Sclerostin antibody treatment enhances metaphyseal bone healing in rats

Sclerostin is the product of the SOST gene. Loss‐of‐function mutations in the SOST gene result in a high‐bone‐mass phenotype, demonstrating that sclerostin is a negative regulator of bone mass. Primarily expressed by osteocytes in bone, sclerostin is reported to bind the LRP5/6 receptor, thereby antagonizing canonical Wnt signaling and negatively regulating bone formation. We therefore investigated whether systemic administration of a sclerostin‐neutralizing antibody would increase the regeneration of traumatized metaphyseal bone in rats. Young male rats had a screw inserted in the proximal tibia and were divided into six groups given 25 mg/kg of sclerostin antibody or control twice a week subcutaneously for 2 or 4 weeks. In four groups, the screws were tested for pull‐out strength. At the time of euthanasia, a similar screw also was inserted in the contralateral tibia and pull‐out tested immediately. Sclerostin antibody significantly increased the pull‐out force by almost 50% compared with controls after 2 and 4 weeks. Also, the screws inserted at the time of euthanasia showed increased pull‐out force. Micro–computed tomography (µCT) of the remaining two groups showed that the antibody led to a 30% increase in bone volume fraction in a region surrounding the screw. There also was a general increase in trabecular thickness in cancellous bone. Thus, as measured by the amount of bone and its mechanical resistance, the sclerostin antibody increased bone formation during metaphyseal repair but also in untraumatized bone. © 2010 American Society for Bone and Mineral Research.     

Anti‐Sclerostin Antibody Treatment in a Rat Model of Progressive Renal Osteodystrophy

Chronic kidney disease (CKD) is associated with abnormalities in bone quantity and quality, leading to increased fractures. Recent studies suggest abnormalities of Wnt signaling in animal models of CKD and elevated sclerostin levels in patients with CKD. The goal of this study was to evaluate the effectiveness of anti‐sclerostin antibody treatment in an animal model of progressive CKD with low and high parathyroid hormone (PTH) levels. Cy/+ male rats (CKD) were treated without or with calcium in the drinking water at 25 weeks of age to stratify the animals into high PTH and low PTH groups, respectively, by 30 weeks. Animals were then treated with anti‐sclerostin antibody at 100 mg/kg i.v. weekly for 5 doses, a single 20‐µg/kg subcutaneous dose of zoledronic acid, or no treatment, and were then euthanized at 35 weeks. As a positive control, the efficacy of anti‐sclerostin antibody treatment was also evaluated in normal littermates. The results demonstrated that the CKD animals with high PTH had lower calcium, higher phosphorus, and lower FGF23 compared to the CKD animals with low PTH. Treatment with anti‐sclerostin antibody had no effect on any of the biochemistries, whereas zoledronic acid lowered dkk‐1 levels. The anti‐sclerostin antibody increased trabecular bone volume/total volume (BV/TV) and trabecular mineralization surface in animals with low PTH, but not in animals with high PTH. Neither anti‐sclerostin antibody nor zoledronic acid improved biomechanical properties in the animals. Cortical porosity was severe in high‐PTH animals and was unaffected by either treatment. In contrast, in normal animals treated with anti‐sclerostin antibody, there was an improvement in bone volume, cortical geometry, and biomechanical properties. In summary, this is the first study to test the efficacy of anti‐sclerostin antibody treatment on animals with advanced CKD. We found efficacy in improving bone properties only when the PTH levels were low. © 2014 American Society for Bone and Mineral Research     

Low‐magnitude whole‐body vibration does not enhance the anabolic skeletal effects of intermittent PTH in adult mice

Whole‐body vibration (WBV) is a low‐magnitude mechanical stimulus that may be anabolic for bone, yet we recently found that WBV did not improve bone properties in adult mice. Because intermittent parathyroid hormone (PTH) enhances the anabolic effects of high‐magnitude skeletal loading, we sought to determine the skeletal effects of WBV in combination with PTH. Seven‐month‐old male BALB/c mice were assigned to six groups (n = 13–14/group) based on magnitude of applied acceleration (0 or 0.3 G) and PTH dose (0, 10, or 40 µg/kg/day). Mice were exposed to WBV (0.3 G, 90 Hz, sine wave) or sham loading (0 G) for 15 min/day, 5 days/week for 8 weeks. Vehicle or hPTH (1–34) was administered prior to each WBV session. Whole‐body bone mineral content increased by ～5% from 0 to 8 weeks in the 40 µg/kg PTH group only, independent of WBV loading. Similarly, PTH treatment increased tibial cortical bone volume by ～5% from 0 to 8 weeks, independent of WBV loading. Neither PTH nor WBV stimulated trabecular bone formation. Consistent with the cortical bone effect, tibias from the 40 µg/kg PTH group had significantly greater ultimate force and energy to failure than tibias in the 0 and 10 µg/kg PTH groups, independent of WBV treatment. In summary, 8 weeks of intermittent PTH treatment increased cortical bone volume and strength in adult male BALB/c mice. Daily exposure to low‐magnitude WBV by itself did not improve skeletal properties and did not enhance the PTH effect. No WBV‐PTH synergy was found in this preclinical study. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:465–472, 2011     

Evaluation of the effects of systemic treatment with a sclerostin neutralizing antibody on bone repair in a rat femoral defect model

Systemic administration of a sclerostin neutralizing antibody (Scl‐Ab) has been shown to enhance fracture callus density and strength in several animal models. In order to further evaluate the potential of Scl‐Ab to improve healing in a bone defect model, we evaluated Scl‐Ab in a 3 mm femoral defect in young male outbred rats. Scl‐Ab was given either continuously for 6 or 12 weeks after surgery or with 2 weeks of delay for 10 weeks. Bone formation was assessed by radiographs, µ‐CT, and histology. Complete bony union was achieved in only a few defects after 12 weeks of healing (Scl‐Ab treated 5/30, vehicle treated 1/15). µ‐CT evaluation demonstrated a significant increase in the BV/TV in the defect in the delayed treatment group (65%, p < 0.05), but a non‐significant increase in the continuous group (35%, p = 0.11) compared to control. However, both regimens induced an anabolic response in the bone proximal and distal to the defect and in the un‐operated femurs. We demonstrate that treatment with Scl‐Ab can enhance bone repair in a bone defect and in the surrounding host bone, but lacks the osteoinductive activity to heal it. This agent seems to be most effective in bone repair scenarios where there is cortical integrity. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:197–203, 2014.     

Two doses of sclerostin antibody in cynomolgus monkeys increases bone formation,bone mineral density,and bone strength

The development of bone‐rebuilding anabolic agents for treating bone‐related conditions has been a long‐standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation. More recently, administration of sclerostin‐neutralizing monoclonal antibodies in rodent studies has shown that pharmacologic inhibition of sclerostin results in increased bone formation, bone mass, and bone strength. To explore the effects of sclerostin inhibition in primates, we administered a humanized sclerostin‐neutralizing monoclonal antibody (Scl‐AbIV) to gonad‐intact female cynomolgus monkeys. Two once‐monthly subcutaneous injections of Scl‐AbIV were administered at three dose levels (3, 10, and 30 mg/kg), with study termination at 2 months. Scl‐AbIV treatment had clear anabolic effects, with marked dose‐dependent increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. Bone densitometry showed that the increases in bone formation with Scl‐AbIV treatment resulted in significant increases in bone mineral content (BMC) and/or bone mineral density (BMD) at several skeletal sites (ie, femoral neck, radial metaphysis, and tibial metaphysis). These increases, expressed as percent changes from baseline were 11 to 29 percentage points higher than those found in the vehicle‐treated group. Additionally, significant increases in trabecular thickness and bone strength were found at the lumbar vertebrae in the highest‐dose group. Taken together, the marked bone‐building effects achieved in this short‐term monkey study suggest that sclerostin inhibition represents a promising new therapeutic approach for medical conditions where increases in bone formation might be desirable, such as in fracture healing and osteoporosis. © 2010 American Society for Bone and Mineral Research     

Inhibition of sclerostin by systemic treatment with sclerostin antibody enhances healing of proximal tibial defects in ovariectomized rats

Recent studies suggest a possible role for inhibitors of sclerostin such as sclerostin antibody (Scl‐Ab) as an anabolic treatment for osteoporosis. Since Scl‐Ab has also been shown to potentiate bone repair, we examined the effect of Scl‐Ab treatment in a metaphyseal defect repair model in ovariectomized (OVX) rats. Four weeks after OVX or sham surgery, 3 mm circular defects were created bilaterally in the proximal tibia of all rats. After defect surgery, Saline or 25 mg/kg Scl‐Ab was administered twice weekly for 3 weeks. Of note, healing was advanced in the 1‐week post‐defect surgery in OVX controls over Sham controls, with increases in bone volume and fluorochrome labeling observed. However, by week 2, OVX controls fell significantly behind in the repair response compared with Sham controls. Scl‐Ab treatment significantly increased bone volume in the defect in OVX rats over the 3‐week time course as examined by either microCT or histology. Significant increases in bone formation via fluorochrome labeling of the new bone were observed with Scl‐Ab treatment, while osteoclast parameters were not different. With its powerful anabolic potential, bone‐specific activity, and potential for low dosing frequency, Scl‐Ab treatment could provide enhanced bone repair, particularly in situations of compromised bone repair such as osteoporotic bone. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1541–1548, 2012     

Sclerostin antibody treatment improves bone mass,bone strength,and bone defect regeneration in rats with type 2 diabetes mellitus

Type 2 diabetes mellitus results in increased risk of fracture and delayed fracture healing. ZDF fa/fa rats are an established model of type 2 diabetes mellitus with low bone mass and delayed bone healing. We tested whether a sclerostin‐neutralizing antibody (Scl‐AbVI) would reverse the skeletal deficits of diabetic ZDF rats. Femoral defects of 3 mm were created in 11‐week‐old diabetic ZDF fa/fa and nondiabetic ZDF +/+ rats and stabilized by an internal plate. Saline or 25 mg/kg Scl‐AbVI was administered subcutaneously (s.c.) twice weekly for 12 weeks (n = 9–10/group). Bone mass and strength were assessed using pQCT, micro–computed tomography (µCT), and biomechanical testing. Bone histomorphometry was used to assess bone formation, and the filling of the bone defect was analyzed by µCT. Diabetic rats displayed lower spinal and femoral bone mass compared to nondiabetic rats, and Scl‐AbVI treatment significantly enhanced bone mass of the femur and the spine of diabetic rats (p < 0.0001). Scl‐AbVI also reversed the deficit in bone strength in the diabetic rats, with 65% and 89% increases in maximum load at the femoral shaft and neck, respectively (p < 0.0001). The lower bone mass in diabetic rats was associated with a 65% decrease in vertebral bone formation rate, which Scl‐AbVI increased by sixfold, consistent with a pronounced anabolic effect. Nondiabetic rats filled 57% of the femoral defect, whereas diabetic rats filled only 21% (p < 0.05). Scl‐AbVI treatment increased defect regeneration by 47% and 74%, respectively (p < 0.05). Sclerostin antibody treatment reverses the adverse effects of type 2 diabetes mellitus on bone mass and strength, and improves bone defect regeneration in rats. © 2013 American Society for Bone and Mineral Research.     

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 OP-1 and PTH in a new experimental model for the study of metaphyseal bone healing.

The purpose of this study was to establish a reliable model of metaphyseal bone healing and to use this model to investigate the effect of recombinant human osteogenic protein 1 (rhOP-1; BMP-7) and parathyroid hormone fragment (PTH 1-34) on healing. A wedge-shaped osteotomy was created in the distal tibia of 16-week-old female New Zealand White rabbits (n = 20) and was bridged with a custom-made external fixator. Five experimental groups of four animals each were investigated. In groups 1-4 the osteotomy gap was filled with tricalcium phosphate (TCP), and the gap was left unfilled in group 5 ("normal healing"). In group 1, 200 microg OP-1 was mixed in with the TCP. Groups 2 and 3 received daily subcutaneous injections of 10 and 40 microg/kg PTH, respectively, beginning on postoperative day 1. Radiographs were taken weekly. Following sacrifice on postoperative day 28, peripheral quantitative computed tomography (pQCT), histology, and mechanical testing (axial compression and torsion) were performed. Only one animal failed to complete the full 4-week time course, and no infections were encountered. Bone healing occurred in all animals. OP-1 stimulated bone formation locally, while the lower dose of PTH enhanced bone formation systemically (p < 0.05). Tibiae treated with OP-1 exhibited higher torsional strength (p = 0.04) than those in the normal healing group. These results indicate that a reliable and reproducible surgical model of metaphyseal healing has been established. In addition, differences in systemic versus local effects of PTH and OP-1 in accelerating metaphyseal fracture healing were found.     

Sclerostin Antibody Treatment Increases Bone Formation,Bone Mass,and Bone Strength in a Rat Model of Postmenopausal Osteoporosis

The development of bone‐rebuilding anabolic agents for potential use in the treatment of bone loss conditions, such as osteoporosis, has been a long‐standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation, although the magnitude and extent of sclerostin's role in the control of bone formation in the aging skeleton is still unclear. To study this unexplored area of sclerostin biology and to assess the pharmacologic effects of sclerostin inhibition, we used a cell culture model of bone formation to identify a sclerostin neutralizing monoclonal antibody (Scl‐AbII) for testing in an aged ovariectomized rat model of postmenopausal osteoporosis. Six‐month‐old female rats were ovariectomized and left untreated for 1 yr to allow for significant estrogen deficiency‐induced bone loss, at which point Scl‐AbII was administered for 5 wk. Scl‐AbII treatment in these animals had robust anabolic effects, with marked increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. This not only resulted in complete reversal, at several skeletal sites, of the 1 yr of estrogen deficiency‐induced bone loss, but also further increased bone mass and bone strength to levels greater than those found in non‐ovariectomized control rats. Taken together, these preclinical results establish sclerostin's role as a pivotal negative regulator of bone formation in the aging skeleton and, furthermore, suggest that antibody‐mediated inhibition of sclerostin represents a promising new therapeutic approach for the anabolic treatment of bone‐related disorders, such as postmenopausal osteoporosis.     

Abaloparatide versus teriparatide: a head to head comparison of effects on fracture healing in mouse models

Background and purpose — Teriparatide accelerates fracture healing in animals and probably in man. Abaloparatide is a new drug with similar although not identical effects on the teriparatide receptor. Given at 4 times the teriparatide dose in a human osteoporosis trial, abaloparatide increased bone density more than teriparatide, and both reduced fracture risk. We investigated in mice whether abaloparatide stimulates fracture healing, and if it does so with the suggested dose effect relation (4:1).

Patients and methods — In a validated mouse model for metaphyseal healing (burr hole with screw pull-out), 96 mice were randomly allocated to 11 groups: control (saline), teriparatide or abaloparatide, where teriparatide and abaloparatide were given at 5 different doses each. In a femoral shaft osteotomy model, 24 mice were randomly allocated to 3 groups: control (saline), teriparatide (15 µg/kg) or abaloparatide (60 µg/kg). Each treatment was given daily via subcutaneous injections. Results were evaluated by mechanical testing and microCT.

Results — In the metaphyseal model, a dose-dependent increase in screw pull-out force could be seen. In a linear regression analysis (r = 0.78) each increase in ln(dose) by 1 (regardless of drug type) was associated with an increase in pull-out force by 1.50 N (SE 0.18) (p < 0.001). Changing drug from teriparatide to abaloparatide increased the force by 1.41 N (SE 0.60; p = 0.02).

In the diaphyseal model, the callus density was 23% (SD 10), 38% (SD 10), and 47% (SD 2) for control, for teriparatide and abaloparatide respectively. Both drugs were significantly different from controls (p = 0.001 and p = 0.008), but not from each other.

Interpretation — Both drugs improve fracture healing, but in these mouse models, the potency per µg of abaloparatide seems only 2.5 times that of teriparatide, rather than the 4:1 relation chosen in the clinical abaloparatide–teriparatide comparison trial.     

Sclerostin Inhibition Prevents Spinal Cord Injury‐Induced Cancellous Bone Loss

Spinal cord injury (SCI) results in rapid and extensive sublesional bone loss. Sclerostin, an osteocyte‐derived glycoprotein that negatively regulates intraskeletal Wnt signaling, is elevated after SCI and may represent a mechanism underlying this excessive bone loss. However, it remains unknown whether pharmacologic sclerostin inhibition ameliorates bone loss subsequent to SCI. Our primary purposes were to determine whether a sclerostin antibody (Scl‐Ab) prevents hindlimb cancellous bone loss in a rodent SCI model and to compare the effects of a Scl‐Ab to that of testosterone‐enanthate (TE), an agent that we have previously shown prevents SCI‐induced bone loss. Fifty‐five (n = 11–19/group) skeletally mature male Sprague‐Dawley rats were randomized to receive: (A) SHAM surgery (T8 laminectomy), (B) moderate‐severe (250 kilodyne) SCI, (C) 250 kilodyne SCI + TE (7.0 mg/wk, im), or (D) 250 kilodyne SCI + Scl‐Ab (25 mg/kg, twice weekly, sc) for 3 weeks. Twenty‐one days post‐injury, SCI animals exhibited reduced hindlimb cancellous bone volume at the proximal tibia (via μCT and histomorphometry) and distal femur (via μCT), characterized by reduced trabecular number and thickness. SCI also reduced trabecular connectivity and platelike trabecular structures, indicating diminished structural integrity of the remaining cancellous network, and produced deficits in cortical bone (femoral diaphysis) strength. Scl‐Ab and TE both prevented SCI‐induced cancellous bone loss, albeit via differing mechanisms. Specifically, Scl‐Ab increased osteoblast surface and bone formation, indicating direct bone anabolic effects, whereas TE reduced osteoclast surface with minimal effect on bone formation, indicating antiresorptive effects. The deleterious microarchitectural alterations in the trabecular network were also prevented in SCI + Scl‐Ab and SCI + TE animals, whereas only Scl‐Ab completely prevented the reduction in cortical bone strength. Our findings provide the first evidence indicating that sclerostin inhibition represents a viable treatment to prevent SCI‐induced cancellous and cortical bone deficits and provides preliminary rationale for future clinical trials focused on evaluating whether Scl‐Ab prevents osteoporosis in the SCI population. © 2014 American Society for Bone and Mineral Research.     

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.     

Continuous elevation of PTH increases the number of osteoblasts via both osteoclast‐dependent and ‐independent mechanisms

Sustained parathyroid hormone (PTH) elevation stimulates bone remodeling (ie, both resorption and formation). The former results from increased RANKL synthesis, but the cause of the latter has not been established. Current hypotheses include release of osteoblastogenic factors from osteoclasts or from the bone matrix during resorption, modulation of the production and activity of osteoblastogenic factors from cells of the osteoblast lineage, and increased angiogenesis. To dissect the contribution of these mechanisms, 6‐month‐old Swiss‐Webster mice were infused for 5 days with 470 ng/h PTH(1‐84) or 525 ng/h soluble RANKL (sRANKL). Both agents increased osteoclasts and osteoblasts in vertebral cancellous bone, but the ratio of osteoblasts to osteoclasts and the increase in bone formation was greater in PTH‐treated mice. Cancellous bone mass was maintained in mice receiving PTH but lost in mice receiving sRANKL, indicating that maintenance of balanced remodeling requires osteoblastogenic effects beyond those mediated by osteoclasts. Consistent with this contention, PTH, but not sRANKL, decreased the level of the Wnt antagonist sclerostin and increased the expression of the Wnt target genes Nkd2, Wisp1, and Twist1. Furthermore, PTH, but not sRANKL, increased the number of blood vessels in the bone marrow. Weekly injections of the RANKL antagonist osteoprotegerin at 10 µg/g for 2 weeks prior to PTH infusion eliminated osteoclasts and osteoblasts and prevented the PTH‐induced increase in osteoclasts, osteoblasts, and blood vessels. These results indicate that PTH stimulates osteoclast‐dependent as well as osteoclast‐independent (Wnt signaling) pro‐osteoblastogenic pathways, both of which are required for balanced focal bone remodeling in cancellous bone. © 2010 American Society for Bone and Mineral Research.     

Parathyroid hormone (PTH)–induced bone gain is blunted in SOST overexpressing and deficient mice

Intermittent parathyroid hormone (PTH) treatment is a potent bone anabolic principle that suppresses expression of the bone formation inhibitor Sost. We addressed the relevance of Sost suppression for PTH‐induced bone anabolism in vivo using mice with altered Sost gene dosage. Six‐month‐old Sost overexpressing and 2‐month‐old Sost deficient male mice and their wild‐type littermates were subjected to daily injections of 100 µg/kg PTH(1–34) or vehicle for a 2‐month period. A follow‐up study was performed in Sost deficient mice using 40 and 80 µg/kg PTH(1–34). Animals were sacrificed 4 hours after the final PTH administration and Sost expression in long bone diaphyses was determined by qPCR. Bone changes were analyzed in vivo in the distal femur metaphysis by pQCT and ex vivo in the tibia and lumbar spine by DXA. Detailed ex vivo analyses of the femur were performed by pQCT, µCT, and histomorphometry. Overexpression of Sost resulted in osteopenia and Sost deletion in high bone mass. As shown before, PTH suppressed Sost in wild‐type mice. PTH treatment induced substantial increases in bone mineral density, content, and cortical thickness and in aging wild‐type mice also led to cancellous bone gain owing to amplified bone formation rates. PTH‐induced bone gain was blunted at all doses and skeletal sites in Sost overexpressing and deficient mice owing to attenuated bone formation rates, whereas bone resorption was not different from that in PTH‐treated wild‐type controls. These data suggest that suppression of the bone formation inhibitor Sost by intermittent PTH treatment contributes to PTH bone anabolism. © 2010 American Society for Bone and Mineral Research     

Parathyroid hormone and mechanical usage have a synergistic effect in rat tibial diaphyseal cortical bone.

Previous reports showed that bone mass and architecture only partially recovered by remobilization (RM) after immobilization (IM)-induced osteopenia, and that parathyroid hormone (PTH) had an anabolic effect on the skeleton. The aim of this study was to determine whether low doses of PTH could restore IM-induced cortical bone loss and whether a combination of PTH plus loading (RM) treatment would be more effective than the PTH in unloaded (IM) limbs. One hundred and sixty 6-month-old rats were divided into aging and IM groups. The right hindlimb of the rat was immobilized by elastic bandage for 18 weeks, and then groups of rats were either kept IM or RM and treated with 30 microgram or 80 microgram of hPTH(1-38)/kg/day for 2, 10, and 20 weeks. Fluorescent-labeled, undecalcified cross-sections of right tibial shafts were studied. We found that RM for 20 weeks after 18 weeks of IM only partially recovered IM-induced muscle weight loss and PTH had no effect on muscle weight in either IM or RM limbs; that RM for 20 weeks after 18 weeks of IM partially restored some minimal cortical width by stimulating periosteal and endocortical bone formation and decreasing endocortical resorption; that PTH treatment of IM limbs completely restored IM-induced cortical bone loss and added extra bone by stimulating bone formation indices on all bone surfaces and depressing bone resorption on endocortical surface; that PTH treatment of RM limbs produced similar anabolic effects as in IM limbs with 30 microgram/kg/day dose but the 80 microgram/kg/day dose-treated limbs had a higher periosteal bone formation rate, which created a larger cross-sectional area, more cortical bone area, and a thicker cortex than the same dose treated IM limbs; and that PTH 80 microgram/kg/day treatment produced more anabolic effect than the 30 microgram/kg/day in both IM and RM limbs. We concluded that reloading the hindlimb by RM after long-term IM could not recover the cortical bone mass. PTH at employed doses was able to completely restore IM-induced cortical bone loss, and this effect was independent of mechanical stimulation. However, when PTH was combined with mechanical loading (RM), a synergistic anabolic effect on periosteal bone formation occurred which increased the cross sectional area that can increase bone strength.     

Dramatic increase in cortical thickness induced by femoral marrow ablation followed by a 3‐month treatment with PTH in rats

We previously reported that following mechanical ablation of the marrow from the midshaft of rat femurs, there is a rapid and abundant but transient growth of bone, and this growth is enhanced and maintained over a 3‐week period by the bone anabolic hormone parathyroid hormone (PTH). Here, we asked whether further treatment with PTH or bisphosphonates can extend the half‐life of the new bone formed in lieu of marrow. We subjected the left femur of rats to mechanical marrow ablation and treated the animals 5 days a week with PTH for 3 weeks (or with vehicle as a control) to replace the marrow by bone. Some rats were euthanized and used as positive controls or treated with vehicle, PTH, or the bisphosphonate alendronate for a further 9 weeks. We subjected both femurs from each rat to soft X‐ray, peripheral quantitative computed tomography (pQCT), micro‐computed tomography (µCT), dynamic histomorphometry analysis, and biomechanical testing. We also determined the concentrations of serum osteocalcin to confirm the efficacy of PTH. Treatment with PTH for 3 months dramatically enhanced endosteal and periosteal bone formation, leading to a 30% increase in cortical thickness. In contrast, alendronate protected the bone that had formed in the femoral marrow cavity after marrow ablation and 3 weeks of treatment with PTH but failed to promote endosteal bone growth or to improve the biomechanical properties of ablated femurs. We further asked whether calcium‐phosphate cements could potentiate the formation of bone after marrow ablation. Marrow cavities from ablated femurs were filled with one of two calcium‐phosphate cements, and rats were treated with PTH or PBS for 84 days. Both cements helped to protect the new bone formed after ablation. To some extent, they promoted the formation of bone after ablation, even in the absence of any anabolic hormone. Our data therefore expand the role of PTH in bone engineering and open new avenues of investigation to the field of regenerative medicine and tissue engineering. Local bone marrow aspiration in conjunction with an anabolic agent, a bisphosphonate, or a calcium‐phosphate cement might provide a new platform for rapid preferential site‐directed bone growth in areas of high bone loss. © 2010 American Society for Bone and Mineral Research     

Inhibition of sclerostin by monoclonal antibody enhances bone healing and improves bone density and strength of nonfractured bones

Therapeutic enhancement of fracture healing would help to prevent the occurrence of orthopedic complications such as nonunion and revision surgery. Sclerostin is a negative regulator of bone formation, and treatment with a sclerostin monoclonal antibody (Scl‐Ab) results in increased bone formation and bone mass in animal models. Our objective was to investigate the effects of systemic administration of Scl‐Ab in two models of fracture healing. In both a closed femoral fracture model in rats and a fibular osteotomy model in cynomolgus monkeys, Scl‐Ab significantly increased bone mass and bone strength at the site of fracture. After 10 weeks of healing in nonhuman primates, the fractures in the Scl‐Ab group had less callus cartilage and smaller fracture gaps containing more bone and less fibrovascular tissue. These improvements at the fracture site corresponded with improvements in bone formation, bone mass, and bone strength at nonfractured cortical and trabecular sites in both studies. Thus the potent anabolic activity of Scl‐Ab throughout the skeleton also was associated with an anabolic effect at the site of fracture. These results support the potential for systemic Scl‐Ab administration to enhance fracture healing in patients. © 2011 American Society for Bone and Mineral Research.     

Enhancement of recombinant human BMP‐7 bone formation with bmp binding peptide in a rodent femoral defect model

Bone morphogenetic binding peptide (BBP) is an 18.5 kDa fragment of a bone matrix protein peptide. A rat femoral defect model was used to test the effect of BBP combined with recombinant human bone morphogenetic protein‐7 (rhBMP‐7) to induced bone healing. Two doses of BBP (500 and 1000 µg) were tested with two doses of rhBMP‐7 (2 and 5 µg), and the results were compared with a positive control (10 µg rhBMP‐7). Bone healing was evaluated by radiology, manual palpation, microcomputed tomography, and histology. The high dose of 10 µg of rhBMP‐7 resulted in a consistent 100% bone union rate and a mature histological appearance on histology, and was used as a positive control. When 1000 µg of BBP was combined with lower doses of BMP‐7 (2 µg rhBMP‐7 or 5 µg rhBMP‐7) significant differences were seen in radiographic scores, manual palpation, and bone volume, when compared to 2 µg rhBMP‐7 or 5 µg rhBMP‐7 alone. The combination of 1000 µg of BBP and 5 µg rhBMP‐7 also achieved 100% fusion rate, induced a larger amount of bone formation, and yielded similar maturity of bone marrow when compared with the high dosage 10 µg rhBMP‐7 group. This study demonstrated that when combined together, BBP can enhance the bone healing of rhBMP‐7. Improved healing imparted by the addition of BBP may result in lesser amounts of rhBMP‐7 needed to achieve union in the clinical setting. © 2010 OrthopaedicResearchSociety.PublishedbyWileyPeriodicals, Inc.JOrthopRes29:753–759,2011     

Accuracy of high‐resolution in vivo micro magnetic resonance imaging for measurements of microstructural and mechanical properties of human distal tibial bone

Micro magnetic resonance imaging (µMRI) is an in vivo imaging method that permits 3D quantification of cortical and trabecular bone microstructure. µMR images can also be used for building microstructural finite element (µFE) models to assess bone stiffness, which highly correlates with bone's resistance to fractures. In order for µMRI‐based microstructural and µFE analyses to become standard clinical tools for assessing bone quality, validation with a current gold standard, namely, high‐resolution micro computed tomography (µCT), is required. Microstructural measurements of 25 human cadaveric distal tibias were performed for the registered µMR and µCT images, respectively. Next, whole bone stiffness, trabecular bone stiffness, and elastic moduli of cubic subvolumes of trabecular bone in both µMR and µCT images were determined by voxel‐based µFE analysis. The bone volume fraction (BV/TV), trabecular number (Tb.N*), trabecular spacing (Tb.Sp*), cortical thickness (Ct.Th), and structure model index (SMI) based on µMRI showed strong correlations with µCT measurements (r² = 0.67 to 0.97), and bone surface‐to‐volume ratio (BS/BV), connectivity density (Conn.D), and degree of anisotropy (DA) had significant but moderate correlations (r² = 0.33 to 0.51). Each of these measurements also contributed to one or many of the µFE‐predicted mechanical properties. However, model‐independent trabecular thickness (Tb.Th*) based on µMRI had no correlation with the µCT measurement and did not contribute to any mechanical measurement. Furthermore, the whole bone and trabecular bone stiffness based on µMRI were highly correlated with those of µCT images (r² = 0.86 and 0.96), suggesting that µMRI‐based µFE analyses can directly and accurately quantify whole bone mechanical competence. In contrast, the elastic moduli of the µMRI trabecular bone subvolume had significant but only moderate correlations with their gold standards (r² = 0.40 to 0.58). We conclude that most microstructural and mechanical properties of the distal tibia can be derived efficiently from µMR images and can provide additional information regarding bone quality. © 2010 American Society for Bone and Mineral Research.