Elizabeth A. Zimmermann

Age-related changes in the plasticity and toughness of human cortical bone at multiple length-scales

The structure of human cortical bone evolves over multiple length scales from its basic constituents of collagen and hydroxyapatite at the nanoscale to osteonal structures at near-millimeter dimensions, which all provide the basis for its mechanical properties. To resist fracture, bone’s toughness is derived intrinsically through plasticity (e.g., fibrillar sliding) at structural scales typically below a micrometer and extrinsically (i.e., during crack growth) through mechanisms (e.g., crack deflection/bridging) generated at larger structural scales. Biological factors such as aging lead to a markedly increased fracture risk, which is often associated with an age-related loss in bone mass (bone quantity). However, we find that age-related structural changes can significantly degrade the fracture resistance (bone quality) over multiple length scales. Using in situ small-angle X-ray scattering and wide-angle X-ray diffraction to characterize submicrometer structural changes and synchrotron X-ray computed tomography and in situ fracture-toughness measurements in the scanning electron microscope to characterize effects at micrometer scales, we show how these age-related structural changes at differing size scales degrade both the intrinsic and extrinsic toughness of bone. Specifically, we attribute the loss in toughness to increased nonenzymatic collagen cross-linking, which suppresses plasticity at nanoscale dimensions, and to an increased osteonal density, which limits the potency of crack-bridging mechanisms at micrometer scales. The link between these processes is that the increased stiffness of the cross-linked collagen requires energy to be absorbed by “plastic” deformation at higher structural levels, which occurs by the process of microcracking.

Natural Flexible Dermal Armor

Fish, reptiles, and mammals can possess flexible dermal armor for protection. Here we seek to find the means by which Nature derives its protection by examining the scales from several fish (Atractosteus spatula, Arapaima gigas, Polypterus senegalus, Morone saxatilis, Cyprinius carpio), and osteoderms from armadillos, alligators, and leatherback turtles. Dermal armor has clearly been developed by convergent evolution in these different species. In general, it has a hierarchical structure with collagen fibers joining more rigid units (scales or osteoderms), thereby increasing flexibility without significantly sacrificing strength, in contrast to rigid monolithic mineral composites. These dermal structures are also multifunctional, with hydrodynamic drag (in fish), coloration for camouflage or intraspecies recognition, temperature and fluid regulation being other important functions. The understanding of such flexible dermal armor is important as it may provide a basis for new synthetic, yet bioinspired, armor materials.

Characterization of the effects of x-ray irradiation on the hierarchical structure and mechanical properties of human cortical bone

Bone comprises a complex structure of primarily collagen, hydroxyapatite and water, where each hierarchical structural level contributes to its strength, ductility and toughness. These properties, however, are degraded by irradiation, arising from medical therapy or bone-allograft sterilization. We provide here a mechanistic framework for how irradiation affects the nature and properties of human cortical bone over a range of characteristic (nano to macro) length-scales, following x-ray exposures up to 630 kGy. Macroscopically, bone strength, ductility and fracture resistance are seen to be progressively degraded with increasing irradiation levels. At the micron-scale, fracture properties, evaluated using insitu scanning electron microscopy and synchrotron x-ray computed micro-tomography, provide mechanistic information on how cracks interact with the bone-matrix structure. At sub-micron scales, strength properties are evaluated with insitu tensile tests in the synchrotron using small-/wide-angle x-ray scattering/diffraction, where strains are simultaneously measured in the macroscopic tissue, collagen fibrils and mineral. Compared to healthy bone, results show that the fibrillar strain is decreased by ∼40% following 70 kGy exposures, consistent with significant stiffening and degradation of the collagen. We attribute the irradiation-induced deterioration in mechanical properties to mechanisms at multiple length-scales, including changes in crack paths at micron-scales, loss of plasticity from suppressed fibrillar sliding at sub-micron scales, and the loss and damage of collagen at the nano-scales, the latter being assessed using Raman and Fourier Transform Infrared spectroscopy and a fluorometric assay.

Glucocorticoid-induced bone loss in mice can be reversed by the actions of parathyroid hormone and risedronate on different pathways for bone formation and mineralization.

OBJECTIVE Glucocorticoid excess decreases bone mineralization and microarchitecture and leads to reduced bone strength. Both anabolic (parathyroid hormone [PTH]) and antiresorptive agents are used to prevent and treat glucocorticoid-induced bone loss, yet these bone-active agents alter bone turnover by very different mechanisms. This study was undertaken to determine how PTH and risedronate alter bone quality following glucocorticoid excess. METHODS Five-month-old male Swiss-Webster mice were treated with the glucocorticoid prednisolone (5 mg/kg in a 60-day slow-release pellet) or placebo. From day 28 to day 56, 2 groups of glucocorticoid-treated animals received either PTH (5 microg/kg) or risedronate (5 microg/kg) 5 times per week. Bone quality and quantity were measured using x-ray tomography for the degree of bone mineralization, microfocal computed tomography for bone microarchitecture, compression testing for trabecular bone strength, and biochemistry and histomorphometry for bone turnover. In addition, real-time polymerase chain reaction (PCR) and immunohistochemistry were performed to monitor the expression of several key genes regulating Wnt signaling (bone formation) and mineralization. RESULTS Compared with placebo, glucocorticoid treatment decreased trabecular bone volume (bone volume/total volume [BV/TV]) and serum osteocalcin, but increased serum CTX and osteoclast surface, with a peak at day 28. Glucocorticoids plus PTH increased BV/TV, and glucocorticoids plus risedronate restored BV/TV to placebo levels after 28 days. The average degree of bone mineralization was decreased after glucocorticoid treatment (-27%), but was restored to placebo levels after treatment with glucocorticoids plus risedronate or glucocorticoids plus PTH. On day 56, RT-PCR revealed that expression of genes that inhibit bone mineralization (Dmp1 and Phex) was increased by continuous exposure to glucocorticoids and glucocorticoids plus PTH and decreased by glucocorticoids plus risedronate, compared with placebo. Wnt signaling antagonists Dkk-1, Sost, and Wif1 were up-regulated by glucocorticoid treatment but down-regulated after glucocorticoid plus PTH treatment. Immunohistochemistry of bone sections showed that glucocorticoids increased N-terminal Dmp-1 staining while PTH treatment increased both N- and C-terminal Dmp-1 staining around osteocytes. CONCLUSION Our findings indicate that both PTH and risedronate improve bone mass, degree of bone mineralization, and bone strength in glucocorticoid-treated mice, and that PTH increases bone formation while risedronate reverses the deterioration of bone mineralization.

Vitamin D Deficiency Induces Early Signs of Aging in Human Bone, Increasing the Risk of Fracture

In addition to decreasing bone mass, vitamin D deficiency causes early aging of the remaining mineralized bone and leads to severe losses in fracture resistance. Vitamin D–Deficient Bone Showing Its Age Vitamin D, which is sometimes called the “sunshine vitamin” because humans can synthesize it in the presence of sunlight, has long been associated with prevention of bone disease. Vitamin D is required for proper absorption of calcium and its uptake into bone, and a lack of vitamin D is known to cause rickets and osteomalacia—diseases in which bone is too soft because of excessive collagenous matrix and its inadequate mineralization. Now, Busse and co-authors provide some evidence that the reverse is also partially true, and vitamin D deficiency can result in areas of overly dense mineralization in the bone. To study the effects of vitamin D deficiency, Busse and colleagues used samples of bone from 30 apparently healthy people. Half of these subjects were deficient in vitamin D, defined by low concentration of vitamin D in the blood and altered macroscopic characteristics of the bone. Through detailed analysis of bone structure and functional tests measuring the bones’ resistance to cracking, the authors characterized the ways in which vitamin D–deficient bone differs from normal. As expected, they found that bones from vitamin D–deficient subjects had a much thicker layer of unmineralized osteoid coating the surface of mineralized bone. However, they also demonstrated that the bone underneath this osteoid layer was more heavily mineralized than normal and had structural characteristics of older and more brittle bone. They explained this phenomenon by noting that osteoclasts, cells that normally remodel the bone, cannot get through the thick osteoid layer. As a result, the areas of bone hidden underneath the osteoid continue to age and mineralize even as the overall bone mineral content progressively decreases. These interesting and unexpected findings about human bone emphasize the negative consequences of vitamin D deficiency, which is all too common, especially at northern latitudes. Additional work will be needed to translate this knowledge into clinical practice, but the detailed understanding of human bone structure may provide some insight into more effective ways to prevent or treat fractures in patients with vitamin D deficiency. Vitamin D deficiency is a widespread medical condition that plays a major role in human bone health. Fracture susceptibility in the context of low vitamin D has been primarily associated with defective mineralization of collagenous matrix (osteoid). However, bone’s fracture resistance is due to toughening mechanisms at various hierarchical levels ranging from the nano- to the microstructure. Thus, we hypothesize that the increase in fracture risk with vitamin D deficiency may be triggered by numerous pathological changes and may not solely derive from the absence of mineralized bone. We found that the characteristic increase in osteoid-covered surfaces in vitamin D–deficient bone hampers remodeling of the remaining mineralized bone tissue. Using spatially resolved synchrotron bone mineral density distribution analyses and spectroscopic techniques, we observed that the bone tissue within the osteoid frame has a higher mineral content with mature collagen and mineral constituents, which are characteristic of aged tissue. In situ fracture mechanics measurements and synchrotron radiation micro–computed tomography of the crack path indicated that vitamin D deficiency increases both the initiation and propagation of cracks by 22 to 31%. Thus, vitamin D deficiency is not simply associated with diminished bone mass. Our analyses reveal the aged nature of the remaining mineralized bone and its greatly decreased fracture resistance. Through a combination of characterization techniques spanning multiple size scales, our study expands the current clinical understanding of the pathophysiology of vitamin D deficiency and helps explain why well-balanced vitamin D levels are essential to maintain bone’s structural integrity.

Mechanical adaptability of the Bouligand-type structure in natural dermal armour

Arapaima gigas, a fresh water fish found in the Amazon Basin, resist predation by piranhas through the strength and toughness of their scales, which act as natural dermal armour. Arapaima scales consist of a hard, mineralized outer shell surrounding a more ductile core. This core region is composed of aligned mineralized collagen fibrils arranged in distinct lamellae. Here we show how the Bouligand-type (twisted plywood) arrangement of collagen fibril lamellae has a key role in developing their unique protective properties, by using in situ synchrotron small-angle X-ray scattering during mechanical tensile tests to observe deformation mechanisms in the fibrils. Specifically, the Bouligand-type structure allows the lamellae to reorient in response to the loading environment; remarkably, most lamellae reorient towards the tensile axis and deform in tension through stretching/sliding mechanisms, whereas other lamellae sympathetically rotate away from the tensile axis and compress, thereby enhancing the scales ductility and toughness to prevent fracture.

Perspectives on the role of nanotechnology in bone tissue engineering

OBJECTIVE This review surveys new developments in bone tissue engineering, specifically focusing on the promising role of nanotechnology and describes future avenues of research. METHODS The review first reinforces the need to fabricate scaffolds with multi-dimensional hierarchies for improved mechanical integrity. Next, new advances to promote bioactivity by manipulating the nanolevel internal surfaces of scaffolds are examined followed by an evaluation of techniques using scaffolds as a vehicle for local drug delivery to promote bone regeneration/integration and methods of seeding cells into the scaffold. RESULTS Through a review of the state of the field, critical questions are posed to guide future research toward producing materials and therapies to bring state-of-the-art technology to clinical settings. SIGNIFICANCE The development of scaffolds for bone regeneration requires a material able to promote rapid bone formation while possessing sufficient strength to prevent fracture under physiological loads. Success in simultaneously achieving mechanical integrity and sufficient bioactivity with a single material has been limited. However, the use of new tools to manipulate and characterize matter down to the nano-scale may enable a new generation of bone scaffolds that will surpass the performance of autologous bone implants.

Mixed-mode fracture of human cortical bone

Although the mode I (tensile opening) fracture toughness has been the focus of most fracture mechanics studies of human cortical bone, bones in vivo are invariably loaded multiaxially. Consequently, an understanding of mixed-mode fracture is necessary to determine whether a mode I fracture toughness test provides the appropriate information to accurately quantify fracture risk. In this study, we examine the mixed-mode fracture of human cortical bone by characterizing the crack-initiation fracture toughness in the transverse (breaking) orientation under combined mode I (tensile opening) plus mode II (shear) loading using samples loaded in symmetric and asymmetric four-point bending. Whereas in most structural materials, the fracture toughness is increased with increasing mode-mixity (i.e., where the shear loading component gets larger), in the transverse orientation of bone the situation is quite different. Indeed, the competition between the maximum applied mechanical mixed-mode driving force and the weakest microstructural paths in bone results in a behavior that is distinctly different to most homogeneous brittle materials. Specifically, in this orientation, the fracture toughness of bone is markedly decreased with increasing mode-mixity.

Structure and fracture resistance of alligator gar (Atractosteus spatula) armored fish scales

The alligator gar is a large fish with flexible armor consisting of ganoid scales. These scales contain a thin layer of ganoine (microhardness ~2.5 GPa) and a bony body (microhardness ~400 MPa), with jagged edges that provide effective protection against predators. We describe here the structure of both ganoine and bony foundation and characterize the mechanical properties and fracture mechanisms. The bony foundation is characterized by two components: a mineralized matrix and parallel arrays of tubules, most of which contain collagen fibers. The spacing of the empty tubules is ~60 μm; the spacing of those filled with collagen fibers is ~7 μm. Using micromechanical testing of such scales in a variable-pressure scanning electron microscope, we identify interactions between propagating cracks and the microstructure, and show that the toughness of the scales increases with crack extension in a classical resistance-curve response from the activation of extrinsic toughening mechanisms. We demonstrate how mechanical damage evolves in these structures, and further identify that the reinforcement of the mineral by the network of collagen fibers is the principal toughening mechanism resisting such damage. Additionally, we define the anisotropy of the toughness of the scales and relate this to the collagen fiber orientation.

How tough is brittle bone? Investigating osteogenesis imperfecta in mouse bone.

The multiscale hierarchical structure of bone is naturally optimized to resist fractures. In osteogenesis imperfecta, or brittle bone disease, genetic mutations affect the quality and/or quantity of collagen, dramatically increasing bone fracture risk. Here we reveal how the collagen defect results in bone fragility in a mouse model of osteogenesis imperfecta (oim), which has homotrimeric α1(I) collagen. At the molecular level, we attribute the loss in toughness to a decrease in the stabilizing enzymatic cross‐links and an increase in nonenzymatic cross‐links, which may break prematurely, inhibiting plasticity. At the tissue level, high vascular canal density reduces the stable crack growth, and extensive woven bone limits the crack‐deflection toughening during crack growth. This demonstrates how modifications at the bone molecular level have ramifications at larger length scales affecting the overall mechanical integrity of the bone; thus, treatment strategies have to address multiscale properties in order to regain bone toughness. In this regard, findings from the heterozygous oim bone, where defective as well as normal collagen are present, suggest that increasing the quantity of healthy collagen in these bones helps to recover toughness at the multiple length scales.