Research Projects: Biomaterials and Tissue Engineering

26 MAGNETIC POSITIONING OF BIOLOGICAL CELLS
Supervisors: Dr D A Allwood and Prof J W Haycock

This project will use the magnetic field from magnetic structures to position magnetically-labelled biological cells. This will be used to investigate nerve tissue generation from Schwann cells positioned with a magnetic ‘template’. Schwann cells initiate nerve growth but do not respond to chemical cues. The magnetic template will allow a physical cue of cell proximity to be introduced and for the effect of inter-cell separation to be studied. A background either in the physical or biological science/engineering is required.

27 DEVELOPMENT OF HYBRID NANOMATERIALS FOR CANCER CELL IMAGING AND THERAPY
Supervisors: Dr B Chen and Professor S MacNeil

Cancer is one of the leading causes of death in the world. Current treatments of cancer mainly involve surgery, chemotherapy and/or radiotherapy, which are highly invasive. This project proposes to develop novel hybrid nanomaterials for a minimally invasive cancer treatment approach and for cancer cell imaging, thus providing simultaneous cancer imaging and treatment for different types of cancer in particular for cutaneous melanoma. This is an interdisciplinary project covering nanomaterials, biomaterials and biology. It will involve synthesis and characterisation of hybrid nanomaterials, and assessment of the materials as potential agents for cancer cell imaging and killing behaviour by using in vitro cell cultures of cancer and normal cells and subsequently 3D melanoma model. Apart from development of the new technology, a sound scientific understanding of the materials and their performance is also expected from the project.

28 MULTIFUNCTIONAL NANOCOMPOSITE HYDROGELS FOR TARGETED DRUG DELIVERY
Supervisors: Dr B Chen and Professor J Haycock

Targeted therapy represents an attractive approach to treatment of diseases because of its potential to improve bioavailability of costly drugs and reduce side effects of toxic drugs. This is relevant for the delivery of agents to tissues in the body e.g. nerve and skin. This project aims to develop novel hydrogels containing stimulus-responsive nanoparticles and organic compounds for targeted drug delivery. A range of experimental techniques will be employed in the project, ranging from materials synthesis, characterisation to evaluation of cytocompatibility and drug delivery behaviour. The project will start by investigating the biological behaviour using in vitro cell cultures of neural, glial, epithelial and mesenchymal cells, which depending on progress will move towards a 3D in vitro tissue model. This research will potentially advance the knowledge and technology of drug delivery as well as the understanding of stimulus-responsive hydrogels as ‘smart’ drug delivery systems.

29 THE EFFECT OF PATCHING ON THE WALL AND FLOW PROPERTIES OF CAROTID ARTERY
Supervisor: Dr C K Chong

Carotid artery endarterectomy (CAE) is one of the most common vascular surgical procedures and it has remained the standard management strategy for significant carotid stenosis. However, there is still uncertainty if patching should be performed. It is unclear if selective patching is preferable to routine patching, how narrow an artery should be before it must be patched, what materials (biological or synthetic) should be used and what should be the width of the patch. These uncertainties, perhaps, are compounded by recurrent stenosis of up to 30% while the true mechanism remains unknown. There is evidence from studies on the pathogenesis of vascular diseases and on bypasses at the coronary and below-knee regions that the vessel geometry and material properties have a strong influence on thrombogenesis, intimal hyperplasia, atherogenesis, the key processes which lead to stenosis in arteries and restenosis in vascular surgery. This project aims to investigate if and how patching alters the vessel wall and flow characteristics, and how these physical parameters could influence the long-term outcome of CAE. This project involves developing a computational fluid-structure interaction model based on physiological data to address and analyse systematically medical problems. The ultimate goal is to develop a guideline for CAE on material selection and the construction of the carotid patch. The project is highly multidisciplinary and would suit an applicant with a strong fluid and solid mechanics background and an outstanding analytical and computational skills/experience with CFD/FEM modeling. Some knowledge of human anatomy/cell biology would be advantageous.

30 DEVELOPMENT AND ASSESSMENT OF SMART MECHANICALLY-RESPONSIVE AND PHARMACOLOGICALLY-ACTIVE STENTS
Supervisor: Dr C K Chong

Restenosis due to thrombosis and neointimal tissue formation are widely believed to be the consequence of vascular injury induced by balloon inflation and stent expansion associated with endovascular stent treatment. To mitigate restenosis, drug-eluting stent (DES) is now commonly-used. To achieve its purpose, this stent must be able to deliver the coated/embedded drugs and deposit strategically and effectively in targeted tissues to suppress inflammation, thrombosis, and neointimal tissue formation while encouraging endothelialisation and healing of the vascular tissues. Such delivery and deposition of drugs, however, could be influenced by interactions between the physical variables e.g. the mass transport process, the structural and morphological features of the stent and vascular wall, as well as the properties of the drugs. This project aims to develop a smart DES with functionalized polymer which could respond to the dynamic environment subjected by the blood flow to meet the therapeutic demand of the vascular tissue. An important feature of the DES would be the ability to provide sustained controlled-release of drugs/pharmacologically-active agents to induce repair of the lesion. The ultimate aim is to develop a “programmable” stent for more effective therapeutic action customised for specific vascular geometry and location. This project would suit an applicant offering strong chemistry/polymer/biomaterials background with outstanding laboratory experience/skills. Some knowledge of fluid/solid mechanics and computational modeling skills/experience would be advantageous.

31 TISSUE-ENGINEERING PRE-SHAPED VASCULAR GRAFT FOR SUSTAINED SURGICAL INTERVENTIONS
Supervisor: Dr C K Chong

Surgical intervention using bypass graft is routinely performed to treat vascular disease. However, the lack of suitable small diameter (< 5mm) vessels that could provide a sustained patency lasting the patient’s lifetime is an outstanding issue. The poor patency is a result of restenosis associated with intimal hyperplasia developing at the distal site where the graft is sutured to the diseased native vessel. While improved surgical techniques could potentially improve its patency, some studies have shown that the material properties of the graft could play an important role, while our studies have also suggested that the performance of these grafts could benefit from a properly defined anastomotic configuration at the distal sites. This project aims to develop tissue-engineered pre-shaped vascular grafts with appropriate biomechanical properties that will provide sustained surgical intervention. The overall challenge will be to develop patient-specific pre-shaped vascular graft based on specific mechanical loadings that can be used to condition tissues that would be fit for implantation at the coronary/peripheral arteries. The project involves designing a modular test-section based on our in-house developed flow-bioreactor system (FBS) for developing pre-shaped vascular grafts, developing/seeding with cells a pre-shaped scaffold and maintaining it in the FBS, characterising structural and biomechanical properties of pre-seeded and post-cultured scaffold. How the structural and mechanical properties of the scaffold and the dynamic loading conditions (e.g. flow waveforms and flowrates) affect the graft development in vitro is of interest. This project is highly multidisciplinary but would suit a student with a solid background in biomedical/mechanical/chemical/materials engineering with some experience in biomechanics/tissue engineering.

32 EFFECTS OF SCAFFOLD STRUCTURES AND MASS TRANSPORT ON REGENERATIVE PROCESSES
Supervisor: Dr C K Chong and Dr F Claeyssens

Scaffold plays an important role in tissue regenerative process which may take place either in vivo (with or without cell seeding) or in vitro (with cell seeding). This regenerative process involves cell attachment, proliferation, migration and extracellular matrix (ECM) deposition. To ensure that these events are taking place effectively, it is important that the cells are in their metabolically-active state and this requires efficient mass transfer. Studies have suggested that cellular events could be influenced by the structural properties of scaffold. However, their exact correlation and the mechanism involved are yet to be established. The proposed project aims to gain more insights on the effects of scaffold structure and mass transport on regenerative process and how tissue regeneration could be optimized. It involves (i) the design, development, and characterisation of scaffold perfusion/construct culture system, (ii) selection, fabrication and characterization (e.g. pore size, porosity, interconnectivity) of stabilized porous scaffolds, (iii) measurement/computation of mass transfer parameters (e.g. diffusivity, flow rate, shear stress distribution), (iv) culture of cell-seeded scaffold and assessment of cellular events (histological studies, metabolic activities and ECM deposition), (v) correlation analysis, and (vi) the formulation of a mathematical model to describe the correlation. This project is highly multidisciplinary allowing student to work at the interface between engineering and life sciences.

33 QUANTITATIVE ANALYSIS OF HAEMODYNAMIC FORCES ON CELLULAR RESPONSE IN CO-CULTURE
Supervisor: Dr C K Chong

Haemodynamic (or biomechanical) forces have been suggested to play important roles in arteriosclerosis, intimal thickening (IT), and restenosis related to stented arteries or surgical anastomosis. Certain flow features e.g. low mean shear stress, oscillating shear stress, abnormal temporal and spatial shear stress gradients, and high particle residence times, are found in the locations where early IT is greatest, suggesting a possible correlation between blood flow features and cellular responses, and failure of the endovascular device or surgical bypasses. This project aims to better understand this correlation quantitatively by deriving biological outputs from physical inputs extracted from well-defined/controlled experimental and computational studies. The scope of the project involves developing and characterising an in vitro EC-SMC co-culture model based on our in-housed developed flow-bioreactor system capable of reproducing highly accurate physiological flow and pressure parameters, and performing parametric studies. The mechanism/pathway involved or how certain key genes are being switched on/off or proteins being expressed/up regulated/down regulated by the biomechanical forces derived from blood flow are of interest. The project is highly multidisciplinary and would suit an applicant with a strong fluid and solid mechanics background and an outstanding computational skills/experience with CFD/FEM modeling. Some background/experience on cell culture/fluorescence microscopy/image processing would be advantageous. Biologists with a strong interest on biomedical engineering and mechanotransduction could also be considered.

34 DIRECT LASER WRITING OF VASCULATURE
Supervisor: Dr F Claeyssens

One of the most important issues problems that need to be solved for successfully tissue engineering of large and complex organs is the inclusion of vasculature in the scaffold to provide oxygen and nutrients to the growing tissue and carry away waste products.
In this project we will investigate building a synthetic analogue to these natural vascular networks via Direct Laser Write (DLW). With this revolutionary production technique, in which we use a short pulse-length (sub-nanosecond) Nd-YAG laser to produce 3D objects via two-photon polymerisation. This technique allows for the formation of tailor-made structures directly from a 3D computer model, via localised photopolymerization of materials. With this technique it is possible to construct micrometer-sized 3D features making this an ideal technique for integrating materials with biology.
We will direct write a proto-vasculature in biodegradable biocompatible polymers and use these tubes to seed a vascular network within a scaffold. This network will incorporate the appropriate growth factors to enhance vascularisation. The tissue engineering scaffold with proto-vascular network will be integrated in a bioreactor which will provide perfusion of the scaffold. The perfusion medium will be seeded with endothelial cells to build up a capillary network. We will specifically concentrate on vascularisation for skin tissue engineering in collaboration with Prof. S. MacNeil.
This interdisciplinary research project will provide the prospective PhD student with experience in polymer/biomaterials synthesis, laser-based production techniques, materials analysis techniques and cell culture.

35 CONTROLLED 3D BIOMATERIALS MANUFACTURE
Supervisor: Dr F Claeyssens

This project has a two-fold aim, (i) production of three-dimensional (3D) objects in biocompatible materials (i.e. materials that do not have toxic or harmful effects to biological systems) and (ii) application of these structures in biology.
The building of biocompatible 3D structures will be achieved via microstereolithography (SL), a laser based direct-write technique. This technique allows for the formation of tailor-made structures directly from a 3D computer model, via localised photopolymerization of materials. With this technique it is possible to construct micrometer-sized 3D features making this an ideal technique for integrating materials with biology.
Applications of this technology in tissue engineering will be investigated during this project, specifically for tissue engineering. The production of a living 3D tissue of its constituent cells starts with providing a 3D scaffold for cells to attach to and to grow in and this project will investigate the production of these ‘tissue scaffolds’. These scaffolds will be built from a biocompatible material and will give the initial rigidity to the engineered tissue, so that the cells can build up their own connective tissue or Extra Cellular Matrix (ECM). Once the ECM is built the engineered scaffold becomes redundant, so this project will concentrate on biodegradable polymers as scaffold material. We are particular interesting in producing scaffolds for neural tissue engineering.
This interdisciplinary research project will provide the prospective PhD student with experience in polymer/biomaterials synthesis, laser-based production techniques, materials analysis techniques and cell culture.

36 LASER BIOPRINTING
Supervisor: Dr F Claeyssens

This project aims to build up a laser-based printing technique for biology. This printing technique utilises the Laser Induced Forward Transfer (LIFT) process to print biological molecules onto surfaces. This versatile technique is able to print solids, viscous liquid materials and powders. Furthermore, previous work has shown that biomolecules (proteins, DNA) and even entire cells can be printed without significant degradation or denaturation. This technique enables to print complex 2D micrometer patterns of biomolecules and cells, and the project will investigate biomolecule printing on biocompatible and biodegradable materials.
This technique will produce in a first iteration 2D patterned tissue sheets, and these will be at a later stage combined to attempt the layer-by-layer build up of 3D tissues. The sheets will be constructed from biodegradable materials, so these sheets will provide an initial structure for cells to grow in, while they build up their own extracellular matrix (ECM).
This interdisciplinary research project will provide the prospective PhD student with experience in polymer/biomaterials synthesis, laser-based production techniques, materials analysis techniques and cell culture.

37 SENSING VIA RESPONSIVE HYDROGELS
Supervisors: Dr F Claeyssens, Dr J Dean and Dr D Allwood

In this project we will study the synthesis of responsive hydrogels. Hydrogels are water swollen cross-linked polymer networks, and these materials can be rendered responsive to given chemical cues via inclusion of different chemical groups in the hydrogels structure. For example via inclusion of acidic or basic groups within the hydrogel these networks become pH responsive, i.e. they change from a swollen to collapsed state with varying pH. The speed of response of these hydrogels to the chemical environment will be critically dependent on the microstructure of the hydrogels. Porous or structured hydrogels will have a fast response to the chemical. In this project we will tune the porosity of the hydrogel to optimise the response time. Additionally we will look at mechanisms to either steer or sense the chemical response.

38 SIMULATING THE CONTROL OF MINERAL GROWTH BY SOFT MATTER
Supervisor: Professor J Harding

Minerals in biological systems (such as shells, teeth and bones) grow into complex shapes, often nothing like the shapes expected from conventional crystal chemistry. Somehow, organic molecules (whether individually or in arrays) are controlling this in the environment where the mineral grows. It is likely that the mineral begins as a soft, hydrated, amorphous material and only later becomes a hard, crystalline materials. This project will use a range of simulation techniques to investigate how biomolecules and arrays of organic molecules can control the growth of minerals such as carbonates and phosphates. This project is linked to collaborations with experimental groups both in the UK and elsewhere. Most of the codes required to do this have already been written, but there will be possibilities for people to develop programming skills if they so wish.

39 TISSUE ENGINEERING OF PERIPHERAL NERVE
Supervisor: Professor JW Haycock

Peripheral nerve tissue has the potential to repair and re-grow following trauma or injury, in contrast to the spinal cord. In practice, repair is frequently not achieved because of a lack of physical and chemical guidance at an injury site. For small gap injuries (typically less than 2cm), the use of nerve guidance conduits have a basic but limited ability for redirecting growth, with early clinical trials showing some promise. Longer gap injuries are always treated by autografting, and this has the major disadvantage of patients losing donor nerve function. This project aims to advance on the design of basic nerve conduit devices by creating unaxial micro-fibres from synthetic biodegradable polymers. Fibres will contain neuronal cells and Schwann cells that are needed for the optimal provision of growth factors for aiding axon growth and myelin production. This project is multidisciplinary with training in 3D cell culture, bioreactor design, biochemical detection, confocal microscopy imaging and biomaterials science.

40 DRUG DELIVERY BY BIOACTIVE SURFACE ADHESION
Supervisor: Professor JW Haycock

This project is based on the use of potent synthetic calixarene-peptide compounds that can be used to treat biomaterial surfaces (or tissue engineered scaffolds) where inflammation is a major problem. An integrated one-stage method for reducing local tissue inflammation is expected to benefit patient health care and have socio-economic benefits. It will involve the design and use of novel analytical techniques in biochemistry / cell and molecular biology for the evaluation, design and synthesis of bioactive materials. Achieving this will therefore require an interdisciplinary approach spanning cell biology and synthetic chemistry / biomaterial science. An understanding on the use of calixarene-peptide chemistry will be expected to have commercial potential, as the proposed generic approach will be applicable to designing other biologically active peptides for many medical device / tissue engineering applications. This project will therefore be suitable for graduates with backgrounds in pharmacy, materials science, biology/biochemistry or chemistry.

41 A 3D IN VITRO MODEL FOR DRUG AND COSMETIC SCREENING
Supervisor: Prof JW Haycock and Prof S MacNeil

Existing approaches that test compounds for irritation, toxicity or inflammation consist largely of very simple cellular tests or inappropriate animal models. There is therefore an increasing need to develop more relevant and accurate reporter systems of cellular stress for developing 3D tissue engineered models for toxicity testing. A European Council Directive (76/768/EEC) will enforce developing alternative tests for irritants and prohibit the use of animals for toxicological testing from 2009. This highlights that alternative methods must replace animals traditionally used for irritation, corrosivity and phototoxicity tests. Such alternative approaches include the use of reconstructed skin equivalents that match the properties of human skin as closely as possible. Using our engineered model of this tissue we will use genetically transfected reported constructs to detect the response of toxic agents in 3D. This will be an interdisciplinary project encompassing biomaterials, cell culture, molecular biology, biochemistry and confocal microscopy.

42 DEVELOPMENT OF A 3D TISSUE ENGINEERING PERIPHERAL NERVE MODEL
Supervisor: Prof JW Haycock

Three-dimensional in vitro cell culture models are seeing a rapid rate of development, principally driven by the need for conducting studies in a more relevant environment compared to the culture of cells in two dimensions, against a background of the 3Rs (replacement refinement, reduction) in regards to animal usage and scientific experimentation. While an increasing number of mammalian tissues have been reconstructed using three dimensional techniques, often by combining scaffolds and the co-culture of cells (e.g. skin), little work has been conducted on peripheral nerve. The development of such models holds considerable value for a breadth of studies, from a basic understanding of neuronal-glial development through to the design of improved scaffolds for nerve tissue reconstruction following injury. We have recently described a controlled process for producing aligned synthetic microfibers with discrete diameters (between 1 µm to and 8 µm) and correlated the response of neuronal cells and Schwann cells separately as single cultures and as glial-neuronal co-cultures. This has also extended to using dorsal root ganglion cultures for forming aligned 3D neuronal-glial co-cultures. Thus the aim for the next stage of research is to develop the organised peripheral nerve structures which are formed and apply the model to the study of disease, in partilcuar de-myelinating condition of the nervous system. This project will be interdisciplinary and encompass biomaterials, cell neuronal culture, molecular biology, biochemistry and confocal microscopy.

43 DELIVERING AND TRACKING LIMBAL EPITHELIAL CELLS TO THE CORNEA FOR TREATING CORNEAL DISEASES
Prof S MacNeil, Dr F Claeyssens, Dr S J Matcher

Over the last 15 years the technology of culturing limbal epithelial stem cells ( LEC) from thecornea to replace damaged corneal tissue has been developed and has reached a reasonable stage of maturity. The culture of the cells does not pose a particular problem but their survival on often damaged and inflamed and aggressive wound beds is a different matter. This project relates to an ongoing Wellcome funded project between the University of Sheffield and LV Prasad Institute of Ophthalmology in Hyderabad, India where around 600 patients have received cultured LEC . The challenge now is to develop ways to improve the survival and long term clinical outcome of these cells.
This project has two distinct objectives towards this aim:
1. To improve the delivery and survival of LEC transplanted to the cornea.
2. To develop methods to track the fate of transplanted LEC on the cornea.
The approaches to delivering cultured cells to the cornea which we wish to undertake are based on producing microfabricated biodegradable scaffolds with in-built limbal stem cell niches. This is technology established in the group of Dr Claeyssens, working with Professor MacNeil. We have developed electrospun scaffolds which degrade over a matter of a few weeks but they are being designed to have a slower degrading outer ring with microfabricated pockets within them to act as artificial stem cell niches. One of the challenges of this project will be to explore different architectures of microfabricated niches and to explore material with different rates of degradation from a permanent non-degradable material through to a material that can degrade rapidly.
Assessment of delivery of LEC from the scaffold with limbal stem cell niches will be by looking at the survival and phenotype of cells within these scaffolds over periods of up to four weeks in culture. Cells will also be assessed for their ability to leave the scaffold and form a new epithelium on an ex-vivo rabbit cornea model which has been deliberately denuded of epithelium for this purpose. With respect to cell tracking, the challenge here is to explore cell tracking agents that can be used for experimental purposes (of which there are many, such as fluorescent dyes) and iron containing nanoparticles through to materials that can hopefully be translated into clinical use. Here we have two possibilities under examination – naturally occurring melanin and some recently described polypyrroles being developed as contrast agents for OCT imaging. Imaging of cells in the limbal stem cell niches and on the cornea will use confocal microscopy and optical coherence tomography.
This is a strongly multidisciplinary based project. It relates to an area of strong clinical need and will give the PhD student a strong background in tissue engineering, electrospinning, microfabrication, confocal microscopy and OCT imaging.

44 USE OF 3D HUMAN TISSUE ENGINEERED SKIN MODEL TO INVESTIGATE THE ROLE OF CALCIUM IN WOUND REPAIR, SKIN CONTRACTION AND THE DEVELOPMENT OF PSORIASIS
Supervisor: Professor S MacNeil

The MacNeil laboratory has developed 3D tissue engineered skin which has gone into clinical use and is also being used to explore many aspects of normal and abnormal skin biology (MacNeil 2007). This 3D skin model will allow us to investigate the role of calcium in normal wound healing, skin graft contraction and a very common skin pathology, psoriasis. The motivation behind undertaking these studies is that a better understanding of calcium signalling in normal and abnormal skin will lend itself to the development of pharmacological approaches to accelerate wound healing, reduce skin graft contraction and ameliorate the symptoms of psoriasis.
Hypotheses to be investigated:
1. That a temporary reduction in intracellular calcium will accelerate wound repair.
2. That temporary reduction in intracellular calcium will reduce skin contraction.
3. That the psoriatic phenotype which we can induce in this model will be associated with a low intracellular calcium and that known agents which elevate calcium (such as vitamin D) will reduce the symptoms of this disease.
This PhD project will suite a candidate with a cell biology, physiology or biochemistry background. The student will acquire a good knowledge of 3D tissue engineering , of calcium signalling and pharmacology and of skin biology and skin pathologies including psoriasis. The project will involve developing a methodology for measuring changes in intracellular calcium in the 3D skin model using multiphoton confocal microscopy. Additionally it is anticipated that the biological data generated in this model will be used to inform a computational model of calcium signalling in human skin to be undertaken in collaboration with the Institute of Bioengineering, University of Auckland.

45 IMMUNE SURVEILLANCE AND TISSUE ENGINEERING HUMAN SKIN
Supervisors: Professor S MacNeil and Prof J Haycock

Background
In normal skin biology immune cells regularly survey the skin and on detecting allergens or inflammation or any cell abnormalities will travel to the lymph node, eventually leading to the activation of T lymphocytes. These will travel back to the skin to for example, kill cancer cells. While 3D tissue engineered skin has been successfully produced and taken to the clinic ( see MacNeil 2007) attempts to introduce immune components into skin are in their infancy. In this project two types of cells will be introduced into the skin model – normal dendritic cells which are capable of responding to antigens, and also selected activated human T lymphocytes (obtained through a collaboration with Professor Rod Dunbar, Department of Biology, University of Auckland, New Zealand). The overall aim is to undertake a feasibility study of introducing immune surveillance into tissue engineered skin to make this a much more physiologically relevant model for studying skin biology and also reducing animal experimentation.
Hypotheses to be investigated:
1. That challenging keratinocytes with common allergens will lead to dendritic cell activation.
2. That reducing intracellular calcium or adding putrescine (see Harrison et al) in this model will induce a psoriatic phenotype in the keratinocytes which will lead to the activation of dendritic cells and possibly T cells when these are added to skin.
3. That introducing activated T cells will induce a psoriatic phenotype in the tissue engineered skin.
This PhD project will suit a candidate with a cell biology, physiology or biochemistry background. This project will involve a good grounding in tissue engineering and in skin immunology and will involve imaging fluorescence labelled cells within 3D skin using multipihoton confocal microscopy and also developing the use of flow bioreactors in which to study immune cell-skin cell interactions. Two conditions will be studied – that of contact dermatitis where allergans such as detergents and chromium will be used as examples of common skin sensitisers and induction of the psoriatic phenotype (using putrescine) will be used to induce a common skin condition, psoriasis, where immune cells are known to be activated.

46 USE OF HUMAN FAT DERIVED ADULT STEM CELLS FOR DEVELOPING MATERIALS FOR REPAIR OF HUMAN FEMAL PELVIC FLOOR
Supervisors: Professor S MacNeil and Prof C Chapple

Stress incontinence and pelvic organ prolapse are depressingly common in woman from middle age onwards. This is due to a weakening of the pelvic floor associated with age and childbirth. The patient’s tissues become stretched and weakened and this leads to prolapse of the uterus and/or vagina and urinary incontinence particularly under any conditions of mild stress. Over the years there have been an impressive array of surgical procedures to tackle pelvic floor weakness and stress incontinence (which often co-exist). Buttressing repair with non autologous biological substitutes is problematic as these are eventually resorbed leaving little trace and can fail quite abruptly, often from 6 months onwards. Synthetic meshes initially provide excellent mechanical strength but as they do not contain any cells they can result in erosion and we don’t have adequate long term follow up data and there is the concern that they will result in morbidity many years later. Certainly, with the reasonable expectation that we are all living longer then a condition such as stress incontinence in the early 40s requires approaches to treatment which will reliably last decades rather than a few years.
To get sustained repair it is clear that there must be cells present. The MacNeil group working with Professor Chapple have recently started to tackle this problem by undertakng a simple approach to making a new tissue engineered tissue by combining the patient’s oral fibroblasts with natural or synthetic scaffolds. This project explores an alternative source of cells for this tissue repair derived from human fat.
Hypothesis:
That cells isolated from human fat will, with relatively simple magnetic bead separation techniques, be able to take on the phenotype of either stromal connective tissue cells or of pre-angiogenic cells to then be combined and introduced into scaffold materials for repair of the weakened tissues of the human female pelvis.
This PhD project will suite a candidate with a cell biology or biomaterials background. This project will involve a good grounding in tissue engineering and adult stem cell biology and will involve imaging fluorescence labelled cells within 3D constructs using multiphoton confocal microscopy as well as electrospinning of biodegradable scaffolds. Recent work from studies of fat derived cells suggest that one can select a population of CD34 cells (as an indicator of stem cell potential) and then further divide these into those cells that express CD31 (as a marker of angiogenesis) and those cells that do not express CD31,indicative of mesenchymal stromal cells respectively. Initial experiments will work with cells selected using magnetic beads to which CD34 and CD31 antibodies are coupled, subsequently characterising the behaviour of these cells and their ability to behave as fibrous tissue producing cells and to behave as cells capable of producing blood vessels. The challenge will then be to combine these cells in appropriate ratios to produce a cell populated tissue (based on electrospun scaffolds) which can be developed for repair of weakened female pelvic tissues.

47 MICROFABRICATION AND EVALUATION OF CORNEAL STEM CELL NICHES
Supervisors: Professor S MacNeil, Dr F Claeyssens and Dr M Evans (CSIRO Melbourne)

In vivo corneal limbal stem cells live within pockets or stem cell niches known as the Palisades of Vogt of approximately 150 x 30 μm. These provide a recessed pocket in which limbal stem cells are thought to proliferate and survive. There is recent evidence (Shortt et al 2007) that these niches become shallower and less well defined in eyes from elderly patients consistent with increased corneal problems with age. One popular theory that has been proposed is that these pockets provide physical protection for the maintenance of a population of slowly dividing corneal stem cells under conditions of physical stress. However there is also conflicting evidence on the role played by stem cell niches in protection of corneal stem cells and it is difficult to study this in vivo. The aim of this project is to get a better understanding of the role, and therefore therapeutic application of, such stem cell niches by using microfabricated pockets to explore the behaviour of corneal limbal stem cells within these pockets.
Hypotheses to be explored:
1. That cells will populate microfabricated pockets made in biocompatible materials and will make an extracellular matrix to assist their attachment and growth within these pockets
2. That pockets will provide physical protection to cells from the shear forces induced by flowing media over the cells in a flow bioreactor
3. That pockets will lead to the generation of cells with a stem cell phenotype which can be cultured for longer than normal without undergoing terminal differentiation.
This interdisciplinary PhD project will suite a candidate with a Biomaterials or a Cell biology background. The student will acquire a good knowledge of microfabrication and of corneal cell biology and of tissue engineering for corneal defects. Microfabrication will be by micro-SL,as described with poly-caprolactone-based polymers by Claeyssens et al. 2009 .The project will also involve developing a methodology for imaging cells in polymeric microfabricated constructs using multiphoton confocal fluorescent imaging and developing a flow bioreactor for studying cells under perfused conditions. Two materials will be used to make microfabricated pockets-polycaprolactone and a patented perfluoropolymer currently used in an implanted contact lens and which will be available for research through a collaboration with Dr Meg Evans CSIRO Melbourne. The perfluoropolymer will be manufactured into a tubed ring for LEC implantation (work to be conducted at CSIRO in Melbourne). Rabbit corneal epithelial cells will be used as a model system looking at how the presence of a microfabricated pocket affects stem cell turnover, response to mechanical trauma (induced by placing cells in a flow bioreactor) and lifespan.

48 OPTICAL MEASUREMENT OF THE MECHANICAL PROPERTIES OF THE STATUM CORNEUM AND CORRELATION WITH THE CLINICAL EFFECTS OF TOPICAL
Supervisors: Dr S J Matcher and Prof M Cork (Medical School).

Optical coherence tomography (OCT) is a biomedical imaging technique related to ultrasound imaging that is widely used in clinical ophthalmology and its use in dermatology is increasing steadily also, because its combination of depth resolution and imaging depth are ideally suited to studying the epidermis and dermis. Atopic eczema is a disease which affects up to 25% of children and arises as a result of breakdown of the skin barrier allowing allergens to gain access to the immune system. Topical treatments for atopic eczema must repair this defective skin barrier in order to control the disease. Many skin medications have a greater effect on the structure of skin that one might realise. The surface stratum corneum in particular can be damaged through a breakdown of corneodesmosomes and lipid lamellae leading to a fall in the cohesive force between corneocytes which form the skin barrier. This can then lead to an exacerbation of the defective skin barrier in atopic eczema and related diseases. Currently the best way of detecting such breakdown is a procedure called tape-strip trans epidermal water loss (tape-strip TEWL). This involves stripping off the surface layers of skin cells using adhesive tape and then measuring how ‘leaky’ the skin is via the rate of evaporation of water. The need to strip of skin cells however makes this procedure unsuitable for use in widespread clinical trials of drugs and so a better alternative is needed. One possibility is to exploit the fact that a fall in cohesive forces between cells in the stratum corneum should cause a fall in its mechanical properties such as the Young’s modulus. A way of determining this number for the stratum corneum in-vivo is thus needed.
Surface elastic waves are a potential solution to this challenge. If the skin surface is subjected to a short mechanical stimulus then viscoelastic shear waves propagate away from the stimulus site at speeds of a few metres per second. The time delay for the surface waves to reach a certain distance can be measured using OCT and this speed is determined by the Young’s modulus of the skin. Theory and measurements suggest that whilst the mechanical properties of the dermis dominate for high-frequency waves (> a few hundred Hz), lower frequency waves have a speed which predominantly reflects the properties of the stratum corneum. If true for very thin layers of stratum corneum (< 40 microns) this could potentially be the ideal tool to non-invasively study the effects of drugs that damage the corneal desmosomes and could provide a surrogate marker for tape-strip TEWL that would be more suited to large-scale clinical trials. The project will combine experiment (dynamic OCT elastography), theory (finite element modelling of elastic waves) and clinical studies in order to deliver the required results.

49 OPTICAL MONITORING OF BONE REGENERATION
Supervisors: Dr S J Matcher and Dr G Reilly

The ability to regenerate bone tissue is a major goal of tissue engineering. Bone tissue is a complex biomaterial consisting of a collagen scaffold on which calcium phosphate crystals are deposited via the process of biomineralization. Current approaches to artificially recreating this material involve seeding polymer scaffolds with mesenchymal stem cells and then stimulating these cells to generate collagen matrix and deposit mineral. This process occurs in a specialised environmental chamber: the 'bioreactor'. Great interest currently surrounds the use of mechanical stimulation to promote collagen and hydroxyapatite formation and there is a recognised need for a tool that can monitor the biomineralization process in real-time and in a bioreactor. In previous work we have demonstrated that the degree to which bone tissue scatters light correlates with bone mineralization. We have also demonstrated that a novel technique, optical coherence tomography (OCT) can measure the light scattering of bone samples in a bioreactor and have related the OCT measurements to a "gold-standard" technique, quantitative x-ray computed tomography (qCT). Interestingly, we have found preliminary evidence that the changes in light scattering measured by OCT might be a more sensitive measurement of the mechanical strength of the bone than the qCT measurements. This raises the important questions a) how do the light scattering properties of bone relate to its microstructure and b) how does the microstructure influence the mechanical strength? This project will investigate the relationship between optical scattering, qCT densitometry, mechanical strength and tissue microstructure in a systematic way. Techniques to be employed include OCT, qCT, SEM and confocal Raman spectroscopy.

50 OPTICAL COHERENCE TOMOGRAPHY MEASUREMENT OF THE COLLAGEN STRUCTURE OF ARTICULAR CARTILAGE AND ITS DEGRADATION IN OSTEOARTHRITIS
Supervisors: Dr S J Matcher and Prof J M Wilkinson (Medical School)

Optical coherence tomography (OCT) is a biomedical imaging technique related to ultrasound imaging that is widely used in clinical ophthalmology and its use in other areas of medicine is increasing steadily also, because of its unique combination of spatial resolution, imaging depth and speed. An area of untapped potential is in orthopaedics and specifically the management of osteoarthritis. Osteoarthritis affects millions of people every year and is a major cause of pain and disability, especially in the old. It arises because of degradation of the articular cartilage that forms the sliding surfaces in articular joints such as the hip, knee and wrist. Articular cartilage is chiefly composed of type-II collagen, water and proteoglycans. It lacks blood vessels and thus when damaged it has a limited ability to repair itself because of a poor supply of blood-borne nutrients and growth factors. The most popular treatments include autologous chondrocyte implantation (which aims to fill small voids and defects in the collagen with a form of scar tissue) and total joint replacement (when the cartilage is so extensively damaged as to be unrepairable).
There is an urgent need to develop improved way to assess the physical condition of articular cartilage. Currently articular joints can be imaged non-invasively using x-ray or magnetic resonance imaging however the resolution is not high enough to reveal the structure of the cartilage itself. Minimally invasive video imaging (arthroscopy) is currently the best available tool to gauge cartilage degradation however it is limited to visually studying the surface of cartilage.
This project will involve developing a new experimental method called polarization-sensitive optical coherence tomography (PS-OCT) to look in detail at the 3-D collagen structure of cartilage in a rapid, non-destructive and minimally invasive way. This technique has the potential to greatly enhance the capability of arthroscopy to detect early structural changes in cartilage. It may also be of value for evaluating tissue-engineered approaches to cartilage repair, as it is a prerequisite of a tissue-engineered construct that it possess a collagen structure that is a close match to that of the native tissue it is replacing.
The project will involve experimental work using a PS-OCT system to characterise normal healthy cartilage as well as cartilage from patient donors undergoing total joint replacement treatments for severe osteoarthritis. Mathematical modelling to extract structural information from the PS-OCT data may also be involved. The goal will be to establish PS-OCT as a new tool to allow orthopaedic surgeons to assess the extent of damage to cartilage before an operation, in order to plan the extent of the joint replacement that is performed.

51 OPTICAL MEASUREMENT OF CELLULAR RESPIRATION IN BIOENGINEERED TISSUES
Supervisors: Dr S Matcher and Professor S MacNeil

Tissue engineering currently lacks reliable, non-destructive and non-invasive tools to monitor the growth and viability of artificial tissues. Optical imaging and spectroscopy offer one technology that is ideally suited to fulfilling this requirement. This project will aim to use optical spectroscopy to monitor the viability of cells cultured in an artificial matrix via bioenergetic signals from respiratory chain enzymes. At the cellular level, fluorescence spectroscopy will be used to determine mitochrondrial redox potential from the nadh/fad fluorescencne ratio. On larger tissue volumes, optical reflectance spectroscopy will be used to determine the redox state of cytochrome-oxidase. The utility of these measurements in improving the quality of artificial skin, oral mucosa, cornea etc will be fully investigated using established tissue models.

52 SPECTROSCOPIC INVESTIGATION OF THE EFFECT OF THERAPEUTIC DRUGS ON METASTATIC MELANOMA CANCER CELLS
Supervisors: Dr. IU. Rehman, Prof. S. MacNeil

This project aims to investigate melanoma cancer cell lines by Raman spectroscopy to demonstrate the biochemical differences between cell types which are resistant or sensitive to chemotherapeutic treatments.
The incidence of melanoma has increased dramatically this century, which is attributed to changed patterns of behaviour of peoples in the sun. Artificial ultraviolet light (UV) exposure allows individuals in colder climates to expose their skin to UV doses hitherto unprecedented, which has potentially grave effects on the incidence of melanoma in these populations.
One of the great challenges is not only the early detection of melanoma, but to treat with the appropriate drug. Metastatic melanoma remains a particularly difficult tumour to treat and the treatment of melanoma is still essentially surgical but there remains considerable controversy about the optimal margins of excision of the primary tumour.
Resistance to chemotherapy is one of the main problems happening during the treatment of cancer, which can lead to relapse of the condition. It is believed that a proper approach in choosing the chemotherapeutic agents can lead to better treatment results. In this study, melanoma cancer cell lines will be analyzed by Raman spectroscopy to demonstrate the biochemical differences between cell types which are resistant or sensitive to chemotherapeutic treatments.
Spectral analysis of the samples provide distinct spectra which can be used to distinguish between the cell types. The main differences will be observed in the area related to cellular proteins and nucleic acids. In addition, simple classifier technique (using specific spectral bands as classification tools) will be employed in distinguishing between resistant and sensitive cell lines.
The project is multidisciplinary in nature – it involves investigating the behaviour of well characterised melanoma cell lines and using these to evaluate how these differ with respect to changes in their proteins, lipids and carbohydrates correlating these to resistant and sensitive cell lines.

53 AN ALTERNATIVE TO ANIMAL TESTING OF BONE GROWTH AROUND IMPLANTS USING TISSUE ENGINEERING
Supervisors: Dr. IU. Rehman, Dr. G. Reilly

The proposed project aims to develop 3D in vitro tissue engineered bone to act as a pre-screening environment for testing implant materials for orthopaedic research. Currently implant materials are evaluated in crude 2D cell culture studies or in small animal models to establish whether they might enable good bone attachment. Our tissue engineered bone would be a bridge between these tests, providing a 3D bone-like matrix containing a co-culture of human cells from bone marrow, in a dynamic environment. We intent to establish whether implant materials with different bone attachment properties can be distinguished in our in vitro system.
Better methods need to be devised to test orthopaedic implant materials in a more representative environment than a 2D model, and alternative methods to animal testing need to be devised to address the limitations of current animal models and future restrictions on the use of animals in scientific research. Therefore, we intend to test the hypothesis that:
Tissue engineered bone grown in a 3D scaffold can be used as an in vitro test system to examine bone matrix growth around orthopaedic implant materials.

54 SYNTHESIS AND CHARACTERISATION OF A NOVEL BIODEGRADABLE MEMBRANE TO BE USED FOR PERIODONTAL TISSUE HEALING
Supervisor: Dr IU. Rehman

There are around more than 500 species of micro-organisms that can harbour the oral cavity, which are initiating factor for a long list of periodontal problems. According to latest survey conducted, the gum diseases or periodontal diseases are now on a hike and becoming more common than problems like fever or flu throughout the world. There prevalence is more than cancer, heart diseases, AIDS, obesity, arthritis and many other diseases in the public eye. Researchers are looking at all aspects to decrease the treatment time and enhance the healing of not just the bone but also the ligaments attached in the tooth socket.
In this project, the aim is to combine the bone regenerative and tissue regenerative agents together in a membrane to enhance healing time and efficacy. The fabrication will be through elecrtospinning, and its chemical, physical, biological and mechanical properties will be evaluated.
This project will focus on the inkjet printing of discrete layers, each of which contains a chemotherapeutic drug mainly 5-Fluorouracil on the surface of polymeric stents, which can be used for the treatment of oesophageal cancer.

55 DESIGN AND DEVELOPMENT OF A NOVEL DRUG ELUTED POLYMERIC BIODEGRADABLE OESOPHAGEAL STENT-GRAFT FOR THE PALLIATIVE TREATMENT OF SQUAMOUS CELL CARCINOMA OF THE PROXIMAL AND MID OESOPHAGUS
Supervisor: Dr IU Rehman

Oesophageal cancer is the ninth leading cause of malignant cancer death and its prognosis remains poor. There is a major clinical need to improve palliative treatment for patients with advanced oesophageal cancer (squamous cell carcinoma) of the proximal and mid-oesophagus. The oesophageal stent acts mechanically by pushing aside the tumour mass, thereby reinstituting a limited oral diet, hence obviating the need for hospitalisation making it an attractive palliative option.
In the past, rigid plastic oesophageal tubes were in use two decades ago and have been replaced by metallic oesophageal stents, as there a number of disadvantages with the metallic stents requiring a dedicated expensive delivery device. In addition, due to the poor radial strength and being compressed within a delivery system, several episodes of post-deployment balloon dilatation is also needed, and still early and late complication rates involved in oesophageal stenting remain high.
Research work would focus on the development of polymeric biodegradable
drug-eluted Auxetic stent-graft as serious long-term complications in oesophageal stenting
can be avoided by the degradable nature of the stent. Various chemotherapeutic, immunosuppressive, and anti-neoplastic agents will be selected and coated on the Auxetic stent-graft for the programmable retarded drug release. Different techniques, such as, inkjet printing will be used for coatings.

56 POROUS TITANIUM FOR IMPROVED ORTHOPAEDIC IMPLANT DESIGN
Supervisors: Dr G Reilly and Dr R Goodall

Over 100,000 people received total joint replacements under the NHS in 2008. These implants frequently fail and require revision surgery partly due to lack of bone in-growth onto the smooth metal surface and because metals have a higher stiffness than bone. The goal of this project is to produce and validate a novel titanium orthopaedic implant structure using graded porosity. Prototype implants will be designed with a dense core that becomes progressively more porous towards the surface using sintering and rapid prototyping techniques. The core will provide mechanical stability while the graded porous architecture will be optimal for in-growth of bone cells and matrix The project will address have three key goals: 1) To compare processing methods for creating a graded interconnected porous architecture in medical grade titanium. 2) To optimise the biofunctionality of prototype implant structures. 3) To elucidate the potential for bone in-growth using a novel 3D culture method. This is a highly interdisciplinary project in which the student will learn materials processing and biological analysis and cell culture techniques.

57 USING TISSUE ENGINEERED BONE TO EXAMINE COLLAGEN AND MINERAL GROWTH AROUND METAL IMPLANTS
Supervisor: Dr G Reilly

There has recently been much interest in the use of tissue engineered structures to test procedures in vitro (outside the body) that are currently tested in vivo. For instance, tissue engineered bone, grown in scaffolds in a bioreactor environment, could be used to test how bone grows around an implant before using this type of implant clinically. This project will use cubes of tissue engineered bone, made by growing cells in 3D polymer scaffolds as a test site for implanting implants made from metals and other clinically utilised materials. The bone/implant construct will be mechanically loaded to simulate the in vivo environment. Bone growth around the implants will be imaged by microCT, confocal microscopy and histology. The results from the project will be compared with finite element simulations being performed at the University of Hull with our collaborator Michael Fagan. This project would best suit someone with a materials/ biomaterials/ engineering background but a student with a good biology background and an interest in bioengineering could also undertake this project.

58 MECHANISMS BY WHICH BONE CELLS RESPOND TO MECHANICAL FORCES
Supervisor: Dr G Reilly

Bone cells respond to mechanical forces such as those induced by exercise, therefore the cell must have a 'mechanotransduction mechanism' - a way of detecting force and transmitting it to a biochemical signal. Previous work in our laboratory has shown a small solitary cilia that sticks out of the cell called the primary cilia may be one of the ways in which force is detected. Another candidate is the coat of the cell or glycocalyx. We have shown that if we remove the primary cilia or the glycocalyx bone cells can not respond to loading so well and less bone matrix is produced. In this project we will investigate how signals from these membrane components of the cells are transmitted within the cell biochemically and how this information can be used to improve bone matrix structure for instance in elderly people suffering form diseases of low bone mass such as osteoporosis. This project will involve cell biology and biochemical techniques, microscopy and the use of fluid flow and compression bioreactors for cell stimulation.