tightly regulated processes of bone formation
and resorption are essential for the achievement
and maintenance of skeletal strength and form.
Circulating hormones are important controlling
factors, but the key influences are locally
generated cytokines, which influence bone cell
function and communication in complex ways,
and often are themselves regulated in turn by
the hormones. For the normal amount of bone
to be maintained, bone formation and resorption
need to be equal. Concepts of the pathogenesis
of osteoporosis have developed that focus on
changes in the number of bone cells, either
over-production of osteoclasts or under-production
of osteoblasts, in the latter case either through
inadequacy of precursors or failure in differentiation.
Bone modelling and remodelling.
formation and resorption proceed throughout
life. The processes are more rapid during skeletal
growth, at which stage the term modelling is
used. Modelling takes place from the beginning
of skeletogenesis during fetal life until the
end of the second decade when the longitudinal
growth of the skeleton is completed. In the
modelling process , bone is formed at a location
different from the sites of resorption, leading
to a change in the shape or macroarchitecture
of the bone. It is responsible for determining
the size and shape of bone, such as the simultaneous
widening of long bone and development of medullary
cavity by bone formation at the periosteal surface
and resorption at the endosteal surface, respectively.
The remodelling process, which continues throughout
adult life, is necessary for the maintenance
of normal bone structure and requires that bone
formation and resorption should be balanced.
The remodelling concept owes much to the ideas
of Frost (1 ), and can be outlined briefly as
follows. Both bone formation and resorption
occur at the same place so that there is no
change in the shape of the bone. After a certain
amount of bone is removed as a result of osteoclastic
resorption and the osteoclasts have moved away
from the site, a reversal phase takes place
in which a cement line is laid down. Osteoblasts
then synthesize matrix, which becomes mineralized
(2). Remodelling thus maintains the mechanical
integrity of the skeleton by replacing old bone
with new bone.
The fact that resorption is followed by an
equal amount of formation is crucial, and has
come to be known as "coupling", with
the uncoupling of resorption from formation
resulting in osteoporosis, the commonest metabolic
bone disease. In the adult human skeleton, approximately
5 to 10% of the existing bone is replaced every
year. The characteristic feature of bone remodelling
is that the process does not occur uniformly
throughout the skeleton. Remodelling of bone
occurs in focal or discrete packets known as
bone remodelling units (BRU) or basic multicellular
units of bone turnover. The cellular sequence
is always initiated by osteoclastic bone resorption
to be followed by osteoblastic new bone formation.
This sequence of events is initiated at asynchronous
sites throughout the skeleton, which are geographically
and chronologically separated from each other.
Bone remodelling is an integral part of the
calcium homeostatic system. It also provides
a mechanism for self-repair and adaptation to
physical stress. The processes of bone resorption
and formation are controlled by systemic hormones
The maintenance of a normal, healthy skeletal
mass depends on interactions between osteoblasts,
osteoclasts and constituents of the bone matrix
to keep the processes of bone resorption and
formation in balance. One of the intriguing
issues of bone cell biology has been to determine
how osteoclast precursors are recruited and
induced to differentiate into mature osteoclasts
and, in turn, how osteoblasts are instructed
to replace just exactly that amount of bone
which has been resorbed.
Osteoclast formation and bone
formation is controlled by several circulating
hormones such as parathyroid hormone, estrogen
and 1,25 dihydroxyvitamin D3 (calcitriol) (3).
The bone marrow microenvironment also plays
an essential role as a source of cytokines such
as tumour necrosis factors and interleukins.
These systemic and local factors regulate osteoclast
formation and function. However, because the
receptors for these systemic and local factors
are expressed in cells of the osteoblast lineage
they need to rely on secondary signals generated
by osteoblasts to mediate their effects (3,4).
For many years it has been recognised that bone-resorbing
factors, in order to produce their effects,
must first act on cells of the osteoblast lineage.
These cells were considered to possess a cell
surface molecule, known as osteoclast differentiation
factor (ODF), which acted upon hemopoietic precursors
to promote osteoclast formation (3-5).
The discovery of osteoprotegerin (OPG), a
soluble member of the TNF receptor superfamily,
revealed it as a very effective inhibitor of
osteoclast formation (6). This provided the
means of identifying and cloning the elusive
ODF, known now as RANK ligand (RANKL), and the
common factor mediating osteoclast formation
in response to all known stimuli (7). Osteoblasts/stromal
cells are also the source of M-CSF, which plays
a crucial role in osteoclast formation by promoting
the proliferation of precursors.
When hemopoietic cells are treated with M-CSF
and RANKL, osteoclasts are formed without any
participation of osteoblasts/stromal cells (8).
The communication with the hemopoietic lineage
results from RANKL binding to its receptor on
the osteoclast lineage, known as RANK.
All of these discoveries have been validated
by studies in genetically altered mice, as follows:
(i) Overexpression of OPG results in mice with
osteopetrosis because of failure to form osteoclasts
(6). Genetic ablation of OPG, on the other hand,
leads to severe osteoporosis (9).
(ii) Genetic ablation of RANKL results in osteopetrosis
because RANKL is necessary for normal osteoclast
(iii) Genetic ablation of RANK leads to osteopetrosis
also because it is the receptor for RANK (11).
Because this signalling pathway is functional
also in immune cells, RANK-null mice have severe
abnormalities in that system, with failure of
lymph node development and impaired immune responses.
Osteoclastic resorption takes place in a
sealed -off microenvironment (12,13). The most
prominent ultrastructural feature of osteoclasts
is the deep folding of the plasma memrane, the
so-called ruffled border, in the area facing
the bone matrix. This structure is surrounded
by a peripheral ring tightly adherent to the
bone matrix, thus sealing off the sub-osteoclastic
resorbing compartment. The mechanism of bone
resorption requires acidification of the resorption
space by the H+ ions produced by the cells,
ressulting ion dissolution of the bone mineral,
thereby exposing the organic matrix to proteolytic
enzymes (12,13). These enzymes, which include
collagenases and cathepsins, are responsible
for the degradation of the organic matrix. This
process explains how bone resorption contributes
to the maintenance of extracellular fluid calcium
and phosphate. It also explains the presence
of biochemical markers of collagen degradation,
such as hydroxyproline and pyridinoline crosslinks,
in plasma and urine, thereby providing an estimate
of the bone resorption rate (14,15).
The discoveries in osteoclast biology of
the recent few years have identified several
new targets for development of drugs that inhibit
bone resorption. Of the existing drugs, calcitonin
inhibits bone resorption by inhibiting osteoclast
activity. The bisphosphonates do so by inhibiting
osteoclast activity, and probably also by enhancing
osteoclast apoptosis (cell death). The mechanisms
by which estrogen inhibits bone resorption are
still not certain. Estrogen withdrawal results
in enhanced production of certain bone-resorbing
cytokines (IL-1, TNFa and IL-6), and enhanced
responsiveness to M-CSF. Any or all of these
effects could contribute to estrogen action
formation results from a complex cascade of
events that involves proliferation of primitive
mesenchymal cells, differentiation into osteoblast
precursor cells (osteoprogenitor, preosteoblast),
maturation of osteoblasts, formation of matrix,
and finally mineralization (2). Although in
its common usage the term osteoblast is used
to describe those cells responsible for the
synthesis of bone matrix, it is clear that the
osteoblast family also includes the osteocyte
and the bone lining cell. The latter are also
called surface osteocytes, resting osteoblasts,
inactive osteoblasts, endosteal lining cells,
and flattened mesenchymal cells. It seems likely
that at the end of the remodelling sequence
when matrix synthesis is no longer required,
osteoblasts lose their synthetic capacity and
become bone lining cells or can become trapped
behind the advancing calcification front, becoming
embedded in bone as osteocytes. The osteocytes
in their lacunae communicate with each other
and with surface osteoblasts or lining cells
by a complex system of cell microprocesses within
Much less is known of the factors which promote
bone formation. Osteoblasts produce powerful
growth factors, TGFb, IGF1 and 2, and FGF and
store these in bone in large amounts. It is
considered likely that production and activation
of these bone growth factors is a vital step
in stimulating bone formation in response to
hormones and to physical processes and drugs
A discovery which is very important for our
understanding of the bone formation process
is that of Cbfa1 (osf2), a transcription factor
which appears to be essential for the progression
of primitive mesenchymal precursor cells through
to osteoblasts(17). Replenishment of osteoblasts
after bone loss is a key requirement in restoring
bone, and Cbfa1 is central to this. Cbfa1 also
plays an important role in maintaining the osteoblast
in a differentiated state (18). Its regulation
is important, and the pharmaceutical industry
rightly views it as a target through which bone-forming
drugs might be developed.
Diseases of Bone - osteoporosis.
involvement of the skeleton in disease results
from disordered bone resorption and formation,
with an excess of one process over the other,
either as localised or generalised effects on
the skeleton. Loss of ovarian function increases
the rate of bone remodelling and leads to an
imbalance between bone resorption and formation,
resulting in net bone loss. As much as 4-8%
of cancellous bone volume may be lost in women
after the menopause, and up to 50% in the first
3 months following ovariectomy in rodents..
There are a number of disorders that can
impair optimal bone mass acquisition during
childhood and adolescence (19). In some disorders,
such as Turner's syndrome, Klinefelter's syndrome,
glucocorticoid excess, hyperthyroidism or growth
hormone deficiency, low bone mass has been attributed
to abnormalities in a single hormone. In diseases
such as anorexia nervosa and exercise-associated
amenorrhea, malnutrition, sex steroid deficiency
and other factors combine to increase the risk
of osteopenia or low bone mass.). This is probably
also the case of various chronic diseases, which
in addition may require therapies that can affect
The onset of substantial bone loss occurs
at the age of 50 and 65 years in females and
males, respectively (see for review: 20). Female
sex hormones appear to be mandatory not only
to the maximal acquisition of bone mass in both
males and females (21-23), but also to the maintenance
of this mass by controlling bone remodelling
during reproductive life in females (24) and
in aging men (25,26). Even a shortening of the
luteal phase could be associated with abnormal
bone loss (64). Other pathological conditions
associated with premature estrogen deficiency,
such as anorexia nervosa, secondary amenorrhea
due to strenuous exercise, or the use of inhibitors
of gonadotropin secretion (19.27,28), support
the concept of a causal link between estrogen
deficiency and accelerated bone loss. By accelerating
bone turnover and uncoupling bone formation
from resorption, estrogen deficiency appears
to be a main cause of osteoporosis observed
in women after the fifth decade, and possibly
in men, and thus is directly implicated in the
age-related increase in the incidence of fragility
fractures (24). It is now clearly established
that bone loss does not attenuate with age,
but continues throughout the whole life, at
least in peripheral skeletal sites (29).
Apart from gonadal deficiency, which is an
important cause of osteoporosis in men, a number
of other endocrine diseases can also lead to
bone loss.. The effect of primary hyperparathyroidism
on bone is to increase the activation frequency
of bone remodelling. This increase in bone turnover
is associated with a reduction in cancellous
bone volume as observed by histomorphometric
technique. Osteodensitometry indicates a decrease
in a BMD at both axial and appendicular sites
(30). An excess of thyroid hormones also increases
the rate of bone remodelling. Thus, bone loss
can occur in hyperthyroidism and in patients
under long-term thyroid replacement therapy
(69). The major net effect of glucocorticoid
excess is the reduction of bone formation. In
addition, there is some evidence that the administration
of glucocorticoids in pharmacological excess
decreases the intestinal absorption of calcium
and perhaps the tubular reabsorption of calcium.
These latter two effects would lead to a negative
calcium balance and consecutive increased bone
resorption through a mechanism which may involve
secondary hyperparathyroidism (37).
Among nutritional factors, deficiencies in
calcium, vitamin D (33-35) - to the extent its
cutaneous production is insufficient to cover
the needs -, and more recently proteins (36)
have been shown to be associated with deficient
skeletal growth or accelerated bone loss. Vitamin
K deficiency has also been shown to be a predictor
of hip fractures (35). . There is solid evidence
sustaining the notion that calcium contributes
to the preservation of the bony tissue during
adulthood, particularly in the elderly. It is
also clear that without an appropriate supply
of vitamin D, from cutaneous and/or exogenous
source, the bioavailability and metabolism of
calcium is disturbed. This results in accelerated
bone loss during adult life. In young adults
and middle-aged premenopausal women there is
evidence for a positive association between
calcium intake and bone mass (35).
In the elderly, several alterations contribute
towards a negative calcium balance. Indeed,
with ageing there is a decrease in: the calcium
intake by reduction in dairy product consumption;
the intestinal absorption of calcium; the absorptive
capacity of the intestinal epithelium to adapt
to a low calcium intake; the exposure to sunlight;
the capacity of the skin to produce vitamin
D; the renal reabsorption of calcium, as well
as the tubular calcium reabsorptive capacity
to respond to the stimulatory effect of parathyroid
hormone (PTH). Furthermore, the mild renal insufficiency
regularly observed in the elderly can contribute
to a state of chronic hyperparathyroidism that
favors negative bone mineral balance and thereby
osteoporosis. Increasing calcium intake is certainly
an important strategy which is relatively easier
to implement than other possible preventive
Osteoclast inhibition as a
number of therapeutic strategies have been developed
or are being explored to inhibit the formation
on activity of osteoclasts in osteoporosis,
cancer - related bone disease, Paget's disease
and inflammatory bone disease of rheumatoid
arthritis and periodontal disease. Inhibition
of bone resorption can be accomplished by reducing
either osteoclast generation (for example with
estrogens) or osteoclast activity (with bisphosphonates
(37). The molecular mechanism of action of
estrogens on bone, as well as on other tissues,
is not fully understood. Two estrogen receptors
(ER's), a and b, have been identified, but their
relative contributions to the various effects
of estrogens are still under investigation.
Broadly, ERa seems to be responsible for most
of estrogen's effects on reproduction and reproductive
organs, which are fully compromised in its absence
in mice. No unique function has yet been assigned
to ERb. The discovery that agents (historically
referred to as antiestrogens) were able to exert
full or partial estrogen agonist effects on
various tissues initiated the development of
a new class of drugs known as SERMs. The first
SERM identified was tamoxifene, a triphenylethylene
compound that was found to prevent bone loss
(38). Raloxifene, considered in the early 1980s
to be a possible treatment for breast cancer,
was found to prevent bone loss induced by estrogen
deficiency in rats and monkeys. In clinical
studies of raloxifene in post-menopausal women,
a 40% reduction in relative risk of vertebral
fractures was achieved, despite the fact that
there was only a 3 to 4% increase in bone density
(39). The mechanism by which SERMs inhibit bone
resorption is likely to be the same as estrogen's
mechanism, that is by blocking production of
cytokines that promote osteoclast differentiation
(16). Although estrogen and the SERM's are effective
at inhibiting bone resorption to produce a therapeutic
benefit in osteoporosis, they are clearly not
sufficiently powerful to inhibit the greatly
increased osteoclast formation that occurs with
the skeletal complications of cancer. The same
almost certainly applies to Paget's disease
and inflammatory bone diseases.Bisphosphonates
are analogs of pyrophosphate (P-O-P) in which
the oxygen in P-O-P has been replaced by a carbon
with various side chains (40). They concentrate
in bone and are, to date, the most effective
inhibitors of bone resorption, a property discovered
empirically during studies of bone mineralization.
Nitrogen-containing BPs are taken up by osteoclasts,
where they inhibit farnesyl diphosphate synthase,
an enzyme in the mevalonate pathway of cholesterol
synthesis (41). This leads to reduction in the
levels of geranylgeranyl diphosphate, which
is required for prenylation of guanosine triphosphate
(GTP)-binding proteins (such as Rho, Rab and
Cdc42) that are essential for osteoclast activity
and survival. Consequently, BPs inactivate osteoclasts,
which then undergo apoptosis, resulting in reduced
bone resorption, lower bone turnover, and a
positive bone balance.
been successful in preventing the osteoclast-mediated
bone loss of osteoporosis, and have reduced
fracture incidence as a result (42). The greatly
increased bone resorption throughout the skeleton
in the HHM syndrome, as well as the increased
osteoclast formation necessary for bone metastasis
formation, can also be treated by bisphosphonates
(36). These drugs have reduced the incidence
of bone metastasis formation in a number of
trials, but so far there are no reports of enhanced
patient survival. Nevertheless bisphosphonates
are the first line of therapy for hypercalcemia
of cancer, metastatic bone disease and Paget's
disease, as well as being used in treatment
of a large proportion of patients with osteoporosis.
New therapeutic approaches
based on mechanistic understanding
are a number of approaches that are being used
in the search for new inhibitors of bone resorption
(37) - although the question might be asked
- we have bisphosphonates, why do we need new
resorption inhibitors? Arguments in favour of
a continued search for such drugs are that we
need a potent, safe, orally active drug that
rapidly reduces bone resorption in a dose-dependent
manner and is no longer effective after treatment
is stopped. Such a drug could even be a candidate
for the prevention of skeletal complications
of cancer, given long-term as an adjuvant in
appropriate patients with breast cancer, for
example. The bisphosphonates are useful drugs,
but are poorly and erratically absorbed from
the intestine, and are extremely stable, becoming
stored in bone so that they exert activity long
after treatment is stopped. Some of the bisphosphonates,
notably those containing amino groups, have
significant gastrointestinal side effects.
New drugs may come
from the greater understanding of osteoclast
biology of the last few years. The discovery
of RANKL, a key factor in osteoclast formation
and activity, RANK, its receptor, and OPG (
the inhibitory "decoy"receptor ) provides
a number of new therapeutic targets for osteoclast
inhibition (6-8, 37). These members of the tumor
necrosis factor ( TNF ) receptor and ligand
families are crucial for osteoclast control
and mediate the regulation of osteoclast formation
and activity by cytokines and hormones. OPG
injected into rats decreases blood calcium in
cancer-induced hypercalcemia (43), and prevents
bone loss following removal of the ovaries (44).
Furthermore, OPG blocks the periarticular bone
destruction in adjuvant-induced arthritis in
mice, without influencing the inflammation in
and around the joint (45), as well as reducing
cancer-induced bone destruction and pain in
identify OPG's interaction with RANKL as a target
for therapeutic intervention. Should the protein
itself be used for therapy? Although apparently
effective, it is a large protein, needs to be
given in substantial doses, and may induce immune
responses and act in organs other than bone.
Physiologically, OPG may accumulate to some
extent in the bone matrix (47) and be able to
block osteoclast formation from there. If the
physiological process requires tight local regulation
of OPG production, then perhaps a logical therapeutic
approach would be to search for ways to modulate
OPG production by bone cells. The possibility
of OPG gene therapy might be considered in the
future. The only study at the time of writing
is one in which ovariectomized mice were injected
with a recombinant adenoviral vector carrying
the cDNA either of full-length OPG or a fusion
protein combining the OPG ligand-binding domain
with the human immunoglobulin constant domain.
Sustained elevation of plasma OPG levels and
prevention of ovariectomy-induced bone loss
was achieved only in mice given the vector containing
OPG-Fc. (48). It is interesting that efficacy
of OPG in the other animal models referred to
above also required injection of OPG-Fc, thereby
complicating this as a definitive therapy.
The pathway to resorption
inhibition through the TNF ligand and receptor
families would mainly be aimed at inhibition
of osteoclast formation, even though RANKL does
promote osteoclast activity also and OPG inhibits
it. It may be that osteoclast inhibition will
be more effective by inhibiting their formation
than inhibiting their activity because inactivated
cells can so readily be replaced. There are
nevertheless considerable efforts being made
by academic and commercial groups to develop
inhibitors of osteoclast activation. These targets
include avb3 integrin, vacuolar H+-ATPase, p60-c-src
kinase, p38 kinase and cathepsin K (reviewed
A recent surprising
finding was that "statin" drugs (simvastatin,
lovastatin etc) are able to promote bone formation
in mice, and prevent the bone loss following
ovariectomy (49). Some early clinical evidence
supports this also. These cholesterol-lowering
drugs mainly target the liver, and their bioavailability
to bone is likely very limited. The important
aspect of this is that as HMGCoA reductase inhibitors,
they draw attention to that pathway as a target
for bone anabolic drugs.
An exciting example
of the great power of mouse genetics in investigation
is the discovery that leptin deficient (ob/ob)
and leptin signalling deficient (db/db) mice
have greatly increased bone mass, despite their
hypogonad and hypercortisol state (50). This
work has raised the intriguing possibility that
leptin, acting in the brain, is a central regulator
of bone formation by reducing the release into
the circulation of a promoter of bone formation.
Finally, the first
apparently effective stimulator of bone formation
in osteoporosis is reaching the end of its first
trials. PTH, long known to be a powerful stimulator
of bone formation in animal experiments, has
been studied in about 1600 patients for 21 months,
with a 65% reduction in vertebral fractures
and 54% reduction in non-traumatic, non-spine
fractures (51). This leads to optimism that
replacement of bone in severely osteoporotic
subjects is a real prospect.
The ability of PTH
to promote bone formation is dependent upon
the hormone being administered intermittently
in a way that yields a peak blood level, which
is not maintained. In that circumstance, processes
are initiated in bone which result in anabolic
effects, presumably as a result of activation
of genes responding specifically to a rapid
increase in PTH or PTHrP. On the other hand,
if PTH or PTHrP is infused, or administered
in such a way that elevated plasma levels are
maintained, the dominant effect is stimulation
of osteoclast formation and bone resorption,
to the extent that these over-ride any anabolic
response. There are recent in vivo studies in
the rat that support this view. Infusion of
PTH into rats caused a robust and sustained
increase in RANKL and decrease in OPG production
in bone, as well as rapid depletion of matrix
stores of OPG, all of which preceded hypercalcemia
and enhanced osteoclast formation. In these
conditions also, sustained elevated levels of
PTH resulted in decreased expression of genes
associated with the bone formation phenotype
of the osteoblast (52). These included cbfa1,
osteocalcin, bone sialoprotein and type 1 collagen.
These observations were in contrast to the findings
with repeated single injections of PTH, which
although they triggered a rapid but transient
increase in the RANKL/OPG ratio, resulted in
increased bone formation and enhanced expression
of the genes associated with bone formation
Much remains to be
learned of the cellular and molecular events
that determine whether the actions of PTH and
PTHrP are resorptive or anabolic. We need to
know whether the anabolic response is predominantly
the result of enhanced differentiation of existing
osteoblast precursors, of of inhibition of apoptosis,
or a combination of the two, or whether specific
genes, such as cbfa1, mediate the PTH/PTHrP
effect. Important as that is, there may be some
relatively simple lessons and hypotheses to
develop from the existing information.
A central role for
PTHrP in bone development and growth is suggested
by the results of the mouse genetic experiments,
including the important observation that PTHrP
haplosufficient mice are osteopenic. What, then,
are the ways in which PTHrP as a paracrine/autocrine
factor in bone, can contribute? The pharmacologic
effects of intermittent versus sustained PTH/PTHrP
treatment are striking and very different. If
the behaviour of osteoblasts in response to
stimulation through PTHR1 requires this type
of variation in delivery of the relevant ligand,
can PTH, as a circulating peptide hormone, achieve
this? That is doubtful. On the other hand, the
regulated, local production of PTHrP could fulfil
this role, with its regulation the result of
hormonal, cytokine or neural control. In the
case of PTHrP there is an added possibility,
that biological activities within the remainder
of the molecule could influence local events,
either through independent processes or by modifying
actions through the PTH1R.
new insights of the last few years into the
control of bone cell function have greatly improved
our understanding of the pathophysiology of
the bone diseases, as well as giving new leads
to the development of interventions to prevent
and treat those diseases. The factors regulating
osteoclast formation and activity, and therefore
bone resorption, are understood to a greater
extent than those influencing bone formation.
The latter is proceeding apace however, and
especially with the excitement surrounding the
first effective anabolic treatment - PTH - and
the discoveries surrounding osteoblast development
and differentiation, we can expect rapid advances
in that field also.
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