Unloading-induced Bone Loss Research Articles

Benign paroxysmal positional vertigo as a complication of 90-day head-down bed rest.

This study aimed to report the occurrence of benign paroxysmal positional vertigo (BPPV) in a 90-day head-down bed rest experiment and evaluate the potential relationship between BPPV-related seizures and bone metabolic changes. Five cases of lateral semicircular canal (LSC) BPPV were diagnosed during a 90-day head-down bed rest experiment. Five age-matched subjects who participated in this experiment and never felt dizziness or vertigo were assigned as controls. The differences between the BPPV and the controls in lumbar bone mineral density, 25-hydroxyvitamin D level, corrected serum calcium, potassium, sodium, phosphorus, iron, uric acid and N-terminal osteocalcin were analyzed to determine the cause of LSC-BPPV. BPPV occurred from Day 17 to Day 42 during head-down bed rest. The occurrences of BPPV were related to low 25-hydroxyvitamin D level (BPPV:20.70 ± 1.95ng/L vs. control: 30.59 ± 2.75ng/L at Day 30 during HDBR, p < 0.05). The relatively longer duration in the prone posture at 6° head down in this experiment may have a potential role in the involvement of the LSC. The maneuver used in the experiment effectively alleviated the acute symptoms of LSC-BPPV. The cases of LSC-BPPV in the early period of 90-day of head-down bed rest were related to the low 25-hydroxyvitamin D level and the 6° head-down posture. These results suggest that the potential role of unloading-induced bone loss on BPPV-related seizures deserves attention in future studies of long-term bed rest.

SUN-335 Abaloparatide Prevents Unloading-Induced Bone Loss in Adult Rats

Disuse osteoporosis (bone loss resulted from a reduction in mechanical loading) occurs in patients due to prolonged bed rest, paralysis and application of braces. Abaloparatide (ABL) is a synthetic peptide analog of PTHrP that has been shown to promote bone formation with limited bone resorption. ABL was approved by the FDA in 2017 to treat osteoporosis in postmenopausal women at high fracture risk. Yet, the ability of ABL to prevent bone loss in disuse is unknown. We hypothesized that ABL would prevent bone loss in the hindlimb unloading (HLU) rat model of disuse osteoporosis.Adult male Wistar rats, 13–14 weeks of age, were assigned to 1 of 4 groups (10 rats/group): ambulatory + vehicle (CON-VEH), ambulatory + ABL (CON-ABL), HLU + vehicle (HLU-VEH) or HLU + ABL (HLU-ABL). The rats received a daily subcutaneous injection of ABL (25µg/kg/day) or vehicle for 28 days. Blood serum was collected on day 0, 7, 14 and 28 to examine the expression of bone markers such as osteocalcin (OCN) and TRAcP5b. pQCT scans were acquired at the proximal tibia at day 0 and 28 to measure changes in the total and trabecular vBMD. Following euthanasia, trabecular (Tb) and cortical (Ct) bone microarchitecture from femurs, tibias and L4 vertebrae were assessed using µCT. Femurs were mechanically tested to failure in 3-point bending to determine ultimate load (N) and stiffness (N/mm). Treatment effects were evaluated using 2-way ANOVA. Effects were considered significant at p < 0.05. Data reported as mean±SD.HLU led to loss of bone density and structure that were prevented by ABL. Longitudinal pQCT revealed significant increases in total vBMD in ABL-CON (52±17%) vs. VEH-CON (20±5%); and in HLU-ABL (24±6%) vs. HLU-VEH (-2±3%) (p<0.001 for both). Significant differences were observed in the µCT analysis of the distal femur: Tb.BV/TV, thickness and BMD were 43.7%, 12.9% and 27.4% lower, respectively, in HLU-VEH compared to CON-VEH (p<0.05 for all). ABL prevented these negative effects, such that Tb.BV/TV, thickness and BMD were 66.5%, 39% and 50.3% higher in HLU-ABL compared to HLU-VEH (p<0.01 for all). A positive impact of ABL on bone morphology was also seen in the CON-VEH rats. CON-ABL had greater femoral stiffness (+22.9%, p=0.03) and ultimate load (+20.5%, p=0.01) than CON-VEH. Vertebral and tibial trabecular parameters mimicked the distal femur parameters. Serum TRAcP5b did not differ among groups, yet both ABL groups had higher OCN levels than the VEH-treated control groups (+63%, p<0.05).We demonstrated positive effects of ABL on BMD, trabecular bone mass and structure in both ambulating and unloaded rats. These results are consistent with prior studies showing positive effects of ABL on bone mass, structure and strength in OVX and ORX rats. Limits include only male rats and 1 dose of ABL. However, the results observed in this study provide a strong rationale for investigating the ability of ABL to prevent disuse bone loss in humans.

Bone-targeted lncRNA OGRU alleviates unloading-induced bone loss via miR-320-3p/Hoxa10 axis

Unloading-induced bone loss is a threat to human health and can eventually result in osteoporotic fractures. Although the underlying molecular mechanism of unloading-induced bone loss has been broadly elucidated, the pathophysiological role of long noncoding RNAs (lncRNAs) in this process is unknown. Here, we identified a novel lncRNA, OGRU, a 1816-nucleotide transcript with significantly decreased levels in bone specimens from hindlimb-unloaded mice and in MC3T3-E1 cells under clinorotation-unloading conditions. OGRU overexpression promoted osteoblast activity and matrix mineralization under normal loading conditions, and attenuated the suppression of MC3T3-E1 cell differentiation induced by clinorotation unloading. Furthermore, this study found that supplementation of pcDNA3.1(+)–OGRU via (DSS)6–liposome delivery to the bone-formation surfaces of hindlimb-unloaded (HLU) mice partially alleviated unloading-induced bone loss. Mechanistic investigations demonstrated that OGRU functions as a competing endogenous RNA (ceRNA) to facilitate the protein expression of Hoxa10 by competitively binding miR-320-3p and subsequently promote osteoblast differentiation and bone formation. Taken together, the results of our study provide the first clarification of the role of lncRNA OGRU in unloading-induced bone loss through the miR-320-3p/Hoxa10 axis, suggesting an efficient anabolic strategy for osteoporosis treatment.

Disorder of Iron Metabolism Inhibits the Recovery of Unloading-Induced Bone Loss in Hypomagnetic Field

Exposure of humans and animals to microgravity in spaceflight results in various deleterious effects on bone health. In addition to microgravity, the hypomagnetic field (HyMF) is also an extreme environment in space, such as on the Moon and Mars; magnetic intensity is far weaker than the geomagnetic field (GMF) on Earth. Recently, we showed that HyMF promoted additional bone loss in hindlimb unloading-induced bone loss, and the underlying mechanism probably involved an increase of body iron storage. Numerous studies have indicated that bone loss induced by mechanical unloading can be largely restored after skeletal reloading in GMF conditions. However, it is unknown whether this bone deficit can return to a healthy state under HyMF condition. Therefore, the purpose of this study is to examine the effects of HyMF on the recovery of microgravity-induced bone loss, and illustrates the changes of body iron storage in this process. Our results showed that there was lower bone mineral content (BMC) in the HyMF reloading group compared to the GMF reloading group. Reloaded mice in the HyMF condition had a worse microstructure of femur than in the GMF condition. Femoral mechanical properties, including elastic modulus, stiffness, and ultimate stress, were poorer and toughness was higher in the HyMF group compared with the GMF group. Simultaneously, more iron content in serum, the tibia, liver, and spleen was found under HyMF reloading than GMF reloading. The iron chelator deferoxamine mesylate (DFO) decreased the iron content in the bone, liver, and spleen, and significantly relieved unloading-induced bone loss under HyMF reloading. These results showed that HyMF inhibits the recovery of microgravity-induced bone loss, probably by suppressing the elevated iron levels' return to physiological level. © 2019 American Society for Bone and Mineral Research.

Focal adhesion proteins Pinch1 and Pinch2 regulate bone homeostasis in mice.

Mammalian focal adhesion proteins Pinch1 and Pinch2 regulate integrin activation and cell-extracellular matrix adhesion and migration. Here, we show that deleting Pinch1 in osteocytes and mature osteoblasts using the 10-kb mouse Dmp1-Cre and Pinch2 globally (double KO; dKO) results in severe osteopenia throughout life, while ablating either gene does not cause bone loss, suggesting a functional redundancy of both factors in bone. Pinch deletion in osteocytes and mature osteoblasts generates signals that inhibit osteoblast and bone formation. Pinch-deficient osteocytes and conditioned media from dKO bone slice cultures contain abundant sclerostin protein and potently suppress osteoblast differentiation in primary BM stromal cells (BMSC) and calvarial cultures. Pinch deletion increases adiposity in the BM cavity. Primary dKO BMSC cultures display decreased osteoblastic but enhanced adipogenic, differentiation capacity. Pinch loss decreases expression of integrin β3, integrin-linked kinase (ILK), and α-parvin and increases that of active caspase-3 and -8 in osteocytes. Pinch loss increases osteocyte apoptosis in vitro and in bone. Pinch loss upregulates expression of both Rankl and Opg in the cortical bone and does not increase osteoclast formation and bone resorption. Finally, Pinch ablation exacerbates hindlimb unloading-induced bone loss and impairs active ulna loading-stimulated bone formation. Thus, we establish a critical role of Pinch in control of bone homeostasis.

Iron overload involved in the enhancement of unloading-induced bone loss by hypomagnetic field.

During deep-space exploration missions, astronauts will be exposed to abnormal space environments including microgravity and hypomagnetic field (HyMF) that is 10,000 times weaker than geomagnetic field (GMF). It is well known that microgravity in space can induce bone loss; however, it is ill-defined whether HyMF involved in this process. Herein, we aimed to investigate the combined effects of HyMF and microgravity on bone loss. A mouse model of hindlimb suspension (HLU) was adopted to simulate microgravity-induced bone loss, that was exposed to a hypomagnetic field of <300 nanotesla (nT) generated by a geomagnetic field-shielding chamber. Besides, a recent study showed that HLU induced bone loss was orchestrated by iron overload. Therefore, the changes of iron content in unloading-induced bone loss under HyMF condition were detected simultaneously. The results showed HyMF exacerbated the loss of bone mineral content (BMC), induced more detrimental effects on microstructure of cancellous bone but not cortical bone and yielded greater negative effects on biomechanical characteristics in mice femur under unloading status. Concomitantly, there was more iron accumulation in serum, liver, spleen and bone in the combined treatment group than in the separate unloading group or HyMF exposure group. These results showed that HyMF promoted additional bone loss in mice femur during mechanical unloading, and the potential mechanism may be involved in inducing iron overload of mice.

Autophagy activation facilitates mechanical stimulation-promoted osteoblast differentiation and ameliorates hindlimb unloading-induced bone loss

Autophagy has been indicated to be involved in regulating bone metabolism. However, little is known about the role of autophagy in mechanical stimulation-influenced osteoblast differentiation and bone formation. In the present study, we first demonstrated that autophagy activation was essential for cyclic mechanical stretching-promoted osteoblast differentiation of bone marrow mesenchymal stem cells. To explore the in vivo role of autophagy in osteoblast differentiation, the hindlimb unloading-induced disuse osteoporosis model was used. Compared to the normal controls, hindlimb unloading led to abundant bone loss as well as lessened autophagy activation of osteoblasts. However, the activation of autophagy by ULK1 overexpression or in the presence of rapamycin significantly increased osteoblast differentiation activity and restored the bone volume. The findings implicate autophagy as a novel mechanosensitive pathway that regulates osteoblast differentiation. The pharmacological activation of autophagy may be an interesting approach for the prevention and treatment of disuse osteoporosis.

Differences in bone structure and unloading-induced bone loss between C57BL/6N and C57BL/6J mice.

The C57BL/6 mouse, the most frequently utilized animal model in biomedical research, is in use as several substrains, all of which differ by a small array of genomic differences. Two of these substrains, C57BL/6J (B6J) and C57BL/6N (B6N), are commonly used but it is unclear how phenotypically similar or different they are. Here, we tested whether adolescent B6N mice have a bone phenotype and respond to the loss of weightbearing differently than B6J. At 9 weeks of age, normally ambulating B6N had lower trabecular bone volume fraction but greater bone formation rates and osteoblast surfaces than corresponding B6J. At 11 weeks of age, differences in trabecular indices persisted between the substrains but differences in cellular activity had ceased. Cortical bone indices were largely similar between the two substrains. Hindlimb unloading (HLU) induced similar degeneration of trabecular architecture and cellular activity in both substrains when comparing 11-week-old HLU mice to 11-week-old controls. However, unloaded B6N mice had smaller cortices than B6J. When comparing HLU to 9 weeks baseline control mice, deterioration in trabecular separation, osteoblast indices, and endocortical variables was significantly greater in B6N than B6J. These data indicate specific developmental differences in bone formation and morphology between B6N and B6J mice, giving rise to a differential response to mechanical unloading that may be modulated, in part, by the genes Herc2, Myo18b, and Acan. Our results emphasize that these substrains cannot be used interchangeably at least for investigations in which the phenotypic makeup and its response to extraneous stimuli are of interest.

The farnesoid X receptor negatively regulates osteoclastogenesis in bone remodeling and pathological bone loss.

Farnesoid X receptor (FXR, NR1H4) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. Since the role of FXR in osteoclast differentiation remains ill-defined, we investigated the biological function of FXR on osteoclastogenesis, using FXR-deficient mice. We demonstrated that FXR deficiency increases osteoclast formation in vitro and in vivo. First, FXR deficiency was found to accelerate osteoclast formation via down-regulation of c-Jun N-terminal kinase (JNK) 1/2 expression. Increased expression of peroxisome proliferator-activated receptor (PPAR)γ and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1)β seems to mediate the pro-osteoclastogenic effect of FXR deficiency via the JNK pathway. In addition, we found that FXR deficiency downregulated the expression of interferon-β (IFN-β), a strong inhibitor of osteoclastogenesis, via receptor activator of nuclear factor-kappaB ligand (RANKL). We further suggested that interference of IFN-β expression by FXR deficiency impaired the downstream JAK3-STAT1 signaling pathways, which in turn increased osteoclast formation. Finally, FXR deficiency accelerated unloading- or ovariectomy-induced bone loss in vivo. Thus, our findings demonstrate that FXR is a negative modulator in osteoclast differentiation and identify FXR as a potential therapeutic target for postmenopausal osteoporosis and unloading-induced bone loss.

Role of endoplasmic reticulum stress in disuse osteoporosis

Osteoporosis is a major skeletal disease with low bone mineral density, which leads to an increased risk of bone fracture. Salubrinal is a synthetic chemical that inhibits dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) in response to endoplasmic reticulum (ER) stress. To understand possible linkage of osteoporosis to ER stress, we employed an unloading mouse model and examined the effects of salubrinal in the pathogenesis of disuse osteoporosis. The results presented several lines of evidence that osteoclastogenesis in the development of osteoporosis was associated with ER stress, and salubrinal suppressed unloading-induced bone loss. Compared to the age-matched control, unloaded mice reduced the trabecular bone area/total area (B.Ar/T.Ar) as well as the number of osteoblasts, and they increased the osteoclasts number on the trabecular bone surface in a time-dependent way. Unloading-induced disuse osteoporosis significantly increased the expression of Bip, p-eIF2α and ATF4 in short-term within 6h of tail suspension, but time-dependent decreased in HU2d to HU14d. Furthermore, a significant correlation of ER stress with the differentiation of osteoblasts and osteoclasts was observed. Administration of salubrinal suppressed the unloading-induced decrease in bone mineral density, B.Ar/T.Ar and mature osteoclast formation. Salubrinal also increased the colony-forming unit-fibroblasts and colony-forming unit-osteoblasts. It reduced the formation of mature osteoclasts, suppressed their migration and adhesion, and increased the expression of Bip, p-eIF2α and ATF4. Electron microscopy showed that rough endoplasmic reticulum expansion and a decreased number of ribosomes on ER membrane were observed in osteoblast of unloading mice, and the abnormal ER expansion was significantly improved by salubrinal treatment. A TUNEL assay together with CCAAT/enhancer binding protein homologous protein (CHOP) expression indicated that ER stress-induced osteoblast apoptosis was rescued by salubrinal. Collectively, the results support the notion that ER stress plays a key role in the pathogenesis of disuse osteoporosis, and salubrinal attenuates unloading-induced bone loss by altering proliferation and differentiation of osteoblasts and osteoclasts via eIF2α signaling.

The regulation of iron metabolism by hepcidin contributes to unloading-induced bone loss

Iron overload inhibits osteoblast function and promotes osteoclastogenesis. Hepcidin plays an important role in this process. The changes in iron content and the regulation of hepcidin under unloading-induced bone loss remain unknown. A hindlimb suspension model was adopted to simulate unloading-induced bone loss in mice. The results showed that iron deposition in both liver and bone was markedly increased in hindlimb unloaded mice, and was accompanied by the upregulation of osteoclast activity and downregulation of osteoblast activity. The iron chelator deferoxamine mesylate (DFO) reduced the iron content in bone and alleviated unloading-induced bone loss. The increased iron content in bone was mainly a result of the upregulation of transferrin receptor 1 (TfR1) and divalent metal transporter 1 with iron response element (DMT1+IRE), rather than changes in the iron transporter ferroportin 1 (FPN1). The hepcidin level in the liver was significantly higher, while the FPN1 level in the duodenum was substantially reduced. However, there were no changes in the FPN1 level in bone tissue. During hindlimb unloading, downregulation of hepcidin by siRNA increased iron uptake in bone and liver, which aggravated unloading-induced bone loss. In summary, these data show that unloading-induced bone loss was orchestrated by iron overload and coupled with the regulation of hepcidin by the liver.

Disruption of glucocorticoid signal in osteoblasts and osteocytes attenuates hindlimb unloading-induced bone loss in mice

Disruption of glucocorticoid signal in osteoblasts and osteocytes attenuates hindlimb unloading-induced bone loss in mice

Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin.

SummaryGlucagon-like peptide 1 (GLP-1) plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4) promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs), but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis.

Elcatonin prevents bone loss caused by skeletal unloading by inhibiting preosteoclast fusion through the unloading-induced high expression of calcitonin receptors in bone marrow cells

This study aimed to clarify whether elcatonin (EL) has a preventive action on bone dynamics in skeletal unloading. Seven-week-old male C57BL/6J mice with either ground control (GC) or tail suspension (TS) were administered EL 20U/kg or a vehicle (veh) three times per week and assigned to one of the following four groups: GCEL, GCveh, TSEL, and TSveh. Blood samples and bilateral femurs and tibias of the mice were obtained for analysis. After 7days of unloading, the trabecular bone mineral density in the distal femur obtained via peripheral quantitative computed tomography and the trabecular bone volume were significantly higher in the TSEL group than in the TSveh group. The bone resorption histomorphometric parameters, such as the osteoclast surface and osteoclast number, were significantly suppressed in the TSEL mice, whereas the number of preosteoclasts was significantly increased. The plasma level of tartrate-resistant acid phosphatase-5b (TRACP-5b) was significantly lower in the TSEL group than in all other groups. In the bone marrow cell culture, the number of TRACP-positive (TRACP+) multinucleated cells was significantly lower in the TSEL mice than in the TSveh mice, whereas the number of TRACP+ mononucleated cells was higher in the TSEL mice. On day 4, the expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), cathepsin K and d2 isoform of vacuolar ATPase V0 domain (ATP6V0D2) mRNA in the bone marrow cells in the TSEL mice was suppressed, and the expression of calcitonin receptor (Calcr) mRNA on day 1 and Calcr antigen on day 4 were significantly higher in the TSveh mice than in the GCveh mice. EL prevented the unloading-induced bone loss associated with the high expression of Calcr in the bone marrow cells of mouse hindlimbs after tail suspension, and it suppressed osteoclast development from preosteoclasts to mature osteoclasts through bone-resorbing activity. This study of EL-treated unloaded mice provides the first in vivo evidence of a physiological role of EL in the inhibition of the differentiation process from preosteoclasts to osteoclasts.

Modulation of unloading-induced bone loss in mice with altered ERK signaling.

Genetic variations mediate skeletal responsiveness to mechanical unloading, with individual space travelers exhibiting large variations in the extent of bone loss. We previously identified genomic regions harboring several hundred genes that can modulate the magnitude of skeletal adaptation to mechanical unloading. Here, bioinformatic filters aided in shortlisting 30 genes with bone-related and mechanoregulatory roles. The genes CD44, FGF2, NOD2, and Fas, all associated with ERK signaling, were then functionally tested in hindlimb-unloaded (HLU) knockout (KO) mice. Compared to their respective normally ambulating wildtype (WT) controls, all KO strains, except Fas mice, had lower trabecular bone volume, bone volume fraction, and/or trabecular number. For cortical bone and compared to ambulatory WT mice, CD44(-/-) had impaired properties while FGF2(-/-) showed enhanced indices. NOD2(-/-) and Fas(-/-) did not have a cortical phenotype. In all KO and WT groups, HLU resulted in impaired trabecular and cortical indices, primarily due to trabecular tissue loss and mitigation of cortical bone growth. The difference in trabecular separation between HLU and ambulatory controls was significantly greater in CD44(-/-) and NOD2(-/-) mice than in WT mice. In cortical bone, differences in cortical thickness, total pore volume, and cortical porosity between HLU and controls were aggravated in CD44(-/-) mice. In contrast, deletion of NOD2 and Fas genes mitigated the differences in Po.V between HLU and control mice. Together, we narrowed a previous list of QTL-derived candidate genes from over 300 to 30, and showed that CD44, NOD2, and Fas have distinct functions in regulating changes in trabecular and cortical bone indices during unloading.

CD44 deficiency inhibits unloading-induced cortical bone loss through downregulation of osteoclast activity.

The CD44 is cellular surface adhesion molecule that is involved in physiological processes such as hematopoiesis, lymphocyte homing and limb development. It plays an important role in a variety of cellular functions including adhesion, migration, invasion and survival. In bone tissue, CD44 is widely expressed in osteoblasts, osteoclasts and osteocytes. However, the mechanisms underlying its role in bone metabolism remain unclear. We found that CD44 expression was upregulated during osteoclastogenesis. CD44 deficiency in vitro significantly inhibited osteoclast activity and function by regulating the NF-κB/NFATc1-mediated pathway. In vivo, CD44 mRNA levels were significantly upregulated in osteoclasts isolated from the hindlimb of tail-suspended mice. CD44 deficiency can reduce osteoclast activity and counteract cortical bone loss in the hindlimb of unloaded mice. These results suggest that therapeutic inhibition of CD44 may protect from unloading induced bone loss by inhibiting osteoclast activity.

The osteogenic effects of swimming on bone mass, strength, and microarchitecture in rats with unloading-induced bone loss.

The effect of nonweight-bearing exercise on osteoporotic bones remains controversial and inconclusive. The purpose of this study was to evaluate the effects of swimming on osteoporotic tibias of rats submitted to hindlimb suspension. Initially, 20 Wistar rats were used to confirm a significant bone loss following 21 days of unloading. Thirty rats were then divided into 3 groups and followed during 51 days: CON (nonsuspended rats), S + WB (suspended rats for 21 days and then released for regular weight-bearing) and, S + Swim (suspended rats for 21 days and then released from suspension and submitted to swimming exercise). We observed that swimming exercise was effective at fully recovering the bone deterioration caused by suspension, with significant increments in BMD, bone strength and bone volume. On the other hand, regular weight-bearing failed at fully restoring the bone loss induced by unloading. These results indicate that swimming exercise may be a potential tool to improve bone density, strength, and trabecular volume in tibias with bone loss induced by mechanical unloading in suspended rats. We conclude that this modality of activity could be beneficial in improving bone mass, strength, and architecture in osteoporotic individuals induced by disuse, such as bed rest or those exposed to microgravity, who may not be able to perform weight-bearing exercises.

Hindlimb-unloading suppresses B cell population in the bone marrow and peripheral circulation associated with OPN expression in circulating blood cells.

Rodent hindlimb unloading (HU) by tail-suspension is a model to investigate disuse-induced bone loss in vivo. Previously, we have shown that osteopontin (OPN, also known as Spp1) is required for unloading-induced bone loss. However, how unloading affects OPN expression in the body is not fully understood. Here, we examined OPN expression in peripheral blood of mice subjected to HU. Real-time RT-PCR analysis indicated that OPN expression is increased in circulating peripheral blood cells. This HU-induced increase in OPN mRNA expression was specific in circulating peripheral blood cells, as OPN was not increased in the blood cells in bone marrow. HU-induced enhancement in OPN expression in peripheral blood cells was associated with an increase in the fraction of monocyte/macrophage lineage cells in the peripheral blood. In contrast, HU decreased the fraction size of B-lymphocytes in the peripheral blood. We further examined if B-lymphogenesis is affected in the mice deficient for osteopontin subjected to HU. In bone marrow, HU decreased the population of the B-lymphocyte lineage cells significantly, whereas it did not alter the population of monocyte/macrophage lineage cells. HU also increased the cells in T-lymphocyte lineage in bone marrow. Interestingly, these changes were observed similarly both in OPN-deficient and wild-type mice. These results indicate for the first time that HU increases OPN expression in circulating cells and suppresses bone marrow B-lymphogenesis.

The application of micro-CT in monitoring bone alterations in tail-suspended rats in vivo

Osteopenia is a pathological process that affects human skeletal health not only on earth but also in long-time spaceflight. Micro-computed tomography (micro-CT) is a nondestructive method for assessing both bone quantity and bone quality. To investigate the characteristics of micro-CT on evaluating the microgravity-induced osteopenia (e.g. early detection time and the sensitive parameters), the bone loss process of tail-suspended rats was monitored by micro-CT in this study. 8-Week-old female Sprague Dawley rats were divided into two groups: tail suspension (TS) and control (CON). Volumetric bone mineral density (vBMD) and microstructure of the femur and tibia were evaluated in vivo by micro-CT at 0, 7, 14, 22days. Biomechanical properties of the femur and tibia were determined by three-point bending test. The ash weight of bone was also investigated. The results showed that (1) bone loss in the proximal tibia appeared earlier than in the distal femur. (2) On day 7, the percent bone volume (BV/TV) of the tibia 15.44% decreased significantly, and the trabecular separation (Tb.Sp) 30.29% increased significantly in TS group, both of which were detected earlier than other parameters. (3) Biomechanical properties (e.g. femur, −22.4% maximum load and −23.75% Young’s modulus vs. CON) and ash weight of the femur and tibia decreased significantly in the TS group in comparison to CON group. (4) vBMD of the femur and tibia were clearly related to bone ash and dry weight (r=0.75–0.87, p<0.05). (5) BV/TV of both femur and tibia were clearly related to maximum load and Young’s modulus (r=0.66–0.87, p<0.05). Similarly, trabecular vBMD and BV/TV of the femur and tibia were clearly related to Young’s modulus (r=0.73–0.89, p<0.05). These indicated that BV/TV and Tb.Sp were more sensitive than other parameters for evaluating bone loss induced by tail suspension, moreover, trabecular vBMD and other parameters might be used to evaluate bone strength. Therefore, micro-CT is a reliable and sensitive method for predicting unloading-induced bone loss in small animals.

β-1 Adrenergic Agonist Mitigates Unloading-Induced Bone Loss by Maintaining Formation

Recent data indicate a direct relationship between the sympathetic nervous system and bone metabolism. The purpose of this study was to evaluate the effects of a beta-1 adrenergic (Adrb1) agonist, dobutamine (DOB), on disuse-induced changes in bone integrity during 28 d of hindlimb unloading (HU). Male Sprague-Dawley rats, age 6 months, were assigned to either a normal cage activity (CC) or HU (n = 24/group). Animals were given one daily bolus dose (4 mg·kg body weight a day) of DOB (n = 12) or an equal volume of saline (VEH, n = 12). In vivo peripheral quantitative computed tomography scans revealed a 9% loss in proximal tibia metaphysis (PTM) volumetric bone mineral density (vBMD) over 28 d of disuse. DOB administration during HU significantly attenuated reductions in PTM vBMD and inhibited reductions in mid-diaphysis tibia cross-sectional moment of inertia. A significant decline in PTM bone formation rate in the HU-VEH group (-56% vs CC-VEH) was completely abolished in the HU-DOB group. Significant reductions in strength of the femoral shaft and neck in the HU-VEH group (14% and 15%, respectively) were prevented with DOB treatment. In conclusion, DOB administration during HU effectively attenuates significant declines in total vBMD at PTM by mitigating associated decrements in bone formation rate. Positive effects of DOB were observed only in unloaded animals, with no effects observed in normal weight-bearing rats. These data provide evidence for the importance of Adrb1 signaling in maintaining osteoblast function during periods of mechanical unloading.