text:
Physiopathology of osteoclast in bone
Xiaomei Gu
A thesis submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Master of Science in the Curriculum
of Oral Biology.
Chapel Hill 2008
Dr. Eric T. Everett
Dr. Patrick Flood
XIAOMEI GU: Physiopathology of Osteoclast in Bone (Under the direction of Dr. Eric T. Everett)
This thesis would not have been possible without the support of so many people. Firs of all, I would like to convey my gratitude to my advisor, Dr. Eric T. Everett, for his invaluable support and suggestions. I am also deeply indebted to the members of my committee, Dr. Patrick Flood and Dr. Ching-Chang Ko for their insightful guidance throughout the writing process.
Besides academic support, I have benefited from the constant moral support and inspiration of a number of persons. Special thanks go to Oral Biology manager, Cindy Blake, for her precious life advice. I am very grateful to my family and friends. I would also like to acknowledge my fellow Oral Biology students.
LIST OF FIGURES ...IX LIST OF ABBREVIATIONS... X
CHAPTER I. BASIC BONE BIOLOGY ...1
1.1. Thesis Introduction ...1
1.2. Embryonic Development of Bone ...2
1.3. Bone Function...3
1.4. Components of Bone...3
1.4.1. Osteoblasts ...4
1.4.2. Osteocytes...5
1.4.3. Osteoclasts ...5
1.5. Bone Structure: Cortical Bone and Trabecular Bone ...6
1.6. Bone Histology: Lamellar Bone and Woven Bone...6
1.7. Bone Remodeling...7
1.7.1. Purposes of Bone Remodeling...7
1.7.3. Difference between Bone Remodeling and Bone Modeling ...8
1.7.4. Regulation of Bone Remodeling...8
1.8. Chapter Summary ...9
Chapter I. Reference List ...10
CHAPTER II. OSTEOCLAST BIOLOGY: ORIGIN AND DIFFERENTIATION ...12
2.1. Osteoclast Origin ...12
2.2. Osteoclast Differentiation ...13
2.2.1. Role of M-CSF in Osteoclast Differentiation ...14
2.2.2. Role of RANKL in Osteoclast Differentiation ...15
2.2.3. Costimulatory Signals for RANK...17
2.3. Regulation of Osteoclast Differentiation ...18
2.3.1. The OPG/RANKL/RANK Regulatory Axis...18
2.3.2. Other Regulatory Mechanisms ...19
2.4. Osteoclast Fusion...20
2.5. Osteoclasts in Periprosthetic Osteolysis and Peri-implantitis...20
2.6. Osteoclasts in Orthodontic Tooth Movement ...22
2.7. Chapter Summary ...24
Chapter II. Reference List...29
3.1. Mechanism of Bone Resorption...37
3.1.1. Dissolving the Mineral Phase ...38
3.1.2. Degrading the Organic Phase...38
3.1.3. Removing Resorption Products ...39
3.2. Role of Osteoclasts in Hematopoiesis...39
3.3. Beyond Bone Resorption: Stimulating Bone Formation ...40
3.3.1. Evidence from the Osteopetrosis Models ...41
3.3.2. Evidence from the Anabolic Action of PTH...42
3.3.3. Evidence from In Vitro Studies ...43
Chapter III. Reference List ...49
CHAPTER IV. OSTEOCLAST PATHOLOGY: BONE LOSS ...51
4.1. Postmenopausal Osteoporosis...51
4.1.1. Introduction...51
4.1.2. Cellular Pathogenesis...53
4.1.3. Indirect Effects of Estrogen Deficiency on Bone Resorption...53
4.1.4. Direct Effects of Estrogen Deficiency on Bone Resorption ...54
4.2. Paget's Disease of Bone...56
4.3. Hyperparathyroidism ...58
4.3.1. Cellular and Molecular Pathogenesis...59
4.3.2. Mechanism of PTH Catabolic Effect on Bone ...60
4.4. Cherubism ...61
4.4.1. Molecular Pathogenesis ...61
Chapter IV. Reference List ...68
CHAPTER V. BONE PHARMACOLOGY: TARGETING THE OSTEOCLAST ...72
5.1. Bisphosphonates ...72
5.1.1. Bone Uptake of Bisphosphonates ...73
5.1.2. Cellular Uptake of Bisphosphonates...73
5.1.3. Biochemical Effects of Bisphosphonates on Osteoclasts ...74
5.1.4. Biochemical Effects of Bisphosphonates on Osteoblasts and Osteocytes...76
5.2. Fluoride ...76
5.2.1. In Vivo Effects on Bone ...77
5.2.2. In Vitro Effects on Osteoblasts ...78
5.2.3. Effects on Osteoclasts and Bone Marrow Cells...79
Figure 2.1. Osteoclastogenesis...25
Figure 2.2. M-CSF/RANKL signaling pathways.. ...26
Figure 2.3. Cooperation of RANKL and ITAM induced signals in osteoclastogenesis...27
Figure 2.4. RANKL-dependent regulation of osteoclast formation. ...28
Figure 3.1. Plasma membrane domains present in a resorbing osteoclast...45
Figure 3.2. Mechanism of osteoclastic bone resorption.. ...46
Figure 4.1. Mutations in components of RANK–NFκB signaling pathway cause Paget's disease of bone and related disorders.. ...65
Figure 4.2. Reciprocal relationship between PTH and extracellular calcium level...66
Figure 5.1. The structure of bisphosphonate and pyrophosphate. ...83
Figure 5.2. Structures of bisphosphonates classified according to their biochemical modes of action...84
Figure 5.3. Formation of AppCp-type metabolites of simple bisphosphonates. ...85
AP activator protein
ALP alkaline phosphatase
ATP or Appp adenosine triphosphate
aPKC atypical protein kinase C
BMC bone mineral content
BMD bone mineral density
BMP bone morphogenetic protein
BMU basic multicellular unit
DXA or DEXA dual energy x-ray absorptiometry
CA II carbonic anhydrase II
CaR calcium receptor
CFU-GEMM colony forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte
CFU-GM colony forming unit-granulocyte,
monocyte/macrophage
CFU-G colony forming unit-granulocyte
CFU-M colony forming unit-monocyte/macrophage
CLC-7 chloride channel 7
CREB cAMP-response element-binding protein
CTK cathepsin K
CTR calcitonin receptor
DC-STAMP dendritic cell-specific transmembrane protein
DMAP dimethylallyl pyrophosphate
ERα estrogen receptor alpha
ERβ estrogen receptor beta
ERK extracellular-signal-regulated kinase
FasL Fas ligand
FPP farnesyl diphosphate
FcRγ Fc receptor common γ subunit
GDP guanosine diphosphate
GTP guanosine triphosphate
GGPP geranylgeranyl diphosphate
G protein guanine nucleotide binding protein
GPCR G protein-coupled protein
GRB2 growth-factor-receptor-bound protein 2
H+ATPase H+-adenosine triphosphatase
HSC hematopoietic stem cell
IκB inhibitor of NFκB
IKK IκB kinase
IL interleukin
IPP isopentenyl pyrophosphate
ITAM immunoreceptor tyrosine-based activation motif
JNK Jun N-terminal kinase
MCP-1 monocyte chemoattractant protein-1
M-CSF macrophage-colony stimulating factor
MGF macrophage growth factor
MGI-IM macrophage and granulocyte inducer IM
MMP 9 metalloproteinase 9
NFκB nuclear factor κ B
NFATc1 nuclear factor of activated T cells, cytoplasmic 1
OCIL osteoclast inhibitory lectin
OCN osteoclacin
OPG osteoprotegerin
OSCAR osteoclast-associated receptor
Osx osterix
Runx2 runt-related transcription factor-2
PDB Paget's disease of bone
PIR-A paired immunoglobulin-like receptor-A
PGE2 prostaglandin E2
PI3K phosphoinositide 3-kinase
PLCγ phospholipase Cγ
PKB protein kinase B
PPi inorganic pyrophosphate
ppm parts per million
PTH parathyroid hormone
RANK receptor activator of nuclear factor κ B RANKL receptor activator of nuclear factor κ B ligand
SCF stem cell factor
SD standard deviation
SDF-1 stromal cell-derived factor-1
sFRP1 secreted frizzled-related protein 1
SIRP β1 signal-regulatory protein β1
TACE metalloprotease disintegrin TNF α convertase
TNF tumor necrosis factor
TGFβ transforming growth factor beta
TRAF TNF receptor-associated factor
TRAP tartrate resistant alkaline phosphatase
TREM-2 triggering receptor expressed on myeloid cells-2
VCP valosin-containing protein
Vitamin D3 1, 25(OH)2D3 25-dihydroxy
CHAPTER I. BASIC BONE BIOLOGY
1.1. Thesis Introduction
Regardless of etiologies, bone loss represents an increase in bone resorption by osteoclasts relative to bone formation by osteoblasts. Understanding osteoclast biology and bone resorption are essential in understanding the pathogeneses and therapies of diseases involving excessive bone destruction. This thesis will begin with a very brief introduction of basic bone biology to orient the readers' understanding of osteoclasts in a context of normal bone remodeling. In the subsequent two chapters, origin, differentiation, and function of osteoclasts will be addressed in greater detail. To help understand osteoclast differentiation and its regulation, the role of osteoclast formation in periprosthetic osteolysis, peri-implantitis, and orthodontic tooth movement will also be discussed. In addition to the mechanism of bone resorption, this thesis will also explore two newly-identified functions of osteoclasts: mobilizing hematopoietic stem cells in bone marrow and stimulating osteoblastic bone formation.
such as bone cells [1, 2]. The anabolic action of PTH will be discussed in a context of osteoclasts being the anabolic signaling sources (Chapter III), whereas the catabolic action of PTH will be discussed in a context of hyperthyroidism pathogenesis. The recent breakthrough in understanding the pathogenesis of a genetic disorder, Cherubism, is a good example of how understanding osteoclast biology can advance our understanding of a disease process.
Osteoclasts are the primary therapeutic targets for conditions with excessive bone resorption. Bisphosphonate is a major class of anti-resorptive drug currently available. In the last chapter, this thesis will discuss the actions of bisphosphonates on bone with a focus on their induction of osteoclast apoptosis. Meanwhile, the actions of fluoride on bone cells will also be addressed. The anabolic action of fluoride on bone has been largely explained by its mitogenic effect on osteoblasts; however, this thesis will mainly focus on the effects of fluoride on osteoclasts. A complete picture of fluoride's effects on the two major cell types in bone—osteoblasts and osteoclasts—is required for the utilization and/or optimization of fluoride's potential in treating diseases with excessive bon resorption. By reviewing the pathophysiology of osteoclasts, this thesis will provide a powerful context to better understand fluoride's effect on osteoclasts.
1.2. Embryonic Development of Bone
Bone development occurs through two distinct developmental processes: intramembranous ossification and endochondral ossification. During intramembranous ossification, mesenchymal cells condense and differentiate directly into osteoblasts to lay down bone matrix [4]. This process occurs in several craniofacial bones and the lateral part of clavicles [5]. During endochondral ossification, mesenchymal cells condense and differentiate directly into chondroblasts to synthesize a cartilage model, which is then replaced by bone and bone marrow [4]. This process occurs in the long bones of the limbs, basal part of the skull, vertebrae, ribs, and medial part of the clavicles [5]. Therefore, the major difference between the two types of ossification is the presence of a cartilaginous precursor template during endochondral ossification.
1.3. Bone Function
Bone tissue has the following functions: 1) it provides structural support for the body; 2) it protects internal organs, such as brain, spinal cord, heart, and lungs; 3) it supports hematopoiesis in bone marrow; 4) it is the basis of posture and locomotion by allowing the attachments of muscles, ligaments, and tendons; 4) it serves as a mineral reservoir and in particular helps regulate calcium homeostasis [6, 7].
1.4. Components of Bone
tissue. The organic matrix is largely composed of type I collagen (95%), and the remaining 5% is composed of proteoglycans and numerous non-collagenous proteins [4, 8]. Three major cell types are present in bone tissue: osteoblasts, osteocytes, and osteoclasts [4].
1.4.1. Osteoblasts
Osteoblasts are the cells responsible for bone formation [7]. They are derived from mesenchymal stem cells in bone marrow [11]. Under appropriate stimuli, these multipotent cells can differentiate along three principal lineages: osteoblastic, adipocytic and chondrocytic lineages [6, 12]. Expression of the transcription factors, runt-related transcription factor-2 (Runx2), is required to shift the progenitors toward the osteoblast lineage and away from adipocytic and chondrocytic lineages. Further differentiation of the preosteoblast into a mature, functional osteoblast phenotype requires the expression of another transcription factor, osterix (Osx). The early stage of osteoblastic differentiation is characterized by markers such as type I collagen and alkaline phosphatase (ALP), while the late stage of osteoblastic differentiation is characterized by markers, such as osteoclacin and mineralization of extracellular matrix [5, 6].
Mature and active osteoblasts synthesize most of the proteins in bone extracellular matrix [6, 7]. Some osteoblasts are buried in bone matrix and become osteocytes, while some osteoblasts become flattened cell on bone surface—bone-lining cells (inactive osteoblasts) [7].
by providing many factors essential for the survival, renewal, maturation and lineage commitment of hematopoietic stem cells [14, 15].
1.4.2. Osteocytes
Osteocytes are the most abundant cells in bone (25,000/mm3 of bone [4]) and yet remain the least characterized [16]. They are terminally differentiated osteoblasts that have become entombed in bone matrix [7, 16]. Osteocytes are strain-sensitive and can transduce mechanical signals. Recent studies have shown that damage-induced osteocyte apoptosis may be involved in the recruitment of osteoclasts and initiation of new bone remodeling [16, 17].
1.4.3. Osteoclasts
1.5. Bone Structure: Cortical Bone and Trabecular Bone
Morphologically, there are two major forms of bone: cortical (compact) and trabecular (cancellous or spongy). In cortical bone, collagen fibrils are densely arranged to form concentric lamellae, with fibrils assembled in perpendicular planes in adjacent lamellae. Trabecular bone is composed of a porous network of thin and mineralized trabeculae with lamellae arranged parallel to each other [4, 8]. The percentage of cortical and cancellous bone varies among different sites in the skeletons system. For example, in the lumbar spine, 66% of the bone is cancellous bone, whereas in the midradius >95% of the bone is cortical bone [4].
1.6. Bone Histology: Lamellar Bone and Woven Bone
1.7. Bone Remodeling
Although it appears to be inert and static, bone tissue is metabolically active. Throughout life, bone is constantly broken down and rebuilt; this continuous process of bone resorption and bone formation is called bone remodeling [6].
1.7.1. Purposes of Bone Remodeling
It has been generally accepted that bone remodeling serves two closely linked purposes. First, some remodeling can be directed towards specific sites to repair fatigue damage, continuously replacing fatigued bone with mechanically competent new bone. So bone remodeling is necessary to maintain the mechanical strength and structural integrity of new bone. Secondly, through continuous bone resorption and formation, bone subserves its metabolic functions as a storehouse of calcium and phosphorus. Therefore, bone remodeling plays a very important role in maintaining mineral homeostasis [22, 23].
1.7.2. Cellular Processes of Bone Remodeling
1.7.3. Difference between Bone Remodeling and Bone Modeling
During bone remodeling, bone resorption is always followed by bone formation at a locus, and these two processes are said to be coupled to one another [17]. In fact, it is the coupling of osteoclast activity to osteoblast activity that makes bone remodeling distinctive from bone modeling. Bone modeling refers to a process in which bone are shaped or reshaped by the independent activities of osteoclasts and osteoblasts [17]. Therefore, unlike remodeling, bone modeling at a single site involves either bone resorption or bone formation, but not both. Bone modeling is most pronounced during bone growth and development; it also occurs in adults bone in response to mechanical loads [17, 22].
1.7.4. Regulation of Bone Remodeling
Bone remodeling process is highly regulated by numerous local factors and systemic hormones. These factors include bone morphogenetic protein (BMP), tumor growth factor β (TGFβ), tumor necrosis factor α (TNFα), interleukins, estrogen, prostaglandin E2 ( PGE2), parathyroid hormone (PTH), calcitonin, and vitamin D [25]. Among them, estrogen is considered the most important hormone in maintaining normal bone turnover [26]. It has a dual effect on bone remodeling: on one hand, it can increase bone formation; on the other hand, it can reduce bone resorption [25].
Increased bone resorption or a relative decrease in bone formation compared to bone resorption can lead to osteoporosis (osteoporosis will be further discussed in Chapter IV). In contrast, increased bone formation or a relative decrease in bone resorption compared to bone formation can lead to osteopetrosis [6].
1.8. Chapter Summary
Chapter I. Reference List
[1] Hamann KL and Lane NE. Parathyroid hormone update. Rheum Dis Clin North Am 2006; 32: 703-719.
[2] Cole DE, Webb S and Chan PC. Update on parathyroid hormone: new tests and new challenges for external quality assessment. Clin Biochem 2007; 40: 585-590.
[3] Sadler TW and Langman J. Langman's Medical Embryology. Philadelphia, Lippincott Williams & Wilkins. 2006.
[4] Favus MJ and American Society for Bone and Mineral Research. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. Philadelphia, Lippincott Williams & Wilkins. 1999.
[5] Nakashima K and de Crombrugghe B. Transcriptional mechanisms in osteoblast differentiation and bone formation. Trends Genet 2003; 19: 458-466.
[6] Cohen MM,Jr. The new bone biology: pathologic, molecular, and clinical correlates. Am J Med Genet A 2006; 140: 2646-2706.
[7] Sommerfeldt DW and Rubin CT. Biology of bone and how it orchestrates the form and function of the skeleton. Eur Spine J 2001; 10 Suppl 2: S86-95.
[8] Bilezikian JP, Raisz LG and Rodan GA. Principles of Bone Biology. San Diego, Academic Press. 2002.
[9] Hernandez CJ, Majeska RJ and Schaffler MB. Osteocyte density in woven bone. Bone 2004; 35: 1095-1099.
[10] Ross MH and Pawlina W. Histology :A Text and Atlas : With Correlated Cell and Molecular Biology. Baltimore, MD, Lippincott Wiliams & Wilkins. 2006.
[11] Ducy P, Schinke T and Karsenty G. The osteoblast: a sophisticated fibroblast under central surveillance. Science 2000; 289: 1501-1504.
[12] Compston JE. Bone marrow and bone: a functional unit. J Endocrinol 2002; 173: 387-394.
[14] Taichman RS. Blood and bone: two tissues whose fates are intertwined to create the hematopoietic stem-cell niche. Blood 2005; 105: 2631-2639.
[15] Haylock DN and Nilsson SK. Stem cell regulation by the hematopoietic stem cell niche. Cell Cycle 2005; 4: 1353-1355.
[16] Kogianni G and Noble BS. The biology of osteocytes. Curr Osteoporos Rep 2007; 5: 81-86.
[17] Robling AG, Castillo AB and Turner CH. Biomechanical and molecular regulation of bone remodeling. Annu Rev Biomed Eng 2006; 8: 455-498.
[18] Teitelbaum SL. Osteoclasts: what do they do and how do they do it? Am J Pathol 2007; 170: 427-435.
[19] Miyamoto T and Suda T. Differentiation and function of osteoclasts. Keio J Med 2003; 52: 1-7.
[20] Takahashi N, Ejiri S, Yanagisawa S and Ozawa H. Regulation of osteoclast polarization. Odontology 2007; 95: 1-9.
[21] Vaananen K. Mechanism of osteoclast mediated bone resorption--rationale for the design of new therapeutics. Adv Drug Deliv Rev 2005; 57: 959-971.
[22] Favus MJ and American Society for Bone and Mineral Research. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. Washington, DC, American Society for Bone and Mineral Research. 2006.
[23] Heaney RP and Weaver CM. Newer perspectives on calcium nutrition and bone quality. J Am Coll Nutr 2005; 24: 574S-81S.
[24] Parfitt AM. Osteonal and hemi-osteonal remodeling: the spatial and temporal framework for signal traffic in adult human bone. J Cell Biochem 1994; 55: 273-286.
[25] Fernandez-Tresguerres-Hernandez-Gil I, Alobera-Gracia MA, del-Canto-Pingarron M and Blanco-Jerez L. Physiological bases of bone regeneration II. The remodeling process. Med Oral Patol Oral Cir Bucal 2006; 11: E151-7.
CHAPTER II. OSTEOCLAST BIOLOGY: ORIGIN AND
Osteoclasts are the exclusive bone resorptive cells. They are large, multinucleated, and terminally differentiated cells formed via the fusion of their mononuclear precursors [1].
2.1. Osteoclast Origin
Controversy concerning the origin of osteoclasts existed until the late 1970s. The pioneering parabiosis experiments where the circulation of a normal rat was connected to an irradiated rat have demonstrated that cells able to differentiate to osteoclasts were present in the circulation and can be recruited to bone-resorbing sites via the blood stream [2]. This observation was further corroborated by studies using osteopetrotic mouse models. The osteopetrotic phenotype of these mice can be rescued after parabiotic union to normal mice, or after transplantation of bone marrow cells from normal mice [2, 3]. The cure of osteopetrosis by bone marrow transplantation has also been established in humans [4]. It is widely accepted that the osteoclast is of hematopoietic origin [2].
stem cells (HSCs) generate all the blood cell types, including the lymphoid and myeloid lineages. The determination of HSCs to the myeloid lineage requires the expression of the transcription factor PU.1 [8, 9]. The myeloid lineage, also known as colony forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), gives rise to CFU-GM (granulocyte, monocyte/macrophage), and CFU-M (monocyte/macrophage), the latter being the common precursors of osteoclasts and macrophages (Figure 2.1.) [3,10]. Interestingly, compared to the more committed CFU-M-derived cells, CFU-GM-derived cells have greater osteoclast differentiating potential in in vitro mouse and human studies [11, 12]. Nevertheless, it has been generally accepted that osteoclasts are derived from CFU-M, the monocyte/macrophage lineage [4].
Even though osteoclasts can be generated in vitro from mononuclear phagocytes of various tissues, osteoclast precursors primarily reside in bone marrow [4, 5, 13]. The formation of osteoclasts only occurs in the close vicinity of bone, and multinucleated osteoclasts are not present in the circulation [13].
2.2. Osteoclast Differentiation
macrophage colony-stimulating factor (M-CSF) [7, 10]. Signaling pathways initiated by these two factors lead to the expression of genes typifying osteoclasts [7, 10]. Mature osteoclasts are characterized by markers such as integrin αVβ3, calcitonin receptor (CTR), cathepsin K (CTK), metalloproteinase-9 (MMP-9), H+-ATPase, and tartrate resistant alkaline phosphatase (TRAP) (Figure 2.1.) [2].
2.2.1. Role of M-CSF in Osteoclast Differentiation
M-CSF, also known as colony-stimulating factor-1 (CSF-1), was the first colony-stimulating factor to be purified [14]. It was formerly known as macrophage growth factor (MGF) or macrophage and granulocyte inducer IM (MGI-IM) [14]. M-CSF is a homodimeric glycoprotein synthesized constitutively by bone marrow stromal cells and osteoblasts [14-16]. It induces proliferation and survival of the common precursors of osteoclasts and macrophages. Additionally, M-CSF signaling stimulates RANK expression in osteoclast precursors, rendering them more responsive to RANKL [16-18]. The pivotal role of M-CSF in osteoclastogenesis has been confirmed by studies of osteopetrotic (op/op) mice. These mice harbor a null mutation in the coding region of M-CSF gene, which leads to the production of a truncated protein. The biologically inactive M-CSF leads to a severe deficiency in osteoclast formation and hence osteopetrotic phenotypes. Moreover, many of the effects of the op/op mutation can be rescued by the administration of soluble M-CSF to neonatal mice [19].
promoter region of c-fms gene and positively regulates its transcription [15]. Mice deficient in PU.1 exhibit an osteopetrotic phenotype similar to the op/op mice [9]. c-fms is a member of the class III receptor tyrosine kinase family and is encoded by the proto-oncogene c-fms (also known as csf1r). It is an integral plasma membrane glycoprotein, expressed primarily on macrophages and osteoclasts [14-16]. The functional linkage between M-CSF and c-fms has been established by the observation that mice lacking c-fms gene exhibit the same major osteopetrotic phenotypes as op/op mice [16, 18].
The binding of M-CSF to c-fms results in the dimerization and the auto-phosphorylation of c-fms. This leads to the recruitment of c-Src kinase, which then phosphorylates the adaptor protein, c-Cbl. c-Cbl recruits a multiprotein complex containing growth-factor-receptor-bound protein 2 (Grb2) and phosphoinositide 3-kinase (PI3K), with the former activating extracellular-signal-regulated kinase (ERK) and the latter the serine/threonine kinase AKT (or protein kinase B, PKB) (Figure 2.2.) [16, 18, 20]. Collectively, M-CSF induces the proliferation of osteoclast precursor cells and supports their survival through PI3K /AKT and Grb2/ERK axis.
2.2.2. Role of RANKL in Osteoclast Differentiation
released from the cell surface by the metalloproteasedisintegrin TNF α convertase (TACE) [26, 27]. In the presence of M-CSF, both membrane-bound and soluble RANKL can support osteoclast differentiation in vitro [25]. However, the membrane-bound may be more efficient than the soluble form [26]. The in vivo biological and pathological significance of soluble RANKL still remains unclear. The importance of RANKL in osteoclast formation has been collaborated by animal studies. RANKL−/− mice display complete failure of osteoclastogenesis and consequently develop severe osteopetrosis [24, 28].
The receptor for RANKL—RANK (receptor activator of NFκB)—is a type I membrane protein and belongs to the TNF receptor superfamily [25]. RANK is also known as TRANCE-R or TNFRSF11A, and is expressed as a transmembrane heterotrimer on the surface of osteoclasts and their precursors [24, 25]. Mice lacking rank have exactly the same osteopetrotic phenotype as rankl−/−mice. However, rank−/− mice have an intrinsic defect in osteoclasts as indicated by the fact that their phenotype can be reversed by bone marrow transplantation from normal mice [25, 29].
to be the master transcription factor for osteoclastogenesis since its induction activates the expression of osteoclast-specific genes [32, 33, 36]. Genetics studies have shown that osteopetrosis occurs in mice lacking key genes involved in RANKL signaling, such as TRAP6 and c-Fos [37-39].
2.2.3. Costimulatory Signals for RANK
Since authentic osteoclasts can be generated in vitro from bone marrow cells in response to recombinant RANKL and M-CSF, it has been generally accepted that RANKL and M-CSF signaling is not only necessary but also sufficient to support osteoclastogenesis [40, 41]. However, recent studies have revealed a more complex picture: additional costimulatory signals are required to activate the transcription factor, NFATc1 [32, 33, 36, 42].
DAP12 into DAP12−/−FcRγ−/− cells can rescue osteoclast differentiation deficiency [33]. These data indicate that ITAM signaling is indispensable for RANKL-induced osteoclastogenesis. However, so far it still remains largely unknown how RANKL/RANK signaling synergizes with ITAM signaling.
2.3. Regulation of Osteoclast Differentiation
Osteoclast differentiation is subject to negative and positive regulation by circulating hormones and locally produced cytokines [4, 43]. Major mechanisms underlying the regulation of osteoclast differentiation are discussed in the following sections.
2.3.1. The OPG/RANKL/RANK Regulatory Axis
OPG (osteoprotegerin, i.e. protector of bone), also known as osteoclastogenesis inhibitory factor (OCIF), is a secreted protein expressed by osteoblasts/stromal cells [44-46]. OPG, like RANK, belongs to the TNF receptor superfamily. However, in contrast to all other members, OPG lacks transmembrane and cytoplasmic domains and is secreted as a soluble protein. OPG functions as a soluble decoy receptor to RANKL, competing with RANK for RANKL binding (Figure 2.4.) [41, 46, 47]. OPG is a negatively regulator of RANKL activity. Genetic studies have found that excessive RANKL activity, as in OPG deficient mice, results in early-onset osteoporosis [48, 49]. In contrast, due to a decreased net RANKL level, transgenic mice overexpressing OPG exhibit osteopetrosis [46].
expression of RANKL and OPG (see reference [46] for a thorough list) [4, 43, 46, 47]. For example, TGF-β released from bone matrix during bone resorption has been shown to upregulate OPG expression, serving as a negative feedback mechanism for bone resorption [24, 26]. Interleukins (IL-1, 6, 11, and 17) can increase RANKL expression [24, 25, 46]. Some factors not only decrease OPG expression but also increase RANKL expression, such as parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), and prostaglandin E2 (PGE2) [25, 46]. Dexamethasone and 25-dihydroxy vitamin D3 (1, 25(OH)2D3, theactive form of vitamin D3) can stimulate RANKL production and are often used in coculture of bone marrow cells and stromal cells to generate osteoclasts in vitro [25, 46]. Therefore, the OPG/RANKL/RANK axis is critical for maintaining the balance between bone formation and resorption by providing a means for controlling osteoblast and osteoclast activity (Figure 2.4.).
2.3.2. Other Regulatory Mechanisms
formation [56]. Taken together, more studies, especially in vivo ones, are necessary to further explore other mechanisms that regulate osteoclast differentiation.
2.4. Osteoclast Fusion
During osteoclastogenesis, preosteoclasts fuse to form multinuclear mature osteoclasts. Studies comparing the gene expression of multinuclear osteoclasts and mononuclear macrophages have identified two molecules critically involved in cell–cell fusion of osteoclasts: the dendritic cell-specific transmembrane protein (DC-STAMP) [57, 58] and the d2 isoform of vacuolar H+-ATPase V0 domain (Atp6v0d2) [59]. Mice deficient in either DC-STAMP or Atp6v0d2 lack multinuclear osteoclasts and develop osteopetrosis [57-59]. Further studies have demonstrated that the expression of DC-STAMP and Atp6v0d2 are induced by transcription factor, NFATc1 [60, 61]. Interestingly, a recent study has suggested a newly-identified protein induced by RANKL, osteoclast stimulatory transmembrane protein (OC-STAMP), is required in the fusion process during osteoclastogenesis [62]. It appears that multiple factors are involved in osteoclast fusion, and more studies are necessary to further understand this process.
2.5. Osteoclasts in Periprosthetic Osteolysis and Peri-implantitis
subsequent osteolysis is largely unclear. The primary cellular response to implants is the phagocytosis of foreign particles, which somehow activates macrophages to release numerous pro-inflammatory cytokines, including TNFα, IL-1α, IL-1β, IL-6, and PGE2 [64, 68, 69]. These cytokines may explain, at least partially, the increased levels of RANKL and RANK in the interface tissue in loosening orthopaedic replacement, leading to increased osteoclastogenesis and bone resorption [64, 70, 71]. In addition to the RANK/RANKL signaling pathway, some cytokines, especially TNFα, may enhance osteoclast differentiation directly [70].
In addition to the involvement of the macrophage-mediated foreign body immune response to particles, T lymphocytes may also play an important role in periprosthetic osteolysis [72, 73]. Immunohistochemical studies have found a large number of T cells in osteolytic tissues [73]. The interplay of immune system and bone has been well established. Activated T cells are well known to produce RANKL and thus directly support osteoclastogenesis [52, 74]. T cells can also produce other osteoclastogenic factors, further contributing to the process of the bone destruction in inflammatory conditions such as rheumatoid arthritis [75]. Whether T cells in osteolytic tissues can secrete other cytokines to influence the RANKL/RANK/OPG system remained to be defined. Collectively, excessive osteoclastogenesis and bone resorption caused by the imbalance of RANKL/RANK/OPG axis plays an important role in aseptic loosening [64, 70, 71].
significantly increased in peri-implant tissue from loosened implants [77]. In order to develop diagnostic and treating strategies for peri-implantitis, effort has been made to determine the levels of several proteins associated with bone resorption in the implant crevicular fluid from patients with failing dental implants. These proteins include PGE2 , soluble RANKL, cathepsin K, and OPG [77-80]. Theses data implicate an important role of osteoclasts in the bone loss during peri-implantitis. Additionally, immunohistochemical studies have found that T cells are the most prominent cell in peri-implantitis tissue [81], suggesting a role of T cells in the pathogenesis of peri-implantitis. Although the involvement of T cells in inflammatory conditions such as rheumatoid arthritis has been established [75], little is known how T cells contribute to peri-implantitis. It appears that excessive bone loss caused by the imbalance of RANKL/RANK/OPG axis plays an important role in peri-implantitis; however, more research is needed to better understand the mechanism of osteoclast activation and bone resorption in peri-implantitis
2.6. Osteoclasts in Orthodontic Tooth Movement
tissue can decrease orthodontic tooth movement [87], while local RANKL gene transfer to the periodontal tissue can increase orthodontic tooth movement [88]. Meanwhile, local administration of OPG can inhibit orthodontic tooth movement [89]. Collectively, these data indicate the pivotal role of OPG/RANKL/RANK regulatory axis in PDL cells during orthodontic tooth movement.
Therefore, hypoxia at the pressure side of a tooth undergoing orthodontic tooth movement leads to bone resorption by directly or indirectly disrupting the balance of the OPG/RANKL/RANK axis in PDL.
M-CSF
RANKL
CFU-M
CFU-GEMM
Osteoclast
PU.1
Osteoblast/Stromal cell
and survival
MAPKs
TRAF6
NFATc1
c-Fos
c-Fos NFATc1 Syk Syk PLCγ Calcium signaling TREM-2 SIRPβ1 DAP12 FcRγ Osteoclast/ osteoclast precursor Osteoclast/osteoclast precursor Osteoblast/stromal cells RANKL RANK M-CSF M-CSF receptor OSCAR PIR-A ITAM ITAM ? TRAF6
c-Fos NFATc1 Syk Syk PLCγ Calcium signaling TREM-2 SIRPβ1 DAP12 FcRγ Osteoclast/ osteoclast precursor Osteoclast/osteoclast precursor Osteoblast/stromal cells RANKL RANK M-CSF M-CSF receptor OSCAR PIR-A ITAM ITAM ?
Differentiation Differentiation
Osteoblast/stromal cells Osteoblast/stromal cells M
M--CSFCSF c c--fmsfms
Cytokines and hormones
Cytokines and hormones Osteoclast Osteoclast precursor precursor Mature Mature osteoclast osteoclast Inhibition
Inhibition OPGOPG Differentiation Differentiation
Cytokines and hormonesCytokines and hormones
Inhibition OPGOPG Inhibition
Inhibition Inhibition
Inhibition OPGOPGOPGOPG
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[38] Johnson RS, Spiegelman BM and Papaioannou V. Pleiotropic effects of a null mutation in the c-fos proto-oncogene. Cell 1992; 71: 577-586.
[39] Wang ZQ, Ovitt C, Grigoriadis AE, Mohle-Steinlein U, Ruther U and Wagner EF. Bone and haematopoietic defects in mice lacking c-fos. Nature 1992; 360: 741-745.
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[44] Simonet WS, Lacey DL, Dunstan CR, et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 1997; 89: 309-319.
[45] Yasuda H, Shima N, Nakagawa N, et al. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 1998; 139: 1329-1337.
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CHAPTER III. OSTEOCLAST BIOLGOY: FUNCTION
Osteoclasts have a number of unique ultrastructural characteristics, including multiple nuclei surrounded by the full-developed Golgi apparatus, abundant mitochondria, and a large number of vacuoles and lysosomes [1]. Functional osteoclasts can be found both in trabecular and cortical bone; they are located on the surface of trabeculi in trabecular bone, and in the cutting edge of the forming osteons in cortical bone [2].
3.1. Mechanism of Bone Resorption
3.1.1. Dissolving the Mineral Phase
The extracellular compartment between the osteoclast and the bone surface is acidified by protons transported by the ATP (adenosine triphosphate)-dependent proton pumps known as the vacuolar H+- adenosine triphosphatase (H+-ATPase) or V-ATPase at the ruffled border. As shown in the reaction below, the low pH value can dissolve the strongly basic hydroxyapatite in solid phase, releasing calcium ions, phosphate ions, and water in solution [4].
[Ca
(PO
Ca(OH)
+ 8H
+ 6HPO
Solid hydroxyapatite Solution
Secretion of protons is accompanied by movement of chloride anions via the CLC-7 chloride channel at the basolateral membrane [2, 3]. Ion equilibrium in the cytoplasm is maintained by the following two processes: 1) the production of protons and bicarbonate ions from CO2 hydration facilitated by carbonic anhydrase II (CA II); and 2) the exchange of bicarbonate to chloride through bicarbonate-chloride (HCO3/Cl) exchanger at the basolateral membrane (Figure 3.2.) [2, 3]. Key enzymes involved in the acidification process are very important for normal bone resorption. Mutations in H+ATPase, CLC-7 chloride channel or carbonic anhydrase II can result in osteopetrosis in mice and humans [3, 5, 6].
3.1.2. Degrading the Organic Phase
proteinase, matrix metalloproteinase-9 (MMP-9), and tartrate resistant alkaline phosphatase (TRAP) (Figure 3.2.) [2, 7-9]. Genetics studies have demonstrated that mice deficient in lysosomal proteases such as TRAP and cathepsin K are osteopetrotic [8, 9].
3.1.3. Removing Resorption Products
In order to continue resorption, osteoclasts must have a mechanism to dispose the resorption products simultaneously with the ongoing secretion of acid and proteolytic enzymes [2]. Studies have demonstrated that degradation products are endocytosed into the cell from the ruffled border, transcytosed through the cell, and finally secreted into the extracellular fluid through a specific plasma membrane domain. This domain is located in the upper part of a resorbing and is called "functional secretory domain" (Figure 3.1.) [2]. It should be noted that the process of transcytosis in osteoclasts is not just a disposal process. It mobilizes the calcium stored in bone, and it releases growth factors such as transforming growth factor-beta (TGF-β ) latent in bone matrix [4].
3.2. Role of Osteoclasts in Hematopoiesis
lipopolysaccharide (LPS) administration and controlled bleeding [13]. Stimulation of osteoclasts by RANKL, bleeding, or LPS results in elevated production of proteolytic enzymes, MMP-9 and CTK, which, in turn, degrade the hematopoietic stem cell niche components, including stromal cell-derived factor-1(SDF-1), stem cell factor (SCF), and osteopontin [13]. This leads to increased mobilization of HSCs into the circulation. In summary, these data associate osteoclasts with the regulation of hematopoiesis and host defense, further emphasizing the function interdependence between bone and bone marrow/ hematopoiesis (Figure 3.3.).
3.3. Beyond Bone Resorption: Stimulating Bone Formation
Osteoclast activity has been traditionally considered as limited to bone resorption; however, accumulating evidence has suggested an alternative function of osteoclast: stimulating bone formation [14-16].
3.3.1. Evidence from the Osteopetrosis Models
In osteopetrosis, bone mass is increased due to reduced bone resorption; however, depending on the etiology, bone formation and resorption is not always coupled. Patients with mutations in the chloride channel CLC-7 or the H+-ATPase display reduced bone resorption due to impaired acidification of the resorption lacunae. However, rather than decreased bone formation as expected from the decreased bone resorption, these patients exhibit normal or even increased bone formation. Moreover, they have increased number of non-resorbing osteoclasts that are otherwise normal [16]. This observation has also been confirmed by a recent finding that bone formation is increased in mice with deficiency in osteoclast fusion [17]. In contrast to patients with defective acidification process, osteopetrosis patients with defective cathepsin K have no apparent uncoupling of formation and resorption, but they have poorly remodeled bone. [16].
In comparison to osteoclast-rich osteopetrosis mentioned above are osteopetrotic models where the osteoclasts are reduced or absent (osteoclast-poor). Studies from op/op mice have shown that in the complete absence of osteoclasts, bone formation is impaired, with disorganized matrix, reduced mineralization, and a lower bone quality [16, 18]. Consistently, c-fos deficient mice, which lack osteoclasts, have markedly decreased serum osteocalcin level, suggesting reduced bone formation [16, 19, 20].
3.3.2. Evidence from the Anabolic Action of PTH
Parathyroid hormone (PTH) is a polypeptide hormone secreted by the chief cells of the parathyroid glands [21, 22]. PTH has a dual action on bone remodeling. Sustained elevations of PTH, as in hyperparathyroidism, have a net catabolic effect on bone, favoring bone resorption [21]. The catabolic action of PTH on bone will be discussed in detail in Chapter V. Interestingly, when administered in an intermittent, rather than a continuous mode, PTH has an anabolic action on bone [21].
This has raised the question whether the resorptive action of PTH is necessary for its anabolic action on bone formation. As indicated by both human and animal studies, the anabolic effect of PTH is not solely dependent on osteoblasts; it also requires signals from osteoclasts linking back to osteoblasts [14, 23].
In addition, the underlying mechanism has been further proposed: PTH can increase production of RANKL, which stimulate the transient activation of osteoclasts. RANKL-activated osteoclasts then provide signals for osteoblast maturation (Figure 3.4.) [14].
In summary, studies from the anabolic action of PTH further emphasize the important role of osteoclasts in providing signals required in coupling bone resorption to bone formation during bone remodeling.
3.3.3. Evidence from In Vitro Studies
Bone lining Osteoblast Bone lining Osteoblast RANKL RANKL Stress Stress Active Osteoclast Active Osteoclast
Anchored stem cells Anchored stem cells Blood vessel Blood vessel Bone Bone Endosteum Endosteum MMP MMP--9 9 Cathepsin CathepsinK K
Mob ilizat ion Mob ilizat ion
Bone lining Osteoblast Bone lining Osteoblast RANKL
RANKL Stress Stress
Active Osteoclast Active OsteoclastActive Osteoclast Active OsteoclastActive Osteoclast Active Osteoclast
Mob ilizat ion Mob ilizat ion Mob ilizat ion Mob ilizat ion
Osteoclast Osteoblast
Bone formation
(transient)
Coupling signal
Chapter III. Reference List
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[23] Koh AJ, Demiralp B, Neiva KG, et al. Cells of the osteoclast lineage as mediators of the anabolic actions of parathyroid hormone in bone. Endocrinology 2005; 146: 4584-4596. [24] Karsdal MA, Neutzsky-Wulff AV, Dziegiel MH, Christiansen C and Henriksen K. Osteoclasts secrete non-bone derived signals that induce bone formation. Biochem Biophys Res Commun 2008; 366: 483-488.
CHAPTER IV. OSTEOCLAST PATHOLOGY: BONE LOSS
Bone resorption plays an important role not only in bone development and physiology, but also in bone pathology. The discovery of the OPG/RANKL/RANK axis as a major regulatory mechanism of osteoclastogenesis has revolutionized our understanding of normal bone biology and a number of bone diseases as well [1-3].
4.1. Postmenopausal Osteoporosis
4.1.1. Introduction
Osteoporosis is a heterogeneous group of metabolic bone diseases [4] and has been defined as "a skeletal disorder characterized by compromised bone strength predisposing a person to an increased risk of fracture" [5]. Although osteoporosis is characterized by low bone mass, the ratio of bone mineral to the organic matrix in osteoporosis normal, as opposed to a decreased ratio of bone mineral to the organic matrix in osteomalacia [6, 7].
compares a patient to a sex-matched, young, healthy population; it is calculated by subtracting the mean BMD of a young adult healthy population from the patient's BMD and then dividing by the standard deviation (SD) of the reference population. A Z score compares a patient to an age-and sex-matched control population; it is calculated by subtracting the mean BMD of an age- and sex-matched control population from the patient's BMD and then dividing by the SD of the reference population [9]. T scores are often reported in postmenopausal women and men of 50 years age and older, while Z scores are often reported in premenopausal women, men under the age of 50, and children [9]. According to the WHO definition, the diagnosis of osteoporosis is established when BMD is 2.5 SDs below the mean for normal Caucasian women (i.e. the T score is at least −2.5 SDs) [5].
However, it has been argued that the measurement of bone mass does not account for another important determinant of bone strength—bone quality [10, 11]. Bone quality is independently influenced by parameters including bone architecture, bone turnover, microdamage, and mineralization [5]. Although BMD measurement is currently the best diagnostic practice, alternative methods that can detect the deterioration in bone quality will provide significant improvement in osteoporosis prevention, diagnosis, and treatment [11].
years of age for healthy women. As a result, the production of ovarian hormones including estrogen is reduced [6]. The pathogenesis of postmenopausal osteoporosis will be discussed below with a focus on the current understanding of estrogen action on osteoclastic bone resorption.
4.1.2. Cellular Pathogenesis
Under normal conditions, bone undergoes constant remodeling, where resorption of the existing bone by osteoclasts is tightly coupled to formation of new bone by osteoblasts [13]. In postmenopausal osteoporosis, the rate of bone remodeling is increased, i.e. increased number of osteoclasts and bone resorption coupled with increased number of osteoblasts and bone formation. However, due to estrogen deficiency, there is an imbalance between bone resorption and bone formation, resulting in a decrease in total bone mass [4, 7].
4.1.3. Indirect Effects of Estrogen Deficiency on Bone Resorption
Figure 2.1.
Osteoclastogenesis. Osteoclastogenesis is a multi-step process regulated by a sequential series of molecular event p.38
M-CSF/RANKL signaling pathways. M-CSF, which is released from osteoblasts/stromal cells, binds to its receptor on osteoclast precursors, c-fms p.39
Cooperation of RANKL and ITAM induced signals in osteoclastogenesis. p.40
RANKL-dependent regulation of osteoclast formation. M-CSF secreted by osteoblasts/stromal cells can drive myeloid progenitors towards osteoclast precursors p.41
Plasma membrane domains present in a resorbing osteoclast. Adapted from reference 2 p.58
Mechanism of osteoclastic bone resorption. The osteoclast adheres to bone surface via integrin αvβ3, and forms a special membrane facing bone: the ruffled border p.59
Osteoclasts mobilize hematopoietic stem cells into circulation. Stimulation of osteoclasts by RANKL or stress situations such as inflammation and injury results in elevated production of proteolytic enzymes, MMP-9 and CTK, which, in turn, deg p.60
Mechanism of the anabolic action of PTH. An anabolic stimulus, PTH, increase transiently RANKL production by osteoblasts, which results in the activation of osteoclasts p.61
Mutations in components of RANK–NFκB signaling pathway cause Paget's disease of bone and related disorders p.78
Reciprocal relationship between PTH and extracellular calcium level. PTH secretion is triggered by hypocalcemia, and suppressed by hypercalcemia p.79
Proposed mechanism of cherubism pathogenesis. Mutation in SH3BP2 gene results in an increased response of myeloid cells to M-CSF and RANKL p.80
The structure of bisphosphonate and pyrophosphate. p.96
Structures of bisphosphonates classified according to their biochemical modes of action p.97
Formation of AppCp-type metabolites of simple bisphosphonates. Upper panel: Mechanism by which aminoacyl-tRNA synthetases catalyze formation of AppCp-type metabolites of bisphosphonates p.98
Mechanism of action of nitrogen-containing bisphosphonates on osteoclasts p.99
Download (N/A - 104 Page - 694.04KB)
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text:
The real story behind the man they called 'the worst Burnley manager ever' / Dave Thomas column
Midwife retires after 37 years
A BURNLEY midwife who has delivered and cared for two generations of babies has retired after 37 years in the health profession.
Saturday, 9th October 2010, 8:22 am
Miss Catherine Nearey (55), who lives in Ightenhill, was joined by more than 70 colleagues and friends at Rosehill House Hotel to mark the milestone.
She began her career in Blackburn as a student nurse in 1973 before becoming a staff nurse at the former Park Lee Hospital. She qualified as a midwife in 1979 and was based at Burnley General Hospital for 32 years.
More recently Sister Nearey was a member of Shuttleworth Team and for many years was a community midwife in Padiham.
She plans to holiday in Cyprus with friends before settling into retirement and said she will miss her job.
"I'm sure I'll miss it. It just feels like being on holiday at the moment. It will be different after working full-time all these years."
And despite carrying out a host of different duties over the years Miss Nearey, who is a keen gardener and enjoys travelling and walking, said she enjoyed all aspects of her job.
"I liked it all. When it's good it's very good. Ante-natal care is all good."
As a sign of her dedication to her career, colleagues and patients, Miss Nearey was presented with jewellery, cards, vouchers, and mother and child ornaments by fellow staff.
"It's just been incredible, and quite exciting.
"I would like to thank my colleagues for celebrating my retirement," she added.
Two men charged with kidnap and robbery after victim flags down member of the public on Pendle Hill
Burnley drugs trafficker who tried to shield himself from prosecution jailed for 11 years
Clitheroe couple celebrating milestone Golden Wedding Anniversary say key to happiness is 'helping each other'
Search on to find new manager for landmark Burnley pub that has been empty for two years
Tributes to much-loved Earby lecturer after courageous battle with Covid
Firefighters rescue person from house fire in Colne
Town gathers to say a final farewell to former Burnley MP who was a voice for the people for over two decades
School reunion anyone? Calling all my former classmates. 2022 is our year / Sue Plunkett column

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: How to detect unused spring beans configured in XML? I have a java project in which many .xml files are present.
All these xml files contain many beans.
The test for unused is:

*

*A bean that is defined but never injected.

*A bean that is injected but is never called in the code.

*A bean that is defined but never loaded into the Spring context.

Questions

*

*How do I identify which bean is used or not?

*Is there a utility to do that?

A: There is no tools for detecting the unused springbeans in xml file. You can use the Spring Tools Suite for detecting. But it is taking too much time for checking.

A: I think that you can use spring actuator to check the opposite problem - which beans have been loaded in your context.
https://docs.spring.io/spring-boot/docs/current/reference/html/production-ready-endpoints.html

Actuator endpoints allow you to monitor and interact with your
  application. Spring Boot includes a number of built-in endpoints and
  you can also add your own. For example the health endpoint provides
  basic application health information.
The way that endpoints are exposed will depend on the type of
  technology that you choose. Most applications choose HTTP monitoring,
  where the ID of the endpoint is mapped to a URL. For example, by
  default, the health endpoint will be mapped to /health.
beans Displays a complete list of all the Spring beans in your application.

Looking for all the beans which are unused requires scanning XML files within application, then you can compare it with the list of beans produced by actuator.

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text:
UKScreen | Moonrise Kingdom – Review
Home / Reviews / Moonrise Kingdom – Review
UKScreen Rating:
Moonrise Kingdom – Review
- Review By: Jason Korsner - - No Comments
Rating: 12A
Starring: Bill Murray, Bruce Willis, Edward Norton, Frances McDormand, Harvey Keitel, Jared Gilman, Jason Schwartzman, Kara Hayward, L.J.Foley, tilda swinton
Scout Master Ward (Edward Norton) sends out a search party when one of his troop, Sam (Jared Gilman), escapes from their camp site.
Walt (Bill Murray) and Laura Bishop (Frances McDormand) call in the local police officer, Captain Sharp (Bruce Willis), when their daughter Suzy (Kara Hayward) disappears.
It turns out that Sam and Suzy have been pen-pals since they met at a school play a year earlier, and they've been planning to elope.
With the police, her parents and his scout troop on their tail, Sam puts his wilderness skills to the test as the pair do their best to avoid capture as they try to get off the island and start a new life together, without any of those pesky adults to make their lives a misery.
WHAT'S IT LIKE?
From the opening tracking shot, which appears to be of the inside of a doll's house, Wes Anderson takes you into his own surreal world in which he appears to fill the background with minor details he couldn't work out how to get into the film otherwise.
It's a children's adventure film whose quirkiness is pushed right to the limit of acceptability, but never quite crosses it.
The story is simple and sweet, the production values – whether cluttered or neat-freak – are immaculate and accurate to the 1960s period and the crackpot characters who inhabit Anderson's world.
The children in the lead roles are tremendous – Sam's a loveable geek and Suzy's a cantankerous teen who hates everything in the world except Sam and her music. Anderson regulars Bill Murray and Jason Schwartzman are on auto-pilot, but no less enjoyable for it, while Frances McDormand's domineering, megaphone-wielding mother is as frightening as she is reassuring.
Tilda Swinton, as an officious social worker who dresses more like an air hostess, also delivers an interesting turn.
Bruce Willis proves that he can still do comedy and it makes a refreshing change to see Ed Norton having some fun for once.
Anderson is one of those love-him-or-hate-him film-makers who tells simple tales with an idiosyncratic twist. Sometimes, he pushes it so far that even those who love him hate him, but with Moonrise Kingdom, he's back on the kind of form he hasn't produced since The Royal Tenenbaums, more than a decade to go.
The film isn't particularly challenging to watch and although it touches on themes such as loneliness and loyalty, it doesn't really have much to say.
But it's thoroughly entertaining, cheekily charming and you'd have to be made of stern stuff not to leave the cinema smiling.
Opens 25 May 2012
Tagged with: Bill Murray • Bruce Willis • Edward Norton • Frances McDormand • Harvey Keitel • Jared Gilman • Jason Schwartzman • Kara Hayward • L.J.Foley • Moonrise Kingdom • tilda swinton • Wes Anderson
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Miramax opens an office in London
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Joaquin Phoenix: Actors don't deserve credit for their performance
Tim Burton: I Love All Monsters
DICKENS' BLEAK HOUSE IS UP FOR SALE
Oprah Winfrey: African American people are real people - Interview
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Have you been contacted by a creditor with regards t a compulsory liquidation. If so we can assist you please call us or apply online for help and assistance with the compulsory liquidation procedure.
Compulsory liquidation or compulsory winding up is when the court makes an order for the company to be wound up on the petition of an appropriate person. If there is more than one director, all the directors must jointly present the winding-up petition - a single director cannot present a winding-up petition.
What are the alternatives to compulsory liquidation?
Informal Arrangement - the company could consider writing to all its creditors to see if a mutually acceptable agreement can be reached. It is advisable to include a timetable of when payments will be made.
Company Voluntary Arrangement (CVA) - this is a formal version of the arrangement described above. The directors would need to apply to the court with the help of an authorised insolvency practitioner, who would supervise the arrangement and pay the creditors in line with the accepted proposals.
Organise a voluntary arrangement or compromise with its creditors; Get a better realisation of assets than would be possible if the company went into liquidation.
The procedure is managed by an administrator, who must be an authorised insolvency practitioner.


Suzuki is running a television advertising campaign in the UK centred around the concept, "Have to Have It", following on from research showing that over half the people who test drive a Suzuki buy a Suzuki. "The Never Ending Drive" and a range of ITV movies sponsorship idents show customers who are so happy with their first experience of the new car that they won't let go. See more on the campaign on the Suzuki UK Facebook page.
Filming was shot by director Andy McLeod (main film) and Austen Humphries (idents) via Rattling Stick with producer Kirsty Dye, director of photography Stuart Graham, editor Saam Hodivala. Post production was done at Big Buoy.
Music, "Tomorrow will be like today" was produced by Money Mark (Mark Ramos-Nishita). Sound was produced at Wave Studios by Parv Thind.
Media was handled at 7 Stars by media planner Nick Maddison.


text:
Q: Need help putting several arrays from a JSONP call into one single array I'm trying to get all viewers (people in chat) from a certain channel on twitch, into an array.
The JSONP call works, but the Twitch API returns several arrays:

*

*moderators

*staff

*admins

*global_mods

*viewers

I want to put all usernames that are in these arrays, into one single array (i.e. "peopleinchat").
My approach was to first declare the array:
var peopleinchat= [];
And then add each subsequent array (group) of people to this array using $.merge(). I can't get it to work however... I get "TypeError: Cannot read property 'length' of undefined". Can you help me out?
Thanks!

$.ajax({
  url: "https://tmi.twitch.tv/group/user/batuhanbuyukakkan/chatters",

  // The name of the callback parameter, as specified by the YQL service
  jsonp: "callback",

  // Tell jQuery we're expecting JSONP
  dataType: "jsonp",

  // Work with the response
  success: function( response ) {

    var peopleinchat = [];

    $.each(response, function(index, data){

      $.each(data.chatters, function(index, group){

        $('.viewers').append(index + ': ' + group);
        $('.viewers').append('
');

        $.merge(peopleinchat, group);

      });

    });

    $('.array_result').append(peopleinchat);

  }
});

A: Almost there - I think your main problem was that you didn't deal with the proper shape of the result parameter to your success callback. The snippet below works:

$.ajax({
  url: "https://tmi.twitch.tv/group/user/batuhanbuyukakkan/chatters",

  // The name of the callback parameter, as specified by the YQL service
  jsonp: "callback",

  // Tell jQuery we're expecting JSONP
  dataType: "jsonp",

  // Work with the response
  success: function( response ) {
    var peopleinchat = [];

    $.each(response.data.chatters, function(index, data){

        $('.viewers').append(index + ': ' + data);
        $('.viewers').append('
');

        $.merge(peopleinchat, data);

    });

    $('.array_result').append(peopleinchat);

  }
});


text:
Q: Check text in a DOM element using Protractor Here's what I'm trying to do while testing an Angular app with Protractor. I would like to get a certain element, which is somewhat like this:

{{textFromBoundModel}}

then get its html, and check whether it contains the text that I expect it to have.
I tried to get this element first by the cssContainingText method, but it didn't quite work (not sure why; maybe because the text within the paragraph is produced dynamically). So now I'm getting this element using just the by.css locator. Next, I'm checking whether it contains the text I'm testing for:
// this is Cucumber.js

this.Then(/^Doing my step"$/, function(callback){
    var el = element(by.css('.someClass'));
    expect(el).to.contain("some interesting string");
    callback();
 });
});

but this doesn't work. Problem is, el is some kind of a locator object, and I can't figure out how to get html of the element it found in order to test against this html. Tried .getText(), with no success.
Any suggestions?

A: Does:
expect(el.getText()).to.eventually.contain("some interesting string");

work?
I believe you need the .eventually to wait for a promise to resolve, and you need the .getText() to get at the content of the div.
See the chai-as-promised stuff at the head of the cucumber sample.

A: Try the below solution worked for me
Solution 1 :
expect(page.getTitleText()).toContain('my app is running!');

Solution 2 :
expect<any>(page.getTitleText()).toEqual('my app is running!');


We are glad to introduce you our «Lesnoe chudo» (trans. "Forest miracle") collection.
We introduce you the collection of three Ivan-tea blends for everyday use. We blended willow-herb with Indian black tea, Chinese green tea and natural briar.
For everyone who wants to be healthy!
Ivan-tea was the most popular natural Russian beverage till the second half of 19th century. Its' toning and healing effects were used to "strengthen the soul and warm the body" for hundreds of years. Today Ivan-tea is gaining back the popularity, as it contains more vitamin C than lemons. More and more people drink it every day to get healthier and stronger. We mixed Ivan-tea with natural briar to make our «Lesnoe chudo » tea that will warm you no matter how cold is outside.
Ingredients: willow-herb (Ivan-tea), briar, thyme.
Using: put one teabag into the cup or teapot, pour the hot water and let it steep for 3-4 minutes.
Ivan-tea was the most popular natural Russian beverage till the second half of 19th century. Its' toning and healing effects were used to "strengthen the soul and warm the body" for hundreds of years. Today Ivan-tea is gaining back the popularity, as it contains more vitamin C than lemons. More and more people drink it every day to get healthier and stronger. We mixed Ivan-tea with natural green tea to make our «Lesnoe chudo» tea and give you the real burst of energy.
Ingredients: willow-herb (Ivan-tea), green tea.
Ivan-tea was the most popular natural Russian beverage till the second half of 19th century. Its' toning and healing effects were used to "strengthen the soul and warm the body" for hundreds of years. Today Ivan-tea is gaining back the popularity, as it contains more vitamin C than lemons. More and more people drink it every day to get healthier and stronger. We mixed Ivan-tea with natural black tea to make our «Lesnoe chudo» tea and give you both health and joy.
Ingredients: willow-herb (Ivan-tea), black tea.


Chris Woakes' maiden Test hundred put England in a dominant position by the end of the third day of the second Test against India, 250 runs ahead with four wickets in hand.
You're 20 years old and you look a lot younger. You're making your Test debut at the most prestigious ground in the world against the best team in the world. You walk out to bat with your side 32-2, batting in the top four and inside the first 10 overs for the first time in your nascent first-class career. Already 12 wickets have fallen for 139 in the game, all but one of them to seam and swing. Your first ball is on a good length on middle, and you play it calmly. The second is even better, honing in on middle stump, testing your technique early. What do you do? If you're Ollie Pope, you lean across and flick it sweetly for four, and then flash an even sweeter smile for good measure.
First impressions can be misleading, and while Pope's eventual 28 was more than Alastair Cook, Joe Root, and Virat Kohli managed, it was also neither here nor there. In truth, we learned little about whether he will make it as a Test batsman. But he at least hinted that the scale of the occasion wouldn't faze him.
As much as the fact that Root was the batsman dismissed and that it left England 84-4, still behind, it was the manner of the dismissal which would have given India hope, Mohammed Shami extracting a devilish bit of low bounce to strike England's captain in front. If they could keep England's lead low and set some sort of target, more uneven bounce could have spelled trouble on the last day. Instead, thanks to the efforts of Woakes and Jonny Bairstow, England will be aiming to use any up and down to bowl India out cheaply in the third innings.
A two-ball passage which summed up Bairstow's innings. First he got in a tangle, playing back when he should have been forward to Ravichandran Ashwin, who was strangley held back until the 38th over when England had a lead of exactly 50. Next ball, which was even fuller, he cracked the off-spinner over mid-on for four.
England's wicket-keeper has been criticised for playing without due care and attention in recent times, and today he edged or missed 22 balls, some through his own sense of adventure, but with the conditions still tilted in favour of the bowlers and an unplayable ball always around the corner, his counter-attack was this time justified. He also put the last ball out of his mind well throughout. He deserved a hundred.
Five Ishant Sharma balls in which Woakes asserted his dominance over India, the conditions, and the Test match. The first was short and carved behind point, the second defended oh-so solidly, the third again played behind square on the off side, this time for a couple. The fourth was crunched gorgeously through long-on, bait which was gobbled up and the hook spat out, and the fifth pulled easily to long-leg for a single which took Woakes to 80 and past Bairstow's score.
This was the best Woakes has looked on the Test stage, fluent, composed and technically all there in conditions in which some of the best in the world struggled. Having made his debut at No.6 he has always been thought of as an all-rounder, and a high score of 66 prior to today didn't do him justice. In this innings, his average nudged above 30, where it belongs.
Not a pivotal moment in the context of the game – Sam Curran entered with England's lead over 200 and the only question was whether a pre-stumps declaration was imminent – but symbolically, left-arm wrist-spinner Kuldeep Yadav serving up full tosses, gleefully dispatched by the 20-year-old, was huge. He was supposed to be a weapon, and England played him with ease.
Playing his first Test of the tour, he struggled to have anywhere near the impact he had in the ODI and T20 series, showing that switching between limited-overs and Test cricket is no easy feat. It also demonstrated the contrasting trajectories of the two sides. India, having won three of the first four games, now face a huge struggle not to lose their fourth in a row.


When you START, COMPLETE and REPEAT a hair program!
receive 3 incredible BONUS GIFTS!
GIFT #1 - Receive a cash back for $57.75 when you START one of our hair programs. Just purchase a 101 Starter Pack ($199.85), register below, and send us your "Before Photos" and receipt of 101 Starter Pack purchase.
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How To Receive Your THREE, Cashback Offer!
1. Complete your details overleaf as soon as possible.
2. Take your "Before Photos". Carefully read the instructions underneath describing how to take your first set of photos. Or you can ask the staff at the outlet you purchased the products if they will take your first set of photos.
3. Send your "Before Photos" to us.
MAIL: Staple your receipt for your 101 Starter Pack to the top, right hand corner of the page overleaf. Mail the form and your "Before Photos" to us at PO Box 138, Ormond, Vic., 3204, Australia.
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This offer is only valid for a) 4 Month Starter Program; b) 4 Month Revive Program; c)8 Month Maintenance Program; or d) 4 Month Alopecia Areata Program.
How Should I Take My First Set Of Photos?
It's very important that you take your photos properly. If your photos are not taken as per the following instructions, it is difficult for us to see if you have received a result with 101 Hair Products.
Ears as close to edge as possible, must see the whole thinning area of scalp, directly in front of subject. Take photo 1.0 meter from subject's head. Separate the hair from the center, making a part in the middle and focus camera shot on most affected area the area.
Ears as close to edge as possible, must see the whole thinning area of scalp, directly behind subject. Take photo 1.0 meter from subject's head. Separate the hair from the center, making a part in the middle and focus camera shot on most affected area.
Nose and back of head close to edge as possible, directly side on, lift hair up with opposite hand to show the thinning area of temple, look up. Take photo 1.0 meter from subject's head and focus camera shot on most affected area.
AFTER PHOTOS" MUST APPROXIMATE "BEFORE PHOTOS" so that we can compare them with accuracy and see if the customer has received a result.
Why is taking my photos essential?
You can only judge if 101 Hair Products have worked for you by comparing your "After Photos" with your "Before Photos" at the end of your 4 Month Hair Program as above. No-one can remember the amount of hair on their scalp prior to treatment.
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* For customers wishing to receive their ongoing Check either $92.25 or $75, only current photos and receipts need to be upload, emailed or mailed to us, as your "Before Photos" will already be at 101's Head Office.


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AccueilTURKISH DRAMACan Yaman and Diletta Leotta, Paolone's backstory on the night at the stadium: 'He was angry'
Can Yaman and Diletta Leotta, Paolone's backstory on the night at the stadium: 'He was angry'
Can Yaman and Diletta Leotta have been at the center of Gossip since the beginning of 2021, that is, since their relationship came to the surface. In the evening of Friday, June 11, the couple went together to the stadium to see the match of the 2020 European Championship Turkey-Italy and the paparazzo Paolone, during an Instagram live with Cristian Alaimo, revealed a backstory about the Turkish actor, known thanks to a friend of his who was at the Olimpico the same evening. According to the photographer Can would have been smiling only when he was being immortalized for social.
Can Yaman at the stadium with Diletta Leotta: Paolone and Alaimo's comments
Can Yaman and Diletta Leotta shared, with millions of followers on social networks, some moments spent during the evening at the Olympic Stadium in Rome. In the videos and in the pictures they published together they appeared happy and very close. The fortune-teller of Ciao Darwin Alaimo, however, during an Instagram live made with the paparazzo Paolone, has reported some reports received from the fans of the Turkish actor, in which it seems that he would have different behavior with the fans, depending on whether he is alone or in the company of Diletta: It will be Diletta who does not want his light obscured?
Can Yaman angry while he is with Diletta Leotta: Paolone's tip
About the evening of June 11th, Paolone revealed that he got a tip, through a friend of his who was at the stadium to watch the match, according to which the protagonist of DayDreamer - Le ali del sogno, while he was with Diletta Leotta, was not in a good mood: ''He was angry this guy''.
The paparazzo then said that the actor would have changed his attitude, showing himself smiling only to take videos and selfies.
Can Yaman continues his relationship with Diletta Leotta
Although there is still someone who doubts the truthfulness of their love story, the relationship between Can Yaman and Diletta Leotta continues at full speed.
In the last period they enjoyed a weekend in a luxury hotel on Lake Como, they went on vacation to the Amalfi Coast and Sicily at her family's house. The Turkey-Italy game at the stadium was just the last of the moments when the two lovers were seen happy and smiling together. All that remains is to wait for the next moves of the couple, to find out what will be their next stop.