Andrew S. Rowlands

Directing osteogenic and myogenic differentiation of MSCs: interplay of stiffness and adhesive ligand presentation

The mechanical properties of the extracellular matrix (ECM) can exert significant influence in determining cell fate. Human mesenchymal stem cells (MSCs) grown on substrates with varying stiffness have been shown to express various cell lineage markers, without the use of toxic DNA demethylation agents or complex cocktails of expensive growth factors. Here we investigated the myogenic and osteogenic potential of various polyacrylamide gel substrates that were coated with covalently bound tissue-specific ECM proteins (collagen I, collagen IV, laminin, or fibronectin). The gel-protein substrates were shown to support the growth and proliferation of MSCs in a stiffness-dependent manner. Higher stiffness substrates encouraged up to a 10-fold increase in cell number over lower stiffness gels. There appears to be definitive interplay between substrate stiffness and ECM protein with regard to the expression of both osteogenic and myogenic transcription factors by MSCs. Of the 16 gel-protein combinations investigated, osteogenic differentiation was found to occur significantly only on collagen I-coated gels with the highest modulus gel tested (80 kPa). Myogenic differentiation occurred on all gel-protein combinations that had stiffnesses >9 kPa but to varying extents as ascertained by MyoD1 expression. Peak MyoD1 expression was seen on gels with a modulus of 25 kPa coated in fibronectin, with similar levels of expression observed on 80-kPa collagen I-coated gels. The modulation of myogenic and osteogenic transcription factors by various ECM proteins demonstrates that substrate stiffness alone does not direct stem cell lineage specification. This has important implications in the development of tailored biomaterial systems that more closely mimic the microenvironment found in native tissues.

Directing phenotype of vascular smooth muscle cells using electrically stimulated conducting polymer

Vascular smooth muscle cells (VSMCs) isolated from rabbit aorta and immortalised A7r5 cells were cultured on conducting polypyrrole (PPy) substrates and were subjected to a 50muA sinusoidal electrical stimulation at 0.05, 5 and 500 Hz. These substrates were doped with hyaluronic acid and coated with collagen IV followed by Matrigel in order to mimic the basement membrane and encourage cell attachment. Increased proliferation and expression of smooth muscle phenotype markers (smooth muscle alpha-actin and smooth muscle myosin heavy chain) were observed in cultures stimulated at 5 and 500 Hz. This increased proliferation and expression of contractile proteins were found to be significantly decreased when L-type voltage-gated calcium channels (VGCC) were blocked with the drug nifedipine. To the best of our knowledge, this is the first work that demonstrates that VSMCs cultured on a conducting polymer substrate and subject to electrical stimulation not only exhibit enhanced proliferation but can be simultaneously encouraged to increase contractile protein expression. This behaviour is somewhat contrary to the classical definition of smooth muscle contractile and synthetic phenotypes that, in general, requires a modulation in phenotype as a prerequisite for smooth muscle proliferation. This interesting result highlights both the inherent plasticity of vascular smooth muscle cells and the potential of electrical stimulation via conducting polymer substrates to manipulate their behaviour.

Surface-bound stem cell factor and the promotion of hematopoietic cell expansion.

In vivo, stem cell factor (SCF) exists in both a bound and soluble isoform. It is believed that the bound form is more potent and fundamentally required for the maintenance of hematopoietic stem cells (HSCs). This theory is supported by the observation that steel-Dickie mice lacking the bound isoform of SCF are unable to maintain hematopoiesis and by the fact that bound SCF displayed on the surface of transgenic cells is better able to maintain c-kit activation than soluble SCF. Further work has shown that recombinant SCF molecules, which include a surface-binding domain, are more potent than their soluble equivalent. It is generally assumed that such an elegant approach is necessary to provide the correct molecular orientation and avoid the pitfalls of random cross-linking or the denaturation associated with the adsorption of proteins to surfaces. However, in this work we demonstrate that SCF physisorbed to tissue culture plastic (TCP) is not only bioactive, but more potent than the soluble equivalent. By contrast, cross-linking of SCF via free amines is shown to compromise its bioactivity. These observations demonstrate that simple surface modification solutions cannot be discounted and with the advent of low-cost pharmaceutical grade proteins, they should not be.

From scrawny to brawny: the quest for neomusculogenesis; smart surfaces and scaffolds for muscle tissue engineering.

The successful generation of functional muscle tissues requires both an in-depth knowledge of muscle tissue physiology and advanced engineering practices. The inherent contractile functionality of muscle is a result of its high-level cellular and matrix organization over a multitude of length scales. While there have been many attempts to produce artificial muscle, a method to fabricate a highly organized construct, comprised of multiple cell types and capable of delivering contractile strengths similar to that of native smooth, skeletal or cardiac muscle has remained elusive. This is largely due to a lack of control over phenotype and spatial organization of cells. This paper covers state-of-the-art approaches to generating both 2D and 3D substrates that provide some form of higher level organization or multiple biochemical, mechanical or electrical cues to cells in order to successfully manipulate their behavior, in a manner that is conducive to the production of contractile muscle tissue. These so-called ‘smart surfaces’ and ‘smart scaffolds’ represent vital steps towards surface-engineered substrates for the engineering of muscle tissues, showing confidently that cellular behavior can be effectively and reproducibly manipulated through the design of the physical, chemical and electrical properties of the substrates on which cells are grown. However, many challenges remain to be overcome prior to reaching the ultimate goal of fully functional 3D vascularized engineered muscle.

Electrochemical Quartz Crystal Microbalance to Monitor Biofilm Growth and Properties during BioElectrochemical System Inoculation and Load Conditions

BioElectrochemical Systems are an emerging technology of interest. Central to the successful operation of these systems is the development of a biofilm on an electrode surface that facilitates the interaction of microorganisms capable of extra-cellular electron transfer. Porous graphite is typically chosen as the electrode material. However, other materials could contribute to a significant improvement in reactor performance. In order to evaluate these novel materials, a platform methodology is required. In this study, E-QCM-D was evaluated using a gold electrode, a mixed biofilm population and a challenging real influent (fermented solubilised sludge). The results gave real-time feedback on both the acoustic and electrochemical impedance of the working electrode, hence providing information about biofilm thickness, viscoelasticity, kinetics and mass transport. The use of E-QCM-D is novel for this field and has the potential to provide a wealth of new data to help optimise the performance of biofilms in these systems.

The University of Queensland refrigerant and supercritical CO2 test loop

The use of organic refrigerants or supercritical CO (sCO ) as a working fluid in closed loop power cycles has the potential to revolutionise power generation. Thermodynamic cycle efficiency can be improved by selecting bespoke working fluids that best suit a given combination of heat source and heat sink temperatures, but thermal efficiency can be maximised by pairing this with a custom made turbine. This work describes the development and design of a new 100kW thermal laboratory-scale test loop at the University of Queensland. The loop has capabil-ities for characterising both simple and recuperated refrigerant and sCO organic Rankine cycles in relation to overall cycle performance and for the experimental characterisation of radial inflow turbines. The aim of this facility is to generate high quality validation data and to gain new insight into overall loop performance, control operation, and loss mechanisms that prevail in all loop components, including radial turbines when operating with supercritical fluids. The paper describes the current test loop and provides details on the available test modes: An organic Rankine cycle mode, a closed loop Brayton cycle mode, and heat exchanger test mode and their respective operating ranges. The bespoke control and data acquisition system has been designed to ensure safe loop operation and shut down and to provide high quality measurement of signals from more than 60 sensors within the loop and test turbine. For each measurement, details of the uncertainty quantification in accordance with ASME standards are provided, ensuring data quality. Data from the commissioning of the facility is provided in this paper. This data confirms controlled operation of the loop and the ability to conduct both cycle characterisation tests and turbomachinery tests.

[Retracted publication] Design and Testing Process for a 7Kw Radial Inflow Refrigerant Turbine At the University of Queensland

The Queensland Geothermal Energy Centre of Excellence (QGECE) has been developing a small 7 kW refrigerant radialinflow turbine assembly. Such turbines, when used with organic fluids (e.g. refrigerants), result in power cycles that can have a superior thermodynamic efficiency compared to traditional power cycles and turbines in the low to medium temperature range (100-250°C). The intended use for the UQ 7kW turbine unit is validation of CFD simulations, characterisation of turbomachinery loss mechanisms, and validation of 1-D design methodologies. This paper describes the structural and aerodynamic design process that has led to completion of the turbine unit. The first generation aerodynamic design (rotor and stator) and operating points were selected using the QGECEs 1-D mean line design software TOPGEN, to obtain a simple and robust turbine. Results from preliminary CFD simulations to verify the volute and stator operation and stage simulations to provide design and off-design performance characteristics and structural loads are presented. The turbine assembly was designed with modularity in mind to allow future turbine design iteration. Design information is provided for the overall turbine concept and the modular sub-components, including volute, magnetic coupling, bearing chamber design, shaft rotordynamics, FEA analysis and the instrumentation scheme. The paper concludes with a summary of the planned tests.