Molecular and Rheological Characterization of Hyaluronic Acid and Equine Synovial Fluid
for the Treatment of Lameness in Horses
Sara TracyAdvisors: Dr. Skip Rochefort PhD and *Dr. Jill Parker VMD DACVS
Department of Chemical Engineering and *College of Veterinary MedicineOregon State University
Corvallis, Oregon
Objective
• Establish baselines for the properties of healthy joints• Correlate concentration and molecular weight of HA to
viscoelastic properties• Study the difference between healthy and diseased
synovial fluid
Motivation
• The equine industry looses up to one billion each year due to lameness
• Up to 14% of the horse population has problems with lameness
• Hyaluronic acid is used to treat lameness and joint pain in both horses and humans
Motivation
Hyaluronic Acid (HA)
Sodium Hyaluronate, HyaluronanSodium Hyaluronate, Hyaluronan• Made up of repeating glucuronic acid and N-Made up of repeating glucuronic acid and N-
acetylglucosamine subunitsacetylglucosamine subunits• High molecular weight: 0.2 to 10 million DaltonHigh molecular weight: 0.2 to 10 million Dalton• Major component of synovial fluidMajor component of synovial fluid• Exhibits viscoelastic propertiesExhibits viscoelastic properties
Synovial FluidMultipurpose fluid surrounding all Multipurpose fluid surrounding all articular jointsarticular joints
Has both viscous and elastic Has both viscous and elastic properties properties
(viscoelastic)(viscoelastic)
ViscousViscous properties - lubrication properties - lubrication
ElasticElastic properties - shock absorption properties - shock absorption
Skeletal View
Common Equine Joint Problems
Degenerative Joint Disease – DJD• 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
Rheological Characterization
Steady shear rate test
•Plate rotates at increasing speeds (shear rate) while rheometer measures shear stress
•Viscosity = Shear rate
Shear stress___________
35-189 Steady Shear Test Results For All Joints
0.01
0.10
1.00
10.00
100.00
0.10 1.00 10.00 100.00 1000.00
Shear Rate (1/s)
Viscosity (P)
RH
LH
LMC
RMC
LS
35-126 RS OCD
Rheological Characterization
Dynamic oscillatory shear test
• Plate oscillates at increasing frequencies and specified strain (amount of rotation) while rheometer measures shear stress
G’ represents elastic (storage) modulus
G’’ represents viscous (loss) modulus
35-189 Dynamic Test Results For All Joints
0.00
0.01
0.10
1.00
10.00
100.00
0.01 0.10 1.00 10.00 100.00
Frequency (Hz)
Modulus (dyn/cm^2)
LS G'
LS G''
LMC G'
LMC G''
RMC G'
RMC G''
LH G'
LH G''
RH G'
RH G''
35-126 RS OCD G'
35-126 RS OCD G''
Molecular Characterization
• Molecules separated by size using porous gel column
• Molecular mass determined using light scattering
• Concentration determined using refractive index detector
SEC - MALLS
-0.1
0.0
0.1
0.2
0.3
0.4
20 30 40 50 60 70
AUX, 90° Detector
T ime (m in)
Alignment - 35-189 LH #3
90°
AUX2
Volume Delay : 0.133 mL
Light Scattering
RI Detector
HA Peak
Protein Peak
35-189 Steady Shear Test Results For All Joints
0.01
0.10
1.00
10.00
100.00
0.10 1.00 10.00 100.00 1000.00
Shear Rate (1/s)
Viscosity (P)
RH
LH
LMC
RMC
LS
35-126 RS OCD
Right Stifle (OCD)Mw=3.6E+06C=0.09 mg/ml
Right HockMw=2.5E+06C=0.57 mg/ml
Right Middle CarpalMw=1.7E+06C=1.00 mg/ml
Left StifleMw=2.3E+06C=0.66 mg/ml
Preliminary Results
• Right and left joints from the same horse have similar rheological curves
• Stifle > Carpal > Hock
• OCD joints appear to have lower HA concentrations that healthy joints
Conclusion
• Both molecular weight and concentration play an important role in the viscoelastic properties of joint fluid
– c*Mw = hydrodynamic interactions parameter
Future Work
• Investigate the repeatability issue of SEC-MALLS with synovial fluid
• Explore the effects of HA supplements for the treatment of lameness on an equine test group
Acknowledgements
• Howard Hughes Medical Institute
• Dr. Kevin Ahern
• Dr. Skip Rochefort, OSU Chemical Engineering Dept
• Dr. Jill Parker, OSU College of Veterinary Medicine
• Matt Wesley
• Katy and Anne Volmert
