Rheological evaluation of the mechanical

properties of equine synovial fluid from tarsocrural joints injected with

hyaluronic acid (HA)

Heidi SchmidtHeidi SchmidtAdvisors:Advisors: Dr. Skip Rochefort PhD and *Dr. Jill Parker VMD Dr. Skip Rochefort PhD and *Dr. Jill Parker VMD

DACVSDACVSDanielle Leiske, Jocelyn Frey, and *Autumn RankinDanielle Leiske, Jocelyn Frey, and *Autumn Rankin

Dept. of Chemical Engineering and *College of Veterinary Medicine Dept. of Chemical Engineering and *College of Veterinary Medicine Oregon State UniversityOregon State University

Corvallis, ORCorvallis, OR

Outline•MotivationMotivation

•BackgroundBackground•What is HAWhat is HA•Horse and synovial fluid overviewHorse and synovial fluid overview•Information about HA supplementation via Hyvisc®Information about HA supplementation via Hyvisc®

•Experimental DesignExperimental Design

•TestsTests•Steady Shear ViscometrySteady Shear Viscometry•Dynamic Oscillatory Shear Rheometry Dynamic Oscillatory Shear Rheometry •Capillary ViscometryCapillary Viscometry

•Results

Motivation• LamenessLameness: Limping, abnormal gait, or : Limping, abnormal gait, or

hobbling resulting from partial loss of hobbling resulting from partial loss of function in a leg function in a leg

• OsteoarthritisOsteoarthritis is a significant cause of is a significant cause of equine lameness, resulting in the equine lameness, resulting in the deterioration of joint cartilage and deterioration of joint cartilage and formation of painful bone spursformation of painful bone spurs

• Economic Impact:Economic Impact: Approximately $1 Approximately $1 billion a year in losses due to lamenessbillion a year in losses due to lameness

Background

• Viscous liquid present Viscous liquid present

in all articular jointsin all articular joints

• PropertiesProperties

• Viscosity relates to joint lubricationViscosity relates to joint lubrication

• Elasticity relates to impact resistance Elasticity relates to impact resistance (i.e. “shock absorbance”) (i.e. “shock absorbance”)

• HA (hyaluronic acid) is the major contributor to HA (hyaluronic acid) is the major contributor to the the viscoelasticviscoelastic properties of synovial fluid. properties of synovial fluid.

Synovial Fluid

Hyaluronic Acid (HA)

• Biopolyelectrolyte from rooster combs, Biopolyelectrolyte from rooster combs, human umbilical cords, and bacterial human umbilical cords, and bacterial fermentation processes 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 Exhibits viscoelasticviscoelastic properties properties

• Intra-articular injection of HA is a common Intra-articular injection of HA is a common treatment for controlling symptoms of treatment for controlling symptoms of osteoarthritis.osteoarthritis.

• Mechanisms are not well understoodMechanisms are not well understood

• Supplementation may stimulate increased Supplementation may stimulate increased production of endogenous high molecular production of endogenous high molecular weight HAweight HA

• It may protect endogenous HA from enzymatic It may protect endogenous HA from enzymatic degradation in osteoarthritic jointsdegradation in osteoarthritic joints

Joint Supplementation

Hyvisc® vs. Synovial Fluid

HA HA ConcentratiConcentrationon

11 mg/ml11 mg/ml 0.5 – 5.0 mg/mL0.5 – 5.0 mg/mL

Molecular Molecular WeightWeight

1.5 – 3 million 1.5 – 3 million DaDa

2 – 10 million 2 – 10 million DaDa

ViscosityViscosity at at

0.10.1 800 P800 P 0.1 – 1.0 P0.1 – 1.0 P

CostCost $50 / 2 mL$50 / 2 mL N / AN / A

SourceSource Rooster CombsRooster Combs Synovial JointsSynovial Joints

Hyvisc® Synovial Fluid

.

• Compare the mechanical properties of Compare the mechanical properties of synovial fluid from horses treated with synovial fluid from horses treated with HA to synovial fluid from untreated HA to synovial fluid from untreated horses. horses.

• Studies on normal, healthy jointsStudies on normal, healthy joints

Objective

Experimental Design

• Five horses (10 healthy hocks) divided into Five horses (10 healthy hocks) divided into three groupsthree groups• Treatment Group Treatment Group (4 hock joints)(4 hock joints)

• Received 2 mL (22 mg) HA supplementation (Hyvisc®)Received 2 mL (22 mg) HA supplementation (Hyvisc®)

• Negative Control Group Negative Control Group (3 hock joints)(3 hock joints)• Received no joint supplementationReceived no joint supplementation

• Positive Control Group Positive Control Group (3 hock joints)(3 hock joints) • Received 2 mL of sterile Lactated Ringers Solution Received 2 mL of sterile Lactated Ringers Solution

(LRS)(LRS)

• Synovial fluid collections:Synovial fluid collections:• Treatment day (before injections)Treatment day (before injections)• 7 days post treatment7 days post treatment• 14 days post treatment14 days post treatment

• The horses were monitored daily for any signs of The horses were monitored daily for any signs of diseasedisease

• Cytology was performed on all of the synovial Cytology was performed on all of the synovial fluid samples to monitor the health of the joints fluid samples to monitor the health of the joints during the study.during the study.

Experimental Design

Experimental DesignExperimental Design

• Test MeasurementsTest Measurements• Steady Shear Tests : Steady Shear Tests : ViscosityViscosity

• Dilute Capillary Viscometer Tests :Dilute Capillary Viscometer Tests :Relative ViscosityRelative Viscosity

• Dynamic Oscillatory Shear Tests:Dynamic Oscillatory Shear Tests:Viscosity & ElasticityViscosity & Elasticity

Geometries

Rheometrics Fluids Spectrometer II

Rheometer

• Bottom plate Bottom plate rotates at a rotates at a constant constant speedspeed

((shear rateshear rate) )

• TorqueTorque measured on measured on upper cone upper cone ((shear stressshear stress)) 0.001

0.010

0.100

1 10 100 1000

Shear Rate (1/s)

Viscosity (Pas)

Newtonian Plateau

Shear Thinning (Non-

Newtonian)

rateshear

stressshearViscosity

−−

=Shear stress

Shear rate

Steady Shear Tests

HA Treated Horse Steady Shear Test

0.01

0.10

1.00

10.00

0.1 1.0 10.0 100.0

Shear Rate (1/s)

Viscosity (P)

Pre-treatment1 Wk. Post Treatment2 Wks. Post Treatment

Data AnalysisData 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 shockabsorber

Dynamic Oscillatory Shear Tests

1

10

100

1000

10000

0.01 0.10 1.00 10.00

Frequency (Hz)

Moduli (dyne/cm^2)

G'G"

Hyvisc®

G’’

G’

G’

G’’

Cross-over

Point

0.001

0.010

0.100

1.000

10.000

100.000

0.10 1.00 10.00 100.00

Frequency (Hz)

Moduli (dyne/cm^2)

G' Pre-treatment G'' Pre-treatmentG' 1 Wk. Post Treatment G'' 1 Wk. Post TreatmentG' 2 Wks. Post Treatment G'' 2 Wks. Post Treatment

HA Treated Horse Dynamic Oscillatory Shear Test

Data Analysis

• Measures Measures relative relative viscosityviscosity in dilute in dilute polymer solutionspolymer solutions

• Flow times differ because Flow times differ because of variations in of variations in concentrationconcentration and and molecular weight molecular weight of HAof HA

Timing Lines

Capillary

Dilute Solution CapillaryViscometer

Relative Viscosity =Solution flow time

Solvent flow time

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0.0 0.1 0.2 0.3 0.4 0.5 0.6

Fraction of Pure Synovial Fluid

Relative Viscosity

Pre-treatment

1 Wk. Post Treatment2 Wks. Post Treatment

HA Treated HorseRelative Viscosity Comparison

Average Relative Viscosity Values at 0.5 Concentration

0

1

2

3

4

5

Week 0 Week 1 Week 2

Weeks After Injection

Relative Viscosity

HA + Control - Control

0.000.200.400.600.801.001.201.40

Week 0 Week 1 Week 2

Weeks After Injection

Viscosity (P)

HA + Control - Control

Average Viscosity at 0.1

-100%

-50%

0%

50%

100%

150%

200%1

Percent Change

HA Group

+ Control Group

- Control Group

G' G'' Viscosity at

?

= 0.1 Viscosity at

?

= 10 Relative viscosity

Percent Change of Synovial Fluid Properties 1 Week Post-Treatment

. .

-100%

-50%

0%

50%

100%

150%

200%

1

Percent Change

HA Group

+ Control

Group

- Control

Group

G' G'' Viscosity at γ= 0.1 Viscosity at γ = 10 Relative viscosity

Percent Change of Synovial Fluid Properties2 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 atdifferent 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