Effect parathyroid hormone administration on dentin formation in mice

Orientador: Marcelo Rocha MarquesDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: A dentina, assim como o osso, é um tecido mineralizado de natureza conjuntiva. Embora já tenha sido relatado que o hormônio paratireóideo (PTH), um dos reguladores da homeostasia do cálcio, participe do processo de formação de dentina, a maioria dos trabalhos envolvendo este hormônio é relacionado ao tecido ósseo. Tal hormônio, que fisiologicamente promove a reabsorção óssea, quando administrado de forma intermitente, pode promover anabolismo ósseo, sendo hoje um dos tratamentos para osteoporose. Por serem ainda pouco conhecidas as funções do PTH na dentinogênese, o objetivo deste estudo foi investigar os efeitos da administração intermitente de PTH sobre a taxa de aposição e características estruturais da dentina em incisivos de camundongos. Para tanto, camundongos A/J Unib foram divididos em quatro grupos: C6, T6, C10 e T10 (n=10). Os animais dos grupos T6 e T10 receberam injeções subcutâneas de 40µg/Kg de hPTH(1-34), durante 6 e 10 dias respectivamente; os dos grupos C6 e C10 receberam injeções subcutâneas do veículo do PTH, também durante 6 e 10 dias respectivamente. Para delimitação da dentina formada no período experimental, os animais dos grupos T6 e C6 receberam injeções intraperitoneais de dois marcadores fluorescentes. Ao término do período experimental, o sangue dos animais dos grupos C6 e T6 foram coletados para quantificação de fosfatase alcalina e, suas hemimandíbulas esquerdas foram removidas para medição das taxas de aposição dentinária. Os animais dos grupos T10 e C10 foram sacrificados e as hemimandíbulas, direita e esquerda, foram processadas para os testes de microdureza knoop da dentina e conteúdo elementar (% de átomos) de cálcio (Ca), fósforo (P), oxigênio (O) e magnésio (Mg) na dentina peritubular e na dentina intertubular por meio da microanálise de energia dispersiva de raios-X (EDX). Após análise estatística pelo teste T de student, verificou-se um aumento significativo de 5% na taxa de aposição de dentina e a análise enzimática detectou um aumento significativo de 25% de fosfatase alcalina no grupo T6 em relação ao grupo C6. Os animais do grupo T10 apresentaram maior (11%) microdureza na dentina do que os animais do grupo C10. A microanálise por EDX demonstrou que o tratamento com PTH (T10) levou ao aumento do conteúdo (% de átomos) de P (23%) e Ca (53%), bem como da relação Ca/P (24%) na dentina peritubular, em relação ao grupo C10 (p<0.01). Estes resultados indicam que a administração intermitente de PTH, teve um efeito anabólico na formação de dentina em camundongos, acompanhado de significativas alterações estruturais na dentina formadaAbstract: Parathyroid hormone (PTH) has been widely studied, especially in the treatment of osteoporosis, since when administered intermittently promotes bone anabolism. Dentin is a mineralized tissue that share certain similarities to the bone. owever, the role of this hormone on dentin formation is poorly known. The purpose f this study was to investigate the effects of intermittent PTH administration on the apposition rate and structural features of dentin from mouse incisors. Forty young male mice A / J Unib were divided into four groups (n = 10): T6 and T10 = animals received subcutaneous injections of 40µg/Kg of hPTH (1-34) diluted in 0.01% acetic acid for 6 and 10 days respectively; C6 and C10 = animals received subcutaneous injections of PTH vehicle, also for 6 and 10 days, which served as control. The animals of C6 and T6 groups received intraperitoneal injections of fluorescent markers for delimitation of dentin formed during the experimental period. Dentin apposition rates, measured by fluorescence microscopy, and the detection of alkaline phosphatase (ALP) plasma levels were evaluated in the animals of T6 and C6 groups. Knoop microhardness testing and element content measurements in atom % of calcium (Ca), phosphorus (P), oxygen (O), and magnesium (Mg) in the peritubular and intertubular dentin by Energy Dispersive ray (EDX) microanalysis via Scanning Electron Microscopy (SEM) were performed in the animals of T10 and C10 groups. Statistical analysis of all tests was completed using unpaired Student's t test (two-tailed). Statistical significance limit was set at 5%, (p<0.05). Histometrical analysis by fluorecence microscopy showed that the animals of T6 group had a significant increase of 5% in the dentin apposition rate when compared with the control animals (C6). In the T6 group, ALP plasma levels were 25% higher than those in the C6 group. The results obtained from the knoop microhardness testing, demonstrated that the animals of T10 group showed greater microhardness than did the control animals (C10) (10%). In addition, EDX microanalysis showed that the P (23%) and Ca (53%) atom % content in the peritubular dentin was increased in the T10 group, compared with C10 group. The Ca/P ratio of T10 animals was also higher than the C10 animals (24%). The chemical composition of intertubular dentin did not differ between the groups. These results indicate that the short-time PTH administration had an anabolic effect on the dentin formation of incisor teeth of young healthy mice; this effect was following by mechanical and composition changes in the dentinMestradoHistologia e EmbriologiaMestre em Biologia Buco-Denta

Efeito do hormônio paratireóideo (PTH) em linhagem de odontoblastos e na dentinogênese terciária em camundongos

Orientador: Marcelo Rocha MarquesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: O hormônio paratireóideo (PTH) é o principal regulador da homeostasia dos íons minerais, cálcio e fosfato, no organismo, exercendo um papel chave no desenvolvimento e homeostase dos tecidos mineralizados. Recentemente nós demonstramos que durante a formação do incisivo de camundongos, a administração intermitente de PTH causou um aumento da taxa de aposição dentinária e resultou em mudanças nas propriedades mecânicas e materiais da dentina formada. Com isso, os estudos apresentados aqui se propuseram a investigar: 1) Os efeitos da exposição transiente (1 ou 24h/ciclo) ou contínua (48h) ao hPTH(1-34) na deposição mineral, na expressão gênica de Fosfatase Alcalina (ALP), Metaloproteinase 2 (MMP2), Biglican (BGN) e Colágeno tipo I (COL1), e na atividade de MMP2 e ALP em odontoblastos MDPC-23; 2) A participação das vias de sinalização dependentes de proteína quinase A (PKA) e proteína quinase C (PKC) na resposta proliferativa e apoptótica de odontoblastos MDPC-23 a diferentes tempos de exposição ao hPTH(1-34) (1, 24 ou 48 horas); 3) O efeito do tratamento do hPTH(1-34) na espessura dos cristais minerais formados durante a odontogênese de incisivos de camundongos. 4) Os efeitos da administração intermitente de hPTH(1-34) na dentinogênese terciária em camundongos. Os resultados mostram que a exposição transiente ao PTH diminuiu a deposição mineral e atividade de ALP. Já as expressões gênicas de ALP, BGN e COL1 estavam aumentadas no grupo 24h/ciclo, enquanto que o grupo Contínuo apresentou um aumento da expressão gênica de BGN e COL1. Também, os níveis de MMP2 secretados estavam aumentados no grupo 1h/ciclo e diminuídos no grupo Contínuo. A exposição ao PTH modula a resposta proliferativa e apoptótica de odontoblastos MDPC-23 de uma maneira dependente do tempo. Além disso, a via PKA atua na resposta a curta exposição ao PTH (1h), mantendo os níveis de proliferação e apoptose das células, enquanto que a via PKC é ativada nos tempos mais longos de exposição (24 e 48hs) levando a um aumento da apoptose e redução da proliferação celular. A administração intermitente de PTH não alterou a espessura dos cristais minerais da dentina de incisivos em formação e nem a quantidade de dentina terciária reacional em molares de camundongos. No entanto, o tratamento com PTH aumentou a concentração de zinco e diminuiu a concentração de cálcio na dentina de molares normais e em molares com indução de dentinogênese terciária. Com isso, pode-se concluir que o hPTH (1-34) modula a mineralização, apoptose e proliferação de odontoblastos MDPC-23 de maneira dependente do tempo, e que as vias de sinalização dependentes de PKA e PKC participam da resposta proliferativa e apoptótica. Além disso, nos modelos utilizados, a administração intermitente de PTH não teve efeito na espessura de cristais da dentina de incisivos, e também não demonstrou ser capaz de aumentar a formação de dentina terciária reacional em molares de camundongosAbstract: Parathyroid hormone (PTH) is the major regulator of the calcium and phosphate ions in and plays a key role in the development and homeostasis of mineralized tissues. Recently we demonstrated that during the formation of the incisor of mice, intermittent PTH administration caused an increase of dentin apposition rate and it was associate to changes in the composition and in the mechanical properties of the formed dentin. Thus, the studies presented here were designed to investigate: 1) The effects of transient exposure (1 or 24h/cycle) or continuous (48h) to hPTH (1-34) in the mineral deposition and in the gene expression of alkaline phosphatase (ALP), metalloproteinase 2 (MMP2), Biglican (BGN) and type I collagen (COL1), and activity of MMP2 and ALP in odontoblast-like cells (MDPC-23); 2) The involvement of signaling pathways dependent of protein kinase A (PKA) and protein kinase C (PKC) in the proliferative and apoptotic response of MDPC-23 at different times of exposure to hPTH (1-34) (1, 24 or 48 hours); 3) The effect of the hPTH (1-34) treatment on the thickness of mineral crystals formed during dentinogenesis of the mice incisors. 4) The effects of intermittent hPTH (1-34) administration on the tertiary dentinogenesis in mice molars. The results showed that the transient exposure to PTH decreased the mineral deposition and ALP activity. The gene expressions of ALP, COL1 and BGN were higher in the group treated with PTH for 24h/cycle, while the PTH continuous administration group (48h) showed an increase in gene expression of COL1 and BGN. Also, the levels of secreted MMP2 were increased in 1h/cycle and decreased in the Continuous group (48h). The exposure to PTH modulates apoptotic and proliferative response of MDPC-23 in a time-dependent manner. Furthermore, the PKA pathway operates in response to short-term exposure to PTH (1h), maintaining levels of proliferation cell and apoptosis, whereas the PKC pathway is activated in longer exposure times (24 and 48h) leading to an increase apoptosis and decreased cell proliferation. The intermittent PTH administration did not change the thickness of the dentin mineral crystals of the incisors mice, and did not alter the deposition volume of the reactional tertiary dentin in the mice molars. However, treatment with PTH increased the concentration of zinc and decreased calcium concentration in normal dentin of molars with or without induction of tertiary dentinogenesis. Thus, it can be concluded that hPTH (1-34) modulates the mineral deposition, proliferation and apoptosis of the odontoblasts MDPC-23 in a time-dependent manner, and the signaling pathways PKA and PKC dependent participate of that proliferative and apoptotic response. Furthermore, in the models used here, the intermittent administration of PTH did not affect the thickness of the incisor dentinal crystals, and also not shown to be able to enhance the formation of reactional tertiary dentin in molar miceDoutoradoHistologia e EmbriologiaDoutor em Biologia Buco-Denta

Effect of ultrasound and dexpanthenol on collagen organization in tegumentary lesions

OBJECTIVE: To analyze the effect of ultrasound (US), dexpanthenol (d-P) and a combination of these treatments (US+d-P) on collagen fiber organization in tegumentary lesions in rats by birefringence analysis. METHODS: Wistar rats (50) were anesthetized (Thionembutal - Sodic = 50mg/Kg), 1cm² of dorsal region skin was removed, and the animals were divided into five groups: control (C), gel (G), US (3 MHz, 0.1 W/cm2, 1 minute, continuous), d-P (10%) and US+d-P. After daily treatment for 7 and 14 days, 6µm thick sections of lesioned areas were stained in picrosirius and measurements of the collagen birefringent area (µm²) were obtained using polarized light microscopy (Zeiss Axiolab-ZEISS- Germany) with histological image analysis software (KS 400 2.0 - Kontrol Eletronics, Munique, Germany). The means were compared by ANOVA followed by the Tukey test (p<0.05). RESULTS: The US+d-P group showed a significantly greater (p<0.001) birefringent area (1586.43±162.14) than the other experimental groups: C (139.36±35.35), US (317.55±129.9) and d-P (192.41±3657) by the 7th day of treatment, indicating acceleration of the wound healing process. By the 14th day of treatment, the US+d-P, US and d-P groups presented greater birefringence than the control group, but did not differ from each other. CONCLUSION: The combination of treatments (US+d-P) accelerated collagen fiber synthesis and organization in the early stages of cutaneous repair.OBJETIVO: Analisar o efeito do ultrassom (US), do dexapantenol (d-P) e da associação dos tratamentos (US+d-P) na organização de fibras colágenas na lesão tegumentar em ratos por meio da análise da birrefringência. MÉTODOS: Foram utilizados 50 ratos Wistar, anestesiados com Thionembutal Sódico (50mg/Kg), dos quais foi retirado 1cm² de pele da região dorsal, divididos em cinco grupos: controle (C), gel (G), US (3 MHz, 0,1 W/cm², 1 minuto, modo contínuo), d-P (10%) e US+d-P. Após sete e 14 dias de tratamento diário, foram removidos segmentos dessas áreas e obtidos cortes de 6µm de espessura que, posteriormente, foram corados em Picrosirius. Os cortes foram observados em microscopia de polarização utilizando um software responsável pela medida de birrefringência das fibras colágenas (KS400 2.0 - Kontrol Eletronics). As médias das áreas birrefringentes (µm²) de cada grupo foram submetidas à análise de variância pela ANOVA, seguida do teste de Tukey (p<0,05). RESULTADOS: A média de área birrefringente do grupo US+d-P (1586,43±162,14) foi maior (p<0,001) que a dos grupos experimentais (C: 139,36±35,35, US: 317,55±129,9 e d-P: 192,41±36,57) no 7º dia de tratamento, indicando uma aceleração na síntese e organização das fibras colágenas na região lesionada. No 14º dia de tratamento, os grupos US+d-P (2858,47±510,17), US (1779,94±482,78) e d-P (2546,88±304,45) apresentaram área birrefringente maior que a do grupo C, porém não diferiram entre si. CONCLUSÃO: A associação dos tratamentos (US+d-P) acelerou a síntese e a organização das fibras colágenas apenas no estágio inicial de reparo tegumentar.Universidade Metodista de Piracicaba Faculdade de Ciências da SaúdeUniversidade Estadual de Campinas Faculdade de Odontologia de PiracicabaUniversidade Federal de São Paulo (UNIFESP) Farmácia e BioquímicaUNIARARASUNIFESP, Farmácia e BioquímicaSciEL

Dinâmica do uso das terras no Cerrado no período de 2002 a 2013

The objective of this work was to analyze land use dynamics in the Brazilian Cerrado region from 2002 to 2013. This analysis was based on the interpretation of Landsat satellite images carried out by the projects Projeto de Conservação e Utilização Sustentável da Diversidade Biológica Brasileira (Probio) and TerraClass Cerrado 2013, both coordinated by Ministério do Meio Ambiente. In 2002, 38.9% of the Cerrado was covered by some type of anthropic activity. In 2013, this percentage increased to 43.4%. One of the main highlights is the emergence of a new agricultural frontier in the northern region of the study area, known as Matopiba.O objetivo deste trabalho foi analisar a dinâmica de uso das terras na região do Cerrado de 2002 a 2013. Essa análise foi baseada nas interpretações de imagens do satélite Landsat realizadas pelo Projeto de Conservação e Utilização Sustentável da Diversidade Biológica Brasileira (Probio) e pelo projeto TerraClass Cerrado 2013, coordenados pelo Ministério do Meio Ambiente. Em 2002, 38,9% do Cerrado eram cobertos por algum tipo de atividade antrópica. Em 2013, essa percentagem passou para 43,4%. Um dos grandes destaques é o surgimento de uma nova fronteira agrícola na região norte da área de estudo, conhecida como Matopiba

Porphyromonas gingivalis LPS stimulation downregulates DNMT1, DNMT3a, and JMJD3 gene expression levels in human HaCaT keratinocytes

Objective: The role of epigenetic regulation in inflammatory diseases such as periodontitis is poorly known. The aim of this study was to assess whether Porphyromonas gingivalis lipopolysaccharide (LPS) can modulate gene expression levels of the some enzymes that promote epigenetic events in cultures of the human keratinocytes and gingival fibroblasts. In addition, the same enzymes were evaluated in gingival samples from healthy and periodontitis-affected individuals. Materials and methods: Primary gingival fibroblast and keratinocyte (HaCaT) cultures were treated with medium containing P. gingivalis LPS or P. gingivalis LPS vehicle for 24 h. After this period, cell viability was assessed by MTT test and total RNA extracted to evaluate gene expression levels of the following enzymes by qRT-PCR: DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), histone demethylases Jumonji domain containing 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX). To evaluate gene expression in healthy and periodontitis-affected individuals, total RNA was extracted from biopsies of gingival tissue from healthy and periodontitis sites, and gene expression of DNMT1, DNAMT3a, JMJD3, and UTX was evaluated by qRT-PCR. Results: No significant differences were found in the gene expression analysis between healthy and periodontitis-affected gingival samples. The results showed that LPS downregulated DNMT1 (p < 0. 05), DNMT3a (p < 0. 05), and JMJD3 (p < 0. 01) gene expression in HaCaT cells, but no modulation was observed in gingival fibroblasts. Conclusion: P. gingivalis LPS exposure to human HaCaT keratinocytes downregulates gene expression of the enzymes that promote epigenetic events. Clinical relevance: The advance knowledge about epigenetic modifications caused by periodontopathogens may to possibly led to the development of new periodontal therapies. © 2012 Springer-Verlag

Development, physical-chemical stability and rheological behavior of silicones formulations containing Dimethylaminoethanol (DMAE)

The aim of this paper was to develop formulations increased of DMAE and evaluate their physical-chemical stability and rheological behavior. Eleven formulations containing 3% DMAE pidolate or 3% DMAE acetamidobenzoate were developed and both preliminary stabilities tests and rheological measurements were carried out. They were considered stable during all period of study. The type of DMAE did not modify the viscosity of the emulsion and all presented pseudoplastic behavior with hysteresis area. An increase of hysteresis area could be observed with DMAE addition. The results point that the type of DMAE can influence the physical stability of the final product

Addition of hydrogen peroxide to methylene blue conjugated to β-cyclodextrin in photodynamic antimicrobial chemotherapy in S. mutans biofilm

This study evaluated the effect of hydrogen peroxide addition on β-cyclodextrin-conjugated methylene blue in antimicrobial photodynamic therapy(a-PDT) in S. mutans biofilm model using laser or light emitting diode (LED) (λ = 660 nm). A preliminary assay was performed to evaluate the cytotoxicity of hydrogen peroxide in oral fibroblasts by the colorimetric method (MTT). Afterwards, groups were divided into (n = 3, in triplicate): C (negative control), CX – chlorhexidine 0.2% (positive control), P (methylene blue/β-cyclodextrin), H (Hydrogen Peroxide at 40 μM), PH, L (Laser), LP, LH (Laser+Hydrogen Peroxide), LPH, LED, LEDP, LEDH, and LEDPH. The biofilm was formed in 24 h with BHI + 1% sucrose (w/v). Light irradiations were conducted with laser, 9 J, 323 J/cm2, 113 s or with LED, 8.1 J, 8.1 J/cm2 for 90 s. Microbial reduction was evaluated by counting the viable microorganisms of the biofilm after the respective treatments, in a selective culture medium, and laser confocal microscopy evaluation. LP, LH, LPH, LEDP, LEDH, and LEDPH groups statistically reduced the counts of S.mutans compared with the C group and the log reductions were of 1.87, 1.94, 2.19, 0.91, 0.92, and 1.33, respectively; the addition of hydrogen peroxide did not potentiate the microbial reductions (LPH and LEDPH) compared with the LP and LEDP groups. The association of hydrogen peroxide with the conjugated β-cyclodextrin nanoparticle as photosensitizer did not result in an enhanced effect of a-PDT; hydrogen peroxide behaved as a photosensitizer, since it reduced the number of S. mutans when associated with laser light28226233CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP132211/2017-30012017/03263-

Fluoride Effect On The Secretory-stage Enamel Organic Extracellular Matrix Of Mice.

The formation of an ordered enamel organic extracellular matrix (EOECM) seems to be a crucial step for the proper formation of the enamel mineral phase. The ordered supramolecular structure of the EOECM in the secretory stage can be analyzed using polarizing microscopy, as it is strongly birefringent. Excessive fluoride (F) ingestion during tooth development can cause enamel fluorosis, leading to increased porosity in mature enamel. We analyzed the effects of F on the birefringence of the EOECM in the A/J, CBA, and DBA/2 strains of mice given 0, 11.25, and 45 ppm of fluoride in drinking water. In the CBA and DBA/2 strains, the 11.25 and 45 ppmF groups presented a significant decrease in optical retardation (OR) when compared with the respective 0 (CBA 11.25 ppmF p = 0.0056 and 45 ppmF p < 0.0001; DBA/2 11.25 and 45 ppmF p < 0.05). ORs in A/J 0 ppmF were significantly higher than in 45 (p < 0.0001). The enamel of the A/J strain was more severely affected by fluoride than it was in the other strains of mice and exhibited the lowest levels of fluoride in plasma, whereas its normal secretory enamel presented a significantly higher protein absorbance than it did in CBA and DBA mice (p = 0.0099 and p = 0.0025, respectively). The results showed that experimental fluorosis can alter the supramolecular organization of EOECM in the secretory stage of amelogenesis and that the susceptibility to dental fluorosis seems to be influenced by the inherent characteristics of the developing enamel.52212-