Chitosan-FastOs® BG membrane-guides for nervous tissue regeneration

Mestrado em Materiais e Dispositivos BiomédicosThree-dimensional (3D) biodegradable composite porous scaffolds made of a biopolymer matrix (chitosan) and a bioactive glass (FastOs®BG-Z4) were fabricated via freeze drying as guides for nerve tissue engineering applications. For this purpose, chitosan was dissolved in aqueous solutions of lactic acid (LA, 1 wt.%) to reach a final concentration of 2 wt.%. Subsequently FastOs®BG-Z4 in powder form was added to chitosan solution in a chitosan/Fasto®BG-Z4 weight ratio of 50/50. The Chitosan/FastOs®BG-Z4 systems were cross-linked via adding different concentrations (0.01, 0.05 and 0.5 wt.%) of two kinds of cross-linking agents, genipin, a natural component, and glutaraldehyde, a synthetic agent, to stiffen the chitosan network. The final mixtures were then frozen at two temperatures, 20ºC and 80ºC followed by freezedrying to obtain porous scaffolds. For achieving the optimal Chitosan/FastOs®BG-Z4 scaffolds, the influences of adding FastOs®BG-Z4 powder and/or different amounts of crosslinking agents on the rheological properties of chitosan/LA solutions were firstly investigated by rheological measurements. The results showed that a strong and stable gel could not be obtained even when the highest amount of cross-linking agents (0.5 wt.%) was added to the 2 wt.% chitosan solution, while effective cross-linking occurred in the presence of FastOs®BG-Z4 powder. Therefore, it was concluded that FastOs®BG-Z4 plays an active role on chitosan complexation. The positive interactions between chitosan and the surface of FastOs®BGZ4 particles and/or the ionic species leached out to the solution needs to be further investigated in future work. The microstructural features of porous scaffolds were investigated by scanning electron microscope (SEM), and the porosity assessment was made by ethanol replacement method. The mechanical properties of porous scaffolds were investigated under compression/swelling tests with samples immersed in phosphate-buffered saline (PBS) solution. In vitro degradation tests were also performed by immersing the samples in iv phosphate-buffered saline (PBS) solution for 2 months tests and the degradation degree was evaluated through the undergone weight changes. The results showed some common features among genipin or glutaraldehyde as crosslinking agents: increasing their amounts from 0.01 to 0.5 wt.% led to reductions in gelling time, porosity fraction, swelling and degradation rate, while cross-linking degree increased. However, their effects on pore size and compression strength of the scaffolds diverged. For genipin pore size decreased and consequently the compression strength increased, while for glutaraldehyde pore size always increased with added amounts, but compression strength was improved with concentration increasing from 0.01 to 0.05 wt.%, decreasing when the added amount was further increased to 0.5 wt.%. Moreover, 20ºC was selected as the most suitable freezing temperature when considering the porous microstructural features and the intended applications.A presente tese relata acerca do fabrico e caracterização de compósitos porosos tridimensionais (3D) biodegradáveis baseados em quitosano, como matriz biopolimérica, carregada com partículas de um vidro bioativo (Fastos®BG-Z4). Para este efeito, o quitosano foi dissolvido em solução aquosa de ácido láctico (LA, 1% em peso) até atingir uma concentração final de 2% em peso. Subsequentemente o Fastos®BG-Z4 em forma de pó foi adicionado à solução de quitosano em uma proporção em peso de quitosano/ Fastos®BG-Z4 de 50/50. Os sistemas quitosano/Fastos®BG-Z4 foram reticulados por meio de adição de diferentes percentagens em peso (0.01, 0.05 e 0.5) de dois tipos de agentes de ligação cruzada, um componente natural, genipin, e um agente sintético, glutaraldeído. As misturas finais foram então reticuladas a 60ºC seguido de congelamento a duas temperaturas diferentes, 20ºC e 80ºC. O gelo foi depois sublimado por liofilização de modo a obter matrizes porosas para aplicações como guias em engenharia de tecidos nervosos periféricos. Com vista à optimização do processo de fabrico e das propriedades das estruturas porosas de suporte (andaimes) de quitosano/Fastos®BG-Z4, estudaram-se os efeitos da adição do Fastos®BG-Z4 em pó e/ou de diferentes quantidades de agentes de reticulação nas propriedades reológicas das soluções de LA/quitosano. Os resultados mostraram a impossibilidade de obter de um gel de quitosano suficientemente forte e estável mesmo quando a quantidade mais elevada de agentes de reticulação (0.5% em peso) foi adicionada à solução de quitosano, em contraste com o que aconteceu com a adição do pó de Fastos®BG-Z4 na ausência de outros agentes de reticulação. Esta descoberta permitiu concluir que o Fastos®BG-Z4 desempenha um papel activo na complexação do quitosano. As interacções positivas entre o quitosano e a superfície das partículas do Fastos®BG-Z4 e/ou as espécies iónicas lixiviadas para a solução precisam de ser melhor investigadas no futuro. vi As características microestruturais dos materiais porosos foram investigadas por microscopia electrónica de varrimento (SEM), e a porosidade foi determinada pelo método de substituição de etanol. As propriedades mecânicas dos compósitos porosos imersos em solução (PBS) de solução salina tamponada com fosfato foram investigadas através de testes de compressão/inchamento. Realizaram-se também testes de degradação in vitro por imersão das amostras na mesma solução de PBS durante 2 meses, e o grau de degradação foi avaliado através das alterações de peso sofridas pelas amostras. Os resultados mostraram algumas características comuns entre o genipin e o glutaraldeído como agentes de reticulação: o aumento das quantidades adicionadas (0.010.5% em peso) levou a reduções no tempo de gelificação, na fracção de porosidade, no grau de inchamento, e na taxa de degradação, enquanto o grau de reticulação aumentou. No entanto, os seus efeitos sobre o tamanho dos poros e a resistência à compressão dos suportes porosos divergiram. O tamanho de poro diminuiu no caso do genipin, o que se traduziu em consequentes aumentos da resistência à compressão; enquanto o tamanho dos poros aumentou sempre com as quantidades adicionadas no caso do glutaraldeído, pelo que só foram registadas melhorias na resistência à compressão na gama de concentrações entre 0.010.05% em peso, diminuindo quando a quantidade adicionada foi aumentada para 0,5 % em peso. Verificou-se ainda que a temperatura de 20ºC era a que permitia obter as microestruturas porosas mais adequadas para as aplicações almejadas

Janus Nanocomposite Hydrogels for Chirality-Dependent Cell Adhesion and Migration

Recently, there has been much interest in the chirality-dependent cell affinity to enantiomorphous nanomaterials (NMs), since, at the nanoscale level, enantiomers of (bio)­molecules have different effects on cell behaviors. In this respect, this study used enantiomorphous NMs with which to generate the Janus nanocomposite (NC) hydrogels as multifunctional biomaterials for studying chirality-dependent cell adhesion and cell migration. These Janus NC hydrogels possess two enantiomorphous NC hydrogels, in which the different halves of the hydrogel contain the opposite enantiomers of a biopolymer functionalized nanomaterials. Thus, the enantiomers contact each other only at the midline of the hydrogel but are otherwise separated, yet they are present in the same system. This advanced system allows us to simultaneously study the impact that each enantiomer of a biopolymer has on cell behavior under the same reaction conditions, at the same time, and using only a single biomaterial. Our results show that cells have higher affinity for and migrate toward the part of the Janus NC hydrogel containing the biopolymer enantiomer that the cells prefer

Functional Nanomaterials on 2D Surfaces and in 3D Nanocomposite Hydrogels for Biomedical Applications

An emerging approach to improve the physicobiochemical properties and the multifunctionality of biomaterials is to incorporate functional nanomaterials (NMs) onto 2D surfaces and into 3D hydrogel networks. This approach is starting to generate promising advanced functional materials such as self-assembled monolayers (SAMs) and nanocomposite (NC) hydrogels of NMs with remarkable properties and tailored functionalities that are beneficial for a variety of biomedical applications, including tissue engineering, drug delivery, and developing biosensors. A wide range of NMs, such as carbon-, metal-, and silica-based NMs, can be integrated into 2D and 3D biomaterial formulations due to their unique characteristics, such as magnetic properties, electrical properties, stimuli responsiveness, hydrophobicity/hydrophilicity, and chemical composition. The highly ordered nano- or microscale assemblies of NMs on surfaces alter the original properties of the NMs and add enhanced and/or synergetic and novel features to the final SAMs of the NM constructs. Furthermore, the incorporation of NMs into polymeric hydrogel networks reinforces the (soft) polymer matrix such that the formed NC hydrogels show extraordinary mechanical properties with superior biological properties

Cell Growth on (“Janus”) Density Gradients of Bifunctional Zeolite L Crystals

Nanoparticle density gradients on surfaces have attracted interest as two-dimensional material surfaces that can mimic the complex nano-/microstructure of the native extracellular matrix, including its chemical and physical gradients, and can therefore be used to systematically study cell–material interactions. In this respect, we report the preparation of density gradients made of bifunctional zeolite L crystals on glass surfaces and the effects of the density gradient and biopolymer functionalization of zeolite L crystals on cell adhesion. We also describe how we created “Janus” density gradient surfaces by gradually depositing two different types of zeolite L crystals that were functionalized and loaded with different chemical groups and guest molecules onto the two distinct sides of the same glass substrate. Our results show that more cells adhered on the density gradient of biopolymer-coated zeolites than on uncoated ones. The number of adhered cells increased up to a certain surface coverage of the glass by the zeolite L crystals, but then it decreased beyond the zeolite density at which a higher surface coverage decreased fibroblast cell adhesion and spreading. Additionally, cell experiments showed that cells gradually internalized the guest-molecule-loaded zeolite L crystals from the underlying density gradient containing bifunctional zeolite L crystals