Damaged cartilage can cause severe pain and restricted mobility, with few long term treatments available. The developing field of tissue engineering offers an alternative to the currently used full joint replacement. Restoring damaged cartilage through tissue engineering would enable an active lifestyle to be recovered and retained, without restrictions to joint mobility. This is increasingly important as the prevalence of osteoarthritis rises. Tissue engineering requires biomaterial scaffolds that mimic the function of the tissue while cells develop, and so the scaffold must provide the appropriate biological, chemical and mechanical stimuli. In this work, methods were developed to enable the design of scaffolds that mimic the microstructure and mechanical properties of articular cartilage. Electrospinning was investigated as a method to mimic the nanoscale collagen fibres within cartilage extracellular matrix. A parametric study was conducted to determine how changes to a gelatin solution affect the mechanical properties of the non-woven fibrous mesh. The solution properties had a clear impact on the morphology of the fibres, but the effect on the mesh mechanical properties was convoluted. The results demonstrated the need for greater understanding of the 3D morphology of electrospun meshes, to establish how these may be altered in order to design scaffolds with desirable mechanical properties. The fracture mechanics of soft materials are complex, and are generally overlooked when designing tissue engineering scaffolds. The complexities have led to a lack of standardised testing, making comparisons between studies impractical. In this work, fracture testing methods were compared, using a viscoelastic polymer to mimic some of the complexities of soft tissue mechanics. Mode III trouser tear tests and mode I pure shear tests were found to provide reliable measurements. Due to the ease of testing small samples, trouser tear testing was concluded to be the most advantageous for determining the fracture resistance of soft tissue engineering scaffolds. Finally, electrospun meshes were combined with hydrogels to create biomimetic scaffolds, which were characterised using tensile and trouser tear fracture tests. Fibre-reinforcement was shown to enhance the mechanical properties of a weak hydrogel, but diminished those of a strong, tough polyacrylamide (PAAm)-alginate hydrogel. The PAAm-alginate hydrogel exhibited mechanical properties close to those of natural articular cartilage, but without the microstructure that would enhance its suitability for use as a cartilage tissue engineering scaffold. An alternative method for reinforcing PAAm-alginate was proposed, which shows promise for producing a biocompatible scaffold that mimics both the mechanics and the microstructure of articular cartilage. Ultimately, this thesis aimed to improve the design of biomimetic scaffolds for cartilage tissue engineering, and advance mechanical characterisation techniques within this field.

This study presents a systematic investigation of two types of soft biomaterials: phospholipid-based microbubbles (MBs) and agarose hydrogels, using atomic force microscopy (AFM) force-distance curves. Microbubbles are used widely in several applications, especially in medical applications, where they are used as ultrasound contrast agents (UCAs) and as vehicles for transporting the drugs and genes to their targets, which is commonly known as drug/gene delivery. Although plenty of attention has been paid to these materials by medical researchers there is a shortage of engineering research on the properties of these materials. The present study tries to address this gap by studying these materials from the engineering perspective; therefore, the aim of this study is to investigate the mechanical properties of MBs and hydrogels. In this research, phospholipid-based microbubbles (MBs), commercially called SonoVue® microbubbles and used as UCAs, were investigated to measure their mechanical properties using an AFM mode of operation called force-distance curves (or force spectroscopy mode); this mode allows for direct mechanical tests to acquire the force-deformation (F-Δ) behaviour of the MBs. The compression tool was a flat (tipless) cantilever moved at constant speed, whereas the variable was MB size. The MBs behaviour was assessed by calculating several mechanical properties, which were the stiffness, Young's modulus (three different models were applied), hysteresis, plasticity, adhesion forces, nonlinearity and instability. The stiffness and the Young's modulus values were measured to be in the same range as found in similar studies. A phenomenon was observed that the local stiffness of the MB increases after each unstable step provided that the MB stays within the linear elastic region. The Young's modulus was calculated applying three models, two for estimating the elastic modulus of the shell and the third for modulus of elasticity of the whole MB. The stretching component of the membrane theory was found to provide the best prediction of the Young's modulus value. To investigate the effect of the tip geometry on the mechanical properties of the MBs, the MBs were studied with different cantilever/tips, including a conical-tipped cantilever. The study concluded that there is no impact of the contact geometry on the mechanical properties of the MBs if the applied forces and the spring constant of the cantilever are the same. The same phenomenon, increasing the local stiffness of the MB after each unstable step, was found however with a higher rate. Hydrogels were also studied in this research using AFM and adopting a nanoindentation technique. The indenter was a conical tip moving toward the sample surface with constant speed and applying similar forces on all samples, where the variable was the gel concentration. In addition to the previous mechanical properties, other properties were investigated, such as hardness, universal hardness and pressure. An effect of the gel concentration on the mechanical properties of the gels was observed. There is a difference in the results compared to those reported in the literature review, where some of the results are in the same range as those found here, while others were either higher or lower, due to the influence of factors such as the indenter geometry, the applied force and the load rate; moreover, it was found that the viscoelastic behaviour of the gels played a significant role.

DNA scaffolds self-assembled by short-stranded synthetic DNA can be tailored to build thermally reversible hydrogels with target binding sites. These hydrogels exhibit highly selective binding properties due to the specificity of DNA and also provide an aqueous environment for various reactions to happen within the network constraints. Hence, a careful study on the assembly mechanism and other physical aspects of DNA hydrogels is required to facilitate the future design and construction of such materials at the precise control. In this thesis, I present the work on well-designed DNA nano-stars as scaffolds for functional bulk materials with potential applications in bio-sensing. Chapter 1 starts with introducing the fundamental properties of DNA molecules, focusing on the advantages of utilising short-stranded DNA to programme and engineer micro- and macro- materials. Then it briefly reviews the field of rheology and micro-rheology, with the diffusing wave spectroscopy (DWS) technique illustrated explicitly as an example passive micro-rheology tool. Afterwards, a critical literature review on computational modelling of DNA systems is present, followed by the thesis outline at the end. Chapter 2 describes a simple DNA dendrimer system self-assembled from three-armed DNA nano-stars. The characterisation tools such as UV-vis spectroscopy, gel electrophoresis and dynamic light scattering (DLS) are introduced to verify the final production of the complex DNA structures. From this practice, we develop a routine for designing DNA scaffolds that yield optimal productivity. Chapter 3 investigates the mechanical properties of DNA hydrogels made of three-armed DNA nano-stars and how they change upon cooling and heating empolying DWS micro-rheology. The resulting viscoelastic moduli over a broad range of frequencies reveal a clear, temperature-reversible percolation transition coinciding with the melting temperature of the system's sticky ends. This indicates that we can achieve precise control in mechanical properties of DNA hydrogels, which is beneficial for designing more sensitive molecular sensing tools and controlled release systems. Chapter 4 develops a coarse-graining computational model of DNA hydrogels that resembles the system in Chapter 3 using LAMMPS, a classical molecular dynamics code. Thermodynamics, structural analysis and rheology tests were taken, qualitatively reproducing the physical phenomena of DNA assembly of the hydrogel network. Chapter 5 studies the internal behaviours of three-armed DNA complexes using oxDNA model also implemented in LAMMPS, with particular focus on the effect of the inert bases in the core and between double-stranded branches and single-stranded sticky ends. A deep insight into sequence-dependent behaviour of such complex structures can guide the parameter optimisation of the individual building blocks for the model described in Chapter 4. Chapter 6 concludes the thesis and presents an outlook for the future work that emerged out of my experimental and numerical studies.

Bile salts are supramolecules with amphiphilic properties. Bile salts form aggregates in aqueous solutions. The primary aggregates of bile salts are hydrophobic and the secondary aggregates which form at higher concentrations are relatively hydrophilic. Among bile salts, sodium deoxycholate (NaDC) has been known to form hydrogels at pHs close to the neutral pH and within a certain concentration range. The aim of this work was to provide insight into the properties of a NaDC hydrogel as a supramolecular gel system through three different projects. Pyrene is a hydrophobic polycyclic aromatic compound which was used as a fluorescent probe and the guest for these projects. 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine perchlorate (DiD) is another fluorescent compound which was used as another guest. The objective of the first project was to understand the mobility of a small guest molecule in NaDC gel in the presence of cucurbit[6]uril (CB[6]) compound as an additive for the gel. Cucurbit[n]urils are macrocyclic compounds with a hydrophobic cavity and two hydrophilic portals. The presence of CB[6] provides another binding site for pyrene in addition to the primary aggregates of the bile salts. The results showed that the mobility of the guest from water and CB[6] to the bile salts network happens when the temperature is raised. The release of the guest back into the water happens when the gel is cooled. The objective of the second project was to investigate the effect of surfaces with different hydrophilicity on the NaDC gel properties. The results of fluorescence correlation spectroscopy (FCS) experiments revealed that either the hydrophilicity of the surface does not affect the NaDC gel network or the FCS is insensitive to the structural changes induced by the hydrophilicity of the surface. These experiments depicted that the aggregates involved in the gel’s network are the same as those formed in the aqueous solutions. Moreover, results of the steady-state and time-resolved fluorescence experiments showed that the bulk gel properties are not affected by the hydrophilicity of the surface. The objective of the last project was to determine the effect of ions on NaDC gel properties. The results showed that cations with different charge density have different effects on the gel formation and properties. The presence of inorganic salts with a monovalent cation leads to the formation of a kinetically favored gel sample within a few hours after sample preparation. The extension of the network occurs overtime and a thermodynamically stable gel forms a couple of days after sample preparation. / Graduate / 2020-10-20

Because engineered tissue constructs are inherently limited by their lack of microvascularization, which is essential to provide oxygen for cell survival, this thesis presents rationally designed materials and cell culture techniques capable of supporting functional tubule formation and stabilization. Combining a synthetic scaffold material with cells and their cell-secreted signals instigated tubule formation throughout the scaffold. Poly(ethylene glycol) (PEG) based hydrogels, biocompatible polymers which resist protein adsorption and subsequent nonspecific cellular adhesion, were modified to induce desired cell characteristics. Human umbilical vein endothelial cells were used as a reproducible and readily available cell type. Several tubule-stabilization signals, including platelet derived growth factor-BB (PDGF-BB) and ephrinA1, were covalently immobilized via conjugation to PEG to enable prolonged bioactive signaling and controlled local delivery. All hydrogels were further tested in a mouse cornea micropocket angiogenesis assay, a naturally avascular tissue for easy imaging in a reproducible and quantifiable assay. Hydrogels containing soluble growth factors induced vessel formation in the hydrogel, and the resulting vessel morphology was modulated using different growth factor concentrations. Immobilized PDGF-BB led to tubule formation in two dimensions, three dimensions, and in the mouse cornea while immobilized ephrinA1 stimulated secretion of extracellular matrix proteins laminin and collagen IV to stabilize the newly formed tubules. Finally, a co-culture of endothelial and pericyte cells encapsulated into hydrogels formed tubules that anastomosed to the host vasculature and contained red blood cells. PEG-based hydrogels represent a promising technique to induce microvascular formation in engineered constructs, leading to stable and functional vessel formation using covalently immobilized growth factors and encapsulated cells. These materials can be used for replacement of damaged or diseased tissues as the current supply of cadaveric donations cannot meet the demand of tissues for the 110,000 people awaiting an organ in the US.

Due to an increased environmental awareness and thus, concerns over the use of fossil-based monomer for polymer production, there is an ongoing effort to find alternatives to non-renewable traditional monomers. This has ushered in the rapid growth in the development of bio-based materials such as green monomers and biodegradable polymers from vegetable and animal resources. Glycerol, as a renewable bio-based monomer, is an interesting candidate for sustainable polymer production. Glycerol is a renewable material that is a by-product of the transesterification of vegetable oils to biodiesel. Utilization of the excess glycerol derived from the growing biodiesel industry is important to oleochemical industries. The main objective of this thesis was to produce high molecular weight polyglycerol from glycerol and synthesize stimuli-responsive polyglycerol hydrogels. The work began with an investigation of the step-growth polymerization of glycerol to relatively high molecular weight polyglycerol using several catalysts. The catalytic reaction mechanisms were compared and the polymer products were fully analyzed. High molecular weight partially branched polyglycerol with multimodal molecular weight distributions was obtained. The polymerization of glycerol proceeded fastest with sulphuric acid as catalyst as indicated by the highest observed conversion of monomer along with the highest molecular weights. Theoretical models were used to predict the gel point and to calculate monomer functionality. High molecular weight polyglycerol was used to synthesize novel stimuli-responsive hydrogels. Real-time monitoring of step-growth polymerization of glycerol was investigated using in-line and off-line Attenuated Total Reflectance/Fourier Transform infrared (ATR-FTIR) technique.

Hydrogels have successfully proved themselves useful for drug delivery applications and several delivery routes have been developed over the years. The particular interest in this work was to design, synthesise and evaluate in situ forming drug eluting hydrogels, which have the potential to ameliorate the healing of cardiovascular diseases. With this aim the anti-inflammatory and immunosuppressant drugs rapamycin (Ra) and dexamethasone (Dex) were made water soluble by conjugation with polyethylene glycol (PEG). Ra was attached pendant from the terminal of PEGs while Dex was incorporated into dendritic structures grown from PEGs. These conjugates were further crosslinked into hydrogels by either conjugate or thiol-ene addition. The gel degradation was tuned to take between 5 and 27 days by using gel building block combinations that induced either 2 or 4 hydrolytically labile bonds per crosslink or by varying the number of crosslinking sites of the building blocks. The use of thiol-ene addition prolonged the degradation time nearly seven folded compared to conjugate addition as a more stable crosslink was formed. Two different formulations for gelling via conjugate addition were used (acrylate-thiol or vinyl sulphone-thiol) to deliver Ra, which was carried by either a 4- or 2-armed PEG. The elution kinetic for the respective gel formulation was of zero order during 15 and 19 days of gel degradation. In addition, Ra was PEGylated via esters, with a distance of either one or two carbons to a nearby thio-ether functionality. The difference in ester conjugation resulted in a slight but significant change in drug-PEG conjugate stability, which was mirrored by the increased time to reach the half amount of total drug elution; from 9.3 to 10.2 days and from 5.1 to 9.7 days for the two gel formulations, respectively. Dexamethasone was incorporated via an ester into dendrons of first and second generation pending from 2- and 4-armed PEGs at loadings of 2, 4 or 6 Dex molecules per carrier molecule. The resulting elution kinetic was of zero order during degradation periods of 5-27 days. Released Dex still possessed biological activity as determined by an in vitro cell assay. The novelties in this thesis are: (A) slow release of rapamycin obtained by covalent incorporation into hydrogels, (B) the use of unique PEG-based dendrimers to incorporate dexamethasone into a hydrogel and (C) zero order sustained release of dexamethasone at physiological pH. / Hydrogeler har framgångsrikt visat sig användbara för att leverera läkemedel och ett flertal metoder har utvecklats de senaste 20 åren. Fokuset i den här avhandlingen ligger på att designa, framställa och utvärdera läkemedelsutsöndrande hydrogeler som spontanhärdar in situ, vilka har potential att förbättra läkningen efter kardiovaskulär sjukdom. Med det syftet gjordes de anti-inflammatoriska och immunsänkande läkemedlen rapamycin (Ra) och dexametason (Dex) vattenlösliga genom att konjugeras med polyetylenglygol (PEG). Ra fästes kovalent längst ut på PEGar medans Dex inkluderades i dendritiska strukturer vilka byggdes från ändpunkten av PEGar. De här konjugaten tvärbands till hydrogeler via antingen konjugerad addition eller radikal polymerisation. Nedbrytningen av gelerna trimmades till att ta mellan 5 och 27 dagar genom att använda kombinationer av gelbyggstenar som bildar antingen 2 eller 4 hydrolyserbara estrar per tvärbindning eller genom att variera antalet tvärbindningspunkter hos byggstenarna. Användandet av radikal polymerisation i sig ledde till att nedbrytningen av geler tog nära sju gånger längre tid jämfört med geler gjorda via konjugerad addition eftersom stabilare tvärbindningar då formas. Två olika kombinationer för härdning via konjugerad addition (akryl-tiol eller vinylsulfon-tiol) användes för att leverera Ra som bars av antingen en 4- eller 2-armad PEG. Utsöndringskinetiken av Ra för de två kombinationerna var av nollte ordningen under de 15 och 19 dagar som gelerna degraderade. Dessutom, Ra PEGylerades via estrar med ett avstånd på antingen ett eller två kol till en närliggande tioeter. Skillnaden i avstånd ledde till en liten men signifikant skillnad i stabiliteten hos Ra-PEG konjugaten, vilket speglades i den förlängda tiden att nå halva mängden av den totala läkemedelsutsöndringen; från 9.3 till 10.2 dagar och från 5.1 till 9.7 dagar för de två respektive gelkombinationerna. Dex kopplades in via en esterbindning till dendroner av första och andra generationen byggda från PEGar med 2 eller 4 armar, vilket resulterade i att 2, 4 eller 6 Dex levererades per bärarmolekyl. Dex eluerade med nollte ordningens kinetik under degraderingsperioder på mellan 5 och 27 dagar. Vidbehålllen biologisk aktivitet av eluerad Dex bekräftades genom cellexperiment in vitro. Nyheterna i den här avhandlingen består av: (A) kontrollerad utsöndring av rapamycin uppnådd genom kovalent inbindning till hydrogeler, (B) användandet av unika PEGbaserade dendrimerer för kovalent inbindning av dexametason till hydrogeler och (C) nollte ordningens utsöndring av dexametason vid fysiologiskt pH. / <p>QC 20130204</p>

Due to an increased environmental awareness and thus, concerns over the use of fossil-based monomer for polymer production, there is an ongoing effort to find alternatives to non-renewable traditional monomers. This has ushered in the rapid growth in the development of bio-based materials such as green monomers and biodegradable polymers from vegetable and animal resources. Glycerol, as a renewable bio-based monomer, is an interesting candidate for sustainable polymer production. Glycerol is a renewable material that is a by-product of the transesterification of vegetable oils to biodiesel. Utilization of the excess glycerol derived from the growing biodiesel industry is important to oleochemical industries. The main objective of this thesis was to produce high molecular weight polyglycerol from glycerol and synthesize stimuli-responsive polyglycerol hydrogels. The work began with an investigation of the step-growth polymerization of glycerol to relatively high molecular weight polyglycerol using several catalysts. The catalytic reaction mechanisms were compared and the polymer products were fully analyzed. High molecular weight partially branched polyglycerol with multimodal molecular weight distributions was obtained. The polymerization of glycerol proceeded fastest with sulphuric acid as catalyst as indicated by the highest observed conversion of monomer along with the highest molecular weights. Theoretical models were used to predict the gel point and to calculate monomer functionality. High molecular weight polyglycerol was used to synthesize novel stimuli-responsive hydrogels. Real-time monitoring of step-growth polymerization of glycerol was investigated using in-line and off-line Attenuated Total Reflectance/Fourier Transform infrared (ATR-FTIR) technique.

Nerve autografts are widely used clinically to repair nerve grafts. However, nerve grafts have many limitations, such as, availability of donor nerve grafts, and loss of function at donor site. To overcome these problems, we have used a tissue engineering approach to design three-dimensional (3D) agarose scaffolds containing gradients of laminin-1 (LN-1) and nerve growth factor (NGF) to mimic in vivo conditions to promote nerve regeneration in rats. To determine the effect of LN-1 gradients on neurite extension in vitro, dorsal root ganglia (DRG) from chick embryos were cultured in 3D hydrogels. A gradient of LN-1 molecules in agarose gels was made by diffusion technique. LN-1 was then immobilized to the agarose hydrogels using a photo-crosslinker, Sulfo-SANPAH (Sulfosuccinimidyl-6-[4-azido-2-nitrophenylamino] hexanoate). Anisotropic scaffolds with three different slopes of LN-1 gradients were used. Isotropic scaffolds with uniform concentrations of LN-1, at various levels, were used as a positive control. DRG cultured in anisotropic scaffolds with optimal slope of LN-1 gradient extended neurites twice as fast as DRG in optimal concentration in isotropic scaffolds. Also, in the anisotropic scaffolds the faster growing neurites were aligned along the direction of LN-1 gradient. To promote nerve regeneration in vivo, tubular polysulfone guidance channels containing agarose hydrogels with gradients of LN-1 and NGF (anisotropic scaffolds) were used to bridge 20-mm nerve gaps in rats. Nerve autografts were used as positive controls and isotropic scaffolds, with uniform concentration of LN-1 and NGF, were used as negative controls. After 4-months, the rats were sacrificed and nerve histology was done to test for nerve regeneration. Only anisotropic scaffolds and nerve autografts contained evidence of axonal regeneration. Both groups had similar numbers of myelinated axons and similar axonal-diameter distribution. However, nerve graft group performed better in functional outcome as measured by relative gastrocnemius muscle weight (RGMW) and electrophysiology. Optimization of performance of anisotropic scaffolds by varying the LN-1 and NGF concentration gradients might lead to development of scaffolds that can perform as well as nerve auotgrafts for nerve regeneration over long nerve gaps.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 157-172).