1. Tissue Engineering Goal: Regenerate or repair tissues Challenge: Understand how tissues are built in-vivo  i.e. what instructions do cells need to organize into tissue and which cells are responsive? Assumption: Employment of natural biology of the system will allow for greater success

2. Tissue Engineering Triad Prosthesis Scaffold CellsSignals e.g. gels, foams, fibers, membranes, ECM components e.g. adult, ES cells, autogeneous, allogenic, engineered cells, migration of cells into scaffold e.g. growth factors, ascorbic acid, mechanical stimuli

3. Cell-Based Therapy: Some Questions to be Addressed  What type of cell?  Pre-cursor vs. differentiated  Source of cells?  How to expand?  How to control differentiation?

4. Cell Source Differentiated Cells Stem Cells Xenogenic Cells Autogenic Cells Syngeneic Cells Allogenic Cells

5. What are Stem Cells? Cells that have the ability to divide for indefinite periods in culture and give rise to specialized cells 2 Hallmarks of stem cells: 1)Self-renewal 2)Potential to differentiate along more than one lineage

6. Example of Stem Cells – Hematopoietic System Hierarchy Palsson, 2004

7. Adult vs. Embryonic Stem Cells Adult Stem Cells  Defined wrt age of donor  Thought to be lineage-specific Embryonic Stem Cells  Derived from early embryo prior to commitment  Can give rise to progeny for any tissue

8. Scaffold Materials Polyglycolic acid Hydrogels Polylactic-co-glycolic acid Alginate Collagen

9. Scaffolds: Mimic Role of the ECM Space formation (hydrogel) –Direct and guide tissue formation and growth Mechanical support –Tension (collagen) –Compression (PGs) –Elasticity (elastin) Cell-cell, cell-matrix interactions –Attachment, proliferation, migration, differentiation –Cell function

10. Design Criteria for Scaffolds Biocompatibility − Material must not be rejected by immune system Diffusion of nutrients/wastes Mechanical integrity –Support loads at implant site Degradability –Non-toxic species easily metabolized –New tissue forms as original graft material degrades Readily processed into (irregular) 3D shapes

11. Concept of TEVG Development Tissue Engineered Vascular Graft * http://www.enduratec.com/pdf/EnduraTEC_BioReactor_Cardiovascular.pdf + Biopsy Cell Expansion Scaffold Culture in Bioreactor Cells Seeded in Scaffold Vascular Cells Animal Trials Surgical Implantation Clinical Trials *

12. Design Process for Vascular Tissue Engineering (VTE) Identify motivation and/or need Understand normal biology and pathologies Identify gold standard Determine design parameters and engineering considerations Develop strategy to repair or regenerate tissue

13. What is the need for Vascular Grafts?

14. Motivation for Vascular Grafts Conduits used to bypass occluded region in treatment of –Atherosclerosis –Aneurysmal disease –Arterio-venous dialysis –Trauma

15. Atherosclerosis http://www.nlm.nih.gov/medlineplus/ency/imagepages/18050.htm

16. Atherosclerosis Normal Coronary Artery Severe Calcific Coronary Atherosclerosis http://medweb.bham.ac.uk/http/depts/path/Teaching/foundat/athero/Athero1.htm

17. What is the current gold standard treatment?

18. Current Gold Standard for Vascular Grafts Large diameter vessels (> 6mm ID) –Aorta –Synthetic grafts Gore-Tex (ePTFE) Dacron Polyurethane Small Diameter vessels (< 6mm ID) –Coronary artery –Autologous tissue Saphenous vein Internal mammary artery

19. Coronary Bypass Graft Surgery (CABG) www.mayoclinic.org/ coronaryartery-jax/ Blockage Internal mammary artery graft Saphenous vein graft Left anterior decending artery Right coronary artery

20. Autologous Small-Diameter Vascular Grafts Advantages –Patency > 50% over 10 years –Resemblance similar to native vessel Disadvantages –Donor site morbidity –Limited supply Previous procedure Peripheral disease Synthetic materials ineffective due to thrombosis and intimal hyperplasia

21. Ideal Blood Vessel Substitute Vascular substitute that mimics the characteristics of native blood vessels –Composition –Structure –Function –Mechanical properties

22. What is the normal biology of a blood vessel?

23. Blood Vessel Structure

24. erl.pathology.iupui.edu/ HISTO/LABEL29.HTM

25. What are the design requirements and engineering considerations?

26. Functions/Requirements of Blood Vessels Transports blood (nutrients, wastes) Resist spontaneous clotting Vasodilates/vasoconstricts Withstand pulsatile flow forces –Pressure (radially = burst pressure) –Shear stress –Cyclic strain

27. VTE Design Considerations Cell source –Stem cells vs. mature vascular cells –Autologous vs. non-autologous – IR Scaffold selection –Natural vs. synthetic –Mechanical properties Signals –Biochemical –Mechanical Endothelialization of grafts Cell and ECM fiber organization, orientation

28. Design Requirements for VTE Scaffolds Biocompatible Nonthrombogenic Elastic – transmit mechanical stimuli Viscoelastic – avoid compliance mismatch Cell-specific interactions (e.g. cell-collagen) Easily, quickly manufactured Minimally, an intimal and media layer likely required for implantation

29. Mechanical Properties for Vascular Grafts Lyons et al., 2003

30. Mechanical Stimuli Influence Vascular Cell Behavior Endothelial cells –Shear stress Smooth muscle cells –Cyclic strain –Shear stress Fibroblasts –Cyclic strain

31. SMC Production of ECM Proteins in Response to Cyclic Stretching Kim et al., Nature Biotechnology 1999

32. Mechanical Strength of SMC-Collagen Constructs Subjected to Cyclic Strain Kim et al., Nature Biotechnology 1999

33. Summary of Cyclic Strain Effects on SMCs Phenotype Orientation ECM production –Collagen –Elastin –Fibronectin –Proteoglycans Growth factor release –bFGF, PDGF, TGF-  MMP-2 secretion Stiffness, strength improved in SMC-seeded constructs

34. Bioreactor Culture for VTE Cells are exposed in vivo to mechanical stimulus, pulsatile flow, which influences their behavior. Vascular grafts can be cultured in a bioreactor to mimic in vivo mechanical environment –shear stress –cyclic strain

35. Concept of TEVG Development Tissue Engineered Vascular Graft * http://www.enduratec.com/pdf/EnduraTEC_BioReactor_Cardiovascular.pdf + Biopsy Cell Expansion Scaffold Culture in Bioreactor Cells Seeded in Scaffold Vascular Cells Animal Trials Surgical Implantation Clinical Trials *

36. PEG Hydrogel Scaffolds for VTE

37. Pulsatile Flow Bioreactor 5% CO 2 Compliance chamber(s) Perfusion Chambers Pulsatile Pump Reservoir P

38. Cyclic Stretch of Tubular PEG Hydrogels in Pulsatile Flow Bioreactor

39. HASMCs Align in Response to 2 Hz Cyclic Strain 50  m Stretched Static Direction of Applied Strain  10% strain for 7 days

40. Outcomes of Bioreactor Culture Enhanced tissue growth –Cell proliferation –ECM protein synthesis Improved tissue organization, orientation Increased mechanical properties Improved function similar to native vessels Do VTE Scaffolds initially require mechanical properties comparable to native vessels? Why?

42. What VTE strategies have been investigated?

43. VTE Approaches 1.Cell-seeded synthetic grafts 2.Acellular matrices 3.Collagen scaffolds 4.Cell sheets 5.PGA scaffolds

44. VTE Strategy #1: Cell-Seeded Synthetic Grafts Eliminate thrombogenecity of material by seeding endothelial cells in lumen Issues –Retention of ECs on surface, particularly when exposed to flow –Formation of uniform cell monolayer –Physical barrier to long term adaptation –No regulation of vasotone  intimal hyperplasia

45. VTE Strategy #2: Acellular Matrices Rolled, small intestinal submucosa treated to remove cells but leave proteins intact and organized Recruitment of cells from surrounding tissue

46. VTE Strategy #3: Collagen Scaffolds Nerem et al., Annu. Rev. Biomed. Eng., 2001

47. VTE Strategy #3 Example 2: Collagen Scaffolds Seliktar et al., 2000

48. VTE Strategy #3 Example 2: Collagen Scaffolds Seliktar et al., 2000

49. VTE Strategy #3: Example of SMC Alignment in Collagen Scaffolds Seliktar et al., 2000

50. VTE Strategy #3: Collagen Fibril Organization from Mechanical Conditioning

51. Seliktar et al., 2000 VTE Strategy #3: Ring Testing

52. Nerem et al., Annu. Rev. Biomed. Eng., 2001 VTE Strategy #3: Mechanical Conditioning of Collagen Constructs

53. VTE Strategy #4: Cell Sheets Nerem et al., Annu. Rev. Biomed. Eng., 2001

54. VTE Strategy #5: PGA Scaffold Nerem et al., Annu. Rev. Biomed. Eng., 2001

55. VTE Strategy #5: Bioreactor System for PGA scaffold Niklason et al., Science 284: 1999

56. Future Challenges for VTE Optimization of in vitro manipulations –Mechanical conditioning –Biochemical supplementation In vivo integration of graft with host tissue Off the shelf availability