International Conference on
Tissue Engineering and
Regenerative Medicine (ICTERM)
27 - 31 AUGUST 2014
Hosted by Tshwane University of Technology,
Department of Biomedical Sciences,
Faculty of Science
Tshwane University
of Technology
We empower people
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Scientific Committee
Dr. Keolebogile Motaung
Prof. Hari Reddi
Prof. Danie du Toit
Prof. Michael Pepper
Prof. Ugo Ripamonti
Dr. Marco Alessandrini
Prof. Sue Kidson
Dr. Janine Scholefield
Dr. Carola Niesler
Prof. Rotimi Sadiku
Organizer
Dr. SCKM Motaung
Department of Biomedical Sciences
Faculty of Science
Arcadia Campus, Nelson Mandela Drive
Tshwane University of Technology
Pretoria, South Africa
Phone: +27 12 382 6265
Fax: +27 12 382 6262
Email: motaungsckm@tut.ac.za
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Our Grateful Thanks to our Sponsors:
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ACKNOWLEDGEMENTS
We express our grateful thanks for the dedicated and outstanding help from:
Mrs Regina Motlhomi
Ms Katlego Sekwaila
Ms Amanda Van Rensburg
Ms Petro Brits
Ms Marinda Van den Berg
Ms Tanya Coetzee
Ms Mari Booysen
Mr Mario Smit
Ms Lizette de Jager
Ms Lebogang Mokgosi
Ms Brenda Chauke
Ms Mapula Razwinani
Ms Nadia Ross
Ms Annelie Bronkhorst
Dr. Abeda Dawood
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Conference programme:
Wednesday, August 27
•
16:00 Registration
PROGRAMME DIRECTORS
Dr. Keolebogile Motaung: Tshwane University of Technology, Pretoria, South Africa
and
Prof. Hari Reddi: UC-Davis Medical Center, Sacramento, USA
•
18:00 - 18:15
•
18:15 -18:30
•
18:30 -19:30Plenary Lecture: Introduction by Prof. Hari Reddi
Prof. Tony Mikos, Rice University, Texas, USA
Tissue Engineering: Challenges and Opportunities
•
19:30 Welcome City of Tshwane Metropolitan Municipality
Representative
Opening and Welcome address:
Vice-Chancellor & Principal, Prof. Nthabiseng Ogude,
Tshwane University of Technology, Pretoria, South Africa
Reception and Dinner
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Thursday, August 28
Scaffolds, Morphogens, Tissue Engineering and Regenerative
Medicine
Chairpersons:
Dr. Keolebogile Motaung: Tshwane University of Technology, Pretoria, South Africa
and
Dr. Marco Alessandrini: University of Pretoria, Pretoria, South Africa
•
08:00 - 08:25Coffee and Registration
•
08:30 - 09: 30
Skeletal Tissue Engineering: the Creation of Biological
Spare Parts:
Prof. Frank P Luyten, KU Leuven, Belgium
•
9:30 - 10:00
The rapid induction of bone formation by the transforming
growth factor-β3: The reality of a nebulous enigmatic myth:
Prof. Ugo Ripamonti, University of Witwatersrand,
Johannesburg, South Africa
•
10:00 - 10:15 Tea/Coffee Break
•
10:15 - 10:30
Enhancing Angiogenesis by Immunomodulators in the Skin
Wound Healing Model:
Gisele Calderon, Swiss Federal Institute of Technology in
Lausanne, Switzerland
•
10:30 - 10:45
The new frontiers in regenerative medicine and bone tissue
engineering: Functionalized surfaces beyond morphogens
and stem cells:
Prof. R. Duarte, University of Witwatersrand Johannesburg,
South Africa
•
10:45 - 11:00
siRNA polymer complexes in enzymatically degradable
synthetic scaffolds: Emma Doubell, University of Cape Town,
Cape Town, South Africa
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•
11:00 - 11:15
Understanding extracellular matrix dynamics is essential in
facilitating functional tissue repair:
Dr. Kyle Peter Goetsch, University of Cape Town, Cape Town,
South Africa
•
11:15 - 11:30
Generation of viable anterior cruciate ligament (ACL)
synthetic grafts from stem cell population for effective
bench to bed ACL rupture repair: Dr. Dimitrios Kouroupis,
Foundation of Research and Technology – Hellas University
Campus, loannina, Greece
•
11:30 - 11:45
Induction of collagen type II in the superficial and middle
zone of articular cartilage by resveratrol:
MJ Maepa, Tshwane University of Technology Tshwane, South
Africa
•
11:45 - 12:00
Of Mice and Mending Broken Hearts:
Megan Masters, University of Oxford, United Kingdom
•
12:00 - 12:15Analysis of the bioactivity of glasses composed by niobium:
an experimental in vivo study:
Dr. José Angelo Camilli, Institute of Biology/State University
of Campinas, Brazil
•
12:15 - 12:30
•
12:30 - 13:30Lunch
Development of Novel Real-Time Methodologies for the In
Vitro Monitoring of Cellular Differentiation:
Dr. Earl Prinsloo, Rhodes University, Grahamstown, South
Africa
Chairpersons:
Prof. Danie du Toit: Tshwane University of Technology, Pretoria, South Africa
and
Prof. Ugo Ripamonti: University of Witwatersrand, Johannesburg, South Africa
•
13:30 - 14:00
Representative from Department of Higher Education and
Training (DHET), South Africa:
Introduction by Dr. Stanley Mukhola
Dr. Engela van Staden
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•
14:00 - 15:00Translation of tissue engineering science into surgical
practice: The oral and maxillofacial surgery Experience:
Prof. Mark E Wong, University of Texas, Houston, USA •
15:00 -15:15
Hydrogels Polymerised with Matrix Metalloproteinase
Cleavage Peptides Allow for Cell Type Specific Invasion:
Dr. Neil Davies, University of Cape Town, Cape Town, South
Africa
•
15:15-15:30
Novel Graphene Oxide based Hydrogel Biocomposite with
Anti-hyperglycemic activity:
S J Owonubi, Tshwane University of Technology, Pretoria,
South Africa
•
15:30-16:00Tea/Coffee Break
Chairpersons:
Prof. Sue Kidson:
University of Cape Town, Cape Town, South Africa
and
Dr. Keolebogile Motaung: Tshwane University of Technology, Pretoria, South Africa
•
16:00 - 17:00
•
17:00 - 18:00Poster Session (participants present posters with even
numbers)
•
19:00Dinner
•
Ministry of Science and Technology: Introduction by Prof. Hari Reddi
•
Minister Naledi Pandor: Department of Science and Technology, South Africa
Articular Cartilage Regeneration: Lubrication in Tissue
Engineered Constructs:
Prof. A. Hari Reddi, UC Davis, Sacramento, USA
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Friday, August 29
Scaffolds, Stem Cells and Tissue Engineering
Chairpersons:
Prof. Hari Reddi: UC-Davis Medical Center, Sacramento, USA
and
Prof. Michael Pepper: University of Pretoria, Pretoria, South Africa
•
08:00 - 08:15Tea/Coffee Break
•
08:15 - 09:15
Mesenchymal Stem Cell Subsets and Sources for Cartilage
Tissue Engineering:
Dr. Kyla Brady, University of Bristol, Bristol, UK
•
09:15 – 9:30 Characterizations and comparison of Mesenchymal stromal
cells isolated from three depots within bone:
Prof. William Ferris, Stellenbosch University, Parrow, South
Africa
•
9:30 - 9:45
Immonogenicity of undifferentiated and differentiated
allogeneic mesenchymal stem cells:
Dr. Blessing Mukonoweshuro, University of Leeds, United
Kingdom
•
9:45 - 10:00 Tea/Coffee Break
•
10:00 - 11:00
The Role of Chondroprogenic Synovial Cells in Cartilage
Repair:
Prof. Ernst B Hunziker, University of Bern, Switzerland
•
11:00 - 11:15
The impact of visceral adiposity of the donor animal on the
differentiation potential of rat adipose-derived stromal cells:
Dr. Hanel Sadie-van Gijsen, Stellenbosch University, Parow,
South Africa
•
11:15 - 12:15
Genetic expression profile of adipose derived stem cells
differentiated into smooth muscle cells in response to low
intensity laser irradiation:
Prof. Heidi Abrahamse, University of Johannesburg,
Johannesburg, South Africa
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•
12:15 - 12:30
The effect of induced oxidative stress and antioxidant
supplementation on the adipogenic differentiation potential
of adipose-derived mesenchymal stem cells:
Danielle de Villiers, University of Pretoria, Pretoria, South
Africa
•
12:30 - 12:45
Bioengineered three-dimensional skeletal muscle tissue:
Dr. Celia Snyman, University of KwaZulu-Natal Scottsville,
South Africa
•
12:45 - 13:00
Growth and potential damage of human bone-derived cells
cultured on fresh and aged C60/Ti films:
Ivana Kopova, Institute of Physiology Academy of Sciences,
Czech Republic
•
13:00Lunch (Lunch bags available)
•
Trip to Apartheid Museum via Vilakazi Street, Tickets will be provided
•
Dinner: Museum
•
All buses leave from Prestige Auditorium at 13:15
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Saturday, August 30
Morphogens, Tissue Engineering and Stem Cells
Chairpersons:
Dr. Keolebogile Motaung: Tshwane University of Technology, Pretoria, South Africa
and
Dr. Janine Scholefield: South African Council and Industrial Research, Pretoria, South Africa
•
08:00 - 08:30
Coffee
•
08:30 - 09:30 Enabling Technologies for Tissue Engineering
Prof. Lydia E Kavraki: Rice University, Houston, USA
•
09:30 - 9:45Wnt/β-catenin and MEK-ERK signaling are required for
matrix-mediated endoderm differentiation of embryonic
stem cells:
Dr. Kevin Dzobo, University of Cape Town, Cape Town, South
Africa
•
9:45 - 10:00
•
10:00 - 10:15Tea/Coffee Break
•
10:15 - 11:15Molecular Mechanism in BMP receptor signalling
Prof. Dr. Petra Knaus, Freie Universität, Berlin
•
11:15 - 11:30
Thiazolidinediones and Wnt signaling in primary
mesenchymal stem cells:
Dr. Mari van de Vyver, Stellenbosch University, Parow, South
Africa
•
11:30 - 11:45
Modeling neural and retinal degeneration in Spinocerebellar
Ataxia type 7, using induced pluripotent tem cells:
Dr. LM Watson, University of Cape Town, South Africa
A multi-colour flow cytometry approach to quantify various
stages during adipocyte differentiation: Comparison
between Nile Red & Bodipy 493/503:
Dr. Chrisna Durandt, University of Pretoria, Pretoria, South
Africa
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•
11:45 - 12:00 Towards a predictive proliferation model for adipose-derived
stem cells:
K.J.F De Bruin, University of Pretoria, Pretoria, South Africa
•
12:00 - 12:15
Derivation of neural crest-like cells from adult corneal
keratocytes and differentiation into corneal endothelium:
Chien-Yu Dennis Lin, University of Cape Town, Cape Town,
South Africa
•
12:15 - 12:30
Targeting the ARNT complex with DMOG and Stemregenin 1
improves the expansion of primitive hematopoietic cells:
Carlo S Jackson, University of Pretoria, Pretoria, South Africa
•
12:30 - 13:30Lunch
Chairpersons:
Prof. Rotimi Sadiku: Tshwane University of Technology, Pretoria, South Africa
and
Dr. Carola Niesler: University of KwaZulu-Natal, Durban, South Africa
•
13:30 - 14:30Microsystems for Shaping and Sensing Cellular Phenotype
Prof. Alexander Revzin: University of California, Davis, USA
•
14:30 - 14:45
Differences in basic biological characteristics exist between
adipose- and Wharton’s jelly-derived mesenchymal stromal
cells:
Karlien Kallmeyer, University of Pretoria, Pretoria, South
Africa
•
14:45 - 15:00
iPSCs for modelling disease in Africa:
Dr. Janine Scholefield, South African Council and Industrial
Research, Pretoria, South Africa
•
15:00 - 15:30
Establishment of a sustainable public cord blood stem cell
bank for South Africa:
Prof. Michael Pepper: University of Pretoria, Pretoria, South
Africa
•
15:30 - 15:45
Conventional HLA Typing Techniques vs. Next Generation
Sequencing in a South African Cohort:
Juanita Mellet, University of Pretoria, Pretoria, South Africa
Page 8
•
15:45 - 16:00
•
16:00 - 16:15Tea/Coffee Break
Clinical indications for mesenchymal stem cell therapy:
Dr. Marco Alessandrini: University of Pretoria, Pretoria, South
Africa
Chairpersons:
Prof. Danie du Toit: Tshwane University of Technology, Pretoria, South Africa
and
Prof. Sue Kidson: University of Cape Town, Cape Town, South Africa
•
16:15 - 16:30
Umbilical Cord Blood Banks: A Perspective:
Carla Dessels, University of Pretoria, Pretoria, South Africa
•
16:30 - 16:45
Questioning the application of the principle of subsidiarity to
human Embryonic Stem Cell research in South Africa:
Fikile Muriel Mnisi, University of Witwatersrand,
Johannesburg, South Africa
•
16:45 - 18:00
Poster Session (participants present posters with odd
numbers)
•
19:00GALA DINNER: MANHATTAN HOTEL
•
Closing Remarks: Vice-Chancellor & Principal, Prof. Nthabiseng Ogude,
Tshwane University of Technology
Sunday, August 31
•
Departure
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Plenary Lecture
Wednesday, August 27, 2014 at 18:30
Prof. Tony Mikos, Rice University, Texas, USA
“Tissue Engineering: Challenges and Opportunities”
Introduced by Prof. Hari Reddi
UC-Davis Medical Center, Sacramento, USA
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Plenary Lecture
Antonios G Mikos
Department of Bioengineering
Rice University, Houston
Texas, USA
Phone: 713.348.5869
Fax: 713.348.5877
Email: mikos@rice.edu
“Biomaterials for Tissue Engineering”
Antonios G Mikos
Department of Bioengineering,
Rice University, Houston, Texas, USA
Biomaterial-based strategies for tissue engineering span a vast spectrum from the production
of scaffolds tailored with appropriate mechanical properties and degradation kinetics to serve
transiently as a bridge to tissue formation to the leverage of biomaterials for the controlled delivery
of biological signals to regenerate tissue in specific sites in the body. For example, our laboratory
has developed a variety of biodegradable polymers for the controlled delivery of bioactive agents
and/or stem cell populations to promote regeneration of tissues such as bone and cartilage. We
have also applied engineered culture of cell populations on three-dimensional scaffolds toward the
development of biologically active hybrid scaffold/extracellular matrix constructs for regenerative
medicine applications as well as testing of anticancer drugs. This talk will present recent examples
of biomaterial-based approaches for the development of tissue engineering technologies to meet
clinical needs.
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Scaffolds, Morphogens, Tissue Engineering
and Regenerative Medicine
Session Presentations
Thursday, 28 August 2014
08:30 – 12:30
Chairpersons:
Dr. Keolebogile Motaung:
Tshwane University of Technology, Pretoria, South Africa
Dr. Marco Alessandrini:
University of Pretoria, Pretoria, South Africa
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Frank P Luyten, MD, PhD
Skeletal Biology and Engineering Research Center &
Prometheus Division of Skeletal Tissue Engineering
KU Leuven, Belgium
Email: Frank.luyten@uzleuven.be
“Skeletal Tissue Engineering:
the Creation of Biological Spare Parts”
Frank P Luyten
Skeletal Biology and Engineering Research Center & Prometheus, Division of Skeletal
Tissue Engineering, KU Leuven, Belgium
The clinical impact of cell based therapeutic approaches for applications in the field of tissue
regeneration is still limited. More information is needed on many aspects of these “Advanced
Therapeutic Medicinal Products” including the choice of cell type and scaffolds, manufacturing
technology, and the mechanisms of action. Indeed, the mechanisms by which stem cells may
contribute to tissue repair are diverse and include trophic effects, anti-inflammatory and immune
modulatory effects and direct tissue repair through engraftment.
In an effort to optimize bone engineering, we have explored the rational selection of components
for a combination product including cells, growth factors such as Bone Morphogenetic Proteins,
and scaffold materials, based on biomaterial properties, and evaluation of their bone formation
capacity. It was found that the cell-factor-material combinations behaved quite differently in vivo,
despite apparent in vitro similarities in gene expression profiles. Our data highlight the importance
of matching a biological matrix with stem cell type and growth factor, and identify parameters that
can be used for the rational selection of combination products for tissue engineering.
We also introduced the “developmental engineering” methodology for the design of robust
manufacturing processes of combination products. This new methodology is based on the design
of in vitro processes consisting of sequential sub-processes corresponding to in vivo developmental
stages. They follow a gradual and coordinated progression of tissue growth and cell differentiation
that leads to organization of cells into intermediate tissue forms. The macroscopic developmental
modularity of tissues can be attributed to a corresponding modularity of the network topology that
describes gene interactions during the developmental process. Microarray studies with a variety
of analysis approaches such as gene expression dynamics and principal component analysis, gene
clustering and osteogenic network analysis provide input on the factors/pathways critical in driving
the bone formation processes.
In conclusion, a second wave of new and more rational approaches for tissue engineering in
general, and bone repair in particular, will hopefully lead to more predictable clinical outcomes.
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U Ripamonti
Bone Research Laboratory, School of Physiology, and Department of Internal Medicine, School of Clinical Medicine
Faculty of Health Sciences, University of the Witwatersrand,
Johannesburg, South Africa
E-Mail: ugo.ripamonti@wits.ac.za
“The rapid induction of bone formation by the transforming
growth factor-β3: The reality of a nebulous enigmatic myth”
U Ripamonti; RM Klar; R Duarte; T Dix-Peek and C Ferretti
Bone Research Laboratory, School of Physiology, Faculty of Health Sciences and
Department of Internal Medicine, School of Clinical Medicine, University of the
Witwatersrand, Johannesburg, South Africa
The last words of our paper for this seminal International Conference on Regenerative Medicine in
the African continent August 2014 re-deploy Marshall Urist’ 1968 Editorial in Clinical Orthopaedics
& Related Research where the famous to be author indicated how bone induction was seen by
several basic scientists as a nebulous enigmatic myth. Almost 47 years later the Bone Research
Laboratory not in Los Angeles but in Johannesburg still grapples with the reality of the nebulous
enigmatic myth of human osteoinduction. 125 µg of recombinant human transforming growth
factor-β3 (TGF-β3) per gr of insoluble collagenous matrix implanted in full thickness mandibular
defects of the Chacma baboon Papio ursinus induce significant and unprecedented restitutio ad
integrum with mineralization of the newly induced cortical plates as early as 30 days. To resolve the
induction of bone formation, 7% hydroxyapatite/calcium carbonate macroporous constructs (7%
HA/CC) were pre-loaded with 125 µg hTGF-β3 or 125 µg hNoggin or a binary application of 125 µg
hTGF-β3 /hNoggin and implanted in the rectus abdominis of muscle of Papio ursinus. 7% HA/CC
were also pre-loaded with 240 µg of the osteoclast inhibitor biphosphonate zoledronate or with 500
µg of the calcium blocker verapamil hydrochloride. Generated tissues on days 15, 60 and 90 were
processed for morphological and molecular analyses by qRT-PCR. hNoggin-treated constructs
at all time periods showed a profoundly delayed tissue patterning with limited collagenous
condensations and bone formation. Biphosphonate zoledronate and calcium blocker verapamil
hydrochloride-treated constructs yielded limited or absent induction molecularly ascribed to
significant up-regulation of Noggin. hTGF-β3 -treated bioreactors significantly up-regulated
BMP-2 on day 60 and 90 as well as BMP-7 together with down-regulation of Noggin on day 60
and 90 correlating with the prominent induction of bone formation. On day 15, hTGF-β3 -treated
bioreactors showed differentiation of fibrin/fibronectin rings structurally providing anchorage for
hyper chromatic differentiating osteoblasts reprogrammed from differentiated invading myoblasts/
pericytes. RUNX-2 and Osteocalcin expression were up-regulated in hTGF-β3 -treated bioreactors
supporting the morphological observations of invading rapidly differentiating cells. Physiological
expression of BMPs genes upon implantation of hTGF-β3 may escape the antagonist expression
of Noggin, whereas direct application of hBMPs, representing a later by-product step of the bone
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induction cascade as set by the TGF-β3 master gene, sets into motion Noggin’ antagonist action, as
shown by the limited effectiveness of hBMPs in clinical contexts. The induction of bone as initiated
by hTGF-β3 is via BMPs expression with hTGF-β3 controlling the induction of bone by regulating
the expression of BMPs via Noggin expression. hTGF-β3 elicits bone induction by up-regulating
endogenous BMPs and is blocked by Noggin providing insights into performance failure of hBMPs
in clinical contexts. The induction of bone by hTGF-β3 has been translated in clinical contexts to
treat a large mandibular defect in a paediatric patient; 30 months after implantation of 250 µg
hTGF-β3 per gram of human demineralized bone matrix, radiographic analyses show the complete
reconstruction of the avulsed large mandibular segment including the induction of the avulsed
coronoid process.
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Gisele Calderon
École Polytechnique Fédérale de Lausanne
(EPFL, Swiss Federal Institute of Technology in Lausanne)
1015 Lausanne, Switzerland
Mobile: 2252239957
E-mail: gcaldero@tulane.edu
“Enhancing Angiogenesis by Immunomodulators in the Skin
Wound Healing Model”
Gisele Calderon; Priscilla Briquez; Jeffrey Hubbell
Swiss Federal Institute of Technology in Lausanne EPFL, Switzerland
Engineering complex, vascularized tissue for regeneration requires a fundamental understanding
of the immunomodulators affecting the stages of healing. Regeneration, the immune system, and
angiogenesis are all interconnected where angiogenesis is tightly mediated by the immune system.
Additionally, angiogenesis is one of the key and initial indications for effective regeneration. Chronic
wounds pose a clinical issue for diabetic and aged patients with limited treatment options. Hence we
aim to rescue chronic wounds to parallel natural wound healing by studying the intercommunication
between immune system and angiogenesis. Wound healing involves several stages beginning with
a fibrin clot formation. The immune system then impacts angiogenesis and tissue regeneration
through initiating a cascade of cytokines, chemokines, and growth factors that induce inflammation.
Subsequently, inflammation and proliferation promote regeneration. Lastly, angiogenesis
vascularizes and feeds the regenerated tissue. Using angiogenesis as a measure of regenerative
capacity, we screened immunomodulators that can positively influence wound healing.
This investigation essentially involves two stories: an in vitro model for determining pro-angiogenic
immunomodulators and an in vivo model for characterizing the immune cell population during
wound healing. For the first portion of the immune system-.angiogenesis crosstalk, we investigated
human umbilical vein endothelial cells (HUVEC) that organize into tubule formation with angiogenic
promoters. We exploited this established in vitro model by monitoring tubule formation deviation
from the organized network formed when HUVECs are seeded on BD MatrigelTM, a rich extracellular
matrix. Images were taken over twenty-four hours for analysis of angiogenic parameters (i.e. total
tubule length). Simultaneously, chemokine families were screened for binding affinity to fibrinogen
to determine whether the immunomodulators can be delivered locally to the wound and remain
attached at the site. Based on this analysis, promising pro-angiogenic immunomodulators will be
delivered in vivo for assessing possible amelioration of skin regeneration in chronic wounds. Moving
onto the identification of immune cell populations, we optimized in vivo characterization in a dorsal
skin wound healing model in C57BL/6 wild-type mice and db/db diabetic mice as our representation
of non-impaired wound healing and chronic wound healing respectively. Briefly, four wounds were
performed on the dorsal skin of the mice using a six mm biopsy punch. Hydrofilms were placed
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over the wounded skin to reduce the chances of infection. The dorsal skin was then digested at the
following time points in order to determine the cell populations with flow cytommetry: 5 hours, 1
day, 3 days, 7 days, and 14 days post- surgery. This tool allowed for a timeline of the immune cell
population infiltrating the wound site with side-by-side comparison of the two mice models. As a
result, we were able to define the deficiency in the chronic wound healing timeline.
Since the immune system has a major role in wound healing, the cell populations at the injured
site impact angiogenesis and therefore the repair process. We identified immunomodulators with
improved angiogenic protential and characterized the immune cells present at different stages of
wound healing. By honing the immunomodulator targets to achieve functional, rapid angiogenesis,
our study has clinical translational potential.
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R Duarte
Bone Research Laboratory, School of Physiology
Faculty of Health Sciences, Department of Internal Medicine,
School of Clinical Medicine
University of the Witwatersrand, Johannesburg, South Africa
E-Mail: Raquel.duarte@wits.ax.za / ugo.ripamonti@wits.ac.za
“The new frontiers in regenerative medicine and bone tissue
engineering: Functionalized surfaces beyond morphogens and
stem cells”
R Duarte; RM Klar; T Dix-Peek and U Ripamonti
Bone Research Laboratory, School of Physiology, Faculty of Health Sciences,
Department of Internal Medicine, School of Clinical Medicine, University of the
Witwatersrand, Johannesburg, South Africa
The central question in developmental biology and thus regenerative medicine is the molecular
basis of pattern formation. What next beyond morphogens and stem cells? Characterization
of structure-function relationship of functionalized surface topographies is the novel tissue
engineering paradigm the 21st century. The fundamental tenet of tissue engineering is to combine
soluble molecular signals with insoluble signals or substrata to erect scaffolds of biomimetic
biomaterial matrices that mimic the supramolecular assembly of the extracellular matrix.
We have modified the tissue engineering paradigm by constructing macroporous biomimetic
matrices that per se, and without exogenous application of the osteogenic molecular signals of
the transforming growth factor-beta (TGF-beta) supergene family, initiate the ripple-like cascade
of bone differentiation by induction. Osteoclastic priming of calcium carbonate/hydroxyapatite
macroporous surfaces sets lacunae, pits and concavities which are the driving morphogenetic and
topographic cues that set into motion the induction of bone formation in a continuum of sequential
phases of resorption/dissolution and induction of bone formation biomimetizing the remodelling
cycle of the cortico-cancellous bone. The driving force of the intrinsic osteoinductivity of biomimetic
matrices is the shape of the implanted scaffold. The language of shape is the language of
geometry; the language of geometry is the language of a sequence of repetitive concavities that
biomimetize the remodelling cycle of the cortico-cancellous bone. To evaluate the role calcium ions
and osteoclastogenesis, calcium carbonate/hydroxyapatite macroporous constructs with limited
conversion to hydroxyapatite (7% HA/CC) were pre-loaded with either 500 cues µg of the calcium
channel blocker verapamil hydrochloride or 240 µg of the osteoclast inhibitor biphosphonate
zoledronate and implanted in the rectus abdominis muscle of Papio ursinus. Generated tissues
on days 15, 60 and 90 were processed for histomorphometry and molecular analyses by qRT-PCR.
Zoledronate-treated specimens showed profoundly delayed and inhibited tissue patterning and
morphogenesis with limited bone formation. Osteoclastic inhibition yielded minimal bone formation
so macroporous constructs pre-treated with the Ca++ channel blocker verapamil hydrochloride.
Page 18
Down-regulation of BMP-2 together with up-regulation of Noggin genes correlated with limited
induction of bone formation. The induction of bone is initiated by a local peak of Ca++ activating stem
cell differentiation, angiogenesis and the induction of bone formation. The concavities biomimetize
the ancestral repetitive multi-tested designs and topographies of Nature; myoblastic/pericytic
perivascular stem cells sense the substratum upon which cells attach and migrate; migrating
and attaching cells onto the substratum are able to convert geometrical and mechanical cues
into triggering gene expression pathways including the BMPs pathway. Functionalization of smart
biomimetic surfaces self-inducing specific gene products which structure-function/activity profile
resulting in tissue morphogenesis regulated by the geometry of functionalized substrata is the true
challenge for regenerative medicine and tissue engineering of the 21st Century.
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Emma Doubell
University of Cape Town
Cape Town
Mobile: +27 846643230
E-mail: dblemm001@myuct.ac.za
“siRNA polymer complexes in enzymatically degradable
synthetic scaffolds”
Emma Doubell and Neil H Davies
University of Cape Town, South Africa
Introduction: RNAi has previously been shown to be a viable treatment for a range of pathologies,
including myocardial infarction (MI). However, a continual obstacle to its use in the clinic is its
successful delivery. One possible novel approach towards generating sustained knockdown might
be through controlled and localised release of siRNA polymer complexes from an injectable scaffold.
We have now begun to investigate the suitability of an injectable synthetic polyethylene glycol (PEG)
gel. Polyethylenimine (PEI) was used as complexing polymer.
Methods: PEI:siRNA complexes were formed at a range of N:P ratios. Complex formation,
RNAse protection and stability were determined by electrophoresis fluorescence quenching. Their
cytotoxicity and transfection efficacy were assayed utilising a cell quantification assay and siRNA
that induce cell death. siRNA:PEI complexes were polymerised within enzymatically degradable PEG
hydrogels. Complex elution and stability within PEG hydrogels were determined by fluorescence
quantification and confocal microscopy. A noval 3D assay was developed to allow for determination
of transfection efficacy in gel.
Results: Complex formation was observed from N:P ratios ≥5:1. The 20:1 and 40:1 ratios were
most stable and protected siRNA from degradation for >4 days. No significant difference was
observed for cytotoxicity between the ratios. The 20:1 ratio displayed substantially higher levels
of transfection efficacy (p<0.001) compared to lower ratios, and had lower levels of cytotoxicity
compared to higher ratios. A PEI/siRNA ratio of 20:1 was determined to be the optimal N/P ratio.
PEI/siRNA complexes were shown to be entrapped within PEG hydrogels for 11 days with <10% of
the siRNA being released. Initial data indicates its potential effectiveness in gel transfection.
Conclusion: PEI:siRNA complexation was optimised. The complexes were found to be efficiently
entrapped within degradable synthetic hydrogels. These hydrogels show potential to act as scaffolds
that are able to release complexed siRNAs.
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Kyle Peter Goetsch
Cardiovascular Research Unit
Department of Health Sciences
University of Cape Town
14 Muswell Hill Rd, Mowbray
Cape Town, 7705
Mobile: 0721869180
E-mail: kpgoetsch@gmail.com / kyle.goetsch@uct.ac.za
“Understanding extracellular matrix dynamics is essential in
facilitating functional tissue repair”
K. P. Goetsch1; C. Chokoza1; C. Snyman2; D. Bezuidenhout1; K. H. Myburgh3;
C. U. Niesler2 and N. H. Davies1
1
Cardiovascular Research Unit, Department of Health Sciences, University of Cape
Town, Cape Town. South Africa; 2Discipline of Biochemistry, School of Life Sciences,
University of KwaZulu-Natal, Pietermaritzburg, South Africa; 3Department of
Physiological Sciences, University of Stellenbosch, Stellenbosch, South Africa
Tissue repair after injury depends on the deposition of a fibrous extracellular matrix (ECM) to
replace lost or damaged tissue. Depending on the severity of the damage, the ECM is then remodelled over time to emulate normal tissue. The ECM is also directly involved in regulating
the behaviour of a wide variety of cell types that are mobilized to the damaged area, modulating
interactions between cells and the ECM in order to regulate key events required for repair; such
as cellular proliferation, migration, and differentiation. The ECM is also highly tissue specific,
recruiting specialized cells for repair via growth factors and cytokines.
Previously, we have demonstrated the highly specific role of the ECM in regulating myoblast
migration during skeletal muscle repair in vitro. In this study we showed decorin, an antifibrotic
proteoglycan, regulated cellular migration in combination with collagen I, the primary ECM
component during skeletal muscle repair, via the Rho/ROCK signalling cascade. We also
demonstrated the importance of investigating the effects of ECM components in combination
providing a holistic point of view during skeletal muscle repair.
Following this study we wished to mimic the ECM with a synthetic hydrogel through which we
could regulate both the mechanical and biological properties of the ECM during cardiovascular
repair. As a mimic we used an engineerable polyethylene glycol (PEG) hydrogel. This synthetic
hydrogel was chosen for its mechanical properties, providing a stiff support scaffold, and is amongst
the most inert synthetic biomaterials at this time. The PEG system utilised also allows for extensive
biological modifications. In this study we assess the effect of the PEG hydrogel’s stiffness and biodegradability on bone marrow-derived stem cell migration. We also attach chemo-attractant growth
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factors within the gel and assess the recruitment potential of the gel, mimicking the native ECM.
Taken together these data demonstrate the important role of the ECM and how understanding the
complex relationship of cell-ECM signalling can contribute to future tissue regenerative therapies
in vivo.
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Dimitrios Kouroupis
Foundation of Research and Technology, Hellas
Institute of Molecular Biology & Biotechnology
Department of Biomedical Research
University Campus, loannina, Greece
Persefonis 16, Ekali
Athens, Greece 14578
Mobile: 00306942577863
E-mail: kouroupis_d@yahoo.gr
“Generation of viable anterior cruciate ligament (ACL) synthetic
grafts from stem cell populations for effective bench to bed ACL
rupture repair”
Dimitrios Kouroupis1; Athena Kyrkou1; Eleni Triantafyllidi3; Michalis
Katsiboulas45; Dimitris Stellas6; George Chalepakis7; Marika Syrrou8;
Apostolos Klinakis6; Anastasios Georgoulis2; Carol Murphy1 & Theodore
Fotsis19
1
Orthopedic Sports Medicine Center, Department of Orthopeadic Surgery, University of
loannina, loannina, Greece; 2Department of Pathology, University of loannina, loannina,
Greece; 3Centre for experimental surgery, Biomedical research foundation of the
Academy of Athens, Athens, Greece; 4Attikon animal hospital, Paiania, Athens, Greece;
5
Center for Basic Research, Biomedical research foundation of the Academy of Athens,
Athens, Greece; 6Department of Biology, Electron microscopy laboratory, University of
Crete, Crete, Greece; 7Laboratory of Biology, Medical School, University of loannina,
loannina, Greece; 8Laboratory of Biological Chemistry, Medical School, University of
loannina, loannina, Greece
Tendon and ligament injuries are the commonest adult health problems accounting for about half
of all musculoskeletal injuries. Anterior cruciate ligament (ACL) is the key knee ligament as ACL
rupture results in the loss of whole joint stability leading to meniscal ruptures, cartilage damage
and early osteoarthritis. Arthroscopic reconstruction techniques with autografts or allografts have
known drawbacks such as ligament laxity, donor site morbidity, and long recovery period. Our
hypothesis is that tissue engineered ACL that consist of, and is regenerated from, living cells is
biomechanically, biochemically and morphologically superior to any artificial construct.
In the present study, we combined stem cell technology with a non-absorbable biomaterial to
reconstruct the ruptured ACL. Towards this purpose, we have generated a 3D tissue engineered
construct by differentiating mesenchymal stem cells derived from subcutaneous human adipose
tissue (hAT) or from induced-pluripotent stem (iPS) cells on the biomaterial for 21 days in vitro.
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Moreover, iPS cells were generated by reprogramming of human foreskin fibroblasts and were
precommitted towards mesodermal fate in vitro (iPS-MSCs). Three-dimensional differentiation
of stem cells on the biomaterial has been performed in a way to closely mimic normal human
ACL’s structure compartmentalisation in vivo. Moreover, simultaneous differentiation of stem cells
towards ligamentous and osseous compartments was achieved by induction with either TGFβ/FGF2
or BMP2/FGF2 growth factor cocktails in different regions of the biomaterial. The tissue engineered
ACL construct has then been implanted in a swine ACL rupture model in place of the extracted
normal ACL. Four months post-implantation, the tissue engineered ACL graft generated a superior
ACL-like tissue showing morphologically and biochemically similar characteristics to normal ACL.
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MJ Maepa
Department of Biomedical Sciences
Tshwane University of Technology
Pretoria, 0001
Email: MaepaMJ@tut.ac.za
“Induction of collagen type II in the superficial and middle zone
of articular cartilage by resveratrol”
MJ Maepa and SCKM Motaung
Department of Biomedical Sciences, Tshwane University of Technology, Pretoria, 0001
Objectives: The aim of the study is to investigate whether resveratrol compound will have the
ability to synthesize collagen type II in superficial and middle zone of articular cartilage.
Background: Articular cartilage is an avascular tissue with limited innate potential for repair
and regeneration. In articular cartilage, superficial zone protein is specifically synthesized and
secreted by superficial zone chondrocytes and collagen type II is synthesized in the middle and
deep zone chondrocytes. Collagen type II is primarily responsible for tensile and compressive
strength in cartilage. It is therefore hypothesized that resveratrol compound would participate in
the differentiation and production of collagen type II in articular chondrocytes.
Materials and Methods: Chondrocytes were isolated from pig joints and cultured as a monolayer
at a density of 1 X 105 cells/well. Cell cultures were treated with various concentration of resveratrol
for 4 days. The levels of inducible collagen type II were measured by western blot and ELISA. The
expressions of collagen type II were determined by RT-PCR.
Results: Resveratrol compound increased collagen type II accumulation in the media. This
increased collagen type II accumulation was also reflected by increase collagen type II mRNA
expression as assessed by RT-PCR. The increase of collagen type II accumulation in the media was
also confirmed by IHC.
Conclusion: This present study reveals that resveratrol is an important regulator of collagen
type II protein in different zones of articular cartilage. Administration of resveratrol compound may
contribute to develop a novel treatment for osteoarthritis
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Megan Masters
Department of Physiology, Anatomy and Genetics
University of Oxford
Mobile: +44 7710268278
Telephone: +44 1865282369
Email: megan.masters@dpag.ox.ac.uk
“Of Mice and Mending Broken Hearts”
Megan Masters and Paul R Riley
Department of Physiology, Anatomy and Genetics, University of Oxford
Coronary heart disease (CHD) remains a leading cause of death worldwide. When a coronary
artery becomes blocked, the contingent heart muscle undergoes ischemic damage a non-contractile
scar forms in its place. Consequently, the heart remodels and chronic heart failure ensues. Despite
the rising incidence of CHD and heart failure, the only effective cure remains a heart transplant;
for which the demand for donor organs far outweighs supply. The gold standard cure would be to
non-invasively restore lost heart muscle (myocardium) and vasculature (coronaries), to regenerate
the organ and recover full function. One approach to achieve this is to stimulate resident cell
populations which are instructive towards myogenesis [1]. The epicardium (the mesothelium which
forms the outer layer of the heart) is one such cell population: it acts as a progenitor/stem cell
pool for numerous cardiac cell types and provides trophic signals instructive to both myocardial
development in mammals and heart regeneration in lower vertebrates.
While lower vertebrates are able to recover their hearts from substantial injury throughout life,
it was long believed that heart regeneration was a capacity lost to mammals through evolution.
However, recent technical leaps showed that newborn mice can regenerate heart muscle and
function within weeks, but only if injured within the first few days of life [2]. If injured on or after
postnatal day seven (P7), scarring and fibrosis are observed and adult like wound healing ensues.
This brief time period after birth has been coined the “neonatal regenerative window”. Through
inducing ischemia in the neonatal mouse heart, we aim to understand the cell and molecular
underpinnings of this ‘switch’ from regeneration to scarring and fibrosis in a mammalian setting;
identify key mechanisms of the former; and extrapolate these findings to regenerating failing
human hearts.
Given the roles of the epicardium in instructing developmental and regenerative myogenesis,
we aimed to investigate the potential role(s) of this cell population and its derivative signals in
mammalian heart regeneration. Previous studies showed that inhibiting one epicardial signal,
retinoic acid (RA) signalling, blocked the myogenesis central to both mammalian heart development
and zebrafish heart regeneration [3, 4]. Here we implicate RA signalling in neonatal heart
regeneration in mammals. We further implicate limited RA signalling in the insufficient regenerative
response of the injured adult heart. We conclude that epicardial RA signalling may be important in
Page 26
modulating the regenerative versus fibrotic response in the mammalian heart; findings which may
be significant in developing novel therapies to treat, or even cure heart failure.
Page 27
José Angelo Camilli
Institute of Biology
State University of Campinas, Brazil
Rua Jacinto Guiraldelli, 789
Bairro Jardim Alvorada
Pederneiras, SP, Brazil
CEP: 17 280 000
Mobile: +55 14997939839
Telephone: 55 1 432 522 673
E-mail: jcamilli@unicamp.br
“Analysis of the bioactivity of glasses composed by niobium:
an experimental in vivo study”
Lucas Pereira Lopes de Souza; Ana Claudia de Souza; Wilson Romero
Nakagaki and José Angelo Camilli
Department of Structural and Functional Biology, Institute of biology, State University
of Campinas, Brazil
The Bioglass® 45S5 is a kind of biomaterial made from the SiO2 – CaO - Na2O complex that has
the capability to form connecting bridges between live tissue and the glass, beyond being bioinert. The
Bioglass® discovery encouraged others researchers to seek other materials that also show the same
properties of Bioglass® 45S5. However, no biomaterial produced by them has enough mechanical
performance to be used under a constant load. Some recent studies have showed that putting niobium
oxide into the glass network would be a solution to improve its mechanic resistance, keeping the
biocompability and bioactivity of the bioglass. Within this context, the aim of this study was to evaluate
the bioactive properties of glasses enriched with Nb2O5. The bioglass, in the powder form, containing
different concentrations of niobium oxide was implanted into tissue muscle of rats. The Bioglass®
45S5 was implanted as a control group. Moreover, bioglasses in the form of dense cylinders,
containing the same niobium oxide concentrations of the powder preparations, were implanted into
defects produced in the tibia of the animals to assay the bioactivity of the biomaterial into the bone.
The animals were euthanized two and four weeks postoperative.
Histological slides were stained with hematoxyin and eosin for qualitative and quantitative
measurements. In addition, an immunohistochemical technique was utilized to assess the material’s
capability to stimulate the angiogenesis and cell proliferation. All the bioglasses showed bioactivity
and biocompatibiliy into the receptor sites of the implants. However, the bioglass with niobium oxide
concentration of 1.3 had the better results regarding angiogenesis and bone formation. The main
conclusion of the experiment was that the BGPN1.3 is a promising material, which could be used as a
bioactive glass to repair defects in bones that support great mechanical load.
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Earl Prinsloo
Biotechnology Innovation Centre
Department of Biochemistry, Microbiology and Biotechnology
PO Box 94
Rhodes University
Grahamstown, 6140
Mobile: +27 718711334
Telephone: +27 466 038 082
Fax: +27 466037576
E-mail: e.prinsloo@ru.ac.za
“Development of Novel Real-Time Methodologies for the In Vitro
Monitoring of Cellular Differentiation”
Earl Prinsloo; Adam H Kramer; Julia Joos-Vandewalle and John Honiball
Biotechnology Innovation Centre, Rhodes University, South Africa
Cellular differentiation is a lineage dependent complex process. Proliferative and morphological
changes that occur are measurable phenomena typically by endpoint analysis.
End point assays are flawed in the potential skewing of data due to preparation of samples. The
rise of real-time technologies permit a global view of these observable events. The availability
of commercial Electric Cell-substrate Impedance Sensors (e.g. ACEA xCELLigence Real-time
Cell Analyser) has facilitated the rapid development of methodology to monitor and optimize
differentiation protocols. Using well established models of differentiation 9adipogenesis,
osteogenesis etc) we investigated the development of novel methods using a real-time ECIS system
and symbolic regression to identify specific patterns that may allow for screening of libraries of
potential small molecule morphogens.
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Session Presentations
Thursday, 28 August, 2014
13:30 – 16:00
Chairpersons:
Prof. Danie du Toit:
Tshwane University of Technology, Pretoria, South Africa
Prof. Ugo Ripamonti:
University of Witwatersrand, Johannesburg, South Africa
Page 30
Mark E Wong, DDS
Oral and Maxillofacial Surgery
University of Texas, Health Science Center
Houston
Email: Mark.E.Wong@uth.tmc.edu
“Translation of tissue engineering science into surgical
practice: the oral and maxillofacial surgery experience”
Prof. Mark E. Wong, DDS
Oral and Maxillofacial Surgery, University of Texas, Health Science Center, Houston
The oral and maxillofacial region in the body represents a highly challenging area for
reconstructive surgery. Defined as the area above the neck and below the cranium, it is a highly
visible part of the human anatomy that contains several organs responsible for the special senses
and hollow structures that are exposed to the external environment. In addition, the oral and
maxillofacial region is composed of multiple paired structures so fidelity of reconstruction is obvious
when compared to the normal or unaffected side. The potential for tissue engineering to replace
autologous or alloplastic reconstructive techniques is well established by the ability to produce
tissue from a combination of cells, scaffolds and biological factors. However, when these techniques
are considered as a viable alternative to current methods of oral and maxillofacial reconstruction,
the ability to regenerate composite structures containing bone, muscle, and epithelial lining tissue
that can support the functions of the facial skeleton and tolerate exposure to air, secretions and
saliva must be evaluated.
This presentation will discuss current reconstructive techniques used in oral and maxillofacial
reconstruction, including the replacement of bone, soft tissue and specialized structures such
as teeth. Strategies to improve on these methods using tissue engineering techniques will be
presented and the challenges facing the translation of these techniques from the laboratory into
clinical practice will be discussed.
Page 31
Neil Davies
University of Cape Town
Cardiovascular Research Unit
Cape Town, South Africa
Mobile: +27 834156645
Telephone: 27 21 4066613
Fax: 27 21 4485945
E-mail: neil.davies@uct.ac.za
“Hydrogels Polymerised with Matrix Metalloproteinase
Cleavage Peptides Allow for Cell Type Specific invasion”
Neil H Davies; Mona Bracher; Deon Bezuidenhout; Thomas Franz and
Peter Zilla
Cardiovascular Research Unit, University of Cape Town, South Africa
Introduction: Precise regulation of cellular ingrowth is a key component of biomaterial based
regenerative scaffolds. Synthetic extracellular mimic hydrogels hold significant potential in this
area and they allow for a systematic approach towards incorporation of biological signals. We have
begun to investigate the ability of these types of hydrogels to selectively influence the invasion of
specific cell types. Invasion of fibroblast and smooth muscle cells (SMC) into hydrogels polymerised
with different matrix metalloproteinase (MMP) cleavage sequences was assayed.
Methods: Polyethylene glycol (PEG) based hydrogels were crosslinked with either a pan-MMP
(sequence cleavable by wide range of MMPs); MMP-14 or MMP-9 specific cleavage peptides. The
hydrogels’ physical properties were characterised with rheology and swelling studies. 3-D cell
spheroids (SMC or fibroblasts) were then cultured in the various PEG hydrogels. All hydrogels
were also modified with the cell binding substrate RGD. Cellular invasion was quantified by image
analysis of phalloiden stained spheroids.
Results: Hydrogels formed with the different MMP substrates had very similar physical properties
and enzyme specificity was preserved after incorporation into hydrogels. Cellular invasion was
completely inhibited by the MMP inhibitor GM6001 demonstrating the absolute dependence of cell
invasion in these hydrogels on the activity of MMPs. The two cell types invaded the MMP permissive
hydrogel to the same extent. However, the invasion of SMC into MMP-14 selective peptide crosslinked
hydrogels was diametrically opposite in nature to that of fibroblasts whereby SMC had a two-fold
increase in sprouting into these hydrogels relative to that observed permissive hydrogels whilst
fibroblasts had a relative two-fold decrease (p<0.01). Both cell types showed a significant decrease
of invasion into MMP-9 crosslinked hydrogels relative to that into pan-MMP crosslinked hydrogels.
Discussion and conclusions: These findings indicate that the use of MMP specific peptide
crosslinkers has the potential to selectively regulate individual cell type invasion and growth. The use
Page 32
of MMP specific cleavage sites may be an additional means towards optimising tissue engineering
matrices. It could also have general utility in exploring cellular mechanisms of invasion.
Page 33
SJ Owonubi
Department of Polymer Technology
Tshwane University of Technology
CSIR Campus, Building 14D
Private Bag X025, Lynwood Ridge 0040
Pretoria, South Africa
E-Mail: oshesan@gmail.com
“Novel Graphene Oxide based Hydrogel Biocomposite with Antihyperglycemic activity”
SJ Owonubi; BA Aderibigbe and ER Sadiku
Department of Polymer Technology, Tshwane University of Technology
The development of any novel drug delivery systems is an essential step toward controlled
drug targeted administration within the body. It is the desire of any drug delivery mechanism to
be introduced into the body with minimal toxicity and also possess the capacity to release the
encompassed drug to the intended location and at a controlled rate. Hydrogel biocomposites
containing a combination of thermally reduced graphene oxide, natural and synthetic polymers were
prepared followed by pH swelling analysis and drug release studies at selected pH values. Drugs
that exhibit anti-hyperglycemic activity were loaded onto the hydrogel biocomposites in selected
ratios. In vitro analysis was performed against 3T3-L1 pre adipocytes cell lines. The biocomposites
were further characterized by Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric
Analysis (TGA), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM)
and RAMAN.
Page 34
Session Presentations
Thursday, 28 August 2014
16:00 – 18:00
Chairpersons:
Dr. Keolebogile Motaung:
Tshwane University of Technology, Pretoria, South Africa
Prof. Sue Kidson:
University of Cape Town, Cape Town, South Africa
Page 35
A Hari Reddi
Lawrence Ellison Center for Tissue Regeneration
Department of Orthopedic Surgery
University of California Davis
School of Medicine
Sacramento CA
USA
Telephone: +1 (916) 734-3311
E-mail: ahreddi@ucdavis.edu
“Articular cartilage regeneration: Lubrication in tissue
engineered constructs”
A Hari Reddi
Lawrence Ellison Center for Tissue Regeneration, Department of Orthopedic Surgery,
University of California Davis, School of Medicine, Sacramento CA, USA
Tissue engineering is the science of design and construction of tissues for the functional
restoration of damaged tissues due to diseases such as arthritis and trauma. Regeneration in
general recapitulates embryonic development and morphogenesis. The form and function of
articular cartilage is altered due to degenerative damage in osteoarthritis.
The three key obligatory ingredients for tissue engineering are: signals for morphogenesis,
stem/progenitor cells that respond to morphogens and scaffolds that are biomimetic of the
extracellular matrix. Thus, the regeneration of articular cartilage is based on the optimal
integration of signals, stem cells and scaffolds. Recent advances in morphogens, stem cells and
scaffolds have improved the bulk mechanical properties of the tissue engineered constructs.
However, the optimal friction coefficient and wear properties of tissue engineered cartilage has
not kept pace. The near friction-free properties and wear-resistance is a prerequisite for the tissue
engineered cartilage constructs.
The recent progress in this area of tribology of tissue engineered constructs will be presented
and discussed.
Page 36
Poster Presentations
Thursday, 28 August 2014
17:00 – 18:00
EVEN NUMBERED
POSTERS
Page 37
Poster
Tariq Ganief
Institute of Infectious Disease and Molecular Medicine
University of Cape Town Medical School
UCT Faculty of Health Sciences
Observatory 7925
South Africa
Mobile: +27 789277334
E-mail: taganief@gmail.com
“A proteomic investigation into the molecular mechanism
of HIV-Tat induced neuronal apoptosis in HIV-associated
dementia”
Tariq Ganief; Shaun Garnett and Jonathan Blackburn
Institute of Infectious Disease and Molecular Medicine,
University of Cape Town Medical School
HIV related neurocognitive disorders affect up to 70% of HIV patients with varying degrees of
severity. While there has been a great deal of work suggesting various viral and host molecules and
pathways which may ultimately result in neuronal apoptosis, there are no all-encompassing data to
consolidate these findings and provide insight as to how they may all function together. To this end,
we performed SILAC based quantitative proteomic analysis on HIV-Tat treated neuroblastoma cells.
Isolated protein was fractionated by PAGE and analysed by nLC-MS/MS on the orbitrap velos. Using
MaxQuant, we identified 2791 unique protein groups with quantitation by minimum two unique
peptides. Using the student’s t-test, we identified 482 differentially regulated proteins, which were
analysed using Ingenuity Pathway Analysis (IPA).
Herein, we present direct proteomic evidence for the entry of HIV-Tat into neurons as well as
kinase cascades, which ultimately results in apoptosis via key HIV-dementia associated signaling
pathways. We also provide evidence for several pathways resulting in known HIV-dementia and
Alzheimer’s disease pathologies which are certainly capable of contributing to the apoptotic
phenotype and warrant investigation as therapeutic targets. Our findings are highly congruent
with literature regarding key molecular features in HIV-dementia. Together, these data provide a
proteomic map of cellular pathways leading to neuronal dysfunction, which can be used to test
the effect of potential treatments and inhibitors used to query the involvement of specific HIV-Tat
dysregulated pathways.
Page 38
Poster
Dimakatso Gumede
University of Cape Town
Department of Human Biology
Institute of Infectious Disease and Molecular Medicine
Cape Town, 7708
Mobile: +27 723631658
Telephone: 27 216 506 462
E-mail: GMDDIM001@myuct.ac.za
“Determining the role a FAM111B mutation in fibrosis using
induced pluripotent stem cells (iPSCs)”
Dimakatso Gumede; Robea Ballo; Bongani Mayosi, and Susan Kidson
Department of Human Biology, Institute of Infectious Disease and Molecular Medicine,
University of Cape Town
Poikilodermas are characterised by mottled pigmentation, epidermal atrophy and telangactasia,
and are commonly caused by autosomal recessive mutations in the RECQ genes involved in DNA
replication. We have identified a South African family with an autosomal dominant form of poikiloderma
accompanied by tendon contracture, myopathy and fibrosis with a novel mutation (c.1861T>G) in the
family with sequence similarity 111B (FAM111B) gene (Khumalo et al. 2006). The function of FAM111B
is unknown and the broad aim of this study is to investigate the role of the FAM111B mutation in
fibrosis using induced pluripotent stem cells (iPSCs). The specific objectives of the study are to a)
reprogramme control and patient fibroblasts iPSCs, b) differentiate the iPSCs to fibroblast- and
myofibrolast-like cells to generate a disease-in-a-dish model of fibrosis, and to c) determine the
expression FAM111B as well as the markers that are activated and up-regulated during fibrogenesis.
Control and patient dermal fibroblasts were infected with the Sendai viral plasmid containing
the Yamanaka four-factor cocktail comprising OCT4, SOX2, c-(MEF) and maintained MYC and KLF4.
Infected cells were passaged onto a mouse embryonic fibroblast feeder layer in human embryonic
stem cell medium with fibroblast growth factor for 30-40days until iPSC-like clones with large nuclei
and defined edges developed. Of the ten cloned picked from patients and control lines, three from
each were characterised by immunocytochemistry, in vitro differentiation, karyotyping and qRT–PCR.
Clones were manually passaged onto MEFs for immunofluorescent staining. Embryoid bodies were
generated for in vitro differentiation and allowed to differentiate into the three germlayers over a
period of 16-20days.
For karyotyping and qRT-PCR iPSC clones were transferred from feeders to feeder free using
Essential 8 medium on geltrex. All of our iPSC clones expressed pluripotency markers (i.e. OCT4,
Page 39
TRA-1-60 & NANOG) and germline (endoderm, mesoderm & ectoderm) markers following in vitro
differentiation indicating their ESC-like phenotype. No chromosomal abnormalities were detected in
any of the clones.
Preliminary qRT-PCR findings showed a difference in FAMIIIB expression between patient
and control dermal fibroblasts. Due to the underlying fibrosis in this condition we are currently
differentiating patient and control iPSC lines into fibroblast cells using human keratinocyte medium
(Hewitt et el. 2009). Twenty-one days after inducing differentiation the expression of fibroblast markers (vimentin,
fibroblast specific protein- 1, & type 1 collagen) will be determined. Gene expression of fibrosis
markers (transforming growth factor- β1, α-SMA, TGF-βR1, type 1 collagen and tissue inhibitors of
metalloproteinases), which are upregulated during fibrogenesis, will also be investigated.
Page 40
Poster
Wenyan Leong
School of Chemical and Biomedical Engineering
Nanyang Technological University
Singapore 637457
Tel: (65) 6514 1030
Email: WLEONG1@e.ntu.edu.sg
“Temperature-cured dissolvable gelatin microsphere carriers
(tDGMCs) delivering non-anchorage dependent therapeutic
cells for tissue engineering applications”
Wenyan Leong; Kai Su; Ting Ting Lau and Dong-An Wang
School of Chemical and Biomedical Engineering, Nanyang Technological University
All kinds of microspheres have been extensively employed as carriers for drug, gene and
therapeutic cell delivery. Most therapeutic cell delivery microspheres rely on a two-step
methodology: fabrication of microspheres and subsequent seeding of cells onto them. In this
study, we have developed a novel one-step cell encapsulation technique using a convenient and
instant water-in-oil single emulsion approach to form cell-encapsulated gelatin microspheres. This
technology is adopted for hyaline cartilage tissue engineering, in which autologous chondrocytes
are used as therapeutic cells. Cell viability was maintained throughout and after the microsphere
formation (75-100 μm diameters) process that avoids involvement of any covalent bonding reactions
or exposure to any further chemicals. Further encapsulation of cell-laden microspheres in alginate
gels were performed under 4°C via a prompt process. Upon the formation of alginate constructs,
they were immediately relocated into CO2 incubator where the temperature was maintained at
37°C; under this temperature, the cell-laden gelatin microspheres dissolved within hours to yield
similarly sized cavities and the chondrocytes were therefore suspended within the cavities inside
the alginate gel bulk. Hence, the gelatin cell-laden microspheres served two roles: as cell delivery
vehicles which can be removable through temperature curing, and as porogens within an alginate
hydrogel construct to provide living space for cell growth and tissue development as well as better
permeability for mutual diffusions. These cell-laden microspheres, namely “temperature-cured
dissolvable gelatin microsphere based cell carriers” (tDGMCs), were further encapsulated in a
chondrocyte-laden alginate scaffold system and analyzed by WST-1, gene expression analyses,
biochemical assays, histology and immunochemistry stains. The positive results consistently
demonstrated the promise of tDGMC technology in delivering these non-anchorage dependent
cells (chondrocytes). It can be further conveniently translated into delivery of other non-anchorage
dependent cell species, including stem cells, progenitors or iPS cells, for regeneration of tissues in
internal organs, such as engineered hepatogenesis or pancreatic regeneration.
Page 41
Poster
Vuyiswa Mkhabela
UJ / CSIR
Department of Applied Chemistry, University of Johannesburg
Doornfontein, 2028
CSIR
1 Meiring Naude Drive, Brummeria, Pretoria, 0001
Mobile: +27 761082463
Telephone: +27 12 8412096
E-mail: VMkhabela@csir.co.za
“In vitro analysis of biodegradable poly(ɛ-caprolactone) /
montmorillonite nanocomposite scaffolds for bone tissue
engineering”
Vuyiswa Jane Mkhabela1,2 and Suprakas Sinha Ray1,2
Department of Applied Chemistry, University of Johannesburg, South Africa; 2DST/
CSIR National Centre for Nanostructured Materials, Council for Scientific and Industrial
Research, South Africa
1
Nanocomposite scaffolds of poly(ε-caprolactone) and chitosan-modified montmorillonite were
studied for applications in bone tissue engineering. The scaffolds were processed by solvent casting
and particulate leaching method, and characterized by scanning electron microscopy, energy
dispersive X-ray spectroscopy and X-ray diffraction. Highly porous scaffolds with interconnected
pores were produced. In vitro degradation and bioresorption studies conducted in simulated
physiological conditions showed that the scaffolds degraded at a very slow and were confirmed
to be bioresorbable. The incorporation of montmorillonite had an influence on the analyses. The
scaffolds obtained demonstrated their potential to be used in bone tissue engineering applications.
Page 42
Poster
Atipat Patharagulpong
Cavendish Laboratory, University of Cambridge
Darwin College, Silver Street
Cambridge, CB3 9EU
Mobile: +44 7795904915
Telephone: 44 1 223 337 200
Fax: 44 1 223 337 000
E-mail: ap774@cam.ac.uk
“Near-field electrospun aligned fibres for neural stem cell
differentiation”
Mr Atipat Patharagulpong1 and Dr. Yan Yan Shery Huang2
1
Department of Physics, University of Cambridge, United Kingdom;
Department of Engineering, University of Cambridge, United Kingdom
2
Near-field electrospinning was employed to provide a topographically aligned scaffold for neural
stem cell differentiation. The fibres were cross-linked to render them insoluble in culture media,
and characterized, where the release of insoluble materials into culture media was found less than
0.1% per day. Neural stem cells were seeded onto the collagen coated fibres in the presence of the
neural growth factor, where the neurite length and the expression of beta-III-tubulin were enhanced
remarkably compared to the seedling on 2-D references, suggesting the guidance of differentiation.
Page 43
Poster
Fiona A van Vollenstee
Department of Immunology and Institute for Cellular and Molecular Medicine
Faculty of Health Sciences
University of Pretoria
Email: fionavanvollenstee@gmail.com
“Immunophenotypical characterization and differentiation
capacity of transduced human adipose derived mesenchymal
stromal cells by optimized GFP positive lentiviral vectors”
Fiona A van Vollenstee1,2; Carlo Jackson1,2; Danie Hoffmann3; Karlien
Kallmeyer1,2; Marnie Potgieter1,2; Chrisna Durandt1,2 and Michael S Pepper1,2
Department of Immunology and 2Institute for Cellular and Molecular Medicine, Faculty
of Health Sciences, University of Pretoria; 3Plastic and Reconstructive Surgeon (Private
Practice)
1
Introduction: It is well known that resident adipose mesenchymal stromal cells (ASCs) are a
heterogeneous population of multipotent cells characterized by (a) their ability to adhere to plastic;
(b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the
capacity to differentiate into cells of mesodermal origin including osteocytes, chondrocytes and
adipocytes. Adipose derived stromal cells (ASCs) can be utilized as biological vehicles for vectorbased gene delivery systems, since they home to sites of inflammation and infection in vivo. In
order to reach the long-term aim of clinical translation of cell-based therapy, preclinical safety
and efficacy need to be shown in animal models. This has motivated the development of an in
vivo tracking system for ASCs and lentiviral vector transduction for a vector-based gene delivery
system.
Methodology: Human ASCs were transduced with green fluorescent protein (GFP)-expressing
lentiviral vectors in vitro. A titration study was performed to optimize the amount of vector needed
to transduce the maximum number of ASCs. The effect of the GFP lentiviral vector on ASCs was
investigated by studying ASC immunophenotypic expression of surface markers as well as their
capacity to differentiate into osteocytes, chondrocytes and adipocytes.
Results: A transduction efficiency in ASC cultures of approximately 80% was observed after
introducing a total of 250 µl of concentrated lentiviral vector suspension per 4.8 x 104 cells. No
immunophenotypic differences were observed between GFP positive and GFP negative cultures.
Flow cytometric analysis revealed an increase in GFP expression following in vitro expansion of
transduced ASCs. Both, GPF negative and GFP positive cultures successfully differentiated into
osteocytes, chondrocytes and asipocytes.
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Discussion: GFP lentiviral vectors produced in our facility demonstrated more than 80%
transduction efficacy of ASCs. In addition, transduced ASCs maintained adherence to plastic,
ASC immunophenotype and were able to differentiate successfully into cells of the mesodermal
lineages. This optimized GFP-ASC transduction technique offers a feasible tracking system as well
as a vector-based gene delivery system for future preclinical studies.
Page 45
Poster
Leah M. Molai
Department of Biomedical Sciences
Faculty of Science
Tshwane University of Technology
Pretoria, South Africa
Email: leahmolai@gmail.com
“Effects of Genistein on Tissue Engineering of
Articular Cartilage”
L Molai and SCKM Motaung
Department of Biomedical Sciences, Tshwane University of Technology
Genistein is an isoflavone also known as a phytoestrogen due to its estrogen-like structure.
Soybeans are the main source of genistein. The aim of the study was to investigate the role of
genistein on accumulation of collagen type II in articular chondrocytes. Articular chondrocytes
were isolated from porcine knee cartilage and cultured as monolayers in serum-free chemically
defined medium (DMEM/F12). Chondrocytes were treated with varying concentrations (5, 15, 25,
50 and 100ᶙM) of genistein for four days. Morphology and cell growth were viewed microscopically.
Cell viability was determined with the trypan blue exclusion assay. Cytotoxicity was evaluated by
xCelligence; confirmed by apoptosis and cell cycle analysis using Mac Quant flow cytometry. RT–
PCR, determined collagen type II mRNA expression.
After four days of treatment, there was an increase in cell growth. The % cell viability was over
70% at the highest concentration of 100 ᶙM. Genistein was not apoptotic nor cytotoxic. It was found
that 5, 15 and 25 ᶙM up regulated collagen type II in the superficial zone, while the middle zone
was up regulated by 15 ᶙM genistein. The present study reveals the biphasic effect of genistein on
the expression of collagen type II in articular chondrocytes and its possible potential role in the
treatment of osteoarthritis.
Page 46
Poster
Lara McGillewie
Department of Immunology,Institute for Cellular and Molecular Medicine (ICMM)
Faculty of Health Sciences, University of Pretoria
5 Bophelo Road, Prinshof Campus
Pathology Building Rm 5-49
Mobile: +27 732230404
Telephone: +27 12 3192107
E-mail: laramcgil@gmail.com
“Does multi-colour flow cytometry allow for the identification
of primitive cord blood-derived hematopoietic stem cells, using
only surface marker staining?”
Lara McGillewie; Carlo Jackson; Chrisna Durandt and Michael S. Pepper
Department of Immunology, Institute for Cellular and Molecular Medicine (ICMM),
University of Pretoria, South Africa
Blood is one of the most regenerative tissues in the human body; red blood cells have a lifespan
of only 120 days whereas some lymphocytes can survive several months to a few years. Adult
hematopoiesis is maintained by the continuous production of vast numbers of blood cells from a
relatively small number of dormant/quiescent hematopoietic stem cells (HSCs), which can selfrenew or differentiate into the multiple blood lineages depending on the stimulus. The regenerative
and self-renewal properties of stem cells have great therapeutic potential. To date, HSCs are the
most widely used stem cells in clinical practice, and hematopoietic stem cell transplants (HSCTs)
are widely used for the treatment of numerous hematologic and non-hematological disorders. In
the clinical setting HSCs can be harvested from umbilical cord blood, bone marrow and mobilized
peripheral blood, and each source has its own advantages and disadvantages. Despite the recent
advances in isolating and characterising HSCs, transplants are not always successful. This in part,
can be attributed to the lack of understanding of the heterogeneity of the primitive hematopoietic
sub-populations. Cord blood is a promising source of HSCs, as it is easily accessible and immune
naïve. However, the most significant limitation when using cord blood in clinical practice, is the
low number of HSC present. Although several studies are underway in an attempt to expand HSCs
in vitro, the success is limited and cannot yet be fully translated into the clinical setting. Thus to
harness the full therapeutic potential of cord blood, investigators need a better understanding of the
heterogeneity of the HSC populations. CD34 is currently used as surrogate marker to identify and
enumerate HSCs. However, it is generally accepted that CD34 might not be the most optimal marker
to identify clinically relevant HSC subpopulation(s), as both primitive stem cells as well as lineage
committed progenitor cells express the CD34 marker (protein). Interestingly, several investigators
Page 47
suggest that the true primitive HSC population does not necessarily express the CD34 surface
marker. The surface antigen profile of these more primitive HSC populations is still unresolved,
and most investigators identify primitive HSCs based on their ability to efflux substrates of the ATPbinding cassette (ABC) transporters, including P-glycoprotein. These cells are designation “side
population” due to their flow cytometric profile. Disadvantages of the side population assay are: (i)
it makes use of an excess of nuclear dye that intercalates into the DNA of the cells of interest. The
excess dyes serves as substrate for the ATP-binding cassette (ABC) efflux proteins; (ii) it is a labour
intensive assay with a relatively slow turnaround time. In this study, several surface markers,
including P-glycoprotein were simultaneously used in order to develop a better understanding of
the surface antigen expression profiles of the most primitive HSC populations.
The results of this study allow investigators to isolate primitive HSCs based on surface marker
expression for further expansion as well in vivo transplantation experiments.
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Scaffolds, Stem Cells,
Tissue Engineering
and
Regenerative Medicine
Session Presentations
Friday, 29 August 2014
8:15 – 13:00
Chairpersons:
Prof. Hari Reddi:
UC-Davis Medical Center, Sacramento, USA
Prof. Michael Pepper:
University of Pretoria, Pretoria, South Africa
Page 49
Kyla Brady
School of Cellular and Molecular Medicine
University of Bristol, Bristol, UK
Email: Kyla.Brady@bristol.ac.uk
“Mesenchymal Stem Cell Subsets and Sources for Cartilage
Tissue Engineering”
Kyla Brady; Sally C Dickinson and Anthony P Hollander
School of Cellular and Molecular Medicine, University of Bristol
Stem cell based transplantation and tissue engineering applications hold great therapeutic
potential for the treatment of a range of human diseases. Methods for deriving chondrocytes
from mesenchymal stem cells (MSCs) have been developed in a number of laboratories, but
translating these processes into therapies has been particularly challenging. By modifying our
optimised cartilage tissue engineering protocol, we were able to show the clinical potential of MSCs
through engineering a functional airway that was successfully implanted into the left bronchus of
a patient with bronchomalacia. In order to progress to the development of a treatment for cartilage
degradation in osteoarthritis, we need to understand how to reduce the variability between MSC
preparations and identify a cell source or subset of cells with effective and consistent chondrogenic
capacity. Using specific cartilage marker assays to monitor the quality of our engineered tissue,
we have shown that human first trimester fetal bone marrow derived MSCs are as effective as
adult MSCs at tissue engineering cartilage with a hyaline-like phenotype. In combination with their
proliferative potential, fetal MSCs would allow for extensive scalability of cells with consistent
chondrogenic potential for use in allogeneic cartilage regeneration strategies. We have also used
MSC cloning and gene array analysis to identify a novel cell surface marker that is predictive of
MSCs with an enhanced capacity for cartilage formation.
This approach is likely to lead to a translatable chondrogenic differentiation method that should
reduce variability of outcome and allow for enrichment of a defined progenitor population for use in
cartilage repair therapies.
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William Ferris
Stellenbosch University, Tygerberg Campus
Room No. 3033 Clinical Building
Francie van Zijl Drive, Parow, 7505
South Africa
E-mail: wferris@sun.ac.za
“Characterization and comparison of Mesenchymal stromal
cells isolated from three depots within bone”
WF Ferris; FA Jacobs; H Sadie-Van Gijsen and FS Hough
Division of Endocrinology, Department of Medicine, Faculty of Medicine and Health
Sciences, Stellenbosch University
Bone marrow is an established source of multipotential mesenchymal stem cells (MSCs). Recently,
a method for isolating MSCs from cortical (compact) bone in the femur diaphysis (shaft) was described.
During isolation of MSCs from bone marrow and cortical bone, the proximal end of the femur (femoral
head) is routinely discarded, although this tissue could potentially harbor MSCs.
Consequently, we investigated whether MSCs can be isolated from the proximal end of the femur,
characterized and these putative proximal end MSCs (peMSCs) compared with MSCs derived from
bone marrow (bmMSCs) and cortical bone (cbMSCs), by means of cell surface marker profiling and
assessment of multi-lineage differentiation potential.
Rat bmMSCs were isolated by flushing the bone marrow from the femur diaphysis and seding
into cell culture dishes. The remaining diaphysis was subsequently utilized for cbMSC isolation. The
diaphysis was cut into fragments and digested with collagenase. The liberated cells were discarded
and the remaining cortical bone fragments were seeded in cell culture dishes.
Putative peMSCs were isolated from the proximal end of the femur, using a method similar to
that for cbMSC isolation. Cells that were plastic adherent and proliferative were characterized by
flow cytometric analyusis examining the following cell surface markers CD90 (a naïve mesenchymal
marker); CD45 a naïve haematopoietic marker); CD26 (a fibroblast marker) and CD106 (a bone MSC
marker).
To demonstrate multi-lineage differentiation, cells were differentiated into either osteoblasts
(bone-forming cells) or adipocytes (fat cells), using lineage-specific differentiation media. Osteoblastic
differentiation was assessed after 7-21 days of culture by Alizarin Red S (ARS) staning of calcified
extracellular matrix, and adipocytic differentiation was assessed by Oil Red O staining of intracellular
lipid droplets.
Both cbMSC and bmMSC populations exhibited high expression (>95%).
Page 51
Blessing Mukonoweshuro
Institute of Medical and Biological Engineering (iMBE)
University of Leeds
United Kingdom
E-mail: fbsbmuk@leeds.ac.uk
“Immunogenicity of undifferentiated and differentiated
allogeneic mesenchymal stem cells”
Blessing Mukonoweshuro; Christopher Brown;
John Fisher and Eileen Ingham
Institute of Medical and Biological Engineering (iMBE), University of Leeds,
United Kingdom
Mesenchymal stem cells (MSC) are multipotential cells with utility in tissue engineering and
regenerative medicine. However, the immunological properties and immunogenicity of allogeneic
MSC remain poorly defined. Recent studies investigating their immunogenicity remain inconclusive
and this has hampered their clinical application. This study investigated the (i) immunogenicity and
(ii) immunomodulatory properties of bone marrow-derived MSC using an allogeneic mouse model
involving Balb/c (responder) and C3H (stimulator) mice.
Dermal fibroblasts (DF) were used as controls for cells of mesenchymal origin. Adaptations of
the lymphocyte transformation assay (LTA) and mixed lymphocyte reaction (MLR) were used to
investigate the immunogenicity and immunomodulatory properties of allogeneic undifferentiated
and chondrogenic-differentiated MSC and DF. Both MSC and DF displayed a similar phenotypic
profile with the exception of lower expression of CD44 and CD105 in DF. Tri-lineage differentiation
of MSC and DF into adipocytes, chondrocytes and osteocytes confirmed their multipotency. In LTA,
both undifferentiated and chondrogenic-differentiated allogeneic MSC stimulated lymphocyte
proliferation. Allogeneic DF were non-stimulatory but chondrogenic–differentiated DF triggered
responder lymphocyte proliferation. In one-way MLR, both allogeneic MSC and DF significantly
suppressed Balb/c lymphocyte proliferation.
The current challenges in distinguishing between MSC and fibroblasts were apparent throughout
the work. These findings support the notion that although MSC possess immunosuppressive
properties, they may not be immunoprivileged. Thus, clinical application of allogeneic MSC should
be taken with due consideration of their potential immunogenicity.
Page 52
Ernst B Hunziker, MD, PhD
Departments of Osteoporosis, Orthopaedic Surgery
and Clinical Research, Inselspital
University of Bern, Switzerland
Email: Ernest.hunziker@dkf.unibe.ch
“The Role of Chondroprogenic Synovial Cells in
Cartilage Repair”
Ernst B Hunziker
Departments of Osteoporosis, Orthopaedic Surgery and Clinical Research, Inselspital,
University of Bern, Switzerland
The tissue-engineering approach to articular cartilage repair has gained in popularity during
recent years, and many different techniques have been elaborated; some, indeed, are already being
applied in clinical orthopaedics. Although different in detail, each of these systems is basically
constructed of the same three essential building blocks, namely, a pool of chondroprogenitor
cells (such as mesenchymal stem cells, periosteal cells, perichondrial cells, synovial cells or
dedifferentiated chondrocytes), signalling substances to induce the differentiation of stem cells into
chondrocytes and their production of a cartilaginous extracellular matrix, and a scaffold to carry
these components. The latter does not serve merely as a convenient means of transporting cells
and signalling substances to the implantation (lesion) site. It is important in spatially defining the
dimensions of the lesion void for the cells that it bears, in optimizing the structural organization of
the repair tissue that it is remodelled into by these cells, and in facilitating the integration of this
repair tissue with native cartilage. In serving as a common carrier for the signalling substances,
the scaffold also mediates the interaction of these agents with the trapped stem cells and thereby
contributes largely to the success of the repair result. In this presentation, the cellular prerequisites
as well as the chemical and physical properties that are required of a scaffold for optimal
chondrocytic differentiation and ideal repair results will be discussed. On a gross level, these
include such features as volume stability, biodegradability and adhesiveness. Finally, promising
directions for the future development of scaffolds and of chondrogenic precursor-cell biology in the
light of articular cartilage repair will be addressed.
Page 53
Hanel Sadie-Van Gijsen
Stellenbosch University
Room 3051, Clinical Building
Department of Medicine
Stellenbosch University, Tygerberg Campus
Francie van Zijl Drive, Parow, 7505
South Africa
Mobile: +27 722461105
Telephone: 27 21 9389542
E-mail: hsadie@sun.ac.za
“The impact of visceral adiposity of the donor animal on the
differentiation potential of rat adipose-derived stromal cells”
Sadie-Van Gijsen; FS Hough and WF Ferris
Division of Endocrinology, Department of Medicine, Faculty of Medicine and Health
Sciences, Tygerberg Campus, Stellenbosch University
Background and aim: We have previously reported that multipotential naïve adipose-derived
stromal cells (ADSCs) isolated from rat subcutaneous adipose tissue (scADSCs) have osteogenic
differentiation potential, whereas ADSCs from perirenal visceral adipose tissue (pvADSCs) do not.
We also observed that pvADSCs had greater adipogenic potential than scADSCs. However, variations
in the perirenel visceral adipose tissue mass (pvATM) of our experimental animals compelled
us, to investigate whether pvATM affected the differentiation potential of pvADCs. In addition we
investigated whether the expression of lineage-specific markers in naïve ADSCs could be correlated
with differentiation potential in these cells.
Methods: Subcutaneous and perirenal visceral adipose tissue samples were harvested from
adult (250 g; ~12 weeks old) male Wistar rats fed on standard laboratory chow. Based on difference
in pvATM weight, the rats were divided into low visceral adipose tissue mass (lowVAT: 0.102% ±
0.023% (SD) of total body weight) and high visceral adipose tissue mass (highVAT: 0.63% ± 0.054%
(SD) of total body weight) groups (n=4; p<0.0001). Confluent ADSC cultures were treated with
either osteoblast differentiation media (OM: standard media plus dexamethasone, ascorbic acid
and beta-glycerophosphate) or adipocyte differentiation media (AM: standard media plus isobutylmethylxanthine, insulin, indomethacin and dexamethasone). Matrix mineralization, as a marker
of osteoblast differentiation, was detected with Alizarin Red S staining, while intracellular lipid
droplets, as a marker of adipocyte differentiation, were stained with Oil Red O. Expression levels
of the pro-osteogenic transcription factors Msx2 and Runx2 were measured by semi-quantitative
RT-PCR.
Results: matrix mineralization occurred in OM-treated cultures of scADSCa and pcADSCs from
Page 54
lowVAT rats within 3-4 weeks, in contrast to pvADSCs from highVAT rats which did not exhibit
an osteogenic response to OM. Both scADSCs and pcADSCs from lowVAT rats differentiated into
adipocytes after approximately 2 weeks of AM treatment, while scADSCs and pvADSCs from highVAT
rats achieved full adipocytic differentiation after only 1 week. Msx2 expression levels in naïve ADSCs
were positively associated with osteogenic potential, and negatively associated with adipogenic
potential. Runx2 expression levels did not show any association with differentiation potential.
Conclusions: These results suggest that visceral adiposity, possible as a surrogate marker
of base-line metabolic status, may influence the differentiation potential of pvADSCs as well as
scADSCs.
Page 55
Heidi Abrahamse
Laser Research Centre, Faculty of Health Sciences
University of Johannesburg,
P.O. Box 17011, Doornfontein
South Africa
Telephone: +27 11 559 6406
Fax: +27 11 559 6884
E-mail: habrahamse@uj.ac.za
Website http://www.uj.ac.za/lrg
“Genetic Expression Profile of Adipose Derived Stem Cells
Differentiated into Smooth Muscle Cells in Response to Low
Intensity Laser Irradiation”
Heidi Abrahamse
Laser Research Centre, Faculty of Health Sciences, University of Johannesburg
Adipose derived stem cells (ADSCs) isolated from adipose tissue are isolated with ease and in
large amounts. Stem cells have two major characteristics of self-renewal and differentiation into
one or more types of specialised cells. These cells are now being used to treat several degenerative
diseases due to their ability to differentiate into different cell types. Our work has focussed on the
potential augmentation of low intensity laser irradiation on ADSCs to differentiate into smooth
muscle cells (SMCs) with the view of using as therapeutic modality in regenerative medicine. The
effect of two different wavelengths of irradiation, 636 and 830 nm as well as three different fluences
viz. 5, 10 and 20 J/cm2 were used to identify laser parameters affecting viability and proliferation
of ADSCs. Throughout the project, suitably recognized stem cell markers (β1 Integrin - CD29 and
Thy-1 - CD90) were used to characterize and confirm stem cells as well as potential differentiation.
In addition, dose responses were performed to determine suitable growth factors (retinoic acid
and transforming growth factor) and concentrations that would induce differentiation into smooth
muscle cells. Our results confirm that 636 nm and 5 J/cm2 (power output 85 mW; power density
9.3 mW/cm2; spot size 9.1 cm2; exposure duration 9 min 10 s) laser irradiation induce increased
viability and proliferation as well as improve the differentiation potential of ADSCs into SMCs using
optimal growth factor combinations. Methodology used include, ATP content and optical density,
flow cytometry, fluorescence microscopy and real-time quantitative polymerase chain reaction RTqPCR profiles. Our results suggest that ADSCs have the ability to differentiate into SMCs while LILI
potentially augments the differentiation potential and need. This further highlights the significant
role that LILI has to offer in the use of ADSC therapy in regenerative medicine.
Page 56
Danielle de Villiers
Department of Immunology and Institute for Cellular and Molecular Medicine (ICMM)
Faculty of Health Sciences, University of Pretoria
5 Touleier Place, Wapadrand
Pretoria, Gauteng, 0050
Mobile: +27 82 3665663
Telephone: +27 12 8071782
E-mail: danielledevilliers@gmail.com
“The effect of induced oxidative stress and antioxidant
supplementation on the adipogenic differentiation potential of
adipose-derived mesenchymal stem cells”
Danielle de Villiers1; Marnie Potgieter2; Chrisna Durandt3 and
Michael S Pepper3
1
Department of Immunology & Institute for Cellular and Molecular Medicine (ICMM),
University of Pretoria, South Africa; 2Department of Physiology, University of Pretoria,
South Africa; 3Department of Immunology & ICMM, University of Pretoria, South Africa
Introduction: The prevalence of adiposity is mounting in both developing and developed countries.
South Africa is ranked the third most obese country after the United States of America and Great
Britain. One of the associated risk factors for obesity is dyslipidemia (abnormal amounts of lipids),
adipose tissue hyperplasia (cell number proliferation and/or differentiation) and hypertrophy (cell size
increase due to lipid droplet accumulation). In obesity, fat accumulation is correlated with increased
oxidative stress and chronic inflammation. Inflammation is associated with the generation of ROS
and the accumulation of ROS leads to oxidative stress. ROS is not only involved in oxidative stress
but is now said to be involved in signal transduction pathways. Adipogenesis is triggered by signaling
molecules leading to the conversion of ASCs to preadipocytes, which further differentiate into mature
adipocytes. It was therefore hypothesized that the addition of exogenous ROS will enhance ASC’s
adipogenic differentiation potential. In order to curb obesity and understand the role of adipose tissue
in this disease it will be necessary to understand the process of adipogenesis in its entirety. Human
ASCs are capable of differentiating down the adipocyte lineage providing a useful human primary cell
model of adipogenesis. An added advantage of studying the effect of ROS is that the target of ASC
therapy, such as diseased or damaged tissue, is often characterized by the presence of inflammation,
and therefore the generation of ROS. Thus, the effect of ROS on the adipogenic differentiation potential
of ASCs was determined.
Methods: ASCs were induced to differentiate into adipocytes using adipogenic-inducing medium
with or without the antioxidant, pyruvate. The use of the pro-oxidant, H2O2, and the antioxidants,
Trolox and CoQ10, supplemented within the media containing pyruvate or without pyruvate allowed for
the modulation of ROS in the ASC cultures. The adipogenic differentiation was quantitatively detected
using flow cytometry and the emission profiles of Nile Red.
Page 57
Results: It was demonstrated that when ROS was added exogenously to ASCs, adipogenesis was
enhanced. It was also observed that H2O2 added to non-induced ASCs grown in medium without
pyruvate caused substantial adipogenic differentiation. Trolox and CoQ10 attenuated the increase in
ROS and thus a decrease in adipogenesis was seen.
Conclusion: The following study demonstrated that in vitro conditioning with ROS mediates
adipocyte differentiation in ASCs. ASCs provide a primary cell culture model for investigating
adipogenesis and oxidative stress, which is relevant for a vast array of metabolic diseases. The study
also revealed that flow cytometry is a powerful technique that can aid in the quantitative detection
of adipogenesis and the cell sub-populations involved in this process.
Page 58
Celia Snyman
Discipline of Biochemistry, School of Life Sciences
University of KwaZulu-Natal
Private Bag X01, Scottville 3209
South Africa
Mobile: +27 835957110
Telephone: 27 33 2605465
Fax: 27 33 2606127
E-mail: snymanc@ukzn.ac.za
“Bioengineered three-dimensional skeletal muscle tissue”
Celia Snyman; Kyle Goetsch; Kathy Myburgh and Carola Niesler
University of KwaZulu-Natal, Discipline of Biochemistry, School of Life Sciences,
South Africa
Bioengineering skeletal muscle often requires customized equipment and intricate casting
techniques. One of the major hurdles when initially trying to establish in vitro tissue engineered
muscle constructs is the lack of consistency across published methodology.
Differences in cell type, number and density, variability in matrix and scaffold usage as well as
inconsistency in the distance between and type of adhesion posts complicates initial establishment
of the technique with confidence. We describe an inexpensive, but readily adaptable silicone
chamber system for the generation of 3D mouse and human skeletal muscle constructs. This
model can be standardized and used to elucidate myoblast behavior in a three-dimensional space.
Muscle generation, regeneration and adaptation in response to changing extracellular conditions
can be investigated in this model, which is more advanced than standard monolayer differentiated
myotubes.
Page 59
Ivana Kopova
Institute of Physiology
Academy of Sciences of the Czech Republic
Czech Republic
Tel.: +42 0233381564
E-mail: ivana.kopova@post.cz
“Growth and potential damage of human bone-derived cells
cultured on fresh and aged C60/Ti films”
Ivana Kopova1; Lucie Bacakova1; Jiri Vacik2 and Vasily Lavrentiev2
1
Institute of Physiology, Academy of Sciences of the Czech Republic, Czech Republic;
Nuclear Physics Institute, Academy of Sciences of the Czech Republic, Czech Republic
2
Thin films of binary C60/Ti composites, with various concentrations of Ti ranging from ~ 25% (i.e.,
25 Ti atoms and 75 C60 molecules) to ~ 70%, were deposited on microscopic glass coverslips in a
micro-patterned form through a metallic mask, and were tested for their potential use in bone tissue
engineering as substrates for the adhesion and growth of bone cells. The novelty of this approach lies
in combination of Ti atoms (widely used biocompatible material for the construction of stomatological
and orthopaedic implants) with atoms of fullerene C60, which can act as very efficient radical
scavengers. This connection has promising therapeutic potential against oxidative stress-associated
conditions and in a treatment of bone and cartilage tissue destruction in arthritis. However, fullerenes
and their derivatives are able to generate harmful reactive oxygen species and to have cytotoxic
effects. In order to prevent possible cytotoxic of fullerenes, the deposition in a compact form of Ti/C60
composites (with various Ti concentrations) was chosen. This innovative approach was expected to
stabilize fullerene molecules, reduce the release of free C60, their penetration into cells and eliminate
the potential negative effects of fullerenes. The reactivity of C60/Ti composites may change in time
due to the physicochemical changes of molecules in an air atmosphere.
In this study, we therefore tested the dependence between the age of C60/Ti films (from one week
to one year) and the adhesion, morphology, proliferation, viability, metabolic activity and potential
DNA damage to human osteosarcoma cells (lines MG-63 and U-2 OS). After 7 days of cultivation, we
did not observe any negative influence of fresh or aged C60/Ti layers on cell behaviour, including the
DNA damage response. Interestingly, in the case of pure fullerene C60 films (previous study), aged
films were better for cell colonization than fresh films, which had a certain negative impact on the
cell spreading, proliferation, viability and activity of mitochondrial enzymes. This difference between
pure fullerene films and C60/Ti composites may lie in the fact that in the composites, changes in
the fullerene molecules, such as fragmentation, polymerization, oxidation and graphitization, occur
not only due to aging of the material, but immediately during C60 and Ti co-deposition due to the
interaction of C60 molecules and Ti atoms. When deposited in the compact form C60/Ti composites
can therefore be considered as promising materials in bone tissue engineering, namely for potential
coating of bone implants.
This study was supported by the Grant Agency of the Czech Republic (grants No. P108/12/1168 and
P107/12/1025).
Page 60
Morphogens, Stem Cells,
Tissue Engineering
and
Regenerative Medicine
Session Presentations
Saturday, 30 August 2014
8:30 – 12:30
Chairpersons:
Dr. Keolebogile Motaung:
Tshwane University of Technology, Pretoria, South Africa
Dr. Janine Scholefield:
South African Council for Scientific and Industrial Research,
Pretoria, South Africa
Page 61
Lydia E Kavraki
Noah Harding Prof. of Computer Science and Bioengineering
Rice University
Dept. of Computer Science, MS 132
Rice University
6100 Main Street
Houston, TX 77005
USA
Tel: +1 (713) 348-5737
Fax: +1 (713) 348-5930
Email: kavraki@rice.edu
“Enabling Technologies for Tissue Engineering”
Lydia E. Kavraki
Rice University
Systems biology is providing researchers with genomic, proteomic, signaling, and metabolomic
information at unprecedented rates. This talk will explore how Tissue Engineering can benefit from
the repositories, analysis tools, and enabling technologies that are becoming available. After a
high-level introduction, the focus of the talk will shift to the computational modeling of receptormediated interactions where the functional epitope is well understood, typically from experiments.
A novel approach will be presented that uses substructure matching methods to identify the given
epitope in proteins deposited in the Protein DataBank. Such data mining is of primary importance
now that high-throughput methods for structure determination have greatly increased the number
of proteins with known structure in the Protein DataBank. Proteins that locally match the epitope,
or a small variant of the epitope, can be efficiently isolated and studied. Identification of proteins of
previously uncharacterized function can aid the design of new therapeutics. Furthermore, a nuanced
and detailed understanding of protein function can also provide insight into the roles proteins play in
signaling networks with yet unexplored implications for Tissue Engineering.
Page 62
Kevin Dzobo
ICGEB/UCT
90 A Main Road
Mowbray
Cape Town
Mobile: +27 842953708
Telephone: +27 21 6507689
E-mail: kd.dzobo@uct.ac.za
“Wnt/β-catenin and MEK-ERK signaling are required for matrixmediated endoderm differentiation of embryonic stem cells”
Kevin Dzobo1,2; Matjaz Vogelsang1 and M Iqbal Parker1,2
1
International Centre for Genetic Engineering and Biotechnology (ICGEB), Cape Town
Component, Wernher and Beit Building (South), UCT Campus, Anzio Road, Observatory
7925, Cape Town, South Africa, 2Division of Medical Biochemistry, Faculty of Health
Sciences, University of Cape Town, Cape Town, South Africa
Human embryonic stem cells (hESCs) have the potential to differentiate into the cells of the three
germ layers, thus making them an attractive source of cells for use in regenerative medicine. The
greatest challenge lies in directing the differentiation of hESCs into specific cell types. Stem cell
behavior is governed by both intrinsic and extrinsic factors. In this study we determined the effect
of a fibroblast-derived extracellular matrix (fd-ECM) on hESCs differentiation. We demonstrate that
growing hESCs on fd-ECM results in hESCs losing their stemness and proliferation abilities. As the
stem cells differentiate they obtain gene expression profiles similar to the primitive streak of the
in vivo embryo. We also show that activation of both the MEK-ERK and Wnt/β-catenin signaling is
required for the fd-ECM-mediated differentiation of hESCs towards the endoderm. The activation of
these signaling pathways involves integrins such as integrin a1, a2, a3 and β1. This study illustrates
the importance of the cellular microenvironment in directing stem cell fate and we propose the use
of fd-ECM as a platform for hESCs differentiation towards endodermal lineage specification.
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Chrisna Durandt
Department of Immunology and Institute of Cellular and Molecular Medicine (ICMM)
Faculty of Health Sciences, University of Pretoria
Department of Immunology, Pathology Building
5 Bophelo Road
Prinshof (Medical) Campus, Faculty of Health Sciences,
University of Pretoria
Telephone: +27 12 319 2161
Fax: +27 12 319 2946
E-mail: chrisna.durandt@up.ac.za
“A multi-colour flow cytometry approach to quantify various
stages during adipocyte differentiation: Comparison between
Nile Red & Bodipy 493/503”
Chrisna Durandt1; Fiona A van Vollenstee1, Carla Dessels1; Karlien
Kallmeyer1; Ilkadim Kiper1, Danielle de Villiers1; Candice Honing1;
Marnie Potgieter1, Danie Hoffman2, and Michael S Pepper1
Department of Immunology & Institute of Cellular and Molecular Medicine (ICMM),
University of Pretoria, South Africa; 2Private Practice, Pretoria, South Africa
1
Obesity is a major contributor to the global burden of disease and South Africa is no exception.
On the other hand, fat grafting is often used in reconstructive surgery. However, the success of
these fat grafts remains highly unpredictable. A better understanding of the processes involved
during adipogenesis (formation and maturation of adipocytes) may assist in understanding the
pathogenesis of obesity and in developing therapies that will contribute to successful long-term
engraftment of the transplanted tissue. The ability of adipose-derived mesenchymal stem cells to
differentiate into adipocytes is potentially a promising model for adipogenesis both for the study of
obesity and to develop such therapies. One of the major challenges is the lack of reliable in vitro
(and in vivo) models to study adipogenesis. Currently, adipogenesis studies are performed using
murine cell lines.
The results of these studies cannot always be translated with confidence to human patients.
The in vitro differentiation of adipose-derived stromal cells is a promising model to study adipocyte
differentiation using human samples. The objective of our study was to develop a reproducible,
quantitative method to study the differentiation of ASCs into mature adipocytes, by comparing two
lipid dyes (Nile Red and Bodipy 493/503) in combination with several surface marker antigens. We
showed that multi-colour flow cytometry enable us to distinguish various adipocyte sub-populations
present during adipose differentiation. Simultaneously, multi-colour flow cytometry allowed us
to develop a better understanding of phenotypic changes that occur during the differentiation of
adipose-derived stromal cells into adipocytes.
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Petra Knaus
Institute of Chemistry and Biochemistry - Biochemistry
Freie Universität Berlin
Thielallee 63
14195 Berlin
Telephone: +49-30-838-52935,
Fax: +49-30-838-52936
Email: knaus@chemie.fu-berlin.de
“Molecular Mechanism in BMP receptor signaling”
Prof. Dr. Petra Knaus
Institute of Chemistry and Biochemistry - Biochemistry, Freie Universitaet Berlin,
Germany
BMPs belong to the TGF superfamily, comprised of structurally related polypeptide growth
factors that regulate central processes in development and disease. BMPs play critical roles in
differentiation, proliferation, chemotaxis, wound healing, tissue regeneration and apoptosis.
Genetic defects in BMP signaling induce multiple phenotypes, ranging from pulmonary arterial
hypertension (PAH) and hereditary hemorrhagic telangiectasia to embryonic lethality. In sporadic
diseases, such as tumors of different etiologies, the altered cellular contexts of diseased cells may
originate from, and be supported by, modified BMP signals. BMPs signal through a complex of type I
and type II transmembrane serine /threonine kinase receptors at the plasma membrane. The mode
of receptor oligomerization as well as activation determines signaling specificity. The interest of my
lab is to understand the molecular mechanism of BMP signal transduction, and to identify common
and distinct mechanistic concepts in different cellular and tissue environments. Applying different
biochemical, biophysical, cell biology and genetic approaches we address fundamental questions
in signaltransduction. Will present data regarding receptor activation in different cellular context,
the lateral mobility of transmembrane receptors and its impact in signaling, compartmentalization
of signaling pathways by cytoskeletal elements, the molecular mechanism of BMP induced cell
migration and chemotaxis, and novel roles of BMPs in the vascular system. The impact of different
BMPs in regeneration of multiple organs will be discussed.
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Mari van de Vyver
Stellenbosch University
Department of Medicine, Division of Endocrinology
Parow
7441
E-Mail: vandevyverm@sun.ac.za
“Thiazolidinediones and Wnt signaling in primary
mesenchymal stem cells”
Mari van de Vyver; Ellen Andrag and Dr. Ingrid Cockburn
Stellenbosch University, Division of Endocrinology, South Africa
Question: Thiazolidinediones (TZDs) have a uniques insulin-sensitizing action; however, the
clinical use of TZDs for the treatment of type 2 diabetes mellitus is currently challenged by an
inadequate safety profile. Understanding the molecular mechanisms underlying TZD induced side
effects is central to the development and successful therapeutic use of these drugs. This study
investigate the effect of two types of TZDs on cell cycle progression and intracellular Wnt signaling.
Methods: Primary mesenchymal stem cells (MSCs) isolated from peri-renal visceral adipose
tissue of male Wistar rats (n=3) were expanded in vitro and semi-confluent MSCs (passage 2) were
treated with rosiglitazone (rosi) or netoglitazone (neto) under standard growth media conditions.
Cell cycle analysis (FACS), total protein content (fluorescent microscopy) and mRNA expression (RT
Profiler Array analysis) were assessed at 24h and 48h.
Results: TZD treatment reduced DNA synthesis and increased G0/G1 phase. β-catenin protein
degradation occurred 24h (rosi) and 48h (neto) treatment. Reductions in gene expression related to
cell cycle (ccnd1, Fosl1, Tcfe2a, PPARd, Wisp1), Wnt negative regulation (ccnd1, DKK3, Fosl1, Nkd2,
nlk, sfrp1), Canonical Wnt signaling (DKK3, Fzd1, Fzd2, Fzd3, Fzd6, Fzd9, Lrp6, Nkd2, Porcn, Pygo2,
sfrp1, Tcfe2a), cell fate (DKK3), cell migration (DKK3, Lrp6), Wnt/Ca2+ signaling (Fzd2) and cell
polarity (Fzd2, Fzd3, Fzd6, Prickle1) were observed. Rosie reduced Nkd2 expression whereas neto
increase the expression of this negative regulator.
Conclusion: Cell cycle arrest, degradation of regulatory protein and reduced Wnt signaling
related gene expression suggest that TZDs predispose MSCs for adipogenic differentiation via
negative regulation of Wnt signaling.
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LM Watson
Division of Human Genetics
Department of Clinical Laboratory Sciences, IDM
Faculty of Health Sciences, University of Cape Town
South Africa
Email: HRTLAU004@myuct.ac.za
“Modeling neural and retinal degeneration in Spinocerebellar
Ataxia type 7, using induced pluripotent stem cells”
LM Watson1; DC Smith1, R Ballo2; J Scholefield3; L Tyers1; SA Cowley4;
MJA Wood5; SH Kidson2 and LJ Greenberg1
Division of Human Genetics, Department of Clinical Laboratory Sciences, IDM, Faculty
of Health Sciences, University of Cape Town, Cape Town, South Africa; 2Depratment of
Human Biology, Faculty of Health Sciences, University of Cape Town, Cape Town, South
Africa; 3Gene Expression & Biophysics Group, Synthetic Biology ERA, CSIR Biosciences,
Pretoria, South Africa; 4Sir William Dunn School of Pathology, University of Oxford,
Oxford, United Kingdom; 5Department of Physiology, Anatomy and Genetics, University
of Oxford, Oxford, United Kingdom
1
Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease, characterized by
selective degeneration of photoreceptors and cerebellar neurons, resulting in blindness and ataxia.
SCA7 is caused by a polyglutamine expansion in the ATXN7 protein, which is known to regulate gene
expression through association with the STAGA transcription coactivator complex. Transcriptional
dysregulation, caused by mutant ATXN7, has thus been identified as an early disease marker.
Intranuclear aggregated of mutant ATXN7, associated with impairment of the ubiquitin-proteasome
system (UPS), are also known to be a common pathological hall mark of the condition. This study
aimed to establish patient-derived induced pluripotent stem cell (iPSC)-based neural and retinal
models of SCA7, for the investigation of disease pathogenesis.
iPSCs were generated through the infection of SCA7 patient and control cultured dermal
fibroblasts with a modified Sendai viral vector carrying four pluripotency transgenes (Oct4, Sox2, Klf4
and c-Myc). Resulting iPSC lines were expanded and characterised in terms of genomic integrity,
pluripotency marker expression and differentiation capacity before undergoing differentiation into
neural stem cells (NSCs), neurons and photoreceptors.
NSCs were generated by culturing iPSC for seven days in neural induction medium containing
3 μM CHIR99021 (GSK3 inhibitor) and 2 μM SB431542 (TGFβ-receptor inhibitor) Mature neurons
were obtained from iPSCs by treatment with 550 ng/ml noggin for 14 days, followed by cultured in
suspension to form neurospheres in the presence of the growth factors EGF (10 ng/ml) and FGF (10
ng/ml).
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After seven days, neurospheres were plated onto poly-L-lysine/laminin-coated dishes, and the
outgrowth of neuronal processes was towards rod and cone photoreceptors, cells were embedded in
a Matrigel matrix and cultured in the presence of additives including retinoic acid (500nM), taurine (1
mM) and recombinant sonic hedgehog protein (3 nM) for 30 days. Following differentiation, diseaseassociated transcriptional alterations were identified by means of quantitative real-time PCR.
Significant changes in the expression of several genes previously implicated in SCA7 pathology,
including HSP27, OLIG1, GRIA2 and the disease-causing gene ATXN7 (p<0.05), were observed
across neural and retinal lines derived from SCA7 patient iPSCs, when compared to control cell
lines.
To determine susceptibility to proteasomal stress, NSCs from SCA7 patients and controls were
subjected to treatment with the proteasome inhibitor MG-132 (10 μM) for 20 hours. Patient NSCs
showed a significantly higher level of ATXN7 aggregation than controls (p=0.001) after treatment,
confirming previous reports of proteasomal impairment in polyglutamine diseases.
The identification of a disease phenotype in iPSC-derived neurons, NSCs and photoreceptors
offers the opportunity to study the molecular pathogenesis of SCA7 in disease-relevant cells from
human patients. Such models may shed light on the mechanisms which render neurons and retinal
photoreceptors particularly susceptible to mutations in ATXN7, and may ultimately serve as models
for screening potential therapies.
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De Bruin, KJF
Department of Immunology,
Faculty of Health Sciences and Institute for Cellular and Molecular Medicine
University of Pretoria
Email: kfdebruin@gmail.com
“Towards a predictive proliferation model
for adipose-derived stem cells”
De Bruin, K.J.F.1,2; De Villiers, J.P2,3; Pepper, M.S.1
Department of Immunology, Faculty of Health Sciences and Institute for Cellular and
Molecular Medicine; 2Department of Electrical, Electronic and Computer Engineering,
EBIT, University of Pretoria; 3Radar and Electronic Warfare Research and Applications
Group, Council for Scientific and Industrial Research
1
Adipose tissue is a source of two important cell populations used in regenerative medicine:
stromal vascular fraction (SVF) cells that are isolated from adipose tissue via collagenase
digestion, and adipose-derived stem cells (ASCs), a plastic-adherent population that is isolated
by culturing SVF cells. ASCs are adult stem cells that have promising applications in cell-based
therapy and regenerative medicine, and are usually expanded to greater numbers in vitro, before
use in treatments. There is substantial inter- and intra-patient variability in the in vitro proliferation
characteristics of ASCs, which is likely to be due in part to the heterogeneity of the SVF. To predict
cell proliferation, a mathematical model is needed that has a sufficient number of measureable
features to define the ASC population at the beginning of culture. If static and reproducible
culture conditions are assumed, then the in vitro expansion of ASCs is a closed system. With this
assumption, the variability in the system is only governed by the initial cell population (i.e. the
initial state). Therefore, it is reasonable to assume that a certain set of features can be measured
at (or near) the start of the culture that will allow the prediction of stem cell growth in vitro. In
pursuit of a predictive model, the well-known Sherley proliferation model was derived from firstprinciples, and modified to a quiescent-state adapted model that satisfies the Markov property.
A mitotic fraction that varies as a function of the cell cycle was then derived using the Verhulst
and generalised logistic equations. Considering that the mitotic fraction can be quantified in vitro,
this derivation will be valuable in experimentally discovering biologically relevant parameters. An
individual cell-based model was then implemented, where cellular automata divide and migrate
according to a random walk algorithm. This resulted in a model with equivalent characteristics
to the quiescent-adapted Sherley population models, with a cycle-varying mitotic fraction that is
governed by spatial constraints. It was discovered that an increase in migration led to an increase
in the growth rate. This could have the profound implication that cell motility is a hidden parameter
in the cycle-varying mitotic fraction of the adapted Sherley model. To quantify the parameters
in the models, and validate their accuracy, various measurements need to be taken during cell
culture. Most basically, flow cytometric counting methods are commonly used to infer the total
number of cells in a culture flask; however, ASCs are adherent when cultured in vitro, and need
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to be trypsinised and placed in suspension before being counted. This precludes the counting of
the same cell culture flask at different time points. Taking into account intra-patient variability,
this is a significant drawback. In an attempt to measure the proliferation of ASCs in a single flask,
from seeding to confluency, automated image processing and machine-learning algorithms were
developed to estimate the percentage confluency, and to count the number of ASCs present in
a micrograph. A live-cell imaging incubator was developed to allow the continuous capture of
micrographs, to further simplify this process. The prediction of ASC growth has proven to be a
complex problem, and the current and future efforts towards this goal will be described.
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Chien-Yu Dennis Lin
IIDMM, Health Sciences Campus
University of Cape Town
Cape Town
Mobile: +27 824446586
E-mail: dennis33lin@hotmail.com
“Derivation of neural crest-like cells from adult corneal
keratocytes and differentiation into corneal endothelium”
Chien-Yu Lin; Robea Ballo; Elizabeth Van der Merwe and Susan Kidson
IIDMM, Health Sciences Campus, University of Cape Town, South Africa
The monolayered corneal endothelium (CE) located in the inner surface of the cornea forms a
boundary between the corneal stroma and the anterior chamber. The function of CE is to maintain
corneal transparency by regulating corneal hydration. CE density is known to decrease with age
and in diseases such as Fuch’s endothelial dystrophy, glaucoma and physical damage can lead to
corneal oedema, corneal opacity and the eventual loss of vision. The lack of ideal treatments for
these conditions points to the need to further our understanding of CE biology and development.
Therefore, the aim of this project is to obtain and characterise a stem cell population from the adult
cornea and differentiate these cells into CE.
Corneas were dissected from mouse eye globes and treated with Dispase (5 mg/ml) to remove
the epithelium and endothelium, and corneal stromas was dissociated with collagenase (20 mg/
ml) to release stromal cells and expanded in DMEM/F12 with 20% FCS. Characterization with
semi-quantitative RT-PCR revealed positive expression of keratocyte-specific markers keratocan,
lumican and ALDH, and positive for stem cell marker Sox10. Having established a heterogeneous
population of cells with keratocyte and stem cell phenotypes, we next wish to establish whether the
keratocytes give rise to the stem cell subpopulation or the stem cell population were already within
the cornea. To enrich the stem cell sub-population, we have shown that the stem cells are able to
form neuro-spheres when cultured containing EGF (20 ng/ml), FGF2 (10 ng/ml) and LIF (103 U/ml)
in non-adherent plates (Yoshida et el., 2006).
Future work includes characterization of stromal cells and neuro-spheres with Foxc1 Foxc2, and
neural crest markers HNK and p75 (FOXC/Foxc1 is known to cause developmental anomalies of the
anterior segment of the eye including corneal endothelium (Kidson et al. 1999)). Differentiation of
neutral crest or stem cells into corneal endothelium will be tested by adding signaling molecules
such as TGF- β followed by ICC staining for the presence of ZO-1.
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Jackson S. Carlo
University of Pretoria
Pretoria
South Africa
E-mail: carlojackson@gmail.com
“Targeting the ARNT complex with DMOG and Stemregenin 1
improves the expansion of primitive hematopoietic cells”
Jackson S Carloa; Durandt Chrisnaa; Praloran Vincentc;
Brunet de la Grange Philippeb; and Pepper S Michaela
University of Pretoria, Pretoria, South Africa
As a result of the limited number of hematopoietic stem and progenitor cells (HSPCs) harvested
from all cord blood samples and from some bone marrow and adult peripheral blood (G-CSF
mobilized) samples, in vitro expansion is envisaged as a means of increasing cell number before
transplantation to adult patients. Culture conditions should ideally favor the in vitro expansion
of HPCs and the self renewal of long-term HSCs. Hypoxia has been shown to be a key culture
parameter in achieving this objective. Under conditions of hypoxia (0.1 to 3% O2), the hydroxylation
and degradation of the HIF-1α transcription factor is blocked which cause the cellular levels of
HIF-1α to increase and which allows HIF-1α to complex with the Aryl hydrocarbon receptor nuclear
translocator (ARNT). The HIF-1α/ARNT complex in turn modulates the expression of numerous
genes and proteins involved in the preservation of primitive hematopoietic cells by regulating their
metabolism (glycolysis and reactive oxygen species (ROS)), cell cycling and self renewal. At 20% O2
the permanent synthesis of HIF-1α is balanced by its hydroxylation and proteasomal degradation
thus favoring HSPC differentiation. HIF-1α degradation can be prevented by culturing CD34+ cord
blood cells in the presence of dimethyloxaloylglycine (DMOG), which has an effect similar to hypoxia
(1% O2). The ARNT nuclear protein also forms protein complexes with the aryl hydrocarbon receptor
(AhR), which promotes differentiation. This differentiation may be reduced by limiting AhR/ARNT
complex formation by culturing the cells in the presence of Stemregenin 1, an antagonist of AhR.
The aim of this project was to investigate how hypoxia, DMOG and Stemregenin 1, alone or in
combination, could improve the in vitro expansion of HSPCs. It was found that DMOG decreased
proliferation but was effective in preserving the number of cells in the primitive hematopoietic subpopulations. Stemregenin 1, however, increased the size of the primitive populations and therefore
favors the expansion of primitive hematopoietic cells in vitro.
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Morphogens, Stem Cells,
Tissue Engineering
and Regenerative Medicine
Session Presentations
Saturday, 30 August 2014
13:30 – 16:00
Chairpersons:
Prof. Rotimi Sadiku:
Tshwane University of Technology,
Pretoria, South Africa
Dr. Carola Niesler:
University of KwaZulu-Natal,
Durban South Africa
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Alexander Revzin
Department of Biomedical Engineering
University of California, Davis
Email: arevzin@ucdavis.edu
“Microsystems for Shaping and Sensing Cellular Phenotype”
Alexander Revzin
Department of Biomedical Engineering, University of California, Davis
Our laboratory employs microfabrication, surface modification and analytical chemistry to create
micropatterned surfaces for cell-based diagnostics and tissue engineering applications. Some of
the recent, exciting projects in the lab relate to the use of microfluidic devices for cultivation of
stem cells and primary hepatocytes. We find dramatic differences in phenotype of the cells confined
within microfluidic devices without convective mixing. Primary hepatocytes may be maintained for
more than two weeks in such devices whereas these cells de-differentiate and die within a few days
on standard tissue culture plastic. Mouse embryonic stem cells in the absence of feeder cells and
inductive signals (e.g. LIF) maintain pluripotency inside confined volume of a microfluidic device,
by producing endogenous morphogens. In addition to obvious utility for cell cultivation, microfluidic
devices may be used to engineer cellular interactions during injury. As an example, I will describe
a co-culture of adult hepatocytes and liver progenitor cells that attempts to model liver stem cell
niche in vivo. Studying responses of this co-culture to alcohol insult we demonstrate that progenitor
cells select biliary epithelial fate in the presence of alcohol but differentiate towards hepatocytes
in its absence. Our observations in vitro confirm outcomes of animal studies and clinical evidence
suggesting impairment of liver stem cell compartment and excessive cholangiocyte differentiation
during alcohol injury. Therefore, one can imagine using cell culture microsystems for tissue injury
engineering and as models of human development / regeneration. Another research thrust in the
lab pertains to developing biosensors for monitoring paracrine signals produced by cells inside
microfluidic devices.
The vision is to combine cell culture microsystems and biosensors to not only engineer cellular
interactions but to also understand the signals involved in cell phenotype and fate selection.
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Karlien Kallmeyer
Department of Immunology
Institute for Cellular and Molecular Medicine (ICMM)
Faculty of Health Sciences
University of Pretoria
5 Bophelo Road, Prinshof Campus
Pathology Building Rm 5-49
Mobile: +27735070103
Telephone: +27 (012) 3192107
E-mail: karlienkallmeyer@gmail.com
“Differences in basic biological characteristics exist between
adipose- and Wharton’s jelly-derived
mesenchymal stromal cells”
Karlien Kallmeyer; Fiona A van Vollenstee; Chrisna Durandt;
Marnie Potgieter and Michael S Pepper
Department of Immunology, Institute for Cellular and Molecular Medicine (ICMM),
University of Pretoria, South Africa
Background: Mesenchymal stromal cells (MSCs) derived from adipose tissue and umbilical cord
(Wharton’s jelly) represent a potential source of adult stem/stromal cells for cell-based therapy and
tissue engineering. One of the concerns when using MSCs therapeutically is a lack of standardization
during isolation and characterization. This study aimed to characterize and compare MSCs from
two human sources on the basis of morphological characteristics; surface antigen expression and
differentiation capacity using standardized isolation protocols.
Methods: Flow cytometric analysis was used to confirm whether the isolated cells displayed
and maintained the MSC immunophenotypic profile during expansion. Histological staining and/or
colorimetric assays were used to determine the multilineage differentiation capacity of the isolated
cells. Two different techniques involving enzymatic digestion or explant cultures were utilized, and
compared for isolating MSCs from Wharton’s jelly.
Results: Proliferation kinetics of isolated MSCs revealed that Wharton’s Jelly derived-MSCs (WJMSCs) had a mean population doubling time (PDT) from passage 1 to 4 that was approximately
3 fold lower than for the adipose-derived MSCs (ASCs). Both WJ-MSCs and ASCs expressed and
maintained the classical MSC immunophenotype (CD73+, CD90+, CD105+, CD34-, and CD45-).
Variability in immunophenotype was observed in early ASC passages, whereas WJ-MSCs seemed
to attain the MSC immunophenotype as early as passage 0 for both isolation techniques. Low levels
of CD34 positive cells remained in the ASCs. The ASCs showed a statistically significantly greater
capacity to differentiate into adipocytes compared to the lack of adipogenic differentiation in WJ-
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MSCs. No significant differences in osteogenic differentiation were observed between ASCs and
WJ-MSCs. Chondrogenic differentiation was observed (but not quantified) for both MSC sources.
Conclusion: This study demonstrated that differences in cell growth, immunophenotypic profiles
and differentiation capacity occurred between MSCs from different sources. There is a need to
determine the right source of MSCs to use for a specific application and/or disease model, and
studies such as this may assist in making the correct choice. The ability of MSCs to be readily
expanded in vitro and their differentiation into adipocytes, osteocytes and chondrocytes, is relevant
for tissue engineering and regenerative medicine applications.
Page 76
Janine Scholefield
Gene Expression and Biophysics Laboratory
Building 20, Biosciences, CSIR
Meiring Naude Road
Pretoria, 0001
Mobile: +27 (0)769639272
E-mail: janine@mhlangalab.org
“iPSCs for modeling disease in Africa”
Janine Scholefield; Samantha Barichievy and Musa Mhlanga
Gene Expression and Biophysics Laboratory, Biosciences, CSIR, South Africa
The ability to reprogram somatic cells into stem cells (induced pluripotent stem cells; iPSCs)
has revolutionized the field of targeted disease research. It has provided scientists with the ability
to produce, with relative ease, previously unimagined, disease-susceptible, patient derived cells in
a dish, on demand.
Whilst not flawless, iPSC derived models provided significant improvements over historically used
cellular models, namely carcinogenic cell lines. iPSCs can be derived from accessible tissue from
almost any individual, thus carrying patient-specific genetic material, which accurately reflects the
human chromosomal contribution.
By the very nature of their inherent pluripotency, they are not bound by the classical Hayflick
limit; furthermore they can be used to generate most (if not all) cell types in the body, from beating
cardiomyocytes to dopaminergic neurons.
In our laboratory we have sought to exploit these cellular models to address questions in the
field of infectious disease, specifically those of interest to South Africa. One such question involves
the unraveling of how a certain group of individuals are able to control viral load after HIV infection,
effectively presenting as healthy individuals for years after infection.
To this end we have embarked on developing a macrophage model from iPSCs generated from
such individuals, enabling us to study HIV susceptibility using a limitless supply of cells of the
immune system. We have further used this model to elucidate how the pathogen hijacks the host
cell machinery to its own advantage. To investigate such mechanisms, it is critical to remove any
variables, including the contributing background genetic differences between individuals – one of
the disadvantages of comparing iPSCs from various genetic backgrounds.
Thus, a major focus of our experimental strategy has included the use of gene editing approaches,
which discretely modify specific genormic regions (even single nucleotides) of interest, including
the CCR5Δ32 mutation associated with HIV susceptibility.
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These modifications generate invaluable cell lines for comparison, by removing the variable of
diverse genome sequences between individuals, and facilitate a more robust comparison of the
defining genetic change, and its specific contribution to disease.
Most research exploiting the potential of iPSC technology is being developed in Western countries,
with less focus on diseases affecting Africa. The advantages of iPSC technology, including the aspect
of retaining patient-specific genetic background in combination with novel gene editing strategies,
are ideally suited to addressing the health concerns of the African diaspora. Here, we present out
approach for and progress towards developing iPSC-based cellular models of disease for Africa.
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Pepper M
Department of Immunology and the Institute for Cellular and Molecular Medicine
University of Pretoria
South Africa
E-mail: marco@mehealth.co.za / Michael.pepper@up.ac.za
Tel: +27 12 420 5317
“Establishment of a sustainable public cord blood stem cell
bank for South Africa”
Alessandrini M and Pepper MS*
Department of Immunology and the Institute for Cellular and Molecular Medicine,
University of Pretoria, South Africa
Bone marrow transplantation is indicated for the treatment of a variety of blood and bloodrelated disorders. These include, amongst others, leukaemia, myeloma, and various anaemias.
The collection of haematopoietic stem cells for transplantation has traditionally been from the
bone marrow itself, but this has changed in recent years, with peripheral blood (containing bone
marrow-derived haematopoietic stem cells) being the predominant source today. As an alternative,
umbilical cord blood is a rich source of haematopoietic stem cells that can be procured with ease
and without discomfort or risk to the donor. The use of cord blood units for transplantation offers
several additional advantages, including a decreased need for strict HLA-matching, a decreased
risk of graft versus host disease, and a lower risk of disease relapse.
Although a local bone marrow registry exists, the majority of South Africans do not have
opportunity to benefit from bone marrow transplantation due to the low probability of obtaining an
HLA-matched donor and cost implications. The establishment of a public cord blood bank would
not only negate the risk of donor attrition from the bone marrow registry, but it will also provide a
readily available resource of HLA-typed stem cell units.
However, there are several challenges to establishing a public cord blood bank, with the most
significant being the question of financial sustainability. In the recent annual report of the World
Marrow Donor Association, it was indicated that more than 90% of public banks globally require
financial support from local governments and/or external funders, while the remaining handful
have managed to reach the point of sustainability.
It was therefore the objective of this study to determine the financial feasibility of establishing a
public cord blood bank in South Africa. A financial model, which incorporates the required capital
expenditure, operational costs and potential for income, was developed. Our findings indicate that
several facets are to be considered when determining the feasibility for such a venture, and that the
viability of maintaining operations is dependent on rational assumptions. Given the local and global
need to service a genetically diverse African diaspora, it is argued that the establishment of an
African public cord blood stem call bank may well be a sustainable venture to explore.
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Juanita Mellet
Department of Immunology
Institute for Cellular and Molecular Medicine (ICMM)
Faculty of Health Sciences
University of Pretoria
5 Bophelo Road, Prinshof Campus
Pathology Building Rm 5-49
Mobile: +27 (0)79 523 6401
Telephone: +27 (0)12 319 2107
E-mail: juanitamellet@yahoo.co.uk
“Conventional HLA Typing Techniques vs. Next Generation
Sequencing in a South African Cohort”
Juanita Mellet1; Clive M. Gray2,1; Prof. Michael S. Pepper1
Department of Immunology, Institute for Cellular and Molecular Medicine (ICMM),
University of Pretoria, South Africa; 2Division of Immunology, Institute of Infectious Diseases
and Molecular Medicine and Clinical Laboratory Sciences, University of Cape Town
1
Background: The human leukocyte antigen (HLA) complex on chromosome 6 is the most
polymorphic region in humans and plays a pivotal role in matching donors and recipients for
hematopoietic stem cell transplantation. The large number of polymorphisms present in the South
African population reduces the probability of finding an adequate HLA-matched donor for individuals
in need of a transplant, and the ambiguous nature of HLA genes makes it challenging to assign
accurate genotypes. Next generation sequencing (NGS) has numerous advantages compared to
conventional typing techniques, such as sequencing of additional exons to resolve ambiguous HLA
typing results. Exons 2 and 3 for HLA-A, -B, and -C as well as exon 2 for HLA-DRB1 and -DQB1 are
routinely genotyped for unrelated bone marrow transplantations. Sequencing of additional exons
could improve the degree of matching between unrelated donors and recipients. The purpose of
this study was to determine: (1) the reliability of NGS HLA typing techniques in determining the
HLA alleles in a cohort of South African individuals; (2) the degree of genomic complexity that this
technique reveals; and (3) the relationship between NGS and conventional HLA typing techniques.
Methods: HLA genotyping was performed using an NGS technique on 20 samples that had
previously been HLA typed by conventional methods. Samples were genotyped for the routinely
sequenced loci and exons, and also included several additional exons to resolve ambiguity.
Results: Genotyping of routinely sequenced loci and exons yielded accurate genotypes for 95.5%
of the five loci of interest compared to 98.5% when additional exons were included.
Conclusion: The results emphasise the importance of sequencing additional exons to resolve
Page 80
ambiguous typing results. In conclusion, NGS techniques are reliable in genotyping South African
individuals. Furthermore, NGS HLA typing techniques that include additional exons are able to
reduce several ambiguous typing results. However, the equipment and reagents for NGS techniques
remain costly and inaccessible to many research and diagnostic institutions in developing countries.
Page 81
Alessandrini M
Department of Immunology and the Institute for Cellular and Molecular Medicine
University of Pretoria
South Africa
E-mail: marco@mehealth.co.za
“Clinical indications for mesenchymal stem cell therapy”
1
Alessandrini M, 2Potgieter M and 1Pepper MS
1
Department of Immunology and the Institute for Cellular and Molecular Medicine,
University of Pretoria, South Africa; 2Department of Physiology, University of Pretoria,
South Africa
The rapidly developing fields of regenerative medicine and cellular therapy have attracted much
attention. Adult stem cells, in the form of haematopoietic stem cells (HSCs) and mesenchymal
stem cells (MSCs), are a non-controversial and easily accessible resource that holds great promise
for the treatment of a variety of indications. Mesenchymal stem cells have several unique properties
that make them attractive for treatment purposes, particularly the fact that they are immuneprivileged, their ability to home to the site of injury, and their involvement in paracrine signalling.
In order to better understand the clinical applications of MSC therapy, it was decided to
investigate the global spectrum of clinical trials that have been initiated to date. The online
resource, Clinicaltrials.gov, and various literature resources were investigated, and a database
of MSC clinical trials established. Only studies where MSCs were used specifically for treatment
purposes were retained, and further information including tissue source, donor type, cell dose and
cycles of treatment was extracted.
As of the end December 2013, MSCs have been or are currently being tested in over 120
different indications, ranging from haematological conditions to cardiovascular disease, and from
autoimmune disorders to dermatological indications. On average, more than 60 new clinical trials
are initiated per annum (five-year average), with most of the interest being in the treatment of
neurological conditions, particularly multiple sclerosis, amyotrophic lateral sclerosis and spinal
cord injury. When considering tissue source and means of preparation, such as cellular expansion
and use of scaffolds, it was determined that over 50 different MSC treatment regimens have been
explored to date.
Our analysis indicates an astonishing lack of consensus across all variables investigated. These
findings are indicative of an early stage technology, for which multiple aspects of MSC therapy
are still to be clarified and the benefits thereof proven in the clinical trial setting, prior to entering
markets as a form of mainstream therapy.
Page 82
Session Presentations
Saturday, 30 August 2014
16:15 – 18:00
Chairpersons:
Prof. Danie du Toit:
Tshwane University of Technology,
Pretoria, South Africa
Prof. Sue Kidson:
University of Cape Town,
Cape Town, South Africa
Page 83
Carla Dessels
Institute for Cellular and Molecular Medicine
University of Pretoria
Pretoria
Mobile: +27 (0)74 404 4989
Email: carla.d.lu@gmail.com
“Umbilical Cord Blood Banks: A Perspective”
Dessels C, Alessandrini M and Pepper MS
Department of Immunology, and the Institute for Cellular and Molecular Medicine,
University of Pretoria, South Africa
Over the past two decades, umbilical cord blood (UCB) has become an attractive source of
hematopoietic stem and progenitor cells for the treatment of blood and immune system diseases.
The scope of treatment has recently expanded to include brain injury and developmental disorders.
Due to the increase in potential therapeutic indications, UCB stem cells can now be harvested at
birth and stored for future use. This is undertaken by either private or public cord blood banks. In
a public cord blood bank, a donation is made by a donor and is available for any recipient to use.
Very strict regulations are in place to ensure the safety of the recipient, and these samples undergo
a series of tests before being banked, after which, neither the donor baby nor the donor family
may retrieve the blood for personal use. One of the major concerns facing public cord banks is the
associated running and maintenance costs. This may be an obstacle to the opening of public banks.
In a private cord blood bank a donation is made by parents whom have made a personal decision
to store their newborn’s cord blood for use either by the donor baby or by a matched relative in the
future. Private banking is controversial mainly due to the very low probability of the donation being
used. Units from public and private banks are being used for both regenerative and transplantation
medicine. In this study, we compare the trends in data seen in public and private banks globally, by
contrasting the use of cord blood units for both regenerative and transplantation medicine between
different bank types.
Page 84
Fikile M Mnisi
Steve Biko Centre for Bioethics
Witwatersrand University
Johannesburg
South Africa
E-mail: mnisimf@gmail.com
Telephone: +27-79-097-2994
“Questioning the application of the principle of subsidiarity to
human Embryonic Stem Cell research in South Africa”
Fikile M Mnisi
Steve Biko Centre for Bioethics, Witwatersrand University, Johannesburg, South Africa
Researchers from all around the world emphasise the enormous possible benefits that stem cells
may have for the treatment of diseases. However, this technology is considered morally problematic
when the source of the stem cell is a human embryo. Nonetheless, there is a consensus that of all
the types of stem cells, human embryonic stem cells (hESC) are the most promising for particular
and important research and therapies. Yet, there are controversial issues regarding ‘killing’
human embryo for stem cell derivation. Penning & Van Steirteghem claim that two general ethical
conditions should govern the instrumental use of embryo; (i) the principle of proportionality, and (ii)
the principle of subsidiarity. In this paper, I focus on the latter principle. According to Devolder the
principle of subsidiarity is defined as ‘a state we have that we have to choose the less contentious
means of achieving the intended goal’. Based on this principle we ought only to use hESC when
there are no other alternatives (stem cells or derivations from other sources) which are less morally
controversially. I argue that criterion for deciding which type of stem cells to use should be their
potential and suitability for advancing scientific knowledge and development of new therapies which
will be greatly beneficial in alleviating human suffering. Subsidiarity is based on assumption that
there is something ethically unsound about the use of hESC. This, in turn, assumes some special
moral status for embryos. I argue that this status is not consistently upheld, since both in-vitro
fertilisation (IVF) and abortion procedures would need to be prohibited if it were. Furthermore, in the
South African law an embryo is not accorded the legal rights of a human child. However, legislation,
regulations and professional guidelines permit research on ‘supernatant’ IVFs embryos but prohibit
the creation of embryos and somatic cell nuclear transfer for research purpose. Given that the
Choice on Termination of Pregnancy Act of 1996 (amended in 2008) permits abortion on demand (at
least in the first 12 weeks of pregnancy) it is clear that there is some inconsistency regarding the
moral status and fetuses in our law. The principle of subsidiarity only makes sense if it is based on
consistently upheld view of moral status of embryo. Therefore, the law should not limit or prohibit
hESC research based on this principle.
Page 85
POSTER PRESENTATIONS
Saturday, 30 August 2014
17:00 – 18:00
ODD NUMBERED
POSTERS
Page 86
Poster
BA Aderibigbe
Department of Chemistry
University of Fort Hare
Alice Campus, Eastern Cape
South Africa
E-mail: blessingaderibigbe@gmail.com
“Synthesis and characterization of polymeric conjugates
containing platinum analogue and procaine”
BA Aderibigbe1 and ER Sadiku2
Department of Chemistry, University of Fort Hare, Alice Campus, Eastern Cape, South
Africa; 2Department of Chemical, Metallurgical and Material Engineering,
Tshwane University of Technology, Pretoria, South Africa
1
Anticancer therapeutic research is aimed at developing cancer therapies that can inhibit
methylation of tumor suppressor genes or by demethylating them and restoring their expression.
Procaine is a DNA-demethylating agent and it exhibit growth-inhibitory effects in human cancer
cells. In this research, polymeric conjugates were prepared by incorporating procaine and an
analogue of platinum via green process. The conjugates were found to be thermally stable. Platinum
and procaine were coupled as pendant groups onto polymeric carrier via selected functionalities.
In principle, the controlled release of the drugs can be achieved by biodegradation of the polymer
or by cleavage of covalently bound polymer-drug conjugates. The conjugates prepared were further
characterized and the preliminary results suggest that these conjugates are potential polymeric
anticancer drugs for combination therapy.
Page 87
Poster
Shaun Garnett
IIDMM, Health Sciences Campus
UCT
Anzio Road
Observatory, 7925
Mobile: +27 (0)824127938
Telephone: 27 274 066 453
E-mail: shaungarnett@gmail.com
“Generating a Proteomic Profile of Neurogenesis, using Human
Foetal Neural Stem Cells”
Shaun Garnett, Susan Kidson, Kathryn Lilley, Austin Smith and
Jonathan Blacburn
IIDMM, Health Sciences Campus, University of Cape Town
Traditionally neural stem cells were cultured as neurospheres, a heterogeneous agglomeration
of neural cells at various stage of differentiation. This heterogeneity prevented accurate quantitative
analysis. In 2008 Sun et al. produced the first non-immortalised human foetal neural stem (NS)
cell line from 9 week old human foetal brain tissue. These cells are cultured as monolayers, have
a radial glia like appearance, self renew and from all three neural cell types, neurons, astrocytes
and oligodendrocytes upon differentiation. More recently human foetal neuroepithelial like (NES)
stem cells have been produced from 5 week old human foetal hindbrain, they resemble neural
plate epithelial, with characteristic rosettes, upon differentiation they form almost pure population
of neurons. These homogeneous monolayer cultures allow detailed quantitative analysis of these
cells.
Using SILAC we are comparing 4406 proteins from proliferating NES and NS cells, cultured under
identical conditions, to better understand the instrinsic differenced within the cells. Interesting
findings include changes in Integrins and Signal Transducer and Activator of Transcription (STAT)
proteins, affecting how they interact with their environment. A timecourse study is underway to
compare the changes occurring in these cells as they differentiate.
This work represent a thorough characterisation of non immortalised human foetal neural
stem cells, providing detailed account of the proteins being expressed by these cells as well as
the molecular changes occurring as they differentiate, it will serve as a useful catalogue to better
understand brain development, aid in regenerative therapies, and the use of these cells as model
systems for better understanding diseases affecting the brain.
Page 88
Poster
Julia Joos-Vandewalle
Biotechnology Innovation Centre
Rhodes University
No 7 Leicester Street
Grahamstown, 6139
Mobile: +27 (0)82 698 1401
Telephone: 27 466 038 082
E-mail: g09V1587@campus.ru.ac.za
“The effect of novel thiazole-derived small molecules on the
self-renewal and differentiation of human multipotent neural
progenitor cells”
Julia Joos Vandewalle1, Emmaneul Pladayo Olawode1,
Bhupesh Samant2 and Dr. Earl Prinsloo3
Biotechnology Innovation Centre, Rhodes University, South Africa; 2Rhodes University,
South Africa; 3Biotechnology Innovation Centre, Rhodes University, South Africa
1
Neural progenitor cells (NPCs) are able to self-renew and differentiate into neurons, astrocytes
and oligodendrocytes; providing an unlimited supply of neuronal cells for use in neurotoxicity-based
research. The cell-permeable small molecule, all-trans-retinoic acid (RA), is well known to actively
prime these NPCs cells into a neuronal lineage; however the biological activity of this compound is
limiting as it remains moderately unstable for routine use in culture. The identification of a novel
small molecule able to selectively further develop NPC differentiation, with an activity comparable
or enhanced to that of RA, would aid in the biotechnological application of neural stem cells.
Thiazole - containing compounds have shown to be active in various neural processes, and it is
therefore hypothesized that human neural progenitor cell fate may be determined through the use
of a noval synthetic thiazole derived small molecule into a well defined neural cell type. In this study,
a library of 30 temperature stable thiazole- containing small molecule compounds was synthesized
and tested for their ability to induce neuronal differentiation. A primary screen identifying compound
cytotoxicity (IC50) was conducted, followed by a secondary screen which identified any differentiation
ability; relating RA-induced differentiation to that of the novel thaizole- containing small molecules.
The xCelligence RTCA system was used for all analysis conducted in real-time followed by endpoint determination of neurite outgrowth, cell viability, proliferation and expression of markers of
mature neuronal cell post-treatment. The SH-SY5Y human neuroblastoma cell line, a widely used
neuronal model of differentiation, was used for all small molecule screening and human neural
progenitor cells for comparative and conclusive analysis. Overall, two lead small molecules were
identified as having optimal neural differentiation activity.
Page 89
Poster
Juanita Mellet
Department of Immunology
Institute for Cellular and Molecular Medicine (ICMM)
Faculty of Health Sciences
University of Pretoria
5 Bophelo Road, Prinshof Campus
Pathology Building Rm 5-49
Mobile: +27 (0)79 523 6401
Telephone: +27 (0)12 319 2107
E-mail: juanitamellet@yahoo.co.uk
“Constituting a public umbilical cord blood bank in South Africa:
Demographics, linguistics, and HLA diversity”
Juanita Mellet and Michael S Pepper
Department of Immunology, Institute for Cellular and Molecular Medicine (ICMM),
University of Pretoria, South Africa
Background: The South African Bone Marrow Registry (SABMR) is one of two such registries in
Africa and caters mostly for Caucasians. The high degree of diversity and the small number of Black
Africans registered with the SABMR decreases the likelihood of finding donors for these individuals.
A public cord blood bank is therefore considered to be necessary on the African continent and may
provide Black Africans with an improved chance of obtaining a human leukocyte antigen (HLA)compatible donor. Umbilical cord blood requires a 4/6 HLA allele match between donors and
recipients for transplantation, which is less than is required for bone marrow transplantation. The
greater degree of HLA disparity allowed for umbilical cord blood further increases the chances of
obtaining an HLA-matched donor. The diversity of the South African population can be classified at
three levels, which are: racial groups, ethnic/linguistic groups, and HLA diversity.
Methods: The aforementioned levels were assessed in order to determine the basis on which
a cord blood bank should be constituted in South Africa to accommodate the diversity of the
population in South Africa. Population and linguistic statistics were obtained from statistics South
Africa regarding the different population groups in the country. HLA statistics for the South African
population were gathered from research that was conducted during the 1980’s and also more
recently published articles.
Conclusion: It is recommended that the first umbilical cord blood bank in South Africa should
be constituted on the basis of either social race or major ethnic groupings (Nguni, Sotho-Tswana,
Venda, and Tsonga). It has been estimated that a minimum number of 10 000 cord blood units would
be needed to initiate the bank.
Page 90
Poster
Zenzele Silla
School of Life Sciences
University of KwaZulu-Natal
Durban, 4001
Mobile: +27 (0)823674918
E-mail: sillazt@gmail.com
“Mapping the development of the corneal endothelium”
Zenzele Silla and Paula Sommer
School of Life Sciences, University of KwaZulu-Natal, Durban
Normal eye development is coordinated by a series of cell-cell interactions that give rise to the
immature lens and presumptive corneal epithelium. Of the 5 layers making up the cornea, the
monolayered corneal endothelium is the innermost and the most important physiologically. It is
formed from precursor peri-ocular mesenchyme (POM) cells that begin to migrate in front of the
developing lens at E12 in mice. These POM cells undergo a transition from a mesenchyme to an
epithelial phenotype and form a monolayer connected by N-cadherin junctions. The formation of the
corneal endothelium is essential for the development of the anterior chamber of the eye. Secreted
signals from the lens epithelium, such as TGFβ2, have been implicated in interactions with the
migrating corneal endothelium precursors (POM cells). The presence the transcription factor Foxc1
is also essential for normal corneal endothelial development.
We, therefore, determined whether Foxc1 and signals from the lens are important for Ncadherin
expression in POM cells derived from E12.5 to E15.5 mice embryos, the developmental time at
which corneal endothelial differentiation is initiated to its completion. N-cadherin expression at the
different developmental times was assessed by qPCR and N-cadherin distribution was assessed by
immunofluorescence.
To determine the effect of the lens, POM cells were cultured in the presence of a lens and
TGFβ2, and N-cadherin expression determined using confocal microscopy. Our data reveal that
the presence of the lens promotes the normal lattice framework of N-cadherin expression. To
determine the effect of Foxc1, N-cadherin expression in cells in which Foxc1 was silenced using
shRNA was measured by qPCR and distribution determined by immunofluorescence. Our data show
that both the presence of Foxc1 and the lens are important in promoting junction formation in the
corneal endothelium. This knowledge will be used to attempt to direct mouse embryonic stem cells
towards a corneal endothelial phenotype.
Page 91
Poster
Fiona A van Vollenstee
Department of Immunology
Faculty of Health Sciences and Institute for Cellular and Molecular Medicine
University of Pretoria
E-mail: fionavanvollenstee@gmail.com
“Establishment of a molecular platform for the detection
of HIV-1 in umbilical cord- and adipose tissue-derived
mesenchymal stem cells”
Fiona A van Vollenstee, 2Lillian Gaggia, 3Danie Hoffmann,
Karlien Kallmeyer, 1Marnie Potgieter, 4Petrus F Coetzee,
2
Marion Vermeulen, 1*Michael S Pepper
1
1
Department of Immunology, Faculty of Health Sciences and Institute for Cellular and
Molecular Medicine, University of Pretoria; 2South African National Blood Service;
3
Plastic and Reconstructive Surgeon in Private Practice, South Africa; and 4Department
of Surgery, Faculty of Health Sciences, School of Medicine and Plastic Surgery Division,
University of Pretoria
1
Background: Research efforts to advance the use of mesenchymal stem cells (MSCs) for
therapeutic purposes face significant challenges. This includes the controversial issue of whether
MSCs can act as a latent reservoir for human immunodeficiency virus (HIV). Routine screening of
autologous and allogeneic human MSCs for HIV and other infectious diseases prior to clinical use is
being recommended especially in high endemic areas such as South Africa. Molecular assays could
prove a useful adjunct to establish pre-clinical safety and also assist ongoing efforts to combat
these diseases through stem cell technology. This study aimed to validate the Procleix Ultrio Plus®
Assay for the detection of HIV-1 in human derived MSC cultures in our setting.
Methods: Human adipose tissue was harvested and umbilical cord tissue collected to isolate
MSCs according to a standardized protocol. Isolated MSCs were submitted to high resolution
sonication to produce a cellular extract (CE). Protein concentration in the conditioned medium
(CM) and CE was determined using a Bradford assay and compared to human serum derived from
whole blood to assess instrument compatibility. Validation was performed by using 6 HIV-1 spiked
positive samples (2 Wharton’s Jelly derived MSC CM samples; 2 ASC derived MSC CM samples; 1
Wharton’s jelly derived MSC CE sample and 1 ASC derived CE sample) as well as unspiked samples
(negatives). All samples were screened prior to spiking. A dilution series was made from the spiked
samples that began at 92 copies/ml and were diluted to 11.5 copies/ml. (positives). Each dilution
was screened once using the Procleix Ultrio Plus® Assay.
Page 92
Results: The Bradford assay confirmed lower protein concentrations for both the CE and CM
compared to human serum. The Procleix Ultrio Plus® Assay detected HIV-1 in all samples spiked
at a viral load concentration of 45 copies per ml. Only the Wharton’s jelly derived MSC CE sample
and the ASC derived CE sample detected all spiked samples as the 1:4 dilution gave invalid results
for the other samples. Wharton’s Jelly derived MSC CM samples tested negative at the 1:8 dilution
giving a sensitivity and specificity of 92% and 98% with the positive and negative predictive values
calculated at 92% and 98% respectively.
Conclusions: The ongoing need to screen all forms of tissue for blood borne viruses and in
particular HIV prior to transplantation has raised the need to establish a suitable testing platform for
stem cells. The lower protein concentration of the CM and CE compared to human serum ensured
compatibility for testing with the ProcleixUltrio Plus® Assay. The platform can be recommended as
a useful tool to detect the presence of HIV-1 in MSCs. False negatives can however occur at very low
viral load concentrations.
Page 93
Poster
GM Molaba
Faculty of Science,
Department of Biomedical Sciences,
Tshwane University of Technology
Pretoria, 0001
“The expression of collagen type II in articular cartilage in
response to resveratrol and bone morphogenetic protein-7”
GM Molaba and SCKM Motaung
Faculty of Science, Department of Biomedical Sciences, Tshwane University of
Technology, Pretoria
OBJECTIVES: To investigate the role of Bone-morphogenetic protein-7 and Resveratrol on the
expression of Collagen Type II in articular cartilage.
BACKGROUND: Damaged articular cartilage requires tissue engineering based therapeutic
methods because of its minimal self-repair capacity. Articular cartilage is a heterogeneous
avascular, aneural tissue that provides a smooth bearing surface for movement. It consists of
superficial, middle, and deep layers with varying matrix composition. The superficial zone is the
site of superficial zone protein synthesis that functions as a boundary lubricant. The middle and
deep layers abound in collagen II and proteoglycan and aggrecan.
MATERIALS AND METHODS: Chondrocytes were plated as monolayers at a density of 1 × 105
cells/well in 12-well culture plates and incubated at 37 ◦C in a humid atmosphere of 5% carbon
dioxide and 95% air. Cell cultures were treated for 4 days with various concentrations of resveratrol
and Bone morphogenetic protein-7. The medium were analyzed for collagen type-II. The cells were
used to isolate RNA for RT-PCR assays for collagen type-II.
RESULTS: The results show that BMP-7 and resveratrol stimulates expression of mRNA and
protein accumulation for collagen type-II in the superficial zone and mild in the middle zone.
Combination of BMP-7 and resveratrol is an important regulator of collagen type-II which may play
an important role in repair of degenerated cartilage.
CONCLUSION: Furthermore, regulation of collagen type-II may be necessary for tissue
engineering to recapitulate the different articular cartilage zone.
Page 94
Poster
Mapula Razwinani
Department of Biomedical Sciences
Tshwane University of Technology
Pretoria 0002
South Africa
Email: Razwinanim@tut.ac.za
“Regulation of collagen type II protein in articular cartilage by
Pleurostylia capensis crude extract”
Mapula Razwinani1, Cornelius Ssemakalu2 and Shirley CKM Motaung1
1
Department of Biomedical Sciences,
Tshwane University of Technology, Pretoria 0002, South Africa
Pleurostylia capensis has been traditional used in Africa for the treatment of various diseases
including colic pain in babies, epilepsy, mental illness and as steam bath, ritual body wash and as
a purgative to treat symptoms of witchcraft. The aim of this study was to investigate the effect of
water extract of P. capensis on the viability of chondrocytes cells and the expression of collagen
type II protein in articular chondrocytes. Growth inhibition was determined by xCelligence, as well
as nucleus changes of cell where observed by fluorescence microscope after PI staining. Cell
cycling distribution and apoptosis were evaluated using Mac Quant flow cytometry. The expression
of collagen type II protein level was detected using ELISA. P. capensis extract indicated the
changing rate of cells within a given time widow, the cell index was decreasing with the increase
in concentration from 0.006 to 0.0039. The extract also induced chondrocytes apoptosis in dosedependent manner where all the concentration maintained over 50% cells viability. The ELISA
results demonstrated that extract expression of collagen type II was directly proportional to the
concentration of the plant extract used. The middle zone cells expressed more collagen type II than
the cells extracted from the surface zone.
In conclusion P. capensis extract was shown to be nontoxic to chondrocytes cells and with no
apoptosis, and also expressed more of collagen type II in both surface and middle zone. The present
study reveals not only the effects of P. capensis extract on tissue regeneration of articular cartilage
but also the therapeutic potential of this plant in treatment of osteoarthritis.
Page 95
Poster
Cornelius C Ssemakalu
Department of Chemistry
Vaal University of Technology
Vanderbijlpark, South Africa
Email: canzazu@gmail.com
“Pterocarpus angolensis crude extracts induce the expression
of collagen type II in articular cartilage”
Cornelius C. Ssemakalu1,2, Mapula Razwinani2, Maepa J. Makwese2 and
Shirley CKM Motaung2
1Department of Chemistry, Vaal University of Technology, Vanderbijlpark, South Africa;
2Department of Biomedical Sciences, Tshwane University of Technology, Pretoria 0002, South
Africa
Pterocarpus angolensis is a large deciduous tree native to the Southern part of the African
continent. This tree has traditionally been used to treat malaria, gonorrhoea, skin inflammation
and wounds. However, the use of Pt. angolensis for tissue regneration of articular cartilage has
not been explored. The aim of the study is to investigate whether Pt. angolensis will have the
ability to synthesize collagen type II in superficial and middle zone of porcine articular cartilage.
Chondrocytes were isolated from pig stiffle joints and cultured as a monolayer at a density of 1
X105 cells/well. Cell cultures were treated with various concentration of Pt. angolensis for 4 days.
The plant extracts from Pt. angolensis where also assessed for their effects on the chondrocyte
viability using xCELLigence assay. Fluorescent nucleus staining and light microscopy was also done
in order to observe the morphology of the chondrocytes. The levels of inducible collagen type II
were measured by ELISA, apoptosis and cell cycle assays were done by Mac Quant flow cytometry.
Results showed no significant difference in the cell index between the controls and treated cells
with the plant extracts at concentrations of 15 and 30 µg/ml. An increase in the expression of collagen
type II protein was observed at concentration of 30 µg/ml. The plant extracts at a concentration of
50 ug/ml neither induce cell cycle arrest nor apoptosis. From this study it was showed that Pt.
angolensis crude extracts have the potential of tissue regeneration of articular cartilage.
Page 96
Poster
Kalliyana Krishnan Venkiteswaran
Sree Chitra Tirunal Institute for Medical Sciences And Technology
Trivandrum, Kerala State, India
9c, Nikunjam Nxt
Jagathy, Trivandrum
Kerala, India 695014
Mobile: +91 944 7134060
Telephone: +91-471-2520221
Fax: +91-471-2341814
E-mail: muralimanasi@gmail.com
“Development and In Vitro Evaluation of Poly (ɛ-caprolactone) –
Quantum dot Composite Scaffolds for Potential Small Diameter
Vascular Constructs”
Kalliyana Krishnan Venkiteswaran, Soumya Columbus
and Lissy K Krishnan
Biomedical Technology Wing, SCTIMST, Trivandrum, Kerala, INDIA 695012
Introduction: Biodegradable polymeric scaffolds can serve as temporary analogues that mimic
native extra-cellular matrix for regeneration of tissues. Incorporation strategies with nanoparticles
like quantum dots (QD) into polymeric systems may provide new platform for inducing antimicrobial
activity, imaging ability etc. [1]. While designing scaffold, porosity characteristics should be properly
optimized with required mechanical properties, degradation characteristics and appropriate cellmaterial interaction. In the present study, we hypothesize that integration of quantum dots into
electrospun polycaprolactone (PCL) scaffolds may introduce antimicrobial activity and imaging
ability to the scaffold. The major objectives are to evaluate (i) the antimicrobial activity of PCL-QD
scaffold, (ii) variation in morphological and mechanical properties QD incorporation into the scaffold
and (iii) cytocompatibility and functionality of the scaffolds for facilitating endothelial growth.
Materials and Methods: PEG protected CdSe-ZnS based quantum dots were synthesized in lab
and characterized [2]. PCL (Mn=80000, Sigma Aldrich) solution was made using dichloromethane
(HPLC grade, Merck). Scaffolds were prepared using electrospinning technique. 0.1% QD was
incorporated in solution before electrospinning. Bare and QD treated scaffolds were characterized by
SEM, porosimetry, contact angle measurements, EDS and ICP/OES. UTM was used for mechanical
testing. Direct contact and MTT assay was carried out for cyto-toxicity estimation. Endothelial
cell adhesion and proliferation studies on bare and treated scaffolds were studied. Quantitative
parameters were analyzed using ANOVA single factor.
Page 97
Results: PCL-QD scaffold was found to have antimicrobial activity compared to control (gentamycin
and bare scaffolds). QD incorporation into PCL/DCM was found to alter fiber morphology and fiber
diameter distribution of PCL-QD scaffolds compared to bare scaffolds (Fig. 1). Distribution and
release profile of QDs from PCL matrix were demonstrated by EDS and ICP analysis respectively.
QD incorporation was found to reduce mechanical properties of electrospun scaffolds significantly
(Table 1). Scaffolds were found to be cyto-compatible and EC adhesion and proliferation were
demonstrated in both scaffolds. For vascular tissue engineering application, growth of smooth
muscle cells is also necessitated, which will be evaluated in future.
Fig.1. SEM images depicting surface morphology of PCL and PCL-QD scaffolds
Scaffold
Tensile strength
(MPa)
Elongation (%)
Modulus (MPa)
PCL
3.15±0.32
830.54±77
19.75±3.4
PCL-QD
2.61±0.30
523.4±58
29.22±2
Artery
1-2
99
12
Table.1. Tensile properties of electrospun scaffolds
Discussion and Conclusions: QD incorporation affected porosity and mechanical properties of
electrospun PCL scaffolds while inducing antimicrobial activity. Scaffolds were found cytocompatible
and supported EC growth.
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