The goal of nerve tissue engineering is to promote and guide axon growth across a site of nerve injury without misdirection. Bioengineered tissue scaffolds have been shown to be promising for the regeneration of damaged peripheral nerves. Schwann cells play a pivotal role following nerve injury by forming aligned “bands of Büngner” that promote and guide axon regeneration into the distal nerve segment. The incorporation of living Schwann cells into various hydrogels has therefore been urged during the fabrication of tissue engineered nerve scaffolds. The aim of this research is to characterize biomaterials suitable for 3D bioplotting of nerve repair scaffolds. Here a novel technique of scaffold fabrication has been optimized to print alginate-based three-dimensional tissue scaffolds containing hyaluronic acid and living Schwann cells. Alginate/hyaluronic acid scaffolds were successfully fabricated with good printability and cell viability. Addition of the polycation polyethyleneimine (PEI) during the fabrication process stabilized the structure of alginate through the formation of a polyelectrolyte complex and had a significant influence on the degree of swelling, degradation rate, mechanical property, and release kinetics of incorporated protein within the scaffolds. A preliminary in vivo study showed the feasibility of implanting 3D printed alginate/hyaluronic acid scaffolds as nerve conduits in Sprague-Dawley (SD) rats with resected sciatic nerves. However alginate/hyaluronic acid scaffolds were found to be unsuitable for axonal regeneration. Further in vitro culture of Schwann cells was performed in collagen type-I, fibrin, fibrin/hyaluronic acid, and their combination with alginate. It was found that Schwann cells had more favorable cell morphology in fibrin/hyaluronic acid or collagen without alginate. Schwann cell proliferation and alignment were better in fibrin/hyaluronic acid. Therefore fibrin/hyaluronic acid is more ideal than most other hydrogel formulations for use in the bioprinting of nerve repair tissue engineering scaffolds, which incorporate cellular elements. As Schwann cells also align along the long axis of the printed fibrin/hyaluronic acid strands, 3D bioprinting of multiple layers of crosslinked fibrin strands can be used to fabricate a nerve conduit mimicking the bands of Büngner.

A multitude of biophysical signals, including chemical, mechanical, and contact guidance cues, are embedded within the extracellular matrix (ECM) to dictate cell behavior and determine cell fate. To understand the complexity of the cell-matrix interaction and how changes to the ECM contribute to the development of tissues or diseases, three-dimensional (3D), culture systems that can decouple the effects of these cues on cell behavior are required. This dissertation describes the development and characterization of approaches based on multiphoton excitation (MPE) to control the chemical, mechanical, and topographical presentation of micro-3D-printed (μ-3DP) protein hydrogels independently. Protein hydrogels were chemically functionalized via the MPE-induced conjugation of benzophenone-biotin without altering the physical properties of the matrix. Complex, immobilized patterns and chemical gradients were generated within protein hydrogels with a high degree of spatial resolution in all axes. Hydrogel surfaces were also labeled with adhesive moieties to promote localized Schwann cell adhesion and polarization. Laser shrinking, a method based on MPE to manipulate the topographical and mechanical presentation of protein hydrogels after fabrication, is also presented. Topographical features on an originally flat substrate are created with depths approaching 6 μm. The Young’s modulus of protein hydrogels can also be increased by 6-fold (~15 – ~90 kPa) using laser shrinking, and parameters can be adjusted to create continuous gradient profiles for studying durotaxis. At determined scan conditions, the two properties can be adjusted independently of each other. Most importantly, the physical properties of the hydrogels can be manipulated in situ to study the effects of dynamic changes to the substrates on cells. As a potential tool to monitor cellular responses to presented cues, fluorescent probes that detect nitric oxide are characterized. Collectively, these technologies represent a key advance in hydrogel tunability, as the platforms presented offer independent, dynamic, and spatiotemporal control of the chemical, mechanical, and topographical features of protein hydrogels. The introduced technologies expand the possibilities of protein hydrogels to clarify underlying factors of cell-matrix interactions that drive morphogenesis and pathogenesis, and are broadly applicable to a multitude of physiological systems. / text

No description available.

Bone marrow-derived mesenchymal stem cells (MSC) have been shown to provide neuroprotection after transplantation into the injured nervous system. The present thesis investigates whether adult human and rat MSC differentiated along a Schwann cell lineage could increase their expression of neurotrophic factors and promote regeneration after transplantation into the injured peripheral nerve and spinal cord. Human and rat mesenchymal stem cells (hMSC and rMSC) expressed characteristic stem cell surface markers, mRNA transcripts for different neurotrophic factors and demonstrated multi-lineage differentiation potential. Following treatment with a cocktail of growth factors, the hMSC and rMSC expressed typical Schwann cells markers at both the transcriptional and translational level and significantly increased production of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Age and time in culture are of relevance for clinical settings and growth-promoting effects of hMSC from young donors (16-18 years) and old donors (67-75 years) were compared. Undifferentiated hMSC from both young and old donors increased total neurite length of cultured dorsal root ganglion (DRG) neurons. Differentiation of hMSC from the young donors, but not the eldery donors, further enhanced the neurite outgrowth. Undifferentiated hMSC were cultured for eleven weeks in order to examine the effect of in vitro expansion time on neurite outgrowth. hMSC from the young donors maintained their proliferation rate and their ability to enhance neurite outgrowth from DRG neurons. Using a sciatic nerve injury model, a 10mm gap was bridged with either an empty tubular fibrin glue conduit, or conduits containing hMSC, with and without cyclosporine treatment. Cells were labeled with PKH26 prior to transplantation. At 3 weeks after injury the conduits with cells and immunosuppression increased regeneration compared with an empty conduit. PKH26 labeled human cells survived in the rat model and the inflammatory reaction could be suppressed by cyclosporine. After cervical C4 hemisection, BrdU/GFP-labeled rMSC were injected into the lateral funiculus rostral and caudal to the spinal cord lesion site. Spinal cords were analyzed 2-8 weeks after transplantation. Transplanted MSC remained at the injection sites and in the trauma zone for several weeks and were often associated with numerous neurofilament-positive axons. Transplanted rMSC induced up-regulation of vascular endothelial growth factor in spinal cord tissue rostral to the injury site, but did not affect expression of brain-derived neurotrophic factor. Although rMSC provided neuroprotection for rubrospinal neurons and significantly attenuated astroglial and microglial reaction, cell transplantation caused aberrant sprouting of calcitonin gene-related peptide immunostained sensory axons in the dorsal horn. In summary these results demonstrate that both rat and human MSC can be differentiated towards the glial cell lineage, and show functional characteristics similar to Schwann cells. hMSC from the young donors represent a more favorable source for neurotransplantation since they maintain proliferation rate and preserve their growth-promoting effects in long-term cultures. The data also suggest that differentiated MSC increase expression of neurotrophic factors and support regeneration after peripheral nerve and spinal cord injury.

Thesis (Ph.D). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2009/06/08). Includes bibliographical references.

No description available.

Orientador: Vânia Niéto Brito de Souza / Resumo: A infecção pelo Mycobacterium leprae (ML) estimula um processo de desdiferenciação e proliferação das células de Schwann (SCs) que pode contribuir para a disseminação do bacilo. Os macrófagos (MOs) são células efetoras da resposta imune que promovem a eliminação de patógenos, entretanto, na hanseníase são colonizados pelo ML. Sabe-se que os MOs podem apresentar uma polarização funcional na qual os MOs M1 apresentam características pró-inflamatórias e microbicidas enquanto os MOs M2 atuam na reparação tecidual e possuem perfil anti-inflamatório. SCs infectadas pelo ML produzem mediadores capazes de interferir com a função dos MOs aumentando sua sobrevida e promovendo sua migração. Embora diferentes programas funcionais tenham sido observados em MOs de pacientes com formas polares da hanseníase a influência de SCs nesse processo não é sabida. Neste estudo avaliamos se SCs infectadas pelo ML podem interferir na polarização de MOs murinos derivados de medula óssea. Para tanto, culturas primárias de SCs murinas foram infectadas experimentalmente com bacilos viáveis e cocultivadas com MOs. Nossos resultados indicam que a produção de óxido nítrico foi baixa nas culturas de MOs após a infecção com o bacilo, mas mostrou-se aumentada nas coculturas de MOs e SCs infectadas pelo ML. A infecção com ML não induziu produção significante das citocinas IL-6, IL-10 e TNF em culturas de MOs e SCs, entretanto, a interação entre MOs e SCs infectadas com o bacilo resultou em aumento na produção de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Mycobacterium leprae (ML) infection stimulates dedifferentiation and proliferation of Schwann cells (SCs) that may contribute to the spread of the bacillus. Macrophages (MOs) are effector cells of the immune response that promote the elimination of pathogens, however, in leprosy they are colonized by ML. It is known that MOs can present a functional polarization in which M1 MOs show pro-inflammatory and microbicidal activities while M2 MOs act in tissue repair presenting an anti-inflammatory profile. SCs infected by ML produce mediators able to interfere with MOs function, increasing their survival and promoting their migration. Although different functional programs have been observed in MOs from patients with polar forms of leprosy, the influence of SCs in this process is not known. In this study we evaluated whether SCs infected with ML could interfere in the polarization of murine MOs derived from bone marrow. For this purpose, primary cultures of murine SCs were experimentally infected with viable bacilli and co-cultivated with MOs. Our results indicate that nitric oxide production was low in cultures of MOs after infection with the bacillus, but it was increased in the co-cultures of MOs and ML-infected SCs. The infection with ML did not induce significant production of IL-10, TNF and IL-6 in cultures of MOs and SCs, however, the interaction between MOs and ML infected-SCs resulted in increased production of cytokines, mainly IL-10, inducing a decrease in the TNF/IL-10 ... (Complete abstract click electronic access below) / Mestre

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

Orientador: Francesco Langone / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T17:39:09Z (GMT). No. of bitstreams: 1 Hussein_Fernanda_M.pdf: 13730743 bytes, checksum: 0ded9b4ee837e3040ae51d3a2c5d6b02 (MD5) Previous issue date: 2007 / Resumo: As células tronco são células indiferenciadas capazes de se auto replicar e de se diferenciar em diversos tipos celulares maduros com funções especializadas. As células tronco de medula óssea são particularmente interessantes por serem células multipotentes, ou seja, geram células de diversos tecidos, são de fácil obtenção através da aspiração da medula óssea femural e eliminam os problemas de rejeição por ser possível realizar transplante autólogo.As células de Schwann são um dos tipos celulares mais importantes do Sistema Nervoso Periférico (SNP). Elas são uma fonte de sinais para a geração e desenvolvimento dos nervos periféricos, da mielinização e, ainda, auxiliam na regeneração axonal no caso de lesões tanto do SNP quanto do SNC. Os estudos enfocando a indução da diferenciação de células progenitoras a tipos celulares específicos desejados estão em seus passos iniciais. Já são conhecidos diversos fatores que favorecem o desenvolvimento de um determinado tipo celular a partir de células progenitoras in vitro. Contudo, permanecem desconhecidos os fatores intrínsecos ao organismo que induzem esse processo. Faz-se necessário portanto que se investigue, ainda in vitro, porém sem manipulações exógenas e mimetizando o microambiente corpóreo, quais fatores endógenos são os responsáveis pela deflagração do processo de diferenciação. Neste sentido, o Meio Condicionado por células de Schwann mostrou exercer efeitos importantes sobre as células mesenquimais de medula óssea, promovendo sua proliferação e possivelmente induzindo o processo de diferenciação nestas células. Ainda, a Eletroforese 2DE comparativa mostrou uma similaridade maior entre CS e CMMO do que com as CMMO cultivadas em MC, indicando que esta última poderia estar sofrendo um processo de diferenciação celular, diferentemente das CS e das CMMO / Abstract: Bone marrow mesenchymal stem cells (BMMSC) are capable to differentiate into several cell types. The differentiation depends on culture media molecules signaling. In this study we investigated the effect of Schwann cell conditioned media on BMMSC morphology and molecular phenotype. Schwann cells (SC) were isolated from adult Wistar rats sciatic nerve (Glia, 17: 327-338, 1996) and were cultivated in DMEM plus 10% FBS (D10). Every 48 hours the SC conditioned media (CM) were collected and stored at -80°C. The BMMSC were isolated from adult Wistar rats femur bone marrow (PNAS, 95:3908-3913,1995). BMMSC were maintained in D10. Subsequently, they were cultivated in CM for 7, 14 and 21 days. After 14 days in CM it was detected changes on the BMMSC typical flattened morphology to the SC bipolar morphology. The frequency of these changes increased after 21 days. These changes have been followed by changes on immunoreactivity and S100b protein localization on BMMSC. Before they were cultivated in CM, the BMMSC showed low immunoreactivity to S100b protein on cytoplasm. Culture in CM generated a S100b label gradual increase and it was found in nuclear compartment. Only the nucleous was S100b labeled, after 21 days. On the other hand, the bipolar morphology cells showed cytoplasmatic and nuclear S100b+. These findings agree with Deloume et al. (Mol. Cell. Neurosci. 27:453-465, 2004) about the S100b immunolabelling during the differentiation of glial progenitor cells into oligodendrocytes. Our results suggest that molecules present in CM are capable of inducing phenotypes changes related with the S100b expression and it roles on differentiation of BMMSC into SC. Moreover, 2DE electrophoresis shown a extent similarity between SC and BMMSC. This result suggests that the BMMSC cultivated in CM could be in a differentiation process / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

OBJETIVO: avaliar o efeito das neuregulinas 1-alfa e 1-beta na regeneração de nervos ciáticos de camundongos C57BL/6J, adultos, machos, através da técnica de tubulização. MÉTODOS: Utilizaram-se 18 animais, divididos em 3 grupos, implantando-se prótese de polietileno em falhas de 4,0 mm no nervo ciático esquerdo: grupo 1 contendo apenas colágeno purificado (Vitrogen®); grupo 2, colágeno associado a neuregulina 1-alfa; grupo 3 com colágeno e neuregulina 1-beta. O grupo controle foi formado por 6 segmentos de nervos ciáticos direitos. Após 4 semanas, os animais foram sacrificados; extraiu-se segmento do ponto médio do nervo regenerado no interior das próteses, padronizaram-se cortes histológicos e confecção das lâminas para análise histomorfométrica. Confrontaram-se os resultados estatisticamente. RESULTADOS: Os animais tratados com neuregulinas tiveram maior número de axônios mielinizados, com diferença estatisticamente significante quando comparados ao grupo colágeno. Não houve diferença estatística entre os grupos de neuregulinas 1-alfa e 1-beta. CONCLUSÃO: a adição de neuregulinas proporcionou aumento significativo do número de fibras mielinizadas. / PURPOSE: To evaluate the effect of neuregulins 1-alpha and 1-beta on the regeneration the sciatic nerves of male adult C57BL/6J mice, using the tubulization technique. METHODS: Eighteen animals were used, divided into three groups. A polyethylene prosthesis was implanted in a 4.0 mm defect of the left sciatic nerve, as follows: group 1 containing only purified collagen (Vitrogen®); group 2, collagen with neuregulin 1-alpha; group 3, collagen with neuregulin 1-beta. The control group was formed by six segments of right sciatic nerves. Four weeks later, the animals were sacrificed. A segment from the midpoint of the nerve regenerated inside the prostheses was extracted, histological sections were standardized and slides were made up for histomorphometric analysis. The results were statistically compared using the Tukey multiple comparisons test and Students t test. RESULTS: The animals treated with neuregulins had greater numbers of myelinized axons, with a statistically significant difference in relation to the collagen-only group. There was no statistical difference between the neuregulin 1-alpha and 1- beta groups. CONCLUSION: It was concluded that the addition of neuregulins provided a significant increase in the number of myelinized fibers.