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

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

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?

Cell Source

Differentiated Cells

Stem Cells

Xenogenic Cells

Autogenic Cells

Syngeneic Cells

Allogenic Cells

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

Example of Stem Cells – Hematopoietic System

Hierarchy

Palsson, 2004

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

Scaffold Materials

Polyglycolic acid

Hydrogels

Polylactic-co-glycolic acid

Alginate

Collagen

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

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

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

*

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

What is the need for Vascular Grafts?

Motivation for Vascular Grafts

• Conduits used to bypass occluded region in treatment of – Atherosclerosis– Aneurysmal disease– Arterio-venous dialysis– Trauma

Atherosclerosis

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

Atherosclerosis

Normal Coronary Artery Severe Calcific Coronary Atherosclerosis

http://medweb.bham.ac.uk/http/depts/path/Teaching/foundat/athero/Athero1.htm

What is the current gold standard treatment?

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

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

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

Ideal Blood Vessel Substitute

• Vascular substitute that mimics the characteristics of native blood vessels– Composition– Structure– Function– Mechanical properties

What is the normal biology of a blood vessel?

ADVENTITIAConnective tissue

fibroblasts capillaries

nerves

MEDIA Smooth muscle cells

elastin fibers

INTIMAEndothelial cell lining

Blood Vessel Structure

Blood Vessel Structure

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

What are the design requirements and

engineering considerations?

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

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

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

Mechanical Properties for Vascular Grafts

Lyons et al., 2003

Mechanical Stimuli Influence Vascular Cell Behavior

• Endothelial cells– Shear stress

• Smooth muscle cells– Cyclic strain– Shear stress

• Fibroblasts– Cyclic strain

SMC Production of ECM Proteins in Response to Cyclic

Stretching

Kim et al., Nature Biotechnology 1999

Mechanical Strength of SMC-Collagen Constructs Subjected to

Cyclic Strain

Kim et al., Nature Biotechnology 1999

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

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

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

*

PEG Hydrogel Scaffolds for VTE

Pulsatile Flow Bioreactor

5% CO2

Compliance chamber(s)

Perfusion Chambers

Pulsatile Pump

Reservoir

P

Cyclic Stretch of Tubular PEG Hydrogels in Pulsatile Flow

Bioreactor

HASMCs Align in Response to

2 Hz Cyclic Strain

50 m 50 m

Stretched Static

Direction of

Applied Strain

10% strain for 7 days

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?

What VTE strategies have been investigated?

VTE Approaches

1. Cell-seeded synthetic grafts2. Acellular matrices3. Collagen scaffolds4. Cell sheets5. PGA scaffolds

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

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

VTE Strategy #3: Collagen Scaffolds

Nerem et al., Annu. Rev. Biomed. Eng., 2001

VTE Strategy #3 Example 2: Collagen Scaffolds

Seliktar et al., 2000

VTE Strategy #3 Example 2: Collagen Scaffolds

Seliktar et al., 2000

VTE Strategy #3: Example of SMC Alignment in Collagen

Scaffolds

Seliktar et al., 2000

Seliktar et al., 2000

VTE Strategy #3: Collagen Fibril Organization from

Mechanical Conditioning

Seliktar et al., 2000

VTE Strategy #3: Ring Testing

Nerem et al., Annu. Rev. Biomed. Eng., 2001

VTE Strategy #3: Mechanical Conditioning of Collagen

Constructs

VTE Strategy #4: Cell Sheets

Nerem et al., Annu. Rev. Biomed. Eng., 2001

VTE Strategy #5: PGA Scaffold

Nerem et al., Annu. Rev. Biomed. Eng., 2001

VTE Strategy #5: Bioreactor System for PGA

scaffold

Niklason et al., Science 284: 1999

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