1. Rheological and Molecular Characterization of Equine Synovial Fluid
Nikki Buck Advisor: Dr. Skip Rochefort Oregon State University School of Chemical, Biological and Environmental Engineering Summer, 2008

2. Objectives
Connect rheological properties to the molecular characterization of equine synovial fluid.
Characterize the properties of hyaluronic acid within synovial fluid.

3. What are Polymers? Compound word derived from Greek Spaghetti Analogy
Poly: many
Meros: part
A polymer is a long chain of repeating units covalently bonded together.
Spaghetti Analogy
One polymer is one noodle entangled within a plate of spaghetti.

4. Polymer- Hyaluronic Acid
the main polymeric component of synovial fluid
Repeating units of hyaluronan

5. Synovial Fluid
Viscoelastic fluid that acts in both lubrication and shock absorption of articular joints.
Equine synovial fluid is being studied from hock and stifle joints of racehorses.

6. Horse Anatomy Stifle (knee) Hock (ankle)

7. Rheology How do we study polymers?
Rheology: The study of the deformation and flow of matter
Elasticity: The ability to return to its natural shape after deformation, restoring force
Viscosity: Resistance to shear or extensional stress

8. Hypothesis: Part I
The molecular weight of synovial fluid makes a difference in the viscosity and elasticity of samples.
Prediction: samples with higher molecular weights will demonstrate more elasticity and viscosity at given shear rates and frequencies.

9. Rheometry: Dynamic Oscillation
The cone oscillates at a specific range of frequencies and the machine measures the viscosity and elasticity of the fluid. G’ = elastic modulus “stored energy” G’’ = viscous modulus “lost energy”

10. Dynamic Oscillation 40mm 2°cone Peltier plate geometry 25°Celcius
G’ Elasticity
G’’ Viscosity

12. Rheometry: Steady Shear Flow
A cone or plate rotates at a constant shear rate (deformation rate), while the machine measures the shear stress exerted on the instrument by the fluid. w
Fluid
Image copywritten from Danielle Lieske, Oregon State University
Viscosity = shear stress
shear rate

13. Steady Shear Flow Hyaluronic Acid
40mm 2°cone
Peltier plate geometry
25°Celcius

15. Comparisons: Closer Look

16. GPC/MALLS Molecular Characterization
Two detector system:
Sample first separated by size exclusion chromatography (porous columns)
Refractive Index detector determines the concentration
Light scattering determines the molecular weight
Gel Permeation Chromatography
Multi-angle laser light scatter
Dn/dc differential refractive index needed to calculate the molecular weight. Change in refractivity as concentration changes.

17. Light Scattering
Detector measures the intensity of light as a function of deflection angle and concentration.
Detector, I()
Detector, Io
Polymer Solution
Light Source 

18. GPC/MALLS hyaluronic acid
Light Scattering RI
Injection volume: 0.2 mL
Flow Rate: 0.2 mL/min

19. GPC/MALLS synovial fluid
Protein Peak
Light Scattering RI
HA Peak
Injection volume: 0.2 mL
Flow Rate: 0.2 mL/min

20. Light Scattering Read-Out
Sample ID: rstifle in 1:10 PBS August 1, 2008
Operator: Nikki Buck
Collection Information
Collection time : Fri Aug 01, :06 AM PST
Solvent name : PBS pH 7
Solvent RI : 1.334
Calibration constants
DAWN : e-06
» AUX2 : e-05
Flow rate : mL/min
Calculation method : dn/dc + AUX Constant
dn/dc (mL/g) :
RESULTS:
Molar Mass Moments (g/mol)
Mw : 3.384e+05 (0.5%) 6.171e+04 (0.17%)

21. Protease
An enzyme that hydrolyzes the peptide bond between amino acids of a protein
Enzyme used: Dipase from Bacillus polymyxa
Protocol:
Dilute synovial fluid 1:3 in PBS
Add 0.78 units Protease per mL synovial fluid
Incubate 15 min in 37°C water bath
Filter
Extract HA using phenol-chloroform

22. Hypothesis: Part 2
Proteins cause the second light scattering peak but do not interfere with the molecular weight reading of GPC/MALLS light scattering.
Prediction: Synovial fluid samples allowed to incubate in protease will not demonstrate a protein peak during light scattering analysis, and will have molecular weights in the same range as that of the undigested samples.

23. Comparison: Pure Vs. Digested
Light Scattering
34089 Right Stifle
MW: 3.384*105 g/mol
34089 Right Stifle digested in Protease
MW: 3.819*105 g/mol RI
Difference = 11.3%
In a single sample we’ve seen differences of over 100%
Light Scattering RI

24. Conclusions
Viscosity and elasticity depend on more than the molecular weight of the hyaluronic acid within the synovial fluid. Samples with higher molecular weights did not necessarily exhibit more viscoelasticity. Concentration of hyaluronic acid must also be taken into account.
Hyaluronic acid in the synovial fluid samples degrade at different rates over time when kept in a laboratory refrigerator. Molecular weights of the samples from horse are significantly lower now than they were two years ago, but this is not evident for or Billie.
Proteins do not interfere with the hyaluronic acid molecular weight reading on a GPC/MALLS system. Protease may be used to digest proteins and purify synovial fluid to focus on the hyaluronic acid peak.

25. Acknowledgements Howard Hughes Medical Institute Dr. Kevin Ahern
Dr. Skip Rochefort, OSU School of Chemical Biological and Environmental Engineering
Sara Tracy, M.S. Chemical Engineering
Dr. Jill Parker, OSU College of Veterinary Medicine
Haley Thompson, Coralie Backlund, and Jesse McKiernan