Cerca de 0,005% da população mundial sofre de lesão medular. O processo regenerativo do tecido nervoso apresenta limitada capacidade para repor as células danificadas (JOHANSSON et al., 1999) e produzir inibidores de crescimento dos axônios associados com a mielina para formação de cicatriz glial (OLSON, 2002). Apesar de resultados promissores, ainda existem controversas quanto ao uso de células-tronco. A eliminação da cicatriz glial, os benefícios de sua formação em diferentes fases e a avaliação da liberação de inibidores de crescimento axonal podem ser parâmetros de análise para o tratamento medular. A enzima condroitinase ABC atua nessa lesão. Neste trabalho avaliamos a interrupção do processo de liberação de inibidores axonais da cicatriz da glia em um tempo não agudo de 7 dias da lesão, sem descartar seus benefícios na fase de formação. Nosso objetivo foi utilizar terapia celular e estabelecer um protocolo de tratamento eficaz, criando uma linha de pesquisa nos estudos da lesão medular. Foi utilizado um grupo de coelhos experimental com realização de hemissecção dorsal e instituído o uso da condroitinase ABC, por aplicação, com micro injeção a curto prazo da lesão. Foi aplicada célula-tronco mesenquimal no foco da lesão após o tratamento da cicatriz da glia com a enzima. Avaliamos por imunohistoquimica a liberação de glial fibrillary acidic protein (GFAP) e sulfato de condroitina proteoglicano (SCPg) nos tecidos após o tratamento no qual foi pretendido fechar algumas lacunas e evitar falhas já descritas, e abrir uma nova esperança no tratamento de pacientes com lesão medular. Nossos resultados ainda mostraram um entendimento superficial sobre a enzima e sua ação sobre cicatrização da glia em associação com o implante celular. Foi aberta uma nova linha de questionamento sobre os benefícios causados à regeneração medular previamente a aplicação de células-tronco. / Around 0,005% of global human population is affected by Spinal Cord Injury (SCI). The regenerative process of neural tissue shows a limited capacity to replace damaged cells (JOHANSSON et al., 1999) and to produce growth inhibitors of associated axons with myelin to create glial scar (OLSON, 2002). Plenty of studies are being developed with stem cell and, despite successful results, there still are controversial opinions. The elimination of the glial scar, the benefits of its growth at different stages and the assessment of axonal growth inhibitors\' release can be parameters of analysis for treating spinal cord. The enzyme chondroitinase ABC acts in this lesion. In this paper we evaluated the release interruption of axonal inhibitors of glial scar in a non-acute 7 days term from injury, not disregarding its benefits during growth. Our goal was to use cell therapy and establish an effective treatment protocol, creating a research line for studies of spinal cord injury and its treatment. A group of rabbits was used under experimental model, conducting dorsal hemisection and application of chondroitinase ABC with micro injection in short-term injury. Mesenchymal stem cells were applied in the lesion focus after the glial scar treatment with the enzyme. Immunohistochemically, we evaluated the release of glial fibrillary acidic protein (GFAP) and sulfate chondroitin proteoglycan (SCPg) in tissues after treatment which was intended to close some gaps and avoid failures described above, and open a new hope in the treatment of patients with spinal cord injury. Our results also showed superficial understanding of the enzyme and its action on glial scarring in association with cell implant. It has opened a new line of questioning about the benefits due to spinal cord regeneration prior to application of stem cells.

This thesis demonstrated the effects of exogenous stimuli on engineered articular cartilage constructs and elucidated mechanisms underlying the responses to these agents. In particular, a series of studies detailed the effects of chondroitinase-ABC (C-ABC), hyaluronic acid (HA), and TGF-β1 on the biochemical and biomechanical properties of self-assembled articular cartilage. Work with C-ABC showed that this catabolic agent can be employed to improve the tensile properties of constructs. When constructs were cultured for 6 weeks, treating with C-ABC at 2 weeks enhanced the tensile stiffness. Furthermore, treating at 2 and 4 weeks synergistically increased tensile properties and allowed compressive stiffness to recover to control levels. Another study showed that combining C-ABC and TGF-β1 synergistically enhanced the biochemical and biomechanical properties of neotissue. Microarray analysis demonstrated that TGF-β1 increased MAPK signaling in self-assembled neocartilage whereas C-ABC had minimal effects on gene expression. SEM analysis showed that C-ABC increased collagen fibril diameter and fibril density, indicating that C-ABC potentially acts via a biophysical mechanism. Constructs treated with C-ABC and TGF-β1 also showed stability and maturation in vivo , exhibiting a tensile stiffness of 3.15±0.47 MPa compared to a pre-implantation stiffness of 1.95±0.62 MPa. To assess the response to HA application, studies were conducted to optimize HA administration and examine its effects in conjunction with TGF-β1. Applying HA increased the compressive stiffness 1-fold and increased GAG content by 35%, with these improvements depending on HA molecular weight, application commencement time, and concentration. Microarray and PCR analyses showed that HA also influenced genetic signaling, up-regulating multiple genes associated with the TGF-β1 pathway. In addition to genetic effects, the enhanced GAG retention due to HA treatment could increase the fixed charge density of the matrix and thereby increase resistance to compressive loading. Additive effects were observed when HA was applied in conjunction with TGF-β1, with the combined treatment increasing compressive stiffness and GAG content by 150% and 65%, respectively. In general, results demonstrated mechanisms underlying C-ABC, HA, and TGF-β1 treatments and showed how these agents can be applied to improve cartilage regeneration efforts.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-05-12T12:43:09Z No. of bitstreams: 1 andreluizdacunha.pdf: 5996425 bytes, checksum: b2672b9464385b63d7ce5aa90c90c7eb (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-12T15:45:22Z (GMT) No. of bitstreams: 1 andreluizdacunha.pdf: 5996425 bytes, checksum: b2672b9464385b63d7ce5aa90c90c7eb (MD5) / Made available in DSpace on 2017-05-12T15:45:22Z (GMT). No. of bitstreams: 1 andreluizdacunha.pdf: 5996425 bytes, checksum: b2672b9464385b63d7ce5aa90c90c7eb (MD5) Previous issue date: 2012-07-31 / Condroitim sulfato é um glicosaminoglicano sulfatado composto por unidades dissacarídicas formadas por ácido D-glucurônico e N-acetil-galactosamina. Este polissacarídeo é utilizado no Brasil em combinação com outros fármacos para tratamento de osteoartrite. Este trabalho teve como objetivo avaliar a qualidade do insumo farmacêutico condroitim sulfato de sódio utilizado em farmácias de manipulação. Para isso foi necessário estabelecer uma técnica de eletroforese em gel de poliacrilamida para análise de açúcares (FACE), bem como produzir enzimas específicas para a digestão de condroitim sulfato. Diferentes dissacarídeos insaturados, monossacarídeos sulfatados e açúcares neutros derivatizados com 2-aminoacridona foram separados por FACE, usando dois sistemas tampão distintos. Fracionamos por cromatografia de interação hidrofóbica três condroitinases de F. heparinum – condroitinase AC, C e B. Por FACE caracterizamos a atividade dessas enzimas e identificamos contaminação de condroitinase AC por sulfatase específica para hexosamina sulfatada na posição 4, que foi inibida por fluoreto de sódio (10 mM). Glucuronidase com atividade específica para dissacarídeo não sulfatado e 6-sulfatado foi detectada em condroitinase C, e sacarolactona (10 mM) foi capaz de impedir somente a degradação de ΔDi0S. Dosagem de condroitim sulfato em amostras farmacêuticas foi realizada por eletroforese em gel de agarose e por titulação fotométrica. Teor superior a 80% foi encontrado em apenas cinco matériasprimas. O peso molecular médio do condroitim sulfato nessas amostras variou entre 16 e 26 kDa, e análise dissacarídica por FACE revelou proporções próximas entre dissacarídeo 4-sulfatado e 6-sulfatado. Onze amostras apresentaram teor inferior a 20%, e análise por FACE demonstrou presença de contaminação por lactose ou polímeros de glicose com diferentes pesos moleculares. Concluímos neste estudo que há grande distorção entre os resultados reportados no certificado de análise de onze insumos analisados e os resultados obtidos nesse trabalho. Torna-se necessário, portanto, maior fiscalização e regulação do comércio deste produto. / Chondroitin sulfate is a sulfated glycosaminoglycan composed of alternate sequences of D-glucuronic acid and N-acetyl-D-galactosamine. This polysaccharide is widely recommended for treatment of osteoarthritis in Brazil, in association with other drugs. The objective of the present study was to evaluate the quality of chondroitim sulfate raw material used for pharmaceutical recipes. A polyacrylamide gel electrophoresis method for carbohydrate analysis (FACE) was established, so as the production of specific enzymes able to digest chondroitin sulfate. Different unsaturated disaccharides, sulfated monosaccharides and neutral sugars derivatized with 2-aminoacridona were resolved by FACE, using two distinct buffer systems. F. heparinum chondroitinase AC, C and B were fractioned by hydrophobic interaction chromatography, and the activity of these enzymes was characterized by FACE. Chondroitinase AC demonstrated contamination by a sulfatase specific for sulfation at position 4 of hexosamine, which was inhibited by 10 mM sodium fluoride. Glucuronidase contamination with specific activity against non sulfated and 6-sulfated disaccharide was detected in chondroitinase C, and 10 mM D-saccharic acid-1,4-lactone inhibited the degradation of only non sulfated disaccharide. Chondroitin sulfate content in pharmaceutical raw materials was determined by agarose gel electrophoresis and by photometric titration. Only five samples showed content greater than 80%. Chondroitin sulfate average molecular weight in these samples varied from 16 to 26 kDa, and disaccharide analysis by FACE revealed similar proportions between 4-sulfated and 6-sulfated disaccharide. Eleven samples showed less than 20% of chondroitin sulfate, and contaminant analysis by FACE detected the presence of lactose or different polymers of glucose. We concluded that there is a large distortion between the data reported at the certificate of analysis of eleven products and the results obtained in this work. Stricter regulation of this raw material should be enforced.

Cerca de 0,005% da população mundial sofre de lesão medular. O processo regenerativo do tecido nervoso apresenta limitada capacidade para repor as células danificadas (JOHANSSON et al., 1999) e produzir inibidores de crescimento dos axônios associados com a mielina para formação de cicatriz glial (OLSON, 2002). Apesar de resultados promissores, ainda existem controversas quanto ao uso de células-tronco. A eliminação da cicatriz glial, os benefícios de sua formação em diferentes fases e a avaliação da liberação de inibidores de crescimento axonal podem ser parâmetros de análise para o tratamento medular. A enzima condroitinase ABC atua nessa lesão. Neste trabalho avaliamos a interrupção do processo de liberação de inibidores axonais da cicatriz da glia em um tempo não agudo de 7 dias da lesão, sem descartar seus benefícios na fase de formação. Nosso objetivo foi utilizar terapia celular e estabelecer um protocolo de tratamento eficaz, criando uma linha de pesquisa nos estudos da lesão medular. Foi utilizado um grupo de coelhos experimental com realização de hemissecção dorsal e instituído o uso da condroitinase ABC, por aplicação, com micro injeção a curto prazo da lesão. Foi aplicada célula-tronco mesenquimal no foco da lesão após o tratamento da cicatriz da glia com a enzima. Avaliamos por imunohistoquimica a liberação de glial fibrillary acidic protein (GFAP) e sulfato de condroitina proteoglicano (SCPg) nos tecidos após o tratamento no qual foi pretendido fechar algumas lacunas e evitar falhas já descritas, e abrir uma nova esperança no tratamento de pacientes com lesão medular. Nossos resultados ainda mostraram um entendimento superficial sobre a enzima e sua ação sobre cicatrização da glia em associação com o implante celular. Foi aberta uma nova linha de questionamento sobre os benefícios causados à regeneração medular previamente a aplicação de células-tronco. / Around 0,005% of global human population is affected by Spinal Cord Injury (SCI). The regenerative process of neural tissue shows a limited capacity to replace damaged cells (JOHANSSON et al., 1999) and to produce growth inhibitors of associated axons with myelin to create glial scar (OLSON, 2002). Plenty of studies are being developed with stem cell and, despite successful results, there still are controversial opinions. The elimination of the glial scar, the benefits of its growth at different stages and the assessment of axonal growth inhibitors\' release can be parameters of analysis for treating spinal cord. The enzyme chondroitinase ABC acts in this lesion. In this paper we evaluated the release interruption of axonal inhibitors of glial scar in a non-acute 7 days term from injury, not disregarding its benefits during growth. Our goal was to use cell therapy and establish an effective treatment protocol, creating a research line for studies of spinal cord injury and its treatment. A group of rabbits was used under experimental model, conducting dorsal hemisection and application of chondroitinase ABC with micro injection in short-term injury. Mesenchymal stem cells were applied in the lesion focus after the glial scar treatment with the enzyme. Immunohistochemically, we evaluated the release of glial fibrillary acidic protein (GFAP) and sulfate chondroitin proteoglycan (SCPg) in tissues after treatment which was intended to close some gaps and avoid failures described above, and open a new hope in the treatment of patients with spinal cord injury. Our results also showed superficial understanding of the enzyme and its action on glial scarring in association with cell implant. It has opened a new line of questioning about the benefits due to spinal cord regeneration prior to application of stem cells.

Chondroitin sulfate proteoglycans (CSPGs) are one major class of axon growth inhibitors that are upregulated and accumulated around the lesion site after spinal cord injury (SCI), and result in regenerative failure. To overcome CSPG-mediated inhibition, digestion of CSPGs with chondroitinase ABC (chABC) has been explored and it has shown promising results. chABC digests glycosaminoglycan chains on CSPGs and can thereby enhance axonal regeneration and promote functional recovery when delivered at the site of injury. However, chABC has a crucial limitation; it is thermally unstable and loses its enzymatic activity rapidly at 37 ºC. Therefore, it necessitates the use of repeated injections or local infusions with a pump for days to weeks to provide fresh chABC to retain its enzymatic activity. Maintaining these infusion systems is invasive and clinically problematic. In this dissertation, three studies are reported that demonstrate our strategy to overcome current limitations of using chABC and develop a delivery system for facilitating chABC treatment after SCI: First, we enhanced the thermostability of chABC by adding trehalose, a protein stabilizer, and developed a system for its sustained local delivery in vivo. Enzymatic activity was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dimethylmethylene blue (DMMB), and conformational change of the enzyme was measured via circular dichroism (CD) with and without trehalose. When stabilized with trehalose, chABC remained enzymatically active at 37 ºC for up to 4 weeks in vitro. We developed a lipid microtube-agarose hydrogel delivery system for a sustained release and showed that chABC released from the delivery system is still functionally active and slowly released over 2 weeks in vitro. Second, the hydrogel-microtube system was used to locally deliver chABC over two weeks at the lesion site following a dorsal over hemisection injury at T10. The scaffold consisting of hydrogel and chABC loaded lipid microtubes was implanted at the top of the lesion site immediately following injury. To determine effectiveness of topical delivery of thermostabilized chABC, animal groups treated with single injection or gel scaffold implantation of chABC and penicillinase (P'ase) were included as controls. Two weeks after surgery, the functionality of released chABC and the cellular responses were examined by immunohistological analysis with 3B3, CS-56, GFAP and Wisteria floribunda agglutinin (WFA). The results demonstrated that thermostabilized chABC was successfully delivered slowly and locally without the need for an indwelling catheter by using the hydrogel-microtube delivery system in vivo. The results demonstrated that released chABC from the gel scaffold effectively digested CSPGs, and therefore, there were significant differences in CSPG digestion at the lesion site between groups treated with chABC loaded microtube-hydrogel scaffolds and controls. Third, a long term in vivo study (45 days) was conducted to examine axonal sprouting/regeneration and functional recovery with both a single treatment each of microtube loaded chABC or Neurotrophin-3 (NT-3), and a combination of them by using the hydrogel-microtube delivery system. Over the long term study period, the treated animals showed significant improvement in locomotor function and more sprouting of cholera toxin B subunit (CTB)-positive ascending dorsal column fibers and 5-HT serotonergic fibers around the lesion site. We demonstrated that this significant improvement of chABC thermostability facilitates the development of a minimally invasive method for sustained, local delivery of chABC that is potentially a useful and effective approach for treating SCI. In addition to that, we demonstrated that combinatorial therapy with chABC and neurotrophic factors could provide a synergistic effect on axonal regrowth and functional recovery after SCI.

Glycosaminoglycans are heterogeneous polysaccharides that mediate important biological functions. There has been considerable interest in deciphering the precise GAG sequences that are responsible for protein interactions. In fact, several GAG oligosaccharides have been discovered to date as targeting proteins with higher level of specificity. Yet, it has been difficult to develop GAG oligosaccharides as drugs. One of the key reasons for this state of art is that GAG synthesis is extremely challenging and is highly structure-specific. Thus, much of the biology and pharmacology of GAG remains unknown and unexploited to date. An alternative approach is to prepare GAG oligosaccharides using enzymatic depolymerization of polymeric GAGs. GAG lyases, including heparinases and chondritinases represent powerful tools that can theoretically generate multiple oligosaccharides in parallel. However, it is difficult to implement such procedures with high consistency. Moreover, GAG lyases can digest GAGs down to disaccharides. A priori, non-polymeric GAGs, or alternatively GAG oligosaccharides containing 4 to 10 residues, would be expected to function better as therapeutic agents because they would be more homogeneous and less non-specific than their polymeric precursors. Thus, we reasoned that immobilization of these enzymes may engineer altered biopolymer processing, which may afford longer oligosaccharides in higher proportions and greater consistency. Heparinase-I and chondroitinase ABC were immobilized on CNBr-activated Sepharose and compared with the free form of the enzyme. Immobilized GAG lyases retained high efficiency of depolymerization over a wide range of pH, temperature and reusability. Most importantly, the immobilized enzyme was found to produce larger proportions of oligosaccharides longer than di- and tetra-saccharides as compared to lyases in the free form. A two dimensional separation involves size exclusion chromatography followed by reversed phase ion-pairing ultra performance liquid chromatography coupled to electrospray ionization mass spectrometry was employed to separate and characterize oligosaccharide structures. We have identified 40 heparin oligosaccharides, including regular and rare structures ranging from dp4 to dp10 and 39 chondroitin sulfate oligosaccharides in high homogeneity and significant yields. Overall, this technology is likely to offer a simple and cost effective route to preparation of larger amounts of sequences that can be expected to bind and modulate protein function.