The Regulatory Role of Matrix Proteins in Mineralization of Bone

C H A P T E R

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The Regulatory Role of Matrix Proteins in Mineralization of Bone Adele L. Boskey1, Pamela Gehron Robey2 1Musculoskeletal

Integrity Program, Hospital for Special Surgery, affiliated with Weill Medical College of Cornell University, New York, NY, USA, 2Craniofacial and Skeletal Diseases Branch, National Institute of Dental Research, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, USA

INTRODUCTION The skeleton is essentially responsible for providing not only structural support and protection to the body’s organs, but also for serving as a reservoir for calcium, magnesium, and phosphate; ions that are of critical importance in physiology. The fabric of bone is a unique composite of living cells embedded in a remarkable three-dimensional structure of extracellular matrix that is stabilized by mineral, which is a carbonate-rich analog of the geologic mineral hydroxyapatite (HA). Because of the vast expansion of literature in this field, this chapter relies primarily on reviews. Readers are referred to the earlier edition of this book for most primary references.

Bone Tissue: Composition During development, mesenchymal cells form the skeleton via two basic pathways [1]. Intramembranous bone is formed by direct differentiation of mesenchymal cells, whereas endochondral bone is formed by an initial condensation of mesenchymal cells that leads to morphogenesis of a cartilaginous structure. Serving as a temporary model, the cartilage becomes calcified and the provisional calcified cartilagenous precursor is subsequently replaced by bone. A third, less recognized, pathway acting primarily in the cranium, but also at other sites in the skeleton, involves regression of unmineralized cartilage by apoptosis, and subsequent remodeling of remaining cells into other tissues, including bone [2]. Invasion by blood vessels brings in the cells that remove bone (osteoclasts) and, in addition, the osteoblastic

Osteoporosis. http://dx.doi.org/10.1016/B978-0-12-415853-5.00011-X

precursors that will replace the calcified cartilage with bona fide bone. The initial bone formed, woven bone, is a rather unorganized conglomeration of collagenous and noncollagenous proteins that induce the precipitation of mineral. Through modeling by osteoclasts, this primordial bone is removed and replaced by the formation of lamellar bone, a more highly organized structure with alternating layers of mineralized extracellular matrix, whose plywood-like structure provides bone with its mechanical strength. Although the mineralized matrices were originally thought to be composed of a unique set of matrix proteins, the majority of these proteins are also synthesized by nonskeletal cells. Bone is composed of 70–90% mineral and only 10–30% protein, with collagenous protein comprising ∼90% of the bone matrix and noncollagenous proteins accounting for the remaining ∼10%. In addition, virtually all of the known collagenous and noncollagenous proteins in bone studied to date differ from those in other tissues in their chemical nature. These diverse forms are a result of alternative splicing of messenger ribonucleic acid (mRNA) and different post-translational modifications, such as glycosylation, phosphorylation, and sulfation. These chemical differences most likely influence the physiological function of these proteins, and the appropriate mixture provides the extracellular matrix with the ability to calcify. Moreover, because most of the extracellular matrix proteins are the secretory products of cells in the osteoblastic lineage, they represent biochemical markers of maturation stages of cells during the formation process (Fig. 11.1), or the resorption process (in their degraded form) of bone.

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FIGURE 11.1  Maturational stage and bone matrix gene expression. Osteoblast cells pass through a series of maturational stages, each of which can be partially characterized by the bone matrix proteins that they produce. In addition, osteoclasts also secrete proteins that become incorporated into mineralized matrix.

Recent comparisons of younger and less mature bone even shows a difference in the nature of the non-collagenous proteins expressed in the same tissues [3]. This chapter describes the major types of proteins synthesized by osteoblastic cells that are present in bone matrix, and discusses their potential roles in the regulation of mineralization.

Bone Mineral The mechanical strength of bone is attributable to the presence of mineral that converts the pliable organic matrix into a more rigid structure [4]. A variety of structural analyses [5] have shown that mineral crystals within bone are analogous to the naturally occurring geologic mineral, HA (Ca10[PO4]6[OH]2) (Fig. 11.2). However, in bone, the mineral includes numerous ions not found in pure HA. This poorly crystalline apatite in bone, because of its small crystal size and large number of lattice-substituted and surface-adsorbed ion impurities, can be dissolved more readily than the larger, more perfect crystals of geologic HA. Moreover, this altered solubility allows bone mineral to play an important role in Ca2+, Mg2+, and PO43− ion homeostasis [6]. Despite claims of the presence of other mineral phases in bone, current evidence supports the view that bone mineral is predominantly apatitic, with numerous, and perhaps unique, impurities. Evidence obtained using cryo-techniques where harvested bone is never exposed to an aqueous environment indicates that initial bone mineral may include some non-structured (amorphous) calcium phosphate [7]. How the initiation of mineral deposition

FIGURE 11.2  Crystal lattice structure. A portion of the apatite structure is depicted as it would be viewed along the length (c axis) of the hydroxyapatite crystal, showing the hexagonal arrangement of the Ca2+ and PO43– ions about the OH– position.

and the growth of mineral crystals are regulated by matrix proteins is discussed below.

COLLAGENOUS PROTEINS In the skeleton, the major (∼90%) structural protein is collagenous in nature. Collagen in bone is predominantly composed of type I collagen, which most likely serves a mechanical function providing tensile strength [8]. Collagen may not directly induce mineral deposition in bone matrix; however, it serves as an important “backbone” in support of initial mineral deposition and the organization of crystal growth by providing appropriate scaffolding and orientation of nucleators of mineralization.

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Collagenous Proteins

Structure of the Molecule Collagen is defined as a trimeric molecule composed of α-chain subunits. A significant feature of the component α chains is that their primary sequence is almost entirely made up of a repeating triplet sequence, ­Gly-X-Y, where X is often proline and Y is often hydroxyproline. Collagenous proteins are either homotrimeric, composed of three identical α chains, or heterotrimeric, with two or three different α chains. Individual α chains of the collagen molecule coil together to form an extended rigid triple helix. The structure is stabilized by hydrogen bonding between OH groups on hydroxyproline and intrachain water, and by aldehyde-derived cross-links [9]. Currently, the collagen family is composed of 29 collagen types of which types I, II, III, VI, XI, X, XXVII are found in bone, calcified cartilage, and matrices associated with the vasculature [9]. Based on their structural features, collagens can be generally divided into two groups: fibrillar and nonfibrillar. Fibrillar collagens are all synthesized as precursors that are proteolytically trimmed of noncollagneous ends to yield mature molecules. Collagen fibrils are formed by the mature collagen molecules via head-to-tail associations and different types of fibrillar collagens share a strong structural similarity in that the major part of each molecule is formed by an uninterrupted triple-helical domain. Once fibers have been formed in the extracellular environment, they are further stabilized by the formation of inter- and intramolecular cross-links. Fibrillar collagens (types I, II, III, V, and XI) are by far the most abundant forms and are formed in the interstitial spaces of connective tissues throughout the body [9]. Type I collagen, the predominant collagen of skin, tendon, and bone, forms the major scaffolding of virtually all connective tissues except cartilage; cartilage contains predominantly type II collagen (α1(II)3) with limited amounts of other collagens. Type III collagen, composed of three identical α1(III) chains, is found in many tissues rich in type I collagen. Quantitatively minor fibrillar collagens, types V and XI, associated with collagen types I and II, respectively, are located on the periphery of the collagen fibrils. Contrary to other fibrillar collagens, their N-terminal extensions are retained and project onto the fibril surface. This feature, together with the correct molar ratios of I/V and II/XI collagens in fibrils, is significant in the regulation of fibril diameter [9]. Structural analysis of fibrillar type I collagen suggests that individual collagen fibrils are aligned in a quarter-staggered array, with a 280-nm periodicity. As a result of the quarter stagger, there are gaps (holes) within the fibrillar structures, and it is these gaps and in the overlapping regions adjacent to them (e band) that bone mineral crystals first appear

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[10,11]. One fibrillar collagen, type XXVII, seems to be involved in the transition from calcified cartilage into bone [12]. The nonfibrillar collagens are characterized by triplehelical domains that are either shorter or longer than those of the fibrillar types, and they may contain stretches of non-triple-helical sequences [9]. Among these ­nonfibrillar collagens, type X collagen is found in calcified cartilage. The localization of type X to hypertrophic chondrocytes is highly specific, but it does not appear to have a major role in cartilage calcification. Type IX is a minor constituent in cartilage. It is composed of three different types of α chains, α1(IX), α2(IX), and α3(IX), which form a short and a long triple helix joined by a flexible hinge region. A glycosaminoglycan chain is also attached to one of the α-chains at the amino terminus, making this collagen a proteoglycan as well. Type IX has been found as a coating of type II collagen fibrils (the major collagen in cartilage) and covalently attached to it. Type XII is similar to type IX, but has three projections extending from the triple helix. This type may also be associated with type I fibrils in tendon. Type XIV (as well as type XII) is structurally related to type IX collagen fibrils, which associate with type II collagen in cartilage. Mice lacking type IX collagen show cartilage and bone abnormalities presumably associated with their altered fibril diameters [13].

Bone Matrix Collagen(s) Bone matrix proper contains a rather limited array of collagen types (Table 11.1). Although bone matrix has been reported to contain predominantly type I collagen, other types are certainly present but at lower levels compared to soft connective tissues. Given the potential role of these low-abundancy collagens in regulating fibril diameter, it is possible that collagen fibrils in bone grow to much larger diameters than in soft tissues due to the reduced proportion of these diameter-regulating collagen types. In all connective tissues, the collagens serve mechanical functions, providing elasticity and strength for the component tissues [8]. The importance of type I collagen in bone is well demonstrated by various forms of osteogenesis imperfecta (OI; brittle bone disease) in humans and in animal models, in which bone fragility has been associated with qualitative and quantitative alterations in the type I collagen genes [14,15] (see Chapter 53). The mineral crystals in the bones of patients and transgenic animals with OI tend to be smaller than those in agematched control bones [15,16]. For example, in a naturally occurring mutant mouse that has an α1(I)3 trimer as opposed to the normal α1(I)2α2(I) trimer, the oim/oim mouse, the pattern of initial mineral deposition and crystal growth along the collagen differs from normal; the

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TABLE 11.1  Collagen Types Found in Bone Matrix Collagen

Location/function

Molecular structure

Type I: α1(1)2α(1) and α1(1)3

Constitutes 90% of matrix in the bone matrix Acts as scaffolding and binds to other proteins that initiate hydroxyapatite deposition

67-nm banded fibrils

Type III: α1(III)3

Present only in trace amounts and can regulate collagen fiber thickness

67-nm banded, coats type I fibrils

Type V: α1(V)2α2(V) and α1(V)α2(V) α3V

Absence can result in collagen fibrils of larger diameter

67-nm banded, coats type I fibrils in some tissues

Type X: α1(X)3

Present in hypertrophic cartilage and can be Probably fishnet-like lattice involved in matrix organization via formation of the template for type I collagen

crystals appear both outside of the collagen matrix and within regions of collagen, which are less mineralized than those in the normal controls [17]. In addition, there are thinner fibrils in OI patients and OI mice that may be insufficient to provide nucleation and scaffolding sites for mineral deposition and can potentially translate into fragile bones. Collagen per se does not initiate mineral deposition; that is, it is not a mineral nucleator since it lacks the appropriate conformation that matches the ion surface of the deposited mineral surface. However, normally collagen has binding sites for the noncollagenous proteins that regulate mineralization, whereas these domains could be missing in mutant tissues [18]. Since these noncollagenous matrix proteins that are “held” within the collagen matrix appear to initiate and regulate the mineral deposition in bone, the data from OI tissues clearly demonstrate the importance of collagen for providing a scaffold to organize the mineral.

INTERMEDIATE CARTILAGE MATRIX Endochondral bone formation is mediated by a cartilage template, and cartilage macromolecules can be in close proximity to forming bone and may actually be incorporated into the initial boney tissue [1]. The basic scaffolding on which cartilage matrix is built is type II collagen. In addition, a number of proteoglycans have been identified in cartilage matrix, primarily the large proteoglycans, such as aggrecan and versican, and small leucine-rich repeat proteoglycans, such as decorin and biglycan, which are also present in bone matrix. Other proteins, including cartilage oligomeric matrix protein (COMP), cartilage-derived retinoic acid-sensitive protein (CD-RAP), chondroadherin, and matrilin-1, are present in cartilage matrix but at much lower levels than type II collagen and aggrecan [19]. Proteoglycans are a class of macromolecules characterized by the covalent attachment of long chains of repeating disaccharides that are often sulfated, termed

glycosaminoglycans (GAGs). Based on the sugar composition of the repeating disaccharides, GAGs are divided into subtypes such as chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparin sulfate (HS), and hyaluronan (HyA; unsulfated, and not bound to a protein core) (Fig. 11.3). Aggrecan is one of the large CS molecules and has the ability to form aggregates with HyA.

Large Proteoglycans Aggrecan Intact human aggrecan has a molecular weight of approximately 2.5 million Da, with a core protein ­ranging in apparent molecular weight between 180 and 370  kDa with slightly more than 100 GAG chains (mostly CS, but with some KS) of approximately 25 kDa. Based on enzymatic cleavage and sequence homology, five domains have been defined in the core protein of aggrecan (Fig. 11.4): three globular (G) domains [20], two of which bind to hyaluronic acid (G1 and G2) with the third one, G3, located at the C terminus; an interglobular domain; and a central domain rich in serine– glycine repeats to which the CS and KS-GAG chains are attached. The G1 domain in the N terminus is structurally homologous to “link protein” [19], a small ­glycoprotein that stabilizes the interaction between the proteoglycan and hyaluronic acid in cartilage, forming a unique gel-like moiety providing resistance to compression in joints [21]. The adjacent G2 domain provides a flexible hinge. The importance of the structure of aggrecan is demonstrated by mutations in mice. Mice with cartilage matrix deficiency (cmd), which is caused by a functional null mutation of aggrecan gene, are characterized by perinatal lethal dwarfism and craniofacial abnormalities, suggesting an important role of this proteoglycan in skeletal development [22]. Mice lacking the ability to make hyaluronic acid in chondrocytes still form aggrecan, but they are unable to form a proper growth plate,

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Intermediate Cartilage Matrix

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FIGURE 11.3  Disaccharide composition of glycosaminoglycans (GAGs). The GAG side chains that are covalently attached to proteoglycan core proteins are composed of repeating disaccharide units. The composition of the disaccharides, along with modifications by acetylation, results in the formation of chondroitin sulfate, which is epimerized to form dermatan sulfate, heparin sulfate, and keratan sulfate. Hyaluronan is the sole GAG the remains unsulfated and is not covalently lined to core proteins.

cannot survive birth, and have major in utero skeletal deformities [23]. Though less severe, mice that lack the ability to add CS to their core protein also have reduced growth plate widths and other skeletal abnormalities [24]. In addition to the hydrodynamic function, aiding in the retention of both water and c­ ations and the exclusion of anions in cartilage, proteoglycans are also responsible for matrix maintenance and organization [19], in part through interactions with the GAG chain of type IX collagen that surround the type II collagen fibrils. Proteoglycans may also play a role in the regulation of cartilage calcification [25]. The large aggregating cartilage proteoglycans can inhibit HA formation and growth in solution (e.g., [26]), and they can also chelate calcium [27] and serve as a source of calcium ions for mineralization if they are degraded into ­non-Ca2+-binding fragments. Although there is debate as to whether this chelation is involved in the inhibition of mineralization, it is clear that proteoglycans and their component GAGs sterically block HA formation and growth [28].

FIGURE 11.4  A representation of the chemical features of the large hyaluronic acid-binding proteoglycan, aggrecan. CRP, C-reactive protein; CS, chondroitin sulfate; EGF, epidermal growth factor; G1, G2, G3, globular domains; GAG, glycosaminoglycan: KS, keratan sulfate.

The amount of aggrecan in bone is much lower than that in cartilage, and whether its presence in bone represents residual calcified cartilage is largely unknown. The presence of elevated amounts of CS proteoglycans in the

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bones of osteopetrotic animals with defective osteoclasts was linked to the inability of these animals to resorb ­calcified cartilage [29]. The functions of aggrecan in bone, if any, are also unknown. Because of its relatively low concentration, it seems likely that its effects are on growth plate (cartilage) calcification, rather than having a direct effect on bone. Versican Versican is another CS proteoglycan related to aggrecan but found at relatively lower levels in both cartilage and bone. Versican has been so termed based on its high variety of forms in a large number of extracellular matrices (“versatility”). The protein core, with a molecular weight of approximately 360 kDa, has a structure similar to that of aggrecan with the exception that it lacks the G2 domain. In addition, versican contains only 12–15 CS side chains (∼45 kDa) in contrast to approximately 100 in aggrecan [30]. A report of rat bone development found that versican was expressed during osteogenesis, where it was more abundant in woven than lamellar bone [31]. The function of versican in cartilage and bone is largely unknown. Potentially, it may serve to capture space destined to become bone, or as a bridge between the extracellular environment and the cell by binding to hyaluronic acid via the amino-terminal binding region and to molecules that have yet to be identified on the cell via the C-terminal domain. In addition, versican stimulates chondrocyte proliferation [32]. Because versican is degraded during matrix mineralization, the EGF-like sequence (G3 domain) may serve to stimulate proliferation of osteoprogenitors because EGF has been reported to stimulate proliferation of osteoblastic cells in vitro [33]. With the exception of one study [34], which showed increased versican degradation products accumulating in mineralizing osteoblast cultures, there are no other studies on the role of versican in mineralization of cartilage or bone.

Small Leucine-Rich Repeat Proteoglycans In addition to aggrecan and versican, another ­family of proteoglycans is represented by a group whose protein core is characterized by a smaller size and a ­leucine-rich repeat sequence (SLRP) that is approximately 20–30 amino acids in length [35]. The SLRP family has been subdivided into five classes based on their similarity in gene and amino acid structures [36]. The class I members include decorin, biglycan, and ­asporin; class II includes fibromodulin, lumican, PRELP, keratocan, and osteoadherin; the class III members are epiphycan/PG-Lb, mimecan/osteoglycin, and opticin; class IV includes chondroadherin, nyctalopin, and tsusuhki; and class V members are podocan and p ­ odocan-like proteoglycans. In cartilage and bone, there are several

members of the small leucine-rich proteoglycans (SLRP) family, predominantly including decorin, biglycan, fibromodulin, osteoadherin, and osteoglycin. Although SLRPs are highly homologous, they exhibit distinctively different patterns of expression and tissue localization, indicative of divergent functions within these tissues. We will only discuss those that are relevant for cartilage calcification or bone mineralization in the following sections.

Class I Small Leucine-Rich Proteoglycans Associated with Mineralization Decorin Decorin, so named for its ability to bind to and “decorate” collagen fibrils, has a core protein of approximately 38 kDa, which includes 10 leucine-rich repeat sequences. Although there are three potential GAG attachment sites, generally only one is utilized for the attachment of a single GAG (Fig. 11.5). The length of the chain, and its extent of sulfation, varies as a function of tissue type and age [37]. Decorin is synthesized with a propetide that is cleaved in bone and other tissues by the enzyme, BMP1 (Tolloid-related bone morphogenetic protein-1) [38]. Decorin like most of the other SLRPs, binds to and regulates collagen fibrillogenesis; however, only decorin can recapitulate the in vivo structure of type I collagen in vitro [39]. In bone, the proposed functions of decorin are the regulation of collagen fibril diameter and fibril orientation, and possibly the prevention of premature osteoid calcification. Targeted disruption of the decorin gene results primarily in skin laxity and fragility in mice, whereas disruption of the biglycan gene (discussed below) results in reduced skeletal growth and bone mass leading to generalized osteopenia [40]. Moreover, the decorin and biglycan double knockout mice have additive deficiency in dermis and synergistic effects in bone, and ultrastructural analysis of these mice reveals a complete loss of the basic fibril geometry with the emergence of marked “serrated fibril” morphology [40]. There is also a decreased expression of decorin in some patients with OI [41], in which abnormal mineral deposition has been detected outside the collagen matrix. A role of decorin in matrix mineralization is suggested by in vitro studies [42,43]. Biglycan Biglycan is another small proteoglycan present in both cartilage and bone that is highly homologous to decorin (Fig. 11.5). The functions of biglycan in cartilage and bone mineralization remain to be determined. In solution, biglycan at low concentrations can promote apatite formation, whereas at higher concentrations it inhibits the growth and proliferation of

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Intermediate Cartilage Matrix

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FIGURE 11.5  The two most abundant proteoglycans present in bone matrix are the small chondroitin sulfate/dermatan sulfate proteoglycans, decorin, and biglycan. The core protein of each is highly homologous to a number of proteins due to the presence of a leucine-rich repeat sequence, as shown for both decorin and biglycan.

mineral crystals [44,45]. These effects appear to be due to the highly specific high-affinity binding of biglycan for apatite. Compared to the decorin knockout mice, the biglycan knockout mice have similar structural abnormalities in collagen fibrils but with more serious impact in bone than in dermis [40]. In addition, the biglycan knockout mice have shorter femora, decreased bone density, and failure in achieving peak bone mass compared to controls. The mineral within these bones has increased crystal size relative to wild-type controls [44], also indicating an inhibitory role of this protein. However, the low amount of biglycan present in bone matrix relative to other mineral nucleators and its absence from bone collagen fibrils suggest that its primary function may not be directly related to mineral deposition of bone. Asporin Asporin, the third member of the type I SLRPs present in bone, cartilage, and the periodontal ligament (all mineralizing tissues), was named so because of the unique aspartic acid repeat at the N terminus of the mature protein [46]. This N terminus prevents asporin from being classified as a true proteoglycan. Asporin, like decorin and biglycan, binds to type I collagen, and

they all compete with one another for the same binding site [42]. Of the three Class 1 SLRPs, only asporin binds calcium, and increases calcium uptake into osteoblastic tissue culture [42].

Class II Small Leucine-Rich Proteoglycans Associated With Mineralization Fibromodulin Fibromodulin is found predominantly in articular cartilage but also exists in bone. The intact protein is approximately 59 kDa, and the core protein shares a high homology with decorin and biglycan, but bears ­KS-GAG chains linked to asparaginyl residues rather than CS or DS linked to serinyl/threonyl residues. Decorin and fibromodulin are the most active collagenbinding proteins in cartilage and bone, reportedly binding to completely different regions on collagen fibrils [47]. Fibromodulin interacts with triple-helical types I and II collagens [47]. In cartilage, the amount of fibromodulin correlates with the size of collagen fibrils [47]. Fibromodulin knockout mice have undermineralized bones and teeth [48,49], revealing a role of fibromodulin in both collagen fibrillogenesis and mineralization.

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OSTEOADHERIN, KERATOCAN, AND LUMICAN Osteoadherin has been isolated as a minor, ­leucineand aspartic acid-rich KS proteoglycan found in the mineralized matrix of bone and dentin. Osteoadherin is synthesized by osteoblasts. In cell culture, it promotes osteoblast mineralization [50]. Keratocan is also expressed by osteoblasts. The keratocan knockout mouse has impaired bone formation, decreased expression of bone specific markers and impaired mineralization [51], implying a role in osteoblast function. Lastly, lumican is also expressed by osteoblasts [52], but to date, the knockout mice [53] were not reported to have any bone phenotype.

Class III Small Leucine-Rich Proteoglycans Involved in Mineralization

enzymes and matrix proteins, may be further modified by post-translational sulfation and phosphorylation.

ALKALINE PHOSPHATASE Although the enzymatic activity of alkaline phosphatase is shared by many types of tissues, there is no doubt that induction of alkaline phosphatase activity in uncommitted progenitors marks the entry of a cell into the osteoblastic lineage and is a hallmark in bone formation. Although alkaline phosphatase is not typically thought of as a matrix protein, studies indicate alkaline phosphatase can be shed from the cell surface of osteogenic cells or in a membrane-bound form (matrix vesicles) [55]. Histological localization of alkaline phosphatase in developing human subperiosteal bone (Fig. 11.6) marked its very specific expression by

Epiphycan Epiphycan is expressed during early development in growth plate and articular cartilage. In its absence, in older mice, femurs are shorter due to abnormalities in the growth plate. They also show a loss of collagen. In aging mice, the absence of epiphycan accelerates the development of osteoarthritis. The epiphycan/biglycan double knockout mouse has a more pronounced phenotype, suggesting some synergistic interaction between these two SLRPs [54].

BONE-ENRICHED MATRIX PROTEINS In bone, the remaining matrix proteins are mainly composed of two major types: glycoproteins and γ-carboxyglutamic acid (Gla)-containing proteins. The most relevant and abundant glycoproteins are represented by alkaline phosphatase, osteonectin, and the cell attachment proteins, which include but are not limited to, the small integrin-binding ligand N-linked glycoprotein (SIBLING) family and additional sialoproteins. Of the Gla-containing proteins, osteocalcin is the major representative. These bone matrix proteins have divergent biochemical properties and play particular roles in the regulation of matrix mineralization.

Glycoproteins

FIGURE 11.6  Alkaline phosphatase in developing bone. By histo-

This class of proteins is characterized by the covalent linkage of sugar moieties attached via asparaginyl or serinyl residues. Collagen also contains another form of glycosylation (galactosyl and glucosyl-galactosylhydroxylysine), which is virtually specific to collagen. These glycoproteins, which include membrane bound

chemical staining for alkaline phosphatase activity during development, areas that are destined to become bone, as shown here in developing human subperiosteal bone, can be clearly illustrated. The fibrous layer (F) of the periosteum is negative, whereas preosteoblasts (POb) and osteoblasts (Ob) produce high levels of activity. Although a glycoprotein with alkaline phosphatase activity has been isolated from the bone matrix, it is not easily detected in mineralized matrix (MM) by this histochemical assay. Source: courtesy of Dr. Paolo Bianco.

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Osteonectin

preosteoblasts and osteoblasts in areas that are destined to become new bone, whereas less expression was found in mineralized matrix [56], suggesting this enzyme is a marker for less mature, osteoprogenitors. Mice with null mutations for the tissue-nonspecific (bone/liver/kidney) alkaline phosphatase provide evidence of the importance of alkaline phosphatase for mineralization [57], showing increased osteoid and defective growth plate development. It is currently believed based on studies with these knockout mice and studies of patients with hypophosphatasia, that alkaline phosphatase functions to regulate pyrophosphate levels, while another enzyme, Phospho-1 is involved in the initiation of mineralization [58,59]. Pyrophosphate is an inhibitor of mineralization that can be hydrolyzed to provide additional phosphate while removing a mineralization inhibitor. Alkaline phosphatase is the major enzyme providing this hydrolysis, although phosphate transport is regulated by a number of other enzymes.

OSTEONECTIN One of the first noncollagenous bone matrix proteins to be isolated and characterized from bone was osteonectin [60]. Osteonectin, also named SPARC (secreted phosphoprotein acidic and rich in cysteine) or BM-40 (basement membrane tumor factor 40), is expressed in a number of tissues during development and by many cell types. In bone, osteonectin can constitute up to 15% of the noncollagenous protein depending on the developmental age and the animal species [61]. Osteonectin has an apparent molecular weight of approximately 35 kDa without reduction of disulfide bonds and appe­ ars to increase in size up to approximately 40–46 kDa­ following reduction, indicative of intrachain disulfide bonds (Fig. 11.7). Due to the nature of the amino acid composition and of the post-translational modifications, osteonectin is acidic. Osteonectin may be differentially glycosylated and/or phosphorylated because there are at least two potential N-glycosylation sites that bear diantennary oligosaccharides (an intermediate between high mannose and complex type oligosaccharides that contains variable amounts of sialic acid and fucose) [62]. Osteonectin is essential for the maintenance of bone mass and for balancing bone formation and resorption in response to parathyroid hormone (PTH). It promotes osteoblast differentiation and cell survival similar to other “matricellular proteins”. The matricellular proteins, originally named so in 1995 as representing “a group of modular, extracellular proteins whose functions are achieved by binding to matrix proteins as well as to cell surface receptors, or to other

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molecules such as cytokines and proteases, that interact in turn, with the cell surface” [63]. These include the TSPs, tenascins, osteonectin, CCN (small secreted cysteine-rich proteins that function as signaling molecules [64]), and the SIBLING proteins [65]. All of these are involved in responses to injury and stress, and the pathogenesis of several chronic diseases of aging. Osteonectin’s effects are tissue specific, including regulation of collagen fibril assembly (skin and bone) and modification of cell shape, migration, ­ proliferation, differentiation, survival, and modulation of cell signaling [66]. Osteonectin-null mice are osteopenic, show increased marrow adiposity, and have c­ ortical bone with decreased mechanical properties and matrix quality [66,67]. The osteonectin knockout was shown to have bones with increased mineral content and crystallinity and increased collagen maturity at all sites, based on Fourier transform infrared (FTIR) microspectroscopy and imaging [68]. The presence of a low turnover form of osteopenia suggested by these data was recently confirmed [67]. These data are consistent with a role in regulating bone formation and remodeling. The spines of mice with targeted deletion of osteonectin similarly showed sclerosis and premature endplate calcification [69].

FIGURE 11.7  The chemical characteristics of osteonectin indicate the presence of two α-helical regions at the amino terminus, along with an ovomucoid like sequence with extensive disulfide bonding, and two EF hand structures that bind to calcium.

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RGD-CONTAINING GLYCOPROTEINS In bone matrix, there are a number of glycoproteins that also have the amino acid sequence Arg-Gly-Asp (RGD). These RGD sequences can be recognized by cell surface receptors as a “cell attachment sequence”, which bridges the attachment between extracellular matrix and cells and thus arranges the cells in matrix. These RGD-containing proteins include collagen (described previously), TSP, fibronectin, vitronectin, and a family of SIBLINGs expressed in bones and teeth. The SIBLINGs have been identified by a cluster of genes including osteopontin (OPN) and bone sialoprotein (BSP), ­dentin matrix protein-1 (DMP-1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE), and enamelin [65,70]. a. Thrombospondin(s). These complex modular glycoproteins are less abundant in mineralized matrix of bone relative to other glycoproteins. They are found in a large variety of connective tissues, particularly in areas of demarcation. The five members of the TSP family are highly expressed during development. They share structural homology due to so-called TSP repeats (Fig. 11.8). The R-spondin family also has these repeats [71]. TSP2 regulates osteoblasts and affects bone mass and geometry, and response to injury. Compound knockout mice of TSP1 and TSP2 have altered craniofacial development.

TSP5 is also known as the COMP. TSP 1/3/5 ­knockout mice display growth plate abnormalities. R-spondin-2 knockout mice also have skeletal defects. These combined data show a role for TSP family molecules in bone development and turnover. b. Fibronectin. Fibronectin is one of the most abundant extracellular matrix proteins in bone and is also a major constituent of serum. It is produced by virtually all connective tissue cells at some stage of development and accumulates in extracellular matrices throughout the body. Although the gene and protein properties of fibronectin indicate a role in matrix deposition and organization by interacting with a number of matrix proteins, its actual function is not clear. Blocking integrin binding by osteoblasts impairs fibronectin and collagen deposition, and mineralization [72], but a direct linkage is not yet established. Earlier studies showed fibronectin supports apatite formation in solution [73]. c. SIBLINGs. The SIBLING family of glycoproteins includes OPN, BSP, DMP-1, DSPP, MEPE, and enamelin, all of which, other than enamelin, are expressed in bone. These genetically related members are clustered on human chromosome 4 (Fig. 11.9), and it is believed to be the result of duplication and subsequent divergent evolution of a single ancient gene. Each of these proteins have been shown or predicted to be “intrinsically ­disordered” [74], having mainly random coil structures,

FIGURE 11.8  Thrombospondin is a disulfide-linked trimer that has globular domains at the amino and carboxy termini, interconnected by a stalk region. Each of these domains has a number of binding sites for other proteins, suggesting numerous potential functions in cell-matrix interactions. The cell attachment consensus sequence, RGD, is in the C-terminal domain; however, its availability depends on the calcium ion concentration, which is known to affect the conformation of this region. EGF: epidermal growth factor.

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RGD-Containing Glycoproteins

which allows them to interact with their different partners (collagen, cell surfaces, and mineral) [75,76]. c.1. Osteopontin. This ubiquitously expressed acidic glycoprotein, which was previously termed BSP-1 in bone, was also described as a secreted phosphoprotein (Spp) and pp66, a protein that is dramatically upregulated by cell transformation and in association with tumor progression. The molecular weight of OPN is in the range of 44–75 kDa depending on the method of

analysis and the extent of post-translational modification (Fig. 11.10). OPN is largely accumulated in bone matrix and is highly enriched at cement lines [77]. Inspection of OPN production at the cellular level during subperiosteal bone formation indicates that it is produced by osteoblasts and, to a lesser extent, by osteocytes, making it a late marker of osteoblastic differentiation and an early marker of matrix mineralization. Phosphorylated OPN inhibits mineralization in vitro and in vivo, but the

Exon Structures Define SIBLING Family OPN Non-coding

Leader + AA

SSEE

PPPP

SSEE

Leader + AA

SSEE

PPPP

SSEE

Leader + AA

SSEE

PPPP

SSEE

RGD

BSP Non-coding

RGD

DMP1 Non-coding

RGD

DSPP Non-coding

Leader + AA

PPPP

SSEE

RGD

PPPP

RGD

MEPE Non-coding

Leader + AA

SSEE

ENAM Non coding Leader? + AA

SEE PP PPPP PPPP SNEE

RGD

S-S

S-S

FIGURE 11.9  Exon structure defines the SIBLING family. The exon structures of the six candidate genes for the SIBLING family are illustrated. Exons are drawn as boxes and introns as connecting lines. Exon 1 is noncoding. For all but ENAM, exon 2 encodes for the leader sequence plus the first two amino acids of the mature protein. Exon 3 often contains the consensus sequence of casein kinase II-mediated phosphorylation (SSEE), as does exon 5. Exon 4 is usually relatively proline rich (PPPP). The last one or two exons encode the vast majority of the protein (figure not drawn to scale), and always contain the integrin-binding tripeptide, Arg-Gly-Asp (RGD). The shadowing of exons illustrates those exons known to be involved in splice variants. ENAM is a more distantly related gene that has two noncoding 5’ regions and is also likely to contain disulfide bonds that other SIBLINGs do not. BSP: bone sialoprotein; DMP: dentin matrix protein; DSPP: dentin sialophosphoprotein; MEPE: matrix extracellular phosphoglycoprotein; OPN: osteopontin; ENAM, enamelin. Source: courtesy of Dr. Larry W. Fisher.

FIGURE 11.10  The osteopontin molecule is composed of numerous stretches of α helix (depicted as cylinders) interconnected in several cases

by α-pleated sheets, one of which contains the cell attachment consensus sequence (RGD). A stretch of polyaspartic acid (Poly Asp), along with phosphorylated residues (PO4), make osteopontin a highly acidic molecule. Source: adapted from Denhardt and Guo (1993) [167].

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dephosphorylated form does not have this effect [78]. The OPN knockout mouse similarly has increased bone mineralization [79], consistent with an in vivo role as a regulator of mineral proliferation. Interestingly, the OPN knockout mouse is also resistant to bone loss induced by ovariectomy, mechanical unloading, etc., suggesting that it plays a role in controlling bone resorption (see [80] for an example and for other references). c.2. Bone sialoprotein. BSP is another major noncollagenous SIBLING that accumulates in cement lines and in spaces between mineralized collagen fibrils. This glycoprotein, somewhat more bone specific than OPN, BSP is a heavily sialylated glycoprotein, formerly known as BSP-II. BSP can comprise up to 10% of the noncollagenous protein of bone, depending on the animal ­species and the type of bone analyzed. BSP has an apparent molecular weight of approximately 75 kDa as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and is composed of 50% carbohydrate (12% sialic acid, 7% glucosamine, and 6% galactosamine) (Fig. 11.11). BSP is rich in aspartic acid, glutamic acid, and glycine. Biosynthesis of BSP is tightly coordinated with the maturational stage of osteoblastic cells, and it is only produced in cultures that are actively mineralizing. In vitro, BSP acts as a HA nucleator [81], and in cell culture it associates with BAG-75 (see c.3 below) to promote mineralization [82]. The BSP knockout mice show impaired bone healing and bone formation, as well as impaired osteoclast activity but the actual function of BSP is difficult to determine because of redundant expression of other SIBLING proteins [83,84]. c.3. Bone acidic glycoprotein-75. Another sialoprotein originally isolated from rat bone that is not a member of

FIGURE 11.11  Sequence analysis of bone sialoprotein predicts the presence of multiple stretches of polyglutamic acid (Poly-Glu) in the first half of the molecule and tyrosine-rich regions in the amino- and carboxy-terminal domains. In the carboxy-terminal region, many of these tyrosines are sulfated. The cell attachment consensus sequence (RGD) is flanked by such regions at the carboxy terminus of the molecule. The molecule is composed of ∼50% carbohydrate, including a high concentration of sialic acid residues. Glycosylation is somewhat restricted to the amino terminal 50% of the molecule. Source: adapted from Fisher et al. (1990) [168].

the SIBLING family has an apparent molecular weight of ∼75 kDa and hence is called bone acidic glycoprotein-75 (BAG-75) [85]. BAG-75 is heavily glycosylated and contains 7% sialic acid and 8% phosphate. Thirty per cent of the residues in this protein are acidic in nature. Whereas in culture, cells from soft connective tissues have been found to synthesize low levels of this protein, BAG-75 is found only in bone, dentin, and growth plate cartilage. The BAG-75 protein binds with high affinity to both HA and Ca2+ ions, as well as to collagen. Immunolocalized next to cells in bone and concentrated in newly formed osteoid, this protein may facilitate the interactions of OPN (a mineralization inhibitor) and BSP (a nucleator) [82]. c.4. Dentin matrix protein-1 Another SIBLING protein, DMP-1, is highly homologous to BAG-75 [86], but also contains a GAG chain. DMP-1 is expressed specifically in mineralized tissue cells including odontoblasts, hypertrophic chondrocytes, osteoblasts, and osteocytes, with the highest levels of expression being in hypertrophic chondrocytes and osteocytes [87,88]. A phosphorylated 57-kDa C-terminal peptide of DMP-1 and an N-terminal peptide were also identified from teeth and was an effective nucleator of HA formation in solution, however, the full-length phosphorylated form of DMP-1, is an effective mineralization inhibitor. A glycosaminoglycan containing peptide derived from DMP-1 is also an effective mineralization inhibitor [76]. The DMP-1 knockout mice have hypomineralized bones and teeth [89], and impaired osteocyte function (see Chapter 10). In addition, these mice were shown to overexpress MEPE (see below), another potential mineralization inhibitor that was found in rodent bones and teeth. Patients with mutations in DMP-1 [90] also have rickets and impaired osteocyte function. Similarly, sheep with DMP-1 mutations have rickets [91]. It was not certain, however, if the impaired mineralization is due to altered expression of other regulatory proteins or to the direct actions of DMP1. The overexpression of DMP-1 in mice resulted in increases in mineralization, bone length, and mechanical properties, in the absence of other major genetic alterations, indicating that DMP-1 is directly involved in the mineralization process [92]. Its calcium binding activity also suggests a role in regulating osteoblast activity [93]. c.5. Dentin sialophosphoprotein. DSPP is a single gene, but an intact protein has not yet been isolated. However, two DSPP products, DSP and DPP, which are differentially phosphorylated and glycosylated, are expressed at moderate levels in bone [94]. DPP has been reported to regulate type I collagen fibrillogenesis and serve as an effective nucleator for HA formation at lower concentrations and an inhibitor at higher concentrations, whereas DSP, which is not phosphorylated and is a glycoprotein is not an effective modulator of in vitro mineralization [95]. Crystals formed in the presence of DPP were larger than

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Osteocalcin

those formed in its absence, suggesting that secondary nucleation is blocked [96]. Furthermore, studies suggest that unphosphorylated DPP has no effects on mineralization, whereas the intact protein is a nucleator, but the sites that must be phosphorylated for mineralization to occur, and for proper interaction with fibrillar collagen, are not known. The DSPP knockout mice have significantly more bone during development, while at maturity (9 months) the amount of bone was decreased. The young knockout animals’ bones had apatite crystals with higher crystallinity (crystal size/perfection) than wild-type controls, but this was reversed in the mature animals [97]. These data suggest a role for the DSPP gene in both dentin and bone mineralization. c.6. Matrix extracellular phosphoglycoprotein. MEPE, another member of the SIBLING family, is 525 residues in length with a short N-terminal signal peptide. This protein was originally identified in oncogenic hypophosphatemic osteomalacia tumors, which are characterized as a bone disease with abnormalities in mineralization [98]. MEPE in solution promotes HA formation when intact, while its fragments, specifically an acidic, s­ erineand aspartic acid-rich motif (ASARM), located in the C terminus of MEPE, is an inhibitor [99,100]. When dephosphorylated, both the intact protein and the ASARM ­peptide fragment had no effect on mineralization, and the phosphorylated ASARM fragment does not bind to HA crystals [100]. Rat and mouse osteoblast cultures lacking MEPE show increased mineralization and human osteoblasts decrease MEPE expression as mineralization progresses [101]. MEPE knockout mice have increased trabecular bone at 1 year, are resistant to remodeling and have increased dentin mineralization compared to control mice at the same age [102]. Transgenic mice overexpressing MEPE show growth and mineralization defects with altered vascularization. There was both a decrease in bone remodeling, and resistance to diet-induced renal calcification. Osteoclastic precursors were unable to differentiate in the presence of osteoblasts, as they normally do [103]. This suggests that MEPE, like many of the other glycoproteins discussed and the other SIBLING proteins, plays multiple roles in bone, regulating both osteoblast function and the mineralization process. MEPE is also involved in regulation of phosphate levels (see Chapter 16).

activity. Osteocalcin is the major ­Gla-containing protein, playing an important role in mineralization and remodeling of bone, whereas MGP is known to be more involved in regulating the calcification of cartilage. Reports also link osteocalcin and other osteoblast produced factors to metabolic functions including, but not limited to, glucose regulation [104,105].

OSTEOCALCIN Osteocalcin (Fig. 11.12) comprises up to 15% of the noncollagenous protein in bone, although the level is variable depending on the animal species, bone type, and age. Osteocalcin was initially reported to be virtually exclusive to bone and was considered the only ­bone-specific protein. It is now known to be expressed in other connective and non-connective tissues as well, including calcified cartilage [106], osteoarthritic articular cartilage [107], the nucleus pulposus of the intervertebral disc [108], dental pulp and newly forming dentin [109], and adipose tissue [110]. The proposed functions for osteocalcin in later stages of bone formation and remodeling have been extensively reviewed [111–113]. During bone development, osteocalcin production is very low and does not reach maximal levels until late stages of mineralization [114,115]. In the osteocalcin knockout mouse, the mineral crystals in the bones fail to mature [116], suggesting a role in regulating bone mineral maturation. The report that the osteocalcin-null

Gla-Containing Proteins Bone contains a number of proteins that are post-­ translationally modified by vitamin K-dependent FIGURE 11.12  Osteocalcin contains two stretches of α helix enzymes to form the amino acid, γ-carboxy glutamic (­depicted as cylinders) and two regions of α-pleated sheet (arrows). acid (Gla). Due to the sequence requirements of the The α-carboxylated residues of glutamic acid in the amino-terminal helix orient the carboxyl groups to the exterior, thereby conferring calcarboxylating enzymes, the Gla proteins of bone share cium ion binding with relatively high affinity. There is one intramosome sequence homology with certain blood coagula- lecular disulfide bridge (C–C) in the middle region of the molecule. tion factors that require γ-carboxylation to maintain their Source: adapted from Hauschka and Carr (1982) [169]. II.  CELLULAR, MOLECULAR, AND DEVELOPMENTAL BIOLOGY OF BONE

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mice were fatter led to the suggestion that osteocalcin is also involved in glucose metabolism [104], a nonbone related function. Osteocalcin has also been suggested to function as a neuropeptide [111]; all of these nonmineralization functions are beyond the scope of this review.

MATRIX GLA PROTEIN The other major Gla-containing protein in the skeleton is MGP, which is predominantly expressed in soft tissues [117]. MGP is a hydrophobic, phosphorylated Gla protein, and is an in vivo inhibitor of both vascular and cartilage mineralization. Mice in which the MGP gene was deleted died prematurely because of massive calcification of their tracheal cartilage and blood vessels [117]. In light of the data in the knockout animals and in cell culture, it seems likely that expression of this protein may be a protective action by the cell against unwanted calcification. This concept is supported by the finding that addition of phosphate stimulates the expression of MGP in osteoblasts, providing a negative feedback loop inhibiting mineralization [118].

PERIOSTIN Periostin, a ∼90-kDa secreted protein, is another matricellular protein that contains Gla, and was so named because of its expression in the periosteum of bone and periodontal ligament [119]. Responsive to mechanical stress, it interacts with extracellular collagen and with cell-bound integrins. One of its major suggested functions is regulation of fibrillogenesis. In periostindeficient mice, cortical bone in the femur shows a decrease in area and thickness [120], associated with a decrease in collagen cross-linking. This in turn is associated with a decrease in the activity of the enzyme, lysyl oxidase, which is required for collagen cross-­ linking. Whether it plays a direct role in mineralization, or whether alterations in mineralization seen in the knockout mice (including decreased expression of other bone matrix proteins associated with mineral formation [121]) are due to the altered collagen fibrillogenesis or a direct effect of periostin remain to be determined. The role of other proteins in regulating mineralization will be summarized in the following section.

FIGURE 11.13  Cell-mediated matrix mineralization in developing chicken bone. Electron micrograph showing a 17-day-old embryonic tibia, stained with uranyl acetate and lead citrate. Mineral clusters (C) outside of the osteoblast (OB) are associated with collagen (thin arrows) and extracellular matrix vesicles (inset). Empty vesicles (thin arrows) as well as vesicles with mineral are seen. Source: courtesy of Dr. Steven B. Doty.

from 20 to 60 A [122]. Bone mineralization is thus distinct from solution-mediated Ca-phosphate precipitation, in which similarly sized, non-oriented small crystals are formed and ripen to appreciably larger sizes [122]. Bone mineral crystals are also distinct from geologic apatite formation, in which high temperatures and pressures yield extremely large single crystals. There are two regulated steps involved in the mineralization process: 1) nucleation, in which the initial crystals are formed via an energy requiring process, and 2) growth and proliferation of these crystals, a process known as crystal growth. Both of these processes have been the subjects of a number of reviews and readers are directed to a selected few of them for further detail [123–128]. To appreciate the mineralization process it must be recognized that while the bone cells direct the process, there is a physical chemical component that must be understood.

Physical Chemistry of Mineralization THE MINERALIZATION OF BONE MATRIX Bone mineral (HA) crystals are arranged in an oriented fashion on a collagen-based matrix (Fig. 11.13), with a very limited size distribution ranging in length

Calcium phosphate precipitation from solution can yield a variety of phases, depending on the pH, the calcium to inorganic phosphate (Ca/Pi) ratio and the solution supersaturation. When the pH is in the physiologic range (7.4–7.8), apatite formation occurs with solution Ca/Pi molar ratios as high as 2:1 and as low as 1:1 as

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The Mineralization of Bone Matrix

long as the solution is supersaturated with respect to apatite (i.e., has a Ca × P product that exceeds the solubility product for apatite, ∼10–35 [129]). Depending on the solution supersaturation, intermediate phases such as amorphous calcium phosphate [130–132], octacalcium phosphate [133], or other transient intermediates may form, instead of a small nanocrystalline imperfect HA structure [134], but in all cases the final product is HA. Equally apparent from requirements for physical mineral deposition is the essential presence of calcium and inorganic phosphate ions. Calcium ions may be supplied from the cells or from circulating or localized calciumbinding proteins. Phosphate ions may be derived from breakdown of pyrophosphate, an abundant metabolic product; from hydrolysis of phosphoesters or phosphoproteins; or from circulating inorganic phosphate ions. Studies have shown the up-regulation of the sodiumdependent Pi transporter, Pit-1 (also named Glvr-1) and NaPi IIb for calcification in cultures [135,136] and for mineralization of endochondral bones during embryonic development [137]. Apatite crystals develop in solution when individual ions or ion clusters associate in the same orientation as in the crystal lattice that they are trying to form. When sufficient ion clusters are correctly oriented, they can persist in solution and can serve as a “critical nucleus” for further crystal growth. Homogeneous nucleation, in which crystals form de novo, is a rare process. Thus, it is likely that in most instances of solution-mediated apatite deposition, nucleation occurs on foreign materials such as dust, scratches on the container, and burette tips. Such heterogeneous nucleation yields the initial crystals, which then facilitate additional growth by the process of secondary nucleation. In secondary nucleation, growth sites on the preformed apatite crystals serve as branch points for the formation of new crystals, analogous in many ways to the branching of the growing glycogen molecule during glycogenesis. Proliferation by secondary nucleation results in numerous small crystals. Crystal growth in the absence of secondary nucleation would result in fewer but larger crystals. This suggests that most of the crystals in bone form by a secondary nucleation-like process or by growth from individual nuclei. Unfortunately, what regulates crystal size in bone cannot be determined from studies of protein-free solutions. Some insight into the factors that regulate mineralization comes from studies of abnormal calcification found in human blood vessels and other soft tissues, and in transgenic animals as indicated below. The studies of vascular and soft tissue calcifications are complicated by the observation that many of the proteins found in these deposits accumulate due to their affinity for HA, and when excessive ­mineral deposits, these proteins may accumulate due to this affinity rather than to a direct role in the calcification process. Confirmation of their roles usually is derived

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from gene expression studies and from studies in transgenic animals.

New Insights into Bone Mineralization Genetic manipulation of mice and their cells, naturally occurring mutations, and chemically modified matrices have begun to shed light onto the mechanism of bone mineralization. Analyses of diseased tissues and tissues from transgenic animals indicate that there are a number of cellular and extracellular factors essential for physiologic mineral deposition. Physiologic bone mineral deposition requires a collagen-based matrix as the HA in bone is oriented on a collagenous matrix. This requirement is emphasized by analyses of bones from animals and humans with OI, a disease associated with either quantitative or qualitative defects in collagen structure [14,15]. These bones have smaller HA ­crystals than ­normal [16,138,139], and an increased relative abundance of mineral that is not associated with collagen [139]. Although in some cases the defective mineralization in the OI bones may also be attributed to altered matrix protein production or retention [140], the phenotypes associated with defective collagen production argues for the importance of collagen for physiologic bone formation. Similarly, since fibronectin forms the basis on which collagen is deposited [72], it must also be a requirement. But the roles of many other factors are not conclusively established. There are several key questions concerning the processes of HA nucleation and growth in bone that are in debate. The first question concerns the nature of the first mineral precipitate. The concept that an unstructured mineral cluster (e.g., amorphous calcium phosphate) could represent the first mineral has been debated for more than forty years [129–131]. Although it is well accepted that the majority of bone mineral is a nanocrystalline HA with many impurities, recent data based on cryomicroscopy has indicated that there may be an amorphous phase in embryonic bone and related tissues [7]. The energy required to form this amorphous cluster would be less than that required to form even the smallest HA nanocrystals. This amorphous calcium phosphate would then transform to poorly crystalline HA, and these HA crystals would then continue to grow and proliferate. A highly hydrated nanocrystal of HA could have properties like an amorphous calcium phosphate [141], hence this question is not completely resolved. The second question is where the initial mineral deposition takes place, be it amorphous or nanocrystalline. Is the initial locale on the collagen matrix as originally suggested almost 50 years ago [142,143] and recently reviewed [144]? Does the first mineral form in mineralization foci that have been observed in mineralizing cell

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culture [82,85,124] that are somehow linked to the collagen matrix? The recent discovery that type XI collagen is found in the mineralization foci along with BSP and BAG-75 suggests that this fibril-binding collagen may link the mineralization foci to the collagen [124]. It has also been suggested that initial mineralization occur in extracellular matrix vesicles [55,125], which may not be directly associated with the collagen matrix, but serve as the template for the mature bone mineral. Extracellular matrix vesicles [145] are formed by osteoblasts with membranes enriched in acidic phospholipids and annexins [146]. They also contain the enzymes needed for matrix breakdown [147] including the matrix metalloproteases [147] and a series of phosphohydrolases (alkaline phosphatase, nucleotide pyrophosphatase/ phosphodiesterase-1) [148] and Phospho-1 [58], which function to regulate phosphate levels. As reviewed elsewhere [58], knockout of each of these phosphatases inhibits mineralization. Similarly, ablation of one of the membrane proteolipids that is upregulated during osteoblast differentiation resulted in decreased matrix vesicle formation along with decreased mineralization, implying a role for the matrix vesicles in the initial calcification process [149]. Bodies resembling matrix vesicles have been isolated from the mineralizing foci described above [124]. If the in situ mineralization process resembles what happens in vitro, this would suggest that the vesicles are produced concurrently with these foci, or as a component of these foci, perhaps to provide some of the enzymes needed to modify the matrix as mineralization takes place. The fact that there is some mineralization even when the matrix vesicle enzymes are absent suggests that they are not solely responsible for the initiation of mineralization. The third question is how the growth of the initial mineral crystals, once formed, is controlled to yield the small range of crystal sizes and high orientation of the collagen-associated HA. Since the mineralization process starts at multiple sites on collagen fibrils, it has been suggested that the spacing of the collagen fibrils controls the size of the mineral [150]. Calculations demonstrate that collagen has charged clusters that could serve as initial binding sites for calcium and directly support HA nucleation [151], as well as regulating growth, without the need for regulation by non-collagenous proteins. However, since most of the non-collagenous proteins discussed above bind to specific sites in the collagen matrix and between the collagen fibrils [18,140], it is equally likely that they could regulate the crystal size and orientation. Collagen appears to act as a scaffold for mineral deposition. As the major (∼90%) structural proteins in matrix of bone and skin, collagens do not by themselves induce the formation of mineral crystals. However, they do serve as scaffold to form a highly orientated “backbone” supplying appropriate sites for retention of noncollagenous proteins

and initiation of mineral deposition. The “­ nucleators” or initiators of mineral crystal formation based on in vitro studies include: BSP [81], bone acidic protein-75 [82], the phosphophoryn component of DSPP [152], fragments of DMP-1 [76], and proteolipids [148]. Such proteins may bind calcium and/or inorganic phosphate ions, forming a surface that resembles the apatite crystal surface. In this manner, the protein serves as an epitaxial (similar surface) nucleator, thereby providing a surface for the start of nucleation. Alternatively, the proteins may participate in the formation of mineralization foci. There appear to be many more noncollagenous proteins that can inhibit HA formation in vitro and in vivo than there are nucleators. This is most likely because much of the collagen in the body is not mineralized, while it is often exposed to elevated levels of circulating calcium and phosphate. Inhibitors of mineral crystal formation include aggrecan [153], matrix gla protein [154], OPN [79], full-length DMP-1 [76], MEPE [99], alpha2-HS glycoprotein (fetuin) [123] and albumin [155] (both adsorbed from serum), and of course pyrophosphate [156]. The proteins can chelate calcium or phosphate ions, reducing the fluid supersaturations, which in turn could prevent crystal nucleation and/or growth. The proteins and ions such as pyrophosphate also can form a protected environment around the crystal nucleus, or one face of the crystal surface, sequestering it and thus preventing crystal growth, or stabilizing the nucleus, protecting it from the external environment. Inhibitors such as osteocalcin, vitronectin, and MGP block the growth of mineral crystals as they bind to one or more faces of the growing crystal because their side chains match positions in the lattice, thereby blocking growth in one or more directions or even blocking growth beyond a specific size. Most of these noncollagenous proteins exist in the form of random coils (intrinsically disordered proteins [74]), enabling them to interact with cells, HA, and collagen with high specificity, adapting their conformation to the structure of their partners. Many of the noncollagenous proteins are known to organize the matrix composition, including: decorin, TSP, fibronectin, vitronectin, versican, and the SIBLINGs. These proteins bind to the collagen backbone of the matrix and other noncollagenous proteins, altering their flexible conformations and their ability to affect crystal nucleation and growth according to the pathways described previously. In addition, these proteins may bind to cell surface via special sequences (RGD) and thus mediate cell–cell and cell–matrix attachments, resulting in a change in the extracellular Ca × Pi concentration or the pH of microenvironments. The ultrastructural studies that combine x-ray crystallographic and electron microscopic techniques provide illustrations for each of these mechanisms for the formation of larger crystals of calcium carbonates, calcium sulfates, brushite, and octacalcium phosphate [157–159]. For example, fibronectin has been shown to bind to

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Acknowledgments

the ionic surfaces of calcite that did not include water molecules, but it does not bind at all to brushite whose surfaces all have bound water [159]. The acidic macromolecules from sea animals have been shown to determine the shape of calcite crystals [160] and other crystals [161,162]. Scanning electron micrographs have similarly been used to identify the binding sites for polyaspartic acid, mollusk shell proteins, and rat dentin phosphoprotein on the surface of octacalcium phosphate [163], while molecular dynamics calculations have been used to predict interactions of matrix proteins with HA crystals [154,162,164,165]. Although there is no direct evidence of the exact nature of the matrix protein–mineral interaction, there are examples of each of these mechanisms from solution studies of apatite formation. Studies of the effects of bone matrix proteins on apatite formation include those in which preformed seed crystals are added to Ca × Pi solutions, and the rate of crystal growth is ­determined at fixed Ca × Pi and fixed pH, or variable Ca × Pi × OH. Other studies have examined the formation (nucleation and growth) of apatite from solutions in the presence of insoluble proteins, proteins immobilized on polyanionic beads, or proteins in solution. Diffusion studies [166], in which the protein is held within an agarose, silicate, fibrillar collagen or denatured collagen gel, have also provided insight into apatite nucleation and growth. From such studies, one can also find examples of the mechanisms listed previously. However, it should be emphasized that because of its affinity for apatite, a protein in low concentrations may act as a nucleator and in higher concentrations may serve to regulate crystal growth. Moreover, promoting or inhibiting mineralization in situ is also dependent on the extent of post-­ translational modification, such as phosphorylation, and on the regulation of collagen fibrillogenesis, which may induce conformational alterations.

ACKNOWLEDGMENTS We thank Drs. Paolo Bianco, Larry W. Fisher and Steven B. Doty for providing photographic materials. Dr. Boskey’s research as discussed in this article was supported by National Institutes of Health (NIH) grants DE04141, AR037661, AR046121, and AR41325. Dr. Robey’s ­research discussed in this article was supported by the DIR, N ­ ational Institute of Dental and Craniofacial Research, of the Intramural ­Research Program, NIH, DHHS.

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