Response of epithelial cells infected by Treponema denticola.

OBJECTIVE: In the gingival crevice, the interaction between epithelial cells and periodontopathic bacteria is important for the development of periodontitis. Treponema denticola is a major pathogen of chronic periodontitis and possesses several virulence factors, such as major surface protein (Msp) and prolyl-phenylalanine-specific protease (dentilisin). Here, we investigated the behaviours of epithelial cells infected with T. denticola by measuring the expression of interleukin (IL)-1ß, IL-6, ß defensin 2 (BD-2) and heat-shock protein 70 (HSP70). METHODS: Epithelial cells were infected with T. denticola wild-type strain, Msp-deficient mutant or dentilisin-deficient mutant, and the expression levels of the above targets were analysed by polymerase chain reaction. RESULTS: Infection with T. denticola wild-type strain and mutants induced the production of IL-6 and HSP70. The level of BD-2 induced by T. denticola wild-type strain at 24 hr was significantly higher than that of the dentilisin-deficient mutant. The level of IL-1ß mRNA in the wild-type strain and dentilisin-deficient mutant was slightly lower than that in the uninfected control. CONCLUSION: These results suggest that the levels of BD-2 were affected by Msp and dentilisin. This effect may contribute to the disruption of the response of epithelial cells to eradicate T. denticola.

Role of mitogen-activated protein kinase pathways in migration of gingival epithelial cells in response to stimulation by cigarette smoke condensate and infection by Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Previous studies have shown that cigarette smoke (CS) and periodontal pathogens could alter wound healing responses of gingival epithelial cells. To elucidate molecular mechanisms leading to these epithelial changes, we studied the signaling pathway involved in the modulation of cell migration by CS condensate (CSC) and the infection by a prominent periodontal pathogen, Porphyromonas gingivalis. MATERIAL AND METHODS: Human gingival epithelial cells (Ca9-22) were treated with CSC or vehicle control for 24 h. Activation of mitogen-activated protein kinases (MAPK) in cells with or without infection by P. gingivalis was assessed by polymerase chain reaction array and immunoblotting using phospho-specific antibodies. Cell migration was assessed using in vitro wound closure model, and specific pharmacologic inhibitors of MAPK pathways were used to characterize further the extent of involvement of the MAPK pathways. RESULTS: Polymerase chain reaction array showed that gene expression of several members of the MAPK, particularly p38 and JNK, was upregulated more than twofold in Ca9-22 cells stimulated with 10 µg/mL CSC. Coincubation with P. gingivalis induced a different pattern of gene expression for MAPK pathways, but it did not suppress the MAPK-related genes upregulated by CSC. A significant phosphorylation of ERK1/2 and p38 was observed in cells stimulated with 10 µg/mL CSC (p < 0.05), whereas coincubation with a higher concentration of CSC (250 µg/mL) evoked no such activation. P. gingivalis infection resulted in a tendency to reduce the phosphorylation of ERK1/2 and p38, which had been enhanced by stimulation with 10 µg/mL CSC. Incubation with ERK1/2 and p38 inhibitors significantly reduced the wound closure of CSC-stimulated cells, by approximately 43% and 46%, respectively (p < 0.05). CONCLUSION: CSC exerts effects on the migration of human gingival epithelial cells through the activation of the MAPK ERK1/2 and p38 signaling pathways. P. gingivalis infection attenuates the CSC-induced migration at least partly by suppressing the phosphorylation of ERK1/2 and p38, but other pathways are likely to be involved in this modulatory process.

Cigarette smoke condensate modulates migration of human gingival epithelial cells and their interactions with Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Epithelial cells are recognized as the first line of defense against bacterial infection and environmental harmful stimuli such as cigarette smoke (CS). Although previous studies explored the effects of nicotine on host cells, mechanisms by which CS affects cellular functions remain uncertain. The present study investigated the effects of CS condensate (CSC) on in vitro wound closure of gingival epithelial cells and their potential interactions with a major periodontal pathogen, Porphyromonas gingivalis. MATERIAL AND METHODS: Human gingival epithelial cells (Ca9-22) were treated with CSC for 24 h. Cell proliferation was determined using a WST-1 assay. Cell migration was assessed using a wound closure model. The expression of integrins was analyzed by confocal scanning laser microscopy and real-time PCR. Intracellular invasion of P. gingivalis was evaluated by confocal scanning laser microscopy and an antibiotic protection assay. RESULTS: Low concentrations (1-10 µg/mL) of CSC showed no significant effect on cell proliferation. CSC demonstrated dual effects on epithelial wound closure of Ca9-22 cells: high concentrations (i.e. 250 µg/mL) significantly inhibited the wound closure whereas low concentrations (i.e. 10 µg/mL) promoted it (p < 0.01). CSC induced distinct changes in cytoskeleton. When CSC-exposed cells were infected with P. gingivalis for 2 h, a significant inhibition of wound closure was observed concurrent with a decrease in integrin α3 expression near the wound area. A significantly increased P. gingivalis invasion into Ca9-22 was observed when exposed to low concentrations of CSC. CONCLUSION: Low concentrations of CSC increased invasion of human gingival epithelial cells by P. gingivalis and induced changes in cytoskeleton and integrin expression, thereby modulating the cell migration.

Effects of epidermal growth factor and/or nerve growth factor on Malassez's epithelial rest cells in vitro: expression of mRNA for osteopontin, bone morphogenetic protein 2 and vascular endothelial growth factor.

BACKGROUND AND OBJECTIVE: Malassez's epithelial rest (MER) cells are involved in the maintenance and homeostasis of the periodontal ligament (PDL). The purpose of this study was to determine the effects of epidermal growth factor (EGF) and/or nerve growth factor (NGF) in vitro on these functions of MER cells. MATERIAL AND METHODS: MER cells from porcine PDL were incubated for 3 or 9 h after the addition of EGF and/or NGF to final concentrations of 10 ng/mL. Cells cultured without those growth factors were used as controls. The expression of mRNA for osteopontin, bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF) was analyzed using quantitative RT-PCR. RESULTS: There was a decrease in the expression of osteopontin mRNA by MER cells treated for 9 h with NGF and the level of mRNA expressed was lower than that of the control and EGF-treated groups. The expression of BMP-2 mRNA by MER cells treated with NGF for 9 h also decreased, and was lower than that of the control and EGF-treated groups. The expression of VEGF mRNA by MER cells treated with EGF for 3 or 9 h was higher than in the control and NGF-treated groups. The expression of VEGF mRNA was lower in MER cells treated with NGF for 3 and 9 h than in the control and EGF-treated groups, and decreased from 3 to 9 h of treatment. EGF stimulated MER cells to secrete VEGF, which suggests that EGF plays an important role in maintaining the homeostasis of the PDL. NGF acts on MER cells to inhibit calcification in the PDL. Furthermore, in the EGF+NGF-treated MER cells, expression of mRNA for BMP-2 and VEGF was similar to that of the NGF-treated group, but cell proliferation and expression of osteopontin mRNA were similar to that of the EGF-treated group. CONCLUSION: EGF and NGF play important roles in maintaining the PDL.

Analysis of side population cells derived from dental pulp tissue.

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 G(0) /G(1) 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.

Cellular responses of rat periodontal ligament cells under hypoxia and re-oxygenation conditions in vitro.

BACKGROUND AND OBJECTIVE: The aim of this study was to investigate the responses of periodontal ligament cells under hypoxia and re-oxygenation conditions in vitro. MATERIAL AND METHODS: Periodontal ligament fibroblasts were isolated from rat incisors. In the hypoxia group, cells were incubated in 2% O(2) for 1-3 d. In the re-oxygenation group, cells were first incubated under the same conditions as the hypoxia group for 24 h and then were returned to normoxic conditions and cultured for 1-2 additional days. RESULTS: Proliferation ratios increased in all groups in a time-dependent manner. Proliferation ratios in both the hypoxia and re-oxygenation groups were significantly higher than in the control group on days 2 and 3. Alkaline phosphatase activity was significantly higher in the hypoxia group than in the control and the re-oxygenation groups. The expression of bone sialoprotein mRNA was significantly higher in the hypoxia group than in the control group on days 1 and 2. The expression of vascular endothelial growth factor mRNA was significantly higher in the hypoxia group than in the control group on days 1 and 2. In the re-oxygenation group, the level of expression of bone sialoprotein mRNA and vascular endothelial growth factor mRNA were similar to those of the control group. The expression of heat shock protein 70 mRNA in the hypoxia group was similar to that in the control group, whereas in the re-oxygenation group it was statistically higher than in the other groups. CONCLUSION: These results suggest that periodontal ligament cells maintain their osteogenic ability in hypoxia and re-oxygenation conditions in vitro.

[Exercise training improves activities of daily living in elderly patients with congestive heart failure].

Exercise training which is one of the multidisciplinary interventions for elderly patients with congestive heart failure, plays an important role for improving the quality of life and reducing the re-admission rate of these patients. We assessed the validity of exercise training for the improvement of patient's skeletal muscle functions and activities of daily living along with monitoring cardiac functions. Exercise training programs were performed in 12 patients with congestive heart failure (New York Heart Association class III or IV), including 5 with valvular disease, 4 with dilated cardiomyopathy and 3 with ischemic cardiomyopathy (mean 79 +/- 9 years). All patients were admitted because of exacerbation of congestive heart failure and were treated conventionally. The exercise training program was started after stabilization of their cardiac condition. The medication was not changed during the training period. After exercise training programs, the cardio-thoracic ratio decreased from 63.8 +/- 7.9% to 60.1 +/- 6.9% (p < 0.01), ejection fraction on echocardiography increased from 47.4 +/- 18.2% to 56.0 +/- 17.5% (p < 0.01), and brain natriuretic peptide decreased from 404.8 +/- 267.5 pg/ml to 313.6 +/- 239.5 pg/ml (p < 0.05). The quadriceps muscle power increased from 0.77 +/- 0.36 Nm/kg to 0.97 +/- 0.41 Nm/kg (p < 0.01). The maximum walking distance on flat surface increased from 149 +/- 164 m to 456 +/- 394 m (p < 0.05). In most patients, the activities of daily living, especially mobility, improved. Appropriate exercise training for the elderly patients with congestive heart failure improves activities of daily living and also reduces the amount of required care by the patients.