Introduction Peripheral nerve injury is common in clinical practice. The usual etiologies are penetrating injury, stretch, compression, crush and ischemia. Outcome of nerve injury depends on the etiology and also the management. Nerve defect is a challenging scenario. The current gold standard of managing a nerve defect is autologous nerve graft. However, due to the selection of nerve graft and donor site morbidity, artificial nerve conduits are gaining popularity. However, there are drawbacks of single hollow conduit such as lack of internal support to prevent conduit collapse and inability so as to recreate the proper native spatial arrangement of cells and extracellular matrix within the conduit. In this study, the biocompatibility and efficacy of five-channel and eight-channel Crosslinked Urethane-doped Polyester Elastomers (CUPEs) as nerve guidance conduit will be evaluated through a rat model with reconstruction of segmental peripheral nerve defect. Material and method Eighteen adult Sprague-Dawley rats were used. Tthey were randomly allocated to three groups: autograft group, five-channel conduit group and eight-channel conduit group with each consisted of six rats. A 10mm nerve defects were created at the right sciatic nerve. They were bridged with reverse autograft, 5-channel conduit and 8-channel conduit. After eight weeks the rats were euthanized and the reconstructed nerves were harvested for histomorphometric analysis. Results All conduits showed regenerated nerve tissue inside. There was no collapse of the conduits. There were no severe tissue reaction or scarring near the reconstructed nerve. No neuroma was formed. Histomorphometric analysis showed nerve regeneration was enhanced with increasing number of channels inside conduit. There was overall drop in fiber density between proximal and distal segment among all groups. Conclusions CUPE nerve guidance conduit is biocompatible and shows good nerve regeneration in reconstructing nerve defect. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Medical Sciences

The golden method of peripheral nerve system injury is the nerve autograft, but it is associated with drawbacks such as donor site morbidity, needs of second incisions and the shortage of nerve grafts. Comparatively, connecting the nerve defect directly is an alternative. Unfortunately, if the defects are long, the induced tension will deteriorate the nerve regeneration. These limitations led to the development of artificial nerve guidance conduit (NGC). The market available NGC have problems of unsatisfactory functional recovery and may collapse after the implantation. These are attributed to material and structural deficiencies. Therefore, there is essential to study a biomaterial, which has excellent biological and physical properties to fit the NGC application. In addition, some studies suggested that the poor functional recovery resulted from the NGC implantation were due to the lack of micro-guidance inside the conduit. Thus, it is necessary to investigate the structural influence on the functional recovery of peripheral nerve injury. Crosslinked urethane-doped polyester elastomer (CUPE) is newly invented for a blood vessel graft because it possesses similar mechanical properties of blood vessel which is similar to nerve as well. Therefore, CUPE was also considered to be the NGC. Its biocompatibility has been proved to be excellent in the previous study done by Dr. Andrew SL, Ip. Targeting on the long peripheral nerve regeneration, the aims of this study are (1) to investigate the biocompatibility of CUPE in in-vitro condition and (2) to study the influence of nerve-like structure on the peripheral nerve system injury in an animal model. The ultimate goal is to enhance the functional recovery of peripheral nerve system injury by implanting a flexible biomaterial, CUPE, which has a nerve-like microarchitecture. It is hypothesized that the nerve-like structure can promote the axonal regeneration. The surface energy and roughness of CUPE were investigated. It showed a relatively low surface energy compared to other conventional biopolymers such that the cell adhesion and also the proliferation were inhibited. Therefore, the CUPE was modified by the immersion into a high glucose DMEM. The change in the hydrophilicity, roughness and cell viability of medium treated CUPE were studied. The hydrophilicity of treated CUPE was increased but the roughness was remaining unchanged whereas the pH of the immersion solution did not cause any effect on the cell activity on the CUPE. In the pilot animal study, five channels along the CUPE-NGC had a similar myelinated fiber density and population compared to the nerve autograft. Also, the channels in the CUPE-NGC were fragmented. In summary, the medium treatment could enhance the hydrophilicity of CUPE and the cell activity on CUPE. Such modifications did not governed by the pH of the medium. The NGC-CUPE with five channels, which imitated a basic nerve structure was shown to have a similar tissue regeneration and the functional recovery as the nerve autograft did. The results proved the hypothesis that the nerve-like structure can promote the functional recovery of peripheral nerve system injury with the use of a new biomaterial, CUPE as the NGC substrate. / published_or_final_version / Orthopaedics and Traumatology / Master / Master of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Not available / text

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Since the discovery of peripheral nerve regeneration nearly a century ago, the mechanisms that guide this regeneration have been elusive. This project aimed to describe how an axon is able to traverse the environment of the body and precisely reinnervate its target cell. Using a novel technique of combining light and electron microscopy, I observed reinnervating axons in transgenic mice to answer the questions of whether Schwann cells are an important guidance cue for the motor neuron and whether the outgrowing axon is fully developed or the process is a step-wise process of activation. The data suggests that Schwann cell contact is important for the tip of the regenerating axon to guide the axon back to its synapse on the muscle fiber. Further, it seems that the tip of the axon is not capable of synaptic transmission as it lacks active zones, suggesting that reinnervation is a step-wise process. / Department of Physiology and Health Science

Orientador: Alexandre Leite Rodrigues de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T22:46:07Z (GMT). No. of bitstreams: 1 Pierucci_Amauri_D.pdf: 4256178 bytes, checksum: fe35fc60e55f7ea9104ff8bf2bc809f5 (MD5) Previous issue date: 2007 / Resumo: A regeneração periférica é um fenômeno intrincado que envolve diferentes tipos celulares, dentre os quais as células de Schwann são os componentes celulares não neurais mais importantes. Após a lesão periférica, as células de Schwann proliferam e, juntamente com os macrófagos, participam na fagocitose dos fragmentos de mielina e dos axônios em degeneração. Essas auxiliam na orientação axonal em direção ao órgão alvo através da formação das bandas de Büngner. Ainda, atuam no rearranjo dos componentes da matriz extracelular do microambiente do nervo lesado, bem como na produção de vários fatores neurotróficos, entre eles, o fator neurotrófico do nervo (NGF), fator neurotrófico derivado do cérebro (BDNF), fator neurotrófico de crescimento ciliar (CNTF), visando à manutenção, desenvolvimento e regeneração dos neurônios após a lesão. As lesões nervosas que acometem no nervo periférico podem ser resultado de traumas como esmagamento, transecção parcial ou completa do nervo. Quando ocorre a transecção completa do nervo, há a perda de continuidade e forma-se uma fenda entre o coto proximal e o coto distal. No sentido de reparar-se o nervo lesionado, foram desenvolvidas diversas técnicas, incluindo-se o emprego de autoenxertos, próteses tubulares não absorvíveis e inertes (polietileno) e biorreabsorvíveis (biomateriais). Essas últimas têm a vantagem de sustentarem o inicio do processo regenerativo, orientando o brotamento axonal em direção ao coto distal, além de serem degradadas à medida que o nervo cresce em diâmetro. Podem ainda ser confeccionadas com as dimensões, formatos e porosidade desejados. Devido às características positivas destas próteses reabsorvíveis, a importância das células de Schwann e dos componentes da matriz extracelular, o presente trabalho tem como objetivos estudar a influência dos biomateriais poli L-ácido láctico e poli caprolactona sobre a expressão, pelas células de Schwann, das cadeias a1, a2 e ß1 que compõem as lamininas tipo I e II, bem como a expressão de colágeno tipo IV, através do emprego das técnicas de imunohistoquímica realizadas após a tubulização e imunocitoquímica através da cultura purificada de células de Schwann sobre os diferentes biomateriais. Além disso, avaliamos o comportamento das células de Schwann sobre os biomateriais, através da microscopia eletrônica de varredura. Já o resultado da regeneração axonal foi estudado através de uma análise morfológica pela microscopia de luz, microscopia eletrônica de transmissão e morfometria dos nervos regenerados. Comparando-se estruturalmente os tubos confeccionados pelo método de extrusão e solvente, pôde-se observar que o último apresentava espessura reduzida em comparação às próteses confeccionadas pelo método de extrusão. Ainda, a transparência dos tubos, ora propostos em nossa metodologia, influenciou positivamente durante o processo de implantação da prótese na tubulização. Após a regeneração, observou-se que o número de fibras regeneradas no interior dos tubos derivados das membranas de PCL foi significantemente maior, 30 e 60 dias após tubulização. Ainda, uma intensa marcação com S-100, colágeno tipo IV e laminina foi observada no nervo regenerado no interior das próteses, em cujos grupos utilizaram-se os biomaterias (PCL e PLLA). De fato, a imunomarcação demonstrou que os biomateriais e o microambiente no interior dos tubos foram capazes de estimular positivamente as células de Schwann em resposta à lesão nervosa periférica. Em conjunto, nossos resultados evidenciam que os tubos de PCL e PLLA derivados da membrana podem ser considerados um método alternativo na preparação de próteses tubulares visando o reparo do nervo periférico / Abstract: The present study proposed a new approach to produce tubular conduits designed for peripheral nerve repair. In this sense, membranes of PLLA and PCL were obtained after solvent evaporation and wrapped around a mandrel. The effectiveness of the nerve regeneration was compared with polyethylene and PCL extruded prosthesis 30 and 60 days after surgery. The comparison between extrusion and solvent tubes cleared shown structural differences which were directly proportional to the hardness and transparency. An important factor to be considered is that the fiber counting indicated that solvent PCL tubes provided a significantly greater number of axons 30 days after repair. Sixty days after operation, the greatest regenerative performance was obtained with PCL, regardless the method of construction of the tube. An intense labeling against S-100, type IV collagen and laminin could be observed in the tissue obtained from solvent PCL and PLLA groups, indicating that such constructions are able to positively stimulate Schwann cell responses. Overall, the present results provide evidence that solvent conduits may be regarded as an alternative preparation method for tubular prosthesis aiming peripheral nerve regeneration. In the in vitro study, PCL and PLLA solvent polymers were used for culturing purified of Schwann cells. The imunolabeling revealed an up-regularion of the expression of collagen IV, laminin I, laminin II and S-100 by the Schwann cells, showing that biodegradable polymers enhance the activity of such cells, positively influencing the peripheral nerve regeneration process / Doutorado / Anatomia / Mestre em Biologia Celular e Estrutural