Hyaluronic acid (HA) is a naturally occurring polymer that holds considerable promise for tissue engineering applications. Current cross-linking chemistries often require a coupling agent, catalyst, or photoinitiator, which may be cytotoxic, or involve a multistep synthesis of functionalized-HA, increasing the complexity of the system. With the goal of designing a simpler one-step , aqueous-based cross-linking system, we synthesized HA hydrogels via Diels-Alder “click” chemistry. Furan-modified HA derivates were synthesized and cross-linked via dimaleimide poly(ethylene glycol). By controlling the furan to maleimide molar ratio, both the mechanical and degradation properties of the resulting Diels-Alder cross-linked hydrogels can be tuned. Rheological and degradation studies demonstrate that the Diels-Alder click reaction is a suitable cross-linking method for HA. These HA cross-linked hydrogels were shown to be cytocompatible and may represent a promising material for soft tissue engineering.

Co-delivery of the embedded growth factor-loaded microspheres and adult stem cells in a hydrogel matrix was studied for its potential as a cell-based therapeutic strategy for cartilage regeneration in partial thickness chondral defects. A photopolymerizable N-methacrylate glycol chitosan (MGC) was employed to form an in situ gel that was embedded with two formulations of growth factor-loaded microspheres and human adipose-derived stem cells (ASC). The polymeric microspheres were used as a delivery vehicle for the controlled release of growth factors to stimulate differentiation of the ASC towards the chondrocyte lineage. The microspheres were made of amphiphilic low molecular weight (Mn < 10,000 Da) poly(1,3-trimethylene carbonate-co--caprolactone)-b-poly(ethylene glycol)-b-poly(1,3-trimethylene carbonate-co--caprolactone) (P(TMC-CL)2-PEG)). This triblock copolymer is solid below 100C, but liquid with a low degree of crystallinity at physiological temperature and degrades slowly, and so acidic degradation products do not accumulate locally. Bone morphogenetic protein-6 (BMP-6) and transforming growth factor-3 (TGF-3) were delivered at 5 ng/day with initial bursts of 14.3 and 23.6%, respectively. Both growth factors were highly bioactive when released, retaining greater than 95% bioactivity for 33 days as measured by cell-based assays. To improve ASC viability within the MGC vehicle, an RGD-containing ligand was grafted to the MGC backbone. Prior to chondrogenic induction within the MGC gel, ASC viability was assessed and greater than 90% of ASC were viable in the gel grafted with cell-adhesive RGD peptides as compared to that in non-RGD grafted gels. For ASC chondrogenesis induced by the sustained release of BMP-6 and TGF-3 in MGC gels, the ASC cellularity and glycosaminosglycan production were similar for 28 days. The ratio of collagen type II to I per cell (normalized to deoxyribonucleic acid content) in the microsphere delivery group was significantly higher than that of non-induced ASC or with soluble growth factor administration in the culture media, and increased with time. Thus, the co-delivery of growth factor-loaded microspheres and ASC in MGC gels successfully induced ASC chondrogenesis and is a promising strategy for cartilage repair. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2011-06-07 19:32:50.94

The expanding field of tissue engineering provides a new approach to regenerative medicine for common ailments such as cardiovascular disease and type-I diabetes. Biomaterials can be administered as a delivery vehicle to introduce therapeutic cells to sites of damaged or diseased tissue. A specific class of biomaterials, termed hydrogels, is suitable for this application as they can provide a biocompatible, biodegradable scaffold that mimics the physical properties of the native soft tissue. Injectable hydrogels are increasingly being developed for biomedical applications due to their ability to be delivered in a minimally invasive manner. One potential use for such materials is in the delivery of therapeutics such as cells or growth factor-releasing particles. In this study, the first aim was to determine the interactive effects between collagen-based hydrogels and additives (cells and microspheres) for cardiac regeneration. The results demonstrated that the addition of either cells or microspheres to a collagen-based hydrogel decreased its gelation time and increased its viscosity. Increased cross-linker concentrations resulted in lower cell viability. However, this cell loss could be minimized by delivering cells with the cross-linker neutralizing agent, glycine. As a potential application of these materials, the second aim of this study was to develop a hydrogel for use as an ectopic islet transplant site. Specifically, collagen-chitosan hydrogels were synthesized and characterized, with and without laminin, and tested for their ability to support angiogenic and islet cell survival and function. Matrices synthesized with lower chitosan content (20:1 collagen:chitosan) displayed greater cell compatibility for both angiogenic cells and for islets and weaker mechanical properties, while matrices with higher chitosan content (10:1 collagen:chitosan) had the opposite effect. Laminin did not affect the physical properties of the matrices, but did improve angiogenic cell and islet survival and function. Overall the proposed collagen-based hydrogels can be tailored to meet the physical property requirements for cardiac and islet tissue engineering applications and demonstrated promising cell support capabilities.

The research described covers fundamental studies of environmentally-responsive microgel-based thin films as a function of film architecture, microgel chemistry, film thickness, and environmental stimulus. Studies of multi-layer microgel thin films were conducted primarily using atomic force microscopy (AFM), quartz crystal microgravimetry (QCM), and surface plasmon resonance (SPR), each of which probed different aspects the film architecture as a function of pH of the environment around the film. Binary thin films were constructed by changing the ratios and composition of the microgels in solution to create multi-functional thin films for surface modification applications and were studied using AFM. The basic understanding of how these components create films at surfaces gives us insight into how the films perform and will allow for greater diversity without the guesswork. The morphology of films created from microgels with a degradable cross-linker was examined by AFM as a function of degradation of the particles structure. This thesis focuses mainly on very thin microgel films (<5 layers) studied using QCM, SPR, and AFM. Additional studies involving the characterization of semi-soft colloidal paint-on photonics are discussed in Appendix A.

Development of optogenetically controllable human neural network models can provide an investigative system that is relevant to the human brain. Conventional cultures of neural networks in two-dimensions (2D) have major limitations of scale. For instance, the soma of neurons in 2D is unrealistically flattened and both axon and dendrite outgrowth is restricted. Using a combination of tissue engineering techniques and the inclusion of optogenetically modified human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs), the development of a three-dimensional (3D) human neural culture model within a defined 3D microenvironment is investigated in this study. Light-sensitive neurons were successfully generated by transducing Channelrhodopsin-2 (ChR2) into human iPSC-derived NPCs and neuroblastoma cells (SH-SY5Y) using lentiviral transduction. The use of neuron specific promoters for synapsin-1 (SYN1) and calcium-calmodulin kinase II (CaMKII) in driving the expression of ChR2-Yellow Florescent Protein (YFP) within the mixed neuronal populations from hiPSC-derived neurons (Axol cells) were compared. Viability of the cells at 7 day-post-infection was 80&percnt; - 97&percnt; in all conditions tested. In line with published literature, transduction efficiency of neurons at day 14 was found to be 3&percnt; - 7&percnt; for plasmids containing the SYN1 promoter and 2&percnt; - 5&percnt; for plasmids containing the CaMKII promoter. An increase in promoter driven ChR2-YFP expression was evaluated over 28 days as the neural subpopulations matured. Stably ChR2 expression continued through-out higher passages (&ge; P₁₀) and possibly for periods up to several months. Both SYN1 and CaMKII promoters were found to drive the expression of ChR2 in Axol cells targeting inhibitory and excitatory neurons, respectively. 3D culture systems to support cell growth and optogenetic application were developed and characterised. Alginate hydrogel functionalised with short peptide sequence arginine-glycine-aspartate (RGD), and small molecules such as Rho Kinase inhibitor (ROCKi) and ZVAD were incorporated to increase the viability of human pluripotent stem cells (hPSCs). Investigation of cell response reveals that a flow rate of 3 ml/min and an alginate concentration of 1.8&percnt; (w/v) are optimal and that stem cell survival is significantly improved through incorporation of RGD and ROCKi. Interestingly, ChR2-YFP expression of Axol and SY5Y cells was detectable when transferred to the 3D culture system. The optogenetically modified neurons were found responsive to light stimulation, showing firing patterns and calcium events typical of early developing neurons (e.g. mixed and burst waves; single and multipeak spikes). Neuronal activities were assessed using calcium imaging. Higher numbers of calcium events were associated with CaMKII driven ChR2-YFP expression than with SYN1 in Axol cells. However, calcium activity in SH-SY5Y cells was most noticeable in neurons expressing ChR2-YFP driven by the SYN1 promoter. In primary rodent neuronal cultures, synchronous calcium firing with repetitive action potentials (APs) resulted from ChR2-YFP expression was driven by both SYN1 and CaMKII promoter upon light stimulation. By combining multi-approaches, we report for the first time on the generation of an in vitro hiPSC-derived neural network model in 3D using functionalised alginate hydrogel and involving optogenetic targeting. Expression of ChR2-YFP was found driven by both SYN1 and CaMKII promoter in the RGD-alginate bead system that cultured with Axol cells.

Protein-based materials are an important area of research for various reasons. Natural protein materials such as spider silk have mechanical properties which compare favourably to artificial or inorganic materials, and in addition are biodegradable and can be produced from easily available feedstocks. It is also possible to produce materials that incorporate the functionality of a natural protein, such as ligand-binding or catalysis of reactions, thus allowing this functionality to be used in the solid rather than solution phase. Two particularly interesting components for protein-based materials are amyloid fibrils and tandem repeat proteins. Amyloid fibrils are exceptionally strong, tough, highly-ordered structures that self-assemble from a wide range of simple building blocks. Meanwhile, tandem repeat proteins are a class of proteins that act as scaffolds to mediate protein-protein interactions and are known to act as elastic springs. Unlike globular proteins, tandem repeat proteins can be designed to bind specific ligands, and their ligand-binding properties and stability can be tuned separately. This work details the synthesis and characterisation of repeat protein and amyloid fibril components for a “smart” hydrogel, the production of these gels, and their characterisation using a microfluidic method that I developed. Although amyloid fibrils have previously been decorated with functional proteins, hitherto, this has usually been done by assembling the fibrils from already-functionalised components. This approach limits the functionality to species that can survive the harsh conditions of amyloid aggregation and do not disturb fibril assembly. Therefore, a method was developed to produce amyloid fibrils that displayed an alkyne functionality on their surface to allow functional proteins or other species to be attached after assembly. This involved the design and synthesis (using solid-phase peptide chemistry) of a peptide based on the previously known TTR105-115 peptide (derived from the amyloidogenic Transthyretin protein). These fibrils were characterised by AFM and TEM and it was then shown that the assembled fibrils could be functionalised using an azide-alkyne “click” reaction. The reaction was shown to work with a variety of ligands including proteins, which were found to retain their structure and function after crosslinking to the fibril. The fibrils with ligands attached were characterised by a variety of methods including LCMS (liquid chromatography-mass spectrometry) and super-resolution optical microscopy. Next, repeat proteins were produced recombinantly containing non-natural azido amino acids at their termini. Incorporation of non-natural amino acids was carried out using a number of different methods including amber codon suppression and methionine replacement. Micron-sized hydrogels were then formed from microfluidic-generated droplets by covalently crosslinking the alkyne-functionalised fibrils with the azide-functionalised repeat proteins. The initial experiments to show proof of principle were carried out with consensus-designed repeat proteins, but repeat proteins based on natural sequences were also used to make hydrogels that could later be tested for potential uptake of peptides known to bind these proteins. These hydrogels could potentially be used for drug delivery or other applications in which a chemical response to a mechanical stimulus is desired. The mechanical properties of the hydrogels were measured using novel microfluidic devices, which were designed and fabricated using standard PDMS-based soft lithography.

Nanoelectrodes are defined as electrodes which have a critical dimension on the order of nanometres. Due to their smaller dimensions they have a reduced iR drop and enhanced mass transport, which results in the rapid establishment of an enhanced steady-state diffusion profile and a greater Faradaic current density, along with a smaller relative double layer capacitance, which together give a significantly increased signal to noise ratio compared to macroelectrodes. This potentially makes nanoelectrodes better sensors and analytical tools than macroelectrodes in terms of their having lower limits of detection and faster detection times. However, due to difficulties with fabrication most nanoelectrode designs are highly irreproducible which has inhibited their characterisation and commercial development. The Mount group has previously reported the design, fabrication and characterisation of a novel nanoelectrode design in conjunction with Engineers from the Scottish Microelectronic Centre (SMC). Microsquare Nanoband Edge Electrode arrays (MNEEs) consist of an array of cavities with nanoscale Pt bands (formed by sandwiching the metal between insulating layers) exposed around their perimeter. MNEEs are fabricated using a photolithographic process so can be reproducibly made in large quantities to high fidelity. The purpose of this work is to develop our understanding of the fundamental electrochemical behaviour of MNEEs for biosensing. First, a quantitative analysis of the cyclic voltammograms (CVs) and Electrochemical Impedance Spectroscopy (EIS) of macroelectrodes, microelectrodes and MNEE are compared and discussed. Second, their fundamental response is compared in terms of their biosensing properties by using a pre-established impedimetric biosensing protocol developed on macroelectrodes. This protocol uses a PNA probe to detect the mecA cassette of methicillin resistant staphylococcus aureus (MRSA). The procedure has been optimised and compared for macroelectrodes, microelectrodes and MNEE so as to compare their performances as biosensors. It was observed that MNEE’s: (a) form thiol films faster than electrodes with larger dimensions, determined by kinetic studies of 6-mercaptohexan-1-ol film formation (b) form films with different packing structures dependant on the electrode bulk to edge ratio (c) can detect the same concentration of target in less time than larger electrodes because of their increased sensitivity. The film packing has also been quantitatively investigated using EIS and it can be seen that films formed n MNEE were better able to incorporate target DNA into their more splayed out structure. Unique to this project has been the establishment of a protocol to form heterogeneous carbazole-alanine hydrogel matrices on nanoelectrodes, whose polymerisation is initiated by a pH swing at the electrode surface induced by the oxidation of hydroquinone. The gels growth pattern follows the diffusion field at the electrode and can be monitored using EIS. This also gives a measure of the permeability of the gel by fitting to the correct equivalent circuit. The gel structure has been imaged using light microscopy, confocal microscopy and scanning electrochemical microscopy (SEM). The results give a visual demonstration that MNEE has enhanced diffusion at the corners of the cavities, which is in agreement with previously published simulations, and give evidence as to the onset of hemispherical diffusion and the conditions at which the diffusion field between neighbouring electrodes begin to overlap, a phenomenon which can be observed visually and correlated to changes in the EIS data. Hydrogels have been grown chronopotentiometrically at different currents and the permittivity (through the diffusion coefficients) has been measured of redox couples through gels grown at different speeds. It was found that the hierarchical structure of the hydrogels can be tuned; potentially opening the door to a new breed of tuneable, biocompatible anti-biofouling matrices on bio-functionalised electrodes. The system was characterised using the same MRSA detection protocol as optimised for the MNEE and the target DNA was found to be able to permeate through the hydrogels and bind to the probe, which resulted in a significant change in impedance.

abstract: Electronic devices based on various stimuli responsive polymers are anticipated to have great potential for applications in innovative electronics due to their inherent intelligence and flexibility. However, the electronic properties of these soft materials are poor and the applications have been limited due to their weak compatibility with functional materials. Therefore, the integration of stimuli responsive polymers with other functional materials like Silicon is strongly demanded. Here, we present successful strategies to integrate environmentally sensitive hydrogels with Silicon, a typical high-performance electronic material, and demonstrate the intelligent and stretchable capability of this system. The goal of this project is to develop integrated smart devices comprising of soft stimuli responsive polymeric-substrates with conventional semiconductor materials such as Silicon, which can respond to various external stimuli like pH, temperature, light etc. Specifically, these devices combine the merits of high quality crystalline semiconductor materials and the mechanical flexibility/stretchability of polymers. Our innovative system consists of ultra-thin Silicon ribbons bonded to an intelligently stretchable substrate which is intended to interpret and exert environmental signals and provide the desired stress relief. As one of the specific examples, we chose as a substrate the standard thermo-sensitive poly(N-isopropylacrylamide) (PNIPAAm) hydrogel with fast response and large deformation. In order to make the surface of the hydrogel waterproof and smooth for high-quality Silicon transfer, we introduced an intermediate layer of poly(dimethylsiloxane) (PDMS) between the substrate and the Silicon ribbons. The optical microscope results have shown that the system enables stiff Silicon ribbons to become adaptive and drivable by the soft environmentally sensitive substrate. Furthermore, we pioneered the development of complex geometries with two different methods: one is using stereolithography to electronically control the patterns and build up their profiles layer by layer; the other is integrating different multifunctional polymers. In this report, we have designed a bilayer structure comprising of a PNIPAAm hydrogel and a hybrid hydrogel of N-isopropylacrylamide (NIPAAm) and acrylic acid (AA). Typical variable curvatures can be obtained by the hydrogels with different dimensional expansion. These structures hold interesting possibilities in the design of electronic devices with tunable curvature. / Dissertation/Thesis / M.S. Chemical Engineering 2012

Mesenchymal stem cells are the stem cells that replace the bone, fat and cartilage tissues of the human body. In addition, these cells can form muscles, ligaments and neurons. This wide multipotency has made mesenchymal stem cells of particular interest in the fields of tissue engineering and regenerative medicine. Furthermore, mesenchymal stem cells can modulate the immune system by reducing factors that increase inflammation and immune recognition. This immune recognition suppression has resulted in their application as part of bone marrow transplantation in the prevention of 'graft versus host‘ disease. There are hundreds of on-going clinical trials using these cells for the treatment of autoimmune diseases such as type I diabetes, arthritis and multiple sclerosis. The increasing importance of these cells has brought in to focus the culture methods used to for their expansion and manipulation. Currently, animal derived components are used as surfaces for their growth and as components in the culture media. This exposes these cells to animal pathogens and antigens that can be passed to the recipients of these cells. In the first part of this thesis, polymer microarrays were employed to identify alternatives to the biological surfaces currently used for mesenchymal stem cell culture. This platform allowed hundreds of polyacrylates/acrylamides and polyurethanes to be simultaneously scrutinised to identify surfaces that could support their growth and maintain their stem cell characteristics. Identified polymer surfaces were monitored in long-term culture (10 passages) and were shown to retain the cell phenotype and capacity to differentiate, thus providing chemically defined substrates for long-term mesenchymal stem cell culture. In the second part of this thesis, a 'smart‘ polymer microarray of hydrophilic cross-linked polymers (hydrogels) were used to remove another key biological component of culture, trypsin. These 'smart‘ hydrogels modulated their properties depending on the temperature. Hydrogels that could trigger mesenchymal stem cell release after a reduction in temperature were identified. A unique passaging system using a modest temperature reduction for 1h was developed as a passaging method. Cells were maintained and monitored for 10 passages using this novel enzyme free passaging method. Analysis of the mesenchymal stem cell phenotype and differentiation capacity revealed this method superior than conventional culturing methods. In the final part of this thesis, a 'knowledge-based‘ small molecule library was designed, which could potentially yield small molecules to manipulate/enhance the mesenchymal stem cell state without the use of biological components. The key protein pathways that control the stem cell state were examine with the bioinformatics tool GeneGo was used to identify compounds that affected these pathways, resulting in selection of 200 small molecules. The effect of the small molecules on the mesenchymal phenotype was examined and 5 small molecules were identified that enhanced the phenotype of these cells. The anti-inflammatory properties associated with the hit compounds led to the investigation of their effects on key surface proteins associated with the immune-modulatory state of the cells. In this preliminary study, two of the small molecules, estriol and spermine, increased the expression of a key mesenchymal stem cell marker STRO-1 and down regulated ICAM-1, a critical component of the immune modulation capacity of this cell type.

Estudos recentes têm demonstrado o efeito das isoflavonas da soja, especialmente da genisteína (GEN), administrada topicamente, na prevenção do fotoenvelhecimento e fotocarcinogênesis. Esse efeito tem sido relacionado com a sua atividade inibidora de tirosina quinase e antioxidante. Recentemente, demonstramos um lento perfil de permeação da GEN in vitro (usando células de difusão de Franz) a partir de nanoemulsões através de pele de orelha suína (SILVA et al., Pharmazie, v. 64, p. 32- 35, 2009). Entretanto, estudos relativos à distribuição da GEN através das camadas da pele ainda não foram realizados. Neste contexto, o principal objetivo deste estudo foi avaliar a distribuição da GEN através da pele suína a partir de nanoemulsões, antes e após incorporação em hidrogéis de ácido acrílico, a fim de ajustar a viscosidade das mesmas para aplicação tópica. Nanoemulsões contendo GEN apresentaram valores de potencial  negativos e baixa viscosidade. Os hidrogéis derivados contendo as nanoemulsões de GEN demonstraram um comportamento não-Newtoniano pseudoplástico. Um método isocrático de cromatografia líquida para determinar a GEN nas camadas da pele de orelha suína foi desenvolvido e validado. O método mostrou ser específico, linear, preciso e exato para a determinação da GEN nas camadas da pele de orelha suína (i.e. stratum corneum, epiderme e derme). Nas condições validadas, o perfil de permeação/retenção da GEN foi claramente influenciado pela natureza do núcleo oleoso empregado. Nanoemulsões constituídas de um núcleo oleoso de triglicerídeos de cadeia média apresentaram uma permeação mais lenta e uma maior retenção da GEN na pele quando comparado com nanoemulsões contendo octildodecanol. A incorporação das nanoemulsões de GEN em hidrogéis aumenta a retenção da GEN, especialmente na epiderme. Tal resultado pode estar relacionado com o aumento da hidratação da epiderme como sugerido nas observações histológicas. Em conclusão, o conjunto dos resultados demonstra o efeito das formulações sobre a distribuição da GEN através das camadas da pele de orelha suína. / Recent studies have shown the effect of soy isoflavones, especially genistein (GEN), topically administrated, in preventing skin photoaging and photocarcinogenesis. This effect has been related to tyrosine kinase inhibition and antioxidant activities. Recently, we have demonstrated a slow permeation profile of GEN in vitro (using Franz type diffusion cells) from nanoemulsions through porcine ear skin (SILVA et al., Pharmazie, v. 64, p. 32-35, 2009). However, studies concerning the distribution of GEN in the skin layers have yet to be performed. In this context, the main purpose of this study was to evaluate the GEN distribution through the porcine skin from topical nanoemulsions, before and after their incorporation into acrylic acid-hydrogels, in order to have their viscosity adjusted to topical applications. Nanoemulsions containing GEN exhibit negative -potential and low viscosity. The derived hydrogels containing GEN-loaded nanoemulsions presents non-Newtonian pseudoplastic behavior. An isocratic liquid chromatographic method to determine GEN in porcine ear skin layers was developed and validated. The method proved to be specific, linear, precise and accurate to determine GEN in the porcine skin layers (i.e. stratum corneum, epidermis and dermis). In the validated conditions, the GEN permeation/ retention profile was clearly influenced by the nature of the oil core used. Nanoemulsions composed by a medium chain triglycerides oil core exhibit both a slower permeation and a higher retention profile of GEN as compared to octyldodecanol nanoemulsions. The incorporation of GEN-loaded nanoemulsion into hydrogels improved the GEN retention, especially in the epidermis. Such a result might be related to the increased hydration of epidermis as suggested in histological observations. In conclusion, the overall results show the effect of the formulation on distribution of GEN through the porcine skin layers.