A novel culture system for modulating single cell geometry in 3D

Dedifferentiation of chondrocytes during in vitro expansion remains an unsolved challenge for repairing serious articular cartilage defects. In this study, a novel culture system was developed to modulate single cell geometry in 3D and investigate its effects on the chondrocyte phenotype. The approach uses 2D micropatterns followed by in situ hydrogel formation to constrain single cell shape and spreading. This enables independent control of cell geometry and extracellular matrix. Using collagen I matrix, we demonstrated the formation of a biomimetic collagenous “basket” enveloping individual chondrocytes cells. By quantitatively monitoring the production by single cells of chondrogenic matrix (e.g. collagen II and aggrecan) during 21-day cultures, we found that if the cell’s volume decreases, then so does its cell resistance to dedifferentiation (even if the cells remain spherical). Conversely, if the volume of spherical cells remains constant (after an initial decrease), then not only do the cells retain their differentiated status, but previously de-differentiated redifferentiate and regain a chondrocyte phenotype. The approach described here can be readily applied to pluripotent cells, offering a versatile platform in the search for niches toward either self-renewal or targeted differentiation

A role for nuclear stretching and NPCs changes in the cytoplasmic-nuclear trafficking of YAP: An experimental and numerical modelling approach

Mechanical forces, acting on eukaryotic cells, are responsible for cell shape, cell proliferation, cell polarity, and cell differentiation thanks to two cells abilities known as mechanosensing and mechanotransduction. Mechanosensing consists of the ability of a cell to sense mechanical cues, while mechanotransduction is the capacity of a cell to respond to these signals by translating mechanical stimuli into biochemical ones. These signals propagate from the extracellular matrix to the nucleus with different well known physical connections, but how the mechanical signals are transduced into biochemical ones remains an open challenge. Recent findings showed that the cell-generated forces affect the translocation of transcription factors (TFs) from the cytoplasm to the nucleus. This mechanism is affected by the features of nuclear pore complexes. Owing to the complex patterns of strains and stresses of the nuclear envelope caused by cytoskeletal forces, it is likely that the morphology of NPC changes as cytoskeleton assemblies’ change. This may ultimately affect molecular transport through the nucleus, hence altering cell functions. Among the various TFs, Yes-associated protein (YAP), which is typically involved in cell proliferation, survival, and differentiation, is able to activate specific pathways when entrapped into the cell nucleus. Here, starting from experimental results, we develop a multiscale finite element (FE) model aimed to simulate the macroscopic cell spreading and consequent changes in the cell mechanical behaviour to be related to the NPCs changes and YAP nuclear transport

Enhancing nerve regeneration in the peripheral nervous system using polymeric scaffolds, stem cell engineering and nanoparticle delivery system

Peripheral nerve regeneration is a complex biological process responsible for regrowth of neural tissue following a nerve injury. The main objective of this project was to enhance peripheral nerve regeneration using interdisciplinary approaches involving polymeric scaffolds, stem cell therapy, drug delivery and high content screening. Biocompatible and biodegradable polymeric materials such as poly (lactic acid) were used for engineering conduits with micropatterns capable of providing mechanical support and orientation to the regenerating axons and polyanhydrides for fabricating nano/microparticles for localized delivery of neurotrophic growth factors and cytokines at the site of injury. Transdifferentiated bone marrow stromal cells or mesenchymal stem cells (MSCs) were used as cellular replacements for lost native Schwann cells (SCs) at the injured nerve tissue. MSCs that have been transdifferentiated into an SC-like phenotype were tested as a substitute for the myelinating SCs. Also, genetically modified MSCs were engineered to hypersecrete brain- derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) to secrete therapeutic factors which Schwann cell secrete. To further enhance the regeneration, nerve growth factor (NGF) and interleukin-4 (IL4) releasing polyanhydrides nano/microparticles were fabricated and characterized in vitro for their efficacy. Synergistic use of these proposed techniques was used for fabricating a multifunctional nerve regeneration conduit which can be used as an efficient tool for enhancing peripheral nerve regeneration

Engineering patterned and dynamic surfaces for the spatio-temporal control of cell behaviour

Stem cell shape and mechanical properties in vitro can be directed by geometrically defined micropatterned adhesion substrates. However conventional methods are limited by the fixed micropattern design, which cannot recapitulate the dynamic changes of the natural cell microenvironment. Recent advancements in microfabrication technologies in combination with the use of light-responsive materials, allow to manipulate the shape of living cells in real-time in a non-invasive Spatio-temporal controlled way to introduce additional geometrically defined adhesion sites and to study relative cell behaviour. Here, the confocal laser technique is exploited for dynamically evaluate the variation over time of the tensional and morphological cell state. This method allows the precise control of specific actin structures that regulate cell architecture. Actin filament bundles, initially randomly organized in circular-shaped cells, are induced to align and distribute to form a rectangular-shaped cell in response to specific dynamic changes in the cell adhesion pattern. The changes in morphology also reflect dramatic changes in FAs distribution, cell mechanics, nuclear morphology, and chromatin conformation. The reported strategy is convenient to explore the cell-substrate interface and the mechanisms through which cell geometry regulates cell signalling in a facile and cost-effective manner and it open new routes to understand how the field of dynamic platforms should potentially contribute to unveil complex biological events such as the modulation of cell shape

A switchable light-responsive azopolymer conjugating protein micropatterns with topography for mechanobiological studies

Stem cell shape and mechanical properties in vitro can be directed by geometrically defined micropatterned adhesion substrates. However conventional methods are limited by the fixed micropattern design, which cannot recapitulate the dynamic changes of the natural cell microenvironment. Current methods to fabricate dynamic platforms usually rely on complex chemical strategies or require specialized apparatuses. Also, with these methods the integration of dynamic signals acting on different length scales is not straightforward, whereas in some applications might be beneficial to act on both a microscale level, i.e. cell shape, and on a nanoscale level, i.e. cell adhesions. Here, we exploited a confocal laser-based technique on a light-responsive azopolymer displaying micropatterns of adhesive islands. The laser light promotes a directed mass migration and the formation of submicrometric topographic relieves. Also, by changing the surface chemistry, the surfacing topography affects cell spreading and shape. This method enabled us to monitor in a non-invasive manner the dynamic changes in focal adhesions, cytoskeleton structures and nucleus conformation that followed the changes in the adhesive characteristic of the substrate. Focal adhesions reconfigured after the surfacing of the topography and the actin filaments reoriented to co-align with the newly formed adhesive island. Changes in cell morphology also affected nucleus shape, chromatin conformation and cell mechanics with different timescales. The reported strategy can be used to investigate mechanotransduction-related events dynamically by controlling cell adhesion at a cell shape and focal adhesion levels. The integrated technique enables achieving a submicrometric resolution in a facile and cost-effective manner

Mikroçevrenin doku mühendisliği uygulamalarına etkisi.

Cues of microenvironment that guide both mature and stem cells determine the success of tissue engineered constructs. To prove and emphasize this expectation, various parameters such as surface topography, scaffold (cell carrier, scaffold) chemistry, 2D vs 3D microenvironments and mechanical stimulation were included into the microenvironment. Surfaces with two distinct physical cues pillar and groove-ridge type micropatterns were transferred to the surfaces of the films by casting the collagen type I and silk fibroin biopolymers on poly(dimethylsiloxane) (PDMS) templates which were replicated from photolithographically produced micropatterns on silicon wafers. Bombyx mori silk fibroin a structural biopolymer, was blended with collagen type I protein, to obtain high mechanical properties and biodegradability. Adipose derived stem cells (ADSCs) were cultured on collagen–silk fibroin films with microchannel and micropillar patterns to investigate the effects of cell morphology changes on osteogenic differentiation. While higher ADSC proliferation profiles were obtained on micropillar patterned film, microchannel patterned films, however, caused twice higher aspect ratio and effective orientation of cells. Alkaline phosphatase activity of ADSCs was several times higher on microchannel surface when the measured activities were normalized to cell number. Effective deposition of collagen type I and mineral after cell culture was observed for patterned and unpatterned films and these extracellular matrix (ECM) components were oriented along the axis of the microchannels. The use of collagen–fibroin blend film with microchannel topography increased the aspect ratio and alignment of cells significantly, and it was also effective in the differentiation of ADSCs into osteogenic lineage. As an additional biochemical cue of microenvironment defining element, elastin-like recombinamer (ELR) with a hydroxyapatite depositing amino acid sequence was incorporated into films of collagen-silk fibroin blend carrying microchannel patterns to stimulate anisotropic cell growth and osteogenesis. The Young's modulus and the ultimate tensile strength (UTS) of unseeded films were 0.58 ± 0.13 MPa and 0.18 ± 0.05 MPa, respectively. After 28 days of cell culture, ADSC seeded films had a Young's modulus of 1.21 ± 0.42 MPa and UTS of 0.32 ± 0.15 MPa which were about 3 fold higher than human osteoblast (HOB) seeded films. The difference in Young's modulus was statistically significant (p = 0.02). ADSCs attached, proliferated and produced calcium phosphate mineral on films better than the HOBs. In the light of these results, ADSCs served as a better cell source than HOBs for bone tissue engineering of collagen-fibroin-ELR based constructs used in this study. In vitro systems generally rely on 2D test media whereas in vivo can best be mimicked if a 3D test medium is used. This is important because the maturation of a tissue engineered product should be different than on a 2D surface due to the kind of interactions and the accumulation of molecular signals in 2D and 3D systems are expected to be different. 3D scaffolds were created by folding long strips of engineered films on a rod to investigate the contributions of 3D microenvironment over mesenchymal stem / multipotent stromal cells (MSCs) by comparing with 2D films. Additionally, the contribution of hypoxia and arterial oxygen pressure was studied further to look into the concept of oxygen limited microenvironments of the native MSCs and osteoblast niche. Hypoxia maintained the stemness of the MSCs on TCPS and 2D scaffold. Interestingly, MSCs had elevated VEGFA level and osteogenic differentiation on 3D construct while preserving their stemness at normoxic conditions. Strikingly, osteogenic and angiogenic marker expressions were 13200 and 266 fold higher, respectively on 3D construct than 2D scaffold at 21% oxygen. At all of the oxygen tensions that were tested, UTS was found to be similar with the unseeded scaffolds at less preferable 2D scaffolds by MSCs. Distinctively, during the transition from hypoxia to normoxia, Young’s modulus and UTS of the MSC seeded 3D scaffolds were enhanced from 1.13 ± 0.33 MPa to 2.16 ± 0.81 MPa and 0.51 ± 0.12 MPa to 1.82 ± 0.27 MPa, respectively, owing to the contribution of cells by secreting their ECM onto the surface of 3D scaffold surface. MSC proliferation was dependent both on oxygen tension and cyclic strain exposure (10%) on collagen hydrogel embedded scaffolds. On the other hand, strain was able to alter MSC shape by elongating them on the force direction and inhibit angiogenic activity both at hypoxia and normoxia. In summary, this study showed that mimicking the bone ECM was possible through the use of microchannel patterns especially in 3D microenvironments that overcome the limitations of 2D substrates. The transition from 2D to 3D microenvironment enhanced the osteogenic and angiogenic activity of MSCs and along with the tensile properties both at normoxia and oxygen limited physiological environments.Ph.D. - Doctoral Progra

Geometric Control of Human Stem Cell Morphology and Differentiation

During tissue morphogenesis, stem cells and progenitor cells migrate, proliferate, and differentiate, with striking changes in cell shape, size, and acting mechanical stresses. The local cellular function depends on the spatial distribution of cytokines as well as local mechanical microenvironments in which the cells reside. In this study, we controlled the organization of human adipose derived stem cells using micro-patterning technologies, to investigate the influence of multi-cellular form on spatial distribution of cellular function at an early stage of cell differentiation. The underlying role of cytoskeletal tension was probed through drug treatment. Our results show that the cultivation of stem cells on geometric patterns resulted in pattern- and position-specific cell morphology, proliferation and differentiation. The highest cell proliferation occurred in the regions with large, spreading cells (such as the outer edge of a ring and the short edges of rectangles). In contrast, stem cell differentiation co-localized with the regions containing small, elongated cells (such as the inner edge of a ring and the regions next to the short edges of rectangles). The application of drugs that inhibit the formation of actomyosin resulted in the lack of geometrically specific differentiation patterns. This study confirms the role of substrate geometry on stem cell differentiation, through associated physical forces, and provides a simple and controllable system for studying biophysical regulation of cell function

Effects of stiffness and cell shape on cellular mechanosensing

Cellular mechanosensing is the process of converting mechanical signals into biological responses. Stem cells are self-renewing cells with the potential to transform into specialized cell types - this differentiation process is influenced by cellular mechanosensing. Cells sense material stiffness, and stiffer environments result in increased cellular mechanosensing and preferential differentiation into bone-producing osteoblasts. Cell shape also plays an important role due to its influence on cytoskeletal contractility, and photopatterning can be used to study the effects of cell shape on cellular mechanosensing. Although the effects of material stiffness and cell shape have been studied, little is known about the joint effects of these factors on stem cell mechanosensing. Taken together, the goal of this research is to develop a biomaterial system to study the combinatorial effects of shape and stiffness on mesenchymal stem cell (MSC) mechanosensing. Hydrogels of three stiffness (5 kPa, 10 kPa, 20 kPa) were photopatterned with shapes (circle, square, octagon) that cause a range of contractile forces in cells. These shapes were made into patterns on a glass photomask, allowing hydrogels placed under the photomask to be photopatterned. Photopatterns were found to over 90% accurate. Highly angular shapes, such as the octagon, and increased stiffness were both seen to influence an increased nuclear localization of mechanosensing protein YAP, with stiffness having a greater influence than shape

Estratégias biomiméticas usando a técnica camada-a-camada para aplicações biomédicas e engenharia de tecidos

The development of a suitable coating or material, which physico-chemical, mechanical or biological properties, that can be tailored according the features of the target tissue, has been gaining increased importance in biomedical and tissue engineering and regenerative medicine (TERM) fields. Biomimetic strategies have contributed significantly for the progress of biomedical field during the last years. This is possible to be achieved at different levels: imitating Nature form or function and mimicking natural processes and systems are the most used biomimetic approaches. In this thesis, Layer-by-Layer (LbL) methodology was used as a hierarchical biomimetic tool to modify surfaces and to produce freestanding membranes based on polyelectrolyte multilayers (PEMs). The possibility to functionalize or engineer biomaterials combined with the ability to incorporate a wide range of building blocks, makes LbL a powerful processing technique in the biomedical field. Synthetic polymers have been used to construct PEMs for biomedical and TERM applications; however, they lack often on adhesive cues for cell attachment and tissue growth. To overcome such issue, biomimetic synthetic polymers have been developed. Elastin-like polypeptides (ELPs) are a class of nature-inspired polymers, nonimmunogenic, genetically encodable and biocompatible. These materials are based on the repetition of short peptides considered to be building blocks in natural elastin and can include specific bioactive sequences, as the tripeptide Arginine-Glycine-Aspartame (RGD) known by promoting cell adhesion. For the first work of this thesis, ELPs were functionalized with azide and alkyne groups to introduce the reactivity required to carry out the 1,3-dipolar cycloaddition under mild biocompatible conditions, with no toxic by-products and in short reaction times. This reaction was done by means of a LbL assembly, driven by covalent interactions instead of being driven by electrostatic interactions, obtaining a bioactive and biomimetic multilayer coating. Moreover, these polymers are characterized by a critical temperature, known as the transition temperature in aqueous solution (Tt), which is related with a conformational reorganization. Thus, below Tt the polymer chains were soluble in water and above Tt they formed nano- and micro-aggregates becoming insoluble in a reversible process, making these coatings stimuli-responsive. In the following chapters, several polysaccharides as chitosan (CHT), alginate (ALG), hyaluronic acid (HA) or chondroitin sulfate (CS) were used to produce freestanding structured membranes through LbL processes, mainly driven by electrostatic interactions. The use of PEMs containing biopolymers are particularly appealing to coat and develop multilayered structures with biochemical functionalities, biocompatibility, and to mimic the interactions observed in native extracellular matrix (ECM). CHI/CS multilayers were used throughout the thesis, revealing some unique properties, when compared with other polysaccharide-based multilayers, such as their elasticity and degradation rate. However, natural origin polymer-based multilayers present low stiffness and higher hydration rates, which hinder cell adhesion. To overcome this, the CHT/CS multilayers were crosslinked with genipin. This is also a natural product, that is extracted from gardenia fruits and presents the ability to improve the mechanical properties, while preserves the biocompatibility and even enhances the cell adhesive properties. The ability to tailor the multilayers properties can be applied during their assembly or postassembly. Upon adjusting cross-linking parameters (e.g., cross-linker concentration and reaction time) the morphology, thickness, water uptake, rate of biodegradation, mechanical properties and cell adhesive properties can be tuned. Studies of shape-memory of these multilayered films, presented promising results regarding their use in biomedical applications. The mechanical properties of the multilayers can be further improved combining covalent and ionic crosslinking, which gives rise to a full interpenetrating polymer network. More interesting, it was possible to create a well-organized patterned topography at the surface of the freestanding multilayered membrane, just by using a different underlying substrate. This strategy envisaged to mimic the topography of the ECM of some tissues, as bone, skin or nerves, creating grooves on the material’s surface at nanoscale. Using this approach, it was possible to control some cellular functions and behavior as alignment and differentiation. Further in this thesis and inspired by the composition of the adhesive proteins in mussels, freestanding multilayered membranes containing dopamine-modified hyaluronic acid (HA-DN) were produced. The presence of DN along with the thickness of the membranes presented better lap-shear adhesion strength than the control membranes (hyaluronic acid and alginate films – two polysaccharides often regarded as good natural adhesives – were assembled together). Moreover, in vitro tests showed an enhanced cell adhesion for the membranes containing HA-DN and ability to use such kind of membranes for different biomedical and TERM applications, particularly for bone regeneration and skin wound healing. Combining different biomimetic concepts, it was also possible to recreate the complex environment of osteoarthritic articular cartilage by preparing human circular discs of superficially damaged articular cartilage from human samples. Herein, the adhesive freestanding multilayered membranes were used as a vehicle to deliver human adipose stem cells (hASCs) to help to repair the damaged cartilage. hASCs temporarily adhered to the adhesive LbL-based membranes, and were transported to the cartilage discs, creating a bridge of cells between the membranes and the surface of the cartilage. The cells started to migrate into the defects of the cartilage, proliferating and secreting factors capable of repairing the cartilage. Overall, the developed work in this thesis shows that LbL is a very versatile technique that provides the means to develop a wide range of solutions to be useful in biomedical and TERM applications.O desenvolvimento de um revestimento ou material cujas propriedades físicoquímica, mecânicas ou biológicas podem ser modificadas de acordo com as propriedades do tecido alvo, tem ganho cada vez mais importância, nomeadamente para fins biomédicos e de engenharia de tecidos e medicina regenerativa. Durante os últimos anos, diferentes estratégias biomiméticas têm contribuído significativamente para o progresso destas áreas. Estas são possíveis de implementar a diferentes níveis: imitar formas e funções existentes na natureza ou mimetizar processos e sistemas naturais. Na presente tese, a técnica camada-a-camada (LbL) foi usada como uma ferramenta biomimética para modificar superfícies ou produzir membranas com base em múltiplas camadas de polieletrólitos. A crescente utilização desta técnica, concretamente na área biomédica, prende-se com a possibilidade de funcionalizar ou produzir biomateriais aliada à capacidade de incorporar uma gama alargada de blocos de construção. Aqui, diferentes polímeros sintéticos e naturais têm sido usados para construir estruturas multicamada; no entanto, a generalidade dos polímeros sintéticos não apresenta naturalmente locais de ligação e adesão celular. Para contornar este obstáculo, algumas modificações químicas aos polímeros sintéticos têm sido sugeridas e novos compostos têm sido desenvolvidos, inspirados na composição de sistemas naturais. Por exemplo, polipéptidos tipo-elastina (ELPs) são uma classe de polímeros inspirados na natureza, que apresentam propriedades não-imunogénicas e biocompatíveis, podendo ser geneticamente programados conforme desejado. A sua composição baseia-se na repetição de pequenos péptidos também presentes na elastina humana, com a possibilidade também de incorporar outras sequências bioativas especificas, como o tripéptido Arginina-GlicinaÁcido Aspártico (RGD), reconhecido por promover a adesão celular. Para esta tese foram produzidos ELPs, que mais tarde foram funcionalizados com grupos azida e alquino para introduzir a reatividade necessária para uma reação 1,3-dipolar de ciclo-adição se realizar em condições biocompatíveis, sem produtos tóxicos resultantes e em curtos tempos de reação. Esta reação foi realizada sob a técnica LbL, mas conduzida por interações covalentes ao invés de electroestáticas, para atuar como revestimento biomédico. Estes polímeros são ainda reconhecidos pela sua temperatura de transição (Tt) em solução aquosa, relacionada com uma reorganização conformacional da cadeia polimérica. Abaixo da Tt as suas cadeias poliméricas são solúveis, mas acima de Tt formam-se micro-agregados; este é um processo reversível que confere propriedades responsivas aos revestimentos. Nos seguintes capítulos, diferentes polissacarídeos como quitosano (CHT), alginato (ALG), sulfato de condroitina (CS) ou ácido hialurónico (HA), foram usados para produzir membranas multicamadas conduzidas maioritariamente via interações electroestáticas. Esta abordagem tem ganho cada vez mais importância para desenvolver materiais com funcionalidade bioquímica, biocompatibilidade e para mimetizar algumas interações observadas na matriz extracelular (ECM). Ao longo desta tese foram usadas membranas multicamada de CHT/CS; estes materiais revelaram algumas propriedades muito particulares, quando comparadas com outros sistemas de multicamada, como a sua elasticidade e taxas de degradação mais rápidas. No entanto, a baixa rigidez e maiores taxas de hidratação, que muitas vezes impedem a adesão celular, surgem frequentemente associados a sistemas multicamada compostos somente por polissacarídeos. Para contornar este obstáculo, as membranas multicamada de CHT/CS foram reticuladas com genipina. De notar que este composto é de origem natural, sendo extraído da fruta da gardénia; a pós-modificação das membranas com genipina resultou na melhoria das propriedades mecânicas e biocompatibilidade, e ainda, no aumentando das propriedades bio-adesivas. Na realidade, a possibilidade de modular as propriedades destes sistemas multicamada por reticulação química pode ser conseguida logo durante a adsorção de cada camada ou no fim do processo. Características dos biomateriais como a morfologia, espessura, taxas de adsorção de água ou biodegradação, propriedades mecânicas e biológicas podem ser moduladas ajustando certos parâmetros de reticulação (por exemplo, agente de reticulação, concentração ou tempo de reação). Para além do mais, estudos de memória de forma destas membranas multicamada mostraram resultados promissores, considerando o seu uso para fins biomédicos. As propriedades mecânicas destes sistemas foram melhoradas combinando as ligações electroestáticas já existentes com ligações covalentes conferidas pela reticulação química, dando origem a uma rede polimérica multicamada, mas interpenetrada. Na continuação deste trabalho foi possível criar uma topografia com padrão bem organizado na superfície das membranas, alterando somente o material onde efetuamos a deposição das multicamadas. Esta estratégia visou mimetizar a topografia da ECM de diferentes tecidos, como o osso, a pele ou os nervos, criando canais alinhados na superfície do material. Usando este tipo de materiais multicamada padronizados foi possível modular funções e comportamentos celulares como o alinhamento ou a diferenciação. Em seguida, inspirados pela composição das proteínas que conferem adesividade aos mexilhões, foram produzidas membranas multicamada contendo HA modificado com dopamina (DN). A presença de DN ao longo da espessura das membranas multicamada parece ter contribuído para uma melhor e maior força de adesão, quando comparadas com as membranas controlo (membranas multicamada CHT/HA e CHT/ALG). Para além do mais, os testes in vitro resultaram em uma significante melhoria da adesão celular às membranas contendo DN. Esta estratégia mostrou ser promissora para diferentes aplicações biomédicas e de engenharia de tecidos, particularmente para a regeneração de tecido ósseo e a cicatrização de feridas da pele. Combinando diferentes estratégias e conceitos biomiméticos, foi também possível recriar um sistema complexo associado à cartilagem articular e concretamente a doenças como a osteoartrite. Assim sendo, na última parte desta tese, estas membranas multicamada com propriedades adesivas foram utilizadas como veículo para transportar células estaminais humanas do tecido adiposo (hASCs) para o local onde a cartilagem se encontra danificada. A presença deste tipo de células tem sido utilizada como tratamento para cartilagem danificada. Aqui, hASCs aderiram temporariamente às membranas multicamada, e foram assim transportadas diretamente para discos de cartilagem humana danificada, permitindo a criação de uma ponte celular entre as membranas e a superfície da cartilagem. Desta forma, estas células começaram a proliferar na superfície da cartilagem começando a migrar para os defeitos (em profundidade), segregando fatores capazes de ajudar na reparação da cartilagem. No geral, o trabalho desenvolvido para a presente tese mostra a grande versatilidade da técnica LbL, que proporciona os meios necessários para desenvolver uma gama alargada de materiais, estratégias e soluções muito necessárias e promissoras para aplicações biomédicas e de engenharia de tecidos e medicina regenerativa.Programa Doutoral em Químic

Evaluating oxygen tensions related to bone marrow and matrix for msc differentiation in 2d and 3d biomimetic lamellar scaffolds

The physiological O microenvironment of mesenchymal stem cells (MSCs) and osteoblasts and the dimensionality of a substrate are known to be important in regulating cell phenotype and function. By providing the physiologically normoxic environments of bone marrow (5%) and matrix (12%), we assessed their potential to maintain stemness, induce osteogenic differentiation, and enhance the material properties in the micropatterned collagen/silk fibroin scaffolds that were produced in 2D or 3D. Expression of osterix (OSX) and vascular endothelial growth factor A (VEGFA) was significantly enhanced in the 3D scaffold in all oxygen environments. At 21% O , OSX and VEGFA expressions in the 3D scaffold were respectively 13,200 and 270 times higher than those of the 2D scaffold. Markers for assessing stemness were significantly more pronounced on tissue culture polystyrene and 2D scaffold incubated at 5% O . At 21% O , we measured significant increases in ultimate tensile strength (p < 0.0001) and Young’s modulus (p = 0.003) of the 3D scaffold compared to the 2D scaffold, whilst 5% O hindered the positive effect of cell seeding on tensile strength. In conclusion, we demonstrated that the 3D culture of MSCs in collagen/silk fibroin scaffolds provided biomimetic cues for bone progenitor cells toward differentiation and enhanced the tensile mechanical properties. 2 2 2 2