Letter pubs.acs.org/NanoLett
Nanomechanics Insights into the Performance of Healthy and Osteoporotic Bones Ying-Ting Wang,† Shou-Yi Chang,*,† Yi-Chung Huang,† Tung-Chou Tsai,‡ Chuan-Mu Chen,‡ and Chwee Teck Lim*,§,∥ †
Department of Materials Science and Engineering, National Chung Hsing University, Taichung 40227, Taiwan Department of Life Sciences, National Chung Hsing University, Taichung 40227, Taiwan § Department of Bioengineering & Department of Mechanical Engineering, National University of Singapore, Singapore 117576, Singapore ∥ Mechanobiology Institute, National University of Singapore, Singapore 117576, Singapore ‡
S Supporting Information *
ABSTRACT: In situ nanoscopic observations of healthy and osteoporotic bone nanopillars under compression were performed. The structural−mechanical property relationship at the atomic scale suggests that cortical bone performance is correlated to the feature, arrangement, movement, distortion, and fracture of hydroxyapatite nanocrystals. Healthy bone comprising tightly bound mineral nanocrystals shows high structural stability with nanoscopic lattice distortions and dislocation activities. On the other hand, osteoporotic bone exhibits brittleness owing to the movements of dispersed minerals in and intergranular fracture along a weak organic matrix. KEYWORDS: Bone, osteoporosis, nanomechanics, in situ TEM indentation, fracture
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indentation have also been performed in the past decade to clarify the roles of different structures,33−35 such as lamellae/ interlamellae36 and trabecular bone.33,37 However, the macroto microscopic tools still do not adequately clarify the nanoscopic mechanical behaviors of bone, in particular the correlation to the hierarchical nanostructures comprising nanoscale hydroxyapatite crystals and collagen fibers. Moreover, elucidating the effects of diseases such as osteoporosis38 on bone structure and strength is important. For example, clarifying the correlations among and changes in bone structures, mechanical properties and deformation and fracture behaviors at the nano- or even atomic scale can not only better explain bone nanomechanics, but also have the potential of assisting in the development of therapeutic treatments of osteoporosis. Osteoporosis, which is caused by a high bone resorption rate due to a marked decrease in estrogen, typically occurs in elderly people and postmenopausal women38,39 and leads to reduced bone mineral densities (BMDs), strengths and toughness, and consequently easy fracture.8,15,39−41 Though nutritional supplements have been developed for enhancing mineral absorption,38,41−43 the influences of the changes in bone nanostructures on the nanoscopic bone deformation and fracture remain unclear. It was reported that even though BMDs were efficiently gained
one is a complex, hierarchical tissue and comprises different levels of structures, from the macroscale cortical and trabecular bones to the nanoscale bundles of mineralized collagen fibers.1,2 At the nanoscale, bone consists of calcium salts (mostly hydroxyapatites in the form of nanocrystalline flakes) grown from an organic matrix (mainly collagen fibers and few protein-based ground substance).3,4 Bone tissue combines the high strength and stiffness of hard minerals and the high toughness and viscoelasticity of compliant collagens, forming a robust natural nanocomposite material.5−8 From a microscopic viewpoint of deformation and fracture, the toughening mechanism of the hierarchical tissue to retard crack propagations5,8−12 has been suggested, dominantly by the consumptions of applied strain energy and the reduced stress intensities at crack fronts through structural deformation, crack deflections, ligament bridging, microcracking, viscoelastic deformation of organic fibers, and fiber pull-out.5,13−16 Nanoscopic sources of bone toughness have been proposed,15−24 but not verified, including fiber sliding,15,17−19 shear deformation of ground substance,17,20 breaking of sacrificial ionic bonds and extension of folded molecules,15,21−23 and uncoiling of tripocollagen molecules associated with the breaking of hydrogen bonds.24 Mechanical characterizations of bone were thus typically performed25 by macroscale tensile tests,26−28 bending tests,5,9,29,30 and also dynamic mechanical analyses31,32 to examine bone strength and stiffness as well as viscoelastic and fracture behaviors.5,8−13 Micro- to nanoscale analyses using instrumented nano© XXXX American Chemical Society
Received: July 23, 2013 Revised: September 16, 2013
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Figure 1. Sample preparations to obtain nanopillars of cortical bone of mouse tibia diaphyses for in situ TEM nanoindentations: (a) schematic illustration of bone specimens cut from tibia diaphyses, (b) FIB-cut thin bone foils from bone specimens for further nanopillar preparation; (c) FIB image (top) and TEM image (bottom) of FIB-cut bone nanopillars on bone foils, (d) schematic illustration of in situ TEM nanoindentations of nanopillars using a flat indenter.
Figure 2. Micro- and nanostructures of cortical bone of mouse tibia diaphyses (a−d: sham mice, e−h: OVX mice): (a, e) TEM bright-field (left) and dark-field images (right) of FIB-cut bone nanopillars, (b, f) high-resolution TEM images (with SAD patterns) of ultrathin bone slices, (c, g) lattice images, (d, h) magnified FFT lattice images (marked regions R1 and R2 around crystals in c, g; dashed lines: differently oriented lattices, CB: crystal boundary, AR: amorphous region).
cortical bone of sham mice comprised long collagen fibers and surrounding mineral nanocrystallite flakes of similar orientation,3,4 whereas the bone of OVX mice exhibited an obvious structural change, under the influences of osteoporosis, with randomly distributed short collagen fibers and loosely dispersed particulate crystals. As seen in the X-ray microcomputed tomography scanning (micro-CT) 3D images of the cortical and cancellous bones (Supplementary Figure S1b,c,e,f), the osteoporotic change in the bone structure of the OVX mice with loose and thin trabecula was obvious though the cortical bone did not exhibit a significant difference. Upon osteoporosis, the BMD of the cancellous bone decreased from 0.49 (±0.03) to 0.39 (±0.02) g/cm3 (p < 0.005), and a smaller variation from 0.86 (±0.01) to 0.76 (±0.02) g/cm3 (p < 0.005) was noticed for the cortical bone, near the values of femur bone.41,45 Micromechanical analyses showed that, by static nanoindentation tests, the elastic modulus and hardness of the tibia cortical bone of the sham mice were determined as 27.9 (±1.73) and 0.89 (±0.07) GPa, respectively, and those of the OVX mice significantly dropped to 18.0 (±2.24) and 0.61 (±0.12) GPa (p < 0.005), in good agreement with literature.41,46 Around the heavily indented regions on cortical bone surfaces (Supplementary Figure S2), two distinct fracture behaviors were observed by atomic force microscopy (AFM),
via clinical treatment of osteoporosis, the bone still exhibited low fracture resistance to small impact.44 In this study, in addition to the characterization of BMDs, nanostructures and mechanical properties and fracture behaviors of bone obtained via nanoindentation tests for healthy (normal, sham) and osteoporotic (variectomized, OVX) mice, we performed in situ nanoscopic examinations of deformation and fracture of bone under indentations using transmission electron microscopy (TEM) to directly clarify the roles of bone nanostructures and the effects of osteoporosis. Cortical bone nanopillars were prepared by focus ion beam (FIB) cutting, and an in situ TEM nanoindentation test of the bone nanopillars was performed, as illustrated in Figure 1 and in Materials and Methods (Supporting Information). For the first time, this technique has helped us to elucidate how bone mechanics is intimately related to the feature, arrangement, movement, distortion, and fracture of hydroxyapatite nanocrystals packed in an organic matrix in the load-bearing cortical bone. Supplementary Figure S1 and Figure 2 show the micro- and nanostructures of cortical bone from the tibia diaphyses of sham and OVX mice adopted in the present study. First from the biological TEM images (Supplementary Figure S1a,d) and the TEM bright- and dark-field analyses of the FIB-cut bone nanopillars (Figure 2a,e), microscopically, it was found that the B
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Figure 3. In situ TEM observation of deformation and fracture of cortical bone nanopillar of sham mouse tibia diaphysis under nanoindentation (maximum indentation displacement of 400 nm, loading rate of 16 nm/s; arrows: moving directions of indenter tip): (a) at the contact of indenter tip with bone nanopillar, (b, c) compression and buckling of bone nanopillar, (d) fracture of bone nanopillar, (e, f) further flexure of fractured nanopillar fragment under continual indentation, (g, h) rebound of nanopillar fragment during indenter tip removed.
and performance between the healthy and osteoporotic groups of mice are comparatively valid. Thus one wonders if the cortical bone performance is not only or simply correlated to BMDs, but also to the simultaneous, substantial changes in the features and arrangements of mineral crystals observed from the nanoscopic examinations. Figure 2b−d and f−h further show the high-resolution TEM images (with selected-area diffraction (SAD) patterns) and lattice images of tibia cortical bone of sham and OVX mice at the nanoscale. Polycrystalline, polygonal minerals with sizes of about 40 nm were densely packed in the cortical bone of the sham mice. From the magnified and fast Fourier transformed (FFT) lattice images in Figure 2c,d (marked region R1 in c), mineral grains were actually composed of ultrafine nanocrystallites of only 5−10 nm in size; the interplanar spacings of 0.26 and 0.28 nm corresponded to the (202) and (211) lattice planes of hydroxyapatite, respectively. For the sham mice, the composing nanocrystallites were tightly adhered to each other with very thin crystal boundaries (CBs); some of them were aligned or preferentially oriented with small-angle (about 6°) mismatches. In comparison, differently featured (particulate or spherical) minerals were randomly dispersed in the bone of the OVX mice. A relatively large fraction of amorphous region (AR, possibly filled with fat or protein-based ground substance,49 as the diffused rings noticed in the SAD pattern) surrounded the mineral nanocrystals; as identified in Figure 2g,h (marked region R2 in g), most of the nanocrystals exhibited different lattice orientations of high angles (about 25°). Under the fast and continual bone resorption upon osteoporosis, bone mineral loss occurred preferentially at the interfaces of mineral nanocrystals and around collagen fibers. Even after remodeling with gained BMDs,44 regenerated hydroxyapatites hardly formed packed and aligned small-angle grains but dispersed particulate crystals with varied orientations of high angles on randomly distributed short collagen fibers within a certain volume of amorphous organic matrix.49 The exploration of lattice and crystalline deformation in loadbearing cortical bone and the structure-mechanical response relationship of healthy and osteoporotic bones, at the nano- to
scanning electron microscopy (SEM), and TEM. To the sham mice, blunt cracks and retarded propagations, with a wide deformation zone, by microcracking, crack deflection, fiber pullout, and the ligament bridging of mineralized fibers similar to the reported microscale toughening mechanisms,5,13−15 were clearly found and yielded a high bone strength and fracture resistance.5−12,15,24,47,48 By contrast, to the OVX mice with lack of these intrinsic or extrinsic toughening mechanism to depress the high stress intensities at crack fronts,15,48 sharp cracks and straight propagations with a relatively narrow deformation zone and brittle fracture with broken fibers and mineral debris were observed.16,41,47,48 It is well-elucidated that a marked decrease in estrogen, typically found in elderly people and postmenopausal women, leads to imbalanced osteoblast and osteoclast activities, and thereafter a high bone resorption rate and eventually osteoporosis38,39 which causes low strength and easy fracture of bone.8,15,39−41 To diagnose osteoporosis and to understand the effectiveness of therapeutic treatments, BMDs have been well-adopted to correlate between BMDs and bone strengths39−41,44 and that the elastic modulus of cortical bone depends mainly on BMDs and less on the cross-linking of ground substance because of a much higher stress concentration on stiff minerals.26 Nevertheless, a concern has been raised that though BMDs are efficiently gained via clinical treatments, the bone still exhibits low fracture resistance to small impact.44 As also examined above, BMDs may be markedly different for cancellous bone but much less for the load-bearing cortical bone when compared between healthy and osteoporotic bones. However, a minor loss (by only 10%) in the BMDs of the cortical bone resulted in a much significant drop (by 40%) in mechanical properties and a ductile-to-brittle transition in fracture behaviors (Supplementary Figure S2). Structural and nanomechanical heterogeneity on the variability of strength and fracture toughness of bone needs to be considered.15,35 However, the variability of bone properties measured parallel to the longitudinal direction is expected to be smaller than that from the transverse direction.35 Also, from the statistical analyses (p < 0.005), the differences in bone density C
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Figure 4. In situ TEM observation of deformation and fracture of cortical bone nanopillar of OVX mouse tibia diaphysis under nanoindentation (maximum indentation displacement of 400 nm, loading rate of 16 nm/s; arrows: moving directions of indenter tip): (a) at the contact of indenter tip with bone nanopillar, (b, c) compression and buckling of bone nanopillar, (d) fracture of bone nanopillar, (e, f) further flexure of fractured nanopillar fragment under continual indentation, (g, h) rebound of nanopillar fragment during indenter tip removed.
Figure 5. In situ TEM observation of deformation of cortical bone nanopillars of mouse tibia diaphyses under nanoindentation: (a, b) TEM brightfield images of bone nanopillar of sham mouse before and after indentation (maximum indentation displacement of 200 nm, loading rate of 8 nm/s), (c, d) TEM bright-field images of bone nanopillar of OVX mouse before and after indentation (displacement of 200 nm, loading rate of 8 nm/s); (e) indenting stress−displacement curves of bone nanopillars of sham and OVX mice (only the stages before obvious pillar buckling shown; blue/green: sham mice, red/pink: OVX mice; displacement of 400/200 nm, loading rate of 16/8 nm/s, respectively; dotted lines: linear fittings at the early elastic stages).
angle only ∼5°); the fragment markedly bounced away, similar to the catastrophic fracture of brittle materials. After removing the indenter tip, the flexed pillar fragment of the sham mouse that was supported by unbroken ligaments47 rebounded elastically and markedly (from 90° back to 45°), whereas the brittle fragment of the OVX mouse rebounded much less (45° to 35°). The high ductility and flexibility of the sham mouse bone was confirmed by tests on other nanopillars (Supplementary Figure S3, bending angle ∼50°), and the structural recoverability was verified from the in situ TEM images of the nanopillars before and after indentations (Figure 5a,b; indentation displacement of 200 nm, undergoing deformation only). Except slight pillar widening, no obvious changes in bone nanostructure and component feature (flake fringes of mineralized fibers) were noticed after deformation. By comparison, the structural instability of the OVX mouse bone
even atomic scales, examined below by the in situ highresolution TEM nanoindentations (Figures 3−6, Supplementary Figure S3, Videos S1 and S2), yield important insights into their overall integrity and performance. Clearly, because of the different features and architecture of bone components, the cortical bone nanopillars of healthy and osteoporotic mice respond to stresses in different ways from both deformation and fracture aspects, as observed from the images captured at different stages (Figures 3 and 4, the time at indenter tip−pillar contacts reset to zero). At the contacts, the pillars were simply compressed but soon began to buckle. The densely packed and fiber-toughened bone pillar of the healthy sham mouse buckled more considerably (bending angle ∼30°) before fracture; the bending angle of the fractured pillar fragment remained unchanged. In comparison, the bone pillar of the OVX mouse abruptly fractured early with less buckling (bending D
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Figure 6. In situ TEM fractographies of cortical bone nanopillars of mouse tibia diaphyses under nanoindentation (a−d: sham mice, e−h: OVX mice; maximum indentation displacement of 400 nm, loading rate of 16 nm/s): (a, e) TEM bright-field images of bone nanopillars around fractures, (b, f) magnified TEM images, (c, g) lattice images around cracks, (d, h) magnified lattice images (marked regions R1 and R2 beneath cracks in c, g); solid arrow lines: crack propagations, dashed lines in d: distorted lattices, circled: dislocations, dashed lines in h: differently oriented lattices, AR: amorphous region.
Figure 6 shows the in situ TEM fractographies of the bone pillars of sham and OVX mice after nanoindentations of 400 nm and having undergone fracture. Though the pillars underwent buckling rather than pure compression, the fracture from the tensile side of the bent pillars was more similar to the typical failure of bone under impact. For the sham mouse bone, a ductile fracture with tissue tearing was observed (Figure 6a,b), while for the OVX mouse bone, a brittle, catastrophic fracture with multiple crack propagation routes was seen (Figure 6e,f). Figure 7 schematically illustrates the nanoscopic deformation
was identified (Figure 5c,d); the movements of bone components (e.g. the circled dark spot of particulate mineral) to another location for a distance of 10 nm and from an angle of 20° to a different angle of 22° (relative to the dashed line and the surface particle) were observed. For the sham mouse bone, the aligned, strongly bound mineral nanocrystals were expected to consume applied energy through their elastic deformation.17,24,34,50 However for the OVX mouse bone, the randomly dispersed mineral crystals within a weak organic matrix underwent with substantial nanoscopic movements and rotations under small stresses, which resulted in the difference in the in situ TEM nanoindenting stress−displacement curves of bone nanopillars as presented in Figure 5e (only early stages before obvious pillar buckling shown; the responses after yielding ignored due to a difference in buckling caused by pillar geometry). The bone nanopillars of the sham mice were found to exhibit a much higher stiffness of about 1.6 times that of the OVX mice (sham 12.6 MPa/nm and OVX 8.1 MPa/nm at a loading rate of 16 nm/s; sham 8.8 MPa/nm and OVX 5.6 MPa/nm at a rate of 8 nm/s). Additionally, the pillar stiffness increased by 1.4 times with a high loading rate because of the effect of strain rate sensitivity on mechanical measurements.28 The drop in the stiffness of the OVX mouse bone by 36%, close to the drop in static mechanical properties by 40%, was not just attributed to the decrease in BMD by only 10% but was also believed to be importantly correlated to the simultaneous changes in the feature and arrangement of bone components at the nanoscale. According to the estimations by using Halpin−Tsai equations51 (see Supporting Information), though the portion of the regenerated organic matrix in the osteoporotic bone was minor, for only 12 wt %, the changes in bone architecture and the role of minerals (from a continuous matrix to one of dispersed reinforcements) would lead to an inefficiency in load transfer and thus a markedly decrease in elastic modulus by more than 60%.
Figure 7. Schematic illustrations of nanoscopic deformation and fracture of mouse cortical bone nanocomposite structures under nanoindentation (close to a bending mode, around crack fronts at a tensile side): (a) sham mice with aligned long collagen fibers embedded in a tightly adhered polygonal mineral matrix with smallangle boundaries; (b) OVX mice with randomly oriented short fibers and particulate minerals distributed in a weak, amorphous organic matrix of ground substance (CF: collagen fiber, HA: hydroxyapatite mineral, AR: amorphous region; arrows: tensile stresses).
and fracture of bone nanocomposite structures. As illustrated in Figure 7a, besides conventional toughening mechanisms including the observed fiber/mineral debonding and unbroken ligament bridging5,13−16 and the literature-proposed fiber stretching, sliding, and friction,15,20,24,50 considerable nanoscopic plastic deformation of the matrix mineral crystals also occurred, as indicated in the lattice images of a decohered mineral grain beneath a crack (Figure 6c,d). Severe lattice E
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straightening, were reported to consume a large part of applied strain energy for the improvement of bone toughness.15,20,24,51 However, the decrease in the mineralization density of collagen fibers, in consequence of repeated nanoscale displacements of bone components, would lead to the formation of porosity and microcracking at the microscale.15,17 Upon osteoporosis, the loss of minerals and the change of collagen cross-linking density at the nanoscale, and the weakened bond strengths between bone components (inter/intraminerals, collagens, and ground substance) at the atomic/molecular scale, are expected to result in the easy displacements of bone components at the nanoscale and the rapid propagations of cracks at the microscale,15,17,23,52,53 as has been observed in the study. However, further investigations on the exact fracture correlations among the different-scale structures of the hierarchical bone tissue are still needed. In summary, the nanoscale deformation and fracture of sham and OVX mouse bones as well as the correlation between the changes in bone structures and the consequent decline in mechanical properties upon osteoporosis were addressed for the first time at the nano and atomic scales via in situ TEM observations of cortical bone nanopillars undergoing compression. We showed that the osteoporotic change in bone nanostructure obtained from micro-CT analyses and BMDs was more significant for cancellous bone than that for loadbearing cortical bone. The structural integrity and mechanical performance of cortical bone can be obtained from understanding of the feature, arrangement, movement, distortion, and fracture of inorganic hydroxyapatite nanocrystals located within an organic matrix. Thus, understanding bone mechanics at the nanoscale can give important insights into how the mechanical response of osteoporotic cortical bones can differ from that of healthy bones.
distortions and dislocation formation which have been seen in typical material deformation but not found in previous microscopic studies of bone deformation also consume a part of applied strain energy and provide the normal bone tissue a high toughness. In comparison as illustrated in Figure 7b, the osteoporotic nanocomposite structure (with randomly oriented short fibers and particulate minerals distributed in an amorphous matrix of ground substance), a nanoscopic intergranular fracture along the organic matrix between the mineral crystals of high-angle orientations was observed (Figure 6g,h). Suggested dominant factors for the brittle fracture and the substantial decrease in toughness include: (1) the nanoscopic movements, rather than lattice distortions and plastic deformation, of the dispersed mineral crystals due to lack of strong bonding (Figure 5), (2) the increase in the crosslinking of nonenzyme-type proteins upon osteoporosis which result in the brittleness of the ground substance,15,50 and (3) most importantly, the change in the roles of bone components, i.e., the weak organic ground substance playing the role of the matrix. Accordingly, rapid and preferential crack propagations through the weak matrix of high strain concentrations and low fracture toughness inactivate other toughness sources,51 provided by the short fibers, the discontinuous minerals and the interfaces, leading to the brittle fracture of the osteoporotic bone. Other than BMDs, cortical bone performance can also be influenced by the changes in nanostructures and can be correlated to the feature and arrangement, and the consequent movement, distortion, and fracture of inorganic hydroxyapatite nanocrystals within an organic matrix. This shows that a weakened binding of bone components and an ineffective load transfer in the “trabecularized cortical bone” will lower the fracture resistance of bone even with efficiently gained BMDs.44 It is proposed but not verified yet that, from an atomic/ molecular perspective of the hierarchical structure of bone, the rotations of collagen side chains, the reversible breaking of hydrogen bonds and ionic bonds in/between proteins, the straightening and uncoiling of tripocollagens, and the stretch and displacement of collagen fibers play important roles in the ductile deformation and fracture of healthy bone.15,21−24 Mineral nanocrystals that nucleate in the gap regions of collagen fibers bond to the fibers by the electrostatic interactions of Ca2+, PO43−, and OH− of hydroxyapatites with lysine (+), arginine (+), glutamic acid (−), and aspartic acid (−) of collagens to form salt bridges for an effective load transfer and energy dissipation.52 Because the present in situ tests were carried out with formaldehyde-fixed and dehydrated bone pillars in a dry environment, the stiffness of collagen fibers was considered to increase (from 0.3−1.2 to 1.8−2.25 GPa) due to an increased arrangement density of collagen fibers with a decreased moisture content of bone tissues.26,50 Nevertheless, a previous study suggests that the elastic modulus of dehydrated whole bone just slightly increases (from 11.5 to 13.9 GPa), and the strain of collagen fibers does not decrease much (from triple to twice the strain of minerals).26 In the fixed, dry tissues, except for the loss of hydrogen bonds and ionic bonds in proteins, the other dominant toughening mechanisms provided by minerals and collagens (ex. molecule straightening) remain functional.26,50,53 Hence, the comparative studies between the healthy and the osteoporotic bones using dry samples will still be meaningful. At the nanoscale, the interface debonding, friction, and displacement of hydroxyapatites, collagen fibers, and ground substance which are dependent on the atomic-scale bond breaking and molecule
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ASSOCIATED CONTENT
S Supporting Information *
Materials and methods, estimation of bone elastic modulus, references and notes, the supplementary figures of bone microstructures, static nanoindenting fractographies and in situ TEM deformation and fracture of bone nanopillars, and the supplementary videos of in situ TEM deformation and fracture. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Phone: +886-422857517. Fax: +886-4-22857017. *E-mail:
[email protected]. Phone: +65-65167801. Fax: +6567791459. Author Contributions
Y.-T.W. and S.-Y.C. contributed equally. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully thank the financial support for this research by the National Science Council, Taiwan, under Grant Nos. NSC-99-2221-E-005-095-MY2 and NSC-100-2628-E-005006-MY3 and in part by the Ministry of Education, Taiwan, under the ATU plan. F
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