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

3D printed muscle-powered bio-bots

Complex biological systems sense, process, and respond to a range of environmental signals in real-time. The ability of such systems to adapt their functional response to dynamic external signals motivates the use of biological materials in other engineering applications. Recent advances in 3D printing have enabled the manufacture of complex structures from biological materials. We have developed a projection stereolithographic 3D printing apparatus capable of patterning cells and biocompatible polymers at physiologically relevant length scales, on the order of single cells. This enables reverse engineering in vitro model systems that recreate the structure and function of native tissue for applications ranging from high-throughput drug testing to regenerative medicine. While reverse engineering native tissues and organs has important implications in biomedical engineering, the ability to “build with biology” presents the next generation of engineers with both a unique design challenge and opportunity. Specifically, we now have the ability to forward engineer bio-hybrid machines and robots (bio-bots) that harness the adaptive functionalities of biological materials to achieve more complex functional behaviors than machines composed of synthetic materials alone. Perhaps the most intuitive demonstration of a “living machine” is a system that can generate force and produce motion. To that end, we have designed and 3D printed locomotive bio-bots, powered by external electrical and optical stimuli. In addition to being the first demonstrations of untethered locomotion in skeletal musclepowered soft robots, these bio-hybrid machines have served as meso-scale models for studying tissue self-assembly, maturation, damage, remodeling, and healing in vitro. Bio-hybrid machines that can dynamically sense and adaptively respond to a range of environmental signals have broad applicability in healthcare applications such as dynamic implants or targeted drug delivery. Advanced research in exoskeletons and hyper-natural functionality could even extend the useful application of such machines to national defense and environmental cleanup. We have developed a modular skeletal muscle bioactuator that can serve as a fundamental building block for such machines, setting the stage for future generations of bio-hybrid machines that can self-assemble, self-heal, and perhaps even self-replicate to target grand engineering challenges. Furthermore, we present a robust optimized protocol for manufacturing 3D printed muscle-powered biological machines, and a mechanism to incorporate biological “building blocks” into the toolbox of the next generation of engineers and scientists

PRELIMINARY FINDINGS OF A POTENZIATED PIEZOSURGERGICAL DEVICE AT THE RABBIT SKULL

The number of available ultrasonic osteotomes has remarkably increased. In vitro and in vivo studies have revealed differences between conventional osteotomes, such as rotating or sawing devices, and ultrasound-supported osteotomes (Piezosurgery®) regarding the micromorphology and roughness values of osteotomized bone surfaces. Objective: the present study compares the micro-morphologies and roughness values of osteotomized bone surfaces after the application of rotating and sawing devices, Piezosurgery Medical® and Piezosurgery Medical New Generation Powerful Handpiece. Methods: Fresh, standard-sized bony samples were taken from a rabbit skull using the following osteotomes: rotating and sawing devices, Piezosurgery Medical® and a Piezosurgery Medical New Generation Powerful Handpiece. The required duration of time for each osteotomy was recorded. Micromorphologies and roughness values to characterize the bone surfaces following the different osteotomy methods were described. The prepared surfaces were examined via light microscopy, environmental surface electron microscopy (ESEM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM) and atomic force microscopy. The selective cutting of mineralized tissues while preserving adjacent soft tissue (dura mater and nervous tissue) was studied. Bone necrosis of the osteotomy sites and the vitality of the osteocytes near the sectional plane were investigated, as well as the proportion of apoptosis or cell degeneration. Results and Conclusions: The potential positive effects on bone healing and reossification associated with different devices were evaluated and the comparative analysis among the different devices used was performed, in order to determine the best osteotomes to be employed during cranio-facial surgery