Mesenchymal Stem Cells Suppress ProInflammatory Cytokine Expression in an

Osteoarthritic Chondrocyte
Co-culture Model

A thesis submitted in partial fulfillment for honors within the Major in Biological
Sciences
By
Tommy Fu

Dr. Alan J. Nixon, PI

Michael S. Scimeca, Research Supervisor

Department of Clinical Sciences, College of Veterinary Medicine

Cornell University, Ithaca, NY

May 13, 2011

Acknowledgements
When I started at the Comparative Orthopaedics Laboratory as a second-semester
freshman, I had never done research in my life. Now, upon completing this project, I
would like to recognize the many people who offered their support during my
undergraduate career.
First, I would like to thank Dr. Nixon for the chance to work in such an amazing
lab with amazing people. The experiences and skills that I have gained will be invaluable
to me in the future.
I would also like to thank Mike Scimeca for all his help in lab. During the
toughest times, Mike always took the time to help me reason up a new solution. Without
his guidance, I never would have complete this thesis.
I would also like to thank the other lab members in Comparative Orthopaedics:
Jeremy Yost, Laila Begum, Kate Morris, and Dr. Ashlee Watts for helping with
equipment, cell culturing, and lab techniques. Additionally I would like to thank the
graduated lab members, Xinran Liu, Yang Gu, and Nasim Khadem, for their help in my
transition to a new research experience.
I would also like to thank the other undergraduates, Nikhita Parandekar and Arjun
Gokhale for making the environment fun and friendly. Also, thanks to Max Meneveau for
his help with data collection.
Lastly, thanks to my family and friends, who never fail to show their undying
support in my endeavors. I am where I am because of their belief in me. In .

Table of Contents
Section

Page Number

I. Abstract...................................3
II. Introduction...........................6
a. Osteoarthritis (OA).......7
b. Inflammation........9
c. Inflammatory Cytokines.......9
d. Cartilage.......12
e. Aggrecan and Aggrecanase..........13
f. MSCs and a New Model for the Treatment of Osteoarthritis......14
III. Materials & Methods..............16
a. Chondrocyte and Mesenchymal Stem Cell Harvesting and Culturing...........17
b. Lipopolysaccharide (LPS) Administration.............17
c. Plating.........................17
d. RNA Isolation and Purification.......19
e. TaqMan Real-Time PCR................19
f. Statistical Analysis and Data Plots..........20
IV. Results..................22
a. Effect of Co-culture on TNF-...........23
b. Effect of Co-culture on IL-1................24
c. Effect of Co-culture on IL-1.........25
d. Effect of Co-culture on IL-6..........26
e. Effect of Co-culture on Aggrecan..............27
f. Effect of Co-culture on Aggrecanase-1 (ADAMTS-4)....................................28

f. Effect of Co-Culture on Aggrecanase-2 (ADAMTS-5)...........29

V. Discussion...................................31
VI. Appendix...........................38
VII. Bibliography........44

I. Abstract

The goal of this study was to evaluate the details of recently proposed treatment
methods for osteoarthritis. Using an equine model, we were interested in the protein
modulatory effects of mesenchymal stem cells on chondrocytes. By co-culturing MSCs
with chondrocytes and using lipopolysaccharide to induce an arthritic-like state, we
looked at the gene expression of several molecules in chondrocytes. We wanted to
evaluate two major effects of MSCs. First, to what degree MSCs can lower the
expression of inflammatory cytokines including TNF-, IL-1, IL-1, and IL-6. Second,
how MSCs affect the expression of the cartilage structural proteins by looking at
aggrecan, Aggrecanase-1 (ADAMTS-4), and Aggrecanase-2 (ADAMTS-5). Overall,
MSCs in the arthritic condition decreased the expression of TNF- and IL-1, and
increased the expression of IL-6. IL-1 was not significantly affected. MSCs did not
significantly affect aggrecan or Aggrecanase-1 expression in chondrocytes. MSCs did
significantly increase Aggrecanase-2 expression. We arrived at several conclusions: first,
MSCs were indeed effective in reducing the expression of pro-inflammatory cytokines
while increasing the expression of anti-inflammatory cytokines. Second, because of the
limited effect on IL-1, however, MSCs alone may not provide ultimate protection
inflammation specific to osteoarthritis cases. Third, because this experiment showed
limited beneficial effects of MSCs on aggrecan and aggrecanase expression, this model
does not seem to be as useful in the anabolic process of rebuilding cartilage. Lastly, much
work still needs to be done looking at the other inflammatory cytokines and structural
proteins. Additionally, this model should also be extrapolated for MSCs to be co-cultured
with other types of cells involved in soft tissue injury and immunity. Most importantly,

being able to isolate the factors responsible for gene expression changes would be greatly
valuable towards understanding more about MSCs.

II. Introduction

Osteoarthritis
Osteoarthritis (OA) is the most common form of arthritis, currently affecting
millions of people within the United States [1]. It is also the most common cause of
lameness in horses and dogs. In fact, joint injury and joint disease together comprise a
large majority of the equine clinicians caseload [2]. Poor performance and early
retirement from strenuous activity are the most common complications from horses
suffering from OA [3]. In addition, the financial cost of providing care and medical
treatment for these horses places a large burden on the owners.
Traditionally, OA was not thought to have biological origins. In fact, until the 20th
century, joint problems were thought to be due to mechanical stress and aging only [4]. In
current medicine, OA is characterized by the degradation of cartilage associated
components in joints, especially type II collagen and specific proteoglycans [5]. The
effects of degradation can also cause damage to the surrounding areas of the joint. Since
cartilage acts as a cushion to the bones during movement, loss of protein strength leads to
wear and inflammation. Figure 1 shows a comparison of a healthy joint to an OA joint.
The cycle of protein degradation and inflammation is complicated and feeds back to
create further complications (Figure 2). Symptoms of OA include joint pain, swelling,
and stiffness. Further complicating OA is the fact that symptoms can be worse either after
strenuous activity or after periods of relative inactivity [2].
OA can be categorized as primary (idiopathic) or secondary. Primary OA is
defined as articular cartilage degeneration which occurs in the absence of a clear
abnormality. An example of this is OA related to aging as most primary cases are seen at
older age. Secondary OA is the result of trauma, repetitive motion, or strenuous activity

[6]. These causes are more related to physical activity or underlying diseases, and are
generally seen at a younger age.

Figure 1. Structural comparison of a healthy joint to an osteoarthritic joint [24].

Figure 2. Diagram of osteoarthritis pathological changes occurring in various joint tissues [2].

Currently accepted OA treatment forms are aimed at pain management and the
delay of progression [4]. Many treatments are largely drug based. These treatments
include nonsteroidal anti-inflammatory drugs (NSAIDS), corticosteroids, hyaluronic acid,
and more [2]. Each drug has its own benefits and drawbacks, and often several drugs are
used simultaneously. If OA damage becomes too extensive, the only end stage treatment
option is the use of endoprostheses [7]. However, while prosthetics are a reasonable
treatment for humans, the same may not apply to horses. Owners may not be willing to
finance such a costly surgical operation or bear the problems associated with surgery.

Inflammation
The atrophy of cartilage causes changes in the underlying bone. The bone
thickens and develops growths, called spurs or osteophytes, towards the epiphyseal ends.
Fragments of bone or cartilage may detach and float loosely in the joint space. Lastly,
cartilage breakdown causes the joint lining, or synovium, to become inflamed.
Inflammation is characterized symptomatically by pain, tenderness, stiffness, effusion,
warmth, and swelling. Molecularly, inflammation is characterized by the interactions of
specific cytokines (inflammatory proteins) and enzymes [8]. Although inflammation is a
natural response to infection and injury, often, complications of inflammation both
disrupt healing and increase discomfort for the horse.

Inflammatory Cytokines
Cytokines are polypeptides or glycoproteins produced by many systemic immune
cell types at the site of injury or infection. Unlike hormones, cytokines are not produced

by specialized tissues and are not preformed molecules. In addition, they function by
paracrine and autocrine mechanisms, appearing rapidly after environmental stressors such
as infection or injury. They bind to cellular receptors that result in the activation of
intracellular signaling pathways, regulating gene transcription. Thus, cytokines can
influence cellular differentiation, proliferation, and activity [9].
Two of the best studied families of cytokines are the Tumor Necrosis Factors
(TNFs) and the Interleukins. TNF- is among the earliest and most potent mediators of
the immune response. Mostly synthesized by monocytes, macrophages, and T cells, TNF is responsible for the generation of other inflammatory mediators and homeostatic
responses [9]. Downstream of the receptor, TNF- primarily activates NF- and MAPK
pathways [10]. Among its functions are neutrophil and macrophage proliferation, tumor
necrosis, and cellular apoptosis. It also initiates a cascade of other cytokines which
downstream, affect vascular permeability, blood clotting, and cellular growth. Because of
its role in several diseases including septic shock, cancer, trauma, and rheumatoid
arthritis, much research has been done concerning TNF therapies [11]. TNF in
osteoarthritis has specific effects including cartilage and bone resorption, inhibition of
collagen and glycoprotein synthesis, and stimulation of chondrocyte apoptosis [3].
Soluble (circulating) TNF receptors (sTNFR) are extracellular domains of TNF receptors
that retain their affinity for TNF- binding. Thus, they compete with cellular receptors for
the binding of TNF-. sTNFRs thus present a counter-regulatory response to TNF-
activity [9].
The Interleukin-1 (IL-1) family can be separated into eleven distinct proteins.
However, IL-1 and IL-1 are the best understood and most relevant to the inflammatory

response. IL-1 is released by macrophages, monocytes, and endothelial cells [9]. While
by themselves, IL-1 and IL-1 can cause an inflammatory response, they also induce the
expression of other pro-inflammatory genes. Most notable of these genes are
cyclooxygenase type 2 (COX-2), inducible nitric oxide synthase (iNOS), and IL-6. IL-1
and TNF also work to stimulate the production of each other, forming an important
amplification loop [9]. IL-1 is generally activated whenever TNF- is activated. IL-1
and TNF- produce similar responses at low doses if administered simultaneously. In
addition, this also accounts for the synergistic roles of TNF and IL-1 in the
inflammatory response [9]. IL-1 is another significant mediator of osteoarthritis. Like
other cytokines, IL-1 is involved in a variety of cellular activities including cell
proliferation, differentiation, and apoptosis. In general it has been more closely associated
to inflammation than IL-1 due to its readily detectable levels in body fluids [12].
Interestingly, IL-1 can induce an arthritic-like state in horses. Synovial fluid samples
from horses with osteoarthritis, infectious arthritis, osteochondritis, and traumatic arthritis
have shown significant IL-1 activity [13, 14]. In the same studies, normal synovial fluid
did not show detectable IL-1 activity. IL-1 receptor antagonist (IL-1ra) works in a similar
manner to sTNFRs, downregulating the IL-1 response. However, contrasting to sTNFRs,
the molecule directly competes with IL-1 for binding to receptors [9].
TNF- and IL-1 are potent inducers of Interleukin-6 (IL-6). IL-6 is quite unique
among cytokines because it has both pro-inflammatory roles and anti-inflammatory roles
[9]. Like TNF- and IL-1, IL-6 also inhibits protein synthesis, namely proteoglycans. In
addition, IL-6 reduces chondrocyte proliferation, increases proteoglycan catabolism and
induces neutrophil activation during inflammation [3,9]. However, IL-6 exerts anti-

inflammatory properties by attenuating TNF- and IL-1while promoting the release of

sTNFRs and IL-1ra [9]. Figure 3 pictures the basic interactions of several inflammatory
cytokines.
TNF- and IL-1 are the most potent mediators of OA. Yet, interestingly, studies
have shown that simply blocking the two cytokines is not enough to stop the disease from
progressing [13]. This suggests that in order to improve OA, a more encompassing
therapy is required.

Figure 3: Diagram of cytokine production after inflammation by various factors [25].

Cartilage
Cartilage has a firm matrix that resists mechanical stress and acts like a cushion
during movement. Specifically, it serves as a load bearing material, absorbing impact
while sustaining shearing forces [15]. Joint articular cartilage provides covering for the

ossified ends of long bones. Articular cartilage is classified as hyaline cartilage,

consisting of several layers of cells and matrix. The primary cells of cartilage are
chondrocytes, which occupy small cavities called lacunae. Chondrocytes secrete the
extracellular matrix, which contains glycosaminoglycans and proteoglycans [16]. These
components, along with collagen fibers, contain large amounts of water to give cartilage
its mechanical properties. In fact, the proteins found in cartilage account for only 20% of
the total wet weight. Water and inorganic salts comprise the rest [15]. Cartilage is
avascular and also lacks nervous and lymphatic networks. It obtains nourishment from
blood vessels surrounding connective tissues by diffusion through the matrix [16]. This
creates problems for healing when cartilage is damaged, and has thus motivated many
new methods for the treatment of cartilage defects.

Aggrecan and Aggrecanase

Proteoglycans are macromolecules composed of a specific core protein
substituted with covalently linked glycosaminoglycan chains. They account for 5-10% of
the tissue wet weight [15]. Aggrecan is one of the main proteoglycan components of the
extracellular matrix of articular cartilage. It is largely responsible for cartilages high
resistance to compression [17]. Aggrecan is also involved in the regulation of cartilage
development, growth, and homeostasis. Aggrecan is classified as a hyalectan
extracellular matrix proteoglycan. This signifies that it can bind hyaluronic acid by its Nterminal globular domain [18]. Hyaluronic acid is a major glycosaminoglycan
polysaccharide in connective tissue, forming portions of the extracellular matrix and
providing support for tissues such as cartilage. It functions as a hygroscopic material, as it

is capable of capturing large amounts of water [19]. Aggrecan is almost exclusively

found in the form of aggregates with hyaluronic acid [18]. Thus, together, hyaluronic acid
and aggrecan contribute largely to the mechanical compressive strength of cartilage.
Aggrecanases are responsible for aggrecan degradation by enzymatic activity. The
activity of aggrecanases is an important manifestation of OA [20]. The most prominent
aggrecanases are Aggrecanase I (ADAMTS-4) and Aggrecanase II (ADAMTS-5). Past
studies have also shown complex interactions between aggrecanases and inflammatory
cytokines. For example, in humans, ADAMTS-4 can be induced following stimulation
with IL-1 and TNF-. Also, ADAMTS-4 in human synoviocytes can be inhibited by
TNF- blockers and IL-1 neutralizing antibodies. ADAMTS-5 does not seem to be
inducible by cytokines in humans, yielding the theory that ADAMTS-5 is constitutively
expressed while ADAMTS-4 is inducible [17]. These aggrecanases have been shown to
be the most efficient in their cleavage of aggrecan, and have been considered to be likely
contributors to the pathological mechanisms of OA. In fact, studies with mice have
shown the deletion of the ADAMTS-5 gene provides protection from proteoglycan
degradation and helped inhibit the progression of OA [21].

Mesenchymal Stem Cells and a New Model for the Treatment of Osteoarthritis
Mesenchymal stem cells (MSCs) are multipotent cells that can be extracted from
the adult bone marrow [21]. They have the potential to differentiate into a variety of cells
including chondroblasts, adipocytes, fibroblasts, mesothelial cells, endothelial cells, and
osteoblasts [16]. MSCs have been used in two general ways to support healing. The first
is by differentiation through exposure to growth factors. For example, MSCs can

differentiate into a chondrocyte-like phenotype by the addition of Transforming Growth

Factor- (TGF-) [22]. The second is by using its immunosuppressive characteristics on
immune cells [21]. While MSCs have long been used as a source of healing for forms of
arthritis, little is known about this cellular mechanism and pathway. One interesting
characteristic of bone marrow MSCs in humans is that they do not produce significant
levels of TNF- when stimulated with LPS. In a study published in 2009, researchers
discovered that the inability to respond to LPS stimulation might make MSCs ideal for
immune-suppressive treatments [23]. It was hypothesized that this was due to
methylation of the promoter region of the TNF gene. This phenomenon is evident only in
some primary and tumor cell lines.
Using this important characteristic of MSCs, we hypothesized that MSCs cocultured with chondrocytes would yield similar immunosuppressive results. Being able to
suppress the actions of TNF- and IL-1 would likely reduce the catabolic imbalance in
cartilage while preventing further damage to cartilage [5]. Also, we were interested in the
effect of MSCs on the proteoglycans in chondrocytes. By administering
lipopolysaccharide to produce an inflammatory response in chondrocytes, we would then
co-culture them with MSCs to view differences in gene expression. The relative gene
expression modulation of TNF-, IL-1, IL-1, and IL-6 were used as indicators of the
degree to which the inflammatory response would be changed by MSCs. In addition, by
looking at the modulation of expression in aggrecan and aggrecanases, we also obtained
data on the effect of MSCs on the structural proteins of chondrocytes.

III. Materials and Methods

Chondrocyte and Mesenchymal Stem Cell (MSC) Harvesting and Culturing

Chondrocytes and MSCs were thawed from liquid nitrogen and plated in T-175
flasks. Chondrocytes were from the left stifle of a clinical case, while the MSCs were
from the bone marrow of a separate horse. One flask contained chondrocytes and one
flask contained MSCs. Each flask contained 2 vials of cells originally frozen at 5 x 106
cells/vial. Respective media was added to each flask to a total volume of 35mL. Protocol
details for harvesting, freezing, and media composition can be found in the Appendix.

Lipopolysaccharide (LPS) Administration

LPS was used to create an arthritic-like response, mimicking a general immune
response. Lipopolysaccharide (Sigma; 055:B5, lyophilized powder form) derived from E.
coli was used at a final concentration of 20g/mL within wells. Powder was resuspended
to a stock solution of 1mg/mL in Minimal Essential Medium (MEM). The stock was
further diluted to final concentration using chondrocyte basal media.

Plating
There were 4 treatment groups for this experiment:
1. Negative Control: Chondrocytes only
2. Positive Control: Chondrocytes + LPS (20g/mL)
3. Chondrocytes (normal well) + MSCs (transwell)
4. Chondrocytes (normal well) + MSCs (transwell) + LPS (20g/mL)
Figure 4 displays a layout of the plate.

Figure 4: 24-well plate treatment layout. MSCs were placed within transwells that share media
with the chondrocytes. Yellow designates the addition of LPS treatment. Gray wells were empty.
MSC transwells were cultured separately from chondrocytes until LPS addition.

After cells were unfrozen and cultured in flasks for 24 hours, they were washed
with Hanks Buffered Salt Solution (HBSS) and treated with trypsin. Cells were counted
using 100L of resuspended cell solution. Cell count was determined using the labs
standard cell counting procedures (see Appendix).
Cells were plated in two 24 well plates with transwells (Millipore 0.4m PTFE
membrane). Each plate allowed for 16 total samples (32 total). Two plates were used for
each molecule, allowing for 8 replicates for each treatment group. Chondrocytes were
plated at a density of 300,000 per well (158,000/cm2). MSCs were plated at a density of
100,000 per transwell (303,000/cm2). At this time, they were still separate from
chondrocytes. Media for respective cells were added to a volume of 1 mL.

After 24 hours, media for respective cells was changed to serum free, antibiotic
free basal media to a volume of 1mL. This was to ensure that any response seen was not
due to cellular response to factors within the serum or antibiotics.
24 hours after the media change, LPS was added to respective wells. At this time,
MSC transwells were also placed into respective co-culture wells. Chondrocyte basal
media was added to all wells. Total volume for all wells was 1mL. Final LPS
concentration for treated wells was 20 g/mL.

RNA Isolation and Purification

Cell monolayers were harvested 24 hours after LPS addition. RNA was isolated
and purified from chondrocytes using the 5 Prime PerfectPure RNA/Tissue Kit-50. 24
hours following LPS addition, media was aspirated and 400 L of provided lysis solution
with 1% TCEP was added to dislodge and lyse cells. Next, the lysis mixture was added to
the provided Clear Purification Columns. RNA purification was done according to the
protocol provided in the kit. RNA was then measured for concentration and purity using
NanoDrop Spectrophotometer ND-1000 (NanoDrop Technologies, Wilmington, DE).
RNA samples were then diluted to 5 ng/mL to standardize concentrations.

TaqMan Real-Time PCR

Purified and diluted RNA samples were analyzed using TaqMan quantitative
real-time polymerase chain reaction (PCR), the TaqMan One-Step RT-PRC (ABI Prism
7900HT Sequence Detection System, Applied Biosystems, Foster City, CA) and
associated SDS 2.1 software to quantify gene expression.

Gene markers analyzed included TNF-, IL-1, IL-1, and IL-6. 18S ribosomal
RNA was also run to use as a normalizing value. For each marker, a master mix was
made containing:
1. 5M forward primer (Integrated DNA Technologies, Coralville, Iowa)
2. 5M reverse primer (Integrated DNA Technologies)
3. 10M fluorescent probe (Integrated DNA Technologies)
4. 2x Universal Master Mix (Applied Biosystems)
5. 40x Multiscribe (Applied Biosystems)
5L of RNA sample was added to 45 L of the mix for each well sample. In
addition, 5L of standards were added to 45 L of the mix (six standards for 18s, seven
standards for other markers). Lastly, a no template control (NTC) was made with blank
solution instead of RNA. These steps were repeated for each marker before running the
PCR cycle. Copy numbers of RNA was measured for each sample based on extrapolation
from the standard curves.

Statistical Analysis and Data Plotting

SDS 2.1 software was used to display real time PCR results on the Taqman
instrument. Data was exported to Microsoft Excel to normalize all gene markers to 18s
values. Statistical analysis was completed using Statistix 8.0 software (Analytical
Software, Tallahassee, FL). This allowed for the determination of group means and
standard deviations. Significant differences and groups were assigned by ANOVA using
the Tukey HSD test for multiple comparisons with a P-value of p<0.05. Plots of this data
was created using SigmaPlot 2000 (SPSS Inc., Chicago, IL). Data is displayed with

group means and +/- standard deviations listed on the error bars. Statistical significances
are labeled with letters at the top of each data column.

IV. Results

The Materials and Methods described above served as a model for inducing an
arthritic-like state in chondrocytes. The culturing method allowed for the isolation of
chondrocyte RNA only. The described treatment groups included the negative control
(chondrocytes only) and the positive control (chondrocytes + LPS). An additional control
(chondrocytes + MSCs, without LPS) was also added. This model allows for the isolation
of variables to determine the actual effect of MSCs on chondrocytes in LPS conditions.
The hypothesis is primarily targeted at comparing the chondrocytes + MSC + LPS group
with the chondrocytes + LPS group, while considering the results of the controls.

Normalized TNF-alpha Expression in Chondrocytes from Chondrocyte/MSC

Co-Culture Treatment Groups
2.5

TNF- Expression
(TNF-/18S Copies)

2.0

1.5

1.0
AB
A
0.5

0.0
Chond

Chond
LPS

Chond
MSC

Chond
LPS
MSC

Treatment

Chondrocytes Only
Chondrocytes + LPS (20g/mL)
Chondrocytes + MSCs
Chondrocytes+ MSCs + LPS (20 g/mL)

Figure 5: TNF- Expression in Chondrocytes by Treatment Group. All treatments groups were
cultured for 24 hours. Different letters indicated statistical significance as determined by Tukey HSD
testing at p<0.05. Values are mean +/- standard deviation. n=8

Figure 5 shows that chondrocytes by themselves express TNF- at low levels

compared to those stimulated with LPS. In fact, LPS stimulated chondrocytes expressed

TNF- at three times the levels of the negative control group. The group of chondrocytes
+ MSCs + LPS showed significantly lower TNF- expression compared to the positive
control group. The presence of MSCs attenuated the TNF- response by approximately
50%. Also, while the presence of MSCs seemed to slightly elevate TNF- levels without
LPS, the response was not statistically significantly different from the negative control.
Normalized IL-1 Expression in Chondrocytes from Chondrocyte/MSC
Co-Culture Treatment Groups
30000

25000
C

IL-1 Expression
(IL-118S copies)

20000

B
15000

10000

5000

A
0

Chond Chond Chond Chond

MSC LPS
LPS
MSC

Treatment
Chondrocytes Only
Chondrocytes + LPS (20g/mL)
Chondrocytes + MSCs
Chondrocytes + MSCs + LPS (20g/mL)

Figure 6: IL-1 Expression in Chondrocytes by Treatment Group. All treatments groups were cultured
for 24 hours. Different letters indicated statistical significance as determined by Tukey HSD testing at
p<0.05. Values are mean +/- standard deviation. n=8

Figure 6 indicates the LPS induction of IL-1 was substantial. The positive
control expressed IL-1 at levels 104 higher than the negative control. For IL-, the coculture of chondrocytes did not significantly affect gene expression without LPS
treatment. Lastly, co-culture with MSCs and LPS significantly decreased IL-1
expression in chondrocytes.

Normalized IL-1 Expression in Chondrocytes from Chondrocyte/MSC

Co-Culture Treatment Groups
160
B
140
B

IL-1 Expression
(IL-1/18S copies)

120
100
80
60
40
20

A
0
Chond

Chond
LPS

Chond
MSC

Chond
LPS
MSC

Treatment

Chondrocytes Only
Chondrocytes + LPS (20g/mL)
Chondrocytes + MSCs
Chondrocytes + MSCs + LPS (20g/mL)

Figure 7: IL-1 Expression in Chondrocytes by Treatment Group. All treatments groups were cultured
for 24 hours. Different letters indicated statistical significance as determined by Tukey HSD testing at
p<0.05. Values are mean +/- standard deviation. n=8

Figure 7 shows that LPS greatly increases IL-1 expression in chondrocytes.

Again, the IL-1 expression was low both in the negative control and the co-culture of
chondrocytes and MSCs without LPS. However, for this cytokine, the addition of MSCs
in the presence of LPS did not significantly alter gene expression.
Normalized Logarithmic IL-6 Expression in Chondrocytes from Chondrocyte/MSC
Co-Culture Treatment Groups

7
D

Log (IL-6) Expression

(IL-6/18S Copies)

B
A

0
Chond

Chond
LPS

Chond
MSC

Treatment

Chond
LPS
MSC

Chondrocytes Only
Chondrocytes + LPS (20g/mL)
Chondrocytes + MSCs
Chondrocytes + MSCs + LPS (20g/mL)

Figure 8: Logarithmic IL-6 Expression in Chondrocytes by Treatment Group. All treatments groups
were cultured for 24 hours. Raw normalized data was transformed into logarithmic data. Different letters
indicated statistical significance as determined by Tukey HSD testing at p<0.05. Values are mean +/standard deviation. n=8

Figure 8 displays the data for IL-6 expression in logarithmic form. For the
previous cytokines, it was evident that the raw data was normally distributed. For IL-6,
the raw data did not show normal distribution. Thus, for this cytokine, the log of all raw

18S-normalized data was taken to produce a more normal distribution. This signified that
IL-6 was being expressed on a log scale.
IL-6 showed significantly different expression for all treatment groups. Again, the
control chondrocytes showed low constitutive IL-6 expression. Co-culture with MSCs
increased IL-6 expression in chondrocytes without LPS treatment. In addition, IL-6
expression is significantly increased by LPS exposure in the positive control. However,
IL-6 expression was significantly increased the most with the addition of MSCs with LPS
exposure.
Normalized Aggrecan Expression in Chondrocytes from Chondrocyte/MSC
Co-Culture Treatment Groups
60000
B

Aggregan Expression
(Aggrecan/18S Copies)

50000

AB

40000

30000

20000

10000

0
Chond

Chond
LPS

Chond
MSC

Treatment

Chond
LPS
MSC

Chondrocytes Only
Chondrocytes + LPS (20g/mL)
Chondrocytes + MSCs
Chondrocytes + MSCs + LPS (20g/mL)

Figure 9: Aggrecan Expression in Chondrocytes by Treatment Group. All treatments groups were
cultured for 24 hours. Different letters indicated statistical significance as determined by Tukey HSD
testing at p<0.05. Values are mean +/- standard deviation. n=8

Aggrecan expression (Figure 9) was highest in the negative control and the
chondrocyte + MSCs treatment group. LPS significantly decreased the aggrecan
expression compared to these two groups. The chondrocyte + MSCs + LPS treatment
group Aggrecan mean was not significantly different than either of the previous
treatments.
Normalized Aggrecanase-1 (ADAM-TS4) Expression in Chondrocytes from
Chondrocyte/MSC Co-Culture Treatment Groups

3.0e+5

Aggrecanase-1 Expression
(Aggrecanase-1/18S Copies)

2.5e+5

2.0e+5

B
1.5e+5

1.0e+5

5.0e+4

A
0.0
Chond

Chond
LPS

Chond
MSC

Chond
LPS
MSC

Treatment

Chondrocytes Only n=4

Chondrocytes + LPS (20g/mL) n=8
Chondrocytes + MSCs n=6
Chondrocytes + MSCs + LPS (20g/mL) n=8

Figure 10: Aggrecancanase-1 (ADAM-TS5) Expression in Chondrocytes by Treatment Group. All

treatments groups were cultured for 24 hours. Different letters indicated statistical significance as
determined by Tukey HSD testing at p<0.05. Values are mean +/- standard deviation. Sample numbers vary
by treatment group.

Aggrecanase-1 expression (Figure 10) was low in the chondrocyte and

chondrocyte + MSC treatment groups. LPS significantly increased Aggrecanase-1
expression to levels above 1.0 x 105 higher than baseline. The addition of MSCs to the
chondrocytes in the LPS condition did not significantly affect Aggrecanase-1 expression.
Normalized Logarithmic Aggrecanase-2 (ADAM-TS5) Expression in Chondrocytes from
Chondrocyte/MSC Co-Culture Treatment Groups

Log (Aggrecanase-2) Expression

(Aggrecanase-2/18S Copies)

3.5

3.0

B
A

2.5
A
2.0

1.5

1.0

0.5

0.0
Chond

Chond
LPS

Chond
MSC

Chond
LPS
MSC

Treatment

Chondrocytes Only
Chondrocytes + LPS (20g/mL)
Chondrocytes + MSCs
Chondrocytes + MSCs+ LPS (20g/mL)

Figure 11: Logarithmic Aggrecanase-2 (ADAM-TS5) Expression in Chondrocytes by Treatment

Group. All treatments groups were cultured for 24 hours. Raw normalized data was transformed into
logarithmic data. Different letters indicated statistical significance as determined by Tukey HSD testing at
p<0.05. Values are mean +/- standard deviation. n=8

Raw data for Aggrecanase-2 (ADAM-TS5) did not show normal distribution. The
log of all raw normalized data for was taken to produce a more normal distribution. This
signified that Aggrecanase-2 was also being expressed on a log scale. Figure 10 shows

aggrecanase was lowest in the negative control and the chondrocyte + MSCs treatment
groups. LPS significantly increases Aggrecanase-2 expression compared to these two
groups. The group containing chondrocytes + MSCs + LPS was significantly different
from the other groups, and expressed aggrecanase at the highest levels.

V. Discussion

TNF- expression in normal chondrocytes and chondrocytes exposed to LPS was

similar to that seen in previous studies [23]. There was a significant difference in the LPS
treated groups by the addition of MSCs in co-culture. MSCs seemed to attenuate the
TNF- response in chondrocytes. Because MSCs have been shown in studies to be
unable to express TNF-, being able to affect other cells has serious implications for how
MSCs can be used in the future [23]. Because only chondrocytes were assayed for gene
expression, MSCs must be able to affect cells through a soluble factor that diffuses into
the media.
IL-1 expression in chondrocytes followed a similar pattern, although the LPS
driven response was much larger than that of TNF-. This could have been due to two
main reasons. First, the relative activity of IL-1 may simply have been larger than other
cytokines. This may reveal a particular sensitivity of IL-1 to stressors such as
inflammation. Second, the large increase in IL-1 expression was only indicative of the
24 hour time point after LPS treatment. Cytokines are expressed at different levels at
different times after exposure to inflammatory stresses [9]. The data certainly shows that
at 24 hours, IL-1 is expressed at much higher levels than the other cytokines tested.
Similar to the TNF- data, the presence of MSCs also decreased chondrocyte IL-1
expression in the presence of LPS. Again, this could only be due to diffusible factors
produced by the MSCs.
LPS significantly increased chondrocyte expression of IL-6 in the chondrocyte +
LPS treatment compared to the chondrocyte only treatment. However, IL-6 has both proinflammatory and anti-inflammatory effects. The presence of MSCs significantly
increased IL-6 expression compared to only chondrocytes in the LPS absent conditions.

IL-6 expression in the chondrocyte + LPS treatment was significantly higher than both
previous treatments. Furthermore IL-6, expression was significantly higher again in the
MSC-chondrocyte co-culture with LPS compared to the chondrocyte + LPS treatment.
This was the opposite effect to TNF- and IL-1 changes in LPS conditioned
chondrocyte/MSC co-cultures. The anti-inflammatory nature of IL-6 was confirmed in
this experimental case. IL-6 expression was increased in an attempt to regulate the
amount of TNF-, IL-, and other pro-inflammatory cytokines. This was to prevent the
overproduction of cytokines without causing damage to surrounding tissues. Thus,
increased production of IL-6 by the addition of MSCs in the LPS treated condition still
supports the use of MSCs to decrease pro-inflammatory cytokines.
IL-1 expression after LPS induction was high, but certainly not as dramatic as
IL-1 levels. However, MSCs did not result in the same IL-1 reduction that occurred
with TNF- and IL-1 in chondrocytes exposed to LPS. In fact, chondrocyte IL-1
expression with MSCs and LPS was barely affected. This was quite surprising
considering the classification of IL-1 as an interleukin and its similarity to IL-1. This
may indicate that the soluble factors from the MSCs that decreased TNF- and IL-1
worked in a separate pathway, leaving IL-1 unaffected. IL-1 and IL-1 share only 24%
of the same amino acid sequence identity, but have largely identical biological functions.
The downstream pathway from these cytokines is complex, and many proteins remain
unstudied. However, it is known that IL-1 primarily signals through an autocrine or
juxtacrine mechanism, while IL-1 acts primarily in a paracrine or systemic pathway
[26]. The differences in the manner of signaling may yield some insight as to why IL-1
expression behaves in this manner.

Disregarding MSC co-culturing for each inflammatory cytokine, LPS had a

significant effect on chondrocytes, increasing gene expression to levels higher than
baseline. IL-1 and IL-1 showed the largest increase in gene expression from the
negative control, at levels 10,000 and 100 times higher, respectively. Thus, a 20g/mL
dose seemed to be a good stimulation of inflammatory cytokines without causing death to
the cells.
It is clear that both MSCs and LPS had a significant effect on the aggrecan
expression in chondrocytes. Aggrecan expression was highest in the chondrocyte only
and chondrocyte + MSCs treatments. Aggrecan expression was significantly lower in the
chondrocyte + LPS condition, signifying decreased structural protein support in
chondrocytes. MSCs did not significantly affect the mean aggrecan expression in
chondrocytes in the presence of LPS compared to the previous arthritic condition. MSCs
would probably have limited effects on increasing the aggrecan framework for cartilage.
Since aggrecanase works to cleave aggrecan, it should be correlated with a more
pathological state. Many of the samples in the chondrocyte only and chondrocyte + MSC
treatments did not amplify during PCR. These were also the two groups with the lowest
Aggrecanase-1 expression. It is very likely that the presence of RNA was so low that no
amplification occurred. Aggrecanase-1 expression was significantly increased in both the
LPS treated conditions compared to the treatments without LPS. MSCs had no effect on
the Aggrecanase-1 expression. Aggrecanse-2 expression in chondrocytes was lowest in
the chondrocyte only and chondrocyte + MSC treatments. The LPS induced state showed
significantly higher aggrecanase than the previous two treatments. However, the

chondrocyte + MSCs + LPS condition showed the highest aggrecanase expression. This
means in an LPS- induced state, MSCs actually increased the degradation of aggrecan.
It is important to remember that using LPS as a model for an arthritic-like state
certainly has limitations. LPS is an endotoxin and most closely resembles an infection by
bacteria. This experiment used the inflammatory aspects of LPS in this experiment to
mimic the possible effects of osteoarthritis. A real clinical case of osteoarthritis would
certainly be more complicated than the stimulus presented here. In addition to the tested
cytokines, osteoarthritis would involve additional genes, proteins, and factors, many of
which are still unknown. However, for the purposes of testing the modulatory effects of
MSCs on certain inflammatory cytokines, the LPS model worked well.
The efficacy of using LPS as a model for cartilage protein damage was confirmed
in this experiment. In general, LPS significantly decreased aggrecan expression while
increasing aggrecanase expression. These results suggest that LPS can be associated with
a weaker protein framework for cartilage. However, the results suggested that MSCs in
co-culture would not be a suitable treatment of OA in terms of aggrecan or aggrecanase
expression. At best, MSCs do not significantly affect aggrecan or aggrecanase. In this
experiment, MSCs actually increased Aggrecanase-2 expression in chondrocytes exposed
to LPS. The results suggest simple exposure to MSCs would not be effective in repairing
structural damage done to cartilage.
Based on the TNF- and IL-1 results, the treatment of OA with MSCs would
probably have mixed results for chondrocytes in osteoarthritis in terms of inflammation.
Because these cytokines are the most significant mediators of osteoarthritis, an optimal
treatment would attenuate the expression of both. However, based on the results of this

experiment, MSCs decreased the TNF- expression, but not IL-1 expression. Despite
the possibility that MSCs may not be an optimal treatment for chondrocytes in
osteoarthritis cases, the experiment certainly showed that MSCs still have antiinflammatory effects. Even if MSCs were used in OA cases, there would likely still be a
decrease in TNF- and IL-1 expression, and an increase in IL-6 expression, most likely
decreasing the complications arising from inflammation. At the very least, MSCs have
proved that they would likely improve inflammatory symptoms in other diseases. The
modulatory effects of TNF-, IL-1, and IL-6 could potentially be used in ailments such
as cancer, injury, infection, and other forms of arthritis wherever cartilage would be
affected. MSCs could decrease the inflammatory response, promoting faster healing
while decreasing undesirable symptoms and discomfort for the horse.
Overall, this experiment has shown that MSCs do have anti-inflammatory
properties for certain cytokines expressed in chondrocytes. There are several further steps
to take in exploring the use of MSCs as treatment. First, studies should be done to
understand why IL-1 behaved differently that the other pro-inflammatory cytokines.
Second, work should be aimed at testing the plethora of cytokines not presented in this
experiment. Based on which cytokines are modulated and the manner in which the genes
are expressed, this would increase our understanding of how the MSCs are really
affecting these changes. Third, work needs to be done to identify the factor(s) responsible
for this change. This would involve isolating the MSC media and assaying for factors that
could be responsible for this change. Fourth, other methods of inducing an arthritic-like
state should be tested. Results would likely be different for each method, but this would
yield a more complete analysis of how genes are actually being modified. Lastly, other

types of cells need to be tested in addition to chondrocytes. Synoviocytes, tenocytes, and

cells found in ligaments would be possible targets for the exploration for healing in
damaged soft tissue. In addition, lymphocytes, neutrophils, monocytes, and macrophages
would also be possible targets for further exploration in immunity and inflammation.
Clearly, there is still much work to be done in this field. As MSCs become increasingly
accepted in clinical cases, it is likely that they will supplement current treatments. In the
future, treatment for osteoarthritis will likely be multi-faceted with MSCs representing
only a single approach.

VI. Appendix

Mesenchymal Stem Cell Isolation

Equipment for Collection:

60 cc syringes

Aspiration Needle (Monoject)*

10,000 U/ml Heparin

Sterile Saline (0.9% NaCl)

Betadine/Alcohol

Gauze and Clippers

3 cc syringes

Needles

*Note: Monoject Bone Marrow Biopsy/Aspiration needles

product number is 8881247111, supplied by Kendall.

Preparation for Collection:

1. Dilute Heparin to 6,000 U/ml:
a. Add 2.65 ml of sterile saline to a 4 ml bottle of 10,000 U/ml Heparin or
b. Remove 60 ml of saline from a 100 ml bottle, then add 15 vials of Heparin
(10,000 U/ml 60 ml)
2. Aseptically draw up 5-10 ml of diluted Heparin (6,000 U/ml) into 60 cc syringes.
3. Collect bone marrow aspirates from the sternum and/or tuber coxae.
4. After harvest, cap syringes with extra needles and then place on ice until processing.

Processing/plating Aspirate:
1. In hood, transfer 15 mls of aspirate to 50 ml conical vials, then add 35 mls of sterile
Earles or Geys BSS and gently rinse.
2. Centrifuge at 260 x g for 15 minutes.
3. Carefully aspirate supernatant.
4. Gently re-suspend pellet with 35 ml MSC medium (see below) and plate cells in T175 tissue culture flasks. Use one T-175 per 30 mls of aspirate collected.
5. Let aspirate/media sit for 48 hours, then aspirate medium, rinse with HANKS BSS, and
replace 35 ml of culture medium.
6. Monitor progress of cell growth and freeze following one passage at 5 million cells per
vial.

Chondrocyte Isolation
Preparation for Cartilage Harvest:
1. Prepare sterile Earles or Geys Balanced Salt Solution with Pen/Strep added.
Aliquot 50 mls to a sterile 100 ml media bottle. Prepare one bottle for each joint
to be isolated (5 for a normal horse) in addition to an additional bottle for rinsing
the cartilage during collection (an additional 150 mls).
2. Tare sterile perti dishes.
Isolation procedure:
1. Transfer the cartilage pieces to a tarred, sterile Petri dish (the swirl and dump
method works the best).
2. Suction off the remaining Earles and Geys BSS completely.
3. Weigh and record the weights of cartilage for each joint and calculate the total
grams of cartilage from all joints.
4. Add 30 mls of chondrocyte medium (F12 with serum and supplements) to each
Petri dish to bathe the cartilage while chopping. Store the Petri dishes with
cartilage and medium in the refrigerator between chopping and digestion
5. Dice the cartilage pieces into small slivers of cartilage about 2-3 mm in width
using a 10 blade (22 blades seem to dull faster). Remember to change the blades
frequently using a pair of sterile hemostats or needle pullers.
Note: Sharp blades make chopping easier and are better for the cells!
6. When all the joints are chopped, prepare the collagenase:
a. Calculate the amount of collagenase medium needed:
- Make 10 mls for every 1 gram of cartilage.
- Add an additional 5% to account for loss in filter, etc.
b. Make chondrocyte media (500 mls F-12, L-glut, Ascorbic Acid, alphaketo, pen/strep, HEPES, 50 mls FBS). You do not need to sterile filter this
prior to adding collagenase.
c. Add 0.75 mg of collagenase per ml of total volume needed (0.075%)
d. Sterile filter the collagenase through a 0.2 m filter.
e. Make sure to record the lot number of the collagenase used; viability and
chondrocyte number can vary by lot.
7. Label Erlenmeyer flasks with the following information: Joint and mls of
collagenase to add. Make sure you choose an appropriate size flask for the volume
of collagenase (use the 250 ml flask for less than 100 mls of collagenase, and the
500 ml Erlenmeyer for anything greater than 100 mls).
8. Fill the Erlenmeyers with the calculated volume of collagenase.
9. In the hood, aspirate medium off of the cartilage slivers in the Petri dishes and
transfer the cartilage using sterile spatulas into the appropriate Erlenmeyer flask.
10. Loosely tighten the Erlenmeyer cap and transfer to the incubator and spin at 100150 rpm overnight on the Corning Slow Stirring Controller.

Note: Do NOT use a normal stir plate, as they produce too much heat and will
cook your cells! If you have to use a normal stir plate, you must insert a
Styrofoam rack of at least 1.5 to insulate the heat.
11. Calculating the amount of time for digestion depends mainly on the age of the
horse, the size of cartilage pieces and collagenase lot, but here are some time
ranges per age of horse:
<1 day old: ~12 hours
< 1 month: ~14 hours
3-6 months: 16-18 hours
~1 year old: 20 hours
** Note: you should monitor the digestion, these times do vary.
12. After most of the cartilage is digested (when there is only a small amount of
cartilage specks in the collagenase), start filtering the cell suspension through a
sterile funnel containing a base layer of 44 m mesh and 4 layers of cheesecloth
into 50 ml conical tubes.
Note: In newborn horses, there will be a good portion that does not digest, as it is
very easy to collect into the A-E complex (you can observe bone specs by seeing
pores in the chunks).
13. Wash out the Erlenmeyer flask and rinse funnel with spare chondrocyte medium.
14. Centrifuge the 50 ml conical tubes at 260xg for 10-15 minutes.
15. Before removing the supernatant look through the tubes. You should be able to
clearly read the numbers through medium. If not, then re-centrifuge the tubes or
spin down the supernatant in another tube.
16. Resuspend the cells from each joint in ~35 mls of chondrocyte medium and
remove a small portion (~0.3 ml) for counting.
17. Viability and Cell Count:
a. In a small test tube combine:

100 l cell suspension

100 l diluted fluorescein diacetate
60 l stock propidium iodide
740 l medium
Mix counting solution well and load a hemocytometer with 10 l and count 10
squares using the fluorescent scope (green=alive, red=dead).
b. Calculate the viability and number of total cells:
Live cells
x 10 x 10,000 (dilution) x Volume of Original Sample
Squares counted

18. Centrifuge cells again at 260g to prepare for freezing.

19. Chondrocytes are frozen at 10 million cells per ml (and 1 ml per cryovial). Start
labeling cryovials with horses code and vial number and prepare freeze medium:
-Freeze Medium (Chondrocyte medium with 10% FCS and 10% DMSO)
For 100 mls: 80 mls F-12 Complete
10 mls FCS

10 mls DMSO
Sterile filter through a 0.2 m filter.
20. Remove supernatant from cell pellets (being careful not to disturb the pellet) and
add the appropriate amount of freeze medium to the pellet. Quickly resuspend the
chondrocytes and aliquot 1 ml per cryovial.
Note: It is important to work as quickly as possible when adding DMSO to the
cells. Also, save the conical tubes when you are done to rinse with medium and
plate in a 6-well plate to check for contamination.
21. After one joint is in cryovials, transfer the cells to a cryovial box and then to a
Styrofoam box (at least 1 in thickness) in the -80C freezer.
22. Let the cells freeze at -80C overnight and then transfer to the liquid nitrogen
tank.
23. When you are done, check through the log to make sure you have written down
the lot number of collagenase and duration of digestion.

Mesenchymal Stem Cell Isolation Media

1. 500mL Low Glucose DMEM containing:
- 5 mL l-glutamine (200mM stock)
- 5mL sodium pyruvate (100mM stock)
2. 12.5mL 1M HEPES Buffer
3. 50mL fetal bovine serum
4. 5mL Penicillin/Streptomycin (10,000 IU/mL Pen, 10,000 g/mL Strep stock)
5. 25L bFGF (basic fibroblast growth factor- 100g/mL stock)
Mesenchymal Stem Cell Basal Media (no antibiotics, no serum)
1. 500mL Low Glucose DMEM containing:
- 5 mL l-glutamine (200mM stock)
- 5mL sodium pyruvate (100mM stock)
2. 12.5mL 1M HEPES Buffer
3. 25L bFGF (basic fibroblast growth factor- 100g/mL stock)
Mesenchymal Stem Cell Freeze Media
1. 80mL Mesenchymal Stem Cell media
2. 10mL fetal bovine serum
3. 10mL Dimethyl Sulfoxide (DMSO)
Chondrocyte Complete Media
1. 500mL Hams F-12 containing
- 5mL l-glutamine (200mM stock)

2. 12.5mL 1M HEPES Buffer

3. 50mL fetal bovine serum
4. 5mL Penicillin/Streptomycin (10,000 IU/mL Pen, 10,000 g/mL Strep stock)
5. 5mL -ketoglutarate (3 mg/mL stock)
6. 5mL Ascorbate (5mg/mL stock)
Chondrocyte Basal Media (no antibiotics, no serum)
1. 500mL Hams F-12 containing
- 5mL l-glutamine (200mM stock)
2. 12.5mL 1M HEPES Buffer
3. 5mL -ketoglutarate (3 mg/mL stock)
4. 5mL Ascorbate (5mg/mL stock)

Chondrocyte Freeze Media

1. 80mL Chondrocyte Complete media
2. 10mL fetal bovine serum
3. 10mL Dimethyl Sulfoxide (DMSO)

Cell Counting/Viability Protocol

1. Label a glass test tube for each cell sample to be counted
2. In a separate glass test tube, mix 2.5 mL of Phosphate Buffer Solution (PBS) or Earls
Balanced Salt Solution (EBSS) with 20 L of FDA stock (50 mg dissolved FDA from
Sigma in 10 mL acetone; stored in foil-wrapped bottle in fridge; stable for about 6
months)
3. Mix in labeled tubs from step 1:
a. 100 L FDA dilution (from step 2)
b. 60 L Propidium Iodide stock (1 mg of PI from sigma dissolved in 50 mL PBS;
stored in foil-wrapped bottle in fridge; stable for about 2 yrs)
c. 740 L F12 or DMEM
d. 100 L cell suspension
4. Mix well and allow to sit briefly (<2 mins)
5. Pipette 10 L into counting chambers of hemacytometer (glass or disposable plastic)
6. Turn on UV light on microscope
7. Count cells in 5 squares on each side (10 squares total); viable cells=green, inviable
cells=orange
8. Calculate the total number of live cells (green)
a. (# green cells counted/# of squares counted) x (dilution factor=10) x
4

10 =Cell/mL
b. (Cells/mL) x (Total volume of cell suspension)= Total # of cells

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