Efeitos do ramipril sobre a doença periodontal induzida experimentalmente em ratos

A doença periodontal (DP) corresponde a um grupo de doenças inflamatórias que acomete as estruturas periodontais de proteção e de suporte e pode levar à perda dentária. A etiologia está relacionada à placa dentobacteriana que leva à produção de grande quantidade de citocinas pró-inflamatórias importantes na destruição tecidual. A angiotensina (Ang) II também pode contribuir para a inflamação e destruição tecidual no periodonto agindo como mediador chave. A utilização de drogas que atuem na cascata do sistema renina-angiotensina (SRA) poderia interferir no estado de saúde ou inflamação do tecido mole, na perda óssea alveolar e na expressão gênica dos componentes do SRA e mediadores inflamatórios. Portanto, o objetivo do presente trabalho foi investigar se o ramipril, um inibidor da enzima conversora de angiotensina (ECA), altera a progressão da DP induzida experimentalmente em ratos. Foi utilizado o modelo de indução da DP por colocação de ligadura ao redor do primeiro molar inferior direito de ratos. Os grupos com 10 animais cada, foram divididos em tratados com ramipril (via gavagem 10 mg/kg/dia) ou água (veículo) durante 14 e 21 dias e o grupo Sham submetido à indução fictícia da DP. Outros quatro grupos foram submetidos ao pré-tratamento com ramipril durante os períodos de 7 e 14 dias e após a indução da DP e tratados por 14 ou 21 dias. As metodologias de avaliação foram: extração de RNA total, transcrição reversa seguida de reação em cadeia da polimerase quantitativa (RTqPCR), análises histológica e da perda óssea alveolar. Os dados foram analisados por meio de gráficos e os resultados foram submetidos à análise unidirecional de variância (ANOVA) e representaram médias e respectivos desvios-padrão. Diferenças entre os grupos foram consideradas estatisticamente significativas quando p < 0,05. Com base nos resultados obtidos pode-se concluir que o ramipril foi capaz de reduzir a progressão da perda óssea no grupo tratado por 21 dias (DP-21d-Rami), entretanto houve aumento do processo inflamatório, além de alteração da expressão de RNAm de ECA-2 e do receptor Mas, alguns mediadores do processo inflamatório, como COX2 e VEGF, e os receptores VEGF-R1 e VEGF-R2.

The use of a magnetic attachment in a removable partial denture of a patient with periodontal tissue loss

O uso dos magnetos em prótese parcial removível é uma alternativa viável para eliminar a estrutura metálica que pode interferir na estética sem perder retenção e estabilidade. Os magnetos podem ser recomendados para pacientes com perda de tecido periodontal, desde que eles diminuam a transmissão de forças ao dente remanescente. O objetivo deste relato de caso clínico foi apresentar uma alternativa de tratamento protético para dentes pilares comprometidos periodontalmente e descrever as vantagens e desvantagens do uso dos magnetos em prótese parcial removível sobre os pontos de vista funcionais, biológicos e estéticos.

Identification of collagenolytic MMP networks as potential biomarkers in progressive chronic periodontitis subjects and MMP-8 null allele model

Chronic periodontitis results from a complex aetiology, including the formation of a subgingival biofilm and the elicitation of the host s immune and inflammatory response. The hallmark of chronic periodontitis is alveolar bone loss and soft periodontal tissue destruction. Evidence supports that periodontitis progresses in dynamic states of exacerbation and remission or quiescence. The major clinical approach to identify disease progression is the tolerance method, based on sequential probing. Collagen degradation is one of the key events in periodontal destructive lesions. Matrix metalloproteinase (MMP)-8 and MMP-13 are the primary collagenolytic MMPs that are associated with the severity of periodontal inflammation and disease, either by a direct breakdown of the collagenised matrix or by the processing of non-matrix bioactive substrates. Despite the numerous host mediators that have been proposed as potential biomarkers for chronic periodontitis, they reflect inflammation rather than the loss of periodontal attachment. The aim of the present study was to determine the key molecular MMP-8 and -13 interactions in gingival crevicular fluid (GCF) and gingival tissue from progressive periodontitis lesions and MMP-8 null allele mouse model. In study (I), GCF and gingival biopsies from active and inactive sites of chronic periodontitis patients, which were determined clinically by the tolerance method, and healthy GCF were analysed for MMP-13 and tissue inhibitor of matrix metalloproteinases (TIMP)-1. Chronic periodontitis was characterised by increased MMP-13 levels and the active sites showed a tendency of decreased TIMP-1 levels associated with increments of MMP-13 and total protein concentration compared to inactive sites. In study (II), we investigated whether MMP-13 activity was associated with TIMP-1, bone collagen breakdown through ICTP levels, as well as the activation rate of MMP-9 in destructive lesions. The active sites demonstrated increased GCF ICTP levels as well as lowered TIMP-1 detection along with elevated MMP-13 activity. MMP-9 activation rate was enhanced by MMP-13 in diseased gingival tissue. In study (III), we analysed the potential association between the levels, molecular forms, isoenzyme distribution and degree of activation of MMP-8, MMP-14, MPO and the inhibitor TIMP-1 in GCF from periodontitis progressive patients at baseline and after periodontal therapy. A positive correlation was found for MPO/MMP-8 and their levels associated with progression episodes and treatment response. Because MMP-8 is activated by hypochlorous acid in vitro, our results suggested an interaction between the MPO oxidative pathway and MMP-8 activation in GCF. Finally, in study (IV), on the basis of the previous finding that MMP-8-deficient mice showed impaired neutrophil responses and severe alveolar bone loss, we aimed to characterise the detection patterns of LIX/CXCL5, SDF-1/CXCL12 and RANKL in P. gingivalis-induced experimental periodontitis and in the MMP-8-/- murine model. The detection of neutrophil-chemoattractant LIX/CXCL5 was restricted to the oral-periodontal interface and its levels were reduced in infected MMP-8 null mice vs. wild type mice, whereas the detection of SDF-1/CXCL12 and RANKL in periodontal tissues increased in experimentally-induced periodontitis, irrespectively from the genotype. Accordingly, MMP-8 might regulate LIX/CXCL5 levels by undetermined mechanisms, and SDF-1/CXCL12 and RANKL might promote the development and/or progression of periodontitis.

Prevalência do polimorfismo -1031 T>C do gene TNFA em um grupo de diabéticos tipo I e sua relação com a periodontite severa

Diabetes mellitus e doenças periodontais são altamente prevalentes na população mundial. Doenças periodontais (DPs) compreendem um grupo de condições crônicas inflamatórias induzidas por microorganismos que levam à inflamação gengival, à destruição tecidual periodontal e à perda óssea alveolar. Diabetes mellitus (DM) é o termo utilizado para descrever um grupo de desordens metabólicas associadas à intolerância à glicose e ao metabolismo inadequado de carboidratos. Uma vez que DPs poderiam agir de forma similar a outros estados infecciosos sistêmicos, aumentando a severidade do diabetes, uma possível relação entre ambas tem sido considerada em todo o mundo. Polimorfismos genéticos de um único nucleotídeo (SNPs) têm sido estudados em diversas doenças. Nas periodontites, acredita-se que possam estar envolvidos na exacerbação da resposta inflamatória frente ao desafio bacteriano, modificando a susceptibilidade do hospedeiro. Neste estudo, a prevalência de periodontite foi avaliada em portadores de diabetes mellitus tipo I. Posteriormente, o SNP localizado na região promotora do gene TNFA (-1031T>C) foi analisado e sua importância para a doença periodontal destrutiva foi avaliada. O grupo teste foi constituído por diabéticos tipo I (DGT, n=113) enquanto o grupo controle por indivíduos não diabéticos (ND, n=73). Para as análises dos polimorfismos genéticos, um subgrupo foi retirado do grupo teste (DG, n=58) e comparado ao grupo ND. Os seguintes parâmetros clínicos e demográficos foram avaliados: percentual de sítios com profundidade de bolsa  6,0 mm (%PBS6,0 mm); índice gengival (IG); perda óssea radiográfica (POR); fumo; duração do diabetes ; idade; índice de massa corpórea (IMC), n de internações e n de dentes presentes. Amostras de sangue e/ou esfregaço bucal foram colhidas de 58 pacientes do grupo teste e de 73 controles. Após a extração do DNA genômico e amplificação da região genômica de interesse por PCR (Polymerase Chain Reaction), o polimorfismo TNFA 1031T>C foi analisado por BbsI RFLP (Restriction Fragment Length Polymorphism). A análise dos produtos de digestão foi feita por eletroforese em gel de poliacrilamida 8%. A análise estatística das freqüências alélica e genotípica juntamente com os dados clínicos e epidemiológicos entre os 2 grupos foi feita através do teste do Mann-Whitney e do Qui-quadrado. Os grupos de estudo obedecem ao princípio de Hardy-Weinberg. No grupo ND, as seguintes freqüências genotípicas foram encontradas: 78,1% (T/T); 20,5% (T/C) e 1,4% (C/C) enquanto no grupo D foram: 42,4%(T/T); 37,3% (T/C) e 20,3% (C/C). A frequência do alelo T no grupo diabético (D) foi de 0,610 ao passo que no grupo ND foi de 0,883. Não foi possível encontrar uma relação entre o polimorfismo -1031 T>C do gene TNFA e a presença de periodontite em diabéticos tipo I. Entretanto, o polimorfismo estudado se mostrou significativamente relacionado (p<0,0001 e OR= 4.85 95%IC 2,271-10,338) à presença do diabetes tipo I.

CCR5 Mediates Pro-osteoclastic and Osteoclastogenic Leukocyte Chemoattraction

Periodontal disease (PD) progression involves the selective leukocyte infiltration into periodontium, supposedly mediated by the chemokine/chemokine receptor system. In this study, we investigated the role of chemokine receptor CCR5 in the immunoregulation of experimental PD in C57BL/6 (WT) and CCR5KO mice. Aggregatibacter actinomycetem comitans infection triggered the chemoattraction of distinct CCR5+ leukocyte subpopulations (determined by flow cytometry): CCR5+F4/80+ leukocytes, which co-express CD14, CCR2, TNF-alpha, and IL-1 beta, indicative of activated macrophages; and CCR5+CD4+ cells, which co-express CXCR3, IFN-gamma, and RANKL, indicative of Th1 lymphocytes, therefore comprising pro-osteoclastic and osteoclastogenic cell subsets, respectively. CCR5KO mice presented a lower PD severity (lower inflammation and alveolar bone loss) when compared with the WT strain, since the migration of F4/80+, TNF-alpha+, CD4+, and RANKL+ cells specifically decreased due to the lack of CCR5. Also, ELISA analysis demonstrated that the production of TNF-alpha, IL-1 beta, IL-6, IFN-gamma, and RANKL in periodontal tissues was significantly decreased in the CCR5KO strain. The periodontal bacterial load and antimicrobial patterns were unaltered in CCR5KO mice. Our results demonstrate that the chemokine receptor is involved in the migration of distinct leukocyte subpopulations throughout experimental PD, being a potential target for therapeutic intervention in PD.

Influence of photodynamic therapy on the development of ligature-induced periodontitis in rats

Background: the purpose of this study was to evaluate, histologically and radiographically, the effect of photodynamic therapy on the progression of experimentally induced periodontal disease in rats.Methods: Ligatures were placed at the first mandibular molar in rats. The animals were divided into four groups: group 1 (C) received no treatment; group 2 was treated topically with methylene blue (MB; 100 mu g/ml); group 3 was treated with low-level laser therapy (LLLT); and group 4 was treated topically with methylene blue followed by LLLT (4.5 J/cm(2)) (photodynamic therapy; PDT). Rats were sacrificed 5, 15, or 30 days postoperatively. Standardized radiographs were taken to measure bone loss around the mesial root surface of the first molar. Data were analyzed statistically (analysis of variance and Tukey test; P < 0.05). A scoring system was used to evaluate the connective tissue, periodontal ligament, and alveolar bone histologically. Data were analyzed statistically (Kruskal-Wallis test; P < 0.05).Results: Radiographic examination showed that there was significantly less bone loss in Group PDT compared to Group C at 5 and 15 days postoperatively. There was no significant difference in bone loss at 30 days. At 15 days, the histologic results showed significant differences in the extent of inflammatory reaction in the gingival tissue, with a greater extent of chronic inflammatory reaction in Group LLLT.Conclusion: PDT transiently reduced the periodontal tissue destruction.

HMGB1 Localization during Experimental Periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Elevated micronucleus frequency in patients with type 2 diabetes, dyslipidemia and periodontitis

The over-production of reactive oxygen species (ROS) can cause oxidative damage to a large number of molecules, including DNA, and has been associated with the pathogenesis of several disorders, such as diabetes mellitus (DM), dyslipidemia and periodontitis (PD). We hypothesise that the presence of these diseases could proportionally increase the DNA damage. The aim of this study was to assess the micronucleus frequency (MNF), as a biomarker for DNA damage, in individuals with type 2 DM, dyslipidemia and PD. One hundred and fifty patients were divided into five groups based upon diabetic, dyslipidemic and periodontal status (Group 1 - poor controlled DM with dyslipidemia and PD; Group 2 - well-controlled DM with dyslipidemia and PD; Group 3 - without DM with dyslipidemia and PD; Group 4 - without DM, without dyslipidemia and with PD; and Group 5 - without DM, dyslipidemia and PD). Blood analyses were carried out for fasting plasma glucose, HbA1c and lipid profile. Periodontal examinations were performed, and venous blood was collected and processed for micronucleus (MN) assay. The frequency of micronuclei was evaluated by cell culture cytokinesis-block MN assay. The general characteristics of each group were described by the mean and standard deviation and the data were submitted to the Mann-Whitney, Kruskal-Wallis, Multiple Logistic Regression and Spearman tests. The Groups 1, 2 and 3 were similarly dyslipidemic presenting increased levels of total cholesterol, low density lipoprotein cholesterol and triglycerides. Periodontal tissue destruction and local inflammation were significantly more severe in diabetics, particularly in Group 1. Frequency of bi-nucleated cells with MN and MNF, as well as nucleoplasmic bridges, were significantly higher for poor controlled diabetics with dyslipidemia and PD in comparison with those systemically healthy, even after adjusting for age, and considering Bonferroni's correction. Elevated frequency of micronuclei was found in patients affected by type 2 diabetes, dyslipidemia and PD. This result suggests that these three pathologies occurring simultaneously promote an additional role to produce DNA impairment. In addition, the micronuclei assay was useful as a biomarker for DNA damage in individuals with chronic degenerative diseases.

Periodontite ulcerativa necrosante: tratamento fisico da superficie radicular com Er YAG laser

Necrotizing ulcerative periodontitis is associated with bacteria and predisposing risk factors. The treatment is centered in elimination and/or control of the bacteria and predisposing risk factors. However the disease acute phase could induce sequelae as periodontal tissue destruction which may be treated by periodontal surgical procedures.

Role of periodontal pathogenic bacteria in RANKL-mediated bone destruction in periodontal disease

Accumulated lines of evidence suggest that hyperimmune responses to periodontal bacteria result in the destruction of periodontal connective tissue and alveolar bone. The etiological roles of periodontal bacteria in the onset and progression of periodontal disease (PD) are well documented. However, the mechanism underlying the engagement of periodontal bacteria in RANKL-mediated alveolar bone resorption remains unclear. Therefore, this review article addresses three critical subjects. First, we discuss earlier studies of immune intervention, ultimately leading to the identification of bacteria-reactive lymphocytes as the cellular source of osteoclast-induction factor lymphokine (now called RANKL) in the context of periodontal bone resorption. Next, we consider (1) the effects of periodontal bacteria on RANKL production from a variety of adaptive immune effector cells, as well as fibroblasts, in inflamed periodontal tissue and (2) the bifunctional roles (upregulation vs. downregulation) of LPS produced from periodontal bacteria in a RANKL-induced osteoclast-signal pathway. Future studies in these two areas could lead to new therapeutic approaches for the management of PD by down-modulating RANKL production and/or RANKL-mediated osteoclastogenesis in the context of host immune responses against periodontal pathogenic bacteria. © 2010 Mikihito Kajiya et al.

Silicate-based bioceramics for periodontal regeneration

Periodontal disease is characterized by the destruction of the tissues that attach the tooth to the alveolar bone. Various methods for regenerative periodontal therapy including the use of barrier membranes, bone replacement grafts, and growth factor delivery have been investigated; however, true regeneration of periodontal tissue is still a significant challenge to scientists and clinicians. The focus on periodontal tissue engineering has shifted from attempting to recreate tissue replacements/constructs to the development of biomaterials that incorporate and release regulatory signals to achieve in situ periodontal regeneration. The release of ions and molecular cues from biomaterials may help to unlock latent regenerative potential in the body by regulating cell proliferation and differentiation towards different lineages (e.g. osteoblasts and cementoblasts). Silicate-based bioactive materials, including bioactive silicate glasses and ceramics, have become the materials of choice for periodontal regeneration, due to their favourable osteoconductivity and bioactivity. This article will focus on the most recent advances in the in vitro and in vivo biological application of silicate-based ceramics, specifically as it relates to periodontal tissue engineering.

The scientific basis and development of a matrix metalloproteinase (MMP) -8 specific chair-side test for monitoring of periodontal health and disease from gingival crevicular fluid

Matrix metalloproteinase (MMP) -8, collagenase-2, is a key mediator of irreversible tissue destruction in chronic periodontitis and detectable in gingival crevicular fluid (GCF). MMP-8 mostly originates from neutrophil leukocytes, the first line of defence cells which exist abundantly in GCF, especially in inflammation. MMP-8 is capable of degrading almost all extra-cellular matrix and basement membrane components and is especially efficient against type I collagen. Thus the expression of MMP-8 in GCF could be valuable in monitoring the activity of periodontitis and possibly offers a diagnostic means to predict progression of periodontitis. In this study the value of MMP-8 detection from GCF in monitoring of periodontal health and disease was evaluated with special reference to its ability to differentiate periodontal health and different disease states of the periodontium and to recognise the progression of periodontitis, i.e. active sites. For chair-side detection of MMP-8 from the GCF or peri-implant sulcus fluid (PISF) samples, a dip-stick test based on immunochromatography involving two monoclonal antibodies was developed. The immunoassay for the detection of MMP-8 from GCF was found to be more suitable for monitoring of periodontitis than detection of GCF elastase concentration or activity. Periodontally healthy subjects and individuals suffering of gingivitis or of periodontitis could be differentiated by means of GCF MMP-8 levels and dipstick testing when the positive threshold value of the MMP-8 chair-side test was set at 1000 µg/l. MMP-8 dipstick test results from periodontally healthy and from subjects with gingivitis were mainly negative while periodontitis patients sites with deep pockets ( 5 mm) and which were bleeding on probing were most often test positive. Periodontitis patients GCF MMP-8 levels decreased with hygiene phase periodontal treatment (scaling and root planing, SRP) and even reduced during the three month maintenance phase. A decrease in GCF MMP-8 levels could be monitored with the MMP-8 test. Agreement between the test stick and the quantitative assay was very good (κ = 0.81) and the test provided a baseline sensitivity of 0.83 and specificity of 0.96. During the 12-month longitudinal maintenance phase, periodontitis patients progressing sites (sites with an increase in attachment loss ≥ 2 mm during the maintenance phase) had elevated GCF MMP-8 levels compared with stable sites. General mean MMP-8 concentrations in smokers (S) sites were lower than in non-smokers (NS) sites but in progressing S and NS sites concentrations were at an equal level. Sites with exceptionally and repeatedly elevated MMP-8 concentrations during the maintenance phase were clustered in smoking patients with poor response to SRP (refractory patients). These sites especially were identified by the MMP-8 test. Subgingival plaque samples from periodontitis patients deep periodontal pockets were examined by polymerase chain reaction (PCR) to find out if periodontal lesions may serve as a niche for Chlamydia pneumoniae. Findings were compared with the clinical periodontal parameters and GCF MMP-8 levels to determine the correlation with periodontal status. Traces of C. pneumoniae were identified from one periodontitis patient s pooled subgingival plaque sample by means of PCR. After periodontal treatment (SRP) the sample was negative for C. pneumoniae. Clinical parameters or biomarkers (MMP-8) of the patient with the positive C. pneumoniae finding did not differ from other study patients. In this study it was concluded that MMP-8 concentrations in GCF of sites from periodontally healthy individuals, subjects with gingivitis or with periodontitis are at different levels. The cut-off value of the developed MMP-8 test is at an optimal level to differentiate between these conditions and can possibly be utilised in identification of individuals at the risk of the transition of gingivitis to periodontitis. In periodontitis patients, repeatedly elevated GCF MMP-8 concentrations may indicate sites at risk of progression of periodontitis as well as patients with poor response to conventional periodontal treatment (SRP). This can be monitored by MMP-8 testing. Despite the lower mean GCF MMP-8 concentrations in smokers, a fraction of smokers sites expressed very high MMP-8 concentrations together with enhanced periodontal activity and could be identified with MMP-8 specific chair-side test. Deep periodontal lesions may be niches for non-periodontopathogenic micro-organisms with systemic effects like C. pneumoniae and possibly play a role in the transmission from one subject to another.

Análise imuno-histoquímica das proteínas VEGF e HIF-1α na doença periodontal

Periodontal disease is a complex inflammatory and infectious condition that immune host, front of the microbial aggressions, can lead to disease progression, resulting in tissue destruction. The tissue damage induces the release of regulatory molecules, which protective roles and / or destructive, including proteins VEGF (vascular endothelial growth factor vascular) and HIF-1 α (hypoxia-induced factor α -1). Thus, this study investigated, quantitatively and comparatively, the immunohistochemical expression of VEGF (vascular endothelial growth factor) and HIF-1 α (hypoxia induced factor 1-α), proteins involved in inflammation, angiogenesis and hypoxia, in human gingival tissues. Therefore, 75 samples of gingival tissues were examined. Thirty samples were chronic periodontitis, 30 with chronic gingivitis and 15 healthy gingival. After sections analysis, positives and negatives inflammatory and endothelial cells in the connective tissue were counted and converted into percentage. Data were statistically analyzed using Kruskal-Wallis test and Spearman correlation. The results showed that both proteins marked. It was observed higher immunoreactivity for HIF-1 α at chronic gingivitis and periodontitis specimens compared to healthy sites, however, no statistically significant differences were observed among them (p>0.05). The VEGF immunostaining showed similarity among the cases of periodontitis, gingivitis and healthy gingiva. Moderate and positive correlation statistically significant differences were verified for the expressions of VEGF and HIF-1α in gingival health (r = 0,529, p = 0.04). Thus, it can be conclude that possibly the route of HIF-1 α, is activated in periodontal disease may have involvement of the protein in pathogenesis and progression of disease, and that activation of VEGF, can be in addition to being triggered transcription by HIF-1 α may be also influenced by other additional factors such as the action of periodontal microorganisms endotoxins