Mödder, Ulrike I.

Abstract There is growing interest in the use of mesenchymal stem cells for bone repair. As a major reason for normal bone remodeling is the removal of fatigue microcracks, advances in our understanding of this process may inform approaches to enhance fracture healing. Increasing evidence now indicates that physiological bone remodeling occurs in close proximity to blood vessels and that these vessels carry perivascular stem cells that differentiate into osteoblasts. Similarly, fracture healing is critically dependent on the ingrowth of blood vessels not only for a nutrient supply but also for the influx of osteoblasts. A number of animal and human studies have now shown the potential benefit of bone marrow-derived mesenchymal stem cells in enhancing bone repair. However, as in other tissues, the question of whether these cells improve fracture healing directly by differentiating into osteoblasts or indirectly by secreting paracrine factors that recruit blood vessels and the accompanying perivascular stem cells remains a major unresolved issue. Moreover, CD34+ cells, which are enriched for endothelial/hematopoietic cells, have also shown efficacy in various bone repair models, at least in part due to the induction of angiogenesis and recruitment of host progenitor cells. Thus, mesenchymal and nonmesenchymal stem/progenitor cells are attractive options for bone repair. It is possible that they contribute directly to bone repair, but it is also likely that they express paracrine factors in the appropriate amounts and combinations that promote and sustain the healing process.

Abstract Sclerostin is a potent inhibitor of Wnt signaling and bone formation. However, there is currently no information on the relation of circulating sclerostin levels to age, gender, or bone mass in humans. Thus we measured serum sclerostin levels in a population-based sample of 362 women [123 premenopausal, 152 postmenopausal not on estrogen treatment (ET), and 87 postmenopausal on ET] and 318 men, aged 21 to 97 years. Sclerostin levels (mean ± SEM) were significantly higher in men than women (33.3 ± 1.0 pmol/L versus 23.7 ± 0.6 pmol/L, p < .001). In pre- and postmenopausal women not on ET combined (n = 275) as well as in men, sclerostin levels were positively associated with age (r = 0.52 and r = 0.64, respectively, p < .001 for both). Over life, serum sclerostin levels increased by 2.4- and 4.6-fold in the women and men, respectively. Moreover, for a given total-body bone mineral content, elderly subjects (age ≥ 60 years) had higher serum sclerostin levels than younger subjects (ages 20 to 39 years). Our data thus demonstrate that (1) men have higher serum sclerostin levels than women, (2) serum sclerostin levels increase markedly with age, and (3) compared with younger subjects, elderly individuals have higher serum sclerostin levels for a given amount of bone mass. Further studies are needed to define the cause of the age-related increase in serum sclerostin levels in humans as well as the potential role of this increase in mediating the known age-related impairment in bone formation. © 2011 American Society for Bone and Mineral Research.

Context: Intermittent PTH treatment stimulates bone formation, but the mechanism(s) of this effect remain unclear. Sclerostin is an inhibitor of Wnt signaling, and animal studies have demonstrated that PTH suppresses sclerostin production. Objective: The objective of the study was to test whether intermittent PTH treatment of postmenopausal women alters circulating sclerostin levels. Design: Prospective study. Setting: The study was conducted at a clinical research unit. Participants and Interventions: Participants included 27 postmenopausal women treated with PTH (1-34) for 14 d and 28 control women. Main Outcome Measures: Serum sclerostin levels were measured. Results: Circulating sclerostin levels decreased significantly in the PTH-treated subjects, from (mean ± SEM) 551 ± 32 to 482 ± 31 pg/ml (−12.7%, P < 0.0001) but did not change in the control women (baseline, 559 ± 34 pg/ml; end point, 537 ± 40 pg/ml, P = 0.207; P = 0.017 for difference in changes between groups). Bone marrow plasma was obtained in a subset of the control and PTH-treated subjects (n = 19 each) at the end of the treatment period, and marrow plasma and peripheral serum sclerostin levels were significantly correlated (R = 0.64, P < 0.0001). Marrow plasma sclerostin levels were 24% lower in PTH-treated compared with control women, but perhaps due to the smaller sample size, this difference was not statistically significant (P = 0.173). Conclusions: Circulating sclerostin levels correlate with bone marrow plasma levels and are reduced by intermittent PTH therapy in postmenopausal women. Further studies are needed to assess the extent to which decreases in sclerostin production contribute to the anabolic skeletal response to PTH.

Osteoporosis is an important clinical problem, affecting more than 50% of people over age 50 yr. Estrogen signaling is critical for maintaining proper bone density, and the identification of an endogenous selective estrogen receptor (ER) modulator, 27-hydroxycholesterol (27HC), suggests a mechanism by which nutritional/metabolic status can influence bone biology. With its levels directly correlated with cholesterol, a new possibility emerges wherein 27HC links estrogen and cholesterol signaling to bone homeostasis. In these studies, we found that increasing concentrations of 27HC, both by genetic and pharmacological means, led to decreased bone mineral density that was associated with decreased bone formation and increased bone resorption. Upon manipulation of endogenous estrogen levels, many of the responses to elevated 27HC were altered in such a way as to implicate ER as a likely mediator. In a model of postmenopausal bone loss, some pathologies associated with elevated 27HC were exacerbated by the absence of endogenous estrogens, suggesting that 27HC may act both in concert with and independently from classic ER signaling. These data provide evidence for interactions between estrogen signaling, cholesterol and metabolic disease, and osteoporosis. Patients with high cholesterol likely also have higher than average 27HC, perhaps putting them at a higher risk for bone loss and fracture. More studies are warranted to fully elucidate the mechanism of action of 27HC in bone and to identify ways to modulate this pathway therapeutically.

Abstract While female mice do not have the equivalent of a menopause, they do undergo reproductive senescence. Thus, to dissociate the effects of aging versus estrogen deficiency on age-related bone loss, we sham-operated, ovariectomized, or ovariectomized and estrogen-replaced female C57/BL6 mice at 6 months of age and followed them to age 18 to 22 months. Lumbar spines and femurs were excised for analysis, and bone marrow hematopoietic lineage negative (lin–) cells (enriched for osteoprogenitor cells) were isolated for gene expression studies. Six-month-old intact control mice were euthanized to define baseline parameters. Compared with young mice, aged/sham-operated mice had a 42% reduction in lumbar spine bone volume/total volume (BV/TV), and maintaining constant estrogen levels over life in ovariectomized/estrogen-treated mice did not prevent age-related trabecular bone loss at this site. By contrast, lifelong estrogen treatment of ovariectomized mice completely prevented the age-related reduction in cortical volumetric bone mineral density (vBMD) and thickness at the tibial diaphysis present in the aged/sham-operated mice. As compared with cells from young mice, lin– cells from aged/sham-operated mice expressed significantly higher mRNA levels for osteoblast differentiation and proliferation marker genes. These data thus demonstrate that, in mice, age-related loss of cortical bone in the appendicular skeleton, but not loss of trabecular bone in the spine, can be prevented by maintaining constant estrogen levels over life. The observed increase in osteoblastic differentiation and proliferation marker gene expression in progenitor bone marrow cells from aged versus young mice may represent a compensatory mechanism in response to ongoing bone loss. © 2010 American Society for Bone and Mineral Research.

Abstract Sex steroids are important regulators of bone turnover, but the mechanisms of their effects on bone remain unclear. Sclerostin is an inhibitor of Wnt signaling, and circulating estrogen (E) levels are inversely associated with sclerostin levels in postmenopausal women. To directly test for sex steroid regulation of sclerostin levels, we examined effects of E treatment of postmenopausal women or selective withdrawal of E versus testosterone (T) in elderly men on circulating sclerostin levels. E treatment of postmenopausal women (n = 17) for 4 weeks led to a 27% decrease in serum sclerostin levels [versus +1% in controls (n = 18), p < .001]. Similarly, in 59 elderly men, we eliminated endogenous E and T production and studied them under conditions of physiologic T and E replacement, and then following withdrawal of T or E, we found that E, but not T, prevented increases in sclerostin levels following induction of sex steroid deficiency. In both sexes, changes in sclerostin levels correlated with changes in bone-resorption, but not bone-formation, markers (r = 0.62, p < .001, and r = 0.33, p = .009, for correlations with changes in serum C-terminal telopeptide of type 1 collagen in the women and men, respectively). Our studies thus establish that in humans, circulating sclerostin levels are reduced by E but not by T. Moreover, consistent with recent data indicating important effects of Wnts on osteoclastic cells, our findings suggest that in humans, changes in sclerostin production may contribute to effects of E on bone resorption. © 2011 American Society for Bone and Mineral Research.

Abstract Recent studies have demonstrated an important role for circulating serotonin in regulating bone mass in rodents. In addition, patients treated with selective serotonin reuptake inhibitors (SSRIs) have reduced areal bone mineral density (aBMD). However, the potential physiologic role of serotonin in regulating bone mass in humans remains unclear. Thus we measured serum serotonin levels in a population-based sample of 275 women and related these to total-body and spine aBMD assessed by dual-energy X-ray absorptiometry, femur neck total and trabecular volumetric BMD (vBMD) and vertebral trabecular vBMD assessed by quantitative computed tomography (QCT), and bone microstructural parameters at the distal radius assessed by high-resolution peripheral QCT (HRpQCT). Serotonin levels were inversely associated with body and spine aBMD (age-adjusted R = −0.17 and −0.16, P < .01, respectively) and with femur neck total and trabecular vBMD (age-adjusted R = −0.17 and −0.25, P < .01 and < .001, respectively) but not lumbar spine vBMD. Bone volume/tissue volume, trabecular number, and trabecular thickness at the radius were inversely associated with serotonin levels (age-adjusted R = −0.16, −0.16, and −0.14, P < .05, respectively). Serotonin levels also were inversely associated with body mass index (BMI; age-adjusted R = −0.23, P < .001). Multivariable models showed that serotonin levels remained significant negative predictors of femur neck total and trabecular vBMD, as well as trabecular thickness at the radius, after adjusting for age and BMI. Collectively, our data provide support for a physiologic role for circulating serotonin in regulating bone mass in humans. © 2010 American Society for Bone and Mineral Research

Abstract Introduction: The mechanism(s) by which sex steroids regulate bone turnover in humans are unclear, and recent studies have suggested that follicle-stimulating hormone (FSH) may play an important role in regulating bone resorption. Materials and Methods: Fifty-nine men (median age, 69 yr) underwent suppression of sex steroids using a gonadotropin-releasing hormone (GnRH) agonist and aromatase blocker and were replaced with testosterone (T; 5 mg/d) and estradiol (E; 37.5 μg/d). After assessment of bone resorption markers (serum C-terminal telopeptide of type I collagen [CTX] and TRACP5b), they were randomized to sex steroid deficiency (−T, −E), E alone (−T, +E), T alone (+T, −E), or both (+T, +E) and restudied 3 wk later. Bone marrow aspirates were obtained to isolate osteoblastic, T, and monocytic cells using magnetic-activated cell sorting. Results: Serum CTX and TRACP5b increased significantly (by 71% and 15%, p < 0.01 and < 0.001, respectively) in the −T, −E group, and these increases occurred despite a 60% suppression of serum FSH levels (p < 0.001) caused by the GnRH agonist. There were significant E (but not T) effects on preventing increases in serum CTx and TRACP levels. There was a nonsignificant trend (p = 0.122) for E to suppress RANKL mRNA levels in bone marrow osteoblastic cells. Changes in mRNA levels for other cytokines (TNF-α, interleukin (IL)-1α, IL-1β, IL-1ra, IFN-γ) in bone marrow cells were not significant. Conclusions: E has greater suppressive effects on bone resorption than T, and increased bone resorption after sex steroid deficiency can occur independently of changes in FSH secretion. E effects on bone resorption may be mediated by regulation of RANKL production by osteoblastic cells, although further studies using more highly purified cells may reduce the variability of the mRNA measurements and allow for clearer definition of the mediators of sex steroid action in vivo.

Abstract ERα acts either through classical (ERE-mediated) or nonclassical (non-ERE) pathways. The generation of mice carrying a mutation that eliminates classical ERα signaling presents a unique opportunity to study the relative roles of these pathways in bone. This study defines the skeletal phenotype and responses to ovariectomy and estrogen replacement in these mice. Introduction: Estrogen receptor α (ERα) can act either through classical estrogen response elements (EREs) or through non-ERE (nonclassical) pathways. To unravel these in bone, we crossed mice heterozygous for a knock-in mutation abolishing ERE binding (nonclassical ERα knock-in [NERKI]) with heterozygote ERα knockout mice and studied the resulting female ERα+/+, ERα+/NERKI, and ERα−/NERKI mice. The only ERα present in ERα−/NERKI mice is incapable of activating EREs but can signal through nonclassical pathways, whereas ERα+/NERKI mice may have a less drastic alteration in the balance between classical and nonclassical estrogen signaling pathways. Materials and Methods: BMD was measured using DXA and pQCT at 3 months of age (n = 46–48/genotype). The mice were randomly assigned to sham surgery, ovariectomy, ovariectomy + estradiol (0.25 μg/day), or ovariectomy + estradiol (1.0 μg/day; n = 10–12/group) and restudied 60 days later. Results and Conclusions: At 3 months of age, both the ERα+/NERKI and ERα−/NERKI mice had deficits in cortical, but not in trabecular, bone. Remarkably, changes in cortical bone after ovariectomy and estrogen replacement in ERα−/NERKI mice were the opposite of those in ERα+/+ mice. Relative to sham mice, ovariectomized ERα−/NERKI mice gained more bone (not less, as in ERα+/+ mice), and estrogen suppressed this increase (whereas augmenting it in ERα+/+ mice). Estrogen also had opposite effects on bone formation and resorption parameters on endocortical surfaces in ERα−/NERKI versus ERα+/+ mice. Collectively, these data show that alteration of the balance between classical and nonclassical ERα signaling pathways leads to deficits in cortical bone and also represent the first demonstration, in any tissue, that complete loss of classical ERE signaling can lead to paradoxical responses to estrogen. Our findings strongly support the hypothesis that there exists a balance between classical and nonclassical ERα signaling pathways, which, when altered, can result in a markedly aberrant response to estrogen.