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Three Structural Roles for Water in Bone Observed by Solid-State NMR
Abstract
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.
... Hydroxyapatite is a bioactive (direct bonding with living tissue) and a biocompatible, non-immunogenic agent, noninflammatory, non-toxic, and osteoconductive [2]. Hydroxyapatite (Ca10(PO4)6OH2) can observed by several synthetic methods and by natural sources ( extracted human teeth, bovine bone, pig teeth and bones, and cuttlefish) [3,4] Hydroxyapatite needs a temperature of more than 1200°C for densification during conventional sintering. The high temperatures sintering consume energy and time. ...
... The high temperatures sintering consume energy and time. On the other hand, it decreases HA's biological activity, deteriorates its structural stability, and causes loss of the hydroxyl group [4]. Several methods are used to solve this problem, including sintering HA at low temperatures, such as spark plasma sintering (SPS), to consolidate the initial powder in an inert atmosphere at temperatures less than 300°C while retaining its bioactivity [5]. ...
... However, Hassan and Ryu have recently reported the synthesis of high-density iodine-substituted Hydroxyapatite without adding any solvent via CSP, which obviously does not conform to the classical dissolution−precipitation mechanism. Nevertheless, they did not pay attention to this important issue [4]. ...
... A reduction of elastic bulk properties was reported as well as a decrease in the ratios between fibril, mineral and macroscopic strains. Water can be found in different regions of the bone matrix influencing the mineral-organic matrix interactions [46,54]. Apart from pore spaces such as lacunae, canaliculi, and Haversian channels, water is present in a bound form in carbonated apatite as well as in the extracellular bone matrix [46,47,54] and small gap regions [55]. ...
... Water can be found in different regions of the bone matrix influencing the mineral-organic matrix interactions [46,54]. Apart from pore spaces such as lacunae, canaliculi, and Haversian channels, water is present in a bound form in carbonated apatite as well as in the extracellular bone matrix [46,47,54] and small gap regions [55]. It is also present as surface water around mineralised collagen fibrils and mineral platelets which is discussed to affect the energy dissipation [46,54], thus, influencing mechanisms at the extracellular matrix level. ...
... Apart from pore spaces such as lacunae, canaliculi, and Haversian channels, water is present in a bound form in carbonated apatite as well as in the extracellular bone matrix [46,47,54] and small gap regions [55]. It is also present as surface water around mineralised collagen fibrils and mineral platelets which is discussed to affect the energy dissipation [46,54], thus, influencing mechanisms at the extracellular matrix level. It is further postulated that dehydration of the nanocomposite bone has different effects on mineral and collagen, which, in turn, leads to changes in the overall tissue behaviour [47]. ...
The hierarchical design of bio-based nanostructured materials such as bone enables them to combine unique structure-mechanical properties. As one of its main components, water plays an important role in bone's material multiscale mechanical interplay. However, its influence has not been quantified at the length-scale of a mineralised collagen fibre. Here, we couple in situ micropillar compression, and simultaneous synchrotron small angle X-ray scattering (SAXS) and X-ray diffraction (XRD) with a statistical constitutive model. Since the synchrotron data contain statistical information on the nanostructure, we establish a direct connection between experiment and model to identify the rehydrated elasto-plastic micro- and nanomechanical fibre behaviour. Rehydration led to a decrease of 65%-75% in fibre yield stress and compressive strength, and 70% in stiffness with a 3x higher effect on stresses than strains. While in agreement with bone extracellular matrix, the decrease is 1.5-3x higher compared to micro-indentation and macro-compression. Hydration influences mineral more than fibril strain with the highest difference to the macroscale when comparing mineral and tissue levels. The effect of hydration seems to be strongly mediated by ultrastructural interfaces while results provide insights towards mechanical consequences of reported water-mediated structuring of bone apatite. The missing reinforcing capacity of surrounding tissue for an excised fibril array is more pronounced in wet than dry conditions, mainly related to fibril swelling. Differences leading to higher compressive strength between mineralised tissues seem not to depend on rehydration while the lack of kink bands supports the role of water as an elastic embedding influencing energy-absorption mechanisms.
... A reduction of elastic bulk properties was reported as well as a decrease in the ratios between fibril, mineral and macroscopic strains. Water can be found in different regions of the bone matrix influencing the mineral-organic matrix interactions [46,54]. Apart from pore spaces such as lacunae, canaliculi, and Haversian channels, water is present in a bound form in carbonated apatite as well as in the extracellular bone matrix [46,47,54] and small gap regions [55]. ...
... Water can be found in different regions of the bone matrix influencing the mineral-organic matrix interactions [46,54]. Apart from pore spaces such as lacunae, canaliculi, and Haversian channels, water is present in a bound form in carbonated apatite as well as in the extracellular bone matrix [46,47,54] and small gap regions [55]. It is also present as surface water around mineralised collagen fibrils and mineral platelets which is discussed to affect the energy dissipation [46, 54], thus, influencing mechanisms at the extracellular matrix. ...
... Third, it supports the suggestion that water is playing an important role in the mediation of mineralorganic matrix interactions [46,54] within the mineralised collagen fibrils. ...
The multiscale architectural design of bio-based nanostructured materials such as bone enables them to combine unique structure-mechanical properties that surpass classical engineering materials. In biological tissues, water as one of the main components plays an important role in the mechanical interplay, but its influence has not been quantified at the length scale of a mineralised collagen fibre. Here, we combine in situ experiments and a statistical constitutive model to identify the elasto-plastic micro- and nanomechanical fibre behaviour under rehydrated conditions. Micropillar compression and simultaneous synchrotron small angle X-ray scattering (SAXS) and X-ray diffraction (XRD) were used to quantify the interplay between fibre, mineralised collagen fibrils and mineral nanocrystals. Rehydration led to a 65% to 75% decrease of fibre yield stress and compressive strength, and a 70% decrease of stiffness with a 3x higher effect on stress than strain values. While in good agreement with bone extracellular matrix, the decrease is 1.5-3x higher compared to micro-indentation and macro-compression. Hydration has a higher influence on mineral than fibril strain while the highest difference to the macroscale was observed comparing mineral and tissue levels. Results suggest that the effect of hydration is strongly mediated by ultrastructural interfaces while corroborating the previously reported water-mediated structuring of bone apatite providing insights towards the mechanical consequences. Results show that the missing reinforcing capacity of surrounding tissue is more pronounced in wet than dry conditions when testing an excised array of fibrils, mainly related to the swelling of fibrils in the matrix. Differences leading to higher compressive strength between mineralised tissues do not seem to depend on the rehydration state while fibril mobilisation follows a similar regime in wet and dry conditions. The lack of kink bands point towards the role of water as an elastic embedding, thus, adapting the way energy is absorbed.
Statement of significance
Characterising structure-property-function relationships of biomaterials helps us to elucidate the underlying mechanisms that enables the unique properties of these architectured materials. Experimental and computational methods can advance our understanding towards their complex behaviour providing invaluable insights towards bio-inspired material development. In our study, we present a novel method for biomaterials characterisation. We close a gap of knowledge at the micro- and nanometre length scale by combining synchrotron experiments and a statistical model to describe the behaviour of a rehydrated single mineralised collagen fibre. Results suggest a high influence of hydration on structural interfaces, and the role of water as an elastic embedding. Using a statistical model, we are able to deduce the differences in wet and dry elasto-plastic properties of fibrils and fibres close to their natural hydration state.
... 54,59 Solid-state NMR also offers a unique view for the characterization of the silica matrix. The 29 Si MAS NMR spectra show signals originating from siloxanes (Q 4 , around −110 ppm), single silanols (Q 3 , around −100 ppm), and geminal silanols (Q 2 , around −90 ppm), the latter being barely visible ( Figure 2, refer to Scheme S1 for a summary of the observable species). The broad line widths are expected for amorphous silica as they are mainly affected by the dispersion of isotropic chemical shifts arising from a distribution of bond angles and distances. ...
... On the contrary, no remarkable changes in the nanostructure of the silica matrix occur after washing with NaCl with a mean radius of the silica primary units of 4 nm as in the starting material. A direct correlation of 13 C MAS NMR signals from the protein to 29 Si MAS NMR signals from the matrix would enable us to reveal an intimate contact between the two components. 65,66 However, detection of such correlation is necessarily limited since both the 13 C nuclei in the protein and the 29 Si nuclei in silica are in natural abundance (1.1% 13 C and 4.7% 29 Si) in the complex, reducing the probability of a coupled 13 C− 29 Si spin pair to 1 in 2000. ...
... A direct correlation of 13 C MAS NMR signals from the protein to 29 Si MAS NMR signals from the matrix would enable us to reveal an intimate contact between the two components. 65,66 However, detection of such correlation is necessarily limited since both the 13 C nuclei in the protein and the 29 Si nuclei in silica are in natural abundance (1.1% 13 C and 4.7% 29 Si) in the complex, reducing the probability of a coupled 13 C− 29 Si spin pair to 1 in 2000. 65 Therefore, we have chosen to work on the comparison of the 1 H traces and to discriminate among the protons that act as polarization sources for the heteronuclear sites. ...
Lysozyme is widely known to promote the formation of condensed silica networks from solutions containing silicic acid, in a reproducible and cost-effective way. However, little is known about the fate of the protein after the formation of the silica particles. Also, the relative arrangement of the different components in the resulting material is a matter of debate. In this study, we investigate the nature of the protein-silica interactions by means of solid-state nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, and electron microscopy. We find that lysozyme and silica are in intimate contact and strongly interacting, but their interaction is neither covalent nor electrostatic: lysozyme is mostly trapped inside the silica by steric effects.
... Elimination de l'eau non constitutionnelle (Figure I-9-A)De la température ambiante à 500 °C, de l'eau non constitutionnelle s'apparentant à de l'eau physisorbée à la surface des grains de l'HA ou faiblement liée à la structure apatitique (chimisorbée), est éliminée. La désorption des molécules d'eau physisorbées s'effectue en dessousde 200 °C [129- 131], tandis qu'une plus grande énergie thermique est nécessaire pour évacuer l'eau chimisorbée[131][132][133][134][135][136][137][138][139] (entre 200 et 500 °C). L'élimination de ces molécules d'eau est un phénomène réversible qui n'impacte pas la structure de l'HA.Déshydratation des sites A (Figure I-9-B) ...
... The IR bands specific to OH group in HA environment at 3572 ( S) and 628 ( L) cm -1 were visible, as the IR bands specific to PO4 group in HA environment at 559 ( 4), 598 ( 4), 962 ( 1), 1016 ( 3), and 1087 ( 3) cm -IR bands were sharp and narrow. No IR band specific to NO group was observed (data not shown).TG curve of sHA under He can be decomposed in four well known[131][132][133][134][135]139,140,150] consecutive steps (Figure 2C) : RT-100 °C, 100 °C-600 °C, 600 °C-1194 °C and 1194 C-1300 °C. The first mass loss of 0.08% is due to release of adsorbed species, such as water, CO2. ...
... The first mass loss of 0.08% is due to release of adsorbed species, such as water, CO2. The second one of 0.29% can be associated to the removal of organic pollutants resulting from grinding and unbounded structural water[130,132,134,135]. The third mass loss of 1.57% was ascribed to the dehydration of sHA A-sites (Eq. ...
L’incorporation d’ions CO3 dans la structure apatitique apparaît comme un levier d’action pertinent pour améliorer la bio-activité de l’hydroxyapatite (HA), et moduler sa cinétique de biodégradation. L’élaboration de biocéramiques en hydroxyapatite carbonatée (CHA) requiert des traitements thermiques mettant en jeu des réactions solide-gaz avec l’atmosphère, nécessairement riche en CO2. Ces échanges ioniques conditionnent à la fois la pureté, la composition chimique et la microstructure de la pièce finale. Pour la première fois, ces échanges ioniques ont été étudiés. Un protocole original de caractérisation de la composition de l’HA a été développé à cette fin. Les atomes d’oxygène en sites A sont identifiés comme sites réactifs préférentiels. La loi cinétique des échanges apparaît fonction de la frittabilité de la poudre : une réduction de surface spécifique agit comme une force motrice des échanges. Ces réactions solide-gaz ont été modélisées par une approche thermodynamique de l’HA et des CbHA. Les modèles qui en découlent permettent de prédire la température de décomposition d’un produit et sa composition après traitement thermique sous atmosphère contrôlée. Ces échanges ioniques désormais maîtrisés, leur impact sur les mouvements de matière a été étudié. Les échanges avec une atmosphère riche en CO2 décalent la densification à plus haute température mais l’accélère à partir de 1050 °C. L’ajout de vapeur d’eau modifie ces échanges ioniques d’une façon encore mal définie, et permet d’obtenir des microstructures fines. Ce travail fournit un cadre solide pour l’élaboration de substituts osseux en CHA personnalisés, de composition et de microstructure contrôlées.
... TEM tomography has most recently shown that the platelet structures are in fact comprised of sideways-aggregated needle-like structures resulting in a "picket-fence"-like morphology [62,63]. NMR spectroscopy of native bone and model mineral phases have been instrumental in explaining the chemical structures that lead to these mineral morphologies [64,65,61]. 2D 1 H-31 P correlation spectra [64,65,61,66,67] have shown that in addition to more crystalline apatitic structures, bone mineral contains substantial quantities of highlyhydrated, disordered non-apatitic calcium phosphate structures, containing hydrogen phosphate as well as orthophosphate. ...
... NMR spectroscopy of native bone and model mineral phases have been instrumental in explaining the chemical structures that lead to these mineral morphologies [64,65,61]. 2D 1 H-31 P correlation spectra [64,65,61,66,67] have shown that in addition to more crystalline apatitic structures, bone mineral contains substantial quantities of highlyhydrated, disordered non-apatitic calcium phosphate structures, containing hydrogen phosphate as well as orthophosphate. If the bone sample is soaked in D 2 O, the 1 H signal from the water in the hydrated, disordered, non-apatitic structures in bone mineral disappears [61], indicating that this water in can be readily exchanged. ...
... If the bone sample is soaked in D 2 O, the 1 H signal from the water in the hydrated, disordered, non-apatitic structures in bone mineral disappears [61], indicating that this water in can be readily exchanged. This along with many previous solid-state NMR studies of bone mineral has led to a chemical model of bone mineral [64,61,68,69] in which the platelet-type structures have a core of more crystalline hydroxyapatite (likely partially substituted at all ionic sites) and a surface layer of highly-hydrated, amorphous calcium phosphate (Fig. 4B). The presence of this hydrated amorphous mineral layer in the mineral platelets is important for the parallel organization of the platelets in bone, an arrangement which is [62,63]. ...
Solid-state NMR spectroscopy has played an important role in multidisciplinary studies of the extracellular matrix. Here we review how solid-state NMR has been used to probe collagen molecular conformations, dynamics, post-translational modifications and non-enzymatic chemical changes, and in calcified tissues, the molecular structure of bone mineral and its interface with collagen. We conclude that NMR spectroscopy can deliver vital information that in combination with data from structural imaging techniques, can result in significant new insight into how the extracellular matrix plays its multiple roles.
... In terms of composition, bone has 32-44 vol% of organic phase, 33-43vol% of inorganic phase, and 15-25% of water (Granke et al., 2015;Nyman et al., 2006;Wilson et al., 2006). The organic phase comprises mostly of type I collagen (~90%) and some non-collagenous proteins (~10%) (Al-Qtaitat, 2014;Gorski, 2011;Roach, 1994). ...
... Water, as another important constituent in bone, resides in three different compartments in bone matrix (Wilson et al., 2006) and may impose significant effects on the bulk mechanical properties of bone. Acting as a plasticizer, water makes bone tougher, but more compliant and weaker (Broz et al., 1993;Nyman et al., 2006). ...
... It has been well known that the toughness of bone is considerably influenced by its hydration state, with the absence of water making bone behave in a brittle manner (Nyman et al., 2006;Samuel et al., 2014). Among the three forms of water in bone, removal of bound water was shown to have a dominant effect on bone toughness and strength (Nyman et al., 2006;Wilson et al., 2006) (Nyman, et al., 2008, Wilson, et al., 2006. A recent study revealed that water molecules in very small gap regions (<4Å in size) imposed the greatest influence on bone toughness, thus ...
Bone is a highly hierarchical material with different structural features encompassing macro to ultrastructural length scales. Bone toughness is understood to originate at the ultrastructural level where the primary architectural feature is one resembling a hybrid nano-composite of an organic matrix comprised mainly of type-I collagen and mineral crystals intertwined with non-collagenous proteins in the presence of water. Understanding the fundamental features of bone at the ultrastructure and their contributions to the bulk behavior is imperative in understanding mechanistic origins of bone fragility at ultrastructural changes levels induced by skeletal disorders and aging.
The mineral phase in bone is highly textured, most likely due to the functional adaptations of the tissue under load, as the mineral crystals are the primary load bearing components of the tissue. Such textured structures contribute to anisotropy of bone tissues, as seen in polycrystalline materials. Another interesting ultrastructural feature of bone is that the mineral crystals can be distinctive based on their spatial location in the matrix. It has been reported that minerals residing inside collagen fibrils show staggered plate-like structures and are preferentially oriented along the longitudinal axis of collagen fibrils, whereas the minerals residing outside the fibrils possess very limited spatial correlations. It is still unclear how the minerals at distinct ultrastructural locations influence the tissue level deformation of bone.
The ultrastructural stresses in mineral and collagen phases are not consistent with the bulk stress of bone due to the complex architecture of bone. Mineral crystals are inherently anisotropic and the contribution of each crystal to the bulk deformation is orientation dependent. In addition, the intrafibrillar and extrafibrillar differences further exacerbate the difficulty in explaining the mechanistic origins of bone fragility at ultrastructure levels. Such complexity of bone architecture has made it very challenging to experimentally evaluate the mechanical behavior of bone at ultrastructural levels. So far, the work in this field has been limited to computational modeling based approaches or to experimental characterization using highly simplified models. However, such models are not capable of capturing the contribution of intrafibrillar and extrafibrillar minerals to the anisotropy and in situ mechanical behavior of bone. In this study, we propose a semi-empirical approach to characterize the in situ strains and stresses of the mineral phase in the distinctive ultrastructural locations using synchrotron X-ray diffraction techniques and optimization-based texture analysis and strain characterization techniques.
... Hahn-Echo 1 H MAS NMR and 2D 31 P( 1 H) HETCOR MAS NMR spectra of SBF-72h are shown in Fig. 11. The 1 H echo spectra, shown in Fig. 11(a), feature three distinct signals: One broad signal at 4.8 ppm corresponding to water and another shoulder at -0.3 ppm, which is usually assigned to hydroxide ions [47,49,50]. As the HETCOR MAS NMR spectrum in Fig. 11(b) confirms, both sites are in proximity to the precipitated orthophosphate, confirming the formation of amorphous HA phases surrounded by surface-bound water. ...
... Another group of signals consists of two sharp lines at 1.1 ppm and 0.7 ppm. While similar signals have also been observed in HA particles [47,49,50] and for other SBF-immersed glass formations [37], they do not give rise to correlation signals in the 2D 31 P{ 1 H} HETCOR MAS NMR spectrum shown in Fig. 11(b). Hence, these signals are assigned to organic residues [49,52]. ...
This study investigates the bioactivity and microstructural properties of borate-based bioactive 13-93B3 glass produced by the solution combustion synthesis (SCS) method using urea and sucrose fuels at different reaction temperatures. The results show these glasses exhibit unique properties compared to conventionally-synthesized borate-based bioactive glasses. The glass particles produced have porous surfaces, with particle sizes below 100 nm. BET measurements reveal that the mesoporous structure of these glasses is characterized by high-specific surface areas, promoting a high ion release and hydroxyapatite layer formation in simulated body fluid (SBF). The crystallization of the amorphous calcium phosphate was investigated by 1H, 31P MAS, and 2D 31P{1H} heteronuclear correlation (HETCOR) spectroscopy, showing that it progresses linearly up to three days. The produced glasses were tested for their effects on the viability of 3T3 cells and were found to have no toxic effects.
Therefore, the produced glasses are promising candidates for tissue engineering applications.
... Additional signals from highly hydrated, disordered ortho-phosphate and hydrogen phosphate anions have been observed previously, and assigned to disordered non-apatitic material on the surfaces of HAp domains. 15,16,20,46,[50][51][52][53][54] The spectrum for the 10-day mixed citrate-lactate material (Figs 4A, B) shows strong similarities to that for mature bone (Figs 4 C, D, top)): the 31 P ~3 ppm -1 H 0 ppm apatitic signal, the water 1 H signal with chemical shift maximum at 5.5 ppm compared with 5.4 ppm in adult bone (in the context of a range of ~4.8 -~6 ppm for water in calcium phosphate minerals 56 ) and the distribution of hydrogen phosphate 1 H chemical shifts. 20 The 31 P spectra correlating with the 5.4 ppm water 1 H and 9.5 ppm HPO4 2-1 H signals for bone (Fig 4D, top) show strong similarities in chemical shift range and intensity distribution to those for the mixed citrate-lactate material (Fig 4B). ...
... Our experimental data is consistent with the HAp needles in the mixed citrate-lactate material having two different types of surface: "internal" surfaces with hydrated, citratecontaining interlayers between HAp needles and external surfaces containing water and hydrogen phosphate similar to those on synthetic nanocrystalline HAp. 15,18,19,46,50,52,53,71 Where bone mineral platelets stack together, these latter external surfaces will form the interplatelet interfaces. It seems likely that metabolic anions are also present in these interplatelet interfaces. ...
Bone mineral has a complex 3D architecture that is essential to its mechanical properties. It is a complex calcium phosphate phase related to hydroxyapatite that also contains significant quantities of cell respiration metabolites, in particular: carbonate, citrate and lactate. An as-yet unanswered question is what, if any, role do these metabolites collectively play in determining the 3D architecture of bone mineral? Here we synthesize apatitic materials by transformation from precursor mineral phases containing citrate, lactate or carbonate so that the synthesis environment mimics the densely-packed ionic environment within which bone mineral forms in vivo, and so that we can understand the mineral factors that may direct bone mineral 3D architecture. We show that incorporating citrate and lactate leads to complex mineral architectures reminiscent of those in bone mineral, including curvature of the mineral crystals. Our results suggest that metabolic acids may assist the moulding of bone mineral to restricted spaces available for mineral in in vivo bone. We find that the incorporation of lactate creates a softer material and inhibits the transformation towards apatitic structures, which may help to explain why foetal bone, necessarily soft, contains considerable quantities of lactate. High levels of plasma citrate have been previously found to correlate with high bone mineral density. Here we find that citrate incorporation leads to mineral crystal curvature modelling that in in vivo bone mineral suggesting its importance in mineral morphology. We conclude that metabolic anions may play an important role in controlling bone mineral physicochemical properties and 3D architecture.
... After collagen and calcium phosphate, water is the third most common compound in bone structures, making up to 31% [1]. Studies of water within bone structures are limited compared to other constituents, despite its fundamental role in the structural makeup [2]. The ability to monitor hydration becomes important when diseases in bones, such as metastatic defects and osteoporosis, can modify the water content of the bone tissue. ...
... Tissue transfer and handling was conducted under approval of the National Research Ethics Service (15/NW/0079) and in accordance with the Human Tissue Act 2004. It should be noted that this study assumes that the dependence of the terahertz complex permittivity of the two main constituent materials of bone, i.e. hydroxyapatite (HA) and collagen [2], with respect to temperature is negligible compared to water. Figure 2 Experimental setup of the TDS system in reflection configuration. ...
... Hardware updates coupled with technological advancement in ultra-fast magic angle spinning (MAS) methods has improved resolution and sensitivity of ssNMR by attenuating the broadening effects of dipolar couplings and chemical shift anisotropy (Brown, 2012;Hodgkinson and Wimperis, 2009), making the direct evaluation of amide regions, collagen, and their associated water molecules a possibility (Mroue et al., 2015). The technique is credited with the discovery of structural water, which had not been determined until ssNMR studies of mineralized tissue were conducted (Casciani, 1971;Wilson et al., 2005Wilson et al., , 2006. Numerous ssNMR experiments have efficaciously probed water across all four of its identified phases and are briefly discussed below. ...
... Finally, 2D 1 H -31 P heteronucleation correlation (HetCor) experiments, based on the distance-dependent heteronuclear dipolar coupling between phosphate and hydrogen, can be used to measure bound water content relative to inorganic content. 2D HetCor was used to document, for the first time, the presence of structural water in three different locations: occupying vacancies within mineral crystal apatite, in the internal portion of the mineral crystal, and at the mineral surface (Wilson et al., 2006). When used in experiments of bone healing, 1 H -31 P HetCor was able to document the earliest stages of biomineral formation (and associated water changes) including the size and shape of mineral apatite (Vyalikh et al., 2017) and, in preclinical studies HetCor has shown it can differentiate bound water with treatment . ...
Water constitutes roughly a quarter of the cortical bone by volume yet can greatly influence mechanical properties and tissue quality. There is a growing appreciation for how water can dynamically change due to age, disease, and treatment. A key emerging area related to bone mechanical and tissue properties lies in differentiating the role of water in its four different compartments, including free/pore water, water loosely bound at the collagen/mineral interfaces, water tightly bound within collagen triple helices, and structural water within the mineral. This review summarizes our current knowledge of bone water across the four functional compartments and discusses how alterations in each compartment relate to mechanical changes. It provides an overview on the advent of- and improvements to- imaging and spectroscopic techniques able to probe nano-and molecular scales of bone water. These technical advances have led to an emerging understanding of how bone water changes in various conditions, of which aging, chronic kidney disease, diabetes, osteoporosis, and osteogenesis imperfecta are reviewed. Finally, it summarizes work focused on therapeutically targeting water to improve mechanical properties.
... Water represents up to 25% of bone [37]. Although room-temperature dehydrated bone samples lose most of the bulk water and surface water layer, strongly bound and structural water remains [38]. Indeed, even after a 225 • C drying process, ∼40% of such tightly bound and structural water remains [38]. ...
... Although room-temperature dehydrated bone samples lose most of the bulk water and surface water layer, strongly bound and structural water remains [38]. Indeed, even after a 225 • C drying process, ∼40% of such tightly bound and structural water remains [38]. Hence, structural water is expected in the bone samples, while the collagen samples are dehydrated [39]. ...
Terahertz imaging is becoming a biological imaging modality in its own right, alongside the more mature infrared and X-ray techniques. Nevertheless, extraction of hyperspectral, biometric information of samples is limited by experimental challenges. Terahertz time domain spectroscopy reflection measurements demand highly precise alignment and suffer from limitations of the sample thickness. In this work, a novel hybrid Kramers-Kronig and Fabry-Pérot based algorithm has been developed to overcome these challenges. While its application is demonstrated through dielectric retrieval of glass-backed human bone slices for prospective characterisation of metastatic defects or osteoporosis, the generality of the algorithm offers itself to wider application towards biological materials.
... The elastic moduli of bone range from 2 to 33 GPa, and the bending strengths range from 27 to 308 MPa [10,16]. Water plays an important role in the mechanical properties of bone [8,[17][18][19][20]. Water removal causes increased stiffness but decreased toughness in bone [21]. ...
Inspired by the process of bone formation in living organisms, many studies have been conducted to develop organic–inorganic composite materials by preparing calcium phosphate crystals within solutions or dispersions of polymers with appropriate functional groups. Bones are composite materials consisting of organic polymers (mainly type I collagen), carbonated apatite, and water, with volume fractions of 35–45%, 35–45%, and 15–25%, respectively. Carbonated apatite in bone contributes to rigidity, while organic polymers and water contribute to toughness. The inorganic crystal, carbonated apatite, is a plate-shaped crystal with dimensions of 50 nm × 25 nm × 1–4 nm, generating a significant organic–inorganic interface, due to its nanoscale size. This interface is believed to absorb externally applied forces to dissipate mechanical energy to thermal energy. Creating such nanometer-scale structures using top-down approaches is challenging, making bottom-up methods, such as the coprecipitation of polymer and inorganic crystals, more suitable. In this account, efforts to develop eco-friendly mechanical materials using biomass, such as cellulose and starch, based on the bottom-up approach to bone-like composites are described.
... The transverse orientation of collagen fibrils is more common in the cortex that experiences compression, whereas their longitudinal orientation predominates in areas that are primarily stretched [3][4][5][6]. The oriented organic molecules of the extracellular matrix of the calcified tissue of vertebrates playing an important role in the structuring of the apatite phase [6][7][8][9][10][11] initiate biologically necessary substitutions and vacancies through the aqueous medium [11][12][13][14] and form the necessary biochemical environment in the mineralization area [6,[15][16][17]. ...
Mechanisms responsible for spatiotemporal changes in the atomic-molecular architecture of the human femur in intact and osteoarthritis-affected areas were studied using high-resolution X-ray diffraction and spectroscopic techniques. Comparison of the experimental data demonstrates strong deviations of core electron-binding energies, lattice constants of hydroxyapatite crystal cells, linear sizes of crystallites, and degrees of crystallinity for both intact and osteoarthritic areas. The quantitative values of these characteristics and their standard deviations in each area are measured and presented. A systematic analysis of the site-dependent deviations was carried out within the framework of the 3D superlattice model. It is argued that the main mechanism responsible for the deviations arises primarily as a result of carbonization and catalytic reactions at the mineral-cartilage interface. The impact of the mechanism is enhanced in the vicinities of the area of sclerosed bone, but not inside the area where mechanical loads are maximum. Restoration of the atomic-molecular architecture of mineralized bone in the sclerosis area is revealed. Statistical aspects of the spatiotemporal changes in mineralized bone under pathogenic conditions are discussed.
... Investigation of the cortical bones via ultrahigh-field MR depends on the clear distinction between different pools of 1 H signals. In principle, distinctions can be made between the spatial distribution of the signal intensity, the T1, T2 and T2* relaxation 20,22,24 , the diffusivity 27-29 , the chemical shift spectrum 12,21,22,38 , and the double-quantum signal 28 . Continuous efforts have been made to develop MR techniques and strategies for characterizing bones in vitro and in vivo 27,39-41 . ...
Compact, mineralized cortical bone tissues are often concealed on magnetic resonance (MR) images. Recent development of MR instruments and pulse techniques has yielded significant advances in acquiring anatomical and physiological information from cortical bone despite its poor 1H signals. This work demonstrates the first MR research on cortical bones under an ultrahigh magnetic field of 14 T. The 1H signals of different mammalian species exhibit multi-exponential decays of three characteristic T2 or T2* values: 0.1–0.5 ms, 1–4 ms, and 4–8 ms. Systematic sample comparisons attribute these T2/T2* value ranges to collagen-bound water, pore water, and lipids, respectively. Ultrashort echo time (UTE) imaging under 14 T yielded spatial resolutions of 20–80 microns, which resolves the three-dimensional anatomy of the Haversian canals. The T2* relaxation characteristics further allow spatial classifications of collagen, pore water and lipids in human specimens. Our study achieves a record of the spatial resolution for MR imaging in bone and shows that ultrahigh-field MR has the unique ability to differentiate the soft and organic compartments in bone tissues.
... Meanwhile, the broad peaks ranging from 5.67 to 6.59 were presumed to be surface water. The 1 H [38], 2 H NMR [39], and 2D 1 H-31 P HETCOR [40,41] experiments revealed that the broad peak corresponded to protons from either adsorbed or structural water. Yesinowski and Eckert identified surface-adsorbed water at the 5.6 ppm peak because this peak was not observed with lower surface areas and its intensity decreased upon exposure to D 2 O (deuterium oxide) [38]. ...
Dental research often uses bovine teeth as a substitute for human teeth. The aim of this study was to evaluate differences in the crystalline nanostructures of enamel and dentin between bovine and human teeth, using X-ray diffraction (XRD) and solid-state nuclear magnetic resonance (NMR). The crystallite size (crystallinity) and microstrains were analyzed using XRD with the Rietveld refinement technique and the Halder–Wagner method. The 31P and 1H NMR chemical environments were analyzed by two-dimensional (2D) 1H-31P heteronuclear-correlation (HETCOR) magic-angle spinning (MAS) NMR spectroscopy. Enamel had a greater crystallite size and fewer microstrains than dentin for both bovine and human teeth. When compared between the species, the bovine apatite had a smaller crystallite size with more microstrains than the human apatite for both dentin and enamel. The 2D HETCOR spectra demonstrated that a water-rich layer and inorganic HPO4− ions were abundant in dentin; meanwhile, the hydroxyl group in the lattice site was more dominant in enamel. A greater intensity of the hydroxyl group was detected in human than in bovine for both dentin and enamel. For 31P projections, bovine dentin and bovine enamel have wider linewidths than human dentin and human enamel, respectively. There are differences in the crystallite profile between human and bovine. The results of dental research should be interpreted with caution when bovine teeth are substituted for human teeth.
... T. Clauzel et al. structure (LeGeros et al., 1978;Wilson et al., 2006;Wang et al., 2013) and rightly contributes to the enamel δ 2 H signature. Moreover, enamel hydroxy-apatite is currently considered as hydroxyl-deficient (Loong et al., 2000;Cho et al., 2003), and δ 2 H enamel "essential contributors" may be HPO 4 3− groups (Loong et al., 2000) and structural H 2 O. ...
Stable hydrogen isotope measurement of body tissues faces analytical and interpretative challenges such as hydrogen exchange with atmosphere or competitive influence of drinking water and food intake. Samples from the Gallic site of Thézy-Glimont, France, have already been investigated isotopically for climate reconstruction and diet investigation of the buried individuals. This allows comparison with the hydrogen isotope composition (δ²H) of bone collagen, tooth enamel and of bulk bone measured for 8 humans and 11 animals. Three of the best-preserved human skeletons were incrementally sampled and show acceptable homogeneity of δ²H values of bone collagen (<5‰) and of bulk bone (<10‰) despite various turnover rates of these tissues. Human tooth enamel records breastfeeding as attested by δ²H values of pre-weaning teeth which are ²H-enriched by +20 to +30‰. We observe that the δ²H of bone collagen and bulk bone are strongly correlated. The δ²H signatures of bone collagen, tooth enamel and bulk bone record both climate conditions and dietary practices, as attested by linear relationships with traditional isotopic proxies (δ¹⁸O, δ¹³C, δ¹⁵N) which were previously measured on the same samples, although interpretations depend on the sampling strategy of each study. Measurements of δ²H in bulk bone and dental tissues are more readily achievable than collagen which requires extraction and purification and could become crucial in studies where bone tissue is scarce or when the only available remains are tooth material.
... While often referred to as "hydroxyapatite", bone mineral is known to be chemically quite different from pure hydroxyapatite [30,35]. First, bone mineral has very little hydroxyl ion content, with so-called structural water occupying most of the hydroxyl sites and some other lattice positions via substitution, and OH − being found extensively only in mature crystallites [30,36]. Low hydroxyl content in bone mineral has been linked to high susceptibility for dissolution and low acid-buffering capability which enables resorption by osteoclasts in vivo [29]. ...
Bone waste is a problematic slaughterhouse waste typically disposed of in landfills. The pyrolyzed product of this waste shows strong potential in mine and industrial waste water remediation and work is needed to identify chemical and structural parameters which drive performance. Diffuse Reflectance Fourier Transform Spectroscopy (DRIFTS) was used to probe carbonate (CO 3 ²⁻ ), phosphate (PO 4 ³⁻ ) and hydroxyl (OH ⁻ ) environments of mineral phases and functional group chemistry in carbonaceous phase, revealing a potentially synergistic functionality between the two in bone char. CO 3 ²⁻ and water substitutions in the mineral lattice were found to persist after pyrolysis to 750 °C, and more soluble non-apatite calcium phosphate phases were observed using second derivative analysis of the v 3 PO 4 ³⁻ band. Nitrogen-rich functional groups were found in the carbonaceous phase which are associated with complexation of aqueous metals, and ordered aromatic clusters identified by Raman spectroscopy indicate a porous carbon skeletal structure to promote metals adsorption and complexation. These results point to unique chemical and structural features of bone char which are not easily replicated by synthetic carbonated apatite or activated carbon and which contribute to the excellent aqueous metals removal power of bone char.
Graphical Abstract
... Despite this correlation between δ 1 H at ∼1 ppm and PO 4 3− detected by 1 H → 31 P HETCOR, the intensity of this correlation is found to be weak and independent of the intensities of H 0.8 and H 1.3 . 39,63,93,94 The 43 Ca MAS NMR studies by Laurencin demonstrated that correlations between H 0.8 and H 1.3 and calcium in the thermally treated rodlike HAP NPs provided additional information that support the assignment of these Ca-associated hydroxyl groups. 95 The chemical shifts of these peaks are detected at ∼1.0 and 1.4 ppm in their study and shift downfield systematically by 0.1 ppm, which could be due to the different standards chosen for referencing. ...
... Both spectra have narrow signals at 0.1 ppm (Figure 3a) and at 0.0 ppm (Figure 3b), respectively. These peaks might result from the hydroxyl groups [37,38]. The signal of the cleaned fibers displays higher intensity than the one of ADH-loaded kapok. ...
Kapok fibers (Ceiba pentandra) were modified for the removal of copper ions from aqueous solutions through adsorption. In this fast and facile method, the polysaccharide-like groups of kapok were oxidized with potassium periodate. The novel modification is the loading of the fibers with adipic dihydrazide (ADH) which contain nitrogen and oxygen atoms for heavy metal ion binding. Adsorption experiments have been carried out and analyzed via atom absorption spectroscopy and ultraviolet/visible spectroscopy. In preliminary adsorption experiments, different kapok-based materials have been analyzed on their adsorption capacity and removal efficiency via atom absorption spectroscopy. ADH-modified fibers showed the best results and an increase of copper removal efficiency by 30% in comparison to untreated kapok fibers and superior adsorption capacity compared to kapok fibers loaded with oxalic dihydrazide (ODH). Moreover, the impact of initial concentration and contact time on the adsorption capacity and on the removal efficiency values of the ADH-modified kapok fibers has been studied. Another comparison of the ADH-modified fibers with raw kapok which was cleaned with Milli-Q water, dichloromethane and ethylene glycol showed that the new adsorbents are best suited for copper solutions with concentration values of under 10 mg/L. The heavy metal adsorption experiments were analyzed through both isotherm models Langmuir and Freundlich. The Langmuir model is found to be a suitable model for copper ions. The value of the maximum adsorption capacity is 4.120 mg/g. The ADH-modified kapok fibers were characterized with attenuated total reflection infrared (ATR-IR) spectroscopy, magic-angle spinning nuclear magnetic resonance (MAS-NMR) spectroscopy and scanning electron microscopy (SEM).
... 15 Moreover, water and its role in bone have been extensively studied by NMR spectroscopy and the relaxation methodology. 9,14,22 Recent advancements in ssNMR, including BioSolids CryoProbe and high-field dynamic nuclear polarization (DNP) based solid-state NMR instrumentation and methodologies, have solved the problem of sensitivity enhancement in bone-like complex materials to a great extent. 23−26 However, exact knowledge about the water-mediated changes inside the bone mineral is lacking, and further work is needed to characterize the role of water in bone mineralization. ...
Bone is a dynamic tissue composed of organic proteins (mainly type I collagen), inorganic components (hydroxyapatite), lipids, and water that undergoes a continuous rebuilding process over the lifespan of human beings. Bone mineral is mainly composed of a crystalline apatitic core surrounded by an amorphous surface layer. The supramolecular arrangement of different constituents gives rise to its unique mechanical properties, which become altered in various bone-related disease conditions. Many of the interactions among the different components are poorly understood. Recently, solid-state nuclear magnetic resonance (ssNMR) has become a popular spectroscopic tool for studying bone. In this article, we present a study probing the interaction of water molecules with amorphous and crystalline parts of the bone mineral through 31P ssNMR relaxation parameters (T 1 and T 2) and dynamics (correlation time). The method was developed to selectively measure the 31P NMR relaxation parameters and dynamics of the crystalline apatitic core and the amorphous surface layer of the bone mineral. The measured 31P correlation times (in the range of 10-6-10-7 s) indicated the different dynamic behaviors of both the mineral components. Additionally, we observed that dehydration affected the apatitic core region more significantly, while H-D exchange showed changes in the amorphous surface layer to a greater extent. Overall, the present work provides a significant understanding of the relaxation and dynamics of bone mineral components inside the bone matrix.
... The NMR assignment for the 1 H signals has previously been reported [35]. Notably, the 1 H-signals corresponding to adsorbed water molecules (strongly and weakly adsorbed, SAW and WAW, respectively) [36,37] were only slightly affected by the incorporation of cis-Pt molecules in the formulation. On the other hand, the 1 H NMR signals of the polymeric content, in which the S-CDs or N-CDs are dispersed, were highly affected by the cis-Pt binding. ...
S or N-doped carbon dots (S-, N-CDs) and their cisplatin (cis-Pt) derivatives were tested on two ovarian cancer cell lines: A2780 and A2780 cells resistant to cis-Pt (A2780R). Several spectroscopic techniques were employed to check S, N-CDs' functionalization with cis-Pt: nuclear magnetic resonance, matrix-assisted laser desorption, ionization time-of-flight mass spectrometry, and X-ray photoelectron spectroscopy. In addition, synchrotron-based Fourier Transformed Infrared (SR-FTIR) spectro-microscopy was used to evaluate the biochemical changes in cells after treating with cis-Pt, S, N-CDs or S-, N-CDs@cis-Pt. Computational chemistry was applied to establish the model for the most stable bond between S and N-CDs and cis-Pt. The results revealed the successful modification of S-, N-CDs with cis-Pt and the formation of a stable composite system that can be used for drug delivery to cancer cells and likewise to overcome acquired
cis-Pt resistance. Treatment of A2780 and A2780R cells led to the changes in the structure of lipids, proteins and nucleic acids depending on the treatment. The results showed that S and N-CDs derivate of cisplatin (cis-Pt) with might overcome the cis-Pt resistance and thus may increase the efficiency of the adenocarcinoma therapy, confirming the potential of S, N-CDs as drug delivery systems.
... Here, we investigate and compare the cleithrum bone from a primordial fish, sturgeon ( Acipenser guldenstatti ), characterized by having osteocytes in its bones, and a cleithrum bone from an anosteocytic neoteleost fish, pike ( Esox lucius of the Esociform order), which lack these cells [20] . Select advanced solid-state NMR (ss-NMR) techniques are used to obtain a high-resolution picture of the bone material constituents [21][22][23][24] . We find molecular differences in the collagen fibrillar composition and organization, the structure of the organic-inorganic junctions in the two bones, and water accessibility to mineral surfaces. ...
Bone is a fascinating biomaterial comprised mostly of type-I collagen fibers as an organic phase, apatite as an inorganic phase, with water molecules residing at the interfaces between these phases. They are hierarchically organized with minor constituents such as non-collagenous proteins, citrate ions and glycosaminoglycans into a composite structure that is mechanically durable yet contains enough porosity to accommodate cells and blood vessels. The nanometer scale organization of the collagen fibrous structure and the mineral constituents in bone were recently extensively scrutinized. However, molecular details at the lowest hierarchical level still need to be unraveled to better understand the exact atomic-level arrangement of all these important components in the context of the integral structure of the bone. In this report, we unfold some of the molecular characteristics differentiating between two load-bearing (cleithrum) bones, one from sturgeon fish, where the matrix contains osteocytes and one from pike fish where the bone tissue is devoid of these bone cells. Using enhanced solid-state NMR measurements, we underpin disparities in the collagen fibril structure and dynamics, the mineral phases, the citrate content at the organic-inorganic interface and water penetrability in the two bones. These findings suggest that different strategies are undertaken in the erection of the mineral-organic interfaces in various bones characterized by dissimilar osteogenesis or remodeling pathways and may have implications to the mechanical properties of the particular bone.
Statement of Significance
Bone boasts unique interactions between collagen fibers and mineral phases through interfaces holding together this bio-composite structure. Over evolution, fish have gone from mineralizing their bones aided by certain bone cells called osteocytes, like tetrapod, to mineralization without these cells. Here, we report atomic level differences in collagen fiber cross linking and organization, porosity of the mineral phases and content of citrate molecules at the bio-mineral interface in bones from modern versus ancient fish. The dissimilar structural features may suggest disparate mechanical properties for the two bones. Fundamental level understanding of the organic and inorganic components in bone and the interfacial interactions holding them together is essential for successful bone repair and for treating better tissue pathologies.
... Water exists in two forms in bone, i.e., water bonded with proteins and free water in pores [35]. It is difficult to separate bounded and free water in bone; the concern is that excessive removal of bounded water can degrade proteins and thus substantially change their mechanical properties [35,36]. By considering bone as an organic-inorganic composite material, the organic and inorganic content can be determined by ashing. ...
Background:
Bone mineral density is widely used by clinicians for screening osteoporosis and assessing bone strength. However, its effectiveness has been reported unsatisfactory. In this study, it is demonstrated that bone organic-inorganic phase ratio is a fundamental determinant of bone material quality measured by stiffness, strength, and toughness.
Methods and results:
Two-hundred standard bone specimens were fabricated from bovine legs, with a specialized manufacturing method that was designed to reduce the effect of bone anisotropy. Bone mechanical properties of the specimens, including Young's modulus, yield stress, peak stress, and energy-to-failure, were measured by mechanical testing. Organic and inorganic mass contents of the specimens were then determined by bone ashing. Bone density and organic-inorganic phase ratio in the specimens were calculated. Statistical methods were applied to study relationships between the measured mechanical properties and the organic-inorganic phase ratios. Statistical characteristics of organic-inorganic phase ratios in the specimens with top material quality were investigated. Bone organic-inorganic phase ratio had strong Spearman correlation with bone material properties. Bone specimens that had the highest material quality had a very narrow scope of organic-inorganic phase ratio, which could be considered as the "optimal" ratio among the tested specimens.
Conclusion:
Bone organic-inorganic phase ratio is a fundamental determinant of bone material quality. There may exist an "optimal" ratio for the bone to achieve top material quality. Deviation from the "optimal" ratio is probably the fundamental cause of various bone diseases. This study suggests that bone organic-inorganic phase ratio should be considered in clinical assessment of fracture risk.
... The collagen fibrils rotate in relation to the lamellar front in 2D, and the mineral planes [9], [11] rotate in relation to both the collagen fibrils and the lamellar front in 3D [12]- [15]. 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]. ...
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.
... Water makes up about a 1/4 of a bone's mass [38]. Bone is also a fluidimbibed material in which the distribution of water affects the mechanical properties of bone. ...
Background
The purpose of the study was to examine how bone mineral density (BMD) is related to body composition depending on the practiced sport (endurance, speed-power, throwing sports) in participants of the World Masters Athletics Championship.
Methods
Dual-energy X-ray absorptiometry (DXA) was used to determine BMD and bone mass (BMC). Body composition was analyzed by means of the JAWON Medical X-scan analyzer using bioelectrical impedance methods. Percentage body fat (%BF), body fat mass (BFM), lean body mass (LBM), total body water (TBW), soft lean mass (SLM), intracellular water (ICW), and extracellular water (ECW) were evaluated.
Results
Among men, the most important variables affecting the BMD norm were LBM (OR = 32.578; p = 0.023), ECW (OR = 0.003; p = 0.016) and ICW (OR = 0.011; p = 0.031), in the distal part and SLM (OR = 5.008; p = 0.020) and ICW (0.354, p = 0.008) in the proximal part. In women, the most important predictors of normal BMD were ICW (OR = 10.174; p = 0.003) and LBM (OR = 0.470; p = 0.020) in the distal part and ICW (OR = 5.254; p = 0.038) in the proximal part.
Conclusion
The representatives of strength based events had the most advantageous BMD levels. The condition of bone tissue evaluated by BMC and BMD of the forearm in masters athletes was strongly determined by the level of lean body components and the type of sports training associated with the track and field event. In the most important predictors of the BMD norm were also hydration components ECW and ICW. However, this relationship requires more research on the nature and mechanisms of these interactions.
This study contributes to advancing the understanding of methylcellulose-based injectable bone substitutes and their underlying mechanisms of gelation and mineralization.
This comprehensive compendium unravels the intricacies of three common and daunting skeletal disorders: osteoporosis, osteoarthritis, and rheumatoid arthritis. These ailments afflict people across all age groups, demanding a deeper understanding of their diagnostic, prognostic, preventive, and therapeutic dimensions.
It presents seven key topics written by medical experts that explore research on these diseases:
Chronic Lung Disease and Osteoporosis
An exploration of the intricate link between chronic lung ailments and osteoporosis.
AI Detection of Knee Osteoarthritis
Recent use of artificial intelligence aiding knee osteoarthritis identification.
Inflammatory Signalling in Rheumatoid Arthritis
Covers the role of cytokines and chemokines in the context of rheumatoid arthritis.
Vitamin D, Immune System, and Bone Health
Unveils the vital implications of Vitamin D on the immune system and bone health.
Bone Water and Hydration Effects
A review of the impact of drugs on bone hydration status through the lens of bone water.
Dietary Patterns and Rheumatoid Arthritis
An analysis of the connection between dietary habits and rheumatoid arthritis.
Quality of Life in Rheumatoid Arthritis Patients (Chapters 112-130):
An examination of the self-perceived quality of life in Rheumatoid Arthritis patients, comparing South Asian and British White populations.
This knowledge-rich treatise is a valuable resource for patients and their families battling these skeletal ailments. It's equally beneficial for medical students, orthopedists, researchers, and anyone eager to grasp the complexities of these widespread skeletal pathologies.
Compact, mineralized cortical bone tissues are often concealed on magnetic resonance (MR) images. Recent development of MR instruments and pulse techniques has yielded significant advances in acquiring anatomical and physiological information from cortical bone despite its poor ¹H signals. This work demonstrates the first MR research on cortical bones under an ultrahigh magnetic field of 14 T. The ¹H signals of different mammalian species exhibit multi‐exponential decays of three characteristic T2 or T2* values: 0.1–0.5 ms, 1–4 ms, and 4–8 ms. Systematic sample comparisons attribute these T2/T2* value ranges to collagen‐bound water, pore water, and lipids, respectively. Ultrashort echo time (UTE) imaging under 14 T yielded spatial resolutions of 20–80 microns, which resolves the 3D anatomy of the Haversian canals. The T2* relaxation characteristics further allow spatial classifications of collagen, pore water and lipids in human specimens. The study achieves a record of the spatial resolution for MR imaging in bone and shows that ultrahigh‐field MR has the unique ability to differentiate the soft and organic compartments in bone tissues.
Raloxifene (RAL) reduces clinical fracture risk despite modest effects on bone mass and density. This reduction in fracture risk may be due to improved material level-mechanical properties through a non-cell mediated increase in bone hydration. Synthetic salmon calcitonin (CAL) has also demonstrated efficacy in reducing fracture risk with only modest bone mass and density improvements. This study aimed to determine if CAL could modify healthy and diseased bone through cell-independent mechanisms that alter hydration similar to RAL. 26-week-old male C57BL/6 mice induced with chronic kidney disease (CKD) beginning at 16 weeks of age via 0.2 % adenine-laced casein-based (0.9 % P, 0.6 % C) chow, and their non-CKD control littermates (Con), were utilized. Upon sacrifice, right femora were randomly assigned to the following ex vivo experimental groups: RAL (2 μM, n = 10 CKD, n = 10 Con), CAL (100 nM, n = 10 CKD, n = 10 Con), or Vehicle (VEH; n = 9 CKD, n = 9 Con). Bones were incubated in PBS + drug solution at 37 °C for 14 days using an established ex vivo soaking methodology. Cortical geometry (μCT) was used to confirm a CKD bone phenotype, including porosity and cortical thinning, at sacrifice. Femora were assessed for mechanical properties (3-point bending) and bone hydration (via solid state nuclear magnetic resonance spectroscopy with magic angle spinning (ssNMR)). Data were analyzed by two-tailed t-tests (μCT) or 2-way ANOVA for main effects of disease, treatment, and their interaction. Tukey's post hoc analyses followed a significant main effect of treatment to determine the source of the effect. Imaging confirmed a cortical phenotype reflective of CKD, including lower cortical thickness (p < 0.0001) and increased cortical porosity (p = 0.02) compared to Con. In addition, CKD resulted in weaker, less deformable bones. In CKD bones, ex vivo exposure to RAL or CAL improved total work (+120 % and +107 %, respectively; p < 0.05), post-yield work (+143 % and +133 %), total displacement (+197 % and +229 %), total strain (+225 % and +243 %), and toughness (+158 % and +119 %) vs. CKD VEH soaked bones. Ex vivo exposure to RAL or CAL did not impact any mechanical properties in Con bone. Matrix-bound water by ssNMR showed CAL treated bones had significantly higher bound water compared to VEH treated bones in both CKD and Con cohorts (p = 0.001 and p = 0.01, respectively). RAL positively modulated bound water in CKD bone compared to VEH (p = 0.002) but not in Con bone. There were no significant differences between bones soaked with CAL vs. RAL for any outcomes measured. RAL and CAL improve important post-yield properties and toughness in a non-cell mediated manner in CKD bone but not in Con bones. While RAL treated CKD bones had higher matrix-bound water content in line with previous reports, both Con and CKD bones exposed to CAL had higher matrix-bound water. Therapeutic modulation of water, specifically the bound water fraction, represents a novel approach to improving mechanical properties and potentially reducing fracture risk.
Terminal sterilization is necessary for bone grafts to prevent infection and disease transmission. Gamma radiation sterilization is currently the accepted method for its convenience and effectiveness in establishing the sterility of bone grafts. Unfortunately, the mechanical properties of bone grafts are also impaired during the gamma radiation sterilization process. Many studies addressed this problem by confining the free radical damage pathway to the bone collagen phase by using free radical scavengers. However, the safety of these free radical scavengers needs to be more carefully investigated before being applied in bone sterilization. Due to the abundance of phenolic compounds in olive leaves and the fact that these compounds are known to be powerful antioxidants, the aim of this study is to examine olive leaves extract (OLE) as a novel, affordable and non-toxic free radical scavenger for bone specimen protection during gamma radiation sterilization. The radioprotective effect of OLE on bone was investigated qualitatively by the dynamic mechanical analysis (DMA) technique. Specimens from the bovine femur were cut, and they were then soaked in previously prepared OLE for 4 days, 7 days, and 10 days at 4°C before being gamma sterilized with 25 KGy. In a 3-point bending configuration, the dynamic mechanical analysis was conducted at 1 Hz in the temperature range of 28–200°C. The research showed that gamma irradiation deteriorates the dynamic mechanical properties of bones. Statistically significant differences (p < 0.0001) in storage and loss moduli were observed between the irradiated group with 25 KGy and those pretreated by soaking in OLE before irradiation. The results obtained from this study proved that the use of OLE as a free radical scavenger before gamma sterilization would allow for biomechanically more stable bone grafts after implementation, and this finding is of great implication in bone banking.
Bone comprises mechanically different materials in a specific hierarchical structure. Mineralized collagen fibrils (MCFs), represented by tropocollagen molecules and hydroxyapatite nanocrystals, are the fundamental unit of bone. The mechanical characterization of MCFs provides the unique adaptive mechanical competence to bone to withstand mechanical load. The structural and mechanical role of MCFs is critical in the deformation mechanisms of bone and the marvelous strength and toughness possessed by bone. However, the role of MCFs in the mechanical behavior of bone across multiple length scales is not fully understood. In the present study, we shed light upon the latest progress regarding bone deformation at multiple hierarchical levels and emphasize the role of MCFs during bone deformation. We propose the concept of hierarchical deformation of bone to describe the interconnected deformation process across multiple length scales of bone under mechanical loading. Furthermore, how the deterioration of bone caused by aging and diseases impairs the hierarchical deformation process of the cortical bone is discussed. The present work expects to provide insights on the characterization of MCFs in the mechanical properties of bone and lays the framework for the understanding of the multiscale deformation mechanics of bone.
Bone is a living tissue made up of organic proteins, inorganic minerals and water. The organic component of bone (mainly made up of Type-I collagen) provides flexibility and tensile strength. Solid - state nuclear magnetic resonance (ssNMR) is one of the few techniques which can provide atomic - level structural insights of such biomaterials in their native state. In the present article, we employed the variable contact time cross-polarization (1 H - 13 C CP) kinetics experiments to study the hydration - dependent atomic - level structural changes in the bone extracellular matrix (ECM). The natural abundant 13 C CP intensity of the bone ECM is measured by varying CP contact time and best fitted to the non - classical kinetic model. Different relaxation parameters were measured by the best - fit equation corresponding to the different hydration conditions of the bone ECM. The associated changes in the measured parameters due to varying levels of hydration observed at different sites of collagen protein have provided its structural arrangements and interaction with water molecules in bone ECM. Overall, the present study reveals a better understanding of the kinetics of the organic part inside the bone ECM that will help in comprehending the disease - associated pathways.
Bone is a hierarchical architecture that consists of both inorganic and organic components. The organic components, including collagen and numerous non-collagenous biomolecules, are crucial for maintaining the mechanical strength and physiological functions of bone. The native structures of organic components and especially the mutual interactions between different components are important questions to be addressed. Among different analytical techniques, solid-state nuclear magnetic resonance (SSNMR) spectroscopy is a powerful tool to reveal the chemical and interactional information at an atomic level. Recent advancements of SSNMR technology and experimental protocols have brought great advances in understanding the molecular details in native bones. In this review, we summarize the progresses on the SSNMR studies of various organic components in the bone matrix. In the first part, we review the studies on collagen from four different aspects: (1) water-associated molecular dynamics; (2) the intrahelical/interhelical interactions in collagen residues; (3) the interactions between collagen and citrate; and (4) the cross-linking between collagen and inorganic surface. In the second part, we review the studies on the non-protein biomolecules including sugar species, citrate, lipids, and nucleic acids. In the end, we propose an outlook of future directions for SSNMR investigations on bones.
X-ray diffraction and inner-shell photoemission studies of rat cortical bone were performed.
The manufacture of carbonated hydroxyapatite-based bioceramics with control of the composition and microstructure remains challenging and reveals our lack of knowledge regarding the thermal behavior of such materials, particularly at high temperatures under reactive atmospheres. This work lays a foundation for addressing this issue by investigating the solid–gas exchange reactions occurring between oxy-hydroxyapatites (OxHA) and a CO2-rich atmosphere during thermal treatment. Accordingly, OxHA reference powders with different oxygen contents (0 ≤ x ≤ 0.79) were produced, extensively characterized and heat-treated under a CO2-rich atmosphere at 950 °C for 5 h. The results of physicochemical, thermal and microstructural analyses showed that the A-site composition of OxHA controls the exchange reactions: a high initial OH content induced concomitant A-site dehydration and carbonation; conversely, a high OH vacancy content induced A-site hydration as a first step. Furthermore, the specific surface area significantly influenced the solid–gas exchange reactions by controlling their kinetic.
Glucocorticoid-induced osteoporosis (GIO) has emerged as a challenge after long-term glucocorticoids (GCs) administration. Exercise has been an important non-pharmacological option, while medications modulate bone remodeling despite adverse effects. In this way, milk Kefir (MK) therapy stands out as a safe alternative to improve bone metabolism. Our study aimed to investigate the effect of MK associated to resistance exercise on bone loss in rats with GIO. For this, sixty male Wistar rats were divided into 2 groups: normal (N) and subjected to GIO, which was subdivided into 4 groups: control (C), milk kefir therapy (K), Exercise (Ex), and Exercise+K (ExK). GIO was induced by dexamethasone (7 mg/kg – i.m.; 1×/wk, 5 wk). MK was administered daily (1×/day; 0.7 ml/animal) and the climb exercise with load was performed 3×/wk; both for 16 wk. Femur was collected for assessment of bone microarchitecture, quality and metabolism. GIO markedly reduced trabecular bone volume density (BV/TV) (−35 %), trabecular thickness (Tb.Th) (−33 %), mineral content of femur (−26 %) as well as bone collagen content (−56 %). Bone strength and its biomechanical properties given by flexural strength (−81 %), fracture load (−80 %), and the number of osteocytes (−84 %) were lowered after GIO. GCs reduced osteoblast number and function while increased osteoclast number, altering bone remodeling (p < 0.05). On the other hand, ExK significantly improved bone microarchitecture and quality, marked by fractal dimension increase (+38 %), cortical volume (+34 %), BV/TV (+34 %), Tb.Th (+33 %), mineral content and collagen maturity, while reduced the space between trabecula (−34 %). The Ex and ExK increased the number of osteocytes (p < 0.05) and they were able to reverse the lower osteoblast number. Both treatments used alone significantly enhanced bone biomechanical properties, but the ExK showed a more significant improvement. ExK ameliorated bone strength and biomechanics (p < 0.05) and stimulated bone formation and modulated bone remodeling (p < 0.05). MK and exercise administered isolated or in association increased the percentage of collagen bone filling after GIO (p < 0.05), but only ExK improved collagen maturity. Our results showed that MK associated to resistance exercise enhanced bone microarchitecture, quality and metabolism, being therefore an interesting tool to improve skeletal response during GIO.
The solid-state 1H, 31P NMR spectra and cross-polarization (CP MAS) kinetics in the series of samples containing amorphous phosphate phase (AMP), composite of AMP + nano-structured calcium hydroxyapatite (nano-CaHA) and high-crystalline nano-CaHA were studied under moderate spinning rates (5-30 kHz). The combined analysis of the solid-state 1H and 31P NMR spectra provides the possibility to determine the hydration numbers of the components and the phase composition index. A broad set of spin dynamics models (isotropic/anisotropic, relaxing/non-relaxing, secular/semi-non-secular) was applied and fitted to the experimental CP MAS data. The anisotropic model with the angular averaging of dipolar coupling was applied for AMP and nano-CaHA for the first time. It was deduced that the spin diffusion in AMP is close to isotropic, whereas it is highly anisotropic in nano-CaHA being close to the Ising-type. This can be caused by the different number of internuclear interactions that must be explicitly considered in the spin system for AMP (I-S spin pair) and nano-CaHA (IN-S spin system with N ≥ 2). The P-H distance in nano-CaHA was found to be significantly shorter than its crystallographic value. An underestimation can be caused by several factors, among those - proton conductivity via a large-amplitude motion of protons (O-H tumbling and the short-range diffusion) that occurs along OH- chains. The P-H distance deduced for AMP, i.e. the compound with HPO42- as the dominant structure, is fairly well matched to the crystallographic data. This means that the CP MAS kinetics is a capable technique to obtain complementary information on the proton localization in H-bonds and the proton transfer in the cases where traditional structure determination methods fail.
The demand for space heating and domestic hot water has caused an increase in building-energy consumption. Thermal energy storage systems can effectively solve the mismatch between heat supply and demand. Phase change materials (PCMs) can be served as the thermal storage media for thermal energy storage systems. In this study, the tailor-made sodium acetate trihydrate (SAT, a kind of PCM) was developed. Multi-wall carbon nanotubes (MWCNTs) was employed as the nucleation agent of SAT, meanwhile other carbon-based materials, including carbon fiber (CF), expanded graphite (EG), and graphene nanoplates (GNPs), were synergistically used as thermal conductivity and photothermal conversion enhancers. Ammonium chloride (NH4Cl) was innovatively adopted as a phase change temperature regulator for SAT. The results showed that the supercooling degree of SAT decreased to 0.9 °C with the presence of MWCNTs. The thermal conductivity of SAT improved by 54.9%, and the photothermal conversion efficiency increased to 89.3% after incorporating GNPs into the SAT/MWCNTs composite. Furthermore, phase change temperature of the SAT/MWCNTs composite, ranged from 57.5 °C to 45.1 °C, could be prepared by adjusting NH4Cl contents for satisfying different application scenarios. The results indicate that the prepared SAT composites can be potentially used in different thermal energy storage systems.
A NMR spin-spin (T2) relaxation technique has been described for determining the porosity, and the bound water distribution in biglycan induced mouse bone and correlate to their mechanical properties. The technique of low-field proton NMR involves spin-spin relaxation and free induction decay (FID) measurements, and the computational inversion methods for decay data analysis. The CPMG T2 relaxation data can be inverted to T2 relaxation distribution and this distribution then can be transformed to a pore size distribution with the longer relaxation times corresponding to larger pores. The FID T2 relaxation data of dried bone (mobile water removed) can be inverted to T2 relaxation distribution and this distribution then can be transformed to bound and solid-like water distribution with the longest relaxation time corresponding to bound water component. These techniques are applied to quantify apparent changes in porosity, and bound water in controlled and biglycan knockout mouse bone. Overall bone porosity from CPMG T2 relaxation is determined using the calibrated NMR fluid volume from the proton relaxation data divided by overall bone volume. Ignore the physical sample differences, from the inversion FID T2 relaxation spectrum, the ratio of the bound to solid-like water components is used to calibrate the bound water inside bone, and the results can be used to correlated bone mechanical properties. Hydration status significantly affects the toughness of bone, and bound water has been considered as a biomarker for prediction of bone fragility fractures. In addition to the collagen phase, recent evidence shows that glycosaminoglycans (GAGs) of proteoglycans (PGs) in the extracellular matrix also play a pivotal role in regulating the tissue-level hydration status of bone, there by affecting the tissue-level toughness of bone. Furthermore, biglycan and decorin are two major types of PGs in bone reports. Biglycan knockout induced changes in GAGs, bound water, as well as bone tissue toughness. Among all subtypes of PGs, biglycan is identified as a major subtype in the bone mineral matrix. In this study, we used a biglycan mouse model and the obtained bone samples were measured by low-field NMR to determine the bone porosity and bound water changes, and used to predict if knockout of biglycan may affect the amount of bound water and subsequently lead to reduce toughness of bone.
Bones represent about 15% of the body’s weight, a figure which doesn’t deserve any interpretation of consequence. Most importantly, ambulation, ventilation, and protection of the human body is bone dependent for a start, highlighting the mechanical function of the bones making them a structural material with mechanical characteristics like any other mineral building material even if the process of discovery and study of the linkage between micro-components and bulk material is opposite for the two types of material. After more than 2000 years of improvements, we know the right components to make a very good steel but we don’t know yet how to fight osteoporosis efficiently in its natural occurrence with age. Following the Wolff’s law: bone remodels stronger where external stress goes higher. Literature shows many studies try and give probable cause of this tenet without the actual key of the phenomenon, which might hopefully be tantamount to unlock the osteoporosis treatment stalemate. Bone cells are no different from the other body cells (at the notable exception of neurons) with constant renewing until the process dramatically seizes up. So, characterization of bone mechanical behavior is not dependent on the bone age, as bone is always “young,” but the age of the individual and biological disorders and mechanical disturbances fall upon it. Beside shape and appearance, bone is classified into two main types: cortical, hard and dense and trabecular, porous and where plentiful biological processes, with some not yet deciphered, are taking place. Following characterization concerns cortical and trabecular separately then the composite of both. The objective of this first chapter is to better understand all the complexity of human bones related to: its structure, its composition, its histology, and its mechanical behavior at different levels of scales.
We provide a critical review of the chemical composition and structure of synthetic and biogenic (bone/dentin mineral) nanocrystalline hydroxy-carbonate apatite (HCA). Such particles exhibit a ”core–shell” organization, where an ordered HCA core is coated by a surface layer, whose nature is best captured by amorphous calcium phosphate (ACP), which is known to be a precursor phase of synthetic HCA, but whose role of bone/dentin mineralization has remained a most controversial subject. After reviewing the structure of each HCA and ACP component, as well as the most recent findings on their in vitroformation mechanisms, we examine the core–shell HCA organization further, with a focus on the disordered surface (”shell”) domain. In most of recent literature, the surface portion is often referred to as the ”hydrated surface layer”, but without identifying its shared chemical and structural features of (synthetic) ACP. Unfortunately, that missing surface-layer/ACP equivalence obscures that the surface layer at the synthetic/biogenic nanocrystallites may simply constitute a remnant of the ACP phase from which the ordered HCA ”core” nucleated. Although many topics reviewed herein have been investigated for more than six decades, several remain unsettled and heavily debated. Notably, decades-old articles offer suggestions that have passed unnoticed by the younger generations of researchers; we contrast and discuss both the latest and early contributions of this field, as well as highlighting several unsettled topics that should be revisited to improve our understanding of the ACP and HCA structures and in vitro/in vivo formation mechanisms.
The identification of markers of the modifications occurring in human bones after death and of the sedimentary and post-sedimentary processes affecting their state of preservation, is of interest for several scientific disciplines. A new index, obtained from spectral deconvolution of the ¹H MAS NMR spectra of bones, relating the number of organic protons to the amount of hydrogen nuclei in the OH⁻ groups of bioapatite, is proposed as indicator of the state of preservation of the organic fraction. In the osteological material from three different archaeological sites, this index resulted positively correlated with the extent of collagen loss derived from infrared spectroscopy. Its sensitivity to changes in the physical and chemical characteristics of bone allows to identify distinct diagenetic pathways specific to each site and to distinguish different trajectories within the same site.
Bone is mineralized tissue constituting the skeletal system, supporting and protecting body organs and tissues. In addition to such fundamental mechanical functions, bone also plays a remarkable role in sound...
Diagenetic modifications in human bones from the early-medieval cemetery discovered in the garden of Vratislavs’ Palace, in the central Malá Strana district of Prague, have been investigated combining histological analysis and instrumental analysis with X-ray diffraction, infrared, and ³¹P NMR spectroscopy. A total of 15 ribs samples were collected for the study. One sample belonged to a child, whereas, of the other samples from adults, 7 belonged to males, 5 to females, and for 2 the sex attribution was uncertain. A diagenetic pathway common to most of the studied samples was considered the result of a burial environment characterized by a nearly static water regime, with limited temperature excursions, moderately oxic to suboxic, and with pH fluctuations around the limit of apatite recrystallization window, in agreement with the fine textured clay-rich soil, its low hydraulic conductivity, and the measured soil pH. A second pattern, related to variations in the microenvironment, interested a limited number of samples with poorer histological preservation. This was interpreted as the result of higher pH and a better oxygenated environment, which favoured mineral recrystallization. Further reactivation of deterioration processes probably occurred later in some of the graves perturbed by works conducted in the seventeenth century. This work highlights the complementarity of the information obtained from the adopted techniques in order to gain insights into the post-mortem fate of the human remains and their sedimentary environment. In this respect, the quantification of the amount of phosphorus in the amorphous hydrated layer of apatite provided a unique type of information on the mineral component of bone and its reorganization during diagenesis, revealing that a relevant fraction can survive diagenesis, at variance with what previously supposed.
Bone mineral comprises nanoparticles of carbonate-substituted bioapatite similar to hydroxylapatite. Yet mechanical values of macroscopic-sized geological hydroxylapatite are often used to model bone properties due to a lack of experimental data for bioapatite. Here, we investigated the effects of carbonate substitution and hydration on biomimetic apatite response to load using in situ hydrostatic pressure loading and synchrotron x-ray diffraction. We find that increasing carbonate levels reduced the bulk modulus and elastic strain ratio. Elastic constants, determined using computational optimization techniques, revealed that compliance values and elastic moduli decreased with increasing carbonate content, likely a result of decreased bond strength due to CO3²⁻ substitution and Ca²⁺ loss. Hydration environment had no clear effects on the elastic properties likely due to dissolution and reprecipitation processes modifying the crystal structure organization. These results reinforce the need to consider carbonate composition when selecting mechanical properties and provide robust data for carbonate-substituted apatite stiffness.
The detailed chemical composition and microstructure of freshly deposited bone mineral, and how these properties change with maturation of the mineral, have been studied intensively and still remain controversial. For example, current analytical technology is inadequate for the unambiguous characterization of the monohydrogen phosphate ions in bone mineral. Using a differential cross polarization/magic angle spinning solid state nuclear magnetic resonance spectroscopy technique, we suppress the dominant orthophosphate (PO4-3) signal to reveal the spectra of the minor phosphate constituents. This method depends upon differences in the cross polarization time constants for phosphorus-31 nuclei in protonated and non-protonated phosphate ions. It is now possible for the first time to directly measure both the proportion of acid phosphate (HPO4-2) as well as the parameters which characterize its isotropic and anisotropic chemical shift. In bone from three species at several developmental stages, we have found a single type of acid phosphate species, identical in all of the specimens examined. The phosphorus-31 isotropic chemical shift of this acid phosphate group in bone mineral corresponds precisely with that of acid phosphate in octacalcium phosphate, and not with that of brushite. In contrast, the bone acid phosphate anisotropic chemical shift parameters are close to those of brushite, and differ significantly from those of octacalcium phosphate. The orthophosphate resonances of bone mineral, synthetic hydroxyapatite and synthetic octacalcium phosphate share identical chemical isotropic shifts, and similar chemical shift anisotropies. The implication of these results is that the intimate structure of the acid phosphate group in bone mineral is unique, and that none of the common synthetic calcium phosphates accounts well for all of the observed solid state phosphorus-31 NMR properties of bone mineral.
Detailed descriptions of the structural features of bone abound in the literature; however, the mechanical properties of bone, in particular those at the micro- and nano-structural level, remain poorly understood. This paper surveys the mechanical data that are available, with an emphasis on the relationship between the complex hierarchical structure of bone and its mechanical properties. Attempts to predict the mechanical properties of bone by applying composite rule of mixtures formulae have been only moderately successful, making it clear that an accurate model should include the molecular interactions or physical mechanisms involved in transfer of load across the bone material subunits. Models of this sort cannot be constructed before more information is available about the interactions between the various organic and inorganic components. Therefore, further investigations of mechanical properties at the 'materials level', in addition to the studies at the 'structural level' are needed to fill the gap in our present knowledge and to achieve a complete understanding of the mechanical properties of bone.
The carbonate and phosphate vibrational modes of different synthetic and biological carbonated apatites were investigated by Raman microspectroscopy, and compared with those of hydroxyapatite. The nu1 phosphate band at 960 cm-1 shifts slightly due to carbonate substitution in both A and B sites. The spectrum of type A carbonated apatite exhibits two nu1 PO43- bands at 947 and 957 cm-1. No significant change was observed in the nu2 and nu4 phosphate mode regions in any carbonated samples. The nu3 PO43- region seems to be more affected by carbonation: two main bands were observed, as in the hydroxyapatite spectrum, but at lower wave numbers. The phosphate spectra of all biominerals apatite were consistent with type AB carbonated apatite. In the enamel spectrum, bands were observed at 3513 and at 3573 cm-1 presumably due to two different hydroxyl environments. Two different bands due to the carbonate nu1 mode were identified depending on the carbonate substitution site A or B, at 1107 and 1070 cm-1, respectively. Our results, compared with the infrared data already reported, suggest that even low levels of carbonate substitution induce modifications of the hydroxyapatite spectrum. Increasing substitution ratios, however, do not bring about any further alteration. The spectra of dentine and bone showed a strong similarity at a micrometric level. This study demonstrates the existence of acidic phosphate, observable by Raman microspectrometry, in mature biominerals. The HPO42- and CO32- contents increase from enamel to dentine and bone, however, these two phenomena do not seem to be correlated.
Natural materials such as bone, tooth, and nacre are nanocomposites of proteins and minerals with superior strength. Why is the nanometer scale so important to such materials? Can we learn from this to produce superior nanomaterials in the laboratory? These questions motivate the present study where we show that the nanocomposites in nature exhibit a generic mechanical structure in which the nanometer size of mineral particles is selected to ensure optimum strength and maximum tolerance of flaws (robustness). We further show that the widely used engineering concept of stress concentration at flaws is no longer valid for nanomaterial design.
While the biomechanical properties of bone are reasonably well understood at many levels of structural hierarchy, surprisingly little is known about the response of bone to loading at the ultrastructural and crystal lattice levels. In this study, our aim was to examine the response (i.e., rate of change of the vibrational frequency of mineral and matrix bands as a function of applied pressure) of murine cortical bone subjected to hydrostatic compression. We determined the relative response during loading and unloading of mineral vs. matrix, and within the mineral, phosphate vs. carbonate, as well as proteinated vs. deproteinated bone. For all mineral species, shifts to higher wave numbers were observed as pressure increased. However, the change in vibrational frequency with pressure for the more rigid carbonate was less than for phosphate, and caused primarily by movement of ions within the unit cell. Deformation of phosphate on the other hand, results from both ionic movement as well as distortion. Changes in vibrational frequencies of organic species with pressure are greater than for mineral species, and are consistent with changes in protein secondary structures such as alterations in interfibril cross-links and helix pitch. Changes in vibrational frequency with pressure are similar between loading and unloading, implying reversibility, as a result of the inability to permanently move water out of the lattice. The use of high pressure Raman microspectroscopy enables a deeper understanding of the response of tissue to mechanical stress and demonstrates that individual mineral and matrix constituents respond differently to pressure.
Properties of the organic matrix of bone as well as its function in the microstructure could be the key to the remarkable mechanical properties of bone. Previously, it was found that on the molecular level, calcium-mediated sacrificial bonds increased stiffness and enhanced energy dissipation in bone constituent molecules. Here we present evidence for how this sacrificial bond and hidden length mechanism contributes to the mechanical properties of the bone composite, by investigating the nanoscale arrangement of the bone constituents and their interactions. We find evidence that bone consists of mineralized collagen fibrils and a non-fibrillar organic matrix, which acts as a 'glue' that holds the mineralized fibrils together. We believe that this glue may resist the separation of mineralized collagen fibrils. As in the case of the sacrificial bonds in single molecules, the effectiveness of this mechanism increases with the presence of Ca2+ ions.
Magic angle spinning (MAS) NMR structure determination is rapidly developing. We demonstrate a method to determine H-1-C-13 distances r(CH) with high precision from Lee-Goldburg cross-polarization (LG-CP) with fast MAS and continuous LG decoupling on uniformly C-13-enriched tyrosine . HCl. The sequence is gamma-encoded, and H-1-C-13 spin-pair interactions are predominantly responsible for the polarization transfer while proton spin diffusion is prevented. When the CP amplitudes are set to a sideband of the Hartmann-Hahn match condition, the LG-CP signal builds up in an oscillatory manner, reflecting coherent heteronuclear transfer. Its Fourier transform yields an effective C-13 frequency response that is very sensitive to the surrounding protons. This C-13 spectrum can be reproduced in detail with MAS Floquet simulations of the spin cluster, based on the positions of the nuclei from the neutron diffraction structure. It is symmetric around omega = 0 and yields two well-resolved maxima. Measurement of CH distances is straightforward, since the separation Delta omega/2 pi between the maxima for a single H-1-C-13 pair is related to the internuclear distance according to r(CH) = a(Delta omega/2 pi)(-1/3), with a = 25.86 +/- 0.01 Angstrom Hz(1/3). For the H-1 directly bonded to a C-13, the magnetization is transferred in a short time of similar to 100 mu s. After this initial rapid transfer period, the COOH, OH, or NH3 that are not directly bonded to a C-13 transfer magnetization over long distances. This offers an attractive route for collecting long-range distance constraints and for the characterization of intermolecular hydrogen bonding.
Full Rietveld refinement of the crystal structure of the synthetic calcium-deficient carbonated apatite Ca13.40[Ca25.90 (NH4)0.10][(PO4)4.95(CO3)1.05(H2O)0.30][(OH)1.65(H2O)0.45] (space group P63/m; a=9.437(1), c=6.888(1) Å Z=1; Rwp=5.23%) was carried out using X-ray powder diffraction data. The use of the model with the split position of O3 atom made it possible to find two orientations of CO3 triangles sharing one of their edges. They occupy randomly the adjacent faces of a PO4 tetrahedron that are parallel to the c axis. O3c atoms coordinating carbon atoms are shifted by 0.37 Å from O3p atoms belonging to PO4 tetrahedra. The charge unbalance occurring when [CO3]2- ions replace [PO4]3- groups is primarily compensated by vacancies in Ca1 sites. The studies of the sample thermal decomposition performed by simultaneous thermal analysis and by X-ray diffraction helped to analyze the localization and the amount of lattice water that enhanced the reliability of the structural model.
Magic angle spinning (MAS) NMR structure determination is rapidly developing. We demonstrate a method to determine 1H−13C distances rCH with high precision from Lee−Goldburg cross-polarization (LG-CP) with fast MAS and continuous LG decoupling on uniformly 13C-enriched tyrosine·HCl. The sequence is γ-encoded, and 1H−13C spin-pair interactions are predominantly responsible for the polarization transfer while proton spin diffusion is prevented. When the CP amplitudes are set to a sideband of the Hartmann−Hahn match condition, the LG-CP signal builds up in an oscillatory manner, reflecting coherent heteronuclear transfer. Its Fourier transform yields an effective 13C frequency response that is very sensitive to the surrounding protons. This 13C spectrum can be reproduced in detail with MAS Floquet simulations of the spin cluster, based on the positions of the nuclei from the neutron diffraction structure. It is symmetric around ω = 0 and yields two well-resolved maxima. Measurement of CH distances is straightforward, since the separation Δω /2π between the maxima for a single 1H−13C pair is related to the internuclear distance according to rCH = a(Δω/2π)-1/3, with a = 25.86 ± 0.01 Å Hz1/3. For the 1H directly bonded to a 13C, the magnetization is transferred in a short time of 100 μs. After this initial rapid transfer period, the COOH, OH, or NH3 that are not directly bonded to a 13C transfer magnetization over long distances. This offers an attractive route for collecting long-range distance constraints and for the characterization of intermolecular hydrogen bonding.
A theory is developed to describe the slow component of the transient decay of transverse spin magnetization, and the central component of the slow-passage absorption spectrum, of a system of spins which is subjected to a periodic and cyclic perturbation. The theory is used to analyze and compare various schemes for high-resolution NMR of solids, including the spinning of the sample about an axis oriented at the "magic angle," the rotating-frame magic-angle experiment of Lee and Goldburg, pulsed versions of the latter, and a number of new pulsed-NMR experiments recently developed in this laboratory. Attention is focused on the factors, both theoretical and practical, which are important in obtaining optimal suppression of static dipole-dipole interactions and quadrupole splittings, and retention of chemical and Knight shifts and scalar spin-spin interactions. Several new experiments are proposed.
The time dependence of different interaction Hamiltonians of nuclear spins as encountered in NMR experiments where the external field is applied at the "magic angle" in the rotating frame is treated with the average Hamiltonian theory. First- and second-order correction terms of the average Hamiltonian are obtained for symmetric and antisymmetric cycles. New types of pulsed "magic-angle" experiments are treated in detail and experiments are performed to show their capability to resolve chemical shifts in solids. It is shown that such magic-angle methods, employed with applied fields of high duty factor, in principle offer advantages in the high-resolution NMR of solids over resonant multiple-pulse schemes. The problem of observing the nuclear-precession signal during applications of the strong fields is solved by "nesting" an observing cycle of low duty factor into the continuous or quasicontinuous irradiation sequence.
It is shown that considerable amounts of water are intimately associated with the crystals of hydroxyl apatite and of bone. Three kinds of studies demonstrated: (a) that this water is only partially removed by high speed centrifugation; (b) that this hydration shell does not contain the electrolytes of the bulk solution; and (c) the crystals adsorb water in accordance with the Brunauer, Emmett and Teller theory. Adult, compact shaft contains little, if any, water that can be removed by centrifugation and there is insufficient water present to furnish complete hydration shells for the mineral crystals.
High-resolution 1H NMR spectra at 200 and 500 MHz of synthetic samples of biologically relevant calcium phosphates have been obtained by using magic-angle spinning (MAS) at spinning speeds up to 8 kHz. The use of high spinning speeds eliminates the need for either isotopic dilution of samples with deuterium or multiple-pulse line-narrowing methods and provides additional structural information, which is absent in the latter two approaches. Structural water molecules in brushite (CaHPO4·2H2O), octacalcium phosphate (Ca8H2(PO4)6·5H 2O), and the model compound gypsum (CaSO4·2H2O) yield 1H MAS NMR spectra with spinning sidebands extending over a 100-kHz range, reflecting the strong, largely inhomogeneous character of the homonuclear dipolar coupling. Structural hydroxyl groups in a series of solid solutions of fluorohydroxyapatites (Ca5Fx(OH)1-x(PO4)3) exhibit discrete peaks whose isotropic chemical shift values can be related to different hydrogen-bonding configurations. Surface-adsorbed water molecules in these samples give rise to a resolvable peak; the weakness of the spinning sidebands associated with this peak indicates substantial isotropic mobility of the water protons on the NMR time scale. The isotropic 1H chemical shifts of protons in calcium phosphates are shown to correlate well with the strength of hydrogen bonding, as measured by O-H⋯O distances. A linear correlation is observed over a chemical shift range of 0-16 ppm; the accurate data reported here agree remarkably well with ab initio calculations of hydrogen-bonded OH groups reported by Ditchfield and co-workers.
The nature of the protein-mineral interface in bone was investigated by using solid-state nuclear magnetic resonance (NMR) spectroscopy. The non-dephased and dephased 13C-{31P} Rotational Echo DOuble Resonance (REDOR) spectra for the bone sample were shown for dephasing times of 4.8, 7.2, and 9.6 ms. The 13C-{31P} REDOR behavior of o-phospho-L-serine under was also investigated. It was shown that in the 13C spectra of the bone sample, there is no 13C signal that correponds to the β-13C of phosphorylated serine. The intramolecular β-13C-31P distance has been determined by X-ray diffraction to be 0.264 nm.
### Tissues and minerals
On a quantitative basis, the most important of the calcium phosphates (Table 1⇓) is an apatite closely related to hydroxylapatite (HAP). This is better described as an impure carbonate-containing apatite (CO3Ap) and forms the inorganic component of bones and teeth. Table 1⇓ also includes calcium phosphates (including two pyrophosphates) that occur in pathological mineralizations and those that are used for the repair of mineralized tissues. Unlike the well-controlled process of normal mineralization in bones and teeth (see later), pathological mineralizations are usually poorly controlled with the result that several calcium phosphates may occur together. In addition, their crystallographic orientations are often random.
View this table:
Table 1.
Occurrence of calcium phosphates.
The solubility isotherms of the calcium phosphates in the system Ca(OH)2-H3PO4-H2O at 37°C are shown in Figure 1⇓. Monetite occurs in the phase diagram, although its occurrence in normal or pathological calcifications has never been confirmed, one contributing reason being that its nucleation and growth is more difficult than brushite under biological conditions, so brushite forms in preference, even though brushite is less stable. β-Ca3(PO4)2 is a high-temperature phase that does not precipitate directly in aqueous systems; however, it is sufficiently stable in water for a solubility product to be determined so that an isotherm can be calculated, which is the origin of the isotherm in Figure 1⇓. However, if the aqueous system contains Mg2+ (1 or 2 mmol/l), the solubility product of “Ca3(PO4)2” is dramatically reduced (more than a thousand-fold) with the result that the structurally related whitlockite can easily precipitate (Hamad and Heughebaert 1986, LeGeros et al. 1989). Fe2+ ions have a similar effect. Whitlockite has superficially the same X-ray diffraction (XRD) …
Unlabelled:
NMR was used to study the nanostructure of bone tissue. Distance measurements show that the first water layer at the surface of the mineral in cortical bone is structured. This water may serve to couple the mineral to the organic matrix and may play a role in deformation.
Introduction:
The unique mechanical characteristics of bone tissue have not yet been satisfactorily connected to the exact molecular architecture of this complex composite material. Recently developed solid-state nuclear magnetic resonance (NMR) techniques are applied here to the mineral component to provide new structural distance constraints at the subnanometer scale.
Materials and methods:
NMR dipolar couplings between structural protons (OH(-) and H(2)O) and phosphorus (PO(4)) or carbon (CO(3)) were measured using the 2D Lee-Goldburg Cross-Polarization under Magic-Angle Spinning (2D LG-CPMAS) pulse sequence, which simultaneously suppresses the much stronger proton-proton dipolar interactions. The NMR dipolar couplings measured provide accurate distances between atoms, e.g., OH and PO(4) in apatites. Excised and powdered femoral cortical bone was used for these experiments. Synthetic carbonate ( approximately 2-4 wt%)-substituted hydroxyapatite was also studied for structural comparison.
Results:
In synthetic apatite, the hydroxide ions are strongly hydrogen bonded to adjacent carbonate or phosphate ions, with hydrogen bond (O-H) distances of approximately 1.96 A observed. The bone tissue sample, in contrast, shows little evidence of ordered hydroxide. Instead, a very ordered (structural) layer of water molecules is identified, which hydrates the small bioapatite crystallites through very close arrangements. Water protons are approximately 2.3-2.55 A from surface phosphorus atoms.
Conclusions:
In synthetic carbonated apatite, strong hydrogen bonds were observed between the hydroxide ions and structural phosphate and carbonate units in the apatite crystal lattice. These hydrogen bonding interactions may contribute to the long-range stability of this mineral structure. The biological apatite in cortical bone tissue shows evidence of hydrogen bonding with an ordered surface water layer at the faces of the mineral particles. This structural water layer has been inferred, but direct spectroscopic evidence of this interstitial water is given here. An ordered structural water layer sandwiched between the mineral and the organic collagen fibers may affect the biomechanical properties of this complex composite material.
A method is described employing 95% hydrazine which completely deproteinates and slightly dehydrates bone under nearly anhydrous conditions with only moderate heating. This method induced only minor chemical changes and no alterations in structural properties of the mineral phase. Physicochemical data are presented demonstrating that although rat bone crystals more closely resemble synthetic controls made in carbonate-rather than hydroxide-rich media, rat bone apatite cannot be interpreted in terms of known or postulated crystal models in any meaningful fashion. CO
32− infrared band assignments made from spectra of whole bone are shown to be in error due to the presence of protein absorption bands. Absorotion of HPO
42− was observed in infrared spectra of young rat bone mineral. Detailed X-ray diffraction comparisons of deproteinated rat bone before and after hydrolysis clearly demonstrated the presence of amorphous calcium phosphate. Electron microscopy indicated that very small apatite crystals were present in rat bone which might also contribute to the overall mineral pool amorphous to X-ray diffraction. Electron microscopy also showed domains in rat bone mineral where plate-like apatite crystals maintained a netc-axis orientation despite the removal of their fibrous matrix.
Solid state nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to characterize the coordination sphere of the selectively observed nucleus in crystalline or amorphous materials. Recent developments of high-resolution NMR techniques for proton in the solid state open the way to obtaining a more accurate description of the structure of proton-bearing materials at different length scales. This can be achieved through the combined use of Lee-Goldburg homonuclear proton decoupling and proton spin-diffusion. In that scope we describe a new 3D experiment that allows direct probing of proton spin-diffusion process between resolved proton spectra obtained under homonuclear decoupling. We anticipate that this class of experiments will soon be used to study poorly crystalline hybrid materials as well as finely divided or porous inorganic materials. (C) 2001 Editions scientifiques et medicales Elsevier SAS. All rights reserved.
A new rf pulse sequence is described for the decoupling of homonuclear
dipolar interactions in solid state NMR. It involves alternate 2π
rotations of opposite signs about the magic-angle axis in spin space, by
π rf phase shifts synchronous with rapid switching of the rf carrier
frequency between opposite sides of resonance; the frequency offsets and
the rf field intensity satisfy the Lee-Goldburg coherent averaging
condition. Direct digital frequency synthesis ensures phase coherence.
The pulse sequence has significant advantages over previous methods in
the case that abundant spins are irradiated while dilute spins are
observed, in an experiment measuring heteronuclear spin-spin couplings.
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Infrared spectral data indicate that both native rat bone mineral and synthetic apatites formed at physiological pH, ionic strength and temperature are extensively deficient in hydroxide ion content; the data also indicate that these biological and synthetic apatites contain considerable internal distortions (lattice defects). In addition, a significant portion of the CO32- ions in rat bone mineral is loosely-structured in either an amorphous or surface environment. Carbonate ions in vacuum-heated bone or solution-ripened synthetic (physiological pH) apatites appear to be in multiple local environments. Internal CO32- in these materials may be substituted in PO43- and (in much lesser amounts) OH− positions, although considerable deviation from or within these sites is probable due to lattice defects. Carbonateapatites produced by thermal conversion (600o) of amorphous calcium phosphates containing 4–9% CO32- exhibit CO32- mainly in OH− environments. Thermal recrystallization of biological and synthetic apatites in an air atmosphere increases OH− content and reorganizes CO32- locales. However, such extremely well-crystallized products are not at all representative of their native apatitic precursors.
Carbonated apatites of known carbonate content were used to develop a method which uses infrared (IR) spectroscopy for quantitative estimation of carbonate. The ratio of the extinction of the IR carbonate band at about 1,415 cm––1 to the extinction of the phosphate band at about 575 cm––1 is linearly related to the carbonate content of the carbonated apatite. Mixtures of BaCO3 and commercially available hydroxyapatite or tricalcium phosphate are used to standardize the apparatus. The method allows carbonate estimation to better than ± 10% in the range 1–12% wt/wt.Copyright © 1984 S. Karger AG, Basel
The question of whether the apatite crystals of bone contain hydroxyl groups was explored using magic angle spinning, proton nuclear magnetic resonance spectroscopy, and resolution enhanced Fourier transform infrared spectroscopy. The powdered bone samples were heated at 300 degrees C in air, in CO2 at 4 bar atmosphere, and at 300 degrees C in air and subsequently at 300 degrees C in CO2, to eliminate the effects of water tightly bound to the crystals and to prevent the degradation of carbonate groups and the elimination of potentially present OH groups. Results confirm earlier findings that bone apatite crystals do not contain detectable amounts of hydroxyl ions.
A new pulse sequence for abundant-spin NMR in solids, called FSLG240W, is demonstrated. The sequence employs phase-coherent frequency-switching of the rf irradiation to induce opposite rotations around the magic-angle axis in the rotating frame. Three 4 pi/3 rotations in one sense are followed by three 4 pi/3 rotations in the opposite sense. Observation windows separate each rotation. The pulse sequence cancels out the average dipolar Hamiltonian, the first-order correction terms, and the most deliterious second-order terms. The pulse sequence has a short cycle time and a high scaling factor kappa = 1/square root of 3, but is sensitive to rf inhomogeneity. We demonstrate slightly better resolution than either MREV8 or BR24 on a small spherical sample of L-alanine.
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 novelty of very large neutron-scattering intensity from the nuclear-spin incoherence in hydrogen has permitted the determination of atomic motion of hydrogen in synthetic hydroxyapatite and in deproteinated isolated apatite crystals of bovine and rat bone without the interference of vibrational modes from other structural units. From an inelastic neutron-scattering experiment, we found no sharp excitations characteristic of the vibrational mode and stretch vibrations of OH ions around 80 and 450 meV (645 and 3630 cm(-1)), respectively, in the isolated, deproteinated crystals of bone apatites; such salient features were clearly seen in micron- and nanometer-size crystals of pure hydroxyapatite powders. Thus, the data provide additional definitive evidence for the lack of OH(-) ions in the crystals of bone apatite. Weak features at 160-180 and 376 meV, which are clearly observed in the apatite crystals of rat bone and possibly in adult mature bovine bone, but to a much lesser degree, but not in the synthetic hydroxyapatite, are assigned to the deformation and stretch modes of OH ions belonging to HPO(4)-like species.
The rate-limiting step in the delivery of nutrients to osteocytes and the removal of cellular waste products is likely diffusion. The transport of osteoid water across the mineralized matrix of bone was studied by proton nuclear magnetic resonance spectroscopy and imaging by measuring the diffusion fluxes of tissue water in cortical bone specimens from the midshaft of rabbit tibiae immersed in deuterium oxide. From the diffusion coefficient (D(a) = (7.8 +/- 1.5) x 10(-7) cm(2)/s) measured at 40 degrees C (close to physiological temperature), it can be inferred that diffusive transport of small molecules from the bone vascular system to the osteocytes occurs within minutes. The activation energy for water diffusion, calculated from D(a) measured at four different temperatures, suggests that the interactions between water molecules and matrix pores present significant energy barriers to diffusion. The spatially resolved profile of D(a) perpendicular to the cortical surface of the tibia, obtained using a finite difference model, indicates that diffusion rates are higher close to the endosteal and periosteal surfaces, decreasing toward the center of the cortex. Finally, the data reveal a water component (approximately 30%) diffusing four orders of magnitude more slowly, which is ascribed to water tightly bound to the organic matrix and mineral phase.
Studies of the apatitic crystals of bone and enamel by a variety of spectroscopic techniques have established clearly that their chemical composition, short-range order, and physical chemical reactivity are distinctly different from those of pure hydroxyapatite. Moreover, these characteristics change with aging and maturation of the bone and enamel crystals. Phosphorus-31 solid state nuclear magnetic resonance (NMR) spin-spin relaxation studies were carried out on bovine bone and dental enamel crystals of different ages and the data were compared with those obtained from pure and carbonated hydroxyapatites. By measuring the 31P Hahn spin echo amplitude as a function of echo time, Van Vleck second moments (expansion coefficients describing the homonuclear dipolar line shape) were obtained and analyzed in terms of the number density of phosphorus nuclei. 31P magnetization prepared by a 90 degree pulse or by proton-phosphorus cross-polarization (CP) yielded different second moments and experienced different degrees of proton spin-spin coupling, suggesting that these two preparation methods sample different regions, possibly the interior and the surface, respectively, of bone mineral crystals. Distinct differences were found between the biological apatites and the synthetic hydroxyapatites and as a function of the age and maturity of the biological apatites. The data provide evidence that a significant fraction of the protonated phosphates (HPO4(-2)) are located on the surfaces of the biological crystals, and the concentration of unprotonated phosphates (PO4(-3)) within the apatitic lattice is elevated with respect to the surface. The total concentration of the surface HPO4(-2) groups is higher in the younger, less mature biological crystals.
Previous measurements of the hydroxyl (OH-) ion content of the calcium phosphate crystals of bone mineral have indicated a substantial depletion or near-absence of OH-, despite its presumed status as a constituent of the hydroxyapatite lattice. Analytical methods for determining bone crystal OH- content have depended on procedures or assumptions that may have biased the results, such as chemical pretreatment to eliminate interference from the organic matrix. We demonstrate a two-dimensional solid-state nuclear magnetic resonance (NMR) spectroscopy technique that detects the proton spectrum of bone crystals while suppressing the interfering matrix signals, eliminating the need for specimen pretreatment other than cryogenic grinding. Results on fresh-frozen and ground whole bone of several mammalian species show that the bone crystal OH- is readily detectable; a rough estimate yields an OH- content of human cortical bone of about 20% of the amount expected in stoichiometric hydroxyapatite. This finding sheds light on the biochemical processes underlying normal and abnormal bone mineral metabolism.
Chemical structure of human bone mineral was studied by solid-state nuclear magnetic resonance (NMR) with magic-angle spinning (MAS). Trabecular and cortical bone samples from adult subjects were compared with mineral standards: hydroxyapatite (HA), hydrated and calcined, carbonatoapatite of type B with 9 wt% of CO
32− (CHA-B), brushite (BRU) and mixtures of HA with BRU. Proton spectra were acquired with excellent spectral resolution provided by ultra-high speed MAS at 40 kHz. 2D 1H-31P NMR heteronuclear correlation was achieved by cross-polarization (CP) under fast MAS at 12 kHz. 31P NMR was applied with CP from protons under slow MAS at 1 kHz. Appearance of 31P rotational sidebands together with their CP kinetics were analyzed. It was suggested that the sidebands of CP spectra are particularly suitable for monitoring the state of apatite crystal surfaces. The bone samples appeared to be deficient in structural hydroxyl groups analogous to those in HA. We found no direct evidence that the HPO
42− brushite-like ions are present in bone mineral. The latter problem is extensively discussed in the literature. The study proves there is a similarity between CHA-B and bone mineral expressed by their similar NMR behavior.
A study on the development of a process to form materials suitable for biomedical xenograft implants from bovine cancellous bone is presented. Bone cubes cut from the condyle portion of bovine femurs sourced from abattoir waste were subjected to a defatting and subsequent deproteination procedure to produce shape-modifiable materials in which the biocompatible mineral calcium hydroxycarbonate apatite component was preserved in the original osseous architecture of the bovine bone. Optimum defatting was achieved by (1) thawing of the precut bone cubes in water, (2) pressure cooking at 15 psi in water, (3) soaking in 0.1 mol l(-1) NaOH followed by a thorough rinse under running water, (4) microwave heating of the bone cubes in water, (5) refluxing in methyl acetate and finally (6) removal of internal liquid from the cubes by shaking and then air drying. Subsequent deproteination of the defatted bone cubes was optimally achieved by (1) soaking in 5% sodium hypochlorite solution at ambient temperature using ultrasonication, (2) thorough rinsing of the cubes in water followed by drying. The final product is a defatted/deproteinated, bleached material that can be molded into various shapes for implant use in the body. The bone specimens were characterized by a suite of analytical techniques (i.e. infrared, 31P and 13C solid magic-angle spinning (MAS) nuclear magnetic resonance (NMR), X-ray photoelectron spectroscopies, atomic absorption (AA) spectrometry, inductively coupled plasma (ICP) spectrometry, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM)) in order to follow compositional changes during the various stages of processing. In general, bovine condyles proved to be the best source of xenograft materials with condyles from other animal species (i.e. deer, sheep and ostrich) being too small to constitute a utilizable source of cancellous bone. This study shows how value can be added to a hitherto underutilized abattoir by-product by using simple processing techniques.
Mineralized bone tissue has a significant water component. Bone water is associated with the collagen fibers or mineral fraction or occurring as pore water of the Haversian and lacuno–canalicular system. Among the multiple physiologic functions that include signaling and providing to bone its viscoelastic properties, bone water enables the transport of ions and nutrients to and waste products from the cells. In addition, it plays a key role during mineralization whereby collagen-bound water is gradually replaced by calcium apatite-like mineral. In this review it is shown how nuclear magnetic resonance (NMR) allows the study of various physiologically relevant properties of bone water nondestructively. Isotope exchange experiments are described from which the apparent water diffusion coefficient can be calculated. The method is based on monitoring the migration of H2O into the D2O after immersion of the specimen in heavy water. Data obtained from rabbit cortical bone in the normal and mineral-depleted skeleton provide evidence for the hypothesized reciprocal relationship between bone water and mineral. Further, from the diffusion coefficient (D
a = (7.8 ± 1.5) × 10−7 cm2/s) measured at 40°C it can be inferred that diffusive transport of small molecules from the bone’s microvascular system to the osteocytes occurs within minutes. Finally, whereas isotope exchange is not feasible in vivo, it is shown that bone water can be imaged by proton MRI.
Hydrogen environments in calcium phosphates
- J P Yesinowski
- H Eckert
Yesinowski, J. P., and H. Eckert. 1987. Hydrogen environments in calcium phosphates: J. Am. Chem. Soc. 109:6274–6282. 1H MAS NMR at high spinning speeds
Regulation of biomineralization by bone proteins and their assembly into extracellular matrices: Implications for implant osseointegration
- McKee
McKee, M. D., and M. T. Kaartinen. 2002. Regulation of biomineralization by bone proteins and their assembly into extracellular
matrices: Implications for implant osseointegration. In Aging, Osteoporosis, and Dental Implants. G. Zarb, U. Lekholm, T. Albrektsson,
and H. Tenenbaum, editors. Quintessence Publishing, Carol Stream,
IL. 191-205.
Materials become insensitive to flaws at nanoscale: Lessons from nature.: Section Title: General Biochemistry
- H Gao
- I L Ji
- E Jager
- P Arzt
- Fratzl
Gao, H., B. Ji, I. L. Jager, E. Arzt, and P. Fratzl. 2003. Materials
become insensitive to flaws at nanoscale: Lessons from nature.: Section
Title: General Biochemistry. Proc. Natl. Acad. Sci. USA. 100:5597-5600.