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

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.

Polymer- Hyaluronic Acid the main polymeric component of synovial fluid http://www.madsci.org/posts/archives/2001-04/986571103.Bc.1.gif Repeating units of hyaluronan

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. http://www.web-books.com/eLibrary/Medicine/Physiology/Skeletal/synovial_joint.jpg&imgrefurl=http://www.web-books.com/eLibrary/Medicine/Physiology/Skeletal/Joint.htm&h=360&w=324&sz=34&hl=en&start=7&um=1&tbnid=2h7AR7IfAA2pSM:&tbnh=121&tbnw=109&prev=/images%3Fq%3Dsynovial%2Bfluid%2Bjoint%26um%3D1%26hl%3Den%26client%3Dfirefox-a%26rls%3Dorg.mozilla:en-US:official%26sa%3DN

Horse Anatomy Stifle (knee) Hock (ankle) http://hippie.nu/~unicorn/tut/img/basics/animalanatomy/horse-skeleton.jpeg

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

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.

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”

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

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

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

Comparisons: Closer Look

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.

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

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

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

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%)

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 http://en.wikipedia.org/wiki/Image:1UTN.png

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.

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

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.

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