From microfluidics to hierarchical hydrogel materials

Over the past two decades, microfluidics has made significant contributions to material and life sciences, particularly via the design of nano-, micro- and mesoscale materials such as nanoparticles, micelles, vesicles, emulsion droplets, and microgels. Unmatched in control over a multitude of material parameters, microfluidics has also shed light on fundamental aspects of material design such as the early stages of nucleation and growth processes as well as structure evolution. Exemplarily, polymer hydrogel particles can be formed via microfluidics with exact control over size, shape, functionalization, compartmentalization, and mechanics that is hardly found in any other processing method. Interestingly, the utilization of microfluidics for material design largely focuses on the fabrication of single entities that act as reaction volume for organic and cell-free biosynthesis, cell mimics, or local environment for cell culturing. In recent years, however, hydrogel design has shifted towards structures that integrate a large variety of functions, e.g., to address the demands for sensing tasks in a complex environment or more closely mimicking architecture and organization of tissue by multiparametric cultures. Hence, this review provides an overview of recent literature that explores microfluidics for fabricating hydrogel materials that go well beyond common length scales as well as the structural and functional complexity of microgels necessary to produce hierarchical hydrogel structures. We focus on examples that utilize microfluidics to design microgel-based assemblies, on microfluidically made polymer microgels for 3D bioprinting, on hydrogels fabricated by microfluidics in a continuous fashion, like fibers, and on hydrogel structures that are shaped by microchannels

Microfluidics and Bio-MEMS for Next Generation Healthcare.

Ph.D. Thesis. University of Hawaiʻi at Mānoa 2018

Engineering novel micro-scaffolds and bottom up strategies for in vitro building of vascularized hybrid tissues

There is a significant demand of de-novo engineered tissue grafts capable of replacing biological tissue and/or organ functions for clinical applications. The major obstacle to achieve this goal is the difficulty of recreating all of the complex biochemical and biomechanical functions of the tissue to regenerate, together with transport limitations into the bulk of these newly synthesized tissues. Oxygen and nutrients are supplied to cells and tissues naturally by the microvasculature, which is composed of branching, variable diameter blood vessels. Replicating the complex architecture and functionalities of native tissue vasculature is therefore one of the most important challenge in tissue engineering strategies. To date, bottom up techniques are strong powerful tools to build large viable tissue constructs by packing and sintering cell-laden scaffold-based micro-modules (μ-scaffolds) in a mould. In fact, after sintering and further μ-scaffolds degradation it is possible to achieve large viable tissues in vitro, replicating the composition and structure of native tissue and suitable for studying biological processes involved in new tissue genesis, maturation and remodelling. The aim of this work is to design and engineering novel μ-scaffolds and bottom up assembly techniques to fabricate vascularized layered tissues and to study the effect of μ-scaffolds spatial distribution and co-culture of human dermal fibroblasts (HDFs) together with human umbilical vein endothelial cells (HUVECs) on new tissue growth and vascularization in vitro. To achieve these aims, in first part of this study, we fabricated porous polycaprolactone (PCL) μ-scaffolds with bioinspired trabecular structure and we demonstrated that these newly developed μ-scaffolds supported the in vitro adhesion, growth, and biosynthesis of HDFs. The μ-scaffolds were fabricated by using a fluidic emulsion/porogen leaching/particle coagulation process and by using polyethylene oxide (PEO) as a biocompatible pore-generating agent. In particular, the effect of the composition of the polymeric solution and the flow rate of the continuous phase on μ-scaffolds size distribution, morphology and architectural properties were assessed with the aim to find the best preparation conditions for biological characterization. In vitro culture of HDFs showed that μ-scaffolds supported cells adhesion, colonization, proliferation and biosynthesis in the entire three-dimensional porosity up to 25 days. The second part of this study involved the development of a soft-lithography approach to control the spatial assembly of μ-scaffolds and to create two distinctive μ-scaffolds patterns, namely ordered and disordered. The as obtained patterns were used as substrate for culturing HDFs and 11 HUVECs aiming to develop viable monolayers and bilayers tissue constructs in vitro. The results of this study demonstrated that μ-scaffolds patterning directed cells colonization and biosynthesis and guided the morphology and distribution of newly formed vasculature. All of the findings reported in this work demonstrated the vital role of μ-scaffolds architectural features and assembly on in vitro tissue growth and, pay the way about the possibility to create in silico-designed vasculatures inside modularly engineered biohybrids tissues

Hydrogels derived from decellularised tissues for nerve repair

Peripheral nerve injury poses a serious clinical problem, with sensory and motor deficits resulting in significant reductions in patient quality of life. Nerve guidance conduits aim to overcome issues with the current gold standard for repair, the autograft. Recent advances in the field of tissue engineering have allowed for the devlopment of biochemically and physically complex constructs that aim to bridge the injury site to provide an environment that favours regeneration. This thesis explores the possibility of extracellualr matrix (ECM) hydrogels derived from decellularised tissues (dECM-h) and their potential for the maintenance of Schwann cells, ability to form anisotropic cellular tissue, and their subsequent ability to promote in vitro and in vivo neurite extension. A number of tissues were decellularised, biochemical properties assessed, and formed into hydrogels that were mechanically characterised. In vitro screening was then performed to assess Schwann cell metabolic activity, contraction, and alignment within three selected dECM-h. Additionally, stabilised dECM-h seeded with Schwann cells were formed and seeded with dorsal root ganglia to assess their in vitro capabilities to promote neurite extension. Hydrogels derived from decellularised cancellous bone (B-ECM) were found to be appropriate to be taken forward into a rat sciatic nerve transection model to be compared to the currently used purified collagen I derived from rat tails. In vivo axonal regeneration was found to be comparable between the two groups, however did not match that observed in nerve autografts. This study brought a portfolio of decellularised materials from generation, through characterisation and in vitro screening, to selction of one candidate that was taken forward into an in vivo model. This has shown, for the first time, that alternatives to the currently used collagen I hydrogels may be employed in the production and utilisation of engineered neural tissue (EngNT)

Microarray Structures for Sensing, Stimuliresponsive Releases, Shaped Microcages and Templating Minified Microstructures.

PhD ThesesMicroarray structure plays a key role in a variety of fields ranging from optical devices, electronics to drug delivery systems due to the special periodic micropatterns, which can not only perform the capability of light diffraction, show the potential as photonic sensors, but also provide the empty space for drug loading when the highly ordered structures are microwells. Especially, the microarray structure can be transferred onto other polymers, after the introduction of microcontact printing techniques. Inspired by the versatility of microarray structures, this work aims at exploring and expanding its potential multidisciplinary applications including multi-sensing platforms, drug delivery vehicles of microchamber array films and microcages, and structuring templates. These can be achieved by functionalizing the microarray structure with extra properties of differently structured materials including polyelectrolytes, polyester, precursor ceramics. To provide a better understanding of the research subjects of this work, an introduction is presented at the beginning of chapter 1, followed by chapter 2 of a literature review. The description of the experimental section including materials, methods and instruments is followed in chapter 3. The results start from chapter 4 which investigates the possibilities of microarray structure for media, pH, ions and thermal sensing using stimuli-responsive polymers. The potential of the microarray structure for preparing drug delivery vehicles is further determined in chapter 5. Biodegradable polymers were fabricated into microchamber array films with the capability to efficiently encapsulate and enzymatically controlled release small hydrophilic molecules. In chapter 6, a novel method for preparing shape and size defined biodegradable microcages for drug delivery based on microarray structure is presented. Moreover, chapter 7 proposed an efficient route to microfabricate proportionally minified microarray structure with the assistance of novel precursor ceramics. Finally, general conclusions of the overall results of this work along with outlooks are summarized in chapter 8

New nanotechnology approaches using dendrimers modified with natural polymers for controlling stem cells behaviour in tissue engineering strategies

Tese de doutoramento em Ciência e Tecnologia de Materiais (ramo de conhecimento em Engenharia de Tecidos - Materiais Híbridos)In the recent years, great progress has been done in the emerging field of tissue engineering. Despite the important advances the performance of cells-scaffold constructs, one of the several tissue engineering approaches, remains limited in part due to the need for optimize cell culture techniques and culture media. Nanocarrier systems have generated a significant amount of interest in the ex vivo cell maintenance, and control of the cellular fate in vivo mainly due to their internalization efficiency, drug loading capacity, and to favorably modulate the solubility and pharmacokinetics of drugs. Dendrimers are synthetic, monodispersive, spherical and highly branched macromolecules that present unique advantages and fulfills most requirements as carriers for drug delivery; however, it has been found that high generation dendrimers are often cytotoxic. Thus, in this thesis we focused our attention in this fundamental problem and explore the development of novel nanobiomaterials based on the grafting of carboxymethylchitosan (CMCht) onto low generation poly(amidoamine) (PAMAM) dendrimers, the socalled CMCht/PAMAM dendrimer nanoparticles. These macromolecular vehicles were developed to explore a new concept consisting on the intracellular and controlled delivery of bioactive molecules aimed at control stem cells functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. Thus, by combining nanotechnology-based systems and traditional tissue engineering strategies, we expect to develop a novel therapeutic solution for the efficient treatment of damage/diseased cells and tissues. To validate this new concept, there is the need to evaluate the performance of the developed nanocarriers, in vitro and in vivo. Firstly, the uptake efficiency and internalization mechanism of fluorescent-labeled CMCht/PAMAM dendrimer nanoparticles was investigated using different cell types. Fluorescence microscopy studies revealed that the developed nanocarriers could be efficiently internalized, either by cell lines and primary cultures, after a few hours. Flow cytometry studies revealed that rat bone marrow stromal cells (RBMSCs) cultured in the presence of colchicine, an alkaloid that inhibits endocytosis, decreased the internalization of the nanoparticles. These data showed that uptake by cells was primarily via an active endocytotic mechanism, but not exclusively. Preliminary studies were also carried out to evaluate the possible applicability of the CMCht/PAMAM dendrimer nanoparticles in the central nervous system. Internalization rate, cell viability and metabolic activity studies were performed using rat post-natal hippocampal neurons and cortical glial cells that revealed their ability for being taken up by these cells and its non cytotoxicity. Complementarily, dexamethasone (Dex), a glucocorticoid known to have important role on the proliferation and expression of osteoblastic differentiation markers, was used as a model drug and incorporated into the bulk of the nanoparticles. Physicochemical characterization and in vitro biological studies have demonstrated that the Dex-loaded CMCht/PAMAM dendrimer nanoparticles were successfully synthesized, were not cytotoxic in the range of concentration below 1 mg.mL-1 and promote osteogenesis (2-D system). To assess the true value of the Dex-loaded CMCht/PAMAM dendrimer nanoparticles systems for application in tissue engineering strategies, we use different biomaterials to develop a set of novel scaffolds both ceramic and polymeric or formulations. These scaffolds were found to be suitable for applications in bone, cartilage and osteochondral tissue engineering. In vitro studies have shown that combination of scaffolds, bone marrow stromal cells and Dex-loaded CMCht/PAMAM dendrimer nanoparticles (3-D system) enhanced osteogenesis. Finally, in vivo studies have shown that the novel Dex-loaded CMCht/PAMAM dendrimer nanoparticles may be beneficial as intracellular nanocarrier, supplying Dex in a regimented manner, while avoiding the need of culturing stem cells for long periods of time in vitro, towards promoting the osteogenic differentiation. Remarkably, the proposed strategy allow modulate and direct stem cells differentiation towards osteogenic phenotype, enhance in vivo proteoglycan extracellular matrix synthesis and promote superior de novo bone formation. This thesis mark the transition from the ‘proof-of-concept’ to useful intracellular nanocarrier tool, as the Dex-loaded CMCht/PAMAM dendrimer nanoparticles show promise for application in direct stem cell to a particular cell fate, in vitro and in vivo.Grandes progressos têm sido feitos nos últimos tempos no emergente campo científico da engenharia de tecidos. No entanto, a eficiência dos sistemas células-matriz tridimensional porosa, uma das estratégias usadas nas abordagens em engenharia de tecidos, tem sido limitada, em parte, pela necessidade de se optimizarem as técnicas e os meios de cultura usados. Sistemas de nanopartículas para o transporte de moléculas bioactivas têm suscitado grande interesse na área da biomedicina, nomeadamente no que respeita à possível aplicação como suplementos em meios de cultura ex vivo, e no controlo das funções celulares in vivo. Isto deve-se, principalmente, à sua eficiência de internalização, elevada capacidade de incorporação de fármacos, e ao facto de favorecerem a solubilidade de moléculas hidrofóbicas e de possibilitarem a modulação da sua farmacocinética. Os dendrímeros são sistemas macromoleculares sintéticos altamente ramificados que apresentam características únicas, tais como monodispersividade e uma estrutura esférica, e que preenchem a maioria dos requisitos para serem usados como veículos para a libertação controlada de fármacos. Não obstante, tem-se verificado que dendrímeros de elevada geração apresentam tipicamente uma citotoxicidade indesejada. Assim, nesta tese a atenção focou-se na resolução deste problema fundamental. Para tal foi explorado o desenvolvimento de novos nanobiomateriais tendo como estratégia a ligação química do polímero carboximetilquitosano (CMCht) a dendrímeros de poliamidoamina (PAMAM) de baixa geração, que se denominaram por nanopartículas de carboximetilquitosano/poliamidoamina (CMCht/PAMAM). Estas macromoléculas foram desenvolvidas e um novo conceito aplicativo foi testado relacionado com a sua aplicação em medicina regenerativa como veículos de libertação controlada e intracelular de moléculas bioactivas, de forma a ser possível controlar efectivamente as actividades celulares, tais como a proliferação e a diferenciação de células estaminais ex vivo, e a manutenção do fenótipo dessas mesmas células após o seu implante. Assim, recorrendo à nanotecnologia e a estratégias usadas na engenharia de tecidos, espera-se que seja possível desenvolver uma nova solução terapêutica que possa possibilitar, de uma forma eficiente, o tratamento de células e tecidos danificados ou que apresentem algum tipo de patologia. De forma a validar este novo conceito, é necessário avaliar o potencial dos nanosistemas desenvolvidos, in vitro e in vivo. Primeiramente, a eficiência e o mecanismo de internalização de nanopartículas de CMCht/PAMAM ligadas a um marcador fluorescente foram investigados, recorrendo a estudos celulares, in vitro. Estudos de microscopia de fluorescência revelaram que as nanopartículas desenvolvidas são internalizadas por diferentes tipos de células após algumas horas em cultura, incluindo linhas celulares e culturas primárias. Estudos envolvendo a citometria de fluxo mostraram que quando células multipotentes do estroma da medula óssea de rato (RBMSCs), foram cultivadas na presença de colchicina, um alcalóide inibidor da endocitose, exibiam menor capacidade de internalização das nanopartículas. Assim, a internalização das nanopartículas pelas células ocorre principalmente por um mecanismo de endocitose, mas que este não é o único. Foram também realizados estudos para determinar a taxa de internalização, viabilidade celular e actividade metabólica, recorrendo a neurónios isolados do hipocampo de rato pós-natais e células da glia. Estes estudos mostraram que as nanopartículas são internalizadas por estas células, e não afectam negativamente a viabilidade da cultura das células. A dexametasona (Dex), uma molécula pertencente à família dos glucocorticóides e conhecida pelo seu papel na modulação da proliferação e expressão dos marcadores de diferenciação osteoblástica, foi usada como fármaco modelo e incorporada nas nanopartículas de CMCht/PAMAM. A caracterização físico-química e estudos biológicos in vitro demonstraram que as nanopartículas de CMCht/PAMAM carregadas com Dex foram sintetizadas com sucesso, não apresentaram citotoxicidade em concentrações até 1 mg.mL-1 e promoveram a osteogénese (sistema de cultura 2-D). Por forma a avaliar o verdadeiro potencial aplicativo das nanopartículas de CMCht/PAMAM carregadas com Dex em estratégias de engenharia de tecidos, diferentes biomateriais foram usados no desenvolvimento de estruturas tridimensionais porosas, incluindo cerâmicos, polímeros e formulações contendo ambos. Estas estruturas tridimensionais porosas mostraram-se adequadas para serem usadas em engenharia de tecidos de osso, cartilagem e defeitos osteocondrais. Estudos in vitro revelaram que a abordagem constituída por estruturas tridimensionais porosas, RBMSCs e nanopartículas de CMCht/PAMAM carregadas com Dex (sistema de cultura 3-D) promoveu um aumento significativo da osteogénese. Por último, estudos in vivo mostraram que as nanopartículas de CMCht/PAMAM carregadas com Dex são um sistema de libertação intracelular altamente eficiente, uma vez que possibilitaram a libertação de Dex com um perfil cinético adequado, permitindo assim evitar os longos períodos de cultura in vitro, necessários à diferenciação osteogénica de células estaminais. A estratégia proposta permite modular e direccionar a diferenciação das células estaminais para o fenótipo osteogénico, aumentar a síntese de proteoglicanos da matriz extracelular e promover a formação de osso num estado de maturação mais avançado. Esta tese marca a transição de “prova de conceito” para uma ferramenta aplicativa das nanopartículas desenvolvidas na libertação intracelular de fármacos, uma vez que as nanopartículas de CMCht/PAMAM carregadas com Dex se mostraram promissoras no direccionamento das células estaminais para um determinado fenótipo, in vitro e in vivo.Fundação para a Ciência e a Tecnologia (FCT) - ref. SFRH/BD/21786/2005 através dos programas POCTI e FEDERRotary Club de Caldas das TaipasFundação para a Ciência e Tecnologia (FCT) - PhD grant Ref. SFRH/BD/21786/2005, POCTI, FEDER programsCanon Foundation in EuropeEuropean NoE EXPERTISSUES (NMP3-CT-2004-500283)European Union HIPPOCRATES STREP Project (NMP3-CT-2003-505758