Bone Vol. 20, No.
3
ELSEVIER
Strain Magnitude Related Changes in Whole Bone
Architecture in Growing Rats
J. R. M O S L E Y , 1 B. M. M A R C H , 1 J. L Y N C H , 2 and L. E. L A N Y O N 1
i Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London, UK
2 Microfocal X-Ray Unit, Medical School Building, United Medical and Dental Schools, Guy's Hospital, London, UK
Short daily periods of controlled dynamic loading were ap-
plied in vivo through the flexed carpus and olecranon to the
intact ulna of 240 g male Sprague-Dawley rats. This tech-
nique involved neither surgical preparation, nor direct load-
ing of the periosteum at a site close to the region of the bone
in which adaptive modeling was subsequently assessed. The
animals used their limbs normally between loading episodes,
thus approximating to the natural situation, in which short
periods of exercise are generally superimposed on longer pe-
riods of less strenuous activity. The strain patterns associated
with normal activities were established for the rat ulna from
strain gauges implanted in vivo. Typical peak strain magni-
tudes during unrestricted locomotion varied between -0.0007
and -0.0012, with peak strain rates between 0.023 and -0.038
sec -~. Stride frequency was 1.5-4.2 Hz. The adaptive re-
sponse to a single 10 min period of loading each day, causing
peak dynamic strains of -0.002 (1200 cycles at 2 Hz, and a
loading/unloading rate of +0.03 sec-~), involved modification
of the normal growth related medial to lateral modeling drift,
simultaneously reducing the rate of lateral periosteal bone
deposition and medial bone resorption. This change to the
normal modeling pattern reduced the total amount of new
bone formation as well as the midshaft curvature of the ulna.
At higher peak strain amplitudes (-0.004), adaptive straight-
ening was accompanied by an increase in bone mass,
achieved by an increase in the mineral apposition rate on the
previously forming lateral face, and arrest of resorption on
the medial ulna surface, with reversal to formation. These
experiments show that the growing rat ulna underwent adap-
tive changes in both bone mass and architecture when short
daily periods of axial loading, producing strains within the
physiological range and with near normal strain distribution,
were superimposed on the loading associated with normal
activity. At moderate peak strain magnitude (-0.002), modi-
fication of drift produced a straighter bone, associated with a
reduced periosteal bone formation. At higher strain magni-
tude (-0.004), adaptive modeling produced a straighter bone
associated with increased periosteal bone formation. (Bone
20:191-198, 1997) 1997 by Elsevier Science Inc. All rights
reserved.
Key Words: Rat; Ulna; Cortical bone; Adaptation; Mechanical
load; Mechanical strain.
Address for correspondence and reprints: John R. Mosley, Department
of Veterinary Basic Sciences, The Royal Veterinary College, Royal
College Street, London, NW1 0TU, United Kingdom. E-mail:
jmosley@rvc.ac.uk
1997 by ElsevierScienceInc.
All fights reserved.
March 1997:191-198
Introduction
It is generally accepted that dynamic functional strains provide
an important input to those ceils of the skeleton responsible for
the maintenance and adjustment of bone architecture. Indeed, of
the many factors that influence bone mass and architecture, it is
probable that only dynamic strain, or one of its derivatives, con-
tains sufficient information to be the input variable for the ap-
propriate control of bone mass and architecture in relation to
loading. 9"14'16 Despite its importance, only limited information is
currently available regarding the contribution provided by each
of the different components of the strain environment within a
bone, to the modeling and remodeling activity responsible for its
architecture. Peak strain magnitude is the most obvious of these
variables, and is the subject of the investigation reported here.
To date, the only study that has directly investigated the effect
of changes in measured dynamic peak strain magnitude on bone
modeling/remodeling activity in vivo is that of Rubin and
Lanyon. 27 This was achieved by the application of controlled
loads of differing magnitudes to a surgical preparation of the ulna
in skeletally mature male turkeys. Two important features of this
model were that the distribution of strain through the ulna cross
section was very different from that recorded during customary
activities, and that the loadable bone segment was isolated from
functional strains between scheduled loading episodes. This en-
sured that the externally applied loads provided the predominant
mechanical stimulus to the bone. While this eliminated the con-
founding influence of unquantified activity between loading epi-
sodes, it is far removed from the more usual in vivo loading
situation, in which bones continually experience functional
strains from normal activities. In a more recent study using the
mature rat, the strains associated with normal activity were
supplemented with a brief period, during which strains of con-
trolled magnitude were applied to the tibia using a four-point
bending technique. 32 The magnitude of the calculated strains was
varied by regulating the applied load. Despite the presence of a
periosteal woven bone reaction, which this method of loading
stimulates through direct periosteal pressure, a lamellar bone
response was documented at the endocortical surface, the mag-
nitude of which increased linearly as the predicted peak strain
magnitude increased above a threshold value of 0.00105. How-
ever, since the sites of endocortical response and periosteal re-
action are in such close proximity, the independence of these two
responses has been the subject of d e b a t e . 24'29"31
The present study investigated the influence of a short, daily
period of controlled, dynamic loading, producing strains of dif-
ferent amplitude, on the development of ulna architecture in the
growing rat. The loading system involved neither surgical inter-
191 8756-3282/97/$17.00
Pll $8756-3282(96)00385-7
 192 J.R. Mosley et al.
Strain magnitude related changes in bone
vention nor direct loading through the periosteum at a site close
to the region of the bone in which the adaptive modeling re-
sponse was subsequently assessed. The strain distribution within
the ulna during loading was similar to that produced during nor-
mal activity to which the bones were exposed for the majority of
the time.
Materials and Methods
Measurement of Functional Strains In Vivo
Male Sprague-Dawley rats were purchased from Charles River
UK Ltd., housed in pairs throughout the experimental period in 53
x 35 cm polypropylene cages, and subjected to a 12-h light/dark
cycle. Room temperature was maintained at 21C + 2C. The
animals were fed a standard rodent diet (RM1; Special Diet
Services Ltd, South Witham, UK), and tap water was available
ad libertum. A 7 day acclimatization period was allowed between
arrival of the animals and the start of each experiment. Each rat
weighed between 250 and 300 g at the start of the study, corre-
sponding to the body mass of the rats during the subsequent
controlled loading period (mean body mass on first day of load-
ing, 238 g, SEM + 1.2 g, and at collection of tissues, 316 g, SEM
+ 2.6 g).
A caudal approach was made to the ulna midshaft by blunt
dissection. The periosteum was removed from the implantation
site, and the bone gently scraped with a scalpel blade to produce
a sound surface for bonding of strain gauges. The bone was
degreased by swabbing with diethyl ether soaked cotton swabs
and, using previously established techniques, 6"~~,~2.19.23 a longi-
tudinally aligned, single element strain gauge (EA-06-015-LA:
Measurements Group, UK) was positioned on either the medial
or lateral ulna diaphysis, directly opposite the center of the in-
terosseous ligament. This marks the ulna midshaft, and serves as
a consistent anatomical landmark. These locations on the ulna
circumference were selected as those experiencing maximum
strain during both in vivo locomotor loading and artificial axial
loading. 23,3
The gauges were initially zeroed while each animal was anes-
thetized, as well as periodically during the course of the record-
ings by briefly lifting the animals so that the gauged limb was
nonweight-bearing, and resetting the zero level once the animal
relaxed.S.11.13.33 Strain recordings were made daily for 7-10 days
during a variety of activities including: free walking on a level
surface; moving at various speeds on a motorized treadmill at a
variety of gradients, both up- and downhill; jumping from a
height of approximately 30 cm; and washing the face with the
forelimbs.
Ex Vivo Strain Calibration
Strain gauges cannot be attached to those bones in which sub-
sequent modeling/remodeling activity is to be assessed. The re-
lationship between the applied axial load, which can be moni-
tored during loading episodes, and the resultant bone strain,
which cannot, was therefore established in a group of minimally
dissected "calibration" limbs (n = 4), taken from rats matched
for weight, and subjected to the same husbandry conditions as the
experimental loading animals. Each strain gauged limb was po-
sitioned and axially loaded in the same way as the limbs sub-
jected to controlled loading in the subsequent in vivo experiment.
This allowed the load necessary to generate the required strain
within the ulna to be predicted for the subsequent in vivo loading
experiments.
Bone Vol. 20, No. 3
March 1997:191-198
In further specimens (n = 4), similar strain recordings were
obtained from three single element strain gauges (EA-06-015-
D J: Measurements Group, UK), positioned in the same plane
around the circumference of the ulna. This permitted an estimate
of the longitudinal strain distribution through the ulna cross-
section to be made.
In Vivo Ulna Loading
Male Sprague-Dawley rats were purchased at a body weight of
185 + 5 g (estimated age 43 days), 1 week before the start of
loading. Animals were randomized into loading groups and
housed in pairs throughout the experimental period. Each group
contained a minimum of seven animals. Anaesthesia was induced
by intraperitoneal injection of xylazine (RompunTM: Bayer, Bury
St Edmunds, UK), 11 mg kg -I, and ketamine hydrochloride
(VetalarfM: Parke, Davis and Co., Pontypool, UK), 55 mg kg i.
The corneas were protected from desiccation by application of a
bland ophthalmic ointment (LacrilubeTM: Allergan, High Wy-
combe, UK).
The anaesthetized animals were placed in dorsal recumbency
on a thermally insulated mat. The left antebrachium was held in
padded cups between the olecranon process of the ulna and the
flexed carpus while the limb was loaded. These cups were
coupled through low friction bearings to a servohydraulic mate-
rial testing machine, arranged in such a way that the actuator
applied a regulated cyclic load to the carpus (Figure 1). Follow-
ing loading, the rats were transferred to insulated polystyrene
boxes for recovery. Consciousness was generally regained within
2OM0 min. Between anaesthetic periods, normal cage activity
was allowed.
Load was applied to the experimental limb in a trapezoidal
pattern on days 1-5 and 8-12 inclusive. The dynamic load range
applied to the forearm was calculated from the ex vivo strain
gauge studies to provide peak dynamic compressive strains at the
medial ulna midshaft of -0.0005, -0.001, -0.002, -0.003, or
-0.004. A standard high-dwell period of 50 msec was used for all
flexed olecranon
carous ulna process
padded radius humerus
cup
ACTUATOR
MOVEMENT
Figure 1. Schematic diagram of the ulna loading system, indicating the
approximate position of the bones during loading. The forearm is held in
padded cups between the flexed carpus and the olecranon. Loads are
applied to the carpus and transmitted through the limb to the olecranon
where they are monitored by a load cell. The ulna is therefore loaded
through the carpal joint, and overlying soft tissues.
Bone Vol. 20, No. 3
March 1997:191-198
loading groups, during which time the load was held at the maxi-
mum level. Each animal was subjected to 1,200 loading cycles,
at a frequency of 2 Hz, and a loading/unloading rate calculated to
give peak strain rates of +0.03 sec -~ at the medial ulna midshaft.
The use of a constant strain rate meant that the time taken to
reach the peak strain was longer for the higher strain magnitude
groups. To maintain a constant frequency therefore, the length of
the low dwell period of the waveform was adjusted as necessary
between loading groups.
Assessment of the Response to Loading
Calcein (Sigma: Poole, Dorset, UK), was administered by intra-
peritoneal injection at a dose rate of 10 mg kg -~, 3 days before
the start of loading, and on the 10th (final) day of loading. Three
days after the final loading period, the animals were killed by
overdose with sodium pentobarbitone (EuthesateTM: Willows
Francis Veterinary, Crawley, UK). Left and right forelimbs were
removed and the radius and ulna dissected free from attendant
soft tissues, leaving the periosteum and all ligamentous attach-
ments between the two bones intact. The length of the ulna was
measured to the nearest 10 i~m from the proximal olecranon to
the lateral styloid process. After fixation in phosphate buffered
formalin, the paired bones were cut transversely at the exact ulna
midshaft, using a low speed, diamond-sintered annular saw (Mi-
croslice II: Cambridge Instruments, Malveru, UK).
The tissues were dehydrated, cleared, and embedded in meth-
yl methacrylate using the technique described by Baron et al. ~
F o l l o w i n g p o l y m e r i s a t i o n , six serial, plane-parallel cross
sections were cut at 1 m m intervals from the ulna midpoint in
both a proximal and distal direction. This represents approxi-
mately 40% of the length of the intact bone. Cutting the bones in
half prior to serial sectioning ensured that the loaded and control
ulnae were sampled at functionally equivalent sites despite any
reduction in longitudinal growth, which may have occurred as a
result of loading. Given the degree of shortening seen in the
loaded ulnae, the greatest misregister between the extreme sec-
tions from loaded and control limbs was less than 250 txm.
The subsurface layer of each section was imaged with a Bio-
Rad MRC-500 confocal laser scanning microscope (CLSM),
(488 nm argon ion laser line excitation, >515 nm emission),
employing a Nikon 4 0.2 NA dry, no coverslip objective. The
intense fluorescence from the incorporated calcein labels ap-
peared superimposed on the much weaker autofluorescence from
the ulna cross section, which provided background contextual
histology.
The response to loading was assessed over the central 12 m m
of the ulna diaphysis by measurement of the periosteal and end-
osteal interlabel areas. Within each loading group the presence of
a loading related effect on bone modeling was established at each
of the 12 sampling levels by comparison of the parameters mea-
sured in the loaded limb with the corresponding measurement
made at the same level in the contralateral control limb, using
paired, two-tailed, Student's t-test. Between groups, pair-wise
comparisons were made at any given site on the bone using
Fisher's protected least significant difference.
To localize the site of any change in periosteal growth, the
mineral apposition rate (MAR) was assessed by measurement of
the separation between periosteal calcein labels in the digital
CLSM fluorescence image. The M A R was determined at 72
separate sites around the bone circumference for the -0.002 and
-0.004 loading groups at a level 2 m m distal to the midshaft,
which corresponded with the site of maximum osteogenic re-
sponse seen at -0.004. Each site for M A R measurement was
J.R. Mosley et al. 193
Strain magnitude related changes in bone
located by superimposition of a 72 point star on the digital image
centered on the medullary cavity.
Assessment of Mean Ulna Curvature
An identical cohort of rats (n = 3), was acquired and loaded
similarly to those above, to peak midshaft strains at the medial
ulna diaphysis of -0.002. Loaded and control ulnae were har-
vested 3 days after the administration of the second fluoro-
chrome, and attached soft tissues removed by incubation with an
enzymatic detergent (Terg-a-zyme: Alconox Inc., NY). This re-
moves periosteum and cellular material, but not osteoid or min-
eralised matrix (A. Boyde, personal communication). The posi-
tion of the bone surfaces therefore effectively represented the
extent of the bone matrix when the animals were killed.
The assessment of curvature was restricted to the medial and
lateral ulna faces, over a distance equal to 10% of the total bone
length around the midshaft. This was chosen as a region where
the curvature was maximal and likely to be reasonably uniform.
The bone was mounted vertically on a rotating stage alongside a
calibration scale, which indicated the position of the midshaft
and the proximal and distal extremes of the length of the diaph-
ysis over which curvature was to be measured. The ulna was then
lit uniformly from behind, and rotated on its long axis until the
shadows cast by the cranial and caudal longitudinal ridges of the
lateral face of the ulna were exactly superimposed at the mid-
shaft. A digital image of the silhouetted bone was captured using
a CCD camera. Automated edge detection 2~ was used to locate
the bone edges over the region of interest, and the mean curva-
ture, expressed as the reciprocal radius of curvature, was calcu-
lated for both the medial and lateral edge of the bone, using
custom written software.
Results
Functional In Vivo Strains
During unrestricted locomotion (running) on a solid level surface
the peak strain magnitude varied between animals from -0.0007
to -0.0012 with the medial ulna face experiencing compression,
and the lateral face tension. Maximum strain rates were in the
range 0.023 to -0.038 sec -1. Stride frequency varied between 1.5
and 4.2 Hz, as the same animals ran with different stride fre-
quencies during separate recording sessions.
The range of activities that rats undertake within their natural
environment is very varied. An attempt to estimate the peak
strain magnitudes during more extreme activities was therefore
made by allowing the rats to fall squarely onto a solid level
surface from a height of approximately 30 cm. This did not cause
any distress, and was usually followed by a period of normal
locomotion. It seems likely that similar loading incidents would
be encountered in the wild rat, whose inquisitive behavior, and
ability to climb and jump will often lead to such unplanned bone
loading "accidents." The peak strain magnitude associated with
such incidents was more than double that associated with normal
locomotor loading, with peak medial face strains in the order of
-0.0025, and a maximum strain rate approaching -0.1 sec -1.
Ex Vivo Strain Validations
During artificial loading, the medial face of the ulna experienced
compression, while the lateral face was subject to tension. The
ratio between lateral and medial face strains at each load was
highly consistent for individual bones. The mean ratio of com-
194 J.R. Mosley et al.
Strain magnitude related changes in bone
pressive to tensile strains at the ulna midshaft, calculated using
the ratio for each specimen at each load was 1.23, (SEM + 0.02;
range 1.10-1.41). If the strain at the ulna midshaft is re-
solved into that due to compression and that due to bending,
over 90% of the midshaft strain is seen to be due to bending,
with only 10% due to axial compression. This resembles the
situation documented for many bones during natural locomotor
loading.2 5,8,17js,25
The strains measured in the calibration bones were applicable
only for the bone architecture on the first day of loading. As the
bones both grow and adapt over the course of the experiment, the
strains produced for any given load will change. Such changes,
although real, are likely to be slight in comparison with other
sources of experimental variation.
Change in Bone Length
For all treatment groups, the loaded ulnae were shorter than their
control, nonloaded pairs. The relative alterations in loaded bone
length compared to paired control bones, are shown by treatment
group in Figure 2. The mean retardation of longitudinal growth
ranged from 0.52% to 3.74% of the control bone length. This
represents a mean absolute shortening between loading groups
ranging from 0.17 mm (SEM + 0.06 mm) to 1.15 mm (SEM +
0.10 mm). Least squares regression analysis demonstrated a
highly significant linear relationship between the magnitude of
the applied load and the degree to which longitudinal growth was
reduced (r = 0.837; p < 0.001).
Change in Periosteal Modeling Patterns
The most marked periosteal response to increasing mechanical
load occurred in the region of the bone just distal to the midshaft
(Figure 3). This pattern of new bone production is representative
of the response assessed between 1 and 4 mm distal to the mid-
shaft. No discemable response is seen in the -0.0005 group. The
3.74%
t~ 1.4
a',b',c',d'.
E apposition rate on the lateral bone face, and conversion of the
1.2
t--
~'~ 1.0
c The periosteal response of the -0.002, and -0.004 treatment
0.8
C -0.002 group, the suppression in periosteal growth which oc-
0.6
.-
0.4
ii1
= 0.2
"1:3
0.0
-500 -1000 -2000 -3000 -4000
peak strain amplitude (xl0 -6)
5 7 11 15 19
applied load amplitude N.
Figure 2. Mean (+SEM) difference in length between control ulnae and
those subjected to axial loads producing strains of increasing magnitude.
Percentage differences are expressed relative to control bones for each
treatment group. Significant differences between groups are indicated by
lower case letters (a significantly different from a' etc.; p < 0.05).
Bone Vol. 20, No. 3
March 1997:191-198
co
E 200 p=0.016
150 .,.,o,IT
9% ~oo
- -
I11 vX
N.S.
5O
L.
c. b ' , ~
a. b.
~ ~ 0
-50
._o N.S. p=0.024 p--O.O02
(-9%) (-11%) (+37%)
-500 -1000 -2000 -3000 -4000
peak strain amplitude (xl0 -6)
Figure 3. Mean (+SEM) difference in periosteal new bone area between
loaded and contralateral control limbs, measured 2 mm distal to the ulna
midshaft in response to loading at increasing peak strain magnitude, p
values indicate the statistical significance of the difference in periosteal
interlabel area between paired loaded and control ulnae within each
loading group (N.S.: not significant). Between loading groups, signifi-
cant differences are indicated by lower case letters (a significantly dif-
ferent from a' etc.; p < 0.05).
-0.001 and -0.002 groups both show a suppression in the rate of
periosteal new bone deposition, representing an 11% (SEM _+
1.9% ), decrease in the rate of periosteal expansion for the -0.002
group. In the animals subjected to higher loads, a reversal in the
direction of this response is seen, resulting in a nonstatistically
significant increase in bone formation at -0.003, and a greatly
increased periosteal new bone area at -0.004. The response to
strains of -0.004 represents a 37% (SEM + 11.6%), increase in
the area of bone deposited over the 14 days between fluoro-
chrome label administration. This large increase in periosteal
new bone deposition is brought about by an increase in mineral
previously resorbing medial face into one that is actively form-
ing organized new bone (Figure 4).
groups over the diaphyseal length is illustrated in Figure 5. In the
curred distal to the midshaft was maintained with a statistically
significant reduction in the amount of periosteal new bone de-
posited along the entire length of the ulna examined. The maxi-
mal increase seen in the -0.004 group occurred 2 mm distal to
the midshaft, and approached zero in both proximal and distal
directions, crossing the x axis proximally, where a significant
reduction in periosteal mineral apposition was observed. The
mean decrease in periosteal mineral apposition for the -0.002
group ranged between - 9 % (SEM + 2.4%), and - 1 7 % (SEM +
5.5%). For the -0.004 loading group, mean changes in periosteal
mineral apposition ranged from - 2 5 % (SEM + 4.7%) to +37%
(SEM + 11.6%).
Despite the large mean increase in bone deposition seen dis-
tally at -0.004, the increase in periosteal new bone area was
variable between subjects, with individual ulnae increasing their
periosteal new bone deposition by as much as 72%. Of the seven
animals in this group, one failed to show any increase in bone
mass and a second mounted only a poor periosteal response. In
all animals, the new bone produced had a characteristically or-
Bone Vol. 20, No. 3
March 1997:191-198
Figure 4. Confocal fluorescence images (488 nm excitation, >515 nm
emission) of paired cross sections through the ulna of a male Sprague-
Dawley rat, taken 2 mm distal to the midshaft. (A) Control, nonloaded
ulna. There is an extensive area of periosteal resorption on the medial
face (arrow heads). Laterally there is deposition of lamellar bone (solid
arrows), with formation of primary osteons at the cranial and caudal bone
edges. (B) Contralateral ulna loaded to peak midshaft strains of -0.004.
Note the complete reversal of resorption to active formation on the me-
dial face (open arrows), and the increased mineral apposition rate on the
lateral face (solid arrows). Scale bar represents 500 ixm.
ganized lamellar appearance and was indistinguishable from the
underlying bone. Woven bone was not observed in any of the
limbs examined. No change in new bone production was mea-
surable on the endosteal envelope. Figure 6 shows the distribu-
tion of the adaptive change in MAR around the circumference of
the ulna for the -0.002, and -0.004 loading groups, 2 mm distal
to the midshaft. It is evident that in the -0.004 group, the ma-
jority of the increase in new bone was due to the response on the
medial and lateral ulna face, with no difference in the MAR of
the cranial and caudal ulna face around the neutral axis. At
-0.002, the change in MAR was distributed around the entire
bone circumference, with the greatest change occurring toward
the neutral axis.
Adjustments in Ulna Curvature
The mean longitudinal curvature measurements are presented in
Table 1, together with the change in curvature in response to
loading at -0.002. In both loaded and control ulnae the medial
face of the bone was longitudinally concave. In all three bone
pairs examined, the medial face straightened in response to load-
ing. Over the central 10% of the ulna length, curvature reduced
significantly, from a mean value of -96.15 m -1 (SEM + 7.33),
J.R. Mosley et al. 195
Strain magnitude related changes in bone
for control bones, to a mean of -38.40 m -~ (SEM + 1.95) for
loaded bones (p < 0.01). The lateral bone face remained rela-
tively straight, and the mean curvature did not change signifi-
cantly as a result of mechanical loading.
Discussion
The nonsurgical rat ulna axial loading model has several advan-
tages over other published models for the determination of the
effect of mechanical loading on functional adaptation in bone.
Since the rat is a mammal, it is perhaps more relevant to the
human situation than are studies in the avian skeleton. Addition-
ally, no surgical preparation is required, and the anatomical cur-
vature of the bone provides a consistent strain distribution which
is similar to that produced during normal locomotion.23 Between
loading episodes, normal activity is allowed that is analogous
to the situation for prescribed exercise regimens.
The relative shortening in length seen in the loaded ulnae is
presumably achieved through a retardation in longitudinal ex-
pansion at the growth plates. The dependency of this effect on the
magnitude of the applied axial load is an interesting observation
that is worthy of further investigation.
From the data presented, it is clear that the growing rat ulna
adapts to axially directed loads producing supraphysiological
strains with increased new bone production. However, the re-
sponse to loads of differing amplitude is not a simple one, in
which increasing peak strain leads to corresponding increases in
bone mass. With strains at and slightly above those experienced
by this bone during normal locomotion (-0.001 to -0.002), there
was a suppression in the rate of periosteal expansion. This is
remarkable, since it means that even short periods of loading,
producing strains that are no higher than usual, and which
appear to be close to the normal distribution, may significantly
influence the development of bone architecture. Indeed, even
when loading at -0.004, a strain level well above that normally
encountered in vivo, the large increase in bone mass seen distally
(1)
o p ~ 0.05
| :~: p s 0.01 T
"" t - - , a , - - -0.002 AT,
~{:~ 1001 _ m _ - 0 . 0 0 4 ,/_L ~,~,T
~E o| TJI' \T
. . . . .,
o ~* **I..=**
o_ 1 2 3 4 5 6 7 8 9 10 11 12
MIDSHAFT
section number
Figure 5. Mean (-+SEM) difference in periosteal new bone production
between ulnae loaded at strains of either -0.002 or -0.004 and their
respective nonloaded controls, calculated over the central length of the
diaphysis. Sections are taken at lmm intervals such that section l is 6
mm proximal to the midshaft and section 12 is 6 mm distal to the
midshaft. Statistical significance of the mean difference at each level on
the bone within each loading group is indicated.
196 J.R. Mosley et al. Bone Vol. 20, No. 3
Strain magnitude related changes in bone March 1997:191-198

54 medial
A. B.
Neutral Axis

1
loaded

E

0
"Q
250

200
]! ........ control
250

200
18 lateral

E m
- ~ 15o

loo ! 100

o
t- 50 -: .... .... ~ T..~ 50
r:, ]ill IT T ~ ~t
~
1"-.
. . ~ : ~
L'k ~g

0 i , l , l l l , l , l l l , l , l , l &", l , I 0
0 6 12 18 24 30 36 42 48 54 ~ 66 72 0 6 12 18 24 30 36 42 48 54 60 66 72

measuring site measuring site

Figure 6. Mean (-SEM) change in periosteal new bone production, measured 2 mm distal to the ulna midshaft in the -0.002 group (A), and the -0.004
group (B), with location around the ulna circumference, as indicated in the schematic ulna cross section.

in the region of maximal peak strain amplitude, is accompanied local strain magnitude decreased toward the neutral axis the
proximally by a reduction in periosteal mineral deposition. From change in the rate of new bone production became less marked.
this it is apparent that across the customary strain range, the This distribution is similar to that observed by Torrance et al. 3
whole bone is responding as a structure in which overall changes A similar relationship did not hold for the -0.002 loading group
in bone shape are as important as local changes in bone mass. however, where the greatest change in new bone formation oc-
The reduction in periosteal new bone production seen in response curred at the neutral axis (Figure 6).
to strains at the limit of the customary strain range for the rat ulna The lateral face of the ulna is relatively straight, and the
could be explained if this bone, which possesses an obvious curvature along this edge did not change detectably in response
medial to lateral longitudinal curvature, was straightening in re- to strains of -0.002, whereas medially the ulna is concave, pro-
sponse to axially directed loads. In this way, the substantial strain moting bending in response to axial loading. Adjustments to
which is due to bending would be reduced, thus lowering the medial edge curvature therefore have the greatest potential to
peak strain magnitude, and perhaps returning it toward some influence bending strains. The medial bone face became less
target strain range. concave over the central 10% of the ulna length in response to
The relatively large increase in bone mass seen in the distal loading at strains of -0.002. The geometric alterations seen at
diaphysis at high strain (-0.004) is brought about by two mecha- this strain level are achieved by a simultaneous reduction in the
nisms; an increase in mineral apposition rate on the lateral face rates of both bone resorption and bone formation. This slows the
of the ulna, and the transformation of the medial resorbing peri- rate of the normal medial to lateral diaphyseal modeling drift,
osteal bone face to one which is actively forming new bone. On resulting in a straighter bone which will experience lower strains
both these faces, the new bone produced was lamellar in appear- for the same applied axial load. Interestingly, this adaptive
ance. The change in MAR brought about by mechanical loading change in bone geometry is associated with a relative reduction
at -0.004 was not uniform around the circumference of the bone, in the rate of periosteal bone formation, and might be incorrectly
rather the majority of the increase was seen along the medial and interpreted as a deleterious effect of mechanical load in exercise
lateral faces of the ulna, away from the neutral axis of bending, related studies, which often rely on measurements of changes in
and consequently in the regions of highest local strain. As the bone mineral density alone. Such adjustment to bone shape re-

Table 1. Curvature of the medial and lateral bone edges, measured over the central 10% of the ulna length. Results are shown for individual animals
(n = 3), together with mean values. Negative values indicate concavity, positive values indicate convexity

Curvature (m-t)
Measurement site Rat A Rat B Rat C Mean SEM p value

Medial edge Loaded -39.90 -40.77 -34.53 -38.40 1.95 0.0092

Control - 106.61 -99.80 -82.03 -96.15 7.33
Lateral edge Loaded 74.40 45.31 - 194.55 -24.90 85.20 NS
Control 18.44 16.04 -26.55 2.60 14.60
Bone Vol. 20, No. 3
March 1997:191-198
quires a coordinated, differential adjustment in the rate of bone
resorption over the ulna face, since a uniform slowing in the rate
of resorption would not adjust curvature, but rather slow the rate
of drift, while maintaining the current degree of curvature. When
loaded to peak midshaft strains o f - 0 . 0 0 4 , the adaptive response
of the growing rat ulna involved alterations in both bone geom-
etry and an increase in periosteal new bone formation.
Torrance and co-workers, 28'3 using a 10 Hz sine wave, noted
a loading-related periosteal osteogenic response when midshaft
strains of -0.00435 were applied to the ulna. This response was
not seen when the dynamic strain magnitude was reduced to
-0.00345. In the absence of any information regarding bone
geometry, this was interpreted as a threshold for production of an
adaptive response. However, the data presented here show that
the absence of an osteogenic response does not necessarily indi-
cate the lack of an adaptive response, but rather that adaptation
may have been occurring through changes in bone geometry
rather than mass. It seems more likely for the growing rat ulna,
where bending strains predominate, that the proposed "thresh-
old" strain of Torrance et al. represents a point on the bone
mass-strain curve (for that particular loading regimen), when the
geometric adaptations to axial load (here associated with a re-
duced bone mass), were counterbalanced by an increasingly large
osteogenic response.
The net reduction in new bone production seen at the distal
diaphysis in response to loads which engender strains up to twice
the magnitude the of customary peak locomotor strains (-0.001
to -0.002), contrasts with the findings of Rubin and Lanyon, 27
who examined the effect of strain magnitude on bone mass in the
functionally isolated avian ulna. By applying 100 loading cycles
per day for 8 weeks, they demonstrated a linear dose-response
relationship, with strains below -0.001 leading to bone loss, and
strains between -0.001 and -0.003 increasing bone mass. It is
intriguing therefore, that the rat ulna does not show this linear
relationship, and only responds to axial loading by increasing
bone mass when strains three to four times the magnitude of
those measured at the ulna midshaft during walking are applied.
Unlike the response to loading documented for other models,
both the geometric change and the site of new bone deposition
are such as to resist the bending strains associated with axially
directed loads. This contrasts with the response reported for the
functionally isolated avian ulna 27 and radius 1 models, where
despite high bending strains, the site of new bone production did
not correlate with the sites of peak strain magnitude, and, in the
case of the avian radius, was better correlated to surface strain
gradients, which are greatest around the neutral axis.
Although there are a number of obvious differences between
the current rat experiment and the previous avian ulna study,
including the fact that the birds were skeletally mature whereas
the rats were not, there are two further differences, which may
explain the differing response. In the avian ulna, the strain pro-
duced by axial loading was very different in distribution from
that produced during natural loading. 26"27 Indeed, since the mag-
nitude of the strains applied to the isolated segment of bone never
exceeded those which were measured during normal activities
for this wing bone, it was hypothesized that the major stimulus
controlling adaptive remodeling was the mismatch between the
distribution of the applied and the habitually encountered strains.
This finding led to the strain distribution error hypoth-
esis,15'26'27 which states that strains presented in a novel distri-
bution are more osteogenic than the equivalent strains presented
in the customary distribution. In the functional rat ulna loading
model, the strains are presented to the bone cell population in a
near normal distribution. If the magnitude of the osteogenic re-
sponse is related to the error strain distribution, then the high
J.R. Mosley et al. 197
Strain magnitude related changes in bone
strain magnitudes necessary to initiate an osteogenic response in
this study may be related to the absence of any significant error
in strain distribution. A further difference between the avian
preparation and the functional rat ulna is that the loaded segment
of avian ulna was isolated from functional strains between load-
ing episodes. It is interesting to speculate that the difference in
pattern and magnitude of response between these two loading
models may reflect a time-averaging of the loading-related
stimulus. 7'2'22 Thus the osteogenic stimulus associated with ar-
tificial loading, would be attenuated by the more numerous in-
tervening load cycles, associated with normal cage activity be-
tween loading episodes.
Conclusion
This study demonstrates that, in response to short daily periods of
axial mechanical loading which produce peak strains and maxi-
mum strain rates within the normal physiological range, long
bones in growing rats adjust both their curvature and their mass.
At peak strain levels of -0.002, the adjustment of bone curvature
is associated with reduced bone formation, whereas at -0.004
this architectural adaptation is accompanied by increased bone
formation. Such structural adaptations, involving local increases
and decreases in bone formation and resorption, are likely to be
as important as changes in bone mass in the local regulation of
the dynamic strain magnitude experienced as a result of bone
loading.
Acknowledgments: The authors would like to thank Dr. Chris Buckland-
Wright and Peter Liepins of UMDS, Guy's Hospital, for their help and
technical assistance. They are also grateful to Professor Alan Boyde of
University College London, who provided access to the confocal micro-
scope. This work was funded by a grant from the Biotechnology and
Biological Sciences Research Council. J.R.M. is currently a Wellcome
Trust Veterinary Research Fellow.
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Date Received: November I 1, 1996
Date Revised: December 13, 1996
Date Acc~7~ted: December 13, 1996