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 VMD DACVS Department of Chemical Engineering and *College of Veterinary Medicine Oregon 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, Hyaluronan Made up of repeating glucuronic acid and N- acetylglucosamine subunitsMade up of repeating glucuronic acid and N- acetylglucosamine 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 Fluid Multipurpose fluid surrounding all articular joints Multipurpose fluid surrounding all articular joints Has both viscous and elastic properties Has both viscous and elastic properties(viscoelastic) Viscous properties - lubrication Elastic 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 ___________

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

Molecular Characterization Molecules separated by size using porous gel column Molecular mass determined using light scattering Concentration determined using refractive index detector SEC - MALLS

Light Scattering RI Detector HA Peak Protein Peak

Right Stifle (OCD) Mw=3.6E+06 C=0.09 mg/ml Right Hock Mw=2.5E+06 C=0.57 mg/ml Right Middle Carpal Mw=1.7E+06 C=1.00 mg/ml Left Stifle Mw=2.3E+06 C=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