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Rheological and Molecular Characterization of Equine Synovial Fluid

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. Objectives.

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Rheological and Molecular Characterization of Equine Synovial Fluid

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  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 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 Hock (ankle) Stifle (knee)

  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

  11. w Fluid 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. Viscosity = shear stress shear rate

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

  13. Comparisons: Closer Look

  14. 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

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

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

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

  18. Light Scattering Read-Out • Sample ID: 34089 rstifle in 1:10 PBS August 1, 2008 Operator: Nikki Buck • Collection Information Collection time : Fri Aug 01, 2008 10:06 AM PST Solvent name : PBS pH 7 Solvent RI : 1.334 Calibration constants DAWN : 8.2930e-06 » AUX2 : 5.1727e-05 Flow rate : 0.200 mL/min Calculation method : dn/dc + AUX Constant dn/dc (mL/g) : 0.167 0.167 • RESULTS: Molar Mass Moments (g/mol) Mw : 3.384e+05 (0.5%) 6.171e+04 (0.17%)

  19. 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 • Filter

  20. 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.

  21. 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 Light Scattering RI

  22. 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 34089 are significantly lower now than they were two years ago, but this is not evident for 34091 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.

  23. 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

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