J Appl Physiol 94: 314324, 2003.

First published September 20, 2002; 10.1152/japplphysiol.00340.2002.

Hindlimb unloading alters ligament healing

PAOLO P. PROVENZANO,1 DANIEL A. MARTINEZ,2 RICHARD E. GRINDELAND,3
KELLEY W. DWYER,1 JOANNE TURNER,1 ARTHUR C. VAILAS,2 AND RAY VANDERBY, JR.1
1
Orthopedic Research Laboratories, Department of Orthopedics and Rehabilitation and Department
of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin 53792-3228; 2Connective
Tissue Physiology Laboratory, Department of Biology and Biochemistry, University of Houston,
Houston, Texas 77204; and 3Life Sciences Research Division, National Aeronautics and Space
Administration-Ames Research Center, Moffett Field, California 94035
Submitted 15 April 2002; accepted in final form 2 September 2002

Provenzano, Paolo P., Daniel A. Martinez, Richard
E. Grindeland, Kelley W. Dwyer, Joanne Turner,
Arthur C. Vailas, and Ray Vanderby, Jr. Hindlimb un-
loading alters ligament healing. J Appl Physiol 94: 314324,
2003. First published September 20, 2002; 10.1152/jappl-
physiol.00340.2002.We investigated the hypothesis that
musculoskeletal system can result in a profound de-
cline in functional ability as well as increased risk of
future pathology (24, 25, 30, 50). Of further concern is
that remobilization does not ensure complete recovery
of preimmobilization mechanical properties of liga-

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hindlimb unloading inhibits healing in fibrous connective
tissue such as ligament. Male rats were assigned to 3- and
7-wk treatment groups with three subgroups each: sham
control, ambulatory healing, and hindlimb-suspended heal-
ing. Ambulatory and suspended animals underwent surgical
rupture of their medial collateral ligaments, whereas sham
surgeries were performed on control animals. After 3 or 7 wk,
mechanical and/or morphological properties were measured
in ligament, muscle, and bone. During mechanical testing,
most suspended ligaments failed in the scar region, indicat-
ing the greatest impairment was to ligament and not to
bone-ligament insertion. Ligament testing revealed signifi-
cant reductions in maximum force, ultimate stress, elastic
modulus, and low-load properties in suspended animals. In
addition, femoral mineral density, femoral strength, gastroc-
nemius mass, and tibialis anterior mass were significantly
reduced. Microscopy revealed abnormal scar formation and
cell distribution in suspended ligaments with extracellular
matrix discontinuities and voids between misaligned, but
well-formed, collagen fiber bundles. Hence, stress levels from
ambulation appear unnecessary for formation of fiber bun-
dles yet required for collagen to form structurally competent
continuous fibers. Results support our hypothesis that hind-
limb unloading impairs healing of fibrous connective tissue.
In addition, this study provides compelling morphological
evidence explaining the altered structure-function relation-
ship in load-deprived healing connective tissue.
hindlimb suspension; biomechanics; disuse; collagen; bone;
muscle
STRESS REDUCTIONS DUE TO MICROGRAVITY (7, 14, 27, 33, 41,
48, 52, 57), hindlimb unloading (2, 8, 18, 47, 49, 51),
bed rest (6, 28, 29, 43, 46), or immobilization (1, 35,
13, 53) result in suppression of skeletal and connective
tissue homeostasis and formation and a reduction in
their mechanical properties. These reductions in the
mentous tissue (53). Compounding this problematic
decline of normal tissue under stress reduction is the
fact that wound healing is also impaired under micro-
gravity and immobilization (15, 19, 23, 26, 44). These
issues become more problematic as the duration of
prolonged spaceflight increases and the probability of
prolonged bed rest grows with the expanding aged
population. Methods to understand and counteract
musculoskeletal degeneration and impaired wound
healing require further study.
Stress reduction is known to adversely affect fibrous
connective tissues, such as ligament and tendon.
Stress shielding in normal patellar tendon results in a
decrease in tensile strength and elastic modulus (31,
36, 59). In addition, immobilization in the form of rigid,
external joint fixation is reported to result in lower
ultimate load and stiffness in healing rat Achilles ten-
don compared with transected tendons without immo-
bilization. In rhesus monkeys, Noyes (35) examined
the effects of 8 wk of immobilization (whole body cast-
ing) and 5 and 12 mo of reconditioning on anterior
cruciate ligaments. Results of his study indicate a
significant decrease in ultimate load in immobilized
ligaments at 8 wk. Neither 5 nor 12 mo of recondition-
ing resulted in full recovery of anterior cruciate liga-
ment ultimate load. Amiel and colleagues (3) studied
biochemical changes associated with joint immobiliza-
tion by using a stainless steel pin to immobilize the
knee joint of rabbits. One end of the pin was inserted
into the tibia while the other end was hooked over the
proximal end of the femur. After pin fixation, the ani-
mals were allowed unrestricted cage activity. This pro-
tocol substantially immobilized the joints; however, it
did not completely shield the joint from load. Results of
their study indicate a reduction in total collagen, in-
creased collagen turnover, and increased collagen syn-

Address for reprint requests and other correspondence: R. The costs of publication of this article were defrayed in part by the
Vanderby, Jr., Dept. of Orthopedic Surgery, Orthopedic Research payment of page charges. The article must therefore be hereby
Laboratories, 600 Highland Ave., Univ. of Wisconsin, Madison, WI marked advertisement in accordance with 18 U.S.C. Section 1734
53792-3228 (E-mail: vanderby@surgery.wisc.edu). solely to indicate this fact.

314 8750-7587/03 $5.00 Copyright 2003 the American Physiological Society http://www.jap.org
 HINDLIMB UNLOADING ALTERS CONNECTIVE TISSUE HEALING
thesis and degradation rate, with degradation rate
exceeding the synthesis rate. Using the same pin tech-
nique, Woo et al. (53) later examined the effect of
immobilization on rabbit medial collateral ligaments
(MCLs). Nine and twelve weeks of immobilization re-
sult in a significant reduction in ultimate load, energy
at failure, and changes in failure mode. Although these
studies examine joint immobilization, they do not ex-
amine the effect of substantial joint stress reduction on
the healing ligament.
To simulate weightlessness, the National Aeronau-
tics and Space Administration (NASA)-Ames institute
developed the hindlimb suspension model (56, 58).
Hindlimb suspension provides a noninvasive model for
tissue disuse. Like microgravity or confined bed rest,
this model eliminates weight bearing and tissue load-
ing from ground reaction forces during mobilization,
which allows substantial hindlimb stress reduction.
Hindlimb suspension for 3 wk reduces the ultimate
stress and tangential modulus of rat Achilles tendon
(2). In addition, Vanderby et al. (51) examined rat
hindlimbs after 7 days of hindlimb suspension and
reported reduced body weight, reduced soleus muscle
mass, as well as reduced ultimate load and stress in
the MCL. Significantly reduced MCL ultimate load,
stiffness, and stress were also present after 2 wk of
hindlimb suspension in rat (49). Although the above
studies have examined changes in normal tendon and
ligament after hindlimb suspension, they do not exam-
ine the effect of hindlimb suspension (i.e., complete loss
of ground reaction force) on healing fibrous connective
tissues such as ligament.
The purpose of this study is to test the hypothesis
that stress reduction through hindlimb unloading de-
lays and impairs the wound-healing response in fi-
brous connective tissue, such as ligament. To examine
this hypothesis, changes in the mechanical properties
and microstructural morphology of healing fibrous con-
nective tissue will be studied by using a rat MCL
model. Mechanical properties are examined at both the
low-load physiological range and failure range. Histol-
ogy and scanning electron microscopy (SEM) are per-
formed to elucidate information regarding the struc-
ture-function relationship of the ligament tissue. In
addition, changes in hindlimb muscle and bone, as well
as total body mass, are examined to more completely
show the physiological effects of hindlimb suspension.
MATERIALS AND METHODS
Animal preparation. This study was approved by the in-
stitutional animal use and care committee and meets Na-
tional Institutes of Health guidelines for animal welfare.
Sixty male Sprague-Dawley rats (245 5 g) were used as an
animal model. These animals were divided into two time
groups (3- and 7-wk healing), each consisting of 30 rats. Each
time group was composed of three subgroups, each contain-
ing 10 randomly selected animals: sham control, ambulatory
healing, and hindlimb suspended healing. Each rat in the
ambulatory healing and hindlimb suspension groups under-
went bilateral disruption of the MCL. Experimental results
show that mechanical properties in healing MCLs are equiv-
alent after either unilateral or bilateral surgical disruption
J Appl Physiol VOL 94 JANUARY 2003
315
(45). Rats were anesthetized with an isofluorane anesthetic
administered by face mask by using a nonrebreathing deliv-
ery system. The incision area was clipped and aseptically
prepared for surgery. All surgeries were performed asepti-
cally. A skin incision 8 mm long was made on the medial
side of the stifle, and fascia was incised to expose the MCL.
The MCL was exposed and transected at the joint line. The
wounds were closed with suture. Sham control animals were
subjected to identical surgical procedures without MCL rup-
ture. All animals were given an analgesic (Tylenol/codeine) in
their water for 72 h postsurgery. Sham control and ambula-
tory healing animals were allowed unrestricted cage move-
ment. Hindlimb suspended healing rats were subjected to
hindlimb unweighting (24 h after surgery), which induced
stress reduction by eliminating ground reaction force by
using the noninvasive tail suspension protocol of the NASA-
Ames center (32, 34, 56, 58). Only the hindlimbs were sus-
pended, and the animals were allowed to move freely within
the cages by using the forelimbs while being restricted from
kicking the sides of the cages. All animals were under a
12:12-h light-dark cycle in a room at 24C. Rats were fed
Purina rat chow, watered ad libitum, and checked twice daily
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for overall health, skin incision healing, food and water
consumption, and the condition of their tails (the harness
should prevent slippage without restricting circulation). Af-
ter either 3 or 7 wk, the animals were euthanized. Of the 20
ligaments per group, 6 were separated for a separate unre-
lated study and will not be included here. The remaining 14
ligaments were distributed as follows. In each of the three
treatment groups for each time point, six ligaments were
used for biomechanical testing, four were used for histology,
and four were used for electron microscopy.
Tissue harvest and biomechanical testing. Immediately
after death, animal hindlimbs were placed in plastic bags
and stored at 80C until testing. It has been shown that
postmortem storage by freezing does not change the biome-
chanical properties of ligament (55). On the day of testing,
hindlimbs were thawed at room temperature. Tissue harvest
and testing were performed by using methods similar to
those previously described (38, 40). Extraneous tissue was
carefully dissected away to expose each MCL. The femur-
MCL-tibia complex was then removed, with care taken not to
disturb the ligament insertion sites. Ligaments were kept
moist in Hanks physiological solution at 25C to prevent
dehydration (pH 7.4), and cross-sectional area was mea-
sured optically. The femur-MCL-tibia complex was then
placed into a custom-designed tissue bath system with spe-
cial structures to hold the femur and tibial sections of the
sample along the longitudinal axis of the MCL where all
fibers appear to load simultaneously (70 flexion). Optical
markers (silicone-impregnated grease) were placed onto the
ligament tissue near the insertion sites for strain measure-
ment. The tissue bath containing the MCL samples was
inserted into our custom-designed testing machine. A small
preload of 0.1 N was applied to obtain a uniform zero point
(i.e., to start the tests from the same relative position). In
displacement control, ligaments were preconditioned (1%
strain for 10 cycles) and pulled to failure at 10%/s. Strains
and strain rates were calculated from videotape (described
below) after displacement controlled tests. Strain rates were
consistently within 5% of the target value. During and after
the test, the failure location was recorded and examined.
Digital images of the location of structural failure were
analyzed to asses failure location. For the case of a potential
tibial avulsion, the ruptured tibial end of the tissue was
examined for bone.
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316 HINDLIMB UNLOADING ALTERS CONNECTIVE TISSUE HEALING
Tissue displacement was obtained by using video dimen-
sional analysis with 10-m resolution (at this level of mag-
nification) to measure the change in distance between mark-
ers. The change in distance between optical markers was
calculated by analyzing stored digital frames with NIH Im-
age software by using a custom macro to calculate the change
in x-y coordinate center (centroid coordinates) of each
marker. Force (resolution of 0.005 N) was displayed on the
video screen and synchronized with displacement. From dis-
placement data, strain was calculated as the change between
stretched length and gauge length [8.5 0.4 mm (mean
SD) at preload] divided by gauge length. Stress was calcu-
lated as the force divided by the initial area. Stress-strain
curves were created and fit with the microstructural model
presented by Hurschler and co-workers (21). Biomechanical
parameters for comparison (n 6 ligaments per group) are
ultimate force, ultimate stress, strain at failure, elastic mod-
ulus (determined from the microstructural model), and stress
and stretch at the toe-to-linear region transition (described
below).
Modeling physiological low-load behavior. The ligament
stress-strain curve displays nonlinear strain-stiffening be-
havior in the toe region, after which a more linear region is
encountered. The model described in this section quantifies
the transition from the toe region to the linear region. This
model has been previously described in detail elsewhere (22).
A brief description will be included here. Stress-stretch data
were fit with the probabilistic microstructural model of Hur-
schler et al. (21), which utilized a Weibull probability density
function (PDF) to represent collagen fiber recruitment and,
hence, load-bearing capability as the tissue is stretched and
the characteristic crimp pattern of the collagen fiber is re-
moved. The model has the form
t
tt Pws33t/sds, t (1)
where Pw is the Weibull PDF as a function of the straight-
ening stretch ratio, defined as the tissue stretch (s) at which
an individual fiber begins to bear load (s fiber length/
tissue reference length), 33 is the longitudinal normal stress
in a fiber, and is the location parameter of the Weibull
distribution and defines the onset of fiber loading (22). Hence,
the stress in the tissue is determined by the overall tissue
stretch (stretch ratio t deformed tissue length/tissue
reference length) and the load-bearing contribution of the
fiber population. After the data have been well described
(R2 0.98) by the model, parameters from the Weibull PDF
are input into the Weibull cumulative distribution function
(CDF), F()
1
t t
s s
Ft Psds exp
where and are the shape and scale parameters of the
Weibull distribution. After integration, Eq. 2 can be algebra-
ically manipulated to yield the inverse CDF
t ln1 F1/
which defines the stretch ratio at which any fraction of the
fibers (F) have been recruited. Based on preliminary studies,
the 85th percentile provides a good representation of com-
plete fiber recruitment and will be used in this study. Hence,
from Eq. 3, we are able to determine the stretch (and hence
J Appl Physiol VOL
stress) at which the tissue exits the nonlinear strain-stiffen-
ing toe region and enters a more linear portion of the curve.
Preparation for histology and SEM. Immediately after
tissue harvest, samples for histology (n 4 ligaments per
group) were fixed in formalin. After standard histology pro-
cedure, 10-m sections underwent hemotoxylin and eosin
staining. Slides were coverslipped and viewed with light
microscopy. Ligament preparation for SEM (n 4 ligaments
per group) was similar to that previously described (39).
MCLs were exposed, and tissues were drip fixed with 2.5%
glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4;
GSCB, Electron Microscopy Sciences, Fort Washington, PA)
at the same knee angle as mechanical testing. After drip
fixation, MCLs were removed and soaked in fixation solution
for at least 1 h. The ligaments were then dehydrated through
a series of ethanol/H2O solutions (30, 50, 75, 90, 100, and
100% ethanol). After dehydration, MCLs were immersed in
liquid nitrogen and subsequently placed on a precooled mi-
croscope slide. Under a dissecting scope, MCL fracture was
initiated by using a microsurgical scalpel blade. The frozen
specimens were carefully fractured in the sagittal plane of
the ligament from the femoral to tibial end, and the femoral
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end was marked for reference. Samples were then critical
point dried (Samdri model 780A, Tousimis Research, Rock-
ville, MA), mounted on 10-mm SEM mounting blocks (JEOL
840, SPI Supplies, Structure Probe, West Chester, PA), gold-
palladium sputter coated to 195 angstroms, and stored in a
vacuum container. The ligaments were imaged with SEM
(JOEL-JSM 6100). At low magnification (approximately
25), the ligament was aligned by orienting the femoral end
of the ligament to the top of the screen. The residual and scar
regions were identified in all healing ligaments. At a magni-
fication of 250, the morphology of normal and scar tissue
was examined, and images were stored digitally.
Analysis of bone mineral density and strength. To assess
how injury and disuse affected bone morphology and strength,
the rat femur was examined. In six limbs per group, from
separate animals that were not used for biomechanical test-
ing, femurs were carefully harvested and kept hydrated with
PBS (1) solution. A Noland XCT Research M system
(STRATEC XCT-960A pQCT, Stratec Medizintechnik GmbH,
Pforzheim, Germany), which employs an X-ray source (47
kVp, 40 keV, beam width 8 keV, 0.3 mA) that produces a fan
beam with an effective width of 2.5 mm, was used to charac-
terize geometry and bone mineral densities of the femurs.
The samples were placed in a custom-designed apparatus to
hold the bones horizontally in the machine while being
scanned by using voxel size E (148 m). The femurs were
scanned at midshaft and at the widest point of the femoral
head. The scan lines were modified by using the scout view
feature. Analysis was performed with separation mode 1, and
images were stored at a resolution of 640 480 pixels. Total
(cortical plus trabecular) bone mineral density was recorded
ds
at the femur midshaft and in the femoral head. To examine
bone strength, the above femurs were mounted in a custom
(2) frame and tested destructively in three-point bending. All
specimens were tested at room temperature at a rate of
0.1667 mm/s. Force was recorded by using Labtech Notebook
digital acquisition software. Maximum load was examined
for comparison between groups.
(3) Measurement of muscle weight. The gastrocnemius and
tibialis anterior muscles were examined. In six limbs per
group, from separate animals that were not used for biome-
chanical testing or SEM, muscles were carefully harvested
and kept hydrated with PBS (1) solution. These muscles
were taken from the same limbs as the femurs discussed in
Analysis of bone mineral density and strength. Muscles were
94 JANUARY 2003 www.jap.org
 HINDLIMB UNLOADING ALTERS CONNECTIVE TISSUE HEALING 317

Table 1. Failure location in rat groups

Failure Midsubstance Tibial Third of Tibial
Location Scar Region (Sham Only) the Ligament Avulsion

Week 3
Sham control N/A 1 5 0
Ambulatory
healing 5 N/A 1 0
Suspended
healing 5 N/A 0 1
Week 7
Sham control N/A 0 6 0
Ambulatory
healing 4 N/A 2 0
Suspended
Fig. 1. Body weight in rats for 3 wk after surgery. Note that body healing 4 N/A 1 1
weight was altered in hindlimb-suspended animals.
N/A, not applicable.

carefully blotted dry to remove any excess fluid and immedi-

ately weighed on a Metler balance scale. all healing ligaments showed a bridging of the rupture
Statistical analysis. Statistical analysis was performed by gap with translucent scar tissue. During MCL harvest

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using a two-way ANOVA followed by pairwise comparison to
examine treatment effect. Data were log transformed to bet-
ter conform to the assumptions of ANOVA. The level of
significance was set at 0.05, and analysis was performed with
SAS PROC MIXED (SAS Institute, Cary, NC). Fishers pro-
tected least significant difference test was used as the post
hoc multicomparison test.
RESULTS
There were no wound infections or other apparent
complications associated with surgery. All of the mobi-
lized animals (sham control and ambulatory healing)
returned to normal cage activity. The health of sus-
pended healing animals was carefully monitored, and
no complications with surgery or suspension were ob-
served. Initial body weights were carefully selected so
that the groups were not different. After surgery, body
weight in the control sham and ambulatory healing
groups proceeded naturally. However, suspended heal-
ing animals had an initial decrease in body weight
followed by attenuated gains in body weight increase
over the study period (Figs. 1 and 2). At tissue harvest
in the 3- and 7-wk suspended animals, tissue adhesion
was present, and in some animals the tissue surround-
ing the surgical site appeared to heal less thoroughly.
In addition, the femur and tibia were noticeably softer
and more fragile, requiring care during harvest of the
femur-MCL-tibia complex.
Examination of failure location in the ligaments re-
vealed that sham tissues fail primarily in the tibial
third of the ligament at both 3 and 7 wk, whereas in
healing tissues from both ambulatory and suspended
animals the majority of tissues failed in the scar region
(Table 1). Only one tissue per time group failed by
tibial avulsion in suspended animals, which indicated
that the dominant decrease in mechanical properties is
because of changes in ligamentous tissue and not the
bone-ligament insertion. Substantial differences in tis-
sue mechanical properties are present between sham

Fig. 3. A typical stress-stretch curve for sham control, ambulatory

healing, and suspended healing rat tissues at 3 wk. Each curve is fit
with the microstructural model of Hurschler and co-workers (21);
Fig. 2. Body weight in rats for 7 wk after surgery. Note that body R2 0.99. Note the significant decrease in tissue properties in
weight was altered in hindlimb-suspended animals. tissues from suspended animals.

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318 HINDLIMB UNLOADING ALTERS CONNECTIVE TISSUE HEALING
Fig. 4. Maximum force in sham control, ambulatory healing, and
suspended healing rat tissues at 3 and 7 wk (means SE). Force
reductions in suspended animals were significantly different from
ambulatory healing animals at 3 wk. Statistical comparison for any
group can be obtained from the intersection of group numbers in the
subset table.
control, ambulatory healing, and suspended healing
(Fig. 3). Maximum force (Fig. 4) was significantly dif-
ferent between sham control and both ambulatory
healing and suspended healing animals after 3 wk of
healing (P 0.0001 and 0.0001, respectively) and after
7 wk of healing (P 0.0064 and 0.0001, respectively).
In addition, maximum force in ambulatory and sus-
pended healing MCLs was significantly different in the
3-wk group (P 0.0011). After 7 wk of healing, maxi-
mum MCL force was substantially reduced in the sus-
pended animals compared with ambulatory animals,
but not significantly (P 0.1433). Full pairwise statis-
tical comparisons for force can be found in Fig. 4.
Ultimate stress (Fig. 5) was significantly decreased
between sham control and both ambulatory healing
Fig. 5. Ultimate stress in sham control, ambulatory healing, and
suspended healing rat tissues at 3 and 7 wk (means SE). Hindlimb
unweighting significantly reduced the ultimate stress in healing
tissue. After 7 wk, ultimate stress values in suspended animals were
comparable to values in ambulatory animals after only 3 wk. Statis-
tical comparison for any group can be obtained from the intersection
of group numbers in the subset table.
J Appl Physiol VOL 94 JANUARY 2003
and suspended healing animals after 3 wk of healing
(P 0.0001 and 0.0001, respectively) and after 7 wk of
healing (P 0.0104 and 0.0001, respectively). Liga-
ments from suspended animals had ultimate stress
values that were significantly lower than ambulatory
animals at both 3 and 7 wk (P 0.0156 and 0.0029,
respectively). In fact, ultimate stress values after 7 wk
of healing in suspended animals was comparable to
3-wk values in the ambulatory animals, which re-
vealed a decrease in rate of recovery. Full pairwise
statistical comparisons for ultimate stress can be found
in Fig. 5. Strain at failure was not significantly differ-
ent between any time or treatment groups: 0.075
0.034, 0.079 0.019, and 0.082 0.028 for 3-wk sham
control, ambulatory healing, and suspended healing
groups, respectively, and 0.086 0.014, 0.082 0.016,
and 0.084 0.011 for 7-wk sham control, ambulatory
healing, and suspended healing groups, respectively.
Elastic modulus was determined from fitting the data
with a microstructural model (21). All data were fit
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well: R2 0.98. Elastic modulus (Fig. 6) was signifi-
cantly decreased between sham control and both am-
bulatory healing and suspended healing animals after
3 wk of healing (P 0.0002 and 0.0001, respectively)
and after 7 wk of healing (P 0.0102 and 0.0001,
respectively). Tissues from suspended animals had
modulus values that were significantly lower than am-
bulatory animals at both 3 and 7 wk (P 0.0082 and
0.0173, respectively). In addition, after 7 wk of healing,
the mean modulus value in the suspended group was
less than the mean modulus in the ambulatory animals
after 3 wk of healing. Full pairwise statistical compar-
isons for elastic modulus can be found in Fig. 6. Exam-
ination of the stretch ratio at the toe region to linear
region transition revealed no significant differences
between any groups (Fig. 7). However, the stress re-
quired to transition from the toe region to the linear
Fig. 6. Elastic modulus in sham control, ambulatory healing, and
suspended healing rat tissues at 3 and 7 wk (means SE). Hindlimb
unweighting significantly reduced the modulus in healing tissue.
After 7 wk, ultimate stress values in suspended animals were com-
parable to values in ambulatory animals after only 3 wk. Statistical
comparison for any group can be obtained from the intersection of
group numbers in the subset table.
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 HINDLIMB UNLOADING ALTERS CONNECTIVE TISSUE HEALING
Fig. 7. Stretch ratio at the transition from the nonlinear strain-
stiffening toe region to the linear region of the stress-strain curve in
ligament (means SE). Transition stretch ratio was not signifi-
cantly different between any study groups. Statistical comparison for
any group can be obtained from the intersection of group numbers in
the subset table.
region was significantly decreased between sham con-
trol and both ambulatory healing and suspended heal-
ing animals after 3 wk of healing (P 0.0005 and
0.0001, respectively) and after 7 wk of healing (P
0.0277 and 0.0001, respectively; Fig. 8). Transition
stress in ambulatory healing animals improved almost
twofold from 3 to 7 wk but was not quite significant
(P 0.0638). Suspended animals showed no improve-
ment in transition stress from 3 to 7 wk (P 0.9142),
indicating a lack of recovery of physiological low-load
behavior in suspended animals. In addition, ambula-
tory animals had significantly larger transition stress
after 7 wk than suspended animals (P 0.0064).
Representative hemotoxylin and eosin sections (Fig.
9) revealed extracellular matrix disorganization and
hypercellularity after injury in tissues from both am-
bulatory and suspended animals. Ligaments from
sham control animals showed the characteristic crimp
pattern, which is associated with normal ligament,
with fibroblasts in between collagen bundles (Fig. 9, A
and B). In contrast to ambulatory animals, which re-
vealed typical scar morphology, tissues from sus-
pended animals after 3 wk of healing revealed pockets
of cell clusters and collagen fibers turning back on
themselves, often forming voids within the tissue (Fig.
9, E and F). After 7 wk of healing, ambulatory animals
showed improvements in collagen fiber orientation and
decreases in cellularity compared with morphology at 3
wk (Fig. 9D). Suspended animals at 7 wk showed
substantial improvement in collagen fiber bundle for-
mation and decreases in cellularity (Fig. 9F). However,
the majority of bundles were not oriented along the
longitudinal axis of the ligament. Collagen fiber bun-
dles in suspended animals were often oriented in di-
rections other than the longitudinal axis of the liga-
ment with some bundles aligning in the transverse
orientation. In addition, different fiber bundles in sus-
pended animals were oriented in different directions,
J Appl Physiol VOL 94 JANUARY 2003
319
creating discontinuities and voids when two differently
oriented bundles came in contact (Fig. 9F).
SEM supported the histological finding that tissues
from suspended animals had good collagen fiber bundle
formation but poor bundle orientation, which resulted
in discontinuities and voids (Fig. 10, AC). Sham con-
trol tissues showed collagen fiber bundle alignment
along the longitudinal axis of the ligament with fibers
having characteristic crimp morphology (Fig. 10A).
Collagen fibers in scar tissue from ambulatory animals
were randomly oriented at 3 wk with substantially
improved alignment and orientation (i.e., more axial)
by 7 wk (Fig. 10B). Yet collagen fibers in ambulatory
animals had not regained normal structure and orga-
nization after 7 wk. Ligaments from suspended ani-
mals had good fiber bundle and aggregation (demon-
strated by fiber grouping and bundle width) at both 3
and 7 wk, but the majority of bundles were not oriented
along the longitudinal axis of the ligament with bun-
dles oriented in separate directions, which created dis-
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continuities and voids. These voids and discontinuities
were created when bundle ends came in contact with
the side of a bundle oriented in a different direction
(Fig. 10C).
Bone and muscle measurements are shown in Table
2. Sham and ambulatory animals had no significant
differences within a time group for a particular prop-
erty, yet properties in ambulatory healing tissues were
often reduced compared with tissues from sham ani-
mals. Midshaft total bone mineral density was reduced
in the suspended animals at 3 wk, but not significantly
until 7 wk. Bone mineral density in the femoral head,
bone strength, gastrocnemius weight, and tibialis an-
terior weight were all significantly reduced in sus-
pended animals compared with sham and ambulatory
groups for both time points.
Fig. 8. Stress at the transition from the nonlinear strain-stiffening
toe region to the linear region of the stress-strain curve in ligament
(means SE). Hindlimb unweighting significantly alter low-load
behavior in healing tissue. After 7 wk, suspended animals showed no
improvement in low behavior, whereas ambulatory animals revealed
an almost twofold increase in transition stress. Statistical compari-
son for any group can be obtained from the intersection of group
numbers in the subset table.
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320 HINDLIMB UNLOADING ALTERS CONNECTIVE TISSUE HEALING

Fig. 9. Representative tissue sections taken from the

midsubstance of control normal tissue (A and B) and
scar tissue (CF). The longitudinal axis of the ligament
is from the top left to bottom right of each image. Tissues
from sham control animals at 3 wk (A) and 7 wk (B)
(200) have the characteristic crimp pattern associated
with normal tissue. Examination of scar tissue from
ambulatory healing animals (200) revealed typical
scar morphology with matrix disorganization and hy-
percellularity after 3 wk (C) that improved after 7 wk
(D), although normal morphology was not completely
recovered. In contrast to ambulatory animals, sus-
pended animals showed abnormal scar formation with
pockets of cell clusters (middle arrow) and collagen
fibers turning back on themselves creating defects (left
and right arrows) at 3 wk (E; 100). After 7 wk, sus-
pended animals showed substantially improved collagen

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fiber bundle formation, but the bundles were not well
oriented, and discontinuities and voids were present
between differently oriented collagen fiber bundles (F;
arrows; 200). Note the fiber orientation in the middle
section (F; between arrows) is transverse to the longitu-
dinal axis of the ligament.

DISCUSSION effects of immobilization with pin fixation, but not

Results of this study support our hypothesis that
stress reduction due to hindlimb unweighting substan-
tially alters the healing of fibrous connective tissue in
rats. In suspended animals, only one tissue at each
time group failed by tibial avulsion, whereas the re-
maining femur-MCL-tibia complexes failed in the lig-
ament proper. These results show impaired ligamen-
tous healing from hindlimb unloading that can occur
with spaceflight or prolonged bed rest. Maximum force,
ultimate stress, and elastic modulus in healing tissues
were all significantly reduced with hindlimb unweight-
ing. Stress at the toe-to-linear region transition was
substantially reduced in suspended animals compared
with ambulatory animals and showed no recovery from
3 to 7 wk. These changes may be due in part to altered
tissue organization during healing. Microscopy and
histology revealed altered cellular and extracellular
matrix organization in suspended animals that was
substantially different from characteristic scar organi-
zation in ambulatory animals. In addition, total bone
mineral density, bone strength, and muscle weight
were reduced in hindlimb suspended animals.
Results of this study are consistent with previous
studies that have shown a reduction in the biological
and mechanical properties of normal ligaments after
immobilization (3, 5, 35, 53) and hindlimb suspension
(49, 51). Woo and co-workers (19, 23, 54) examined the
J Appl Physiol VOL
complete stress deprivation of the joint after MCL
rupture, and concluded that long-term immobilization
impedes MCL healing. However, in their studies, am-
bulatory animals did not undergo surgical repair of the
MCL, whereas surgical repair was performed on im-
mobilized ligaments, making it difficult to directly com-
pare their work and data presented herein. Yet, re-
gardless of the surgical procedure, stress reduction
(through joint immobilization or hindlimb suspension)
diminishes healing of fibrous connective tissue leading
to impaired tissue function. In addition, Woo et al. (54)
reported increased varus-valgus laxity in healing ani-
mals that was recovered in ambulatory animals but not
in immobilized animals. These results are in agree-
ment with results obtained by using mathematical
modeling of the toe region. Stress-deprived animals
required a substantially lower magnitude of stress to
leave the physiological toe region and enter the linear
portion of the stress-strain curve and revealed no re-
covery of these low-load properties after 7 wk (Fig. 8).
The fact that low-load properties are not recovered in
remobilized animals raises an interesting question in
light of previous work in our laboratory. We previously
identified a strain-based tissue damage threshold in
normal rat MCLs of 4060% of the failure strain (38).
Because suspended animals require lower amounts of
stress to enter linear region of the stress-strain curve
94 JANUARY 2003 www.jap.org
 HINDLIMB UNLOADING ALTERS CONNECTIVE TISSUE HEALING 321

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Fig. 10. A: scanning electron micrograph of sham control tissue at 3 wk (2 left panels) and 7 wk (2 right panels).
Collagen fibers are oriented along the longitudinal axis of the ligament and possess characteristic crimp morphol-
ogy. B: scanning electron micrograph of ambulatory healing tissue at 3 wk (2 left panels) and 7 wk (2 right panels).
Collagen fibers are randomly oriented at 3 wk with substantially improved alignment and orientation along the
longitudinal axis of the ligament by 7 wk. However, collagen structure, organization, and alignment have not
regained normal appearance. C: scanning electron micrograph of suspended healing tissue at 3 wk (2 left panels)
and 7 wk (2 right panels). At 3 wk, tissue shows good fiber bundle formation and aggegation, but fiber bundles are
not well oriented along the longitudinal axis of the ligament with fiber bundles of different orientations coming
together without fiber continuity to form discontinuities (arrows) and voids (V) in the extracellular matrix. The
3-wk micrographs show regions where misaligned bundles do not connect (arrows). After 7 wk, collagen bundle
formation and aggregation continue to improve, yet voids (V) and discontinuities (arrows) are still present between
misaligned collagen fiber bundles.

and, hence, the damage threshold (which may be lower
in healing tissue), remobilization in the form of normal
activity may result in further tissue microdamage lead-
ing to difficult or incomplete recovery of presuspended
properties. Further microstructural and mechanical
studies are required to understand the residual effects
of suspension on healing tissues in remobilized ani-
mals.
Results of this study agree, in general, with struc-
ture-function studies in fibrous connective tissue that
reveal a strong relationship between altered extracel-
lular matrix structure and organization after injury
and reduced mechanical properties. Previous studies
have shown that extracellular matrix flaw size reduces
with healing time and has a strong association with
changes in failure stress and modulus (42), whereas
changes in collagen fibril diameter in scar tissue do not
J Appl Physiol VOL
correlate well with increased mechanical properties
over the time course of healing (17). In studies utilizing
SEM, Padgett and Dahners (37) reported a larger num-
ber of matrix defects in immobilized healing rat MCLs
than in mobilized tissue, whereas Frank et al. (16)
reported better matrix alignment in immobilized rab-
bit MCLs. These studies (16, 37) do not necessarily
conflict with each other. The study by Frank et al. (16)
examined the extracellular matrix at a relatively high
magnification (4,000), whereas the work by Padgett
and Dahners (37) was performed at lower magnifica-
tion (100). Evaluation at high magnification (relative
to collagen fiber organization) does not readily allow
identification of voids and defects, and Frank et al. (16)
do not give information on overall matrix organization.
Regardless, it is possible that both studies support the
results obtained in this study using histology and
94 JANUARY 2003 www.jap.org
322 HINDLIMB UNLOADING ALTERS CONNECTIVE TISSUE HEALING

Table 2. Bone and muscle properties fibrils join continuously with residual fibrils in ambu-
latory animals and are suspected to be a necessary
Bone and Muscle Ambulatory Suspended
Properties Sham Control Healing Healing
means of transferring force between newly formed scar
tissue and residual tissue. This continuity is largely
Midshaft total not present in suspended animals. As such, we propose
bone density,
mg/cm3
collagen bundle discontinuities in suspended tissue are
Week 3 897.7 20.4 903.3 29.8 837.6 41.5 a major contributor to the reduced mechanical proper-
Week 7 929.0 22.8 953.8 36.5 841.4 14.5* ties of healing tissue in suspended animals. Interest-
Femoral head ingly, stress levels associated with normal ambulation
total bone appear to be unnecessary for the formation of fiber
density,
mg/cm3 bundles (orchestrated by fibroblasts first with the for-
Week 3 695.0 10.0 654.1 13.3 574.1 12.4* mation of the collagen fibrils), but this stress appears
Week 7 758.7 16.4 702.1 33.0 626.8 19.3* necessary to align and promote fusion between new
Bone strength, N fiber bundles to form structurally competent fiber con-
Week 3 116.9 9.9 104.6 5.3 62.8 5.4*
Week 7 130.4 3.7 119.2 13.5 94.6 3.8*
tinuity. However, further study is required to under-
Gastocnemius stand the mechanisms of collagen formation and orga-
weight, mg nization in scar tissue and to answer the above-stated
Week 3 2,258.2 73.0 2,160.5 83.9 1,607.9 57.4* question. In addition, further studies into the biochem-
Week 7 2,710.0 26.6 2,469.5 215.5 1,817.5 77.0* ical composition of the matrix and more intense exam-
Tibialis anterior,
mg ination of cell behavior and organization need to be

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Week 3 815.1 30.2 748.2 31.9 646.0 37.9*
Week 7 940.1 28.8 858.2 83.3 670.2 57.8*
Values are means SE. * Significant (P 0.05) difference from
both sham control and ambulatory healing groups.
SEM. In this study, stress reduction from hindlimb
suspension altered scar morphology and resulted in
voids and discontinuities where well-formed collagen
bundle ends (demonstrated by fiber grouping and bun-
dle width) came in contact with the side of a bundle
oriented in a different direction. These results are in
agreement with work by Padgett and Dahners (37)
that increased voids are present in immobilized tis-
sues. Because of their use of only 10 images per tissue
at higher magnification, Frank et al. (16) may have
been viewing these well-formed bundles that we have
shown to be grossly or slightly misaligned (Fig. 10) to
create discontinuities and voids in stress-altered tis-
sue. Hence, Frank et al. may have viewed slightly
misaligned, well-formed bundles, which led to the con-
clusion of increased matrix alignment in immobilized
tissues.
Examination of the altered structure in tissue from
suspended hindlimbs leads to an interesting question:
How do such high-quality collagen fiber bundles form
in ligaments with substantially reduced mechanical
stress and yet not form a continuous force coupling
with surrounding well-formed collagen bundles? Birk
and Trelstad (10) studied tendon morphogenesis in
chick embryos and showed that formation of collagen
fibrils and fibril bundles occur within cytoplasmic re-
cesses of fibroblasts. In addition, Birk and co-workers
(9, 11, 12) showed that discontinuous fibril segments
can fuse to form longer fibril in developing embryonic
chick tendon. During development, these fibril fusions
can occur through collagen fibril end-to-end fusion (20).
Previous examination of the transition from scar to
residual tissue in MCLs from ambulatory mature ani-
mals has shown collagen fibril and fiber continuity
between scar and residual tissues (39). Hence, new
J Appl Physiol VOL 94 JANUARY 2003
performed to better understand the root causes of the
altered composition- and structure-function changes
associated with fibrous connective tissue disuse.
Several limitations must be borne in mind when
considering the results of this study. First, the rat
model may be limited in its ability to model adult
humans under stress-altered conditions such as space-
flight or prolonged best rest. However, the results of
this study are consistent with related studies that
show reduction in ligament, bone, and muscle proper-
ties with disuse. Second, the rat hindlimb suspension
model does not inhibit muscle activity, hence some
knee motion and joint loading can occur. Observation
and tissue atrophy suggest that such motion and loads
are substantially reduced. Third, transected ligaments
do not replicate stretch-induced injuries, but they are
very reproducible for comparative purposes. Fourth,
the effects of short periods of therapeutic exercise, such
as passive and active motion, to counteract the effects
of limb disuse were not studied. Such treatments
would be more similar to conditions of human tissue
healing. Fifth, low-load behavior was described from
an ex vivo preloaded condition, and further under-
standing of the ex vivo vs. in situ reference position is
required. Sixth, sample preparation for SEM can in-
duce a shrinkage artifact. However, all specimens were
handled and prepared in the same controlled manner.
Because the morphological portions of this study are
qualitative in nature, shrinkage error most likely had
little effect on our observations. Last, although further
biological, biochemical, and histomorphometric studies
are ongoing, further understanding of the biological
and physical mechanisms driving the compelling
changes associated with tissue disuse is required.
Despite these limitations, our results support our
hypothesis that stress reduction through hindlimb un-
loading impairs healing of fibrous connective tissue.
Maximum stress, ultimate stress, elastic modulus, and
physiological low-load behavior were altered by hind-
limb suspension. Examination of extracellular matrix
organization revealed novel and important changes in
www.jap.org
 HINDLIMB UNLOADING ALTERS CONNECTIVE TISSUE HEALING
stress-deprived tissues with voids and discontinuities
between well-organized, but misaligned, collagen bun-
dles in suspended animals. This morphology was sub-
stantially different from matrix organization in ambu-
latory animals and opens further questions regarding
the essential role of mechanical stress for collagen
matrix organization in connective tissues.
The authors thank Dr. Dennis Heisey for statistical insight. In
addition, the authors thank individuals at the NASA-Ames center
who contributed to animal care: Tom Wang, Fang Yuan, Megan
Moran, Lisa Baer, Cyra Fung, David Garcia, and Guilia Gurun.
This work was funded by NASA (Grant NAG9-1152) and a Wis-
consin Space Consortium scholarship (to J. Turner).
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