I-1
THE ROLE OF BONE QUALITY IN THE PATHOPHYSIOLOGY OF OSTEOPOROSIS
M. L. Bouxsein
Orthopedic Biomechanics Laboratory, Beth Israel Deaconess Medical Center
& Harvard Medical School, Boston, USA
The first Consensus Conference on Osteoporosis of the new millennium
proposed a new definition of osteoporosis as 'a skeletal disorder characterized by
compromised bone strength predisposing to an increased risk of fracture.' In this
statement, bone density and bone quality were cited as primary contributors to bone
strength. Whereas a precise definition of bone quality remains elusive, evidence is
mounting to support the concept that characteristics independent of bone density, such as
trabecular architecture, bone turnover, the organic and inorganic composition of bone
matrix, damage accumulation, and cell viability, may be important in the pathophysiology
of osteoporosis and in the mechanisms that underlie the anti-fracture effects of
osteoporosis therapies. For example, the concept of bone quality has been invoked to
explain observations from clinical trials that small changes in BMD result in greater than
predicted reductions in fracture risk, that therapy-induced changes in BMD explain only a
small proportion of the variance in fracture risk reduction, and that reductions in
fracture risk are evident long before changes in BMD are observed. Ultimately, the effect
of these potential contributors to bone quality must be reflected in the mechanical
behavior of bone. In this presentation, the ways in which these factors may contribute to
bone strength will be described, with an emphasis on discussing several plausible
mechanisms by which osteoporosis therapies may affect bone strength, and therefore
fracture risk, independently or synergistically with their effects on bone density.
[Programme]
I-2
PREDICTION OF OSTEOPOROTIC FRACTURES BY CLINICAL RISK FACTOR ANALYSIS
C. E. De Laet1*, H. A. P. Pols2
1Institute for Medical Technology Assessment, Erasmus Medical
Center, Rotterdam, The Netherlands
2Dept of Internal Medicine, Erasmus Medical Center, Rotterdam,
The Netherlands
Although it is believed that most fractures occurring in the elderly are
related to osteoporosis, the most important fractures are those of the hip, wrist and
vertebrae. In most areas in the world osteoporotic fractures occur more frequently in
women than in men. For targeting therapeutic interventions the crucial question is not to
diagnose osteoporosis, but to evaluate fracture risk. Fracture risk assessment can be
based on the use of Clinical Risk Factors (CRF's), on an assessment of the quantity or
quality of bone for example by bone mineral density (BMD), or on a combination of both. A
large number of risk factors for osteoporotic fractures have been identified. In most
studies age, BMD, previous fractures steroid use and frailty related parameters are strong
predictors of a future osteoporotic fracture. Between men and women no large differences
appear to exist in the predictive power of these risk factors For the Dutch osteoporosis
guidelines we evaluated the 10-year fracture risk. Based on evidence from literature we
proposed a simple risk score based on 5 easy to use risk indicators and calculated, taking
into account mortality, 10-year fracture risk for hip, wrist and vertebral fractures at
several ages. Moreover, we integrated the effect on quality of life of several fracture
types in one score using the concept of hip fracture equivalents. When based on this 'a
priori' risk an intervention is considered, an additional assessment of bone metabolism
(BMD, ultrasound, bone turnover markers) can be carried out. It is clear that epidemiology
is not restricted anymore to the description of the size of the problem, but also
contributes to a better understanding of the aetiology of osteoporosis in both men and
women. Currently a worldwide effort to find reliable and consistent clinical risk factors
for fractures is underway through a meta-analysis of several large cohort studies. The
results can then be used to evaluate medium term fracture risk in specific populations and
validate the current risk scores.
[Programme]
I-3
INTERACTIONS BETWEEN RANKL, TNF-ALPHA AND TNF-BETA IN THE REGULATION OF
OSTEOCLAST FORMATION AND FUNCTION
T. J. Chambers
St George's Hospital Medical School, London, UK
It has been known for many years that osteoclasts derive from
M-CSF-dependent precursors of the mononuclear phagocyte series, and that their formation
and function are regulated by cells of the osteoblastic lineage. There has recently been
an explosion of information concerning the molecular basis for this regulation.
Osteoblastic cells express M-CSF, which is required for proliferation, survival, and the
induction of RANK in precursors. Osteoblastic cells induce osteoclastic differentiation
and resorptive activity through expression of RANKL. Osteoclastic regulation is assisted
through secretion by osteoblastic and other cells of osteoprotegerin (OPG), a soluble
decoy receptor for RANKL. TGF-beta appears to be an essential cofactor for osteoclast
formation. Thus, TGF-beta not only powerfully synergizes with RANKL, but osteoclast
formation is abolished by soluble TGF-beta receptors. TGF-beta seems to act partly by
preventing the development of resistance to OCL-induction that otherwise occurs when
precursors are incubated in M-CSF, and opposes the ability of agents such as IFN-gamma to
induce alternative macrophagic lineages. TGF-beta also suppresses apoptosis in
osteoclasts.
Surprisingly, not only RANKL but TNF-alpha can induce osteoclast
formation in-vitro, especially in the presence of TGF-beta. Thus, TNF-alpha can induce
inflammatory/cytocidal macrophages or osteoclasts, depending upon the presence or absence
of cofactors such as interferons or TGF-beta. This suggests a model in which lineage is
activated by RANKL/TNF-alpha, but determined by the cofactors.
TNF-alpha enhances bone resorption by several pathways. Not only does it
induce RANKL in osteoblastic cells, and directly stimulate osteoclastic differentiation in
precursors, but it also stimulates bone resorption in mature osteoclasts, in the absence
of osteoblastic cells. TNF-alpha is equipotent with RANKL for this action. Interestingly,
TNF activates osteoclasts at concentrations an order of magnitude below those required for
osteoclastic differentiation. Moreover, TNF-alpha strongly synergizes with RANKL, such
that miniscule concentrations of TNF-alpha are sufficient to substantially augment
osteoclast activation. The extreme sensitivity of osteoclasts to activation by TNF-alpha,
suggests that the most sensitive osteolytic response of bone to TNF-alpha is through
activation of existing osteoclasts; and the strong synergy with RANKL provides a mechanism
whereby increased osteolysis can be achieved without disturbance to the underlying pattern
of osteoclastic localisation.
[Programme]
I-4
THE MEVALONATE PATHWAY IN OSTEOCLAST SIGNALLING
M. J. Rogers*, F. P. Coxon
Dept of Medicine & Therapeutics, University of Aberdeen, UK
The mevalonate pathway is responsible for the synthesis of cholesterol
and a variety of isoprenoid lipids (such as farnesyl diphosphate/FPP and
geranylgeranyldiphosphate/GGPP). FPP and GGPP are substrates for prenyl:protein
transferases that attach the isoprenoid lipid onto the C-terminus of small GTPases. The
latter are a large family of highly conserved proteins that act as molecular switches. The
binding of GTP causes allosteric changes that allow interaction with effector proteins (eg
protein kinases), thereby activating mutiple downstream signalling pathways.
Post-translational prenylation of small GTPases with a farnesyl or geranylgeranyl group is
essential for their correct function since it allows association with subcellular
membranes and, in some cases, interaction with other proteins.
Recently, the importance of prenylated small GTPases in osteoclasts was
highlighted by the discovery that nitrogen-containing bisphosphonates (N-BPs) are potent
inhibitors of FPP synthase. Inhibition of this enzyme causes loss of FPP and, indirectly,
GGPP, thereby inhibiting prenylation of small GTPases. In particular, geranylgeranylated
GTPases appear to be essential in osteoclasts, since restoring geranylgeranylation, but
not farnesylation, rescues osteoclasts from the effects of N-BPs. Furthermore, the effects
of N-BPs on osteoclasts are mimicked in vitro by GGT8, a selective inhibitor of protein
geranylgeranylation.
The role of geranylgeranylated GTPases in osteoclasts is poorly
understood, although they are probably fundamental for the intracellular control of
osteoclast activity. For example, the Rho family GTPases (Rho, Rac and Cdc42) are involved
in cytoskeletal organisation. In osteoclasts, inhibition of Rho or Rac function prevents
the formation of podosomes and F-actin rings and inhibits resorption, although the exact
signalling pathways upstream and downstream are largely unclear. Cdc42 appears to regulate
podosome assembly in other cell types but its role in osteoclasts is unknown. Rab GTPases,
the largest subfamily, are involved in membrane and vesicular trafficking. Rab7 is
localised to the ruffled border, and antisense inhibition of Rab7 disrupts ruffled border
formation and inhibits bone resorption. The roles of other Rab proteins in osteoclasts are
unknown, although specific inhibition of Rab prenylation inhibits resorption and causes
morphological changes in the osteoclast membrane.
The mevalonate pathway is therefore of fundamental importance in
osteoclasts for providing metabolites necessary for the correct function of small GTPase
signalling proteins. Inhibition of this pathway is the basis for existing and potentially
novel anti-resorptive agents.
[Programme]
I-5
OSTEOGENESIS IMPERFECTA: PATHOPHYSIOLOGY AND MANAGEMENT
F. H. Glorieux
Shriners Hospital for Children and McGill University, Montreal, Canada
Osteogenesis imperfecta (OI) is a heritable disorder characterized by
increased bone fragility. Four different types are commonly distinguished that range from
a mild condition (type I) to a lethal one (type II). Types III and IV are the severe forms
surviving the neonatal period. In most cases, there is a reduction in the production of
normal type I collagen or the synthesis of abnormal collagen as a result of mutations in
the type I collagen genes. There are instances, however, where alterations in bone matrix
components, other than type I collagen, are the likely basic abnormalities. We have
identified three such discrete types by histomorphometric evaluation (type V and VI) and
linkage analysis (type VII). They provide evidence for the as yet poorly understood
complexity of the phenotype-genotype correlations in OI.
In all severe forms, histologic and densitometric evaluations have
documented an increase in osteoclastic activity and a reduction in the formation of new
bone. We thus designed studies to assess the effects of bisphosphonate (potent inhibitors
of bone resorption) treatment in children with severe OI. Pamidronate administered
intravenously at 2-4 month intervals resulted in significant increase in bone mineral
density, decrease in chronic pain and fracture incidence, and improved mobility and
ambulation. Patients received annual doses of 9-16 mg/kg. There was no adverse effects on
growth, fracture healing or bone modeling. The benefits observed were more marked in
children under 2 years of age, with reshaping of vertebral bodies and long bones, probably
correlated to rapid skeletal growth in the young infant. The major impact on bone is
cortical thickening, with the long term effect on remodeling remaining to be evaluated.
Although not a cure for OI, this therapeutic approach is the first one that permits to
significantly alter the natural course of events in severe OI, characterized by relentless
progression of crippling deformities and chronic pain. This approach will remain the
therapy of choice until targeted gene therapy will become a reality.
[Programme]
I-6
THE MUTATIONAL SPECTRUM OF HUMAN MALIGNANT AUTOSOMAL RECESSIVE
OSTEOPETROSIS
A. Frattini1, C. Sobacchi1, P. Orchard2,
I. Tezcan3, P. Vezzoni1, I. Baric4, S. Dupuis-Girod5,
A. Musio1, M. Mirolo1, A. Villa1*
1Institute of Biomedical Technologies, CNR, Segrate (MI),
Italy
2Department of Pediatrics, Division of Bone Marrow
Transplantation, University of Minnesota, Minneapolis, Minnesota, USA
3Hacettepe University Children's Hospital, Ankara, Turkey
4Department of Pediatrics, University Hospital Center, Zagreb,
Croatia
5Unite d'Immonulogie et d'Hematologie Pediatriques, Hopital
Necker-Enfant Malades, Paris, France
Human malignant infantile osteopetrosis (arOP) is a genetically
heterogenous autosomal recessive disorder of bone metabolism, which, if untreated, has a
fatal outcome. Most human arOP patients have normal to elevated number of osteoclasts in
bone biopsies, suggesting that osteopetrosis is due to a defect in genes affecting the
function and not the differentiation of osteoclasts. This notion has been recently
supported by the finding of a defect in the vacuolar proton pump (VPP) in about 50% of the
patients and by the demonstration that ClC-7, a chloride channel which acts in concert
with the VPP, was mutated in one of 12 families studied. These data confirm that
acidification is the most sensitive step in bone resorption. Our group, as well as others
have recently found that mutations in the ATP6i gene, encoding the a3 subunit of the VPP,
are responsible for a subset of arOP. By sequencing the ATP6i gene in arOP patients from
44 unrelated families with a worldwide distribution we have now confirmed that ATP6i
mutations are responsible for approximately one-half of patients affected by arOP. The
vast majority of these mutations (40 out of 42 alleles, including 7 deletions, 2
insertions, 10 nonsense substitutions and 21 mutations in splice sites) are predicted to
cause severe abnormalities in the protein product and are likely to represent null
alleles. In addition, we have also analysed 9 unrelated arOP patients from Costa Rica,
where this disease is apparently much more frequent than elsewhere. All these patients
bore either or both of two missense mutations (G405R and R444L) in amino acid residues
evolutionary conserved from yeast to humans. The identification of ATP6i gene mutations in
two families allowed us for the first time to perform prenatal diagnosis: both fetuses
were predicted not to be affected and two healthy babies were born. We also examined
whether ClC-7 gene is mutated in our ATP6i-independent arOP patients. Our results suggest
that this gene is only rarely responsible for arOP. This study contributes to the
determination of genetic heterogeneity of arOP and allows further delineation of the other
genetic defects causing this severe condition.
[Programme]
I-7
FIBRODYSPLASIA OSSIFICANS PROGRESSIVA: CLUES TO THE FORMATION OF A
HETEROTOPIC SKELETON
F. S. Kaplan*, J. Ahn, L. Serrano de la Pena, E. M. Shore
School of Medicine, University of Pennsylvania, Philadelphia, USA
Fibrodysplasia ossificans progressiva (FOP), a rare autosomal dominant
genetic disorder, is the most disabling form of heterotopic ossification in humans. FOP is
characterized by anterior-patterning malformations of the great toes and by progressive
induction of endochondral osteogenesis at ectopic sites. Bone morphogenetic protein 4
(BMP4) mRNA and protein are uniquely over-expressed in lymphocytes and lesional cells from
patients who have FOP. New findings suggest that defects in the BMP4 pathway in FOP cells
severely limit their ability to express multiple BMP antagonists in response to a BMP4
stimulus. The BMP4 gene is not mutated in FOP, and the BMP4 locus has been excluded from
linkage to the condition. These data suggest that the primary defect in FOP is not in the
BMP4 gene itself, but in a component of the BMP4 pathway or interacting pathway that leads
to resistance of BMP4 antagonist expression. Our data suggest that a loss of negative
feedback due to an insufficient BMP antagonist response may account in part for increased
BMP4 activity in FOP. Such a defect suggests the loss of a critical negative feedback
mechanism by which cells normally regulate the magnitude and boundaries of ambient
morphogenetic signals. These findings from a disabling human disease illustrate the
importance of a critical balance between an inductive morphogen and its secreted
antagonists in the formation of an ectopic organ system and suggest the potential of BMP
antagonist-based strategies in the therapy of FOP.
[Programme]
I-8
11BETA-HYDROXYSTEROID DEHYDROGENASE IN GLUCOCORTICOID SIGNALLING IN BONE
M. Hewison*, M. S. Cooper, P. M. Stewart
Division of Medical Sciences, The University of Birmingham, Queen
Elizabeth Hospital, Birmingham B15 2TH, UK
Glucocorticoids play an essential role in skeletal development but are
detrimental in excess. Despite this, relatively little is known about the molecular
mechanisms involved in determining the sensitivity of bone cells to glucocorticoids.
Susceptibility to the bone-specific effects of glucocorticoids correlates only weakly with
circulating hormone levels or target cell glucocorticoid receptor (GR) expression. We have
therefore proposed that tissue-specific 'prereceptor' regulation may act as a pivotal
determinant of the impact of glucocorticoids on bone. Previous studies have shown that in
several tissues glucocorticoid action is dependent on the expression of
11beta-hydroxysteroid dehydrogenase (11beta-HSD) isozymes, which interconvert active
cortisol and inactive cortisone. 11beta-HSD1 is located in tissues which express
relatively high levels of GR such as liver and adipose where it functions by increasing
local concentrations of cortisol. 11beta-HSD2 is located predominantly in tissues with
high mineralocorticoid receptor (MR) expression such as kidney and colon. In these tissues
11beta-HSD2 acts to protect the MR from illicit occupancy by cortisol by metabolising it
to cortisone. Immunohistochemical, Western blot and RT-PCR analyses of normal human bone
and primary osteoblast cultures showed predominant expression of 11beta-HSD1. Conversely,
fetal bone and osteosarcoma cells exhibited predominant 11beta-HSD2 expression. In each
case the presence of an isozyme was associated with GR expression. Subsequent enzyme
activity studies have shown that primary cultures of human osteoblasts are able to
synthesise cortisol from cortisone. This was potently stimulated by inflammatory cytokines
such as TNFalpha and IL-1, suggesting a role for 11beta-HSD1 as a modulator of
inflammatory responses. Paradoxically, osteoblast 11beta-HSD1 activity was also stimulated
by therapeutic corticosteroids such as prednisone and prednisolone. Functional studies in
vitro using stable transfectant models have shown that the major impact of 11beta-HSD
isozymes is on osteoblast proliferation, with 11beta-HSD1 being antiproliferative and
11beta-HSD2 proproliferative. However, studies using an 11beta-HSD inhibitor suggest that
altered bone resorption may be the chief consequence of 11beta-HSD1 action in vivo. These
data confirm that prereceptor regulation is a crucial determinant of the impact of
glucocorticoids on bone. In particular, 11beta-HSD1 may play an important role in the
pathophysiology of steroid-induced bone disease.
[Programme]
I-9
ANALYSIS OF THE REGULATION OF BONE FORMATION BY LEPTIN
G. Karsenty
Baylor College of Medicine, Houston, TX, USA
We have shown earlier that leptin regulates following its binding to
hypothalamic receptors. This initial study however did not provide any clues about the
molecular mechanisms accounting for this action of leptin.
In an effort to analyze how leptin can regulate bone formation in vivo we
asked three questions. The first question is what is the identity of the neurons
controlling bone formation. We performed chemical leisoning in wild-type mice followed by
leptin infusion in the third ventricle to answer this question. This allowed us to
identify a population of hypothalamic neurons that act as the central center of bone
formation. To determine how leptin signals to this center we generated various transgenic
mouse models expressing leptin either in ob/and in liver as well or mice overexpressing
leptin receptors. Results of the analysis of these mice will be presented at the meeting.
Finally, we followed several different approaches to determine the nature and the identity
of the mediators of leptin action on bone formation. These investigations indicated that
one group of molecules are responsible for this action. This part of the investigation
will also be presented at the meeting.
[Programme]
I-10
THE BIOCHEMICAL AND CELLULAR CHARACTERIZATION OF SCLEROSTIN, THE
CAUSATIVE GENE FOR SCLEROSTEOSIS
J. A. Latham
Gene Function and Target Validation, Celltech R&D Inc, Bothell WA,
USA
Sclerosteosis is a recessive disorder restricted to the skeleton that
afflicts patients with excessive bone matrix deposition. The matrix material, both
cortical and cancellous, is extremely strong and of superior quality. This disease
provides an excellent opportunity through the application of the drug surrogate hypothesis
for the identification of therapeutic targets for use in treating diseases associated with
low bone mass. In this strategy, the combination of recessive genetics inferring loss of
function and a restricted phenotype converse from the diseased population, provides a
direct route to a therapeutic intervention opportunity. Positional cloning methods have
identified the causative gene, which has been called Sclerostin, a novel family member of
the soluble BMP antagonists. This group of proteins, which includes Chordin, Noggin,
Cerberus and Gremlin, form high affinity noncovalent complexes with BMPs. These
protein/protein complexes perturb the signaling property of the BMP and therefore act to
suppress the mitogenic properties of this protein. Data will be presented that describes
the characterization of this gene product in biochemical and cellular systems with an eye
to understanding the role of the gene in bone matrix deposition and its applicability in
treating osteoporosis.
[Programme]
I-11
ROLE OF FOS PROTEINS IN OSTEOBLAST DIFFERENTIATION AND FUNCTION
E. F. Wagner, et al.
Research Institute of Molecular Pathology (I.M.P.), Dr. Bohr-Gasse 7,
A-1030 Vienna, Austria
Fos proteins are transcription factors belonging to the AP-1 complex,
which acts like a biosensor for many cells and is causally involved in many developmental
processes, but also in cell differentiation and disease. One of the four members of the
Fos proteins is c-Fos, which is a key regulator of bone development (1). Transgenic mice
expressing exogenous c-Fos develop bone tumors, characterised by transformation of
osteoblasts. In contrast, mice lacking c-Fos are osteopetrotic due to a differentiation
block in bone resorbing osteoclasts (2). We were also interested to study how c-Fos and
its related protein Fra-1, which is c-Fos inducible, control osteoblast proliferation and
osteoclast differentiation (3). Gene deletion experiments in mice demonstrated that Fra-1
is an essential gene for development (4) and genetic rescue experiments suggest that Fra-1
is not essential for osteoblast and osteoclast differentiation. However, transgenic mice
overexpressing Fra-1 develop the bone disease osteosclerosis, which is due to increased
bone formation (5). The primary cause of the disease in Fra-1 transgenic mice is an
osteoblast differentiation defect, although the transgenic osteoclasts are hyperactive in
vitro. To test whether Fra-1 can substitute for c-Fos, we generated knock-in mice that
express Fra-1 in place of c-Fos. Fra-1 rescues c-Fos dependent functions in bone
development, which appeared to be gene-dosage dependent (6). However, Fra-1 failed to
substitute for c-Fos in inducing expression of target genes in vitro. We are using
these systems to identify novel Fos target genes by microarrays and with the help of
bone-specific conditional alleles of c-Fos and Fra-1, we are studying the molecular
mechanisms how Fos proteins govern bone cell development and differentiation.
1. Wagner, E.F. and Karsenty, G. (2001) Genetic control of skeletal
development. Curr Opin. Genetics & Development, 11, 527-533.
2. Grigoriadis, A.E., Wang, Z.-Q., Cecchini, M.G., Hofstetter, W., Felix,
R., Fleisch H.A. and Wagner, E.F. (1994) c-Fos is a key regulator of osteoclast/
macrophage lineage determination and bone remodelling. Science 266, 443-448.
3. Matsuo, K., Owens, J.M., Tonko, M., Elliot, C. Chambers, T.J. and
Wagner, E.F. (2000) Osteoclast differentiation by the c-Fos target gene Fra-1, Nature
Genetics 24, 184-187.
4. Schreiber, M., Wang, Z.Q., Jochum W., Fetka, I. Elliott, C. and
Wagner, E.F. (2000). Placental vascularization requires the AP-1 component Fra1.
Development 127, 4937-4948.
5. Jochum, W., David, J.P., Elliot, C., Wutz, A., Plenk, H., Matsuo, K.
and Wagner, E.F. (2000) Increased bone formation in transgenic mice expressing the
transcription factor Fra-1, Nature Medicine 6, 980-984.
6. Fleischmann, A., Hafezi, F., Elliott, C., Remé, C.E., Rüther, U. and
Wagner, E.F. (2000). Fra-1 replaces c-Fos-dependent functions in mice. Genes &
Development 14, 2695-2700.
[Programme]
I-12
QUANTITATIVE ULTRASOUND: METHODS AND CLINICAL APPLICATIONS
C. C. Glüer*, R. Barkmann
Medizinische Physik, Diagnostische Radiologie, Universitätsklinikum
Kiel, Germany
Quantitative Ultrasound (QUS) approaches have been developed for the
assessment of bone status in osteoporosis and other skeletal disorders. Today a number of
different QUS devices are commercially available that assess partially different aspects
of bone status. One needs to recognise the difference between the measurement parameters
Speed of Sound (SOS) and Broadband Ultrasound Attenuation (BUA), the different measurement
sites (calcaneus, finger phalanges, and radius) and the different technologies employed
(transverse transmission, axial transmission). For example, one could expect substantial
differences between BUA results at a trabecular weight bearing bone measured in transverse
transmission and SOS measured at a cortical non weight bearing bone measured in axial
transmission. Today the main area for use of QUS approaches is the assessment of a
fracture risk. For use in monitoring and diagnosis, further studies and methodological
clarification to determine diagnostic criteria suitable for peripheral devices are
required. Special applications, e.g. in paediatrics or rheumatology, have been
investigated with promising results. In clinical practice, adequate and regularly
performed quality assurance measures and in-depth knowledge about the general and device
specific limitations of the approach used are of substantial importance. When used
appropriately, QUS devices can play an important role in the assessment of bone status.
Ultrasound interaction with bone is strongly affected by bone mass but also by other
factors related to elasticity and strength of bone. Bone microstructure and material
properties both have been shown to affect QUS parameters.
[Programme]
I-13
PERIPHERAL OR CENTRAL DENSITOMETRY: DOES IT MATTER WHICH TECHNIQUE WE
USE?
I. Fogelman*, G. M. Blake
GKT Medical School, Guy's Hospital, London, UK
Over the past decade, bone density scans have assumed an essential role
in the diagnosis of osteoporosis. Although dual X-ray absorptiometry (DXA) scans of the
central skeleton remain widely used, a variety of different types of equipment for
measuring peripheral sites is now available. However, the poor correlation between
different types of measurement and a lack of consensus on how results from peripheral
sites should be interpreted have proved a barrier to the more widespread use of these
devices. These issues prompt the following questions: Which technique best identifies
patients at risk of fracture? What approaches to scan interpretation ensure the closest
agreement between different methods? Does it matter if different patients are selected for
treatment on the basis of different techniques? The relative risk (RR) of fracture derived
from prospective studies is a key parameter for comparing the clinical value of different
techniques. Recent reports confirm the advantages of hip bone mineral density compared
with peripheral measurements for predicting hip fracture risk, although for fractures at
other sites the differences are inconclusive. Using receiver operating characteristic
curves, we show that the guidelines adopted for scan interpretation are of crucial
importance for ensuring that the information is used effectively. The closest agreement
between different techniques is achieved by setting thresholds for peripheral devices that
target either the same percentage of the population or the same percentage of future
fracture cases as femur DXA. Different methods select different groups of individuals from
the total pool of patients who will later sustain a fracture, with the most successful
technique being the one with the largest RR value. The emphasis placed by many studies on
validating new techniques by studying their correlation with DXA may lead to the clinical
value of peripheral devices being underestimated when the key datum is the RR value
inferred from prospective fracture studies.
[Programme]
I-14
ADVANCED IMAGING OF BONE MACRO AND MICRO STRUCTURE
H. K. Genant
Osteoporosis and Arthritis Research Group, University of California, San
Francisco CA, USA
In the past decade, considerable progress has been made in the
development of methods for assessing the skeleton noninvasively so that osteoporosis can
be detected early, its progression and response to therapy carefully monitored, or the
risk of fracture effectively ascertained. Bone mineral density (BMD) of the peripheral,
central or entire skeleton as well as the trabecular or cortical bone envelopes can now be
evaluated with a high degree of accuracy and precision and the strength of bone and the
propensity to fracture can be reliably estimated.
Noninvasive and/or nondestructive techniques are capable of providing
macro or micro structural information about bone, beyond simple bone densitometry. While
the latter provides important information about osteoporotic fracture risk, numerous
studies indicate that bone strength is only partially explained by BMD. Quantitative
assessment of macrostructural characteristics such as geometry, and microstructural
features such as relative trabecular volume, trabecular spacing and connectivity may
improve our ability to estimate bone strength.
The methods available for quantitatively assessing macrostructure include
(besides conventional radiographs, which are not considered here) computed tomography, and
particularly volumetric quantitative computed tomography (vQCT). Methods for assessing
microstructure of trabecular bone noninvasively and/or nondestructively include high
resolution computed tomography (hrCT), micro computed tomography (µCT), high resolution
magnetic resonance (hrMR), and micro magnetic resonance µMR. Volumetric QCT, hrCT and
hrMR are generally applicable in vivo, and µCT and µMR are principally applicable in
vitro.
From a clinical perspective, the challenges for bone imaging include
balancing the relative advantages of simple bone densitometry versus the more complex
architectural features of bone or similarly the deeper research requirements versus the
broader clinical needs. The considerable potential biological differences between the
peripheral appendicular skeleton and the central axial skeleton must be further examined.
Finally, the relative merits of these sophisticated imaging techniques have to be weighed
with respect to their applications as diagnostic procedures requiring high accuracy or
reliability versus their monitoring applications requiring high precision or
reproducibility.
[Programme]
I-15
PROSPECTS FOR CARTILAGE ENGINEERING
A. P. Hollander1*, W. Z. Kafienah1, S. C. Dickinson1,
T. J. Sims1, I. Martin2, M. D. Barker3, A. Frazer3
1Academic Rheumatology, University of Bristol, Bristol, UK
2Department of Surgery, University of Basel, Basel,
Switzerland
3Division of Genomic Medicine, Sheffield University,
Sheffield, UK
There have been significant advances in the techniques required for
successful tissue engineering of cartilage. However a number of challenges remain to be
resolved in advance of clinical application.
Cartilage engineering to date has mostly relied on the use of cells
isolated from young animals. There have been few attempts at developing a clinically
useful methodology. An autologous cell source will be essential for the treatment of
cartilage lesions, however the clinical use of human chondrocytes for tissue engineering
will require expansion of cell numbers from a small donor site biopsy. Therefore clinical
application will be limited by the chondrogenic potential of cells after proliferation. We
have now demonstrated that adult human articular chondrocytes that have been expanded in
monolayer culture and then used for cartilage engineering have poor chondrogenic
potential. In contrast, adult nasal septum chondrocytes are relatively effective when used
under identical conditions. We therefore propose that nasal chondrocytes should be used to
repair articular cartilage lesions. An alternative would be to use autologous mesenchymal
stem cells and promote their differentiation to chondrocytes, however a key challenge will
be to avoid the generation of hypertrophic chondrocytes, which may result in calcification
of cartilage at the articular surface.
Techniques must be developed to ensure that implanted engineered
cartilage integrates well with the surrounding natural tissue. In order to achieve
short-term mechanical stability it may be necessary to engineer osteochondral implants
that can be inserted into the subchondral bone. Subsequent integration of implant with
surrounding cartilage must involve migration of chondrocytes between the two surfaces and
techniques for enhancing this process are under development.
Successful clinical trials of implanted engineered cartilage must include
effective outcome measures. In addition to a range of clinical assessment techniques, it
will be essential to use quantitative methods for analysing follow-up biopsies taken
months or years after implantation. We have developed methods for determining the specific
collagen content as well as collagen crosslink maturity in very small tissue biopsies.
In summary, the technique of cartilage engineering is now advanced enough
that we must consider how to use it successfully in the clinical setting.
[Programme]
I-16
PROSPECTS FOR CELL ENGINEERING IN BONE REPAIR
A. K. Smith
Osiris Therapeutics, Inc., Baltimore MD, USA
Human mesenchymal stem cells (MSCs) can be isolated and expanded ex vivo
as a non-differentiated adult stem cell population. These cells are capable of
multilineage differentiation into diverse tissue types such as bone marrow stromal cells,
bone, adipose tissue, cartilage, tendon and ligament and muscle.
Osiris Therapeutics has evaluated the potential to use cryopreserved MSCs
attached to matrix materials for their combined ability to repair critically sized bone
defects. Matrix materials evaluated include hydroxyapatite/tricalcium phosphate composites
as well as modified allograft bone. Preclinical studies have demonstrated successful bone
repair in large critical gap osteoperiosteal defects in canine and non-human primate
models. Results from the animal studies show inclusion of MSCs results in superior bone
formation compared to matrix alone which showed incomplete healing. Other issues
associated with use of matrix alone included suboptimal integration with the host bone
tissue and/or lack of mechanical integrity.
Preclinical animal studies have also demonstrated the potential to use an
allogeneic MSC preparation in tissue engineering. Studies show that bone formation is
accomplished to the same extent using allogeneic MSCs, in the absence of any
immunosuppression, as is seen with autologous MSCs. There is no evidence of either a
cellular or humoral immune response to the allogeneic MSCs in preclinical animal studies
even with multiple delivery of MSC doses. MSCs can be cryopreserved and successfully
stored for long periods of time. The combined storage potential and the ability to achieve
equivalent efficacy with an allogeneic MSC observations support the potential to use
allogeneic MSC preparations support the potential for an 'off-the-shelf' or 'universal'
MSC product for tissue engineering applications. Clinical studies are currently underway
for bone repair using an MSC matrix combination.
Preclinical models have also demonstrated regenerative potential for use
of MSCs in the arthritic knee joint, where these cells repair damaged meniscus and retard
articular cartilage and subchondral bone damage. MSCs have also been successfully used in
preclinical studies to treat cardiac infarct and in human clinical trials for improved
hematopoietic transplantation support. Preclinical and previous human clinical data
support the safe and widespread potential of adult-derived hMSCs in regenerative medicine.
[Programme]
I-17
THE REPAIR OF SKELETAL DEFECTS WITH BMPS
T. A. Einhorn
Boston University School of Medicine, 720 Harrison Avenue, Boston MA, USA
Bone has the capacity to undergo regeneration as part of a repair
process. While bone regeneration may involve several independent molecular events, a key
regulator of bone regeneration is the TGF-b superfamily of molecules which include the
bone morphogenetic proteins (BMPs). In addition to identifying specific molecules and
cloning them for biotechnology applications, the receptors and signal transduction
pathways of BMPs have also been elucidated. Although much more can be learned regarding
the biology of bone regeneration and, in particular, BMP-induced osteogenesis, technology
in this field has advanced to the point where clinical applications have already been
made.
The use of recombinant BMP-2 and BMP-7, in several types of delivery
systems, and in several animal models, has suggested that recombinant gene technology may
lead to the application of BMPs in the repair and restoration of skeletal defects, and in
the treatment of degenerative conditions of the spine. Clinical studies on the application
of BMP-2 in fresh fractures of the tibia, and BMP-7 in tibial nonunions have yielded
impressive results. Indeed, BMP-7 (known as osteogenic protein-1; OP-1) is now available
for clinical use in the treatment of nonunions in several European countries as well as
Australia. More recently, the US Food and Drug Administration has issued an HDE
(Humanitarian Device Exception) for the use of OP-1 in the treatment of recalcitrant
nonunions of long bones. Future investigations on the clinical applicability of BMPs will
concentrate on improved clinical experimental design and the development of novel carriers
for enhanced delivery of the active molecules. In particular, the role of gene therapy in
the delivery of BMP in patients should be explored. The few reports that have demonstrated
enhanced bone regeneration when BMP-2 is delivered in an adenoviral vector have been
impressive. Innovative methods for gene delivery could yield safe and effective therapies
within the foreseeable future.
[Programme]
I-18
MANAGEMENT OF CANCER ASSOCIATED BONE DISEASES: NEW DEVELOPMENTS
R. E. Coleman
Cancer Research Centre, Weston Park Hospital, Sheffield, UK
The bisphosphonates are an important class of agents for the treatment of
metastatic bone disease. Randomised controlled trials have clearly demonstrated that
bisphosphonate treatment is effective in reducing skeletal morbidity in breast cancer and
multiple myeloma. Attention is now focussing on when to start treatment and how long to
continue, as well as the use of bone markers to predict those patients most likely to
benefit.
At present clodronate, usually given orally, and infusions of pamidronate
are the two most widely used bisphosphonates in oncology. New more potent bisphosphonates
are now available and could be expected to simplify treatment and possibly improve the
therapeutic effectiveness of bisphosphonate therapy. Zoledronic acid (Zometa) is the
most potent, and has shown superior efficacy in hypercalcaemia of malignancy. Three large
(n=>3000) phase III trials in treatment of bone metastases have been completed. In
patients with multiple myeloma and breast cancer patients, non-inferiority of zoledronic
acid compared with pamidronate has been confirmed, with advantages in terms of convenience
and some of the secondary endpoints. Importantly, in placebo-controlled trials, a
reduction in skeletal complications has been shown in other solid tumours affecting bone
including prostate cancer, confirming the role of bisphosphonates across the range of
tumours affecting bone.
Although many studies indicate that bisphosphonates prevent the
development of metastatic bone disease in animals, the clinical role of prophylactic
bisphosphonates in early breast cancer is not defined. Three studies with oral clodronate
have been published, two of them showing a protective effect on the development of bone
metastases and improved survival, and one suggesting a disadvantage to the use of adjuvant
clodronate. Further large adjuvant trials with clodronate and zoledronate are in progress.
Recently, the biological importance of the RANK-OPG system has been
defined. Osteoprotogerin (OPG), has been identified as a decoy receptor to RANK ligand,
inhibiting the osteoclast stimulatory effects of this molecule. Phase I studies of
synthetic OPG are complete and open up a range of new therapeutic avenues for metastatic
bone disease.
[Programme]
I-19
OSTEOMALACIA TUMOURS, RICKETS AND NOVEL BONE-RENAL MOLECULAR PATHWAYS
P. S. N. Rowe
University of Texas Health Science Center at San Antonio, Molecular
Medicine,
Institute for Drug Development CTRC,14960 Omicron Drive San Antonio, TX
78245, USA
Oncogenic hypophosphatemic osteomalacia (OHO), X-linked hypophosphatemic
rickets (HYP) and autosomal dominant hypophosphatemic rickets (ADHR) are characterized by
rachitic and osteomalacic bone defects, hypophosphatemia, abnormal renal phosphate (Pi)
handling, and renal defects in vitamin D metabolism. Also, all three diseases (familial
and tumor), are associated with primary or secondary secretion of novel uncharacterized
factors (phosphatonins), that have direct impact on pathophysiology.
HYP is primarily caused by a defective novel zinc-metalloendopeptidase
(PHEX). This has prompted a search for a putative PHEX phosphatonin-substrate (PTN). PHEX
is thought to either activate or inactivate PTN(s) initiating a regulatory bone-renal Pi
cascade. Physiology experiments in Hyp-mouse models support the PTN model and indicate
that PTN(s) are circulating osteoblast-derived factor(s). Moreover, the apparent
overlapping pathophysiology between HYP and OHO suggests that OHO-tumours may secrete
inappropriately processed form(s) of PTN. Candidate PTNs have been discovered and these
molecules could potentially play major roles in the mineralisation of osteoid, renal
phosphate handling and cancer metastasis to bone.
Fibroblast-growth-factor (FGF23) is an example of a good PTN candidate.
Activating mutations in specific FGF23 C-terminal residues occur in patients with ADHR and
the protein is expressed in some OHO-tumours. Also, CHO-cells expressing FGF23 cause
rickets when heterotransplanted into nude mice. Interestingly no FGF23 inhibition of
Pi-uptake occurs in-vitro in renal cell-lines and FGF23 is not expressed in bone. Another
good PTN candidate, MEPE, is a novel glycosylated protein with RGD/ASARM motifs and is a
member of the SIBLING proteins (DMP-1, OPN, DSPP, BSP-I). This protein was cloned from
OHO-tumours and is not expressed in non-phosphaturic tumours. MEPE is found in plasma and
is expressed in osteoblasts/osteocytes in rodents and humans. MEPE and PHEX are
developmentally co-expressed in osteoblasts. Also, MEPE expression is markedly increased
during osteoblast-mediated matrix mineralisation and vastly elevated levels occur in bone
and osteoblasts derived from Hyp mice. MEPE peptides inhibit phosphate uptake in human
renal cell-lines and alter vitamin D metabolism. Moreover, MEPE peptides increase bone
mineralisation in bone organ-culture and increase osteoblast numbers. Further
experimentation is needed to unravel the abundance of factors and molecular-interactions
involved in the regulation of Pi and the mineralisation of osteoid.
[Programme]
I-20
TREASURE YOUR EXCEPTIONS: MAPPING HUMAN SKELETAL DYSPLASIAS
C. A. Francomano
National Institute on Aging, Baltimore MD, USA
The human skeletal dysplasias are a genetically and clinically diverse
group of disorders, representing over 200 distinct conditions. Knowledge of the genes
underlying this widely diverse group of disorders has the potential to inform our
understanding of the genetic and biochemical pathways critical to skeletal growth and
development. It is likely that many of the genes found to cause the rare skeletal
dysplasias also play a role in the pathogenesis of more common disorders of skeletogenesis
and skeletal aging. To date, over 70 loci known to cause skeletal phenotypes have been
mapped. These include loci encoding structural proteins of the extracellular matrix,
enzymes involved in the modification of the structural proteins, molecules involved in
signal transduction, and genes encoding regulatory proteins and RNA products. Current
methodology and tools to further analyze the pathogenesis of these conditions will be
presented, including the Skeletal Gene Database, a searchable listing of all genes known
to be involved in skeletal growth and development. This database includes genes from 11
different bone libraries represented in the National Center for Biotechnology Informatics
databases, with a total of 28,218 expressed sequence tags (ESTs) represented.
To illustrate many of the issues involved in mapping and analyzing the
skeletal dysplasias, this presentation will focus on the family of disorders caused by
mutations in Fibroblast Growth Factor Receptor 3 (FGFR3). Clinical and molecular features
of this family of disorders, which includes achondroplasia, hypochondroplasia,
thanatophoric dysplasia, and Severe Achondroplasia with Acanthosis Nigricans and
Developmental Delay (SADDAN) will be discussed. The pivotal role of the Human Genome
Project in allowing investigators to move from a map location to a disease gene will be
illustrated using the example of achondroplasia. The role of microarrays, genomic and
proteomic approaches to further understanding pathogenesis will be presented. The utility
of animal models to dissecting molecular pathways will also be discussed. Finally, an
overview of the utility of knowledge about skeletal dysplasias and how that knowledge may
be applied to understanding more common disorders of skeletal growth and development will
be presented.
[Programme]
I-21
PROGRESS IN MAPPING OSTEOPOROSIS GENES IN MICE
R. F. Klein
Oregon Health & Science University and Portland VA Medical Center,
Portland, Oregon, USA
Osteoporosis is one of the most common bone and mineral disorders in both
aging men and women. Susceptibility to osteoporosis appears to involve the interaction of
multiple environmental and genetic factors. Considerable past research has centered on the
influence of reproductive, nutritional and/or life-style factors on the development of
osteoporosis. With the advent of new molecular genetic approaches, the focus of research
has recently shifted towards genetic factors. The number, location and effects of the
individual genes contributing to osteoporosis risk are all unknown. The extreme difficulty
of dissecting out environmental factors from genetic ones in humans has motivated the
investigation of animal models. Recent analyses of inbred strain databases are beginning
to reveal important aspects of the genetic regulation of bone mass acquisition and
maintenance. Advances in genetic mapping of complex traits, such as quantitative trait
locus (QTL) mapping, are especially promising. A major strength of this approach is that
it enables the provisional identification of candidate genes in the absence of any prior
hypothesis about the mechanism by which the phenotype is expressed. QTL mapping offers an
attractive interface between forward and reverse genetics. As candidate genes are
identified as having important skeletal functions, the tools of molecular biology will
allow the genetic diversity underlying their expression and function to be more fully
examined. Discoveries made with animal models can often set the stage for skeletal
research in human subjects to augment the results from animals and confirm their relevance
to our own species. Perhaps the most versatile aspect of animal model systems is in their
use as a proving ground for hypotheses regarding the genetic as well as the epigenetic
basis of osteoporosis. Old ideas regarding disease mechanisms can now be rigorously tested
in vivo, and perhaps more importantly, provocative new concepts can emerge.
[Programme]
I-22
HARVESTING BIOLOGICAL DATA USING BIOINFORMATICS: PREDICTION OF HUMAN
DISEASE GENES
J. A. M. Leunissen1,2*, M. A. van Driel1, K.
Cuelenaere2, P. Neerincx1, G. H. Degenhart1, H. G.
Brunner3
1CMBI, University of Nijmegen, Nijmegen, Netherlands
2Dalicon BV, Nijmegen, Netherlands
3Human Genetics, UMCN, Nijmegen, Netherlands
We have developed a tool integrating different genetic web-based
databases in order to quickly generate a list of candidate genes for human diseases. Given
a cytogenetic localisation and phenotypic data for input, the tool extracts gene names
from several distributed databases and returns only genes that match the two
prerequisites. The system provides researchers of human genetic diseases with plausible
candidate genes based on phenotype as well as genotype.
The tool is written in Perl and has a modular set-up; new genetic
databases can easily be added, as long as the database supports a 'bulk query' strategy,
i.e. being able to return all known or appropriate gene names in one query. The power of
the tool is based upon the fact that gene names are mostly standardised, thus allowing
them to be used as unique identifiers when integrating search results. Several filters are
included in order to allow man to mouse homologies to be included in the search
strategies. Notably, an 'Oxford grid' is constructed to query the relevant sections of
mouse genetic databases, and a list of gene synonyms is included to deal with gene naming
inconsistencies between human and mouse. Recent additions include EST database searching
to verify the expression location 'experimentally', and the use of pathway databases to be
able to include those genes where the relationship between expression profile and
localisation is indirect, i.e. linked through a common pathway.
In its present form the program is mainly suited for syndromes, for which
it is valid to assume that the disease gene has an aberrant or absent gene expression in
the affected tissues. Through the addition of pathway information, as well as by expanding
the number of databases, we will increase both its sensitivity and its versatility.
[Programme]
I-23
APPLICATION OF PROTEOMICS TO ELUCIDATE MECHANISMS OF DISEASE
L. C. Lawrie1,2*, G. I. Murray2, J. E. Fothergill1
1Dept. of Molecular & Cell Biology, University of Aberdeen,
Scotland, UK
2Dept. of Pathology, University of Aberdeen, Scotland, UK
Proteomics offers a powerful analytical tool for the post-genomic era.
Proteome is the word used to describe the total protein complement expressed by an
organism, a cell, or a tissue type. By studying the differences in protein expression
profiles between normal and disease/treated samples proteomics can be used to elucidate
molecular mechanisms of disease, to identify diagnostic markers or therapeutic targets and
also to provide an understanding of the mechanisms of drug toxicity.
The first part of this presentation will describe the basic principles
involved in proteomic analysis including the separation and visualisation of proteins by
2D gel electrophoresis, followed by the characterisation and identification of these
proteins by mass spectrometry and database searching.
The second part of the presentation will concentrate on the application
of proteomics in oncology. Cancer is characterised by multiple genetic abnormalities. Some
of these are crucial to the development of individual types of cancer, whereas others
appear to be the consequence of tumour development. It is important to characterise the
functional consequences of these gene changes in terms of protein expression, to elucidate
more fully the processes of tumour development and progression and also the response of
established tumours to therapy. There is a need to identify biomarkers of cancer risk, of
early diagnosis, and of disease progression, and to pinpoint targets for therapeutic
intervention, diagnosis, or imaging. It is also important to develop methods of cancer
classification which more accurately predict the clinical behaviour of tumours. The
application of proteomics is expected to have a major impact on oncology, both for our
understanding of tumour development and progression, and in providing improved diagnosis
and treatment.
[Programme]
I-24
PROBING GENE FUNCTION BY TRANSGENIC APPROACHES
M. F. Young
CSDB, National Institute of Dental Research, NIH, Bethesda, MD, USA
Genetically engineered mice are critical tools to uncover the function of
a protein in mineralized tissue. At the same time they provide valuable experimental
models for prevalent calcified tissue diseases such as osteoporosis, osteoarthritis and
ectopic calcification. They can also be used to study the function of a protein in the
context of external influences such as impact (running), hormone imbalance, nutritional
variation or pharmaceutical intervention.
Technical advances in cell and molecular biology have resulted in a
corresponding increase in the ways that mice can be genetically altered. The goal of the
workshop will be to present a brief outline of the types of transgenic mice available with
emphasis on the concepts and techniques used to create them. The advantages and
disadvantages of each type of genetic alteration will be addressed. Examples from the
literature and from the author's own experience will be used to illustrate key points such
as ways to circumvent gene redundancy or lethality. The workshop will discuss conventional
transgenics that contain genes 'driven' by a promoter either ectopically or, in a tissue
specific fashion. The multi-faceted nature of creating mice with a single gene deficiency
using gene targeting and homologous recombination (knockout or KO) will be detailed.
Despite the technical power of the gene targeting method it is restricted
by the fact that some genes when knocked out from conception are lethal. Although these
observations clearly show the importance of a protein, they limit examining its function
later in life. In this regard, new techniques developed to abrogate genes at a specific
time and location in vivo will be discussed. The ways that one can determine the function
of a gene in any tissue for which a specific promoter is available will be presented. A
new development in this area is to combine spatial specificity with controlled temporal
reduction using chemicals that can be injected into the genetically engineered mutant
mice. The workshop will conclude with commentary on the newest advances in calcified
tissue research using transgenic strategies.
[Programme]
I-25
BONE CELLS' RESPONSES TO MECHANICAL STRAIN: IMPLICATIONS FOR
POSTMENOPAUSAL OSTEOPOROSIS
L. E. Lanyon
Royal Veterinary College, London, UK
Bones' ability to withstand load-bearing without damage is achieved and
maintained by a homeostatic mechanism in which bone cells adjust bone architecture to
control the strains engendered by functional loading. The mechano-responsive cells are
most likely osteocytes and osteoblasts.
The increased fracture incidence characteristic of post-menopausal
osteoporosis is associated with a decline in bone mass despite continued functional
activity. This implies reduced responsiveness to the increased strains which the bone loss
should engender.
In cultures of rat primary osteoblasts, and in ROS 17/2.8 cells, both a
short period of strain and estrogen stimulate proliferation. The size of the proliferative
response to strain and estrogen together equals the addition of the two individual maximal
responses separately. The estrogen antagonist ICI 182,780 reduces, and tamoxifen abolishes
the proliferative response to strain and reduces that due to estrogen. In human primary
osteoblasts and in TE85 cells ICI 182,780 similarly blocks strain-related proliferation
but tamoxifen does not.
In rat primary osteoblasts and ROS17/2.8 cells estrogen and strain cause
phosphorylation of serine 118 in the amino terminal of ERalpha which is the consensus
phosphorylation site for MAPK. This response is eliminated by inhibitors of ERK-1 and
ERK-2, the immediate upstream activators of MAPK.
In ROS cells increasing ERalpha number by transfection increases the
proliferation stimulated by both estrogen and strain, both of which are inhibited by ICI
182,780. Strain and estrogen also both upregulate ERE activity which is also blocked by
ICI.
In osteoblast-like cells early proliferative responses to strain and
estrogen appear to involve the MAPK pathway, Estrogen Receptor alpha and EREs. At the cell
level strain and estrogen neither enhance nor obstruct each other's effects on osteoblast
proliferation although different interactions may occur at the tissue and organ level.
Proliferation of osteoblasts is only one of resident bone cells' adaptive responses to
strain but it may be representative of others.
The etiology of post-menopausal osteoporosis could be explained by less
effective strain-related control of bone architecture associated with the down-regulation
of ER number which others have reported to follow the withdrawal of estrogen.
[Programme]
I-26
WHAT TYPE OF EXERCISE PROTECTS THE SKELETON?
A. Heinonen
UKK Institute for Health Promotion Research, Tampere, Finland
In human exercise studies the changes in bone mass are small, and local
rather than generalised, and the attainable changes take place slowly. Different exercise
modalities and sports load the skeleton at different sites and different ways, and thus
the training response may vary. In terms of skeletal loading, weightlifting, for instance,
creates extreme torques in the upper extremities whereas the lower extremities experience
large compressive stresses. The bone mass values of the intensively training female and
male weightlifters (i.e., high-magnitude loading) were more than 10% higher than those of
comparable referents in the previous studies. However, prospective weight-training studies
have demonstrated only small to moderate (0.8-3.8%) gain in regional bone mineral mass.
The most commonly practised sports that involves a large number of repetitive loading is
running and walking. However, the difference is not as great as between athletes in
strength sports and control subjects. Nevertheless, earlier randomised controlled clinical
trials have shown that multi-exercise endurance training at a relatively high intensity
maintained bone mass at the femoral neck and lumbar spine in healthy postmenopausal women.
Previous studies have reported that athletes engaged in sports producing high strain rates
via versatile impact-type movements have much higher bone mass (9-40%), geometry and
structure (the largest differences over 50%) than their sedentary controls. Adaptation to
this loading seemed to occur in a site-specific fashion by gross-geometric changes,
structural or architectural changes or by their combination. Clinical trials also support
the high-impact loading concept. In our recent randomised controlled study, we showed that
the 18-month high impact training (jumping) resulted in significant increase (1.4-3.7%) in
bone mass in the loaded sites. Cumulative evidence shows that the important components of
osteocenic exercise stimulus are high-impact and high-magnitude loading in versatile
movements. Greater differences in BMC and BMD between playing and non-playing arm sites in
squash and tennis supports the osteogenic value of high-impact loading. When the loading
magnitude is low, as may occur in the elderly, the number of loading cycles becomes more
important. In the elderly, progressive strength training is likely to be safe and may be
effective in reducing the risk factors for falling, although having less effect on BMD.
[Programme]
I-27
IDENTIFYING PATIENTS WHO NEED TREATMENT FOR OSTEOPOROSIS
J. A. Kanis
WHO Collaborating Centre for Metabolic Bone Diseases, University of
Sheffield Medical School, Sheffield, UK
In the past 10 years there have been significant advances in the field of
osteoporosis, including the development of diagnostic techniques, a greater understanding
of the pathogenesis of osteoporosis and the evolution of effective treatments. There are,
however, large gaps in our ability to implement effective therapeutic strategies.
Strategies may be based on a global approach where the intention is to shift the
distribution of bone mineral density (BMD) in the whole population, for example by the
promotion of exercise, smoking cessation or manipulating the dietary intake of calcium.
However, evidence for the efficacy of such approaches is lacking, and their feasibility
has never been tested. The alternative approach is the 'high risk' strategy whereby
segments of the population most at risk are targeted for intervention, for example, mass
population screening of women at the time of the menopause. There are many reasons why
this is not feasible at present.
A particular problem with BMD is that despite its high specificity, its
sensitivity (detection rate) for fracture outcomes is low over most reasonable
assumptions. For this reason, treatment strategies need to take into account the risk
factors that operate independently of BMD and thereby enhance the predictive value of the
test. These factors include age, family history of hip fracture, prior fragility fracture,
high rates of bone turnover, low body mass index and neuromuscular incompetence. The
presence of such factors increases fracture risk over and above that which can be
explained on the basis of BMD. Therefore, diagnostic thresholds differ from intervention
thresholds. Intervention thresholds should be based on the absolute risk of the clinically
significant outcomes.
Fracture risk is optimally determined as a fracture probability over a
relatively long time-frame (eg 10 years). The setting of intervention thresholds is
ultimately dependent upon health economic considerations that have been evaluated in the
Swedish population. These require modification in different countries to take account of
different costs and risks that vary markedly in different regions of the world.
[Programme]
I-28
OSTEOPOROSIS TREATMENT WITH PARATHYROID HORMONE
R. Neer
Massachusetts General Hospital, Boston, MA 02114, USA
Anti-resorptive agents rarely cure osteoporosis, because bone formation
is too slow, and such drugs slow it even more. Agents that stimulate bone formation may
overcome this limitation. Human parathyroid hormone (hPTH) and its amino-terminal
fragments and analogs increase bone formation, and can increase bone mass and strength in
animals. The new bone produced by hPTH treatment is histologically, chemically, and
mechanically normal. There have now been more than 14 prospective clinical trials of
hPTH-(1-34) or hPTH-(1-84) in osteoporotic women or men. In a randomized
placebo-controlled multi-national trial in 1,637 postmenopausal women with a prior
vertebral fracture, hPTH-(1-34) reduced the risk of a first additional vertebral fracture
61-69%, and reduced the risk of a first new non-vertebral fracture 50%, while increasing
bone mineral density (BMD) 9-13% in the spine, 3-6% in the femoral neck, and 2-4% in the
total body, and decreasing forearm cortical BMD non-progressively by 1-2%. These fracture
reductions exceed those reported for anti-resorptive agents. In prospective, randomized,
direct comparisons, hPTH-(1-34) also increases spine and hip BMD substantially more than
alendronate or hormone replacement therapy in postmenopausal osteoporotic women. In
osteoporotic men, hPTH-(1-34) increases spine and hip BMD more than anti-resorptive drugs
are reported to do.
hPTH routinely cures estrogen-deficiency osteoporosis in rodents, but
does so far less often in humans, in part because humans cannot tolerate the high hPTH
doses needed in rodents. Combining hPTH with an anti-resorptive drug might overcome this
limitation, and might also enhance the effectiveness of hPTH in humans. Several clinical
trials are now exploring this possibility.
In humans hPTH has no major adverse effects and causes only minor side
effects. In rodents, high doses of hPTH cause marked osteosclerosis, and nearly life-long
high-dose hPTH treatment causes osteosarcomas. Neither complication has been observed in
humans treated with hPTH or its amino-terminal fragments or analogs, nor is the incidence
of this rare malignancy increased in humans with primary or secondary hyperparathyroidism.
In the near future, hPTH may be marketed as a treatment for established osteoporosis.
[Programme]
I-29
STATINS AND BONE: MYTH OR REALITY?
T. D. Spector
St Thomas' Hospital, London, UK
The actions of statins on serum cholesterol concentrations are well
documented and are associated with decreased cardiovascular events and death. In late 1999
experimental work produced the unexpected finding that statins may/could stimulate bone
formation when administered locally to bony sites or when given systemically in rats. A
number of other animal studies have confirmed an effect of most but not all statins.
Since these findings a number of epidemiological studies have been
published recently that explore the effects of statin use on bone mineral density (BMD)
and fracture risk in humans. These studies have produced mixed results even disagreeing in
the same dataset. Whilst unequivocal evidence for a therapeutic effect in the doses used
in humans is lacking, many statins do influence bone via a number of potential pathways
including stimulating osteoblasts, inhibiting osteoclasts, altering apoptosis,
angiogenesis, and inhibiting inflammation. Understanding these mechanisms should prove
helpful in new therapeutic approaches and understanding of osteoporosis.
[Programme] |