1 (USF-1) specifically mediated metformin-induced SHP gene expression. In addition, metformin-induced AMPK activation increased the level of Runx2 mRNA and protein. However, USF-1 and SHP were not involved in metformin-induced Runx2 expression. Transient transfection and chromatin immunoprecipitation assays confirmed that metformin-induced SHP interacts physically and forms a complex with Runx2 on the osteocalcin gene promoter in MC3T3E1 cells. These results suggest that metformin may stimulate osteoblast differentiation through the transactivation of Runx2 via AMPK/USF-1/SHP regulatory cascade in mouse calvaria-derived cells.

Background/Aims: Cartilage oligomeric matrix protein (COMP)-angiopoietin 1 (Ang1) is a soluble and stable form of Ang1 which plays important roles in vessel formation and the survival of endothelial cells, neurons and cardiomyocytes. However, the effects of COMP-Ang1 on the survival of mesenchymal cells are unknown. Mesenchymal cells have been transplanted with some scaffolds for bone tissue regeneration, but they occasionally underwent cell death due to a lack of nutrient supply. This study examined the effects of COMP-Ang1 on the survival of mesenchymal cells under nutrient-deprived conditions. Methods: Primary and C3H10T1/2 mesenchymal cells were cultured under serum deprivation with or without COMP-Ang1. The effects of COMP-Ang1 on mesenchymal cell survival and its molecular mechanism were determined using a viability test, RT-PCR, Western blotting and fluorescence-activated cell sorting analysis. Results and Conclusion: COMP-Ang1 inhibited the nutrient-deprived apoptotic cell death of mesenchymal cells through the Akt, p38 and extracellular-signal-regulated kinase (ERK) pathways. In addition, COMP-Ang1 reversed the nutrient-deprived suppression of cyclin D1 mRNA expression. These results suggest that COMP-Ang1 has a protective role in the survival of nutrient-deprived mesenchymal cells. The use of COMP-Ang1 with some scaffolds might be useful for bone tissue engineering.

Tooth development occurs through a complex and intricate set of gene-expression cascades. Although its early events have been examined extensively, there are fewer reports on the late events, such as dental hard-tissue formation. This study searched for genes involved in the late stages of tooth development. Differential display-polymerase chain reaction revealed myelin basic protein (MBP) mRNA to be expressed differentially in the second molar root stage germs compared with the third molar cap/early bell stage germs. The MBP expressed during hard tissue formation was confirmed to be a 21.5 kDa molecule by Western blotting. Immunoreactivity of MBP in the third molar (cap/early bell stage) germs was barely detectable in the dental papilla and inner enamel epithelia, whereas strong reactivity was noted in the differentiating and differentiated ameloblasts and odontoblasts in a temporospatial pattern. However, after complete formation of the full-thickness enamel, no reactivity was observed in the maturation-stage and protection stage ameloblasts. Myelin basic protein immunoreactive nerve fibers were also observed near the developing molar germs. This is the first report showing the presence of MBP in dental hard tissue cells, and its functional implications should be studied further.

Bisphosphonates have been reported to have chondroprotective activities in addition to its original functions. However, mechanisms for these just began to be elucidated. Under the hypothesis that bisphosphonates may regulate expression and activities of matrix enzymes during degradation of cartilage for bone formation, we administrated an alendronate (1 mg/kg) to newborn rats subcutaneously once a day for 4, 7, and 10 days. To identify the effects of alendronate on cartilage, thickness of cartilage layer was measured by histomorphometry on the proximal epiphysis of tibia. Immunofluorescence staining and RT-PCR were performed to investigate the expressions of matrix enzymes in both in vitro and in vivo. MTS assay revealed that at 10 -3 M in concentration, alendronate significantly reduced viability of chondrocytes. The mRNA expressions of MMP-1, MMP-9, EMMPRIN, and TIMP-3 in primary chondrocytes were decreased by the alendronate treatment. Interestingly, TIMP-1 mRNA expression was significantly increased, whereas a constitutive form, TIMP-2 was relatively unchanged by the treatment. The thickness of proliferating layer at postnatal day 7 was not significantly different, whereas thickness of hypertrophied layer was significantly thicker in the alendronate group than in the control (p These results suggest that the alendronate have chondroprotective properties by down-regulation of MMPs and up-regulation of TIMPs during endochondral ossification.

The aim of this study was to investigate the characteristics of mandibular premolars regarding size and morphology in Koreans. Moreover, comparisons of gender difference in mandibular premolars were examined to expand anatomical database in Koreans. Data was obtained from students in School of Dentistry, Chonnam National University, Gwangju, Korea. The total number of participants was 66 (33 men and 33 women) and dental casts were fabricated. A total of nine items was investigated using a digital measuring software. Five measurements were performed including intercuspal distance (ID), buccolingual diameter (BL), mesiodistal diameter (MD), total crown area, and each cusp area. One item as each cusp area ratio was calculated, and three items were observed including the number of lingual cusp, occlusal groove patterns, and mesiolingual developmental groove. Comparison measurements were analyzed using paired t-tests, independent t-tests and Pearson correlation tests. Average values in mandibular second premolars were larger than first premolars in most of measurements with a significance with the exception of mesiodistal diameter (p=0.223). Overall average values were significantly higher in male than in female except intercuspal distance (p=0.607) and lingual cusp area (p=0.070) in mandibular premolars. The presence of mesiolingual developmental grooves in the first premolars was 59.1% (male 51.5%, female 66.7%). The most common occlusal groove patterns of the second premolar were a Y pattern, followed in order by H and U patterns. These results provide valuable morphological characteristics of mandibular premolars in Koreans.

This study aimed to investigate the cusp size and morphological characteristics of permanent mandibular molars in Koreans with reference to the hypoconulid, and to analyze the differences and correlations between both sexes as well as between first and second mandibular molars. We obtained data from dental casts of 110 adults (78 males and 32 females). Mesiodistal and buccolingual diameters of first and second mandibular molars, the area of five cusps (protoconid, metaconid, hypoconid, entoconid, and hypoconulid), as well as the total cusp area and occlusal table area were measured. Paired t-test was performed to analyze the morphological differences between first and second mandibular molars and the sex differences between both sexes using SPSS program. Crown diameters and cusp areas of mandibular first molars were larger than those of mandibular second molars in both sexes. The hypoconulid was the most variable in size and morphological pattern among the five cusps, and the first molars showed a higher incidence of hypoconulid than the second molars. Except for the entoconid area of the first molar (p=0.06) and the hypoconulid area of the second molar (p=0.24), all other mean values were larger in males than in females, demonstrating a significant sexual dimorphism. These data suggest that the teeth which develop late in ontogeny tend to be smaller in size and more variable in morphological characteristics.

Understanding the genetic control of tooth eruption is one of the major issues in tooth development. Thus far, it is known that eruption-related molecules are secreted from follicular cells surrounding the germs and are related mainly to osteoclast formation. This study examined the involvement of CD147 and its downstream molecules in the eruption of rat developing molars using immunohistochemistry, RT-PCR and histomorphometry. CD147 was expressed differentially in the cap (3rd molar germs) and root formation (2nd molar germs) stages in tooth development. CD147 was localized immunohistochemically in the follicular cells and osteoclasts as well as in the ameloblasts and odontoblasts. The expression pattern of CD147 and mmps was investigated because CD147 is an mmp inducer. The expression of both mmp-2 and -9 increased at the root formation stage compared to that at the cap stage and increased in a stage dependent manner. However, the level of mmp-13 was not changed notably. The histomorphometrical study suggested that the number of osteoclasts that appeared occlusal to the molar germs for the resorption of alveolar bone increased significantly during development. These results suggest that CD147 may play an important role in the formation of the eruption pathway along with the mmps.

This study was performed to verify the possibility of MTA and calcium sulfate as a pulp capping agent through comparing the dental pulp response in dogs after capping with MTA, calcium sulfate, and calcium hydroxide. 24 teeth of 2 dogs, 8 month old, were used in this study. Under general anesthesia, cervical cavities were prepared and pulp was exposed with sterilized #2 round bur in a high speed handpiece. MTA calcium hydroxide, and calcium sulfate were applied on the exposed pulp. Then the coronal openin,fs were sealed with IRM and light-cured composite. Two months after treatment, the animals were sacrificed. The extracted teeth were fixed in 10% neutral-buffered formalin solution and were decalcified in formic acid-sodium citrate. They were prepared for histological examination in the usual manner. The sections were stained with haematoxylin and eosin. In MTA group, a hard tissue bridges formation and newly formed odontoblasts layer was observed. There was no sign of pulp inflammatory reaction in pulp tissue. In calcium hydroxide group, there was no odontoblast layer below the dentin bridge. In pulpal tissue, chronic inflammatory reaction with variable intensity and extension occurred in all samples. In calcium sulfate group, newly formed odontoblast layer was observed below the bridge. Mild chronic inflammation with a few neutrophil infiltrations was observed on pulp tissue. These results suggest that MTA is more biocompatible on pulp tissue than calcium hydroxide or calcium sulfate.

Nitric oxide (NO) gas has been recognized to diffuse readily across the membrane and bind directly to molecule (s) inside target cells. In the salivary gland, eNOS and nNOS are constitutively expressed. iNOS was also reported to express in neoplastic salivary tissues. Regarding the role of NO in the salivary gland, it has been suggested that it may control blood flow to the glands and furthermore involve in growth and development of the gland. The present study hypothesized that denervation of parasympathetic secretomotor fibers may lead to salivary secretion dysfunction, changing NOS expression. The gland weight on the denervated side significantly decreased from 3 days after the denervation, comparing the control (p＜0.01). Some atrophic and hyperchromatic changes, but no inflammatory reactions were found for the whole period of the experiment. All three kinds of NOS were mainly expressed in the ducts of the gland in both the control and experimental sides. Immunoreactivities of nNOS and eNOS were not noticeably different from those of the control. However, iNOS was also detected in ducts in the normal submandibular gland by immunohistochemical staining. The iNOS expression increased more than 2 times at denervated side of the gland than the control. These results suggest that NOS isoforms, especially iNOS following chorda-lingual denervation may lead to matrix loss or cell death in the salivary gland.

Electrical characteristics of Pt/Ti/Pt/Au and Pd/Zn/Pd/Au contacts to p-type InGaAs grown on an InP substrate have been characterized as a function of the doping concentration and the annealing temperature. The Pt/Ti/Pt/Au contacts produced the specific contact resistance as low as , when heat-treated at an annealing temperature of . Comparison of the Pt/Ti/Pt/Au and Ti/Pt/Au contacts showed that the first Pt layer plays an important role in reducing the contact resistivity probably by lowering energy barrier at the metal-semiconductor interface. For the Pd/Zn/Pd/Au contacts, the contact resistivity remained virtually unchanged with increasing annealing temperature. The specific contact resistivity as low as was obtained. The results indicate that the Pt/Ti/Pt/Au and Pd/Zn/Pd/Au schemes could be potentially important for the fabrication of InP-based optoelectronic devices, such as photodetector and optical modulator.

Bisphosphonates have similar chemical structures to endogenous inorganic pyrophosphate which inhibits mineral deposition in biological tissues. Even though their clinical applications have been attempted, their molecular mechanism and cellular effects on dental hard tissue development remain to be elucidated. The present study was performed to investigate their effects on the development of the tooth and their mode of action. Alendronate, a synthetic derivative of bisphosphonates was subcutaneously daily injected in postnatal day 1 Sprague Dawley rats for successive 10 days. Animals were sacrificed at 3, 12 and 40 days after the final administration and light microscopy, RT-PCR and TUNEL were used for the analyses. Alendronate inhibited the teeth development and retarded their eruption. In the alendronate group, the mandibular first and second molars were under-developed, compared with those in the control group at day 3. Ameloblasts in the mandibular 1st molar were discontinuous in several parts. The development of the mandibular 2nd molar was deterred by the woven bone growing into the dental papilla. The third molar tooth germ did not appear. The TUNEL positive cells were rarely seen in the normally developing hard tissue cells. But in the alendronate group, the positive cells appeared frequently in layers of ameloblasts. Furthermore, the expression of alkaline phosphatase mRNA was downregulated in the alendronate group, suggesting that osteoblastic activity was decreased. These results suggested that bisphosphonates may act on dental hard tissue cells, preventing tooth development and eruption.

Hypoxic/ischemic condition induces the neuronal apoptotic events, consequently resulting in neuronal damages. Cobalt chloride (CoCl₂) could mimic the hypoxic condition including the production of reactive oxygen species (ROS). This study aimed to investigate the roles of Bcl-2 family and caspases as central regulators of apoptosis, in CoCl₂-induced apoptosis of PC12 cells. Cell viability was determined by MTT assay and DNA fragmentation was detected by DNA laddering. The expression levels of Bcl-2, Bax, Bid, cytochrome c and Fas/APO-1 were determined by RT-PCR or Western blotting analysis in CoCl₂-treated PC12 cells. Caspase-9 and caspase-3 activities were assessed using spectrophotometry and caspase-8 activity was measured with fluorospectrocytometry. Administration of CoCl₂ decreased viability of cells in a dose- and time-dependent manner. Furthermore, fragmentation of the genomic DNA and apoptotic bodies were induced in CoCl₂-treated PC12 cells. Bcl-2, an anti-apoptotic Bcl-2 family, was downregulated, whereas Bax, pro-apoptotic molecule, was upregulated in CoCl₂-treated cells. Treatment of CoCl₂ augumented the release of cytochrome c into the cytoplasm and increase of caspase-8, -9, and -3 activities. In addition, CoCl₂ upregulated Fas and downregulated pro-Bid, which are known to be correlated with death receptor-mediated apoptotic signaling pathway. Therefore, these results suggest that Bcl-2 family and caspase play crucial roles in CoCl₂-induced apoptosis through mitochondria- and death receptor-dependent pathways in PC12 cells.

One fifth dilution of formocresol is usually used for pulpotomy of the primary teeth and emergency pulpotomy of the permanent teeth. However the use of formaldehyde has been subjected to criticism because it may be absorbed into the blood stream and become distributed systemically, it nay also alter the pulp tissue rendering it immunologically active, and have carcinogenic potential. Recently Depulpin(VoCo., Germany) gains popularity as a devitalizing agent during root canal therapy in spite of high concentration of 49 % paraformaldehyde because it facilitate devitalization of pulp and make root canal therapy easier But there have been not enough publications about the reaction of pulp and periapical tissue caused by Depulpin. This study was performed to evaluate the histological changes in pulp and periapical tissue of rats after pulpotomy using formocresol and Depulpin and to elucidate the toxic effects of these agents. Thirty six Sprague-Dawley rats were anesthetized by intraperitoneal injection of ketamine Maxillary first molar teeth were used for pulpotomy with formocresol and Depulpin. Rats were sacrificed after 2 days, 4 days, 1 week, 2 weeks, 3 weeks and 4 weeks respectively. Specimens were histologically observed by light microscope changes in pulp and periapical tissue. The obtained results were as follows. 1. Formocresol group A zone of fixed tissue. in which odontoblasts could clearly be defined, was present directly underneath the pulpotomy dressing in almost all teeth of this group. This was followed by an area of necrotic tissue which resembled dried out fibrous tissue with no cellular detail except some pyknotic nuclei. In the specimens of after 2 days, 4 days, 1 week, 2 weeks in which vital tissue was present, it was separated from the fibrous area by a zone of inflammation. In the specimens of after 3 weeks and after 4 weeks, inflammatory infiltrate was in the periodontal ligament adjacent to the apical foramina of the teeth. 2. Depulpin group The area of necrotic tissue which had no cells and fibers, was present adjacent to the dressing. This was followed by dried out fibrous tissue with no cellular details except some pyknotic nuclei, A short stump of vital pulp with odontoblasts was present at the end of the canal after 2 days. Inflammatory infiltrate was in the periodontal ligament after 4 days and after 1week. Severe root resolution and necrosis of periapical tissue opposite the root resorption site were defined after 2 weeks and after 3 weeks. Periapical lesion which consist of necrotic tissue surrounded by a fibrous connective wall, was found after 4 weeks. The results indicated that Depulpin can cause more adverse reaction to the dental pulp and periapical tissue than formocresol, and further studies are needed for its clinical use with safety.