Osteopenia and sarcopenia develops rapidly during disuse. The study investigated whether intermittent parathyroid hormone (1-34) (PTH) and growth hormone (GH) administered alone or in combination could prevent or mitigate disuse osteopenia and sarcopenia in rats. Disuse was achieved by injecting 4IU botulinum toxin A (BTX) into the right hindlimb musculature of 12-14-week-old female Wistar rats. Seventy-two rats were divided into six groups: 1. Baseline; 2. Ctrl; 3. BTX; 4. BTX+GH; 5. BTX+PTH; 6. BTX+PTH+GH. PTH (1-34) (60μg/kg/day) and GH (5mg/kg/day). The animals were sacrificed after 6weeks of treatment. Sarcopenia was established by histomorphometry, while the skeletal properties were determined using DXA, μCT, mechanical testing, and dynamic bone histomorphometry. Disuse resulted in lower muscle mass (-63%, p<0.05), trabecular BV/TV (-28%, p<0.05), Tb.Th (-11%, p<0.05), lower diaphyseal cortical thickness (-10%, p<0.001), and lower bone strength at the distal femoral metaphysis (-27%, p<0.001) compared to Ctrl animals. PTH fully counteracted the immobilization-induced lower BV/TV, Tb.Th, and distal femoral metaphyseal strength. GH increased muscle mass (+17%, p<0.05) compared to BTX, but did not prevent the immobilization-induced loss of bone strength, BV/TV, and cortical trabecular thickness. Combination of PTH and GH increased distal femoral metaphyseal bone strength (+45%, p<0.001), BV/TV (+50%, p<0.05), Tb.Th (+40%, p<0.05), and whole femoral aBMD (+15%, p<0.001) compared to BTX and muscle mass (+21%, p<0.05) compared to BTX+PTH. In conclusion, PTH and GH in combination is more efficient at preventing the disuse-related deterioration of bone strength, density, and micro-architecture than either PTH or GH given as monotherapy. Furthermore, GH, either alone or in combination with PTH, attenuated disuse-induced loss of muscle mass. The combination of PTH and GH resulted in a more effective treatment than PTH and GH as monotherapy.

Damage of the lower motor neuron cell bodies or their axons results in reduced or abolished voluntary movement accompanied by a substantial loss of bone and muscle mass. Intermittent parathyroid hormone 1-34 (PTH) (teriparatide) is one of the most potent bone-anabolic treatment regimens. ActRIIA-mFc is an activin type IIA decoy receptor that increases bone mass mediated by inhibition of the activin receptor signaling pathway. We investigated whether PTH or ActRIIA-mFc alone or in combination could prevent loss of bone and muscle mass induced by injecting botulinum toxin A (BTX) into the right hind limb in mice. Seventy-two 16-week-old female C57BL/6 mice were allocated to the following groups: Baseline, Control, BTX, BTX + ActRIIA-mFc (10 mg/kg), BTX + PTH (100 μg/kg), and BTX + ActRIIA-mFc + PTH. The mice were sacrificed after three weeks of disuse and treatment. In contrast to monotherapy with PTH, ActRIIA-mFc alone or in combination with PTH was able partly or completely to prevent disuse-induced loss of whole femoral bone mass, trabecular thickness, and bone strength. Moreover, an additive effect of ActRIIA-mFc and PTH on areal bone mineral density and trabecular bone volume was found. In summary, ActRIIA-mFc and PTH in combination were more effective in preventing disuse-induced bone loss and deterioration of trabecular micro-architecture than either treatment alone.

Mountaineers at high altitude are at increased risk of acute mountain sickness as well as high altitude pulmonary and cerebral edema. A densitometric study in mountaineers has suggested that expeditions at high altitude decrease bone mineral density. Surprisingly, the in vivo skeletal effects of hypobaric hypoxia are largely unknown, and have not been studied using advanced contemporary methods to assess bone microstructure. Eighty-four 22-week-old female mice were divided into seven groups with 12 mice in each group: 1. Baseline; 2. Normobaric, 4 weeks; 3. Hypobaric hypoxia, 4 weeks; 4. Normobaric, 8 weeks; 5. Hypobaric hypoxia, 8 weeks; 6. Normobaric, 12 weeks; and 7. Hypobaric hypoxia, 12 weeks. Hypobaric hypoxia mice were housed in hypobaric chambers at an ambient pressure of 500 mbar (5500 m altitude), while normobaric mice were housed at sea level atmospheric pressure for 4, 8, or 12 weeks, respectively. Hypobaric hypoxia had a profound impact on femoral cortical bone and L4 trabecular bone, while the effect on femoral trabecular bone was less pronounced. Hypobaric hypoxia reduced the bone strength of the femoral mid-diaphysis and L4 at all time-points. At femoral cortical bone, hypobaric hypoxia reduced bone formation through fewer mineralizing surfaces and lower bone formation rate after 2 weeks. In addition, bone strength decreased, and C-terminal telopeptide of type I collagen (CTX-I) increased independently of the duration of exposure to simulated high altitude. At L4, hypobaric hypoxia resulted in a substantial reduction in bone volume fraction, trabecular thickness, and trabecular number after 4 weeks of exposure. Hypobaric hypoxia reduced bone strength and femoral bone mass, while femoral trabecular bone was much less affected, indicating the skeletal response to hypobaric hypoxia differ between cortical and trabecular bone. These findings provide initial preclinical support for future clinical studies in mountaineers to assess bone status and bone strength after exposure to prolonged high altitude exposure.

In rodents, lactation is associated with a considerable and very rapid bone loss, which almost completely recovers after weaning. The aim of the present study was to investigate whether the bisphosphonate Zoledronate (Zln) can inhibit lactation induced bone loss, and if Zln interferes with recovery of bone mass after lactation has ceased. Seventy-six 10-weeks-old NMRI mice were divided into the following groups: Baseline, Pregnant, Lactation, Lactation+Zln, Recovery, Recovery+Zln, and Virgin Control (age-matched). The lactation period was 12days, then the pups were removed, and thereafter recovery took place for 28days. Zln, 100μg/kg, was given s.c. on the day of delivery, and again 4 and 8days later. Mechanical testing, μCT, and dynamic histomorphometry were performed. At L4, lactation resulted in a substantial loss of bone strength (-55% vs. Pregnant, p<0.01), BV/TV (-40% vs. Pregnant, p<0.01), and trabecular thickness (Tb.Th) (-29% vs. Pregnant, p<0.001). Treatment with Zln completely prevented lactation induced loss of bone strength, BV/TV, and Tb.Th at L4. Full recovery of micro-architectural and mechanical properties was found 28days after weaning in vehicle-treated mice. Interestingly, the recovery group treated with Zln during the lactation period had higher BV/TV (+45%, p<0.01) and Tb.Th (+16%, p<0.05) compared with virgin controls. Similar results were found at the proximal tibia and femur. This indicates that Zln did not interfere with the bone formation taking place after weaning. On this background, we conclude that post-lactation bone formation is not dependent on a preceding lactation induced bone loss.

Immobilization is known to cause a rapid bone loss due to increased osteoclastic bone resorption and decreased osteoblastic bone formation. Zoledronate (Zln) is a potent anti-resorptive pharmaceutical, while intermittent PTH is a potent bone anabolic agent. The aim of the present study was to investigate whether PTH or Zln alone or in combination could prevent immobilization-induced osteopenia. Immobilization was achieved by injecting 4IU Botox (BTX) into the right hind limb musculature. Seventy-two 16-week-old female Wistar rats were randomized into 6 groups; baseline (Base), control (Ctrl), BTX, BTX+PTH, BTX+Zln, and BTX+PTH+Zln. PTH (1-34) (80μg/kg) was given 5days/week and Zln (100μg/kg) was given once at study start. The animals were killed after 4weeks of treatment. The bone properties were evaluated using DEXA, μCT, dynamic bone histomorphometry, and mechanical testing. BTX resulted in lower femoral trabecular bone volume fraction (BV/TV) (-25%, p<0.05), lower tibial trabecular bone formation rate (BFR/BS) (-29%, p<0.05), and lower bone strength (Fmax) at the distal femur (-19%, p<0.001) compared with Ctrl. BTX+PTH resulted in higher femoral BV/TV (+31%, p<0.05), higher tibial trabecular BFR/BS (+297%, p<0.05), and higher Fmax at the distal femur (+11%, p<0.05) compared with BTX. BTX+Zln resulted in higher femoral BV/TV (+36%, p<0.05), lower tibial trabecular BFR/BS (-93%, p<0.05), and higher Fmax at the distal femur (+10%, p<0.05) compared with BTX. BTX+PTH+Zln resulted in higher femoral BV/TV (+70%, p<0.001), higher tibial trabecular BFR/BS (+59%, p<0.05), and higher Fmax at the distal femur (+32%, p<0.001) compared with BTX. In conclusion, BTX-induced immobilization led to lower BV/TV, BFR/BS, and Fmax. In general, PTH or Zln alone prevented the BTX-induced osteopenia, whereas PTH and Zln given in combination not only prevented, but also increased BV/TV and BFR/BS, and maintained Fmax at the distal femoral metaphysis compared with Ctrl.

Prolonged disuse and substantial mechanical unloading are particularly damaging to skeletal integrity. Preclinical studies in rodents and clinical studies have highlighted the need for potent bone anabolic drugs to counteract disuse-induced osteopenia. The aim of present study was to compare the efficacy of romosozumab (Scl-Ab) and abaloparatide (ABL), alone or in combination, to prevent botulinum toxin (BTX) induced bone loss in a rat model. Eighty female Wistar rats were divided into the following six groups: 1. Baseline (n = 12); 2. Control (Ctrl) (n = 12); 3. BTX (n = 12); 4. BTX + Scl-Ab (n = 16); 5. BTX + ABL (n = 12); and 6. BTX + Scl-Ab + ABL (n = 16). Disuse was achieved by injecting 4 IU BTX into the hind limb musculature at study start. Scl-Ab (25 mg/kg) was injected s.c. twice weekly, while ABL (80 μg/kg) was injected s.c. five days a week for four weeks. Hind limb disuse dramatically decreased muscle mass and skeletal integrity and deteriorated the cortical morphology and trabecular microstructure. Treatment with Scl-Ab alone prevented most of the adverse cortical and trabecular effects of disuse, while ABL monotherapy mainly attenuated the disuse-induced loss of femoral areal bone mineral density (aBMD). Moreover, the combination of Scl-Ab and ABL not only counteracted most of the negative skeletal effects of unloading, but also increased aBMD (+10% and +20%), epiphyseal trabecular bone volume fraction (BV/TV) (+25% and +73%), and metaphyseal bone strength (+18% and +30%) significantly above that of Scl-Ab or ABL monotherapy, respectively. The potent and additive osteoanabolic effect of Scl-Ab and ABL, when given in combination, is highly intriguing and underlines that an osteoanabolic bone gain can be maximized by utilizing two pharmaceuticals targeting different cellular signaling pathways. From a clinical perspective, a combination treatment may be warranted in patients where the osteoanabolic effect of either monotherapy is not sufficient, or if a dose-reduction is required due to adverse effects.

The ability of osteocytes to demineralize the perilacunar matrix, osteocytic osteolysis, and thereby participate directly in bone metabolism, is an aspect of osteocyte biology that has received increasing attention during the last couple of years. The aim of the present work was to investigate whether osteocyte lacunar properties change during immobilization and subsequent recovery. A rat cortical bone model with negligible Haversian remodeling effects was used, with temporary immobilization of one hindlimb induced by botulinum toxin. Several complementary techniques covering multiple length scales enabled correlation of osteocyte lacunar properties to changes observed on the organ and tissue level of femoral bone. Bone structural parameters measured by μCT and mechanical properties were compared to sub-micrometer resolution SR μCT data mapping an unprecedented number (1.85 million) of osteocyte lacunae. Immobilization induced a significant reduction in aBMD, bone volume, tissue volume, and load to fracture, as well as the muscle mass of rectus femoris. During the subsequent recovery period, the bone structural and mechanical properties were only partly regained in spite of a long-term (28weeks) study period. No significant changes in osteocyte lacunar volume, density, oblateness, stretch, or orientation were detected upon immobilization or subsequent recovery. In conclusion, the bone architecture and not osteocyte lacunar properties or bone material characteristics dominate the immobilization response as well as the subsequent recovery.

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.