Longitudinal Examination of Bone Loss in Male Rats After Moderate-Severe Contusion Spinal Cord Injury.

To elucidate mechanisms of bone loss after spinal cord injury (SCI), we evaluated the time-course of cancellous and cortical bone microarchitectural deterioration via microcomputed tomography, measured histomorphometric and circulating bone turnover indices, and characterized the development of whole bone mechanical deficits in a clinically relevant experimental SCI model. 16-weeks-old male Sprague-Dawley rats received T9 laminectomy (SHAM, n = 50) or moderate-severe contusion SCI (n = 52). Outcomes were assessed at 2-weeks, 1-month, 2-months, and 3-months post-surgery. SCI produced immediate sublesional paralysis and persistent hindlimb locomotor impairment. Higher circulating tartrate-resistant acid phosphatase 5b (bone resorption marker) and lower osteoblast bone surface and histomorphometric cancellous bone formation indices were present in SCI animals at 2-weeks post-surgery, suggesting uncoupled cancellous bone turnover. Distal femoral and proximal tibial cancellous bone volume, trabecular thickness, and trabecular number were markedly lower after SCI, with the residual cancellous network exhibiting less trabecular connectivity. Periosteal bone formation indices were lower at 2-weeks and 1-month post-SCI, preceding femoral cortical bone loss and the development of bone mechanical deficits at the distal femur and femoral diaphysis. SCI animals also exhibited lower serum testosterone than SHAM, until 2-months post-surgery, and lower serum leptin throughout. Our moderate-severe contusion SCI model displayed rapid cancellous bone deterioration and more gradual cortical bone loss and development of whole bone mechanical deficits, which likely resulted from a temporal uncoupling of bone turnover, similar to the sequalae observed in the motor-complete SCI population. Low testosterone and/or leptin may contribute to the molecular mechanisms underlying bone deterioration after SCI.

Testosterone and Adult Male Bone: Actions Independent of 5α-Reductase and Aromatase.

Androgens and estrogens influence skeletal development and maintenance in males. However, the relative contributions of the circulating sex steroid hormones that originate from testicular/adrenal secretion versus those produced locally in bone via intracrine action require further elucidation. Our novel hypothesis is that testosterone exerts direct protective effects on the adult male skeleton independently of the actions of 5α-reductase or aromatase.

The soluble interleukin-6 receptor is a mediator of hematopoietic and skeletal actions of parathyroid hormone.

Both PTH and IL-6 signaling play pivotal roles in hematopoiesis and skeletal biology, but their interdependence is unclear. The purpose of this study was to evaluate the effect of IL-6 and soluble IL-6 receptor (sIL-6R) on hematopoietic and skeletal actions of PTH. In the bone microenvironment, PTH stimulated sIL-6R protein levels in primary osteoblast cultures in vitro and bone marrow in vivo in both IL-6(+/+) and IL-6(-/-) mice. PTH-mediated hematopoietic cell expansion was attenuated in IL-6(-/-) compared with IL-6(+/+) bone marrow, whereas sIL-6R treatment amplified PTH actions in IL-6(-/-) earlier than IL-6(+/+) marrow cultures. Blocking sIL-6R signaling with sgp130 (soluble glycoprotein 130 receptor) inhibited PTH-dependent hematopoietic cell expansion in IL-6(-/-) marrow. In the skeletal system, although intermittent PTH administration to IL-6(+/+) and IL-6(-/-) mice resulted in similar anabolic actions, blocking sIL-6R significantly attenuated PTH anabolic actions. sIL-6R showed no direct effects on osteoblast proliferation or differentiation in vitro; however, it up-regulated myeloid cell expansion and production of the mesenchymal stem cell recruiting agent, TGF-ß1 in the bone marrow microenvironment. Collectively, sIL-6R demonstrated orphan function and mediated PTH anabolic actions in bone in association with support of myeloid lineage cells in the hematopoietic system.

CXCL12/CXCR4 signaling in the osteoblast regulates the mesenchymal stem cell and osteoclast lineage populations.

The chemokine CXCL12 and its receptor CXCR4 play a key role in regulation of hematopoietic stem cells and cell migratory function during morphogenesis. Osteoblasts express both the ligand and the receptor, but little is known about the role of CXCL12-CXCR4 signaling in maintaining skeletal homeostasis. Using Cre-Lox technology to delete CXCR4 in mature osteoblasts in mice, we show here a significant decrease in bone mass and alterations in cancellous bone structure. CXCR4 gene ablation increased the number of colony-forming units (CFU), CFU-positive for alkaline phosphatase (CFU-AP(+)), and mineralizing nodules in bone marrow stromal cell (BMSC) cultures. The adipocyte precursor population decreased in BMSCs harvested from the KO animals. The nonadherent population of BMSCs harvested from the long bone diaphysis of KO animals formed more osteoclasts, a finding that was associated with increased circulatory levels of pyridinoline, a marker of bone resorption. Our data show that osteoblast-specific CXCR4 deletion has profound effects on the mesenchymal stem cell pool and allocation to the osteoblastic and adipocytic cell lineages. They also show that CXCL12/CXCR4 signaling in the mature osteoblast can feedback to regulate the osteoclast precursor pool size and play a multifunctional role in regulating bone formation and resorption.

Celebrating 50-years: the history and future of the International Society of Bone Morphometry.

The International Society of Bone Morphometry (ISBM) is dedicated to advancing research, education, and clinical practice for osteoporosis and other bone disorders by developing and improving tools for the quantitative imaging and analysis of bone. Its initial core mission was to promote the proper use of morphometric techniques in bone research and to educate and train clinicians and basic scientists in bone morphometry. This article chronicles the evolution of the ISBM and the history and development of bone morphometric techniques for the past 50-years, starting with workshops on bone morphometry in 1973, to the formal incorporation of the ISBM in 1996, to today. We also provide a framework and vision for the coming decades. This effort was led by ISBM presidents Dr Erica L. Scheller (2022-2024) and Dr Thomas J. Wronski (2009-2012) in collaboration with all other living ISBM presidents. Though the underlying techniques and questions have changed over time, the need for standardization of established tools and discovery of novel approaches for bone morphometry remains a constant. The ISBM fulfills this need by providing a forum for the exchange of ideas, with a philosophy that encourages the open discussion of pitfalls and challenges among clinicians, scientists, and industry partners. This facilitates the rapid development and adaptation of tools to meet emerging demands within the field of bone health at a high level.

Enoxacin directly inhibits osteoclastogenesis without inducing apoptosis.

Enoxacin has been identified as a small molecule inhibitor of binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments. It inhibits bone resorption by calcitriol-stimulated mouse marrow cultures. We hypothesized that enoxacin acts directly and specifically on osteoclasts by disrupting the interaction between plasma membrane-directed V-ATPases, which contain the osteoclast-selective a3-subunit of V-ATPase, and microfilaments. Consistent with this hypothesis, enoxacin dose-dependently reduced the number of multinuclear cells expressing tartrate-resistant acid phosphatase (TRAP) activity produced by RANK-L-stimulated osteoclast precursors. Enoxacin (50 µM) did not induce apoptosis as measured by TUNEL and caspase-3 assays. V-ATPases containing the a3-subunit, but not the "housekeeping" a1-subunit, were isolated bound to actin. Treatment with enoxacin reduced the association of V-ATPase subunits with the detergent-insoluble cytoskeleton. Quantitative PCR revealed that enoxacin triggered significant reductions in several osteoclast-selective mRNAs, but levels of various osteoclast proteins were not reduced, as determined by quantitative immunoblots, even when their mRNA levels were reduced. Immunoblots demonstrated that proteolytic processing of TRAP5b and the cytoskeletal protein L-plastin was altered in cells treated with 50 µM enoxacin. Flow cytometry revealed that enoxacin treatment favored the expression of high levels of DC-STAMP on the surface of osteoclasts. Our data show that enoxacin directly inhibits osteoclast formation without affecting cell viability by a novel mechanism that involves changes in posttranslational processing and trafficking of several proteins with known roles in osteoclast function. We propose that these effects are downstream to blocking the binding interaction between a3-containing V-ATPases and microfilaments.

17ß-Hydroxyestra-4,9,11-trien-3-one (trenbolone) exhibits tissue selective anabolic activity: effects on muscle, bone, adiposity, hemoglobin, and prostate.

Selective androgen receptor modulators (SARMs) now under development can protect against muscle and bone loss without causing prostate growth or polycythemia. 17ß-Hydroxyestra-4,9,11-trien-3-one (trenbolone), a potent testosterone analog, may have SARM-like actions because, unlike testosterone, trenbolone does not undergo tissue-specific 5α-reduction to form more potent androgens. We tested the hypothesis that trenbolone-enanthate (TREN) might prevent orchiectomy-induced losses in muscle and bone and visceral fat accumulation without increasing prostate mass or resulting in adverse hemoglobin elevations. Male F344 rats aged 3 mo underwent orchiectomy or remained intact and were administered graded doses of TREN, supraphysiological testosterone-enanthate, or vehicle for 29 days. In both intact and orchiectomized animals, all TREN doses and supraphysiological testosterone-enanthate augmented androgen-sensitive levator ani/bulbocavernosus muscle mass by 35-40% above shams (P ≤ 0.001) and produced a dose-dependent partial protection against orchiectomy-induced total and trabecular bone mineral density losses (P < 0.05) and visceral fat accumulation (P < 0.05). The lowest doses of TREN successfully maintained prostate mass and hemoglobin concentrations at sham levels in both intact and orchiectomized animals, whereas supraphysiological testosterone-enanthate and high-dose TREN elevated prostate mass by 84 and 68%, respectively (P < 0.01). In summary, low-dose administration of the non-5α-reducible androgen TREN maintains prostate mass and hemoglobin concentrations near the level of shams while producing potent myotrophic actions in skeletal muscle and partial protection against orchiectomy-induced bone loss and visceral fat accumulation. Our findings indicate that TREN has advantages over supraphysiological testosterone and supports the need for future preclinical studies examining the viability of TREN as an option for androgen replacement therapy.

The beta-glucuronidase intracisternal A particle insertion model results in similar overall MPSVII phenotype as the single base deletion model when on the same C57BL/6J mouse background.

Two unique gene mutations in the enzyme beta-glucuronidase (GUSB) that result in the lysosomal storage disease Mucopolysaccharidosis (MPS) type VII had previously been reported to have differing disease phenotype severities when compared on differing mouse strains. The MPSVII mouse has proven to be a highly efficacious model to study mucopolysaccharidoses and for evaluating potential gene or stem cell therapies for lysosomal storage diseases. We examined the single base pair deletion (MPSVII) and the intracisternal A particle element insertion (MPSVII2J) in GUSB compared with control animals by skeletal measures, electroretinography, auditory-evoked brainstem response and life span on a C57BL/6J background strain. In all measures, both mutations result in either a trend toward or significant changes from the background strain control. In all measures, there is no significant phenotypic difference between the two mutations. The 2J variant is a more easily genotyped and equally affected phenotype, which holds promise for further studies of chimerism and stem cell therapy approaches.

Methods to quantify sex steroid hormones in bone: applications to the study of androgen ablation and administration.

Bone may contain an intraskeletal reservoir of sex steroids that is capable of producing biological effects. The purposes of these experiments were to 1) establish and validate methods to extract and measure intraskeletal sex hormones, 2) compare serum and intraskeletal sex hormone abundance, and 3) determine the impact of testosterone-enanthate administration and orchiectomy on intraskeletal sex hormone concentrations. Tibiae from male F344 rats were crushed, suspended in an aqueous buffer, disrupted mechanically and sonically, extracted with organic solvents, dried, and reconstituted in assay buffer appropriate for measurement of testosterone, dihydrotestosterone, and estradiol by immunoassay. Prior to extraction, bone homogenate was spiked with [³H]testosterone, [³H]dihydrotestosterone, or [³H]estradiol, and >80% of each ³H-labeled sex hormone was recovered. Extracted bone samples were also assayed with and without known amounts of unlabeled sex hormones, and >97% of the expected hormone concentrations were measured. Administration of testosterone-enanthate increased intraskeletal testosterone 11-fold and intraskeletal dihydrotestosterone by 82% without altering intraskeletal estradiol (P < 0.01). Conversely, orchiectomy did not alter intraskeletal testosterone or estradiol but increased intraskeletal dihydrotestosterone by 39% (P < 0.05). In intact rats, intraskeletal testosterone and dihydrotestosterone were directionally higher than in serum, whereas intraskeletal estradiol was directionally lower than serum. Serum androgens were positively correlated with intraskeletal androgens (r = 0.74-0.96, P < 0.001); however, neither serum nor intraskeletal androgens nor serum estradiol were correlated with intraskeletal estradiol. We report the validation of a novel method for measuring intraskeletal sex hormones. Our findings demonstrate that the intraskeletal sex steroid reservoirs are modifiable and only partially influenced by circulating sex hormones.

Dietary dried plum increases bone mass in adult and aged male mice.

Bone is progressively lost with advancing age. Therapies are limited and the only effective proanabolic regimen presently available to restore bone is intermittent treatment with teriparatide (parathyroid hormone 1-34). Recent evidence suggests that dietary supplementation with dried plum (DP) can prevent bone loss due to estrogen deficiency. To determine whether dietary DP supplementation can prevent the loss of bone with aging and whether bone that has already been lost can be restored, adult (6 mo) and old (18 mo) male mice were fed a normal diet or isoenergetic, isonitrogenous diets supplemented with DP (0, 15, and 25% DP by weight) for 6 mo. MicroCT analysis and bone histomorphometry were used to assess bone volume, structure, and metabolic activity before, during, and after dietary supplementation. Mice fed the 0% DP diet (control diet) lost bone, whereas both adult and old mice fed the 25% DP-supplemented diet gained bone. Adult but not old mice fed the 15% diet also gained bone. Cancellous bone volume in mice receiving 25% DP exceeded baseline levels by 40-50%. Trabecular structure varied with diet and age and responses in old mice were generally blunted. Trabecular, but not cortical, mineral density varied with age and measures of bone anabolic activity were lower in aged mice. Our findings suggest that DP contains proanabolic factors that can dramatically increase bone volume and restore bone that has already been lost due to aging. In turn, DP may provide effective prophylactic and therapeutic agents for the treatment of osteoporosis.

Murine model for cystic fibrosis bone disease demonstrates osteopenia and sex-related differences in bone formation.

As the incidence of cystic fibrosis (CF) bone disease is increasing, we analyzed CF transmembrane conductance regulator (CFTR) deficient mice (CF mice) to gain pathogenic insights. In these studies comparing adult (14 wk) CF and C57BL/6J mice, both bone length and total area were decreased in CF mice. Metaphyseal trabecular and cortical density were also decreased, as well as diaphyseal cortical and total density. Trabecular bone volume was diminished in CF mice. Female CF mice revealed decreased trabecular width and number compared with C57BL/6J, whereas males demonstrated no difference in trabecular number. Female CF mice had reduced mineralizing surface and bone formation rates. Conversely, male CF mice had increased mineralizing surface, mineral apposition, and bone formation rates compared with C57BL/6J males. Bone formation rate was greater in males compared with female CF mice. Smaller bones with decreased density in CF, despite absent differences in osteoblast and osteoclast surfaces, suggest CF transmembrane conductance regulator influences bone cell activity rather than number. Differences in bone formation rate in CF mice are suggestive of inadequate bone formation in females but increased bone formation in males. This proanabolic observation in male CF mice is consistent with other clinical sex differences in CF.

Role for beta1 integrins in cortical osteocytes during acute musculoskeletal disuse.

The mammalian skeleton adjusts bone structure and strength in response to changes in mechanical loading, however the molecular and cellular mechanisms governing this process in vivo are unknown. Terminally differentiated osteoblasts, the osteocytes, are presumptive mechanosensory cells for bone, and cell culture studies demonstrate that beta1 integrins participate in mechanical signaling. To determine the role of beta1 integrins in osteoblasts in vivo, we used the Cre-lox system to delete beta1 integrin from cells committed to the osteoblast lineage. While pCol2.3 Cre-mediated recombination was widespread in bones from Colalpha1(I)-Cre+/beta1fl/fl conditional knockout mice (cKO), beta1 integrin protein was depleted from cortical osteocytes, but not from cancellous osteocytes or cells lining bone surfaces in adults. Bones from cKO mice that were normally loaded were similar in structure to WT littermates. However, hindlimb unloading of adult cKO mice for one week intended to cause bone loss (disuse osteopenia), resulted in unexpected, rapid changes in the geometry of cortical bone; hindlimb unloading increased the cross-sectional area, marrow area, and moments of inertia in cKO, but not WT mice. Furthermore, these hindlimb unloading-induced geometric changes in cortical bone of cKO mice resulted in increased whole bone bending stiffness and strength of the femur. Together, these results confirmed the concept that osteocytes are mechanosensory cells and showed beta1 integrins in cortical osteocytes limited changes in cortical geometry in response to disuse, thus providing the first in vivo evidence that beta1 integrins on osteocytes mediate specific aspects of mechanotransduction.

Histological analysis of bone.

Bone is an important target tissue for alcohol. Moderate alcohol consumption may slow bone loss during aging, but alcohol consumption inhibits bone growth during adolescence, and alcohol abuse in adults is an important risk factor for osteoporosis. Various techniques have been applied for evaluating the impact of alcohol on bone, including densitometry for assessment of bone mass and density, computed tomography for evaluation of bone microarchitecture, serum biochemistry for measurement of markers of global bone resorption and formation, and histomorphometry for assessment of cellular activity. Of these methods, histomorphometry is the gold standard for assessing bone because it is the only method for the direct in situ analysis of bone cells and their activities. The procedures described in this chapter provide tools for the histomorphometric characterization of the effects of alcohol on cancellous and cortical bone growth and turnover. Specifically detailed are processes for embedding, cutting, staining, and evaluating histological bone specimens with a focus on rodent models.

Sclerostin Antibody Reverses Bone Loss by Increasing Bone Formation and Decreasing Bone Resorption in a Rat Model of Male Osteoporosis.

Sclerostin antibody (Scl-Ab) restored bone mass and strength in the ovariectomized rat model of postmenopausal osteoporosis. Increased bone mineral density (BMD) and decreased skeletal fragility fracture risk have been reported in postmenopausal osteoporotic women receiving Scl-Ab. In males, loss of androgen leads to rapid decreases in BMD and an increased risk of fragility fractures. We hypothesized that Scl-Ab could reverse the loss of bone mass and strength caused by androgen ablation in the orchiectomized (ORX) rat model of male osteoporosis. We treated 9-month-old ORX Sprague Dawley rats (3 months after ORX) subcutaneously twice weekly with vehicle or Scl-Ab (5 or 25 mg/kg) for 6 weeks (n = 10 per group). Both doses of Scl-Ab fully reversed the BMD deficit in the lumbar spine and femur and tibia in ORX rats. Microcomputed tomography showed that the bone mass in the fifth lumbar vertebral body, femur diaphysis, and femoral neck were dose-dependently restored by Scl-Ab. The bone strength at these sites increased significantly with Scl-Ab to levels matching those of sham-operated controls and correlated positively with improvements in bone mineral content, demonstrating bone quality maintenance. Dynamic histomorphometry of the tibial diaphysis and second lumbar vertebral body demonstrated that Scl-Ab significantly increased bone formation on periosteal, endocortical, and trabecular surfaces and significantly decreased bone resorption on endocortical and trabecular surfaces. The effects of Scl-Ab on increasing bone formation and decreasing bone resorption led to restoration of bone mass and strength in androgen-deficient rats. These findings support the ongoing evaluation of Scl-Ab as a potential therapeutic agent for osteoporosis in men.

PTH stimulates bone formation in mice deficient in Lrp5.

UNLABELLED: Lrp5 deficiency decreases bone formation and results in low bone mass. This study evaluated the bone anabolic response to intermittent PTH treatment in Lrp5-deficient mice. Our results indicate that Lrp5 is not essential for the stimulatory effect of PTH on cancellous and cortical bone formation. INTRODUCTION: Low-density lipoprotein receptor-related protein 5 (Lrp5), a co-receptor in canonical Wnt signaling, increases osteoblast proliferation, differentiation, and function. The purpose of this study was to use Lrp5-deficient mice to evaluate the potential role of this gene in mediating the bone anabolic effects of PTH. MATERIALS AND METHODS: Adult wildtype (WT, 23 male and 25 female) and Lrp5 knockout (KO, 27 male and 26 female) mice were treated subcutaneously with either vehicle or 80 microg/kg human PTH(1-34) on alternate days for 6 weeks. Femoral BMC and BMD were determined using DXA. Lumbar vertebrae were processed for quantitative bone histomorphometry. Bone architecture was evaluated by microCT. Data were analyzed using a multiway ANOVA. RESULTS: Cancellous and cortical bone mass were decreased with Lrp5 deficiency. Compared with WT mice, cancellous bone volume in the distal femur and the lumbar vertebra in Lrp5 KO mice was 54% and 38% lower, respectively (p<0.0001), whereas femoral cortical thickness was 11% lower in the KO mice (p<0.0001). The decrease in cancellous bone volume in the lumbar vertebrae was associated with a 45% decrease in osteoblast surface (p<0.0001) and a comparable decrease in bone formation rate (p<0.0001). Osteoclast surface, an index of bone resorption, was 24% lower in Lrp5 KO compared with WT mice (p<0.007). Treatment of mice with PTH for 6 weeks resulted in a 59% increase in osteoblast surface (p<0.0001) and a 19% increase in osteoclast surface (p=0.053) in both genotypes, but did not augment cancellous bone volume in either genotype. Femur cortical thickness was 11% higher in PTH-treated mice in comparison with vehicle-treated mice (p<0.0001), regardless of genotype. CONCLUSIONS: Whereas disruption of Lrp5 results in decreased bone mass because of decreased bone formation, Lrp5 does not seem to be essential for the stimulatory effects of PTH on cancellous and cortical bone formation.

Effects of basic fibroblast growth factor and a prostaglandin E2 receptor subtype 4 agonist on osteoblastogenesis and adipogenesis in aged ovariectomized rats.

UNLABELLED: bFGF stimulates osteo- and adipogenesis concurrently at skeletal sites with red but not with fatty marrow, whereas a PGE2 receptor subtype 4 agonist has bone anabolic effects at both skeletal sites and decreases adipose tissue within red and fatty marrow. INTRODUCTION: Basic fibroblast growth factor (bFGF) stimulates osteogenesis at skeletal sites with hematopoietic but not with fatty marrow. The prostaglandin E2 (PGE2) receptor subtype 4 agonist (EP4A) stimulates osteogenesis at the former skeletal sites, but its effects at fatty marrow sites are unknown. In addition, both bFGF and PGE2 through the EP4 receptor have also been implicated in adipogenesis. However, their specific effects on bone marrow adipogenesis and the inter-relationship with osteogenesis have never been studied in vivo. MATERIALS AND METHODS: Female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated and maintained for 1 yr after surgery. OVX rats were then injected daily with bFGF or with EP4A SC for 3 wk. The osteo- and adipogenic effects of these agents were assessed by histomorphometry and by determining changes in expression of genes associated with these events by real-time PCR in the lumbar and caudal vertebrae, bones with a predominance of hematopoietic and fatty marrow, respectively. Expression of FGFR1-4 and the EP4 receptor were also evaluated by real-time PCR and immunocytochemistry. RESULTS: bFGF and EP4A stimulated bone formation at skeletal sites with hematopoietic marrow, but only the later anabolic agent is also effective at fatty marrow sites. The diminished bone anabolic effect of bFGF at the fatty marrow site was not caused by a lack of cell surface receptors for the growth factor at this site. Interestingly, whereas EP4A decreased fatty marrow area and the number of adipocytes, bFGF increased osteogenesis and adipogenesis within the bone marrow. CONCLUSIONS: bFGF can stimulate osteogenesis and bone marrow adipogenesis concurrently at red marrow sites, but not at fatty marrow sites. In contrast, EP4A stimulates bone formation at skeletal sites with hematopoietic and fatty marrow and simultaneously decreased fatty marrow area and the number of adipocytes in the bone marrow, suggesting that osteogenesis occurs at the expense of adipogenesis.

Treatment With a Soluble Bone Morphogenetic Protein Type 1A Receptor (BMPR1A) Fusion Protein Increases Bone Mass and Bone Formation in Mice Subjected to Hindlimb Unloading.

Previous work has shown that the soluble murine BMPR1A-fusion protein (mBMPR1A-mFc) binds to BMP2 and BMP4 with high affinity, preventing downstream signaling. Further, treatment of intact and ovariectomized mice with mBMPR1A-mFc leads to increased bone mass, and improved bone microarchitecture and strength, via increased bone formation and reduced resorption. In this study, we tested the effects of mBMPR1A-mFc on disuse-induced bone loss caused by 21 days of hindlimb unloading (HLU) via tail suspension versus cage controls (CONs). Adult female C57BL/6J mice (12 weeks old) were assigned to one of four groups (n = 10 each): CON-VEH; CON-mBMPR1A-mFc; HLU-VEH; and HLU-mBMPR1A-mFc. Mice were injected subcutaneously with VEH or mBMPR1A-mFc (4.5 mg/kg, 2×/week). Leg BMD declined in the HLU-VEH group (-5.3% ± 1.3%), whereas it was unchanged in HLU-mBMPR1A-mFc (-0.3% ± 0.9%, p < 0.05 versus HLU-VEH). Leg BMD increased significantly more in CON-mBMPR1A-mFc than CON-VEH (10.2% ± 0.6% versus 4.4% ± 0.8%). In the femur, trabecular, and cortical bone microarchitecture was worse in the HLU-VEH compared to CON-VEH mice, whereas mBMPR1A-mFc treatment for 3 weeks led to greater Tb.BV/TV, Tb.Th, and midshaft Ct.Th in both the HLU and CON groups compared to comparable VEH-treated counterparts (p < 0.05). HLU-mBMPR1A-mFc mice also had 21% greater failure load (p < 0.05) compared to their VEH-treated counterparts. Dynamic histomorphometry indicated that treatment with mBMPR1A-mFc led to significantly greater mineralizing surface and mineral apposition rate, resulting in a 3.5-fold and fivefold higher bone formation rate in the mBMPR1A-mFc-treated CON and HLU animals versus VEH groups, respectively. mBMPR1A-mFc-treated mice had a similar osteoblast surface but significantly lower osteoclast surface than VEH-treated animals in both the CON and HLU groups. Altogether, these findings suggest that treatment with the soluble BMPR1A fusion protein may be useful for maintenance of skeletal integrity in the setting of disuse-induced bone loss.

Strain-dependent variations in the response of cancellous bone to ovariectomy in mice.

UNLABELLED: The goal of this study was to characterize the skeletal response to ovariectomy in mice (129P3, C57BL/6, and B6129PF2) commonly used in gene manipulation studies to evaluate their potential as preclinical models of postmenopausal osteoporosis. The magnitude of cancellous bone loss and cellular indices of increased bone turnover in response to ovariectomy varied with mouse type and skeletal site, but in general, were less pronounced and less consistent than in Sprague-Dawley rats, the established preclinical model for postmenopausal bone loss. INTRODUCTION: The ovariectomized (OVX) rat is the most widely used preclinical rodent model for postmenopausal osteoporosis. However, the underlying mechanisms of bone disorders, including osteoporosis, have been explored predominantly in the mouse. The purpose of this study was to evaluate mice (129P3 and C57BL/6 inbred strains and their F2 hybrid offspring, B6129PF2), commonly used for gene knockout and overexpression studies, for their potential as preclinical models of postmenopausal bone loss. MATERIALS AND METHODS: The mice were OVX or sham-operated at 4 months of age and killed at 1 or 3 months after surgery. Lumbar vertebrae and distal femora were subjected to histomorphometric assessment. RESULTS: Mice in the two strains and the F2 hybrids (will be referred to as strain for the remainder of the abstract) lost vertebral cancellous bone after OVX; bone volume (BV/TV) was 20% and 27% lower at 1 and 3 months after surgery, respectively. The decreased cancellous BV/TV was associated with an increase in osteoclast surface at 1 month after OVX in the 129P3 strain only. Osteoblast surface was increased by 20% with OVX at both 1 and 3 months after surgery, irrespective of mouse strain. However, bone formation rate was not altered by OVX in any of the mouse strains. In contrast to the lumbar vertebrae, cancellous bone loss in response to OVX differed in the distal femur among the three mouse strains. OVX had no significant effect on distal femur BV/TV in the B6129PF2 mouse strain. In the C57BL/6 strain, cancellous BV/TV was reduced by OVX at 1 month after surgery but not at 3 months after surgery, whereas distal femur BV/TV in 129P3 mice was reduced at 3 months after surgery. Osteoclast surface was not affected by OVX at either time-point in the C57BL/6 strain, but was increased by 116% at 1 month after surgery in the 129P3 strain. Osteoblast surface was increased with OVX at 1 month after surgery, irrespective of strain, whereas bone formation rate was not altered by OVX at either time-point in any of the strains. CONCLUSIONS: The magnitude of cancellous bone loss and cellular indices of increased bone turnover in response to OVX varied with mouse strain and skeletal site, but in general, were less pronounced and less consistent than in the Sprague-Dawley rat. Although mouse models will continue to provide insights into genetic influences on bone mass and turnover, caution should be exercised when using 129P3 and C57BL/6 mice, and their F2 hybrids, as models for postmenopausal bone loss and preclinical testing of potential therapies for osteoporosis.

Sequential treatment of ovariectomized mice with bFGF and risedronate restored trabecular bone microarchitecture and mineralization.

Basic fibroblast growth factor (bFGF), a potent mitogen, has been found to restore trabecular bone mass and connectivity in osteopenic rats. The purpose of this study was to determine how sequential treatment of ovariectomized (OVXed) mice with bFGF followed by risedronate would restore trabecular microarchitecture and improve bone strength through alterations in bone mineralization. Six-month old female Swiss-Webster mice were OVXed or sham-operated and left untreated for 4 weeks to develop osteopenia. At week 5, a group of Sham and OVXed mice were treated with vehicle, and 3 other groups of OVXed mice were treated with bFGF (1 mg/kg daily, s.c., 5x/week) for 3 weeks. At week 8, one group of bFGF-treated mice was sacrificed and the other two bFGF-treated groups were treated with vehicle or risedronate (Ris, 5 microg/kg, s.c., 3x/week) for an additional 6 weeks. Study endpoints included trabecular microarchitecture by microCT, histomorphometry, bone turnover, degree of bone mineralization (DBM), and whole bone strength for the lumbar vertebral body. Compared to sham-operated animals, OVXed mice had significant reductions in trabecular bone volume, connectivity density, DBM, and bone biomechanical properties (P < 0.05). Treatment with bFGF resulted in higher trabecular bone structure and bone strength compared to pre-treatment sham control (P < 0.05). Treatment of OVXed mice with bFGF for 3 weeks followed by 6 weeks Ris maintained the trabecular microarchitecture gained by bFGF treatment, and DBM and bone strength were restored to baseline control levels. Also compared to Sham-operated animals, serum TGF-beta1 was transiently increased after OVX, increased an additional 100% after bFGF withdrawal, and decreased by 30% with risedronate. In addition, DBM was the strongest predictor for bone biomechanical properties (R2 > 0.7, P < 0.001). Serum TGF-beta1 correlated with bone turnover (DPD/Cr, osteocalcin) and was negatively correlated to DBM. Thus, in osteopenic mice, sequential treatment with bFGF followed by risedronate increased trabecular bone microarchitecture, DBM, and bone strength. In addition, suppression of the serum TGF-beta1 with risedronate was associated with increased DBM. Therefore, sequential treatment with bFGF and Ris restores trabecular architecture and allows mineralization of bone to increase, which appears to be beneficial to bone strength.

Dietary dried plum increases bone mass, suppresses proinflammatory cytokines and promotes attainment of peak bone mass in male mice.

Nutrition is an important determinant of bone health and attainment of peak bone mass. Diets containing dried plum (DP) have been shown to increase bone volume and strength. These effects may be linked to the immune system and DP-specific polyphenols. To better understand these relationships, we studied DP in skeletally mature (6-month-old) and growing (1- and 2-month-old) C57Bl/6 male mice. In adult mice, DP rapidly (<2 weeks) increased bone volume (+32%) and trabecular thickness (+24%). These changes were associated with decreased osteoclast surface (Oc.S/BS) and decreased serum CTX, a marker of bone resorption. The reduction in Oc.S/BS was associated with a reduction in the osteoclast precursor pool. Osteoblast surface (Ob.S/BS) and bone formation rate were also decreased suggesting that the gain in bone in adult mice is a consequence of diminished bone resorption and formation, but resorption is reduced more than formation. The effects of DP on bone were accompanied by a decline in interleukins, TNF and MCP-1, suggesting that DP is acting in part through the immune system to suppress inflammatory activity and reduce the size of the osteoclast precursor pool. Feeding DP was accompanied by an increase in plasma phenolics, some of which have been shown to stimulate bone accrual. In growing and young adult mice DP at levels as low as 5% of diet (w/w) increased bone volume. At higher levels (DP 25%), bone volume was increased by as much as 94%. These data demonstrate that DP feeding dramatically increases peak bone mass during growth.