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Probing the role of water in lamellar bone by dehydration in the environmental scanning electron microscope
Abstract
Water, collagen and mineral are the three major components of bone. The structural organization of water and its functions within the bone were investigated using the environmental scanning electron microscope and by analyzing dimensional changes that occur when fresh equine osteonal bone is dehydrated and then rehydrated. These changes are attributed mainly to loss of bulk and weakly bound water. In longitudinal sections a contraction of 1.2% was observed perpendicular to the lamellae, whereas no contraction occurred parallel to the lamellae. In transverse sections a contraction of 1.4% was observed both parallel and perpendicular to the lamellae. SEM back scattered electron images showed that about half of an individual lamella is less mineralized, and thus has more water than the other half. We therefore propose that contractions perpendicular to lamellae are due to the presence of more water-filled rather than mineral-filled channels within the mineralized collagen fibril arrays. As these channels are also aligned with the crystal planes, the crystal arrays, oriented as depicted in the rotated plywood model for lamellar bone, facilitate or hinder contraction in different directions.
... The 23 wt% collagen fibrils and the 65 wt% mineral are hydrated by 12 wt% bone fluid [5], [12], [16]- [20], present in the gaps within the fibril, between the fibrils and the fibers [13], [21]- [26]. The collagenous layers [8], [2], [23], the level of mineralization as well as the mineral plate orientation [27]- [28] determine bone mechanical behavior as displayed in the nanoindentation studies [29]- [31]. ...
... The dehydration caused dimensional changes in bone have been studied in the environmental scanning electron microscope (ESEM) as wet, dry and rehydrated samples [8]. The results of that study have indicated that in the longitudinal (Plane 2 in Fig. 1) and transverse (Plane 1 in Fig. 1) cross-sections of bone, if the specified length of a given segment is measured perpendicular and parallel to the lamellae before and after dehydration, the difference in length varies as a function of the orientation of that segment. ...
... and tangentially 1.41±0.57% change in dimension has been observed at 28% relative humidity under the ESEM [8]. The axial contraction results have displayed a high standard deviation and a wide range (-4.64% to 4.52%). ...
In this study, the effect of orientation of mineralized collagen fibrils on bone mechanical properties relating to bone anisotropy was studied using data obtained from rehydrated lamellar bone samples. The dehydration-rehydration based and experimentally determined contraction, observed in orientations parallel and perpendicular to the osteonal axis was used to calculate bone anisotropy. The sublamellar bone model, with the layered mineralized collagen fibrils rotating at 5° was used. Following this model, the mineralized collagen layers were transformed at 5° relative to the orthogonal axes using a transform matrix. With dehydration, fibril diameter was reduced towards the mineral, forming contraction vectors. The x, y and z intercepts for these vectors were then calculated to give the u, v and w displacements, which gave anisotropy ratios ranging from 0.15266 to 6.55054. Compared with the experimental nanoindentation findings in the literature, there may be an indication of a correlation with the results of sublamellar arrangement at 20° angles. As the lateral indentation used in the anisotropy experiments may involve varying amounts of u and v displacements, the aspect angle of lateral indentation was evaluated in relation to the structural features of the model. This evaluation indicated the larger contribution of v displacement and thus relatively much smaller contribution of u displacement to lateral contraction. These findings indicate the significant effect of the mineralized collagen fibril arrangement on bone anisotropy.
... Bone is a hierarchically structured, anisotropic, fiber-reinforced natural composite, consisting mainly of organic (Type I collagen, glycosaminoglycans (GAGs) and non-collagenous proteins) and inorganic (carbonated hydroxyapatite (CHA)) components within a hydrating medium (Robinson and Watson, 1952;Ascenzi and Bonucci, 1967;Nomura et al., 1977;Weiner and Wagner, 1998;Cowin, 2002;Ziv et al., 1996;Fratzl and Weinkamer, 2007;Utku et al., 2008;Reznikov et al., 2014a). ...
... There are differing views on the constituents of lamellar organization of mineralized collagen fibrils. Based on scanning electron microscope (SEM) images, lamellar bone may be organized as rotated plywood-like collagen fibril layers with uniform density (Giraud-Guille, 1988), or as alternating collagenrich and collagen-poor layers (Marotti, 1993), or, based on environmental scanning electron microscope (ESEM) and back-scattered electron (BSE) microscope images, as uniform density fibrils appearing as more or less concentrated zones of mineralization within a lamella (Utku et al., 2008;Reznikov et al., 2014a). ...
... Studies on the mineralized and non-mineralized wet and dry collagen fibrils display lateral contraction on the transverse plane with comparatively less change in the axial direction (Eanes et al., 1976;Bonar et al., 1985;Fratzl et al., 1993;Ascenzi, 1999;Utku et al., 2008;Currey, 2002). In transverse sections of lamellar bone, radial (v) and tangential (u) contraction ( Fig. 1) are thought to be due to not only the orientation of the humidity-responsive structural layers arranged parallel to the lamellae (Utku et al., 2008;Eanes et al., 1976;Ascenzi and Bonucci, 1967), but also the extent of mineralization and mineral plate orientation (Fois et al., 2001;Utku et al., 2008). ...
In this study, the effect of dehydration-hydration based dimensional change in the sublamellae on bone anisotropy was used as a tool to understand sublamellar organization of mineralized collagen fibrils. Bone consists of hydroxyapatite, Type I Collagen, mucopolysaccharides and bone fluid, which associates with bone constituents and improves the mechanical properties of bone. Knowing that dehydration causes dimensional changes comparable to those observed in the mechanical testing of a bone sample, here, the dehydrated organic component of lamellar bone was modelled to contract towards the mineral, forming a contraction vector as the surface normal of the mineral plate. The amount of dehydration based contraction in rotated collagen fibrils was calculated for two models of sublamellar arrangements, namely A and B, where the mineral plate of the 0° (axial, [001]) sublamellar collagen fibril was oriented along either (010) or (001) planes. Projections of sublamellar contraction vectors were denoted as u, v and w displacements at 10°-20°-30° angles and summed to give the lamellar total. Using the total displacements, anisotropy ratios of properties in directions parallel (w) versus perpendicular (u or v) to the osteonal axis were calculated. With dehydration, the osteonal lamellae in Model A (behaving as positive Poisson’s ratio material) may display maximal planar expansion (at 1.4%) and peraxial contraction (-0.24%), which may even cause sample warping. Correlation of only some of the wet and dry bone anisotropy ratios of the models with the literature demonstrates the effect of collagen orientation on bone mechanics.
... As biomineralization in bone occurs by replacing water, bone with low mineral content will contain more water than highly mineralized bone. Dehydration processes will therefore cause more structural, and thus mechanical, changes in bone with relatively low mineral content (Currey, 1999a;Utku et al., 2008). While the studies detailed above examine the dehydration of bone and the corresponding effects on its mechanical behaviour, little work has been done to examine the effects of vacuum conditions on bone structure. ...
... While the studies detailed above examine the dehydration of bone and the corresponding effects on its mechanical behaviour, little work has been done to examine the effects of vacuum conditions on bone structure. Observation of water loss from various regions resulting in dimensional contractions in bone has been directly observed by environmental scanning electron microscopy (Utku et al., 2008) but the corresponding effects on mechanical behaviour have been lacking. The evaluation of bone mechanics using techniques involving SEM has distinct advantages compared to previous works. ...
... Previous literature has highlighted the use of focused ion beam (FIB) microscopy to isolate micron-sized cantilevers from teeth for subsequent bending tests (Chan et al., 2009). The dual beam system setup allows FIB technology to be combined with the structural analysis of a scanning electron microscope (SEM) (Utku et al., 2008). The capacity to isolate discrete bone volumes within a dual beam system using the FIB and observe structural changes using SEM is therefore persuasive but mechanical testing capability within the SEM is required. ...
The lamellar unit is a critical component in defining the overall mechanical properties of bone. In this paper, micro-beams of bone with dimensions comparable to the lamellar unit were fabricated using focused ion beam (FIB) microscopy and mechanically tested in bending to failure using atomic force microscopy (AFM). A variation in the mechanical properties, including elastic modulus, strength and work to fracture of the micro-beams was observed and related to the collagen fibril orientation inferred from back-scattered scanning electron microscopy (SEM) imaging. Established mechanical models were further applied to describe the relationship between collagen fibril orientation and mechanical behaviour of the lamellar unit. Our results highlight the ability to measure mechanical properties of discrete bone volumes directly and correlate with structural orientation of collagen fibrils.
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... Nomura et al. (1977) identified four types of water: two nonfreezable, corresponding to the structural and bound water, and two freezable, corresponding to the unbound bulk water. Moreover, recent studies suggest that the amount of water varies between the different lamellar sublayers (Reznikov et al., 2012;Utku et al., 2008), raising new questions about the role of water on local structure and properties. ...
... Structurally, recent studies suggest (Reznikov et al., 2012) that fibrils have denser mineralized packing in dry samples than in high pressure frozen samples, however, their orientation and organization remains similar. Utku et al. (2008) measured the shrinkage of osteonal lamellae in different directions, comparing environmental SEM images under 100% humidity and dry conditions. They found a contraction of 1.2% perpendicular to the lamellae but no contraction parallel the lamellae, in longitudinal sections. ...
... In that case, the change in fiber orientation from wet to dry state is the mechanism responsible for pods opening. In bone, the hypothesis of a change in fibril orientation upon hydration is strengthened in light of the findings of Utku et al.(2008). According to their study, whereas in plane 2 (they use the term "longitudinal section") the drying contraction is different in parallel and perpendicular directions to the lamellae, in plane 1 (they use the term "transverse section") it is equal in all directions. ...
The effect of hydration on the mechanical properties of osteonal bone, in directions parallel and perpendicular to the bone axis, was studied on three length scales: (i) the mineralized fibril level (∼100 nm), (ii) the lamellar level (∼6 µm); (iii) the osteon level (up to ∼30 µm).We used a number of techniques, namely atomic force microscopy (AFM), nanoindentation and microindentation. The mechanical properties (stiffness, modulus and/or hardness) have been studied under dry and wet conditions. On all three length scales the mechanical properties under dry conditions were found to be higher by 30–50% compared to wet conditions. Also the mechanical anisotropy, represented by the ratio between the properties in directions parallel and perpendicular to the osteon axis (Anisotropy Ratio, designated here by AnR), surprisingly decreased somewhat upon hydration. AFM imaging of osteonal lamellae revealed a disappearance of the distinctive lamellar structure under wet conditions. Altogether, these results suggest that a change in mineralized fibril orientation takes place upon hydration.
... As biomineralization in bone occurs by replacing water, bone with low mineral content will contain more water than highly mineralized bone. Dehydration processes will therefore cause more structural, and thus mechanical , changes in bone with relatively low mineral content (Currey, 1999a; Utku et al., 2008). While the studies detailed above examine the dehydration of bone and the corresponding effects on its mechanical behaviour, little work has been done to examine the effects of vacuum conditions on bone structure. ...
... While the studies detailed above examine the dehydration of bone and the corresponding effects on its mechanical behaviour, little work has been done to examine the effects of vacuum conditions on bone structure. Observation of water loss from various regions resulting in dimensional contractions in bone has been directly observed by environmental scanning electron microscopy (Utku et al., 2008) but the corresponding effects on mechanical behaviour have been lacking. The evaluation of bone mechanics using techniques involving SEM has distinct advantages compared to previous works. ...
... Previous literature has highlighted the use of focused ion beam (FIB) microscopy to isolate micron-sized cantilevers from teeth for subsequent bending tests (Chan et al., 2009). The dual beam system setup allows FIB technology to be combined with the structural analysis of a scanning electron microscope (SEM) (Utku et al., 2008). The capacity to isolate discrete bone volumes within a dual beam system using the FIB and observe structural changes using SEM is therefore persuasive but mechanical testing capability within the SEM is required. ...
The mechanical properties of rat bone at micron length scales have been evaluated as a function of environmental conditions using an in situ atomic force microscope (AFM) setup while observing using scanning electron microscopy (SEM). Focused ion beam fabricated rat bone cantilever samples were tested in both low and high vacuum conditions in the SEM as well as wet in air using the AFM to measure their elastic modulus. The elastic modulus of rat bone at micron length scales is shown to be independent of the environmental testing conditions and indicates water is bound to bone material even under relatively high vacuum conditions. Our work therefore shows how in situ mechanical testing of bone while observing using high resolution SEM can provide results similar to testing wet in air.
... A similar mechanical heterogeneity in the cortical bone has also been observed by other micro-mechano-sensitive techniques, such as nano-indentation and scanning acoustic microscopy, but the explanation about the origin of this modulation is still unclear. In fact, in some works, it has been ascribed to the presence of arrays of mineralized bundles with different orientations [16,47], while, in other works, it has been attributed to alternative lamellae with different degrees of mineralization or to an alternate degree of packing of mineralized fibrils in the bundles (the so-called dense-loose motif) [48][49][50][51]. The contrast between these different interpretations is the cause of an objective difficulty in elaborating an exhaustive model of the osteon's lamellar microstructure. ...
... An extremely complex scenario in the mechanical properties of the mineralized matrix was revealed-a scenario that is induced by the anisotropy of the micrometric lamellar pattern of cortical bone. The combined Brillouin and Raman analyses, supported by EDX elemental analysis, show that the modulation of the elastic modulus in the lamellar pattern is not due to a different degree of mineralization, as proposed in previous mechanical investigations [50,51], but rather to the oscillating orientation of bundles when passing through adjacent lamellae, thus supporting the notion previously suggested by scanning acoustic microscopy [16]. Finally, the appearance of transverse acoustic modes in the Brillouin spectra, which can be ascribed to the anisotropy of the mineralized bundles, can considerably improve the elastic characterization of the tissue. ...
Bone has a sophisticated architecture characterized by a hierarchical organization, starting at the sub-micrometre level. Thus, the analysis of the mechanical and structural properties of bone at this scale is essential to understand the relationship between its physiology, physical properties and chemical composition. Here, we unveil the potential of Brillouin-Raman microspectroscopy (BRaMS), an emerging correlative optical approach that can simultaneously assess bone mechanics and chemistry with micrometric resolution. Correlative hyperspectral imaging, performed on a human diaphyseal ring, reveals a complex microarchitecture that is reflected in extremely rich and informative spectra. An innovative method for mechanical properties analysis is proposed, mapping the intermixing of soft and hard tissue areas and revealing the coexistence of regions involved in remodelling processes, nutrient transportation and structural support. The mineralized regions appear elastically inhomogeneous, resembling the pattern of the osteons' lamellae, while Raman and energy-dispersive X-ray images through scanning electron microscopy show an overall uniform distribution of the mineral content, suggesting that other structural factors are responsible for lamellar micromechanical heterogeneity. These results, besides giving an important insight into cortical bone tissue properties, highlight the potential of BRaMS to access the origin of anisotropic mechanical properties, which are almost ubiquitous in other biological tissues.
... During the past decade, several innovative surface engineering approaches, which are based on our understanding of the complex hierarchical design and diverse functionality of bone and dental tissues, have been developed (Fratzl and Weinkamer, 2007;Aizenberg et al., 2005;Sarikaya et al., 2003). The surface engineering approaches include various deposition-based methods, eg, mineral coatings Zhang et al., 2009;Leeuwenburgh et al., 2006;Shirkhanzadeh, 1998;Munch et al., 2008;Deville et al., 2006a;Lowenstam, 1981;Addadi and Weiner, 1992;Niemeyer, 2001;Woesz et al., 2006;Gungormus et al., 2012;Utku et al., 2008). Among these approaches, solid-binding peptides, ie, peptides that have affinity and selectivity for the inorganic surfaces, have attracted increasing interest. ...
... Bone, a product of vertebrate biomineralization, is a hierarchically structured, graded composite material of high complexity (Reznikov et al., 2014;Weiner and Wagner, 1998;Fratzl et al., 2004). Bone consists of type I collagen, extracellular matrix (ECM) proteins and molecules, glycoaminoglycans, and noncollagenous proteins, carbonated HA, and water (Weiner and Traub, 1986;Cowin, 1999;Nomura et al., 1977;Robinson, 1952;Utku et al., 2008). Modeling of bone as a hierarchically organized material involves the complexity of a nine-level scheme (Reznikov et al., 2014). ...
Biological structures have been a constant source of inspiration for the design of practical materials and systems. With a growing understanding of the processes involved, biological principles are now revisited as novel routes in materials assembly and fabrication for various technological applications. In Nature, molecular recognition is the means to guide the organization of hierarchical structures through self-assembly; self-assembly is achieved through the precise interactions of components with the surrounding molecules. The development of robust molecular tool kits for designing, engineering, and controlling the orientation, and the confinement of the biomolecules on the desired material surfaces can be a game changer in the next generation of biomaterials. Inspired by nature's elegant hierarchical organization, we mimic the molecular-scale interactions that are built upon molecular recognition and self-assembly at surfaces and interfaces. Our approach incorporates solid material–binding peptides that are engineered to have affinity and specificity to target inorganic surfaces. Using engineered peptides, we create self-organized surfaces and interfaces starting at the molecular scale. In this chapter, we summarize solid-binding peptide-based approaches specifically for restorative biomaterials. Starting from their design, we provide various examples of the application of these approaches. We also discuss the use of engineered peptides as chimeric molecules that link solid recognition ability to the existing repertoire of bioactive peptides, integrin-binding domains, as well as proteins, eg, green fluorescence, protein to monitor mineralization. The chapter ends with a future perspective on the utilization of peptide engineering approaches to design smart biomaterials–biomaterials that restore dental tissues by providing self-healing at the interface.
... Experiments using quantitative backscattered electron imaging (qBEI) and nanoindentation [19] suggested locally varying mineral contents in osteonal lamellae and concluded that compositional variations may contribute to the oscillating lamellar mechanical properties, as well. Similarly, intralamellar variations of the mineralization were observed in a study based on backscattered electron microscopy [21]. Contrary to these propositions, experimental evidence on the homogeneity of the lamellar composition was presented by means of polarized Raman spectroscopy (PRS) [4,22,23] and synchrotron X-ray phase nano tomography (SR-nanoCT) [14,24]. ...
... A comparative parameter profile analysis of several adjacent interstitial lamellae in two interstitial domains (Figs. 5, S5) confirmed the compositional markers MTMR, CTPR, crystallinity, and the local mass density to be independent of lamellar modulations. In contrast to our findings, others [19,21] reported slight lamellar modulations of the calcium content in human lamellar bone by means of qBEI. However, the orientation distribution of calcium-containing apatite crystals in the intraand extrafibrillar spaces may have an impact on the amplitude of the backscatter signal, which should be further investigated in future studies. ...
Statement of significance:
Bone is a complex structured composite material consisting of collagen fibrils and mineral particles. Various studies have shown that not only composition, maturation, and packing of its components, but also their structural arrangement determine the mechanical performance of the tissue. However, prominent methodologies are usually not able to concurrently describe these factors on the micron scale and complementary tissue characterization remains challenging. In this study we combine X-ray nanoCT, polarized Raman imaging and scanning acoustic microscopy and propose a protocol for fast and easy assessment of predominant fibril orientations in bone. Based on our site-matched analysis of cortical bone, we conclude that the elastic modulations of bone lamellae are mainly determined by the fibril arrangement.
... 36 37 38 39 1. Introduction 40 The material bone is actually a family of mineralized connective 41 tissues that share some common structural characteristics and ful- 42 fil diverse mechanical functions. A characteristic elementary unit 43 of bone is the mineralized collagen fibril. The mineralized fibril, 44 together with non-collagenous proteins and water, arranged in a 45 complex hierarchical structure, are ultimately responsible for the 46 mechanical properties of the material [1][2][3]. ...
... 3). This indicates 225 that, even in mineralized bone, liquid water is present in the colla-226 gen channels[43]. Unbound water is presumably present in the 227 canaliculi, lacunae and blood vessels. For an overview of water in 228 bone and in mineralization, see Ref.[44].229 ...
Bone is a complex hierarchically structured family of materials that includes a network of cells and their interconnected cell processes. New insights into the 3D structure of various bone materials (mainly rat and human lamellar bone and minipig fibrolamellar bone) were obtained using the focused ion beam electron microscope and the serial surface view method. These studies revealed the presence of two different materials: the major material being the well-known ordered arrays of mineralized collagen fibrils and associated macromolecules, and the minor component being a relatively disordered material composed of individual collagen fibrils with no preferred orientation, crystals inside and possibly between fibrils and extensive ground mass. Significantly the canaliculi and their cell processes are confined within the disordered material. Here we present a new hierarchical scheme for several bone tissue types that incorporates these two materials. The new scheme updates the hierarchical scheme presented by Weiner et Wagner (1998). We discuss the structures at different hierarchical levels with the aim of obtaining further insights into structure-function related questions, as well as defining some remaining unanswered questions.
... İnnovatif fonksiyonel biyoteknolojik kompozit nanomalzemeler ve sistemlerin tasarım ve üretimi biomineralizasyon gibi biyolojik süreçlerin benzetimi yoluyla mümkündür [1]. Doğada, biyomineralizasyon mineral, organik malzeme ve suyun, hiyerarşik yapıda bir kompozit malzeme oluşturacak şekilde entegrasyonunu içerir [2]. Biyomineral kaplı implant tasarımında biyomineralizasyonun benzetim yokuyla elde edilmesi, kaplama işlemini, kaplama türünü ve biyouyumlu implant malzemesini içerir. ...
... Yeni implant malzemesi olan nanotübüler titanyum oksit plakaların CaP bileşikleriyle kaplanması zor olsa da, anataz yapıdaki titanyum oksitin CaP oluşumu için daha iyi bir temel teşkil ettiği belirtilmiştir [21]. Dalgalı elektrokimyasal çöktürme yöntemi, CaP bileşiklerinin titanyum oksit nanotüpler boyunca oluşturulabilmesi ve difüzyona ilişkin kısıtlamaları gidermek amacıyla kullanılmaya başlanmıştır [2,8,22]. Bu çalışmada, saf titanyum, anodize titanyum oksit ve nanotübüler titanyum oksit plakaların dalgalı elektrokimyasal çöktürme yöntemiyle elde edilen kalsiyum fosfat fazların morfolojilerine ve kimyasal yapılarına olan etkilerinin incelenmesi amaçlanmıştır. ...
Titanium and its alloys are used in dental and orthopaedic applications. Chemical and physical properties of implant surfaces are important determinants of implant stability and osteointegration. In this study, pure titanium, anodized titania and ordered titanium dioxide nanotubular plates were coated with calcium phosphate using a modified SBF solution and pulsed electrodeposition process at 80°C, with a current density of -10mA/cm2. Calcium phosphate deposition was characterized using XRD, FTIR and FE-SEM. Although carbonated hydroxyapatite and calcium deficient hydroxyapatite phases were deposited on all surface types, the deposition on nanoporous titania displayed significant differences from those on anodized titania and flat titanium. Our results indicated that ordered titanium oxide nanotubes providing a larger surface area for hydroxide ion generation, enabled deposition of carbonated hydroxyapatite phases, which flat and anodized titania plates do not to the same extent under same reaction conditions.
... Both nanoindentation modulus and acoustic impedance showed a periodic lamellar pattern undulating between low and high values along the radial direction in cross sections of osteons. Previous reports suggest that the observed lamellar modulation of microelastic properties is either related to a variation of mineral content measured by quantitative backscattered electron imaging (qBEI) [18], [20], [21] or to changes in lamellar sublayers orientation inferred from Raman spectroscopy [11], synchrotron radiation phase nanotomography [7] and serial backscattered electron microscopy measurements [21]. These reports suggest that both anisotropy and variation in composition could explain the lamellar level modulation of the elastic properties [22]. ...
... Here, we point out that, although the tissue mineralization is known to be correlated with tissue stiffness [18], [35], it does not necessarily explain the prominent variation in elasticity at the Fiber Orientation on Bone Microelastic Properties lamellar scale, confirming our search for other factors. Moreover, it should be noted that small intra-lamellar variations of tissue mineralization, as reported by others [20], [21] can also be seen in the DMB maps, e.g. in the lower part ofFig. 3B, directly adjacent to the Haversian canal. ...
The elastic properties of bone tissue determine the biomechanical behavior of bone at the organ level. It is now widely accepted that the nanoscale structure of bone plays an important role to determine the elastic properties at the tissue level. Hence, in addition to the mineral density, the structure and organization of the mineral nanoparticles and of the collagen microfibrils appear as potential key factors governing the elasticity. Many studies exist on the role of the organization of collagen microfibril and mineral nanocrystals in strongly remodeled bone. However, there is no direct experimental proof to support the theoretical calculations. Here, we provide such evidence through a novel approach combining several high resolution imaging techniques: scanning acoustic microscopy, quantitative scanning small-Angle X-ray scattering imaging and synchrotron radiation computed microtomography. We find that the periodic modulations of elasticity across osteonal bone are essentially determined by the orientation of the mineral nanoparticles and to a lesser extent only by the particle size and density. Based on the strong correlation between the orientation of the mineral nanoparticles and the collagen molecules, we conclude that the microfibril orientation is the main determinant of the observed undulations of microelastic properties in regions of constant mineralization in osteonal lamellar bone. This multimodal approach could be applied to a much broader range of fibrous biological materials for the purpose of biomimetic technologies.
... Lastly, the exclusion of the lacuna-canalicular network and the water inside the pores of the extrafibrillar matrix is worth noting. Additionally, given the time-dependent properties of water, future investigations should consider its effects on bone mechanical properties, especially in relation to loading velocity and hydration status Utku et al., 2008). ...
Bone is a naturally occurring composite material composed of a stiff mineral phase and a compliant organic matrix of collagen and non-collagenous proteins (NCP). While diverse mineral morphologies such as platelets and grains have been documented, the precise role of individual constituents, and their morphology, remains poorly understood. To understand the role of constituent morphology on the fracture behaviour of lamellar bone, a damage based representative volume element (RVE) was developed, which considered various mineral morphologies and mineralised collagen fibril (MCF) configurations. This model framework incorporated a novel phase-field damage model to predict the onset and evolution of damage at mineral-mineral and mineral-MCF interfaces. It was found that platelet-based mineral morphologies had superior mechanical performance over their granular counterparts, owing to their higher load-bearing capacity, resulting from a higher aspect ratio. It was also found that MCFs had a remarkable capacity for energy dissipation under axial loading, with these fibrillar structures acting as barriers to crack propagation, thereby enhancing overall elongation and toughness. Interestingly, the presence of extrafibrillar platelet-based minerals also provided an additional toughening through a similar mechanism, whereby these structures also inhibited crack propagation. These findings demonstrate that the two primary constituent materials of lamellar bone play a key role in its toughening behaviour, with combined effect by both mineral and MCFs to inhibit crack propagation at this scale. These results have provided novel insight into the fracture behaviour of lamellar bone, enhancing our understanding of microstructure-property relationships at the sub-tissue level.
... Finally, the presence of water and its corresponding time-dependent properties were neglected in this work. It has been reported that bone effective properties are highly dependent on the loading velocity and hydration status, therefore, it is indispensable to study the viscosity contributions in bone mechanical properties (Peruzzi et al., 2021;Utku et al., 2008). ...
At the tissue-scale and above, there are now well-established structure-property relationships that provide good approximations of the biomechanical performance of bone through, for example, power-law relationships that relate tissue mineral density to elastic properties. However, below the tissue-level, the individual role of the constituents becomes prominent and these simple relationships tend to break down, with more detailed theoretical and computational models are required to describe the mechanical response. In this study, a two-dimensional micromechanics damage-based representative volume element (RVE) of lamellar bone was developed, which included a novel implementation of a phase-field damage model to describe the behaviour of non-collagenous proteins at mineral-mineral and mineral-fibril interface regions. It was found that, while the stiffness of the tissue was governed by the relative proportion of extra-fibrillar mineral and mineralised collagen fibrils, the strength and toughness of the tissue in transverse direction relied on the interactions occurring at mineral-mineral and mineral-fibril interfaces, highlighting the prominence of non-collagenous proteins in determine fracture-based processes at this scale. While fractures tended to initiate in mineral rich areas of the extra-fibrillar mineral matrix, it was found that the presence of mineralised collagen fibrils at low density did not provide a substantial contribution to crack propagation behaviour under transverse loading. However, at physiological volume fraction (
), different scenarios could arise depending on the relative strength value of the interphase around the MCFs (
) to the interphase between individual minerals (
): (i) When
, MCFs appear to facilitate crack propagation with MCF-mineral debonding being the dominant failure mode; (ii) once
, the MCFs hinder the microcracks, leading to inhibition of crack propagation, which can be regarded as an energy dissipation mechanism. The effective fracture properties of the tissue also experience a sudden increase in fracture work density (J-integral) once the crack is arrested by MCFs or severely deflected. Collectively, the predicted behaviour of the model compared well to those reported through experimental and computational methods, highlighting its potential to provide further understanding into the mechanistic response of bone ultrastructure alterations related to the structural and compositional changes resulting from disease and aging.
... For the hydrated samples tested in air, it is estimated that they would have lost ~3% water by mass, including pore and bound water, based on work by Nyman et al. (2013). The loss of loosely collagen-bound water is associated with embrittlement under dehydration conditions (Broz et al., 1993;Nyman et al., 2006Nyman et al., , 2013Nyman et al., , 2013Yan et al., 2008;Melvin and Evans, 1973;Kahler et al., 2003;Shin et al., 2022;Li et al., 2013;Abdel-Wahab et al., 2012;Vergani et al., 2014;Burr, 2019;Samuel et al., 2014Samuel et al., , 2016Utku et al., 2008). While the exact water content could not be measured during the SEM experiments, most of the water was removed from the tissue to achieve the vacuum pressure levels in Table 1 since the SEM will not permit operation if the sample is still heavily outgassing. ...
A 2D plane strain extended finite element method (XFEM) model was developed to simulate three-point bending fracture toughness tests for human bone conducted in hydrated and dehydrated conditions. Bone microstructures and crack paths observed by micro-CT imaging were simulated using an XFEM damage model. Critical damage strains for the osteons, matrix, and cement lines were deduced for both hydrated and dehydrated conditions and it was found that dehydration decreases the critical damage strains by about 50%. Subsequent parametric studies using the various microstructural models were performed to understand the impact of individual critical damage strain variations on the fracture behavior. The study revealed the significant impact of the cement line critical damage strains on the crack paths and fracture toughness during the early stages of crack growth. Furthermore, a significant sensitivity of crack growth resistance and crack paths on critical strain values of the cement lines was found to exist for the hydrated environments where a small change in critical strain values of the cement lines can alter the crack path to give a significant reduction in fracture resistance. In contrast, in the dehydrated state where toughness is low, the sensitivity to changes in critical strain values of the cement lines is low. Overall, our XFEM model was able to provide new insights into how dehydration affects the micromechanisms of fracture in bone and this approach could be further extended to study the effects of aging, disease, and medical therapies on bone fracture.
... This result is in accordance with previous studies on deformation in bone that indicate dehydration promotes brittle behavior and a reduction in plasticity that is associated with a loss of water that is loosely bound to collagen. (Burr, 2019;Nyman et al., 2006Nyman et al., , 2013Samuel et al., 2014Samuel et al., , 2016Utku et al., 2008;Yan et al., 2008). According to Nyman et al. (2013), air drying at 23 • for 3-10 min results in 1.4-5.7% ...
... This result is in accordance with previous studies on deformation in bone that indicate dehydration promotes brittle behavior and a reduction in plasticity that is associated with a loss of water that is loosely bound to collagen. (Burr, 2019;Nyman et al., 2013;Nyman et al., 2006a;Samuel et al., 2016;Samuel et al., 2014;Utku et al., 2008;Yan et al., 2008). According to Nyman et al. (Nyman et al., 2013), air drying at 23 degrees for 3-10 min results in 1.4-5.7% ...
Water is a crucial component of bone, affecting the interplay of collagen and minerals and contributing to bone’s high strength and ductility. Dehydration has been shown to significantly effect osseous mechanical properties; however, studies comparing the effects of various dehydrating environments on fracture toughness of bone are scarce. Accordingly, the crack resistance curve (R-curve) behavior of human and sheep cortical bone was characterized in a bio-bath, in ambient pressure air, and in scanning electron microscopes (SEMs) under three different environmental conditions (water vapor pressure, air pressure, and high-vacuum). The aim of this work was to better understand the impact of test environment on both intrinsic and extrinsic toughening and hence crack initiation toughness, K0 and crack growth resistance, dK/da. Results show significantly lower K0 values for samples that were tested inside SEM combined with pronounced extrinsic toughening through microcracking and crack path deflections out of the mode I plane. Importantly, all three SEM test environments gave similar results, and thus it does not matter which type of SEM is used. Ex situ testing of hydrated samples revealed similar K0 for both environments but elevated crack growth resistance for testing in ambient air relative to the bio-bath. Our data reveals the experimental difficulties to directly observe microscale crack propagation in cortical bone that resembles the in vivo situation. Ex situ testing immersed in Hanks’ Balanced Salt Solution (HBSS) with subsequent crack path analysis, while tedious, is thought to presents the most realistic picture of the in vivo structure-fracture property relations in biological tissue.
... [101] The local water concentration is not homogeneous and bears on the bone anisotropy. [102] In a study of the effect of hydration on bone mechanics, we found that the water content influences the properties at each hierarchical level. [103] The different size-scales were interrogated using AFM at the fibrillar level of about 100 nm, instrumented nanoindentation at the lamellar level (several μm), and microindentation at the osteonal level (tens of μm). ...
Biological organisms are inherently complex. Although investigations of the function and design of living organisms have existed since the dawn of science, only in recent years have the tools existed to extend such studies to the nanoscale. Progress in nanomechanics of biological systems has enabled our understanding of intricate mechanical designs which nature has optimized for specific functions. This review provides an overview of the field of bionanomechanics, emphasizing the manner in which fundamental mechanical concepts are expressed in the design of a wide spectrum of biological specimens. We show that diverse species exploit common concepts to achieve desired function; a principle that extends over large scales of size and mechanical properties. The powerful techniques that enable such studies, particularly atomic force microscopy and instrumented nanoindentation, as well as common analytical approaches are given special attention.
... It is interesting to compare the micromorphological patterns of Figs. 3 to 9 to microscopic observations reported in the literature, in view of the terminology associated with bone tissue development: Elongated, ellipsoidal vascular pores as seen in the micrographs of cow, ostrich, and pig tissues are reminiscent of so-called fibro-lamellar bone, as shown e.g. for the femora of minipig [3]; the circular vascular pores seen in rabbit and horse bone are typical for canonical osteonal bone as preferentially depicted in most textbooks on anatomy; they are in line with many earlier observations, e.g. see [115,121]. The emu tissue, in turn, seems to lie somewhat halfway between osteonal and fibrolamellar bone. ...
It is very well known that bone is a hierarchically organized material produced by bone cells residing in the fluid environments filling (larger) vascular pores and (smaller) lacunar pores. The extracellular space consists of hydroxyapatite crystals, collagen type I molecules, and water with non-collageneous organics. It is less known to which extent the associated quantities (mineral, organic, and water concentrations; vascular, lacunar, and extracellular porosities) vary across species, organs, and ages. We here investigate the aforementioned quantities across femoral shaft tissues from cow, horse, emu, frog, ostrich, pig, and rabbit; by means of light microscopy and dehydration-demineralization tests; thereby revealing interesting invariances: The extracellular volume fractions of organic matter turn out to be similar across all tested non-amphibian tissues; as do the extracellular volume fractions of hydroxyapatite across all tested mammals. Hence, the chemical composition of the femoral extracellular bone matrix is remarkably “invariant” across differently aged mammals; while the water content shows significant variations, as does the partitions of water between the different pore spaces. The latter exhibit strikingly varying morphologies as well. This finding adds to the ample “universal patterns” in the sense of evolutionary developmental biology; and it provides interesting design requirements for the development of novel biomimetic tissue engineering solutions.
... A graft polymerization and covalent modification of inorganic components can enhance their surface solubility with a polymer matrix, which can partly overcome the interfacial compatibility problem 18-22 . Relative to complicated designs based on the principle of similar compatibility, an evaluation of nature shows exquisite designs of tough bone and seashell connected by crystal-surface-bound water as a transition phase to connect the organic and inorganic components that have a large surface energy difference [23][24][25] . In ideal bone tissue, 65 wt% inorganic hydroxyapatite provides strength and hardness and 25 wt% organic collagen provides ductility and toughness 26,27 . ...
With the world’s focus on wearable electronics, the scientific community has anticipated the plasticine-like processability of electrolytes and electrodes. A bioinspired composite of polymer and phase-changing salt with the similar bonding structure to that of natural bones is a suitable electrolyte candidate. Here, we report a water-mediated composite electrolyte by simple thermal mixing of crystallohydrate and polymer. The processable phase-change composites have significantly high mechanical strength and high ionic mobility. The wide operating voltage range and high faradic capacity of the composite both contribute to the maximum energy density. The convenient assembly and high thermal-shock resistance of our device are due to the mechanical interlocking and endothermic phase-change effect. As of now, no other non-liquid electrolytes, including those made from ceramics, polymers, or hydrogels, possess all of these features. Our work provides a universal strategy to fabricate various thermally manageable devices via phase-change electrolytes. Here the authors report composite electrolytes combining polymer chains and hydrated salts with a similar bonding structure to that of natural bones. The design breaks the trade-off between strength and ionic mobility of solid electrolytes and allows for good electrochemical performance in supercapacitors.
... The loss of bulk and weakly bound water leads to 1.2%-1.4% contraction, which has been attributed to the presence of more water-containing rather than mineral-containing spaces within mineralised collagen fibril arrays. 52 However, even in the most potentially dehydrating environment inside the SEM, elastic modulus values of bone remain independent of vacuum conditions (tested up to 5.25 × 10 −4 Pa pressure and 2 h exposure time). 53 Interfaces between bone and fibrous connective tissues (e.g., ligaments and tendons) are highly interesting from a biomechanical point of view. ...
Bone is an architecturally complex system that constantly undergoes structural and functional optimisation through renewal and repair. The scanning electron microscope (SEM) is among the most frequently used instruments for examining bone. It offers the key advantage of very high spatial resolution coupled with a large depth of field and wide field of view. Interactions between incident electrons and atoms on the sample surface generate backscattered electrons, secondary electrons, and various other signals including X-rays that relay compositional and topographical information. Through selective removal or preservation of specific tissue components (organic, inorganic, cellular, vascular), their individual contribution(s) to the overall functional competence can be elucidated. With few restrictions on sample geometry and a variety of applicable sample-processing routes, a given sample may be conveniently adapted for multiple analytical methods. While a conventional SEM operates at high vacuum conditions that demand clean, dry, and electrically conductive samples, non-conductive materials (e.g., bone) can be imaged without significant modification from the natural state using an environmental scanning electron microscope. This review highlights important insights gained into bone microstructure and pathophysiology, bone response to implanted biomaterials, elemental analysis, SEM in paleoarchaeology, 3D imaging using focused ion beam techniques, correlative microscopy and in situ experiments. The capacity to image seamlessly across multiple length scales within the meso-micro-nano-continuum, the SEM lends itself to many unique and diverse applications, which attest to the versatility and user-friendly nature of this instrument for studying bone. Significant technological developments are anticipated for analysing bone using the SEM.
... Bones provide not only calcium, magnesium, and phosphate ions for the vertebrates, but also enable their mobility, protect the visceral organs, and act as sites of blood cell production [1]. As hierarchically organized biological structures, bones consist of hydroxyapatite (HA), Type I Collagen, proteoglycan and water [2]. In the treatment of bone and joint fractures, in addition to metal implants [4], autograft, allograft, xenograft and alloplastic materials [3] are used. ...
Mineralized natural protein based novel bone replacement materials are investigated for tissue engineering. Mineralized silk fibroin composite foams and films display excellent biocompatibility. In this study, the biomimetic and electrochemical mineralization of orderly oriented silk fibroin scaffolds was studied. Commercially obtained pure silk woven fabric was boiled in 0.02 M Na2CO3 for 20 min. Calcium phosphate was deposited at 37°C for twenty minutes in seven sequential immersion steps, using 250 mM CaCl2 2H2O and 120 mM K2HPO4, containing 0.15 M NaCl and 50mM TRIS-HCl, pH 7.4, followed by electrochemical treatment in modified SBF solution at 40°C at a current density of -25mA/cm² for 60 min. The amount of biomimetically deposited Ca-P increased with the number of immersion steps. SEM images and XRD analysis of the Ca-P deposit indicated the initial formation of brushite with its monoclinic crystal structure and characteristic peak at 11.76 2θ, and electrochemical conversion of brushite to hydroxyapatite on silk after electrochemical cathodization as confirmed by XRD and SEM analysis. Thus, a silk-fibroin-hydroxyapatite composite material prepared as a xenograft consisting of biocompatible components, and easily prepared as an economical bone segment replacement material with highly oriented fibers.
... Bone surfaces were prepared as described and imaged with wet mode AFM. Bone specimens were kept wet at all times, to avoid structural modifications due to dehydration [46]. Overall, this surface characterization indicates that the bone surfaces we have created contain multiple topographic signals at different lateral scales. ...
Bone homeostasis is continuously regulated by the coordinated action of bone-resorbing osteoclasts and bone-forming osteoblasts. Imbalance between these two cell populations leads to pathological bone diseases such as osteoporosis and osteopetrosis. Osteoclast functionality relies on the formation of sealing zone (SZ) rings that define the resorption lacuna. It is commonly assumed that the structure and dynamic properties of the SZ depend on the physical and chemical properties of the substrate. Considering the unique complex structure of native bone, elucidation of the relevant parameters affecting SZ formation and stability is challenging. In this study, we examined in detail the dynamic response of the SZ to the microtopography of devitalized bone surfaces, taken from the same area in cattle femur. We show that there is a significant enrichment in large and stable SZs (diameter larger than 14 mm; lifespan of hours) in cells cultured on rough bone surfaces, compared with small and fast turning over SZ rings (diameter below 7 mm; lifespan approx. 7 min) formed on smooth bone surfaces. Based on these results, we propose that the surface roughness of the physiologically relevant substrate of osteoclasts, namely bone, affects primarily the local stability of growing SZs. © 2017 The Author(s) Published by the Royal Society. All rights reserved.
... positioned at the boundaries of 2-3μmthick ordered lamellae with a distinct orientation of collagen fibrils. The disordered layers at the interlamellar boundaries are less mineralized, in addition to the loose packing of collagen and higher hydration 73 . This contributes to microscale fluctuations of Young's modulus; the numerous inter faces of varying stiffness, together with alternating col lagen alignment, explain the observation that a crack in healthy bone never follows a straight line (that is, an energetically lowcost path). ...
From an engineering perspective, skeletal tissues are remarkable structures because they are lightweight, stiff and tough, yet produced at ambient conditions. The biomechanical success of skeletal tissues is largely attributable to the process of biomineralization — a tightly regulated, cell-driven formation of billions of inorganic nanocrystals formed from ions found abundantly in body fluids. In this Review, we discuss nature's strategies to produce and sustain appropriate biomechanical properties in mineralizing (by the promotion of mineralization) and non-mineralizing (by the inhibition of mineralization) tissues. We review how perturbations of biomineralization are controlled over a continuum that spans from the desirable (or defective in disease) mineralization of the skeleton to pathological cardiovascular mineralization, and to mineralization of bioengineered constructs. A materials science vision of mineralization is presented with an emphasis on the micro- and nanostructure of mineralized tissues recently revealed by state-of-the-art analytical methods, and on how biomineralization-inspired designs are influencing the field of synthetic materials.
... [32][33][34][35] Mimicking these natural systems, innovative nanomaterials have been developed using various deposition methods including mineral coatings. [27][28][29][30][31][36][37][38][39][40][41][42][43] Recently, biological materials with nanostructured topography have been developed with controlled and arranged surface porosity from macro to nanoscale, 44 that is, from the macroscale of titanium metal, maintaining bone integrity, down to the micro-nanoporous metal-oxide level interdigitating the implant with the bone tissue, to the nanocrystalline bioactive mineral layer enabling bone regeneration by the bone forming cells of the body. 45 The metal oxide-tissue interface can be enriched with the use of biomolecules that can undertake an interfacial role. ...
Realising controllable interactions at the bio-nanomaterial interfaces are vital in developing next-generation engineered implant materials. Titanium-based implants are key materials in biomedical engineering due to excellent bulk mechanical properties and biocompatibilities. Advanced bio-interfaces resolving nanostructured modulated surfaces that allow manipulation with the biological molecules is one of the keys to enhance favourable interactions with the surrounding biological species. Here, we developed a protein-mediated hydroxyapatite composite layer on nanotubular titania surface. Green fluorescence protein, engineered to contain hydroxyapatite binding peptides (GFPuv-HABP), was co-deposited with the hydroxyapatite precursors onto the titania nanotubes that are formed by anodisation. Ordered titanium dioxide nanotubular surfaces were coated with hydroxyapatite at physiological pH and temperature using simulated body fluid and pulsed electrochemical cathodisation. The hydroxyapatite deposit interdigitated into the nanotubes, producing a metal oxide-mineral composite. The engineered GFPuv-HABP protein was then self-assembled on the hydroxyapatite, forming a bio-modulated interface. Additionally, the engineered proteins were co-deposited with the precursor ions of hydroxyapatite mineral on the nanotubular titania plate. Bio-mediated assembly resulted in formation of a hybrid composite as an integrated interface on the nanotubular surface. Biomolecular assisted fabrication of hybrid composite interface on metal oxide substrate offers wide range of opportunities to design novel interfaces.
... Biomineralization organizes the mineral, the organic material and the tissue fluid hierarchically to form a natural composite material. Biomimicking biomineralization has enabled the design and production of innovative nanomaterials and systems that contain a bioactive, biocompatible and osteoconductive calcium phosphate (Ca-P) layer, produced using an appropriate deposition method [1][2][3][4][5][6][7][8][9][10][11]. ...
The effect of temperature and nanotubular surface morphology on calcium phosphate deposition was investigated using a modified simulated body fluid and electrochemistry. Ordered nanotubular titanium oxide plates were coated by pulsed electrochemical deposition process, while titanium oxide and pure titanium surfaces were used as controls at 80 °C and 37 °C. The calcium phosphate deposit was characterized using XRD, FT-IR and FE-SEM. Carbonated hydroxyapatite was deposited at the physiological temperature of 37 °C on nanotubular surfaces, which provided a large surface area for hydroxide ion generation and a small volume for the confinement and concentration of hydroxide ions. Compounds containing carbonates and hydrogen phosphates were deposited on porous titanium oxide surfaces and flat titanium surfaces as the control group. This study demonstrates deposition of hydroxyapatite at physiological temperatures, which is essential for codeposition of organic bioceramics for medical use.
... Regarding the measured linear expansion coefficients, the minor dimensional change along the axial direction is evident, which corresponds to the axis of the mineralized collagen fibrils, while the major swelling comes out along the thickness and width directions. This fairly agrees with the results of similar studies [54,55]. ...
We firstly measured the swelling of single trabeculae from human femur heads during water imbibition. Since the swelling is caused by water diffusing from external surfaces to the core of the sample, by measuring the sample swelling over time, we obtained direct information about the transport of fluids through the intimate constituents of bone, where the mineralization process takes place. We developed an apparatus to measure the free expansion of the tissue during the imbibition. In particular, we measured the swelling along three natural axes (length L, width W, and thickness T) of plate-like trabeculae. For this aim, we developed a 3D analytical model of the water uptake by the sample that was performed according to Fickian transport mechanism. The results were then utilized to predict the swelling over time along the three sample directions (L, W, T) and the apparent diffusion coefficients D T , D W , and D L .
... The disordered material forms a continuous phase and the canaliculi, which course through the lamellar structure, are embedded in the disordered material. Both these materials are mineralized in vivo, possibly to different extents [16,21]. Together they form a composite material, in which the disordered component can be regarded as an isotropic "matrix" and the rods are the reinforcing "fibers". ...
Lamellar bone is the most common bone type in humans. The predominant components of individual lamellae are plywood-like arrays of mineralized collagen fibrils aligned in different directions. Using a dual-beam electron microscope and the Serial Surface View (SSV) method we previously identified a small, but significantly different layer in rat lamellar bone, namely a disordered layer with collagen fibrils showing little or no preferred orientation. Here we present a 3D structural analysis of 12 SSV volumes (25 complete lamellae) from femora of 3 differently aged human individuals. We identify the ordered and disordered motifs in human bone as in the rat, with several significant differences. The ordered motif shows two major preferred orientations, perpendicular to the lamellar bone is the most common bone type in humans. The predominant components of individual lamellae are plywood-like arrays of mineralized collagen fibrils aligned in different directions. Using a dual-beam electron microscope and the Serial Surface View (SSV) method we previously identified a small, but significantly different layer in rat lamellar bone, namely a disordered layer with collagen fibrils showing little or no preferred orientation. Here we present a 3D structural analysis of 12 SSV volumes (25 complete lamellae) from femora of 3 differently aged human individuals. We identify the ordered and disordered motifs in human bone as in the rat, with several significant differences. The ordered motif shows two major preferred orientations, perpendicular to the long axis of the bone, and aligned within 10-20˚ of the long axis, as well as fanning arrays. At a higher organizational level, arrays of ordered collagen fibrils are organized into 'rods' around 2 to 3 microns in diameter, and the long axes of these 'rods' are parallel to the lamellar boundaries. Human bone also contains a disordered component that envelopes the rods and fills in the spaces between them. The disordered motif is especially well-defined between adjacent layers of rods. The disordered motif and its interfibrillar substance stain heavily with osmium tetroxide and alcian blue indicating the presence of another organic component in addition to collagen. The canalicular network is confined to the disordered material, along with voids and individual collagen fibrils, some of which are also aligned more or less perpendicular to the lamellar boundaries. The organization of the ordered fibril arrays into rods enveloped in the continuous disordered structure was not observed in rat lamellar bone. We thus conclude that human lamellar bone is comprised of two distinct materials, an ordered material and a disordered material, and contains an additional hierarchical level of organization composed of arrays of ordered collagen fibrils, referred to as rods. This new structural information on human lamellar bone will improve our understanding of structure-mechanical function relations, mechanisms of mechano-sensing and the characterizations of bone pathologies.
... In York the specimens were weighed dry, and then placed in Hanks balanced saline solution for three days. This is much more than enough time to allow the specimens to become fully saturated with the solution, this process taking only a few hours [3,8]. Imbibing water causes bones to swell slightly, so the width and depth of the hydrated specimens were re-measured. ...
We have shown recently that antlers of red deer, at the time that they are used in fights in the rut, are essentially dry. We have also shown that dry antler material has a lower impact energy absorption than that of wet antler, a property which, in a fight, is probably very important in the first clashing of antlers. However, dry antler has a much higher Young's modulus (stiffness) than wet antler, and this property will be important in the pushing match that follows the initial impact. These values were compared with those of wet bone, and it was found that although dry antler had a somewhat lower Young's modulus than wet bone, it had a much higher impact energy absorption. In that paper we did not consider the properties of dry bone. We now rectify that. The present paper compares the Young's modulus and impact energy absorption of wet and dry antler with that of wet and dry long bone of deer. It is found that the Young's modulus of dry antler is slightly less (12%) than that of wet long bone, which is in turn slightly less (7%) than that of dry long bone. On the other hand the impact energy absorption of dry antler is much (x6.6) greater than that of wet bone and even greater (x14.3) than that of dry bone. (The impact energy absorption of wet antler could not be measured consistently; it was certainly very high). We suggest that the material properties of antler (which is necessarily dry when used in fights) are superior to those of dry long bone material because, although they have somewhat lower (18%) Young's modulus, they have a much greater (x14) impact energy absorption. The properties of wet long bone and wet antler material are given for comparison, but neither could be used in reality.
... Recent investigations revealed that neither the orthogonal nor the twisted plywood models are feasible from a micro-mechanical point of view [25] and that models similar to the rotated and helicoidal plywood arrangements better explain the experimentally observed variation in mechanical properties [9,25]. The arsenal of previously applied methods of investigation includes polarized light [13,26] and confocal [27,28] microscopy, transmission and backscattered scanning electron microscopy [15,17,293031, contact microradiography [32] , scanning nanoinden- tation [10,33], Raman microscopy [34] , scanning acoustic micros- copy [9,23], small angle and wide angle X-ray scattering [35,36], wide angle X-ray diffraction [24], time of flight secondary ion mass spectrometry [37], or combinations thereof [9,33,38], which have provided insights into the lamellar composition, size and orientation of collagen molecules and mineral platelets, as well as the anisotropic elastic properties of the mineralized fibrils. However, most of these techniques either probe the tissue surface in reflection mode or the bulk material in transmission mode. ...
We investigate the 3D organization of mineralized collagen fibrils in human cortical bone based on synchrotron X-ray phase nano-tomography images. In lamellar bone the collagen fibrils are assumed to have a plywood-like arrangement, but due to experimental limitations the three-dimensional fibril structure has only been deduced from section surfaces so far and the findings have been discussed controversially. Breakthroughs in synchrotron tomographic imaging give access to direct 3D information on the bone structure at the nanoscale. Using an autocorrelation based orientation measure, we confirm that the fibrils are unidirectional in quasi-planes of sub-lamellae and find two specific dominating patterns, oscillating and twisted plywood coexisting in a single osteon. Both patterns exhibit smooth orientation changes between adjacent quasi-planes. Moreover, we find the periodic changes of collagen fibril orientation to be independent of fluctuations of local mas density. These data improve our understanding of the lamellar arrangement in bone and allow more detailed investigations of structure-function relationships at this scale, providing templates for bio-inspired materials. The presented methodology can be applied to non-destructive 3D characterization of the sub-micron scale structure of other natural and artificial mineralized biomaterials.
... The presence of a thin disordered layer in the lamellar structure raises the question of whether or not this layer has a different mineral density compared to the rest of the lamellar structure, which in turn could have important mechanical implications. Utku et al. [20] also noticed changes in mineral content within one lamella of mini-pig bone, but could not associate this to a particular structure.Fig. 4 shows a BSE image of a transverse section of a rat tibia. ...
... (6) It is therefore understandable that the nature and function of BW has been studied extensively. (3,(7)(8)(9)(10)(11)(12) Knowledge of BW is of substantial interest as it can provide insight into mechanical properties of bone. BW can be found in the pore space within bone (ie, ''pore BW'') and in the bone matrix, where it is associated with the mineral (13)(14)(15) and organic phases. ...
Bone water (BW) plays a pivotal role in nutrient transport and conferring bone with its viscoelastic mechanical properties. BW is partitioned between the pore spaces of the Haversian and lacuno-canalicular system, and water predominantly bound to the matrix proteins (essentially collagen). The general model of BW is that the former predominantly experiences fast isotropic molecular reorientation, whereas water in the bone matrix undergoes slower anisotropic rotational diffusion. Here, we provide direct evidence for the correctness of this model and show that unambiguous quantification in situ of these two functionally and dynamically different BW fractions is possible. The approach chosen relies on nuclear magnetic resonance (NMR) of deuterium ((2) H) that unambiguously separates and quantifies the two fractions on the basis of their distinguishing microdynamic properties. Twenty-four specimens of the human tibial cortex from six donors (3 male, 3 female, ages 27-83 years) were cored and (2) H spectra recorded at 62 MHz (9.4 Tesla) on a Bruker Instruments DMX 400 spectrometer after exchange of native BW with (2) H(2) O. Spectra consisted of a doublet signal resulting from quadrupole interaction of water bound to collagen. Doublet splittings were found to depend on the orientation of the osteonal axis with respect to the magnetic field direction (8.2 and 4.3 kHz for parallel and perpendicular orientation, respectively). In contrast, the isotropically reorienting pore-resident water yielded a single resonance line superimposed on the doublet. Nulling of the singlet resonance allowed separation of the two fractions. The results indicate that in human cortical bone 60-80% of detectable BW is collagen-bound. Porosity determined as the difference between total BW and collagen bound water fraction was found to strongly parallel µCT based measurements (R(2) = 0.91). Our method provides means for direct validation of emerging relaxation-based measurements of cortical bone porosity by proton MRI. © 2012 American Society for Bone and Mineral Research.
... This can be associated to a local reorganisation in the sample that corresponds to a water loss associated to the sample collapsing The enthalpy of water ordering in the sample can be derived from the recorded image series as reported by Sievers et al. The effect of dehydration on lamellar bones was also studied by in situ ESEM experiments (Utku et al., 2008). The obtained results indicate that dehydration affects the dimensions of lamellar bone in an anisotropic manner in longitudinal sections, whereas in transverse sections the extent of contraction is almost the same in both the radial and tangential directions. ...
... The interaction of water with mineral and collagen is critical to the mechanical behavior of fresh bone [3] . There are many types of water which are present in bone [4][5][6][7] , such as free water, the structural water forming hydrogen bonds within the triple helix of collagen molecules and crystal water bonding to apatite surface or in the crystal lattice, etc. Evidence for an architecture and distribution of water in bone by nuclear magnetic resonance (NMR) has recently been published [8][9][10][11] . ...
To comparatively investigate the inorganic composition and crystallographic properties of cortical and cancellous bone via thermal treatment under 700 °C.
Thermogravimetric measurement, infrared spectrometer, X-ray diffraction, chemical analysis and X-ray photo-electron spectrometer were used to test the physical and chemical properties of cortical and cancellous bone at room temperature 250 °C, 450 °C, and 650 °C, respectively.
The process of heat treatment induced an extension in the a-lattice parameter and changes of the c-lattice parameter, and an increase in the crystallinity reflecting lattice rearrangement after release of lattice carbonate and possible lattice water. The mineral content in cortical and cancellous bone was 73.2wt% and 71.5wt%, respectively. For cortical bone, the weight loss was 6.7% at the temperature from 60 °C to 250 °C, 17.4% from 250 °C to 450 °C, and 2.7% from 450 °C to 700 °C. While the weight loss for the cancellous bone was 5.8%, 19.9%, and 2.8 % at each temperature range, the Ca/P ratio of cortical bone was 1.69 which is higher than the 1.67 of stoichiometric HA due to the B-type CO₃²⁻ substitution in apatite lattice. The Ca/P ratio of cancellous bone was lower than 1.67, suggesting the presence of more calcium deficient apatite.
The collagen fibers of cortical bone were arrayed more orderly than those of cancellous bone, while their mineralized fibers ollkded similar. The minerals in both cortical and cancellous bone are composed of poorly crystallized nano-size apatite crystals with lattice carbonate and possible lattice water. The process of heat treatment induces a change of the lattice parameter, resulting in lattice rearrangement after the release of lattice carbonate and lattice water and causing an increase in crystal size and crystallinity. This finding is helpful for future biomaterial design, preparation and application.
... As a result, the ratio of the hydrated/dehydrated apparent moduli was equivalent to the ratio of hydrated/dehydrated nanoindentation-derived tissue moduli (as would be expected from [34]) due to the failure to account for changes in architecture. The current study has treated the structural changes as isotropic; however, dehydration-induced shrinkage in bone has been shown to be anisotropic at the macroscopic [13] and at the microstructural level [33]. This anisotropy will result in architectural changes that cannot be captured using the methodology described herein. ...
Dehydration is known to cause an increase in the elastic modulus of bone tissue. However, it also causes structural changes (i.e. shrinkage) which can themselves significantly alter the mechanical properties, particularly in cancellous bone. The current study attempts to estimate the contribution of these two competing factors to the net change of dehydration on the apparent modulus of bovine cancellous bone. Cylindrical cores from the lumbar vertebrae were tested in tension, while hydrated and again after dehydration. The bone volume fractions (BV/TV) were measured in both conditions. The results indicate that the average overall increase in the apparent modulus after dehydration is 14±14% (mean±SD), which represents the net effect of a 27% increase in modulus due to increased tissue modulus offset by a modulus decrease of 13% due to reductions in bone volume fraction. These observations underscore the need to consider both structural and material changes when comparing hydrated and dehydrated mechanical behaviour.
The aim of this study was to investigate the influence of moisture content in frozen and embalmed human cadavers on the detection of dentinal microcracks using micro-computed tomography (micro-CT).
The group of embalmed specimens included three mandibular and two maxillary segments each containing one tooth. The group of frozen cadavers consisted of two frozen mandibular bone-blocks with two teeth and one mandibular segment containing one tooth. The final number of teeth for each preservation method was n = 5. All specimens were scanned with eight different moisture conditions: 48 h wet, 2 h dry, 48 h wet, 24 h dry, 48 h wet, 1 wk dry, 48 h wet, 1 wk dry. Micro-CT images were screened for the presence of dentinal microcracks. Statistical analysis was performed by nonparametric analysis of variance (α = 5%).
Only few microcracks were observed in wet and in 2 h dried bone-blocks with no significant differences (p = 0.63 and p = 0.23, respectively). There was a significant and steady increase of microcracks within the groups of dried specimens as follows: 2 h dry < 24 h dry < first wk dry < second wk dry (all p < 0.008). Preservation method had no significant influence on the visibility of microcracks (p = 0.98).
Identification of dentinal microcracks on micro-CT images is influenced by moisture content of cadaveric bone-blocks irrespective of the preservation method.
This study presents a new protocol for preparing bone samples for scanning electron microscopy (SEM) using a room temperature ionic liquid (RTIL) treatment method. RTIL-based solutions can be adopted as an alternative to lengthy and laborious traditional means of preparation for SEM due to their unique low-vapour pressure and conductive properties. Applied to biological samples, RTILs can be used quickly and efficiently to observe hydrated, unfixed structures in typical SEM systems. This first-time feasibility study of the optimization of this protocol for bone was explored through various SEM modalities using two distinct ionic liquids, 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMI][BF4]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMI][BF4]), at varying concentrations of 5, 10, and 25% v/v in aqueous solution through an addition-based method. Based on qualitative observations in the SEM, a 60-second solution addition treatment of 10% v/v [BMI][BF4] performed the best in imaging hydrated, unfixed bone samples, resulting in minimal charge buildup and no solution pooling on the surface. The treatment was applied effectively to a variety of bone samples, notably flat and polished, as well as highly topographical bone fracture surfaces of both healthy and osteoporotic human bone samples. In comparison to conventionally dehydrated bone, the RTIL treatment better preserved the natural bone structure, resulting in minimal microcracking in observed structures.
As the application of Raman spectroscopy to study bone has grown over the past decade, making it a peer technology to FTIR spectroscopy, it has become critical to understand their complimentary roles. Recent technological advancements have allowed these techniques to collect grids of spectra in a spatially resolved fashion to generate compositional images. The advantage of imaging with these techniques is that it allows the heterogenous bone tissue composition to be resolved and quantified. In this review we compare, for non-experts in the field of vibrational spectroscopy, the instrumentation and underlying physical principles of FTIR imaging (FTIRI) and Raman imaging. Additionally, we discuss the strengths and limitations of FTIR and Raman spectroscopy, address sample preparation, and discuss outcomes to provide researchers insight into which techniques are best suited for a given research question. We then briefly discuss previous applications of FTIRI and Raman imaging to characterize bone tissue composition and relationships of compositional outcomes with mechanical performance. Finally, we discuss emerging technical developments in FTIRI and Raman imaging which provide new opportunities to identify changes in bone tissue composition with disease, age, and drug treatment.
Animal bones are a high-quality source of protein and comprehensive nutrients and improper handling can cause resource wasting and environmental issues. Pretreatment before enzymatic hydrolysis of bone could significantly improve the enzymolytic efficiency, which is an essential step to achieve high value-added utilization of bones. This study investigated the effect of lipase pretreatment on the enzymatic hydrolysis of bones. The degree of hydrolysis after lipase pretreatment was 12.58%, which was 8.19% higher than that without pretreatment. Lipase pretreatment was optimal at 9% substrate concentration and initial pH 7.5, with 0.08% lipase, followed by 4 h incubation at 40 °C. Mechanism analysis indicated that lipase pretreatment improved the enzymolytic efficiency by significantly decreasing the lipid content, and changing the surface structure and surface element content of C, N, and O, promoting the attachment of alkaline protease onto the sample. Overall, lipase pretreatment was an effective method to reduce the costs of production.
Environmental-scanning electron microscopy (ESEM) is routinely applied to various biological samples due to its ability to maintain a wet environment while imaging; moreover, the technique obviates the need for sample coating. However, there is limited research carried out on electron-beam (e-beam) induced tissue damage resulting from using the ESEM. In this paper, we use quantitative second-harmonic generation (SHG) microscopy to examine the effects of e-beam exposure from the ESEM on collagenous tissue samples prepared as either fixed, frozen, wet or dehydrated. Quantitative SHG analysis of tissues, before and after ESEM e-beam exposure in low-vacuum mode, reveals evidence of cross-linking of collagen fibers, however there are no structural differences observed in fixed tissue. Meanwhile wet-mode ESEM appears to radically alter the structure from a regular fibrous arrangement to a more random fiber orientation. We also confirm that ESEM images of collagenous tissues show higher spatial resolution compared to SHG microscopy, but the relative tradeoff with collagen specificity reduces its effectiveness in quantifying collagen fiber organization. Our work provides insight on both the limitations of the ESEM for tissue imaging, and the potential opportunity to use as a complementary technique when imaging fine features in the non-collagenous regions of tissue samples.
Interfaces provide the structural basis of essential bone functions. In the hierarchical structure of bone tissue, heterogeneities such as porosity or boundaries are found at scales ranging from nanometers to millimeters, all of which contributing to macroscopic properties. To date, however, the complexity or limitations of currently used imaging methods restrict our understanding of this functional integration. Here we address this issue using label-free third-harmonic generation (THG) microscopy. We find that the porous lacuno-canalicular network (LCN), revealing the geometry of osteocytes in the bone matrix, can be directly visualized in 3D with submicron precision over millimetric fields of view compatible with histology. THG also reveals interfaces delineating volumes formed at successive remodeling stages. Finally, we show that the structure of the LCN can be analyzed in relation with that of the extracellular matrix and larger-scale structures by simultaneously recording THG and second-harmonic
Collagen is the most abundant protein in mammals and its primary role is to serve as mechanical support in many extracellular matrices such as those of bones, tendons, skin or blood vessels. Water is an integral part of the collagen structure, but its role is still poorly understood, though it is well-known that the mechanical properties of collagen depend on hydration. Recently, it was shown that the conformation of the collagen triple helix changes upon water removal, leading to a contraction of the molecule with considerable forces. Here we investigate the influence of mineralization on this effect by studying bone and turkey leg tendon (TLT) as model systems. Indeed, TLT partially mineralizes so that well-aligned collagen with various mineral contents can be found in the same tendon. We show that water removal leads to collagen contraction in all cases generating tensile stresses up to 80MPa. Moreover, this contraction of collagen puts mineral particles under compression leading to strains of around 1%, which implies localized compressive loads in mineral of up to 800MPa. This suggests that collagen dehydration upon mineralization is at the origin of the compressive pre-strains commonly observed in bone mineral.
Copyright © 2015 Elsevier Ltd. All rights reserved.
In this paper we firstly illustrate the measurement of the swelling of a single trabecula from human femur heads during water imbibition. Moreover, since the swelling is caused by water diffusing from external surfaces to the core of the sample, by measuring the sample swelling over time, we obtained direct information about the transport of fluids through the intimate constituents of bone, where the mineralization process takes place. We developed an apparatus to measure the free expansion of the tissue during the imbibition. In particular, we measured the swelling along three natural axes (length L, width W, and thickness T) of the trabecula. To this aim, a 3D analytical model of the water uptake by the sample was performed according to Fickian transport mechanism. The results have been utilized to fit the measured swelling along the three sample directions (L, W, T) and the apparent diffusion coefficients DT, DW, and DL.
Synchrotron X-ray scattering is used to measure average strains in the two main nanoscale phases of cortical bone – hydroxyapatite (HAP) platelets and collagen fibrils – under tensile loading at body temperature (37 °C) and under completely hydrated conditions. Dog-bone shaped specimens from bovine femoral cortical bone were prepared from three anatomical quadrants: anterio-medial, anterio-lateral, and posterio-lateral. The apparent HAP and fibrillar elastic moduli – ratios of tensile stress as applied externally and phase strains as measured by diffraction – exhibit significant correlations with the (i) femur quadrant from which the samples are obtained, (ii) properties obtained at the micro-scale using micro-computed tomography, i.e., microstructure, porosity and attenuation coefficient, and (iii) properties at the macro-scale using thermo-gravimetry and tensile testing, i.e., volume fraction and Young's modulus. Comparison of these tensile apparent moduli with compressive apparent moduli (previously published for samples from the same animal and tested under the same temperature and irradiation conditions) indicates that collagen deforms plastically to a greater extent in tension. Greater strains in the collagen fibril and concomitant greater load transfer to the HAP result in apparent moduli that are significantly lower in tension than in compression for both phases. However, tensile and compressive Young's moduli measured macroscopically are not significantly different during uniaxial testing.
The swelling of air-dried single trabeculae from human femur heads was obtained by complete immersion in water and the dimensional changes of the samples were measured over time. The experimental results were analyzed under the viewpoint of the diffusion through a porous material. The dimensional changes of the single trabeculae were 0.26 ± 0.15 percent (length), 0.45 ± 0.25 percent (width) and 1.86 ± 0.97 percent (thickness). The diffusion coefficients were then calculated from the swelling recorded over time and a value of (4.12 ± 0.8) x 10−10(m2s−1) (mean ± standard deviation) was found.
Since the dimensional variations of the specimens is due to the swelling of the collagen bone matrix, this technique could offer new insights for (1) a selective characterization of bone microstructure at the collagen matrix level and (2) the dynamics of diffusion through bone tissue.
Chemical pretreatment has been the prevailing sample preparation procedure for infrared (IR) spectroscopic studies on bone. However, experiments have indicated that chemical pretreatment can potentially affect the interactions between the components. Typically the IR techniques have involved transmission experiments. Here we report experimental studies using photoacoustic Fourier transform infrared spectroscopy (PA-FTIR). As a nondestructive technique, PA-FTIR can detect absorbance spectrum from a sample at controllable sampling depth and with little or no sample preparation. Additionally, the coupling inert gas, helium, which is utilized in the PA-FTIR system, can inhibit bacteria growth of bone by displacing oxygen. Therefore, we used this technique to study the undisturbed human cortical bone. It is found that photoacoustic mode (linear-scan, LS-PA-FTIR) can obtain basically similar spectra of bone as compared to the traditional transmission mode, but it seems more sensitive to amide III and ν(2) carbonate bands. The ν(3) phosphate band is indicative of detailed mineral structure and symmetry of native bone. The PA-FTIR depth profiling experiments on human cortical bone also indicate the influence of water on OH band and the cutting effects on amide I and mineral bands. Our results indicate that phosphate ion geometry appears less symmetric in its undisturbed state as detected by the PA-FTIR as compared to higher symmetry observed using transmission techniques on disturbed samples. Moreover, the PA-FTIR spectra indicate a band at 1747cm(-1) possibly resulting from CO stretching of lipids, cholesterol esters, and triglycerides from the arteries. Comparison of the spectra in transverse and longitudinal cross-sections demonstrates that, the surface area of the longitudinal section bone appears to have more organic matrix exposed and with higher mineral stoichiometry.
The mechanical and structural properties of the sub-layers of osteonal lamellae were studied. Young's modulus (E) of adjacent individual lamellae was measured by nanoindentation of parallel slices every 1-3μm, in planes parallel and perpendicular to the osteon axis (OA). In planes parallel to the OA, the modulus of a lamella could vary significantly between sequential slices. Significant modulus variations were also sometimes found on opposing sides of the osteonal canal for the same lamella. These results are rationalized by considerations involving the microstructural organization of the collagen fibrils in the lamellae. Scanning electron microscope imaging of freeze fractured surfaces revealed that the sub-structure of a single lamella can vary significantly on the opposing sides of the osteonal axis. Using a serial surface view method, parallel planes were exposed every 8-10nm using a dual beam microscope (FIB-SEM). Analysis of the orientations of fibrils revealed that the structure is rotated plywood like, consisting of unidirectional sub-layers of fibrils of several orientations, with occasional randomly oriented sub-layers. The dependence of the measured mechanical properties of the lamellae on the indentation location may be explained by the observed structure, as well as by the curvature of the osteonal lamellae through simple geometrical-structural considerations. Mechanical advantages arising from the curved laminate structure are discussed.
Most natural materials are composites based on biopolymers and some minerals. Despite the relative paucity of these constituents, their combination yields materials with outstanding properties and a great variation in functionality. A particular characteristic of biological composites is their multifunctionality. The basis for achieving this property is usually a complex hierarchical architecture in which an adaptation to the function(s) is possible at different structural levels. Only a few biological composites have been thoroughly studied from a materials science perspective; nacre is a prominent example. Fueled by the increasing interest in bioinspired materials research, biological composites are now studied more widely, and it has become apparent that Nature often solves materials problems in an unexpected way. This review discusses some striking examples. Many more are likely to emerge in the near future.
Biomineralization is a dehydration process in which water from the intrafibrillar compartments of collagen fibrils are progressively replaced by apatites. As water is an important element that induces a lack of durability of resin-dentin bonds, this study has examined the use of a biomimetic remineralization strategy as a progressive dehydration mechanism to preserve joint integrity and maintain adhesive strength after ageing. Human dentin surfaces were bonded with dentin adhesives, restored with resin composites and sectioned into sticks containing the adhesive joint. Experimental specimens were aged in a biomimetic analog-containing remineralizing medium and control specimens in simulated body fluid for up to 12 months. Specimens retrieved after the designated periods were examined by transmission electron microscopy for the presence of water-rich regions using a silver tracer and for collagen degradation within the adhesive joints. Tensile testing was performed to determine the potential loss of bond integrity after ageing. Control specimens exhibited severe collagen degradation within the adhesive joint after ageing. Remineralized specimens exhibited progressive dehydration, as manifested by silver tracer reduction and partial remineralization of water-filled microchannels within the adhesive joint, as well as intrafibrillar remineralization of collagen fibrils that were demineralized initially as part of the bonding procedure. Biomimetic remineralization as a progressive dehydration mechanism of water-rich, resin-sparse collagen matrices enables these adhesive joints to resist degradation over a 12-month ageing period, as verified by the conservation of their tensile bond strength. The ability of the proof of concept biomimetic remineralization strategy to prevent bond degradation warrants further development of clinically relevant delivery systems.
▪ Abstract The term bone refers to a family of materials, all of which are built up of mineralized collagen fibrils. They have highly complex structures, described in terms of up to 7 hierarchical levels of organization. These materials have evolved to fulfill a variety of mechanical functions, for which the structures are presumably fine-tuned. Matching structure to function is a challenge. Here we review the structure-mechanical relations at each of the hierarchical levels of organization, highlighting wherever possible both underlying strategies and gaps in our knowledge. The insights gained from the study of these fascinating materials are not only important biologically, but may well provide novel ideas that can be applied to the design of synthetic materials.
The purpose of this study was to explore the effects of changes in Type I collagen on the viscoelasticity of bone. Bone coupons were heated at either 100 or 200 degrees C to induce the thermal denaturation of Type I collagen. Half of these specimens were rehydrated after heat treatment; the other half were tested in a dry condition. The degree of denatured collagen (DC%) was analyzed by a selective digestion technique with the use of alpha-chymotrypsin. Isothermal (37 degrees C) and variable temperature tests (scans from 35 to 200 degrees C) were performed with the use of a dynamic mechanical analyzer to evaluate changes in bone viscoelastic properties as a function of collagen damage, specifically, changes in the loss factor (tan delta) and storage modulus (E') were assessed. Significant collagen denaturation occurred only when bone was heated at 200 degrees C irrespective of the hydration condition. Also, DC% did not show a significant effect on tan delta. However, higher values of tan delta were observed in wet samples compared to dry specimens. The temperature-scan tests revealed that the hydration condition, but not DC%, significantly affected the behavior of tan delta. However, E' was not strongly influenced either by DC% or by water content. These results suggest that at a constant frequency the denaturation of collagen triple-helical molecules may have few effects on the viscoelasticity of bone, but moisture may play a prominent role in determining this property.
Relevant mechanical properties of bone The mechanical properties of bone material are determined by the relative amounts of its 3 major constituents: mineral, water, and organics (mainly type I collagen); by the quality of these components; and by how the resulting material is arranged in space. For our purposes, the mechanical properties of bone can be summed up as follows: modulus of elasticity, yield stress and yield strain, post-yield stress and post-yield strain, and the total area under the stress-strain curve. Also important are some fracture mechanics properties, but these are not discussed here. A typical tensile stress-strain curve for a bone specimen is shown in Fig. 1. The modulus of elasticity shows how stiff the bone material is. Indeed, stiffness is the prime property of bone, distinguishing it from tendon, which has much less tensile stiffness, almost no shear stiffness, but which is nearly as strong and is much tougher. Yield stress and strain determine how much energy can be absorbed before irreversible changes take place. Post-yield stress and strain determine mainly how much energy can be absorbed after yield but before fracture. Irreversible changes take place at yield, caused by microdamage. The total area under the stress-strain curve is equivalent to the work that must be done per unit volume on the specimen before it breaks. Fracture mechanics properties show the extent to which bone is resistant to crack initiation and to crack travel (which are different things and governed by somewhat different features). In fact, crack travel resistance is given rather well by post-yield stress and strain.
The extracellular matrix provides an environment for cells. It is produced, assembled and modified by cells and in turn, it modifies the functions and behavior of the cells it encounters. The molecules that make up the matrix are diverse in both structure and function. This well-illustrated unit provides an introduction to the structure and function of the major components of matrix and serves as a background for the other units in the chapter which include protocols for isolation and analysis of individual components.
Collagen is the principal organic matrix in bone. The triple helical region of the molecule is 1014 amino acids long. In fibrils these molecules are staggered axially by integers of 234 residues or 68 nm (D). This axial shift occurs by self-assembly and can be understood in terms of a periodicity in the occurrence of apolar and polar residues in the amino acid sequence. Because the molecular length L = 4.47 D, there are gaps 1.5 x 36.5 nm regularly arrayed throughout the fibrils. The three-dimensional molecular arrangement is a quasi-hexagonal lattice with three distinct values for the principal interplanar spacings. Analysis of the intensity distribution in the mediumangle X-ray diffraction patterns from tendons has produced the following picture of the molecular arrangement in fibrils (Fraser et al. 1983). The molecular helices have a coherent length of 32 nm and are tilted parallel to a specific place within the lattice. A regular azimuthal interaction exists between these helices. This crystalline region could be the overlap region with a non-crystalline gap region. However, the gap is still regular axially and the molecular helices retain their structure; their lateral packing is perturbed although they retain a `gap'. Neutron and X-ray scattering experiments have shown that calcium hydroxyapatite crystals occur in the gap and are nucleated at a specific though unknown location within the gap. The c-axis of the apatite crystals is parallel to the fibril axis and its length c = 0.688 nm is close to the axial periodicity in a protein with an extended beta-conformation. If the telopeptides at the end of a collagen molecule do have this conformation they would either have a highly heterogeneous conformation or exist in a folded manner because the overall length of the telopeptides is shorter than a regular collagen repeat of 0.029 nm would allow.
In this study, FTIR photoacoustic spectroscopy is utilized to investigate collagen in the solid state. A resolution enhancement of the amide I band in collagen The presence of bound water, probably hydrogen bonded, in chick skin type I collagen has been shown from resolution enhancement of the amide I region of FTIR spectrum of collagen. However, as noted earlier, in the case of collagen, 20,35 the principle molecular vibrations of the polypeptide backbone are confined by stereochemical constraints to certain narrow regions in the spectra, viz., amide I, 1636-1661 cm-1, amide II, 1549-1558 cm-1 which differ from the characteristic vibrations for α-helical and β-sheet structures.
The knowledge of human bone viscoelasticity is an important issue for defining semirigid calcified tissues implants. A very sensitive technique was used to investigate bone viscoelasticity: the thermally stimulated creep method. A study of demineralized human bone was performed to determine the molecular origin of bone viscoelasticity. The thermally stimulated creep spectra of bone and demineralized bone, at the hydrated state, present a similar shape with one main retardation mode located at −133 and −120°C, respectively. This mode is shifted toward higher temperatures after dehydration, revealing the existence of another mode at around −155°C. The analysis of elementary spectra of bone and demineralized bone has shown that retardation times follow an Arrhenius equation, and that two compensation phenomena are observed with comparable compensation parameters. The first compensation phenomenon, which corresponds to the main retardation mode, was attributed to motions of water molecules located inside the collagen triple helix. The second compensation phenomenon, which reveals the existence of another relaxation mode at higher temperatures, was assigned to movements of hydrophilic side chains bound to water molecules. As for the mode observed at around −155°C, it was associated with motions of aliphatic side chains. Overall, bone viscoelasticity originates from the organic matrix. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 79: 2527–2533, 2001
The preservation of nineteenth-century albumen prints is of great concern to collection managers and to conservators of photographic materials. In the field of art conservation, preservation techniques incorporating aqueous treatments are often used to enhance the long- and short-term stability of historical artifacts or art objects. In a study of the interaction of water with albumen photographs, experiments were carried out in the ESEM to follow the real time effects of water on the prints. The experiments were designed to observe the effects of a range of relative humidities and liquid water on samples of expendable historic albumen prints, utilizing the advantages of imaging in the presence of water vapor. All albumen photographs exhibit a fine network of cracks in the albumen protein layer. Average crack width is approximately 10 μm. As observed in the ESEM, a 4.25-fold increase in the width of a single crack (at 50% RH), viewed normal to the surface, resulted from a single controlled excursion to high relative humidity and immersion. In an extraordinary series of images, a print viewed in cross-section exhibited a 22% swelling and shrinkage in thickness, and a 5% and 9% swelling and shrinkage along the width of a fragment of the albumen/image layer when the sample was immersed in water and dried. The visual information gained through the use of the ESEM helped to focus a materials investigation and served as a foundation for a study which shows that aqueous treatment causes increased cracking of both unsupported albumen and the albumen/image layer in prints. © 1993 Wiley-Liss, Inc.
Types of H2O in human enamel and in precipitated apatites are characterized using X-ray diffraction, infrared (IR) absorption spectroscopic and thermogravimetric analyses. Changes in lattice parameters (principally in the -axis dimensions) and in the character of the IR absorption bands are correlated with weight losses at pyrolysis temperatures of 100 to 400C and with effect of rehydration and reignition of previously ignited samples.This study demonstrated that the loss of H2O below 200C is reversible and causes no significant change in the lattice parameter of these apatites, whereas loss of H2O between 200 and 400C is irreversible and causes a contraction in the -axis dimension. It is proposed that two general types of H2O are present in these apatites: (a)adsorbed H
2O—characterized by reversibility, thermal instability below 200C, and lack of effect on lattice parameters; and (b)lattice H
2O—characterized by irreversibility, thermal instability between 200 and 400C, and induction of expansion in the -axis dimensions of human enamel and precipitated apatites. Lattice H2O is assumed to be due to H2O-for-OH and/or HPO4-for-PO4 substitutions in these apatites. Loss of adsorbed H2O caused sharpening of the OH absorption bands in the spectra of these apatites. Loss of lattice H2O caused the appearance of P–O–P absorption bands (due to the presence of P2O7
4– group) in precipitated apatites containing small amounts of CO3
2–.The observed larger -axis of human enamel apatite, i.e., 9.4450.003A, compared to that of the mineral or synthetic (prepared at 1000C) OH-apatite, i.e., 9.442A, may be attributed to the presence of lattice H2O, Cl-for-OH, and concerted substitutions of larger cations (e.g., Sr, Ba, Pb, K) for Ca in this apatite.
Most biological tissues are built with polymeric fibres. Two of the most abundant fibres found in Nature are cellulose and collagen. Cellulose is predominately found in plants, but is also produced by bacteria, for instance. It is one of the major constituents of the plant cell wall and confers rigidity to the plant body. Recent advances give a better insight into the relation between the arrangement of cellulose fibrils inside the cell wall and its mechanical properties. One of the most important questions in this context is the way in which the cellulose architecture is assembled and controlled by the cell. Despite some recent discoveries relating to cellulose biosynthesis, the full understanding of the self-assembly of cellulose fibrils into larger scale structures remains a challenge. Collagen is a major constituent of animal bodies and can be found in large quantities in tendon, bone, skin, cornea, cartilage. A long-standing debate on the packing of collagen molecules into fibrils has led in recent years to a consensus on the collagen fibril structure. Progress has also been made in describing the relation between structure and deformation mechanisms of collagen-rich tissues. The principles for the self-assembly of collagen fibrils into larger scale structures still remain a mystery, though the importance of liquid crystal-like arrangements have been highlighted by recent experiments.
Water bound in bone of rat tail vertebrae was investigated by 1H NMR spectroscopy at 210–300 K and by the thermally stimulated depolarization current (TSDC) method at 190–265 K. The 1H NMR spectra of water clusters were calculated by the GIAO method with the B3LYP/6-31G(d,p) basis set, and the solvent effects were analyzed by the HF/SM5.45/6-31G(d) method. The 1H NMR spectra of water in bone tissue include two signals that can be assigned to typical water (chemical shift of proton resonance δH = 4–5 ppm) and unusual water (δH = 1.2–1.7 ppm). According to the quantum chemical calculations, the latter can be attributed to water molecules without the hydrogen bonds through the hydrogen atoms, e.g., interacting with hydrophobic environment. An increase in the amount of water in bone leads to an increase in the amount of typical water, which is characterized by higher associativity (i.e., a larger average number of hydrogen bonds per molecule) and fills larger pores, cavities and pockets in bone tissue.
A relatively small amount of bone tissue is present in the human body in view of the tissue's structural and chemical importance. The disparity between the constant appearance of mineralized bone matrix, regardless of source, under the electron microscope and the variability of previously reported analyses of bone specimens of a much larger size was disturbing and led to studies of the content and distribution of water in bone tissue and in whole bone specimens. Water in premineralized bone matrix is largely replaced by the non-crystalline (amorphous) and crystalline mineral phase without changes in the overall volume of bone matrix or that of whole bones, nor in the volume and position of collagen fibrils. This resistance to volume and fibril position change during mineralization is probably unique to bone tissue, and permitted critical water analyses of whole bone specimens of varying porosity. When marrow and bone tissue weight and volume analyses were compared with the specific gravity of whole bone specimens, constancy of the composition of bone tissue in these specimens was demonstrated. This result led to a concentration of our studies on the cells that synthesize and control this unique tissue. Currently, the investigations of this laboratory are oriented to study of the enzyme chemistry of isolated bone cells and of whole bone specimens, as well as the localization of matrix-bound and membrane-bound enzymes in bone tissue by histochemical techniques suitable for electron microscopy.
The influence of hydration of rat-tail tendons has been studied by measuring the heat involved in the water fixation deplending on the degree of hydration. The modulus and damping dependences have been measured when changing the temperature for different water uptakes. In situ hydrations have been realized. Evidence of several states of water absorption has been dervied from both calorimetic and dynamic mechanical experiments. A model has been proposed taking into account the energies corresponding to the different regimes of fixation.
Types of "H2O" in human enamel and in precipitated apatites are characterized using X-ray diffraction, infrared (IR) absorption spectroscopic and thermogravimetric analyses. Changes in lattice parameters (principally in the a-axis dimensions) and in the character of the IR absorption bands are correlated with weight losses at pyrolysis temperatures of 100 degrees to 400 degrees C and with effect of rehydration and reignition of previously ignited samples. This study demonstrated that the loss of "H2O" below 200 degrees C is reversible and causes no significant change in the lattice parameter of these apatites, whereas loss of "H2O" between 200 degrees and 400 degrees C is irreversible and causes a contraction in the a-axis dimension. It is proposed that two general types of "H2O" are present in these apatites: (a) adsorbed H2O--characterized by reversibility, thermal instability below 200 degrees C, and lack of effect on lattice parameters; and (b) lattice H2O--characterized by irreversibility, thermal instability between 200 and 400 degrees C, and induction of expansion in the a-axis dimensions of human enamel and precipitated apatites. Lattice H2O is assumed to be due to H2O-for-OH and/or HPO4-for-PO4 substitutions in these apatites. Loss of adsorbed H2O caused sharpening of the OH absorption bands in the spectra of these apatites. Loss of lattice H2O caused the appearance of P-O-P absorption bands (due to the presence of P2O74- group) in precipitated apatites containing small amounts of CO32-.
The interaction of water with collagenous tissue was investigated using dynamic mechanical spectroscopy and cryogenic X-ray techniques. The loss spectrum was found to be very sensitive to water which is highly associated with the macromolecule. Two water-sensitive loss peaks were observed below 0°C: the β2 or “water dispersion” at 150°K and the β1 at 200°K which is attributed to motion of polar side chains. Changes in peak temperature and intensity were not continuous with water content, but exhibited regimes in behavior which were associated with two types of nonfreezable water, structural and bound water. In cryogenic X-ray experiments, specimens which contained some freezable water exhibited reflections identified with the cubic form of ice. These ice crystals underwent an irreversible transition to the more common hexagonal form when warmed above 200°K. On the basis of these experiments, a model for the hydration of native collagenous tissue was proposed.
A short review is given of the water in bone. Various analyses of bone water content are discussed, and its possible location is considered in relation to the behaviour of water in isolated components of bone. Some of the difficulties encountered in examining such microscopic phenomena as water structure in a heterogeneous system such as bone are also discussed.
X-ray diffraction and water sorption data are presented which show that the extracellular water in calcified turkey leg tendon is associated principally with the collagen component.
The mechanical properties of bones are governed by the same principles as those of man-made load-bearing structures, but the organism is able to adapt its bone structure to changes in skeletal loading. In this overview of the determinants of the strength and stiffness of bone, a continuum approach has been taken, in which the behavior of a macroscopic structure depends on its shape and size, and on the mechanical properties of the material within. The latter are assumed to depend on the composition (porosity and mineralization) and organization (trabecular or cortical bone architecture, collagen fiber orientation, fatigue damage) of the bone. The effects of each of these factors are reviewed. Also, the possible means of non-invasively estimating the strength or other mechanical properties of a bone are reviewed, including quantitative computed tomography, photon absorptiometry, and ultrasonic measurements. The best estimates of strength have been obtained with photon absorptiometry and computed tomography, which at best are capable of accounting for 90% of the strength variability in a simple in vitro test, but results from different laboratories have been highly variable.
Compact bone specimens from a wide variety of reptiles, birds, and mammals were tested in tension, and their failure properties related to mineral volume fraction, porosity and histological orientation. The principal findings were that the ultimate strain and the work under the stress-strain curve declined sharply with mineralisation, as did the stress and strain appearing after the specimen had yielded. Ultimate tensile strength was not simply related to any combination of the possible explanatory variables, but some relatively poorly mineralised bones, notably antlers, had high stresses at failure. These high strengths were allowed by a great increase in stress after the bones had yielded at quite low stresses.
Crystal-collagen relationships in calcified turkey leg tendons and cortical bone and dentin of the rat were studied by bright field and selected-area dark field electron microscopy. The latter imaging technique enables the specific and direct visualization of apatite crystal sizes and their crystallographic orientations within collagen fibrils. Cortical bone possessed the longest mean c-axial length (170 +/- 50 A), then the tendon (142 +/- 43 A) and the smallest was dentin (110 +/- 30 A). Crystallographic orientations of apatite were found to alternate between a,b- and c-axial planes along the axial period of longitudinally sectioned collagen. This distribution of apatite may reflect a crystal alignment with collagen molecules as they spiral in superhelical fashion along the long axis of the collagen fibril. Apatite crystals were localized within both the gap and overlap zones of collagen fibrils even at very early stages of mineralization. The relative amounts of mineral within single collagen periods were determined as a function of electron absorbency. In the tendon at the onset of mineralization 80% of the mineral was located in the gap zone and 20% in the overlap zone; with further mineralization these relative amounts changed to 55% in the gap zone and 45% in the overlap zone. This 55/45% ratio observed in the heavily mineralized tendon was also observed in both cortical bone and dentin. The implications of these findings are discussed in view of collagen molecular ordering and the spread of apatite along collagen fibrils.
This investigation determined the relative importance of collagen fiber orientation, porosity, density, and mineralization in determining the tensile strength of bovine cortical bone. Thirty-nine specimens were tested for failure stress and the values of eight histologic and compositional parameters: collagen fiber orientation, wet and dry apparent density, percent mineralization of the bone matrix, and several components of porosity (Haversian canals, Volkmann's canals, and plexiform vascular spaces). Linear regression analysis showed that collagen fiber orientation was consistently the single best predictor of strength. Mineralization of the bone matrix was generally a poor predictor of strength. Density and porosity ranked between these variables in importance. Multiple regression equations containing all significantly correlated variables achieved correlation coefficients of 0.607 for plexiform bone and 0.881 for osteonal bone. Also, separate analysis of plexiform and osteonal specimens showed that the latter group was weaker even though it was less porous, apparently because it had collagen fibers which were less longitudinally oriented. This study suggests it is feasible to develop better empirical formulae for the prediction of cortical bone strength than are currently available if a variety of variables is introduced. Additional data are needed to confirm these results.
Neutron diffraction measurements have been made of the equatorial and meridional spacings of collagen in fully mineralized mature bovine bone and demineralized bone collagen, in both wet and dry conditions. The collagen equatorial spacing in wet mineralized bovine bone is 1.24 nm, substantially lower than the 1.53 nm value observed in wet demineralized bovine bone collagen. Corresponding spacings for dry bone and demineralized bone collagen are 1.16 nm and 1.12 nm, respectively. The collagen meridional long spacing in mineralized bovine bone is 63.6 nm wet and 63.4 nm dry. These data indicate that collagen in fully mineralized bovine bone is considerably more closely packed than had been assumed previously, with a packing density similar to that of the relatively crystalline collagens such as wet rat tail tendon. The data also suggest that less space is available for mineral within the collagen fibrils in bovine bone than had previously been assumed, and that the major portion of the mineral in this bone must be located outside the fibrils.
X-ray diffraction studies were conducted on calcified turkey leg tendon to establish the effect of mineralization on some of the structural properties of collagen. The principal finding was that the first equatorial reflection of collagen in freshly-excised calcified tendon had a d-spacing intermediate between the values for dried collagen and fresh unmineralized collagen. Since this spacing is a sensitive monitor of moisture levels in collagen, the data suggest that mineralization reduces the amount of water than can associatein vivo with the collagen component in tissue. It was also found that the presence of mineral appears to increase the resistance of collagen to permanent thermal denaturation.
There is a considerable variation in the mineralization of bone; normal, non-pathological compact bone has ash masses ranging from 45 to 85% by mass. This range of mineralization results in an even greater range of mechanical properties. The Young modulus of elasticity can range from 4 to 32 GPa, bending strength from 50 to 300 MPa, and the work of fracture from 200 to 7000 Jm-2. It is not possible for any one type of bone to have high values for all three properties. Very high values of mineralization produce high values of Young modulus but low values of work of fracture (which is a measure of fracture toughness). Rather low values of mineralization are associated with high values of work of fracture but low values of Young modulus and intermediate values of bending strength. The reason for the high value for the Young modulus associated with high mineralization is intuitively obvious, but has not yet been rigorously modelled. The low fracture toughness associated with high mineralization may be caused by the failure of various crack-stopping mechanisms that can act when the mineral crystals in bone have not coalesced, but which become ineffective when the volume fraction of mineral becomes too high. The adoption of different degrees of mineralization by different bones, leading to different sets of mechanical properties, is shown to be adaptive in most cases studied, but some puzzles still remain.
Backscattered electron (BSE) imaging is considered to be a useful technique for determining relative differences in bone tissue density. However, it is not clear how graylevel variations seen in BSE images of bone tissue, which are primarily dependent on the tissue's average atomic number, correlate to tissue density (g/cm3) and mineral content. Simulated bone tissues, ranging from 32-50% mineral by volume, were made by mixing synthetic hydroxyapatite with a simulated organic matrix. This technique allowed mineral content to be varied while mineral composition and crystallography remained constant. The densities of the simulated tissues were determined using Archimedes' principle. Average atomic numbers of the simulated tissues were interpolated from a regression of BSE graylevel against average atomic numbers of pure standard materials. A strong positive correlation was found to exist between mineral content and density (r2 = 0.978) as well as between mineral content and atomic number (r2 = 0.965). The average graylevel in the BSE image also exhibited a positive correlation to mineral content (r2 = 0.965) and density (r2 = 0.923). Graylevel variations in BSE images of simulated bone tissue were shown to be strongly correlated to density and mineral content, but only as a coincidence of their association with atomic number.
Several recent results are suggesting that the collagen packing in mineralized tissues is much less regular than in the case of other nonmineralizing collagen, e.g., rat tail tendon. To clarify this question we have investigated the molecular arrangement in mineralized and unmineralized turkey leg tendon as a model for the collagen of mineralized tissues. Using a combination of diffuse x-ray scattering and computer simulation, it could be shown quantitatively that, although the collagen fibril structure is periodic in the axial direction, it is similar to a two-dimensional fluid in the lateral plane. This has important consequences for the understanding of the mineralization process, which is also discussed.
To define the ultrastructural accommodation of mineral crystals by collagen fibrils and other organic matrix components during vertebrate calcification, electron microscopic 3-D reconstructions were generated from the normally mineralizing leg tendons from the domestic turkey, Meleagris gallopavo. Embedded specimens containing initial collagen mineralizing sites were cut into 0.5-micron-thick sections and viewed and photographed at 1.0 MV in the Albany AEI-EM7 high-voltage electron microscope. Tomographic 3-D reconstructions were computed from a 2 degree tilt series of micrographs taken over a minimum angular range of +/- 60 degrees. Reconstructions of longitudinal tendon profiles confirm the presence of irregularly shaped mineral platelets, whose crystallographic c-axes are oriented generally parallel to one another and directed along the collagen long axes. The reconstructions also corroborate observations of a variable crystal length (up to 170 nm measured along crystallographic c-axes), the presence of crystals initially in either the hole or overlap zones of collagen, and crystal growth in the c-axis direction beyond these zones into adjacent overlap and other hole regions. Tomography shows for the first time that crystal width varies (30-45 nm) but crystal thickness is uniform (approximately 4-6 nm at the resolution limit of tomography); more crystals are located in the collagen hole zones than in the overlap regions at the earliest stages of tendon mineralization; the crystallographic c-axes of the platelets lie within +/- 15-20 degrees of one another rather than being perfectly parallel; adjacent platelets are spatially separated by a minimum of 4.2 +/- 1.0 nm; crystals apparently fuse in coplanar alignment to form larger platelets; development of crystals in width occurs to dimensions beyond single collagen hole zones; and a thin envelope of organic origin may be present along or just beneath the surfaces of individual mineral platelets. Implicit in the results is that the formation of crystals occurs at different sites and times by independent nucleation events in local regions of collagen. These data provide the first direct visual evidence from 3-D imaging describing the size, shape, orientation, and growth of mineral crystals in association with collagen of a normally mineralizing vertebrate tissue. They support concepts that c-axial crystal growth is unhindered by collage hole zone dimensions, that crystals are organized in the tendon in a series of generally parallel platelets, and that crystal growth in width across collagen fibrils may follow channels or grooves formed by adjacent hole zones in register.
A basic structural motif of lamellar bone is the arrays of parallel collagen fibrils, with successive arrays having different orientations to form a plywood-like structure. Measurements of the angles between adjacent arrays from cryomicrotomed and vitrified thin sections of demineralized rat bone, cut approximately parallel to the lamellar boundary plane, show that most angles are around 30 degrees, although a subset are around 70 degrees. A structural model for collagen organization based on these measurements is proposed in which an individual lamellar unit (thick and thin lamellae together with transition zones) is composed of five arrays of parallel collagen fibrils, each offset by 30 degrees.
Poroelasticity is a well-developed theory for the interaction of fluid and solid phases of a fluid-saturated porous medium. It is widely used in geomechanics and has been applied to bone by many authors in the last 30 years. The purpose of this work is, first, to review the literature related to the application of poroelasticity to the interstitial bone fluid and, second, to describe the specific physical and modeling considerations that establish poroelasticity as an effective and useful model for deformation-driven bone fluid movement in bone tissue. The application of poroelasticity to bone differs from its application to soft tissues in two important ways. First, the deformations of bone are small while those of soft tissues are generally large. Second, the bulk modulus of the mineralized bone matrix is about six times stiffer than that of the fluid in the pores while the bulk moduli of the soft tissue matrix and the pore water are almost the same. Poroelasticity and electrokinetics can be used to explain strain-generated potentials in wet bone. It is noted that strain-generated potentials can be used as an effective tool in the experimental study of local bone fluid flow, and that the knowledge of this technique will contribute to the answers of a number of questions concerning bone mineralization, osteocyte nutrition and the bone mechanosensory system.
The term "bone" refers to a family of materials that have complex hierarchically organized structures. These structures are primarily adapted to the variety of mechanical functions that bone fulfills. Here we review the structure-mechanical relations of one bone structural type, lamellar bone. This is the most abundant type in many mammals, including humans. A lamellar unit is composed of five sublayers. Each sublayer is an array of aligned mineralized collagen fibrils. The orientations of these arrays differ in each sublayer with respect to both collagen fibril axes and crystal layers, such that a complex rotated plywood-like structure is formed. Specific functions for lamellar bone, as opposed to the other bone types, could not be identified. It is therefore proposed that the lamellar structure is multifunctional-the "concrete" of the bone family of materials. Experimentally measured mechanical properties of lamellar bone demonstrate a clear-cut anisotropy with respect to the axis direction of long bones. A comparison of the elastic and ultimate properties of parallel arrays of lamellar units formed in primary bone with cylindrically shaped osteonal structures in secondary formed bone shows that most of the intrinsic mechanical properties are built into the lamellar structure. The major advantages of osteonal bone are its fracture properties. Mathematical modeling of the elastic properties based on the lamellar structure and using a rule-of-mixtures approach can closely simulate the measured mechanical properties, providing greater insight into the structure-mechanical relations of lamellar bone.
Mechanical properties of single lamellae of human compact and trabecular bone tissue were measured with a combined atomic force microscopy (AFM) and nanoindentation technique. This combination allows for both characterization of bone surface topography and indentation of the bone extracellular matrix (ECM) with depths of between 100 and 600 nm. Four bone structural units (BSUs) were tested with 400 indents under dry conditions, and four BSUs with 160 indents were tested in a liquid cell under physiological conditions. A correspondence was established between the optical appearance of bone lamellae and the topography of the polished bone surface. The indentation modulus and hardness of bone ECM were investigated as a function of lamella type and indentation depth under wet and dry conditions. For low depth indents, thick lamellae showed a higher indentation modulus than thin lamellae. With increasing indentation depth, thick lamellae exhibited a significant decrease in indentation modulus and hardness, whereas, for thin lamellae, the effect of indentation depth was much less significant. These trends were similar for dry and physiological conditions and support compositional and/or ultrastructural differences between thick and thin lamellae.
NMR was used to measure matrix water content in normal and hypomineralized cortical bone. Water content showed an inverse relationship with mineral content, suggesting it could serve as a surrogate measure for the bone's degree of mineralization.
So far, true bone mineral density (DMB; degree of mineralization of bone) can not be measured nondestructively.
Here, a new technique combining 1H nuclear magnetic resonance (NMR) spectroscopy and deuterium isotope exchange was used to measure water content in cortical bone from two groups of rabbits: a control group and a group fed a low-phosphorus (P) diet to induce hypomineralization of the bone matrix.
NMR-derived water content was higher in the P-depleted group and showed an inverse relationship with mineral content (measured gravimetrically and by 31p NMR). Hypomineralized bone was found to be weaker than normal bone as demonstrated by mechanical testing. More importantly, the data showed a strong inverse correlation between water content and bone mechanical properties, which indicates that water content could be predictive of the bone's mechanical competence.
Water content could potentially serve as a surrogate measure for the bone's degree of mineralization, and this technique could be used to study other disorders of mineral homeostasis known to alter the mineralization state of the matrix. Although the method presented here is not suitable for in vivo measurements of bone water content, the authors have previously shown that 1H NMR images of bone can be acquired; thus, noninvasive quantification of bone water may be feasible.
Dielectric spectroscopy has been applied to study aspects of the organization of water in selected animal tissues (tendon, bone and horn). The measurements of the relative permittivity epsilon' and the dielectric loss epsilon'' were carried over the frequency range of 10-100 kHz and at temperatures from 22 to 240 degrees C. The water content was 10% for bone and horn, and 22% for tendon by mass at room temperature at a relative humidity of 70%. The temperature dependencies of epsilon' and epsilon'' reveal distinctively the temperature ranges corresponding to the release of water in temperatures up to about 200 degrees C for all tissues and the melting of the crystalline structure only for tendon and horn, above this temperature. The frequency dependencies of epsilon' and epsilon'' show a remarkable dispersion in the low-frequency at selected temperatures up to 200 degrees C for all tissues due to the release of the loosely and strongly bound water. The results were discussed in terms of the interfacial (Maxwell-Wagner) polarization and polarization mechanism involving hopping charge carriers interacting with the bound water molecules. The information on the effect of temperature, water content and frequency of the electromagnetic field on the dielectric behaviour of the tissues studied is of importance in the design and construction of medical diagnostic or therapeutic instruments based on the use of electric signals.
Dentine is a porous hydrated composite structure that forms the major bulk of the human tooth. The aim of this study was to investigate the role of free water on the in-plane, mechanical strain response in dentine structure. A digital moire interferometry was used for this purpose. It was observed from this experiment that structural dentine demonstrated distinct strain gradients in the axial (perpendicular to the dentinal tubules) and lateral (parallel to the dentinal tubules) directions. The hydrated dentine displayed significant increase in strain with stress in the direction perpendicular to the dentinal tubules, and this response was characteristic of a tough material. On the contrary, the dehydrated dentine, which was dehydrated at 24 degrees C, 55% relative humidity for 72 h showed a strain response characteristic of a brittle material. The strains formed in the direction parallel to the dentinal tubules for hydrated dentine were consistent and did not vary much with increase in applied loads. Upon dehydration, the outer dentine experienced higher strains, and the difference between the outer and inner dentine became more conspicuous with increase in loads. This experiment highlights hydration-induced, distinct in-plane strain gradients in the directions perpendicular and parallel to the dentinal tubules in the dentine structure.
Although the effects of dehydration on the mechanical behavior of cortical bone are known, the underlying mechanisms for such effects are not clear. We hypothesize that the interactions of water with the collagen and mineral phases each have a unique influence on mechanical behavior. To study this, strength, toughness, and stiffness were measured with three-point bend specimens made from the mid-diaphysis of human cadaveric femurs and divided into six test groups: control (hydrated), drying in a vacuum oven at room temperature (21 degrees C) for 30 min and at 21, 50, 70, or 110 degrees C for 4 h. The experimental data indicated that water loss significantly increased with each increase in drying condition. Bone strength increased with a 5% loss of water by weight, which was caused by drying at 21 degrees C for 4 h. With water loss exceeding 9%, caused by higher drying temperatures (> or =70 degrees C), strength actually decreased. Drying at 21 degrees C (irrespective of time in vacuum) significantly decreased bone toughness through a loss of plasticity. However, drying at 70 degrees C and above caused toughness to decrease through decreases in strength and fracture strain. Stiffness linearly increased with an increase in water loss. From an energy perspective, the water-mineral interaction is removed at higher temperatures than the water-collagen interaction. Therefore, we speculate that loss of water in the collagen phase decreases the toughness of bone, whereas loss of water associated with the mineral phase decreases both bone strength and toughness.
Hydrogen-bearing species in the bone mineral environment were investigated using solid-state NMR spectroscopy of powdered bone, deproteinated bone, and B-type carbonated apatite. Using magic-angle spinning and cross-polarization techniques three types of structurally-bound water were observed in these materials. Two of these water types occupy vacancies within the apatitic mineral crystal in synthetic carbonated apatite and deproteinated bone and serve to stabilize these defect-containing crystals. The third water was observed at the mineral surface in unmodified bone but not in deproteinated bone, suggesting a role for this water in mediating mineral-organic matrix interactions. Direct evidence of monohydrogen phosphate in a (1)H NMR spectrum of unmodified bone is presented for the first time. We obtained clear evidence for the presence of hydroxide ion in deproteinated bone by (1)H MAS NMR. A (1)H-(31)P heteronuclear correlation experiment provided unambiguous evidence for hydroxide ion in unmodified bone as well. Hydroxide ion in both unmodified and deproteinated bone mineral was found to participate in hydrogen bonding with neighboring water molecules and ions. In unmodified bone mineral hydroxide ion was found, through a (1)H-(31)P heteronuclear correlation experiment, to be confined to a small portion of the mineral crystal, probably the internal portion.
Water bound in bone of rat tail vertebrae was investigated by 1H NMR spectroscopy at 210-300 K and by the thermally stimulated depolarization current (TSDC) method at 190-265 K. The 1H NMR spectra of water clusters were calculated by the GIAO method with the B3LYP/6-31G(d,p) basis set, and the solvent effects were analyzed by the HF/SM5.45/6-31G(d) method. The 1H NMR spectra of water in bone tissue include two signals that can be assigned to typical water (chemical shift of proton resonance deltaH=4-5 ppm) and unusual water (deltaH=1.2-1.7 ppm). According to the quantum chemical calculations, the latter can be attributed to water molecules without the hydrogen bonds through the hydrogen atoms, e.g., interacting with hydrophobic environment. An increase in the amount of water in bone leads to an increase in the amount of typical water, which is characterized by higher associativity (i.e., a larger average number of hydrogen bonds per molecule) and fills larger pores, cavities and pockets in bone tissue.
The onset of water gelation around a collagen-like triple helix peptide was studied at ambient temperature and pressure by performing Molecular Dynamics simulations. The radial distribution functions of the oxygen and hydrogen atoms of water are distorted below 4 A from the peptide. The distortion is accompanied by the breakdown of the tetrahedral coordination of the hydrogen-bonded network of water molecules. The water shell around the peptide consists of alternating regions of higher and lower density. In agreement with experiments we find that the first hydration shell is kinetically labile, with a residence time in the order of picoseconds for a water molecule. From the computed diffusion coefficient, a key measure of the collective dynamics, we estimate the average diffusion speed decreases by a factor of 1.5 close to the peptide compared to the liquid. Our results give new insight in gel formation and structure on a molecular level.
- J D Currey
Currey, J.D., 2002. Bones, First ed. Structure and Mechanics, Princeton
University Press, New Jersey.
- P A Timmins
- J C Wall
Timmins, P.A., Wall, J.C., 1977. Bone water. Calcif. Tis. Res. 23, 1-5.