Sclerostin-neutralizing Antibody Research Articles

A mechanism of action-reflective, dual cell-based bioassay for determining the bioactivity of sclerostin-neutralizing antibodies

Osteoporosis is a major threat to the elderly worldwide. The Wnt signaling pathway plays a critical role in bone development and homeostasis. Sclerostin, a Wnt ligand inhibitor, competes with Wnt ligands for low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) on osteoblasts, thereby suppressing bone formation. Sclerostin-neutralizing monoclonal antibodies (mAbs) have emerged as a potential bone-forming therapy for osteoporosis. A cell-based bioassay which determines the relative activity of a product, related to its mechanism of action, is of great importance from drug discovery to quality control and batch release. Currently used cell-based bioassays for sclerostin-neutralizing mAbs usually use Wnt1 or Wnt3a to stimulate the Wnt pathway; sclerostin is a direct inhibitor of Wnt1 but not Wnt3a. Wnt1 is a highly hydrophobic protein that binds to the producing cell membrane and acts in a juxtacrine manner to stimulate the Wnt pathway in neighboring cells. Bioassays for drugs that induce Wnt1 signaling should be performed in a juxtacrine manner. Here, we present a mechanism of action-reflective, dual cell-based reporter gene assay. In this assay, Wnt1 producer cells are co-cultured with cells containing the Wnt reporter genes, Wnt1 on the producer cells activates the Wnt signaling pathway in the reporter cells that are in direct cell-to-cell contact, and sclerostin-neutralizing mAbs specifically and effectively antagonize the sclerostin-mediated Wnt reporter gene suppression. This bioassay demonstrates good specificity, accuracy, linearity, and precision and is suitable for quality control, stability testing, batch release, and biosimilarity assessment of sclerostin-neutralizing mAbs.

O-GlcNAcylation mediates Wnt-stimulated bone formation by rewiring aerobic glycolysis

Wnt signaling is an important target for anabolic therapies in osteoporosis. A sclerostin-neutralizing antibody (Scl-Ab), that blocks the Wnt signaling inhibitor (sclerostin), has been shown to promote bone mass in animal models and clinical studies. However, the cellular mechanisms by which Wnt signaling promotes osteogenesis remain to be further investigated. O-GlcNAcylation, a dynamic post-translational modification of proteins, controls multiple critical biological processes including transcription, translation, and cell fate determination. Here, we report that Wnt3a either induces O-GlcNAcylation rapidly via the Ca2+-PKA-Gfat1 axis, or increases it in a Wnt-β-catenin-dependent manner following prolonged stimulation. Importantly, we find O-GlcNAcylation indispensable for osteoblastogenesis both in vivo and in vitro. Genetic ablation of O-GlcNAcylation in the osteoblast-lineage diminishes bone formation and delays bone fracture healing in response to Wnt stimulation in vivo. Mechanistically, Wnt3a induces O-GlcNAcylation at Serine 174 of PDK1 to stabilize the protein, resulting in increased glycolysis and osteogenesis. These findings highlight O-GlcNAcylation as an important mechanism regulating Wnt-induced glucose metabolism and bone anabolism.

Sclerostin antibody corrects periodontal disease in type 2 diabetic mice.

Type 2 diabetes (T2D) is on the rise worldwide and is associated with various complications in the oral cavity. Using an adult-onset diabetes preclinical model, we demonstrated profound periodontal alterations in T2D mice, including inflamed gingiva, disintegrated periodontal ligaments (PDLs), marked alveolar bone loss, and unbalanced bone remodeling due to decreased formation and increased resorption. Notably, we observed elevated levels of the Wnt signaling inhibitor sclerostin in the alveolar bone of T2D mice. Motivated by these findings, we investigated whether a sclerostin-neutralizing antibody (Scl-Ab) could rescue the compromised periodontium in T2D mice. Administering Scl-Ab subcutaneously once a week for 4 weeks, starting 4 weeks after T2D induction, led to substantial increases in bone mass. This effect was attributed to the inhibition of osteoclasts and promotion of osteoblasts in both control and T2D mice, effectively reversing the bone loss caused by T2D. Furthermore, Scl-Ab stimulated PDL cell proliferation, partially restored the PDL fibers, and mitigated inflammation in the periodontium. Our study thus established a T2D-induced periodontitis mouse model characterized by inflammation and tissue degeneration. Scl-Ab emerged as a promising intervention to counteract the detrimental effects of T2D on the periodontium, exhibiting limited side effects on other craniofacial hard tissues.

Bone matrix properties in adults with osteogenesis imperfecta are not adversely affected by setrusumab-a sclerostin neutralizing antibody.

Osteogenesis imperfecta (OI) is a skeletal dysplasia characterized by low bone mass and frequent fractures. Children with OI are commonly treated with bisphosphonates to reduce fracture rate, but treatment options for adults are limited. In the Phase 2b ASTEROID trial, setrusumab (a sclerostin neutralizing antibody, SclAb) improved bone density and strength in adults with type I, III, and IV OI. Here, we investigate bone matrix material properties in tetracycline-labeled trans iliac biopsies from 3 groups: (1) control: individuals with no metabolic bone disease, (2) OI: individuals with OI, (3) SclAb-OI: individuals with OI after 6 mo of setrusumab treatment (as part of the ASTEROID trial). In addition to bone histomorphometry, bone mineral and matrix properties were evaluated with nanoindentation, Raman spectroscopy, second harmonic generation imaging, quantitative backscatter electron imaging, and small-angle X-ray scattering. Spatial locations of fluorochrome labels were identified to differentiate inter-label bone of the same tissue age and intra-cortical bone. No difference in collagen orientation was found between the groups. The bone mineral density distribution and analysis of Raman spectra indicate that OI groups have greater mean mineralization, greater relative mineral content, and lower crystallinity than the control group, which was not altered by SclAb treatment. Finally, a lower modulus and hardness were measured in the inter-label bone of the OI-SclAb group compared to the OI group. Previous studies suggest that even though bone from OI has a higher mineral content, the extracellular matrix (ECM) has comparable mechanical properties. Therefore, fragility in OI may stem from contributions from other yet unexplored aspects of bone organization at higher length scales. We conclude that SclAb treatment leads to increased bone mass while not adversely affecting bone matrix properties in individuals with OI.

Sclerostin blockade inhibits bone resorption through PDGF receptor signaling in osteoblast lineage cells.

While sclerostin-neutralizing antibodies (Scl-Abs) transiently stimulate bone formation by activating Wnt signaling in osteoblast lineage cells, they exert sustained inhibition of bone resorption, suggesting an alternate signaling pathway by which Scl-Abs control osteoclast activity. Since sclerostin can activate platelet-derived growth factor receptors (PDGFRs) in osteoblast lineage cells in vitro and PDGFR signaling in these cells induces bone resorption through M-CSF secretion, we hypothesized that the prolonged anticatabolic effect of Scl-Abs could result from PDGFR inhibition. We show here that inhibition of PDGFR signaling in osteoblast lineage cells is sufficient and necessary to mediate prolonged Scl-Ab effects on M-CSF secretion and osteoclast activity in mice. Indeed, sclerostin coactivates PDGFRs independently of Wnt/β-catenin signaling inhibition, by forming a ternary complex with LRP6 and PDGFRs in preosteoblasts. In turn, Scl-Ab prevents sclerostin-mediated coactivation of PDGFR signaling and consequent M-CSF upregulation in preosteoblast cultures, thereby inhibiting osteoclast activity in preosteoblast/osteoclast coculture assays. These results provide a potential mechanism explaining the dissociation between anabolic and antiresorptive effects of long-term Scl-Ab.

Affinity targeting of therapeutic proteins to the bone surface-local delivery of sclerostin-neutralizing antibody enhances efficacy.

Currently available biotherapeutics for the treatment of osteoporosis lack explicit mechanisms for bone localization, potentially limiting efficacy and inducing off-target toxicities. While various strategies have been explored for targeting the bone surface, critical aspects remain poorly understood, including the optimal affinity ligand, the role of binding avidity and circulation time, and, most importantly, whether or not this strategy can enhance the functional activity of clinically relevant protein therapeutics. To investigate, we generated fluorescent proteins (eg, mCherry) with site-specifically attached small molecule (bisphosphonate) or peptide (deca-aspartate, D10) affinity ligands. While both affinity ligands successfully anchored fluorescent protein to the bone surface, quantitative radiotracing revealed only modest femoral and vertebral accumulation and suggested a need for enhanced circulation time. To achieve this, we fused mCherry to the Fc fragment of human IgG1 and attached D10 peptides to each C-terminus. The mCherry-Fc-D10 demonstrated an ~80-fold increase in plasma exposure and marked increases in femoral and vertebral accumulation (13.6% ± 1.4% and 11.4% ± 1.3% of the injected dose/g [%ID/g] at 24h, respectively). To determine if bone surface targeting could enhance the efficacy of a clinically relevant therapeutic, we generated a bone-targeted sclerostin-neutralizing antibody, anti-sclerostin-D10. The targeted antibody demonstrated marked increases in bone accumulation and retention (20.9 ± 2.5% and 19.5 ± 2.5% ID/g in femur and vertebrae at 7days) and enhanced effects in a murine model of ovariectomy-induced bone loss (bone volume/total volume, connectivity density, and structure model index all increased [P< .001] vs untargeted anti-sclerostin). Collectively, our results indicate the importance of both bone affinity and circulation time in achieving robust targeting of therapeutic proteins to the bone surface and suggest that this approach may enable lower doses and/or longer dosing intervals without reduction in biotherapeutic efficacy. Future studies will be needed to determine the translational potential of this strategy and its potential impact on off-site toxicities.

Role of Wnt signaling and sclerostin in bone and as therapeutic targets in skeletal disorders.

Wnt signaling is a key regulator of skeletal development and homeostasis; germinal mutations affecting genes encoding components, inhibitors, and enhancers of the Wnt pathways were shown to be responsible for the development of rare congenital metabolic bone disorders. Sclerostin is a bone tissue-specific inhibitor of the Wnt/β-catenin pathway, secreted by osteocytes, negatively regulating osteogenic differentiation and bone formation, and promoting osteoclastogenesis and bone resorption. Here, we reviewed current knowledge on the role of sclerostin and Wnt pathways in bone metabolism and skeletal disorders, and on the state of the art of therapy with sclerostin-neutralizing antibodies in low-bone-mass diseases. Various in vivo studies on animal models of human low-bone-mass diseases showed that targeting sclerostin to recover bone mass, restore bone strength, and prevent fragility fracture was safe and effective in osteoporosis, osteogenesis imperfecta, and osteoporosis pseudoglioma. Currently, only treatment with romosozumab, a humanized monoclonal anti-sclerostin antibody, has been approved in human clinical practice for the treatment of osteoporosis, showing a valuable capability to increase BMD at various skeletal sites and reduce the occurrence of new vertebral, non-vertebral, and hip fragility fractures in treated male and female osteoporotic patients. Preclinical studies demonstrated safety and efficacy of therapy with anti-sclerostin monoclonal antibodies in the preservation/restoration of bone mass and prevention of fragility fractures in low-bone-mass clinical phenotypes, other than osteoporosis, to be validated by clinical studies for their approved translation into prevalent clinical practice.

Role of Sclerostin in Multiple Myeloma Progression

Multiple Myeloma (MM) is a tumor of plasma cells that originates and expands within bone marrow (BM). The BM microenvironment, including its constituent stromal cells and cytokines contribute to disease progression. The BM stromal cells include osteolineage cells, osteoclasts, adipocytes, endothelial cells etc. Among these, cells of the osteoblast lineage have been identified as the most versatile regulators of hematopoietic cells. The osteolineage cells include preosteoblasts, committed osteoblasts, mature osteoblasts, and the terminally differentiated osteocytes, each with its own characteristic biological activity. The osteolineage cells have been shown to regulate commitment and differentiation of hematopoietic lineage cells including hematopoietic stem cells, B-cells, and myeloid lineage cells.Previously published data have shown that loss of parathyroid hormone (PTH) signaling in osteolineage cells adversely affects B-cell development (Panaroni et al., 2016). PTH is also a strong inhibitor of sclerostin, a Wnt-signaling inhibitor secreted by osteocytes. Moreover, mice lacking osteocytes show decreased lymphopoiesis, which may in part be regulated by sclerostin. A sclerostin-neutralizing antibody has shown promise for treatment for age-induced osteoporosis. We have recently demonstrated that MM patients have increased levels of sclerostin and that treatment with a SOST neutralizing antibody resulted in increased osteoblastic differentiation (Eda et al., 2015) and decreased tumor burden. Based upon these findings, we hypothesized that the osteolineage cells contribute to MM progression, and that this may in part occur via sclerostin. To test this hypothesis, we have used genetic mouse models of osteolineage or sclerostin deficiency. Mouse MM cell line 5TGM1 was used to examine MM progression in these mouse models.Global sclerostin deficient (SOST KO) mice were generated as described previously. Given that sclerostin acts as a negative regulator of bone formation, SOST KO mice displayed robust increase in trabecular and cortical bone density as analyzed by micro-computed tomography. Co-culture with 5TGM1 MM cells showed that the BM stromal cells from SOST KO mice significantly decreased proliferation of MM cells raising the possibility that sclerostin deficiency may decrease MM progression in vivo. The SOST KO mice showed significantly reduced BM cellularity compared to the controls. However, relevant to MM progression, overall B-lymphopoiesis, proliferation or apoptosis was unchanged during any stages of B-cell development in BM of 3-month old SOST KO mice.To determine if sclerostin deficiency alters MM engraftment in vivo, 5TGM1 cells were injected into the tibia of 3-month old control or SOST KO mice and engraftment was followed for 2-months. Complete blood counts (CBC) showed no significant changes between the control and SOST KO mice injected with the MM cells. The 5TGM1 cells were fluorescently tagged before in vivo injections allowing us to track the cells by whole body bioluminescence imaging and by contralateral tibia BM flow cytometry analysis. Both the analyses showed no changes in the distant MM engraftment between the control and the SOST KO mice. Ongoing work is examining local MM engraftment in the tibia with injected MM cells.These data suggest that the sclerostin deficiency does not alter systemic MM engraftment after 2 months of follow-up observations. It is possible, however, that the local progression of MM cells may have altered. We are investigating this possibility. We are also examining MM engraftment in mice lacking mature osteoblasts and osteocytes to identify sclerostin-dependent and independent factors controlling MM progression. DisclosuresRaje:Millenium: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Onyx: Consultancy.

Sclerostin antibody stimulates periodontal regeneration in large alveolar bone defects

Destruction of the alveolar bone in the jaws can occur due to periodontitis, trauma or following tumor resection. Common reconstructive therapy can include the use of bone grafts with limited predictability and efficacy. Romosozumab, approved by the FDA in 2019, is a humanized sclerostin-neutralizing antibody (Scl-Ab) indicated in postmenopausal women with osteoporosis at high risk for fracture. Preclinical models show that Scl-Ab administration preserves bone volume during periodontal disease, repairs bone defects surrounding dental implants, and reverses alveolar bone loss following extraction socket remodeling. To date, there are no studies evaluating Scl-Ab to repair osseous defects around teeth or to identify the efficacy of locally-delivered Scl-Ab for targeted drug delivery. In this investigation, the use of systemically-delivered versus low dose locally-delivered Scl-Ab via poly(lactic-co-glycolic) acid (PLGA) microspheres (MSs) was compared at experimentally-created alveolar bone defects in rats. Systemic Scl-Ab administration improved bone regeneration and tended to increase cementogenesis measured by histology and microcomputed tomography, while Scl-Ab delivered by MSs did not result in enhancements in bone or cemental repair compared to MSs alone or control. In conclusion, systemic administration of Scl-Ab promotes bone and cemental regeneration while local, low dose delivery did not heal periodontal osseous defects in this study.

Sclerostin-Neutralizing Antibody Treatment Rescues Negative Effects of Rosiglitazone on Mouse Bone Parameters.

Obesity, a growing pandemic, is a risk factor for many cancers and causes increased bone marrow adipose tissue (BMAT). in vitro studies and obese animal models suggest that BMAT contributes to cancer progression, but there is a lack of preclinical models to directly test BMAT's role in cancer. Overactivation of peroxisome-proliferator-activated receptor-γ (PPARγ) can skew bone formation and resorption rates, resulting in increased BMAT and trabecular bone loss. Thiazolidinediones (eg, rosiglitazone) are anti-diabetic therapies that promote adipogenesis through PPARγ activation. We investigated if rosiglitazone increases BMAT in an immunocompromised model, commonly used in cancer research, and if these effects could be reversed by co-administering a bone anabolic agent (sclerostin-neutralizing antibody [Scl-Ab]), which has been shown to inhibit adipogenesis, using DXA, μCT, OsO4 μCT, and dynamic histomorphometry. Four weeks of rosiglitazone in female SCID Beige mice (cohort 1) significantly decreased trabecular bone volume (BV/TV) by about one-half, through increased osteoclast and suppressed osteoblast activity, and significantly increased BMAT. In cohort 2, mice were administered rosiglitazone ± Scl-Ab for 4 weeks, and then rosiglitazone was discontinued and Scl-Ab or vehicle were continued for 6 weeks. Scl-Ab significantly increased bone parameters (eg, BV/TV, N.Ob/B.Pm, and MS/BS) in both groups. Scl-Ab also overcame many negative effects of rosiglitazone (eg, effects on trabecular bone parameters, increased mineralization lag time [MLT], and decreased bone formation rate [BFR]). Interestingly, Scl-Ab significantly decreased rosiglitazone-induced BMAT in the femur, mostly due to a reduction in adipocyte size, but had a much weaker effect on tibial BMAT. These data suggest targeting sclerostin can prevent rosiglitazone-induced bone loss and reduce BM adiposity, in some, but not all BMAT locations. Collectively, our data demonstrate that rosiglitazone increases BMAT in SCID Beige mice, but concomitant changes in bone may confound its use to specifically determine BMAT's role in tumor models. © 2020 American Society for Bone and Mineral Research (ASBMR).

Single-Cell RNA Sequencing of Calvarial and Long-Bone Endocortical Cells.

Single-cell RNA sequencing (scRNA-Seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-Seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-Seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-Seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-Seq on freshly recovered long bone endocortical cells from mice that received either vehicle or sclerostin-neutralizing antibody for 1 week. We were unable to detect significant changes in bone anabolism-associated transcripts in immature and mature osteoblasts recovered from mice treated with sclerostin-neutralizing antibody; this might be a consequence of being underpowered to detect modest changes in gene expression, because only 7% of the sequenced endocortical cells were osteoblasts and a limited portion of their transcriptomes were sampled. We conclude that scRNA-Seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single-cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required. © 2020 American Society for Bone and Mineral Research.

Sclerostin-Antibody Treatment Decreases Fracture Rates in Axial Skeleton and Improves the Skeletal Phenotype in Growing oim/oim Mice.

In osteogenesis imperfecta (OI), vertebrae brittleness causes thorax deformations and leads to cardiopulmonary failure. As sclerostin-neutralizing antibodies increase bone mass and strength in animal models of osteoporosis, their administration in two murine models of severe OI enhanced the strength of vertebrae in growing female Crtap-/- mice but not in growing male Col1a1Jrt/+ mice. However, these two studies ignored the impact of antibodies on spine growth, fracture rates, and compressive mechanical properties. Here, we conducted a randomized controlled trial in oim/oim mice, an established model of human severe OI type III due to a mutation in Col1a2. Five-week-old female WT and oim/oim mice received either PBS or sclerostin antibody (Scl-Ab) for 9weeks. Analyses included radiography, histomorphometry, pQCT, microcomputed tomography, and biomechanical testing. Though it did not modify vertebral axial growth, Scl-Ab treatment markedly reduced the fracture prevalence in the pelvis and caudal vertebrae, enhanced osteoblast activity (L4), increased cervico-sacral spine BMD, and improved the lumbosacral spine bone cross-sectional area. Scl-Ab did not impact vertebral height and body size but enhanced the cortical thickness and trabecular bone volume significantly in the two Scl-Ab groups. At lumbar vertebrae and tibial metaphysis, the absolute increase in cortical and trabecular bone mass was higher in Scl-Ab WT than in Scl-Ab oim/oim. The effects on trabecular bone mass were mainly due to changes in trabecular number at vertebrae and in trabecular thickness at metaphyses. Additionally, Scl-Ab did not restore a standard trabecular network, but improved bone compressive ultimate load with more robust effects at vertebrae than at metaphysis. Overall, Scl-Ab treatment may be beneficial for reducing vertebral fractures and spine deformities in patients with severe OI.

Finite element analysis of bone strength in osteogenesis imperfecta.

As a dedicated experimentalist, John Currey praised the high potential of finite element (FE) analysis but also recognized its critical limitations. The application of the FE methodology to bone tissue is reviewed in the light of his enthusiastic and colorful statements. In the past decades, FE analysis contributed substantially to the understanding of structure-function properties in the hierarchical organization of bone and to the simulation of bone adaptation. The systematic experimental validation of FE analysis of bone strength in anatomical locations at risk of fracture led to its application in clinical studies to evaluate efficacy of antiresorptive or anabolic treatment of bone fragility. Beyond the successful analyses of healthy or osteoporotic bone, FE analysis becomes increasingly involved in the investigation of other fragility-related bone diseases. The case of osteogenesis imperfecta (OI) is exposed, the multiscale alterations of the bone tissue and the effect of treatment summarized. A few FE analyses attempting to answer open questions in OI are then reported. An original study is finally presented that explored the structural properties of the Brtl/+ murine model of OI type IV subjected to sclerostin neutralizing antibody treatment using microFE analysis. The use of identical material properties in the four-point bending FE simulations of the femora reproduced not only the experimental values but also the statistical comparisons examining the effect of disease and treatment. Further efforts are needed to build upon the extraordinary legacy of John Currey and clarify the impact of different bone diseases on the hierarchical mechanical properties of bone.

Inverse correlation between trabecular bone volume and bone marrow adipose tissue in rats treated with osteoanabolic agents.

There is currently an unmet clinical need for improved treatments for skeletal diseases such as osteoporosis and cancer-induced bone disease. This is due in part to a paucity of novel targets and an incomplete understanding of the mechanisms of action for established therapies. We defined the effects of anabolic treatments on bone and the bone marrow adipocyte (BMA). Sclerostin-neutralizing antibodies (Scl-Ab), romosozumab, human parathyroid hormone (hPTH, 1-34), and hPTH/hPTHrP analogues (e.g. teriparatide and abaloparatide) stimulate bone formation and have been studied in clinical trials for severe osteoporosis. In this study, eight-week-old male and female rats were administered vehicle, Scl-Ab (3 mg/kg or 50 mg/kg) weekly, or hPTH (1-34) (75 μg/kg) daily for 4 or 26 weeks. Histological analyses of distal femura were performed using a novel ImageJ method for trabecular bone and bone marrow adipose tissue (BMAT). Adipocyte number, circumference, and total adipose area were compared within the tissue area (T.Ar) or the marrow area (Ma.Ar), (defined as the T.Ar minus the trabecular bone area). After 26 weeks of treatment, a significant inverse correlation between bone and tissue adiposity (total adipocyte area divided by T.Ar) were observed in males and females (p < 0.0001). However, there were no significant correlations between bone and marrow adiposity (total adipocyte area divided by Ma.Ar) for either sex after 26 weeks of treatments. Scl-Ab treatments also resulted in no effect on adipocytes based on marrow adiposity for either sex after 26 weeks. However, chronic hPTH treatments significantly reduced adipocyte number and adiposity within the T.Ar and within the Ma.Ar in males. Overall, our data suggest that with long-term treatment, Scl-Abs decrease total tissue adiposity mainly by increasing trabecular bone, resulting in an overall reduction in the space in which adipocytes can reside. These findings were determined by developing and comparing two different methods of assessment of the marrow cavity, defined to either include or exclude trabecular bone. Thus, researchers should consider which adiposity measurement is more informative and relevant for their studies. Overall, our findings should help design improved therapies or combination treatments to target a potential new contributor to bone diseases: the bone marrow adipocyte.

Differential changes in bone strength of two inbred mouse strains following administration of a sclerostin-neutralizing antibody during growth.

Administration of sclerostin-neutralizing antibody (Scl-Ab) treatment has been shown to elicit an anabolic bone response in growing and adult mice. Prior work characterized the response of individual mouse strains but did not establish whether the impact of Scl-Ab on whole bone strength would vary across different inbred mouse strains. Herein, we tested the hypothesis that two inbred mouse strains (A/J and C57BL/6J (B6)) will show different whole bone strength outcomes following sclerostin-neutralizing antibody (Scl-Ab) treatment during growth (4.5–8.5 weeks of age). Treated B6 femurs showed a significantly greater stiffness (S) (68.8% vs. 46.0%) and maximum load (ML) (84.7% vs. 44.8%) compared to A/J. Although treated A/J and B6 femurs showed greater cortical area (Ct.Ar) similarly relative to their controls (37.7% in A/J and 41.1% in B6), the location of new bone deposition responsible for the greater mass differed between strains and may explain the greater whole bone strength observed in treated B6 mice. A/J femurs showed periosteal expansion and endocortical infilling, while B6 femurs showed periosteal expansion. Post-yield displacement (PYD) was smaller in treated A/J femurs (-61.2%, p < 0.001) resulting in greater brittleness compared to controls; an effect not present in B6 mice. Inter-strain differences in S, ML, and PYD led to divergent changes in work-to-fracture (Work). Work was 27.2% (p = 0.366) lower in treated A/J mice and 66.2% (p < 0.001) greater in treated B6 mice relative to controls. Our data confirmed the anabolic response to Scl-Ab shown by others, and provided evidence suggesting the mechanical benefits of Scl-Ab administration may be modulated by genetic background, with intrinsic growth patterns of these mice guiding the location of new bone deposition. Whether these differential outcomes will persist in adult and elderly mice remains to be determined.

Advanced glycation end-product 2 and Porphyromonas gingivalis lipopolysaccharide increase sclerostin expression in mouse osteocyte-like cells

Sclerostin is a secreted glycoprotein that is mainly expressed in osteocytes, exerts negative effects on bone formation, and is present at elevated levels in diabetes mellitus (DM). Periodontitis is an infectious disease caused by periodontopathic bacteria, a complication of DM, and sometimes associated with severe inflammation and alveolar bone resorption. Advanced glycation end-products (AGEs) are a major pathogen in DM complications and adversely influence periodontitis in DM patients. In the present study, the effects of AGE2 and Porphyromonas gingivalis lipopolysaccharide (P-LPS) on the expression of sclerostin in mouse osteocyte-like cells (MLO-Y4-A2 cells) and its function in osteoblast differentiation were investigated. AGE2 and P-LPS up-regulated the expressions of receptor of AGE (RAGE) and Toll-like receptor 2 (TLR2), respectively, and significantly up-regulated that of sclerostin and interleukin 6 (IL-6) in osteocytes. Sclerostin, RAGE and TLR2 levels were synergistically increased by AGE2 and P-LPS. The siRNAs of RAGE and TLR2 significantly inhibited AGE2- and P-LPS-induced sclerostin expression. AGE2 up-regulated sclerostin expression in osteocyte-like cells via the RAGE, ERK and JNK, and NF-κB signal pathways. On the other hand, P-LPS elevated sclerostin levels via the TLR2, JNK and p38, and NF-κB signal pathways. When osteocytes pre-treated with AGE2 and P-LPS and osteoblastic cells (MC3T3-E1) were co-cultured in the medium with a sclerostin-neutralizing antibody, AGE2- and P-LPS-induced decreases in alkaline phosphatase activity and Runx2 expression in osteoblastic cells were significantly inhibited by the sclerostin-neutralizing antibody. These results suggest that AGE2 and P-LPS influence bone metabolism and inflammation through the regulation of sclerostin expression, and may aggravate periodontitis with DM.

Sclerostin-Neutralizing Antibody Enhances Bone Regeneration Around Oral Implants.

Dental implants are an important option for replacement of missing teeth. A major clinical challenge is how best to accelerate bone regeneration and reduce the healing time for functional restoration after implant placement. A sclerostin-neutralizing antibody (Scl-Ab) has been shown to enhance alveolar bone formation and fracture repair. The aim of this study was to investigate the effects of systemic administration of Scl-Ab on dental implant osseointegration and bone regeneration in an experimental alveolar ridge tooth extraction model. To investigate the effects of Scl-Ab on bone regeneration and dental implant osseointegration, an experimental alveolar bone osteotomy rat model was adopted. One month after extraction of maxillary right first molars, osteotomy defects were created at the coronal aspect of each of the extraction sites, and 1 × 2-mm custom titanium implants were installed into the osteotomies. Coincident with implant placement, Scl-Ab was administered subcutaneously at a dose of 25 mg/kg twice weekly for 10-28 days and compared with a vehicle control. Animals were sacrificed 10, 14, and 28 days after surgery, and maxillae were harvested and analyzed by microcomputed tomography (microCT), histology, and histomorphometry. microCT analysis demonstrated that the maxillary bone volume fraction was approximately 2- to 2.5-fold greater in Scl-Ab-treated animals compared with vehicle alone at days 14 and 28. Consistent with those findings, two-dimensional bone fill percentages within the coronal osteotomy sites were highest in Scl-Ab treatment groups at 28 days. In addition, bone-implant contact at 28 days was approximately twofold greater in the Scl-Ab group compared with the vehicle control. These results indicate that systemic Scl-Ab administration enhances osseointegration and bone regeneration around dental implants. This approach offers potential as a treatment modality for patients with low bone mass or bone defects to achieve more predictable bone regeneration at alveolar bone defects and to enhance dental implant osseointegration.

Diminished Canonical β-Catenin Signaling During Osteoblast Differentiation Contributes to Osteopenia in Progeria.

Patients with Hutchinson-Gilford progeria syndrome (HGPS) have low bone mass and an atypical skeletal geometry that manifests in a high risk of fractures. Using both in vitro and in vivo models of HGPS, we demonstrate that defects in the canonical WNT/β-catenin pathway, seemingly at the level of the efficiency of nuclear import of β-catenin, impair osteoblast differentiation and that restoring β-catenin activity rescues osteoblast differentiation and significantly improves bone mass. Specifically, we show that HGPS patient-derived iPSCs display defects in osteoblast differentiation, characterized by a decreased alkaline phosphatase activity and mineralizing capacity. We demonstrate that the canonical WNT/β-catenin pathway, a major signaling cascade involved in skeletal homeostasis, is impaired by progerin, causing a reduction in the active β-catenin in the nucleus and thus decreased transcriptional activity, and its reciprocal cytoplasmic accumulation. Blocking farnesylation of progerin restores active β-catenin accumulation in the nucleus, increasing signaling, and ameliorates the defective osteogenesis. Moreover, in vivo analysis of the Zmpste24-/- HGPS mouse model demonstrates that treatment with a sclerostin-neutralizing antibody (SclAb), which targets an antagonist of canonical WNT/β-catenin signaling pathway, fully rescues the low bone mass phenotype to wild-type levels. Together, this study reveals that the β-catenin signaling cascade is a therapeutic target for restoring defective skeletal microarchitecture in HGPS. © 2018 American Society for Bone and Mineral Research.

Sclerostin neutralization unleashes the osteoanabolic effects of Dkk1 inhibition.

The WNT pathway has become an attractive target for skeletal therapies. High-bone-mass phenotypes in patients with loss-of-function mutations in the LRP5/6 inhibitor Sost (sclerosteosis), or in its downstream enhancer region (van Buchem disease), highlight the utility of targeting Sost/sclerostin to improve bone properties. Sclerostin-neutralizing antibody is highly osteoanabolic in animal models and in human clinical trials, but antibody-based inhibition of another potent LRP5/6 antagonist, Dkk1, is largely inefficacious for building bone in the unperturbed adult skeleton. Here, we show that conditional deletion of Dkk1 from bone also has negligible effects on bone mass. Dkk1 inhibition increases Sost expression, suggesting a potential compensatory mechanism that might explain why Dkk1 suppression lacks anabolic action. To test this concept, we deleted Sost from osteocytes in, or administered sclerostin neutralizing antibody to, mice with a Dkk1-deficient skeleton. A robust anabolic response to Dkk1 deletion was manifest only when Sost/sclerostin was impaired. Whole-body DXA scans, μCT measurements of the femur and spine, histomorphometric measures of femoral bone formation rates, and biomechanical properties of whole bones confirmed the anabolic potential of Dkk1 inhibition in the absence of sclerostin. Further, combined administration of sclerostin and Dkk1 antibody in WT mice produced a synergistic effect on bone gain that greatly exceeded individual or additive effects of the therapies, confirming the therapeutic potential of inhibiting multiple WNT antagonists for skeletal health. In conclusion, the osteoanabolic effects of Dkk1 inhibition can be realized if sclerostin upregulation is prevented. Anabolic therapies for patients with low bone mass might benefit from a strategy that accounts for the compensatory milieu of WNT inhibitors in bone tissue.

Role of Osteocyte-PDL Crosstalk in Tooth Movement via SOST/Sclerostin

Sclerostin (Scl) negatively regulates bone formation and favors bone resorption. Osteocytes, the cells responsible for mechanosensing, are known as the primary source of Scl and are a key regulator of bone remodeling through the induction of receptor activator of NF-κB ligand (RANKL). However, the spatiotemporal patterns of Scl in response to mechanical stimuli and their regulatory mechanisms remain unknown. We investigated the regulatory dynamics of the SOST/Scl expression generated by orthodontic tooth movement (OTM) in vivo and in vitro. In 8-wk-old male mice, coil springs were used to move the first molar mesially for 0, 1, 5, or 10 d. A regional histogram and the distribution patterns of the Scl expression showed that the Scl expression in the alveolar bone was increased on the compression side and peaked on day 5, with a gradual increase in the degree of significance. On day 10, the expression around the periodontal ligament (PDL)–alveolar bone boundary returned to the control level. Conversely, the expression of Scl on the tension side was only significantly decreased on day 1. Compressive force biphasically modulated the SOST/Scl expression in the isolated human PDL and thereby upregulated osteocytic SOST via paracrine activation in an osteocyte-PDL co-culture system designed to mimic OTM. This system did not affect the RANKL or OPG expression in osteocytes, suggesting that the bone resorption pathways are acted upon in a PDL-dependent and osteocyte-independent manner through RANKL/OPG signaling. Moreover, sclerostin neutralizing antibody significantly attenuated the upregulation of SOST that was induced by compressive force. In conclusion, our results provide evidence to support that factors secreted by the PDL, including SOST/Scl, control alveolar bone remodeling through osteocytic SOST/Scl in OTM.