Bio-Inspired Materials For Parsing Matrix Physicochemical Control Of Cell Migration: A Review

Cell motility is ubiquitous in both normal and pathophysiological processes. It is a complex biophysical response elicited via the integration of diverse extracellular physicochemical cues. The extracellular matrix directs cell motilityvia gradients in morphogens (a.k.a. chemotaxis), adhesive proteins (haptotaxis), and stiffness (durotaxis). Three-dimensional geometrical and proteolytic cues also constitute key regulators of motility. Therefore, cells process a variety of physicochemical signals simultaneously, while making informed decisions about migration viaintracellular processing. Over the last few decades, bioengineers have created and refined natural and synthetic in vitro platforms in an attempt to isolate these extracellular cues and tease out how cells are able to translate this complex array of dynamic biochemical and biophysical features into functional motility. Here, we review how biomaterials have played a key role in the development of these types of model systems, and how recent advances in engineered materials have significantly contributed to our current understanding of the mechanisms of cell migration

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

Fabrication and characterization of a polymeric nanofluidic device for DNA analysis

The growing needs for cheaper and faster sequencing of long biopolymers such as DNA and RNA have prompted the development of new technologies. Among the novel techniques for analyzing these biopolymers, an approach using nanochannel based fluidic devices is attractive because it is a label-free, amplification-free, single-molecule method that can be scaled for high-throughput analysis. Despite recent demonstrations of nanochannel based fluidic devices for analyzing physical properties of such biopolymers, most of the devices have been fabricated in inorganic materials such as silicon, silicon nitride and glass using expensive high end nanofabrication techniques such as focused ion beam and electron beam lithography. In order to use the nanochannel based fluidic devices for a variety of bioanalyses, it is imperative to develop a technology for low cost and high through fabrication of such devices and demonstrate the feasibility of the fabricated nanochannel based fluidic devices in obtaining information on biopolymers. We developed a low cost and high throughput method to build polymer-based nanofluidic devices with sub-100 nm nanochannels using direct imprinting into polymer substrates. Imprinting with the polymer stamps showed good replication fidelity for multiple replication processes, preventing damage of the expensive nanopatterned master and reducing undesirable deformation in the molded polymer substrate. This approach opened up a possibility to build cheap and disposable polymer nanofluidic devices for single molecule analysis. The ion transportation and DNA motion in nanofluidic systems were studied. Simulation and experiment results indicate that fast degeneration of the electric field at micro/nano interface plays a major role, in addition to the bulk flow in the microfluidic networks. Inlet structures and bypass microchannels were designed and built, the use of which has proven to enable enhancing the DNA capture rate by over 500 %. Attributed to the improved capture rate, the blockade current of DNA translocation though a nanochannel was also measured. We observed in the current versus time curves both current increase and decrease in the existence of a DNA molecule in the nanochannel, which we attributed to the ion channel blockage and electrical double layer formed around the DNA molecule, respectively

Three Dimensional (3D) Printable Gel-Inks for Skin Tissue Regeneration

Recent and rapid progression in three-dimensional (3D) printing techniques has revolutionized conventional therapies in medicine; 3D printed constructs are gradually being recognized as common substitutes for the replacement of skin wounds. As gel-inks, large numbers of natural and synthetic (e.g., collagen and polyurethane, respectively) substances were used to be printed into different shapes and sizes for managing both acute and chronic skin wounds. The resultant 3D printed scaffolds not only provide physical support but also act as supporting niches for improving immunomodulation and vascularization and subsequent accelerated wound healing. Recently, the use of thermosensitive and pH-responsive gels has made it possible to prepare 3D printed constructs with the ability to facilitate in situ crosslinking within the biopolymer and with native wound edge tissue as well as to fill the exact shape of wound damage. In this chapter, we aim to introduce the current state of 3D printable gel-inks utilized for skin wound treatment and illustrate future prospects in this amazing area of science

Evaluation of the adhesion forces between dust particles and photovoltaic module surfaces

Soiling of Photovoltaic (PV) modules is a growing area of concern due to the adverse effect of dust accumulation on PV performance and reliability. In this work, we report on four fundamental adhesion forces that take place at the first stage of soiling process. These are capillary, van der Waal, electrostatic and gravitational forces. It is found that under high relative humidity, the adhesion mechanism between dust particles and PV module surfaces is dominated by capillary force, while van der Waal force dominates under dry conditions. Moreover, real field data for long soiling periods over solar panels in Qatar were investigated and resulted in proposing a novel modified sigmoid function that predicts a relative humidity inflexion value at which transition in the particulate matter deposition rate takes place from low to high values. Moreover, the effect of surface roughness was investigated by measuring adhesion force over clean glass versus substrates that are coated with in-house developed anti-dust titania thin films

Fundamental Investigation of Biological Interactions for Applications in Infection Prevention and Biomaterial Development

Bacterial infections persist as a public threat due to the ease by which bacteria adapt to commonly used antibiotics. In addition, bacteria on surfaces develop protective communities called biofilms that hinder the ability of antibiotics to completely eliminate the pathogens. The rapid development of bacterial resistance to antibiotics has made pharmaceutical companies reluctant to fund new antibiotics research. Hence, novel approaches to prevent and treat infections are needed. The development of infections can be divided into three steps: adhesion, invasion and multiplication. Antibiotics target at the latter two step and are prone to bacterial resistance as passive strategies. Bacterial adhesion to host cells/implanted medical devices is the first step leading to following invasion and multiplication. However, fundamental understanding of bacterial adhesion process is still lacking. The current studies are aimed to systematically investigate biological interactions between pathogenic bacteria and host cell, proteins and biomaterials with both macro and micro scale approaches. The macro scale methods include bacterial adhesion assay, viability studies, and thermodynamic modeling. The micro scale methods include direct adhesion force measurements, ultra surface visualization via atomic force microscopy (AFM) and surface structure modeling. Our work combines experiments and modeling aimed at understanding the initial steps of the bacterial adhesion process, focusing on two case studies: 1) Mechanisms by which cranberry can prevent urinary tract infections through interfering with bacterial adhesion; and 2) Design of anti-adhesive and antimicrobial coatings for biomaterials. We make direct adhesion force measurements between bacteria and substrates with an atomic force microscope (AFM), and combine such experiments with thermodynamic calculations, in order to develop a set of tools that allows for the prediction of whether bacteria will attach to a given surface. These fundamental investigations of the bacterial adhesion process help elucidate the underlying mechanisms behind bacterial adhesion, thus leading to improved clinical outcomes for a number of biomedical applications

The development of optical nanomachines for studying molecules : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Mechatronics Engineering at Massey University, Palmerston North, New Zealand

Chapter 3 is ©2020 IEEE. Accepted manuscript is reprinted, with permission, from 2020 IEEE/ASME International Conference on Advanced Intelligent Mechatronics (AIM). Chapter 5 is ©2022 IEEE. Accepted manuscript is reprinted, with permission, from 2022 International Conference on Manipulation, Automation and Robotics at Small Scales (MARSS).Optical tweezers have been used for a number of applications since their invention by Arthur Ashkin in 1986, and are particularly useful for biological and biophysical studies due to their exceptionally high spatial and force-based resolution. The same intense laser focus that allows light to be used as a tool for micro-nanoscale manipulation also has the potential to damage the objects being studied, and the extremely high force resolution is coupled with the limitation of very low forces. There is potential to overcome these drawbacks of optical manipulation through making use of another laser based technique: two-photon absorption polymerisation (TPAP). This thesis has brought these together to demonstrate the uses of optical nanomachines as helpful tools for optical tweezer studies. The project was highly interdisciplinary, concerning the intersection of optical trapping, 3D micromachine design and development, and DNA stretching. The thesis was based around the strategy of first developing microrobots and demonstrating their manipulation using optical tweezers, then adjusting the design for specific applications. Microlevers were developed for lever-assisted DNA stretching and amplification of optical forces. The influence of design features and TPAP parameters on microlever functionality was investigated; particularly the influence of overlapping area and presence of supports, and the effects of differently shaped "trapping handles". These features were important as lever functionality was tested in solutions of different ionic strength, and stable trapping of the levers was required for force amplification. DNA stretching was chosen as a target application for distanced-application of optical forces due to its status as a well-known and characterised example of single-molecule studies with optical tweezers. Amplification of optical forces was also seen as an application that could demonstrate the utility of optical micromachines, and microlevers with a 2:1 lever arm ratio were developed to produce consistent, two-fold amplification of optical forces, in a first for unsupported, pin-jointed optical microrobotics. It is hoped that in the future fully-remote, micromachine-assisted studies will extend optical tweezer studies of laser-sensitive subjects, as well as increasing the forces that can be applied, and the results obtained in this thesis are encouraging. All in all, the thesis confirms the potential of optical micromachines for aiding studies using optical tweezers, and demonstrates concrete progress in both design and application