ABSTRACT
Unlike treatments for most rickets, the treatment using 1,25-(OH)2 vitamin D3 has little efficacy on patients with hypophosphatemic rickets, a set of rare genetic diseases. Thus, understanding the local cause for osteomalacia in hypophosphatemic rickets and developing an effective treatment to restore mineralization in this rare disease has been a longstanding goal in medicine. Here, we used Dmp1 knockout (KO) mice (whose mutations led to the same type of autosomal recessive hypophosphatemic rickets in humans) as the model in which the monoclonal antibody of sclerostin (Scl-Ab) was tested in two age groups for 8weeks: the prevention group (starting at age 4weeks) and the treatment group (starting at age 12weeks). Applications of Scl-Ab greatly improved the osteomalacia phenotype (>15%) and the biomechanical properties (3-point bending, ~60%) in the treated long-bone group. Our studies not only showed improvement of the osteomalacia in the alveolar bone, which has the highest bone metabolism rate, as well as the long bone phenotypes in treated mice. All these improvements attributed to the use of Scl-Ab are independent of the change in serum levels of phosphorus and FGF23, since Scl-Ab had little efficacy on those parameters. Finally, we propose a model to explain how Scl-Ab can improve the Dmp1 KO osteomalacia phenotype, in which the sclerostin level is already low.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Extracellular Matrix Proteins/genetics , Gene Knockout Techniques , Glycoproteins/immunology , Osteomalacia/drug therapy , Adaptor Proteins, Signal Transducing , Animals , Antibodies, Monoclonal/pharmacology , Biomechanical Phenomena/drug effects , Disease Models, Animal , Fibroblast Growth Factor-23 , Fibroblast Growth Factors/blood , Humans , Intercellular Signaling Peptides and Proteins , Mice , Mice, Knockout , Osteomalacia/genetics , Phosphorus/blood , Treatment OutcomeABSTRACT
The inhibition of sclerostin by the systemic administration of a monoclonal antibody (Scl-Ab) significantly increased bone mass and strength in fractured bones in animal models and non-fractured bones in ovariectomised (OVX) rats. In this study, the effects of Scl-Ab on healing were examined in a closed fracture model in OVX rats. Sixty Sprague-Dawley rats underwent an ovariectomy or a sham operation at 4 months of age, and a closed fracture of the right femur was performed 3 months later. Subcutaneous injections with Scl-Ab (25 mg/kg) or saline were then administered on day 1 after the fracture and twice a week for 8 weeks (n = 20 per group), at which time the fractured femurs were harvested for micro-computed tomography analysis, four-point bending mechanical testing and histomorphometric analysis to examine bone mass, bone strength and dynamic bone formation at the fracture site. The angiogenesis at the fracture site was also examined. Bone marrow stem cells were also isolated from the fractured bone to perform a colony-forming unit (CFU) assay and an alkaline phosphatase-positive (ALP(+)) CFU assay. OVX rats treated with Scl-Ab for 8 weeks had significantly increased bone mineral density and relative bone volume compared with OVX rats treated with saline. Similarly, maximum loading, energy to maximum load and stiffness in Scl-Ab-treated OVX rats were significantly higher than those in saline controls. The mineral apposition rate (MAR), mineralising surface (MS/BS) and bone formation rate (BFR/BS) were also significantly increased in Scl-Ab-treated group compared with the saline-treated group in OVX rats. Furthermore, the Scl-Ab-treated group had more CFUs and ALP(+) CFUs than the saline-treated group in OVX rats. No significant difference in angiogenesis at the fracture site was found between the groups. Our study demonstrated that Scl-Ab helped to increase bone mass, bone strength and bone formation at the fracture site in a closed femoral fracture model in OVX rats. Bone marrow stem cells in OVX rats injected with Scl-Ab also had increased CFUs and ALP(+) CFUs.
Subject(s)
Antibodies/chemistry , Bone Morphogenetic Proteins/chemistry , Femoral Fractures/immunology , Alkaline Phosphatase/metabolism , Animals , Body Weight , Bone Marrow Cells/cytology , Female , Femoral Fractures/diagnostic imaging , Fracture Healing , Genetic Markers , Injections, Subcutaneous , Neovascularization, Pathologic , Osteogenesis , Ovariectomy , Rats , Rats, Sprague-Dawley , Stem Cells/cytology , Stress, Mechanical , X-Ray MicrotomographyABSTRACT
INTRODUCTION: Periodontitis and osteoporosis are bone destructive diseases with a high prevalence in the adult population. The concomitant presence of osteoporosis may be a risk factor of progression of periodontal destruction. We studied the effect of sclerostin-neutralizing monoclonal antibody (Scl-Ab) on alveolar bone endpoints in an ovariectomized (OVX) rat model of induced experimental periodontitis. METHODS: Sixty female, 4-month-old Sprague-Dawley rats underwent sham operation or bilateral OVX and were left untreated for 2 months. Experimental periodontitis (ligature) was established by placing silk sutures subgingival to the right maxillary first and second molar teeth for 4 weeks, and feeding the rats food and high-sugar drinking water during this period. Thereafter, ligatures were removed and 25mg/kg vehicle or Scl-Ab was administered subcutaneously twice weekly for 6 weeks. Rats were randomized into four groups: (1) Control (Sham+Vehicle), (2) Sham+Ligature+Vehicle, (3) OVX+Ligature+Vehicle, and (4) OVX + Ligature + Scl-Ab. Terminal blood and right maxilla specimens were collected for analyses. RESULTS: Group 3 rats showed lower bone volume fraction (BVF) of alveolar bone with higher bone resorption and lower bone formation than Group 2 rats. Group 4 rats had higher alveolar crest height, as assessed by linear distance of cementoenamel junction to the alveolar bone crest and greater alveolar bone mass using Micro CT, than Group 3 rats. Significantly higher values of mineral apposition rate (MAR) and mineralizing surface/bone surface (MS/BS) were also observed in Group 4 rats by analyzing polychrome sequential labeling data. Increased serum osteocalcin and osteoprotegerin, and deceased serum tartrate-resistant acid phosphatase and CTx-1 illustrate the ability of Scl-Ab to increase alveolar bone mass by enhancing bone formation and decreasing bone resorption in an animal model of estrogen deficiency osteopenia plus periodontitis. CONCLUSION: Scl-Ab could be a potential bone anabolic agent for improving alveolar crest height and higher alveolar bone mass in conditions where alveolar bone loss in periodontitis is compounded by estrogen deficiency osteopenia.
Subject(s)
Alveolar Process/pathology , Antibodies, Neutralizing/therapeutic use , Bone Density , Bone Morphogenetic Proteins/immunology , Disease Models, Animal , Genetic Markers/immunology , Ovariectomy , Periodontitis/therapy , Alveolar Process/diagnostic imaging , Animals , Female , Osteocalcin/blood , Osteoprotegerin/blood , Periodontitis/diagnostic imaging , Rats , Rats, Sprague-Dawley , X-Ray MicrotomographyABSTRACT
Understanding periodontal ligament (PDL) biology and developing an effective treatment for bone and PDL damage due to periodontitis have been long-standing aims in dental medicine. Here, we first demonstrated by cell lineage tracing and mineral double-labeling approaches that murine PDL progenitor cells display a 2- and 3-fold higher mineral deposition rate than the periosteum and endosteum at the age of 4 weeks, respectively. We next proved that the pathologic changes in osteocytes (Ocys; changes from a spindle shape to round shape with a >50% reduction in the dendrite number/length, and an increase in SOST) are the key pathologic factors responsible for bone and PDL damage in periostin-null mice (a periodontitis animal model) using a newly developed 3-dimensional FITC-Imaris technique. Importantly, we proved that deleting the Sost gene (a potent inhibitor of WNT signaling) or blocking sclerostin function by using the mAb in this periodontitis model significantly restores bone and PDL defects (n = 4-5; P < 0.05). Together, identification of the key contribution of the PDL in normal alveolar bone formation, the pathologic changes of the Ocys in periodontitis bone loss, and the novel link between sclerostin and Wnt signaling in the PDL will aid future drug development in the treatment of patients with periodontitis.
Subject(s)
Cell Adhesion Molecules/deficiency , Glycoproteins/deficiency , Periodontitis/therapy , Adaptor Proteins, Signal Transducing , Alveolar Bone Loss/pathology , Alveolar Bone Loss/physiopathology , Alveolar Bone Loss/therapy , Animals , Antibodies, Monoclonal , Cell Adhesion Molecules/genetics , Cell Lineage , Collagen/metabolism , Disease Models, Animal , Glycoproteins/antagonists & inhibitors , Glycoproteins/genetics , Intercellular Signaling Peptides and Proteins , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteocytes/pathology , Periodontal Ligament/pathology , Periodontitis/pathology , Periodontitis/physiopathology , Phenotype , Wnt Signaling PathwayABSTRACT
In this study we investigated if Wnt/ß-catenin signaling in mesenchymal progenitor cells plays a role in bone fracture repair and if DKK1-Ab promotes fracture healing through activation of ß-catenin signaling. Unilateral open transverse tibial fractures were created in CD1 mice and in ß-catenin(Prx1ER) conditional knockout (KO) and Cre-negative control mice (C57BL/6 background). Bone fracture callus tissues were collected and analyzed by radiography, micro-CT (µCT), histology, biomechanical testing and gene expression analysis. The results demonstrated that treatment with DKK1-Ab promoted bone callus formation and increased mechanical strength during the fracture healing process in CD1 mice. DKK1-Ab enhanced fracture repair by activation of endochondral ossification. The normal rate of bone repair was delayed when the ß-catenin gene was conditionally deleted in mesenchymal progenitor cells during the early stages of fracture healing. DKK1-Ab appeared to act through ß-catenin signaling to enhance bone repair since the beneficial effect of DKK1-Ab was abrogated in ß-catenin(Prx1ER) conditional KO mice. Further understanding of the signaling mechanism of DKK1-Ab in bone formation and bone regeneration may facilitate the clinical translation of this anabolic agent into therapeutic intervention.
Subject(s)
Antibodies/pharmacology , Fracture Healing/drug effects , Fractures, Bone/pathology , Intercellular Signaling Peptides and Proteins/immunology , Signal Transduction/drug effects , beta Catenin/metabolism , Animals , Biomarkers/metabolism , Biomechanical Phenomena/drug effects , Bony Callus/drug effects , Bony Callus/pathology , Cartilage/drug effects , Cartilage/pathology , Fractures, Bone/diagnostic imaging , Gene Expression Regulation/drug effects , Integrases/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Organ Size/drug effects , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteoclasts/pathology , Radiography , Recombination, Genetic/geneticsABSTRACT
OBJECTIVE: To test whether inhibition of sclerostin by a targeted monoclonal antibody (Scl-Ab) protects from bone and cartilage damage in inflammatory arthritis. Sclerostin is a potent inhibitor of bone formation and may be responsible for the low level of bone repair in patients with rheumatoid arthritis. METHODS: Human tumour necrosis factor transgenic mice (hTNFtg mice) developing inflammatory arthritis and local and bone loss were administered either vehicle, anti-TNF antibody, Scl-Ab, or a combination of both agents. Inflammation, systemic and periarticular bone loss, bone erosion and cartilage damage were evaluated at baseline (week 8) and after 3 weeks of treatment by clinical assessment, micro-CT and histology. RESULTS: Scl-Ab did not affect joint swelling or synovitis. Systemic bone loss in the spine and periarticular bone loss in the proximal tibia were completely blocked and partially reversed by inhibition of sclerostin but not by inhibition of TNF. Moreover, Scl-Ab completely arrested the progression of bone erosion in hTNFtg mice and in combination with TNF inhibition even led to significant regression of cortical bone erosions. Protective effects of Scl-Ab were also observed for the articular cartilage. CONCLUSIONS: These data suggest that sclerostin inhibition is a powerful tool to enhance bone repair in inflammatory arthritis.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Arthritis, Experimental/complications , Bone Diseases, Metabolic/drug therapy , Glycoproteins/antagonists & inhibitors , Adaptor Proteins, Signal Transducing , Animals , Bone Diseases, Metabolic/etiology , Bone Diseases, Metabolic/pathology , Bone Regeneration/drug effects , Cartilage Diseases/pathology , Cartilage Diseases/prevention & control , Cartilage, Articular/pathology , Disease Progression , Drug Evaluation, Preclinical/methods , Female , Intercellular Signaling Peptides and Proteins , Mice , Mice, Transgenic , Tumor Necrosis Factor-alpha/antagonists & inhibitorsABSTRACT
During development and repair of bone, two distinct yet complementary mechanisms, intramembranous and endochondral, mediate new bone formation via osteoblasts. Because mechanical bone marrow ablation leads to the rapid and transient formation of new bone in the marrow cavity, we postulated that parathyroid hormone (PTH), which is a bone anabolic hormone, enhances the formation of new bone that forms after marrow ablation. We subjected the left femur of rats to mechanical marrow ablation, or sham operation, and injected the animals daily with PTH or vehicle for 1, 2, or 3 weeks in a first experiment, then with PTH, parathyroid hormone-related peptide (PTHrP), or vehicle for 3 weeks in a second experiment. We subjected both femurs from each rat to soft X-ray, peripheral quantitative computed tomography, computed tomography on a microscale, and histological analysis, and determined the concentration of serum osteocalcin. In addition, in the second experiment, we determined the serum concentration of calcium, tartrate-resistant acid phosphatase (TRAP), and receptor activator of NF-kappaB ligand (RANKL) at 3 weeks, and subjected femurs to biomechanical testing. Following treatment with PTH or PTHrP for 3 weeks, bone filled the marrow cavity of the shafts whose marrow had been ablated. PTH increased trabecular density in the right femur, but failed to induce bone formation in the medullary region of the right unoperated femoral shafts. The newly formed bone endowed left femoral shafts with improved biomechanical properties when compared to those of right femurs and left femurs from control, sham-operated, and vehicle-treated rats. PTHrP, like PTH, increased serum osteocalcin, but neither increased serum calcium, TRAP, or RANKL at 3 weeks. Our results reveal that the newly formed bone that follows marrow ablation is responsive to PTH, expand the role of PTH in bone, and might open new avenues of investigations to the field of regenerative medicine and tissue engineering. Local bone marrow removal in conjunction with pharmacologic intervention with an anabolic agent might provide a technique for rapid preferential site-directed bone growth in areas of high bone loss.
Subject(s)
Bone Marrow/surgery , Femur/surgery , Tissue Engineering/methods , Acid Phosphatase/blood , Animals , Bioreactors , Bone Development/drug effects , Bone Marrow/drug effects , Bone and Bones , Calcium/blood , Femur/growth & development , Humans , Isoenzymes/blood , Male , Parathyroid Hormone/pharmacology , Parathyroid Hormone-Related Protein/pharmacology , RANK Ligand/blood , Rats , Tartrate-Resistant Acid PhosphataseABSTRACT
Heptanoic acid lactams, exemplified by 2, were identified as highly selective EP4 agonists via high throughput screening. Lead optimization led to the identification of lactams with a 30-fold increase in EP4 potency in vitro. Compounds demonstrated robust bone anabolic effects when administered in vivo in rat models of osteoporosis.
Subject(s)
Bone Density/drug effects , Bone Development , Bone and Bones/drug effects , Organ Size/drug effects , Ovariectomy , Receptors, Prostaglandin E/agonists , Animals , Female , Osteoporosis , Rats , Rats, Sprague-Dawley , Receptors, Prostaglandin E, EP4 SubtypeABSTRACT
Macrophages seed all tissues in which they have the ability, in specific and rare instances, to fuse with themselves and to differentiate into osteoclasts in bone or into giant cells in chronic inflammatory reactions. Although these cells play a central role in osteoporosis and in foreign body rejection, respectively, the molecular mechanism used by macrophages to fuse remains poorly understood. Macrophages might also fuse with somatic and tumor cells to promote tissue repair and metastasis, respectively. We reported that CD44 expression is highly induced in macrophages at the onset of fusion in which it plays a role. We report now that the intracellular domain of CD44 (CD44ICD) is cleaved in macrophages undergoing fusion and that presenilin inhibitors prevent the release of CD44ICD and fusion. We also show that CD44ICD promotes the fusion of tissue macrophages and bone marrow-derived macrophages. Finally, we report that CD44ICD is localized in the nucleus of macrophages in which it promotes the activation of NF-kappaB. These observations open avenues to study the role of CD44ICD in blood cells and tumors.
Subject(s)
Hyaluronan Receptors/metabolism , Macrophages, Alveolar/metabolism , Macrophages, Peritoneal/metabolism , Membrane Proteins/antagonists & inhibitors , Animals , Bone Marrow/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Fusion , Cell Nucleus/metabolism , Electrophoretic Mobility Shift Assay , Hyaluronan Receptors/genetics , Luciferases/metabolism , Macrophages, Alveolar/cytology , Macrophages, Peritoneal/cytology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Mice , NF-kappa B/genetics , NF-kappa B/metabolism , Presenilin-1 , Protein Structure, Tertiary , RANK Ligand , Rats , Rats, Inbred F344 , Receptor Activator of Nuclear Factor-kappa B , Retroviridae/genetics , Subcellular FractionsABSTRACT
A series of ligands with varying heterocyclic cores and substituents that display a range of selectivity's (up to >100x) for ER-beta over ER-alpha are reported.