|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online January 31, 2007
Journal of Experimental Biology 210, 602-613 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02675
Variation in estradiol level affects cortical bone growth in response to mechanical loading in sheep
Department of Anthropology, Peabody Museum, Harvard University, 11 Divinity Avenue, Cambridge, MA 02138, USA
* Author for correspondence (e-mail: mdevlin{at}fas.harvard.edu)
Accepted 29 November 2006
 |
Summary |
|---|
 TOP Summary IntroductionMaterials and methodsResultsDiscussionList of symbols and...References |
|---|
Key words: bone, estradiol, estrogen receptor-alpha, periosteal modeling, sheep, strain
|
Introduction |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of symbols and...References |
|---|
;
Lanyon and Rubin, 1984;
Lanyon and Rubin, 1985;
Martin et al., 1998;
Carter and Beaupré,
2001; Currey,
2002; Pearson and Lieberman,
2004). Strain often stimulates formation of new cortical bone
(modeling), but may also provoke no response (stasis), Haversian remodeling,
or bone resorption by osteoclasts, depending on the magnitude, frequency and
duration of the strain signal and the age of the individual (for a review, see
Currey, 2002). Bones are
particularly sensitive to high-magnitude cyclic loads and to intermittent
loading bouts (Hsieh and Turner,
2001; Robling et al., 2001), and exhibit the greatest growth
response prior to sexual maturity (Jones
et al., 1977; Lieberman et
al., 2003; Pearson and
Lieberman, 2004).
In addition, there is evidence for a trade-off between growth and repair in
tapered limbs, with more modeling in response to strain in proximal limb
elements and more Haversian remodeling in distal limb elements
(Lieberman and Crompton, 1998;
Lieberman and Pearson, 2001;
Lieberman et al., 2003). This
trade-off may reduce the kinetic energy cost of accelerating additional bone
mass in distal segments (e.g. Hildebrand,
1985; Myers and Steudel,
1985; Bertram and Biewener,
1988; Marsh et al.,
2004), but at a cost of higher strains and higher potential for
fatigue-induced microcracks, which may be repaired via Haversian
remodeling. For example, in juvenile sheep, average total strain magnitudes in
the metatarsal are over 50% higher than in the tibia (1850±132
µ
vs 1162±122 µ), and rates of Haversian
remodeling are about 250% higher in the metatarsal than in the tibia
(16.31±4.71 vs 4.67±2.79 secondary osteons
mm–2) (Lieberman et al.,
2004).
At the cellular level, there are multiple pathways by which strain
influences osteoblasts and osteocytes. Potential sensory mechanisms include
fluid flow and communication at gap junctions between osteocyte canaliculi
(Cowin et al., 1995;
Saunders et al., 2001;
Cherian et al., 2003),
Ca2+ flux through stretch-activated ion channels in osteoblast cell
membranes (Guggino et al.,
1989; Davidson et al.,
1996), small electrical charges known as strain-generated
potentials (SGPs) (Cowin and Moss,
2001), and the primary cilium, a cell process involved in
mechanosensation in other tissues
(Whitfield, 2003). Such
extracellular signals initiate a variety of osteogenic intracellular responses
within bone cells, including production of nitrous oxide (NO) and
prostaglandin E2 (PGE2)
(Bakker et al., 2001;
Bakker et al., 2003;
Jessop et al., 2002), and
upregulation of Runx2 (also known as Cbfa1), a transcription factor necessary
for cortical bone matrix secretion and osteoblast differentiation from
precursor cells (Karsenty,
1999; Olsen et al.,
2000).
|
), estradiol
(E2) and strain. Recent in vitro experiments indicate that
strain in osteoblasts causes phosphorylation of ER-, allowing it to
function as a mechanosensory structure
(Damien et al., 1998;
Damien et al., 2000;
Zaman et al., 2000;
Zaman et al., 2006;
Jessop et al., 2001;
Cheng et al., 2002).
Osteoblast response to mechanical stimuli also varies with ER- density.
Transfecting osteoblasts with additional ER- increases strain-induced
proliferation by 40%, while ER- knockout (ERKO) mice exhibit markedly
reduced cortical growth in response to in vivo mechanical loading,
compared to normal controls (Zaman et al.,
2000; Lee et al.,
2003). The relationship between ER- number and strain
sensitivity in osteoblasts is of particular interest because ER-
transcription depends in part on E2 level. ER- transcription
is decreased by estrogen deficiency and increased by E2 treatment
in humans (Hoyland et al.,
1999) and murine models (Lim
et al., 1999; Zhou et al.,
2001; Zaman et al.,
2006).
Although more research is needed on the effects of E2 on
ER- transcription, particularly in cortical bone, there is evidence
that more estrogen leads to more estrogen receptors
(Hoyland et al., 1999;
Zhou et al., 2001;
Zaman et al., 2006), with more
receptors generally producing a greater osteogenic response to strain
(Zaman et al., 2000;
Lee et al., 2003). A
reasonable prediction that follows from these results is that variation in
E2 and ER- could alter cortical bone response to mechanical
stimuli. If so, then the same strain stimulus could produce a range of
osteogenic responses in different individuals, depending on their
E2 levels and ER- density.
Here we test a model of the effects of variation in E2 on
in vivo cortical responses to strain in limb bone midshafts
(Fig. 1). We focus on the
midshaft because it is the site of maximum bending within a diaphysis
(Biewener et al., 1986), and
because previous studies have measured strain distributions at the midshafts
of the tibia and metatarsal in sheep
(Lieberman et al., 2003;
Lieberman et al., 2004),
allowing comparisons between local strain environment and bone growth
(Fig. 2). The general
hypothesis is that estradiol (E2) affects osteoblast responses to
loading by increasing their sensitivity to strain signals. Although this
effect presumably occcurs via upregulation of ER- density, it
is important to note that this study does not include direct measurement of
ER-, but rather tests for correlations between E2 level and
cortical response to loading. Future studies will include direct
quantification of ER-.
|
Hypotheses to be tested
The general hypothesis that estradiol (E2) affects the capacity
of osteoblasts to respond to mechanical loading in vivo leads to two
sets of hypotheses. The first is that there will be interactions between
E2 level and mechanical loading that have varying effects on
cortical bone growth, depending on skeletal location.
Effects on periosteal appositional bone growth
Hypothesis 1. There will be an interaction between E2
and mechanical loading. Exercised animals with elevated E2 levels
will have more bone growth than those with normal or suppressed E2
levels. Sedentary animals will exhibit little difference in periosteal bone
growth, regardless of E2 level, because of the low levels of strain
stimulus.
Hypothesis 2. Interactions between E2 and mechanical loading will vary by skeletal location. In mammals such as sheep with tapered limbs, such interactions will follow a proximo-distal gradient, with the greatest growth response in the femur and the least in the metatarsal. Although one might generally expect the most periosteal growth in the bones subject to the greatest loads, previous studies on sheep limbs reveal the opposite pattern, with more modeling proximally and less modeling distally, regardless of applied loads (see discussion above). Accordingly, we predict that interactions between estrogen, strain, and bone growth will follow the same overall pattern.
Effects on midshaft cross-sectional geometry
A second set of hypotheses relates to how interactions between
E2 and strain affect bone strength. The general hypothesis is that
by increasing mechanosensitivity at the level of the osteoblast, E2
may allow localized effects of strain on cortical growth, which leads to the
following specific predictions.
Hypothesis 3. There will be an interaction between E2 and bone strength. Exercised animals with higher E2 levels will have greater overall resistance to deformation, as measured by section moduli, than those with normal or low E2 levels. Sedentary animals will exhibit little difference in resistance to bending deformation, regardless of E2 level, because of the low levels of strain stimulus.
Hypothesis 4. Interactions between E2 and mechanical
loading will be larger on surfaces subjected to tension and compression during
locomotion, and smaller near the neutral axis of bending, where strains are
lower during locomotion. For this study, periosteal growth was measured on the
outer surface of the cortex at the furthest distance from the neutral axis
(NA) at peak strain, for two reasons. First, it is reasonable to expect the
largest growth responses will occur where strains are highest, i.e.
perpendicular to the NA at peak strain. Second, although the position of the
NA rotates counterclockwise and migrates caudally during stance phase
(Fig. 2), the cortex in the
measured locations remains in tension or compression throughout stance phase
(Lieberman et al., 2003;
Lieberman et al., 2004).
|
Materials and methods |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of symbols and...References |
|---|
;
Lieberman et al., 2004); (2)
the animals are of sufficient size and body mass to conduct in vivo
loading experiments; (3) sheep are easily trained to run on the treadmill; and
(4) their rapid pubertal growth involves some of the same hormones that
mediate human growth, including growth hormone (GH), insulin-like growth
factor (IGF) and E2 (Turner,
2002). The animals were housed in an outdoor paddock at the
Concord Field Station, Harvard University. All animals received the same diet
of hay and high-protein grain (Rumilab®, PMI Nutrition International, St
Louis, MO, USA), and water ad libitum. The protocol was approved by
the Harvard University IACUC, protocol #22-13.
|
Hormonal treatment
The 32 sheep were divided into two E2 treatment groups in
Experiment 1, low E2 (N=8) and high E2
(N=8), and three treatment groups in Experiment 2, low E2
(N=4), normal E2 (N=8) and high E2
(N=4) (Table 1). The
low-E2 animals were vaccinated [4 ml intramuscularly (IM)] on day 1
and day 22 against GnRH (gonadotrophin releasing hormone) using Protherics
immunoneutering vaccine (Protherics PLC, Cheshire, UK). In previous studies,
vaccination against GnRH suppressed production of gonadal steroids, including
E2 (Brown et al.,
1995). The high-E2 animals were implanted on day 1 with
subcutaneous capsules that release 61.5 µg E2
day–1 (Encore©, VetLife, Inc, Norcross, GA, USA). No
side effects were observed from the vaccine or the estradiol implant, and all
treated animals exhibited normal appetite, activity levels and weight
gain.
Exercise treatment
Half of the animals in each E2 treatment group were sedentary
and half were exercised, for a total of six treatment groups: low
E2-sedentary (LS), normal E2-sedentary (NS), high
E2-sedentary (HS), low E2-exercised (LE), normal
E2-exercised (NE), and high E2-exercised (HE)
(Table 1). Prior to the start
of the experiment, animals assigned to exercise groups were habituated to
running in an enclosed box on a treadmill at a moderate trot, a Froude number
of approximately 0.5 (1.67 m s–1)
(Alexander, 1977). During the
experiment, animals exercised for 40 min day–1, generating
approximately 4000 loading cycles per limb per day. Exercise was divided into
two bouts of 20 min, separated by 4–6 h, as bone cells lose their
sensitivity to mechanical stimuli after 20–30 min, and only regain this
sensitivity after several hours' rest
(Robling et al., 2002). The
sedentary animals were not exercised.
Growth measurements
Cortical bone growth during the treatment period was labeled using calcein
(20 mg kg–1 on day 1), a fluorochrome dye that incorporates
into bone mineral. All animals were weighed biweekly on a digital scale. Blood
samples were collected at the beginning, midpoint and conclusion of the
experiment for measuring serum E2 levels.
Analysis
Histology
At the end of the treatment period, the animals were euthanized and their
limbs prepared for histological analysis. Lengths of the femur, tibia and
metatarsal were measured postmortem using digital calipers. Femoral
length was measured from the most proximal point on the femoral head to the
line connecting the two distal condyles; tibial length was measured from the
center of the lateral condylar surface to the center of the distal articular
surface; metatarsal length was measured from the center of the proximal
articular surface to the most distal point of the distal articular surface.
Midshaft cortical bone sections were prepared following the protocol in
Lieberman et al. (Lieberman et al.,
2003). Specifically, a 1-cm cylinder was cut from the midshafts of
the femur, tibia and metatarsal, cleaned of soft tissue, fixed in ethanol and
cleared in xylene, and embedded in Epotek 301 epoxy resin (Epoxy Technology,
Billerica, MA, USA). Two sections were cut from each embedded midshaft using
an Isomet 1000 low-speed saw (Buehler Ltd, Lake Bluff, IL, USA), mounted on
slides, ground and polished to a thickness of 100 µm using a Buehler
Petrothin grinder, and coverslips placed on top.
Images of each slide were captured at 3.5–11.25x under fluorescent light using a Retiga 1300 camera (QImaging, Burnaby, BC, Canada) attached to an Olympus SZH10 stereozoom microscope (Olympus, Melville, NY, USA) and imported into IPLab imaging software (Scanalytics, Rockville, MD, USA).
Bone growth
Periosteal appositional bone growth during the treatment period was
measured as the total area added (mm2), from the initial calcein
line marking day 1 to the outer surface of the bone, in IPLab (Scanalytics,
Rockville, MD, USA).
Cross-sectional properties
Midshaft cross-sectional properties were measured in NIH Image 1.63
(http://rsb.info.nih.gov/nih-image/)
for the tibia and metatarsal using the experimentally determined neutral axis
(Fig. 2) and a custom NIH Image
macro (for details, see Lieberman et al.,
2004). Second moments of area, IN and
INy, and the polar moment of area, JN,
were calculated by the macro. The section moduli of tension and compression,
ZNc and ZNt, were calculated as
IN/ac and
IN/at, where ac
and at are the greatest perpendicular distances from the
experimentally derived NA to the outer perimeter subject to compression and
tension in the plane of bending. Linear cortical bone growth was measured from
the calcein line to the outer cortex, at the points where the neutral axis
(NA) and the perpendicular axis intersect the bone surface, in IPLab.
Hormonal assays
Serum estradiol measurements were obtained at the beginning, midpoint, and
conclusion of the experiment via radioimmunoassay (Prairie Diagnostic
Services, University of Saskatchewan, SK, Canada).
Standardization and data pooling
Histological measurements were standardized by body mass (proportional to
volume). Therefore, areas were standardized to body mass
(Mb)0.67, while linear measurements were
standardized to Mb0.33. Because the animals
gained mass rapidly during the treatment period, we standardized periosteal
bone area added by average body mass at the midpoint (mMb)
and conclusion (cMb) of the experiment
[(mMb+cMb)/2]0.67, and
linear bone growth by
[(mMb+cMb)/2]0.33.
Cross-sectional properties were standardized by
{[(mMb+cMb)/2]xlimb length}
(Lieberman et al., 2003).
The data reported here come from two separate experiments of identical duration, exercise protocol, and hormonal treatments, using subjects of the same age and breed (Table 1). Although there is a significant difference in initial and final body mass between the experiments (Table 1), all measurements of bone growth are standardized by body mass, allowing us to pool data from the two experiments in all analyses.
Hypothesis testing
Given the interactions examined here, ANOVA and pairwise comparisons with
Fisher's LSD tests in Statistica (Statsoft, Tulsa, OK, USA) were primarily
used for hypothesis testing, using E2 treatment and exercise as
nominal variables and bone growth (standardized by body mass) as a continuous
variable. In addition, bone growth was regressed against average body mass in
Statview (SAS, Cary, NC, USA) to obtain the residual for each individual.
ANOVA was then used to test for significant differences among treatment
groups, using E2 treatment and exercise as nominal variables and
the residual of bone growth vs body mass as a continuous
variable.
|
Results |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of symbols and...References |
|---|
;
Bartlewski et al., 1999b). This
unexpected finding has several implications for our results, which are
discussed below.
|
Periosteal growth
Periosteal appositional growth varied with activity level, with estrogen
treatment, and with interactions between activity and estrogen. Overall,
exercised animals grew more bone than did sedentary animals
(Table 2). Exercise increased
bone growth by 27% in the femur (P=0.09, ANOVA, Fisher's LSD), 46% in
the tibia (P<0.05), and 35% in the metatarsal (P=0.11)
relative to sedentary controls. The overall effects of estrogen treatment on
cortical growth, with animals of differing activity levels pooled, were less
clear (Table 2). As noted
above, circulating E2 levels were actually higher in vaccinated
than in untreated animals (6 pg ml–1 vs 3 pg
ml–1). However, vaccinated animals had 34–49% less
cortical growth in the femur, tibia and metatarsal than untreated controls
(P<0.05, ANOVA, Fisher's LSD). High estrogen levels had similarly
complex overall effects. Periosteal growth in high-E2 animals was
similar to that of normal controls in the femur, but 35–40% less than
normal controls in the tibia (P=0.06) and in the metatarsal
(P<0.05). To summarize, while exercise clearly stimulates
periosteal bone growth, it would appear that estrogen has no effect, or a
suppressive effect, on bone growth.
|
|
In the tibia, the pattern was less consistent. The HE group added, on average, 6% (range –7% to +143%) more bone than the other groups, but the relationship between E2 dose and bone growth was less clear. While there was 46% more bone growth in HE vs LE animals (P<0.05, ANOVA, Fisher's LSD), there was 6–7% less growth in HE vs NE or NS animals (Table 2). Also, both the HE and NE animals added significantly more bone than did LS and HS animals (P<0.05).
In the metatarsal, interactions between E2 dose and bone growth were more modest. The HE group added about 14% (range –15 to +171%) more bone than the other groups, and in particular, 42% more bone than the LE group (P<0.05, ANOVA, Fisher's LSD). However, as in the tibia, the greatest growth response occurred in the NS and NE groups, both of which had about 15% more growth than the HE group. As a result, both HE and NE animals had significantly more growth than did LS and HS animals (P<0.05).
Given the significant difference in body mass between animals in experiments 1 and 2, we regressed cortical bone added vs body mass (Table 2, Fig. 3A–C) and compared the residuals for each experimental group (Table 2, Fig. 4A–C). Although body mass explained 53–65% of the variance in added bone (Fig. 3A–C), box plots of the residuals demonstrate that they were not randomly distributed among the treatment groups, but instead revealed a significant interaction between E2 and exercise (Fig. 4A–C). In the femur (Fig. 4A), the residual of bone added vs body mass was highest in the HE group, which differed significantly from the sedentary groups and the LE group (P<0.01, ANOVA), but not the NE group (P=0.13). In the tibia, the pattern was more varied (Fig. 4B), with a significantly higher residual in the HE group vs the LS and HS groups (P<0.01), but not the NS (P=0.07) or LE (P=0.08) groups. Finally, in the metatarsal (Fig. 4C), the HE group did not differ significantly from the other groups except for the HS group (P<0.01).
Cross-sectional properties
Table 3 and Figs
5,
6,
7,
8 present cross-sectional
properties in the tibia and the metatarsal, for which midshaft strains in
sheep of similar size and age have been experimentally determined
(Fig. 2)
(Lieberman et al., 2003;
Lieberman et al., 2004). The
analysis excluded the femur, for which in vivo strain data are
unavailable.
|
|
|
|
|
In general, midshaft cross-sectional properties were somewhat elevated in exercised animals, particularly at higher E2 doses. In the tibia, moments of area and section moduli in the HE group were, on average, 8% greater (range 0–14%) than in the other groups (Table 3, Fig. 5A,B), although these differences were not statistically significant. Within each treatment group, tibial second moments of area about the neutral axis (IN) and about the axis perpendicular to the neutral axis (INy) were similar, suggesting equal resistance to deformation in the anteroposterior plane, which is tensed and compressed at midstance, and in the mediolateral plane, in which strains are low at midstance (Fig. 5A). However, about the neutral axis, the section modulus of tension, ZNt, was higher than the section modulus of compression, ZNc, indicating increased resistance to tension vs compression during bending (Fig. 5B).
To compare bone growth to strain distribution, cortical apposition from the
calcein label to the outer bone surface was measured at the neutral axis
(IN) and the perpendicular axis (INy).
On all tibial bone surfaces, there was generally more periosteal apposition
with exercise and with increasing E2 dose
(Table 3), with an average of
95% more growth in the HE group than in other groups (range 0 to +275%,
P<0.05 where indicated; Fig.
6). However, HE animals did not grow significantly more bone than
NE or NS animals on any surface, and local rates of periosteal bone apposition
within the cortex did not appear to be correlated with strain distribution at
midstance. Within each treatment group, the extent of bone growth on the
anterior (cranial) and posterior (caudal) surfaces, which are, respectively,
tensed and compressed during stance phase, was similar to the extent of growth
on the medial and lateral surfaces, where strains are about 50% lower
(0–300 µ vs 300–800 µ,
Fig. 2).
In the metatarsal, in contrast to the tibia, there was no trend toward greater resistance to deformation with increasing E2 or exercise (Table 3). In fact, moments of area and section moduli were 40–50% higher in the NS group than in the HE group (P<0.05, Table 3, Fig. 7A,B). As in the tibia, IN and INy were similar in the metatarsal, suggesting comparable resistance to anteroposterior and mediolateral deformation, and the section modulus of tension, ZNt, was higher than the section modulus of compression, ZNc, indicating greater resistance to tension than to compression during bending.
In terms of linear bone growth at the intersection of the cortex with the neutral axis (IN) and the perpendicular axis (INy), there was less apparent effect of E2 or exercise in the metatarsal. Overall, the HE group added an average of 54% more bone (range –78% to +160%, P<0.05 where indicated, Fig. 8) than did the other groups. However, there were no significant differences between HE, NE, and NS animals. As in the tibia, within each group, similar growth occurred on the posterior (tensile) surface and on the medial and lateral surfaces near IN (Table 3, Fig. 8), thereby maintaining a rounded cross-sectional shape.
|
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of symbols and...References |
|---|
). The hypothesis is that exercise-induced
mechanical loading will have greater effects on bone growth and
cross-sectional properties in individuals with higher E2 levels,
due to increased osteoblast sensitivity to strain stimuli. The data indicate
support for three of the four specific components of this hypothesis.
Hypothesis 1, that exercised animals with higher E2 levels (HE)
will have more bone growth than those with normal E2 levels, is
strongly supported in the femur, in which HE animals had 16–92% more
bone growth than any other treatment group. However, the effects of reduced
E2 level on bone growth remain ambiguous. Estrogen levels in the
vaccinated and normal E2 groups were similar, yet exercise-induced
bone growth was reduced by up to 50% in vaccinated animals. It is not clear
how the anti-GnRH vaccine caused diminished bone growth without affecting
circulating E2. Although the mechanism for diminished growth in the
vaccinated animals requires further study, it is clear that increasing
estrogen availability makes bone more sensitive to strain, as predicted.
Hypothesis 2, that the effects of E2 will follow a
proximo-distal gradient within a limb, is also supported. Residual plots
demonstrate significant differences in growth response among treatment groups
in the femur, with more moderate differences among groups in the tibia and the
metatarsal (Fig. 4). In other
words, the interaction between E2 and mechanical loading is
extensive in the femur, intermediate in the tibia, and minimal in the
metatarsal, despite the fact that bone strains are likely lowest in the femur,
intermediate in the tibia, and highest in the metatarsal. Thus
estrogen-mediated bone growth follows the trade-off previously documented in
tapered sheep limbs between increased resistance to deformation and increased
cost of locomotion (Lieberman et al.,
2003). This pattern suggests that in the periosteum, at least,
estrogen (and perhaps ER-) has a specific role in mediating skeletal
responses to strain, as opposed to upregulating overall bone growth.
In terms of cross-sectional geometry, Hypothesis 3, that exercised animals
with higher E2 levels (HE) will have greater overall resistance to
deformation, as measured by cross-sectional geometry, than normal controls
(NE) or vaccinated (LE) animals, is modestly supported in the tibia, but not
in the metatarsal. In the tibia, there is an 8% average increase in resistance
to deformation in the HE group compared to the other groups, although this
difference did not reach significance. In the metatarsal, the greatest
resistance to deformation was actually in the normal, sedentary (NS) animals.
Finally, the data relevant to Hypothesis 4, that E2-induced
periosteal bone growth will coincide with areas of higher strain during
locomotion, are more difficult to interpret. While the HE group generally
added more bone than did the other treatment groups in both the tibia and the
metatarsal, this effect occurred on all bone surfaces, rather than
corresponding to areas subjected to high tensile or compressive loads, as
predicted by the hypothesis. As a result, in both bones the second moments of
area in the anteroposterior plane, IN, and the
perpendicular plane, INy, are similar, despite the fact
that strains are much higher anteroposteriorly than mediolaterally during the
stance phase of locomotion. There are several possible reasons for this
similarity. Although strains on the medial and lateral surfaces are about 50%
lower than on the anterior and posterior surfaces (0–300 µ
vs 300-800 µ), they may be sufficient to stimulate some bone
growth. Additionally, the experimental animals were growing rapidly during the
treatment period, and the apposition we observed around the entire tibia and
metatarsal may simply reflect normal cortical drift.
Overall, our results support the hypothesized interaction between estrogen,
strain and bone growth. Raising circulating E2 levels increases the
sensitivity of growing bone to mechanical signals, but has little effect on
bone growth in sedentary animals, in the absence of strain signals
(Fig. 4). To our knowledge,
this is the first in vivo study to demonstrate that physiological
variation in E2 level among individuals can produce differential
growth responses to an identical mechanical loading regime. The finding that
interactions between E2 and mechanical loading follow a
proximo-distal gradient, with larger effects in the femur than in the
metatarsal, warrants future study. If, as we hypothesize, E2
increases the sensitivity of osteoblasts to mechanical stimuli via
upregulation of ER-, then ER- transcription may vary within a
limb, with more receptors in proximal elements and fewer in distal elements.
Such a mechanism may underlie the observed higher modeling rates in proximal
vs distal segments (Lieberman et
al., 2003), a hypothesis that must be tested in future
experiments.
Finally, the results presented here are interesting to consider in light of
two well-documented trends in human skeletal evolution: that recent humans are
less robust than earlier modern humans, and that humans from warm climates
have less robust limbs than humans from cold climates
(Ruff et al., 1993;
Trinkaus, 1997;
Pearson, 2000). If osteogenic
responses to mechanical loading vary among individuals or populations, perhaps
because of differences in hormone levels (e.g.
Churchill, 1998), then there
may not be a simple relationship between patterns of skeletal robusticity and
individual loading history. This finding has significant implications for
attempts to model the relationship between environmental strain and bone
morphology.
|
List of symbols and abbreviations |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of symbols and...References |
|---|
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of symbols and...References |
|---|
Alexander, R. M. (1977). Terrestrial locomotion. In Mechanics and Energetics of Animal Locomotion (ed. R. M. Alexander and G. Goldspink), pp.168 -203. London: Chapman & Hall.
Bakker, A. D., Soejima, K., Klein-Nulend, J. and Burger, E. H. (2001). The production of nitric oxide and prostaglandin E(2) by primary bone cells is shear stress dependent. J. Biomech. 234,671 -677.
Bakker, A. D., Joldersma, M., Klein-Nulend, J. and Burger, E. H. (2003). Interactive effects of PTH and mechanical stress on nitric oxide and PGE2 production by primary mouse osteoblastic cells. Am. J. Physiol. 285,E608 -E613.
Bartlewski, P. M., Beard, A. P. and Rawlings, N. C. (1999a). Ovarian function in ewes at the onset of the breeding season. Anim. Reprod. Sci. 57, 67-88.[CrossRef][Medline]
Bartlewski, P. M., Beard, A. P. and Rawlings, N. C. (1999b). Ovarian function in ewes during the transition from breeding season to anoestrus. Anim. Reprod. Sci. 57, 51-66.[CrossRef][Medline]
Bertram, J. E. A. and Biewener, A. A. (1988). Bone curvature: sacrificing strength for load predictability? J. Theor. Biol. 131,75 -92.[CrossRef][Medline]
Biewener, A. A., Swartz, S. M. and Bertram, J. E. A. (1986). Bone modeling during growth: dynamic strain equilibrium in the chick tibiotarsus. Calcif. Tissue Int. 39,390 -395.[Medline]
Brown, B. W., Mattner, P. E., Carroll, P. A., Hoskinson, R. M. and Rigby, R. D. (1995). Immunization of sheep against GnRH early in life: effects on reproductive function and hormones in ewes. J. Reprod. Fertil. 103,131 -135.[Abstract]
Carter, D. R. and Beaupré, G. S. (2001). Skeletal Form and Function: Mechanobiology of Skeletal Development, Aging and Regeneration. Cambridge: Cambridge University Press.
Cheng, M. Z., Rawlinson, S. C., Pitsillides, A. A., Zaman, G., Mohan, S., Baylink, D. J. and Lanyon, L. E. (2002). Human osteoblasts' proliferative responses to strain and 17beta-estradiol are mediated by the estrogen receptor and the receptor for insulin-like growth factor I. J. Bone Miner. Res. 17,593 -602.[CrossRef][Medline]
Cherian, P. P., Cheng, B., Gu, S., Sprague, E., Bonewald, L. F.
and Jiang, J. X. (2003). Effects of mechanical strain on the
function of Gap junctions in osteocytes are mediated through the prostaglandin
EP2 receptor. J. Biol. Chem.
278,43146
-43156.
Churchill, S. (1998). Cold adaptation, heterochrony, and Neanderthals. Evol. Anthropol. 47, 46-60.[CrossRef]
Cowin, S. C. and Moss, M. L. (2001). Mechanosensory mechanisms in bone. In Bone Biomechanics Handbook. 2nd edn (ed. S. C. Cowin), pp.29 -1–29-17. Boca Raton: CRC Press.
Cowin, S. C., Weinbaum, S. and Zeng, Y. (1995). A case for bone canaliculi as the anatomical site of strain generated potentials. J. Biomech. 28,1281 -1297.[CrossRef][Medline]
Currey, J. D. (2002). Bones: Structure and Mechanics. Princeton: Princeton University Press.
Damien, E., Price, J. S. and Lanyon, L. E. (1998). The estrogen receptor's involvement in osteoblasts' adaptive response to mechanical strain. J. Bone Miner. Res. 13,1275 -1282.[CrossRef][Medline]
Damien, E., Price, J. S. and Lanyon, L. E. (2000). Mechanical strain stimulates osteoblast proliferation through the estrogen receptor in males as well as females. J. Bone Miner. Res. 15,2169 -2177.[CrossRef][Medline]
Davidson, R. M., Lingenbrink, P. A. and Norton, L. A. (1996). Continuous mechanical loading alters properties of mechanosensitive channels in G292 osteoblastic cells. Calcif. Tissue Int. 59,500 -504.[Medline]
Guggino, S. E., Lajeunesse, D., Wagner, J. A. and Snyder, S.
H. (1989). Bone remodeling signaled by a dihydropyridine- and
phenylalkylamine-sensitive calcium channel. Proc. Natl. Acad. Sci.
USA 86,2957
-2960.
Hildebrand, M. (1985). Walking and running. In Functional Vertebrate Morphology (ed. M. Hildebrand, D. M. Bramble, K. F. Liem and D. B. Wake), pp. 38-57. Cambridge: Harvard University Press.
Hoyland, J. A., Baris, C., Wood, L., Baird, P., Selby, P. L., Freemont, A. J. and Braidman, I. P. (1999). Effect of ovarian steroid deficiency on oestrogen receptor alpha expression in bone. J. Pathol. 188,294 -303.[CrossRef][Medline]
Hsieh, Y. F. and Turner, C. H. (2001). Effects of loading frequency on mechanically induced bone formation. J. Bone Miner. Res. 16,918 -924.[CrossRef][Medline]
Jessop, H. L., Sjoberg, M., Cheng, M. Z., Zaman, G., Wheeler-Jones, C. P. and Lanyon, L. E. (2001). Mechanical strain and estrogen activate estrogen receptor alpha in bone cells. J. Bone Miner. Res. 16,1045 -1055.[CrossRef][Medline]
Jessop, H. L., Rawlinson, S. C., Pitsillides, A. A. and Lanyon, L. E. (2002). Mechanical strain and fluid movement both activate extracellular regulated kinase (ERK) in osteoblast-like cells but via different signaling pathways. Bone 31,186 -194.[Medline]
Jones, H. N., Priest, J. D., Hayes, W. C., Tichenor, C. C. and
Nagel, D. A. (1977). Humeral hypertrophy in response to
exercise. J. Bone Joint Surg. Am.
59,204
-208.
Karsenty, G. (1999). The genetic transformation
of bone biology. Genes Dev.
13,3037
-3051.
Lanyon, L. E. and Rubin, C. T. (1984). Static vs dynamic loads as an influence on bone remodelling. J. Biomech. 17,897 -905.[CrossRef][Medline]
Lanyon, L. E. and Rubin, C. T. (1985). Functional adaptation to load-bearing in bone tissue. In Functional Vertebrate Morphology (ed. M. Hildebrand, D. M. Bramble, K. F. Liem and D. B. Wake), pp. 1-26. Cambridge, MA: Harvard University Press.
Lee, K., Jessop, H., Suswillo, R., Zaman, G. and Lanyon, L. (2003). Endocrinology: bone adaptation requires oestrogen receptor-alpha. Nature 424, 389.[Medline]
Lieberman, D. E. and Crompton, A. W. (1998). Responses of bone to stress. In Principles of Biological Design: The Optimization and Symmorphosis Debate (ed. E. Weibel, C. R. Taylor and L. Bolis), pp. 78-86. Cambridge: Cambridge University Press.
Lieberman, D. E. and Pearson, O. M. (2001). Trade-off between modeling and remodeling responses to loading in the mammalian limb. Bull. Mus. Comp. Zool. 156,269 -282.
Lieberman, D. E., Pearson, O. M., Polk, J. D., Demes, B. and
Crompton, A. W. (2003). Optimization of bone growth and
remodeling in response to loading in tapered mammalian limbs. J.
Exp. Biol. 206,3125
-3138.
Lieberman, D. E., Polk, J. D. and Demes, B. (2004). Predicting long bone loading from cross-sectional geometry. Am. J. Phys. Anthropol. 123,156 -171.[CrossRef][Medline]
Lim, S. K., Won, Y. J., Lee, H. C., Huh, K. B. and Park, Y. S. (1999). A PCR analysis of ERalpha and ERbeta mRNA abundance in rats and the effect of ovariectomy. J. Bone Miner. Res. 14,1189 -1196.[CrossRef][Medline]
Marsh, R. L., Ellerby, D. J., Carr, J. A., Henry, H. T. and
Buchanan, C. I. (2004). Partitioning the energetics of
walking and running: swinging the limbs is expensive.
Science 303,80
-83.
Martin, R. B., Sharkey, N. A. and Burr, D. B. (1998). Skeletal Tissue Mechanics. New York: Springer-Verlag.
Myers, M. J. and Steudel, K. (1985). Effect of
limb mass and its distribution on the energetic cost of running. J.
Exp. Biol. 116,363
-373.
Olsen, B. R., Reginato, A. M. and Wang, W. (2000). Bone development. Annu. Rev. Cell Dev. Biol. 16,191 -220.[CrossRef][Medline]
Pauwels, F. (1980). Biomechanics of the Locomotor Apparatus. Berlin: Springer-Verlag.
Pearson, O. M. (2000). Activity, climate, and postcranial robusticity: implications for modern human origins and scenarios of adaptive change. Curr. Anthropol. 41,569 -607.[CrossRef][Medline]
Pearson, O. M. and Lieberman, D. E. (2004). The aging of Wolff's `law': ontogeny and responses to mechanical loading in cortical bone. Am. J. Phys. Anthropol. 39, Suppl.63 -99.
Robling, A. G., Hinant, F. M., Burr, D. B. and Turner, C. H. (2002). Improved bone structure and strength after long-term mechanical loading is greatest if loading is separated into short bouts. J. Bone Miner. Res. 17,1545 -1554.[CrossRef][Medline]
Ruff, C. B., Trinkaus, E., Walker, A. and Larsen, C. S. (1993). Postcranial robusticity in Homo. I. Temporal trends and mechanical interpretation. Am. J. Phys. Anthropol. 91,21 -53.[CrossRef][Medline]
Saunders, M. M., You, J., Trosko, J. E., Yamasaki, H., Li, Z., Donahue, H. J. and Jacobs, C. R. (2001). Gap junctions and fluid flow response in MC3T3-E1 cells. Am. J. Physiol. 281,C1917 -C1925.
Trinkaus, E. (1997). Appendicular robusticity
and the paleobiology of modern human emergence. Proc. Natl. Acad.
Sci. USA 94,13367
-13373.
Turner, A. S. (2002). The sheep as a model for osteoporosis in humans. Vet. J. 163,232 -239.[CrossRef][Medline]
Whitfield, J. F. (2003). Primary cilium – is it an osteocyte's strain-sensing flowmeter? J. Cell. Biochem. 89,233 -237.[CrossRef][Medline]
Zaman, G., Cheng, M. Z., Jessop, H. L., White, R. and Lanyon, L. E. (2000). Mechanical strain activates estrogen response elements in bone cells. Bone 27,233 -239.[Medline]
Zaman, G., Jessop, H. L., Muzylak, M., De Souza, R. L., Pitsillides, A. A., Price, J. S. and Lanyon, L. E. (2006). Osteocytes use estrogen receptor alpha to respond to strain but their ERalpha content is regulated by estrogen. J. Bone Miner. Res. 21,1297 -1306.[CrossRef][Medline]
Zhou, S., Zilberman, Y., Wassermann, K., Bain, S. D., Sadovsky, Y. and Gazit, D. (2001). Estrogen modulates estrogen receptor alpha and beta expression, osteogenic activity, and apoptosis in mesenchymal stem cells (MSCs) of osteoporotic mice. J. Cell. Biochem. 81,144 -155.[CrossRef]
This article has been cited by other articles:
|
|
 |
|
  K. J. Carlson and S. Judex Increased non-linear locomotion alters diaphyseal bone shape J. Exp. Biol., September 1, 2007; 210(17): 3117 - 3125. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
