Bone's Material Constituents and their Contribution to Bone Strength in Health, Disease, and Treatment.

Calcif Tissue Int DOI 10.1007/s00223-015-9971-y

REVIEW

Bone’s Material Constituents and their Contribution to Bone Strength in Health, Disease, and Treatment Y. Bala • E. Seeman

Received: 8 January 2015 / Accepted: 11 February 2015 Ó Springer Science+Business Media New York 2015

Abstract Type 1 collagen matrix volume, its degree of completeness of its mineralization, the extent of collagen crosslinking and water content, and the non-collagenous proteins like osteopontin and osteocalcin comprise the main constituents of bone’s material composition. Each influences material strength and change in different ways during advancing age, health, disease, and drug therapy. These traits are not quantifiable using bone densitometry and their plurality is better captured by the term bone ‘qualities’ than ‘quality’. These qualities are the subject of this manuscript. Keywords Bone water Collagen Mineralization Osteopontin Osteocalcin Pentosidine Qualities of bone

Introduction Bone ‘density’ is an ambiguous term. The ‘bone density’ of a region is its ‘apparent’ density as quantified by the net attenuation of photons produced by the volume of mineralized matrix volume, the completeness of its mineralization, its spatial distribution, porosity, and other factors. Y. Bala Laboratoire Vibrations Acoustique, Institut National des Sciences Applique´es de Lyon, Campus LyonTech la Doua, Villeurbanne, France Y. Bala INSERM UMR1033, Universite´ de Lyon, Lyon, France E. Seeman (&) Departments of Endocrinology and Medicine, Centaur Wing, Repatriation Campus, Austin Health, University of Melbourne, Waterdale rd, Heidelberg, Melbourne 3081, Australia e-mail: [email protected]

Each constituent may influence whole bone’s compressive, tensile and shear strengths in different, and sometimes opposite, ways. Bone ‘quality’ is also ambiguous. Indeed, the word contains many phenotypes like the material composition, microarchitecture, geometry, remodeling, and micro-damage so that it is better used in the plural, bone qualities as this better serves the need to then define each constituent of bone’s material composition so that its contribution to material strength can be quantified. We avoid the vagaries of ‘density’ and ‘quality’ and confine our discussion to specified components of bone’s material composition; the building blocks used to assemble its macro- and micro-scopic structure, and examine the role of these constituents in determining the strength of bone during advancing age, disease, and drug therapy. An analysis of bone’s macro- and micro-scopic structure; its size, shape, the spatial configuration of differing mineralized matrix and void volumes forming cortical, transitional and trabecular bone, and their contributions to structural strength are outside the scope of this manuscript.

Bone’s Material Composition in Healthy and Young Adulthood The Organic Phase Type 1 Collagen Mineralized bone matrix at the nanoscale level includes the organic matrix which comprises 90 % type I collagen and 10 % of non-collagen proteins (e.g., osteopontin and osteocalcin) [1]. Type I collagen consists of two a1 and one a2 polypeptide chains assembled as a tropocollagen triple

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Y. Bala, E. Seeman: Bone Strength in Health, Disease and Treatment

helix. These molecules are assembled in a staggered fashion into long ([1 mm) and thin (20–50 nm) fibrils stabilized by post-translational modifications forming intraand inter-fibrillary crosslinks [2, 3]. This material can elongate elastically (i.e., reversibly) in tension by uncoiling of the collagen molecule and intermolecular sliding. Eventually, fibril stiffening occurs with the stretching of the tropocollagen molecules until rupture. Deformation by lengthening is, in part, dependent on the density and the type of crosslinks [4].

decrease in trivalent mature crosslinks reduced the tensile strength of the fibril by 45 % and impaired post-yield strength [4]. Advanced glycation end products (AGEs) do not rely on enzymatic reactions but rather on a reduction of a reactive sugar. The sugar reacts with free amino groups in lysine or arginine of collagen fibrils. Pentosidine, the most studied AGE, is derived from spontaneous condensation of glucose with lysine and arginine [8]. Accumulation of the AGEs stiffens the collagen network limiting intermolecular sliding of the fibrils and so reduces toughness [12–14].

Enzymatic and Non-enzymatic Crosslinking Enzymatic intermolecular crosslinks within and between fibrils stabilize the collagen and improve its mechanical properties while non-enzymatic crosslinks are associated with impaired mechanical properties [5–7]. Enzymatic crosslinks are formed by the action of lysyl oxidase that establishes immature divalent molecular links between the lysine amino acids of the end of two collagen molecules (e.g., hydroxy or dihydroxy lysinonorleucine) [2, 8]. Over time, these divalent crosslinks react with aldehyde groups forming a mature, stiffer trivalent bond [4, 8]. [See Saito and Marumo, this issue]. Both the total amount of enzymatic crosslinks and the ratio of immature to mature forms of the enzymatic crosslinks affect mechanical properties (Fig. 1). An increase in the content of the mature crosslinks confers higher ultimate stress and higher post-yield energy absorption in bending or compression [9, 10]. Lathyrism in rats produces a 50 % decrease in trivalent crosslinks and a *30 % reduction in bending strength [11]. Using computational modeling, Depalle et al. reported that a similar Fig. 1 As tissue mineral content increases stiffness increases but toughness decreases. Adapted from Ref. [177] with permission

Mineral-Collagen Interface, Non-collagenous Proteins, Sacrificial Bonds, and Hidden Length Non-collagenous proteins like osteopontin and osteocalcin account for 10 % of bone’s organic phase. These proteins contribute to toughening at the nano-structural level by forming a ‘glue’ that creates collagen–mineral and collagen–collagen interfaces. Sacrificial bonds of the protein ‘glue’ like osteopontin allow its helical structure to unwind and lengthen with dissipation of energy as the bonds of this molecule are sacrificed to permit lengthening of the collagen fibrils protecting the tropocollagen backbone (Fig. 2) [15]. The uncoiling of osteopontin and fibrillary lengthening reduces the stress imposed upon mineral platelets, the structures that confer stiffness but yet are the source of brittleness. The elongation is reversible with reformation of sacrificial bonds during relaxation [16]. Osteopontin also participates in toughening by connecting two osteocalcin molecules bound to mineral platelets through calcium ions. These osteocalcin-osteopontin-osteocalcin complexes

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Tension Fig. 2 a The mineralized fiber contains mineralized fibrils bound by interfibrillary matrix. b During lengthening in tension, helical proteins like osteopontin uncoil. Platelets of mineral are protected by

lengthening of these interfibrillary proteins which is achieved by breaking sacrificial hydrogen bonds. See text. Insets adapted from Ref. [16] with permission

unfold in tension and provide additional lengthening and toughening [17]. Mutant mice deficient in osteopontin, osteocalcin or both have decreases in toughness by 30 % independent of changes in mineralized bone matrix mass [17, 18].

within few weeks [19–21]. This is followed by a slower maturation by secondary mineralization which takes months to years [19, 22]. The chemical properties and structure of the mineral influence the mechanical properties of the matrix. Strength in compression is achieved by mineralization. Mineral also brings stiffness in tension but dramatically reduces toughness (Fig. 1). As crystal size increases, the surface area of interaction with the collagen increases limiting fibril deformation. In human cortical bone, the variability in crystallinity, reflecting the size and the perfection of the crystals, explained 20–25 % of the variance in tensile mechanical properties—higher crystallinity conferred a higher elastic modulus, the ability to deform reversibly but a lower strain to failure (Fig. 3) [23]. During maturation, mineral interacts with the local ionic environment [24]. Phosphate or hydroxyl groups can be

The Inorganic Phase Bone mineral phase is composed of poorly-crystallized non-stoichiometric apatite. The deviation from stoichiometry confers the ability for elements of the lattice to be substituted by others. The mineral is arranged as 50 to150nm stacks of 2.5 to 4-nm-thick platelets that nucleate and propagate in organic matrix after its deposition. The mineralization of osteoid is biphasic with a rapid increase in crystal number and size during the first few days of primary mineralization reaching 70 % of maximum mineralization

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Y. Bala, E. Seeman: Bone Strength in Health, Disease and Treatment Fig. 3 Longer crystals increase the surface of interaction with collagen decreasing its ability to deform (Left panel). This stiffens the matrix decreasing the ability to deform in tension without fracturing. Adapted from Ref. [23] with permission

replaced by carbonates in the crystal lattice [25]. While the carbonate content is low crystal growth is facilitated; longer crystal form leading to a larger surface of interaction with collagen and increased material stiffness [23, 26]. Once in the crystal lattice, carbonates create strains and structural irregularities that may limit crystal growth because the energy needed for crystal growth increases [26]. Advancing age is associated with a reduction in replacement of phosphonate and hydroxyl by carbonate that may lead to enlargement of crystals and appearance of brittleness [27–29]. Water Content of Bone The water content of matrix bound to collagen and mineral accounts for 10–20 % of cortical wet weight (excluding water in Haversian and Volkmann canals) [30]. Collagen bound water influences its three-dimensional structure. A decrease in water content is associated with a reduction in collagen fiber diameter and loss of toughness [31–33].

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Water binds mineral particles to each other and to organic matrix allowing movement and deformation of the components during loading. In vitro, loss of water affects viscoelasticity and plastic toughness [34, 35]. At the crystal level, the apatite core is surrounded by a water layer that allows exchanges between it and both the extracellular fluids and the crystalline core. These exchanges permit crystal growth and maturation during mineralization. A loss of water decreases the distances between successive lattices of apatite crystals decreasing their size [36]. Within the physiological range of crystal size, smaller crystals are associated with lower stiffness [23]. Mineralization increases the number and size of crystals which replace water in the fiber volume occupied by water. Therefore as mineral is deposited and crystals grow in matrix, hydration decreases until ions are no longer able to diffuse. Mineralization stops at *95 %, the maximum mineral content organic matrix can reach [37]. The decrease with age in water content is associated with loss of elasticity [31, 38].


Osteons: Bone Structural Units (BSUs) The building blocks of mineralized matrix material used to assemble bone’s macrostructure have a microstructural organization. In humans and some animals, cortical bone is fashioned by remodeling into osteons or bone structural units (BSUs). Cortical osteons and trabecular hemiosteons are morphological ‘fingerprints’ of completed remodeling cycles. Within each BSU, bone matrix is organized into a lamellar structure of successive layers of fibrils differing in orientation in their long axis. Osteons are composed of 5–30 circular lamellae surrounding a Haversian canal. Trabecular bone is also lamellar. Reznikov et al. report that compared with cortical bone of the femoral shaft, human trabecular lamellar bone from the proximal femur had the same organization of ordered and disordered (loose) lamellae with the canalicular network confined to loose lamellae [39]. In trabecular lamellae, a proportion of the ordered collagen fibril arrays aligned with the long axis of the trabeculae. The remaining ordered collagen fibrils are offset from the axis by about 30° or 70°. Within interconnected trabeculae, the same lamellar structure will have a component of fibrils aligned with their long axes. The differing proportions of ordered fibrils might be important in determining mechanical function for load redistribution among trabecular elements [39]. During early mineralization, mineral platelets orient along the long axis of the collagen fibrils [40]. Mineral particles also orient parallel to each other when water is present [41]. The orientation between collagen and mineral platelets varies from lamella to lamella. In osteons, greater stiffness is achieved when the collagen and mineral long axes run parallel to the main loading direction [42, 43]. Individual osteons show different mechanical behavior depending on the dominant orientation of the mineral and collagen of its lamellae. Longitudinal lamellae confer tensile and bending strength while transverse lamellae confer compressive strength [44, 45]. The diameter of the osteon, or the wall width of any BSU, reflects the greatest depth achieved during the resorptive phase of a remodeling cycle. The Haversian canal diameter reflects the volume of bone deposited during the refilling phase [46]. The cement line defines the interface between the interstitial bone (formed months to years earlier) and the osteonal bone. Cement lines have little or no collagen [47]. They are reported to have a high mineral density or a low mineral density [48, 49]. They act as a barrier minimizing crack propagation [50]. In bone samples tested to rupture in fatigue, small cracks (\100 lm) are stopped, intermediate length cracks (100–300 lm) are deflected while cracks penetrating the osteons are longer ([400 lm) [51].

Remodeling rate and remodeling balance determine osteonal size, number per unit matrix volume, and total cement line length [52]. Heterogeneity in material composition is partly the result of differences in the timing and the location of remodeling events, and differences in the duration of the resorptive, reversal, and formation phases of each remodeling event (Fig. 4). For example, at any time, in cortical bone, osteoclasts of a BMU initiate resorption at a point upon a Haversian canal or endocortical surface resorbing matrix during *3 weeks [53, 54]. Elsewhere, BMUs initiated weeks earlier are in their reversal phase of *1 week [55]. As the formation phase of remodeling is slow, *3 months, BMUs initiated months earlier at other locations are in their formation phase while at yet other locations where remodeling was initiated months to years earlier, the fully formed BSUs undergo secondary mineralization, the slowest component of the remodeling cycle, taking 12–36 months [19, 56, 57]. Heterogeneity in matrix composition is established by variation in matrix age. The younger more recently formed osteons are composed of less mature, less crosslinked organic matrix, they have a lower mineral content and have a lower stiffness [58]. Older osteons and surrounding interstitial bone established by remodeling months to years earlier are more completely mineralized with a higher crosslink content, more mature mineral with bigger crystals. Heterogeneity in the completeness of mineralization is beneficial as it increases the energy needed for a crack to propagate.

Changes in Material Composition Associated with Advancing Age During young adulthood, remodeling is slow and in steady state; the number of BMUs initiated at points upon the inner or endosteal surfaces (intracortical, endocortical, trabecular) approximately equals the number of BMUs in various stages of completeness of their formation phase. There is little or no net bone loss, no net structural decay and material composition is stable because remodeling is balanced; each BMU removes and deposits equal volumes of bone. Nevertheless, as the formation phase of remodeling is delayed in onset and proceeds slowly, refilling is not instantaneous so that the unfilled excavated cavities upon Haversian canal, endocortical, and trabecular surfaces may form transitory stress risers or stress concentrators until the cavities are eventually completely refilled [59]. For this reason, high rates of remodeling before menopause may increase fracture risk independent of any bone loss or structural deterioration [60].

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Fig. 4 Effects of remodeling rate (increase: light gray arrows, decrease: dark gray arrows) on tissue mineral density: a Increased activation frequency in hyperparathyroidism decreases mean tissue mineral density and increases its heterogeneity by accumulating

younger and less fully mineralized osteons. Units (b & c). The opposite occurs when remodeling rate is reduced during bisphosphonate therapy. Adapted from Ref. [24] with permission

Menopause-Related Abnormalities in Remodeling and Micro-Morphology

produce more cement lines that defend against crack propagation. If the volume of bone deposited in the smaller cavity also decreases this may result in osteons with fewer lamellae and a larger central Haversian canal (high porosity). Over years, cortical osteonal heterogeneity increases as more osteons of different sizes accumulate with an increasing proportion of void volume leading to an increase in the number and the size of the stress concentrators impairing fracture resistance [68, 69]. Mean tissue mineral density may not change across menopause or as age advances but there is evidence of an increase in heterogeneity in the degree of mineralization as the result of the increased numbers of recently completed osteons formed due to high remodeling [70, 71]. Other studies suggest that mean tissue mineralization is higher due to the preferential loss of less completely mineralized osteons and hemiosteons as well as a higher proportion of interstitial bone producing more brittle material [71–73]. Currey et al. reported increased ash content with age, an effect that produced high stiffness and reduced toughness [74, 75].

Around midlife, the volume of bone deposited by each BMU decreases and the rate of remodeling increases. The negative BMU balance and rapidity of remodeling cause changes in material composition and structural deterioration [61–64]. There is evidence that osteonal diameter decreases as age advances, reflecting a decline in the maximum volume of matrix resorbed during each remodeling cycle [65–67]. Relative interstitial volume increases unless the increase in remodeling rate produces more small osteons. As total bone matrix volume decreases and total void volume increases due to bone loss, the diminishing total mineralized matrix volume may be composed of a higher relative amount of interstitial bone and a lower relative volume of osteonal bone. Interstitial bone has a higher mineral density, AGE crosslinking and fewer osteocytes; features contributing to brittleness of bone. Alternatively, larger numbers of smaller osteons may

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Enzymatic and Non-enzymatic Crosslinking of Osteons, Interstitial Bone and the Collagen-Mineral Interface Aging is associated with a decrease in the enzymatic crosslink content and an accumulation of nonenzymatic crosslinks like pentosidine in both sexes regardless of the skeletal site [2]. Pentosidine content is higher in more mineralized older BSUs [76, 77]. This is observed in both high or low remodeling states [2]. Matrix deposited during young adulthood may undergo different patterns of crosslinking than matrix deposited later in life. Nyman et al. reported that pentosidine content was higher in osteonal and interstitial bone of 70 to 90-year-old women than in 40 to 60-year-old women. Pentosidine content was higher in interstitial than osteonal bone in younger women, but was similar in these two regions in older women suggesting non-enzymatic crosslinking may be faster as age advances [78]. This higher AGE content stiffens the collagen fibrils [13]. AGEs influence strain distribution between tissue, collagen, and mineral platelets. For a given tissue strain, collagen fibrils in young bone take on a higher proportion of the strain protecting the mineral platelets. The accumulation of AGEs with aging decreases strain taken up by collagen by *25 % so more strain is imposed upon the mineral platelets, the brittle component of matrix (Fig. 5) [13]. Advancing age is also associated with collagen fibers becoming more longitudinally oriented relative to the axis of the loading, with reduced heterogeneity in orientation [79]. Osteons having lamellae with more longitudinally oriented fibers may impair compressive strength [45]. Fragility Fractures and Material Composition Unbalanced and rapid remodeling compromise bone’s material composition as well as bone microstructure. However, studies of differences in material composition in persons with versus those without fractures are limited, in part, because assessment of material composition requires invasive procedures like bone biopsy. High remodeling removes more fully mineralized matrix replacing it with younger less fully mineralized matrix [24]. Most studies suggest that patients with fractures have a lower mean tissue mineral density and greater heterogeneity in the distribution of the tissue mineral density [80–83]. At the proximal femur, the decrease in tissue mineralization density is more pronounced in the superior cortex of the femoral neck, a common location of origination of femoral neck fracture during sideways falls [84]. Although heterogeneity in matrix mineral density distribution increases the energy needed to propagate a crack, the higher prevalence of low matrix mineral density

decreases the stress needed to initiate permanent deformation [85]. In addition, increased oxidative stresses associated with estrogen deficiency promote formation of AGEs which reduce resistance to crack growth (loss of toughness) [86, 87]. AGEs also accumulate in patients with low rates of bone remodeling and may contribute to bone fragility [76, 88].

Diseases Produced by Disorders of the Organic Phase Osteogenesis Imperfecta Osteogenesis imperfecta (OI) or brittle bone disease encompasses a range of inherited disorders primarily affecting the collagen matrix. In more than 90 % of cases, the patients have dominant mutations in COL1A1 or COL1A2, the genes coding for the type I collagen a-chains. These mutations may cause a 50 % reduction in normal type I collagen synthesis (quantitative mutations) or alterations in collagen structure (qualitative mutations). Based on animal models using oim/oim mice and biopsy studies in human subjects, the mutations result in abnormalities in both organic and mineral components of the matrix. Regardless of the disease severity, collagen fibrils have a smaller diameter [89]. The crosslinking profile is not affected in human subjects but in the oim model, accumulation of non-enzymatic crosslinks and a decrease in enzymatic crosslinks is reported [90]. Most studies report an increased tissue mineral density and a loss of heterogeneity in tissue mineral density in animals and humans [90–94]. Mineral composition may also vary with a lower Ca/P ratio in infantile OI due to an increase in phosphorous but not calcium content [95]. Alteration in collagen fiber structure is held to be responsible for the changes in mineralization by there being more fluid-filled space for the mineral to grow [93, 96]. Other investigators suggest that the increase in tissue mineral density is independent of the severity of OI and collagen mutations (Fig. 6) [92, 94]. Some studies suggest mineral particle size in OI is normal or reduced, not increased. The high tissue mineral density is held to be due to smaller more densely packed particles contained within a similar volume of collagen fibrils [92]. Electron microscopy in the oim model support this view [97]. This pattern of mineralization is likely to contribute to counterintuitive micromechanical behavior in oim mice and in children with OI; despite the high tissue mineral density, tissue stiffness assessed by nanoindentation is lower, hardness is lower and plastic deformation is reduced [91, 97], the opposite of what is reported in normal bone [58]. These changes are directly associated with the brittle behavior of bone in oim mice [90].

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Fig. 5 a With increasing age, the number of osteons and the diameter of their Haversian canal increases (the dark region corresponds to the notch created for toughness evaluation). b At the nanoscale, collagen accumulates non-enzymatic crosslinks that stiffen the matrix. c Matrix

is less able to deform for a given stress. These changes in material composition at different scale levels lead to brittle behavior. (AGE advanced glycation end products). Adapted from Ref. [13] with permission

Diseases Produced by Disorders of the Inorganic Phase

mineralized collagen fibrils in animals have greater extensibility and lower stiffness [101]. In the Hpr model, during growth, the crystals are unable to orient their long axis along the direction of loading [102, 103]. These alterations in mineral quantity, quality, and orientation account for the skeletal deformities observed at the macroscale in rickets/osteomalacia.

Rickets, Osteomalacia Rickets in children and osteomalacia in adults are disorders of primary mineralization caused by vitamin D deficiency due to lack of sunlight exposure, intestinal malabsorption, or disorders of vitamin D metabolism. Hypophosphatemia also leads to osteomalacia with normal vitamin D status but this is usually due to mutations in genes coding for proteins participating in the phosphate absorption in the gut [98]. Mineralization lag time is increased in human subjects with osteomalacia and the matrix is lamellar in organization with a modest increase in crosslinks [99, 100]. The Hpr model of human X-linked hypophosphatemia rickets demonstrates lower tissue mineral density and greater heterogeneity in its distribution [101]. The incompletely

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Fluorosis Depending on the dose and the time of the exposure, fluoride (F-) has differing effects on material composition. Low doses of F- are used to prevent dental caries while high cumulative exposure in drinking water or drug therapy lead to fluorosis. Fluoride accumulates in bone formed during the period of exposure and substitutes for hydroxyl in apatite [104]. This substitution is associated with


Fig. 6 a In osteogenesis imperfecta (OI) matrix accumulates smaller more densely packed crystals in a collagen (W width). b Despite a higher tissue mineral density, the bone has decreased stiffness. c Changes in material composition in osteogenesis imperfecta

produce brittle bone with a reduced ability do deflect cracks as seen in the oim/oim mouse model of human osteogenesis imperfecta. Adapted from references [90–92] with permission

increases in crystal width that account for a large proportion of the variance in ultimate stress as assessed in mice. The higher the crystal width the lower the ultimate stress [105]. Fluoride retards mineralization halving mineral apposition rate and increasing mineralization lag time 30-fold [106]. At the tissue level, the heterogeneity of the mineral density is decreased [105]. In humans, the dose response to fluoride varies with greater sensitivity in some populations [107]. Altered sensitivity is also reported in various mouse strains such that similar exposure to NaF produces increases in crystal width and impairment in mechanical properties, while resistant strains have no changes in mechanical properties despite exposure to high doses [105]. Mousny et al., suggest there is genetically determined variation in the way collagen and mineral interface [105]. Heritable factors may modulate the strength of mineral-collagen bonding which in turn regulates the flux of ions such as fluoride to mineral limiting its impact on crystal structure.

diseases are associated with high BMD, thickened cortices, and trabeculae but an increased risk for appendicular fractures [98, 108]. Osteopetrosis is the result of inactivation of the gene encoding for the chloride-channel responsible for the acidification essential for dissolution of mineral and collagen degradation to create the resorption lacunae [109]. Tissue mineral density is increased as predicted by the low remodeling activity [110]. Abnormalities occur in several components of the matrix. For instance, in the toothless (tl/tl) osteopetrotic rat, content of mature enzymatic crosslinks is increased and concentration of immature crosslinks is decreased. The mineral content is higher in the tl/tl animals while the crystallinity is lower [111]. Boskey et al. reported higher mineral to matrix ratio but a lower crystallite size and perfection in the incisors-absent (ai/ai) osteopetrotic rat confirming a delayed maturation of the mineral phase [112]. These alterations in collagen maturation and mineral components confer lower bending and compressive strength [113]. In humans, osteopetrotic matrix has a high osteocalcin content, especially in the thickened cement lines [114]. The higher osteocalcin content may increase matrix stiffness and alter mechanical properties of the cement lines because osteocalcin binds apatite crystals by interaction between the Gla residues and calcium [17]. Higher mineral content of the cement line is reported in some, but not all studies [48, 49]. This may compromise the role of the cement line as an interface which attenuates microcrack propagation [50]. Loss of the ability to deflect cracks or preventing

Disorders of Remodeling Affecting Material Composition Osteopetrosis, Pycnodysostosis Osteopetrosis (Albers-Scho¨nberg disease) and pycnodysostosis are genetic diseases associated with low remodeling and reduced bone resorption due to absent osteoclasts or ineffective osteoclast functions. Both

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osteon debonding from matrix may compromise toughness [49, 115]. In pycnodysostosis, a mutation of the gene encoding for cathepsin-K results in failure of collagen degradation, reduced bone resorption with excavation of shallow pits rather than resorption trenches. Bone formation continues or is increased, in part perhaps due to sphingosine 1 phosphate-mediated osteoblastogenesis [108, 116]. Bone tissue from patients with pycnodysostosis contains fragments of unresorbed hypermineralized cartilage, inclusions that increase the heterogeneity of mineralization but not mean tissue mineral density (Fig. 7) [117]. Matrix is more mature due to low remodeling. Platelets of mineral are thicker than in controls. The crystals also lack a preferential orientation due to a chaotic lamellar orientation. This limits the ability of the material to adapt to mechanical loading leading to increased bone fragility [117]. Paget’s Bone Disease Paget’s disease of bone produces focal abnormalities in bone macro- and micro-structure and abnormalities in material composition due to focal increases in remodeling with accumulation of a non-lamellar and under-mineralized rapidly deposited matrix [118, 119]. The organic matrix accumulates hydroxylysinorleucine (HLNL), an immature and less stiff divalent crosslink [120]. Tissue mineral density is reduced and has a heterogeneous distribution [121]. The mineral has characteristics of younger matrix with fewer carbonate substitutions. In 49 patients with Paget’s disease and 49 controls, Zimmerman et al. reported

loss of osteonal and lamellar organization, features likely to reduce the ability of the matrix to deflect crack progression while the higher heterogeneity in tissue mineral density may improve it. Large and straight cracks are reported [121]. Stiffness is reduced in relation to the decrease in mineral content. However, toughness is maintained, probably due to the lower mineral content and greater heterogeneity, which result in deformation and microfractures but not catastrophic structural failure. Primary Hyperparathyroidism Excessive secretion of PTH in primary hyperparathyroidism results in hypercalcemia and low trauma fractures [98, 122, 123]. High remodeling is associated with bone loss if remodeling is unbalanced. While several studies suggest trabecular bone is spared, this is the result of errors in segmentation (separation) of the cortical and trabecular compartments during image analysis [124, 125]. Indeed, remodeling occurs on trabecular, endo- and intra-cortical surfaces, the latter leading to increased porosity and fragmentation of the inner cortex leaving cortical remnants that look like trabeculae erroneously apportioned into the medullary cavity and regarded as trabecular bone. This underestimates trabecular deterioration [126, 127]. In addition, the high remodeling produces increased numbers of newly formed BSUs which account for about 2 % of the decrease in cortical vBMD because tissue mineralization density is reduced in younger osteons [126]. High remodeling is accompanied by a reduced mean tissue mineral density and an increased heterogeneity in

Fig. 7 Left panel highly mineralized cartilage inclusion in the bone matrix of patient with pycnodysostosis. Right panel (a and b) Chaotic orientation of mineral (white bars) in pycnodysostosis compared to (c) normal orientation in a control. Adapted from Ref. [117] with permission

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mineralization as well as a lower ratio of mature to immature enzymatic crosslinks [128, 129]. The heterogeneity in material composition increases resistance to crack propagation. However, the accumulation of recently deposited matrix with immature collagen and mineral may have lower material stiffness. The changes in material composition are reversed after surgical treatment [126, 128, 129]. Sclerostin Deficiency Sclerostin is an inhibitor of bone formation produced by osteocytes and coded by the SOST gene that antagonizes WNT signaling [130]. Mutations of the SOST genes lead to sclerosteosis. Bone mineral density is high due to increased bone formation and the fracture risk is low even after major trauma [131, 132]. Whether increased formation is due to increased modeling and/or positive balance at the BMU level is not known. Hassler et al. reported Sost knock out mice had delayed mineral accretion and maturation [133]. A similar pattern was observed in human subjects: accumulation of younger incompletely mineralized matrix with smaller crystals and increased heterogeneity in the mineralization. In sclerosteosis, the high BMD phenotype is due to increase of bone matrix volume with normal tissue mineral density and preserved heterogeneity producing bone with greater strength. In osteopetrosis or pycnodysostosis, a high BMD is due to an accumulation of matrix with higher mineral content and lower heterogeneity produces brittleness [110, 117, 133]. Diabetes Type 1 and type 2 diabetes mellitus (T1DM, T2DM) are associated with increased fracture risk at levels of BMD higher than the risk predicted by the same BMD in nondiabetic subjects [134]. This finding suggests that there are differences in the material composition of bone that may account for the increased bone fragility [135]. In T2DM, using in vivo indentation testing, Farr et al. reported impaired ‘‘material strength’’ (i.e., increased indentation depth) in diabetic patients compared to controls independent of BMD [136]. Indentation depth is related to the energy needed to propagate a crack but this measurement is sensitive to the direction of loading. The observation remains difficult to attribute to a specific material property [137]. Likely candidates are the increase in AGEs and decrease in enzymatic crosslinks [138]. T2DM is associated with metabolic changes that may up-regulate AGE formation and down-regulate the formation of enzymatic crosslinks [139]. For example, T2DM is associated with (i) vitamin B6 deficiency, a co-factor of the lysyl oxidase involved in the production of enzymatic crosslinks and (ii)

increased levels of homocysteine that may promote AGE production directly and/or through increased oxidative stresses [140, 141]. Patients with diabetes may have reduced rates of bone remodeling so more complete secondary mineralization of matrix may contribute to fragility but little data is available to address this mechanism. Farlay et al. report higher tissue mineral density and lower heterogeneity in femoral neck bone of patients with T2DM and osteoarthritis compared to controls with osteoarthritis [142]. Tissue mineral density in bone from patients with T1DM with fracture was higher than that in controls and patients with T1DM without fractures. The AGE content was also higher among the patients with fracture than controls [143]. Material Composition Changes During Drug Therapy Antiresorptive Therapy Antiresorptive treatments slow the rate of remodeling and thereby slow, but do not abolish, structural deterioration produced by the remodeling and negative BMU balance that persists despite compliance with therapy. These drugs should slow the decline in BMD; they increase BMD because steady state remodeling is transiently perturbed at the onset of therapy; the number of new BMUs appearing decreases by about 50–70 % but the many more BMUs initiated prior treatment complete remodeling. Net BMD increases because (i) the osteoid deposited in the many more refilling sites undergoes primary then slower secondary mineralization, (ii) osteons now not resorbed complete secondary mineralization, and (iii) osteons formed months to years earlier also complete secondary mineralization (Fig. 8). Similar changes occur in the hemiosteons and interstitial bone of trabeculae. Bisphosphonates do not abolish remodeling, in part, because those with high matrix affinity may access and distribute less within deeper cortical matrix than they do in trabecular bone matrix with its thin plate-like structure. For example, Smith et al. report ibandronate administration reduced remodeling upon trabecular and endocortical surfaces but not Haversian canal surfaces in non-human primates. The concentration of ibandronate was lower in cortical than trabecular bone and improvement in bone strength was confined to the trabecular compartment [144]. Remodeling, bone loss, and structural decay continue so secondary mineralization progresses in an everdecreasing and deteriorated total bone matrix volume which is likely to be permissive for crack propagation [145–148]. Bisphosphonates alter zeta potential and interfacial tension, parameters that alter crystal growth and shape [149]. For example, during treatment with zoledronate or

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Fig. 8 Decreasing remodeling with alendronate leads to accumulation of advanced glycation end products (top left panel: ALN Alendronate, CTL control), increased tissue mineral density and

homogeneity (right panel). These alterations in material composition decrease post-yield work to fracture of bone (lower panel). Adapted from references [154, 156, 157] with permission

alendronate, matrix mineral deposited during primary mineralization has lower crystallinity. During alendronate treatment, after completion of secondary mineralization crystallinity was no different to that in the placebo group [150, 151]. The explanation for this is not apparent. However, in another study, women treated for *8 years with alendronate had lower matrix mineral crystallinity and less ability to dissipate energy by plastic deformation [152, 153]. These alterations in material composition are due to the bisphosphonate-mineral interactions independent of remodeling. Nevertheless, the benefits achieved by slowing structural deterioration are likely to outweigh the compromise in material composition, at least in most individuals [154, 155]. In addition, there is increased pyridinoline/ deoxypyridinoline and pentosidine content by up to 50 % [156]. In human and canine bone treated with bisphosphonates, the crosslink maturity (enzymatic mature/ immature forms) has a narrow distribution because of its antiresorptive activity, i.e., on average bone matrix is older and contains more trivalent mature crosslinks but additionally, alendronate accelerates the conversion of enzymatic immature forms into mature forms [157–159].

Dogs treated for 3 years with incadronate accumulate the non-enzymatic crosslink pentosidine in proportion to the reduction in activation frequency. The levels also are associated with microdamage density and length [160]. Tjhia et al. report an increase in resistance to plastic deformation independent of changes in tissue mineral density in postmenopausal osteoporotic women treated with bisphosphonate [161]. Further studies are needed to determine the contributions of crosslinking and matrix mineral properties to bone strength during antiresorptive therapy. Denosumab suppresses remodeling more rapidly and more completely than bisphosphonates like alendronate. This treatment reduces the birth of new osteoclasts, but it also reduces the life span and resorptive activity of osteoclasts in BMUs existing at the time of treatment. This profound suppression of remodeling should lead to more rapid and more complete secondary mineralization of BSUs in cortical and trabecular bone and higher content of AGEs. In ovariectomized non-human primates, Denosumab increased bone strength [162]. However, material mechanical properties did not differ between treated and untreated animals. Despite the absence of direct assessment of material composition, these results suggested that most

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of the changes in strength were predominantly explained by differences in bone structure [162, 163]. No published data in human subjects is available regarding material composition associated with denosumab treatment. In one study published in abstract form only, after 6 months denosumab treatment in adult mice dosed twice during 6 months, midshaft cortical and vertebral trabecular mean or peak values for bone mineralization density distribution did not change, but decreased heterogeneity in mineral distribution was observed as a reduction in the width of the distribution curve by 14.2 % with denosumab, which was greater than the 7 % reduction observed with alendronate. No reduction in toughness was observed in this brief study [164]. Water content of bone has implications in therapy. Gallant et al. reported raloxifene increases bound water content which increased post-yield toughness and total energy to failure in bending. [165]. The authors hypothesized that increased bound water facilitates slipping between mineral and collagen, increasing the ability of the matrix to deform post-yield. By increasing water retention, raloxifene alters the transfer of the loads between collagen and the mineral crystals suggesting that there may be changes in the mineral-collagen interfaces. [165]. Another example of the role of water is a report suggesting bone from rats treated with strontium ranelate has higher stiffness, hardness, and surrogate of toughness when testing in hydrated conditions [166]. Strontium is present in the hydrated layer of bone, not the mineral phase but how this influences bone strength is not known. Anabolic Therapy Intermittent administration of teriparatide (PTH1-34) stimulates the deposition of osteoid upon quiescent and actively remodeling intracortical, endocortical, trabecular, and periosteal surfaces. This newly deposited osteoid undergoes primary and then secondary mineralization. The deposition of younger matrix upon quiescent and remodeling surfaces, and the rapid replacement of older more fully mineralized matrix with younger matrix produces bone with a lower tissue mineral content, at least initially. In OVX monkeys, 18 months of PTH (1–34) was associated with a higher collagen content, higher enzymatic crosslink levels, a lower mature/immature enzymatic crosslinks ratio, and lower levels of pentosidine [167]. Only the higher levels of enzymatic crosslinks correlated with greater ultimate load, breaking energy, and stiffness in compression in vertebrae [159, 167, 168]. In addition, the accumulation of younger and less completely mineralized matrix decreases mean tissue mineral density and increases heterogeneity in mineral density distribution [169]. After 12 or 24 months of

teriparatide, Paschallis et al. reported that crystallinity was lower after treatment [168, 169]. Lower mean tissue mineral density and lower crystallinity should reduce stiffness and strength, and increase toughness. However, most preclinical data suggest improved stiffness, strength, and toughness [170–173]. Romosozumab, a sclerostin antibody under investigation as an anabolic therapy with antiresorptive properties has little effect on bone’s material properties as assessed in rats and monkeys. Despite increased bone formation and a 50 % increase in matrix volume, neither the crosslinking profile nor the mean tissue mineral density was altered. Only a modest increase in the number of pixels containing matrix of low mineralization was observed as well as an unexpected decrease in its heterogeneity. The reason for this may be that romosozumab may increase kinetics of primary mineralization permitting matrix to achieve 70–80 % of its mineral content rapidly [174, 175].

Summary Bone achieves paradoxical properties of strength yet lightness, and stiffness yet flexibility by varying the organic and inorganic constituents of its matrix and by varying the spatial distribution of this mineralized matrix using void volume to form its cortical, transitional, and trabecular compartments. Larger tubular bones assembled by bone modeling and remodeling use less material relative to their cross-sectional size; wider bones have cortices that are thinner, more porous and have a lower matrix mineralization density relative to more narrow tubular bones. Smaller structures are assembled with more material relative to their size, and material that is more fully mineralized. Individuals achieve a given set of traits that confer sufficient strength and stiffness to accommodate prevailing loading circumstances [176]. Longevity, disease or drug therapy modify one or more of these traits largely as a result of abnormalities in bone modeling and remodeling, the final common cellular pathway mediating all genetic and environmental influences on the skeleton. Deterioration in bone microstructure and compromises in its matrix constituents such as its collagen content and orientation, mineral content, crystal size and purity, enzymatic and non-enzymatic cross links, and water content, compromise compression, bending and shear strength.

Future Research The challenge for future research is to accurately quantify the individual, relative, and combined contributions of

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traits at nano-, micro- and macro-scopic levels to bone fragility of a region in individuals. Currently, patients with fractures are treated using similar drug regimens even though the abnormalities in bone modeling and remodeling, material composition, micro- and macro-structure, responsible for bone fragility vary from individual to individual. More sensitive and specific identification of individuals at risk and not at risk for fracture, respectively, is likely to be achieved using this approach. It is also likely that using pathogenesis and morphology to assess the need for single, combined or sequential therapy will improve treatment success, and allow modification of therapy when changes in morphology such as worsening of cortical porosity, trabecular deterioration, increases in tissue mineralization, or pentosidine suggest treatment has failed. Conflict of interest conflicts of interest.

Bala and Seeman declare that they have no

Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

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Sclerostin Antibody Treatment Increases Bone Formation, Bone Mass, and Bone Strength of Intact Bones in Adult Male Rats.

The contribution of collagen crosslinks to bone strength.

Mechanical basis of bone strength: influence of bone material, bone structure and muscle action.

Cranberries and their bioactive constituents in human health.

Bone Marrow Stem Cell Contribution to Pulmonary Homeostasis and Disease.

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Women with previous stress fractures show reduced bone material strength.

Bond strength of restorative material to dentin submitted to bleaching and Er:YAG laser post-treatment.

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Antibacterial efficacy of local plants and their contribution to public health in rural Ethiopia.

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Effects of fluoride treatment on bone strength.

The mitochondrion, its genome and their contribution to well-being and disease.

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Religion's contribution to health.

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An integrative review of attention biases and their contribution to treatment for anxiety disorders.