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DIETARY SUPPLEMENTS AND THEIR EFFECT ON
STRESSED CARTILAGE EXPLANTS

presented by

Robert Harlan

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DIETARY SUPPLEMENTS AND THEIR EFFECT ON
STRESSED CARTILAGE EXPLANT S

By

Robert Harlan

A THESIS

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE
Animal Science

2006

ABSTRACT

DIETARY SUPPLEMENTS AND THEIR EFFECT ON STRESSED
CARTILAGE EXPLANT S

By Robert Harlan

We performed two experiments: for the ﬁrst experiment, three culture schemes:
1) Interleukin-1 (IL-1) (50 ng/ml) treatment with and without GLN + CS for 48 h (short-
term non-impact), 2) IL-1 plus mechanical trauma (15 mega pascal) with GLN + CS for
48 h (short-term impact), 3) two-week cultures were cultured with GLN + CS and were
exposed to IL-1 (50 ng/ml) on day 2 and day 10 (long-term non-impact). For the second
experiment, SAMe was tested at concentrations of 1.0, 0.1, and 0.01 ug/ml (high,
medium, and low dose) and ASU was tested at concentrations 10.0, 1.0, and 0.1 ug/rnl

(high, medium, and low dose). Interleukin-l (IL-1) at 15 ng/ml was used to initiate

inﬂammatory stress. Nitric oxide (NO) and prostaglandin E2 (PGEZ) were measured as

indicators of inﬂammatory response for both experiments. Glycosaminoglycans (GAG)

were measured as an indicator of cartilage turnover for both experiments.

For the ﬁrst experiment, GLN + CS did not affect NO, PGEz, or GAG

concentrations in the non-impact short-term model. When mechanical impact was
combined with IL-1 , the NO concentration in the GLN + CS treatment was lower than the
IL-1 treatment and did not differ from control. The GAG release was also lower in the

GLN + CS treatment than in the IL-1 treatment. The non-impact long-term cultures

demonstrated that GLN + CS treatment did not affect NO or PGEZ concentration or GAG

content in the explants at termination. The second experiment showed no signiﬁcant

treatment effects for either SAMe or ASU.

Capyrigh‘t by
Robert Harlan
2006

I would like to dedicate this thesis to my parents for all their unconditional love and
support.

iv

ACKNOWLEDGEMENTS

I would like to thank the members Dr. Orth’s laboratory, past and present, for all
of their help and support. I would like to thank Cara O’Conner for all the help with
experiments, spread sheets, and generally bringing my research from chaos to
organization. Thanks you to Peggy Wolf for her help and training and for all the good
lunch conversations that we had. Thanks also to Pooi-See Chan for answering my
questions and training me with RT-PCR. Thanks to Kirsten Neil for her help with RT-
PCR and giving me primers when I needed them. Thanks to John Wheeler for all his
help with assays, cleaning my lab bench, and cracking jokes. Thanks also to Stacy
Endres and Jessica Partlow for all their help with assays. I would also like to thank all of
my graduate student friends that I have known through the Animal Science Graduate
Student Association for their friendship.

Thank you to Dr. Orth for all his guidance and for giving me the opportunity to
study and perform research in his laboratory and for being available and encouraging me
along the way. Thank you also to Dr. Nielsen, Dr. Bursian, and Dr. Caron for serving on
my committee. Thank you to Dr. Tempelmen for helping me with all the statistical
analysis of the project.

I would like to thank my friends from Spartan Christian Fellowship, Grace
Immanuel Reformed Baptist Church, and the Grace Orthodox Presbyterian Church for
their friendship, prayers and spiritual support. I would also like to thank my parents for

their unconditional love and support through the good times and the bad. Finally, I

would like to thank God, my heavenly father, for giving me the strength to make it

through when my strength was gone.

vi

TABLE OF CONTENTS

LIST OF TABLES ................................................................... ix
LIST OF FIGURES ................................................................... x
LIST OF ABBREVIATIONS ....................................................... xi
INTRODUCTION ..................................................................... 1
CHAPTER 1 .................................................................................................... 4
LITERATURE REVIEW ................................................................................ 4
The importance to the horse industry .................................................... 4
Articular cartilage .................................................................................. 4
Chondrocytes ........................................................................................ 5
Extra cellular matrix ................................................... 6
Osteoarthritis ................................................................... 7
Onset of disease ........................................................ 7
Inﬂammatory mediators .............................................. 8
Treatment ....................................................................... 9
Conventional treatments .............................................. 9
Glucosamine and chondroitin sulfate .............................. 10
S-adenosylmethionine ................................................ l4
Avocado/soybean unsaponiﬁables ................................. 15
Rationale for experiments ................................................... 16
References ..................................................................... 18
CHAPTER 2 .......................................................................... 26
The Effect of Biologically Relevant Concentrations of Glucosamine and
Chondroitin Sulfate on Stressed Equine Cartilage Explants ................... 26
Summary ....................................................................... 26
Introduction .................................................................... 27
Materials and Methods ...................................................... 28
Results .......................................................................... 31
Discussion ..................................................................... 32
References ..................................................................... 49
CHAPTER 3 .......................................................................... 52

vii

The Effect of Avacado and Soy Unsaponiﬁables and S-Adenosyl-L-

Methionine on Inﬂammatory Mediators in Bovine Cartilage .................. 52
Summary ...................................................................... 52
Introduction ................................................................... 53
Materials and Methods ...................................................... 55
Results ......................................................................... 57
Discussion ..................................................................... 57
References ..................................................................... 69

viii

LIST OF TABLES

Chapter 2
Table I. Agents added to short-term explant cultures .......................... 38
Table 11. Agents added to long-term explant cultures ........................... 39

Table III. Mean NO concentration (ng/ml) 2!: SE with n=3 for d 4, 12, and
cumulative over 14 days ............................................................ 46

Table IV. Mean GAG concentration (ng/ml) i SE with n=3 for d 4, 12, and

cumulative over 14 days ............................................................ 47
Chapter 3

Table 1. Agents added to explant cultures-explants #1 ........................ 61

Table 11. Agents added to explant cultures-explants #2 ........................ 62

ix

LIST OF FIGURES

Chapter 2

Fig 1: Mean (iSE, n=6 for medium dose, n=4 for low dose) NO, GAG, and
PGE2 concentrations in media samples at day 0 and 1 (A, C) or at day 1 (B).
IL-1 (50 ng/ml) was added to each treatment except for control. The
experimental treatments contained 5 ug/ml GLN + 20 ug/ml CS (medium
dose) or 1 ug/ml GLN + 5 itng CS (low dose) ................................. 40

Fig 2: Mean (:tSE, n=3) NO, PGEZ, and GAG concentrations in media
samples at day 0 and 1 (A, C) or at day l (B). IL—1 (50 ng/ml) + Impact was
added to each treatment except for control. The experimental treatment

contained 5 ug/rnl GLN + 20 ug/ml CS (medium dose) ....................... 43
Fig 3: Mean (iSE, n=3) ﬁnal GAG concentration in papain digest of
cartilage explants at day 14. ....................................................... 48
Chapter 3

Fig I: Mean (iSE, n=4) NO, PGEZ, and GAG concentrations in media
samples at day l (A-C). IL-1 (15 rig/ml) was added to each treatment except
for control. The experimental treatments contained 1 ug/ml SAMe (high
dose); 0.1 ug/ml SAMe (medium dose); 0.01 ug/ml SAMe (low dose); 0.3%
EthOH (EthOH control); 10 ug/ml ASU (high dose); or 1 ug/ml ASU
(medium dose) ........................................................................ 63

Fig 2: Mean (iSE, n=4) NO, PGEZ, and GAG concentrations in media
samples at day 1 (A-C). IL-1 (15 ng/ml) was added to each treatment except
for control. The treatments contained: 0.3% EthOH (EthOH control); 10
ug/ml ASU (high dose); 1 ug/ml ASU (medium dose); 0.01 ug/ml ASU
(low dose) ............................................................................. 66

LIST OF ABBREVIATIONS

A disintegrin and metalloproteinase with thrombospondin motifs .................. ADAMTS
Avocado and Soy Unsaponiﬁables ............................................................. ASU
Chondroitin Sulfate ................................................................................. CS
Cyclooxygenase ................................................................................. COX
Cyclooxygenase-l ............................................................................. COX-l
Cyclooxygenase-2 ............................................................................. COX-2
Dirnethylene blue ............................................................................... DMB
Enzyme-Linked Irnmunosorbent Assay ..................................................... ELISA
Fetal bovine serum ................................................................................ FBS
Glucosamine ...................................................................................... GLN
Glycosaminoglycan .............................................................................. GAG
Inducible nitric oxide synthase ................................................................ iNOS
Insulin transferrin selenite ........................................................................ ITS
Interleukin-l ....................................................................................... IL-l
Microsomal prostaglandin E synthase-1 .................................................. mPGEsl
Mega pascal ....................................................................................... MPa

Matrix metalloproteinase ....................................................................... MMP
Nitric Oxide ......................................................................................... NO
Nonsteroidal anti-inﬂammatory drugs .................................................... NSAIDs
Osteoarthritis ........................................................................................ OA
Prostaglandin E2 ................................................................................. PGE2

xi

S-Adenosyl-L-Methionine ..................................................................... SAMe

Tumor necrosis factor-alpha ...................................................................... TNFa

xii

INTRODUCTION

Osteoarthritis (0A) is a degenerative joint disease marked by progressive
cartilage tissue loss, pain, inﬂammation, and eventual loss of joint function (F elson et a1.
1998; Hashimoto er al. 1998). The signiﬁcance of 0A to the horse racing industry is that
it results in lameness. The onset of OA can be initiated by normal exercise of the animal
(Stashak 2002). Since no known way to cure or prevent OA exists, better therapies are
needed. In recent years, dietary supplements (now known as nutraceuticals) have been
used to treat 0A in both humans and animals. Glucosamine (GLN) and chondroitin
sulfate (CS), S-adenosyl-L-methionine (SAMe), and avocado soy unsaponiﬁables (ASU)
have been among some of the dietary supplements taken for joint pain.

This project consisted of two objectives: 1) our ﬁrst objective was to test the anti-
inﬂarnmatory effects of biologically relevant concentrations of GLN plus CS in three
different equine cartilage cultures that included conditions for inducing stress; 2) The
second objective was to test concentrations of ASU and SAMe in bovine cartilage
explant cultures for their ability to mitigate cytokine-induced stress. Explant tissue
culture provides an excellent system for testing chondroprotective agents because of the
convenience and accuracy of the data collection. Mechanical loading is known to be a
risk factor for CA in horses and the combination of GLN and CS at biologically relevant
concentrations have neverbeen tested in a mechanical loading tissue culture system.

In the ﬁrst series of experiments, we provide evidence that biologically relevant
concentrations of GLN + CS may be effective against harmful inﬂammatory responses to

trauma in equine cartilage tissue. We exposed equine cartilage explant cultures to three

different types of conditions: 1) a short-term non-impact culture system utilizing
(interleukin-1) IL-1 (50 ng/ml) as the inducer of inﬂammatory stress with and without
GLN + C8, 2) a short-term impact utilizing IL-l plus mechanical trauma to induce
inﬂammatory stress with and without GLN + CS 3) a two-week culture (long term) with
continuous exposure to GLN + CS and introduction of IL-1 (50 ng/ml) on day 2 and day
10.

In the second series of experiments, we used the short-term culture system
mentioned above to test varying concentrations of SAMe and ASU in bovine explants.
Both experiments measure tissue inﬂammatory and breakdown products as the response
variables. Similar types of culture systems have been used in our lab previously (Chan et
al. 2005a; Chan et al. 2005b; Chan et al. 2005c), and in this study we use an in vitro
cartilage explant approach to test GLN and CS, SAMe, and ASU and their ability to

mitigate inﬂammatory responses.

REFERENCES

Chan, P.S., Caron, J .P. and Orth, M.W. (2005a) Effect of glucosamine and chondroitin
sulfate on regulation of gene expression of proteolytic enzymes and their
inhibitors in interleukin-l-challenged bovine articular cartilage explants. Am J Vet
Res 66, 1870-1876. '

Chan, P.S., Caron, J .P., Rosa, G.J. and Orth, M.W. (2005b) Glucosamine and chondroitin
sulfate regulate gene expression and synthesis of nitric oxide and prostaglandin
E(2) in articular cartilage explants. Osteoarthritis Cart 13, 387-394.

Chan, P.S., Schlueter, A.E., Coussens, P.M., Rosa, G.J., Haut, RC. and Orth, M.W.
(2005c) Gene expression proﬁle of mechanically impacted bovine articular
cartilage explants. J Orthop Res 23, 1 146-1151.

Felson, D.T. and Zhang, Y. (1998) An update on the epidemiology of knee and hip
osteoarthritis with a view to prevention. Arthritis Rheum 41, 1343-1355.

Hashirnoto, S., Ochs, R.L., Rosen, F., Quach, J ., McCabe, G., Solan, J., Seegmiller, J .E.,
Terkeltaub, R. and Lotz, M. (1998) Chondrocyte-derived apoptotic bodies and
calciﬁcation of articular cartilage. Proc Natl Acad Sci U S A 95, 3094-3099.

Stashak, TS (2002) Examination for lameness. In: Adams' Lameness in Horses, 5 edn.,
Ed: D. Troy, Williams and Wilkins, Philadelphia. pp 113-183.

Chapter 1
Literature Review
Importance of dietary supplements to the horse industry

The American Horse Council reported that the equine industry provides 1.4
million full-time jobs in the US. and that there are 9.2 million horses nation-wide (2005).
The same report stated that the direct contribution of the horse industry to the US.
economy is $39.9 billion annually. Also, they found that 4.6 million people are involved
in the equine industry in a professional or volunteer capacity.

The signiﬁcance of osteoarthritis (CA) to the racing industry is that OA results in
lameness, reduced performance, and wastage (Murray et a1. 2005); and one of the main
factors that determines the value and usefulness of a race horse is soundness of the legs
(Jeffcott et al. 1982; Rossdale et al. 1985; Fubini et al. 1999). Lameness is the most
signiﬁcant limiting factor for race horses; the greatest numbers of days lost to training are
caused by lameness (Rossdale et al. 1985), and failure to race is most commonly due to
lameness (Jeffcott et al. 1982).

The presence of 0A is pervasive in the American horse population. A survey of
veterinary schools found that at veterinary schools, 33% of equine patients had OA
lesions (Rose 1977). In another survey of 140 2-year-old Thoroughbred horses, only
23% were sound (Jeffcott et a1. 1982). The onset of OA can be initiated by injuries
sustained during normal exercise normal exercise of the animal and has been linked to
activities such as barrel racing, pole bending, dressage, and other types of competions
(Stashak 2002). Since no known way to cure or prevent OA exists, better therapies are

needed.

Articular Cartilage
Chondrocytes

Articular cartilage tissue at the ends of long bones provides a frictionless surface
that allows for joint mobility. Cartilage also provides a shock-absorbing cushion that
protects the ends of bones. The tissue is composed of chondrocytes, which are
surrounded by an extracellular matrix. These cells only constitute 1% of the tissue
volume (Buckwalter and Lane 1997). In healthy cartilage, chondrocytes are capable of
breaking down and synthesizing extracellular matrix to maintain a stable equilibrium of
catabolism and anabolism (Bullough 1992a). Chondrocytes are also capable of
responding to mechanical stimuli, which cause these cells to modulate their metabolism
and matrix production to meet the demands of external forces (Buschrnann and
Grodzinsky 1995). Extracellular matrix mediates cell signaling and maintenance of
phenotype (Bullough 1992a; Blanco et al. 1995). This is substantiated when
chondrocytes are removed from the extracellular matrix and cultured in a monolayer, the
cells begin to lose their spherical morphology and chondrocyte-speciﬁc markers (Domm
et al. 2004; Marlovits et al. 2004). Adult cartilage tissue is avascular and therefore
chondrocytes absorb nutrients from the synovial ﬂuid, which ﬂows through the matrix as
a result of mechanical forces on the joint (Mankin 1984). Chondrocytes absorb oxygen
solely from the synovial ﬂuid and carry on a combination of aerobic and glycolytic
metabolism (Clegg et al. 2006).

Chondrocytes are organized into four distinct zones within the cartilage:

superﬁcial, transitional, radial, and calciﬁed zones. Within the superﬁcial zone,

chondrocytes have a ﬂattened morphology and are arranged randomly within the matrix
(Buckwalter and Mankin 1997). In the transitional zone, they are spherical and are also
arranged in a random pattern in the matrix (Buckwalter and Mankin 1997).
Chondrocytes in the radial zone are arranged in columns and also demonstrate a spherical
morphology (Buckwalter and Mankin 1997). Chondrocytes in the calciﬁed zone have a

spherical shape and are arranged in a random pattern in the matrix (Bullough 19923).

Extracellular matrix

The extracellular matrix of cartilage consists largely of collagen surrounded by
proteoglycans. Collagen gives cartilage its tensile strength and provides a rigid
framework, which prevents the tissue from swelling to its fullest extent (Maroudas 1976).
Collagen ﬁbrils have characteristics appropriate for the each zone of cartilage tissue
(Bullough 1992b). Collagen ﬁbrils are arranged parallel to the surface in the superﬁcial
zone while in the transitional zone the ﬁbrils appear to be randomly arranged (Bullough
1992b). In the radial zone, the collagen ﬁbrils are arranged perpendicular to the articular
surface (Bullough 1992b). The collagen ﬁbrils in the calciﬁed zone however, are
calciﬁed by the surrounding chondrocytes (Bullough 1992b). Most of the collagen in the
matrix is type II, but type, VI, IX, and XI are also present (Bruckner 1994).

Proteoglycans consist of a protein core with glycosaminoglycan (GAG) side
chains in a radial arrangement giving the molecule a “bottle brush” type structure (10220
1998). The speciﬁc proteoglycan that is produced by chondrocytes is called aggrecan,
which contains chondroitin sulfate (CS) as the predominant GAG side chain. This GAG

is composed of repeating disaccharide units of N-acetyl galactosarnine and glucuronic

acid residues. The negatively charged sulfur groups of this disaccharide allow hydrogen
bonding with water molecules. The resulting hydration endows aggrecan with the
cushioning properties that are typical of articular cartilage (Prydz and Dalen 2000). As
mechanical forces are applied to the cartilage tissue, ﬂuid is expelled from between the
aggrecan side chains and from the tissue. As the force is removed, ﬂuid returns to the
matrix and re-hydrates the negatively charged aggrecan side chains, thus yielding the

compressive elasticity and shock absorbing properties of the cartilage matrix.

Osteoarthritis
Onset of disease

Osteoarthritis is a degenerative disease marked by progressive cartilage tissue
loss, pain, inﬂammation, and eventual loss of joint function (Felson et al. 1998;
Hashirnoto et al. 1998). When injury or disruption to a joint occurs, enzymes are
synthesized that break down the proteoglycan component of the matrix (Spiers et al.
1994a; Spiers et al. 1994b). Chondrocytes respond to this stress by proliferating and
increasing proteoglycan synthesis; but this response is sometimes insufﬁcient to
overcome the degradation. As a result, the equilibrium is shiﬁed toward a gradual loss of
total proteoglycan, leading to cartilage destruction (Morales and Roberts 1988; Hedbom
and Hauselmann 2002).

Onset of OA may be initiated by trauma or chronic mechanical stress (Quinn et
al. 2001; Patwari et al. 2003). 0A in horses is believed to usually be the result of trauma
resulting from over use of the joint (Mackay-Smith 1962). Factors that can induce OA

include intense exercise and fatigue (Hodgson and Rose 1989). The pressure on a joint

that a race horse can generate during a race can be millions of foot-pounds per mile
Wackay-Smith 1962). Another risk factor in horses is subchondral bone sclerosis, which
results from excessive bone remodeling in response to mechanical forces on the bone
(Kawcak 2000). Cartilage that is covering areas of the bone affected by sclerosis is
vulnerable to the development of OA because the subchondral bone changes shape and
increases in density (Kawcak et al. 2001).

Acute trauma to a joint can also lead to OA. Also injury to the anterior cruciate
ligament can cause OA (Roos et al. 1995). For example, damage to intra-articular
ligaments can lead to 0A. This has been demonstrated in dogs, rabbits, and in horses by
transection of the cranial cruciate ligament and lateral collateral and lateral collateral
sesamoidean ligaments in horses, which leads to destabilization of the joint and cartilage
wear (Pond and Nuki 1973; Troyer 1982; Simmons et al. 1999; Clegg et al. 2006).
Injury to the meniscus is also a common cause for joint destabilization leading to 0A in
humans (Roos et al. 1998; Clegg et al. 2006). Increased concentrations of proteoglycan
fragments accumulate in the synovial ﬂuid after injury to the anterior cruciate ligament or
meniscus (Lohmander et al. 1989). Stromelysin, an enzyme that breaks down cartilage,

can increase up to 40-fold over time in an injured joint in (Lohmander et al. 1993).

Inﬂammatory mediators

Joint injury is often accompanied by an inﬂammatory response that includes the
production of pro-inﬂammatory cytokines such as interleukin-1 beta (ILal). This
cytokine is secreted by macrophages, monocytes, and synoviocytes (Kirker-Head 2000;

Femandes et al. 2002) and is implicated in the inﬂammatory symptoms of OA. Another

example of an inﬂammatory cytokine that is implicated in 0A is tumor necrosis factor-
alpha (TNFu) (Hardingham et al. 1992; Fernandes et al. 2002; Lopez-Armada et al.
2006), which is produced by macrophages and synoviocytes. The inﬂammatory activity

of IL-1 is mediated by signaling molecules such as nitric oxide (NO) and prostaglandin

E2 (PGEZ) (Smalley et al. 1995; Grabowski et al. 1997). IL-l inhibits proteoglycan

synthesis and increases proteoglycan breakdown. When inﬂammation in the joint is
prolonged, it leads to a chronic imbalance in tissue turnover, which results in the eventual
destruction of the cartilage tissue (Hardingham et al. 1992; Goldring et al. 1994). The
imbalance is mediated by enzymes in the joint called matrix metalloproteinases (MMPs)
and a disintegrin and metalloproteinase with a thrombospondin type 1 motif (ADAMTS),
which are responsible for breakdown of matrix components such as aggrecan and
collagen (Struglics et al. 2006). Some examples of enzymes of these types that have
been implicated in OA include, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5

(Patwari et al. 2003; Neil et al. 2005; Struglics et al. 2006).

Treatment
Conventional treatments

Conventional treatments for CA include nonsteroidal anti-inﬂammatory drugs
(NSAIDs) and intra-articular corticosteroids (Hochberg et al. 1995b, 1995a). These
treatments are effective for decreasing joint pain, but they do not affect the progression of
the disease. They also have signiﬁcant side effects (Hochberg et al. 1995a).
Nonsteroidal anti-inﬂammatory drugs, amoung other things, inhibit two enzymes known

as cyclooxygenase-l and -2 (COX-1 and COX-2) (Harkins et al. 1993). Both of these

enzymes produce PGEz, which mediates pain and inﬂammation in the joint. However,

COX-1 is also an important enzyme for maintaining the gastrointestinal lining. As a
result of COX-1 inhibition, NSAIDs eventually cause gastrointestinal ulceration if they
are taken long-term, which results in thousands of deaths each year (Hochberg et al.
1995a; Griffin 1998). A common NSAID used in horses, called phenylbutazone, is
associated with gastric ulcers, weight loss, and diarrhea (T raub et al. 1983). The
development of a COX-2-speciﬁc inhibitor seemed to be a signiﬁcant advance promising
to become a NSAIDs treatment without signiﬁcant side effects. Inhibiting COX-2
exclusively, however, resulted in cardiovascular side effects in patients. Clinical trials of
some of these drugs demonstrated that adverse cardiovascular events were '3 times more
frequent after coronary bypass surgery when COX-2 inhibitors were administered (Ott et
al. 2003; Nussmeier et al. 2005). Reports also detail increased risk of cardiovascular

events and serious skin reactions (Sibbald 2005; Talhari et al. 2005). This cardiovascular

effect may be because COX-2 produces prostaglandin 12 in vascular epithelial tissue,

which inhibits platelet aggregation, neutrophil adhesion, and dilatation of bronchial and
vascular smooth muscles (Lin 2005). Modulation of such physiological processes could
cause thromboembolic events, which is a risk especially to patients with a history of
cardiovascular disease (Fitzgerald 2004; Warner et al. 2004).

Synthetic corticosteroids can be injected intra-articularly for treatment of OA and
have shown efficacy in dramatically decreasing inﬂammation in horses and in humans
(Harkins et al. 1993; Bellamy et al. 2005). These treatments also decrease the production
of prostaglandins by COX-2 enzymes (Moses et al. 2001; Frean et al. 2002; Tung et al.

2002). The side effects are eventually damaging to the cartilage, which may include the

10

formation of calcium deposits on the surface of the cartilage, ﬁssuring of the cartilage,

and decreased cartilage elasticity (Harkins et al. 1993).

Glucosamine and chondroitin sulfate

In recent years, dietary supplements (now known as nutraccuticals) have been
used to treat OA in both humans and animals. Glucosamine (GLN) and CS have been
among the most popular supplements. GLN is a precursor molecule of various
components of the cartilage matrix (Hamennan 1989; Muller-Fassbender et al. 1994).
Chondroitin sulfate is a component of the cartilage proteoglycan called aggrecan (Conte
et al. 1991). Some evidence suggests that glucosarnine is as effective as ibuprofen at
relieving pain (Muller-Fassbender et al. 1994) and that the combination of GLN and CS
is effective at relieving moderate to severe joint pain in OA patients (Clegg et al. 2006).
GLN has an excellent safety proﬁle, when taken orally, based on studies done in humans,
dogs, and rats (Pujalte et al. 1980; Tapadinhas et al. 1982; Muller-Fassbender et al.
1994). CS also has an excellent safety proﬁle in humans, dogs, and rats (Conte et al.
1995; Bucsi and Poor 1998; Ronca et al. 1998; Volpi 2002). Dosing in humans is usually
1500 mg of GLN and 1200 mg of CS daily (Clegg et al. 2006) and in horses the dosage is
9 g of GLN and 3 g of CS (Du et al. 2004).

Work has been done in animal models to show the effectiveness of these agents.

For instance, work done in cultures systems with IL-1 stimulated cartilage explants from

Holstein steers show that GLN and CS will decrease cartilage NO and PGE2 synthesis at

concentrations found in the blood (Chan et al. 2005b). Another study used ﬁbronectin

fragments to initiate cartilage degradation in bovine cartilage explants and showed that

11

mixture of GLN and CS at biologically relevant concentrations was effective at
decreasing expression of catabolic mediators MMP-3 and MMP-13 and promoting repair

(Homandberg et al. 2006). Studies in equine cartilage explant cultures have shown that

GLN and CS are effective at decreasing NO, PGEz , MMP-9, and MMP-13, though the

concentrations used here were higher than what is normally found in viva (Orth et al.
2002). Studies in equine chondrocyte cell culture (pellet culture) have shown that
relevant concentrations of GLN decrease expression of catabolic mediators such as
MMP-13, aggrecanase-1, inducible nitric oxide synthase (iNOS), and COX-2 (Neil et al.
2005)

The mechanism of action of these two substances has received signiﬁcant
attention lately. Based on the biochemistry of GLN and CS and GAG synthesis is
examined, it appears feasible that these molecules could increase the synthesis of GAG
(Mch et al. 2000). McCarty et al. proposed that GLN supplementation provides the
rate-limiting substrate for GAG (McCarty et al. 2000). Evidence for this theory was
demonstrated by a study in which over-expression of glutamine fructose-6-phosphate
aminotransferase, an important rate limiting enzyme in the synthesis of GLN, caused
resistance to IL-1 inhibition of GAG synthesis (Gouze et al. 2001). In a study involving
dogs with a transected cranial cruciate ligament, a combination of GLN and CS increased
GAG synthesis (Johnson et al. 2001). One of the mechanisms for the preservation of

cartilage tissue by GLN maybe the inhibition of aggrecanase (Patwari et al. 2003; Chan

et al. 2005a). GLN alone decreases IL-l -induced production of NO and PGE2 and IL-1-

induced proteoglycan breakdown (F enton et al. 2000; Gouze et al. 2001; F enton et al.

2002; Chan et al. 2005b). The mechanism of decreasing proteoglycan breakdown is in

12

part due to the inhibition of MMPs, aggrecanases, and collagenase (Fenton et al. 2000;
Piperno et al. 2000; Fenton et al. 2002; Dodge and Jimenez 2003; Chan et al. 2005a).
GLN can also decrease the gene expression of MMP-1, MMP-3, and MMP-13 (Byron et
al. 2003; Dodge and Jimenez 2003; Chan et al. 2005a). CS May stimulate GAG
production by binding to membrane proteins of synovial cells (Mch et al. 2000). CS
increase proteoglycan synthesis and decreases proteoglycan breakdown (Bassleer et al.
1992; Nerucci et al. 2000). CS reduces proteoglycan loss from articular cartilage in
humans (Uebelhart et al. 1998) and rats (Omata et al. 1999) when taken orally. CS may

reduce proteoglycan loss in part by decreasing aggrecanase-1 synthesis (Chan et al.

2005a). CS can also decrease IL-l-induced PGE2 and NO production (Bassleer et al.

1992; Chan et al. 2005b). The mechanism for this appears to be that CS decreases the
expression of iNOS and microsomal prostaglandin E synthase-l (Chan et al. 2005b).

If GLN and CS are taken together, they appear to have a synergistic effect in
mitigating the clinical signs of OA. An in vivo study that used a rabbit instability model
of osteoarthrosis showed that animals given GLN and CS did not develop OA lesions as
severe as rabbits fed GLN or CS alone (Lippiello et al. 2000). In the in vitro portion of
the study, the combination of glucosarnine hydrochloride and chondroitin sulfate acted
synergistically in stimulating GAG synthesis (Lippiello et al. 2000). GLN and CS also
both increase chondrocyte response to mechanical stress and prevent the loss of GAG
(Lippiello 2003). The ﬁbronectin ﬁagrnents study also showed a synergistic relationship
between GLN and CS for reversing cartilage damage and promoting repair (Homandberg
et al. 2006). GLN and CS have the ability to modify expression of genes that regulate

inﬂammatory mediators such as iNOS, COX-2, and mPGEsl (Chan et al. 2005b; Neil et

13

al. 2005). The concentrations used in these studies (Chan "et al. 2005b; Neil et al. 2005)
were within range of those found in the blood of horses after oral administration (Du et
al. 2004), and within GLN levels achieved in the synovial ﬂuid of horses when

administed by intravenous infusion (Laverty et al. 2005).

S-adenosylmethionine

S-Adenosyl-L-methionine (SAMe) is synthesized by the body from the essential
the amino acid L-methionine and the nucleoside adenosine triphosphate. SAMe acts as a
methyl donor for reactions important in the synthesis of proteins, phospholipids, and
hormones. It has a variety of roles and is involved in membrane ﬁmction, gene
regulation, and brain function (Chiang et al. 1996). SAMe was ﬁrst sold over the counter
in 1999 for treatment of joint pain. The common human dosage is usually between 400
and 1600 mg daily. SAMe has a good safety record, with few side effects and an absence
of generation of toxic metabolites (Kagan et al. 1990; Goren et al. 2004). The
concentration attained in the synovial ﬂuid in OA patients given SAMe at 400 mg daily
for 7 days is 30 to 80 ng/ml (Stramentinoli 1987). Clinical trials show that SAMe
supplementation decreases pain and inﬂammation (Polli et al. 1975; Caruso and
Pietrogrande 1987; Soeken et al. 2002). A randomized, double-blind, cross-over study,
comparing SAMe with celecoxib (Celebrex) showed that SAMe, over the long term, is
just as effective at relieving pain as Celecoxib (Najm et al. 2004).

The exact mechanism of action is not known. SAMe also upregulates the
proteoglycan synthesis of chondrocytes, and some believe that it may function as a signal

of sulfur availability (McCarty and Russell 1999). A study used human chondrocytes in

14

a thick-layer culture model and showed that SAMe increases proteoglycan synthesis
(Harmand et al. 1987). SAMe has also demonstrated anti-inﬂammatory effects by
suppressing the effects of TNFa and transcriptional activation of iNOS and COX-2
(Hevia et al. 2004). IL-l suppresses the production of SAMe in the joint. Therefore,
since IL-1 production in the joint is upregulated in the disease state of 0A, a
supplementation of SAMe may compensate for this deﬁcit (McCarty and Russell 1999).
SAMe also decreases with aging in human and rats (di Padova 1987; Stramentinoli
1987). Since aging is a risk factor for 0A, the two may be related and supplementation

with age may be a beneﬁt.

Avocado/soybean unsaponiﬁables

Avocado soybean unsaponiﬁables (ASU) are a mixture of vegetable extracts
obtained from avocado and soy bean oils that are normally taken orally. Clinical trials
with ASU show that it has a good safety record (Lequesne et al. 2002; Little and Parsons
2002), though more study is needed to conﬁrm this. These extracts are obtained when
the oil is saponiﬁed (heated and broken down by alkaline hydrolysis). The small fraction
that is not broken down is known as the “unsaponiﬁable” fraction (Bassleer et al. 1992).
It relieves pain when taken orally, and is usually taken at 300 mg daily (Reginster et al.
2000; Walker—Bone 2003; Soeken 2004).

This decrease in pain is attributed to the ability of ASU to decrease inﬂammatory
mediators such as IL-1, NO, and PGE2 as seen in human chondrocyte studies (Bassleer et
al. 1992). A study using human chondrocytes in an alginate bead tissue culture system

showed that ASU increased the accumulation of GAG in the alginate beads in a dose

15

dependent manner (Bassleer et al. 1992). Avocado soy unsaponiﬁables also prevent
collagen breakdown and thus potentially save cartilage from degradation (Mauviel et al.
1989). This is partially mediated by the ability of ASU to inhibit MMP production, such
as MMP-3, as it did in a study of human chondrocytes in culture (Bassleer et al. 1992).
There has been no pharrnacokinetic studies done on this supplement and the exact

mechanism of action is unknown.

Rationale for experiments

One objective is to test biologically relevant concentrations of GLN and CS in
various equine cultures: 1) a short-term (2 d) in vitro culture model designed for
cartilage explants in a 24-wellplate format, 2) a similar short-term culture model
including mechanical loading, 3) a long-term culture system (without loading) designed
to test the effects of GLN and CS over a 2-week period. Explant tissue culture provides
an excellent system for testing chondroprotective agents because of the convenience and
accuracy of the data collection. Mechanical loading is known to be a risk factor for CA
in horses and the combination of GLN and CS at biologically relevant concentrations
have never been tested in a mechanical loading tissue culture system. IL-l will be used
to model inﬂammatory stress in each model and a hydraulic stressor with IL-1 will be
used to model mechanical loading in the short-term impact culture. The response
variables will consist of concentrations of inﬂammatory mediators NO and PGE2 in the
media. Glycosaminoglycan concentration in the media and within cartilage explants will

be measured as an indicator of cartilage breakdown. The concentrations of GLN and CS

16

that will be used in these cultures will be in the range of what is measured in the blood of
dogs and horses after oral administration (Adebowale et al. 2002; Du et al. 2004).
Another objective is to test varying concentrations of SAMe and ASU in bovine
cartilage explant cultures. The system will be a short term 2 (1 culture with IL-1 used to
model inﬂammatory stress. The response variables will consist of NO, PGEz, and GAG
concentrations in the media and concentration of GAG in cartilage explants at
termination of the experiment. Bovine tissue culture is used here because cartilage tissue
from bovine species is readily available. Bovine tissue culture is often used as a model

for testing agents that have relevance to human treatment.

17

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28

Chapter 2
The Effect of Biologically Relevant Concentrations of Glucosamine and Chondroitin

Sulfate on Stressed Equine Cartilage Explants

Summary

Reasons for performing study: In the past decade, dietary supplements such as
glucosamine (GLN) and chondroitin sulfate (CS) have gained popularity in treating
joint pain and inﬂammation due to osteoarthritis (0A) in humans and animals.
Although GLN and CS have established anti-inﬂammatory properties, to our
knowledge, no study has tested biologically relevant concentrations of these molecules
in equine cartilage explants.

Hypothesis: Glucosamine and CS have anti-inﬂammatory properties at biologically
relevant concentrations when tested in equine cartilage explant cultures.

Methods: Three culture schemes were used: 1) IL-1 (50 ng/ml) treatment with and
without GLN + CS (short-term non-impact) 2) IL-1 plus mechanical trauma with and
without GLN + CS (short-term impact) 3) two-week cultures were cultured with and

without GLN + CS and were exposed to IL-1 (50 ng/ml) on day 2 and day 10 (long-

term non-impact). Nitric oxide (N O) and prostaglandin E2 (PGE2) were measured as

indicators of inﬂammatory response. Glycosarninoglycans (GAG) were measured as an

indicator of cartilage turnover.

Results: Glucosamine + CS did not affect NO, PGEZ, or GAG concentrations in the non-

impact short-term model. When mechanical impact was combined with IL-1 the NO

production in the GLN + CS treatment was lower than the IL-1 treatment and did not

29

differ from control. The GAG release was also lower in the GLN + CS treatment than

the IL-1 treatment, although the PGE2 concentration was not affected by GLN + CS.

The non-impact long-term cultures demonstrated that GLN + CS treatment decreased

GAG release and did not affect NO or PGE2 concentration or GAG content in the

explants at termination.

Conclusions: This study suggests that biologically relevant concentrations of GLN + CS
may have protective effects against some indicators of mechanically-induced trauma in
explants.

Potential relevance: These in vitro data provide support that GLN and CS may be

beneﬁcial for equine joint health.

Introduction

In recent years, dietary supplements (now known as nutraceuticals) have been
used to treat osteoarthritis (0A) in both humans and animals. Glucosamine (GLN) and
chondroitin sulfate (CS) have been among the most popular dietary supplements taken for
joint pain. Glucosamine is a precursor of N-acetyl galactosarnine, which is a component
of glycosaminoglycans (GAG). Glycosaminoglycans, such as CS, are components of a
proteoglycan called aggrecan, which gives cartilage its distinctive properties. Both of
these molecules increase synthesis (Bassleer et al. 1992; Lippiello et al. 2000) and
decrease breakdown (Orth et al. 2002; Lippiello 2003) of GAG in cartilage tissue, and
they may be an effective treatment for decreasing joint pain in clinical trials (Clegg et al.

2006; Qui et al. 2005).

30

We use three different culture systems with equine” cartilage explants: short-term
non-impact used previously in our lab (Chan et al. 2005b), short-term impact system
(Ewers et al. 2001), and a long-term non-impact system. Our objective was to test the
anti-inﬂammatory effects of biologically relevant concentrations of GLN and CS in these
three different equine cartilage cultures that. We provide evidence that biologically
relevant concentrations of GLN and CS may be effective against harmful inﬂammatory

responses to trauma in equine cartilage tissue.

Materials and methods
Explant Cultures

Articular cartilage was isolated from antebrachial-carpal and middle carpal joints
of 2- to 9- year-old horses, of several breeds (Thoroughbred, Appaloosa, Tennessee
Walking Horse, Arabian, and Paint). Horses were euthanized at The Diagnostic Center
for Population and Animal Health at Michigan State University for reasons other than

lameness. Cartilage discs (6 mm in diameter) were isolated and randomly distributed to

be cultured in 24-well Falcon platesb with two discs per well. Each well contained 1 m1

of media. The medium (Ham media: 1:] Dulbecco’s Modiﬁed Eagles Medium: nutrient

mixture F-12)a was supplemented as previously described (Fenton et al. 2000). Medium

was also supplemented with all 20 amino acids6 at 25% the concentration as previously

described (Rosselot et al. 1992).

Human recombinant interleukin-1 betae (IL-1) at a concentration of 50 ng/ml was

used to induce inﬂammatory stress. Glucosamine Hle (1, 5, or 10 rig/ml) and low

31

molecular weight (16.9 kilodaltons) chondroitin sulfatef (5, 20, or 50 ug/ml) were all

within the range of concentrations known to occur in equine blood after oral

administration (Adebowale et al. 2002; Du et al. 2004).

Short- Term Non-Impact Model

Explants for short-term experiments were harvested from six different horses and
equilibrated for 2 d in media without fetal bovine serum21 (FBS). Media was removed
and replaced with fresh media 1 d after harvesting. Cultures were then incubated in

media supplemented with 10% FBS3 and treatments 1-4 in Table 1 for 2 d. There were 6

to 12 wells per treatment depending on the availability of cartilage from a given horse.
Treatments were non-impact treatments were assigned as listed in Table 1. Media were

replaced daily until termination of the experiment at 4 d after harvest.

Short-term Impact Model
Explants from three horses were mechanically impacted as a single acute load of

15 mega pascal (MPa) for 50 msec using a Servo-Hydraulic Testing Machine (Model

1331).g The unconﬁned explants were compressed between highly polished stainless

steel plates and peak load was recorded electronically. There were 6 wells per treatment

and the impact treatments were assigned as listed in Table 1.

Long- Term Model

32

Explants in long-term cultures were equilibrated as above for 2 d and the media

was changed at 24 h after harvesting. Cultures were then incubated in serum-free media

supplemented with 1 ul/ml insulin transferring seleniteh (ITS) to termination. There were

9 wells per treatment assigned as listed in Table 11. Media were replaced every 2 (1 until
termination of the experiment at 14 d. The cartilage explants were then stored at -20° C
for later papain digestion and GAG analysis.

All conditioned media samples were stored at 4° C for later assay. All cultures
were maintained at 37° C in a humidiﬁed incubator with 7% C02. For the short-term
non-impact system, there were six horses used (11 = 6) for all treatments except the low
dose, which only had four horses (n = 4). For both the short-term impact and the long-

terrn non-impact there were three horses (n = 3).

Biochemical Analyses:
Nitrite levels in the media were assayed as previously described (Blanco et al.

1995; Chan et al. 2005b). Absorbance was detected at 540 nm by a Spectromax 300
plate reader.l Results are expressed as nmol NO/ml.
GAG release into the media was assayed by dimethylene blueJ (DMB) assay as

previously described (Chandrasekhar 1987). The total amount of GAG in the explant

was obtained from papainc-digested cartilage. Results of explant DMB assays are

expressed as pg GAG/mg wet weight of cartilage.

Prostaglandin E2 was assayed using a commercially available kite according to the

manufacturer’s instructions concentrations are reported in pg/ml. Indomethacinc (10

33

ug/ml) was added to the media at collection and samples were stored at -20° C until

analysis. To bring the samples within range of the standard curve, samples were diluted

1:10 or 1:20 depending on the PGE2 level of the sample, and PGE2 was detected at 405

nm with a wavelength correction set at 590 nm with a spectrometer.

Statistical Analysis

Data were analyzed using Fisher’s Least Signiﬁcant Difference in the PROC

MIXED procedure of SAS software.k The data from all wells were pooled according to

treatment, with individual horses as replicates. The random effects included horse,
treatrnent*horse, and horse*day. The long-term data had the same random effects and
were analyzed for each day using Fisher’s Least Signiﬁcant Difference in the PROC
MDIED procedure by using daily average and the repeated measures option. We also
analyzed the long-term cumulative total for GAG and NO for all days of the long term
using Fisher’s Least Signiﬁcant Difference, except that the horse*day effect and the
repeated measure options were omitted since only one measurement was being analyzed
for a total time period. GAG content in explants were also measured with Fisher Least
Signiﬁcant Difference with no horse*day effect and no repeated measures option. A p

value of <0.05 was considered signiﬁcant and p values <0.1 were considered a trend.

Results

Short- Term Non-Impact Model

34

The explants in the IL-l-only treatment released signiﬁcantly more NO, PGE2,

and GAG compared with the control. The NO concentration in the media in the 5 ug/ml
GLN + 20 ug/ml CS (medium dose), and 1 rig/ml GLN + 5 ug/ml CS (low dose) did not

differ from the IL-l-only treatment (Fig 1A). The low dose did not differ from the

control (Fig 1A). The nutraceutical treatments had no effect on PGEZ or GAG release

into the media. (Fig 18, C).

Short-term Impact Model

The IL-l + impact explants released more NO (p = 0.03) and GAG (p = 0.02)
than the control (Fig 2A-C). The impact model included only the medium dose of GLN
+ CS because of lack of available tissue. The NO concentration in the IL-1 + impact
treatment containing GLN + CS was lower than in the IL-1 + impact treatment (p =
0.013) and did not differ from the control (Fig 2A). Neither the IL-1 nor the GLN + CS
treatment had a signiﬁcant effect on PGE2 release into the media (Fig 2B). GAG release
in the GLN + CS treatment was lower than in the IL-1 + impact treatment (p = 0.023)

(Fig 2C).

Long- Term Model

The IL-1 treatment resulted in increased NO release in all treatments with IL-1 on
d 4, d 12, and total for the two week accumulation (Table 3). The GLN + CS treatments
did not affect the IL-l-induced NO release (Table 3).

The GAG release was higher in all treatments with IL-1 than those without IL-1

on d 4, but the IL-1 did not effect the GAG release on d 12 (Table 4). The high dose

35

GLN and CS treatment had lower GAG release than the IL-1 treatment on d 4 (p < 0.001)
(Table 4). For the cumulative totals, all treatments containing IL-1 were lower than the
IL-l-only, except for the control, which tended to be lower (p = 0.093) (Table 4). The
high dose GLN and CS treatment had lower GAG release over the two-week period (p =
0.001) and the low dose tended to be lower (p = 0.055).

The assay for GAG content in explants did not show any difference between the
control and the IL-l-only treatment (Fig 3). Furthermore, the IL-l-only treatment did not

differ signiﬁcantly from the control or any other treatment.

Discussion

We used equine cartilage explant cultures including short-term, long-term, and
mechanical trauma (impact) culture systems. The short-term model has been used befor
in out laboratory to test these agents (Chan et al. 20053-c). To our knowledge, this is the
ﬁrst study to test physiologically relevant concentrations of GLN + CS in equine articular
cartilage using trauma to stress the explants. This is a relevant way to induce stress is to
apply mechanical impact, since OA often results from acute trauma to the joint and
subsequent inﬂammation (Quinn et al. 2001; Patwari et al. 2003). We modeled acute
trauma to the cartilage using a hydraulic impact concomitant with IL-1 treatment as
previously reported (Quinn et al. 2001; Patwari et al. 2003). A previous study
(Dvoracek-Driksna 2001) has demonstrated that impact treatment at 15 MPa pressure
causes ﬁssuring and cell death in the superﬁcial tangential zone of bovine cartilage. We
used this treatment concomitantly with IL-1, to model trauma and inﬂammatory stress

found in vivo. The long-term study was used to see if there were long-term effects of

36

GLN and CS on cartilage explants. The advantage of the long-term system is that total
release of NO and GAG can be calculated at the end of an experiment as well as the
average concentration of GAG within the explant for each treatment. This cumulative
effect of the treatments may reveal effects that may not be seen over the short-term,
especially since these agents are known to be slow acting when compared with
conventional treatments (Muller-Fassbender et al. 1994).

NO is an inorganic free radical released into the media by the cartilage tissue.
Studies using rabbit and human cartilage have shown that NO can increase GAG loss and
decrease GAG synthesis (Taskiran et al. 1994; Hardy et al. 2002). Nitric oxide promotes
inﬂammation and cartilage breakdown by increasing cytokine production, suppressing
matrix synthesis, and increasing matrix metalloproteinase (MMP) synthesis (T askiran et
al. 1994; Evens 1995; Murrell et al. 1995). We demonstrated that the NO release was
lower in cartilage explants treated with GLN + CS and impact + IL-l than in cartilage
explants treated with impact + IL-l alone, though GLN + CS were not effective without

impact as they were in a previous study (Chan et al. 2005b).

Prostaglandin E2 was also measured because it is implicated in the pain,

inﬂammation, and cartilage breakdown observed in OA (Hardy et al. 2002; Kirker-Head

et al. 2000; Schueter and Orth 2004). Why the GLN and CS treatments were ineffective

at decreasing the PGE2 release or why concentration of PGE2 was so much higher in the
GLN and CS low dose treatment than even in the IL- l—only treatment in the short-term
non-impact model is not known (Fig 1B). In contrast, a previous study has demonstrated

success in effecting a change in PGEz release with GLN + CS at these concentrations in

explants from Holstein steers (Chan et al. 2005b). Other studies that used equine

37

cartilage demonstrated positive affects of GLN (alone) on PGEz, but at much higher (up
to 100 times) concentrations of GLN used in the present study (Orth et al. 2002;
Schlueter and Orth 2004).

IL-l-induced MMP synthesis causes cartilage matrix breakdown, which we
quantiﬁed by measuring GAG release (Chan et al. 2005a). GLN alone decreases IL-l-
induced proteoglycan breakdown (Fenton et al. 2000; Gouze et al. 2001; Fenton et al.
2002). CS also may reduce proteoglycan loss in part by decreasing MMP-13 (Chan et al.
2005a). Another study showed a synergistic relationship between GLN and CS for
reversing cartilage damage and promoting repair (Homandberg et al. 2006). In the
current study, the high dose was most effective at protecting cartilage against the loss of
GAG (Table 4), and there was also an effective protection using the medium dose in the
impact system Gig 2 C). There was no treatment effect on the GAG content in the
explants at termination (Fig 4).

Chan et al. (2005b) have previously demonstrated that genes responsible for NO
and PGE2 synthesis are down-regulated when bovine explant cultures are exposed to
physiologically relevant concentrations of GLN + CS. Metalloprotienases responsible for
breakdown of the cartilage matrix are also down regulated (Chan et al. 20053). The
present study tested the same physiologically relevant concentrations of GLN and CS as
those used by Chan et al. (2005a-b). There were instances in this study where the GLN +

CS treatments were less effective than reported in earlier studies. It is unknown why the

PGE2 concentration was unaffected by the GLN + CS treatment in either the short-term

impact experiment or the short-term non-impact experiments. It is also difﬁcult to explain

the lack of responsiveness of NO and GAG level to GLN + CS treatment in the short-

38

term non-impact experiment. Signiﬁcant differences may not have been found because
the GLN + CS concentrations may have been too low to effect a change. Also, the
variation between horses, which differed with respect to breed and age, may have been
too high to detect a statistically signiﬁcant difference between treatments. The Chan et
al. (2005a-b) studies used bovine species of a single breed and common age, which may
have decreased the variation in these studies and made ﬁnding differences more

statistically possible.

Conclusion

Our hypothesis was that GLN + CS would decrease inﬂammatory markers and
cartilage breakdown at physiologically relevant concentrations. Our results indicate that
GLN + C8 are effective in mitigating harmful inﬂammatory responses due to mechanical
impact + IL-1 in equine cartilage tissue. The advantage of this impact model is the
combination of impact and IL-1. The reason for this is that 0A is often initiated by
trauma to the joint, which is then followed by IL-l-mediated inﬂammation (Quinn et al.
2001; Patwari et al. 2003). This makes the conditions in this system somewhat similar to
those found in vivo. We demonstrated that the NO release was lower in cartilage explants
treated with GLN + CS and impact + IL-l than in cartilage explants treated with impact +
IL-l alone. We have also shown that the GAG release was lower in cartilage explants
treated with GLN + CS and impact + IL-l than in cartilage explants treated with impact +
IL-l alone. We also demonstrated that, over a two-week period, the GAG release was
lower in cartilage explants treated with GLN + CS and IL-1 than in cartilage explants

treated with IL-1 alone. This study provide some support these supplements are

39

beneﬁcial for horses, in maintaining equine joint health, although in vivo research should

be done to conﬁrm the ﬁndings.

Manufacturer's address

aGibco, Grand Island, New York, USA.

bFisher Scientiﬁc, Pittsburgh, Pennsylvania, USA.

cSigma Chemical, St Louis, Missouri, USA.

dJ.T. Baker, Phillipsburg, New Jersey, USA.

eR&D Systems, Minneapolis, Minnesota, USA.

{NutIamax Laboratories, Edgewood, Maryland, USA.
gInstron Corrperation, Canto, Massachusetts, USA.

hRoche Diagnostics Corporation, Indianapolis, Indiana, USA.
iMolecular Devices, Sunnyvale, California, USA.
jPolyscience, Inc., Warrington, Pennsylvania, USA.

kSAS Institute, Inc., Cary, North Carolina, USA.

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Fig I: Mean (iSE, n=6 for medium dose, n# for low dose) NO, GAG, and PGE2
concentrations in media samples at d 1 (A, C) or at d 1 (B). IL-1 (50 ng/ml) was added to
each treatment except for control. The experimental treatments contained 5 11ng GLN

+ 20 11me CS (medium dose) or 1 ug/ml GLN + 5 ug/ml CS (low dose).

45

 

 

 

  

 

 

 

Fig 2: Mean (iSE, n=3) NO, PGEz, and GAG concentrations in media samples at d O
and 1 (A, C) or at d 1 (B). IL-1 (50 ng/ml) + Impact was added to each treatment except
for control. The experimental treatment contained 5 ug/ml GLN + 20 ug/ml CS (medium
dose). GLN + CS treatment differed from IL-l-only treatment in NO (p = 0.0013) and

GAG (p = 0.023) concentrations.

48

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51

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Adebowale, A., Du, J ., Liang, Z., Leslie, J .L. and Eddington, ND. (2002) The
bioavailability and pharmacokinetics of glucosamine hydrochloride and low
molecular weight chondroitin sulfate after single and multiple doses to beagle
dogs. Biopharm Drug Dispos 23, 217-225.

Bassleer, C., Henrotin, Y. and Franchimont, P. (1992) In-vitro evaluation of drugs
proposed as chondroprotective agents. Int J Tissue React 14, 231-241.

Blanco, F .J ., Geng, Y. and Lotz, M. (1995) Diﬁ‘erentiation-dependent effects of IL-1 and
TGF-beta on human articular chondrocyte proliferation are related to inducible
nitric oxide synthase expression. J Immunol 154, 4018-4026.

Chan, P.S., Caron, J .P. and Orth, M.W. (2005a) Effect of glucosamine and chondroitin
sulfate on regulation of gene expression of proteolytic enzymes and their
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Res 66, 1870-1876.

Chan, P.S., Caron, J.P., Rosa, G.J. and Orth, M.W. (2005b) Glucosamine and chondroitin
sulfate regulate gene expression and synthesis of nitric oxide and prostaglandin
E(2) in articular cartilage explants. Osteoarthritis Cart 13, 387-394.

Chan, P.S., Schlueter, A.E., Coussens, P.M., Rosa, G.J., Haut, R.C. and Orth, M.W.
(2005c) Gene expression proﬁle of mechanically impacted bovine articular
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Chandrasekhar, S., Esterman, M.A. and Hoffman, H.A. (1987) Microdetermination of
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Clegg, D.O., Reda, D.J., Harris, C.L., Klein, M.A., O'Dell, J .R., Hooper, M.M., Bradley,
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Moreland, L.W., Schumacher, H.R., Jr., Oddis, C.V., Wolfe, F., Molitor, J .A.,
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Moskowitz, R.W. and Williams, H.J. (2006) Glucosamine, chondroitin sulfate,
and the two in combination for painful knee osteoarthritis. N Engl J Med 354,
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Du, J ., White, N. and Eddington, ND. (2004) The bioavailability and pharmacokinetics

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Dvoracek-Driksna, D. (2001) Effects of mechanical impact on a bovine articular
cartilage explant system, Michigan State University, East Lansing.

Evens, CH. (1995) Nitric oxide: what role does it play in inﬂammation and tissue
distruction? Agents Actions Suppl 47, 107-1 16.

Ewers, B.J., Dvoracek-Driksna, D., Orth, M.W. and Haut, R.C. (2001) The extent of
matrix damage and chondrocyte death in mechanically traumatized articular
cartilage explants depends on rate of loading. J Orthop Res 19, 779-784.

Fenton, J .I., Chlebek-Brown, K.A., Caron, J .P. and Orth, M.W. (2002) Effect of
glucosamine on interleukin-l -conditioned articular cartilage. Equine Vet J Suppl,
219-223. '

Fenton, J .I., Chlebek-Brown, K.A., Peters, T.L., Caron, J .P. and Orth, M.W. (2000)
Glucosamine HCl reduces equine articular cartilage degradation in explant
culture. Osteoarthritis Cart 8, 258-265.

Gouze, J.N., Bordji, K., Gulberti, S., Terlain, B., Netter, P., Magdalou, J ., Fournel-
Gigleux, S. and Ouzzine, M. (2001) Interleukin-lbeta down-regulates the
expression of glucuronosyltransferase I, a key enzyme priming
glycosaminoglycan biosynthesis: inﬂuence of glucosamine on interleukin-lbeta-
mediated effects in rat chondrocytes. Arthritis Rheum 44, 351-360.

Homandberg, G.A., Guo, D., Ray, L.M. and Ding, L. (2006) Mixtures of glucosamine
and chondroitin sulfate reverse ﬁbronectin fragment mediated damage to cartilage
more effectively than either agent alone. Osteoarthritis Cart. (Available Online).

Kirker-Head, C.A., Chandna, V.K., Agarwal, R.K., Morris, E.A., Tidewell, A.,
O’Calaghan, M.W., Rand, W. and Kumar, M.S. (2000) Concnetrations of
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Lippiello, L. (2003) Glucosamine and chondroitin sulfate: biological response modiﬁers
of chondrocytes under simulated conditions of j oint stress. Osteoarthritis Cart 11,
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Lippiello, L., Woodward, J ., Karpman, R. and Hammad, T.A. (2000) In vivo
chondroprotection and metabolic synergy of glucosamine and chondroitin sulfate.
Clin Orthop Relat Res, 229-240.

Moses, V.S., Hardy, J ., Bertone, A.L. and Weisbrode, SE. (2001) Effects of anti-

inﬂarnmatory drugs on lipopolysaccharide-challenged and -unchallenged equine
synovial explants. Am J Vet Res 62, 54-60.

53

Muller-Fassbender, H., Bach, G.L., Haase, W., Rovati, L.C."and Setnikar, 1. (1994)
Glucosamine sulfate compared to ibuprofen in osteoarthritis of the knee.
Osteoarthritis Cart 2, 61-69.

Murrell, G.A., Jang, D. and Williams, R.J. (1995) Nitric oxide activates metalloprotease
enzymes in articular cartilage. Biochem Biophys Res Commun 206, 15-21.

Orth, M.W., Peters, T.L. and Hawkins, J .N. (2002) Inhibition of articular cartilage
degradation by glucosamine-HCI and chondroitin sulphate. Equine Vet J Suppl,
224-229.

Patwari, P., Cook, M.N., DiMicco, M.A., Blake, S.M., James, I.E., Kumar, S., Cole,
A.A., Lark, M.W. and Grodzinsky, A.J. (2003) Proteoglycan degradation aﬁer
injurious compression of bovine and human articular cartilage in vitro: interaction
with exogenous cytokines. Arthritis Rheum 48, 1292-1301.

Qiu, G.X., Weng, X.S., Zhang, K., Zhou, Y.X., Lou, S.Q., Wang, Y.P., Li, W., Zhang,
H., Liu, Y. (2005) A multi-central, randomized, controlled clinical trial of
glucosamine hydrochloride/ sulfate in the treatment of knee osteoarthritis.
Zhonghua Yi Xue Za Zhi 85, 3067-3070.

Quinn, T.M., Allen, R.G., Schalet, B.J., Perumbuli, P. and Hunziker, EB. (2001) Matrix
and cell injury due to sub-impact loading of adult bovine articular cartilage
explants: eﬁects of strain rate and peak stress. J Orthop Res 19, 242-249.

Rosselot, G., Reginate, A.M., Leach, RM. (1 992) Development of a serum-free system
to study the effect of growth hormone and insulinlike growth factor-1 on cultured
postembryonic growth plate chondrocytes. In Vitro Cell Dev Biol 28A, 235-244.

Schlueter, A.E. and Orth, M.W. (2004) Further studies on the ability of glucosamine and
chondroitin sulphate to regulate catabolic mediators in vitro. Equine Vet J36,
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Biochem Biophys Res Commun 200, 142-148.

54

Chapter 3
The Effect of Avocado and Soy Unsaponiﬁables and

S-Adenosyl-L-Methionine on Inﬂammatory Mediators in Bovine Cartilage

Summary

Reasons for performing study: Alternative ways of treating osteoarthritis (OA) have
become popular in the past decade. S-Adenosyl-L-Methionine (SAMe) and avocado
soy unsaponiﬁables (ASU) are among the supplements that are available for controlling
pain and inﬂammation due to CA. We used bovine cartilage explant cultures to
measure the effect of both of these agents on inﬂammatory mediators and cartilage
breakdown.

Hypothesis: SAMe and ASU have anti-inﬂammatory properties and will decrease
inﬂammatory mediators and cartilage breakdown in a dose dependent manner.

Methods: Two experiments were performed: the ﬁrst tested SAMe at concentrations of
1.0, 0.1, and 0.01 ug/ml (high, medium, and low dose) and ASU at concentrations 10.0
and 1.0 ug/ml (high dose and medium dose); the second tested ASU at concentrations
of 10.0, 1.0, and 0.1 uyml (high, medium, and low dose). Interleukin-1 (IL-1) at 15
ng/ml was used to initiate inﬂammatory stress. Nitric oxide (NO) and Prostaglandin E2
(PGEz) were measured as indicators of inﬂammatory response. Glycosaminoglycans
(GAG) were measured as an indicator of cartilage turnover.

Results: The ﬁrst experiment showed no signiﬁcant treatment effects for either SAMe or

ASU. However, the medium dose of SAMe tended to have a lower PGEz

55

concentration than the IL-l—only (p = 0.09). In the second experiment, ASU again did
not signiﬁcantly decrease inﬂammatory mediators or cartilage breakdown.

Conclusions: This study yields little evidence that SAMe or ASU affects inﬂammation or
cartilage breakdown at these concentrations.

Potential relevance: Further testing with different in vitro models is needed to determine
whether these agents affect inﬂammation and cartilage turnover and at what

concentrations.

Introduction

Osteoarthritis (0A) is a degenerative joint disease marked by progressive
cartilage tissue loss, pain, inﬂammation, and eventual loss of joint function (Felson et al.
1998; Hashirnoto et a1. 1998). Onset of 0A may be initiated by trauma or chronic
mechanical stress and is marked by production of proinﬂammatory cytokines such as
interleukin 1 beta (IL-1) (Quinn et al. 2001; Patwari et al. 2003). The inﬂammatory

activity of these cytokines is mediated by signaling molecules such as nitric oxide (NO)

and prostaglandin E2 (PGEZ) (Smalley et al. 1995; Grabowski et al. 1997). IL-l inhibits

glycosaminoglycan (GAG) synthesis and increases GAG catabolism. Since GAG is an
important component of cartilage extracellular matrix, this imbalance in tissue turnover
leads to ultimate destruction of the cartilage tissue and joint disability (Hardingham et al.
1992; Goldring et al. 1994).

GLN and CS are the most popular nutraceuticals for management of 0A; however
other nutraceuticals such as S-Adenosyl-L-Methionine (SAMe) seem effective against

the symptoms of 0A. SAMe was ﬁrst sold over the counter in 1999 for treatment of joint

56

pain. It is a compound that is synthesized by the body from the essential the amino acid
L-methionine and adenosine triphosphate. Clinical trials show that SAMe
supplementation decreases joint pain and inﬂammation (Polli et al. 1975; Caruso and
Pietrogrande 1987; Soeken et al. 2002) and increases proteoglycan synthesis (Harmand et
al. 1987), which is important since proteoglycan is responsible for the shock absorbing
properties of cartilage tissue. The exact mechanism of these protective effects is not
known.

Avocado soy unsaponiﬁables (ASU) are a mixture of natural vegetable extracts
obtained from avocado and soy bean oils. These extracts are obtained when the oil is
saponiﬁed (heated and broken down by alkaline hydrolysis). The small fraction that is
not broken down is known as the “unsaponiﬁable” fraction (Bassleer et al. 1992), which
may relieve pain when taken orally (Reginster et al. 2000; Walker-Bone 2003; Soeken

2004). This decrease in pain is attributed to the ability of ASU to decrease inﬂammatory
mediators such as IL-1 , NO, and PGE2 seen in human chondrocyte studies (Bassleer et al.

1992). Avocado soy unsaponiﬁables also increase synthesis and decrease breakdown of
GAG, thus potentially preserving cartilage ﬁom breakdown (Mauviel et al. 1989;
Bassleer et al. 1992).

Our objective was to test biologically relevant concentrations of SAMe and ASU
in bovine cartilage explant cultures. We have utilized this type of culture system to study
GLN and CS (Chan et al. 2005a; Chan et al. 2005b; Chan et al. 2005c), and in this study
we use a similar approach to test SAMe and ASU and there ability to mitigate

inﬂammatory responses to IL-1.

57

Materials and methods
Explant Cultures

Articular cartilage was isolated from antebrachial-carpal and middle carpal joints
of Holstein steers (18-24 mo old) from a local abattoir within 3 hours of slaughter. The

cartilage was obtained in 6 mm discs, which were washed three times in media. The

discs were cultured in 24-well Falcon platesb with two discs per well. Each well

contained 1 ml of media (1 :1 Dulbecco’s Modiﬁed Eagles Medium: nutrient mixture F-

12).a The medium was supplemented as previously described (Fenton et al. 2000).

Media was also supplemented with all 20 amino acidsc at the concentrations used by

Chan et al. (2005b). All cultures were maintained at 37° C in a humidiﬁed incubator with

7% C02.
Human recombinant IL-1 betae (IL-1) at a concentration of 15 ng/ml was used to

induce inﬂammatory stress. S-Adenosyl-L-Methionine concentrations were 1.0, 0.1, and
0.01 ug/ml (high, medium, and low doses, respectively). These concentrations,
especially the lower two, approximate those in the blood and synovial ﬂuid when orally
administered (Stramentinoli 1987). Avocado soy unsaponiﬁables concentrations were
10, 1, 0.1 ug/ml (high, medium, and low doses, respectively). Concentrations of ASU are
achieved in the synovial ﬂuid after oral administration are not known, but these
concentrations were chosen because past studies have found effects using similar

concentrations (Bassleer et al. 1992).

Explants were equilibrated in media without fetal bovine serumﬁl (FBS) for 2 d

with the media being changed at 24 hours. The media without PBS was replaced with

58

media with F BS and treatments at 48 h. At 72 h the media was replaced again with F BS
and treatments in the media. The conditioned media was stored at 4° C until analysis for
NO and GAG content. A small fraction of the media was isolated for PGE2 analysis.
The cartilage explants were stored at -20° C for later papain digestion and GAG analysis.
Two experiments were performed: (1) the ﬁrst included concentrations for both
SAMe and ASU and (2) in the second only ASU concentrations. There were six wells
per treatment for each experiment, which were assigned to one of eight treatments for the
ﬁrst experiment (Table I) and one of six treatments for the second experiment (Table II).
Each experiment was replicated four times with cartilage being isolated from four

different Holstein steers.

Biochemical Analyses:

Nitrite levels in the media were assayed as previously described (Blanco et al.

1995). Absorbance was detected at 540 nm by a Spectromax 300 plate reader.l Results

are expressed as nmol NOz/ml. GAG release into the media was assayed by the

dimethylene blueJ (DMB) assay as previously described (Chandrasekhar 1987). The
digest contained 1 ug papainc per mg cartilage digest. Results of explant DMB assays
are expressed as ug/mg wet weight for cartilage. PGEZ was assayed using a

commercially available kit.e Indomethacinc (10 ug/ml) was added to the media at

collection and samples were stored at -20° C until analysis. Media samples were assayed

for PGEZ on media samples from the ﬁrst 24 h of both experiments according to the

59

manufacturer’s instructions and are reported in pg/ml. To bring the samples within range

of the standard curve, samples were diluted 1:10 or 1:20 depending on the PGEZ level of

the sample. PGEz was detected at 405 nm with a wavelength correction set at 590 nm.

Statistical Analysis

Data were analyzed using the PROC MIXED procedure of SAS software.k The

data from all wells were pooled according to treatment, with individual cows as
replicates. The random effects included cows, neatrnent‘cow, and cow“ day. A p value

of <0.05 was considered signiﬁcant and p values <0.1 were considered a trend.

Results
The explants in the IL-I-only treatment released signiﬁcantly more NO, PGEz,
and GAG than control explants, as expected. The SAMe treatments did not have lower

NO, PGEZ, or GAG concentration than the IL-l-only treatments (Fig lA-C). However,

the PGEz concentration in the medium dose of SAMe showed a trend (p = 0.09) of being

lower compared to the IL-l-only treatment (Fig 1B). The treatments also had no effect

on ﬁnal GAG content in the explants (data not shown).

None of the ASU treatments decreased NO, PGEz, or GAG concentration in the

media (Fig l and 2). The GAG concentration was signiﬁcantly affected by the ethanol
since the ethanol control released more GAG than did the IL-l-only treatment. The

medium concentration of ASU increased rather than decreased the GAG concentration

60

(Fig 2C). The ASU treatments also had no effect on ﬁnal" GAG content in the explants

(data not shown).

Discussion

SAMe has the ability to decrease the transcriptional activation of genes that effect
NO and PGE2 level (iNOS and COX-2, respectively) (Hevia et al. 2004). Our study did
show a trend that the medium dose of SAMe was lower than the IL-l-only. Our study
did not show the effects of SAMe the Hevia et al. 2004 study showed. However, the
current study differed from the Hevia et al. (2004) study in that 5'-methylthioadenosine
(MTA), a product of SAMe metabolism, was used instead of SAMe (Hevia et al. 2004).
Another difference was that the Hevia et al. study use a murine macrophage cell line and
rat hepatocytes (Hevia et al. 2004). They also used LPS to model inﬂammation rather
than IL-l. It may be that the anti-inﬂammatory effects of SAMe are mediated by the
breakdown product MTA. If this is the case, then MTA, as a downstream molecule, may
be more potent than SAMe as an anti-inﬂammatory. SAMe has also been reported to
increase proteoglycan synthesis in human chondrocytes in vitro at concentrations used in
the current study (1 to 10 ug/ml) (Harmand et al. 1987). Hannand et al. (1987) used
human articular osteoarthritic chondrocytes in a thick-layer culture model. Using
chondrocytes in culture without the extracellular matrix may yield a more responsive
culture system with respect to SAMe supplementation than using explants in culture. A
clinical study showed that SAMe was just as effective as a known anti-inﬂammatory
(celecoxib) at relieving pain, however SAMe took a much longer time to elicit the same

effects as celecoxib (Najm et al. 2004). Because SAMe is involved in so many different

61

reactions, our experiments may not have been carried on long enough to detect the
delayed nature of the SAMe.

ASU increased cartilage proteoglycan content and thickness in sheep (Cake et al.
2000). In that study, 0A was induced by meniscectomy and ASU was administered
orally. ASU also inhibited the breakdown of rat cartilage implanted beneath the skin of
mice (Khayyal and el-Ghazaly 1998). The concentrations of ASU achieved in the blood
or in the synovial ﬂuid in either of these studies, which may account for the discrepancy
between the results from Khayyal and el-Ghazaly and the current study was unknown.
ASU also had anti-inﬂammatory effects in two studies using human chondrocytes: IL-I-
induced PGEz was reduced by 40 to 50% (Bassleer et al. 1992), and ASU decreased basal
production of NO and PGE2 in hmnan OA chondrocytes (Bassleer et al. 1992).

In the case of both the SAMe studies and the ASU studies, a species other than
bovine species was used, which raises the possibility of species-speciﬁc effects. Bovine
cartilage tissue may be less responsive to SAMe and ASU than cartilage tissue from other
species. Another consideration is that all of the tissue culture experiments reported have
used monolayers rather than explants. Cells may respond differently to these

supplements when cultured without the natural extra cellular matrix surrounding the cells.

Conclusion
The results presented here using bovine explants do not support the usefulness of
ASU and SAMe as chondroprotective molecules. Further work is needed to elucidate the

concentration of ASU in the blood and synovial ﬂuid upon oral administration. Further

62

work is also need to delineate the exact mechanism of these two supplements as the

mechanism of both of these supplements remains largely unknown.

Manufacturer's address:

aGibco, Grand Island, New York, USA.

bFisher Scientiﬁc, Pittsburgh, Pennsylvania, USA.

cSigma Chemical, St Louis, Missouri, U.S.A.

dJ.T. Baker, Phillipsburg, New Jersey, USA.

eR&D Systems, Minneapolis, Minnesota, USA.

fNutrarnax Laboratories, Edgewood, Maryland, USA.
gRoche Diagnostics Corporation, Indianapolis, Indiana, USA.
hMolecular Devices, Sunnyvale, California, USA.
iPolyscience, Inc., Warrington, Pennsylvania, USA.

JSAS Institute, Inc., Cary, North Carolina, USA.

63

 

 

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7O

Fig 2." Mean (iSE, n=4) NO, PGEZ, and GAG concentrations in media samples at day 1
(A-C). IL-1 (15 ng/ml) was added to each treatment except for control. The treatments
contained: 0.3% EthOH (EthOH control); 10 ug/ml ASU (high dose); 1 ug/ml ASU

(medium dose); 0.01 ug/ml ASU (low dose).

71

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