Mineralization in Mammals

C H A P T E R

23 Mineralization in Mammals Adele L. Boskey

†

Hospital for Special Surgery, New York, NY, United States; Weill Medical College, New York, NY, United States; Graduate School of Cornell University, New York, NY, United States

O U T L I N E The Influence of Vitamin D on Bone Matrix Proteins

Introduction383 Definitions383 Direct and Indirect Effects of Vitamin D and Vitamin D Metabolites on Mineralization 384 Physical Chemistry of Mineralization 384 The Nature of Vertebrate Mineral 385 Mineralization Mechanisms in Bone, Cartilage, Dentin, and Pulp 386 Methods for Quantifying Tissue Mineralization Where Is the Tissue Mineralized? How Much Mineral Is Present? Is the Mineral Characteristic of Physiologic Mineral (Is It a Poorly Crystalline Apatite)? Methods for Studying In Vitro Mineralization

Mechanism of Effects of Vitamin D on Mineralization 391 Physicochemical Effects 391 Effects of Vitamin D on Cells and Matrix Molecules 393

INTRODUCTION Definitions “Biologic mineralization,” is the physicochemical process leading to deposition of inorganic crystals (minerals) on an organic matrix within the cell or outside it. This term is more specific than “mineralization,” as biologic mineralization implies a relation between the cells, organic matrix, and the mineral. The cell-mediated biomineralization process comprises an oriented deposition of mineral within, or adjacent † Deceased,

Growth Factors

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Mineralization and Mineral Properties in Systems With Vitamin D Alterations 394 Vitamin D Deficiency 394 Vitamin D Receptor Alterations

Vitamin D-Binding Protein Knockout 1α-Hydroxylase Knockout Osteoporosis and Osteomalacia in Humans

387 387 388 389 391

395

396 396 397

Conclusions397 Acknowledgments398 References398 Further Reading

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to cells, or on an extracellular matrix, reviewed elsewhere [1]. Examples of the former include iron oxides and sulfides in magnetotactic bacteria and silicates in diatoms; examples of the latter include calcium carbonates in shells or exoskeletons and calcium phosphates in bones and teeth. The mineral in physiologically calcified vertebrate tissues is an analogs of the geologic mineral hydroxyapatite (HA) (Fig. 23.1). Physiologic HA crystals are 10–60 nm in their largest dimension [2], far smaller than those found in geologic deposits, and stoichiometries different from the predicted 10 Ca: 6 PO4:2 OH of geologic mineral. It is for that reason that biologic

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Vitamin D, Volume 1: Biochemistry, Physiology and Diagnostics, Fourth Edition http://dx.doi.org/10.1016/B978-0-12-809965-0.00023-9

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Collagen393 Noncollagenous Proteins 394

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© 2018 Elsevier Inc. All rights reserved.

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TABLE 23.1  Pathologic Mineral Deposits

FIGURE 23.1  The hydroxyapatite unit cell showing the major substituents occurring in biologic apatites and the ions for which they substitute.

vertebrate mineral is often referred to as “apatite” or “apatitic” meaning “like hydroxyapatite.” This chapter will focus on physiologic and aberrant apatite formation in situ and in culture, as well as on the effects of vitamin D on mineral formation. Crystalline deposits may form by several different mechanisms [1]. De novo crystal deposition occurs when the solution supersaturation exceeds the solubility of the precipitating phase. Supersaturation refers to the ratio of the solution ion product to the solubility product of the phase in question. The process starts with “nucleation,” in which several ions or ion clusters (prenucleation clusters) come together in solution, with the orientation they will have in the final crystal. This first step requires a great deal of energy, and is facilitated by increasing the ion product or reducing ionic diffusion in solution. Prenucleation clusters can form an initial noncrystalline or amorphous phase, which can be stabilized or transformed to a crystalline phase [3]. Most biomineralization is epitaxial or heterogeneous— occurring on the surfaces of preexisting crystals or on protein and lipid templates, which resemble the surface of the crystal [4]. These processes use much less energy than the de novo mineralization process, and require less supersaturation. Crystal growth occurs as ions and ion clusters add on to the surface of the initial nuclei or other preexisting crystals. Crystal growth requires less energy than nucleation and, is limited in the case of biologic mineralization, by the template on which the crystals are deposited. Crystals can grow in all dimensions by the addition of ions [4], by agglomeration [5] in which crystals accumulate, not always in an oriented fashion, or by secondary nucleation. Secondary nucleation, seen with HA crystals maturing in solutions in the presence of phosphophoryn, a dentin phosphoprotein [6], is a branching process in which new nuclei form on the surfaces of existing crystals, resulting in a new population of immature crystals. Among the vertebrate mineralized tissues, enamel contains the largest apatite crystals, which do contain carbonate [7] and other environmental contaminants (e.g., strontium, fluoride, lead, etc. [8]). Bone and tendon-bone insertions contain the smallest apatite mineral crystals [9] with 6%–10% carbonate [10], as well as adsorbed and incorporated citrate, fluoride, and other trace impurities. Bone apatite crystals are also hydroxide-deficient [11], but are not totally devoid of them. Cementum and dentin mineral crystals have intermediate sizes and accumulation of foreign ions. In each of these tissues, crystals that are formed initially are smaller than those found in mature tissues due to both growth of existing crystals

Mineral Phase

Found In

Affected by Vitamin Da

Apatite

Blood vessels Kidney and bladder stones Salivary stones Pulp stones Muscle Skin Hyaline and articular cartilage

+ + + + + + +

Calcium carbonate (aragonite) (calcite)

Pancreatic stones Calcified intrauterine devices

0

Oxalates

Kidney and salivary stones Soft tissues

+

Pyrophosphates

Hyaline and articular cartilage

0

Urates

Hyaline and articular cartilage Kidney stones

+ +

aDeposits

influenced by vitamin D are shown with +; those where there is no reported effect by 0.

and the removal of smaller crystals during osteoclast remodeling. Mineral crystal size and perfection (absence of vacancies or impurities), therefore, increases with age [12]. In humans, mineral also deposits at abnormal (pathologic) locations [1,13]. This mineral is frequently apatitic, however, calcite (calcium carbonate) is found in pancreatic stones alone [14]. Monosodium urate, sodium pyrophosphate, and apatite [13] are found in cartilage, whereas brushite, oxalates, and uric acid are found in kidney and salivary stones (Table 23.1). The mineral in pathologic calcifications may be formed by physiologic processes or may be associated with dying cells (dystrophic calcification), or metastasis [13]. An example of dystrophic calcification induced by vitamin D toxicity is seen in vasculature, and is mediated by the vitamin D receptor (VDR) (see Chapter 9) [15]. In most cases the tissue becomes bone-like, and genes associated with osteogenesis are activated [16–18]. Of note, blocking VDR activation decreases this osteogeniclike calcification in culture [19]. Dystrophic calcifications are distinct from physiologic mineralization, as in the latter, viable cells are required; while in the former, cells die, releasing calcium, phosphate, and degradative enzymes [20].

DIRECT AND INDIRECT EFFECTS OF VITAMIN D AND VITAMIN D METABOLITES ON MINERALIZATION Physical Chemistry of Mineralization De novo apatite formation, requires Ca+2, PO4 − 3, and OH− ions to come together in the correct orientation with sufficient energy and in sufficient numbers to form the first stable apatite crystal (nucleus). Following formation, additional ions can be added on to these small crystals (nuclei) triggering crystal growth. As crystals become larger, new nuclei can branch off the surface, a phenomenon termed secondary nucleation, in a fashion analogous to glycogen formation. These new nuclei

III. MINERAL AND BONE HOMEOSTASIS

Direct and Indirect Effects of Vitamin D and Vitamin D Metabolites on Mineralization

grow and form additional secondary nuclei in an exponential fashion. During cell-mediated biologic apatite formation, matrix molecules provide sites for the accumulation of ions and serve as templates to orient mineral growth. Some molecules may act as heterogeneous nucleators facilitating deposition of the mineral. Others, may bind to the crystals and regulate their shape and size by limiting growth along specific crystal faces [1]. Nucleation occurs at multiple sites along these templates, and crystal habit (shape) and size are regulated by the template and by other matrix proteins, discussed later, that bind to the surface of the apatite crystals. In cartilage, bone, dentin, cementum, tendon, and ligaments, mineral crystals form on a collagenous matrix. This collagen functions as a “template” [21] for initial mineral formation and crystal growth; recent studies further suggest that initial nucleation occurs at specific regions within the collagen fibrils [22]. The mineralization of the collagen matrix gives it increased mechanical strength [23]. Differences in distribution, posttranslational modification, and conformation [1,24] of extracellular matrix molecules associated with fibrillar collagen influence the crystal size and site of initial deposition. In enamel, which does not have a collagen component, amelogenin, a large hydrophobic intrinsically disordered protein (IDP) regulates mineral formation [1]. IDPs assume different conformation when they bind to their partners; for amelogenin these include HA and the protein, enamelin. Amelogenin associates into nanospheres and linear polymers, depending on binding partner, and induces initial in vitro calcification [25,26], facilitates the ordering and agglomeration of crystals, and regulates the size to which these crystals grow [27]. There are several additional proteins in enamel that initiate and regulate mineralization [1]; mice lacking amelogenin, however, form less enamel [28] and the enamel formed is disorganized and smaller in size than that present in wild type. Amelogenin expression is regulated by vitamin D [29], thus early enamel maturation and mineralization and dentin hypomineralization were observed in VDR knockout (KO) mice (VDR−/−) [30]. As discussed latter the early enamel maturation in these mice may be indicative of altered local calcium and phosphate levels. One of the ways in which vitamin D influences mineralization is by stimulating the formation of matrix proteins and the enzymes responsible for their posttranslational modification [31]. The genes for many of these proteins, as well as many of the enzymes that posttranslationally modify them, have vitamin D-responsive elements (VDRE) [30]. The processing of collagen itself is mediated in vitro [32] and in vivo [33] by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), which regulates the activity of lysyl hydroxylase (LH) and lysyl oxidase (LOX), which, in turn, increases the number of collagen cross-links and stabilizes the collagen fibrils, thus facilitating collagenbased mineralization [34]. Several of the phospholipases, reviewed in detail elsewhere [35], including phospholipase A2 [36], phospholipase D [37], phospholipase C [38], and sphingomyelinase [39], important not only as a source of inorganic phosphate (providing substrates for alkaline phosphatase), but also as regulators of cell shape and function, are reported to have VDREs. Extracellular matrix proteins that in solution,

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or in culture, affect the formation of apatitic mineral along with enzymes and growth factors known to affect mineralization, which have VDREs listed in Table 23.2. The major way in which vitamin D affects mineralization is by increasing local calcium and phosphate concentrations, possibly regulating magnesium [60] and pyrophosphate concentrations as well. 1,25(OH)2D reduces pyrophosphate levels (an inhibitor of HA deposition) by controlling transcription of ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) and 3 (Enpp3) and progressive ankylosing (inorganic pyrophosphate transport regulator, Ank). Vitamin D also tends to reduce tissue nonspecific alkaline phosphatase messenger RNA (mRNA) levels when VDR is functional [54]. In VDR deficient mice, in contrast, 1,25(OH)2D3 increases pyrophosphate levels by regulating the proteins involved in pyrophosphate metabolism, which in turn suppresses mineralization [54]. It is important to remember that vitamin D-deficient animals can have their mineralization phenotype “rescued” by treatment with calcium and phosphate. VDR KO mice, however, are not totally rescued by such treatment implying that the protein factors discussed above are not activated in this model [36]. These data indicate the redundancy of the vitamin D system in the control of the mineralization process. Cells, both directly and indirectly control mineralization by regulating local calcium and phosphate concentrations and pH [61] as well as by the production and posttranslational modification of collagen, noncollagenous proteins [1,24], and transcription factors, which guide and regulate initial mineral deposition and crystal growth. There is also a physicochemical process by which supersaturated solutions lead to mineral deposition in unwanted sites. One example of this physicochemical effect is the hypervitaminosis D syndrome [62], where injections of vitamin D into animals cause an elevation of circulating calcium and result in arterial and kidney calcification.

The Nature of Vertebrate Mineral An analogs of the geologic mineral, HA, is found in physiologically mineralized tissues (calcified cartilage, bone, dentin, cementum, and enamel) and many aberrant calcifications. The chemical formula for HA is Ca10(PO4)6(OH)2. With the exception of enamel, physiologic HA is deposited in an oriented fashion on a collagen template. Unlike geologic HA crystals, which are quite large and visible to the naked eye, the physiologic mineral crystals are microscopic in size, <20 nm in their longest dimension in collagen-based tissues, and ∼1000 nm in the longest dimension in enamel. Because of the small crystal size (some crystals contain only a few unit cells) and the relatively large surface area, the physiologic HA crystals contain a large number of surface impurities and vacancies. The presence of such imperfections tends to make the crystals more soluble than geologic HA [63]. This solubility is essential so as to enable remodeling and facilitate mineral homeostasis. The mechanisms of this homeostasis are highly dependent on vitamin D metabolites reviewed in Introduction section.

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TABLE 23.2  Extracellular Matrix Proteins and Enzymes Associated With Mineralization and Regulated by Vitamin D: Solution Data Effect on Mineralization

Regulated by Vitamin Da

Amelogenin

Regulator of crystal growth [27]

+ [29]

Biglycan

Nucleator [40]

+ [41]

Bone sialoprotein (BSP)

Nucleator [42]

+ [43–45]

Collagen I

Template

Protein EXTRACELLULAR MATRIX PROTEINS

Nucleator [21]

+ [32,33]

Dentin matrix protein-1 (DMP1)

Nucleator/inhibitor [46]

+ [47]

Dentin phosphophoryn

Nucleator [6]

+ [48]

Dentin sialoprotein

Weak nucleator/inhibitor [1]

0 [48]

Matrix gla protein (MGP)

Inhibitor

+ [32]

Matrix extracellular phospho-glycoprotein (MEPE)

Inhibitor

+ [32]

Osteopontin

Nucleator/Inhibitor [49,50]

+ [50]

Osteocalcin

Inhibitor [51]

+ [52]

ATPase

Increase local Pi concentration

+ [53]

Progressive Ankylosing (ANK)

Transport pyrophosphate thru membranes

+ [36]

Ectonucleotide pyrophosphatase phosphodiesterase (ENPP1)

Forms pyrophosphate, an inhibitor of HA formation

+ [54]

Matrix metalloproteinases Stromolysin MMP3 Gelatinase MMP2

Removes/degrades proteoglycan inhibitors

+ [54]

Cystathionine β-synthase

Degrade matrix molecules that inhibit mineralization

+ [55]

PHEX

Decrease enzymatic collagen cross links

+ [56]

SOST

Inhibits matrix formation

+ [57]

Activin A

Regulates Mineralization

+ [58]

LRP5

LRP5, the Frizzled coreceptor-initiating canonical Wnt signaling

+ [59]

β-catenin

Stimulates osteoblast gene expression, inter alia

+ [59]

RUNX2

Osteoblast master-gene; effect depends on species

+ [59]

ENZYMES

TRANSCRIPTION FACTORS

HA, hydroxyapatite; PHEX, phosphate-regulating gene with homologies to endopeptidase on the X chromosome. aRegulation by vitamin D depends on concentration, cell type and cell maturity; hence + indicates that there is an effect or that the gene contains a vitamin D responsive element (VDRE). Readers are referred to other chapters and references provided to see precise effects.

Mineralization Mechanisms in Bone, Cartilage, Dentin, and Pulp In general, body fluids are supersaturated with respect to HA. In other words, the ion product [Ca]10 × [PO4]6 × [OH]2 exceeds the HA solubility product of 10−58 [63]. Biologic apatite, on the other hand, only deposits in the so-called “mineralized tissues” and generally not in skin, hyaline cartilage, blood, or other soft tissues. This is because body fluids and tissues contain numerous mineralization inhibitors. These inhibitors prevent de novo precipitation, protect the cells from

becoming engulfed in mineral, and regulate the size and shape of the crystals that do form. Mineralization inhibitors as mentioned above include both anionic molecules that can chelate calcium (citrate, ATP, pyrophosphate, glycosaminoglycans) and proteins that bind specifically to HA crystals (osteonectin, osteopontin, matrix-gla-protein, fetuin, and albumin) [1]. The deposition of mineral in bones and teeth occurs at specific sites where the barriers to crystal deposition are diminished either by elevating CaxPO4 concentrations, removing the inhibitors, or by exposing matrix molecules or structures that facilitate mineral deposition.

III. MINERAL AND BONE HOMEOSTASIS

Methods for Quantifying Tissue Mineralization

There are several steps in the formation of physiologically mineralized tissues, common to all the tissues, however, the details and the sequence vary across tissues. The first step is the differentiation of the precursor cells (marrow stromal cells, preosteoblasts, etc.) into a mature cell. These cells then secrete a matrix that can be mineralized. For bone, dentin, and cartilage it is predominantly a collagen matrix. For enamel it is mainly an amelogenin matrix. These matrices are then posttranslationally modified so that mineral deposition can be favored at specific sites along the collagen fibers (or on the amelogenin nanospheres). There is no physiologic mineral deposition until the matrix is deposited and modified. Mineralization begins simultaneously at multiple sites in a polarized direction by the nucleation of crystals. The molecular factors responsible for nucleation and the sequence in which they act are uncertain. In some mineralizing tissues, extracellular matrix vesicles provide a site for the accumulation of mineral ions away from inhibitors. In other tissues, mineralization appears to start on the collagen fibrils. Extracellular matrix vesicles [64], the site of initial mineral deposition in calcifying cartilage and mantle dentin (the first site of dentin mineral deposition), are exosomes [65,66] providing protected sites for the accumulation of calcium and phosphate ions and enzymes involved in deposition. Their membranes are also rich in the enzymes required to disrupt mineralization inhibitors in the extracellular matrix, such as ATPase, pyrophosphatase, alkaline phosphatase, and matrix metalloproteinases. Vesicle membrane structures are modulated by phospholipases [35], which in turn are regulated by VDREs. While the first few mineral crystals might be formed within the vesicle, most physiologic mineral is believed not to be formed by a vesicle-mediated process. When 1,25(OH)2D3 is added to osteoblast cultures before they start to mineralize, however, the extent of mineralization is increased due to the enhanced production of mature matrix vesicles [67]. It is not known whether vesicle-mediated calcification is a mandatory step in the calcification process, or just one that may facilitate the process. 1,25(OH)2D3 plays a crucial role in regulating both bone metabolism and mineralization of skeletal and dental tissues, where it promotes osteoblast [68] and odontoblast differentiation [69], by regulating extracellular signal-regulated kinases (ERKs) that control both differentiation of mesenchymal stem cells and skeletal development [70,71], along with differentiation of pulp cells [72]. 1α,25(OH)2D3 increases cell viability and enhances mineralization via phosphorylation of ERK leading to increased expression of alkaline phosphatase, dentin sialophosphoprotein, and dentin matrix protein 1 (DMP1), even in the absence of osteogenic promoters. Most physiologic mineral forms at multiple discrete locations on type I collagen fibrils, with the long axis of the crystals parallel to the length of the collagen fibrils [73]. It is generally thought that collagen itself does not support de novo apatite formation, although there are in vitro studies suggesting collagen can nucleate mineral particles [21,22]. The counterargument is that specific anionic noncollagenous proteins that associate with collagen [74], can bind to and stabilize the initially formed crystals and regulate the mineralization process

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[1]. In solutions and in cell culture, several of these proteins have been proven to be nucleators of HA and regulate HA growth and proliferation (Table 23.2). Their effects in the presence of fibrillar collagen are quite often different from effects in its absence [40,49,75,76]. These in vitro observations have been validated in animals in which these proteins are ablated (knocked out) or overexpressed (Table 23.3). One of the challenges associated with deciphering the mechanism of biologic mineralization is in determining which proteins are absolutely essential for initiation of mineralization and which are redundant, then finding the order in which these proteins are expressed in different mineralizing tissues. Also important is the modification or removal of the inhibitors of mineralization [94]. Genetic and proteomics studies that report the temporal expression of cartilage, bone, and tooth matrix proteins, are providing insights into these challenges as they have for the calcite forming sea urchin [95], the avian egg shell [96], and mature human enamel [97].

METHODS FOR QUANTIFYING TISSUE MINERALIZATION Several important questions must be addressed when examining an existing mineralized tissue or one in the process of being mineralized. Where is the tissue mineralized and to what extent? Is the mineral present characteristic of a physiologic one? (Is the apatite poorly crystallized and is it properly aligned with the collagen matrix? Is its composition different from that in an age- and background-matched control tissue? What size are the crystals?). Multiple techniques can be used to address these questions; these are illustrated by examples from vitamin D-related studies.

Where Is the Tissue Mineralized? Radiographic methods, including plain films, dual energy X-ray absorptiometry (DEXA), peripheral quantitative computed tomography (pQCT), high resolution pQCT, and microcomputed tomography (μCT), show changes in X-ray attenuation when a mineral is present. For example, plain films of rachitic (vitamin D-deficient) growing animals show enlarged epiphyses and reduced tissue density. Plain X-ray films are routinely used to reveal the presence of decreased mineralization and bowing bones, characteristic of vitamin D-deficient osteomalacia. Vitamin D toxicity is similarly recognized radiographically, as prematurely closed epiphyses and denser bones than normal in both animals and humans. The use of plain film radiographs and DEXA to assess reduced bone mineral content are well documented [98]. Here we focus on three-dimensional (3D) quantitative methods such as pQCT and μCT, which are capable of providing more detail on bone mineral content and bone density. QCT and its high-resolution peripheral counterpart (HR-pQCT) enable in vivo measurement of both cortical and trabecular density. The effects of vitamin D and calcium on bone morphology have been determined by pQCT [99,100], HR-pQCT [101,102] in humans, and μCT in humans [103].

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TABLE 23.3  Proteins Associated With Mineralization That Are Regulated by Vitamin D: Mineral Properties in Knockout (KO), Overexpression (Tg), and Mutant (Mu) Mice or Humans (h) Protein

Model(s)

Phenotype

Alkaline phosphatase (tissue nonspecific)

KO

Decreased mineral content; no detected change in crystal size [77]

Amelogenin

KO, Tg

Altered enamel density, defective enamel mineralization and organization [27,28]

Biglycan

KO

Decreased bone mineral content, larger crystals in bone and dentin [78]

Bone sialoprotein (BSP)

KO Tg

Decreased mineral content at young age; decreased bone formation in older animals Increased remodeling [79,80]

Collagen I

Osteogenesis imperfecta Mu (h)

Brittle bones; smaller crystals [81]

Dentin Matrix Protein 1 (DMP1)

Mu (h) KO Tg

Decreased mineral content Decreased mineral content, increased crystal size [82,83] Increased mineral content, decreased CO3/PO4, decreased ductility [84]

Dentin sialophosphoprotein (DSPP)

Dentin dysplasias Mu(h) KO Tg

Impaired dentin formation [1] Larger bone mineral crystals [85] Inhibited skeletal development [86]

Matrix extracellular phosphoglycoprotein (MEPE)

KO

Increased mineralization, decreased turnover [87]

Osteocalcin

KO

Increased mineral content, larger crystals [51]

Osteonectin

KO

Increased collagen maturity, larger crystals [88]

Osteopontin (OPN)

KO

Increased mineral content, larger crystals [89]

PHEX

Mu

Decreased mineral content, larger crystals [90]

Phospholipase A2

KO

Accelerated age related bone mass [91]

MMP2

KO

Decreased early bone formation. Decreased dentin mineralization, increased OPN expression [92]

SOST

KO, Mu (h)

Reduced mineral content and crystallinity [93]

MMP2, matrix metalloproteinase; PHEX, phosphate-regulating gene with homologies to endopeptidase on the X chromosome.

Both μCT and PET/μCT [104,105] were used in animal models. All of these studies consistently demonstrate improved trabecular and cortical architecture following treatment with 1,25(OH)2D3 thereby increasing bone strength, even though the association between vitamin D and trabecular microarchitecture was weak [102]. The principal advantage of using HR-pQCT is that trabecular bone structure can be resolved. Consequently, morphological parameters such as bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) can be calculated. To date, the highest resolution is achieved using μ-CT provides 3D ex vivo characterization of trabecular microarchitecture and tissue density with isotropic resolution as small as 6 μm. Bone structural data, made possible using this technique includes: BV/TV, Tb.Th, Tb.Sp, Tb.N, trabecular connectivity, and tissue mineral density (TMD, mass mineral/volume bone tissue), in good, but not perfect agreement with parameters

measured by 2D histomorphometry [106]. μCT is capable of detecting changes in both cortical geometry and trabecular microarchitecture. For example, the effect of a variant of vitamin D on rat bone growth was monitored using in vivo μCT at 0 and 8 months [107]. The ability to make measurements using living animals at multiple time points is enabling longitudinal studies of skeletal development, adaptation, and treatment response [108].

How Much Mineral Is Present? The standard in vitro method for quantifying the amount of mineral in a tissue is a gravimetric measurement of the weight of the residue left after the tissue is dried (110°C) and the organic components removed by ashing at 600°C. The ash weight or the ash density, if expressed per volume, reveals the total amount of mineral in the tissue. Chemical analysis of Ca and PO4 content has also been used to calculate mineral

III. MINERAL AND BONE HOMEOSTASIS

Methods for Quantifying Tissue Mineralization

content, these values, however, may be inaccurate due to the high organic phosphate content of these tissues. Bone mineral content can also be mapped in biopsied tissue with quantitative backscattered electron imaging (qBEI) in a scanning electron microscope [109]. qBEI can be used to characterize the quantity and spatial distribution of mineral in a biopsy. As the intensity of the backscattered signal is proportional to the atomic number of the specimen, the gray level intensity values in a BSE image can be related to TMD by comparing the intensity of each pixel in the image to that of osteoid and HA standards, from which, calcium weight percent values are calculated. This technique has been used to describe mineral distribution in vitamin D-deficient patients [110] and in VDR transgenic mice [111]. Interestingly, the above patient biopsies had an increased mineral content, characteristic of older bone, revealing an inability to deposit new mineral. The D-deficient mouse bones also had increased microcrack propagation [111]. Finally, μCT can also be used to quantify the mass and 3D spatial distribution of bone mineral in addition to tissue morphology. The mass of mineral within each bone voxel can be calculated from the X-ray attenuation using calibration phantoms. The TMD is calculated as the mass of mineral/bone tissue volume (g/cm3) [111].

Is the Mineral Characteristic of Physiologic Mineral (Is It a Poorly Crystalline Apatite)? The gold standard for determining the presence of bone mineral, for that matter any mineral phase, is X-ray diffraction (XRD). The X-rays, incident on a powdered sample, are reflected at specific angles related to the spacing between lattice planes, which are characteristic of a particular mineral phase (Fig. 23.2A). While the small crystal size and numerous imperfections in the physiologic apatite crystals make their diffraction patterns quite broad, they are, nonetheless, easily recognizable as apatite. Additionally, the broadening of individual peaks due to small size and imperfections in a particular dimension can be used to calculate an average crystallite size and perfection. Information on mineral particle thickness and alignment can be obtained by scanning small-angle X-ray scattering (scanning-SAXS). SAXS detects changes in particle sizes in the range of 2–25 nm, while XRD detects changes smaller than 1 nm. The data obtained agree with the crystal size predictions generated by infrared (IR) techniques [3]. Scanning SAXS has been used to characterize bone mineral in transgenic mice that overexpress the VDR [111]. Transmission electron microscopy (TEM) shows the size and shape of the mineral crystals and collagen fibers and selected area electron diffraction, analogous to XRD yields a characteristic pattern that can be used to quantify crystallite size and composition. The advantage of TEM is that mineral crystals can be observed along with collagen fibrils, and physiologic mineral parallel to the collagen fibers can be distinguished from dystrophic mineral, which may not always have that appearance. Both XRD and electron diffraction have proven useful for characterizing mineral associated

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with matrices and matrix vesicles developed in culture. The amount of mineral present in culture cannot be quantified by either XRD or electron diffraction. Such quantitation can be achieved by quantitative analysis (chemical determination by microprobe energy-dispersive X-ray analysis (EDAX) or other chemical analysis) or by ashing. EDAX can provide information on the chemical composition of the surface. Coupled to scanning electron microscopes, EDAX enables the observation of the morphology of the mineral deposit and simultaneous determination of the Ca:PO4 ratios of the mineral using appropriate chemical standards. Atomic force microscopy (AFM) [3,112], enables examination of the surface structure of a variety of materials and can be used to image individual bone crystals as well as collagen fibers. This high-resolution technique depends on the use of a probe to visualize individual crystals. For in situ measurements of bone crystal size, however, the organic matrix must be etched from the tissue surface, a challenging process that may cause a breakdown of some crystals. To date, there are no AFM analyses of any of the bones or soft tissues of animals with vitamin D abnormalities, despite the use of AFM in characterize the osseointegration of bone into oral implants in the presence of different concentrations of 1,25(OH)2D3 [113]. Vibrational spectroscopic techniques can provide detailed information on the structure and chemical environment of both mineral and collagen phases in bone tissue. Vibrations of atoms in molecules are determined by both the bonds in which they are located and by the environment surrounding these moieties. Vibrations that affect the dipole moment and those which are symmetric, detected by IR and Raman techniques, respectively, can be used for quantitative analysis of mineral composition and content. As in X-ray and electron diffraction, where poorly mineralized biologic apatite has broad diffraction peaks, the IR and Raman spectra characteristic of physiologic apatite are also broadened (Fig. 23.2B). Analysis of the relative phosphate to protein peak area ratios (v1/amide I) in Raman; v3/amide I in IR is used to calculate the relative amount of mineral present in the tissue. Curve-fitting or chemometric analysis of specific subbands of the compound’s spectral features can be used to reveal mineral crystal size and perfection (defined as crystallinity), along with acid phosphate content and carbonate substitution [12]. IR and Raman spectroscopic analysis of mineral to matrix ratio correlates linearly with ash weight of synthetic mixtures [114,115] and relates to mechanical properties [12]. Despite this, there are few studies using FTIR or Raman spectroscopy to study vitamin D or VDRE effects in animal bones [111,116,117]. FTIR spectra in a talcinduced osteoporotic rabbit model, supplemented with calcium alone, or calcium and vitamin D, reported based on FTIR that vitamin D alone was not as effective in promoting mineralization as was calcium and vitamin D. Vitamin D treatment in this model, however, increased labile carbonate content, indicative of new bone formation [116]. In another study, Vitamin D deficiency was used to create a model of reduced mineral content, without changes in collagen, to

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23.  MINERALIZATION IN MAMMALS

(A)

Bone

211

300 112 002 202

210 102

20

21

22

23

24

25

26

27

28

HA

29

30

31

32

33

34

35

36

37

38

39

40

Degrees 2θ

(B)

PO4 vibrations Protein vibrations

Bone

HA

1900 1800 1700 1600 1500 1400 1300 1200 1100 1000

900

800

700

600

500

Wavenumber (cm-1)

FIGURE 23.2  (A) X-ray diffraction pattern of a highly crystalline synthetic hydroxyapatite (HA) (dotted lines) and bone mineral (solid lines) showing the major peaks used for mineral analysis. The broadening of the 002 (c-axis) reflection (arrow) is routinely used to estimate crystallite size and perfection. (B) Infrared spectrum of the same samples as shown in (A), showing the bands characteristic of mineral phosphate and protein.

enable mechanical testing. Short-term vitamin D deficiency reduced whole bone stiffness (∼35%) and strength (∼30%). TMD alone was reduced by ∼10% compared to controls [117]. Average tissue level mechanical properties measured by Raman spectroscopy did not correlate with whole bone mechanical behavior, however, mineral-to-matrix ratio and

B-type carbonate substitution for individual indents, did correlate with indentation modulus [117]. Finally, an earlier study of mice overexpressing VDR, showed increased mineral content without any change in crystallinity, using a combination of methods including BMD measurements, XRD, and FTIR spectroscopy [111].

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Magnetic resonance imaging (MRI) and nuclear magnetic resonance (NMR) methods can also reveal the distribution of acid phosphate and features of the mineral crystal structure. Solid-state NMR spectroscopy has long been used for 1H, 13C, and 31P studies of bone mineral and calcium phosphates [118]. MRI methods have been used to measure porosity and other compositional features of calcified tissues, alone, or in combination with finite element models [119–123]. Magic angle spinning (MAS), coupled with cross polarization (CP-MAS) have been used to characterize initial mineral and matrix compositions in developing bones and in disease models [121,122]. Newer methodology (decay due to diffusion in the internal field or DDIF MRI) provides data on microstructures of porous materials such as trabecular bone [124]. MRI/NMR studies on the effects of vitamin D on bone structure are limited, however, a micro-MRI investigation of nine patients with hereditary 1,25(OH)2D3-resistant rickets (HVDRR) and seven controls [125] illustrates this application. Patients in this study, with mutations in VDR and hence presenting with hypocalcaemia, hypophosphatemia, and osteomalacia, were all treated with elevated amounts of calcium at a young age, ceasing treatment after puberty. No significant differences, as compared to controls, were noted for cortical bone, trabecular bone fraction, Tb.N, Tb.Th, and Tb.Sp.

Methods for Studying In Vitro Mineralization Studies in cell and organ culture have been used extensively to assess the function of vitamin D metabolites and the VDR in the mineralization process. While it is popular to use 10 mM β-glycerophosphate as a substrate in these studies [43,126,127], 5–10 mM of organic phosphate can cause aberrant mineral deposition, even in the absence of cells or matrix, as long as alkaline phosphatase activity is present [127,128]. Two mM of organic phosphate were demonstrated to be sufficient, in the presence of vitamin C, to cause physiologic mineral deposition in both mouse and rat osteoblast cultures [129]. HA mineral formed in cultures can be characterized by measuring changes of calcium or phosphate concentrations in solution, at times using of radiolabeled ions, and the amount of this mineral in the matrix using chemical analysis, diffraction, or spectroscopic methods discussed above. More recently it has become very popular to quantitate calcium and phosphate accumulations in culture, using histochemical stains. At times, these values are measured by extracting the solubilized moiety of the stained culture. One of the problems with this histochemical approach is that these stains are often not specific for HA mineral. For example, von Kossa staining for phosphate [130] can equally label inorganic phosphates and organic phosphate containing matrix molecules, particularly, cell membranes [131]. Alizarin red, used to stain calcium can indicate the presence of this ion not only in calcium phosphates, but also calcium bound to proteoglycans. Conclusive proof of the presence of HA mineral in a culture experiment comes not from labeled indications, but from diffraction methods, electron microscopy, or spectroscopic techniques as discussed above.

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MECHANISM OF EFFECTS OF VITAMIN D ON MINERALIZATION Physicochemical Effects Vitamin D’s principal effect is to increase serum calcium levels by stimulating renal calcium retention, intestinal calcium absorption, and osteoclastic resorption. This, in turn, leads to an elevation in localized calcium concentration and often an elevation in phosphate. The increased circulating levels of calcium and phosphate, while not causing marked hypercalcemia or hyperphosphatemia (due to counter effects of parathyroid hormone (PTH)), are thought to be sufficient to raise serum levels enough so as to promote dystrophic calcification, especially in blood vessels [62,66] and in the kidney [15,18,19]. Injection of vitamin D into animals can cause formation of apatite deposits in articular and growth cartilage as well as in bone. There is also a case report showing that milk, accidentally fortified with excessive vitamin D, caused dental pulp calcification in a child [132], demonstrating that mineralizing tissues other than bone or cartilage can be similarly affected. The VDR KO mouse has an impaired bone formation phenotype (see below), that can be rescued by calcium treatment [133]. Similarly, vitamin D-deficient osteomalacia in animals can be cured by increasing serum calcium (lactose and calcium infusion) without the addition of vitamin D [134,135]. This suggests that the increased mineralization associated with vitamin D, may simply reflect an increase in calcium or in calcium and phosphate levels. The VDR KO mouse, similarly, suffers defective tooth mineralization that can be partially corrected by increasing serum calcium and phosphate levels or by replacing the intestinal VDR [136]. From a physical chemistry perspective, in the precipitation a basic calcium phosphate such as bone apatite, the solution needs to have an appropriate pH and sufficient calcium and phosphate concentrations. In solution, at a pH of 7.4, when the CaxPO4 mM2 product exceeds 5.5 mM2, precipitation will occur despite the absence of a nucleator or a template for mineral deposition [137], thus, raising local calcium or phosphate concentration, even slightly, can cause precipitation, all other factors being equal. This is extremely important when considering cell culture studies where ion concentrations are elevated beyond physiological levels. In vitamin D-deficient animals (with rickets and/or osteomalacia), the mineral content of the bulk tissue, reflected by histochemical stains or ash weights, is decreased relative to controls (Fig. 23.3). HA crystals present in the epiphysis and metaphysis of these animals (newly formed crystals) tend to be larger than those in age- and gender-matched controls. It is not certain as to whether this is because osteoclast activity is increased or because existing crystals grow at the expense of new crystals being formed in the absence of an appropriate matrix or sufficient calcium phosphate levels. Increased osteoclast activity has been noted in some cases of human hypophosphatemic (HYP) oncogenic osteomalacia [139]. The effects of vitamin D metabolites on extracellular matrix formation and composition suggest that impaired formation may be a more important factor.

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(A) D-deficient

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FIGURE 23.3  Mineral analyses in models of vitamin D deficiency. (A) Vitamin D-deficient rats. Male Sprague-Dawley rats (21 days old) were maintained in the dark on a vitamin D- and phosphate-deficient diet for 3 weeks. The first panel shows their serum Ca and PO4 levels. 1,25(OH)2 vitamin D levels were not detectable in the vitamin D-deficient animals. Tibias removed at sacrifice were used for analysis of ash weight and β002 (1/crystallinity). (B) Female mice with hypophosphatemic rickets (Hyp) and their age- and sex-matched controls were 35 days old at sacrifice. Femora were used for analysis of ash weight, β002, and Ca/PO4 ratio of the ash. (C) Second-generation vitamin D-deficient rats and their age-matched controls were 7 weeks old at sacrifice. Femora were used for analysis of ash weight and β002 and Ca/PO4 ratio of the ash. (A and B) Reproduced, with permission from Springer-Verlag, Boskey AL, Di Carlo EF, Gilder H, Donnelly R, Weintroub S. The effect of short-term treatment with vitamin D metabolites on bone lipid and mineral composition in healing vitamin D-deficient rats. Bone 1988;9:309–18. (C) Reprinted, with permission from the third edition of this book.

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Effects of Vitamin D on Cells and Matrix Molecules The effects of vitamin D metabolites on cell differentiation are reviewed elsewhere in this book (See chapters in Section III of this book). In terms of mineralization, however, it is important to note that fetal development is normal both in vitamin D-deficient rats [134] and VDR-deficient mice [140], implying vitamin D and the VDR are not critical for skeletal formation. Rickets develops postnatally, but can be corrected by supplying increased calcium and phosphate, likely due to redundancy and the interaction of vitamin D with so many other pathways. The effects of various vitamin D metabolites, when examined in culture or in animal models, depends on species, stage of differentiation, and on the interactions with the various pathways. For example, in the rat 24,25(OH)2D3 stimulates the proliferation of resting zone cells in the growth plate, while 1,25(OH)2D3 stimulates the proliferation of hypertrophic and proliferating cells [141], presumably favoring mineralization. Overexpressing VDR in mature osteoblasts also leads to increased bone mass [111]. Mature chondrocytes and osteoblasts synthesize the mineralizable matrix and provide the enzymes necessary to modulate the matrix so as to facilitate mineralization. An indirect effect of vitamin D, here, might be related to the differentiation and proliferation of the cells responsible for controlling mineralization. Consequently, it is interesting to note that excess 1,25(OH)2D surprisingly blocks mineralization leading to hyperosteoidosis in rats [142], explained by the genomic and nongenomic actions (see Chapter 24) of this metabolite on osteoblast proliferation [31,143,144].

The Influence of Vitamin D on Bone Matrix Proteins Vitamin D modulates the production of several molecules (Table 23.2) that are crucial for the synthesis and function of bone tissue, despite the lack of evidence that direct effects of vitamin D are required for normal bone mineralization. The actions of these proteins have been described in detail elsewhere [1], hence, only a few representative proteins are described in this section. Readers are referred to references in Tables 23.2 and 23.3 for more detail on this subject.

Collagen The scaffold of bone matrix is made predominantly of type I collagen molecules. These are produced early on in bone tissue formation and their particular morphology supports the mineral deposition in later stages. Additionally, collagen molecules provide the elasticity characteristic of the bone tissue, which is essential for normal function in response to mechanical forces. Synthesis of collagen is a complex process, regulated by several factors including vitamin D. In a variety of cell culture systems, 1,25(OH)2D3 downregulates the transcription of α1(I) collagen by osteoblasts [145,146]. This decrease in type I collagen expression appears to be cellstage-dependent, occurring in early bone nodules, formed in culture, but not in intermediate and mature ones [145]. Adding

FIGURE 23.4  Effect of 10−10 M 1,25(OH)2D3 on mineralization in differ-

entiating chick limb bud mesenchymal cell micromass cultures. Data are expressed as μg Ca/μg DNA. Continuous vitamin D treatment was started either at day 5 (when cartilage nodules formed), at day 7 (prior to the start of visible chondrocyte hypertrophy), or at day 9 (2 days before start of mineralization). Control cultures (red outline, open bars) with 1M inorganic phosphate (P) received a comparable volume (20 μL) of ethanol, the carrier for 1,25(OH)2D3. On days 12–21, Ca and DNA contents were determined in control and mineralizing cultures. Mineralization was promoted by the addition of 4M inorganic phosphate (4P) from day 2 (grey bars). The DNA contents of mineralizing and control (nonmineralizing) cultures were not significantly different (not shown); 1,25(OH)2D3 treatment did not significantly accelerate proliferation. There was a slight increase in DNA content of cultures treated on day 5 (Cyan bars), but neither this increase nor the gradual increase in DNA content with time was significant, indicating that the cultures had already achieved their plateau phase of proliferation. In contrast, cultures treated on day 7 (blue bars) and day 9 (purple bars) showed increases in Ca content relative to the other cultures.

1,25(OH)2D3 after the completion of extracellular matrix formation has been reported to inhibit further mineralization in chick osteoblast cultures [44], suggesting that 1,25(OH)2D3 affects the metabolic processes by which osteoblasts control mineralization independent of its effects on the formation of the collagenous matrix. Matsumoto, however, showed that 1,25(OH)2D3 stimulated alkaline phosphatase activity in both early and late osteoblast cultures and reported enhanced mineral deposition based on electron microscopy and 45Ca uptake, when 10−10 M 1,25(OH)2D3 was added following the start of mineralization [147]. Similarly (Fig. 23.4), in differentiating chick limb-bud mesenchymal cell micromass cultures, which form a mineralizable matrix starting at day 9, 1,25(OH)2D3 added continuously to cultures following matrix formation and the onset of mineralization was started (day 9), increased the rate of mineral accumulation compared to those cultures that did not receive exogenous vitamin D and contained no organic-phosphate additives. This increase was in contrast to cultures to which vitamin D was added continuously, while cells were differentiating (day 5), or when the cells were first beginning to deposit a cartilage matrix (day 7). Vitamin D also regulates the expression of the noncollagenous proteins in mineralized tissues. Many of these proteins

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are believed to play direct roles in the regulation of biomineralization [1] thus they account in part for the indirect effect of vitamin D on mineralization.

Noncollagenous Proteins Osteocalcin (OCN) was one of the first matrix proteins whose expression was shown to be upregulated by vitamin D [52]. OCN is a member of a large family of hepatic and skeletal vitamin K-dependent proteins which undergo posttranslational modification and γ-carboxylation at key glutamic acid (Gla) residues and have mineral-binding capacities. Mature OCN protein contains three Gla residues and accumulates in bone due to its high affinity for HA. Initial osteoblastic expression of OCN occurs after the onset of extracellular matrix mineralization, and increases with progressive mineralization and maturation of the osteoblast to a terminally differentiated state. OCN is a regulator of bone mineralization; and also, as reviewed elsewhere [51], modulates osteoblast and osteoclast activity and the functions of other organs [148,149]. Bone sialoprotein (BSP), a member of the small-integrin binding N-glycosylated (SIBLING) family of proteins is an in vitro apatite nucleator [42] and a mineralization regulator whose expression is suppressed by addition of 1,25(OH)2D3 to osteoblast cultures [43–45]. In osteoblast cultures without exogenous vitamin D supplementation, BSP is expressed at its highest levels prior to onset of extracellular matrix mineralization [45]. Its suppression, in the presence of 10−8 M 1,25(OH)2D3, may reflect a compensatory mechanism, preventing excessive initial mineralization. As reviewed in detail elsewhere [79], data from mice lacking BSP or overexpressing it, suggests that BSP is important for bone turnover and bone repair. Lack of BSP causes an overexpression of osteopontin, a mineralization inhibitor, which may explain the lack of mineral deposition in young BSP KO mice [80]. Osteopontin (OPN), another SIBLING protein produced in a number of tissues, is upregulated by 1,25(OH)2D3 in proliferating and differentiated mouse and rat osteoblasts among other tissues [50]. In bone, OPN mediates autocrine and paracrine functions in the regulation of tissue formation. OPN is important for recruiting osteoclasts for bone remodeling [50] and it acts as a signaling protein in many tissues. OPN is an in vitro inhibitor of mineralization in soft tissues and bone, and because of its affinity for HA accumulates in pathologic HA deposits [50]. The expression of another SIBLING protein, DMP1 is also inhibited by 1,25(OH)2D3. DMP1 is a major protein produced by the osteocyte [150]. In vitro DMP1 both promotes and inhibits the formation of HA crystals, depending on the posttranslational history (phosphorylation, cleavage, etc.) of DMP1 [46]. DMP1 gene mutations cause autosomal recessive HYP rickets in humans and sheep [82] and a similar rachitic phenotype in both bone-specific and global DMP1 KO mice [83].

Growth Factors VDRE’s are present in a number of growth factors [29]. Few have been studied in terms of effects on mineralization.

The SOST gene, which leads to sclerostin production, is upregulated by 1,25(OH)2D3 in human osteoblasts [57]. SOST is an inhibitor of osteoblastic matrix production, hence, inhibits the extent of mineral deposition. Activin A, a member of the transforming growth factor beta family is also stimulated by 1,25(OH)2D3; in cultures of human osteoblasts and murine cell lines, 1,25(OH)2D3 causes increased activin A production as well as production of an agonist, suggesting that it acts in culture, to limit the extent of new mineral deposition. Activin A also decreases OCN production in these cells [58].

MINERALIZATION AND MINERAL PROPERTIES IN SYSTEMS WITH VITAMIN D ALTERATIONS The effects on mineralization of 1,25(OH)2D3 and other vitamin D metabolites vary with dose and type, as well as with the system being studied. Even in similar species, the effects depend on animal age, gender, route of delivery, and method of analysis. In cell culture, results are also dependent on the maturity of cells and tissues, concentration of metabolites, and whether these metabolites are given once or continuously. Vitamin D deficiency or insufficiency causes rickets, which is the result of failure of the growth plate cartilage to mineralize, also osteomalacia which is demonstrated by the failure of the osteoid to mineralize [151]. Contrastingly, vitamin D toxicity (hypervitaminosis D) results in hypercalcemia and frequently dystrophic calcifications [62,66,152]. Reviewed, in this section, are how different models of D-deficiency provide insight into the role of vitamin D metabolites in mineralization mechanisms.

Vitamin D Deficiency Animal models of vitamin D deficiency arise from either dietary intervention or genetic alterations. Examples of the former, include vitamin D-deficient rats [117,131,134,142] and second-generation vitamin D-deficient rats born to vitamin D-deficient mothers [138], where the latter category includes HYP mice [153] and animals created by genetic manipulation (conditional and global knockouts or knockins). Animals in all of these models have negligible levels of 1,25(OH)2D3 and reduced levels of 25-hydroxyvitamin D3 (25(OH)D3). Generally, in the bones of these D-deficient animals, mineral content is decreased and crystal size is increased relative to age-matched controls, Ca/P ratios, however, are vary greatly (Fig. 23.3). The effect of first-compared with second-generation vitamin D deficiency is also inconsistent. In second-generation vitamin D-deficient rats, only small differences in bone ash weight, crystallinity, and Ca/P ratios exist when compared to age-matched controls. These differences are greater in firstgeneration vitamin D-deficient animals matched for age, gender, and background. Initial electron microscopic studies of vitamin D-deficient bones [154], revealed that the total number of extracellular

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matrix vesicles was not different from that in the vitamin D-sufficient rats, the vesicles, however, contained less mineral and an extracellular matrix devoid of mineral. Healing of the rickets, in these vitamin D-deficient animals, by treatment with vitamin D and phosphate, was associated with increases in both matrix vesicle and extracellular matrix mineralization [155]. In the HYP mouse [153], in which, failure to mineralize the growth plate and osteoid is due to a genetic abnormality in the endopeptidase known as PHEX (phosphate-regulating gene with homologies to endopeptidase on the X chromosome (see Chapter 66 (vol. 2 of this book)) The hypocalcemic disorders, Thomas Carpenter); Ca/P ratios are elevated, probably due to abnormalities in renal phosphate transport. Additionally, there is less bone mineral and, those mineral crystals present, tend to be larger and more perfect. A mechanism can be postulated to explain the mineralization abnormalities in HYP animals’ bones. Vitamin D is inhibiting mineralization by preventing PHEX-dependent [154] degradation of matrix proteins. This concept is based on the observations that matrix extracellular phosphoglycoprotein (MEPE) [155–157] and OPN are substrates for PHEX that accumulate in the matrix of HYP mouse bones [158,159]. Because OPN is an inhibitor of mineral proliferation, the decreased mineral content in these animals is likely attributed to the accumulation of inhibitory matrix proteins. The mineral properties of vitamin D insufficiency in human children with rickets have not been characterized in consequence of ethical reasons. Mineral in adults with osteomalacia, however, has been subjected to a more detailed analysis. An autopsy study of 675 bone biopsies and serum levels from a German population known to be vitamin D-deficient, sought to define a cutoff in vitamin D levels associated with osteomalacia (increased osteoid volume). Biopsies came from males and females (ages 30–100), in all of which diseases causing secondary bone disorders were excluded. Mineralization defects were present in 20% of the population analyzed with serum 25(OH)D serum levels were below 75 nmol/L(30 ng/mL) [160].

Vitamin D Receptor Alterations The nuclear VDR binds to 1,25(OH)2D with high affinity and selectivity. Animals lacking VDR have normal circulating levels of vitamin D and reduced levels of 1,25(OH)2D [140,161]. They are slightly hypocalcaemic with elevated PTH levels due to the inability of vitamin D target organs to regulate calcium influx. The histologic appearance of the bone tissue of VDR KO animals resembles that of an animal with vitamin D deficiency. These mice have a normal skeleton at birth but develop hypocalcaemia and hyperparathyroidism shortly after weaning, which is comparable to human rickets type II. The rachitic malformation, growth retardation, and impaired structural performance in the bones of the VDR KO animals, can be rescued by dietary supplementation of calcium, restriction of phosphorus intake, or with high-dose vitamin 1,25(OH)2D3 [30,162], suggesting that VDR signaling in bone is not needed when sufficient calcium is present. However, while vitamin D replenishment corrects the mineralization deficit, it does not

395

alter PTH levels [163]. Prior to rescue, the VDR KO animals have lower mineral content in their bones compared with the wild-type and rescued KO, even though their bone geometries are not altered during gestation and lactation. The VDRnull animals’ dentin is also under mineralized; their enamel matures more rapidly relative to those of wild-type controls [30]. VDR KO animals have extended growth plates and an increased number of hypertrophic chondrocytes. The hypertrophic chondrocytes express the correct relative amounts of phenotypic markers of chondrocyte maturity, type X collagen, vascular endothelial growth factor, and osteopontin, but show decreased staining for annexin V-phosphatidyl serine, an early marker of apoptosis [163]. This suggests that impaired mineralization may be due to the failure of chondrocytes to undergo apoptosis, which, however, at least in culture, is not required for mineralization in avians [164] or mice [165]. A more likely possibility is that both calcium and phosphate levels are not sufficient to mineralize the growth plates, with apoptosis following rather than preceding mineralization. Additionally, both annexin and phosphatidyl serine are membrane components of extracellular matrix vesicles; thus, decreased staining for these components might suggest that the chondrocytes are not producing sufficient vesicles, or that vesicles lacking these components are not functional. Osteoclast numbers are normal in the VDR KO, arguing against excessive remodeling as the origin of the mineralization defect. However, even when hypocalcaemia and hyperparathyroidism are prevented by a high-calcium diet; osteoblast number, mineralization rate, and bone volume are reported not to be fully corrected [136]. Conditional KOs of VDR either in the intestine (to impair Ca resorption) or in the osteocytes (to maintain circulating Ca levels) when contrasted with the VDR global KO provide some important insights [54]. In this model, when intestinal VDR is not functional, the skeletal mineral is lost in an attempt to normalize serum calcium. More significantly, when intestinal VDR KO mice are treated with 1,25(OH)2D3, remodeling is further increased and the mice bones become osteopenic and may fracture. Contrastingly, in the conditional skeletal KO, the bones are preserved to a greater extent. This emphasizes the importance of maintaining normal serum calcium levels, and explains many of the abovementioned observations, specifically, the intestinal deletion of VDR reduces Ca absorption but had no effect on serum Ca levels. On the other hand, the global VDR KO had hypocalcemia. Intestinal ablation of VDR did not affect the growth plate or bone growth in these animals but did show a decrease in BV/TV, a decrease in calcium content and bone mineral density (BMD), increase in porosity and bone resorption, and hyperosteoidosis, coupled with normal calcium and phosphate levels. Calcium storage in bone was therefore reduced to maintain normocalcemia. These changes were also associated with increases in the levels of OPN, PHEX, and enzymes regulating pyrophosphate/phosphate levels. Contrastingly, in the osteocyte-specific VDR KO, serum calcium, phosphate, bone mass, and bone remodeling were not altered when the animals were fed a Ca-deficient diet. Expression of ENPP3, ANK, and OPN (each of which inhibits mineral formation) were increased, while PHEX was decreased. These studies show that

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maintaining normocalcemia for all body functions takes precedence over maintaining bone mass, and that 1,25(OH)2D regulates the shift of calcium from the skeleton toward the serum by controlling the level of mineralization inhibitors. Bone mineralization is also affected by vitamin D analogs. These provide further insight into the mechanism of vitamin D action. For example, treatment of nonhuman primates with an analogs of 1,25(OH)2D3, Eldecalcitol (1α,25-dihydroxy-2β-(3hydroxypropyloxy) vitamin D3, ELD), decreased the remodeling and increased bone formation in ovariectomized animals [166]. Specifically, ELD treatment for 6 months increased lumbar spine BMD, suppressed ovariectomy-induced increases in bone turnover markers, and improved biomechanical properties of lumbar vertebrae in ovariectomized cynomolgus monkeys [166]. Trabecular connectivity of the lumbar vertebra was also increased as was the formation of immature divalent and mature trivalent enzymatic collagen cross-links, by directly stimulating LH or LOX gene expression in bones. Advanced glycation end products, whose accumulation weakens bones, were decreased, although the mechanism of this effect was not determined [167]. In humans, HVDRR known as vitamin-D-dependent rickets type II, is a rare autosomal recessive disease that arises as a result of mutations in the gene encoding VDR. Hypomineralized teeth in these patients have elevated OPN and MEPE contents [168] analogous to the VDR KO mice. There has been only one study of mineral properties in patients with HYP rickets, however, these had been treated with phosphate and vitamin D for various periods of time [169]. Nevertheless, the study showed that trabecular volumetric BMD (vBMD) was elevated where cortical vBMD was lower in patients with XLH caused by PHEX mutations. Children receiving treatment had high trabecular vBMD, whereas trabecular vBMD was lower in older patients who discontinued treatment or who never received it. Cortical vBMD was low at all ages and treatment groups, but z-scores were relatively higher in currently treated children than in the other two groups of patients.

normal tissue levels of metabolites of vitamin D are maintained despite the absence of the DBP, hence the absence of a mineral phenotype is not surprising [173].

1α-Hydroxylase Knockout Hepatic enzymes having 25-hydroxylase activity, CYP2R1, CYP3A4, and l CYP27A1 of which CYP2R1 is the major enzyme controlling hydroxylation vitamin D to 25(OH)D. The second hydroxylation step occurs mainly in the kidney, where 25(OH)D is hydroxylated by the mitochondrial 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) to a biologically active hormone, 1, 25(OH)2D, which binds to and activates the VDR. Patients lacking the 1α-hydroxylase have pseudo-deficiency rickets or vitamin D-deficiency rickets type 1, with secondary hyperparathyroidism, growth retardation, rickets, and osteomalacia [174]. Mice that lack the 1α-hydroxylase also present with hypocalcaemia, growth retardation, hyperosteoidosis, impaired mineralization, and increased matrix synthesis (rickets and osteomalacia) [175]. Similar to the VDR KO mice, there are no differences in osteoclast number in the control and KO mice given normal or calcium-enriched diet, suggesting that the reduced mineral content arises from decreased bone formation rather than from elevated resorption. These 1α-hydroxylase-deficient mice can also be rescued by treatment with high-dose (10 or 20 × the normal mouse requirement) vitamin D. Mice rescued with high-dose vitamin D had a complete recovery of cortical bone as evidenced by μCT, however, only partial recovery of trabecular bone (Fig. 23.5). Such data indicate that high-serum levels of 25(OH)D influence Ca and bone metabolism independent of its conversion to 1,25(OH)2D [176]. Mice with a chondrocyte-specific KO of the 1-hydroxylase have impaired endochondral ossification with delayed cellular maturation and delayed mineralization; mice overexpressing the 1-hydroxylase had the opposite phenotype with accelerated endochondral ossification [177].

Vitamin D-Binding Protein Knockout Vitamin D metabolites are carried to the receptor (VDR) in the target tissues, by the vitamin D-binding protein (DBP) [170]. Mineral properties in the DBP KO mice have not been elucidated, however, these KO mice have an interesting bone phenotype [171]. The DBP KO mice have low levels of serum vitamin D metabolites, and otherwise appear normal, presenting with none of the bone abnormalities seen in vitamin D-deficiency rickets or osteomalacia. When depleted of vitamin D, these mice develop hyperparathyroidism. When stressed with very high doses of exogenous vitamin D to induce vitamin D toxicity, they present with elevated serum calcium levels, but did not show toxic effects found in wildtype animals [172]. Kidney mineral deposits were found in the wild-type but not in the DBP KO mice. The observation that individual tissues can maintain sufficient levels of 1,25(OH)2D3 for all key processes is significant. Apparently,

FIGURE 23.5  Infrared microspectroscopy of the effect of threefold VDR overexpression on the tibia of 4-month-old mice shows slight but not significant increase in mineral content (mineral:matrix ratio). Data were collected from cortical bone on the periosteal side (CP), in the center (CC), and on the endosteal side (CE) as well as within individual trabeculae (T). Reprinted with permission from the third edition of this book.

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Conclusions

Osteoporosis and Osteomalacia in Humans Many individuals with osteoporosis may be Ca-deficient, demonstrate vitamin D insufficiency and show histochemical evidence of osteomalacia [178]. Based on FTIR microspectroscopic analysis, osteoporotic bones without evidence of osteomalacia have modified mineral content and consistent increased mineral crystallinity and enzymatic collagen cross-links [12,179– 182]. Because it is likely that some of these osteoporotic patients could have osteomalacia, we tested the hypothesis that the mineral in osteomalacic bone was different from that in normal bone. In tissue sections from female patients with osteomalacia

(n = 11, age 22–72), mineralization rates determined by histomorphometry were small relative to controls (n = 7, age 36–57), whereas the osteoid fraction was larger, revealing defective primary mineralization. In these sections, the degree of mineralization measured by qBEI in old bone (calcified tissue) was slightly higher than in four of the controls, reflecting a normal evolution of the secondary mineralization of bone tissue. FTIRI analysis of these biopsies [182] showed decreased mineral content in both cortical and trabecular bone of osteomalacic patients and no differences in mineral crystallinity or collagen maturity (Fig. 23.6). These findings support the hypothesis that the reduced strength of osteomalacic bone originates in delayed primary mineralization and reduced mineralized tissue volume.

CONCLUSIONS

FIGURE 23.6  Infrared imaging comparing the mean mineral content (mineral:matrix ratio) and crystallinity in normal female patients to female patients with osteomalacia. Values are mean ± SD (n = 7 and 4, respectively) as averaged from 3 to 5 fields from each biopsy. Reprinted with permission from the third edition of this book.

Vitamin D affects bone, tooth, and cartilage mineralization, predominantly indirectly, by regulating local calcium and phosphate levels. Additionally, there is a direct action on bone, tooth, and cartilage mineralization because vitamin D has genomic and nongenomic effects on cells of mineralizing tissues. Vitamin D’s actions on membrane properties, enzyme activity, and matrix protein expression and phosphorylation can affect the mineral that is formed in its presence or absence. Vitamin D and its various forms alter the composition of the matrix as to maintain normocalcemia. In doing so, vitamin D inhibits some mineralization pathways and stimulates others (Fig. 23.7). Because the mineralization process is critical to the life of the species, it must have redundant controls, as illustrated here the vitamin D dependent pathways are one of them.

FIGURE 23.7  Outline of the direct and indirect effects of Vitamin D and its metabolites on the physiological process of biomineralization. Items in green are promoters and those in red are inhibitors of mineralization. ANK, progressive ankylosing; BSP, bone sialoprotein; DMP, dentin matrix protein; ENPP1, ectonucleotide pyrophosphatase phosphodiesterase; HA, hydroxyapatite; MEPE, matrix extracellular phosphoglycoprotein; MMP2, matrix metalloproteinase; PHEX, phosphate-regulating gene with homologies to endopeptidase on the X chromosome.

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Acknowledgments Dr. Boskey’s research described in this chapter was supported by NIH grants DE04141 and AR041325. She appreciates the contributions made by Dr. Eve Donnelly to the prior version of this chapter, and thanks Dr. Judah Gerstein for his editorial review of the current chapter.

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Further Reading

[179] Donnelly E, Meredith DS, Nguyen JT, Boskey AL. Bone tissue composition varies across anatomic sites in the proximal femur and the iliac crest. J Orthop Res 2012;30:700–6. [180] Gourion-Arsiquaud S, Faibish D, Myers E, Spevak L, Compston J, Hodsman A, Shane E, Recker RR, Boskey ER, Boskey AL. Use of FTIR spectroscopic imaging to identify parameters associated with fragility fracture. J Bone Miner Res 2009;24:1565–71. [181] Gourion-Arsiquaud S, Lukashova L, Power J, Loveridge N, Reeve J, Boskey AL. Fourier transformed infra-red imaging of femoral neck bone: reduced heterogeneity of mineral-to-matrix and carbonateto-phosphate and more variable crystallinity in treatment-naïve fracture cases compared to fracture-free controls. J Bone Miner Res 2013;28:150–61. [182] Faibish D, Gomes A, Boivin G, Binderman I, Boskey A. Infrared imaging of calcified tissue in bone biopsies from adults with osteomalacia. Bone 2005;36:6–12.

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Further Reading [1] Kazakia GJ, Burghardt AJ, Cheung S, Majumdar S. Assessment of bone tissue mineralization by conventional x-ray microcomputed tomography: comparison with synchrotron radiation microcomputed tomography and ash measurements. Med Phys 2008;35:3170–9. [2] Camacho NP, Rinnerthaler S, Paschalis EP, Mendelsohn R, Boskey AL, Fratzl P. Complementary information on bone ultrastructure from scanning small angle X-ray scattering and Fourier-transform infrared microspectroscopy. Bone 1999;25:287–93.

III.  MINERAL AND BONE HOMEOSTASIS