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Extracellular Matrix and Actin Cytoskeleton - the Control Unit of Interstitial Fluid VolumeReyhani, Vahid January 2014 (has links)
The regulation of fluid (water) volume in the body is crucial for tissue homeostasis. The interstitial fluid, which comprises almost 20% of the body fluid, is stored in the loose connective tissue and its volume is actively regulated by components of this tissue. The loose connective tissue provides a path for fluid flow from capillaries to the tissue and lymphatics. This fluid is partially stored in the interstitium and the remainder is directed to the lymphatics. The fibroblasts in the loose connective tissue actively compact the fibrous extracellular matrix (ECM) through mechanotransduction via integrins. This in turn, maintains the interstitial fluid pressure and keeps the ground substance underhydrated. The interstitial fluid pressure is part of the forces that regulate the efflux of fluid from capillaries and keep the ground substance underhydrated. The underhydrated ground substance has a potential to take up fluid 3-fold the plasma volume. Therefore, the active contraction of the ECM via fibroblasts is crucial to prevent the risk of evacuation of fluid from capillaries. During pathologies, such as inflammation and carcinogenesis, the interstitial fluid pressure and hence the interstitial fluid volume is altered. The results presented in this thesis show that the signaling events downstream of αVβ3 integrin, collagen-binding β1 integrins, and platelet-derived growth factor receptor β, that induce cell-mediated matrix contraction, included paired function of PI3K and PLCγ, cofilin activation, actin turnover, and generation of actomyosin forces. Furthermore, the results highlight new potential roles for fibrin and αVβ3 integrins, for instance during clearance of edema. Notably, fibrin extravasation at inflammatory sites induced αVβ3 integrin-dependent matrix contraction, leading to normalization of the altered interstitial fluid volume. It also reprograms the expression of ECM-related genes and hence induces ECM turnover. Taken together, these results provide further insight into the regulatory mechanism through which the loose connective tissue actively regulates the interstitial fluid volume.
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A mathematical model of wound healing and subsequent scarringCumming, Benjamin Donald January 2006 (has links)
Wound healing is governed by a complex cascade of related processes, involving cells, extracellular matrix and cytokines. In adults this always results in a scar whilst embryonic wound healing is scarless and extensive research worldwide is aimed at reducing scarring in adults. A mathematical framework for problems in dermal wound healing is developed that incorporates models of the individual processes involved. Cells are modelled as discrete individuals. Cytokines and other biologically active factors are modelled as continua. A novel tensorial approach is taken to modelling the extracellular matrix. The numeric and computational challenges associated with combining models for the individual processes are identified and investigated. These include the development of data structures and numeric methods for the continuous and discrete species. Effective visualisation methods for the large amounts of data generated by the model are also discussed. The possibilities offered by high performance computing in mathematical biology are highlighted in this work. The final part of this thesis gives an example of a combined model of the inflammatory and proliferative phases of dermal wound healing using the new computational framework. Both quantitative and qualitative methods are used to analyse the information-rich data sets generated by the model, offering insight into the dynamic systems that can be modelled using the new approach.
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Influence de la nature du fibrinogène sur la structure et la mécanique du caillot de fibrine / Influence of the nature of fibrinogen on the structure and mechanics of fibrin clotsGarcia gonzalez, Xabel 14 December 2016 (has links)
La formation du caillot de fibrine, processus clé de la coagulation sanguine, implique la polymérisation des monomères de fibrine en un réseau de fibres. Ce réseau contrôle les propriétés mécaniques du caillot et constitue le squelette sur lequel se base la cicatrisation. Si l’influence des conditions de réaction (pH, concentration, …) est bien connue, le rôle de la composition du fibrinogène sur la structure de la fibrine est inexploré. Cet aspect pourrait être important pour les pathologies cardiovasculaires qui présentent toutes une structure de fibrine anormale.Nous avons étudié la relation entre la composition de plusieurs fibrinogènes et les propriétés structurelles nano- et micro-métriques ainsi que la mécanique des caillots de fibrine. La composition en protéines co-purifiées de ces fibrinogènes a peu d’influence, alors que le profil de polydispersité contrôle la structure multi-échelle de la fibrine. Des mesures de diffusion des rayons x, de spectrophotométrie multi-longueur d’ondes et de microscopie confocale ont mis en évidence que les fibres provenant des fibrinogènes monodisperses sont quasi-cristallines, droites et rigides. Les fibres provenant de fibrinogènes polydisperses sont, elles, beaucoup moins organisées, courbées, avec un module de rigidité faible. Enfin, les propriétés mécaniques de la fibrine ont montré que la réponse des caillots aux déformations, aussi que les scenarios de rupture, sont directement liés à sa structure et donc significativement dépendants du profil de polydispersité des fibrinogènes. Ces résultats ouvrent de nouvelles perspectives dans plusieurs domaines, que ce soit pour l’utilisation optimale des fibrinogènes pour les dysfibrinogénémies et hémorragies, mais également pour la reconstruction tissulaire, ainsi que la compréhension du lien entre la structure anormale des caillots et les maladies cardiovasculaires. / Fibrin clot formation is one of the major processes leading to blood clotting. It involves the polymerization of fibrin monomers into a network of fibrin fibres. This network controls the mechanical properties of the clot and serves as a skeleton for wound healing. Environmental factors (pH, concentration, …) have been proved to influence polymerization, however the role of fibrinogen composition on the structure of fibrin remains unexplored. This aspect might be important for the case of cardiovascular pathologies, which present abnormal fibrin structures.We have determined the relation between different sources of fibrinogen with the nano- and micro-metric structural and mechanical properties of fibrin clots. The composition in co-purified proteins of the fibrinogens has no significant importance, however the polydispersity profile controls the multiscale properties of fibrin. Indeed, x-ray scattering, multi-wavelength spectrophotometry and confocal microscopy measurements have proved that fibres from monodisperse fibrinogens are quasi-crystalline, straight and rigid. Fibres from polydisperse fibrinogens are less organised, curbed and less rigid. Finally, the mechanical properties of fibrin showed that the response of clots to deformation, as well as the scenarios of rupture are closely related to the structure, and consequently related to the profiles of polydispersity. This opens outstanding perspectives in many fields such the optimisation of fibrinogen’s use on dysfibrinogenemias or haemorrhages, tissue regeneration or the understanding between the abnormal structure of clots and cardiovascular diseases.
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The in vitro antimicrobial activity of advanced platelet rich fibrin (A-PRF) against microorganisms of the oral cavityBhamjee, Feheem January 2017 (has links)
Magister Chirurgiae Dentium - MChD (Oral Medicine and Periodontics) / In recent years, the development and use of autologous platelet rich concentrates (PC's) has
gained traction within the rapidly progressive, multidisciplinary field of regenerative
medicine. A PC subtype, marketed as advanced platelet rich fibrin (A- PRF), is a recent
advancement of the original PRF protocol and promoted as a "blood concentrate"
containing platelets, leukocytes, circulating stem cells and endothelial cells. A-PRF in the
form of membranes, plugs, or even shredded particulates are increasingly being used as
surgical adjuncts in areas of previous infection or left exposed within the microbial rich oral
environment. Although recent literature has noted the biologic benefits of this material
within the context of wound healing and regeneration, the antimicrobial potential of APRF
has remained unexplored. The aim of this investigation is to determine if A-PRF
displays antimicrobial activity against microbes of the oral cavity with a null hypothesis that
its activity is no different to a clot of unprocessed venous blood.
Methodology: A-PRF and whole blood samples were obtained from consenting individuals
and utilised to conduct an in-vitro agar disk diffusion investigation to determine their
antimicrobial activity. Standardised samples of A-PRF, unprocessed clotted blood and 0.2%
chlorhexidine gluconate (CHX) were tested against organisms cultured from fresh oral rinse
samples and pure cultures of candida albicans, streptococcus mutans, staphylococcus
aureus and enterococcus faecalis. The antimicrobial activity was assessed in accordance to
the established principles of the agar disk diffusion method and measurement of inhibition
zones.
Results: A-PRF displayed antimicrobial activity against all of the individual organisms
tested within this study following a 24 hour incubation period. However, no significant
differences were noted between A-PRF and a natural clot of blood when tested against
cultures of the oral rinse sample. Finally, the antimicrobial activity of A-PRF is significantly
inferior to an equal volume of the CHX preparation.
Conclusion: Although A-PRF displays antimicrobial activity; its strength, spectrum and
biologic activity within a polymicrobial environment requires further investigation.
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Conception et élaboration de matériaux à biodégradabilité contrôlée pour la médecine régénérative / Design and development of materials with controlled biodegradability for regenerative medicineGoczkowski, Mathieu 18 December 2017 (has links)
Les gels de fibrine présentent un fort intérêt en médecine régénérative, puisqu’ils miment la matrice temporaire créée lors de la cicatrisation. Cependant, quand préparés à concentration physiologique, ils ne sont pas manipulables, ni conservables à sec. Pour contrer ces désavantages, ils peuvent être associés à un autre réseau de polymères, dans une architecture de Réseaux Interpénétrés de Polymères (RIP). Cette approche a été utilisée pour associer à un réseau de fibrine, un coréseau semi-synthétique d’albumine de sérum bovin (BSA) et de poly(oxyde d’éthylène) (POE), obtenu par photopolymérisation de BSA et PEG modifiés avec des fonctions méthacrylate (BSAm, PEGDM).Il a été démontré par des tests ex vivo et in vitro que ces matériaux ont de multiples applications potentielles, puisqu’ils supportent à leur surface, la croissance de nombreux types cellulaires. De plus, il a été observé que ces matériaux peuvent servir comme vecteurs pour la délivrance de molécules d’intérêt thérapeutique.La technologie a d’ailleurs été optimisée, en utilisant non plus des précurseurs modifiés avec des fonctions méthacrylate, mais acrylate. Cette modification a permis de réduire la toxicité du procédé de synthèse, tout en conservant les performances des matériaux. Il a également été démontré que divers matériaux optimisés ont des mécanismes de dégradation différents, et contrôlables par leur formulation initiale.Enfin, deux nouvelles familles de RIPs à base de fibrine ont été développées, en associant à un réseau de fibrine, un autre réseau de protéine, la fibroïne de soie. Des RIPs parfaitement manipulables ont été obtenus, supportant à leur surface la culture de fibroblastes. Ces matériaux sont donc prometteurs pour l’ingénierie tissulaire de la peau et d’autres applications. / Fibrin gels are of interest in regenerative medicine, as they mimic the provisory matrix synthesized during wound healing process. However, when prepared at physiologic concentration, these gels cannot be handled, nor stocked in dry state. To face these drawbacks, they can be associated with another polymer network, in an Interpenetrating Polymer Network (IPN). This strategy was used to associate to a fibrin network, a semi-synthetic conetwork composed of bovine serum albumin (BSA) and poly(ethylene oxide) (PEO), obtained by photopolymerization of methacrylate-modified BSA and PEG.It was demonstrated through ex vivo and in in vitro experiments that these materials have numerous potential applications, as they support on their surface, the culture of numerous cell types. Moreover, it was observed that they may be used as drug carrier for drug release applications.Moreover, the technology was optimized by modifying the methacrylate functions on the precursors for acrylate functions. This modification allowed to reduce the toxicity of the process, while preserving materials performances. It was also demonstrated that these optimized materials have different degradation mechanisms, which are controllable by their initial formulation.Finally, 2 new groups of fibrin-based IPNs were developed, by associating to a fibrin network, another protein network, the silk fibroin. Perfectly handable IPNs were obtained, which support on their surface the culture of fibroblasts. These materials are then very promising for skin tissue engineering, and most likely other applications.
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Cola de fibrina canina produzida com fibrinogênio obtido por crioprecipitação e precipitação com protamina a partir de diferentes categorias de plasma pobre em plaquetas / Canine fibrin glue produced with fibrinogen concentrated from cryo- and protamine precipitation using different platelet poor plasma categoriesGonçalves, Monalyza Cadori January 2015 (has links)
A cola de fibrina tem sido utilizada em diferentes procedimentos cirúrgicos como agente hemostático, selante e de suporte adesivo. No entanto, seu emprego na Veterinária ainda é limitado devido à falta de formulações não dependentes dos componentes de origem humana e de validação baseada em necessidades e condições cirúrgicas de animais. Objetivou-se avaliar a viabilidade da produção de cola de fibrina canina com fibrinogênio obtido por crioprecipitação (crio) e precipitação por protamina a partir de fontes plasmáticas mais disponíveis em bancos de sangue e centros hospitalares. Quatro categorias de plasma pobre em plaquetas foram utilizadas: plasma fresco (FR), congelado dentro de oito horas da colheita e processado em menos de uma semana após congelamento; plasma fresco congelado (FFP), com armazenamento inferior a um ano; plasma fresco congelado que ultrapassou um ano de armazenamento (eFFP), e plasma congelado com período entre colheita e congelamento maior que oito horas e de armazenamento superior a um ano (FP). No estudo in vitro de cada técnica, avaliou-se a concentração de fibrinogênio precipitado por meio do método de Clauss, as propriedades reológicas do gel por tromboelastografia (TEG) e as características estruturais do coágulo por microscopia eletrônica de varredura (SEM). O estudo in vivo consistiu da avaliação da praticidade de aplicação e das propriedades hemostáticas e adesivas das colas de fibrina resultantes em fígado e intestino de coelho (Oryctolagus cuniculus). Em avaliação prévia do protocolo de crio quanto ao uso de bolsas ou tubos, o aproveitamento do material inicial não diferiu, mas os tubos se mostraram mais simples, rápidos e homogêneos para o processamento, além de permitirem aumento da concentração final. O protocolo crio em comparação ao de protamina foi superior na precipitação de fibrinogênio coagulável nas avaliações de Clauss e TEG. Os coágulos formados se mostraram semelhantes entre os dois protocolos na SEM, no modelo de hemostasia hepática e na adesão à serosa intestinal. O uso de aprotinina com o protocolo de protamina não prejudicou a aplicação da cola sobre o intestino. Na crio, plasmas com maior tempo de armazenamento (eFFP, FP) se mostraram significativamente superiores aos mais frescos (FFP, FR) nas análises por Clauss e TEG. Não foi possível identificar diferenças estatísticas entre os tipos de plasma no protocolo protamina em nenhum dos parâmetros avaliados. Estudos adicionais e ajustes nos testes para avaliação de soluções concentradas são necessários para determinação do efeito dos protocolos e tempo de armazenamento do plasma congelado sobre o fibrinogênio precipitado e demais componentes plasmáticos na cola de fibrina. Adequações e pesquisas ainda são necessárias para aproveitamento da precipitação de fibrinogênio por protamina e a partir de plasma fresco com a finalidade de obtenção rápida de cola de fibrina. Bolsas de plasma menos requisitadas em bancos de sangue veterinários representam uma fonte importante de fibrinogênio para a produção de cola de fibrina canina em centros hospitalares apropriadamente equipados, viabilizando seu uso em diferentes aplicações cirúrgicas e pesquisas relacionadas. / Fibrin glue (FG) has been widely used in surgery for hemostatic, adhesive, sealant, and would healing support. In veterinary surgery, however, its use has been hindered by lack of specie-specific formulations and validation of its properties and biological characteristics. This study evaluated methods of fibrinogen precipitation from canine plasma envisioning autologous and allogeneic FG production for surgical use. The efficacy of cryo and protamine fibrinogen precipitation methods in producing canine FG was assessed by analysis on feasibility of each protocol with most available canine plasma sources, rheological and structural characteristics of the resultant FG clot and the hemostatic and adhesive properties of FG during in vivo application. The plasma categories studied included fresh plasma (FR), obtained and frozen within 8 hours from blood collection and processed within a week; fresh frozen plasma (FFP), frozen within 8 hours from blood collection and stored for up to a year; expired fresh frozen plasma (eFFP), plasma frozen within 8 hours from blood collection but stored for more than a year; and, frozen plasma (FP), which was frozen after 8 hours from collection and stored for more than a year. Comparison of cryoprecipitation among plasma types was previously performed in both 120-mL bags and 50-mL tubes and analyzed by Clauss. Total precipitation capacity did not differ significantly between bags and tubes. Nevertheless, the processing was more easily and homogeneously performed in tubes and allowed tailoring the final concentration. Cryoprecipitation generated better results in Clauss and TEG in comparison to protamine protocol. The resultant fibrin glue clots of cryo- and protamine-precipitation showed similar ultrastructure in scanning electron microscopy (SEM) and performance in the in vivo evaluations with the rabbit hepatic and intestinal incision models. The use of aprotinin in the protamine clot seemed beneficial in the intestinal evaluation. With cryoprecipitation, eFFP and FP were superior to FFP in the assessments performed by Clauss and TEG. Fresh plasma performed poorly with cryoprecipitation. Significant differences were not detected among plasma categories processed with protamine precipitation in any of the assays performed. While cryoprecipitation was more reliable regarding homogeneity and capacity to increase final fibrinogen concentration, protamine protocol was faster and simpler considering the equipment required. Although, older plasma units generated significantly more cryoprecipitated and/or clottable fibrinogen, further studies are needed to validate the assays with such high concentrated solutions and to elucidate the effect of freezing storage on precipitation and clottability of fibrinogen intended for FG production. Adjustments on protamine protocol and improvements on fibrinogen precipitation from fresher plasma sources would support the use of autologous or allogeneic plasma for on-site production of canine FG. Veterinary hospitals, blood banks, and patients can benefit from usage of surplus plasma units for FG production aiming surgical and scientific needs.
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Nanostructure des fibres de fibrine / Nanostruture of fibrin fibers.Yeromonahos, Christelle 12 October 2011 (has links)
La formation d'un caillot de fibrine, processus clé de la coagulation sanguine, implique la polymérisation des monomères de fibrinogène en un réseau de fibres de fibrine. Bien que ce réseau contrôle l'ensemble des propriétés mécaniques du caillot et constitue le squelette sur lequel se base la reconstruction des tissus, sa structure aux échelles inférieures au micron est très mal caractérisée. Nous avons démontré que l'analyse du spectre de lumière visible transmis à travers un caillot permet de déterminer simultanément, quantitativement et en conditions quasi-physiologiques, plusieurs paramètres essentiels de cette nanostructure, à savoir le rayon et la concentration interne en protéines des fibres. Cette méthode de spectrophotométrie a montré le caractère extraordinairement poreux de ces fibres et comment l'environnement de la réaction (concentrations en fibrinogène, en thrombine, température, force ionique) influe sur leur dimension et leur porosité. Cette méthode a ensuite permis de caractériser les effets respectifs sur cette structure de différentes molécules anti-coagulantes, montrant l'action spécifique de l'enoxaparine par rapport aux héparines non-fractionnées et au pentasaccharide. Enfin, nous avons construit un prototype à vocation hospitalière (spectrophotomètre) afin d'étudier la cinétique de polymérisation de la fibrine, non seulement en système purifié en combinaison avec nos spectres de diffusion de rayons X, mais également sur des plasmas de patients présentant des troubles de l'hémostase. Des discussions sont en cours avec un laboratoire pharmaceutique afin d'intégrer cette méthode sur des appareils de diagnostic. / The formation of a fibrin clot is one of the major processes leading to blood coagulation. It involves the polymerization of fibrinogen monomers into a network of fibrin fibers. This network controls the overall mechanical properties of the clot and serves as a scaffold to promote wound healing. However its structure at scales less than one micron is very poorly characterized. We demonstrated that an analysis of the visible light spectra transmitted through fibrin clots enables the simultaneous determination, in quantitative terms and in conditions near physiological, of several key parameters of this nanostructure, i.e. the radius and the protein content of the fibers. This spectrophotometry technique has shown the extraordinary porous nature of these fibers and how the reaction parameters (fibrinogen and thrombin concentrations, temperature, ionic strength) control their size and their porosity. This method was then used to characterize the respective effects on the structure of different anticoagulant molecules, showing the specific action of enoxaparin compared with unfractionated heparin and pentasaccharide. We built a prototype (spectrophotometer), used at hospital, to study the kinetics of fibrin polymerization, not only in purified system in combination with our X-ray spectra, but also in plasmas of patients with bleeding disorders. Discussions are underway with a pharmaceutical company to integrate this method on diagnostic equipment.
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Efeito da raspagem com laser de Er, Cr:YSGG nas superfícies radiculares: estudos in vitroOliveira, Guilherme José Pimentel Lopes de [UNESP] 29 March 2010 (has links) (PDF)
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oliveira_gjpl_me_arafo.pdf: 645436 bytes, checksum: 36e8a12c99620e4820f3710a3bb8b0d3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Foram utilizados 60 dentes humanos nessa pesquisa. No primeiro experimento, 15 dentes extraídos por doença periodontal foram selecionados para avaliar a influência da irradiação com laser de Er,Cr: YSGG sobre a morfologia e adesão de células sanguíneas sobre as superfícies radiculares. As 60 amostras provenientes desses dentes foram divididos em 3 grupos de acordo com o tipo de tratamento aplicado: Grupo 1- RAR; Grupo 2- Irradiação com laser de Er,Cr: YSGG; Grupo 3- RAR e irradiação com o laser de Er,Cr: YSGG. 10 amostras de cada grupo foram avaliados quanto a adesão de elementos sanguíneos, e as outras 10 amostras foram avaliados quanto a morfologia da superfície radicular por MEV. Os testes de Kruskall-Wallis e Mann-Whitney foram utilizados para avaliar os resultados. Em relação à adesão de elementos sanguíneos, este estudo não demonstrou diferenças estatísticas entre os grupos (p=0.359), a análise morfológica demonstrou que as superfícies radiculares irradiadas com o laser de Er-Cr:YSGG foram mais rugosas que as do grupo controle (G2-G1: p=0.0003 e G3-G1: p=0.0003). No segundo experimento, 20 dentes foram utilizados para avaliar a influência do ângulo de irradiação do laser de Er,Cr:YSGG sobre a rugosidade e o desgaste das superfícies radiculares. Cada face proximal foi dividida em 3 áreas, sendo que a área superior foi tratada com raspagem e alisamento radicular, a área média não foi submetida a nenhum tipo de tratamento e a área inferior foi irradiada com o laser de Er,Cr:YSGG. Os dentes foram divididos em 4 grupos ,com 5 dentes cada, a depender da angulação da aplicação da irradiação do laser de Er,Cr:YSGG na área ( 30º, 45º, 60º, 90º). A rugosidade das áreas foram avaliadas através de um rugosímetro e posteriormente os dentes foram submetidos a processamento histológico... / 60 human teeth were used in that research. In the first experiment, 15 extracted teeth for periodontal disease were selected to evaluated the effect of Er,Cr:YSGG irradiation on root surfaces for adhesion of blood components and morphology. 60 root surface specimens were obtained by selecting four from each tooth. Samples were divided into three groups of 20 each, according to treatments. Group 1 (G1) was treated by scaling and root planing (SRP), Group 2 (G2) was irradiated by Er,Cr:YSGG laser and Group 3 (G3) was treated by SRP and Er,Cr:YSGG laser irradiation. Blood was placed on each of 10 specimens from each of the three groups, to evaluate adhesion of blood components to the root surfaces. A morphological analysis was made of the root surfaces of the other 10 specimens from each group by scanning electron microscope (SEM). Statistical processing was done with the Kruskal-Wallis and Mann-Whitney tests. No statistical differences for adhesion of blood components to root surfaces were found between the groups (p = 0.359). However, morphological analysis disclosed that all root surfaces irradiated by Er,Cr:YSGG laser (100%) were rougher than surfaces that were not irradiated (G1-G2: p = 0.0003 and G1-G3: p = 0.0003). In the second experiment, 20 teeth were used to evaluated the effect of the working tip angulations on root roughness and substance removal using Er,Cr:YSGG radiation. The distal and mesial surfaces of each tooth was divided in 3 areas. The upper area was treated with scaling and root planing. The medium area was not submitted to any treatment and the lower area was irradiated with Er,Cr:YSGG laser. The teeh were divided in 4 groups, with 5 teeth each depending the working tip angulations using Er,Cr:YSGG at the lower area (30º, 45º, 60º, 90º).The roughness surfaces were evaluated by a profilometer, and... (Complete abstract, click electronic access below)
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Cola de fibrina canina produzida com fibrinogênio obtido por crioprecipitação e precipitação com protamina a partir de diferentes categorias de plasma pobre em plaquetas / Canine fibrin glue produced with fibrinogen concentrated from cryo- and protamine precipitation using different platelet poor plasma categoriesGonçalves, Monalyza Cadori January 2015 (has links)
A cola de fibrina tem sido utilizada em diferentes procedimentos cirúrgicos como agente hemostático, selante e de suporte adesivo. No entanto, seu emprego na Veterinária ainda é limitado devido à falta de formulações não dependentes dos componentes de origem humana e de validação baseada em necessidades e condições cirúrgicas de animais. Objetivou-se avaliar a viabilidade da produção de cola de fibrina canina com fibrinogênio obtido por crioprecipitação (crio) e precipitação por protamina a partir de fontes plasmáticas mais disponíveis em bancos de sangue e centros hospitalares. Quatro categorias de plasma pobre em plaquetas foram utilizadas: plasma fresco (FR), congelado dentro de oito horas da colheita e processado em menos de uma semana após congelamento; plasma fresco congelado (FFP), com armazenamento inferior a um ano; plasma fresco congelado que ultrapassou um ano de armazenamento (eFFP), e plasma congelado com período entre colheita e congelamento maior que oito horas e de armazenamento superior a um ano (FP). No estudo in vitro de cada técnica, avaliou-se a concentração de fibrinogênio precipitado por meio do método de Clauss, as propriedades reológicas do gel por tromboelastografia (TEG) e as características estruturais do coágulo por microscopia eletrônica de varredura (SEM). O estudo in vivo consistiu da avaliação da praticidade de aplicação e das propriedades hemostáticas e adesivas das colas de fibrina resultantes em fígado e intestino de coelho (Oryctolagus cuniculus). Em avaliação prévia do protocolo de crio quanto ao uso de bolsas ou tubos, o aproveitamento do material inicial não diferiu, mas os tubos se mostraram mais simples, rápidos e homogêneos para o processamento, além de permitirem aumento da concentração final. O protocolo crio em comparação ao de protamina foi superior na precipitação de fibrinogênio coagulável nas avaliações de Clauss e TEG. Os coágulos formados se mostraram semelhantes entre os dois protocolos na SEM, no modelo de hemostasia hepática e na adesão à serosa intestinal. O uso de aprotinina com o protocolo de protamina não prejudicou a aplicação da cola sobre o intestino. Na crio, plasmas com maior tempo de armazenamento (eFFP, FP) se mostraram significativamente superiores aos mais frescos (FFP, FR) nas análises por Clauss e TEG. Não foi possível identificar diferenças estatísticas entre os tipos de plasma no protocolo protamina em nenhum dos parâmetros avaliados. Estudos adicionais e ajustes nos testes para avaliação de soluções concentradas são necessários para determinação do efeito dos protocolos e tempo de armazenamento do plasma congelado sobre o fibrinogênio precipitado e demais componentes plasmáticos na cola de fibrina. Adequações e pesquisas ainda são necessárias para aproveitamento da precipitação de fibrinogênio por protamina e a partir de plasma fresco com a finalidade de obtenção rápida de cola de fibrina. Bolsas de plasma menos requisitadas em bancos de sangue veterinários representam uma fonte importante de fibrinogênio para a produção de cola de fibrina canina em centros hospitalares apropriadamente equipados, viabilizando seu uso em diferentes aplicações cirúrgicas e pesquisas relacionadas. / Fibrin glue (FG) has been widely used in surgery for hemostatic, adhesive, sealant, and would healing support. In veterinary surgery, however, its use has been hindered by lack of specie-specific formulations and validation of its properties and biological characteristics. This study evaluated methods of fibrinogen precipitation from canine plasma envisioning autologous and allogeneic FG production for surgical use. The efficacy of cryo and protamine fibrinogen precipitation methods in producing canine FG was assessed by analysis on feasibility of each protocol with most available canine plasma sources, rheological and structural characteristics of the resultant FG clot and the hemostatic and adhesive properties of FG during in vivo application. The plasma categories studied included fresh plasma (FR), obtained and frozen within 8 hours from blood collection and processed within a week; fresh frozen plasma (FFP), frozen within 8 hours from blood collection and stored for up to a year; expired fresh frozen plasma (eFFP), plasma frozen within 8 hours from blood collection but stored for more than a year; and, frozen plasma (FP), which was frozen after 8 hours from collection and stored for more than a year. Comparison of cryoprecipitation among plasma types was previously performed in both 120-mL bags and 50-mL tubes and analyzed by Clauss. Total precipitation capacity did not differ significantly between bags and tubes. Nevertheless, the processing was more easily and homogeneously performed in tubes and allowed tailoring the final concentration. Cryoprecipitation generated better results in Clauss and TEG in comparison to protamine protocol. The resultant fibrin glue clots of cryo- and protamine-precipitation showed similar ultrastructure in scanning electron microscopy (SEM) and performance in the in vivo evaluations with the rabbit hepatic and intestinal incision models. The use of aprotinin in the protamine clot seemed beneficial in the intestinal evaluation. With cryoprecipitation, eFFP and FP were superior to FFP in the assessments performed by Clauss and TEG. Fresh plasma performed poorly with cryoprecipitation. Significant differences were not detected among plasma categories processed with protamine precipitation in any of the assays performed. While cryoprecipitation was more reliable regarding homogeneity and capacity to increase final fibrinogen concentration, protamine protocol was faster and simpler considering the equipment required. Although, older plasma units generated significantly more cryoprecipitated and/or clottable fibrinogen, further studies are needed to validate the assays with such high concentrated solutions and to elucidate the effect of freezing storage on precipitation and clottability of fibrinogen intended for FG production. Adjustments on protamine protocol and improvements on fibrinogen precipitation from fresher plasma sources would support the use of autologous or allogeneic plasma for on-site production of canine FG. Veterinary hospitals, blood banks, and patients can benefit from usage of surplus plasma units for FG production aiming surgical and scientific needs.
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Cola de fibrina canina produzida com fibrinogênio obtido por crioprecipitação e precipitação com protamina a partir de diferentes categorias de plasma pobre em plaquetas / Canine fibrin glue produced with fibrinogen concentrated from cryo- and protamine precipitation using different platelet poor plasma categoriesGonçalves, Monalyza Cadori January 2015 (has links)
A cola de fibrina tem sido utilizada em diferentes procedimentos cirúrgicos como agente hemostático, selante e de suporte adesivo. No entanto, seu emprego na Veterinária ainda é limitado devido à falta de formulações não dependentes dos componentes de origem humana e de validação baseada em necessidades e condições cirúrgicas de animais. Objetivou-se avaliar a viabilidade da produção de cola de fibrina canina com fibrinogênio obtido por crioprecipitação (crio) e precipitação por protamina a partir de fontes plasmáticas mais disponíveis em bancos de sangue e centros hospitalares. Quatro categorias de plasma pobre em plaquetas foram utilizadas: plasma fresco (FR), congelado dentro de oito horas da colheita e processado em menos de uma semana após congelamento; plasma fresco congelado (FFP), com armazenamento inferior a um ano; plasma fresco congelado que ultrapassou um ano de armazenamento (eFFP), e plasma congelado com período entre colheita e congelamento maior que oito horas e de armazenamento superior a um ano (FP). No estudo in vitro de cada técnica, avaliou-se a concentração de fibrinogênio precipitado por meio do método de Clauss, as propriedades reológicas do gel por tromboelastografia (TEG) e as características estruturais do coágulo por microscopia eletrônica de varredura (SEM). O estudo in vivo consistiu da avaliação da praticidade de aplicação e das propriedades hemostáticas e adesivas das colas de fibrina resultantes em fígado e intestino de coelho (Oryctolagus cuniculus). Em avaliação prévia do protocolo de crio quanto ao uso de bolsas ou tubos, o aproveitamento do material inicial não diferiu, mas os tubos se mostraram mais simples, rápidos e homogêneos para o processamento, além de permitirem aumento da concentração final. O protocolo crio em comparação ao de protamina foi superior na precipitação de fibrinogênio coagulável nas avaliações de Clauss e TEG. Os coágulos formados se mostraram semelhantes entre os dois protocolos na SEM, no modelo de hemostasia hepática e na adesão à serosa intestinal. O uso de aprotinina com o protocolo de protamina não prejudicou a aplicação da cola sobre o intestino. Na crio, plasmas com maior tempo de armazenamento (eFFP, FP) se mostraram significativamente superiores aos mais frescos (FFP, FR) nas análises por Clauss e TEG. Não foi possível identificar diferenças estatísticas entre os tipos de plasma no protocolo protamina em nenhum dos parâmetros avaliados. Estudos adicionais e ajustes nos testes para avaliação de soluções concentradas são necessários para determinação do efeito dos protocolos e tempo de armazenamento do plasma congelado sobre o fibrinogênio precipitado e demais componentes plasmáticos na cola de fibrina. Adequações e pesquisas ainda são necessárias para aproveitamento da precipitação de fibrinogênio por protamina e a partir de plasma fresco com a finalidade de obtenção rápida de cola de fibrina. Bolsas de plasma menos requisitadas em bancos de sangue veterinários representam uma fonte importante de fibrinogênio para a produção de cola de fibrina canina em centros hospitalares apropriadamente equipados, viabilizando seu uso em diferentes aplicações cirúrgicas e pesquisas relacionadas. / Fibrin glue (FG) has been widely used in surgery for hemostatic, adhesive, sealant, and would healing support. In veterinary surgery, however, its use has been hindered by lack of specie-specific formulations and validation of its properties and biological characteristics. This study evaluated methods of fibrinogen precipitation from canine plasma envisioning autologous and allogeneic FG production for surgical use. The efficacy of cryo and protamine fibrinogen precipitation methods in producing canine FG was assessed by analysis on feasibility of each protocol with most available canine plasma sources, rheological and structural characteristics of the resultant FG clot and the hemostatic and adhesive properties of FG during in vivo application. The plasma categories studied included fresh plasma (FR), obtained and frozen within 8 hours from blood collection and processed within a week; fresh frozen plasma (FFP), frozen within 8 hours from blood collection and stored for up to a year; expired fresh frozen plasma (eFFP), plasma frozen within 8 hours from blood collection but stored for more than a year; and, frozen plasma (FP), which was frozen after 8 hours from collection and stored for more than a year. Comparison of cryoprecipitation among plasma types was previously performed in both 120-mL bags and 50-mL tubes and analyzed by Clauss. Total precipitation capacity did not differ significantly between bags and tubes. Nevertheless, the processing was more easily and homogeneously performed in tubes and allowed tailoring the final concentration. Cryoprecipitation generated better results in Clauss and TEG in comparison to protamine protocol. The resultant fibrin glue clots of cryo- and protamine-precipitation showed similar ultrastructure in scanning electron microscopy (SEM) and performance in the in vivo evaluations with the rabbit hepatic and intestinal incision models. The use of aprotinin in the protamine clot seemed beneficial in the intestinal evaluation. With cryoprecipitation, eFFP and FP were superior to FFP in the assessments performed by Clauss and TEG. Fresh plasma performed poorly with cryoprecipitation. Significant differences were not detected among plasma categories processed with protamine precipitation in any of the assays performed. While cryoprecipitation was more reliable regarding homogeneity and capacity to increase final fibrinogen concentration, protamine protocol was faster and simpler considering the equipment required. Although, older plasma units generated significantly more cryoprecipitated and/or clottable fibrinogen, further studies are needed to validate the assays with such high concentrated solutions and to elucidate the effect of freezing storage on precipitation and clottability of fibrinogen intended for FG production. Adjustments on protamine protocol and improvements on fibrinogen precipitation from fresher plasma sources would support the use of autologous or allogeneic plasma for on-site production of canine FG. Veterinary hospitals, blood banks, and patients can benefit from usage of surplus plasma units for FG production aiming surgical and scientific needs.
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