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									    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

•Background
    •What is HA
    •Horse and synovial fluid overview
    •Information about HA supplementation via Hyvisc®

•Experimental Design

•Tests
    •Steady Shear Viscometry
    •Dynamic Oscillatory Shear Rheometry
    •Capillary Viscometry

•Results
Motivation
•   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

•   Economic Impact: Approximately $1 billion a
    year in losses due to lameness
Background
Synovial Fluid
  • Viscous liquid present
  in all articular joints
  • Properties
     • Viscosity relates to joint lubrication
     • Elasticity relates to impact resistance
       (i.e. “shock absorbance”)
  • HA (hyaluronic acid) is the major contributor
    to the viscoelastic properties of synovial fluid.
Hyaluronic Acid (HA)


•   Biopolyelectrolyte from rooster combs,
    human umbilical cords, and bacterial
    fermentation processes
•   High molecular weight (0.2-2 million Da)
•   $450,000/lb (from Sigma Chemicals)
•   Exhibits viscoelastic properties
Joint Supplementation
•   Intra-articular injection of HA is a common
    treatment for controlling symptoms of
    osteoarthritis.

•   Mechanisms are not well understood

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

    • It may protect endogenous HA from enzymatic
      degradation in osteoarthritic joints
Hyvisc ®        vs. Synovial Fluid
                  Hyvisc®            Synovial Fluid
HA
                  11 mg/ml           0.5 – 5.0 mg/mL
Concentration
Molecular
                1.5 – 3 million Da   2 – 10 million Da
Weight
Viscosity at
.
g = 0.1
                     800 P              0.1 – 1.0 P

Cost               $50 / 2 mL              N/A

Source          Rooster Combs         Synovial Joints
Objective
•   Compare the mechanical properties of
    synovial fluid from horses treated with HA
    to synovial fluid from untreated horses.

•   Studies on normal, healthy joints
    Experimental Design
•   Five horses (10 healthy hocks) divided into
    three groups
    • Treatment Group (4 hock joints)
       •   Received 2 mL (22 mg) HA supplementation (Hyvisc®)


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


    • Positive Control Group (3 hock joints)
       •   Received 2 mL of sterile Lactated Ringers Solution (LRS)
Experimental Design
•   Synovial fluid collections:
     • Treatment day (before injections)
     • 7 days post treatment
     • 14 days post treatment

•   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.
Experimental Design

•   Test Measurements
    • Steady Shear Tests :
    Viscosity

    • Dilute Capillary Viscometer Tests :
    Relative Viscosity

    • Dynamic Oscillatory Shear Tests:
    Viscosity & Elasticity
Rheometer
                Geometries




        Rheometrics Fluids Spectrometer II
       Steady Shear Tests
•     Bottom plate
      rotates at a                               0.100

      constant                                               Newtonian Plateau
      speed
    (shear rate)               Viscosity (Pas)

                                                 0.010

•    Torque
     measured on                                                   Shear Thinning
                                                                  (Non-Newtonian)
     upper cone
     (shear stress)                              0.001
                                                         1          10             100   1000
              shear  stress
               Shear stress
Viscosity =                                                          Shear Rate (1/s)
              shear  rate
               Shear rate
                                          HA Treated Horse
                                          Steady Shear Test
                10.00                                                        Pre-treatment
                                                                             1 Wk. Post Treatment
                                                                             2 Wks. Post Treatment
Viscosity (P)




                 1.00


                 0.10
                          Data Analysis
                                                                  Data Analysis
                 0.01
                        0.1                  1.0                      10.0                  100.0
                                                   Shear Rate (1/s)
                            Dynamic Oscillatory
                                Shear Tests
                                                                      •Cone oscillates
                                      Hyvisc®
              10000                                                   •Measures strain, stress
                                                                      •G’: Elastic Modulus
                                                           G’
                                                                      •G”: Viscous Modulus
Moduli (dyne/cm^2)




                     1000


                                                            G’’
                     100
                               G’’                                    •G’<G”: Viscous
                               G’                  Cross-over           •Acts as a lubricant
                      10
                                                   Point              •G’>G”: Elastic
                                                              G'

                       1
                                                              G"        •Behaves like a shock
                        0.01         0.10          1.00         10.00
                                                                        absorber
                                       Frequency (Hz)
                 HA Treated Horse
            Dynamic Oscillatory Shear Test
Moduli (dyne/cm^2)




                     100.000
                      10.000
                       1.000
                       0.100
                       0.010            Data Analysis
                       0.001
                           0.10            1.00             10.00          100.00
                                               Frequency (Hz)


                          G' Pre-treatment               G'' Pre-treatment
                          G' 1 Wk. Post Treatment        G'' 1 Wk. Post Treatment
                          G' 2 Wks. Post Treatment       G'' 2 Wks. Post Treatment
Dilute Solution Capillary
       Viscometer
                  •   Measures relative viscosity
                      in dilute polymer solutions
                  •   Flow times differ because
      Timing
      Lines
                      of variations in
                      concentration and
                      molecular weight of HA
      Capillary

                               Solution flow time
       Relative Viscosity =
                                Solvent flow time
       HA Treated Horse
Relative Viscosity Comparison
                     4.5
                                 Pre-treatment
                     4.0         1 Wk. Post Treatment
                                 2 Wks. Post Treatment
                     3.5
Relative Viscosity




                     3.0

                     2.5

                     2.0

                     1.5

                     1.0
                           0.0    0.1       0.2          0.3     0.4       0.5   0.6
                                         Fraction of Pure Synovial Fluid
Average Relative Viscosity Values
      at 0.5 Concentration
                      5
 Relative Viscosity



                      4
                      3
                      2
                      1
                      0
                            Week 0        Week 1      Week 2
                                  Weeks After Injection
       HA                 + Control   - Control
Average Viscosity at g = 0.1
                 1.40
                 1.20
 Viscosity (P)



                 1.00
                 0.80
                 0.60
                 0.40
                 0.20
                 0.00
                           Week 0      Week 1      Week 2
                                Weeks After Injection
       HA               + Control   - Control
Percent Change of Synovial Fluid Properties
         1 Week Post-Treatment
                                                               1
                  200%
                                  HA Group
                  150%
 Percent Change




                  100%
                                                       + Control Group
                   50%
                                                                              - Control Group
                    0%

                  -50%

                  -100%
                                             .                      .
                   G'     G''   Viscosity at γ = 0.1   Viscosity at γ = 10   Relative viscosity
Percent Change of Synovial Fluid Properties
         2 Weeks Post-Treatment
                  200%

                  150%
 Percent Change




                  100%
                                                       + Control               - Control
                   50%
                                  HA Group             Group                   Group
                    0%
                                                            1


                  -50%

                  -100%

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

								
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