Immobilization-induced Bone Loss Research Articles

Cell-Free Fat Extract Prevents Tail Suspension-Induced Bone Loss by Inhibiting Osteocyte Apoptosis.

Introduction: As the space field has developed and our population ages, people engaged in space travel and those on prolonged bed rest are at increasing risk for bone loss and fractures. Disuse osteoporosis occurs frequently in these instances, for which the currently available anti-osteoporosis agents are far from satisfactory and have undesirable side effects. CEFFE is a cell-free fraction isolated from nanofat that is enriched with a variety of growth factors, and we aim to investigate its potential therapeutic effects on disuse osteoporosis. Methods: A tail suspension–induced osteoporosis model was applied in this study. Three weeks after tail suspension, CEFFE was intraperitoneally injected, and PBS was used as a control. The trabecular and cortical bone microstructures of the tibia in each group were assessed by μCT after 4 weeks of administration. Osteocyte lacunar-canalicularity was observed by HE and silver staining. In vitro, MLO-Y4 cell apoptosis was induced by reactive oxygen species (ROSUP). TUNEL staining and flow cytometry were used to detect apoptosis. CCK-8 was used to detect cell proliferation, and Western blotting was used to detect MAPK signaling pathway changes. Results: CEFFE increased the bone volume (BV/TV) and trabecular number (Tb.N) of the trabecular bone and increased the thickness of the cortical bone. HE and silver staining results showed that CEFFE reduced the number of empty lacunae and improved the lacuna-canalicular structure. CEFFE promoted osteocyte proliferative capacity in a dose-dependent manner. CEFFE protected MLO-Y4 from apoptosis by activating the serine/threonine-selective protein kinase (ERK) signaling pathways. Conclusion: CEFFE attenuated immobilization-induced bone loss by decreasing osteocyte apoptosis. CEFFE increased the survival of osteocytes and inhibited osteocyte apoptosis by activating the ERK signaling pathway in vitro.

The Efficacy of PTH and Abaloparatide to Counteract Immobilization-Induced Osteopenia Is in General Similar.

Immobilization results in a substantial bone loss and increased fracture risk. Powerful bone anabolic therapies are necessary to counteract the bone loss and reduce fracture risk during periods with immobilization. Intermittent parathyroid hormone 1−34 (PTH) (teriparatide) and PTH related peptide analog abaloparatide (ABL) are potent bone anabolic therapies acting through the same receptor, but induce different durations of signaling response. We investigated the efficacy of PTH or ABL in preventing immobilization-induced bone loss in rats in a direct mole-to-mole comparison. Immobilization was achieved by injecting botulinum toxin type A (BTX) into the right hindlimb musculature. Sixty 14-week-old female Wistar rats were allocated to the following groups: Baseline, Control, BTX, BTX + PTH (80 μg/kg/day), and BTX + ABL (77 μg/kg/day). Immobilization resulted in a substantial and significant reduction in bone mineral density (aBMD), metaphyseal and epiphyseal trabecular bone volume fraction (BV/TV) and trabecular thickness (Tb.Th), metaphyseal trabecular number (Tb.N), and femoral neck bone strength. Both PTH and ABL prevented the immobilization-induced decrease in aBMD, metaphyseal and epiphyseal Tb.Th, and metaphyseal Tb.N. In addition, PTH rescued the reduction in metaphyseal BV/TV and femoral neck strength, while ABL did not. However, the effect of PTH and ABL did not differ significantly for serum calcium, aBMD, metaphyseal, and epiphyseal BV/TV, Tb.Th, or Tb.N. In conclusion, in a mole-to-mole comparison the efficacy of PTH and ABL is similar in counteracting immobilization-induced reduction in bone mineral density, deterioration in trabecular microarchitecture, and decrease in bone strength.

Long-Term High-Dose Resveratrol Supplementation Reduces Bone Mass and Fracture Strength in Rats.

Resveratrol (RSV) is a natural polyphenolic compound. A recent study suggests a positive effect on BMD in men; however, the underlying changes in microstructure and strength remain unknown. We aimed to investigate the effects of RSV on the skeleton in hindlimb-immobilized and non-immobilized rats. Seventy-two female Wistar rats were divided into six groups. Two baseline (BSL) groups underwent short-term diet intervention for 4weeks before sacrifice [phytoestrogen-deficient diet (PD) (BSL+PD) or RSV diet (600mg/kg body weight/day) (BSL+RSV)]. Four groups were injected in the right hindlimb with botulinum toxin (BTX) (immobilized) or saline (non-immobilized), and fed either PD diet or RSV diet 4 weeks pre-injection and 6weeks post-injection before sacrifice (BTX+PD, BTX+RSV, PD, and RSV, respectively). DXA, µCT, dynamic histomorphometry, and mechanical tests were performed. Short-term RSV treatment did not affect bone parameters, whereas long-term RSV exposure had a consistent negative impact on non-immobilized rats (RSV vs. PD); whole femoral aBMD (p=0.01) and distal femoral metaphyseal Tb.N (p=0.01), Tb.Sp (p=0.02), and BV/TV (p=0.07). At the femoral mid-diaphysis, RSV increased periosteal resorption (p=0.01) and increased endosteal formation (p=0.02), while mineralization was unaffected. In addition, RSV reduced femoral mid-diaphyseal three-point bending strength (p=0.03) and stiffness (p=0.04). BTX-induced immobilization resulted in significant bone loss and reduced bone strength; however, RSV supplementation was unable to prevent this. In conclusion, long-term high-dose RSV reduced bone mass and fracture strength and did not prevent immobilization-induced bone loss in rats.

Daily parathyroid hormone administration enhances bone turnover and preserves bone structure after severe immobilization-induced bone loss.

Immobilization, as a result of motor‐complete spinal cord injury (SCI), is associated with severe osteoporosis. Whether parathyroid hormone (PTH) administration would reduce bone loss after SCI remains unclear. Thus, female mice underwent sham or surgery to produce complete spinal cord transection. PTH (80 μg/kg) or vehicle was injected subcutaneously (SC) daily starting on the day of surgery and continued for 35 days. Isolated tibias and femurs were examined by microcomputed tomography scanning (micro‐CT) and histology and serum markers of bone turnover were measured. Micro‐CT analysis of tibial metaphysis revealed that the SCI‐vehicle animals exhibited 49% reduction in fractional trabecular bone volume and 18% in trabecular thickness compared to sham‐vehicle controls. SCI‐vehicle animals also had 15% lower femoral cortical thickness and 16% higher cortical porosity than sham‐vehicle counterparts. Interestingly, PTH administration to SCI animals restored 78% of bone volume, increased connectivity to 366%, and lowered structure model index by 10% compared to sham‐vehicle animals. PTH further favorably attenuated femoral cortical bone loss to 5% and prevented the SCI‐associated cortical porosity. Histomorphometry evaluation of femurs of SCI‐vehicle animals demonstrated a marked 49% and 38% decline in osteoblast and osteoclast number, respectively, and 35% reduction in bone formation rate. In contrast, SCI‐PTH animals showed preserved osteoblast and osteoclast numbers and enhanced bone formation rate. Furthermore, SCI‐PTH animals had higher levels of bone formation and resorption markers than either SCI‐ or sham‐vehicle groups. Collectively, these findings suggest that intermittent PTH receptor activation is an effective therapeutic strategy to preserve bone integrity after severe immobilization.

Ibandronate concomitantly blocks immobilization-induced bone and muscle atrophy

Both bone and muscle volume is concomitantly reduced under immobilization conditions; however, no single drug is currently available to block these outcomes simultaneously. Bisphosphonates are utilized clinically to inhibit osteoclast-dependent bone resorption, but their effects on muscle are largely unknown. Here we show that skeletal muscle is a direct target of the bisphosphonate ibandronate (IBN) and that reduced muscle volume and induction of Atrogin-1 and MuRF1, both atrogenes, are significantly inhibited by IBN administration in vivo using a mouse model of muscle atrophy. IBN treatment also significantly blocked immobilization-induced bone loss in vivo. We also report that expression of Atrogin-1 and MuRF1 and accumulation of Smad2/3 proteins, which are upstream of atrogines, occurred following serum starvation of myogenic C2C12 cells in vitro, effects significantly inhibited by IBN treatment. Interestingly, IBN effects on C2C12 cells were abrogated by MG132, an ubiquitin/proteasome inhibitor, suggesting that IBN functions via the ubiquitin-proteasome system. Our findings lend new insight into the role of IBN in preventing muscle atrophy.

Effects of zoledronic acid on bone fusion in osteoporotic patients after lumbar fusion.

Treatment with zoledronic acid in osteoporotic patients with spinal fusion shortens the duration of time to fusion, improves the fusion rate, prevents the subsequent adjacent vertebral compression fractures, improves the clinical outcomes, and prevents immobilization-induced bone loss in the hip. The objective of the study was to explore the effects of zoledronic acid on the healing process in osteoporotic patients following spinal fusion in a randomized, placebo-controlled, and triple-blinded study. Seventy-nine osteoporotic patients with single-level degenerative spondylolisthesis were randomly assigned to receive either zoledronic acid infusion (zoledronic acid group) or saline infusion (controls) after spinal fusion. Functional radiography and CT scans were used to evaluate fusion status. Bone formation was graded into three categories: Grade A (bridging bone bonding with adjacent vertebral bodies), Grade B (bridging bone bonding with either superior or inferior vertebral body), or Grade C (incomplete bony bridging). A solid fusion was defined as less than 5° of angular motion with Grade A or B bone formation. Adjacent vertebral compression fractures (VCF) were assessed on MRI at 12months after surgery. Serum level of carboxy terminal cross-linked telopeptide of type I collagen (β-CTX) and amino-terminal propeptide of type I procollagen (PINP) was measured. Bone mineral density (BMD) was measured by DXA. Oswestry Disability Index (ODI) was used to assess the clinical outcomes. Grade A or B bridging bone was more frequently observed in zoledronic acid group at 3, 6, and 9months post-operation compared to the control group (p < 0.05). At 12 -months post-operation, bridging bone and solid fusion were not significantly different between groups. No patients in zoledronic acid group showed aVCF, whereas six patients (17%) in the control group did (p < 0.05). Both β-CTX and PINP were suppressed in zoledronic acid group. BMD at the femoral neck decreased rapidly and did not return to the preoperative level in the controls at 3 (-1.4%), 6 (-2.5%), and 12 (-0.8%) months after surgery. Zoledronic acid prevented this immobilization-induced bone loss and increased BMD. ODI showed the improved clinical outcomes compared with controls at 9 and 12months post-surgery. Treatment with zoledronic acid in osteoporotic patients with spinal fusion shortens the time to fusion, improves the fusion rate, prevents subsequent aVCFs, and improves clinical outcomes.

PTH (1–34), but not strontium ranelate counteract loss of trabecular thickness and bone strength in disuse osteopenic rats

PTH and strontium ranelate (SrR) have both been shown to reduce bone loss induced by immobilization. PTH is a potent bone anabolic agent, whereas SrR has been suggested to be an antiresorptive as well as a bone anabolic agent.The aim of the study was to investigate whether PTH, SrR, and PTH and SrR in combination could counteract immobilization-induced bone loss in a rat model.Immobilization was induced by injecting 4IU Botox (BTX) into the muscles of the right hind limb. Seventy-two female Wistar rats, 3-months-old, were divided into the following groups: Baseline, Controls, BTX, BTX+PTH, BTX+SrR, and BTX+PTH+SrR (n=12 in each group). PTH was given as injections (SC) at a dosage of 60μg/kg/d, and SrR as 900mg/kg/d in the diet. The experiment lasted for 4weeks.BTX resulted in lower trabecular bone formation rate (−68%) and periosteal bone formation rate (−91%), and a higher fraction of osteoclast-covered surfaces (+53%) compared with controls. This was accompanied by significantly lower trabecular bone volume fraction (−24%), trabecular thickness (−16%), and bone strength (−14% to −32% depending on site). PTH alone counteracted immobilization-induced losses in trabecular (4-fold increase vs. BTX) and periosteal (5-fold increase vs. BTX) bone formation rate, trabecular thickness (+25% vs. BTX) and femoral neck strength (+24% vs. BTX). In contrast, SrR did not influence BTX-induced loss of bone formation rate, trabecular bone volume fraction, trabecular thickness, or bone strength. Finally, no additive effect was found when PTH and SrR treatments were combined.In conclusion, PTH counteracted loss in bone architecture and bone strength in immobilized rats, whereas as no effect of SrR was found. Moreover, no additional effect was found by combining PTH with SrR.

Immobilization induced bone loss is strain specific in mice

Immobilization induced bone loss is strain specific in mice

Increased Sclerostin Serum Levels Associated with Bone Formation and Resorption Markers in Patients with Immobilization-Induced Bone Loss

Sclerostin, a Wnt signaling antagonist on the osteoblasts produced by osteocytes, is regulated by mechanical strain and is implicated in the pathogenesis of disuse bone loss. There are no data on sclerostin in humans. The aim of the study was to evaluate sclerostin in patients immobilized after stroke, compared with control subjects, and to analyze its relationship with markers of bone formation and resorption. This was a cross-sectional study. We studied 40 postmenopausal women immobilized after a single episode of stroke 6 months or longer after onset, and 40 postmenopausal women from the general community. Bone status was assessed by quantitative ultrasound measurements at the calcaneus. Bone alkaline phosphatase (b-AP), carboxy-terminal telopeptide of type I collagen (CrossLaps), and sclerostin were evaluated by ELISA. We also used ELISA to measure serum levels of Dickkopf-1, another soluble inhibitor of Wnt/beta-catenin signaling, highly expressed by osteocytes. Immobilized patients had higher sclerostin serum levels (median 0.975 ng/ml; 25th to 75th percentiles 0.662-1.490) than controls (median 0.300 ng/ml; 25th to 75th percentiles 0.165-0.400: P < 0.0001) and an increased bone turnover with a more significant rise in bone resorption (CrossLaps) than formation (b-AP) markers. Sclerostin correlated negatively with b-AP (r = -0.911; P < 0.0001) and positively with CrossLaps (r = 0.391; P = 0.012). Dickkopf-1 did not significantly differ between the groups. Patients also had quantitative ultrasound measurements index lower than controls (P < 0.001). This study shows for the first time that long-term immobilized patients present hypersclerostinemia associated with reduced bone formation, and suggests that sclerostin could be a link between mechanical unloading and disuse osteoporosis in humans.

Bone strength is maintained after 8 months of inactivity in hibernating golden-mantled ground squirrels, Spermophilus lateralis

Prolonged inactivity leads to disuse atrophy, a loss of muscle and bone mass. Hibernating mammals are inactive for 6-9 months per year but must return to full activity immediately after completing hibernation. This necessity for immediate recovery presents an intriguing conundrum, as many mammals require two to three times the period of inactivity to recover full bone strength. Therefore, if hibernators experience typical levels of bone disuse atrophy during hibernation, there would be inadequate time available to recover during the summer active season. We examined whether there were mechanical consequences as a result of the extended inactivity of hibernation. We dissected femur and tibia bones from squirrels in various stages of the annual hibernation cycle and measured the amount of force required to fracture these bones. Three groups were investigated; summer active animals were captured during the summer and immediately killed, animals in the 1 month detraining group were captured in the summer and killed following a 1-month period of restricted mobility, hibernating animals were killed after 8 months of inactivity. A three-point bend test was employed to measure the force required to break the bones. Apparent flexural strength and apparent flexural modulus (material stiffness) were calculated for femurs. There were no differences between groups for femur fracture force, tibia fracture force, or femur flexural strength. Femur flexural modulus was significantly less for the 1 month detraining group than for the hibernation and summer active groups. Thus, hibernators seem resistant to the deleterious effects of prolonged inactivity during the winter. However, they may be susceptible to immobilization-induced bone loss during the summer.

Bone loss in the lower leg during 35 days of bed rest is predominantly from the cortical compartment

Immobilization-induced bone loss is usually greater in the epiphyses than in the diaphyses. The larger fraction of trabecular bone in the epiphyses than in the diaphyses offers an intuitive explanation to account for this phenomenon. However, recent evidence contradicts this notion and suggests that immobilization-induced bone loss from the distal tibia epiphysis is mainly from the cortical compartment. The aim of this study was to establish whether this pattern of bone loss was a general rule during immobilization. We monitored various skeletal sites with different tissue composition during 5 weeks of immobilization.Ten healthy male volunteers with mean age of 24.3 years (SD 2.6 years) underwent strict horizontal bed rest. Bone scans were obtained during baseline data collection, at the end of bed rest and after 14 days of recovery by peripheral Quantitative Computed Tomography (pQCT). Sectional images were obtained from the distal tibia epiphysis (at 4% of the tibia's length), from the diaphysis (at 38%), from the proximal metaphysis (at 93%) and from the proximal epiphysis (at 98%), as well as from the distal femur epiphysis (at 4% of the femur's length) and from the patella.Relative bone losses were largest at the patella, where they amounted to −3.2% (SD 1.8%, p<0.001) of the baseline values, and smallest at the tibia diaphysis, where they amounted to −0.7% (SD 1.0%, p=0.019). The relative losses were generally larger from cortical than from trabecular compartments (p=0.004), and whilst all skeletal sites depicted such cortical losses, substantial trabecular losses were found only from the proximal tibia epiphysis.Results confirm that the differential losses from the various skeletal sites cannot be explained on the basis of trabecular vs. cortical tissue composition differences, but that endocortical circumference can account for the different amounts of bone loss in the tibia. The present study therefore supports the suggestion of the subendocortical layer as a transitional zone, which can readily be transformed into trabecular bone in response to immobilization. The latter will lead to cortical thinning, a factor that has been associated with the risk of fracture and with osteoarthritis.

Thermostability of bone tissue after immobilization induced osteopenia in a rat model.

Immobilization of load-bearing bones results in imbalance of bone turnover followed by bone loss and impairment of its mechanical function. The question is whether immobilization induced bone loss is accompanied by deterioration of properties of the bone tissue components. Thermally induced transformations of collagen reflect the overall condition of the structure and cross-links in collagen network. The aim of the present study was to investigate whether immobilization induced osteopenia effects stability of collagen in bone tissue. Bone loss was developed by unilateral hindlimb immobilization in adult rats. Effects of unloading on cortical tissue from tibiae were studied after three weeks of unloading (I3R0) and four weeks after remobilization and free convalescence (I3R4) in both tibiae. Thermodynamic parameters of collagen degradation in bone were determined from differential scanning calorimetry (DSC) analysis of partially dehydrated cortical bone samples from tibiae in the range of temperatures from 60 degrees C up to 300 degrees C. All bone samples were thermally very stable showing first clear endothermal process with a peak temperature within a range from 150 degrees C to 169 degrees C, for different samples. The next endotherm, wider and flatter, was observed between 245-298 degrees C with a peak at 255 degrees C - 260 degrees C. There were significant side-to-side (right to left) differences for both endothermal processes in tibiae samples from experimental groups: I3R0 and I3R4. Immobilization of load-bearing bones influences stability of collagen in bone tissue. Free remobilization was not sufficient for recovery of thermal parameters of bone.

Protective effect of female gender against bone loss in the forearm following clean-cut tendon injuries, repair, and passive mobilization

The aim of this study was to investigate whether there is any significant difference in bone loss between female and male patients as a result of early passive mobilization after surgery for acute tendon-artery-nerve clean-cut injuries at the wrist level. A total of 51 patients who underwent such operations were enrolled in this study. Group I consisted of 40 male patients (mean age 30.2 +/- 9.1 years, age range 15-44 years) and group II consisted of 11 female patients (mean age 24.1 +/- 9.9 years, age range 14-43 years); postmenopausal women were not included in this group. Twenty-one patients in group I and six patients in group II were operated on for clean-cut tendon injury combined with nerve injury, artery injuries, or both. Bone mineral density (BMD) measurements of injuired forearms were obtained at 1 week, 6 weeks, 3 months, and 12 months after operation. The BMD of the radius and ulna did not change significantly throughout the follow-up period in group II (P > 0.05). In group I, BMD values were reduced significantly in the distal regions of the ulna and in the ultradistal region of the radius at the follow-up measurements when compared to the values at week 1 and the reduction was higher in month 3 than at other times. On the other hand, after 12 months, the BMD values of the ultra-distal region of the ulna and radius were higher than those after 3 months. In conclusion, our results showed that there is considerable bone loss of the radius and ulna in patients operated on for acute clean-cut tendon injuries on the volar side of the wrist at postoperative month 3 in male patients. These bone losses were recovered after 12 months. Additionally, female patients are less likely to experience immobilization-induced bone loss than male patients.

Serum Markers of Bone Metabolism Show Bone Loss in Hibernating Bears

Disuse osteopenia was studied in hibernating black bears (Ursus americanus) using serum markers of bone metabolism. Blood samples were collected from male and female, wild black bears during winter denning and active summer periods. Radioimmunoassays were done to determine serum concentrations of cortisol, the carboxy-terminal cross-linked telopeptide, and the carboxy-terminal propeptide of Type I procollagen, which are markers of bone resorption and formation, respectively. The bone resorption marker was significantly higher during winter hibernation than it was in the active summer months, but the bone formation marker was unchanged, suggesting an imbalance in bone remodeling and a net bone loss during disuse. Serum cortisol was significantly correlated with the bone resorption marker, but not with the bone formation marker. The bone formation marker was four- to fivefold higher in an adolescent and a 17-year-old bear early in the remobilization period compared with the later summer months. These findings raise the possibility that hibernating black bears may minimize bone loss during disuse by maintaining osteoblastic function and have a more efficient compensatory mechanism for recovering immobilization-induced bone loss than that of humans or other animals.

Treatment with risedronate or alendronate prevents hind-limb immobilization-induced loss of bone density and strength in adult female rats

Immobilization leads to rapid loss of bone mass and mechanical competence, and long-term immobilization or repeated periods of short-term immobilization can have serious skeletal consequences and may lead to increased fracture liability. The aim of the present preclinical study was, therefore, to assess whether two antiresorptive agents, risedronate (Ris) or alendronate (Aln), would be capable of preventing immobilization-induced loss of bone mass and strength in rats. The study was designed as a dose-response study, and the site-specific effects of immobilization and of treatment are described. Four-month-old virgin female Sprague-Dawley rats were divided into eight groups with 12 animals in each group: (1) immobilized (Imm) control; (2) normal control; (3) Imm + Ris 0.1 mg/kg body weight/day (b.w./day); (4) Imm + Ris 0.2 mg/kg b.w./day; (5) Imm + Ris 1.0 mg/kg b.w./day; (6) Imm + Aln 0.2 mg/kg b.w./day; (7) Imm + Aln 1.0 mg/kg b.w./day; and (8) Imm + Aln 2.0 mg/kg b.w./day. In groups 1 and 3–8, the right hind leg was immobilized with an elastic bandage. The study period was 28 days. The effects of unilateral hind-limb immobilization and of treatment were determined by dual-energy X-ray absorptiometry (DEXA) measurements on tibiae and by biomechanical testing of femora at three different sites: diaphysis; femoral neck; and distal metaphysis. Bilateral measurements were performed (on the immobilized and nonimmobilized legs). Immobilization induced a significant loss of bone mineral density (BMD) at the proximal tibial metaphysis, but no change at the middiaphysis. Furthermore, immobilization induced a loss of bone strength at the two femoral metaphyses, but no change was seen in three-point bending of the diaphysis. Both risedronate and alendronate treatment showed a dose-dependent protection against the immobilization-induced loss of bone density and strength at the metaphyses. We conclude that, in rats, short-term hind-limb immobilization affects only the metaphyses and that no changes are seen in the diaphysis. Both risedronate and alendronate can prevent immobilization-induced bone loss at the metaphyses. The present study confirms the importance of examining several skeletal sites when testing the efficacy of therapeutic agents.

Short term immobilization-induced cancellous bone loss is limited to regions undergoing high turnover and/or modeling in mature rats

Estrogen and calcium deficiencies increase both bone resorption and formation, whereas immobilization mainly decreases bone formation. How these functionally different risk factors for bone loss interact in cancellous bone undergoing modeling or remodeling activity is not well understood. Mature (6-month-old) female rats were subjected to sham operation (sham), ovariectomy (ovx), dietary calcium deficiency (LoCa, 0.1% Ca), and sciatic and femoral denervation (IM), ovx + IM, or LoCa + IM for 4 weeks. The primary spongiosa, the region of active modeling within 1 mm of the growth plate, in ovx, LoCa, and IM groups showed a decrease in cancellous bone volume, trabecular number, and connectivity when compared to sham controls. Groups combining two risk factors exhibited additive changes when compared with single risk factor groups. In the secondary spongiosa, an area with little modeling activity, ovx and LoCa groups, as expected, lost bone. In contrast with the primary spongiosa, IM alone did not induce bone loss in the secondary spongiosa, and the groups with a combination of IM and ovx or IM and LoCa showed a greater bone loss than either ovx or LoCa alone. Ovx and LoCa groups showed increases in both bone formation rate and eroded surface in the secondary spongiosa, while IM groups showed a decrease in bone formation rate. Combining IM with either ovx or LoCa resulted in increased eroded surface. The effects on cortical bone were assessed at the tibio-fibular junction. A trend toward decreased percentage of cortical bone area and an increase in marrow cavity area were observed in the combined deficiency groups only. These changes were the result of a statistically significant increase in endosteal eroded surface in IM + ovx and IM + LoCa groups. Our results demonstrate that immobilization-induced bone loss is restricted to the primary spongiosa where most modeling events occur. However, the inhibitory effect of IM on bone formation in the secondary spongiosa is unmasked in remodeling sites when a high turnover state is provided by either estrogen or dietary calcium deficiency. These results suggest that the presence of a risk factor, such as immobilization, which in the short term causes inhibition of bone formation, does not predispose the skeleton to rapid cancellous bone loss except when accompanied by modeling or high turnover.

Demonstration of feasibility of automated osteoblastic line culture in space flight

There is a large body of evidence that microgravit- or immobilization-induced bone loss is mainly related to osteoblastic cell impairment. Osteoblasts are sensitive to increased mechanical stress and could therefore be responsible for unloading-induced bone changes. However, the nature of osteoblast involvement remains unclear. The effects of the space environment on cells have been studied extensively, but little information about anchorage-dependent cell cultures of the 25 different cell types flown in space has been published. We studied the effects of long-term weightlessness on the cell shape of cultured osteoblasts during the Russian Bion 10 space-flight. This experiment required the development of special automatic culture devices (the plunger-box culture system) finalized with the constructors. Multiple feasibility experiments were performed to allow osteoblast culture for 6 days in microgravity. The study revealed plunger-box biocompatibility; optimization of ROS 17/2.8 (mammalian adherent cells) culture under closed conditions (without gas exchange); and transport of viable cells for 5 days. During the 6 days of microgravity, the growth curves of ground controls and cells in space were roughly similar. Alkaline phosphatase activity was enhanced twofold in microgravity. ROS 17/2.8 cell morphology began to change significantly after 4 days of microgravity; they became rounder and covered with microvilli. At the end of the flight, the cells exhibited mixed morphological types, piling cells, stellar shape, and spread out cells, resembling ground controls or 1g flight controls (centrifuge). We demonstrated that ROS 17/2.8 cells were viable during a 6 day automatic culture in space and were sensitive to space related conditions. They adapted their structure and function to this environment, characterized by loss of mechanical stimuli.

The effect of training on the recovery from immobilization-induced bone loss in rats.

We studied the effect of exercise on the recovery from osteoporosis induced by immobilization in growing laboratory rats. The right hind limb of male rats was immobilized by cast for 2 weeks. The rats were then divided into control and exercise groups. The controls were allowed to move freely in their cages and the exercise group was running for 1 hour every day. After 2 weeks immobilization, the ash weight of the immobilized tibia decreased to 85.2 +/- 0.6% (P less than 0.001) from that of contralateral tibia. After removal of the cast, the bone mass recovered and, in 9 weeks, the decrease of ash weight was reduced to 2.9 +/- 0.8% and 4.2 +/- 0.3% in the control and exercise groups, respectively. There was no statistical difference in ash weights between the running and control groups and the trabecular bone volume in the distal femur was also equal in both groups. Immobilization reduced the incorporation of [45Ca] into the tibia. After remobilization, the [45Ca]-incorporation was found to be significantly higher in the recovering leg than in the control leg. Exercise further stimulated [45Ca]-incorporation into the recovering tibia.

Prostaglandin E 2 prevents disuse-induced cortical bone loss

The object of this study was to determine whether prostaglandin E 2 (PGE 2) can prevent disuse (underloaded)-induced cortical bone loss as well as add extra bone to underloaded bones. Thirteen-month-old retired female Sprague-Dawley breeders served as controls or were subjected to simultaneous right hindlimb immobilization by bandaging and daily subcutaneous doses of 0,1,3, or 6 mg PGE 2/kg/d for two and six weeks. Histomorphometric analyses were performed on double-fluorescent labeled undecalcified tibial shaft sections (proximal to the tibiofibular junction). Disuse-induced cortical bone loss occurred by enlarging the marrow cavity and increasing intracortical porosity. PGE 2 treatment of disuse shafts further increased intracortical porosity above that in disuse alone controls. This bone loss was counteracted by enhancement of periosteal and corticoendosteal bone formation. Stimulation of periosteal and corticoendosteal bone formation slightly enlarged the total tissue (cross-sectional) area and inhibited marrow cavity enlargement. These PGE 2-induced activities netted the same percentage of cortical bone with a different distribution than the beginning and agerelated controls. These findings indicate the PGE 2-induced increase in bone formation compensated for the disuse and PGE 2-induced bone loss, and thus prevented immobilizationinduced bone loss.