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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