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Frank Baaijens et al.Laboratory for Tissue Biomechanics and Tissue Engineering, Department of Biomedical Engineering, TU/e
Simon Hoerstrup et al.Laboratory for Tissue Engineering and Cell Transplantation, Clinic for Cardiovascular Surgery, University Hospital Zurich
Bert Meijer et al.Laboratory for Macro-Molecular and Organic Chemistry, Department of Biomedical Engineering, TU/e
Jan Feijen et al.Polymer Chemistry and Biomaterials, Department of Chemical Engineering, UT
Polymers for health carePolymers for functional tissue engineering
of cardiovascular substitutes
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• All procedures that restore missing tissue in patients require some type of replacement structure.
• Traditionally: totally artificial substitutes, nonliving processed tissue, or transplantation.
• New alternative, tissue engineering: the replacement of living tissue with living tissue, designed and constructed for each individual patient.
• Cardiovascular substitutes market estimated at 80 B€.
Tissue Engineering (The Lancet)
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Small diameter vascular graft
Tunica media
• Cardiovascular disease leading cause of adult death
• No synthetic vascular graft available for diameters < 6mm
Thrombogenicity
Neo-intima hyperplasia (excessive proliferation of SMCs)
external elastic lamina
smooth muscle cells
internal elastic lamina
endothelium
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Aortic heart valve
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Valve replacements
Artificial durability remarks
mechanical life-long trombogenic, noise
synthetic ? mechanical and hemodynamical behaviour ok
Biological
xenograft 7-10 yr Chemical fixation
allograft 7-10 yr Donor dependent
autograft > 15 yr Pulmonary valve transplant
No growth, no repair and adaptation to functional demands
• 300,000 heart valve replacements each year
• Open and close 100,000 times each day, 3 billion in a lifetime
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TE valves: Chain-of-Knowledge
Implantation
Cells Scaffold(Mechanical)preconditioning
Tissue formation,matrix remodelling
Implantation/Model system
Isolation of cells from vessels
Seeding in scaffold Culture, conditioning
Tissue formation
vsmc endothelial cells
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Challenges
Create functional, living cardiovascular tissues: strong: collagen structure elastic: elastin network non-thrombogenic: endothelial lining three dimensional tissue architecture
external elastic lamina
smooth muscle cells
internal elastic lamina
endothelium
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Role of scaffold
• Initial attachment of cells (shape)
• Supply the tissue with sufficient strength
• Bioactivity to control 3D architecture modulate proliferation and differentiation modulate ECM synthesis and degradation stimulate angiogenesis (vasculature)
time
scaffold degradation
ECM remodeling
load
bea
ring
prop
.
implantation ?
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Tissue engineering of heart valves
• Successfully implanted at pulmonary site in juvenile sheep
• Not suitable for implantation at aortic site
• In-vivo tissue maturation takes 20 weeksHoerstrup et al., Circulation (2000)
Tissue engineered heart valve 6 weeks 16 weeks 20 weeks
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• Optimal cell source?
• Design requirements of scaffold?
• What is optimal loading protocol in bioreactor for optimal tissue (collagen) architecture?
• What is mechanical load on tissue?
• How to test functionality of tissue-engineered valves?
How to improve strength?
Cells Scaffold(Mechanical)preconditioning
Tissue formation,matrix remodelling
Implantation/Model system
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Mechanical load on valve: systole
Cells Scaffold(Mechanical)preconditioning
Tissue formation,matrix remodelling
Implantation/Model system
De Hart et al, J. Biomechanics (2003)
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In-vitro testing of life tissue
Bioreactor: Physiological flow and pressure
Bio-prosthetic valve
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MRI: velocity profiles in bioprosthesis
Rutten et al (2003)
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MRI: velocity profiles in bioprosthesis
Rutten et al (2003)
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Heart valve collagen orientation prediction
Driessen et al (2003) Diastole
Computational study of collagen synthesis, alignment and distribution in response to mechanical loading:
• Loading in closed configuration is optimal
• 10 % straining needed
Cells Scaffold(Mechanical)preconditioning
Tissue formation,matrix remodelling
Implantation/Model system
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Impact of cyclic straining on ECM
StaticScaffold Cyclic straining
600 10 20 30 40 500.00
0.05
0.10
0.15
0.20
Str
ess
(MP
a)
Strain (%)
Static
10 % straining (optimal)
A. Mol et al, Thorac. Cardiovasc. Surg., (2003)
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Bioreactor design
Diastole is critical to obtain proper collagen structure
• Change of paradigm for in-vitro mechanical conditioning protocol: new bioreactor design
Mol et al, van Lieshout et al (2003)
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Design requirements of scaffold
• ‘Trivial’: biocompatible, cell attachment, biodegradable, etc
• Elasticity: accommodate cyclic strains of order 10 %
• Strength: stresses of order 1 MPa
• Bioactive to control tissue architecture
• Degradation: both fast (~ 2 weeks) and slow (~ 20 weeks)
• Bio-mimicking: appropriate micro-environment
Cells Scaffold(Mechanical)preconditioning
Tissue formation,matrix remodelling
Implantation/Model system
Collagen structure in arterial wall
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Bioactive scaffolds
Building blocks: PGA, PCL, PTMC, etc
• PGA (‘golden’ standard)
fast degradation (~ 2 weeks)
brittle
• PCL
slow degradation (> 20 weeks)
elastic, ductile, strong
• PTMC
enzymatic in-vivo degradation
elastic, strong
surface erosion: controlled drug release
Meijer et al, Feijen et al (2003)
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Bioactive scaffolds
Building blocks: PGA, PCL, PTMC, etc
Bioactive supramolecular polymer
ureido-pyrimidinone (UPy) polymers
UPy-GRGDS & UPy-PHSRNUPy-GRGDS UPy-PHSRN
UPy-GRGDS
UPy-PHSRN
Synergistic effect on cell-attachment
Dankers et al (2003)
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Electro spinning of bio-mimicking scaffolds
PCL scaffold 1 week culture 2 weeks, confluent
Vaz et al (2003)
Multiple layers for site specific bioactivity
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ECM organization: 6-12 months!
6 weeks
20 weeks
time
scaffold degradation
ECM remodelling
load
bea
ring
prop
.
implantation
PGA
Hybrid scaffold
PGA+PCL
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• First, successful, trial with bone marrow derived mesenchymal stem cells
• Electrospinning of strong, elastic and bioactive scaffolds
• New bioreactor design and loading protocol, extensive in-vitro studies in Zurich and Eindhoven
• In-vitro testing capabilities
• Animal studies in Zurich in progress (pulmonary)
• First human implantation, upon successful completion of animal and in-vitro tests, in pediatric age group
Summary & Outlook
Cells Scaffold(Mechanical)preconditioning
Tissue formation,matrix remodelling
Implantation/Model system
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Acknowledgements
• Core DPI program BioPolymers R-0d
• TU/e ‘Bio-Initiative’ grant
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Hybrid scaffold for vascular graft
• Slow formation of elastin > aneurysm
Porous, elastic support
• Neo-intima hyperplasia
Compliance matching
• Thrombogenicity
Confluent endothelial lining
Fast degradation
Slow degradation
Elastic support
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‘Golden standard’: Coated PGA scaffold
Deformation PGA/P4HB
0
10
20
30
40
50
60
0 2 4 6 8 10 12 14 16
Applied strain (%)
Def
orm
atio
n (%
of a
pplie
d st
rain
)
Biocompatible +
Cell attachment +
Highly porous (98 %) +
Complex shapes -
Mechanical strength -
Elasticity -
Bioactivity -
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Elastic biopolymer: TMC
TMC
Low Tg
in-vivo degradable
cross-linked: no-creep
Example: Scaffold for vascular graft
Inner layer P(TMC)
Particulate leaching
Pore size: 1-10 m
Outer layer P(TMC-CL) (10:90)
Fiber winding
Pore size: 20-60 m Feijen, Grijpma
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DPI Biopolymers for TE program
Hybrid Scaffolds
Baaijens et al. TU/e
Supramolecular Bioactive Polymers
Meijer et al. TU/e
Elastic TMC
Feijen et al. UT
DPI
Biopolymers for Medicine
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Effect of mechanical conditioning
0 10 20 30 40 50 600.00
0.05
0.10
0.15
0.20
Str
ess
(MP
a)
Strain (%)
Control Stretched
Cyclic straining results in :
more pronounced and organized tissue formation
increased load-bearing properties
trend towards cell orientation parallel to the applied strain
tissue strength/stiffness proportional to strain magnitude
0
50
100
150
200
250
DNA GAG HP
%
*** *
StaticMax. 7% strain
Max. 9% strain
Max. 10% strain
A. Mol et al, Thorac. Cardiovasc. Surg., (2003)