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