frank baaijens et al. laboratory for tissue biomechanics and tissue engineering, department of...

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

• 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)

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

Aortic heart valve

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

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

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

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 ?

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

• 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

Mechanical load on valve: systole

Cells Scaffold(Mechanical)preconditioning

Tissue formation,matrix remodelling

Implantation/Model system

De Hart et al, J. Biomechanics (2003)

In-vitro testing of life tissue

Bioreactor: Physiological flow and pressure

Bio-prosthetic valve

MRI: velocity profiles in bioprosthesis

Rutten et al (2003)

MRI: velocity profiles in bioprosthesis

Rutten et al (2003)

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

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)

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)

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

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)

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)

Electro spinning of bio-mimicking scaffolds

PCL scaffold 1 week culture 2 weeks, confluent

Vaz et al (2003)

Multiple layers for site specific bioactivity

ECM organization: 6-12 months!

6 weeks

20 weeks

time

scaffold degradation

ECM remodelling

load

bea

ring

prop

.

implantation

PGA

Hybrid scaffold

PGA+PCL

• 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

Acknowledgements

• Core DPI program BioPolymers R-0d

• TU/e ‘Bio-Initiative’ grant

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

‘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 -

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

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

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)