Molecular and Rheological Characterization of Hyaluronic Acid and Equine Synovial Fluid for the Treatment of Lameness in Horses

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

Sara Tracy

Objective

Establish normal properties of equine synovial fluid

Compare to properties of hyaluronic acid solutions of various concentrations

Motivation

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. Hyaluronic acid is used to treat joint problems in both horses and humans.

Background
Synovial Fluid

Multipurpose fluid surrounding all articular joints Synovial fluid has both viscous and elastic properties (viscoelastic)

Viscosity relates to lubrication

Elasticity relates to shock absorption

Background
Hyaluronic Acid (HA)

High molecular weight biopolyelectrolyte made up of repeating glucuronic acid and N-acetylglucosamine subunits
Major component of synovial fluid Exhibits viscoelastic properties Commonly obtained from: Chicken combs Human umbilical cords Bacterial fermentation processes
Molecular weight around

0.5 to 10 million Da

Joints

Joints of focus

Skeletal view of the joints

Joints

Joints

Common problems with equine joints:

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

Joints
Synovial Fluid Average HA concentration

Hyaluronic Acid Joint Supplementation

0.5 to 5.0 mg/mL 2 to 10 million Da

Average molecular weight

Intra-articular injection Intravenous injection Oral administration

Viscosity at a shear rate of 0.1/second

Synthovial 7/Hyalun

0.1 to 1.0 Poise

Molecular weight - around 1.5 million Da Concentration 3 to 5 mg/mL

Tests
Rheology

The rheometer measures both ELASTICITY and VISCOSITY

Rheometrics RFS II

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

Steady Shear Test

Tests

Plate rotates at increasing shear rates Torque, or shear stress, is measured by the rheometer

Viscosity = shear stress

shear rate

Tests

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

Steady Shear Test - Synthovial 7

10.00

Viscosity (P)

1.00

0.10
1 mg/mL 2 mg/mL 3 mg/mL

0.01 0.10

1.00

10.00

100.00

Rate (1/s)

Steady Shear Test - Hocks

1.00

Viscosity (P)

0.10

0.01 0.10

022602 RH 22-160 RH 22-160 LH 34-089 LH 34-089 RH 34-092 RH OCD

1 mg/mL 2 mg/mL

1.00

10.00

100.00

Rate (1/s)

Steady Shear Test - Stifle

10.00

Viscosity (P)

1.00

0.10 071602 RS 34-089 LS 34-089 RS 022602 RS DJD 0.01 0.10 1.00

1 mg/mL 2 mg/mL 3 mg/mL

10.00

100.00

Rate (1/s)

Tests
Dynamic Oscillatory Shear Test

Plate oscillates at increasing frequencies Strain and stress are measured to determine G and G

When G > G the fluid behaves more elastic When G < G the fluid behaves more viscous

G represents the elastic (storage) modulus G represents the viscous (loss) modulus

Tests

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

Dynamic Test - Synthovial 7

100.00

Modulus (dyn/cm2)

10.00

1.00 G' 1 mg/mL G'' 1 mg/mL G' 2 mg/mL G'' 2 mg/mL G' 3 mg/mL G'' 3 mg/mL 1.00 10.00 100.00

0.10

0.01 0.10

Frequency (Hz)

Dynamic Test - Hocks

100.00

Modulus (dyn/cm2)

10.00

1.00

0.10
G' 34-089 RH G'' 34-089 RH G' 34-089 LH G'' 34-089 LH G' 34-092 RH OCD G'' 34-092 RH OCD

0.01

G' 1 mg/mL G'' 1 mg/mL G' 2 mg/mL G'' 2 mg/mL

0.00 0.10 1.00 10.00 100.00

Frequency (Hz)

Dynamic Test - Stifles

100.00

Modulus (dyn/cm2)

10.00

1.00 G' 34-089 LS G'' 34-089 LS G' 34-089 RS G'' 34-089 RS G' 022602 RS DJD G'' 022602 RS DJD 10.00 100.00

0.10
G' 2 mg/mL

0.01

G'' 2 mg/mL G' 3 mg/mL G'' 3 mg/mL

0.00 0.10

1.00

Frequency (Hz)

Tests
Capillary Viscometry

Known dilutions of the fluid are used and the flow times are measured The flow times are used to measure relative viscosity

Relative viscosity = solution flow time solvent flow time

Relative Viscosity of Synthovial 7

6

y = 8.8644x + 0.7198 2 R = 0.9854

5

Relative Viscosity (t/ts)

y = 3.2303x + 1.1314 R2 = 0.9867

2

2 mg/mL
0 0 0.1 0.2 0.3

1 mg/mL
0.4 0.5 0.6

Fractional Concentration of HA Solution

Relative Viscosity of Synovial Fluid

7

Relative Viscosity (t/ts)

022602 RH 22-160 LH

101102 LS

2 mg/mL
0 0 0.1 0.2 0.3

1 mg/mL
0.4

101102 RS
0.5 0.6

Fractional Concentration of SF

Conclusion

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

Future Work

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
Explore the effects of varying molecular weights and concentrations of HA supplements on an equine test group

Acknowledgements

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