Skeletal Unloading Research Articles

Whole-body vibration and resistance exercise prevent long-term hindlimb unloading-induced bone loss: independent and interactive effects

Skeletal unloading induced by disuse or immobilization causes a decrease in bone mass and strength. We investigated the relationship between whole-body vibration (WBV) and resistance exercise (RE) in preventing bone loss induced by 8-week hindlimb unloading in young male rats. Sixty male Wistar rats were assigned randomly to 6 groups: age-matched control group (CON, n=10), hindlimb unloading group (HU, n=10), hindlimb unloading+standing group (HU+ST, n=10), hindlimb unloading+WBV group (HU+WBV, n=10), hindlimb unloading+RE group (HU+RE, n=10) and hindlimb unloading+WBV+RE group (HU+WBV+RE, n=10). After 8-week hindlimb unloading, micro-CT scanning and three-point bending test were performed in the femur. Sera were collected for analysis of bone formation and resorption markers. Compared with HU group, WBV, RE and the combination of WBV and RE (WBV+RE) significantly improved (P<0.01) one repetition maximum (1RM) (expressed as the percentage change from baseline, HU: -23%, HU+WBV: 21%, HU+RE: 48%, HU+WBV+RE: 51%), and maintained (P<0.05) cancellous volumetric bone mineral density (vBMD) and trabecular structure. No difference of cortical vBMD was found among all groups (P>0.05). WBV had no effects on biomechanical properties of the femur diaphysis (P>0.05). RE and WBV+RE significantly increased maximum load and cross-sectional moment of inertia of the femur diaphysis in hindlimb unloading rats (P<0.05). There was an interaction between WBV and RE in improving cancellous bone. These results demonstrate that WBV and RE interactively maintain cancellous structure and vBMD, and independently partially mitigate the reduction of bone strength in long-term hindlimb unloading rats.

Impaired osteoblastogenesis potential of progenitor cells in skeletal unloading is associated with alterations in angiogenic and energy metabolism profile

Skeletal unloading provokes bone loss. These bone alterations have been shown to be associated with impairment of osteoblastic activity. In the present study, we evaluated the effect of skeletal unloading on bone marrow progenitor cells, for exploration of the underlying mechanism. Wistar rats were randomized to be either hindlimb unloaded for 9 days or to act as controls. Micro-CT was used to detect tibial trabecular architecture changes in response to skeletal unloading. Microgravity conditions for 9 days resulted in a decreased number and an increased spacing of the bone trabeculae in the proximal tibia. The proliferative capacity of the femoral bone marrow samples was assessed (fibroblast-colony-forming assay). By using qPCR, the expression of selected markers of vascularization (Vegfa; Hif1a; Angpt1), energy metabolism (Prkaa2; Mtor), bone formation (Runx2; Alp; Bglap; Bmp2; Bmp4; Bmp7) and bone resorption (Acp5; Tnfsf11; Tnfrsf11b) in these bone marrow suspensions was measured. We demonstrated a striking decrease in the number of fibroblastic progenitors in response to hindlimb unloading. This deficit in proliferation was shown to be accompanied by altered hindlimb perfusion and cellular energy homeostasis. Ex vivo culture assays of the bone marrow-derived progenitor cells screened for osteogenic (Runx2; Alp; Bglap) and adipogenic (Pparg; Fabp4) differentiation alterations in response to microgravity. Induced progenitor cells from unloaded rats showed a delay in osteogenic differentiation and impaired adipogenic differentiation compared to control. The data of this multi-level approach demonstrate that skeletal unloading significantly affects the bone tissue and its metabolism at the progenitor stage. The molecular expressions of the bone marrow population support a role of cellular metabolic stresses in skeletal alterations induced by inactivity.

Stimulation of Osteogenesis in Bone Defects Implanted with Biodegradable Hydroxyapatite Composed of Rod-Shaped Particles under Mechanical Unloading

The aim of this study was to evaluate the influence of mechanical unloading on the repair of bone defects with implantation of biodegradable bone substitutes. Spherical granules of biodegradable hydroxyapatite composed of rod-shaped particles (RHA) or beta-tricalcium phosphate composed of rod-shaped particles (RTCP) were implanted into a bone defect created in the distal end of the right femur of 8-week-old Wistar rats. Two, 6, 10, and 22 weeks after implantation, part of the sciatic nerve in the thigh was resected and exposed to mechanical unloading for 2 weeks. Then, 4, 8, 12 and 24 weeks after implantation, repair of the bone defect was analyzed. As a control, the bone defect without implantation of ceramic granules was also analyzed. Both RHA and RTCP tended to be reduced, but the reduction was not obvious during the experimental period. At 12 and 24 weeks after implantation, the amount of newly formed bone in the animal implanted with RHA was significantly greater than that at 4 weeks after implantation, but that in the animal implanted with RTCP or without implantation was not significantly different. The number of osteoclasts in the region implanted with RHA was significantly larger than that of the region implanted with RTCP or without implantation at 12 and 24 weeks. The activities of alkaline phosphatase in osteoblasts and tartrate-resistant acid phosphatase in osteoclasts were remarkably increased in the bone defects with implantation compared with those in the bone defects without implantation. These results suggested that RHA stimulated osteogenesis and osteoclastogenesis even after 2 weeks of mechanical unloading, and that RHA could be expected to improve the repair of bone defects in patients under the condition of skeletal unloading.

Deterioration of Bone Quality in the Tibia and Fibula in Growing Mice During Skeletal Unloading: Gender-Related Differences

Skeletal unloading causes bone loss in both men and women; however, only a few studies have been performed on the effects of gender differences on bone quality during skeletal unloading. Moreover, although the fibula also plays an important role in load bearing and ankle stability, the effects of unloading on the fibula have been rarely investigated. The present study aimed to investigate the effects of skeletal unloading on bone quality of the tibia and fibula in growing animals and to determine whether differences existed between genders. Six-week-old female and male mice were randomly allocated into two groups. The right hindlimb of each mouse in the skeletal unloading group was subjected to sciatic neurectomy. After two weeks of skeletal unloading, the structural characteristics of the tibia and fibula in both genders were worsened. In addition, the bone mineralization density distribution (MDD) of the tibia and fibula in both genders were altered. However, the magnitude of deterioration and alteration of the MDD in the bones of females were larger than in those of males. These results demonstrate that skeletal unloading diminishes bone quality in the tibia and fibula, leading to an increase in bone fracture risks, particularly in females.

Skeletal unloading–induced insulin-like growth factor 1 (IGF-1) Nonresponsiveness is not shared by platelet-derived growth factor: The selective role of integrins in IGF-1 signaling

Integrin receptors bind extracellular matrix proteins, and this link between the cell membrane and the surrounding matrix may translate skeletal loading to biologic activity in osteoprogenitor cells. The interaction between integrin and growth factor receptors allows for mechanically induced regulation of growth factor signaling. Skeletal unloading leads to decreased bone formation and osteoblast proliferation that can be explained in part by a failure of insulin-like growth factor 1 (IGF-1) to activate its signaling pathways in unloaded bone. The aim of this study is to determine whether unloading-induced resistance is specific for IGF-1 or common to other skeletal growth factors, and to examine the regulatory role of integrins in IGF-1 signaling. Bone marrow osteoprogenitor (BMOp) cells were isolated from control or hindlimb suspended rats. Unloaded BMOp cells treated with IGF-1 failed to respond with increased proliferation, receptor phosphorylation, or signaling activation in the setting of intact ligand binding, whereas the platelet-derived growth factor (PDGF) response was fully intact. Pretreatment of control BMOp cells with an integrin inhibitor, echistatin, failed to disrupt PDGF signaling but blocked IGF-1 signaling. Recovery of IGF-1 signaling in unloaded BMOp cells followed the recovery of marked reduction in integrin expression induced by skeletal unloading. Selective targeting of integrin subunits with siRNA oligonucleotides revealed that integrin β1 and β3 are required for normal IGF-1 receptor phosphorylation. We conclude that integrins, in particular integrin β3, are regulators of IGF-1, but not PDGF, signaling in osteoblasts, suggesting that PDGF could be considered for investigation in prevention and/or treatment of bone loss during immobilization and other forms of skeletal unloading.

Investigating the mechanism for maintaining eucalcemia despite immobility and anuria in the hibernating American black bear (Ursus americanus)

Ursine hibernation uniquely combines prolonged skeletal unloading, anuria, pregnancy, lactation, protein recycling, and lipolysis. This study presents a radiographic and biochemical picture of bone metabolism in free-ranging, female American black bears (Ursus americanus) that were active (spring bears and autumn bears) or hibernating (hibernating bears). Hibernating bears included lactating and non-lactating individuals. We measured serum calcium, albumin, inorganic phosphate, creatinine, bone specific alkaline phosphatase (BSALP), CTX, parathyroid hormone, insulin-like growth factor-I (IGF-l), leptin, 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)2D] and sclerostin from 35 to 50 tranquilized hibernating bears and 14 to 35 tranquilized spring bears. We compared metacarpal cortical indices (MCI), measured by digital X-ray radiogrammetry, from 60 hunter-killed autumn bears and 79 tranquilized, hibernating bears. MCI was greater in autumn than winter in younger bears, but showed no seasonal difference in older bears. During hibernation eucalcemia was maintained, BSALP was suppressed, and CTX was in the range expected for anuria. During hibernation 1,25(OH)2D was produced despite anuria. 1,25(OH)2D and IGF-I were less in hibernating than spring bears. In a quarter of hibernating bears, sclerostin was elevated. Leptin was greater in hibernating than spring bears. In hibernating bears, leptin correlated positively with BSALP in non-lactating bears and with CTX in lactating bears. Taken together the biochemical and radiographic findings indicate that during hibernation, bone turnover was persistent, balanced, and suppressed; bone resorption was lower than expected for an unloaded skeleton; and there was no unloading-induced bone loss. The skeleton appears to perceive that it was loaded when it was actually unloaded during hibernation. However, at the level of sclerostin, the skeleton recognized that it was unloaded. During hibernation leptin appeared anabolic in non-lactating bears and catabolic in lactating bears. We hypothesize that ursine hibernation may represent a natural model in which suppression of the sympathetic nervous system prevents unloading-induced bone loss by influencing leptin's skeletal effects and preventing transmission of loading information.

Connexin43 deficiency reduces the sensitivity of cortical bone to the effects of muscle paralysis.

We have shown previously that the effect of mechanical loading on bone depends in part on connexin43 (Cx43). To determine whether Cx43 is also involved in the effect of mechanical unloading, we have used botulinum toxin A (BtxA) to induce reversible muscle paralysis in mice with a conditional deletion of the Cx43 gene in osteoblasts and osteocytes (cKO). BtxA injection in hind limb muscles of wild-type (WT) mice resulted in significant muscle atrophy and rapid loss of trabecular bone. Bone loss reached a nadir of about 40% at 3 weeks after injection, followed by a slow recovery. A similar degree of trabecular bone loss was observed in cKO mice. By contrast, BtxA injection in WT mice significantly increased marrow area and endocortical osteoclast number and decreased cortical thickness and bone strength. These changes did not occur in cKO mice, whose marrow area is larger, osteoclast number higher, and cortical thickness and bone strength lower relative to WT mice in basal conditions. Changes in cortical structure occurring in WT mice had not recovered 19 weeks after BtxA injection despite correction of the early osteoclast activation and a modest increase in periosteal bone formation. Thus BtxA-induced muscle paralysis leads to rapid loss of trabecular bone and to changes in structural and biomechanical properties of cortical bone, neither of which are fully reversed after 19 weeks. Osteoblast/osteocyte Cx43 is involved in the adaptive responses to skeletal unloading selectively in the cortical bone via modulation of osteoclastogenesis on the endocortical surface.

Longitudinal Changes in Apparent Young Modulus and Bone Volume by Denervation: Subject-Specific Finite Element Analysis Considering Heterogeneous Tissue Properties

This study aimed to predict bone fracture risk during skeletal unloading through a subject-specific finite element (FE) analysis considering heterogeneous tissue modulus. Twelve male and 14 female Institute of Cancer Research (ICR) mice (6 weeks old) were allocated into skeletal unloading and normal groups (each gender in both groups). The right hind tibia of each mouse in the skeletal unloading groups was subjected to sciatic neurectomy (denervation) and was scanned before and at 2 week after denervation using µCT. Bone volume (BV) and the distribution of Young's moduli (E) were measured from µCT images. The apparent modulus (Eapp) was calculated using the subject-specific (FE) analysis considering the heterogeneous tissue modulus derived by Hounsfield unit. At 2 weeks after denervation, the Eapp was significantly decreased in both genders (p < 0.05), and the distributions of E differed between the skeletal unloading and normal groups. However, the BV in females was significantly decreased (p < 0.05), while that in male was unchanged (p &gt 0.05). These results indicated that skeletal unloading reduced bone strength, leading to increased bone fracture risk.

Laser Acupuncture and Prevention of Bone Loss in Tail-Suspended Rats

Skeletal unloading during spaceflight results in bone loss. This study investigated whether laser acupuncture could be an effective countermeasure to prevent unloading-induced bone loss in rats. There were 18 rats that were randomly assigned into three groups: a control group, a tail-suspended group (TS), and a tail-suspended with laser acupuncture treatment group (TSA). The rats in the TSA group were treated with laser acupuncture at the KI1 (Yong Quan) and ST36 (Zu San Li) acupoints of the left leg for 3 min per day. Bone mineral density (BMD), biomechanical properties, and histomorphometry of both tibiae were determined after the animals were euthanized at the end of week 4. Compared with the control group, BMD in the TS group significantly decreased by 12.3% in cortical bone and 15.1% in cancellous bone, whereas BMD in the TSA group decreased by only 3.1% in cortical bone and 9.0% in cancellous bone. The hardness of cortical bone dropped 44.1% in the TS group and 22.3% in the TSA group compared with the control group. The histomorphometry data were in accordance with BMD measurements. Although acupuncture treatment was applied only to the left side, we observed similar changes between the measurements of both the left and right tibiae. Laser acupuncture on KI1 and ST36 can inhibit bone loss in rats subjected to unloading. The fact that similar changes between the right and left sides when only the left limbs were treated suggests that the preventive effect of laser acupuncture occurs via a systemic regulation.

REBUTTAL FROM STEWART AND PELL

REBUTTAL FROM STEWART AND PELL

Validation of a skeletal unloading model for assessment of the Impact of bone quality on initial implant osseointegration

Validation of a skeletal unloading model for assessment of the Impact of bone quality on initial implant osseointegration

Heavy ion irradiation and unloading effects on mouse lumbar vertebral microarchitecture, mechanical properties and tissue stresses

Astronauts are exposed to both musculoskeletal disuse and heavy ion radiation in space. Disuse alters the magnitude and direction of forces placed upon the skeleton causing bone remodeling, while energy deposited by ionizing radiation causes free radical formation and can lead to DNA strand breaks and oxidative damage to tissues. Radiation and disuse each result in a net loss of mineralized tissue in the adult, although the combined effects, subsequent consequences for mechanical properties and potential for recovery may differ. First, we examined how a high dose (2 Gy) of heavy ion radiation ( 56Fe) causes loss of mineralized tissue in the lumbar vertebrae of skeletally mature (4 months old), male, C57BL/6 mice using microcomputed tomography and determined the influence of structural changes on mechanical properties using whole bone compression tests and finite element analyses. Next, we tested if a low dose (0.5 Gy) of heavy particle radiation prevents skeletal recovery from a 14-day period of hindlimb unloading. Irradiation with a high dose of 56Fe (2 Gy) caused bone loss (−14%) in the cancellous-rich centrum of the fourth lumbar vertebra (L4) 1 month later, increased trabecular stresses (+ 27%), increased the propensity for trabecular buckling and shifted stresses to the cortex. As expected, hindlimb unloading (14 days) alone adversely affected microarchitectural and mechanical stiffness of lumbar vertebrae, although the reduction in yield force was not statistically significant (−17%). Irradiation with a low dose of 56Fe (0.5 Gy) did not affect vertebrae in normally loaded mice, but significantly reduced compressive yield force in vertebrae of unloaded mice relative to sham-irradiated controls (−24%). Irradiation did not impair the recovery of trabecular bone volume fraction that occurs after hindlimb unloaded mice are released to ambulate normally, although microarchitectural differences persisted 28 days later (96% increase in ratio of rod- to plate-like trabeculae). In summary, 56Fe irradiation (0.5 Gy) of unloaded mice contributed to a reduction in compressive strength and partially prevented recovery of cancellous microarchitecture from adaptive responses of lumbar vertebrae to skeletal unloading. Thus, irradiation with heavy ions may accelerate or worsen the loss of skeletal integrity triggered by musculoskeletal disuse.

Psychotropic drugs have contrasting skeletal effects that are independent of their effects on physical activity levels

Popular psychotropic drugs, like the antidepressant selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs), and the mood stabilizer lithium, may have skeletal effects. In particular, preclinical observations suggest a direct negative effect of SSRIs on the skeleton. A potential caveat in studies of the skeletal effects of psychotropic drugs is the hypoactive (skeletal unloading) phenotype they induce. The aim of this study was to investigate the contribution of physical inactivity to the skeletal effects of psychotropic drugs by studying bone changes in cage control and tail suspended mice treated with either vehicle, SSRI, TCA or lithium. Tail suspension was used to control for drug differences on physical activity levels by normalizing skeletal loading between groups. The psychotropic drugs were found to have contrasting skeletal effects which were independent of drug effects on animal physical activity levels. The latter was evident by an absence of statistical interactions between the activity and drug groups. Pharmacological inhibition of the 5-hydroxytryptamine (5-HT) transporter (5-HTT) using a SSRI reduced in vivo gains in lower extremity BMD, and negatively altered ex vivo measures of femoral and spinal bone density, architecture and mechanical properties. These effects were mediated by a decrease in bone formation without a change in bone resorption suggesting that the SSRI had anti-anabolic skeletal effects. In contrast, glycogen synthase kinase-3[beta] (GSK-3[beta]) inhibition using lithium had anabolic effects improving in vivo gains in BMD via an increase in bone formation, while TCA-mediated inhibition of the norepinephrine transporter had minimal skeletal effect. The observed negative skeletal effect of 5-HTT inhibition, combined with recent findings of direct and indirect effects of 5-HT on bone formation, are of interest given the frequent prescription of SSRIs for the treatment of depression and other affective disorders. Likewise, the anabolic effect of GSK-3[beta] inhibition using lithium reconfirms the importance of Wnt/beta-catenin signaling in the skeleton and it's targeting by recent drug discovery efforts. In conclusion, the current study demonstrates that different psychotropic drugs with differing underlying mechanisms of action have contrasting skeletal effects and that these effects do not result indirectly via the generation of animal physical inactivity.

Role of nutritional zinc in the prevention of osteoporosis

Zinc is known as an essential nutritional factor in the growth of the human and animals. Bone growth retardation is a common finding in various conditions associated with dietary zinc deficiency. Bone zinc content has been shown to decrease in aging, skeletal unloading, and postmenopausal conditions, suggesting its role in bone disorder. Zinc has been demonstrated to have a stimulatory effect on osteoblastic bone formation and mineralization; the metal directly activates aminoacyl-tRNA synthetase, a rate-limiting enzyme at translational process of protein synthesis, in the cells, and it stimulates cellular protein synthesis. Zinc has been shown to stimulate gene expression of the transcription factors runt-related transcription factor 2 (Runx2) that is related to differentiation into osteoblastic cells. Moreover, zinc has been shown to inhibit osteoclastic bone resorption due to inhibiting osteoclast-like cell formation from bone marrow cells and stimulating apoptotic cell death of mature osteoclasts. Zinc has a suppressive effect on the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-induced osteoclastogenesis. Zinc transporter has been shown to express in osteoblastic and osteoclastic cells. Zinc protein is involved in transcription. The intake of dietary zinc causes an increase in bone mass. beta-Alanyl-L: -histidinato zinc (AHZ) is a zinc compound, in which zinc is chelated to beta-alanyl-L: -histidine. The stimulatory effect of AHZ on bone formation is more intensive than that of zinc sulfate. Zinc acexamate has also been shown to have a potent-anabolic effect on bone. The oral administration of AHZ or zinc acexamate has the restorative effect on bone loss under various pathophysiologic conditions including aging, skeletal unloading, aluminum bone toxicity, calcium- and vitamin D-deficiency, adjuvant arthritis, estrogen deficiency, diabetes, and fracture healing. Zinc compounds may be designed as new supplementation factor in the prevention and therapy of osteoporosis.

Skeletal Effects of Long-Duration Head-Down Bed Rest

Skeletal unloading during spaceflight causes regional loss of bone mineral density (BMD), primarily in the spine and lower body regions. This loss of skeletal mass could adversely affect crew health during and after spaceflight and jeopardize mission success. Bed rest has long been used as a spaceflight analog to study the effects of disuse on many body systems, including the skeleton. This study was undertaken by the NASA Flight Analogs Project (FAP) to collect control data for upcoming countermeasure studies. There were 13 subjects who participated in 42, 44, 49, 52, 60, or 90 d of continuous, head-down bed rest. DXA scans (dual-energy X-ray absorptiometry) were obtained before and after bed rest to measure changes in BMD of the whole body, lumbar spine, hip, heel, and wrist; the 90-d subjects were also scanned at the 60-d time point. Follow-up DXA scans were performed after 6 mo and 12 mo of reambulation to assess BMD recovery. BMD changes were consistent with earlier bed rest and spaceflight studies, with statistically significant losses averaging 1% per month in the hip, pelvis, and heel. Recovery data were also consistent with data obtained after spaceflight. Bone biomarker data are described, and support the findings of previous studies. Specifically, the process of normal bone remodeling is uncoupled: increased bone resorption with no concomitant change in bone formation. The FAP appears to be a valid test bed for skeletal disuse studies, and should provide a useful research platform for evaluating countermeasures to spaceflight-induced bone loss.

Whole-Body Vibration Effects on Bone Before and After Hind-Limb Unloading in Rats

Skeletal unloading during spaceflight leads to bone loss. Following long-duration flight, such loss may be very slow or impossible to fully recover. Therefore, it is important to seek countermeasures to prevent the loss. We studied the effects on bone of whole-body vibration (WBV) with variable parameters (10-60 Hz and 0.1-1 g) using hind-limb unloading (HLU) in the rat as a model for microgravity. The bone mineral densities (BMD) of the femur, tibia, and lumbar spines were measured and the mechanical properties of the femur were determined by mechanical testing. Serum alkaline phosphatase (ALP) and pyridinoline (PYD) levels were analyzed. After 28 d of HLU, WBV reduced the losses of BMD in the femur and tibia from 18.8 to 10.1% and from 16.7 to 7.1%, respectively, and prevented the loss in stiffness of the femur after HLU. However, WBV had no effect on the lumbar spine. ALP levels were suppressed by HLU and maintained by WBV (28.3% increase). There was no significant difference in PYD among groups. In the recovery period following HLU, there were no significant effects of WBV on BMD, and the elastic modulus of the femur returned to normal. Mechanical stimulation in the form of WBV limited reduction of bone density when it was applied during the unloading, but not afterward. It is of interest that the mechanical properties of the femur were influenced by WBV in the control femurs as well as in the unloaded bones of this rat model.

Viewpoint: Dried plum, an emerging functional food that may effectively improve bone health

Osteoporosis is a debilitating disorder that affects both female and male, albeit to a greater extent in women than men. As the demographic shift to a more aged population continues, a growing number of men and women will be afflicted with osteoporosis and a search for potential non-pharmacological alternative therapies for osteoporosis is of prime interest. Aside from existing drug therapies, certain lifestyle and nutritional factors are known to reduce the risk of osteoporosis. Among nutritional factors, recent observations suggest that dried plum, or prunes ( Prunus domestica L.) is the most effective fruit in both preventing and reversing bone loss. Animal studies and a 3-month clinical trial conducted in our laboratories have shown that dried plum has positive effects on bone indices. The animal data indicate that dried plum not only protects against but more importantly reverses bone loss in two separate models of osteopenia. Our initial animal study indicated that dried plum prevented the ovariectomy-induced reduction in bone mineral density (BMD) of the femur and lumbar vertebra. In another study, to mimic established osteoporosis, rats were ovariectomized and allowed to lose bone before the initiation of treatment. Dried plum as low as 5% (w/w) restored BMD to the level of intact rats. More importantly, dried plum reversed the loss of trabecular architectural properties such as trabecular number and connectivity density, and trabecular separation. We have also shown the effectiveness of dried plum in reversal of bone loss due to skeletal unloading. Analysis of BMD and trabecular bone structure by microcomputed tomography (μCT) revealed that dried plum enhanced bone recovery during re-ambulation following skeletal unloading and had comparable effects to parathyroid hormone. In addition to the animal studies, our 3-month clinical trial indicated that the consumption of dried plum daily by postmenopausal women significantly increased serum markers of bone formation, total alkaline phosphatase, bone-specific alkaline phosphatase and insulin-like growth factor-I by 12, 6, and 17%, respectively. This review summarizes the findings of studies published to date which examine the beneficial effects of dried plum on bone in both female and male animal models of osteoporosis as well as the only published clinical study.

Skeletal unloading induces a full-thickness patellar cartilage defect with increase of urinary collagen II CTx degradation marker in growing rats

Mechanical stress plays an important role in tissue morphogenesis and extracellular matrix metabolism. However, little is known about the effects of reduced loading without restriction of joint motion on the patella. We investigated the effects of long-term skeletal unloading on patellar cartilage and subchondral bone and systemic collagen II metabolism. Nine-week-old male F344/N rats ( n = 128) were randomly divided into two groups: caged control (C) and tail suspended (TS). Hindlimbs of the TS rats were subjected to unloading for up to 12 weeks. Sequential changes in the patellar cartilage and subchondral bone were analyzed macroscopically, by pathological findings and histomorphologically. All animals received double tidemark fluorochrome labeling prior to sacrifice. Glycosaminoglycan (GAG) content in patellar cartilage, cross-linked C-telopeptide of type II collagen (CTx-II) in 24-h urine and type II procollagen-C-peptide (pCol-II-C) in sera were also measured by DMB assay, ELISA and EIA, respectively. In the TS group, GAG content was significantly reduced only during the first 3 weeks. No further significant decrease was found. Alkaline phosphatase (ALP) activity was increased, especially at the deep zone. Tidemark mineral apposition rate (MAR) was temporally increased, which resulted in an increase in the ratio of calcified cartilage to the entire cartilage. In the medial part, in addition, thickness of the entire cartilage was decreased by temporal acceleration of subchondral ossification advancement and, in the medial margin, a full-thickness cartilage defect was revealed in 88.6% of TS rats. However, the remaining articular surface was free from fibrillation. While urinary CTx-II was significantly increased during the experimental periods, serum pCol-II-C was significantly decreased at the early phase. There were significant correlations between the urinary CTx-II levels and tidemark MAR. Our results provided evidence that skeletal unloading increased ALP activity at the deep zone and temporally accelerated tidemark advancement associated with a decrease in proteoglycan content. In addition, skeletal unloading temporally accelerated subchondral ossification advancement in the medial part of the patella and finally induced a full-thickness patellar cartilage defect without any fibrillation at the remaining articular surface by additional subchondral bone modeling and possible retarded cartilage growth, which was through a different mechanism than overloading.

Transforming growth factor-β prevents osteoblast apoptosis induced by skeletal unloading via PI3K/Akt, Bcl-2, and phospho-Bad signaling

Loss of mechanical loading induces rapid bone loss resulting from reduced osteoblastogenesis and decreased bone formation. The signaling mechanisms involved in this deleterious effect on skeletal metabolism remain poorly understood. We have previously shown that hindlimb suspension in rats increases osteoblast apoptosis associated with decreased phosphatidylinositol 3-kinase (PI3K) signaling. In this study, we investigated whether transforming growth factor (TGF)-beta2 may prevent the altered signaling and osteoblast apoptosis induced by skeletal unloading in vivo. Hindlimb suspension-induced decreased bone volume was associated with reduced alpha(5)beta(1)-integrin protein levels and PI3K/Akt signaling in unloaded bone. Continuous administration of TGF-beta2 using osmotic minipumps prevented the decreased alpha(5)beta(1)-integrin expression and the reduced PI3K/Akt signaling in unloaded bone, resulting in the prevention of osteoblast apoptosis. We also show that TGF-beta2 prevented the decreased Bcl-2 levels induced by unloading, which suggests that TGF-beta2 targets Bcl-2 via PI3K/Akt to prevent osteoblast apoptosis in unloaded bone. Furthermore, we show that TGF-beta2 prevented the decrease in phosphorylated Bad, the inactive form of the proapoptotic protein Bad, induced by unloading. These results identify a protective role for TGF-beta2 in osteoblast apoptosis induced by mechanical unloading via the alpha(5)beta(1)/PI3K/Akt signaling cascade and downstream Bcl-2 and phospho-Bad survival proteins. We thus propose a novel role for TGF-beta2 in protection from unloading-induced apoptosis in vivo.

Integrins, insulin like growth factors, and the skeletal response to load

Bone loss during skeletal unloading, whether due to neurotrauma resulting in paralysis or prolonged immobilization due to a variety of medical illnesses, accelerates bone loss. In this review the evidence that skeletal unloading leads to bone loss, at least in part, due to disrupted insulin like growth factor (IGF) signaling, resulting in reduced osteoblast proliferation and differentiation, will be examined. The mechanism underlying this disruption in IGF signaling appears to involve integrins, the expression of which is reduced during skeletal unloading. Integrins play an important, albeit not well defined, role in facilitating signaling not only by IGF but also by other growth factors. However, the interaction between selected integrins such as alphaupsilonbeta3 and beta1 integrins and the IGF receptor are of especial importance with respect to the ability of bone to respond to mechanical load. Disruption of this interaction blocks IGF signaling and results in bone loss.