Rheological evaluation of the mechanical properties of equine synovial fluid from tarsocrural joints injected with hyaluronic acid (HA) Heidi Schmidt Advisors: Dr. Skip Rochefort PhD and *Dr. Jill Parker VMD DACVS Danielle Leiske, Jocelyn Frey, and *Autumn Rankin Dept. of Chemical Engineering and *College of Veterinary Medicine Oregon State University Corvallis, OR

Outline Motivation Motivation Background Background What is HA What is HA Horse and synovial fluid overview Horse and synovial fluid overview Information about HA supplementation via Hyvisc® Information about HA supplementation via Hyvisc® Experimental Design Experimental Design Tests Tests Steady Shear Viscometry Steady Shear Viscometry Dynamic Oscillatory Shear Rheometry Dynamic Oscillatory Shear Rheometry Capillary Viscometry Capillary Viscometry Results

Motivation Lameness : Limping, abnormal gait, or hobbling resulting from partial loss of function in a leg Lameness : Limping, abnormal gait, or hobbling resulting from partial loss of function in a leg Osteoarthritis is a significant cause of equine lameness, resulting in the deterioration of joint cartilage and formation of painful bone spurs Osteoarthritis is a significant cause of equine lameness, resulting in the deterioration of joint cartilage and formation of painful bone spurs Economic Impact: Approximately $1 billion a year in losses due to lameness Economic Impact: Approximately $1 billion a year in losses due to lameness

Background Viscous liquid present Viscous liquid present in all articular joints Properties Properties Viscosity relates to joint lubrication Viscosity relates to joint lubrication Elasticity relates to impact resistance (i.e. “shock absorbance”) Elasticity relates to impact resistance (i.e. “shock absorbance”) HA (hyaluronic acid) is the major contributor to the viscoelastic properties of synovial fluid. HA (hyaluronic acid) is the major contributor to the viscoelastic properties of synovial fluid. Synovial Fluid

Hyaluronic Acid (HA) Biopolyelectrolyte from rooster combs, human umbilical cords, and bacterial fermentation processes Biopolyelectrolyte from rooster combs, human umbilical cords, and bacterial fermentation processes High molecular weight (0.2-2 million Da) High molecular weight (0.2-2 million Da) $450,000/lb (from Sigma Chemicals) $450,000/lb (from Sigma Chemicals) Exhibits viscoelastic properties Exhibits viscoelastic properties

Intra-articular injection of HA is a common treatment for controlling symptoms of osteoarthritis. Intra-articular injection of HA is a common treatment for controlling symptoms of osteoarthritis. Mechanisms are not well understood Mechanisms are not well understood Supplementation may stimulate increased production of endogenous high molecular weight HA Supplementation may stimulate increased production of endogenous high molecular weight HA It may protect endogenous HA from enzymatic degradation in osteoarthritic joints It may protect endogenous HA from enzymatic degradation in osteoarthritic joints Joint Supplementation

Hyvisc ® vs. Synovial Fluid HA Concentration 11 mg/ml 11 mg/ml 0.5 – 5.0 mg/mL 0.5 – 5.0 mg/mL Molecular Weight 1.5 – 3 million Da 2 – 10 million Da 2 – 10 million Da Viscosity at  P 800 P 0.1 – 1.0 P 0.1 – 1.0 P Cost $50 / 2 mL $50 / 2 mL N / A N / A Source Rooster Combs Synovial Joints Synovial Joints Hyvisc ® Synovial Fluid.

Compare the mechanical properties of synovial fluid from horses treated with HA to synovial fluid from untreated horses. Compare the mechanical properties of synovial fluid from horses treated with HA to synovial fluid from untreated horses. Studies on normal, healthy joints Studies on normal, healthy joints Objective

Experimental Design Five horses (10 healthy hocks) divided into three groups Five horses (10 healthy hocks) divided into three groups Treatment Group (4 hock joints) Treatment Group (4 hock joints) Received 2 mL (22 mg) HA supplementation (Hyvisc®) Received 2 mL (22 mg) HA supplementation (Hyvisc®) Negative Control Group (3 hock joints) Negative Control Group (3 hock joints) Received no joint supplementation Received no joint supplementation Positive Control Group (3 hock joints) Positive Control Group (3 hock joints) Received 2 mL of sterile Lactated Ringers Solution (LRS) Received 2 mL of sterile Lactated Ringers Solution (LRS)

Synovial fluid collections: Synovial fluid collections: Treatment day (before injections) Treatment day (before injections) 7 days post treatment 7 days post treatment 14 days post treatment 14 days post treatment The horses were monitored daily for any signs of disease The horses were monitored daily for any signs of disease Cytology was performed on all of the synovial fluid samples to monitor the health of the joints during the study. Cytology was performed on all of the synovial fluid samples to monitor the health of the joints during the study. Experimental Design

Test Measurements Test Measurements Steady Shear Tests : Steady Shear Tests :Viscosity Dilute Capillary Viscometer Tests : Dilute Capillary Viscometer Tests : Relative Viscosity Dynamic Oscillatory Shear Tests: Dynamic Oscillatory Shear Tests: Viscosity & Elasticity

Geometries Rheometrics Fluids Spectrometer II Rheometer

Bottom plate rotates at a constant speed Bottom plate rotates at a constant speed (shear rate) (shear rate) Torque measured on upper cone (shear stress) Torque measured on upper cone (shear stress) Newtonian Plateau Shear Thinning (Non-Newtonian) Shear stress Shear rate Steady Shear Tests

Data Analysis

Cone oscillates Measures strain, stress G’: Elastic Modulus G”: Viscous Modulus G’<G”: Viscous Acts as a lubricant G’>G”: Elastic Behaves like a shock absorber Dynamic Oscillatory Shear Tests Hyvisc ® G’’ G’ G’’ Cross-over Point

HA Treated Horse Dynamic Oscillatory Shear Test Data Analysis

Measures relative viscosity in dilute polymer solutions Measures relative viscosity in dilute polymer solutions Flow times differ because of variations in concentration and molecular weight of HA Flow times differ because of variations in concentration and molecular weight of HA Timing Lines Capillary Dilute Solution Capillary Viscometer Relative Viscosity = Solution flow time Solvent flow time

HA Treated Horse Relative Viscosity Comparison

Average Relative Viscosity Values at 0.5 Concentration

 Average Viscosity at  0.1

Percent Change of Synovial Fluid Properties 1 Week Post-Treatment..

G'G'' Viscosity at γ = 0.1 Viscosity at γ = 10 Relative viscosity Percent Change of Synovial Fluid Properties 2 Weeks Post-Treatment..

Conclusion Hyaluronic Acid supplementation has a positive affect on the rheological properties of synovial fluid one week post-treatment

Future Work Extend research to a larger test group Study the effects of intravenous HA injections Perform light scattering tests to find specific HA concentration and molecular weight Explore the effects of HA supplements at different molecular weights

Acknowledgements Howard Hughes Medical Institute Dr. Skip Rochefort, OSU ChE Dept Danielle Leiske and Jocelyn Frey Dr. Jill Parker and Autumn Rankin, OSU College of Veterinary Medicine Kevin Ahern

Questions? Danielle Leiske and Autumn Rankin Preparing the joint for an injection