Eitoyo Kokubu

Modulation of human gingival fibroblast adhesion, morphology, tyrosine phosphorylation, and ERK 1/2 localization on polished, grooved and SLA substratum topographies

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.

Synergy in biofilm formation between Fusobacterium nucleatum and Prevotella species.

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.

Formation of bone-like tissue by dental follicle cells co-cultured with dental papilla cells.

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.

Porphyromonas gingivalis entry into gingival epithelial cells modulated by Fusobacterium nucleatum is dependent on lipid rafts

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.

Analysis of side population cells derived from dental pulp tissue

Kenmotsu M, Matsuzaka K, Kokubu E, Azuma T, Inoue T. Analysis of side population cells derived from dental pulp tissue. International Endodontic Journal. Aim To investigate the characteristics of side population (SP) cells derived from the dental pulp of young and aged rats. Methodology Maxillary and mandibular incisors were extracted from 5-week-old (young) rats and 60- to 80-week-old (aged) rats. Coronal pulp tissue was removed mechanically, and single-cell suspensions were prepared using collagenase and dispase. Cells were stained with Hoechst 33342 and sorted with an fluorescence-activated cell sorter (FACS). Isolated SP and main population (MP) cells were analysed by real-time reverse transcription polymerase chain reaction, immunohistochemical localization and cell cycle determination. Two-way analysis of variance and the multiple comparison Scheffè test were used for statistical analysis (P < 0.05). Results Approximately 0.40% of pulp cells in young rats and 0.11% in aged rats comprised SP cells. SP cells expressed a higher mRNA level of ATP-binding cassette transporter G2 (ABCG2), but lower mRNA levels of nestin, alkaline phosphatase, p16 and p57 than MP cells in both age groups. Immunohistochemical observation revealed ABCG2-positive cells localized in the cell-rich zone and nestin in the odontoblastic layer in both groups. Furthermore, the majority of both young and aged SP and MP cells were in growth arrest of the G0/G1 phase. Conclusion The FACS analysis revealed a decrease in the proportion of SP cells with age, whilst p16 mRNA expression indicated an increase in cell senescence. The cell cycles of SP and MP cells from both young and aged dental pulp were generally in the G0/G1 phase.

Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation

ABSTRACT Capnocytophaga ochracea is a Gram-negative, rod-shaped bacterium that demonstrates gliding motility when cultured on solid agar surfaces. C. ochracea possesses the ability to form biofilms; however, factors involved in biofilm formation by this bacterium are unclear. A type IX secretion system (T9SS) in Flavobacterium johnsoniae was shown to be involved in the transport of proteins (e.g., several adhesins) to the cell surface. Genes orthologous to those encoding T9SS proteins in F. johnsoniae have been identified in the genome of C. ochracea; therefore, the T9SS may be involved in biofilm formation by C. ochracea. Here we constructed three ortholog-deficient C. ochracea mutants lacking sprB (which encodes a gliding motility adhesin) or gldK or sprT (which encode T9SS proteins in F. johnsoniae). Gliding motility was lost in each mutant, suggesting that, in C. ochracea, the proteins encoded by sprB, gldK, and sprT are necessary for gliding motility, and SprB is transported to the cell surface by the T9SS. For the ΔgldK, ΔsprT, and ΔsprB strains, the amounts of crystal violet-associated biofilm, relative to wild-type values, were 49%, 34%, and 65%, respectively, at 48 h. Confocal laser scanning and scanning electron microscopy revealed that the biofilms formed by wild-type C. ochracea were denser and bacterial cells were closer together than in those formed by the mutant strains. Together, these results indicate that proteins exported by the T9SS are key elements of the gliding motility and biofilm formation of C. ochracea.

Role of extracytoplasmic function sigma factors in biofilm formation of Porphyromonas gingivalis

BackgroundPorphyromonas gingivalis has been implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis biofilm formation in the subgingival crevice plays an important role in the ability of the bacteria to tolerate stress signals outside the cytoplasmic membrane. Some bacteria use a distinct subfamily of sigma factors to regulate their extracytoplasmic functions (the ECF subfamily). The objective of this study was to determine if P. gingivalis ECF sigma factors affect P. gingivalis biofilm formation.MethodsTo elucidate the role of ECF sigma factors in P. gingivalis, chromosomal mutants carrying a disruption of each ECF sigma factor-encoding gene were constructed. Bacterial growth curves were measured by determining the turbidity of bacterial cultures. The quantity of biofilm growing on plates was evaluated by crystal violet staining.ResultsComparison of the growth curves of wild-type P. gingivalis strain 33277 and the ECF mutants indicated that the growth rate of the mutants was slightly lower than that of the wild-type strain. The PGN_0274- and PGN_1740-defective mutants had increased biofilm formation compared with the wild-type (p < 0.001); however, the other ECF sigma factor mutants or the complemented strains did not enhance biofilm formation.ConclusionThese results suggest that PGN_0274 and PGN_1740 play a key role in biofilm formation by P. gingivalis.

Vinculin and Rab5 Complex Is Requited for Uptake of Staphyrococcus aureus and Interleukin-6 Expression

Vinculin, a 116-kDa membrane cytoskeletal protein, is an important molecule for cell adhesion; however, little is known about its other cellular functions. Here, we demonstrated that vinculin binds to Rab5 and is required for Staphylococcus aureus (S. aureus) uptake in cells. Viunculin directly bound to Rab5 and enhanced the activation of S. aureus uptake. Over-expression of active vinculin mutants enhanced S. aureus uptake, whereas over-expression of an inactive vinculin mutant decreased S. aureus uptake. Vinculin bound to Rab5 at the N-terminal region (1-258) of vinculin. Vinculin and Rab5 were involved in the S. aureus-induced phosphorylation of MAP kinases (p38, Erk, and JNK) and IL-6 expression. Finally, vinculin and Rab5 knockdown reduced infection of S. aureus, phosphorylation of MAPKs and IL-6 expression in murine lungs. Our results suggest that vinculin binds to Rab5 and that these two molecules cooperatively enhance bacterial infection and the inflammatory response.

An artificial fusion protein between bone morphogenetic protein 2 and titanium-binding peptide is functional in vivo.

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 vivostudy, 5-μm thick Ti was coated with electron cyclotron resonance sputtering on a porous carbon scaffold which was then dipped in one of three 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 messenger RNAs (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 alkaline phosphatase, bone sialoprotein, and runt-related gene 2 mRNAs was significantly increased. These results suggest that the scaffold of the collagen gel with AFP-TBP-BMP-2 accelerates osteogenesis in vivo.

Synergistic effect on biofilm formation between Fusobacterium nucleatum and Capnocytophaga ochracea

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.