EPSRC Centres for Doctoral Training in Tissue
Engineering and Regenerative Medicine
Joint Conference 2015
Poster Abstracts
Year 1
University of Leeds
Conference Auditorium 2/Sports Hall 2
Friday 10th July 2015
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Sumaiya Al Hinai
University of Leeds
Year 1
Comparing mesenchymal stem cell (MSC)
abnormalities in knee and hip osteoarthritis
Sumaiya Al Hinai
Osteoarthritis, MSC, CD271 antibody
Osteoarthritis (OA) is a heterogeneous disease with a multifactorial
pathogenesis. A final common feature of disease is severe articular
cartilage loss, variable bone attrition and joint failure. Although hip
and knee OA appear to share many of the aforementioned features
it is noteworthy that hip joint disease typically progresses much
faster than in knee. There is increasing evidence that endogenous
multipotential stromal cells also termed mesenchymal stem cells
(MSCs) that have high proliferative potentials and the ability to
differentiate into bone and cartilage and to control osteoclast
activation, could be key players in the pathogenesis of OA.
The biology of native in vivo joint and bone MSCs could be critical
determinates in a dysregulated homeostatic repair mechanisms
between hip and knee joint disease in OA. Combining
histopatholgoical techniques with knowledge of native in vivo MSCs
allows for the MSC responses to be interrogated in both of these
joints.
Aim:
1. To investigate the functional characteristics of subchondral
bone MSCs and their role in the pathogenesis of
osteoarthritis in hip and knee.
2. To compare functional characteristics of subchondral bone
MSCs with synovial fluid MSCs and superficial cartilage
MSCs from the same donors.
The overarching hypothesis is that cartilage regeneration may by a
stem cell mediated “bottom up” or a “top down” down
pheneomonon.
The key cellular protagonists in these putative
processes may be the synovial fluid and superficial cartilage MSCs
or subchondral MSCs.
Methodologies used:
Histology and immunohistochemistry on EDTA-decalcified tissues to
study the topography of MSCs (using CD271 antibody); collagenase
bone tissue digest and purification of MSCs by cell sorting based on
CD271 marker, transcriptional profile of freshly sorted MSCs; clonal
MSC culture from bone and synovial fluid and superficial cartilage
followed by differentiation and osteoclast activation assay.
Benefits and Impact
Understanding the changes in MSCs function will improve our
understanding to the prognosis of the disease and help finding
better or preventive way of treatment. Using knowledge of joint
mechanics to augment endogenous repair processes.
ABSTRACT
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Abstract
Catherine Colquhoun
University of Leeds
Year 1
Investigating the role of ependymal cells in spinal cord
injury repair using a 3D hydrogel model
Catherine Colquhoun, Joanne Tipper, Richard Hall,
David Wood, Susan Deuchars, Jim Deuchars.
Spinal cord injury; ependymal cells; hydrogel.
Glial scar formation follows spinal cord injury (SCI), having both
beneficial and detrimental effects on patient recovery. It has been
found that the majority of the astrocytes responsible for this scar
formation are generated by ependymal cells (EC), which are the
neural stem cells found within the central canal of the spinal cord
and lining the ventricular walls in the brain. Ependymal cells have
also been shown to generate myelinating oligodendrocytes and
neurones, crucial to the repair of SCI.
It is believed that if these cells can be controlled within an SCI
site, to increase and direct axonal regeneration, improved clinical
outcomes may be achieved.
Various 3D models of the spinal cord currently exist, including an
advanced tissue engineering construct utilising collagen as
matrix. The response of cells in the collagen model are readily
monitored and the environment can be controlled to some extent,
however there are limitations. It is proposed that this project will
include the development of an alternative 3D advanced hydrogel
matrix that can be used as a homogenous, chemically defined
alternative to currently used gels in the construction of central
nervous system (CNS) models.
The aims of this project are to investigate the use of different
advanced hydrogels, with defined matrix stiffness and
functionalised with biological additives in order to enhance
ependymal cell attachment, and induce stem cell differentiation.
Investigation into the effect of matrix stiffness on stem cell
differentiation, neural cell phenotype and neuron-glial interactions
will be carried out.
It is envisioned that by understanding the effect of matrix
stiffness on stem cell differentiation in more detail this project will
lead to advances in neural tissue repair and regeneration. It is
also thought that with the addition of specific ligands we will be
able to direct and influence ependymal cells, as desired, for SCI
repair.
ABSTRACT
Name:
Gavin Day
University:
University of Leeds
Year of Study:
Year 1
Abstract Title:
Development of variable finite element vertebrae
models to predict the mechanical outcomes of
vertebroplasty.
Authors:
Gavin Day, Alison Jones, Ruth Wilcox
Keywords
Vertebroplasty, Modelling, PCA
Abstract
Vertebral fractures account for over 700,000 reported fractures each
year in the United States, more than hip and ankle fractures
combined. These fractures have many adverse effects, including
pain, loss of vertebral height due to its collapse and kyphosis, which
often results in degeneration of the vertebral disc and increased risk
of fracture in the adjacent levels. The use of vertebroplasty has
allowed large reductions in the pain experienced, especially in
patients with chronic symptoms and those with specific
characteristics. However, some controversy remains over outcomes
including which patient groups will benefit most. The project outlined
here has the potential to enable vertebroplasty outcomes to be
predicted in the clinical scenario, along with an understanding of the
patient characteristics that determine the success, allowing clinicians
to decide which patients vertebroplasty is most suited to. Finite
Element (FE) models of vertebrae can be used to acquire
information regarding failure prediction and patient characteristics,
however these models are usually derived from single patients and
therefore do not describe the general population. An option to
generate a range of models based on a large database of micro CT
scans comes from a mathematical procedure named Principal
Component Analysis (PCA). PCA allows models to be generated at
various standard deviations away from the mean, creating a range of
models that can describe the majority of the population, including
those at the extremes. The objectives of the project involve the initial
development of FE vertebrae models using the PCA tools followed
by validation against existing FE models and against experimental
test data. These new models can be used to examine how different
patient variables may affect the mechanical outcomes of
vertebroplasty and models representing longer sections of the spine
can identify the effects of vertebroplasty on adjacent levels.
ABSTRACT
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Abstract
Catherine Disney
University of Manchester
Year 1
High speed X-ray micro computed tomography for
characterising soft tissues and evaluating tissue
engineered constructs.
Catherine Disney, Judith Hoyland, Peter Lee, Michael
Sherratt
X-ray micro tomography, soft tissue imaging, dynamic
loading
Quality of life during old age is increasingly determined by structural
and mechanical changes in soft tissues such as arteriosclerosis of
blood vessels, fibrosis of the lungs, compositional changes in tendon
and intervertebral disc degeneration. Dynamic structural studies of
tissues have the potential to: i) increase our understanding of normal
function, ii) characterise localised pathological remodelling and iii)
assess the success of tissue engineering strategies and implanted
biomaterial.
Several tissue components and structures in soft tissues are optimised
to perform under physiological loading conditions. Mechanical
behaviour is directly related to the complex and sophisticated
microstructures created by constituent cells and extracellular proteins.
In general, traditional biological imaging techniques require the tissue to
be mechanically sectioned in order to visualise the internal
microstructure.
This research project focusses on developing high-speed imaging
methods to visualise the 3D structure of intact (non-sectioned) soft
tissues. Studies will be carried out using laboratory and synchrotron
source X-ray micro computed tomography (microCT). The developed
imaging techniques will be applied to a wide field of dynamic tissue
studies.
Micro-structural changes have been identified in unpressurised and
pressurised arteries using phase contrast laboratory microCT (Walton
et al., 2015). These arteries were fixed and paraffin embedded and so
this method is not suitable for dynamic loading. Visualising structural
changes during dynamic loading requires rapid scan times using a
synchrotron source. Advances are therefore required to obtain high
resolution imaging of unfixed tissues at high speed.
The intervertebral disc has been chosen as a clinically relevant model.
Structural degeneration causes low back pain which affects millions of
older people in the UK. Finite element analysis can be used to identify
strain heterogeneities and develop a refined model for evaluating
mechanical behaviour. The mechanical behaviour of physiologically
loaded healthy, degenerated and tissue engineered replacement IVDs
will be studied using the developed technique.
References
Walton, L. A., Bradley, R. S., Withers, P. J., Newton, V. L., Watson, R.
E., Austin, C. & Sherratt, M. J. 2015. Morphological Characterisation of
Unstained and Intact Tissue Micro-architecture by X-ray Computed
Micro-and Nano-Tomography. Scientific reports, 5.
ABSTRACT
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Abstract
James Holland
University of Leeds
Year 1
Development of sterilisation strategies for acellular
nerve grafts
James Holland, Dr Paul Rooney, Dr Stacy-Paul Wilshaw
Nerve, Sterilisation, Acellular
Peripheral nerve injuries are highly prevalent in the UK, resulting in long
term patient morbidities and disabilities and an associated economic
burden. Microsurgical implantation of autologous graft material is the
current standard intervention, however full functional restoration
through such procedures is rare. This can partly be attributed to a
limited supply of appropriate autograft material. Alternative materials for
repair have been considered, including nerve guidance conduits which
aim to promote tissue repair by facilitating directional growth of
regenerating axons through physical and chemical means. Whilst
synthetic materials (e.g. PLA, PCL/PGA) have frequently been
employed for this purpose resulting in a number of commercial
products, they are inadequate substrates for supporting cell growth and
therefore full restoration of function.
A scaffold produced through the decellularisation of peripheral nerve
tissue offers considerable potential as an alternative material for grafts,
demonstrated by clinical evidence supporting the use of decellularised
human nerves. One barrier to their widespread adoption is the
sterilisation of such scaffolds. Little is known about the effect of tissue
sterilisation methods on the biological and mechanical properties of
acellular extracellular matrix scaffolds. A process has been developed
to remove the cells from porcine peripheral nerves using low
concentration SDS. The composition and biomechanical properties of
the acellular nerves have been shown to be similar to fresh nerves,
indicating significant promise in this approach. 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. The hypothesis is
that one or more of the sterilisation strategies will maintain the
biochemical and biomechanical properties of the tissue. This study will
also aim to elucidate the effects of sterilisation methods on extracellular
matrices and cell attachment & differentiation.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
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Abstract
Kwan Choi Kwan (Raymond)
University of Leeds
Year 1
Engineering Mechanically Competent Constructs for
Cartilage Repair
Kwan Choi Kwan (Raymond)
Tissue engineering, cartilage, compressive loading
Cartilage defects affect a large proportion of the population
worldwide with osteoarthritis being the eleventh leading cause of
global disability in the 2010 global burden of disease report.
Approximately 10,000 people undertake surgical treatment due
to severe cartilage defects annually in the UK. Cartilage is
difficult to treat as it lacks the inherent ability to repair. If
untreated, cartilage is susceptible to further degeneration that
eventually leads to osteoarthritis. A number of surgical
techniques are currently available that attempt to treat chondral
lesions, but these treatments present limitations and have a high
risk of defect recurrence. Thus, there is a significant demand for
an effective solution. One possible solution is to implant a tissue
engineered construct as a direct, functional replacement for the
damaged cartilage. A new method has been recently devised
that uses compressive loading to produce cartilage with similar
mechanical and histochemical properties to native cartilage.
Utilising this method, this project aims to determine if there is a
specific range of cellular deformation that 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
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University:
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Abstract
Kenny Man
University of Leeds
Year 1
Pre-treatment of stem cells with HDAC inhibitors to
enhance bone tissue engineering efficacy in a bone
defect model in vivo
Kenny Man, Ronald Grigg, Lin-Hua Jiang, Xuebin Yang
Epigenetics, Osteogenic differentiation, HDACs
Our continuously ageing population suffers from bone damage
caused by trauma, cancer, congenital defects or common ageassociated diseases, such as osteoporosis. The ability to generate
new bone is still a major clinical need.
The key for bone tissue engineering strategies is to effectively
control and divert stem cell differentiation down the osteogenic
lineage. One of the steps for controlling stem cell specific gene
expression is the control of the coiling and uncoiling of DNA around
histones.
Histone deacetylase (HDAC) proteins play a key role in epigenetics
and their inhibitor compounds (HDACi) are well researched for
cancer treatments. HDACi may have the potential to control and
direct stem cells differentiation. Novel HDAC3 selective inhibitor
MI192, has proven its potential in leukaemia, and rheumatoid
arthritis treatments. Inhibition of HDAC3 is linked to osteogenic
differentiation.
This project aims to optimise the pre-treatment condition of MI192
on stem cells both in vitro and in vivo to enhance the efficacy of
bone tissue regeneration.
We will also utilise different in vitro (such as MicroTissue system)
and in vivo models will be used for this study. The MicroTissue
system enhances the cell seeding efficiency on scaffolds creating a
construct with a higher osteogenic potential compared to
conventional seeding methods. In vivo we will evaluate the
repairing/of critical bone defects using HDACi pre-treated stem cells
and 3D porous scaffold in a long bone defect model.
Sample
evaluation
will
utilise
histology,
biochemistry,
immunohistochemistry, RT-PCR, FACS, confocal microscope, SEM,
microCT.
The pre-treatment of stem cells with HDACi to enhance
osteogenesis is a novel solution for bone regeneration that could be
patented and clinically translated, directly improving patient’s quality
of life and help to relieve the related social and economic burden on
our society.
ABSTRACT
Name:
University:
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Keywords
Abstract
Katrina Moisley
University of Leeds
Year 1
Membranes for delivery of mesenchymal stem cells for
complex fracture repair
Katrina Moisley
Bone regeneration, MSCs
Clinically, bone regeneration is limited by a lack of
containment of grafted material to the site of fracture.
However, it recently has already been shown that the
naturally induced periosteal membrane holds great potential
for fracture repair as a rich source of mesenchymal stem
cells (MSCs). Using this knowledge, our study aims to
characterise the naturally induced membrane, as well as
engineer a biomimetic membrane to act as a guided
regeneration device. For optimal regeneration, membranes
need osteogenic MSCs and growth factors which can be
sourced from both bone marrow aspirate and platelet rich
plasma (PRP). MSCs will be cultured from bone marrow
aspirates whilst immunohistochemistry will be used to
characterise the naturally induced membrane. Three
prototype collagen membranes will be developed and their
properties such as biocompatibility will be compared
against a commercial membrane. The membranes will then
be loaded with bone marrow aspirate or PRP and MSC
attachment and proliferation in long-term culture will be
compared to conclude which is the optimal combination.
This project will contribute to the development of a novel
membrane to improve complex fracture repair which will
reduce recovery times for patients as well as provide an
alternative solution to orthopaedic surgeons.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Halina Norbertczak
University of Leeds
Year 1
Evaluation of the decellularisation process for the
intervertebral disc for disc repair
Halina Norbertczak, Ruth Wilcox and Eileen Ingham
(Institute of Medical and Biological Engineering, University of
Leeds, Leeds, LS2 9JT)
Keywords
Decellularisation, Intervertebral Disc
(and Tissue Engineering)
Abstract
The intervertebral discs, located between the vertebrae of the
spine, play a vital role in load transmission and joint articulation.
Disc degeneration occurs as a result of the natural ageing
process, trauma or a combination of the two. As a result the
mechanical properties of the discs deteriorate and back, neck
and referred pain may result, adversely impacting on the
individual’s quality of life. Current treatments such as surgical
fusion of the vertebrae and total disc replacements 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 cellular components of native tissues, leaving an
acellular extracellular matrix scaffold which is non-immunogenic
when implanted into the recipient. The use of acellular scaffolds
has been successful in the replacement of defective heart valves
and in vascular repair.
This project aims to extend the decellularisation technology to
the production of acellular xenogeneic intervertebral discs for the
future treatment of human degenerative discs. 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.
A suitable size matched
animal model will be selected and a non-damaging extraction
method will be employed. A decellularisation protocol will be
developed utilising a combination of physical and chemical
treatments. The successful protocol will be reproducible, remove
all cellular content and will not significantly alter the biological
and mechanical properties of the tissue.
Qualitative and
quantitative tests will assess protocol effectiveness; biochemical,
histological, immunohistochemical and biomechanical tests will
be used to achieve this. In vitro biocompatibility testing will be
carried out to assess the potential for recipient host cells to
infiltrate the implant.
ABSTRACT
Name:
University:
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Abstract
Heather Owston
University of Leeds
Year 1
Building New Bone and Periosteum Using Collagen
Based Scaffolds
Bone, Periosteum, Scaffold
Critical size bone defects following severe trauma or post cancer
treatment remain difficult to treat. Current methods include the
‘Masquelet’ technique, a two step procedure resulting in the
defect being surrounded by an ‘induced periosteum’ membrane.
An alternative method could involve a collagen-based membrane
enclosing the defect area, mimicking periosteum and acting as a
containment factor for a synthetic scaffold loaded with the
patient’s bone marrow, a source of regenerative mesenchymal
stem cells (MSC).
The project aims are to investigate
interactions between MSCs, a bone scaffold and a collagenbased membrane. Followed by further examination into the
different loading methods of the MSCs onto the scaffold and the
effects this has on the membranes colonisation and maturation to
resemble native periosteum.
Samples of bone marrow and
periosteum will be expanded and co-cultured with a bone
scaffold. Optimisation of loading of the bone scaffolds with bone
marrow will be carried out using uncultured samples, with or
without allowing fibrin clots to form. Assessment of cellular
attachment, differentiation and proliferation onto the bone
scaffold will be carried out using flow cytometry, scanning
electron microscopy and confocal microscopy. Based on the
outcomes of histological evaluation of periosteum, collagen
based membranes of similar architecture will be produced, using
wet spinning. Co-culture of bone marrow loaded bone scaffolds
with the membrane will be used to investigate the colonisation
dynamics of the membrane. Histology will then be used to
compare the ‘neo periosteum’ membrane to normal periosteum.
This research hopes to lay down the framework to developing a
new one-step surgical technique for the treatment of critical size
bone defects, whilst reducing surgical time and the need for
further surgeries.
ABSTRACT
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Abstract
Jayna Patel
University of Leeds
Year 1
Extending the Lifetime of Hip Replacements: Is a
silicon nitride coating the solution to biocompatibility
concerns?
Jayna Patel
Hip Replacement Coating
Adverse biological responses to wear debris generated by Total Hip
Replacements (THRs) limit the lifetime of such devices. This has led
to the development of coatings for application onto metal THRs.
Silicon nitride (SiN) is a ceramic that has been investigated for use
as a coating due to high wear resistance of the material, a
favourable biocompatibility profile, and the unique property of any
resultant wear debris to dissolve.
The aim of this project is to assess the use of SiN as a coating for a
metal THR in vivo. Specifically, the project aims to characterise
relevant wear debris, as part of an investigation into biocompatibility
of the coating. A novel method to isolate SiN wear debris from
tissues using density gradient ultracentrifugation will be developed
and used.
Thus far, the method has been used to successfully isolate SiN
particles from 25% foetal bovine serum (FBS), with a recovery rate
of up to 94%, and low levels of contamination. Some variability in
recovery rates has been observed due to the method of using
filtration to assess particle recovery. The particles have been
imaged before and after isolation using scanning electron
microscopy (SEM), and characterised using ImageJ software.
Particles appear similar in size and morphology, though slight
changes may be due to particle dissolution during the isolation
procedure. This method is currently being adapted for particle
retrieval from tissue. Clinically relevant cobalt chrome wear debris
have been generated using a pin-on-plate rig and visualised using
SEM, and these will be used alongside SiN particles for isolation
experiments from tissue, to validate the efficacy of the method for
both types of material, since cobalt chrome will be employed as a
control material.
Later in the project, tissues from in vivo studies will be used to
assess biocompatibility histologically.
ABSTRACT
Name:
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Abstract
Katherine Pitrolino
University of Nottingham
Year 1
Development of a Biphasic Osteochondral plug for
Joint Repair
Katherine Pitrolino
Osteochondral, Scaffold Mesenchymal
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 using process mapping
techniques, which help identify the key process stages and
highlight areas of variability to produce a more robust and
repeatable output
The current area of focus is around a successful regulatory
approval. This involves improving the scaffold toxicity and
creating robust end product testing techniques.
Cell viability has been improved by changing the polymer
cross-linker.
Luciferase testing using a transgenically
modified reporter cell line has quantified osteocalcin
production to support bone differentiation from human
mesenchymal stem cells up to day 14. This test has also
demonstrated a difference between human serum and fetal
calf serum indicating further work is needed to remove
xenogenous factors from the process.
ABSTRACT
Name:
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Abstract
Georgina Webster
University of Leeds
Year 1
Development of acellular allogeneic nerve grafts
Georgina Webster, Paul Rooney, John Haycock, Stacy-Paul
Wilshaw
Acellular, nerve,
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, normal sensory and motor function is
rarely restored, leading to prolonged disability. The poor
outcome reflects microsurgical failure to adequately
address nerve regeneration at the cellular level, as
insufficient autograft is available for major reconstruction.
Implantable nerve guides direct regenerating axons by
topographic guidance, and synthetic materials have been
studied extensively for this purpose, including PLA,
PCL/PGA and PLA/PGA. Although these materials have
appropriate breakdown rates they are not particularly
suitable substrates for supporting neuronal and Schwann
cell growth. The use of a decellularised peripheral nerve
scaffold offers potential as an alternative to both autografts
and nerve guides. This has not been considered in detail
but some clinical evidence supports the use of
decellularised human nerves (Avance® nerve graft).
However, the ability of the Avance® scaffold to support
axon regeneration is unknown. Such a strategy using
acellular allogeneic nerve tissue would address
regeneration of proximal axons at the cellular level, while
simultaneously encouraging proximal regeneration towards
the distal stump within a native guidance environment. The
aim of the project is to develop physically compatible, nonimmunogenic nerve grafts to restore sensory and motor
function following injury. This will be achieved using novel
yet simple proprietary techniques to decellularise allogeneic
peripheral nerve. It will then be used as a basis for the
study of perfused flow within the tissue for the introduction
of primary Schwann cells versus adipose-derived stem cells
as the delivery of such cell types is reported to improve
nerve cell development. Key questions can additionally be
asked using this as a model, including the influence of a
native 3D environment on cell development and stem cell
differentiation.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
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Abstract
Aaron Zammit-Wheeler
University of Leeds
Year 1
Enrichment of Autologous Minimally Manipulated Stem
Cells using Biosensor-based Technology
Aaron Zammit-Wheeler, Jennifer Kirkham, Christoph
Wälti, Giles Davies, Michael McPherson
Biosensor Stem Cells Microfluidics
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 coupled
microbeads. 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. The novel solution to sort these cells
from tissue, which would otherwise be discarded, offers
great clinical benefit with minimal effort. 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.
EPSRC Centres for Doctoral Training in Tissue
Engineering and Regenerative Medicine
Joint Conference 2015
Poster Abstracts
Year 2
University of Leeds
Conference Auditorium 2/Sports Hall 2
Friday 10th July 2015
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Imran Mohammed Asif
University of Leeds
Year 2
Isolation of very low volumes of zirconia-toughened
alumina ceramic particles from serum lubricant using
density gradient ultra-centrifugation
Imran M. Asif, Sophie Williams, John Fisher, Mazen AlHajjar, James Anderson, Saurabh Lal, Joanne L. Tipper
Ceramic; Wear; Isolation
Understanding wear particle characteristics and their biological
activity is an essential step for the pre-clinical testing of joint
replacements. Many studies have reported the wear performance
of the latest composite ceramic-on-ceramic (CoC) total hip
replacements such as BIOLOX delta, however very few studies
reported the characteristics and biological response of the wear
debris generated from these bearings. Current particle isolation
methods lack the sensitivity to isolate very low wear volumes
such as those produced by BIOLOX delta CoC bearings. The
aim of this study was to assess and validate the sensitivity of a
newly developed ceramic wear particle isolation method for the
isolation of very low volumes of ceramic wear particles from hip
simulator lubricants.
Foetal bovine serum (25% (v/v) was spiked with a range of
volumes of ZTA particles (5mm3, 1mm3, 0.5mm3, 0.1mm3 and
0.05mm3), which were subsequently digested with proteinase K.
The digested serum was centrifuged on a sodium polytungstate
gradient at 270,000g. The ZTA particles were filtered onto preweighed 0.015µm filter membranes which were subsequently
dried and weighed to obtain the recovery weight of the ZTA
particles. The ZTA particles were characterised in terms of size
and morphology using high resolution field emission gun
scanning electron microscopy (FEGSEM), EDX analysis and
image analysis software.
The ZTA particles were recovered with an average percentage
recovery of >70% for all particle volumes, even from serum
spiked with very low volume of ZTA particles (0.05mm3).
FEGSEM analysis of the ZTA particles revealed a bimodal size
distribution, whereby the large polygonal shard-like alumina
particles were up to 3 microns in size and the small round
zirconia granular particles appeared to range from 10-140nm in
size. In addition, analysis of the characteristics of the ZTA
particles showed that the proteolytic digestion did not affect the
size or morphology of the ZTA particles.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Anne Canning
University of Nottingham
Year 2
Cell responsive interfaces for dynamic stem cell niches
Authors:
Anne Canning, Mischa Zelzer, Jon Aylott and Lee
Buttery
Enzyme responsive surfaces
Keywords
Abstract
Enzyme catalysed reactions are highly efficient and selective to the
substrate and operate under physiological conditions. They are
involved in many biological and metabolic processes, thus present
vast opportunities and uses as biological stimuli for the design of
responsive (bio)materials. They have been investigated in
applications such as drug delivery, diagnostics and biosensors. The
substrate, often synthesised from amino acids can be effectively
tailored to undergo a response in presence of the specific enzyme
through rational and sophisticated system design.
Diseased
biological systems often up/ down regulate certain enzymes which
can be used as targets for the ERM application. This work focuses
on surfaces and design routes to achieve the desired composition
and conformation in the substrate to elucidate a response on
enzymatic action focusing on phosphatases as a trigger. Here,
polymers containing serine (a target for phosphorylation) and
glutamic acid at different ratios have been successfully synthesised
and characterised in solution, by GPC, FTIR and NMR. Progress
has been made to obtain these polymers on surfaces via grafting to
and grafting from methods. Water contact angle (WCA), Time of
flight Secondary ion mass spectrometry (ToF SIMS) and atomic
force microscopy (AFM) techniques have been used to characterise
the modified surfaces. A relationship has been successfully
established between composition and structure and work is
underway to translate this onto a surface to study cell material
interactions.
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Philippa Clarkson
University of Leeds
Year 2
Mechanical Influences on Neural Stem Cells
Philippa Clarkson, Joanne Tipper, Giuseppe Tronci,
David Wood, James Phillips, Richard Hall
Hydrogel nerve cell
Stem cells are responsive not only to their biochemical
environment, but also to physical inputs. By changing the
stiffness of the substrate of mesenchymal stem cells, the
fate of the cells can be manipulated. This could provide a
means for better, targeted treatment. By encapsulating
neural stem cells within a 3D matrix, the present project
aims to control the fate of the cells to remain quiescent,
proliferate or differentiate.
Research to date has involved the creation of suitable 3D
matrices, crosslinking collagen fibrils to create hydrogels
and seeding cells onto these. The C6 glial cell line has
been used so far to determine the feasibility of using these
gels as stem cell niches. The mechanical properties of the
gels are characterised by measuring the viscoelastic
response to shear stresses. Future work seeks to optimise
the hydrogel networks, seed neural stem cells and monitor
the mechanical properties of these gels.
ABSTRACT
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Ruth Coe
University of Leeds
Year 2
Simulation of the Fatigue Performance of Vertebrae
after Augmentation
Ruth Coe, Sebastian Sikora, David Barton, Ruth Wilcox
Vertebroplasty, Finite element, Fatigue
It is estimated that over 25% of the population will suffer a
vertebral fracture over the course of their life. The most
frequently occurring of these are compression fractures,
which can be caused by osteoporosis or trauma.
Percutaneous vertebroplasty is a surgical treatment option
for compression fractures involving the injection of bone
cement into the fracture space to relieve pain and stabilise
the fracture. Despite this method being known to restore
mechanical properties of the vertebral body, there is still
considerable debate over the efficacy of the procedure. A
number of studies have been conducted to investigate the
mechanical properties of augmented vertebrae in vitro;
however these are typically short term or static loading
cases. Additionally considerable work has been done in the
development of finite element (FE) methods for modelling
the mechanics of vertebrae, particularly with respect to
changes caused by augmentation. It is important to
understand the longer term effects of vertebral
augmentation, so this project aims to investigate the fatigue
performance of vertebroplasty treatment through the use of
a combined computational and experimental approach.
Work so far has concentrated on developing an in vitro
large animal fracture model as a basis for further
experimental and FE work. Ovine vertebrae were dissected
individually and prepared with cement endcaps to provide
flat loading surfaces. Specimens were loaded centrally and
anteriorly up to 10kN for 10 cycles in an attempt to create
fractures. Using this method, specimens only displayed
elastic behaviour so consequently were pre-damaged with
a notch cut into the anterior surface. This allowed for
repeatable failure to be observed at approximately 5kN.
An existing method of creating specimen specific FE
models from micro CT scans has been used to develop prefracture models of ovine specimens. Stiffness was
compared with experimental stiffness values and local
errors of up to 20% were seen.
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Robert Cooper
University of Leeds
Year 2
Geometric Parameterisation for Computational Models
of Cam-type Femoroacetabular Impingement
Robert Cooper, Marlène Mengoni, Philip Robinson,
Sophie Williams, Alison Jones
FEA, Parameterisation, Femoroacetabular impingement
Abnormal geometry of the hip joint can lead to femoroacetabular
impingement, resulting in pain and joint damage. One such
geometric feature is a bony lump on the femoral neck, known as
a cam deformity. Cam hips can cause damage to the soft tissue
in the joint, including delamination of the cartilage from the
underlying bone. The damage mechanisms are not fully
understood and it is unclear why some cam hips cause pain and
damage while others do not.
Finite element models can be used to investigate the effects of
geometric variation in hip joints on their contact mechanics.
Image-based models utilising patient scans have shown high
contact pressures in areas corresponding with clinical damage,
but are time consuming to generate and analyse. Parameterised
models - more generic models incorporating simple geometric
features to capture key aspects of hip morphology - could be an
alternative. It is possible to automatically generate variations of
parameterised models and isolate the effects of individual
parameters by varying each parameter separately, whilst in
image-based models it may not possible to tell which aspects of
joint morphology cause differences in contact mechanics. It is
challenging however to identify the ideal set of parameters
capable of producing distinct subject-specific results.
In this work, an initial set of geometric parameters to describe
simplified proximal femur geometry, with cam deformity, have
been defined. Bone surfaces were segmented from pre-operative
CT scans of patients who underwent surgery for cam FAI. A
computational procedure was developed to systematically extract
hip geometry parameter values from these surfaces, and these
values were used to automatically generate parameterised
versions of the surface geometry.
ABSTRACT
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Matthew Dunn
Keele University
Year 2
A neural biomimetic model of the basal ganglia
Matthew Dunn, Munyaradzi Kamudzandu, Rosemary
Fricker, Paul Roach
Neural, microfluidic, circuit
Cell damage or dysfunction within the brain can result in a
number of debilitating and progressive neurodegenerative
disorders. One example is the basal ganglia: losing dopamine
neurons in this area is responsible for Parkinson’s and
Huntington’s diseases. However, the basal ganglia is deep within
the midbrain, and difficult to access without causing damage to
outer parts of the brain. Even so, 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 much easier to
study, but can a complex functioning circuit be created in vitro?
In this study, a five-port biomimetic device has been developed in
an attempt to replicate a simplified version of the basal ganglia
circuitry using different neural cell subtypes. By growing primary
cells from the striatum, SNc, SNr, cortex and globus pallidus, and
forcing them to connect from otherwise isolated ports via
channels large enough for axons, a controlled network can be
produced. The cells can be functionally assessed to determine
any electrophysiological activity within defined populations and
across the entire circuit. The complexity can be gradually
increased to introduce surface chemical coatings, physical
surface features (micro/nanotopographies) and surface
mechanical properties similar to those presented to cells within
the brain niche.
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Kyle Efendi
Loughborough University
Year 2
The creation of segmented collagen gels to replicate a
muscle-tendon-bone interface
Efendi KK, Player D, Martin N, Lewis MP
Collagen, MTB, musculoskeletal
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.
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Ama Frimpong
University of Leeds
Year 2
Preliminary in-vitro assessment of torque in total hip
replacements
Ama Frimpong, John Fisher, Mazen Al-Hajjar, Philippa
Withers, Sophie Williams
Hip, Torque, Simulator
Following recent reports of metal-on-metal hip replacement failure,
there has been a need for more extensive in-vitro hip replacement
testing to assess all possible failure mechanisms. There is currently
however limited understanding of the effects of frictional and offset
torques on the performance of total hip replacements (THRs). Invitro studies have been conducted on single axis simulators and
have limited clinical relevance. The aim of this study was to
generate preliminary data on a single axis pendulum friction hip
simulator for comparison with data from a multi-axis single station
hip simulator (SSHS) for the in-vitro investigation of frictional and
offset torques in total hip replacements. Friction tests were
conducted on 36mm metal-on-polymer (MOP, n=3) and 36mm
ceramic-on-ceramic (COC, n=3) bearings. Constant (1kN, 2kN) and
dynamic loads (2kN peak and 100N swing phase) were applied to
the bearings with a flexion/extension range of ±10º and ±25º. Tests
were run for 125 seconds and 1hour, at a frequency of 1Hz. 25%
(v/v) bovine calf serum was used as a lubricant.
For the short tests, friction factors were significantly higher for MOP
bearings than COC (p<0.05) for all loading conditions except for the
1kN constant load and ±10º of flexion/extension. The friction factors
were also generally lower under dynamic loading conditions than
under constant loading.
COC bearings however exhibited a greater change in friction factor
over an hour than the MOP bearings.
These preliminary tests have therefore created a basis for
comparison with data from the SSHS to ascertain its ability to
accurately detect torque in THRs.
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John Garcia
Keele University
Year 2
Comparison of the chondrogenic potential of human
donor-matched chondrocytes, bone marrow and
adipose derived mesenchymal stromal cells from
arthritic joint.
John Garcia, Claire Mennan, James Richardson, Sally
Roberts, Karina Wright.
Stem cells, cartilage repair
Autologous chondrocyte implantation (ACI) is a cell based
therapy option used to treat defects in articular cartilage.
However, donor sight morbidity is a potential risk associated with
harvesting healthy cartilage for ACI, which could contribute to
joint degeneration. As a result, cells obtained from easily
accessible alternative tissue sources such as bone marrow and
adipose tissue are currently being assessed for chondrogenic
potency.
Human chondrocytes, bone marrow mesenchymal stromal cells
(BM-MSCs), subcutaneous adipose stromal cells (SC-ASCs) and
infrapatellar fat pad adipose stromal cells (FP-ASCs) were
obtained from 4 donors undergoing total knee replacement. At
passage 3, cells were analysed by flow cytometry for MSCs
lineage markers, a panel of chondrogenic markers (CD44,
CD166, CD49c, CD271, CD151, CD39, FGFR3 and ROR2) and
the
immunomodulatory
marker
CD106.
Chondrogenic
differentiation was performed for 28 days in 3D pellet cultures,
after which toluidine blue staining was used to assess the
presence of glycosaminoglycans (GAG) in pellet sections.
Chondrocytes, BMSCs, SC-ASCs and FP-ASCs adhered to
immunoprofile criteria for MSCs. All cell types were constitutively
high levels of CD44 and CD15, while CD271, FGFR3 and ROR2
showed relatively low positivity. Interestingly, CD166, CD49c,
CD39 and CD106 were differentially expressed between cell
types and across donors. Histological assessment showed that
chondrocytes and FP-ASCs produced larger pellets with a higher
GAG content compared to BMSCs and SC-ASCs. Preliminary
analyses suggest that CD49c and CD39 immunopositivity on
chondrocytes and FP-ASCs may indicate enhanced
chondrogenic potency.
Our results indicate that FP-ASCs show the most promise as an
alternative cell resource for autologous cartilage therapies, due
to their apparent superior ability to produce GAG in pellet culture
compared to BMSCs and SC-ASCs. CD49c and CD39 are
interesting markers which may be used to select subpopulations
of cells with high chondrogenic potency.
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Carlos Granja
Loughborough University
Year 2
Anharmonic acoustic sensing for mechanical
characterization of cell-based therapies.
Carlos Granja, Sourav Ghosh
Process analytical technology
The cell therapy industry (CTI) is a rapidly growing
multibillion sector of healthcare. Similarly to other
biopharmaceuticals, CTI is encouraged by regulatory
authorities to adopt process analytical technology (PAT) to
monitor and control in-process product quality. This could
greatly impact different stages of product development,
leading to successful realization of cell therapy products.
The Anharmonic Detection Technique (ADT) has been
shown to complement traditional quartz resonators, widely
used for biosensing applications. This constitutes a
valuable opportunity to assess the performance of nonlinear
acoustic sensing in the context of manufacturing and
distribution of cell-based therapeutics.
This project envisages to explore the ADT by first
optimizing the detection of IgE-coated microbeads in an
immunoassay. The outcome of these experiments will
facilitate modelling of the detection parameters of
micrometre-sized bodies, to latter be implemented in
characterization studies of larger mammalian cells.
Moreover, the nature of the work required to fulfil the
objectives of the project opens additional technology
development streams that can meet close requirements
and specifications of other healthcare sectors.
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Sam Hollings
University of York
Year 2
The mechanism behind the induced osteogenesis of
MSCs on strontium bioactive glass scaffolds
Sam Hollings, Y. Hancock, David Wood, Paul Genever,
Apatite-Wollastonite, Strontium, MSC
New bone regeneration strategies are required due to the
inadequacies of the current gold standard (autografts) and more
widely due to the ageing population and elevated risk of
osteoporosis. There is evidence that strontium has bone forming
effects, and that it is able to encourage a patient’s own resident
mesenchymal stromal cells (MSCs) down a differentiation path
into becoming bone producing osteoblasts. This project aims to
test strontium apatite-wollastonite glass-ceramic (Sr-AWGC) as a
material to induce bone formation in human MSCs, and to
determine the intracellular mechanisms involved in the MSC
response. Initially, this will involve screening for the optimum
strontium content in the AWGC to induce osteogenesis. This will
use an immortalised MSC cell line developed at York, shown to
have good growth properties and trilineage differentiation
capacity. Various other techniques will be used to inform on the
biological mechanisms. One such is Raman spectroscopy, which
will be used to measure non-invasively the biomolecular content
of the MSCs allowing their differentiation on the various glassceramic compositions to be monitored. This will require the
spectra of both the MSCs and the Sr-AWGC to be taken and
interpreted individually as some features will overlap, making
their interpretation challenging, but enabling elucidation of the
cellular response to different strontium content
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Roisin Holmes
University of Leeds
Year 2
Investigation of collagen hydrogels as construct
materials for alveolar bone tissue engineering
Roisin Holmes, Giuseppe Tronci, Xuebin Yang, David
Wood
Collagen, Hydrogel, Bone
Periodontal diseases such as periodontitis can range in
severity from mild gum inflammation to complete
destruction of the periodontium. Functional reconstruction
of the alveolar bone is important for full restoration of the
periodontium. An autologous bone graft is considered to be
the gold-standard approach although it results in donor site
morbidity and the graft can be difficult to shape. Alveolar
bone tissue engineering has been promoted as an
alternative approach. The project aims to assess the effect
of hydrogel scaffold stiffness and microenvironment on cell
differentiation of dental pulp stromal cells (DPSCs) down
the osteogenic lineage. The recent collaborative work
between the School of Dentistry and the School of Design
at the University of Leeds has led to the preparation of
novel collagen hydrogels with high stiffness and
compressive modulus. So far in the project, this has led to
the preparation of functionalized collagen hydrogels with
compressive modulus of 28 ± 6 kPa similar to that of natural
pre-calcified bone. These hydrogels have been assessed
using a range of chemical, physical and mechanical testing
parameters and are to be used for in vitro cell assays to
examine differentiation and proliferation of DPSCs and to
assess the viability of the scaffold. This will eventually lead
into an in vivo subcutaneous mouse model. A favourable
stance with this construct material would be to create an
injectable hydrogel with sufficient mechanical properties
similar to that of pre-calcified alveolar bone
ABSTRACT
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Helen Jesson
Loughborough University
Year 2
Preservation Requirements for Cell
Therapy/Regenerative Medicine Commercialisation: A
Superficial Zone Chondrocyte Case Study
Helen Jesson, Rob Thomas, Nick Medcalf
Preservation, Chondrocytes, Commercialisation
One of the biggest challenges for the commercialisation of cellular
therapies/regenerative medicine products is securing the therapeutic
potential of the cells. This requires cellular preservation to be
managed throughout the manufacturing and distribution process in
order to ensure a quality and efficacious product is delivered to the
patient.
The project aims to provide effective modes of production and
supply for cellular therapies by improving the reproducibility and
reducing the constraints on the preservation steps and the
successful integration with a theoretical model of production for
industry-friendly practice.
By mapping the manufacturing process, as unit operations, the
areas that require biopreservation have been identified. These
areas can be divided into three categories: i) cryopreserved cell
banks, ii) inter-process pooling, iii) cell therapy distribution.
Reconstructive treatments for damaged diarthodial joints offer a high
value business proposition and due to the global market size,
commercialisation will be required. A side population of cells
resident in the superficial zone of articular cartilage, display ‘stemlike’ properties and once isolated, these hold great promise as a
suitable cell source for cartilage repair.
This work will define criteria for the characterisation of superficial
zone chondrocytes (SZCs) in order to identify variation between
donors and the effect of preservation on the critical quality attributes
(CQAs). Using this cell strain as a case study, the areas and
features of biopreservation that exert most influence over cost and
operability in the business model will be examined. The current
challenges associated with bedside processing will be examined
and the use of technology and alternative preservation solutions will
be explored. The suitability of process automation, scalability and
the successful integration into manufacturing facilities and the
supporting distribution network will also be investigated.
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George Joseph
Keele University
Year 2
Machine learning techniques informing surface
engineering for in vitro neural stem cell control
George Joseph, Paul Roach, Rosemary Fricker,
Theocharis Kyriacou
modelling, cell, responses
INTRODUCTION: The drive for this project is to generate enhanced
neural cell populations from stem cells through the study of the cellmaterial 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 variation. The many permutations of these factors pose
a major challenge in the optimisation of biomaterial design1. Here
we aim to use machine learning techniques to assess the impact of
surface properties on the biological micro-environment; protein
adsorption and subsequent mediation of cell interaction/response
will be used as outputs, with input variables being derived from
material properties such as surface chemistry (charge, density,
wettability, etc.) and topography (nano and micro-scale, aspect ratio,
etc)2.
METHODS: Glass surfaces were modified to present a range of
chemical moieties using self-assembly molecules. Surface
chemistry was characterised with FTIR and XPS analysis. Neural
stem cells from rat embryos were cultured as neurospheres and
then seeded on modified surfaces. Cell responses were investigated
at 3 time points using fluorescence and bright-field microscopy to
evaluate qualitative and quantitative differences. These included
neurosphere attachment area, neuron and glia counts and neural
cell morphology elongation. Prediction models were constructed
from observed surface properties and cell responses.
RESULTS: Co-variance tests between surface properties and cell
response variables indicated the existence of relationships. Glia
fibre length decreased as surface acidity (pKa) decreased. A
positive correlation was seen between surface characteristics and
neuron density, glial density and neurosphere area, which all
increased as the partition constant of tethered surface molecules
(logP) increased. Neurite length was inversely proportional to logP.
Neurite length and glia density were modelled with derived
estimates close to experimental results.
DISCUSSION & CONCLUSIONS: Some relationships between the
components of the cell culture system were uncovered and
described computationally. Predictions of some cell responses from
surface property data were made with good accuracy compared to
experimental results.
REFERENCES: 1 Roach, P., Parker, T. et al, (2010) Surface
strategies for control of neuronal cell adhesion: A review. Surface
Science Reports, 65(6), 145–173. 2 Roach, P., Fricker, R., &
Kyriacou, T. (2013). Computational methods for optimising stem cell
differentiation. 2013, 1–3.
ABSTRACT
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Elizabeth Kapasa
University of Leeds
Year 2
Bone Tissue Engineering in vitro and in vivo using a
Novel Multilayer Cell Sheet Technology
Elizabeth KAPASA1, Peter GIANNOUDIS2, Xiaodong
JIA3, Paul HATTON4, Xuebin YANG1
1
School of Dentistry, University of Leeds, UK
School of Medicine, University of Leeds, UK
3
School of Process, Environmental & Materials Engineering, University
of Leeds, UK
4
School of Clinical Dentistry, University of Sheffield, UK
2
Keywords
Abstract
Bone, regeneration, tissue engineering,
Introduction:
There is a major clinical need to restore damaged bone structure and
function. In 2020 the direct medical costs of osteoporotic fractures in
the UK is predicted to reach £2.2+ billion (1). Current treatments have
limitations, which has lead research to develop alternative approaches
for bone regeneration.
The aim of this project is to investigate the potential of using multi-layer
cell sheet (MLCS) technology and stem cell therapy to improve the
efficacy of bone tissue engineering.
MLCS technology provides intact monolayer sheets of confluent stem
cells that are simply detached by altering the temperature and stacked
together to make a 3D tissue graft.
Method:
Human dental pulp stem cells (HDPSCs) were cultured in temperatureresponsive dishes for up to 1-3 weeks to form a confluent monolayer
cell sheet. MLCS (3-6 layers) were assembled using various
stacking/handling techniques before further culture and subsequent
analysis.
Results:
1. Cell densities and various stacking/handling techniques were
explored and optimised including a gelatin gel stamp, a cell shifter
membrane and a polyglycolic acid mesh.
2. Live/dead labelling and fluorescent imaging revealed good cell
viability is maintained within the MLCS.
3. Two weeks post-culture in osteogenic and basal medium
respectively, alkaline phosphatase (ALP) staining showed a
stronger ALP positive staining in the osteogenic culture group
compared to the basal control. This was confirmed by ALP
biochemical quantitative assay (P<0.05).
Conclusion:
Current results have demonstrated the feasibility of using monolayer
cell sheets to assemble MLCS. HDPSCs within the MLCS are viable
after 3-6 weeks in culture and maintain their osteogenic phenotype.
These results indicate the potential of MLCS technology as an
efficacious cell-based therapy for bone tissue engineering.
Future work plan:
Bone tissue engineering in vitro and in vivo will be investigated using
MLCS with human bone marrow-derived mesenchymal stem cells and
in combination with biocompatible scaffolds.
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.
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Jessica Kirk
University of Leeds
Year 2
Development of a 3D spinal cord model to investigate
the role of the mechanical properties of the matrix in
spinal cord injury
Jessica Kirk, Professor Richard Hall, Dr James Philips,
Dr Giuseppe Tronci, Professor David Wood, Professor
Joanne Tipper
Spinal Cord Injury, hydrogels, matrix stiffness
The mechanical properties of the matrix are gaining recognition as
important factors for driving stem cell fate decisions and more
recently for affecting how cells behave in culture. We propose to
develop an advanced 3D hydrogel spinal cord multicellular model
that will be chemically defined and allow the matrix stiffness to be
modified.
The objectives of this study are to investigate a range of proprietary
and commercially available hydrogels to make a 3D in vitro spinal
cord injury model using a tethered gel system and primary rat cocultures of astrocytes, dorsal root ganglion neurones and microglia.
The mechanical properties of the model will then be modified by
physical and chemical means to measure the response of the cells
to changes in matrix stiffness and later to incorporate injury by
impaction.
The results to date indicate that hydrogels synthesised from natural
materials such as collagen or self-assembling peptides are more
biocompatible than synthetic hydrogels such as poly(ethylene
glycol). The natural materials have shown good cellular adhesion,
high cell metabolism determined by ATP-Lite assay over 3 days and
validated using live-dead staining. However, there are limitations to
the use of natural materials such as the difficulties in manipulating
the mechanical properties of the matrix, the derision from animal
tissues and therefore generally expensive to synthesise. Future
work will aim to develop a synthetic hydrogel functionalised with
natural molecules which will improve the biocompatibility of a
synthetic hydrogel whilst maintaining the ease of manipulation
mechanically.
This model has the potential to reduce the number of animals used
in drug testing and elucidate a potential therapeutic target to
manipulate the mechanical properties of the matrix to enhance
spinal cord nerve regeneration in vivo.
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Diana Maradze
Loughborough University
Year 2
Effect of Magnesium corrosion products on skeletal
tissue
Diana Maradze
Magnesium, corrosion, biocompatibility
Studies have shown that corrosion behaviour of magnesium
(Mg) alloys observed in in vitro studies is not comparable to
that observed in vivo. The aim of the current study is to
generate a knowledge base for correlation of carefullydesigned in vitro studies emulating in vivo environment to
increase in vitro predictability. Furthermore we aim to
understand the response of cells in skeletal tissue
including; osteoblast, myoblasts and mesenchymal stem
cells (hMSCs) to the corrosion of Mg biomaterial at local
and systematic level. Results have shown that the presence
of corrosion products in high concentration reduces cell
proliferation but increases osteogenic marker expression.
Cells are able to tolerate up to 16mM Mg ion concentration,
with the concentration between 9-10mM being favourable
for cell growth and proliferation. This data can be used to
build a knowledge base of magnesium corrosion behaviour
that would allow the design of Mg-based implants that can
function efficiently as orthopaedic implants.
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Pritesh Mistry
University of Nottingham
Year 2
Reconstituting organ level liver function by 3D printing
P. Mistry, A. Bennett, K. Shakesheff, M. Alexander, J.
Yang
3D Printing Liver
A major challenge in the discovery of new therapeutics is
the assessment of their toxicity. The liver is the major site of
drug detoxification and it is here where a large portion of
toxicity occurs. 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 reduced and decaying function, which is far from
comparable with in vivo data. One reason may be due to
their lack of 3D architecture. There is a clear necessity to
develop a 3D human liver model which can reliably predict
the toxicity in new therapeutics.
Bioprinted human liver models may be used to bridge the
gap between preclinical testing and clinical trials to reduce
attrition rate at expensive clinical stages. Furthermore,
these models may be used to aid the production of
extracorporeal liver devices or implants. The aim of this
project is to print a 3D human liver tissue which mimics the
in vivo liver architecture and demonstrates improved cell
survival, maintained state of differentiation and prolonged
liver-specific functions. This work will be achieved by (i)
screening an array of materials to find those that improve
hepatocyte survival and function, (ii) incorporating a
perfusable network that can sufficiently vascularise the
printed tissue, enabling the fabrication of larger liver
constructs, and (iii) patterning parenchymal and nonparenchymal cells to mimic the in vivo architecture.
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Diogo Mosqueira
University of Nottingham
Year 2
Modelling hypertrophic cardiomyopathy using
CRISPR/Cas9 genome editing technology in human
pluripotent stem cell-cardiomyocytes
Diogo Mosqueira, Viola Borgdorff, James Smith, Vinoj
George, Maria Barbadillo-Muñoz, Thomas Eschenhagen
and Chris Denning
CRISPR/ Cas9; Cardiomyopathy; hPSCCardiomyocytes
Hypertrophic cardiomyopathy (HCM) is a prevalent genetic
cardiovascular disease affecting 1:500 individuals whose cardiac
function is deteriorated due to thickening of the left ventricle of
the heart, mostly owing to mutations in sarcomeric genes.
Modelling HCM in vitro using human-pluripotent stem
cellcardiomyocytes (hPSC-CM) provides a pathophysiologicallyrelevant platform to further investigate the disease mechanisms,
ultimately enhancing drug discovery efforts.
Herein, we harnessed CRISPR/Cas9 genome editing technology
to introduce pathological mutations in the MYH7 sarcomeric gene
(coding for β-myosin heavy chain) in healthy hPSC lines. Guide
RNAs were designed to specifically direct Nickase Cas9 to the
MYH7 gene, towards the introduction of the R453C mutation, by
co-transfection with a targeting construct. Positive selection of
targeted cells was performed via antibiotic resistance, and the
selection cassette was thereafter excised by flippase
transfection.
We produced monoclonal hPSC lines either heterozygous or
homozygous for the R453C mutation in the MYH7 gene. PCRbased off-target analysis revealed that the genome editing was
performed with high specificity, and one of the lines also
displayed a frameshift mutation in the highly homologous MYH6
gene, thereby generating another interesting cell line where both
myosin genes are mutated. The generated hPSC lines will now
be differentiated into cardiomyocytes and cardiac function will be
analysed by comparing mutated cells with their healthy controls.
Phenotyping these cells will encompass integration in 3Dhuman
engineered heart tissues whereby contractility will be evaluated
in response to distinct pharmacological agents.
ABSTRACT
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Abstract
Oscar O’Dwyer Lancaster-Jones
University of Leeds
Year 2
Effect of implant positioning on edge loading
occurrence and severity
Wear, hip, ceramic
Edge loading, where the femoral head contact the rim of the
acetabular cup, has shown to be a factor which greatly impacts
the wear in hard-on-hard hip replacement bearings. There are
different factors that can contribute to the occurrence of edge
loading conditions.
The aim of this research study was to determine the effect of the
acetabular cup inclination angle with different levels of the
reconstructed head and cup rotational centre mismatch on the
occurrence and severity of edge loading and the resultant wear
rates in a hip joint simulator.
Ceramic-on-ceramic bearings (BIOLOX® delta, DePuy Synthes,
36 mm) were tested on the Leeds II hip joint simulator. Three
levels of mismatch (2, 3 and 4 mm) between the reconstructed
rotational centres of the head and the cup were considered in the
medial-lateral axis. Two acetabular cup inclination angles were
investigated; equivalent to 45° and 65° in-vivo. A total of six
conditions (n=6 for each condition) were tested for three million
cycles. The wear of the bearings were assessed gravimetrically.
Dynamic microseparation was measured using a linear variable
transformer. Mean wear rates and 95% confidence limits were
determined, and statistical analysis was performed using one
way ANOVA (significance taken at p<0.05).
The results demonstrate that with a higher level of centre
mismatch, the wear increased for all cup inclination angles.
However, under a steeper angle the severity of edge loading
increased significantly for the same level of centre mismatch in
comparison to the lower cup inclination angle.
The acetabular cups positioned at an inclination of 45° exhibited
greater resistance to dynamic microseparation for any given
mismatch condition medial-laterally. These results indicate how
different conditions can alter the severity of edge loading, and
highlight the necessity of understanding how the surgical position
can affect the occurrence of edge loading conditions and wear.
ABSTRACT
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Abstract
Laura Oakes
University of Leeds
Year 2
Using acellular liver extracellular matrix to study
hepatocyte differentiation and proliferation
Laura Oakes1, Stephen Griffin2, Raj Prassad3, StacyPaul Wilshaw1
1
School of Biomedical Sciences, University of Leeds;
2
Leeds Institute of Cancer and Pathology; 3School of
Medicine, University of Leeds
Liver, Decellularisation, cell therapy
In the United Kingdom, liver disease affects over two million
people and is the 5th most common cause of death. The
incidence of liver disease has increased by 40 %, due to
increased alcohol consumption, viral infection, and obesity. Liverassist devices and cell therapies have been proposed as bridging
therapies until effective transplants are found. These therapies
require large cell numbers and are limited by hepatocyte
differentiation.
The aim of this study was to develop an acellular porcine liver
scaffold to promote proliferation, prevent differentiation, and
maintain hepatocyte function.
A decellularisation process was developed utilising hypotonic
buffer and low concentration SDS in combination with nuclease
enzymes. This process was applied to defined cores taken from
different regions throughout the porcine liver.
H&E and DAPI staining of decellularised samples indicated
retention of native liver architecture and lack of nuclear
structures. DNA levels were reduced by >90 % (w/w) when
compared to native liver, the DNA content varied between all
lobes and locations therein. Contact and extract cytotoxicity
testing indicated these acellular scaffolds to be biocompatible.
Additional histological staining using Masson’s Trichrome,
reticulin, periodic acid schiff (with/without diastase) and
immunohistochemical labelling for Collagens I and III did not
reveal any changes in extracellular matrix composition.
This study characterised native porcine liver and was able to
demonstrate a significant variation in DNA content between
different liver lobes. Furthermore cores of porcine liver were
successfully decellularised using low concentration SDS in
combination with hypotonic buffers resulting in a biocompatible
acellular matrix, which had a similar histioarchitecture to native
liver.
Future work will focus on the interaction of a hepatocyte cell line
and primary hepatocytes with an acellular porcine liver scaffold.
ABSTRACT
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Abstract
Gizem Osman
University of Nottingham
Year 2
GET proteins for efficient gene transfer
Osman G, Denning C, Shakesheff K, Dixon J
A major scientific goal is the development of non-viral drug
delivery platforms for the delivery of exogenous DNA to cell
nuclei. Many of these technologies cannot overcome
challenges in low transfection efficiency, cytotoxicity and/or
serum-inhibition limiting in vivo efficacy. If these issues
could be resolved then powerful technologies such as in
vivo cell programming or gene correction could be
employed therapeutically. Cell penetrating peptides (CPPs)
are intracellular delivery vehicles that can 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
termed P21 LK15 8R. The reporter gene (pSIN GFP) was
delivered into cells. 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 serum-resistant transfection system that could
potentially be applied in-vivo which is not cytotoxic and
doesn’t affect proliferation/expansion of cells. We believe
that our system could facilitate new approaches for in vivo
cell programming, gene correction, and in regenerative
medicine.
ABSTRACT
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Abstract
Flora Ruiter
University of Nottingham
Year 2
A Design for Manufacture approach for Electrospun
Scaffolds.
FAA Ruiter; M Mather; C Alexander; JI Segal; FRAJ
Rose
Electrospun 3D scaffolds, Design of Experiments,
Optimisation
Electrospinning is an affected method to obtain 3D fibre
scaffolds for tissue engineering. However, little is known
about the interaction of the parameters used in this
process. A design of Experiment (DoE) optimisation
approach can provide this understanding and produce a
prediction of the process outputs in relation to the
parameter inputs. poly-lactic acid PLA electrospun scaffolds
for enzymatic-free cell culture passage purposes were
optimised with a DoE approach. Applied voltage, distance
nozzle-to-mandrel, flow rate and viscosity of the polymer
solution showed the most impact on the electrospinning
process and therefore were used as DoE inputs. Mean fibre
diameter and standard deviation were considered DoE
outputs. Three morphologies were observed during
preliminary studies, showing big beads with small strings,
beads-on strings and uniform fibres. It was assumed that
standard deviation (stdev) represent these different
electrospun scaffold fibre morphologies. This was
confirmed by SEM of different scaffold morphologies fibre
calculations. These showed a higher stdev (± 1200 nm)
when more beads-on-string formation were observed and
lower stdev (± 400 nm) for uniform fibres.
ABSTRACT
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Authors:
Alexandra Smyth
University of Leeds
Year 2
Wear of Total Ankle Replacements
Alexandra Smyth, John Fisher, Alison Traynor, Claire
Brockett
Abstract
Introduction
Total ankle replacements (TAR) are a much debated alternative to ankle
fusion for treatment of end stage arthritis. Compared with hip and knee
replacements these are implanted in small numbers with less than 500 per
year recorded by the joint registry for England and Wales. The small
numbers are a likely result of typically low mid-term survival rates, as well
as extensive contra-indications for surgery. There have been multiple
generations of TARs consisting of both constrained and unconstrained
designs but due to device classification pre-clinical testing has been
minimal.
Method
Five Zenith (Corin Group PLC), Titanium Nitride coated, unconstrained
TARs with conventional polyethylene inserts were tested in an adapted
knee simulator (Simulator Solutions, UK) for six million cycles (MC). The
input parameters were taken from available literature as there is no
recognised ISO standard in place. The range of motion included 30degrees
flexion, 10degrees rotation, 9mm displacement and a peak load of 3.1kN.
A parametric study with three conditions was conducted to understand the
impact of kinematic inputs on the polyethylene wear rate. These conditions
aimed to understand the effect of both linear wear with isolated flexion then
multidirectional motion by implementing a rotational input with and without
anterior/posterior (AP) displacement (Table 1). Each condition was run for
two MC.
Stage One: Flexion and Load
Stage Two: Flexion, Load, Rotation and Displacement
Stage Three: Flexion, Load and Displacement
A lubricant of 25% bovine serum, 0.03% Sodium Azide solution was used
to replicate the protein content of the natural joint capsule. The wear was
measured gravimetrically every million cycles and surface measurements
taken with a contacting profilometer.
Results
The wear tests showed that under solely flexion and loading there was a
low wear rate of 1.1±0.5 mm³/MC. With the addition of rotation and a 10mm
AP displacement in Stage two the wear rate increased to 25.8±3.1
mm³/MC. When the displacement was removed in Stage three the wear
rate decreased significantly to 15.2±2.5 mm³/MC (Figure 2).
Discussion
Wear of the TAR was shown to vary significantly with kinematic input. As
observed with other polymer total joint replacement articulations,
unidirectional motion of the ankle yielded minimal wear of the Zenith TAR.
Using an extreme anterior/posterior displacement motion significantly
increased the wear in combination with the rotation.
After these wear stages the TiN tibials show obvious signs of the region of
polyethylene contact. The counter surface of the polyethylene insert
showed both linear and radial scratches whereas as the curved surface of
the insert and the talar component solely showed unidirectional wear lines.
Conclusions
The design allows a large range of motion within the simulator. The wear
rate presented by the Zenith TAR is similar to previous tests and highly
dependent on the kinematic conditions.
ABSTRACT
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Abstract
Ashley Stratton-Powell
University of Leeds
Year 2
Determining the Failure of Total Ankle Replacements
Ashley Stratton-Powell, Anthony Redmond, Sophie
Williams, Joanne Tipper, Claire Brockett
Ankle; Osteolysis; Joint Replacement
Over the past 40 years, total ankle replacement has not
developed to the same extent as other lower limb joint
replacements. The anatomy is complex, the first two
generations of devices had poor clinical outcomes and little
is known about the mechanisms of device failure.
The multitude of factors associated with ankle replacement
failure make determining the initial cause of failure a
challenge. This project will retrospectively analyse the:
retrieved implant, surrounding tissue and medical images
for a consecutive case series of patients with failed total
ankle replacements.
Explant analyses include examining the surface
characteristics of both the fixation and articulating surfaces.
Topographic information may reveal damage modes
incident of failure.
Tissue analyses uncover the cell types surrounding the
device at the point of failure. Wear debris is known to
aggravate immunological responses, but is this relevant in
the ankle? Sensitive particle isolation methods will be used
to determine the probability of a particle-induced reaction.
The presence of osteolysis, a common problem in replaced
ankles, may undermine the device’s fixation integrity.
Medical imaging will be used to measure the extent of
osteolysis and how this relates the implants’ wear and
tissue characteristics.
This holistic approach to implant failure analysis will
comprehensively characterise the circumstances under
which failure was apparent; an approach to understanding
ankle replacement yet to be taken.
ABSTRACT
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Abstract
Samantha Swarbrick
Loughborough University
Year 2
MiRNA Biomarkers for Alzheimer’s disease
S. Swarbrick, Dr S Ghosh and Dr A Stolzing
Alzheimer’s Disease,
One milestone in Alzheimer’s research is to develop a more
accurate system for diagnosis or prognosis of Alzheimer’s. One
way in which this can be achieved is through valid biomarkers.
There is growing interest in utilising the changes in stable
circulating miRNA in blood as a biomarker. Therefore in order to
assess the specific miRNA de-regulated and the signalling
pathways they correspond with a literature search was
conducted using pubmed and web of knowledge. In a second
step the miRNA’s selected were assessed for their viability as a
biomarker by looking at their regulation to anticipate whether deregulation in blood can be reliably attributed to Alzheimer’s.
From the information gathered a schematic was produced which
highlights the key signalling pathways involved, the cells
attributed to these pathways and how miRNA can be deregulated by changes in these processes to become part of the
Alzheimer’s pathology. The pathological process generally
seems to involve amyloid beta activating microglia toll like
receptors which causes the release of pro-inflammatory
cytokines. The cytokines can cause oxidative stress and the
initiation of akt/mapk pathways causing tau hyperphosphorylation and neuronal apoptosis. The miRNA seem to
antagonise these processes by causing alterations in the
mediators involved.
ABSTRACT
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Abstract
Sam Whitworth
University of Leeds
Year 2
Self-assembling peptide nano-apatite hybrid material
for dentine mineralisation
Sam Whitworth
Remineralisation, Nano-hydroxyapatite, Peptides
Dentine Hypersensitivity (DH, “sensitive teeth”) is a condition
which continues to bring pain and discomfort to many, especially
those in the older age group as people keep their natural
dentition for longer. Dentine is the porous, bone-like tissue that
underlies the enamel crown of the tooth and covers the tooth
root. 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 and there is still a
clinical need to develop new, more effective DH treatments. The
aim of this project is to determine the effectiveness of a
biomimetic hybrid material which has the potential to treat DH
through promoting dentine tubule mineralization, occluding the
tissue pores. 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, which have been
shown to be effective at infiltrating the dentine tubules. SAPs
were designed and selected for this purpose based upon their
assembly behavior under physiological conditions and tested for
their ability to promote mineralization ± nano-HAP using an in
vitro steady state mineralization assay developed in-house.
Nano-HAP was synthesized by hydrothermal synthesis and
characterized using electron microscopy and X ray
crystallography. 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 and nano-CT as well as a dentine
permeability tests including ac-impedance spectroscopy.
ABSTRACT
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Abstract
Shiraz Ziya
Keele University
Year 2
WP1-2: Investigating the clinical utility of human
umbilical cord-derived mesenchymal stromal cells for
bone repair
Shiraz Ziya1,2, Karina Wright1, Claire Mennan1, Nick
Medcalf2 & Sally Roberts1
1
Spinal Studies, ISTM (Keele University), Robert Jones
& Agnes Hunt Orthopaedic Hospital
2
EPSRC Centre for Innovative Manufacturing in
Regenerative Medicine, Loughborough University
Umbilical, mesenchymal, osteogenic
Introduction
Umbilical cords (UC) represent a promising alternative source of
mesenchymal stromal cells (MSCs) for allogeneic off-the-shelf
therapies. UC-MSCs are ethically accessible, highly proliferative and
have an immune privileged status. Previous work in our group
showed that MSCs can be isolated from all regions of the UC. This
project aims to help transition these cells into the clinic by testing
their growth, differentiation and potency in alternative supplements
to foetal calf serum (FCS). The osteogenic potential of MSCs from
different cord regions will also be assessed with the aim of
evaluating the use of these cells for regenerating bone e.g. nonunion fractures.
Methods
UC-MSCs were isolated from vein, artery and Wharton’s Jelly (WJ)
and cultured as tissue explants. MSCs from whole cord-tissue were
isolated by enzymatic digest, cultivated in basal media (DMEM-F12,
10% FCS). Whole cord-tissue digest and WJ explants were
cultured in basal media supplemented with either 10% FCS,
autologous cord blood serum (CBS), or pooled human platelet
lysate (hPL; Stemulate, Cook).
UC-MSC and Bone Marrow (BM) MSCs were cultured in standard
osteogenic media for 7-14 days. Differentiation was assessed by
alkaline phosphatase (ALP) staining, gene expression (RT-qPCR)
and flow cytometry for relevant cell markers. Cell cycle analysis was
performed using flow cytometry on cell populations at each passage
and following osteogenic induction with the BD Cycletest™ DNA kit
(BD Biosciences).
Results
MSC-like cells were seen after ~1-2 days following enzymatic
digestion and after ~7 days from tissue explants. Preliminary data
showed higher proliferation in hPL cultures compared to FCS
cultures. Initial results from osteogenic-induced UC-MSCs suggest
that stronger ALP staining may correlate to higher levels of CD146.
This data will be combined with RT-qPCR to help identify
populations of cells more suited to the regeneration of bone.
EPSRC Centres for Doctoral Training in Tissue
Engineering and Regenerative Medicine
Joint Conference 2015
Poster Abstracts
Year 3
University of Leeds
Conference Auditorium 2/Sports Hall 2
Friday 10th July 2015
ABSTRACT
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Abstract
Joss Atkinson
University of Sheffield
Year 2
Functionalisation of Hydroxyapatite Surfaces using a
Covalently Coupled Biologically Active Peptide
Joss Atkinson, Piergiorgio Gentile, Paul Hatton &
Cheryl Miller
Bone, Peptide, Hydroxyapatite
Introduction
Bone tissue loss remains a significant challenge in medicine
today, affecting the quality of life of millions of people
throughout the world. Calcium phosphates such as
hydroxyapatite (HAP) are arguably the most successful group
of bone graft substitutes, but clinical performance is often poor
in ageing or compromised patients. While some progress has
been reported in the field of orthobiologics (e.g. the use of
bone morphogenic proteins), this approach is both expensive
and faces a more complex regulatory pathway. New strategies
are therefore being considered to enhance bone tissue
regeneration without the costs and risks associated with
orthobiologic substances. In this study we report progress with
the preparation of a medical grade hydroxyapatite that has
been enhanced with the anchoring of a heparin-bonding
peptide to the surface using carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) covalent coupling.
Materials and Methods
Gelcasting was used to prepare HAP discs. The as-sintered
HAP surface was characterised by X-ray diffraction (XRD) and
x-ray
photoelectron
spectroscopy
(XPS).
Plasma
polymerisation with acrylic acid was then used to generate
carboxyl functional groups on the surface. These functional
groups were then activated for covalent peptide attachment
using EDC/NHS. Peptide grafting at the surface of HAP was
investigated using XPS, and peptide release into distilled
water was characterised using reverse-phase high
performance liquid chromatography (HPLC).
Results and Discussion
XRD and XPS results revealed a high purity of HAP with the
remaining component material suggested to be β-tricalcium
phosphate. After plasma polymerisation, the HAP surface was
covered by a layer of polyacrylic acid, as confirmed via XPS
survey scans. Once the EDC/NHS treatment was applied,
XPS detected the presence of nitrogen (~3 At%). After peptide
grafting, XPS data suggested the presence of peptide as the
concentration of nitrogen detected increased (~7 At%).
Release studies showed that peptide retention was
significantly increased on HAP samples treated using plasma
polymerisation and activated for covalent coupling using
EDC/NHS.
ABSTRACT
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Abstract
Andres Barco
University of Leeds
Year 3
Rheological properties of peptide-based hydrogelglycosaminoglycan mixtures.
Andres Barco*, J. Fisher, E. Ingham, H. Fermor, R.P.W.
Davies
Cartilage, Peptides, Osteoarthritis
Peptide-based hydrogels are of high interest as a class of
biomaterials for their potential use in regenerative medicine.
Mixing these hydrogels with materials that may enhance
their properties, such as glycosaminoglycans (GAGs), has
the potential to extend their range of applications.
The aims of this study were to investigate the physical
properties of self-assembling peptide hydrogels based on
three peptides of the P-11 series in combination with
chondroitin sulphate using rheology.
The hydrogel mixtures of the three peptides were
investigated in two different salt solutions at a temperature
of 37oC, in order to determine their suitability for a range of
applications. Peptide alone, peptide in combination with
chondroitin sulphate at two different molar ratios and
chondroitin sulphate alone were investigated. A Malvern
Kinexus pro rheometer was used to carry out the
measurements. An amplitude sweep at two frequencies
(1Hz & 20Hz) was run to determine a suitable strain value
within the linear viscoelastic region (LVER). This strain
value was used to run a frequency sweep across a range of
frequencies (1-20Hz) to determine the elastic and viscous
modulus of the material.
The results indicated that all of the variables (peptide, salt
concentration, and chondroitin sulphate molar ratio) had a
significant effect on the mechanical properties of the
hydrogels. However, one of the peptide-hydrogel mixtures,
P11-8, showed greater mechanical strength in both salt
solutions and molar ratios when compared to other peptide
hydrogel mixtures. This peptide-hydrogel mixture will be
investigated further in glycosaminoglycan depleted model
tissues.
ABSTRACT
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Abstract
Sam Beckett
University of Sheffield
Year 3
A biodegradable delivery system for delivering adipose
derived stem cells to chronic wounds
Sam Beckett, Sheila MacNeil, Stephen Matcher
MSCs, Wound Healing
Introduction:
With an aging population and prevalence of diabetes the number
of patients with non-healing wounds is rising. Chronic wounds
severely impact the quality of life and carry a high amputation
risk. Conventional therapies are ineffective and expensive,
therefore there is a need for an alternative treatment.
Mesenchymal stem cells (MSC) are increasingly being used to
stimulate wound healing- they promote angiogenesis and
decrease inflammation.
Objective:
The aim of this project is to develop a biodegradable, easy to use
carrier system for delivering MSCs to promote wound healing.
An electrospun scaffold of PLGA was produced and a 3D human
skin wound model is being used to assess the effects MSCs on
wound healing in this model.
Methods:
3D tissue engineered skin (TE skin) was produced before
wounding. Wound healing was assessed by conventional
histology and also non-invasively by optical coherence
tomography following delivery of MSC cells. Biodegradable
electrospun scaffolds of PLGA were produced as a cell carrier.
Results:
The addition of MSCs to TE skin wound models increases the
rate of wound healing compared to controls. Additionally,
collagen production in MSC treated wounds increased. Wounds
not treated with MSCs showed no additional collagen production.
Biodegradable electrospun scaffolds of PLGA were produced
and MSCs were able to proliferate on these. These scaffolds
degrade within a couple of weeks and can be gamma irradiated,
vacuum packed and stored under sterile conditions for at least 2
years.
Conclusion:
Many studies now show that MSCs can increase the rate of
wound healing. We have established a human skin wound
model and methodologies for imaging wound healing noninvasively and shown that MSCs benefit wound healing in this
model and will grow well on biodegradable PLGA scaffolds.
Future work will focus on exploring the efficiency of cell delivery
to wounds and the rate of scaffold breakdown. We conclude that
the development of an electrospun biodegradable synthetic
scaffold presents a promising approach to the transport and
delivery of MSCs for chronic wounds for the promotion of wound
healing.
ABSTRACT
Name:
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Abstract Title:
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Abstract
Ayesha Bint-E-Siddiq
University of Leeds
Year 3
Mechanical Characterisation and Computational
Modelling of Spinal Ligaments
Purpose
The focus of this study is the spinal ligaments which provide
passive stability to spine and play a major mechanical role
within the physiological range of motion. However, existing
studies investigating their mechanical properties have
revealed large variation in behaviour depending on
specimen preparation and testing method. This study is
unique in combining experimental and computational
approaches to characterise the ligamentous spinal
structures and identify their importance in functional spinal
unit models (FSUs).
Experimental Work: Ovine thoracic spines were dissected
into FSUs and imaged under microCT using NaI gel to
visualise the soft tissue structures. A tensile testing
machine (3365, Instron, UK) was used to test the FSUs
under tension to obtain load displacement curves. Tests
were initially undertaken on the FSU with all the ligaments
attached, consecutively removing ligaments and retesting.
Further tests were undertaken on only the anterior section
of the FSU to minimise the effects of the facet joints.
Computational work: The image data from each FSU was
exported to an image processing package (Scan IP,
Simpleware, UK) which enabled the images to be
segmented and the volume of the ligaments to be identified.
A finite element (FE) mesh was generated based on direct
voxel to element conversion. Preliminary FE models of
ligaments were built in Abaqus (Simulia Corp, USA) to
assess attachment site replication and element types to be
used in future modelling, and to evaluate the mechanical
role of these ligaments.
Results
The tensile tests data was post-processed to derive the
load-extension curves and hence tensile modulus for each
ligament. Whole FSU results indicated that the outcome
was heavily controlled by the presence of facet joints.
Results from the anterior sections showed the characteristic
non-linear behaviour of ligaments for both the posterior and
anterior longitudinal ligament (ALL), with the ALL being
stiffer and stronger.
ABSTRACT
Name:
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Abstract
Joseph Collier
University of Leeds
Year 3
Meniscus Biomechanics and Extrusion Measurement
Joseph Collier, Martin Stanley, Eileen Ingham, John
Fisher
Meniscus, Extrusion, Biomechanics
The meniscus performs a critical biomechanical function in
the human knee joint; aiding stability, distributing forces and
shielding cartilage from excessive stresses. Meniscal injury
can affect all age groups, from sports related damage in the
young to tissue degeneration in the elderly. A common link
between traumatic and degenerative knee injuries is the
extrusion of the meniscus from the joint.
The cause and impact of meniscal extrusion have not been
widely studied in-vitro, but clinical observation and
assessment has linked extrusion to the development of
whole knee joint degeneration. Key to understanding the
process of extrusion further, its impact on joint
biomechanics and link to degeneration is a method of
reliably measuring meniscal extrusion in-vitro.
A method for measuring the extrusion of the meniscus from
a porcine knee joint, and measuring the loading of the tibial
plateau was developed. Specimens were mounted in a
uniaxial compression tester (Instron 3365), with custom
photo markers placed at the widest point of the medial
meniscus. A camera was mounted behind the knee joint in
line with, and focused onto the photo marker. The meniscus
was loaded with 500N for 1000 seconds, and the process
recorded on camera. Frames were extracted at set time
points to analyse the movement of the photomarker using
ImageJ software.
The aim of the test was to demonstrate the ability to track
the extrusion of the meniscus from the joint during loading.
Two states were investigated, one with the intact meniscus,
and one with the outer tension band of the meniscus
disrupted with an incision near the anterior horn
attachment.
Results demonstrated measureable extrusion of the
meniscus from the joint during loading of 0.5mm in its intact
state, and 1.5mm in the disrupted state; peak contact
pressure on the tibial plateau increased up to 0.9MPa.
ABSTRACT
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Abstract
Karl Firth
University of Nottingham
Year 3
Engineered pseudo 3-D hPSC-cardiomyocyte platforms
to detect cardiovascular safety liabilities
Karl Firth, Diogo Mosqueira, James Smith, Chris
Denning
Drug screening, engineered cardiac tissue
The development and screening of new therapeutic entities
is often a lengthy and costly process, where it has been
estimated to take between 10 – 15 years to bring a drug to
market at a cost of up to $1.8 billion. The pharmaceutical
industry also relies heavily on the use of animal models for
measuring drug safety liabilities, however these have been
shown to be unreliable. The ability to differentiate human
pluripotent stem cells (hPSC) into cardiomyocytes could
provide an alternative approach for drug testing without the
need for animal use. We are developing a pseudo-3D
muscular thin film (MTF) platform with tuneable elastic
properties and customisable surface micro-patterning for
use in drug screening assays. The platform will allow for
key parameters such as calcium imaging, electrophysiology
and contractile force measurement to be measured using a
single assay. Our aim is to develop clinically relevant
cardiovascular models that are not only cost effective and
will help to reduce the reliance on animal testing but will
deliver improved patient safety. Current work has focused
on developing MTFs with an elastic modulus which
replicates healthy (30kPa) as well as diseased (100kPa)
human adult cardiac tissue, optimisation of the substrate
micro-patterning has produced synchronised contraction of
anisotropically aligned cardiomyocytes. Future work will
focus on the development of an optical system and
associated software to allow for image movement analysis
as well as contraction force measurements.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Atichart Kwanyuang
University of Leeds
Year 3
Development of a Finite Element Model of Total Ankle
Replacement to Examine Bone Failure Following
Implantation
Atichart Kwanyuang, Qingen Meng, Claire Brockett,
John Fisher, Ruth Wilcox
Ankle, Computational Modelling
Globally, 1% of the population is affected by arthritis of the
foot and ankle. Total ankle replacement (TAR) was
developed as an alternative to arthrodesis to treat endstage arthritis, but failure rates are relatively high and are
often related to bony damage. Knowledge of the failure
mechanisms of TAR is limited to date, therefore, finite
element (FE) analysis may be useful for investigating the
damage to the bone surrounding the implant.
The objective of this study is to develop a FE model of a
TAR which is a sufficiently accurate representation of the
real-world for further use to examine the failure
mechanisms.
Zenith™ TARs (Corin, UK) were inserted into polyurethane
foam blocks (Sawbones, USA) representing the tibial and
talar bones. The constructs were loaded in an
electromechanical testing machine (Instron, USA) with
increasing compressive force until reaching plastic
deformation. A corresponding FE model was generated and
analyzed using Abaqus (Dassault Systèmes, France).
Experimental contact pressure, which was captured by
pressure mapping sensor (Tekscan, USA), and plastic
deformation data were used to validate the FE results.
Good agreement was found in the location of damage (the
plastic deformation of the synthetic bone was located at the
posterior region of the talar bone-implant interface) and the
depth of plastic deformation in the synthetic bone (average
experimental depth was 2.8% smaller than the
corresponding value obtained from the FE analysis).
Experimentally, there was some difference in deformation
laterally, which corresponded to a difference in contact
pressures measured on either side of the mid-plane,
illustrating that there was some degree of inclination
between the interfaces of the TAR. The good agreement
indicates the FE model can be used for further predictions
of bone failure. Importantly, the model will be used to
investigate clinically relevant misalignment, which this study
shows may have an effect on bone damage.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Panagiota Moutsatsou
Loughborough University
Year 3
Determining factors of the electrospinning window for
conducting PANI fibers
Panagiota Moutsatsou, Stella Georgiadou
Electrospinning, conductive polymers, process
parameters
Polyaniline doped with CSA / PEO conductive nanofibers
were produced by electrospinning. The electrospinning
window was determined by using a three level, full factorial
experimental design and the combined effect of the
humidity, voltage and flow rate was examined
demonstrating that the ambient humidity is the critical factor
affecting the electrospinning process and determining the
electrospinning window. Low humidity favors the formation
of defect free fibers while high humidity either hinders fiber
formation or causes the formation of defects on the fibers
either due to jet discharge or due to water absorption and
phase separation. The role of voltage and flow rate
variation on the diameter and morphology of the
electrospun fibers is discussed. High level of doping with
CSA led to the formation of crystalline structures. Data
fitting was used to explore the behavior of conducting
polymers in electrospinning and very good agreement
between experimental and theoretical predictions was
obtained for only a limited range of experimental conditions,
whereas deviation was observed for all other sets of
conditions.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Samand Pashneh-Tala
Sheffield
Year 3
Novel photocurable poly(glycerol sebacate) as a
material for degradable polymer scaffolds for tissue
engineered blood vessels
Samand Pashneh-Tala, Sheila MacNeil and Frederik
Claeyssens
Vascular, polymer, scaffold
Poly(glycerol sebacate) (PGS) has been successfully used
to produce polymer scaffolds for various soft tissue
engineering applications. In the in vitro production of tissue
engineered blood vessels, for use as vascular grafts, the
elasticity and rapid degradation rate of this scaffold material
has been associated with improved extracellular matrix
deposition rates and quality, particularly relating to elastin
fibres. However, manufacturing scaffolds from PGS
requires the application of high temperatures and extended
reaction times to crosslink and cure the polymer, limiting its
utility.
Here, we have developed a novel photocurable form of
PGS with improved processing capabilities: PGS-M. By
methacrylating the secondary hydroxyl group of the glycerol
units in the PGS polymer chain, the material is rendered
photocurable and can be crosslinked rapidly on exposure to
UV light at ambient temperatures. Our results have shown
that both the polymer’s molecular weight and the degree of
methacrylation can be controlled independently and that the
mechanical properties of the crosslinked material are
critically dependent on these two parameters. The polymer
has also demonstrated acceptable biocompatibility and
rapid degradation under physiological conditions.
Using a mould-based porogen leaching method, porous
tubular scaffolds have been produced from PGS-M with
suitable structure, porosity and mechanical properties for
use in vascular graft tissue engineering. There is also the
additional potential to generate bespoke scaffolds using an
additive manufacturing process. Future work is set to
examine these scaffolds in vascular graft tissue engineering
using a pulsatile flow bioreactor system.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Kinga Pasko
University of Leeds
Year 3
Silicon nitride coatings for cervical total disc
replacement
K.M. Pasko, R.M. Hall, A. Neville, J.L. Tipper
Cervical disc replacement, wear, particles, silicon
nitride
Surgical interventions for the treatment of chronic neck
pain, which affects 330 million people globally, include
fusion and cervical total disc replacement (CTDR). While
fusion is the current gold standard of surgical treatment, it is
often associated with degeneration of adjacent vertebrae.
Theoretical advantages of CTDRs are to decrease
abnormal biomechanical forces at adjacent segments,
thereby decreasing the risk of degeneration and need for
subsequent surgery. However, current CTDRs can be
associated with issues similar to those affecting other joint
replacement devices, including excessive wear and wear
particle-related inflammation. Currently, there is little known
about the characteristics of wear debris produced by CTDR
devices, and any potential adverse effects of the particles
on tissues and cells in the immediate vicinity of the spinal
cord. Therefore, there is a clear need for improved
materials and material combinations, as well investigations
focusing on the effects of wear particles produced by the
CTDR devices on the surrounding tissues and cells.
The project aims to investigate a novel silicon nitride
coating for CTDR applications, focusing on characterisation
of the wear and wear particles produced by coated devices.
Thus far, preliminary silicon nitride coatings deposited on
cobalt chromium were characterised in terms of mechanical
and compositional properties. The results have revealed
that these preliminary coatings demonstrated low surface
roughness and high hardness. The coatings demonstrated
columnar and amorphous microstructure, and consisted
mainly on Si-N bonds. Results of preliminary investigation
of biological responses of L929 murine fibroblasts to model
silicon nitride particles, obtained from ATP viability assay
showed no cytotoxic effects of the silicon nitride model
particles after 6 days of co-culture.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Joshua Price
Keele University
Year 3
The influence of cyclic hydrostatic pressure on in vitro
bone growth
Joshua Price, Prof Alicia El Haj
Bone, Pre-conditioning, hMSC
My project involves the study of cellular responses to in
vitro mechanical loading prior to implantation to treat bone
defects that do not naturally heal. We have characterised a
hydrostatic force bioreactor developed in collaboration with
Instron/TGT and found both 2D and 3D growth
environments respond to external hydrostatic forces by
increasing synthesis of key ECM components that
contribute to bone development. We have also developed a
fracture repair model using an embryonic chick femur to
test the hypothesis that mechanically preconditioned hMSC
seeded scaffolds can be used as effective treatments for
bone repair.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Preeti Puntambekar
Loughborough University
Year 3
Investigation of Corneal Cell Migration in Response to
Mechanical Stimulation
Preeti Puntambekar, Yang Liu, Pablo D. Ruiz
Cornea, migration, biomechanics.
Cells respond to mechanical changes in their extracellular
environment, as reflected by various cell behaviours and
observed through changes in the tissue biomechanics. Cell
migration is a key part of many biological processes
including corneal wound repair. It is important to understand
the mechanisms involved in cell migration and the
differences in cell behaviour between 2D and 3D
environments.
Rabbit Corneal Epithelial (RCE) cells have been used to
perform scratch wound assays, which gave an indication of
the speed and direction of cell migration of the RCE cells in
response to injury. Collagen gels have been used to
provide the 2D and 3D environments for studying RCE cell
migration in response to stimuli. Furthermore, other
materials have been studied for this application and
currently, Polydimethylsiloxane (PDMS) is being tested as a
substrate to be used for the mechanical stimulation of the
RCE cells. The use of cold atmospheric plasma treatment
on the surface of the PDMS has shown to improve RCE cell
attachment to PDMS and this process has been optimised
for mechanical stimulation.
The aim of this work is to understand corneal cell migration
in correlation with extracellular matrix (ECM) displacement
and by observing the migratory behaviour of corneal cells
through fluorescent microscopy. By using 3D imaging, cell
tracking, advanced imaging processing and measuring
cellular strain response to mechanical stimulation, studies
could improve our understanding of corneal epithelial cell
behaviour.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Laura Shallcross
Sheffield
Year 3
Nanotechnology and Polyelectrolytes for Medical and
Dental Applications
Laura Shallcross
Biomaterials, polymers, nanoparticles
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 made to disperse,
this would increase their functionality and make them
applicable 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.
Branched polymers of poly(acrylic acid), were synthesised
by
RAFT
polymerisation
using
4-vinylbenzyl
pyrrolecarbodithioate or 4-vinylbenzyl dithiobenzoate as
chain transfer agents. Samples of polymer were introduced
into the synthesis of hydroxyapatite (HA) nanoparticles.
Analysis by transmission electron microscopy (TEM)
showed a difference in the morphology of the polymertreated HA when compared to control samples of HA
nanoparticles. Spectroscopic techniques including: fourier
transform infra-red (FTIR) and x-ray diffraction (XRD)
proved HA had been synthesised with extra peaks
corresponding to the COO- of the PAA. Thermogravimetric
analysis (TGA) of samples of the composite material,
polymers and pure nanoHA supported this showing the
inclusion of polymer in the composite.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Michael Taylor
University of Nottingham
Year 3
3D Chemical Characterisation of Frozen Hydrated Hydrogels
Using ToF-SIMS with Argon Cluster Sputter Depth Profiling
Taylor. M, Scurr. D, Lutolf. M, Buttery. L, Zelzer. M,
Alexander. M R.
Hydrogels, Chemical Analysis, ToF-SIMS
Over the last decade the beneficial properties of hydrogels as
artificial cell culture supports have been extensively investigated1.
Certain synthetic hydrogels have been proposed to be similar in
composition and structure to the native extracellular matrix of the
stem cell niche, their in vivo cell habitat, which is a powerful
component in controlling stem cell fate2. The choice of stem cell
developmental pathway taken is modulated by number of factors.
When culturing cells within or upon hydrogel cell culture supports
this choice can be strongly dependent on the underlying 3D
hydrogel chemistry which governs hydrogel-cell interactions3. The
interrelationship between hydrogel chemistry and that of
biomolecules in controlling cellular response requires methods to
characterise the chemistry without labels and often in 3D. Time-offlight secondary ion mass spectrometry has the potential to be
utilised for through thickness characterisation of hydrogels. Full
through thickness chemical characterisation of hydrogels ideally
involves a sample format where the material in question is frozenhydrated to minimise changes associated with dehydration or the
chemical complexity of ‘fixation’, a challenging aspect in vacuum
analysis conditions4. Deposition of gaseous species on a cooled
sample, most particularly water, can occur evidenced by
macroscopic ice crystals on the surface preventing analysis of the
native material5. Frozen-hydrated full-through thickness chemical
characterisation is demonstrated here on a poly(2-hhydroxyethyl
methacrylate) hydrogel film where a protein, lysozyme, is
incorporated to demonstrated how biomolecule distribution within
hydrogels can be determined. A comparison of lysozyme
incorporation is made between the situation where the protein is
present in the polymer dip coating solution and lysozyme is a
component of the incubation medium. It is shown that protonated
water clusters H(H2O)n+ where n=5-11 that are indicative of ice are
detected through the entire thickness of the pHEMA and the
lysozyme distribution through the pHEMA hydrogel films can be
determined using the intensity of characteristic fragment secondary
ions.
(1)
Lutolf, M. P. Biomaterials: Spotlight on Hydrogels. Nat. Mater. 2009, 8
(6), 451–453.
(2)
Kobel, S.; Lutolf, M. P. Biomaterials Meet Microfluidics: Building the
next Generation of Artificial Niches. Curr. Opin. Biotechnol. 2011, 22 (5), 690–
697.
(3)
Yang, C.; Tibbitt, M. W.; Basta, L.; Anseth, K. S. Mechanical Memory
and Dosing Influence Stem Cell Fate. Nat. Mater. 2014, 13 (June), 645–652.
(4)
Robinson, M. a; Castner, D. G. Characterization of Sample
Preparation Methods of NIH/3T3 Fibroblasts for ToF-SIMS Analysis.
Biointerphases 2013, 8 (1), 15.
(5)
Piwowar, a.; Fletcher, J.; Lockyer, N.; Vickerman, J. Investigating the
Effect of Temperature on Depth Profiles of Biological Material Using ToF-SIMS.
Surf. Interface Anal. 2011, 43 (1-2), 207–210.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
James Warren
University of Leeds
Year 3
Self-assembling peptide gels for articular cartilage
repair
James Warren, Eileen Ingham, John Fisher, Stuart
Warriner
Self-assembly peptides cartilage
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.
Experimental approach
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 while an optimal OA damage
model will be designed and generated. This model will then
be used to develop methods of delivery of the selected
SAP-GAGs to GAG depleted cartilage will be investigated.
Optimal SAP-GAGs will then be tested for their ability to self
assemble within and restore biotribological function to GAG
depleted cartilage in vitro.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Simon Whittingham
University of Sheffield
Year 3
In-Vitro Model for Quantifying Cell-Based Therapies for
Peripheral Nerve Injury
S.Whittingham, J.W.Haycock, F.Claeyssens
Nerve, Regeneration, Model
Nerve guidance conduits are an alternative to autologous
nerve grafts for large injury gap peripheral nerve injuries,
where regeneration can’t be achieved via end-to-end
suturing. Autologous nerve grafts are the ‘golden standard’
for peripheral nerve regeneration, but this procedure has
inherent problems, needing a donor site for harvesting the
autologous nerve which can lead to donor site morbidity.
Many alternatives are being explored such as nerve
guidance conduits of both natural and synthetic origins and
cell- based therapies both individually and utilising a
combination of approaches.
Cell-based therapies are a potential approach for
regenerative medicine; however several intrinsic issues
have reduced their impact of the research. For peripheral
nerve regeneration, addition of Schwann cells to the injury
site has displayed increased regeneration potential,
however disadvantages of slow culture times in-vitro and
autologous donor extraction limits application of the therapy
in a clinical environment. Recent research showing
differentiation of adipose-derived stem cells into Schwann
cell phenotypes indicates a potential methodology of a cell
therapy approach to nerve injury which negates the
previous drawbacks due to the abundance of adipose
tissue.
However, the quantification for cell numbers to be included
at the site of injury remains unknown. Highly accurate and
rapidly produced platforms for neuronal-glia cell co-cultures
via laser based two photon polymerisation fabrication are
produced which aim to characterise the aforementioned cell
therapy approach offering insights which may be used to
inform future clinical progression of the therapy.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Matthew Worrallo
Loughborough University
Year 3
Immobilised Growth Factors for Scalable
Haematopoietic Cell Expansion
M. Worrallo, Dr K. Glen, Dr R. Thomas
HSCs, Scale-Up, Growth Factors
The major limiting factors for the manufacture of cell
therapies are cost and process control. Growth factors
represent a significant proportion of the total process cost
and are, in part, responsible for cell lineage control.
Immobilising growth factors provide many benefits over
soluble growth factors, such as reduced total quantities
(and therefore cost), increased potency and receptor costimulation. We have shown that a 1500 fold reduction in
growth factor when immobilised achieves equivalent
responses to soluble growth factors and the quantity of
immobilised growth factor can be precisely controlled. A 30
minute exposure period is only necessary to sustain cell
growth for over 24 hours and no benefits are observed for
continuous exposure. We also demonstrate that the
immobilised growth factors are re-usable, significantly
reducing waste. In conclusion, immobilised growth factors
offer significant economical, quality and environmental
advantages over their soluble counterparts.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Lauren Yarrow
University of Leeds
Year 3
Can we use RAW cells to model inflammatory response
to antioxidant doped UHMWPE wear debris?
Lauren Yarrow, Professor Joanne Tipper, Dr Sophie
Williams and Dr Eric Hewitt
Antioxidant, Macrophage, UHMWPE.
Ultra-high molecular weight polyethylene (UHMWPE) has
undergone several modifications, such as radiative crosslinking and
antioxidant doping, in order to improve its physical qualities as a
joint replacement material. The addition of antioxidants has no effect
on the volume of wear debris produced in comparison to crosslinked only UHMWPE (Gowland, 2014) however antioxidant doped
UHMWPE does appear to exhibit anti-inflammatory properties
(Bladen et al 2013a, Bladen et al 2013b). To date these effects are
not fully understood i.e. which inflammatory pathways are affected
and how. U937 macrophages (a cell line) and peripheral blood
mononucleocytes (PBMNC’s) have been used to model the
response to antioxidant doped crosslinked UHMWPE through
assessment of cytokine production. However both of these cell
types have proven difficult to use. U937 macrophages are
suspension cells and need to be made adherent and activated
before they can be cultured with particles. Furthermore, donor
variability and lack of adequate donor response have proven
problematic with the use of PBMNC’s. This study examines the use
of an alternate macrophage cell type – RAW 264.7 murine
macrophage cell line – to model cellular responses to UHMWPE
wear debris and assesses whether this is an appropriate choice of
cell to model this effect. The cells were initially challenged with
ceridust particles (a low molecular weight polyethylene and a wellestablished model of wear debris) to assess their response. Not all
types of macrophages respond adequately in vitro to challenge with
wear debris and therefore not all types of macrophage are suitable
for modelling this response (Matthews et al, 2001). The validity of a
RAW 264.7 murine macrophage model for wear debris response will
be evaluated.
References:
Gowland, N. (2014). Ph.D thesis. University of Leeds.
Bladen, C. et al. (2013a). “In vitro analysis of the cytotoxic and antiinflammatory effects of antoxidant compounds used as additives in
ultra-high molecular weight polyethylene in total joint replacement
components”, J Biomed Mater Res Part B 2013 101B: 407 – 413.
Bladen, Catherine et al (2013b). Analysis of wear, wear particles,
and reduced inflammatory potential of vitamin E ultra-high molecular
weight polyethylene for use in total joint replacement. J Biomed
Mater Res Part B 2013 101B: 458 – 466.
Matthews, J. et al. (2001). Comparison of the response of three
human monocytic cell lines to challenge with polyethylene particles
of known size and dose. Journal of material science: materials in
medicine 12: 249 – 258.
EPSRC Centres for Doctoral Training in Tissue
Engineering and Regenerative Medicine
Joint Conference 2015
Poster Abstracts
Year 4
University of Leeds
Conference Auditorium 2/Sports Hall 2
Friday 10th July 2015
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Divya Baji
University of Leeds
Year 4
Developing a methodology to study the biomechanics
and biotribology of osteochondral substitutions in the
natural patellofemoral joint.
Divya Baji, Prof John Fisher, Prof Eileen Ingham, Dr
Louise Jennings
Biotribology, knee, patello femoral
Joint replacement is the common end-stage treatment for
damaged and degenerated joints. It relieves pain and restores
function of the joint. Regenerative treatments such as
osteochondral substitutions are minimally invasive and
conservative treatments that hold the future.
The aim of this project was to develop and apply novel
experimental methodologies to investigate the biomechanics
and biotribology of the natural patellar femoral joint (PFJ).
A methodology has been developed to investigate the
biomechanics of the natural PFJ using Tekscan pressure
sensors in a single station knee simulator. This method is
currently being used to study the contact mechanics of the
porcine PFJ under varying degrees of flexion and load. The
mechanical properties of the cartilage in the porcine patella
and femoral groove have been characterised in order to
support the understanding of the contact mechanics and wear
of the cartilage. The elastic modulus, permeability and
thickness of the cartilage in the patella and groove were
measured. The material properties of human cartilage in the
patella and groove are currently also being be characterised.
A methodology has been developed to investigate the
biotribology of the natural PFJ using an in-vitro porcine model
in a single station knee simulator. This involved determination
of the relative position of patella with respect to the femur at
the start of the gait cycle, determination of centre of rotation of
the patella groove and a cement mounting method to finalise
the in-vitro animal model. This has been tested on a porcine
PFJ under the PFJ gait cycle for 7hrs. The wear of the joint
will be graded and compared to the wear from a positive
control prepared with a natural patella articulating against an
artificial femur.
The developed methods can be used to investigate the
performance of grafts by applying the same methods to the
joint implanted with the tissue substitutions.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Philippa Bowland
University of Leeds
Year 4
The Stability and Biotribology of Osteochondral Grafts
in the Natural Knee
P Bowland, E Ingham, L Jennings, J Fisher
Biotribology, knee, osteochondral graft
Introduction
To deliver successful regenerative osteochondral grafts to the
patient, there is the requirement to develop robust preclinical test
methods incorporating functional biomechanical and tribological
simulations to assess the performance of grafts in the natural
knee environment. The study aimed to: (1) investigate the
effects of graft and defect geometry, implantation location and
graft harvest method on the stability of osteochondral allografts;
(2) develop a method to establish whether osteochondral
allografts have an effect on the local biotribology of the joint postimplantation, utilising a small-scale model of the natural joint.
Methods
For stability testing osteochondral grafts were harvested from
bovine femoral condyles and implanted into defects prepared on
recipient femoral condyles. Grafts were then compressed in situ
at a rate of 4mm/s using an Instron Testing Machine (3365).Test
groups included grafts of: (1) 8.5 mm and (2) 6.5 mm diameter;
(3) grafts implanted in defects of equal length and (4) unequal
length; (5) grafts harvested with a chisel and (6) grafts harvested
with a power trephine (n=12 for each test group). The method for
local biotribology assessment was developed using porcine
cartilage-bone pins reciprocating against bovine cartilage-bone
plates on a reciprocating friction rig. A range of loads / contact
pressures were tested until optimal friction and wear was
obtained for the cartilage on cartilage controls
Results
The most significant factor determining graft stability post
implantation was the ratio between graft and defect length.
Grafts implanted into defects longer than the graft length were
less inherently stable and subject to subsidence below
congruency at considerably lower loads.
Discussion and Significance
The approach adopted in implanting osteochondral grafts plays a
significant role in determining graft stability and maintaining joint
surface congruency post implantation. Inadequate implantation
of grafts and excessive loads can directly result in graft failure,
incongruent articulating surfaces and therefore disruption to the
local biotribology of the joint.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Jack Bridge
University of Nottingham
Year 4
Developing an in vitro model of smooth muscle
contraction
JC Bridge, GE Morris, JW Aylott, MP Lewis, FRAJ Rose
Electrospinning, Smooth muscle
Smooth muscle (SM) tissue is found in many parts of the
body, primarily in sheets or bundles surrounding hollow
organs. Its main function being the regulation of organ tone
via its contractile state. Dysfunction of SM in diseases such
as asthma and atherosclerosis affect millions worldwide.
Current methods for studying SM primarily rely on ex vivo
animal tissues or 2D in vitro models. These 2D models are
cultured on stiff surfaces lacking the elastic properties and
3D morphology found in natural extracellular matrix in vivo.
Therefore it is desirable to develop both an in vitro model of
SM that possesses the ability to contract and a method in
which this contraction can be measured.
In order to achieve this, primary rat aortic SM cells were
cultured in collagen hydrogels and cultured under tension in
order to generate aligned SM collagen constructs. When
stimulated with contractile agonists, these tissues contract
in a uniaxial fashion. The design of the constructs allows
them to be attached to a force transducer allowing the
physical force of contraction to be measured. Reproducible
force measurements have been made across multiple gels.
Electrospun scaffolds provide another method of aligning
cells and have the advantage of forming a confluent layer of
cells in which gap junctions can be formed, allowing the
cells to behave as a syncytium. By adapting the previously
described hydrogel model to use aligned electrospun
gelatin based scaffolds, a reliable, reproducible method for
measuring the contractile force generated by SM in vitro
has been developed
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
James Butler
University of York
Year 4
Simulation modelling of autoimmune disease
pathology
James Butler, Jon Timmis, Mark Coles
Autoimmune Disease, Computational Biology,
Lymphoid Tissue
Tertiary lymphoid tissues (TLT) form in response to
localized production of inflammatory cytokines and
chemokines. These highly organised structures consist of B
cell follicles surrounded by T cells, supported by a stromal
networks consisting of follicular dendritic cells (FDC) and
fibroblastic reticular cells (FRC). In autoimmune disease, it
is thought that germinal center reactions within the B cell
follicle ultimately result in a sustained auto-antibody
formation driving disease pathology. Therefore, it is
important to develop a detailed mechanistic understanding
of the processes that mediate TLT induction and formation,
which could lead to novel therapeutic approaches.
TLT formation is a complex emergent phenomenon
resulting from interactions between large numbers of cells
and signalling molecules, difficult to fully examine using
reductionist in vivo and in vitro techniques due to the role of
these molecules in immune system development. From our
in vivo & in vitro models, we hypothesise that TLT formation
may be described by T cell migration in response to an
inflammatory trigger, initiating a stromal-lymphocyte crosstalk feedback loop mediating CXCL13 up-regulation driving
B cell recruitment and follicle induction. To test this
hypothesis, and to inform further experiments, we
developed a multi-scale hybrid mathematical and
computational model of TLT formation using a transparent
and principled approach, paramaterised using in vivo
imaging, flow cytometry, histology and gene expression
datasets. Using the simulation we determined minimum
requirements for TLT induction, surprisingly identifying a
role for CXCR5 receptor dynamics on B cells regulating this
process. In particular, the internalisation of CXCR5
maximises B cell motility in the developing follicle.
Additionally the model predicts that receptor dynamics
regulate efficient T - B cell interactions necessary for
efficient GC formation.
ABSTRACT
Name:
University:
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Authors:
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Abstract
Alex Chan
Loughborough University
Year 4
Development of immunosuppression and angiogenic in
vitro potency assays for human mesenchymal stem
cells.
Alexander K. C. Chan, Emma Neale-Edwards, Karen
Coopman, Christopher J. Hewitt
hMSC, Potency, Assay
Human mesenchymal stem cells (hMSCs) are being increasingly
applied for various cell-based therapies. The challenge ahead for
this field is to develop assays that are predictive of potency and
quality that can be linked to in vivo efficacy. Potency assays must
be developed to provide a measurable readout of relevant
biological properties with respect to clinical function. They must
also allow for comparability assessment during changes in
manufacturing processes and new donor input material. The
work here describes the development of two individual assays
that can be deployed to identify a higher quality hMSC line in
terms of in vitro biological activity.
It has been shown that hMSCs can suppress and modulate the
immune system, this property makes them an ideal candidate for
cell-based therapies in inflammatory and autoimmune disorders.
The work here describes the development of a hMSC-mediated
T-cell suppression assay that can be deployed across multiple
hMSC donors. The suppression effect is linked to indoleamine
2,3-dioxygenase (IDO) enzyme activity.
hMSCs are also in clinical trials for their pro-angiogenic effect
following myocardial infarction among other cardiovascular
conditions. This action is in part by their ability to produce
angiogenic cytokines, production of which can be altered when
cultured in a lower dissolved oxygen environment. Here using a
HUVEC tube formation assay we investigate the optimal in vitro
measurement systems for successful angiogenesis and the
parameters to determine hMSC potency.
These defined assays allow for comparability throughout culture
and through a manufacturing process. For both allogeneic and
autologous cell-based therapies the quality characterisation of
input material from individual donors will be necessary to ensure
an effective treatment. This work highlights the need for the
application of quality and potency in vitro assays that can provide
a meaningful representation of in vivo activity.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Emily Clark
University of Leeds
Year 4
Temporal evaluation of SMC phenotype and function in
a bioreactor model of AAA
Emily Clark, Louise Jennings, Julian Scott, Karen
Porter
Bioreactor, vascular, phenotype
Abdominal aortic aneurysm (AAA) is a progressive,
asymptomatic aortic dilatation with high mortality when
rupture occurs. Smooth muscle cells (SMC) cultured from
elective AAA repair patients exhibit phenotypic and
functional aberrancies. This study aimed to develop an ex
vivo model of “early” and “end” stage AAA in a novel
bioreactor and characterise SMC derived from the vessels
and the whole vessel itself.
Following protease pre-treatment, porcine arteries were
cultured under steady flow for either 12 days (END) or 3
days (EARLY). SMC were cultured from retrieved tissue
and characterised in terms of morphology (image analysis),
proliferation (cell counting) and senescence (bgalactosidase). Tissue was also analysed using
immunohistochemistry (α-SMA and Miller’s elastin).
Uniaxial tensile testing and dilation/burst pressure testing
determined mechanical properties of intact vessels.
END SMC displayed increased circularity (2.32-fold,
p<0.001 n=2) yet the morphology of EARLY SMC was
indistinguishable from SMC cultured from freshly isolated
(FI) arteries (spindle). Proliferation of END SMC was
impaired by 46% (p<0.001), conversely it increased by 41%
in EARLY SMC (p<0.001, n=6 both vs. FI). Senescence
was greater in END SMC (2.1-fold, p<0.05, n=6) than in
EARLY SMC. Vessels which underwent the protease pretreatment were less compliant and exhibited a 92%
reduction in burst pressure strength (p<0.05, n=5 vs.
vehicle pre-treatment).
END SMC are phenotypically comparable to human endstage AAA-SMC with a rhomboid, aberrant morphology,
high levels of senescence and impaired proliferative
capacity. Prior to this phenotypic “switch”, SMC exhibit a
period of rapid proliferation. Understanding the mechanism
that drives this detrimental “switch” may reveal targets for
novel therapeutics.
ABSTRACT
Name:
University:
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Authors:
Keywords
Abstract
Ruth Craven
University of Leeds
Year 4
Mechanisms of membrane toxicity of cobalt chromium and
alumina ceramic wear particles generated from total hip
replacements
Ruth Craven, Lars Jeuken, Sophie Williams, Joanne L.
Tipper
Toxicology; Membrane; Hip
Total hip replacements (THRs) have been hugely successful at
improving mobility in patients. Ceramic-on-ceramic and cobalt
chromium (CoCr) metal-on-metal THRs, under optimum conditions,
have low wear rates with the generation of predominately nanoscale
wear particles 1, 2. However, adverse biological reactions to the wear
particles, both in vitro and in vivo have been reported especially for
CoCr wear particles, which have been associated with
hypersensitivity reactions and pseudotumours 3,4. The cellular
effects of the wear particles are not fully understood, such as how
the particles interact with the plasma membrane. This project aims
to investigate how CoCr and alumina wear particles affect the cell
plasma membrane integrity and particle binding.
The effect of clinically relevant CoCr and alumina wear particles on
the integrity of a model phospholipid membrane was assessed using
vesicle leakage assays. Phospholipid vesicles encapsulating a
fluorescent dye were exposed to 10% (v/v) foetal bovine serum,
followed by incremental concentrations from 1 – 100 µg.ml-1 of
particles and membrane damage resulted in the release of the
fluorescent dye.
The ability of the particles to bind to the cell plasma membrane was
measured using quartz crystal microbalance with dissipation (QCMD). The frequency and dissipation of the oscillating quartz crystal
was measured as an indicator of particle binding.
Alumina and CoCr particles induced vesicle leakage in a dose
dependent manner. However only the highest concentration of
ceramic particles (100 µg.ml-1) had a significant effect on vesicle
leakage. QCM-D results suggested that the wear particles interacted
very weakly with the membrane. Further analysis using surface
Plasmon resonance techniques confirmed that in fact the wear
particles did not bind to the model membrane.
This study has demonstrated that CoCr and alumina wear particles
do not interact non-specifically to a model membrane. Future work
will investigate the presence of membrane proteins and particle
interactions.
References:
[1] Dowson. D. et al. 2004. J Arthroplast 19:118-123.
[2] Nevelos JE, et al. 2001. J Mater Sci-Mater Med 12:141-144.
[3] Gill HS. et al. 2012. Trends Mol Med 18:145-155.
[4] Brown et al. 2006. Proc Inst Mech Eng Part H – J Eng Med
220:355-369.
ABSTRACT
Name:
University:
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Abstract
Stephen Goode
University of Leeds
Year 4
Development of a Computational Spinal Cord Injury
Model using the Material Point Method
Stephen Goode, Joanne Tipper, Richard M Hall, and
Jon Summers
Spinal Cord Injury, Biomechanics, Computational
Introduction
Spinal cord injury (SCI) is characterized by permanent loss of
motor and sensory function. The primary damage from the
initial mechanical insult is exacerbated by the secondary
patho-physiological cascade. Computational SCI models form
part of a wider effort to investigate the link between the
biomechanics of the primary mechanical insult and the
subsequent evolution of the secondary injury. Due to the
presence of the cerebrospinal fluid (CSF) this is a fluidstructure interaction (FSI) problem. To date, these models
have relied on finite element (FE) methods, however; the
complexity of FE models is limited by difficulties in coping with
large rapid deformations due to mesh tangling, incorporation
of the FSI, and parallel scalability. The Material Point Method
(MPM) is an alternative, “mesh-free”, computational technique
that avoids these limitations, with the potential to enable more
complex SCI models to be created going forward.
Methods
A MPM SCI model, including neural tissue and dura mater,
was created. A simulated bone fragment transversely
impacted the cord surface, representative of a vertebral burst
fracture. Results were validated against existing FE and
experimental results. The simulation, containing 332,640
material points, was run using 640 cores in parallel.
Results
The max deformation for the MPM model was 4.41 mm, vs
5.58 mm, and 5.69 mm (SEM±0.21 mm) for the FE and
experimental models respectively. The time to max
deformation for the MPM model was 2.24 ms, compared to
2.50 ms for the FE model, and 2.97 ms (SEM±0.11 ms) for the
experiments. The MPM model result underestimates the max
deformation by ~1.28 mm and subsequently underestimates
the time to max deformation.
Conclusions
Results suggest that MPM is a suitable substitute for FEM,
whilst also being highly suited to parallelisation. Future work
will seek to optimize the MPM model, bringing the results
closer to the experimental mean, and to incorporate the CSF.
ABSTRACT
Name:
University:
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Abstract Title:
Authors:
Keywords
Abstract
Helen Lee
University of Leeds
Year 4
The Cellular Response of Primary Astrocytes to Cobalt
Chrome Wear Debris.
Helen Lee, Dr James Phillips, Professor Richard Hall,
Professor Joanne Tipper.
Astrocyte, Debris, Cytotoxicity.
Back pain associated with a degenerated intervertebral disc
is a major public health concern. The current treatment
options to treat such pain are: a spinal fusion procedure
which is the current gold standard or the use of a total disc
replacement (a motion preservation device). As a result of
clinical complications associated with a fusion procedure
there has been an increased interest in the use of total disc
replacements (TDRs). The longevity of these devices is
compromised by wear and there is growing concern within
the orthopaedic community that metal debris from these
devices could impact and alter the functionality of
periprosthetic tissues in particular the spinal cord.
This research aims to provide an insight into the biological
response of CNS cells to clinically relevant cobalt chrome
and stainless steel wear debris. Specifically focusing on the
effect the particles have on cell viability (using a live dead
stain), metabolic activity (measuring ATP production) and
reactivity (Glial fibrillary acidic protein production- GFAP).
The Cobalt chrome wear particles significantly reduced the
viability of primary astrocytes after 48 hours in 3D culture
at both the 50m3 and 5m3 per cell doses (when
compared to a cell only negative control). The effect was
not significant for the lowest 0. 5m3 per cell dose. Upregulation of GFAP expression was also observed when
primary astrocytes were cultured with cobalt chrome wear
particles for 48 hours (the effect was more pronounced
when cultured with the highest 50m3 per cell dose).
Up-regulation in GFAP expression in response to particles
is indicative of reactive astrogliosis, which is associated
with glial scar formation and a severe reduction in axonal
regrowth in CNS injury. Additional longer time points will be
investigated to further understand the biological response of
these particles to primary astrocytes.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
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Abstract
Atra Malayeri
University of Sheffield
Year 4
Biocompatibility of Poly High Internal Phase Emulsion
Scaffolds prepared using Stereolithography
Atra Malayeri, Ilida Ortega, Frederik Claeyssens, Colin
Sherborne, Neil R Cameron, Paul V. Hatton
Solid Freeform Fabrication technology, Bone Tissue
Engineering.
Complex bone defects in the head and face would benefit
significantly from the use of custom-shaped devices or
scaffolds that stimulated tissue regeneration after
implantation. Despite some promising data, resorbable
polymeric biomaterials have not yet been optimised for
preparation of custom structures using contemporary
advanced manufacturing methods.
Poly high internal
phase emulsions (PolyHIPEs) have been reported to
provide structures with interconnected micropores that have
the potential to enhance biocompatibility. Despite their
promise, only a limited volume of work has been reported
on the biocompatibility of PolyHIPEs prepared using 2ethylhexyl acrylate and isoboronyl acrylate, and only
recently the fabrication of simple 3D structures via
stereolithography has been reported1. The aim of this study
was therefore to evaluate the biocompatibility of this
polyHIPE system using cultured bone cells, and to
manufacture more complex “woodpile” three-dimensional
scaffolds using stereolithography. It was concluded the
acrylate-based PolyHIPEs described here supported the
growth of bone cells in vitro, and that plasma polymerisation
appeared to further improve cell response.
Using
stereolithography, it was possible to prepare more even
more complex 3D scaffolds or devices that have great
potential for tissue engineered craniofacial surgery.
ABSTRACT
Name:
University:
Rachel Pallan
University of Leeds
Year of Study:
Abstract Title:
Year 4
Biological and Mechanical Characterisation of the
Porcine Labrum
R. L. Pallan, J. L. Tipper, J. Fisher, S. Williams
Labrum, Cartilage, Characterisation
Femoroacetabular impingement (FAI) is thought to be a
precursor to osteoarthritis in the hip. Bony growths on the
femoral head-neck and/or the acetabular rim can alter the
loading patterns through the hip, resulting in damage to the
tribological interface. Labral damage is often seen in FAI
cases, where the labrum becomes impinged upon by the
bony growth, resulting in either tears and/or labral-cartilage
separation. Although labral repair is becoming more
favoured than excision, scientific rationale for interventions
is limited. This study characterised the labrum in terms of its
structure, constituents and mechanical properties. The
porcine labrum forms an apex at the rim and transitions to
the exterior of the acetabulum, on the articulating surface it
connects directly to the acetabular cartilage through a clear
transition zone. The labrum is primarily composed of type I
collagen and has a significantly lower water and
glycosaminoglycan (GAG) content and significantly higher
collagen content compared to articular cartilage. Indentation
tests were used to determine the mechanical properties of
the porcine labrum. The compressive modulus (0.185 +/0.037 MPa) and permeability (7.96 x10-15 +/- 2.43 x10-14 m4
N.s) of the porcine labrum were comparable to that of
bovine labrum. The creep (0.466 +/- 0.107mm),
deformation (15.5% +/- 3.6%) and stiffness (1.604 +/-0.314
N/mm) properties were also determined for the porcine
labrum.
Authors:
Keywords
Abstract
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Lindsey Parker
University of Leeds
Year 4
Mechanotransduction in Multipotential Mesenchymal
Stromal Cells
Lindsey Parker, Eileen Ingham, John Fisher and Daniel
Thomas
Mechanotransduction, hMSC
Mechanical stimuli have been shown to affect gene expression in
a range of differentiated cell types. There have, however been
very few studies of mechanotransduction in multipotential
mesenchymal stromal cells (MSC)1,2, and the role of physical
stimuli in the differentiation of these cells is not known. The aims
of this project are to investigate the differentiation of MSC
following the application of cyclic tensile strain, and identify strain
regimes that promote differentiation towards a smooth muscle
lineage.
An acellular porcine pericardium scaffold will be used for 3D
culture of human MSC under cyclic tensile strain (2-12%) in the
Tencell bioreactor. Porcine pericardial samples (35) were
decellularized and validated using an established proprietary
method, and an appropriate seeding method devised. Tencell
was calibrated with regard to evaporation, temperature
maintenance and arm displacement. A set of 15 primers
corresponding to genes of interest typically expressed in cells
differentiating towards the chondrogenic, osteogenic, adipogenic,
tenogenic and smooth muscle lineages were designed and
validated with respect to primer pair specificity and PCR
efficiency.
Decellularized pericardial tissue contained no cellular debris and
very low levels of residual DNA (˂ 50 ng.mg-1 dry tissue). There
was no evidence of cytotoxicity or microbial contamination in
treated tissue. Significant issues with media evaporation and
temperature variation in Tencell were resolved using humidifying
apparatus and an improved temperature regulator. Seeding rings
designed for use in conjunction with Tencell were found to be
cytotoxic and an alternative seeding method was developed.
Primer pairs leading to one amplicon of the expected size and
with PCR efficiency in the range 90-110% were selected for
future use. In future work, gene expression analyses will be
performed on seeded scaffolds subjected to uniaxial strain.
1
2
Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E.
Matrix Elasticity Directs Stem Cell Lineage Specification.
Cell 126, 677-689 (2006).
Even-Ram, S., Artym, V. & Yamada, K. M. Matrix control
of stem cell fate. Cell 126, 645-647(2006).
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Nathalie Robinson
Loughborough University
Year 4
Low Temperature Cell Pausing: An Alternative ShortTerm Preservation Method for Use in Cell Therapies
Nathalie J Robinson & Karen Coopman
Cell preservation, cell therapies, hypothermia
With encouraging advancements in cell therapies, a
requirement for an effective short-term cell preservation
method, enabling time for quality assurance testing and
transport to their clinical destination is required. This project
aims to ‘pause’ cells at ambient temperatures, whilst
maintaining viability and function post-preservation.
Ambient cell preservation bypasses ice crystal exposure
and the high solute concentrations experienced with
cryogenic storage. It also avoids the use of toxic
cryoprotectants and aims to greatly reduce costs and
reliability on specialist machinery.
Early work using HOS TE85 cells (derived from an
osteosarcoma) as a model, paused cells at ambient
temperatures for up to 144 hours. Post-preservation, HOS
TE85 cells effectively recovered in terms of morphology,
membrane integrity and fold growth expansion, when
atmospheric factors were controlled (viability >90%).
Without atmospheric control, addition of the buffering agent
HEPES (25mM) to cell medium was required to keep
viability above 70% and maintain cell yield and proliferative
capacity. Stable pH is vital during pausing and additional
antioxidants may be required to quench free radical
production during continued hypothermic stress ( ≥ 96
hours).
Current work is investigating the potential for pausing
therapeutically relevant cells (human mesenchymal stem
cells) in suspension to make the process clinically
applicable.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Normalina Sandora
University of Leeds
Year 4
Seeding of human MSC onto acellular porcine patella
tendon scaffolds
Normalina Sandora
Acellular scaffold, human MSCs, seeding
Tears of the anterior cruciate ligament fail to heal and require
surgical intervention to prevent or delay the onset of
osteoarthritis. Current surgical replacements all have limitations.
An acellular scaffold produced from porcine patellar tendon
(PPT) is proposed for ligament replacement. The overall aims of
this study are to explore the regeneration of acellular PPT with
human MSC under cyclic uniaxial strain in vitro. To this end, the
acellular PPT have been prepared and characterised biologically
and biomechanically. Human MSC have also been shown to be
capable of tri-lineage differentiation and their growth in vitro
characterised. The aim of this part of the study was to determine
the viability of the human MSC following seeding onto the
acellular PPT scaffold under different conditions. Scaffolds were
shaped into 20 x 10 x 0.6 -0.8 mm3 samples. Five groups (n=3)
and preconditioned overnight at 370C, 5% (v/v) CO2 in air. Four
groups of scaffolds (n=3) were seeded with 1x105 cells.cm-2
human MSCs passage 6 and incubated under the same
conditions for 24 hours. One group of acellular samples was left
unseeded and used as a negative control. One seeded group of
scaffolds was harvested and analysed immediately (time zero).
The remaining three groups of scaffolds were cultured at 370C,
5% (v/v) CO2 in air under different conditions (a) untethered in a
6-well plate (b) uniaxially tethered in Tencell wells and incubated
in an incubator and (c) uniaxially tethered in Tencell wells and
incubated in Tencell (without the application of strain). Following
24 h incubation, the scaffolds were divided into two halves. One
half was analysed using LIVE/DEAD cell staining and other half
by ATP assay. The Live/Dead staining showed that the
percentage of live cells post-seeding was 69.17%.After 24 hours
incubation untethered in the six well plate had 67.33% of the
cells were viable. Scaffolds tethered in the Tencell well had
40.08% viable cells, and those tethered in Tencell in the Tencell
rig, 43.00% viable cells. The ATPlite assay data showed a 20
fold increase in ATP content of the scaffolds incubated
untethered and a three 3 fold increase in ATP levels in tethered
scaffolds under both conditions, compared to the ATP levels
post-seeding.
Further studies are required to determine whether the increase in
ATP levels of the untethered seeded scaffolds was a result of
rapid cell proliferation in 3D or an increase in cell metabolism.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Ryan Taylor
University of Sheffield
Year 4
Polymer Scaffolds for Peripheral Nerve Regeneration
Ryan Taylor, Fred Claeyssens and John Haycock
PolyHIPE peptide nerve
The ‘gold standard’ for the repair of nerves is an autologous
graft[1]. This is not without its downsides i.e. donor site
morbidity and limited availability of nerves due to the size of
the donor nerve having to match that of the site of injury.
The interaction between integrin sites on cells and proteins
on the extracellular matrix is one which allows cells to bind,
migrate and form tissues. Arg-Gly-Asp (RGD) containing
peptides mimic the binding of the extracellular matrix
protein fibronectin to the α5β1 integrin site and this can help
to promote adhesion of cells to biomaterials, creating a
completely synthetic ‘extracellular matrix’.
Functionalized biomaterials are able to direct and enhance
cellular growth. Therefore, the ability to be able to
functionalize a polymer structure is crucial when looking to
control the other properties of the material (i.e. Young’s
modulus, degradation rate, degree of swelling etc.). Without
using functionalized biomaterials the number of materials
viable for cell adhesion is vastly reduced, meaning a match
of materials properties and degree of adhesion is more
difficult to achieve.
One such material to be used is a PolyHIPE material
comprised of Polycaprolactone (PCL). This gives a
degradable porous structure which can be functionalised
with acid end groups using plasma polymerisation and then
further functionalised with peptide sequences. The effect of
this is to be determined on Schwann cells.
These polyHIPE materials have been synthesised and
functionalised with great success which leaves many
opportunities synthesise other polymer materials such as
polyurethane type materials using similar techniques.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Rachel Thompson
University of Sheffield
Year 4
Lock-in Amplified Lens Vibration Spontaneous Raman
Spectroscopy for Tissue Engineering Applications
Rachel Thompson, Ben Varcoe, Aileen Crawford & Paul
Hatton
Raman Spectroscopy
Raman spectroscopy is a technique with the potential to
meet the growing need for non-destructive analysis of
tissue engineered constructs [1]. One possible barrier to the
use of Raman spectroscopy is the requirement to use a low
laser irradiance in order to minimise of damage to the cells
and DNA by thermal effects [2]. Current methods to
minimise irradiance involve using complex subtraction
algorithms or the use of lock-in amplification via sample
vibration [3-5]. However, these solutions are sub optimal for
biological spectroscopy.
Here we present a new Raman spectrometer based upon
vibrating the lens which focuses the laser onto a sample,
combined with a lock-in amplifier to amplify the Raman
spectrum, whilst suppressing the background signal. The
spectrometer has been applied to silica, various
hydroxyapatites and scanning electron microscopy
processed bone cell cultures and has produced promising
initial results.
Lens-vibration Raman spectroscopy is an interesting new
implementation of the technique and we believe that it is
optimised for biological applications. It exploits underlying
physical methods to produce clear spectra from a low laser
power.
1.
Short, K.W., et al. Biophysical Journal, 2005. 88(6):
p. 4274-4288.
2.
Mohanty, S.K., et al., Radiation Research, 2002.
157(4): p. 378-385.
3.
Tzinis, C., et al., Review of Scientific Instruments,
1978. 49(12): p. 1725-1728.
4.
Rusciano, G., A.C. De Luca, and A. Sasso,
Analytical Chemistry, 2007. 79(10): p. 3708-3715.
5.
Rusciano, G., et al., Applied Physics Letters, 2006.
89: p. 261116.
ABSTRACT
Name:
University:
Year of Study:
Abstract Title:
Authors:
Keywords
Abstract
Jamie Thurman-Newell
University of Loughborough
Year 4
A Meta-analysis of Biological Variation in Blood-based
Therapy as a Precursor to Bio-Manufacturing.
Jamie A. Thurman-Newell, Jon N. Petzing, David J.
Williams
HSCT, variation, optimisation
Currently cellular therapies, such as haematopoietic stem
cell transplantation, are produced at a small scale on caseby-case basis, usually in a clinical or near-clinical setting.
Meeting the demand for future cellular therapies will require
a robust and scalable manufacturing process that is either
designed around, or controls the variation associated with
biological starting materials.
Understanding variation requires both a measure of the
allowable variation ( that does not negatively affect patient
outcome ) and the achievable variation ( with current
technology ). The prevalence of HSCT makes it an ideal
case study to prepare for more complex biological
manufacturing
with
more
challenging
regulatory
classifications.
A systematic meta-analysis of the medical literature
surrounding HSCT has been completed of which the key
outcomes are;
• The range transplanted CD34+ cells / kg can be up to 6
orders of magnitude around the median for allogeneic and 4
orders for autologous procedures
• No improvement in variation encountered over a period of
thirty years
• As study size increases the amount of variation
encountered increases
• A more detailed, stratified source from a controlled single
site clinical centre is required to further define a control
strategy for manufacture of biologics.
ABSTRACT
Name:
University:
Year of Study:
Ammar Wahid
University of Leeds
Year 4
Abstract Title:
Sensitivity Analysis of marker misplacement in the Plug-inGait model when analysing hip motion of Leg Length Inequality
Patients
Ammar Wahid, Neil Messenger, Todd Stewart
Authors:
Abstract
During 2014, 93000 total hip replacements (THR) were undertaken, the
majority of these providing high levels of satisfaction for patients. Up to
10% may be symptomatic for a leg length inequality (LLI) which can
lead to discomfort, nerve palsy, and the potential for revision surgery.
For LLI, gait analysis is a common functional outcome measure as LLI
can lead to reduced flexion (Budenberg, 2012). One of the best known
clinical techniques to assess clinical gait is “Plug-in-Gait” (PiG) due to
its quick and easy clinical implementation. In this study a sensitivity
analysis was undertaken to investigate how the misplacement of
markers on the thigh can lead to errors in the kinematics of the hip in
patients with LLI.
For the sensitivity analysis gait analysis data was collected for a typical
male THR patient with a BMI of 28.1 and a symptomatic LLI of 40 mm
(measured from the ASIS to medial malleoli). A PiG model was built in
Visual3D (C-motion) with a total of 16 markers used (4 at the pelvis, two
on each thigh, two on each shank and two on each foot) with each
segment having 6DOF. Two points were selected for the sensitivity
analysis; the lateral thigh marker (THI), the lateral knee marker (KNE).
Analysis was undertaken by producing a virtual marker in the static
human trial offset by 10mm to the original marker. The lateral knee and
thigh markers were moved in the anterior-posterior direction. The joint
angle at initial heel strike and toe off together with the standard
deviation were extracted from Visual3D where both gait events had
been predefined by the user through visual inspection of the motion trial
of the model. A t-test was used to compare the hip kinematic changes
which occurred following moving the marker position 10 mm.
Moving the lateral thigh marker 10mm led to a 1.8˚ and 1.9˚ increase in
hip flexion angle during heel strike and toe off with p<0.08 and p<0.04
respectively, with the latter being statistically significant at the 5% level.
Similarly, statistically significant results were also produced for hip
internal-external rotation with heel strike (p<0.01) leading to an 11.6˚
change in rotation (p<0.01) and toe off a 11.5˚ change. There was no
statistical difference found in terms of abduction-adduction at heel strike
(p<0.87) or toe off(p<0.88) with changes of 0.34˚ and 0.39˚
respectively.
Moving the lateral knee marker 10mm led to a non-statistically
significant (p<0.31) increase in hip flexion-extension at heel strike and
toe off (p<0.06) with differences of 2.7˚ and 2.8˚ respectively. This is
also the case for abduction adduction with angle changes of 0.55˚ and
0.32˚ being statistically not significant with p<0.85 at heel strike and
p<0.96 at toe off. Hip rotation was however very statistically significant
with a 11.7˚(p<0.01) increase following a 10mm movement of the knee
marker at heel strike and a 11.6˚ increase at toe off(p<0.01)
The results of the study found that misplacements of the thigh marker
are likely to introduce significant artefacts in hip rotation of up to 11.7˚.
Hip joint rotation angles were also found to be equally sensitive to the
placement of the lateral knee marker. Conclusions which can be drawn
from this study indicate that the misplacement of markers on the thigh
in the anterior-posterior direction can lead to unreliable results in terms
of hip rotation.
ABSTRACT
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University:
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Abstract Title:
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Keywords
Abstract
Leyla Zilic
University of Sheffield
Year 4
Development of an in vitro peripheral nerve model
using xenogeneic nerve tissue
Peripheral nerve; Extracellular matrix; Schwann cells
Peripheral nerve injuries affect 1 in 1000 of the population [1].
Injuries greater than 1-2cm are normally bridged using
autografts, which direct regenerating axons, by topographic
guidance [2]. Commercially available products such as nerve
guide conduits are not particularly suitable as they lack
architecture similar to that of the native ECM of the nerve. It is
hypothesized that an acellular nerve would facilitate
regeneration of axons at the cellular level, encouraging
regeneration within a native microenvironment. The present
aim is to develop compatible, non-immunogenic, nerve grafts
using low concentration SDS to decellularise porcine
peripheral nerves. The acellular nerve will then be used as a
basis for the study of perfused flow within the tissue for the
introduction of Schwann cells - as the delivery of such cell
types is reported to improve nerve cell development.
Decellularised porcine peripheral nerves were characterised
using histological, immunohistochemical staining, quantitative
biochemical assays and biomechanical testing. The acellular
nerves were repopulated with primary rat Schwann cells under
perfused flow using a bioreactor. Decellularisation of nerves
resulted in a 95% DNA reduction with the preservation and
retention of the native nerve architecture and ECM
components. Biomechanical testing indicated that the
decellularisation process had little effect on the mechanical
properties. Characterisation of the repopulated nerve showed
Schwann cells distributed through the tissue.
The acellular model can be used as a basis for the study of
perfused flow within the tissue for the introduction of Schwann
cells. Key questions can additionally be asked using this as a
model, including the influence of a native 3D environment on
cell migration and development.
References
1. Dunning C, McArthur SL, Haycock JW. Three-dimensional
alignment of Schwann cells using hydrolysable microfiber
scaffolds: Strategies for peripheral nerve repair. Methods Mol
Biol 695, 155, 2010.
2. Bell JHA, Haycock JW. Next generation nerve guides materials, fabrication, growth factors and cell delivery.
Tissue Engineering 18,116, 2012.