epsrc and mrc cdts in tissue engineering and ......journal nanomedicine and sits on the editorial...

EPSRC and MRC CDTs in Tissue Engineering and Regenerative

Medicine

Joint Conference 2016

Friday 8 July University of Manchester

Sponsored by

PROGRAMME

9.00 Registration and refreshments

9.30 Conference Session 1: Chair – Christopher Bullock

Welcome: Prof Nicola Tirelli - Director, EPSRC-MRC CDT in Regenerative Medicine, University of Manchester

9.40 Keynote speaker: Prof Liam Grover – Professor of Biomaterials Science, University of Birmingham

10.10

Research presentations 10.10: Catherine Disney – ‘3D Structural and Mechanical Characterisation of the Intervertebral Disc’ 10.30: Gizem Osman – ‘GET Proteins for Efficient Gene Transfer’ 10.50: Joss Atkinson – ‘Bioactive Peptides Coupled to Functionalised Hydroxyapatite’

11.10 Coffee Break

11.40 Conference Session 2: Chair - Olivera Rajkovic

Keynote speaker: Prof Kostas Kostarelos – Professor of Nanomedicine, University of Manchester

12.10

Research presentations 12.10: Emma Neale-Edwards – ‘The Development of Assays for the Scale-up of Cell Manufacturing’ 12.30: Ayesha Bint-E-Siddique - ‘Mechanical Characterisation of Spinal Ligaments – An Anterior Section Study of Human and Ovine Spine’

12.50 Lunch

1.30 Poster Presentation A – Odd-numbered posters

2.15 Poster Presentation B – Even-numbered posters

3.00 Coffee break

3.20 Conference Session 3: Chair - Catriona Cunningham

Keynote speaker: Dr Saba Alzabin – Senior Inflammation Research Scientist, Epistem Ltd

3.40

Research presentations 3.40: Yein Nam – ‘Nanomaterial-based Delivery of miRNA for in vivo Brain Reprogramming and Regeneration’ 4.00: Laura Oakes – ‘Optimisation, Validation and Characterisation of Acellular Porcine Liver Cores to Study the Differentiation of HepaRG Cells’ 4.20: Matthew Dunn – ‘A Pre-clinical in vitro Model of the Basal Ganglia’

4.40 Feedback and prizes

5.00 Social reception

6.30 Close

KEYNOTE

PRESENTATION

ABSTRACTS

ABSTRACT

A Flexible Platform for the Growth of Ligament, Tendon and Bone

Professor Liam M Grover

University of Birmingham

Abstract

The majority of skeletal tissues are formed from a collagenous matrix that has been organised to withstand the mechanical loads to which it is exposed. This talk will describe how it is possible to generate such highly organised tissue-like structures in vitro by using a simple structural analogue of healing tissues. It will describe how cells within this structure respond to local chemical and physical cues and remodel the organic newly forming tissue accordingly, going from a simple homogenous material to a highly specialised heterogenous tissue. Making small changes to the composition of this structure can result in the formation of ligament-like tissue, tendon or bone within a period of only a few weeks of culture. These tissue-like structures have been used to identify novel rehabilitation regimes, and molecules that can be used for the treatment of a host of pathological conditions. The importance of environment in shaping tissue formation has suggested novel methods to regenerate tissues in vivo and to prevent the formation of a number of pathologies in the musculoskeletal system.

Biography

Prof. Grover is a multidisciplinary scientist whose research focuses on designing materials that enhance the tissue regeneration process. He studied for both his undergraduate degree (biomedical materials science) and his PhD on the development of a novel bioresponsive ceramic (under the supervision of Jake Barralet) at the University of Birmingham, UK. On completion of his PhD, Prof. Grover spent two years working in the labs of Jake Barralet and Marc McKee at McGill University, Montreal, where he was awarded a CIHR Skeletal Health Scholarship to study the role of a range of proteins and condensed phosphates in the formation of minerals. He returned to the University of Birmingham in 2006 to establish a research group in the School of Chemical Engineering. In the time since, Prof. Grover has held funding from a multitude of funding bodies, including: the EPSRC, BBSRC, MRC, NC3Rs, the Wellcome Trust, Orthopaedics Research UK, the MoD, the EU, the Drummond Foundation and the NIHR, as well as numerous industrial partners. Since 2006, he has been involved in raising more than £20m of research funding that has provided the University of Birmingham with an exceptional infrastructure for the development of medical technologies. He has published in excess of 100 peer reviewed publications, more than 200 conference contributions, 4 book chapters, has been an inventor on five patent applications and has made more than 35 invited presentations. He was made a Fellow of the Institute of Materials at 30 and was made one of the youngest full Professors in the

history of the University of Birmingham at 32. He is a visiting Professor at the University of Sao Paolo.

ABSTRACT

A Strategy to Enhance Tissue Regeneration by Transient in vivo Cell Reprogramming towards Pluripotency

Professor Kostas Kostarelos

University of Manchester

Abstract

The differentiated status of somatic cells can be reverted to pluripotency in vivo by forced expression of reprogramming transcription factors, in spite of the pro-differentiation signals present in the tissue microenvironment. While sustained expression of such factors leads to widespread tumorigenesis, strategies relying on transient in vivo reprogramming have shown to induce pluripotency and proliferation only temporarily, without the appearance of teratomas. This lecture will illustrate that transient reprogramming can be achieved within mouse adult tissues and will further develop our hypothesis that such an approach can be utilised to enhance the regeneration capability of the tissues after injury. In vivo reprogrammed skeletal muscle showed signs of enhanced regeneration, including rapid appearance of centronucleated myofibers, reduced fibrotic scarring in the injured site and improved recovery of muscle force. In conclusion, reprogramming of somatic cells to a pluripotent-like state in vivo led to transient cell proliferation in the absence of tumorigenesis. We propose such strategy to enhance regeneration and improve functional recovery of skeletal muscle following injury.

Biography

Kostas obtained his Diploma in Chemical Engineering and PhD from the Department of Chemical Engineering at Imperial College London, studying the steric stabilization of liposomes using block copolymer molecules. He carried out his postdoctoral training in various medical institutions in the United States and has worked closely with Professors Th.F. Tadros (ICI plc, UK), P.F. Luckham (Imperial College London), D. Papahadjopoulos (UCSF, USA), G. Sgouros (Memorial Sloan-Kettering, NY, USA) and R.G. Crystal (Weill Medical College of Cornell University, NY, USA).

He was Assistant Professor of Genetic Medicine & Chemical Engineering in Medicine at Cornell University Weill Medical College when he relocated to the UK as the Deputy Director of Imperial College Genetic Therapies Centre in 2002. In 2003 Professor Kostarelos joined the Centre for Drug Delivery Research at the UCL School of Pharmacy as the Deputy Head of the Centre. He was promoted to the Personal Chair of Nanomedicine and Head of the Centre in 2007. The entire Nanomedicine Lab was embedded within the Faculty of Medical and Human Sciences and the National Graphene Institute at the University of Manchester in 2013. Prof. Kostarelos is currently Professor of Nanomedicine at the University of Manchester and Visiting Professor at

UCL Faculty of Life Sciences.

He has been invited Fellow of the Royal Society of Medicine, Fellow of the Institute of Nanotechnology and Fellow of the Royal Society of Arts all in the United Kingdom. In 2010 he was awarded the Japanese Society for the Promotion of Science (JSPS) Professorial Fellowship with the National Institute of Advanced Industrial Science and Technology (AIST) in Tsukuba, Japan. He is the Founding and Senior Editor of the journal Nanomedicine and sits on the Editorial Board of Archives in Toxicology, 2D Materials, Nanoscale Horizons, Journal of Visualized Experiments (JoVE), Applied Materials Today, The Journal of Liposome.

ABSTRACT

The Role of Industry in Promoting Basic Research for Pre-clinical Validation

Dr Saba Alzabin

Epistem Ltd

Abstract

Research and development (R&D) outsourcing is widely used by pharmaceutical and biotechnology related industries to augment research and development and achieve objectives in a more cost and time effective manner. In anticipation of major blockbuster drugs coming off patent, the pharmaceutical industry has responded by cutting down internal R&D efforts, resulting in outsourcing to specialists. Outsourcing is rapidly growing as pharmaceutical companies look to the expertise of CROs to improve productivity, increase speed and flexibility, minimize risks, and to gain access to technology and facilities.

Epistem Ltd. is predominantly a contract research organisation with 16 years of experience offering pre-clinical services and R&D to the pharmaceutical industry. In my presentation, I will talk about the basic pre-clinical research that is covered by the scientists at Epistem with a focus on how the field of immunology helps the pharmaceutical industry validate candidate drugs that target inflammatory diseases and cancer.

Biography

Saba Alzabin was granted a PhD in Basic Medical Research from New York University with a focus on molecular oncology and immunology where she characterised a novel immune-suppressive pathway. As a post-doctoral associate, Dr. Alzabin joined the Kennedy Institute of Rheumatology where, under the tutelage of Prof. Sir Mark Feldmann and Prof. Richard Williams, she conducted a translational research project that resulted in the identification of the basis for non-responders to the most widely used biologic for the treatment of autoimmune diseases. Her findings resulted in the investment of major pharmaceutical companies in the development of bi-specific antibodies for the treatment of arthritis and psoriasis.

In 2012, Dr. Alzabin joined Epistem Ltd, which was predominately an oncology-focused contract research organisation to help them expand on their preclinical services to include inflammatory diseases. She is now a group director with a focus on offering pre-clinical services for drugs targeting rheumatoid arthritis, psoriasis and the anti-tumour immune response to cancer. Saba is also actively engaged in the promotion of academic-industrial collaboration, and holds a post as a visiting scientist at the University of Manchester.

ORAL PRESENTATION

ABSTRACTS

ABSTRACT

Name Catherine Disney

University University of Manchester

Year of Study 2

Title 3D Structural and Mechanical Characterisation of the Intervertebral Disc

Authors C. Disney, J.A. Hoyland, P.D. Lee and M.J. Sherratt

Key words MicroCT, Intervertebral Disc, 3D Strain Map

Abstract

Mechanical performance and associated biological response of implanted constructs is dependent on microstructure. This must be studied at a local level and in 3D in order to understand the complex micromechanical environment. Extra cellular rich native intervertebral disc (IVD) tissue has been imaged using synchrotron X-ray micro tomography (Diamond Light Source) whilst under increasing sequential load. This imaging method does not require tissue sectioning or staining and so the tissue closely retains native mechanics. Synchrotron tomography of unstained tissues resolved not only major anatomical structures (nucleus pulposus, annulus fibrosus and constituent lamellae) but also finer structural details including collagen bundle orientation and cross-bridges. The microstructure between two scans (± load) has been tracked to quantify internal displacement using digital volume correlation (DVC). DVC analysis has provided 3D strain maps at micron resolution with high tracking accuracy. These methods will be applied to degenerated IVD to evaluate stress/strain concentrations due to age-related remodelling. Importantly, visualising the microstructure and mapping strain will be used to assess the efficacy of tissue engineered constructs in a 3D mechanically loaded environment.

ABSTRACT

Name Gizem Osman

University University of Nottingham

Year of Study 4

Title GET Proteins for Efficient Gene Transfer

Authors G. Osman, K. Shakesheff and J. Dixon

Abstract

Cell penetrating peptides (CPPs) are intracellular delivery vehicles that carry biologically active molecules into cells. In previous work we demonstrated that the modification of CPPs to include a heparan sulphate-glycosaminoglycan (HS-GAG) cell surface binding region increased uptake of a reporter protein by 2 orders of magnitude. This phenomenon was termed GAG-binding enhanced transduction (GET). Due to their ability to deliver cargo into cells much more efficiently than unmodified CPPs, GET proteins may provide a powerful tool for the delivery of exogenous DNA into cells. We designed and synthesised a DNA-binding GET protein for the transfection of the reporter gene (pSIN GFP). Results demonstrated GET-mediated transfection efficiencies of 38.1 ± 1.8% in serum conditions. We also compared the transfection of GET protein with a commercial transfection reagent Lipofectamine 2000. Cells treated with Lipofectamine 2000 showed inhibited cell growth and lower cell viability than cells treated with P21 LK15 8R. Following a 3 day serial transfection, 4-fold more GFP positive cells were detected using GET gene-transfer compared to Lipofectamine 2000. In conclusion we have developed a transfection system which we believe that our system could facilitate new approaches for in vitro and in vivo cell programming, gene correction, and in regenerative medicine.

ABSTRACT

Name Joss Atkinson

University University of Sheffield

Year of Study 4

Title Bioactive Peptides coupled to Functionalised Hydroxyapatite

Authors J.K. Atkinson, P.V. Hatton, P. Gentile and C.A. Miller

Key words Peptides, Functionalised, Hydroxyaptite

Abstract

Calcium phosphates such as hydroxyapatite (HAP) are arguably the most successful group of bone graft substitutes, but clinical performance is more limited in ageing or otherwise compromised patients. New strategies are being investigated to enhance bone tissue regeneration without the costs and risks associated with orthobiologic substances, with one promising route described here being the coupling of bio-functional peptides to implant surfaces.

Sintered HAP discs were produced via gelcasting. HAP surfaces were functionalised using acrylic acid plasma polymerisation. The carboxyl groups generated at the surface were then activated for peptide attachment. Finally, the HAP discs were exposed to a heparin binding peptide KRSR [3]. X-ray photoelectron spectroscopy (XPS) and high performance liquid chromatography (HPLC) were used for surface characterisation and peptide release studies.

When analysed using XPS, survey scans from the sintered gelcast HAP discs showed a calcium/phosphate ratio of ~1.63 which was close to stoichiometric HAP (1.67). After full treatment XPS survey scans detected ~7 at% nitrogen at the HAP surface indicating the presence of peptides and therefore successful functionalisation.

The results indicated that the gelcasting and sintering procedures used here were successful in generating near pure HAP scaffolds. The XPS and HPLC results demonstrated that the plasma polymerisation process successfully generated functional groups that could be used to couple peptides to the surface of the scaffolds. It was concluded that peptide coupling provided a highly promising route to produce stable, bio-functional modifications to calcium phosphate biomaterials.

ABSTRACT

Name Emma Neale-Edwards

University Loughborough University

Year of Study 4

Title The Development of Assays for the Scale Up of Cell Manufacturing

Authors E.C. Neale-Edwards

Key words Bioanalytics, Critical Quality Attributes, hMSCs

Abstract

Human Mesenchymal Stem Cells (hMSCs) are being increasingly utilised for their role in tissue repair and immunomodulation. Angiogenesis is an important mechanism involved in tissue repair, and hMSCs have been shown to promote this process (Kwon et al. 2014; Lee et al. 2013). However, our work has shown that the effect of hMSC on angiogenesis varies between donors, and is not directly related to growth rate of an hMSC cell line. This contradicts the common practice of only using hMSCs with higher growth rates in the scale up manufacturing process.

Over all our work demonstrates the need for pre-process characterisation of hMSC lines based on the critical quality attributes relevant to the end treatment. We have improved on the current tube formation assay, which is commonly used to assess angiogenesis in vitro by imaging more regularly and consistently over a larger area. With these improvements we can now asses three criteria: time taken for maximum tube and node formation, the peak number of nodes and tube length formed, and the duration of tube formation before degeneration of the structures. We have also assessed the variation in cytokine production between cells lines and have related these results back to the tube formation assay performance. Furthermore, we have also developed a rapid ELISA testing platform for near time monitoring of cytokine production in the dynamic bioreactor scale up environment, in order to pin point optimum cytokine production by hMSCs.

Kwon, H.M. et al., 2014. Multiple paracrine factors secreted by mesenchymal stem cells contribute to angiogenesis. Vascular pharmacology, 63(1), pp.19–28.

Lee, E.J. et al., 2013. Potentiated therapeutic angiogenesis by primed human mesenchymal stem cells in a mouse model of hindlimb ischemia. Regenerative medicine, 8(3), pp.283–93.

ABSTRACT

Name Ayesha Bint-E-Siddiq

University University of Leeds

Year of Study 4

Title Mechanical Characterisation of Spinal Ligaments – An Anterior Section Study of Human and Ovine Spine

Authors A. Bint-E-Siddiq, M. Mengoni, A.C. Jones and R.K. Wilcox

Key words Anterior Longitudinal Ligament, Posterior Longitudinal Lligament, Stiffness

Abstract

Introduction: The focus of this study is an often overlooked aspect of the spinal motion segment: the spinal ligaments. These ligaments provide passive stability to spine and some studies have suggested that they play a major mechanical role within the physiological range of motion. However, existing studies investigating the mechanical properties of spinal ligaments have revealed large variation in behaviour depending on specimen preparation and testing method.

Methods: Ovine and human thoracic spines were dissected into functional spinal units (FSUs) and separated from the posterior elements. Tests were undertaken on the anterior FSU with all the soft tissue except either the anterior longitudinal ligament (ALL) or the posterior longitudinal ligament (PLL) was removed.

Results: The load-extension curves were used to calculate the stiffness of each ligament. Results showed the characteristic non-linear behaviour of both ligaments, with the ALL being stiffer and stronger.

Conclusion: Ovine ligaments were shown to have a higher stiffness than the human with ALL calculated to be 172 ± 93 N/mm & PLL as 61 ± 25 N/mm. There is some variability in the stiffness across specimens for each ligament and this is considered to be the natural variation due to the difference in location and animal.

ABSTRACT

Name Yein Nam


Year of Study 3

Title Nanomaterial-based Delivery of miRNA for in vivo Brain Reprogramming and Regeneration

Authors Y. Nam and K. Kostarelos

Key words Brain, Nanomedicine, Reprogramming

Abstract

In vivo reprogramming of endogenous cells has emerged as a novel strategy to regenerate the brain by bypassing issues associated with ex vivo manipulation and classical cell transplantation. Despite the success and popularity of viral-based reprogramming, biosafety concerns remain. We utilised two non-viral nanoscale vector systems, carbon nanotubes and cationic liposomes, to stereotactically deliver non-integrative pluripotency-inducing miRNA mimics (miR302s and miR367) with the aim of transiently reprogramming adult mouse striata. We show that both vectors can effectively transfect non-coding RNAs into mouse striata up to 7 days post-injection. The delivery of miR302s or miR302s/367 cocktails complexed with nanoparticles can trigger a significant induction of Ki67+ proliferating cells and upregulation of the neurogenic factor NeuroD1, confirming the presence of a transient proliferative stage in the postmitotic microenvironment. This correlated with a significant reduction of GFAP+ astrocytes suggesting reprogramming of astrocytes into proliferative cells. TUNEL staining revealed that these proliferative cells may have re-differentiated and re-integrated into the host tissues without apoptosis. This study reveals preliminary evidence for transient generation of proliferating cells by reprogramming astrocytes within a glial cell-rich environment for its future application in chronic neural injuries.

ABSTRACT

Name Laura Oakes


Year of Study 3

Title Optimisation, Validation and Characterisation of Acellular Porcine Liver Cores to Study the Differentiation of HepaRG Cells

Authors L. Oakes, S. Griffin and S.P. Wilshaw

Key words Liver, Decellularisation, Characterisation

Abstract

In the United Kingdom, liver disease affects over two million people and is the 5th most common cause of death. Between 2001 and 2012, rates have increased 40%, primarily due to rising alcohol consumption, viral infection and obesity. Liver-assist devices and cell therapies can bridge until transplants are found, although the large cell numbers required, loss of differentiated cell phenotype and clogging of membranes limits clinical application. This project aims to optimise the production of a porcine liver acellular matrix, in order to study the differentiation of HepaRG cells.

The current study characterises native porcine liver tissue to identify tissue variation using histological stains and immunohistochemistry (Collagens I, III and IV, fibronectin and laminin). No gross macroscopic variations were noted, however DNA content varied between all lobes and locations studied. Cells and cellular components were subsequently removed using a decellularisation process with low concentrations of SDS (n=6), validated using H&E, DAPI and DNA quantification.

H&E and DAPI staining indicate the retention of native liver architecture and lack of nuclear structures. DNA quantification indicates a significant reduction in nuclear material. In addition, Reticulin, Masson’s trichrome staining and immunohistochemical labelling using Collagen’s I, III, IV, fibronectin and laminin was performed. Results indicate the retention of Fibronectin, and Collagens I and III, and loss of Laminin and Collagen IV. Extract and contact cytotoxicity indicate that cells grow up to, and in contact with, acellular cores and an absence of cytotoxic components.

An acellular porcine liver core has been developed and subsequently validated. Current work towards the optimisation of culture conditions within acellular cores are ongoing.

ABSTRACT

Name Matthew Dunn

University University of Keele

Year of Study 3

Title A Pre-clinical In Vitro Model of the Basal Ganglia

Authors M. Dunn, M. Kamudzandu, R. Fricker and P. Roach

Key words Model, Parkinson’s, Huntington’s

Abstract

Cell damage or dysfunction of neurons within the basal ganglia can lead to Parkinson’s and Huntington’s diseases. However, the basal ganglia is difficult to access (located in the centre of the midbrain) and the neurons within are densely interconnected, meaning neural study in vivo is very challenging. By re-creating the basal ganglia in vitro, it will be significantly easier to study, but can a complex functioning neural circuit be created in vitro? In this study, a model has been developed with five separate areas for culture of primary cells from five main functional areas of the basal ganglia; namely the cortex, striatum, substantia nigra (pars compacta and pars reticulata) and the globus pallidus. These areas (or ports) are connected by microchannels of varying widths through which axons can grow, connecting the cell populations and creating a circuit mimicking the in vivo basal ganglia. In addition, the whole circuit is cultured on a multi-electrode array (MEA), so that the external electrical signals of the cells can be measured to ensure a functional connection between neural cell populations. Data can be gathered concerning Parkinson’s and Huntington’s by testing pharmaceuticals, neuroprotective factors and stem cell therapies with this novel in vitro neural model.

POSTER

PRESENTATION

ABSTRACTS

ABSTRACT 1

Name Aaron Zammit-Wheeler


Year of Study 2

Title Enrichment of Autologous Minimally Manipulated Stem Cells for Skeletal Repair using Biosensor based Technology

Authors A. Zammit-Wheeler, M. McPherson, C. Wälti and J. Kirkham

Key words Stem Cells, Biosensor, Bone Repair

Abstract

Autologous mesenchymal stem cell (MSC) therapies in regenerative medicine offer potential solutions to complex clinical challenges, such as fracture non-union and repair of critical bone defects. Current techniques used to isolate MSCs rely on centrifugation or antibody based techniques. These have limitations due to high costs, lack of specificity and unknown cellular effects in solid tissue. This project aims to develop a device which is able to separate MSCs from a mixed population derived from bone marrow or orthopedic “surgical waste” tissue in intraoperative time. An end product of an enriched population of MSCs with minimal cell manipulation will be delivered. This project comprises four distinct objectives, the development of selective binding and subsequent controlled cell release, development of fluidics-based technology for cell enrichment, identification of further binders to alternative MSC surface markers and characterisation of enriched cell populations at each stage. The implantation of MSCs with a bone lineage potential will promote healing within the patient without the need for surgical intervention. Identification of other MSC surface markers offers a platform technology for marker-specific cell enrichment, which potentially offers healing for a wide range of tissue types.

ABSTRACT 2

Name Africa Smith de Diego


Year of Study 1

Title Rapid Label-free Separation of Specific Stromal Cell Populations for Autologous Cell Therapy in Musculoskeletal Disease

Authors A. Smith de Diego, J. Kirkham, C. Walti, X. Yang and C. Wood

Key words Dielectrophoresis, Acoustic Waves, Cell Separation

Abstract

Musculoskeletal diseases are the second greatest cause of disability worldwide. Future treatment strategies include use of autologous stem cells to seed engineered scaffolds for tissue repair. Currently, patients’ cells are usually harvested and expanded in vitro to obtain clinically relevant cell numbers for seeding. This process is both time consuming and requires cell manipulation which can impact on patient safety and final costs of the therapy. There is therefore a need for devices enabling label-free separation of stem cells in intra-operative time. By using remote dielectrophoresis, where an electric field is coupled into a microfluidic channel using surface acoustic waves, cells can be separated. Proof of concept of this technology has already been demonstrated using dental pulp stromal cells. Our aim is to scale up this device to deliver rapid cell separation whilst maintaining osteogenic potential. The device will be first optimised following finite element modelling of microfluidics and tested with a complex (known) mixture of cells. After separation, the cells will be seeded on scaffolds and the expression of osteogenic markers will be determined. After in vitro validation, human stem cell-seeded scaffolds will be implanted into athymic rats with calvarial defects to determine efficacy of bone repair.

ABSTRACT 3

Name Agatha Joseph


Year of Study 4

Title 3D In-vitro Diagnostic Tissue Models for Potential for Drug Screening

Authors A. Joseph, S. Ahmed, D. Gardner, D. Rodenhizer, A.P. McGuigan and J.W. Aylott

Key words In Situ Sensing, 3D Scaffold, In-vitro Model

Abstract

Within the field of regenerative medicine and tissue engineering, there is a lack of tools for in-situ real-time monitoring of in-vitro cell response. To overcome this, we have developed self-reporting scaffolds by incorporating pH-sensitive polyacrylamide nanosensors in optically transparent scaffolds. Fluorescent polyacrylamide nanosensors are ratiometric and pH-sensitive across the physiological range (pH 3-8). They comprise a polyacrylamide matrix and contain three entrapped fluorophores; Oregon Green (OG), 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA).

Scaffolds for the in-vitro model can be made by electrospinning or via gelation of gelatin and collagen to form optically transparent scaffolds. By incorporation of pH-sensitive nanosensors and control of gelatin concentration and cross-linking conditions allow an optimal self-reporting scaffold to be prepared, capable of reporting changes in pH while maintaining the structural requirements to support cell growth and proliferation.

Experimental results demonstrate that the self-reporting scaffolds can support cell growth and proliferation while fluorescent and live-cell microscopy methods are used to simultaneously track cell migration and to follow changes in the pH microenvironment. This new diagnostic scaffold has shown pH change within in-vitro models of cancer and wound healing. This model can enable an improved understanding within in-vivo conditions for tissue regeneration (Wound healing and Acute kidney injury) and drug testing (Cancer).

ABSTRACT 4

Name Alice Mortimer


Year of Study 1 (MRes TERM)

Title The Role of Glial Growth Factor in the Differentiation of Adipose Derived Stem Cells towards Schwann Cells

Authors A. Mortimer, A. Faroni and A. Reid

Key words Peripheral Nerve Injury, Stem Cells, Gene Expression

Abstract

Background: Differentiating adipose derived stem cells (ASCs) towards Schwann cells produces an unstable phenotype when stimulating factors are withdrawn. Here we examine the role of glial growth factor in the differentiation process.

Methods: Human ASCs were differentiated toward Schwann cells using previously described methods. Following differentiation, growth factors were withdrawn as follows: group one cells remained in normal medium, group two were maintained with only glial growth factor and group three with all factors except glial growth factor. Undifferentiated ASCs were the control group. 72 hours-post withdrawal, reverse transcription real-time polymerase chain reaction and enzyme-linked immunosorbent assays were performed.

Results: Group two cells showed significantly higher gene expression of glial derived neurotrophic factor (P=<0.02) and Krox20 (P=<0.02). Expression of cJun was significantly higher in group two compared to group three (P=<0.03). Protein analysis revealed that glial derived neurotrophic factor levels were not significantly different between groups, but brain derived neurotrophic factor was significantly lower in group two. Group two cells also reverted to a stem cell-like morphology.

Conclusions: Glial growth factor is unlikely to be involved in the morphological transition of ASCs but appears to have effects on gene expression that may render cells favourable for a nerve injury environment.

ABSTRACT 5

Name Alice Philipson


Year of Study 1

Title Rapid Label-free Separation of Specific Stem Cell Populations for Autologous Cell Therapies

Authors A. Philipson, C. Wälti, C. Wood, J. Kirkham and M. McPherson

Key words Stem Cells, Cell Sorting, Minimal Manipulation

Abstract

There are vast potential uses for stem cells in regenerative therapies addressing clinical challenges in oncology, Parkinson’s disease, spinal cord injuries, bone, muscle or cartilage defects and diabetes. Using autologous stem cells is advantageous due to their accessibility and safety but a major limitation is the isolation of the rare stem cell population in adult tissues containing many other different cells. The ability to separate and concentrate autologous cell subpopulations intraoperatively and without the need for labelling would avoid in vitro cell manipulation and costly cell expansion possibly providing an accelerated translation to clinic. This project will use remote dielectophoresis, where an electric field is coupled into a microfluidic channel using surface acoustic waves, combined with biosensor-based technology using protein-binding proteins (Adhirons) to capture stem cells via surface marker recognition. The methodology proposed will use nanofabrication/photolithography/fluidic channel prototyping to manufacture the device, as well as establishing multi-lineage potential of the isolated cell cultures to determine the specificity and selectivity of the device. This research will benefit researchers and patients alike, as the isolation of specific stem cell populations will assist in the elucidation of regenerative mechanisms and deliver a minimally manipulated, enriched population of specific stem cells for therapeutic purposes.

ABSTRACT 6

Name Aljona Kolmogorova


Year of Study 1

Title Using Nano-bioprinting to Investigate Mechanisms of Axon Pathfinding and Regeneration

Authors A. Kolmogorova, C. Ballestrem, A. Reid and B. Derby

Key words Extracellular Matrix, Bioprinting, Nerve Repair

Abstract

Peripheral nervous system (PNS), unlike the central nervous system (CNS) has the ability to recover and regenerate after injury. Axon outgrowth is an essential requirement for traversing the severed nerve gap and consequently regaining back nerve function.

In general, the extracellular matrix (ECM) serves as a reservoir for growth factors, mechanical support and influences cellular survival and motility processes. However, it is also the main factor contributing to growth orientation of neurons.

Specific biochemical cues in the ECM act as axonal guidance factors by stabilising the growth cone machinery, and consequently directing axonal outgrowth.

Therefore, the project aims to determine these guidance factors in order to manipulate and facilitate axon outgrowth for a rapid nerve recovery.

With modern ink-jet bioprinting technology, we will directly create ECM patterns and controllable environmental input for neurons. This will be used to study the specific contributions of various ECM components. Knowledge about the mechanism and factors for axon directionality can then be used to improve currently used artificial nerve scaffolds, such as nerve conduits.

ABSTRACT 7

Name Ama Frimpong


Year of Study 3

Title Effects of Variations in Surgical Positioning on Torque in Total Hip Replacements

Authors A. Frimpong, J. Fisher, M. Al-Hajjar, P. Withers and S. Williams

Key words Hip, Torque, Simulator

Abstract

There is currently a limited understanding of the effects of frictional and offset torques on the performance of total hip replacements (THRs), particularly following speculation about possible involvement in metal-on-metal implant failures.

Studies have previously been conducted using pendulum friction simulators which evaluate frictional torque about the primary flexion-extension (FE) axis. Although these simulators are functionally limited, results from these studies have been validated as accurate.

A single station hip simulator (SSHS) provides the means of assessing THRs under clinically relevant conditions. The aim of this study was to determine the ability of this newer SSHS to measure torques, by conducting comparative tests on both simulators.

Constant (1kN) and dynamic loads (2kN peak and 100N swing phase) were applied to 36mm metal-on-polymer bearings (n=6) with an FE range of ±10º and ±25º. There was no significant difference (p>0.05) between the torques obtained from both simulators under the different loading conditions except for the 2kN peak 100N swing phase loading at ±10º FE.

Further analysis involving the derivation of equations to calculate the frictional torque from the load cell output of the SSHS showed an inability of the SSHS to accurately determine the frictional torque of the MOP bearings tested.

ABSTRACT 8

Name Amy Smith


Year of Study 1

Title Tissue Repair Capacity of Prototype Antimicrobial-Releasing Scaffold

Authors A.Smith, M. Raxworthy, S. Wood, G. Tronci

Key words Regenerative, Biomaterial, Antimicrobial

Abstract

Advanced biomaterials have been designed to aid the regeneration of soft tissue. They are expected to play key roles in the future of periodontology. Bacterial infection is a common complication which can prevent successful tissue integration. Misuse of antibiotics has led to the concerning spread of antimicrobial resistance (AMR) so alternative approaches are required to minimise the risk of bacterial contamination.

PhotoTherixTM is an advanced bioresorbable polymer scaffold equipped with photodynamic therapy (PDT) technology aimed for use in maxillofacial applications. Typically, an antimicrobial PDT agent is loaded in its inert form and then activated locally through a light source if infection should occur.

To enable translation to clinical use, this study aims to investigate how the scaffold architecture and antimicrobial functionality are affected by its chemical, physical and mechanical properties. Evaluation of the viability of the cells populating the scaffold will be performed to determine tissue regenerating capabilities. If the technology is established, it could also be extended to the treatment of chronic wounds.

The resulting prototype would have a great impact on the health industry by improving patient outcomes, reducing the health economic burden and controlling the spread of AMR.

ABSTRACT 9

Name Ana Kyoseva


Year of Study 1

Biography

Research interests: Tissue engineering, biomaterials and tissue scaffolds.

Aligned project: A tissue engineering strategy to improve lung function and clinical outcome in patients with emphysema.

ABSTRACT 10

Name Anne Canning


Year of Study 3

Title Synthesis of Enzyme Responsive Surfaces – Towards a Smart Cell-Material Interface

Authors A. Canning, M. Zelzer, J. Aylott and L. Buttery

Key words Enzyme, Responsive, Materials

Abstract

Enzyme responsive materials (ERMs) are a promising new class of stimuli responsive materials with potential in the biological field. Recent progress includes the use of enzymes as triggers in molecular self-assembly and in enzyme-mediated release of therapeutics. We aim to design reversible enzyme responsive surfaces that undergo an optically monitorable conformational change on the action of cell secreted enzymes. This response will demonstrate dynamic interactions at the bio-interface, with future potential to modulate cell behaviour. N-carboxy anhydride ring opening polymerisation (NCA ROP) is a versatile facile technique to synthesise polymers of long chain lengths. Here in, homo and copolymers of Glutamic acid and Serine with tuneable conformations have been synthesised via ROP. Composition / conformation relationships in solution have been identified and reaction kinetics have been used to gain insight into how polypeptide properties can be controlled. This was then translated to the preparation of enzyme responsive polypeptide surfaces which have been analysed and tested for suitability to support the culture of two cell types. Enzyme responsive behaviour was demonstrated by measuring casein kinase II induced surface phosphorylation with ToF-SIMS.

ABSTRACT 11

Name Ashley Stratton-Powell


Year of Study 3

Title A Retrieval Analysis of 22 AES Total Ankle Replacement Explants

Authors A. Stratton-Powell, J.L. Tipper, S. Williams, A. Redmond and C.L Brockett

Key words Retrieval Analysis, Total Ankle Replacement, Explants

Abstract

Total ankle replacement (TAR) is less successful than other joint replacements with a 77% survivorship at 10 years. Osteolysis and aseptic loosening are commonly cited TAR failure modes, the mechanisms of which are unknown. This study investigated whether or not TAR explants exhibit similar damage modes and indicators of failure to those recognised in other total joint replacements. Twenty-two Ankle Evolution System (AES) TARs were implanted and retrieved by the same surgeon. Mean implantation time was 7.8 yrs (5.3 to 12.1 range). Macro photography, an Alicona Infinite microscope and the Hood/Wasielewski scale were used to classify damage modes on the polyethylene insert. Scanning electron microscopy with energy dispersive X-ray spectroscopy was used to determine the composition of third body debris. Mean damage score was 185.4 (± 40.0 SD). Burnishing, scratching, pitting and abrasion were prevalent damage modes on both the superior and inferior insert surfaces. Titanium particles, hydroxyapatite fragments and bone debris were embedded in the insert surface. Fixation surface delamination was identified by the ongrowth of tissue between the cobalt chromium substrate and titanium alloy coating. Damage modes indicative of high levels of wear and deformation were evident. Pitting caused by third body debris was abundant which suggested fixation surface wear.

ABSTRACT 12

Name Beenish Siddique


Year of Study 2

Title Double Network Hydrogels via pH-photopolymerisation Technique for Cell Culture

Authors B. Siddique, L.A. Castillo, M.A. Elsawy, A.F. Miller and A. Saiani

Key words Hydrogels, Cell, pH-photopolymerisation

Abstract

The use of self-assembling peptide β-sheet rich fibrous network hydrogels which mimic the extracellular matrix (ECM) of the cells is limited in various medical applications due to their weak mechanical strength. From this perspective, we have used double network (DN) design rational to prepare polymer-peptide hydrogels with tuneable mechanical strength, via a pH photopolymerisation technique. FEFKFEFK (F8) or FEFKFEFKK (F9) / poly (ethylene glycol) (PEGDA) DN hydrogels were prepared. The first network of fabricated DN hydrogels comprised of a fibrous network of pH responsive β-sheet forming peptide, while the second networks is a crosslinked network of PEGDA. The presences of fibrous and polymeric networks in DN hydrogels were confirmed by FTIR spectroscopy. Moreover, due to presence of unique contrasting networks in a single system the storage modulus (G’) of fabricated DN hydrogels was superior to parent single network hydrogels of peptide or PEGDA monomers. The biocompatibility of the F8/PEG and F9/PEG DN hydrogels was assessed using murine 3T3 and stem cells respectively. The viability assay showed that most of the 3T3 cells remained alive under both 2D and 3D cell culture conditions over 48hrs. While the stem cells also shows good viability under 2D cell culture conditions over 21 days.

ABSTRACT 13

Name Benjamin Diffey


Year of Study 1

Biography

Research interests: 1st year CDT regenerative medicine student, BSc in Cell and Molecular Biology.

My interests include stem cell differentiation, transdifferentiation, the role of the immune system in regenerative medicine (both influencing and being influenced by), HSCs and work on neuromuscular junctions. I am currently uncertain exactly which area I am expecting to go in to.

ABSTRACT 14

Name Ben Golland


Year of Study 1

Title Mimicking Extracellular Matrix (ECM) to Promote Nerve Growth for Spinal Cord Injury (SCI) Repair

Authors B.Golland, G. Tronci, J. Tipper, R. Hall and S. Russell

Key words Self-assembling Peptide, ECM-mimicking, Hydrogel

Abstract

Clinical need: Spinal cord injury (SCI) affects around 1000 people in the UK yearly (NHS England, 2013) with treatment cost approximately £1 billion (Spinal Research, 2011). Symptoms stretch beyond impaired bodily functions to poor mental health (WHO, 2013) and currently no recognised treatment to regenerate damaged spinal cord exists.

SCI is characterised by initial trauma and secondary scar formation. Being both fibrotic and glial (Yuan & He, 2013), axons are chemically inhibited due to proteoglycan overexpression and physically blocked (Jones et al., 2001). Attempting to control the mechanical environment using material-based regenerative therapies may lead to enhanced repair.

Research challenges: An ECM-mimicking polymeric device will be engineered at nano-, micro- and macroscopic levels of material organisation. Research strategies include:

• Identifying a self-assembling peptide (SAP) system with inherent biofunctionality and nanofibre-forming capability

• Combination with a nano-/microfibre manufacturing process to create a porous, fibrous architecture for appropriate fibre orientation and morphology

• Integration into a novel hydrogel for superior mechanical performance and tissue-like behaviour

The target outcome is structural support within a mechanically-tuneable, hydrated environment. The research involves development of a new sub-micron fibre spinning technology capable of supporting peptide self-assembly, materials characterisation and development of new structure-property relationships and investigation of neural cell responses.

ABSTRACT 15

Name Catherine Colquhoun


Year of Study 2

Title Manipulating the Properties of Hydrogels to Promote Ependymal Cell Migration, Proliferation and Differentiation for Spinal Cord Repair

Authors C. Colquhoun, J. Deuchars, S. Deuchars, G. Tronci, D. Wood and J. Tipper

Key words Spinal Cord Injury, Ependymal Cells, Hydrogels

Abstract

Despite the prevalence of spinal cord injury and its costly impact on society, treatment remains limited. Following injury, the main obstacles to repair include neuronal loss, demyelination of axons via oligodendrocyte reduction, and the formation of a glial scar comprised of astrocytes (1).

Ependymal cells (ECs) have been identified as potential targets for the repair of such injuries as these have shown the ability to differentiate into astrocytes, oligodendrocytes and potentially neurons following injury to the central nervous system (2).

This projects aims to direct the migration, proliferation, and differentiation of endogenous ECs along favorable lineages using hydrogels with varied mechanical properties, functionalisation and drug release.

(1) Grossman, S.D. et al. Temporal-spatial pattern of acute neuronal and glial loss after spinal cord contusion. Exp Neurol. 2001. 168. Pp.273–282.

(2) Meletis, K. et al. Spinal cord injury reveals multilineage differentiation of ependymal cells. PLoS Biology. 2008. 6(7). Pp. 1494-1497.

ABSTRACT 16

Name Catriona Cunningham


Year of Study 2

Title Characterising 3D Spheroid Cultured Human Mesenchymal Stem Cells for Transplantation into a Mouse Model of Stroke

Authors C. Cunningham, E. Redondo-Castro, L. Martuscelli, E.Pinteaux and S. Allan

Key words Stroke, Stem Cells

Abstract

Stroke is a major health problem causing significant morbidity and mortality worldwide. Post-stroke depression (PSD) is common, affecting 30-50% of patients, and is associated with poorer functional outcomes after rehabilitation. The mechanisms underlying PSD are not well understood. One hypothesis is that pro-inflammatory cytokines such as interleukin-1 (IL-1) and IL-6, upregulated after ischaemia, mediate the behavioural and neuroendocrine changes observed. There is a wealth of data suggesting stem cells promote the recovery of sensorimotor function in animal models of stroke but a paucity of literature on PSD. Therefore, the overall aim of this project is to investigate the effect of human mesenchymal stem cells (hMSCs) on PSD and inflammation in mouse model of ischaemic stroke. Our most recent work has focused on characterising hMSCs cultured in 3D as spheroids before transplantation.

ABSTRACT 17

Name Chris Gabbott


Year of Study 2

Title Investigation of Engineered Tissues or Scaffolds in Simulated ‘Host Environments’ by Developing Miniaturised Bioreactor Systems

Authors C. Gabbott and T. Sun

Key words Tissue Engineering, Bioreactors, Host Integration

Abstract

A bioreactor system was designed to suspend 8 nickel mesh grids in medium, to observe human dermal fibroblasts (HDF) on a free standing scaffold. Cells were seeded onto the nickel scaffolds and observed via phase contrast microscopy (PCM), fluorescent microscopy and scanning electron microscopy (SEM). It was observed that HDF cells were able to bridge the 100 μm square diameter void on the nickel scaffolds. This work follows on from previous experimentation conducted by Sun, and will look to take the work a step further into imaging through transmission electron microscopy (TEM). To observe in greater detail the bridging mechanisms of cells, and the changes to the cytoskeleton of cells during void migration.

ABSTRACT 18

Name Christopher Bullock


Year of Study 2

Title Electrical Stimulation of Cells through Novel Graphene-based Culture Substrate

Authors C.J. Bullock, K. Kostarelos and C. Bussy

Key words Electrical Stimulation, Stem Cells

Abstract

There is a clinical and industrial need to improve the ability to control the behaviours of pluripotent stem cells in vitro. To this end, novel avenues to artificially stimulate stem cells should be explored. Owing to the innate electrical activity of cells, with particular reference to the to the clear disparity in transmembranal voltage between stem cells and differentiated cells, there is great potential for the use of electrical stimulation to direct stem cell behaviours. In order to facilitate systematic experimentation in this area, a novel cell electrical-stimulation system has been designed and commissioned. Key to this system are conductive reduced graphene oxide, rGO, cell culture substrates, which have been developed and characterised in terms of their surface chemistry and topography. The ability of these rGO substrates to support cell growth was later confirmed and quantified using flow cytometry. No significant difference was found between the viability of NIH/3T3 cells on rGO and an uncoated control after 24 hours of culture, validating this approach. Work is currently underway to systematically determine the impact of different electrical stimulation regimes on a differentiated model cell type in order to guide future experiments using pluripotent stem cells.

ABSTRACT 19

Name Cian Vyas


Year of Study 1

Title 3D Printing and Characterisation of Geometrically Complex Multi-Material Scaffolds for Skeletal Tissue Regeneration

Authors C. Vyas, P. Bartolo, J. Hoyland and G. Poologasundarampillai

Key words Bioprinting, Cartilage

Abstract

Degenerative diseases of the skeletal tissues are increasingly causing a significant burden on healthcare systems due to ageing population worldwide. Current clinical treatments can only delay disease progression and subsequently require surgical intervention. New treatments are required to improve the current clinical standard.

Using 3D printing to fabricate geometrically complex composite bioactive scaffolds will allow more effective tissue engineering strategies to be developed which could be translated into the clinic. By altering material composition, fibre size, printing parameters and morphology of 3D printed scaffolds, the cellular-material interactions can be tuned thus generating a library of scaffolds with properties corresponding to different tissues. The scaffold can be optimised to incorporate the required graded properties of both bone and cartilage, allowing a single scaffold to be fabricated avoiding the structural weakness of separate tissue specific scaffolds and improving integration with the underlying bone tissue.

This project aims to optimise the design parameters to develop a scaffold that closely resembles the properties of the different regions in cartilage tissue and can induce a desired cellular response. This requires a combination of multiple materials and techniques. The principles developed here will be versatile and translate into scaffold designs for other tissues.

ABSTRACT 20

Name Daniel Merryweather


Year of Study 1

Title Mesenchymal Stromal Cells Growth Environment and Culture Dynamics

Authors D. Merryweather, E. Cheeseman and M. McCall

Key words Mesenchymal Growth Dynamics

Abstract

High quality MSCs sourced from RoosterBio™ were labelled with FITC and CellTracker™ Violet to determine if sub-populations with varying growth rates could be identified within a single culture. The population was processed using flow cytometry to provide high throughput analysis. Over the course of four days of in vitro culture a significant increase in the variation of label fluorescence was observed, indicating that MSC in vitro population expansion is the product of a sub-population within the wider culture of quiescent cells. This raises questions as to the validity of measurements of population expansion such as PDL and growth rate calculations as these assume a consistent growth rate across the whole population. The staining process was found to be cytotoxic, resulting in a significantly reduced growth rate.

ABSTRACT 21

Name Daniel Secker


Year of Study 1

Title Fabrication of Novel Electrically Conductive Scaffolds for Spinal Cord Tissue Engineering

Authors D. Secker, L-H. Jiang, X. Jia and X. Yang

Key words Neural, Hydrogel, Hypoxia

Abstract

Subsequent to primary traumatic injury, most neuronal death related to spinal cord injury (SCI) occurs during a secondary phase due to ischemia, anoxia, free-radical formation, excitotoxicity and inflammation. Ischemia is characterised by deprivation of oxygen and nutrients, and in order to investigate the effects of oxygen on neuronal cells, a hypoxic workstation (provided by CASE partner – Don Whitley Scientific) will be used to investigate the behaviour and viability of neuronal cells and also neural tissue forming stem cells at both physiologically relevant and hypoxic oxygen concentrations. This knowledge will then be used in the design, fabrication, and optimisation of a hydrogel scaffold which will be functionalised with gold nanoparticles to improve electrical conductivity, building on the work of Das, S., et al (2015), who have demonstrated their use in functionalisation of peripheral nerve conduits.

ABSTRACT 22

Name David Richards


Year of Study 1

Title Photo-Tuneable Hydrogel for Studying Mesenchymal Stem Cell Response to Substrate Stiffness Modulation

Authors D. Richards, O. Dobre, I-N. Lee, L.S. Wong, J. Swift and S. Richardson

Key words Mesenchymal Stem Cells, Mechanobiology, Photo-Tuneable Stiffness

Abstract

Mesenchymal stem cells (MSCs) are a promising cell source for tissue engineering and regenerative medicine. Understanding how to control and maintain their phenotype and lineage commitment is paramount. MSCs are partly regulated by the physical niche environment, such as matrix stiffness. Exactly how MSCs sense, transduce and respond to matrix stiffness is not fully understood.

Most studies utilise static stiffness hydrogels to investigate MSCs response to substrate stiffness. However, elucidation of mechanotransductive mechanisms employed by MSCs and their role in regulating phenotype and lineage commitment requires time-resolved modulation of substrate stiffness.

We have developed a photo-responsive hydrogel, optimised for MSC culture, where stiffness can be reduced and then increased upon photo-irradiation with UV and blue light, respectively.

Initial results indicate MSC spread area increases as a function of increasing substrate stiffness. This suggests MSCs sense and respond to substrate stiffness modulation and that our hydrogel provides a useful culture substrate to probe MSC response to dynamic stiffness over biologically relevant timescales.

We aim to further optimise our hydrogel for reversible stiffness modulation and study its influence on MSC phenotype and lineage commitment. Improved understanding is essential for informing the development of superior tissue engineering and regenerative medicine therapies.

ABSTRACT 23

Name David Rochelle


Year of Study 1

Title Development of a Biomechanical Foot and Ankle Simulator for Evaluation of Interventions for Chronic Ankle Instability

Authors D. Rochelle, A. Herbert, A. Redmond, D. White and C. Brockett

Key words Chronic Ankle Instability, Sprain, Ligament

Abstract

Two million people suffer an ankle sprain annually in the US, 40% of whom suffer chronic ankle instability. This project aims to evaluate: i) the joint contact forces and which ligaments are most affected during a sprain, ii) how the impairment of ligaments affects joint stability and iii) how synthetic reconstruction can improve stability.

Firstly, the ligaments within the ankle will be characterised mechanically. The biomechanics of the ankle in normal and unstable conditions will then be determined to assist the development of an experimental ankle simulator to assess the loading and motion under normal and adverse loading conditions. The effectiveness of synthetic ligament repair to restore joint stability and biomechanics will be evaluated through a parametric study exploring intervention size, position and fixation. This work will further our understanding of the mechanical environment in the ankle and the strains within the tissues under adverse conditions which is critical for informing future treatment strategies. The application of these models to synthetic repair materials affords the development of tools to predict pre-clinically the mechanical function of interventions.

ABSTRACT 24

Name Dimitris Tampakis


Year of Study 1

Title Epigenetic Modifications in Mesenchymal Stem Cells Induced by Aging

Authors D. Tampakis

Key words MSCs, Aging, Epigenetics

Abstract

Mesenchymal stem cells (MSCs) are very promising for the treatment of many diseases in clinical trials. The expansion of MSCs in cultures is fundamental in order to produce adequate amount of cells for therapies and tissue engineering. However, advanced age of donors that cells are isolated from and culture expansion of MSCs, have a detrimental effect on their differentiation and proliferation potential. The molecular mechanisms involved are not yet fully understood. It is believed that epigenetic factors play an important role in major modifications happening to the MSCs in aging. Our project aims to identify these alterations to the epigenome, such as changes in histone modification and DNA methylation that affect the gene expression of MSCs, upon aging. For this purpose, dental-pulp MSCs from donors with various ages (13 – 48 yrs) have been expanding in cultures until senescence occurs and specific epigenetic markers will be tested for any differences upon organismal (in vivo) and cellular (in vitro) aging. The ultimate goal of this project is to understand how epigenetics work in aging of MSCs and to find an epigenetic aging signature that will be used as biomarker to define the state of cellular aging.

ABSTRACT 25

Name Elizabeth Kapasa


Year of Study 3

Title Bone Tissue Engineering in vitro and in vivo using a Novel Multilayer Cell Sheet Technology

Authors E. Kapasa, P. Giannoudis, X. Jia, P. Hatton, X. Yang

Key words Bone, Regeneration, Tissue Engineering

Abstract

Introduction: UK osteoporotic fractures alone are predicted to cost £2.2+ billion by 2020 (1). There is a major clinical need to restore damaged bone structure and function. Multi-layer cell sheet (MLCS) technology provides intact monolayer sheets of stem cells that are simply detached by altering the temperature and stacked together to make a 3D tissue graft. This project aims to investigate the potential of using MLCS technology and stem cell therapy to improve the efficacy of bone tissue engineering.

Method: Human dental pulp stem cells (HDPSCs) were characterised, then cultured to form monolayer cell sheets. MLCS were assembled (3-6 layers) using various stacking techniques before further culture and subsequent analysis.

Results:

1. HDPSC-monolayers maintained an osteogenic phenotype, which was confirmed by alkaline phosphatase (ALP) staining and ALP-specific activity.

2. Increasing cell density and culture duration in osteogenic medium enhanced the size of detached monolayers.

3. Various stacking techniques successfully formed MLCS. 4. SEM and histology confirmed MLCS assembly; the test group

formed thicker tissues and stained positive for collagen and calcium deposition.

5. Live/dead-labelling revealed good cell viability after 3-6 weeks.

Conclusion: Current results demonstrate the feasibility of using osteogenic monolayers to assemble MLCS, indicating the potential of using this novel technology for bone regeneration. Future studies will apply MLCS in combination with/without biocompatible scaffolds in vitro and in vivo.

1. Burge, R.T. et al. The cost of osteoporotic fractures in the UK: projections for 2000-2020. Journal of Medical Economics. 2001, 4(1-4), pp.51-62.

ABSTRACT 26

Name Emily Davies


Year of Study 1

Title Characterisation and Modelling of Clinically Relevant Mechanical Damage of the IVD

Authors E Davies, J.L. Tipper, R.K. Wilcox and M. Mengoni

Key words Spinal Surgical Intervention, Computational Model, Experimental Model

Abstract

Back pain is a common problem, causing around 19 million physician visits and costing approximately $20 million in the US alone per year. Intervertebral disc (IVD) disorders are thought to be a contributor to back pain.

Methods for diagnosing back pain include magnetic resonance imaging (MRI) and discography: the injection of a radio-opaque dye into the nucleus pulposus (NP). This does not take into account the effects of the needle during this invasive procedure (i.e. through puncture). It has recently been suggested that this technique contributes to degeneration in the disc. A number of other surgical interventions, such as repair of annular tears and microdiscectomy, can also lead to unwanted damage.

The aim of this project is to enable the opportunity to find an optimal approach to such invasive procedures, with the goal to reduce the degeneration effects linked to physical damage. The objectives are to produce clinically-relevant experimental models using either animal tissue or donor tissue, to develop corresponding computational models based on image data, and to evaluate the effect of extend of damage or surgical procedure on the biomechanics of the intervertebral disc. The image-based computational models can be validated with the corresponding experimental data.

ABSTRACT 27

Name Floor Agnes Andrea Ruiter


Year of Study 3

Title The Identification of Interacting Electrospinning Parameter Combinations that influence Fibre Morphology using a Design of Experiments Approach

Authors F.A.A Ruiter, C. Alexander, F.R.A.J. Rose and J. Segal

Key words Electrospun Scaffolds, Design of Experiments, Enzymatic-free Cell Culture

Abstract

Electrospinning is a well-known but complex process used to produce fibrous materials [1]. Their similarity to the morphology of the extracellular matrix makes them of particular interest in the field of regenerative medicine. Although well-established, interactions between process parameters and their influence on the fibre morphology are not yet fully understood. A design of experiments (DoE) model was used to determine how combinations of process parameters result in significant effects on fibre morphology. Three different fibre morphologies were investigated within the model; 1) ‘big beads’, 2) ‘beads-on-string’ and 3) ‘uniform’ (defect-free) fibres. Voltage, concentration and flow rate and conc. combined were identified as the main influencing parameters on the average fibre diameter. However, standard deviation was only significantly influenced by the concentration, whereas, the number of beads per µm2 was influenced by both conc. and flow rate. As a result of the DoE experiments linear regression equations were obtained and tested as predictive models. All tested scaffolds fall within the 95% confidence interval of estimates of these equations. The scaffolds produced were then used for studies of 3T3 fibroblast proliferation. No difference in cell growth was observed between uniform and beads-on-strings morphology, however, significantly more growth was observed for big beads morphology. This may be due to the scaffolds mechanical properties.

References

1R. J. Wade, E. J. Bassin, C. B.Rodell and J. A. Burdick, Nat Commun, 2015

ABSTRACT 28

Name Gavin Day


Year of Study 2

Title Development of Experimental and Computation Methods to Model Vertebroplasty

Authors G. Day, A. Jones, R. Wilcox

Key words Vertebroplasty, Simulation Methods

Abstract

Vertebral fractures account for over 700,000 reported fractures each year in the United States, causing pain, loss of vertebral height due to its collapse and increased risk of fracture in the adjacent levels. Using vertebroplasty has allowed large reductions in the pain experienced, however, some controversy remains over outcomes including which patient groups benefit most. To tackle this Finite Element (FE) analysis can be used to understand the characteristics of patient subsets. The work presented details the development of methods both experimental and computational to model augmentation of bovine tail vertebra. The former includes the creation of vertebral fractures using material testing machines, acquiring the stiffness of the vertebrae and augmenting the vertebrae with PMMA cement. Computationally the methods involve an automated approach to converting µCT scans into 3D FE models and modelling augmented vertebrae. Currently a good agreement has been found between experimentally and computationally derived stiffness's for intact vertebrae. Vertebroplasty methods have been able to augment vertebrae with clinically relevant volumes of cement which are able to be masked and models generated. The methods developed will allow for human in vitro vertebroplasty studies to be carried out and accurate augmented vertebral models to be generated.

ABSTRACT 29

Name George Joseph


Year of Study 3

Title Computational Techniques informing Surface Engineering for in vitro Neural Stem Cell Control

Authors G. Joseph, P. Roach, R. Fricker and T. Kyriacou

Key words Material, Modelling, Stem Cells

Abstract

The interest in the clinical use of stem cell therapies is increasing rapidly, with a need for more control over cell populations cultured/expanded in vitro. This is particularly relevant for the treatment of neurological disorders such as Parkinson's disease where positive outcome measures of clinical trials will be limited by the number of derived neurons and their specific sub-types. The aim is to generate enhanced neural cell populations from stem cells through the design of the cell-material interface.

The niche micro-environment is complex, being responsible for cell attachment, proliferation and differentiation. Material engineering approaches to better control cell responses have looked towards surface chemical, topographical and mechanical cues. The many permutations of these factors poses a major challenge in the optimisation of biomaterial design1. Machine learning techniques will be used to assess the impact of surface properties on the biological micro-environment.

Cell interaction/response provides computational outputs, with input variables being derived from material properties such as surface chemical characteristics (logP, charge, density, wettability, etc.) and topography (nano- and micro-scale, aspect ratio, etc)2. The aim is to uncover the relationship between cells and biomaterial surface of in vitro cell culture. In vitro experiments and in silico modelling will continually inform each other towards the optimisation of neural cell characteristic responses.

References

1. Roach, P et al. Surface strategies for control of neuronal cell adhesion. Surf. Sci. Rep. 6: 145–173, 2010.

2. Roach, P et al. Computational methods for optimising stem cell differentiation, 2013.

ABSTRACT 30

Name Georgina Webster


Year of Study 2

Title Development of Acellular Allogeneic Peripheral Nerve Grafts for Peripheral Nerve Reconstruction

Authors G. Webster, P. Rooney, J. Haycock, R. Hall and S-P. Wilshaw

Key words Decellularised Peripheral Nerve

Abstract

Peripheral nerve injuries affect 1 in 1000 of the population. The most common form of intervention is microsurgical repair, with autografts used to bridge defects greater than 1-2 cm. However, autografts are associated with significant limitations, including sacrificing a functional nerve, and adequate sensory and motor function is rarely restored. The poor outcome reflects insufficient autograft availability for major reconstruction and microsurgical failure to address regeneration at the cellular level. Implantable nerve guides aim to provide physical guidance to regenerating axons and concentrate neurotrophic signals. Synthetic and natural materials have been studied extensively for this purpose, but the number of commercially available products is confined to a relatively small number of biodegradable materials, including collagen I, III and IV, PLA, PGA and PLGA. Decellularised peripheral nerve grafts offer potential as an alternative to both autografts and nerve guides, providing a physically compatible, non-immunogenic scaffold for regeneration. Although this has not been considered in detail, some clinical evidence supports the use of decellularised human nerves (Avance® nerve graft). This project aims to develop a decellularised human peripheral nerve graft that retains the biological and mechanical properties of native ECM, providing a native guidance environment for peripheral nerve regeneration.

ABSTRACT 31

Name Halina Norbertczak


Year of Study 2

Title Evaluation of Decellularisation Processes for the Intervertebral Disc for Disc Replacement

Authors H. Norbertczak, R.Wilcox and E. Ingham

Key words Intervertebral Disc, Decellularised Natural Scaffold

Abstract

The intervertebral discs play a vital role in load transmission and articulation of the spine. Disc degeneration occurs as a result of the natural ageing process and/or trauma. The mechanical properties of the discs deteriorate which may cause pain and adversely impact quality of life. Current treatments have limitations and this has led to research and development of tissue engineered solutions with the aim of producing biological implants for the repair and replacement of degenerate discs. One approach is to utilise tissue decellularisation technologies to remove the DNA and other cellular components of native tissues, leaving an acellular extracellular matrix scaffold which is non-immunogenic when implanted into recipients.

This project aims to extend the decellularisation technology to the production of acellular xenogeneic intervertebral discs. A decellularisation protocol is under development which utilises a combination of physical, chemical and enzymatic treatments. The resulting method should not significantly alter the biological and mechanical properties of the tissue. Qualitative and quantitative tests will assess protocol effectiveness for example; biochemical, histological, immunohistochemical and biomechanical tests. It is anticipated that an acellular biological implant will restore mechanical function and allow infiltration of the recipient’s cells which will remodel and maintain the scaffold.

ABSTRACT 32

Name Heather Owston


Year of Study 2

Title Investigating the Potential of Periosteal Cells for Bone Regeneration

Authors H. Owston, G. Tronci, S. Russell, P. Giannoudis and E. Jones

Key words Periosteum, Bone Regeneration

Abstract

Surgical interventions for large bone defects remain suboptimal. Mesenchymal stem cells (MSC) can be combined with a bone scaffold and contained to a defect site by a membrane. MSCs are pivotal to the bone regeneration process therefore, periosteal MSCs (P-MSC) are investigated as a new source of bone regenerating cells.

P-MSCs are more proliferative than donor-matched bone marrow MSCs (BM-MSC) in 2D tissue culture (n=4 donors). After the first passage BM-MSC population doublings (PD) plateau, whereas, P-MSCs continue to divide up to 20 PD. Flow cytometry compared the antigen expression of P and BM-MSCs, known positive MSC markers (CD105, CD73, CD90) were expressed on P-MSCs and negative markers (CD45, CD14, CD34) were not. Three extra markers (MSCA-1, SUSD2, CD271) were tested to investigate differential marker expression between P and BM-MSCs, which could be used in the future for cell tracking studies. However, no definitive differences were shown at this time. Samples of iliac crest bone (autograft material) were imaged histologically and using SEM in comparison to commercial bone scaffolds as controls for future 3D experiments.

In conclusion, P-MSCs have been shown to have greater proliferative potential compared to BM-MSCs, suggestive this source should be investigated for use in clinic.

ABSTRACT 33

Name Henry Cox


Year of Study 1

Title The Self-assembly of Amphiphilic Peptide I3K

Authors H. Cox, P. Georgiades, T. Waigh and J. Lu

Key words Hydrogel, Peptide, Self-assembly

Abstract

Materials made from self-assembling materials, such as synthetic peptides, are a topic of active research due to potential therapeutic applications like tissue engineering or drug delivery. I3K is a small synthetic peptide which can self-assemble to form a hydrogel constructed from a network of self-assembled nanotubes. Passive particle tracking micro-rheology is used to determine the properties of I3K solutions and gels. The technique relates the thermal forces felt by microscopic beads embedded in a medium with the physical properties of the medium itself. Preliminary results suggest that a phase change occurs between 1 mM and 10 mM concentrations in pure water. Moreover, a nematic liquid crystalline phase may exist above 20 mM concentrations. The physical size of I3K aggregates have been observed with STORM super resolution fluorescence microscopy and small angle neutron scattering. The results confirm that twisted cylindrical structures are formed of length on order microns and diameter tens of nanometres. Furthermore, the fibres remain stable under dilution and incubation for a week at room temperature.

ABSTRACT 34

Name Iwan Vaughan Roberts


Year of Study 1

Title An in vitro Developmental Model for Functional Muscle Fibres

Authors I. Roberts, N. Tirelli, G. Cossu, A. Tirella, R. Donno and F. Galli

Key words Biomaterials, Differentiation, Muscle

Abstract

We aim to study biomaterial-instructed differentiation processes in the context of the generation of muscle fibres from human myo-progenitor cells (mesoangioblasts). This will start with the use of hydrogels as 2D substrates with controlled modulus/density of integrin-binding sites. This will then progress into the production of contractile and innervated muscle fibres in a controlled 3D environment.

Using genetic editing it will be possible to use this in vitro model to replicate dystrophic fibres seen in Duchenne muscular dystrophy. This model is amenable to medium-to-high-throughput screening. It will be utilised to optimise the conditions for a genetically engineered cell-based therapy. This can then be used to inform upcoming clinical trials and minimise the variables that need to be tested in a clinical or in vivo setting.

ABSTRACT 35

Name Jack Brewster


Year of Study 1

Title Neural Tissue Engineering on Cell Instructive Graphene Scaffolds

Authors J. Brewster, L.H. Jiang, X. Yang, X. Jia and H. Zhao

Key words Graphene, Neuron, Scaffold

Abstract

There is an unmet clinical demand for neural tissue repair and replacement due to the increasing prevalence of neurodegeneration in our aging society, in addition to treatment of severe damage to neural tissues. However, regenerating neural tissues remains a highly challenging task. Studies from ours and other groups have recently shown the mechanical properties of neuron-supporting substrates in vitro and extracellular matrix in vivo can have profound influence on neuronal cell functions. Such findings bear important implications to the development of new generations of biomaterials and particularly fabrication of novel neuronal cell-instructive scaffolds in advanced neural tissue engineering. Graphene is a two dimensional material, and its lateral dimensions can be readily adjusted with different thickness tuned from single to multiple monolayers, porous structure and adaptable surface chemistry. Due to its mechanical properties and electrical conductivity, graphene has been considered to be attractive in fabricating biomedical materials, and indeed has been demonstrated for its use in biomedical applications and potential for neural interface.

ABSTRACT 36

Name Jack Hart


Year of Study 1

Title Passive Microrheology of Staphylococcus aureus Biofilms

Authors J. Hart, T. Waigh, I. Roberts and J. Lu

Key words Graphene, Biofilm, Microrheology

Abstract

Following adhesion to a surface, bacteria will create an external architecture of polysaccharides, proteins and nucleic acids. The resulting microenvironment, known as a ‘biofilm’, protects bacteria from external antibiotic assault. Biofilms can form on natural and artificial surfaces within the body, such as teeth, heart values and intravenous catheters, leading to chronic infection. Probing the physical and mechanical properties of bacterial biofilms remains a challenging area in biophysics due to their innate complex chemical and structural heterogeneity.

Using a flow-cell chemostat, Staphylococcus aureus biofilms were grown and analysed using optical microscopy. Tracking of individual bacteria was employed to show that biofilms obey power-law rheology. Ripley’s K-function was used to investigate the clustering density of bacteria within the biofilm.

Future experiments will study the influence on biofilm development of novel surface coatings, including graphene oxide, antibacterial peptides and synthetic polymers. Furthermore, super-resolution fluorescence microscopy techniques will be used to explore the relationships between the structural components of the biofilm and bacterial membrane.

ABSTRACT 37

Name James Holland


Year of Study 2

Title Development of Sterilisation Strategies for Acellular Nerve Grafts

Authors J. Holland, P. Rooney, H. Berry, L. Jennings and S-P. Wilshaw

Key words Nerve, Sterilisation, Acellular

Abstract

Peripheral nerve injuries are highly prevalent in the UK, resulting in long term patient morbidities and an associated economic burden. Microsurgical autologous grafting is effective but is limited by the quantity of appropriate material available and causes significant donor site morbidity. Alternative materials for repair include nerve guidance conduits which facilitate directional growth of regenerating axons through physical and chemical means.

A process has been developed to remove the cells from porcine peripheral nerves using low concentration SDS, producing an extracellular matrix (ECM) based scaffold. The composition and properties of the acellular nerves have been shown to be similar to native tissue, indicating significant promise in this approach.

One barrier to the widespread adoption of ECM based scaffolds is the lack of knowledge regarding the effect of tissue sterilisation methods on their biological and mechanical properties. This project aims to determine the effect of different sterilisation strategies on acellular nerves, including both established (e.g. γ-irradiation, E-beam, ethylene oxide) and novel (e.g. supercritical CO2) methods.

Initial work has focussed upon optimisation of the decellularisation protocol, histological and biomechanical characterisation of native and acellular peripheral nerves and the preliminary characterisation of the effects of the sterilant peracetic acid on acellular nerve tissue.

ABSTRACT 38

Name James Kusena


Year of Study 1

Biography

Research interests: Having a chemistry background, I am interested by the chemical/biological interface and how that links to medicine, therapeutics and biotechnology. In addition, I am intrigued by work that focuses on the translational space between scientific discovery and production of cell/biologically based products, techniques and therapies.

ABSTRACT 39

Name James Warren


Year of Study 4

Title Self-assembling Peptides for Articular Cartilage Repair

Authors J. Warren, E. Ingham, J. Fisher and S. Warriner

Key words Osteoarthritis, Self-assembling Peptides

Abstract

Background: Osteoarthritic (OA) cartilage is associated with a loss of glycosaminoglycans (GAGs) and biotribological function. There is a clinical need for early intervention treatments to restore cartilage function and delay the progression of cartilage degeneration. Previous studies have shown that the delivery of the self-assembling peptide (SAP), P11-4 together with P11-4 covalently linked to chondroitin sulphate may have utility in the restoration of biomechanical function to GAG depleted cartilage.

Aims: The aims of this project are to systematically evaluate two rationally designed SAPs for delivery of GAGs to GAG depleted cartilage with a view to restoration of biotribiological function.

Results: Two SAPs were selected, based upon the primary sequence and alternating overall charge. The selected SAPs were covalently linked to chondroitin sulphate/ synthetic GAG using click chemistry. The effects of mixing different molar ratios of each SAP-GAG and SAP on the self-assembling and mechanical properties of the SAP have been determined through the use of TEM, AFM, NMR and SEC-MALLS experiments. The penetration and integration of the SAP-GAG gels into the cartilage will be determined through biochemical assays and microscopy while the effect of integration is assessed by mechanical testing such as indentation testing.

ABSTRACT 40

Name Jayna Patel


Year of Study 2

Title Development of a Novel Method for the Isolation of Low Volumes of SiN Wear Debris

Authors J. Patel, S.P. Wilshaw, R.M. Hall and J.L. Tipper

Key words SiN, Wear, Biocompatibility

Abstract

Adverse biological responses to wear debris limit the lifetime of hip replacements. This has led to the development of a biocompatible silicon nitride (SiN) coating for prostheses, which produces soluble wear debris. The aim of this research is to develop a sensitive method for isolating low volumes of SiN wear debris from periprosthetic tissue.

Commercial SiN particles were incubated with formalin fixed sheep synovium at a volume of 0.01mm3 /g of tissue (n=3). The tissue was digested enzymatically and samples were subjected to density gradient ultracentrifugation to remove proteins. Control tissue samples, to which no particles were added, were also subjected to the procedure. Particles were washed, filtered and imaged by scanning electron microscopy before and after the isolation procedure. Imaging software was used to determine size distributions and morphological parameters of particles.

The particle size distributions of isolated and non-isolated particles were similar. Morphology in terms of roundness and aspect ratio was unchanged by the procedure (KS test, p<0.05). Future work aims to test the method on titanium and cobalt chrome wear debris, and then apply the method to isolate and characterise particles from in vivo studies of novel SiN coated prostheses in a rabbit and sheep model.

ABSTRACT 41

Name Jekaterina Maksimcuka


Year of Study 1

Title Multimodal Imaging of Vascularisation within a 3D Fibrous Bioactive Construct

Authors J. Maksimcuka, O. Tsigkou, G. Poologasundarampillai and P. Withers

Key words Vasculature, 3D-Imaging, Scaffolds

Abstract

Bioactive glasses have great potential for bone regeneration. Once implanted, they form a surface layer of hydroxyl carbonate apatite, which is similar to bone mineral. Recently, bioactive glass of 70S30Ca (70 mol% SiO2, 30 mol% CaO) composition was electrospun into cotton-wool-like fibres, which promote cell infiltration and resemble the supramolecular structure of collagen bundles in ECM.

We aim to develop a vascularised bioactive glass-collagen composite, where bioactive glass is a structural and osteogenic element, while the latter serves as a cellular vehicle and angiogenic element. Induced pluripotent stem cell-derived endothelial cells and mesenchymal stem cells will be used to develop bone tissue and vasculature throughout the scaffold. Micro-computed tomography, electron and confocal microscopy, paired with biochemical and molecular biology methodologies will reveal the scaffold’s 3D structure and vascularised bone tissue formation.

Advanced staining approaches will be developed to target specific cell markers for imaging cell-fibre interactions. High magnification imaging, to study bioactive glass fibres, the collagenous matrix and cell morphology in 3D. Distinct atomic contrasts and morphology of the materials will allow segmentation of individual glass fibres, collagen fibres and cells. Obtained information will connect cell and fibre morphology to molecular and biochemical events occurring during vascularization and bone formation.

ABSTRACT 42

Name Jessica Bratt


Year of Study 2

Title Bronchopulmonary Dysplasia: An in vitro Model

Authors J. Bratt, N. Forsyth, A. El Haj and P. Wu

Key words Lung, Collagen, Model

Abstract

Bronchopulmonary dysplasia (BPD) is a neonatal disease affecting the lungs of premature infants. The disease develops due to abnormal development of the alveoli and pulmonary vasculature. The pathogenesis of BPD remains poorly understood and currently relies on animal models for investigation. We aim to develop an in vitro model of the lung alveoli using cell seeded collagen-elastin hydrogels using a previously described protocol. Collagen and elastin are key mediators in the lung biomechanics, however irregular deposition of collagen and elastin is a hallmark of BPD. The hydrogels provide a 3D environment representative of the lung ECM. The constructs can be exposed to hydrostatic stimulation to represent the ventilation which BPD patients are exposed to. The initial stages of this study focuses on the potential co-culture of lung fibroblasts and endothelial cells in collagen gels to represent the lung alveoli.

ABSTRACT 43

Name Jessica Kirk


Year of Study 3

Title Development of a Synthetic Hydrogel System with Tunable Mechanical Properties to Support a 3D Spinal Cord Injury Model

Authors J.S.L. Kirk, R.M. Hall, J.B. Philipps, G. Tronci, D. Wood and J.L. Tipper

Key words Stiffness, Hydrogel, CNS

Abstract

Injury to the spinal cord can have devastating consequences, such as paralysis. Many advances have been made in the development of biomaterials for cellular therapies to treat spinal cord injury. However, most therapies emphasise the importance of the biochemical environment, whilst little is understood about the biomechanical properties of the matrix and how the mechanical factors affect cellular behaviour in spinal cord injury and nerve regeneration.

A synthetic hydrogel system has been developed using an acrylate-PEG-NHS Ester to chemically attach biological molecules to a Poly(ethylene glycol) diacrylate backbone which polymerises under UV light in the presence of a photoinitiator. The biological molecules contain RGD motifs which allow cellular adhesion and survival. The synthetic nature of the polymer allows flexible tuning of the elastic modulus from 40Pa to 40kPa, by one or all of the following mechanisms:

Molecular weight of the polymer monomer (PEGDA),

Molecular weight of the biological molecule (i.e. gelatin bloom, protein fragment size)

UV polymerisation time

Degree of functionalization of the biological molecule (measured by TNBS assay)

Polymer (PEGDA) concentration (3-20%)

Biological molecule concentration (mg/ml)

Biological molecule type (i.e. gelatin, laminin, RGD, fibronectin)

ABSTRACT 44

Name Jeya Balachandra


Year of Study 2

Title PKC Modifies the Aldara Psoriasis Skin Phenotype

Authors J.P Balachandra, J.L Pennock, S. Alzabin, C.E Griffiths and M. Hardman

Key words Psoriasis, PKC, Aldara

Abstract

Psoriasis is an autoimmune inflammatory disease affecting 2- 3% of the world’s population, for which there is no cure. The disease manifests in a number of ways and presents with several comorbidity. Psoriasis-like lesions were found to occur as a side effect on patients with non-melanoma skin cancer (NMSC) when treated with topical application of Aldara, a cream that contains 5% of the toll-like receptor 7 (TLR7)-agonist imiquimod (IMQ, Walter et al., 2013). Presently, repeated topical application of Aldara is considered a valued psoriasis mouse model. Several reports suggest that the PKC signalling in psoriasis is altered (Rasmussen and Celis, 1993, Fisher et al., 1993). We therefore co-treated the dorsal skin of wild-type C57BL/6 mice with Aldara and tetradecanoylphorbol acetate (TPA: PKC activator) or Gö6976 (PKC inhibitor) to manipulate the levels of PKCα (Cataisson et al., 2003).

We confirmed that topical application of Aldara only on C57BL/6 wild type mouse skin resulted in phenotype and histological characteristics similar to human psoriasis. Immunohistochemical staining and analysis revealed epidermal hyperproliferation indicating acanthosis, increased dermal thickness with infiltration of immune cells, presence of microabscesses, and parakeratosis/hyperkeratosis indicating altered epidermal differentiation. Most interestingly, when co-treated with PKC inhibitor, the epidermal thickness was significantly lower than co-treatment with vehicle demonstrating that inhibition of PKCα may be a beneficial and potential therapy for psoriasis. Future work will confirm the link between PKCα and psoriasis, testing the therapeutic potential of PKC modulation in a psoriatic humanised mouse model.

ABSTRACT 45

Name Jing Zhang


Year of Study 3

Title Development of Innovative 3D Small Intestinal Cell Model

Authors J. Zhang, J.R Lu and J. Penny

Key words Three-dimensional, Intestine, Model

Abstract

As the main site for absorbing nutrients and drugs, small intestine has attracted more and more attentions over last decades. Various models have been developed for better understanding of absorption and metabolism mechanisms in small intestine, among which Caco-2 cell model is the most widely used one. However, this monolayer cell model may lead inaccurate experiment results and faulty conclusions due to its over-simplification. In present study, an innovative three-dimensional cell model which simulates in vivo small intestine epithelium, subepithelial fibroblast network and extracellular matrix was developed to improve drug absorption research. In comparison with Caco-2 monolayer cell model, the 3D model shows reduced transepithelial electrical resistance (TEER) and higher levels of alkaline phosphatase (ALP) activity in microvilli. Two main efflux transporters – P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) were also studied, the decreased P-gp activity and increased BCRP activity indicated a better simulation of in vivo intestine. In summary, this reconstructed 3D cell model is optimised and much closer to human small intestine in structure, physiological and functional levels.

ABSTRACT 46

Name John Garcia

University RJAH Orthopaedic Hospital-University of Keele

Year of Study 4

Title Characterisation of Joint Macrophages: Implications for Osteoarthritis and Cell Based Therapies

Authors J. Garcia, W. Wei, K. Wright, C. Mennan, S. Roberts, J. Richardson, G.J.V.M. van Osch and Y. Bastiaansen-Jenniskens

Key words Inflammation, Immunomodulation, Osteoarthritis

Abstract

Inflammation in the synovium is a feature of osteoarthritis (OA) that contributes to the degradation of the joint. The infrapatellar fat pad (IPFP) is an adipose tissue that is also a source of inflammation. We have characterised the M1 (pro-inflammatory) or M2 (ant-inflammatory) phenotype of macrophages in donor-matched IPFP and synovium.

Tissues were obtained from patients with various arthropathies and were sectioned and stained for CD68 (pan macrophage marker), CD86 (M1), CD206 (M2) and arginase-1 (M2). IPFP explants were treated with the corticosteroid triamcinolone, after which cells were extracted by enzymatic digestion. Flow cytometry was used to assess the phenotype of CD14 positive cells (pan macrophage marker). The IPFP and synovium contained CD14+ cells that were immunopositive for M1 and M2 macrophages markers. There was a strong correlation between synovium and stroma positivity of CD68 and CD206, but not between the synovium or stroma and IPFP. More CD14+CD163+ and CD14+CD86+ cells were present in the cells from IPFP explants treated with triamcinolone, compared to untreated controls. Our data suggests that the IPFP and synovium/stroma may function differentially with regard to macrophage associated-inflammation. We have also demonstrated that the phenotype of IPFP macrophages can be modulated in situ, which could have implications in developing therapies to target joint inflammation in OA.

ABSTRACT 47

Name Julen Zabala Mancebo


Year of Study 2

Abstract Title Nanofabricated Cell Culture Surfaces to Control Self-renewal and Differentiation of Mesenchymal Stem Cells

Authors J. Zabala Mancebo, L.S. Wong, J.A. Hunt, J.M. Curran and S.M. Richardson

Key words Biomaterials, MSCs, Nanofabrication

Abstract

Mesenchymal stem cells (MSCs) have been proposed as a promising cell source for cartilage tissue engineering as they can differentiate towards the chondrogenic lineage. Up until now, MSC chondrogenesis has been achieved by using 3D cultures supplemented with bioactive substances (e.g. TGF-β), which can also act as limiting factors when larger cultures are needed. With these limitations in mind, material induced control of cell responses, via control of initial cell adhesion is a promising tool.

The aim of this work is to generate surfaces with defined chemical features and associated nanotopographical profiles by varying the chain length of a silane used in the production of a self-assembled monolayer (SAM) and, assess their effect on MSC self-renewal and differentiation.

SAM characterisation demonstrated subtle differences in dynamic contact angle and surface roughness. Although surfaces did not show any statistical significance in cell viability or morphology, PCR results demonstrated that ODDMCS induced the highest chondrogenic marker gene expression. Morphology wise, chondrocytes exhibited differences in cell shape depending on which SAM they were cultured on.

While an optimal surface chemistry has not been identified yet, the cell compatibility and influence on phenotype of the surfaces allows experiments to be initiated to assess how OH and NH2 SAMs affect MSC responses.

ABSTRACT 48

Name Katherine Pitrolino


Year of Study 2 (part-time)

Title Development of a Biphasic Osteochondral Plug for Joint Repair

Authors K. Pitrolino, V. Sottile, D. Grant, C. Scotchford, G. Roberts and N. Medcalf

Key words Osteochondral, Scaffold, Mesenchymal

Abstract

Disease and injury to the joints are increasingly common in an ageing population. Current tissue engineering approaches are limited in their capacity to regenerate the bone/cartilage interface, which is important to provide enough strength for the repaired tissue to bear sufficient loads to be fully functional. An osteochondral scaffold is being developed to promote dual differentiation of mesenchymal stem cells and specifically to replicate the in vivo bone/cartilage environment.

The aim of this project is to optimise the current scaffold and its production process to facilitate successful translation to the clinic. Process mapping techniques, which help identify the key process stages and highlight areas of variability, have been used to produce a more robust and repeatable output.

The main functional criteria of the scaffold have been investigated and tests performed to determine how well the current product meets the criteria. Work has been performed to improve the scaffold in terms of cell viability, mechanical strength and improved degradation profile.

ABSTRACT 49

Name Katherine Timms


Year of Study 1

Title Spinal Cord Injury: How does Stabilisation affect Outcomes?

Authors K. Timms, J. Tipper, R. Ichiyama and R. Hall

Key words Spinal cord injury; stabilisation; experimental models

Abstract

Background: In the UK, 1000 people/year get spinal cord injuries. Initial injury is followed by a biological cascade resulting in lifelong disability. Treatment costs £1 billion per year, yet causes few clinical improvements.

Following injury, the spine is often surgically stabilised. However, scientific evidence supporting stabilisation is controversial. Changing the mechanical environment of the spine could have consequences on the cord.

Aim: This project aims to replicate spinal cord injury and subsequent stabilisation using 3D cellular and animal models. Results will help ascertain whether stabilisation is beneficial post-injury.

Methods: Experimental methods to mimic cord injury will be evaluated. Ideal methods would cause reproducible injuries of a pre-determined extent.

Then, the effects of stabilisation on this injury will be explored. In-vitro, this will comprise applying different mechanical stresses and/or strain. In-vivo, this encompasses design and manufacture of stabilisation device(s) with pre-determined rigidities.

Different stabilisation conditions will be compared to each other, and unstabilised controls. This will help determine whether stabilisation has benefits or negative impacts upon damage and regeneration.

Outcomes: Overall, understanding whether stabilisation is beneficial will help guide clinical practice. If changes to stabilisation cause better functional outcomes, this may help improve function in spinal cord injury patients.

ABSTRACT 50

Name Katrina Moisley


Year of Study 2

Title Bone Repair using Collagen Membranes and Autologous Cells: Comparison of Bone Marrow Aspirate and Platelet Rich Plasma

Authors K. Moisley, P. Giannoudis, E. Jones, G. Tronci and S. Russel

Key words BMSC, Collagen, PRP

Abstract

The average cost for patients suffering from fractures has been estimated at £10,000 per case. The fastest, most effective current treatment involves a two-stage reconstructive process, but there is a strong clinical need to streamline the procedure. One solution could be to use the induced membrane which forms around a PMMA cement spacer (to stabilise the joint) as a basis for a biomimetic scaffold. This scaffold will act as a guided bone regeneration device that can be loaded with bone marrow aspirate, source of mesenchymal stem cells (MSCs), and platelet-rich plasma (PRP), a source of osteogenic growth factors.

Histological analysis uncovered the IM’s bi-layer structure including a dense cellular layer and an open spongy fibrous layer. Clinical analysis of bone marrow aspirate based on donor age, gender, aspirated volume and nucleated cell count were unable to predict MSC quantity which also proved to be a very rare and highly variable cell population. We have also been able to optimize the PRP method, generating a ‘pure’ platelet plasma which has a 10-fold enrichment of platelets compared to a four-fold increase in commercial products. Commercial PRP was also found to enrich leukocytes and neutrophils making it not only suboptimal, but also potentially detrimental to bone regeneration.

ABSTRACT 51

Name Kavit Amin


Year of Study 1

Title Preservation of Porcine Extremities with Ex-Vivo Perfusion

Authors K. Amin, M. Hardman, J. Fildes and J. Wong

Key words Transplant, Rejection, Ischemic Reperfusion Injury

Abstract

Limb amputation most commonly occurs as a result of trauma and accounts for 5000 cases in the UK annually. This is followed by surgical amputation in pathological disease, primarily accounted for by infection and tumours. This has a significant impact on psychological and functional outcomes. Current reconstructive options are limited in restoring function. When the limb is from a donor, the immunological response is severe, rendering it functionless and is debatable in non life-threatening conditions due to the side effects of immunosuppressive therapy. Prolongation of limb preservation prior to transplant and minimization of ischaemia reperfusion injury (IRI) following revascularisation will help increase the donor pool and improve outcomes.

We have developed a unique ex-vivo limb perfusion (EVcaP) system that involves attaching the amputated extremity to a device that pumps a bespoke oxygenated blood based solution within a closed circuit to maintain tissue viability.

We hope to address whether (i) EVcaP can be used to effectively deplete the graft of donor immune cells prior to transplantation, (ii) If exhausting the pro-inflammatory response of the donor extremity on the EVcaP circuit drives a tolerogenic phenotype following transplantation, (iii) If EVcaP protects the donor extremity from IRI following revascularisation.

ABSTRACT 52

Name Kelly Wemyss


Year of Study 1

Title Employing Parasitic Worm Products to Improve Chronic Wound Healing

Authors K. Wemyss, K. Mace, N. Tirelli, K. Else and J. Grainger

Key words Parasite, Wound Healing, Immune Response

Abstract

Parasitic worms are the best-described inducers of type 2 immune responses, which are driven by parasite-induced damage and the secretion of excreted/secreted (ES) products with immunomodulatory effects. During infection parasites are able to target a number of host cells, including macrophages, leading to their alternative activation and subsequently elimination of infection, tissue repair and wound healing.

Parasites and their ES products have been shown to reverse or reduce disease in models of allergy, autoimmunity and sepsis and may have beneficial applications in other inflammatory conditions, such as wound healing. In some disease states, such as diabetes, the precise sequence of events involved in normal healing can be disrupted with macrophages existing in their damaging inflammatory form for an extended period of time. Persistence of pro-inflammatory macrophages can lead to the development of a chronic, non-healing wound.

Exploring the local and systemic effects of parasite infection and their associated products will allow us to understand the potential therapeutic benefit of parasitic worms for chronic wounds. We hypothesise that local delivery of ES products to wounds, using nanoparticle technology to target macrophage populations, will provide a powerful new tool in promoting a wound healing phenotype in macrophages and thus, facilitating tissue repair.

ABSTRACT 53

Name Kenny Man


Year of Study 2

Title Pre-treatment of Human Dental Pulp Stem Cells with Novel Histone Deacetylase Inhibitors to enhance Bone Tissue Engineering Efficacy

Authors K. Man, B. Schon, R. Grigg, R. Foster, L.H. Jiang, T. Woodfield and X.B. Yang

Key words Epigenetics, MSCs, Bone

Abstract

Ageing population suffers from bone damage due to numerous causes. The key for bone tissue engineering is to effectively control mesenchymal stem cell osteogenic differentiation. Histone deacetylases (HDAC) play a key role in epigenetics and inhibition of HDAC3 is linked to osteogenic differentiation. Therefore, this study aims to evaluate the effects of HDAC3 selective inhibitor MI192 on cell viability and osteogenic differentiation of human dental pulp stem cells (HDPSC). Cell viability was evaluated using MTS assay after cells treated with MI192 (1,5,10,20,50µM) over 24, 48 and 72 hours. Treatments with MI192 at ≥20 µM after 24 hours, ≥5µM after 48 hours, or ≥1µM after 72 hours significantly reduced cell viability. In further studies, HDPSCs were treated with MI192 at 1,2,5µM for 48 hours prior to osteogenic culture for 14 days and alkaline phosphatase (ALP) was examined by histological staining and biochemical quantitative assays. The ALP expression was significantly enhanced in cells treated for 48 hours with 2µM MI192 compared to non-treated cells and cells treated with 1µM MI192. Similarly, enhanced expression of ALP was observed in 3D pellet culture from MI192 treated HDPSCs compared to non-treated controls. Our results demonstrate the potential of MI192 in enhancing bone tissue engineering efficacy.

ABSTRACT 54

Name Kwan Choi Kwan


Year of Study 2

Title Engineering Mechanically Competent Constructs for Cartilage Repair

Authors K.C. Kwan (Raymond), J. Kirkham, D. Wood, B. Seedhom and S. Finlay

Key words Cartilage, tissue engineering, compressive loading

Abstract

Cartilage has limited repair capacity; any damage untreated could worsen and eventually leads to osteoarthritis. One possible strategy is to implant a functional tissue engineered construct as a replacement for the damaged cartilage. A new method has been recently devised that uses compressive loading to generate constructs with similar mechanical and histochemical properties to native cartilage. Utilising this method, this project aims to determine whether a specific range of cellular deformation promotes optimal matrix deposition in maturing constructs. In addition, the corresponding overall construct strain used to produce desired cellular deformation will be defined. To investigate these objectives, constructs will be grown by seeding bovine synoviocytes on non-woven polyethylene terephthalate scaffolds and subjecting them to compressive loading using a bioreactor. The mechanical and biochemical characteristics of the constructs will be measured at different time points during construct development. Matrix quality and quantity will be determined at each time point, along with confocal visualisation of the constructs under compression, to determine whether a specific range of cellular deformation produces optimal matrix deposition in relation to the corresponding construct strain. Such findings could improve the maturation of constructs and the understanding of the use of mechanical stimulation in cartilage tissue engineering.

ABSTRACT 55

Name Kyle Burgess


Year of Study 2

Title Self-assembling Peptide-based Hydrogels for Myocardial Regeneration

Authors K. Burgess, D. Oceandy and A. Saiani

Key words Cardiovascular, Hydrogel, Cell Therapy

Abstract

Self-assembling peptide based hydrogels have emerged as a promising avenue for in situ cardiac repair following myocardial infarction. In particular, our focus is on a family of peptides based on the alternation of hydrophilic/ hydrophobic residues which form well-hydrated, injectable nanofibrous scaffolds with tuneable properties for the targeted delivery of exogenous cells.

We have shown that these peptide hydrogels can support the in vitro culture of human pluripotent stem cells; which progress from loose, disorganised cell clusters, typically 50μm in diameter, to smooth spheroid aggregates around 250-300μm in size; which retain their pluripotent nature. This opens up the idea of using these 3D matrices as a means to utilise mechanical cues to differentiate stem cells into cardiomyocytes in a way that better mimics their native microenvironment. Alternatively, we have shown stem cell-derived cardiomyocytes, differentiated in 2D, remain viable following 3D encapsulation for subsequent in vivo delivery.

Moreover, this paper demonstrates how these peptide hydrogels can be tagged with a fluorescent fluorophore and successfully injected directly into a healthy mouse myocardium for ease of tracking. Together, these preliminary findings highlight the potential for these peptide hydrogels to act as a temporary cellular scaffold for myocardial regeneration.

ABSTRACT 56

Name Kyle Efendi


Year of Study 3

Title Creation of a Muscle-Tendon-Bone Interface Model using Segmented Collagen Gels

Authors K.K. Efendi, D.J. Player, N.M. Martin, A.J. Capel and M.P. Lewis

Key words Muscle, Tendon, Bone

Abstract

3D collagen based models of skeletal muscle have previously been created using mechanically induced tension. This project now aims to take the first steps into combining these skeletal muscle constructs with a suitable model of tissue engineered bone and in doing so, to create and intermediate ‘tendon’ region as an interface between the two engineered tissue types. In order to achieve this aim, preliminary experiments were conducted whereby a collagen gel consisting of three separate regions was cast with the intention of seeding each section with different cell types. Collagen constructs were cast as previously described into a modified 8-well plate to create standard gels and were adapted by adding bespoke sylgard barriers to create a segmented construct. The altered setup involved setting acellular gels first at the peripheral regions of the mould nearest the A-frames and once set, removing the central piece of sylgard and setting a cellular collagen gel in between them. Constructs were cultured for 14 days before being fixed and stained for the muscle specific proteins desmin and phalloidin. This method has shown promise and further developments are underway to create the MTB junction.

ABSTRACT 57

Name Laura Shallcross


Year of Study 4

Title Investigating the Interactions of Nanoscale Calcium Phosphates with Polymer Additives

Authors L. Shallcross, S. Spain and P. Hatton

Key words Hydroxyapatite, Nanocomposites

Abstract

Current challenges involved in the design and preparation of functional nanoparticles include the difficulty of overcoming nanoparticle aggregation. The formation of aggregates is thermodynamically favourable; however it prevents the full functionality of nanoparticles from being expressed. If the nanoparticles could be dispersed, this would increase their functionality and improve their application to medicine and dentistry. The project aims to encapsulate apatite nanoparticles with functional polymer coatings to demonstrate an enabling technology with the possibility of opening new opportunities in medicine, dentistry and other non-health sectors.

Commercial low and high molecular weight poly(acrylic acid) was added to the synthesis of fluorhydroxyapatite (FHA) and hydroxyapatite (HA) nanoparticles. Analysis via FTIR, XRD, TGA and TEM showed the inclusion of the polymer in the (F)HA samples had an effect on the morphology and size of the particles. Other polymer additives including linear copolymers of poly(ethylene glycol-b-hydroxyethyl acrylamide), synthesised by SET LRP polymerisation were included in the synthesis of (F)HA showing varying effects on the morphology and size of these particles. Ongoing work is looking into the effects of other polyelectrolytes and phosphorylated polymers to determine their effect on the growth of (F)HA nanoparticles.

ABSTRACT 58

Name Leon Edney


Year of Study 4

Title The Use of Inkjet Printing and Thermal Phase Change Inks to Create Sacrificial Prevascular Networks

Authors L. Edney, P.J. Smith and P.V. Hatton

Key words Tissue Engineering, Additive Manufacturing, Inkjet Printing

Abstract

Within Tissue Engineering one of the greatest challenges is providing cells in an engineered tissue with nutrients and oxygen via blood or serum (Auger et al. 2013). A spectrum of research approaches ranging from cell-based therapies that attempt to induce spontaneous ‘budding’ of capillary structures to scaffold based therapies that provide a spatial ‘prevascular’ structure to guide the formation of the vascular network are currently being investigated (Novosel et al. 2011).

This contribution discusses the research that has been undertaken to indirectly fabricate prevascular scaffolds. Inkjet printing is used to make sacrificial three-dimensional microstructures that will be employed in the production of the prevascular scaffolds. Inkjet printing is of particular interest when it is considered that the feature resolution range for Inkjet printing (20 -100 micron) corresponds well with the lumen sizes present in microvasculature (10 – 100 micron). Furthermore inkjet output is circular, which is ideal for capillary shaped sacrificial microstructures.

The process is complimented by the use of low temperature thermal phase change materials e.g. Hexadecane and Octadecane, as inks that enable structurally sound three-dimensional microstructures to be constructed, and then sacrificed without damage to the biocompatible materials (gelatin with a variety of cross linking mechanisms) used for the prevascular scaffold.

Auger, F.A., Gibot, L. & Lacroix, D., 2013. The Pivotal Role of Vascularization in Tissue Engineering. Annual Review of Biomedical Engineering, Vol 15, 15(1), pp.177-200

Novosel, E.C., Kleinhans, C. & Kluger, P.J., 2011. Vascularization is the key challenge in tissue engineering. Advanced drug delivery reviews, 63(4- 5), pp.300–311

ABSTRACT 59

Name Lia Andrea Blokpoel Ferreras


Year of Study 2

Title Characterising the Enhanced Intracellular Delivery of Magnetic Nanoparticles by GET

Authors L.A. Blokpoel Ferreras, K.M. Shakesheff and J.E. Dixon

Key words GET, MNP, Delivery

Abstract

Magnetic nanoparticles (MNP) small size enables them to interact at cellular and molecular levels, furthermore their paramagnetic properties makes them suitable for manipulation through a magnetic field opening a whole range of different of applications in biomedicine including cell tracking, cell labelling, hyperthermia or drug delivery. GET (GAG-binding enhanced transduction) system is based on a novel fusion protein that couples a membrane docking peptide to heparan sulfate glycosaminoglycans (GAGs) with a Protein Transduction Domain (PTD) and has been previously reported to improve delivery of different cargoes into cells. The aim of this work was to develop and characterize a delivery system for MNP to cells. We have demonstrated that GET enhances MNP uptake on cells in vitro.

ABSTRACT 60

Name Lucia Marani


Year of Study 2

Title Developing Three-dimensional Muscle-nerve Constructs using Human Induced Pluripotent Stem Cells

Authors L. Marani, M. Pardo-Figuerez, D.J. Player, A. Stolzing and M.P. Lewis

Key words Tissue Engineering, Skeletal Muscle, iPSCs, Motor Neurons, ALS, SMA

Abstract

Motor neurons (MNs) are specialised cells of the nervous system which innervate skeletal muscles and allow locomotion. These cells undergo deterioration in diseases such as Amyotrophic Lateral Sclerosis (ALS)

and Spinal Muscular Atrophy (SMA). As a consequence, patients experience progressive paralysis due to the associated atrophy of the muscle and the malfunction of the neuromuscular junction (NMJ). Developing a human in vitro model of the NMJ is a challenge yet to overcome. Previous work based on animal-derived cells or human embryonic stem cells was done in monolayer, lacking the three-dimensional in vivo features. Deriving MNs from human induced pluripotent stem cells (hiPSCs) and co-culturing them with human skeletal muscle cells would represent an innovative disease model. It would allow drug screening and understanding of the cellular and molecular mechanisms that contribute to disease aetiology and pathology. This model will be associated with tissue engineering constructs which are suitable for physiology studies, mechanical stimulation of the cells and analysis of their functional properties. Creating a fully human model will reproduce a functional 3D environment. Along the advantages of the use of hiPSCs we acknowledge higher availability and reduced ethical problems which are typically related to animal-derived or embryonic stem cells.

ABSTRACT 61

Name Luke Souter


Year of Study 1

Title Mechanical Properties and in vivo Imaging of Perineuronal Nets

Authors L. Souter, R. Wall and J. Kwok

Key words Perineuronal Nets, Neuroscience

Abstract

Perineuronal nets are structures that primarily surround the cell body of subpopulations of neurons in the central nervous system. They form a compact, lattice-like matrix layer on the surface of neurons.

Recovery from spinal cord injuries, as well as memory retention in mice with Alzheimer’s disease has been enhanced when perineuronal nets are removed. This suggests perineuronal nets block new connection from forming, therefore removing them may enhance the formation of new neuronal connections.

Regulating perineuronal net formation could present a promising therapeutic target. The benefit will go beyond spinal cord injury and Alzheimer’s disease, as it may also provide potential therapies to overcome addiction.

Currently, studies have removed perineuronal nets by digesting chondroitin sulfate using an enzyme. This project aims to tweak the properties of the perineuronal nets to achieve the same enhanced plasticity, without the need for completely removing the perineuronal nets.

To do this regional differences in biochemical and biophysical properties will be characterised and the results will be correlated to understand the relationship between these properties. The information collected will allow the fabrication of an artificial biomimetic surface that simulates perineuronal nets, allowing the relationship between neuronal behaviour and biophysical properties to be studied.

ABSTRACT 62

Name Mathew Hollingworth


Year of Study 2

Title Osteoblast Chemotaxis: Differential Responses to PDGF-BB and TGF-β1 at Defined Stages of Differentiation

Authors M. Hollingworth, J. Ware and L. Buttery

Key words Chemotaxis, Osteoblast

Abstract

During bone remodelling the migration of bone cells and their progenitors is regulated in part by numerous growth factors, allowing temporal recruitment of specific cell types throughout the process of bone turnover. Previous studies have revealed PDGF-BB as a potent chemoattractant for osteoblasts, whilst TGF-β influences migration of osteoblast progenitors. This study describes the chemotactic responses of osteoblasts to growth factors at varying stages of differentiation, helping to characterise the migration of bone cells throughout their development.

Tracking of MPC migration revealed chemotaxis towards PDGF-BB, whilst controls showed no directional movement. Net cell movement showed migration towards PDGF-BB increased after culture in osteogenic medium for 7 days, whilst migration towards TGF-B1 decreased. MPCs also showed a dose-dependent chemotactic response to PDGF-BB. Immunocytochemical staining of MPCs at 0 & 7 days revealed alteration in key osteogenic markers (bone sialoprotein, osteocalcin) suggesting different states of differentiation.

Chemotactic responses to PDGF-BB and TGF-β1 were significantly altered following exposure to osteogenic media. These results contribute to a better understanding of cell recruitment during bone turnover and repair, and have implications on the use of bone cells during cell therapies, which rely upon effective cell recruitment upon delivery.

ABSTRACT 63

Name Megan Hanson


Year of Study 1

Title Musculoskeletal Analysis of Activity for Healthy Aging

Authors M. Hanson, T. Stewart, N. Messenger and B. Cox

Abstract

With an aging population, the ability to maintain good health throughout life is a priority of the BBSRC, EPSRC and MRC as a way to reduce the ever increasing costs of health care. The main focus of this project is to understand how aging influences the musculoskeletal system in order to maintain physical activity for longer, promoting healthy ageing. It is known that as BMI and age increases, the range of motion of joints and power output decreases. This may lead to pain, and therefore reduction in activity and weight gain causing the cycle to repeat leading to an increased risk of heart disease, stroke and diabetes amongst other illnesses.

Working with industrial partners, a unique rowing apparatus will be tested and developed in order to optimise function of the musculoskeletal system with ageing. The project will use detailed biomechanical motion analysis to assess how to reduce adverse loading during rowing in order to prevent damage to joints by mechanical changes to sporting equipment so as to benefit not only the user but also the manufacturer of such equipment.

ABSTRACT 64

Name Mi Zhou


Title Graphene Oxide can act as a Growth Factor Delivery Carrier to promote Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Hydrogels

Authors M. Zhou, N. Lozano, J. Wychowaniec, K. Kostarelos and J.A. Hoyland

Key words Graphene Oxide, MSC Differentiation, TGF-beta3 Delivery

Abstract

The use of graphene (G) and its oxides (GO) to form cell substrates and guide appropriate cell function is a relatively new topic. Formats of 2D G/GO-coated surfaces, 3D chemical-vapour deposited foams and GO dispersions within cell pellets have generated fascinating results particularly in the area of directing stem cell differentiation into specific lineages. Here we report the use of GO flakes dispersed in collagen hydrogels to deliver a chondrogenic factor 3-dimensionally for inducing chondrogenic differentiation of human mesenchymal stem cells (hMSCs). GO TGF-β3-loaded flakes were dispersed in a collagen I hydrogel with hMSCs simultaneously encapsulated. Interestingly, the flakes can adsorb >99 % TGF-β3 with <0.1 % released over time. > 99 % encapsulated cells remained alive indicating the system’s excellent biocompatibility. TGF pre-adsorbed on GO induced cell differentiation into a chondrocytic phenotype; the expression of chondrogenic genes in these gels exceeded the expression in gels with TGF exogenously supplied, and deposition of cartilage-specific extracellular matrix were enhanced. Our results show that GO acts as an efficient growth factor delivery carrier to supply a matrix-bound growth factor effectively. This then creates a platform for potentially loading multiple factors to construct bioactive 3D cell-scaffold for various tissue engineering purposes.

ABSTRACT 65

Name Nathaniel C. Y. Ng


Year of Study 1

Title The Role and Regulation of ROS during Tissue Formation, Repair and Regeneration

Authors N.C.Y. Ng, E. Amaya and A.J. Fielding

Key words Xenopus, ROS, Development/Regeneration

Abstract

It has recently been shown that tail regeneration in Xenopus tadpoles require a sustained increase in the production of reactive oxygen species (ROS). We have also found that embryos are associated with sustained high levels of ROS, which are also needed for tissue development to proceed. It is not entirely clear what is the source of ROS during embryogenesis and following injury, and whether it is identical for both processes. In addition, the identity(ies) of ROS produced are unknown. One of the primary aims of this project will be to identify the source of ROS and their nature, during embryogenesis and following injury. A second primary aim will be to determine how the change in redox state affects processes at the cellular/molecular level, particularly during tissue formation, repair and regeneration. We plan to investigate this question using a model with high regenerative capacity, namely frog embryos and tadpoles. Characterisation of ROS involved is made possible through the use of the electron paramagnetic resonance (EPR) technique, spin trapping. Ultimately, we would like to explore whether manipulating ROS levels might provide a mechanism by which we may induce and sustain a better and more complete regenerative response in humans, following injury.

ABSTRACT 66

Name Nicholas Poulson


Year of Study 1

Biography

Research interests: Previous experience in Tissue Engineering, in particular, expansion and differentiation of human mesenchymal stem cells derived from different tissues. Interests have developed to move towards biomaterials for tissue engineering and resulting differentiation of stem cells.

ABSTRACT 67

Name Nicola Foster


Year of Study 2

Title 3D Models of Chondrogenesis in a Dynamic Environment

Authors N. Foster, H.Markides, S. Kimber and A. El Haj

Key words Chondrogenesis, hESC, MSC

Abstract

The high incidence of diseases such as osteoarthritis, combined with the poor regenerative capacity of native cartilage, mean there is a huge clinical demand for tissue engineered constructs. Chondrocytes can be derived from sources such as embryonic stem cells (ESC) and mesenchymal stem cells (MSC). Successful differentiation to a chondrogenic lineage requires a 3D environment, which allows for close cell-cell contact. This can be achieved with pellet culture or by seeding cells into a gel such as fibrin. Fibrin gels, seeded with hESC-derived chondrocytes or hMSC, are stimulated in the hydrostatic bioreactor for 1 hour/day, 5 days/week at a pressure of 270 kPa and a frequency of 1 Hz for 1 week. Ovine or human MSC, labelled with magnetic nanoparticles, are cultured as pellets and stimulated in the magnetic force bioreactor for 1 hour/day, 5 days/week for 3 weeks. Results from the first experiment suggest that magnetic stimulation is having a positive effect.

ABSTRACT 68

Name Nigel de Melo


Year of Study 1

Biography

Background: 1st Year CDT student, second miniproject. BSc Biotechnology and MSc in Stem Cell Technology

Research interests: Worked on highly versatile peptide based delivery systems. Engineered for switch activity at a specific pH for anti-cancer drug delivery and light based activation of delivery for use in regenerative medical applications.

ABSTRACT 69

Name Nivedah Kuganenderan


Year of Study 4

Title Mechanical Characterisation of Porcine Ankle Cartilage

Authors N. Kuganenderan, J. Tipper, J. Fisher and C. Brockett

Key words Porcine Ankle Cartilage, Cartilage Deformation, Cartilage Thickness

Abstract

Background: Ankle arthritis affects approximately 4% of the global adult population. There has been limited research to characterise the properties of ankle cartilage.

Aims: The aim of this study was to investigate the mechanical properties and thickness of porcine ankle cartilage from the tibio-talar joint.

Methods: Ankle tissue between 3 to 6 month old porcine legs was dissected. Osteochondral plugs of 8.5mm diameter were taken from two locations on each of the tibial and talar joint surfaces (n=6 for both studies). Cartilage thickness measurements were conducted using a needle-probe technique on an Instron 3365 (Instron, UK). Indentation testing, with a load of 0.24N, was conducted for a period of one hour to assess deformation, and computational models were used to derive the elastic modulus and cartilage permeability.

Results: Cartilage thickness was significantly different across both joint surfaces, with a mean thickness of 0.8mm for the tibial joint surface, and 1.0mm for the talar joint surface (ANOVA, p=0.00001). Overall, cartilage deformation across both surfaces showed no statistical significance. The talar ankle cartilage was typically higher in modulus and lower in permeability than the tibial cartilage, but this was not statistically significant (ANOVA, p>0.05).

Summary/Conclusions: In comparison to previously reported data for porcine cartilage in the hip and knee, the ankle cartilage that was thinner, had a comparable modulus to the knee (lower than the hip) and higher cartilage permeability. This study provides an initial model for the mechanical characterisation of ankle cartilage and will be followed with a similar human-tissue based study.

ABSTRACT 70

Name Olivera Rajkovic


Year of Study 2

Title Therapeutic Action of Nanoparticles in Stroke

Authors O. Rajkovic, C. Gourmel, N. Tirelli and E. Pinteaux

Key words Stroke, Inflammation, Nanoparticles

Abstract

Stroke is caused by an inadequate blood supply to the brain due to arteriolar rupture and parenchymal bleeding (haemorrhagic stroke) or an abrupt interruption of blood flow within a cerebral artery due to a blood clot or embolism (ischaemic stroke). Currently, tissue plasminogen activator (tPA) and rapid mechanical thrombectomy (clot retrieval) are the only clinically approved therapies for acute ischaemic stroke. Thus, there is an urgent need for effective medical treatment for acute stroke. One approach is to investigate anti-inflammatory strategies that limit brain injury. This is important as ischaemic stroke is characterised by a potent inflammatory response that contributes to brain injury. Recently, nanoparticles have been developed and we tested the hypothesis that these nanoparticles are effective at limiting inflammation and protect the brain tissue after ischaemic stroke.

Primary mouse mixed glial cells (astrocytes and microglia) were treated with 0.5 μg/ml of inflammatory mediator lipopolysaccharide (LPS). Nanoparticles significantly decreased LPS-induced production of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α), detected with enzyme linked immunosorbent assay (ELISA).

Nanoparticles caused a concentration-dependent decrease of IL-6 and TNF-α production. These data suggest that nanoparticles decrease production of inflammatory cytokines by brain cells. Therefore, nanoparticles can be considered as anti-neuroinflammatory therapy for ischaemic stroke.

ABSTRACT 71

Name Panagiota Moutsatsou


Year of Study 3

Title Novel Chitosan Electrospun Nanofibres incorporating Conductive Polyaniline

Authors P. Moutsatsou and S. Georgiadou

Abstract

Aim of this work is the production of electrospun nanofibrous mats combining chitosan, a biocompatible polymer with antibacterial properties [1] and polyaniline, conductive polymer with tunable properties for tissue engineering applications [2]. CSA doped polyaniline was either blend into chitosan solution or grafted on the chitosan backbone in different ratios to produce chitosan-Pani nanofibers with the electrospinning method. The effect of different process and environmental parameters on the electrospinnability and nanofiber morphology was investigated. Humidity was determined as one of the important parameters affecting the electrospinnability of chitosan- PANI blend solutions followed by the applied voltage. Chitosan grafted PANI (ChgPANI) solutions were found to be not electrospinnable throughout the range of the parameters tested in the current study. The produced nanofibrous mats were then water-proofed with the aid of basic solution and further tested for their biocompatibility on human osteoblasts. Pure chitosan nanofibers were used as a positive control.

References

[1] R. Jayakumar, M. Prabaharan, R. L. Reis, and J. F. Mano, “Graft copolymerized chitosan—present status and applications,” Carbohydrate Polymers, vol. 62, no. 2, pp. 142–158, Nov. 2005.

[2] N. K. Guimard, N. Gomez, and C. E. Schmidt, “Conducting polymers in biomedical engineering,” Progress in Polymer Science, vol. 32, no. 8–9, pp. 876–921, Aug. 2007.

ABSTRACT 72

Name Philippa Clarkson


Year of Study 3

Title Effects of Mechanical Properties and Physical Inputs on Cells of the Central Nervous System

Authors P. Clarkson, J. Tipper, G. Tronci, J. Phillips, D. Wood and R. Hall

Key words Neural Stem Cells

Abstract

It is known that stem cells can respond to the mechanical properties of their surroundings by differentiating accordingly [1]. There are few studies which have explored this effect in neural stem cells, and none of these studies have used constructs with mechanical properties that are biomimetic of natural tissue [2-4]. The CNS is one of the only tissues in the body to be entirely surrounded by bone. Inflammation of this tissue due to trauma or infection creates high ICP in the system which can last for several weeks [5]. After other injuries, stem cells are known to migrate into the peripheral blood supply within 2-3 days after the initial trauma [6]. The CNS is notoriously bad at self-repair, however it is known that neural stem cells are known to be highly migratory and are present in the spinal cord [7]. It is not known if raised ICP affects the repair process. This project aims to understand the effects of elastic modulus and of raised hydrostatic pressure on the differentiation and migration of neural stem cells in a 3D environment.

[1] Engler 2006. Cell 126, 677-689.

[2] Banerjee 2009. Biomaterials 30(27) pp.4695-4699

[3] Her 2013. Acta Biomater 9(2) pp.5170-5180

[4] Bozza 2014. Biomaterials 35(16) pp.4636-4645

[5]Stocchetti 2007. Neurotrauma 24(8) pp1339-1346

[6] Hamou 2009. Plast Reconstr Surg 123(2) pp.45S-55S

[7] Weiss 1996. Neuroscience 16(23) pp.7599-609

ABSTRACT 73

Name Pritesh Mistry


Year of Study 3

Title Reconstructing Organ-level Liver Functions by 3D Printing

Authors P. Mistry, A. Aied, O. Hamid, M. Alexander, K. Shakesheff, A. Bennett and J.Yang

Key words 3D printing, Liver, Hydrogels

Abstract

A major challenge in the discovery of new therapeutics is the assessment of their toxicity. The liver is a vital organ and the major site of drug metabolism and is therefore a key target for these studies. Unfortunately, hepatocytes are extremely sensitive cells and rapidly lose their in vivo functions when removed from their native environment. Current toxicity data is taken from 2D cell culture models and animal models, neither of which can be reliably extrapolated to humans. 2D models are limited by their decaying function, which may be due to their lack of 3D architecture. Here, the use of core-shell fibres in the fabrication of a 3D printed in vitro liver model has been explored. Core-shell fibres present an interesting strategy to build larger constructs as they resemble several in vivo structures. We have combined hydrogels and demonstrated greater mechanical properties of the resultant hybrid gels. We have also demonstrated the printability of these core-shell fibres into complex 3D structures. Finally we have shown high cell viability for the printed cells for over two weeks. The cells were able to proliferate, self-arrange and demonstrate functionality.

ABSTRACT 74

Name Rachel Gater


Year of Study 2

Title Development of an Improved 3D Organotypic Retinal Tissue System for Better Retinal Disease Study and Treatment

Authors R. Gater, D.Nguyen, A. El Haj and Y. Yang

Key words Retinal Tissue Engineering, Glaucoma, Macular Degeneration

Abstract

Ocular diseases such as glaucoma and macular degeneration can lead to significant sight problems and blindness if left untreated. Current treatments are highly invasive, have a low success rate and lack ability to regenerate damaged tissue. Requirement for more effective treatments is therefore high. Stem cell therapies and tissue engineering techniques for retinal disease have advanced significantly over recent years with promising results, but challenges still remain. Therefore, both clinical research and the field of regenerative medicine still require complex in vitro or ex vivo retinal models, for study of disease mechanisms and testing of neuroregenerative materials. In this study, we establish an efficient and reproducible 3D organotypic retinal tissue system using porcine eyes. Our methodology allows us to better preserve all intact layers of the retinal tissue with minimal distortion. Results indicate that using biologically original substrates better preserves the layered morphology of the retinal tissue. We also explore optical coherence tomography (OCT) imaging modality to non-destructively monitor the integrity of the retinal tissue during culture. With further development, we anticipate that this in vivo model would provide better opportunities to research new treatments for retinal disease, such as cell therapies and drug intervention.

ABSTRACT 75

Name Rebecca Grant


Year of Study 1

Biography

Research interests:

Understand the current scale and context of metrology in the clinical laboratory

Determine influence of operators and “best practice” on the metrology confidence

Define confidence in current metrology and characterization of SCT clinical production

Understand ex vivo process distributions and the consequence for manufacturing process control

Evaluate the relevance of Six Sigma and SPC to understand metrology processes and data sets

Use Flow Cytometry and Near-Infrared Spectroscopy as exemplars

Determine the validity of traceability chains to the cell measurement process

Develop initial specifications for metrological and statistical characterization.

ABSTRACT 76

Name Rebecca Morgan


Year of Study 1

Biography

Background: I am a first year PhD student doing the Loughborough/ Nottingham/ Keele CDT in regenerative medicine programme. Prior to this programme, I completed an undergraduate degree in Biochemistry (with a Year in Industry) at the University of York.

Research interests: My research interests include: glycobiology, functional biointerfaces and tissue engineering.

ABSTRACT 77

Name Richa Gandhi


Year of Study 1

Title In Vivo Imaging of Pathological Vascular Remodelling in Rodents using Positron Emission Tomography

Authors R. Gandhi, M. Bailey and C. Tsoumpas

Key words Abdominal Aortic Aneurysms, Atherosclerosis, 18F-fluorothymidine PET/CT

Abstract

In vivo molecular imaging is being increasingly implemented in the context of cardiovascular diseases. Considering both atherosclerosis and abdominal aortic aneurysm (AAA) disease, current medical imaging techniques are typically limited to the visualisation of pathological changes in the vasculature after the disease has progressed to more severe stages. This investigation aims to determine the potential utility of pre-clinical combined positron emission tomography and computed tomography (PET/CT) imaging in studying the molecular mechanisms that promote the manifestation of atherosclerosis and AAA in rodent models. PET/CT imaging will be used in these models to localise and monitor pathological vascular remodelling non-invasively with 18F-fluorothymidine (18F-FLT). 18F-FLT is a marker for cell proliferation, as its pharmacokinetic behaviour correlates with the synthesis phase of the cell cycle, and use of this tracer will be demonstrated for the first time in AAA models. Quantitative analysis of the PET/CT images will potentially contribute to predicting therapeutic response by building upon an approach using novel calcium channel inhibitors to minimise the progression of AAA in rodent models. Evaluating the efficacy of 18F-FLT may hence represent a promising step forward in better understanding pathological cell proliferation and vascular remodelling in AAA disease, whilst expediting treatment development and patient management.

ABSTRACT 78

Name Robert Cooper


Year of Study 3

Title Geometric Parameterisation of Cam Impingement

Authors R. Cooper, M. Mengoni, S. Williams, P. Robinson and A. Jones

Key words Femoroacetabular Impingement

Abstract

Abnormal morphology of the hip joint is a factor explaining soft tissue damage caused by femoroacetabular impingement. Two-dimensional radiographic measurements used in the diagnosis of hip pain do not capture the full bony geometry of the hip. A geometric parameterisation tool was developed to capture key variations in the geometry of proximal femurs with cam deformity, and of articular surfaces of the acetabulum. This tool was used to derive measurements to quantify impingement risk based on three-dimensional bone shape, and applied to a set of 20 patients to assess for differences in deformities between males and females. The systematic method of assessing impingement risk based on three-dimensional bone geometry allowed capture of both the size and position of cam lesions, and differences between subjects were identified that could not be seen in two-dimensional images.

ABSTRACT 79

Name Robert Harrison


Year of Study 2

Title The Role of Toll-Like Receptors in Diabetic Wound Healing

Authors R. Harrison and K. Mace

Key words Wound, Inflammation, Diabetes

Abstract

Wound healing is a complex process defined by three distinct yet overlapping stages: inflammation, proliferation and remodelling. In approximately 4-10% of diabetic patients, aberrant inflammation leads to the formation of non-healing diabetic foot ulcers (DFUs). Macrophages contribute to all three stages of wound healing, by exhibiting pro-inflammatory ‘M1-like’ states and anti-inflammatory ‘M2-like’ states. One reason for DFU formation is a failure to switch between the M1 and M2 states. Toll-like receptors (TLRs) play a key role in triggering inflammation, and as such are a potential therapeutic target. I found that one of the TLRs, Tlr4, is expressed at a slightly lower level in diabetic-derived macrophages, compared to non-diabetic-derived macrophages. However, there was no difference in cell-surface Tlr4-Md2 expression. Early analysis of other TLRs also exhibited differential expression between non-diabetic and diabetic macrophages. Further analysis of these TLRs will be continued in future. Finally, diabetic mice with a haematopoietic cell-specific knockout of TLR function had delayed wound healing. These results demonstrate that TLRs may play an important role in DFU formation. Future work will focus on what impact aberrant TLR signalling has on epigenetic changes, through analysis of their downstream signalling target genes.

ABSTRACT 80

Name Roisin Holmes


Year of Study 3

Title Green Light Initiated Collagen Hydrogels as Cell-hydrogel Constructs in Regenerative Medicine

Authors R. Holmes, G. Tronci, X. Yang and D.Wood

Key words Biomaterials, Tissue Engineering

Abstract

The microenvironment design can possess features that induce appropriate gene expression and thereby direct cell fate as a result of cell-matrix interactions. Collagen hydrogels can be compared to the natural, hydrated tissues in the body (ECM) with a microenvironment largely dependent on the degree of functionalization, cross-linker type, chemical net-point reactivity, cross-link density and porosity. Green light-induced gelation was performed using eosin Y as the photo-catalyst and triethanolamine (TEOA) as a sacrificial reducing agent (539 nm). This was compared to UV light-induced gelation with Irgacure 2959 (365 nm) as the initiator. The physical and mechanical properties of the hydrogels were investigated by compression, swelling and degradation tests. ATP assays were used to examine cell viability in gels after 1, 3 and 7 days and LIVE/DEAD stains with confocal microscopy examined cell encapsulation and cell spreading with dental pulp stem cells (DPSCs). Cell viability in response to UV light resulted in a third less viable cells than when exposed to green light. The mechanical and physical properties were varied by changing the initiator type and the concentration of the prepolymer solution.

ABSTRACT 81

Name Ruth Coe


Year of Study 3

Title Simulation of the Fatigue Performance of Spinal Vertebrae after Fracture

Authors R.H. Coe, S.N.F. Sikora, D.C. Barton and R.K. Wilcox

Key words Vertebroplasty, Fatigue, Finite Element

Abstract

Over 25% of the population are expected to suffer a vertebral fracture during the course of their life. These are often caused by osteoporosis and are particularly seen in post-menopausal women. Treatment options for vertebral fractures can range from analgesics to surgical interventions, such as vertebroplasty and kyphoplasty. This is the injection of bone cement into the vertebra to stabilise the fracture. There is still considerable debate over the efficacy of cement augmentation, and further investigation into the mechanisms behind the treatment and the longer term effects could increase understanding of its suitability.

The aim of this project is to investigate the fatigue performance of spinal fractures treated with vertebroplasty through a combined experimental and finite element approach. This work will inform on the suitability of vertebroplasty for different patient groups.

Using a bovine tail model, a new method has been developed to fatigue test vertebrae, with loading levels based on the fracture strength. This work will form the basis for fatigue testing augmented vertebrae. In addition, specimen-specific finite element models of the specimens have been developed and validated against the experimental data, showing good levels of agreement.

The fatigue tests of augmented vertebrae will be used to validate equivalent finite element models. These can then be used to investigate the effect of vertebroplasty on specific patient groups.

ABSTRACT 82

Name Sam Beckett


Year of Study 4

Title ADSC Delivery to Chronic Skin Wounds

Authors S. Beckett, S. MacNeil and S. Matcher

Key words ADSC, Skin Wounds

Abstract

Adipose derived mesenchymal stem cells (AD-MSCs) are being investigated as a potential therapy for chronic wounds as they are pro-angiogenic and have anti-inflammatory effects both in vitro and in vivo.

Current attempts to transport cells from a GMP certified lab to the clinic often fail to keep cells viable for more than 24 hours, this is due to lack of temperature control or pH instability of the cell media. Maintaining the pH of cell media by use of a 5% CO2 gassed container can retain cell viability for up to 3 days when out of incubated conditions. Gassing cells with 5% CO2 in combination with a fibrin gel is being investigated as a method of delivering cells to dermal wounds.

Working with a GMP certified lab we have shown AD-MSCs retain stem cell markers up to 3 days and are viable using FACS analysis, when cells are brought out of incubated conditions, and show cells migrate through dermal tissue after application. It is our aim that we can culture cells in a GMP certified lab before delivering cells to patient in an easy to use fashion without any need for surgical expertise.

ABSTRACT 83

Name Sam Hollings

University University of York

Year of Study 2

Title Determining the Short Range Order of Strontium Bioactive Glasses with Neutron Diffraction

Authors S. Hollings, Y. Hancock, D. Wood, P. Genever and R. Martin

Key words Biomaterial, Bioactive Glass

Abstract

Strontium doped apatite wollastonite (Sr-AW) glass-ceramic is a promising bone tissue engineering scaffold because of its strength and the established bone regenerative effects of strontium, however it is not known how the addition of strontium affects the structure of the glass and thus its mechanical and properties and how it responds to physiological fluids is unknown. This is difficult to determine a priori, hence neutron diffraction, MAS-NMR and Raman spectroscopy, along with isomorphic substitution analysis were used to determine how Sr affects the short range order of six different compositions of SrAW glass with increasing strontium for calcium substitution. It was found that the atomic spacings largely agreed with similar glasses, as did the silicon and phosphorus speciation, indicating a largely Q2 and Q3 Si-O network, with orthophosphate. The addition of strontium did not significantly alter the atomic spacings or speciation of silicon or phosphorus, suggesting that Sr simply replaced calcium and that the glass network was unchanged, with any residual changes a result of strontium's slightly increased radius. This is a promising result, and supports the continued development of SrAW glass, and derived glass ceramics, as tissue engineering biomaterials.

ABSTRACT 84

Name Sam Whitworth


Year of Study 2

Title Self-assembling Peptide Nano-apatite Hybrid Material for Dentine Mineralisation

Authors J. Kirkham, D. Wood and R. Davies

Key words Remineralisation, Nano-hydroxyapatite, Peptides

Abstract

Dentine Hypersensitivity (DH, “sensitive teeth”) is a condition which continues to bring pain and discomfort to many. DH is caused by movement of tissue fluid in the dentinal tubules in response to physical stimulation. There are a plethora of treatment strategies available to a DH sufferer but as yet no gold standard treatment has been produced.

The aim of this project is to determine the effectiveness of a biomimetic hybrid material which has the potential to promote dentine tubule mineralization. The strategy will combine a self-assembling peptide (SAP), which is designed to mimic the biological macromolecular structure found in mineralised tissues’ extracellular matrices, with nano-sized hydroxyapatite (HAP) seed crystals. Nano-HAP was synthesized by hydrothermal synthesis and characterized using electron microscopy, dynamic light scattering and X ray crystallography. Seeding density optimization has been completed, using a validated in vitro steady state mineralization assay developed in-house, with 50µg/ml being found to be the lowest concentration to produce statically increased mineralization compared to PGA control.

SAP-nano-HAP combinations will be tested in situ using human dentine and the extent of mineralisation within the dentine tubules assessed using a variety of advanced techniques including FIB-SEM, nano-CT and dentine permeability tests including fluid filtration.

ABSTRACT 85

Name Samira Sadat Hosseini Alghaderi


Year of Study 1

Title The Role of Deltex Modulation of Notch Signalling in Regulating Homoeostasis of the Drosophila Midgut

Authors S.H. Alghaderi, M. Wilkin and M. Baron

Key words Drosophila, Intestinal Stem Cell, Notch Signalling

Abstract

The adult Drosophila midgut is maintained by intestinal multipotent stem cells (ISC). These ISCs divide asymmetrically to produce one daughter cell, which maintains ISC self-renewal, and one enteroblast daughter, which terminally differentiates into either an absorptive-enterocyte or a secretory enteroendocrine cell. Notch signalling is an evolutionary conserved pathway and is known to regulate ISC maintenance and differentiation in the fly and mammal intestine. We found that Deltex, a mediator of Notch ligand-independent signalling could alter midgut homeostasis. We show that in an adult flies lacking Deltex protein, ISC divisions produce tumour-like un-differentiated cells that express a reporter for Notch signalling but remain adhered in a clump to the Delta expressing stem cell. As the fly ages, these Notch signalling cells, progressively take over the midgut. The onset of this phenotype can be accelerated by feeding dextran sulphate with a regime that does not noticeably impair the wild-type gut.

ABSTRACT 86

Name Shiraz Ziya


Year of Study 3

Title Assessing the Operational Readiness of the NHS for the Local Manufacture and Delivery of Autologous Cellular Therapies

Authors S. Ziya, N. Medcalf, A. El Haj, S. Roberts and K. Wright

Key words Operational, Manufacturing, Service Delivery

Abstract

Cellular therapies have proven regenerative capacity in many chronic diseases, each requiring different cell types. It is perceived that to enable widespread availability as a treatment option, the production of such therapies will require selection of an appropriate operational model to ensure successful delivery to patients. We suggest that cellular therapies will cause disruption to the existing medicines supply chain established for traditional pharmaceuticals.

The current research explores the possibility to establish cell therapy as a feasible and cost-effective treatment option, closely integrated within the clinic. We consider whether the local manufacture of an autologous cell-based therapy for knee cartilage repair would be feasible at other treatment centres within the NHS. For the study we consider the requirements of the service delivery, operation and resources in the context of delivering the autologous chondrocyte implantation (ACI) product. We take particular focus on the flow of information and activities through the ‘needle-to-needle’ process. Utilising principles from systems engineering, the fundamental activities that contribute to the delivery of the medicine to the patient, rather than the biological mechanisms and efficacy, will be better understood. Comparison with other sites undergoing studies at similar stages will help clarify the challenges experienced when establishing manufacturing facilities.

ABSTRACT 87

Name Soukaina Bahsoun


Year of Study 1

Biography

Research interests:

Stem cell biology

Cell therapy development

ABSTRACT 88

Name Steffan Llewellyn


Year of Study 1

Title Exploiting Nano-functionalised Graphene and Human Adipose-derived Stem Cells towards Directed Nerve Regeneration and Interfacing

Authors S. Llewellyn, A. Faroni, A. Francesco Verre, A. Vijayaraghavan and A. Reid

Key words Nerve Regeneration, Graphene

Abstract

The exploitation of stem cell technology is poised to become a novel therapeutic intervention for peripheral nerve injury. Adipose-derived stem cells (ASCs) originate from a ubiquitous source, with low patient morbidity, and can differentiate towards Schwann-like phenotype; cells that enhance axon recovery, making ASC therapy desirable 1. Stem cell differentiation can be influenced by nanoscale topography of artificial substrates mimicking extracellular matrix proteins 2. Graphene and its derivatives provides a desirable platform for topographical functionalisation, due to its large surface area and versatile chemistry 3. This project sets out to culture ASCs on nano-patterned graphene scaffolds, functionalised with desirable growth factors and peptide sequences to promote, as well as maintain a differentiated Schwann like cell state, with the aim of directing nerve outgrowth.

1. P. G. di Summa, P. J. Kingham, W. Raffoul, M. Wiberg, G. Terenghi and D. F. Kalbermatten, Journal of Plastic, Reconstructive & Aesthetic Surgery, 2010, 63, 1544-1552.

2. S. Martino, F. D'Angelo, I. Armentano, J. M. Kenny and A. Orlacchio, Biotechnology Advances, 2012, 30, 338-351.

3. G.-Y. Chen, D.-P. Pang, S.-M. Hwang, H.-Y. Tuan and Y.-C. Hu, Biomaterials, 2012, 33, 418-427.

ABSTRACT 89

Name Steven Ruck


Year of Study 3

Title Development of a Microcarrier-based Culture Process for the Expansion of Human Mesenchymal Stem Cells

Authors S. Ruck, K. Coopman, C. Hewitt, A. Nienow, B. Rees and M. Szczypka

Key words Microcarriers, Bioreactors, hMSCs

Abstract

With cell-based therapies moving towards commercialisation there is a drive to develop expansion technologies that can yield cells at the number required for clinical scale manufacture.

Microcarrier-based culture systems offer a promising scale-up solution to addressing this demand.

This project, in collaboration with Pall Life Sciences and Cook Regentec aims to evaluate, characterise and optimise a scalable, controlled microcarrier-based culture process for the expansion of human mesenchymal stem cells in xeno-free conditions.

ABSTRACT 90

Name Sumaiya Al-Hinai


Year of Study 2

Title Histological and Immunohistochemistry (IHC) Comparison of Mesenchymal Stem Cell (MSC) in Knee Osteoarthritis

Authors S. Al Hinai, D. McGonagle, E. Jones, T. Baboolal, and L. Jennings

Key words MSC, OA, IHC

Abstract

Introduction: Osteoarthritis (OA) is a biomechanically related progressive degenerative disease that results in cartilage fibrillation, fissuring, associated subcondoral bone (SCB) changes and joint inflammation of soft tissue. Mesenchymal stem cells (MSCs) are promising source for cellular therapy that can regenerate and restore cartilage however the effect of resident MSCs in OA development is poorly understood.

Aim: To compare histological features and distribution of CD271+ MSCs between medial (more damaged) and lateral (less damaged) areas of OA tibial plateaus.

Methods: Tibial plateaus from total knee replacement (TKR) for OA (n=7) were fixed at 10°C in 4% formalin and decalcified in EDTA. Decalcification was monitored by X-Ray sample flexibility and followed by another fixation. The samples were further dissected before wax embedding. Following embedding 5 μm sections were cut and slides were stained with H/E (for gross morphology) or immunohistochemistry with anti-CD271.

Result: Characteristic features of cartilage damage were observed in each sample. Medial areas showed fissures, flaking of cartilage and areas of complete cartilage loss. Cartilage depleted areas corresponded with area of SCB thickening.

Initial results indicate CD271+ MSCs are responding to cartilage damage and associated SCB changes.

Future work: CD271+ MSCs can be harvested from knee SCB for sorting, expansion and transcriptional profile analysis.

ABSTRACT 91

Name Thomas Kisby


Year of Study 1

Title In vivo Reprogramming towards Pluripotency to Enhance Tissue Repair and Regeneration

Authors T. Kisby, I. de Lazaro, M. Buggio and K. Kostarelos

Key words Cell Reprogramming, Pluripotency, Gene-therapy

Abstract

Somatic cells can be reprogrammed towards pluripotency in vivo through forced expression of four transcription factors; Oct3/4, Klf4, Sox2 and c-Myc (OKSM). Previous investigations in our laboratory have utilised plasmid pDNA to drive transient expression of OKSM in mouse liver and skeletal muscle in vivo, which resulted in the generation of pluripotent-like cells which appeared to differentiate and re-integrate into the tissues. However, limited reprogramming efficiency associated with poor pDNA uptake lead to only a modest improvement in regenerative capacity. We hypothesize that the use of more suitable vectors could provide more efficient gene delivery and in turn produce a more clinically relevant enhancement of tissue repair and regeneration. In this project, we aim to develop suitable viral and non-viral vectors for the delivery and transient expression of reprogramming factors in desired tissues in vivo. We will test such systems in clinically relevant models of injury or disease to demonstrate safety and efficacy in the form of functional recovery. With this strategy we hope to show that in vivo reprogramming can bring together gene and cell therapy to provide a clinically translatable strategy for enhancing tissue regeneration.

ABSTRACT 92

Name Trang Kim Nguyen


Year of Study 1

Title Engineering Biological Bridges to Reconnect a Severed Spinal Cord

Authors T.K. Nguyen, R.M. Ichiyama, S-P.Wilshaw, R.M. Hall, J. Kwok and J.L. Tipper

Key words Spinal Cord Injury (SCI), Spinal Cord Transection, Decellularised Peripheral Nerve Graft (DPNG)

Abstract

Spinal cord injury (SCI) creates immediate and long-term physical and emotional pain for 1,000 patients every year, in the UK. The poor intrinsic regenerative potential and inhibitory microenvironment of the lesion, after injury, has resulted in slow progress in the development of effective treatments.

This project seeks to enhance the regenerative process and recovery of sensorimotor function by use of a decellularised peripheral nerve graft to bridge complete spinal cord transections. In a combinatorial approach, the graft will be used alongside vertebral fixation, electrical stimulation, and chondroitinase ABC in a rodent model. Development of in vitro and in vivo assays will be used to test and optimise the regeneration conditions around the lesion. Also, electrophysiological, immunohistochemical, mechanical analysis, and functional rehabilitation techniques will also be considered.

Particular research challenges include the transfer of peripheral nerve repair research into the central nervous system and limited implementation of a vertebral fixation device in a rodent model. Thus, a bespoke device will need to be designed and biomechanically evaluated.

ABSTRACT 93

Name William Mitchell


Year of Study 1

Biography

Background: I am in the first year of the Loughborough/Nottingham/Keele CDT in Regenerative Medicine.

Research interests: My undergraduate degree is in electronics and computer systems engineering. I am interested in developing sensors for stem cell manufacturing.

ABSTRACT 94

Name Zach Welshman


Year of Study 1

Title Characterising Variations in Foot and Ankle Mechanics using Statistical Shape Modelling and a Novel Multibody Model

Authors Z. Welshman, C. Brockett, G. Chapman and A. Redmond

Key words Foot, Ankle, Modelling

Abstract

Problems associated with variations in foot posture and function affects a large proportion of the population ranging from the sedentary with a high body-mass-index to active athletes1, 2. Both “high-arch” and “flat” foot-types have been linked to acute lower extremity injuries and chronic conditions such as knee osteoarthritis3. The precise relationship between foot posture and pain remains unclear however.

The aim is to develop a method to characterise variations from normal in foot and ankle biomechanics, using a statistical shape model and a multibody model.

Statistical shape modeling has been used to segment structures and geometric variations in hip and knee images 4 but this technique has not been applied to the foot and ankle previously, due to the complexity of the multiple anatomical structures present.

Musculoskeletal multibody modeling has the potential to model internal pathological changes in foot and ankle mechanics. With inputs from medical imaging, motion capture, and force plate data, it is possible to characterise normal and potentially pathological variation in the foot and ankle with the potential for identifying future treatment targets.

References

1. Butterworth, P. A., et al. 2015 Gait Posture 41(2): 465-469. 2 Burns, J., et al. 2005 J Am Podiatr Med Assoc 95(3): 235-241. 3. Abourazzak et al. (2014) Open Rheumatol J 8: 96-99. 4. Baldwin et al., Computer methods and programs in biomedicine 2010 97 (3) 232-40.

ABSTRACT 95

Name Holly Wilkinson


Year of Study 1

Title Exploring the Role of Metal Ions in Wound Healing

Authors H.N. Wilkinson, K. Theis, P. Lythgoe, K. Mace, M.J. Hardman

Key words Skin, Wound Healing, Metals

Abstract

Our skin has evolved a tightly co-ordinated and efficient ability to heal wounds. As we age, or in conditions such as diabetes, this ability is attenuated. In fact, age and diabetes are major risk factors for developing chronic skin wounds that fail to heal entirely. While numerous studies have identified genes and proteins important for efficient wound healing, much less is known about the role of metals in normal and pathological repair. Here, we hypothesise that changes in the skin metallome (the global profile of metal ions) are functionally important for pathological wound healing. To test this we have used techniques such as ICP-MS to carefully profile the metal levels in skin and wounds from normal and pathological healing models. Our pilot data now suggest, for the first time, that alterations in the metallome are linked to pathological healing. Current studies are: i) testing the mechanistic role of specific metals in vitro and in vivo and; ii) exploring methods to restore metallome defects in pathological healing. Ultimately, this work will reveal new insights into the underlying causes of chronic wounds, and provide new avenues for future therapeutic exploitation.

epsrc and mrc cdts in tissue engineering and ......journal nanomedicine and sits on the editorial...

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