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Designing Vascularized Soft Tissue Constructs for Transport

Designing Vascularized Soft Tissue Constructs for Transport. EID 121 Biotransport EID 327 Tissue Engineering David Wootton The Cooper Union. Acknowledgement and Disclaimer. This material is based upon work supported in part by the National Science Foundation under Grant No. 0654244

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Designing Vascularized Soft Tissue Constructs for Transport

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  1. Designing Vascularized Soft Tissue Constructs for Transport EID 121 Biotransport EID 327 Tissue Engineering David Wootton The Cooper Union

  2. Acknowledgement and Disclaimer • This material is based upon work supported in part by the National Science Foundation under Grant No. 0654244 • Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation

  3. Challenge • Develop a CAD model for printing a hydrogel tissue engineering construct for soft tissue • Vascular template • Sufficient oxygen delivery • Model validation/justification

  4. Learning Objectives • Tissue Engineering (for EID 121) • Oxygen Transport • With oxygen carriers • Vascular Anatomy • Biomanufacturing for Tissue Engineering • Bulk Methods • Computer-aided Manufacturing • Organ printing

  5. Overview of Tissue Engineering • Working definition (1988): “The application of the principles and methods of engineering and life sciences toward the fundamental understanding of structure-function relationships in normal and pathological mammalian tissue and the development of biological substitutes to restore, maintain, or improve tissue function.” • Where we are already: • Robust research area • Tissue Engineered Medical Products – several approved • Expansion to biological model systems • Many unsolved challenges remain • Science base is rather weak for engineering (fundamental laws?)

  6. A Famous Picture of TE Polymer Ear shape Bovine chondro-cytes Implant in Nude Mouse

  7. Potential TE Applications

  8. Tissue Engineering Market Size • Costs of tissue-related disease procedures: $400 B (1993) • 70+ companies • Average $10 M/year • Organ transplant waiting lists are growing (doubled in 6 years) $$

  9. One Famous TE Paradigm

  10. Your Design Challenge • Overcome practical size limit on engineered tissue • Diffusion is not sufficient for oxygenation in thick tissues • Compare 3 Approaches: • No flow (diffusion only) • Porous scaffold with permeation flow • Hydrogel with vascular channels

  11. Design Challenge • Example: engineer a 1 cm3 liver tissue construct • Scaffold + hepatocytes • How will you make the scaffold? • How will you assure oxygenation? • What else do you need to know? Polysaccarid • Questions for instructor? • Discuss in groups of 3 http://licensing.inserm.fr/upload/ 270109_140959_PEEL_U5UFfJ.gif Polysacchiride scaffold Cell-seeded scaffold

  12. Design Challenge • What else do you need to know? • Formulate biotransport problem • Hepatocyte (cell) properties • Oxygen transport properties • Dimensions • Is there a vascular system?

  13. Oxygen Transport • References: • Truskey, Yuan, and Katz. Transport Phenomena in Biological Systems. 2nd Ed., 2009. (Section 13.5) • RL Fournier. Basic Transport Phenomena in Biomedical Engineering. 2nded, 2006. (Ch. 6) • O2 Readily crosses cell membranes • Transport Mechanisms: diffusion, convection • Metabolic demand and cell density control oxygen concentration

  14. Oxygen Diffusion Transport • Simplest Approach: diffusion only • Use 1D slab for simplicity • How deep can O2 penetrate? tissue

  15. Oxygen Diffusion Transport • Half-slab model (thickness 2L, max concentration on top and bottom) • Dissolved O2 in medium via Henry’s Law • O2 in blood at 37ºC, H = 0.74 mmHg/mM • Typical air pO2 = 140mmHg, CO2 = 190mM 0 tissue L x

  16. Oxygen Diffusion Transport • O2 uptake rate RO2 or Gmetabolic • Expect Michealis-Menten kinetics, e.g. • Usually pO2 >> Km, so ~ zero order: C = C0 = 190mM 0 tissue L Symmetry: x C = C0 = 190mM

  17. Oxygen Diffusion Transport • Diffusion flux = uptake (1-D): Hepatocytes: Vmax = 0.4 nmol/106 cells/sec Km = 0.5 mmHg Cell diameter = 20 mm Density up to rcells = 108 cell/cm3 Oxygen: H = 0.74 mmHg/mM De = 2 x 10-5 cm2/s • Effective Diffusivity, De • Uptake rate • Cell seeding density, r C = C0 = 190mM 0 tissue L Symmetry: x C = C0 = 190mM

  18. Oxygen Diffusion Transport Hepatocytes: Vmax = 0.4 nmol/106 cells/sec Km = 0.5 mmHg Cell diameter d = 20 mm Density up to rcells = 108 cell/cm3 Oxygen: H = 0.74 mmHg/mM De = 2 x 10-5 cm2/s • Diffusion flux = uptake (1-D): • Void volume, e • Effective Diffusivity, De C = C0 = 190mM 0 tissue L Symmetry: x C = C0 = 190mM

  19. Oxygen Diffusion Transport • Work in small groups • What is the O2 uptake rate in the tissue? • What is the concentration distribution? • How thick could the construct be? • Check vs. following solution

  20. Oxygen DiffusionTransport solution Hepatocytes: Vmax = 0.4 nmol/106 cells/sec Km = 0.5 mmHg Cell diameter d = 20 mm Density up to rcells = 108 cell/cm3 Oxygen: H = 0.74 mmHg/mM De = 2 x 10-5 cm2/s • Uptake rate: • Solution: • Maximum thickness • Set C(L) to zero: • Example gives Lmax = 138 mm • How far would you need to reduce cell density to compensate, for 1 cm construct?

  21. Oxygen Diffusion Transport • Simplest Approach: diffusion only • Use axisymmetric cylinder for simplicity • How deep can O2 penetrate?

  22. Oxygen Diffusion Transport • Cylinder model (radius Rc, max concentration on surface) • Dissolved O2 in medium via Henry’s Law • O2 in blood at 37ºC, H = 0.74 mmHg/mM • Typical air pO2 = 140mmHg, CO2 = 190mM r Rc tissue 0

  23. Oxygen Diffusion Transport • O2 uptake rate RO2 • Expect Michealis-Menten kinetics, • Usually pO2 >> Km, so ~ zero order r C = C0 = 190mM Rc tissue 0 Symmetry:

  24. Oxygen Diffusion Transport • Diffusion flux = uptake (axisymmetric): Hepatocytes: Vmax = 0.4 nmol/106 cells/sec Km = 0.5 mmHg Cell diameter = 20 mm Density up to rcells = 108 cell/cm3 Oxygen: H = 0.74 mmHg/mM De = 2 x 10-5 cm2/s • Effective Diffusivity, De r C = C0 = 190mM Rc tissue 0 Symmetry:

  25. Oxygen Diffusion Transport Hepatocytes: Vmax = 0.4 nmol/106 cells/sec Km = 0.5 mmHg Cell diameter d = 20 mm Density up to rcells = 108 cell/cm3 Oxygen: H = 0.74 mmHg/mM De = 2 x 10-5 cm2/s • Diffusion flux = uptake (1-D): • Void volume, e • Effective Diffusivity, De r C = C0 = 190mM Rc tissue 0 Symmetry:

  26. Oxygen Diffusion Transport • Work in small groups • What is the O2 uptake rate in the tissue? • What is the concentration distribution? • How thick could the construct be? • Check vs. following solution

  27. Oxygen DiffusionTransport solution Hepatocytes: Vmax = 0.4 nmol/106 cells/sec Km = 0.5 mmHg Cell diameter d = 20 mm Density up to rcells = 108 cell/cm3 Oxygen: H = 0.74 mmHg/mM De = 2 x 10-5 cm2/s • Uptake rate: • Solution:

  28. Oxygen DiffusionTransport solution Hepatocytes: Vmax = 0.4 nmol/106 cells/sec Km = 0.5 mmHg Cell diameter d = 20 mm Density up to rcells = 108 cell/cm3 Oxygen: H = 0.74 mmHg/mM De = 2 x 10-5 cm2/s • Uptake rate: • Solution: • Maximum thickness • Set C(0) to zero: • Example gives Rmax = 195 mm • How far would you need to reduce cell density to compensate, for 1 cm construct?

  29. Checking your learning progress • What is diffusion transport? • Diffusion is fast over short distances, slow over long distances • Why? • How does oxygen uptake reaction affect oxygen penetration into tissue • Dimensionless transport-reaction parameter (see Krogh cylinder model F)

  30. Class Discussion Time • Q&A about diffusion transport • Make suggestions to improve oxygen transport rate

  31. Oxygen Transport Problem • We can improve transport with flow (convection) through thick direction • Four approaches to consider • Tissue in to spinner flask • Drive permeation flow through pores • Tissue with engineered vascular channels • Let tissue form vascular system

  32. Oxygen Transport Problem • Spinner flask doesn’t help much • Minimal medium flow due to small pressure gradients • Best model: diffusion through tissue • Permeation flow • Manufacturing methods needed to control pores • Characterize scaffold media flow • Can scaffold withstand pressure required? • Implantation issue: source of pressure?

  33. Oxygen Transport Problem • Engineered vascular system • How to manufacture? • Current research subject • Proposed solutions use computer-aided manufacturing (CAM) and design (CAD) • What are the mass transport requirements for the vascular system?

  34. Tissue Engineering Manufacturing Overview • How to make tissues more efficiently? • How to improve control of tissue constructs? • Use modern manufacturing methods

  35. Bulk Scaffold Manufacturing Methods • First consider “Bulk” scaffold manufacturing methods • Widely used: • Relatively easy to replicate • Relatively fast • Good control of material biochemical properties • Recipes influence scaffold architectural properties (indirect control)

  36. Bulk Scaffold Manufacturing Examples • Electrospinning • Salt Leaching • Freeze Drying • Phase Separation • Gas Foaming • Gel Casting

  37. Electrospinning http://www.centropede.com/UKSB2006/ePoster/images/background/ElectrospinFigure.jpg

  38. Salt Leaching Agrawal CM et al, eds, Synthetic Bioabsorbable Polymers for Implants, STP 1396, ASTM, 2000

  39. Freeze Drying

  40. Phase Separation

  41. Bulk methods pros and cons + Relatively fast batch processing + Often low investment required - Non optimal microstructures: • High porosity (required for connectedness) • Permeability often low (especially foams) • Low strength (eg too low to replace bone) • Modest control of pore shape

  42. Computer-aided manufacturing • Top-down control of scaffold • CAD models • Reverse engineering (from medical images) • Based on existing technology • Inkjet/bubblejet/laserjet printers • Rapid prototyping machines • Electronics and MEMS manufacturing • Often compatible with bulk methods

  43. Photopatterning Surface Chemistry

  44. Microcontact and Microfluidic Printing

  45. Micromachining, Soft Lithography Soft Lithography

  46. 3D Printing Spread powder layer Print powder binder

  47. Solid Freeform Fabrication http://www.msoe.edu/rpc/graphics/fdm_process.gif • Make arbitrary shapes • Limited resolution • Incrementally build • Layer by layer • Fuse Layers to get 3D part • Several processes including • Fused deposition • Drop on demand • Laser sintering http://www-ferp.ucsd.edu/LIB/REPORT/ CONF/SOFE99/waganer/fig-2.gif

  48. CAD-based Porogen Method Mondrinos M et al, Biomaterials 27 (2006) 4399–4408

  49. Dead Live Current Research on Scaffolds • EWOD Video Clips

  50. EWOD Microarrays Mounted on X-Y Moving Planar Arm Material Delivery System Hydrogel Reservoir Cell Microarray Hydrogel Microarray Crosslinker Microarray Growth Factor Microarray Crosslinker Reservoir X-Y Moving Control System Scaffold Cell Reservoir EWOD Microarrays Control System Growth Factor Reservoir Z Moving Control System Moving Direction Moving Table Current Research on Scaffolds • Drexel, Duke, Cooper Union collaboration • Electrowetting tissue manufacturing • CAD model • Print components • Hydrogel • Crosslinker • Cells • Growth Factor • Web site: http://www.mem.drexel.edu/zhou2/research/electro-wetting-on-di-electric-printing

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