J Bone Miner Metab (2005) 23[Suppl]:16-22 © Springer-Verlag Tokyo 2005

Charles H . Turner • A l e x a n d e r G. R o b l i n g

Mechanisms by which exercise improves bone strength

A b s t r a c t Certain exercises can induce osteogenesis and that directs bone formation in response to high mechanical
improve bone strength, yet the biological processes in- stresses (or strains), thus strengthening the skeleton in
volved in bone mechanotransduction are only beginning to highly stressed regions. This system, sometimes called the
be understood. Several pathways are emerging from current "mechanostat" [1], involves the resident cells within bone
research, including calcium signaling associated with mem- tissue that detect and respond to mechanical loads. In re-
brane ion channels, adenosine triphosphate signaling, sec- gions of high stresses, bone formation is increased, particu-
ond messengers such as prostaglandins and nitric oxide, and larly at periosteal bone surfaces, and bone turnover is
signaling involving mitogen-activated protein kinase. One reduced, reducing bone porosity. Consequently, mechanical
characteristic of the mechanosensing apparatus that has loading can caused bones to increase their cross-sectional
only recently been studied is the important role of desensi- area and strengthen the bone tissue by decreasing porosity
tization. Experimental protocols that insert ~rest" periods and consequently improving tissue density.
to reduce the effects of desensitization can double anabolic Efficiency of structures is enhanced if mass can be re-
responses to mechanical loading. Exercises that reduce duced as much as possible without compromising strength
desensitization may provide an effective means to build or rigidity. Hence, bone should not be formed in regions of
bone strength. low stress, as this adds little structural benefit and increases
the risk of inappropriate bone formation that might impinge
Key words Bone mineral density. Calcium channel. A T P . on nerves or other adjacent tissues, whereas adding bone
Prostaglandins • Nitric oxide to regions under high stress greatly improves bone strength.
A recent study [2] shows how long bones apply this algo-
rithm in response to loading. Cyclic mechanical loads were
applied axially to the forelimbs of adult rats three times per
Introduction week for 16 weeks. The rat ulna has a natural curvature in
the mediolateral direction, so axial loads induce bending of
Mechanical stresses, and the resulting tissue deformation the bone (Fig. 1). Under load, the medial periosteal surface
(strain), result from the loads carried by the skeleton. of the bone was subject to compressive stresses and the
Stresses are not uniform throughout the bone but can be lateral periosteal surface was under tension. The pattern of
concentrated in certain regions, e.g., muscle attachments. bone formation induced by loading resembles the strain
Various exercises create different stress patterns in the skel- distribution, with more bone formation where the strains
eton. For instance, racquet sports selectively overload the are greatest. The improvement in bone structure is evi-
dominant arm whereas sports that require jumping, such denced by a 69% increase in second moment of area (I).
as volleyball, generate high stresses in the lower legs. The The ulnar bone strength in loaded limbs was 64% greater
skeleton possesses an inherent biological control system than control and energy absorbed before fracture increased
by 94%, yet the improvement in areal bone mineral density
(BMD) was only a modest 5%. Therefore, loading caused
dramatic improvements in bone biomeehanical properties,
even with small changes in BMD. The structural efficiency
C.H. Turner ([]) • A.G. Robling
Orthopaedic Research Laboratories and Biomechanics and of the ulna was improved by bone formation preferentially
Biomaterials Research Center, Indiana University Purdue University on the periosteal surface, with more bone formation in
at Indianapolis, 541 Clinical Drive, Room 541. Indianapolis, IN highly stressed areas where it was most needed.
46202, USA
Tel. +1-317-274-3226; Fax +1-317-274-3702
e-mail: [email protected]
 17

c,uo, . LEFT (NONLOADED) RIGHT (LOADED)

medial

; 0
2400 tastrain
lateral

Fig. 1. Cross sections through the midshaft of the rat ulna. The rat ulna (control) ulna, but the loaded ulna showed increased bone formation
is strained more on the medial (top) surface when loaded. The bottom mostly in regions where the strains were of greatest magnitude.
figure shows the strain profile across the loaded ulna. The strains are (Reprinted from Robling AG, Hinant FM, Burr DB, Turner CH.
designated in units of microstrain (~tstrain). Positive values are tensile Improved bone structure and strength after long-term mechanical
strain and negative values are compressive strain. Bone formation is loading is greatest if loading is separated into short bouts. J Bone
shown in the right panel. The red lines within the bone show labels Miner Res 2002; 17:1545-1554. Used with permission from the
at the beginning of loading. Little new bone was formed in the left American Society for Bone and Mineral Research)

experiments in which animals were exposed to brief (1-50

Exercise as a therapy for osteoporosis
cycles), moderate (50-100 cycles), or long (100-1600 cycles)
periods of mechanical loading on a daily basis [10,11]. These
As illustrated in Fig. 1, mechanical loading can effectively experiments demonstrate that bone "tunes out" mechanical
strengthen bones and mechanical loading is an important signals after a couple of dozen exercise cycles so that further
regulator of skeletal development. However, bones become exercise adds no further anabolic response (Fig. 3).
less sensitive to mechanical loading after skeletal maturity Strategies have been proposed to increase the anabolic
is reached (age 18-25 years). The addition of exercise to effect of exercise by developing exercise regimens that are
everyday activities provides a relatively ineffective treat- more effective and easier to accomplish. Studies in animals
ment for adult osteoporosis [3]. A recent meta-analysis of have shown that mechanical loading is much more anabolic
controlled exercise trials likened exercise to calcium supple- if applied at a higher frequency. For instance, loading
mentation in its effect on bone, i.e., both cause a modest of rat forelimbs produced more than tenfold more bone
reduction of bone resorption, resulting in only 1 % - 2 % gain formation when applied at 10 cycles/s (Hz), compared to
in B M D per year [4]. It has proven to be very difficult to application at 1 Hz [12]. In another study~ small loads were
translate the bone-strengthening effects of mechanical load- applied to sheep hindlimbs at 30Hz, 20min per day for a
ing seen in lab animals (Fig. 1) into effective exercise thera- year [13]. In this experiment, the loading was applied by a
pies for osteoporosis. vibrating plate that the sheep stood on. Trabecular bone
Osteogenesis is initiated only if mechanical loading is volume was increased by more than 30% in the femur (one
applied rapidly. Static loads do not induce osteogenesis in of the bones subjected to loading), but areal B M D was not
animal models whereas dynamic loading can be an effective increased in the loaded bones, nor was cortical bone mass
stimulus for bone formation [5-7] (Fig. 2). In addition, ani- increased. The limited anabolic response in cortical bone to
mal studies have shown that the rate of loading or applied 30-Hz vibration could be due to the fact that peak strains in
strain influences the osteogenic capacity of exercise [8]. the bone were very small [about 5 microstrain (~tstrain)].
Consequently, exercises that involve impact (such as jump- Alternatively, it is possible that the bone tissue rapidly
ing) are best for building bones [9]. Unfortunately, high- desensitized to the 30-Hz loading signal, thus limiting the
impact exercises are difficult for the frail and elderly and anabolic response.
can aggravate osteoarthritis in the joints. A n o t h e r challenge Recently, several studies have addressed the problem of
for anabolic exercise therapy is that bone tissue desensitizes bone cell desensitization to mechanical loading. The mecha-
to mechanical loading after a relatively few exercise repeti- nisms of desensitization are much less well characterized for
tions. Perhaps the best example of mechanosensory desen- bone than for other systems, but certainly the desensitiza-
sitization in bone tissue is illustrated by two separate tion pathways elucidated in other tissues can provide clues
 18

CONTROL LOADED

STATIC

DYNAMIC

Fig. 2. Effect of static and dynamic loading applied to the rat ulna. rightpanel received dynamic loading at 2 cycles per second for 10min/
Dynamic loading induced a potent osteogenic stimulus whereas there day over the same study period. The yellow lines are calcein labels
was a slight suppression of bone formation after exposure to static given 5 and 12 days after loading began. (Adapted from Robling AG,
loading. The ulnar cross section in the top right panel received an axial et aL Modulation of appositional and longitudinal bone growth in the
load of 17N for 10min/day for 2 weeks, whereas the ulna in the bottom rat ulna by applied static and dynamic force. Bone 2001:29:105-113)

A
m 140 //// sitization to mechanical stimulation: Ca 2+ influx and G-
160
p r o t e i n signaling a p p e a r to be required for m e c h a n o -
C
130 2 transduction to occur [14,15].
q- C
0 140 o To illustrate mechanodesensitization in bone, we and
120 - - T - - N,,.
O
others have p e r f o r m e d a series of experiments showing that
.-e recovery periods, which allow the tissue to resensitize, en-
120 hance the effectiveness of mechanical loading on osteogen-
:~ 110 o
C ii1 esis. Moreover, we have shown that there are different time
0
1oo
scales, ranging from seconds to hours, required for resensi-
m 100
axis) _¢ tization to occur. In one experiment, rats were a d m i n i s t e r e d
zx Ulnar BMC (right a x i s )
.Q 36 loading cycles p e r day in a single bout, 5 days a week for
~" 90 i i i i i i //// i 80 2 weeks. Some of the rats were given the 36 daily cycles
0 20 40 60 80 100 360 380 consecutively, with no time between each cycle (back-to-
Load Cycles Per Day back cycles), while o t h e r rats were given different durations
of rest between each of their 36 daily cycles. Rats given 14s
Fig. 3. Bone mass in the tibia of rats (closed circles [11]) or ulna of b e t w e e n each load cycle f o r m e d 67% m o r e bone on the
turkeys (open triangles [10]) increases after applied mechanical load-
ing. However, the anabolic effect of loading saturates as the number of endocortical surface than rats administered b a c k - t o - b a c k
loading cycles increases. There is limited benefit of additional loading cycles [16]. Similar e x p e r i m e n t s from other groups have
cycles above a couple dozen cycles per day. (From Burr DB, Robling confirmed the osteogenic benefit of short-term (on the
AG, Turner CH. Effects of biomechanical stress on bones in animals. o r d e r of seconds) recovery periods in restoring sensitivity to
Bone 2002;30:781-786. Reprinted with permission from Elsevier)
l o a d e d bone [17,18]. A t this point, the m o l e c u l a r mecha-
nism of this desensitization remains unknown, but the
fact that this p h e n o m e n o n occurs on such a short time
for possible mechanisms in bone. F o r example, pressure scale rules out several classical desensitization mechanisms,
sensitivity in skin baroreceptors (e.g., Pacinian corpuscle) including receptor p h o s p h o r y l a t i o n or internalization.
involves a change in m e m b r a n e potential (depolarization), A m o r e likely candidate would involve electrophysiological
but continued pressure by a constant-intensity stimulus effects within the cell (e.g., cytosolic versus endoplasmic
quickly reduces the generator potential and returns the reticulum Ca 2+ levels) or m e m b r a n e permeability to ions.
m e m b r a n e potential to its resting state. A n o t h e r possible W e have also o b s e r v e d resensitization of mechanically
pathway is G - p r o t e i n - c o u p l e d r e c e p t o r desensitization via l o a d e d bone that requires several hours. W e subjected the
receptor p h o s p h o r y l a t i o n by several classes of kinases right tibiae of rats to four separate bouts of loading p e r day
and eventually receptor sequestration/downregulation. for 3 nonconsecutive days [16]. Each bout comprised 90
Both these mechanisms could play a role in bone cell desen- load cycles; the only difference among the six groups of rats
 19

was the amount of time allotted between each of the four Signaling Events in Osteoblasts
daily loading bouts. The rats received 8, 4, 2, 1, 0.5, or Oh Os ~20s <lm ~5m ~15m >lhr
between loading bouts, and their bone formation response
was assessed 2 weeks later. The 8-h recovery group exhib- T T T T T T
ited - 1 0 0 % more bone formation that the 0- or 0.5-h recov- ~o ^~,~o~
ery group, suggesting that the recovery periods restored a
significant amount of mechanosensitivity to the bone; 4-8h
recovery appeared to fully restore mechanosensitivity. We
Fig. 4. Sequence of molecular events that occur after a mechanical
repeated a portion of this experimental design in a long- stimulus and precede bone formation. AT[', adenosine triphosphate;
term loading experiment using the rat ulna loading model PG, prostagtandin: NO, nitric oxide, ERK, extracellular signal-related
developed previously by Torrance et al. [19]. Here we com- kinase
pared a standard loading protocol (360 consecutive cycles
of loading) with a 3-h recovery protocol (four bouts of
90 cycles with 3h between bouts) over a 16-week loading An increase in intracellular calcium is observed in osteo-
period, and found that both protocols significantly im- blastic cells seconds after a mechanical stimulus. The
proved the bone strength in the loaded ulnae, but the combined effects of increased open frequency of membrane
ulnae from the recovery group exhibited 75% greater calcium channels (L-type voltage-operated and stretch-
work to failure [20,21]. The loss and subsequent return of activated) and increased release of Ca 2+ from intracellular
mechanosensitivity that occurs on the order of hours might stores mobilize intracellular calcium [22,27,28]. The inositol
be dependent on cell structural changes (e.g., architecture 1,4,5-triphosphate pathway plays a key role in intracellular
of the actin cytoskeleton) or on kinase-induced receptor calcium release after a mechanical stimulus [22,28], and
phosphorylation and/or internalization. In vitro, reorgani- intracellular calcium signaling appears to be requisite for
zation of the osteoblast's actin cytoskeleton into stress fiber expression of bone matrix proteins. Intracellular calcium
bundles is required for fluid shear-induced expression of mobilization triggers a mitogen-activated protein kinase
genes linked to mechanotransduction and bone formation (MAPK) signaling pathway, which is linked to the expres-
[22,23]. Reorganization of the actin cytoskeleton results in sion of osteopontin [28]. M A P K signaling involves the
an increase in whole-cell stiffness, which might make detec- activation of extracellular signal-regulated kinase (ERK)
tion of mechanical signals less effective. Alternatively, the within about 15rain after mechanical loading [29]. E R K
signal transduction cascades activated by mechanical sti- activation might be initiated by prostaglandins or nitric
mulation, which appear to involve autocraine/paracrine oxide [30], which are released following mechanical load-
loops, involve several G-protein-coupled receptors (e.g., ing. In addition, activation of focal adhesion kinase (FAK)
prostanoid receptors, adenosine triphosphate receptors) may be required for E R K phosphorylation (activation) [31].
that might undergo uncoupling to their G-protein messen- Osteoblastic cells attach to the bone matrix through the
gers by any of several kinases known to be expressed in integrin-cytoskeleton complex. Integrins are heterodimeric
bone cells. transmembrane proteins that bind to the extracellular
matrix (ECM) on the outside of cells and are linked to the
actin cytoskeleton via the short cytoplasmic domain of the
[~-subunit on the inside of cells at specialized sites known
The characteristics of the skeletal as focal adhesions. Several lines of evidence obtained with
mechanotransducer various cell types, including fibroblasts, epithelial cells,
endothelial cells, and neutrophils, as well as osteoblasts,
Mechanotransduction in bone involves several cell types. indicate that a key molecule in mediating linkage of
The cells that ultimately form or resorb bone may not be the actin filaments to integrin cytoplasmic domains is the pro-
same ones that transduce and respond to mechanical sig- tein ct-actinin [23,32]. Microinjection into osteoblasts of
nals. Mechanotransduction might involve signaling through a dominant negative c~-actinin causes the competitive
mechanically activated ion channels in the cell membrane, displacement of the endogenous ct-actinin from focal adhe-
focal adhesions of the cytoskeleton, or a G-protein-coupled sions and blocks fluid flow-induced gene expression [23].
mechanoreceptor. In cultured osteoblastic cells, fluid
flow increases intracellular calcium within minutes, and this
response is suppressed by gadolinium, a blocker of the
stretch-activated calcium channel [24] (Fig. 4). In addition, Autocrine and paracrine messengers for
the L-type voltage-operated calcium channel probably skeletal mechanotransduction
plays a role in bone cell mechanotransduction. Studies using
bone explants showed that gadolinium abolished loading- In cultured osteoblasts, fluid flow causes release of adenos-
related responses in osteocytes, whereas a blocker of L-type ine triphosphate (ATP), prostaglandins, and nitric oxide
calcium channels inhibited loading-related responses in os- within minutes [33-37]. Mechanical stimuli also affect
teoblasts [25]. In addition, two blockers of L-type calcium osteoclasts, but this effect appears to be indirect. When
channels, verapamil and nifedipine, strongly suppress me- exposed to strain, marrow stromal cells (of osteoblastic lin-
chanically induced bone formation in rats [14,26]. eage) reduce expression of osteoclast differentiation factor
20
(a.k.a. R A N K L or TRANCE), which in turn decreases os- suppressed bone formation by 67% in the rat tibia, whereas
teoclast number [38,39]. Consequently, cells of osteoblastic administration of the drug 30min after loading had no
lineage appear to be mediators of the suppressive effects of significant effect [52]. These findings demonstrate that pros-
mechanical stimuli on bone resorption. taglandin synthesis is most important before loading, sug-
Adenosine triphosphate (ATP) signaling plays a role in gesting that prostaglandins must be available at the time of
skeletal mechanotransduction. Osteoblastic cells can com- loading to potentiate the osteogenic response.
municate through autocrine or paracrine activity of se- Nitric oxide release from bone cells appears to be in-
creted ATP on P2Y2 purinergic receptors [40], and P2Y2 volved in cellular mechanotransduction because the nitric
signaling appears to be mechanosensitive [33]. A local oxide synthesis inhibitor L-NAME (N~-nitro-i.-arginine
mechanical stimulus initiates intercellular calcium signaling, methylester) suppresses mechanically induced bone forma-
mediated by ATP receptors, which rapidly propagates from tion in vivo [53]. The endothelial isoform of nitric oxide
cell to cell. In addition, the P2X family of receptors is prob- synthase (NOS-3) is thought to mediate the effects of me-
ably an important target for mechanically derived signals. chanical forces in bone tissue [37]. However, it is not clear
P2X7 receptor knockout mice have an osteopenic pheno- how nitric oxide affects intracellular signaling pathways in
type that resembles the skeleton of animals subjected to osteoblastic cells. In other cell types, nitric oxide binds to
chronic disuse. P2X7 signaling is important for promoting soluble guanylyl cyclase, thus stimulating the enzyme and
osteoblastic activity and bone formation, whereas P2X7 sig- increasing intracellular cyclic guanosine monophosphate
naling suppresses osteoclastic bone resorption [41]. (GMP). Cyclic GMP has a number of effects in different cell
The ultimate effect of released prostaglandins on bone types and has been suggested as a mediator of mechanical
biology involves a tangled web of interactions that is ex- loading in osteoblastic cells [54]. Alternatively, nitric oxide
tremely complicated. Prostaglandins might (1) recruit new may have a more important role as a mediator of the sup-
osteoblasts from marrow stroma, (2) amplify their own re- pressive effects of mechanical loading on osteoclasts. Nitric
lease by stimulating expression of prostaglandin synthases, oxide is known to be a strong inhibitor of osteoclast activity
(3) improve cell-to-cell communication through cellular gap [55-57] and has been shown to decrease expression of re-
junctions, (4) reduce apoptosis in osteoblasts, and (5) am- ceptor activator of NFkB ligand (RANKL, a.k.a., osteoclast
plify the loading-related increase of osteoblastic expression differentiation factor) and increases expression of osteopro-
of matrix proteins. It is exceedingly difficult to sort out the tegerin (OPG, an inhibitor of osteoclast differentiation),
relative importance of each of these effects. which in turn leads to decreased recruitment of osteoclasts
The two most active prostaglandins (PG) in bone cells [58]. Therefore, it appears that local release of nitric
are PGE2 and PGI2. Both are released from osteoblasts oxide enhances bone formation and suppresses bone re-
or osteocytes shortly after mechanical loading and have sorption, suggesting that nitric oxide potentiates an ana-
numerous effects on bone, including the recruitment of bolic response.
osteoblasts from marrow stroma [42]. Exogenous PGE.
administered in rats is strongly osteogenic and results in
increased recruitment of osteoblasts and accelerated
osteoblastic activity [43]. The E family of prostaglandins
Conclusions
also have the ability to amplify their own production
[35,44]. This autoamplification effect is mediated through Exercise can build bone strength by activating the intrinsic
the EPj prostaglandin receptor [44], indicating that EP1 is mechanotransduction machinery that directs bone for-
linked to expression of prostaglandin synthase. However, mation to where it is most needed. We are beginning to
the anabolic effects of PGE2 are mediated through the E P 4 understand the important role desensitization plays in
prostaglandin receptor [45], suggesting that signaling down- mechanotransduction. Experimental protocols that insert
stream from E P 4 is important in bone matrix synthesis. "rest" periods to reduce the effects of desensitization can
Prostaglandin signaling through the EP2 receptor improves double anabolic responses to mechanical loading. Several
cell-to-cell communication through cellular gap junctions key pathways are emerging from skeletal mechano-
[46-48], and prostaglandins reduce apoptosis in osteoblasts transduction research including membrane ion channels,
[49] by inhibiting caspase 3. ATP signaling, and second messengers such as prostaglan-
There are two isoforms of prostaglandin synthase dins and nitric oxide.
(cyclooxygenase), constitutive (COX-l) and inducible
(COX-2). Selective inhibition of COX-2 using NS-398 is
considerably more effective in blocking loading-induced References
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