Alignment of the Fibrin Network Within an Autologous Plasma Clot.

Autologous plasma clots with longitudinally aligned fibrin fibers could serve as a scaffold for longitudinal axonal regrowth in cases of traumatic peripheral nerve injuries. Three different techniques for assembling longitudinally oriented fibrin fibers during the fibrin polymerization process were investigated as follows: fiber alignment was induced by the application of either a magnetic field or-as a novel approach-electric field or by the induction of orientated flow. Fiber alignment was characterized by scanning electron microscopy analysis followed by image processing using fast Fourier transformation (FFT). Besides FFT output images, area xmin to xmax, as well as full width at half maximum (FWHM) of the FFT graph plot peaks, was calculated to determine the relative degree of fiber alignment. In addition, fluorescently labeled human fibrinogen and mesenchymal stem cells (MSCs) were used to visualize fibrin and cell orientation in aligned and nonaligned plasma clots. Varying degrees of fiber alignment were achieved by the three different methods, with the electric field application producing the highest degree of fiber alignment. The embedded MSCs showed a longitudinal orientation in the electric field-aligned plasma clots. The key feature of this study is the ability to produce autologous plasma clots with aligned fibrin fibers using physical techniques. This orientated internal structure of an autologous biomaterial is promising for distinct therapeutic applications, such as a guiding structure for cell migration and growth dynamics.

Effect of instrumentation using curettes, piezoelectric ultrasonic scaler and Er,Cr:YSGG laser on the morphology and adhesion of blood components on root surfaces: a SEM study.

This study used scanning electron microscopy (SEM) to evaluate the morphology and adhesion of blood components on root surfaces instrumented by curettes, piezoelectric ultrasonic scaler and Er,Cr:YSGG laser. One hundred samples from 25 teeth were divided into 5 groups: 1) Curettes; 2) Piezoelectric ultrasonic scaler; 3) Curettes plus piezoelectric ultrasonic scaler; 4) Er,Cr:YSGG laser; 5) Curettes plus Er,Cr:YSGG laser. Ten samples from each group were used for analysis of root morphology and the other 10 were used for analysis of adhesion of blood components on root surface. The results were analyzed statistically by the Kruskall-Wallis and Mann-Whitney tests with a significance level of 5%. The group treated with curettes showed smoother surfaces when compared to the groups were instrumented with piezoelectric ultrasonic scaler and the Er,Cr:YSGG laser. The surfaces instrumented with piezoelectric ultrasonic scaler and Er,Cr:YSGG laser, alone or in combination with hand scaling and root planing, did not differ significantly (p>0.05) among themselves. No statistically significant differences (p>0.05) among groups were found as to the adhesion of blood components on root surface. Ultrasonic instrumentation and Er,Cr:YSGG irradiation produced rougher root surfaces than the use of curettes, but there were no differences among treatments with respect to the adhesion of blood components.

Effect of instrumentation using curettes, piezoelectric ultrasonic scaler and Er,Cr:YSGG laser on the morphology and adhesion of blood components on root surfaces: a SEM study

This study used scanning electron microscopy (SEM) to evaluate the morphology and adhesion of blood components on root surfaces instrumented by curettes, piezoelectric ultrasonic scaler and Er,Cr:YSGG laser. One hundred samples from 25 teeth were divided into 5 groups: 1) Curettes; 2) Piezoelectric ultrasonic scaler; 3) Curettes plus piezoelectric ultrasonic scaler; 4) Er,Cr:YSGG laser; 5) Curettes plus Er,Cr:YSGG laser. Ten samples from each group were used for analysis of root morphology and the other 10 were used for analysis of adhesion of blood components on root surface. The results were analyzed statistically by the Kruskall-Wallis and Mann-Whitney tests with a significance level of 5 percent. The group treated with curettes showed smoother surfaces when compared to the groups were instrumented with piezoelectric ultrasonic scaler and the Er,Cr:YSGG laser. The surfaces instrumented with piezoelectric ultrasonic scaler and Er,Cr:YSGG laser, alone or in combination with hand scaling and root planing, did not differ significantly (p>0.05) among themselves. No statistically significant differences (p>0.05) among groups were found as to the adhesion of blood components on root surface. Ultrasonic instrumentation and Er,Cr:YSGG irradiation produced rougher root surfaces than the use of curettes, but there were no differences among treatments with respect to the adhesion of blood components.

Esse estudo utilizou microscopia eletrônica de varredura (MEV) para avaliar a morfologia e a adesão de elementos sanguíneos em superfícies radiculares instrumentadas com curetas, ultrassom piezoelétrico e laser de Er,Cr:YSGG. Foram utilizadas no presente estudo 100 amostras provenientes de 25 dentes que foram divididas em 5 grupos: 1) Raspagem manual com curetas; 2) Raspagem com ultrassom; 3) Associação instrumento manual e ultrassom; 4)Irradiação do laser de Er,Cr:YSGG;5)Associação raspagem manual com irradiação com laser de Er,Cr:YSGG. Dez amostras de cada grupo foram utilizadas para análise da morfologia e as outras 10 foram utilizadas para a análise de adesão de elementos sanguíneos. As eletromicrografias foram analisadas através dos escores de adesão de elementos sanguíneos e pelo índice de morfologia radicular e os resultados foram analisados estatisticamente através dos testes de Kruskall-Wallis e de Mann-Whitney com nível de significância de 5 por cento. O grupo que foi tratado com instrumentos manuais apresentou superfície mais lisa em relação aos grupos que foram instrumentados com ultrassom e com o laser de Er,Cr:YSGG. As superfícies instrumentadas com ultrassom e com o laser de Er,Cr:YSGG de forma isolada ou associada a raspagem manual não apresentaram diferenças estatísticas entre si (p>0,05). Não houve diferenças estatísticas entre os grupos em relação a adesão de elementos sanguíneos(p>0,05). A instrumentação ultrassônica e a irradiação com o laser de Er,Cr:YSGG produziram superfícies radiculares mais rugosas em relação a raspagem com curetas, porém não houve diferenças entre os tratamentos com relação à adesão de elementos sanguíneos.

Using F0F1-ATPase motors as micro-mixers accelerates thrombolysis.

We have developed a novel micro-mixer using a biological molecular ATP motor. The micro-mixer was constructed from arrays of chromatophore-embedded delta-free F(0)F(1)-ATPases, where the delta-free F(1) part acted as a rotator to mix solutions, and the F(0) part was driven by light. Confocal microscope studies indicated that the micro-mixer did not touch directly on the fibrin labeled with FITC. The nanomechanical force generated by the motor induced drug movement in the solution and accelerated the fibrinolysis process. All results strongly suggest that the micro-mixers generated a nanomechanical force which accelerated the fibrinolysis process in the presence of lower concentrations of lumbrokinase.

Concurrent application of charge using a novel circuit prevents heat-related coagulum formation during radiofrequency ablation.

INTRODUCTION: Thromboembolism resulting from coagulum formation on the catheter and electrode surfaces is a serious complication with radiofrequency ablation procedures for heart rhythm disorders. Why coagulum occurs despite therapeutic heparinization is unclear. In this report, we demonstrate a novel approach to minimize coagulum formation based on the electromolecular characteristics of fibrinogen. METHODS AND RESULTS: Atomic force microscopy was used to establish that fibrinogen deposited on surfaces underwent conformational changes that resulted in spontaneous clot formation. We then immersed ablation catheters that were uncharged, negatively, or positively charged in heparinized blood for 30 minutes and noted the extent of clot formation. In separate experiments, ablation catheters were sutured to the ventricle of an excised porcine heart immersed in whole, heparinized blood and radiofrequency ablation performed for 5 minutes with and without charge delivered to the catheter tips. Electron microscopy of the catheter tips showed surface coverage of fibrin clot of the catheter to be 33.8% for negatively charged catheters, compared with 84.7% (P = 0.01) in noncharged catheters. There was no significant difference in surface coverage of fibrin clot between positively charged catheters (93.8%) and noncharged catheters (84.7%, P = ns). In contrast, the thickness of surface clot coverage for positively charged catheters was 87.5%, compared with 28.45% (P= 0.03) for noncharged catheters and 11.25% (P = 0.03) for negatively charged catheters, compared with noncharged catheters. CONCLUSIONS: We describe a novel method of placing negative charge on electrodes during ablation that reduced coagulum formation. This may decrease thromboembolism-related complications with radiofrequency ablation procedures.

The role of ascorbate and histidine in fibrinogen protection against changes following exposure to a sterilizing dose of gamma-irradiation.

Sodium ascorbate and histidine were employed to protect fibrinogen against modifications followed by a gamma-irradiation process that could potentially inactivate the blood-borne viruses in plasma-derived products. Fibrinogen was irradiated (50 kGy total dose, on dry ice) using a 60Co source. Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot. Carbonyl groups were measured by the 2,4-dinitrophenylhydrazine-coupled method, and the fibrinogen clotting activity was assessed by different functional assays. In irradiated fibrinogen, the carbonyl group concentration was elevated three-fold versus control; and moderate fragmentation of largely Aalpha and Bbeta chains was revealed. The rate of thrombin-catalyzed fibrinogen polymerization was inhibited (average 50%) with normal fibrinopeptide release and with a minor decrease of total clottable fibrinogen and alpha-polymer formation. Ascorbate reduced the incorporation of carbonyls to the fibrinogen molecule (by > 50% at 50 mmol/l; P < 0.001). Contrary to ascorbate, which alone delayed the fibrinogen polymerization rate, histidine abolished irradiation-induced inhibition of fibrinogen polymerization (by 80% at 50 mmol/l; P < 0.001). In conclusion, even though ascorbate effectively protects fibrinogen from oxidation due to its adverse effects on fibrinogen function, it may not serve as a suitable radioprotective. On the contrary, the first definite evidence is provided that radiation-sterilized fibrinogen in the presence of histidine greatly retains its clotting capability.

Late arterial responses (6 and 12 months) after (32)P beta-emitting stent placement: sustained intimal suppression with incomplete healing.

BACKGROUND: Three-month studies of stent-delivered brachytherapy in the rabbit model show reduced neointimal growth. However, intimal healing is delayed, raising the possibility that intimal inhibition is merely delayed rather than prevented. The purpose of this study was to explore the long-term histological changes after placement of beta-emitting radioactive stents in normal rabbit iliac arteries. METHODS AND RESULTS: Three-millimeter beta-emitting (32)P stents (6, 24, and 48 microCi) were placed in normal rabbit iliac arteries with nonradioactive stents as controls. Animals were euthanatized at 6 and 12 months, and histological assessment, morphometry, and analysis of endothelialization were performed. Morphometric measurements demonstrated a >50% reduction in intimal growth and percent lumen stenosis within 24- and 48-microCi stents versus control nonradioactive stents at both 6 and 12 months. However, the 24- and 48-microCi stents also showed delayed healing of the intimal surface, characterized by persistent fibrin thrombus with nonconfluent areas of matrix, incomplete endothelialization, and increased intimal cellular proliferation. Stent edge stenosis was present at 12 months in the 24- and 48-microCi stent groups, characterized by both intimal thickening and negative arterial remodeling. CONCLUSIONS: Inhibition of intimal growth is maintained 6 and 12 months after (32)P beta-emitting stent placement. However, delayed arterial healing, incomplete endothelialization, and edge effects are present.

Photosensitized labeling of solvent-exposed parts of proteins. Studies on fibrinogen and the fibrinogen-fibrin conversion.

A new technique for surface labeling of biological structures based on dye-sensitized photochemical coupling of labeled low molecular weight substances has been applied to studies on fibrinogen. When a solution of fibrinogen containing fluorescein and 3H-labeled tryptophan was illuminated with visible light, radioactive labeling of fibrinogen occurred in proportion to the illumination time. Determination of the relative labeling of the various peptide chains after reduction and separation by polyacrylamide gel electrophoresis showed the following labeling pattern; alpha : beta : gamma = 2.7 : 1.0 : 1.0, suggesting that the alpha chain is relatively more exposed to solvent than the other chains. When the illumination was performed in the presence of urea the labeling pattern was changed to 1.0 : 1.9 : 1.5, showing that the labeling is sensitive to conformation. When fibrinogen was transformed to fibrin and then labeled the labeling ratio was 3.0 : 1.3 : 1.0.

The binding of tumor localizing porphyrins to a fibrin matrix and their effects following photoirradiation.

The binding of two tumor localizing porphyrins, meso-tetra (4-carboxyphenyl) porphine (TCPP) and meso-tetra (4-sulfonatophenyl) porphine (TPPS4) to fibrin clots was determined in vitro. Both TCPP and TPPS4 were found to bind extensively, although weakly, to fibrin. No appreciable binding by Hematoporphyrin IX to the fibrin matrix was detectable. Similar results were obtained whether the clot was non-crosslinked or crosslinked with factor XIII. Photoirradiation of a porphyrin-impregnated non-crosslinked clot rendered it urea insoluble. Electrophoretic analysis indicated that the alpha chain of the fibrinogen molecule was most affected by photoirradiation. This was manifested as a loss of the alpha chain intensity and a concomitant increase of high molecular weight components, suggesting the formation of crosslinks. No substantial differences were noted in susceptibility to plasmin lysis between photoirradiated and non-irradiated clots. Photoirradiated clots were, however, significantly more resistant to lysis induced by the plasminogen activators urokinase and streptokinase, suggesting that inactivation of plasminogen within the clot matrix had occurred during photoirradiation. The relevance of porphyrin binding to fibrin with regard to tumor localization and destruction is also discussed.

Ultraviolet light inhibition of oriented fibrin formation in psoriasis.

Radially-oriented fibrin crystallization was induced by incubation of blood from psoriatic patients with killed Pseudomonas aeruginosa. The phenomenon was inhibited by irradiation of the buffy coat in plasma with UV-B. It was not inhibited by UV-A unless trioxsalen had been added. Addition of UV-A-irradiated plasma to non-irradiated buffy coat also inhibits the fibrin formation.