2.10 Bone as a Material☆

2.10 Bone as a Material☆ LM McNamara, National University of Ireland Galway, Galway, Ireland r 2017 Elsevier Ltd. All rights reserved.

2.10.1 2.10.2 2.10.2.1 2.10.2.2 2.10.2.3 2.10.2.4 2.10.2.4.1 2.10.2.4.2 2.10.2.4.3 2.10.2.4.4 2.10.3 2.10.3.1 2.10.3.2 2.10.4 2.10.4.1 2.10.4.2 2.10.5 2.10.5.1 2.10.5.2 2.10.5.3 2.10.6 2.10.6.1 2.10.6.2 2.10.6.3 2.10.6.4 2.10.7 2.10.7.1 2.10.7.1.1 2.10.7.1.2 2.10.7.1.3 2.10.7.1.4 2.10.8 References

Introduction Bone Composition Collagen Mineral Noncollagenous Proteins Bone Cells Mesenchymal stem cells and osteoprogenitors Osteoblasts and bone lining cells Osteocytes Osteoclasts Bone Formation Endochondral Ossification Intramembranous Ossification Bone Structure and Hierarchical Organization Bone Tissue Structure and Organization Organ Level Bone Mechanical Behavior Structural Mechanical Behavior of Trabecular Bone Tissue Mechanical Behavior of Bone Fracture Behavior Bone as a Dynamic Adaptive Material Bone Modeling Bone Remodeling Fracture Healing Mechanobiology Bone as a Material During Disease and Drug Treatment Osteoporosis Bone cell biology during osteoporosis Mechanical behavior and structure during osteoporosis Mechanobiology and osteoporosis Approaches for treatment of osteoporosis Conclusion

Abbreviations BGP Bone Gla protein BMP Bone morphogenetic protein BMU Basic multi-cellular unit μCT Micro-CT scanning ER Estrogen receptor FGF Fibroblast growth factor M-CSF Macrophage colony-stimulating factor MSC Mesenchymal stem cell NCP Noncollagenous protein OCIF Osteoclastogenesis inhibitory factor

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OPG Osteoprotegerin PDGF Platelet-derived growth factor PTH Parathyroid hormone RANK-L Receptor for Activation of Nuclear Factor Kappa B Ligand SERM Selective estrogen receptor modulator STEAR Selective tissue estrogenic activity regulator TGF-β Transforming growth factor-beta TNF Tumor necrosis factor VEGF Vascular endothelial growth factor

Change History: August 2016. L.M. McNamara updated sections 2.10.2.4.3, 2.10.5.2, 2.10.5.3, 2.10.6.4, 2.10.7.1, and 2.10.7.1.1–2.10.7.1.4; added new Fig. 8.

This is an update of L. McNamara, 2.210 – Bone as a Material. In Comprehensive Biomaterials, edited by Paul Ducheyne, Elsevier, Oxford, 2011, pp. 169–186.

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doi:10.1016/B978-0-12-803581-8.10127-4

Bone as a Material

Glossary Angiogenesis Angiogenesis is the physiological process by which blood vessels are formed. Apoptosis Apoptosis is the process by which cells die in response to a variety of stimuli in a controlled, regulated fashion, and is often referred to as programmed cell death. Cytokine Cytokine is a signaling molecule, such as a protein or peptide, that is secreted by immune cells to regulate the activity of other biological cells. Differentiation Differentiation is the process by which a biological cell alters its size, shape, membrane potential, metabolic activity, and gene expression in response to signals to become a more specialized cell. Hematoma Hematoma is a collection of blood within a body tissue in response to injury of a blood vessel.

2.10.1

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Hematopoietic stem cell Hematopoietic stem cell an unspecialized cell from the blood or bone marrow that can renew itself and differentiate to a variety of specialized cells. Mesenchyme Mesenchyme is loose reticular connective tissue which is derived from all three germ layers and located within the embryo. Morphogenesis Morphogenesis is a biological process that causes a tissue or organ to develop its shape by controlling the spatial distribution of cells during embryonic development. Morphology Morphology the form or shape of an organism (ie, cell, tissue, organ). Osteogenesis Osteogenesis is the term used for the biological process by osteoblasts produce bone tissue.

Introduction

The skeletal system gives the body its structure, provides support for the heart and lungs, protects internal organs, such as the brain, kidneys and uterus, and facilitates movement by acting as a system of kinematic links to which muscles can attach. Bone tissue is the primary structural element that forms the skeletal system. It is an exceptional material that is lightweight to allow efficient movement but also exhibits excellent strength and stiffness. This unique mechanical behavior is imparted by a composite material of organic proteins and mineral crystals, which are intricately organized on many scales to create the material properties that allow bone to serve these functions under the variety of loading conditions experienced during everyday activities (Currey, 1984). Furthermore, healthy bone is a living, growing, dynamic material that has the capacity to renew itself, and adapt its architecture, so that it can maintain strength and continue to serve its functions throughout life. This adaptive behavior is facilitated by specialized biological cells from bone surfaces and marrow that continuously digest aged or damaged bone and reform new bone tissue in its place. During normal physiology bone cells can also repair fractures that occur. Each of these characteristics is fundamental to the normal physiological function of bone tissue and is discussed in detail below.

2.10.2

Bone Composition

Bone tissue is a porous composite material, consisting of a mineral phase and an organic phase. The organic phase comprises approximately 35% of the total mass of bone, of which 90% is a collagen matrix and the remainder is composed of other noncollagenous proteins, water lipids and cells. The mineral phase of bone is composed mainly of calcium and phosphorus in the form of hydroxyapatite crystals and accounts for approximately 25% of the total bone volume and 50% of the bone mass. The structure and mechanical behavior of bone is determined by the quantity and mechanical integrity of each of these phases, the structural organization of the different phases, and the physical interaction between them.

2.10.2.1

Collagen

Collagen is a ubiquitous constitutive protein that forms the fundamental matrix upon which all connective tissues are assembled. Its primary function is to provide structural integrity and shape to biological tissues. The tropocollagen molecule is the basic structural unit of collagen and is comprised of three polypeptide strands (a-chains) which are twisted together into a right-handed coiled coil known as a triple helix (Fig. 1(d)). In bone tissue tropocollagen molecules are synthesized by osteoblast cells during the initial phase of bone formation (Section 2.10.3). The formation of tropocollagen initiates when the polypeptide chains are produced by the nucleus of osteoblast cells (Fig. 1(a)). Next, these chains are modified in the endoplasmic reticulum of the osteoblast when enzymes (proteases) break the peptide bonds between amino acid side chains (proline and lysine) of the polypeptide. This process is known as proteolytic cleavage (Fig. 1(b)). These chains are assembled into a triple helix tropocollagen structure that is stabilized by hydrogen bonds. Within the space at the center of the triple helix there is a repeating amino acid sequence consisting of glycine molecules and amino acids (eg, proline and hydroxyproline). This process produces triple helical collagen molecules that are approximately 1.5 nm in diameter and 300 nm long and have short non-helical regions called telopeptides at each end, known as amino (N) and carboxyl (C) telopeptides, which facilitate later assembly into fibrils. The tropocollagen molecules are processed and packaged in the Golgi apparatus (Fig. 1(c)) and then secreted to the extracellular matrix in the form of soluble precursors called procollagens (Fig. 1(d)). During secretion the propeptides are removed by procollagen N and C proteinases and this triggers these molecules to aggregate together extracellularly and assemble to form collagen fibrils (Fig. 1(e)). Intermolecular cross-links are formed between the non-helical domains and the helical domain of adjacent collagen

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Fig. 1 Intracellular synthesis and assembly of Type I collagen in the osteoblast (a–c) and extracellular formation of collagen fibrils (d–e).

Fig. 2 Transmission electron imaging of collagen fibrils in bone tissue; (a) collagen fibrils are oriented parallel to each other; (b) collagen fibrils in adjacent lamellae are oriented at an angle to each other ( indicates interface between lamellae).

molecules. These molecules are staggered from each other at a space of approximately 67 nm in fibrillar collagen (Minary-Jolandan and Yu, 2009). The newly formed collagen fibers are stabilized by the formation of covalent cross-links between neighboring collagen molecules. Immature ketomine and aldimine cross-links are first formed, which contribute to the later formation of mature pyridinium or pyrrole cross-links. Together these crosslinks result in fibril formation and these fibrils act as a scaffold for bone minerals and provide strength (Eyre et al., 1988; Viguet-Carrin et al., 2006) and are responsible for the post-yield behavior of bone tissue (Reilly and Burstein, 1975). There are more than 27 forms of collagen in biological tissues and each type is distinguished by roman numerals (eg, Types I, II, III, V, and XI). The differences between each form of collagen lies in the manner in which the tropocollagen molecules and fibrils are arranged. Depending on the collagen type, the triple helix can be homotrimeric, ie, comprised of three identical a-chains (eg, Type III collagen), or heterotrimeric, whereby at least one of the polypeptides is not identical to the others (eg, Type I collagen). The collagen in bone is primarily Type I collagen ([a1(I)]2a2(I)), which comprises 95% of the collagen content and 80% of the total proteins present in bone (Niyibizi and Eyre, 1994), but types III, IV, and VI are also present (Miller, 1984, 1969). In contrast to ligament and tendon, where fibrils are arranged in parallel bundles, the collagen fibrils in bone are arranged concentrically into packages known as lamellae (Section 2.10.4.1). Collagen fibers within the same lamella are parallel to one another (Fig. 2(a)), whereas collagen fibers in adjacent lamellae may oriented at an angle of up to 901 relative to each other (Fig. 2(b)). It is through the intricate arrangement of collagen molecules at the nanoscale and fibrils at the microscale that collagen provides tensile strength, elasticity and toughness (capacity to absorb energy) to bone (Ascenzi and Bonucci, 1976). Collagen forms the basis of immature bone tissue (osteoid) and acts as a template upon which proteins and mineral crystal are deposited (Glimcher, 1959) and are interspersed in the spaces between molecules and between adjacent fibrils (Fig. 3(a)).

2.10.2.2

Mineral

The mineral component of mature bone tissue consists of small crystals of impure hydroxyapatite (Ca10(PO4)6(OH)2), that are bound within and between the collagen fibrils in an ordered manner. This mineral is produced by osteoblast cells on the calcification front after the organic matrix is deposited, when calcium and phosphate ions bind in the presence of non-collagenous

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Fig. 3 (a) Distribution of mineral and non-collagenous proteins within and between collagen fibrils. (b) Color enhanced image depicting variation in tissue mineral content with age; mineral content is lower in the superficial region.

proteins (Section 2.10.2.3). These cells also regulate the binding of circulating calcium and phosphorus to the extracellular matrix. Over time the size of the mineral particles increases slowly (secondary mineralization) so that within 5–10 days the matrix is 70% mineralized. Complete mineralization at any site takes between 3–5 months (Cowin, 2001) and mineralization of the entire skeleton does not occur for years. The mineral crystals are located within and between the spaces between collagen fibrils (Fig. 3(a)) and are approximately 100 nm in length and have a thickness of 2–4 nm (Landis, 1995). The distribution and growth of these minerals, as well as their orientation, is governed by the structure and spatial limitations of the underlying collagen matrix (Paschalis et al., 1996), and the distribution of non-collagenous proteins (Section 2.10.2.3) which together interact with and regulate the growth of the mineral crystals (Landis et al., 1996). Some proteins can inhibit crystal formation by binding to specific surfaces (Section 2.10.2.3), blocking potential nucleation sites and thereby decrease crystal length (Boskey et al., 1993). Changes in local ionic concentration can also influence the rate of mineral deposition (Boskey and Adele, 2001). Together these events regulate the formation of inorganic mineral crystals at distinct sites within bone tissue, which are aligned in the same orientation as the collagen fibrils (Cowin, 2001). The osteoid seam is a thin layer of unmineralized organic matrix that remains at the interface between newly formed osteoid and mineralized bone prior to mineralization of newly formed matrix. The degree of bone tissue mineralization is vital for mechanical integrity of the skeleton, its load bearing strength and its stiffness (Currey, 1984). The quantity of bone mineral varies considerably from different skeletal regions (Brennan et al., 2014a), within an individual and from person to person. In particular sex, race and age-dependent differences have been reported (Parfitt et al., 1997; Handschin and Stern, 1994). The normal aging process increases mean mineralization and mineral heterogeneity at a trabecular level (Brennan et al., 2014a). The overall mineral content of bone tissue increases with age by an average of 5–10% (Reid and Boyde, 1987), but after 60 years of age it has been shown to decrease again in males and females (Currey et al., 1996). Such changes alter the stiffness and strength of bone tissue (Currey, 1988). At high mineral contents bone tissue becomes brittle and allows cracks to propagate easily leading to a reduction in toughness and impact strength (Currey et al., 1996; Currey, 1984). The mechanical properties of bone are highly sensitive to small changes in mineral; based on published relationships between calcium content and ash fraction and between ash fraction and mechanical strength an increase as small as 0.5% (wt% Ca) equates to an increase in strength of 4.5%. Genetic disorders that lead to defects in the underlying collagen structure can result in an altered mineral composition (Section 2.10.7).

2.10.2.3

Noncollagenous Proteins

There are many noncollagenous proteins (NCP’s) in the extracellular matrix of bone and these comprise approximately 10% of the entire bone by mass. The most prominent proteins are osteocalcin, osteopontin, alkaline phosphatase, fibronectin, bone sialoprotein, osteoprotegerin, osteonectin and thrombospondin. These proteins fulfill numerous functions, including organization of the collagenous matrix, mediating cell attachment, regulating the rate of growth and regulating the stability of the mineral phase crystals (Luchinetti, 2001), as is outlined in detail below. Osteocalcin, also known as bone Gla protein (BGP), constitutes 20 to 25% of non-collagen bone protein and is ubiquitous in bone tissue. It is produced by osteoblast cells and binds to the calcium of hydroxyapatite to regulate the growth of mineral crystals in bone (Heinegård and Oldberg, 1989). In particular it is believed to be able to slow down the growth of HA crystals and thereby act as a mineralization inhibitor (Roach, 1994). Other studies propose that osteocalcin may act as a signal to osteoclast precursors to begin differentiating into mature osteoclasts and initiate the process of bone resorption (Roach, 1994; Glowacki et al., 1991; Robey, 1996). Osteopontin is a phosphorylated protein that is found in abundance in cement lines, in the spaces between mineralized collagen fibrils and along the canalilcular wall of osteocyte cells (Nanci, 1999; Sodek and McKee, 2000; McKee and Nanci, 1996; Devoll et al., 1997). It mediates cell attachment by binding cell surface integrin receptors. Therefore it is believed that osteopontin acts as a regulator of osteoclast activity during bone resorption (Robey, 1996). It is able to influence cell dynamics, and it is proposed that it may also promote adhesion between opposing surfaces at cement lines in bone (McKee and Nanci, 1996). Furthermore it has been proposed that osteopontin might facilitate osteocyte mechanical sensing (Section 2.10.6.4;

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Yoshitake et al., 1999a; McNamara et al., 2009) by binding the extracellular matrix to an integrin receptor (avb3) that is present along osteocyte cell processes (McNamara et al., 2009). In the absence of osteopontin the response of bone to underloading is altered (Yoshitake et al., 1999a). Osteopontin also plays an important role in the formation of new blood vessels (angiogenesis) and prevents apatite formation and growth due to its high affinity for apatite crystals (Boskey et al., 1993). Thrombospondin is a protein that is synthesized by osteoblasts (Robey et al., 1989) during the early stages of bone formation and as such is ubiquitous in unmineralized osteoid (Robey, 1996). Bone sialoprotein is a member of the small integrin-binding ligand, N-linked glycoproteins family (SIBLING proteins) that is produced by osteoblasts at a late stage of maturation. The protein co-distributes with osteopontin and accumulates in cement lines and inter-fibrillar collagen spaces (Nanci, 1999). Fibronectin is an extracellular matrix glycoprotein that is relatively abundant in bone tissue. Thrombospondin, bone sialoprotein and fibronectin all mediate binding of osteoblasts to the collagen, mineral and other proteoglycans in bone tissue (Heinegård and Oldberg, 1989). Bone sialoprotein is also believed to act to promote the mineralization process (Robey, 1996) whereas fibronectin regulates the differentiation of osteoblasts (Moursi et al., 1997). Alkaline phosphatase is an enzyme glycoprotein that is produced by osteoblasts during the early stages of bone formation. It functions to remove phosphate groups (hydrolyze) from mineral deposition inhibitor proteins (Clarke, 2008) and thereby provides an initial attachment site for mineral nucleation along the collagen fibrils and is vital for increasing local phosphate concentration (Boskey and Adele, 2001). Osteoprotegerin (OPG), also known as osteoclastogenesis inhibitory factor (OCIF), is a cytokine that is a member of the tumor necrosis factor (TNF) receptor superfamily (Kostenuik and Shalhoub, 2001; Feige, 2001). It is believed to play a role in inhibiting the formation of mature osteoclasts by blocking the binding of RANKL to RANK (Boyle et al., 2003). Therefore the primary role of OPG is to act as a regulator of osteoclastic bone resorption (Kostenuik and Shalhoub, 2001). Osteonectin is the most abundant noncollagenous protein in mineralized bone matrix. It contains calcium binding domains and plays a role in regulating the bone mineralization process (Heinegård and Oldberg, 1989). It also binds varyingly to collagen and it has been shown to play roles in regulating the cell cycle, cell–matrix interactions and cell morphology (Robey, 1996). It has long been established that many proteins regulate matrix organization by acting as mineral crystal nucleation sites that bind mineral crystals to the collagen matrix (Tye et al., 2003, 2005; Hunter and Goldberg, 1993; Qiu et al., 2004). Proteoglycans have a high negative fixed charge density and readily bind to Ca2 þ ions. They are also know to play an important role in facilitating cell attachment (Fantner et al., 2005) and thereby regulate cellular activity and maintenance of the collagen–mineral interface. They are believed to be responsible for regulating the mechanical properties of the collagen–mineral interface between modulating the formation of bonds between collagen and mineral (Luchinetti, 2001). Recent studies now suggest that NCPs form tough bonds between the mineralized fibers (Fantner et al., 2005; Zappone et al., 2008), and that these bonds have molecular selfhealing abilities (Thurner et al., 2008; Fantner et al., 2007). Interestingly in vitro studies have shown that these bonds may have both energy dissipation (Fantner et al., 2005) and energy storage capabilities (Zappone et al., 2008; Fantner et al., 2005). There are substantial local variations in the content and distribution of bone proteins (Nanci, 1999) and it has been proposed that such compositional differences may influence cell dynamics and remodeling (Ingram et al., 1993). Therefore collectively the NCP’s in bone are important for regulating the mechanical strength of bone by several different means.

2.10.2.4

Bone Cells

Bone tissue is made and maintained by cells of the osteogenic and phagocytic lineage. There are five main types of bone cells in bone tissue osteoprogenitors, osteoblasts, osteocytes, osteoclasts and bone lining cells, each of which are crucial for bone growth, healing, and remodeling. Osteoprogenitors, osteoblasts and osteocytes are responsible for the production of bone matrix and mineral, whereas osteoclasts and bone lining cells are fundamental for the maintenance of this tissue throughout life. Each cell type has a different morphology and internal structure, they interact varyingly with their external matrix and these differences play a large role in dictating the different physiological functions of these bone cells and more importantly the unique behavior of bone as a material.

2.10.2.4.1

Mesenchymal stem cells and osteoprogenitors

Mesenchymal stem cells (MSC’s) are unspecialized multipotent cells that have the potential to mature into various cell types. MCS’s are present in the primary cells of the embryo (germ layers) and are responsible for early skeletal development, but they also persist in mature adult bones where they are found in the periosteum, endosteum and bone marrow (Fig. 4(a)). The MSC has a basic cell morphology characterized by a small cell body, comprised of a large, round nucleus with a prominent nucleolus that is surrounded by finely dispersed chromatin particles, a Golgi apparatus, rough endoplasmic reticulum, mitochondria, and polyribosomes. Within the cytoplasm there is a three-dimensional network of proteins known as the cytoskeleton, which consists of microtubules, intermediate filaments and actin filaments that extend across the entire cytoplasm (Rodríguez et al., 2004). The cytoskeleton dictates the morphology and shape of cells and MSC’s extend their cytoplasm into the surrounding matrix in which they reside by means of cell processes. At the beginning of the bone formation process (osteogensis) MSC’s proliferate to form a dense nodule of cells and differentiate to become osteochondral progenitors (osteoprogenitors) under the influence of growth factors, cytokines and physical stimuli. Osteoprogenitors are cells that have committed to differentiating along the osteochondral lineage and from this stage on they only have the ability to differentiate into chondrogenic (cartilage) or osteogenic (bone) cells. Morphologically osteoprogenitors have large cell bodies, with more Golgi apparatus and rough endoplasmic reticulum than MSC’s

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Fig. 4 Transmission electron microscopy images of bone cells; (a) bone marrow cells containing blood cells, platelets, and progenitor cells and osteoblasts lining the bone surface, (b) osteoblasts on the surface become embedded in newly formed osteoid and develop cytoplasmic cell processes (↑↑) as they differentiate into osteocytes, (c) osteocyte embedded in bone cells with cytoplasmic cell processes encased in bone canaliculi (↑↑), and (d) osteoclast cells with ruffled border (↑↑↑) and multiple nuclei.

and do not have cell processes. Whether they ultimately become chondroblast or osteoblast cells is dependant on the presence of growth factors and the surrounding physical environment. Bone morphogenetic proteins (BMPs), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-b) are believed to promote the division of osteoprogenitors and increase differentiation along the osteogenic lineage to become osteoblasts.

2.10.2.4.2

Osteoblasts and bone lining cells

Osteoblasts are the bone cells that are primarily responsible for synthesizing bone matrix proteins and minerals during early bone formation in the embryo, but also control bone formation and mineralization throughout life. They are found in areas of high metabolism where new bone formation is occurring. They have differentiated from either osteoprogenitors in the mesenchyme of the embryo or osteoprogenitors near bone surfaces in mature bone to become cuboidal, columnar cells that have a large Golgi apparatus, rough endoplasmic reticulum and a central nucleus, see Fig. 4(a) and (b). During embryonic bone formation osteoblasts secrete collagen and non-collagenous proteins that act as a template for the bone, but also produce minerals and regulate the precipitation and binding of minerals from the blood to the newly formed osteoid to facilitate the formation of a mature mineralized bone tissue and regulate the flux of ions into the extracellular environment during mineralization. Osteoblasts also function throughout life to produce new bone that is required to replace aged or damaged bone (Section 2.10.6.2) or repair bone fractures (Section 2.10.6.3). The formation period typically persists for about 100–150 days (Eriksen and Kassem, 1992). Osteoblasts express a range of genetic markers and synthesis various proteins including Type I collagen, bone sialoprotein, M-CSF, RUNX2, alkaline phosphate, osteocalcin, osteopontin, and osteonectin. The mode of differentiation, recruitment and inhibition of osteoblasts is controlled by numerous hormonal and growth factors (Jee, 2001) including vitamin D, estrogen, and parathyroid hormone. Estrogens and PTH can increase the number of osteoblasts, which subsequently increases collagen production. Vitamin D also plays a part in the mineralization of bone matrix and a lack of Vitamin D results in osteomalacia (impaired mineralization). Osteoblasts have the ability to communicate with neighboring cells and osteocytes via gap junctions and they secrete factors that activate osteoclasts (RANK-ligand). After osteoblasts have produced newly formed osteoid, a certain amount of the cells become encased in this matrix and differentiate to become osteocytes distributed throughout the bone matrix. Those that are not embedded remain on the new bone surface as quiescent osteoblasts, known as bone lining cells, or undergo apoptosis (programmed cell death). Bone-lining cells have similar morphology to osteoblasts and serve to regulate the movement of calcium and phosphate into and out of the bone and provide access for osteoclasts during bone resorption.

2.10.2.4.3

Osteocytes

Osteocytes are the most mature and abundant cell in bone tissue and are formed when some osteoblasts become embedded in their secreted osteoid and begin to extend cytoplasmic cell processes to interconnect with each other (Fig. 4(b)). During this time the cells begin to differentiate into osteocytes and alter their morphology, their anabolic activity and lose much of the organelle of osteoblasts. Mature osteocytes reside in a fluid filled space in the extracellular matrix known as a lacuna and have a large nucleus, very little cytoplasm, and the extend many elongated processes into their extracellular matrix (Fig. 4(c)) to facilitate communication with other embedded osteocytes, bone lining cells and osteoblasts. These cell processes reside in fluid filled channels known as canaliculi, which radiate from the osteocyte lacunae to the osteonic (haversian) canal to provide passageways for nutrient supply through the impermeable matrix (Cowin, 2001). The cell body and processes are surrounded by a glycocalyx (You et al., 2001) and the cell processes are attached to the ECM by means of punctate integrin-based attachments (McNamara et al., 2009). The cells are distributed throughout the extracellular matrix at a spacing of approximately 1600 mm2. Osteocyte canaliculi traverse the cement line in human cortical bone, allowing communication with other osteons or interstitial bone (Milovanovic et al., 2013). Osteocytes play an important role in controlling the extracellular concentration of calcium and phosphate in bone tissue over time, by means of a process known as secondary mineralization whereby they control the growth in the crystal size of mineral

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(Frost, 1960a; Inoue et al., 2006; Gluhak-Heinrich et al., 2007). Some studies also suggest that osteocytes might be capable of controlling calcium release from the bone tissue to the blood in a process known as osteocyte osteolysis, although this theory remains contentious (Teti and Zallone, 2009). It is also believed that osteocytes play an integral role in sensing mechanical signals and transducing these into biochemical signals to osteoclasts and osteoblasts to alter bone mass (Cowin et al., 1991; Lanyon, 1993; Bonewald, 2002). Their ubiquitous spatial distribution throughout bone and their extensive communication network is ideal to facilitate intercellular communication. They have a variety of biological mechanisms, by they monitor the mechanical environment and communicate the requirements for adaptation of the skeleton under altered mechanical demands or repair due to damage (Section 2.10.6.4). Osteocytes also have the ability to sense changes in interstitial fluid and the levels of hormones that circulate in it (Manolagas, 2000) and stabilize bone mineral by maintaining an appropriate local ionic environment (Cowin, 2001). Biochemical signaling from both osteocytes and osteoblasts, has been shown to direct osteogenic differentiation of MSCs, however osteocytes are more influential than osteoblasts in stimulating osteogenesis in MSCs (Birmingham et al., 2012). Osteocytes also secrete large amounts of factors known to stimulate osteoclast resorption (M-CSF, RANKL) along their cell processes (Zhao et al., 2002). The activity of the cells is governed by estrogen, parathyroid hormone, glucocorticoids, vitamin D, calcitonin, and prostaglandin (Rodan, 1992).

2.10.2.4.4

Osteoclasts

Osteoclasts are derived from precursor stem cells of the macrophage lineage, known as bone marrow stromal cells or monocytes, which reside in the hematopoietic bone marrow. The primary function of the osteoclast is to digest aged, damaged or disused bone during the physiological processes of modeling and remodeling. This process is known as bone resorption and begins when bonelining cells degrade unmineralized osteoid and increase expression of growth factors to recruit preosteoclasts from the bone marrow. Under the influence of cytokine and growth factors the monocytes fuse and differentiate to form mature active osteoclasts that have multiple nuclei and a large cytoplasm (Fig. 4(d)) that has many vesicles and vacuoles (Holtrop and King, 1977). The mature osteoclast attaches to bone surface via integrin-based attachment proteins, which involves binding of the integrin avb3 to vitronectin, to form a specialized cell membrane known as the ruffled border (Fig. 4(d)). This membrane seals off the resorption site of the underlying matrix and it is through this membrane that hydrogen ions and enzymes are secreted to acidify and dissolve the calcium and phosphate minerals in the matrix and thereby facilitate bone resorption. The function of the ruffled border is to maximize the surface through which hydrogen ions are released. The digested material is absorbed into small vesicles and these are released into the extracellular fluid as waste products. After resorption osteoclasts undergo apoptosis. Osteoclast differentiation and function is controlled primarily by three factors: Macrophage Colony-Stimulating Factor (MCSF), Receptor for Activation of Nuclear Factor Kappa B Ligand (RANK-L) and Osteoprotegerin (OPG). M-CSF is produced by osteoblasts and is required for survival and differentiation of osteoclast precursors. RANK is a receptor that is present on the surface of osteoclast precursor cells. RANKL is expressed on the surface of osteoblasts and lining cells and binds to RANK, which leads to the differentiation and maturation of the osteoclast precursor into mature multinucleated osteoclasts. OPG is made by osteoblasts and blocks both osteoblast formation and bone resorption. Osteoclast recruitment and differentiation is also governed by hormonal and growth factors (Jee, 2001) including estrogen, Vitamin D and parathyroid hormone (PTH). Estrogen's effect on osteoclasts is inhibitory; estrogen activates osteoclast receptors to decrease formation of mature osteoclasts (Oursler et al., 1991) and increases osteoclast apoptosis (Hughes et al., 1996). Vitamin D and PTH can increase the recruitment of osteoclasts and the activity of osteoclasts stimulating bone resorption and resulting in an increase in blood calcium levels. Other growth factors and cytokines also regulate osteoclast activity; tumor necrosis factor (TNF-a), the interleukin cytokines (IL-1, IL-6, IL-7), transforming Growth factor (TGF-b), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) (Lerner, 2006; Teitelbaum, 2006; Lam et al., 2000). As a result the osteoclast resorption process is sensitive to hormonal status and drug treatments.

2.10.3

Bone Formation

During embryonic development bone formation occurs by two distinct mechanisms; either endochondral ossification or intramembranous ossification. Bone formation also persists throughout life to alter the geometry of bones and provide larger bones that are sufficient to withstand changes in mechanical loading (modeling), or to replace aged or damaged bone (remodeling) or fill fracture gaps (Section 2.10.6). Bone formation is typically a two-step process whereby an organic matrix (osteoid/cartilage template) is initially laid down by osteoblasts, and then mineral crystals are precipitated and grow slowly over time to produce the composite material.

2.10.3.1

Endochondral Ossification

Endochondral ossification is the process by which bone tissue is formed in early fetal development. It begins when mesenchymal stem cells (MSC’s) start to produce a cartilage template of long bones, such as the femur and the tibia, upon which bone morphogenesis occurs (Ortega et al., 2004). The process initiates when MSC cells differentiate to become chondroblast cells (Fig. 5(a)) and form a membrane around the template known as the perichondrium. This template grows in length (interstitial growth) and thickness (appositional growth) when the chondroblasts proliferate or more chondroblasts are recruited from the

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Fig. 5 Schematic diagram of endochondral ossification.

perichondrium (Fig. 5(b)). Together these cells secrete an extracellular matrix comprised mainly of collagen and proteoglycans. Over time the chondroblasts differentiate to become chondrocytes and begin to secrete alkaline phosphatase (Mackie et al., 2008; Kronenberg, 2003), which is an enzyme that acts as a nucleator for deposition of minerals on the template, but also secrete growth factors to promote invasion of blood vessels into the perichondrium (vascularization) and thereby form the outer membrane (periosteum) of the bone (Fig. 5(c)). The periosteum is an important a source for undifferentiated osteoprogenitor cells (Gerber and Ferrara, 2000). It is divided into an outer fibrous layer, which is a source for fibroblasts, and an inner osteogenic layer, which is a source of osteoprogenitor cells that develop into osteoblasts. The periosteum also provides sites for attachment for ligaments, tendons and muscles. This process begins in the middle of the template, which is known as the primary center of ossification (Fig. 5(c)). During mineralization chondrocytes undergo hypertrophy and apoptosis and the cavities that remain are invaded by blood vessels from the perichondrium. The hypertrophic chondrocytes direct mineralization of their surrounding matrix and attract blood vessels by producing vascular endothelial growth factor (VEGF). These blood vessels are a source for hemopoietic cells that form the bone marrow and osteoprogenitor cells, which differentiate to become osteoblast cells and secrete bone proteins and minerals. Endothelial cells (ECs) on the lining of blood vessels produce essential growth factors that control the recruitment, proliferation and differentiation of osteoblasts (Sumpio et al., 2002; Kronenberg, 2003). Therefore vascularization is an essential requirement for bone formation (Gerber and Ferrara, 2000; Collin-Osdoby, 1994). A number of other factors regulate the formation of blood vessels including oxygen tension, mechanical loading, nutrients and growth factors (Brandi and Collin-Osdoby, 2006). Osteoclasts are also recruited during this time to remodel the template and form a cavity for bone marrow (medullary cavity) and together these events provide the first bone tissue during fetal development. At birth a secondary ossification center appears in the epiphyses of long bones, which is vascularized and forms a cartilage layer known as the growth plate (Fig. 5(d)). The formation and growth of bones is ongoing throughout childhood and is regulated by the epiphyseal or growth plate (Fig. 5(d)), which continues to produce new cartilage, which is replaced by bone, and thereby facilitates lengthening of bones. In adults lengthening of bones stops and the growth plate fuses and is replaced by bone, known as the epiphyseal line. Bones can continue to grow in diameter around the diaphysis by deposition of bone by osteoblasts beneath the periosteum and simultaneously osteoclasts on the interior surface (endosteum) resorb bone to maintain a lightweight structure. The coordinated process of endochondral ossification is essential to the development and growth of long bones of the body, but also regulates fracture repair, as is discussed in Section 2.10.6.3.

2.10.3.2

Intramembranous Ossification

During embryonic development bone formation also occurs by means of a process known as intramembranous ossification, which regulates the formation of non-long bones, such as the bones of the skull and clavicle. The primary difference between intramembranous and endochondral ossification, is that the intramembranous process does not rely on the formation of a cartilage template. Instead embryonic stem cells (MSCs) within mesenchymal tissue of the embryo, derived from primary tissue (germ layers), begin to proliferate and condense to form an aggregate of MSC cells. This nodule is surrounded by a membrane and MSCs within the membrane begin to differentiate to first become osteoprogenitor cells and then osteoblasts. These osteoblasts line the nodule and secrete an extracellular matrix consisting of Type-I collagen fibrils within the center of the nodule. Some osteoblasts become embedded within the newly formed matrix and in this environment they differentiate and form interconnecting cytoplasmic processes to become osteocytes. The cells on the outer surface form a periosteum and bone growth continues at the surface of trabeculae. At this time the nodule is mineralized to form rudimentary bone tissue that is populated by osteocytes and lined by active osteoblasts (Jee, 2001). This tissue is known as a bone spicule and many spicules fuse to form trabeculae, known as primary spongiosa, which then fuse to form woven bone. Over time this woven bone is remodeled to become lamellar bone, with

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concentric lamellae surrounding haversian systems in what is known as an osteon (Jee, 2001; Chung et al., 2004; Kanczler and Oreffo, 2008).

2.10.4

Bone Structure and Hierarchical Organization

Bone architecture is hierarchical and complex, consisting of component phases at different levels of structural organization (Rho et al., 1998) and these can be classified from the organ level, to the macrostructural, microstructural and nanostructural level. This intricate and complex hierarchical organization of bone is fundamental to its function.

2.10.4.1

Bone Tissue Structure and Organization

The bone that comprises that adult skeleton is organized into two main types; approximately 80% of the mass is a bone tissue known as cortical bone and the remaining 20% is known as trabecular bone, which is also known as cancellous or spongy bone. The microstructure of bone is organized differently depending on the anatomical location and the type of bone involved; however, the basis of the differences between bone types arises predominantly from the microstrucural organization of layers, or lamellae, of bone tissue. Cortical bone is a dense bone tissue that is characterized by a low porosity of approximately 5–10%. It is most commonly found in the shafts of long bones, and also forms a shell around the ends of bones such as the vertebrae, scapula and pelvis (Fig. 6(d)). It serves to bear load, transmit mechanical forces from musculature and provide levers to facilitate movement. Cortical bone can be further classified into either woven or lamellar bone and the tissue is organized into structural units known as lamellae and osteons. Woven bone is the term used for immature bone that has a highly irregular organization wherein there is no distinct organization of the collagen fiber bundles or osteocyte cells, but rather they are oriented at random in a meshwork. Due to the lack of structural organization woven bone exhibits low strength. Woven bone is present in newly formed bones and is gradually replaced by lamellar bone as growth continues by remodeling (Section 2.10.6.2), but is maintained at tendon insertions and tooth sockets. In lamellar bone collagen fibrils and osteocyte lacunae are organized into parallel layers known as lamellae, and within each lamella the collagen fibrils are oriented in the same direction, but collagen fibrils in adjacent lamellae are oriented by 901 relative to each other (Fig. 2). This organization of lamellae differs in certain regions in the bone; lamellae that are organized parallel to periosteal and endosteal surfaces are known as circumferential lamellae whereas in regions of bone tissue that contain blood vessels lamellae are organized in concentric rings around a Haversian canal that contains a central vessel and each structural unit is known as an osteon (Haversian system). These blood vessels are essential for providing a nutrient supply to the bone cells that reside in the impermeable bone matrix. The Haversian canal is approximately 70 mm in diameter and the blood vessel is 15 mm (Burr and Martin, 1989). Primary osteons have few lamellae and small vascular channels and are formed in the embryo during mineralization of the cartilage template. Secondary osteons are formed by replacement of existing bone tissue during remodeling (Section 2.10.4) and are 200 mm in diameter. A significant feature of secondary osteons is the presence of cement lines that mark the interface between adjacent osteons. It is a matter of debate whether this material is less or more mineralized than the osteon (Schaffler et al., 1987; Burr et al., 1988; Skedros et al., 2005), but NCPs (Section 2.10.2.3), in particular bone sialoprotein and osteopontin, have been shown to accumulate in cement lines (Nanci, 1999; Barthelat et al., 2016). It has been proposed that the cement lines act as mechanical bonds between

Fig. 6 Schematic of bone tissue organization in cortical and trabecular bone.

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211

osteons or transfer energy between osteons to slow crack growth (Burr et al., 1988; Barthelat et al., 2016). There are multiple types of microstructural pores within cortical bone tissue, vascular pores, lacunar–canalicular pores and collagen–apatite pores (Rho et al., 1998; Cowin, 1999). These pores provide a network to transport nutrients, mineral ions and waste products to and from the vascular system to bone cells. It is also believed that fluid flow, within these pores, provides essential mechanical stimulation to the cells (Section 2.10.6.4). Trabecular bone is a highly porous (typically 75–95%) form of bone tissue that is organized into a network of interconnected rods and plates called trabeculae which surround pores that are filled with bone marrow. This bone tissue is found at the ends of long bones such as the femur, and also in the vertebrae. In trabecular bone, the lamellae are organized into single trabeculae, which are the structural units of trabecular bone and are not vascularized. The intervening space between these trabeculae is filled with bone marrow. Human trabeculae are typically 150–300 mm in diameter and can be up to 2000 mm in length and their shape is determined by their anatomical location and loading situation. It has been hypothesized that these trabeculae are directed along the force trajectories created by weight-bearing forces (Wolff, 1892). This highly specialized hierarchical organization lends bone its unique strength and stiffness to withstand the complex loading experienced during normal activities, but also ensures that bone is sufficiently lightweight to facilitate efficient movement in response to the forces generated by muscles. Extensive research has been carried out to characterize the exact morphology of the trabecular architecture (Nicholson et al., 1997; Odgaard, 1997; Kothari et al., 1999). Micro-architecture is a term which refers primarily to the microscopic morphology and organization of trabecular bone. Bone micro-architecture is typically characterized using techniques such as micro-CT scanning (mCT), which relies on exposing bone to an X-ray source, and using an X-ray detector to obtain 3D representations of the trabecular architecture at various resolutions. Such techniques have allowed the morphology of trabecular bone to be analyzed at resolutions ranging from 10–250 mm (Bauer and Link, 2009; Waarsing et al., 2004, 2006). Quantitative parameters have been defined to characterize the structure of trabecular bone, such as bone volume, trabecular thickness, trabecular separation, number of trabeculae per mm2 and trabecular length. It has been reported that more than 80% of the variance of trabecular bone strength and modulus can be explained by measures of density and trabecular orientation (Goldstein et al., 1993). The degree of porosity of trabecular bone varies with anatomical location. Besides the porosity associated with the marrow-filled cavities in the trabecular structure, trabecular bone tissue also has a lacunar–canalicular porosity and the collagen–apatite porosity (Cowin, 1999). These different levels of porosity serve to exchange bone fluid with marrow, to provide mechanical stimuli for bone cells (Section 2.10.6.4), and to transport nutrients and waste products to and from the marrow to the bone cells.

2.10.4.2

Organ Level

The skeleton consists of 206 bones, which are classified broadly into either the axial skeleton or the appendicular skeleton. The appendicular skeleton is composed of 126 bones, which are essential for locomotion and manipulation of objects in the environment. These bones include the long bones such as the femur, tibia, fibula of the legs and the humerus, ulna and radius bones of the arm. Long bones are tubular in structure, and have a shaft comprised of dense cortical bone known as the diaphysis, which surrounds a medullary cavity filled with bone marrow. Porous trabecular bone is found in the extremities (epiphysis) and is covered by a thin shell of cortical bone. The epiphyseal plate is a hyaline cartilage plate in the metaphysis of long bones and is the site from where the bone grows in length. Hyaline cartilage covers the ends of bones and serves to prevent friction and absorb shock. Each of these bones is covered by vascular membranes known as the periosteum on the outside surface in regions where there is no hyaline cartilage, which attaches to ligaments and tendons, and an endosteum on the inner surface. Each of these linings is a source for bone cells that are essential for maintenance and remodeling. The axial skeleton consists of 80 bones in the human skull, the rib cage, the vertebral column, the ossicles of the inner ear and the hyoid bone of the throat. The vertebrae are primarily composed of trabecular bone and are covered by a thin layer of cortical bone. In contrast to long bones, vertebrae have no medullar cavity. Short bones are comprised predominately of trabecular bone and have a thin shell of cortical bone surrounding. These bones include the bones of the wrist and ankle, such as the tarsal bones of the feet, the carpal and metacarpal bones of the hands, the phalanges, the pectoral girdles, clavicles, scapula, pelvis and sesamoid bones.

2.10.5

Bone Mechanical Behavior

As outlined above, bone is a highly specialized tissue that is comprised of a composite material which is organized in a complex hierarchical structure and its biomechanical behavior is accordingly intricate and varies greatly depending on the length scale (ie, tissue level, organ level) at which it is assessed. Given this complexity, the mechanical behavior has been investigated at different length scales and resolutions to provide a holistic understanding of the biomechanical behavior of bone. In general the mechanical behavior can be segregated into the whole bone mechanical behavior, the structural mechanical behavior and the tissue-level mechanical behavior. The structural mechanical properties are determined by mechanical testing of samples that are comprised of multiple trabeculae and thereby the measured mechanical behavior encompasses the trabecular architecture and bone mass of the sample (Section 2.10.5.1). In contrast the properties of the tissue itself are independent of bone mass and architecture and are typically characterized by testing single trabeculae or microscopic samples of bone tissue (Section 2.10.5.2). These studies have

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provided information regarding the complex hierarchical mechanical behavior of bone at the microscopic and nanoscopic levels. At each scale bone can be classified as an anisotropic, heterogenous, non-linear material and the material properties of trabecular bone vary considerably at different anatomical locations and the type and direction of loading that is applied.

2.10.5.1

Structural Mechanical Behavior of Trabecular Bone

The structural behavior (also known as the apparent behavior) is determined by mechanically testing cylinders (typically 5 mm in diameter) or cubes (5 mm3) of trabecular bone. Samples of this size represent the continuum mechanical properties of the bone tissue but are sufficiently large to overcome limitations of the continuum assumption near biologic interfaces (within three to five trabeculae of an interface), and in areas of large stress gradients (Harrigan et al., 1988). Mechanical testing of volumes of trabecular bone from the proximal and distal femur, the vertebral body or the proximal tibia direction (Nicholson et al., 1997; Kopperdahl and Keaveny, 1998; Keaveny et al., 1997, 1993; Torres et al., 2016) have provided an important understanding of the heterogeneous mechanical behavior of trabecular bone. At this scale the heterogeneity is largely due to variations in the bone mass (volume fraction) and the three dimensional architecture of the trabeculae (Keaveny et al., 2001), but variations in tissue composition and organization also contribute to this behavior (Section 2.10.5.2). The trabecular micro-architecture is an important determinant of bone strength and susceptibility to fracture (Dalle Carbonare and Giannini, 2004; Recker, 1993). The apparent elastic modulus of trabecular bone varies considerably within a single vertebral bone with modulus values ranging from 165 MPa in the supero-inferior direction to 43 MPa in the lateral direction (Nicholson et al., 1997). It has also been reported that the mean yield strength of trabecular bone in tension is 15.6 MPa and that this is 30% lower than the yield strength under compressive loading (Keaveny et al., 1994). The apparent yield strain of trabecular bone is approximately 0.8% (Kopperdahl and Keaveny, 1998). The fatigue failure criterion of the trabecular bone matrix has also been investigated by testing within normal physiological frequencies that range from 0.5 and 3 Hz, and it has been reported that the number of cycles to failure ranged from 20 cycles at 2.1% strain to 400,000 at 0.8% strain (Michel et al., 1993). The mechanical behavior of trabecular bone varies across anatomical locations (Goldstein et al., 1983, 1993; Brown and Ferguson, 1980) and further variations arise between different bones and species. The mechanical behavior of trabecular bone deteriorates with the process of aging and it has been reported that the ultimate stress of trabecular bone from the human femur and spine are reduced by approximately 10% per decade in the adult skeleton (McCalden et al., 1997; Mosekilde and Danielsen, 1987; Ding et al., 2002). Due to the structure of trabecular bone, the material properties obtained by testing volumes of trabecular bone are highly dependent on the architecture of the matrix and do not represent the properties of the mineralized tissue itself.

2.10.5.2

Tissue Mechanical Behavior of Bone

As bone is a hierarchical material, the properties of the tissue must be known to fully understand the mechanical behavior of bone. It is often widely assumed that the mechanical properties of trabecular bone are the same as cortical bone at the tissue level, and that it is their architecture that distinguishes between them. Mechanical testing of trabecular and cortical bone specimens has been carried out using a variety of methods such as 3/4-point bending (Choi et al., 1990), buckling (Runkle and Pugh, 1975; Townsend et al., 1975), cantilever beam tests (Mente and Lewis, 1989), micro-tensile testing (Rho et al., 1993; Ryan and Williams, 1989; Samelin et al., 1996; McNamara et al., 2005, 2006a) and ultrasonic measurement (Rho et al., 1993). The details of these studies are summarized in Table 1. Reported values for the elastic modulus of trabecular tissue from these studies have conflicted, with values ranging from 0.75 to 20 GPa. One study applied both microtensile testing and an ultrasonic technique to compare the elastic modulus of individual trabeculae and microspecimens of cortical bone (Rho et al., 1993) and reported that the elastic modulus of individual trabeculae (10.473.5 GPa) is significantly lower (po0.0001) that that of cortical bone (18.673.5 GPa). A system was designed to measure the tensile strength and elastic modulus of individual trabeculae, which overcame previous limitations by eliminating inaccuracies due to sample alignment, geometry and gripping (Luchinetti, 2001). The results of this study indicated that the elastic modulus of trabecular bone is significantly lower than that of cortical bone with an approximate elastic modulus of 2.8 GPa (McNamara et al., 2005, 2006a). Three and four-point bending tests of individual trabeculae have been applied to investigate the fatigue properties of individual trabeculae from the human proximal tibia and reported that the fatigue strength of trabecular bone tissue is 100–140 MPa and that at these stress levels the number of cycles to failure is approximately 100,000 (Choi and Goldstein, 1992). The failure strain values of individual trabeculae have been investigated and are within the range of 20–36% (Yeh and Keaveny, 2001). It has been shown that homogeneity of mineral distribution within individual trabeculae is biomechanically beneficial and that nano-sized particles and collagen fibers may affect the fracture characteristic of trabeculae (Busse et al., 2009). The tissue mechanical properties of trabecular bone have also been assessed using the nanoindentation technique, which can characterize the sub-microstructural (1–5 mm resolution) mechanical properties (elastic modulus, hardness) of bone tissue (Brennan et al., 2009). Atomic force microscopy or scanning electron microscopy techniques can be used to image the indentation and accurately estimate the indent area. This technique has been extensively applied to compare the nano-mechanical properties of bone tissue (Rho et al., 1999, 1997; Ozcivici et al., 2008; Ferguson et al., 2003; Brennan et al., 2011b, 2009; Harrison et al., 2008). The stiffness calculated has ranged from 7–25 GPa for cortical, trabecular and interstitial tissue (Rho et al., 1997; Ferguson et al., 2003; Zysset et al., 1999; Hoffler et al., 2000). The variability between different studies arises as result of differences in the method

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Table 1

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Previous studies to determine the mechanical properties of bone tissue

Study

Property

Mechanical test

McNamara et al. (2005, 2006a,b) Townsend et al. (1975) Runkle and Pugh (1975)

Elastic modulus, yield strength, post yield Tensile testing Strain Elastic modulus Buckling Elastic modulus Buckling

Mente and Lewis (1989)

Elastic modulus

Cantilever beam

Ryan and Williams (1989) Ryan and Williams (1989) Kuhn et al. (1989) Choi and Goldstein (1992) Choi et al. (1990) Rho et al. (1993) Choi and Goldstein (1992) van Rietbergen et al. (1995)

Elastic modulus Elastic modulus Elastic modulus Fatigue strength Elastic modulus Elastic modulus S–N curve Elastic modulus

Rho et al. (1997) Turner et al. (1999)

Elastic modulus Elastic modulus

Tensile Compression Three-point bending Three-point bending Three-point bending Ultrasonic/microtensile Four-point bending High-resolution FE modeling Nanoindentation Nanoindentation Acoustic microscopy

Specimen origin

Elastic modulus (GPa) mean (SD)

Rat proximal tibia

2.8172.09

Human medial tibia Human subchondral bone Dried human femur Fresh human tibia Bovine distal femurs Bovine femora Human iliac crest Proximal tibia Proximal tibia. Proximal human tibia Proximal tibia Proximal human tibia

14.1 (dry); 11.3 (wet) 8.7 (3.2)

Human vertebrae Human distal femur

6.2 (1.2) 11.2 (10.1) – 0.8 (0.4) 3.8 – 4.6 (1.3) 10.4 (3.5) – 5.91 13.5 (2.0) 18.14 (1.7) 17.5 (1.12)

of material preparation and degree of moisture of the sample (Rho et al., 1997; Rho and Pharr, 1999; Turner et al., 1999). Furthermore nanoindentation systems are particularly sensitive to changes in temperature, humidity and environmental vibrations Acoustic microscopy (30–60 mm resolution) can also be used to evaluate the microstructural elastic properties of trabecular bone. This method involves producing an acoustic wave in a tiny area just in the vicinity of surface (near-field area) through different interaction mechanisms. The acoustic properties of materials can be determined at a high resolution from the acoustic wave detected. Turner et al. (1999) used both nano-indentation and acoustic microscopy to compare the Young's moduli of trabecular and cortical bone tissues from a common human donor (Turner et al., 1999). They reported that the Young's modulus of cortical bone in the longitudinal direction was about 40% greater than (po0.01) the Young's modulus in the transverse direction. The Young's modulus of trabecular bone tissue was slightly higher than the transverse Young's modulus of cortical bone, but substantially lower than the longitudinal Young's modulus of cortical bone. These findings were consistent for both measurement methods and suggest that elasticity of trabecular tissue is within the range of that of cortical bone tissue.

2.10.5.3

Fracture Behavior

Bone is a composite material and as a result its fracture behavior is intricate. Physiological bone fractures occur under creep, fatigue and impact loading conditions. The fracture and damage occurring in bone can be segregated into macro-level cracks and diffuse microdamage, which occur in bone during both normal and extreme loading conditions. Studies have investigated the route of propagation of major cracks (Robertson et al., 1978; Melvin, 1993; Koester et al., 2008) and it has been shown that for the most part fracture occurs at the interfaces between separate bone tissue lamellae (Peterlik et al., 2006). It is believed that this interface provides a toughening mechanism for the bone tissue by absorbing energy and resisting the growth of macro-level cracks. This energy is dissipated elastically to deform the collagen–mineral matrix, to deflect cracks along lamellar interfaces, or by the formation of microcracks prior to catastrophic fracture. In particular it has been demonstrated that, in the presence of a large propagating cracks, smaller microcracks nucleate, grow and coalesce to absorb energy from larger cracks in bone (Vashishth et al., 1996). Other studies have shown that toughening occurs in bone when microcracks are bridged by collagen fibrils that span the width of the crack, and that this acts to increase fracture resistance in bone tissue (Nalla et al., 2004b, 2005; Kruzic et al., 2006; Zimmermann et al., 2011). The fracture behavior of bone is anisotropic and crack growth occurs by brittle mechanisms in the longitudinal direction, becomes deflected in the tangential direction, and is toughened by microcracking in the radial direction (Peterlik et al., 2006). This is due to the organization of the collagen fibril matrix which resist cracks that attempt to drive across the grain (fiber direction) that those that are oriented parallel to the collagen fibril axis (Peterlik et al., 2006). One of the most important features of bone is that it has the ability to tolerate a certain degree of microdamage without causing macroscopic failure. Microdamage accumulates in bone tissue from the cyclic loading situations created by everyday activities such as walking and running throughout our lifetime (Lee et al., 2002; Vashishth et al., 2000). Microcrack accumulation impairs the mechanical properties of bone by reducing its elastic modulus (Burr et al., 1998). The ability of bone to renew is believed to be vital to remove this microdamage and maintain strength (Section 2.10.6.2). The fracture toughness of bone gives a measure of the ability of the material to resist crack growth. It has been shown that young bone is tough under impact loading, which is less mineralized (Currey et al., 1996), but the fracture toughness of bone deteriorates significantly with age (Zioupos and Currey, 1998;

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Nalla et al., 2004a; Zimmermann et al., 2011). At high strain rates the post-yield region, the capacity for energy absorption, and the microcracking damage reduce significantly (Hansen et al., 2008; Zioupos et al., 2008). Relatively small amounts of microdamage are associated with large reductions in reload mechanical properties of trabecular bone, whereby 1.50% damage volume fraction was associated with substantial decrease in Young's modulus (41.073.2%), yield strength (63.174.5%) and ultimate strength (52.774.0%) (Hernandez et al., 2014). A few large microdamage sites account for 70% of all microdamage caused by cyclic loading and the number of large microdamage sites is a better predictor of reductions in Young's modulus caused by cyclic loading than overall damage volume fraction (Goff et al., 2015). The majority of microdamage volume occurred primarily within interstitial regions of trabecular bone. Microdamage accumulation in trabecular bone is dominated by heterogeneity in tissue material properties, associated with increased ductility of strut surfaces, rather than stress concentrations arising due to micro-scale geometric features, in particular resorption cavities (Goff et al., 2015; Torres et al., 2016). The more ductile surfaces of cancellous bone are a result of reduced accumulation of advanced glycation end products compared with the strut interior, and render cancellous bone more tolerant of stress concentrations at strut surfaces (Torres et al., 2016).

2.10.6

Bone as a Dynamic Adaptive Material

Bone is a particularly fascinating dynamic material that has the capacity to adapt its shape, mass and microstructural architecture throughout life by means of the physiological processes of bone modeling and remodeling. Bone modeling involves growth and adaptation of bones to produce a mechanically-functional architecture (Jee, 2001) whereas bone remodeling is a process whereby bone is renewed continuously so that it continues to maintain strength throughout life. Both processes are facilitated by the coupled action of osteoclast and osteoblast cells. Bone is also able to renew itself following fracture through a process known as fracture healing, which is regulated by stem cells, cartilage cells and bone cells. These adaptive processes are fundamental to the normal physiological function of the skeleton as they allow bones to survive and adapt under the variety of loading conditions experienced.

2.10.6.1

Bone Modeling

Modeling is a process that facilitates the growth and change in shape of bones and predominantly occurs during childhood and adolescence. Modeling is defined as the process by which bone resorption by osteoclasts and bone formation by osteoblasts occur simultaneously, but on different surfaces of the bone, to alter the overall bone shape or dimensions (Frost, 1990). For example, the cells operate to increase the diameter of long bones and develop a marrow cavity when osteoclasts resorb the endosteum while osteoblasts form new bone at the periosteum. This process results in an overall change in the bone morphology and is distinct from bone remodeling (Frost, 1990). Modeling also occurs in the adult skeleton and serves to regulate overall changes in bone morphology in response to altered mechanical loading. During unloading bone resorption is not followed by formation and during overloading bone formation occurs in the absence of bone resorption. Both processes result in changes in both the microarchitecture and whole bone shape (Mosekilde, 1990).

2.10.6.2

Bone Remodeling

Remodeling is a coordinated physiological process, which is regulated by osteoclast cells that digest aged or damaged bone tissue and osteoblasts that reform new bone tissue in its place. In contrast to modeling, osteoclasts and osteoblasts operate concurrently on the same surfaces and are known collectively as a Basic Multi-cellular Unit (BMUs). The action of these cells appears to be coupled, with osteoclasts traveling along trabecular surfaces or tunneling into cortical bone resorbing unwanted or damage bone tissue and osteoblasts subsequently filling in the resorption cavity with new tissue (Parfitt, 1994; see Fig. 7). The process initiates when bone-lining cells degrade unmineralized osteoid and increase expression of growth factors to recruit bone resorbing osteoclasts from the bone marrow that attach to bone surfaces and secrete acids and enzymes to digest bone matrix. Osteoclasts resorb at a longitudinal rate of 40 mm per day and a typical resorption cavity depth is 40–60 mm (Jaworski et al., 1975; Cohen-Solal et al., 1991). At any one spot on the surface the resorption lasts approximately 2 weeks and the entire resorptive sequence lasts for 40–50 days (Eriksen and Kassem, 1992). After resorption, the osteoclasts undergo apoptosis, and additional growth factors are released which recruit osteoblasts to form layers of osteoid and slowly refill the cavity (Parfitt, 2005). In normal remodeling, there is a small bone mass loss (  1 mm) with age as osteoblasts deposit less bone tissue than the osteoclasts have resorbed (Eriksen and Kassem, 1992). These cells also produce growth factors that inhibit further resorption by osteoclasts. During normal physiology, this continuous cellular activity is co-ordinated so that bone mass and strength are maintained to allow bones to bear normal physiological loading.

2.10.6.3

Fracture Healing

Even though bone has the ability to repair and remodel itself continuously throughout life, certain traumatic injuries and various pathological diseases, such as osteoporosis, osteogenesis imperfecta and Paget’s disease, can impair these normal functions and lead to bone fractures (Section 2.10.7). Fracture healing is a physiological process by which bone fractures are repaired and occurs

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215

Fig. 7 Schematic illustrating cellular activity during the bone remodeling process; (1,2) osteoclasts resorb damaged tissue and (3,4) osteoblasts refill the cavity with newly formed osteoid, which is subsequently mineralized.

through the coordinated activity of osteoprogenitor, chondroblast and osteoblast cells (Mckibbin, 1978). The process initiates when bleeding results in the formation of a hematoma and inflammatory cells are recruited to the fracture site. Next cells within the bone and platelets within the hematoma produce cytokines that recruit fibroblasts and MSCs from the periosteum and marrow to the fracture site. These cells produce a granulation tissue, consisting of cells and blood vessels, at the site of fracture. Immune cells, known as macrophages, are also recruited to remove damaged tissues and other debris. The inflammatory period persists for 3–7 days and, after this initial period, the reparative phase begins whereby cells in the fracture site begin to differentiate into either osteoblasts or fibroblasts. Osteoblasts produce woven bone, which quickly forms a periosteal and marrow callus that fills the fracture gap, but has a low mechanical stiffness (Tsiridis et al., 2007). In sites of poor blood supply fibroblasts within the granulation tissue differentiate to become chondrocytes and form hyaline cartilage (Tsiridis et al., 2007). The cartilage and woven bone together form the fracture callus. The callus has a larger cross-sectional area than the native fractured bone to compensate for the presence of fibrous tissue and woven bone, which reduce the callus stiffness. Over time the woven bone is remodeled to become lamellar bone and the cartilage is replaced by means of endochondral ossification. The lamellar bone is vascularized by the invasion of vascular channels. This reparative phase lasts for approximately 1 month and produces new bone that has similar mechanical properties to that prior to fracture. However the enlarged cross-sectional area at the site of the fracture callus has implications in the efficiency of movement. Therefore the callus is continuously modeled and remodeled by osteoclasts and osteoblasts until the bone approaches its original geometry, strength and stiffness. This remodeling process depends to a large extent on the mechanical forces applied to bone. In some cases, for example, in excessively large fractures or during disease, fracture healing is not activated and as a result bone fractures do not repair (non-unions).

2.10.6.4

Mechanobiology

It has long been established that bone can adapt to its mechanical environment and there is ample evidence of bone loss (atrophy) during disuse and bone formation (hypertrophy) during increased physical exercise (Woo et al., 1981). The exact mechanical stimulus for such changes has been much debated, as has the mechanism by which bone can detect and communicate the need for bone repair. Bone formation in the week following a short term mechanical stimulus occurs near regions of bone tissue experiencing high tissue strain energy density (Cresswell et al., 2016). It is most commonly believed that bone adaptation is response to mechanical loading is regulated by mechanosensitive osteocyte cells that have the ability to direct osteoclasts and osteoblasts to alter bone mass and optimize strength (el Haj et al., 1990; Cowin et al., 1991; Lanyon, 1993; Klein-Nulend et al., 1995; Bonewald, 2002). It has been postulated that osteocytes are capable of sensing several stimuli; matrix strain, periceullular fluid flow or physical damage. In particular is believed that bone adapts to changes in the matrix strain to optimize the bone mass and architecture so that the bone is sufficiently strong to bear the loads it experiences but has a minimal mass. Experimental evidence has demonstrated that,

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at low strains, net bone resorption occurs and that, at elevated strains, net bone formation occurs (Carter, 1984). It has been shown that cells of the osteoblast lineage (osteoprogenitors, osteoblasts, osteocytes) are capable of transducing strain-based mechanical signals into biochemical stimuli (el Haj et al., 1990). Based on such experimental evidence the theory of adaptive elasticity was proposed, which stated that bone has the capacity to adapt its architecture to attain a remodeling equilibrium strain state (Cowin and Hegedus, 1976). When the loading conditions deviate from normal the density and mechanical stiffness of the material or the geometry of the bone is adapted to return to the equilibrium strain state. This theory was capable of predicting net surface remodeling of bone according to adaptive elasticity theory (Cowin and Van Buskirk, 1979). A number of strain-based mechanoregulation theories have been formulated upon the theory of adaptive elasticity to predict the adaptive behavior of bone. These have been applied in computational simulations to successfully predict bone adaptation under various conditions (Huiskes et al., 1987, 2000; Carter et al., 1989; Weinans et al., 1992; Beaupré et al., 1990a,b). This theory has also been used to test the hypothesis that the osteocyte network has a mechano-sensory function and is capable of signaling bone cells to adapt trabecular architecture (Mullender et al., 1994; Mullender and Huiskes, 1997). Loading-induced fluid flow around the osteocyte is widely understood to act as the primary stimulus for bone growth (Owan et al., 1997; Smalt et al., 1997; You et al., 2000; Bacabac et al., 2004; Bakker et al., 2001). Theoretical studies have predicted that loading drives flow of interstitial fluid within the lacunar–canalicular network and imparts a shear stress (0.8–3 Pa) on the osteocyte cell membrane, which acts as a stimulus for biochemical signaling (Weinbaum et al., 1994; Han et al., 2004; Wang et al., 2000, 2005, 2007; You et al., 2001; Knothe Tate and Knothe, 2000; Knothe Tate et al., 1998a,b, 2000; Bakker et al., 2001; Zeng et al., 1994). Computational finite element and fluid mechanics modeling techniques have also been applied to idealized models of the lacunar–canalicular system to predict the mechanical environment within the canaliculi (Mak et al., 1997; Anderson et al., 2005). Ultra high voltage electron microscopes (UHVEM) have been used to develop highly detailed computational models of 80 nm long sections of osteocyte canaliculi and these have been applied to fluid flow around osteocyte cell processes (Kamioka et al., 2012). Recent studies have pushed the limits of computational representation of bone, by developing multiscale models (organ, tissue and cellular), multi-physics (fluid, solid) models (Vaughan et al., 2013a,b; Verbruggen et al., 2012, 2014; Birmingham et al., 2013) and anatomically representative models (Verbruggen et al., 2012) to further understand bone mechanobiology (Fig. 8). Although osteocytes are regarded to be the primary mechanosensors in bone, the precise mechanism by which they can sense mechanical strain is unknown. It has been hypothesized that deformation of the osteocyte lacuna, strain energy density in the bone tissue (Mullender et al., 1994; Huiskes et al., 2000) or strain-derived fluid flow in the osteocyte canalicular channels (Cowin et al., 1995) might stimulate the cell membrane. Experiments have supported such theories by demonstrating that fluid flow (Bakker et al., 2001; McGarry et al., 2005a,b) and matrix strain (Owan et al., 1997; You et al., 2000) activate an anabolic response by osteoblastic cells in vitro. Latest research found that osteocytes may sense matrix strain and fluid flow in bone canaliculi via integrin-based (avb3) focal attachments between their cell processes and the extracellular matrix (McNamara et al., 2009; Wang et al., 2007). In vitro studies which blocked the integrin avb3 were shown to prevent osteocytes from developing the cell processes characteristic of osteocytes and also disrupted the expression of COX-2 and PGE2 release by those cells when they were exposed to mechanical stimulation in the form of fluid shear stress (Haugh et al., 2013). Theoretical models have predicted that these attachments may participate in osteocyte mechanotransduction by amplifying fluid flow-induced and matrix stresses on osteocyte cell processes (Wang et al., 2007). Another study identified cellular structures comprised of microtubules known as primary cilia in

Fig. 8 (a) Multiscale models (organ, tissue and cellular) and (b) multi-physics (fluid, solid) and anatomically representative models to understand bone mechanobiology (Verbruggen et al., 2012, 2014; Vaughan et al., 2013b).

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bone tissue (Tonna and Lampen, 1972); primary cilia are understood to act as fluid flow sensors in other tissues in particular the kidney (Schwartz et al., 1997). Therefore it was proposed that primary cilia may facilitate bone cells to sense strain-derived fluid flow (Whitfield, 2003, 2008). In vitro studies have supported this premise by identifying primary cilia on osteoblastic cell lines [MC3T3, MLYO4] in cell culture (Xiao et al., 2006; Malone et al., 2007), and one study has shown that these cilia play a role in production of proteins associated with osteogenic and bone resorptive responses under in vitro fluid flow (Malone et al., 2007). However, there are conflicting reports regarding in vivo expression of cilia in osteocytes in cortical bone. One study reported that 94% of osteocytes in rat tibia exhibited immunohistochemical staining for the primary cilia marker, acetylated a-tubulin, but very few (410%) of those cells exhibited a distinct cilia-like structure when examined by transmission electron microscopy (Uzbekov et al., 2012). Others have reported that only 4% of osteocytes in aging mice possess primary cilia using transmission electron microscopy (TEM) (Tonna and Lampen, 1972) and that a recent quantitative analysis, accounting for the length of positive immunostaining for acetylated a-tubulin, reported that 4% of osteocytes in sheep trabecular bone have a cilia-like structure (Coughlin et al., 2015). It is known that microdamage accumulates in trabecular bone, from the cyclic loading situations created by everyday activities such as walking, running throughout our lifetime. In vivo microdamage has been identified in bone by a number of researchers (Lee et al., 2002; Vashishth et al., 2000). Microcrack accumulation impairs the mechanical properties of bone by reducing its elastic modulus (Burr et al., 1998). Therefore, it has been proposed that bone remodeling may be targeted to maintain bone strength by resorbing damaged bone and replacing it with new bone thereby preventing accumulation of damage (Martin, 2002; Burr et al., 1985; Lee et al., 2006; Prendergast and Huiskes, 1996; McNamara and Prendergast, 2007; McNamara et al., 2006b; Frost, 1960b). Experimental evidence has shown that resorption cavities occur preferentially in regions of microdamage (Burr et al., 1985, 1997; Mori and Burr, 1993), which would suggest that resorption cavities might indeed be initiated in sites where microdamage is present. Studies observed that microcracks, resorption cavities and osteon formation sites are located near the periosteal surface of the original cortex, but that there were no microcracks in new fibrolamellar bone at periosteal or endosteal surfaces (Lee et al., 2000). In a later study they observed that the average microcrack length is consistent between sites and across species, which supported the idea of a repair mechanism triggered beyond a critical crack length (Lee et al., 2002). Prendergast and Taylor (1994) developed a damage-adaptive law for bone remodeling. They hypothesized that, even at remodeling equilibrium, there exists a homeostatic burden of microdamage in the form of microcracks within bone tissue. If the amount of damage changed from this equilibrium amount then a stimulus for bone remodeling was generated. The repair rate for bone remodeling was determined from the homeostatic stress. Application of the theory was capable of giving physically reasonable predictions of the adaptive response of a bone diaphysis under a change in torsional load. It has been proposed that physical damage acts as a stimulus to bone cells by rupturing cell processes and interrupting cell signaling (Burr et al., 1985, 1997), by shearing osteocytes cell processes (Hazenberg et al., 2006) or by causing apoptotic death of the osteocyte, which in turn signals for removal of the dead cell (Lee et al., 2006; Prendergast and Huiskes, 1996; McNamara and Prendergast, 2005, 2007; McNamara et al., 2006b). In an in vitro simulated bone environment, MLO-Y4 cell networks subjected to microdamage were shown to alter concentrations of cytokines regulating bone remodeling (RANKL, OPG) in a manner which depended on the size of the induced damage and the amount of time following application of injury (Mulcahy et al., 2011). It is likely that bone cells are responsive to both strain and damage based remodeling stimuli, in order to simultaneously maintain bone mass and prevent fracture. Mechanoregulatory computational models have been developed and have predicted that a regulatory system capable of responding to changes in either strain or microdamage, but which prioritizes removal of damaged bone, can successfully predict the bone remodeling process (resorption, reversal, refilling) at the cellular level (Mulvihill et al., 2008; McNamara and Prendergast, 2005, 2007). It was proposed that osteocyte processes can sense changes in strain and fluid flow but when excessive microdamage occurs this damage interferes with the signaling mechanism, or causes osteocyte apoptosis so that a remodeling response occurs to remove the dead osteocytes.

2.10.7

Bone as a Material During Disease and Drug Treatment

The process of aging and various pathological diseases can impair the capacity for bone to perform fundamental mechanical functions. The most prominent diseases affecting bone are osteoporosis, osteogenesis imperfecta, Paget's disease (Osteitis deformans), osteomalacia, rickets (Vitamin D deficiency) and cancer of the bone (osteosarcoma). Each of these conditions severely impairs the ability of bone and leads to bone fractures, and often these fractures do not repair (non-unions) or cause immobility, severe pain and deformity.

2.10.7.1

Osteoporosis

Osteoporosis is disease, which is most commonly manifested in post-menopausal women, that degrades bone mass and architecture. Physiologically, osteoporosis is manifested as an imbalance in bone cell activity during the remodeling process (Section 2.10.6.2) whereby excessive resorption occurs without adequate new bone formation. The basic bone units (trabeculae) become thin and eventually fracture and resorb altogether (Compston et al., 1989). As a consequence bone mass is reduced, the trabecular architecture is severely degraded (Fig. 9), its mechanical strength is reduced and bones become more susceptible to fracture. Fractures of the hip, wrist or vertebra are most prevalent and lead to severe pain and spinal deformity (Cummings and

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Fig. 9 Micro-CT scanning image depicting the deterioration of bone microarchitecture during osteoporosis; (a1, a2) bone trabeculae are interconnected in normal bone tissue; (b1, b2) bone loss and microarchitectural deterioration (loss of connectivity, trabecular thinning) in osteoporotic bone.

Melton, 2002). Approximately 40% of women over 50 are at risk of developing the disease (Melton et al., 2005) and the disease is the second most significant health threat to women, after breast cancer, with normal mortality rates increasing by 10–20% within a year of experiencing an osteoporotic fracture (Melton et al., 2005; Cummings and Melton, 2002). There is one osteoporotic fracture every three seconds (Johnell and Kanis, 2006) and in 2010, the economic cost of incident and prior fragility fractures was estimated at € 37 billion in the European Union (Hernlund et al., 2013).

2.10.7.1.1

Bone cell biology during osteoporosis

During menopause the levels of circulating estrogen in the blood are deficient and this deficiency is believed to play a role in the imbalance in the cellular activity during osteoporosis. Osteoclasts, osteoblasts, and osteocytes all possess estrogen receptors (ERa, ERb) that are activated to permit protein synthesis when they bind to estrogen (Braidman et al., 2001). The effect of estrogen deficiency on bone cell activity has been studied to identify pathways by which post-menopausal osteoporosis is manifested. It has been found that osteoclastic activity is enhanced by estrogen deficiency, and that the reduced levels of circulating estrogen during osteoporosis are associated with increased rates of bone turnover and bone loss due. These changes occur as a result of an increase in the number and activity of osteoclasts (Bell et al., 1996; Rosen, 2000). The effect of estrogen deficiency on osteoclast behavior has been extensively characterized; (1) the activation frequency of bone multicellular units (BMU’s) and turnover rate is markedly increased (Brockstedt et al., 1993; Eriksen et al., 1999), (2) more osteoclasts are recruited (Rosen, 2000), (3) osteoclast apoptosis is inhibited (Hughes et al., 1996), and as a result (4) the osteoclasts resorption persists for longer resulting in deeper resorption cavities and trabecular perforation (Bell et al., 1996). The perforated trabeculae are removed by further remodeling and this loss of bone mass, as is illustrated in Fig. 8, increases the fragility of bones, resulting in a greater propensity for fracture (Dalle Carbonare and Giannini, 2004). The effects of estrogen deficiency are not restricted to osteoclast activity as osteoblastogenesis also increases (Rosen, 2000; Bell et al., 1996). It has been suggested that estrogen allows the normal response of osteoblasts and osteocytes to loading (Lanyon, 1996). As both cell types possess receptors for estrogen (Braidman et al., 2001), their function may be affected when estrogen production is deficient during post-menopausal osteoporosis. Previous findings regarding the effects of estrogen (E2) on osteoblasts are inconsistent; E2 enhances differentiation and mineralized bone nodule formation in-vitro (Rao et al., 2003), but alkaline phosphatase and osteocalcin expression are stimulated, inhibited, or unaffected (Rickard et al., 2002). Recent studies have found for the first time that complex tissue levels changes in bone composition might be explained by alterations in bone cell biology, in particular the mechanobiological responses (Brennan et al., 2012a–c, 2014a,b). Osteoblastic cells deprived of estrogen display deficient osteogenic responses to mechanical stimuli in vitro (Sterck et al., 1998; Jessop et al., 2004). The osteoblasts may be no longer capable of completely refilling the resorbed space, resulting in irreversible bone loss. Estrogen deficiency induces osteocyte apoptosis (Kousteni et al., 2001; Tomkinson et al., 1997), which might result in hypermineralization of the surrounding tissue (Frost, 1960a; Boyde, 2003; Kingsmill and Boyde, 1998). In addition the organization of the osteocyte network is altered (Knothe Tate et al., 2004) and osteocyte density is reduced (Mullender et al., 1996), which may reduce the mechanoresponsiveness of the tissue (Tatsumi et al., 2007).

2.10.7.1.2

Mechanical behavior and structure during osteoporosis

The main concern with the disease of osteoporosis is that bone fractures occur unpredictably and with little force. For this reason, a number of studies have been carried out to characterize the biomechanical behavior of bone during osteoporosis by assessing the structural degradation of the trabecular architecture and the mechanical consequences of bone loss. At the onset of osteoporosis, an irreversible reduction in bone mass and volume occurs, leading to thinning and micro-fracture of trabecular struts and loss of trabecular connectivity (Parfitt, 1987; Compston et al., 1989; Lane et al., 1998). Fractured trabeculae

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are eventually resorbed completely resulting in an overall degradation in the trabecular network, see Fig. 8(b). Furthermore, the trabecular architecture of osteoporotic bone is significantly more anisotropic than normal bone, with fewer trabeculae transverse to the primary load axis (Ciarelli et al., 2000). Until recently it was believed that bone loss occurred systemically, ie, bone resorption occurred throughout the trabecular architecture to same extent, and that this loss was irreversible. However, recent studies have shown that, following an initial increase in bone loss and trabecular thinning in OVX rats, the few remaining trabeculae subsequently slowly increase in thickness (Waarsing et al., 2004, 2006). Thus the authors proposed that, rather than describing estrogen depletion as a state of bone loss, the condition should be referred to as accelerated bone metabolism or mechanically driven, bone adaptation (Waarsing et al., 2004, 2006). Assessment of the effects of osteoporosis on the mechanical behavior of bone has been carried out previously using whole bone testing of vertebrae and femora (Ederveen et al., 2001; Kasugai et al., 1998; Yoshitake et al., 1999b; Hirano et al., 2000; Bourrin et al., 2002) or testing of volumes of cancellous bone (Mosekilde et al., 1995; Hu et al., 2002; Teramura et al., 2002; Sugita et al., 1999) from rat and dog models. Using ovariectomy to induce osteoporosis in the rat, it has been found, for example, that the compressive strength of rat vertebral bodies and the bending strength of rat femora are significantly compared to control bone (Ederveen et al., 2001; Kasugai et al., 1998; Yoshitake et al., 1999b). In human bone it has been reported that the maximum modulus and ultimate stress is reduced in the femur following an osteoporotic fracture (Zysset et al., 1999; Hasegawa et al., 1993). Such studies can explain the increase in fracture susceptibility at these sites. However, these observations were accompanied by significant decreases in trabecular bone volume and thus cannot discriminate whether changes were due only to the reduction in bone mass and loss of architecture, or whether a reduction in tissue strength also contributed. It has been proposed that an initial increase in bone turnover is accompanied by a change in bone composition, and is followed by a continued increase in bone resorption and relative reduction in bone formation, leading to deterioration in bone microarchitecture. Ultimately, these cumulative changes led to a significant reduction in the compressive strength of bones following 31 months of estrogen deficiency (Brennan et al., 2012c). It is essential to develop an understanding of the material behavior at the bone tissue level during osteoporosis to seek to fully address the issues of bone fracture during osteoporosis. It has been reported that trabecular bone tissue from osteoporotic patients had a lower apparent density, stiffness and strength than age matched patients with no evidence of disease (Li and Aspden, 1997). In contrast other studies have demonstrated that, while overall bone mass and bone mineral density are reduced, tissue (single trabeculae) stiffness and strength of ovariectomized rats are increased by 40–90% (McNamara et al., 2005, 2006a). In an ovine model of osteoporosis, the tissue modulus, as measured by nanoindentation, was significantly less than in the control bone (Brennan et al., 2009). While variations in experimental methods, animal model or anatomical location might explain the discrepancies between previous studies, it is still unclear how bone mechanical properties are altered during osteoporosis. In human cortical bone a lower elastic modulus was measured in the osteoporosis in comparison to younger cases (Zimmermann et al., 2016). Osteoporosis cases were shown to have significantly less resistance to plasticity and a lower strength, as measured by three-point bending strength tests (Zimmermann et al., 2016). Such studies suggest that the primary constituents of bone tissue, namely collagen, mineral and noncollagenous proteins, may be altered during osteoporosis. A number of studies have been carried out to investigate the effects of osteoporosis on the mineral content in the bone tissue, with conflicting findings. Some studies report that the mineral content is unchanged or slightly lower in osteoporotic bone tissue (Gadeleta et al., 2000; Li and Aspden, 1997), and others reveal an increase in mineral content and a lack of collagen (Dickenson et al., 1981; Boyde et al., 1998; Zioupos and Aspden, 2000). A lower level of cortical mineralization has been reported for human osteoporotic bone (Zimmermann et al., 2016). Studies using a micro-CT system calibrated for bone mineral content assessment in individual trabeculae showed that a significant increase (11%) in mineral content of remaining trabeculae (following bone loss) corroborated unexpected changes in tissue properties (McNamara et al., 2005, 2006a). Bone tissue composition changes have been confirmed at the microscopic level using quantitative back-scattered electron microscopy, which were undetectable by conventional diagnostic techniques (DEXA) (McNamara et al., 2006a; Brennan et al., 2011a,b, 2012c). In particular it has been recently shown that bone loss and tissue mineral changes during estrogen deficiency do not occur ubiquitously, but are more prevalent at specific anatomical regions within the femora of rat and ovine models of osteoporosis (Brennan et al., 2011a). Moreover, while normal aging increases mean mineralization and mineral heterogeneity at a trabecular level, prolonged estrogen deficiency leads to significantly decreased mean mineralization and further exacerbates increases in mineral heterogeneity (Brennan et al., 2014a). In a study of human osteoporotic bone cortical and cancellous collagen maturity, cortical mineral/matrix ratio and cancellous crystallinity all increased with increased fracture risk, albeit that the heterogeneity of crystallinity also increased (Gourion-Arsiquaud et al., 2013). Significantly higher amounts of mineralized osteocyte lacunae have been identified in the human femoral cortex with aging and osteoporosis, which is attributed to increased osteocyte apoptosis (Milovanovic et al., 2015). The mineral distribution in the femoral osteoporotic cortical bone was lower in comparison to young cases, which was proposed to reflect the increased surface area undergoing remodeling and insufficient secondary mineralization (Milovanovic et al., 2015). Various changes in the compositional properties of the collagen have been reported at the onset of postmenopausal osteoporosis, in particular that changes in the amount of Type I, Type VI and Type III collagen occur (Bailey et al., 1993). The nature and quantity of the collagen cross-links that is altered (Batge et al., 1992; Kowitz et al., 1997), in particular the quantity of ketoimine, pyridinium (Mansell and Bailey, 2003), aldimine (Bailey et al., 1993; Oxlund et al., 1996) and pyrrole cross links are decreased in osteoporotic bone (Knott et al., 1995). Interestingly, these changes more prevalent in the femoral neck, a site which is highly susceptible to fracture (Bailey et al., 1993), which indicate that such changes increase fracture susceptibility either by altering the strength of the collagen matrix or by secondary changes to the mineralization of this matrix (Bailey et al., 1993;

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Bailey and Knott, 1999). Fibril deformation is lower in osteoporotic human cortical bone than in young cases, which was proposed to explain why the tissue has a lower strength and is more susceptible to fragility fractures (Zimmermann et al., 2016). Moreover it was proposed that post-translational modifications to collagen (eg, hydroxylation of lysine) and changes in noncollagenous proteins (Bailey et al., 1992, 1999) could affect deformation of the fibril (Zimmermann et al., 2016). There is some evidence that extent of microdamage is increased during estrogen deficiency (Dai et al., 2004), which suggest that the tissue may be more brittle and less resistant to crack growth (Burr, 2003; Schaffler, 2003; Mashiba et al., 2001). Alternatively the remaining bone tissue may be more susceptible to damage following significant bone loss. Increased remodeling during osteoporosis has been proposed to be related to an accelerated removal of damaged tissue (Burr et al., 1997).

2.10.7.1.3

Mechanobiology and osteoporosis

Studies have shown that estrogen plays an important role in the responsiveness of bone cells to in vitro mechanical stimuli (Bakker et al., 2005; Joldersma et al., 2001; Yeh et al., 2010; Damien et al., 1998; Cheng et al., 2002) and that osteoblasts from osteoporotic bone alter their biochemical response to loading (Neidlinger-Wilke et al., 1995; Sterck et al., 1998). Recent studies have found for the first time that complex tissue levels changes in bone composition might be explained by alterations in bone cell biology, in particular the mechanobiological responses (Brennan et al., 2014b, 2012d). Specifically mechanosensors expression (b3 integrin subunit) is altered in bone cells from osteoporotic animals compared to controls (Voisin and McNamara, 2015). Moreover, the mechanical environment of osteoporotic bone cells is altered; osteocytes are exposed to higher strains than healthy bone cells after short durations of estrogen deficiency (5 weeks), whereas there is no significant difference in the mechanical stimulation of bone cells in healthy and osteoporotic bone in long-term estrogen deficiency (34 weeks) (Verbruggen et al., 2015). Thus it has been proposed that the mechanical environment of bone cells is altered during early-stage osteoporosis, and that mechanobiological responses act to restore the mechanical environment of the bone tissue after it has been perturbed by ovariectomy (Verbruggen et al., 2015), perhaps in an attempt to restore homeostasis (Verbruggen et al., 2016). In aged individuals, there is a diminished osteocyte network, characterized by fewer canaliculi per osteocyte lacuna, connections within the osteon and interosteon connections (Milovanovic et al., 2013). This depleted network might degrade the mechanosensitivity of osteocytes (Milovanovic et al., 2013).

2.10.7.1.4

Approaches for treatment of osteoporosis

The primary aim of drug treatments for osteoporosis is to reduce fracture incidence and a variety of different treatments have been formulated which are based on the use of hormone replacement therapy, anti-resorptive therapy or anabolic agents. Hormone replacement therapy involves a pharmacological treatment with estrogen, which is often combined with progesterone, as a treatment for postmenopausal osteoporosis. This treatment has been shown to increase the bone mineral density of sufferers and thereby reduces the risk of hip and vertebral fractures in healthy postmenopausal women (Cauley et al., 2003). However hormone therapy is no longer a popular choice of treatment as it increases the risk of stroke, venous thromboembolism, coronary heart disease, and breast cancer. There are a wide range of anti-resorptive therapies, such as bisphosphonates, selective tissue estrogenic activity regulators (STEARs), selective estrogen receptor modulators (SERMs), calcitonins, calcium, vitamin D and metabolites and these represent a popular choice of treatment. While drugs of these types have different modes of operation, the common aim of anti-resorptive drugs is to maintain bone mass and architecture by restoring the remodeling imbalance through inhibition of osteoclast activity and reducing bone resorption (Kloosterboer and Ederveen, 2002). The efficacy of anti-resorptive drug treatments has been assessed and it has been found, for example, that treatment with Tibolone (Kasugai et al., 1998; Yoshitake et al., 1999b; Ederveen et al., 2001) and other anti-resorptive drugs such as Risedronate (Mosekilde et al., 1995), Etidronate (Hirano et al., 2000), Alendronate (Hu et al., 2002), Pamidronate (Bourrin et al., 2002) and Incadronate (Teramura et al., 2002) increases the structural or bulk trabecular bone strength (Mosekilde et al., 1995; Hu et al., 2002; Teramura et al., 2002) and the whole bone strength (Kasugai et al., 1998; Yoshitake et al., 1999b; Ederveen et al., 2001; Hirano et al., 2000; Bourrin et al., 2002) compared to untreated osteoporotic bone by maintaining bone mass and trabecular architecture. Treatment of ovariectomized sheep with the bisphosphonate zoledronic acid increased the mineral content and tissue modulus and was also found to alter the mineral and modulus gradients normally associated with healthy bone tissue (Brennan et al., 2011b). Zoledronic acid significantly reduced tissue mineral variability, both at a trabecular level and between femoral regions, and thereby acted to reverse the increased mineral heterogeneity occurring during estrogen deficiency, which may contribute to its capacity to reduce osteoporotic fractures (Brennan et al., 2014a). Using an in vitro microinjury model, the effect of two commonly utilized aminobisphosphonate treatments for osteoporosis, alendronate and zoledronate, on osteocytes, osteoclasts and osteoblasts was investigated (Mulcahy et al., 2015). Osteocyte networks treated with bisphosphonates exhibited altered RANKL and OPG activity, osteoclastogenesis was reduced, but the osteogenic potential increased following microinjury (Mulcahy et al., 2015). Parathyroid hormone (PTH) was shown to increase BMD and bone mineral content in ovarectomized monkeys (Brommage et al., 1999). PTH alone or in combination with anti-resorptive medications reduces fracture risk of osteoporosis patients by increasing BMD (Vestgaard et al., 2007). While these drugs are capable of reducing the propensity to fracture by approximately 50%, even with continuous use of drug treatment fractures still arise (Randell et al., 2002). It has been found that treatment with high doses of certain anti-resorptive drugs is associated with an increase in the rate of spontaneous fractures of the thoracic spinous process, ribs and pelvic fractures in animal models of osteoporosis (Flora et al., 1981; Hirano et al., 2000). A concern with some drugs for osteoporosis is that inhibiting the remodeling process prevents the necessary osteoclastic bone resorption, which serves as a repair mechanism to remove aged damaged bone tissue (Burr et al., 1985; Lee et al., 2006; Prendergast and Huiskes, 1996;

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McNamara and Prendergast, 2005, 2007; McNamara et al., 2006b). This may allow microdamage to accumulate in the bone tissue, so that, although bone mass and architecture are maintained, bone strength at the tissue level may be impaired.

2.10.8

Conclusion

Even with the vast amount of research, the complex biological and mechanical behavior of bone is still intriguing as the pathogenesis of bone disease is not fully understood and the occurrence of bone fractures remains a significant clinical issue. This fascinating material will continue to be studied until the normal physiological and mechanical function of bone has been delineated and the pathogenesis of fracture is characterized.

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