Transcript Molecular and Rheological Characterization of Hyaluronic Acid and Equine Synovial Fluid for the Treatment of Lameness in Horses Sara Tracy Advisors: Dr.

Molecular and Rheological Characterization
of Hyaluronic Acid and Equine Synovial Fluid
for the Treatment of Lameness in Horses
Sara Tracy
Advisors: Dr. Skip Rochefort PhD and *Dr. Jill Parker DVM
Danielle Leiske
Dept. of Chemical Engineering and *College of Veterinary Medicine
Oregon State University
Corvallis, Oregon
Objective


Establish normal properties of equine
synovial fluid
Compare to properties of hyaluronic acid
solutions of various concentrations
Motivation



The horse industry loses around 1 billion
dollars annually due to lameness.
Lameness: An indication of a structural or
functional disorder in one or more limbs
that is evident during movement or in the
standing position.
Hyaluronic acid is used to treat joint
problems in both horses and humans.
Background
Synovial Fluid


Multipurpose fluid surrounding all
articular joints
Synovial fluid has both viscous and
elastic properties (viscoelastic)

Viscosity relates to lubrication

Elasticity relates to shock absorption
Background
Hyaluronic Acid (HA)

High molecular weight biopolyelectrolyte made up of repeating
glucuronic acid and N-acetylglucosamine subunits

Major component of synovial fluid

Exhibits viscoelastic properties


Commonly obtained from:
 Chicken combs
 Human umbilical cords
 Bacterial fermentation processes
Molecular weight around

0.5 to 10 million Da
Joints

Joints of focus
Joints

Skeletal view of the joints
Joints

Common problems with equine joints:

Degenerative Joint Disease – DJD
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Damage to the articular cartilage
Commonly affects heavily worked and aged horses
Osteochondritis Dissecans – OCD


Failure of the bone underlying the smooth articular cartilage
to form properly
Commonly affects young horses
Joints
Synovial Fluid
 Average HA concentration


0.5 to 5.0 mg/mL



2 to 10 million Da
Viscosity at a shear rate
of 0.1/second
0.1 to 1.0 Poise
Hyaluronic Acid Joint
Supplementation

Average molecular weight




Intra-articular injection
Intravenous injection
Oral administration
Synthovial 7/Hyalun


Molecular weight - around
1.5 million Da
Concentration – 3 to 5
mg/mL
Tests
Rheology

The rheometer measures both
ELASTICITY and VISCOSITY
Rheometrics RFS II


Steady shear test measures the viscosity as a
function of shear rate
Dynamic oscillatory shear test measures the
viscous modulus and elastic modulus as a function
of frequency
Tests
Steady Shear Test

Plate rotates at increasing shear rates

Torque, or shear stress, is measured by the
rheometer

Viscosity = shear stress
shear rate
Tests



Bottom plate rotates at
increasing shear rates
Shear stress is measured
on the top cone
Viscosity decreases with
increasing shear rates
due to molecular
disentanglement
Steady Shear Test - Synthovial 7
Viscosity (P)
10.00
1.00
0.10
1 mg/mL
2 mg/mL
3 mg/mL
0.01
0.10
1.00
10.00
Rate (1/s)
100.00
Steady Shear Test - Hocks
Viscosity (P)
1.00
0.10
0.01
0.10
022602 RH
22-160 RH
22-160 LH
34-089 LH
34-089 RH
34-092 RH OCD
1 mg/mL
2 mg/mL
1.00
10.00
Rate (1/s)
100.00
Steady Shear Test - Stifle
Viscosity (P)
10.00
1.00
0.10
071602 RS
34-089 LS
34-089 RS
022602 RS DJD
0.01
0.10
1.00
1 mg/mL
2 mg/mL
3 mg/mL
10.00
Rate (1/s)
100.00
Tests
Dynamic Oscillatory Shear Test


Plate oscillates at increasing
frequencies
Strain and stress are measured
to determine G’ and G’’




G’ represents the elastic (storage)
modulus
G’’ represents the viscous (loss)
modulus
When G’ > G’’ the fluid behaves
more elastic
When G’ < G’’ the fluid behaves
more viscous
Tests




strain = imposed
stress = measured
G’ (elastic modulus)
= in phase
G’’ (viscous modulus)
= out of phase
Dynamic Test - Synthovial 7
Modulus (dyn/cm2)
100.00
10.00
1.00
G' 1 mg/mL
G'' 1 mg/mL
G' 2 mg/mL
G'' 2 mg/mL
G' 3 mg/mL
G'' 3 mg/mL
0.10
0.01
0.10
1.00
10.00
Frequency (Hz)
100.00
Dynamic Test - Hocks
Modulus (dyn/cm2)
100.00
10.00
1.00
0.10
G' 1 mg/mL
0.01
G'' 1 mg/mL
G' 2 mg/mL
G'' 2 mg/mL
G' 34-089 RH
G'' 34-089 RH
G' 34-089 LH
G'' 34-089 LH
G' 34-092 RH OCD
G'' 34-092 RH OCD
0.00
0.10
1.00
10.00
Frequency (Hz)
100.00
Dynamic Test - Stifles
Modulus (dyn/cm2)
100.00
10.00
1.00
0.10
G' 2 mg/mL
0.01
G'' 2 mg/mL
G' 3 mg/mL
G'' 3 mg/mL
0.00
0.10
1.00
G' 34-089 LS
G'' 34-089 LS
G' 34-089 RS
G'' 34-089 RS
G' 022602 RS DJD
G'' 022602 RS DJD
10.00
Frequency (Hz)
100.00
Tests
Capillary Viscometry


Known dilutions of the fluid are used and the
flow times are measured
The flow times are used to measure relative
viscosity

Relative viscosity = solution flow time
solvent flow time
Relative Viscosity of Synthovial 7
6
y = 8.8644x + 0.7198
2
R = 0.9854
Relative Viscosity (t/ts)
5
4
3
y = 3.2303x + 1.1314
R2 = 0.9867
2
1
2 mg/mL
1 mg/mL
0
0
0.1
0.2
0.3
0.4
Fractional Concentration of HA Solution
0.5
0.6
Relative Viscosity of Synovial Fluid
7
Relative Viscosity (t/ts)
6
5
4
3
022602 RH
2
22-160 LH
1
101102 LS
2 mg/mL
1 mg/mL
101102 RS
0
0
0.1
0.2
0.3
0.4
Fractional Concentration of SF
0.5
0.6
Conclusion


Different joints have different rheological
properties, which could mean different HA
concentrations
Synovial fluid obtained from the hock joint
behaves like a 1 to 2 mg/mL HA solution,
and stifle joint fluid behaves like a 2 to 3
mg/mL HA solution
Future Work



Perform light scattering to determine the specific
concentration and molecular weight of HA in
synovial fluid samples
Study the effectiveness of different forms of
administering HA
Explore the effects of varying molecular weights
and concentrations of HA supplements on an
equine test group
Acknowledgements


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


Howard Hughes
Medical Institute
URISC
Dr. Skip Rochefort,
OSU ChE Dept
Dr. Jill Parker, OSU
College of Veterinary
Medicine
Danielle Leiske and
Erica Zaworski
Dr. Kevin Ahern