Stem Cells and Regenerative Medicine For Osteoarthritis

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Stem Cells and Regenerative Medicine For
Osteoarthritis
Farshid Guilak, Ph.D. Departments of Surgery,
Biomedical Engineering, and Mechanical
Engineering Materials Science Duke University
Medical Center
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Tissue Engineering The Oversimplified Paradigm

• Cells
• Biomaterial template
• Growth factors

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Tissue Engineering The Emerging Paradigm

• Cell isolation
• Cell selection and expansion in culture
• Genetic manipulation of cells
• Biomaterial template
• Multiple cell and tissue types, innervation,
  vascularization
• Growth factors and soluble mediators
• Biophysical stimulation in bioreactors
• Packaging, shipping, storage
• Host disease state, inflammation, co-morbidities
• Surgical Implantation and integration
• Rehabilitation
• Regulatory
• Marketing and sales

growing list
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Challenges in Treating Cartilage Defects

• Lack of cell source cell harvest damages native
  tissues
• Inability to restore original structure and
  congruity (40 of ACIs show hypertrophy)
• Cartilage defect market is small
• Lack of adhesion between new repair cartilage and
  original tissue
• Most scaffolds do not possess adequate mechanical
  properties for early load-bearing
• Long FDA cycle for new materials

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Challenges in Treating Cartilage Defects
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Sites of Stem Cells in the AdultLearning from
Natures Mistakes

• Bone marrow
• Hematopoietic
• Mesenchymal
• Muscle
• Bone
• Skin
• Cartilage
• Brain
• Fat

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Are these Cells Really Stem Cells?- Assumptions
in the Definition -

• Self-renew
• Give rise to specialized cell(s)

• Assumptions
• Within the body
• Physiologic conditions and purpose
• Renew indefinitely (?)
• In tissue engineering, we are allowed to violate
  these assumption.

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Isolation of Adipose Stem Cells
Adipose tissue
Collagenase, centrifugation
Stromal-vascular cells
Primary adipocytes
Selective culture
(floaters)
Other cells
Adipose Stem Cells
1mm
1mm
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Adipose-Derived Adult Stromal (ADAS) Cells
Subcutaneous Fat
Adipose-derived Adult Stromal Cells
Halvorsen et al, 2002, Erickson et al, 2002,
Wickham et al, 2002, Safford et al, 2002
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ADAS Cell Surface Phenotype

• Positive Markers CD9, CD10, CD13, CD29, CD34,
  CD44, CD49, CD54, CD55, CD59, CD90, CD105, CD106,
  CD146, CD166, HLA-ABC
• Negative Markers CD11b, CD14, CD18, CD31, CD45,
  CD50, CD62e, Stro1, HLA-DR

Gronthos et al., J. Cell. Physiol., 2001 Wickham
et al., CORR, 2003
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Adipose Stem CellsAggrecan Expression
Estes et al., 2006
p lt 0.0005 relative to all other groups
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Adipose Stem CellsCollagen X Expression



100




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Fold Difference vs. Day 0 /- Sp

1
TGF-ß1
TGF-ß3
DEX TGF-ß1
BMP-6
Base Medium
Day 0 Control
TGF-ß1 IGF-1
TGF-ß1 IGF-1
BMP-6
0.1
p lt 0.0005 relative to all other groups p lt
0.05 relative to Day 0 Control p lt 0.05
relative to Base Medium at Day 7
Estes et al., 2006
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Scaffold MaterialsEffects on Morphology and
Phenotype
10 mm
Gelatin Sponge
2 Agarose
2 Sodium Alginate
Awad et al., 2003
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Tissue Biomechanical Function

• Sub-failure properties
• Inhomogeneous (properties vary with site)
• Anisotropic (properties vary with direction)
• Viscoelastic (behavior is time-dependent,
  biphasic)
• Nonlinear (stress-strain curve is not linear)
• Tribological properties (friction, wear)
• Transport properties (diffusion, convection)
• Failure Properties
• Yield and ultimate stress, fatigue properties
• Structural Properties
• Congruence, surface roughness, size

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What Mechanical Properties are Necessary?
Huang et al., 2005
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Mechanical Properties of Scaffolds

• Gels
• Conducive to chondrogenesis
• Okay in compression, weaker in tension
• Nonwoven fibrous polymers
• Good in tension
• Anisotropy and inhomogeneity are difficult to
  control
• Composite materials
• Combinations of dissimilar materials
• Fibers, gel, etc.

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Developing 3D Woven Scaffolds

• Stacked 2D layers
• 3D Woven

Moutos et al, Nature Materials, 2007
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3-D Woven Scaffolds for Tissue Engineering

• 400 individual fiber bundles
• 50-100 µm diam.
• Vacuum infiltration of gel
• 30-50 void fraction

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3-D Woven Scaffolds for Tissue Engineering
Freed, Moutos, and Guilak
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Mechanical Properties of Composite Matrices
PGA-Agarose
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Tissue Engineering for Osteoarthritis
Liposuction to acquire autologous stem cells
Isolate cells
Mold and prepare 3D scaffold
Culture in bioreactor (?)
Surgical implantation
www.steaman-hawkins.com
Seed cells in 3D Woven fiber scaffold
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Challenges

• Tissue engineered repair must often overcome an
  underlying disease state - how do we predict
  success or patient variability?
• Commercial success and scientific success are
  different issues and may depend on the choice of
  a target
• Bridge the gap between stem cell biologists and
  tissue engineers
• Scale of the problem is larger than we are used
  to dealing with in our field (but not
  unprecedented)

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Systems-Based Approaches

• Research or design questions that are too complex
  for one investigator or team to address alone
• Require understanding the structure-function,
  biology, and regulation of multiple components,
  different disciplines
• Require multiple, interacting, interdisciplinary
  teams (ERCs, industry)

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The Crystal Ball

• Immunologically silent cell for allogeneic
  transplantation
• Methods for cryopreservation (more off-the-shelf
  products)
• Biologically based joint replacement
• Cellular and acellular therapies for arthritis

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Orthopaedic Bioengineering Lab
Staff Robert Nielsen, B.S. Steve Johnson,
LATG Bridgette Furman, B.S. Mimi Phan,
B.S. Elaine Campbell, M.S.
Post-docs and faculty Andrea Clark, Ph.D. Catrin
Davies, Ph.D. Eric Darling, Ph.D. Beverley
Fermor, Ph.D. Tim Griffin, Ph.D. Amy Mcnulty,
Ph.D. Sharon Lubkin, Ph.D. Graduate
Students Bryce Davis, B.S. Susan Christensen,
B.S. Brad Estes, M.S. John Finan, M.S. Will
Garrigues, B.S. Holly Leddy, M.S. Frank Moutos,
M.S.
Undergraduate Students Becca Wilusz Chelsea
He Dennis Cattel Johannah Sanchez-Adams Melanie
Kolkin
Medical Students Ben Ward, B.S.
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Acknowledgments
Collaborators Jeffrey Gimble, M.D., Ph.D. Lori
Setton, Ph.D. Virginia Kraus, M.D., Ph.D. Lisa
Freed, M.D., Ph.D. Tad Vail, M.D. Brice Weinberg,
M.D. Ashutosh Chilkoti, Ph.D.
Funding National Institutes of Health T32
EB01630 VA Research Service National Institutes
of Health P01 AR50245 NCBC National Institutes of
Health R01 AG15768 NASA National Institutes of
Health R01 AR48182 NSF National Institutes of
Health R21 AR49294 Arthritis Foundation GlaxoSmith
Kline, Inc.