Artigo 2 - Effects of aging and exercise training on the histological and mechanical properties of articular structures in knee joints of male rat

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RESEARCH ARTICLE
Effects of aging and exercise training on the histological
and mechanical properties of articular structures in knee
joints of male rat
Hideki Moriyama • Naohiko Kanemura • Inge Brouns •
Isabel Pintelon • Dirk Adriaensen • Jean-Pierre Timmermans •
Junya Ozawa • Nobuhiro Kito • Toshiaki Gomi • Masataka Deie
Received: 13 December 2011 / Accepted: 12 April 2012
Ó Springer Science+Business Media B.V. 2012
Abstract The impact of aging on joints can have a
profound effect on an individual’s functioning. Our
objectives were to assess the histological and mechan-
ical properties of the knee joint capsule and articular
cartilage with aging, and to examine the effects of
exercise on age-related changes in the knee joint.
2-year-old Wistar rats were divided into a sedentary
control group and an exercise-trained group. 10-week-
old animals were used to investigate the changes with
aging. The joint capsule and cartilage were evaluated
with histological, histomorphometric, immunohisto-
chemical, and mechanical analyses. Severe degenera-
tive changes in articular cartilage were observed with
aging, whereas exercise apparently did not have a
significant effect. The articular cartilage of aged rats
was characterized by damage to the cartilage surface,
cell clustering, and an abnormal cartilage matrix.
Histomorphometric analysis further revealed changes
in cartilage thickness as well as a decreased number of
chondrocytes. Aging led to stiffness of the articular
cartilage and reduced the ability to dissipate the load
and distribute the strain generated within the joint.
Joint stiffness with aging was independent of capsular
stiffness and synovitis was not a characteristic feature
of the aging joint. This study confirms that aging alone
eventually leads to joint degeneration in a rat model.
The lack of recovery in aging joint changes may be due
to several factors, such as the duration of the interven-
tion and the regeneration ability of the cartilage.
Keywords Aging � Joint capsule � Cartilage �
Exercise � Rat
Introduction
Aging joint and muscle changes can have a tremen-
dous impact on an individual’s overall functioning
(Ahmed et al. 2005). Although the morphological,
metabolic, and contractile properties of aging muscle
Hideki Moriyama and Naohiko Kanemura contributed equally
to this work.
H. Moriyama (&) � M. Deie
Graduate School of Health Sciences, Hiroshima
University, Hiroshima 734-8551, Japan
e-mail: morihide@harbor.kobe-u.ac.jp
Present Address:
H. Moriyama
Graduate School of Health Sciences, Kobe University,
Tomogaoka 7-10-2, Suma-ku, Kobe 654-0142, Japan
N. Kanemura � T. Gomi
School of Health and Social Services, Saitama Prefectural
University, Saitama 343-8540, Japan
I. Brouns � I. Pintelon � D. Adriaensen �
J.-P. Timmermans
Laboratory of Cell Biology and Histology, University
of Antwerp, 2020 Antwerp, Belgium
J. Ozawa � N. Kito
Department of Physical Therapy, Hiroshima International
University, Hiroshima 739-2695, Japan
123
Biogerontology
DOI 10.1007/s10522-012-9381-8
have been extensively studied in rat models, much less
information is available on age-related changes in the
lower extremity joints. While the thickness of tibial
cartilage did not substantially decrease in 26-month-
old rats, its histological structure was changed when
compared to young adult rats (Gyarmati et al. 1987).
Similar results were found in 24-month-old Wistar
rats, 68 % of which displayed minimal to mild lesions
in the medial tibial plateau that were characterized by
loss of proteoglycan staining in the superficial zone
accompanied by loss of chondrocytes (Smale et al.
1995). Furthermore, the thickness and cellular density
of articular cartilage in the femoral trochlea was found
to be diminished in 32-month-old rats (Oda et al.
2007) and elevated chondrocyte apoptosis rates were
found in the calcified layer of knee cartilage in
24-month-old rats (Adams and Horton 1998). In the
lower limb joints, the articular surface of the femoral
heads in 21-month-old rats was characterized by
empty lacunae of chondrocytes and numerous exposed
collagen fibrils (Gattone et al. 1982). These studies
have been focused on the age-dependent alterations in
the articular cartilage and on only one of morpholog-
ical changes taking place in the main components of
the cartilage matrix. Osteoarthritis (OA) increases in
prevalence with aging and is a degenerative joint
disease generally characterized by progressive carti-
lage degeneration (Martin and Buckwalter 2002);
therefore, these studies have been described in asso-
ciation with OA. Joints are composed of several
different tissues (cartilage, capsule, synovium, and
ligament) that interact in unknown ways to allow
joints to function relatively well (Burr 2004). These
tissues are all important to the optimal functioning of
joints, and when one tissue begins to deteriorate, it
inevitably affects on the others (Burr 2004). Taken
together, earlier experiments in only part of the
cartilage are insufficient to understand the changes in
the joint with advancing age.
Joint stiffness is frequently observed in elderly
people (Trichard et al. 1982) and individuals with knee
OA (Dixon et al. 2010), and increases the joint’s
vulnerability to injury (Ahmed et al. 2005). The joint
capsule is known to be of vital importance to the
function of a synovial joint. It forms part of the seal that
keeps lubricating synovial fluid in position, provides
passive stability by limiting jointmovements, and active
stability via its proprioceptive nerve endings (Ralphs
and Benjamin 1994). Earlier studies emphasized the
pathogenetic role of muscles causing joint stiffness
(Ochi et al. 2008), whereas tissues within the articular
structures have been poorly documented. We hypoth-
esize that joint stiffness results from capsular stiffness.
It is widely accepted that appropriate exercise
counteracts the progressive loss of muscle mass,
muscle strength, and quality (Larsson and Ramamur-
thy 2000). Even short-term (1 month) treadmill exer-
cise training started at an advanced age was able to
reverse age-related skeletal muscle apoptosis and thus
proved to be effective strategy for improving physical
performance and muscle strength in old rats (Marzetti
et al. 2008). On the other hand, the effects of exercise
training on the age-related changes in rat joints have
not been studied experimentally. Animal studies in
healthy young joints have shown that short-term
moderate or strenuous exercise does not cause cartilage
degeneration and does not have a deleterious effect on
the mechanical properties of canine cartilage (Newton
et al. 1997). Additionally, animal studies have shown
that disruption of proprioception in a joint leads to
advanced and accelerated degenerative changes of the
joint (O’Connor and Brandt 1993). Studies comparing
knee proprioception in active elderly and sedentary
elderly participants suggested that regular exercise
attenuated the decline in proprioceptive seen with
aging (Petrella et al. 1997). Exercise intervention also
has been reported to influence joint stiffness to varying
effects depending on age (Ochi et al. 2008).
The main aims of the present study were to inves-
tigate the histological and biomechanical changes
occurring in the knee joint capsule and articular cartilage
with (physiological) aging, and to examine the effects of
exercise on these age-related changes. Biomechanical
studies are essential since structural and biochemical
changes have been associated with alterations in the
mechanical properties of the tissue (Akizuki et al. 1986).The functional biomechanical properties of a tissue,
therefore, best reflect the complexity of fundamental
structural changes (Poole et al. 2010).
Materials and methods
Experimental design
The protocols for the experiments were approved by
the Committee of Research Facilities for Laboratory
Animal Science of Hiroshima University School of
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123
Medicine. Animal model systems represent an impor-
tant surrogate for studies of aging in humans (Aigner
et al. 2010). Given the high cost of maintaining larger
species, smaller animal models are preferred for
preliminary screening, and rat models are frequently
used for initial assessment of in vivo efficacy. Themost
efficacious compound in a rat model may subsequently
be tested in a larger animal model prior to setting up
human trials (Gerwin et al. 2010). In the present study,
10 old male Wistar rats (CLEA Japan Inc., Tokyo,
Japan; age: 24 months; weight 400–520 g) were used
and divided into a sedentary control group and an
exercise group. The half-mortality age ofWistar rats is
24 months and the maximal lifespan is 36 months
(Larsson and Ansved 1995). Animals of 24 months of
age were used to avoid the unpredictable effects of
disease processes. Four animalswere used as sedentary
controls. The remaining six rats were trained on a
treadmill or a custom balance exercise device for
1 month (5 times a week). Three rats were introduced
daily to the treadmill walking at a speed of 11.8 m/min
for 1 h. Walking speed and the duration of exercise
were calculated so as to reflect a work rate of*40 %
maximal O2 uptake in the animals (Lawler et al. 1993).
Three animals were placed on a moving platform
(angle of inclination, ±7°; number of revolutions,
25 rpm) for 1 h a day to attenuate the decline in
proprioception seen with aging (Ahmed et al. 2005;
Petrella et al. 1997). The balance exercise programwas
intended to recruit a combination of isometric, con-
centric, and eccentric activities of the lower-extremity
musculature (Brown and Taylor 2005). In addition,
four adolescent male Wistar rats (age: 10 weeks;
weight: 172–186 g) were used as a young group with
healthy joints to investigate age-related changes. All
animals were housed in roomswith a temperature set at
23 ± 1 °C, relative humidity between 50 and 60 %,
and a 12/12 h light/dark cycle. Only active rats with
proper food intake and without evident motor deficits
or visible pathological signs were admitted to the
study.
Histological evaluation
At the end of the maintenance intervals, the animals
were killed by exsanguination under anesthesia. The
femur and tibia were dissected free of soft tissues and
disarticulated at the hip and ankle. The knee joints
spontaneously assumed an extended position at 125°
when the restraint of surrounding soft tissues was
removed (O’Connor 1997). We prepared undecalci-
fied frozen sections following the protocols estab-
lished by Kawamoto (2003). The unilateral knee
joints including the patella and joint capsule were
resected and immediately frozen in a mixture of
hexane and isopentane cooled with a cooling appara-
tus (UT2000F, FINETEC, Tokyo, Japan). Each
sample was embedded in 5 % carboxymethyl cellu-
lose gel and completely frozen. Each undecalcified
frozen block was then attached to the sample stage of a
cryomicrotome (CM3050S, Leica Microsystems,
Tokyo, Japan) in a cryochamber. The block was
covered with a polyvinylidene chloride film (CF0114,
Leica Microsystems) and sectioned with the film at a
thickness of 5 lm using a disposable tungsten carbide
blade (Jung TC-65, Leica Microsystems). The knees
were aligned so that sagittal plane sections were cut in
the proximal-to-distal direction at the medial and
lateral mid-condylar level in the sagittal plane. These
sections were stained with aldehyde fuchsin–Masson
Goldner for the joint capsule, and toluidine blue or
safranin-O/fast green for the articular cartilage.
Quantitative histology
Determination of measurement sites
We quantified the histological changes in articular
cartilage as described previously (Moriyama et al.
2008, 2009). Cartilage alterations after immobilization
(Hagiwara et al. 2009; Trudel et al. 2005) or unloading
(O’Connor 1997) may differ between different carti-
lage plates of the knee, based on the different mechan-
ical conditions specific to each site. Therefore, changes
in femoral and tibial cartilage at the medial and lateral
mid-condylar level were determined in the 12 regions
by slight modification of the method applied in our
previous study (Moriyama et al. 2008, 2009). Cartilage
regions were defined according to their positions in
embedded joints where the knee joint was positioned at
an angle of 125°. The anterior femoral (FA) andmiddle
tibial (TM) regions were defined as the regions of
articular cartilage located between the inner edges of
the anterior and posterior meniscal horns. The edge
of the posterior femoral (FP) region was located 20 lm
beyond the outer edge of the posterior meniscal horn,
and the middle femoral (FM) cartilage region was
situated between the FA and FP regions. The anterior
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123
tibial (TA) region was located adjacent to the anterior
horn of the meniscus, and the posterior tibial (TP)
region was adjacent to the posterior horn.
Cartilage thickness
Articular cartilage thickness was measured on sections
stained with toluidine blue, which provides excellent
color discrimination between bone and calcified
cartilage, as well as a distinct basophilic line that
marks the location of the tidemark (O’Connor 1997).
Cartilage thickness was defined as the distance
between the cartilage surface and the osteochondral
junction. Because of the undulating nature of the
osteochondral junction, and the consequent large site-
dependent variability in cartilage thickness, we mea-
sured an area of cartilage and calculated the mean
thickness over that area (Trudel et al. 2005). Histo-
logical sections were digitized by a 49 microscope
objective with a light microscope (BX51, Olympus,
Tokyo, Japan) and a camera (DP70, Olympus). In each
region, a 400-lm-long stretch of the cartilage surface
was defined and the cartilage area under this stretch
was measured, following the osteochondral junction.
The mean thickness of the cartilage was calculated by
dividing the area by 400 lm.
Number of cells
Histological sections stained with safranin-O/fast
green were digitized by a 209 microscope objective
with a light microscope and a camera. Rectangles
100 lm deep and 400 lm long were superimposed
over histologic sections in each of the 12 regions.
Chondrocytes were manually counted within the rect-
angular field.
Immunohistochemical analysis
We assessed age- and exercise-related changes in
immunohistological staining patterns at the joint
capsule and in the articular cartilage. Frozen sections
were air-dried, fixed in ethanol for 2 min, and
rehydrated in 0.01 M phosphate-buffered saline
(PBS, pH 7.4) for 5 min. Sections were treated with
0.5 % bovine testicular hyaluronidase (H3506, Sigma-
Aldrich Co., MO, USA) in PBS for 60 min at room
temperature. After two rinses with PBS for 5 min
each, endogenous peroxidase was inactivated by
incubation of the sections in methanol containing
0.3 % H2O2 for 20 min. Non-specific binding sites
were blocked by treating the sections with 1 % normal
serum from the host species of the secondary anti-
body (horse or goat) in PBS. After removal of the
blocking solution, sections were incubated with mouse
monoclonal anti-collagen type I (diluted 1:4,000;
C2456, Sigma-Aldrich Co.), anti-collagentype III
(diluted 1:8,000; C7805, Sigma-Aldrich Co.), anti-
CD31 (diluted 1:250; MCA1334GA, AbD Serotec,
Oxford, UK), or anti-pentosidine (diluted 1:50;
KH012, Trams Genic Inc., Kumamoto, Japan) anti-
bodies for the joint capsule, and mouse monoclonal
anti-collagen type II (diluted 1:1,000; F-57, Daiichi
Fine Chemical Co., Toyama, Japan) or goat polyclonal
anti-matrix metalloproteinase 13 (MMP-13; diluted
1:2,000; AB8120, Millipore Co., Billerica, MA, USA)
antibodies for the articular cartilage overnight at 4 °C.
Sections were then rinsed in PBS and reacted with
horse biotinylated anti-mouse IgG (diluted 1:250; BA-
2001, Vector Laboratories, Burlingame, CA, USA) or
rabbit biotinylated anti-goat IgG (diluted 1:30; Histo-
fine 416022, Nichirei Biosciences Inc., Tokyo, Japan)
for 1 h at room temperature. A subsequent reaction
was made by the streptavidin–biotin-peroxidase com-
plex technique using an Elite ABC kit (diluted 1:50;
Vector Laboratories) for 30 min. Immunoreactivity
was visualized with 3,30-diaminobenzidine tetrahy-
drochloride (K3466, Dako Japan, Tokyo, Japan),
followed by counterstaining with methylene green.
The primary antibody was omitted for the negative
controls.
Mechanical analysis
Each specimen assigned to the mechanical analysis of
the contralateral knee joint was stored at-80 °C until
mechanical tests. We performed two series of mechan-
ical tests as described by published studies (Akai et al.
1993; Usuba et al. 2007). These tests were nonde-
structive, dynamic forced vibration methods to exam-
ine viscoelasticity (phase lag and dynamic stiffness),
and a static destruction test with tensile force. The
specimens were thawed at room temperature just
before mechanical analysis and were kept moist with
normal saline at all times. Testing was completed
within 4 h of thawing.
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123
Mechanical testing of the viscoelasticity
of the articular structure
The first mechanical test evaluated the viscoelasticity
of the joint capsule and the articular cartilage by
spectral analysis based on the fast fourier transform of
displacement. A spectrum analyzer (3582A, Hewlett
Packard, Palo Alto, CA, USA) served as a dual-
channel fast fourier transform machine and provided
electrical driving signals to the viscoelastic spectrom-
eter (DDV-VMF, Orientec Co., Tokyo, Japan). A
small rod, which was connected to the load cell of the
spectrometer, applied the forced vibration at a fre-
quency of 1 Hz to the distal end of the tibia and 10 Hz
to the cartilage surface (weight-bearing area) of the
femur or tibia at the medial and lateral mid-condylar
level. The transfer functions, the mathematic relation
between both input and output signals through the
specimen, indicated a delay of phase (phase lag) and
structural stiffness (dynamic stiffness). Phase lag (tan
d) indicated the shock-absorbing ability and dynamic
stiffness (N/mm) referred to the transform resistance
at the primary phase of lengthening.
Static destruction testing of the periarticular
connective tissue
Another mechanical test, the tensile test of the bone-
joint-bone complex, was determined from the load-
deformation curves. A load cell, which was attached to
a traction device (Tensilon UTM-10T, Orientec Co.)
that moved to hyperextend the joint at 3 mm/min, was
applied to the distal end of the tibia. The load
and deformation of each joint were continuously
recorded with a xy-recorder. The liner slope of the
load-deformation curve was used to calculate peak
maximum load (N) and stiffness (N/mm) of the joint.
Statistical analysis
The software program JMP 7 (SAS Institute, Cary,
NC, USA) was used for the statistical analysis.
Descriptive statistics were calculated as median and
interquartile range (IQR). An alpha level of 0.05 was
used for all statistical tests, and two-tailed tests were
applied. The Kruskall–Wallis nonparametric test was
used to evaluate the differences among the groups
which were not normally distributed (Zar 2010).When
statistical significance was achieved, a post hocWelch
test was used to further specify the difference between
the groups. For the post hoc analysis, the Shaffer
correction was applied to adjust the a priori alpha level
to the number of comparisons performed.
Results
Given the lack of differences between the treadmill
and balance exercise groups, the results for all
outcome measures were pooled into one comparison
(exercise-trained) group for proper comparison of the
effects of aging on all outcome measures.
Histological findings
Microscopic examination showed severe degenerative
changes in the articular cartilage with aging, although
no difference was observed between the sedentary and
exercise-trained groups in any region. Histological
findings revealed fissures, enlarged lacunae without
nuclei corresponding to chondrons, and clustering of
chondrocytes, particularly in the medial tibial carti-
lage (Fig. 1a–c). On the other hand, evidence of
inflammation, such as the presence of inflammatory
cells (e.g., polynucleated cells, lymphocytes, macro-
phages), and proliferation of connective tissue was not
observed at the joint capsule. No apparent degenera-
tion of the joint capsule or articular cartilage was
found in any of the specimens from young rats.
Total cartilage thickness (including both uncalci-
fied and calcified layers) did not differ among the
groups in any region of the femur or tibia at the medial
or lateral levels (P[ 0.10). At the medial mid-
condylar level of the knee, only the uncalcified layer
in the FA cartilage region was thinner in the sedentary
(P = 0.046) and exercise-trained (P = 0.016) aged
groups than in the young group. Accordingly, at the
lateral mid-condylar level, the uncalcified layer in
both the FA (P = 0.0002) and FP (P = 0.006)
cartilage regions was thinner in exercise-trained old
rats than in young rats, although cartilage thickness in
sedentary rats was comparable with that in young rats.
The calcified layers in the FA, FM, FP, and TP
cartilage regions were thicker in the sedentary and
exercise-trained groups than in the young group
(P\ 0.038) at medial level. The lateral level, unlike
the medial level, did not show any significant differ-
ences in femoral or tibial calcified cartilage among the
Biogerontology
123
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123
groups (P[ 0.05). The mean uncalcified layers
expressed as a percentage of total cartilage thickness
are shown in Table 1. The tidemark advanced toward
the joint surface with aging, leading to a shift in the
proportion of uncalcified versus calcified cartilage
(Fig. 1d–f). As compared with the young group, the
proportion of uncalcified cartilage was significantly
smaller in the sedentary and exercise-trained groups
in the FA, FM, FP, and TP cartilage regions (all
P\ 0.05).
Table 2 shows the number of chondrocytes in
femoral and tibial cartilage. The number of chondro-
cytes differed among the various cartilage plates of the
knee. At the medial level, there were significantly fewer
chondrocytes in the sedentary and exercise-trained old
knee joints than in young knee joints in FA, FP, and TP
cartilage (P\ 0.029). In contrast, no statistically
significant differenceswere found at the lateral cartilage
level among the different groups (P[ 0.05).
Immunohistochemical observations
Only weak immunolabeling for collagen type II was
observed over all layers of the articular cartilage with
aging at both the medial and lateral levels, whereas
collagen type II was abundantly expressed in young
cartilage (Fig. 1g–i). MMP-13 immunostaining showed
intense expression in pericellularregions from the
middle to deep cartilage layers in both the sedentary
and exercise-trained groups (Fig. 1j–l). Staining inten-
sity and the expression pattern of collagen types I and
III did not appear to differ among the groups in any
joint capsule (data not shown). Accordingly, pentos-
idine labeling showed no difference among the groups
either. CD31-immunoreactive blood vessels were
predominantly distributed throughout the synovial
membrane of young knees, while only weak reactivity
Table 1 Mean uncalcified layer expressed as a percentage of whole cartilage thickness
Regions Young Sedentary old Exercised old P values Power
Medial
FA 90.13 (86.22, 93.43) 61.75 (55.88, 66.12)* 62.21 (51.16, 64.28)* 0.015 0.996
FM 89.15 (88.29, 90.21) 68.77 (60.19, 69.36)* 62.24 (61.79, 64.33)* 0.017 0.936
FP 93.15 (91.97, 94.98) 62.18 (57.64, 65.65)* 59.46 (44.80, 75.83)* 0.018 0.878
TA 69.50 (63.43, 75.14) 52.22 (50.98, 55.66) 67.57 (43.11, 76.41) 0.304 0.148
TM 75.84 (64.93, 86.29) 55.62 (45.62, 65.46) 55.42 (49.94, 65.21) 0.291 0.277
TP 87.54 (81.75, 88.91) 48.29 (41.90, 53.90)* 55.93 (54.84, 58.36)* 0.012 0.889
Lateral
FA 94.09 (87.48, 96.24) 64.75 (56.15, 74.29) 53.70 (43.24, 72.97)* 0.047 0.689
FM 82.59 (77.70, 86.56) 58.56 (47.81, 69.30) 61.25 (57.59, 72.29) 0.075 0.480
FP 89.24 (82.67, 93.93) 58.50 (48.32, 67.05)* 54.86 (42.96, 60.05)* 0.039 0.830
TA 73.07 (70.05, 77.40) 65.59 (49.58, 77.12) 66.83 (57.90, 74.35) 0.695 0.142
TM 83.01 (79.75, 87.73) 47.12 (30.69, 68.02) 66.13 (63.26, 72.99) 0.045 0.616
TP 85.59 (83.11, 89.88) 54.27 (45.22, 61.79)* 50.11 (45.22, 60.28)* 0.018 0.983
Displacement values are given as median (IQR) %
P value by Kruskal–Wallis H statistic
* Significantly different from young group
Fig. 1 Typical histopathological changes in rat knees. Evi-
dence of fissures on the cartilage surface and cluster formation
are obvious in an old rat (compared with the young rats) (a–c).
Aging induced advancement of the tidemark (arrowheads)
toward the joint surface, resulting in a thinning of the uncalcified
cartilage layer and a concurrent thickening of the calcified layer
(d–f). a–c: Medial TM cartilage; Safranin-O and light green
staining. d–f: Lateral TP cartilage; Toluidine blue staining.
Immunohistochemical detection of the distribution of collagen
type II in the lateral TA cartilage (g–i), MMP-13 in the medial
TP cartilage (j–l), and CD31 in the lateral synovial membrane
between the patellar tendon and femur (m–o). Collagen type II
was present in large quantities over all layers in young cartilage
(g) but decreased with aging (h, i). Cartilage in the young group
solely expressed MMP-13 (j), whereas the pericellular regions
in the old rat groups were clearly stained (arrows) (k, l).
Immunostaining for CD31 showed intense labeling in the young
group (m) but weak labeling in the old groups (n, o). Scale bars
100 lm (a–c, j–o) and 200 lm (d–i). AC articular cartilage, SB
subchondral bone
b
Biogerontology
123
was found in the old knees (Fig. 1m–o). Exercise had
no significant effect on these staining patterns.
Mechanical properties
Viscoelasticity (phase lag and dynamic stiffness) and
static destruction (peak maximum load and stiffness) of
sedentary old and exercise-trained old knees were com-
parable to those of young knees (P = 0.180, power =
0.431;P = 0.374, power = 0.133;P = 0.873, power =
0.085; P = 0.341, power = 0.272; respectively) in
periarticular connective tissue (data not shown).
The viscoelasticity of articular cartilage significantly
differed among the groups (P\ 0.05). Although no
significant effect of exercise was detected in articular
cartilage (P[ 0.05), dynamic stiffness was greater in
old rats at the medial femur (P = 0.013, power =
0.999) (Fig. 2a) and phase lag was lower at the medial
(P = 0.022, power = 0.884) (Fig. 2b) and lateral tibia
(P = 0.013, power = 0.967) (Fig. 2c).
Discussion
In this study, we characterized the natural history of the
joint capsule and cartilage in rat knee joints, and
examined the effects of exercise training on age-related
changes to joints. Aged animals had a high incidence of
cartilage lesions, although no effect of exercise was
detected.
We found a percentage reduction in thickness of the
uncalcified layer aswell as advancement of the tidemark
with aging.Tidemark advancementmay accelerate after
the age of 60 in humans (Lane andBullough 1980) and it
has been suggested that the tidemarkmay be reactivated
by trauma, joint unweighting, or duringOA, leading to a
thinning of the uncalcified layer of cartilage (Bullough
1981; O’Connor 1997; Radin et al. 1991). Articular
cartilage calcification is a well-known phenomenon
observed in late-stageOA (Ea et al. 2011). The results of
the present study are at least partially consistent with
these earlier findings. In a non-diseased joint, although
tidemark advancement makes the calcified cartilage
thicker, remodeling at the osteochondral junction occurs
more quickly, which causes the calcified cartilage to
become thinner (Burr 2004).With aging, both tidemark
advancement and subchondral remodeling accelerate
resulting in a thicker calcified cartilage layer (Burr
2004). This process is accompanied by a reduction in the
thickness of uncalcified cartilage. Mechanical stresses
in the articular cartilage are likely to increase as this
process progresses. Under normal conditions, articular
cartilage and subchondral bone act together in trans-
mitting load pressure through joints; therefore, the
Table 2 Number of chondrocytes in a 100 lm by 400 lm area of articular cartilage
Regions Young Sedentary old Exercised old P values Power
Medial
FA 74.00 (68.25, 75.75) 34.50 (23.50, 48.75)* 35.00 (23.50, 39.00)* 0.032 0.869
FM 80.50 (68.75, 86.50) 42.00 (30.50, 51.00) 52.00 (42.00, 58.50) 0.068 0.689
FP 105.50 (91.00, 112.50) 36.50 (27.75, 50.00)* 42.00 (40.50, 49.00)* 0.029 0.974
TA 106.00 (98.75, 115.50) 78.00 (66.25, 86.00) 60.00 (56.00, 74.50) 0.034 0.759
TM 122.00 (101.25, 130.00) 64.00 (37.00, 88.50) 47.00 (33.00, 60.50) 0.055 0.880
TP 102.50 (92.50, 108.50) 41.00 (33.00, 53.75)* 51.00 (50.50, 62.00)* 0.018 0.964
Lateral
FA 94.50 (78.75, 107.00) 51.50 (35.25, 65.50) 59.00 (40.00, 84.50) 0.114 0.446
FM 98.00 (89.00, 108.25) 53.50 (36.25, 71.75) 65.00 (57.00, 71.50) 0.084 0.731
FP 97.50 (75.00, 120.75) 67.50 (45.50, 84.75) 62.00 (51.00, 86.50) 0.321 0.253
TA 107.00 (101.25, 118.00) 70.00 (43.50, 96.25) 69.00 (65.00, 92.50) 0.056 0.464
TM 96.00 (84.25, 108.25) 64.50 (39.50, 86.50) 71.00 (57.50, 89.00) 0.214 0.303
TP 80.00 (68.00, 100.50) 69.50 (59.00, 76.25) 68.00 (61.50, 78.50) 0.416 0.284
Displacement values are given as median (IQR)
P value by Kruskal–Wallis H statistic
* Significantly different from young group
Biogerontology
123
integrity of both tissues is required for adequate function
(Bobinac et al. 2003; Eckstein et al. 1992, 1998). This
study demonstrated that aging leads to stiffening of
articular cartilage (medial femoral condyle) and reduces
the ability to dissipate the load and distribute the strain
generated within the joint (tibial plateaus). From a
mechanical perspective, this would increase mechani-
cal stresses, thereby increasing damage to articular
cartilage.
Aging is associated with progressively reduced
cellularity in articular cartilage (Barbero et al. 2004;
Oda et al. 2007; Temple et al. 2007), probably as a
consequence of cell death over time (Adams and
Horton 1998; Grogan and D’Lima 2010). The ability
of chondrocytes to proliferate, and hence also to repair
and maintain the cartilage matrix seems to decrease
with age (Ahmed et al. 2005). Furthermore, the
multifaceted nature ofjoint pathologies suggests that
the contribution of cell death and cluster formation is
an important factor in early- and late-stages OA
(Grogan and D’Lima 2010). In addition to earlier
reported age-related changes in articular cartilage
(Adams and Horton 1998; Gyarmati et al. 1987; Smale
et al. 1995), we also confirm marked differences
between the medial and lateral knee joint. Changes in
mechanical loading of chondrocytes caused by dam-
age to and loss of matrix molecules contribute to the
degenerative pathology, because excessive and non-
cyclic loading can stimulate cartilage degeneration in
vitro (Poole et al. 2010). Therefore, the decline in cell
numbers observed in the medial region only may be
explained by articular surface incongruity resulting
from changes in cartilage thickness.
Collagen type II, the main collagen type of hyaline
cartilage responsible for the stability and cell biolog-
ical functions of healthy articular cartilage (Poole
1999; Prockop et al. 1979), originates from chondro-
cytes (Doherty et al. 1998; Hagiwara et al. 2010a).
MMP-13 is capable of degrading type II collagen at a
much higher rate than other collagenases (Bramono
et al. 2004), and its expression is highly increased in
articular cartilage in response to joint injury (Hayami
et al. 2004). We observed collagen damage at the
articular surface extending deeper into the cartilage in
the aging joint as well as increased immunostaining of
MMP-13 at the pericellular regions from the middle to
deep cartilage layers, which was not seen in younger
cartilage. Damage to the collagen type II meshwork is
a critical event in the pathology of OA (Hagiwara et al.
2010a; Henrotin et al. 2007) and increased expression
of MMP-13 in human OA cartilage and in OA animal
models has been well documented (Ando et al. 2009).
Determining the levels of structural collagens is a
vital element in understanding the elastic changes
A
B
C
160
140
120
100
80
60
40
20
0
Young Sedentary old
D
yn
am
ic 
st
iff
ne
ss
 (N
/m
m)
P = 0.019
0.20
Young Sedentary old Exercised old
Ph
as
e 
la
g 
(ta
n δ
)
0.12
0.14
0.16
0.18
0.10
0.08
0.00
0.02
0.04
0.06
0.20
Young Sedentary old Exercised old
Ph
as
e 
la
g 
(ta
n δ
)
0.12
0.14
0.16
0.18
0.10
0.08
0.00
0.02
0.04
0.06
P = 0.001
P = 0.021 P = 0.020
P = 0.012
P = 0.004
Exercised old
Fig. 2 Viscoelastic analysis (phase lag and dynamic stiffness)
in each group. Dynamic stiffness of medial femoral cartilage (a).
Phase lag of medial tibial cartilage (b). Phase lag of lateral tibial
cartilage (c). The horizontal bars indicate the median and the
vertical bars the range. The horizontal boundaries of the boxes
represent the first and third quartiles
Biogerontology
123
taking place in the capsule (Hagiwara et al. 2010b;
Moriyama et al. 2007). The major structural collagens
of the capsule are collagen types I and III, with the
former accounting for 83 % of all collagen present
(Hagiwara et al. 2010b; Kleftogiannis et al. 1994).
Collagen type I is found in tissues requiring high levels
of mechanical strength (Hayashi and Nagai 1981) and
its expression is increased at sites where new fibrosis
and connective tissue proliferation occur (Matsumoto
et al. 2002; Schollmeier et al. 1996). Increased
collagen cross-linking is central to stiffening of the
collagen network and loss of flexibility in articular
structures (Ahmed et al. 2005). Earlier studies have
reported that the concentration of pentosidine, one of
the key proteins associated with joint stiffness,
increased with age, giving rise to increased collagen
cross-linking (Bank et al. 1998). Unexpectedly, the
results of this study suggest that accumulation of
collagen type I and pentosidine did not cause joint
stiffness and that changes in the capsule did not result
from proliferative fibrosis. The mechanical properties
of the capsule remained unchanged with aging, which
further indicates that joint stiffness with aging is
independent of capsular stiffness. Joint stiffness
probably results from a combination of age-related
changes seen in articular structures (e.g., muscles and
articular cartilage) other than the joint capsule.
Collagen type III is abundant in tissues that require
high levels of mechanical compliance (Bornstein and
Sage 1980; Hayashi and Nagai 1981). It is a major
constituent of normal synovial membranes (Bland and
Ashhurst 1997) and is also present in the inflamed and
rheumatoid synovial membrane (Adam et al. 1976;
Hagiwara et al. 2010b; Weiss et al. 1975). Synovial
angiogenesis has been linked to histological synovitis,
in particular macrophage infiltration of the synovium
(Haywood et al. 2003; Walsh et al. 2007), and is
stimulated during the inflammatory response that
accompanies the pathological progression of OA
(Appleton et al. 2007; Im et al. 2010). Blood vessel
formation has been found to be substantially increased
in the synovium in knee joints of late-stage human OA
(Im et al. 2010). In line with these earlier reports, our
histological and immunohistochemical findings indi-
cate that synovitis is not a characteristic feature of the
aging joint.
Animal models of OA have provided biological
insights into OA-induced progressive pathological
changes in knee joint structures (Im et al. 2010).
Spontaneous OA is uncommon in rats (Gerwin et al.
2010), although minor foci of articular cartilage
degeneration have been observed (Adams and Horton
1998; Gyarmati et al. 1987; Smale et al. 1995). Aging
and the development of cartilage degeneration involve
many factors, which either alone or in combination
may accelerate the onset of OA (Grogan and D’Lima
2010). OA increases in prevalence with increasing age
to becoming almost ubiquitous in elderly populations,
making it difficult to be easily distinguished from age-
related changes (Mapp et al. 2008). However, our
work emphasizes that it is important to understand the
difference between the effects of natural aging and the
manifestation of OA in joints. The changes seen in
articular cartilage with aging followed a pattern
similar to that of OA, while no sign of synovitis was
observed. The extracellular matrix and cell functions
of articular cartilage considered the most important
factors in the development and progression of OA
were found to change with aging. Although it is
recommended that much older rats are used to mimic
the development of OA in humans as closely as
possible for all rat models of OA (Gerwin et al. 2010),
future studies should require more attention to over-
estimating these severities. Also, prevalence of knee
OA was higher in women than in men (Pereira et al.
2011). The female hormones have the potential to alter
the properties of the periarticular connective tissues
(Ohtera et al. 2002); therefore we used male rats.
Further study is needed to clarify whether similar
results are expected in females or not.
The age-related changes seen in articular cartilage
(with aging) were not abolished in our exercised old
rats. This lack of cartilage recovery may be due to
several factors, including the duration of the interven-
tion, the regenerative capacity of the cartilage, and the
small sample size. The exercise intervention period in
the present study was identical to that of earlier studies
investigating the effect of treadmill exercise training
on physical performance and skeletal muscle apopto-
sis in old rats (Marzetti et al. 2008). Balance training
can be effective for postural and neuromuscular
control improvements (Zech et al. 2010).Muscle and
neuromuscular improvements may take much longer
to have a beneficial effect on age-related changes in
the joint. With little or no potential for cartilage
regeneration, additional research on this possibility is
warranted. The statistical power of the present findings
was partly reduced by the small sample size due to the
Biogerontology
123
half-mortality age of rats, which does not allow ruling
out chance findings. Parameters with low statistical
power are likely to generate/produce type II errors,
necessitating larger sample sizes in future studies.
In conclusion, our study confirms that aging alone, as
investigated in mechanical tests and by light micros-
copy, results in joint degeneration, eventually leading to
OA, in a male rat model. Joint degeneration do not
appear to be the inevitable consequence of aging, but
instead is brought about by alterations in the aging joint
that make it more susceptible to degeneration.
Acknowledgments This study was supported in part by
Grant-in-Aid for Scientific Research (21500483) and for
Young Scientists (21700545) from the Japanese Ministry of
Education, Culture, Sports, Science, and Technology. We thank
Dr. Yoshiko Tobimatsu and Professor Seiichi Kawamata for
their advice and expertise; Mr. Yoshio Shirasaki, Mr. Francis
Terloo, Ms. Sofie Thys, and Mr. Dominique De Rijck for their
skilled technical assistance; and Dr. Hidetaka Imagita and Mr.
Tomoyuki Kurose for helpful discussions.
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