Homogalactanas sulfatadas da alga Codium isthmocladum com atividade anticoagulante

Since the first description of sulfated polysaccharides from seaweeds, the biological activities of these compounds have been evaluated under different aspects and experimental procedures. Among the broad biological activities presented by seaweed polysaccharides, anticoagulant action appears as a promising function. In this present study we have obtained sulfated polysaccharides from the green seaweed Codium isthmocladium by proteolytic digestion, followed by separation into five fractions (0.3, 0.5, 0.7, 0.9 and 1.2) by sequential acetone precipitation. The chemical analyses have demonstrated that all fractions are composed mainly by sulfated polysaccharides. The anticoagulant activity of these fractions was determined by activated partial thromboplastin time (aPTT) and prothrombin time test (PT) using citrate normal human plasma. None fraction has shown anticoagulant activity by PT test. Furthermore, all of them have shown anticoagulant activity by aPTT test. These results indicated that the molecular targets of these sulfated polysaccharides are mainly in the intrinsic via of the coagulation cascade. Agarose gel electrophoresis in 1,3-diaminopropane acetate buffer, pH 9.0, stained with 0.1% toluidine blue showed the presence of two or three bands in several fractions while the fraction 0.9 showed a single spot. By anion exchange chromatography, the acid polysaccharides from the 0.9 acetone fraction were separated into two new fractions eluted respectively with 2.0 and 3.0 M NaCl. These compounds showed a molecular weight of 6.4 and 7.4 kDa respectively. Chemical analyses and infrared spectroscopy showed that Gal 1 and Gal 2 are sulfated homogalactans and differ one from the other in degree and localization of sulfate groups. aPPT test demonstrated that fractions 2,0 and 3,0M (Gal1 and Gal 2, respectively) have anticoagulant activity. This is the first time that anticoagulant sulfated homogalatans have been isolated from green algae. To prolong the coagulation time to double the baseline value in the aPTT, the required amount of sulfated galactan 1 (6,3mg) was similar to low molecular heparin Clexane®, whereas only 0,7mg of sulfated galactan 2 was needed to obtain the same effect. Sulfated galactan 2 in high doses (250mg) induces platelet aggregation. These results suggest that these galactans from C. isthmocladum have a potential application as an anticoagulant drug

Efeito in vitro da terapia fotodinâmica sobre bactérias orais em suspensão e na formação de biofilme : ensaio com azul de metileno e toluidina ativados por luz halógena

Photodynamic therapy (PDT) has been proposed as an alternative method for the treatment of biofilm-dependent oral diseases like dental caries. This therapy consists of simultaneous action of a visible light (L) and a photosensitizer (FS) in the presence of oxygen, which leads to production of different reactive oxygen species that can interact with the bacterial cell components, and promote cell death. This study aims to evaluate the antimicrobial action of PDT on oral bacteria in suspension, as well as the formation of mono and multi-species biofilms, in vitro, from a standard strain of Streptococcus mutans (ATCC 25175) and saliva samples, respectively. The dye methylene blue (MB) and toluidine blue (TB) were used at a concentration of 100 mg/ L and activated by halogen light (600 to 750 nm) from a modified hand held photopolymerizer (Ultralux ®, Dabi Atlante, Ribeirão Preto , São Paulo, Brazil.). Planktonic cultures were prepared and submitted to different experimental conditions: 1. PDT using TB 2. PDT using MB, 3. L+ FS- , 4. TB + L - ; 5. MB+ L-; 6. L- FS- (negative control) and 7. administration of 0.12% chlorhexidine digluconate (positive control) (Periogard ®, Colgate-Palmolive Company, New York, USA). The immediate and mediated action of PDT on bacterial suspensions, as well as its effect on biofilm formation were observed from the number of colony-forming units per milliliter (CFU/mL) and measures optical density (OD). The data were statistically analyzed using the Kruskal-Wallis and Mann-Whitney test for the significance level of 5%. According to the results, the PDT showed no antibacterial action on suspensions of S. mutans, regardless of the dye used. PDT with MB activated by halogen light was able to reduce 86.6% CFU/mL multi-species planktonic cultures, however, this reduction was not significant (p > 0.05). PDT showed antibacterial effect, mediate on multi-species planktonic cultures with TB (p < 0.001) and MB (p < 0.001), activated by halogen light. PDT was able to prevent the formation of multispecies biofilm, through the activation of TB by halogen light (p = 0.01). We conclude that activation of the dye toluidine blue and methylene blue, by halogen light (PDT) showed antimicrobial activity, compared to multi-species planktonic cultures prepared from saliva samples

Lethal photosensitization and guided bone regeneration in treatment of peri-implantitis: an experimental study in dogs

The purpose of this study was to evaluate the effect of lethal photosensitization and guided bone regeneration (GBR) on the treatment of ligature-induced peri-implantitis in different implant surfaces. The treatment outcome was evaluated by clinical and histometric methods. A total of 40 dental implants with four different surface coatings (10 commercially pure titanium surface (cpTi); 10 titanium plasma-sprayed (TPS); 10 acid-etched surface; 10 surface-oxide sandblasted) were inserted into five mongrel dogs. After 3 months, the animals with ligature-induced peri-implantitis were subjected to surgical treatment using a split-mouth design. The controls were treated by debridment and GBR, while the test side received an additional therapy with photosensitization, using a GaAlAs diode laser, with a wavelength of 830 nm and a power output of 50 mW for 80 s (4 J/cm(2)), and sensitized toluidine blue O (100 mu g/ml). The animals were sacrificed 5 months after therapy. The control sites presented an earlier exposition of the membranes on all coating surfaces, while the test group presented a higher bone height gain. Re-osseointegration ranged between 41.9% for the cpTi surface and 31.19% for the TPS surface in the test sites; however differences were not achieved between the surfaces. The lethal photosensitization associated with GBR allowed for better re-osseointegration at the area adjacent to the peri-implant defect regardless of the implant surface.

Lethal photosensitization in microbiological treatment of ligature-induced peri-implantitis: a preliminary study in dogs

This pilot study evaluated, by culture testing, the effectiveness of lethal photosensitization for the microbiological treatment of peri-implantitis in dogs. Experimental peri-implantitis was induced by ligature placement for 2 months. Following ligature removal, plaque control was instituted by scrubbing with 0.12% chlorhexidine daily for 12 months. Subsequently, mucoperiosteal flaps were elevated for scaling the implant surface. Microbial samples were obtained with paper points before and after treatment of implant surfaces by means of 100 microg/ml toluidine blue O (TBO,) and were exposed, for 80 s, to light with a wavelength of 685 nm from a 50 mW GaAlAs diode laser. The mean initial and final bacterial counts were 7.22 +/- 0.20 and 6.84 +/- 0.44 CFU/ml, respectively for TVC (P < 0.0001); 6.19 +/- 0.45 and 3.14 +/- 3.29 CFU/ml for P. intermedia/nigrescens (P = 0.001); 5.98 +/- 0.38 and 1.69 +/- 2.90 CFU/ml for Fusobacterium spp. (P = 0.001); and 6.07 +/- 0.22 to 1.69 +/- 2.94 CFU/ml for beta-hemolytic Streptococcus (P = 0.0039). It may be concluded that lethal photosensitization resulted in a reduction of the bacterial count. Complete elimination of bacteria was achieved in some samples.

Effect of antimicrobial photodynamic therapy on periodontally infected tooth sockets in rats

The aim of the present study was to evaluate the antimicrobial effect of antimicrobial photodynamic therapy (aPDT) in alveolar treatment of areas with induced periodontitis. Thirty male Wistar rats were subjected to ligature-induced periodontal disease (PD) in the first left inferior molars, while the right side molars did not receive ligatures. After 7 days of PD evolution, ligatures were removed from the left side, and the first left and right mandibular molars were extracted. Afterwards, animals were divided into groups according to the following treatments: control (C)-no treatment; mechanical debridement (MD)-mechanical debridement and irrigation with saline solution; and aPDT-mechanical debridement, irrigation with toluidine blue O (TBO), and 1 min of laser irradiation (GaAlAs, 660 nm, 30 mW, 32 J/cm2, 60 s). Ligatures were removed and samples of the alveolar content after extraction and after each treatment were collected for microbial processing by real-time polymerase chain reaction with specific primers for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Treponema denticola. Data were submitted to statistical analysis by multiple comparison tests (McNemar test; p < 0.05). T. denticola was not found in the collected samples. A. actinomycetemcomitans and P. gingivalis were found in ligature samples. Tooth socket samples without periodontitis induction presented lesser microbial charge than samples with induced periodontitis (p < 0.05). aPDT significantly reduced A. actinomycetemcomitans levels on the left side (p < 0.05). It was concluded that aPDT was an effective antimicrobial treatment for tooth sockets in areas affected by induced periodontitis. © 2013 Springer-Verlag London.

Estudos histopatológicos e ciclos biológicos de Meloidogyne mayaguensis e M. javanica em tomateiros com gene Mi

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análises biomecânica e histológica da interface de implantes osseointegrados submetidos à carga mastigatória imediata em mandíbulas de cães

Pós-graduação em Odontologia - FOA

Streptococcus Mutans Photoinactivation by Combination of Short Exposure of a Broad-Spectrum Visible Light and Low Concentrations of Photosensitizers

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Assessment of in vitro sperm characteristics and their importance in the prediction of conception rate in a bovine timed-AI program

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito da aPDT na osseointegração de implantes em alvéolos contaminados

Objective: The aim of this study is to evaluate by histometric and immunohistochemistry analysis the effects of antimicrobial photodynamic therapy (aPDT) in alveolar prior to placement of mini-implants in animals with or without induced periodontal disease. Material and method: Thirty-two rats were used. Periodontal disease (PD) was induced by ligature in the lower left first molar. After 7 days of PD evolution, it was performed removal of the ligature and extraction of the left lower first molars in all animals. Thus, animals were divided into 2 groups (n=16) according to the treatments in the dental alveolus before immediate implant installation: MD (control) : mechanical debridement (MD), irrigation with 1 ml of saline solution followed by implant installation; aPDT- MD, irrigation with 1 ml of Toluidine Blue-O and low intensity laser (LLLT) and implant installation. Eight animals from each group were euthanized at 15 and 30 days after the installation of mini-implants. Specimens were processed for histologic, immunohistochemical and histometric analysis. The histometric data were processed for statistical analysis (Kruskall-Wallis and Dunn test; p <0.05). Result: In treatment analysis, results indicated that there was a greater BIC in implants installed in uncontaminated alveolus treated with a PDT ( p<0,05) and greater imuno-reactivity to OPG in bone issue treated with aPDT. Conclusion: The aPDT proved to be effective in bone loss control in no contaminated area and it has increased the bone loss and metabolic activity in alveolus irradiated prior to implant installation.

Antimicrobial photodynamic therapy in the non-surgical treatment of aggressive periodontitis: microbiological profile

The aim of this trial was to investigate changes occurring in the subgingival microbiological composition of subjects with aggressive periodontitis, treated with antimicrobial photodynamic therapy (aPDT), in a single episode, or scaling and root planing (SRP), in a split-mouth design on -7, 0, and +90 days. Ten patients were randomly assigned to either aPDT using a laser source in conjunction with a photosensitizer or SRP with hand instruments. Subgingival plaque samples were collected and the counts of 40 subgingival species were determined using checkerboard DNA-DNA hybridization. The data were analyzed using the method of generalized estimating equations (GEE) to test the associations between treatments, evaluated parameters, and experimental times (alpha = .05). The results indicated that aPDT and SRP affects different bacterial species, with aPDT being effective in reducing numbers of A. actinomycetemcomitans than SRP. On the other hand, SRP was more efficient than aPDT in reducing the presence of periodontal pathogens of the Red Complex. Additionally, a recolonization in the sites treated by aPDT was observed, especially for T. forsythia and P. gingivalis. Under our experimental conditions, this trial demonstrates that aPDT and SRP affected different groups of bacteria, suggesting that their association may be beneficial for the non-surgical treatment of aggressive periodontitis.

Haemostatic agents used in periradicular surgery: an experimental study of their efficacy and tissue reactions

AIM: To evaluate the haemostatic efficacy and the histologic tissue responses after the application of different haemostatic agents used in periradicular surgery. METHODOLOGY: The study was conducted in the calvarium of six rabbits. Standardized bone defects (diameter 4 mm) were trephined, and different haemostatic agents were applied and compared with control defects: bone wax (left for 10 min), Stasis (ferric sulphate, left for 5 s), Expasyl (aluminium chloride, left for 2 min and left permanently in situ), and a combination of Expasyl (2 min) and Stasis (5 s). The sites were photographed before the application and after the removal of the haemostatic agents. Three independent examiners judged the initial and final bleeding (on the photographs) using a bleeding score for each site and treatment. The results were compared using Wilcoxon's signed rank test. For the histologic analysis, three animals were killed after 3 weeks and three animals after 12 weeks. Transverse, nondecalcified sections were stained with combined basic fuchsin and toluidine blue for descriptive histology. RESULTS: The most efficient haemorrhage control was provided by Expasyl in combination with Stasis and by Expasyl alone, whereas bone wax had the weakest bleeding reduction effect. The histologic analysis after 3 weeks demonstrated an inflammatory and foreign body tissue response towards all haemostatic agents. At 12 weeks, this tissue response was less pronounced but still present in sites treated with bone wax or Expasyl. In general, the inflammatory tissue reactions were limited to the bone defects, and never extended into the surrounding tissues. CONCLUSIONS: Expasyl alone or in combination with Stasis appeared to be the most efficient of tested agents to control the bleeding within the bony defects created in a rabbit calvarium model.

Cytotoxicity of topical antimicrobial agents used in burn wounds in Australasia

Background: Burn sepsis is a leading cause of mortality and morbidity in patients with major burns. The use of topical antimicrobial agents has helped improve the survival of these patients. Silvazine (Sigma Pharmaceuticals, Melbourne, Australia) (1% silver sulphadiazine and 0.2% chlorhexidine digluconate) is used exclusively in Australasia, and there is no published study on its cytotoxicity. This study compared the relative cytotoxicity of Silvazine with 1% silver sulphadiazine (Flamazine (Smith & Nephew Healthcare. Hull. UK)) and a silver-based dressing (Acticoat (Smith & Nephew Healthcare, Hull, UK)). Methods: Dressings were applied to the centre of culture plates that were then seeded with keratinocytes at an estimated 25% confluence. The plates were incubated for 72 h and culture medium and dressings then removed. Toluidine blue was added to stain the remaining keratinocytes. Following removal of the dye, the plates were photographed under standard conditions and these digital images were analysed using image analysis software. Data was analysed using Student's t-test. Results: In the present study, Silvazine is the most cytotoxic agent. Seventy-two hour exposure to Silvazine in the present study results in almost no keratinocyte survival at all and a highly statistically significant reduction in cell survival relative to control, Acticoat and Flamazine (P

Caracterização estrutural e atividades farmacológicas do alginato obtido da alga Dictyopteris delicatula (J.V. Lamouroux., 1809) e seu derivado sulfatado

Marine algae are rich sources of various structural compounds which recently has been increasingly studied as a new source of bioactive substances. The alginate, as come as fucans, are considered the main acidic polysaccharides found in brown seaweed. This molecule consists a linear natural polysaccharide, non-sulfated, and presents monosaccharides: acid β-D-mannuronic (M) and α-L-guluronic acid (G); in a vast amount compositions and threads. Alginate has been widely applied in food and pharmaceutical industries because of its ability to retain water, forming films and gels as well as thickening, stabilizing and form emulsions. In this work we aimed to extract, structurally characterize, compare and analyze the possible pharmacological activities of native alginate molecule obtained from brown seaweed Dyctiopteris delicatula (DYN), and its chemically sulfated derivative (DYS). The alginate structure and composition molecule can be proven through chemical dosing, that showed low protein contamination and high sugar level, existence and separation of M and G blocks in the descending paper chromatography, infrared spectroscopy and nuclear magnetic resonance. Molecule sulfation was proven with sulphate dosage, resulting in 28.56% sulphate in molecule; electrophoresis, verify metachromasia with toluidine blue; and infrared spectroscopy, that showed a characteristic band at 1221cm-1 corresponding a sulfate group vibration. For the pharmacological activities the tests was: antioxidant activity, changes in cell function (MTT test) and anticoagulant test. In the antioxidant activity we observed that DYN showed better results in the kidnapping of hydroxyl radicals and ferric chelation compared to DYS, this had the best result in the total antioxidant capacity. Both showed similar activity in reducing power and the kidnapping radicals DPPH. In MTT test DYN and DYS had not proliferative and cytotoxic activity in fibroblast cells (3T3) and showed antiproliferative and cytotoxic activity in cancer cell lines HeLa and B16 melanoma. In anticoagulant assay DYN showed good activity in the intrinsic pathway of blood coagulation, and a small activity in the extrinsic pathway, in the other hand DYS showed only a very small activity in the extrinsic pathway, but cannot come to be regarded as an anticoagulant agent. From these results it can be concluded that the alginate was extracted and sulfated, revealing a potential compound to be used in the pharmaceutical industry as an anticoagulant agent, antioxidant and antitumor and the sulfation has not been conclusively important to performance in the tested pharmacological activities

Aplicação de porfirinas como fotossensibilizadores para a inativação fotodinâmica de microrganismos da cavidade oral

A cavidade oral é um habitat favorável ao desenvolvimento de microrganismos, alguns dos quais podem causar doenças, sendo Enterococcus faecalis uma bactéria frequentemente encontrada em biofilmes instalados em diferentes nichos da cavidade oral. Este trabalho teve como objetivo testar a aplicabilidade da inativação fotodinâmica (PDI), usando porfirinas como fotossensibilizadores, como estratégia de controlo de biofilmes da cavidade oral, tomando E. faecalis como microrganismo modelo. Como fotossensibilizadores, foram testadas as porfirinas catiónicas Tetra-Py+-Me, Tri-Py+-Me-PF, PCat 2, PCat 3, PCat 4 e o corante azul de toluidina O (TBO), incluído como fotossensibilizador de referência. Os biofilmes de E. faecalis foram irradiados com luz branca (270 J.cm-2) a uma intensidade de 150 mW.cm-2, na presença de até 50 µM de porfirina ou até 20 µM de TBO. A cinética de inativação foi caracterizada pela variação da concentração de células viáveis ao longo da experiência. Foi também testada a inativação de células na forma livre, em condições equivalentes. Os biofilmes de E. faecalis mostraram-se muito resistentes à PDI com qualquer dos PS testados, não tendo sido conseguidos fatores de inativação superiores a 2 log com a concentração máxima de PS (50 µM) e a dose máxima de luz (270 J.cm-2). Na forma livre as células foram inativadas até ao limite de quantificação com concentrações de PS de 0,5 µM e doses de luz até 108 J.cm-2, com uma intensidade de 10 mW.cm-2. No entanto, a eficiência de ligação dos PS às células livres não foi maior do que aos biofilmes. Embora os fatores de inativação obtidos não permitam ainda considerar que a PDI com os compostos testados seja uma abordagem antimicrobiana eficiente contra biofilmes de E. faecalis, o facto de se confirmar uma relação entre as propriedades químicas e físicas do PS e a sua eficiência, bem como os resultados muito promissores obtidos com uma das famílias de porfirinas testadas apenas em células livres, justifica a prossecução do desenvolvimento de novos PS para o controle de biofilmes bacterianos na cavidade oral.