Cyclic strain disrupts endothelial network formation on Matrigel

Most forms of tissue healing depend critically on revascularisation. In soft tissues and in vitro, mechanical stimuli have been shown to promote vessel-forming activity. However, in bone defects, increased interfragmentary motion impairs vascular regeneration. Because these effects seem contradictory, we aimed to determine whether a range of mechanical stimuli exists in which angiogenesis is favoured. A series of cyclic strain magnitudes were applied to a Matrigel-based “tube formation” assay and the total lengths of networks formed by human microvascular endothelial cells measured at 24 h. Network lengths were reduced at all strain levels, compared to unstretched controls. However, the levels of pro-angiogenic matrix metalloproteases-2 and -9 in the corresponding conditioned media were unchanged by strain, and vascular endothelial growth factor was uniformly elevated in stretched conditions. By repeating the assay with the addition of conditioned media from mesenchymal stem cells cultivated in similar conditions, paracrine stimuli were shown to increase network lengths, but not to alter the negative effect of cyclic stretching. Together, these results demonstrate that directly applied periodic strains can inhibit endothelial organisation in vitro, and suggest that this may be due to physical disruption rather than biochemical modulation. Most importantly, the results indicate that the straining of endothelial cells and their assembly into vascular-like structures must be studied simultaneously to adequately characterise the mechanical influence on vessel formation.

Mechanical influences on endothelial cell network formation in vitro

While both the restoration of the blood supply and an appropriate local mechanical environment are critical for uneventful bone healing, their influence on each other remains unclear. Human bone fracture haematomas (<72h post-trauma) were cultivated for 3 days in fibrin matrices, with or without cyclic compression. Conditioned medium from these cultures enhanced the formation of vessel-like networks by HMEC-1 cells, and mechanical loading further elevated it, without affecting the cells’ metabolic activity. While haematomas released the angiogenesis-regulators, VEGF and TGF-β1, their concentrations were not affected by mechanical loading. However, direct cyclic stretching of the HMEC-1 cells decreased network formation. The appearance of the networks and a trend towards elevated VEGF under strain suggested physical disruption rather than biochemical modulation as the responsible mechanism. Thus, early fracture haematomas and their mechanical loading increase the paracrine stimulation of endothelial organisation in vitro, but direct periodic strains may disrupt or impair vessel assembly in otherwise favourable conditions.

Poly(2-oxazoline) hydrogel monoliths via thiol-ene coupling

Copoly(2-oxazoline)s, prepared by cationic ring-opening polymerization of 2-(dec-9-enyl)-2-oxazoline with either 2-methyl-2-oxazoline or 2-ethyl-2-oxazoline, have been crosslinked with small dithiol molecules under UV-irradiation to form homogeneous networks. In-situ monitoring of the crosslinking reaction by photo-rheology revealed network formation within minutes. The degree of swelling in water was found to be tunable by the hydrophilicity of the starting macromers and the proportion of alkene side arms. Furthermore, degradable hydrogels have been prepared based on a hydrolytically cleavable dithiol crosslinker.

STOF Model : critical design issues and critical success factors

In this chapter we will make the transition towards the design of business models and the related critical issues. We develop a model that helps us understand the causalities that play a role in understanding the viability and feasibility of the business models, i.e. long-term profitability and market adoption. We argue that designing viable business models requires balancing the requirements and interests of the actors involved, within and between the various business model domains. Requirements in the service domain guide the design choices in the technology domain, which in turn affect network formation and the financial arrangements. It is important to understand the Critical Design Issues (CDIs) involved in business models and their interdependencies. In this chapter, we present the Critical Design Issues involved in designing mobile service business models, and demonstrate how they are linked to the Critical Success Factors (CSFs) with regard to business model viability. This results in a causal model for understanding business model viability, as well as providing grounding for the business model design approach outlined in Chapter 5.

Theoretical investigation of mechanisms of formation and interaction of nanoparticles

In this thesis an investigation into theoretical models for formation and interaction of nanoparticles is presented. The work presented includes a literature review of current models followed by a series of five chapters of original research. This thesis has been submitted in partial fulfilment of the requirements for the degree of doctor of philosophy by publication and therefore each of the five chapters consist of a peer-reviewed journal article. The thesis is then concluded with a discussion of what has been achieved during the PhD candidature, the potential applications for this research and ways in which the research could be extended in the future. In this thesis we explore stochastic models pertaining to the interaction and evolution mechanisms of nanoparticles. In particular, we explore in depth the stochastic evaporation of molecules due to thermal activation and its ultimate effect on nanoparticles sizes and concentrations. Secondly, we analyse the thermal vibrations of nanoparticles suspended in a fluid and subject to standing oscillating drag forces (as would occur in a standing sound wave) and finally on lattice surfaces in the presence of high heat gradients. We have described in this thesis a number of new models for the description of multicompartment networks joined by a multiple, stochastically evaporating, links. The primary motivation for this work is in the description of thermal fragmentation in which multiple molecules holding parts of a carbonaceous nanoparticle may evaporate. Ultimately, these models predict the rate at which the network or aggregate fragments into smaller networks/aggregates and with what aggregate size distribution. The models are highly analytic and describe the fragmentation of a link holding multiple bonds using Markov processes that best describe different physical situations and these processes have been analysed using a number of mathematical methods. The fragmentation of the network/aggregate is then predicted using combinatorial arguments. Whilst there is some scepticism in the scientific community pertaining to the proposed mechanism of thermal fragmentation,we have presented compelling evidence in this thesis supporting the currently proposed mechanism and shown that our models can accurately match experimental results. This was achieved using a realistic simulation of the fragmentation of the fractal carbonaceous aggregate structure using our models. Furthermore, in this thesis a method of manipulation using acoustic standing waves is investigated. In our investigation we analysed the effect of frequency and particle size on the ability for the particle to be manipulated by means of a standing acoustic wave. In our results, we report the existence of a critical frequency for a particular particle size. This frequency is inversely proportional to the Stokes time of the particle in the fluid. We also find that for large frequencies the subtle Brownian motion of even larger particles plays a significant role in the efficacy of the manipulation. This is due to the decreasing size of the boundary layer between acoustic nodes. Our model utilises a multiple time scale approach to calculating the long term effects of the standing acoustic field on the particles that are interacting with the sound. These effects are then combined with the effects of Brownian motion in order to obtain a complete mathematical description of the particle dynamics in such acoustic fields. Finally, in this thesis, we develop a numerical routine for the description of "thermal tweezers". Currently, the technique of thermal tweezers is predominantly theoretical however there has been a handful of successful experiments which demonstrate the effect it practise. Thermal tweezers is the name given to the way in which particles can be easily manipulated on a lattice surface by careful selection of a heat distribution over the surface. Typically, the theoretical simulations of the effect can be rather time consuming with supercomputer facilities processing data over days or even weeks. Our alternative numerical method for the simulation of particle distributions pertaining to the thermal tweezers effect use the Fokker-Planck equation to derive a quick numerical method for the calculation of the effective diffusion constant as a result of the lattice and the temperature. We then use this diffusion constant and solve the diffusion equation numerically using the finite volume method. This saves the algorithm from calculating many individual particle trajectories since it is describes the flow of the probability distribution of particles in a continuous manner. The alternative method that is outlined in this thesis can produce a larger quantity of accurate results on a household PC in a matter of hours which is much better than was previously achieveable.

Contexts, hybrids and network governance : a comparison of three case-studies in infrastructure governance

While hybrid governance arrangements have been a major element of organisational architecture for some time, the contemporary operating environment has brought to the fore new conditions and expectations for the governance of entities that span conventional public sector departments, private firms and community organisations or groups. These conditions have resulted in a broader array of mixed governance configurations including Public Private Partnerships, alliances, and formal and informal collaborations. In some such arrangements, market based or ‘complete’ contractual relationships have been introduced to replace or supplement existing traditional ‘hierarchical’ and/or newer relational ‘network-oriented’ institutional associations. While there has been a greater reliance on collaborative or relational contracts as an underpinning institutional model, other modes of hierarchy and market may remain in operation. The success of these emergent hybrid forms has been mixed. There are examples of hybrids that have been well adopted, achieving the desired goals of efficiency, effectiveness and financial accountability; while others have experienced implementation problems which have undermined their results. This paper postulates that the cultural and institutional context within which hybrids operate may contribute to the implementation processes employed and the level of success attained. The paper explores hybrid arrangements through three cases of the use of inter-organisational arrangements in three different national contexts. Distilling the various elements of hybrids and the impact of institutional context will provide important insights for those charged with the responsibility for the formation and key infrastructure and public value development.

SimTrust : a new method of trust network generation

Trust can be used for neighbor formation to generate automated recommendations. User assigned explicit rating data can be used for this purpose. However, the explicit rating data is not always available. In this paper we present a new method of generating trust network based on user’s interest similarity. To identify the interest similarity, we use user’s personalized tag information. This trust network can be used to find the neighbors to make automated recommendation. Our experiment result shows that the precision of the proposed method outperforms the traditional collaborative filtering approach.

A collagen network phase improves cell seeding of open-pore structure scaffolds under perfusion

Scaffolds with open-pore morphologies offer several advantages in cell-based tissue engineering, but their use is limited by a low cell seeding efficiency. We hypothesized that inclusion of a collagen network as filling material within the open-pore architecture of polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds increases human bone marrow stromal cells (hBMSC) seeding efficiency under perfusion and in vivo osteogenic capacity of the resulting constructs. PCL-TCP scaffolds, rapid prototyped with a honeycomb-like architecture, were filled with a collagen gel and subsequently lyophilized, with or without final crosslinking. Collagen-free scaffolds were used as controls. The seeding efficiency was assessed after overnight perfusion of expanded hBMSC directly through the scaffold pores using a bioreactor system. By seeding and culturing freshly harvested hBMSC under perfusion for 3 weeks, the osteogenic capacity of generated constructs was tested by ectopic implantation in nude mice. The presence of the collagen network, independently of the crosslinking process, significantly increased the cell seeding efficiency (2.5-fold), and reduced the loss of clonogenic cells in the supernatant. Although no implant generated frank bone tissue, possibly due to the mineral distribution within the scaffold polymer phase, the presence of a non crosslinked collagen phase led to in vivo formation of scattered structures of dense osteoids. Our findings verify that the inclusion of a collagen network within open morphology porous scaffolds improves cell retention under perfusion seeding. In the context of cell-based therapies, collagen-filled porous scaffolds are expected to yield superior cell utilization, and could be combined with perfusion-based bioreactor devices to streamline graft manufacture.

Porous scaffold architecture guides tissue formation

Critical-sized bone defect regeneration is a remaining clinical concern. Numerous scaffold-based strategies are currently being investigated to enable in vivo bone defect healing. However, a deeper understanding of how a scaffold influences the tissue formation process and how this compares to endogenous bone formation or to regular fracture healing is missing. It is hypothesized that the porous scaffold architecture can serve as a guiding substrate to enable the formation of a structured fibrous network as a prerequirement for later bone formation. An ovine, tibial, 30-mm critical-sized defect is used as a model system to better understand the effect of the scaffold architecture on cell organization, fibrous tissue, and mineralized tissue formation mechanisms in vivo. Tissue regeneration patterns within two geometrically distinct macroscopic regions of a specific scaffold design, the scaffold wall and the endosteal cavity, are compared with tissue formation in an empty defect (negative control) and with cortical bone (positive control). Histology, backscattered electron imaging, scanning small-angle X-ray scattering, and nanoindentation are used to assess the morphology of fibrous and mineralized tissue, to measure the average mineral particle thickness and the degree of alignment, and to map the local elastic indentation modulus. The scaffold proves to function as a guiding substrate to the tissue formation process. It enables the arrangement of a structured fibrous tissue across the entire defect, which acts as a secondary supporting network for cells. Mineralization can then initiate along the fibrous network, resulting in bone ingrowth into a critical-sized defect, although not in complete bridging of the defect. The fibrous network morphology, which in turn is guided by the scaffold architecture, influences the microstructure of the newly formed bone. These results allow a deeper understanding of the mode of mineral tissue formation and the way this is influenced by the scaffold architecture. Copyright © 2012 American Society for Bone and Mineral Research.

Mastering the canonical loop of serine protease inhibitors : enhancing potency by optimising the internal hydrogen bond network

Background Canonical serine protease inhibitors commonly bind to their targets through a rigid loop stabilised by an internal hydrogen bond network and disulfide bond(s). The smallest of these is sunflower trypsin inhibitor (SFTI-1), a potent and broad-range protease inhibitor. Recently, we re-engineered the contact β-sheet of SFTI-1 to produce a selective inhibitor of kallikrein-related peptidase 4 (KLK4), a protease associated with prostate cancer progression. However, modifications in the binding loop to achieve specificity may compromise structural rigidity and prevent re-engineered inhibitors from reaching optimal binding affinity. Methodology/Principal Findings In this study, the effect of amino acid substitutions on the internal hydrogen bonding network of SFTI were investigated using an in silico screen of inhibitor variants in complex with KLK4 or trypsin. Substitutions favouring internal hydrogen bond formation directly correlated with increased potency of inhibition in vitro. This produced a second generation inhibitor (SFTI-FCQR Asn14) which displayed both a 125-fold increased capacity to inhibit KLK4 (Ki = 0.0386±0.0060 nM) and enhanced selectivity over off-target serine proteases. Further, SFTI-FCQR Asn14 was stable in cell culture and bioavailable in mice when administered by intraperitoneal perfusion. Conclusion/Significance These findings highlight the importance of conserving structural rigidity of the binding loop in addition to optimising protease/inhibitor contacts when re-engineering canonical serine protease inhibitors.

Governing self : SNSs as tools for self-formation

This thesis investigates how modern individuals relate to themselves and others in the service of shaping their ethical conduct and governing themselves. It considers the use of online social networking sites (SNSs) as one particular practice through which people manage their day-to-day conduct and understandings of self. Current research on the use of SNSs has conceptualised them as tools for communication, information-sharing and self-presentation. This thesis suggests a different way of thinking about these sites as tools for self-formation. A Foucaultian genealogical, historical and problematising approach is applied in order to explore processes of subjectivation and historical backgrounds involved in the use of SNSs. This is complimented with an ANT-based understanding of the role that technologies play in shaping human action. Drawing new connections between three factors will show how they contribute to the ways in which people become selves today. These factors are, one, the psychologisation and rationalisation of modern life that lead people to confess and talk about themselves in order to improve and perfect themselves, two, the transparency or publicness of modern life that incites people to reveal themselves constantly to a public audience and, three, the techno-social hybrid character of Western societies. This thesis will show how some older practices of self-formation have been translated into the context of modern technologised societies and how the care of self has been reinvigorated and combined with the notion of baring self in public. This thesis contributes a different way of thinking about self and the internet that does not seek to define what the modern self is and how it is staged online but rather accounts for the multiple, contingent and historically conditioned processes of subjectivation through which individuals relate to themselves and others in the service of governing their daily conduct.

Electrochemically-induced TCNQ/Mn TCNQ 2 (H2O) 2 (TCNQ= 7, 7, 8, 8-Tetracyanoquinodimethane) solid-solid interconversion : two voltammetrically distinct processes that allow selective generation of nanofiber or nanorod network morphologies

Unlike the case with other divalent transition metal M\[TCNQ](2)(H(2)O)(2) (M = Fe, Co, Ni) analogues, the electrochemically induced solid-solid phase interconversion of TCNQ microcrystals (TCNQ = 7,7,8,8-tetracyanoquinodimethane) to Mn\[TCNQ](2)(H(2)O)(2) occurs via two voltammetrically distinct, time dependent processes that generate the coordination polymer in nanofiber or rod-like morphologies. Careful manipulation of the voltammetric scan rate, electrolysis time, Mn(2+)((aq)) concentration, and the method of electrode modification with solid TCNQ allows selective generation of either morphology. Detailed ex situ spectroscopic (IR, Raman), scanning electron microscopy (SEM), and X-ray powder diffraction (XRD) characterization clearly establish that differences in the electrochemically synthesized Mn-TCNQ material are confined to morphology. Generation of the nanofiber form is proposed to take place rapidly via formation and reduction of a Mn-stabilized anionic dimer intermediate, \[(Mn(2+))(TCNQ-TCNQ)(2)(*-)], formed as a result of radical-substrate coupling between TCNQ(*-) and neutral TCNQ, accompanied by ingress of Mn(2+) ions from the aqueous solution at the triple phase TCNQ/electrode/electrolyte boundary. In contrast, formation of the nanorod form is much slower and is postulated to arise from disproportionation of the \[(Mn(2+))(TCNQ-TCNQ)(*-)(2)] intermediate. Thus, identification of the time dependent pathways via the solid-solid state electrochemical approach allows the crystal size of the Mn\[TCNQ](2)(H(2)O)(2) material to be tuned and provides new mechanistic insights into the formation of different morphologies.

Electrochemical formation of porous copper 7, 7, 8, 8-tetracyanoquinodimethane and copper 2, 3, 5, 6-tetrafluoro-7, 7, 8, 8-tetracyanoquinodimethane honeycomb surfaces with superhydrophobic properties

The electrochemical formation of highly porous CuTCNQ (TCNQ = 7,7,8,8-tetracyanoquinodimethane) and CuTCNQF4 (TCNQF4 = 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane) materials was undertaken via the spontaneous redox reaction between a porous copper template, created using a hydrogen bubbling template technique, and an acetonitrile solution containing TCNQ or TCNQF4. It was found that activation of the surface via vigorous hydrogen evolution that occurs during porous copper deposition and TCNQ mass transport being hindered through the porous network of the copper template influenced the growth of CuTCNQ and CuTCNQF4. This approach resulted in the fabrication of a honeycomb layered type structure where the internal walls consist of very fine crystalline needles or spikes. This combination of microscopic and nanoscopic roughness was found to be extremely beneficial for anti-wetting properties where superhydrophobic materials with contact angles as high as 177° were created. Given that CuTCNQ and CuTCNQF4 have shown potential as molecular based electronic materials in the area of switching and field emission, the creation of a surface that is moisture resistant may be of applied interest.

Effect of dangling chains on the structure and physical properties of a tightly crosslinked poly(ethylene glycol) network

Major imperfections in crosslinked polymers include loose or dangling chain ends that lower the crosslink d., thereby reducing elastic recovery and increasing the solvent swelling. These imperfections are hard to detect, quantify and control when the network is initiated by free radical reactions. As an alternative approach, the sol-​gel synthesis of a model poly(ethylene glycol) (PEG-​2000) network is described using controlled amts. of bis- and mono-​triethoxy silyl Pr urethane PEG precursors to give silsesquioxane (SSQ, R-​SiO1.5) structures as crosslink junctions with a controlled no. of dangling chains. The effect of the no. of dangling chains on the structure and connectivity of the dried SSQ networks has been detd. by step-​crystn. differential scanning calorimetry. The role that micelle formation plays in controlling the sol-​gel PEG network connectivity has been studied by dynamic light scattering of the bis- and mono-​triethoxy silyl precursors and the networks have been characterized by 29Si solid state NMR, sol fraction and swelling measurements. These show that the dangling chains will increase the mesh size and water uptake. Compared to other end-​linked PEG hydrogels, the SSQ-​crosslinked networks show a low sol fraction and high connectivity, which reduces solvent swelling, degree of crystallinity and the crystal transition temp. The increased degree of freedom in segment movement on the addn. of dangling chains in the SSQ-​crosslinked network facilitates the packing process in crystn. of the dry network and, in the hydrogel, helps to accommodate more water mols. before reaching equil.

Phosphorus-based functional groups as hydrogen bonding templates for rotaxane formation

We report on the use of the hydrogen bond acceptor properties of some phosphorus-containing functional groups for the assembly of a series of [2]rotaxanes. Phosphinamides, and the homologous thio– and selenophosphinamides, act as hydrogen bond acceptors that, in conjunction with an appropriately positioned amide group on the thread, direct the assembly of amide-based macrocycles around the axle to form rotaxanes in up to 60% yields. Employing solely phosphorus-based functional groups as the hydrogen bond accepting groups on the thread, a bis(phosphinamide) template and a phosphine oxide-phosphinamide template afforded the corresponding rotaxanes in 18 and 15 % yields, respectively. X-Ray crystallography of the rotaxanes shows the presence of up to four intercomponent hydrogen bonds between the amide groups of the macrocycle and various hydrogen bond accepting groups on the thread, including rare examples of amide-to-phosphonamide, -thiophosphinamide and -selenophosphinamide groups. With a phosphine oxide-phosphinamide thread, the solid state structure of the rotaxane is remarkable, featuring no direct intercomponent hydrogen bonds but rather a hydrogen bond network involving water molecules that bridge the H-bonding groups of the macrocycle and thread through bifurcated hydrogen bonds. The incorporation of phosphorus-based functional groups into rotaxanes may prove useful for the development of molecular shuttles in which the macrocycle can be used to hinder or expose binding ligating sites for metal-based catalysts.