Transcript Nikki Buck Advisor: Dr. Skip Rochefort Oregon State University School of Chemical, Biological and Environmental Engineering Summer, 2008

Nikki Buck
Advisor: Dr. Skip Rochefort
Oregon State University
School of Chemical, Biological
and Environmental Engineering
Summer, 2008


Connect rheological
properties to the molecular
characterization of equine
synovial fluid.
Characterize the properties
of hyaluronic acid within
synovial fluid.

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.
the main polymeric component of
synovial fluid
Repeating units of hyaluronan


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.
Stifle
Hock
(knee)
(ankle)

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


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.
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”
40mm 2°cone
Peltier plate geometry
25°Celcius
3 mg/mL Dynamic Oscillation
6
5
Molulus (Pa)
4
3
G'
G''
2
1
0
0
2
4
6
8
10
Frequency (Hz)
12
14
16
18
Concentration Comparison
4.00E+00
3.50E+00
Cross-over
Point
~12.5 Hz
3.00E+00
Molulus (Pa)
2.50E+00
3 mg/mL G'
2.00E+00
3 mg/mL G''
1 mg/mL G'
1.50E+00
Cross-over
Point ~11.5
Hz
1.00E+00
5.00E-01
0.00E+00
0
2
4
6
8
Frequency (Hz)
10
12
14
1mg/mL G''
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
w
Fluid
40mm 2°cone
Peltier plate geometry
25°Celcius
3 mg/mL
1.00E+03
Viscosity (pa*s)
1.00E+02
1.00E+01
Viscosity
1.00E+00
1.00E-01
1.00E-02
1.00E-03
1.00E-02
1.00E-01
1.00E+00
Shear Rate (1/s)
1.00E+01
1.00E+02
Steady Shear Flow Comparisons
1.00E+00
Viscosity (Pa*s)
Billie RH old
Billie RH new
1.00E-01
34089 LS old
34089 LS new
34089 RS old
34089 RS new
34091 RS old
1.00E-02
1.00E-03
1.00E-02
34091 RS new
1.00E-01
1.00E+00
1.00E+01
Shear Rate (1/s)
1.00E+02
1.00E+03
34-089 RS Steady Shear Comparison
1
Viscosity (Pa*s)
0.1
New 34-089 RS
Old 34-089 RS
0.01
0.001
0.01
0.1
1
10
Shear Rate (1/s)
100

Two detector system:
◦ Sample first separated by size exclusion
chromatography (porous columns)
◦ Refractive Index detector determines the
concentration
◦ Light scattering determines the molecular weight

Detector measures the intensity of light as a
function of deflection angle and
concentration.
Polymer Solution
Light Source

Detector, Io
Detector, I()
Alignment - Hyalun 0
AUX, 90° Detector
0.4
Volume Delay : 0.179 mL
90°
AUX2
0.3
Light
Scattering
0.2
RI
0.1
0.0
-0.1
20
40
60
Time (min)
80
Injection volume: 0.2 mL
Flow Rate: 0.2 mL/min
100
GPC/MALLS synovial fluid
Alignment - 34089 rstifle 8
0.8
Volume Delay : 0.179 mL
AUX, 90° Detector
0.6
0.4
Protein Peak
90°
AUX2
Light
Scattering
RI
0.2
HA Peak
0.0
-0.2
20
40
60
Time (min)
80
Injection volume: 0.2 mL
Flow Rate: 0.2 mL/min
100
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
Filter
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.
Alignment - 34089 rs tifle 8
0.8
Volume Delay : 0.179 mL
34089 Right Stifle
MW:
3.384*105
g/mol
AUX, 90° Detector
0.6
90°
AUX2
Light
Scattering
0.4
RI
0.2
0.0
-0.2
20
40
60
Time (min)
80
100
Alignment - 34089 rstifle 8
34089 Right Stifle
digested in Protease
MW: 3.819*105 g/mol
AUX, 90° Detector
0.12
Volume Delay : 0.179 mL
0.08
90°
AUX2
Light
Scattering
RI
0.04
0.00
-0.04
20
40
60
Time (min)
80
100



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.
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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