a comprehensive details of functioning of BMU

1. Metabolic Bone DiseasesThe remodeling sequence Vinod Naneria Consultant orthopaedic surgeon Choithram Hospital & Research Centre Indore , India

2. Basics – Remodeling stages There are more than 25 recognized steps involved in one cycle of remodeling. BMU Cells activation, Resorption of Matrix and Minerals –2 -3 Wks. Re activation of Bone forming unit or reversal, New bone formation Mineralization start after 2 weeks Maximum mineralization 3 - 12 months Maturation takes up to 3 years.

3. Basics… Bone loss through osteoclast-mediated bone resorption and bone replacement through osteoblast-mediated bone formation are tightly coupled processes. Resorbed bone is precisely replaced in location and amount by new bone.

4. Basics… Osteoblasts direct osteoclasts differentiation. Osteoclasts play a crucial role in the promotion of bone formation. Osteoclast conditioned medium stimulates human mesenchymal stem (hMS) cell migration and differentiation toward the osteoblast lineage as measured by mineralized nodule formation in vitro.

5. Bone remodeling - sequences Micro fracture – Mechanical stress Signals to brain, Activation of stem cells from mesenchymal origin, Maturation of pre osteoblasts, Maturation of pre osteoclasts, Rank-L secretion by osteoblast, Fusion of pre osteoclast in to a multi nucleated mature Osteoclast, Osteoclast adherence to hydroxyapatite crystals, Formation of sealing mechanism, Formation of podosomes or Ruffled border

6. 11. Matrix absorption, 12. Mineral absorption 13. Trans cytoplasmic calcium transportation to ECF, 14. Ph regulation at ruffled border, 15. Formation of lacunae, 16. Reversal to bone formation 17. Cross talk with mature osteoblasts, (coupling) 18. Separation/ migration or apoptosis of osteoclasts, 19. Role of osteoprotegerin, 20. Migration of osteoblasts in the lacunae, 21. Secretion of new matrix, 22. Role of Sclerostatins, 23. Mineralization of matrix, 24. Role of Inorganic phosphates in regulation of mineralization, 25. Maturation of bone architecture,

7. Micro-crackSequence of bone repair

9. Quiescent stage This is a quiescent bone surface. The embedded osteocytes in the bone is actively secreting “Sclerostin”, a protein which inhibits Wnt-signaling in cells near the surface. The preosteoclasts are circulating in the blood vessels. Wnt – siganaling is the key for osteogenesis.

10. The photograph is showing a micro-crack due to sudden stress. Osteocytes are alive

11. Osteocytes Osteocytes are the mechanosensory cells of bone, play a pivotal role in functional adaptation of bone. Periosteocytic space filled with Extracellular fluid. Sensation of mechanical load is perceived as fluid shear stress on bone surface. apoptosis of Osteocytes generate signals that activate osteoclast resorption.

12. Detection of the crack - micro fracture The osteocyte functions as a mechanoreceptor that translates mechanical stimuli into biochemical signals. The surrounding osteocytes detect strain and start secreting growth factors, prostaglandins and nitric oxide. The expression of PGE2 by loaded osteocytes and the consequent activation of the cAMP/PKA pathway, together with stabilization of β-catenin, may permit cross talk with the canonical Wnt/β-catenin pathway, the activation of which is increased by the load-dependent reduction of the expression of DKK1 and of sclerostin.

13. Detection of the crack - micro fracture These signal pathways, as well as TGF-β and BMP expression, stimulate the differentiation and activity of osteoblasts, reduce their apoptosis, and enhance bone formation. The expressions of MEPE, FGF-23 and DMP-1 further contribute to the osteocytic control of bone metabolism. Periosteocytic osteolysis, if definitively demonstrated, would constitute another fundamental function of osteocytes.

14. Due to crack, osteocytes near the surface goes in to apoptosis.

15. Crack Death of Osteocytes

16. Canopy The lining cells pull away from the bone matrix and form a canopy which mergers with the blood vessels. Some of the canopy cells look like pre-osteoblasts. The stromal cells are now released from the inhibitory effect of Sclerostin and are exposed to the factors like IL-1. They generate pre-osteoblasts. Stromal cells also secrete M-CSF ( macrophage colony stimulating factor) which help generate pre-osteoclast.

17. Canopy

18. Sclerostin Sclerostin produced by the osteocytes It blocks the mineralization beyond lining cells. Defects in the SOST gene -absence or reduced production of Sclerostin, causes Sclerosteosis and Van Buchem diseases, hypertrophic bones which are fracture resistant. Sclerostin binds to LRP5 and antagonizes the Wnt pathway.

20. Preosteoblast proliferation

21. Pre Osteoblast The pre-osteoblasts proliferate and secrete more factors, such as Wnt, ILs, and BMPs. All these factors are responsible for maturation of osteoblasts.

22. Wnt – Osteoblast maturation Signals originating from members of the wnt (wingless-type mouse mammary tumor virus integration site) family of paracrine factors are important. Paracrine is a “inter cellular communication” system. The central role of Wnt signaling is in regulating osteoblast lineage specification, expansion, and terminal differentiation. Humans and mice missing a wnt-family co-receptor, LRP5 (lipoprotein receptor–related protein 5), have osteoporosis. Remarkably, humans with an overactive form of LPR5 have increased bone mass.

23. +promote Osteogenesis Chondrogenesis Adipogenesis -inhibits

24. Osteoblast maturation BMP-2 + ALP + Mineralization + Wnt autocrine loop 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. LRP5 acts as a co-receptor 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).

25. BMP-2 + ALP + Mineralization + Wnt autocrine loop 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.

26. Rank-L secretion

27. RANK =Receptor activator for nuclear factor kb The pre-osteoblasts start to express RANK-L on their surfaces. The pre-osteoclasts have RANK receptors on their surface. For maturation of pre-osteoclast into osteoclast RANK-L play a crucial role. RANK is a member of TNF family of receptors expressed mainly on cells of macrophages / monocytes lineage such as Pre-osteoclasts When this receptor binds its specific ligand (RANK-L) through cell- cell contact , osteoclastogenesis is initiated.

28. RANK-L RANK-L is produced by and expressed on the cell membranes of osteoblast & marrow stromal cells Its major role is stimulation of osteoclast formation , fusion, differentiation, activation , survival. In chemistry, a ligand is either an atom, ion, or molecule (functional group) that binds to a central metal to produce a coordination complex.

29. The discovery and characterization of RANK-L, 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. With the appearance of RANK-L, the pre-osteoclasts start fusing with each other and maturing into a functional multi-nucleated “Osteoclast” cell. RANK & RANK - L

30. RANK – L + RANK Osteoclast Maturation

31. Resorption Cycle From the formation of functional osteoclasts to resorption of bone matrix and excretion of degraded products into extra cellular spaces, it needs a step wise “resorption cycle”. This cycle starts with the death of osteocytes due to stress or strain and ends with the reinstallation of “osteoblasts” in the resorption pits.

32. Resorption Cycle The non-mineralized osteoid covers the mineralized bone matrix in vivo, preventing its resorption by osteoclasts. Thus, before osteoclasts can begin the actual resorption in vivo, the osteoid must be dissolved or mineralized, after which osteoclasts can attach to the mineralized matrix and initiate bone resorption. Proteases released by osteoblastic cells have been shown to be responsible for dissolving the osteoid and thereby inducing osteoclastic resorption and determining its location

33. Osteopontin (OPN) – adhesion to matrix 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.

34. Osteopontin (OPN) – adhesion to matrix 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.

35. Osteoclast - Sealing Zone In the sealing zone, also called as clear zone, the cell membrane forms a tight attachment to the bone surface, thereby isolating the resorption lacuna from the extracellular fluid and permitting the maintenance of a specific microenvironment in the lacuna. The sealing zone is seen as an electro-dense area free of organelles and it has a striated appearance in electron microscopy, with alternating dark and light areas orientated perpendicular to the bone surface, consisting of bundles of actin filaments. Ruffled border (RB) is a highly convoluted plasma membrane domain under which the actual bone resorption takes place.

36. Sealing Zone & Ruffled Border (RB) Sealing zone surrounds the RB, thus defining the outlines of the resorption lacuna. Acidic intracellular vesicles fuse to the cell membrane facing the bone matrix, thereby forming RB membrane. RB does not fit to any type of previously described plasma membrane domains, because it has both lysosomal and endosomal features. Bone degradation occurs in the extracellular space between the bone matrix and the RB, which is called the resorption lacuna. In the middle of the basolateral membrane is the fourth membrane domain, the functional secretory domain (FSD), which appears when matrix degradation is started.

37. Osteoclastic membrane domains Polarization Sealing zone or clear zone (SZ) Ruffled border (RB) Basolateral membrane (BLM) Functional secretory domain (FSZ)

38. Mature Osteoclast

39. Normal Multinucleated Osteoclast Tightly Adherent to Bone

40. Normal Multinucleated Osteoclast tightly adherent to bone. The interface with the bone perimeter is marked by a ruffled or striated border of micro-villi (white arrow), which is best visualized by electron microscopy. The osteoclast–bone interface creates a unique extracellular microenvironment, ideal for bone resorption. The lysosome-rich vacuolar zone (black arrow), composed of acidified vesicles containing Cathepsin K and matrix metalloproteinases, is distinctly visualized. Polarized away from the bone surface are six nuclei with prominent nucleoli.

41. Sealing mechanism A number of cell surface glycoproteins have been identified as intercellular adhesion molecules. These are classified into three major molecular families, the Immunoglobulin (Ig) super-family, 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 .

42. Sealing mechanism - Vitronectin receptor Osteoclasts express two members of Integrin super family, the Vitronectin receptor αvβ 3 and α2β 1. 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. 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 .

43. Vitronectin receptor The first indications of the importance of vitronectin receptor came from the experiments in which monoclonal antibodies against osteoclasts, later identified as anti-vitronectin receptor antibodies, inhibited osteoclastic bone resorption in vitro. It has been shown that peptides containing the RGD sequence potentially inhibit bone resorption. It has also been shown that osteoclasts adhere in vitro to a wide range of RGD peptide containing proteins, including bone sialoproteins, via a β 3 integrin.

44. Bone Absorption The osteoclasts bind to the bone matrix with Integrins. They secrete acid and Cathepsin K at its ruffled border to absorb the bone. Bone re-absorption at this spot take about 2 weeks. Bone – derived growth factors IGF & TGF-b are released.

45. Bone Absorption

47. Bone absorption At this stage osteoclast actually transforms in to multi-functional unit with three different types of cytoplasmic membranes. A ruffled border secrete acid and dissolve the bone matrix (organic and inorganic both), the Basolateral border maintains the intracellular Ph by active exchange of ions and prevent the damage to intra cellular structures. The third Functional border becomes an excretory membrane. It disperse the large vesicles containing material adsorbed by the ruff border in to extra cellular spaces by trans-cytoplasmic route

48. Functional Osteoclast

49. Schematic view of a bone resorbing osteoclast. Extensive vesicular trafficking involving several specific membrane domains is a hallmark of actively resorbing cells. BL, basolateral domain (blue); FSD, functional secretory domain (red); SZ, sealing zone (green); RB, ruffled border (black). Brown vesicles illustrate vesicular pathways from the trans-Golgi network and the basolateral membrane to RB, and yellow vesicles illustrate the transcytotic route from the RB to the FSD. Vesicular pathways from the trans-Golgi network to the apical (black vesicles) and basolateral (blue vesicles) membrane domains are shown. HA, haemagglutinin; VSV-G, vesicular stomatitis virus G protein. RL, resorption lacuna (white).

50. 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.

51. osteoclast undergoes apoptosis

52. RESORPTION CYCLE - Summary 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. αvb3 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 αvb3 are present at the ruffled border and denatured type I collagen has a high affinity for αvb3.

53. The Ruffled Border 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.

54. The Ruffled Border 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.

55. Ion channel pumps 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.

56. Ion channel pumps 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.

57. MMP-9 (gelatinase B) and Cathepsin K 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.

58. Excretion - Transcytosis 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.

59. Transcytosis 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. 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 .

60. The Ph regulation in Osteoclasts 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.

61. The osteoclast acidifies the resorption lacunae by active secretion of protons through a V-ATPase, which is followed by passive transport of chloride ions, thereby maintaining electroneutrality. The chloride channel ClC-7 is responsible for this process. Mutations or inhibition of either ClC-7 or the V-ATPase will lead to reduced acidification and therefore inhibition of bone resorption. The disruption of ClC-7 and loss of function mutations in the a3 subunit of the VATPase leads to a defect in bone degradation, resulting in a severe form of osteopetrosis, which leads to an increase in the bone mass. Calcitonin has long been known to suppress bone resorption inhibiting the activity of osteoclasts. However, the exact mode of action of calcitonin is still unknown. Preliminary data suggest that calcitonin may be the long sought physiology modulator of intracellular pH in the osteoclasts.

62. 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.

63. .Carbonic anhydrase II (CA II) 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) 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 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.

64. Carbonic anhydrase II (CA II) 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.

65. Osteoclast apoptosis

66. OPG as Decoy The pre-osteoblasts matures into osteoblasts, which stop making RANK-L, and start secreting OPG (osteoprotegerin) The OPG binds to RANK-L acts as a decoy receptor, thus block the activation of Osteoclasts.

67. Regulation of osteoclastic bone resorptionRole of Genes 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 .

68. Regulation of osteoclastic bone resorption Systemic stimulators of bone resorption include PTH, interleukin-1, tumor necrosis factor , transforming growth factor α and 1,25-dihydroxyvitamin D3 . PTH and D3 stimulate bone resorption by increasing the activity of existing osteoclasts and by promoting the differentiation of osteoclast precursors into mature multinucleated osteoclasts and D3 or other vitamin D metabolites seem to play a role in the correction of calcium malabsorption, Osteoblasts mediate the effects of PTH and D3 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 . Calcitonin, gamma interferon and transforming growth factor β are extremely potent in inhibiting osteoclast differentiation and activity.

69. OPG Osteoprotegerin is a soluble protein member of TNF family Produced by bone , hematopoetic marrow , immune cells OPG blocks action of RANKL , inhibits osteoclastogenesis by acting as a decoy receptor that binds to RANKL , thus preventing interaction between RANK & RANKL Therefore interplay between bone cells & these molecules permits osteoblasts and stromal cells to control osteoclasts development.

70. Bone Resorption

71. Reversal Phase During the reversal phase macrophages may appear on the resorption surface. The roles of these macrophages are not known, but it has been suggested that they complete the task of bone resorption. Alternatively, they might produce factors that help initiate osteoblastic bone formation. If the fate of osteoclasts at the reversal site is apoptosis, the function of macrophages could be related to destruction of osteoclast corpses.

72. Coupling mechanism The mechanism by which osteoblasts are summoned into the resorption lacuna is uncertain, but it is probable that a number of paracrine factors produced in or around the remodeling site are involved. These “coupling factors” could be elaborated by cells involved in the activation of resorption (lining cells) or by osteoclasts themselves or some other cell types present in the resorption lacunae. The factors could also be released from the bone matrix during the resorption phase. Osteoclasts are highly motile and actively migrating cells, so that after completion of one resorption lacuna, they can move along the bone surface to another site and restart the resorption phase.

73. “coupling factor” – Change over The coupling factor is derived from 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.

74. Maturation of Osteoblasts Creeping of Osteoblast

77. The mature osteoblasts secrete osteoid and then mineralize it to fill in the cavity in 3 – 4 months. The matrix also contains other proteins and growth factors such as IGF and TGF-b During mineralization and filling of the cavity, some osteoblasts turns into osteocytes, some into lining cells, and the rest undergo apoptosis.

78. Bone Formation Bone formation is a two-stage process beginning with the deposition of osteoid, an organic matrix consisting primarily of type I collagen and various other components. In normal adult bone, osteoid is laid down in discrete lamellae about 3 m thick. The second stage in bone formation is mineralization of the organic matrix, which occurs after a delay of about 20 days called the mineralization lag time.

79. Nucleation - theory The nucleation theory holds that the type I collagen fibril is the major site of crystal nucleation where calcium phosphate crystals are deposited in the hole regions of these fibrils. Originally it was believed that the initiation of bone mineralization is mainly an extracellular, biochemical process where the presence of collagens, certain glycosaminoglycans or lipids could trigger the initiation of calcification. The first line of evidence that cellular activity might be needed for the mineralization of bone arose out of the finding that mitochondria accumulate calcium. Later it was shown that mitochondria of bone-forming cells might produce a local rise in the levels of mineral ions, and also a matrix, which was capable of being mineralized.

80. Matrix - theory The matrix vesicles theory states that bone-forming cells produce small 100-200 nm in diameter organelles that have been observed in mineralizing tissues and cell cultures, often in contact with the initial mineral crystals These vesicles have high alkaline phosphatase, alkaline pyrophosphatase and ATPase content, but hardly any acid phosphatase, and are thus not lysosomal origin. Plausible evidence has been demonstrated to support the matrix vesicle theory, showing that membrane bound vesicles bud from the long processes of osteoblastic cells and that the mineralization is induced by the release of Ca2+-containing vesicles.

81. Osteonectin Osteonectin is a glycoprotein in the bone that binds calcium. It is secreted by osteoblasts during bone formation, initiating mineralization and promoting mineral crystal formation. Osteonectinshows affinity for collagen in addition to bone mineral calcium. Osteonectinincreases the production and activity of matrix metalloproteinases, a function important to invading cancer cells within bone. Over expression of osteonectin is reported in many human cancers such as breast, prostate and colon. Additional functions of osteonectin beneficial to tumor cells include angiogenesis, proliferation and migration.

82. Mineralization However, later it has been speculated that the two mechanisms for bone mineralization are not alternative but parallel; one is predominant in both calcified cartilage and primitive woven bone, the other in lamellar bone. The rate of mineralization in both woven bone and in lamellar bone seems to depend on the presence of inhibitor molecules (e.g. pyrophosphate and acidic NCPs), which in solution seem to regulate the kinetics of the mineralization process. The cell buds off organelles capable of mineral accumulation and then synthesizes proteins that can control the rate at which crystallization proceeds

84. Mineralization After the mineralization process is triggered, the mineral content of the matrix increases rapidly over the first few days to 75% of the final mineral content (primary mineralization), but it takes from 3 months up to a year for the matrix to reach its maximum mineral content (secondary mineralization). The principal component of the mature mineral phase is hydroxyapatite.

85. Formation of lining cells and mature osteocytes

86. Osteocytes

87. The newly formed osteocytes re-establish its network with the surrounding osteocytes and lining cells.

88. Maturation of Mineralization 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. The complete maturation of the newly form matrix takes about 3 years.

90. Mineralization - Inorganic pyrophosphate The regulation of mineralization relies largely on inorganic pyrophosphate, which inhibits abnormal calcification. It is a very small molecule, but it is an important inhibitor of calcification. It is involved in controlling the right rate, the right pace of calcification in the normal skeleton. Levels of this important bone regulator are controlled by at least three other molecules: nucleotide pyrophosphatasephosphodiesterase 1 (NPP1), which produces pyrophosphate outside the cells; ankylosis protein (ANK), which further contributes to the extracelluar pool of pyrophosphate by transporting it from the cell's interior to the cell surface; and tissue nonspecific alkaline phosphatase (TNAP), which breaks down pyrophosphate in the extracellular environment, keeping its levels in check.