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ABSTRACT: The purpose of this study was to investigate osteogenesis using an artificial fusion protein (AFP) composed of modified bone morphogenetic protein-2 (BMP-2) with a titanium (Ti) binding peptide (TBP) motif on a Ti surface in vivo. In the in vivo study, 5 μm thick Ti was coated with electron cyclotron resonance sputtering on a porous carbon scaffold which was then dipped in 1 of 3 different mixtures of collagen gel: 1, collagen gel only, 2, collagen gel with TBP, and 3, collagen gel with the AFP between BMP-2 and the TBP motif (AFP-TBP-BMP-2). These scaffolds were then implanted into rat abdominal muscles and were studied histologically at various times and the expression of several bone-related protein mRNAs was also analyzed. The Ti-coated scaffold of the collagen gel with AFP-TBP-BMP-2 produced cartilage in the muscle and the expression of ALP, BSP and Runx2 mRNAs was significantly increased. These results suggest that the scaffold of the collagen gel with AFP-TBP-BMP-2 accelerates osteogenesis in vivo.
Journal of Biomedical Materials Research Part A 04/2013; · 2.63 Impact Factor
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ABSTRACT: Host cell invasion by a major periodontal pathogen, Porphyromonas gingivalis, has been proposed as an important mechanism involved in host-pathogen interactions in periodontal and cardiovascular diseases. The present study sought to gain insight into the underlying mechanism(s) involved in previously demonstrated fusobacterial modulation of host cell invasion by P. gingivalis. An immortalized human gingival cell line Ca9-22 was dually infected with P. gingivalis ATCC 33277 and Fusobacterium nucleatum TDC 100, and intracellular invasion was assessed by scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). SEM observation showed that P. gingivalis and F. nucleatum formed consortia and were in the process of penetrating into Ca9-22 by 30-60 min after infection. In CSLM, Ca9-22 cells that contained both P. gingivalis and F. nucleatum were frequently observed after 2 h, although cells that contained exclusively P. gingivalis were also found. Infection by P. gingivalis and/or F. nucleatum revealed evident colocalization with a lipid raft marker, GM1-containing membrane microdomains. In an antibiotic protection assay, depletion of epithelial plasma membrane cholesterol resulted in a significant reduction of recovered P. gingivalis or F. nucleatum (∼33% of untreated control; p < 0.001). This inhibition was also confirmed by CSLM. Sequential infection experiments showed that timing of infection by each species could critically influence the invasion profile. Co-infection with F. nucleatum significantly enhanced host cell invasion by P. gingivalis 33277, its serine phophatase SerB mutant and complemented strains, suggesting that the SerB does not play a major role in this fusobacterial enhancement of P. gingivalis invasion. Thus, the interaction between F. nucleatum and host cells may be important in the fusobacterial enhancement of P. gingivalis invasion. Collectively, these results suggest that lipid raft-mediated process is at least one of the potential mechanisms involved in fusobacterium-modulated host cell invasion by P. gingivalis.
Microbial Pathogenesis 08/2012; 53(5-6):234-42. · 1.94 Impact Factor
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ABSTRACT: Abstract - The purpose of this study was to evaluate the characteristics of dental pulp cells for tissue engineering derived from the fractured incisal portion of tooth crowns. Thirty Sprague-Dawley rats were used for histological and immunohistochemical analysis of nestin protein expression and to measure levels of mRNAs encoding osteocalcin, osteopontin, bone sialoprotein (BSP), dentin sialoprotein (DSP), heat shock protein (HSP) 27, vascular endothelial growth factor (VEGF), ATP-binding cassette transporter G2 (ABCG2), nestin, and p57(Kip2) . Odontoblasts at the incisal portion in the control group were oriented in a regular pattern, but those in the experimental group were randomly stratified. Immunohistochemically, only a few odontoblasts were positive for nestin at the incisal portion in the experimental group at 2 days. Some cells in the inner area in the control group were positive for nestin, but nestin-positive cells in the experimental group at the incisal portion were not observed. The mRNA expression for osteogenic or odontogenic markers in the experimental group was higher than in the control group. HSP27 mRNA expression in the experimental group at 2 days was higher than in the control group and in the experimental group at 7 days. mRNA expression of stem cell markers, such as ABCG2 and nestin, in the experimental group tended to decrease compared with the control. In conclusion, this study demonstrates that dental pulp stem cells derived from fractured teeth differentiate to osteogenic or odontogenic cells.
Dental Traumatology 03/2012; · 1.20 Impact Factor
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ABSTRACT: The formation of dental plaque biofilm by specific Gram-negative rods and spirochetes plays an important role in the development of periodontal disease. The aim of this study was to characterize biofilm formation by Fusobacterium nucleatum and Capnocytophaga ochracea. Coaggregation between F. nucleatum and Capnocytophaga species was determined by visual assay. Biofilm formation was assessed by crystal violet staining. Enhancement of biofilm formation by F. nucleatum via soluble factor of C. ochracea was evaluated by addition of culture supernatant and a two-compartment separated co-culture system. Production of autoinducer-2 by the tested organisms was evaluated using Vibrio harveyi BB170. F. nucleatum strains coaggregated with C. ochracea ATCC 33596 or ONO-26 strains. Ethylenediamine tetraacetic acid, N-acetyl-d-galactosamine or lysine inhibited coaggregation. Heating or proteinase K treatment of F. nucleatum cells affected coaggregation, whereas the same treatment of C. ochracea cells did not. Co-culture of F. nucleatum with C. ochracea in the same well resulted in a statistically significant increase in biofilm formation. Enhancement of F. nucleatum biofilm formation by a soluble component of C. ochracea was observed using the two-compartment co-culture system (P < 0.05) and confirmed by addition of culture supernatant of C. ochracea (P < 0.01). The present findings indicate that induction of coaggregation and intracellular interaction by release of a diffusible molecule by C. ochracea play a significant role in the formation of biofilm by F. nucleatum and C. ochracea.
Anaerobe 02/2012; 18(1):157-61. · 2.41 Impact Factor
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ABSTRACT: The formation of biofilm by anaerobic, Gram-negative bacteria in the subgingival crevice plays an important role in the development of chronic periodontitis. The aim of this study was to characterize the role of coaggregation between Fusobacterium nucleatum and Prevotella species in biofilm formation. Coaggregation between F. nucleatum and Prevotella species was determined by visual assay. Effect of co-culture of the species on biofilm formation was assessed by crystal violet staining. Effect of soluble factor on biofilm formation was also examined using culture supernatant and two-compartment co-culture separated by a porous membrane. Production of autoinducer-2 (AI-2) by the organisms was evaluated using Vibrio harveyi BB170. Cells of all F. nucleatum strains coaggregated with Prevotella intermedia or Prevotella nigrescens with a score of 1-4. Addition of ethylenediamine tetraacetic acid or l-lysine inhibited coaggregation. Coaggregation disappeared after heating of P. intermedia or P. nigrescens cells, or Proteinase K treatment of P. nigrescens cells. Co-culture of F. nucleatum ATCC 25586 with P. intermedia or P. nigrescens strains increased biofilm formation compared with single culture (p < 0.01); co-culture with culture supernatant of these strains, however, did not enhance biofilm formation by F. nucleatum. Production of AI-2 in Prevotella species was not related to enhancement of biofilm formation by F. nucleatum. These findings indicate that physical contact by coaggregation of F. nucleatum strains with P. intermedia or P. nigrescens plays a key role in the formation of biofilm by these strains.
Anaerobe 09/2011; 18(1):110-6. · 2.41 Impact Factor
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ABSTRACT: The aim of this study was to investigate cellular activity of the cervical portion of peri-implant tissue due to immediate loading after implant placement following tooth extraction from the dog mandible, in terms of morphological, immunohistochemical and molecular characteristics.
A sand-blasted implant was inserted into the root septum bone of each extraction socket and was connected to a superstructure made from resin and then covered with an expanded polytetrafluoroethylene membrane. Implants without the superstructure were used as the non-loading control group. Animals were sacrificed 1-3 weeks later and specimens were observed using light microscopy and mRNA levels were analyzed by real-time polymerase chain reaction.
The new bone formation ratio in the loading group at 3 weeks was significantly higher than in the non-loading group. Alkaline phosphatase (ALP)-positive cells were observed in tissues around the implant surface in both groups at each of the time periods. More osteocalcin (OCN)-positive cells were observed in the non-loading group than in the loading group at 2 weeks. The expression of ALP mRNA in the loading group was significantly up-regulated compared with the non-loading group (P<0.05). The expression of OCN mRNA in the loading group was significantly up-regulated compared with the non-loading group at 2 weeks (P<0.05).
These results suggest that immediate loading after implant placement following tooth extraction osteogenic affects cellular activity of cervical portion of peri-implant tissue.
Clinical Oral Implants Research 03/2011; 22(12):1372-8. · 2.51 Impact Factor
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ABSTRACT: The protective effect of DNA vaccines expressing the Arg-gingipain A domain against bone loss induced by Porphyromonas gingivalis infection was investigated in a murine model. phgp44, which expresses the 44-kDa adhesion/hemagglutinin domain of Arg-gingipain A, prevented P. gingivalis-induced alveolar bone loss. The results indicate that phgp44 could be a candidate antigen for a vaccine against P. gingivalis infection.
Clinical and vaccine immunology: CVI 03/2011; 18(5):888-91. · 2.37 Impact Factor
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ABSTRACT: During tooth root formation, dental follicle cells (DFCs) differentiate into osteoblasts/cementoblasts when they are in contact with pre-existing dentin. Since some factors of dentin matrix were also produced by dental papilla cells (DPCs) and could induce DFCs differentiation, we hypothesized that DPCs can directly promote DFCs differentiation and that differentiation could occur in a co-culture model. To test this hypothesis, we investigated the characteristics of DFCs that are influenced by DPCs in an in vitro co-culture and in vivo heterotopic transplant model. One week into the co-culture, there were significant increases in the mRNA level of bone morphogenetic protein 2 (BMP2), osteoprotegerin (OPG), bone sialoprotein (BSP) and osteocalcin (OCN), and a decrease of the receptor activator of nuclear factor κB ligand (RANKL). Additionally, the number of BMP2-, OPG-, BSP- and OCN-positive DFCs increased whereas RANKL-positive DFCs decreased. Three weeks after co-culture, DFCs produced calcified nodules, accompanied with increased sub-cellular organelles for protein synthesis and secretion. In the heterotopic transplant model, the adult male rats were used as hosts, DFCs were transplanted into the omentum. In vivo 5-week growth of DFCs in the presence of DPCs led to the formation of bone-like tissues, positive for BSP, OCN and BMP2. In contrast, DFCs alone led to fibrous-like tissues. These results indicated that in the absence of pre-existing dentin, DPCs can stimulate osteogenesis and inhibit osteoclastogenesis in DFCs and suggested a novel strategy to promote DFCs differentiation.
Cell and Tissue Research 10/2010; 342(2):221-31. · 3.11 Impact Factor
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ABSTRACT: Attachment of connective tissue to dental implants, which is influenced by surface topography, is an important determinant of implant success. Approaches employed to alter topography include acid etching or blasting to produce roughened surfaces, and production of precisely defined topographies using microfabrication techniques. The aim of this study was to assess the influence of polished, microgrooved, and sand-blasted, large grit, acid-etched (SLA) topographies on fibroblast adhesion, morphology, activation, and ERK 1/2 phosphorylation and localization. Human gingival fibroblasts (HGFs) spread on all tested surfaces within 2 h, and topography influenced the pattern of phosphotyrosine localization. Fibrillar adhesion formation was prominent in HGFs cultured on microgrooves and SLA at 24 h compared with smooth. No significant difference in ERK 1/2 phosphorylation was observed at 2 or 24 h, but nuclear localization depended on culture time and substratum topography. Nuclear localization of ERK 1/2 occurred at 2 h on polished surfaces, but was not evident at 1 week. In contrast, cells on SLA and grooved surfaces did not exhibit nuclear localization of ERK 1/2 at early times, but did at 1 week. The results of this study suggest that rough and microfabricated topographies influence fibroblast adhesion and intracellular signaling through focal adhesion/integrin-dependent mechanisms in a time-dependent manner.
Journal of Biomedical Materials Research Part A 12/2008; 91(3):663-70. · 2.63 Impact Factor
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ABSTRACT: The aim of this study was to investigate the behavior of rat periodontal ligament (PDL) cells cultured on fibroblast growth factor-2 (FGF-2)-immobilized titanium surfaces treated by oxygen (O2) plasma. We used cell disks (15 mm in diameter), and 35-mm culture dishes sputter-coated with titanium. These were treated with oxygen plasma and dipped in FGF-2 solution. Immobilized FGF-2 was visualized with a confocal laser-scanning microscope, and its weight was calculated to be approximately 22.6 ng/cm(2) using a quartz crystal microbalance-dissipation apparatus. The PDL cells were obtained from rat incisors. Cells from fourth subculture were seeded onto the FGF-2-immobilized titanium surface. Proliferation ratio, alkaline phosphatase (ALP) activity, and expressions of type I collagen and vascular endothelial growth factor (VEGF) mRNAs were evaluated. Proliferation ratio and expressions of type I collagen and VEGF mRNAs were significantly higher, whereas ALP activity was significantly lower in FGF-2-immobilized cells than in control group (p < 0.05). These findings suggest that oxygen plasma modification can immobilize FGF-2 onto a titanium surface. Immobilized FGF-2, although inferior to culture medium with FGF-2, influenced the proliferation of PDL cells and might have promoted collagen and vascular synthesis.
Journal of Biomedical Materials Research Part A 09/2008; 91(1):69-75. · 2.63 Impact Factor
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Kenichi Matsuzaka,
Morito Tsuruoka, Eitoyo Kokubu,
Akira Katakura,
Takayuki Endo,
Yoshihiro Shibukawa,
Masuro Shintani,
Masakazu Tazaki,
Kazuyuki Ishihara,
Sadamitsu Hashimoto,
Masao Yoshinari,
Takashi Inoue
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ABSTRACT: The purpose of this study was to evaluate age-related differences in expression of vascular endothelial growth factor (VEGF) by periodontal ligament (PDL) cells. PDL cells were obtained from Wistar male rats weighing approximately 150 g each in the young group and 350 g each in the old group. PDL cells derived from upper and lower incisors were seeded in 35-mm culture dishes after primary culture. For cell proliferation assays, cells were detached and counted at 1, 3, 5, 7, 11 and 14 days after culture. VEGF mRNA expression was analyzed with TaqMan. The number of cells in both groups increased day by day, but the rate of increase in the young group was higher than that in the old group. VEGF mRNA expression in the young group increased from 3 to 14 days, but in the old group increased only slightly over the same time period. Expression ratios in the young group were higher than those in the old group, and there were significant differences between the young and old groups at 7 and 14 days of culture. In conclusion, the data revealed that PDL cells varied with age, and suggest that in view of such changes in cell proliferation and VEGF mRNA expression, age should be taken into consideration in periodontal treatment.
The Bulletin of Tokyo Dental College 09/2007; 48(3):143-6.
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ABSTRACT: We report a rare case of a papillary cystadenoma arising from the upper lip. This tumor was not distinctly encapsulated and had proliferated replacing the ductal epithelium. Mast cells were found not only in the stroma but also in the oncocytic epithelial layer. There was a strong immunoreaction with mitochondrial antibody in the epithelial layer. Only one case (0.9%) of papillary cystadenoma has occurred among the 110 benign intraoral salivary gland tumors seen in our hospital from 1966 through September 2003.
The Bulletin of Tokyo Dental College 12/2003; 44(4):213-6.
