Mechanical loading attenuates bone loss due to

immobilization and calcium deficiency

Cynthia L. Inman, Gordon L. Warren, Harry A. Hogan and Susan A. Bloomfield
J Appl Physiol 87:189-195, 1999.

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Mechanical loading attenuates bone loss due
to immobilization and calcium deficiency
CYNTHIA L. INMAN,1 GORDON L. WARREN,1 HARRY A. HOGAN,2
AND SUSAN A. BLOOMFIELD1
1Department of Health and Kinesiology and 2Department of Mechanical Engineering,

Texas A&M University, College Station, Texas 77843

Inman, Cynthia L., Gordon L. Warren, Harry A. bone loss that is similar in many respects to that
Hogan, and Susan A. Bloomfield. Mechanical loading observed with osteoporosis (12–14, 20). Unilateral hind-
attenuates bone loss due to immobilization and calcium limb immobilization in the adult rat has several advan-
deficiency. J. Appl. Physiol. 87(1): 189–195, 1999.—Our pur- tages as a model for postmenopausal or aging-related
pose was to determine the effects of a mechanical loading bone loss. Immobilization effectively stimulates resorp-
intervention on mass, geometry, and strength of rat cortical tion to exceed the rate of bone formation, resulting in a
bone during a period of disuse concurrent with calcium loss of both cancellous and cortical bone (12, 13). If
deficiency (CD). Adult female rats were assigned to unilateral calcium deficiency is superimposed on the effects of
hindlimb immobilization, immobilized-loaded, or control (stan-
disuse, the rate of bone loss is accelerated because of
dard chow, 1.85% calcium) treatments. Both immobilized
the resultant increase in serum PTH (20, 23). To our
groups were fed a CD rat chow (0.01% calcium) to induce high
knowledge, only one previous study has addressed the

Downloaded from jap.physiology.org on October 16, 2006

bone turnover. Three times weekly, immobilized-loaded rats
were subjected to 36 cycles of 4-point bending of the immobi- combined effects of calcium deficiency and immobiliza-
lized lower leg. After 6 wk, the immobilized rats exhibited tion in skeletally mature rats (20).
decreased tibial shaft bone mineral density (⫺12%), ultimate If this disuse-induced loss of bone mass could be
load (⫺19%), and stiffness (⫺20%; tested in 3-point bending slowed or prevented altogether, one would expect that
to failure) vs. control rats. Loading prevented this decline in significant decrements in bone strength and the in-
bone density and attenuated decreases in ultimate load and creased risk of fracture could be minimized. Because
stiffness. Elastic modulus was unaffected by disuse or load- weight-bearing activities can slow the loss of bone with
ing. Bone cross-sectional area in the immobilized-loaded rats aging (9, 17), mechanical loading during a period of
was equivalent to that of control animals, even though disuse might also be effective in minimizing deleterious
endocortical resorption continued unabated. On the medial changes in bone mass and bone geometry affecting
periosteum, percent mineralizing surface doubled vs. that in fracture risk. Several means of producing quantifiable
immobilized rats. This loading regimen stimulated periosteal mechanical loading have been developed for use in
mineralization and maintained bone mineral density, thereby animal models (4, 10, 19). The purpose of this investiga-
attenuating the loss in bone strength incurred with disuse tion was to test the hypothesis that in vivo mechanical
and concurrent calcium deficiency. loading in adult female rats can attenuate losses of
bone density; adult rats; disuse; mechanical properties cortical bone mass and strength during unilateral
hindlimb immobilization concurrent with calcium defi-
ciency.

OSTEOPOROSIS is a multifactorial disease, with contribu- METHODS

tions from genetic factors, lack of physical activity,
inadequate nutritional intake, and endocrine deficien- Experiment 1. Twenty-eight female Sprague-Dawley re-
tired breeder rats, aged 5 mo (300 ⫾ 3.3 g; Harlan, Indianapo-
cies. For example, the declines in vitamin D status,
lis, IN) were randomly assigned to one of three groups:
dietary calcium intake, and calcium absorption effi- control, immobilized, and immobilized-loaded. The control
ciency typically observed in the elderly may account, in rats (n ⫽ 9) were fed a standard rat chow (1.85% calcium,
part, for the increases in serum parathyroid hormone Harlan Teklad, Madison, WI) and were free to move about
(PTH) observed in many older individuals (7). Chroni- their cages without restriction. After a 1-wk acclimation
cally elevated serum PTH can contribute to increased period, four animals consuming normal rat chow were killed
rates of bone resorption and loss of bone mass at the to serve as the baseline control. The right hindlimb of rats in
the immobilized group (n ⫽ 9) was immobilized for 6 wk by
femoral neck in the elderly (16). Many older individuals taping, as previously described (13); this group was simulta-
also decrease voluntary physical activity or may even neously fed a calcium-deficient diet (TD no. 93278; 0.01%
be subjected to prolonged bed rest, further exacerbat- calcium, Harlan Teklad). The immobilized-loaded group (n ⫽
ing bone loss (11). 10) experienced external mechanical loading of the immobi-
The decrease in mechanical loading incurred with lized tibia three times per week, beginning 1 wk after the
experimental limb immobilization results in localized start of immobilization. In addition, they were fed the calcium-
deficient diet. All rats were housed two per cage; the lighting
schedule was 12 h on, 12 h off, and deionized water was
The costs of publication of this article were defrayed in part by the provided. Rats were allowed to consume their assigned rat
payment of page charges. The article must therefore be hereby chow ad libitum. Rat chow for each cage was weighed daily;
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 total chow consumed per day was halved to estimate food
solely to indicate this fact. consumption per rat. After 6 wk, all rats were killed by a fatal

http://www.jap.org 8750-7587/99 $5.00 Copyright r 1999 the American Physiological Society 189
190 LOADING ATTENUATES DISUSE-INDUCED BONE LOSS

dose of pentobarbital sodium (150 mg/kg). Left and right mineral density (BMD) and bone mineral content (BMC) in
tibiae were removed, cleaned of soft tissue, and stored in three regions of interest on both immobilized (right) and
saline at ⫺80°C. These samples were used for densitometry weight-bearing (left) tibiae. Thawed bones, cleaned of soft
and for mechanical testing. The left and right tibialis anterior tissue, were placed on top of a Lucite phantom block, medial
and soleus muscles, along with the spleen, were removed, and side down. The proximal tibia region included the full width
wet weights were recorded. of the most proximal 8 mm of the tibia. The midshaft tibia
Experiment 2. Thirty female Sprague-Dawley retired region extended from 4.0 to 13.5 mm proximal to the tibiofibu-
breeder rats, aged 5 mo old (306 ⫾ 4.1 g; Harlan Sprague lar junction, including the full width of the tibial diaphysis,
Dawley) were randomly assigned to baseline control (n ⫽ 7), corresponding to that region experiencing bending strains
aging cage-activity control (n ⫽ 8), immobilized (n ⫽ 8), and during the in vivo four-point bending (Fig. 1). The distal tibia
immobilized-loaded (n ⫽ 7) groups. Control rats ate standard region included the full width of the most distal 6 mm of each
chow, and both immobilized groups consumed the calcium- bone. Scanning speed was 2 mm/s, with resolution set at 0.5 ⫻
deficient chow as described above. Ten and three days before 0.5 mm. Coefficients of variation were determined from five
euthanasia, all rats received intraperitoneal injections of repeat scans on two tibiae over several days, with reposition-
calcein in saline (8 mg/kg; Sigma Chemical) at pH 6.8 to label ing for each scan. The average coefficients of variation across
mineralizing bone surfaces. After 6 wk, rats were killed by a all three regions were 2.8% for BMC and 2.2% for BMD.
fatal dose of pentobarbital sodium. The right tibiae were Mechanical testing. Cortical bone strength at midshaft was
removed, cleaned of soft tissue, and stored in 70% ethanol at determined by using a three-point bending-to-failure proce-
4°C. These samples were used for histomorphometric analy- dure on an Instron machine (model 1125). The area tested
was at the midpoint of the region loaded during in vivo
ses. The study protocol and animal procedures for both
four-point bending. The tibia was positioned lateral side down
experiments met the guidelines of the Texas A&M University
on the custom-made supports (pins 4 mm in diameter)
Laboratory Animal Care Committee.

Downloaded from jap.physiology.org on October 16, 2006

positioned 18 mm apart. A 50-lb. load cell was used with
Immobilization method. Before immobilization of the right quasi-static loading (2.54 mm/min) applied to the medial
hindlimb, rats were anesthetized with methoxyflurane (Meto- surface of the tibia at midshaft. The small displacements of
fane, Pitman-Moore), and each animal’s lower torso and right the servo-controlled Instron were monitored by a linear
hindlimb were shaved of all hair. A protective skin coating variable differential transformer interfaced with a personal
(AllKare protective barrier wipe, ConvaTec) was applied to computer unit. Load vs. displacement plots were recorded by
the exposed skin. The right hindlimb was immobilized against using Gardener Systems software. Ultimate load was defined
the abdomen with the hip joint in flexion and the knee and as the highest load (N) recorded just before the first decline in
ankle joints in extension, using four to five layers of elastic load as displacement increased. Stiffness was determined as
tape (Johnson & Johnson Elastikon). Within 24 h, the rats the slope of the linear portion of the load vs. displacement curve,
were able to ambulate on three legs with no obvious discom- using TableCurve 2.0 (Jandel Scientific, San Rafael, CA).
fort. Throughout the 6-wk treatment period the animals were Cross-sectional geometry was determined by embedding
checked daily for evidence of discomfort, sores, or swelling. It the distal half of each bone in black polyester resin. Trans-
was necessary to regularly reapply the tape bandaging one to verse sections were cut by a low-speed diamond wafering saw
two times per week to maintain immobilization. During (Buehler Isomet, Lake Bluff, IL). The bones consistently
retaping the animal was anesthetized, and the leg was fractured beneath the upper loading point in the middiaphy-
massaged and stretched before reapplication of the tape. sis region, and each section was cut as near to the fracture
Mechanical loading. Commencing 1 wk after the start of point as possible. A Wild M420 macroscope was used to
immobilization, immobilized-loaded rats had their right hind- capture the image of the cross-sectional image by using
limb untaped and the tibia was subjected to external loading Bioscan Optimas software. Jandel SigmaScan software (ver-
three times per week. The in vivo loading was accomplished sion 1.20.09) was used to determine cross-sectional moment
by using a four-point loading device previously described (1).
Briefly, bending loads were applied by a lever system powered
by a stepper motor; the applied load (in N) was determined by
measuring strains induced on a calibrated load cell. Applied
external loads of ⬃32 N (bending moments of ⬃96 N/mm)
generated peak strains of 1,000–1,400 microstrain (µ⑀) on the
tibia’s lateral surface. These strain magnitudes are biologi-
cally relevant because significant bone modeling responses
have been demonstrated in bone experiencing these strain
magnitudes in rats (14, 19), turkeys (10), dogs (3), and
sheep (5).
On loading days, each rat was anesthetized, the tape was
removed, and the immobilized limb was stretched; the rat’s
right hindlimb was then loaded for 36 cycles at 2 Hz with an
external load of 32 N. A similar regimen has been shown to
produce new bone formation on the medial periosteal surface Fig. 1. Loaded region of tibia with in vivo 4-point bending extends
of the tibial diaphysis after 3 wk of alternate-day loading (19). from 3.5 to 14.5 mm proximal to tibiofibular junction (TFJ). Lateral
After each loading protocol was complete, the rat’s hindlimb surface experiences compression, medial surface tension, in bending.
was retaped, and the rat was returned to its cage. At no time Loading is applied on alternate days, 36 cycles at 2 Hz; strains on
lateral surface as measured by uniaxial strain gauges are ⬃1,200
during this procedure was the rat allowed to bear weight on
microstrain (µ⑀) (Inman and Bloomfield, unpublished observations).
the immobilized hindlimb. Stippled area, scanned for midshaft bone mineral density (BMD) and
Densitometry. The Norland XR 2600 dual-energy X-ray bone mineral content (BMC; experiment 1); solid area, separate tibiae
absorptiometer (Norland, Ft. Atkinson, WI) with Small Sub- sectioned for histomorphometric analyses (experiment 2; 5–7 mm
ject software (version 2.5, Norland) was used to estimate bone proximal to TFJ).
 LOADING ATTENUATES DISUSE-INDUCED BONE LOSS 191

of inertia (CSMI) about the anterior-posterior axis. Determi-

nation of modulus of elasticity (E) was estimated by employ-
ing classic beam theory by using the equation E ⫽
[(K*L3 )*1,000]/(48*CSMI), where K is stiffness and L is
bottom support span (18 mm). Ultimate stress (␴) of each
sample was calculated by using the equation ␴ ⫽ (UL*L*OD)/
(8*CSMI), where UL is ultimate load, L is bottom support
span (18 mm), and OD is outer diameter of bone at failure
site.
Histomorphometry. The right tibiae from rats in experi-
ment 2 were block stained in Villanueva stain (Polysciences)
for 72 h and then progressively dehydrated in ethanol and
acetone before embedding in methyl methacrylate. Cross
sections of 120–150 µm were cut on a low-speed diamond
wafer saw (Buehler Isomet) starting 5 mm proximal to the
tibiofibular junction, which is centered in the region of
maximal bending for those tibiae receiving mechanical load-
ing. After sections were ground to a thickness of 80–100 µm
and mounted on slides, image analysis for areas and labeled
perimeters was performed by using BioQuant TrueColor
Windows, version 2.0 (R&M Biometrics, Nashville, TN). Total
tissue area (inside periosteal perimeter) and marrow area

Downloaded from jap.physiology.org on October 16, 2006

(Ma.Ar) were measured at an objective lens magnification of
⫻2; cortical bone area (Ct.Ar) was calculated as the difference
between these two areas. Each cross section was divided into
medial and lateral regions as previously described (19). By
using epifluorescent illumination, total periosteal and endo-
cortical perimeters and single-labeled perimeters were quan-
tified for each region. No double-labeled perimeters were
observed on cortical bone surfaces in all samples; therefore,
we could not calculate bone formation rate or mineral apposi-
tion rate. Uptake of fluorochrome labels was confirmed by the
presence of double labels in cancellous bone samples from the
same animals. Samples from two control rats showed no
evidence of calcein uptake and were excluded from the
determination of single-labeled perimeters. Single-labeled
perimeters are expressed as a percentage of the total bone
perimeter. All histomorphometric nomenclature conforms to
that recommended by Parfitt et al. (18).
Statistical analyses. Group differences were evaluated by
using one-way ANOVA. When a significant main effect was
detected, group means were further compared by using
Student-Newman-Keuls or Duncan’s multiple-range post hoc Fig. 2. Body weight and food intake (means ⫾ SE) over 6 wk of
tests. If normality or equal variance assumptions were vio- unilateral hindlimb immobilization (Immobilized) or immobiliza-
lated, a Kruskal-Wallis ANOVA on ranks was performed. tion ⫹ external loading of immobilized tibia (Immob-Loaded).
* P ⬍ 0.05 vs. control at same time point. ⫹ P ⬍ 0.05 vs 0-wk time
Unpaired t-tests were performed on data from the baseline point within group.
control rats and the aging control rats in both experiments to
assess changes over 6 wk of aging. An alpha level of P ⬍ 0.05
was used to determine statistical significance. All statistical
tests were done by using SigmaStat software (Jandel Scien- weight loss over 6 wk amounted to a mean of 54.5 ⫾
tific). 3.7 g in the immobilized group and 42.3 ⫾ 6.6 g in the
immobilized-loaded group (Fig. 2A). Food consumption
RESULTS in both immobilized groups was significantly less than
There were no significant differences between the that of the control group (Fig. 2B). Observations during
two control groups in experiment 1 (baseline and aging daily monitoring suggested reduced voluntary physical
control) for most variables. Therefore, results from the activity of immobilized animals. Given the different
two control groups were pooled (n ⫽ 9 after pooling) for body weights of immobilized groups vs. control rats,
the remainder of the statistical analyses in both experi- tissue weights are expressed relative to total body
ments for all but the histomorphometric variables. One weight (Table 1). Normalized spleen weight did not
immobilized-loaded rat in experiment 2 died prema- vary among groups. Effective immobilization was con-
turely because of an overdose of methoxyflurane during firmed by the 43 and 50% decrements in tibialis ante-
an external loading and retaping procedure. Immobi- rior and soleus muscle normalized weights, respec-
lized and immobilized-loaded groups experienced simi- tively, of the right hindlimb (Table 1). The loading
lar weight loss during the experimental period. Total regimen had no effect on muscle or spleen weights.
192 LOADING ATTENUATES DISUSE-INDUCED BONE LOSS

Table 1. Body and tissue weights (normalized to body weight) of rats at euthanasia
Tissue Weights, mg/g BW
Body
Group n Weight, g Spleen R. tibialis ant. L. tibialis ant. R. soleus L. soleus

Control 9 307 ⫾ 5 2.14 ⫾ 0.06 1.99 ⫾ 0.03 1.99 ⫾ 0.04 0.42 ⫾ 0.01 0.41 ⫾ 0.02
Immob 8 248 ⫾ 6* 2.12 ⫾ 0.08 1.13 ⫾ 0.08* 1.95 ⫾ 0.02 0.21 ⫾ 0.01* 0.45 ⫾ 0.01
Immob-Load 10 253 ⫾ 4* 2.11 ⫾ 0.12 0.96 ⫾ 0.07* 1.91 ⫾ 0.05 0.20 ⫾ 0.01* 0.45 ⫾ 0.02
Values are means ⫾ SE. n, No. of rats; BW, body weight; Control, cage activity controls, killed at 0 and at 6 wk; Immob, right hindlimb
immobilized for 6 wk; Immob-Load, immobilized tibia subjected to mechanical loading 3 times/wk; R, right; L, left; ant., anterior. * P ⬍ 0.05 vs.
Control.

BMD and BMC of the immobilized rats’ tibial mid- Histomorphometric analyses (Table 3) of tibial middi-
shaft were 12 and 10% lower, respectively, than those of aphysis from age-matched rats in experiment 2 allowed
control rats (Table 2). The proximal tibia appeared for tibial bone Ct.Ar to be determined in a consistent
most affected by immobilization, with 23–28% reduc- anatomic location (5–6 mm proximal to the tibiofibular
tions in BMD or BMC vs. control. At the distal tibia junction), which was within 2 mm of the fracture site
BMC was unaffected, whereas BMD was reduced by for those tibiae subjected to three-point bending to
10%. Five weeks of mechanical loading prevented the failure. Ct.Ar was 7% lower in immobilized tibiae than
decline in both BMD and BMC of the tibial midshaft in aging control rat tibiae (P ⬍ 0.05), whereas loading of
seen with immobilization. The decreases in BMD and immobilized tibiae effectively maintained Ct.Ar. Ma.Ar

Downloaded from jap.physiology.org on October 16, 2006

BMC were proportionately smaller than were the de- at this site increased significantly in both immobilized
creases in total body weight, as evidenced by 6–19% groups, with a greater increase observed in the immobi-
increases in BMD and BMC per gram of body weight in lized-loaded tibiae. Although increases in periosteal
these groups. The loading regimen utilized did not alter and endocortical surfaces exhibiting single fluoro-
BMD or BMC of the proximal or distal tibia. chrome label (%single-labeled surface) in immobilized-
Measures of mechanical strength and geometry of loaded tibiae ranged from 25 to 110% vs. unloaded
the tibial midshaft are illustrated in Fig. 3. Ultimate immobilized tibiae, none of these increases was statisti-
load of the tibia determined in three-point bending to cally significant.
failure was 19% lower in the immobilized group than in
the control group. This decrease in ultimate load at
failure was halved by the loading regimen. A similar
pattern is observed for changes in stiffness. Loading
effectively attenuated the 20% decline in stiffness ob-
served with immobilization alone, reducing this deficit
in stiffness to ⫺8.5%, which was not significantly
different from the value for control rats. Intergroup
comparisons of CSMI nearly achieved significance (P ⫽
0.056). CSMI in the immobilized and immobilized-
loaded groups was 20 and 8% lower, respectively, than
was mean CSMI in control animals. There were no
significant differences among the three groups for
modulus of elasticity nor for ultimate stress at the site
of failure in these specimens.

Table 2. Bone densitometry data on proximal,

midshaft, and distal tibia of the immobilized hindlimb
BMC, BMC, BMD, BMD, mg · cm2 · g
mg mg/g BW mg/cm2 BW⫺1

Proximal tibia
Control 76 ⫾ 3 0.25 ⫾ 0.01 133 ⫾ 4 ⫹1.0 ⫾ 1.5
Immob 55 ⫾ 2* 0.22 ⫾ 0.01 102 ⫾ 3* ⫺12.4 ⫾ 1.0*
Immob-Load 58 ⫾ 1* 0.23 ⫾ 0.005 108 ⫾ 2* ⫺11.5 ⫾ 1.5*
Midshaft tibia
Control 48 ⫾ 1 0.16 ⫾ 0.01 95 ⫾ 2 ⫺0.4 ⫾ 1.6
Immob 43 ⫾ 1* 0.17 ⫾ 0.004* 84 ⫾ 2* ⫺7.9 ⫾ 1.1*
Immob-Load 48 ⫾ 1† 0.19 ⫾ 0.004*† 94 ⫾ 1† ⫺0.2 ⫾ 1.2†
Distal tibia
Control 31 ⫾ 1 0.10 ⫾ 0.003 105 ⫾ 2 ⫺0.2 ⫾ 1.3
Immob 30 ⫾ 1 0.12 ⫾ 0.01* 94 ⫾ 2* ⫺7.1 ⫾ 1.3* Fig. 3. Comparisons among experimental groups for cross-sectional
Immob-Load 29 ⫾ 1 0.12 ⫾ 0.002* 100 ⫾ 2* ⫺4.7 ⫾ 1.2* moment of inertia (CSMI) and mechanical properties [ultimate (Ult.)
load, stiffness, ultimate stress, and elastic modulus] of tibial shaft.
Values are means ⫾ SE. BMC, bone mineral content; BMD, bone Values are means ⫾ SE. * P ⬍ 0.05 vs. Control. ⫹ P ⬍ 0.05 vs.
mineral density. * P ⬍ 0.05 vs. Control. † P ⬍ 0.05 vs. Immob. Immobilized.
 LOADING ATTENUATES DISUSE-INDUCED BONE LOSS 193

Table 3. Histomorphometric data on middiaphyseal tibial bone

Area, mm2 Periosteal, %sLS Endocortical, %sLS

Group Bone Medullary Medial Lateral Medial Lateral

Control-0 4.14 ⫾ 0.08 2.17 ⫾ 0.07 20.0 ⫾ 5.0 30.9 ⫾ 6.4 19.2 ⫾ 7.3 17.7 ⫾ 7.0
Control-6 4.19 ⫾ 0.08 2.14 ⫾ 0.06 29.7 ⫾ 10.3 19.9 ⫾ 5.5 2.2 ⫾ 1.7 1.3 ⫾ 0.8
Immob 3.90 ⫾ 0.09* 2.66 ⫾ 0.08† 15.8 ⫾ 4.0 36.0 ⫾ 8.7 9.1 ⫾ 3.7 5.7 ⫾ 3.8
Immob-Load 4.23 ⫾ 0.10‡ 2.87 ⫾ 0.07†‡ 33.2 ⫾ 4.5 45.1 ⫾ 4.7 16.2 ⫾ 5.1 11.5 ⫾ 4.6
Values are means ⫾ SE; n ⫽ 7 (Control-0), 7 (Control-6 areas), 5 [Control-6 % single-labeled surface (sLS)], 8 (Immob), and 6 (Immob-Load)
rats. Control-0, baseline control group killed, at 0 wk; Control-6, aging Control killed at 6 wk. * P ⬍ 0.05 vs. Control-6. † P ⬍ 0.05 vs. both
Control groups. ‡ P ⬍ 0.05 vs. Immob.

DISCUSSION not subjected to immobilization, this loading procedure

induces compressive strains on the lateral tibial sur-
Acute calcium deficiency concurrent with immobiliza-
tion effectively ensures a high turnover state in bone face of 1,000–1,400 µ⑀ (Inman and Bloomfield, unpub-
due to elevated serum PTH. The subsequent loss of lished observations), ⬃50% higher than strains pro-
bone with this combined disuse and calcium deficiency duced during normal ambulation in the rat (8).
is dramatic; in immature rats (⬃2 mo old), femoral ash This loading intervention maintained bone cross-
weight declines by 46% after only 72 h (23). The age of sectional area and possibly geometry (i.e., CSMI),
the animal at the onset of immobilization and calcium thereby contributing to the maintenance or attenuated

Downloaded from jap.physiology.org on October 16, 2006

deficiency appears to have a large impact on the decline in bone mechanical properties during a period
magnitude and rapidity of the response. We observed of immobilization. The decrease in ultimate load of
smaller decreases of 28 and 10% in proximal tibial midshaft tibial bone seen with immobilization was
BMC and midshaft BMC, respectively, in our 5-mo-old halved by the in vivo four-point bending regimen; tibial
rats subjected to immobilization and a low-calcium stiffness was effectively maintained. For small animals
diet. The larger effect at the proximal tibia is likely due such as the rat, stiffness of appendicular bones may be
to the higher content of cancellous bone at this site, the more important factor, because bone stiffness is
which experiences a more dramatic decrease in bone critical in optimizing muscle function during locomo-
volume than does cortical bone with prolonged immobi- tion and the peak loads typically imposed on the bone
lization or spaceflight (15, 22). We also observed in- are small (2).
creased Ma.Ar, suggesting increased endocortical re- When ultimate load and stiffness (structural proper-
sorption, and a significant 6.5% decrease in Ct.Ar after ties) are normalized to cross-sectional geometry, the
6 wk of this treatment. resulting indexes (ultimate stress and modulus) yield
The most significant findings of these studies are information about material properties of the tissue
that mechanical loading imposed on immobilized corti- independent of area or CSMI. There were no significant
cal bone experiencing high turnover effectively main- differences for modulus of elasticity or for ultimate
tained BMD and tibial cross-sectional area in that stress with immobilization or with immobilization plus
region experiencing bending forces during loading. loading. If bone material properties are unchanged in
Furthermore, the loss of mechanical strength normally the face of declining whole bone stiffness, the remain-
observed with disuse was attenuated with this loading
ing possibility is that the quantity and/or geometry of
regimen. BMD remained significantly depressed at the
the bone changed with disuse and calcium deficiency.
proximal and distal tibial sites in immobilized hind-
The mean Ct.Ar declined 7% in immobilized tibiae.
limbs undergoing in vivo four-point bending. Resorp-
tion at the endocortical surfaces persisted or increased Shen et al. (20) observed a similar reduction (⫺9%) in
in immobilized-loaded tibiae, as suggested by marrow Ct.Ar and a 44-fold increase in endocortical eroded
cavity areas that were larger than in unloaded immobi- surface in denervated hindlimbs of adult rats fed a
lized tibiae. This endocortical expansion was counterbal- calcium-deficient diet. Loading in the present study
anced in the loaded bones by a suggestion of increased was effective in preventing the decline in Ct.Ar due to
bone formation activity (implied from single-labeled disuse and calcium deficiency. The larger Ma.Ar in the
surfaces) at the medial periosteal surface. In ambula- immobilized-loaded animals vs. those in immobilized
tory rats, this same loading regimen produces a rapid rats also imply greater endocortical resorption, perios-
increase in bone formation in the maximum bending teal expansion, and larger CSMI. Although changes in
area of the tibia after only 2 wk of daily loading (6, 19). bone CSMI among groups did not achieve statistical
In a comparison of the bending moments between in significance, the attenuated declines in ultimate load
vivo four-point loading (estimated at 96 N/mm) and in and stiffness seen with loading suggest that the smaller
vitro three-point bending before failure (estimated at decline in CSMI in loaded tibiae vs. that in unloaded
360 N/mm), it was determined that the strains pro- immobilized tibiae was functionally significant.
duced during in vivo loading were within the elastic A similar beneficial effect of an external loading
portion of the load-deformation curve. Hence our four- intervention has been demonstrated in functionally
point bending regimen imposed loads within the rat isolated (and therefore immobilized) avian cortical bone,
tibia’s usual functional loading ranges. In adult rats in which the usual loss of bone area was exacerbated by
194 LOADING ATTENUATES DISUSE-INDUCED BONE LOSS

dietary calcium deficiency. A daily loading regimen of Address for reprint requests and other correspondence: S. A.
100 cycles generating 2,000 u⑀ on bone surface effec- Bloomfield, Texas A&M Univ., 276E Read Bldg., College Station, TX
77843-4243 (E-mail: [email protected]).
tively attenuated a 32% decrease in cross-sectional
Received 2 June 1998; accepted in final form 24 February 1999.
area observed in untreated isolated ulnae of egg-laying
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 LOADING ATTENUATES DISUSE-INDUCED BONE LOSS 195

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