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Tissue Engineering
• Goal: Regenerate or repair tissues• Challenge: Understand how tissues
are built in-vivoi.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
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Tissue Engineering Triad
Prosthesis
Scaffold
Cells Signals
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
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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?
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Cell Source
Differentiated Cells
Stem Cells
Xenogenic Cells
Autogenic Cells
Syngeneic Cells
Allogenic Cells
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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-renewal2) Potential to differentiate along more than
one lineage
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Example of Stem Cells – Hematopoietic System
Hierarchy
Palsson, 2004
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Adult vs. Embryonic Stem Cells
• Adult Stem CellsDefined wrt age of donor Thought to be lineage-specific
• Embryonic Stem CellsDerived from early embryo prior to
commitment Can give rise to progeny for any
tissue
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Scaffold Materials
Polyglycolic acid
Hydrogels
Polylactic-co-glycolic acid
Alginate
Collagen
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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
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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
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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
*
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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
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What is the need for Vascular Grafts?
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Motivation for Vascular Grafts
• Conduits used to bypass occluded region in treatment of – Atherosclerosis– Aneurysmal disease– Arterio-venous dialysis– Trauma
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Atherosclerosis
http://www.nlm.nih.gov/medlineplus/ency/imagepages/18050.htm
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Atherosclerosis
Normal Coronary Artery Severe Calcific Coronary Atherosclerosis
http://medweb.bham.ac.uk/http/depts/path/Teaching/foundat/athero/Athero1.htm
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What is the current gold standard treatment?
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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
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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
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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
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Ideal Blood Vessel Substitute
• Vascular substitute that mimics the characteristics of native blood vessels– Composition– Structure– Function– Mechanical properties
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What is the normal biology of a blood vessel?
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ADVENTITIAConnective tissue
fibroblasts capillaries
nerves
MEDIA Smooth muscle cells
elastin fibers
INTIMAEndothelial cell lining
Blood Vessel Structure
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Blood Vessel Structure
erl.pathology.iupui.edu/ HISTO/LABEL29.HTM
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What are the design requirements and
engineering considerations?
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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
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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
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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
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Mechanical Properties for Vascular Grafts
Lyons et al., 2003
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Mechanical Stimuli Influence Vascular Cell Behavior
• Endothelial cells– Shear stress
• Smooth muscle cells– Cyclic strain– Shear stress
• Fibroblasts– Cyclic strain
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SMC Production of ECM Proteins in Response to Cyclic
Stretching
Kim et al., Nature Biotechnology 1999
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Mechanical Strength of SMC-Collagen Constructs Subjected to
Cyclic Strain
Kim et al., Nature Biotechnology 1999
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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
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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
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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
*
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PEG Hydrogel Scaffolds for VTE
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Pulsatile Flow Bioreactor
5% CO2
Compliance chamber(s)
Perfusion Chambers
Pulsatile Pump
Reservoir
P
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Cyclic Stretch of Tubular PEG Hydrogels in Pulsatile Flow
Bioreactor
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HASMCs Align in Response to
2 Hz Cyclic Strain
50 m 50 m
Stretched Static
Direction of
Applied Strain
10% strain for 7 days
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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?
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What VTE strategies have been investigated?
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VTE Approaches
1. Cell-seeded synthetic grafts2. Acellular matrices3. Collagen scaffolds4. Cell sheets5. PGA scaffolds
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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
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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
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VTE Strategy #3: Collagen Scaffolds
Nerem et al., Annu. Rev. Biomed. Eng., 2001
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VTE Strategy #3 Example 2: Collagen Scaffolds
Seliktar et al., 2000
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VTE Strategy #3 Example 2: Collagen Scaffolds
Seliktar et al., 2000
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VTE Strategy #3: Example of SMC Alignment in Collagen
Scaffolds
Seliktar et al., 2000
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Seliktar et al., 2000
VTE Strategy #3: Collagen Fibril Organization from
Mechanical Conditioning
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Seliktar et al., 2000
VTE Strategy #3: Ring Testing
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Nerem et al., Annu. Rev. Biomed. Eng., 2001
VTE Strategy #3: Mechanical Conditioning of Collagen
Constructs
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VTE Strategy #4: Cell Sheets
Nerem et al., Annu. Rev. Biomed. Eng., 2001
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VTE Strategy #5: PGA Scaffold
Nerem et al., Annu. Rev. Biomed. Eng., 2001
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VTE Strategy #5: Bioreactor System for PGA
scaffold
Niklason et al., Science 284: 1999
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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