1. 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 care Polymers for functional tissue engineering of cardiovascular substitutes

2. 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)

3. 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

4. Aortic heart valve

5. Valve replacements 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

6. TE valves: Chain-of-Knowledge Implantation CellsScaffold (Mechanical) preconditioning Tissue formation, matrix remodelling Implantation/ Model system Isolation of cells from vessels Seeding in scaffoldCulture, conditioning Tissue formation vsmc endothelial cells

7. 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

8. 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 bearing prop. implantation ?

9. 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 weeks Hoerstrup et al., Circulation (2000) Tissue engineered heart valve 6 weeks16 weeks20 weeks

10. 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? CellsScaffold (Mechanical) preconditioning Tissue formation, matrix remodelling Implantation/ Model system

11. Mechanical load on valve: systole CellsScaffold (Mechanical) preconditioning Tissue formation, matrix remodelling Implantation/ Model system De Hart et al, J. Biomechanics (2003)

12. In-vitro testing of life tissue Bioreactor: Physiological flow and pressure Bio-prosthetic valve

13. MRI: velocity profiles in bioprosthesis Rutten et al (2003)

14. MRI: velocity profiles in bioprosthesis Rutten et al (2003)

15. 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 CellsScaffold (Mechanical) preconditioning Tissue formation, matrix remodelling Implantation/ Model system

16. Impact of cyclic straining on ECM StaticScaffoldCyclic straining 60 01020304050 0.00 0.05 0.10 0.15 0.20 Stress (MPa) Strain (%) Static 10 % straining (optimal) A. Mol et al, Thorac. Cardiovasc. Surg., (2003)

17. 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)

18. 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 CellsScaffold (Mechanical) preconditioning Tissue formation, matrix remodelling Implantation/ Model system Collagen structure in arterial wall

19. 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)

20. Bioactive scaffolds Building blocks: PGA, PCL, PTMC, etc Bioactive supramolecular polymer ureido-pyrimidinone (UPy) polymers UPy-GRGDS & UPy-PHSRNUPy-GRGDSUPy-PHSRN UPy-GRGDS UPy-PHSRN Synergistic effect on cell-attachment Dankers et al (2003)

21. Electro spinning of bio-mimicking scaffolds PCL scaffold1 week culture2 weeks, confluent Vaz et al (2003) Multiple layers for site specific bioactivity

22. ECM organization: 6-12 months! 6 weeks 20 weeks time scaffold degradation ECM remodelling load bearing prop. implantation PGA Hybrid scaffold PGA+PCL

23. 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 CellsScaffold (Mechanical) preconditioning Tissue formation, matrix remodelling Implantation/ Model system

24. Acknowledgements Core DPI program BioPolymers R-0d TU/e ‘Bio-Initiative’ grant

26. 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

27. ‘Golden standard’: Coated PGA scaffold Deformation PGA/P4HB 0 10 20 30 40 50 60 0246810121416 Applied strain (%) Deformation (% of applied strain )  Biocompatible+  Cell attachment+  Highly porous (98 %)+  Complex shapes-  Mechanical strength-  Elasticity-  Bioactivity-

28. 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

29. 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

30. Effect of mechanical conditioning 0102030405060 0.00 0.05 0.10 0.15 0.20 Stress (MPa) 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 DNAGAGHP % * * * * Static Max. 7% strain Max. 9% strain Max. 10% strain A. Mol et al, Thorac. Cardiovasc. Surg., (2003)