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ABSTRACT: ABSTRACT Objective: The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS). Material and Methods: Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0 - 10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results. Results: Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF. Conclusion: These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation.
Journal of applied oral science: revista FOB 01/2013; 21(2). · 0.39 Impact Factor
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ABSTRACT: Background: In a previous report, it was shown that Toll-like receptor (TLR) 2 knockdown modulates Interleukin (IL)-6 and IL-8 but not the chemokine CXCL12, an important mediator with inflammatory and proangiogenic effects, in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). This study investigated whether knocking down two important TLRs adaptor molecules such as myeloid differentiation protein 88 (MyD88) and TRIF-related adaptor molecule (TRAM) could affect mRNA expression of Interleukin (IL)-6, IL-8 and CXCL12 in HGF and HPLF. Methods: After small interfering RNA-mediated silencing of MyD88 or TRAM, HGF and HPLF were stimulated with Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) or two synthetic ligands of TLR2 (Pam2CSK4 and Pam3CSK4) for 6 hours. IL-6, IL-8 and CXCL12 mRNA were evaluated by quantitative polymerase chain reaction. Results: Knockdown of MyD88 or TRAM partially impaired the IL-8 mRNA upregulation in both fibroblasts subpopulations. Similarly, IL-6 upregulation was partially prevented by siMyD88 or siTRAM in HGF stimulated with PgLPS as well as in both fibroblasts subtypes challenged with Pam2CSK4. Conversely, constitutive CXCL12 mRNA levels were upregulated by MyD88 or TRAM knockdown in non-stimulated cells. Conclusion: These results suggest that TLRs adaptor molecules knockdown, such as MyD88 or TRAM can decrease IL-6 and IL-8 mRNA and increase CXCL12 mRNA expression, in HGF and HPLF. This can be an important step for the better understanding of the mechanisms that control the inflammatory cytokines and chemokines expression which in turn contribute to periodontal pathogenesis.
Journal of Periodontology 11/2012; · 2.60 Impact Factor
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ABSTRACT: Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen associated molecular patterns and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual since different studies have reported that it can be an agonist for TLR2 and an antagonist or agonist for TLR4. This study investigated and compared whether signaling through TLR2 or TLR4 could affect the secretion of Interleukin (IL)-6, IL-8 and Stromal Derived Factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPLF from the same donors were stimulated with Porphyromonas gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, Pam2CSK4 and Pam3CSK4 in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.
Journal of Periodontology 06/2012; · 2.60 Impact Factor
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ABSTRACT: This study investigated the role of periodontal disease in the development of stroke or cerebral infarction in patients by evaluating the clinical periodontal conditions and the subgingival levels of periodontopathogens.
Twenty patients with ischemic (I-CVA) or hemorrhagic (H-CVA) cerebrovascular episodes (test group) and 60 systemically healthy patients (control group) were evaluated for: probing depth, clinical attachment level, bleeding on probing and plaque index. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were both identified and quantified in subgingival plaque samples by conventional and real-time PCR, respectively.
The test group showed a significant increase in each of the following parameters: pocket depth, clinical attachment loss, bleeding on probing, plaque index and number of missing teeth when compared to control values (p<0.05, unpaired t-test). Likewise, the test group had increased numbers of sites that were contaminated with P. gingivalis (60%x10%; p<0.001; chi-squared test) and displayed greater prevalence of periodontal disease, with an odds ratio of 48.06 (95% CI: 5.96-387.72; p<0.001). Notably, a positive correlation between probing depth and the levels of P. gingivalis in ischemic stroke was found (r=0.60; p=0.03; Spearman's rank correlation coefficient test). A. actinomycetemcomitans DNA was not detected in any of the groups by conventional or real-time PCR.
Stroke patients had deeper pockets, more severe attachment loss, increased bleeding on probing, increased plaque indexes, and in their pockets harbored increased levels of P. gingivalis. These findings suggest that periodontal disease is a risk factor for the development of cerebral hemorrhage or infarction. Early treatment of periodontitis may counteract the development of cerebrovascular episodes.
Journal of applied oral science: revista FOB 02/2012; 20(1):104-12. · 0.39 Impact Factor
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ABSTRACT: The aim of this study was to quantify and compare the production of transforming growth factor beta (TGF-β), interleukin (IL)-8 and IL-10 by human cultured periodontal ligament and gingival fibroblasts both obtained from the same donors challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Fibroblasts were exposed to 0.1-10 µg/mL of LPS from P. gingivalis and after 24 h the supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA). TGF-β protein production was upregulated in a concentration-dependent manner, mainly in gingival fibroblasts, which was statistically significant when challenged by 10 µg/mL LPS. Additionally, at this concentration, gingival fibroblasts had almost a two-fold increase in the amount of TGF-β when compared to periodontal ligament fibroblasts. Both periodontal ligament and gingival fibroblasts showed an increase in IL-8 production when challenged with 1 µg/mL and 10 µg/mL LPS. IL-10 production remained unaffected when challenged by any of the LPS concentrations tested in either periodontal ligament or gingival fibroblasts. Our results demonstrate that periodontal ligament and gingival fibroblasts when challenged by LPS from P. gingivalis with 24 h may play a critical role in producing TGF-β and IL-8 but not IL-10.
Brazilian oral research 04/2011; 25(2):157-62.
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ABSTRACT: Chemokines and chemokine receptors have been implicated in the selective migration of leukocyte subsets to periodontal tissues, which consequently influences the disease outcome. Among these chemoattractants, the chemokines CCL3, CCL4 and CCL5 and its receptors, CCR1 and CCR5, have been associated with increased disease severity in mice and humans. Therefore, in this study we investigated the modulation of experimental periodontitis outcome by the treatment with a specific antagonist of CCR1 and 5 receptors, called met-RANTES. C57Bl/6 mice was orally infected with Aggregatibacter actinomycetemcomitans and treated with 0.05, 0.1, 0.5, 1.5 and 5 mg doses of met-RANTES on alternate days, and evaluated by morphometric, cellular, enzymatic and molecular methods. At 0.5 mg up to 5 mg doses, a strong reduction in the alveolar bone loss and inflammatory cell migration were observed. Interestingly, 5 mg dose treatment resulted in the maximum inhibition of inflammatory cell migration, but resulted in a similar inhibition of bone loss when compared with the lower doses, and also resulted in increased bacterial load and CRP response. When 0.5 and 5 mg therapy regimens were compared it was observed that both therapeutic protocols were able to downregulate the levels of pro-inflammatory, Th1-type and osteoclastogenic cytokines, and CD3+ and F4/80+ cells migration to periodontal tissues, but the high dose modulates host response in a more pronounced and unspecific and excessive way, interfering also with the production of antimicrobial mediators such as MPO, iNOS and IgG, and with GR1+ and CD19+ cells migration. Our results demonstrate a thin line between beneficial immunoregulation and impaired host defense during experimental periodontitis, and the determination of the exact equilibrium point is mandatory for the improvement of immune-targeted therapy of periodontitis.
PLoS ONE 01/2011; 6(7):e22526. · 4.09 Impact Factor
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ABSTRACT: Aggregatibacter actinomycetemcomitans is an oral Gram-negative bacterium that contributes to periodontitis progression. Isolated antigens from A. actinomycetemcomitans could be activating innate immune cells through Toll-like receptors (TLRs). In this study, we evaluated the role of TLR4 in the control of A. actinomycetemcomitans infection.
We examined the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR4(-/-) mice. The production of cytokines was evaluated by ELISA. The bacterial load was determined by counting the number of colony-forming units per gram of tissue.
The results showed that TLR4-deficient mice developed less severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly lower bone loss and inflammatory cell migration to periodontal tissues. However, the absence of TLR4 facilitated the A. actinomycetemcomitans dissemination. Myeloperoxidase activity was diminished in the periodontal tissue of TLR4(-/-) mice. We observed a significant reduction in the production of tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta in the periodontal tissue of TLR4(-/-) mice.
The results of this study highlighted the role of TLR4 in controlling A. actinomycetemcomitans infection.
Journal Of Clinical Periodontology 03/2010; 37(3):248-54. · 3.00 Impact Factor
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Valéria Gelani,
Ana Paula Fernandes,
Thaís Helena Gasparoto,
Thiago Pompermaier Garlet,
Tânia Mary Cestari,
Hayana Ramos Lima,
Erivan Schnaider Ramos,
Tatiana Salles de Souza Malaspina, Carlos Ferreira Santos,
Gustavo Pompermaier Garlet,
João Santana da Silva,
Ana Paula Campanelli
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ABSTRACT: Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) is a Gram-negative bacterium present in the oral cavity and is usually associated with localized aggressive periodontitis. Isolated antigens from A. actinomycetemcomitans can activate innate immune cells through Toll-like receptors (TLRs), which are molecules that recognize structural components conserved among microorganisms. In this study, we evaluate the role of TLR2 in the recognition of A. actinomycetemcomitans.
Macrophages and neutrophils from knockout mice with targeted disruption of TLR2 (TLR2(-/-) mice) and wild-type mice were collected and used for the subsequent assays. The production of cytokines and chemokines was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of apoptotic cells was determined by flow cytometry. In addition, the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR2(-/-) mice were examined.
The results show that TLR2-deficient mice developed more severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly higher bone loss and inflammatory cell migration to periodontal tissues. The inflammatory cell influx into the peritoneal cavities of TLR2(-/-) mice was three-fold lower than that observed for the littermate controls. A significantly diminished production of the cytokines tumor necrosis factor-alpha and interleukin-1beta as well as the chemokine CC-ligand-5 in the peritoneal cavities of TLR2(-/-) mice was observed. In addition, a high frequency of apoptotic cells in the inflammatory exudates from TLR2(-/-) mice was observed. Phagocytosis and nitric oxide production was diminished in cells from TLR2(-/-) mice, facilitating the dissemination of the pathogen to the spleen.
The results of this study highlight the involvement of TLR2 in recognizing A. actinomycetemcomitans and its essential role in controlling A. actinomycetemcomitans infection.
Journal of Periodontology 12/2009; 80(12):2010-9. · 2.60 Impact Factor
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ABSTRACT: Inhibitory signals mediated via molecules such as programmed death-1 (PD-1) play a critical role in downmodulating immune responses and maintaining peripheral tolerance. We investigated the involvement of cytokines and PD-1 engagement in mediating the T-cell unresponsiveness to bacterial and ubiquitous antigens in periodontal diseases.
Gingival and peripheral blood samples from healthy individuals and patients with chronic periodontitis were collected and used for the subsequent assays. Leukocytes in the lesion site and blood were evaluated using flow cytometry. The production of interferon-gamma, interleukin-10, and transforming growth factor-beta proteins was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of PD-1+ cells in the inflamed gingiva was confirmed by immunofluorescence confocal microscopy for CD4 and PD-1 colocalization.
T cells from patients with chronic periodontitis proliferated poorly in response to Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) antigen. T-cell unresponsiveness was not associated with imbalanced cytokine production. However, T cells from patients with chronic periodontitis expressed significantly higher levels of PD-1 either upon isolation or after culture with antigens. Moreover, PD-1 blocking did not result in significant T-cell proliferation in cells cultured with phytohemagglutinin or bacterial antigens. The blockade of PD-1 resulted in the increased production of IFN-gamma. In addition, CD4+ and CD8+ T cells expressing PD-1 accumulated in lesions with chronic periodontitis.
These data show that PD-1 engagement could be involved in the modulation of IFN-gamma production by T cells in patients with chronic periodontitis.
Journal of Periodontology 11/2009; 80(11):1833-44. · 2.60 Impact Factor
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ABSTRACT: This study is intended to verify the correlation among clinical indices of the peri-implant soft tissues, the histological condition and the presence of 3 pathogens commonly associated with peri-implant diseases (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia).
Four clinical indices, Gingival Index (GI), Sulcus Bleeding Index, GI modified by Mombelli, and Plaque Index modified by Mombelli (mPI) were evaluated around 1 dental implant of each subject (n = 10). Subgingival plaque was collected for bacterial analysis (polymerase chain reaction) and a biopsy of peri-implant soft tissues for histological analysis was harvested. The clinical indices and detected pathogens correlated with a developed histological index (HI).
There was no statistically significant relationship between the clinical indices (GI, Sulcus Bleeding Index, and GI modified by Mombelli) and the HI, except for the mPI on the central area of lingual aspects (r = 0.85, P = 0.0029). There was a tendency for a positive correlation between the mPI on the central area of buccal aspects and the HI (r = 0.63, P = 0.0544). The counting of lymphocytes and plasmocytes correlated positively with HI, thus suggesting the index reliability. The prevalence of A. actinomycetemcomitans, P. gingivalis, and T. forsythia did not present a significant relationship with the HI.
Despite the small number of samples and the poor statistical significance, the mPI seems to be useful for evaluation of inflammatory severity on soft tissue around dental implants as demonstrated by its relationship with the HI. Further studies are necessary to elucidate this subject.
Implant dentistry 09/2009; 18(4):334-44. · 1.51 Impact Factor
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The Cleft Palate-Craniofacial Journal 09/2009; 46(5):481-6. · 0.82 Impact Factor
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ABSTRACT: Candida albicans is considered the most important Candida species able to cause oral infections in denture wearers. In recent years, Candida dubliniensis has emerged as a pathogenic yeast in humans. The close phenotypic similarities of C. albicans and C. dubliniensis have led to the misidentification of these species. In this work, our aim was to verify through PCR the presence of C. dubliniensis in palate and maxillary denture samples from 112 denture wearers presenting with or without denture-related stomatitis (DRS). C. dubliniensis was isolated at low rates from both palate (5.3 % and 10.7 %) and maxillary denture (5.3 % and 8.9 %) samples from wearers regardless of the presence of the disease. However, when C. dubliniensis was detected in individuals with DRS, it was always associated with C. albicans. In addition, our results showed that C. albicans was the most commonly identified candidal species in maxillary denture and hard palate samples from DRS patients (78.5 % and 89.2 %, respectively) as well as from controls (31.2 % and 28.5 %, respectively). In conclusion, C. dubliniensis was detected in the oral environment of denture wearers. The association of C. dubliniensis with C. albicans occurred in approximately 10 % of the DRS cases.
Journal of Medical Microbiology 08/2009; 58(Pt 7):959-62. · 2.50 Impact Factor
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ABSTRACT: There are several studies about the cytotoxic effects of dental materials in contact with the pulp tissue, such as calcium hydroxide (CH), adhesive systems, resin composite and glass ionomer cements. The aim of this review article was to summarize and discuss the cytotoxicity and biocompatibility of materials used for protection of the dentin-pulp complex, some components of resin composites and adhesive systems when placed in direct or indirect contact with the pulp tissue. A large number of dental materials present cytotoxic effects when applied close or directly to the pulp, and the only material that seems to stimulate early pulp repair and dentin hard tissue barrier formation is CH.
Journal of applied oral science: revista FOB 05/2009; 17(6):544-54. · 0.39 Impact Factor
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ABSTRACT: The aim was to elucidate the changes occurring in temporomandibular joint (TMJ) after surgical mandibular advancement with different fixation materials: bicortical screws and miniplates.
Eighteen minipigs were randomly divided into 3 groups: group I (control), nonoperated animals; group II, animals submitted to surgical advancement surgery and osteosynthesis by bicortical screws; and group III, animals submitted to surgical advancement surgery and osteosynthesis by miniplates. Four months after the surgeries, TMJs were collected and histologically prepared after computerized tomography (CT) scanning for the blind detection of erosion, flattening, and osteophyte.
The CT analysis revealed significant alterations in the shape of the condyles (erosion: P = .0010; flattening: P < .0001) for group II compared with groups I and III. Descriptive histologic analysis was compatible with the CT findings.
The results indicated that bicortical screw fixation resulted in more pronounced condylar alterations in the shape of the condyles than miniplate osteosynthesis. However, further clinical studies are necessary to confirm these data.
Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 01/2009; 107(4):477-84. · 1.50 Impact Factor
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ABSTRACT: This case report outlines the sequel and possible management of a permanent tooth traumatized through the predecessor, a maxillary right primary central incisor that was avulsed and replanted by a dentist 1 h after the trauma in a 3-year-old girl. Three years later, discoloration and fistula were present, so the primary tooth was extracted. The patient did not come to the scheduled follow-ups to perform a clinical and radiographic control of the succeeding permanent incisor, and only returned when she was 10 years old. At that moment, the impaction and dilaceration of the maxillary right permanent central incisor were observed through radiographic examination. The dilacerated permanent tooth was then surgically removed, and an esthetic fixed appliance was constructed with the crown of the extracted tooth. Positive psychological influence of the treatment on this patient was also observed.
Dental Traumatology 11/2008; 24(5):569-73. · 1.20 Impact Factor
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ABSTRACT: A case of extensive crown fracture associated with intrusion of the permanent maxillary central incisors in an 8-year-old boy is reported. The treatment of both injured teeth included attempts of apexification and arrest of root resorption with calcium hydroxide. After 8 months of the trauma, there was no calcified barrier formation in the apex. Mineral trioxide aggregate (MTA) was then used as a filling material. At 15-month follow up, the teeth were asymptomatic and correctly sealed, the external inflammatory root resorption had stopped, and the radiolucent image had disappeared, which meant the initial healing of the periapical lesion. MTA may be considered as an alternative option for the treatment of traumatized and immature permanent teeth.
Dental Traumatology 11/2008; 24(5):565-8. · 1.20 Impact Factor
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ABSTRACT: The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.
Journal of Molecular Histology 07/2008; 39(3):275-82. · 1.48 Impact Factor
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ABSTRACT: The objective of this study was to elucidate the changes occurring in the temporomandibular joint (TMJ) after surgical mandibular advancement with different fixation techniques: bicortical screws (rigid fixation) and miniplates (semi-rigid fixation).
Eighteen minipigs were equally and randomly divided into 3 groups: Group I (control), nonoperated animals; Group II, animals submitted to surgical advancement surgery and osteosynthesis by bicortical screws; and Group III, animals submitted to surgical advancement surgery and osteosynthesis by miniplates. Four months after the surgeries, the presence of interleukin (IL)-6 and IL-10 in synovial fluid samples was assessed in ELISA experiments. TMJs were histologically prepared.
Higher levels of IL-10 (P = .0436) were found for Group II. Descriptive histological analysis was compatible with the ELISA findings.
Rigid fixation evokes more pronounced signs of bone remodeling in the TMJ, whereas malleable fixation promotes a more intense inflammatory activity. Therefore, rigid fixation seems to transmit a higher impact of postoperative masticatory forces to the TMJ.
Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 07/2008; 106(3):331-8. · 1.50 Impact Factor
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ABSTRACT: Dental fractures of the permanent maxillary anterior teeth are relatively frequent accidents during childhood. Placement of direct composite restorations allows clinicians a conservative method of restoring fractured anterior teeth to the original shape and color. A methodical protocol for the restoration of a fractured maxillary left central incisor of a 7-year-old girl is presented. As a temporary treatment, pulpal protection and the use of an acetate crown with glass-ionomer cement were performed. As a definitive treatment, diagnostic waxing and a silicone matrix formed the basis for successful reconstruction of the fractured tooth with composite resin. After finishing and polishing, an esthetic and natural-looking restoration was achieved, which completely satisfied the functional and esthetic expectation of the patient and dental team.
Dental Traumatology 01/2008; 23(6):371-5. · 1.20 Impact Factor
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ABSTRACT: This case report outlines an alternative oral rehabilitation treatment of a 4-year-old boy with hypodontia and conical tooth shape, which along with other characteristics led to a suspicious ectodermal dysplasia diagnosis. Diagnostic waxup and a silicone matrix formed the basis to the successful reconstruction of the anterior teeth with resin composite. Additionally, removable partial dentures were fabricated to improve esthetics and chewing ability. Positive psychologic influence of this alternative treatment on this patient is also discussed.
Quintessence international 11/2006; 37(9):725-30. · 0.76 Impact Factor
