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1.
Methods Mol Biol ; 1914: 507-531, 2019.
Article in English | MEDLINE | ID: mdl-30729484

ABSTRACT

This chapter describes the use of micro-computed tomography scanning for analyzing bone structure, focussing on rodent bone. It discusses sample preparation, the correct setup of the scanner, the impact of some of the important scanner settings and new applications.


Subject(s)
Bone and Bones/diagnostic imaging , Imaging, Three-Dimensional/methods , X-Ray Microtomography/methods , Animals , Biopsy , Bone Density , Bone and Bones/anatomy & histology , Bone and Bones/pathology , Female , Histocytological Preparation Techniques/instrumentation , Histocytological Preparation Techniques/methods , Humans , Imaging, Three-Dimensional/instrumentation , Mice , Models, Animal , Rats , Software , X-Ray Microtomography/instrumentation
2.
Dis Model Mech ; 11(9)2018 08 23.
Article in English | MEDLINE | ID: mdl-30154079

ABSTRACT

Paget's disease of bone (PDB) is an age-related metabolic bone disorder, characterised by focally increased and disorganised bone remodelling initiated by abnormal and hyperactive osteoclasts. The germline P392L mutation of SQSTM1 (encoding p62) is a strong genetic risk factor for PDB in humans, and the equivalent mutation in mice (P394L) causes a PDB-like disorder. However, it is unclear why pagetic lesions become more common with age. Here, we assessed the effect of the p62 P394L mutation on osteoclastogenesis and bone morphometry in relation to ageing, the natural history of lesion progression in p62P394L mice and the effect of zoledronic acid (ZA) on lesion development. p62P394L+/+ osteoclast precursors had increased sensitivity to RANKL (also known as TNFSF11) compared with wild-type (WT) cells, and the sensitivity further increased in both genotypes with ageing. Osteoclastogenesis from 12-month-old p62P394L+/+ mice was twofold greater than that from 3-month-old p62P394L+/+ mice (P<0.001) and three-fold greater than that from age-matched WT littermates. The p62P394L+/+ mice lost 33% more trabecular bone volume in the long bones by 12 months compared with WT mice (P<0.01), and developed pagetic-like lesions in the long bones which progressed with ageing. ZA prevented the development of pagetic-like lesions, and increased trabecular bone volume tenfold compared with vehicle by 12 months of age (P<0.01). This demonstrates that ageing has a pro-osteoclastogenic effect, which is further enhanced by the p62 P394L mutation, providing an explanation for the increased penetrance of bone lesions with age in this model. Lesions are prevented by ZA, providing a rationale for early intervention in humans.


Subject(s)
Bone Resorption/pathology , Osteitis Deformans/drug therapy , Osteitis Deformans/prevention & control , Sequestosome-1 Protein/genetics , Zoledronic Acid/therapeutic use , Aging/pathology , Animals , Bone Resorption/complications , Bone and Bones/diagnostic imaging , Bone and Bones/drug effects , Bone and Bones/pathology , Calcification, Physiologic/drug effects , Disease Models, Animal , Mice, Inbred C57BL , Mice, Mutant Strains , Mutation/genetics , Organ Size , Osteitis Deformans/complications , Osteitis Deformans/pathology , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteoclasts/pathology , Phenotype , Zoledronic Acid/pharmacology
3.
Bioorg Med Chem Lett ; 23(3): 816-20, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23265887

ABSTRACT

Triaylsulfonamides were identified as novel anti-inflammatory agents, acting by inhibition of RANKL and TNFα signaling. Structure-activity studies led to the identification of compounds with in vitro potencies of <100 nM against J774 macrophages and osteoclasts, but with little activity against osteoblasts or hepatocytes (IC(50) >50 µM). A representative compound (4k, ABD455) was able to completely prevent inflammation in vivo in a prevention model and was highly effective at controlling inflammation in a treatment model.


Subject(s)
Anti-Inflammatory Agents/chemical synthesis , Biphenyl Compounds/chemical synthesis , Sulfonamides/chemical synthesis , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , Caco-2 Cells , Disease Models, Animal , Humans , Molecular Structure , RANK Ligand/metabolism , Signal Transduction/drug effects , Sulfonamides/chemistry , Sulfonamides/pharmacology , Tumor Necrosis Factor-alpha/metabolism
4.
Methods Mol Biol ; 816: 177-86, 2012.
Article in English | MEDLINE | ID: mdl-22130929

ABSTRACT

The murine co-culture assay is used to generate mature osteoclasts from bone marrow precursors by culturing them with osteoblasts that are stimulated with 1,25-dihydroxy vitamin D(3) and prostaglandin E(2). This assay is used particularly to analyse osteoblast-osteoclast interactions and to determine the cell type affected in knock-out or transgenic mice. This chapter describes also the isolation of bone marrow cells from mice and the methods to purify and replate mature osteoclasts.


Subject(s)
Bone Marrow Cells/cytology , Coculture Techniques/methods , Osteoblasts/cytology , Osteoclasts/cytology , Acid Phosphatase/analysis , Animals , Bone Marrow Cells/metabolism , Calcitriol/metabolism , Cell Separation/methods , Cells, Cultured , Dinoprostone/metabolism , Isoenzymes/analysis , Mice , Osteoblasts/metabolism , Osteoclasts/metabolism , Staining and Labeling/methods , Tartrate-Resistant Acid Phosphatase
5.
Methods Mol Biol ; 816: 461-76, 2012.
Article in English | MEDLINE | ID: mdl-22130944

ABSTRACT

This chapter describes the use of microcomputed tomography scanning for analysing bone structure, focussing on rodent bone. It also discusses sample preparation, the correct set-up of the scanner, and the impact of some of the important scanner settings.


Subject(s)
Bone and Bones/ultrastructure , X-Ray Microtomography/methods , Animals , Bone and Bones/anatomy & histology , Image Processing, Computer-Assisted , Mice , Specimen Handling/instrumentation , Specimen Handling/methods , X-Ray Microtomography/instrumentation
6.
Methods Mol Biol ; 816: 537-44, 2012.
Article in English | MEDLINE | ID: mdl-22130950

ABSTRACT

This chapter describes the calvarial injection method, whereby the effect of a substance on bone is tested by subcutaneous injection over the calvarium of a mouse. This assay allows testing of the effect of substances on both bone resorption and bone formation in a relatively simple in vivo model. The analysis is carried out by histological means, usually in glycolmethacrylate-embedded tissue, allowing for histochemical analysis and for a variety of different histological staining methods which are also described in detail.


Subject(s)
Bone Morphogenetic Protein 2/administration & dosage , Bone Resorption/drug therapy , Drug Evaluation, Preclinical/methods , Interleukin-1alpha/administration & dosage , Osteogenesis/drug effects , Skull/drug effects , Animals , Bone Morphogenetic Protein 2/therapeutic use , Bone Resorption/pathology , Injections , Interleukin-1alpha/therapeutic use , Mice , Recombinant Proteins/administration & dosage , Recombinant Proteins/therapeutic use , Skull/pathology , Staining and Labeling/methods
7.
Hum Mol Genet ; 20(14): 2734-44, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21515589

ABSTRACT

Mutations of SQSTM1 occur in about10% of patients with Paget's disease of bone (PDB), but it is unclear whether they play a causal role or regulate susceptibility to an environmental trigger. Here we show that mice with a proline to leucine mutation at codon 394 of mouse sqstm1 (P394L), equivalent to the P392L SQSTM1 mutation in humans, develop a bone disorder with remarkable similarity to PDB. The P394L mutant mice developed focal bone lesions with increasing age and by 12 months, 14/18 (77%) heterozygotes and 20/21 (95%) homozygotes had lesions, compared with 0/18 (0%) wild-type littermates (P< 0.001). Lesions predominantly affected the lower limbs in an asymmetric manner and were characterized by focal increases in bone turnover, with increased bone resorption and formation, disruption of the normal bone architecture and accumulation of woven bone. Osteoclasts within lesions were larger and more nucleated than normal and some contained nuclear inclusions similar to those observed in human PDB. Osteoclast precursors from P394L mutant mice had increased sensitivity to RANKL in vitro resulting in the generation of osteoclasts that were larger and more nucleated than those generated from wild-type littermates. There was increased expression of sqstm1, autophagy-related gene 5 (atg5) and light chain 3 gene (lc3) in osteoclast precursors and increased LC3-II protein levels in Bafilomycin-treated osteoclasts from P394L mutant mice compared with wild-type suggesting dysregulation of autophagy and enhanced autophagosome formation. These studies demonstrate that SQSTM1 mutations can cause a PDB-like skeletal disorder in the absence of an additional trigger and provide a new disease model for PDB.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Bone Resorption/metabolism , Heat-Shock Proteins/metabolism , Osteitis Deformans/metabolism , Osteogenesis , Point Mutation , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Substitution , Animals , Autophagy/genetics , Autophagy-Related Protein 5 , Bone Resorption/genetics , Bone Resorption/pathology , Disease Models, Animal , Heat-Shock Proteins/genetics , Humans , Mice , Mice, Mutant Strains , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Osteitis Deformans/genetics , Osteitis Deformans/pathology , Osteoclasts/metabolism , Osteoclasts/pathology , RANK Ligand/genetics , RANK Ligand/metabolism , Sequestosome-1 Protein
8.
Endocrinology ; 152(6): 2141-9, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21447627

ABSTRACT

The type 2 cannabinoid receptor (CB2) has been reported to regulate bone mass and bone turnover but the mechanisms responsible are incompletely understood. In this study we investigated the role that the CB2 pathway plays in bone metabolism using a combination of genetic and pharmacological approaches. Bone mass and turnover were normal in young mice with targeted inactivation of CB2 receptor (CB2(-/-)), but by 12 months of age, they had developed high-turnover osteoporosis with relative uncoupling of bone resorption from bone formation. Primary osteoblasts from CB2(-/-) mice had a reduced capacity to form bone nodules in vitro when compared with cells from wild-type littermates and also had impaired PTH-induced alkaline phosphatase (ALP) activity. The CB2-selective agonist HU308 stimulated bone nodule formation in wild-type osteoblasts but had no effect in CB2(-/-) osteoblasts. Further studies in MC3T3-E1 osteoblast like cells showed that HU308 promoted cell migration and activated ERK phosphorylation, and these effects were blocked by the CB2 selective inverse agonist AM630. Finally, HU308 partially protected against ovariectomy induced bone loss in wild-type mice in vivo, primarily by stimulating bone formation, whereas no protective effects were observed in ovariectomized CB2(-/-) mice. These studies indicate that the CB2 regulates osteoblast differentiation in vitro and bone formation in vivo.


Subject(s)
Cell Differentiation , Osteoblasts/cytology , Osteogenesis , Osteoporosis/metabolism , Receptor, Cannabinoid, CB2/metabolism , Animals , Cells, Cultured , Disease Models, Animal , Gene Expression Regulation , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteoblasts/metabolism , Osteoporosis/etiology , Osteoporosis/genetics , Osteoporosis/physiopathology , Ovariectomy/adverse effects , Receptor, Cannabinoid, CB2/genetics
9.
Cell Metab ; 10(2): 139-47, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19656492

ABSTRACT

Age-related osteoporosis is characterized by reduced bone formation and accumulation of fat in the bone marrow compartment. Here, we report that the type 1 cannabinoid receptor (CB1) regulates this process. Mice with CB1 deficiency (CB1(-/-)) had increased peak bone mass due to reduced bone resorption, but developed age-related osteoporosis with reduced bone formation and accumulation of adipocytes in the bone marrow space. Marrow stromal cells from CB1(-/-) mice had an enhanced capacity for adipocyte differentiation, a reduced capacity for osteoblast differentiation, and increased expression of phosphorylated CREB (pCREB) and PPARgamma. Pharmacological blockade of CB1 receptors stimulated adipocyte differentiation, inhibited osteoblast differentiation, and increased cAMP and pCREB in osteoblast and adipocyte precursors. The CB1 receptor is therefore unique in that it regulates peak bone mass through an effect on osteoclast activity, but protects against age-related bone loss by regulating adipocyte and osteoblast differentiation of bone marrow stromal cells.


Subject(s)
Adipocytes/cytology , Cell Differentiation , Osteoblasts/cytology , Osteoporosis/metabolism , Receptor, Cannabinoid, CB1/metabolism , Animals , Bone Density , Cells, Cultured , Cyclic AMP Response Element-Binding Protein/metabolism , Female , Male , Mice , Mice, Knockout , PPAR gamma/metabolism , Receptor, Cannabinoid, CB1/deficiency , Receptor, Cannabinoid, CB1/genetics , Stromal Cells/cytology
10.
Endocrinology ; 149(11): 5619-26, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18635663

ABSTRACT

The endocannabinoid system has recently been shown to play a role in the regulation of bone metabolism. The type 2 cannabinoid receptor (CB2) has been reported to regulate bone mass, but conflicting results have been reported with regard to its effects on bone resorption and osteoclast function. Here we investigated the role that CB2 plays in regulating bone mass and osteoclast function using a combination of pharmacological and genetic approaches. The CB2-selective antagonist/inverse agonist AM630 inhibited osteoclast formation and activity in vitro, whereas the CB2-selective agonists JWH133 and HU308 stimulated osteoclast formation. Osteoclasts generated from CB2 knockout mice (CB2-/-) were resistant to the inhibitory effects of AM630 in vitro, consistent with a CB2-mediated effect. There was no significant difference in peak bone mass between CB2-/- mice and wild-type littermates, but after ovariectomy, bone was lost to a greater extent in wild-type compared with CB2-/- mice. Furthermore, AM630 protected against bone loss in wild-type mice, but the effect was blunted in CB2-/- mice. We conclude that CB2 regulates osteoclast formation and bone resorption in vitro and that under conditions of increased bone turnover, such as after ovariectomy, CB2 regulates bone loss. These observations indicate that CB2 regulates osteoclast formation and contributes to ovariectomy-induced bone loss and demonstrate that cannabinoid receptor antagonists/inverse agonists may be of value in the treatment of bone diseases characterized by increased osteoclast activity.


Subject(s)
Bone Resorption/genetics , Bone and Bones/anatomy & histology , Osteoclasts/physiology , Receptor, Cannabinoid, CB2/physiology , Animals , Bone Density/genetics , Bone Marrow Cells/drug effects , Bone Resorption/etiology , Cannabinoids/pharmacology , Cells, Cultured , Coculture Techniques , Drug Evaluation, Preclinical , Drug Inverse Agonism , Female , Indoles/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Organ Size/genetics , Osteoclasts/drug effects , Osteoclasts/metabolism , Ovariectomy/adverse effects , Piperidines/pharmacology , Pyrazoles/pharmacology , Receptor, Cannabinoid, CB2/agonists , Receptor, Cannabinoid, CB2/antagonists & inhibitors , Receptor, Cannabinoid, CB2/genetics
11.
Nat Med ; 11(7): 774-9, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15908955

ABSTRACT

Accelerated osteoclastic bone resorption has a central role in the pathogenesis of osteoporosis and other bone diseases. Identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and to the development of new treatments. Here we show that mice with inactivation of cannabinoid type 1 (CB1) receptors have increased bone mass and are protected from ovariectomy-induced bone loss. Pharmacological antagonists of CB1 and CB2 receptors prevented ovariectomy-induced bone loss in vivo and caused osteoclast inhibition in vitro by promoting osteoclast apoptosis and inhibiting production of several osteoclast survival factors. These studies show that the CB1 receptor has a role in the regulation of bone mass and ovariectomy-induced bone loss and that CB1- and CB2-selective cannabinoid receptor antagonists are a new class of osteoclast inhibitors that may be of value in the treatment of osteoporosis and other bone diseases.


Subject(s)
Bone Resorption , Osteoclasts/metabolism , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/metabolism , Animals , Apoptosis/drug effects , Bone Density/genetics , Bone and Bones/anatomy & histology , Bone and Bones/drug effects , Camphanes/pharmacology , Carrier Proteins/drug effects , Carrier Proteins/metabolism , Cells, Cultured , Female , Indoles/pharmacology , Ligands , Membrane Glycoproteins/drug effects , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteoclasts/drug effects , Ovariectomy , Piperidines/pharmacology , Pyrazoles/pharmacology , RANK Ligand , Rabbits , Receptor Activator of Nuclear Factor-kappa B , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB1/genetics , Receptor, Cannabinoid, CB2/antagonists & inhibitors
12.
Endocrinology ; 145(11): 5068-74, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15297441

ABSTRACT

Nitric oxide (NO) is produced by NO synthase (NOS) and plays an important role in the regulation of bone cell function. The endothelial NOS isoform is essential for normal osteoblast function, whereas the inducible NOS isoform acts as a mediator of cytokine effects in bone. The role of the neuronal isoform of NOS (nNOS) in bone has been studied little thus far. Therefore, we investigated the role of nNOS in bone metabolism by studying mice with targeted inactivation of the nNOS gene. Bone mineral density (BMD) was significantly higher in nNOS knockout (KO) mice compared with wild-type controls, particularly the trabecular BMD (P < 0.01). The difference in BMD between nNOS KO and control mice was confirmed by histomorphometric analysis, which showed a 67% increase in trabecular bone volume in nNOS KO mice when compared with controls (P < 0.001). This was accompanied by reduced bone remodeling, with a significant reduction in osteoblast numbers and bone formation surfaces and a reduction in osteoclast numbers and bone resorption surfaces. Osteoblasts from nNOS KO mice, however, showed increased levels of alkaline phosphatase and no defects in proliferation or bone nodule formation in vitro, whereas osteoclastogenesis was increased in nNOS KO bone marrow cultures. These studies indicate that nNOS plays a hitherto unrecognized but important physiological role as a stimulator of bone turnover. The low level of nNOS expression in bone and the in vitro behavior of nNOS KO bone cells indicate that these actions are indirect and possibly mediated by a neurogenic relay.


Subject(s)
Bone Remodeling/physiology , Bone and Bones/cytology , Nitric Oxide Synthase/metabolism , Osteoblasts/enzymology , Animals , Bone Density , Bone and Bones/physiology , Mice , Mice, Knockout , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type I
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