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The role of osteocytes and bone microstructure in preventing osteoporotic

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Osteoporos Int (2007) 18:1–8
DOI 10.1007/s00198-006-0222-y
REVIEW
The role of osteocytes and bone microstructure in preventing
osteoporotic fractures
Jan G. Hazenberg & David Taylor & T. Clive Lee
Received: 30 March 2006 / Accepted: 8 August 2006 / Published online: 14 September 2006
# International Osteoporosis Foundation and National Osteoporosis Foundation 2006
Abstract The skeleton alters its geometry following trauma,
the introduction of artificial defects and of fatigue-induced
microcracks. The precise mechanism by which the skele-
ton adapts remains unclear. Microcracks might directly
affect the cell by damaging the osteocyte cell network or
causing apoptosis. Bone microstructure may play an
important role in these processes by diverting and
arresting propagating microcracks and so prevent fracture
failure. This paper discusses the effects of microstructure
on propagating cracks, how microdamage may act as a
stimulus for bone adaptation and its potential effects on
bone biochemistry.
Keywords Bone adaptation . Microdamage . Osteocytes .
Osteoporotic fracture . RANKL
Introduction
Bones are subjected each day to cyclic tensile, compressive
and torsional loading, making them very susceptible to
microdamage accumulation. The amount of microdamage
increases as our bones get older and as the number of bone
cells decrease. Caucasian bones are more susceptible to
damage accumulation than those of African Americans [1,
2]. Fortunately, bone contains a mechanism which is able to
J. G. Hazenberg (*) : T. C. Lee
Department of Anatomy, Royal College of Surgeons in Ireland,
St. Stephen’s Green,
Dublin 2, Ireland
e-mail: jhazenberg@rcsi.ie
D. Taylor : T. C. Lee
Trinity Centre for Bioengineering, Trinity College,
Dublin 2, Ireland
repair defects. Unfortunately, as we grow older it tends to
work against us, especially in woman after the age of 40,
resulting in osteoporosis. Osteoporosis is a progressive
bone disease in which bone tissue is normally mineralized,
but the amount of bone is decreased and the structural
integrity is impaired, becoming more brittle, thinner and
more prone to fracture [3]. Annually, 1.5-million fragility
fractures occur in the USA, including 700,000 vertebral
fractures, 300,000 hip fractures, 250,000 Colles’ fractures,
and 250,000 at other skeletal sites [4]. The annual cost
associated with this has been estimated at $17 billion. Hip
fractures are associated with the greatest morbidity, with
50% of patients unable to walk without assistance and 25%
requiring long-term domiciliary care, and mortality, with
10–20% dying within 6 months [5]. In this paper, we
review how bone morphology affects damage accumulation
and the role that osteocytes play in preventing fracture in
bone, from microstructure to cell signalling.
Bone microstructure and crack arrest
Bone consists of a hard mineral phase, mainly comprised of
hydroxyapatite, and a fibrous scaffold, mainly composed of
type I collagen. The stiffness caused by the mineral phase
provides resistance to compressive stresses, while the
collagen provides bone with toughness and resistance to
tensile stresses. Throughout the matrix are vascular canals
that supply the bone cells, embedded in the hard matrix,
with nutrients. The main vascular canals (Haversian canals)
run approximately parallel to the long axis of the bone,
whereas the smaller Volkmann’s canals radiate tangentially.
Is it the sole function of these vascular canals to provide
nutrients to the bone or might they have a secondary
function in maintaining mechanical integrity?
2 Osteoporos Int (2007) 18:1–8
It has been known for over 40 years that bone in vivo
contains small cracks [6, 7]. These cracks are the visual
manifestation of fatigue damage, caused by the cycles of
stress that occur during daily activities. To date, most
studies have focused either on detecting cracks formed in
vivo in humans and animals [8, 9] or on experiments to
study the initiation and propagation of cracks in specimens
of dead bone during either brittle fracture [10–12] or fatigue
[13–15]. Experimental measurements of cracks tend to be
made from histological sections oriented transversely to the
bone’s long axis. Individual cracks have an average length
(measured tip-to-tip) of 60–100 μm. In longitudinal
sections, which have rarely been used, crack lengths appear
significantly longer than those in transverse sections [9,
16]. This implies that they are taking advantage of a
relatively easy growth direction afforded by the anisotropic
‘grain’ of the material. Observations have been made of
different microcrack appearances in tension and compres-
sion. Cracks induced by compression are usually relatively
long, straight and start at obvious stress concentrators, such
as channels and lacunae [17, 18]. Figure 1 shows a
scanning electron microscope (SEM) image of this phe-
nomenon. A microcrack has propagated towards and then
through an osteocyte lacuna in a bone subjected to a
uniform load, while a second microcrack has initiated from
a neighbouring lacuna. It shows that once cracks accumu-
late, secondary damage formation occurs, which weakens
the bone structure and increases fracture risk. Tensile
cracks, on the other hand, are often orientated with the
grain of the bone and are associated with planes of
weakness, such as cement lines and inter-lamellar debond-
ing [19, 20]. This is illustrated in Fig. 2. In general, crack
Fig. 1 A scanning electron microscope image of a microcrack (A),
which has propagated towards and through an osteocyte lacuna. A
second microcrack has initiated from a neighbouring osteocyte lacuna
propagation requires strain energy, which causes the crack
to increase in length. It seems that shorter cracks tend to
stabilise and become dormant after reaching some critical
length, whereas those longer than the critical length are
self-propagating and may cause fracture [21]. A number of
researchers have noted that the vast majority of the cracks
can be found in older bone, located between the osteons.
The percentages of cracks found in interstitial bone by
various researchers [22–24] are, respectively, 87%, 62%
and 85%. According to Martin and Burr [25], microcracks
formed in interstitial bone propagate to the cement line or
concentric lamellae and debond or separate the osteon from
the surrounding bone. Furthermore, propagating micro-
cracks under uniaxial tension exhibit an intermitted crack
growth pattern [26, 27]. Using optical microscopy to
monitor propagating cracks, it was shown that this
propagation behaviour was related to the cracks encounter-
ing vascular canals, which either caused crack growth rates
to decrease or arrested the crack completely [27]. The
available data support the theory that cement lines,
surrounding the osteons and other vascular canals act as
microcrack-arresting features or decrease their propagation
rate. These microscopic observations have shown that the
micro-mechanisms of crack propagation and toughening are
complex and involve contributions from several different
factors, including microdamage zones ahead of the crack tip
and bridging actions across the crack faces [28–30].
Experiments conducted on bone demonstrate that fracture
resistance is not constant but increases with crack length
[29, 31, 32]. Others have argued that microdamage, in the
form of small cracks, or diffuse damage, in front of larger
microcracks acts as an energy-dissipating mechanism which
may cause crack arrest [33–35]. Observations of collagen
fibres crossing the crack faces could be another mechanism
by which crack propagation is controlled. This extrinsic
mechanism [36], could explain the observed increase in
toughness with crack length [37]. As the crack propagates,
more ligaments will be left in its wake, limiting the opening
and shear displacements between the crack faces [38].
Furthermore, microdamage accumulation and propagation
in cortical bone and trabecular bone might be completely
different in nature. Microdamage in cortical bone involves
cracks that are embedded in a solid matrix, whereas cracks
in trabecular bone are typically surface cracks. A surface
crack of the same length, under similar loading conditions,
will have more strain energy to propagate than those in
cortical bone. The consequences of a single fractured
trabecula might not pose a threat to the overall mechanical
integrity, while a propagating crack in cortical bone could
result in fracture of the complete structure. It is evident
from these studies that microcrack propagation in bone is a
multi-scale problem.
Osteoporos Int (2007) 18:1–8
Fig. 2 Three examples (a, b, c) of microcracks propagating from a notch (N). They preferred to propagate in a direction which is parallel to the
osteonal direction instead of taking a direction perpendicular to the maximum principal stresses
Bone adaptation
Bone adaptation is a dynamic process which involves both
modelling and remodelling. Modelling is a process by
which bone changes in length and diameter during growth
and development. Unlike modelling, which involves either
resorption or formation, bone remodelling follows an
activation, resorption, formation sequence [39]. Remodel-
ling of bone requires a complex arrangement and interac-
tion of cells, collectively called basic multicellular units
(BMUs). Both processes are tightly controlled by a number
of cells. The four main types of cells which can be found in
bone are bone lining cells, osteoblasts, osteocytes and
osteoclasts. Bone lining cells, osteoblasts and osteoclasts
are located on the bone surfaces, whereas the osteocytes are
in the bone matrix. Palumbo et al. [40] proposed that pre-
osteoblasts mature into osteoblasts, some of which are
committed to differentiate further. These committed osteo-
blasts start to lose their activity and are buried inside the
bone matrix by adjacent osteoblasts. Consequently, pre-
osteocytes start the formation of cellular processes and
become osteoid osteocytes. The mature osteocyte radiates
processes in all directions, forming a network inside the
hard mineralized matrix. There is great interest in osteo-
cytes as potential regulators of damage and repair [41–45].
Osteocytes
Human cortical bone contains between 13,900 and 19,400
osteocytes per mm3 and trabecular bone roughly 13,000
osteocytes per mm3 [46, 47]. These osteocytes form an
interconnected network through their dendritic processes,
allowing communication between individual osteocytes and
3
the surface bone lining cells. Although the functional role
of the osteocyte remains partly unknown, a key role in the
regulation of remodelling the skeletal architecture in
response to mechanical load has been suggested. Marotti
et al. [48] suggested that an inhibitory signal is generated
by the osteocytes, which is passed through their cell
processes to osteoclasts to prevent bone resorption. Martin
[49] extended this concept by developing a hypothetical
concept for the down-regulation of remodelling by osteo-
cytes. He proposed that the strength of the inhibitory signal
perceived by each osteoclast is proportional to the number
of osteocytes connected to lining cells located on quiescent
bone surfaces, and inversely proportional to the distance
from the surface. Regions of bone exhibiting high rates of
remodelling were shown to correlate with relatively low
numbers of viable osteocytes, as low osteocyte density
reduced this inhibition of remodelling [50]. This is
interesting since it has been reported that osteocytes in
woven bone appear at an early stage of bone repair and
develop shorter processes which are irregularly distributed
in the osteoid matrix. Osteocytes in lamellar bone form
long cell processes that are regularly distributed in mature
bone matrix. Based on the pattern of development of bone
processes by the osteocytes [51], woven bone osteocytes
may be necessary for induction of lamellar bone osteocytes
followed by active appositional growth of lamellar bone at
an early stage of bone repair. It has been estimated that the
osteocyte population in woven bone is approximately four
to eight times as large as that in lamellar bone [52], which
might indicate that woven bone with increased lacunar
density may undergo remodelling at an accelerated rate
[53]. Given the potentially important role of osteocytes,
immuno-fluorescence microscopy has been used to study
the morphology of these cells [54–56]. Osteocytes contain
4 Osteoporos Int (2007) 18:1–8
between 40 and 60 cell processes per osteocyte with a cell-
to-cell distance between 20 and 30 μm [56] and a diameter
varying from 50 to 410 nm [57]. However, there are still
questions which remain to be answered. For example,
African Americans have higher BMD indexes [2], lower
osteon densities [58], higher osteocyte densities [59] and
are less prone to stress fractures [60] compared with white
Americans. Do these various parameters contribute to a
reduction in the accumulation and initiation of micro-
damage and therefore of stress and fragility fractures?
Microdamage-stimulated bone adaptation
Other researchers have developed theoretical models based
on the idea that adaptation is controlled by the level of
fatigue damage, i.e. by the number and length of cracks
[42, 61]. Martin [61], for example, used the amount of
damage (mean crack length times crack density) as the
stimulus for bone remodelling and the activation frequency
of BMUs is directly related to the amount of damage
present in a local area. As a consequence of damage
formation, BMUs are activated, which increases the
porosity resulting in increased strain. This promotes
damage accumulation, which is not dangerous while the
load remains below a critical level. The increasing damage
removal overtakes the rising damage formation rate,
resulting in a new state of equilibrium. On the other hand,
when the effective strain levels are below a critical value,
BMUs are activated, causing resorption of bone. This idea
is an attractive one because the level of damage provides a
direct measure of the potential risk of failure. If the damage,
and particularly its rate of increase, is greater than can be
repaired, then failure will occur unless adaptation is
initiated to reduce the stress level. An advantage of this
approach is that it automatically accounts for the dynamic
loading history as the driving force for the remodelling
process. However, there remains a lack of knowledge of
how bone detects damage and ‘decides’ to initiate repair or
adaptation.
Detection of microdamage by osteocytes
Recently, Qui et al. [62] found that regions with osteocyte
densities <728/mm2, such as interstitial bone, are 3.8-times
more likely to contain microdamage than regions with
higher osteocyte densities. The questions remain: if micro-
damage can trigger bone adaptation and what may be the
role of osteocytes in this process? Using the isolated ulna
loading model, intracortical bone remodelling in rats was
shown by Bentolila et al. [45] to be triggered by fatigue
loading. In this experiment, 14 out of a total of 16 rats
showed microcracks in the bone cortex. After 10 days of
fatigue loading, resorption cavities were observed. Two rats
had no microdamage following fatigue loading and no
resorption cavities were observed. Hsieh et al. [63] and Lee
at al. [64] also used the isolated ulna loading model and
reported bone deposition, but no evidence of microdamage
formation was found. This is rather surprising, given the
loading amplitude and frequency used for the experiments.
However, what these studies have shown is that it is
irrelevant how many load cycles to which bones are
subjected, the stimulus caused by the induced strain
saturates. In these studies, there was an asymptotic
approach to saturation as the duration of loading was
increased from 36 to 720 cycles per day. Increasing the
duration of a loading bout therefore results in diminishing
returns in bone formation, again suggesting that cells may
become non-responsive to repeated mechanical stimuli
[65]. Even if these studies show that the effective altered
strain would diminish, microdamage might indirectly cause
a sustained local stimulus. Microdamage causes a local
stress concentrator around its perimeter. If strain levels do
not decrease, cracks will continue to propagate and
therefore the local strains, caused by these cracks, will
continue to increase [66]. It has been suggested that
increased strain levels cause osteocytic apoptosis due to
the presence of microcracks which affect osteocyte homeo-
stasis [22, 67, 68]. Recent experimental work which has
tried to investigate this phenomenon has indicated that
osteocyte apoptosis increases osteoclastic activity [69].
Various in vitro studies have focused on the role of
osteocytes in bone regulation, especially regarding inhibi-
tion and activation of osteoclastic resorption [70–74]. As
soon as an osteocyte network is damaged and cells undergo
apoptosis, the inhibitory signal is abolished and osteoclasts
are released for resorption [75].
If bone is the activation signal to trigger BMU activity,
how can bone cells detect the presence of cracks? If all
microcracks were removed from the matrix, the mechanical
integrity of bone might be compromised since tunnelling
BMUs would weaken the structure. On the other hand, if
microdamage remained undetected, various microcracks
might propagate to form macrocracks leading to fracture.
So how do the osteocytes decide whether a crack is
dangerous enough to require repair? If this principle were
extended, and the rate of microdamage accumulation were
so high that the remodelling process could not keep up,
then the only way to reduce propagation would be to
deposit new bone at surfaces in order to reduce strain
levels. But how do the osteocytes decide whether the level
of damage is high enough to require surface adaptation? A
possible answer to these questions might lie in damage to
the cell processes. Recently, a theoretical model was
developed based on this principle.
Osteoporos Int (2007) 18:1–8
A typical microcrack is elliptical in shape with the minor
axis having a length of 100 μm and the major axis
measuring lengths up to 700 μm, orientated at approxi-
mately 20° to the longitudinal axis of the bone [9]. Given
that human cortical bone contains between 13,900 and
19,400 osteocytes per mm3 [46], with 40–60 cell processes
each, it is likely that the microcrack crosses this network.
Since microcracks tend to propagate in a direction similar
to the local lamellar orientation, it would mean that cracks
subjected to tensile loading open up and produce a shear
displacement. Cracks subjected to compressive loading
would result in compression of the fracture surfaces and
an additional shear displacement, resulting in the rupture of
cell processes. Using linear elastic fracture mechanics and
various osteocyte densities, it was predicted that small
microcracks (<30 μm) do not produce enough shear
displacement to rupture cell processes, independent of the
stress level to which these cracks were subjected. Micro-
cracks with a typical length of 100 μm show ruptured cell
processes at stress levels exceeding 28 MPa. If the stress
levels are increased for cracks of this length, several
hundred cell processes will rupture. Extremely large cracks
(>300 μm) tend to rupture several thousand cell processes
even at extremely low stress levels [66]. Given that hardly
any crack lengths in excess of 100 μm are reported in the
literature, it might indicate that cell process rupture plays an
important role in monitoring local microdamage accumula-
tion. Experimental evidence, in which propagating cracks
were monitored under a UV-epiflourescence microscope
with additional staining of the cytoskeleton, showed that
the majority of cell processes rupture between the two crack
faces. Only near the crack tip, a region where crack-face
displacements are negligible, did cell processes remain
intact [76].
Biochemistry of bone cell signalling
The response of osteocytes to damaged cell processes
influences the cell-to-cell interaction and, therefore, the
cellular network biochemical homeostasis in bone. The
bone remodelling cycle is controlled by various systemic
factors (see Fig. 3). TGF-β, for example, is released and
activated by resorbing osteoclasts [77] and promotes
different stages of maturation of osteoblasts [78], which is
also controlled by IGF [79]. Osteoblasts produce osteopro-
tegerin (OPG) and express RANKL on their surfaces. The
secretion of OPG and the binding of RANK-RANKL
stimulate the differentiation and activation of osteoclasts
[80]. While osteoclasts, osteoblasts and the signalling
pathways between them have received much attention
[81–85], osteocytes are less well understood since they
5
Fig. 3 A new schematic proposal for bone cell signalling in damage-
stimulated bone remodelling
are embedded in the hard mineralised matrix, limiting our
knowledge regarding bone regulation. The development of
the MLO-Y4 osteocyte cell line has provided more data for
this particular. For example, Kuratu et al. [86] used a novel
approach to investigate the effect of microdamage on the
cellular network. In their study, damage was introduced to a
three-dimensional osteocyte cell culture with a 21-gauge
needle. Microdamage was found to have no significant
effect on the number of dead cells compared with the
control. However, it was found that the bone marrow cells,
which were seeded on top of the gel, were TRACP-positive
in a region close to the damage site. Furthermore, it was
shown, using an ELISA assay, that the gel-embedded
osteocytes secreted significant amounts of both M-CSF
and RANKL. In other words, if damage to the osteocyte
processes causes up-regulation of RANKL, then this would
provide a new pathway to activate osteoclastic activity (see
Fig. 3) and so link microdamage, resorption and formation
of BMUs.
In summary, it has been know for half a century that
bones contain microcracks [6]. Their initiation, propagation
and arrest are affected by microstructural features [20, 87]
and the nano-composition of bone [37]. There is evidence
to suggest that microcracks are repaired by a targeted
remodelling process [43, 45, 88]. How this process is
regulated at a cellular level still remains unknown. One
proposed mechanism has focused on ruptured osteocyte cell
processes affecting signalling pathways [66, 89] and
causing up-regulation of RANKL [86]. The second mech-
anism proposed is that microdamage accumulation causes
osteocyte apoptosis, which triggers osteoclastic activity [22,
67, 75]. Although these two mechanisms differ, a common
pathway might be present where ruptured cell processes
trigger osteocyte apoptosis, causing an up-regulation of
RANKL which stimulates osteoclastic activity and the
formation of BMUs.
Acknowledgement This work was financially supported through the
EMBARK Postdoctoral Fellowship by the Irish Research Council for
Science.
6 Osteoporos Int (2007) 18:1–8
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