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6f07 plelRC/DateDueindd-p.1

THE MECHANOTRANSDUCTION RESPONSE
OF TENDON CELLS TO TEN SILE LOADING

By

Michael Lavagnino

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Mechanical Engineering

2007

ABSTRACT

THE MECHANOTRANSDUCTION RESPONSE
OF TEN DON CELLS TO TENSILE LOADING

By
Michael Lavagnino

The ability of tendon cells to sense and respond to load is central to the concept of
mechanotransduction and the maintenance of tendon homeostasis. Tendon cells sense
load through a mechano—electrochemical sensory system(s) that detects mechanical load
signals through the deformation of the cellular membrane and/or the cytoskeleton. This
cellular deformation produces tension in the cytoskeleton, which can be sensed by the
cell nucleus through a mechano-sensory tensegrity system to elicit a metabolic response.
While the precise level (magnitude, frequency/rate, and duration) of mechanobiological
stimulation required to maintain normal tendon homeostasis is not currently known, it is
very likely that an abnormal level(s) of stimulation may play a role in the
etiopathogenesis of tendinopathy. Although tendinopathy has been well described
pathologically, the precise etiopathogenesis of this condition remains unsettled.
Classically, the etiology of tendinopathy has been linked to the performance of repetitive
activities (so-called overuse injuries). This has led many investigators to suggest that it is
the mechanobiologic over-stimulation of tendon cells from repetitive loading that is the
initial stimulus for the degradative processes that have been shown to accompany
tendinopathy. Although several studies have been able to demonstrate that the in vitro
over-stimulation of tendon cells in monolayer can result in a pattern(s) of gene expression
seen in clinical cases of tendinopathy the strain magnitudes and durations used in these in

vitro studies, as well as the model systems, may not be clinically relevant. Using an in

vitro rat tail tendon model, the objective of this research was to study the
mechanobiologic response of tendon cells in situ (within their normal extracellular
matrix), to various tensile loading regimes. The studies have shown that the gene
response of tendon cells to load is both frequency and amplitude dependent and that
tendon cells appear to be “programmed” to sense a certain level of stress. Model
analyses combined with the experimental results have demonstrated that both strain rate
and strain amplitude are able to independently alter rat interstitial collagenase gene
expression through increases in ﬂuid-ﬂow-induced shear stress and matrix-induced cell
deformation respectively. The studies have also shown that the absence of stress has a
profound effect on the catabolic response of tendon cells, which in turn decreases the
mechanical properties of the tendon independent of its collagen ﬁber distribution. The
studies have shown that isolated fibrillar damage can occur within tendons and produce a
localized upregulation of interstitial collagenase in response to altered (decreased) tendon
cell stimulation. This weakens the tendon and may put more of the extracellular matrix at
risk for further damage with subsequent loading. From these studies the hypothesis is
forwarded that the etiopathogenic stimulus for the degenerative cascade that precedes the
overt pathologic development of tendinopathy is the catabolic response of tendon cells to
mechanobiologic under-stimulation as a result of microscopic damage to the collagen
ﬁbers of the tendon. This dissertation is a collection of research involving the response
of tendon cells to changing load conditions and the examination of the implications of

these responses as a potential etiopathogenic mechanism for the onset of tendinopathy.

Copyright by
MICHAEL LAVAGNINO
2007

DEDICATION

To myjoy, my beloved, my wife, Nicole.

ACKNOWLEDGEMENTS

I would like to thank God for the many people in my life that have made this
dissertation possible. I am especially grateful for:

My advisor, Dr. Roger C. Haut, whose guidance and encouragement has helped
me through each step of the way. I am thankful for his invaluable expertise that he has
shared with me in the ﬁeld of orthopaedic biomechanics.

My mentor, Dr. Steven P. Arnoczky, whose scientiﬁc excellence and vision has
been the inspiration for my work. I am grateful for his friendship, patience, and
generosity in providing the opportunity to pursue this degree.

My committee members: Drs. Dahsin Liu and Tom Pence for their time,
guidance, and assistance.

My co-workers: Keri Gardner, Oscar Caballero, Dr. Monika Egerbacher, Jean
Kilfoyle, and Tao Tian for their friendship, talents, and assistance with my research.

My fellow students: Katie Frank, Eugene Kepich, Eric Meyer, Shrishail Nashi,
Erin Robertson, and Drs. Meghan Burns, Kevin Riutort, Erika Sorge, and Zachary
Vaupel for their help.

Staff: Ralph Common, Mike McLean, and Shirley Owens for their expertise.

Faculty: Drs. Steve Elder, Niell Elvin, Steve Goldstein, Jim Kimura, Robert
Malinowski, Steve Shaw, and Joanne Whallon for their consultation, help, and advise.

My family-in-law: Michael and Diane Sheehy, D. Johnson, Denise and Jimmy

Deardorff, and Steve Sheehy for their prayers and love.

vi

My extended family: All my uncles, aunts and cousins for their continuous
interest and support, especially my Uncle Mike Lavagnino, who always wanted to have a
business card with Mike Lavagnino, PhD.

My Godparents: Uncle Al and Aunt Betty Lavagnino for their love and example.

My brothers and sister-in-law: Steve, Albert, and Ayn Lavagnino who fostered
my desire to learn and learn quickly, as well as for their encouragement and example.

My parents: John and Anna Lavagnino, for their loving sacriﬁce, inspiration, and
motivation for my education.

My children: Clare and John for their joy and laughter.

Finally I would like to offer my deepest appreciation to my wife, Nicole, who has

been my constant support and encouragement from the beginning.

I am grateful for the ﬁnancial support I received from the Laboratory for
Comparative Orthopaedic Research and the Wade 0. Brinker Endowed Chair in the
College of Veterinary Medicine at Michigan State University. In addition, I would like to
acknowledge the College of Veterinary Medicine at Michigan State University for

providing me with a Graduate School Dissertation Completion Fellowship.

vii

TABLE OF CONTENTS

LIST OF TABLES ...................................................... x
LIST OF FIGURES .................................................... xi
INTRODUCTION ...................................................... 1
CHAPTER 1 — Effect of Amplitude and Frequency of Cyclic Tensile Strain on

CHAPTER 2 —

CHAPTER 3 —

the Inhibition of MMP-l3 mRNA Expression in Tendon Cells:

an in vitro study ....................................... 28
ABSTRACT ........................................... 29
INTRODUCTION ...................................... 30
MATERIALS AND METHODS ........................... 31
RESULTS ............................................ 36
DISCUSSION ......................................... 39
REFERENCES ........................................ 44
In vitro Alterations in Cytoskeletal Tensional Homeostasis

Control Gene Expression in Tendon Cells ................. 48
ABSTRACT ........................................... 49
INTRODUCTION ...................................... 50
MATERIALS AND METHODS ........................... 52
RESULTS ............................................ 57
DISCUSSION ......................................... 63
ACKNOWLEDGEMENTS ............................... 67
REFERENCES ........................................ 68

Collagen F ibril Diameter Distribution
Does Not Reflect Changes in the Mechanical Properties

of in vitro Stress-Deprived Tendons ....................... 73
ABSTRACT ........................................... 74
INTRODUCTION ...................................... 75
MATERIALS AND METHODS ........................... 76
RESULTS ............................................ 80
DISCUSSION ......................................... 86
ACKNOWLEDGEMENTS ............................... 90
REFERENCES ........................................ 91

viii

CHAPTER 4 — Isolated F ibrillar Damage in Tendons Stimulates Local

Collagenase mRNA Expression and Protein Synthesis ....... 95
ABSTRACT ........................................... 96
INTRODUCTION ...................................... 97
MATERIALS AND METHODS ........................... 99
RESULTS ........................................... 104
DISCUSSION ........................................ 107
REFERENCES ....................................... 113

CHAPTER 5 — A Finite Element Model Predicts the
Mechanotransduction Response of Tendon Cells

to Cyclic Tensile Loading .............................. 116
ABSTRACT ......................................... 117
INTRODUCTION ..................................... 1 18
MODEL DEVELOPMENT .............................. 121
FINlTE ELEMENT METHOD ........................... 124
EXPERIMENTAL METHODS ........................... 130
RESULTS ........................................... 132
DISCUSSION ........................................ 137
REFERENCES ....................................... 144
CONCLUSIONS ..................................................... 150

APPENDIX A — Ex vivo static tensile loading inhibits MMP-l expression
in rat tail tendon cells through a cytoskeletally based

mechanotransduction mechanism ........................ 153
ABSTRACT .......................................... 154
INTRODUCTION .................................... 155
MATERIALS AND METHODS .......................... 156
RESULTS ........................................... 160
DISCUSSION ........................................ 165
REFERENCES ....................................... 170
APPENDIX B — Computational input ﬁles
B.1 Global Model for 1% strain at 2% strain/minute .......... 173
8.2 Global Model for 1% strain at 20% strain/minute ......... 180
B3 Global Model for 3% strain at 6% strain/minute .......... 180
B4 Global Model for 3% strain at 2% strain/minute .......... 180
B5 Submodel for 1% strain at 2% strain/minute ............. 181
B6 Submodel for 1% strain at 20% strain/minute ............ 196
B7 Submodel for 3% strain at 6% strain/minute ........... 196
3.8 Submodel for 3% strain at 2% strain/minute ............. 196

ix

LIST OF TABLES

Table 3.1 Mean : standard deviation for control and stress-deprived ﬁbril
number, mean ﬁbril diameter and mean ﬁbril density. Resulting p-value
from paired t-test with signiﬁcance set at p<0.05 ..................... 82

Table 3.2 Comparison of the cross-sectional area, tensile modulus, failure stress,
and failure strain of control and stress-deprived rat tail tendons.
TCross—sectional area measurements were paired from the same tendon.
The control tendon area was used for both groups to calculate failure

stress. * signiﬁcantly different than control specimens, p<0.05 ......... 85
Table 5.1 Global matrix material properties ................................ 127
Table 5.2 Submodel material properties ................................... 130
Table 5.3 Global model and submodel strain and shear stress values ............ 135
Table A.1 The effect of static stress on MMP-l expression .................... 161

LIST OF FIGURES

Figure 1 The hierarchical collagen network of tendon (Kastelic et al. 1978) ........ 3

Figure 2 Extension of an elastic collagen ﬁber zig-zag crimp with apex points
of inﬁnite rigidity (Diamant et al. 1972) ............................. 4

Figure 3 Advancements in crimp deﬁnition using a blunted zig-zag (b: crimp
blunting factor) used in the SSL model (Kastelic et al. 1980).
The undeformed and deformed conﬁgurations of collagen crimp with
spring apex points (Stouffer et al. 1985) ............................. 5

Figure 4 A schematic diagram showing the bilinear stress-strain curve for a
single collagen ﬁbril. Fibrils can vary by strain at which the crimp
is fully stretched (S) or at which ultimate failure occurs (U)
(Kwan and Woo 1989) .......................................... 6

Figure 5 Spring-element model showing nonlinear spring elements (ﬁbers)
overlaid on the continuum elements (matrix) (Wilson et a1. 1997) ........ 8

Figure 6 (a) Schematic representation of the hierarchical structure of a collagen
tendon. If (a ﬁbre of) the tendon is stretched by an amount 81', this is
distributed between the collagen ﬁbrils (cf) with a tensile strain 80 and
the proteoglycan-rich matrix (pg), which is mainly sheared. Covalent
cross-links between molecules are drawn schematically within the
collagen ﬁbrils. (b) An illustration representing a mechanical model,
where ﬁbrils and matrix are considered as viscoelastic systems arranged
in series. ED is the elastic modulus of the ﬁbrils that depends critically
on the covalent cross-links. n0 is the viscosity of the ﬁbrils, possibly
due to friction between molecules. EM is the effective elastic modulus
of the matrix and 11M is the viscosity of the matrix (Puxkandl et al. 2002). . 9

Figure 7 Conceptualization of the geometry of a fascicle used as the basis
of a ﬁnite element model (Atkinson et al. 1997) ..................... 11

Figure 8 A ﬁnite-element model of tendon as a two-phase linear elastic ﬁber
reinforced composite using the rebar formulation of ABAQUS
(Giori et al. 1993) ............................................. 12

Figure 1.1 A: Photograph of the computer-controlled, stepper motor driven,
cyclic loading system. The system permits ﬁve tendons to be loaded
simultaneously while in media. B: Close up view of the testing system
showing rat tail tendons in place .................................. 33

xi

Figure 1.2

Figure 1.3

Figure 1.4

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Representative Northern blot gel from the amplitude experiment

illustrating the relative expression of MMP—l3 mRNA expression in fresh
control tendons (lane 1); immobile for 24 hours (lane 2); 1% cyclic strain

at 0.017Hz for 24 hours (lane 3); 3% cyclic strain at 0.017Hz for 24

hours (lane 4); 6% cyclic strain at 0.017Hz for 24 hours (lane 5).

GAPDH was used as an internal control. Experiments were performed

three times and a representative result is shown ...................... 36

Representative Northern blot gel from the frequency experiment

illustrating the relative expression of MMP-l3 mRNA expression in fresh
control tendons (lane 1); immobile for 24 hours (lane 2); 1% cyclic strain

at 0.017Hz for 24 hours (lane 3); 1% cyclic strain at 0.17Hz for 24

hours (lane 4); 1% cyclic strain at 1.0Hz for 24 hours (lane 5).

GAPDH was used as an internal control. Experiments were performed

three times and a representative result is shown ...................... 37

Representative Northern blot gel from the cytochalasin D experiment
illustrating the relative expression of MMP—13 mRNA expression in fresh
control tendons (lane 1); immobile for 24 hours (lane 2); 6% cyclic strain

at 0.017Hz for 24 hours (lane 3); 6% cyclic strain at 0.017Hz for 24

hours with 10 uM of cytochalasin D for 24 hours (lane 4).

GAPDH was used as an internal control. Experiments were performed

three times and a representative result is shown ...................... 38

Photograph showing the 3-point traction devices ..................... 54

Representative rhodamine-phalloidin stained cell images under confocal
microscopy (40x) of A) elongated cells in adhered gels at 48 hours
containing well-organized actin stress ﬁbers, B) the addition of

cytochalasin D to the adhered gels or C) the physical release of the gels

from the culture dish resulted in an immediate loss of actin stress

ﬁber organization ............................................. 57

Photograph showing a representative gel after 48 hours of attachment to
its culture dish (A) and the contraction of the gel following release after
24 hrs (B), 10 days (C), and 14 days (D). (Scale bar = 10 mm) ......... 58

Representative Northern blot analysis of rat interstitial collagenase and

(11(1) collagen with GAPDH as a control. Lanes represent 1) adhered to

dish for 24 hours, 2) cytochalasin D for 24 hours, 3) 24 hours contraction,

4) 10 days contraction, 5) 10 days contraction plus cytochalasin D

for additional 24 hours, 6) 14 days contraction ...................... 59

xii

Figure 2.5

Figure 2.6

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 4.1

Photograph showing a representative gel with a 3—point traction device in
place immediately after release (A) and after 24 hrs (B) and 10 days (C)

of gel contraction around the three pins. Removal of the traction devices
(and the opposing tractional resistance they provided) permitted

further contraction of the gels (D). (Scale bar = 10 mm) .............. 61

Representative Northern blot analysis of rat interstitial collagenase and

(11(1) collagen with GAPDH as a control. Lanes represent 1) adhered to

dish for 24 hours, 2) 24 hours contraction around 3pt traction pins,

3) 10 days contraction around pins, 4) 10 days contraction around pins

plus free contraction for an additional 24 hours ...................... 62

Representative transmission electron microscope image of A: control rat
tail tendon cross-section and of B: a cross-section of a rat tail tendon
stress-deprived for 21 days (x 19,000). Scale bar = 500 nm ........... 81

Histogram illustrating collagen ﬁbril density in control and 21 day stress-
deprived rat tail tendons for each of the six paired tendons measured and

for all the control and stress-deprived tendons combined. There was

no signiﬁcant differences between tendons, p>0.05 ................... 82

Histogram illustrating relative frequencies of collagen ﬁbril diameters in
control and 21 day stress-deprived rat tail tendons. There was no
signiﬁcant differences between tendons within each bin, p>0.05 ........ 83

Representative stress versus strain curves from paired control and 21 day
stress-deprived rat tail tendons of one ﬁbril ......................... 84

Northern blot gel illustrating the relative expression of MMP-l3 mRN A
expression in fresh control tendons (lane 1) and stress-deprived for 21
days (lane 2). GAPDH was used as an internal control ................ 85

Representative stress-strain curve of a rat tail tendon fascicle (dark

solid line) demonstrating the point at which ﬁbrillar damage occurred

(point C), and the unloading of the tendon to 100g (dashed line). A
representative curve of the tendon loaded to failure displaying the

negative slope in the stress strain curve that eventually ends in total

tendon failure (Lavagnino et al. 2005) has also been included (light

solid line). Points A-D on the stress-strain curve correspond to the

images of the fascicle at those points in Figure 4.2A-D ............... 101

xiii

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 5.1

Figure 5.2

Images of a rat tail tendon fascicle at various points throughout the

testing protocol: A) Prior to loading (the crimp pattern is clearly visible).

B) During loading in the linear portion of the stress-strain curve
demonstrating the elimination of the crimp pattern. C) Onset of

ﬁbrillar damage as manifested by a change in the reﬂectivity of the

damaged ﬁbrils (arrows). D) Unloading of the tendon to 100g

and the reoccurrence of the crimp pattern within the damaged

ﬁbrils (arrows). (bar = 200 microns) ............................. 105

Representative images of a rat tail tendon fascicle following ﬁbrillar

damage. A) The presence of the crimp pattern on the bottom of the

tendon fascicle (arrows) indicates the site of isolated ﬁbrillar damage.

B) In situ hybridization of the tendon fascicle reveals interstitial

collagenase mRNA expression in those cells associated with the

damaged ﬁbril(s). The borders of the tendon fascicle are delineated

by broken lines. (bar = 100 microns) ............................. 106

Representative image of a control (unloaded [stress deprived] for

24 hours) rat tail tendon fascicle demonstrating interstitial collagenase
mRNA expression by cells throughout the entire fascicle.

(bar = lOOmicrons) ........................................... 106

A) Representative photomicrograph of an injured rat tail tendon fascicle
showing the damaged ﬁbrils (denoted by the presence of crimp)

immediately adjacent to uninjured ﬁbrils. (bar = 20 microns)

B) Photornicrograph of the same ﬁeld under ﬂuorescent light

demonstrating the positive (light gray) staining of MMP-13 protein in

the cytoplasm of only those cells within the damaged ﬁbrils. The nuclei

have been counterstained with DAPI (white) to help identify the cells.

(bar = 20 microns) ............................................ 107

Axisymmetric global poroelastic model of the rat tail tendon

(20mm x 0.15mm), divided into 300 4-noded axisymmetric

elements (0.2mm x 0.05mm) with radially variant nonlinear spring

elements attached at the nodes of the matrix elements (springs),

zero pore pressure on the outer boundary (circles), constrained

at the tendon center (triangles), and loaded at the tendon end as

per previous experimental conditions (arrows). The darkened

element boundary indicates the location of the submodel ............. 125

Reaction force (N) plotted against strain (%) to show the radial

variation in ﬁber recruitment from the outer boundary to the inner
or center that predicts the nonlinear response of the global tendon ...... 126

xiv

Figure 5.3

Figure 5.4

Figure 5.5

Figure 5.6

Figure 5.7

Figure 5.8

Figure A.1

Figure A.2

Figure A.3

Figure A.4

Submodel of the rat tail tendon, the size of a global element, composed
of an ovoid-shaped cell, cell membrane, pericellular matrix (PCM),
extracellular matrix (ECM), and collagen ﬁbers .................... 128

Comparison of tendon model to actual tendon stress-strain response
(3% strain at 6% strain/minute) ................................. 133

Plot of the ﬂuid velocity magnitude (mm/s) showing ﬂuid ﬂow in the
positive direction (arrow) out of the tendon (3% strain at 6% strain/min).
The curved lines (springs) represent the collagen ﬁbers .............. 133

Plots of the ﬂuid velocity resultant around the cell and out of the tendon
(3% strain at 6% strain/min) .................................... 134

Graph of shear-stress induced by ﬂuid ﬂow. Note the marked increase
in at the polar ends of the cell ................................... 135

Gene expression levels of rat interstitial collagenase (MMP-13) as
determined by Real-Time Quantitative PCR. All experimental samples
were quantiﬁed relative to the fresh (0 hour) control ................. 136

Photograph of the static tensile loading system. Individual tendons were
suspended in 50 ml test tubes ﬁlled with culture media containing
10% FBS and calibrated stainless steel weights were attached by clips. . . 157

Representative Northern blot gel illustrating the relative expression of
MMP-1 mRNA as a function of applied stress for 24 h. G3PDH was

used as an internal control. Experiments were performed three times

and a representative result is shown .............................. 161

Composite polynomial regression of graph of the three experimental
replicates plotting MMP-l mRNA expression (normalized as a ratio of
MMP-1 to G3PDH) against static stress. There was a strong (r2 = 0.78)

and signiﬁcant (p < 0.0001) inverse correlation between MMP-l mRNA
expression and static stress ..................................... 163

Western blot analysis illustrating the absence of pro-MMP-l and MMP-1
protein expression in freshly harvested rat tail tendons. There was a
signiﬁcant up-regulation of pro-MMP-l and MMP-1 protein expression

in the tendon cells after 24 h of in vitro load deprivation .............. 163

XV

Figure A.5 Representative Northern blot gel illustrating the relative expression of
MMP—1 mRNA in fresh control tendons (lane 1): 24 h load deprived at
0 MPa (lane 2); 24 h at 2.60 MPa static tensile stress (lane 3): 24 h at
2.60 MPa static tensile stress in 10 pM cytochalasin D (lane 4).
G3PDH was used as an internal control. Experiments were performed
three times and a representative result is shown ..................... 164

Figure A.6 Confocal overlay images of rat tail tendon cells stained with rhodamine
phalloidin to label actin ﬁlaments. (A) Fresh control tendon: note
presence of actin stress ﬁbers (arrows) in cytoskeleton. (B) Tendon
treated with 10 uM cytochalasin D for 1 h; note the absence of
organized actin stress ﬁbers. (confocal 40x oil immersion: 2x zoom). . . . 164

xvi

INTRODUCTION

Tendinopathy, a syndrome of tendon pain, localized tenderness, and impaired
performance, is a common and major health issue in workers and athletes who perform
repetitive activities (Renstrom and Woo 2007). Although the pathology of tendinopathy
has been well described, the precise etiopathogenesis of this condition remains unsettled
(Renstrom and Woo 2007). Classically, the etiology of tendinopathy has been linked to
the performance of repetitive activities (overuse injuries) (Almekinders et al. 1993). A
proposed algorithm for the onset of overuse tendinopathy involves altered cell-matrix
interactions in response to repetitive loading (Archambault et al. 1995). In this scenario,
repeated strains below the injury threshold of the tendon induce degenerative changes in
the tendon-matrix composition and organization (Jarvinen et al. 1997; Jones et al. 2006;
Jozsa and Kannus 1997). The degeneration of the extracellular matrix leads to a transient
weakness of the tissue making it more susceptible to damage from continued loading.
This damage then accumulates until the overt pathology of tendinopathy develops
(Archambault et a1. 1995). While this is a feasible algorithm for the development of
overuse tendinopathy, the precise mechanism(s) which lead to altered cell-matrix
interactions have not been described. To better understand the mechanical association
between tendon matrix and tendon cells, it is necessary to understand the following: 1)
the composition of the tendon extracellular matrix, 2) the contribution(s) of these
extracellular components in deﬁning the material properties of the tendon, 3) the
mechanisms by which mechanical signals are transmitted through the extracellular matrix

to the tendon cells, and 4) the cellular response to these mechanical signals.

Tendon composition

Tendons are described as soft connective tissues which link bone to muscle and
consist of solid (collagen ﬁbers, cells, and matrix constituents of proteoglycans and
glycoproteins) and ﬂuid phases (Kannus 2000; Riley 2005; Woo et al. 1997). Type I
collagen is the main component of the solid phase (65-80% dry weight) and is organized
within the tendon in parallel ﬁber bundles with hierarchies of ﬁbrillar arrangement down
to microﬁbril size (Kannus 2000; Kastelic et al. 1978) (Figure 1). Under polarized light
microscopy, tendon collagen ﬁbrils appear in a sinusoidal wave pattern referred to as
crimp (Diamant et al. 1972). Collagen has the ability to form covalent intramolecular and
intermolecular cross-links, which are the keys to its tensile strength characteristics and
resistance to chemical or enzymatic breakdown (Tanzer 1973; Woo et al. 1997). The
matrix, or ground substance that surrounds the collagen, consists of proteoglycans,
glycosaminoglycans (GAGs), and glycoproteins. Proteoglycans and GAGs only make up
a small percentage of the total dry tendon weight, but due to their highly negative charge,
these molecules attract and limit the movement of water, which represents 60-80% of the
total wet weight (Woo et al. 1997). This water binding capacity improves the mechanical
properties of tendon against shear and compressive forces (Kannus 2000). Glycoproteins
in tendons include both structural and adhesive molecules. The structural glycoproteins
(elastin, ﬁbrillin) provide elastic properties of tendon while the adhesive glycoproteins
mediate cell-matrix interactions (e. g. tenascin-C, ﬁbronectin, thrombospondin)(Riley
2005). Each tendon component (collagen, crosslinks, crimp, matrix, water) has
mechanical signiﬁcance and together they form a well-organized tissue for optimal load

distribution and response (Woo et al. 1997).

"éii at x;

 

     
  

 

- . 93'.
MICRO— SUB- '.-tr.‘;v" TENDON
l"'31“! ~ FIBRIL . cm
. “3“"?
TROPO- keg}? .
COLLAGEN -’u’ I
35:153me 640A ‘
penodrcrty
reticbular
- - mem rane
fibroblasts wavg£orm f as ci cul a r
I ' I crimp structure membrane
I I I
15 A 35 A 100-200 A 500-5000 A 5030011 1005000
' SIZE SCALE
Figure l The hierarchical collagen network of tendon (Kastelic et al. 1978).

Tendon Computational Models

Based on these structural components (collagen ﬁbers, proteoglycan matrix, and
ﬂuid) computational models of tendon have been created to further understand the
mechanical response of tendon to applied strain. Although no models have examined the
role of tendon mechanical load on cell deformation, many structural and continuum

models exist that accurately model overall tendon mechanical behavior.

Collagen Fibers

Initial structural and microstructural models investigated parameters (orientation,
number, distribution) of the collagen fibril, the main solid constituent (70-80% dry
weight) of tendon, that relate to tissue morphology to describe the mechanical function of
the tendon (Belkoff and Haut 1992; Comninou and Yannas 1976; Diamant et a1. 1972;
Kastelic et al. 1980; Kwan and Woo 1989; Lanir 1983; Stouffer et a1. 1985; Viidik 1972).
These models assumed that the toe-in region occurs due to a structural feature of the
tissue, i.e., the gradual removal of crimp during deformation leads to increasing stiffness.
The earliest structural model described tendon as a cable consisting of these crimped
strands of microﬁbrils where the stress behavior is determined by the properties of the
individual strands (Diamant et a1. 1972). These microﬁbrils were modeled as having

elastic segments joined by rigid hinges based on the elastica problem in mechanics

 

(Figure 2).
A A
l
(a) A
A A
Figure 2 Extension of an elastic collagen ﬁber zig-zag crimp with apex points of

inﬁnite rigidity (Diamant et al. 1972).

Investigators advanced this model to explain additional structural features of crimp
including the disappearance of crimp apices and the variability in crimp angle along the
depth and length of the tendon using linearly elastic collagen ﬁbers (Comninou and

Yannas 1976; Kastelic et al. 1980; Stouffer et al. 1985) (Figure 3).

 

 

Figure3 Advancements in crimp deﬁnition using a blunted zig-zag (b: crimp
blunting factor) used in the SSL model (Kastelic et al. 1980). The
undeformed and deformed conﬁgurations of collagen crimp with spring
apex points (Stouffer et al. 1985).

Including crimp angle variations throughout the tendon was ﬁrst incorporated in the

sequential straightening and loading (SSL) model of collagen crimp (Kastelic et al.

1980). This model suggested that crimped ﬁbrils are assumed to have a negligible

resistance to extension while loaded ﬁbrils resist deformation through a linear elastic

relationship, thus resistance arises only from the elasticity of already straightened ﬁbrils

(Kastelic et al. 1980). This distribution of ﬁbril angle in the tendon leads to a sequential

ﬁbril straightening and loading that result in the initial nonlinearity of the toe region

(Kastelic et al. 1980). Investigators than described crimp using a modiﬁed SSL model,

with varying initial ﬁber lengths of a bilinear elastic collagen ﬁber that have a speciﬁed

failure stretch/strain (Figure 4) (Belkoff and Haut 1992; Crisco and Panjabi 1996;
Hurschler et al. 1997; Kwan and Woo 1989; Liao and Belkoff 1999).

A

0'

 

8:

 

8s 8u

Figure 4 A schematic diagram showing the bilinear stress-strain curve for a single
collagen ﬁbril. Fibrils can vary by strain at which the crimp is fully
stretched (S) or at which ultimate failure occurs (U) (Kwan and Woo
1989).

These models deﬁned varying initial ﬁbril lengths and varying failure lengths'either

arbitrarily (Kwan and Woo 1989), based on normal distributions (Crisco and Panjabi

1996), or extensively on the organization of the collagen microstructure (ﬁber density

and distribution) (Hurschler et al. 1997) to determine how collagen affects the material

properties. Other studies have further reﬁned the model by using a quasi-linear
viscoelastity law incorporating a relaxation function and a nonlinear time independent

elastic response for collagen ﬁbers (Decraemer et al. 1980; Frisen et al. 1969; Fung 1967;

Haut and Little 1972; Lanir 1980; Viidik and Ekholm 1968). One proposed viscoelastic

model, the single integral ﬁnite strain (SIFS), utilizes the concept of constitutive

branching to allow different constitutive equations at different elongations and the model
is fully nonlinear and reduces to classic viscoelasticity (Mooney-Rivlin) if linearized
(Johnson et al. 1996). Thus instead of a bilinear elastic model to describe crimp as shown
previously (Kwan and Woo 1989), this model describes the change in micromechanism
caused by the onset or cessation of collagen ﬁber recruitment as the tendon is extended as
a change in constitutive equations. This model appears very accurate at modeling stress-
relaxation, peak cyclic stresses, and stress strain curves (Johnson et al. 1996).
Proteoglycan Matrix

Although these models accurately depict the whole range of tissue behavior from
initial load to failure, by only taking into account collagen structure and properties they
lack potential mechanical properties from the remainder of the tendon constituents
(extraﬁbrillar matrix and water). Tendons have been previously modeled as a composite
material to incorporate both collagen ﬁber and matrix properties (Ault and Hoffman
1992; Ault and Hoffman 1992; Luo et a1. 1998; Puxkandl et a1. 2002; Redaelli et al.
2003; Scott 2003; Wilson et al. 1997; Wren and Carter 1998). One such composite
model, similar to the pure collagen model proposed by Hurschler (Hurschler et al. 1997),
included both extensive ﬁber and matrix properties (Wren and Carter 1998). This model
allowed for uncrimping, stretching, and breaking of collagen ﬁbers, but in addition
described the ﬁbers ability to rotate in the matrix as fully, partially, or un- constrained.
Another researcher investigated the relationship between the matrix and the ﬁbers and the
likelihood of potential “links” between ﬁbers (Wilson et al. 1997). This model utilized a
plane stress ﬁnite element model with nonlinear spring elements (ﬁbers) overlaid on the

continuum elements (matrix) to study the possibility of shear stress within the tissue

(Figure 5) (Wilson et al. 1997). The non-linear springs provide the model with the ability

to simulate the nonlinear behavior attributed to the crimp of the collagen ﬁbers.

9——\/‘—7——\/‘—0—\/‘——3

 

 

 

 

o—ﬁﬂ—o—w—é—w—b

Figure 5 Spring-element model showing nonlinear spring elements (ﬁbers) overlaid
on the continuum elements (matrix) (Wilson et al. 1997).
This model determined that shear transfer may occur from linking between collagen
ﬁbers, but unfortunately only small deformations were modeled. The idea that a model
should include ﬁber-ﬁber and ﬁber proteoglycan interaction began a subset of models
that delve into modeling collagen ﬁbril crosslinks from the molecular standpoint of
matrix components, most notably glycosaminoglycan (GAG) chains. A sliding GAG
ﬁlament model was proposed in which the GAG chain can slide and re-engage and thus
provide mechanical stability (Scott 2003). These GAG chains, located on proteoglycans
play a role in retaining water as well as creating a bond between collagen ﬁbrils. One

model incorporated this ﬁber-matrix interaction as a composite material with collagen

ﬁbrils embedded in a proteoglycan rich matrix, where the matrix is mostly loaded under

shear (Figure 6a) (Puxkandl et al. 2002). Deformation in the tendon occurs through

extensibility of collagen ﬁbrils and nonﬁbril (crosslink, matrix) deformation. The

composite structure was modeled in series with collagen ﬁbril and proteoglycan matrix as

viscoelastic elements taking into account molecular friction, cross links, viscous

relaxation, and matrix shearing (Figure 6b) (Puxkandl et al. 2002).

 

Figure 6

 

 

(b) collagen fibril pg matrix
molecular viscous
friction relaxation

 

£0.50 EM.EM n

effective elastic modulus
and tensile strain due to:

W

molecular matrix
cross-links shearing

 

 

(a) Schematic representation of the hierarchical structure of a collagen
tendon. If (a ﬁbre of) the tendon is stretched by an amount 81, this is
distributed between the collagen ﬁbrils (cf) with a tensile strain 8D and the
proteoglycan—rich matrix (pg), which is mainly sheared. Covalent cross—
links between molecules are drawn schematically within the collagen
ﬁbrils. (b) An illustration representing a mechanical model, where ﬁbrils
and matrix are considered as viscoelastic systems arranged in series. ED is
the elastic modulus of the ﬁbrils that depends critically on the covalent
cross-links. 11D is the viscosity of the ﬁbrils, possibly due to friction
between molecules. EM is the effective elastic modulus of the matrix and
nMis the viscosity of the matrix (Puxkandl et al. 2002).

A similar model of proteoglycan links was also proposed with 40% of elongation due to

ﬁbril length change, and the remainder suggested due to relative movement of ﬁbrils

(Redaelli et al. 2003). Shear stress was assumed responsible for force transfer from ﬁbril

to ﬁbril. Fibril length, diameter, and interfibrillar distance were used as variables. Using
this model, it was found that a stress-transfer matrix with low elastic modulus was
sufﬁcient and an increase in collagen ﬁbril diameter alone cannot explain tendon
mechanical properties (Redaelli et a1. 2003).
Interstitial ﬂuid ﬂow

None of the previously mentioned collagen or collagen-matrix structural models
include the permeability of the tendon or look at ﬂuid exudation (Hannaﬁn and Arnoczky
1994) and its role in the stress strain response (Haut and Haut 1997) within the tendon. A
general 3D viscoelastic model for ﬁbrous tissue was proposed that takes into account
ﬂuid ﬂow, ﬁber density, and ﬁber orientation (Lanir 1983). The collagen ﬁbers were
assumed to only be subjected to uniaxial strain along the length of the ﬁber, with no
compressive strength and the ﬂuid ﬂow through the matrix was due to a hydrostatic
pressure differential (Lanir 1983). The model accurately predicted that the unfolding of
the ﬁber leads to increased hydrostatic pressure or ﬂuid ﬂow through the matrix (Lanir
1983). Multiple continuum theories include tendon permeability and interstitial ﬂuid
ﬂow in tendon models (Adeeb et al. 2004; Atkinson et al. 1997; Butler et al. 1997; Chen
and Ingber 1999; Yin and Elliott 2004). These continuum models have added the
structural morphology of the tendon extracellular matrix with ﬂuid using a biphasic
approach (Atkinson et al. 1997; Giori et al. 1993; Luo et al. 1998; Wakabayashi et al.
2003; Yin and Elliott 2004). One such biphasic model assumed that tendon was
transversely isotropic in the ﬁber direction and the ﬁbers are assumed to be piecewise
linear elastic (Yin and Elliott 2004). Each of these models has predicted the importance

of ﬂuid ﬂow and permeability in determining intertendonous strains and the exudation of

10

ﬂuid ﬂow from the tendon. These theories however do not take into account the
nonhomogeneity of the crimp pattern (Hansen et al. 2002) nor the viscoelastic properties
of the collagen ﬁbers (Haut and Little 1972).

Collagen, matrix, and ﬂuid ﬂow

There are relatively few tendon models that take into account collagen structure, and a
porous matrix that allows for ﬂuid ﬂow. One ﬁnite element model assumed the collagen
crimp as an outer helical arrangement of 62° inclination (Figure 7) (Atkinson et al. 1997).
The central core was then modeled as nonlinear poroelastic material with varying
permeabilities (Atkinson et al. 1997). This model predicted ﬂuid exudation and ﬁber

straightening by having the outer collagen ﬁbers wring out the ﬂuid containing central

 

 

 

portion.

water based l<737ﬁx106—J

matrix

sealed w

surfaces <

/ 10.0x10-6
62°

collagen /

fibers

. fiber

fixed base orientation
Figure 7 Conceptualization of the geometry of a fascicle used as the basis of a ﬁnite

element model (Atkinson et al. 1997).

Another model examining the role of ﬂuid in tissue and potential causes of ﬂuid
exudation addressed three mechanisms: 1) crimp straightening, 2) Poisson’s ratio —

determined by cross links, and 3) osmotic pressure (Adeeb et al. 2004). Another

11

investigator used a ﬁnite element system to track changes in ﬂuid pressure and collagen
orientation to examine the alterations in tendon composition with mechanical load (Giori
et al. 1993). The maintenance and rearrangement of the tendon’s ﬁbrous extracellular
matrix was associated with regions where stretching and distortion of cells take place as
the tendon was physiologically loaded (tendon wrapped around bone). The tendon was
considered to be a two-phase linear elastic ﬁber-reinforced composite deﬁned using the
rebar formulation of the ABAQUS FEM code (Figure 8) (Giori et al. 1993). This model
assumed the tendon had no crimp initially (Giori et al. 1993). Results from this study
showed that the hydrostatic stress and distortional strain are important tissue level
mechanical stimuli regulating the composition of connective tissue and gene expression

(Giori et al. 1993).

16 MPa
Average Stress

  
    

goo-u...— 0....
I

"3

0 0‘.
Illlllllllllllllllll o
IIIIIIIIIIIIIIIIHHP'.‘
llllllllllllllllllll".
llllll‘llllllllllllll‘ -
mummium -
lllllllllllllllllill 9.
IIIIIIIIIIIIIIIIIIII (O
IIIIIIIIIIIIIIIlIIII .
IIIIIIIIIIIIIIIIIIII
lIIlIIIIIIIIIIIIlIll
IIIIIIIIIIIIIIIIIIII
IIIIIIIOIIIIIIIIIIII
IIIIIIIIIIIIIIINIII
“II"HIIINIIIIIII
IIII lllllll
IIIIIIIHHIIIIHIIl
lllllIl'lIllllllllll
III” M"

  

:::: Frictionless
:::::: ':': Rigid Surface

 

 

 

 

Figure8 A ﬁnite-element model of tendon as a two-phase linear elastic ﬁber
reinforced composite using the rebar formulation of ABAQUS (Giori et al.
1993).

12

Other biphasic tissue studies have suggested the importance of both the ﬂuid and solid
phases in the transmission of strain from the tissue to the cell, or mechanotransduction
(Baer et al. 2003; Guilak and Mow 2000). Currently there are no tendon models that
model the mechanical response of tendon strain using the solid and ﬂuid components of
tendon and then take that response to determine what is happening on the cellular level.
Although each component is known to play an important role in the mechanical response
of tendon to load, their inﬂuence on cellular mechanotransduction, or the transmission of
strain through the extracellular matrix to the cell, within the tendon is unknown.
Cell-Matrix Interactions

The ability of tendon cells to sense and respond to a physical stress with a
biological response, the concept of mechanotransduction, is vital in maintaining tendon
homeostasis (Banes et al. 1995; Ingber 1997; Wang and Ingber 1994). Tendon cells
sense physical stress through a mechano-biological sensory system(s) that detects
mechanical load signals through the deformation of the cellular membrane and/or the
cytoskeleton (Adams 1992; Banes et al. 1995; Brown et al. 1998; Ingber 1997; Wang
2006; Wang et al. 1993; Wang and Ingber 1994; Watson 1991). Cell membrane
deformations may open or close stretch-activated ion channels, which control the inﬂux
of second-messenger molecules such as calcium and inositol triphosphate (1P3) (Banes et
al. 1995; Sachs 1988; Sachs 1988; Shirakura et al. 1995). These second messengers can
activate a wide array of cellular machinery including DNA synthesis, mitosis, cell
differentiation, and gene expression (Binderman et al. 1984; Ryan 1989; Sachs 1988;
Sachs 1988). Cellular deformation also alters the cytoskeletal tension, which in turn, can

be sensed by the cell nucleus through a mechano-sensory tensegrity system to elicit a

13

metabolic response (Adams 1992; Arnoczky et al. 2002; Banes et al. 1995; Ben—Ze'ev
1991; Ingber 1997; Wang 2006; Wang et al. 1993; Wang and Ingber 1994; Watson
1991). For both of these signaling pathways, change in cell shape with applied stress is
thought to occur through the binding of the cell to extracellular matrix proteins such as
collagen and ﬁbronectin (Banes et a1. 1995; Rosales et al. 1995; Sung et al. 1996). The
binding between the extracellular matrix proteins and the interior cytoskeleton of the cell
occurs through the integrin family of cell surface receptors (Banes et al. 1995; Ingber
1991; Janmey 1998; Rosales et al. 1995; Shyy and Chien 1997; Sung et al. 1996; Wang et
al. 1993). Thus mechanotransduction, in response to tendon load, is likely mediated
through the deformation of the extracellular matrix, which in turn would result in in situ
cell deformation (Banes et al. 1995; Sachs 1988; Watson 1991). The results of a recent
study support the hypothesis that mechanical loads placed on tendons result in a
concomitant in situ deformation of the cell nucleus (Arnoczky et al. 2002). As has been
proposed in cartilage, this nuclear deformation may play a signiﬁcant role in the
mechanotransduction of these tissue loads into intracellular signals (Guilak 1995; Guilak
and Mow 2000; Guilak et al. 1995).
Mechanotransduction, Homeostasis, and Pathology

The importance of load (stress) in the homeostasis of connective tissues has been
well documented (Akeson et al. 1974; Hannaﬁn et al. 1995; Noyes 1977; Woo et al.
1982; Woo et al. 1997; Yasuda and Hayashi 1999). Several studies have shown that
stress deprivation in ligaments and tendons results in signiﬁcant alterations in their
structural and functional properties (Akeson et al. 1974; Hannaﬁn et al. 1995; Noyes

1977; Woo et al. 1982; Woo et al. 1997; Yasuda and Hayashi 1999). While these

14

alterations in the extra-cellular matrix appear to be cell mediated, the exact mechanism by
which tissue load (or lack thereof) affects this process is unclear. Numerous in vitro
investigations have demonstrated that when a deformable substrate on which adherent
cells have been cultured is cyclically strained this extrinsic deformation activates a wide
array of cellular machinery including DNA synthesis, mitosis, gene expression, and cell
differentiation (Almekinders et al. 1993; Banes et al. 1994; Banes et al. 1995; Banes et al.
1995; Bhargava et al. 1999; Binderman et al. 1984; Birukov et al. 1995; Brighton et al.
1991; Brown 2000; Cheng et al. 1996; Hsieh et al. 2000; Matyas et al. 1995; Sumpio et
a1. 1990). Understanding the relationship between tendon strain and cell deformation
may provide insight into the role of physical stress in the homeostasis of normal tissue.
While the precise level (magnitude, frequency, and duration) of mechanobiological
stimulation required to maintain normal tendon homeostasis in not currently known, it is
very likely that an abnormal level(s) of stimulation may play a role in the
etiopathogenesis of tendinopathy (Archambault et al. 1995).
Over-stimulation

Several investigators have suggested that tendinopathy is initiated by repetitive
loading which over—stimulates the tendon cells leading to a mechano-biologic response of
degeneration (Archambault et al. 2002; Archambault et a1. 2001; Skutek et al. 2001;
Tsuzaki et al. 2003; Wang et al. 2003). Over-stimulation of tendon cells in vitro has been
shown to induce increases in inﬂammatory cytokines and degenerative enzymes
(Almekinders et al. 1993; Archambault et al. 2002; Banes et al. 1999; Banes et al. 1995;
Tsuzaki et al. 2003; Wang et al. 2003). The majority of these in vitro studies are based on

the response of large numbers of tendon cells cultured on artiﬁcial substrates to various

15

regimes of mechanical loading (Almekinders et al. 1993; Archambault et al. 2002;
Arnoczky et al. 2002; Banes et al. 1999; Banes et al. 1995; Tsuzaki et al. 2003).

However these monolayer cell cultures may not replicate the normal in situ
environmental conditions of tendon cells within a three dimensional, extracellular,
collagenous matrix (Arnoczky et al. 2007). Since mechanotransduction signals are
known to be mediated through the pericellular matrix to the nucleus via integrin based
cell-matrix connections (Banes et al. 1995; Ritty et al. 2003; Sachs 1988; Wang et al.
1993; Watson 1991) it is not clear how, or even if, these complex cell-matrix interactions
are maintained or recreated in cell cultures. In addition, the strain magnitudes (> 8%) and
durations (> 20 hours) used to elicit an up-regulation in the expression of these
inﬂammatory and catabolic genes may not be clinically relevant (Almekinders et al.
1993; Bhargava et al. 2004; Wang et al. 2003). Because tendon cell strain in situ has
been shown to be appreciably less than whole tendon strain (Arnoczky et al. 2002), it is
unlikely that such high levels of repetitive tendon cell strain could be reached and
maintained in vivo without signiﬁcant damage occurring within the extracellular matrix
of the tendon (Woo 1982). Also, since tendons are known to exhibit non-homogeneous
strain patterns in response to tensile load (Kastelic et al. 1978), it would seem impossible
to precisely recreate the complex and varied patterns of strain amplitudes experienced by
a population of tendon cells in situ by uniformly straining a large population of isolated
tenocytes in monolayer. Studies have shown that in rat tail tendons even local tissue
strain is nonhomogenous throughout the depth of the tendon (Arnoczky et al. 2002;
Hansen et al. 2002). Thus, the in vitro application of high magnitudes of cyclic cellular

strain to tendon cells in monolayer for excessively long durations may have little bearing

16

on what is actually occurring to tendon cells in situ and the clinical relevance of these
studies must be called into question (Arnoczky et al. 2007).
Hypothesis

To better understand how the mechanotransduction response of tendon cells under
tensile load affects gene expression and may contribute to the etiopathogenesis of
tendinopathy, an in Sllll rat tail tendon model has been utilized in an effort to maintain the
tendon cells’ natural cell-matrix interactions as well as the naturally occurring strain
ﬁelds that are developed in response to tensile loading (Arnoczky et al. 2004; Lavagnino
et al. 2006; Lavagnino et al. 2005; Lavagnino et al. 2003). In this dissertation the
mechanobiological response of tendon cells to changing loading patterns is examined and
a hypothesis is forwarded that it is a mechanobiological under-stimulation resulting from
altered cell-matrix interactions and not a repetitive over-stimulation of tendon cells that is
the etiopathogenic stimulus for the degenerative cascade which may eventually lead to
tendinopathy.
Chapter Overviews

Chapter 1 documents rat interstitial collagenase mRNA expression in an in situ
tendon cell model in response to various cyclic loading regimes. In addition, the effect of
chemically disrupting a segment of the mechanotransduction mechanism (cytoskeleton)
on interstitial collagenase mRNA expression was also examined. This study suggested
that removing tendons from their normal mechanical environment could signiﬁcantly
alter the homeostatic tension within the cytoskeletal tensegrity system and be responsible
for the up-regulation of collagenase mRN A expression seen following 24 hours of stress-

deprivation. In addition, the study demonstrated that interstitial collagenase mRNA

17

expression in tendon cells in situ was inhibited or even eliminated by cyclic tensile strain
in a dose-dependent manner (both amplitude and frequency), presumably through a
cytoskeletally based mechanotransduction pathway.

Chapter 2 investigated the potential that tendon cells may have a threshold, or set-
point with regard to their mechanoresponsiveness to tensile loading. A collagen gel
matrix model system was used to investigate if changes in the cytoskeletal tensional
homeostasis of tendon cells was related to the control of gene expression and to
determine the ability of tendon cells to re-establish their cytoskeletal tensional
homeostasis in response to a changing mechanical environment. Changes in cytoskeletal
tension control a reciprocal expression of anabolic and catabolic genes by tendon cells.
Of particular interest in this study was the apparent ability of the tendon cells to re-
establish their baseline level of internal cytoskeletal tension (as evidenced by a return to
baseline gene expression) following the loss of opposing external forces offered by the
collagen matrices following release.

Chapter 3 examined if an association exists between the tensile properties and the
collagen ﬁbril diameter distribution in in vitro stress-deprived rat tail tendons. The
results of this study demonstrated that the decrease in mechanical properties observed in
in vitro stress-deprived rat tail tendons was not correlated with the collagen ﬁbril
diameter distribution and, therefore, the collagen ﬁbril diameter distribution does not, by
itself, dictate the decrease in mechanical properties observed in in vitro stress-deprived
rat tail tendons.

Chapter 4 examined the ability to create isolated collagen ﬁbril damage and

subsequent altered cell-matrix interactions at the damaged site using the rat tail tendon

18

fascicle model. This study demonstrated the creation of isolated tendon ﬁbrillar damage
within an otherwise intact tendon fascicle results in an up—regulation of collagenase
mRNA expression and protein synthesis by only those tendon cells associated with the
damaged ﬁbrils. This would suggest a loss of load-transmitting function in the damaged
ﬁbril(s) and a subsequent altered cell-matrix interaction within the affected area.

Chapter 5 builds on the experimental study of Chapter 1 with the creation of a
multiscale computational tendon model composed of both matrix and ﬂuid phases to
examine how global tendon loading may affect ﬂuid-ﬂow-induced shear stresses and
membrane strains at the cellular level. The model analysis, combined with additional
experimental results, demonstrated that both strain rate and strain amplitude are able to
independently alter rat interstitial collagenase gene expression through increases in ﬂuid-

ﬂow-induced shear stress and matrix-induced cell deformation respectively.

19

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27

CHAPTER 1

Effect of Amplitude and Frequency of Cyclic Tensile Strain
on the Inhibition of MMP-l3 mRNA Expression in Tendon Cells:
an in vitro study

Michael Lavagnino
Steven P. Arnoczky
Tao Tian
Zachary Vaupel

From the Laboratory for Comparative Orthopaedic Research
College of Veterinary Medicine, Michigan State University,
East Lansing, Michigan 48824, USA

Lavagnino, M, Arnoczky, SP, Tian, T and Vaupel, Z (2003) Effect of amplitude and
frequency of cyclic tensile strain on the inhibition of MMP-1 mRNA expression in
tendon cells: an in vitro study. Connect Tissue Res 44:181-187.

28

ABSTRACT

To determine the effect of cyclic strain amplitude and frequency on MMP-13
(interstitial collagenase) expression in tendon cells, rat tail tendons (RTT) were
immobilized or cyclically displaced to various amplitudes (1, 3, or 6% strain at 0.017 H2)
or frequencies (1% strain at 0.017, 0.17, or 1.0 Hz) for 24 hr. Stress-deprivation for 24 hr
resulted in a marked upregulation in MMP-l3 expression. Cyclic tensile loading at 0.017
Hz was found to signiﬁcantly inhibit, but not completely eliminate, MMP—l3 expression
at 1% strain. MMP-13 expression was completely eliminated at 3 and 6% strain.
Increasing the frequency of application of the 1% strain to 0.17 or 1.0 Hz completely
eliminated MMP-13 expression. Disruption of the actin cytoskeleton with cytochalasin D
abolished all inhibitory effects of cyclic strain on MMP-13 expression. The results of our
study demonstrate that MMP-13 expression in tendon cells can be modulated by varying
amplitudes and frequencies of cyclic tensile strain, presumably through a cytoskeletally

based mechanotransduction pathway.

29

INTRODUCTION

Stress deprivation has been shown to have deleterious effects on the structural and
functional prOperties of ligaments and tendons (Amiel et al. 1982; Boorman et al. 1998;
Gamble et al. 1984; Goomer et al. 1999; Hannaﬁn et al. 1995; Loitz et al. 1989; Majima
et al. 2000; Majima et al. 1994; Noyes 1977). These effects appear to be cell mediated
(Hannaﬁn et al. 1995) and are thought to involve interstitial collagenase based alterations
of the extracellular matrix (Goomer et al. 1999; Loitz et al. 1989). Previous in vitro
studies have implicated a stress-suppressible effect of tensile stress on interstitial
collagenase expression in ligaments (Hannaﬁn et al. 1995; Loitz et al. 1989; Majima et
al. 2000). A recent study from our lab has demonstrated that in situ stress-deprivation of
rat tail tendon cells resulted in an immediate up-regulation of rat interstitial collagenase
(MMP-l3) mRNA expression (Arnoczky et al. 2004). Application of static tensile
loading produced a dose-dependent inhibition of MMP-l3 mRNA expression through a
cytoskeletally based mechanotransduction mechanism (Arnoczky et al. 2004). However,
this inhibition was incomplete at the physiological stresses examined (Arnoczky et a1.
2004).

In bone, cyclic loading has been shown to be more effective than static loading in
inhibiting catabolic activity and stimulating anabolic processes of cells (Burger and
Klein-Nulen 1999; Burger and Klein-Nulend 1999; Jacobs et a1. 1998). Cyclic loading of
bone produces oscillatory ﬂuid ﬂow within the lacunar/canalicular network (Burger and
Klein-Nulend 1999). This ﬂuid ﬂow, and the resulting shear stress, has been shown to be
an important physical signal that inﬂuences bone cell metabolism and bone adaptations to

mechanical loading (Burger and Klein-Nulen 1999; Burger and Klein-Nulend 1999;

Hsieh and Turner 2001; Hung et al. 1995; Jacobs et al. 1998; Owan et al. 1997; You et al.
2000). Indeed, in vitro studies have shown that, at low levels (<0.2%) of tissue strain,
ﬂuid ﬂow is a more potent stimulator of bone cells than is matrix deformation itself (You
et al. 2000). These ﬁndings have led to the successful application of low amplitude, high
frequency mechanical stimulation in bone to inhibit catabolism (Rubin et al. 2001).

Cyclic loading of tendons has been shown to produce interstitial ﬂuid ﬂow (Butler
et al. 1997; Chen et al. 1998; Hannaﬁn and Arnoczky 1994; Lanir et al. 1988), which in
turn, has been linked to gene expression through a mechanotransduction process
involving a cytoskeletal tensegrity system (Archambault et al. 2002). It is possible that
lower amplitudes and increased frequency of repetitive cyclic tensile loading may have a
more profound effect on maintaining tendon health than higher amplitudes of low
frequency or static loading.

Therefore, the purpose of this study was to examine the effect of various
amplitudes and frequencies of cyclic tensile strain on the regulation of MMP-l3
expression in rat tail tendon cells. It was our hypothesis that cyclic tensile strain will
inhibit MMP-13 expression in tendon cells in a dose dependent (amplitude and
frequency) manner through a cytoskeletally based mechanotransduction pathway.
MATERIALS AND METHODS
Drugs and Chemicals

Dulbecco’s modiﬁed Eagle medium (DMEM), fetal bovine serum (FBS),
Ascorbate, gentamicin, and penicillin-streptomycin-fungizone solution were obtained

from Gibco (Grand Island, NY, USA). Cytochalasin D, a fungal product that

31

depolymerizes actin ﬁlaments of the cytoskeleton, was obtained from SIGMA (St. Louis,
MO, USA).
Tendon Culture

Following institutional animal care and use approval, tendons were obtained from
the tails of adult Sprague Dawley rats. The tendons were removed immediately after
euthanasia and maintained in DMEM media supplemented with 10% FBS,
antibiotic/antimycotic solution, and Ascorbate at 37°C and 10% CO; for the duration of
the experiments.
Experimental Groups

Tendons were divided into groups for three experiments. Each experiment had a
zero time control group of fresh rat tail tendons. The ﬁrst experiment varied cyclic strain
amplitude as follows: Group 1: stress-deprived (no strain) for 24 hours; Group 2: 1%
cyclic strain at 0.017Hz for 24 hours; Group 3: 3% cyclic strain at 0.017Hz for 24 hours;
Group 4: 6% cyclic strain at 0.017Hz for 24 hours. The second experiment varied cyclic
strain frequency as follows: Group I: stress—deprived (no strain) for 24 hours; Group 2:
1% cyclic strain at 0.017Hz for 24 hours; Group 3: 1% cyclic strain at 0.17Hz for 24
hours; Group 4: 1% cyclic strain at 1.0Hz for 24 hours. The third experiment evaluated
the effect of cytochalasin D (SIGMA) as follows: Group 1: stress-deprived (no strain)
for 24 hours; Group 2: 6% cyclic strain at 0.017Hz for 24 hours; and Group 3: 6% cyclic
strain at 0.017Hz with lOuM cytochalasin D for 24 hours. There were twenty tendons
per group and each experiment was repeated three times.

Testing Setup

32

Stress-deprived tendons were kept in a 60mL dish in complete media and

conditions as described above for 24 hours. A sawtooth-shaped waveform of cyclic

strain was applied to tendons using a custom made, computer-controlled, stepper motor-

driven device (Figure 1.1). The grip-to—grip length was set to 40mm using digital calipers

 

Figure 1.1

A: Photograph of the computer-controlled, stepper motor driven, cyclic
loading system. The system permits ﬁve tendons to be loaded
simultaneously while in media. B: Close up view of the testing system
showing rat tail tendons in place.

33

The grip-to-grip length was set to 40mm using digital calipers (Mitutoyo, Tokyo, Japan).
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 25pm and a rate of 0.017, 0.17, or le. At 1%
strain, a frequency of 0.017Hz corresponds to a strain rate of 0.033% strain/second and a
total of 1440 load events over the 24-hour testing period. At strains of 3 and 6% this
strain rate increases to 0.1 and 0.2% strain/second, respectively. Increasing the frequency
to 0.17Hz results in a strain rate of 0.33% strain/second and a total of 14,400 load events,
whereas a frequency of 1.0Hz corresponds to a strain rate of 2% strain/second and results
in a total of 86,400 load events during the 24-hour test period.
Tendon Analysis

At the end of the experimental period, the unclamped tendon segment (~40 mm)
of the cyclically strained tendons and the entire length of the stress-deprived tendons
were collected and total cellular RNA was hybridized with a DNA probe for rat
interstitial collagenase (MMP-13) generated in our lab and a human GAPDH cDNA
control probe. The exposed ﬁlms were scanned and MMP-l3 expression was quantiﬁed
by optical density measurements and standardized as a ratio of GAPDH expression. The
effect of strain amplitude, strain frequency, and cytochalasin D on MMP-13 expression
was evaluated using an ANOVA and Tukey’s post-hoe test. Signiﬁcance was set at
p<0.05.
Construction of MMP-13 DNA Plasmid

To prepare the probe, MMP-13 DNA (GeneBank M60616) was ampliﬁed by

polymerase chain reaction (PCR) using rat genomic DNA template. The primers used

34

were 5’-GCC CAT ACA GTT TGA ATA CAG TAT CTG-3’ and 5’-CCA GTT TAA
TAA ACA CCA TCT CTT GA-3’. PCR product (1167-bp) was subjected to
electrophoresis on 1% agarose gel and recovered using Qiaex H Kit (QIAGEN Inc.,
Valencia, CA, USA) and then cloned into pCR®2.1-TOPO® plasmid using TOPO TA
Cloning kit (Invitrogen Living Science, Carlsbad, CA, USA). The plasmid was
transformed into competent E. coli with selection for kanamycin and ampicillin resistance.
The construction was conﬁrmed by restriction enzyme digestion (EcoR I, EcoR V, and
Hind III) and polymerase chain reaction.

RNA Extraction and Northern Blot Analysis

 

Total cellular RNA was isolated from rat tail tendons by the acid guanidine
thiocyanate-phenol—chloroform procedure (totally RNA kit, Ambion Inc., Austin, TX,
USA). The RNA samples were subjected to electrophoresis on 1.2% agarose gels
containing 0.66M formaldehyde and MOPS, then transferred to a nylon membrane
(Pierce Corp, Rockford, IL, USA) for 1 hour in TAE at 300mA. Following transfer, the
membrane was air—dried and UV cross-linked at 10 Joules/cmz.

The MMP-13 probe and human GAPDH cDNA control probe (Clontech
Laboratories, Inc., Palo Alto, CA, USA) were labeled with biotin using the North2South
Direct HRP Labeling and Detection Kit (Pierce Corp). The RNA blots were hybridized
with labeled probes (10ng/ml hybridization solution) at 55°C for 1 hour. The membrane
was then washed with 40 ml (~0.5 mL per cmz) 2x SSC/0.1% SDS at 55°C (3 x 5
minutes) and washed with 40 ml (~0.5 mL per cm2) of 2x SSC at room temperature.

Following wash, the membrane was incubated with chemiluminescent working

solution for 5 minutes and exposed to ﬁlms for 5-10 minutes. The ﬁlms were scanned

35

with a laser ﬁlm digitizer (Lumiscan 75, Lumisys Inc., Sunnyvale, CA, USA) and MMP—
13 mRNA expression was quantitated by optical density measurements using Scion
Image (Scion Corporation, Frederick, MD, USA).
RESULTS

In all experiments, stress-deprivation for 24 hours resulted in a signiﬁcant
(p<0.05) up-regulation of MMP—l3 expression in tendon cells compared to fresh tendons

(Figures 1.2-1.4).

 

MMP—13
GAPDH

 

 

Lane 1 fresh tendon (0 time)

Lane 2 no strain -24 hrs

Lane 3 1% cyclic strain @0.017Hz -24hrs
Lane 4 3% cyclic strain @0.017Hz -24hrs
Lane 5 6% cyclic strain @0.017Hz -24hrs

 

 

 

Figure 1.2 Representative Northern blot gel from the amplitude experiment
illustrating the relative expression of MMP-13 mRNA expression in fresh
control tendons (lane 1); immobile for 24 hours (lane 2); 1% cyclic strain
at 0.017Hz for 24 hours (lane 3); 3% cyclic strain at 0.017Hz for 24 hours
(lane 4); 6% cyclic strain at 0.017Hz for 24 hours (lane 5). GAPDH was
used as an internal control. Experiments were performed three times and a
representative result is shown.

36

Amplitude

A low cyclic strain amplitude of 1% at 0.017Hz resulted in a significant (p<0.05),
but incomplete, inhibition of MMP-13 expression. Increasing the cyclic amplitude to 3
or 6% strain at 0.017Hz completely eliminated MMP-13 expression (Figure 1.2).
Frequency

A low cyclic strain frequency of 0.017Hz at 1% strain again resulted in a
signiﬁcant (p<0.05), but incomplete, inhibition of MMP-l3 expression. Increasing the
cyclic frequency to 0.17 or 1.0Hz completely eliminated MMP-13 expression (Figure

1.3).

1 2 3 4 5

MMP—13 - 4.
GAPDH O--- -

Lane 1 fresh tendon (0 time)

Lane 2 no strain -24 hrs

Lane 3 1% cyclic strain @0.017Hz -24hrs
Lane 4 1% cyclic strain @0.170Hz -24hrs
Lane 5 1% cyclic strain @1.000Hz -24hrs

 

 

 

 

 

Figure 1.3 Representative Northern blot gel from the frequency experiment
illustrating the relative expression of MMP-l3 mRNA expression in fresh
control tendons (lane 1); immobile for 24 hours (lane 2); 1% cyclic strain
at 0.017Hz for 24 hours (lane 3); 1% cyclic strain at 0.17Hz for 24 hours
(lane 4); 1% cyclic strain at 1.0Hz for 24 hours (lane 5). GAPDH was
used as an internal control. Experiments were performed three times and a
representative result is shown.

37

Cytocghalasin D

Disruption of the actin cytoskeleton in rat tail tendon cells abrogated the
inhibitory effect of cyclic loading on MMP-13 expression. There was no signiﬁcant
(p=0.56) difference in MMP-13 expression between tendons stress—deprived for 24 hours
and tendons exposed to 6% cyclic strain at 0.017Hz and lOuM cytochalasin D for 24

hours (Figure 1.4).

1 2 3 4
WIMP-13 - -

GAPDH - -.- -

 

 

Lane 1 fresh tendon (0 time)
Lane 2 no strain -24 hrs
Lane 3 6% cyclic strain @0.017Hz -24hrs
Lane 4 6% cyclic strain @0.017Hz
+ cytochalasin -24hrs

 

 

 

Figure 1.4 Representative Northern blot gel from the cytochalasin D experiment
illustrating the relative expression of MMP-13 mRNA expression in fresh
control tendons (lane 1); immobile for 24 hours (lane 2); 6% cyclic strain
at 0.017Hz for 24 hours (lane 3); 6% cyclic strain at 0.017Hz for 24 hours
with 10 uM of cytochalasin D for 24 hours (lane 4). GAPDH was used as
an internal control. Experiments were performed three times and a
representative result is shown.

38

DISCUSSION

Application of load to bone (Brighton et al. 1991; Burger and Klein-Nulen 1999;
Hsieh and Turner 2001; Huiskes et al. 2000; Neidlinger—Wilke et al. 1994; Rubin et al.
2001), ligament (Hannafin et al. 2006; Hsieh et al. 2000), and tendon (Arnoczky et al.
2004; Arnoczky et al. 2002; Banes et al. 1995; Hannaﬁn et al. 1995) has been implicated
in the maintenance of tissue homeostasis. This is thought to occur through the transfer of
tissue strain to the cell cytoskeleton that, in turn, initiates a mechanotransduction
signaling response (Banes et al. 1995; Ingber et al. 1995; Sachs 1988). Transmission of
tendon strain to the extracellular matrix and cells has not been completely determined,
although substrate (extracellular matrix) strain and ﬂuid ﬂow are potential mechanisms
(Archambault et al. 2002; Takai et al. 1991).

Previous in vitro studies in ligaments and tendons have shown that tensile loading
produces substrate (extracellular matrix) strain that, in turn, alters cell shape (Arnoczky et
al. 2002; Matyas et al. 1994). Changes in cell shape and the resulting alterations in the
actin cytoskeleton are key components in the mechanotransduction response(s) of cells
(Banes et al. 1995; Ingber et al. 1995; Sachs 1988; Wang et al. 1993; Watson 1991). A
recent study has shown that when static tensile load is applied to rat tail tendons, rat
MMP-l (MMP-13) mRNA expression in tendon cells is inhibited in a dose dependent
manner (Arnoczky et al. 2004). This amplitude-dependent inhibition of MMP-13
expression appears to correlate with the progressive loss of collagen crimp (from the
surface to the center of the rat tail tendon) and the increase in ﬁber recruitment reported
in tendon fascicles with increasing stresses (Hansen et al. 2002). Thus, sequential

increases in substrate strain likely result in an increasing number of cells being deformed

39

(Arnoczky et al. 2002). However, because MMP-13 mRNA expression was only
inhibited and not totally eliminated with what would appear to be physiologic levels of
static stress, substrate deformation may not be the sole factor, or even the most important
factor involved in tendon cell signaling and subsequent gene expression with tensile load.

Fluid ﬂow and the resultant shear stress are thought to be other important
mechanisms for the transmission of tissue strain to cells (You et al. 2000). Shear strain
and an up-regulation in gene expression result when musculoskeletal cells in monolayer
are exposed to ﬂuid ﬂow (Archambault et al. 2002; Burger and Klein-Nulen 1999; Hung
et a1. 1997; Hung et al. 1995; Jacobs et al. 1998; Owan et al. 1997; Xu et al. 2000; You et
al. 2000). With bone cells, ﬂuid ﬂow is thought to play a signiﬁcantly greater role than
substrate deformation in activating gene expression (Owan et al. 1997; You et al. 2000).
While cyclic strain-induced ﬂuid ﬂow in bone occurs through a patent and well-
developed canalicular network, the ﬂow of interstitial ﬂuid in response to cyclic loading
is less deﬁned in tendons (Archambault et al. 2002; Hannaﬁn and Arnoczky 1994).
Researchers modeling the interstitial ﬂuid ﬂow in tendons have proposed that tensile
loading, and the resulting ﬂuid ﬂow, exert a mechanotranduction effect on the tendon
cells through shear strain and pressure; however, the exact levels of ﬂuid-induced shear
stress have not been identiﬁed (Butler et al. 1997; Chen et al. 1998). More research is
necessary to determine the precise role of cyclic tensile loading, ﬂuid ﬂow, and shear
stress in the mechanotransduction response of tendon cells in situ.

In the current study, increasing the cyclic strain frequency totally eliminated
MMP-13 mRNA expression at low amplitude strain levels. Similar effects have been

reported in other cell types (Yang et al. 1998). Low amplitude (1%) cyclic straining at

40

le is effective in suppressing interstitial collagenase production in human vascular
smooth muscle cells (Yang et al. 1998). Whereas the enhanced response of the cells in
the current study may be directly related to the increase in frequency of tensile loading, it
also may be a result of the increase in strain rate and/or the increase in the total number of
load events associated with increases in loading frequency. Frequency, strain rate, and/or
number of loading cycles potentially could have a threshold effect on MMP-13 mRNA
expression.

While the role of strain rate and number of loading cycles on MMP-13 mRNA
expression remains to be elucidated, an in vivo study examining the role of cyclic loading
in tendon healing suggests increased loading frequency and not an increase in total load
events is responsible for improved mechanical properties in healing tissues (Takai et al.
1991). In that study, loading frequencies (0.017 and 0.2Hz) similar to those used in the
current study were compared, but the number of loading events for each frequency
remained constant (Takai et al. 1991). The authors reported a signiﬁcant improvement in
the mechanical properties of tendons loaded at the higher frequency (Takai et al. 1991).
However, the exact mechanism for this improvement (i.e., inhibition of catabolism or
stimulation of anabolism) was not determined.

The loading frequencies and amplitudes utilized in this study were commensurate
with those used in previous in vivo (Majima et al. 2000; Takai et al. 1991) and in vitro
(Hannaﬁn et al. 1995; Yang et al. 1998) studies and reﬂect accepted physiologic levels
for normal tendon activity (Viidik 1990). Since tendons are known to exhibit
nonhomeogeneous strain patterns in response to tensile load (Kastelic et al. 1978), it is

impossible to determine precise amplitudes of strain experienced by the cells based on

41

overall tendon strain. Studies have shown that in rat tail tendons even local tissue strain
is nonhomogenous throughout the depth of the tendon (Arnoczky et al. 2002; Hansen et
al. 2002). The overall tissue strains used in this study are within the normal functional
range of tendons and the response of interstitial collagenase mRNA expression to tensile
load reported in the current study is similar to those reported for an in vivo ligament and
tendon study (Majima et al. 2000).

As stated, the strain-induced mechanotransduction response is thought to be
mediated through the cytoskeleton (You et al. 2000). Changes in cell shape, speciﬁcally
the loss of actin stress ﬁber organization, has been strongly correlated with collagenase
gene expression (Aggeler et al. 1984; Arnoczky et al. 2004). Procollagenase expression
in rabbit synovial ﬁbroblasts was induced by treatments that modiﬁed cellular actin
(Aggeler et al. 1984). Collagenase synthesis was upregulated within six hours of
cytoskeletal alteration (Aggeler et al. 1984) and would suggest that a change in the state
of actin assembly has a rapid inﬂuence on the kinetics of collagenase mRNA expression.
In the current study, treatment cytochalasin D, a fungal product that depolymerizes actin
ﬁlaments, completely abrogated the cyclic strain-induced inhibition of MMP-l3 mRNA
expression. These results match similar ﬁndings with static load (Arnoczky et al. 2004)
and further support the role of a cytoskeletally based mechanosensory tensegrity system
in the control of MMP-13 mRNA expression in tendon cells.

A limitation of this study is that only MMP-13 mRNA expression and not protein
synthesis was examined. Since gene expression may not correlate directly with synthesis
of the active enzyme, the association between stress-deprivation induced MMP—13

expression and extracellular matrix degradation was not examined in this study. While

42

stress-deprivation and cyclic load may have an effect on the regulation of other MMPs,
previous in vivo studies have suggested that interstitial collagenase appears to be the
major matrix metalloproteinase associated with immobilization-induced alterations of
tendons and ligaments (Goomer et al. 1999; Majima et al. 2000). Finally, the current
study only examined the ability of cyclic load to inhibit the upregulation of the MMP-l3
gene. Future studies are needed to determine the effects of cyclic loading on
downregulating MMP-l3 mRNA expression after its expression has been upregulated.
The results of our study demonstrate that MMP-13 mRNA expression in tendon
cells in situ can be modulated by cyclic tensile strain in a dose-dependent manner (both
amplitude and frequency), presumably through a cytoskeletally based
mechanotransduction pathway. Understanding the role of exercise on gene expression
may help determine optimal exercise protocols for both injured and healthy tissues
through the controlled application of load and frequency. It is possible that lower
amplitudes and increased frequency of repetitive cyclic tensile loading may have a more
profound effect on maintaining tendon health than higher amplitudes of low frequency or
static loading. This could lead to advances in overuse injury prevention and optimal

rehabilitation protocols following tendon injury and repair.

43

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47

CHAPTER 2

In vitro Alterations in Cytoskeletal Tensional Homeostasis
Control Gene Expression in Tendon Cells

Michael Lavagnino
Steven P. Arnoczky

From the Laboratory for Comparative Orthopaedic Research
College of Veterinary Medicine, Michigan State University,
East Lansing, Michigan 48824, USA

Lavagnino, M and Arnoczky, SP (2005) In vitro alterations in cytoskeletal tensional
homeostasis control gene expression in tendon cells. J Orthop Res 23: 121 1-1218.

48

ABSTRACT

An in vitro collagen gel system was used to determine the effect of alterations in
cytoskeletal tensional homeostasis on gene expression in tendon cells. Collagen gel
matrices, seeded with rat tail tendon cells, underwent cytochalasin D and gel contraction
treatments designed to alter the internal cytoskeletal homeostasis of the cells. Gels were
examined for cytoskeletal organization using a rhodamine phalloidin stain for actin. The
effect of altered cytoskeletal organization on mRN A expression of a catabolic (interstitial
collagenase) and anabolic (alphal(I) collagen) gene was examined using northern blot
analysis. Tendon cells in adhered gels demonstrated a highly organized cytoskeleton and
showed evidence of alpha1(I) collagen mRNA expression but no evidence of collagenase
mRNA expression. Treatment of the attached gel with cytochalasin D disrupted the
cytoskeletal organization and resulted in the up-regulation of collagenase mRNA and the
inhibition of alpha1(I) collagen mRNA expression. Release of the gels resulted in a cell
mediated gel contraction, an immediate loss of cytoskeletal organization, and an mRNA
expression pattern similar to that seen with cytochalasin D treatment. Isometric
contraction of the gel on itself or around a 3-point traction device resulted in an mRNA
expression pattern similar to the adhered gel. Gene expression in the contracted gels
could be reversed through chemical cytoskeletal disruption or removal of the traction
device which permitted further gel contraction. The results of the study suggest that
tendon cells can establish an internal cytoskeletal tension through interactions with their
local extracellular environment. Alterations in this tension appear to control the

expression of both catabolic and anabolic genes in a reciprocal manner.

49

INTRODUCTION

Mechanoresponsiveness is a fundamental feature of all living tissues (Brown et al.
1998; Ingber 1991; Ingber 1997) and tendons are no exception (Banes et al. 1995).
Experiments with cultured tendon cells in monolayer conﬁrm that mechanical stresses
can regulate a wide variety of cellular processes including signal transduction, gene
expression, and proliferation (Almekinders et al. 1993; Archambault et al. 2002;
Arnoczky et al. 2002; Banes et al. 1999; Banes et al. 1994; Banes et al. 1995; Tsuzaki et
a1. 2003; Tsuzaki et al. 2003). However, the precise level of mechanical load required to
initiate (or inhibit) specific cell processes has not been rigorously investigated.

Recent in vitro studies have shown that stress deprivation of tendon cells in situ
results in an immediate up-regulation of rat interstitial collagenase via a cytoskeletally
based mechanotransduction mechanism (Arnoczky et al. 2004; Lavagnino et al. 2003).
Conversely, application of a tensile load has been shown to inhibit mRNA expression of
interstitial collagenase in a dose dependent manner; presumably, through the same
cytoskeletally based mechanism (Arnoczky et al. 2004). These results suggest that
tendon cells may have a threshold, or set-point, with regard to their
mechanoresponsiveness to tensile loading.

Frost ﬁrst proposed the concept of the mechanostat set-point to explain the
mechanoresponsiveness of bone cells in controlling bone mass (Frost 1987). He
theorized that bone cells are programmed to sense a certain level of strain induced
signals. If the signal was below the set-point the cell would activate catabolic
mechanisms that decrease bone mass (Frost 1987). Conversely, if the strain signal

exceeded the set-point, anabolic mechanisms would be activated to increase bone mass

50

(Frost 1987). While this concept provides an explanation of how bone mass adapts to
gross overloading and underloading, a recent study has suggested that bone cells can also
autoregulate their sensitivity to a strain-induced signal by altering their local
microenvironment (Rubin et al. 1999). It was theorized that in response to subtle changes
in mechanical stress bone cells could actively tune their microenvironment to maintain
their idealized strain environment (Rubin et al. 1999).

A similar response has been observed in ﬁbroblasts seeded into collagen gels.
These cells have been shown to generate a homeostatic contractile force within their
extracellular collagenous matrix (Brown et al. 1998). This is achieved through the
creation of tension within the internal cytoskeleton via an actomysin ﬁlament sliding
mechanism (Chen and Ingber 1999; Dickinson et al. 1994; Skalak et al. 1994). The cells
reciprocally increased or decreased their endogenous contractions against changes to
opposing external loads (Brown et al. 1998). This response allowed the ﬁbroblasts to
respond to perceived changes in mechanical loading in a way that maintained tensional
homeostasis between the cell and its surrounding extracellular matrix (Brown et al.
1998). It is probable that cytoskeletal tensional homeostasis is the mechanism by which
tendon cells establish and attempt to maintain their mechanostat set-point.

The purpose of this study was to determine if changes in the cytoskeletal tensional
homeostasis of tendon cells are related to the control of gene expression and to determine
the ability of tendon cells to re-establish their cytoskeletal tensional homeostasis in
response to a changing mechanical environment. Our hypotheses were that tendon cells
can generate an internal tensional homeostasis which calibrates the cell with respect to

gene expression (rat interstitial collagenase and (11(1) collagen) and that alterations in this

51

internal stress cause a reciprocal change in the expression of catabolic (interstitial
collagenase) and anabolic (ul(I) collagen) genes.
MATERIALS AND METHODS
Cell Culture

Rat tail tendon cells were harvested via primary explant cultures from adult
Sprague-Dawley rats euthanized for another unrelated study. The cells were expanded to
passage 3 in 75 cm2 tissue culture ﬂasks in Dulbecco’s modiﬁed Eagle medium, 10%
fetal bovine serum, Ascorbate (150 mg/ml), 0.01 mg/ml gentamicin, and 1%
antibiotic/antimycotic solution (Gibco, Grand Island, NY, USA) at 37°C in a 10% C02
atmosphere.
Collagen Gel

Collagen gels made of 2.4 mg/ml type 1 bovine collagen (Vitrogen, Cohesion
Technologies, Palo Alto, CA, USA) were seeded with rat tail tendon cells at a
concentration of 400,000 cells/mL. Two milliliter gels were created in individual 60 mm
culture dishes and allowed to adhere to the culture dishes for at least 24 hours before
treatment. The gels were incubated in complete media composed of Dulbecco’s modiﬁed
Eagle medium, 10% fetal bovine serum, Ascorbate, and penicillin-streptomycin-
fungizone (Gibco, Grand Island, NY, USA) at 37°C and 10% C03. The gels were
divided into groups to examine the effects of either chemical or physical treatments on
cytoskeletal organization and subsequent gene expression. At the end of the study, gels
were frozen at —80°C until processed for Northern blot analysis. Each group consisted of

ﬁve gels and was repeated three times. Additional gels were used to examine cytoskeletal

52

organization in attached, cytochalasin D treated, and released gels using a rhodamine
phalloidin stain.
Contraction and Chemical Alteration of the Cytoskeletal Tension

Seeded collagen gels were allowed to attach to the culture dishes for 24 hours and
then treated as follows: Group 1: left attached to the culture dishes for an additional 24
hours; Group 2: attached to the culture dishes for an additional 24 hours while exposed
to 10 uM cytochalasin D (SIGMA, St. Louis, MO, USA) to disrupt the cellular
cytoskeleton; Group 3: released from the culture dishes and allowed to contract for 24
hours; Group 4: released and allowed to contract for 10 days; Group 5: released from
the culture dishes and allowed to contract for 10 days, and then exposed to 10uM
cytochalasin D to disrupt the cellular cytoskeleton for an additional 24 hours; Group 6:
released from the culture dishes and allowed to contract for 14 days.
Contraction and Physical Alteration of the C ytoskeletal Tension

To study response of tendon cells to changing extracellular environments an
external 3-point traction device was inserted into the gels immediately following release
of the gels from the bottom of the culture dishes, such that the gels contracted around the
three stainless steel pins (Figure 2.1). The pins in the device were arranged in an
equilateral triangular fashion with 12 mm between each pin. This experiment consisted
of allowing seeded collagen gels to attach to the culture dishes for 24 hours and then
treating them as follows: Group 1: left attached to the culture dishes for an additional 24
hours; Group 2: released from the culture dishes and allowed to contract for 24 hours
around the 3-point traction pins; Group 3: released from the culture dishes and allowed to

contract for 10 days around 3-point traction pins; Group 4: released from the culture

53

dishes, allowed to contract for 10 days around 3-point traction pins, and then removal of

the pins from the gel allowing for an additional 24 hours of contraction.

 

Figure 2.1 Photograph showing the 3-point traction devices.

Construction of DNA plasmids to rat interstitial collagenase and rat at] (I) collagen

To prepare the collagenase probe, rat interstitial collagenase DNA (GeneBank
M60616) was ampliﬁed by polymerase chain reaction using rat genomic DNA template.
The primers used were 5’-GCC CAT ACA GTT TGA ATA CAG TAT CTG-3’ and 5’-
CCA G'I'I‘ TAA TAA ACA CCA TCT CT'I‘ GA-3’. PCR product (1167-bp) was
subjected to electrophoresis on 1% agarose gel and recovered using Qiaex II Kit
(QIAGEN Inc., Valencia, CA, USA) and then cloned into pCR®2.l-TOP0® plasmid
using TOPO TA Cloning kit (Invitrogen Living Science, Carlsbad, CA, USA). The
plasmid was transformed into competent E. coli with selection for kanamycin and
ampicillin resistance. The construction was conﬁrmed by restriction enzyme digestion

(EcoR I, EcoR V, and Hind HI) and polymerase chain reaction.

54

To prepare the collagen probe, rat (11(1) collagen DNA (GeneBank M60616) was
ampliﬁed by reverse transcription-polymerase chain reaction with total RNA puriﬁed
from cultured rat tail tendon ﬁbroblasts. The oligonucleotide primers used were 5’-GTC
CAT TCC GAA TTC CTG GTC—3’ and 5’GTC GCA CTG GCG ATA GTG G-3’. PCR
product (866-bp) was subjected to electrophoresis on 1% agarose gel and recovered using
a Qiaex II Kit (QIAGEN Inc., Valencia, CA, USA), and then cloned into pZErOTM-2
plasmid using a Zero BackgroundTM/Kan Cloning kit (Invitrogen Living Science,
Carlsbad, CA, USA). The plasmid was transformed into competent E. coli with selection
for kanamycin resistance. The construction was conﬁrmed by restriction enzyme
digestion (ECOR I and Hind III) and polymerase chain reaction.

RNA Extraction and Northern Blot Analysis

A Northern blot analysis was performed to assay for rat interstitial collagenase
mRNA expression and (11(1) collagen mRNA expression. The gels within each
experimental group were combined and total cellular RNA isolated by the acid guanidine
thiocyanate-phenol-chloroform procedure (totally RNA kit, Ambion Inc., Austin, TX,
USA). The RNA samples were subjected to electrophoresis on 1.2% agarose gels
containing 0.66M formaldehyde and MOPS, then transferred to a nylon membrane
(Pierce Corp, Rockford, IL, USA) for 1 hour in TAE at 300 mA. Following transfer, the
membrane was air—dried and UV cross-linked at 10 J/cmz.

The rat interstitial collagenase probe and (11(1) collagen probe, as well as a human
GAPDH cDNA control probe (Clontech Laboratories, Inc., Palo Alto, CA, USA) were
labeled with biotin using the North2South Direct HRP Labeling and Detection Kit (Pierce

Corp, Rockford, IL, USA). The RNA blots were hybridized with labeled probes (10

55

ng/ml hybridization solution) at 55°C for 1 hour. The membrane was then washed with
40 mL (~ 0.5 mL/cmz) 2x SSC/0.1% SDS at 55°C (3x 5 minutes) and washed with 40
mL (~ 0.5 mL/cmz) of 2x SSC at room temperature. Following the wash procedure, the
membrane was incubated with chemiluminescent working solution for 5 minutes and
exposed to ﬁlms for 5-10 minutes.
C ytoskeletal Organization

To evaluate alterations in cytoskeletal organization following chemical or
physical manipulations, additional gels were prepared for actin staining and confocal
laser microscopy. Gels were ﬁxed in 10% phosphate buffered formalin and stained with
rhodamine phalloidin (5 units/mL) (Molecular Probes, Eugene, Oregon, USA) to
examine the actin ﬁlament structure of the cells after the following treatments: a gel
adhered to a culture dish for 48 hours, a gel adhered to a culture dish for 24 hours and
then exposed to lOum cytochalasin D for 1 hour, and a gel adhered to a culture dish for
24 hours and then released and allowed to contract for 5 minutes. The gels were wet
mounted on a glass slide for viewing with a Zeiss Pascal Laser Scanning Confocal
microscope (Carl Zeiss, Thornwood, NY, USA). Observations were made using a 40x
oil immersion objective without a coverslip. Fluorescent images were obtained using a
HeNe 543 nm laser with a long pass 560 nm emission ﬁlter. Cell shape and actin stress
ﬁber organization were observed in cells throughout the gel. After localizing a

representative cell in each gel, a 2x zoom was used to obtain an image.

56

RESULTS
Cytoskeletal Organization

In gels adhered to the culture dish for 48 hours, the tendon cells appeared
elongated and their cytoskeletons contained well-organized actin stress ﬁbers (Figure
2.2A). The addition of cytochalasin D to the adhered gels or the physical release of the

gels from the culture dish resulted in an immediate loss of this actin stress ﬁber

organization (Figure 2.2B and C).

 

Figure 2.2 Representative rhodamine-phalloidin stained cell images under confocal
microscopy (40x) of A) elongated cells in adhered gels at 48 hours
containing well-organized actin stress ﬁbers, B) the addition of
cytochalasin D to the adhered gels or C) the physical release of the gels
from the culture dish resulted in an immediate loss of actin stress ﬁber
organization.

57

Contraction and Chemical Alteration of the Cytoskeletal Tension

Upon release from their attachment to their individual culture dishes, the tendon
cell seeded collagen gels were contracted by the tendon cells (Figure 2.3). The
contraction continued over the next two weeks condensing the gel into a dense, circular

bead at 14 days (Figure 2.3).

 

Figure 2.3 Photograph showing a representative gel after 48 hours of attachment to
its culture dish (A) and the contraction of the gel following release after 24
hrs (B), 10 days (C), and 14 days (D). (Scale bar = 10 mm).

In each of the groups examined, it was apparent that there was a reciprocal
relationship between rat interstitial collagenase and (11(1) collagen mRNA expression
such that when one was expressed, the other was not (Figure 2.4). Tendon cells in
adhered gels demonstrated expression of (11(1) collagen mRNA but no measurable
expression of rat interstitial collagenase mRNA (Lane 1). Disruption of cytoskeletal

stress ﬁber organization from exposure to cytochalasin D (lane 2) or gel release and

58

contraction for 24 hours (lane 3) produced an up-regulation of rat interstitial collagenase

mRNA expression and an inhibition of (11(1) collagen mRNA expression compared to the

adheredgel.
D
i:
'7: 8 m D 0
(u (u (n 0 (n
'o E 2 8 E. 8
09 .c a: — O —
d) O h L. + L.
5 s s s s a
< 0 N 1- 1- 1-
' m Interstitial
Collagenase mRNA
C .3 .CC C GAPDH

     

Type I a1

- W i Collagen mRNA

...CCCGAPDH
1 2 3 4 5 6

 

Figure 2.4 Representative Northern blot analysis of rat interstitial collagenase and
(11(1) collagen with GAPDH as a control. Lanes represent 1) adhered to
dish for 24 hours, 2) cytochalasin D for 24 hours, 3) 24 hours contraction,
4) 10 days contraction, 5) 10 days contraction plus cytochalasin D for
additional 24 hours, 6) 14 days contraction.

59

Following release and 10 days of contraction (lane 4) the gels became more
condensed and the cells demonstrated a down-regulation of interstitial collagenase
mRNA expression and an up-regulation of (11(1) collagen mRNA expression (compared
to actively contracting gels (lane 3)). The disruption of the cytoskeleton with cytochalasin
D following 10 days of gel contraction (lane 5) produced the same mRNA expression
pattern seen following cytoskeletal alteration in adhered gels (lane 2). This demonstrated
that the cells still possessed the ability to respond to cytoskeletal alteration after 10 days.
In gels that were released and allowed to contract for 14 days (lane 6), the gels reached an
asymptotic contraction (based on previous studies (Arnoczky et al. 2004)) and the mRNA
expression of rat interstitial collagenase and (11(1) collagen was similar to the adhered gel.
Contraction and Physical Alteration of the C ytoskeletal Tension

As noted previously, the tendon seeded collagen gels began to contract upon
release from their individual culture dishes. However, instead of contracting into dense
beads the gels contracted around the 3-point traction devices forming equilateral
triangular structures comprised of bands of condensing collagen gel (Figure 2.5).
Removal of the traction devices (and the opposing tractional resistance they provided)

permitted further contraction of the gels (Figure 2.5).

60

 

Figure 2.5 Photograph showing a representative gel with a 3-point traction device in
place immediately after release (A) and after 24 hrs (B) and 10 days (C) of
gel contraction around the three pins. Removal of the traction devices (and
the opposing tractional resistance they provided) permitted further
contraction of the gels (D). (Scale bar = 10 mm).

In each of the groups examined, it was apparent that there was a reciprocal
relationship between the rat interstitial collagenase mRNA and (11(1) collagen mRNA
(Figure 2.6). As noted above, gels adhered to the culture dish for 24 hours (lane 1)
produced no measurable expression of rat interstitial collagenase mRNA and a positive
expression of (11(1) collagen mRNA. Releasing the gel and allowing it to contract around
the 3-point pins for 24 hours (lane 2) caused an immediate up-regulation of rat interstitial
collagenase and an inhibition of (11(1) collagen mRNA expression compared to the
adhered gel. After releasing the gel and allowing a steady state of contraction around the

3—point pins after 10 days (lane 3) the cells regained similar mRNA expression to the

adhered gel. The removal of the 3-point pins after 10 days of gel contraction (lane 4)

61

allowed additional contraction to occur (Figure 2.5). This apparent alteration in cell
matrix interaction resulted in an up-regulation in the expression of interstitial collagenase

and an inhibition of (11(1) collagen mRNA expression.

(D

2 m c

._ 0’ '5.

U a IE '5 o
09 h B 3

< N 1- 1- +

V},
D

Interstitial
Collagenase mRNA

GAPDH

 

Type I (11
A Collagen mRNA

. GAPDH

1 2 3 4

   

   

Figure 2.6 Representative Northern blot analysis of rat interstitial collagenase and
(11(1) collagen with GAPDH as a control. Lanes represent 1) adhered to
dish for 24 hours, 2) 24 hours contraction around 3pt traction pins, 3) 10
days contraction around pins, 4) 10 days contraction around pins plus free
contraction for an additional 24 hours.

62

DISCUSSION

The results of the current study suggest that changes in cytoskeletal tension
control a reciprocal expression of anabolic and catabolic genes by tendon cells. It has
been suggested that the cellular regulation of biological function lies in the ability of cells
to sense, generate, and balance mechanical forces (Chicurel et al. 1998). This
mechanoresponsiveness has been shown to be mediated through a tensegrity apparatus
comprised of the cell’s cytoskeleton as well as its attachment(s) to the extracellular
matrix (Chen and Ingber 1999; Ingber 1991; Ingber 1997; Kolodney and Wysolmerski
1992; Pourati et al. 1998; Ralphs et al. 2002; Roy et al. 1999; Tomasek et al. 1992).
Previous studies have shown that cells can generate an internal tension within their
cytoskeleton by way of an actomysin ﬁlament sliding mechanism (Dickinson et a1. 1994;
Skalak et al. 1994). This mechanism allows a cell to maintain a constant cytoskeletal
tension in response to changes in external loading (Brown et al. 1998; Sims et al. 1992;
Takakuda and Miyairi 1996). This has been termed cytoskeletal homeostasis and is
thought to be the mechanism by which a cell maintains a pre-set level of sensitivity to
external loading (Brown et al. 1998).

In the current study, a previously described collagen gel matrix model system was
used to examine the effect of cytoskeletal tension on gene expression (Brown et al. 1998;
Brown et al. 1996; Eastwood et al. 1994; Eastwood et al. 1998; Eastwood et al. 1996;
Fringer and Grinnell 2001; Grinnell 1994; Grinnell 1999; Grinnell et al. 1999; He and
Grinnell 1994; Lee et al. 1993; Lin and Grinnell 1993; Rosenfeldt et al. 1998). In this
system, tendon cells seeded into the collagen gels were able to establish a cytoskeletal

tensional homeostasis through an isometric contraction against collagen gel matrices left

63

attached to their culture dishes. This was characterized by the presence of organized
stress ﬁbers within the cytoskeleton and an up-regulation of the anabolic gene (0t1(I)
collagen). Loss of cytoskeletal organization through chemical disruption or a
detachment of the gel resulted in an up-regulation in the expression of the catabolic gene
(interstitial collagenase) and an inhibition in the expression of the anabolic gene (0tl(I)
collagen). Previous studies have shown that alterations in cell shape, secondary to cell
detachment (Aggeler et al. 1984; Unemori and Werb 1986), or loss of extracellular
matrix tension produce an increase in interstitial collagenase expression (Arnoczky et al.
2004; Goomer et al. 1999; Lambert et al. 1992; Langholz et a1. 1995; Lavagnino et al.
2005; Lavagnino et al. 2003; Loitz et al. 1989; Mauch et al. 1989; Prajapati et al. 2000;
Unemori and Werb 1986) and a decrease in collagen production (Lambert et al. 1992;
Mauch et al. 1989; Mochitate et a1. 1991; Prajapati et al. 2000; Unemori and Werb 1986).

Detachment of the tendon cell seeded collagen gels was followed by a contraction
of the matrices by the cells, either on themselves or around the three point traction
devices. The phenomenon of collagen gel matrix contraction by ﬁbroblasts has been
documented in numerous studies (Brown et al. 1998; Brown et al. 1996; Eastwood et a].
1994; Eastwood et a1. 1998; Eastwood et a1. 1996; Fringer and Grinnell 2001; Grinnell
1994; Grinnell 1999; Grinnell et al. 1999; He and Grinnell 1994). The ability of the cells
to re-align their extracellular matrix by way of an internal contractile mechanism has
been implicated in the process of wound contracture and connective tissue
morphogenesis (Grinnell 1994; Grinnell 1999; Grinnell 2000; Harris et al. 1981; Stopak
and Harris 1982). In both instances, interstitial collagenase is thought to play a critical

role in the remodeling process of the extracellular matrix (Stemlicht and Werb 2001;

64

Werb and Chin 1998). During contraction of the gels the cells are thought to exert an
isotonic force against the contracting extra-cellular matrix (Grinnell 2000). As
contraction slows and the remodeling matrix is able to produce an opposing external
force (either by contracting against ﬁxed points or condensing upon itself) the forces
exerted by the cells becomes isometric and a homeostatic internal tension develops
(Brown et a1. 1998; Grinnell 2000).

In the current study, interstitial collagenase expression was partially inhibited
after 10 days in the freely contracting collagen matrices as the gels condensed and began
to provide resistance to the cellular tension and completely inhibited after 14 days when
the freely contracting gels reached an asymptotic level of contraction (Arnoczky et al.
2004). Contraction of the gels around the 3-point traction devices at 10 days resulted in a
complete inhibition of interstitial collagenase. Removal of the traction devices permitted
additional gel contraction and initiated an immediate up-regulation of interstitial
collagenase expression. In all cases, a decrease in interstitial collagenase expression was
accompanied by a reciprocal increase in 0t1(I) collagen expression. Thus, the cells were
able to return to their baseline level of gene expression (seen in the attached collagen gel
matrices) by altering their local extracellular environment through contraction of the gels.

The ability of cells to actively “tune” their microenvironment in order to
manipulate the degree to which they sense strain is thought to be the mechanism by
which tissues adapt to large variations in mechanical loading over time (Frost 1987;
Rubin et al. 1999). A recent study in bone suggested that osteocytes can actively
modulate their response to external loading by regulating their connections to the

extracellular matrix in both a positive (collagen) and negative (collagenase) manner

65

(Rubin et a1. 1999). It was theorized that through this “autoregulation” the osteocytes
were able to adjust their exposure to strain stimulus through the regulation of the physical
constraints of their own microenvironment (Rubin et al. 1999). This would permit a cell
to maintain a “set-point” of activation with respect to the level of mechanical stress
required to initiate a biologic response. This has been termed the “mechanostat set-point”
in bone cells (Frost 1987) and a similar phenomenon may occur in tendon cells.

Of particular interest in this study was the apparent ability of the tendon cells to
re—establish their base-line level of internal cytoskeletal tension (as evidenced by a return
to baseline gene expressions) following the loss of opposing external forces offered by
the collagen matrices following release. This suggests that tendon cells can play an active
role in “recalibrating” their sensitivity to changes in external stresses. While this
“recalibration” was accomplished by a gross reorganization and contraction of the pliable
collagen gel matrices over a 10 day time period such alterations in cell-matrix
interactions may be more localized (i.e. the pericellular matrix) and/or require more time
(chronic exposure to altered matrix strains) in a mature connective tissue setting.

The current study only examined the response of tendon cells to alterations in
cytoskeletal tensional homeostasis caused by a decrease in opposing external forces. The
application of increased external forces (above the presumed set-point of the tendon cells)
was not examined. A previous study has suggested that dermal ﬁbroblasts can respond to
increases in opposing external matrix strains by a reciprocal decrease in contractile force,
which maintains a homeostatic tension (Brown et al. 1998). The same may hold true for
tendon cells. However, the upper and lower limits of the external forces against which the

cell can maintain tensional homeostasis is likely dependent on a myriad of factors

66

including cell type and local extracellular matrix composition as well as the frequency
and rate of external stress application.

The relationship between internal cytoskeletal tension and gene expression would
appear to be a key factor in understanding the ability (or inability) of cells to adapt their
extracellular matrix to changing external stresses. The results of this study suggest that
tendon cells can establish an internal cytoskeletal tension through interactions with their
local extracellular environment. Alterations in this tension appear to control the
expression of both catabolic and anabolic genes in a reciprocal manner. Although
mechanotranduction signals are mediated through a variety of means (focal adhesions,
integrins, cytoskeleton) (Banes et al. 1995), the current study focused on how
cytoskeletal transformations lead to changes in gene expression. Additional studies are
needed to examine the role of cell-matrix connections in establishing and maintaining
tensional homeostasis in tendon cells and to determine the precise mechanical/physical
threshold (set-point) that is required to activate various catabolic and anabolic genes.
ACKNOWLEDGEMENTS
The authors would like to thank Keri Gardner and Tao Tian, PhD for their technical

expertise.

67

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72

CHAPTER 3

Collagen Fibril Diameter Distribution Does Not Reﬂect Changes in the
Mechanical Properties of in vitro Stress-Deprived Tendons

Michael Lavagnino
Steven P. Arnoczky
Katherine Frank2
Tao Tian

From the Laboratory for Comparative Orthopaedic Research
College of Veterinary Medicine, Michigan State University,
East Lansing, Michigan 48824, USA
2Kalamazoo College, Kalamazoo, Michigan 49006, USA

Lavagnino, M, Arnoczky, SP, Frank, K and Tian, T (2005) Collagen ﬁbril diameter
distribution does not reﬂect changes in the mechanical properties of in vitro stress-
deprived tendons. J Biomech 38:69-75.

73

ABSTRACT

The purpose of this study was to determine if an association exists between the
tensile properties and the collagen ﬁbril diameter distribution in in vitro stress-deprived
rat tail tendons. Rat tail tendons were paired into two groups of 21 day stress-deprived
and 0 time controls and compared using transmission electron microscopy (n = 6) to
measure collagen ﬁbril diameter distribution and density, and mechanical testing (n =6)
to determine ultimate stress and tensile modulus. There was a statistically signiﬁcant
decrease in both ultimate tensile strength (control: 17.95+/-3.99 MPa, stress-deprived:
6.79+/-3.91 MPa) and tensile modulus (control: 312.8+/-89.5 MPa, stress-deprived:
176.0+/-52.7 MPa) in the in vitro stress-deprived tendons compared to controls.
However, there was no signiﬁcant difference between control and stress-deprived
tendons in the number of ﬁbrils per tendon counted, mean ﬁbril diameter, mean ﬁbril
density, or ﬁbril size distribution. The results of this study demonstrate that the decrease
in mechanical properties observed in in vitro stress-deprived rat tail tendons is not
correlated with the collagen ﬁbril diameter distribution and, therefore, the collagen ﬁbril
diameter distribution does not, by itself, dictate the decrease in mechanical properties

observed in in vitro stress-deprived rat tail tendons.

74

INTRODUCTION

Tendons are composite aggregations of Type I collagen, elastin, proteoglycans,
glycolipids, water, and cells. The collagen ﬁbrils and their hierarchial arrangement are
thought to play an important role in the tensile properties of the tendon (Kastelic et a1.
1978; Viidik 1972). Previous research has documented changes in collagen ﬁbril
diameter distributions in tendons and ligaments as a result of aging (Derwin and
Soslowsky 1999; Parry et a1. 1978), stress (Cherdchutham et al. 2001; Zachos et al.
2002), stress deprivation (Binkley and Peat 1986; Nakagawa et al. 1989), genetically
induced alterations in the extracellular matrix (Clark et al. 2001; Derwin and Soslowsky
1999), healing (Christel and Gibbons 1993; Frank et al. 1992), and anterior cruciate
ligament graft remodeling (Frogameni et al. 1993; Oakes 1993).

Several studies have correlated a decrease in mean collagen ﬁbril diameter with a
loss of mechanical properties in healing and remodeling ligaments and tendons
(Frogameni et al. 1993; LaPrade et a1. 1997; Oakes 1993). Most authors attribute this to
an increase in the production of small diameter collagen ﬁbers associated with an
increase in type III collagen synthesis (Shino et a1. 1995). However, a recent in vitro
study has suggested that the decrease in mean collagen ﬁbril diameter seen in remodeling
tendon grafts may, in part, be a result of enzymatic (collagenase) degradation of
endogenous large collagen ﬁbrils (Cunningham et a1. 1999). In that study, rabbit medial
collateral ligaments exposed to bacterial collagenase for 72 or 144 hours demonstrated a
significant reduction in mean collagen ﬁbril diameter.

Interstitial collagenase has been implicated in the loss of material properties

associated with immobilization of ligament and tendons (Goomer et a1. 1999; Loitz et al.

75

1989). Several experimental studies have documented an increase in interstitial
collagenase mRNA expression following in vivo immobilization or in vitro stress-
deprivation of ligaments and tendons (Arnoczky et al. 2004; Lavagnino et al. 2003;
Majima et al. 2000). Thus, it is possible that interstitial collagenase may affect the
mechanical properties of stress-deprived tendons and ligaments by enzymatically
degrading endogenous collagen ﬁbers.

The purpose of this study was to determine if an association exists between the
tensile properties and the collagen ﬁbril diameter distribution in a previously described in
vitro, stress-deprived, rat tail tendon model (Arnoczky et al. 2004). hi this model, in vitro
stress deprivation has been shown to result in a signiﬁcant increase in rat interstitial
collagenase (MMP-13) mRNA and protein expression (Arnoczky et a1. 2004). We
hypothesized that in vitro stress deprivation for 21 days would result in a decrease in
tensile properties (tensile modulus and failure stress) and that this change in material
properties would reﬂect a decrease in mean collagen ﬁbril diameter.

MATERIALS AND METHODS
Drugs and Chemicals

Dulbecco’s modiﬁed Eagle medium (DMEM), fetal bovine serum (FBS),
Ascorbate, gentamicin, and penicil1in-streptomycin-fungizone solution were obtained
from Gibco (Grand Island, New York, USA).

Rat Tail Tendons

Following institutional animal care and use approval, tendons were obtained for

this investigation from the tails of two 13-month-old Sprague-Dawley rats, euthanized

with sodium pentobarbital injection. The tendons were removed immediately after

76

euthanasia. Using a sterile scalpel blade, the tail was cut between coccygeal vertebrae at
both the base and at the distal tip of the tail for a total length of approximately 120 mm.
Tendons were gently teased from the distal portion of each tail with forceps and placed
into a petri dish containing DMEM media supplemented with 10% FBS,
antibiotic/antimycotic solution and Ascorbate. The rat tail tendons were then cut in half
and paired into two groups: stress-deprived and control. The control group was
immediately processed for either transmission electron microscopy (n = 6) or mechanical
testing (n = 6). The stress-deprived tendon group were maintained in the above media
incubated at 37°C and 10% C03 for 21 days, with media changed three times per week,
before processing for either transmission electron microscopy (n = 6) or mechanical
testing (n = 6). An additional 30 tendons were divided into control (0 time) and stress-
deprived (21 days) groups for a Northern blot analysis to assay for MMP-13 mRNA
expression.
Transmission Electron Microscggy

Tendons were placed in 4% paraformaldehyde for 24 hours, transferred to 4%
formalin, and then sent to be cut and embedded for transmission electron microscopy
(TEM). The tendons were cut in cross section three times, with the location of the cuts
equally distributed along the length of the tendon. The cross sections (100 nm thick) of
each ﬁxed tendon were rinsed in 0.1M phosphate buffer then placed in 1% osmium
tetroxide in 0.1M phosphate buffer for 3 hours. They were then dehydrated in graded
ethanol solutions (30%, 50%, 65%, 75%, 95%, 100%), and transferred to propylene
oxide. Tendons were then inﬁltrated with an Epon-type resin (Poly/Bed 812; Araldite,

and dodenyl succinic anhydride [DDSA] in ratios of 5:4:12 [Polysciences, Inc,

77

Warrington, PA, USA]). The resin was inﬁltrated in three steps (50%, 75%, and 100%
resin: propylene oxide). Each inﬁltration process was performed for 12 hours, and then
the specimens were hardened at 60°C for 48 hours. Thin sections, three from the center
of each rat tail tendon, were stained with aqueous uranyl acetate and lead citrate, and then
scanned with a Philips 301 TEM (Philips Electronic Instrument Co. Roselle, IL, USA).
Three random ﬁelds were photographed from each section at a magniﬁcation of X 19,000.
Image Analysis

All images were scanned using a Hewlett Packard ScanJet Ich (Palo Alto, CA,
USA), and then quantitatively analyzed using Scion Image Beta 4.0.2 software (Scion
Corporation, Frederick, MD, USA). All ﬁbrils within each scanned ﬁeld were analyzed.
To eliminate any potential error due to non-perpendicular cuts, the collagen ﬁbril minor
diameter was used to represent actual collagen ﬁbril diameter. The area of each collagen
ﬁbril was measured and used to calculate the collagen ﬁbril density, deﬁned as the sum
of the area of collagen ﬁbrils in the image divided by the total image area.
Mechanical Testing

Tendons were frozen at -80°C in media until testing. At the time of testing,
tendons were thawed to room temperature and a 5 mm portion of each tendon dissected
and placed in a saline ﬁlled 12-well plate to measure initial tendon diameter with a
calibrated microscope. The tendon diameter was determined by taking the mean of four
measurements perpendicular to the long axis of the tendon and a circular cross section
was assumed for the computation of initial area (Haut 1983; Haut 1985; Haut 1986;
Rowe 1985). The remaining portion of the tendon was gripped at the ends with saw—

tooth clamps for a 40 mm gage length. The portion of the tendon under each clamp was

78

ﬁrst air-dried and placed between two pieces of emery board while the midsection (test
area) was kept moistened with saline (Haut 1983; Haut 1985; Haut 1986). Since
dehydration increases the strength of rat tail tendons by nearly 5 times (Betsch and Baer
1980), premature fracture at the grip was eliminated. The gripped tendon was then
mounted onto a custom—made material testing system. The system was equipped with a 5
lb load cell (Sensotec, Columbus, OH, USA), a linear variable differential transformer
(LVDT) (Lucas Schaevitz, Pennsauken, NJ, USA) to measure grip-to-grip tendon
displacement, and a motion controller (Newport, Fountain Valley, CA, USA) to strain the
tendons at a constant rate of 0.168 mm/s (~0.42% strain/s). Each tendon was preloaded
to 10 g then loaded at the above rate to failure in a phosphate buffered saline (PBS) bath
at room temperature. The load and displacement values were recorded using an analog-
to-digital computer data acquisition system. Failure stress, tensile modulus, and failure
strain were computed from the load-deformation data.
RNA Extraction and Northern Blot Analysis

A Northern blot analysis was performed to assay for MMP-l3 mRNA expression
after 21-days of stress deprivation in 30 tendons (15 control and 15 stress-deprived).
Total cellular RNA was isolated from the rat tail tendons in each group (control and
stress-deprived) by the acid guanidine thiocyanate-phenol-chloroform procedure (totally
RNA kit, Ambion Inc., Austin, TX, USA) and pooled. The RNA samples were subjected
to electrophoresis on 1.2% agarose gels containing 0.66M formaldehyde and MOPS, then
transferred to a nylon membrane (Pierce Corp, Rockford, IL, USA) for 1 hour in TAE at
300 mA. Following transfer, the membrane was air-dried and UV cross-linked at 10

Joules/cmz.

79

An MMP-13 probe (Lavagnino et al. 2003) and a human GAPDH cDNA control
probe (Clontech Laboratories, Inc., Palo Alto, CA, USA) were labeled with biotin using
the North2South Direct HRP Labeling and Detection Kit (Pierce Corp, Rockford, IL,
USA). The RNA blots were hybridized with labeled probes (lOng/ml hybridization
solution) at 55°C for 1 hour. The membrane was then washed with 40 mL (~ 0.5 mL per
cm?) 2x SSC/0.1% SDS at 55°C (3 x 5minutes) and washed with 40 mL (~ 0.5 mL per
cm2) of 2x SSC at room temperature. Following the wash procedure, the membrane was
incubated with chemiluminescent working solution for 5 minutes and exposed to ﬁlms
for 5-10 minutes.

Statistical Analysis

Paired t—tests were used to determine any signiﬁcant statistical difference between
control and deprived tendons for the mean ﬁbril diameter and density. A three factor
ANOVA was used to determine if there were any differences in collagen diameter
distribution between control and deprived tendons, with treatment and size as ﬁxed
factors and tendon as a random factor. Paired t-tests were also performed to obtain the
statistical difference in both the tensile modulus and ultimate failure strength from the
mechanical tests. Statistical signiﬁcance was set at p<0.05 for all tests.

RESULTS
Image Analysis

Fibril distributions for normal rat-tail tendons exhibit a near equal distribution of

both small (<100 nm 52.7%) and large (>100 nm 47.3%) ﬁbrils. There was no

signiﬁcant difference between control and stress-deprived tendons in the number of

80

ﬁbrils per tendon counted, mean ﬁbril diameter, or mean ﬁbril density (Figures 3.1-2,

Table 3.1).

 

Figure 3.1 Representative transmission electron microscope image of A: control rat
tail tendon cross-section and of B: a cross-section of a rat tail tendon
stress-deprived for 21 days (x 19,000). Scale bar = 500 nm.

81

  

 

 

 

 

 

 

0.5 -
a? o 4 L Egg
2 1 $1
0 L3:
2 1‘ C t I
E 0.3 - on ro
E ii . [Deprived
: 3.4% .
o a
g 0.2 §
3 "11‘
o .i‘

0.1 -

0.0 “"79 7‘ {3M ‘ $3; Ema;

Tendon 1 Tendon 2 Tendon 3 Tendon 4 Tendon 5 Tendon 6 Tendon
All

Figure 3.2 Histogram illustrating collagen ﬁbril density in control and 21 day stress-
deprived rat tail tendons for each of the six paired tendons measured and
for all the control and stress-deprived tendons combined. There was no
signiﬁcant differences between tendons, p>0.05.

Table 3.1 Mean i standard deviation for control and stress—deprived ﬁbril number,
mean ﬁbril diameter and mean ﬁbril density. Resulting p-value from
paired t-test with signiﬁcance set at p<0.05.

Fibril Number Mean Fibril Mean Fibril Density,
Diameter, nm nmzlnm2
Control 2951 :t 247 150.01 :1: 10.9 0.464 :1: 0.016
Stress-Deprived 2404 1 224 153.65 i 7.11 0.473 :t 0.029
p value 0.17 0.45 0.59

 

82

There was no signiﬁcant difference in the ﬁbril size distribution between control and

deprived tendons within each ﬁbril diameter bin (Figure 3.3).

 

 

 

 

 

 

 

 

 

 

I Control
- -- ——- I Deprived

 

 

 

 

 

 

 

 

Percentage of Total Fibrils (%)

 

 

 
     

g l
O O O O O O O O O O 0
to 0 LO 0 LO 0 ID 0 10 O In
Lt.) '— 1— N N CO CO V V to ID
I I I I I I I I I I

(V) v- v- 1- 1- 1- 1- 1- v- 1- 1-
1!) O l!) O ID 0 l0 0 LO 0

v— V- N N ('3 CO V V LO

Fibril ﬂameter (nm)

Figure 3.3 Histogram illustrating relative frequencies of collagen ﬁbril diameters in
control and 21 day stress-deprived rat tail tendons. There was no
signiﬁcant differences between tendons within each bin, p>0.05.

Mechanical Testing

There was a statistically signiﬁcant decrease in both ultimate tensile strength

(control: 17.95 i 3.99 MPa, stress-deprived: 6.79 i 3.91 MPa) and tensile modulus

(control: 312.8 1- 89.5 MPa, stress—deprived: 176.0 3: 52.7 MPa) in the stress-deprived

tendons compared to controls. A representative stress-strain curve from both groups is

shown in Figure 3.4. A comparison of tensile modulus, failure stress, and failure strain

for each pair of tendon samples is listed in Table 3.2.

83

Rat Tail Tendon Failure Testing

 

 

   
    

 

 

 

 

 

 

25 (0.168 mm / second)
20-
,
Control
[is 15 — ---- Deprived
2
a)"
(I)
9
<75 10 —
1
5-
O‘i lllll'T'l‘r'lT'I
0.06 0.08 0.10

 

0.02 0.04

0.00
Strain, mm/mm

Representative stress versus strain curves from paired control and 21 day

Figure 3.4
stress-deprived rat tail tendons of one ﬁbril.

84

Table 3.2

Comparison of the cross-sectional area, tensile modulus, failure stress, and
failure strain of control and stress-deprived rat tail tendons. TCross-
sectional area measurements were paired from the same tendon. The
control tendon area was used for both groups to calculate failure stress.
* signiﬁcantly different than control specimens, p<0.05.

 

 

 

 

 

Control Stress-deprived

Tendon Silgjrslal Tensile Failure Failure 85:31:28 Tensile Failure Failure
ID Area Modulus Stress Strain Area Modulus Stress Strain

(m2) (MPa) (MPa) (%) (mmz) (MPa) (MPa) (%)
1 0.078 455 23.46 6.31 0.078 191 2.90 2.59
4 0.195 262 17.85 9.32 0.195 182 9.72 8.15
5 0.160 230 13.99 7.24 0.160 96 1.14 2.61
6 0.201 227 13.39 7.96 0.201 171 10.35 8.12
9 0.129 346 21.47 7.63 0.129 259 9.69 5.05
11 0.114 357 17.56 7.36 0.114 159 6.93 7.37
Avg. 0.146 312.8 17 .95 7.60 0.146 176.0* 6.79* 5.60“
SD 0.048 89.5 3.99 0.99 0.048 52.7 3.91 2.57

 

 

 

Northern Blot

 

The Northern blot gel demonstrated a complete absence of MMP-l3 mRNA

expression in the pooled control tendons. However, MMP-13 mRNA expression was

clearly demonstrated in the pooled in vitro stress deprived tendons at 21 days (Figure

3.5).

Figure 3.5

MMP- 13 ”W

GAPDH C C

1 Control
2 Stress-deprived 21 days

Northern blot gel illustrating the relative expression of MMP-13 mRNA
expression in fresh control tendons (lane 1) and stress—deprived for 21
days (lane 2). GAPDH was used as an internal control.

85

DISCUSSION

Stress deprivation has been shown to have deleterious effects on the structural and
functional properties of ligaments and tendons both in vivo and in vitro (Gamble et al.
1984; Hannaﬁn et al. 1995; Majima et al. 1994; Noyes 1977). These cell mediated
effects of stress deprivation are thought to produce multiple alterations in the biochemical
and biomechanical character of these tissues including an increase in interstitial
collagenase mRNA expression (Arnoczky et al. 2004; Goomer et a1. 1999), an increase in
collagen degradation and synthesis leading to an increase in collagen turnover and a
decrease in collagen mass (Amiel et al. 1983), a decrease in proteoglycans and thus water
content (Gamble et al. 1984; Woo et al. 1997), a decrease in ultimate failure stress and
tensile modulus (Binkley and Peat 1986; Noyes 1977; Woo et al. 1982), and an increase
in reducible collagen crosslinks (Akeson et al. 1987).

The results of this 21—day in vitro stress deprivation study demonstrate no
signiﬁcant difference in mean collagen diameter, mean collagen density, or collagen
ﬁbril diameter distribution between control and in vitro stress-deprived rat tail tendons.
Previous 40-day in vivo stress deprivation studies have produced conﬂicting
ultrastructural results. Binkley and Peat (Binkley and Peat 1986) found a signiﬁcant
increase in the proportion of larger ﬁbrils and a signiﬁcant decrease in the proportion of
smaller ﬁbrils in immobilized rat medial collateral ligaments compared with controls.
Nakagawa et a1. (Nakagawa et al. 1989) however, found signiﬁcantly smaller mean
collagen ﬁbril diameter using a hindlimb disuse model of the rat Achilles tendon
compared with control tendons. The disparity observed between the results of these

studies as well as the results of the current study may be due to differences in

86

methodology, a variability in the degree of stress deprivation actually achieved, or
differences in the tissues used. It is also possible that the increase in small diameter
collagen ﬁbrils observed in one in vivo study (Nakagawa et al. 1989) could actually be
due to an anabolic production of new small diameter ﬁbrils associated with tendon
remodeling (Christel and Gibbons 1993; Frogameni et al. 1993; Oakes 1993) rather than
a collagenase-induced catabolic deconstruction of large diameter ﬁbrils as suggested by
others (Cunningham et al. 1999). In remodeling tissues such as tendon grafts, small
diameter ﬁbrils are predominant and are thought to represent newly synthesized collagen
(Shino et al. 1995).

The mechanical results of the current in vitro stress deprivation study agree with
those from several in vivo immobilization studies (Binkley and Peat 1986; Noyes 1977;
Woo et al. 1982), which demonstrated signiﬁcant decreases in tensile modulus and
ultimate tensile stress following immobilization. The absence of any signiﬁcant
difference in collagen ﬁbril diameter distribution in the presence of signiﬁcantly altered
material properties seen in the present study suggests that collagen ﬁbril diameter
distribution does not solely determine the mechanical properties of rat tail tendons. The
ultrastructural results of in vivo stress deprivation studies (Binkley and Peat 1986;
Nakagawa et a1. 1989) also lend support to this conclusion as they demonstrate that
regardless of the collagen ﬁbril diameter distribution (increase or decrease in small
diameter collagen ﬁbrils) the mechanical properties of these stress-deprived tissues
diminished over time.

Although some studies have correlated changes in collagen ﬁbril diameter with

changes in mechanical properties of tendons (Parry et al. 1978) and ligaments (Binkley

87

and Peat 1986), other investigations have suggested that collagen ﬁbril diameter
distribution alone cannot predict the material and structural properties of tendons (Derwin
and Soslowsky 1999; Derwin et al. 2001) and ligaments (Bay et al. 1993). Researchers
have shown other factors beside collagen ﬁbril diameter distribution are important for
mechanical integrity and strength of the tendon (Clark et al. 2001; Derwin et al. 2001;
Elliott et al. 2003; Haut 1985). Proteoglycans help to regulate the extracellular matrix
assembly and structure and are thought to play a role in the structure function relation in
tendons (Clark et a1. 2001; Derwin et al. 2001; Elliott et a1. 2003). An increased level of
collagen crosslinking has been shown to inhibit the rate of collagenase activity (Woessner
and Nagase 2002) and appears to increase the tensile modulus and reduce the strain to
failure of collagen (Haut 1985; Thompson and Czernuszka 1995). Other factors that may
play a role in determining the structure function relationship of tendons are the crimp
pattern (Hansen et al. 2002), the length of the collagen ﬁbril (Trotter and Wofsy 1989),
the cell volume fraction or other sources of collagen irregularity (Derwin and Soslowsky
1999; Shrive et a1. 1995) as well as the distribution and amount of proteoglycans and type
I collagen (Kronick and Sacks 1994).

The results of the current study also demonstrate an increase in levels of rat
interstitial collagenase mRN A expression following 21 days of in vitro stress deprivation.
Other studies have shown increases in interstitial collagenase mRNA expression in
tendons after 24 hours (Arnoczky et al. 2004) and 12 weeks (Goomer et a1. 1999) of
stress deprivation. A previous study from our lab using the same rat tail tendon model
has demonstrated that MMP-13 mRNA expression correlated with MMP-l3 protein

synthesis (Arnoczky et al. 2004). Although MMP-13 expression in this study was clearly

88

present after 21 days of stress deprivation, there was no associated change in the collagen
ﬁbril diameter. Thus, the effect of cell-produced collagenase on collagen ﬁbril diameter
may not be as immediate or as profound as the previously published effects with bacterial
collagenase (Cunningham et al. 1999). This is perhaps due to the impurities of other
non-speciﬁc proteinases found within bacterial collagenase as well as its different mode
of action in degrading type I collagen compared to interstitial collagenase (Woessner and
Nagase 2002).

The failure strain of the normal control tendons in the current study was similar to
that in other investigations (Haut 1985). The decrease in failure strain coupled with the
decrease in tensile modulus observed following in vitro stress deprivation in the current
study suggests a degradation in the properties of the collagen ﬁbril itself rather than an
increase in ﬁbril sliding. However, the mechanism of stress deprivation and MMP-13
expression on the ultrastructural components of extracellular matrix and the subsequent
correlation to the mechanical properties of stress-deprived rat tail tendons has yet to be
determined.

Care must always be taken when comparing similarities and differences between
in vivo and in vitro models at both the functional and molecular levels (Hart et al. 2002).
It is likely that the level of stress deprivation achieved in the current in vitro system is
more complete and more precisely duplicated when compared to the immobilization
methods employed in other in vivo systems (Binkley and Peat 1986; Majima et al. 2000;
Noyes 1977; Woo et al. 1982). While this may limit the ability to precisely compare the
biochemical and physiological sequelae of altered stress conditions in the two systems

(owing to the inability to precisely determine the levels of stress deprivation provided by

89

the various immobilization methods) we believe the in vitro stress deprivation model
used in the current study is appropriate to test the hypothesis that changes in material
properties are related to changes in collagen ﬁbril diameter distribution.

In summary, the current study demonstrates that the changes in tensile properties
observed in in vitro stress-deprived rat tail tendons are not related to changes in the
collagen ﬁbril diameter proﬁle of the tissue. Furthermore, the results of the current study
also suggest that, contrary to prior speculation (Cunningham et a1. 1999), the presence of
endogenous rat interstitial collagenase (MMP-l3) does not produce an increase in small
diameter collagen ﬁbrils in these in vitro stress-deprived tissues. While in vitro stress
deprivation in rat tail tendons has been shown to be associated with an increase in MMP-
13 mRNA and protein expression and a decrease in tensile properties, the precise
relationship between these sequela have yet to be determined.
ACKNOWLEDGEMENTS

The authors wish to acknowledge the technical expertise of Ralph Common in
obtaining the transmission electron microscope images. The authors would also like to
acknowledge the help of Zachary Vaupel and Erika Sorge in their help in analyzing the

electron microscope images.

90

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94

CHAPTER 4

Isolated Fibrillar Damage in Tendons Stimulates
Local Collagenase mRNA Expression and Protein Synthesis

Michael Lavagnino

Steven P. Arnoczky

Monika Egerbacher
Keri L. Gardner
Meghan E. Burns

From the Laboratory for Comparative Orthopaedic Research
College of Veterinary Medicine, Michigan State University,
East Lansing, Michigan 48824, USA

Lavagnino, M, Arnoczky, SP, Egerbacher, M, Gardner, KL and Burns, ME (2006)
Isolated ﬁbrillar damage in tendons stimulates local collagenase mRNA expression and
protein synthesis. J Biomech 39:2355—2362.

95

ABSTRACT

The etiology of repetitive stress injuries in tendons has not been clearly identified.
While minor trauma has been implicated as an inciting factor, the precise magnitude and
structural level of tissue injury that initiates this degenerative cascade has not been
determined. The purpose of this study was to determine if isolated tendon ﬁbril damage
could initiate an upregulation of interstitial collagenase (MMP13) mRNA and protein in
tendon cells associated with the injured ﬁbril(s). Rat tail tendon fascicles were subjected
to in vitro tensile loading until isolated ﬁbrillar damage was documented. Once ﬁbrillar
damage occurred, the tendons were immediately unloaded to 100g and maintained at that
displacement for 24h under tissue culture conditions. In addition, non—injured tendon
fascicles were maintained under unloaded (stress-deprived) conditions in culture for 24h
to act as positive controls. In situ hybridization or immunohistochemistry was then
performed to localize collagenase mRNA expression or protein synthesis, respectively.
Fibrillar damage occurred at a similar stress (41.13+/—5.94MPa) and strain (13.24+/-
1.94%) in the experimental tendons. In situ hybridization and immunohistochemistry
demonstrated an upregulation of interstitial collagenase mRN A and protein, respectively,
in only those cells associated with the damaged ﬁbril(s). In the control (stress-deprived)
specimens, collagenase mRNA expression and protein synthesis were observed
throughout the fascicle. The results suggest that isolated ﬁbrillar damage and the resultant
upregulation of collagenase mRNA and protein in this damaged area occurs through a
mechanobiological understimulation of tendon cells. This collagenase production may
weaken the tendon and put more of the extracellular matrix at risk for further damage

during subsequent loading.

96

INTRODUCTION

It 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 a1. 1995). However, the precise magnitude and
structural level of tissue injury that is required to initiate this degenerative cascade has
not been determined. While some investigators have implicated overstimulation of
tendon cells as a mechanobiological stimulus for inﬂammatory cytokine production and
matrix degradation (Almekinders et al. 1993; Archambault et al. 2002; Tsuzaki et al.
2003)((Bhargava et al. 2004; Wang et al. 2003) these studies have been performed in
monolayer culture and often include high strain magnitudes (Almekinders et a1. 1993;
Bhargava et al. 2004; Wang et al. 2003), long loading histories (Archambault et al. 2002;
Bhargava et al. 2004; Wang et al. 2003), and the addition of biochemical factors
(Archambault et al. 2002; Tsuzaki et al. 2003). Thus, the clinical relevance of such
studies is unclear.

Recent investigations have demonstrated that ﬁbroblasts are capable of
establishing a cytoskeletal tensional homeostasis through interactions with their local
extracellular environment (Brown et al. 1998; Lavagnino and 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 and Arnoczky 2005; Lavagnino et al. 2003).

97

A decrease in extracellular strain in tendons (below homeostatic levels) has been
associated with an increase in the upregulation of interstitial collagenase and a
subsequent decrease in the tensile properties of these tissues (Arnoczky et al. 2004;
Lavagnino et al. 2005). Therefore, it is possible that mechanobiological
understimulation 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 (Kastelic et al. 1980; Viidik 1972; 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 matrix interactions secondary to isolated ﬁbrillar damage could cause a
mechanobiological understimulation of tendon cells which has been shown to result in an
up-regulation of collagenase mRNA expression and protein synthesis (Lavagnino and
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 al. 2005). 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).

The purpose of the current study was to induce an isolated ﬁbrillar failure in an in
vitro rat tail tendon model system and determine if isolated ﬁbrillar failure results in an

up-regulation of collagenase mRNA expression and protein synthesis. The hypothesis

98

was that a mechanobiological understimulation of tendon cells secondary to isolated
ﬁbrillar damage in rat tail tendons would result in a local up—regulation in interstitial
collagenase mRNA expression and protein synthesis in these cells.
MATERIALS AND METHODS
Rat Tail Tendon

Following institutional animal care and use approval, tendon fascicles were
obtained from the tail tendons of adult Sprague-Dawley rats. The fascicles were removed
immediately after euthanasia. Using a sterile scalpel blade, the tail was cut between
coccygeal vertebrae at both the base and at the distal tip of the tail. Tendon fascicles
were gently teased from the distal portion of each tail with forceps and then stored for
less than four hours in a 100mm culture dish containing Dulbecco’s Modiﬁed Eagle
medium (DMEM) supplemented with an antibiotic/antimycotic solution until induction
of ﬁbrillar damage testing. During the ﬁbrillar damage testing, the tendon fascicles were
maintained in a complete media solution of DMEM supplemented with 10% fetal bovine
serum, antibiotic/antimycotic solution, and Ascorbate at room temperature. Ten rat tail
tendon fascicles from ten different rats were used to create the isolated ﬁbrillar damage.
To conﬁrm that collagenase mRNA expression and protein synthesis was due to ﬁbrillar
damage and a resulting lack of mechanobiological stimulus, additional rat tail tendon
fascicles were either processed immediately after harvest as negative controls (n=10) or
maintained under non-loaded (stress-deprived) conditions in tissue culture for 24 hours as

positive controls (n=10).

99

Induction of F ibrillar Damage

At the time of testing, a single rat tail tendon fascicle was mounted with a small
amount of slack in a custom-designed, low-load, tensile testing apparatus (Arnoczky et
al. 2002). The device was equipped with a 5 lb load cell (Sensotec, Columbus, OH,
USA) and a linear variable differential transducer (LVDT) (Lucas Schaevitz,
Pennsauken, NJ, USA) to measure grip-to-grip tendon displacement. Observations and
fascicle diameter (268 i 72 um) measurements were made using a stereomicroscope
(Olympus, Melville, NY, USA) with a 7x objective and recorded through the microscope
using a CCD camera (Javelin Systems, Torrance, CA, USA). The initial fascicle length
(40.4 :I; 0.8 mm) was established by consecutively applying a known displacement (0.001
mm) through a computer controlled stepper motor until the slack was removed and the
crimp pattern just began to diminish. The tendon fascicles were loaded starting from this
crimped state (0% strain, 0 MPa) at a displacement rate of 20 urn/s until ﬁbrillar damage
occurred. Fibrillar damage in the fascicle was determined by visible ﬁbril sliding and a
change in the reﬂectivity of the damaged ﬁbrils as a “crimp” pattern returned to these lax
ﬁbrils (Hansen et al. 2002; Kastelic et al. 1978; Viidik and Ekholm 1968). Previous
studies have shown that reﬂective light can be used to delineate loaded (taut) from
unloaded (lax) collagen ﬁbers (Hansen et al. 2002; Kastelic et a1. 1978; Viidik and
Ekholm 1968). The alteration in reﬂectivity (and the associated reappearance of crimp)
was used to delineate the location and extent of the damaged ﬁbrils. Additionally,
ﬁbrillar damage was determined to occur when increases in strain produced a decrease in
stress on the recorded stress-strain curve (Figure 4.1). Only those tendons in which

isolated ﬁbrillar damage could be documented on both imaging and the stress strain curve

100

were utilized. Immediately following visible ﬁbrillar damage, the tendon fascicles were
unloaded to 100 g (Figure 4.1) and maintained at that displacement while incubated at
37°C and 10% C02 for 24 hours. Following this 24 hour incubation period, the tendons
were ﬁxed in 4% paraformaldehyde at 4°C until processed for in situ hybridization or

immunohistochemistry.

50

 

.‘(C

40

03
O

llllllllllml

N
0

Stress (MPa)
03
\

1O

lllLllllllll

 

 

 

O 1 Cr T T I T f l l I an f l

0.05 0.1 0.15

0

Strain (mm/mm)

Figure 4.1 Representative stress-strain curve of a rat tail tendon fascicle (dark solid
line) demonstrating the point at which ﬁbrillar damage occurred (point C),
and the unloading of the tendon to 100g (dashed line). A representative
curve of the tendon loaded to failure displaying the negative slope in the
stress strain curve that eventually ends in total tendon failure (Lavagnino
et al. 2005) has also been included (light solid line). Points A-D on the
stress-strain curve correspond to the images of the fascicle at those points
in Figure 4.2A-D.

101

In situ hybridization

To determine the location of cells expressing rat interstitial collagenase (MMP-
13), in situ hybridization was performed on the ﬁxed tendon fascicles (5 injured, 5 fresh,
5 stress—deprived) to identify collagenase mRNA activity. The rat tail tendon fascicle
was cut into 15 mm pieces to localize the injury site and reduce the tissue size to facilitate
processing. The tendon fascicle segments were incubated in the following solutions: 0.2
N HCl (20 minutes), 3% hydrogen peroxide (20 minutes), RNase-free proteinase K (10
uL/mL for 10 minutes at 37°C) and 4% formalin in PBS. Subsequently, acetylation was
performed with acetic anhydride in 0.1 M triethanolamine. The segments were rinsed in
2x SSC (1x SSC containing 150 mM NaCl and 15 mM sodium citrate) at 37°C. For
hybridization, antisense and sense probes for rat interstitial collagenase were diluted in
hybridization solution (50% deionized formamide, 2x SSC, 50lrg/ml yeast RNA, SmM
EDTA, 0.2% Tween 20, 0.5% CHAPS and 100ug/ml Heparin) to a concentration of
2.5llg/mL, and incubated at 90°C for 3 minutes, then on ice for 5 minutes. The
prehybridization was carried out at 60°C for 1 hour. The hybridization solution was then
layered onto the fascicles and hybridized overnight at 37°C in a humid chamber.
Posthybridization washes were performed at 60°C for 3x 15 rrrinutes in 2x SSC with 50%
formamide and 10% Chaps, then washed with 2x SSC for 2 minutes. The segments were
incubated with block solution (Roche Diagnostics) for 1 hour at room temperature. The
digoxigenin-labeled hybrids were detected by antibody incubation performed according
to the manufacturer’s instructions (Roche Diagnostics) with the following modiﬁcations.
A 1:10 dilution of anti-digoxigenin (Fab) conjugated to Rhodamine was used for

overnight incubation at room temperature. Then an extra washing step of 0.025% Tween

102

in Tris-buffered saline (pH 7.5) was performed. The fascicle segments were wet
mounted on a glass slide for viewing with an Axiovert 200M ﬂuorescent microscope
(Carl Zeiss, Thornwood, NY, USA).

Preparation of RNA probe

A 1167-bp rat interstitial collagenase cDNA fragment was cloned into the pZErOTM-2
vector (Invitrogen Life Technologies). The construct was then linearized by cutting it
with EcoRI. In vitro transcription was performed using the appropriate RNA
polymerases (T7 RNA polymerase for the anti-sense probe and Sp6 RNA polymerase for
the sense probe) in the presence of digoxigenin (DIG)-linked UTP in the reaction mixture
(DIG RNA Labeling Kit, Roche Diagnostics).

Immunohistochemistry

The rat tail tendon fascicles (5 injured, 5 fresh, 5 stress-deprived) were ﬁxed in 4%
paraformaldehyde for 24 hours, and rinsed in 70% ethanol and distilled water. The ﬁxed
tissues were permeabilized in 0.2% Triton X-100 for 1 hour and antigen retrieval was
performed with an unmasking solution (H-3300, VECTOR Lab, Burlingame, CA, USA)
for 1 hour at 65°C followed by cooling at room temperature for 20 minutes. Unspeciﬁc
protein binding was blocked with a solution consisting of 2% horse serum, 1% BSA,
0.1% Triton X-100, and 0.05% Tween-20 in PBS for 1 hour. Anti- MMP-13 (Ab-5,
NeoMarkers, Labvision Corp, Fremont, CA, USA) was applied to the fascicles overnight
at 4°C at a dilution of 1:80. The primary antibody was detected with anti-rabbit Texas red
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:300. Tendon

fascicles were mounted in Vectashield® mounting solution with DAPI (H-1200,

103

VECTOR Lab) and viewed under transmitted and ﬂuorescent light on a Zeiss Axioplan
microscope.
Statistical Analysis

The association between fresh control, injured, and stress-deprived rat tail tendon
fascicles and interstitial collagenase gene expression and protein synthesis was evaluated
for signiﬁcance using a minimum chi-square test. Minimum chi-square values were
calculated for both gene expression and protein synthesis among the three groups with a
critical chi-square value of 5.99 (p=0.05) for signiﬁcance.
RESULTS
The crimp pattern on the rat tail tendon fascicles, apparent at 0% strain (Figure 4.2A),
disappeared with increasing tension (Figure 4.2B). Fibrillar damage was manifested as a
visible sliding of ﬁbrils and a resultant change in the reﬂectivity of the damaged ﬁbrils
(Figure 4.2C). This also coincided with a sudden decrease in stress on the stress-strain
curve (Figure 4.1C). The precise location of the isolated ﬁbrillar damage varied between
samples. Fibrillar damage occurred at similar stress (41.13 i 5.94 MPa) and strain (13.24
i 1.94 %) values in all experimental tendons. This ﬁbrillar damage occurred at
approximately 79% of the failure strain of rat tail tendon fascicles loaded to failure in a
previous study (Lavagnino et al. 2005). Upon unloading the tendon fascicles to 100
grams the crimp pattern reappeared within the area of ﬁbrillar injury, but not in the
remaining, uninjured, portion of the fascicles (Figure 4.2D). Isolated ﬁbrillar damage did
not appear to affect the ability of the remaining intact fascicle to support the 100g tensile
loads. Thus, while ﬁbrils within the tendon fascicle were damaged, the fascicle remained

structurally intact.

104

 

Figure 4.2

Images of a rat tail tendon fascicle at various points throughout the testing
protocol: A) Prior to loading (the crimp pattern is clearly visible). B)
During loading in the linear portion of the stress-strain curve
demonstrating the elimination of the crimp pattern. C) Onset of ﬁbrillar
damage as manifested by a change in the reﬂectivity of the damaged
ﬁbrils (arrows). D) Unloading of the tendon to 100g and the reoccurrence
of the crimp pattern within the damaged ﬁbrils (arrows). (bar = 200
microns)

In situ hybridization of all the injured rat tail tendons indicated an up-regulation

of MMP-13 mRNA in the cells within the damaged ﬁbril(s) (Figure 4.3). Cells within the

remaining, undamaged portion of the tendon fascicle showed no evidence of collagenase

mRNA expression. Fresh control fascicles showed no evidence of collagenase mRNA

expression while in the unloaded (stress-deprived) control samples, cells throughout the

entire fascicle were positive for collagenase mRNA expression (Figure 4.4).

105

100 um

 

Figure 4.3 Representative images of a rat tail tendon fascicle following ﬁbrillar
damage. A) The presence of the crimp pattern on the bottom of the tendon
fascicle (arrows) indicates the site of isolated ﬁbrillar damage. B) In situ
hybridization of the tendon fascicle reveals interstitial collagenase mRNA
expression in those cells associated with the damaged ﬁbril(s). The
borders of the tendon fascicle are delineated by broken lines. (bar = 100
microns)

100 um

 

Figure 4.4 Representative image of a control (unloaded [stress deprived] for 24
hours) rat tail tendon fascicle demonstrating interstitial collagenase mRNA
expression by cells throughout the entire fascicle. (bar = lOOmicrons)

Irnmunohistochemical staining for interstitial collagenase protein produced results
similar to that seen for collagenase mRNA. Cells within the damaged ﬁbrils stained
positive for collagenase protein while cells in the undamaged portion of the tendon
fascicle were negative (Figure 4.5). Fresh control fascicles showed no evidence of
collagenase protein synthesis while in the unloaded (stress-deprived) control samples,

cells throughout the entire fascicle were positive for collagenase protein synthesis.

106

d injured uninjured

1:":

up

injured uninjure

pa

   

 

 

Figure 4.5 A) Representative photomicrograph of an injured rat tail tendon fascicle

showing the damaged ﬁbrils (denoted by the presence of crimp) immediately
adjacent to uninjured fibrils. (bar = 20 microns)
B) Photomicrograph of the same ﬁeld under ﬂuorescent light demonstrating
the positive (light gray) staining of MMP-l3 protein in the cytoplasm of only
those cells within the damaged ﬁbrils. The nuclei have been counterstained
with DAPI (white) to help identify the cells. (bar = 20 microns)

A signiﬁcant association existed between control, injured, and stress deprived
tendons and interstitial collagenase mRNA expression (Min. x2 = 14.14, p=0.001) and
collagenase protein synthesis (Min. x2 = 14.14, p=0.001).

The absence of collagenase mRNA expression and protein synthesis in the fresh
control fascicles suggests that the process of removing the rat tail tendon fascicles from
the animal did not, in itself, contribute to the increase in collagenase gene expression and
protein synthesis.

DISCUSSION

It is well known that increasing tensile loading alters the structure of tendons

through the progressive loss of collagen crimp and the increase in ﬁbril recruitment

107

(Diamant et al. 1972; Hansen et al. 2002; Kastelic et al. 1980; Viidik 1972). At the
extremes of physiologic loading, ﬁbril sliding and ﬁbrillar damage occur prior to
complete structural failure of the tissue (Kastelic et al. 1980; Viidik 1972; Woo et al.
1982). In the current study, ﬁbrillar damage occurred at a similar strain (13.24 + 1.94%)
in all experimental tendons and was documented by a sudden decrease in load as seen on
the load deformation curve. This is similar to a recent study which documented ﬁbril
sliding in rat tail tendons using confocal laser microscopy (Screen et al. 2004). In that
study, the initiation of ﬁbril sliding and damage took place at a median value of 13%
strain and was found to coincide with a decrease in tensile stress (Screen et al. 2004).

The strain at which ﬁbrillar damage occurred in the current study was
approximately 80% of failure strain previously reported for rat tail tendon fascicles (Haut
1985; Lavagnino et a1. 2005). Studies in ligaments have shown that while loading the
tissue to 80% of the failure deformation did cause isolated ﬁbrillar damage, this isolated
ﬁbrillar damage had no effect on the mechanical properties above 80% deformation
including failure load and failure deformation (Panjabi and Courtney 2001; Panjabi et al.
1999; Panjabi et a1. 1996). The investigators suggested that the isolated injury could be
sufﬁcient to stimulate a biological response without sacriﬁcing the gross mechanical
behavior of the ligament (Panjabi and Courtney 2001).

The ability to produce isolated ﬁbril failure within an otherwise intact tendon
fascicle may be attributable to the multicomposite structure of the tissue (Kastelic et al.
1980; Viidik 1980). The sequential straightening and loading of crimped collagen ﬁbrils,
as well as interﬁbrillar sliding and shear between ﬁbers and/or ﬁbrils, produce a non-

linear load-deformation behavior of tendons that may put certain ﬁbrils “at risk” for

108

damage before others (Viidik 1980; Viidik 1990). Indeed, previous studies have
demonstrated that individual tendon ﬁber and ﬁbril failure begins to occur near the end of
the linear range of the stress strain curve prior to complete failure (Viidik 1980; Viidik
1990). While the isolated ﬁbril damage seen in the current study still permitted the
remaining tendon to withstand loading (100g) within the linear range of the load
deformation curve of the fascicles, the mode of ﬁbril injury or the effect of this injury on
the overall mechanical properties of the fascicles was not determined. Further studies are
needed to elucidate these micro-injury mechanisms and their impact on tendon material
and structural properties.

A previous study has shown that isolated ﬁbrillar damage in tendons has been
followed by a relaxation of the damaged ﬁbrils (Knorzer et al. 1986). This relaxation (or
laxity) of the damaged fibrils was identiﬁed by a change in the reﬂectivity of incident
light by these lax collagen ﬁbrils when compared to adjacent loaded ﬁbrils (Hansen et al.
2002; Kastelic et al. 1978; Viidik and Ekholm 1968). The sudden change in light
reﬂectivity (and the reappearance of crimp) in isolated collagen ﬁbrils coincided with a
decrease in strain on the stress strain curve conﬁrming ﬁbril damage.

In the current study, the reappearance of crimp within the damaged ﬁbril(s) was
observed immediately after injury suggesting that these ﬁbrils were no longer
transmitting load. The importance of mechanical stress on gene expression in tendon
cells has been the subject of several recent investigations (Arnoczky et a1. 2004;
Lavagnino and Arnoczky 2005; Lavagnino et al. 2005; Lavagnino et al. 2003). These
studies have shown that loss of a homeostatic tensile load on tendon cells results in an

immediate upregulation of interstitial collagenase mRNA and protein synthesis

109

(Arnoczky et al. 2004; Lavagnino and Arnoczky 2005 ; Lavagnino et al. 2005; Lavagnino
et al. 2003). In the current study, isolated ﬁbrillar damage resulted in the immediate
upregulation of collagenase mRNA expression in those tendon cells within the damaged
ﬁbril(s). This would suggest an altered cell-matrix interaction within the damaged portion
of the fascicle. While this alteration (decrease) in mechanotransduction stimuli is most
likely a result of damage to the extracellular matrix (e.g., collagen ﬁbrils, collagen
crosslinks, etc.) of the tendon fascicle and a subsequent loss of load-transmitting
function, it could also be a result of damage to other components of the
mechanotransduction pathway. Fibril sliding in loaded tendons produces strong local
shear forces (Knorzer et al. 1986) which could damage cell-matrix adhesions (focal
complexes, focal adhesions, or ﬁbrillar adhesions) and/or alter the cytoskeleton. Indeed,
a previous study has demonstrated that disruption of the cytoskeleton resulted in an
immediate upregulation of collagenase mRNA expression in tendon cells within a loaded
tendon (Arnoczky et al. 2004). Additional studies are needed to determine what, if any,
portion(s) of the mechanotransduction pathway could be damaged by this ﬁbrillar injury
mechanism.

Collagenase expression, secondary to mechanobiological understimulation in
tendons has been associated with a signiﬁcant decrease in tensile properties (Lavagnino
et al. 2005; Majima et al. 1994). Stress deprivation of rat tail tendons resulted in a
signiﬁcant increase in interstitial collagenase mRNA and protein expression as well as a
decrease in both tensile modulus and tensile strength (Lavagnino et al. 2005). A clinical
study examining matrix metalloproteinase activity in ruptured human tendons

demonstrated a signiﬁcant increase in interstitial collagenase activity when compared to

110

normal controls (Riley et al. 2002). This increase in collagenase activity was associated
with a deterioration in the quality of the collagen network. Another study which
examined the histopathology of ruptured and tendinopathic Achilles tendons suggested
that while the ruptured tendons were significantly more degenerated than the
tendinopathic tendons, the general pattern of tendon degeneration was common to both
groups (Tallon et al. 2001). This implies a common pathological mechanism acting on
both tendon populations (Tallon et al. 2001) and suggests that tendon degeneration is an
active, cell mediated process that may result from a failure to regulate speciﬁc matrix-
metalloproteinase activities in response to injury (Riley et a1. 2002).

The results of the current study demonstrate that isolated ﬁbrillar damage in
tendon fascicles results in an upregulation of interstitial collagenase mRNA expression
and protein synthesis in those tendon cells associated with the damaged ﬁbril(s). This is
thought to occur due to a mechanobiological understimulation of the tendon cells as a
result of altered cell-matrix interactions. The upregulation of interstitial collagenase
mRNA and protein by tendon cells throughout the unloaded (stress-deprived) control
tendon fascicles further supports the role of mechanobiological understimulation of
tendon cells in gene expression.

The resultant upregulation of interstitial collagenase mRNA expression and
protein synthesis may weaken the tendon and put more of the extracellular matrix at risk
for further damage with subsequent loading (Lavagnino et al. 2005). Therefore, it is
possible that isolated collagen ﬁbrillar damage may have more of an impact on long-term
tendon health by altering homeostatic mechanotransduction interactions between the cell

and the extracellular matrix rather than from any initial structural weakness of the tendon.

lll

While any reparative response(s) secondary to the isolated ﬁbrillar damage could
not be examined in the in vitro system used in the current study, understanding the nature
and extent of such responses would be critical in determining the ability of tendons to

recover from this localized ﬁbrillar damage.

112

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Archambault, J, Tsuzaki, M, Herzog, W and Banes, AJ (2002) Stretch and interleukin-
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Archambault, JM, Wiley, JP and Bray, RC (1995) Exercise loading of tendons and the
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Arnoczky, SP, Lavagnino, M, Whallon, JH and Hoonjan, A (2002) In situ cell nucleus
deformation in tendons under tensile load; a morphological analysis using
confocal laser microscopy. J Orthop Res 20229-35.

Arnoczky, SP, Tian, T, Lavagnino, M and Gardner, K (2004) Ex vivo static tensile
loading inhibits MMP-l expression in rat tail tendon cells through a cytoskeletally
based mechanotransduction mechanism. J Orthop Res 22:328-333.

Bhargava, M, Attia, ET and Hannaﬁn, JA (2004) The effect of cyclic tensile strain on
MMPs, collagen, and casein degrading activities of ﬁbroblasts isolated from

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Brown, RA, Prajapati, R, McGrouther, DA, Yannas, IV and Eastwood, M (1998)
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Diamant, J, Keller, A, Baer, E, Litt, M and Arridge, RG (1972) Collagen; ultrastructure
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Hansen, KA, Weiss, J A and Barton, J K (2002) Recruitment of tendon crimp with applied
tensile strain. J Biomech Eng 124:72-77.

Haut, RC (1985) The effect of a lathyritic diet on the sensitivity of tendon to strain rate. J
Biomech Eng 107:166-174.

Kastelic, J, Galeski, A and Baer, E (1978) The multicomposite structure of tendon.
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Kastelic, J, Palley, I and Baer, E (1980) A structural mechanical model for tendon
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Knorzer, E, Folkhard, W, Geercken, W, Boschert, C, Koch, MH, Hilbert, B, Krahl, H,
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Lavagnino, M and Arnoczky, SP (2005) In vitro alterations in cytoskeletal tensional
homeostasis control gene expression in tendon cells. J Orthop Res 23:1211-1218.

Lavagnino, M, Arnoczky, SP, Frank, K and Tian, T (2005) Collagen ﬁbril diameter
distribution does not reﬂect changes in the mechanical properties of in vitro
stress-deprived tendons. J Biomech 38:69-75.

Lavagnino, M, Arnoczky, SP, Tian, T and Vaupel, Z (2003) Effect of amplitude and
frequency of cyclic tensile strain on the inhibition of MMP-1 mRNA expression
in tendon cells: an in vitro study. Connect Tissue Res 44:181-187.

Majima, T, Yasuda, K, Yamamoto, N, Kaneda, K and Hayashi, K (1994) Deterioration of
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Panjabi, MM and Courtney, TW (2001) High-speed subfailure stretch of rabbit anterior

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Panjabi, MM, Moy, P, Oxland, TR and Cholewicki, J (1999) Subfailure injury affects the
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Panjabi, MM, Yoldas, E, Oxland, TR and Crisco, JJ, 3rd (1996) Subfailure injury of the
rabbit anterior cruciate ligament. J Orthop Res 14:216-222.

Riley, GP, Curry, V, DeGroot, J, van El, B, Verzijl, N, Hazleman, BL and Bank, RA
(2002) Matrix metalloproteinase activities and their relationship with collagen
remodelling in tendon pathology. Matrix Biol 21:185-195.

Screen, HR, Lee, DA, Bader, DL and Shelton, JC (2004) An investigation into the effects
of the hierarchical structure of tendon fascicles on micromechanical properties.
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Tallon, C, Maffulli, N and Ewen, SW (2001) Ruptured Achilles tendons are signiﬁcantly
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Tsuzaki, M, Bynum, D, Almekinders, L, Yang, X, Faber, J and Banes, AJ (2003) ATP

modulates load-inducible IL-lbeta, COX 2, and MMP-3 gene expression in
human tendon cells. J Cell Biochem 89:556—562.

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Viidik, A (1972) Interdependence between structure and function in collagenous tissues.
In: A Viidik and J Vaust (Ed.), Biology of Collagen. Academic Press, New York,

pp. Pages.

Viidik, A (1980) Mechanical properties of parallel-ﬁbered collagenous tissues. In:
Biology of Collagen, A Viidik and J Vuust (eds), pp. 237-255. London:
Academic Press.

Viidik, A (1990) Structure and function of normal and healing tendon and ligaments. In:
Biomechanics of Diarthroidal Joints, VC Mow, A Ratcliffe and SL Woo (eds.),
pp. 3-38. New York: Springer.

Viidik, A and Ekholm, R (1968) Light and electron microscopic studies of callagen ﬁbers
under strain. Z Anat Entwicklungsgesch 127:154-164.

Wang, JH, Jia, F, Yang, G, Yang, S, Campbell, BH, Stone, D and Woo, SL (2003) Cyclic
mechanical stretching of human tendon ﬁbroblasts increases the production of
prostaglandin E2 and levels of cyclooxygenase expression: a novel in vitro model
study. Connect Tissue Res 44:128—133.

Woo, SL, Gomez, MA, Woo, YK and Akeson, WH (1982) Mechanical properties of
tendons and ligaments. II. The relationships of immobilization and exercise on
tissue remodeling. Biorheology 19:397-408.

115

CHAPTER 5

A Finite Element Model Predicts the Mechanotransduction Response of
Tendon Cells to Cyclic Tensile Loading

Michael Lavagninol
Steven P. Arnoczkyl
Eugene Kepich2
Oscar Caballerol
Roger C. Haut2

(1) Laboratory for Comparative Orthopaedic Research, College of Veterinary Medicine,
Michigan State University, East Lansing, Michigan 48824, USA
(2) Orthopaedic Biomechanics Laboratories, College of Osteopathic Medicine,
Michigan State University, East Lansing, MI 48824, USA

Lavagnino, M, Arnoczky, SP, Kepich, E, Caballero, O, Haut, RC (2007) A Finite
Element Model Predicts the Mechanotransduction Response of Tendon Cells to Cyclic
Tensile Loading. Biomechanics and Modeling in Mechanobiology: Submitted

116

ABSTRACT

The importance of ﬂuid—ﬂow-induced shear stress and matrix-induced cell
deformation in transmitting the global tendon load into a cellular mechanotransduction
response has yet to be determined. A multiscale computational tendon model composed
of both matrix and ﬂuid phases was created to examine how global tendon loading may
affect ﬂuid-ﬂow-induced shear stresses and membrane strains at the cellular level. The
model was then used to develop a quantitative experiment to help better understand the
roles of membrane strains and ﬂuid-induced shear stresses on the biological response of
individual cells. The model was able to predict the global response of tendon to applied
strain (stress, ﬂuid exudation), as well as the associated cellular response of increased
ﬂuid-ﬂow-induced shear stress with strain rate and matrix-induced cell deformation with
strain amplitude. The model analysis, combined with the experimental results,
demonstrated that both strain rate and strain amplitude are able to independently alter rat
interstitial collagenase gene expression through increases in ﬂuid-ﬂow-induced shear

stress and matrix-induced cell deformation respectively.

117

1 INTRODUCTION

Cells are known to respond to physical signals by way of a mechanotransduction
tensegrity system, which connects the cell nucleus to the extracellular matrix through a
cytoskeleton (Banes et al. 1995; Brown et al. 1998; Ingber 1997). Deformation of the
cytoskeleton, via membrane anchored attachment proteins (integrins), or stimulation of
other transmembrane proteins (G-protein receptors, receptor kinases, mitogen-activated
protein kinases) (Wang 2006) initiates a cascade of gene expressions activating catabolic
and/or anabolic cell responses (Lambert et al. 1992; Lavagnino and Arnoczky 2005;
Mochitate et a1. 1991). For tendons, the importance of physical stimuli in maintaining
homeostasis has been well documented (Hannaﬁn et a1. 1995; Lavagnino et al. 2005;
Yasuda and Hayashi 1999). Recent in vitro studies have shown that stress deprivation of
tendon cells in situ results in an immediate up-regulation of rat interstitial collagenase
mRNA expression (Arnoczky et al. 2004; Lavagnino et al. 2003). Conversely,
application of static stress (Arnoczky et al. 2004) or cyclic strain (Lavagnino et a1. 2003)
has been shown to inhibit interstitial collagenase mRN A expression in this in vitro tendon
model through a cytoskeletally based mechanism. While this gene inhibition has been
shown to occur in an amplitude and frequency, dose-dependent manner (Lavagnino et al.
2003), the effect of cyclic loading rate on gene expression has not been explored.

Previous studies in bone have demonstrated that both matrix deformation and
ﬂuid ﬂow are important regulators of gene expression (Han et al. 2004; Mullender et al.
2004; You et al. 2000). Application of tensile strain to tendons leads to a progressive loss
of collagen crimp, an increase in ﬁber recruitment, and a subsequent deformation of the

extracellular matrix (Hansen et al. 2002; Kastelic et al. 1980). Tendon tensile strain has

118

also been shown to modulate tendon cell deformation in a dose-dependent manner
(Arnoczky et al. 2002). Cellular deformation has also been implicated as a cell signaling
mechanism in tendon cells through a calcium-based pathway (Arnoczky et al. 2007).

In addition to matrix deformation, experimental studies have demonstrated
interstitial ﬂuid ﬂow in response to cyclic tensile loading of tendons (Hannaﬁn and
Arnoczky 1994; Helmer et al. 2006; Lanir et al. 1988). Fluid ﬂow has been shown to
control gene expression in a monolayer of tendon cells, presumably through a
mechanotransduction mechanism, but without the signiﬁcant calcium inﬂux seen in
deformed cells (Archambault et al. 2002). Therefore, it is possible that the dose-
dependent inhibition of collagenase gene expression observed with increasing cyclic load
amplitudes and/or frequencies may be related to concomitant increase in deformation and
ﬂuid ﬂow induced shear stress on individual cells in the tendon (Lavagnino et a1. 2003).
However, the exact levels of ﬂuid-induced shear stress on tendon cells in vivo have not
been identiﬁed (Archambault et al. 2002).

While external loading is known to be an important regulator of bone metabolism,
it has been suggested that ﬂuid ﬂow may actually play more of a role than matrix
deformation in bone cell mechanotransduction (You et a1. 2000). In tendons, a biphasic
model has suggested the importance of distortional strain and hydrostatic stress as
important mechanical stimuli regulating the composition of tendon and gene expression
(Giori et al. 1993). This and other biphasic tendon models have successfully modeled
global tendon properties in response to strain (Atkinson et al. 1997; Giori et al. 1993; Yin
and Elliott 2004), but none have predicted local strains or ﬂuid-induced shear stresses on

the cell.

119

The mechanical response of tendon to load is determined by its solid (collagen
matrix) and ﬂuid components (Atkinson et al. 1997). Collagen ﬁbers and their crimp
formation are thought to play a signiﬁcant role in determining the toe-in region of the
nonlinear stress—strain response of tendon (Kastelic et al. 1980). Glycosaminoglycans
have also been cited as strong determinants of mechanical properties due to their role in
ﬁbril-ﬁbril binding (Redaelli et al. 2003) and their interaction with the ﬂuid in the
extracellular matrix (Robinson et a1. 2004). The ﬂuid content of a tendon, or its
hydration, is known to alter the mechanical response of tendons to strain rate (Haut and
Haut 1997). Although each of these components (extra-cellular matrix deformation and
ﬂuid ﬂow) is known to play an important role in the mechanical response of tendon to
load, their inﬂuence on cellular mechanotransduction is unknown.

Therefore, the purpose of this study was to create a multiscale computational
tendon model composed of both matrix and ﬂuid phases to examine how global tendon
loading may affect stresses and strains at the cellular level. We hypothesized that
mechanotransduction signaling in tendon cells can result from either increases in ﬂuid
ﬂow induced shear stresses or membrane strains on the tendon cells. The model was
developed to study the results of previous experiments (Lavagnino 2003) as well as to
guide the development of new experiments to help better understand the roles of
membrane strains and ﬂuid-induced shear stresses on the biological response of

individual cells.

120

2 MODEL DEVELOPMENT

A multi-scale modeling approach was utilized in this study to help predict the levels of
ﬂuid-ﬂow-induced shear stress on cells and cell membrane strain generated under various
amplitudes and rates of global tendon loading.

2.1 Global Model

The global model was based on the geometry and composition of a rat tail tendon (RTT).
Tendons are made of three main components: type I collagen (70-80% of dry weight), an
extraﬁbrillar matrix (proteoglycans, glycolipids, cells), and water (60-80% of wet weight)
(Woo et al. 1997). Each of these components has mechanically signiﬁcant functions in
the response of tendon to load (Woo et al. 1997).

2.1.1 Collagen Fibers

Collagen ﬁbers have been previously modeled as linear elastic (Diamant et al. 1972;
Kastelic et al. 1980; Stouffer et al. 1985), bilinear elastic (Belkoff and Haut 1992;
Hurschler et a1. 1997; Kwan and Woo 1989), and linear viscoelastic (Johnson et a1. 1996;
Lanir 1980; Wang et al. 1997; Wilson et al. 2004). To represent the nonlinear effect of
collagen recruitment in the current model, collagen ﬁbers were simulated using axially
oriented nonlinear spring elements attached at the nodes of the matrix elements, similar to
the model of Wilson et al. (Wilson et al. 1997). The tissue strain at which tendon ﬁbers
became uncrimped and began load bearing was assumed to vary linearly in the radial
direction, with the largest strain required at the RTT center and the smallest strain at the
outer boundary of the RTT (Hansen et a1. 2002). The collagen ﬁber distribution in
tendon was assumed to be uniform with no variation in collagen ﬁber density. Crosslinks

between collagen ﬁbers are formed by secondary collagen ﬁbers (types VI, XII, XIV)

121

and glycosaminoglycans. Although crosslinks are thought to play a role in the
mechanical properties of tendon (Haut 1985; Puxkandl et al. 2002; Redaelli et al. 2003;
Robinson et al. 2004), they are only indirectly (Poisson’s ratio, low permeability)
included in the current model.

2.1.2 Transversely Isotropic Matrix

Following the experimental demonstration of ﬂuid exudation from tendon during cyclic
tensile loading (Hannaﬁn and Arnoczky 1994; Lanir et al. 1988), computational models
have incorporated the effects of permeability and interstitial ﬂuid ﬂow (Adeeb et al.
2004; Atkinson et al. 1997; Butler et a1. 1997; Chen et al. 1998; Yin and Elliott 2004).
To adequately model ﬂuid ﬂow in the current model, the RTT matrix was assumed to be
transversely isotropic in the ﬁber or 2-axis direction, with a zero pore pressure enforced
on the outer boundary to allow free ﬂuid ﬂow out of the tendon. The transversely
isotropic model, with Poisson’s ratio greater than 0.5, has been measured in ligament and
shown to be necessary to generate ﬂuid exudation in response to tensile load (Adeeb et al.
2004; Hewitt et a1. 2001; Yin and Elliott 2004). Orthotropic materials may have higher
Poisson’s ratios (>0.5) than isotropic materials, as long as thermodynamic constraints are
met (Adeeb et al. 2004). A Poisson’s ratio greater than 0.5 is required for volume loss
with uniaxial tension, and this volume loss is presumably due to ﬂuid exudation from
tendon (Yin and Elliott 2004). The high Poisson’s ratio in tendon may be due to the
organization of ﬁbers and their crosslinks and not because of high Poisson’s ratios of the
individual tendon components (Adeeb et a1. 2004). The Poisson’s ratio in this model was

chosen to reﬂect the ﬂuid exudation effect of the whole tendon.

122

2.1.3 Tendon Permeability

A major determinant of ﬂuid ﬂow is permeability, but the permeability of tendon is not
known. However, it has been suggested that permeability is strain (Lai et al. 1981; Weiss
and Maakestad 2006; Yin and Elliott 2004), porosity (Chen et al. 1998) and/or voids ratio
(van der Voet 1997) dependent. Higher water content in the peripheral rim compared to
the tendon core suggests that the porosity or voids ratio may also be depth dependent
within a tendon (Wellen et al. 2005). The permeability in tendon is also believed to be
anisotropic with a higher permeability along the ﬁber direction than across the ﬁbers
(Butler et al. 1997; Chen et al. 1998; Han et al. 2000). Although the directional
dependence within tendon is known, the actual values are still in question. Therefore, for
the current model, permeability and voids ratio were assumed to be uniform throughout
the tendon.

2.2 Submodel

A submodeling function was utilized to provide the output from the global RTT model as
boundary conditions in the cell model. The submodel consisted of an ovoid-shaped cell,
a low-permeable cell membrane, pericellular and extracellular matrices, and nonlinear
collagen ﬁbers. Although tendon cells are often arranged in columns, the current model
used a lone cell located on the outer boundary of the tendon. The outer boundary of the
tendon was of interest, as cells in this area were assumed to be subject to the largest
matrix deformations and ﬂuid ﬂows. In addition, a previous in situ study documented the
cell nuclei deformation in this location following applied strain (Arnoczky et al. 2002).
The membrane of the cell was assumed to have a low permeability, thus diverting ﬂuid to

ﬂow around and not through the cell (Ateshian et al. 2007). Surrounding the cell and cell

123

membrane was the pericellular matrix. The tendon pericellular matrix appears similar in
composition to that of cartilage pericellular matrix in that they both contain versican and
type VI collagen (Ritty et al. 2003). In cartilage, the pericellular matrix signiﬁcantly
alters the mechanical environment of the cell and thus plays a role in
mechanotransduction (Guilak and Mow 2000). In addition, the submodel includes the
transversly isotropic extracellular matrix and nonlinear collagen ﬁbers as described for

the global model.

3 FINITE ELEMENT METHOD

3.1 Global Model

The RTT was assumed to be cylindrically shaped with symmetry along the radial and
axial directions. Thus, a global, poroelastic model of the RTT was created 20mm long
and 0.15mm in width (Figure 5.1). The RTT was divided into 300, 4-noded,
axisymmetric elements (0.2mm x 0.05mm) using the commercial code ABAQUS 6.3
(ABAQUS, Inc. Providence, RI).

3.1.1 Collagen Fibers

To represent the nonlinear effect of collagen recruitment, collagen ﬁbers were simulated
using axially oriented spring elements attached at the nodes of the matrix elements with a
bilinear collagen spring stiffness of ON/mm during ﬁber straightening and 20N/mm after

ﬁber straightening (Wilson et al. 1997).

124

Figure 5.1

 

__1\__
if
/\
\/

 

 

?>>

-_ll_____ll__
/\
/\

 

 

 

<>
P>>
<<
>
$

 

I

20mm ll

II

“4— 0.15mm —>

 

 

 

 

 

 

 

 

 

Axisymmetric global poroelastic model of the rat tail tendon (20mm x
0.15mm), divided into 300 4-noded axisymmetric elements (0.2mm x
0.05mm) with radially variant nonlinear spring elements attached at the
nodes of the matrix elements (springs), zero pore pressure on the outer
boundary (circles), constrained at the tendon center (triangles), and loaded
at the tendon end as per previous experimental conditions (arrows). The
darkened element boundary indicates the location of the submodel.

125

 

 

Total Reaction Force]

 

 

Outer ------- outer Middle —- - —— — - Center Middle — - — -— - Center

 

 

 

 

 

 

 

 

'l
I
0.15: / -—
0.1 *-
l o
005 w'
. ,p'
. ,o" ,l'
: 4'. 1’ r
l ,0" v’ o’
0‘ o’ o’
0'. v’ a’
,o" v’ 4’
‘f 0’ 1’
I .a. ’7’ ’0’
0 l .‘M r CAI-4’4 -" -I' r

0 0.5 1 1.5 2 2.5 3 3.5
Strain (%)

Reaction Force (N)

 

Figure 5.2 Reaction force (N) plotted against strain (%) to show the radial variation
in ﬁber recruitment from the outer boundary to the inner or center that
predicts the nonlinear response of the global tendon.

The tendon deformation at which the ﬁber became uncrimped and began load bearing

varied in the radial direction, with the largest strain required at the RTT center and the

smallest strain at the outer boundary of the RTT (Figure 5.2) (Hansen et al. 2002). A

sensitivity analysis of the global tendon response to each parameter (data not shown)

made it apparent that the collagen parameters (stiffness and uncrimping deformation)
were dominant determinants of resultant force compared to matrix parameters. Therefore
these collagen parameters were manually adjusted to ﬁt the experimental stress-strain

curve of a tendon loaded to 3% strain at 6% strain/minute. A constant ﬁber density of 4

columns of springs was used to mimic all collagen ﬁbers.

126

3.1.2 Transversely Isotropic Matrix

To adequately model ﬂuid ﬂow in the current model, the RTT matrix was assumed to be
transversely isotropic in the ﬁber or 2-axis direction. Material properties for the matrix
were taken from the range of previous models and experiments on tendon and ligament
(Table 5.1) (Adeeb et al. 2004; Atkinson et a1. 1997; Gupta and Haut Donahue 2006;

Haridas et al. 1999; Hewitt et al. 2001; Yin and Elliott 2004).

 

 

 

Table 5.1 Global matrix material properties.

E2 E1=E3 V21=V23 V13=V31 G12=G23 k VOid
Parameters (MP3) (MP3) (MP3) (m4/Ns) Ratio
Matrix 1.0 0.0457 1.7 0.7 0.1 3.086-14 2.0
Reference 00457.10 00457.10 07.2.73 0.3.09 0157-50 5.5e-18-3.98e-l4 1.0-2.33
Range

 

 

(Adeeb et al. 2004; Atkinson et al. 1997; Gupta and Haut Donahue 2006; Haridas et al.
1999; Hewitt et a1. 2001; Yin and Elliott 2004)

 

3.1.3 Boundary Conditions

Boundary conditions on the global model included zero pore pressures on the outer
boundary to allow free ﬂuid ﬂow out of the tendon (Figure 5.1 circles) and constraint in
the ﬁber direction at the RTT center to account for radial symmetry (Figure 5.1 triangles).
The RTT was elongated with an applied displacement to a peak strain in the ﬁrst cycle at
the same applied strain rate as previous experiments: 1% strain at 2% strain per minute
(0.017Hz); 1% strain at 20% strain per minute (0.17Hz); 3% strain at 6% strain per
minute (0.017Hz) (Figure 5.1 arrows) (Lavagnino et a1. 2003). Additionally the model
was also run in the current study at 3% strain and 2% strain per minute (0.0056Hz) to
help understand the individual roles of ﬂuid-induced shear stress and membrane strain on

interstitial collagenase mRN A expression of tendon cells.

127

3.2 Submodel

The submodel was not axisymmetric and was located in the midportion along the length
of the global model on the outer boundary (Figure 5.1 darkened element) and composed
of 4-node bilinear displacement and pore pressure (CPE4P) elements.

3.2.1 Submodel Components

A submodeling function was utilized to provide the output from the global RTT model
(displacements, reaction forces, pore pressure) for boundary conditions in the cell model.
The submodel was the size of the global element and it consisted of an ovoid-shaped cell,
a low-permeable cell membrane, pericellular and extracellular matrices, and nonlinear

collagen ﬁbers (Figure 5.3).

 

 

7%"
T

 

 

 

"ix
__[., +
200%. 250 . \ 0pm.
_L_ \ +

’ 2

—> 10 um.

 

 

 

 

 

 

 

 

‘—5O l.lm.—-*

Figure 5.3 Submodel of the rat tail tendon, the size of a global element, composed of
an ovoid-shaped cell, cell membrane, pericellular matrix (PCM),
extracellular matrix (ECM), and collagen ﬁbers.

128

The cell was chosen as an ovoid shape with a major axis length of 24pm and minor axis
length of 4pm. The cell membrane in this model was used as a low—permeable barrier
between the pericellular matrix and the cell to prevent ﬂuid ﬂow through the cell
(Ateshian et a1. 2007). The thickness of the membrane (500nm) was chosen in an attempt
to maintain a similar element size as the rest of the submodel and thus maintain
numerical stability. This same cell membrane thickness was also used in another
submodeling study (Gupta and Haut Donahue, 2006). Two limitations associated with
using a thicker membrane than normal (5-10nm) include altered permeability and
stiffness values. Having a thicker cell membrane may make the membrane stiffer and
thus less deformable. The permeability deﬁned for the cell membrane in this study is
higher by a factor of 20 than that previously prescribed to represent the permeability for a
500nm cell membrane (Ateshian et al. 2006). In taking the thickness of the cell
membrane into effect we have potentially increased the cell stiffness and permeability
and therefore predicted lower cell membrane deformation and ﬂuid ﬂow induced shear
stress than if using a normal membrane size (lOnm). Thus, by using a larger membrane
thickness to help maintain element stability in the submodel and altering its theoretical
permeability, the cell membrane deformations and ﬂuid induced shear stresses were
likely altered in an the same direction to preserve the relationship and help validate the
overall conclusions of this ﬁrst study. Some of these issues would necessarily have to be
further investigated in future studies to develop this mechanotransduction computational
effort. Surrounding the cell and cell membrane is the pericellular matrix with a 2.5um
thickness (Gupta and Haut Donahue 2006; Ritty et a1. 2003). Linear poroelastic

continuum elements were used to create the cell model with material properties taken

129

from the range of previous models (Table 5.2) (Guilak and Mow 2000; Gupta and Haut
Donahue 2006). The transversely isotropic extracellular matrix, as described for the
global model, surrounds the pericellular matrix. Nonlinear springs, as collagen ﬁbers,

connect only the central and outside edges of the cell model (Figure 5.3).

Table 5.2 Submodel material properties

 

 

 

 

Parameter E (MPa) v k (m4/Ns) Void Ratio
Pericellular Matrix 1.0 0.490 3.924e-14 2.0
Cell Membrane 1 .0 0.490 4.905e-19 2.0
Cell 0.5 0.069 4.415e-14 2.0

 

 

 

 

 

(Guilak and Mow 2000; Gupta and Haut Donahue 2006)

 

3.2.2 Submodel Analysis

Coupled pore ﬂuid diffusion and stress analysis were used to analyze the fluid ﬂow
velocities, stresses and strains within the model. Membrane strain was calculated by the
change in the perimeter length divided by the original perimeter length as follows:

Alf .
8:7. Fluid ﬂow induced shear stress was calculated by the following equation

1' = #331 , where p was the viscosity of the ﬂuid (0.001Pa-s) and gl- was the change in
v v

the ﬂuid velocity perpendicular to the surface of the cell (Gupta and Haut Donahue

2006).

4 EXPERIMENTAL METHODS
To compare the predicted cellular stresses and strains to the interstitial collagenase
mRNA inhibition of tendon cells in situ, the following in vitro experiment was

performed.

130

4.1 Cyclic Tensile Loading

After Institutional Animal Care and Use Committee approval was granted, rat tail
tendons were harvested from adult Sprague-Dawley rats immediately after euthanasia.
Using a sterile scalpel blade, the tail was cut between coccygeal vertebrae at both the
base and at the distal tip of the tail for a total length of approximately 120 mm. Tendons
were gently teased from the distal portion of each tail with forceps and maintained in
DMEM media supplemented with 10% FBS, antibiotic/antimycotic solution and
ascorbate incubated at 37°C and 10% C02. The rat tail tendons were divided into 6
groups as follows: Group A: 1% cyclic strain at 2% strain per minute (0.017Hz) for 24
hours; Group B: 1% cyclic strain at 20% strain per minute (0.17Hz) for 24 hours; Group
C: 3% cyclic strain at 6% strain per minute (0.017Hz) for 24 hours; Group D: 3% cyclic
strain at 2% strain per minute (0.0056Hz) for 24 hours; Group E: stress-deprived for 24
hours; Group F: fresh (0 hour) control. All experimental input parameters, except Group
D, were the same as in previous studies (Lavagnino et al. 2003). Stress-deprived tendons
were maintained in a culture dish in complete media under tissue culture conditions.
Cyclic strain was applied to tendons in complete media under tissue culture conditions
using a custom made, computer controlled, motor driven device (Lavagnino et a1. 2003).
While in the previous study Northern blots were used to show alterations in collagenase
expression with various global loading parameters, in the current study matrix
metalloproteinase (MMP)-l3 (rat interstitial collagenase) mRNA expression was

quantiﬁed using quantitative real-time polymerase chain reaction. (Q-PCR).

131

4.2 Quantitative Polymerase Chain Reaction

At the end of each experimental period, ten tendons from each group were placed in
1.0mL of RNAlaterTM (Qiagen, Valencia, CA) for a period of at least twenty-four hours
at 4°C before processing. Total RNA was then extracted using the Qiagen RNEasy Kit
(Valencia, CA) with the protocol provided for ﬁbrous tissues. RNA (200-400ng), once
concentrations were normalized, was converted into cDNA using the Invitrogen
SuperScript III Reverse Transcription system (Carlsbad, CA). Real Time Quantitative
PCR was performed using the TaqMan Gene Expression Assay from Applied Biosystems
(ABI, Foster City, CA). Samples were run in a 96-well plate (20ltl ﬁnal volume per
reaction) on an ABI 7500-Fast Q-PCR apparatus. The endogenous control used for all Q-
PCR experiments was 185 rRNA. Results were analyzed using the Sequence Detection
System software available from ABI. TaqMan probe and primer sets were obtained for
MMP-l3 (Rn01448197_m1) and 18s rRNA (Hs99999901_sl) from ABI’s Gene

Expression Assay database (http://allgenes.com).

5 RESULTS

5.1 Finite Element Results

The global model analysis closely predicted the nonlinear stress-strain response of the
tendon to 3% strain at 6% strain/minute (Figure 5.4). In addition, the global model also
indicated ﬂuid ﬂow in a radial direction leaving the tendon during tensile stretch (Figure
5.5). On the submodel level, this ﬂuid was predicted to ﬂow around the cell with the

highest velocity occurring at the cell poles (Figure 5.6).

132

 

- . Model - - - Experiment

Stress, MPa

 

0 0.5 1 1.5 2 2.5 3
Strain, °/o

Figure 5.4 Comparison of tendon model to actual tendon stress-strain response (3%
strain at 6% strain/minute).

 

 

FLVIL. Haqnimd 9
(Ave Crib: 7511

UVWV

 

 

 

 

 

 

Figure 5.5 Plot of the ﬂuid velocity magnitude (mm/s) showing ﬂuid ﬂow in the
positive direction (arrow) out of the tendon (3% strain at 6% strain/min).
The curved lines (springs) represent the collagen ﬁbers.

133

ﬁzz-3H / . jar-- \t\\-
-—9—-"—’ I“; 1.— . /. " ¥ \\\
3%? a a"- em -~ ‘\ \
33,. ”11‘ . ‘1'. ., T- “V
WWW?‘ \ ' \‘
Mme-Less -~ \. \\\
2;; s33 \ *0 \
“tier; \3‘ \ ‘Vi‘ \‘ \
:2: ".2 -- . \ :
2- 3m.» . s ' \.'\-\ \ -
_._., _.:.._ _ , m I} j \ \\ ~
# W... / .. \ \ \

 

Figure 5.6 Plots of the ﬂuid velocity resultant around the cell and out of the tendon
(3% strain at 6% strain/min).

The tendon model predicted that increasing both the global strain rate (2% to 6%
strain per minute) and strain amplitude (1% to 3%) (group A versus C) would result in an
increase in both the maximum shear stress (142%) and cell membrane strain (191%)
(Figure 5.7, Table 5.3). With an increase in the applied strain rate from 2% to 20% strain
per minute at 1% global model strain (group A versus B), the model predicted a
corresponding 124% increase in the maximum localized shear stress at the cell poles, but
only a modest (5%) increase in cell membrane strain (Figure 5.7, Table 5.3). In
contrast, the theoretical responses of the tendon model for inputs of 1% strain at 20%
strain per minute (group B) versus 3% strain at 6% strain per minute (group C) generated
similar maximum shear stress values (8% difference). However, the cell membrane
strain levels were increased by 175%. The model was then exercised in an attempt to
deﬁne an experiment to further explore the effects of membrane strain and shear stress.
For a 3% strain at 2% strain per rrrinute (group D), the model predicted a cell membrane
strain within 8% of group C, with a maximum shear stress that was 145% less than group

C (Figure 5.7, Table 5.3).

134

2.5E-03 ——— —

 

 

 

 

 

 

 

 

 

 

 

I
ZOE-03 -——-—— e e
1; I
a. II. _ _ _ _ __ _—
V : 'I' 0 0/0} ' 'A
‘0 1.5E-03 f "V _n l 1 /o @ 2 , mm
3 . : I 3 1% @ 20%/min -B
I; I ‘ '- - - 3% @ 6°/o/min -C
I
3 1.0E-03 . — —— - 3% @ 2°/o/min -D
0 I.
l
L as:

 

 

 

 

 

5.05-04 ‘ . ,l m
0.0E+OO é ? . k 3 A: - . . . . . - l
0 30 60 90 120 150 180 210 240 270 300 330 360
angle (deg)
Figure 5.7 Graph of shear-stress induced by ﬂuid ﬂow. Note the marked increase in

at the polar ends of the cell.

Table 5.3 Global model and submodel strain and shear stress values.

 

Group A Group B Group C Group D

 

Global Model Strain, % 1.00 1.00 3.00 3.00
Global Model Strain Rate, % strain/minute 2.00 20.0 6.00 2.00
Cell Membrane Strain. % 1.26 1.33 3.67 3.97
Maximum Shear Stress at 270°. mPa 0.414 0.927 1.004 0.409
Average Shear Stress, mPa 0.157 0.346 0.459 0.179
Collagenase Expression (Lavagnino et al. 2003) Inhibited Eliminated Eliminated NA

 

5.2 Q-PCR Results

The Q-PCR results from each group of tendons suggested direct correlations could be
established between the levels of membrane strain and shear stress predicted in the model
and the inhibition of collagenase mRNA. These data were calculated relative to the fresh
control (0 hours) sample (Figure 5.8). Tendon cells exposed to a higher global strain rate
and strain amplitude (group A versus C), expressed a signiﬁcantly reduced amount of rat
interstitial collagenase mRNA (Figure 5.8, Table 5.3). Increasing the global strain rate
from 2% to 20% strain per minute at 1% global model strain (group A versus B) also

signiﬁcantly reduced this expression. On the other hand, increasing the amplitude of

135

global strain (1% to 3%), with a reduction of global strain rate from 20% to 6% per
minute (group B versus C), had little effect on the expression of rat interstitial
collagenase mRNA expression (Figure 5.8, Table 5.3). These data were thus suggestive
that enhanced shear stress, rather than increased cell membrane strain was primarily
responsible for inhibition of collagenase expression. Furthermore, it was seen that in
comparing the results of group C to D, where the ﬂuid induced shear stress on the cell
was signiﬁcantly reduced at 3% strain, the expression of rat interstitial collagenase
mRNA was increased. Alternatively, if the Q-PCR results of group A versus D were
compared, the collagenase expression was signiﬁcantly reduced (Figure 5.8). This
biological response of the tendon was associated with a theoretical increase in cell

membrane strain and little change in the ﬂuid-induced shear stress on the cell (Table 5.3).

 

3500
2851

 

3000 7 -7 7-

2500 A r---)“

 

 

2000 -C, ,

 

1000 -7

 

Relative Quantification of MMP-13

 

j 33 81 1 54

0 l .r __ J _
Group A Group B Group C Group D Group E Group F
1% strain 1% strain 3% strain 3% strain Stress- Fresh (Oh)
(2%/min) (20%/min) (6°/o/min) (2%/min) Deprived Control

1

 

 

 

.__rn

Figure 5.8 Gene expression levels of rat interstitial collagenase (MMP-l3) as
determined by Real-Time Quantitative PCR. All experimental samples
were quantiﬁed relative to the fresh (0 hour) control.

136

6 DISCUSSION

The results of the initial study showed a strong association between the amount of ﬂuid-
ﬂow-induced shear stress on the cell and the inhibition of collagenase mRNA expression,
regardless of cell strain. The importance of ﬂuid-ﬂow-induced shear stress was
conﬁrmed when the additional boundary condition was predicted and showed that a
decrease in shear stress with the same cell membrane strain resulted in less collagenase
mRN A inhibition (3% strain at 6% strain per minute [C] versus 3% strain at 2% strain per
minute [D]). However, the additional boundary condition also showed that increasing the
membrane strain at similar low ﬂuid-induced shear stress also resulted in a decrease in
collagenase mRNA expression (1% strain at 2% strain per minute [A] versus 3% strain at
2% strain per minute [D]). Therefore, increased ﬂuid-ﬂow-induced shear stress alone
and cell membrane strain alone were shown to inhibit the expression of rat interstitial
collagenase mRNA. Thus, the results of this study conﬁrm our hypothesis that ﬂuid-
ﬂow-induced shear stresses and matrix-induced cell deformation are able to alter
catabolic gene expression, presumably through a mechanotransduction pathway.

The interstitial ﬂuid ﬂow and extrusion out of tendon predicted by the model have
been documented to occur in tendon following application of static and cyclic tensile
loading (Hannaﬁn and Arnoczky 1994; Helmer et al. 2006; Lanir et al. 1988) although
the exact amount of ﬂuid exuded per stretch remains unknown. The increased ﬂuid ﬂow
predicted around the cell with increased loading rate may explain why collagenase
mRNA expression decreases (Lavagnino et al. 2003). Similar to matrix-induced cell
deformation, ﬂuid ﬂow has also been suggested to affect cell function and gene

expression; however, the method of action has yet to be determined (Hannaﬁn and

137

Arnoczky 1994). A previous study has shown that the ﬂuid ﬂow induced by cyclic or
static load did not alter the diffusion of low molecular weight solutes compared to
unloaded controls (Hannaﬁn and Arnoczky 1994). These conclusions suggested that any
biological beneﬁt of cyclic loading (and resultant ﬂuid ﬂow) is probably not due to an
increase in cell nutrition (Hannaﬁn and Arnoczky 1994). Fluid ﬂow, as suggested in
bone, may alter gene expression as it induces a drag gradient on the pericellular matrix
ﬁbers (Han et al. 2004). The drag gradient on the pericellular ﬁbers, which are connected
to the cell membrane, in turn create a drag force on the cell, thereby creating strain
ampliﬁcation from ﬂuid (Han et al. 2004). While the current model does not include
pericellular matrix ﬁbers, the increased ﬂuid velocity with increased strain rate suggests
that a similar phenomenon could occur in tendons.

An increase in ﬂuid-induced shear stress on the cell could also explain the
decreased collagenase expression with increased ﬂuid ﬂow. Fluid ﬂow induces shear
stress on the cell membrane and can mechanically stimulate cells to alter their gene
expression (Archambault et al. 2002; Jin et al. 2000; Ng et al. 2005; You et al. 2000).
The current model predicts that cyclic strain at low rate and amplitude results in an
average shear stress of 0.157mPa. The magnitude of the shear stress increases
signiﬁcantly with increased rate (0.346mPa) or amplitude and rate (0.459mPa)
corresponding with the inhibition of collagenase mRNA expression. While, the amount
of ﬂuid-induced shear stress on tendon cells under physiological conditions in vivo is
unknown, both cartilage and tendon models have suggested that interstitial ﬂuid ﬂow
may be in the range of 54nm/s to lOum/s, resulting in ﬂuid-induced shear of ~65mPa

(Ateshian et al. 2007; Frank and Grodzinsky 1987; Ng et al. 2005). In a low ﬂow

138

(63an5) study, myoﬁbroblasts seeded in a 3D collagen gel scaffold were subjected to an
estimated average ﬂuid shear stress on the cells that varied between 15 and 33mPa, which
were considered superphysiological (Ng et al. 2005). These values of shear stress from
interstitial ﬂow have been shown to strongly induce an anabolic response by ﬁbroblasts
(Ng et al. 2005). Therefore, the levels of ﬂuid-induced shear stress predicted in this study
appear to sufﬁciently approximate the values needed to inhibit catabolic gene expression.
Previous studies have applied no load (stress-deprivation) (Arnoczky et al. 2004;
Lavagnino et al. 2003) or much higher levels of ﬂuid-induced shear stress to cells (0.1-
2.5Pa) to induce a catabolic response (Archambault et al. 2002; Jin et al. 2000).
Although suggested by the current study, additional studies are needed to determine
whether a range of shear stress may exist that result in a homeostatic mechanobiological
response, where an imbalance above or below that shear stress range would result in a
catabolic response.

The matrix-induced cell deformation predicted by the model has also been
documented to occur in tendon following application of static tensile load (Arnoczky et
a1. 2002; Hansen et al. 2002; Kastelic et a1. 1980). In addition, static tendon load is
known to correlate with nuclear strain (Arnoczky et al. 2002) and has been shown to have
a dose-dependent effect on gene expression due to cytoskeletal deformation (Arnoczky et
al. 2004). Cell deformation and the resulting alterations in the cytoskeleton are key
components in the mechanotransduction response(s) of cells (Banes et al. 1995). The
dose dependent inhibition of rat interstitial collagenase mRNA with cyclic strain
amplitude shown in this study continues to support the mechanosensitive response of

cells to deformation.

139

Therefore, both strain rate and strain amplitude appear to play a role in altering
cellular gene expression in tendon. This mechanoresponsiveness of tendon cells is vital
to maintaining tendon homeostasis (Lavagnino and Arnoczky 2005). The importance of
both the ﬂuid and solid components in transmitting mechanical signals to cells has been
suggested in other biphasic tissue studies (Guilak and Mow 2000; Gupta and Haut
Donahue 2006; You et al. 2000). Although this cellular response to global load has been
studied in cartilage and meniscus (Guilak and Mow 2000; Gupta and Haut Donahue
2006), this is the ﬁrst study to predict the mechanical environment of tendon cells to
tensile loading.

Although the current study suggests that interstitial collagenase gene expression
has both rate and amplitude dose dependence, it was not possible to determine whether
rate or amplitude plays a more signiﬁcant role in maintaining homeostasis. Studies in
bone cells suggest ﬂuid ﬂow plays a signiﬁcantly greater role than substrate deformation
in activating gene expression (You et al. 2000). One reason for this dichotomy may be
due to how ﬂuid-shear and matrix strains play a role in mechanotransduction. With
tendon cell deformation, calcium inﬂux is a ﬁrst and primary responder prior to
alterations in gene expression (Archambault et al. 2002). However, even at high ﬂuid-
shear rates and with the induction of collagenase, there is no signiﬁcant calcium inﬂux in
tendon cells (Archambault et al. 2002). Thus, gene expression may be activated through
different mechanotransduction mechanisms (kinases, stretch-activated ion channels)
depending on the mechanical signal experienced by the cell (Archambault et al. 2002).
Indeed, kinases are cell membrane proteins that are phosphorylated when subjected to

cyclic stretching or shear stress (Arnoczky et al. 2002; Iwasaki et al. 2000; Wang 2006).

140

A limitation of this study is that permeability is assumed constant throughout the
tendon. Previous computational models and experiments have suggested that tendon
permeability is anisotropic and strain dependent (Butler et al. 1997; Chen et al. 1998; Han
et al. 2000; Yin and Elliott 2004; You et al. 2000). The use of constant permeability may
limit the model from determining strain dependent changes in ﬂuid ﬂow that could help
explain the weaker rate dependence of interstitial collagenase mRNA expression at
higher strain amplitudes seen in this study. Future studies should investigate how the
anisotropic, strain-dependent permeability of tendon would affect the magnitude of ﬂuid
ﬂow around the cells.

Permeability was further investigated over the range of values seen in the
literature (data not shown). The results of this analysis showed that the reduced
permeability signiﬁcantly lowered the ﬂuid-ﬂow induced shear stress predicted on the
cell, while only modestly lowering the cell membrane strain. However, this change in
permeability did not alter the overall relationship of shear stress between groups. The
permeability of the PCM would also seem to have a potentially important impact on the
shear stress developed on the cell membrane from ﬂuid ﬂow. Thus, this additional
analysis demonstrates the importance of determining the material properties of tendon
both at the global and cellular level to accurately model tendon mechanical signals on the
cell.

The current model assumes that the collagen ﬁbers act as springs and therefore
the model does not allow for load transfer between ﬁbers or between the ﬁbers and the
matrix and cell. Collagen crosslinking with both covalent (Haut 1985) and GAG bonds

(Redaelli et al. 2003; Scott 2003) are thought to play an important role in tendon

141

mechanics. A recent study though, does not support the theory that sulfated GAG cross-
links inﬂuence continuum-level mechanical behavior during quasi-static tensile loading
(Lujan et a1. 2007). However, the dermatin sulfate may still contribute to the viscoelastic
response when other loading rates or protocols are used (Lujan et al. 2007). The ﬂuid
content of tendon determines the strain-rate-sensitive stiffness of tendon (Haut and Haut
1997). Therefore, although the crosslinking effect of GAGs may not be as important as
previously theorized, the water content of tendon due to the GAG concentration may still
contribute, to both the mechanical properties and the cell response of tendons. Therefore,
although the collagen ﬁbers as springs may be able to closely predict the global tendon
response, they remain a limitation through their inability to transfer load to the matrix and
thus alter the ﬂuid response of the tendon upon loading.

The cellular stresses and strains in this study are only evaluated following the
global applied displacement to peak strain in the ﬁrst cycle. Therefore, the correlations
between global loading, cell loading and the corresponding gene expression are all based
on this one time point. The peak strain of the ﬁrst cycle was assumed to validly represent
the peak values of deformation and shear stress experienced by the cell. However, this
assumption is another limitation of this study and additional studies are required to
determine how tendon hysterisis and/or stress relaxation may affect mechanical signals at
the cellular level over a 24-hour time period of cyclic strain.

In conclusion, the model was able to predict the global response of tendon to
applied strain (stress, ﬂuid exudation), as well as the associated cellular response of
increased ﬂuid-ﬂow-induced shear stress with strain rate and matrix-induced cell

deformation with strain amplitude. The model analysis, combined with the experimental

142

results, showed that both strain rate and strain amplitude are able to independently alter
catabolic gene expression through increases in ﬂuid-ﬂow-induced shear stress and
matrix-induced cell deformation respectively. Although shear stress and cell deformation
appear to alter gene expression through mechanotransduction pathways, additional
studies are required to separate the importance of these mechanical factors in

homeostasis, injury, or rehabilitation.

143

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148

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149

CONCLUSIONS

The goal of this dissertation was to investigate the role of cell mechanobiology in
tendon health. The response of tendon cells to changing loading conditions has
signiﬁcant implications in unraveling the etiopathogenesis of tendinopathy (Arnoczky
IJEP). This dissertation supports the hypothesis that the etiopathogenic stimulus for
tendinopathy may occur from the mechanobiologic under-stimulation of tendon cells
resulting from altered cell-matrix interactions.

Stress-deprivation of rat tail tendons results in the under-stimulation of tendon
cells in situ and an immediate up-regulation of interstitial collagenease mRNA expression
and protein synthesis [Appendix 1, Chapter 1] (Arnoczky et al. 2004; Lavagnino et al.
2003). Application of cyclic strain to tendons inhibits the up-regulation of this catabolic
gene expression in a dose-dependent manner (both amplitude and frequency), presumably
through a cytoskeletally based mechanotransduction pathway [Chapter 1] (Lavagnino et
al. 2003). The applied strain rate and strain amplitude are able to independently alter
interstitial collagenase gene expression through increases in ﬂuid-ﬂow—induced shear
stress and matrix-induced cell deformation respectively [Chapter 5]. This ability of
cyclic strain to inhibit interstitial collagenase, a degenerative enzyme involved in
tendinopathy, may lead to advances in overuse injury prevention and optimal
rehabilitation protocols following tendon injury and repair.

These results suggest that tendon cells may have a threshold, or set-point, with
regard to their sensitivity to tensile loading and the resulting change in gene expression.
Further investigation determined that tendon cells, when seeded into collagen gels, were

able to demonstrate a mechanostat set point as changes in their cytoskeletal tension

150

(established or lost) corresponded to the alterations of anabolic (collagen) and catabolic
(collagenase) gene expression in a reciprocal manner [Chapter 2] (Lavagnino and
Arnoczky 2005). The long-term loss of cytoskeletal tension (21-day stress deprivation)
in tendons results in a maintained up-regulation of interstitial collagenase, which, in turn,
was shown to cause a signiﬁcant loss of material properties of the tendon [Chapter 3]
(Lavagnino et al. 2005). It was also shown that at high strains, isolated ﬁbril damage
could occur in tendons where the damaged ﬁbril(s) are no longer able to transmit
extracellular matrix loads to the tendon cells [Chapter 4] (Lavagnino et al. 2006). The
localized stress-deprivation in this damaged area, will result in an under-stimulation of
these cells [Chapter 1] (Lavagnino et a1. 2003) which, initiates a catabolic response that
can weaken the tendon [Chapter 3] (Lavagnino et al. 2005) making it more susceptible to
damage from subsequent loading [Chapter 4] (Lavagnino et a1. 2006). However the
partially damaged tendon fascicle maintained its ability to bear load (Lavagnino et al.
2006). Therefore, it is possible that during a series of repetitive loading cycles a single
abnormal loading cycle could produce strains sufﬁcient enough to induce isolated ﬁbril
damage, but not cause clinical injury. Thus, while repetitive loading, per se, may not be
responsible for initiating the cascade of events that lead to tendinopathy, it is likely that
continued loading of the compromised tissue plays a signiﬁcant role in the progression of
the pathological process. Then when a critical level of damage has been reached the
clinical and histological signs of tendinopathy may become evident (Arnoczky et al.
2007). Therefore, in conclusion this dissertation suggests that it is actually an absence of
mechanical stimuli, secondary to microtrauma that is the mechanobiological stimulus for

the degenerative cascade that leads to tendinopathy.

151

REFERENCES

Arnoczky, SP, Lavagnino, M and Egerbacher, M (2007) The Mechanobiological
Etiopathogenesis of Tendinopathy: Is it the over-stimulation or the under-
stimulation of tendon cells? International Journal of Experimental Pathology In
Press.

Arnoczky, SP, Tian, T, Lavagnino, M and Gardner, K (2004) Ex vivo static tensile
loading inhibits MMP-l expression in rat tail tendon cells through a cytoskeletally
based mechanotransduction mechanism. J Orthop Res 22:328-333.

Lavagnino, M and Arnoczky, SP (2005) In vitro alterations in cytoskeletal tensional
homeostasis control gene expression in tendon cells. J Orthop Res 23:1211-1218.

Lavagnino, M, Arnoczky, SP, Egerbacher, M, Gardner, KL and Burns, ME (2006)
Isolated ﬁbrillar damage in tendons stimulates local collagenase mRNA
expression and protein synthesis. J Biomech 39:2355-2362.

Lavagnino, M, Arnoczky, SP, Frank, K and Tian, T (2005) Collagen ﬁbril diameter
distribution does not reﬂect changes in the mechanical properties of in vitro
stress-deprived tendons. J Biomech 38:69-75.

Lavagnino, M, Arnoczky, SP, Tian, T and Vaupel, Z (2003) Effect of amplitude and
frequency of cyclic tensile strain on the inhibition of MMP-1 mRNA expression
in tendon cells: an in vitro study. Connect Tissue Res 44:181-187.

152

APPENDIX A

Ex vivo static tensile loading inhibits MMP-l expression
in rat tail tendon cells through a cytoskeletally based
mechanotransduction mechanism

Steven P. Arnoczky
Tao Tian
Michael Lavagnino
Keri L. Gardner

From the Laboratory for Comparative Orthopaedic Research
College of Veterinary Medicine, Michigan State University,
East Lansing, Michigan 48824, USA

Arnoczky, SP, Tian, T, Lavagnino, M, and Gardner, KL (2004) Ex vivo static tensile
loading inhibits MMP-l expression in rat tail tendon cells through a cytoskeletally based
mechanotransduction mechanism J Ortho Res 22:328-333.

153

ABSTRACT

To determine the effect of various degrees of ex vivo static tensile loading on the
expression of collagenase (MMP-l) in tendon cells, rat tail tendons were statically loaded
in tension at 0.16, 0.77, 1.38 or 2.6 MPa for 24 h. Northern blot analysis was used to
assay for mRNA expression of MMP-1 in freshly harvested, 24 h load deprived, and 24 h
statically loaded tendons. Western blot analysis was used to assay for pro-MMP-l and
MMP-1 protein expression in fresh and 24 h load deprived tendons. Freshly harvested rat
tail tendons demonstrated no evidence of MMP-1 mRNA expression and no evidence of
the pro-MMP-l or MMP-l protein. Ex vivo load deprivation for 24 h resulted in a
marked increase in the mRNA expression of MMP-1 which coincided with a marked
increase of both pro-MMP-l and MMP-1 protein expression. When tendons were
subjected to ex vivo static tensile loading during the 24 h culture period, a signiﬁcant
inhibition of this upregulation of MMP-1 mRNA expression was found with increasing
load (p<0.05). A strong (r2=0.78) and signiﬁcant (p<0.001) inverse correlation existed
between the level of static tensile load and the expression of MMP-1. Disruption of the
actin cytoskeleton with cytochalasin D abolished the inhibitory effect of ex vivo static
tensile loading on MMP-l expression. The results of this study suggest that up-regulation
of MMP-1 expression in tendon cells ex vivo can be inhibited by static tensile loading,

presumably through a cytoskeletally based mechanotransduction pathway.

154

INTRODUCTION

The deleterious effects of immobilization on the structural and functional
properties of ligaments and tendons are well-known [3,11,16,17,26-28]. While these
alterations in the extracellular matrix appear to be cell mediated [17], the exact
mechanism by which load affects tissue homeostasis is not completely understood.
Previous work from our laboratory has demonstrated a progressive and signiﬁcant
decrease in the tensile modulus of load-deprived tendons in an in vitro system [17].
However, this effect was inhibited by application of low level (frequency and magnitude)
cyclic strain [17]. Although the exact mechanism by which load deprivation decreases
tensile modulus is still unclear, several investigators have implicated the role of
interstitial collagenase (MMP-l) in this process [16,27]. A recent study has demonstrated
that immobilization-induced up-regulation in the mRNA expression of MMP-1 in rabbit
medial collateral ligaments could be inhibited by in vitro tensile loading [27]. Other in
vitro studies have suggested that changes in cell shape can be correlated with a program
of gene expression which is manifested by the degradation and synthesis of extracellular
macromolecules [2,25,32,33]. Indeed, collagenase expression in ﬁbroblasts has been
shown to be up-regulated following chemically induced alterations in cell shape and a
reorganization of the actin cytoskeleton [2,33].

A recent study has demonstrated that tensile loading of rat tail tendons results in
an alteration of cell nuclear shape in a dose dependent manner [5]. Changes in cell and
nuclear shape associated with changes in tendon tensile load are thought to be mediated
through the extracellular matrix to the nucleus via transmembrane integrins and the

cytoskeleton [8,15,31,34,35]. Thus, MMP-l gene expression in tendons and ligament

155

cells could be inﬂuenced by mechanical load trhough such “mechanosensory”
cytoskeletal tensegrity system(s). Thus, we hypothesized that MMP-l gene expression in
tendon cells varies with the magnitude of tensile load applied to the tendon and that this
effect is mediated through the cytoskeleton.
MATERIALS AND METHODS

Tendons were obtained from the tails of adult Sprague Dawley rats. The tendons
were removed immediately after euthanasia and maintained in Dulbecco’s modiﬁed
Eagle medium media supplemented with 10%, FBS, antibiotic/antimycotic solution, and
ascorbate at 37 °C and 10% C02 for the duration of the experiments. Tendons were
divided into the following groups: Group 1: freshly harvested tendons (time zero);
Group 2: load deprived at 0 MPa for 24 h; Group 3: static tensile stress at 0.16 MPa for
24 h: Group 4: static tensile stress at 0.77 MPa for 24 h; Group 5: static tensile stress at
1.38 MPa for 24 h and Group 6: static tensile stress at 2.60 MPa for 24 h.

Static tensile load was applied by attaching clips (weighing 1.2 g) alone or clips
with individual, calibrated stainless steel weights (5, 10, or 20 g) to an approximately 50
mm length of tendon (Figure 1). Stress values were calculated from representative
microscopic measurements of the rat tail tendons (average diameter 320 pm). The
weighted tendons were suspended in individual test tubes containing the above described
media while load deprived tendons were placed in a 60 mm culture dish with the same
media. All specimens were incubated for 24 h at 37 °C and 10% C0;

To determine the role of the cytoskeleton on the tensile load-modulated inhibition
of MMP-1 mRNA expression, statically loaded (2.6 MPa) rat tail tendons were incubated

in media containing 10 uM cytochalasin D (SIGMA, St Louis, MO).

156

 

Figure A.l Photograph of the static tensile loading system. Individual tendons were
suspended in 50 ml test tubes ﬁlled with culture media containing 10%
FBS and calibrated stainless steel weights were attached by clips.
Construction of rat MMP-I3 DNA plasmid: To prepare the probe, rat MMP-l
DNA (GeneBank M60616) was ampliﬁed by polymerase chain reaction using rat

genomic DNA template. The oligonucleotide primers used were 5 ’-GCC CAT ACA GTT

TGA ATA CAG TAT CTG-3’ and 5’-CCA GTT TAA TAA ACA CCA TCT CTT GA-

157

3’. PCR product (1167-bp) was subjected to electrophoresis on 1% agarose gel and
recovered using Qiaex II Kit (QIAGEN Inc., Valencia. CA), and then cloned into PCR
®2.1-T0P0® plasmid using TOPO TA cloning kit (Invitrogen Living Science, Carlsbad,
CA). The plasmid was transformed into competent E. coli with selection for kanamvcin
and ampicillin resistance. The construction was conﬁrmed by restriction enzyme
digestion (ECOR 1, EcoR V and Hind III) and polymerase chain reaction.

RNA extraction und northern blot analysis: Twenty rat tail tendons per group per
experiment were used, and the experiment was repeated three times. At the end of the
experimental period an approximately 40 mm long segment (which did not include any
portion of the tendon in contact with the clips used to attach the weight or to suspend the
tendon) of the statically loaded tendons and the entire length of the load deprived tendons
were collected. Total cellular RNA was extracted from the 20 tendons from each group
by the acid guanidine thiocyanate-phenol-chloroform procedure (totally RNA kit,
Ambion Inc., Austin, TX) and pooled. The RNA samples were loaded (8 jig/lane) onto a
1.2% agarose gel containing 0.66 M formaldehyde and MOPS, subjected to
electrophoresis, and transferred to a nylon membrane (Pierce Corp. Rockford. IL) for 1 h
in TAE at 300 mA. Following transfer, the membrane was air-dried and UV cross-linked
at 10 J/cmz.

The RNA blots were hybridized with a DNA probe for rat MMP-l mRNA
generated in our lab and a human G3PDH cDNA control probe (Clontech Laboratories,
Inc. Palo Alto, CA). The probes were labeled with biotin using the North2South Direct
HRP Labeling and Detection Kit (Pierce Corp, Rockford, IL). The RNA blots were

hybridized with labeled probes (10 ng/ml hybridization solution) at 55 °C for 1 h and

158

then washed. The membrane was then incubated with a chemiluminescent working
solution for 5 min and exposed to ﬁlms for 5-10 min. The ﬁlms were scanned with a laser
ﬁlm digitizer (Lumiscan 75, Lumisys, Inc., Sunnyvale, CA), and MMP-1 mRNA
expression was quantiﬁed by optical density measurements using Scion Image (Scion
Corporation, Frederick, MD) as a ratio of G3PDH expression. The effect of load
magnitude was evaluated using a Kruskal-Wallis test, followed by a Mann-Whitney U
post hoc test, and the correlation between tensile load and mRNA expression of MMP-1
was examined using a polynomial regression analysis (f = I/a + bx). Signiﬁcance was
set at p<0.05.

Western blot analysis: To verify that MMP-l mRNA expression coincided with
the expression of the MMP-l protein, rat tail tendons (freshly harvested [n = 20] or load-
deprived for 24 h [n = 20] as noted above) were processed for Western immunoblot
staining. Total protein was extracted from each group of pooled tendons by
homogenizing the tendons and lysing the cells overnight with a buffer containing 25 mM
HEPES, 0.5 mM Na3V04, 50 mM sodium ﬂuoride, 5 mM sodium pyrophosphate, 10 mM
sodium B-glycerophosphate, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 1% Triton, 0.1
mM PMSF, 1 pg aprotinin, 1 uM microcystin, 1 rig/ml pepstatin, and 1 pg/ml leupeptin.
The lysates were centrifuged (104 RPM at 4 °C for 30 min), and the protein quantiﬁed
(BioRad Protein Assay. BioRad Labs, Hercules, CA) and electrophoresed on a 12.5%
SDS-polyacrylamide gel. The separated proteins were transferred to a nitrocellulose
membrane and analyzed for pro-MMP-l protein and MMP-1 protein using a mouse

monoclonal antibody (Oncogene, Boston, MA) and chemiluminescence detection (New

159

England BioLabs, Beverly, MA). Human MMP-l protein (Sigma, St. Louis, MO) was
used as the positive control.

Actin staining/confocal laser microscopy: To verify in situ disruption of the actin
cytoskeleton by cytochalasin D, additional rat tail tendons were prepared for actin
staining and confocal laser microscopy. Ten millimeter segments of control (n = 5) and
experimental (n = 5 treated for 1 h with 10 11M cytochalasin D) rat tail tendons were ﬁxed
in 10% phosphate buffered formalin and stained with rhodamine phalloidin (5 units/ml)
(Molecular Probes, Eugene, Oregon). The tendons were wet mounted on a glass slide for
viewing with a Zeiss Pascal Laser Scanning Confocal microscope (Carl Zeiss,
Thornwood. NY). Observations were made using a 40x oil immersion objective with a
coverslip. Fluorescent images were obtained using a HeNe 543 nm laser with a long pass
560 nm emission ﬁlter. After localizing a representative cell, a 2x zoom was used to
obtain the image. Overlay images were achieved by multiple confocal 0.6 um thick '2-
sectioning' through the depth of the cell and then combining the images into a single
overlay image.

Drugs and Chemicals: Except as noted, all drugs and chemicals (Dulbecco‘s
modiﬁed Eagle medium (DMEM), fetal bovine serum (FBS), ascorbate, gentamicin, and
penicillin/streptomycin-fungizone solution) were obtained from Gibco, Grand Island,
New York.

RESULTS
The yield of total RNA extracted from each group was 0.2 rig/mg wet weight and

was not signiﬁcantly different among groups.

160

Freshly harvested rat tail tendons (Group 1) demonstrated no appreciable MMP-l
mRNA expression on Northern blots. Tendons in the load-deprived group (Group 2)
cultured for 24 h demonstrated substantial mRNA expression of MMP-1 (Figure 2).
When tendons were subjected to static tensile loading during the same 24 h culture
period, MMP-l mRNA expression decreased signiﬁcantly with increasing load (p <

0.05) (Figure 2 and Table 1).

Control 0MPa 0.16MPa 0.77MPa 1.38MPa 2.6MPa

 

Figure A.2 Representative Northern blot gel illustrating the relative expression of
MMP-1 mRNA as a function of applied stress for 24 h. G3PDH was used
as an internal control. Experiments were performed three times and a
representative result is shown.

Table A.1 The effect of static stress on MMP-l expression

 

 

STRESS MMP-l/G3PDH
Fresh tendonsa Obcdef

0.00 MPa for 24 hb 0.670 : 0.266adef
0.16 MPa for 24 h“ 0.680 :1: 0.038aef
0.77 MPa for 24 hd 0.333 : 0.153ab
1.38 MPa for 24 he 0.116 t 0.050abc
2.60 MPa for 24 hf 0.077 e 0.017abc

 

Superscripts a-e indicate Si gniﬁcantly different corresponding data pairs (p < 0.05).

161

A strong (r2 = 0.78) and signiﬁcant (p < 0.001) inverse correlation was found
between the level of static tensile load and the expression of MMP-1 mRNA (Figure 3).
However, MMP-l mRNA expression was not completely abolished at the highest stress

(2.6 MPa) examined.

Western imrnunoblot staining revealed no appreciable pro-MMP-l or MMP-l
protein expression in the freshly harvested rat tail tendons. However, following 24 h of
load deprivation, substantial pro-MMP-l and MMP-1 protein expression was detected in

the tendons (Figure 4).

Treatment with cytochalasin D did not inﬂuence MMP-l mRNA expression in the
load deprived tendons (Group 2) (p > 0.05). However, cytochalasin D treatment did
Signiﬁcantly (p< 0.05) increase MMP-l mRNA expression in tendons exposed to a

tensile loading stress of 2.60 MPa (Figure 5).

Actin staining and confocal laser microscopic examination demonstrated the
presence of actin stress ﬁlaments within the cells of the time zero control tendons (Figure
6A). However, in those tendons incubated for 1 h in 10 M cytochalasin D, the actin

appeared globular, and no evidence of organized actin stress ﬁlaments was found (Figure

6B).

162

 

 

 

 

 

 

1.0
o
#:078
o 0.8 - . p<0.0001
1175'
I
I 0.6 -
D
O.
8
E 0.4 -
O.
E
E
0.2 .
0.0 I I I I I I
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Stress (MPa)

Figure A.3 Composite polynomial regression of graph of the three experimental
replicates plotting MMP-l mRNA expression (normalized as a ratio of
MMP-1 to G3PDH) against static stress. There was a strong (r2 = 0.78)
and signiﬁcant (p < 0.0001) inverse correlation between MMP-l mRNA
expression and Static stress.

1 2 3
Pro-MMP1 m C 57kDa
MMP1 a“: w 48kDa
1) fresh control tendon
2) 24 hr load deprived tendon
3) positive control
Figure A.4 Western blot analysis illustrating the absence of pro-MMP-l and MMP-1

protein expression in freshly harvested rat tail tendons. There was a
signiﬁcant up-regulation of pro-MMP-l and MMP-1 protein expression in
the tendon cells after 24 h of in vitro load deprivation.

163

MMP-1

G3PDH '

Figure A.5

   

 

Fresh Control

24 hr 0MPa Static Tensile Stress

24 hr 2.6MPa Static Tensile Stress

24 hr 2.6MPa Static Tensile Stress in 10uM
cytochalasin

Representative Northern blot gel illustrating the relative expression of
MMP-1 mRNA in fresh control tendons (lane 1): 24 h load deprived at 0
MPa (lane 2); 24 h at 2.60 MPa static tensile Stress (lane 3): 24 h at 2.60
MPa static tensile Stress in 10 uM cytochalasin D (lane 4). G3PDH was
used as an internal control. Experiments were performed three times and a
representative result is Shown.

 

Figure A.6

Confocal overlay images of rat tail tendon cells stained with rhodamine
phalloidin to label actin ﬁlaments. (A) Fresh control tendon: note presence
of actin stress ﬁbers (arrows) in cytoskeleton. (B) Tendon treated with 10
uM cytochalasin D for 1 h; note the absence of organized actin stress
ﬁbers. (confocal 40x oil immersion: 2x zoom).

164

DISCUSSION

The strain-induced signaling of cells through mechanotransduction pathways is
thought to play a signiﬁcant role in maintaining the normal homeostasis of connective
tissues [1,8,31,34,35]. While various stress levels have been shown to induce anabolic
responses in bone [12,21,22,29,30], ligament [9,20,23], and tendon cells [6,7], stress
deprivation has been associated with tissue catabolism [1,3,10,16,17,30]. These catabolic
changes have been attributed to degradation of the extracellular matrix by matrix
metalloproteinases (MMPS) [3,16,27]. The results of the current study demonstrate that,
when compared to freshly harvested control tendons, ex vivo load deprivation results in
an immediate (within 24 h) increase in MMP-l mRNA expression and MMP-1 protein
expression in the tendon cells. While in vivo studies have reported similar ﬁndings in
immobilized ligaments and tendons [16,27], the precise mechanism by which load
deprivation triggers MMP-l gene expression in these tissues is unclear.

A proposed mechanism of mechanotransduction theorizes that extracellular
matrix strain imparts a strain to cells through their integrin based cell-matrix connections
[8,31,34,35]. The resulting cellular deformation creates changes in tension within the
cytoskeleton, which can be sensed by the cell nucleus through a mechanosensory
tensegrity system and results in speciﬁc cellular responses [8]. While most studies have
focused on the effect of increased (over normal resting levels) strain on the
mechanotransduction signaling responses of tendon cells in monolayer [6,7], the impact
of total load deprivation on this process has not been examined in whole tendons ex vivo.
A recent study suggested that cells are capable of establishing a constant level of tension

between themselves and their extracellular matrix through a cytoskeletal tensegrity

165

system [13]. This tensional homeostasis is thought play an important role in the
mechanotransduction response of the cell [13]. Removal of tendons from their normal
mechanical environment could signiﬁcantly alter this homeostatic tension within the
cytoskeletal tensegrity system and be responsible for up—regulation of MMP-13 mRNA
expression seen following 24 h of load deprivation.

An in vitro study has Shown that MMP-l expression in ﬁbroblasts is Significantly
higher in free (contracting) gels than in anchored gels [25]. This suggests that the
mechanical forces that are generated by the cell between itself and its surrounding matrix
may, in part, regulate MMP-l expression. Sustained loss of normal cell-matrix tension, as
could occur when tendons have been removed from their origins and insertions and
placed in load deprived environment for 24 h, could have played a role in triggering the
MMP-l expression seen in this study.

Changes in cell shape, Speciﬁcally the loss of actin stress ﬁber organization, has
been strongly correlated with collagenase gene expression [2,33]. It has been Shown that
procollagenase expression in rabbit synovial ﬁbroblasts was only induced by treatments
that modiﬁed cellular actin [2,33]. This would suggest that a change in the state of actin
assembly may inﬂuence collagenase expression. In the current study, treatment with
cytochalasin D, a fungal product which depolymerizes actin ﬁlaments, completely
abrogated the load induced inhibition of MMP-1 mRNA expression. This further supports
the role of a cytoskeletally based mechanosensory tensegrity system in the control of
MMP-1 mRNA expression in tendon cells.

The application of a tensile load to the tendons in culture resulted in a decrease in

MMP-l mRNA expression with increasing load. While MMP-l mRNA expression ex

166

vivo was decreased by increasing load, this inhibitory effect was non-linear and was not
completely eliminated at the loads examined. A plausible explanation for this observation
is the non-linear stress—strain behavior exhibited by rat tail tendons [18]. It is likely that
with increasing load an increasing number of cells undergo nuclear (and presumably
cytoskeletal) deformation [5]. Thus, the increase in the number of cells receiving a
mechanotransduction stimulus with increasing load may reﬂect the increased inhibition
of MMP-1 mRNA expression. The inability to completely eliminate MMP-l mRNA
expression at the loads sampled could be explained by the fact that the loads examined in
this study were all within the reported “toe” region (<3% strain) of the stress-strain curve
for rat tail tendon fascicles [18]. A recent study has shown that collagen crimp within the
center of the fascicles might not be completely eliminated until 2.8% strain [18]. This
could mean that, in the current Study, cells within the center of the fascicle were not
exposed to stresses sufﬁcient to activate mechanotranduction responses that could affect
(inhibit) MMP— 1 mRNA expression.

Finally, the exact inﬂuence of an ex vivo environment on the expression of MMP-
1 mRNA in rat tail tendon cells is unknown. While the experimental design of this study
would suggest that under similar ex vivo conditions static tensile loading does inhibit the
expression of MMP-1 mRNA when compared to load-deprived tendons, the incomplete
inhibition of this expression with what would appear to be physiologic loads may also be
related to the ex vivo environment. A recent review article suggests that the absence of
innervation and vascularization in explant culture systems may also contribute to the
regulation of speciﬁc gene expression in connective tissues [19]. Thus, the interpretation

of mechanotransduction-induced results from such culture systems must be tempered

167

with the possibility that the gene response seen ex vivo may not be reﬂective of that
which occurs in vivo [19]. However, the effect of rigorously controlled loading and load
deprivation conditions on MMP-l mRNA expression in tendon cells ex vivo seen in the
current study are similar to those reported for in vivo immobilized ligaments and tendons
[16,27] and in vitro cyclically loaded ligaments [27] and ﬁbrochondrocytes [1]. A
comparison of these results would suggest that the mechanostimulation of tendon cells
(whether in vivo or ex vivo) does play a signiﬁcant role in the regulation of MMP-1
mRNA expression.

The results obtained from this ex vivo test system demonstrate that MMP-l
mRNA expression in tendon cells can be affected by static tensile load, presumably
through a cytoskeletally based mechanotransduction pathway. MMP-l was chosen as the
gene of interest based on a previous in vitro study that documented an increase in MMP-l
mRNA expression and not MMP-13 mRNA following immobilization [27]. While this
study only examined the effects of various levels of static tensile load on this Signaling
system, cyclic loading (which reﬂects a more relevant in vivo loading environment) and
the resulting ﬂuid ﬂow have also been Shown to play a key role in the
mechanotransduction response of connective tissues [4,14,23,24]. This is especially true
in bone where ﬂuid ﬂow is believed to be an important physical Signal that inﬂuences
bone cell metabolism and bone adaptation to mechanical loading [14,24,30]. The effect
of cyclic load and ﬂuid ﬂow on the cytoskeletal components of the mechanosensory
tensegrity system of tendon cells must also be investigated to completely understand the

interaction of mechanical loading and tendon physiology. As in bone [21], the effect of

168

loading history on cytoskeletal adaptations and matrix interactions could provide a key to

understanding the effect of various mechanical loading regimes on tendon health.

169

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[l9] Hart DA, Natsu-ume T, Sciore P, Tasevski V, Frank CB, Shrive NG.
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[21] Hsieh YF, Turner CH. Effects of loading frequency on mechanically induced bone
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[22] Huiskes R, Ruimerman R, van Lenthe GH, Janssen JD. Effects of mechanical forces
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extracellular matrix macromolecules and collagenase expression in ﬁbroblasts by
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[26] Loitz BJ, Zemicke RF, Vailas AC, Kody MH, Meals RA. Effects of Short-term
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[27] Majima T, Matchuk LL, Shrive NG, Frank CB, Hart DA. In vitro cyclic tensile
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[28] Majima T, Yasuda K, Yamamoto N. Deterioration of mechanical properties of the
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[29] Neidlinger-Wilke C, Wilke H-J, Claes L. Cyclic stretching of human osteoblasts
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[32] Tremble P, Damsky CH, Werb Z. Components of the nuclear cascade that regulate
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[35] Watson PA. Function follows form: generation of intracellular Signals by cell
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172

APPENDIX B

 

 

2 2 4. 4 6 6 8 8 2 2 4 4 6 6 8 8 2 2 4. 4 6
o '2 a :4 u 06 n :8 o O .2 o .4 I .6 o .8 o o o .2 o .4 n .6
.525 .545 .565 .585 .5 6 .626 .646 .666 .686 .6 7 .727 .747 .767 .
5 . 5 . 5 . 5 . 5 . 6 . 6 . 6 . 6 . 6 . 7 . 7 . 7 . 7
I5 I I5 I I5 I ’5 I I6 I I6 I I6 I '6 I I6 I I7 I I7 I I7 I I7 I
I5 5 I5 5 15 5 I5 5 I5 5 IS 5 I5 5 I5 5 I5 5 IS 5 15 5 I5 5 I5 5 I
111. 1011 (011 1011 I011 I011 I011 I011 Inualﬁ.l_ 1011 I011 1011 I011 I01
00000000000000000000000000000000000000000000000000000
34567890123456789012345678901234567890123456789012345
00000001111111111222222222233333333334444444444555555
1111111111111111111111111111111111111llllllllllllllll
4 4 6 6 8 8 2 2 4 4 6 6 8 8 2 2 4 4 6 6 8 8
'4 n .6 n .8 a o a :2 n 54 o .6 I .8 n a o .2 o .4 n .6 a .8 a o
2 .262 .282 .2 3 .323 .343 .363 .383 .3 4 .424 .444. .464 .484 .4 5
2 o 2 o 2 n 3 n 3 o 3 o 3 a 3 n 4 o 4 a 4 u 4 n 4 o
I [2 I I2 I I3 I I3 l I3 I I3 I I3 I I4 I I4 I I4 I I4 I I4 I I5 I
5 IS 5 15 5 15 5 I5 5 I5 5 15 5 I5 5 15 5 IS 5 :5 5 IS 5 I5 5 IS 5
011 1011 I011 I011 I011 I011 I011 IOll I011 I011 1011 lOll 1011 I0
00000000000000000000000000000000000000000000000000000
01234567890123456789012345678901234567890123456789012
55555555556666666666777777777788888888889999999999000
111
m
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1.“
rm 2 2 4. 4 6 6 8 8 2 2 4 4 6 6 8 8 2 2
mm . .2 . .4 . .6 . .8 . . .2 . .4 . .6 . .8 . . .2 .
O .020 .040 .060 .080 .0 1 .121 .141 .161 .181 .1 2 .222 .24
dew. . O . 0 . O . 0 . 0 . l . l . 1 . l . .1. . 2 . 2 .
dr 0 I I0 I I0 I I0 I I0 I I1 I I1 I I1 I I1 I I1 I I2 I 12 I I2
0t 5 I5 5 I5 5 I5 5 I5 5 I5 5 l5 5 I5 5 I5 5 I5 5 I5 5 I5 5 IS
MS I011 1011 I011 I011 1011 I011 I011 I011 I011 I011 (011 I011
l% 0000000000000000000000000OOOOOOOOOOOOOOOOOOOOOOOO
azg IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
l “.10.. lll1111111222222222233333333334444444444
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B S. *

173

156,
157,
158,
159,
160,
161,
162,
163,
164,
165,
166,
167,
168,
169,
170,
171,
172,
173,
174,
175,
176,
177,
178,
179,
180,
181,
182,
183,
184,
185,
186,
187,
188,
189,
190,
191,
192,
193,
194,
195,
196,
197,
198,
199,
200,
201,
202,
203,
204,
205,
206,
207,
208,
209,
210,
211,
212,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.15,
., 10.
.05,
.1,

.15,
., 10.2
.05,
.l,

.15,
., 10.4
.05,
.l,

.15,

10.
10.
10.

10.2
10.2
10.2

10.4
10.4
10.4

 

213,
214,
215,
216,
217,
218,
219,
220,
221,
222,
223,
224,
225,
226,
227,
228,
229,
230,
231,
232,
233,
234,
235,
236,
237,
238,
239,
240,
241,
242,
243,
244,
245,
246,
247,
248,
249,
250,
251,
252,
253,
254,
255,
256,
257,
258,
259,
260,
261,
262,
263,
264,
265,
266,
267,
268,
269,

., 10.6
.05, 10.6
.1, 10.6
.15, 10.6
.,10.8
.05, 10.8
.1, 10.8
.15,10.8
., 11.
.05,11.
.1,11.
.15,11.
., 11.2
.05, 11.2
.1, 11.2
.15,11.2
., 11.4
.05, 11.4
.1,11.4
.15,11.4
., 11.6
.05, 11.6
.1, 11.6
.15, 11.6
., 11.8
.05, 11.8
.1,11.8
.15,11.8
., 12.
.05,12.
.1, 12.
.15,12.
., 12.2
.05, 12.2
.1, 12.2
.15, 12.2
., 12.4
.05, 12.4
.1, 12.4
.15, 12.4
., 12.6
.05, 12.6
.1, 12.6
.15, 12.6
., 12.8
.05,12.8
.1,12.8
.15,12.8
., 13.
.05, 13.
.1,13.
.15, 13.
., 13.2
.05, 13.2
.1, 13.2
.15, 13.2
., 13.4

174

 

270,
271,
272,
273,
274,
275,
276,
277,
278,
279,
280,
281,
282,
283,
284,
285,
286,
287,
288,
289,
290,
291,
292,
293,
294,
295,
296,
297,
298,
299,
300,
301,
302,
303,
304,
305,
306,
307,
308,
309,
310,
311,
312,
313,
314,
315,
316,
317,
318,
319,
320,
321,
322,
323,
324,
325,
326,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.05, 13.4
.1, 13.4
.15, 13.4
., 13.6
.05, 13.6
.1, 13.6
.15, 13.6
., 13.8
.05, 13.8
.1, 13.8
.15, 13.8
., 14.
.05, 14.
.1, 14.
.15, 14.
., 14.2
.05, 14.2
.1, 14.2
.15, 14.2
., 14.4
.05, 14.4
.1, 14.4
.15, 14.4
., 14.6
.05, 14.6
.1, 14.6
.15, 14.6
., 14.8
.05, 14.8
.1, 14.8
.15, 14.8
., 15.
.05, 15.
.1, 15.
.15, 15.
., 15.2
.05, 15.2
.1, 15.2
.15, 15.2
., 15.4
.05, 15.4
.1, 15.4
.15, 15.4
., 15.6
.05, 15.6
.1, 15.6
.15, 15.6
., 15.8
.05, 15.8
.1, 15.8
.15, 15.8
., 16.
.05, 16.
.1, 16.
.15, 16.
., 16.2
.05, 16.2

327, 0.1, 16.2 384, 0.15, 19.
328, 0.15, 16.2 385, 0., 19.2
329, 0., 16.4 386, 0.05, 19.2
330, 0.05, 16.4 387, 0.1, 19.2
331, 0.1, 16.4 388, 0.15, 19.2
332, 0.15, 16.4 389, 0., 19.4
333, 0., 16.6 390, 0.05, 19.4
334, 0.05, 16.6 391, 0.1, 19.4
335, 0.1, 16.6 392, 0.15, 19.4
336, 0.15, 16.6 393, 0., 19.6
337, 0., 16.8 394, 0.05, 19.6
338, 0.05, 16.8 395, 0.1, 19.6
339, 0.1, 16.8 396, 0.15, 19.6
340, 0.15, 16.8 397, 0., 19.8
341, 0., 17. 398, 0.05, 19.8
342, 0.05, 17. 399, 0.1, 19.8
343, 0.1, 17. 400, 0.15, 19.8
344, 0.15, 17. 401, 0., 20.
345, 0., 17.2 402, 0.05, 20.
346, 0.05, 17.2 403, 0.1, 20.
347, 0.1, 17.2 404, 0.15, 20.
348, 0.15, 17.2 *Element, type=CAX4P
349, 0., 17.4 1, 1, 2, 6,
350, 0.05, 17.4 2, 2, 3, 7,
351, 0.1, 17.4 3, 3, 4, 8,
352, 0.15, 17.4 4, 5, 6, 10,
353, 0., 17.6 5, 6, 7, 11,
354, 0.05, 17.6 6, 7, 8, 12,
355, 0.1, 17.6 7, 9, 10, 14,
356, 0.15, 17.6 8, 10, 11, 15,
357, 0., 17.8 9, 11, 12, 16,
358, 0.05, 17.8 10, 13, 14, 18,
359, 0.1, 17.8 11, 14, 15, 19,
360, 0.15, 17.8 12, 15, 16, 20,
361, 0., 18. 13, 17, 18, 22,
362, 0.05, 18. 14, 18, 19, 23,
363, 0.1, 18. 15, 19, 20, 24,
364, 0.15, 18. 16, 21, 22, 26,
365, 0., 18.2 17, 22, 23, 27,
366, 0.05, 18.2 18, 23, 24, 28,
367, 0.1, 18.2 19, 25, 26, 30,
368, 0.15, 18.2 20, 26, 27, 31,
369, 0., 18.4 21, 27, 28, 32,
370, 0.05, 18.4 22, 29, 30, 34,
371, 0.1, 18.4 23, 30, 31, 35,
372, 0.15, 18.4 24, 31, 32, 36,
373, 0., 18.6 25, 33, 34, 38,
374, 0.05, 18.6 26, 34, 35, 39,
375, 0.1, 18.6 27, 35, 36, 40,
376, 0.15, 18.6 28, 37, 38, 42,
377, 0., 18.8 29, 38, 39, 43,
378, 0.05, 18.8 30, 39, 40, 44,
379, 0.1, 18.8 31, 41, 42, 46,
380, 0.15, 18.8 32, 42, 43, 47,
381, 0., 19. 33, 43, 44, 48,
382, 0.05, 19. 34, 45, 46, 50,
383, 0.1, 19. 35, 46, 47, 51,

 

175

\lmU'l

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10

13
14
15
17
18
19
21
22
23
25
26
27
29
30
31
33
34
35
37
38
39
41
42
43
45
46
47
49
50

 

36,
37,
38,
39,
40,
41,
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44,
45,
46,
47,
48,
49,
50,
51,
52,
53,
54,
55,
56,
57,
58,
59,
60,
61,
62,
63,
64,
65,
66,
67,
68,
69,
70,
71,
72,
73,
74,
75,
76,
77,
78,
79,
80,
81,
82,
83,
84,
85,
86,
87,
88,
89,
90,
91,
92,

47,
49,
50,
51,
53,
54,
55,
57,
58,
59,
61,
62,
63,
65,
66,
67,
69,
70,
71,
73,
74,
75,
77,
78,
79,
81,
82,
83,
85,
86,
87,
89,
9o,
91,
93,
94,
95,
97,
98,
99,
101,
102,
103,
105,
106,
107,
109,
110,
111,
113,
114,
115,
117,
118,
119,
121,
122,

48,
50,
51,
52,
54,
55,
56,
58,
59,
60,
62,
63,
64,
66,
67,
68,
70,
71,
72,
74,
75,
76,
78,
79,
80,
82,
83,
84,
86,
87,
88,
90,
91,
92,
94,
95,
96,
98,
99,
100,
102,
103,
104,
106,
107,
108,
110,
111,
112,
114,
115,
116,
118,
119,
120,
122,
123,

52,
54,
55,
56,
58,
59,
60,
62,
63,
64,
66,
67,
68,
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71,
72,
74,
75,
76,
78,
79,
80,
82,
83,
84,
86,
87,
88,
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91,
92,
94,
95,
96,
98,
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100,
102,
103,
104,
106,
107,
108,
110,
111,
112,
114,
115,
116,
118,
119,
120,
122,
123,
124,
126,
127,

51
53
54
55
57
58
59
61
62
63
65
66
67
69
70
71
73
74
75
77
78
79
81
82
83
85
86
87
89
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91
93
94
95
97
98
99
101
102
103
105
106
107
109
110
111
113
114
115
117
118
119
121
122
123
125
126

93,

94,

95,

96,

97,

98,

99,
100,
101,
102,
103,
104,
105,
106,
107,
108,
109,
110,
111,
112,
113,
114,
115,
116,
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118,
119,
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121,
122,
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125,
126,
127,
128,
129,
130,
131,
132,
133,
134,
135,
136,
137,
138,
139,
140,
141,
142,
143,
144,
145,
146,
147,
148,
149,

123,
125,
126,
127,
129,
130,
131,
133,
134,
135,
137,
138,
139,
141,
142,
143,
145,
146,
147,
149,
150,
151,
153,
154,
155,
157,
158,
159,
161,
162,
163,
165,
166,
167,
169,
170,
171,
173,
174,
175,
177,
178,
179,
181,
182,
183,
185,
186,
187,
189,
190,
191,
193,
194,
195,
197,
198,

124,
126,
127,
128,
130,
131,
132,
134,
135,
136,
138,
139,
140,
142,
143,
144,
146,
147,
148,
150,
151,
152,
154,
155,
156,
158,
159,
160,
162,
163,
164,
166,
167,
168,
170,
171,
172,
174,
175,
176,
178,
179,
180,
182,
183,
184,
186,
187,
188,
190,
191,
192,
194,
195,
196,
198,
199,

128,
130,
131,
132,
134,
135,
136,
138,
139,
140,
142,
143,
144,
146,
147,
148,
150,
151,
152,
154,
155,
156,
158,
159,
160,
162,
163,
164,
166,
167,
168,
170,
171,
172,
174,
175,
176,
178,
179,
180,
182,
183,
184,
186,
187,
188,
190,
191,
192,
194,
195,
196,
198,
199,
200,
202,
203,

127
129
130
131
133
134
135
137
138
139
141
142
143
145
146
147
149
150
151
153
154
155
157
158
159
161
162
163
165
166
167
169
170
171
173
174
175
177
178
179
181
182
183
185
186
187
189
190
191
193
194
195
197
198
199
201
202

 

150,
151,
152,
153,
154,
155,
156,
157,
158,
159,
160,
161,
162,
163,
164,
165,
166,
167,
168,
169,
170,
171,
172,
173,
174,
175,
176,
177,
178,
179,
180,
181,
182,
183,
184,
185,
186,
187,
188,
189,
190,
191,
192,
193,
194,
195,
196,
197,
198,
199,
200,
201,
202,
203,
204,
205,
206,

199,
201,
202,
203,
205,
206,
207,
209,
210,
211,
213,
214,
215,
217,
218,
219,
221,
222,
223,
225,
226,
227,
229,
230,
231,
233,
234,
235,
237,
238,
239,
241,
242,
243,
245,
246,
247,
249,
250,
251,
253,
254,
255,
257,
258,
259,
261,
262,
263,
265,
266,
267,
269,
270,
271,
273,
274,

200,
202,
203,
204,
206,
207,
208,
210,
211,
212,
214,
215,
216,
218,
219,
220,
222,
223,
224,
226,
227,
228,
230,
231,
232,
234,
235,
236,
238,
239,
240,
242,
243,
244,
246,
247,
248,
250,
251,
252,
254,
255,
256,
258,
259,
260,
262,
263,
264,
266,
267,
268,
270,
271,
272,
274,
275,

176

204,
206,
207,
208,
210,
211,
212,
214,
215,
216,
218,
219,
220,
222,
223,
224,
226,
227,
228,
230,
231,
232,
234,
235,
236,
238,
239,
240,
242,
243,
244,
246,
247,
248,
250,
251,
252,
254,
255,
256,
258,
259,
260,
262,
263,
264,
266,
267,
268,
270,
271,
272,
274,
275,
276,
278,
279,

203
205
206
207
209
210
211
213
214
215
217
218
219
221
222
223
225
226
227
229
230
231
233
234
235
237
238
239
241
242
243
245
246
247
249
250
251
253
254
255
257
258
259
261
262
263
265
266
267
269
270
271
273
274
275
277
278

 

207,
208,
209,
210,
211,
212,
213,
214,
215,
216,
217,
218,
219,
220,
221,
222,
223,
224,
225,
226,
227,
228,
229,
230,
231,
232,
233,
234,
235,
236,
237,
238,
239,
240,
241,
242,
243,
244,
245,
246,
247,
248,
249,
250,
251,
252,
253,
254,
255,
256,
257,
258,
259,
260,
261,
262,
263,

275,
277,
278,
279,
281,
282,
283,
285,
286,
287,
289,
290,
291,
293,
294,
295,
297,
298,
299,
301,
302,
303,
305,
306,
307,
309,
310,
311,
313,
314,
315,
317,
318,
319,
321,
322,
323,
325,
326,
327,
329,
330,
331,
333,
334,
335,
337,
338,
339,
341,
342,
343,
345,
346,
347,
349,
350,

276,
278,
279,
280,
282,
283,
284,
286,
287,
288,
290,
291,
292,
294,
295,
296,
298,
299,
300,
302,
303,
304,
306,
307,
308,
310,
311,
312,
314,
315,
316,
318,
319,
320,
322,
323,
324,
326,
327,
328,
330,
331,
332,
334,
335,
336,
338,
339,
340,
342,
343,
344,
346,
347,
348,
350,
351,

280,
282,
283,
284,
286,
287,
288,
290,
291,
292,
294,
295,
296,
298,
299,
300,
302,
303,
304,
306,
307,
308,
310,
311,
312,
314,
315,
316,
318,
319,
320,
322,
323,
324,
326,
327,
328,
330,
331,
332,
334,
335,
336,
338,
339,
340,
342,
343,
344,
346,
347,
348,
350,
351,
352,
354,
355,

279
281
282
283
285
286
287
289
290
291
293
294
295
297
298
299
301
302
303
305
306
307
309
310
311
313
314
315
317
318
319
321
322
323
325
326
327
329
330
331
333
334
335
337
338
339
341
342
343
345
346
347
349
350
351
353
354

264,
265,
266,
267,
268,
269,
270,
271,
272,
273,
274,
275,
276,
277,
278,
279,
280,
281,
282,
283,
284,
285,
286,
287,
288,
289,
290,
291,
292,
293,
294,
295,
296,
297,
298,
299,
300,

351,
353,
354,
355,
357,
358,
359,
361,
362,
363,
365,
366,
367,
369,
370,
371,
373,
374,
375,
377,
378,
379,
381,
382,
383,
385,
386,
387,
389,
390,
391,
393,
394,
395,
397,
398,
399,

*Element,
elset=High

301,
305,
309,
313,
317,
321,
325,
329,
333,
337,
341,
345,
349,
353,
357,
361,
365,
369,

352, 356, 355
354, 358, 357
355, 359, 358
356, 360, 359
358, 362, 361
359, 363, 362
360, 364, 363
362, 366, 365
363, 367, 366
364, 368, 367
366, 370, 369
367, 371, 370
368, 372, 371
370, 374, 373
371, 375, 374
372, 376, 375
374, 378, 377
375, 379, 378
376, 380, 379
378, 382, 381
379, 383, 382
380, 384, 383
382, 386, 385
383, 387, 386
384, 388, 387
386, 390, 389
387, 391, 390
388, 392, 391
390, 394, 393
391, 395, 394
392, 396, 395
394, 398, 397
395, 399, 398
396, 400, 399
398, 402, 401
399, 403, 402
400, 404, 403
type=SPRINGA,

1, 5

5, 9

9, 13

13, 17
17, 21
21, 25
25, 29
29, 33
33, 37
37, 41
41, 45
45, 49
49, 53
53, 57
57, 61
61, 65
65, 69
69, 73

 

373,
377,
381,
385,
389,
393,
397,
401,
405,
409,
413,
417,
421,
425,
429,
433,
437,
441,
445,
449,
453,
457,
461,
465,
469,
473,
477,
481,
485,
489,
493,
497,
501,
505,
509,
513,
517,
521,
525,
529,
533,
537,
541,
545,
549,
553,
557,
561,
565,
569,
573,
577,
581,
585,
589,
593,
597,

73,

77,

81,

85,

89,

93,

97,

101,
105,
109,
113,
117,
121,
125,
129,
133,
137,
141,
145,
149,
153,
157,
161,
165,
169,
173,
177,
181,
185,
189,
193,
197,
201,
205,
209,
213,
217,
221,
225,
229,
233,
237,
241,
245,
249,
253,
257,
261,
265,
269,
273,
277,
281,
285,
289,
293,
297,

77
81
85
89
93
97
101
105
109
113
117
121
125
129
133
137
141
145
149
153
157
161
165
169
173
177
181
185
189
193
197
201
205
209
213
217
221
225
229
233
237
241
245
249
253
257
261
265
269
273
277
281
285
289
293
297
301

177

 

601,
605,
609,
613,
617,
621,
625,
629,
633,
637,
641,
645,
649,
653,
657,
661,
665,
669,
673,
677,
681,
685,
689,
693,
697,

301,
305,
309,
313,
317,
321,
325,
329,
333,
337,
341,
345,
349,
353,
357,
361,
365,
369,
373,
377,
381,
385,
389,
393,
397,

*Element,
elset=MedHigh

302,
306,
310,
314,
318,
322,
326,
330,
334,
338,
342,
346,
350,
354,
358,
362,
366,
370,
374,
378,
382,
386,
390,
394,
398,
402,
406,
410,
414,
418,

2, 6
6, 1
10,
14,
18,
22,
26,
30,
34,
38,
42,
46,
50,
54,
58,
62,
66,
7o,
74,
78,
82,
86,
90,
94,
98,
102,
106,
110,
114,
118,

305
309
313
317
321
325
329
333
337
341
345
349
353
357
361
365
369
373
377
381
385
389
393
397
401
type=SPRINGA,

O
14
18
22
26
3O
34
38
42
46
50
54
58
62
66
7O
74
78
82
86
9O
94
98
102
106
110
114
118
122

422,
426,
430,
434,
438,
442,
446,
450,
454,
458,
462,
466,
470,
474,
478,
482,
486,
490,
494,
498,
502,
506,
510,
514,
518,
522,
526,
530,
534,
538,
542,
546,
550,
554,
558,
562,
566,
570,
574,
578,
582,
586,
590,
594,
598,
602,
606,
610,
614,
618,
622,
626,
630,
634,
638,
642,
646,

122,
126,
130,
134,
138,
142,
146,
150,
154,
158,
162,
166,
170,
174,
178,
182,
186,
190,
194,
198,
202,
206,
210,
214,
218,
222,
226,
230,
234,
238,
242,
246,
250,
254,
258,
262,
266,
270,
274,
278,
282,
286,
290,
294,
298,
302,
306,
310,
314,
318,
322,
326,
330,
334,
338,
342,
346,

126
130
134
138
142
146
150
154
158
162
166
170
174
178
182
186
190
194
198
202
206
210
214
218
222
226
230
234
238
242
246
250
254
258
262
266
270
274
278
282
286
290
294
298
302
306
310
314
318
322
326
330
334
338
342
346
350

 

650, 350, 354
654, 354, 358
658, 358, 362
662, 362, 366
666, 366, 370
670, 370, 374
674, 374, 378
678, 378, 382
682, 382, 386
686, 386, 390
690, 390, 394
694, 394, 398
698, 398, 402
*Element, type=SPRINGA,
elset=MedLow
303, 3, 7
307, 7, 11
311, 11, 15
315, 15, 19
319, 19, 23
323, 23, 27
327, 27, 31
331, 31, 35
335, 35, 39
339, 39, 43
343, 43, 47
347, 47, 51
351, 51, 55
355, 55, 59
359, 59, 63
363, 63, 67
367, 67, 71
371, 71, 75
375, 75, 79
379, 79, 83
383, 83, 87
387, 87, 91
391, 91, 95
395, 95, 99
399, 99, 103
403, 103, 107
407, 107, 111
411, 111, 115
415, 115, 119
419, 119, 123
423, 123, 127
427, 127, 131
431, 131, 135
435, 135, 139
439, 139, 143
443, 143, 147
447, 147, 151
451, 151, 155
455, 155, 159
459, 159, 163
463, 163, 167
467, 167, 171

178

 

471,
475,
479,
483,
487,
491,
495,
499,
503,
507,
511,
515,
519,
523,
527,
531,
535,
539,
543,
547,
551,
555,
559,
563,
567,
571,
575,
579,
583,
587,
591,
595,
599,
603,
607,
611,
615,
619,
623,
627,
631,
635,
639,
643,
647,
651,
655,
659,
663,
667,
671,
675,
679,
683,
687,
691,
695,

171,
175,
179,
183,
187,
191,
195,
199,
203,
207,
211,
215,
219,
223,
227,
231,
235,
239,
243,
247,
251,
255,
259,
263,
267,
271,
275,
279,
283,
287,
291,
295,
299,
303,
307,
311,
315,
319,
323,
327,
331,
335,
339,
343,
347,
351,
355,
359,
363,
367,
371,
375,
379,
383,
387,
391,
395,

175
179
183
187
191
195
199
203
207
211
215
219
223
227
231
235
239
243
247
251
255
259
263
267
271
275
279
283
287
291
295
299
303
307
311
315
319
323
327
331
335
339
343
347
351
355
359
363
367
371
375
379
383
387
391
395
399

699, 399,
*Element,
elset=Low
304, 4, 8
308, 8, 1
312, 12,
316, 16,
320, 20,
324, 24,
328, 28,
332, 32,
336, 36,
340, 40,
344, 44,
348, 48,
352, 52,
356, 56,
360, 60,
364, 64,
368, 68,
372, 72,
376, 76,
380, 80,
384, 84,
388, 88,
392, 92,
396, 96,
400, 100,
404, 104,
408, 108,
412, 112,
416, 116,
420, 120,
424, 124,
428, 128,
432, 132,
436, 136,
440, 140,
444, 144,
448, 148,
452, 152,
456, 156,
460, 160,
464, 164,
468, 168,
472, 172,
476, 176,
480, 180,
484, 184,
488, 188,
492, 192,
496, 196,
500, 200,
504, 204,
508, 208,
512, 212,
516, 216,

403
type=SPRINGA,

2
16
2O
24
28
32
36
40
44
48
52
56
60
64
68
72
76
80
84
88
92
96
100
104
108
112
116
120
124
128
132
136
140
144
148
152
156
160
164
168
172
176
180
184
188
192
196
200
204
208
212
216
220

 

520, 220,
524, 224,
528, 228,
532, 232,
536, 236,
540, 240,
544, 244,
548, 248,
552, 252,
556, 256,
560, 260,
564, 264,
568, 268,
572, 272,
576, 276,
580, 280,
584, 284,
588, 288,
592, 292,
596, 296,
600, 300,
604, 304,
608, 308,
612, 312,
616, 316,
620, 320,
624, 324,
628, 328,
632, 332,
636, 336,
640, 340,
644, 344,
648, 348,
652, 352,
656, 356,
660, 360,
664, 364,
668, 368,
672, 372,
676, 376,
680, 380,
684, 384,
688, 388,
692, 392,
696, 396,
700, 400,
*Spring,
nonlinear
0, 0

0, 0.0044
400, 20
*Spring,
nonlinear
0, 0

0, 0.004

224
228
232
236
240
244
248
252
256
260
264
268
272
276
280
284
288
292
296
300
304
308
312
316
320
324
328
332
336
340
344
348
352
356
360
364
368
372
376
380
384
388
392
396
400
404
elset=High,

elset=MedHigh,

179

 

400, 20
*Spring, elset=MedLow,
nonlinear

0, 0

0, 0.0032

400, 20

*Spring, elsetzLow,
nonlinear

0,0

0, 0.0006

400, 20

*Orientation, name=Ori-
1

0., 1., 0.
2, 0.
** Region: (Section-
1:Picked), (Material
Orientation:Picked)
*Elset, elset=_I1,
generate

l, 300, 1
** Section: Section-1
*Solid Section,
elset=_I1,
orientation=Ori-1,
materia1=YINmatrix
1.,

*Nset,
nset=_PickedSet24,
generate
4, 404, 4
*Elset,
elset=_PickedSet24,
generate
3, 300, 3
*Nset,
nset=_PickedSet26,
generate
401, 404, 1
*Elset,
elset=_PickedSet26,
generate
298, 300, 1
*Nset, nset=NALL,
generate
1, 404, 1
*Elset, elset=NALL,
generate
l, 300, l
*Nset,
nset=_PickedSet34,
generate
1, 4, 1

*Elset,
elset=_PickedSet34,

generate

1, 3, 1

*Nset, nset=_halfLine,
generate

201, 204, 1

*‘k

** MATERIALS

**

*Material,

name=YINmatrix
*Elastic,

type=ENGINEERING

CONSTANTS
0.0457, 1.,
0.07769, 0.7,
0.013441, 0.1

*Permeability,

specific=9.81e-06
3.1392e-09,2.

** BOUNDARY CONDITIONS
** Name: Free Edge

Type: Pore pressure

*Boundary

_PickedSet24, 8, 8

** Name: Radial Type:

Displacement/Rotation

*Boundary

_PickedSet34, 2, 2

*Boundary

NALL, 3, 3

*INITIAL CONDITIONS,

TYPE=RATIO

NALL, 2.

**

0.0457,
1.7, 0.1,

** 1% strain at 2%
strain/minute

** STEP: Step-l

**
*Step,
nlgeom,
inc=1000

*Soils, consolidation,
end=PERIOD

1., 30., , ,

*El Print, elset=_Il,
SUMMARY=YES

COORD, FLVEL
*CONTROLS,
PARAMETERS=FIELD,
FIELD=PORE FLUID
PRESSURE

,,6.329E—06

** BOUNDARY CONDITIONS
** Name: Displacement

name=Step-1,
amplitude=RAMP,

_PickedSet26, 2,

 

Type:
Displacement/Rotation
*Boundary

_PickedSet26, 2, 2, 0.2

**

** OUTPUT REQUESTS
*Restart, write,
frequency=1

**

** FIELD OUTPUT: F-
Output—1

**

*Output, field
*Node Output

U, RF, POR

*Element Output

S, LE, VOIDR, SAT,
FLVEL

**

** HISTORY OUTPUT: H—
Output-1

**

*Output, history,
variable=PRESELECT
*El Print, freq=999999
*Node Print,
freq=999999

*FILE FORMAT, ZERO
INCREMENT

*End Step

B.2 Global Model for 1 %
strain at 20% strain/min

* * ....................
** 1% strain at 20%
strain/minute

** STEP: Step—1

*Step, name=Step-1,
nlgeom, amplitude=RAMP,
inc=1000

*Soils, consolidation,
end=PERIOD

0.1, 3., , ,

*CONTROLS,

PARAMETERS=FIELD,
FIELD=PORE FLUID
PRESSURE

,,6.329E-06

** BOUNDARY CONDITIONS
** Name: Displacement
Type:
Displacement/Rotation
*Boundary

180

 

B.3 Global Model for 3%
strain at 6% strain/minute

**

** 3% strain at 6%
strain/minute

** STEP: Step-1

*Step, name=Step-1,
nlgeom, amplitude=RAMP,
inc=1000

*Soils, consolidation,
end=PERIOD

1., 30., , ,

*CONTROLS,
PARAMETERS=FIELD,
FIELD=PORE FLUID
PRESSURE

,,6.329E-06

** BOUNDARY CONDITIONS
** Name: Displacement
Type:
Displacement/Rotation
*Boundary

_PickedSet26, 2, 2, 0.6

BA Global Model for 3%
strain at 2% strain/minute

** 3% strain at 2%
strain/minute

** STEP: Step-1

*Step, name=Step-1,
nlgeom, amplitude=RAMP,
inc=1000

*Soils, consolidation,
end=PERIOD

1., 130., , ,
*CONTROLS,
PARAMETERS=FIELD,
FIELD=PORE FLUID
PRESSURE

,,6.329E-06

** BOUNDARY CONDITIONS
** Name: Displacement
Type:
Displacement/Rotation
*Boundary
_PickedSet26, 2,

———~—-———————a—————~a—

B.5 Submodel for 1%

strain at 2% strain/minute

*Heading

*Node

1, 0.12, 9.89

2, 0.1225, 9.89

3, 0.1225, 9.91

4, 0.12, 9.91

5, 0.13, 9.89

6, 0.1275, 9.89

7, 0.1275, 9.91

8, 0.13, 9.91

9, 0.127, 9.91

10, 0.123, 9.91

11, 0.123, 9.89

12, 0.127, 9.89

13, 0.1, 9.8

14, 0.15, 9.8

15, 0.15, 10.

16, 0.1, 10.

17, 0.1273756, 9.889221
18, 0.1270097, 9.888513
19, 0.1264553, 9.887967
20, 0.125782, 9.887626
21, 0.1250419, 9.887501
22, 0.1243278, 9.887592
23, 0.1237166, 9.887855
24, 0.123218, 9.888247
25, 0.1228318, 9.888756
26, 0.1225847, 9.889355
27, 0.1225, 9.891253
28, 0.1225, 9.892502
29, 0.1225, 9.893751
30, 0.1225, 9.895

31, 0.1225, 9.89625

32, 0.1225, 9.8975

33, 0.1225, 9.89875

34, 0.1225, 9.9

35, 0.1225, 9.90125

36, 0.1225, 9.9025

37, 0.1225, 9.90375

38, 0.1225, 9.904999
39, 0.1225, 9.906248
40, 0.1225, 9.907496
41, 0.1225, 9.908743
42, 0.122596, 9.910686
43, 0.1228836, 9.911331
44, 0.1233149, 9.911847
45, 0.1238201, 9.912204
46, 0.1245113, 9.912452
47, 0.1252149, 9.912491
48, 0.1259368, 9.912318
49, 0.1265545, 9.911958
50, 0.1270634, 9.911411
51, 0.1273919, 9.910728
52, 0.1275, 9.908745

 

53,
54,
55,
56,
57,
58,
59,
60,
61,
62,
63,
64,
65,
66,
67,
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83,
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86,
87,
88,
89,
90,
91,
92,
93,
94,
95,
96,
97,
98,
99,
100,
101,
102,
103,
104,
105,
106,
107,
108,
109,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.1275,
.1275,
.1275,
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.1275,
.1275,
.1275,
.1275,
.1275,
.1275,
.1275,
.1275,
.1275,
.1201603, 9

.1206616,
.1214864,
.1225794,
.1238598,
.1252736,
.1266752,
.1279844,
.1290676,
.1297744, 9

.13, 9.
.892498
.893749
.895
.89625
.897499
.89875
.9
.90125
.9025
.903751
.905005
.906258
.907513
.13, 9.

.13,
.13,
.13,
.13,
.13,
.13,
.13,
.13,
.13,
.13,
.13,
.13,
.13,

.129833,
.129309,
.1284311,
.1272283,
.1258984,
.1244844,
.1230736,
.1218183, 9.
.1207713,9.
.1201865,9.
9.

0000000000

\OKDKOKJKOKDKOKDWWWKDKO

.12,
.12,
.12,
.12,
.12,
.12,
.12,
.12,

\DKDKOKOkOKOLO

WOKOkOtOkOKDKDODKOkOKD

\0

.907496
.906248
.905
.90375
.9025
.90125
.9
.89875
.8975
.89625
.895
.89375
.892503
.891255

.888744
.887514
.886443

.885625
.885132
.885008
.885289
.885988
.887093
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891246

koxoxoxotokoxoko

908789
9.911282
9.912537
.913637
.914476
.914919
.914973
.914614
913857
912668
911353
908752
.907501
.90625

.905

.90375
.9025
.90125
.900001

\OKDKOKOKO

1'81

 

110,
111,
112,
113,
114,
115,
116,
117,
118,
119,
120,
121,
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123,
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133,
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135,
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144,
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146,
147,
148,
149,
150,
151,
152,
153,
154,
155,
156,
157,
158,
159,
160,
161,
162,
163,
164,
165,
166,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.12,
.12,
.12,
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.12, 9

\DKOKDKDLDKD

.89875
.8975
.89625
.895
.89375
.8925
.891251

.1230789,9.
.1233067,9.
.1236304,9.
.1239892,9.
.124416,
.1250051
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9.
9.888

I

889443
888936
888542
888274
888087

.1260631,9.888306

.126531, 9.888713
.1268786,9.889314
.127, 9.891258
.127, 9.892504
.127, 9.893751
.127, 9.895

.127, 9.89625
.127, 9.8975
.127, 9.89875
.127, 9.9

.127, 9.90125
.127, 9.9025
.127, 9.90375
.127, 9.905

.127, 9.906248
.127, 9.907495
.127, 9.908741
.1268968,9.910634
.1265889,9.911215
.1261494,9.911636
.1256743,9.911883
.1251192,9.911997
.1245382,9.911946
.1239607,9.911709
.1236536,9.911479
.1233289,9.911098
.1230874,9.910584
.123, 9.908737
.123, 9.907492
.123, 9.906246
.123, 9.904999
.123, 9.903749
.123, 9.9025
.123, 9.90125
.123, 9.9

.123, 9.89875
.123, 9.8975
.123, 9.89625
.123, 9.895

.123, 9.893751
.123, 9.892506
.123, 9.891265

167,
168,
169,
170,
171,
172,
173,
174,
175,
176,
177,
178,
179,
180,
181,
182,
183,
184,
185,
186,
187,
188,
189,
190,
191,
192,
193,
194,
195,
196,
197,
198,
199,
200,
201,
202,
203,
204,
205,
206,
207,
208,
209,
210,
211,
212,
213,
214,
215,
216,
217,
218,
219,
220,
221,
222,
223,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.105,
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.115,
.12,
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.135,
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.145,
.15,
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9

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9

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\OKDOKOkDKOKDkDKOKDKDKDkaDkD\DkaOWKDKOKOKO\DKOKOkOKDkO\D\O\O\O\O\O\O\OKO\O\OKO\DKOKOKO\O\O\O

9

.8
8
8
8
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8
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8
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.805024
.81005
.815056
.820071
.825154
.830199
.835218
.840233
.84523
.850204
.855147
.860065
.865031
.869888
.874309
.878421
.881973
.885245
.887938
.890355
.892571
.89453
.896206
.897494
.898749
.900001
.901252
.902507
.903771
.90537
.907381
.909738
.912341
.915176
.918248
.92154
.924954
.928956
.932903
.936989
.941261
.94559
.950184
.954956
.959836
.964806
.9698
.974839

 

224,
225,
226,
227,
228,
229,
230,
231,
232,
233,
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235,
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243,
244,
245,
246,
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248,
250,
251,
252,
253,
254,
255,
256,
257,
258,
259,
260,
261,
262,
263,
264,
265,
266,
267,
268,
269,
270,
271,
272,
273,
274,
275,
276,
277,
278,
279,
280,
281,
282,

H

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.15,
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.1428571,
.1392857,
.1357143,
.1321429,
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.1178572,
.1142857,
.1107143,
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9.

\ ‘ S ‘ ‘ ‘ § § § ‘ ~ ‘ ‘ ‘ § § ‘ §

\

‘ ‘ \ \ ‘ ‘ ‘ ‘ ‘ ‘ ~ ‘ ‘ ‘ ~ ‘ ~ ‘

H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H
m m m m m m m m m m m m m m m m m m m m m m w m m m m m m m m m m m m m m m m

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.937828
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10.
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283,
284,
285,
286,
288,
290,
291,
292,
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294,
295,
296,
297,
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321,
322,
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324,
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326,
327,
328,
329,
330,
331,
332,
333,
334,
335,
336,
337,
338,
339,
340,
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.1, 9.
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‘

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342,
343,
344,
345,
346,
347,
348,
349,
350,
351,
352,
353,
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355,
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358,
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395,
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398,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.1218188,
.1254165,9.913175
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.1262947,9.886303
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.1273307,9.886866
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.1283313,9.893751
.1291583,9.892503
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9.88919

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912106

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9.91293

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9.9
9.9
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9.89625
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9.895

 

399,
400,
401,
402,
403,
404,
405,
406,
407,
408,
409,
410,
411,
412,
413,
414,
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417,
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420,
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434,
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439,
440,
441,
442,
443,
444,
445,
446,
447,
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OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

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.1262, 9.9
.1254, 9.9
.1246, 9.9
.1238, 9.9
.1262, 9.89875
.1254, 9.89875
.1246, 9.89875
.1238, 9.89875
.1262, 9.897499
.1254, 9.897499
.1246, 9.897499

183

 

456,
457,
458,
459,
460,
461,
462,
463,
464,
465,
466,
467,
468,
469,
470,
471,
472,
473,
474,
475,
476,
477,
478,
479,
480,
481,
482,
483,
484,
485,
486,
487,
488,
489,
490,
491,
492,
493,
494,
495,
496,
497,
498,
499,
500,
501,
502,
503,
504,
505,
506,
507,
508,
509,
510,
511,
512,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.1238,
.1262,
.1254,
.1246,
.1238, 9.
.1261999,
.1253998,9.895001
.1245998,9.895002
.1237999,9.895002
.1261994,9.893755
.1253991,
.1245993,9.893763
.1237997,9.893761
.1261982,9.892517
.1253973,9.892532
.124598, 9.
.1237994,
.1261953,9.891291
.1253935,9.891325
.1245958,9.891342
.1237997,9.891317
.1254858,9.911613
.1250823,9.911595
.1260817,9.910472
.1258958,9.910967
.1256716,9.911354
.1251406,9.911226
.1252392,9.910832
.1253242,9.910385
.1241713,9.911494
.1240816,
.1239427,9.910886
.1238549,9.910442
.1241872,9.888486
.1238417,
.1239246,9.889132
.1240677,9.888741
.1254048,9.888359
.1250043,9.888385
.1256022,9.888604
.125843, 9.
.1260521,9.889493
.1252972,9.889603
.125187, 9.
.1250805,9.888731
.1251679,9.855898
.1249347,9.946147
.1201744,
.1151425,
.1101005,9.856346
.1050535,9.856426
.1450486,9.855179
.1400837,9.855508
.1351184,9.855652
.1301433,9.855773
.12525,
.1253804,9.865154

.897499
.89625
.89625
.89625
89625
9.895

\OUJLOKO

9.89376

89254
9.89253

9.91126

9.88959

888987

889132

9.8561
9.85638

9.860677

513,
514,
515,
516,
517,
518,
519,
520,
521,
522,
523,
524,
525,
526,
527,
528,
529,
530,
531,
532,
533,
534,
535,
536,
537,
538,
539,
540,
541,
542,
543,
544,
545,
546,
547,
548,
549,
550,
551,
552,
553,
554,
555,
556,
557,
558,
559,
560,
561,
562,
563,
564,
565,
566,
567,
568,
569,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.1255717,9.869333
.1257468,9.873148
.1257962,9.876843
.1256901,9.879618
.1255477,9.881708
.1254307,9.883379
.1175374,9.889952
.1154984,9.889482
.1134595,9.888799
.1113164,9.887873
.1089269,9.886832
.1062072,9.885845
.1031775,9.885231
.1468403,9.885336
.1437661,
.1408746,9.886351
.1383104,9.887238
.1360513,9.888116
.1340202,9.888878
.1321107,9.889471
.1176932,9.910164
.1159885,9.910698
.1141984,9.911491
.1120286,9.912323
.1095356,9.913473
.1063889,9.914026
.1031867,9.914318
.1464794,9.914962
.1430041,9.914434
.140153, 9.
.1377452,9.912246
.1356355,9.911454
.1337084,
.1318848,
.1244525,9.915964
.1244569,
.1244739,9.918481
.1244928,9.920108
.12452,
.1245608,9.923951
.1246456,9.926343
.1247237,9.928861
.1247986,9.931556
.1248462,9.934562
.124881, 9.
.1249095,9.942087
.1213575,9.946206
.1177838,9.946188
.114214, 9.
.1106495,9.946076
.107092, 9.
.1035435,9.946094
.1464283,9.945506
.142849, 9.
.1392655,
.1356802,9.945755
.1320961,9.945908

9.88573

913254

9.91084

9.91037

9.91704

9.921928

938158

946131

946071

945532
9.94561

 

570,
571,
572,
573,
574,
575,
576,
577,
578,
579,
580,
581,
582,
583,
584,
585,
586,
587,
588,
589,
590,
591,
592,
593,
594,
595,
596,
597,
598,
599,
600,
601,
602,
603,
604,
605,
606,
607,
608,
609,
610,
611,
612,
613,
614,
615,
616,
617,
618,
619,
620,
621,
622,
623,
624,
625,
626,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.1285141,9.946049
.1050278,9.851227
.1050137,9.846064
.1050064,9.840889
.1050027,9.835724
.1050008,
.1050003,
.1050001,9.820381
.1050001,9.815293
.105,
.105,
.110044, 9.
.1100205,9.846174
.1100092,9.841063
.110004, 9.
.1100017,9.830806
.1100007,9.825674
.1100003,9.820541
.1100001,9.815409
.11,
.11,
.1150731,9.851378
.1150368,
.1150155,
.1150055,9.836206
.1150024,9.831039
.115001,
.1150004,9.820691
.1150002,9.815516
.1150001,9.810341
.115,
.1201007,9.851l98
.1200551,9.846235
.1200248,9.841275
.1200087,9.836271
.1200039,9.831108
.1200011,9.825985
.1200005,
.1200002,9.815578
.1200001,9.810379
.12,
.1251042,9.851041
.125058,

.1250272,
.1250118,9.836168
.1250052,9.831052
.1250015,9.825944
.1250005,9.820782
.1250002,9.815576
.1250001,9.810375
.125,
.l300901,9.850926
.1300507,9.846029
.1300237,9.841093
.1300096,9.836077
.1300037,9.830986
.1300011,9.825873

9.83056
9.82547

9.810202
9.805104
851257

835938

9.810274
9.805138

9.84633
9.84129

9.825866

9.805168

9.82078

9.805185

9.84614
9.8412

9.805183

184

 

627,
628,
629,
630,
631,
632,
633,
634,
635,
636,
637,
638,
639,
640,
641,
642,
643,
644,
645,
646,
647,
648,
649,
650,
651,
652,
653,
654,
655,
656,
657,
658,
659,
660,
661,
662,
663,
664,
665,
666,
667,
668,
669,
670,
671,
672,
673,
674,
675,
676,
677,
678,
679,
680,
681,
682,
683,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.1300005,9.820687
.1300002,9.815505
.1300001,9.810329
.13,
.1350663,9.850823
.1350309,9.845955
.1350116,9.841017
.1350055,9.835914
.1350025,9.830791
.1350012,9.825657
.l350005,9.820522
.1350002,9.815388
.1350001,9.810254
.135,
.1400378,9.850653
.1400195,9.845636
.1400098,9.840592
.1400047,9.835531
.1400022,9.830462
.140001, 9.
.1400005,9.820328
.1400002,9.815252
.1400001,
.14,
.1450245,
.1450125,9.845258
.1450063,9.840252
.1450031,9.835237
.1450015,9.830225
.1450007,9.825193
.1450003,9.820142
.1450001,9.815135
.1450001,9.810101
.145,
.1107116,9.873552
.1101853,9.861538
.1103606,9.867079
.104999,
.1208478,
.1161493,9.871035
.1202417,9.885108
.1191315,9.883805
.117807,
.1162192,
.1139727,9.877419
.1070493,
.1083724,9.878796
.1051043,9.867197
.1050857,9.861694
.1152809,9.861296
.1202895,9.860893
.1155376,9.866225
.1204571,9.865458
.1216608,9.873728
.1223483,9.877121
.1228565,9.879744
.1232394,9.881777

9.805161

9.805124

825393

9.81017
9.805085
9.85024

9.805052

9.872904
9.8696

9.882193
9.8801

9.88258

684,
685,
686,
687,
688,
689,
690,
691,
692,
693,
694,
695,
696,
697,
698,
699,
700,
701,
702,
703,
704,
705,
706,
707,
708,
709,
710,
711,
712,
713,
714,
715,
716,
717,
718,
719,
720,
721,
722,
723,
724,
725,
726,
727,
728,
729,
730,
731,
732,
733,
734,
735,
736,
737,
738,
739,
740,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.1235388,9.883355
.ll77046,9.875055
.1190799,
.1201908,9.880621
.1210629,9.882492
.1217759,9.883947
.1189644,9.886529
.1180427,9.888242
.1175222,9.885546
.1163976,9.887438
.1158578,9.884308
.1145351,9.886467
.1140171,9.88262
.1124644,9.8852l9
.1115812,9.880709
.1100044,9.883847
.1036167,9.881796
.1042433,9.877834
.1356599,
.1351836,9.860476
.1352672,9.865288
.1354349,9.870108
.1302797,
.1456014,9.874557
.1410072,9.875127
.1398581,9.883514
.1383183,9.880283
.1290355,9.884704
.1300215,9.883231
.1312946,9.881398
.13302,
.1303119,
.1302496,9.865171
.1302034,9.860549
.1401481,9.860394
.1450936,9.860091
.1402809,9.865186
.1451795,9.865044
.1405316,9.870129
.1453342,9.87
.1460573,9.878643
.146491, 9.
.1421306,9.879326
.1430666,9.882719
.137173, 9
.134959, 9.
.1331235,9.886932
.1315948,9.887863
.1355158,9.882025
.1334493,9.883652
.1318114,9.884992
.1305189,9.886071
.1273108,9.883775
.12786,
.1285599,9.880175
.1294116,9.877542
.1028988,9.888037

9.87817

9.87584

9.87397

9.879214
9.86943

882168

.884703
885872

9.882222

 

741,
742,
743,
744,
745,
746,
747,
748,
749,
750,
751,
752,
753,
754,
755,
756,
757,
758,
759,
760,
761,
762,
763,
764,
765,
766,
767,
768,
769,
770,
771,
772,
773,
774,
775,
776,
777,
778,
779,
780,
781,
782,
783,
784,
785,
786,
787,
788,
789,
790,
791,
792,
793,
794,
795,
796,
797,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.1057024,9.888452
.1083303,9.889072
.110756, 9.
.113008, 9.
.1152467,9.890943
.1175055,9.891241
.1027154,
.1053734,
.1079364,9.891212
.1103702,9.891693
.1127083,9.892159
.1151284,9.892315
.1175262,9.892462
.1025909,9.892895
.1051434,9.893103
.1076773,9.893194
.1101042,9.893513
.1125244,9.893699
.1150326,9.893698
.1175051,9.893741
.102519, 9.
.1050144,9.894938
.1075372,9.894886
.1100169,9.894957
.1125119,9.894973
.1150068,9.894985
.1175032,9.894994
.1025028,9.896231
.1050056,9.896225
.1075071,9.896226
.1100071,9.896231
.1125055,9.896236
.1150035,9.896242
.1175018,9.896246
.102501, 9.
.1050021,9.897491
.1075028,9.897491
.1100029,9.897491
.1125024,9.897494
.1150017,9.897496
.1175009,9.897498
.1025004,9.898748
.1050008,9.898746
.1075011,9.898746
.1100012,9.898746
.112501,
.1150008,9.898748
.1175004,9.898748
.1025001,
.1050003,9.899999
.1075004,9.899999
.1100005,9.899999

889786
890496

9.89057
9.89085

894836

897493

9.898748

9.9

.1125004, 9.9
.1150003, 9.9
.1175002, 9.9
.1025, 9.901251
.1050001, 9.90125

185

 

798,
799,
800,
801,
802,
803,
804,
805,
806,
807,
808,
809,
810,
811,
812,
813,
814,
815,
816,
817,
818,
819,
820,
821,
822,
823,
824,
825,
826,
827,
828,
829,
830,
831,
832,
833,
834,
835,
836,
837,
838,
839,
840,
841,
842,
843,
844,
845,
846,
847,
848,
849,
850,
851,
852,
853,
854,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.1075002,
.1100002,
.1125002,
.1150001,
.1175001,
.1025,
.105,

.1075001,
.1100001,
.1125001,
.1150001,
.1175,
.1025002,9.
.1050006,9.
.1075008,9.
.1100009,9.
.1125007,9.
.1150005,9.
.1175003,9.
.1025188,
.1050073,9.
.1075115,9.
.1100128,9.
.1125082,9.
.1150043,9.
.1175021,9.
.1026081,
.1051775,9.
.1077127,9.
.1101901,9.
.1126029,9.
.115039,
.1175182,9.
.1027591,9.
.1054776,9.
.108112,
.1106027,9.
.1129512,9.
.115213,
.1175589,9.
.1029544,9.
.105872,
.1086677,9.
.1111743,9.
.1134557,
.1155313,9.
.1176096,9.
.146859,
.1438222,9.
.1410762,9.
.1386052,9.
.1363501,9.
.1342404,9.
.1321494,9.
.1471345,9.
.1443491,9.
.1417162,9.

\O\O&D\D

9.
9.902504
9.902501

9
9
9
9

9

9

9.

9.

9.

9.

9

9.

.90125
.90125
.90125
.90125
90125

.9025
.9025
.9025
.9025

9.9025

903763
903757
903753
903752
903751
903751
903751
.9052

905041
905033
905025
905014
905006
905003
.90706
906847
906702
906548
906378
906293
906269
909286
909056
908765
908403
908028
907703
907555
911718
911434
910966
910325
.90975
909206
908844
912149
911717
911035
910375
909803
909328
908999
909578
909268
908859

855,
856,
857,
858,
859,
860,
861,
862,
863,
864,
865,
866,
867,
868,
869,
870,
871,
872,
873,
874,
875,
876,
877,
878,
879,
880,
881,
882,
883,
884,
885,
886,
887,
888,
889,
890,
891,
892,
893,
894,
895,
896,
897,
898,
899,
900,
901,
902,
903,
904,
905,
906,
907,
908,
909,
910,
911,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.1392237,
.1369032,
.1345779,
.1323334,
.1473269,
.1447156,
.1421779,
.1396701,
.1372803,
.1348348,
.1324085,
.1474449,
.1449248,
.1423945,
.139882,

.1373917,
.134916,

.1324519,
.1474851,
.1449679,
.1424535,
.1399461,
.1374485,
.1349587,
.1324756,
.147494,

.1449867,
.1424801,
.1399762,
.1374763,
.1349803,
.1324881,
.1474977,
.1449947,
.1424918,
.1399898,
.1374895,
.1349909,
.1324944,
.1474991,
.1449979,
.1424967,
.1399957,
.1374954,
.1349959,
.1324974,
.1474997,
.1449992,
.1424987,
.1399982,
.1374981,
.1349982,
.1324988,
.1474999,
.1449997,
.1424995,
.1399993,

9.908501
9.908091

9.90786
9.907627
9.907253
9.907004
9.906796
9.906679
9.906474
9.906397
9.906328
9.905268
9.905179
9.905178
9.905155
9.905119
9.905081
9.905045
9.903806
9.903826
9.903831
9.903824

9.90381
9.903793
9.903774
9.902521
9.902531
9.902535
9.902534
9.902529
9.902521
9.902513
9.901258
9.901262
9.901265
9.901264
9.901263
9.901259
9.901256
9.900002
9.900005
9.900005
9.900005
9.900005
9.900004
9.900002

9.89875
9.898751
9.898751
9.898751
9.898751
9.898751

9.89875
9.897497
9.897499
9.897499
9.897499

 

912,
913,
914,
915,
916,
917,
918,
919,
920,
921,
922,
923,
924,
925,
926,
927,
928,
929,
930,
931,
932,
933,
934,
935,
936,
937,
938,
939,
940,
941,
942,
943,
944,
945,
946,
947,
948,
949,
950,
951,
952,
953,
954,
955,
956,
957,
958,
959,
960,
961,
962,
963,
964,
965,
966,
967,
968,

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

.1374992,
.1349992,
.1324995,
.1474999,
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186

9.897499
9.897499
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9.916774
9.941801
9.937749
9.933907
9.930395
9.927011
9.923742
9.920314
9.918882
9.920525
9.921429
9.917135
9.917007
9.911798
9.91249
9.913341
9.914528
9.92361
9.926044

 

969, 0

970, 0.1212301,
971, 0

972, 0

973, 0.1213313,
974, 0.1178275,9.
975, 0.1176724,9.
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978, O.1l76819,9.
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980, 0.1177553,9.
981, 0.1142109,9.
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1000,0.1069961,9.
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1003,0.1209963,9.
9.
1005,0.1185653,9.
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9.
1012,0.1189316,9.
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9.
1017,0.1428255,9.
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1004, 0.12266,

1011, 0.11618,

1016,0.142837,

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9.9314
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9.9421

922902
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911715
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921516
925011
929011
932888
93692

941099
941419
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93765

937901
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931128
931465
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921614
919862
91831

91694

915874
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915353
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917771
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91439

917727
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131775,9.913864

1305955,9.
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913102
949978
954803

 

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1464286,9.
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1356968,9.
1357079,9.

959748
96474

969755
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989896
994944
94985

954432
959416
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974579
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984736
98982

994907
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95903

964106
969212
974333
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984592
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135714,9.958808

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1285711,9.
1285713,9.
1285714,9.
1285714,9.
1285714,9.

187

96377

968919
97409

979269
984451
989635
994818
950035
954357
958854
96358

968685
973894
979114
984338
989561
994783
95015

954483
958914
963531
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973781
979024
984272
989519

 

1140,0.
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1285714,9.
1249564,9.
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994762
950305
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124987,9.959026

1249962,9.
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963605
968537
973766
979012

125,9.984262
125,9.989513
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1213804,9.

95039

121399,9.954689

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959133
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97385

979077
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968841
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117857,9.984408

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989609
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989695
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96437

969418
974493
979585
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98979

994895
950452
954925

107135,9.959752

1071389,9.

96472

107l41,9.969725
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1197,0.
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1209,0.
1210,0.

*Element,

1 I
I
I

I

‘

‘

OD\]O\U"I|I>UUN

\D

I

10,
11,
12,
13,
14,
15,
16,
17,
18,
19,
20,
21,
22,
23,
24,
25,
26,
27,
28,
29,
30,
31,
32,
33,
34,
35,
36,
37,
38,
39,
40,
41,
42,

1, 293,

293,
294,
116,
305,
306,
115,
307,
308,
114,
309,
310,
113,
311,
312,
112,
313,
314,
111,
315,
316,
110,
317,
318,
109,
319,
320,
108,
321,
322,
107,
323,
324,
106,
325,
326,
105,
327,
328,
104,
329,
330,

1071425,9.979773
1071427,9.984821
1071428,9.989878
1071428,9.994938
103555,9.95049

1035628,9.955174
1035668,9.960047
1035691,9.964996
1035703,9.969962
1035709,9.974937
1035712,9.979918
1035713,9.984922
1035714,9.989943
1035714,9.994969

type=CPE4P
305, 116
294, 306, 305
2, 27, 306
305, 307, 115
306, 308, 307
27, 28, 308
307, 309, 114
308, 310, 309
28, 29, 310
309, 311, 113
310, 312, 311
29, 30, 312
311, 313, 112
312, 314, 313
30, 31, 314
313, 315, 111
314, 316, 315
31, 32, 316
315, 317, 110
316, 318, 317
32, 33, 318
317, 319, 109
318, 320, 319
33, 34, 320
319, 321, 108
320, 322, 321
34, 35, 322
321, 323, 107
322, 324, 323
35, 36, 324
323, 325, 106
324, 326, 325
36, 37, 326
325, 327, 105
326, 328, 327
37, 38, 328
327, 329, 104
328, 330, 329
38, 39, 330
329, 331, 103
330, 332, 331
39, 40, 332

 

43,
44,
45,
46,
47,
48,
49,
50,
51,
52,
53,
54,
55,
56,
57,
58,
59,
60,
61,
62,
63,
64,
65,
66,
67,
68,
69,
70,
71,
72,
73,
74,
75,
76,
77,
78,
79,
80,
81,
82,
83,
84,
85,
86,
87,
88,
89,
90,
91,
92,
93,
94,
95,
96,
97,
98,
99,

103, 331, 333, 102
331, 332, 334, 333
332, 40, 41, 334
102, 333, 296, 4
333, 334, 295, 296
334, 41, 3, 295
71, 297, 335, 70
297, 298, 336, 335
298, 22, 23, 336
70, 335, 337, 69
335, 336, 338, 337
336, 23, 24, 338
69, 337, 339, 68
337, 338, 340, 339
338, 24, 25, 340
68, 339, 341, 67
339, 340, 342, 341
340, 25, 26, 342
67, 341, 293, 1
341, 342, 294, 293
342, 26, 2, 294
48, 299, 343, 47
299, 300, 344, 343
300, 95, 96, 344
47, 343, 345, 46
343, 344, 346, 345
344, 96, 97, 346
46, 345, 347, 45
345, 346, 348, 347
346, 97, 98, 348
45, 347, 349, 44
347, 348, 350, 349
348, 98, 99, 350
44, 349, 351, 43
349, 350, 352, 351
350, 99, 100, 352
43, 351, 353, 42
351, 352, 354, 353
352, 100, 101, 354
42, 353, 295, 3
353, 354, 296, 295
354, 101, 4, 296
22, 298, 355, 21
298, 297, 356, 355
297, 71, 72, 356
21, 355, 357, 20
355, 356, 358, 357
356, 72, 73, 358
20, 357, 359, 19
357, 358, 360, 359
358, 73, 74, 360
19, 359, 361, 18
359, 360, 362, 361
360, 74, 75, 362
18, 361, 363, 17
361, 362, 364, 363
362, 75, 76, 364

188

 

100,
101,
102,
103,
104,
105,
106,
107,
108,
109,
110,
111,
112,
113,
114,
115,
116,
117,
118,
119,
120,
121,
122,
123,
124,
125,
126,
127,
128,
129,
130,
131,
132,
133,
134,
135,
136,
137,
138,
139,
140,
141,
142,
143,
144,
145,
146,
147,
148,
149,
150,
151,
152,
153,
154,
155,
156,

17, 363, 302, 6
363, 364, 301, 302
364, 76, 5, 301
95, 300, 365, 94
300, 299, 366, 365
299, 48, 49, 366
94, 365, 367, 93
365, 366, 368, 367
366, 49, 50, 368
93, 367, 369, 92
367, 368, 370, 369
368, 50, 51, 370
92, 369, 304, 8
369, 370, 303, 304
370, 51, 7, 303

8, 304, 371, 91
304, 303, 372, 371
303, 7, 52, 372
91, 371, 373, 90
371, 372, 374, 373
372, 52, 53, 374
90, 373, 375, 89
373, 374, 376, 375
374, 53, 54, 376
89, 375, 377, 88
375, 376, 378, 377
376, 54, 55, 378
88, 377, 379, 87
377, 378, 380, 379
378, 55, 56, 380
87, 379, 381, 86
379, 380, 382, 381
380, 56, 57, 382
86, 381, 383, 85
381, 382, 384, 383
382, 57, 58, 384
85, 383, 385, 84
383, 384, 386, 385
384, 58, 59, 386
84, 385, 387, 83
385, 386, 388, 387
386, 59, 60, 388
83, 387, 389, 82
387, 388, 390, 389
388, 60, 61, 390
82, 389, 391, 81
389, 390, 392, 391
390, 61, 62, 392
81, 391, 393, 80
391, 392, 394, 393
392, 62, 63, 394
80, 393, 395, 79
393, 394, 396, 395
394, 63, 64, 396
79, 395, 397, 78
395, 396, 398, 397
396, 64, 65, 398

157,
158,
159,
160,
161,
162,
163,
164,
165,
166,
167,
168,
169,
170,
171,
172,
173,
174,
175,
176,
177,
178,
179,
180,
181,
182,
183,
184,
185,
186,
187,
188,
189,
190,
191,
192,
193,
194,
195,
196,
197,
198,
199,
200,
201,
202,
203,
204,
205,
206,
207,
208,
209,
210,
211,
212,
213,

78, 397, 399, 77
397, 398, 400, 399
398, 65, 66, 400
77, 399, 301, 5
399, 400, 302, 301
400, 66, 6, 302

9, 403, 417, 141
403, 404, 418, 417
404, 401, 419, 418
401, 405, 420, 419
405, 10, 152, 420
141, 417, 421, 140
417, 418, 422, 421
418, 419, 423, 422
419, 420, 424, 423
420, 152, 153, 424
140, 421, 425, 139
421, 422, 426, 425
422, 423, 427, 426
423, 424, 428, 427
424, 153, 154, 428
139, 425, 429, 138
425, 426, 430, 429
426, 427, 431, 430
427, 428, 432, 431
428, 154, 155, 432
138, 429, 433, 137
429, 430, 434, 433
430, 431, 435, 434
431, 432, 436, 435
432, 155, 156, 436
137, 433, 437, 136
433, 434, 438, 437
434, 435, 439, 438
435, 436, 440, 439
436, 156, 157, 440
136, 437, 441, 135
437, 438, 442, 441
438, 439, 443, 442
439, 440, 444, 443
440, 157, 158, 444
135, 441, 445, 134
441, 442, 446, 445
442, 443, 447, 446
443, 444, 448, 447
444, 158, 159, 448
134, 445, 449, 133
445, 446, 450, 449
446, 447, 451, 450
447, 448, 452, 451
448, 159, 160, 452
133, 449, 453, 132
449, 450, 454, 453
450, 451, 455, 454
451, 452, 456, 455
452, 160, 161, 456
132, 453, 457, 131

 

214,
215,
216,
217,
218,
219,
220,
221,
222,
223,
224,
225,
226,
227,
228,
229,
230,
231,
232,
233,
234,
235,
236,
237,
238,
239,
240,
241,
242,
243,
244,
245,
246,
247,
248,
249,
250,
251,
252,
253,
254,
255,
256,
257,
258,
259,
260,
261,
262,
263,
264,
265,
266,
267,
268,
269,
270,

453,
454,
455,
456,
131,
457,
458,
459,
460,
130,
461,
462,
463,
464,
129,
465,
466,
467,
468,
128,
469,
470,
471,
472,
127,
473,
474,
475,
476,
145,
146,
409,
410,
411,
412,
478,
482,
483,
484,
403,
479,
480,
481,
405,
488,
487,
486,
409,
10,

151,
150,
149,
120,
413,
414,
415,
416,

454, 458, 457
455, 459, 458
456, 460, 459
161, 162, 460
457, 461, 130
458, 462, 461
459, 463, 462
460, 464, 463
162, 163, 464
461, 465, 129
462, 466, 465
463, 467, 466
464, 468, 467
163, 164, 468
465, 469, 128
466, 470, 469
467, 471, 470
468, 472, 471
164, 165, 472
469, 473, 127
470, 474, 473
471, 475, 474
472, 476, 475
165, 166, 476
473, 407, 12
474, 406, 407
475, 402, 406
476, 408, 402
166, 11, 408
146, 478, 477
147, 409, 478
410, 482, 478
411, 483, 482
412, 484, 483
401, 404, 484
482, 481, 477
483, 480, 481
484, 479, 480
404, 403, 479
9, 142, 479
142, 143, 480
143, 144, 481
144, 145, 477
401, 412, 488
412, 411, 487
411, 410, 486
410, 409, 485
147, 148, 485
405, 488, 151
488, 487, 150
487, 486, 149
486, 485, 148
121, 413, 489
414, 492, 489
415, 491, 492
416, 490, 491
402, 408, 490

189

 

271,
272,
273,
274,
275,
276,
277,
278,
279,
280,
281,
282,
283,
284,
285,
286,
287,
288,
289,
290,
291,
292,
293,
294,
295,
296,
297,
298,
299,
300,
301,
302,
303,
304,
305,
306,
307,
308,
309,
310,
311,
312,
313,
314,
315,
316,
317,
318,
319,
320,
321,
322,
323,
324,
325,
326,
327,

408, 11, 117, 490
490, 117, 118, 491
491, 118, 119, 492
492, 119, 120, 489
407, 406, 498, 497
406, 402, 416, 498
497, 498, 499, 496
498, 416, 415, 499
496, 499, 500, 495
499, 415, 414, 500
495, 500, 494, 493
500, 414, 413, 494
413, 121, 122, 494
494, 122, 123, 493
123, 124, 495, 493
124, 125, 496, 495
125, 126, 497, 496
126, 12, 407, 497
22, 121, 120, 23
23, 120, 119, 24
24, 119, 118, 25
25, 118, 117, 26
26, 117, 11, 2
121, 22, 21, 122
122, 21, 20, 123
123, 20, 19, 124
124, 19, 18, 125
125, 18, 17, 126
126, 17, 6, 12

2, 11, 166, 27

27, 166, 165, 28
28, 165, 164, 29
29, 164, 163, 30
30, 163, 162, 31
31, 162, 161, 32
32, 161, 160, 33
33, 160, 159, 34
34, 159, 158, 35
35, 158, 157, 36
36, 157, 156, 37
37, 156, 155, 38
38, 155, 154, 39
39, 154, 153, 40
40, 153, 152, 41
41, 152, 10, 3

12, 6, 66, 127
127, 66, 65, 128
128, 65, 64, 129
129, 64, 63, 130
130, 63, 62, 131
131, 62, 61, 132
132, 61, 60, 133
133, 60, 59, 134
134, 59, 58, 135
135, 58, 57, 136
136, 57, 56, 137
137, 56, 55, 138

328,
329,
330,
331,
332,
333,
334,
335,
336,
337,
338,
339,
340,
341,
342,
343,
344,
345,
346,
347,
348,
349,
350,
351,
352,
353,
354,
355,
356,
357,
358,
359,
360,
361,
362,
363,
364,
365,
366,
367,
368,
369,
370,
371,
372,
373,
374,
375,
376,
377,
378,
379,
380,
381,
382,
383,
384,

138,
139,
140,
141,
149,
150,
151,
49,

50,

51,

144,
145,
146,
147,
148,
282,
283,
284,
285,
286,
287,
288,
289,
290,
291,
292,
506,
571,
572,
573,
574,
575,
576,
577,
578,
579,
580,
505,
581,
582,
583,
584,
585,
586,
587,
588,
589,
590,
504,
591,
592,
593,
594,
595,
596,
597,
598,

55,
54,
53,
52,
44,
43,
42,
144,
143,
142,
49,
48,
47,
46,
45,
283,
284,
285,
286,
287,
288,
289,
290,
291,
292,
13,
571,
572,
573,
574,
575,
576,
577,
578,
579,
580,
167,
581,
582,
583,
584,
585,
586,
587,
588,
589,
590,
168,
591,
592,
593,
594,
595,
596,
597,
598,
599,

54, 139
53, 140
52, 141
7, 9
43, 150
42, 151
3, 10
143, 50
142, 51
9, 7
48, 145
47, 146
46, 147
45, 148
44, 149
571, 506
572, 571
573, 572
574, 573
575, 574
576, 575
577, 576
578, 577
579, 578
580, 579
167, 580
581, 505
582, 581
583, 582
584, 583
585, 584
586, 585
587, 586
588, 587
589, 588
590, 589
168, 590
591, 504
592, 591
593, 592
594, 593
595, 594
596, 595
597, 596
598, 597
599, 598
600, 599
169, 600
601, 503
602, 601
603, 602
604, 603
605, 604
606, 605
607, 606
608, 607
609, 608

 

385,
386,
387,
388,
389,
390,
391,
392,
393,
394,
395,
396,
397,
398,
399,
400,
401,
402,
403,
404,
405,
406,
407,
408,
409,
410,
411,
412,
413,
414,
415,
416,
417,
418,
419,
420,
421,
422,
423,
424,
425,
426,
427,
428,
429,
430,
431,
432,
433,
434,
435,
436,
437,
438,
439,
440,
441,

599,
600,
503,
601,
602,
603,
604,
605,
606,
607,
608,
609,
610,
501,
611,
612,
613,
614,
615,
616,
617,
618,
619,
620,
510,
621,
622,
623,
624,
625,
626,
627,
628,
629,
630,
509,
631,
632,
633,
634,
635,
636,
637,
638,
639,
640,
508,
641,
642,
643,
644,
645,
646,
647,
648,
649,
650,

600,
169,
601,
602,
603,
604,
605,
606,
607,
608,
609,
610,
170,
611,
612,
613,
614,
615,
616,
617,
618,
619,
620,
171,
621,
622,
623,
624,
625,
626,
627,
628,
629,
630,
172,
631,
632,
633,
634,
635,
636,
637,
638,
639,
640,
173,
641,
642,
643,
644,
645,
646,
647,
648,
649,
650,
174,

190

610,
170,
611,
612,
613,
614,
615,
616,
617,
618,
619,
620,
171,
621,
622,
623,
624,
625,
626,
627,
628,
629,
630,
172,
631,
632,
633,
634,
635,
636,
637,
638,
639,
640,
173,
641,
642,
643,
644,
645,
646,
647,
648,
649,
650,
174,
651,
652,
653,
654,
655,
656,
657,
658,
659,
660,
175,

609
610
501
611
612
613
614
615
616
617
618
619
620
510
621
622
623
624
625
626
627
628
629
630
509
631
632
633
634
635
636
637
638
639
640
508
641
642
643
644
645
646
647
648
649
650
507
651
652
653
654
655
656
657
658
659
660

 

442,
443,
444,
445,
446,
447,
448,
449,
450,
451,
452,
453,
454,
455,
456,
457,
458,
459,
460,
461,
462,
463,
464,
465,
466,
467,
468,
469,
470,
471,
472,
473,
474,
475,
476,
477,
478,
479,
480,
481,
482,
483,
484,
485,
486,
487,
488,
489,
490,
491,
492,
493,
494,
495,
496,
497,
498,

507,
651,
652,
653,
654,
655,
656,
657,
658,
659,
660,
279,
280,
281,
664,
674,
675,
505,
504,
503,
662,
676,
677,
663,
678,
679,
513,
514,
515,
516,
517,
518,
665,
680,
681,
682,
683,
684,
666,
685,
686,
687,
688,
689,
69,

68,

67,

667,
690,
691,
668,
692,
693,
669,
694,
695,
670,

651,
652,
653,
654,
655,
656,
657,
658,
659,
660,
175,
280,
281,
282,
674,
675,
506,
504,
503,
501,
676,
677,
511,
678,
679,
512,
514,
515,
516,
517,
518,
72,
680,
681,
682,
683,
684,
71,
685,
686,
687,
688,
689,
70,
68,
67,
11
690,
691,
519,
692,
693,
520,
694,
695,
521,
696,

690,
691,
519,

185,
184,
183,
182,
181,
180,
179,
178,
177,
176,
14,
674,
675,
506,
663,
662,
505,
676,
677,
511,
678,
679,
512,
666,
665,
513,
680,
681,
682,
683,
684,
71,
685,
686,
687,
688,
689,
70,
671,
670,
669,
668,
667,
69,

692,
693,
520,
694,
695,
521,
696,
697,
522,
698,

186
185
184
183
182
181
180
179
178
177
176
664
674
675
661
663
662
662
676
677
663
678
679
661
666
665
665
680
681
682
683

684

666
685
686
687
688

689

661
671
670
669
668

667

667

690
691

668
692
693
669
694
695
670
696
697
671

499,
500,
501,
502,
503,
504,
505,
506,
507,
508,
509,
510,
511,
512,
513,
514,
515,
516,
517,
518,
519,
520,
521,
522,
523,
524,
525,
526,
527,
528,
529,
530,
531,
532,
533,
534,
535,
536,
537,
538,
539,
540,
541,
542,
543,
544,
545,
546,
547,
548,
549,
550,
551,
552,
553,
554,
555,

696,
697,
671,
698,
699,
524,
525,
672,
700,
673,
701,
514,
513,
512,
511,
706,
715,
716,
717,
509,
508,
507,
703,
718,
719,
704,
720,
721,
705,
722,
723,
190,
191,
192,
707,
724,
725,
708,
726,
727,
528,
529,
530,
531,
532,
709,
728,
729,
730,
731,
710,
732,
733,
734,
735,
74,

73,

697, 699, 698
522, 523, 699
698, 673, 661
699, 672, 673
523, 524, 672
525, 700, 672
276, 277, 700
700, 701, 673
277, 278, 701
701, 664, 661
278, 279, 664
513, 715, 706
512, 716, 715
511, 717, 716
501, 510, 717
715, 705, 702
716, 704, 705
717, 703, 704
510, 509, 703
508, 718, 703
507, 719, 718
186, 187, 719
718, 720, 704
719, 721, 720
187, 188, 721
720, 722, 705
721, 723, 722
188, 189, 723
722, 708, 702
723, 707, 708
189, 190, 707
191, 724, 707
192, 725, 724
193, 526, 725
724, 726, 708
725, 727, 726
526, 527, 727
726, 710, 702
727, 709, 710
527, 528, 709
529, 728, 709
530, 729, 728
531, 730, 729
532, 731, 730
5, 76, 731
728, 732, 710
729, 733, 732
730, 734, 733
731, 735, 734
76, 75, 735
732, 714, 702
733, 713, 714
734, 712, 713
735, 711, 712
75, 74, 711
73, 736, 711
72, 518, 736

 

556,
557,
558,
559,
560,
561,
562,
563,
564,
565,
566,
567,
568,
569,
570,
571,
572,
573,
574,
575,
576,
577,
578,
579,
580,
581,
582,
583,
584,
585,
586,
587,
588,
589,
590,
591,
592,
593,
594,
595,
596,
597,
598,
599,
600,
601,
602,
603,
604,
605,
606,
607,
608,
609,
610,
611,
612,

711,
736,
712,
737,
713,
738,
714,
739,
276,
525,
524,
523,
522,
521,
520,
519,
275,
740,
741,
742,
743,
744,
745,
746,
274,
747,
748,
749,
750,
751,
752,
753,
273,
754,
755,
756,
757,
758,
759,
760,
272,
761,
762,
763,
764,
765,
766,
767,
271,
768,
769,
770,
771,
772,
773,
774,
270,

736, 737, 712
518, 517, 737
737, 738, 713
517, 516, 738
738, 739, 714
516, 515, 739
739, 706, 702
515, 514, 706
525, 740, 275
524, 741, 740
523, 742, 741
522, 743, 742
521, 744, 743
520, 745, 744
519, 746, 745
1, 116, 746

740, 747, 274
741, 748, 747
742, 749, 748
743, 750, 749
744, 751, 750
745, 752, 751
746, 753, 752
116, 115, 753
747, 754, 273
748, 755, 754
749, 756, 755
750, 757, 756
751, 758, 757
752, 759, 758
753, 760, 759
115, 114, 760
754, 761, 272
755, 762, 761
756, 763, 762
757, 764, 763
758, 765, 764
759, 766, 765
760, 767, 766
114, 113, 767
761, 768, 271
762, 769, 768
763, 770, 769
764, 771, 770
765, 772, 771
766, 773, 772
767, 774, 773
113, 112, 774
768, 775, 270
769, 776, 775
770, 777, 776
771, 778, 777
772, 779, 778
773, 780, 779
774, 781, 780
112, 111, 781
775, 782, 269

191

 

613,
614,
615,
616,
617,
618,
619,
620,
621,
622,
623,
624,
625,
626,
627,
628,
629,
630,
631,
632,
633,
634,
635,
636,
637,
638,
639,
640,
641,
642,
643,
644,
645,
646,
647,
648,
649,
650,
651,
652,
653,
654,
655,
656,
657,
658,
659,
660,
661,
662,
663,
664,
665,
666,
667,
668,
669,

775,
776,
777,
778,
779,
780,
781,
269,
782,
783,
784,
785,
786,
787,
788,
268,
789,
790,
791,
792,
793,
794,
795,
267,
796,
797,
798,
799,
800,
801,
802,
266,
803,
804,
805,
806,
807,
808,
809,
265,
810,
811,
812,
813,
814,
815,
816,
264,
817,
818,
819,
820,
821,
822,
823,
263,
824,

776,
777,
778,
779,
780,
781,
111,
782,
783,
784,
785,
786,
787,
788,
110,
789,
790,
791,
792,
793,
794,
795,
109,
796,
797,
798,
799,
800,
801,
802,
108,
803,
804,
805,
806,
807,
808,
809,
107,
810,
811,
812,
813,
814,
815,
816,
106,
817,
818,
819,
820,
821,
822,
823,
105,
824,
825,

783,
784,
785,
786,
787,
788,
110,
789,
790,
791,
792,
793,
794,
795,
109,
796,
797,
798,
799,
800,
801,
802,
108,
803,
804,
805,
806,
807,
808,
809,
107,
810,
811,
812,
813,
814,
815,
816,
106,
817,
818,
819,
820,
821,
822,
823,
105,
824,
825,
826,
827,
828,
829,
830,
104,
831,
832,

782
783
784
785
786
787
788
268
789
790
791
792
793
794
795
267
796
797
798
799
800
801
802
266
803
804
805
806
807
808
809
265
810
811
812
813
814
815
816
264
817
818
819
820
821
822
823
263
824
825
826
827
828
829
830
262
831

670,
671,
672,
673,
674,
675,
676,
677,
678,
679,
680,
681,
682,
683,
684,
685,
686,
687,
688,
689,
690,
691,
692,
693,
694,
695,
696,
697,
698,
699,
700,
701,
702,
703,
704,
705,
706,
707,
708,
709,
710,
711,
712,
713,
714,
715,
716,
717,
718,
719,
720,
721,
722,
723,
724,
725,
726,

825,
826,
827,
828,
829,
830,
262,
831,
832,
833,
834,
835,
836,
837,
261,
838,
839,
840,
841,
842,
843,
844,
209,
540,
541,
542,
543,
544,
545,
546,
208,
845,
846,
847,
848,
849,
850,
851,
207,
852,
853,
854,
855,
856,
857,
858,
206,
859,
860,
861,
862,
863,
864,
865,
205,
866,
867,

826, 833, 832
827, 834, 833
828, 835, 834
829, 836, 835
830, 837, 836
104, 103, 837
831, 838, 261
832, 839, 838
833, 840, 839
834, 841, 840
835, 842, 841
836, 843, 842
837, 844, 843
103, 102, 844
838, 539, 260
839, 538, 539
840, 537, 538
841, 536, 537
842, 535, 536
843, 534, 535
844, 533, 534
102, 4, 533

540, 845, 208
541, 846, 845
542, 847, 846
543, 848, 847
544, 849, 848
545, 850, 849
546, 851, 850
8, 91, 851

845, 852, 207
846, 853, 852
847, 854, 853
848, 855, 854
849, 856, 855
850, 857, 856
851, 858, 857
91, 90, 858

852, 859, 206
853, 860, 859
854, 861, 860
855, 862, 861
856, 863, 862
857, 864, 863
858, 865, 864
90, 89, 865

859, 866, 205
860, 867, 866
861, 868, 867
862, 869, 868
863, 870, 869
864, 871, 870
865, 872, 871
89, 88, 872

866, 873, 204
867, 874, 873
868, 875, 874

 

727,
728,
729,
730,
731,
732,
733,
734,
735,
736,
737,
738,
739,
740,
741,
742,
743,
744,
745,
746,
747,
748,
749,
750,
751,
752,
753,
754,
755,
756,
757,
758,
759,
760,
761,
762,
763,
764,
765,
766,
767,
768,
769,
770,
771,
772,
773,
774,
775,
776,
777,
778,
779,
780,
781,
782,
783,

868,
869,
870,
871,
872,
204,
873,
874,
875,
876,
877,
878,
879,
203,
880,
881,
882,
883,
884,
885,
886,
202,
887,
888,
889,
890,
891,
892,
893,
201,
894,
895,
896,
897,
898,
899,
900,
200,
901,
902,
903,
904,
905,
906,
907,
199,
908,
909,
910,
911,
912,
913,
914,
198,
915,
916,
917,

869,
870,
871,
872,
88,

873,
874,
875,
876,
877,
878,
879,
87,

880,
881,
882,
883,
884,
885,
886,
86,

887,
888,
889,
890,
891,
892,
893,
85,

894,
895,
896,
897,
898,
899,
900,
84,

901,
902,
903,
904,
905,
906,
907,
83,

908,
909,
910,
911,
912,
913,
914,
82,

915,
916,
917,
918,

192

87,

86,

85,

84,

83,

82,

81,

876,
877,
878,
879,

880,
881,
882,
883,
884,
885,
886,

887,
888,
889,
890,
891,
892,
893,

894,
895,
896,
897,
898,
899,
900,

901,
902,
903,
904,
905,
906,
907,

908,
909,
910,
911,
912,
913,
914,

915,
916,
917,
918,
919,
920,
921,

922,
923,
924,
925,

875
876
877
878

879

203
880
881
882
883
884
885

886

202
887
888
889
890
891
892

893

201
894
895
896
897
898
899

900

200
901
902
903
904
905
906

907

199
908
909
910
911
912
913

914

198
915
916
917
918
919
920

921

197
922
923
924

 

784,
785,
786,
787,
788,
789,
790,
791,
792,
793,
794,
795,
796,
797,
798,
799,
800,
801,
802,
803,
804,
805,
806,
807,
808,
809,
810,
811,
812,
813,
814,
815,
816,
817,
818,
819,
820,
821,
822,
823,
824,
825,
826,
827,
828,
829,
830,
831,
832,
833,
834,
835,
836,
837,
838,
839,
840,

918,
919,
920,
921,
197,
922,
923,
924,
925,
926,
927,
928,
196,
929,
930,
931,
932,
933,
934,
935,
195,
936,
937,
938,
939,
940,
941,
942,
194,
943,
944,
945,
946,
947,
948,
949,
551,
552,
553,
554,
555,
556,
557,
558,
960,
967,
968,
969,
970,
971,
972,
973,
959,
974,
975,
976,
977,

919,
920,
921,
81,

922,
923,
924,
925,
926,
927,
928,
80,

929,
930,
931,
932,
933,
934,
935,
79,

936,
937,
938,
939,
940,
941,
942,
78,

943,
944,
945,
946,
947,
948,
949,
77,

552,
553,
554,
555,
556,
557,
558,
502,
967,
968,
969,
970,
971,
972,
973,
559,
974,
975,
976,
977,
978,

926,
927,
928,
80,
929,
930,
931,
932,
933,
934,
935,
79,
936,
937,
938,
939,
940,
941,
942,
78,
943,
944,
945,
946,
947,
948,
949,
77,
526,
527,
528,
529,
530,
531,
532,
5,
967,
968,
969,
970,
971,
972,
973,
559,
974,
975,
976,
977,
978,
979,
980,
560,
981,
982,
983,
984,
985,

532

925
926
927

928

196
929
930
931
932
933
934

935

195
936
937
938
939
940
941

942

194
943
944
945
946
947
948

949

193
526
527
528
529
530
531

960
967
968
969
970
971
972
973
959
974
975
976
977
978
979
980
958
981
982
983
984

841, 978, 979, 986, 985
842, 979, 980, 987, 986
843, 980, 560, 561, 987
844, 958, 981, 957, 950
845, 981, 982, 956, 957
846, 982, 983, 955, 956
847, 983, 984, 954, 955
848, 984, 985, 953, 954
849, 985, 986, 952, 953
850, 986, 987, 951, 952
851, 987, 561, 562, 951
852, 562, 563, 988, 951
853, 563, 564, 989, 988
854, 564, 251, 252, 989
855, 951, 988, 990, 952
856, 988, 989, 991, 990
857, 989, 252, 253, 991
858, 952, 990, 992, 953
859, 990, 991, 993, 992
860, 991, 253, 254, 993
861, 953, 992, 994, 954
862, 992, 993, 995, 994
863, 993, 254, 255, 995
864, 954, 994, 996, 955
865, 994, 995, 997, 996
866, 995, 255, 256, 997
867, 955, 996, 998, 956
868, 996, 997, 999, 998
869, 997, 256, 257, 999

870,956,998,1000,957
871,998,999,1001,1000
872,999,257,258,1001
873,957,1000,962,950
874,1000,1001,961,962
875,1001,258,259,961
876,259,260,539,961
877,961,539,538,962
878,962,538,537,950
879,537,536,966,950
880,536,535,965,966
881,535,534,964,965
882,534,533,963,964
883,533,4,101,963
884,101,100,1002,963
885,100,99,1003,1002
886,99,98,1004,1003
887,98,97,547,1004
888,963,1002,1005,964
889,1002,1003,1006,1005
890,1003,1004,1007,1006
891,1004,547,548,1007
892,964,1005,1008,965
893,1005,1006,1009,1008
894,1006,1007,1010,1009
895,1007,548,549,1010
896,965,1008,1011,966
897,1008,1009,1012,1011

 

898,1009,1010,1013,1012
899,1010,549,550,1013
900,966,101l,958,950
901,1011,1012,959,958
902,1012,1013,960,959
903,1013,550,551,960
904,210,211,1031,1021
905,211,212,1032,1031
906,212,213,1033,1032
907,213,214,1034,1033
908,214,215,1035,1034
909,215,216,1036,1035
910,216,217,565,1036
911,1021,1031,1020,1014
912,1031,1032,1019,1020
913,1032,1033,1018,1019
914,1033,1034,1017,1018
915,1034,1035,1016,1017
916,1035,1036,1015,1016
917,1036,565,566,1015
918,566,567,1037,1015
919,567,568,1038,1037
920,568,569,1039,1038
921,569,570,1040,1039
922,570,502,558,1040
923,1015,1037,1041,1016
924,1037,1038,1042,1041
925,1038,1039,1043,1042
926,1039,1040,1044,1043
927,1040,558,557,1044
928,1016,1041,1045,1017
929,1041,1042,1046,1045
930,1042,1043,1047,1046
931,1043,1044,1048,1047
932,1044,557,556,1048
933,1017,1045,1049,1018
934,1045,1046,1050,1049
935,1046,1047,1051,1050
936,1047,1048,1052,1051
937,1048,556,555,1052
938,1018,1049,1053,1019
939,1049,1050,1054,1053
940,1050,1051,1055,1054
941,1051,1052,1056,1055
942,1052,555,554,1056
943,1019,1053,1057,1020
944,1053,1054,1058,1057
945,1054,1055,1059,1058
946,1055,1056,1060,1059
947,1056,554,553,1060
948,1020,1057,1025,1014
949,1057,1058,1024,1025
950,1058,1059,1023,1024
951,1059,1060,1022,1023
952,1060,553,552,1022
953,552,551,1061,1022
954,551,550,1062,1061

193

 

955,550,549,1063,1062
956,549,548,1064,1063
957,548,547,1065,1064
958,547,97,96,1065
959,1022,1061,1066,1023
960,1061,1062,1067,1066
961,1062,1063,1068,1067
962,1063,1064,1069,1068
963,1064,1065,1070,1069
964,1065,96,95,1070
965,1023,1066,1071,1024
966,1066,1067,1072,1071
967,1067,1068,1073,1072
968,1068,1069,1074,1073
969,1069,1070,1075,1074
970,1070,95,94,1075
971,1024,1071,1076,1025
972,1071,1072,1077,1076
973,1072,1073,1078,1077
974,1073,1074,1079,1078
975,1074,1075,1080,1079
976,1075,94,93,1080
977,1025,1076,1030,1014
978,1076,1077,1029,1030
979,1077,1078,1028,1029
980,1078,1079,1027,1028
981,1079,1080,1026,1027
982,1080,93,92,1026
983,92,8,546,1026
984,1026,546,545,1027
985,1027,545,544,1028
986,1028,544,543,1029
987,1029,543,542,1030
988,1030,542,541,1014
989,541,540,1021,1014
990,540,209,210,1021
99l,217,218,1081,565
992,218,219,1082,1081
993,219,220,1083,1082
994,220,221,1084,1083
995,221,222,1085,1084
996,222,223,1086,1085
997,223,224,1087,1086
998,224,225,1088,1087
999,225,226,1089,1088
1000,226,227,1090,1089
1001,227,15,228,1090
1002,565,1081,1091,566
1003,1081,1082,1092,1091
1004,1082,1083,1093,1092
1005,1083,1084,1094,1093
1006,1084,1085,1095,1094
1007,1085,1086,1096,1095
1008,1086,1087,1097,1096
1009,1087,1088,1098,1097
1010,1088,1089,1099,1098
1011,1089,1090,1100,1099

1012,1090,228,229,1100
1013,566,109l,1101,567

1014,1091.
1015,1092.
1016,1093.
1017,1094.
1018,1095.
1019,1096.
1020,1097.
1021,1098.
1022,1099.

1092.
1093.
1094.
1095.
1096.
1097
1098.
1099.
1100.

1102,1101
1103,1102
1104,1103
1105,1104
1106,1105

.1107,1106

1108,1107
1109,1108
1110,1109

1023,1100,229,230,1110
1024,567,1101,1111,568

1025,1101.
1026,1102.
1027,1103.
1028,1104.
1029,1105,
1030,1106.
1031,1107.
1032,1108.
1033,1109,

1102.
1103.
1104
1105.
1106.
1107.
1108
1109.
1110.

.1114.

.1118.

1112.
1113.

1111
1112
1113
1114
1115
1116
1117
1118
1119

1115.
1116.
1117.

1119.
1120.

1034,1110,230,231,1120
1035,568,1111,1121,569

1036,1111.
1037,1112,
1038,1113.
1039,1114.
1040,1115.
1041,1116.
1042,1117.
1043,1118.
1044,1119.
1045,1120.

1047,1121.
1048,1122.
1049,1123.
1050,1124.
1051,1125.
1052,1126.
1053,1127.
1054,1128.
1055,1129.
1056,1130.

1058,1131.
1059,1132.
1060,1133.
1061,1134.
1062,1135.
1063,1136.
1064,1137.
1065,1138.
1066,1139.
1067,1140.

1112.
1113.
1114
1115.
1116.
1117
1118.
1119.
1120.

.1124.

.1127.

1122.
1123.

1121
1122
1123
1124
1125
1126
1127
1128
1129

1125.
1126.

1128.
1129.
1130.

231,232,1130
1046,569,1121,1131,570

1122.
1123.
1124
1125.
1126.
1127
1128.
1129.
1130.

1132,1131
1133,1132

.1134,1133

1135,1134
1136,1135

.1137.1136

1138,1137
1139,1138
1140,1139

232.233.1140
1057,570,1131,1141,502

1132.
1133.
1134.
1135.
1136.
1137.
1138.
1139.
1140.

1142,1141
1143,1142
1144,1143
1145,1144
1146,1145
1147,1146
1148,1147
1149,1148
1150,1149

233,234,1150
1068,502,1141,1151,559

 

1069.
1070.
1071.
1072.
1073.
1074.
1075.
1076.
1077,
1078.
1079.
1080.
1081.
1082.
1083.
1084.
1085.
1086.
1087.
1088.
1089.
1090.
1091.
1092.
1093.
1094.
1095.
1096.
1097.
1098.
1099.
1100.
1101.
1102.
1103.
1104.
1105.
1106.
1107.
1108.
1109.
1110.
1111.
1112.
1113.
1114.
1115.
1116.
1117.
1118.
1119.
1120.
1121.
1122.
1123.
1124.
1125.

1141.
1142.
1143.
1144.
1145.
1146.
1147.
1148.
1149.
1150.

1142.
1143.
1144
1145.
1146.
1147.
1148.
1149.
1150.

.1154.

1152.
1153.

1151
1152
1153
1154
1155
1156
1157
1158
1159

1155.
1156.
1157.
1158.
1159.
1160.

234,235,1160

559,1151,1161,560

1151.
1152.
1153.
1154.
1155.
1156.
1157,1158.
1158,1159.
1159,1160.

1152.
1153.
1154.

1156.

1155.

1157.

1162,1161
1163,1162
1164,1163
1165,1164
1166,1165
1167,1166
1168,1167
1169,1168
1170,1169

1160,235,236,1170
560,1161,1171.561

1161.
1162.
1163.
1164.
1165.
1166.
1167.
1168.
1169.
1170.

1162.
1163.
1164.
1165.
1166.
1167,
1168.
1169.
1170.

1172,1171
1173,1172
1174,1173
1175,1174
1176,1175
1177,1176
1178,1177
1179,1178
1180,1179

236,237,1180

561,1171,1181,562

1171.
1172.
1173.
1174.
1175,1176
1176,1177.
1177,1178.
1178,1179.
1179,1180.

1172.
1173.
1174.
1175.

1182,1181
1183,1182
1184,1183
1185,1184

.1186,1185

1187,1186
1188,1187
1189,1188
1190,1189

1180,237,238,1190
562,1181,1191,563

1181.
1182.
1183.
1184.
1185.
1186.
1187.
1188.
1189.
1190.

1182.
1183.
1184.
1185.
1186.
1187.
1188.
1189.
1190.

1192,1191
1193,1192
1194,1193
1195,1194
1196,1195
1197,1196
1198,1197
1199,1198
1200,1199

238,239,1200

563,1191,1201,564

1191,1192.
1192,1193,

194

1202,1201
1203,1202

 

1126,1193,1194,1204,1203
1127,1194,1195,1205,1204
1128,1195,1196,1206,1205
1129,1196,1197,1207,1206
1130,1197,1198,1208,1207
1131,1198,1199,1209,1208
1132,1199,1200,1210,1209
1133,1200,239,240,1210
1134,564,1201,250,251
1135,1201,1202,249,250
1136,1202,1203,248,249
1137,1203,1204,247,248
1138,1204,1205,246,247
1139,1205,1206,245,246
ll40,1206,1207,244,245
1141,1207,1208,243,244
1142,1208,1209,242,243
1143,1209,1210,241,242
1144,1210,240,16,241
*Element, type=SPRINGA,
Elset=MedLow

1145, 13, 16

*Element, type=SPRINGA,
Elset=Low
1146, 14,
*Spring,
nonlinear

15
Elset=MedLow,

O, O

0, 0.0032
400, 20
*Spring,
nonlinear

Elset=Low,

0,0
0, 0.0006
400, 20
*Orientation,name=0ri-3
1., 0., 0.,

0., 1., 0.
2, 0.
** Region: (PCM-
Section:Picked),
(Material
OrientationzPicked)
*Elset, elset=_I1,
generate

1, 162, 1
** Section: PCM-Section
*Solid Section,
elset=_I1,
orientation=Ori-3,
material=YINPCM
1.,
** Region: (Cell—
Section:Picked),

 

 

(Material
Orientation:Picked)
*Elset, elset=_12,
generate

163, 288, 1

** Section: Cell—
Section

*Solid Section,
elset=_I2,
orientation=Ori—3,
materia1=YINCELL
1.,

** Region: (membrane-
sectionzPicked),
(Material
OrientationzPicked)
*Elset, elset=_I3,
generate

289, 342, 1

** Section: membrane—
section

*Solid Section,
elset=_I3,
orientation=Ori-3,
material=YINMEM

1.,

** Region: (ECM—
Section:Picked),

(Material
OrientationzPicked)
*Elset, elset=_I4,
generate

343, 1144, 1

** Section: ECM-Section
*Solid Section,
elset=_I4,

orientation=Ori—3,
material=YINMatrix

elset=shearstress

1.,
*Elset,
289, 290,
293, 294,
297, 298,
301, 302,
305, 306,
309, 310,
313, 314,
317, 318,
321, 322,
324, 325,
328, 329,
332, 333,
336, 337,
340, 341,
6, 9,
21, 24,

291,
295,
299,
303,
307,
311,
315,
319,
323,
326,
330,
334,
338,
342,
12,
27,

292,
296,
300,
304
308,
312,
316,
320
323,
327,
331,
335
339,
3.
15,
30,

18,
33,

 

36, 39, 42, 45, 48,
51, 54, 57, 60, 63,
64, 67, 70, 73, 76,
79, 82, 85, 88, 91,
94, 97, 100, 105, 108,
111, 114, 117, 120,
123, 126, 129, 132,
135, 138, 141, 144,
147, 150, 153, 156,
159, 162
*Nset, nset=NSUB,
generate
1, 1210, 1
*Elset, elset=NSUB,
generate
1, 1144, 1
*Nset, nset=_bottom,
generate
13, 14, 1
*Nset,
nset=_PickedSet76
13, 14, 15, 16,
167, 168, 169, 170,
171, 172, 173, 174,
175, 176, 177, 178
179, 180, 181, 182,
183, 184, 185, 186,
187, 188, 189, 190,
191, 192, 193, 194
195, 196, 197, 198,
199, 200, 201, 202,
203, 204, 205, 206,
207, 208, 209, 210
211, 212, 213, 214,
215, 216, 217, 218,
219, 220, 221, 222,
223, 224, 225, 226
227, 228, 229, 230,
231, 232, 233, 234,
235, 236, 237, 238,
239, 240, 241, 242
243, 244, 245, 246,
247, 248, 249, 250,
251, 252, 253, 254,
255, 256, 257, 258
259, 260, 261, 262,
263, 264, 265, 266,
267, 268, 269, 270,
271, 272, 273, 274
275, 276, 277, 278,
279, 280, 281, 282,
283, 284, 285, 286,
287, 288, 289, 290,
291, 292
*Elset,

elset=_PickedSet76

195

343, 344,
347, 348,
351, 352,
375, 386,
419, 430,
443, 444,
447, 448,
451, 452,
455, 505,
520, 523,
530, 531,
572, 580,
604, 612,
636, 644,
668, 676,
700, 708,
732, 740,
764, 772,
796, 804,
857, 860,
869, 872,
904, 905,
908, 909,
991, 992,
995, 996,
999, 1000,
1023, 1034,
1067, 1078,
1111, 1122,
1135, 1136,
1139, 1140,
1143, 1144
*Submodel,

345,
349,
353,
397,
441,
445,
449,
453,
507,
526,
532,
588,
620,
652,
684,
716,
748,
780,
812,
863,
875,
906,
910,
993,
997,

1001,

1045,
1089,
1133,
1137,
1141,

346,
350,
364,
408,
442,
446,
450,
454,
509,
529,
564,
596,
628,
660,
692,
724,
756,
788,
854,
866,
876,
907,
990,
994,
998,

1012,

1056,
1100,
1134,
1138,
1142,

exteriorTolerance=0.01

**

_PickedSet76,

 

 

** MATERIALS

*Material, name=YINCELL
*Elastic

0.5, 0.069
*Permeability,
specific=9.81e~06
4.5e-09,2.
*Material,
*Elastic
1., 0.49
*Permeability,
Specific=9.81e—O6

5e-14,2.
*Material,
name=YINMatrix
*Elastic,
type=ENGINEERING
CONSTANTS

0.0457, 1., 0.0457,
0.07769, 0.7, 1.7,
0.5, 0.013441, 0.5,

name=YINMEM

 

*Permeability,
specific=9.81e-O6
3.1392e-09,2.

*Material, name=YINPCM
*Elastic
1., 0.49
*Permeability,
specific=9.81e—06
4e-09,2.
*Material,
name=membrane
*Elastic,
type=ENGINEERING
CONSTANTS
1., 70., 1.,
0.01, 0.3, 0.7,
5., 0.385, 5.,
*Permeability,
specific=9.81e-06
9.81e—08,2.

*INITIAL CONDITIONS,
TYPE=RATIO

NSUB, 2.

*Boundary

_bottom, 3, 3

**

** 1% strain at 2%
strain/minute

** STEP: Step—1

**
*Step,
nlgeom,
inc=1000

*Soils, consolidation,
end=PERIOD

5., 30., , ,

*El Print,
elset=shearstress,
FREQUENCY=30

COORD, FLVEL
*CONTROLS,
PARAMETERS=FIELD,
FIELD=PORE FLUID
PRESSURE

,,0.001

**

name=Step-l,
amplitude=RAMP,

** _____________________
**
** BOUNDARY CONDITIONS

**

** Name:

Submodel

*Boundary, submodel,
step=1, timescale
_PickedSet76, 1, 1
_PickedSet76, 2, 2

Sub Type:

 

_PickedSet76, 8, 8

**

** OUTPUT REQUESTS

**

*Restart, write,
frequency=1

**

** FIELD OUTPUT: F—
Output-1

**

*Output, field
*Node Output

U, RF, POR

*Element Output
S, LE, VOIDR, SAT,
FLVEL, COORD

**

** HISTORY OUTPUT: H-
Output-1

**

*Output, history,
variab1e=PRESELECT

*El Print, freq=999999

*Node Print,
freq=999999
*FILE FORMAT,
INCREMENT
*End Step

ZERO

B.6 Submodel for 1%
strain at 20% strain/min

**

** ....................
**

** STEP: Step—l

**

*Step, name=Step-1,
nlgeom, amplitude=RAMP,
inc=1000

*Soils, consolidation,
end=PERIOD

0.1, 3., , ,

*El Print,

elset=shearstress,
FREQUENCY;3O
COORD, FLVEL
*CONTROLS,
PARAMETERS=FIELD,
FIELD=PORE FLUID
PRESSURE

196

 

B.7 Submodel for 3%
strain at 6% strain/minute

**

** ____________________
**

** STEP: Step-1

**

*Step, name=Step-1,
nlgeom, amplitude=RAMP,
inc=1000

*Soils, consolidation,
end=PERIOD

1., 30., , ,

*El Print,

elset=shearstress,
FREQUENCY=30
COORD, FLVEL
*CONTROLS,
PARAMETERS=FIELD,
FIELD=PORE FLUID
PRESSURE

B.8 Submodel for 3%
strain at 2% strain/minute

**

** STEP: Step-l
**
*Step,
nlgeom,
inc=1000

*Soils, consolidation,
end=PERIOD

1., 130., , ,

*El Print,
elset=shearstress,
FREQUENCY=30

COORD, FLVEL
*CONTROLS,
PARAMETERS=FIELD,
FIELD=PORE FLUID
PRESSURE

name=Step—l,
amplitude=RAMP,

_--——_--—_-_-—--_-—_

 

TE

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