Matrix elasticity directs stem cell lineage — Soluble factors that limit osteogenesis

Abstracts / Bone 44 (2009) S200–S210

Typically RCTs are not powered to address subset hypotheses or treatment effects over long periods of time. Only the intent-to-treat comparisons in a RCT ensure that the treated and untreated groups were fully comparable at entry. Hence, population science research must rely on observational studies, a mainstay epidemiologic approach to the study of the association between various exposures and behaviors and subsequent chronic disease incidence and mortality. Advantages of cohort studies include their prospective design with large sample sizes and long durations that can lead to a large number of outcomes and ability to test multiple hypotheses. The main weakness is residual confounding by unmeasured factors. Important information about osteoporosis has comes from large observational studies including the magnitude of the problem, secular trends, risk factors and identification of high risk women e. g., FRAX and the consequences of osteoporosis. But, the Women’s Health Initiative (WHI) showed largely discrepant results between the RCT and cohort studies of hormone therapy (HT) and cardiovascular disease (CVD) but consistent results for fracture. Reanalysis of the WHI Observational study (OS) showed a lower risk of CVD among HT users consistent with other cohort studies. However control for time from HT initiation resulted in closer agreement between the two study designs. Women in the RCT all initiated treatment at randomization while many women in the OS had initiated HT years ago. Effects of HT on CVD not fracture differs across initiation of use. The complimentary roles fulfilled by RCTs and observational studies and their joint analyses can result in improved analytical approaches to address confounding and bias ultimately improving our understanding of disease. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.022

IS17 Sex steroids in the regulation of bone metabolism in men C. Ohlsson Centre For Bone Research, Institute of Medicine, Gothenburg, Sweden Osteoporosis-related fractures constitute a major health concern not only in women but also in men. The relative contribution of estrogens and androgens for the male skeleton remains unclear. Most epidemiological studies demonstrate that serum estradiol is a stronger predictor of bone mineral density than serum testosterone. However, conflicting results have been presented regarding the impact of serum sex steroids for fracture risk in men, probably because previous studies have been underpowered and have analyzed the serum sex steroid levels using immunoassay-based techniques with a questionable specificity at lower concentrations. We recently showed that elderly men with low serum estradiol have an increased risk of fractures in the large population-based MrOS Sweden study, with serum sex steroids analyzed by the specific gas chromatography-mass spectrometry technique. In contrast, serum testosterone was not an independent predictor of fracture risk. There are two main sources of sex steroids in elderly men, the testicles and the adrenals. Interestingly, we found that low serum DHEA was related to fracture risk independently of serum sex steroids in the MrOS Sweden study, indicating that adrenalderived DHEA, which is locally converted to estradiol and/or testosterone, has an impact on fracture risk. Experiments using mice with inactivated sex steroid receptors demonstrated that both activation of the estrogen receptor (ER)alpha; and activation of the androgen receptor (AR) result in a stimulatory effect on the cancellous bone mass in males. ERbeta; was of no importance for the skeleton in male mice while it modulated the ERalpha-action on cancellous bone in females. In vitro studies demonstrated that the G-protein coupled receptor GPR30 is a functional ER. Our recent in vivo analyses of GPR30inactivated mice revealed no function of GPR30 for cancellous bone

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mass but it is involved in the regulation of longitudinal bone growth. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.023

IS18 The benefits of combining cohorts for mega-analysis E.V. McCloskey Academic Unit of Bone Metabolism, University of Sheffield, Sheffield, UK Very many risk factors for fracture have been proposed which arise from observations in cross-sectional and case-control studies or prospective cohorts. For many of these factors, their general validity on an international basis has been less secure. Meta-analyses are, however, available for the dependence of fracture risk on smoking, and BMD, use of oral glucocorticoids, as well as the influence of a prior fragility fracture on the risk of a future fracture. In the development of risk models, it is critical that interactions between risk factors are taken into account. For example, in the case of BMD, account needs to be taken of age and the prevailing BMD value, since the risk of osteoporotic fracture is dependent on both variables. Several other risk factors have been identified which add to risk independently of BMD and have been incorporated into risk assessment algorithms. While it is usual to report their predictive value independently of age and BMD, uncertainties remain concerning differences between men and women, dependence upon age, and attenuation of the predictive value of the risk factors with time. In addition, potential interactions can be masked by assuming linearity of effect. An example would be BMI, where an increase of 1 unit from a low value (e.g. from 20 kg/m2 to 21 kg/m2) is more protective than from a high value (e.g. 30 kg/m2 to 31 kg/m2).In the derivation and synthesis of risk models, it is therefore necessary to determine the interactions between each of the risk factors, e.g. to what extent does the risk of glucocorticoid treatment depend upon smoking and how does this affect the interaction of smoking with age. This requires a meta-analysis of the meta-analyses (referred to as a mega-analysis), and has been the technique used in the development of the FRAX algorithms for estimating 10-year fracture probabilities (www.shef.ac.uk/FRAX). The WHO Collaborating Centre at Sheffield examined a series of candidate risk factors from 12 prospectively studied international cohorts drawn from the general population, using the primary data from each study, comprising almost 60 000 men and women and a total follow-up somewhat over 250 000 person–years. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.024

IS19 Matrix elasticity directs stem cell lineage — Soluble factors that limit osteogenesis D.E. Discher Molecular and Cell Biophysics Lab, University of Pennsylvania, Philadelphia, USA Stem cells appear to sense their microenvironment through adhesion-coupled cytoskeletal interactions. Naive mesenchymal stem cells (MSCs) from human bone marrow will be shown to specify lineage and commit to phenotypes with extreme sensitivity to tissue level elasticity. Soft matrices that mimic brain have appeared neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone seem osteogenic. Inhibition of myosin blocks all elasticity directed lineage

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Abstracts / Bone 44 (2009) S200–S210

specification — without strongly perturbing many other aspects of cell function and shape. A “Cysteine Shotgun” Mass Spectrometry method for in situ labeling of the ‘foldome’ reveals distinct structural differences attributable to unfolding and/or dissociation of cellular proteins. The results have significant implications for understanding physical effects of the in vivo microenvironment and also for therapeutic uses of stem cells. Lastly, since fibrotic regions are stiff and tend to favor osteogenesis, soluble factors are being evaluated to limit such processes and thus selectively block some lineages and not others. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.025

IS20 Clinical applications of mesenchymal stem cells F. Jakob Orthopaedic Department, University of Wuerzburg, Wuerzburg, Germany Mesenchymal stem cells (MSC) are a multipotent population of skeletal precursor cells which are capable of self-renewal and can give rise to several other mesenchymal phenotypes like bone, cartilage, fat and tendon. Bone marrow stroma derived populations can be easily obtained, but are not completely homogenous. There is quite robust proof for their capability of giving rise to colony forming units in vitro and their differentiation towards the above discussed pathways as well as their immunomodulating attitudes and low immunogenicity. This renders them attractive candidates to be addressed and used for tissue regeneration in both in vivo and ex vivo settings. Although there are promising in vitro and preclinical data as well as clinical case reports in humans and despite of more than a decade of intensive research on tissue engineering (TE) and regeneration using MSC, no application has yet become a routine clinical procedure. Moreover in many of the applications tested and even in clinical pilot trials, no robust proof has yet been reported of exogenous retransplanted MSC substantially participating in tissue regeneration as surviving differentiated cells. To this end the best evidence for MSC participation in (partial) ex vivo TE and survival in vivo comes from preclinical studies for cartilage replacement. But in this setting the main problem is still the achievement and maintenance of a mature chondrocyte phenotype. The debatable evidence for enhanced tissue regeneration and healing under the influence of transplanted MSC in e.g. bone regeneration and healing (e.g. osteonecrosis), myocardial infarction and diabetes may also be due to a supportive role of MSC, e.g. serving as – albeit rapidly dying – delivery devices for growth and differentiation factors. Recent developments for rapid translation of cell-based therapies for several reasons favor the use of crude mixtures of freshly isolated bone marrow cells and of such mixtures after ex vivo expansion for several weeks. These mixtures comprise variable amounts of MSC, but also endothelial precursors. There is growing casuistic evidence of enhanced healing and regeneration, but convincing superiority when compared with state of the art strategies is missing. Future developments will have to consider several key problems and aspects of clinical settings that have not yet been clarified. From a developmental perspective MSC may in fact differ quite remarkably depending on their localization, because they arise from different compartments during the course of developmental patterning, e.g. from axial, paraxial, lateral and from neuroectodermal mesoderm and there is evidence for a positional memory in various MSC populations. Thus MSC population may not be alike in terms of regeneration and differentiation capacity if not being reprogrammed. Alternatively we need to add other adult stem cells and progenitors for TE since MSC alone cannot effectively enough give rise to

endothelial cells and neurons. Given the need of nutrient supply of constructs and regenerative tissue, scaffolds and complex ex vivo constructs will have to be enhanced. We will also have to go back from bedside to bench to characterize the cell biology of MSC niches, unravel the potential of MSC for reprogramming, learn to modulate processes of angio- and neurogenesis and to decide if – except for rare cases of large tissue defects – in situ guided tissue regeneration is perhaps a better option. Moreover, in musculoskeletal research the majority of the applications will be and has to be performed in the elderly and oldest old and we will have to adopt regenerative applications to an ageing, and in many cases diseased organism. We will have to get more knowledge about cellular ageing and senescence and their countermeasures, especially during ex vivo procedures. In conclusion there is huge potential for the future, but also a huge amount of research still to be done. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.026

IS21 Is long-term suppression of bone turnover associated with increased fracture risk? J.E. Zerwekh Internal Medicine, University of Texas Southwestern Medical Center, Dallas, USA Accumulation of microdamage (microcracks) in engineering materials generally signifies an impending failure. Bone behaves in a similar fashion and cyclic loading leads to incremental failure through a process known as fatigue. If bone cannot repair the microcracks, microdamage can accumulate yielding a bone with reduced fracture resistance. It is generally accepted that one function of bone remodeling is to remove and replace bone microdamage with new, biomechanically competent bone. Thus, an excessive reduction in bone turnover may result in inadequate microdamage repair and cause fracture. This suggestion is supported from several studies demonstrating reduced bone biomechanical properties in bisphosphonate-treated animal models. However, this finding has not been universal. There have been even fewer studies in humans. The role of reduced bone remodeling and increased fracture risk is clearly evident in patients with renal disease and the adynamic form of renal osteodystrophy. It is also supported from recent clinical reports of patients on long-term bisphosphonate therapy who present with atypical femoral diaphyseal fractures. There has been limited histological examination of bone in such patients but when performed has been consistent with severe suppression of bone turnover. Another potential mechanism for explaining increased fracture risk during suppression of bone remodeling is through hypermineralization of bone. During bisphosphonate therapy, there is a significant shift in bone mineral density fraction to a more dense value, probably the result of suppressed bone turnover. Such increases in mineral density can lead to the formation of well mineralized bone but with reduced biomechanical strength. This mechanism may also help explain the finding of atypical diaphyseal fractures in bisphosphonate-treated women with severe suppression of bone turnover, despite radiographic evidence of thick, adequately mineralized, cortices. Thus, it appears that long-term suppression of bone remodeling is associated with an increased fracture risk. To better understand which of the aforementioned mechanisms may be responsible for increased fracture risk will require careful studies in patients using bone histomorphometry as well as assessment of biomechanical strength through finite element analysis of bone. With continued use of anti-resorptive agents, it is possible that a sufficiently large patient population will become available for such