The enamel protein amelotin is a promoter of hydroxyapatite mineralization.

Amelotin (AMTN) is a recently discovered protein that is specifically expressed during the maturation stage of dental enamel formation. It is localized at the interface between the enamel surface and the apical surface of ameloblasts. AMTN knock-out mice have hypomineralized enamel, whereas transgenic mice overexpressing AMTN have a compact but disorganized enamel hydroxyapatite (HA) microstructure, indicating a possible involvement of AMTN in regulating HA mineralization directly. In this study, we demonstrated that recombinant human (rh) AMTN dissolved in a metastable buffer system, based on light scattering measurements, promotes HA precipitation. The mineral precipitates were characterized by scanning and transmission electron microscopy and electron diffraction. Colloidal gold immunolabeling of AMTN in the mineral deposits showed that protein molecules were associated with HA crystals. The binding affinity of rh-AMTN to HA was found to be comparable to that of amelogenin, the major protein of the forming enamel matrix. Overexpression of AMTN in mouse calvaria cells also increased the formation of calcium deposits in the culture medium. Overexpression of AMTN during the secretory stage of enamel formation in vivo resulted in rapid and uncontrolled enamel mineralization. Site-specific mutagenesis of the potential serine phosphorylation motif SSEEL reduced the in vitro mineral precipitation to less than 25%, revealing that this motif is important for the HA mineralizing function of the protein. A synthetic short peptide containing the SSEEL motif was only able to facilitate mineralization in its phosphorylated form ((P)S(P) SEEL), indicating that this motif is necessary but not sufficient for the mineralizing properties of AMTN. These findings demonstrate that AMTN has a direct influence on biomineralization by promoting HA mineralization and suggest a critical role for AMTN in the formation of the compact aprismatic enamel surface layer during the maturation stage of amelogenesis.

Cell-matrix and cell-cell interactions of human gingival fibroblasts on three-dimensional nanofibrous gelatin scaffolds.

An in-depth understanding of the interactions between cells and three-dimensional (3D) matrices (scaffolds) is pivotal to the development of novel biomaterials for tissue regeneration. However, it remains a challenge to find suitable biomimetic substrates and tools to observe cell-material and cell-cell interactions on 3D matrices. In the present study, we developed biomimetic nanofibrous 3D gelatin scaffolds (3D-NF-GS) and utilized confocal microscopy combined with a quantitative analysis approach to explore cell-matrix and cell-cell interactions on the 3D-NF-GS. Human gingival fibroblasts (HGFs) migrated throughout the 3D-NF-GS by 5 days and formed stable focal adhesions by 14 days. The focal adhesions were detected using integrin-ß1, phospho-paxillin and vinculin expression, which were quantified from specific wavelength photon data generated using a spectral separation confocal microscope. As the cells became more confluent after 14 days of culture, cell-cell communication via gap junctions increased significantly. Collagen I matrix production by HGFs on 3D-NF-GS was visualized and quantified using a novel approach incorporating TRITC label in the scaffolds. Based on confocal microscopy, this study has developed qualitative and quantitative methods to study cell-matrix and cell-cell interactions on biomimetic 3D matrices, which provides valuable insights for the development of appropriate scaffolds for tissue regeneration.

Antioxidant combinations protect oral fibroblasts against metal-induced toxicity.

OBJECTIVE: In dentistry, the use of metals in fillings, braces, implants, bridges and other prosthodontic restorations is a common practice. Previous studies revealed that zinc (Zn) and copper (Cu) released from gold alloys, and nickel (Ni) released from nickel-chromium alloys, have a highly cytotoxic effect on fibroblast cell cultures. Our working hypothesis is that oral fibroblasts are susceptible to damage from metals because they elevate reaction oxygen species (ROS). In this study, we investigated specific antioxidant (AO) combinations to determine if they counteract the effects of Cu, Ni and Zn on cultured oral fibroblast proliferation and oxidative damage. METHODS: Oral fibroblasts were pretreated with Cu, Ni and Zn for 60min. Thereafter, cells were treated with 10(-5)M combinations of bioactive AO resveratrol (R), ferulic acid (F), phloretin (P) and tetrahydrocurcuminoids (T) (RFT, PFR, PFT) for 24h. Cell viability and DNA synthesis were monitored by 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium (MTS) and 5-bromo-2-deoxyuridine (BrDU) assays. ROS was measured using the fluorescence response of dichlorodihydrofluorescein diacetate (DCF). RESULTS: AO compounds increased recovery of cells exposed to Cu and Zn. Moreover, AO treatment induced DNA synthesis in the presence of the metal stressors. Cu and Ni stimulated production of ROS. PFR treatment decreased ROS in the presence of Cu, Ni and Zn. SIGNIFICANCE: These data indicate that pure AOs counteracted the detrimental effects of Cu, Ni, Zn on oral fibroblasts in vitro by increasing cell viability, and DNA synthesis and decreasing ROS activity.

Ephrin-B stimulation of calvarial bone formation.

INTRODUCTION: Ephrin-B2 on osteoclasts was reported to promote bone formation as part of homeostasis by activating the EphB4 tyrosine kinase receptor on osteoblasts. Little is known about the role of ephrin-B signaling to EphBs in developmental bone formation. RESULTS: We observed expression of an ephrin-B2 LacZ chimeric allele in the periosteum, sutural bone fronts, and dura mater of embryonic and neonatal mice. Expression in the adult skull was confined to sutures, but was heavily upregulated at sites of bone injury. Culture of embryonic calvariae with soluble recombinant ephrin-B2/Fc doubled their bone content without altering suture width or overall skull morphology. Ephrin-B2/Fc also stimulated osteoblast marker gene expression in cultured MC3T3 preosteoblastic cells without the need for type 1 collagen-induced differentiation. EphB4 was absent in embryonic and adult skulls. However, EphB1 and EphB2, both physiological receptors for ephrin-Bs, were expressed at sites of osteogenesis, and EphB1 knockout mice displayed a reduction in calvarial bone content compared to controls. CONCLUSIONS: These data support a role for ephrin-B2 in the development and healing of bone through activation of osteoblast-specific gene expression. EphB1 and EphB2 are likely candidates receptors for the ephrin-B2 in bone.

Bioactive polyphenol antioxidants protect oral fibroblasts from ROS-inducing agents.

BACKGROUND: Oxidative damage to soft oral tissues may result from exposure to the chemicals or biochemicals found in teeth-whitening products, dental restorations, tobacco, and alcohol. Our working hypothesis is that oral tissues are susceptible to the toxic effects of stressors such as hydrogen peroxide (H(2)O(2)), ethanol (EtOH) and nicotine (Nic), which decrease cell viability/DNA synthesis and elevate reactive oxygen species (ROS). In this study, we investigated specific polyphenols and turmeric derivative antioxidants (AO) in combinations that counteracted the effects of these stressors on cultured oral fibroblast proliferation and ROS production. METHODS: Oral fibroblasts were exposed to stressors for 30 min and then treated with 10(-5) M of bioactive AO mixtures [resveratrol, ferulic acid and tetrahydrocurcuminoid (RFT), phloretin, ferulic acid and resveratrol (PFR), phloretin, ferulic acid and tetrahydrocurcuminoid (PFT)] for 24 h. Cell viability and DNA synthesis were monitored using incorporated 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulphophenyl]-2H-tetrazolium (MTS) and 5-bromo-2-deoxyuridine (BrdU) assays, respectively. Total ROS was measured with dichlorodihydrofluorescein diacetate (H(2)DCFDA). RESULTS: Incubation of oral fibroblasts in the stressors for 30 min resulted in a dose-dependent decrease of DNA synthesis and number of viable cells, and an increased total ROS activity. AO treatment counteracted the insults by restoring DNA synthesis levels and cell viability, and decreasing the total ROS activity. CONCLUSION: The AO combinations of RFT, PFR and PFT protected the oral fibroblasts from the detrimental effects of H(2)O(2), EtOH and Nic by decreasing total ROS and increasing cell viability and DNA synthesis.

Bioactive antioxidant mixtures promote proliferation and migration on human oral fibroblasts.

OBJECTIVE: Antioxidants (AOs) are the first line of defence against free radical damage and are critical for maintaining optimum health and well being. The need for AOs becomes even more critical with increased exposure to free radicals generated by pollution, cigarette smoke, drugs, illness, stress and exercise. Antioxidant supplementation is an excellent way of improving free radical protection. The aim of this study was to provide cytotoxicity, proliferation and migration data on the in vitro effects of bioactive AO mixtures on human oral fibroblasts. METHODS: Human oral fibroblasts were obtained from human gingival (HGF) and periodontal (HPDL) tissues. Each of these oral fibroblasts was cultured separately in three concentrations of the bioactive pure polyphenol and turmeric derivative mixtures; resveratrol (R), ferulic acid (F), phloretin (P) and tetrahydrocurcuminoids (T); [(RFT), (PFR), and (PFT)]. Cell viability, proliferation, morphology and migratory behaviour were analysed in vitro using high throughput in vitro 96 well plate wound assay. RESULTS: RFT decreased (10(-3)M) and increased (10(-5)M) cell number in HGF cells. Three concentrations (10(-3), 10(-4), and 10(-5)M) of PFR and PFT increased DNA synthesis in HGF cells. PFT promoted cell migration but PFR and RFT had no significant change in HGF wound healing rates in a 96 well plate assay monolayer wound. In the HPDL cells, the 10(-4)M concentration of both RFT and PFT increased cell number at 72 h and 96 h whereas the lower concentration 10(-5)M of RFT significantly stimulated cell number at 96 h. PFR (10(-3)M and 10(-5)M) and PFT (10(-3)M) increased DNA synthesis after 48 h treatment in HPDL cells. CONCLUSIONS: High and low concentrations (10(-3)-10(-5)M) of these AOs (RFT, PFR) may have beneficial effects on functional mechanisms regulating fibroblast migration and proliferation during gingival healing or periodontal repair.

Ephrin reverse signaling controls palate fusion via a PI3 kinase-dependent mechanism.

Secondary palate fusion requires adhesion and epithelial-to-mesenchymal transition (EMT) of the epithelial layers on opposing palatal shelves. This EMT requires transforming growth factor ß3 (TGFß3), and its failure results in cleft palate. Ephrins, and their receptors, the Ephs, are responsible for migration, adhesion, and midline closure events throughout development. Ephrins can also act as signal-transducing receptors in these processes, with the Ephs serving as ligands (termed "reverse" signaling). We found that activation of ephrin reverse signaling in chicken palates induced fusion in the absence of TGFß3, and that PI3K inhibition abrogated this effect. Further, blockage of reverse signaling inhibited TGFß3-induced fusion in the chicken and natural fusion in the mouse. Thus, ephrin reverse signaling is necessary and sufficient to induce palate fusion independent of TGFß3. These data describe both a novel role for ephrins in palate morphogenesis, and a previously unknown mechanism of ephrin signaling.

Use of antioxidants in oral healthcare.

There is increasing attention to the potential benefit from the use of antioxidants in the field of dental medicine. In general, antioxidants may be available through oral ingestion, diet or vitamin supplements, and in nutraceuticals. In addition, treatment of oral and dental health problems may include drug-free, natural antioxidant remedies that are available in topical oral applications such as mouth rinse, gel, paste, gum, or lozenge compositions. These topical antioxidant remedies help reduce free-radical or reactive-oxygen species, which are causative inflammatory factors in the progression of gingival and periodontal maladies. This review focuses on relationships between antioxidants and free-radical/reactive-oxygen species in the oral environment.

Reactive oxygen species and antioxidant defense mechanisms in the oral cavity: a literature review.

Through dental procedures and environment, periodontal tissues are exposed to many types of reactive oxygen species (ROS). Recently, various forms of antioxidants have been introduced as an approach to fight dental diseases and improve general gingival health. This article focuses on the classification of antioxidants and the link between oxidative stress and periodontal disease. The protective mechanisms of antioxidants and how routine dental procedures may increase ROS is discussed. The final section reviews the effect of tobacco products on gingival health and disease.

Use of antioxidants in oral healthcare.

There is increasing attention to the potential benefit from the use of antioxidants in the field of dental medicine. In general, antioxidants may be available through oral ingestion, diet or vitamin supplements, and in nutraceuticals. In addition, treatment of oral and dental health problems may include drug-free, natural antioxidant remedies that are available in topical oral applications such as mouth rinse, gel, paste, gum, or lozenge compositions. These topical antioxidant remedies help reduce free-radical or reactive-oxygen species, which are causative inflammatory factors in the progression of gingival and periodontal maladies. This review focuses on relationships between antioxidants and free-radical/reactive-oxygen species in the oral environment.

Antioxidants counteract nicotine and promote migration via RacGTP in oral fibroblast cells.

BACKGROUND: Smoking is associated with an increased risk of oral health and dental problems. The aim of this study is to address the hypothesis that nicotine impairs wound healing by increasing reactive oxygen species and inhibiting cell migration, and antioxidants (AOs) may counteract nicotine effects. METHODS: Primary human gingival fibroblasts (HGFs) and human periodontal ligament (HPDL) fibroblasts were grown to confluence, pretreated with 6 mM nicotine for 2 hours, and treated with AOs in the presence of nicotine. The pure AO compounds ferulic acid (F), phloretin (P), tetrahydrocurcuminoid Cockroft Gault (T), and resveratrol (R) were tested in single, double, or triple combinations (10(-5) M). The migratory behavior at a scratch-wound edge was recorded every 15 minutes for 10 hours by using live-cell imaging. The active form of the Rho-associated protein (Rac) and guanosine triphosphate (GTP) (RacGTP) was immunolabeled and analyzed using confocal microscopy. RESULTS: Combinations of double and triple AOs had a greater effect than single AOs on migration rates and Rac activation. The triple combinations PFR and RFT clearly and unambiguously counteracted the effects of nicotine and significantly increased migration rates in HGF and HPDL fibroblast. CONCLUSIONS: Treatment with AO combinations clearly counteracted the effects of nicotine by restoring and increasing cell-migration rates. We found the combination of PFR was the most effective in HGFs, whereas, RFT was the most effective combination in HPDL fibroblast. These results clearly demonstrate that PF, RFT, and PFR counteract the negative effects of nicotine on cultured oral fibroblasts via the RacGTP signal-transduction pathway.

The effects of extracts from periodontopathic bacteria on human periodontal fibroblasts stimulated with mineralization supplements.

Bacterial effects on in vitro mineralization of human periodontal fibroblasts (HPF) have not yet been examined in great detail. In our study, we investigated the effects of soluble extracts of the periodontopathic bacteria Porphyromonas gingivalis, Bacteroides forsythus and, Treponema denticola on cell proliferation, mineralization, as well as on osteoblastic markers present in HPF cultured in vitro, such as alkaline phosphatase (ALP) activity and collagen content. Periodontal fibroblasts stimulated by B-glycerophosphate, ascorbic acid and dexamethasone (BAD) or by dexamethasone and ascorbic acid (DA) were compared to unstimulated cells. During the cultivation period, the stimulation of HPF by combined dexamethasone and ascorbic acid (DA) had a strong inductive effect on proliferation, ALP activity and collagen formation. The extracts obtained from the periodontal pathogens had a suppressing effect on the proliferation rate of HPF. The extracts from P. gingivalis, B. forsythus and T. denticola caused a decrease in ALP activity within 24 h of application. While extracts obtained from P. gingivalis and B. forsythus induced a reduction in collagen content in BAD- and DA-stimulated HPF cells, T. denticola extracts led to an increase in collagen. Our data suggest that specific periodontopathic bacteria may suppress tissue regeneration in vivo not only by activating host defense mechanisms but also directly via a suppression of growth and differentiation of HPF and a reduction in the extracellular collagen matrix. For the process of pocket formation, not even the direct influence of viable bacteria seems to be necessary. Additionally, long-distance effects of bacteria harboured in periodontal pockets or in root canals may be of importance.