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Caracterização e avaliação in-vitro de biovidro experimental em microparticulas para o tratamento da hipersensibilidade dentinária cervical / Characterization and In-vitro evaluation of an experimental Microparticulate Bioglass for treatment of cervical dentinalAcevedo, Luisa Fernanda Alegria 22 February 2016 (has links)
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Previous issue date: 2016-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O biovidro tem-se mostrado com um material que por sua bioatividade com organismo pode ser utilizado em diferentes condições clinicas, como é a hipersensibilidade dentinária cervical (HDSC). Os objetivos deste trabalho foram caracterizar as propriedades físicas, químicas, biológicas e redução da
permeabilidade dentinária in vitro, de biovidros experimentais em micropartículas. Os biovidro com formulação 2Na2O.1CaO.3SiO2- 6%P2O5 (Bv1) e suas diferentes composições, 2Na2O.1CaO.3SiO2- 6%P2O5- K2CO3 (Bv2); 2Na2O.1CaO.3SiO2-6%P2O5-SrO (Bv3), sintetizados pelo método de fusão. Os biovidro comerciais 45S5® (Bv4) e o Biosilicato®(Bv5) foram usados como controle. Todos os biovidros foram caraterizados em microscopia eletrônica de varredura por efeito de campo,
espectrofotometria no infravermelho com transformação de Fourier, difração de raio X e espectroscopia de micro-Raman para confirmar sua composição química, a cristalinidade e formato das micropartículas. Nos testes biológicos em cultura de células com fibroblastos 3T3; os biovidros foram colocados em contato com o meio de cultura em 3 concentrações (100, 10 e 1 mg/mL) e foi usado o sobrenadante
como meio de cultura nas placas de 96 poços, avaliou se a proliferação celular em presença dos matérias. O efeito antimicrobiano foi avaliado com as amostras dos patógenos Staphylococcus aureus ATCC® 25923, Escherichia coli ATCC® 25922, Pseudomona aeruginosa ATCC 27853. Foi realizado o teste de microdiluição e avaliou-se a concentração inibitória mínima (CIB) e concentração bactericida mínima (CBM). Na redução da permeabilidade dentinária foi usado dentina bovina. Foi avaliada a permeabilidade mínima, permeabilidade máxima (após aplicação EDTA 24%) e permeabilidade final (após aplicação do materiais). As amostras foram armazenadas em estufa (37ºC) em placas de 24 poços com saliva artificial, sendo submetidas ou não à escovação simulada. Os testes mostraram que todos os
materiais presentavam características compatíveis com o biovidro. Na cultura celular houve uma aumento na proliferação, com uma diferencia significativa (P<0,0001) entre os grupos Bv1–10 e 1 mg/mL, Bv4 – 10 mg/mL e Bv5 – 1 mg/mL. Os biovidro Bv3 apresentou efeito antimicrobiano, no entanto não foi estatisticamente significativo comparado ao controle positivo (clorexidina 0,12%). Todos os materiais
conseguiram reduzir a porcentagem de permeabilidade dentinária (diferença significativa) considerando os fatores grupo (biovidros) e tratamento (com e sem escovação simulada). Conclui-se que os biovidros não foram citotóxicos em baixas concentrações, não apresentaram grande potencial antimicrobiano. Os biovidros apresentaram redução da permeabilidade dentinária. / Bioglass has been shown as a bioactive material, in contact with human body. It can be used in several clinical dental conditions, such as cervical dentine hypersensitivity (CDHS). The objectives of this present study were to characterize the physical, chemical, biological behavior and dentin permeability reduction. The bioglass powder formulation: 2Na2O.1CaO.3SiO2- 6% P2O5 (BV1) and their different compositions,
2Na2O.1CaO.3SiO2- 6% P2O5- K2CO3 (BV2); 2Na2O.1CaO.3SiO2- 6% P2O5-SrO (Bv3) were synthesized by the melt method. Commercial bioglass 45S5® (BV4) and Biosilicate® (BV5) were used as controls positive. All bioactive glasses were characterized by scanning electron microscopy by field effect, infrared spectroscopy with Fourier transform, X-ray diffraction and spectroscopy micro-Raman to confirm
the crystallinity and microstructure and chemical composition. Biocompatibility test: cell culture (3T3 fibroblasts); the bioactive glasses were placed in culture medium in three concentrations (100, 10 and 1 mg/mL) and the supernatant was used to evaluate cell proliferation. The antimicrobial effect was evaluated with Staphylococcus aureus (ATCC® 25923), Escherichia coli (ATCC® 25922), Pseudomonas aeruginosa (ATCC 27853) were tested by microdilution, minimum inhibitory concentration (CIB) and minimum bactericidal concentration (MBC). The dentin permeability reduction was evaluated on bovine dentine in 3 stages: minimum permeability, maximum permeability (after EDTA 24%) and permeability final (after
biomaterial). The samples were stored in 24-well plates (37.5oC) in artificial saliva with and no simulated brushing. The results showed all biomaterials had similar bioglass characteristics. In cell culture there was an increase in proliferation (significant difference between the Bv1-10 groups and 1 mg / ml, BV4 - 10 mg / ml and BV5 - 1 mg / ml. The bioglass Bv3 presented antimicrobial effect not statistically significant compare to the positive control (0.12% chlorhexidine). All biomaterials were promoted dentin permeability reduction, however there was a significant difference considering the group factors (bioactive glasses) and treatment (with and without brushing simulated). Considering the results it can be conclude that bioactive
glasses were not cytotoxic; not show a broad spectrum antimicrobial however should be carried out other methodologies, all experimental bioglasses show dentin permeability reduction.
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Resposta celular e molecular do tecido conjuntivo de camundongos e medicações intracanal / Cellular and molecular response of mice connective tissue to intracanal dressingsMaristela Soares Swerts Pereira 22 March 2012 (has links)
Substâncias contendo clorexidina (CHX) ou associação de antibióticos têm sido pesquisadas como medicações intracanal. Os objetivos do presente estudo foram: Capítulo 1- Caracterizar a resposta do tecido conjuntivo subcutâneo de camundongos à pasta triantibiótica (Trimix), por microscopia óptica convencional e por RT-PCR em tempo real; e Capítulo 2 - Comparar a resposta do tecido conjuntivo subcutâneo de camundongos a medicações intracanal contendo CHX por microscopia óptica convencional. No Capítulo 1, a resposta do tecido conjuntivo subcutâneo de camundongos foi avaliada por meio da implantação de tubos de polietilieno vazios ou contendo uma das substâncias avaliadas: Trimix ou Calen. Como controle adicional, foram utilizados animais que não receberam a implantação dos tubos. Para a avaliação histopatológica, decorridos os períodos experimentais de 7, 21 e 63 dias, os implantes (n=10) contendo Trimix ou Calen foram removidos juntamente com o tecido conjuntivo subcutâneo e a pele adjacente e submetidos ao processamento histotécnico, sendo os cortes corados pelo método de hematoxilina e eosina e picrosírius vermelho. Foram efetuadas análises qualitativa, determinando os parâmetros de resposta biológica e quantitativa, onde foram avaliados o número de células inflamatórias e de vasos, a área e a densidade vascular, além do percentual relativo de colágeno. As reações de RT-PCR em tempo real foram realizadas nos grupos tubo vazio, pasta Calen, Trimix e controle adicional nos períodos experimentais de 7 e 21 dias. Foi realizada a detecção e quantificação das citocinas pró-inflamatórias (IL-1β, TNF-α e IL- 17) e anti-inflamatória (TGF-β), fator crescimento endotelial vascular (VEGF), fator induzido por hipóxia (HIF-1α), metaloproteinases (MMP-2 e -9) e inibidores de metaloproteinases (TIMP-1 e - 2). Os resultados foram comparados empregando teste t de Student e ANOVA, seguida do pósteste de Tukey. No Capítulo 2, foi efetuada a comparação da resposta do tecido conjuntivo subcutâneo de camundongos às pastas Calen+CHX a 0,5%, Calen+CHX a 2,0%, ao gel de CHX a 2,0% e à pasta Calen (controle) utilizando metodologia semelhante à empregada para avaliação histopatológica no Capítulo 1. Os resultados obtidos foram analisados por meio da ANOVA, seguida do pós-teste de Tukey. O nível de significancia adotado em todas as análises estatísticas foi de 5%. Com base nos resultados obtidos, pôde-se concluir que: 1) A resposta do tecido conjuntivo subcutâneo de camundongos à pasta Trimix caracterizou-se por reação inflamatória aguda persistente e ausência de reparo no período estudado de 63 dias, o que foi suportado pela maior expressão gênica dos biomarcadores relacionados à resposta inflamatória e angiogênica, comparado à pasta Calen; 2) Quando comparadas as medicações contendo CHX, os resultados evidenciaram que a Calen+CHX a 0,5% exibiu resposta tecidual reparativa, em contraste com a Calen+CHX a 2,0% e o gel de CHX a 2,0% que propiciaram resposta inflamatória persistente, apontando para agressividade destes materiais. Considerando a Calen+CHX a 2,0% e o gel de CHX a 2,0%, este apresentou resposta inflamatória de maior intensidade. Desse modo, o presente estudo fornece indícios que as pastas Trimix, Calen+CHX a 2,0% e o gel de CHX a 2,0% não deveriam ser empregadas como medicação intracanal. / Substances containing chlorhexidine (CHX) or combination of antibiotics have been investigated as intracanal dressings. The aim of this study were: Chapter 1 - To characterize the response of mice subcutaneous connective tissue to triantibiotic paste (Trimix), by conventional optical microscopy and real-time RT-PCR; and Chapter 2 - Compare the response of mice subcutaneous connective tissue to intracanal dressings containing CHX by conventional optical microscopy. In Chapter 1, the response of mice subcutaneous connective tissue was assessed by implantation of polyethylene tubes empty or containing one of the substances evaluated: Trimix or Calen. As additional control, animals that not received implantation of the tubes were used. For histopathological evaluation, after the experimental periods of 7, 21 and 63 days, the implants (n=10) containing Trimix or Calen were removed along with the subcutaneous connective tissue and adjacent skin and subjected to processing histotechnical, and the sections were stained with hematoxylin and eosin or picrosirius red. It was carried out a qualitative analysis, determining the biological parameters and a quantitative response, assessing the number of inflammatory cells and vessels, the area and vascular density, and the relative percentage of collagen. The real-time RT-PCR reactions were performed in the empty tube group, pastes Calen, Trimix and additional control at the experimental periods of 7 and 21 days. The detection and quantification of proinflammatory (IL-1β, TNF-α and IL-17) and antiinflammatory cytokines (TGF-β), vascular endothelial growth factor (VEGF), hypoxia-induced factor (HIF-1α), metalloproteinases (MMP-2 and -9) and metalloproteinases inhibitors (TIMP-1 and -2) were performed. The results were compared using Student\'s t test and ANOVA followed by Tukey test. In Chapter 2, it was compared the response of mice subcutaneous connective tissue to Calen+0.5% CHX, Calen+2.0% CHX, 2.0% CHX gel and paste Calen (control) using methodology similar to that one used for histopathologic evaluation in Chapter 1. The results were analyzed by ANOVA followed by Tukey test. The significance level for all statistical analysis was 5%. It was concluded: 1) The response of mice subcutaneous connective tissue to Trimix paste was characterized by persistent acute inflammatory reaction with no repair during the studied period of 63 days, which was supported by the higher gene expression of biomarkers related to inflammation and angiogenesis, compared to Calen paste; 2) The Calen+0.5% CHX showed reparative tissue response, in contrast to Calen+2.0% CHX and 2.0% CHX gel that have led to persistent inflammatory response, indicating aggressiveness of these materials. Considering the Calen+2.0% CHX and CHX 2.0% gel, this induced more intense inflammatory response. Thus, this study provides evidence that the pastes Trimix, Calen+2.0% CHX and 2.0% CHX gel should not be used as intracanal dressing.
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Avaliação do peptídeo LL-37 em contato com células-tronco da polpa dentária / Evaluation of LL-37 Peptide in contact with stem cells from dental pulpMilhan, Noala Vicensoto Moreira [UNESP] 16 January 2017 (has links)
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Previous issue date: 2017-01-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O peptídeo LL-37 (catelicidina derivada de humano), é liberado por algumas células humanas e capaz de neutralizar os tecidos com lipopolissacarídeo (LPS), além de atrair células da polpa, e induzir a angiogênese, características que o tornam um possível adjunto para a regeneração do complexo dentino-pulpar. O objetivo desse trabalho foi avaliar in vitro a biocompatibilidade do peptídeo LL-37 nas concentrações de 5 e 10 μg/mL, e sua possível atuação na diferenciação de células-tronco da polpa dentária (DPSC) para odontoblastos- like. Com esse propósito, foram avaliados: (a) a citotoxicidade, pelo teste MTT; (b) a genotoxicidade, através do ensaio do micronúcleo; (c) a produção e quantificação de óxido nítrico; (d) as fases do ciclo celular, por citometria; (e) a expressão de alguns genes associados à formação de tecido mineralizado, através do teste qRT-PCR; (f) o conteúdo de proteína total; (g) a atividade de fosfatase alcalina (ALP); e (h) a produção de sialofosfoproteína dentinária (DSPP), pelo ensaio imunoenzimático ELISA. Foi observado que as concentrações de 5 e 10 μg/mL de LL-37 não foram citotóxicas e ainda aumentaram, em geral, a viabilidade celular (p<0,05), sendo que os maiores valores de absorbância foram observados no 3° dia de contato. As concentrações testadas também não induziram genotoxicidade, após 7 dias de contato, tendo sido genotóxico apenas o grupo controle positivo (EMS) (p<0,05). Ainda, não foi observado diferença estatisticamente significativa na produção de nitrito, pelas células expostas ao LL-37 após 7 dias, em ambas as concentrações. A análise do ciclo celular, evidenciou maior porcentual de células na fase G0/G1, em todos os grupos (p<0,05). Quando estes foram comparados, foi observado maior quantidade de células na fase G0/G1 na concentração de 10 μg/mL de LL- 37 comparada ao grupo controle (p<0,05). Por outro lado, o grupo controle exibiu mais células na fase G2 e em mitose (M) que os grupos tratados com 5 e 10 μg/mL de LL-37 (p<0,05), e mais células na interfase (S) que o grupo tratado com 10 μg/mL de LL-37 (p<0,05). A análise da expressão gênica demonstrou que não houve aumento de expressão dos genes fosfatase alcalina, osteocalcina, osteopontina e Runx2 após tratamento com ambas as concentrações do peptídeo, no 3° dia. Além disso, não foi observado diferença estatisticamente significativa na ALP nos grupos tratados e controle, após 3 e 14 dias, enquanto o conteúdo de proteína total foi maior aos 14 dias nos grupos tratados com LL-37 (p<0,05). Ainda, aos 3 dias, a produção da proteína DSPP foi maior no grupo tratado com 10 μg/mL de LL-37 (p<0,05). Com base nesses resultados, pode-se concluir que o LL-37 é biocompatível nas concentrações testadas nesse trabalho, e ainda aumenta o número de células viáveis, principalmente em período inicial. Além disso, aos 3 dias, na concentração de 10 μg/mL, ele retarda o ciclo celular e aumenta a expressão da proteína DSPP, além de aumentar a síntese proteica aos 14 dias, o que indica que esse peptídeo pode desempenhar algum tipo de função na diferenciação odontoblástica. / The LL-37 peptide (human derived cathelicidin) is released by some human cells and able of neutralizing the tissues that present lipopolysaccharide (LPS), as well as, attracts pulp cells and induces angiogenesis; characteristics that makes it a possible adjunct for regeneration of the dentin-pulp complex. The aim of this study was evaluate in vitro the biocompatibility of LL-37 in the concentrations of 5 and 10 μg/mL, and its possible performance in the differentiation of dental pulp stem cells (DPSC) into odontoblasts-like cells. For this purpose, it was evaluated: (a) the cytotoxicity by MTT assay; (b) the genotoxicity by the micronucleus test; (c) the production and quantification of nitric oxide; (d) the cell cycle, by flow cytometry; (e) the expression of genes associated with the mineralization by qRT-PCR; (f) the total protein content; (g) the alkaline phosphatase activity (ALP); and (h) the production of dentine sialofosfoprotein (DSPP) by indirect enzyme-linked immunosorbent assay (ELISA). It was observed that the concentrations of 5 and 10 μg/ml of LL-37 were not cytotoxic, in addition to they increased, in general, the cell viability (p<0,05). Moreover, higher absorbance values were observed on 3rd day of contact. After 7 days, the tested concentrations also did not induce genotoxicity, (p<0,05); only the positive control group (EMS) was genotoxic (p<0.05). Furthermore, there was not statistical significance in the nitrite production by the cells exposed to LL-37 for 7 days, in both concentrations. The cell cycle test showed higher percentage of cells in the phase G0/G1 in all groups (p<0.05). When they were compared, it was noticied that concentration of 10 ug/ml of LL-37 arrested the cells in G0/G1 compared to the control group (p<0.05). On the other hand, the control group, exhibited higher amount of cells in G2 and mitosis (M) than the others (p<0.05) and also higher number of cells in interfase (S) than the group treated with 10 μg/mL of LL-37 (p<0.05). On the 3rd day, the analysis of gene expression demonstrated no increase in the expression of the genes alkaline phosphatase, osteocalcin, osteopontin and Runx2, after treatment with both peptide concentrations. Furthermore, it was not observed statistical significance in the ALP in the treated and control groups after 3 and 14 days, while total protein content was higher in the groups treated with LL-37, at 14 days (p<0.05). On the 3rd day, the production of DSPP protein was higher in the group treated with 10 μg/mL of LL-37 (p<0.05). Based on these results, it can be concluded that LL-37 is biocompatible at these concentrations and increases the number of viable cells, especially in the initial period. Moreover, on the 3rd day, the concentration of 10 μg/mL arrests the cell cycle, and increases the expression of DSPP protein, in addition to raising the protein content at 14 days, which indicates that this peptide may present some kind of function in the odontoblastic differentiation.
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Materiais obturadores de canais radiculares de dentes decíduos: avaliação da atividade antibacteriana in vitro e da compatibilidade tecidual in vivo / Root canal filling materials for primary yeeth: evaluation of the in vitro antibacterial activity and in vivo tissue compatibilityAlexandra Mussolino de Queiroz 29 August 2008 (has links)
Avaliou-se a atividade antibacteriana e a compatibilidade tecidual dos seguintes materiais obturadores de canais radiculares de dentes decíduos: cimento de óxido de zinco e eugenol, pasta Calen® espessada com óxido de zinco, cimento Sealapex® e cimento EndoREZTM. A atividade antibacteriana foi avaliada in vitro, por meio do teste de difusão em ágar, frente a 5 microrganismos indicadores (Kocuria rizophila, Enterococcus faecalis, Streptococcus mutans, Escherichia coli e Staphylococcus aureus), sendo os resultados submetidos à análise de variância (ANOVA) e ao pósteste de Tukey. A compatibilidade tecidual foi avaliada in vivo, por implantação subcutânea, em camundongos isogênicos BALB/c, de tubos de polietileno contendo os materiais obturadores. Foram efetuadas análises descritiva e semi-quantitativa do fibrosamento e do infiltrado inflamatório e quantitativa da área e maior espessura do tecido reacional granulomatoso. Os resultados foram submetidos à análise estatística por meio dos testes de Kruskal-Wallis, análise de variância (ANOVA) e pós-teste de Tukey. Para todos os testes o nível de significância foi de 5%. Com relação à atividade antibacteriana, evidenciaram-se diferenças estatisticamente significantes (p<0,0001) entre os halos de inibição ocasionados pelos diferentes materiais, para todos os microrganismos avaliados. A Kocuria rizophila foi inibida mais eficazmente pelo cimento de óxido de zinco e eugenol (p<0,05), enquanto que o Enterococcus faecalis foi inibido mais eficazmente pela pasta Calen® espessada com óxido de zinco (p<0,05). O Streptococcus mutans foi inibido pela pasta Calen® espessada com óxido de zinco, cimento Sealapex® e cimento de óxido de zinco e eugenol na mesma intensidade (p>0,05). A Escherichia coli foi inibida mais eficazmente pelo cimento de óxido de zinco e eugenol, seguido pela pasta Calen® espessada com óxido de zinco e pelo cimento Sealapex® (p<0,05). O Staphylococcus aureus foi inibido pela pasta Calen® espessada com óxido de zinco e pelo cimento de óxido de zinco e eugenol na mesma intensidade (p>0,05), e menos intensamente pelo cimento Sealapex® (p<0,05). O cimento EndoREZTM apresentou atividade antibacteriana apenas frente a Kocuria rizophila e ao Staphylococcus aureus. Com relação à compatibilidade tecidual, não observou-se diferença entre os materiais, com relação ao fibrosamento e à maior espessura do tecido reacional granulomatoso (p>0,05). A pasta Calen® espessada com óxido de zinco foi o material que apresentou infiltrado inflamatório de menor intensidade (p<0,05). A área do tecido reacional granulomatoso foi menor para a pasta Calen® espessada com óxido de zinco e para o cimento Sealapex® (p>0,05) em comparação aos cimentos EndoREZTM e óxido de zinco e eugenol (p<0,05). Com base nos resultados obtidos nas metodologias empregadas pôde-se concluir que: 1- A atividade antibacteriana, in vitro, em ordem decrescente foi: cimento de óxido de zinco e eugenol, pasta Calen® espessada com óxido de zinco, cimento Sealapex® e cimento EndoREZTM; 2- A pasta Calen® espessada com óxido de zinco foi o material que apresentou a melhor compatibilidade tecidual, seguido pelos cimentos Sealapex® e de óxido de zinco e eugenol. O cimento EndoREZTM apresentou resposta tecidual insatisfatória. / This study evaluated the antibacterial activity and tissue compatibility of the following root canal filling materials for primary teeth: zinc oxide and eugenol cement, Calen® paste thickened with zinc oxide, Sealapex® sealer and EndoREZTM sealer. The antibacterial activity of these materials against 5 indicator microorganisms (Kocuria rizophila, Enterococcus faecalis, Streptococcus mutans, Escherichia coli and Staphylococcus aureus) was evaluated in vitro using the agar diffusion test. The results were analyzed statistically by the analysis of variance (ANOVA) and Tukeys post-hoc test. Tissue compatibility was evaluated in vivo by the implantation of polyethylene tubes containing the root canal filling materials in the subcutaneous tissue of isogenic BALB/c mice. Descriptive and semi-quantitative analyses of the rate of fibrosis and the inflammatory infiltrate were performed, as well as a quantitative analysis of the area and the greater thickness of the granulomatous reactionary tissue. The results were analyzed statistically by the Kruskal-Wallis test, ANOVA and Tukeys post-hoc test. For all tests, the significance level was set at 5%. Regarding the antibacterial activity, there were statistically significant differences (p<0.0001) among the zones of inhibition of microbial growth produced by the different materials for all target microorganisms. Kocuria rizophila was inhibited more effectively by the zinc oxide and eugenol cement (p<0.05), while Enterococcus faecalis was inhibited more effectively by the Calen® paste thickened with zinc oxide (p<0.05). Streptococcus mutans was inhibited by the Calen® paste thickened with zinc oxide, Sealapex® sealer and zinc oxide and eugenol cement in the same intensity (p>0.05). Escherichia coli was inhibited more effectively by the zinc oxide and eugenol cement, followed by the Calen® paste thickened with zinc oxide and Sealapex® sealer (p<0.05). Staphylococcus aureus was inhibited by the Calen® paste thickened with zinc oxide and by the zinc oxide and eugenol cement in the same intensity (p>0.05), and was less effectively inhibited by Sealapex® sealer (p<0.05). EndoREZTM sealer presented antibacterial activity only against Kocuria rizophila and Staphylococcus aureus. Regarding tissue compatibility, there was no statistically significant difference among the materials with respect to the rate of fibrosis or the greater thickness of the granulomatous reactionary tissue (p>0.05). The Calen® paste thickened with zinc oxide produced the inflammatory infiltrate with lowest intensity of all materials (p<0.05). The area of the granulomatous reactionary tissue was smaller for the Calen® paste thickened with zinc oxide and Sealapex® sealer (p>0.05) compared to EndoREZTM sealer and the zinc oxide and eugenol cement (p<0.05). Based on the results obtained with the methodologies employed in the present study, it may be concluded that: 1- The in vitro antibacterial activity in a decreasing order was: zinc oxide and eugenol cement, Calen® paste thickened with zinc oxide, Sealapex® sealer and EndoREZTM sealer; 2- The in vivo tissue compatibility in a decreasing order was: Calen® paste thickened with zinc oxide, Sealapex® sealer, zinc oxide and eugenol cement and EndoREZTM sealer. The Calen® paste thickened with zinc oxide permitted the occurrence of an adequate tissue response, while Sealapex® sealer and the zinc oxide and eugenol cement produced a fair tissue response. EndoREZTM sealer did not induce tissue repair.
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Biocompatibility evaluation and synthesis of macrocyclic compounds / Synthèse et étude de la biocompatibilité de composés macrocycliquesRoka, Eszter 21 February 2017 (has links)
La faible solubilité de certains médicaments cause des problèmes majeurs dans les formulations pharmaceutiques, puisque la solubilité dans l'eau est un critère indispensable pour la biodisponibilité. Les composés macrocycliques tels que les CDs et les calixarènes ont une cavité relativement hydrophobe, leur permettant ainsi d'encapsuler de nombreuses molécules. Les CDs ont déjà été utilisées comme excipients pharmaceutiques pour l'amélioration de la solubilité. La structure de ces macrocycles permet d'effectuer de nombreuses modifications, qui causent des changements tant au niveau de leurs caractéristiques physico-chimiques que sur leurs effets sur les organismes vivants. Ainsi, l'évaluation de la biocompatibilité de ces dérivés est primordiale en vue de leur utilisation en pharmacie. Puisque l'étude de la biocompatibilité de plusieurs dérivés de β-CD a déjà été étudiée, l'objectif de cette recherche était d'étendre ces expériences à des dérivés de l'α-CD qui sont disponibles dans le commerce. Nous nous sommes intéressés aux relations entre structure et toxicité. Ainsi les dérivés alkyl éther d'α-CD, avec des chaînes alkyle de longueur croissante et substitués sur différentes positions, ont été synthétisés et leur toxicité étudiée. Les para-sulphonato-calix[n]-arènes quant à eux, ont souvent été étudiés et ont montré une forte capacité à complexer de nombreux médicaments. Ils ont aussi démontré une activité biologique polyvalente. Néanmoins, leurs effets sur le mécanisme de transport paracellulaire n'a jamais été évaluée. Les tests de viabilité cellulaire et d'hémolyse nous ont permis d'une part de classer les α-CDs et de choisir les dérivés les plus sûrs, et d'autre part de comparer leur effets toxiques dans des systèmes différents. La comparaison des α- et ß-CDs portant les mêmes modifications chimiques nous a montré l'importance du nombre d'unités de construction. Le rapport entre l'effet cytotoxique et le nombre de groupes hydroxyles libres est également très important. Les dérivés portant de longues chaînes alkyles possèdent une faible solubilité, ce qui nous a conduits vers d'autres modifications chimiques : la sulfonation de ces derniers dérivés semble avoir un impact bénéfique sur la biocompatibilité de CDs. Elle a aussi amélioré la solubilité des calixarènes. Les calix[4] et [8]arène sulphonates ont prouvé leur effet positif sur l'absorption paracellulaire, tandis que le calix[6]arène sulphonate n'a pas eu d'effet similaire. Notre recherche conclut que les changements structurels sur les anneaux macrocycliques peuvent avoir un impact majeur sur la biocompatibilité. Comme les possibilités de modification sont pratiquement illimitées, l'évaluation de la structure et de l'activité est indispensable pour faciliter les choix les plus sûrs dans les applications pharmaceutiques à venir / The low solubility of drug candidates cause a major problem in pharmaceutical formulations, as the aqueous solubility is an indispensable criterion for appropriate bioavailability. Macrocyclic compounds possess a relatively hydrophobic cavity, which is suitable for guest molecule inclusion. Cyclodextrins and calixarenes are widely studied organic host-compounds, and CDs have already been used as pharmaceutical excipients for solubility enhancement. The macrocycles’ chemical structure allows their versatile modification, which eventuates changes not only in physicochemical characteristics, but in their effects on living organisms, as well. Thus, the biocompatibility evaluation of the derivatives is fundamental. Owing to the already performed assessment of numerous β-CD derivatives’ biocompatibility, the aim of this research was to extend these experiments to commercially available α-CDs. They have been used less frequently, however several derivatives, which have not been tested yet in vitro, have the possibility of future pharmaceutical use. Their importance is also certified by their benefits in nanoparticle formation. We have been interested in concrete structure-toxicity correlations, thus alkyl ether α-CD derivatives were synthetized bearing increasing length alkyl chains, in different positions. Para-sulphonato-calix[n]-arenes have already been widely examined due to their efficient drug complexation and versatile biological activity, however, their effects on paracellular transport mechanism have not been evaluated until now.The cell viability and hemolysis tests have allowed us to rank the α-CDs and to choose the safest derivatives, also to compare their toxic effects in different systems. The comparison of α- and β-CDs bearing the same chemical modifications highlighted the importance of the number of building units. Important information has been evaluated regarding the connection between the cytotoxic effect and the number of free hydroxyl groups. Derivatives with long alkyl chains possess low solubility, which led us towards further chemical modifications. Sulfonation seemed to have beneficial impact on the biocompatibility. Sulfonation also improved the solubility of calixarenes. C4S and C8S proved their positive effect on paracellular absorption in a non-toxic concentration range, however C6S had no similar effect, thus their behaviour in in vitro absorption model system arose forward-looking questions.Our research concludes, that the structural changes on the macrocyclic rings may have major impact on the biocompatibility. As the modification possibilities are practically unlimited, the evaluation of structure and activity cannot be avoided, facilitating the safest choice for further pharmaceutical use / A gyógyszerhatóanyagok rossz vízoldékonysága nagy kihívást jelent formulálásuk során, ugyanis a vízoldékonyság elengedhetetlen feltétele a megfelelő biohasznosulásnak. A makrociklusos vegyületek belső ürege viszonylag hidrofób, ez alkalmassá teszi őket vendégmolekulákkal való komplexképzésre. A ciklodextrinek és kalixarének széles körben tanulmányozott vegyületek, egyes CD-ek bejegyzett oldékonyságnövelő segédanyagok. A makrociklusok felépítése számos kémiai módosításra ad lehetőséget, amelyek nem csupán a fiziko-kémiai tulajdonságok változását eredményezik, hanem az élő organizmusokra kifejtett hatásokat is módosítják. Ezen származékok biokompatibilitás vizsgálata tehát elengedhetetlen. Számos β-CD származék biokompatibilitása ismert már, így kutatásunk célul tűzte ki ezen vizsgálatok α-CD-ekre történő kiterjesztését. Az α-CD-ek alkalmazása ritkább, azonban vannak származékok, amelyek in vitro vizsgálata még nem történt meg, de jelentőségük a nanopartikulum-képzésben már igazolt. A szerkezet-toxicitás összefüggések feltárása érdekében olyan alkil-éter CD származékokat szintetizáltunk, amelyek növekvő szénatomszámú alkil-csoportokkal rendelkeznek, eltérő pozíciókban. A para-szulfonáto-kalix[n]aréneket hatóanyag-komplexáló tulajdonságuk, valamint sokoldalú biológiai aktivitásuk miatt széles körben tanulmányozták már, azonban a paracelluláris anyagtranszportra gyakorolt hatásuk ezidáig még nem volt ismert. A sejtéletképességi és hemolízis vizsgálatok hozzásegítettek az egyes α-CD-ek rangsorolásához, továbbá a vegyületek különböző rendszerekben mért toxikussága is összevethetővé vált. A megegyező kémiai módosításokon átesett α- és β-CD-ek biokompatibilitása rávilágított a CD-gyűrű mértének jelentőségére. Egyértelmű összefüggést fedeztünk fel a toxicitás és a szabad hidroxil-csoportok száma között. A hosszú alkil-csoporttal rendelkező CD-ek rossz oldékonysága további kémiai módosításokat tett szükségszerűvé; a szulfát csoportok jelenléte jótékony hatással volt az oldhatóságra, és a citotoxicitásra is. A szulfatálás a kalixarének oldékonyságát is növelte. A C4S és C8S vegyületek növelték a paracelluláris felszívódás mértékét szubtoxikus koncentrációban, azonban a C6S nem mutatott hasonló hatást. Ezen eredmények további kérdéseket vetnek fel a pontos hatásmechanizmusról. Eredményeink rávilágítanak a makrociklusok szerkezetének és biokompatibilitásának összefüggéseire, valamint ezen ismeretek fontosságára annak érdekében, hogy minden formulációban a legbiztonságosabb segédanyagok legyenek alkalmazhatóak
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Nano-porous Alumina, a Potential Bone Implant CoatingKarlsson, Marjam January 2004 (has links)
<p>This thesis describes a method of growing a highly adherent nano-porous alumina coating on titanium implant materials, a design which might be useful in hard tissue replacement. Alumina layers were formed by anodisation of aluminium, which had been deposited on titanium and titanium alloys by electron beam evaporation. Mechanical testing showed the coatings’ shear and tensile strength to be ~20MPa and ~10MPa respectively. </p><p>Human osteoblasts were cultured on purchased membranes, produced in the same way with similar characteristics as the coating mentioned above. Cell viability, proliferation and phenotype were assessed by measuring redox reactions, DNA, tritiated thymidine incorporation and alkaline phosphatase production. Results showed normal osteoblastic growth patterns with increasing cell numbers the first two weeks after which cell growth decreased and alkaline phosphatase production increased, indicating that osteoblastic phenotype was retained on the alumina. Flattened cell morphology with filipodia attached to the pores of the material was seen. </p><p>Implants frequently trigger inflammatory responses due to accumulation and activation of cells such as polymorphonuclear granulocytes (PMN), also called neutrophils. Activation and morphology of human PMN in response to nano-porous alumina with two pore sizes (20 and 200 nm) was investigated by luminol-amplified chemiluminescence, granule enzyme deposition measurement, optical and scanning electron microscopy. Activation was observed on both membrane types, however less pronounced on the 200 nm alumina. For both membranes a decrease in activation was seen after coating with fibrinogen, collagen I and serum (more pronounced for the two latter). On fibrinogen-coated alumina many flattened cells were observed, indicating frustrated phagocytosis. Finally when culturing osteoblasts on non-coated and collagen-coated membranes (after exposure to PMN) many more cells had established on the protein-coated surface after 24 h. </p><p>The overall results indicate that it might be possible to produce a novel bone implant coating by anodisation of aluminium deposited on titanium and that this material will support osteoblast adhesion and proliferation. Furthermore neutrophil activation can be suppressed when coating the alumina with collagen I, which is beneficial considering the fact that this protein also is essential for bone formation.</p>
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Laser Textured Calcium Phosphate Bio-Ceramic Coatings on Ti-6Al-4V for Improved Wettability and Bone Cell CompatibilityPaital, Sameer R 01 August 2010 (has links)
The interaction at the surfaces of load bearing implant biomaterials with tissues and physiological fluids is an area of crucial importance to all kinds of medical technologies. To achieve the best clinical outcome and restore the function of the diseased tissue, several surface engineering strategies have been discussed by scientific community throughout the world. In the current work, we are focusing on one such technique based on laser surface engineering to achieve the appropriate surface morphology and surface chemistry. Here by using a pulsed and continuous wave laser direct melting techniques we synthesize three dimensional textured surfaces of calcium phosphate (Ca-P) based surface chemistry on Ti-6Al-4V. The influence of each processing type on the micro texture and phase evolution and thereby its associated effect on wettability, in vitro bioactivity, and in vitro biocompatibility are systematically discussed. For samples processed using the pulsed laser, it was realized that with increasing laser scan speed and laser pulse frequency there was a transition from surface textures with sharp circular grooves to surface textures with radial grooves and thereby improved hydrophilicity. For CW laser processing the results demonstrated improved hydrophilicity for the samples processed at 100 μm line spacing as compared to the samples processed at 200 μm line spacing. Owing to the importance of Si for cartilage and hard tissue repair, a preliminary effort for synthesizing Ca-P-SiO2 composite coating on Ti-6Al-4V surface were also conducted. As a future potential technique we also explored the Laser Interference Patterning (LIP) technique to achieve the textured surfaces and developed understanding on their wetting behavior. In the current work, by adjusting the laser processing parameters we were able to synthesize textured coatings with biocompatible phases. The in vitro bioactivity and in vitro vi biocompatibility of the coatings were proved by the precipitation of an apatite like phase following immersion in simulated body fluid (SBF), and increased proliferation and spreading of the MC3T3-E1 like cells. The results and understanding of the current research is encouraging in terms of looking at other bio-ceramic precursor compositions and laser process parameter window for synthesizing better textured biocompatible coatings.
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Atmospheric Pressure Plasma Synthesis of Biocompatible Poly(ethylene glycol)-like CoatingsNisol, Bernard 26 May 2011 (has links)
The role of a protein-repelling coating is to limit the interaction between a device and its physiological environment. Plasma-polymerized-PEG (pp-PEG) surfaces are of great interest since they are known to avoid protein adsorption. and cell attachment. However, in all the studies previously published in the literature, the PEG coatings have been prepared using low pressure processes.
In this thesis, we synthesize biocompatible pp-PEG coatings using atmospheric pressure plasma. Two original methods are developed to obtain these pp-PEG films. 1. Atmospheric pressure plasma liquid deposition (APPLD) consists in the injection of the precursor, tetra(ethylene glycol)dimethylether (tetraglyme), by means of a liquid spray, directly in the post-discharge of an atmospheric argon plasma torch. 2. In atmospheric pressure plasma-enhanced chemical vapor deposition (APPECVD), tetraglyme vapors are brought in the post-discharge trough a heating sprinkler. The chemical composition, as well as the non-fouling properties of the APPLD and APPECVD films, are compared to those of PEG coatings synthesized by conventional low pressure plasma processes.
In the first part of the study, the effect of the power on the chemical composition of the films has been investigated by infrared reflection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS) and secondary ions mass spectroscopy (SIMS).
The surface analysis reveals that for the APPECVD samples, the fragmentation of the precursor increases as the power of the treatment is increased. In other terms, the lower the plasma power is, the higher the “PEG character” of the resulting films is. Indeed, the C-O component (286.5 eV) of the XPS C 1s peak is decreasing while the hydrocarbon component (285 eV) is increasing as the power of the plasma is increased. The same conclusion can be drawn from the signature ToF-SIMS peaks (m/z = 45 (CH3OCH2+ and +CH2CH2OH), 59 (CH3OCH2CH2+), 103 (CH3(OCH2CH2)2+)) that are decreasing in the case of high power treatments. Accordingly, IRRAS measurements show that the C-O stretching band is decreasing for high power plasma deposition. This is in agreement with the observations made from the analysis of the LP PECVD coatings and from the literature.
The films deposited by the APPLD process do not show the same behavior. Indeed, whatever the power injected into the discharge is, we are able to achieve films with a relatively high PEG character (83 %).
The second part of this study is dedicated to the evaluation of the non-fouling properties of the coatings by exposing them to proteins (bovine serum albumin and human fibrinogen) and cells (mouse fibroblasts (L929 and MEF)) and controlling the adsorption with XPS (proteins) and SEM (cells).
For the APPECVD samples, a low plasma power (30 W) leads to an important reduction of protein adsorption and cell adhesion (over 85%). However, higher-powered treatments tend to reduce the non-fouling ability of the surfaces (around 50% of reduction for a 80 W deposition).
The same order of magnitude (over 90% reduction of the adsorption) is obtained for the APPLD surfaces, whatever is the power of the treatment.
Those results show an important difference between the two processes in terms of power of the plasma treatment, and a strong relationship between the surface chemistry and the adsorption behavior: the more the PEG character is preserved, the more protein-repellent and cell-repellent is the surface. / Le rôle d’une couche empêchant l’adsorption de protéines est de limiter les interactions entre un implant et le milieu physiologique auquel il est exposé. Les films de poly(éthylène glycol) polymérisés par plasma (pp-PEG) sont d’intérêt majeur car ils sont connus pour empêcher l’adsorption de protéines ainsi que l’attachement cellulaire. Cependant, dans toutes les études publiées précédemment, les couches de type PEG ont été réalisées sous vide.
Dans cette thèse de doctorat, nous synthétisons des couches de type pp-PEG biocompatibles par plasmas à pression atmosphérique. A cette fin, deux méthodes originales ont été développées. 1. La première méthode consiste en l’injection du précurseur, le tetra(éthylène glycol) diméthyl éther (tetraglyme), en phase liquide, en nébulisant ce dernier au moyen d’un spray, directement dans la post-décharge d’une torche à plasma atmosphérique fonctionnant à l’argon. En anglais, nous appelons ce procédé « Atmospheric pressure plasma liquid deposition (APPLD) ». 2. Dans la deuxième méthode, appelée en anglais « Atmospheric pressure plasma-enhanced chemical vapor deposition (APPECVD)», le tetraglyme est amené en phase vapeur dans la post-décharge, au moyen d’un diffuseur chauffant. La composition chimique des dépôts de type APPLD et APPECVD, ainsi que leurs propriétés d’anti-adsorption sont évaluées, et comparées aux dépôts pp-PEG obtenus par les méthodes à basse pression conventionnelles.
Dans la première partie de cette étude, nous nous focalisons sur la composition chimique des films déposés, et plus particulièrement sur l’influence de la puissance injectée dans le plasma sur cette composition chimique. A cette fin, nous avons fait appel à des techniques d’analyse telles que la spectroscopie de réflexion-absorption infrarouge (IRRAS), la spectroscopie des photoélectrons X (XPS) et la spectrométrie de masse des ions secondaires (SIMS).
Il en ressort que les films de type APPECVD perdent progressivement leur « caractère PEG » à mesure que la puissance de la décharge plasma est élevée. Cela serait dû à une plus grande fragmentation du précurseur dans la post-décharge d’un plasma plus énergétique. Cette tendance est cohérente avec ce que nous avons observé pour les dépôts à basse pression ainsi que dans la littérature.
Dans le cas des films de type APPLD, un tel comportement n’a pas été mis en évidence : quelle que soit la puissance dissipée dans le plasma, les films présentent un « caractère PEG » relativement élevé.
La deuxième partie de cette thèse est dédiée à l’évaluation des propriétés d’anti-adsorption des films synthétisés, en les exposant à des protéines (albumine de sérum bovin et fibrinogène humain) et des cellules (fibroblastes de souris, L929 et MEF). L’adsorption de protéines est contrôlée par XPS tandis que l’attachement cellulaire est contrôlé par imagerie SEM.
Pour les échantillons de type APPECVD, un dépôt à faible puissance (30 W) mène à une importante réduction de l’adsorption de protéines et de cellules (> 85%) tandis qu’à de plus hautes puissances (80 W), l’anti-adsorption est sensiblement diminuée (50% de réduction). Dans le cas des dépôts de type APPLD, quelle que soit la puissance du plasma, une forte diminution de l’adsorption de protéines et de cellules est observée (> 90 %).
Ces résultats montrent une différence majeure entre les deux procédés quant à l’influence de la puissance du plasma ainsi qu’une forte relation entre la composition chimique de la surface synthétisée et son pouvoir d’anti-adsorption : plus le « caractère PEG » du dépôt est conservé, plus la surface empêchera l’interaction avec les protéines et les cellules.
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Nano-porous Alumina, a Potential Bone Implant CoatingKarlsson, Marjam January 2004 (has links)
This thesis describes a method of growing a highly adherent nano-porous alumina coating on titanium implant materials, a design which might be useful in hard tissue replacement. Alumina layers were formed by anodisation of aluminium, which had been deposited on titanium and titanium alloys by electron beam evaporation. Mechanical testing showed the coatings’ shear and tensile strength to be ~20MPa and ~10MPa respectively. Human osteoblasts were cultured on purchased membranes, produced in the same way with similar characteristics as the coating mentioned above. Cell viability, proliferation and phenotype were assessed by measuring redox reactions, DNA, tritiated thymidine incorporation and alkaline phosphatase production. Results showed normal osteoblastic growth patterns with increasing cell numbers the first two weeks after which cell growth decreased and alkaline phosphatase production increased, indicating that osteoblastic phenotype was retained on the alumina. Flattened cell morphology with filipodia attached to the pores of the material was seen. Implants frequently trigger inflammatory responses due to accumulation and activation of cells such as polymorphonuclear granulocytes (PMN), also called neutrophils. Activation and morphology of human PMN in response to nano-porous alumina with two pore sizes (20 and 200 nm) was investigated by luminol-amplified chemiluminescence, granule enzyme deposition measurement, optical and scanning electron microscopy. Activation was observed on both membrane types, however less pronounced on the 200 nm alumina. For both membranes a decrease in activation was seen after coating with fibrinogen, collagen I and serum (more pronounced for the two latter). On fibrinogen-coated alumina many flattened cells were observed, indicating frustrated phagocytosis. Finally when culturing osteoblasts on non-coated and collagen-coated membranes (after exposure to PMN) many more cells had established on the protein-coated surface after 24 h. The overall results indicate that it might be possible to produce a novel bone implant coating by anodisation of aluminium deposited on titanium and that this material will support osteoblast adhesion and proliferation. Furthermore neutrophil activation can be suppressed when coating the alumina with collagen I, which is beneficial considering the fact that this protein also is essential for bone formation.
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Silica-Supported Organic Catalysts For The Synthesis Of Biodegradable PolymersWilson, Benn Charles 06 December 2004 (has links)
Aliphatic polyesters such as polycaprolactone and polylactide have received more attention in recent years for their use in biomedical applications because of their biodegradable nature. These polymers are often synthesized using homogeneous metal complexes. Unfortunately, using homogeneous metals as catalysts leads to metal contamination in the product polymer, a result which is highly undesirable in a polymer intended for biomedical use.
More recent work has shown that these polymers can be synthesized using homogeneous metal-free complexes. These catatlysts are generally less active than metal catalysts, and although they do not contaminate the polymer with metal residue, they are still difficult to recover and hence recycle for further use.
In this work, we attempted to create a metal-free, silica-supported catalyst for use in the synthesis of polycaprolactone or polylactide.
Ultimately, n-propylsulfonic acid-functionalized porous and nonporous silica materials are evaluated in the ring-opening polymerization of epsilon-caprolactone. All catalysts allow for the controlled polymerization of the monomer, producing polymers with controlled molecular weights and narrow polydispersities. Polymerization rates are low, with site-time-yields generally one to three orders of magnitude lower than metal-based systems. The catalysts are easily recovered from the polymerization solution after use and are shown to contain significant residual adsorbed polymer. Solvent extraction techniques are useful for removing most of the polymer, although the extracted solids are not effective catalysts in recycle experiments. These new materials represent a green alternative to traditional metal-based catalysts, as they are recoverable and leave no metal residues in the polymer.
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