Regenerative Medicine
for Musculoskeletal Disease
Jennifer G. Barrett, DVM, PhD, Diplomate ACVS
Assistant Professor of Equine Surgery
Form and Function
• Tendon
– Transmit energy from muscle to bone
– Absorbs energy of impact and releases it
in next stride
– Increases energy efficiency in gait
Form and Function
• Joints
– Subchondral Bone
– Cartilage
• Nearly friction-free
• Shock absorber
• Allows movement
– Ligament
• Stabilize joints
• Stabilize tendons
– Capsule
• Synovial membrane
• Synovial fluid
Form and Function
• Bone
– Protects organs
– Gives shape and frame for body
– Calcium storage
– Allows locomotion:
• Muscles
• Tendons
• Joints
So, what’s the problem?
Cartilage and tendon are
well-adapted for their functions
But, they are poorly adapted for healing and
repair  scar and lose functionality
Although bone heals well, horses have
special problems associated with
fractures
Regenerative Medicine
• Healing of injured tissue back to
original quality
• No scar formation (ideally)
• Efficient healing
• During embryonic development these
tissues are built “from scratch”
– Efficiently
– Normal function
Musculoskeletal Targets
• Tissues that scar and are not
functional after healing
• Tissues that heal slowly
– Tendon
– Ligament
– Cartilage
– Joint
– Bone
Mimic Embryonic Development
• Stem cells
• Growth factors
– Cells talk to each other
• Matrix and Environment
– Matrix is material outside cells that they
attach to (proteins and other molecules)
– Mechanical stresses
Regenerative Techniques
• Cell therapy
• Growth Factor Therapy
• Matrix
• Combinations and specialized sources
of the above
• Gene therapy to enhance cell
proliferation and differentiation
Stem Cells in Development
• Embryonic v. Adult Stem Cells
– All tissues v. some tissues
– Generalized and full of potential v. partially
specialized
• Musculoskeletal tissues come from the same
type of stem cell: Mesenchymal Stem Cells
Stem Cell Roles & Functions
•
•
•
•
•
•
•
Contribute to generating new tissue
Call more stem cells to the area
Supply growth factors
Make matrix
Angiogenesis (new blood supply)
Anti-apoptosis (stop cell death)
Anti-inflammatory
Growth Factors
• Small protein molecules
• Made by certain types of our own cells
• Cells talk to each other with growth
factors
• Signals that tell cells:
– Divide and grow
– Make products (matrix)
– Become a certain type of tissue
Cells & Matrix
Cells
• Adhere to surfaces
• Respond to their environment
• Take shape or migrate
• Grow in one layer or in 3 dimensions
• Create their matrix and tissue based on
all of these factors
Type of matrix affects what cells make
and how it is organized
Cell Therapy
• Embryonic stem cells
• Adult stem cells
– Adipose
– Bone Marrow
– Tendon
– Umbilical Cord Blood
• Tissue cells
– Cartilage
– Tendon
Adult Stem Cell Sources
•
•
•
•
•
Bone Marrow
Muscle
Fat
Synovium
Tendon
• Autologous
Cell Therapy
• Tendon and ligament healing
– MSCs from BM or fat
– Tendon cells/Tendon progenitor cells
• Joint resurfacing / treatment
– Chondrocytes (cartilage tissue cells)
– Mesenchymal stem cells from BM or fat
• Bone healing
– Bone graft
– MSCs from BM or fat
Bone Marrow MSCs
• Improved chance of racing
without re-injury of tendon
• Used in many species
• Studies in model species
have shown increase in
strength of repair in ligament
• Calcification is reported
Equine Vet J 35:99-102, 2003
J Orthop Res. 16:406-413, 1998
J Orthop Res. 22:998-1003, 2004
Bone Marrow MSCs
• Small percentage of BM
cells
• Most studied cell type
• Able to make certain
tissues more easily than
other stem cells
Equine Vet J 35:99-102, 2003
J Orthop Res. 16:406-413, 1998
J Orthop Res. 22:998-1003, 2004
BM MSCs to Tendon
CONTROL
MSC
Schnabel et al. J Orthop Res. 2009 Oct;27(10):1392
MSC-IGF-1
Adipose Cells
• Vet-Stem cell fraction
– Quickly available
– Mixture of cell types
– Less characterized
Vet-Stem Adipose Cells
Saline
Control
Treated
-reduce infiltrate
-improved collagen crimping
-improved collagen fibers
-improved linearity
Nixon, AJVR, 2008
Adipose Progenitor Cells
• Cultured adipose progenitor cells
– More homogeneous population
– More characterization
Embryonic Stem Cells
• ES cell  MSC  Tenocyte
H and E
Masson
Chen et al. Stem Cells. 2009 Jun;27(6):1276-87
• Equine ES cells
– Not autologous, needs FDA approval
TEM
Growth Factors
• IGF-1
• TGF-b
• Autologous conditioned
serum (IRAPTM)
• Platelet Rich Plasma
J Orthop Res. 20:910-919, 2002
Equine Vet J. 35:99-102, 2003
J Orthop Res. 16:406-413, 1998
J Orthop Res. 22:998-1003, 2004
Growth Factors
• Platelet Rich Plasma
•
•
•
•
Enriched platelet fraction from whole blood
Autologous
Can perform this at time of treatment
Potent source of growth factors:
–
–
–
–
–
PDGF
VEGF
TGF
IGF
etc
• Can also release GFs: IRAPTM, and ACS
Platelet Rich Plasma
•
•
•
•
Source of many growth factors
Sustained release?
Possible trophic mediators
Plasma  gel
• Differences between preparations
• Differences between techniques
• Differences between species
Do we want this profile of GFs in every
situation that needs augmentation of healing???
Platelet Rich Plasma
In human patients, used for:
• Tendon injury
• Ligament injury
• Cartilage resurfacing
• Bone healing augmentation
• Intra-articular use
Platelet Rich Plasma
Used for:
• Arthrodesis / tibiotalar fusions
• Endodontics
• Plastic reconstructions
• Wound healing
PRP Growth Factors
PRP
Vol
Platelet #
Fold
Increase
PDGF-BB
TGF-b
b-FGF
VEGF
EGF
IGF-1
0.1
1.4
0.4
1.6
36
27
112
88
Human Preps:
Regen
Fibrinet
HM
Plateltex
5.0
1.2
1.9
2.0
EMC-RMS 7
430
1358
1196
1160
1.65
3.9
4.4
4.4
1016
8
2.3
3.6
11.4
14.3
6.2
8.8
29.8
40.4
13
31
95
3.5
0.1
0.3
0.6
0.7
Mazzucco et al. Vox Sang. 2009 Apr 8
PRP: Canine Ligament
• Lesions created in ligament
• Effect of collagen-PRP hydrogel on
healing
• Ligaments w/ PRP/ and collagen
– greater filling of lesion
– Increased fibrinogen, fibronectin, PDGF-A,
TGF-b1, FGF-2
Murray et al, J Orthop Res. 25(8):1007-17
PRP and Trophism
• PRP injected into tendon recruits cells
to the tendon
• Mouse study:
– Chimeric mouse with GFP-BM cells
– Injected PRP into patellar tendon
– Increased numbers of GFP cells in tendon
Morihara. J Cell Physiol. 2008 Jun;215(3):837-45.
PRP and Bone Healing
• Use PRP with MSCs in canine bone
defect
• Radiography, histology, and
histomorphometry
• MSCs/PRP mixture is useful to promote
bone healing
Yamada, et al. Tissue Engineering 10(5/6), 2004
PRP Effects on Synovium
• Joint cells from arthritic patients
• Release growth factors from PRP
• PRP GFs increased HA secretion
compared with PPP
Anitua et al. Rheumatology 2007;46:1769–1772
Matrix
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•
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•
•
•
•
Fibrin (alone or from PRP)
Collagen
Hyaluronic Acid
Complexed hyaluronic acid
Urinary Bladder Matrix
Human umbilical vein
Synthetic absorbable
Matrix
• AcellTM
– Porcine urinary bladder matrix
– Collagen, basement membrane, growth
factors, etc
– Shows promise, but can cause
inflammatory response
Tissue Regeneration
CELLS
Growth
Factors
Matrix
Mechanical Load
Regenerative Medicine Service
• Bone Marrow Mesenchymal Stem Cells
– Equine
• Adipose Progenitor Cells
– Equine
– Canine
• Platelet Rich Plasma
– Equine
– Canine
Platelet Rich Plasma
• EMC Regenerative Medicine Service
– 7-15 fold higher platelets
– 0.01-0.1 fold lower White Blood Cells
– Suspended in plasma
• Treat:
– Suspensory desmitis
– Small tendon lesions
– Collateral ligament desmitis
BM Stem Cells
• Tendinitis
– Superficial digital flexor tendon
– Deep digital flexor tendon
• Ligament lesions
– Suspensory core lesions
– Collateral ligament lesions
• Joint disease
MRI Guided Injection
• Stem cell / PRP treatment within the hoof
• Collateral ligament desmitis of the coffin joint
EMC-RMS: Future Directions
• Umbilical Cord Blood Stem Cells
– Equine
– Canine
• Tendon Progenitor Cells
– Equine
• Chondrocyte Grafting
– Equine
• ACS
– Equine
– Canine
Thank you for your attention!