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 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 Establish normal properties of equine synovial fluid Compare to properties of hyaluronic acid solutions of various concentrations Compare to properties of hyaluronic acid solutions of various concentrations

Motivation The horse industry loses around 1 billion dollars annually due to lameness. 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. 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. Hyaluronic acid is used to treat joint problems in both horses and humans.

Background Synovial Fluid Multipurpose fluid surrounding all articular joints Multipurpose fluid surrounding all articular joints Synovial fluid has both viscous and elastic properties (viscoelastic) Synovial fluid has both viscous and elastic properties (viscoelastic) Viscosity relates to lubrication Viscosity relates to lubrication Elasticity relates to shock absorption Elasticity relates to shock absorption

Background Hyaluronic Acid (HA) High molecular weight biopolyelectrolyte made up of repeating glucuronic acid and N-acetylglucosamine subunits High molecular weight biopolyelectrolyte made up of repeating glucuronic acid and N-acetylglucosamine subunits Major component of synovial fluid Major component of synovial fluid Exhibits viscoelastic properties Exhibits viscoelastic properties Commonly obtained from: Commonly obtained from: Chicken combs Chicken combs Human umbilical cords Human umbilical cords Bacterial fermentation processes Bacterial fermentation processes Molecular weight around Molecular weight around 0.5 to 10 million Da 0.5 to 10 million Da

Joints Joints of focus Joints of focus

Joints Skeletal view of the joints Skeletal view of the joints

Joints Common problems with equine joints: Common problems with equine joints: Degenerative Joint Disease – DJD Degenerative Joint Disease – DJD Damage to the articular cartilage Damage to the articular cartilage Commonly affects heavily worked and aged horses Commonly affects heavily worked and aged horses Osteochondritis Dissecans – OCD Osteochondritis Dissecans – OCD Failure of the bone underlying the smooth articular cartilage to form properly Failure of the bone underlying the smooth articular cartilage to form properly Commonly affects young horses Commonly affects young horses

Joints Synovial Fluid Average HA concentration Average HA concentration 0.5 to 5.0 mg/mL 0.5 to 5.0 mg/mL Average molecular weight Average molecular weight 2 to 10 million Da 2 to 10 million Da Viscosity at a shear rate of  0.1/second Viscosity at a shear rate of  0.1/second 0.1 to 1.0 Poise 0.1 to 1.0 Poise Hyaluronic Acid Joint Supplementation Hyaluronic Acid Joint Supplementation Intra-articular injection Intravenous injection Oral administration Synthovial 7/Hyalun Synthovial 7/Hyalun Molecular weight - around 1.5 million Da Concentration – 3 to 5 mg/mL

Tests Rheology The rheometer measures both The rheometer measures both ELASTICITY and VISCOSITY ELASTICITY and VISCOSITY Steady shear test measures the viscosity as a function of shear rate 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 Dynamic oscillatory shear test measures the viscous modulus and elastic modulus as a function of frequency Rheometrics RFS II

Tests Steady Shear Test Plate rotates at increasing shear rates Plate rotates at increasing shear rates Torque, or shear stress, is measured by the rheometer Torque, or shear stress, is measured by the rheometer Viscosity = Viscosity = shear stress shearrate shear rate

Tests Bottom plate rotates at increasing shear rates Bottom plate rotates at increasing shear rates Shear stress is measured on the top cone Shear stress is measured on the top cone Viscosity decreases with increasing shear rates due to molecular disentanglement Viscosity decreases with increasing shear rates due to molecular disentanglement

Tests Plate oscillates at increasing frequencies Plate oscillates at increasing frequencies Strain and stress are measured to determine G’ and G’’ Strain and stress are measured to determine G’ and G’’ G’ represents the elastic (storage) modulus G’ represents the elastic (storage) modulus G’’ represents the viscous (loss) modulus G’’ represents the viscous (loss) modulus When G’ > G’’ the fluid behaves more elastic When G’ > G’’ the fluid behaves more elastic When G’ < G’’ the fluid behaves more viscous When G’ < G’’ the fluid behaves more viscous Dynamic Oscillatory Shear Test

Tests strain = imposed strain = imposed stress = measured stress = measured G’ (elastic modulus) = in phase G’ (elastic modulus) = in phase G’’ (viscous modulus) = out of phase G’’ (viscous modulus) = out of phase

Tests Capillary Viscometry Known dilutions of the fluid are used and the flow times are measured Known dilutions of the fluid are used and the flow times are measured The flow times are used to measure relative viscosity The flow times are used to measure relative viscosity Relative viscosity = Relative viscosity = solution flow time solvent flow time

Conclusion Different joints have different rheological properties, which could mean different HA concentrations 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 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 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 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 Explore the effects of varying molecular weights and concentrations of HA supplements on an equine test group

Acknowledgements Howard Hughes Medical Institute Howard Hughes Medical Institute URISC URISC Dr. Skip Rochefort, OSU ChE Dept Dr. Skip Rochefort, OSU ChE Dept Dr. Jill Parker, OSU College of Veterinary Medicine Dr. Jill Parker, OSU College of Veterinary Medicine Danielle Leiske and Erica Zaworski Danielle Leiske and Erica Zaworski Dr. Kevin Ahern Dr. Kevin Ahern