Group Title: effects of glucosamine and chondroitin on the viscosity of synovial fluid in patients with osteoarthritis
Title: The effects of glucosamine and chondroitin on the viscosity of synovial fluid in patients with osteoarthritis
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Title: The effects of glucosamine and chondroitin on the viscosity of synovial fluid in patients with osteoarthritis
Physical Description: Book
Language: English
Creator: Conrad, Bryan Preston
Publisher: University of Florida
Place of Publication: Gainesville Fla
Gainesville, Fla
Publication Date: 2001
Copyright Date: 2001
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Subject: Biomedical Engineering thesis, M.E   ( lcsh )
Dissertations, Academic -- Biomedical Engineering -- UF   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
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Summary: ABSTRACT: Osteoarthritis (OA), a disease resulting in the degeneration of articular cartilage, is a debilitating condition afflicting millions of people around the world. Current therapeutic approaches using non-steroidal anti-inflammatory drugs are ineffective in altering the course of the disease and are known to cause severe side effects in some patients. There is a dire need for treatments that stop or at least delay the degeneration of articular cartilage. Healthy synovial fluid is highly viscous allowing it to act as a natural lubricant in the joint. In persons with OA, the synovial fluid has been shown to have a decreased viscosity and thus has less effective lubrication. The objective of this thesis was to evaluate whether glucosamine and chondroitin sulfate (GCS) are effective in treating OA. Efficacy will be determined by measuring the changes in the viscosity of synovial fluid with an increase in viscosity demonstrating a positive response to the treatment. A secondary goal was to establish a method for measuring highly viscous fluid at different shear rates using a cylinder microrheometer. Twenty-six osteoarthritic subjects were randomized into one of two groups, either to receive GCS or placebo. Treatments were blinded to both the investigator and the patient. Synovial fluid was aspirated from the subjects' knee at weeks 0, 4, 12 and 20. The viscosity of the synovial fluid was measured using a Brookfield cone-plate viscometer and the microrheometer. At four weeks, patients taking GCS had a higher synovial fluid viscosity than patients on placebo. Also, at later time points it was found that patients on placebo had a decreased viscosity while patients taking GCS had not changed.
Summary: ABSTRACT (cont.): However these differences are not statistically significant (p= about 0.5). In addition it was noted that synovial fluid exhibits shear thinning behavior. The results indicate a need for a study with a larger number of subjects. The trend observed, nevertheless, suggests that GCS may have a beneficial effect on some patients, resulting in an initial increase in viscosity over the first month of treatment and eventually returning to pretreatment condition after that. By demonstrating that viscosity can be used to track changes in osteoarthritis, this thesis provides an important tool for future OA research. The next step of this research will be to correlate the synovial fluid viscosity with other clinical parameters, such as pain, function, and joint space narrowing. This finding could have a great impact on the evaluation of treatments for OA and in the assessment of its severity.
Summary: KEYWORDS: osteoarthritis, glucosamine, chondroitin, synovial fluid, viscosity, microrheometer
Thesis: Thesis (M.E.)--University of Florida, 2001.
Bibliography: Includes bibliographical references (p. 64-68).
System Details: System requirements: World Wide Web browser and PDF reader.
System Details: Mode of access: World Wide Web.
Statement of Responsibility: by Bryan Preston Conrad.
General Note: Title from first page of PDF file.
General Note: Document formatted into pages; contains xi, 69 p.; also contains graphics.
General Note: Vita.
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Bibliographic ID: UF00100737
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Resource Identifier: oclc - 48991769
alephbibnum - 002766279
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THE EFFECTS OF GLUCOSAMINE AND CHONDROITIN ON THE VISCOSITY OF
SYNOVIAL FLUID IN PATIENTS WITH OSTEOARTHRITIS

















By

BRYAN PRESTON CONRAD


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF ENGINEERING

UNIVERSITY OF FLORIDA


2001




























Copyright 2001

by

Bryan Preston Conrad


























Not only my words and my works, but my entire life I dedicate to Suzanne.
















ACKNOWLEDGMENTS

I extend my sincerest gratitude to Dr. Roger Tran-Son-Tay for his thoughtful and

caring guidance throughout my graduate career. He has taught me much about research

and even more about life. I would also like to acknowledge the contribution of my

supervisory committee: Dr. Cristescu, Dr. Fregly and Dr. Horodyski. I am indebted to

Dr. Cristescu for his immense abilities in the area of theoretical fluid analysis. Dr. Fregly

has always been ready with encouragement and enthusiasm. By never settling for

mediocrity, Dr. Horodyski has challenged me to explore the greater depths of learning

and always challenged me to determine the relevance of my research. Because of their

input, advice and challenges I have matured as a researcher and as a student. I would

also like to extend my gratitude to Dr. Levy for his help in aspirating the synovial fluid.

During my research I had the pleasure of working in two different labs, in orthopaedics

and biorheology. With the help of my coworkers and friends even the tedium became

bearable. I thank Randy, Tim, Butch, Joanne, Suny, Narcisse, Becky, Cecile and Wil. I

extend a special thanks to my family Mom, Dad and Annie. They have definitely been

encouraging me for the longest! They will always be dear to my heart. I would be

negligent to not mention the contributions and sacrifices of my wife, Suzanne. Through

all of my research and studies she has endured the ups and downs and has never faltered

in her support and encouragement. Her love lifts me. I thank God for all He has done for

me.
















TABLE OF CONTENTS

Page

A C K N O W L E D G M E N T S ...................... .. .. ............. ....................................................iv

LIST OF TABLES ................. ................ ............ ........ .. ............ vii

LIST OF FIGURES ................. ... ........ ......... ............... ..... ...... viii

A B S T R A C T ......................................................... ................ .. x

CHAPTERS

1 IN TR O D U C TIO N ....................... .... .............. ..................... ..............

W h at is O steoarthritis?........................................................................ . ...... ...... 1
W hy is Synovial Fluid Im portant? .......................... ........ ................................. 2
Specific Aim s ..................................... ................................. .......... 2
Stu dy D esig n ...................................... .................................... .............. 2

2 BACKGROUND ............... ................. .....................................4.

Anatomy and Physiology of the Synovial Joint..................... ................................ 4
Subchondral Bone ........ ...... ................... ...... ........ ........ 4
Synovium ............................................................................................................. 5
A rticular C cartilage ....................................................... .. .......... .. 6
Synovial Fluid ................................... ........................... 7
Normal Structure and Function of Synovial Fluid.................................................. 7
Pathology of O steoarthtitis ......................................................................... 9
Pathologic Synovial Fluid ................................................... .................................. 12
Management and Treatment of Osteoarthritis..................................................... 16
Outcome Measures in Osteoarthritis Research and Clinical Practice..................... 19
R adiograp hic A n aly sis .......................................................................................... 2 0
Pain Q questionnaires .............................................. ........ .. .......... .. 20
M agnetic R esonance Im aging........................................................... .............. 21
Proteoglycan Assay.......... .................. ......... ...................... 21
V isco sity ...................................................................................................... 2 1

3 M ATERIALS AND M ETHODS ........................................ ........................... 22

E xperim mental Setup .... ........................ ........ .............................. .............. 22









Strength T testing ..................................................... 23
M agnetic R esonance Im aging........................................................... .............. 23
P ain Q u estionn aire ...... .............. ........................ ... ................ .............. ...... 2 3
Proteoglycan A ssay........... ....................................................... .............. 23
V isco sity ...................................................................................................... 24
Microrheometer.................. .. ................................. 25
Background ......................................... 25
A apparatus ........................................ 26
Experim ents.......................... ........... .............. 28

4 RESU LTS ......... ......... ....... ...... ..................... ............... 31

M icrorheom eter V alidation .................................................................................. 31
Clinical Data.................................... .............. 35

5 D IS C U S S IO N ........................................................................................................ 3 8

M icrorheom eter .................................................................. 3 8
C lin ical ......... ................................................................................. ............... 3 8

6 C O N C LU SIO N .........................................44.............................

APPENDICES

A FALLING CYLINDER THEORY ................................. ...............47

Formulation of the Problem .......................................... 47
Velocity Profile for Open Tube................................................... .................... 50
Velocity Profile for Closed Tube ............................................................ 52

B R A W D A T A .......................................................................................................... 5 5

L IST O F R E FE R E N C E S ............................................................................................. 64

BIOGRAPHICAL SKETCH .................. ............. .. ............... 69















LIST OF TABLES


Table Page

1. Summary of studies analyzing synovial fluid viscosity. ........................ .....................15

2. Newtonian fluids tested on both the microrheometer (MR) and the
cone-plate rheometer (CP). N is the number of samples tested on
the MR. SD is the standard deviation of the MR viscosity
measurement. Accuracy = ((MR-CP)/CP*100%). Variability =
SD /M R viscosity*100% ......... ................. ........................................ ............... 32

3. Non-Newtonian HA fluid tested on both the microrheometer (MR) and
cone-plate viscometer (CP). MR shear rate is given for the
measured viscosity. HA was tested over a range of shear rates in
th e C P ...................................... ....................................................... 3 2

4. Changes in synovial fluid viscosity between first and last sample (cone-
plate viscometer, shear rate = 1/s) for both experimental groups.
Effect size = change/baseline viscosity. a Two subjects in the
placebo group withdrew from the study, one to due a TKA, the
other due to lack of benefit from study medication. b Effect size is
not statistically different (t=0.45)........................................ ........................... 36

5. Change in synovial fluid viscosity between week 0 and 4. One
subject in placebo group withdrew due to TKA. d Effect size is not
statistically different (p=0.50) ....................................................... ............... 36

6. R aw data from synovial fluid ......... ......................... .......................... ............... 55
















LIST OF FIGURES


Figure Page

1. D iagram of the structures of the knee ........................................ .......................... 5

2. Schematic of Microrheometer (a sample tube, b -
cylinder, c piezoelectric crystal, d ultrasound
transducer, e water jacket, f electromagnet) ..................................................27

3. Main bang is the large signal on the left, the echo of the cylinder is the
smaller signal on the right (tf time of flight, time for ultrasound
pulse to travel to the cylinder and back to the transducer.)............................28

4. Oscilloscope plot of the cylinder's displacement vs. time as it falls
through the sample tube. The arrow indicates the path of the
cylinder. From this output the speed of sedimentation (of falling
velocity) of the cylinder is m measured. ............................................. ............... 30

5. Viscosity plotted against shear rate for non-newtonian HA samples
tested on the cone-plate viscometer (log-log plot)................... .......................... 33

6. Viscosity of 4M MW Hyaluronic acid plotted against shear rate................................. 34

7. Viscosity of 4M MW HA plotted against shear rate. Measured using
Brookfield cone-plate viscom eter. ........................................ ........................ 34

8. Shear rate versus viscosity for a subject in GCS group. This particular
subject experienced a decrease in viscosity the first 12 weeks
which somewhat recovered by week 23. Notice that the fluid
displays newtonian behavior at low shear rates at week 12....................................37

9. Viscosity versus shear rate for patient Id. ............................... ................................. 57

10. Viscosity versus shear rate for patient fh (fh withdrew from study due
to receiving a total knee replacement after week 4)............................................57

11. Viscosity versus shear rate for patient ka ................ ..... ..... ..... .... ............... 58

12. Viscosity versus shear rate for patient fr ............................ .... ... ............... 58









13. Viscosity versus shear rate for patient jd. ......................................................................59

14. Viscosity versus shear rate for patient aa......................... ... .................... ........... 59

15. Viscosity versus shear rate for patient wz (the only patient for whom
synovial fluid samples are available at all four time points).............................. 60

16. Viscosity versus shear rate for patient dw............................................................ 60

17. Viscosity versus shear rate for patient cm (Patient withdrew from
study due to lack of efficacy of treatm ent)............................................................ 61

18. Viscosity versus shear rate for patient jb. ............................... .......................... 61

19. Viscosity versus shear rate for patient ss. ............................................ ............... 62

20. Viscosity versus shear rate for patient w................................................................... 62

21. V iscosity versus shear rate for patient a ...................................................... ............. 63















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Engineering

THE EFFECTS OF GLUCOSAMINE AND
CHONDROITIN ON THE VISCOSITY OF SYNOVIAL
FLUID IN PATIENTS WITH OSTEOARTHRITIS

By

Bryan Preston Conrad

August 2001

Chairman: Dr. Roger Tran-Son-Tay
Major Department: Biomedical Engineering

Osteoarthritis (OA), a disease resulting in the degeneration of articular cartilage,

is a debilitating condition afflicting millions of people around the world. Current

therapeutic approaches using non-steroidal anti-inflammatory drugs are ineffective in

altering the course of the disease and are known to cause severe side effects in some

patients. There is a dire need for treatments that stop or at least delay the degeneration of

articular cartilage. Healthy synovial fluid is highly viscous allowing it to act as a natural

lubricant in the joint. In persons with OA, the synovial fluid has been shown to have a

decreased viscosity and thus has less effective lubrication.

The objective of this thesis was to evaluate whether glucosamine and chondroitin

sulfate (GCS) are effective in treating OA. Efficacy will be determined by measuring the

changes in the viscosity of synovial fluid with an increase in viscosity demonstrating a









positive response to the treatment. A secondary goal was to establish a method for

measuring highly viscous fluid at different shear rates using a cylinder microrheometer.

Twenty-six osteoarthritic subjects were randomized into one of two groups, either

to receive GCS or placebo. Treatments were blinded to both the investigator and the

patient. Synovial fluid was aspirated from the subjects' knee at weeks 0, 4, 12 and 20.

The viscosity of the synovial fluid was measured using a Brookfield cone-plate

viscometer and the microrheometer.

At four weeks, patients taking GCS had a higher synovial fluid viscosity than

patients on placebo. Also, at later time points it was found that patients on placebo had a

decreased viscosity while patients taking GCS had not changed. However these

differences are not statistically significant (p= about 0.5). In addition it was noted that

synovial fluid exhibits shear thinning behavior.

The results indicate a need for a study with a larger number of subjects. The trend

observed, nevertheless, suggests that GCS may have a beneficial effect on some patients,

resulting in an initial increase in viscosity over the first month of treatment and

eventually returning to pretreatment condition after that. By demonstrating that viscosity

can be used to track changes in osteoarthritis, this thesis provides an important tool for

future OA research. The next step of this research will be to correlate the synovial fluid

viscosity with other clinical parameters, such as pain, function, and joint space

narrowing. This finding could have a great impact on the evaluation of treatments for

OA and in the assessment of its severity.














CHAPTER 1
INTRODUCTION


What is Osteoarthritis?

Osteoarthritis (OA) is a disease characterized by erosion of articular cartilage,

which becomes soft, frayed and thinned with outgrowths of marginal osteophytes

(nodules of bone forming on the joint surface). The most common symptoms are pain

and loss of function. OA mainly affects weight-bearing joints and is more common in

overweight and older persons [1]. However, such a concise definition of OA belies its

complex and multifaceted nature. In recent years OA has become a generic term for a

variety of "overlapping and distinct diseases" [2] that affect the entire joint, not only the

articular cartilage. Due to many interwoven factors, it is difficult to predict the individual

possibility for a patient's disease to progress.

Osteoarthritis (OA) is the most prevalent disease associated with aging, affecting

the quality of life of approximately 10 million Americans. However, diagnosing OA can

be an enigmatic process in itself. There are many definitions of OA and clinicians

dispute which signs and symptoms truly signify the onset or progression of OA. While it

is widely accepted that end stage OA is represented by full thickness loss of articular

cartilage, early OA is much more ambiguous. OA can be defined by any combination of

the following: joint pain, crepitus on active motion (noisy joints), morning stiffness, joint

space narrowing, presence of osteophytes at joint margin, sclerosis (thickening) of

subchondral bone, subchondral cysts and loss of function.












Why is Synovial Fluid Important?

Synovial fluid is a highly viscous plasma-like fluid excreted by cells that line the

joint. One of its functions is to aid in lubrication of the joint. In the healthy joint,

synovial fluid lubrication does an excellent job of protecting the articular cartilage from

wear. However in the diseased joint, synovial fluid becomes less viscous and therefore

less effective in lubrication. In fact, diseases can be differentiated by measuring the

viscosity of synovial fluid (rheumatoid arthritis < osteoarthritis < normal). It is

postulated that by monitoring the viscosity of synovial fluid, one can track the

progression of joint disease.




Specific Aims

The specific aims of this thesis are to:

1. Validate the use of a custom-built small-volume rheometer (microrheometer) in
measuring the viscosity of synovial fluid from patients with OA.

2. Determine whether orally administered glucosamine and chondroitin sulfate has an
effect on the viscosity of synovial fluid in patients with osteoarthritis of the knee.



Study Design

The microrheometer will be evaluated in two steps. First, the viscosity of four

newtonian oils will be measured on both the microrheometer and on a standard cone-

plate viscometer (Brookfield Inc., MA). By comparing the values gathered from the two

devices, it will be determined whether the microrheometer yields comparable results with

the standard cone-plate viscometer. Second, the viscosity of four non- newtonian fluids









will be measured at various shear rates on the cone-plate viscometer and compared to the

values calculated from the microrheometer. This step is vital since synovial fluid has

demonstrated a shear thinning effect; that is it has a lower viscosity at higher shear rates.

A clinical study will be conducted to evaluate the effectiveness of glucosamine

and chondroitin (GCS). Twenty-six osteoarthritic subjects will be enrolled in the study

and randomized into one of two groups (GCS or placebo). At baseline (week 0) all

subjects will have a synovial fluid sample drawn from their study knee. Subjects will

return to the clinic at weeks 4, 12 and 20 for follow-up. At each return visit, an

additional synovial fluid sample will be collected from all subjects. The viscosity of fluid

samples with a volume of greater than 0.5 mL will be measured on both the

microrheometer and the cone-plate viscometer. Since the cone-plate viscometer requires

0.5 mL of fluid to test, samples less than 0.5 mL will only be measured on the

microrheometer. The change in viscosity between week 0 and week 20 will be used to

determine the effect of GCS. It is hypothesized that GCS will have a beneficial effect on

OA. Additionally it is expected that this beneficial effect will be accompanied by an

increase in synovial fluid viscosity.














CHAPTER 2
BACKGROUND




Anatomy and Physiology of the Synovial Joint

Synovial joints are found in locations that require a wide range of motion. In the

human body knee, hip, ankle and shoulder joints fall into this category. These joints

consist of a closed system made up of the two articulating surfaces surrounded by a

capsule and bathed in fluid. The following section will describe how the mechanical and

biological properties of the tissues of the knee that enable it to be an extremely effective

joint under a wide range of loads over millions of cycles. In particular, the tissues that

will be discussed will be the subchondral bone, synovium, articular cartilage, and

synovial fluid, Figure 2.1. Although there are many complex biochemical interactions at

work in the knee, they will not be addressed in great detail in this thesis. The purpose of

this background is to justify the methods employed in the experimental section of this

work.

Subchondral Bone

Between articular cartilage and underlying cancellous bone is a thin plate of

subchondral bone. With a Young's modulus between 1.0-15.0 GPa, the material

properties of subchondral bone lie between those of cortical and cancellous bone [3]. It is

thought that by pairing the compliant cancellous bone with the denser and stiffer

subchondral bone allows optimal congruence of the joint under high loads [4].










Ouodrinctp Tendon
Fermur
SoBuI
Cortical Bone L


SfM. eniscus
Articular Cartiloqe .aPoielr Uigoaer
Fibrous Copu -- Joi n CopsuLe
Articulor Cortiloqe Concellous Bone



t CoAica l Bone

A Tibia

Conlcllwus Bone
/ and Mrrow

Joint Cavity Articular Cartilage
cu Meniscus











Figure 2.1. Diagram of the structures of the knee. Source: [3].
Synovi Fluum
Surrounding the knee joint is a tough cartilage capsule that creates a closed
system. This capsule is lined with fluid producing mesenchymal cells called the
', -Subchaondra COrtex

Cancellous Bone V00d
and Morrow




Figure 2.1. Diagram of the structures of the knee. Source: [3].



Synovium

Surrounding the knee joint is a tough cartilage capsule that creates a closed

system. This capsule is lined with fluid producing mesenchymal cells called the

synovium [5]. The synovium secretes the synovial fluid that bathes the joint. Since

articular cartilage is devoid of a blood supply, the joint relies on the well vascularized

synovium to import raw materials and remove waste products from the joint.









Articular Cartilage

Articular cartilage is a biphasic material consisting of a type II collagen matrix

infused with water and proteoglycans [6]. This unique structure enables articular

cartilage to function as both a shock absorber and as a superb load bearing surface. One

of the main tasks of the articular cartilage is to provide a near frictionless bearing surface

as the femur and tibia articulate. This is no easy task considering the large loads the joint

experiences in even ordinary activity such as walking (3-5 times body weight) and the

enormous number of cycles that the joint will experience in a lifetime (well over 50

million). Yet despite these severe conditions the joint performs the task admirably and

undergoes remarkably little wear [3, 7].

One primary difference between biological joints and engineering bearings is that

the body utilizes self repair mechanisms that enable microdamage to be repaired before it

progresses to cause failure of the joint. There is a continuous balance between damage

accumulation and repair and until instances of microfractures begin to outpace the

biologic repair, the joint will remain healthy and effective. In articular cartilage,

chondrocytes are responsible for responding to various stimuli and synthesizing new

cartilage. The precise stimuli and how they signal chondrocytes is an active area of

research.

Cartilage is also designed to provide attenuation of impact loads, thereby reducing

the stress that is transmitted to the bones. It accomplishes this task because of its

biphasic nature and the high concentration of high molecular weight polymers trapped in

the collagen matrix. The viscoelastic properties of cartilage are very important in this

task in that the tissue response is determined by the rate of loading. Since mechanical









insults are known to be risk factors for osteoarthritis it is important that the joint retain its

ability to both lubricate and cushion in order to maintain health.

Synovial Fluid

Normal synovial fluid is a clear to yellow highly viscous fluid composed

primarily of serum [8], which is similar to blood plasma except there are fewer proteins.

Long chains of hyaluronic acid give synovial fluid many beneficial theological properties

[9, 10]. Synovial fluid is non-newtonian, in that it demonstrates a shear thinning effect,

normal stress effect perpendicular to the shear direction and an elastic component [11].

The viscoelastic properties that synovial fluid exhibits are particularly well suited to the

joint since the fluid is highly viscous at low shear rates such as during gait, and highly

elastic during high shear rates, such as a in jumping or trauma. The joint's synovial fluid

is at work in many complex mechanisms within the joint to maintain efficient motion.

Synovial fluid is a primary component of the lubrication process. In both boundary layer

and fluid film theories it is essential that synovial fluid provide necessary lubricating

molecules and viscous properties, respectively. Synovial fluid and articular cartilage are

closely linked in providing lubrication and changes in either will have an effect on the

performance of the joint.





Normal Structure and Function of Synovial Fluid

Healthy joints contain hyaluronic acid (HA) with a molecular weight between 1

and 10 million [12-15]. In 1953 Ogston and Stanier showed that HA concentration has a

marked influence on the theological properties of synovial fluid [11]. They also

postulated that it is the HA that facilitates both the static and dynamic lubrication in the

joint. Through a unique experiment Ogston and Stanier were also able to demonstrate









that as synovial fluid is compressed it behaves in a very elastic manner up to a point at

which it will no longer compress under a given load, thus preventing the two loading

surfaces from coming in contact. This effect, presumably due to the presence of HA, has

significant implications in the lubricating ability of synovial fluid and will be discussed in

greater detail later. By engineering standards, the efficiency of the human knee joint is

astounding, with a coefficient of friction lower than ice on ice or Teflon on Teflon. There

are few man made materials that even approach the low coefficient of friction that has

been measured between articulating cartilage. Synovial fluid further enhances the

lubricating abilities of the knee joint. Synovial fluid is extremely viscous at low shear

rates and extremely elastic at high shear rates. Additionally it contains lubricating

molecules that contribute to even lower frictional properties. These properties allow

synovial fluid to act as an ideal lubricant to prevent friction and wear of cartilage surfaces

[16].

The study of the interactions during motion at the joint surfaces is known as

tribiology, which encompasses friction, lubrication and wear. Two theories have been

developed to describe the lubrication mechanisms that occur at the joint surface. These

two models are boundary lubrication and fluid film lubrication. In boundary

layer lubrication a monolayer of glycoproteins (most likely lubricin, a component of

synovial fluid) [17] is absorbed into the articular cartilage thus reducing the friction as the

opposing surfaces articulate across each other [3]. Due to solid-solid contact, this theory

does not depend on the viscosity of synovial fluid. However, experiments have shown

that the coefficient of friction predicted by this model alone is one to two orders of

magnitude greater than what has been measured in vitro. Because of this it is expected

that another mechanism is at work as well. Fluid film lubrication theory is more useful









for explaining the extremely low coefficient of friction that is observed in fluid film

lubrication, a thin layer of viscous fluid is present between the ends of bones and when

joint loads force the ends closer together, the fluid is squeezed out of the joint space thus

inducing a pressure that acts to support the bones. There are several hypotheses for fluid-

film lubrication, including hydrodynamic lubrication, self-generating mechanism,

squeeze-film lubrication, hydrostatic lubrication and boosted lubrication [3]. In reality

the joint probably experiences phases during which each theory is valid (as during gait).

One composite of these two theories is that as articular cartilage is deformed, pocket of

fluid may be trapped creating areas of solid-solid contact as well as areas that are

separated by a thin fluid film [3].





Pathology of Osteoarthtitis

Osteoarthritis (OA) is the most prevalent disease associated with aging [6, 18].

The quality of life of approximately 10 million Americans is adversely affected by OA

with about 70% of these individuals in the 65 and over age group [19]. Approximately

80% of people over the age of 55 show radiographic evidence of OA. An alarming

statistic considering the swelling elderly population is that the frequency of OA increases

with advancing age and does so exponentially after the age of 50 [20]. Perhaps the most

ubiquitous risk factor for OA is age itself. Over 80% of individuals over the age of 60

have radiographic evidence of OA [20]. However, there are subtle differences between

natural degradation associated with aging and true OA and without careful analysis, the

distinction can be difficult to make [21].

Current research has focused on the mechanical insults that can contribute to the

initiation and progression of OA [22-24]. In knees, repetitive high impact loading and









traumatic loading are two sources that have relatively clear ties to initiating OA.

Increased loading can have two effects on the joint: 1) The long-term repetition will

increase the density of the subchondral bone thereby making it less effective in absorbing

future impacts and in turn transferring more stress to the articular cartilage. 2) Traumatic

loading can damage the underlying bone of the joint and lead to incongruencies of the

joint surface that may eventually become stress concentrations. Other risk factors include

obesity, genetics (although no genetic linkage has been found in idiopathic knee and hip

OA), gender, race, high bone density, estrogen deficiency and nutritional deficiency

(vitamins D and C).

The primary characteristic of OA is cartilage degeneration, sometimes referred to

as wear and tear of the joint [18]. One theory for articular cartilage degeneration is that

repeated high impact loads cause localized remodeling of the subchondral bone.

Evidence of this remodeling can be seen radiographically in the form of subchondral

sclerosis, or thickening. During remodeling of the subchondral bone layer it is possible

for areas of increased stiffness to develop. Since these areas alter the compliance and

congruency of the joint their presence tends to result in stress concentrations. Increased

stresses in certain areas of the joint are then transferred to the articular cartilage which,

when subjected to high stress, begins to deteriorate. Additionally, as articular cartilage

degenerates, even greater forces are transmitted to the underlying bone thus causing

accelerated remodeling [26].

OA generally progresses in three stages: damage to the cartilage matrix, response

of the chondrocytes to damage and the decline of tissue synthesis and the degeneration of

tissue [26]. In the first stage, as the matrix is compromised, it becomes more infused

with water at the same time the concentration and length of glycosaminoglycan (GAG)









chains decreases. The main GAGs in articular cartilage are keratan sulfate and

chondroitin 4- and 6-sulfates [26]. As the water concentration in articular cartilage

increases, the matrix becomes less stiff and more susceptible to mechanical damage [26].

It is the synovial fluid, in conjunction with articular cartilage, which acts as a

lubricating, cushioning, and protective barrier between the ends of the bones [10].

Specifically, it is the presence of long chains of hyaluronic acid produced by the cartilage

matrix that gives the fluid its beneficial viscoelastic properties [15]. Damage to the

cartilage matrix will result in decreased molecular weight and concentration of HA and

eventually result in deficient theological properties in the joint. There are at least three

factors that act to dilute the concentration of HA. One cause is that the joints increase

production of HA in response to the degradation, however the fluid is produced under

abnormal conditions and contains primarily low molecular weight HA, thus leading to a

dilution of the concentration of normal, high molecular weight hyaluronan in synovial

fluid [13]. Another cause is that the release of enzymes, such as

matrixmetalloprotienases, by the damaged articular cartilage will result in the digestion of

long hyaluronan chains, leaving shorter molecules [26]. A third cause is the mechanical

destruction of long HA chains causing them to be torn into smaller fragments. All of

these factors will lead to the result of decreased viscoelastic properties of the synovial

fluid and therefore less effective in protecting against mechanical forces especially in the

model of fluid film lubrication. As the synovial fluid is degraded, a feedback cycle is

initiated resulting in more damage to the articular cartilage.

Although in the normal joint wear is minimal even over long periods, the

conditions in an arthritic joint accelerate the process. There are two main mechanisms

for wear in the joint: fatigue and interfacial [3]. Fatigue wear is a result of accumulation









of small cracks caused by the everyday repeated loading of the joint. If the cracks build

up faster than the chondrocytes can synthesize new cartilage, eventually the damage will

reach to the surface of the cartilage and manifest itself as fibrillation. Interfacial wear is a

result of the contact between the two surfaces of the joint and can be subdivided into two

classes: abrasive and adhesive. Abrasive wear occurs when a soft material comes in

contact with a harder one which cuts the soft material. Adhesive wear occurs when two

surfaces come into contact with each other and form a bond that is stronger than the bond

within either material. When the surfaces are separated, material is pulled off of the

softer material resulting in pitting [3]. Fatigue wear does not depend on the lubricating

properties of the joint, so it is unaffected by changes in synovial fluid viscosity, but

adhesive wear is influenced by the joint lubrication and may be accelerated by changes in

synovial fluid viscosity.





Pathologic Synovial Fluid

Interest in the rheologic properties of synovial fluid has been documented back to

1925 [25]. Even when using a rather unsophisticated capillary viscometer, Schneider was

able to conclude that chronic illnesses were associated with a lower than normal

viscosity. However, due to his study population and test methods he encountered large

variation. Over the past 80 years, there has been considerable discussion about whether

synovial fluid viscosity is altered in patients with OA. At the 1968 Hebreden Society

meeting [27] several scientists reported successfully measuring the viscosity of

pathological fluids including rheumatoid arthritis (RA) and OA. However, no one had

been able to accurately determine the viscosity of healthy synovial fluid. The problem

was the fact that healthy people generally do not have a large volume of synovial fluid









(around 0.3 mL) whereas diseased joints are typically effusive (1-5 mL). Therefore, the

researchers were forced to make comparisons based on the properties of normal bovine

synovial fluid, which has a lower viscosity than normal human fluid [7] leading some

researchers to mistakenly conclude that there are no differences between normal and OA

synovial fluid.

By 1976 at least two papers reported results comparing normal and pathological

synovial fluid [10, 30]. Unfortunately, Wright and Dowson do not give any indication of

the source of their data, so it is not possible to discuss their methods of classifying

"normal" and "pathological". Reimann boldly stated that "in all pathological joints there

is something wrong with the synovial fluid." In his series of experiments, Reimann used

a custom made rheometer to show that there are significant differences in the viscosity of

synovial fluid from patients that had RA, OA (mostly damaged cartilage) and torn

meniscus (mostly normal cartilage). Riemann also noticed a correlation between the

boundary lubricating ability of synovial fluid and the degree of degeneration of articular

cartilage. Although Reimann claims that viscosity of synovial fluid plays a role in

boundary lubrication, it seems unlikely due to the evidence that hyaluronic acid, the main

component of synovial fluid viscosity, has no effect on this form of lubrication [15]. It

seems probable that synovial fluid plays a more vital role in fluid film lubrication,

especially in light of the viscoelastic properties demonstrated by Ogston and Stanier.

In 1978, Davis and colleagues somewhat contradicted Reimann by reporting that

that the viscosity of the fluid was not well correlated to the level of joint degeneration

[28]. However, in their population, all fluid was taken post-mortem and most of the

subjects had been ambulatory prior to dying suddenly opposed to Reimann's groups of

severely osteoarthritic patients. Additionally, it has been shown repeatedly that there is









no correlation between observable joint condition and clinical symptoms, which casts

doubt about whether Davis' subjects ever complained of OA before their deaths.

The subject of normal versus pathological synovial fluid was again addressed in

1979 by Anadere and colleagues [16]. They used an oscillating capillary viscometer to

compare the viscous and elastic components of viscoelasticity from patients with various

joint diseases to "normal" knees (patients with meniscus tears). Both components were

decreased in the diseased fluids with the most pronounced changes observed in fluids

from rheumatic and traumatic knees and somewhat more moderated changes noted in OA

fluids. Unfortunately it is unclear what criteria these researchers used to differentiate

patients with OA.

Schurz and Ribitsch made further contributions to the field of synovial fluid

rheology in 1987 [29]. They evaluated more than 200 knee joints to determine the

theological properties of normal and pathologic synovial fluids. Measurements were

carried out on a Contraves low shear rotational viscometer and indicated a decrease of

about one order of magnitude between normal and pathological synovial fluid. The

authors in this study did not they describe their criteria for diagnosis of pathological

fluids. Additionally, the normal fluids were gathered post-mortem but no mention was

made concerning the selection of "normal" subjects. In conclusions similar to Reimann

(1976); Schurz and Ribitisch claim that synovial fluid viscosity can be used to

differentially diagnose degenerative and inflammatory joint disease. A summary of the

literature reviewed is provided in Table 2.1. An excellent review of the early synovial

fluid research (1925-1971) is presented by Blair [25].










Table 2.1. Summary of studies analyzing synovial fluid viscosity.
Study Temp Viscometer Source of Subject Shear Rate Viscosity
(C) Fluid Condition (1/s) (cp)
Wright (1975) ? ? ? Normal 10 1000
[10]
[10] OA 10 80-150

Davis (1978) 35 Couette Post-mortem OA 40 85-505
[28]
Schurz (1987) 25 Contraves Post-mortem Normal 10 1000-40,000
[29] Rotational
SR Diseased OA 10 100-1000
human knee
Caygill (1969) 28 Weissenberg Post-mortem Normal 0.05-0.08 20,000-
[8] Rheogoniometer 60,000
Reimann 22 Custom made Patients with Near .15-2.5 30-170
(1976) torn meniscus Normal (complex)
[30] Diseased OA .15-2.5 22-108
human knee (complex)
Anadere 23 Oscillating Patients with Near 10 40
(1979) Capillary meniscus Normal (complex)
[16] defects
Diseased OA 10 31
Human knee (complex)
Safari (1990) 27 Bohlin VOR Arthroscopy Normal 2 366-513
[31] Parallel Plate not related to (complex)
OA (ACL,
meniscus)
Diseased OA 2 137-262
human knee (complex)
Ogston (1953) 25 Couette Bovine Normal 10 8,000-
[11] Viscometer 80,000



Traditionally laboratory analysis of synovial fluid has consisted of determining

the type and number of cells present in the fluid, the volume of fluid aspirated and

qualitative analysis of rheologic behavior of the fluid. While it has been shown that there

is an association between viscosity and joint pathology [10, 16, 29, 30, 31], clinical use of

viscosity in diagnosis remains minimal. One reason why more clinicians do not utilize

viscosity is the lack of easy to use low volume viscometers in the clinical setting.

Despite the lack of precise instrumentation some physicians have found it useful to

describe the qualitative properties of synovial fluid. In the process of "synovialysis", a

qualitative measure of fluid viscosity is one of the tools used to differentially diagnosis

joint disease [32].









Currently it is feasible to collect sufficient amounts of pathological fluid for

analysis, yet in patients with good health to mild OA, it is sometimes impossible to

collect enough fluid to perform a viscosity measurement. This limitation has prevented

viscosity from becoming a more widely accessible outcome measure for OA. Some

researchers have noted that some patients may have sufficient fluid at the onset of clinical

trials, but will have little synovial fluid volume if the experimental treatment is effective

(Personal communication, Dr. Philip Brand 2000). It is because of this problem that the

microrheometer is such a valuable tool in the study of synovial fluid.

It has long been suspected that HA is responsible for the viscoelastic properties of

synovial fluid and in pathological conditions it has a noticeably lower molecular weight,

which in turn results in lower viscosity. Considerable theological data were amassed

concerning HA over the decades and for nearly half a century it has been controversial as

to whether destruction of HA macromolecules is a cause of OA or merely a consequence

of the disease. The controversy is partly rooted in how one interprets the lubrication of

the joint. If it is assumed that boundary lubrication is the primary mechanism, then there

is no reason to draw a link between lack of viscosity (due to HA breakdown) and OA

progression. However, as discussed earlier it is dubious to rely on boundary lubrication

alone, and numerous studies have shown that at least some phases of gait are better

explained by fluid film lubrication. Therefore, if one accepts the plausibility of fluid film

lubrication, then the viscosity, and the HA size, is inherently linked to the healthy motion

of the joint.


Management and Treatment of Osteoarthritis

While OA is not life threatening, it can certainly be a debilitating condition. The

cost of OA is significant because the disease is both widespread and has at least a









moderate impact on the affected individual. Pain and loss of function resulting from OA

can lead to direct costs of treatment and also to substantial indirect cost due to lost wages.

In a study of the economic impact of musculoskeletal diseases in the US, it was found

that arthritis carries a burden of $13 billion in direct costs and $42 billion in indirect costs

[19]. The national costs are expected to rise as the median age of the population

increases.

Weight loss and physical therapy are generally prescribed to treat mild OA.

Traditionally more advanced OA is treated with non-steroidal anti-inflammatory drugs

(NSAIDS) until the drugs no longer provide relief from pain at which point the treatment

usually progresses to surgery. While NSAIDS are the most commonly prescribed

medications for OA, they do not seem to alter the natural course of the disease and they

produce significant gastrointestinal side effects due to their inhibition of gastric

prostaglandin synthesis [6]. These severe gastric side effects have recently led to the

development of a new class of drugs know as cyclooxygenase-2 (cox-2) inhibitors. Cox-

2 inhibitors are intended to act on only the pathways that cause pain and decrease effects

on the gastrointestinal tract. Current surgical techniques include load reducing

osteotomy, tenotomy, hemiarthroplasty, and total joint replacement. There are also

inherent risks associated with surgery. The risk for dangerous side effects from this

standard of care has led to the use and development of many new and not so new

treatments for OA.

There are a growing number of non-traditional approaches becoming used

throughout the world. In Europe and Asia, glucosamine and chondroitin sulfate (GCS)

have long been used to treat arthritis. In Europe GCS is regulated as a pharmaceutical,

thus subject to strict quality standards and requiring a prescription. Glucosamine is a









constituent of glycosaminoglycans (GAGs) and proteoglycans that are naturally found in

cartilage and synovium. It is also a building block for hyaluronic acid, which forms the

backbone of the proteoglycans embedded in the articular cartilage. Chondroitin 4- and 6-

sulfates are the main GAGs of articular cartilage [26]. In theory, by providing the

building blocks for healthy articular cartilage, the joint will be able to repair and

regenerate cartilage, but this ability of chondroprotection is unproven. Clinical studies

evaluating GCS have suggested that they are effective in relieving symptoms but have

been criticized for reporting bias and low quality [34]. American physicians have been

slow to adopt the use of dietary supplements. However, there seems to be a changing

opinion among both clinicians and researchers that the use of nutraceuticals might be

beneficial for some patients [35-37]. Despite decades of anecdotal evidence and at least a

dozen clinical studies, very little is known about the actual mechanisms through which

GCS may work. In studies of radioisotope labeled GCS, it has been shown that when

orally ingested GCS does indeed reach the joint. But it is not known what the effective

levels must be in the joint to attain clinical improvement nor is it clear what happens to

the compounds ounce they are in the joint.

Another non-surgical treatment for OA is intraarticular injections of hyaluronic

acid (HA) known as viscosupplementation (VS). Balazs [13] has advocated the use of

artificial synovial fluid since 1960's and is truly a pioneer in the field. VS is a procedure

in which a hyaluronic acid derivative is injected directly into the affected joint. The goal

of viscosupplementation is to recreate the environment of a healthy joint in an effort to

encourage the joint to escape the negative feedback cycle of OA progression. Once

normal viscoelastic properties are returned to the joint fluid, the cartilage lining the joint

will increase production of healthy synovial fluid. Initially the mode of action is mainly









mechanical, but it is suspected that additional biologic benefits are eventually levied.

The half-life of the injected fluid is only a few days. Therefore after one week there is no

longer a significant amount of artificial fluid in the joint. However, clinical studies have

shown that the benefits of viscosupplementation can last from 6 months up to a year after

the last injection [37]. Researchers are attempting to elucidate the mechanism by which

VS provides intermediate-term benefits. It is believed that because of the transient nature

of the HA within the joint, it is not actually the restoration of joint viscosity that is the

cause of the noted changes [38]More likely, the benefits observed in VS are due to

interactions on the molecular level between the HA and pain receptors in the joint.

However, since the cause for the initial onset of arthritis is not addressed (subchondral

sclerosis, varus/valgus deformity, obesity) the benefits of viscosupplementation are not

and will never be long term. Studies evaluating the use of repeated HA injection over

long periods of time are not yet completed.




Outcome Measures in Osteoarthritis Research and Clinical Practice

In many studies OA populations are determined through radiographic diagnosis

(joint space narrowing, presence of osteophytes at joint margin, sclerosis of subchondral

bone, subchondral cysts [36].However, many of these subjects have no other symptoms

of OA, and only 10-20% have any limitations in function due to OA. Alternatively, in

some patients that have joint pain most days of the month, there is no radiographic

evidence of OA [6]. Therefore, several procedures have been developed to screen for

OA. One popular and well-validated tool is the WOMAC (Western Ontario and

McMaster Universities) [36, 39] scale which utilizes a combination of visual analog pain

scales and functional ability scores to arrive at a quantitative index of level of OA. Other









algorithms use risk factors such as age, obesity and level of activity in conjunction with

clinical, radiographic and laboratory findings to determine levels of OA.

Radiographic Analysis

The use of radiographic analysis is perhaps the most widely used indicator of OA

progression. Using this method is advantageous because there are at least four criteria

that can be observed on an x-ray image.

1. Joint space narrowing can be measured as either the mean distance between

the femur and tibia as determined by computerized image analysis or by

measuring the minimum distance between the bones manually.

2. Subchondral sclerosis can be recognized as an area of low radiolucency

signifying the presence of dense bone.

3. Subchondral cysts can form within the cancellous bone around the joint.

4. Osteophytes may be observed at the periphery of the joint, another symptom

of the hypertrophic conditions in the joint.

Pain Questionnaires

Questionnaires are a well validated clinical tool for assessing the functionality of

patients and the pain associated with routine tasks such as standing up from a chair and

going up and down stairs. Various test utilize different form of functionality

measurement but the most popular is the visual analog scale (VAS) in which a patient

places a vertical mark on a 10 cm horizontal line corresponding to their level of pain or

functionality.









Magnetic Resonance Imaging

Magnetic resonance imaging is an excellent tool for analyzing the soft tissues of

the joint. Currently there are experiments underway at our institution attempting to

quantify the microstructure of articular cartilage using a 3T magnet.

Proteoglycan Assay

Synovial fluid aspirations can be analyzed for their molecular content. Important

markers such as chondroitin (4- and 6-) sulfate, keratin sulfate and

matrixmetalloprotienases (MMP) can signify that the joint is experiencing high levels of

catabolism due to cartilage damage.

Viscosity

It has been shown that pathologic changes in joints will result in alterations in the

synovial fluid viscosity [30]. However, in very few studies has viscosity been used as an

outcome measure for OA treatment [9, 14]. If, according to Peyron and Balazs, the loss

of viscoelasticity is directly related to the severity of OA and that by restoring the

viscoelasticity, the joint function can be improved, then viscosity should be a good

correlation to the level of disease. One limitation of viscosity measurements is the

availability of synovial fluid. Pathological joints generally produce larger amounts of

fluid (2-5 mL) than normal joints (0.3 1 mL) but the amount available for aspiration is

highly variable even for the same patient. Therefore it would be useful to have access to

a specialized low-volume viscometer to perform viscosity analysis. Such a device has

been developed in our laboratory.














CHAPTER 3
MATERIALS AND METHODS



Experimental Setup

In this double blind placebo controlled randomized clinical study, 26 subjects

were recruited from a population of patients at the VA. The inclusion criteria were:

1. Diagnosis of OA based on loss of motion associated with pain and loss of joint space

on x-rays compared to the contralateral knee.

2. Currently taking NSAIDs with no relief.

Subjects were excluded if they had surgery on their study knee within ten years prior to

enrollment in study or were currently taking GCS supplements. After providing

informed consent, subjects were randomly assigned to one of two experimental groups,

GCS or Placebo. A commercial formulation of GCS containing 500 mg glucosamine

hydrochloride and 400 mg chondroitin sulfate (GCS; Osteo-Bi-flex, Sundown

Vitamins, Boca Raton, FL) was used. The placebo was also manufactured by Sundown

Vitamins and was identical in appearance to the GCS capsule. Beginning week 0,

subjects were instructed to take three capsules daily. NSAID use was not restricted, but

subjects were asked to keep a "pill diary" to record their level of pain and NSAID

consumption daily.

At the time of enrollment (week 0), all subjects had an MRI taken of their study

knee, filled out SF-36 (pain and functionality questionnaire), performed strength testing

and had theirjoint aspirated. Aspirations were collected and stored at 0C for testing at a









later date. Subjects returned to the study center at weeks 4, 12, and 20 for follow up, at

each return visit subjects had their knee aspirated and performed strength evaluation. On

the first and last visits, subjects received a MRI of the study knee. In this thesis only the

preliminary viscosity data will be analyzed and discussed.

Strength Testing

Prior to strength testing, range of motion was collected from subjects in the supine

position. Subjects were then allowed to sit in a chair and comfortably restrained to

prevent injury during strength testing. Concentric/concentric knee exercises were

performed on both the study knee and the contralateral knee using a BTE Primus testing

machine. Each test consisted of three sets of five repetitions (knee extension and flexion

= 1 repetition). The resistance of the testing machine for each set was randomly

determined prior to testing using a Latin square method. The peak force exerted and

average work done for each set was recorded.

Magnetic Resonance Imaging

Subjects received a 3T MRI of their study knee. The MRI's were read by an

experienced radiologist for quantification of articular cartilage microstructure and

documentation of structural changes

Pain Questionnaire

A pain and functionality questionnaire was used to determine the quality of life

change that the subjects experienced.

Proteoglycan Assay

Synovial fluid aspirates was analyzed for proteoglycan content using ELISA

techniques. Proteoglycans are released into the joint by articular cartilage at an

accelerated rate in patients with OA.









Viscosity

The viscosity of subjects with less than 0.5 mL synovial fluid was tested using a

unique microrheometer that has been developed in our lab. In subjects that provided

more than 0.5 mL of synovial fluid aspirate, viscosity measurements were also performed

on a Brookfield cone-plate viscometer. Microrheometer measurements were made with a

1.01 mm diameter magnetic iron cylinder (Fisher Scientific) and a 1.61 mm inner

diameter glass sample tube also at 250 C. All cone-plate measurements were made with a

cp-52 cone (30 angle, 0.5 mL fluid) at 250 C. Viscosity was measured at 6 different shear

rates on the cone-plate viscometer to evaluate non- newtonian behavior of the synovial

fluids. The most relevant difference between the two rheometers is that the

microrheometer applies a known force to a cylinder (gravity) and measures the change in

shear rate (falling velocity), whereas the cone-plate viscometer applies a known shear

rate to the sample and measures the torque response through the spindle. Therefore, for

non-newtonian fluids, a correction must be made to compare the apparent viscosity

derived from the microrheometer and that measured from cone-plate viscometer. To

compensate for the difference, the viscosity versus shear rate curve plotted from the cone-

plate viscometer must be extrapolated to the shear rate of the microrheometer and at that

shear rate the two values can be compared. The microrheometer is discussed in much

further detail in the next section.

To validate and calibrate the results from the microrheometer, the viscosity of

standardized newtonian and non-newtonian fluids of known properties was measured

with both the microrheometer and the cone-plate viscometer and the results were

compared. The accuracy of the microrheometer was based on its correlation to the cone-

plate viscometer. The known newtonian fluids tested were standardized oils of differing









viscosity. Non- newtonian samples of four different molecular weight (MW) hyaluronic

acids were tested on both devices to determine the feasibility of measuring non-

newtonian viscosity using the microrheometer.




Microrheometer

Background

The Microrheometer is a unique device that was designed to measure the viscosity

and viscoelasticity of small samples of biological fluids. Often in medical applications it

is difficult, if not impossible, to collect large volumes of samples for testing. In response

to this challenge, Tran-Son-Tay [51] developed an acoustically tracked falling ball

rheometer. The theory for this rheometer is based on the Navier-Stokes solution for a

ball falling through a finite tube. By tracking the displacement of the ball with an

ultrasound transducer, the falling velocity can be calculated. Since the diameter of the

ball, the diameter of the tube, the density of the ball and the density of the fluid are

known, the viscosity can be calculated. By oscillating the ball with an electromagnet, the

phase shift between the input current to the magnet and the motion of the ball can be

determined and correlated to the viscoelastic components of the fluid. The

microrheometer setup has recently been modified by replacing the ball with a magnetic

cylinder to allow for more consistent applied shear rate.

The most common viscometers and rheometers presently in use are those based

on the measurement of stress on a fixed surface while a parallel or opposing surface is

moved with a known applied strain rate. However, as already mentioned, these devices

require fairly large sample volumes. Often in medical applications it is difficult, if not









impossible, to collect large volumes of samples for testing. The Microrheometer (Figure

3.1) is a unique device that was designed to measure the viscosity and viscoelasticity of

small samples of biological fluids by using a spherical steel ball that is concentrically

located in a small cylindrical tube with a volume of about 20 tiL. The ball falls either

under the force of gravity or is levitated and oscillated with a force produced by a

magnetic field. The position of the ball is tracked by ultrasonic pulse-echo method.

Furthermore, the small volume permits accurate temperature control and rapid

temperature changes to be effected in the sample under study. Recent modifications have

been made to the microrheometer so that a constant shear rate will be exhibited across the

fluid, that is, a falling cylinder is used in place of a ball.

Apparatus

A 20 ptL sample is loaded, by retrograde injection, into a disposable glass tube

with an inner diameter of 1.6 mm and a height of 10 mm. Once loaded, the tube is

centered inside a cylindrical, Plexiglas, water-jacket chamber. The ultrasonic transducer

is housed at the bottom center of the Plexiglas chamber. An O-ring forms a watertight

seal with the base of the sample tube and with the transducer. A plastic cap is screwed

into place at the top of the chamber and provides a seal that completely protects the

sample. A schematic of the microrheometer is given in Figure 3.1.







































Figure 3.1. Schematic of Microrheometer (a sample tube, b cylinder, c -
piezoelectric crystal, d ultrasound transducer, e water jacket, f electromagnet).



A small electromagnet coupled with a micromanipulator is used to position and

drop a 1.01 mm diameter cylinder in the center of the tube, where the strongest echo

occurs. The pulse-echo mode is used to locate and track the falling cylinder. A single

sound pulse is transmitted into the fluid medium by pulsing an ultrasound transducer that

also acts as a receiver. Any returning echoes from the cylinder cause a voltage rise

across the transducer that is amplified by the ultrasonic pulser/receiver unit (Panametrics

5052 PR). Figure 3.2 shows the main bang followed by a pulse reflected from the top of

the cylinder. The time for the sound waves to travel to and back from the cylinder, and







28


the speed of sound in the fluid are measured. From that information, the distance

between the transducer and the cylinder, i.e., the location of the falling cylinder, is

determined.


Tek Run; 10.0MS/s Sample
[-"*4 "-~----


Ih 5. 0 dd vb ch2 solomyO vis.OOps ch4 --doom4 24 Iblay 200'1
113 500mV [EE~ 200mV 16:59:5~3


Figure 3.2. Main bang is the large signal on the left, the echo of the cylinder is the
smaller signal on the right (tf time of flight, time for ultrasound pulse to travel to the
cylinder and back to the transducer.)





Experiments

Two types of measurements can be performed with the microrheometer: (1) a

speed-of-sound evaluation, and (2) a steady state viscosity. The parameters of interest

are c, the local speed of sound in the medium and q, the bulk suspension viscosity.

Speed of Sound. In order to measure the speed of sound in the fluid, the

instrument needs to be calibrated. To accomplish this, the sample chamber is first filled


-- ni L L ; L -- -- . J | I I I I 1 | 1 I !
.. . . . . . . . . . .. $ . . . . . . .
: + -^~~~------ --------\-------.-----*-----
!_=_-__ 1 - - ___ - 1 . .- .'0-


.. . . . . . . . . . . . . '_ . . . . . .
2' ^ | . ,, :.-. *,. ^, :





: 1: : $ : : : :


t'
,, M O iP l n g : . . : ... : . .. .. . : . . .. . . . . . .
I Ethho of Delay Tip I Echo ,f Cylinder
-' ' l '- J !_. ' ' : ' :: .


.............-..... -1


: 33.21JS
6: 33.2,S









to the top with distilled water and then capped with a glass cover slip to assure a fixed

sample height h. This distance is given by

T *C
h = f(7o) h2 (3.1)
2

where Tf(12o) is the measured time of flight for the sound to travel to the top of the sample

chamber and back in distilled water, and Co, is the known speed of sound in water.

Once the chamber height is determined, the water is removed and replaced by the fluid

sample. The local speed of sound in the medium Cfis then determined by the

relationship

2*h
C, = (3.2)


where Tfis the measured time of flight in the fluid sample.



Viscosity. The fluid viscosity is determined from the speed of sedimentation of

the falling cylinder, Figure 3.3. The method used to determine the falling velocity of the

cylinder is presented in Appendix A.










Tek lMl 10O S/s


S Ch2 Zoom: 2.0~ Vert .OX HOrz A: 75mV
,L* A: 385ms
........... ....... .. @ : -37m V
i.' --
.. . . . . ..... .. . .




.. . . . .


. . .. ... - - - -. .

t -
-t + :

CM .UU OmV V L' M 2b50ms1 Chi VL iV 24 May 2001
17:13:27


Figure 3.3. Oscilloscope plot of the cylinder's displacement vs. time as it falls through
the sample tube. The arrow indicates the path of the cylinder. From this output the speed
of sedimentation (of falling velocity) of the cylinder is measured.














CHAPTER 4
RESULTS




Microrheometer Validation

Results from the preliminary calibration and validation experiments that were

carried out on the microrheometer and the Brookfield cone-plate viscometer are

presented in Tables 4.1 and 4.2. The viscosities determined from both devices are quite close

to the specified standards for each fluid. The microrheometer has greater measurement

variability than the cone-plate viscometer. Shear rate, j, for the microrheometer was

calculated based on the falling velocity, v, and the gap between the cylinder and the

v
sample tube wall, h, using the following equation: j = -. Due to the inherent properties
h

of the microrheometer, it is not possible to control shear rate therefore each fluid will be

sheared at a different rate. The accuracy of the microrheometer was established by

comparing it to measurements of the same fluids made with the cone-plate rheometer. In

the newtonian fluids, summarized in Table 4.1, the viscosity is independent of shear rate.

However in the non-newtonian fluids Table 4.2 confirms the dramatic effect of shear

thinning, where the viscosity decreases as the shear rate increases. Shear rate is plotted

against viscosity for four non-newtonian HA samples (Hylan A, Biomatrix, NJ) in Figure

4.1. The viscosity of non- newtonian HA samples measured on the cone-plate viscometer

correlates well to what has been reported in the literature Figures 4.2 and 4.3.














Table 4.1. Newtonian fluids tested on both the microrheometer (MR) and the cone-plate
rheometer (CP). N is the number of samples tested on the MR. SD is the standard
deviation of the MR viscosity measurement. Accuracy = ((MR-CP)/CP*100%).
Variability = SD/MR viscosity* 100%.
Fluid CP Viscosity N MR Viscosity SD Accuracy % Variability %
(cp) (cp)
1000 1005 16 1067 87 6.16 8.15
3000 3085 17 3274 241 6.13 7.36
10000 9549 37 9442 1564 -1.12 16.56
12500 12120 16 10560 960 12.87 9.09


Table 4.2. Non-Newtonian HA fluid tested on both the microrheometer (MR) and cone-
plate viscometer (CP). MR shear rate is given for the measured viscosity. HA was tested
over a range of shear rates in the CP.
MR CP
Shear Rate (1/s)
Fluid Shear Viscosity 1 2 5 10 20 40 100 200
Rate (1/s) (cp)

0.75M MW 4.82 296.5 393.2 294.9 275.2 275.2 265.4 250.7 214.3 179.9
Hylan A
(1.1%
Polymer
Content)
2MMW Hylan 0.162 8985 10810 8650 5780 3952 2536 1558 792.3 466.9
A (1.0%
Polymer
Content)
4M MW Hylan 0.0105 170200 79030 43350 18830 9339 4925 2644 1121 581
A (1.0%
Polymer
Content)
5MMW Hylan 0.0064 244000 81890 46690 20960 10640 5583 3018 1292 670.4
A (1.0%
Polymer
Content)____














100000


10000 --- -4MMW
--A---4MMW
~ <* 5M MW
0.
S1000 -

U,

100
100-




10





1 10 100 1000
Shear rate (1/s)

Figure 4.1. Viscosity plotted against shear rate for non-newtonian HA samples tested on
the cone-plate viscometer (log-log plot).



High molecular weight HA samples showed significant shear thinning effects,

however, the 0.75 M molecular weight fluid displayed nearly newtonian behavior. It is

expected that the theological properties of high molecular weight HA solutions are

representative of synovial fluid from healthy joints.


'u











IDOOOO


10000



10000


Low viscos;ty
at hich shear


100 ,- -
0.001 0,01 0.1 1 10 100 1.000
Shear rate {s' 1




Figure 4.2. Viscosity of 4M MW Hyaluronic acid plotted against shear rate. Source:
[40]


1000000


100000


10000


1000


1 10 100
Shear Rate (1/s)


1000


Figure 4.3. Viscosity of 4M MW HA plotted against shear rate. Measured using
Brookfield cone-plate viscometer.












Clinical Data

This thesis is a preliminary evaluation of the synovial fluid viscosity from the first

14 subjects enrolled in the study. A summary of the results from synovial fluid testing on

the cone-plate viscometer is presented in Table 4.3. In some patients it was impossible to

draw any volume of fluid, thus there is no way to determine synovial fluid viscosity at

those time points. Due to these missing data points, all subjects could not be compared

over all time points. This problem was dealt with in the following method: treatment

effect was defined as the change in viscosity (cone-plate, 1/s) between the earliest and the

latest time available time point divided by the earliest viscosity. Treatment effect was

calculated for each patient. Positive values mean that the synovial fluid viscosity

increased and negative values mean that the synovial fluid viscosity decreased. A second

analysis was made of only subjects from which both week 0 and week 4 data were

available. This analysis is presented in Table 4.4.

Viscosity data from a representative subject is shown in Figure 4.2 (the only subject

from whom synovial fluid was obtained at each visit). Notice that the viscosity initially

decreases over the fist 12 weeks, but then increases over the next 11 weeks.









Table 4.3. Changes in synovial fluid viscosity between first and last sample (cone-plate
viscometer, shear rate = 1/s) for both experimental groups. Effect size = change/baseline
viscosity. a Two subjects in the placebo group withdrew from the study, one to due a
TKA, the other due to lack of benefit from study medication. b Effect size is not
statistically different (t=0.45).
GCS Placebo a
(n=8) (n=3)
Increased 25.0% 0.0%

No Change 12.5% 66.6%

Decreased 62.5% 33.3%

Effect Size b 0.04 (0.83) -0.29 (0.51)
Mean (SD)


Table 4.4. Change in synovial fluid viscosity between week 0 and 4. One subject in
placebo group withdrew due to TKA. d Effect size is not statistically different (p=0.50).
GCS Placebo c
(n=5) (n=2)
Increased 40.0% 0.0%

No Change 20.0% 100.0%

Decreased 40.0% 0.0%

Effect Size d 0.22 (0.867) 0.0 (0.0)
Mean (SD)












1000 -

-X-week 0
-- week 4
h week 12
S--. -*-week 23

100 -




0



10








1 -
1 10 100 1000
Shear Rate (1/s)

Figure 4.2. Shear rate versus viscosity for a subject in GCS group. This particular
subject experienced a decrease in viscosity the first 12 weeks which somewhat recovered
by week 23. Notice that the fluid displays newtonian behavior at low shear rates at week
12.














CHAPTER 5
DISCUSSION




Microrheometer

The validation of the microrheometer revealed that the device is fairly accurate in

measuring the viscosity of newtonian fluids, as compared to a cone-plate rheometer. An

unexpected improvement for the microrheometer was conceived during testing on highly

viscous fluids. A new method was created in to adjust the shear rate at which fluids are

tested. By generating an external magnetic field, an increased force can be applied to the

magnetic cylinder. Varying the current that supplies the electromagnet will allow the

shear rate to be adjusted. This concept was included in the theory for a closed form

solution for a falling cylinder [41] as a "local density" term (Appendix A). In most

samples (29 out of 34) it was possible to evaluate the synovial fluid using the cone-plate

viscometer, which allowed accurate plotting of viscosity versus shear rate measurements.




Clinical

At four weeks, patients taking GCS had a 22% increase in synovial fluid viscosity

compared to no change in patients on placebo. Also, at later time points it was found that

patients on placebo had a decreased viscosity while patients taking GCS had not changed.

However these differences are not statistically significant (p= 0.5, p=0.45 respectively).

In addition it was noted that synovial fluid exhibits shear thinning behavior.









The results indicate a need for a study with a larger number of subjects. The trend

observed, nevertheless, suggests that GCS may have a beneficial effect on some patients,

resulting in an initial increase in viscosity over the first month of treatment and

eventually returning to pretreatment condition after that

These results fit well with what has been reported elsewhere. Glucosamine and

chondroitin are touted as a "cure for arthritis" in the lay literature [42]. Theodasakis is

partly responsible for the current vogue of GCS supplements for OA. In his popular

book, he extols the benefits of GCS and supplies bountiful anecdotal evidence of their

effectiveness. Indeed, a number of clinical trials have found that GCS supplements have

at least a modest effect on the symptoms of OA [39, 43-48], but the scientific community

remains divided concerning the efficacy of GCS [49]. In a recent review by McAlindon

and colleagues [34] 17 clinical trials evaluating either glucosamine or chondroitin were

identified in the literature. They analyzed the quality of 15 of the studies using

standardized methods and compared the effects of treatment using predetermined

significant outcomes. McAlindon found that clinical trials demonstrate glucosamine and

chondroitin to have a modest to large effect, but cautioned that methodological flaws may

exaggerate the benefit. In particular a trend developed that higher quality studies

reported smaller effects compared to lower quality studies. This has been a common

theme in the criticism of GCS clinical studies. It is interesting to note McAlindon's

treatment of the one clinical trial conducted in North America [39]. In this study, Houpt

concludes that glucosamine hydrochloride is not effective in relieving OA pain as

compared to placebo. However in the McAlindon meta-analysis, the Houpt study is

presented as having a small to moderate effect on improving pain. This discrepancy is









presumably attributable to the structured and systematic review protocol applied by

McAlindon, yet the strikingly contrary conclusions seem dubious on McAlindon's part.

The study by Houpt is unique in several regards: it is the only published clinical study

trial conducted in North America, it is the only study to evaluate the glucosamine

hydrochloride formulation, and it is the only study that does not report improvement in

subjects taking glucosamine. It is possible that the formulation of the study compounds

has an effect on the efficacy, especially since in North America glucosamine and

chondroitin are considered nutritional supplements and therefore not as strictly regulated

as pharmaceuticals. In Europe and Asia, glucosamine and chondroitin are classified as

pharmaceuticals thus ensuring that quality is held to a higher standard.

In an interesting study that has been published after McAlindon's review,

Reginster, et al. [36] it was found that joint space in subjects taking GCS did not change

over the course of a 3 year treatment compared to the placebo group which experienced

significant joint space narrowing. This study is important due to its large sample size,

106 subjects per group, and long term follow up, 3 years. However, the use of standing

film x-rays has been called into question due to alignment problems that may alter the

measurements of joint space and due to bias introduced by pain in the joint causing

subjects to have difficulty standing in a standardized position. This study is also valuable

because it demonstrates the long-term safety of GCS.

There are three main arguments against the use of GCS in the treatment of OA:

lack of definitive proof of the mechanism of action, quality of clinical trials to date and

regulatory issues concerning purity and optimal compound dosaging [49]. GCS is

theorized to work by supplying the joint with the rate limiting compound in cartilage









synthesis. By increasing the amount of building blocks of cartilage, it is assumed that the

production of cartilage will increase. Since most GCS studies have only evaluated the

oral formulation of the compound, it also must be clearly demonstrated that the active

agents can be absorbed by the intestine, without being degraded, and then reach the joint

in significant amounts. Clinical trial quality has been questioned primarily because

nearly all have been sponsored by GCS manufacturers. In fact most have been sponsored

by the same Italian pharmaceutical company, Rotta. In response to this issue, the

National Institutes of Health has recently funded a clinical trial of GCS, which will most

likely be of the highest quality. Due to its classification as a dietary supplement is the

US, GCS is not regulated by the Food and Drug Administration as are pharmaceuticals.

Therefore until universal standards are adopted and enforced, the quality of GCS

products sold in the US cannot be ensured.

As mentioned earlier, the high viscosity of synovial fluid is caused by the

presence of long chains of chains of hyaluronic acid (HA). In a diseased joint, where

cartilage is being destroyed, enzymes are released that break down HA, thus reducing the

viscosity of synovial fluid. Since glucosamine is a constituent of HA, providing more

glucosamine to the joint may increase the production of HA. However, this mechanism

has not been definitively proven and the mechanism of action, indeed if any, of GCS

remains unclear. In addition to beneficial viscoelastic properties, HA is thought to also

have an analgesic effect by insulating the pain fibers in the synovium [50]. Although at

this time it appears as if the synovial fluid viscosity is not affected by GCS, it is possible

that other symptoms, such as pain and joint space may respond to treatment.









Since this is the first trial to use viscosity as an outcome measure in a GCS study,

there is no data in the literature available to compare. In fact there have only been two

studies identified that used viscosity as a measurement for any treatment. In one trial, the

viscosity of fluid from four joints was measured before and one week after HA (Healon)

injections [9]. They found that the viscosity increased by about 15% after treatment. In

this study no placebo was used. Synovial fluid from normal donors was also tested

however, all but three of these samples had to be pooled due to low volume.

Additionally, this study only measured the intrinsic viscosity of the fluid. Intrinsic

viscosity is measured at a defined shear rate on consecutively diluted samples and is the

value obtained by extrapolating the resulting curve to 0 concentration [38]. The intrinsic

viscosity is useful in measuring the theological properties that are not dependent on

concentration, but it is difficult to relate this value to what has been reported elsewhere.

In the second study that examined viscosity [14] viscosity was measured in 20 patients

before and after receiving 5 weekly injections of HA (Hyalgan) or saline. The viscosity

of the HA group increased relative to baseline, but was unaltered in the saline group.

During the course of the trial two subjects have withdrawn. Both were in the

placebo group. One subject received a total knee arthroplasty in the study knee. The

second subject withdrew due to lack of efficacy of treatment. This subject had an 88%

decrease in synovial fluid viscosity, the largest decrease in either group. No adverse

events were reported for either group.

This study has several limitations. The population that was selected for this study

consisted of patients with severe OA, to the point that their physician has suggested a

total knee arthroplasty. It is generally accepted that GCS is most effective in subjects









with mild OA. Therefore, the disease state of this population may have progressed

beyond the realm of efficacy of GCS before the study began. Another limitation of this

study is that at some time points it was impossible to aspirate any of synovial fluid from

the subjects (in only one subject was synovial fluid collected at all time points), thus

making viscosity testing impossible at those time point. This loss of data led to

incomplete analysis over the total 20-week course of the study. Since this thesis is an

interim analysis of the data, it is likely that during the conclusion of the trial additional

data will be collected, thus providing a more complete picture of the effects of GCS. The

synovial fluid collected before July 1, 2000 was stored in plain plastic test tubes. All

later synovial fluid was stored in EDTA tubes. An analysis was done to determine if the

EDTA had an effect on viscosity. GCS and placebo groups were equally mixed between

the two different tubes. There was no difference between the mean viscosity (cone-plate,

1/s) of synovial fluid in the plain tubes and that in the EDTA tubes (p=0.45). At this time

only the viscosity data has been analyzed, therefore it is not yet possible to evaluate the

clinical effects of GCS. It will be interesting to determine how the viscosity data relates

to clinical parameters such as pain and joint space. To date there have been no studies

that have attempted to correlate viscosity to clinical symptoms of OA, making this

contribution significant.














CHAPTER 6
CONCLUSION



From this study two primary conclusions can be drawn. Although not statistically

different, there are observable changes to the synovial fluid viscosity after GCS

consumption at this interim analysis. More patients need to be enrolled to allow stronger

conclusions. Also, the microrheometer has been shown to be a valuable instrument for

measuring viscosity when only small fluid volumes are available. The novel concept of

altering the shear rate of the microrheometer using an external magnetic field promises to

be a valuable development in the analysis of synovial fluid due to its non- newtonian

nature. This research demonstrates the feasibility of using synovial fluid viscosity as an

outcome measure for OA trials.



Obviously the next step in this research is to compete enrollment of subjects for

the current study and to evaluate the remainder of the data at study conclusion. At that

time all of viscosity data will be compiled and re-analyzed. A more complete picture of

the efficacy of GCS will be drawn once all clinical outcomes are analyzed.

Modifications are being made to the microrheometer that will enable the measurement of

viscoelastic properties of synovial fluid. Viscoelasticity can be calculated by oscillating

the magnetic cylinder within the fluid using a function generator wired to an

electromagnet and measuring the phase shift between the applied current to the

electromagnet and the response of the magnetic cylinder. This will be an important step,









since viscoelastic properties of synovial fluid may play an even greater role in joint

mechanics and lubrication than viscosity. A separate improvement is being developed

that will allow the shear rate of the microrheometer to be adjusted. This will be

accomplished by pulling the magnetic cylinder through the fluid in the sample tube with

an electromagnet. By adjusting the current supplied to the electromagnet, the force by

which the magnet is pulled can be changed thus changing the shear rate. The difference

in applied electromagnetic force from that of gravity will be accounted for in a "magnetic

density' term in the viscosity equation derived Cristescu [41].

Further clinical trials need to be conducted to establish viscosity as a reliable

outcome measure for OA. It remains to be determined how sensitive viscosity is to

changes in clinical symptoms. It would be interesting to track the changes in synovial

fluid viscoelasticity over the natural course of disease to better determine how abnormal

lubrication relates to cartilage degeneration. In future work it would useful to complete

more in depth analysis on normal synovial fluid in an attempt to elucidate the properties

that enable it to be such an effective lubricant. The variation in normal and diseased fluid

needs to be examined more closely, so that significant changes in viscosity can more

accurately be detected. In order to accomplish this, it will be necessary to do long term

studies on large groups.

Due to the questions that have been raised concerning many of the previously

conducted clinical trials, much work remains to be done to complete high quality, long

term, independent trials with reliable outcome measures. The NIH has recently funded

such trials and the results will be received with great interest. Considerable work also

remains to elucidate the precise mechanism of action of GCS. Specifically, the









biochemical interactions that take place at the cellular level between GCS and

chondrocytes, synoviocytes and fibroblasts must be explained. Also, the roles of

aggrecan, lubricin and HA are only crudely understood and it must be determined if their

synthesis is affected by GCS. The process of cartilage regeneration is an important area

in the search for a cure for OA and whether GCS is capable of stimulating synthesis or

whether it is merely an analgesic compound is an important question. Such studies will

most likely involve animal models, and therefore more reliable animal models need to be

developed. The current gold standard OA animal model, ligamentously unstable dogs, is

unlikely to accurately reflect the progression of the disease in humans.














APPENDIX A
FALLING CYLINDER THEORY




Formulation of the Problem.

In what follows we use cylindrical coordinates and bodies the

notations shown on the figure we use: p cylinder density, p fluid density, v1 -

velocity of cylinder.

Assumptions made are:

Al. The flow is laminar and telescopic; the velocity components are

V, = vo = 0, v = v = f(r) (A.1)


with r = x2+ y2

A2. The boundary conditions at the cylinder wall

t > r= R,: v(R,)=- v (A.2)

i.e. the fluid adhere to the cylinder wall.

A3. The boundary condition at the tube wall:

t> 0, r = R2: v(R2)= 0 (A.3)

i.e., the fluid adhere to the tube wall.

A4. The fluid is a Newtonian viscous fluid:

T, = 2 7 D, (A.4)









where T is the Cauchy stress deviator, DU is the strain rate tensor and ri the

viscosity coefficient, may be depending on z, if the fluid is nonhomogeneous.



Taking into account (A. 1) the only nonzero strain rate component is


Dr = f' (r) (A.5)
2

and from (A.4) we get for the stresses

T, =Too =T = T z = ToB = 0 T = 1(z)f'(r). (A.6)

Since v is varying with r and assuming that this variation is of the kind shown in

Fig.A. we expect for Tr:

[ positive if R1

Tr, = r(z) is < negative in ro Or

S0 for r = r

if at r = r the velocity reaches a maximum and the stress Tr is zero.



From the equilibrium equations written in cylindrical coordinates, since the

problem is with cylindrical symmetry, follows for our case:

9a 9o 9T 9o T
=- 0, + +- +pb =0 (A.8)
9r 960 r dz r

where b. is the body force component. Thus o(z) depends on z alone, and

from the last equation (A.8) and (A.6) we get









d(r f') (do
Sd+ +-pb
dr ddz


r =0.


Integrating once with respect to r, we get


T, p d b r C,
dz 2 r


Integrating a second time with respect to r we obtain


do r
d z r 2
77f + + pb = C, Ir +C2
d z )4


The integration constants can be determined from the boundary conditions A2 and


(da
+dz


b R2 R
P}) 4


1 R2
Ri


C2 = +dpb
dz


ddo
17 V + d
Idz


Thus the velocity distribution is obtained as


de l R22-r2 1 d l R -R12 r
V= -+pbRzR2 + 1 + p1 In R
dz 4 R2 dz 4 R2
R1 -


(A.13)


if the viscosity coefficient is known and do /d z is the pressure gradient along


the tube.


(A.9)


(A.10)


(A.11)


R 4
4


S R 2 -R 2
+ pb, 24L


R,
1R


In R2


(A.12)









Let us write now a global equilibrium condition for the cylinder: the projection on

the z-axis of all forces acting on the cylinder are zero; i.e. gravitational force + buoyancy

force + shearing force on the cylinder wall = 0. Thus


T ? R c -p+m) (A.14)
Trz R1 2

with m the "local density" of the magnetic force. With (A.9) this relation

becomes

S2 22z
rV1= -+dpbz R1 R2 R -R1 i [g(pc -p+m)] (A.15)
dz 2 R1 4 2 R1


do
which is a relation between 7 v, + pb z and m.
dz




Velocity Profile for Open Tube

First let us consider an open vertical tube (or an "infinite" tube) in which can

move a heavy cylinder and a pressure gradient exists. Let us assign various values to the

pressure gradient d a /d z in order to find out the various possible velocity profiles.



Example 1. From (A. 14) follows that if the pressure gradient satisfies the relation


Ri IR2 [g(c P+m)]
do 2 R1
-+ pb (A. 16)
dz R2 R2 R 2-
2 R1 4









the cylinder is stationary ( v1 = 0). In other words under this pressure gradient the

shearing forces of the fluid flowing upwards will keep stationary the cylinder. Figure A.2

shows an example computed for r = 1.005 N s / m2 (1005 cP) and

da+pbz = 9.36x104 Pam- (A.17)
dz

The velocity distribution shown was obtained with (A. 12).



Example 2. If the pressure gradient is greater than that obtained from (A. 16), the

shearing force of the fluid flowing up is able to move upwards the cylinder. In order to

give an example, Figure A.3 is showing the velocity profile of the fluid for

S+ p b = 1.2 x 10' Pa xm '. If this term reaches the value x 106 Pax m-1 the
dz

velocity of the fluid in contact with the cylinder is equal with that of the cylinder itself.

This case can be obtained from (A. 12) by putting the condition that the maximum

velocity of the fluid be reached for r = R1.



Example 3. This example considers the most common case when term

(d o /d z) + p bz has a smaller value than that obtained from (A. 15). Figure A.4 shows an

example obtained with the value (do /dz)+p bz = 8.0 x 104 Pa m1 and same n as

before. In this case most of the fluid is moving upwards, but a thin layer of fluids

neighboring the cylinder, the fluid is moving downwards. In the present example ? v ?

= 0.000528 m/s (down) is obtained from (A.14).









Example 4. The last example corresponds to very small values for

(d o /d z) + p bz. Let us consider the case when the pressure gradient is zero d a/ d z = 0.

Introducing this value in (A. 14) together with p g = 9.807 x 103 kg /(m2 s2) and same


value for n as above, we get

v = 0.00363 m/s (down). The velocity profile follows from (A.12). This time

the falling cylinder induces into the fluid a shearing stress which will put it to flow

downwards (see Figure A.5). The velocity of the fluid in contact with the tube is

certainly zero.




Velocity Profile for Closed Tube

Let us consider now the case when the bottom of the tube is closed and the

pressure gradient is generated by the falling cylinder (Figrs.A.6 and A.7). We can write

down the condition that the volume of the fluid displaced by the falling cylinder is equal

to the volume of the fluid flowing (up and down) between the cylinder and the tube:

R2
Sv2i r dr = v, R2 (A.18)
R,

Introducing here v (= f) from (A. 11) we get


2f a+pb -+Clrlnr+C2r dr =v, R (A.19)

with the values of C and C2 m (A.14) and the notations
with the values of C, and C, from (A. 14) and the notations









R4- R4R R22 R12 n R In R R R R 22 -R
2 = + R2 -_ R + in -R
16 4nR 2 4 1 2 4) 4 2 n R 2
R, R,


1 I[2n R2 In R 1 In R2 R R R
V =- R-. R +- +Ri (A.20)
InR. R2 2 In 4 R2 2 2
R, R,

we finally obtain

do V
-d +p b, = Iv (A.21)
dz Y

which established another relationship between i7 vl and (do / dz)+ p b,

obtained from a kinematic assumption. However, the term 77 v, as obtained from (A.21)

and from (A. 15) must be equal. From this condition follows

2n [g (p p + m)]
do 2 R
d- + P =b (A.22)
dz R2 R R2 R2
V 2 R, 4

This relation determines (da / d z) + p b The magnetic force is involved as a

"local density", i.e. if pushing down it is equivalent with a heavier cylinder. After

calibration, this observation will be used to speed up some tests with very viscous fluids,

and using the very same magnetic cylinder. Introducing this value into (A.21) we obtain a

formula for the determination of the viscosity coefficient


=- v- d + p b, (A.23)
V v, [dz


We can consider the expression










1g ln
S2 R1
a =-- (A.24)
V R12 R2 R2 R2
V 2 R, 4

to be a viscometer constant. We have introduced / as a correction parameter,

mainly because the cylindrical magnet has sometimes no perfect cylindrical shape, and

also to take into account the end effects. Thus the formula for the determination of the

viscosity coefficient (A.23) writes

Pc p +m
7= =a (A.25)
Ivl


















APPENDIX B
RAW DATA








Table B.1. Raw data from synovial fluid.
Viscosityl
Shear Rate
Study Joint Fluid Temp. Date -
Patient Treatment Number Volume During Test Fluid Appearance Week Aspiration 1 2 5 10 20 40 100 200
Idl p 1 0 3/2/00
Id2 p 1 4 3/27/00
ld3 p 1 12 5/25/00
Id4 p 1 10ml 25.5 clear 19 7/13/00 196.6 196.6 118 98.3 68.8 49.2 31.5 22.6
Change
fhl p 2 6.2ml 25 light yel cloudy 0 3/2/00 196.6 294.9 157.3 118 78.6 59 35.4 26.5
fh2 p 2 6.2ml 24.5 lightyelcloudy 4 3/27/00 196.6 196.6 118 98.3 59 44.2 29.5 21.6
Drop Out due to
fh3 ** TKA ***
Change _0 -98.3 -39.3 -19.7 -19.6 -14.8 -5.9 -4.9
kal qcs 3 .5 ml 25.5 palepink 0 4/4/00 1179.6 786.4 550.5 393.2 275.2 181.9 106.2 69.8
ka2 qcs 3 1.5ml 25.5 brightred 6 5/16/00 786.4 688.1 471.8 334.2 235.9 157.3 92.4 60.9
ka3 gcs 3 13 7/17/00
ka4 qcs 3 20 9/7/00
Change -393.2 -98.3 -78.7 -59 -39.3 -24.6 -13.8 -8.9
red dry not enough
frl gcs 4 trace to test 0 4/4/00
pale yel. Slightly
fr2 gcs 4 8.2 ml 25 cloudy 6 5/16/00 393.2 294.9 196.6 137.6 88.5 59 37.4 25.6
fr3 gcs 4 2.3cm 25.5 paleyel. Clear 26 9/19/00 196.6 196.6 118 98.3 68.8 49.2 33.4 23.6
Change -196.6 -98.3 -78.6 -39.3 -19.7 -9.8 -4 -2
id1 qcs 5 1-2 drops reddish MR 0 4/13/00
jd2 gcs 5 4.5ml 25.5 yel. Fairly clear 12 6/22/00 393.2 393.2 275.2 196.6 137.6 98.3 61 41.3
jd3 gcs 5 1.3cm 26 yel. Very clear 21 9/7/00 196.6 98.3 78.6 59 49.2 34.4 27.5 20.6
Change -196.6 -294.9 -196.6 -137.6 -88.4 -63.9 -33.5 -20.7
aal gcs 6 0 0 4/18/00
aa2 gcs 6 5.0ml 24.5 paletranslu. Yel. 6 6/1/00 983 688.1 471.8 314.6 196.6 122.9 68.8 45.2
aa3 gcs 6 2.8cm 26.5 paletranslu. Yel. 14 7/31/00 196.6 196.6 118 98.3 78.6 63.9 43.3 31.5
Change -786.4 -491.5 -353.8 -216.3 -118 -59 -25.5 -13.7
aa4 cs 6 2.8cm 25.5 paletranslu. Yel. 19 8/29/00 196.6 196.6 117.96 98.3 88.47 63.6 45.22 33.4
Change -786.4 -491.5 -353.8 -216.3 -108.1 -59.3 -23.58 -11.8













wzI non
study knee 8 9 ml 25.5 lightyel 0 5/2/00 196.6 196.6 118 78.6 59 44.2 27.5 19.7

wz1 gcs 8 7.7 ml 24.5 lightyel 0 5/2/00 983 688.1 471.8 314.6 196.6 122.9 68.8 45.2
wz2 gcs 8 5.5 ml 25.5 lightyel 4 6/6/00 786.4 491.5 353.9 235.9 147.5 98.3 57 37.4
Change -196.6 -196.6 -117.9 -78.7 -49.1 -24.6 -11.8 -7.8
light yel, ver slight
wz3 ,cs 8 5.1 cm 24 pink tinge 12 8/15/00 196.6 196.6 157.3 118 88.5 68.8 45.2 31.5
Change -786.4 -491.5 -314.5 -196.6 -108.1 -54.1 -23.6 -13.7
wz4 gcs 8 3.5 cm 26 lightyel 23 10/12/00 786.4 589.8 393.2 255.6 167.1 108.1 61 40.3
Change -196.6 -98.3 -78.6 -59 -29.5 -14.8 -7.8 -4.9
pale yel. Slightly
dwl qcs 9 3.8 ml 26 cloudy 0 5/4/00 598.8 393.2 314.6 216.3 137.6 93.4 57 38.3

dw2 ,cs 9 1.5 ml 26 very pale yel. Clear 4 6/1/00 786.4 688.1 432.5 314.6 206.4 132.7 74.7 49.2
Change 187.6 294.9 117.9 98.3 68.8 39.3 17.7 10.9
dw3 gcs 9 0 8 6/30/00
pale yel. Clear.
Bubbles due to
dw4 qcs 9 .5cm 26.5 small volume 19 9/14/00 1376.2 983 598.8 373.5 245.8 152.4 84.5 55
Change 777.4 589.8 284.2 157.2 108.2 59 27.5 16.7
bml 0cs 11 0 0 6/6/00
red clear must test
bm2 gcs 11 .3cm on MR 4 7/10/00
clear must test on
bm3 qcs 11 .4cm MR 13 9/19/00

pale yel clear must
bm4 gcs 11 .4cm be tested on MR 20 11/14/00
light pink
cml p 12 1.7 ml 25 somewhat cloudy 0 6/20/00 1769.4 1277.9 786.4 530.8 353.9 226.1 125.8 80.6
yel/orange slighty
cm2 p 12 1.7 cm 25 cloudy 11 9/1/00 196.6 196.6 118 118 88.5 73.7 53.1 41.3
Drop Out at
cm4 ***** *** request of patient **
Change -1572.8 -1081.3 -668.4 -412.8 -265.4 -152.4 -727 -39.3
pale yel clear
ibl p .7cm 26 *reused for MR 0 4/12/01 786.4 589.8 353.9 255.6 176.9 118 68.8 45.2
b2 p .6cm 25.5 pale elclear 4 5/10/01 786.4 589.8 393.2 275.2 186.8 122.9 74.7 49.2
Change 0 0 39.3 19.6 9.9 4.9 5.9 4
ssl gcs 1.2 cm 26 pale orange clear 0 4/12/01 589.8 393.2 275.2 176.9 118 78.6 45.2 30.5
ss2 qcs 2.2 cm 25.5 pale orange clear 4 5/10/01 589.8 393.2 235.9 176.9 108.1 73.7 43.3 29.5
Change 0 0 -39.3 0 -9.9 -4.9 -1.9 -1
w1 p 15 .7cm 26 paleyel. Clear 0 4/13/01 196.6 196.6 157.3 118 88.5 63.9 39.3 28.5

very pale yel. Clear
w2 p 15 .4cm must test on MR 4 5/11/01
bubbles due to
al gcs 17 .6cm 25.5 small vol 0 4/13/01 589.8 393.2 235.9 176.9 108.1 73.7 43.3 29.5
a2 qcs 17 1.0cm 25 red/orange clear 4 1376.2 1081.3 707.8 471.8 304.7 191.7 106.2 66.8
Change 786.4 688.1 471.9 294.9 196.6 118 62.9 37.3














1000-


1 10 100
Shear Rate (1/s)



Figure B.1. Viscosity versus shear rate for patient Id.


1000


1000


1 10 100 1000
Shear Rate (11s)


Figure B.2. Viscosity versus shear rate for patient fh (fh withdrew from study due to
receiving a total knee replacement after week 4).


-4- Week 19








58






10000


-*- week 0
0-- week 6
1000


4L

'7 100

0


10




1 -
1 10 100 1000
Shear Rate (1/s)


Figure B.3. Viscosity versus shear rate for patient ka.








1000

-*-week 6



100
U


0

10-






1
1 10 100 1000
Shear Rate (1/s)


Figure B.4. Viscosity versus shear rate for patient fr.
















1000






- 100
0

o

1








10
8f
(lO


1000


Shear Rate (1/s)


Figure B.5. Viscosity versus shear rate for patient jd.


Shear Rate (1/s)


Figure B.6. Viscosity versus shear rate for patient aa.


- week 12
week 21


-week 6
-*-week 14
w--eek 19


I I















1000






100


o
C,


10








1


1 10 100
Shear Rate (1/s)


Figure B.7. Viscosity versus shear rate for patient wz (the only patient for whom
synovial fluid samples are available at all four time points).


1 10 100
Shear Rate (1/s)


1000


Figure B.8. Viscosity versus shear rate for patient dw.














10000




1000


Shear Rate (1/s)


Figure B.9. Viscosity versus shear rate for patient cm (Patient withdrew from study due
to lack of efficacy of treatment).


1000





100


0







1


1 10 100
Shear Rate (1/s)


Figure B.10. Viscosity versus shear rate for patient jb.


-*week 0
- week 11/















1000





- 100


o
0

10





1


10 100
Shear Rate (1/s)


Figure B.11. Viscosity versus shear rate for patient ss.


1000


Shear Rate (1/s)


Figure B.12. Viscosity versus shear rate for patient w.


1000


-*-Week 0














10000




1000


1 10 100
Shear Rate (1/s)


Figure B.13. Viscosity versus shear rate for patient a.


1000













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68


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BIOGRAPHICAL SKETCH

The author was born in Little Rock, Arkansas to an Air Force navigator and an

emergency room nurse. He has one very special sibling, an incredibly intelligent and

beautiful sister named Annie. After the wonderful experience of living overseas for four

years, the author's family settled down in Charleston, South Carolina. It was here that

the author was introduced to many of the things that have shaped his life. He turned to

Jesus Christ as his personal Savior and was later baptized at Old Fort Baptist Church. It

was at this church that he met his future wife, Suzanne. The author was married on May

27, 2000, midway through his graduate school studies. His wife has been a constant

source of inspiration and a fountain of encouragement. Blessings may come and go out

with the tide but her love will always be by my side.




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