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THE STIMULUS AND INHIBITION OF MMP-l3 IN
RAT TAIL TENDONS: THE ROLE OF MECHANICAL
STIMULUS AND EXOGENOUS INHIBITORS
By

Keri Lynn Gardner

A THESIS
Submitted to
Michigan State University

In partial fulfillment of the requirements
For the degree of

MASTER OF SCIENCE

Department of Small Animal Clinical Sciences

2007

THE STIMULUS AND INHIBITION OF MMP-l 3 IN RAT TAIL TENDONS: THE
ROLE OF MECHANICAL STIMULUS AND EXOGENOUS INHIBITORS

By

Keri Lynn Gardner

Tendinopathy, a degenerative condition of tendons associated with overuse injuries, is
characterized by catabolic alterations in the extracellular matrix secondary to increases in
matrix metalloproteinase (MMP) synthesis. While the precise etiology of tendinopathy is
unknown, some investigators believe that changes in the mechanobiological signaling
pathyways, which control the tendon cell’s response to tensile loading forces, may play a
significant role in regulating MMP gene expression. This research focuses on the role of
tendon cell load in the expression and inhibition of a specific MMP (rat interstitial
collagenase; MMP-13) mRNA in an in vitro rat tail tendon model. In addition, the
effects of exogenous and endogenous MMP inhibitors on ameliorating the degenerative
effects of MMP-13 in the model were also examined. The results of the study suggest
that loss of normal tendon cell signaling due to alterations in cell matrix interactions
result in an increase in MMP-13 mRNA expression and protein synthesis and a
subsequent degeneration of tendon material properties. These degenerative changes can
be inhibited by the application of exogenous MMP inhibitors or through an increase in
endogenous tissue inhibitors of metalloproteinases (TIMPs) secondary to cyclic tensile
loading. These findings may have significant implications in the development of

therapeutic modalities for the prevention and treatment of tendinopathy.

ACKNOWLEDGEMENTS

I would first like to acknowledge my mentor Dr. Steven Amoczky, who has guided
me through my master degree studies balancing independence and guidance along the
way. I would also like to acknowledge my master’s committee members Drs. Vilma
Yuzbasiyan-Gurkan, Cheryl Swenson, and Clifford Les who have furthered my scientific
development during my degree. Additionally, I would like to acknowledge Dr. Monika
Egerbacher, Michael Lavagnino, and Oscar Caballero for technical expertise and
experimental problem solving and support; Dr. Michael Orth for designing an
extracellular matrix special studies course; Victoria Hoelzer-Maddox for all her
assistance; and my family for supporting me in my decision to return to school.

This work was supported by The Laboratory for Comparative Orthopaedic Research
and The Wade 0. Brinker Endowed Chair of Veterinary Surgery in the College of

Veterinary Medicine at Michigan State University.

iii

TABLE OF CONTENTS

Page
LIST OF FIGURES ........................................................................... v
CHAPTERS:
I. INTRODUCTION ........................................................................ 1
References .................................................................... 5
II. LITERATURE SURVEY ............................................................... 8
Tendon Anatomy ............................................................ 8
Tendon Biochemistry ....................................................... 9
Tendon Mechanical Function ............................................. 10
Tendinopathy (Epidemiology) ............................................. 12
Tendinopathy (Pathogenesis) .............................................. 13
Tendinopathy (Pathology) ................................................. 16
Mechanotransduction ....................................................... 17
Research Models ............................................................ 20
Tendinous Injuries Overusernderuse ................................... 21
Loss of Homeostatic Tension ............................................. 23
References ................................................................... 30
III. THE EFFECT OF THE LOSS OF HOMEOSTATIC TENSION
AND EXERCISE ON THE RATIO OF TIMP/MMP mRNA
EXPRESSSION IN TENDONS: AN IN- VITRO EXPERIMENTAL
STUDY .................................................................................... 45
Introduction .................................................................. 46
Material and Methods ...................................................... 48
Results ........................................................................ 5 1
Discussion .................................................................... 52
References .................................................................... 61
IV. DISCUSSION ............................................................................. 66
Future Research Directions ................................................ 68
References .................................................................... 69

iv

Figure 2.1

Figure 2.2

Figure 2.3

Figure 3.1

Figure 3.2

Figure 3.3

LIST OF FIGURES

The division of tendons into fascicles and fibrils is shown in
this diagram

Schematic presentation of development of chronic tendon
Disorders

Algorithum of inadequate cell matrix response leading to
transient tendon weakness and continual loading exceeding
the tendon’s healing capacity resulting in an overuse injury

Graph of TIMP-l/MMP-l 3 mRN A expression ratios of O, 24hr,
48hr, and 72hr stress-deprived tendon

Graph of TIMP-l/MMP-l 3 mRNA expression ratios with
Ilomastat treatment for 24hrs

Graph of TIMP-l/MMP-l3 mRNA expression ratios with
increasing cyclic strain

Page

27

28

29

58

59

60

I. INTRODUCTION

Tendinopathy is a generic term used to describe a common condition affecting the
tendons, which causes pain, swelling, or impaired performance. It includes tendinosis,
tendonitis, paratendonitis and paratendonitis with tendinosis (Khan et al. 1999).
Tendinopathy remains one of the most common injuries encountered in sports or at the
workplace and describes clinical conditions in and around tendons resulting from over-
load or overuse. It is a major medical problem associated with sports and physical
activity accounting for 30-50% of all sports injuries and more than 48% of occupational
problems (Renstrom et al. 1991, NIOSH 1996). Treatment of tendinopathy is difficult
and problem management is often based on trial and error. The basic cell biology of
tendons is still not fully understood, and the management of tendon injury poses a
considerable challenge for clinicians.

Tendons connect muscle to bone and allow transmission of forces generated by
muscle to bone, resulting in joint movement (Sharma 2005). They have a fibroelastic
texture, which includes tenoblasts and tenocytes within the extracellular matrix network.
These cell types constitute about 90-95% of the cellular element of tendons (Kannus
2000). Collagen type I accounts for 65%-80% of the dry mass of tendons (O’Brien M,
1997) and is arranged in hierarchical levels of increasing complexity. Tropocollagen
(triple-helix polypeptide chain) unites into fibrils. Fibrils are considered the primary
bundles with fascicles being the secondary bundles, then tertiary bundles and then the
tendon itself (AnstromM 1997, Jozsa LG 1997, Movin T 1998). The ground substance

of the extracellular matrix network surrounding the collagen and the tenocytes is

composed of proteoglycans, glycosaminoglycans, glycoproteins and several other small
molecules (Kannus 2000).

When tendons transmit force from muscle to bone they act as a buffer by absorbing
external forces to limit muscle damage (Best et al 1994). The mechanical behavior of
collagen depends on the number and types of intramolecular and intermolecular bonds
(Fyfe et al 1992). A stress-strain curve helps to demonstrate the behavior of tendon.
Microscopic failure occurs when the strain exceeds 4% and beyond 8-10% strain
macroscopic failure occurs from intrafibril damage (O’Brien 1992, Butler DL 1978,
Kastelic J 1980). The tensile strength of tendons is positively related to thickness and
collagen content (Oakes BW 1998, Shadwich RE 1990).

Tendon injuries can be acute or chronic and are caused by intrinsic or extrinsic factors,
either alone or in combination (Pankaj Shanna et al 2005). It has been claimed that
intrinsic factors such as alignment and biomechanical faults (muscle weakness and
imbalance) play a causative role in two-thirds of Achilles tendon disorders in athletes
(Kvist 1991, 1994). Tendon damage may occur from stresses within physiological
limits, as frequent cumulative microtrauma (small tears or stress) may not allow enough
time for repair (Selvannett, A et al 1997). The oxygen consumption of tendons is 7.5
times lower than that of skeletal muscle (V ailas et al 1978) and this low metabolic rate
results in slow healing after injury (Williams JG, 1986). Microtrauma can also result
from non-uniform stress with the tendons, producing abnormal load concentrations and
frictional forces. between the fibrils causing localized fiber damage (Amdt AN, et a1

1998). Localized fibril damage and subsequent altered cell matrix interactions could lead

to a catabolic cascade initiated by the understimulation of the tendon cells in the damaged
portion of the tendon (Amoczky et al 2007-book).

The ability of tendon cells to sense and respond to load is central to the concept of
mechanotransduction and maintenance of tissue homeostasis (Wang & Ingber 1994,
Banes et al 1995, Ingber 1997). The precise level (magnitude, frequency, and duration)
of mechanobiological stimulation required to maintain normal tendon homeostasis is not
currently known. The tendon cells sense load through a mechanoelectrochemical sensory
system(s), which detects mechanical load through the deformation of the cellular
membrane and/or the cytoskeleton (Ben-Ze’Ev 1991; Watson 1991; Adams 1992; Wang
et al 1993, 1994; Banes et al 1995b; Ingber 1997; Brown et a1 1998; Wang 2006).

Studies have demonstrated that in situ deformation of rat tail tendon cells occurs in
response to tensile load (Amoczky et al 2002a). The relationship between changes in cell
morphology and tissue strain is thought to occur through the binding of the cell to
extracellular matrix proteins such as collagen and fibronectin (Banes et al 1995b; Rosales
et al 1995, Sung et al 1996).

Studies have suggested that tendon cells are capable of establishing an internal
cytoskeletal tensional homeostasis through interactions with their local extracellular
environment (Lavagnino 2005). Alterations in the cytoskeletal tension, secondary to
changes in the extracellular matrix strain have been shown to regulate gene response in a
reciprocal manner (Lavagnino 2005; Amoczky 2004). One of the genes known to have
increased expression with tendinosis is interstitial collagenase (Fu SC 2002; Riley 2002;
Jones GC 2006). An increase in interstitial collagenase and the resulting degradation of

the extracellular matrix collagen has been implicated in the pathogenesis of tendinopathy.

Interstitial collagenase is known as matrix metalloproteinase—l (MMP-1) in humans
and matrix metalloproteinase-13 (MMP-13) in rats. Matrix metalloproteinases (MMPs),
a family of zinc and calcium dependent endopeptidases, are involved in the remodeling of
extracellular matrix (ECM) through broad proteolytic capability (Parks & Mecham 1998;
Woessner & Nagase 2000; Oblander 2003). Degradation of collagen in tendon ECM is
initiated by MMPs (Riley 2002) and they are crucial to the development and maintenance
of healthy tendon. An imbalance in the expression of MMPs and their inhibitors, tissue
inhibitors of metalloproteinases (TIMPs), can lead to pathologic conditions such as
rheumatoid arthritis (Green MJ), metastasis (Gomez DE 1997) and tendinopathy (Fu et a1
2002). In normal tendons, it is thought that a balance between TIMPs and MMPs is
required to maintain tissue homeostasis (Magra 2005) and an imbalance of TIMP/MMP
has been implicated in tendinopathy (Jones et al 2006).

In processes that involve excessive proteolytic ECM breakdown, TIMPs can counteract
MMP-mediated tissue destruction.

A better understanding of the interactions that occur at a cellular and molecular level
during tendondegeneration may allow the development of more targeted therapeutics and
result in a greater number of positive clinical outcomes. This research will focus on the
effect of the loss of homeostatic tension and exercise on the ratio of TIMP/MMP mRNA

expression in tendons.

References

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to chemical environment and mechanical loading. J Cell Biol, 117:83-93, 1992.

Amdt, A.N., Komi, P.V., Gruggemann, G.P., Lukkariniemi, J ., Individual muscle
contributions to the in vivo Achilles tendon force. Clin Biomech (Bristol, Avon), 13:5 32-
541, 1998.

Amoczky, S.P., Lavagnino, M., Whallon, J.H., Hoonjan, A., In situ cell nucleus
deformation under tensile load: a morphologic analysis using confocal laser microscopy.
J Orthop Res, 20:29-35, 2002a.

Amoczky, S.P., Lavagnino, M., Egerbacher, M., The response of tendon cells to
changing loads: implications in the etiopathogenesis of tendinopathy. Tendinopathy in
Athletes, Blackwell Publishing, 46-59, 2007.

Banes, A.J., Tsuzaki, M., Hu, P., Brigman, B., Brown, T., Almekinders, L., Lawrence,
W.T., Fischer, T., Cyclic mechanical load and growth factors stimulate DNA synthesis in
avian tendon cells. J Biomech, 28: 1505-1513, 1995a.

Banes, A.J., Tsuzaki, M., Yamamoto, M., et al., Mechanoreception at the cellular level:
the detections interpretation, and diversity of responses to mechanical signals. Biochem
Cell Biol, 73:349-365, 1995b.

Brown, R.A., Prajapati, R., McGrouther, D.A., Yannus, I.V., Eastwook M., Tensional
homeostasis in dermal fibroblasts: mechanical responses to mechanical loading in three
dimensional substrates. J Cell Phys, 175:323-332,1998.

Ben-Ze’ev, A., Animal cell shape changes and gene expression. Bioessays, 13:207-212,
1991.

Best, T.M., Garrett, W.E., Basic science of soft tissue: muscle and tendon. In:
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Butler, D.L., Grood, E.S., Noyes, F .R., Zernicke, R.F., Biomechanics of ligaments and
tendons. Exerc Sport Sci Rev, 6:125-181, 1978.

Fu, S.C., Chan, B.P., Wang, W., Pau, H.M., Chan, K.M., Rolf, C.G., Increased
expression of matrix metalloproteinase 1 (MMPI) in 11 patients with patellar tendinosis.
Acta Ortho Scand, 73(6):658-662, 2002.

Gomez, D.E., Alonso, D.R., Yoshiji, H., Thorgeirsson, U.P., Tissue inhibitors of
metalloproteinases: structure, regulation and biological functions. Eur J Cell Biol,
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F yfe, I., Stanish, W.D., The use of eccentric training and stretching in the treatment and
prevention of tendon injuries. Clin Sports Med, 11:601-624, 1992.

Green, M.J., Gough, A.K., Devlin, J ., Smith, J ., Astin, P., Taylor, D., Emery, P., Serum
MMP-3 and MMP-1 and progression of joint damage in early rheumatoid arthritis.
Rheumatology (Oxford), 42(1):83-88, 2003.

Ingber, D.E., Tensegrity: the architectural basis of cellular mechanotransduction. Ann
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Jones, G.C., Corps, A.N., Pennington, C.J., Clark, I.M., Edwards, D.R., Bradley, M.M.,
Hazleman, B.L., Riley, G.P., Expression profiling of metalloproteinases and tissue
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Khan, K.M., Cook, J .L., Bonar, F ., Harcourt, P., Astrom, M., Histopathology of common
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Kvist, M., Achilles tendon overuse injuries: a clinical and pathophysiological study in
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Lavagnino, M., Arnoczky, S.P., In vitro alterations in cytoskeletal tensional homeostasis
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Magra, M., Maffulli, N., Molecular events in tendinopathy: a role for metalloproteinases.
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Oakes, B.W., Singleton, C., Aut, R.C., Correlation of collagen fibril morphology and
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Oblander, S.A., Somerville, R.P.T., Apte, 8.8., An update on metalloproteinases in the
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Matrix metalloproteinase activities and their relationship with collagen remodeling in
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Wang, N., Ingber, D.E., Control of cytoskeletal mechanics by extracellular matrix, cell
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I]. L ITERATURE SURVEY

Tendon An_atomy

A complete understanding of the structure and metabolism of normal tendons is
required for understanding the pathogenesis of tendon injuries and diseases. Tendon is a
soft connective tissue that usually experiences longitudinal/tensile forces as it transmits
the contraction of muscle to bone, making joint movement possible. Each muscle has
two tendons, proximal and distal. The point of union with a muscle is called the
myotendinous junction and the point of union with a bone is the osteotendinous junction.
Tendons vary considerably in shape and in the way they are attached to bone. They
range from wide and flat to cylindrical, fan-shaped, and ribbon-shaped (Jozsa, L &
Kannus, P., 1997). Tendons receive their blood supply from three main sources: the
intrinsic systems at the myotendinous junction and osteotendinous junction, and the
extrinsic system through the paratenon or the synovial sheath (Carr AJ, 1989; Kvist M,
1995)

The fibrous structure of a tendon begins with triple helical collagen molecules
assembled into fibrils and the fibrils are assembled into fibers. Bundles of these parallel
fibers are sometimes called fascicles and the whole tendon is composed of several fiber
bundles enclosed by the endotenon, which enables the tendon to glide against adjacent
tissue (V ogal 2003). The division of tendons into fibrils ensures that minor damage does
not necessarily spread to the entire tendon, and also provides a high total structural

strength (Kjaer 2004) (Figure 2.1).

Tendon Biochemistry

The major constituents of tendon are water (~ 55% of wet weight) and Type I collagen
(~ 85% of dry weight). The dominant cells of the tendon are fibroblasts, which are also
called tenocytes. They are very elongated and found in the spaces between the fibrils,
lying along the long axis (Jozsa & Kannus 1997). Tenocytes are responsible for the
production and maintenance of tendon collagen and non-collagenous constituents
(O’Brien 1997; Jozsa et al 1979; Kvist et al 1987) that are in the extracellular matrix. In
general the synthetic activity of these components is high during growth and diminishes
with age, although the activity pattern can change drastically with pathological conditions
(O’Brien 1997; Jozsa et al 1979; Kvist et a1 1987).

Collagen cross-links, discovered by David Eyre (Eyre 1997), increase as the tendon
ages, and can be used to estimate turnover rates. The cross-links within and between
mature collagen molecules provide mechanical stiffness (Puxkandl et al 2002; Avery &
Bailey 2005). They also provide resistance to breakdown by proteases, which contributes
to the remarkably long half-life of collagen in tendon (>100days)(Sell 1995). The critical
importance of cross-linking is demonstrated by a complete absence of mechanical
strength of the collagen fiber following inhibition of the cross-linking enzyme (Puxkandl
et a1 2002).

The ground substance of tendon extracellular matrix network is composed of
proteoglycans, glycosaminoglycans, glycoproteins, and several other small molecules
(Kannus et al 2000; Vogel 2003; Merrilees & Flint 1980; Jarvinen et al 1991; Kvist et al

1991). The proteoglycans are strongly hydrophilic, enabling rapid diffusion of water-

soluble molecules and migration of cells (Sharma & Maffulli 2005; Jozsa & Kannus
1997; Wolfe 1993). Mature collagen and its associated proteoglycans provide tendon
with its tensile strength and shield the intratendinous cells from injury (Scott A et al
2007). Adhesive glycoproteins, such as fibronectin, laminin and thrombospondin,
participate in repair and regeneration processes in tendon and ligament (Balint BJ 1978;
Lawler J. 1986; Miller RR, 1991; Jozsa et al 1991a). These macromolecules are able to
bind either other macromolecules together or cell surfaces together (J ozsa & Kannus
1997). Fibronectin participates in both degenerative and repair processes of tendons

(J ozsa et al 1991b). Thrombospondin, like laminin, is a multi-domain adhesive
glycoprotein that can interact with fibrinogen, fibronectin, plasminogen, glycolipids and
calcium (Lawler 1986) and is a good candidate for mediating cell-matrix interactions.

Tendon cells (fibroblasts) are responsible for tendon homeostasis, which includes
production of the components of the extracellular matrix J ozsa & Kannus). Internal
tendon cells are organized into linear arrays along the axis of the tendon, parallel to the
fascicles, separated from load-bearing collagen (Ritty et a1 2003). They have a distinct
pericellular matrix that contains the cells within descrete multicellular units in this
connective tissue (Ritty et al 2003).

In total, the complex architecture of the fibroblasts and their connection to the
extracellular matrix provides a three-dimensional network. This allows for transmission
of force through the extracellular matrix to the cell resulting in a cell signaling response.
Tendon Mechanical Function

To transmit force from muscle to bone, the tendon must be capable of resisting high

tensile forces with limited elongation (Kannus 2000; Best & Garrett 1994). Because the

10

collagen fibers within tendon are aligned in a parallel orientation, they transmit force with
minimal energy loss and deformation (Squier & Bausch 1984; Jozsa, L. & Kannus, P,
1997)

Tendon rotation, if present, can play an important role in tendon mechanics and
function. For example, Achilles tendon twists as it descends and the forces are in two
planes. These combined stresses can create unequal tensile forces on different parts of
the tendon (Reynolds & Worrell, 1991) leading to torsional ischemia and possible
damage (Clement, Taunton & Smart 1984; Smart et a1 1980). In addition to the load-
transmitting role, tendons must be flexible enough to bend at joints and they must absorb
sudden shock to limit damage to muscle (Best & Garrett 1994).

The mechanical properties of tendons are usually measured by in vitro tensile tests.
The tissue is elongated to failure at a prescribed rate while changes in force are recorded.
The force is plotted against time, but a constant displacement rate is normally used.
From this testing, tendon stiffness, load, strain time, strain to failure and energy to failure
can be measured (Butler 1978). The graphical curve is adjusted to the tendon’s cross
sectional area and length (Stone 1992; Viidik 1966). The stress-strain curve obtained
from such studies allows the calculation of various mechanical parameters such as
stiffness or modulus and is a valuable tool for studying mechanical properties of tendon.

Tendons are remarkably strong. A tendon with a cross-sectional area of 1cm2 is
capable of supporting a weight of SOO-IOOOkg. The tensile strength of tendon is actually
stronger than that of compact bone (Barnett et a1 1961) and it has more than twice the
strength of its attached muscle (Frey & Shereff 1988; Kvist ,1991). Forces that place the

highest stress on the tendon occur during eccentric muscle contractions vs. concentric

11

contractions (Fyfe & Stanish 1992; Komi 1984; Stanish et al 1985). An example of this
kind of contraction would be when stepping up a stair, the heel slides back off the edge of
the stair and drops below the foot. During eccentric contraction the whole muscle-tendon
unit lengthens and thus most strain injuries occur during such contraction (Zarins &
Ciullo 1983; Best & Garrett 1994; Noonan & Garrett 1992).

In tendinous injuries, “overuse” implies that then tendon has been strained repeatedly
until it is unable to endure further tension, then injury occurs (Curwin & Stanish 1984,
Hess et a1 1989, Renstrom & Johnson 1985). According to current concepts, repetitive
strain (3-5-8% strain) may lead to cumulative fiber microtrauma (Kannus 1997) (Figure
2.2). The microscopic structure of the tendon is disrupted by the repetitive strain and if
damage progresses, tendinosis, partial tears or complete ruptures may occur.
Tendinopathy (Epidemiology)

Since tendons allow transmission of forces generated by muscle to bone, tendon
injuries produce considerable disability that may last for months, despite appropriate
management (Almekinders & Almekinders 1994). Such injuries can lead to loss of
normal morphologic and functional characteristics of the tendon. This could lead to
altered performance and increased risk of recurrent injury (Sharma et al 2005; J arvinen et
al 2005; Jozsa et a1 1994).

Tendon overuse injury normally occurs as a result of work-induced or sports-induced
repetitive overloading of the muscle-tendon unit. Work-induced overuse injuries usually
occur in the upper extremities of workers who perform monotonous repetitive
movements (J ozsa 1997). In textile workers there was an 11.6% prevalence of overuse

tendonitis and related disorders (McCormack et al 1990). Among musicians, the

12

prevalence of performance-impairing medical problems was 76% of which two-thirds
were of musculotendinous origin (Fishbein et a1 1988).

About 10% of traumatic injuries treated in the emergency room of industrialized
countries are sustained in sports. Chronic problems caused by sports-related overuse of
tendons probably account for 30% of all running-related injuries (James et a1 1978), and
the prevalence of elbow tendinopathy in tennis players can be as high as 40% (Gruchow
& Pelletier 1979). The exact incidence of overuse injuries is complicated by difficult
diagnosis and the population at-risk is unknown, although absolute numbers of overuse
injuries has increased dramatically during the last decades due to a general increase in
sporting activities (Jarvinen 1992, Kannus el al 1988, Markison 1990, 1992,). About
one-third of sports injuries that are treated at outpatient clinics involve the knee and many
are due to overuse (Kannus 1987,1988,1989; Kujala et a1 1986; Kvist & Jarvinen 1978).
Tendinopathy (Pathogenesis)

Tendinopathy encompasses various tendon injuries including tendinosis, tendonitis,
and paratenonitis (Khan et al 1999). It can be defined as a syndrome of tendon pain,
localized tenderness, and swelling that impairs performance and is a major medical
problem associated with sports and physical activity in active people over 25 years of age
(Renstrom & Woo 2007).

Many factors contribute to the development of tendinopathy. These factors can be
extrinsic factors, such as excessive distance, intensity, technique, equipment, playing
surface and fatigue or intrinsic factors, such as malalignment, overweight, age and gender
(Jozsa et al 1989a; Malmivaara et al 1995; Nirschl 1992; Lorentzon 1988; Stanish 1984;

Renstrom & Woo 2007). Extrinsic means all factors that act externally on the body

13

(N igg 1998). It is thought that the most common extrinsic factor is excessive loads on
the body due to training errors. In a runner, a force of three to five times the body weight
ascends the lower extremity at heel strike (Mann 1982) and in a long distance runner this
heel strike could happen about three million times a year (Renstrom 1988). It is not
surprising that these causal factors result in injuries.

Intrinsic factors related to tendon injuries are entirely or partly genetically determined
(J ozsa et al 1989; Malmivaara et a1 1995; Nirschl 1992). The most common
malalignment in the foot is hyperpronation where the ankle is medially angled placing
excessive stress on the Achilles tendon (Jozsa & Kannus 1997). James et a1 (1978)
noticed that increased pronation was present in 60% of a group of injured runners.

Currently, the cause of tendinopathy is not known. The etiopathogenesis remains
unclear and many causes have been theorized. Etiology can be viewed as a failure of cell
matrix to adapt to a variety of stresses as a result of an imbalance between matrix
degeneration and synthesis (Selvanetti et al 1997; Leadbetter et a1 1992). Additionally, it
has been suggested that the destructive mechanism that precedes pathological
development may be the catabolic response of cells to the loss of homeostatic strain as a
result of microscopic tendon damage (Amoczky et al 2007; Jones et a1 2006). Proposed
etiopathologic mechanisms for tendinopathy such as: repetitive (chronic) loading
(Leadbetter 1992), ischemia reperfusion (Gordon 1991), microtrauma (Curwin & Stanish
1984; Renstrom & Johnson 1985; Ker 2002), hypoxia (Kraus-Hansen et al 1992),
hyperthermia (Wilson & Goodship 1994), and reactive oxygen species (Kannus 1997;

Bestwick & Maffulli 2000) have been suggested.

14

The intensive repetitive activity, which ofien is eccentric by nature, may lead to
cumulative microtrauma, which firrther weakens the collagen cross-linking, non-
collagenous matrix, and vascular elements of the tendon (Stanish et al 1985). Overuse has
also been speculated to cause chronic tendon problems, by disturbing the micro- and
macrovasculature of the tendon and resulting in insufficiency in the local blood
circulation. Decreased blood flow simultaneous with an increased activity may result in
local tissue hypoxia, impaired nutrition and energy metabolism, and increase in reactive
oxygen species (Clement et al 1984; Rathbun & Macnab 1970). Together these factors
are likely to play an important role in the sequence of events leading to tendon
degeneration (J arvinen 1997).

It is believed that repetitive loading and microtrauma are the most rational
etiopathologic mechanisms for tendinopathy (Kannus 1997; Kvist 1994, 1991; Jarvinen
et al 2001). Numerous investigators have suggested that overstirnulation of tendon cells,
secondary to repetitive loading, results in a gene expression pattern that can lead to
tendinopathy (Almekinders et al 1993; Skutek et al 2001; Archambault et al 2002;
Tsuzaki et al 2003; Wang et al 2003). Although pathological changes exist in
tendinopathy, the etiopathogenesis of it remains unknown (Cook & Khan 2007).

An algorithum has been theorized that overuse injuries in tendons are caused by a
failure of the tissue to adapt to repetitive microtrauma and a resultant deterioration of the
extracellular matrix over time (Archambault et al 1995). In this algorithum, repetitive
loading of a tendon is thought to elicit a cellular matrix response. This cell matrix
response can be adequate or inadequate. If this response is adequate the tissue will adapt

to repetitive load. However, an inadequate cell matrix response leads to transient

15

weakness in the tendon and continual loading will exceed the tendon’s healing capacity
resulting in an overuse injury (Figure 2.3).
Tendinopathy (Pathologv)

Tendon pathology can be examined using macroscopic features or molecular biology
techniques. Gene expression can be measured in normal and pathologic tendons (Ireland
et al 2001; Alfiedson et al 2003). The extent of tendon pathology can also be described
or quantified according to functional outcomes or by mechanical testing (Sano et a1
1998). Tendinosis involves altered expression of matrix proteins, growth factors, and
cytokines, pointing to a pathologic process involving the resident tenocytes, endotheliurn,
perivascular cells, and neurons (Maffulli et a1 1998, 2003; Khan et al 2002). Type I and
III collagen synthesis is increased concomitantly with altered matrix metalloprotease
(MMP) activity leading to pathologic state of remodeling that fails to restore tendon
architecture and function (Maffulli et al 2000; Ireland et a1 2001; Fu et al 2002a,b; Riley
et a1 2002; Tillander et al 2002; Alfredson et al 2003b).

Matrix metalloproteinases, such as interstitial collagenase, are a family of zinc- and
calcium-dependent endopeptidases involved in the normal remodeling of the extracellular
matrix of connective tissues through their broad proteolytic capabilities (Bramono et a1
2004; Parks et a1 1998; Magra & Maffulli 2005). The activity of endogenous MMPs is
normally inhibited by endogenous tissue inhibitors of metalloproteinases (TIMPs)
(Bramono et a1 2004; Parks & Mecham1998). The metabolic balance between the
activities of endogenous MMPs and TIMPs is responsible for regulating normal tendon
remodeling (Bramono et a1 2004; Dalton et a1 1995; Parks et al 1998). An excess of

MMP activity can lead to a progressive degeneration and weakening of the extracellular

l6

matrix of tendons (Lavagnino et a1 2005). Some researchers say that there is an increase
in the expression of TIMP-1 and MMP-1 in injured tendon (Jones et al 2006). ACL
injury synovial fluid samples have shown increased MMP-3 and TIMP-1 (Higuchi et al
2006), while others say there is a decrease in the expression of TIMP-1 with tendinosis
(F u et a1 2002). Still others describe an increase in serum or peritendinous fluid TIMP-1
levels with exercise (Koskinen et a1 2001 , 2004; Mackey et al 2004; Tyebjee et a1 2005).

In other tissues such as osteoarthritic cartilage, there is an increased expression of
MMPs and a decreased expression of TIMP-1. The processes that occur in cartilage may
be the same as those occurring in tendon. This disturbed balance, resulting in an excess of
metalloproteinase over TIMP, underlies pathologic cartilage destruction (Kavorkian et al
2004). From the literature it is apparent that the local balance of metalloproteinase and
TIMP activities is pivotal in regulating tissue homeostasis.
Meflanotransduction

Mechanotransduction is the process by which cells elicit a metabolic action in
response to mechanically derived signals secondary to extracellular matrix strain (Kjaer
2004; Wang 2006; Swartz et a1 2001). The ability of tendon cells to respond to load is
central to the concept of mechanotransduction and subsequent maintenance of tissue
homeostasis (Wang & Ingber 1994; Banes et al 1995b; Ingber 1997). Progressive
rehabilitation or increasingly intense rehabilitation is widely used following tendon and
ligament injuries and surgery (Gamble et a1 1984; Noyes 1977; Woo et al 1982).
However, the amount of loading necessary to improve/accelerate the healing process
without causing damage to the healing tissue remains unclear (Steadman et a1 1989;

Zeichen et al 2000).

17

Tendon cells are able to detect mechanical signals from deformation of their
cellular membrane and /or cytoskeleton through a mechanoelectrochemical sensory
system resulting in specific cellular responses (Banes et al 1995b). Previous work in our
lab demonstrated that tensile loading of rat tail tendons resulted in an alteration of cell
nuclear shape in a dose dependent manner. The cells were stained with acridine orange
and strains of 0, 2, 4, and 6% were used to deform the tendon cells resulting in thinner,
longer cells with increasing strain. Using confocal laser microscopy we were able to
demonstrate a significant, but weak, correlation between cellular (nuclear) strain and
tendon strain (Amoczky et al 2002a).

Deformation of cellular membranes can affect the control of second-messenger
molecules that can, in turn, activate a wide array of cellular machinery (Sachs 1998;
Banes et al 1995b). Another study from our laboratory has shown that cytosolic calcium
is increased in tendon cells in response to in situ deformation. As the strain is increased
(0%, 2%, 4%, 6%) the cells increased in brightness due to calcium influx. Also strain
from 0-4% produced significant cell elongation. The increase in cytosolic calcium
associated with cell deformation supports a calcium channel mediated signaling system in
tendon cells that is responsive to load (Shirakura et a1 1995). These second messengers
can activate a wide array of cellular machinery including DNA synthesis, mitosis, cell
differentiation and gene expression (Sachs 1998, Banes et a1 1995).

Mechanotransduction signals are known to be mediated through the pericellular
matrix to the nucleus via integrin-based cell matrix connections (Sachs 1988; Ingber
1991; Watson 1991; Wang et al 1993, 2007; Banes et al 1995b; Ritty et al 2003). Large

modular extracellular matrix (ECM) proteins, such as fibronectin, tenascin, laminin and

18

thrombospondin, have regions that participate in protein—protein interactions, as well as
sites that can be bound by cell-surface receptors. Each of these proteins contain both
integrin and heparin/heparan sulphate (HS)-binding sites (Labat-Robert et al 1990;
Mostafavi-Pour et al 2003; Aumailley & Krieg 1996; Aumailley & Smyth 1998;
Chowdhury et al 2003; Yurchenco et a1 1994).

Integrins participate in a wide-range of biological interactions including development,
tissue repair, angiogenesis, inflammation and hemostasis. Fibroblasts anchor to ECM by
focal adhesions that physically link with actin cytoskeleton via integrin family of ECM
receptors. Stretching induced conformational changes in the ECM may alter integrin
structure, which leads to activation of several secondary messenger pathways in the cells
(Katsumi et a1 2005). Hence, integrins are regarded as receptors capable of inducing
biochemical signals that regulate gene expression (Ingber, 1991; Schwartz et al., 1995;
Ross, 2004).

Besides playing the role of indirect mechanosensors, integrins can act as direct
mechanosensors, physically connecting the cytoskeleton to ECM, thus transmitting
mechanical loads acting on the ECM (Hynes 1992). The extracellular domain of
integrins binds to ECM proteins and functions as signaling receptors for them. Besides
integrins, other various potential biochemical mediators of mechanotransduction such as
mechanosensitive cell surface receptors (RTKs, G proteins) have also been suggested in
fibroblasts (Ruwhof & van der Laarse 2000; Wang 2006; Wang & Tharnpatty 2006).
While the cell matrix response is important to cellular homeostasis, it does not explain

the mechanisms that result in transient weakness seen in tendinopathy.

19

Research Models

Although the pathology is well characterized, the pathogenesis is still unclear.
Research models for tendinopathy have basically been divided into two groups: in vitro
and in vivo. In vitro cell culture monolayers have been used to examine the response of
tendon cells to various stimuli outside their extracellular matrix (Banes et al 1995,1999;
Tsuzaki et al 2003). In vivo models have utilized rabbits, rats, and horses to document
changes associated with repetitive loading (Nakama et a1 2005; Scott et al 2007;
Dahlgren et a1 2002). However, these models lack the ability to precisely control
confounding variables such as the ability to control or measure load and the effects of
biological variation (genes, physical shape). In vitro studies can be conducted using
monolayer cell cultures, but in this environment the cells are not in their natural cell
matrix and normal cell interactions are not duplicated. Uniform local strains are present
in monolayers, but not in a normal tendon (Berenson et al 1996; Fallon et al 2002) and
high cell numbers with increased intracellular communication may introduce a paracrine
stimulus. Conversely, in situ tissue culture would allow for natural cell matrix
interactions and visco-elasticity. The cell number and relationship are those dictated by
nature.

While some investigators have implicated overstirnulation of tendon cells as a
mechanobiological stimulus for inflammatory cytokine production and matrix
degradation (Almekinders et a1 1993; Archambault et al 2002; Tsuzaki et al 2003; Wang
et a1 2003; Bhargava et al 2004), these studies have been performed in monolayer
cultures, often including high strain magnitudes (Almekinders et al 1993; Wang et al

2003; Bhargava et a1 2004), long loading histories (Archambault et al 2002; Wang et al

20

2003; Bhargava et al 2004), and the addition of biochemical factors (Archambault et a1
2002; Tsuzaki eta12003). Thus, the clinical relevance of such studies is unclear.
Because of these reasons, we decided to concentrate on the rat tail tendon model.

This allows us to use cells in an in situ environment, numerous antibodies and techniques
optimized for rat species, and maintain tissue culture viability because of the small size of
the RTT (approximately 3x the thickness of a human hair).
Tendinous Iniuries Overuse/Underuse

In tendinous injuries “overuse” implies a repeated strain of 4-8% on a tendon that
results in the inability of the tendon to endure further tension, whereby injury occurs
(Curwin & Stanish 1984; Hess et al 1989; Micheli & Fehlandt 1992; Renstrom &
Johnson 1985). Central to this theory is the concept that excessive loading of tendons
during vigorous physical activity is the main pathologic stimulation for degeneration of
the extracellular matrix (Selvanetti et a1 1997). Some investigators have suggested that it
is the tendon cell’s response to excessive loading that initiates the degenerative cascade
that leads to tendinopathy (Skutek et al 2001; Archambault et al 2002; Tsuzaki et al 2003;
Wang et a1 2003). A recent study found that a general pattern of collagen deterioration
and tissue degeneration was common to both ruptured and tendinopathic tendons
suggesting a common, but as yet unidentified, cell-mediated, pathological mechanism
acting on both of these tendon populations (Tallon et a1 2001). However, the relationship
between the tendon overuse and loss of tendon cell tension is unclear. The level of stimuli
required to elicit these cellular responses is not clinically relevant and has only been
demonstrated in cultured cells on artificial substrates (Almekinders et a1 1993; Wang et al

2003; Bhargava et a1 2004).

21

Several animal models have been utilized attemping to create overload of tendons.
Such models would allow for evaluation at early stages of tissue overloading. One
stimulation regimen in adult rabbits over several weeks did not result in any
inflammatory or degenerative changes within or around the loaded Achilles tendon
(Archambault et al 1998). Messner et al subjected rats to repetitive electrical-induced
eccentric exercise and found that only half of the animals achieved histological changes
in and around the tendon (Messner et a1 1999). Only horses and greyhound dogs, which
are undergoing forced regimens of exercise, display overuse injuries similar to those in
humans, whereas other animals only display tendon overuse when extreme regimens are
imposed (Patterson-Kane et a1 1997; Kjaer 2004).

Experimental studies in our laboratory have documented an increase in caspase-3 gene
expression and protein synthesis as well as an increase in the number of apoptotic cells
following 24 hours of stress-deprivation (loss of homeostatic tension) (unpublished data).
Some studies suggest that apoptosis may have a role in the pathogenesis of tendinopathy
(Yuan et al 2002,2003; Hosaka et a1 2005). One ex-vivo study was able to induce
apoptosis in tendon cells following high strains (20%), but it is probable that the high
strain used in the investigation damaged tendon fibers or fibrils (Scott et al 2005). This
could result in the understimulation or underuse of the tendon cells associated with the
damaged tissue and subsequent induction of apoptosis secondary to the loss of
homeostatic tension (Grinnell et al 1999). Understimulation due to damaged rat tail
tendon fasicles in our laboratory produced an up-regulation of MMP mRN A expression
in the damaged area (Lavagnino et al 2006). The up-regulation of MMP by tendon cells

supports the role of mechanobiological under-stimulation (loss of hemeostatic tension) of

22

tendon cells in gene expression which could weaken the tendon putting the ECM at risk
for further damage with subsequent loading (Lavagino et a1 2005).
Loss of Homeostatic Tension

Tendon fibroblasts are capable of establishing a cytoskeletal tensional homeostasis
through interactions with their local extracellular environment (Brown et a1 1998;
Lavagnino & Arnoczky 2005). This internal cellular tension has been shown to regulate
gene expression in tendon cells and establish the cell’s “calibration point” (Lavagnino
and Arnoczky 2005). Mechanical forces, which exert additional tension (above and
beyond this homeostatic calibration point) to the cytoskeleton, will elicit an anabolic
response gene, while an absence of mechanical stimuli (or a decrease below the
homeostatic level) will elicit a catabolic gene response (Arnoczky et al 2004; Lavagnino
et a1 2003; Lavagnino & Arnoczky 2005). A decrease in extracellular strain in tendons
(loss of homeostatic tension) has been associated with an increase in the up-regulation of
interstitial collagenase and a subsequent decrease in the tensile properties of these tissues
(Arnoczky et al 2004; Lavagnino et a1 2005). Therefore, it is possible that
mechanobiological understimulation or underuse of tendon cells, due to an alteration in
cell—matrix interactions, could also be an inciting factor in the etiology of overuse
injuries.

Previous biomechanical studies have suggested that isolated collagen fibril damage
occurs near the end of the linear portion of the load deformation curves of ligaments and
tendons (Viidik 1972; Kastelic et al 1980; Woo et al 1982). While this damage may not
affect the ultimate tensile strength of the tissues (Panjabi et al 1996) it could alter the

cell—matrix interactions within the damaged portion of the tendon. The alteration of cell-—

23

matrix interactions secondary to isolated fibrillar damage could cause a
mechanobiological understimulation (underuse) of tendon cells, which has been shown to
result in an upregulation of interstitial collagenase mRNA expression and protein
synthesis (Lavagnino & Arnoczky 2005). This, in turn, could weaken the tendon and put
more of the extracellular matrix at risk for further damage with subsequent loading
(Lavagnino et a1 2005).

As noted previously Matrix metalloproteinases (MMPs) have been shown to be
increased in equine and human tendinopathy cases (Clegg et al 2005; Riley et al 2005;
Jones et a1 2006). Increased expression of MMPs has been associated with a decrease in
the material properties of tendons and other connective tissues (Lavagnino et al 2005;
Zernicke et al 1995; Smith et a1 1997; Hanemaaijer et a1 1998). Therefore, inhibition of
MMPs may play a role in limiting the degradative effects of MMPs (Woessner & Nagase
2002; Baker et al 2002).

MMP inhibitors have been shown to limit articular cartilage degradation (Billinghurst
et al 1999; Smith et al 1998; Yu et al 1992) and have been used extensively in treating
periodontal disease (Ryan et al 1996). Also, they have been used as promising candidates
for prognostic markers in cancer as well as possible treatments. The proteolytic enzyme
systems, which involve MMPS, are responsible for the turnover of the ECM in normal
physiological systems but it is believed that overexpression of the enzymes in tumors
render the cells capable of breaking down the ECM allowing cells to invade surrounding
tissue (Wurtz et a1 2005). The expression and activation of MMPs are increased in
almost all human cancers (Chenard et al 1998; Nakopoulou et al 2002a; Ranuncolo et a1

2003; Talvensaari-Mattila et al 2003; Leppa et al 2004) and TIMP-1 has been shown to

24

be upregulated in numerous malignancies including breast cancer (Ree et al 1997;
McCarthy et al 1999; Nakopoulou et al 2002b; Schrohl et al 2003, 2004). The
upregulation of TIMP-1 in cancer together with the association to a poor prognosis could
reflect the fact that the balance between expression of MMPs and TIMP-1, although still
favoring MMPS, is at a higher overall level than in non-cancerous conditions (Wurtz et al
2005). Therefore, high levels of TIMP-1 levels would be associated with a worse
prognosis but would not be the cause of it (Egeblad & Werb 2002). Exogenous MMP
inhibitors such as Batimastat and Merimastat have been developed for therapeutic
benefits but the growth of the MMP family has made the task complex. Some tumor
types will depend more heavily on MMPs for their invasive growth than others (Parks &
Mecham 1998). Hydroxamate inhibitors (G1168, GIl73) caused significant inhibition
and necrosis of subcutaneous prostate tumors in nude mice (Conway et al 1996). These
new treatments will target tumor reliance on its collaboration with nonmalignant tissue,
which may be cancer’s “Achilles heel” (Parks & Mecham 1998).

A study from our lab questioned whether exogenous MMP inhibitors could prevent
the decrease in material properties associated with the loss of homeostatic tension in rat
tail tendons. The results of the study demonstrated that short-term (7d) administration of
broad-spectrum MMP inhibitors minimized the loss of material properties associated with
the mechanobiological under-stimulation of tendons in vitro (Arnoczky et al 2007). One
clinical study demonstrated the beneficial effects following the local application of a
protease inhibitor in the treatment of Achilles tendinopathy (Capasso et al 1993). The
results of these studies support the beneficial effects of exogenous MMP inhibitors on

limiting the degradative effects of endogenous MMPs on tendons.

25

Therefore, the purpose of this study was to determine the effect of in vitro loss of
homeostatic tension or cyclic loading (exercise) on the gene expression ratio of TIMP
(endogenous inhibitor) to MMP in rat tail tendons since the metabolic balance between
the activities of endogenous MMPs and TIMPS is responsible for regulating normal
tendon remodeling (Bramono et a1 2004; Dalton et a1 1995; Parks et al 1998). These
exercise regimes have focused on the use of eccentric loading techniques, which

increases tendon strain (Bramono et al 2004; Dalton et al 1995; Parks et a1 1998).

26

T ndo

Tendon Structure 6 n g ,
Tertiary -

fiberb ndle -.-

 
 
 
  
 
 
 
  
  

Seconda
fiber bund e .
(fascicle) "
Primary . ~
fiber bundle frjg‘jj-‘f .
I ' “

Collagen
fiber

Figge 2.1 The division of tendons into fascicles and fibrils is shown in this diagram

(Shanna & Maffulli 2005).

27

 

 

 

 

 

 

 

 

tendon rupture
Stress overuse injury M
5 - 8% / g E
3- 5% /
physiologic =-__=
‘1— » .
1-3% /
O - 1%
W
t .1 l i . l l l —>
1 2 3 4 5 6 7 8 Strain (%)

 

 

 

Figgre 2.2 A schematic presentation of the development of chronic tendon disorders.
According to current concepts, repetitive tendon strain (3-5-8% strain) may lead to

cumulative fiber microtrauma. If the reparative capacity of the tendon tissue is exceeded,

overuse injury can occur (Kannus 1997).

28

 

 

 

Repetitive mechanical loading of a tendon

~l
‘

 

 

 

 

Cellularlmatrix response

 

    

 

 

 

 

 

Inadequate
Transient '
weakness
Adafieptatlon In tendon

 

 

.1.

Continued loading exceeding
the tendon's heallng capability

 

 

 

 

 

OVERUSE INJURYIJ

 

 

Figure 2.3 In this algorithum, repetitive loading of a tendon is thought to elicit a cellular
matrix response. This cell matrix response can be adequate or inadequate. If this response
is adequate the tissue will adapt to repetitive load. However, an inadequate cell matrix
response leads to transient weakness in the tendon and continual loading will exceed the
tendon’s healing capacity resulting in an overuse injury (adapted from Archambault

1995).

29

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44

Chapter III

The effect of stress-deprivation and cyclic loading
on the TIMP/MMP ratio in tendon cells: an
in-vitro experimental study

Keri Gardner, Steven P. Arnoczky, Oscar Caballero, Michael Lavagnino

45

Introduction

Tendinopathy, a syndrome of tendon pain, localized tenderness, and swelling that
impairs performance, is a major medical problem that accounts for 30-50% of all sports-
related injuries (Renstrom 1991) and 48% of reported occupational maladies (N IOSH
1996). While many factors have been linked to the development of tendinopathy, very
little is understood about its pathogenesis. Recent clinical and experimental studies have
suggested that matrix metalloproteinases (MMPs) may play a significant role in the
catabolic cascade that has been associated with tendinopathy (Alfredson et a1 2000; Fu et
al 2002; Ireland et a1 2001; Jones et al 2006; Lo et a1 2004; Magra & Maffulli 2005; Riley
2004; Riley et al 2002; Sharma & Maffulli 2005). While a variety of MMPs have been
shown to be increased in clinical cases of tendinopathy (Alfredson et a1 2000; F 11 et a1
2002; Ireland et a1 2001; Jones et a1 2006; Lo et al 2004; Magra & Maffulli 2005; Riley
2004; Riley et a1 2002; Shanna & Maffulli 2005; Riley 2005), the mechanism(s) that
trigger this increase are unclear. It has been suggested that the destructive mechanism(s)
that precedes overt pathological development of tendinopathy may, in fact, be a MMP-
mediated catabolic response by the tendon cells to the local loss of homeostatic strain as a
result of isolated, microscopic, collagen fiber damage (Jones et al 2006; Arnoczky et a1;
Arnoczky et a1 2007). Several in vitro experimental studies have demonstrated an
increase in MMP-13 (interstitial collagenase) mRNA and protein expression in rat tail
tendon cells following loss of homeostatic tension secondary to stress-deprivation
(Arnoczky et al 2007; Arnoczky et a1 2004; Lavagnino et a1 2005; Lavagnino et al 2003).
This increase in interstitial collagenase activity in stress-deprived tendons has produced a

series of catabolic events (collagen degeneration and disarray as well as significant loss

46

of material properties), which have been reported to occur in tendinopathy (Jarvinen et al
1997; Jozsa et a1 1990; Kannus & Jozsa 1991).

The regulation of extracellular matrix catabolism in connective tissues is mediated
by a family of homologous proteins referred to as tissue inhibitors of metalloproteinases
(TIMPS) (Brew et a1 2000). TIMPS are multifunctional proteins that inhibit the activation
of MMPs by binding in 121 stoichiometric reversible complexes with the MMPs
(Willenbrock & Murphey 1994). MMPs and TIMPS are involved in a wide variety of
physiological and pathological conditions that involve the turnover of the extracellular
matrix (Koskinen et a1 2004) and an imbalance in the TIMP/MMP ratio has been
implicated in the etiopathogenesis of tendinopathy (Fu et a1 2002; Jones et al 2006; Lo et
al 2004; Dalton et a1 1995; Magra & Maffulli 2005). A clinical study found a higher
percentage of TIMP-1 positive cells compared to MMP-1 (human interstitial collagenase)
positive cells in normal human patellar tendons (Fu et a1 2002). However, this ratio was
reversed when patellar tendons from tendinopathy patients were examined (F u et al
2002)

A recent experimental study has demonstrated that exogenous broad-spectrum MMP
inhibitors could be used to prevent the catabolic effects caused by excessive levels of
interstitial collagenase in stress-deprived tendons (Arnoczky et a1 2007). A clinical study
has also demonstrated beneficial effects following the local application of a protease
inhibitor in the treatment of Achilles tendinopathy (Capasso et a1 1993). These results
would suggest that increasing endogenous TIMP expression in tendons might be
beneficial in the prevention and/or treatment of tendinopathy. Clinical studies have

demonstrated an increase in levels of TIMP-1 in the serum and peritendinous space

47

following exercise (Koskinen et al 2004; Mackey et a1 2004; Tayabjee et a1 2005),
suggesting that cyclic loading may stimulate TIMP-1 mRNA and protein expression in
tendon cells. Such a response could reverse the imbalance of TIMP/MMP seen in
tendinopathy (Fu et al 2002; Jones et al 2006) and provide a mechanistic explanation for
the beneficial effects seen in some exercise regimes used to treat tendinopathy (Canell et
al 2001; Jensen & Di Fabio; Jonsson & Alfredson 2005; Kongsgaard et al 2006; Purdam
et al 2004; Young et a1 2005).

The purpose of this study was to determine the effect of in vitro stress deprivation
(loss of tendon cell homeostatic tension) or cyclic loading on the TIMP-I/MMP-13 (rat
interstitial collagenase) mRNA expression ratio in rat tail tendon cells. We hypothesized
that normal tendons have a positive TIMP-1/MMP-13 ratio and that loss of homeostatic
tension (stress-deprivation) will inverse this ratio through a significant increase in MMP-
13 expression. We also hypothesize that cyclic strain will increase the ratio of TIMP-1/
MMP-13 mRNA expression above basal levels in a dose dependent manner.

Materials and Methods
Drugs and Chemicals

Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS),
gentamicin, and penicillin streptomycin-fungizone solution were obtained fi'om Gibco
(Grand Island, NY). Ascorbate was from Sigma (St. Louis, MO) and ilomastat was
obtained from BIOMOL International L.P. (Plymouth Meeting, Pa).

Rat Tail Tendons
Following institutional animal care and use approval, tendons were obtained from the

tails of euthanized adult Sprague-Dawley rats that were approximately six months old.

48

The tendons were gently teased from the distal portion of each tail with forceps and
placed into a petri dish containing complete media, which consists of Dulbecco’s
Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1%
antibiotic/antimycotic solution, 0.2% gentamicin and 7.5g/L ascorbate. The tendons were
then allocated to the following experiments:
TIMP-1/MMP-I3 mRNA expression ratio with stress deprivation

To determine the effect of stress deprivation on the TIMP-l/MMP-13 ratio, tendons
were divided into 4 groups with 10 tendons per group; (1) 0 time (fresh) controls, (2)
stress-deprived 24hrs, (3) stress-deprived 48hrs, and (4) stress deprived 72hrs. Stress-
deprived tendons were maintained in the above noted media under tissue culture
conditions at 37°C and 10% C02. The experiment was repeated four times. Significant
differences between groups were determined by running multiple (6) paired t-tests with
significance set at p<0.008.
TIMP-I/MMP-I3 mRNA expression ratio with stress deprivation and 5 0M ilomastat

To determine if the increase in MMP-13 mRNA expression seen with stress-
deprivation seen in previous studies (Lavagnino et al 2003; Arnoczky et al 2004) had any
effect on TIMP-1 mRNA expression, tendons were stress-deprived in the presence or
absence of SOuM ilomastat (BIOMOL, International L.P., Plymouth Meeting, PA). A
previous study has shown that ilomastat, a broad-spectrum MMP inhibitor, will inhibit
MMP-13 mRNA expression in stress-deprived RTTs (Arnoczky et al 2007). Ten tendons
were examined in each group (1) 0 time (fresh) controls, (2) stress-deprived 24hrs, and

(3) stress-deprived 24hrs in the presence of SOuM ilomastat. Tendons were maintained in

the above noted media under tissue culture conditions at 37°C and 10% C02. The

49

experiment was repeated three times. Significant differences between groups were
determined by running an ANOVA with significance set at p<0.05.
TIMP-1/MMP-13 mRNA expression ratio with exercise

To determine the effect of cyclic loading on the TIMP-I/MMP-l3 ratio, RTTs (10 per
group) were cyclically loaded at 0.17Hz at one of three strains: 1%, 3%, or 6% for 24hrs
and compared to fresh (0 time) controls and 24hrs of stress deprivation. A sawtooth-
shaped waveform of cyclic strain was applied to the tendons using a custom-designed,
computer-controlled, stepper motor-driven device (Lavagnino et a1 2003). The grip-to-
grip length was set to 40mm using digital calipers. Tendons were placed in the device
until all visible slack was removed to approximate 0% strain. Tendons were then clamped
in the grips to prevent slipping before undergoing 1, 3 or 6% cyclic strain with a step size
of 25 um and a frequency of 0.17Hz. Tendons were maintained in complete media and
under tissue culture conditions during the loading period and the experiment repeated 3
times. At the end of the loading protocol, the entire lengths of the unclamped tendon
segments (~40mm) were collected for RNA extraction. To determine the significance of
loading on the TIMP-lfMMP-13 ratio, a linear regression analysis was performed with
significance set at p<0.05.
T IMP-1 and MMP-13 mRNA Expression

Rat interstitial collagenase (MMP-13) and TIMP—1 mRNA were evaluated for each
treatment group. Ten tendons from each group were placed in 1.0ml of RNAlater®
(Quiagen, Valencia, CA) at 4°C for at least 24hrs prior to processing. Total RNA was
extracted using the Quiagen RN Easy kit with protocol provided for fibrous tissues. RNA

(200-400ng) was converted into cDNA using the Invitrogen SuperScript III Reverse

50

Transcription system (Carlsbad, CA). Real Time Quantitative PCR was performed using
TaqMan Gene Expression Assay from Applied Biosystems (ABI, Foster City, CA).
Samples were run in a 96-well plate (20ul final volume per reaction) on an ABI 7500-
Fast Q-PCR apparatus. The endogenous control used for all Q-PCR experiments was 18S
rRNA. Results were analyzed using the Sequence Detection System software from ABI.
TaqMan probe and primer sets were obtained for MMP-13 (Rn01448197_m1), TIMP-1
(Rn00587558_m1) and 18s rRNA (Hs99999901_sl) from ABI’s Gene Expression Assay
database (http2//allgenes.com).
TIMP-I/MMP-I3 Ratio Calculations

Assuming optimal amplification reaction efficiency, it is estimated that ten copies of a
gene are generated every 3.32 PCR cycles. Based on the difference between a
hypothetical Ct value equivalent to one gene copy, 39, and the Ct value for the 0hrs
(control) samples, an estimated copy number can be calculated for the 0 hours samples
(Livak & Schmittgen 2001). This then allows for the estimation of the number of gene
COpies for the treated samples. The TIMP/MMP ratios were calculated by dividing the
number of TIMP-1 copies by the number of MMP-l3 copies for each group.
3.653115».
TIMP-1/MMP-13 mRNA expression ratio with stress deprivation

The ratio of TIMP-1/MMP-13 mRNA expression in fresh rat tail tendons was 3.73:1 i
0.73. Twenty-four hours of stress deprivation resulted in a significant decrease in (0.25:1
: 0.04), and an inversion of, the TIMP-1/MMP-13 ratio (Figure 1). The TIMP-1/MMP-

13 ratio continued to decrease significantly with increasing time of stress deprivation

(Figure 1).

51

T IMP-I/MMP—I 3 mRNA expression ratio with stress deprivation and 5 0M ilomastat

The ratio of TIMP-1/MMP-l3 mRNA expression in stress-deprived tendons treated
with 50uM ilomastat was 2.50:] 1: 1.17. This was not statistically different from fresh
controls. Tendons that were stress-deprived for 24hrs in the absence of ilomastat had an
inversed TIMP-1/MMP-13 ratio (0.37:1 : 0.35) (Figure 2).
TIMP-1/MMP-13 mRNA expression ratio with exercise

Cyclic loading at 1, 3, or 6% strain at 0.17Hz for 24hrs resulted in a weak (r2=0.35),
but significant (p=0.016) dose-dependent increase in the TIMP-1/MMP-l3 mRNA
expression ratio when compared to zero hour controls (Figure 3).
Discussion

Matrix metalloproteinases (MMPs) belong to a diverse group of enzymes that are not
only involved in restructuring the extracellular matrix, but also play a major role in
various pathophysiological conditions by virtue of their complicated expression,
activation, and regulation processes (Acharya et al 2004). The activity of MMPs is
inhibited reversibly by tissue inhibitors of metalloproteinases (TIMPs), which are
endogenous inhibitors of MMPs (Bramono et a1 2004). The balance between TIMPS and
MMPs regulates extracellular matrix turnover and remodeling during normal
development and pathology (Chirco et a1 2006). Disruption of the TIMP-MMP balance
has been associated with a number of pathological processes including tumor invasion
and metastasis, rheumatoid arthritis, osteoarthritis, and ligament injuries (Fehrenbacher et
a1 2003; Hijilla et al 2003; Kleiner & Stetler—Stevenson 1999; Sprindler et al 1996). An
imbalance in the TIMP/MMP ratio has also been implicated in the pathogenesis of

tendinopathy (Fu et a1 2002; Jones et a1 2006; Lo et al 2004; Dalton et al 1995; Magra &

52

Maffulli 2005) and the resultant increase in MMP activity reported in tendinopathy
(Alfredson et a1 2000; Fu et a1 2002; Ireland et a1 2001; Jones et a1 2006; Lo et a1 2004;
Magra & Maffulli 2005; Riley 2004; Riley et a1 2002; Shanna & Maffulli 2005) is
thought to be responsible for the progression of extracellular matrix catabolism seen in
tendinopathy.

A clinical study examining tissues from normal and tendinopathy patients found a
higher percentage of TIMP-1 immunopositive cells compared to MMP-1 (human
interstitial collagenase) positive cells (24% to 18% respectively) in healthy control
tendons (Fu et a1 2002). However, this positive TIMP-l/MMP-l ratio became inversed
(3.4% to 47%) when tendinopathy tissues were examined (F u et a1 2002). A recent study
profiling gene expression in tendinopathy patients also noted a 1000 fold increase in
MMP-l expression in ruptured tendons compared to only an 8 fold increase in TIMP
(Jones et a1 2006). The imbalance of TIMPS and MMPs in favor of the MMPs seen in
tendinopathy tissues may explain the collagenolysis reported in the histological
evaluations of these pathologic tissues (Jarvinen et a1 1997; Jozsa et a1 1990; Kannus &
J ozsa 1991). Because absolute values of TIMP and/or MMP mRNA expression can differ
between individuals and with the extent of pathology (Jones et a1 2006; Riley et a1 2002),
some investigators have suggested that the relative expression of TIMPS and MMPs may
be more important in determining the presence of collagenolysis in patients with
tendinosis (F u et a1 2002). Therefore, in the current study, we chose to express the results
as the ratio between TIMP-1 and MMP-13 (rat interstitial collagenase) mRNA

expression.

53

While the TIMP family consists of four distinct members, TIMP-1, TIMP-2, TIMP-3,
and TIMP-4, we chose TIMP-1 as the representative TIMP in our study. TIMP-1
expression is known to be inducible (Chirco et a1 2006), has broad MMP inhibitory
activity, and has a specific effect in inhibiting interstitial collagenase activity (Bramono
et a1 2004; Cawston et a1 1994). Previous studies from our laboratory utilizing a rat tail
tendon model have documented an increase in both MMP-13 mRNA and protein
expression following loss of cellular homeostatic tension (Arnoczky et a1 2004;
Lavagnino et a1 2005; Lavagnino et a1 2003; Lavagnino et al 2006). We have postulated
that the etiopathologic stimulus for the degenerative cascade that precedes the overt
pathological development of tendinopathy is the catabolic response of tendon cells to
mechanobiologic understimulation as a result of microscopic damage to the collagen
fibers of the tendon and loss of homeostatic cellular tension (Arnoczky et a1 2007).
Therefore, we chose interstitial collagenase (MMP-13) as our representative MMP in this
study.

The results of the current study reveal that, similar to results reported in healthy
human tendons (Fu et al 2002), there is a positive ratio of TIMP-1 to MMP-13 mRNA
expression in normal rat tail tendons. However, following only 24 hours of stress
deprivation, this ratio becomes inversed. The change in TIMP-1/MMP-l3 mRNA
expression seen with stress-deprivation appears to be as a result of a significant increase
in MMP-13 mRNA expression and not a decrease in TIMP-1 expression. Indeed,
inhibiting MMP-13 mRNA expression with a broad-spectrum MMP inhibitor, ilomastat,
did not result in any significant change in TIMP-1 expression in the stress-deprived

tendons. The inversion in the ratio of TIMP-1fMMP-13 mRNA expression has been

54

shown to have deleterious effects on the mechanical properties of these stress-deprived
tendons over time (Lavagnino et a1 2005) and has been implicated in the pathogenesis of
tendinopathy (Fu et a1 2002; Jones et a1 2006; Lo et a1 2004; Dalton et a1 1995; Magra &
Maffulli 2005). Therefore, inhibition of MMP activity in tendinopathy patients via
exogenous and/or endogenous routes may represent a novel therapeutic approach to this
problem.

A recent in vitro experimental study has shown that two broad-spectrum MMP
inhibitors, doxycycline and ilomastat, were able to prevent the degradative histological
and biomechanical changes in stress-deprived rat tail tendons (Arnoczky et a1 2007).
Presumably, this was accomplished through the inhibition of MMP activation by these
compounds. However, the ability of exogenous MMP inhibitors to minimize
collagenolysis in tendinopathy patients has yet to be determined. A preliminary clinical
study has reported significant improvement in patients with Achilles tendinopathy with
the use of a locally administered aprotinin, a protease inhibitor (Capasso et a1 1997).
However, the exact mechanism(s) (inhibition of proteolytic enzymes, anti-inflarnmatory
effect, etc.) by which aprotinin exerted this beneficial effect has yet to be determined.

Increasing the expression of endogenous TIMPS in patients with tendinopathy may
also be beneficial. The results of the current study demonstrated a significant effect of
cyclic loading in increasing TIMP-I/MMP-l3 mRNA expression ratio. Previous clinical
studies have demonstrated an increase in TIMP-1 protein synthesis in the blood and
peritendinous space in individuals following treadmill exercise (Koskinen et a1 2004;
Tayebjee et al 2005). Another clinical study has shown that a single bout of high force,

eccentric exercise increased serum levels of TIMP-1 (Mackey et al 2004). These clinical

55

observations, coupled with the results of the current experimental study, could provide a
mechanistic explanation for the reported beneficial effects of some rehabilitation regimes
that have been recently advocated in the non-operative treatment of tendinopathy patients
(Cannell et a1 2001; Jensen & Di Fabio 1989; Jonsson & Alfredson 2005; Kongsgaard et
a1 2006; Purdam et a1 2004; Young et a1 2005). Recent studies have documented the
clinical efficacy of eccentric exercise programs in the non-operative treatment of
tendinopathy (Cannell et a1 2001; Jensen & Di Fabio 1989; Jonsson & Alfredson 2005;
Kongsgaard et a1 2006; Purdam et a1 2004; Young et a1 2005). Several of these studies
(Jonsson & Alfredson 2005; Purdam et a1 2004; Young et a1 2005) have demonstrated
significant clinical improvement in patients with patellar tendinopathy following 12
weeks of eccentric training performed on a 25° decline board when compared to standard
eccentric exercise performed on a flat surface. However, the precise physiological
mechanisms responsible for this improvement have not been identified. A recent study
has demonstrated that the use of a 25° decline during eccentric loading actually increases
the load and strain on the patellar tendon when compared to eccentric loading on a flat
surface (Kongsgaard et a1 2006). The results of the current study suggest that one
potential benefit of increased tendon strain in the treatment of tendinopathy may be
related to an increase in the TIMP/MMP ratio seen with increasing tendon strain. In
tendinopathy patients, the higher tendon strains produced during eccentric loading may
inhibit further catabolism and encourage homeostasis of the extracellular matrix by re-
establishing a normal, positive TIMP/MMP ratio. Additional clinical studies are needed

to monitor the TIMP/MMP ratios in tendinopathy patients to determine if there is a

56

correlation between this ratio and the extent of pathology and/or the level of clinical
improvement following therapy.

As noted previously, the current experimental study focused only on the relationship
between TIMP-1 and MMP-13 (rat interstitial collagenase) mRNA expression. While
these two genes represent the major inhibitor and effector, respectively, of type I
collagenolysis, the significance of potential imbalances between other MMP and TIMP

levels in the pathogenesis of tendinopathy must still be explored.

57

TlMPIMMP-13 mRNA expression-Stress Deprivation

 

1° 2,3,4

 

TIMP-1/MMP-l3 Ratio
0

 

_O
O
H

 

 

 

0'00] 0hr (1) 24hrSD (2) 48hrSD(3) 72hrSD (4)

Figge 3.1 Graph of TIMP-1/MMP-13 mRNA expression ratios based on transcript
numbers calculated from quantitative PCR, (1) 0 hour stress-deprived tendons, (2) 24
hour stress-deprived tendons, (3) 48 hour stress-deprived tendons, and (4) 72 hour stress-
deprived tendon. Ten tendons per treatment group and the experiment was replicated
three times. A ratio of 1 represents equal amounts of TIMP-1 and MMP-13 expression.
Samples with bars above 1 indicate a greater expression of TIMP-1 than MMP-13. Bars
below 1 indicate less expression of TIMP-1 than MMP-l3. The numbers above each bar

represent statistically significant differences.

58

TIMP-l/MMP-l3 mRNA Expression-Ilomastat

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

10

2 2
I T

.2 I

:3

a:

E

g 0.1 - -—-Au~—~- ———W -

'7

3.1

2 aL

E‘ 0.01? -~-——— --—-#

0.001

0H SD 24H SD 24H SD + ILOMASTAT
(1) (2) (3)

Figure 3.3 Graph of TIMP-1/MMP-13 mRNA expression ratios based on transcript
numbers calculated from quantitative PCR, (1) 0 hour stress-deprived tendons, (2) 24
hour stress-deprived tendons, and (3) 24 hour stress-deprived tendons treated with 50uM

ilomastat. The numbers above each bar represent statistically significant differences.

59

TIMP-I/MMP-l3 mRNA Expression-Exercise

 

100 r2: 0.35

p=0.016

 

 

TIMP-l/MMP-l3 Ratio

001-, - ,, , i - fl -- -

 

 

 

0.001
0hr SD 24hr SD 24hr Ex1% 24hr Ex3% 24hr Ex6%

Figure 3.2 Graph of TIMP-1/MMP-13 mRNA expression ratios of control (0hr), 24hr
stress-deprived (SD), and cyclically loaded for 24 hr at 1%, 3%, and 6% strain based on
transcript numbers calculated from quantitative PCR. Ten tendons per treatment and the

experiment was replicated three times.

60

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IV. DISCUSSION

Understanding the etiology of tendinopathy is a critical aspect of improving
knowledge to help with the debilitating symptoms in this disease. Many causes have
been theorized (Jozsa & Kannus 1997; Shanna & Maffulli 2005), but central to these
theories is the concept that excessive loading of tendons during rigorous physical activity
is the main pathologic stimulation for degeneration of the extracellular matrix (Selvanetti
et a1 1997). Data from this study and others from our laboratory suggest that it is actually
an absence of mechanical stimuli, secondary to microtrauma, which is the
mechanobiologic stimulus for the degradative cascade that leads to tendinopathy.

The overall conclusions from this study coincide with the observations that tissues
from tendinopathy patients have demonstrated an increase in MMP expression and
degeneration of the extracellular matrix. Our research has sought to provide a
mechanistic explanation for these clinical findings. Based on previous research from our
laboratory we concluded that the loss of tendon cell homeostatic strain has been shown to
result in an increase in MMP mRNA expression and protein synthesis (Arnoczky et a1
2004, Lavagnino & Arnoczky 2006). Also, isolated tendon fibril failure secondary to
tendon over-strain results in a localized alteration of cell matrix interactions and
subsequent loss of tendon cell homeostatic strain. This loss of tendon cell homeostatic
strain within the isolated fibril leads to a localized increase in MMP mRNA expression
and protein synthesis (Lavagnino et al 2006). In this current research, the loss of tendon
cell homeostatic strain has led to an inverse TIMP-1/MMP-13 mRNA expression ratio.
The imbalance in the TIMP/MMP mRN A expression ratio may be responsible for the

degenerative changes in the extracellular matrix associated with loss of tendon cell

66

homeostatic strain. Therefore, we theorize that during repetitive activities one or more
abnormal cycles may occur, during which individual collagen fibrils can be damaged
leading to a loss of tendon cell homeostatic strain. This in turn initiates the catabolic
cascade, which has been associated with tendinopathy.

Results from previous studies suggest there are several potential treatment modalities
for tendinopathy that may be a consequence of our research. We have shown that
exogenous MMP inhibitors minimize the degradative loss of tendon material properties
associated with the loss of tendon cell homeostatic strain (Arnoczky et a1 2006). We
have shown that cyclic loading increases the ratio of TIMP-I/MMP-l 3 mRNA
expression due to increased levels of endogenous tissue inhibitor of metalloproteinase-1
(TIMP-1) in these current studies. This suggests that the increased TIMP levels seen
with exercise may provide a scientific basis for the beneficial effects seen with the
eccentric loading rehabilitation regimes for tendinopathy, which favor increased strain
amplitudes (Cannell et a1 2001; Jensen & Di Fabio 1989; Jonsson & Alfredson 2005;
Kongsgaard et al 2006; Purdam et al 2004; Young et a1 2005).

This research can assist in our understanding some of the clinical applications for the
treatment of tendinopathy. Therapeutic regulation of MMPs for the treatment of various
orthopedic conditions, including tendinopathies, is currently being explored. Initial
clinical studies using paratendinous injections of aprotinin, a polyvalent inhibitor of
MMPS, have shown promising results in limiting extracellular matrix degeneration and
thus decreasing pain (Capasso et a1 1993, 1997; Sayana & Maffulli 2007). Therefore,
increasing the local production of TIMPS or partial inhibition the MMPs may be

alternative therapeutic options. In conclusion, my research has Shown that the stimulus

67

and inhibition of MMP-13 production in tendon cells may have key implications in the
etiopathogenesis and treatment of tendinopathy.
Future Research Direction_s_

Investigations into the effect of age on TIMP/MMP mRNA expression ratios would
make an interesting study, now that is has been established that this ratio decreases with
loss of homeostatic tension and reverses with cyclic loading. A hypothesis could be
formed stating that, the TIMP/MMP ratio decreases with increasing age, predisposing
older animals to tendinopathy. This study could be set up with three age groups of rats,
young (30 days), middle age (6 mo), and old (12 mo). Ratios of TIMP-l/MMP-l 3
mRN A expression could be determined using quantitative PCR. Arr age related
TIMP/MMP study could lead to further investigation into the benefit of exercise in older
rats. Older rats are likely more sedentary than younger rats, therefore, controlled exercise
protocols may increase the TIMP/MMP ratios, decreasing the predisposition of older rats
for tendinopathy. These are just a few studies that could continue the examination of
TIMP/MMP ratios in tendons. Various other ratio studies extending from this research
could examine the effects of exercise protocols (in vivo and in vitro), other age ranges,

alternate tendons, and blood versus tissue levels of TIMP/MMP.

68

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