Metabolic Bone DiseasesMolecular Biology

Vinod NaneriaConsultant orthopaedic surgeon

Choithram Hospital & Research Centre

Indore , India

Recent Trend In

Metabolic Bone Disease

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.

The molecular triad OPG/RANK/RANKL: Orchestration of patho-physiological bone remodeling.

The recent identification of the receptor activator of nuclear factor kappaB ligand (RANKL), its cognate receptor RANK, and its decoy receptor osteoprotegerin (OPG) has led to a new molecular perspective on osteoclast biology and bone homeostasis. Specifically, the interaction between RANKL and RANK has been shown to be required for osteoclast differentiation. The third protagonist, OPG, acts as a soluble receptor antagonist for RANKL that prevents it from binding to and activating RANK.

Basics of Skeletal Functions

• Modeling and Remodeling of bone structure as a part of repair, maintenance process or as directed by the “Mechanical stress-sensor” mechanism.

• Maintenance of critical level of Ionic Ca in the extra cellular fluid.

• Hematopoiesis

Bone Anatomy

Osteogenic cells

Osteocytes• Osteocytes - terminally differentiated bone-

forming long lived most abundant cells in bone.• Stimulated by calcitonin; inhibited by PTH• Osteocytes- actively involved in bone turnover; • Osteocyte network is through its large cell-matrix

contact surface involved in ion exchange;

Osteocyte with Cytoplasmic Extensions

Osteoblasts

• Synthesize organic components of matrix (collagen type I, proteoglycans, glycoproteins.)

• Collagen forms osteoids: strands of spiral fibers that form matrix

• Influence deposit of Ca++, PO4.• Active vs inactive osteoblasts• Estrogen, PTH stimulate activity• Mesenchymal linage

Osteoclasts• Derived from monocytes; engulf bony material

• Active osteoblasts stimulate osteoclast activity

• Large, branched, motile cells

• Secrete enzymes that digest matrix

• Heamopiotic linage.

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.

Bone Matrix Organic part of bone is about 20% of the dry weight, and

counts in, other than osteopontin, collagen type-I  osteocalcin, osteonectin, bone sialoprotein and alkaline phosphatase. Collagen type I counts for 90% of the protein mass. The inorganic part of bone is the mineral hydroxyapatite, Ca10(PO4)6(OH)2.

Bone sialoprotein (BSP) is a component of mineralized tissues such as bone, dentin, and calcified cartilage. BSP is a significant component of the bone extracellular matrix and has been suggested to constitute approximately 8% of all non-collagenous proteins found in bone.

BSP acts as a nucleus for the formation of the first apatite crystals. As the apatite forms along the collagen fibres within the extracellular matrix, BSP could then help direct, redirect or inhibit the crystal growth.

Collagen type I, alpha 1, also known as COL1A1, is a human gene that encodes the major component of type I collagen

The COL1A1 gene produces a component of type I collagen, called the pro-alpha1(I) chain. This chain combines with another pro-alpha1(I) chain and also with a pro-alpha2(I) chain (produced by the COL1A2 gene) to make a molecule of type I procollagen. These triple-stranded, rope-like procollagen molecules must be processed by enzymes outside the cell. Once these molecules are processed, they arrange themselves into long, thin fibrils that cross-link to one another in the spaces around cells. The cross-links result in the formation of very strong mature type I collagen fibers.

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.

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. Osteonectin also shows affinity for collagen in addition to bone mineral calcium.

• Osteonectin is an acidic, secreted extracellular matrix glycoprotein that plays a vital role in bone mineralization, cell-matrix interactions, and collagen binding. Osteonectin also increases 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.

Basics – Remodeling stages

• BMU Cells activation,

• Resorption of Matrix and Minerals

• Re activation of Bone forming unit or reversal

• New bone formation

• Mineralization and Maturation

Leptin

Sclerostin

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

LeptinLeptin indirectly restrains but directly stimulates bone formation.

Leptin from fat cells or from late-stage, matrix-mineralizing osteoblasts inhibits osteoclast generation by stimulating the production of the anti-osteoclastogenic osteoprotegerin (OPG) and reciprocally reducing the pro-osteoclastogenesis RANK by marrow osteoblastic cells.

Leptin also causes osteoblastic cells to make IGF-I and TGF-betas, which in turn stimulate the proliferation of osteoprogenitor cells (osteoprogenitor), stimulate bone matrix mineralization, and prevent osteoblasts and osteocytes from committing apoptotic suicide.

Leptin also stimulates hypothalamus cells to make some kind of hypothalamic osteoblast inhibitory factor (HOBIF) or trigger some neural process that restrains osteoblasts' matrix-making activity.

Leptin inhibits the release of neuropeptide Y (NPY), which prevents the activation of Y2R receptors, signals that would otherwise mimic HOBIF's osteoblast-restraining activity.

Leptin -- and with it, HOBIF production -- should decrease, NPY production would surge, and the osteoclast-suppressing signals from Y2R receptors would take over from HOBIF.

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

• Numerous other growth-regulatory factors affect osteoblast function, including the three closely related transforming growth factors, fibroblast growth factors (FGFs) 2 and 18, platelet-derived growth factor, and insulin-like growth factors (IGFs) I and II.

• Hormones such as parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1,25(OH)2D] activate

receptors expressed by osteoblasts to assure mineral homeostasis and to influence a variety of bone cell functions.

Other Growth factors in bone formation

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)2D 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.

Maturation Pathway

Osteoblasts

Osteoclasts

Sequence of bone repair

Activation

• Selection of the site for BRU.

• Signaling mechanism from Osteocytes

• Recruitment of Osteoclast precursors

• Fusion

• Formation of Functional Osteoclast

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.

Sclerostin• Sclerostin produced by the osteocytes blocks the

mineralization at the later stages.• osteocytes main source of sclerostin.• osteocytes play a major role in regulating bone

remodeling.• 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.

Wnt• Signals originating from members of the wnt

(wingless-type mouse mammary tumor virus integration site) family of paracrine factors are important.

• 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.

+ promote

- inhibits

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.

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.

RANK =Receptor activator for nuclear factor kb

• RANK is a member of TNF family of receptors expressed mainly on cells of macrophages / monocytes lineage such as preosteoclasts

• When this receptor binds its specific ligand (RANKL) through cell- cell contact , osteoclastogenesis is initiated

• RANKL 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.

The cell biology of functional osteoclast

• Resorption cycle

• Osteoclasts attach to bone matrix through the sealing zone

• Resorbing osteoclasts are polarized

• Bone matrix is degraded in the resorption lacuna

• Degradation products are removed by transcytosis

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

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.

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.

Osteoclastic membrane domains

• Polarization• Sealing zone or clear zone• ruffled border, • basolateral membrane• functional secretory domain

Normal Multinucleated Osteoclast Tightly Adherent to Bone

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 αvβ 3 and α2β 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 .

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 

Resorption cycle Functional Osteoclast

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.

RESORPTION CYCLE - Summary

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

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

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

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 .

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

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.

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)

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)

Regulation of osteoclastic bone resorptionA 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 D3 .

On the other hand, calcitonin, gamma interferon and transforming growth factor β are extremely potent in inhibiting osteoclast differentiation and activity.

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, 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 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 .

OPG• Osteoprotegrin 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.

Bone Resorption

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.

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. 

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.

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.

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.

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

•Resorbed bone is nearly precisely replaced in location and amount by new bone. •Bone loss through osteoclast-mediated bone resorption and bone replacement through osteoblast-mediated bone formation are tightly coupled processes. •Osteoblasts direct osteoclast 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.

Mineralization - Inorganic pyrophosphate

• The regulation of mineralization relies largely on a substance called 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 pyrophosphatase phosphodiesterase 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.

Life cycle of Osteoclast

Osteoclast : target organ

• Blocked of RANK ligend by human antibody to RANK ligand:- Denosumab

• Cathepsin K- deficiency:- Picnodisostosis• Corbonic anhydrase deficiency: – Osteopetrosis• RANKL decoy by Osteoprotegrin:- ↓ Osteoclastosis• Over production of RANKL by parathyroid harmones:

↑ osteoclastosis – Brown lesions.• Sclerostatin by osteocytes : prevents extra new bone

formation at the site of repair.

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

How is the close pairing of the osteoclast and osteoblast

activity regulated?

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.

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

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

• Osteoblasts regulate osteoclast formation via the RANKL–RANK and the M-CSF–OPG mechanism, but there is no known direct feedback of osteoclasts on osteoblasts.

• The coupling mechanism is proposed as a link where “cross talk” via paracrine or autocrine osteoclast signals the osteoblasts to creep into the lacune.

• The whole bone remodeling process is primarily under endocrine control.

• Parathyroid hormone accelerates bone resorption and estrogens slow bone resorption by inhibiting the production of bone-eroding cytokines.

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..

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.

RANK Ligand and Osteoprotegerin. Paracrine Regulators of Bone Metabolism and Vascular

Function Receptor activator of nuclear factor (NF-kappaB) ligand

(RANKL), its cellular receptor, receptor activator of NF-kappaB (RANK), and the decoy receptor osteoprotegerin (OPG) constitute a novel cytokine system.

RANKL produced by osteoblastic lineage cells and activated T lymphocytes is the essential factor for osteoclast formation, fusion, activation, and survival, thus resulting in bone resorption and bone loss.

RANKL activates its specific receptor, RANK located on osteoclasts and dendritic cells, and its signaling cascade involves stimulation of the c-jun, NF-kappaB, and serine/threonine kinase PKB/Akt pathways..

Cont…… The effects of RANKL are counteracted by OPG which acts

as a soluble neutralizing receptor.

RANKL and OPG are regulated by various hormones (glucocorticoids, vitamin D, estrogen), cytokines (tumor necrosis factor alpha, interleukins 1, 4, 6, 11, and 17), and various mesenchymal transcription factors (such as cbfa-1, peroxisome proliferator-activated receptor gamma, and Indian hedgehog).

Transgenic and knock-out mice with excessive or defective production of RANKL, RANK, and OPG display the extremes of skeletal phenotypes, osteoporosis and osteopetrosis

Abnormalities of the RANKL/OPG system have been implicated in the pathogenesis of: -

• postmenopausal osteoporosis, • rheumatoid arthritis, • Paget's disease, • periodontal disease, • benign and malignant bone tumors, • bone metastases, • hypercalcemia of malignancy.

Administration of OPG has been demonstrated to prevent or mitigate these disorders in animal models.

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.

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

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

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

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

progenitors.

canonical Wnt signaling pathway

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

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

paracrine communication

• Paracrine system is essential to bone metabolism • The paracrine signaling molecule, Indian hedgehog

(Ihh), plays a critical role in osteoblast development, as evidenced by Ihh-deficient mice that lack osteoblasts in bone formed on a cartilage mold (endochondral ossification).

• Signals originating from members of the wnt (wingless-type mouse mammary tumor virus integration site) family of paracrine factors are also important. 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.

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

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

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 D3 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 D3 in the presence and absence of recombinant human BMP-4 or BMP6. PTH and vitamin D3 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 D3. Both BMPs enhanced calcium formation in MSC cultures and this response was potentiated by PTH and vitamin D3. The present results revealed a novel potent effect of PTH and vitamin D3 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)

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

DISCLAIMER.• It is intended for use only by the students of orthopaedic surgery. •Many GIF files are taken from Internet.• Views and opinion expressed in this presentation are personal opinion..• For any confusion please contact the sole author for clarification.• Every body is allowed to copy or download and use the material best suited to him. I am not responsible for any controversies arise out of this presentation.• For any correction or suggestion please contact naneria@yahoo.com

IMPORTANT INFORMATIONAll animation slides are taken from, “Osteoporosis and Bone Physiology” web site, 1999 - 2006 http://courses.washington.edu/bonephys of Dr. Susan Marie Ott, MD. Medical staff of University of Washington Medical Center.

And the summary of BMU mechanism is based on the thesis“Attachment, polarity and communication characteristics of bone cells”Joanna IlvesaroDepartment of Anatomy and Cell Biology and Biocenter Oulu, P.O. B. 5000, FIN-90014 University of Oulu, Finland, http://herkules.oulu.fi/isbn9514259351/html/index.html

Bon Voyage