Rheological evaluation of the mechanical
properties of equine synovial fluid from
tarsocrural joints injected with
hyaluronic acid (HA)
Heidi Schmidt
Advisors: Dr. Skip Rochefort PhD and *Dr. Jill Parker VMD DACVS
Danielle Leiske, Jocelyn Frey, and *Autumn Rankin
Dept. of Chemical Engineering and *College of Veterinary Medicine
Oregon State University
Corvallis, OR
Outline
•Motivation
•Background
•What is HA
•Horse and synovial fluid overview
•Information about HA supplementation via Hyvisc®
•Experimental Design
•Tests
•Steady Shear Viscometry
•Dynamic Oscillatory Shear Rheometry
•Capillary Viscometry
•Results
Motivation
•
•
•
Lameness: Limping, abnormal gait, or
hobbling resulting from partial loss of function
in a leg
Osteoarthritis is a significant cause of equine
lameness, resulting in the deterioration of joint
cartilage and formation of painful bone
spurs
Economic Impact: Approximately $1 billion a
year in losses due to lameness
Background
Synovial Fluid
• Viscous liquid present
in all articular joints
• Properties
• Viscosity relates to joint lubrication
• Elasticity relates to impact resistance
(i.e. “shock absorbance”)
• HA (hyaluronic acid) is the major contributor
to the viscoelastic properties of synovial fluid.
Hyaluronic Acid (HA)
•
•
•
•
Biopolyelectrolyte from rooster combs,
human umbilical cords, and bacterial
fermentation processes
High molecular weight (0.2-2 million Da)
$450,000/lb (from Sigma Chemicals)
Exhibits viscoelastic properties
Joint Supplementation
•
•
Intra-articular injection of HA is a common
treatment for controlling symptoms of
osteoarthritis.
Mechanisms are not well understood
• Supplementation may stimulate increased
production of endogenous high molecular
weight HA
• It may protect endogenous HA from enzymatic
degradation in osteoarthritic joints
®
Hyvisc
HA
Concentration
Molecular
Weight
Viscosity at
g = 0.1
.
Cost
Source
vs. Synovial Fluid
Hyvisc®
Synovial Fluid
11 mg/ml
0.5 – 5.0 mg/mL
1.5 – 3 million Da
2 – 10 million Da
800 P
0.1 – 1.0 P
$50 / 2 mL
N/A
Rooster Combs
Synovial Joints
Objective
•
•
Compare the mechanical properties of
synovial fluid from horses treated with HA
to synovial fluid from untreated horses.
Studies on normal, healthy joints
Experimental Design
•
Five horses (10 healthy hocks) divided into
three groups
• Treatment Group (4 hock joints)
•
Received 2 mL (22 mg) HA supplementation (Hyvisc®)
• Negative Control Group (3 hock joints)
•
Received no joint supplementation
• Positive Control Group (3 hock joints)
•
Received 2 mL of sterile Lactated Ringers Solution (LRS)
Experimental Design
•
•
•
Synovial fluid collections:
• Treatment day (before injections)
• 7 days post treatment
• 14 days post treatment
The horses were monitored daily for any signs of
disease
Cytology was performed on all of the synovial fluid
samples to monitor the health of the joints during
the study.
Experimental Design
•
Test Measurements
• Steady Shear Tests :
Viscosity
• Dilute Capillary Viscometer Tests :
Relative Viscosity
• Dynamic Oscillatory Shear Tests:
Viscosity & Elasticity
Rheometer
Geometries
Rheometrics Fluids Spectrometer II
Steady Shear Tests
•
Bottom plate
rotates at a
constant
speed
(shear rate)
Torque
measured on
upper cone
(shear stress)
Viscosity =
Shearstress
shear
stress
shear
rate
Shearrate
0.100
Newtonian Plateau
Viscosity (Pas)
•
0.010
Shear Thinning
(Non-Newtonian)
0.001
1
10
100
Shear Rate (1/s)
1000
Viscosity (P)
HA Treated Horse
Steady Shear Test
Pre-treatment
1 Wk. Post Treatment
2 Wks. Post Treatment
10.00
1.00
0.10
Data Analysis
Data Analysis
0.01
0.1
1.0
10.0
Shear Rate (1/s)
100.0
Dynamic Oscillatory
Shear Tests
Hyvisc®
Moduli (dyne/cm^2)
10000
1000
100
G’’
G’
10
1
0.01
0.10
•Cone oscillates
•Measures strain, stress
•G’: Elastic Modulus
G’
•G”: Viscous Modulus
G’’
•G’<G”: Viscous
•Acts as a lubricant
Cross-over
•G’>G”: Elastic
Point
G'
G"
•Behaves like a shock
1.00
10.00
absorber
Frequency (Hz)
Moduli (dyne/cm^2)
HA Treated Horse
Dynamic Oscillatory Shear Test
100.000
10.000
1.000
0.100
0.010
Data Analysis
0.001
0.10
1.00
10.00
100.00
Frequency (Hz)
G' Pre-treatment
G' 1 Wk. Post Treatment
G' 2 Wks. Post Treatment
G'' Pre-treatment
G'' 1 Wk. Post Treatment
G'' 2 Wks. Post Treatment
Dilute Solution Capillary
Viscometer
•
Timing
Lines
•
Measures relative viscosity
in dilute polymer solutions
Flow times differ because
of variations in
concentration and
molecular weight of HA
Capillary
Relative Viscosity =
Solution flow time
Solvent flow time
HA Treated Horse
Relative Viscosity Comparison
4.5
Pre-treatment
1 Wk. Post Treatment
2 Wks. Post Treatment
Relative Viscosity
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.0
0.1
0.2
0.3
0.4
Fraction of Pure Synovial Fluid
0.5
0.6
Relative Viscosity
Average Relative Viscosity Values
at 0.5 Concentration
5
4
3
2
1
0
Week 0
Week 1
Week 2
Weeks After Injection
HA
+ Control
- Control
Viscosity (P)
Average Viscosity at g = 0.1
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
Week 0
Week 1
Week 2
Weeks After Injection
HA
+ Control
- Control
Percent Change of Synovial Fluid Properties
1 Week Post-Treatment
1
200%
HA Group
Percent Change
150%
100%
+ Control Group
50%
- Control Group
0%
-50%
-100%
G'
G''
.
Viscosity at γ = 0.1
.
Viscosity at γ = 10
Relative viscosity
Percent Change of Synovial Fluid Properties
2 Weeks Post-Treatment
Percent Change
200%
150%
100%
50%
HA Group
0%
+ Control
Group
- Control
Group
1
-50%
-100%
G'
G''
.
Viscosity at γ= 0.1
.
Viscosity at γ = 10
Relative viscosity
Conclusion
Hyaluronic Acid supplementation has
a positive affect on the rheological
properties of synovial fluid one week
post-treatment
Future Work
•Extend research to a larger test group
•Study the effects of intravenous HA
injections
•Perform light scattering tests to find specific
HA concentration and molecular weight
•Explore the effects of HA supplements at
different molecular weights
Acknowledgements
•Howard Hughes Medical Institute
•Dr. Skip Rochefort, OSU ChE Dept
•Danielle Leiske and Jocelyn Frey
•Dr. Jill Parker and Autumn Rankin,
OSU College of Veterinary Medicine
•Kevin Ahern
Questions?
Danielle Leiske and Autumn Rankin
Preparing the joint for an injection