Scl-Ab Treatment Research Articles

Dkk1 KO Mice Treated with Sclerostin Antibody Have Additional Increases in Bone Volume.

Dickkopf-1 (DKK1) and sclerostin are antagonists of the Wnt/β-catenin pathway and decreased expression of either results in increased bone formation and mass. As both affect the same signaling pathway, we aimed to elucidate the redundancy and/or compensation of sclerostin and DKK1. Weekly sclerostin antibody (Scl-Ab) was used to treat 9-week-old female Dkk1 KO (Dkk1-/-:Wnt3+/-) mice and compared to Scl-Ab-treated wild-type mice as well as vehicle-treated Dkk1 KO and wild-type animals. While Wnt3 heterozygote (Wnt3+/-) mice show no bone phenotype, Scl-Ab and vehicle-treated control groups of this genotype were included. Specimens were harvested after 3 weeks for microCT, bone histomorphometry, anti-sclerostin immunohistochemistry, and biomechanical testing. Scl-Ab enhanced bone anabolism in all treatment groups, but with synergistic enhancement seen in the cancellous compartment of Dkk1 KO mice (bone volume + 55% Dkk1 KO p < 0.01; + 22% wild type p < 0.05). Scl-Ab treatment produced less marked increases in cortical bone of the tibiae, with anabolic effects similar across genotypes. Mechanical testing confirmed that Scl-Ab improved strength across all genotypes; however, no enhancement was seen within Dkk1 KO mice. Dynamic bone labeling showed that Scl-Ab treatment was associated with increased bone formation, regardless of genotype. Immunohistochemical staining for sclerostin protein indicated no differences in the Dkk1 KO mice, indicating that the increased Wnt signaling associated with DKK1 deficiency was not compensated by upregulation of sclerostin protein. These data suggest complex interactions between Wnt signaling factors in bone, but critically illustrate synergy between DKK1 deficiency and Scl-Ab treatment. These data support the application of dual-targeted therapeutics in the modulation of bone anabolism.

Sclerostin Neutralizing Antibody Treatment Enhances Bone Formation but Does Not Rescue Mechanically Induced Delayed Healing

During bone healing, tissue formation processes are governed by mechanical strain. Sost/sclerostin, a key Wnt signaling inhibitor and mechano-sensitive pathway, is downregulated in response to mechanical loading. Sclerostin neutralizing antibody (SclAb) increases bone formation. Nevertheless, it remains unclear whether sclerostin inhibition can rescue bone healing in situations of mechanical instability, which otherwise delay healing. We investigated SclAb's influence on tissue formation in a mouse femoral osteotomy, stabilized with rigid or semirigid external fixation. The different fixations allowed different magnitudes of interfragmentary movement during weight bearing, thereby influencing healing outcome. SclAb or vehicle (veh) was administeredand bone healing was assessed at multiple time points up to day 21 postoperatively by in vivo micro-computed tomography, histomorphometry, biomechanical testing, immunohistochemistry, and gene expression. Our results show that SclAb treatment caused a greater bone volume than veh. However, SclAb could not overcome the characteristic delayed healing of semirigid fixation. Indeed, semirigid fixation resulted in delayed healing with a prolonged endochondral ossification phase characterized by increased cartilage, lower bone volume fraction, and less bony bridging across the osteotomy gap than rigid fixation. In a control setting, SclAb negatively affected later stages of healing under rigid fixation, evidenced by the high degree of endosteal bridging at 21 days in the rigid-SclAb group compared with rigid-veh, indicating delayed fracture callus remodeling and bone marrow reconstitution. Under rigid fixation, Sost and sclerostin expression at the gene and protein level, respectively, were increased in SclAb compared with veh-treated bones, suggesting a negative feedback mechanism. Our results suggest that SclAb could be used to enhance overall bone mass but should be carefully considered in bone healing. SclAb may help to increase bone formation early in the healing process but not during advanced stages of fracture callus remodeling and not to overcome delayed healing in semirigid fixation. © 2018 American Society for Bone and Mineral Research.

Differential time-dependent transcriptional changes in the osteoblast lineage in cortical bone associated with sclerostin antibody treatment in ovariectomized rats

Inhibition of sclerostin with sclerostin antibody (Scl-Ab) results in stimulation of bone formation on cancellous (Cn), endocortical (Ec), and periosteal (Ps) surfaces in rodents and non-human primates. With long-term dosing of Scl-Ab, the increase in bone formation is not sustained, attenuating first on Cn surfaces and later on Ec and Ps surfaces. In Cn bone, the attenuation in bone formation (self-regulation) is associated with transcriptional changes in the osteocyte (OCy) that would limit mitogenesis and are sustained with continued dosing. The expression changes in Cn OCy occur coincident with a decrease in osteoprogenitor (OP) numbers that may directly or indirectly be a consequence of the transcriptional changes in the OCy to limit OP proliferation. To characterize the Scl-Ab–mediated changes in cortical (Ct) bone and compare these changes to Cn bone, densitometric, histomorphometric, and transcriptional analyses were performed on femur diaphyses from aged ovariectomized rats. Animals were administered 50 mg/kg/wk of Scl-Ab or vehicle for up to 6 months (183 days), followed by a treatment-free period (up to 126 days). Scl-Ab increased Ct mass and area through day 183, which declined slightly when treatment was discontinued. Ps and Ec bone formation was sustained through the dosing on both Ct surfaces, with evidence of a decline in bone formation only at day 183 on the Ec surface. This is in contrast to Cn bone, where reduced bone formation was observed after day 29. TaqMan analysis of 60 genes with functional roles in the bone using mRNA isolated from laser capture micro-dissection samples enriched for Ec osteoblasts and Ct OCy suggest a pattern of gene expression in Ct bone that differed from Cn, especially in the OCy, and that corresponded to observed differences in the timing of phenotypic changes. Notable with Scl-Ab treatment was a “transcriptional switch” in Ct OCy at day 183, coincident with the initial decline in bone formation on the endocortex. A consistent sustained increase of expression for most genes in response to Scl-Ab was observed from day 8 through day 85 at the times of maximal bone formation on both Ct surfaces; however, at day 183, this increase was reversed, with expression of these genes generally returning to control values or decreasing compared to vehicle. Genes exhibiting this pattern included Wnt inhibitors Sost and Dkk1, though both had been up-regulated until the end of dosing in Cn OCy. Changes in cell cycle genes such as Cdkn1a and Ndrg1 in Ct OCy suggested up-regulation of p53 signaling, as observed in Cn OCy; however, unlike in Cn bone, p53 signaling was not associated with decreased bone formation and was absent at day 183, when bone formation began to decline on the Ec surface. These data demonstrate involvement of similar molecular pathways in Ct and Cn bone in response to Scl-Ab but with a different temporal relationship to bone formation and suggest that the specific mechanism underlying self-regulation of Scl-Ab–induced bone formation may be different between Cn and Ct bone.

Decreased osteoprogenitor proliferation precedes attenuation of cancellous bone formation in ovariectomized rats treated with sclerostin antibody

Sclerostin antibody (Scl-Ab) stimulates bone formation, which with long-term treatment, attenuates over time. The cellular and molecular mechanisms responsible for the attenuation of bone formation are not well understood, but in aged ovariectomized (OVX) rats, the reduction in vertebral cancellous bone formation is preceded by a reduction in osteoprogenitor (OP) number and significant induction of signaling pathways known to suppress mitogenesis and cell cycle progression in the osteocyte (OCy) (Taylor et al., 2016). To determine if the reduction in OP number is associated with a decrease in proliferation, aged OVX rats were administered vehicle or Scl-Ab for 9 or 29 days and implanted with continuous-delivery 5-bromo-2′-deoxyuridine (BrdU) mini-osmotic pumps 5 days prior to necropsy. The total number of BrdU-labeled osteoblasts (OB) was quantified in vertebral cancellous bone to indirectly assess the effects of Scl-Ab treatment on OP proliferation at the time of activation of modeling-based bone formation at day 9 and at the time of maximal mineralizing surface, initial decrease in OP number, and transcriptional changes in the OCy at day 29. Compared with vehicle, Scl-Ab resulted in an increase in the total number of BrdU-positive OB (+260%) at day 9 that decreased with continued treatment (+50%) at day 29. These differences in proliferation occurred at time points when the increase in total OB number was significant and similar in magnitude. These findings suggest that reduced OP proliferation contributes to the decrease in OP numbers, an effect that would limit the OB pool and contribute to the attenuation of bone formation that occurs with long-term Scl-Ab treatment.

Sclerostin antibody enhances bone formation in a rat model of distraction osteogenesis.

Neutralizing monoclonal sclerostin antibodies are effective in promoting bone formation at a systemic level and in orthopedic scenarios including closed fracture repair. In this study we examined the effects of sclerostin antibody (Scl-Ab) treatment on regenerate volume, density, and strength in a rat model of distraction osteogenesis. Surgical osteotomy was performed on 179 Sprague Dawley rats. After 1 week, rats underwent distraction for 2 weeks, followed by 6 weeks for consolidation. Two treatment groups received biweekly subcutaneous Scl-AbIII (a rodent form of Scl-Ab; 25 mg/kg), either from the start of distraction onward or restricted to the consolidation phase. These groups were compared to controls receiving saline. Measurement modalities included longitudinal DXA, ex vivo QCT, and microCT, tissue histology, and biomechanical four-point bending tests. Bone volume was increased in both Scl-Ab treatments regimens by the end of consolidation (+26-38%, p < 0.05), as assessed by microCT. This was associated with increased mineral apposition. Importantly, Scl-Ab led to increased strength in united bones, and this reached statistical significance in animals receiving Scl-Ab during consolidation only (+177%, p < 0.01, maximum load to failure). These data demonstrate that Scl-Ab treatment increases bone formation, leading to regenerates with higher bone volume and improved strength. Our data also suggest that the optimal effects of Scl-Ab treatment are achieved in the latter stages of distraction osteogenesis. These findings support further investigation into the potential clinical application of sclerostin antibody to augment bone distraction, such as limb lengthening, particularly in the prevention of refracture. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1106-1113, 2018.

Sclerostin Antibody Treatment Stimulates Bone Formation to Normalize Bone Mass in Male Down Syndrome Mice.

ABSTRACTDown syndrome (DS), characterized by trisomy of human chromosome 21, is associated with a variety of endocrine disorders as well as profound skeletal abnormalities. The low bone mass phenotype in DS is defined by low bone turnover due to decreased osteoclast and osteoblast activity, decreasing the utility of antiresorptive agents in people with DS. Sclerostin antibody (SclAb) is a therapeutic candidate currently being evaluated as a bone anabolic agent. Scl, the product of the sclerostin gene (SOST), inhibits bone formation through its inhibition of Wnt signaling. SclAb increases bone mass by suppressing the action of the endogenous inhibitor of bone formation, Scl. To examine the effects of SclAb on the DS bone phenotype, 8‐week‐old male wild‐type (WT) andTs65Dn DS mice were treated with 4 weekly iv injections of 100 mg/kg SclAb. Dual‐energy X‐ray absorptiometry (DXA), microCT, and dynamic histomorphometry analyses revealed that SclAb had a significant anabolic effect on both age‐matched WT littermate controls and Ts65Dn DS mice that was osteoblast mediated, without significant changes in osteoclast parameters. SclAb treatment significantly increased both cortical and trabecular bone mass at multiple sites; SclAb treatment resulted in the normalization of Ts65Dn bone mineral density (BMD) to WT levels in the proximal tibia, distal femur, and whole body. Ex vivo bone marrow cultures demonstrated that SclAb increased the recruitment of the mesenchymal progenitors into the osteoblast lineage, as indicated by increased alkaline phosphatase–positive colonies, with no effect on osteoclast differentiation. Together, in the setting of a murine model of DS and decreased bone turnover, SclAb had a potent anabolic effect. SclAb stimulated bone formation and increased osteoblastogenesis without affecting osteoclastogenesis or bone resorption. These data suggest that SclAb is a promising new therapy to improve bone mass and reduce fracture risk in the face of the low bone mass and turnover prevalent in the DS population. © 2017 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Sclerostin Antibody Augments the Anabolic Bone Formation Response in a Mouse Model of Mechanical Tibial Loading.

Decreased activity or expression of sclerostin, an endogenous inhibitor of Wnt/β-catenin signaling, results in increased bone formation and mass. Antibodies targeting and neutralizing sclerostin (Scl-Ab) have been shown to increase bone mass and reduce fracture risk. Sclerostin is also important in modulating the response of bone to changes in its biomechanical environment. However, the effects of Scl-Ab on mechanotransduction are unclear, and it was speculated that the loading response may be altered for individuals receiving Scl-Ab therapy. To address this, we carried out a 2-week study of tibial cyclic compressive loading on C57Bl/6 mice treated with vehicle or 100 mg/kg/wk Scl-Ab. Increases in bone volume, density, and dynamic bone formation were found with loading, and the anabolic response was further increased by the combination of load and Scl-Ab. To investigate the underlying mechanism, gene profiling by RNA sequencing (RNAseq) was performed on tibias isolated from mice from all four experimental groups. Major alterations in Wnt/β-catenin gene expression were found with tibial loading, however not with Scl-Ab treatment alone. Notably, the combination of load and Scl-Ab elicited a synergistic response from a number of specific Wnt-related and mechanotransduction factors. An unexpected finding was significant upregulation of factors in the Rho GTPase signaling pathway with combination treatment. In summary, combination therapy had a more profound anabolic response than either Scl-Ab or loading treatment alone. The Wnt/β-catenin and Rho GTPase pathways were implicated within bone mechanotransduction and support the concept that bone mechanotransduction is likely to encompass a number of interconnected signaling pathways. © 2017 American Society for Bone and Mineral Research.

Sclerostin Antibody Treatment Enhances Rotator Cuff Tendon-to-Bone Healing in an Animal Model.

Rotator cuff tears are a common source of pain and disability, and poor healing after repair leads to high retear rates. Bone loss in the humeral head before and after repair has been associated with poor healing. The purpose of the current study was to mitigate bone loss near the repaired cuff and improve healing outcomes. Sclerostin antibody (Scl-Ab) treatment, previously shown to increase bone formation and strength in the setting of osteoporosis, was used in the current study to address bone loss and enhance rotator cuff healing in an animal model. Scl-Ab was administered subcutaneously at the time of rotator cuff repair and every 2 weeks until the animals were sacrificed. The effect of Scl-Ab treatment was evaluated after 2, 4, and 8 weeks of healing, using bone morphometric analysis, biomechanical evaluation, histological analysis, and gene expression outcomes. Injury and repair led to a reduction in bone mineral density after 2 and 4 weeks of healing in the control and Scl-Ab treatment groups. After 8 weeks of healing, animals receiving Scl-Ab treatment had 30% greater bone mineral density than the controls. A decrease in biomechanical properties was observed in both groups after 4 weeks of healing compared with healthy tendon-to-bone attachments. After 8 weeks of healing, Scl-Ab-treated animals had improved strength (38%) and stiffness (43%) compared with control animals. Histological assessment showed that Scl-Ab promoted better integration of tendon and bone by 8 weeks of healing. Scl-Ab had significant effects on gene expression in bone, indicative of enhanced bone formation, and no effect on the expression of genes in tendon. This study provides evidence that Scl-Ab treatment improves tendon-to-bone healing at the rotator cuff by increasing attachment-site bone mineral density, leading to improved biomechanical properties. Scl-Ab treatment may improve outcomes after rotator cuff repair.

Combination sclerostin antibody and zoledronic acid treatment outperforms either treatment alone in a mouse model of osteogenesis imperfecta

In this study, we examined the therapeutic potential of anti-Sclerostin Antibody (Scl-Ab) and bisphosphonate treatments for the bone fragility disorder Osteogenesis Imperfecta (OI). Mice with the Amish OI mutation (Col1a2 G610C mice) and control wild type littermates (WT) were treated from week 5 to week 9 of life with (1) saline (control), (2) zoledronic acid given 0.025mg/kg s.c. weekly (ZA), (3) Scl-Ab given 50mg/kg IV weekly (Scl-Ab), or (4) a combination of both (Scl-Ab/ZA). Functional outcomes were prioritized and included bone mineral density (BMD), bone microarchitecture, long bone bending strength, and vertebral compression strength. By dual-energy absorptiometry, Scl-Ab treatment alone had no effect on tibial BMD, while ZA and Scl-Ab/ZA significantly enhanced BMD by week 4 (+16% and +27% respectively, P<0.05). Scl-Ab/ZA treatment also led to increases in cortical thickness and tissue mineral density, and restored the tibial 4-point bending strength to that of control WT mice. In the spine, all treatments increased compression strength over controls, but only the combined group reached the strength of WT controls. Scl-Ab showed greater anabolic effects in the trabecular bone than in cortical bone. In summary, the Scl-Ab/ZA intervention was superior to either treatment alone in this OI mouse model, however further studies are required to establish its efficacy in other preclinical and clinical scenarios.

Sclerostin Antibody Increases Callus Size and Strength but does not Improve Fracture Union in a Challenged Open Rat Fracture Model.

Open fractures remain a challenge in orthopedics. Current strategies to intervene are often inadequate, particularly in severe fractures or when treatment is delayed. Sclerostin is a negative regulator of bone growth and sclerostin-neutralizing antibodies (Scl-Ab) can increase bone mass and strength. The application of these antibodies to improve orthopedic repair has shown varied results, and may be dependent on the location and severity of the bony injury. We examined Scl-Ab treatment within an established rat osteotomy model with periosteal stripping analogous to open fracture repair. In one study, Scl-Ab was given 25mg/kg bi-weekly, either from the time of fracture or from 3 weeks post-fracture up to an end-point of 12weeks. A second study treated only delayed union open fractures that did not show radiographic union by week 6 post-fracture. Outcome measures included radiographic union, microCT analysis of bone volume and architecture, and histology. In the first study, Scl-Ab given from either 0 or 3weeks significantly improved callus bone volume (+52%, p < 0.05 and +58%, p < 0.01) at 12weeks, as well as strength (+48%, p < 0.05 and +70%, p < 0.05). Despite these improvements, union rate was not changed. In the second study treating only established delayed fractures, bony callus volume was similarly increased by Scl-Ab treatment; however, this did not translate to increased biomechanical strength or union improvement. Sclerostin antibody treatment has limited effects on the healing of challenging open fractures with periosteal stripping, but shows the greatest benefits on callus size and strength with earlier intervention.

Sclerostin Antibody Administration Converts Bone Lining Cells Into Active Osteoblasts.

Sclerostin antibody (Scl-Ab) increases osteoblast activity, in part through increasing modeling-based bone formation on previously quiescent surfaces. Histomorphometric studies have suggested that this might occur through conversion of bone lining cells into active osteoblasts. However, direct data demonstrating Scl-Ab-induced conversion of lining cells into active osteoblasts are lacking. Here, we used in vivo lineage tracing to determine if Scl-Ab promotes the conversion of lining cells into osteoblasts on periosteal and endocortical bone surfaces in mice. Two independent, tamoxifen-inducible lineage-tracing strategies were used to label mature osteoblasts and their progeny using the DMP1 and osteocalcin promoters. After a prolonged "chase" period, the majority of labeled cells on bone surfaces assumed a thin, quiescent morphology. Then, mice were treated with either vehicle or Scl-Ab (25 mg/kg) twice over the course of the subsequent week. After euthanization, marked cells were enumerated, their thickness quantified, and proliferation and apoptosis examined. Scl-Ab led to a significant increase in the average thickness of labeled cells on periosteal and endocortical bone surfaces, consistent with osteoblast activation. Scl-Ab did not induce proliferation of labeled cells, and Scl-Ab did not regulate apoptosis of labeled cells. Therefore, direct reactivation of quiescent bone lining cells contributes to the acute increase in osteoblast numbers after Scl-Ab treatment in mice. © 2016 American Society for Bone and Mineral Research.

Effects of sclerostin antibodies in animal models of osteoporosis

There is an unmet need for therapies that can restore bone strength and reduce fracture risk among patients at high risk of osteoporotic fracture. To address this need, bone-forming therapies that increase osteoblast activity are required to help restore bone structure and strength. Sclerostin is now recognized as a target for osteoporosis therapy. Sclerostin is predominantly secreted by the osteocyte and acts as an extracellular inhibitor of canonical Wnt signaling by binding to the receptors lipoprotein receptor-related protein-4, 5 and 6. Monoclonal antibodies to sclerostin (Scl-Ab) have been used in both clinical and in preclinical studies of osteoporosis with beneficial outcomes for bone density, structure, strength and fracture risk reduction. In this review paper, we summarize the current literature describing the effects of Scl-Ab in animal models of osteoporosis. In addition, we report new pharmacologic data from three animal studies of Scl-Ab: 1) a 12-month study evaluating bone quality in ovariectomized (OVX) rats; 2) a 6-month study evaluating bone structure and strength in adolescent cynomolgus monkeys; and 3) the effects of transition from Scl-Ab to vehicle or the RANKL inhibitor osteoprotegerin-Fc in OVX rats. Together, these results demonstrate that inhibition of sclerostin by Scl-Ab increased bone formation, and decreased bone resorption, leading to improved bone structure, bone mass and bone strength while maintaining bone quality in multiple animal models of osteoporosis. Further, gains in bone mass induced by Scl-Ab treatment were preserved by antiresorptive agents such as a RANKL inhibitor as a follow-on therapy. The bone-forming effects of Scl-Ab were unaffected by pre- or co-treatment with a bisphosphonate, and were restored following a treatment-free period after initial dosing. These data support the clinical development of Scl-Ab for treatment of conditions with low bone mass such as postmenopausal and male osteoporosis.

Suppression of Sclerostin Alleviates Radiation-Induced Bone Loss by Protecting Bone-Forming Cells and Their Progenitors Through Distinct Mechanisms.

Focal radiotherapy is frequently associated with skeletal damage within the radiation field. Our previous in vitro study showed that activation of Wnt/β-catenin pathway can overcome radiation-induced DNA damage and apoptosis of osteoblastic cells. Neutralization of circulating sclerostin with a monoclonal antibody (Scl-Ab) is an innovative approach for treating osteoporosis by enhancing Wnt/β-catenin signaling in bone. Together with the fact that focal radiation increases sclerostin amount in bone, we sought to determine whether weekly treatment with Scl-Ab would prevent focal radiotherapy-induced osteoporosis in mice. Micro-CT and histomorphometric analyses demonstrated that Scl-Ab blocked trabecular bone structural deterioration after radiation by partially preserving osteoblast number and activity. Consistently, trabecular bone in sclerostin null mice was resistant to radiation via the same mechanism. Scl-Ab accelerated DNA repair in osteoblasts after radiation by reducing the number of γ-H2AX foci, a DNA double-strand break marker, and increasing the amount of Ku70, a DNA repair protein, thus protecting osteoblasts from radiation-induced apoptosis. In osteocytes, apart from using similar DNA repair mechanism to rescue osteocyte apoptosis, Scl-Ab restored the osteocyte canaliculi structure that was otherwise damaged by radiation. Using a lineage tracing approach that labels all mesenchymal lineage cells in the endosteal bone marrow, we demonstrated that radiation damage to mesenchymal progenitors mainly involves shifting their fate to adipocytes and arresting their proliferation ability but not inducing apoptosis, which are different mechanisms from radiation damage to mature bone forming cells. Scl-Ab treatment partially blocked the lineage shift but had no effect on the loss of proliferation potential. Taken together, our studies provide proof-of-principle evidence for a novel use of Scl-Ab as a therapeutic treatment for radiation-induced osteoporosis and establish molecular and cellular mechanisms that support such treatment. © 2016 American Society for Bone and Mineral Research.

Rictor is required for optimal bone accrual in response to anti-sclerostin therapy in the mouse

Wnt signaling has emerged as a major target pathway for the development of novel bone anabolic therapies. Neutralizing antibodies against the secreted Wnt antagonist sclerostin (Scl-Ab) increase bone mass in both animal models and humans. Because we have previously shown that Rictor-dependent mTORC2 activity contributes to Wnt signaling, we test here whether Rictor is required for Scl-Ab to promote bone anabolism. Mice with Rictor deleted in the early embryonic limb mesenchyme (Prx1-Cre;Rictorf/f, hereafter RiCKO) were subjected to Scl-Ab treatment for 5weeks starting at 4months of age. In vivo micro–computed tomography (μCT) analyses before the treatment showed that the RiCKO mice displayed normal trabecular, but less cortical bone mass than the littermate controls. After 5weeks of treatment, Scl-Ab dose-dependently increased trabecular and cortical bone mass in both control and RiCKO mice, but the increase was significantly blunted in the latter. Dynamic histomorphometry revealed that the RiCKO mice formed less bone than the control in response to Scl-Ab. In addition, the RiCKO mice possessed fewer osteoclasts than normal under the basal condition and exhibited lesser suppression in osteoclast number by Scl-Ab. Consistent with the fewer osteoclasts in vivo, bone marrow stromal cells (BMSC) from the RiCKO mice expressed less Rankl but normal levels of Opg or M-CSF, and were less effective than the control cells in supporting osteoclastogenesis in vitro. The reliance of Rankl on Rictor appeared to be independent of Wnt-β-catenin or Wnt-mTORC2 signaling as Wnt3a had no effect on Rankl expression by BMSC from either control or RICKO mice. Overall, Rictor in the limb mesenchymal lineage is required for the normal response to the anti-sclerostin therapy in both bone formation and resorption.

Sclerostin Antibody Treatment Improves the Bone Phenotype of Crtap–/– Mice, a Model of Recessive Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is characterized by low bone mass, poor bone quality, and fractures. Standard treatment for OI patients is limited to bisphosphonates, which only incompletely correct the bone phenotype, and seem to be less effective in adults. Sclerostin-neutralizing antibodies (Scl-Ab) have been shown to be beneficial in animal models of osteoporosis, and dominant OI resulting from mutations in the genes encoding type I collagen. However, Scl-Ab treatment has not been studied in models of recessive OI. Cartilage-associated protein (CRTAP) is involved in posttranslational type I collagen modification, and its loss of function results in recessive OI. In this study, we treated 1-week-old and 6-week-old Crtap(-/-) mice with Scl-Ab for 6 weeks (25 mg/kg, s.c., twice per week), to determine the effects on the bone phenotype in models of "pediatric" and "young adult" recessive OI. Vehicle-treated Crtap(-/-) and wild-type (WT) mice served as controls. Compared with control Crtap(-/-) mice, micro-computed tomography (μCT) analyses showed significant increases in bone volume and improved trabecular microarchitecture in Scl-Ab-treated Crtap(-/-) mice in both age cohorts, in both vertebrae and femurs. Additionally, Scl-Ab improved femoral cortical parameters in both age cohorts. Biomechanical testing showed that Scl-Ab improved parameters of whole-bone strength in Crtap(-/-) mice, with more robust effects in the week 6 to 12 cohort, but did not affect the increased bone brittleness. Additionally, Scl-Ab normalized the increased osteoclast numbers, stimulated bone formation rate (week 6 to 12 cohort only), but did not affect osteocyte density. Overall, our findings suggest that Scl-Ab treatment may be beneficial in the treatment of recessive OI caused by defects in collagen posttranslational modification. © 2015 American Society for Bone and Mineral Research.

Time-dependent cellular and transcriptional changes in the osteoblast lineage associated with sclerostin antibody treatment in ovariectomized rats

Inhibition of sclerostin with sclerostin antibody (Scl-Ab) has been shown to stimulate bone formation, decrease bone resorption, and increase bone mass in both animals and humans. To obtain insight into the temporal cellular and transcriptional changes in the osteoblast (OB) lineage associated with long-term Scl-Ab treatment, stereological and transcriptional analyses of the OB lineage were performed on lumbar vertebrae from aged ovariectomized rats. Animals were administered Scl-Ab 3 or 50mg/kg/wk or vehicle (VEH) for up to 26weeks (d183), followed by a treatment-free period (TFP). At 50mg/kg/wk, bone volume (BV/total volume [TV]) increased through d183 and declined during the TFP. Bone formation rate (BFR/bone surface [BS]) and total OB number increased through d29, then progressively declined, coincident with a decrease in total osteoprogenitor (OP) numbers from d29 through d183. Analysis of differentially expressed genes (DEGs) from microarray analysis of mRNA isolated from laser capture microdissection samples enriched for OB, lining cells, and osteocytes (OCy) revealed modules of genes that correlated with BFR/BS, BV/TV, and osteoblastic surface (Ob.S)/BS. Expression change of canonical Wnt target genes was similar in all three cell types at d8, including upregulation of Twist1 and Wisp1. At d29, the pattern of Wnt target gene expression changed in the OCy, with Twist1 returning to VEH level, sustained upregulation of Wisp1, and upregulation of several other Wnt targets that continued into the TFP. Predicted activation of pathways recognized to integrate with and regulate canonical Wnt signaling were also activated at d29 in the OCy. The most significantly affected pathways represented transcription factor signaling known to inhibit cell cycle progression (notably p53) and mitogenesis (notably c-Myc). These changes occurred at the time of peak BFR/BS and continued as BFR/BS declined during treatment, then trended toward VEH level in the TFP. Concurrent with this transcriptional switch was a reduction in OP numbers, an effect that would ultimately limit bone formation. This study confirms that the initial transcriptional response in response to Scl-Ab is activation of canonical Wnt signaling and the data demonstrate that there is induction of additional regulatory pathways in OCy with long-term treatment. The interactions between Wnt and p53/c-Myc signaling may be key in limiting OP populations, thus contributing to self-regulation of bone formation with continued Scl-Ab administration.

Sclerostin Antibody Treatment Increases Bone Formation, Bone Mass, and Bone Strength of Intact Bones in Adult Male Rats.

We investigated the systemic effect of sclerostin monoclonal antibody (Scl-Ab) treatment on intact non-operated bones in an open osteotomy male Sprague Dawley (SD) rat model. Six-month-old male SD rats were subjected to transverse osteotomy at the right femur mid-shaft. Rats were injected subcutaneously with vehicle or Scl-Ab (25 mg/kg, 2 times per week) treatment for 9 weeks. Compared with vehicle control, Scl-Ab treatment significantly improved trabecular and cortical bone mass and microarchitecture at L5 vertebrae and left femora by micro-CT at week 6 and 9. Mechanical testing showed that Scl-Ab treatment resulted in significantly higher stiffness, energy to failure and ultimate load at the femora at week 9. Mineral apposition rate, mineralizing surface and bone formation rate on the trabecular bone in the distal femora was significantly increased in Scl-Ab group at week 6 and 9. The administered Scl-Ab was localized in the osteocytes and beta-catenin was strongly expressed in osteoblasts. Scl-Ab treatment significantly increased serum P1NP level and there was no between-group difference in serum level of CTX-1. In conclusion, Scl-Ab treatment could induce rapid and sustained increase in bone formation, bone mass and bone strength in non-operated bones. Sclerostin inhibition might be advantageous to prevent secondary fracture(s).

Sclerostin antibody and interval treadmill training effects in a rodent model of glucocorticoid-induced osteopenia

Glucocorticoids have a beneficial anti-inflammatory and immunosuppressive effect, but their use is associated with decreased bone formation, bone mass and bone quality, resulting in an elevated fracture risk. Exercise and sclerostin antibody (Scl-Ab) administration have both been shown to increase bone formation and bone mass, therefore the ability of these treatments to inhibit glucocorticoid-induced osteopenia alone or in combination were assessed in a rodent model. Adult (4months-old) male Wistar rats were allocated to a control group (C) or one of 4 groups injected subcutaneously with methylprednisolone (5mg/kg/day, 5days/week). Methylprednisolone treated rats were injected subcutaneously 2days/week with vehicle (M) or Scl-Ab-VI (M+S: 25mg/kg/day) and were submitted or not to treadmill interval training exercise (1h/day, 5days/week) for 9weeks (M+E, M+E+S). Methylprednisolone treatment increased % fat mass and % apoptotic osteocytes, reduced whole body and femoral bone mineral content (BMC), reduced femoral bone mineral density (BMD) and osteocyte lacunae occupancy. This effect was associated with lower trabecular bone volume (BV/TV) at the distal femur. Exercise increased BV/TV, osteocyte lacunae occupancy, while reducing fat mass, the bone resorption marker NTx, and osteocyte apoptosis. Exercise did not affect BMC or cortical microarchitectural parameters. Scl-Ab increased the bone formation marker osteocalcin and prevented the deleterious effects of M on bone mass, further increasing BMC, BMD and BV/TV to levels above the C group. Scl-Ab increased femoral cortical bone parameters at distal part and midshaft. Scl-Ab prevented the decrease in osteocyte lacunae occupancy and the increase in osteocyte apoptosis induced by M. The addition of exercise to Scl-Ab treatment did not result in additional improvements in bone mass or bone strength parameters. These data suggest that although our exercise regimen did prevent some of the bone deleterious effects of glucocorticoid treatment, particularly in trabecular bone volume and osteocyte apoptosis, Scl-Ab treatment resulted in marked improvements in bone mass across the skeleton and in osteocyte viability, resulting in decreased bone fragility.

Sclerostin antibody treatment improves implant fixation in a model of severe osteoporosis.

The mechanical fixation of orthopaedic and dental implants is compromised by diminished bone volume, such as with osteoporosis. Systemic administration of sclerostin antibody (Scl-Ab) has been shown to enhance implant fixation in normal animals. In the present study, we tested whether Scl-Ab can improve implant fixation in established osteoporosis in a rat model. We used an ovariectomized (ovx) rat model, in which we found a 78% decrease in trabecular bone volume at the time of implant surgery; sham-ovx, age-matched rats were used as controls. After placement of a titanium implant in the medullary cavity of the distal aspect of the femur, the rats were maintained for four, eight, or twelve weeks and treated biweekly with Scl-Ab or with the delivery vehicle alone. Outcomes were measured with use of microcomputed tomography, mechanical testing, and static and dynamic histomorphometry. Scl-Ab treatment doubled implant fixation strength in both the sham-ovx and ovx groups, although the enhancement was delayed in the ovx group. Scl-Ab treatment also enhanced bone-implant contact; increased peri-implant trabecular thickness and volume; and increased cortical thickness. These structural changes were associated with an approximately five to sevenfold increase in the bone-formation rate and a >50% depression in the eroded surface following Scl-Ab treatment. Trabecular bone thickness and bone-implant contact accounted for two-thirds of the variance in fixation strength. In this model of severe osteoporosis, Scl-Ab treatment enhanced implant fixation by stimulating bone formation and suppressing bone resorption, leading to enhanced bone-implant contact and improved trabecular bone volume and architecture. Systemic administration of anti-sclerostin antibodies might be a useful strategy in total joint replacement when bone mass is deficient.

Systemic administration of sclerostin monoclonal antibody accelerates fracture healing in the femoral osteotomy model of young rats.

Genetic studies have demonstrated that sclerostin was a key negative regulator of bone formation. Sclerostin monoclonal antibody (Scl-Ab) treatment enhanced bone healing in experimental fracture healing. The purpose was to investigate the effects of systemic Scl-Ab administration on open fracture healing in young rats. Unilateral femoral fractures were generated in eight-week-old Sprague-Dawley rats. Rats were treated with vehicle or Scl-Ab for 6weeks. Fracture healing was evaluated by western blotting, immunohistochemistry, histology, radiography, micro-CT, and biomechanical testing. In addition, the bone mass of intact femur was also evaluated by micro-CT. The results showed that, at 1 and 2weeks after fracture, proliferating cell nuclear antigen (PCNA) score and bone morphogenetic protein-2 (BMP-2) expression in the Scl-Ab group were significantly increased compared with the control group. A decrease in cartilage in the Scl-Ab group was also observed after fracture, and this was accompanied by more rapider fracture healing. At 4 and 6weeks, there were significant increases in bone mass and mechanical properties in the calluses from Scl-Ab group compared with control group. In addition, Scl-Ab treatment also showed significant anabolic effects in intact femur. In conclusion, systemic Scl-Ab administration has a significant enhancement in a rat femoral osteotomy model. These results support the therapeutic potential of Scl-Ab as a noninvasive strategy to enhance open fracture healing.