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It has been shown that Emdogain Gel (Emd-Gel) containing enamel matrix proteins promotes biomineralization, such as osteogenesis and cementogenesis, during the regeneration of periodontal tissues. However, the growth factors involved in these activities of Emd-Gel remain unclear. In this study, Emd-Gel was fractionated into 22 sub-fractions by size exclusion chromatography. The osteoinductive factors, TGF-beta and BMP, were examined by a specific luciferase reporter gene assay. In the unfractionated Emd-Gel, TGF-beta-like activity was detected, while BMP activity was not. In contrast, in the fractionated Emd-Gel samples, TGF-beta-like activity was detected from fractions 8 to 13, and BMP-like activity was detected from fractions 4 to 6. Also, it was confirmed that the BMP-like activity in Emd-Gel was inhibited by authentic TGF-beta1 and TGF-beta-like activity. These results indicate that Emd-Gel contains both TGF-beta- and BMP-like growth factors that contribute to the induction of biomineralization during periodontal regeneration. [ABSTRACT FROM AUTHOR]

Background and Objective The periodontal ligament ( PDL) is characterized by rapid turnover, high remodeling capacity and high inherent regenerative potential compared with other connective tissues. Periostin, which is highly expressed in the fibroblasts in the PDL, has been widely discussed in relation to collagen fibrillogenesis in the PDL. Recently, several reports have indicated periostin in cell migration. The aim of this study was to examine whether human PDL fibroblasts ( hPDLFs) with high levels of periostin expression promote the migration of human bone marrow mesenchymal stem cells ( hMSCs). Material and Methods The migration of hMSCs was examined by transwell chamber migration assay under different conditions: medium alone, hPDLFs, human dermal fibroblasts, recombinant periostin, integrin αvβ3 blocking antibody (anti- CD51/61 antibody) and inhibitors of FAK ( PF431396) and PI3 K ( LY294002). Phosphorylation of FAK and Akt in hMSCs under stimulation of periostin was examined by western blotting. Results The migration assay revealed that the number of migrated hMSCs by hPDLFs was significantly larger than those by dermal fibroblasts, periostin small interfering RNA hPDLFs and medium alone. Furthermore, recombinant periostin also strongly induced hMSC migration. The addition of anti- CD51/61 antibody, PF431396 and LY294002 caused a significant reduction in the number of migrated hMSCs respectively. The anti- CD51/61 antibody inhibited both FAK and Akt phosphorylations under periostin stimulation. PF431396 inhibited both FAK and Akt phosphorylations. LY294002 inhibited only Akt phosphorylation, and FAK phosphorylation was not influenced under periostin stimulation. Conclusion Periostin expression in hPDLFs promotes the migration of hMSCs through the αvβ3 integrin/ FAK/ PI3 K/ Akt pathway in vitro. [ABSTRACT FROM AUTHOR]

Porcine dentin sialophosphoprotein (DSPP) is the most abundant non-collagenous protein in dentin. It is processed by proteases into 3 independent proteins: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We fractionated DPP and DSP along with TGF-β activity by ion exchange (IE) chromatography from developing pig molars and measured their alkaline phosphatase (ALP)-stimulating activity in human periodontal (HPDL) cells with or without TGF-β receptor inhibitor. We then purified TGF-β-unbound or -bound DPP and DSP by reverse-phase high-performance liquid chromatography (RP-HPLC) using the ALP-HPDL system. The TGF-β isoform bound to DPP and DSP was identified as being TGF-β1 by both ELISA and LC-MS/MS analysis. We incubated carrier-free human recombinant TGF-β1 (CF-hTGF-β1) with TGF-β-unbound DPP or DSP and characterized the binding on IE-HPLC using the ALP-HPDL system. When only CF-hTGF-β1 was incubated, approximately 3.6% of the ALP-stimulating activity remained. DPP and DSP rescued the loss of TGF-β1 activity. Approximately 19% and 10% of the ALP stimulating activities were retained by the binding of TGF-β to DPP and DSP, respectively. The type I collagen infrequently bound to CF-hTGF-β1. We conclude that both DPP and DSP help retain TGF-β1 activity in porcine dentin. [ABSTRACT FROM AUTHOR]

We describe a process for the growth of a single, electronically decoupled graphene layer on SiC(0001). The method involves annealing in disilane to (1) prepare flat, clean substrates, (2) grow a single graphene layer, and (3) electronically decouple the graphene from the substrate. This approach uses a single process gas, at μTorr pressures, with modest substrate temperatures, thus affecting a drastic simplification over other processes described in the literature. [ABSTRACT FROM AUTHOR]

The linearity of the radio frequency response of graphene field-effect transistors has been measured as a function of gate bias using the two-tone method. Two kinds of transistors, which differ in both the graphene source material and the device structure, have been compared. Both devices show high linearity compared to contemporary silicon transistors. The physical origins of this behavior are analyzed and discussed. [ABSTRACT FROM AUTHOR]

It has been shown that Emdogain® Gel (Emd-Gel) containing enamel matrix proteins promotes biomineralization, such as osteogenesis and cementogenesis, during the regeneration of periodontal tissues. However, the growth factors involved in these activities of Emd-Gel remain unclear. In this study, Emd-Gel was fractionated into 22 sub-fractions by size exclusion chromatography. The osteoinductive factors, TGF-β and BMP, were examined by a specific luciferase reporter gene assay. In the unfractionated Emd-Gel, TGF-β-like activity was detected, while BMP activity was not. In contrast, in the fractionated Emd-Gel samples, TGF-β-like activity was detected from fractions 8 to 13, and BMP-like activity was detected from fractions 4 to 6. Also, it was confirmed that the BMP-like activity in Emd-Gel was inhibited by authentic TGF-β1 and TGF-β-like activity. These results indicate that Emd-Gel contains both TGF-β- and BMP-like growth factors that contribute to the induction of biomineralization during periodontal regeneration. [ABSTRACT FROM AUTHOR]

Amelogenin is the major enamel matrix component in developing teeth. In eutherian mammals, amelogenin is expressed from the X chromosome only, or from both the X and Y chromosomes. Two classes of porcine amelogenin cDNA clones have been characterized, but the chromosomal localization of the gene(s) encoding them is unknown. To determine if there are sex-based differences in the expression of porcine amelogenin, we paired PCR primers for exons 1a, 1b, 7a, and 7b, and amplified enamel organ-derived cDNA separately from porcine males and females. The results show that exons 1a/2a and 7a are always together and can be amplified from both males (XY) and females (XX). Exons 1b/2b and 7b are also always paired, but can be amplified only from females. We conclude that porcine amelogenin is expressed from separate genes on the X and Y chromosomes, and not, as previously proposed, from a single gene with two promoters. [ABSTRACT FROM AUTHOR]

Amelogenin, enamelin, sheathlin (ameloblastin/ amelin), enamelysin (MMP-20), and KLK4 (EMSP-1) are the major structural proteins and proteinases in developing tooth enamel. Recently, odontoblasts were reported to express amelogenin, the most abundant enamel protein. In this study, we hypothesized that odontoblasts express all enamel proteins and proteases, and we measured their relative mRNA levels in enamel organ epithelia and odontoblasts associated with porcine secretory- and maturation-stage enamel by RTPCR, using a LightCycler instrument. The results showed that amelogenin mRNA in secretory-stage EOE is 320-fold higher than in odontoblasts beneath secretory-stage enamel, and over 20,000- fold higher than in odontoblasts under maturationstage enamel. Similar results were obtained for enamelin and sheathlin. Enamelysin mRNA levels were equivalent in these two tissues, while KLK4 mRNA was higher in odontoblasts than in secretory-stage EOE. These results support the conclusion that odontoblasts are involved in the formation of the enamel layer adjacent to enameldentin junction. [ABSTRACT FROM AUTHOR]

The temporal expression patterns and activity distributions of enamelysin and EMSP1, which are the major proteinases in immature enamel, were characterized. Extracellular matrix fractions from developing porcine incisors, individually comprised of predentin, dentin, and four secretory-stage enamel samples, including the highly mineralized enamel (HME) at the enamel-dentin junction (EDJ), were isolated, and their resident proteinases were identified by zymography. Softtissue fractions, which included cells from the extension site of enamel formation (ESEF), secretory- and maturation-stage ameloblasts, and odontoblasts, were characterized histologically and by RT-PCR for their expression of enamelysin and EMSP1. A significant finding was that EMSP1, expressed by odontoblasts, concentrates in the HME, but is not detected in predentin or dentin. We conclude that odontoblasts deposit EMSP1 via their cell processes into the deepest enamel layer, which facilitates the hardening of this layer and contributes significantly to the functional properties of the EDJ. [ABSTRACT FROM AUTHOR]

Enamel extracts induce biomineralization such as osteogenesis and cementogenesis, but the molecular component responsible for this activity remains uncertain. We fractionated enamel extracts from developing pig teeth and isolated the osteoinductive fraction. Proteins from pig enamel scrapings were extracted under alkaline conditions (pH 10.8) and fractionated with the use of a Sephadex G-100 (size exclusion) column. The ability of each fraction to enhance alkaline phosphatase (ALP) activity was assayed in ST2 cells, a mouse bone marrow stromal cell line. The osteoinductive fraction of enamel extracts (OFE) was found in fractions 44 and 45, which induced ST2 cells to express the phenotype of boneforming osteoblasts, and to form mineralized nodules. Furthermore, the ALP activity of ST2 cells exposed to OFE was reduced by noggin, an antagonist of BMPs, and OFE reacted with BMP-2/4 antibody in dot-blot analysis. These results indicate that OFE contains BMPs that contribute to the induction of biomineralization. [ABSTRACT FROM AUTHOR]

Amelogenin is the major organic component in the enamel matrix of developing teeth and plays an important role in enamel biomineralization. Amelogenin has been reported to be a specific secretory product of ameloblasts. In this study, we examined amelogenin gene expression in various cell layers prepared from a porcine permanent tooth germ using reverse transcription-polymerase chainreaction (RT-PCR). Amelogenin amplification products were detected only in the secretory ameloblast layer after 20 cycles of PCR. After 30 cycles of PCR, amelogenin amplification products were detected in secretory and maturation-stage ameloblasts and in odontoblasts. The relative levels of amelogenin gene expression in secretory and maturation-stage ameloblasts and odontoblasts were determined. Secretory ameloblasts expressed over 1000 times the level of amelogenin mRNA found in odontoblasts. Amelogenin gene expression in odontoblasts was confirmed in an erupted porcine permanent first molar, which has no ameloblasts. Amelogenin PCR amplification products were identified from 4 different alternatively spliced transcripts in the ameloblast samples, and the same spliced forms were detected in the odontoblast samples. [ABSTRACT FROM AUTHOR]

There have been many immunohistochemical studies of enamel proteins during root formation. In the present article, the detection and expression of enamel proteins in tissue samples prepared from the apical portion of the forming root (APFR) in porcine permanent incisor tooth germs were studied. Amelogenin, enamelin, and sheathlin were detected by immunoblot analysis, but only in small amounts. The detection of their derivatives indicated their degradation. It is, at present, unclear as to which proteinases are involved in these degradations, because activity of enamel matrix serine proteinase 1 and enamelysin was not detected on gelatin and casein zymograms. The expression of enamel proteins was also proved in the APFR sample by the detection of polymerase chain reaction products of their cDNAs, and this may be related to cells of fragmentized Hertwig's epithelial root sheath. Amelogenin expression was not greater than that of enamelin and sheathlin. It was different from the expression pattern of secretory ameloblasts involved in enamel matrix formation. These results suggest that the amelogenins found in the APFR do not form a three-dimensional structure of amelogenin micelles, which has been proposed for the secretory enamel matrix structure. In this case, the enamel proteins could spread out easily following degradation into the matrix of future cementum. Some of their derivatives may play a role in the formation of the cementum. [ABSTRACT FROM AUTHOR]

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 (S1OOA4 and S10OA2). In Northern blotting analysis, the highest expression of S10OA4 was detected in PDL from erupted teeth (PDLE). PDL from teeth under eruption (PDLU) showed a lower expression of S10OA4, 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. S10OA2 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 β-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 5100A4 could be a useful marker for distinguishing cells from these two tissues. [ABSTRACT FROM AUTHOR]

Tissue-nonspecific-type alkaline phosphatase is found in the bone, liver, kidney, and other tissues, and its gene consists of 12 exons with the coding sequence beginning in the second exon. Recently, an alternative non- coding first exon was identified in the liver message which differed from that of the previously known osteoblast-derived cDNA sequence. Although these two mRNAs produce an identical protein, they have different promoter regions. The periodontal ligament tissue expresses a high level of alkaline phosphatase activity. To identify its mRNA type, we isolated a full-length cDNA for alkaline phosphatase from a cultured human periodontal ligament cell expression library, using bone-derived tissue-nonspecific alkaline phosphatase cDNA as a hybridization probe. The size of this clone was 2.5 kb, and its 5′ and 3′ untranslated sequences were identical to those of the human tissue-nonspecific type isolated from osteoblastic cells but not to those of the liver type. In addition, the same fragments as in bone-derived tissue-nonspecific-type cDNA were detected by the treatment of the cDNA clone with restriction enzymes Hinc II and Pst I. The results suggest that expression of the same alkaline phosphatase isozyme in human periodontal ligament cells may be regulated by the same transcriptional mechanism as in bone. [ABSTRACT FROM AUTHOR]

In this study, the effects of guanidine extracts from demineralized bovine dentin matrix on rat mesenchymal cells were investigated by use of an agarose gel culture. The dentin extracts were divided into water-soluble and -insoluble fractions. Rat mesenchymal cells obtained from the cultivation of skeletal muscle tissue and embedded in agarose gel were treated with these two fractions. After three weeks of cultivation, the treated cells formed colonies that were stained metachromatically with toluidine blue in a dose-dependent manner. The activity necessary to form chondrocyte-like colonies by the water-insoluble fraction was significantly higher than that by the water-soluble fraction. Each chromatographic fraction of the water-insoluble part of dentin extracts on tandem Sephacryl S-200 High-resolution columns was also investigated. Chondrocyte-like colony-forming activity was concentrated in a single fraction. However, the electrophoretic pattern of this fraction revealed that there were still some bands of molecular weight between 18 and 30 kDa. According to the Western blot analysis of this fraction, there was a band corresponding to purified transforming growth factor β (TGF-β) under the non-reducing condition. After reduction, this band disappeared and we found a band corresponding to a component of 13 kDa as well as TGF-β. These findings suggest that TGF-β is present not only in bone but also in the dentin matrix. [ABSTRACT FROM AUTHOR]

Enzymatic and immunological properties of alkaline phosphatase [ALP; orthophosphoric monoesterphosphohydrolase, alkaline optimum, EC 3.1.3.1.] in the human dental pulp were investigated. In inhibition and thermal inactivation studies, dental pulp ALP showed properties of universal-type ALP (kidney/bone/liver type). Dental pulp ALP cross-reacted with polyclonal and monoclonal antibodies against purified swine-kidney ALP, and with monoclonal antibody against ALP of human osteoblast-like cells in the same manner as ALPs of human bone and kidney. The sodium dodecyl sulfate-gel electrophoretic pattern showed a 140,000-Mr native protein band. These data suggest that dental pulp ALP can be classified as a universal-type ALP having antigenic determinants common to ALP of the kidney and bone. [ABSTRACT FROM AUTHOR]

We describe a simple method to detect the formation of graphene during Si sublimation from SiC surfaces at elevated temperature. The method exploits differences in the thermionic emission current density between graphene and SiC. When graphene forms, the thermionic current from the sample increases by a factor of about 20. The increase in thermionic emission can be detected in situ using a biased plate located near the sample. The ability to detect when graphene forms during processing is useful in optimizing graphene synthesis processes. [ABSTRACT FROM AUTHOR]

Depth profiling of graphene with high-resolution ion beam analysis is a practical method for analysis of monolayer thicknesses of graphene. Not only is the energy resolution sufficient to resolve graphene from underlying SiC but by use of isotope labeling it is possible to tag graphene generated from reacted ethylene. Furthermore, we are able to analyze graphene supported by oxidized Si(100) substrates, allowing the study of graphene films grown by chemical vapor deposition on metal and transferred to silicon. This introduces a powerful method to explore the fundamentals of graphene formation. [ABSTRACT FROM AUTHOR]