Impact of smoking on protein levels of beta-defensins in periodontal disease

Abstract Antimicrobial peptides (AMPs) are important components of the host response against invading pathogens. In addition to their direct antimicrobial activity, they can also participate in the immune system modulation. However, the role of AMPs in the etiopathogenesis of periodontal disease and the risk factors that may influence their expression in the oral cavity are not fully understood. The aim of this study was to determine the impact of smoking on beta-defensin (hBD) 1 and 2 levels analyzing samples from periodontitis patients. Fifty patients with periodontitis, 25 smokers and 25 non-smokers, and 20 periodontally healthy patients were recruited. After periodontal clinical evaluation, gingival crevicular fluid (GCF) samples were collected from healthy sites of patients without periodontal disease and from healthy and diseased sites of patients with periodontitis. Peptides quantification was performed by sandwich ELISA technique. Smokers showed reduced GCF hBD 1 levels and increased hBD 2 levels compared to non-smokers in diseased sites (p <0.05). Higher levels of hBD 1 were observed in healthy sites of patients without periodontal disease than in healthy sites of patients with periodontitis (p<0.0001). Diseased sites of non-smokers presented higher levels of hBD 2 than healthy sites (p <0.05). These results reveal that protein levels of hBDs 1 and 2 can be impaired by cigarette smoking in the presence of periodontal disease.

Resumo Peptídeos antimicrobianos (PAMs) são componentes importantes da resposta do hospedeiro contra patógenos invasores. Além de sua atividade antimicrobiana direta, eles também podem participar da modulação do sistema imunológico. No entanto, o papel dos PAMs na etiopatogenia da doença periodontal e os fatores de risco que podem influenciar a sua expressão na cavidade oral não são totalmente compreendidos. O objetivo deste estudo foi determinar o impacto do tabagismo nos níveis de beta-defensina (hBD) 1 e 2 analisando amostras de pacientes com periodontite. Cinquenta pacientes com periodontite, 25 fumantes e 25 não fumantes e 20 pacientes periodontalmente saudáveis foram recrutados. Após avaliação clínica periodontal, amostras de fluido crevicular gengival (FCG) foram coletadas de sítios saudáveis de pacientes sem doença periodontal e de sítios saudáveis e doentes de pacientes com periodontite. A quantificação dos peptídeos foi realizada pela técnica de ELISA sanduíche. Fumantes apresentaram níveis reduzidos de hBD 1 no FCG e níveis aumentados de hBD 2 em comparação com não fumantes em locais doentes (p <0,05). Níveis mais elevados de hBD 1 foram observados em sítios saudáveis de pacientes sem doença periodontal do que em sítios saudáveis de pacientes com periodontite (p<0,0001). Os sítios doentes de não fumantes apresentaram níveis mais elevados de hBD 2 do que os sítios saudáveis (p<0,05). Esses resultados revelam que os níveis das hBDs 1 e 2 podem ser prejudicados pelo tabagismo na presença de doença periodontal.

Proteolytic activity and degradation of bovine versus human dentin matrices

Abstract Non-human teeth have been commonly used in research as replacements for human teeth, and potential dissimilarities between the dental tissues should be considered when interpreting the outcomes. Objective: To compare the proteolytic activity and degradation rate of bovine and human dentin matrices. Methodology: Dentin beam specimens were obtained from human molars (n=30) and bovine incisors (n=30). The beams were weighed hydrated and after complete dehydration to obtain the mineralized wet and dry masses. Then, the beams were demineralized in 10 wt% phosphoric acid. Next, 15 beams from each substrate were randomly selected and again dehydrated and weighed to obtain the initial demineralized dry mass (DM). Then, the beams were stored in saliva-like buffer solution (SLBS) for 7, 14 and 21 days. SLBS was used to evaluate hydroxyproline (HYP) release after each storage period. The remaining beams of each substrate (n=15) were tested for initial MMP activity using a colorimetric assay and then also stored in SLBS. DM and MMP activity were reassessed after 7, 14 and 21 days of incubation. The data were subjected to two-way ANOVA tests with repeated measures complemented by Bonferroni's tests. Unpaired two-tailed t-tests were also used (p<0.05). Results: Similar water and inorganic fractions were found in human and bovine dentin, while human dentin had a higher protein content. The most intense proteolytic activity and matrix deterioration occurred short after dentin was demineralized. Both substrates exhibited a sharp reduction in MMP activity after seven days of incubation. Although human dentin had higher MMP activity levels, greater HYP release and DM loss after seven days than bovine dentin, after 14 and 21 days, the outcomes were not statistically different. Conclusion: Bovine dentin is a suitable substrate for long-term studies involving the degradation of dentin matrices.

Bioactivity effects of extracellular matrix proteins on apical papilla cells

Abstract Potent signaling agents stimulate and guide pulp tissue regeneration, especially in endodontic treatment of teeth with incomplete root formation. Objective This study evaluated the bioactive properties of low concentrations of extracellular matrix proteins on human apical papilla cells (hAPCs). Methodology Different concentrations (1, 5, and 10 µg/mL) of fibronectin (FN), laminin (LM), and type I collagen (COL) were applied to the bottom of non-treated wells of sterilized 96-well plates. Non-treated and pre-treated wells were used as negative (NC) and positive (PC) controls. After seeding the hAPCs (5×103 cells/well) on the different substrates, we assessed the following parameters: adhesion, proliferation, spreading, total collagen/type I collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1) (ANOVA/Tukey; α=0.05). Results We observed greater attachment potential for cells on the FN substrate, with the effect depending on concentration. Concentrations of 5 and 10 µg/mL of FN yielded the highest cell proliferation, spreading and collagen synthesis values with 10 µg/mL concentration increasing the ITGA5, ITGAV, and COL1A1 expression compared with PC. LM (5 and 10 µg/mL) showed higher bioactivity values than NC, but those were lower than PC, and COL showed no bioactivity at all. Conclusion We conclude that FN at 10 µg/mL concentration exerted the most intense bioactive effects on hAPCs.

Comparação de dados clínicos e imunológicos entre indivíduos geneticamente suscetíveis à periodontite pela presença de um haplótipo no gene da IL-4 e indivíduos geneticamente protegidos contra a periodontite / Comparison of clinical and immunologic data between individuals genetically susceptible to periodontitis and genetically protected against periodontitis

A Doença Periodontal (DP), desencadeada pela infecção de microrganismos periodontopatogênicos tem sua progressão e morbidade grandemente influenciada pela suscetibilidade genética do hospedeiro. Dentre as várias citocinas produzidas pelo periodonto inflamado está a Interleucina 4 (IL-4). Estudos recentes realizados por este grupo demonstraram que os polimorfismos -590(T/A), +33(T/G) e VNTR (Inserção/Deleção) no gene IL4, bem como haplótipos formados por eles, estavam associados à periodontite crônica, mesmo após ajustes para idade, gênero, cor da pele e hábito de fumar. O objetivo do presente estudo foi testar a hipótese de que há diferença nos índices clínicos periodontais e nos níveis de IL-4 no fluido sulcular gengival de pacientes suscetíveis e protegidos, e como segundo objetivo foi avaliar a resposta ao tratamento periodontal não-cirúrgico, em função da referida carga genética. Foram selecionados 62 pacientes que carregavam um haplótipo no gene IL4 que confere suscetibilidade genética à periodontite crônica ou que confere proteção contra o desenvolvimento desta, tendo sido subdivididos quanto à presença ou ausência da periodontite crônica. Cada paciente foi submetido a exame clínico periodontal e coleta de fluido sulcular (FS) antes (baseline), 45 e 90 dias após finalizado o tratamento periodontal não-cirúrgico. A citocina IL-4 foi quantificada por meio de teste imunoenzimático (ELISA). Como resultado verificou-se que, comparando indivíduos geneticamente suscetíveis à DP com os geneticamente protegidos, no período baseline, houve diferença estatisticamente significante dos índices de placa visível, sangramento marginal e sangramento à sondagem considerando tais parâmetros clínicos de boca toda. Não houve diferença significante quanto à resposta ao tratamento periodontal, avaliando os índices clínicos periodontais. A quantidade total e a concentração de IL-4 foi significativamente diferentes entre os indivíduos geneticamente suscetíveis à DP e os geneticamente protegidos, nos períodos de 45 e 90 dias após o tratamento periodontal nãocirúrgico. A presença do haplótipo que confere suscetibilidade genética à DP ou proteção contra o desenvolvimento desta correlacionou-se com determinados parâmetros clínicos periodontais em função da produção de IL-4 ou do volume de FS. Concluiu-se que a suscetibilidade genética à DP conferida pela presença de haplótipos no gene IL4 influenciou no período baseline alguns dos índices clínicos periodontais avaliados, bem como a produção da citocina IL-4 após 45 e 90 dias de finalizado o tratamento periodontal não-cirúrgico. Além disso, a correlação de parâmetros clínicos periodontais com a produção de IL-4 ou com o volume de FS foi significativa em grupos com diferentes cargas genéticas. Futuros estudos in vitro poderão contribuir para compreender como os diferentes haplótipos influenciam a taxa de transcrição e tradução da proteína IL-4, bem como isso poderia ser influenciado pela ação de microrganismos, tal como ocorre na doença periodontal

Periodontal disease (PD) is an encompasses multifactorial disease. Genes belonging to the immune response have been investigated as a potential factor influencing the susceptibility to PD. Among the various cytokines produced by the inflamed periodontium is interleukin 4 (IL-4), which is a potent downregulator of macrophage function that inhibits the secretion of proinflammatory cytokines Previously, we identified a haplotype formed by - 590(T/A), +33(T/G) and VNTR (variable number of tandem repeats; I/D) polymorphisms in the IL4 gene that conferred susceptibility to or protection against PD. The aim of this study was to investigate whether subjects with genetic susceptibility [S] to PD compared to genetically protected [P] individuals would present both differences in the clinical parameters and levels of IL-4 in sulcular fluid, and if these were different 45 and 90 days after non-surgical periodontal treatment. Those patients were subdivided in: genetically susceptible without PD (healthy) (SH, n=16), genetically susceptible with PD (SPD, n=16), genetically protected without PD (PH, n=20), and genetically protected with PD (PPD, n=10). Periodontal clinical examination and collection of periodontal sulcular fluid were performed for each patient at baseline, 45 and 90 days after finishing the non-surgical periodontal treatment. The cytokine IL-4 was measured by the use of Enzyme Immunoassay (ELISA). The results showed that comparing the genetically susceptible with the genetically protected individuals to PD, there were significant differences in clinical parameters: plaque index (p=0.013), bleeding index (p=0.005), bleeding on probing (p=0.015) at baseline. There were no significant differences in clinical parameters 45 and 90 days after completion of the periodontal treatment. The total amount and concentration of IL-4 was different between individuals after non-surgical treatment. The presence of haplotype that conferred susceptibility to or protection against PD was correlated with clinical periodontal parameters based on sulcular fluid production of IL-4. It was concluded that genetic susceptibility to PD conferred by the presence of haplotypes in the gene IL4 influenced at baseline some of the clinical periodontal parameters evaluated, well as the production of the cytokine IL-4 after 45 and 90 days of the nonsurgical finalized periodontal treatment. Furthermore, the correlation of clinical periodontal parameters with the production of IL-4 or with the volume of FS was significant in groups with different genetic load. Future in vitro studies could contribute to understand how different haplotypes could influence the rate of transcription and protein translation of IL-4, as well as how this could be influenced by the action of microorganisms, such as it occurs in the periodontal disease