Skeletal unloading induces resistance to insulin-like growth factor-I (IGF-I) by inhibiting activation of the IGF-I signaling pathways.

We showed that unloading markedly diminished the effects of IGF-I to activate its signaling pathways, and the disintegrin echistatin showed a similar block in osteoprogenitor cells. Furthermore, unloading decreased alphaVbeta3 integrin expression. These results show that skeletal unloading induces resistance to IGF-I by inhibiting activation of the IGF-I signaling pathways at least in part through downregulation of integrin signaling. INTRODUCTION: We have previously reported that skeletal unloading induces resistance to insulin-like growth factor-I (IGF-I) with respect to bone formation. However, the underlying mechanism remains unclear. The aim of this study was to clarify how skeletal unloading induces resistance to the effects of IGF-I administration in vivo and in vitro with respect to bone formation. MATERIALS AND METHODS: We first determined the response of bone to IGF-I administration in vivo during skeletal unloading. We then evaluated the response of osteoprogenitor cells isolated from unloaded bones to IGF-I treatment in vitro with respect to activation of the IGF-I signaling pathways. Finally we examined the potential role of integrins in mediating the responsiveness of osteoprogenitor cells to IGF-I. RESULTS: IGF-I administration in vivo significantly increased proliferation of osteoblasts. Unloading markedly decreased proliferation and blocked the ability of IGF-I to increase proliferation. On a cellular level, IGF-I treatment in vitro stimulated the activation of its receptor, Ras, ERK1/2 (p44/42 MAPK), and Akt in cultured osteoprogenitor cells from normally loaded bones, but these effects were markedly diminished in cells from unloaded bones. These results were not caused by altered phosphatase activity or changes in receptor binding to IGF-I. Inhibition of the Ras/MAPK pathway was more impacted by unloading than that of Akt. The disintegrin echistatin (an antagonist of the alphaVbeta3 integrin) blocked the ability of IGF-I to stimulate its receptor phosphorylation and osteoblast proliferation, similar to that seen in cells from unloaded bone. Furthermore, unloading significantly decreased the mRNA levels both of alphaV and beta3 integrin subunits in osteoprogenitor cells. CONCLUSION: These results indicate that skeletal unloading induces resistance to IGF-I by inhibiting the activation of IGF-I signaling pathways, at least in part, through downregulation of integrin signaling, resulting in decreased proliferation of osteoblasts and their precursors.     

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.     

Early Response of Bone Marrow Osteoprogenitors to Skeletal Unloading and Sclerostin Antibody

Sclerostin functions as an antagonist to Wnt signaling and inhibits bone-forming activity. We studied the effects of skeletal unloading and treatment with sclerostin antibody (Scl-Ab) on mesenchymal stem cell, osteoprogenitor and osteoclast precursor pools, and their relationship to bone formation and resorption. Male C57BL/6 mice (5-months-old) were hind limb unloaded for 1 week or allowed normal ambulation and treated with Scl-Ab (25 mg/kg, s.c. injections on days 1 and 4) or placebo. Unloading decreased the serum concentration of bone formation marker P1NP (-35 %), number of colony-forming units (CFU) (-38 %), alkaline phosphatase-positive CFUs (CFU-AP+) (-51 %), and calcified nodules (-35 %); and resulted in a fourfold increase in the number of osteoclast precursors. The effects of Scl-Ab treatment on unloaded and normally loaded mice were nearly identical; Scl-Ab increased serum P1NP and the number of CFU, CFU-AP+, and calcified nodules in ex vivo cultures; and increased osteoblast and bone mineralizing surfaces in vivo. Although the marrow-derived osteoclast precursor population increased with Scl-Ab, the bone osteoclast surface did not change, and the serum concentration of osteoclast activity marker TRACP5b decreased. Our data suggest that short-term Scl-Ab treatment can prevent the decrease in osteoprogenitor population associated with skeletal unloading and increase osteoblast surface and bone mineralizing surface in unloaded animals. The anabolic effects of Scl-Ab treatment on bone are preserved during skeletal unloading. These findings suggest that Scl-Ab treatment can both increase bone formation and decrease bone resorption, and provide a new means for prevention and treatment of disuse osteoporosis.     

The effect of reloading on bone volume, osteoblast number, and osteoprogenitor characteristics: studies in hind limb unloaded rats

Skeletal unloading during space flight results in bone loss. In astronauts the extent to which bone is lost varies greatly between different bones of the skeleton as well as between different individuals. Following return to earth, recovery of bone mass during reloading also varies between different bones and different individuals. Due to this variability and the limited number of subjects it is difficult to study the effects of unloading/reloading on bone in humans. A viable alternative is to use the rat model of hind limb unloading developed at NASA. We have previously demonstrated that, in 6-week-old male rats, 14 days of unloading result in a decrease in osteoprogenitor number in cell populations isolated from the proximal femur. The goal of the present study was to determine the number of osteoprogenitor cells present in cell populations derived from the proximal femur of young rats after 14 days of unloading followed by 14 days of reloading and to characterize their proliferative capacity. To do this, we determined the number of alkaline phosphatase-positive colony forming units (CFU-AP) and osteoblast CFU (CFU-O). To establish whether the effects of unloading and reloading were specific for cells of the osteoblast lineage, we also determined the number of fibroblastic CFU (CFU-F). Effects on proliferation were evaluated by measuring the size of CFU-O. Unloading resulted in a 66% reduction in CFU-AP. CFU-O numbers were decreased by 76% and mean colony size was 33% less than controls. The decrease in osteogenic and osteoprogenitor cells in vitro paralleled the decrease in bone volume (- 50%), osteoblast number (- 35%), and bone formation rate (- 46%) observed in the proximal tibial metaphysis of unloaded rats. Unloading had no effect on osteoclast number or surface. Subsequent reloading for 14 days restored CFU-AP. CFU-O numbers were only partially restored at 14 days (83% of controls) but nodule size was 1.2-fold greater than controls. Neither unloading nor reloading had an effect on the total number of progenitors (CFU-F). Reloading restored bone volume back to control values, but osteoblast number and bone formation rate were still lower than those in corresponding controls. Both osteoclast number and surface were lower in reloaded animals than in age-matched controls. Our results indicate that 14 days of unloading result in a decrease in osteoprogenitor number and that reloading for 14 subsequent days completely restores CFU-AP and bone volume to control levels, while the number of CFU-O in vitro and osteoblasts in vivo were partially recovered but still lower than corresponding controls. Strikingly, osteoclastic bone resorption after 14 days of reloading was greatly reduced compared to controls, suggesting a significant contribution of this to the recovery process.     

Ubiquitin ligase Cbl-b downregulates bone formation through suppression of IGF-I signaling in osteoblasts during denervation.

Unloading can prevent bone formation by osteoblasts. To study this mechanism, we focused on a ubiquitin ligase, Cbl-b, which was highly expressed in osteoblastic cells during denervation. Our results suggest that Cbl-b may mediate denervation-induced osteopenia by inhibiting IGF-I signaling in osteoblasts. INTRODUCTION: Unloading, such as denervation (sciatic neurectomy) and spaceflight, suppresses bone formation by osteoblasts, leading to osteopenia. The resistance of osteoblasts to growth factors contributes to such unloading-mediated osteopenia. However, a detailed mechanism of this resistance is unknown. We first found that a RING-type ubiquitin ligase, Cbl-b, was highly expressed in osteoblastic cells after sciatic neurectomy in mice. In this study, we reasoned that Cbl-b played an important role in the resistance of osteoblasts to IGF-I. Materials AND METHODS: Cbl-b-deficient (Cbl-b(-/-)) or wildtype (Cbl-b(+/+)) mice were subjected to sciatic neurectomy. Bone formation in these mice was assessed by calcein labeling and histomorphometric analyses. We examined IGF-I signaling molecules in femora of these mice by Western blot and immunohistochemical analyses. We also examined the mitogenic response of Cbl-b-overexpressing or -deficient osteoblastic cells to various growth factors. RESULTS: In Cbl-b(+/+) mice, denervation decreased femur mass and bone formation, whereas it increased the expression of Cbl-b protein in osteoprogenitor cells and in osteocalcin-positive cells (osteoblastic cells) in hindlimb bone. In contrast, in Cbl-b(-/-) mice, bone mass and bone formation were sustained during denervation. Denervation inhibited the mitogenic response of osteoprogenitor cells most significantly to IGF-I. Therefore, we focused on Cbl-b-mediated modification of IGF-I signaling. Denervation decreased the amounts of insulin receptor substrate-1 (IRS-1), phosphatidly inositol 3-phosphate kinase (PI3K), and Akt-1 proteins in femora of Cbl-b(+/+) mice, whereas the amounts of these IGF-I signaling molecules in femora of Cbl-b(-/-) mice were constant after denervation. On a cellular level, primary osteoblastic cells from Cbl-b(-/-) mice were more stimulated to proliferate by IGF-I treatment compared with those from Cbl-b(+/+) mice. Furthermore, overexpression of Cbl-b increased ubiquitination and degradation of IRS-1 in primary Cbl-b(-/-) osteoblastic cells, leading to their impaired mitogenic response to IGF-I. CONCLUSIONS: These results suggest that Cbl-b induces resistance of osteoblasts to IGF-I during denervation by increasing IRS-1 degradation and that Cbl-b-mediated modification of IGF-I signaling may contribute to decreased bone formation during denervation.     

Protracted Systemic Changes in Bone Biology after Segmented Unloading in the Rat

To investigate whether the decreased bone formation observed in most experimental situations of disuse was caused by an increased inhibition by the bone microenvironment of osteoblast (OB) proliferation, we studied the inhibiting power on ROS 17/2.8 proliferation of the bone marrow extracellular fluid (IPEF) in loaded and unloaded bones of rats submitted to two situations of partial disuse: tail suspension (TS) for 3 days to 2 weeks and around the knee tenectomy (KT) for 2–10 weeks. Histomorphometric parameters and osteoblast precursors dynamics were studied in parallel. Bone volume was lost in the unloaded bones, but not in loaded bones, in both experimental situations. Bone formation was low at early times (7 - 14 days) in TS rats. However, in KT at later times (4 - 10 weeks), the osteoblastic index of the unloaded tibia was increased. IPEF was not increased in the unloaded bones 3–7 days after TS. It was decreased later in the course of unloading (after 2 weeks of TS and 2–10 weeks after KT). This decrease was observed in the loaded bones as well. Unexpectedly, we also found that the number of FCFUs was decreased in both loaded and unloaded limbs in TS and KT, and that the yield of cells obtained in primary culture from tibial metaphysis was decreased in both tibiae from KT animals. These data show that an increased IPEF does not play a role in the early inhibition of bone formation responsible for the loss of bone after unloading in the TS model. Its later decrease could be permissive for the increased osteoblastic index observed in the KT model. They also show that, contrary to the usual assumptions, bone biology is changed all over the skeleton after partial unloading, even if the changes result in bone loss in the unloaded bones only. Thus, as yet, unidentified systemic factors probably superimpose on the local factors that control bone volume.     

Mechanical Load Increases in Bone Formation via a Sclerostin‐Independent Pathway

Sclerostin, encoded by the Sost gene, is an important negative regulator of bone formation that has been proposed to have a key role in regulating the response to mechanical loading. To investigate the effect of long‐term Sclerostin deficiency on mechanotransduction in bone, we performed experiments on unloaded or loaded tibiae of 10 week old female Sost?/? and wild type mice. Unloading was induced via 0.5U botulinum toxin (BTX) injections into the right quadriceps and calf muscles, causing muscle paralysis and limb disuse. On a separate group of mice, increased loading was performed on the left tibiae through unilateral cyclic axial compression of equivalent strains (+1200 µe) at 1200 cycles/day, 5 days/week. Another cohort of mice receiving equivalent loads (?9.0 N) also were assessed. Contralateral tibiae served as normal load controls. Loaded/unloaded and normal load tibiae were assessed at day 14 for bone volume (BV) and formation changes. Loss of BV was seen in the unloaded tibiae of wild type mice, but BV was not different between normal load and unloaded Sost?/? tibiae. An increase in BV was seen in the loaded tibiae of wild type and Sost?/? mice over their normal load controls. The increased BV was associated with significantly increased mid‐shaft periosteal mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS), and endosteal MAR and BFR/BS. Notably, loading induced a greater increase in periosteal MAR and BFR/BS in Sost?/? mice than in wild type controls. Thus, long‐term Sclerostin deficiency inhibits the bone loss normally induced with decreased mechanical load, but it can augment the increase in bone formation with increased load. © 2014 American Society for Bone and Mineral Research.     

Skeletal unloading alleviates the anabolic action of intermittent PTH(1-34) in mouse tibia in association with inhibition of PTH-induced increase in c-fos mRNA in bone marrow cells.

We analyzed the effect of unloading by tail suspension on the anabolic action of intermittent PTH in the tibia of growing mice. Unloading alleviated the PTH-induced increase of bone formation and accelerated bone resorption, consequently reducing bone mass. Reduction of the PTH-induced anabolic actions on bone was associated with unloading, which was apparently related to suppression of c-fos mRNA expression in bone marrow. INTRODUCTION: The effects of intermittent parathyroid hormone (PTH) administration on unloading bone have not been well elucidated at the cellular and molecular levels. We tested the effects of PTH on unloaded tibias of tail-suspended mice. MATERIALS AND METHODS: Eighty male C57BL/6J mice, 8 weeks of age, were divided into four groups with loading or unloading and administration of PTH (40 microg/kg body weight) or vehicle five times per week. Mice were killed at 8 or 15 days, and both tibias were obtained. Bone histomorphometry of the trabecular bone in the proximal tibia, development of osteogenic cells, and mRNA expression of osteogenic molecules in bone marrow cells were assessed. RESULTS AND CONCLUSIONS: At 15 days of unloading, bone volume decreased in PTH-treated mice. The increase in the bone formation rate by PTH was depressed, and the osteoclast surface was thoroughly increased. The increase in alkaline phosphatase-positive colony-forming units-fibroblastic (CFU-f) colonies induced by PTH was maintained and that of TRACP+ multinucleated cells enhanced. The PTH-induced increase in c-fos mRNA was depressed, but the increases in Osterix and RANKL mRNA were maintained. Unloading promoted the PTH-associated osteoclastogenesis and seemed to delay the progression of osteogenic differentiation in association with reduction of the PTH-dependent increase of c-fos mRNA in bone marrow cells.     

Trabecular bone turnover and bone marrow cell development in tail-suspended mice.

To clarify the relationship between the changes of trabecular bone turnover and bone marrow cell development during mechanical unloading and reloading, we performed experiments with tail-suspended mice. At 8 weeks of age, 150 male ddY mice were divided into three body weight-matched groups. Mice of group 1 were euthanized at the start of tail suspension (day 0) as a baseline control. The mice of group 2 were subjected to hindlimb unloading by tail suspension for 14 days and reloading for the subsequent 14 days. The mice of group 3 were normally loaded as age-matched controls. Mice of groups 2 and 3 were sacrificed at 7, 14, and 28 days after the start of the experiment. In the first experiment (histomorphometric study of tibiae), unloading for 7 and 14 days and reloading for the subsequent 14 days significantly decreased the bone volume compared with that in the age-matched controls, respectively. Unloading for 7 and 14 days also significantly reduced the bone formation rate (BFR/BS), respectively, but reloading for the subsequent 14 days restored BFR/BS to the control level. While the unloading for 7 and 14 days significantly increased both the osteoclast surface (Oc.S/BS) and the osteoclast number (Oc.N/BS), the reloading for the subsequent 14 days decreased Oc.S/BS and Oc. N/BS, respectively. In the second experiment (bone marrow cell culture study of tibiae), unloading for 7 and 14 days reduced the adherent stromal cell number, without significance. Unloading for 7 days significantly decreased the mineralized nodule formation. Reloading for the subsequent 14 days markedly increased the adherent stromal cell number and the mineralized nodule formation. Unloading for 7 days significantly increased the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. These data clearly demonstrate that unloading reduces bone formation and increases bone resorption, and subsequent reloading restores reduced bone formation and suppresses increased bone resorption, closely associated with the changes in adherent stromal cell number, mineralized nodule formation, and the number of TRAP-positive multinucleated cells.     

Osteoclastogenesis Inhibitory Factor/Osteoprotegerin Reduced Bone Loss Induced by Mechanical Unloading

Skeletal unloading resulting from space flight and prolonged immobilization causes bone loss. Such bone loss ostensibly results from a rapid increase in bone resorption and subsequent sustained reduction in bone formation, but this mechanism remains unclear. Osteoclastogenesis inhibitory factor/osteoprotegerin (OCIF/OPG) is a recently identified potent inhibitor of osteoclast formation. We studied effects of OPG administration on tail-suspended growing rats to explore the therapeutic potential of OPG in the treatment and prevention of bone loss during mechanical unloading, such as that which occurs during space flight. Treatment with OPG in tail suspension increased the total bone mineral content (BMC g) of the tibia and femur and the total bone mineral density (BMD g/cm²) of the tibia. Moreover, treatment with OPG prevented reduction not only of BMC and BMD, but also of bone strength occurring through femoral diaphysis. Treatment with OPG in tail-suspended rats improved BMC, BMD and bone strength to levels of normally loaded rats treated with vehicle. Treatment with OPG in normally loaded rats significantly decreased urinary excretion of deoxypyridinoline, but the effect of OPG in tail suspension was unclear. These results indicate that OPG may be useful in inhibiting bone loss-engendered mechanical unloading.     

Histomorphometric, physical, and mechanical effects of spaceflight and insulin-like growth factor-I on rat long bones

Previous experiments have shown that skeletal unloading resulting from exposure to microgravity induces osteopenia in rats. In maturing rats, this is primarily a function of reduced formation, rather than increased resorption. Insulin-like growth factor-I (IGF-I) stimulates bone formation by increasing collagen synthesis by osteoblasts. The ability of IGF-I to prevent osteopenia otherwise caused by spaceflight was investigated in 12 rats flown for 10 days aboard the Space Shuttle, STS-77. The effect IGF-I had on cortical bone metabolism was generally anabolic. For example, humerus periosteal bone formation increased a significant 37.6% for the spaceflight animals treated with IGF-I, whereas the ground controls increased 24.7%. This increase in humeral bone formation at the periosteum is a result of an increased percent mineralizing perimeter (%Min.Pm), rather than mineral apposition rate (MAR), for both spaceflight and ground control rats. However, IGF-I did inhibit humerus endocortical bone formation in both the spaceflight and ground control rats (38.1% and 39.2%, respectively) by limiting MAR. This effect was verified in a separate ground-based study. Similar histomorphometric results for spaceflight and ground control rats suggest that IGF-I effects occur during normal weight bearing and during spaceflight. Microhardness measurements of the newly formed bone indicate that the quality of the bone formed during IGF-I treatment or spaceflight was not adversely altered. Spaceflight did not consistently change the structural (force-deflection) properties of the femur or humerus when tested in three-point bending. IGF-I significantly increased femoral maximum and fracture strength.     

Human PTH (1–34) induces longitudinal bone growth in rats

The growth plate is a specialized structure that is responsible for longitudinal bone growth (LGR). Growth plate organization is altered with loading in rats. Parathyroid hormone (PTH) is known to induce mitogenic effect on chondrocytes in vitro. Type I PTH/PTH related peptide (rP) receptor is expressed in growth plate cartilage in rats. We therefore investigated the effect of PTH administration on the organization and longitudinal growth rate of the growth plate in rats. We also investigated the effect of PTH on the changes induced by unloading in the organization and growth of the growth plate. Thirty 6-week-old and 30 15-week-old male Sprague-Dawley rats were randomly assigned to five groups (n = 6 per group), i.e., basal controls, control (i.e., normally loaded), PTH-treated control (i.e., PTH-treated under normal loading), unloaded, and PTH-treated under unloading. PTH-treated animals received human PTH (1–34) at a dose of 80 μg/kg per day five times per week for 3 weeks, for the duration of unloading. In young loaded rats treated with the systemic administration of PTH, growth plate thickness, chondrocyte number, and LGR were increased in the proximal tibiae compared with findings in young loaded rats without PTH administration. Hindlimb unloading induced a reduction in growth plate thickness, chondrocyte number, and LGR. In young rats, systemic administration of PTH partly prevented these changes induced by unloading. These preventive effects of PTH were observed only in young rats; not in adult rats. These results show that the systemic administration of PTH stimulates longitudinal bone growth, and diminishes the reduction in growth plate growth induced by unloading in young rats. Received: August 9, 2001 / Accepted: November 9, 2001     

The effect of PTH(1‐34) on fracture healing during different loading conditions

Parathyroid hormone (PTH) and PTH(1‐34) have been shown to promote bone healing in several animal studies. It is known that the mechanical environment is important in fracture healing. Furthermore, PTH and mechanical loading has been suggested to have synergistic effects on intact bone. The aim of the present study was to investigate whether the effect of PTH(1‐34) on fracture healing in rats was influenced by reduced mechanical loading. For this purpose, we used female, 25‐week‐old ovariectomized rats. Animals were subjected to closed midshaft fracture of the right tibia 10 weeks after ovariectomy. Five days before fracture, half of the animals received Botulinum Toxin A injections in the muscles of the fractured leg to induce muscle paralysis (unloaded group), whereas the other half received saline injections (control group). For the following 8 weeks, half of the animals in each group received injections of hPTH(1‐34) (20 µg/kg/day) and the other half received vehicle treatment. Fracture healing was assessed by radiology, dual‐energy X‐ray absorptiometry (DXA), histology, and bone strength analysis. We found that unloading reduced callus area significantly, whereas no effects of PTH(1‐34) on callus area were seen in neither normally nor unloaded animals. PTH(1‐34) increased callus bone mineral density (BMD) and bone mineral content (BMC) significantly, whereas unloading decreased callus BMD and BMC significantly. PTH(1‐34) treatment increased bone volume of the callus in both unloaded and control animals. PTH(1‐34) treatment increased ultimate force of the fracture by 63% in both control and unloaded animals and no interaction of the two interventions could be detected. PTH(1‐34) was able to stimulate bone formation in normally loaded as well as unloaded intact bone. In conclusion, the study confirms the stimulatory effect of PTH(1‐34) on fracture healing, and our data suggest that PTH(1‐34) is able to promote fracture healing, as well as intact bone formation during conditions of reduced mechanical loading. © 2013 American Society for Bone and Mineral Research.     

Calcium homeostasis and bone pathology in magnesium deficient rats

Summary Calcium homeostasis and bone pathology were studied in weanling rats fed a low (70 ppm) magnesium diet for 2–21 days. The rats developed significant, progressive hypercalcemia after 6 days on the diet. The increase in blood calcium was accompanied by progressive hypoactivity of the parathyroid gland (PTG), as determined by histologic and morphometric analyses. Thus hyperactivity of the PTG could not have been responsible for the hypercalcemia observed. Histologic examination of femora and humeri from magnesium-deficient rats showed progressive subperiosteal hyperplasia, consisting of undifferentiated osteoprogenitor cells and fibrous tissue, after 7 days of deficiency. The presence of unmineralized osteoid tissue in the metaphyses indicated that mineralization was not proceeding normally. The alterations in differentiation of osteoprogenitor cells, together with the failure of mineralization, resulted in significantly lower rates of bone formation (as measured by fluorochrome labeling) in the magnesium-deficient rats. Basophilic cementing lines and inactive osteocytes in the cortices of bones from magnesium-deficient rats indicated that bone resorption was also severely reduced in magnesium deficiency. We postulate that bone magnesium depletion (66% by day 21) has a direct negative effect on osteoblastic and osteocytic activity, and may explain, in part, the decreased responsiveness of bone to parathyroid hormone (PTH) that has been observed in magnesium-deficient animals.     

IGF-I receptor is required for the anabolic actions of parathyroid hormone on bone.

We showed that the IGF-IR-null mutation in mature osteoblasts leads to less bone and decreased periosteal bone formation and impaired the stimulatory effects of PTH on osteoprogenitor cell proliferation and differentiation. INTRODUCTION: This study was carried out to examine the role of IGF-I signaling in mediating the actions of PTH on bone. MATERIALS AND METHODS: Three-month-old mice with an osteoblast-specific IGF-I receptor null mutation (IGF-IR OBKO) and their normal littermates were treated with vehicle or PTH (80 microg/kg body weight/d for 2 wk). Structural measurements of the proximal and midshaft of the tibia were made by microCT. Trabecular and cortical bone formation was measured by bone histomorphometry. Bone marrow stromal cells (BMSCs) were obtained to assess the effects of PTH on osteoprogenitor number and differentiation. RESULTS: The fat-free weight of bone normalized to body weight (FFW/BW), bone volume (BV/TV), and cortical thickness (C.Th) in both proximal tibia and shaft were all less in the IGF-IR OBKO mice compared with controls. PTH decreased FFW/BW of the proximal tibia more substantially in controls than in IGF-IR OBKO mice. The increase in C.Th after PTH in the proximal tibia was comparable in both control and IGF-IR OBKO mice. Although trabecular and periosteal bone formation was markedly lower in the IGF-IR OBKO mice than in the control mice, endosteal bone formation was comparable in control and IGF-IR OBKO mice. PTH stimulated endosteal bone formation only in the control animals. Compared with BMSCs from control mice, BMSCs from IGF-IR OBKO mice showed equal alkaline phosphatase (ALP)(+) colonies on day 14, but fewer mineralized nodules on day 28. Administration of PTH increased the number of ALP(+) colonies and mineralized nodules on days 14 and 28 in BMSCs from control mice, but not in BMSCs from IGF-IR OBKO mice. CONCLUSIONS: Our results indicate that the IGF-IR null mutation in mature osteoblasts leads to less bone and decreased bone formation, in part because of the requirement for the IGF-IR in mature osteoblasts to enable PTH to stimulate osteoprogenitor cell proliferation and differentiation.     

Low dose PTH improves metaphyseal bone healing more when muscles are paralyzed

Stimulation of bone formation by PTH is related to mechanosensitivity. The response to PTH treatment in intact bone could therefore be blunted by unloading. We studied the effects of mechanical loading on the response to PTH treatment in bone healing. Most fractures occur in the metaphyses, therefor we used a model for metaphyseal bone injury.One hind leg of 20 male SD rats was unloaded via intramuscular botulinum toxin injections. Two weeks later, the proximal unloaded tibia had lost 78% of its trabecular contents. At this time-point, the rats received bilateral proximal tibiae screw implants. Ten of the 20 rats were given daily injections of 5 μg/kg PTH (1–34). After two weeks of healing, screw fixation was measured by pull-out, and microCT of the distal femur cancellous compartment was performed. Pull-out force provided an estimate for cancellous bone formation after trauma.PTH more than doubled the pull-out force in the unloaded limbs (from 14 to 30 N), but increased it by less than half in the loaded ones (from 30 to 44 N). In relative terms, PTH had a stronger effect on pull-out force in unloaded bone than in loaded bone (p = 0.03).The results suggest that PTH treatment for stimulation of bone healing does not require simultaneous mechanical stimulation.     

Effects of bisphosphate treatment and mechanical loading on bone modeling in the rat tibia.

Bisphosphonate treatment has been shown to decrease endosteal bone formation and increase periosteal bone apposition in the rat tibial diaphysis. This study tested the hypothesis that the increase in periosteal apposition is a compensatory attempt to maintain skeletal mass appropriate for the mechanical load. Twenty-four rats were divided into four groups. In two of the groups, one hindlimb in each rat was immobilized with a sling device to increase the mechanical load on the opposite limb. Daily injections of dichloromethylene bisphosphonate (Cl2MBP) were given at 10 mg/kg to one immobilized group and one mobile group. The other two groups were given daily injections of normal saline. Fluorescent bone labels were administered at two-week intervals. All rats were killed after ten weeks of treatment, and calcified tibial cross sections were prepared for fluorescence microscopy. Bone dimensions and periosteal and endosteal apposition rates were calculated. When compared with saline controls, Cl2MBP treatment decreased endosteal apposition rate in all tibias. Periosteal apposition rate was increased with Cl2MBP treatment in all tibias except the unloaded limb of immobilized rats. The Cl2MBP-induced increase in periosteal apposition rate was greatest in loaded limbs and was proportional to the relative amount of body weight supported.     

Development of an in vivo rabbit ulnar loading model

Ulnar and tibial cyclic compression in rats and mice have become the preferred animal models for investigating the effects of mechanical loading on bone modeling/remodeling. Unlike rodents, rabbits provide a larger bone volume and normally exhibit intracortical Haversian remodeling, which may be advantageous for investigating mechanobiology and pharmaceutical interventions in cortical bone. Therefore, the objective of this study was to develop and validate an in vivo rabbit ulnar loading model. Ulnar tissue strains during loading of intact forelimbs were characterized and calibrated to applied loads using strain gauge measurements and specimen-specific finite element models. Periosteal bone formation in response to varying strain levels was measured by dynamic histomorphometry at the location of maximum strain in the ulnar diaphysis. Ulnae loaded at 3000 microstrain did not exhibit periosteal bone formation greater than the contralateral controls. Ulnae loaded at 3500, 4000, and 4500 microstrain exhibited a dose-dependent increase in periosteal mineralizing surface (MS/BS) compared with contralateral controls during the second week of loading. Ulnae loaded at 4500 microstrain exhibited the most robust response with significantly increased MS/BS at multiple time points extending at least 2 weeks after loading was ceased. Ulnae loaded at 5250 microstrain exhibited significant woven bone formation. Rabbits required greater strain levels to produce lamellar and woven bone on periosteal surfaces compared with rats and mice, perhaps due to lower basal levels of MS/BS. In summary, bone adaptation during rabbit ulnar loading was tightly controlled and may provide a translatable model for human bone biology in preclinical investigations of metabolic bone disease and pharmacological treatments.     

Regional alterations of type I collagen in rat tibia induced by skeletal unloading.

Skeletal unloading induces loss of mineral density in weight-bearing bones that leads to inferior bone mechanical strength. This appears to be caused by a failure of bone formation; however, its mechanisms still are not well understood. The objective of this study was to characterize collagen, the predominant matrix protein in bone, in various regions of tibia of rats that were subjected to skeletal unloading by 4 weeks tail suspension. Sixteen male Sprague-Dawley rats (4 months old) were divided into tail suspension and ambulatory controls (eight rats each). After the tail suspension, tibias from each animal were collected and divided into five regions and collagen was analyzed. The collagen cross-linking and the extent of lysine (Lys) hydroxylation in unloaded bones were significantly altered in proximal epiphysis, diaphysis, and, in particular, proximal metaphysis but not in distal regions. The pool of immature/nonmineralized collagen measured by its extractability with a chaotropic solvent was significantly increased in proximal metaphysis. These results suggest that skeletal unloading induced an accumulation of post-translationally altered nonmineralized collagen and that these changes are bone region specific. These alterations might be caused by impaired osteoblastic function/differentiation resulting in a mineralization defect.     

Continuous parathyroid hormone induces cortical porosity in the rat: effects on bone turnover and mechanical properties.

We examined the time course effects of continuous PTH on cortical bone and mechanical properties. PTH increased cortical bone turnover and induced intracortical porosity with no deleterious effect on bone strength. Withdrawal of PTH increased maximum torque to failure and stiffness with no change in energy absorbed. INTRODUCTION: The skeletal response of cortical bone to parathyroid hormone (PTH) is complex and species dependent. Intermittent administration of PTH to rats increases periosteal and endocortical bone formation but has no known effects on intracortical bone turnover. The effects of continuous PTH on cortical bone are not clearly established. MATERIALS AND METHODS: Eighty-four 6-month-old female Sprague-Dawley rats were divided into three control, six PTH, and two PTH withdrawal (WD) groups. They were subcutaneously implanted with osmotic pumps loaded with vehicle or 40 microg/kg BW/day human PTH(1-34) for 1, 3, 5, 7, 14, and 28 days. After 7 days, PTH was withdrawn from two groups of animals for 7 (7d-PTH/7d-WD) and 21 days (7d-PTH/21d-WD). Histomorphometry was performed on periosteal and endocortical surfaces of the tibial diaphysis in all groups. microCT of tibias and mechanical testing by torsion of femora were performed on 28d-PTH and 7d-PTH/21d-WD animals. RESULTS AND CONCLUSIONS: Continuous PTH increased periosteal and endocortical bone formation, endocortical osteoclast perimeter, and cortical porosity in a time-dependent manner, but did not change the mechanical properties of the femur, possibly because of addition of new bone onto periosteal and endocortical surfaces. Additionally, withdrawal of PTH restored normal cortical porosity and increased maximum torque to failure and stiffness. We conclude that continuous administration of PTH increased cortical porosity in rats without having a detrimental effect on bone mechanical properties.