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Mammalian Limb Loading and Chondral Modeling During Ontogeny.

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THE ANATOMICAL RECORD 293:658–670 (2010)
Mammalian Limb Loading and Chondral
Modeling During Ontogeny
ASHLEY S. HAMMOND, JIE NING, CAROL V. WARD, AND MATTHEW J. RAVOSA*
Department of Pathology and Anatomical Sciences, University of Missouri School of
Medicine, Columbia, Missouri
ABSTRACT
The adaptive growth response of cartilage, or chondral modeling, can
result in changes in joint and limb proportions during ontogeny and ultimately contribute to the adult form. Despite Hamrick’s (1999) reevaluation of the mechanisms of chondral modeling, the process of chondral
modeling remains poorly studied in animal models. Here, we characterize
the macro- and microanatomical responses of the femoral growth plate,
articular cartilage, and bone in 15 juvenile Sus scrofa domestica subjected
to different locomotor activity patterns. The exercised animals exhibit
thinner cartilage zones, greater cellularity and larger proliferative chondrocyte areas in the growth plate, as well as larger femoral dimensions
and a more elongate femoral head compared with sedentary controls. In
general, the growth plate demonstrates greater adaptive changes than
articular cartilage. Moreover, chondrocyte hypertrophy and proliferation
were found to be responsive to locomotor loading and thus more important
factors in chondral modeling than the extracellular matrix variables that
were examined herein. In sum, the underlying mechanisms of adaptive
chondrogenesis and bone plasticity are key to informing evolutionary and
translational studies regarding determinants of variation in joint form
and function. Given the disparity between the predictions of chondral
modeling theory and our experimental findings, this suggests a need for
further evaluation of chondral modeling responses during ontogeny. Anat
C 2010 Wiley-Liss, Inc.
Rec, 293:658–670, 2010. V
Key words: chondral modeling; cartilage composition; exercise;
ontogeny; joint plasticity; pig; femur
Growing bones and joints are dynamic structures,
transforming in dimensions, mass, and physical properties in response to altered mechanical forces and/or loading environments, a process referred to as adaptive or
plastic phenotypic response (Gotthard and Nylin, 1995).
While the modeling and remodeling capabilities of long
bones have been extensively investigated (e.g., Lanyon
and Rubin, 1985; Biewener et al., 1986; Biewener and
Bertram, 1993; Judex and Zernicke, 2000; Hamrick
et al., 2006; Robling et al., 2006), the adaptive postnatal
responses of cartilage, the most fundamental tissue
involved in skeletal and joint formation, and its influence on skeletal morphology remains poorly studied
(Frost, 1979, 1999; Hamrick, 1999; Plochocki et al.,
2009).
Chondral modeling is the adaptive growth response of
cartilage via changes in shape, size, and composition to
create a phenotype that is presumably better suited to
C 2010 WILEY-LISS, INC.
V
altered mechanical environments during ontogeny
(Frost, 1979, 1999; Hamrick, 1999; Plochocki et al.,
2009). To maintain the functionality of a skeletal element or joint system, chondral modeling must facilitate
normal joint and bone movements as well as minimize
potentially damaging tissue contact stresses (Hamrick,
1999; Plochocki et al., 2009). Putative chondral modeling
Grant sponsor: National Institute of Health; Grant number:
NIH # PO1 HL52490.
*Correspondence to: Matthew J. Ravosa, M303 Medical Sciences Building, University of Missouri, Columbia, MO 65212. Fax:
573-884-4612. E-mail: ravosam@missouri.edu
Received 7 January 2010; Accepted 11 January 2010
DOI 10.1002/ar.21136
Published online in Wiley InterScience (www.interscience.wiley.
com).
CHONDRAL MODELING AND LIMB JOINT PLASTICITY
sites include the articular surfaces, the physeal cartilage, and sites of fascial, ligamentous or tendonous
insertion (Frost, 1979). The chondral modeling response
is posited to include regional or widespread cartilage
thickening, changes in cartilage cellular and extracellular matrix (ECM) composition and organization, and
potential for increased calcification and ossification.
Chondral modeling may result in differential mineralization and ossification of the deepest hyaline cartilage
layers (i.e., the calcified layer of articular cartilage, hypertrophic zone of growth plate). As such, it may directly
contribute to the form and proportions of bones via influences on subchondral and diaphyseal bone.
Frost (1979) outlined the basis of chondral modeling
within cartilaginous tissues using several observations.
Similar to the case for modeling and remodeling of bony
elements (Lanyon and Rubin, 1985; Biewener and Bertram, 1993), Frost concluded that a physiological loading
range must exist to maximally stimulate regional cartilage growth. Cartilage growth is generally reduced
under routinely high compressive loads yet enhanced
under moderate forces, although the specific magnitude
and frequency of such loads was unclear. Under Frost’s
model, negative feedback from unequal mechanical loads
is responsible for chondral modeling. For example, high
load-bearing joint cartilage will cease growth yet compensatory growth will occur in adjacent areas to more
equally distribute the load.
The changes associated with chondral modeling,
including tissue thickness, content, organization, and
production rates of extracellular matrix components, are
regulated by chondrocytes (Kiviranta et al., 1992). Furthermore, chondrocyte proliferation and metabolism, as
well as the morphological variables under their control
(e.g., ECM synthesis, proteoglycan production) are
known to be influenced by mechanical loading (Eggli
et al., 1988; Kiviranta et al., 1988; Urban, 1994; Wu and
Chen, 2000; Liu et al., 2001; Carter and Wong, 2003;
Ravosa et al., 2007, 2008a,b). Hamrick (1999) reevaluated the chondral modeling theory, identified the optimum range and frequency of hydrostatic pressure that
stimulates chondral modeling during ontogeny, and proposed specific ways that cartilage will adaptively
respond to moderate levels of mechanical stimuli. In
order to produce uniform hydrostatic pressure throughout the tissue, Hamrick (1999) predicted that cartilage
should respond to altered mechanical loads through differential chondrocyte division and cartilage matrix
synthesis.
Despite Hamrick’s extensive review of the theory
underlying chondral modeling, no published studies
have explicitly tested the model’s adaptive response
mechanisms in vivo. Thus, we tested certain predictions
of the chondral modeling theory via measures for altered
matrix and cellularity in a group of exercised and sedentary pigs. Based on modern chondral modeling theory,
we expected to find increased ECM, increased viscoelasticity through elevated proteoglycan content, increased
cellularity, increased average cell size, larger femoral
dimensions, and flatter joints in the exercised group
(Frost, 1979; Paukkonen et al., 1985; Eggli et al., 1988;
Urban, 1994; Hamrick, 1999; Plochocki et al., 2006,
2009). Increases in chondrocyte proliferation indicate the
availability for differential mineralization, increased
bone growth, and an increased ability to alter the mor-
659
phological variables of cartilage (Hunziker and Schenk,
1989). Although cell size is expected to vary throughout
the depth of cartilage itself, an increase in average cell
size in high-load areas has been suggested to represent
either enhanced physical properties or a metabolic functional adaptation to loading (Paukkonen et al., 1985;
Eggli et al., 1988; Freeman et al., 1994). Holding cell
size and cell number constant, increased ECM and
changes in the composition (e.g., proteoglycan content)
will physically alter the cartilage’s thickness and viscoelasticity, and therefore alter its ability to withstand loading. Here we characterize the effects of endurance
running on femoral head growth plate, articular cartilage, and bone in relation to the predictions of chondral
modeling theory. Such information is critical to a more
complete understanding of the process of chondral modeling and the role of ontogenetic variation in mechanical
loading on intra- and interspecific variation in joint and
limb proportions.
MATERIALS AND METHODS
Sample
Procedures performed in this experiment were
approved by the University of Missouri Animal Care and
Use Committee under protocol 472–2. Fifteen castrated
male juvenile miniature swine (Sus scrofa domesticus)
were used in this study. The pigs were housed in contiguous plastic fence enclosures with concrete flooring, limiting physical but not visual and acoustic access to other
pigs. The dimensions of the individual crates were 1.5 0.9 m (1.4 m2). The swine were supplied with water ad
libitum and fed a high-fat diet provided once daily. All
animals were fed the same amount of food, regardless of
participation in the exercise regime or being sedentary.
It should be noted that a high-fat diet has the potential
to slow bone mineralization and cartilage regeneration
(see Silberberg and Silberberg, 1950; Zernicke et al.,
1995; Wohl et al., 1998), reducing the potential to induce
and document major gross and histomorphometric
changes.
Pigs are not skeletally mature until 5 to 6 years of
age, with the femoral proximal growth plate remaining
unfused until 3 years old (Barone, 1999; Dyce et al.,
2002). The juvenile pigs began the protocol at 8 months
of age and were sacrificed after seventeen weeks of participation in the experiment. The pigs were divided into
two groups comprised of seven exercised and eight sedentary animals. The 15 pigs comprising the sample came
from eight different litters, and brother pigs were divided between experimental groups as equally as possible. During the experimental period, exercised swine
completed treadmill running to exertion limit 5 days a
week, while the sedentary cohort was raised without exposure to exercise for the same seventeen week period.
The exercise training was done on electric motorized
ClubTrack 3.0 PLUS treadmills (Quinton; Bothell, WA).
Dynamic treadmill running consisted of four stages:
warm-up (2.0–2.5 mph), a high-intensity sprint (4.0–7.0
mph), endurance running (3.0–5.0 mph), and cool-down
(1.5–2.5 mph). A 5 min warm-up was followed by a 15
min sprint, a variable-length high-intensity endurance
run, and was finished by a 5 min cool-down. The pigs
were unable to maintain high-intensity speed and duration in the beginning of the experiment due to lower
660
HAMMOND ET AL.
TABLE 1. Mean exercise regime changes over 17 weeks for all 7 exercised pigs (6 SD)
Week
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Mean sprint
MPH
Mean sprint
time (min.)
4.00
4.24
4.65
5.17
5.46
5.63
5.72
5.97
6.14
6.26
6.36
6.14
6.17
5.91
6.12
5.66
12.50
15.00
15.00
15.00
15.00
15.00
15.00
15.00
15.00
15.00
15.00
15.00
15.00
15.00
15.00
14.83
(0.00)
(0.18)
(0.30)
(0.18)
(0.09)
(0.24)
(0.23)
(0.45)
(0.61)
(0.63)
(0.46)
(0.63)
(0.62)
(0.99)
(0.77)
(1.08)
a
Mean endurance
MPH
(2.10)
(0.00)
(0.00)
(0.00)
(0.00)
(0.00)
(0.00)
(0.00)
(0.00)
(0.00)
(0.00)
(0.00)
(0.00)
(0.00)
(0.00)
(3.12)
a
3.00
3.00
3.31
3.50
3.50
3.47
3.50
3.49
3.65
3.66
4.04
4.28
4.28
4.19
4.15
3.88
(0.00)
(0.02)
(0.26)
(0.00)
(0.00)
(0.17)
(0.00)
(0.03)
(0.15)
(0.18)
(0.26)
(0.37)
(0.56)
(0.63)
(0.54)
(0.66)
a
Mean endurance
Time (min.)
22.17
26.57
32.00
36.47
39.83
42.17
45.00
47.30
49.33
50.48
55.70
56.55
58.17
56.33
54.52
50.56
(2.45)
(1.64)
(1.86)
(1.57)
(0.91)
(1.70)
(0.00)
(1.76)
(3.65)
(7.98)
(4.15)
(10.19)
(4.04)
(6.42)
(11.29)
(14.45)
a
Mean total
distance (mi.)
11.57
13.87
16.53
18.95
20.41
21.08
22.27
22.82
24.58
25.05
28.91
29.02
31.18
29.12
28.93
24.98
(1.02)
(0.32)
(0.57)
(0.53)
(0.31)
(0.43)
(0.28)
(1.40)
(1.32)
(1.80)
(1.79)
(3.45)
(2.35)
(4.83)
(5.21)
(7.59)
a
The non-variable daily components of the exercise regime, warm up and cool down, averaged 2.4 mph/5 min and 2.3 mph/5
min.
a
Sacrifice week represents an incomplete exercise regime week.
fitness levels and, thus, variation in duration in total
work-out time and speed was necessary. Total endurance
running times were variable, building from 45 min at
the start of the protocol to 80 min in weeks 11–15. Average distance run built from 2 miles per day at the start
of the protocol to over 5 miles per day by week 11 with a
simultaneous increase in proportion of the run spent at
high-intensity speeds (Table 1). It is believed that the
pig joints experienced moderate to high levels of loading
given the intensity of the experimental protocol.
All exercised pigs were exercised the day before sacrifice, and all pigs were weighed the day of sacrifice. Pigs
were euthanized using a Telazol (5 mg/kg) and Xylazine
(2.25 mg/kg) injection, followed by thiopental (25 mg/kg)
to deep anesthesia. The secondary means of assuring
death was through exsanguination and removal of the
heart. Following sacrifice, the swine femora, including
intact articular cartilage, were immediately dissected
from the pelvis and surrounding tissues.
Measurements
Thirteen femoral measurements (Table 2) were taken
with digital calipers on the left femur (Fig. 1a), intact
with cartilage, following Ruff (2002) (see footnote). Bones
were fixed by immersion in 10% neutral buffered formalin. A DELTA band saw (DELTA; Jackson, TN) was used
to remove the proximal end of the femur, which was
then immersed in Surgipath Decalcifier II decalcification
solution (Surgipath Medical Ind., Inc.; Richmond, IL) for
2 weeks. The left femoral head was divided coronally
into 5 mm thick sections (Fig. 1b), photographed, dehydrated and paraffin imbedded for histological sectioning.
Linear digital measures designed to quantify femoral
head curvature were taken from images of the thin-sectioned femoral heads (Fig. 1c; Table 2). A ratio of (45
dorsal chord/45 ventral chord) captures the femoral
head’s departure from roundness, whereas the ratios
(subchondral arc width/midpoint chord length) and
(articular arc width/articular chord) show change influ-
enced by epiphyseal bone shape and whole joint shape,
respectively. Articular surface area was estimated based
on Ruff ’s (2002) formula for a partial sphere
1.57*FHDP* (FHSIþFHAP). It should be noted that this
surface area estimation is biased towards linear measures and not shape changes, which may influence articular area.
A Reichert-Jung 2040 Autocut microtome (ReichertJung, Inc.; West Germany) was used to obtain histological sections at 4 lm, which were then floated in a water
bath, deparaffinized, and stained. Both groups were
stained concurrently to reduce temporal variability in
staining intensity. Hematoxylin and Eosin (H&E) staining was employed to identify cartilage zones for histomorphometric analyses. A second set of slides was
stained with Safranin-O, a proteoglycan indicator which
was used to qualitatively evaluate variation in proteoglycan content of the ECM. All things being equal, cartilage
with higher amounts of proteoglycans (glycosaminoglycans - GAGs - bound to a protein core) typically has
enhanced tissue stiffness and viscoelasticity (Jurvelin
et al., 1986; Kiviranta et al., 1987; Tanaka et al., 2003).
Slides were analyzed with an Olympus BX41 microscope (Olympus Corp.; Tokyo, Japan). Eight sites on
each H&E slide were assessed, four sites per physeal
and four per articular cartilage (Fig. 1d). These sites
were anatomically-determined and chosen by a single
observer (ASH) for their repeatability on all slides. It
should be noted that the epiphyseal plate exhibited
stereotypical undulations (in coronal section) and the
locations of measurement sites were selected based on
FHAP is a measure of the anteroposterior femoral head maximum width, when the observer orients the bone vertically from
a supporting surface. FHSI is a measure of the superoinferior
femoral head maximum width, when the observer orients the
bone vertically from a supporting surface. FHDP is a measure
taken perpendicular to FHSI through the center of the head to
its intersection with the lateral border of the articular surface,
with the anterior (or cranial) surface facing the observer. All
linear measure definitions from Ruff (2002).
661
CHONDRAL MODELING AND LIMB JOINT PLASTICITY
TABLE 2. Body size measurement means (6SD) of control (sedentary) and exercised pigs
Mass (kg)
Mass al start of experiment
Mass at sacrifice
Femoral linear msmt. (mm)
Femoral head height (FHDP)
Femoral head S-I breadth (FHSI)
Femoral head A-P breadth (FHAP)
Neck S-I breadth
Neck A-P breadth
M-L breadth of medial condyle
M-L breadth of lateral condyle
M-L condylar surface
S-I breadth of medial condyle
S-I breadth of lateral condyle
M-L mid-diaphyseal breadth
A-P mid-diaphyseal breadth
Length (g. troch to lat. condyle)
Subchondral bone msmt.
Epiphyses cross-sectional area (mm2)
Max subchondral width
Subchondral arc width
Midpoint chord length
Articular arc width
Articular chord (from MP of width)
Dorsal chord 45 degrees from midpoint (mm)
Ventral chord 45 degrees from midpoint (mm)
Dorsal chord/ventral chord
Joint Size and Curvature Ratios
Joint size surrogate (FHSI * FHAP, mm2)
Femoral head surface area (mm2)
Subchondral arc width/midpoint chord length
Articular arc width/articular chord
Sedentary (N ¼ 8)
Trend
Exercised (N ¼ 7)
% Difference
P-Value
36.59 (8.34)
74.50 (9.03)
¼
>
36.95 (1.17)
45.50 (2.51)
0.98
38.93
0.35
0.22
18.91
26.70
25.45
25.73
21.37
16.77
17.02
42.21
29.08
27.67
18.63
20.40
168.06
(1.68)
(1.59)
(1.27)
(1.66)
(0.86)
(1.26)
(1.35)
(1.68)
(2.23)
(1.73)
(1.31)
(1.11)
(10.11)
>
¼
¼
>
<
<
<
<
<
<
<
¼
<
18.57
26.83
25.65
25.27
22.05
17.18
17.33
42.84
29.56
29.04
19.32
20.34
171.00
(0.81)
(0.75)
(0.48)
(0.48)
(1.18)
(0.78)
(0.69)
(0.92)
(0.62)
(0.82)
(0.73)
(0.69)
(2.84)
1.80
0.49
0.79
1.79
3.18
2.44
1.82
1.49
1.65
4.95
3.70
0.29
1.75
0.73
0.64
0.73
0.11
0.30
0.25
0.42
0.30
0.42
0.06
0.30
1.00
1.00
176.94
23.85
22.40
11.55
25.24
12.29
11.40
11.47
0.97
(21.08)
(1.51)
(1.42)
(0.71)
(1.56)
(0.70)
(0.47)
(0.83)
(0.04)
<
¼
>
<
>
<
>
<
>
186.01
23.75
21.63
12.01
24.95
12.76
11.16
12.01
0.93
(21.20)
(0.39)
(1.49)
(0.93)
(1.34)
(0.59)
(1.04)
(0.41)
(0.07)
5.13
0.42
3.44
3.98
1.15
3.82
2.11
4.71
4.12
0.56
0.95
0.30
0.20
0.49
0.18
0.64
0.15
0.05a
680.87
1553.94
1.94
2.05
(67.67)
(206.44)
(0.08)
(0.08)
<
>
>
>
688.14
1530.22
1.80
1.96
(22.90)
(79.49)
(0.10)
(0.05)
1.07
1.53
7.22
4.39
1.00
0.56
0.02a
0.02a
P-values were calculated from the sedentary and exercised individuals for each measure.
a
Significance P < 0.05. Percent difference is calculated as the absolute value of (exercised mean - sedentary mean) divided
by the sedentary average. Less than 1% difference is considered approximately equal. Cartilage height, or thickness, is
equal to layer area/300 lm. Cell area is approximated by the equation (0.5 h * 0.51 * p).
equidistance between a characteristic inferior undulation
(site VII in Fig. 1d) and growth plate articular cartilage.
Digital images of all sites analyzed were saved in Tagged
Image File Format (*.tiff) for histomorphometric analysis. All size values taken from the images were calibrated from a microscopy scale bar to pixels. A single
observer (ASH) collected all measurements and was
blinded to the experimental regime of each specimen
during data collection.
NIH ImageJ 1.40 (National Institutes of Health; Bethesda, M.D.) was used to align the images so that the
cartilage-bone interface was parallel to the horizontal
axes. A standard longitudinal column 300 lm wide was
selected from the center of each image. Within these
standardized images, boundaries of the cartilage were
delimited and defined as the superior cartilage border to
the inferior edge of the ‘‘tidemark,’’ a boundary between
the uncalcified and calcified cartilage that stains deeply
with hematoxylin. Three layers (hypertrophic, proliferative, reserve) within the growth plate hyaline cartilage
were manually delimited based on cell morphology
(Niehoff et al., 2004). Cell morphology is an indicator for
the behavior of the cells. The hypertrophic layer (HZ)
was characterized by voluminous hypertrophying cells,
where width approximated cell height, and included cells
undergoing calcification and resorption. The proliferative
layer (PZ) was defined by elongated, homogeneous cells
whose width was typically twice cell height. The proliferative cells were further characterized by their distinct
columnar configuration, oriented perpendicular to the
horizontal axis of the growth plate. The reserve zone
(RZ) was defined here by the start of the proliferative
zone to the beginning of the epiphyseal bone and contained round stem-like chondrocytes. As in Fig. 2, the
three layers of chondrocytes appear large and round in
the hypertrophic layer, stacked and flat in the proliferative layer, and small and erratically-arranged in the
reserve layer.
Unlike in the growth plate, chondrocyte morphology
within the articular cartilage was not clearly identifiable. While raw thickness and cellularity were calculated,
histomorphometric measures involving individual layers
of the articular cartilage were not computed.
An average thickness was calculated for the articular
cartilage and average thicknesses were calculated for
each cartilage layer of the growth plate based on the
formula ‘‘height’’ ¼ area/300 lm. Cell counts were
computed by individually numbering and counting all
chondrocytes within the standard image frames. Cell
counts were then scaled to the ‘‘height’’ of the cartilage
(‘‘cell count/height’’). ‘‘Cell area’’ was calculated in the
growth plate from cellular dimensions of 6 cells by the
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HAMMOND ET AL.
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Fig. 1. Methods. (a) A laser scanned Sus scrofa domesticus femoral head to show morphology (posterior view). (b) The femoral head,
demonstrating the coronal plane where sections of bone were
removed for histological preparation (indicated by green line). (c) A
sectioned Sus scrofa domesticus femoral head demonstrating measures used to determine joint curvature, from sedentary pig # 12–3.
The red line is the ‘‘subchondral arc width’’, measured at the inferiormost aspect of the epiphyseal subchondral bone. The black chord, or
‘‘midpoint chord length,’’ is a midpoint measure taken 90 to the subchondral width and terminating at the surface of the bone. A dorsal
and ventral chord length was measured at 45 to the midpoint chord
length, indicated by the dashed lines. The dorsal chord length terminated at the subchondral-articular cartilage interface, and the ventral
chord length terminated where the subchondral-articular cartilage
interface would be if the natural curve of the bone was continued over
the fovea capitis. The ‘‘articular arc width,’’ represented in yellow, is
the width of the articular surface. The ‘‘articular chord’’ is the midpoint
90 vertical height to articular surface is blue. When scaled as subchondral arc width/midpoint chord length and articular arc width/articular chord, these are measures for the subchondral and articular
surface curvature. (d) Hematoxylin and Eosin (H&E) preparation showing eight sample sites for histomorphometric analysis on the femoral
head of exercised pig 15–4. Sites I-IV are articular cartilage sample
sites, V-VIII are growth plate cartilage samples. The white arrows for
(a,c,d) indicates the dorsal loading surface; note the difference in
shape of the dorsal loading surface. (a-d) are oriented to the reference, where D is dorsal and V is ventral.
formula cell area ¼ (0.5 h * 0.5 L * p). We did not use a
randomizer to select cells in order to avoid oversampling
cells from the same chondrocyte parent line. Instead, the
cells used to compute cell area were selected throughout
the image frame in order to ensure sampling from multiple cell lineages.
uated qualitatively. Histological sample sites that were
deemed unsuitable for inclusion in the analysis due
to damage during histological preparation were not
included in the statistical analyses. This is reflected in
varying sample size values listed in variables used for
histomorphometrics in Tables 3 and 4.
RESULTS
Statistical Analyses
Between-group comparisons of linear metrics, ratios
and cell counts were compared via a series of discriminant function analyses. Due to the small sample size,
nonparametric ANOVA was used to assess variation in
specific parameters (P < 0.05) between groups. The nonparametric ANOVAs also show directionality to facilitate
the interpretation of discriminant function analyses
between groups. Safranin-O staining intensity was eval-
Body Size
No significant difference in body mass was found
between the exercised and sedentary control groups at
the start or conclusion of the experiment (Table 2).
There was more variability in body mass in the sedentary sample, with both the largest and smallest weight
values observed in the sedentary group. Nonetheless,
due to the lack of mean differences, between-group
CHONDRAL MODELING AND LIMB JOINT PLASTICITY
663
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Fig. 2. Examples of cartilage histology sample sites in the proximal
femur of juvenile Sus scrofa domesticus. (a, b) are articular cartilage
H&E preparations of region III from sedentary pig #15–1 and exercised
pig #15–4. (c, d) show a Safranin-O preparation of site VI on the
growth plate from sedentary pig #15–1 and exercised pig #15–4.
Safranin-O staining results for articular cartilage were negligible and
are not included. (e, f) shows H&E preparations of the growth plates
of region VI for sedentary pig #11–1 and exercised pig #10–1. (e) demonstrates the cell types used for layer identification with reserve
(RCZ), proliferative (PZ), and hypertrophic chondrocytes (HCZ) identified in an insert. Scale: All images presented in 10 objective except
(e, f) which were imaged at 20 objective and the inset to (e) which
was imaged at 40. All scale bars represent 50 lm.
variation in joint and limb form is more readily attributable to the exercise regimen than body mass.
matrices. The articular cartilage zone thickness was not
highly distinct between groups with only 67% classifying
correctly in a discriminant function analysis (Table 3, 5).
The exercised group demonstrated thinner growth plate
cartilage zones, with 93% of the sample classifying correctly in a discriminant function analysis (Table 4, 6).
This was confirmed by the univariate analyses, where
the growth plate of exercised swine tended to be thinner,
ECM Thickness
Data for articular cartilage and growth plate cartilage
are summarized in Tables 3 and 4, respectively. Tables 5
and 6 summarize discriminant function classification
664
HAMMOND ET AL.
TABLE 3. Articular cartilage measurement means (6SD) of sedentary and exercised pigs
Sedentary1,2,3
Cartilage thickness (‘height’’)
I Total height raw (lm)
II Total height raw (lm)
III Total height raw (lm)
IV Total height raw (lm)
Cell count scaled to cartilage thickness
I Total cell count: total height
II Total cell count: total height
III Total cell count: total height
IV Total cell count: total height
782.33
593.66
774.77
627.57
0.26
0.27
0.22
0.24
Trend
Exercised3,4,5
(170.02)3
(174.09)1
(162.50)3
(128.90)2
>
>
<
¼
662.02
554.80
843.68
624.71
(0.04)3
(0.08)2
(0.04)1
(0.06)1
<
<
<
>
0.28
0.29
0.24
0.22
Number of individuals is indicated by superscript numbers.
individuals, 5 ¼ 3 individuals.
1
¼ 8 individuals,
2
% Difference
P-Value
15.38
6.55
8.89
0.46
0.29
0.57
0.68
0.80
7.69
7.41
9.09
8.33
0.83
0.57
0.41
0.52
(140.43)4
(140.31)3
(307.75)4
(140.16)5
(0.08)4
(0.07)5
(0.05)5
(0.04)3
¼ 7 individuals,
3
¼ 6 individuals,
4
¼ 4
TABLE 4. Growth plate measurement means (6SD) of sedentary and exercised pigs
Sedentary1
Cartilage layer height
V height reserve zone (lm)
VI height reserve zone (lm)
VII height reserve zone (lm)
VIII height reserve zone (lm)
V height proliferative zone (lm)
VI height proliferative zone (lm)
VII height proliferative zone (lm)
VIII height proliferative zone (lm)
V height hypertrophic zone (lm)
VI height hypertrophic zone (lm)
VII height hypertrophic zone (lm)
VIII height hvpertroohic zone (lm)
Cell count scaled to layer height
V reserve cell count: height reserve zone
VI reserve cell count: height reserve zone
VII reserve cell count: height reserve zone
VIII reserve cell count: height reserve zone
V proliferative cell count: height proliferative zone
VI proliferative cell count: height proliferative zone
VII proliferative cell count: height proliferative zone
VIII proliferative cell count: height proliferative zone
V hypertrophic cell count: height hypertrophic zone
VI hypertrophic cell count: height hypertrophic zone
VII hypertrophic cell count: height hypertrophic zone
VIII hypertrophic cell count: height hypertrophic zone
Average cell area
V reserve cell area (lm2)
VI reserve cell area (lm2)
VII reserve cell area (lm2)
VIII reserve cell area (lm2)
V proliferative cell area (lm2)
VI proliferative cell area (lm2)
VII proliferative cell area (lm2)
VIII proliferative cell area (lm2)
V hypertrophic cell area (lm2)
VI hypertrophic cell area (lm2)
VII hypertrophic cell area (lm2)
VIII hypertrophic cell area (lm2)
98.52
126.14
135.76
122.82
185.35
260.22
211.64
132.35
104.42
98.35
90.56
80.61
0.34
0.31
0.30
0.34
0.61
0.61
0.77
0.85
0.55
0.48
0.66
0.60
52.58
48.75
42.57
45.53
56.31
45.83
41.13
52.18
201.53
186.52
178.29
208.87
Number of individuals is indicated by superscript numbers.
individuals, 5 ¼ 3 individuals.
with reductions in average thickness localized in the
proliferative zone.
Cartilage ECM Composition
Micrographs of articular and physeal cartilage in the
two groups are shown in Figs. 2a–d. High levels of
1
Trend
Exercised2,3
(34.09)1
(45.06)1
(40.75)1
(48.04)1
(49.22)1
(122.53)1
(30.78)1
(36.14)1
(24.07)1
(43.35)1
(51.48)1
(19.25)1
<
>
>
>
>
>
>
>
<
>
>
<
131.08
122.72
120.13
112.83
154.82
176.29
161.50
122.56
105.90
80.54
66.29
83.72
(0.14)1
(0.09)1
(0.08)1
(0.13)1
(0.16)1
(0.22)1
(0.25)1
(0.19)1
(0.21)1
(0.18)1
(0.27)1
(0.17)1
>
<
<
>
<
<
<
>
<
<
<
¼
0.33
0.37
0.35
0.32
0.73
0.72
0.88
0.80
0.63
0.67
0.74
0.60
(20.53)1
(19.41)1
(16.94)1
(15.82)1
(18.57)1
(17.14)1
(13.11)1
(11.14)1
(47.46)1
(28.08)1
(95.85)1
(88.92)1
>
>
<
<
<
<
<
<
>
>
>
¼
43.04
42.33
48.33
49.02
64.94
47.43
44.75
59.90
180.88
182.65
147.16
209.28
¼ 8 individuals,
2
% Difference P-Value
(56.07)3
(15.91)2
(31.01)2
(54.27)2
(71.78)3
(52.36)2
(46.62)2
(22.55)2
(56.20)3
(12.50)2
(13.07)2
(30.57)2
33.05
2.71
11.51
8.13
16.47
32.25
23.69
7.40
1.42
18.11
26.80
3.86
0.30
0.64
0.56
0.56
0.37
0.20
0.06
0.49
0.70
0.91
0.64
0.91
(0.03)3
(0.12)2
(0.09)2
(0.12)2
(0.28)3
(0.19)2
(0.17)2
(0.22)2
(0.23)3
(0.18)2
(0.17)2
(0.19)2
2.94
19.35
16.67
5.88
19.67
18.03
14.29
5.88
14.55
39.58
12.12
0.00
0.44
0.20
0.49
0.56
0.37
0.42
0.42
0.56
0.52
0.06
0.91
1.00
(8.64)3
(12.28)2
(10.84)2
(18.36)2
(17.08)3
(8.05)2
(12.48)2
(24.13)2
(68.11)3
(28.67)2
(20.65)2
(22.46)2
18.14
13.17
13.53
7.67
15.33
3.49
8.80
14.79
10.25
2.07
17.46
0.20
0.61
0.48
0.36
0.64
0.37
0.49
0.73
0.82
0.61
0.82
0.91
0.36
¼ 7 individuals,
3
¼ 6 individuals,
4
¼ 4
safranin staining were observed in the physeal cartilage in the exercised and sedentary groups, with both
groups appearing very similar in overall staining characteristics and matrix composition (Figs. 2c,d). Maximum positive staining occurred in the hypertrophic
region in both groups, with moderate staining in all
other regions. Negligible Safranin-O staining was
665
CHONDRAL MODELING AND LIMB JOINT PLASTICITY
TABLE 5. Articular cartilage discriminant
function classification matrices
Predicted
sedentary
Total height (Sites I–IV)
Sedentary (N ¼ 6)
Exercised (N ¼ 3)
Total
Total cell count scaled to
total height (Sites I-IV)
Sedentary (N ¼ 6)
Exercised (N ¼ 3)
Total
TABLE 7. Classification matrix for 13 femoral
linear measurements
Predicted
exercised
%
Correct
4
1
5
2
2
4
67
67
67
2
1
3
4
2
6
33
67
44
The canonical correlations are 0.638 for heights raw and
0.320 for total cell count scaled to total height.
Number of individuals is indicated by superscript numbers.
TABLE 6. Growth plate discriminant function
classification matrices
Predicted
sedentary
Predicted
exercised
%
Correct
Zone heights raw
(Sites V–VIII; reserve,
proliferative,
hypertrophic)
Sedentary (N ¼ 8)
Exercised (N ¼ 6)
Total
7
0
7
1
6
7
88
100
93
Cell counts scaled
by section height
(Sites V–VIII; reserve,
proliferative,
hypertrophic)
Sedentary (N ¼ 8)
Exercised (n ¼ 6)
Total
7
1
8
1
5
6
88
83
86
Average cell area
(Sites V–VIII; reserve,
proliferative,
hypertrophic)
Sedentary (N ¼ 8)
Exercised (N ¼ 6)
Total
8
0
8
0
6
6
100
100
100
Sedentary (N ¼ 8)
Exercised (N ¼ 7)
Total
Predicted
sedentary
Predicted
exercised
%
Correct
8
0
8
0
7
7
100
100
100
The canonical correlation value for the femoral measures is
0.895.
Number of individuals is indicated by superscript numbers.
Measures of cellularity within each growth plate cartilage layer were accurate indicators of exercise treatment
group (Table 4). Cellularity measures classified the samples correctly 86% of the time (Table 6). The proliferative
and hypertrophic layers showed the most consistency,
with three of four sample sites each having an average
increase in cell density in the exercised group. The proliferative chondrocytes had a larger average cell area in
the exercised group as well (also see Figs. 2e, f), with
analyses of chondrocyte size correctly classifying all
members (100%) of each locomotor treatment group.
Femoral Size and Shape
The canonical correlations are 0.793 for zone heights raw,
0.819 for cell counts scaled by section height, and 0.996 for
average cell area.
Number of individuals is indicated by superscript numbers.
present in the articular cartilage for both treatment
groups.
Cellularity
Like articular cartilage thickness, articular cellularity
is a poor discriminator as well, with only 44% classifying
correctly (Table 3, 5). Articular cartilage cellularity
tended to be higher in the exercised group, however,
with all dorsally sampled areas (e.g., sites I-III, the
lunate surface contact site) displaying an increased cellularity signal.
All members of the exercised and sedentary locomotor
groups were correctly classified in a discriminant function analysis using the 13 femoral linear measurements
(Table 7). In univariate comparisons, the exercised treatment group tended to exhibit larger femoral dimensions
than the sedentary group in 8 of 13 measures (Table 2).
While joint size did not vary between groups, the exercised cohort had relatively taller epiphyses which created more expansive dorsal subchondral and articular
surfaces. The measure of epiphyseal curvature indicated
by the ratio (45 dorsal chord/45 ventral chord) was significantly different between groups, with the smaller ratio indicative of dorsal flattening found in the exercised
treatment group.
DISCUSSION
The primary function of chondral modeling is to maintain a morphology that maximizes the ability of bone
and cartilage to resist dynamic mechanical loads while
ensuring the overall functional integrity and congruence
of the structures or joint system (Frost, 1979, 1999;
Hamrick, 1999). Chondral modeling has been proposed
to occur through differential chondrocyte mitosis and
synthesis of the ECM, and this signal should be evidenced by (1) increased cartilage thickness and differences in extracellular composition, (2) increased
chondrocyte proliferation and average cell size, and (3)
differences in gross limb dimensions and shape. We only
found support for differential physeal chondrocyte proliferation and altered morphology as well as bone growth,
suggesting that chondral modeling theory may have
understated key implications for adaptive chondrogenesis in bone growth. These findings and their considerations for postcranial bone and joint form are considered
below.
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HAMMOND ET AL.
Extracellular Matrix
Both indicators for ECM activity, cartilage thickness
(‘‘height’’) and proteoglycan content via Safranin-O
staining were not clearly different between groups in the
articular cartilage. When considering the sample sites
most directly affected by loading, however, we may see
evidence of chondral modeling. As Frost (1979) predicted, articular cartilage thickness decreased in highly
loaded dorsal sample sites (I-II) with a simultaneous
increase in thickness adjacent to the high-load areas
(III-IV). It is unclear whether these results indicate an
adaptive ECM response to loading or that articular cartilage is a poor indicator for modeling. Other possibilities
include that the loading regime was not within the optimum threshold for adaptive chondrogenesis or that our
articular cartilage sample was comprised largely of adult
chondrocytes less capable of ECM synthesis.
The exercised and sedentary articular cartilage had
similar matrix composition (e.g., proteoglycan content),
with a minimal Safranin-O staining intensity. Articular
cartilage is known to display a reduction in proteoglycans under high intensity exercise regimes (Kivranta
et al., 1992; Ravosa et al., 2007) or from connective tissue pathology (Archer, 1994; Ostergaard et al., 1999;
LeRoux et al., 2001). These do not explain the poor proteoglycan content in the articular cartilage of this sample, however, as both the exercised and sedentary
treatment groups displayed equally low GAG levels. We
are investigating alternative theories for lack of proteoglycan content in the articular cartilage, including an
examination of the collagen fiber orientation and other
matrix constituents. Nonetheless, it is worth noting that
Safranin-O histological pilot data from exercised and
sedentary juvenile hypercholesterolemic miniature pigs
has yielded identical results.
In the growth plate, the similar Safranin-O staining
between groups qualitatively indicates similar matrix
composition and presumably tissue viscoelasticity levels
for both treatment groups. There was consistently high
safranin staining throughout the growth plate in both
groups, especially in the hypertrophic zone ECM. Strong
hypertrophic staining does not indicate zonal enhanced
viscoelasticity, however, and is a result of proteoglycan
concentration in the hypertrophic zone due to the reduction in matrix volume and calcification of structures that
accompanies normal cell hypertrophy (Alini et al., 1992).
Further exploration of other matrix constituents (e.g.,
water, collagen, noncollagenous proteins) may implicate
other tissue microstructures and/or their organization as
part of the phenotypic response.
Growth plate cartilage layer thickness, an indicator of
differential ECM synthesis, was negatively responsive to
the loading regime. Contrary to expectations of chondral
modeling theory (Hamrick, 1999), our study does not
demonstrate an increase in ECM via cartilage thickness
in loaded animals and, in fact, shows the opposite signal.
It is possible that our exercise regime exceeded the optimum loading threshold to elicit ECM synthesis associated with chondral modeling and in fact inhibited the
production of ECM, an avenue that should be explored
with additional experimentation in mild to moderate
exercise tasks. ECM synthesis was considered an indicator of chondral modeling for a variety of reasons, including known ECM decreases associated with cartilage
degradation and/or chondrocyte deformation as a result
of loading (Hamrick, 1999). Furthermore, chondrocytes
increase their matrix production in the proliferating
stage, thus it was expected that we would see increased
ECM production downstream from this stage (e.g., the
proliferative and hypertrophic zones). The sedentary
group tended to be thicker and was unambiguously
thicker in proliferative height, however. This suggests
that ECM production in this region was either not
mechanoresponsive or displayed cartilage degradation
without repair. Either explanation questions the predicted role of ECM synthesis in chondral modeling.
Chondrocyte Activity
Both the exercised articular cartilage and the growth
plate cartilage displayed elevated cellularity levels when
scaled to cartilage layer thickness, confirming that chondrocyte proliferation plays a key role in joint mechanobiology. The growth plate in the exercised group exhibited
relative increases chondrocyte numbers in the proliferative and hypertrophic zones. Previous research has
shown altered mechanical loading to increase proliferative chondrocytes (Wu and Chen, 2000), as well as
increases in hypertrophic chondrocytes related to
increased subchondral mineralization (Ravosa et al.,
2007). This directly supports predictions of the chondral
modeling theory regarding chondrocyte mitosis (Frost,
1999; Hamrick, 1999), although it should be noted that
the research herein does not directly document an
increase in chondrocyte mitosis. As the proliferative and
hypertrophic layers of growth plate cartilage are responsible for mitosis and calcification of chondrocytes, respectively, increases in chondrocytes in these regions
indicates an elevated number of cells available for mineralization. Cellularity increases in the growth plate,
especially increased hypertrophic chondrocyte density,
are typically reflected in limb elongation (Hunziker and
Schenk, 1989).
An additional factor to consider is increased cell size
in the proliferative zone of the growth plate. The relationship between cell size and altered loading, while
poorly studied in physeal cartilage, has been examined
thoroughly in articular cartilage (Paukkonen et al.,
1985; Eggli et al., 1988; Freeman et al., 1994; Stokes
et al., 2006). Chondrocytes and matrix regularly undergo
deformation as a result of loading, particularly in the
upper levels of articular cartilage (e.g., the superficial
zone - Guilak, 2000; Carter and Wong, 2003; Grodzinsky
et al., 2006). Our experimental model shows that the
proliferative chondrocytes, in addition to increasing in
density, are relatively larger in the exercised treatment
group. The significance of this plasticity response in proliferative cell size is unclear. However, increases in articular chondrocyte size during loading have been
attributed to altered physiological states, changes in intracellular composition, and changes in viscoelasticity,
osmotic and hydrostatic pressure (Paukkonen et al.,
1985; Eggli et al., 1988; Freeman et al., 1994; Guilak,
2000; Stokes et al., 2006). As the hypertrophic chondrocyte stage follows the proliferative stage, it may be logical to assume that the exercised hypertrophic cells
would also be increased in size compared with the sedentary control cells. In fact, such hypertrophic chondrocytes were smaller than those in the sedentary group,
CHONDRAL MODELING AND LIMB JOINT PLASTICITY
which leaves one to speculate that this is due to an
increased turnover rate in the hypertrophic cells of the
exercise group.
Bone Dimensions and Joint Shape
Our results demonstrate that relative length and
shape of postcranial elements in growing mammals is
indeed differentially influenced by postnatal variation in
loading behavior. This has been linked to an increase in
physeal cellular proliferation and hypertrophy, which
initiates a cascade of cellular and molecular events that
are crucial for bone growth (e.g., apoptosis, resorption,
ossification). Interestingly, while there is an apparent
relationship between cartilage cellularity and bone
measures, our findings show that the correspondence
among loading, bone growth, and growth plate thickness
are not necessarily complementary. Niehoff et al., (2004),
who studied the effects of varying levels of exercise on
distal femoral growth plates in rats, observed that
growth plate height and proliferative zone height were
lower in association with exercise yet the femur lacked
length changes. Robling et al., (2001) found longitudinal
bone growth and growth plate cartilage thickness
uncoupled as well, although differing results for physeal
cartilage thickness response to altered loading. Our
results demonstrate that long bone growth and growth
plate thickness do not necessarily reflect increased loading (Hunziker and Schenk, 1989; Robling et al., 2001;
Niehoff et al., 2004; but see also Seinsheimer and
Sledge, 1981). That is, elevated loading does not result
in a correspondingly larger growth plate, and a larger
growth plate is not essential for greater longitudinal
bone growth.
The human femoral head is slightly nonspherical to
maximize contact with the acetabulum during high loading and reduce vertical resultant forces (Radin, 1980;
Afoke et al., 1984; Adams, 2006). Ratios reflective of
changes in epiphyseal height and flattening of the dorsal
loading surface characterized the exercised treatment
group, which seemingly corresponds with predictions
that joint surfaces should become flatter with increased
loading (Latimer and Lovejoy, 1989; Plochocki et al.,
2006, 2009). The shape changes in the exercised femoral
heads are a direct result of taller epiphyses (i.e., the
mean distance between growth plate and articular surface is larger) and a less-spherical femoral head (45 dorsal chord/45 ventral chord), likely due to a combination
of bone modeling and adaptive chondrogenic activity. A
taller epiphysis may create a more expansive dorsal
loading surface or an increased range of motion (Eckstein et al., 1994, 1997; Steppacher et al., 2008). As the
exercised pigs maintained their normal adducted and
extended femoral position during loading, the relatively
taller epiphyses may be functioning to create a larger
loading surface rather than enhance mobility. If the pig
femora mirror the biomechanical constraints of humans,
less-spherical femoral heads will lessen forces transmitted through the hip by distributing joint loads normally
across a larger joint surface during high loading (Afoke
et al., 1984; Eckstein et al., 1994). Comparison of
changes in the complementary lunate surface would be
necessary to further evaluate this hypothesis. This suggestion would likewise benefit from experimental data
667
regarding femoral head position during peak ground
reaction forces.
Joints where flattening has been hypothesized to occur
have largely been hinge joints, such as the tibiotalar
joint or the knee joint, not the highly integrated rotational ball-and-socket type joint (e.g., Latimer and Lovejoy, 1989; Plochocki et al., 2009; but see Plochocki et al.,
2006). Major flattening of the femoral head is unlikely to
be phenotypically adaptive and is more commonly associated with pathologies such as femoracetabular impingement and hip dysplasia (Lequesne et al., 2004;
Steppacher et al., 2008). In the condyles of the human
distal femur, however, a flatter surface increases joint
contact area and creates a larger surface for loads to
pass normal to the joint during bipedal locomotion than
a highly grooved or curved surface (Heiple and Lovejoy,
1971; Latimer et al., 1987; Organ and Ward, 2006; Sylvester and Organ, 2010). Interestingly, the femoral condyles appear to become both mediolaterally wider and
superoinferiorly taller in the exercised pig group, showing changes in joint form in the distal femoral articular
surface as well (Table 2). Thus, the postnatal plasticity
response of joints due to altered loading may depend on
joint type and mobility requirements, and include adaptive shape changes rather than global increases in size.
Indeed, while we investigate our findings vis-à-vis
chondral modeling theory, generalized bone plasticity
responses should not be overlooked as a contributing factor to changes in skeletal morphology.
Evolutionary Implications
High adaptive plasticity responses in bones and cartilage, including altered joint shapes, can be achieved if
stimulated during early growth (Frost, 1979, 1999;
Robling et al., 2001; Niehoff et al., 2004; Ravosa et al.,
2007, 2008a,b). The altered macro- and microanatomical
variables produced here in a larger experimental model
animal with an extended limb posture loaded with moderate to high loading largely corresponds with work
done in small animals with habitually flexed hips. This
suggests that there may be a pattern of adaptive
changes in mammalian joint form despite inherent anatomical or postural differences (Robling et al., 2001;
Niehoff et al., 2004; Plochocki et al., 2006). It would be
interesting to conduct an interspecific comparison of
effects of loading on joint shape, growth and, potentially,
altered locomotion. Our experimental model, while
unable to show clear altered locomotor adaptations from
chondral modeling, did result in differing joint morphologies for a cohort of male pigs engaged in differing intensities of their normal locomotor activity. These results
appear to suggest that adaptive chondrogenesis and
bone plasticity during ontogeny is likely involved with
intra- and interspecific variation in joint and bone
dimensions in fossil mammals as well.
Despite the overall increase towards larger dimensions
in the exercised group, joint size remained similar and a
slightly smaller average articular surface area was
found in the exercised group. Our results correspond
well with the earlier findings of Lieberman et al., (2001),
who found articular surface area to be conserved regardless of loading regime. As we failed to demonstrate a
plastic phenotypic increase in hip joint size or articular
surface area associated with loading, this has
668
HAMMOND ET AL.
provocative implications for unproven associations
between endurance exercise, its supposed anatomical
correlates, and the evolution of Homo (see Bramble and
Lieberman, 2004). If enlarged joint size or area is an anatomical correlate for this major behavioral adaptation,
then it is more likely to have been a result of directional
genetic changes, rather than phenotypically plastic
response to altered behaviors during postnatal
development.
CONCLUSIONS
Chondral modeling has been theorized to maintain
joint congruence in altered loading environments by
increasing cellularity and cartilage ECM production
(Hamrick 1999; also Frost, 1979, 1999). Despite different
joint shapes in the two experimental groups, ECM synthesis and cartilage viscoelasticity do not appear to
increase in response to an exercise loading regime,
showing that extracellular matrix synthesis and ECM
proteoglycans may not be fundamental in chondral modeling processes. The articular cartilage demonstrated a
poor response to mechanical loading, with minimal differences between treatment groups, and it appears that
the articular cartilage itself can vary greatly in thickness and cell counts even within an experimental group.
It is surprising that the joint morphology was altered
with small histomorphometric differences in the articular cartilage, although it should be noted that the
increased
articular
histomorphometric
measures
occurred in low load areas as Frost (1979) predicted.
Given the lack of a perichondrium on the articular surface, bony articular surface shape changes are primarily
due to articular cartilage modeling activities, although
the bony remodeling activities of subchondral bone
should also be considered during postnatal loading and
adaptive chondral responses (see Rubin and Lanyon,
1984; Murray et al., 2001; Robling et al., 2006).
The growth plate was mechanoresponsive and showed
that chondrocyte hypertrophy and proliferation are important processes in adaptive chondrogenesis and, potentially, in bone plasticity and growth. Overall, the growth
plate appears more responsive to exercise-induced loading than articular cartilage, due likely to higher metabolic activities, increased vascular supply as well as
differentially greater involvement in limb elongation.
These findings may reflect the inherent nature of these
two forms of hyaline cartilage (primarily limb development vs. joint function) as well as their innate responsiveness
to
mechanical
stimuli
(sensitive
vs.
conservative). A greater understanding of how the hierarchically organized structures of the proximal femur
behave under different loading regimes and ultimately
contribute to morphological variation may provide a better interpretation of locomotor behavior in living and fossil species.
While ‘‘chondral modeling’’ may be an appropriate
description of the adaptive plasticity of cartilage related
to altered joint function, it is unclear if the specific
mechanisms, tissue/cellular responses, signal pathways,
etc. previously linked to this hypothesis indeed apply
equally to all types of cartilage, joint configurations, species, ages, and types of loading (Paukkonen et al., 1985;
Kiviranta et al., 1987; Eggli et al., 1988; Kiviranta et al.
1992; Urban, 1994; Sibonga et al., 2000; LeRoux et al.,
2001; Robling et al., 2001; Niehoff et al., 2004; Plochocki
et al., 2006; Ravosa et al., 2007, 2008a,b). Experimental
models have shown variable plasticity responses for cartilage thickness, cellularity, chondrocyte size, proteoglycan content, and skeletal correlates, although one thing
remains clear: cartilage and the morphological parameters influenced by cartilage are in turn modulated by mechanical loading. Therefore, it may be more appropriate
to consider chondral modeling a form of adaptive chondrogenesis owing to the role of postnatal chondral modeling
in both adult cartilage and skeletal morphology.
Given the disparity between our findings and certain
predictions, one should examine a variety of joint types
from different species so as to better gauge the broader
applicability of chondral modeling to notions about
cartilage plasticity. Arguably, a long-term integrative
perspective should be employed so as to more fully characterize the coordinated series of changes at the gross,
cellular and molecular level that facilitate the adaptive
process of chondral modeling (Ravosa et al., 2007,
2008a,b). Moreover, as plasticity responses decrease with
age in a wide range of organisms (Hinton and McNamara, 1984; Meyer, 1987; Bouvier, 1988; Rubin et al.,
1992; Ravosa et al., 2008b), appropriate controls should
be employed in comparing developmental data across
taxa. It would also be informative to examine chondrogenic response at sites of tendonous and ligamentous
insertion and how it may contribute to skeletal morphology. The roles of subchondral modeling and remodeling
are also integral to the shape changes associated with
chondral modeling, yet subchondral and chondral modeling and remodeling have only been examined independently and without a unifying synthesis. Moreover,
despite its known involvement in the formation of subchondral bone and articular cartilage, the role of adaptive chondrogenesis in osteoarthritis remains poorly
studied (Arokoski et al., 2000; Aspden 2008).
The chondral modeling response, perhaps more accurately termed adaptive chondrogenesis, appears to be
complex, site-specific, and highly variable even within
hyaline cartilage, hinting at the importance of intrinsic
cellular mechanisms that underlie the process. This
study suggests that greater insight into adaptive chondrogenesis would profit considerably from research
directed at understanding the nature of joint loads in
vivo. In sum, our experimental analyses regarding joint
plasticity in a high endurance environment have implications for the mechanobiology of limb growth and form,
specifically in terms of identifying which connective tissue components are most responsive to exercise stimuli
and thus potentially linked to normal and abnormal
phenotypes.
ACKNOWLEDGMENTS
Jason Organ, Valerie DeLeon, Timothy Smith, and
Qian Wang are thanked for inviting us to contribute to
their volume on experimental approaches to morphology.
Sincere gratitude to the Laughlin Lab for providing the
experimental specimens, especially Dave Harah and Dr.
Harold M. Laughlin. Specimens were acquired with the
assistance of NIH grant PO1-HL52490 to HML. Stephanie Child and Ian George are thanked as well as
two anonymous reviewers. The Veterinary Medical Diagnostic Lab (VDML) assisted with certain histological
CHONDRAL MODELING AND LIMB JOINT PLASTICITY
methods. ASH was supported by a MU Life Sciences Fellowship and a Life Sciences Travel Award.
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