Effect of ultrastructural changes on the toughness of bone

Micron 36 (2005) 566–582 www.elsevier.com/locate/micron

Review

Effect of ultrastructural changes on the toughness of bone Jeffry S. Nymana, Michael Reyesb, Xiaodu Wanga,b,* a

Mechanical Engineering and Biomechanics, University of Texas at San Antonio, 6900 North Loop 1604 West, San Antonio, TX 78249, USA b Biomedical Engineering, University of Texas at San Antonio, 6900 North Loop 1604 West, San Antonio, TX 78249, USA

Abstract The ultrastructure of bone can be considered as a conjunction between the biology and the biomechanics of the tissue. It is the result of cellular and molecular activities of bone formation, and its organization dominates the mechanical behavior of bone. Following this perspective, the objective of this review is to provide a current understanding of bone ultrastructure and its relationships with the toughness of the tissue. Therefore, we first provide a discussion on the organization of bone constituents, namely collagen, mineral, and water. Then, we present evidence on how the toughness of bone relates to its ultrastructure through the formation of microdamage. In addition, attention is given to how damage accumulation serves as a toughening mechanism. Finally, we describe how changes in the ultrastructure-caused by osteogenesis imperfecta, gamma irradiation, fluoride treatment, and aging affect the toughness and competence of bone. q 2005 Elsevier Ltd. All rights reserved. Keywords: Collagen; Mineral; Microdamage; Water; Aging; Osteogenesis imperfecta; Mechanical properties

Contents 1. 2.

3.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bone hierarchy and composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Organic matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Collagen cross-links . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Mineral phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Bone mineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Primary versus secondary mineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Lamellar organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction of microdamage with ultrastructure influences bone toughness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Microdamage in bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Post-yield behavior of bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Damage accumulation as a toughening mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ultrastructure affects bone mechanical behavior: examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Osteogenesis imperfecta (OI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Gamma irradiation of bone allograft . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Fluoride substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Aging effects on bone ultrastructure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1. Age-related changes in cross-links . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2. Age-related changes in collagen fibrillar orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.3. Age-related changes in mineral phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Address: Mechanical Engineering and Biomechanics, University of Texas at San Antonio, 6900 North Loop 1604 West, San Antonio, TX 78249, USA. Tel.: C1 210 458 5565; fax: C1 210 458 6504. E-mail address: [email protected] (X. Wang).

0968-4328/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2005.07.004

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4.4.4. Potential role of water in the age-related decrease in bone toughness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction There is a high susceptibility of bone fracture among the elderly population, and such fractures necessitate rather costly treatment and cause significant morbidity and mortality (Johnell et al., 2004; Melton, 2003). For example, two population studies have estimated that the lifetime risk of fracture is between 40 and 46% for Caucasian women at the age of 50 years and is between 13 and 22% for Caucasian men at the same age (Kanis et al., 2000; Melton et al., 1992). Furthermore, every year there is a rise in the number of bone fractures, and such fractures sustained world-wide could reach 4.5 million by 2050, increasing from 1.26 million as estimated in 1990 (Gullberg et al., 1997). Thus, understanding the structure–function behavior of bone is of considerable interest to both scientists and health care providers. Bone is a highly hierarchical structure (Fig. 1). Detrimental changes in the organization at any level (e.g. fenestration of trabeculae, abnormal mineralization, defective collagen molecules, etc.) can increase the risk of bone fracture. Recently, the toughness of bone has drawn more attention as an important indicator of bone quality, independent of its size or mass (Currey, 2001). Much of the quality of bone originates at the sub-micron and molecular levels. Because of this, certain changes in bone ultrastructure could lead to a deterioration of bone quality, which is reflected by its toughness and other physical and chemical properties.

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By definition, toughness is the mechanical energy absorbed by a material when deformed until failure. For bone, most of this energy is dissipated after yielding (when permanent deformation occurs). This energy is related to fracture toughness, which quantifies the resistance to crack propagation. Microdamage formation (e.g. microcrack and diffuse damage) is a major pathway for bone to dissipate energy during post-yield deformation. It is also evident that energy absorption could be dissipated mainly through permanent alterations in the ultrastructure and material properties of bone constituents (e.g. denaturing of collagen and cracking of mineral phase, etc.). Thus, the ability of bone to resist catastrophic failure is dependent on the ability of its microarchitecture and the ultrastructure to dissipate energy.

2. Bone hierarchy and composition Following the lead of others (Rho et al., 1998; Weiner and Wagner, 1998), the organization of bone can be described in five hierarchical levels as shown in Fig. 1: (1) cortical bone and cancellous bone at the macrostructural level; (2) Haversian systems (osteons)/interstitial tissue or trabeculae at the sub-millimeter scale; (3) the lamellae (including lacunae and canaliculi) at the micron level; (4) mineral matrix and collagen fibrils at the submicron scale; and (5) mineral crystals, collagen and water molecules at the nanometer level. There are also other important features in bone such as bone cells,

Fig. 1. At each level of hierarchy of the femur, there are: cortical and cancellous bone; osteons and trabeculae (not shown); lamellae; assemblies of collagen fibrils; and collagen molecules, mineral crystals, and water. Reprinted from Medical Engineering and Physics, vol. 20, Rho et al., Mechanical properties and the hierarchical structure of bone, pp. 92–102, 1998 with permission from The Institute of Physics and Engineering in Medicine.

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Fig. 2. After the terminal ends are cleaved, the collagen molecule begins to assemble into a fibril via enzymatic cross-links. q 1996 From ‘The major matrix molecule in mineralized tissues’ in Phosphorus in Health and Disease (JJB Anderson and SC Garner, eds) by Yamauchi, M. Reproduced by permission of Routledge/Taylor & Francis Group, LLC.

non-collagenous proteins, and damage in the forms of linear microcracks and diffuse patches. Nonetheless, the organic matrix (mainly collagen), apatite mineral, and water predominately make up bone and so a description of each follows.

in diameter) via cross-links, forming a staggered arrangement (Fig. 3(A)). First described by Hodge and Petruska (1963), a distance of 35–40 nm separates the head and tail of any two aligned collagen molecules as shown in Fig. 3(A). A neighboring helix aligns in parallel but is offset by 64–70 nm (Hodge, 1989). The resulting overlap (w27 nm) gives rise to the striation pattern that is seen when viewing collagen fibrils with electron microscopy or atomic force microscopy (Fig. 3(B)). Packing of the microfibril is not entirely clear, but recently Hulmes et al. (1995), presented a concentric model of packing that fits with mechanisms for the control of fibril diameter (Hulmes, 2002). Corroborating evidence for the organized packing of collagen molecules comes from the work of Yamauchi et al. (1989). Aldehyde-derived cross-links predominately occurred in the carboxyl terminal between an a1 chain and either an a1 or an a2 chain on a neighboring molecule at a ratio of 3.5:1. This indicates a stereospecific (i.e. not random) packing of collagen because otherwise the ratio would be 2:1. Groupings of the amino acid residues (e.g. polar, acidic, etc.) at regular intervals also facilitate organization of the collagen molecules into fibrils (Miller, 1984).

2.1. Organic matrix The organic matrix of bone occupies about 32% of the volume and mainly consists of type I collagen (90%) with small, but important amounts of non-collagenous proteins like osteocalcin and osteonectin, which aid in the mineralization process. Secreted by bone forming cells known as osteoblasts, procollagen is a helical rod (Fig. 2) of three intertwining polypeptide chains (two identical a1 helices and one different a2 helix) (Yamauchi, 1996). Each chain contains approximately 1000 amino acids in which every third residue is glycine and is positioned toward the center of the super-coil (Nimni, 1993; Traub and Piez, 1971). Proline typically occupies the next position, and there is an abundance of hydroxyproline in the third position (Hannig and Nordwig, 1967). The latter residue facilitates thermal stability through intramolecular hydrogen bonding (Berg and Prockop, 1973). Hydroxylysine is also a characteristic residue of bone collagen and gives rise to cross-linking. Upon cleavage of the non-helical amino (N) and carboxyl (C) terminals, procollagen begins to selfassemble into a collagen fibril (300 nm in length and 1.2 nm

2.1.1. Collagen cross-links The collagen cross-links are a salient feature of bone because they not only organize fibrillation, but also contribute to the mineralization process, thus affecting the mechanical behavior of bone. Detailed reviews of the chemistry and function of collagen cross-links have been given by Knott and Bailey (1998); Bailey et al. (1998). Briefly, the enzymatic control of cross-links occurs through the lysyl and hydroxylysyl residues, which are located at both C- and N-terminal ends (though cross-linking in the carboxyl domain is more prevalent in bone) (Yamauchi, 1996). Given an aldehyde group by lysyl hydroxylase (copper dependent enzyme), these residues spontaneously condense with a hydroxylysyl residue on a neighboring collagen molecule. This divalent bond (referred to as a ketoimine cross-link) is reducible (Yamauchi, 1996) and occurs between the N- and C-terminals of two staggered helices (Fig. 3(A)) (Bailey et al., 1980). With time, a keto-imine may react with yet another neighboring hydroxylysyl aldehyde (maturing into a trivalent cross-link or pyridinoline), or they may devolve (Fig. 4). There are more divalent

Fig. 3. Collagen molecules arrange into fibrils in a staggered fashion with cross-links connecting the C-terminal to the N-terminal of neighboring molecule (A). The overlap regions of molecules create a banding pattern visible with atomic force microscopy (B).

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(Ca5(PO4CO3)3 (Weiner and Wagner, 1998). There are various reasons for the indistinct diffraction pattern of bone, including non-stoichiometric ratio of calcium to phosphorous, presence of strongly bound water, and deposition of amorphous mineral (tricalcium or octacalcium phosphate) (Glimcher, 1987). In addition, bone crystals are quite small (possibly the smallest formed biologically). Plate-like in shape, the apatite crystals have length, width, and thickness of 50 nm!25 nm!1.5–4 nm, respectively (Fratzl et al., 1992; Landis and Glimcher, 1978; Wachtel and Weiner, 1994). Given the right concentration and a nucleation agent, mineral (irrespective of composition) will thermodynamically precipitate in vitro. In the mammalian skeleton, bone mineralization follows collagen organization. Fig. 4. Cross-linking provides a stable template for mineralization and directs the compaction of molecules as mineral accumulates within the fibril. Some cross-links are thought to dissociated while others mature. q 1996 From ‘The major matrix molecule in mineralized tissues’ in Phosphorus in Health and Disease (JJB Anderson and SC Garner, eds) by Yamauchi, M. Reproduced by permission of Routledge/Taylor & Francis Group, LLC.

cross-links than trivalent over a lifetime, but the discrepancy decreases with skeletal maturation (Eyre et al., 1988). Another non-reducible, enzymatic cross-link is the pyrrole, formed when hydroxylysyl aldehyde reacts with nonhydroxynated lysine (i.e. tends to occur with reduced hydroxylation) (Bailey and Knott, 1999). Cross-links in collagen may also occur through nonenzymatic pathways, namely glycation. An amino group (e.g. lysine and arginine) of long-lived proteins is susceptible to reaction with glucose, specifically its aldehyde (Bunn et al., 1978). The resulting aldimine linkage rearranges into a more stable keto-imine linkage producing what is called the Amadori product. Though more stable, oxidative breakdown occurs over time causing reactions with other amino acid residues. This produces advanced glycation end-products (AGE), (Bailey et al., 1998), the anagram being relevant since such products increase with age. 2.2. Mineral phase The mineral phase makes up about 43% of the volume of bone and contains primarily calcium and phosphate with small but significant amounts of carbonate as well as other impurities (sodium, magnesium, potassium, citrate, fluoride, and HPO3K) (Glimcher, 1987). Early X-ray diffraction studies identified the bone mineral as a hydroxyapatite (Posner, 1969; Roseberry et al., 1931). However, the reflection patterns of bone are not as distinct as those of synthetic hydroxyapatite (Boskey, 2003). Furthermore, there is an absence of hydroxyl groups when bone is analyzed with Fourier Transform Infrared Spectroscopy (FTIR) or Nuclear Magnetic Resonance (NMR) (Loong et al., 2000; Rey et al., 1995). Thus, the mineral phase may be more precisely classified as a carbonated apatite

2.2.1. Bone mineralization Initially, water fills the void space within the organic framework of the osteoid (i.e. collagen matrix). Crystals then begin to form and displace water (Robinson, 1975). Mineralization may start with extracellular matrix vesicles or with collagen, in which the former is more associated with rapid bone formation (woven bone) and the latter is more associated with organized bone formation (lamellar bone) (Lowenstam and Weiner, 1989). With regard to collagen-based mineralization, crystals are preferentially associated with contiguous gaps in the collagen network (Landis et al., 1996a,b; Robinson and Watson, 1952; Traub et al., 1989; Weiner and Traub, 1986). This is apparent from transmission electron microscope (TEM) images of turkey tendons that show similar banding patterns in both stained, unmineralized collagen fibrils and non-stained, mineralized collagen fibrils. The association of mineral deposits with the contiguous gaps (also referred to as grooves) has also been observed in three dimensions in embryonic chick bone (Landis et al., 1996a). The grooves provide enough width for crystals to fit in the gap. Furthermore, the long axis of the plate-like crystal (the crystallographic c-axes) aligns with the long axis of the fibril (Landis et al., 1993; Traub et al., 1989; Weiner and Traub, 1986). Evidence for the dependence of mineralization on the organization of the collagen network also comes from in vitro studies which found that calcium–phosphate crystals only nucleated when the collagen fibrils had aggregated like their native counterpart (i.e. gaps at a 67–70 nm period) (Glimcher, 1960). Nonetheless, a gap is not necessarily sufficient to serve as a heterogeneous nucleation site, for there is evidence that non-organic proteins regulate mineralization as indicated by a number of in vitro studies (Cowles et al., 1998; Endo and Glimcher, 1989; Glimcher, 1989; Hunter and Goldberg, 1993; Termine et al., 1981). Following crystal nucleation at multiple, independent gaps within the collagen fibril as well as on the surface of fibrils (Landis et al., 1996b), crystal growth continues into the overlap zones that exist between collagen molecules (Arsenault, 1991a,b; Maitland and Arsenault, 1991). Yamauchi (1996) has suggested that the dissociation of

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certain cross-links and maturation of others directs the compaction of collagen molecules (Fig. 4). In other words, the cross-linking pattern facilitates mineral accumulation between collagen molecules. The use of mineralizing turkey tendon to understand the mechanism of biomineralization begs the question of whether a similar process (i.e. predominately intrafibrillar mineral accumulation) occurs in bone. Another possible explanation for compaction of collagen molecules is a second loci of mineralization between the fibrils (Lees and Prostak, 1988). Estimations of intrafibrillar volume in the literature are conflicting (Bonar et al., 1985; Katz and Li, 1973). Early studies of mineralization by Robinson et al. (Robinson, 1979; 1975; Sheldon and Robinson, 1957) provide some clarification on the location of mineral: (1) using their data and that of Bear (Bear, 1956), they estimated that 40% of the water in the bone matrix was located between the fibrils and fibers, and moreover, that this extrafibrillar volume was not adequate to contain all crystallites (Robinson, 1979); (2) electron microscopy of early mineralization revealed the presence of crystals within and between collagen fibrils (Sheldon and Robinson, 1957); (3) mineral accumulation did not grossly displace the fibrils or push them together (not to be confused with local compacting of the collagen molecules) (Robinson, 1975). It would seem that the mineral occupies two compartments in the extracellular matrix of bone tissue, and answering the question of whether the majority of the mineral resides intra- or extra-fibrillarly depends on a better understanding of how collagen molecules pack into fibrils and how densely fibrils aggregate. This, in turn, depends on the nature of the inter- and intra-fibrillar cross-links, which constrain the expansion of both the intra- and extra-fibrillar volume. Interestingly, a recent model of collagen packing has indicated that the average concentration of mineral is equal between the two compartments (Hellmich and Ulm, 2003) 2.2.2. Primary versus secondary mineralization One of the unique aspects of bone in the skeletons of larger animals is that old tissue is continually being replaced with new tissue in a process called bone remodeling. Thus, a microradiograph for a thin cross-section of bone can reveal a heterogeneity of mineralization as newly formed osteons have less mineral content than primary osteons or interstitial bone. Because there is such a variety in the absorption of X-rays across osteons within the same bone sample, bone is thought to mineralize in two stages. Mineralization in the primary stage occurs rapidly (over a few days) following deposition of osteoid (Wergedal and Baylink, 1974) while the secondary stage takes much longer and its rate of mineral accumulation decreases with time (Marotti et al., 1972). An early study by Amprino (1958) reported that the period of secondary mineralization was several months. However, a more recent study with Raman spectroscopy has found that mineral maturation can occur over two decades

(Akkus et al., 2003). Thus, the state of the bone ultrastructure depends on the remodeling activity. One implication of this connection is that drug treatments, which suppress remodeling (e.g. bisphosphonates) may facilitate more mineralized and homogenous bone tissue, both of which could decrease the fracture toughness of the tissue. 2.3. Lamellar organization The lamellae, 3–7 mm thick, are a central feature of nearly all bone tissue (Martin and Burr, 1989). With reflective light microscopy, they appear as white bands of varying thickness separated by a thin dark layer. Electron microscopy has revealed that this banding results from the difference in the orientation of collagen fibrils between neighboring lamella (e.g. see the pattern shifts between lamella in the sub-microstructure of Fig. 1). Fibrils may intermingle across lamellae, but there is a distinct and preferred orientation for any given layer (Frasca et al., 1981). Initially, the orientations of collagen fibrils in lamellae were determined from polarized microscopic views of osteons: transverse fiber bundles had an orientation parallel to the cross-sectional plane, longitudinal fibers aligned parallel to the longitudinal axis of the osteon, and intermediate fibers alternated from transverse to longitudinal (Ascenzi and Bonucci, 1967). This concept evolved when thin sections of decalcified human cortical bone were viewed with TEM. In doing so, Giraud-Guille (1988) found two general architectures of collagen fibers: (1) ‘orthogonal plywood’ with alternating orthogonal orientations of fibrils, and (2) ‘twisted plywood’ with continually changing orientation of fibrils in which the pattern repeats itself through 1808 cycles. There are also TEM as well as scanning electron microscope (SEM) observations of parallel fibrils rotating at a plywood angle of w308 through five successive sub-layers of varying thickness in a lamella (Weiner et al., 1997; 1999). In general, the fibrils in lamellae have a preferred but shifting orientation, and such a structure may prevent microcrack propagation. 2.4. Water Water occupies up to 25% of the volume of bone and is distributed throughout the tissue in various forms: freely mobile in vascular-lacunar-canalicular space, bound to the collagen network, and bound to the mineral phase. For example, Robinson (1979) found that free water residing in the marrow-vascular-osteocytic space takes up 13.74– 22.84% of volume, whereas the bound water that is attached to collagen or mineral occupies an additional 6.97% of the volume of canine cortical bone. In arriving at this distribution, it was assumed that drying at 50 8C would remove all water associated with the marrow-vascularosteocytic space. However, other studies suggest that lower temperatures may remove bound water along with mobile water (Nyman et al., 2005; Timmins and Wall, 1977).

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Fig. 5. Water helps stabilize the collagen by creating inter- and intrahydrogen bonds with hydrophilic residues.

Nonetheless, such estimations match expected results when considering the area of Haversian canals, lacunae, and canaliculi in microradiographs of cortical bone. Using a different approach by measuring the dielectric constant and loss as a function of hydration, Marino et al. (1967) presented a critical hydration value for free and bound water in bone. Their results suggest that water first situates in the mineral and collagen phase, and once exceeding saturation, it starts to occupy void spaces. H2O is by nature a polar molecule and naturally 2C associates itself with mineral (POK ) and collagen 4 or Ca (glycine, hydroxyproline, carboxyl, and hydroxylysine), as depicted in Fig. 5. There have been studies done on the hydration of collagenous tissue (human dura mater and rattail tendons) with dynamic mechanical spectroscopy, and they indicate that water does associate with collagen at two levels (Nomura et al., 1977; Pineri et al., 1978):

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hydrogen bonding occurs on and between collagen molecules (due to the hydroxyl group of hydroxyproline and the polar side chains, respectively). In addition, NMR analysis of enamel, dentine, cementum, and bone has revealed the presence of crystalline water (Casciani, 1971). Because the absorption of radio frequency energy of a proton in a magnetic field depends on the closeness of neighboring protons, several distinct proton resonance spectra are detected in bone. One such spectrum has been considered the crystalline hydrate form of water and can be eliminated by ashing at 540 8C (Casciani, 1971). More recently, an analysis of the spin relaxation components in an NMR study of dentin has exhibited that 30% of the water was strongly bound to the apatite (crystalline water), 52% was hydrating a large molecule (at the surfaces of the crystallites or within collagen fibrils), and the remaining 18% was residing in the tubules (akin to the canaliculi of bone) (Schreiner et al., 1991). This estimation of free water is considerably less than what would be expected in bone because dentin does not have an extensive vascular space like bone.

3. Interaction of microdamage with ultrastructure influences bone toughness 3.1. Microdamage in bone Daily loading activities generate damage in the extracellular matrix of bone (Frost, 1960). Two general types of damage (microcracks and diffuse damage as shown in Fig. 6) have been identified with the use of chelating fluorochromes (e.g. basic fuchsin or xylenol orange) (Burr and Stafford, 1990; Fazzalari et al., 1998). Diffuse damage (C in Fig. 6) is a patch of extensive networks of fine, ultrastructural-level cracks (Boyce et al., 1998) that are visible in more detail with a laser scanning confocal microscope at high magnification, whereas a microcrack (A in Fig. 6) is a linear defect (O100 mm) that is readily visible with a light microscope at low magnification.

Fig. 6. Linear microcracks tend to form between to osteons in cortical bone (A), but fatigue loading can cause macrocracks to cut through them (B). Diffuse patches of ultra-structural cracks tend to develop when bone is loaded in tension (C).

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A number of studies have provided evidence showing osteoclastic resorption serves to remove damage in bone (Bentolila et al., 1998; Burr et al., 1985; Mori and Burr, 1993). Such biological action strongly suggests that damage is recognized by the body as a threat to bone quality. Moreover, bone damage interacts with both the micron and sub-micron level of organization. For instance, linear microcracks are often observed in the interstitial tissue suggesting that the osteons act as barriers to crack progression. In addition, these cracks most likely nucleate from a defect at the sub-micron level, such as lacunae, which have been implicated as generators of high strain (Nicolella and Lankford, 2002). Furthermore, diffuse damage or linear microcracks dissipate energy by rupturing mineral crystals and collagen fibrils. 3.2. Post-yield behavior of bone With aging, there are likely changes in the ultrastructure that allow microdamage to nucleate and develop more readily. Courtney et al. (1996) loaded human cortical bone tensile specimens (both young and old) to a change in elongation of 1%, which caused a deformation beyond yielding (i.e. when stress and strain are no longer linearly related), but not before fracture. The specimens were then unloaded and re-loaded back to 1% strain. Microdamage was analyzed in test (loaded) and control specimens (not loaded). The elderly bone had 50% more microcracks in the test specimens than in the control, while the younger bone exhibited no difference between test and control. Further analysis revealed that damage accumulation is the likely cause of the stiffness reduction that occurs when bone is reloaded after being subjected to permanent deformation or yielding (Fig. 7). Microcrack orientation to loading has also

been found to be important to the degradation of bone stiffness caused by damage accumulation (Akkus et al., 2003). Lastly, Kotha and Guzelsu (2003) also investigated the effect of induced or de novo damage on the properties of bone. Tensile specimens that had been cyclically loaded several times beyond the yield point had significantly less ductility than control specimens (Table 1). Toughness of bone appears to depend on the level of microdamage, and less energy is required to generate microdamage in older bone than in younger bone. There are also a number of other induced changes in the bone ultrastructure that affect bone toughness, as given in Table 1. For example, a decline in collagen integrity is associated with decrease in toughness. From a damage mechanics point of view, the energy dissipation of bone due to the microdamage accumulation during post-yield deformation is most likely associated with a decreased elastic modulus (increased compliance of the tissue) and residual permanent strain (3p in Fig. 7). Increased energy dissipation associated with microdamage accumulation is likely due to the formation of new crack surfaces (i.e. surface energy). However, the release of surface energy with microcrack formation alone cannot explain the energy dissipation in the plastic deformation (residual strain) during the post-yielding of bone. Addressing this issue, Fondrk et al. (1999) proposed a rheological damage model to describe the non-linear tensile behavior of bone by considering both increased compliance and permanent (plastic) strains associated with the post-yield deformation of bone. The increased plastic (residual) strain during the post-yield deformation of bone is most theoretically due to the contribution of the collagen phase. This is reasonable given that the mineral phase has minimal influence on the plastic deformation of bone (Zioupos, 2001b). Thus, two ultrastructural effects, cracking of the mineral phase and rupturing of the collagen network, likely contribute to postyield toughness of bone. 3.3. Damage accumulation as a toughening mechanism

Fig. 7. Post-yield deformation causes a stiffness loss (E/E0!1) and permanent strain (3p) as shown when bone is reloaded after having been previously loaded beyond its yield point in which stress-strain relationship is no longer linear.

While increases in bone microdamage have been associated with decreases in bone toughness (among other bone parameters as given in Table 2), the accumulation of damage during deformation is also thought to act as a toughening mechanism. With compact tension (CT) specimens of bovine and human cortical bone, Vashishth et al. (1997) investigated the suggestions from other studies (Wright et al., 1981; Zioupos et al., 1994) that microcracking toughens bone. Fracture toughness and damage accumulation were observed to increase with crack extension. Based on other observations in the fracture of ceramics and their own observations of damage in bone fracture specimens, Vashishth (2000, 2003) proposed that microcrack formation occurs in two stages: the formation of a frontal process zone (Stage I) and the formation of a process zone wake (Stage II). Upon the application of a load, a zone of microdamage (the frontal process zone)

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573

Table 1 A number of changes in the ultrastructure of bone affect toughness Cause of change

Change in ultrastructure

Effect

Reference

Heat treatment (O120 8C) Heat treatment (350 8C)

[ In denatured collagen Small Y in carbonate concentration No change in crystallinity [ In denatured collagen

Y In work-to-fracture Y In ultimate strain

Wang et al. (2001) Catanese et al. (1999)

Y In work-to-fracture

Wang et al. (2002)

[ In non-calcified denatured collagen (4%) [ In pentosidine [ In bound water Y In mineral [ In non-enzymatic glycation products Loss of copper implies loss of crosslinking Less microdamage at given stress intensity Impairment of damage accumulation during fracture Implied [ in diffuse damage Y In mineral Y In electrostatic bonds

Y In work-to-fracture

Wang et al. (2003)

[ In ultimate strain

Broz et al. (1995)

Y In damage fraction

Vashishth et al. (2001)

Y In torque-to-fracture

Jonas et al. (1993)

Y In fatigue crack propagation resistance Y In fracture toughness and work-to-fracture Y In ultimate strain [ In toughness [ In ductility

Mitchell et al. (2004)

Cleavage of collagen of demineralized bone Age

Demineralization in EDTA Ribose-induced glycation In vivo copper deficiency Gamma irradiation Gamma irradiation Mechanical-induced damage Fluoride treatment Fluoride treatment

Akkus and Rimnac (2001) Kotha and Guzelsu (2003) DePaula et al. (2002) Joseph Catanes and Keaveny (1996)

and monitored the diffuse damage in the frontal process zone by laser scanning confocal microscopy. Diffuse damage was found to consistently form and accumulate in the matrix between canaliculi and in the vicinity of lacunae. The canalicular–lacunar network appeared to deflect microcrack growth. Fracture toughness undulated as a function of the density of diffuse damage because spatially distinct small cracks accumulated until exhausting the local region of mineral, thus creating a larger crack, which in turn initiated a new set of diffuse damage formation. Beyond crack-tip shielding, there are other possible toughening mechanisms that involve damage. At the microstructure level, cement lines are thought to deflect the crack path (Yeni and Norman, 2000).

forms around a crack tip in bone. Increasing the load coalesces the microdamage into a propagating crack, which can then accelerate through the frontal process zone. As this main crack continues propagating forward, new microdamage is formed around its tip. The formation of these microcracks absorbs energy, thereby decelerating the crack propagation. When the region ahead of the main crack becomes saturated with microdamage, it accelerates again and repeats the aforementioned process. The toughening mechanism proposed by Vashishth et al. is supported by an independent study. Parsamian and Norman (2001) subjected CT specimens of human tibial cortical bone to fatigue loading (without inducing macrocrack growth)

Table 2 Decreases in work to fracture (Wf), impact energy (impact), critical tensile strain energy release rate (GIc), critical shear strain energy release rate (GIIc), critical stress intensity factor (Kc), and plastic fracture toughness (J-integral) have been statistically correlated (p!0.05) with an increase in age. These properties are also associated with age-related changes in certain human femora, cortical bone parameters Parameter Age Ash content Porosity Osteon density Microcrack density Wet bone density Collagen contraction rate Collagen strength Pentosidine a b c d e f g

Currey et al. (1996). Yeni et al. (1997). Zioupos (2001a). Norman et al. (1998). Yeni et al. (1998). Zioupos et al. (1999). Wang et al. (2002).

Wf

Impact a

K0.60 K0.53a

0.83f 0.54g K0.62g

a

K0.57 K0.52a

GIc

GIIc b

Kc b

J-integra1 c

K0.40

K0.42

K0.84

K0.63c

K0.56b 0.79b K0.16d 0.43e

K0.55b 0.66b

K0.78c

K0.76c

K0.74c

K0.82c

0.65f

0.86f

0.53e

K0.48g

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At the ultrastructure level, collagen fibers can bridge the crack, thus retarding its growth (Nalla et al., 2003b).

4. Ultrastructure affects bone mechanical behavior: examples Since the ultrastructure influences microdamage formation and further affects bone toughness, changes in the structural integrity of collagen molecules, in collagen crosslinks, in mineral dissolution, and in lattice structure would affect the toughness of bone. The following are examples of such relationships in the cases of osteogenesis imperfecta, irradiation and fluoride treatments, and the aging process. 4.1. Osteogenesis imperfecta (OI) OI is a heritable syndrome that affects both mineralized and non-mineralized tissues, and clinically, it causes increased bone fragility and low bone mass (Rauch and Glorieux, 2004). The basis of the disease is a genetic defect that affects the ability of osteoblasts to produce normal collagen. Mutations of the procollagen genes (COL1A1 and COL1A2) affect either the synthesis or structure of type I collagen. A substitution of the glycine residue in the a1 or a2 chain is an example of a structural mutation (Byers et al., 1991). Other mutations in COL1A1 and COL1A2 consist of exon skipping, deletions, insertions, duplications, and frameshifts. The inability of the collagen molecules to form normally disrupts the packing of the molecules into fibrils. Since collagen acts as a scaffold upon which bone mineralization occurs, it is not surprising that bone from humans with OI as well as animal models of the disease exhibit ultrastructure that differs from normal controls. The calcium to phosphorous (Ca/P) ratio of apatite, is generally, lower than normal in humans with OI (Cassella and Ali, 1992) and animal models of the disease (Cassella et al.,

1994) with the degree to which the Ca/P ratio is reduced reflecting the severity of the disease (Cassella et al., 1994; Sarathchandra et al., 1999). The length of mineral crystals in OI bones is also shortest in the more severe forms of the disease, though crystal length increases with increasing age (Vetter et al., 1991). In the OI rodent model, homozygous mice (two non-functional alleles) exhibit shorter and thinner crystals than heterozygous mice (one functional and one non-functional allele) and both types less than normal controls (two functional alleles) (Camacho et al., 1999; Fratzl et al., 1996; Grabner et al., 2001; Traub et al., 1994). Crystals of apatite have also been shown to be less oriented to the long axis of bone in homozygous mice compared to their heterozygous counterparts (Fratzl et al., 1996). Studies indicate that OI bones have abnormally low collagen contents (Camacho et al., 1999) and differ in their compositions of minor collagens (types III and V) and noncollagenous proteins (Brenner et al., 1989; Vetter et al., 1991). Intermolecular collagen cross-linking has been shown to be normal in the presence of reduced mineral crystallinity (Vetter et al., 1993). Variations exist regarding fibril diameter with some studies indicating larger than normal diameter in OI osteoid (Cassella and Ali, 1992; Cassella et al., 1994). A good deal of normal lamellar bone structure composed of normally mineralized fibrils has been reported even for severe types of OI (Traub et al., 1994). However, human bone specimens have also demonstrated a poor lamellar pattern with lamellae that were thinner and more disrupted. OI osteoid exhibited areas having collagen fibrils of various diameters, without regular orientation, and with short lengths (Cassella et al., 1996). OI, which involves a single change in the coding of gylcine in collagen, has a profound affect on the gross mechanical properties of bone, as listed in Table 3. As with changes in the ultrastructure, differences in mechanical properties between OI and normal bone reflect the severity of the phenotype. For example, microhardness measurements of OI mouse bone exhibited increased hardness for

Table 3 The effect of OI is apparent when comparing the mechanical properties of OI bone to those of normal or control bone Property

Percent difference from normal

Change in microstructure

Test method

OI Type

Reference

Maximum strength

42% Y

Y In collagen fibril diameter

Tensile test of rat-tail tendon

Mild and severe

Misof et al. (1997)

Maximum strain Microhardness

54% Y 39%[

[ Mineral content; thinner crystals; less oriented

22% Y

Mild and severe Mild

Grabner et al. (2001)

Yield strength

Vicker’s microhardness of mouse tibia and femur specimens 4-point bending of machined mouse femur specimens

Bending strength Fatigue life Torsional strength

25% Y Y 60% Y

[ Damage accumulation Y Mineral content and crystallinity

Torsion of whole mouse femur

Mild and severe

Camacho et al. (1999)

Torsional stiffness Post yield deflection

47% Y 61% Y

Y Collagen and mineral content;[ ash content

4-point bending of whole mouse femur

Mild

Jepsen et al. (1996)

Jepsen et al. (1997)

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the homozygous compared to the heterozygous phenotype, with both types having harder mineral than normal bone. These measurements correlated with increased mineral content compared to normal bone, and the mineral crystals were, generally, thinner and less oriented to bone axis in the OI phenotypes (Grabner et al., 2001). The yield and bending strength have been found to be less for OI bone than for normal mouse bone. Furthermore, OI bone has significantly less fatigue life. Fatigue damage accumulates faster and at lower stress levels. The threshold stress at which damage accumulation initiated was 22–27% lower than controls (Jepsen et al., 1997). The reductions of mechanical properties have been found to depend on the severity of the phenotype, with heterozygous mice exhibiting properties intermediate to homozygous and normal mice (Camacho et al., 1999). Analysis of OI bone fracture surfaces revealed smooth cortical faces, in contradistinction to the fracture surfaces of normal bones, which demonstrated extensive longitudinal splitting and interlamellar cleavage (Jepsen et al., 1996). This suggests that collagen fibril orientation in normal bone deflects crack growth. A single mutation for the coding of one amino acid has drastic effects on bone toughness because the proper packing of collagen is vital for proper bone mineralization. 4.2. Gamma irradiation of bone allograft Gamma irradiation provides another example of an ultrastructural change that causes a change in toughness and does so without the confounding influences of cells. Clinically, gamma irradiation is necessary to prevent infection when human bone allografts are implanted to help reconstruct osseous defects. Such allografts are often placed under mechanical demands. In a clinical study, 38% of the patients who had received irradiated allografts (between 10 and 30 kGy) suffered allograft fracture compared to 18% of those who had received non-irradiated allografts (although infection rate was lower in the irradiated group) (Lietman et al., 2000). Ex vivo studies of cortical bone allografts have demonstrated that irradiation (17–35 kGy) decreases the yield strength, ultimate strain, fracture toughness, and workto-failure (i.e. toughness) of bone (Akkus and Rimnac, 2001; Currey et al., 1997; Hamer et al., 1996; Komender, 1976; Pelker et al., 1983). In general, the stiffness of bone is not affected by irradiation, and the loss of strength and toughness is more severe with an increase in each of the following doses: 17, 29.5 and 94 kGy (Currey et al., 1997). Irradiation affects the collagen network, even at common dosages used to sterilize allografts (25–35 kGy). The radiolysis of water creates free radicals, which then denature collagen molecules. Thus, freezing allografts at K78 8C could reduce the collagen damage by lifting the threshold of the energy required for radiolysis of water, thus alleviating the brittle effect (Hamer et al., 1999). Scission of the collagen molecule caused by irradiation is also associated with a decrease in hydration (Kubisz et al.,

575

2003). This may have consequences on the proper function of collagen because water stabilizes collagen through hydrogen bonding. As for a loss in fracture resistance, Mitchell et al. (2004) speculate that irradiation-induced changes in the collagen inhibit the formation of microdamage at the tip of cracks (i.e. loosing the capability to dissipate energy). This allows cracks to propagate more easily, thereby reducing the toughness of bone. However, the changes in collagen that prevent damage formation are not entirely clear. A recent Fourier Transform Infrared (FTIR) examination of cortical bone before and after X-ray irradiation found that irradiation decreased the size of the mineral crystals and decreased the association between collagen side chains and mineral (Hubner et al., 2005). 4.3. Fluoride substitution Proper fluoride treatment (i.e. low dosage of 50 mg per day with a 2 month period of no fluoride drug after 1 year of intake) appears to reduce spinal bone fracture in women who had at least one prior fracture (Rubin et al., 2001). Such treatment has also been used as a means to explore the effect of ultrastructural changes on the mechanical properties of bone. Fluoride cannot only stimulate bone formation, but also prolong the time period of mineralization. Thus, when rats were given a high dosage of fluoridated water, they developed osteomalacia (i.e. 20-fold increase in vertebral osteoid volume) (Turner et al., 1996). The evidence also indicates that fluoride produces bone mineral with intrinsically weaker material properties than normal mineral. For example, treating both rats and rabbits with fluoride (at a dosage that did not affect mineralization) was found to decrease bone strength, despite increases in bone mass and bone volume fraction (Sogaard et al., 1995). Fluoride treatment of ex vivo mouse bones, which are similar histologically to rat bone, produced similar effects on the mechanical properties. Soaking femora in buffered sodium fluoride (1.5 M) for 12 h decreased the torsional strength and stiffness of the whole bone and increased the displacement at fracture (Silva and Ulrich, 2000). Similar effects were observed for machined cortical bone specimens (bovine) that were tested uniaxially: sodium fluoride soaked bone (0.145, 0.5, or 2.0 M) had less strength and stiffness but greater strain at fraction than untreated bone in both tension (DePaula et al., 2002; Kotha et al., 1998) and compression (Walsh and Guzelsu, 1994). At higher doses of fluoride (2.0 M NaF), a significant increase was observed in toughness as well (Table 1) (DePaula et al., 2002). Fluoride concentration, pH, and time of emersion all affect the mechanical and ultra-structural response. Despite the similar effects on the mechanical properties of bone (e.g. lower strength) between in vivo and in vitro fluoride treatment, the ultrastructural changes are not necessarily the same. For example, fluoride influences cellular activity (i.e. anabolic stimulation of bone

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formation), which does not occur when soaking ex vivo bone in fluoride. In addition, level of fluorine interacting with bone mineral is much greater in vitro. FTIR spectroscopy of fluoride treated mouse bone (in vivo) and control bone found the former to have higher crystallinity, lower Type A carbonate (CO3 replaces OH in the apatite), and a different unit cell size (Grynpas and Rey, 1992). Furthermore, X-ray diffraction analysis of fluoride treated rabbit bone has found an increase in crystal width (Chachra et al., 1999). More importantly, the experimental evidence has suggested that fluoride alters the interfacial bonding between the mineral and collagen (Walsh and Guzelsu, 1994; Walsh et al., 1994). In solution, lower levels of fluoride concentration give rise to fluorahydroxyapatite type mineral, while higher concentrations cause partial dissolution of the bone mineral with reprecipitation into CaF2 (effectively decreasing normal bone apatite). Also, the increase in water content accompanying the loss of normal mineral could possibly cause the increase in ductility of fluoride treated bone. 4.4. Aging effects on bone ultrastructure The aging effect is not only reflected in changes in the collagen phase, but also in changes in the mineral phase of bone. Therefore, we describe age-related changes in both the collagen (i.e. cross-links and re-orientation) and mineral phases (i.e. crystallinity and mineral content). In addition, attention is also given to the aging effect on the water distribution in bone. 4.4.1. Age-related changes in cross-links By studying the concentration of enzymatic cross-links in bone samples from a range of aged human donors (1 month to 80 years), Eyre et al. (1988) found that immature cross-links consistently decreased until around the age of 28 years when the concentration leveled off at w0.7 mol per mol of collagen. In contrast, the mature cross-links increased up to 0.5 mol per mole of collagen until 28 years of age, and then it slightly decreased after 50 years of age. These trends most likely reflect agerelated changes in bone remodeling activity. For example, the concentration of mature cross-links was less in cancellous bone, which has higher remodeling rate, than in cortical bone (Eyre et al., 1988). In fact, the bone remodeling process confounds the study of the contribution of mature cross-links to bone toughness because old tissue with mature cross-links is continually replaced with new tissue with reducible cross-links. To investigate whether a maturing cross-link profile would stiffen and reduce the ductility of bone, small specimens of mainly interstitial bone (2 mm!0.2 mm) would need to be mechanically tested. This poses rather difficult technical challenges. There is evidence from animal studies showing the importance of cross-links to the mechanical properties of

bone, namely strength. With an avian bone model with and without osteoporosis, Knott et al. (1995) found a significant linear relationship between three-point bend strength and pyrrole concentration. They also showed that giving young birds lathyritic agents, which inhibit crosslinking, reduces bone strength. A similar observation was made by Oxlund et al. (1995) when they measured bone strength after injecting beta-amino-propionitrile into female rats. There was a decrease in strength and deflection at fracture following a 45% decrease in pyridinoline crosslinks and no change in mineral content. While the research of Knott et al. has emphasized the importance of the pyrrole cross-link over the pyridinoline cross-links in the strength of bone (Knott and Bailey, 1998), a study by Rath et al. (1999) of broiler breeder chickens of varying age did find a strong association between pyridinoline cross-links and bone strength. Both pyrrole and the pyridinoline cross-links as well as the divalent, immature ones are important to the mechanical properties of bone, but age-related changes in them are likely not great contributors to decreases in strength and toughness. For example, age-related collagen content was strongly associated with strength or work-tofracture but not with any of the enzymatic cross-links (Bailey et al., 1999). Bone remodeling appears to maintain an optimal balance between the reducible and mature crosslinks with age. Yet, diseases such as osteoporosis disturb the remodeling process and so changes in cross-link profiles have been found to affect bone quality (Oxlund et al., 1996). Moreover, thermal stability of collagen may change with age (Danielsen, 1990), although marked declines in human bone have only been observed in iliac crest biopsies from elderly males (O65 years) (Danielsen et al., 1994). Although there is no demonstrated association between maturing cross-links and strength or toughness of bone, there is evidence that glycation-induced cross-links may decrease the toughness of bone. Wang et al. (2002) performed mechanical tests and collagen analysis on cortical bone from donors of varying age (19–89 years) and found associations between an increase in pentosidine, a non-enzymatic cross-link, and a decrease in strength, toughness, and fracture toughness of noncalcified collagen. The mechanical properties of mineralized bone depended on the intracortical porosity and strength of the collagen network (Table 2). Thus, nonenzymatic glycation end-products that increase with age possibly decrease bone toughness by weakening the collagen network. In a different study on aging effects on bone toughness, Zioupos et al. (1999) also found no correlation with mature cross-links and age-related decline in toughness. They did, however, find such an association with a decreasing maximum rate of collagen contraction (Table 2). This shrinkage behavior is indicative of poor stability of collagen network due to the condition of the cross-links. Again, the ultrastructural organization of bone, such as the integrity of collagen cross-linking, is important to the overall toughness of bone.

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4.4.2. Age-related changes in collagen fibrillar orientation A preferred orientation of collagen fibrils is a significant contributor to the mechanical properties of bone (Evans and Vincentelli, 1969; Martin et al., 1996; Riggs et al., 1993b). Moreover, in the largest sample size to date, circularly polarized light microscopy of cross-sections from the midshaft of cadaveric femurs revealed age and gender differences in collagen fiber orientation (Goldman et al., 2003). This suggests that age-related changes in bone mechanical properties could be related to changes in fiber orientation. However, there was no unique motif for collagen orientation throughout the cross-section in any of the gender or age groups. Thus, if spatial distribution of collagen fiber orientation depends on mechanical demands (i.e. functional adaptation), as suggested by strain data from horse radius (Boyde and Riggs, 1990; Mason et al., 1995; Riggs et al., 1993a), then loading conditions of long bones (joint reaction forces and muscle forces) varies among the population. The idea that collagen orientation is functionally adapted to mechanical loading gained credence after experiments on osteons, which are close to the ultra-structural level of bone. Ascenzi and Bonucci (1967 and 1968) and later Ascenzi et al. (1990, 1994) mechanically tested osteons in tension, compression, bending, and torsion as well as examined collagen orientation of the specimens with polarized microscopy. The results indicated that osteons with longitudinal fibers had higher tensile strength than those with alternating fibers but lower ductility. Osteons with transverse fibers had the greatest compressive strength and ultimate strain of three osteonal types. In bending, osteons with longitudinal fibers had greater deformation and were less rigid than osteons with alternating fibers. In torsion, the longitudinal osteons had greater energy absorbed to failure than the alternating osteons. Cyclically loading osteons does not appear to affect orientation of collagen fibers, but it does disorient apatite crystals, especially longitudinal osteons (Ascenzi et al., 1998). As for fracture toughness, failure has been observed to occur more readily when the crack is oriented parallel to the fibers in dentin (Nalla et al., 2003a). An increase in the homogeneity of collagen fiber orientation, as observed in human femurs (Goldman et al., 2003), may decrease the toughness of bone assuming cracks will move greater distances before hitting a perpendicular fiber, which can bridge the crack. 4.4.3. Age-related changes in mineral phase Although the bone remodeling process is continually replenishing old bone tissue with new tissue throughout life, there are still increases in crystallinity, crystal size, Ca/P ratio, and carbonate with age (Grynpas, 1993). Observed increases in the Ca/P ratio with age may actually reflect changes in carbonate concentration and treatments in the processing method (e.g. solvents, heat, and acid). Recent measurements of Ca/P have been done with a less invasive technique than chemical treatment in which freeze dried bone, washed with acetone and alcohol, is analyzed by

577

radiation (neutron activation analysis). Gender and aging effects were not observed in human femoral neck nor rib bone, although specimens from donors older than 55 years were analyzed (Tzaphlidou and Zaichick, 2002; Zaichick and Tzaphlidou, 2002). Density fractionation analysis of human cortical bone has indicated that the proportion of highly mineralized bone increases at the expense of less mineralized bone with age (Simmons et al., 1991). Up to approximately 30 years of age (end of skeletal maturity), this increase in mineral can be explained by a reduction in remodeling activity. However, since porosity in the midshaft of the femur increases in the later stages of life, remodeling activity must rise with age, at least after 35 years. This suggests that there is a pool of bone tissue that does not get replaced with new, less mineralized matrix. Since toughness declines with age (Table 2), any of the aforementioned changes in mineral with age could cause a decline in bone quality. The relationship between ash content and toughness is complex with the maximum toughness occurring at an optimal mineral content (Currey, 1969). In general, hypermineralized bones have low toughness (Currey, 1984). Whether it is normal or not, it is suspected but not well established that age-related increases in mineralization affect toughness. Currey et al. (1996) have observed a decrease in impact energy and work-to-fracture coinciding with an increase in ash content. However, the observed increase in mineralization mainly occurred between the younger ages (5–30 years) with later years (40–80 years) showing high variability in ash content. In fact, the statistical association was done on the log of age. The studies by Zioupos (2001a,b) have found stronger associations between toughness and collagen integrity as well as microdamage than between toughness and mineral content (Table 2). Interestingly, Raman micro-spectroscopy and mechanical testing of rat bone found that increases in mineralization, crystallinity, and carbonate substitution significantly correlated with a decrease in the elastic deformation capacity of bone with age (Akkus et al., 2004). 4.4.4. Potential role of water in the age-related decrease in bone toughness The contribution of the mineral phase to toughness would appear to be related to its resistance to cracking. For example, Currey (1979) speculated that higher mineral content lowers the plastic deformation at a crack tip (i.e. crack can propagate more readily). However, mineral may influence toughness through its relationship with water. Early studies have demonstrated that bone toughness decreases following the dehydration of bone tissues (Dempster and Liddicoat, 1952; Evans, 1973; Evans and Lebow, 1951; Sedlin and Hirsch, 1966; Smith and Walmsley, 1959; Yamada and Evans, 1970). Recently, Nyman et al. (2005) found that low levels of water removal (caused by drying at room temperature) decreased bone toughness (Fig. 8). Moreover, this loss occurred through a loss of plasticity (post-yield deformation was removed)

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Fig. 8. Drying bone at room temperature (for 30 min or 4 h) caused a decrease in toughness which was likely associated with decline of collagen hydration. Drying at elevated temperatures caused further decreases in toughness suggesting mineral hydration contributes to elastic toughness (Nyman et al., 2005).

and was most likely associated with the removal of loosely bound water in the collagen phase. Given that mineral displaces water volume for volume (Robinson, 1979) and that the state of collagen is dependent on hydration (Lees, 1981; Nomura et al., 1977; Pineri et al., 1978), we speculate then that mineralization may decrease toughness by decreasing the water in the collagen phase. Such a decrease would contract fibril diameter, stiffen collagen, and thereby reduce ductility. It has been observed that equatorial spacing of collagen molecules decrease (i.e. pack more closely together) with a decrease in water content and with the presence of mineral (Bonar et al., 1985; Lees and Hukins, 1992; Lees and Mook, 1986). There would appear to be an inverse relationship between intermolecular spacing and amount of mineral (Lees, 1984). Such changes could affect collagen-mineral interaction, and thus, mineral content may affect toughness by affecting the hydration of collagen. In addition, water associated with the mineral phase may also contribute to toughness. There was a significant decrease in toughness when drying bone at high temperatures (Fig. 8) that was presumably affecting water strongly bound to the mineral. Removal of such water has been found to alter the crystallographic structure of synthetic apatites (LeGeros et al., 1978). Therefore, crystalline water may give rise to an optimal mineral structure. Observed decreases in water content in bone with age (Jonsson et al., 1985; Mueller et al., 1966) may explain age-related decline in bone toughness.

5. Concluding remarks The toughness of bone is directly related to the ultrastructure and composition of bone constituents. Aging and bone diseases can result in abnormal cellular and molecular functions in bone, which in turn causes adverse ultra-structural and micro-structural changes in the tissue

and leads to deteriorated bone quality. Thus, understanding the relationship of bone ultrastructure with its toughness is of significant importance to elucidate the underlying mechanisms of such clinically significant issues. Microdamage contributes to the toughness of bone by its interaction with the collagen and mineral phases of bone: the breaking of collagen and the cracking of mineral both dissipate energy. The mechanisms by which such changes in ultrastructure lead to the weakening of bone are not entirely clear. With aging, the extracellular matrix of bone becomes more susceptible to microdamage formation and so microdamage occurs more readily and dissipates less energy. However, more detailed investigation seems necessary to determine whether this is due to a loss in collagen integrity (e.g. denatured collagen molecules, overly cross-linked, homogenized fibril orientation, etc.), changes in the mineral phase, or both. In addition, it is important to understand the conditions of the ultrastructure that maximize energy dissipation by exploring the underlying mechanism of microdamage formation and its relationship with the ultrastructure of bone.

Acknowledgements We thank Professor Ozan Akkus for suggesting examples of how the ultrastructure influences toughness. We also thank Dr Daniel Nicollela and Don Moravits for providing quality images of collagen fibrils and microdamage. Lastly, the San Antonio Area Foundation provided the funds to secure the use of previously published figures.

References Akkus, O., Rimnac, C.M., 2001. Fracture resistance of gamma radiation sterilized cortical bone allografts. Journal of Orthopaedic Research 19, 927–934. Akkus, O., Knott, D.F., Jepsen, K.J., Davy, D.T., Rimnac, C.M., 2003a. Relationship between damage accumulation and mechanical property degradation in cortical bone: microcrack orientation is important. Journal of Biomedical Materials Research Part A 65, 482–488. Akkus, O., Polyakova-Akkus, A., Adar, F., Schaffler, M.B., 2003b. Aging of microstructural compartments in human compact bone. Journal of Bone and Mineral Research 18, 1012–1019. Akkus, O., Adar, F., Schaffler, M.B., 2004. Age-related changes in physicochemical properties of mineral crystals are related to impaired mechanical function of cortical bone. Bone 34, 443–453. Amprino, R., 1958. Investigations on some physical properties of bone tissue. Acta Anatomica 34, 161–186. Arsenault, A.L., 1991a. Image analysis of collagen-associated mineral distribution in cryogenically prepared turkey leg tendons. Calcified Tissue International 48, 56–62. Arsenault, A.L., 1991b. Image analysis of mineralized and non-mineralized type I collagen fibrils. Journal of Electron Microscopy Technique 18, 262–268. Ascenzi, A., Bonucci, E., 1967. The tensile properties of single osteons. The Anatomical Record 158, 375–386. Ascenzi, A., Bonucci, E., 1968. The compressive properties of single osteons. The Anatomical Record 161, 377–391.

J.S. Nyman et al. / Micron 36 (2005) 566–582 Ascenzi, A., Baschieri, P., Benvenuti, A., 1990. The bending properties of single osteons. Journal of Biomechanics 23, 763–771. Ascenzi, A., Baschieri, P., Benvenuti, A., 1994. The torsional properties of single selected osteons. Journal of Biomechanics 27, 875–884. Ascenzi, A., Benvenuti, A., Bigi, A., Foresti, E., Koch, M.H., Mango, F., Ripamonti, A., Roveri, N., 1998. X-ray diffraction on cyclically loaded osteons. Calcified Tissue International 62, 266–273. Bailey, A.J., Knott, L., 1999. Molecular changes in bone collagen in osteoporosis and osteoarthritis in the elderly. Experimental Gerontology 34, 337–351. Bailey, A.J., Light, N.D., Atkins, E.D., 1980. Chemical cross-linking restrictions on models for the molecular organization of the collagen fibre. Nature 288, 408–410. Bailey, A.J., Paul, R.G., Knott, L., 1998. Mechanisms of maturation and ageing of collagen. Mechanisms of Ageing and Development 106, 1–56. Bailey, A.J., Sims, T.J., Ebbesen, E.N., Mansell, J.P., Thomsen, J.S., Mosekilde, L., 1999. Age-related changes in the biochemical properties of human cancellous bone collagen: Relationship to bone strength. Calcified Tissue International 65, 203–210. Bear, R.S., 1956. The structure of collagen molecules and fibrils. Journal of Biophysical and Biochemical Cytology 2, 363–368. Bentolila, V., Boyce, T.M., Fyhrie, D.P., Drumb, R., Skerry, T.M., Schaffler, M.B., 1998. Intracortical remodeling in adult rat long bones after fatigue loading. Bone 23, 275–281. Berg, R.A., Prockop, D.J., 1973. The thermal transition of a nonhydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen. Biochemical and Biophysical Research Communications 52, 115–120. Bonar, L.C., Lees, S., Mook, H.A., 1985. Neutron diffraction studies of collagen in fully mineralized bone. Journal of Molecular Biology 181, 265–270. Boskey, A., 2003. Bone mineral crystal size. Osteoporosis International 14, S16–S21. Boyce, T.M., Fyhrie, D.P., Glotkowski, M.C., Radin, E.L., Schaffler, M.B., 1998. Damage type and strain mode associations in human compact bone bending fatigue. Journal of Orthopaedic Research 16, 322–329. Boyde, A., Riggs, C.M., 1990. The quantitative study of the orientation of collagen in compact bone slices. Bone 11, 35–39. Brenner, R.E., Vetter, U., Nerlich, A., Worsdorfer, O., Teller, W.M., Muller, P.K., 1989. Osteogenesis imperfecta: Insufficient collagen synthesis in early childhood as evidenced by analysis of compact bone and fibroblast cultures. European Journal of Clinical Investigation 19, 159–166. Broz, J.J., Simske, S.J., Greenberg, A.R., 1995. Material and compositional properties of selectively demineralized cortical bone. Journal of Biomechanics 28, 1357–1368. Bunn, H.F., Gabbay, K.H., Gallop, P.M., 1978. The glycosylation of hemoglobin: relevance to diabetes mellitus. Science 200, 21–27. Burr, D.B., Stafford, T., 1990. Validity of the bulk-staining technique to separate artifactual from in vivo bone microdamage. Clinical Orthopaedics and Related Research 260, 305–308. Burr, D.B., Martin, R.B., Schaffler, M.B., Radin, E.L., 1985. Bone remodeling in response to in vivo fatigue microdamage. Journal of Biomechanics 18, 189–200. Byers, P.H., Wallis, G.A., Willing, M.C., 1991. Osteogenesis imperfecta: translation of mutation to phenotype. Journal of Medical Genetics 28, 433–442. Camacho, N.P., Hou, L., Toledano, T.R., Ilg, W.A., Brayton, C.F., Raggio, C.L., Root, L., Boskey, A.L., 1999. The material basis for reduced mechanical properties in oim mice bones. Journal of Bone and Mineral Research 14, 264–272. Casciani, F.S., 1971. Identification of hydrate water in enamel, dentine, cementum, and bone. In: Fearnhead, R.W., Stack, M.V. (Eds.), Tooth Enamel II: Its Composition, Properties, and Fundamental Structure. John Wright & Sons, Ltd., Bristol, pp. 14–23.

579

Cassella, J.P., Ali, S.Y., 1992. Abnormal collagen and mineral formation in osteogenesis imperfecta. Bone and Mineral 17, 123–128. Cassella, J.P., Pereira, R., Khillan, J.S., Prockop, D.J., Garrington, N., Ali, S.Y., 1994. An ultrastructural, microanalytical, and spectroscopic study of bone from a transgenic mouse with a COL1.A1 pro-alpha-1 mutation. Bone 15, 611–619. Cassella, J.P., Stamp, T.C., Ali, S.Y., 1996. A morphological and ultrastructural study of bone in osteogenesis imperfecta. Calcified Tissue International 58, 155–165. Catanese 3rd., J., Featherstone, J.D., Keaveny, T.M., 1999. Characterization of the mechanical and ultrastructural properties of heat-treated cortical bone for use as a bone substitute. Journal of Biomedical Materials Research 45, 327–336. Chachra, D., Turner, C.H., Dunipace, A.J., Grynpas, M.D., 1999. The effect of fluoride treatment on bone mineral in rabbits. Calcified Tissue International 64, 345–351. Courtney, A.C., Hayes, W.C., Gibson, L.J., 1996. Age-related differences in post-yield damage in human cortical bone. Experiment and model. Journal of Biomechanics 29, 1463–1471. Cowles, E.A., DeRome, M.E., Pastizzo, G., Brailey, L.L., Gronowicz, G.A., 1998. Mineralization and the expression of matrix proteins during in vivo bone development. Calcified Tissue International 62, 74–82. Currey, J.D., 1969. The mechanical consequences of variation in the mineral content of bone. Journal of Biomechanics 2, 1–11. Currey, J.D., 1979. Changes in the impact energy absorption of bone with age. Journal of Biomechanics 12, 459–469. Currey, J.D., 1984. Effects of differences in mineralization on the mechanical properties of bone. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences 304, 509–518. Currey, J.D., 2001. Bone strength: What are we trying to measure? Calcified Tissue International 68, 205–210. Currey, J.D., Brear, K., Zioupos, P., 1996. The effects of ageing and changes in mineral content in degrading the toughness of human femora. Journal of Biomechanics 29, 257–260. Currey, J.D., Foreman, J., Laketic, I., Mitchell, J., Pegg, D.E., Reilly, G.C., 1997. Effects of ionizing radiation on the mechanical properties of human bone. Journal of Orthopaedic Research 15, 111–117. Danielsen, C.C., 1990. Age-related thermal stability and susceptibility to proteolysis of rat bone collagen. Biochemical Journal 272, 697–701. Danielsen, C.C., Mosekilde, L., Bollersleve, J., Mosekilde, L., 1994. Thermal stability of cortical bone collagen in relation to age in normal individuals and in individuals with osteoporosis. Bone 19, 91–96. Dempster, W.T., Liddicoat, R.T., 1952. Compact bone as a non-isotropic material. American Journal of Anatomy 91, 331–362. DePaula, C.A., Abjornson, C., Pan, Y., Kotha, S.P., Koike, K., Guzelsu, N., 2002. Changing the structurally effective mineral content of bone with in vitro fluoride treatment. Journal of Biomechanics 35, 355–361. Endo, A., Glimcher, M.J., 1989. The effect of complexing phosphoproteins to decalcified collagen on in vitro calcification. Connective Tissue Research 21, 179–190 (discussion 191-176). Evans, F.G., 1973. Mechanical Properties of Bone. Thomas, Springfield. Evans, F.G., Lebow, M., 1951. Regional differences in some of the physical properties of the human femur. Journal of Applied Physiology 3, 563–572. Evans, F.G., Vincentelli, R., 1969. Relation of collagen fiber orientation to some mechanical properties of cortical bone. Journal of Biomechanics 2, 63–71. Eyre, D.R., Dickson, I.R., Van Ness, K., 1988. Collagen cross-linking in human bone and articular cartilage. Age-related changes in the content of mature hydroxypyridinium residues. Biochemical Journal 252, 495–500. Fazzalari, N.L., Forwood, M.R., Manthey, B.A., Smith, K., Kolesik, P., 1998. Three-Dimensional confocal images of microdamage in cancellous bone. Bone 23, 373–378. Fondrk, M.T., Bahniuk, E.H., Davy, D.T., 1999. A damage model for nonlinear tensile behavior of cortical bone. Journal of Biomechanical Engineering 121, 533–541.

580

J.S. Nyman et al. / Micron 36 (2005) 566–582

Frasca, P., Harper, R.A., Katz, J.L., 1981. Scanning electron microscopy studies of collagen, mineral and ground substance in human cortical bone. Scanning Electron Microscopy, 339–346. Fratzl, P., Groschner, M., Vogl, G., Plenk Jr.., H., Eschberger, J., FratzlZelman, N., Koller, K., Klaushofer, K., 1992. Mineral crystals in calcified tissues: a comparative study by SAXS. Journal of Bone and Mineral Research 7, 329–334. Fratzl, P., Paris, O., Klaushofer, K., Landis, W.J., 1996. Bone mineralization in an osteogenesis imperfecta mouse model studied by small-angle x-ray scattering. The Journal of Clinical Investigation 97, 396–402. Frost, H.M., 1960. Presence of microscopic cracks in vivo in bone. Henry Ford Hospital Medical Bulletin 8, 25–35. Giraud-Guille, M.M., 1988. Twisted plywood architecture of collagen fibrils in human compact bone osteons. Calcified Tissue International 42, 167–180. Glimcher, M.J., 1960. Specificity of the molecular structure of organic matrices in mineralization. In: Sognnaes, R.F. (Ed.), Calcification in Biologic Systems. American Association for the Advancement of Science, Washington, pp. 421–487. Glimcher, M.J., 1987. The nature of the mineral component of bone and the mechanism of calcification. Instructional Course Lectures 36, 49–69. Glimcher, M.J., 1989. Mechanism of calcification: Role of collagen fibrils and collagen-phosphoprotein complexes in vitro and in vivo. The Anatomical Record 224, 139–153. Goldman, H.M., Bromage, T.G., Thomas, C.D., Clement, J.G., 2003. Preferred collagen fiber orientation in the human mid-shaft femur. The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology 272, 434–445. Grabner, B., Landis, W.J., Roschger, P., Rinnerthaler, S., Peterlik, H., Klaushofer, K., Fratzl, P., 2001. Age- and genotype-dependence of bone material properties in the osteogenesis imperfecta murine model (oim). Bone 29, 453–457. Grynpas, M., 1993. Age and disease-related changes in the mineral of bone. Calcified Tissue International 53 Suppl 1, S57–S64. Grynpas, M.D., Rey, C., 1992. The effect of fluoride treatment on bone mineral crystals in the rat. Bone 13, 423–429. Gullberg, B., Johnell, O., Kanis, J.A., 1997. World-wide projections for hip fracture. Osteoporosis International 7, 407–413. Hamer, A.J., Strachan, J.R., Black, M.M., Ibbotson, C.J., Stockley, I., Elson, R.A., 1996. Biochemical properties of cortical allograft bone using a new method of bone strength measurement. A comparison of fresh, fresh-frozen and irradiated bone. The Journal of Bone and Joint Surgery British 78, 363–368. Hamer, A.J., Stockley, I., Elson, R.A., 1999. Changes in allograft bone irradiated at different temperatures. The Journal of Bone and Joint Surgery British Volume 81, 342–344. Hannig, K., Nordwig, A., 1967. Amino acid sequences in collagen. In: Ramachandran, G.N. (Ed.), Treatise on collagen. New York Academic Press, London, pp. 73–101. Hellmich, C., Ulm, F.J., 2003. Average hydroxyapatite concentration is uniform in the extracollagenous ultrastructure of mineralized tissues: Evidence at the 1-10-microm scale. Biomechanics and Modeling in Mechanobiology 2, 21–36. Hodge, A.J., 1989. Molecular models illustrating the possible distributions of ’holes’ in simple systematically staggered arrays of type I collagen molecules in native-type fibrils. Connective Tissue Research 21, 137–147. Hodge, A.J., Petruska, J.A., 1963. Recent studies with the electron microscope on ordered aggregates of the tropocollagen macromolecule. In: Ramachandran, G.N. (Ed.), Aspects of protein structure. Academic Press, New York, pp. 299–300. Hubner, W., Blume, A., Pushnjakova, R., Dekhtyar, Y., Hein, H.J., 2005. The influence of X-ray radiation on the mineral/organic matrix interaction of bone tissue: An FT-IR microscopic investigation. The International Journal of Artificial Organs 28, 66–73.

Hulmes, D.J., 2002. Building collagen molecules, fibrils, and suprafibrillar structures. Journal of Structural Biology 137, 2–10. Hulmes, D.J., Wess, T.J., Prockop, D.J., Fratzl, P., 1995. Radial packing, order, and disorder in collagen fibrils. Biophysical Journal 68, 1661–1670. Hunter, G.K., Goldberg, H.A., 1993. Nucleation of hydroxyapatite by bone sialoprotein. Proceedings of the National Academy of Sciences of the United States of America 90, 8562–8565. Jepsen, K.J., Goldstein, S.A., Kuhn, J.L., Schaffler, M.B., Bonadio, J., 1996. Type-I collagen mutation compromises the post-yield behavior of Mov13 long bone. Journal of Orthopaedic Research 14, 493–499. Jepsen, K.J., Schaffler, M.B., Kuhn, J.L., Goulet, R.W., Bonadio, J., Goldstein, S.A., 1997. Type I collagen mutation alters the strength and fatigue behavior of Mov13 cortical tissue. Journal of Biomechanics 30, 1141–1147. Johnell, O., Kanis, J.A., Oden, A., Sernbo, I., Redlund-Johnell, I., Petterson, C., De Laet, C., Jonsson, B., 2004. Mortality after osteoporotic fractures. Osteoporosis International 15, 38–42. Jonas, J., Burns, J., Abel, E.W., Cresswell, M.J., Strain, J.J., Paterson, C.R., 1993. Impaired mechanical strength of bone in experimental copper deficiency. Annals of Nutrition & Metabolism 37, 245–252. Jonsson, U., Ranta, H., Stromberg, L., 1985. Growth changes of collagen cross-linking, calcium, and water content in bone. Archives of Orthopaedic and Trauma Surgery 104, 89–93. Joseph Catanes, I., Keaveny, T.M., 1996. Role of collagen and hydroxyapatite in the mechanical behavior of bone tissue. Journal of Bone and Mineral Research 11, S295. Kanis, J.A., Johnell, O., Oden, A., Sembo, I., Redlund-Johnell, I., Dawson, A., De Laet, C., Jonsson, B., 2000. Long-term risk of osteoporotic fracture in Malmo. Osteoporosis International 11, 669–674. Katz, E.P., Li, S.T., 1973. Structure and function of bone collagen fibrils. Journal of Molecular Biology 80, 1–15. Knott, L., Bailey, A.J., 1998. Collagen cross-links in mineralizing tissues: A review of their chemistry, function, and clinical relevance. Bone 22, 181–187. Knott, L., Whitehead, C.C., Fleming, R.H., Bailey, A.J., 1995. Biochemical changes in the collagenous matrix of osteoporotic avian bone. Biochemical Journal 310, 1045–1051. Komender, A., 1976. Influence of preservation on some mechanical properties of human haversian bone. Materia Medica Polona 8, 13–17. Kotha, S.P., Guzelsu, N., 2003. Tensile damage and its effects on cortical bone. Journal of Biomechanics 36, 1683–1689. Kotha, S.P., Walsh, W.R., Pan, Y., Guzelsu, N., 1998. Varying the mechanical properties of bone tissue by changing the amount of its structurally effective bone mineral content. Bio-medical Materials and Engineering 8, 321–334. Kubisz, L., Mielcarek, S., Jaroszyk, F., 2003. Changes in thermal and electrical properties of bone as a result of 1 MGy-dose gammairradiation. International Journal of Biological Macromolecules 33, 89–93. Landis, W.J., Glimcher, M.J., 1978. Electron diffraction and electron probe microanalysis of the mineral phase of bone tissue prepared by anhydrous techniques. Journal of Ultrastructure Research 63, 188–223. Landis, W.J., Song, M.J., Leith, A., McEwen, L., McEwen, B.F., 1993. Mineral and organic matrix interaction in normally calcifying tendon visualized in three dimensions by high-voltage electron microscopic tomography and graphic image reconstruction. Journal of Structural Biology 110, 39–54. Landis, W.J., Hodgens, K.J., Arena, J., Song, M.J., McEwen, B.F., 1996a. Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography. Microscopy Research and Technique 33, 192–202. Landis, W.J., Hodgens, K.J., Song, M.J., Arena, J., Kiyonaga, S., Marko, M., Owen, C., McEwen, B.F., 1996b. Mineralization of collagen may occur on fibril surfaces: Evidence from conventional and high-voltage electron microscopy and three-dimensional imaging. Journal of Structural Biology 117, 24–35.

J.S. Nyman et al. / Micron 36 (2005) 566–582 Lees, S., 1981. A mixed packing model for bone collagen. Calcified Tissue International 33, 591–602. Lees, S., Bonar, L.C., Mook, H.A., 1984. A study of dense mineralized tissue by neutron diffraction. International Journal of Biological Macromolecules 6, 321–326. Lees, S., Mook, H.A., 1986. Equatorial diffraction spacing as a function of water content in fully mineralized cow bone determined by neutron diffraction. Calcified Tissue International 39, 291–292. Lees, S., Prostak, K., 1988. The locus of mineral crystallites in bone. Connective Tissue Research 18, 41–54. Lees, S., Hukins, D.W., 1992. X-ray diffraction by collagen in the fully mineralized cortical bone of cow tibia. Bone and Mineral 17, 59–63. LeGeros, R.Z., Bonel, G., Legros, R., 1978. Types of “H2O” in human enamel and in precipitated apatites. Calcified Tissue Research 26, 111–118. Lietman, S.A., Tomford, W.W., Gebhardt, M.C., Springfield, D.S., Mankin, H.J., 2000. Complications of irradiated allografts in orthopaedic tumor surgery. Clinical Orthopaedics and Related Research 375, 214–217. Loong, C.K., Rey, C., Kuhn, L.T., Combes, C., Wu, Y., Chen, S., Glimcher, M.J., 2000. Evidence of hydroxyl-ion deficiency in bone apatites: An inelastic neutron-scattering study. Bone 26, 599–602. Lowenstam, H.A., Weiner, S., 1989. On Biomineralization. Oxford University Press, Inc., New York. Maitland, M.E., Arsenault, A.L., 1991. A correlation between the distribution of biological apatite and amino acid sequence of type I collagen. Calcified Tissue International 48, 341–352. Marino, A.A., Becker, R.O., Bachman, C.H., 1967. Dielectric determination of bound water of bone. Physics in Medicine and Biology 12, 367–378. Marotti, G., Favia, A., Zallone, A.Z., 1972. Quantitative analysis on the rate of secondary bone mineralization. Calcified Tissue Research 10, 67–81. Martin, R.B., Burr, D.B., 1989. Structure, Function, and Adaptation of Compact Bone. Raven Press, New York. Martin, R.B., Lau, S.T., Mathews, P.V., Gibson, V.A., Stover, S.M., 1996. Collagen fiber organization is related to mechanical properties and remodeling in equine bone. A comparison of two methods. Journal of Biomechanics 29, 1515–1521. Mason, M.W., Skedros, J.G., Bloebaum, R.D., 1995. Evidence of strainmode-related cortical adaptation in the diaphysis of the horse radius. Bone 17, 229–237. Melton 3rd.., L.J., Chrischilles, E.A., Cooper, C., Lane, A.W., Riggs, B.L., 1992. Perspective. How many women have osteoporosis? Journal of Bone and Mineral Research 7, 1005–1010. Melton 3rd.., L.J., 2003. Adverse outcomes of osteoporotic fractures in the general population. Journal of Bone and Mineral Research 18, 1139–1141. Miller, A., 1984. Collagen: the organic matrix of bone. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences 304, 455–477. Misof, K., Landis, W.J., Klaushofer, K., Fratzl, P., 1997. Collagen from the osteogenesis imperfecta mouse model (oim) shows reduced resistance against tensile stress. The Journal of Clinical Investigation 100, 40–45. Mitchell, E.J., Stawarz, A.M., Kayacan, R., Rimnac, C.M., 2004. The effect of gamma radiation sterilization on the fatigue crack propagation resistance of human cortical bone. The Journal of Bone and Joint Surgery American Volume 86, 2648–2657. Mori, S., Burr, D.B., 1993. Increased intracortical remodeling following fatigue damage. Bone 14, 103–109. Mueller, K.H., Trias, A., Ray, R.D., 1966. Bone density and compostiton. Age-related and pathological changes in water and mineral content. The Journal of Bone and Joint Surgery American Volume 48, 140–148. Nalla, R.K., Kinney, J.H., Ritchie, R.O., 2003a. Effect of orientation on the in vitro fracture toughness of dentin: the role of toughening mechanisms. Biomaterials 24, 3955–3968. Nalla, R.K., Kinney, J.H., Ritchie, R.O., 2003b. Mechanistic fracture criteria for the failure of human cortical bone. Nature Materials 2, 164–168.

581

Nicolella, D.P., Lankford, J., 2002. Microstructural strain near osteocyte lacuna in cortical bone in vitro. Journal of Musculoskeletal & Neuronal Interactions 2, 261–263. Nimni, M.E., 1993. Fibrillar Collagens: Their Biosynthesis, Molecular Structure, and Mode of Assembly. In: Zern, M.A., Reid, L.M. (Eds.), Extracellular Matrix: Chemistry, Biology, and Pathobiology with Emphasis on the Liver. Marcel Dekker, Inc., New York, pp. 121–148. Nomura, S., Hiltner, A., Lando, J.B., Baer, E., 1977. Interaction of water with native collagen. Biopolymers 16, 231–246. Norman, T.L., Yeni, Y.N., Brown, C.U., Wang, Z., 1998. Influence of microdamage on fracture toughness of the human femur and tibia. Bone 23, 303–306. Nyman, J. S., Roy, A., Shen, X.-m., Acuna, R. L., Tyler, J. H., Wang, X., 2005. The influence of water removal on the strength and toughness of cortical bone. Journal of Biomechanics, In press. Oxlund, H., Barckman, M., Ortoft, G., Andreassen, T.T., 1995. Reduced concentrations of collagen cross-links are associated with reduced strength of bone. Bone 17, 365S–371S. Oxlund, H., Mosekilde, L., Ortoft, G., 1996. Reduced concentration of collagen reducible cross links in human trabecular bone with respect to age and osteoporosis. Bone 19, 479–484. Parsamian, G.P., Norman, T.L., 2001. Diffuse damage accumulation in the fracture process zone of human cortical bone specimens and its influence on fracture toughness. Journal of Materials Science Materials in Medicine 12, 779–783. Pelker, R.R., Friedlaender, G.E., Markham, T.C., 1983. Biomechanical properties of bone allografts. Clinical Orthopaedics and Related Research 174, 54–57. Pineri, M.H., Escoubes, M., Roche, G., 1978. Water–collagen interactions: calorimetric and mechanical experiments. Biopolymers 17, 2799–2815. Posner, A.S., 1969. Crystal chemistry of bone mineral. Physiological Reviews 49, 760–792. Rath, N.C., Balog, J.M., Huff, W.E., Huff, G.R., Kulkarni, G.B., Tierce, J.F., 1999. Comparative differences in the composition and biomechanical properties of tibiae of seven- and seventy-two-week-old male and female broiler breeder chickens. Poultry Science 78, 1232–1239. Rauch, F., Glorieux, F.H., 2004. Osteogenesis imperfecta. Lancet 363, 1377–1385. Rey, C., Miquel, J.L., Facchini, L., Legrand, A.P., Glimcher, M.J., 1995. Hydroxyl groups in bone mineral. Bone 16, 583–586. Rho, J.Y., Kuhn-Spearing, L., Zioupos, P., 1998. Mechanical properties and the hierarchical structure of bone. Medical Engineering & Physics 20, 92–102. Riggs, C.M., Lanyon, L.E., Boyde, A., 1993a. Functional associations between collagen fibre orientation and locomotor strain direction in cortical bone of the equine radius. Anatomy and Embryology 187, 231–238. Riggs, C.M., Vaughan, L.C., Evans, G.P., Lanyon, L.E., Boyde, A., 1993b. Mechanical implications of collagen fibre orientation in cortical bone of the equine radius. Anatomy and Embryology 187, 239–248. Robinson, R.A., 1975. Physiocochemical structure of bone. Clinical Orthopaedics and Related Research 125, 263–315. Robinson, R.A., 1979. Bone tissue: composition and function. Johns Hopkins Medical Journal 145, 10–24. Robinson, R.A., Watson, M.L., 1952. Collagen-crystal relationships in bone as seen in the electron microscope. The Anatomical Record 114, 383–409. Roseberry, H., Hastings, A., Morse, J., 1931. X-ray analysis of bone and teeth. The Journal of Biological Chemistry 90, 395–407. Rubin, C.D., Pak, C.Y., Adams-Huet, B., Genant, H.K., Li, J., Rao, D.S., 2001. Sustained-release sodium fluoride in the treatment of the elderly with established osteoporosis. Archives of Internal Medicine 161, 2325–2333. Sarathchandra, P., Kayser, M.V., Ali, S.Y., 1999. Abnormal mineral composition of osteogenesis imperfecta bone as determined by electron probe X-ray microanalysis on conventional and cryosections. Calcified Tissue International 65, 11–15.

582

J.S. Nyman et al. / Micron 36 (2005) 566–582

Schreiner, L.J., Cameron, I.G., Funduk, N., Miljkovic, L., Pintar, M.M., Kydon, D.N., 1991. Proton NMR spin grouping and exchange in dentin. Biophysical Journal 59, 629–639. Sedlin, E.D., Hirsch, C., 1966. Factors affecting the determination of the physical properties of femoral cortical bone. Acta Orthopaedica Scandinavica 37, 29–48. Sheldon, H., Robinson, R.A., 1957. Electron microscope studies of crystalcollagen relationships in bone. IV. The occurrence of crystals within collagen fibrils. Journal of Biophysical and Biochemical Cytology 3, 1011–1016. Silva, M.J., Ulrich, S.R., 2000. In vitro sodium fluoride exposure decreases torsional and bending strength and increases ductility of mouse femora. Journal of Biomechanics 33, 231–234. Simmons Jr.., E.D., Pritzker, K.P., Grynpas, M.D., 1991. Age-related changes in the human femoral cortex. Journal of Orthopaedic Research 9, 155–167. Smith, J.W., Walmsley, R., 1959. Factors affecting the elasticity of bone. Journal of Anatomy 93, 503–523. Sogaard, C.H., Mosekilde, L., Schwartz, W., Leidig, G., Minne, H.W., Ziegler, R., 1995. Effects of fluoride on rat vertebral body biomechanical competence and bone mass. Bone 16, 163–169. Termine, J.D., Kleinman, H.K., Whitson, S.W., Conn, K.M., McGarvey, M.L., Martin, G.R., 1981. Osteonectin, a bone-specific protein linking mineral to collagen. Cell 26, 99–105. Timmins, P.A., Wall, J.C., 1977. Bone water. Calcified Tissue Research 23, 1–5. Traub, W., Piez, K.A., 1971. The chemistry and structure of collagen. Advances in Protein Chemistry 25, 243–352. Traub, W., Arad, T., Weiner, S., 1989. Three-dimensional ordered distribution of crystals in turkey tendon collagen fibers. Proceedings of the National Academy of Sciences of the United States of America 86, 9822–9826. Traub, W., Arad, T., Vetter, U., Weiner, S., 1994. Ultrastructural studies of bones from patients with osteogenesis imperfecta. Matrix Biology 14, 337–345. Turner, C.H., Owan, I., Brizendine, E.J., Zhang, W., Wilson, M.E., Dunipace, A.J., 1996. High fluoride intakes cause osteomalacia and diminished bone strength in rats with renal deficiency. Bone 19, 595–601. Tzaphlidou, M., Zaichick, V., 2002. Neutron activation analysis of calcium/phosphorus ratio in rib bone of healthy humans. Applied Radiation and Isotopes 57, 779–783. Vashishth, D., Behiri, J.C., Bonfield, W., 1997. Crack growth resistance in cortical bone: Concept of microcrack toughening. Journal of Biomechanics 30, 763–769. Vashishth, D., Tanner, K.E., Bonfield, W., 2000. Contribution, development and morphology of microcracking in cortical bone during crack propagation. Journal of Biomechanics 33, 1169–1174. Vashishth, D., Gibson, G.J., Khoury, J.I., Schaffler, M.B., Kimura, J., Fyhrie, D.P., 2001. Influence of nonenzymatic glycation on biomechanical properties of cortical bone. Bone 28, 195–201. Vashishth, D., Tanner, K.E., Bonfield, W., 2003. Experimental validation of a microcracking-based toughening mechanism for cortical bone. Journal of Biomechanics 36, 121–124. Vetter, U., Fisher, L.W., Mintz, K.P., Kopp, J.B., Tuross, N., Termine, J.D., Robey, P.G., 1991. Osteogenesis imperfecta: changes in noncollagenous proteins in bone. Journal of Bone and Mineral Research 6, 501–505. Vetter, U., Weis, M.A., Morike, M., Eanes, E.D., Eyre, D.R., 1993. Collagen crosslinks and mineral crystallinity in bone of patients with osteogenesis imperfecta. Journal of Bone and Mineral Research 8, 133–137. Wachtel, E., Weiner, S., 1994. Small-angle x-ray scattering study of dispersed crystals from bone and tendon. Journal of Bone and Mineral Research 9, 1651–1655. Walsh, W.R., Guzelsu, N., 1994. Compressive properties of cortical bone: Mineral-organic interfacial bonding. Biomaterials 15, 137–145.

Walsh, W.R., Labrador, D.P., Kim, H.D., Guzelsu, N., 1994. The effect of in vitro fluoride ion treatment on the ultrasonic properties of cortical bone. Annals of Biomedical Engineering 22, 404–415. Wang, X., Bank, R.A., TeKoppele, J.M., Agrawal, C.M., 2001. The role of collagen in determining bone mechanical properties. Journal of Orthopaedic Research 19, 1021–1026. Wang, X., Li, X., Bank, R.A., Agrawal, C.M., 2002a. Effects of collagen unwinding and cleavage on the mechanical integrity of the collagen network in bone. Calcified Tissue International 71, 186–192. Wang, X., Shen, X., Li, X., Agrawal, C.M., 2002b. Age-related changes in the collagen network and toughness of bone. Bone 31, 1–7. Wang, X., Li, X., Shen, X., Agrawal, C.M., 2003. Age-related changes of noncalcified collagen in human cortical bone. Annals of Biomedical Engineering 31, 1365–1371. Weiner, S., Arad, T., Sabanay, I., Traub, W., 1997. Rotated plywood structure of primary lamellar bone in the rat: orientations of the collagen fibril arrays. Bone 20, 509–514. Weiner, S., Traub, W., 1986. Organization of hydroxyapatite crystals within collagen fibrils. FEBS Letters 206, 262–266. Weiner, S., Wagner, H.D., 1998. The material bone: Structure-mechanical function relations. Annual Review of Material Science 28, 271–298. Weiner, S., Traub, W., Wagner, H.D., 1999. Lamellar bone: Structurefunction relations. Journal of Structural Biology 126, 241–255. Wergedal, J.E., Baylink, D.J., 1974. Electron microprobe measurements of bone mineralization rate in vivo. American Journal of Physiology 226, 345–352. Wright, T.M., Vosburgh, F., Burstein, A.H., 1981. Permanent deformation of compact bone monitored by acoustic emission. Journal of Biomechanics 14, 405–409. Yamada, H., Evans, F.G.,, 1970. Strength of Biological Materials. Williams & Wilkins, Baltimore. Yamauchi, M., 1996. Collagen: The major matrix molecule in mineralized tissues. In: Anderson, J.J., Garner, S.C. (Eds.), Calcium and Phosphorus in Health and Disease. CRC Press, Inc., Boca Raton, pp. 127–145. Yamauchi, M., Katz, E.P., Otsubo, K., Teraoka, K., Mechanic, G.L., 1989. Cross-linking and stereospecific structure of collagen in mineralized and nonmineralized skeletal tissues. Connective Tissue Research 21, 159–167. Yeni, Y.N., Norman, T.L., 2000. Calculation of porosity and osteonal cement line effects on the effective fracture toughness of cortical bone in longitudinal crack growth. Journal of Biomedical Materials Research 51, 504–509. Yeni, Y.N., Brown, C.U., Wang, Z., Norman, T.L., 1997. The influence of bone morphology on fracture toughness of the human femur and tibia. Bone 21, 453–459. Yeni, Y.N., Brown, C.U., Norman, T.L., 1998. Influence of bone composition and apparent density on fracture toughness of the human femur and tibia. Bone 22, 79–84. Zaichick, V., Tzaphlidou, M., 2002. Determination of calcium, phosphorus, and the calcium/phosphorus ratio in cortical bone from the human femoral neck by neutron activation analysis. Applied Radiation and Isotopes 56, 781–786. Zioupos, P., 2001a. Accumulation of in-vivo fatigue microdamage and its relation to biomechanical properties in ageing human cortical bone. Journal of Microscopy 201, 270–278. Zioupos, P., 2001b. Ageing human bone: Factors affecting its biomechanical properties and the role of collagen. Journal of Biomaterials Applications 15, 187–229. Zioupos, P., Currey, J.D., Sedman, A.J., 1994. An examination of the micromechanics of failure of bone and antler by acoustic emission tests and laser scanning confocal microscopy. Medical Engineering & Physics 16, 203–212. Zioupos, P., Currey, J.D., Hamer, A.J., 1999. The role of collagen in the declining mechanical properties of aging human cortical bone. Journal of Biomedical Materials Research 45, 108–116.