BMU, osteoclasts, osteoblasts, molecular biology of bone multi cellular unit..

1. Metabolic Bone Diseases Molecular Biology Vinod Naneria Consultant orthopaedic surgeon Choithram Hospital & Research Centre Indore , India

5. Bone Anatomy

6. Osteogenic cells

8. Osteocyte with Cytoplasmic Extensions

11. In this photo you can actually see the contact between an osteoclast. Osteoclasts produce hydrogen ions that acidify and dissolve the bone surface, as well as hydrolytic enzymes osteoclast at breakfast, basically shows an osteoclast with some similarities to a snail- leaving behind not a trail of mucus but rather of eaten bone.

14. Type-I collagen is the most abundant collagen of the human body. It is present in scar tissue, the end product when tissue heals by repair. It is found in tendons, myofibrils and the bone, cartilage, skin and sclera. Collagen is a protein that strengthens and supports many tissues.

17. Leptin Sclerostin

18. Core-binding factor A1 (CBFA1, also called Runx2), is a transcription factor expressed specifically in osteoblast progenitors, as well as in mature osteoblasts. Runx2 regulates the expression of several important osteoblast proteins including osterix (another transcription factor needed for osteoblast maturation), osteopontin, bone sialoprotein, type I collagen, osteocalcin, and receptor-activator of NFB (RANK) ligand. Runx2 expression is regulated, in part, by bone morphogenic proteins (BMPs). Runx2-deficient mice are devoid of osteoblasts. whereas mice with a deletion of only one allele (Runx2 +/–) exhibit a delay in formation of the clavicles and some cranial bones. The latter abnormalities are similar to those in the human disorder cleidocranial dysplasia, RUNX2

20. Macrophage colony-stimulating factor (M-CSF) plays a critical role during several steps in the pathway and ultimately leads to fusion of osteoclast progenitor cells to form multinucleated, active osteoclasts. RANK ligand, a member of the tumor necrosis factor (TNF) family, is expressed on the surface of osteoblast progenitors and stromal fibroblasts. In a process involving cell-cell interactions, RANK ligand binds to the RANK receptor on osteoclast progenitors, stimulating osteoclast differentiation and activation. Several growth factors and cytokines (including interleukins 1, 6, and 11; TNF; and interferon) modulate osteoclast differentiation and function. M-CSF,TNF,RANK, IL 1,6,11, & Interferon

22. hormones that influence osteoclast function do not directly target this cell but instead influence M-CSF and RANK ligand signaling by osteoblasts. Both PTH and 1,25(OH) 2 D increase osteoclast number and activity, estrogen decreases osteoclast number and activity by this indirect mechanism. Calcitonin, in contrast, binds to its receptor on the basal surface of osteoclasts and directly inhibits osteoclast function .

23. Maturation Pathway Osteoblasts Osteoclasts

24. Sequence of bone repair

36. + promote - inhibits

37. Wnt/beta-catenin signaling is involved in bone biology. Wnt autocrine loop mediates the induction of alkaline phosphatase and mineralization by BMP-2 in pre-osteoblastic cells. Loss of function of LRP5 leads to osteoporosis (OPPG syndrome), and a specific point mutation in this same receptor results in high bone mass (HBM). LRP5 acts as a coreceptor for Wnt proteins, and plays crucial role for Wnt signaling in bone biology. In mesenchymal cells, only Wnt's capable of stabilizing beta-catenin induced the expression of alkaline phosphatase (ALP). BMP-2 + alkaline phosphatase + mineralization + Wnt autocrine loop.

38. Canonical beta-catenin signaling is a responsible factor for inhibition of Wnt-3 activity. The induction of ALP by Wnt is independent of morphogenetic proteins and does not require de novo protein synthesis. Blocking Wnt/LRP5 signaling or protein synthesis inhibited the ability of both BMP-2 and Shh to induce ALP in mesenchymal cells. BMP-2 enhanced Wntl and Wnt3a expression in cells. The capacity of BMP-2 and Shh to induce ALP relies on Wnt expression and the Wnt/LRP5 signaling cascade. Moreover the effects of BMP-2 on extracellular matrix mineralization by osteoblasts are mediated, at least in part, by the induction of a Wnt autocrine/paracrine loop. BMP-2 + alkaline phosphatase + mineralization + Wnt autocrine loop.

43. Osteopontin (OPN) is a highly phosphorylated sialoprotein that is a prominent component of the mineralized extracellular matrices of bones. OPN is characterized by the presence of a polyaspartic acid sequence and sites of Ser/Thr phosphorylation that mediate hydroxyapatite binding, and a highly conserved RGD motif that mediates cell attachment/signaling. Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. OPN is a required osteoclast motility factor mediating surface expression of CD44 receptor. Also, OPN secreted into the resorption pit is required for adhesion during bone resorption. Osteopontin (OPN) – adhesion to matrix

44. Known immunologic functions of OPN. OPN binds to several integrin receptors including α4β1, α9β1, and α9β4 expressed by leukocytes and are known to induce cell adhesion, migration, and survival in immune cells including neutrophils, macrophages, T cells, mast cells, and osteoclasts.

45. The fact that OPN interacts with multiple cell surface receptors which are ubiquitously expressed makes it an active player in many physiological and pathological processes including wound healing, bone turnover, tumorigenesis, inflammation, ischemia and immune responses. Therefore, manipulation of plasma OPN levels may be useful in the treatment of autoimmune diseases, cancer metastasis, osteoporosis and some forms of stress. OPN has been implicated in pathogenesis of rheumatoid arthritis. Researchers found that OPN-R, the thrombin-cleaved form of OPN, was elevated in the rheumatoid arthritis joint. It was found that OPN knock-out mice were protected against arthritis, while others were not able to reproduce this observation. OPN has been found to play a role in other autoimmune diseases including autoimmune hepatitis, allergic airway disease, and multiple sclerosis. It has been shown that OPN drives IL-17 production; OPN is over expressed in a variety of cancers, including lung cancer, breast cancer, colorectal cancer, stomach cancer, ovarian cancer, melanoma and mesothelioma; OPN contributes both glomerulonephritis and tubulointestinal nephritis; and OPN is found in atheromatous plaques within arteries. Thus, manipulation of plasma OPN levels may be useful in the treatment of autoimmune diseases, cancer metastasis, osteoporosis and some forms of stress. Research has implicated osteopontin in excessive scar-forming and a gel has been developed to inhibit its effect.

47. Normal Multinucleated Osteoclast Tightly Adherent to Bone

48. Sealing mechanism A number of cell surface glycoproteins have been identified as intercellular adhesion molecules, and these have been classified into at least three major molecular families, the immunoglobulin (Ig) superfamily, the integrin superfamily, and the cadherin family. Integrins have been identified as a family of cell surface receptors that recognize extracellular matrices. Integrins are adhesion molecules that mediate cell attachment to the substrate by binding to an arginine-glycine-aspartic acid (RGD) consensus sequence in their ligands . Osteoclasts express two members of integrin superfamily, the vitronectin receptor α v β 3 and α 2 β 1 . It has been suggested that vitronectin receptor mediates the tight attachment of osteoclasts to bone matrix and that osteopontin, a bone matrix component containing the RGD sequence is the ligand of the osteoclast vitronectin receptor. Nevertheless, VNR has also been shown to be located only in the ruffled border, basolateral membranes and intracellular vesicles of osteoclasts, but missing from the area of the tight sealing zone .

50. Integrins are receptors that mediate attachment between a cell and the tissues surrounding it, which may be other cells or the extracellular matrix (ECM). They also play a role in cell signaling and thereby define cellular shape, mobility, and regulate the cell cycle. Typically, receptors inform a cell of the molecules in its environment and the cell evokes a response. Not only do integrins perform this outside-in signalling, but they also operate an inside-out mode. Thus, they transduce information from the ECM to the cell as well as reveal the status of the cell to the outside, allowing rapid and flexible responses to changes in the environment, for example to allow blood coagulation by platelets. Integrins

53. Resorption cycle Functional Osteoclast

54. Resorbing osteoclasts are highly polarized cells. osteoclasts contain not only the sealing zone but also at least three other specialized membrane domains: a ruffled border, a functional secretory domain and a basolateral membrane. Resorption requires cellular activities: migration of the osteoclast to the resorption site, its attachment to bone, polarization and formation of new membrane domains, dissolution of hydroxyapatite, degradation of organic matrix, removal of degradation products from the resorption lacuna, and finally either apoptosis of the osteoclasts or their return to the non-resorbing stage. α vb3 is highly expressed in osteoclasts and is found both at the plasma membrane and in various intracellular vacuoles. The integrin could play a role both in adhesion and migration of osteoclasts and in endocytosis of resorption products. High amounts of α vb3 are present at the ruffled border and denatured type I collagen has a high affinity for α vb3. RESORPTION CYCLE - Summary

55. The ruffled border is a resorbing organelle, and it is formed by fusion of intracellular acidic vesicles with the region of plasma membrane facing the bone. During this fusion process much internal membrane is transferred, and forms long, finger-like projections that penetrate the bone matrix. Several late endosomal markers, such as Rab7, Vtype H-ATPase and lgp110, are densely concentrated at the ruffled border. Basolateral domain of the resorbing osteoclast is divided into two distinct domains and that the centrally located domain is an equivalent to the apical membrane of epithelial cells. the basal membrane represents homogeneous membrane area. The apical domain (also known as the functional secretory domain) in this unexpected site might function as a site for exocytosis of resorbed and transcytosed matrix-degradation products. Before proteolytic enzymes can reach and degrade collageneous bone matrix, tightly packed hydroxyapatite crystals must be dissolved. The dissolution of mineral occurs by targeted secretion of HCl through the ruffled border into the resorption lacuna. This is an extracellular space between the ruffled border membrane and the bone matrix, and is sealed from the extracellular fluid by the sealing zone . The Ruffled Border

56. The low pH in the resorption lacuna is achieved by the action of ATP-consuming vacuolar proton pumps both at the ruffled border membrane and in intracellular vacuoles. Acidic extracellular compartments lie beneath the resorbing cells and also that there is a high density of acidic intracellular compartments inside non-resorbing Osteoclasts . Concomitant with the appearance of the ruffled border, the number of intracellular acidic compartments promptly decreases as the vesicles containing proton pumps are transported to the ruffled border. resorption lacuna is further acidified by direct secretion of protons through the ruffled border. Protons for the proton pump are produced by cytoplasmic carbonic anhydrase II, high levels of which are synthesized in osteoclasts. Excess cytoplasmic bicarbonate is removed via the chloride-bicarbonate exchanger located in the basolateral membrane. There is a high number of chloride channels in the ruffled border, which allows a flow of chloride anions into the resorption lacuna to maintain electroneutrality . Ion channel pumps

57. After solubilization of the mineral phase, several proteolytic enzymes degrade the organic bone matrix, two major classes of proteolytic enzymes, lysosomal cysteine proteinases and matrix metalloproteinases (MMPs), The high levels both of expression of MMP-9 (gelatinase B) and cathepsin K and of their secretion into the resorption lacuna suggest that these enzymes play a central role in the resorption process. After matrix degradation, the degradation products are removed from the resorption lacuna through a transcytotic vesicular pathway from the ruffled border to the functional secretory domain, where they are liberated into the extracellular space. Tartrate-resistant acid phosphatase (TRAP), is localised in the transcytotic vesicles of resorbing osteoclasts, and that it can generate highly destructive reactive oxygen species able to destroy collagen. This activity, together with the co-localisation of TRAP and collagen fragments in transcytotic vesicles, suggests that TRAP functions in further destruction of matrix-degradation products in the transcytotic vesicles . MMP-9 (gelatinase B) and cathepsin K

58. Transcytosis osteoclasts remove the degradation products of bone matrix from the resorption lacuna by transcytosis. The degradation products, both organic and inorganic, are endocytosed to the resorbing osteoclast, transported through the cell in large vesicles, and finally released into the extracellular space through the FSD. This enables that osteoclasts can remove large amounts of degradation products via transcytosis without detaching from the bone surface and loosing the tight attachment of the sealing zone to the bone surface. This further supports the osteoclastic penetration deeper into the bone. The transcytosis route provides a possibility for osteoclasts to further process the endocytosed degradation products intracellularly during their passage through the cell. The bone-specific enzyme TRAP is located in cytoplasmic vesicles, which fuse to the transcytotic vesicles and participates in destroying the endocytosed material in the transcytotic route .

59. Osteoclasts resorb bone by attaching to the surface and then secreting protons into an extracellular compartment formed between osteoclast and bone surface. This secretion is necessary for bone mineral solubilization and the digestion of organic bone matrix by acid proteases. The primary mechanism responsible for acidification of the osteoclast-bone interface is vacuolar h +- Adenosine triphosphatase (atpase) coupled with cl− conductance localized to the ruffled membrane. Carbonic anhydrase II (CAII) provides the proton source for extracellular acidification by H+-atpase and the HCO3− source for the HCO3−/cl− exchanger. Whereas some transporters are responsible for the bone resorption process, others are essential for ph regulation in the osteoclast. The Ph regulation in Osteoclasts

60. The Ph regulation in Osteoclasts The HCO3−/cl− exchanger, in association with CAII, is the major transporter for maintenance of normal intracellular ph. An na+/H+ antiporter may also contribute to the recovery of intracellular ph during early osteoclast activation. Once this mechanism has been rendered inoperative, another conductive pathway translocates the protons and modulates cytoplasmic ph. Inward-rectifying K+ channels may also be involved by compensating for the external acidification due to H+ transport. These different effects of transport processes, either on bone resorption or ph homeostasis, increase the number of possible sites for pharmacological intervention in the treatment of metabolic bone diseases.

61. Osteoclasts resorb bone by generating a pH gradient between the cell and bone surface . An acidic pH favors the dissolving of the bone mineral (hydroxyapatite) and provides optimal conditions for the action of the proteinases secreted by osteoclasts. Inorganic bone matrix is dissolved by the acidic environment in the resorption lacuna revealing the organic collagen network. Carbonic anhydrase II (CA II) is a cytoplasmic enzyme hydrolyzing carbon dioxide into bicarbonate and protons . CA II is suggested to be the main source of protons for the acidification of the resorption lacuna . A vacuolar-type proton pump, V-ATPase, is present in high amounts in the membranes of a population of intracellular vesicles. V-ATPase transports the protons generated by CA II into these vesicles, which are then transported and fused to the RB membrane releasing their proton content to the lacuna. . Carbonic anhydrase II (CA II)

62. Acidification of the extracellular resorption lacuna is completed by passive, potential-driven chloride transport . RB thus contains large amounts of V-ATPase, which probably continues to function by transporting protons directly from the cytoplasm to the lacuna. Hydroxyapatite is first solubilized in the acidified lacuna, after which various proteolytic enzymes, such as lysosomal enzymes and bone-derived collagenases secreted by osteoclasts through RB, digest the exposed organic matrix. When the osteoclast stops resorption and moves away from the resorption lacuna, phagocytes clean up the remains and make room for osteoblasts to begin bone formation in the newly formed resorption cavity. Carbonic anhydrase II (CA II)

64. Regulation of osteoclastic bone resorption A balance between bone formation and bone resorption is a necessity for the normal function of bone. Although many acquired or environmental factors are known to affect the adult bone mineral density (BMD) , genetic factors play a major role as determinants of variation in BMD . It has been estimated that up to 80% of BMD is genetically controlled, and it is the rate of bone formation rather than the rate of bone resorption that is influenced by genes . Systemic stimulators of bone resorption include PTH, interleukin-1, tumor necrosis factor , transforming growth factor α and 1,25-dihydroxyvitamin D 3 . On the other hand, calcitonin, gamma interferon and transforming growth factor β are extremely potent in inhibiting osteoclast differentiation and activity. PTH and D 3 stimulate bone resorption by increasing the activity of existing osteoclasts and by promoting the differentiation of osteoclast precursors into mature multinucleated osteoclasts and D 3 or other vitamin D metabolites seem to play a role in the correction of calcium malabsorption, which is a common feature in osteoporosis . It has also been shown that peak bone mass is associated with vitamin D receptor polymorphism and is also related to the rate of bone loss. Osteoblasts mediate the effects of PTH and D 3 on the osteoclasts . Calcitonin, regulates blood calcium and phosphate levels by causing short-lived falls in the plasma calcium. It does that by its effects to inhibit osteoclastic bone resorption and to promote renal calcium excretion .

66. Bone Resorption

79. calcium phosphates were formed through a multistage assembly process, during which an initial amorphous phase DPCD was followed by a phase transformation into a crystalline phase and then the most stable hydroxyapatite (HAp). This provided new insights into the template−biomineral interaction and a mechanism for biomineralization. Maturation of Mineralization

83. Life cycle of Osteoclast

86. The resorbing osteoclast is an exceptional cell that secretes large amounts of acid through the coupled activity of a v-type H(+)-ATPase and a chloride channel that both reside in the ruffled membrane. Impairment of this acid secretion machinery by genetic mutations can abolish bone resorption activity, resulting in osteopetrotic phenotypes. Another key feature of osteoclasts is the transport of high amounts of calcium and phosphate from the resorption lacuna to the basolateral plasma membrane. Evidence exists that this occurs in part through entry of these ions into the osteoclast cytosol. Handling of such large amounts of a cellular messenger requires elaborate mechanisms. Membrane proteins that regulate osteoclast calcium homeostasis and the effect of calcium on osteoclast function and survival are of clinical interest. Osteoclasts

88. How is the close pairing of the osteoclast and osteoblast activity regulated?

89. Bone Remodeling. The remodeling process of bone comprises the coupled activity of bone resorbing osteoclasts and bone forming osteoblasts. This system is tightly controlled by a number of soluble regulatory factors and through cellular interactions within the bone microenvironment.

90. The link between bone resorption by osteoclasts to subsequent bone formation by osteoblasts. The coupling factor is derived from one or many growth factors such as IGF-I and -II and TGF-β that are released by osteoclastic proteolytic digestion of bone matrix during bone resorption, and are thus made available for stimulating osteoblast precursors to form osteoblasts and new bone. Osteoclasts secrete anabolic growth factors that mediate osteoblast chemotaxis, proliferation, differentiation, and mineralization. Some of the osteoclast-secreted factors that may enhance osteoblast activity include TGF-β , IGF-1, TRAP (tartrate-resistant acid phosphatase), and BMPs. "coupling factor“ – Change over

91. BMPs are members of the TGF-β superfamily that have highly conserved seven-cysteine repeats in their carboxy terminus. BMPs have well-established roles in pattern formation, organogenesis, and skeletal morphogenesis during vertebrate development. At the cellular level, BMPs regulate cell proliferation, differentiation, and apoptosis in embryonic and postnatal chondrocytes, osteoblasts, and osteoclasts. BMP dimers bind to one of the two types of serine and threonine kinase membrane receptors, and ligand receptor binding initiates an intracellular signaling cascade mediated by Smad proteins, ultimately leading to regulation of target genes. BMPs are thought to be key regulators of embryonic skeletogenesis, endochondral ossification, bone remodeling, fracture repair, and bone regeneration. Role of BMPs

94. Induction of sphingosine kinase 1 (SPHK1), which catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), in mature multinucleated osteoclasts as compared with preosteoclasts. S1P induces osteoblast precursor recruitment and promotes mature cell survival. Wnt10b and BMP6 also were significantly increased in mature osteoclasts, whereas sclerostin levels decreased during differentiation. Stimulation of hMS cell nodule formation by osteoclast conditioned media was attenuated by the Wnt antagonist Dkk1, a BMP6-neutralizing antibody, and by a S1P antagonist. BMP6 antibodies and the S1P antagonist, but not Dkk1, reduced osteoclast conditioned media-induced hMS chemokinesis. .

95. Sphingolipids are a class of lipids derived from the aliphatic amino alcohol sphingosine. They play important roles in signal transmission and cell recognition. Sphingolipidoses, or disorders of sphingolipid metabolism, have particular impact on neural tissue. Sphingolipids are believed to protect the cell surface against harmful environmental factors by forming a mechanically stable and chemically resistant outer leaflet of the plasma membrane lipid bilayer.

99. RANKL and OPG are also important regulators of vascular biology and calcification and of the development of a lactating mammary gland during pregnancy, indicating a crucial role for this system in extraskeletal calcium handling. The discovery and characterization of RANKL, RANK, and OPG and subsequent studies have changed the concepts of bone and calcium metabolism, have led to a detailed understanding of the pathogenesis of metabolic bone diseases, and may form the basis of innovative therapeutic strategies.

100. Factors that modulate osteoblast function during development fall in two categories. The first category includes those that have direct effects on osteoblast function, such as the bone morphogenetic proteins (BMPs); transforming growth factor-β1 (TGFβ1) and TGFβ2; insulin-like growth factor 1 (IGF1) and IGF2; fibroblast growth factor (FGF); platelet-derived growth factor (PDGF); and WNT. The second category includes those that affect osteoblast function indirectly, by modifying the bone microenvironment, such as vascular endothelial growth factor (VEGF). All these factors regulate osteoblast function by activating signalling pathways involved in the regulation of osteoblast proliferation and differentiation. A transcription factor essential for osteoblast differentiation is RUNX2 (also called core binding factor 1). In mice, inactivation of Runx2 results in formation of a skeleton composed entirely of cartilage without normal bone, indicating that RUNX2 is essential for bone formation. Another transcription factor that controls osteogenesis is osterix . Mice without osterix do not develop bones. BMP2 and FGF have been shown to stimulate osteoblast function by activating RUNX2 and BMP2 also upregulates osterix expression. Osteoblast as target cell for research

101. Signal-transduction pathways that regulate osteoblast function. Binding of bone morphogenetic protein (BMP) to its receptor induces the formation of a complex in which the type II BMP receptor phosphorylates and activates the type I BMP receptor. The type I BMP receptor then propagates its signal by phosphorylating the SMAD1 and SMAD5 proteins. Phosphorylation of the SMAD proteins leads to the upregulation of RUNX2 and osterix, two transcription factors that control osteogenesis. BMP2 activate p38 mitogen-activated protein kinase (MAPK), leading to an increase in RUNX2 transcription. TGFβ regulates RUNX2 transcription by phosphorylating SMAD2 and SMAD3 as well as by activating p38 MAPK. Fibroblast growth factors (FGFs) signal through a group of high-affinity transmembrane receptors (FGFR1 to FGFR4), which have intrinsic tyrosine kinase activities. Insulin-like growth factor 1 (IGF1) and endothelin-1 (ET1), which bind to the receptor tyrosine kinases IGF1R and G-protein-coupled receptor ETA, respectively, have both been shown to activate the MAPK pathway in osteoblasts. Osteoblast as target cell for research

102. Signal transduction from these growth factors results in the activation of RUNX2 and/or osterix. In osteoblasts, the interactions between FGF2 and its receptors induce dimerization and autophosphorylation of these receptors, which in turn activate p42/44 MAPK and protein kinase C (PKC). Activation of PKC leads to an increase in RUNX2 transcription, whereas phosphorylation and activation of p42/44 MAPK leads to RUNX2 protein phosphorylation and activation. Activation of RUNX2 and/or osterix leads to increased expression of osteoblast-specific genes, such as alkaline phosphatase and osteocalcin. This results in increased bone formation. The WNT proteins, on the other hand, interact with WNT receptor frizzled and co-receptor LRP5 or LRP6 to activate a signalling pathway that stabilizes cytoplamic β-catenin. Stabilized β-catenin is then translocated to the nucleus to regulate as-yet-unidentified genes that promote bone formation. Osteoblast as target cell for research

105. Proposed model for the role of Hh and canonical Wnt signaling in regulating the differentiation of skeletal progenitors.

106. canonical Wnt signaling pathway

107. The formation process of inorganic crystals in biological systems (biomineralization) is the fruitage of an extended period of fine-tuning by evolution and is replete with material scientific key considerations. It has been known that inorganic crystals precipitate onto organic matrix surfaces in biomineralization processes. In bone formation, osteoblasts first secrete the proteins of the bone matrix, or osteoid, which acquires mineral after forming as a histologically distinct layer. Several proteins have been identified with the property of inhibiting matrix mineralization, suggesting that the potential for precipitation of mineral is inherent in the physiological milieu, and that a counterbalancing inhibition is required to prevent inappropriate formation of insoluble crystals. Steps of mineralization

108. Three kinds of Langmuir monolayers formed by dipalmitoylphosphatidylcholine (DPPC), arachidic acid (AA), and octadecylamine (ODA) were used as templates to study the initial stage of nucleation and crystallization of calcium phosphates. It was demonstrated that the combination of calcium ions (or phosphates) to the monolayer/subphase interface is a prerequisite for subsequent nucleation. It was found that calcium phosphate dihydrate (DPCD) formed at 25.0 °C for 12 h has a biphasic structure containing both amorphous and crystalline phases. These results showed that calcium phosphates were formed through a multistage assembly process, during which an initial amorphous phase DPCD was followed by a phase transformation into a crystalline phase and then the most stable hydroxyapatite (HAp). This provided new insights into the template−biomineral interaction and a mechanism for biomineralization. Ca+ PO4 Amorphous Calcium phosphate Crystalline Calcium phosphate

110. in the past decades, it has become apparent that the skeleton is a major target tissue for reproductive steroid hormones, estrogen, androgen, testosterone, and to a lesser extent, progestin. The sex steroids, especially estrogen and testosterone, play a major role in the sexual dimorphism of the skeleton in mineral homeostasis during reproduction and bone balance in adults. Estradiol and progesterone are the principal circulating sex steroids in females but also function in males . Sex steroids are essential for maintenance of normal bone volume and estrogen deficiency is an important risk factor for osteoporosis. Ovariectomy leads to a deficit in ash weight and bone mineral density in adult rats and monkeys and it is generally believed that treatment with estrogen of ovariectomized rats prevents the increase of bone resorption. Reproductive steroid hormones

111. Vacuolar ATPase (V-ATPase) has been proposed as a drug target in lytic bone diseases. Studies of Bafilomycin derivatives suggest that the key issue regarding the therapeutic usefulness of V-ATPase inhibitors is selective inhibition of osteoclast V-ATPase. Previous efforts to develop therapeutic inhibitors of osteoclast V-ATPase have been frustrated by a lack of synthetically tractable and biologically selective leads. Therefore, we tried to find novel potent and specific V-ATPase inhibitors, which have new structural features and inhibition selectivity, from random screening using osteoclast microsomes. Finally, a novel V-ATPase inhibitor, FR167356, was obtained through chemical modification of a parental hit compound. A novel inhibitor of vacuolar ATPase, FR167356, which can discriminate between osteoclast vacuolar ATPase and lysosomal vacuolar ATPase Kazuaki Niikura, Mikiko Takano, and Masae Sawada

112. Human bone marrow-derived mesenchymal stem cells (MSCs) represent an ideal source for cell therapy for inherited and degenerative diseases, bone and cartilage repair, and as target for gene therapy. The role of the combination of human parathyroid hormone (PTH) and vitamin D 3 in bone formation and mineralization has been established in several osteoblast cell culture studies. Human MSC derived from adult normal bone marrow that are positive for CD29, CD44, CD105, and CD166 and negative for CD14, CD34, and CD45, were treated with the PTH and 1,25-dihydroxyvitamin D 3 in the presence and absence of recombinant human BMP-4 or BMP6. PTH and vitamin D 3 induced high levels of expression of two key markers of bone formation: osteocalcin and alkaline phosphatase by MSCs. BMP-6 but not BMP-4 increased osteocalcin expression induced by PTH and vitamin D 3 . Both BMPs enhanced calcium formation in MSC cultures and this response was potentiated by PTH and vitamin D 3 . The present results revealed a novel potent effect of PTH and vitamin D 3 plus BMPs in inducing bone development by human MSCs. These results may facilitate therapeutic utility of MSCs for bone disease and help clarify mechanisms involved in stem cell-mediated bone development. Mesenchymal Stem cells Therapy(MSCs)

113. Denosumab is the first fully human monoclonal antibody in late stage clinical development that specifically targets RANK Ligand, an essential regulator of osteoclasts. Denosumab is being investigated for its potential to inhibit all stages of osteoclast activity through a targeted mechanism. Denosumab is being studied in a range of bone loss conditions including PMO, rheumatoid arthritis, and bone loss in patients undergoing hormone ablation for prostate and breast cancer, as well as for its potential to delay bone metastases and inhibit and treat bone destruction across many stages of cancer . Denosumab – Antibody against RANK-L

115. Bon Voyage