As lesões que envolvem nervos periféricos, especialmente os traumatismos facias, são muito comuns e decorrentes principalmente de acidentes com veículos motorizados, lesões acidentais e quedas, que levam a fraturas do osso temporal ou lacerações da face e consequentemente lesões do nervo facial. A principal meta no estudo da regeneração nervosa é descobrir uma técnica adequada de reparo em lesões de nervos periféricos que traga como resultado a recuperação funcional das estruturas por eles inervadas. A sutura epineural é um método muito utilizado para recuperação de lesões nervosas, assim como o uso do adesivo de fibrina, que requer menor destreza do cirurgião. O adesivo derivado do veneno de serpente (CEVAP/UNESP, Botucatu-SP) é um selante biológico e biodegradável, pois não produz reações adversas, não contém sangue humano, apresenta uma boa capacidade adesiva, não transmite doenças infecciosas, e pode ser utilizado como coadjuvante em procedimentos de sutura convencional. Sendo assim, o objetivo deste trabalho foi comparar duas técnicas de recuperação de nervos periféricos lesionados: a sutura epineural término-terminal e o adesivo de fibrina derivado do veneno de serpente, e observar se o uso da laserterapia de baixa potência influencia esse processo de regeneração. Para isso, foram utilizados 42 ratos machos (Rattus norvegicus, Wistar), com 60 dias de vida, separados aleatoriamente em um Grupo Controle e quatro Grupos Experimentais, assim formados: Grupo Controle (GC, n=10), em que foi coletado o nervo facial íntegro aos 95 e 135 dias de vida; Grupo Experimental Sutura (GES, n=16) e Grupo Experimental Adesivo de Fibrina (GEF, n=16), onde no lado direito da face o ramo bucal do nervo facial foi seccionado e realizado a sutura epineural término-terminal e, no lado esquerdo da face, o ramo bucal do nervo facial foi seccionado e utilizado o adesivo de fibrina para coaptação das extremidades; Grupo Experimental Sutura e Laserterapia (GESL, n=16) e Grupo Experimental Adesivo de Fibrina e Laserterapia (GEFL, n=16), submetidos aos mesmos procedimentos de GES e GEF, adicionada a aplicação de Laser de Arseneto de Gálio Alumínio (GaAlAs) de pulso contínuo, comprimento de onda de 830 nm, 6J/cm2, por 24 segundos, três vezes por semana durante cinco semanas, em três pontos dos locais operados. Os animais dos Grupos Experimentais foram eutanasiados com 95 dias (cinco semanas pós-cirurgia) e 135 dias (dez semanas pós-cirurgia). As amostras coletadas foram submetidas à análise morfológica por microscopia óptica e eletrônica de transmissão, além de análise morfométrica da área e diâmetro da fibra, área e diâmetro do axônio, espessura e diâmetro da bainha de mielina. Observou-se brotamento axonal no coto distal do nervo facial em todos os Grupos Experimentais, com morfologia semelhante às fibras do Grupo Controle, e predomínio de fibras mielínicas sobre as amielínicas. Pode-se concluir que a reparação por meio da sutura epineural término-terminal apresentou melhores resultados em relação ao adesivo de fibrina e a laserterapia de baixa potência não influenciou o processo de regeneração. / The injuries involving peripheral nerves, especially facial traumatisms are very common and serious and longstanding facial paralysis lead to significant deterioration in the quality of the individuals life. The main goal in the study of nerve regeneration is finding a suitable repair technique for peripheral nerve injuries that bring results in the functional recovery of the structures innervated by them. Therefore, the aim of this study was to compare two techniques for recovery of injured peripheral nerves: the end-to-end epineural suture and fibrin adhesive derived from snake venom (CEVAP / UNESP, Botucatu-SP), and observeif the use of low-level laser therapy influences this regeneration process. For this purpose, 42 male rats (Rattus norvegicus, Wistar) were used , with 60 days of life, were randomly separated into a control group and four experimental groups, which were formed this way: Control Group (CG , n = 10), in which the intact facial nerve was collected at 95 and 135 days of life; Experimental Suture Group (ESG, n = 16 ) and Experimental Fibrin Adhesive Group (EFG, n = 16), where the right side of the face the buccal branch of the facial nerve was transectioned and the epineural end-to-end suture was performed and the left side of the face, the buccal branch of the facial nerve was transectioned and the fibrin glue was used for coaptation of the edges; Experimental Suture Laser Therapy Group (ESLG, n = 16) and Experimental Fibrin Adhesive Laser Therapy Group (EFLG, n = 16) underwent the same procedures of ESG and EFG , included the application of Laser Gallium- Aluminum-Arsenide (GaAlAs) by an 830 nm wavelength pulse continuous, 6 J/cm2, for 24 seconds, three times a week during five weeks at three points of the operated areas. The animals in the experimental groups were euthanized at 95 days (five weeks post-surgery) and 135 days (ten weeks post-surgery). The collected samples were subjected to morphological analysis by optical microscopy and transmission electronic microscopy, besides the morphometric analysis of the area and diameter of the fiber, area and diameter of the axon, thickness and diameter of the myelin sheath. It was observed axonal sprouting in the distal stump of the facial nerve in all experimental groups, with similar morphology to the fibers of the control group, and predominance of myelinated fibers to amyelinated. It can be concluded that the repair by epineural end-to-end suture showed better results in relation to fibrin adhesive and the low-level laser therapy did not influence the regeneration process

O adesivo de fibrina derivado do veneno de serpente é um selante biológico, constituído por componentes provenientes do plasma sanguíneo cujo mecanismo de ação se assemelha à última fase da coagulação fisiológica (formação do fibrinogênio). Ele tem sido utilizado no tratamento de lesões como colagem de tecidos moles, no entanto não existem evidências suficientes sobre a sua aplicação na estabilização de enxertos ósseos. Os enxertos, para serem efetivos no seu propósito, de reconstruir perdas ósseas, necessitam de vascularização e estabilização. Visto isso, este trabalho teve por objetivo avaliar, por meio de análise histológica e histomorfométrica, se o adesivo de fibrina promove fixação de enxerto do tipo onlay em calvária de ratos, e observar se o uso da laserterapia de baixa potência auxilia nesse processo. Foram utilizados 40 ratos da linhagem Wistar (Rattus norvegicus), separados aleatoriamente em dois grupos (EI e EII), nos quais foi realizada uma secção circular com uma broca trefina de 5 milímetros no osso parietal direito e a descorticalização do osso parietal esquerdo com uma broca esférica número 6. No grupo EI foi realizada a colagem do fragmento retirado do lado direito sobre o osso parietal esquerdo com adesivo de fibrina, e no Grupo EII os mesmos procedimentos do Grupo EI, associando-se a terapia por laser de baixa potência. Cinco animais de cada grupo foram eutanasiados nos períodos de 10, 20, 30 e 40 dias após a cirurgia. Após inclusão histológica de rotina, as peças foram submetidas à análise histológica e histomorfométrica. Observou-se, em ambos os grupos, a presença de tecido conjuntivo entre o enxerto autógeno e o leito receptor, que foi gradualmente transformando-se em tecido osteóide. No período final de análise o tecido conjuntivo encontrava-se quase ausente, sem evidência de reação inflamatória e tecido ósseo neoformado unindo o enxerto ao leito receptor em grande área da interface, ocorrendo uma regeneração óssea parcial. Na análise histomorfométrica, observou-se um melhor desempenho do laser no processo de formação de novo osso quando se comparou o Grupo EII com Grupo EI, nos mesmos períodos, demonstrando diferenças significativas em todos os períodos analisados. Conclui-se que o adesivo de fibrina derivado do veneno de serpente promoveu regeneração óssea parcial em enxerto do tipo onlay fixado em calvária, e que a laserterapia de baixa potência promoveu uma melhora nos resultados desse processo de regeneração. / The fibrin tissue adhesive derived from snake venom is a biological sealant constituted of components from blood plasma whose mechanism of action is similar to the last phase of physiological coagulation (formation of the fibrinogen). It has been used to treat injuries such as collage soft tissue; however there is insufficient evidence on its application in the stabilization of bone grafts. A graft, to be effective to this purpose, to rebuild bone loss requires vascularization and stabilization. Seen it, this study aimed to evaluate, by means of histological and histomorphometric analysis, if the fibrin glue promotes fixation of the onlay graft type in rat calvaria, and observe whether the use of low level laser therapy assists in this process. 40 Wistar rats (Rattus norvegicus) were randomly separated into two groups (EI and EII), in which was performed a circular perforation with a trephine drill of 5 mm in the right parietal bone section and nine small perforations the left parietal bone using a spherical drill number 6. In EI group the fragment removed of the right side was glued over the left parietal bone with fibrin tissue adhesive, and the same procedures of group EI was made in the EII group, associating with low-level laser therapy. Five animals from each group were euthanized on days 10, 20, 30 and 40 days after surgery. After histological routine inclusion, the pieces were subjected to histological and histomorphometric analysis. It was observed in both groups, the presence of the connective tissue between the allograft and the recipient bed, which was gradually transformed into osteoid tissue. In the final period of analysis, the connective tissue was almost absent, with no evidence of inflammatory reaction and neoformed bone uniting the graft to the recipient bed in great area of the interface, occurring a partial bone regeneration. In histomorphometric analysis, we observed a better laser performance in the training process of new bone when compared Group EII with EI Group, in the same periods, demonstrating significant differences in all periods analyzed. The conclusion of this study is that the fibrin adhesive derived from snake venom promoted partial bone regeneration in onlay graft type fixed in calvaria, and that low-level laser therapy promoted an improvement in the results of the regeneration process.

Orientador: Rui Seabra Ferreira Junior / Resumo: Atualmente são muitos casos de pacientes que perdem estrutura óssea em acidentes ou reabsorção patológica. A bioengenharia óssea é um tratamento promissor que visa reconstruir estas estruturas sem a morbidade do enxerto autógeno. O tecido ósseo é um conjuntivo especializado com a função principal de proteção e sustentação dos tecidos moles, mas também é responsável pela produção de tipos celulares e homeostase de minerais. Sua reparação é complexa com diferentes tipos celulares e agentes quimiotáticos que funcionam de forma orquestrada até a reparação. As terapias celulares vêm sendo estudadas para promover a reparação de defeitos que o organismo por si não consegue resolver. Células menos especializadas como as células-tronco embrionárias (ESCs) possuem grande potencial terapêutico, mas são complicadas eticamente. Já as células-tronco mesenquimais (MSC) podem ser autólogas, o que minimiza o risco de imunogenicidade mas necessitam área doadora do paciente. Atualmente ainda não há consenso quanto ao uso de células tronco na terapia regenerativa pois há grandes variáveis como a melhor forma de aplicação, a quantidade correta e o melhor tipo celular para a regeneração óssea. As células produzem mediadores químicos no local enxertado, que segundo pesquisas recentes é o principal mecanismo de reparação tecidual. Estes mediadores são depositados em abundância no meio de cultura durante a cultura celular e usados na bioengenharia com a ajuda de scaffolds. Os biopolímeros de fibrina ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Chapter I present a review about bone repair and its biological events, bioengineering, cells, fibrin biopolymer as scaffold and the secretoma derived from cell culture. Many patients nowadays lose bone structure in accidents or pathological reabsorption. Bone bioengineering is a promising treatment that aims to reconstruct these structures without autogenous graft morbidity. Bone tissue is a specialized connective tissue specialized in protecting and supporting soft tissues, but it is also responsible for the production of cell types and mineral homeostasis. The bone healing is a complex process where different cell types and chemotactic agents work in an orchestrated way. The cell therapies can promote the repair of defects that the body cannot solve. Less specialized cells like embryonic stem cells (ESCs) have great therapeutic potential, but are ethically complicated. In contrast, mesenchymal stem cells (MSCs) may be autologous, which minimizes the risk of immunogenicity but requires a patient's donor area. Currently there is still no consensus regarding the use of stem cells in regenerative therapy, studies uses different methods, cells and biomaterials for bone regeneration. Recent researches advocate that paracrine secretions by cells are main mechanism of tissue repair. These mediators are deposited in abundance in the culture medium during cell culture. Fibrin biopolymers (BF) are natural biomaterials to the body and can function as drug delivery of growth factors, c... (Complete abstract click electronic access below) / Doutor

Volumetric muscle loss (VML) typically results from traumatic incidents; such as those presented from combat missions, where soft-tissue extremity injuries account for approximately 63% of diagnoses. These injuries lead to a devastating loss of function due to the complete destruction of large amounts of tissue and its native basement membrane, removing important biochemical cues such as hepatocyte growth factor (HGF), which initiates endogenous muscle regeneration by recruiting progenitor cells. Clinical strategies to treat these injuries consist of autologous tissue transfer techniques, requiring large amounts of healthy donor tissue and extensive surgical procedures that can result in donor site morbidity and limited functional recovery. As such, there is a clinical need for an off-the-shelf, bioactive scaffold that directs patient’s cells to align and differentiate into muscle tissue in situ. In this thesis, we developed fibrin microthreads, scaffolds composed of aligned fibrin material that direct cell alignment along the longitudinal axis of the microthread structure, with specific structural and biochemical properties to recreate structural cues lost in VML injuries. We hypothesized that fibrin microthreads with an increased resistance to proteolytic degradation and loaded with HGF would enhance the functional, mechanical regeneration of skeletal muscle tissue in a VML injury. We developed a crosslinking strategy to increase fibrin microthread resistance to enzymatic degradation, and increased their tensile strength and stiffness two- to three-fold. This crosslinking strategy enhanced the adsorption of HGF, facilitated its rapid release from microthreads for 2 to 3 days, and increased the chemotactic response of myoblasts twofold in 2D and 3D assays. Finally, we implanted HGF-loaded, crosslinked (EDCn-HGF) microthreads into a mouse model of VML to evaluate tissue regeneration and functional recovery. Fourteen days post-injury, we observed more muscle ingrowth along EDCn-HGF microthreads than untreated controls, suggesting that released HGF recruited additional progenitor cells to the injury site. Sixty days post-injury, EDCn-HGF microthreads guided mature, organized muscle to replace the microthreads in the wound site. Further, EDCn-HGF microthreads restored the contractile mechanical strength of the tissue to pre-injured values. In summary, we designed fibrin microthreads that recapitulate regenerative cues lost in VML injuries and enhance the functional regeneration of skeletal muscle.

A incompetência glótica ainda representa um desafio para a laringologia. Implantes de biomateriais no espaço de Reinke ou no espaço paraglótico estão entre as opções de tratamento, e suturas e confecção de bolsões subepiteliais são normalmente utilizados para fixação desses implantes. Alternativamente, a cola biológica pode ser usada como adesivo nesses casos. A cola de fibrina (CF) é produto da reação de dois componentes do sistema de coagulação: o fibrinogênio e a trombina, que formam uma rede de fibrina, responsável, entre outros, pela adesão dos tecidos. Entretanto, além do efeito adesivo, a CF e seus componentes podem interferir no processo cicatricial, atuando sobre citocinas como o fator de crescimento transformador-beta (TGF-beta). O objetivo deste estudo é avaliar o efeito da cola de fibrina na deposição de colágeno após enxertia de fáscia em pregas vocais de coelhos. Dezoito coelhos foram submetidos a enxerto de fáscia em ambas as pregas vocais, sendo o lado esquerdo fixado com CF. Os coelhos foram sacrificados após 7, 30 e 90 dias. As laringes foram removidas e as pregas vocais preparadas para estudo histomorfométrico através da coloração picrossirius, a fim de avaliar a deposição de colágeno total em torno do enxerto. Foi observado um aumento estatisticamente significativo na densidade de colágeno em torno dos enxertos de fáscia nas pregas vocais que receberam a CF (p=0,0102) após 90 dias, em comparação com as pregas vocais controles. A aplicação da CF interferiu na deposição de colágeno em torno dos enxertos de fáscia, resultando em um aumento significativo na densidade de colágeno após 90 dias, possivelmente em decorrência da interação de seus componentes com citocinas e células envolvidas no processo de cicatrização / The glottal incompetence is still a challenge in Laryngology. Implants of biomaterials in Reinkes space or paraglottic space are among the treatment options, and sutures and pockets dissections are usually used to set these implants. Alternatively, biological glue can be used as adhesive in these cases. Fibrin glue (FG) is the product reaction of two components of the coagulation system: the fibrinogen and thrombin that form a fibrin net, responsible for the tissues adhesion, among other functions. However, the FG and its components may interfere in wound healing, interacting with cytokines as the transforming growth factor-beta (TGF-beta). The objective is to study the effect of fibrin glue on collagen deposition after fascia grafting on the rabbits vocal folds. Eighteen rabbits were submitted to the fascia graft on both vocal folds, being the left side fixed with FG. The rabbits were sacrificed after 7, 30 and 90 days. The larynx were removed and vocal folds prepared for histomorphometric study through Picrosirius Red stain, in order to evaluate the collagen deposition around the graft. There was a significant increase in collagen density around the grafts on the vocal folds that received FG (p=0.0102) after 90 days, compared with the control. FG application interfered in collagen deposition around fascia grafts, resulting in a significantly increase of collagen density after 90 days, which possibly resulted from the interaction of FG and its components with the cytokines and cells involved in the wound healing

Les brûlures cutanées graves dont les lésions profondes et étendues des tissus ne permettent pas d’envisager un traitement classique par autogreffes, bénéficient aujourd’hui de substituts épidermiques autologues pour leur recouvrement. En effet l’ingénierie tissulaire permet l’obtention d’épiderme de culture autologue (CEA) par l’amplification in vitro de kératinocytes à partir d’une petite biopsie de peau saine. Si ces CEAs ont permis la survie de nombreux patients depuis les années 1980, ils n’en restent pas moins largement perfectibles du point de vue de l’esthétique et de la fonctionnalité de l’épiderme et de la jonction dermo-épidermique régénérés mais aussi en termes de coûts et de prévention des zoonoses.Ainsi, ce travail de thèse s’inscrit dans le cadre d’une recherche translationnelle et porte sur l’élaboration d’un épiderme de culture par ingénierie tissulaire en vue d’une application clinique chez les grands brûlés, afin de favoriser la cicatrisation et la régénération épidermique. Ce travail porte également sur l’évaluation non clinique de notre substitut épidermique sur un modèle animal de brûlure cutanée.Après avoir développé un nouveau substitut épidermique sur fibrine de plasma humain (hPBES : human Plasma-Based Epidermal Substitute), nous avons procédé à l’évaluation détaillée de ses caractéristiques phénotypiques et fonctionnelles en comparaison au substitut épidermique de référence Epicel® (Genzyme, US). Nous avons montré des propriétés plus intéressantes en termes de clonogénicité et de caractère souche, de prévention de la différenciation, de prolifération, de potentiel de migration, et de conservation des protéines de la membrane basale avec notre produit par rapport à Epicel®.L’apport de la matrice de fibrine de plasma sur la culture des kératinocytes en comparaison à une matrice de fibrine issue de fibrinogène purifié et à une culture sans matrice a été étudié notamment sous l’angle des facteurs libérés par les matrices. Nous avons observé une amélioration des propriétés des substituts épidermiques par la fibrine : diminution de la différenciation terminale, augmentation du potentiel migratoire et sécrétion de facteurs pro-cicatrisants (GM-CSF et PDGF-BB). En outre, la fibrine de plasma améliore spécifiquement la morphogénèse épidermique, la prolifération kératinocytaire et la clonogénicité.Le procédé de culture de ce nouveau substitut a été adapté aux exigences réglementaires sans altération du potentiel régénératif: remplacement des cellules nourricières murines par des fibroblastes dermiques humains, adaptation du milieu de culture et sécurisation du plasma par traitement à l’amotosalen.Toutes les étapes d’un modèle de brûlure chez le rat nude reproduisant la technique chirurgicale employée pour la greffe de CEA ont été mises au point, mais un rejet du greffon épidermique n’a pas permis l’évaluation d’hPBES sur ce modèle. Nous avons donc évalué le substitut épidermique développé hPBES chez la souris NOD-SCID après greffe au fascia et mis en évidence une bonne prise de greffe épidermique permettant la régénération d’un épiderme humain aux caractéristiques proches d’une peau saine avec une réorganisation de la jonction dermo-épidermique des 2 semaines après greffe. / The deep and large injuries caused by massive burns prevent the use of split-thickness skin autografts. Today, autologous epidermal substitutes constitute an alternative treatment for skin regeneration. In fact tissue engineering allows the production of Cultured Epithelial Autografts (CEA) by in vitro keratinocyte amplification from a small healthy skin biopsy. The clinical use of CEA since the 1980’s has allowed an increase in the survival rate of burnt patients. However, CEAs present numerous drawbacks, among them the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction leading to heavy cosmetic and functional sequelae.This thesis focuses on the bioengineering of epidermal substitute for clinical burn treatment to enhance wound healing and skin regeneration, from a translational research point of view. This work also includes the development of an animal model of third degree burn for the evaluation of epidermal substitute.We first developed a new epidermal substitute cultured on a fibrin matrix from human plasma (hPBES: human Plasma-Based Epidermal Substitute). Then we characterized this new substitute with phenotypical and functional analyses, using as a reference the epidermal substitute Epicel® (Genzyme, US). We observed properties more favorable with hPBES than with Epicel® in terms of clonogenicity, stemness, differentiation level, proliferation, migration potential and basement membrane protein conservation.The influence of the plasma-based fibrin matrix on keratinocytes was studied in comparison with a culture on a fibrin from purified fibrinogen, and with a culture with no matrix. For this study we focused our analysis on the role of the released factors from fibrin matrices during epidermal substitute culture. Both fibrin matrices improved epidermal substitute properties: reduction of terminal differentiation, enhancement of migration potential, secretion of wound healing enhancing factors. Besides, plasma-based fibrin specifically promote epidermal morphogenesis, keratinocyte proliferation and clonogenicity.The culture process was adapted to European regulation requirements without diminishing its regenerative potential: substitution of murine feeder cells by human dermal fibroblasts, adaptation of culture medium and plasma viral inactivation using amotosalen treatment.A burn model including all the surgical steps used in clinics for CEA grating was developed on nude rats. However the evaluation of hPBES on this model could not be achieved due to graft rejection. Therefore we evaluated epidermal regeneration after hPBES graft on acute wounds on NOD-SCID mice. We showed a good graft rate, with the regeneration of a human epidermis close to healthy epidermis with a well-organized dermal-epidermal junction two weeks after hPBES grafting.

As lesões na face estão mais comuns na sociedade atual, envolvendo acidentes automobilísticos, quedas ou consequências iatrogênicas após procedimentos. Essas lesões podem afetar os nervos responsáveis pela musculatura facial repercutindo em alterações físicas, emocionais e psicossociais, por exemplo, no sistema estomatognático, na voz, na expressão e estética faciais, nos sentimentos e convívio social do indivíduo. Pesquisas atuais estão almejando melhores técnicas para o processo de reparo nervoso periférico e reabilitação funcional dessas lesões. As técnicas tradicionais como sutura epineural término-lateral e coaptação por meio de selante de fibrina são utilizadas com essa finalidade. O objetivo deste estudo foi avaliar a reparação do ramo bucal do nervo facial lesionado por meio da técnica términolateral de duas diferentes formas: sutura epineural e o novo selante heterólogo de fibrina, associadas ou não ao tratamento com laser de baixa potência. Foram utilizados trinta e dois ratos (Rattus norvegiccus, Wistar) com 80 dias de vida, distribuídos aleatoriamente em Grupo Controle (GC; n=8), e Grupos Experimentais (Grupo Experimental Sutura - GES e Grupo Experimental Fibrina GEF; n=12; Grupo Experimental Sutura Laser GESL e Grupo Experimental Fibrina Laser GEFL; n=12). Os animais do GC não receberam intervenção cirúrgica; no GES foi realizado, no lado direito da face, a secção do ramo bucal do nervo facial, onde o coto proximal foi suturado à tela subcutânea e o coto distal suturado de forma término-lateral ao ramo zigomático do nervo facial; no GEF, no lado esquerdo da face, foram realizados os mesmos procedimentos do GES, porém foi utilizada a coaptação com selante de fibrina do coto distal. Os grupos GESL e GEFL, além das técnicas descritas, receberam tratamento com aplicação de Laser Arseneto de Gálio Alumínio (GaAlAs), pulso contínuo, comprimento de onda de 830 nm, 6 J/cm2, por 24 segundos, três vezes por semana durante cinco semanas, em três pontos dos locais operados de ambos os lados. Foi realizada também a análise funcional do movimento das vibrissas de todos os grupos na 5ª e 10ª semanas pós-cirúrgica. Os animais foram eutanasiados 10 semanas após a cirurgia e as fibras nervosas foram analisadas morfologicamente, utilizando microscopia óptica e eletrônica, e morfometricamente por meio das medidas da área e diâmetro da fibra, área e diâmetro do axônio, espessura e diâmetro da bainha de mielina. Foi possível observar a regeneração no coto distal de fibras mielínicas e amielínicas. Nas variáveis mensuradas, ocorreu diferença significante na área da fibra nervosa entre os grupos GEF e GEFL. A recuperação funcional dos movimentos das vibrissas apresentou melhores resultados nos grupos com uso do selante de fibrina (GEF e GEFL), sendo que os grupos associados à laserterapia foram os que mais se aproximaram do GC. Portanto, concluiu-se que os dois métodos analisados para reparação nervosa periférica foram efetivos, permitindo o brotamento axonal no coto distal, e que a laserterapia proporcionou melhores resultados, tanto morfométricos quanto funcionais. / The injuries on the face are more common in today\'s society, involving motor vehicle accidents, falls or iatrogenic consequences after procedures. These injuries can affect the nerves responsible for facial muscles reflecting in physical, emotional and psychosocial changes, for example in the stomatognathic system, in voice, facial expression and aesthetics, feelings and social life of the individual. Current researches are aiming best techniques to the process of peripheral nerve repair and functional rehabilitation of these injuries. Traditional techniques such as end-to-side epineural suture coaptation by fibrin sealants are used for this purpose. The objective of this study was to evaluate the repair of buccal branch of the facial nerve injured by end-toside technique in two different ways: epineural suture and the new heterologous fibrin sealant, associated or not to treatment with low power laser. Thirty-two rats (Rattus norvegicus, Wistar) were used. They were 80 days old and were randomly divided into Control Group (CG, n=8) and Experimental Groups (Experimental Suture Group ESG and Experimental Fibrin Group - EFG; n=12; Experimental Suture Laser Group ESLG and Experimental Fibrin Laser Group - EFLG; n=12). The CG animals did not receive surgery; the ESG was performed on the right side of the face, the section of the buccal branch of the facial nerve, where the proximal stump was sutured to the subcutaneous tissue and the distal stump sutured end-to-side portion of the zygomatic branch of the facial nerve; EFG on the left side of the face, the same procedures were carried ESG, but was used coaptation with fibrin sealant of the distal stump. The groups ESLG and EFLG, and the techniques described, have been treated with the application of Laser Gallium Aluminum Arsenide (GaAlAs), continuous pulse, wave length 830 nm, 6J /cm2 for 24 seconds, three times per week for five weeks, at three points of the local operated from both sides. It also performed a functional analysis of the movement of the vibrissae of all groups in the 5th and 10th post-operativeweeks. The animals were euthanized 10 weeks after surgery and the fibers were morphologically analyzed, using light and electron microscopy, and morphometrically by means measures the area and diameter of the fiber, area and diameter of the axon, thickness and diameter of the myelin sheath. It was possible to observe regeneration in the distal stump of myelinated and unmyelinated fibers. The measured variables, there was a significant difference in the area of the nerve fiber between the EFG and EFLG groups. Functional recovery of the movements of the vibrissae the best results in the groups with fibrin sealant use (EFG and EFLG), and the groups associated with laser therapy were the most nearly CG. Therefore, it follows that the two methods discussed for peripheral nerve repair was effective, allowing for axonal sprouting in the distal stump, and that the laser treatment yielded better results, both morphometric and functional.

Considerando que os dados epidemiológicos apontam uma elevação alarmante na incidência de diabetes na população mundial, muitos estudos vêm sendo realizados buscando estabelecer propostas preventivas e terapêuticas para esta patologia e suas complicações, principalmente em relação a cicatrização de feridas. A maneira convencional de unir os tecidos e as margens de feridas, utilizando suturas pode causar problemas como fístula e formação de granulomas, devido a uma incompatibilidade do tecido com os materiais de sutura, deiscência quando os materiais de sutura absorvível mostram desintegração precoce e isquemia tecidual com conseqüente necrose de borda da ferida quando uma sutura é muito apertada. Dentre os novos meios de adesão tecidual que têm sido idealizados, um deles é o adesivo biológico, tendo como objetivo selar tecidos e feridas sem produzir qualquer tipo de trauma O adesivo cirúrgico derivado do veneno de serpente é um produto biológico e biodegradável que não produz reações adversas, não contém produtos derivados do sangue humano, portanto não favorece transmissão de doenças infecciosas, tem uma capacidade de bom adesivo, e pode ser usado como coadjuvante nos procedimentos de sutura convencional. Este trabalho analisou, portanto, em feridas cirúrgicas feitas no dorso de ratos Wistar machos de três meses de idade diabéticos e não diabéticos três materiais utilizados como coadjuvantes na cicatrização de feridas cirúrgicas: sutura de nylon 5.0, a cola de fibrina comercial Tissucol® e o adesivo cirúrgico do veneno de serpente. Os animais foram eutanasiados nos períodos de três e sete dias do pós-operatório e as áreas das feridas foram analisadas microscopicamente através das colorações de Hematoxilina & Eosina e Tricrômio de Mallory. No experimento realizado constatamos que a reepitelização no grupo controle foi favorecida pelo material Tissucol®. A presença de infiltrado inflamatório mononuclear foi significativamente proeminente pelo uso do adesivo cirúrgico derivado do veneno de cobra. Adicionalmente, a proliferação vascular foi significativamente favorecida pelo uso do adesivo cirúrgico derivado do veneno de cobra no grupo controle aos sete dias de proservação. As técnicas de sutura realizadas até hoje somente para manter a coaptação entre as margens das feridas podem e devem ser melhoradas com o uso de outros materiais disponíveis a fim de acelerar as fases da cicatrização e viabilizar o conforto e bem estar do paciente. / Whereas epidemiological data point to an alarming increase in the incidence of diabetes in the world population, many studies have been conducted to establish preventive and therapeutic proposals for this disease and its complications, especially in relation to wound healing. The conventional way of joining tissue and wound edges, using sutures may cause problems such as fistulas and granulomas due to incompatibility with the materials of the fabric of suture dehiscence when the absorbable suture materials show premature disintegration and tissue ischemia with subsequent necrosis of the wound edge when a suture is too tight. Among the new means of tissue adhesion that have been devised, one of them is the biological adhesive, aiming to \"paste\" and injured tissues without producing any kind of trauma. The surgical adhesive derived from snake venom is a biological product that is not biodegradable and produce adverse reactions, it does not contain human blood therefore does not favor the transmission of infectious diseases, have a good adhesive ability , and can be used as an adjunct to conventional suturing procedures. This study therefore considered in surgical wounds made in the back of male Wistar rats three months old diabetic and non-diabetic three materials used as adjuvants in the healing of surgical wounds: 5.0 nylon suture, the commercial fibrin seletante Tissucol® and surgical adhesive snake venom. The animals were sacrificed at three and seven days after surgery and wound areas were analyzed microscopically by staining with hematoxylin & eosin and Mallory trichrome . In the experiment we found that re-epithelialization in the control group was favored by the material Tissucol®. The presence of mononuclear cell infiltration was prominent significantly by the use of surgical adhesive derived from snake venom. Additionally, vascular proliferation was significantly enhanced by the use of surgical adhesive derived from snake venom in the control group at seven days of follow up. The suture techniques performed today only to maintain coaptation between the margins of the wounds can and should be improved with the use of other materials available to accelerate the stages of healing and facilitate the comfort and wellbeing of the patient.

Tourniquet mediated Ischemia Reperfusion (I/R) injury causes damage to skeletal muscle, often resulting in prolonged functional impairment. The current study utilizes immunohistochemistry (IHC) to determine whether the controlled release of the anabolic factor, insulin-like growth factor-I (IGF-I), from the biodegradable PEGylated fibrin gel matrix can facilitate the recovery of skeletal muscle from I/R. Treatment groups following a 2-hour tourniquet applied to the limb of 6-9 month rats, included intramuscular injections of saline, PEGylated fibrin gel (PEG-Fib) only and IGF-I conjugated to PEGylated fibrin gel (PEG-Fib-IGF). Expression of the myogenic regulatory factors MyoD and myogenin detected via IHC in the PEG-Fib-IGF group was significantly lower compared to the saline group, showing a 1.4±0.8% nuclear co-localization for MyoD and a 2.0±0.8% nuclear co-localization for myogenin at 14 days of recovery. The saline group showed higher values, 31.4±4.4% and 44.1±7.3% for MyoD and myogenin nuclear co-localization respectively. A significantly greater percentage, 88.8±3.7% of Desmin positive myofibers was seen at 14 days of recovery, while a lower percentage of fibers expressing neonatal myosin, 7.7±2.7% was seen in the PEG-Fib-IGF group compared to the saline treatment group. These results indicate that IGF-I delivered intramuscularly via PEGylated fibrin gel, functions therapeutically in skeletal muscle recovery, from I/R mediated damage. In a separate injury model that deals with volumetric muscle loss, IHC analyses were performed to test the efficacy of a novel tissue engineering strategy utilizing extracellular matrix (ECM) as a scaffold. In this model, also called the defect model, a 1.0 X 1.0 cm piece of the lateral gastrocnemius was removed and replaced with a muscle-derived ECM. The constructs were then seeded with bone marrow derived cells (BMSCs), adipose derived stem cells (ADSCs) or the peroneal nerve was relocated to the area of the ECM implant. 42 days post recovery IHC analysis was performed on the ECM implants. The quantification of desmin-positive regenerating myofibers bearing centrally located nuclei, showed significantly greater values in the top, middle and bottom region of the ECM implants that received peroneal nerve relocation, when compared to the experimental group that received the ECM implant alone. Blood vessel density increases were seen within the middle region of the ECM implant groups that received BMSC+Nerve treatment and the bottom region of the ECM implant groups that received ADSC+Nerve treatment. Thus, these results corroborate the therapeutic effect of peroneal nerve relocation, which stimulated an increase in myofiber regeneration and vascular maintenance within the construct. / text

Previous work has shown that thrombin activatable fibrinolysis inhibitor (TAFI) was unable to prolong lysis of purified clots in the presence of Lys-plasminogen (Lys-Pg), indicating a possible mechanism for fibrinolysis to circumvent prolongation mediated by activated TAFI (TAFIa). Therefore, the effects of TAFIa on Lys-Pg activation and Lys-plasmin (Lys-Pn) inhibition by antiplasmin (AP) were quantitatively investigated using a fluorescently labeled recombinant Pg mutant which does not produce active Pn. High molecular weight fibrin degradation products (HMW-FDPs), a soluble fibrin surrogate that models Pn modified fibrin, treated with TAFIa decreased the catalytic efficiency (kcat/Km) of 5IAF-Glu-Pg cleavage by 417-fold and of 5IAF-Lys-Pg cleavage by 55-fold. A previously devised intact clot system was used to measure the apparent second order rate constant (k2) for Pn inhibition by AP over time. While TAFIa was able to abolish the protection associated with Pn modified fibrin in clots formed with Glu-Pg, it was not able to abolish the protection in clots formed with Lys-Pg. However, TAFIa was still able to prolong the lysis of clots formed with Lys-Pg. TAFIa prolongs clot lysis by removing the positive feedback loop for Pn generation. The effect of TAFIa modification of the HMW-FDPs on the rate of tissue type plasminogen activator (tPA) inhibition by plasminogen activator inhibitor type 1 (PAI-1) was investigated using a previously devised end point assay. HMW-FDPs decreased the k2 for tPA inhibition rate by 3-fold. Thus, HMW-FDPs protect tPA from PAI-1. TAFIa treatment of the HMW-FDPs resulted in no change in protection. Vitronectin also did not appreciably affect tPA inhibition by PAI-1. Pg, in conjunction with HMW-FDPs, decreased the k2 for tPA inhibition by 30-fold. Hence, Pg, when bound to HMW-FDPs, protects tPA by an additional 10-fold. TAFIa treatment of the HMW-FDPs completely removed this additional protection provided by Pg. In conclusion, an additional mechanism was identified whereby TAFIa can prolong clot lysis by increasing the rate of tPA inhibition by PAI-1 by eliminating the protective effects of Pn-modified fibrin and Pg. Because TAFIa can suppress Lys-Pg activation but cannot attenuate Lys-Pn inhibition by AP, the Glu- to Lys-Pg/Pn conversion is able to act as a fibrinolytic switch to ultimately lyse the clot. / Thesis (Master, Biochemistry) -- Queen's University, 2008-01-31 17:04:50.447