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Probing the role of water in lamellar bone by dehydration in the environmental scanning electron microscope
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Structural Biology Journal of Structural Biology 162 (2008) 361–367 www.elsevier.com/locate/yjsbi
Probing the role of water in lamellar bone by dehydration in the environmental scanning electron microscope F. Sermin Utku a,*, Eugenia Klein b, Hale Saybasili a, Can A. Yucesoy a, Steve Weiner c a
Bog˘azicßi University, Biomedical Engineering Institute, Kuzey Kampus, Kare Bina, RumeliHisarustu, Bebek, 34342 Istanbul, Turkey b The Weizmann Institute of Science, Department of Research Support, Rehovot, Israel c The Weizmann Institute of Science, Department of Structural Biology, Rehovot, Israel Received 3 January 2008; accepted 4 January 2008 Available online 11 January 2008
Abstract Water, collagen and mineral are the three major components of bone. The structural organization of water and its functions within the bone were investigated using the environmental scanning electron microscope and by analyzing dimensional changes that occur when fresh equine osteonal bone is dehydrated and then rehydrated. These changes are attributed mainly to loss of bulk and weakly bound water. In longitudinal sections a contraction of 1.2% was observed perpendicular to the lamellae, whereas no contraction occurred parallel to the lamellae. In transverse sections a contraction of 1.4% was observed both parallel and perpendicular to the lamellae. SEM back scattered electron images showed that about half of an individual lamella is less mineralized, and thus has more water than the other half. We therefore propose that contractions perpendicular to lamellae are due to the presence of more water-filled rather than mineral-filled channels within the mineralized collagen fibril arrays. As these channels are also aligned with the crystal planes, the crystal arrays, oriented as depicted in the rotated plywood model for lamellar bone, facilitate or hinder contraction in different directions. Ó 2008 Published by Elsevier Inc. Keywords: ESEM; Bone; Mineralization; Water; Lamellar structure
1. Introduction Bone is a viscoelastic, fiber reinforced, anisotropic composite material. The three major components of mature bone are type I collagen (the bulk of the organic matrix), carbonated hydroxyapatite (the mineral) and water. These are organized in an hierarchical structure. Furthermore, the relative proportions of the three major components vary within the bone, thus making it also a graded material (Weiner and Wagner, 1998). The mineral and matrix components have been well-investigated both with respect to their structural organization and in terms of their contributions to the mechanical properties of bone (Martin and Ishida, 1989; Martin, 1991; Currey, 1984, 2002, 2003; Nyman et al., 2006). However, the third component, water, is less
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well-understood. Water is clearly an integral part of the structure (Nomura et al., 1977; Pineri et al., 1978; Robinson and Elliot, 1957; Robinson, 1979) and plays an important role in the mechanical properties of bone and teeth (Currey, 1990; Yamashita et al., 2002; Kishen and Asundi, 2005; Fois et al., 2001; Fernandez-Seara et al., 2004). The water content of bone varies significantly within a species as a function of age and sex of the individual and type of bone (Timmins and Wall, 1977). The water component of cortical bone comprises on the average 10–12 wt% and 20% of the bone matrix volume consists of water. In general, water can be an integral part of the mineral phase (LeGeros et al., 1978) and the organic matrix (Nomura et al., 1977; Pineri et al., 1978). It can also be bound to the organic matrix and crystal surfaces (Wilson et al., 2006), and can be present as bulk water that fills the pores, canaliculi and vascular system (Cowin, 1999; Turov et al., 2006). Nomura et al. (1977) identified four types of water: structural water forming hydrogen bonds within the triple
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helix of collagen molecules (0–0.07 g/g), water bound to the polar side chains and located in the interhelical regions (0.07–0.25 g/g), water in a transition region where both bound and free water are absorbed (0.25–0.45 g/g), and free water (>0.45 g/g). Pineri et al. (1978) added a fifth group (0.010 g/g), extractable at 104 Torr at 100 °C. Marzec and Warchol (2005) using dielectric spectroscopy also identified four types of water at different temperatures. Structural water cannot be removed even at high temperatures and under vacuum (Renugopalakrishnan et al., 1989). A key point is that during mineralization mineral replaces some of the water (Robinson and Elliot, 1957), and thus the mineral content of bone is inversely proportional to the water content. Eanes et al. (1970) showed using X-ray diffraction, that the spacing between triple helical molecules of mineralized turkey leg tendon collagen has intermediate values between fresh and dry non-mineralized collagen. This indicated that as water is replaced with mineral, the lateral spacing of the collagen fibrils becomes more compact due to the reduction in water-filled space between molecules. Additional studies of the lateral spacing of mineralized and non-mineralized collagen fibrils and the contraction observed between wet and dry tissues also showed a lateral contraction and change in dimensions of the mineralized turkey leg tendon without any change in the axial direction (Eanes et al., 1970; Bonar et al., 1985; Fratzl et al., 1993). Experiments involving the dehydration and rehydration of bone showed that water is mainly associated with the organic phase (Eanes et al., 1976). In this study, we further explore the structural role of water in lamellae by measuring the extent of contraction of osteonal bone with respect to lamellar orientations as a function of dehydration. During dehydration, structural water is not expected to be removed, due to its strong association with the triple helix molecules and the mineral lattice. On the other hand, water that is located within the collagen fibril but not strongly bound to its surface or to other matrix macromolecules, such as proteoglycans (Mecham, 1998) or water that is present in the different sized pores (especially canaliculi), is expected to be partially removed by dehydration (Cowin, 1999). As the water content of bone is inversely proportional to its level of mineralization, we also studied the extent of mineralization within a lamella using back scattered electron (BSE) analysis; a method capable of differentiating between small differences in mineral content in bone (Boyde et al., 1993; Skedros et al., 1993). 2. Materials and methods 2.1. Materials Both fresh frozen and fresh osteonal bone samples obtained from the cranial section of the third metatarsal of a 5-year-old female Arab horse and from an 11 year old male horse of mixed breed were used. The fresh bone was prepared and analysed within 5 days after the horse
had died and was maintained wet in a refrigerator with no preservatives. 2.2. Sample preparation Bone samples of approximately 1 mm3 were prepared by initially cutting the sample with a water-cooled diamond blade saw, grinding the surface of interest and then polishing with 5-lm diamond paste, then 1-lm diamond paste and finally 0.05-lm alumina paste. The samples prepared as longitudinal sections (cut parallel to the bone long axis and the outer surface) and transverse sections (cut perpendicular to the bone long axis) were then kept refrigerated in a small amount of water. 2.3. ESEM analyses An XL30 ESEM-FEG (FEI) was used to study the effects of dehydration on structural changes in bone as a function of changing relative humidity (rh). This was achieved by controlling vapor pressure in the observation chamber, while keeping the other conditions stationary at 5 °C and at 10 kV, making use of the factory settings that have been programmed into the ESEM using an isobar that associates the pressure, temperature and relative humidity (Messier and Vitale, 1993). In each wet sample 3–10 sites were selected for imaging at 350 and/or 1200 magnifications. The exact coordinates for these sites were recorded for re-imaging under the rehydration conditions mentioned below. These specific sites were first imaged wet at 100% rh (6.5 Torr) (referred to as wet). They were then dried at 0% rh (0.1 Torr) for 60 min (referred to as dry). The same sites were re-imaged as the samples were rehydrated either to 28% rh (1.9 Torr) or to 42% rh (2.9 Torr) (referred to as rehydrated). After each change in the conditions, the samples were allowed to equilibrate for 45 min, which was experimentally determined to be an adequate time for samples to equilibrate with the environment, by taking images of the rehydrating sample between 0 and 60 min, at five minute intervals. All samples were fully rehydrated back to 100% rh (6.5 Torr) (referred to as completely rehydrated) and imaged. 2.4. Image analysis The changes in the lamellar structure of five longitudinal and two transverse sections were studied. Data points, as percent change in length, were defined based on the structure using Adobe Photoshop from 59 sites (n = 1492) and 20 sites (n = 672) from longitudinal and transverse sections, respectively. Haversian canals, lacunae and lamellar striations were used as landmarks to choose the two points between which the distance was measured (referred to as measurement). Sites in which osteons intersected the section obliquely were avoided in order to observe only orthogonally sectioned lamellae. Only measurements longer than 15 lm were included in data analysis as the precision decreased sig-
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Fig. 1. (A) A longitudinal equine bone section. Solid and dashed lines indicate directions parallel and perpendicular to the lamellae, respectively. Lacunae orientation was used as an indicator of lamellar orientation. Note that directions parallel and perpendicular to the lamellae are axial and radial to the third metatarsal bone shaft, respectively. (B) Transverse equine bone section. Solid and dashed lines indicate tangential and radial orientations, respectively. Note that in a transverse section radial and tangential segments are the respective equivalent of segments perpendicular and parallel to the lamellae in a longitudinal section. Scale bars: 50 lm.
nificantly for shorter distances. In longitudinal sections (Fig. 1A), segments perpendicular to lamellae (radial to the long axis of the bone), and parallel to the lamellae (along the long axis of the bone); and in transverse sections (Fig. 1B), segments radial (perpendicular to lamellae) and tangential (parallel to lamellae) to the osteon were measured. As the lamellae are curved, we regarded segments that intercepted the lamellar front at an angle between 70° to 110° as being perpendicular to the lamellae, and between 20° and 20° as being parallel to the lamellae. The length of a measurement in a rehydrated sample (Fig. 2B) was subtracted from its wet length (Fig. 2A). The difference was divided by the original wet length and multiplied by 100 to obtain a value for percent change in length. The percent changes in length for each bone sample were then averaged and standard deviations were calculated. Positive values indicated contraction of tissue with dehydration. % Change in length ¼ ðLw Lr =Lw Þ100 where Lw and Lr refer to wet and rehydrated length, respectively. Note that our experiments did not involve the appli-
cation of any external forces. We therefore do not use the term ‘‘strain”, but refer to the percent change per length due to dehydration. 2.5. Statistical analysis Averages, standard deviations (SD), and the minima and maxima of the measurements under each condition were determined. As the homogeneity of variance was established between the compared groups, data with normal and skewed distributions were statistically analyzed using a parametric test, Student’s t-test for large samples, and a non-parametric test, Mann–Whitney U-test, respectively. Differences were considered significant at p < 0.05. 2.6. Back scattered electron image analysis One of the longitudinally sectioned samples was ground to 20–30 lm pieces and was embedded in a high-conductive epoxy resin (Polymer adhesive EP75-1, Master Bond Inc., Hackensack, New Jersey) in order to minimize electron
Fig. 2. A longitudinally cut equine bone imaged as (A) wet and (B) at 28% relative humidity. In these images, the distance between a pair of encircled points was measured. In these images, measurements I–III and II–IV were perpendicular to the lamellae, and measurements I–II and III–IV were parallel to the lamellae. Scale bars: 20 lm.
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charging. It was then polished following the procedure described above for ESEM sample preparation and was coated with a 2 nm layer of chromium and imaged in a Zeiss Ultra 55 (Carl Zeiss NTS GmbH) with a BSE detector, Quadra (QBSD; a solid state detector able to detect electrons with energies 5 keV and higher).
of 0.37% ± 1.24% (n = 110) perpendicular to the lamellae and 0.19% ± 1.31% (n = 91) parallel to the lamellae. In transverse sections, the average radial change between wet and completely rehydrated samples was 0.11% ± 0.32% (n = 37). 3.1. Longitudinal sections
3. Results A key issue in this experiment is the reproducibility of the measurements. This depends on the time it takes for a sample to reach equilibrium after each change in relative humidity conditions, and on the imaging quality in the ESEM, which is a function of both the relative humidity and the measurement reproducibility. Therefore, two different sets of control data were obtained: for measurement reproducibility and experimental reproducibility. Data used to determine measurement reproducibility were obtained from a total of 19 sites (n = 251) and 4 sites (n = 125) from longitudinal and transverse sections, respectively. The measurement reproducibility was determined using Adobe Photoshop within an image and between images taken under the same conditions to determine the presence of any drifts in experimental settings. The error of measurement within an image showed a standard deviation of ±0.09% (n = 25) for a wet sample and ±0.07% (n = 25) for a rehydrated sample. The error of measurement between the two images of the same site taken under the same rh conditions was 0.19% ± 0.20% (n = 10). Images of wet and completely rehydrated samples were compared (referred to as control data) to ensure that length changes were reversible and that the samples had reached equilibrium with the new relative humidity conditions. Various equilibration times (5–60 min) at a specific site were tested at five minute intervals and it was determined that no further change took place after 35 min. Another indication that equilibrium was reached was that the reproducibility of these measurements is lower than that obtained without changing humidity conditions. In longitudinal sections, the control data showed an average change in length
The length changes observed during rehydration perpendicular to the lamellae were significantly greater than the reproducibility of the measurements between wet and completely rehydrated samples. However, the length change parallel to the lamellae was almost the same as the control data. Table 1 compares these length changes and the significance values for measurements perpendicular and parallel to lamellae. The angular dependence vis-a-vis the lamellar boundaries is consistent with the highest contraction being perpendicular to the lamellar planes. 3.2. Transverse sections In contrast to the results of the longitudinal sections, in transverse sections significant contraction took place in both the radial and tangential directions, such that at 28% rh, they were almost equal (pT < 0.45) (Table 2). Please note that in transverse sections, radial measurements are the equivalent of those perpendicular to the lamellae in longitudinal sections and tangential measurements are almost equivalent to those parallel to the lamellae (Fig. 2). Therefore, the average percent change in length between 42% rh and 28% rh observed in longitudinal sections perpendicular to the lamellae and in transverse sections radial to the lamellae, were almost the same. 3.3. Mineral content variations as an indication of the distribution of water in the lamellar structure One possible reason for the differences in the contraction behavior of longitudinal and transverse sections and the structural basis for it was examined using polished longitu-
Table 1 Length changes in longitudinal sections as a function of relative humidity Parallel to Lamellae (=axial)
Perpendicular to Lamellae (=radial in transverse sections) Relative Humidity fresh refrigerated fresh frozen
28% 28% 42%
Significance
Average (%)
Min. (%)
Max. (%)
Average (%)
Min.(%)
Max. (%)
(T Test) p < 0.0005 (U Test) p < 0.0005 (U Test) p < 0.0005
1.51 ± 0.72 (n = 22) 1.19 ± 0.78 (n = 359) 0.97 ± 1.02 (n = 74)
0.65 0.30 1.20
3.71 8.02 4.45
0.20 ± 0.32 (n = 19) 0.12 ± 0.75 (n = 357) 0.08 ± 0.83 (n = 67)
0.72 4.64 2.68
0.52 4.52 1.49
Table 2 Length changes in transverse sections as a function of relative humidity Radial
Tangential
Relative Humidity
Significance
Average (%)
Min. (%)
Max. (%)
Average (%)
Min. (%)
Max. (%)
fresh frozen
(T Test) p < 0.45 (U Test) p < 0.007
1.39 ± 0.87 (n = 143) 1.11 ± 0.79 (n = 90)
0.46 0.21
2.87 3.43
1.41 ± 0.57 (n = 75) 0.91 ± 0.36 (n = 46)
0.46 0.49
2.38 1.45
28% 42%
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dinal sections in the SEM with an in-lens backscattered electron (BSE) detector that samples the material very close to the surface (up to about 300 nm). Fig. 3a and b clearly show darker and lighter stripes that are well-aligned with the lamellar structure. A pair of light and dark stripes has a thickness of 3–5 lm. Lamellae usually have a thickness of around 3 lm if the section is exactly perpendicular to the lamellar boundary (Weiner et al., 1999). As these are cut and polished surfaces it is almost impossible to prepare specimens exactly perpendicular to the lamellae, and hence thicknesses ranging from 3 to 5 lm, are consistent with a pair of stripes being derived from one lamella. As BSE images are sensitive to material density (and hence mineral content) (Boyde et al., 1993; Skedros et al., 1993), this implies that about half of one lamella is more mineralized than the other half. This in turn implies that the less mineralized part of a lamella contains more water in the channels (Robinson and Elliot, 1957). 4. Discussion This study shows that dehydration affects the dimensions of lamellar bone in an anisotropic manner in longitudinal sections, whereas in transverse sections the extent of contraction is almost the same in both the radial and tangential directions. Back scattered electron imaging shows that each lamella has a less mineralized portion. This implies that this part of the lamella has more water and hence may account for the observed contraction behavior during dehydration. As much of this water can be removed during the dehydration process it is not structurally bound. It is therefore conceivable that when the bone is mechanically loaded, this water moves to some other location. 4.1. Implications with regard to water in the lamellar structure In globular proteins, water molecules can be bound to specific sites within the molecule: strongly bound to the surface of the molecule (first hydration shell) and less strongly bound at greater distances, as the water molecules
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grade into bulk water (Handgraaf and Zerbetto, 2006). However, for type I collagen, this classification is less obvious; specific sites within the structure where water molecules are tightly bound are not known. Nevertheless, the staggered-array structure of the fibril results in extended intrafibrillar grooves (Miller, 1984). These grooves have a thickness of about 1.5 nm and extend along the length of the fibril axis for about 40 nm. Their widths depend on the 3-dimensional organization of the fibril, which judging from the structure of mineralized collagen fibrils, could extend across the fibril diameter, i.e. about 80 nm (Fratzl, 2003; Weiner et al., 1999). The fact that crystal growth continues and is not inhibited by the collagen, implies that the interface is not dominated by charged interactions. Wilson et al. (2006) have shown using solid state NMR that a layer of water exists between the crystal surface and the collagen surface. They suggest that it might act as a mechanical couple between the two phases and hence as a cushion during application of stress. This in turn implies that when the crystals grow, they only displace the water molecules located in the middle of the groove. Water molecules present in the groove would be bound to the protein surfaces exposed in the grooves (analogous to the first hydration shell). Modeling of the water bound to the surface of the collagen triple helical molecule shows that it forms a layer with a thickness of about 0.4 nm (Handgraaf and Zerbetto, 2006). This implies that about half of the groove volume is filled with such bound structural water. The crystals nucleate in the grooves and then continue to grow initially within the grooves and later extend between the layers of collagen triple helical molecules (Arsenault, 1989; Landis et al., 1993). In unmineralized collagen only the water molecules in the center of the groove would be removed by dehydration. Assuming this involves two water layers some 0.8 nm thick, the highest amount of contraction that would occur for a parallel array of triple helical molecules aligned in 3-dimensions to form a groove across the entire fibril would be about 9.5% (the groove constitutes a fifth of the volume and only the half of the groove that contains bulk water contracts). This is similar to the amount of contraction that occurs
Fig. 3. Back scattered electron (BSE) images of polished surfaces of longitudinally cut equine bone analysed. (a) A low magnification image using a shallow penetrating BSE detector. As the BSE images are sensitive to material density and hence mineral content, the darker and lighter stripes show differences in mineral density. As the stripes are aligned with the lamellae and a pair of stripes is between 3 and 5 lm, we infer that each stripe is a part of a single lamella (normally around 3 lm thick if the section is exactly perpendicular to the lamellar boundaries). Scale bar: 10 lm. (b) Higher magnification image showing the light and dark stripes. Scale bar: 2 lm.
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upon dehydration of unmineralized collagen fibrils (Fratzl et al., 1993) or between unmineralized and mineralized collagen fibrils (Bonar et al., 1985) based on X-ray and neutron diffraction measurements of the distances between triple helical molecules, respectively. If however all the groove volume was filled with mineral then no contraction upon dehydration would occur, as no extractable water exists and the water molecules between the collagen and the crystal surfaces remain. The experiments that showed the presence of water between the collagen and the mineral were in fact performed under dehydrated conditions (Wilson et al. (2006). The BSE images showed that part of a single lamella is less mineralized than the other part. This implies that some of the grooves are not filled with mineral in the less mineralized part of a lamella. Thus the much smaller extent of contraction we observe presumably relates to the proportion of unmineralized grooves within a single lamella. The presence of more mineralized and less mineralized zones within a lamella are consistent with the nano-indentation results of Hengsberger et al. (2002), which showed that the thicker sub-lamellae of a single lamella have a higher elastic modulus than the thin sub-lamellae. It is conceivable that the stripes imaged in Fig. 3 could be ascribed to the different orientations of the mineralized collagen fibrils within a lamella (Weiner et al., 1997) intersecting with the observed surface, and thus revealing more or less collagen relative to mineral at the surface. This is however unlikely as the mineral and collagen alternate at length scales of a few nanometers and the depth from which the BSE signal is collected, is much more than that. We therefore conclude that these changes in contrast are due to different mineral densities. 4.2. Structural implications The morphological difference between the longitudinal and the transverse sections is that in the longitudinal section, the osteons are cut such that lamellae essentially form half cylinders, whereas in the transverse sections, the cylinders remain whole (Fig. 4). In the dehydrated longitudinal sections, the half cylinders are packed closer together by contraction perpendicular to the lamellae, without a change in dimensions parallel to the lamellae. This would occur if, as demonstrated, part of the structure responsive to drying was also arranged in layers parallel to the lamellae. On the other hand, in the transverse sections, the internal forces arising from dehydration have induced the lamellae of the intact cylinders to come closer together. For this to happen, the lamellae would have to also change dimensions in the direction parallel to the lamellar boundaries. Based on the rotated plywood model of the lamella structure, we can deduce which half of the lamella is most likely to contract more during dehydration. In one half of a lamella most of the collagen fibrils have their grooves (inferred from the orientation of the crystal plane) aligned
Fig. 4. Schematic illustration showing the observed contraction directions in the transverse (left hand side) and longitudinal (right hand side) sections. Note that in the latter the cylindrically shaped lamellae are cut.
with the lamellar boundary to within about 30°. In the other half the grooves are orthogonal to or close to orthogonal to the lamellar boundary. If this part of the lamellae was less mineralized, then most of the contraction should occur parallel to the lamellar boundary plane and not perpendicular to the lamellar boundary plane, as was observed in longitudinal sections. From this we infer that it is most likely that the less mineralized part of a lamella is the part where the crystal layers and hence the grooves are approximately aligned with the lamellar boundary. Therefore, we propose (in the nomenclature of the rotated plywood model (Weiner et al., 1997, 1999)) that the more mineralized part of the lamellar unit corresponds to the 3rd and 4th sub-lamellae of the model and that 1st, 2nd and 5th sub-lamellae are less mineralized. In the transverse sections, the lamellae also contract during dehydration in the direction parallel to the lamellar boundary. This implies that the lamellar structure is not continuous along the lamella. We do not know what structural feature of the lamellae could enable this contraction behavior. We do note however that in the higher magnification BSE image in Fig. 4b, the darker and hence less mineralized portions are not continuous along the lamellae. The fact that this contraction does not occur when the osteons are disrupted in the longitudinal sections implies that in this direction the water content is not as easily removed and hence contraction does not occur independently.
5. Conclusions The water component of the lamellar structure clearly fulfills important structural and mechanical functions. About half of an individual lamella is less mineralized than the other half, and hence must have a higher proportion of channels within the collagen fibrils that are filled with water and not mineral. We suggest that it is the removal of the water from these channels that probably accounts for much
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of the observed contraction of lamellae upon dehydration in the ESEM. Acknowledgments We thank Dr. John Currey, University of York, Department of Biology, Dr. Ron Shahar, Hebrew University, Faculty of Veterinary Sciences and Dr. Meir Barak and Dr. Yurong Ma, The Weizmann Institute of Science and Dr. Sebnem Ozupek, Bogazici University, Department of Mechanical Engineering for their help and advice. This study has been supported by the Bogazici University Scientific Research Projects Grant No. 05X101D. Stephen Weiner is the incumbent of the Dr. Walter and Dr. Trude Burchardt Professorial Chair of Structural Biology. Support for this research was provided from grant RO1 DE006954 from the National Institute of Dental and Craniofacial Research to Dr. Stephen Weiner, The Weizmann Institute of Science. References Arsenault, A.L., 1989. A comparative electron microscopic study of apatite crystals in collagen fibrils of rat bone, dentin and calcified turkey leg tendons.. Bone and Mineral 6, 165–177. Boyde, A., Elliot, J.C., Jones, S.J., 1993. Stereology and histogram analysis of backscattered electron images: age changes in bone. Bone 14, 205–210. Bonar, L.C., Lees, S., Mook, H.A., 1985. Neutron diffraction studies of collagen in fully mineralized bone. J. Mol. Biol. 181, 265–270. Cowin, S.C., 1999. Bone poroelasticity. J. Biomech. 32, 218–238. Currey, J.D., 1984. Effects of differences in mineralization on the mechanical-properties of bone. Phil. Trans. R. Soc. B-Biol. Sci. 304, 509–518. Currey, J.D., 1990. Physical characteristics affecting the tensile failure properties of compact bone. J. Biomech. 23, 837–844. Currey, J.D., 2003. Role of collagen and other organics in the mechanical properties of bone. Osteo Int. 14, S29–S36. Currey, J.D., 2002. Bones, First ed. Structure and Mechanics, Princeton University Press, New Jersey. Eanes, E.D., Lundy, D.R., Martin, G.N., 1970. X-ray diffraction study of mineralization of turkey leg tendon. Calcif. Tis. Res. 6, 239–248. Eanes, E.D., Martin, G.N., Lundy, D.R., 1976. Distribution of water in calcified turkey leg tendon. Calcif. Tis. Res. 20, 313–316. Fernandez-Seara, M.A., Wehrli, S.L., Takahashi, M., Wehrli, F.W., 2004. Water content measured by proton–deuteron exchange NMR predicts bone mineral density and mechanical properties. J. Bone Min. Res. 19, 289–296. Fois, M., Lamure, A., Fauran, M.J., Lacabanne, C., 2001. Study of human cortical bone and demineralized human cortical bone viscoelasticity. J. App. Poul. Sci. 79, 2527–2533. Fratzl, P., Fratzl-Zelman, N., Klaushofer, K., 1993. Collagen packing and mineralization. An x-ray scattering investigation of turkey leg tendon. Biophys. J. 64, 260–266. Fratzl, P., 2003. Cellulose and collagen: from fibres to tissues. Curr. Opin. Coll. Interf. Sci. 8, 32–39. Handgraaf, J.W., Zerbetto, F., 2006. Molecular dynamics study of onset of water gelation around the collagen triple helix. Proteins: Struct. Funct. Bioinf. 64, 711–718.
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Hengsberger, S., Kulik, A., Zysset, P.H., 2002. Nanoindentation discriminates the elastic properties of individual human bone lamellae under dry and physiological conditions. Bone 30, 178–184. Kishen, A., Asundi, A., 2005. Experimental investigation on the role of water in the mechanical behavior of structural dentine. J. Biomed. Mater. Res. 73A, 192–200. Landis, W.J., Song, M.J., Leith, A., McEwen, L., McEwen, B.F., 1993. Mineral and organic matrix interaction in normally calcifying tendon visualized in three dimensions by high-voltage electron microscopic tomography and graphic image reconstruction. J. Struc. Biol. 110, 39– 54. LeGeros, R.Z., Bonel, G., Legros, R., 1978. Types of ‘‘H2O” in human enamel and in precipitated apatites. Calcif. Tis. Res. 26, 111–118. Martin, R.B., Ishida, J., 1989. The relative effects of collagen fiber orientation porosity density and mineralization on bone strength. J. Biomech. 22, 419–426. Martin, R.B., 1991. Determinants of the mechanical properties of bones. J. Biomech. 24, 79–88. Marzec, E., Warchol, W., 2005. Dielectric properties of a protein–water system in selected animal tissues. Bioelectrochemistry 65, 89–94. Mecham, R.P., 1998. Overview of extracellular matrix. Curr Protocols Cell Biol. 10.1.1–10.1.14. Messier, P.M., Vitale, T., 1993. Cracking in albumen photographs: an ESEM investigation. Micr. Res. Tech. 25, 374–383. Miller, A., 1984. Collagen—the organic matrix of bone. Phil. Trans. R. Soc. London Ser. B. 304, 455–477. Nomura, S., Hiltner, A., Lando, J.B., Baer, E., 1977. Interaction of water with native collagen. Biopolymers 16, 231–246. Nyman, J.S., Roy, A., Shen, X., Acuna, R.L., Tyler, J.H., Wang, X., 2006. The influence of water removal on the strength and toughness of cortical bone. J. Biomech. 39, 931–938. Pineri, M.H., Escoubes, M., Roche, G., 1978. Water–collagen interactions: calorimetric and mechanical experiments. Biopolymers 17, 2799– 2815. Renugopalakrishnan, V., Chandrakasan, G., Moore, S., Hutson, T.B., Berney, C.V., Bhatnagar, R., 1989. Bound water in collagen. Evidence from Fourier transform infrared and Fourier transform infrared photoacoustic spectroscopic study. Macromolecules 22, 4121–4124. Robinson, R.A., Elliot, M.D., 1957. The water content of bone. J. Bone Joint Surg. 39A, 167–188. Robinson, R.A., 1979. Bone tissue: composition and function. The Johns Hopkins Med. J. 145, 10–24. Skedros, J.G., Bloebaum, R.D., Bachus, K.N., Boyce, T.M., 1993. The meaning of graylevels in backscattered electron images of bone. J. Biomed. Mater. Res. 27, 47–56. Timmins, P.A., Wall, J.C., 1977. Bone water. Calcif. Tis. Res. 23, 1–5. Turov, V.V., Gun’ko, V.M., Zarko, V.I., Leboda, R., Jablonski, M., Gorzelak, M., Jagiello-Wojtowicz, E., 2006. Weakly and strongly associated nonfreezable water bound in bones. Colloids Surf. B: Biointerfaces 48, 167–175. Weiner, S., Traub, W., Wagner, H.D., 1999. Lamellar bone: structure– function relations. J. Struct. Biol. 126, 241–255. Weiner, S., Wagner, H.D., 1998. The material bone: structure–mechanical function relations. Annu. Rev. Mater. Sci. 28, 271–298. Weiner, S., Arad, L.T., Sabanay, I., Traub, W., 1997. Rotated plywood structure of primary lamellar bone in the rat: orientations of the collagen fibril arrays. Bone 20, 509–514. Wilson, E.E., Awonusi, A., Morris, M.D., Kohn, D.H., Tecklenburg, M.M.J., Beck, L.W., 2006. Three structural roles for water in bone observed by solid state NMR. Biophys. J. 90, 3722–3731. Yamashita, J., Li, X., Furman, B.R., Rawls, H.R., Wang, X., Agrawal, C.M., 2002. Collagen and bone viscoelasticity: a dynamic mechanical analysis. J. Biomed. Mater. Res. 63, 31–36.
Structural Biology Journal of Structural Biology 162 (2008) 361–367 www.elsevier.com/locate/yjsbi
Probing the role of water in lamellar bone by dehydration in the environmental scanning electron microscope F. Sermin Utku a,*, Eugenia Klein b, Hale Saybasili a, Can A. Yucesoy a, Steve Weiner c a
Bog˘azicßi University, Biomedical Engineering Institute, Kuzey Kampus, Kare Bina, RumeliHisarustu, Bebek, 34342 Istanbul, Turkey b The Weizmann Institute of Science, Department of Research Support, Rehovot, Israel c The Weizmann Institute of Science, Department of Structural Biology, Rehovot, Israel Received 3 January 2008; accepted 4 January 2008 Available online 11 January 2008
Abstract Water, collagen and mineral are the three major components of bone. The structural organization of water and its functions within the bone were investigated using the environmental scanning electron microscope and by analyzing dimensional changes that occur when fresh equine osteonal bone is dehydrated and then rehydrated. These changes are attributed mainly to loss of bulk and weakly bound water. In longitudinal sections a contraction of 1.2% was observed perpendicular to the lamellae, whereas no contraction occurred parallel to the lamellae. In transverse sections a contraction of 1.4% was observed both parallel and perpendicular to the lamellae. SEM back scattered electron images showed that about half of an individual lamella is less mineralized, and thus has more water than the other half. We therefore propose that contractions perpendicular to lamellae are due to the presence of more water-filled rather than mineral-filled channels within the mineralized collagen fibril arrays. As these channels are also aligned with the crystal planes, the crystal arrays, oriented as depicted in the rotated plywood model for lamellar bone, facilitate or hinder contraction in different directions. Ó 2008 Published by Elsevier Inc. Keywords: ESEM; Bone; Mineralization; Water; Lamellar structure
1. Introduction Bone is a viscoelastic, fiber reinforced, anisotropic composite material. The three major components of mature bone are type I collagen (the bulk of the organic matrix), carbonated hydroxyapatite (the mineral) and water. These are organized in an hierarchical structure. Furthermore, the relative proportions of the three major components vary within the bone, thus making it also a graded material (Weiner and Wagner, 1998). The mineral and matrix components have been well-investigated both with respect to their structural organization and in terms of their contributions to the mechanical properties of bone (Martin and Ishida, 1989; Martin, 1991; Currey, 1984, 2002, 2003; Nyman et al., 2006). However, the third component, water, is less
*
Corresponding author. Fax: +90 212 257 50 30. E-mail address: [email protected] (F.S. Utku).
1047-8477/$ - see front matter Ó 2008 Published by Elsevier Inc. doi:10.1016/j.jsb.2008.01.004
well-understood. Water is clearly an integral part of the structure (Nomura et al., 1977; Pineri et al., 1978; Robinson and Elliot, 1957; Robinson, 1979) and plays an important role in the mechanical properties of bone and teeth (Currey, 1990; Yamashita et al., 2002; Kishen and Asundi, 2005; Fois et al., 2001; Fernandez-Seara et al., 2004). The water content of bone varies significantly within a species as a function of age and sex of the individual and type of bone (Timmins and Wall, 1977). The water component of cortical bone comprises on the average 10–12 wt% and 20% of the bone matrix volume consists of water. In general, water can be an integral part of the mineral phase (LeGeros et al., 1978) and the organic matrix (Nomura et al., 1977; Pineri et al., 1978). It can also be bound to the organic matrix and crystal surfaces (Wilson et al., 2006), and can be present as bulk water that fills the pores, canaliculi and vascular system (Cowin, 1999; Turov et al., 2006). Nomura et al. (1977) identified four types of water: structural water forming hydrogen bonds within the triple
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helix of collagen molecules (0–0.07 g/g), water bound to the polar side chains and located in the interhelical regions (0.07–0.25 g/g), water in a transition region where both bound and free water are absorbed (0.25–0.45 g/g), and free water (>0.45 g/g). Pineri et al. (1978) added a fifth group (0.010 g/g), extractable at 104 Torr at 100 °C. Marzec and Warchol (2005) using dielectric spectroscopy also identified four types of water at different temperatures. Structural water cannot be removed even at high temperatures and under vacuum (Renugopalakrishnan et al., 1989). A key point is that during mineralization mineral replaces some of the water (Robinson and Elliot, 1957), and thus the mineral content of bone is inversely proportional to the water content. Eanes et al. (1970) showed using X-ray diffraction, that the spacing between triple helical molecules of mineralized turkey leg tendon collagen has intermediate values between fresh and dry non-mineralized collagen. This indicated that as water is replaced with mineral, the lateral spacing of the collagen fibrils becomes more compact due to the reduction in water-filled space between molecules. Additional studies of the lateral spacing of mineralized and non-mineralized collagen fibrils and the contraction observed between wet and dry tissues also showed a lateral contraction and change in dimensions of the mineralized turkey leg tendon without any change in the axial direction (Eanes et al., 1970; Bonar et al., 1985; Fratzl et al., 1993). Experiments involving the dehydration and rehydration of bone showed that water is mainly associated with the organic phase (Eanes et al., 1976). In this study, we further explore the structural role of water in lamellae by measuring the extent of contraction of osteonal bone with respect to lamellar orientations as a function of dehydration. During dehydration, structural water is not expected to be removed, due to its strong association with the triple helix molecules and the mineral lattice. On the other hand, water that is located within the collagen fibril but not strongly bound to its surface or to other matrix macromolecules, such as proteoglycans (Mecham, 1998) or water that is present in the different sized pores (especially canaliculi), is expected to be partially removed by dehydration (Cowin, 1999). As the water content of bone is inversely proportional to its level of mineralization, we also studied the extent of mineralization within a lamella using back scattered electron (BSE) analysis; a method capable of differentiating between small differences in mineral content in bone (Boyde et al., 1993; Skedros et al., 1993). 2. Materials and methods 2.1. Materials Both fresh frozen and fresh osteonal bone samples obtained from the cranial section of the third metatarsal of a 5-year-old female Arab horse and from an 11 year old male horse of mixed breed were used. The fresh bone was prepared and analysed within 5 days after the horse
had died and was maintained wet in a refrigerator with no preservatives. 2.2. Sample preparation Bone samples of approximately 1 mm3 were prepared by initially cutting the sample with a water-cooled diamond blade saw, grinding the surface of interest and then polishing with 5-lm diamond paste, then 1-lm diamond paste and finally 0.05-lm alumina paste. The samples prepared as longitudinal sections (cut parallel to the bone long axis and the outer surface) and transverse sections (cut perpendicular to the bone long axis) were then kept refrigerated in a small amount of water. 2.3. ESEM analyses An XL30 ESEM-FEG (FEI) was used to study the effects of dehydration on structural changes in bone as a function of changing relative humidity (rh). This was achieved by controlling vapor pressure in the observation chamber, while keeping the other conditions stationary at 5 °C and at 10 kV, making use of the factory settings that have been programmed into the ESEM using an isobar that associates the pressure, temperature and relative humidity (Messier and Vitale, 1993). In each wet sample 3–10 sites were selected for imaging at 350 and/or 1200 magnifications. The exact coordinates for these sites were recorded for re-imaging under the rehydration conditions mentioned below. These specific sites were first imaged wet at 100% rh (6.5 Torr) (referred to as wet). They were then dried at 0% rh (0.1 Torr) for 60 min (referred to as dry). The same sites were re-imaged as the samples were rehydrated either to 28% rh (1.9 Torr) or to 42% rh (2.9 Torr) (referred to as rehydrated). After each change in the conditions, the samples were allowed to equilibrate for 45 min, which was experimentally determined to be an adequate time for samples to equilibrate with the environment, by taking images of the rehydrating sample between 0 and 60 min, at five minute intervals. All samples were fully rehydrated back to 100% rh (6.5 Torr) (referred to as completely rehydrated) and imaged. 2.4. Image analysis The changes in the lamellar structure of five longitudinal and two transverse sections were studied. Data points, as percent change in length, were defined based on the structure using Adobe Photoshop from 59 sites (n = 1492) and 20 sites (n = 672) from longitudinal and transverse sections, respectively. Haversian canals, lacunae and lamellar striations were used as landmarks to choose the two points between which the distance was measured (referred to as measurement). Sites in which osteons intersected the section obliquely were avoided in order to observe only orthogonally sectioned lamellae. Only measurements longer than 15 lm were included in data analysis as the precision decreased sig-
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Fig. 1. (A) A longitudinal equine bone section. Solid and dashed lines indicate directions parallel and perpendicular to the lamellae, respectively. Lacunae orientation was used as an indicator of lamellar orientation. Note that directions parallel and perpendicular to the lamellae are axial and radial to the third metatarsal bone shaft, respectively. (B) Transverse equine bone section. Solid and dashed lines indicate tangential and radial orientations, respectively. Note that in a transverse section radial and tangential segments are the respective equivalent of segments perpendicular and parallel to the lamellae in a longitudinal section. Scale bars: 50 lm.
nificantly for shorter distances. In longitudinal sections (Fig. 1A), segments perpendicular to lamellae (radial to the long axis of the bone), and parallel to the lamellae (along the long axis of the bone); and in transverse sections (Fig. 1B), segments radial (perpendicular to lamellae) and tangential (parallel to lamellae) to the osteon were measured. As the lamellae are curved, we regarded segments that intercepted the lamellar front at an angle between 70° to 110° as being perpendicular to the lamellae, and between 20° and 20° as being parallel to the lamellae. The length of a measurement in a rehydrated sample (Fig. 2B) was subtracted from its wet length (Fig. 2A). The difference was divided by the original wet length and multiplied by 100 to obtain a value for percent change in length. The percent changes in length for each bone sample were then averaged and standard deviations were calculated. Positive values indicated contraction of tissue with dehydration. % Change in length ¼ ðLw Lr =Lw Þ100 where Lw and Lr refer to wet and rehydrated length, respectively. Note that our experiments did not involve the appli-
cation of any external forces. We therefore do not use the term ‘‘strain”, but refer to the percent change per length due to dehydration. 2.5. Statistical analysis Averages, standard deviations (SD), and the minima and maxima of the measurements under each condition were determined. As the homogeneity of variance was established between the compared groups, data with normal and skewed distributions were statistically analyzed using a parametric test, Student’s t-test for large samples, and a non-parametric test, Mann–Whitney U-test, respectively. Differences were considered significant at p < 0.05. 2.6. Back scattered electron image analysis One of the longitudinally sectioned samples was ground to 20–30 lm pieces and was embedded in a high-conductive epoxy resin (Polymer adhesive EP75-1, Master Bond Inc., Hackensack, New Jersey) in order to minimize electron
Fig. 2. A longitudinally cut equine bone imaged as (A) wet and (B) at 28% relative humidity. In these images, the distance between a pair of encircled points was measured. In these images, measurements I–III and II–IV were perpendicular to the lamellae, and measurements I–II and III–IV were parallel to the lamellae. Scale bars: 20 lm.
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charging. It was then polished following the procedure described above for ESEM sample preparation and was coated with a 2 nm layer of chromium and imaged in a Zeiss Ultra 55 (Carl Zeiss NTS GmbH) with a BSE detector, Quadra (QBSD; a solid state detector able to detect electrons with energies 5 keV and higher).
of 0.37% ± 1.24% (n = 110) perpendicular to the lamellae and 0.19% ± 1.31% (n = 91) parallel to the lamellae. In transverse sections, the average radial change between wet and completely rehydrated samples was 0.11% ± 0.32% (n = 37). 3.1. Longitudinal sections
3. Results A key issue in this experiment is the reproducibility of the measurements. This depends on the time it takes for a sample to reach equilibrium after each change in relative humidity conditions, and on the imaging quality in the ESEM, which is a function of both the relative humidity and the measurement reproducibility. Therefore, two different sets of control data were obtained: for measurement reproducibility and experimental reproducibility. Data used to determine measurement reproducibility were obtained from a total of 19 sites (n = 251) and 4 sites (n = 125) from longitudinal and transverse sections, respectively. The measurement reproducibility was determined using Adobe Photoshop within an image and between images taken under the same conditions to determine the presence of any drifts in experimental settings. The error of measurement within an image showed a standard deviation of ±0.09% (n = 25) for a wet sample and ±0.07% (n = 25) for a rehydrated sample. The error of measurement between the two images of the same site taken under the same rh conditions was 0.19% ± 0.20% (n = 10). Images of wet and completely rehydrated samples were compared (referred to as control data) to ensure that length changes were reversible and that the samples had reached equilibrium with the new relative humidity conditions. Various equilibration times (5–60 min) at a specific site were tested at five minute intervals and it was determined that no further change took place after 35 min. Another indication that equilibrium was reached was that the reproducibility of these measurements is lower than that obtained without changing humidity conditions. In longitudinal sections, the control data showed an average change in length
The length changes observed during rehydration perpendicular to the lamellae were significantly greater than the reproducibility of the measurements between wet and completely rehydrated samples. However, the length change parallel to the lamellae was almost the same as the control data. Table 1 compares these length changes and the significance values for measurements perpendicular and parallel to lamellae. The angular dependence vis-a-vis the lamellar boundaries is consistent with the highest contraction being perpendicular to the lamellar planes. 3.2. Transverse sections In contrast to the results of the longitudinal sections, in transverse sections significant contraction took place in both the radial and tangential directions, such that at 28% rh, they were almost equal (pT < 0.45) (Table 2). Please note that in transverse sections, radial measurements are the equivalent of those perpendicular to the lamellae in longitudinal sections and tangential measurements are almost equivalent to those parallel to the lamellae (Fig. 2). Therefore, the average percent change in length between 42% rh and 28% rh observed in longitudinal sections perpendicular to the lamellae and in transverse sections radial to the lamellae, were almost the same. 3.3. Mineral content variations as an indication of the distribution of water in the lamellar structure One possible reason for the differences in the contraction behavior of longitudinal and transverse sections and the structural basis for it was examined using polished longitu-
Table 1 Length changes in longitudinal sections as a function of relative humidity Parallel to Lamellae (=axial)
Perpendicular to Lamellae (=radial in transverse sections) Relative Humidity fresh refrigerated fresh frozen
28% 28% 42%
Significance
Average (%)
Min. (%)
Max. (%)
Average (%)
Min.(%)
Max. (%)
(T Test) p < 0.0005 (U Test) p < 0.0005 (U Test) p < 0.0005
1.51 ± 0.72 (n = 22) 1.19 ± 0.78 (n = 359) 0.97 ± 1.02 (n = 74)
0.65 0.30 1.20
3.71 8.02 4.45
0.20 ± 0.32 (n = 19) 0.12 ± 0.75 (n = 357) 0.08 ± 0.83 (n = 67)
0.72 4.64 2.68
0.52 4.52 1.49
Table 2 Length changes in transverse sections as a function of relative humidity Radial
Tangential
Relative Humidity
Significance
Average (%)
Min. (%)
Max. (%)
Average (%)
Min. (%)
Max. (%)
fresh frozen
(T Test) p < 0.45 (U Test) p < 0.007
1.39 ± 0.87 (n = 143) 1.11 ± 0.79 (n = 90)
0.46 0.21
2.87 3.43
1.41 ± 0.57 (n = 75) 0.91 ± 0.36 (n = 46)
0.46 0.49
2.38 1.45
28% 42%
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dinal sections in the SEM with an in-lens backscattered electron (BSE) detector that samples the material very close to the surface (up to about 300 nm). Fig. 3a and b clearly show darker and lighter stripes that are well-aligned with the lamellar structure. A pair of light and dark stripes has a thickness of 3–5 lm. Lamellae usually have a thickness of around 3 lm if the section is exactly perpendicular to the lamellar boundary (Weiner et al., 1999). As these are cut and polished surfaces it is almost impossible to prepare specimens exactly perpendicular to the lamellae, and hence thicknesses ranging from 3 to 5 lm, are consistent with a pair of stripes being derived from one lamella. As BSE images are sensitive to material density (and hence mineral content) (Boyde et al., 1993; Skedros et al., 1993), this implies that about half of one lamella is more mineralized than the other half. This in turn implies that the less mineralized part of a lamella contains more water in the channels (Robinson and Elliot, 1957). 4. Discussion This study shows that dehydration affects the dimensions of lamellar bone in an anisotropic manner in longitudinal sections, whereas in transverse sections the extent of contraction is almost the same in both the radial and tangential directions. Back scattered electron imaging shows that each lamella has a less mineralized portion. This implies that this part of the lamella has more water and hence may account for the observed contraction behavior during dehydration. As much of this water can be removed during the dehydration process it is not structurally bound. It is therefore conceivable that when the bone is mechanically loaded, this water moves to some other location. 4.1. Implications with regard to water in the lamellar structure In globular proteins, water molecules can be bound to specific sites within the molecule: strongly bound to the surface of the molecule (first hydration shell) and less strongly bound at greater distances, as the water molecules
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grade into bulk water (Handgraaf and Zerbetto, 2006). However, for type I collagen, this classification is less obvious; specific sites within the structure where water molecules are tightly bound are not known. Nevertheless, the staggered-array structure of the fibril results in extended intrafibrillar grooves (Miller, 1984). These grooves have a thickness of about 1.5 nm and extend along the length of the fibril axis for about 40 nm. Their widths depend on the 3-dimensional organization of the fibril, which judging from the structure of mineralized collagen fibrils, could extend across the fibril diameter, i.e. about 80 nm (Fratzl, 2003; Weiner et al., 1999). The fact that crystal growth continues and is not inhibited by the collagen, implies that the interface is not dominated by charged interactions. Wilson et al. (2006) have shown using solid state NMR that a layer of water exists between the crystal surface and the collagen surface. They suggest that it might act as a mechanical couple between the two phases and hence as a cushion during application of stress. This in turn implies that when the crystals grow, they only displace the water molecules located in the middle of the groove. Water molecules present in the groove would be bound to the protein surfaces exposed in the grooves (analogous to the first hydration shell). Modeling of the water bound to the surface of the collagen triple helical molecule shows that it forms a layer with a thickness of about 0.4 nm (Handgraaf and Zerbetto, 2006). This implies that about half of the groove volume is filled with such bound structural water. The crystals nucleate in the grooves and then continue to grow initially within the grooves and later extend between the layers of collagen triple helical molecules (Arsenault, 1989; Landis et al., 1993). In unmineralized collagen only the water molecules in the center of the groove would be removed by dehydration. Assuming this involves two water layers some 0.8 nm thick, the highest amount of contraction that would occur for a parallel array of triple helical molecules aligned in 3-dimensions to form a groove across the entire fibril would be about 9.5% (the groove constitutes a fifth of the volume and only the half of the groove that contains bulk water contracts). This is similar to the amount of contraction that occurs
Fig. 3. Back scattered electron (BSE) images of polished surfaces of longitudinally cut equine bone analysed. (a) A low magnification image using a shallow penetrating BSE detector. As the BSE images are sensitive to material density and hence mineral content, the darker and lighter stripes show differences in mineral density. As the stripes are aligned with the lamellae and a pair of stripes is between 3 and 5 lm, we infer that each stripe is a part of a single lamella (normally around 3 lm thick if the section is exactly perpendicular to the lamellar boundaries). Scale bar: 10 lm. (b) Higher magnification image showing the light and dark stripes. Scale bar: 2 lm.
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upon dehydration of unmineralized collagen fibrils (Fratzl et al., 1993) or between unmineralized and mineralized collagen fibrils (Bonar et al., 1985) based on X-ray and neutron diffraction measurements of the distances between triple helical molecules, respectively. If however all the groove volume was filled with mineral then no contraction upon dehydration would occur, as no extractable water exists and the water molecules between the collagen and the crystal surfaces remain. The experiments that showed the presence of water between the collagen and the mineral were in fact performed under dehydrated conditions (Wilson et al. (2006). The BSE images showed that part of a single lamella is less mineralized than the other part. This implies that some of the grooves are not filled with mineral in the less mineralized part of a lamella. Thus the much smaller extent of contraction we observe presumably relates to the proportion of unmineralized grooves within a single lamella. The presence of more mineralized and less mineralized zones within a lamella are consistent with the nano-indentation results of Hengsberger et al. (2002), which showed that the thicker sub-lamellae of a single lamella have a higher elastic modulus than the thin sub-lamellae. It is conceivable that the stripes imaged in Fig. 3 could be ascribed to the different orientations of the mineralized collagen fibrils within a lamella (Weiner et al., 1997) intersecting with the observed surface, and thus revealing more or less collagen relative to mineral at the surface. This is however unlikely as the mineral and collagen alternate at length scales of a few nanometers and the depth from which the BSE signal is collected, is much more than that. We therefore conclude that these changes in contrast are due to different mineral densities. 4.2. Structural implications The morphological difference between the longitudinal and the transverse sections is that in the longitudinal section, the osteons are cut such that lamellae essentially form half cylinders, whereas in the transverse sections, the cylinders remain whole (Fig. 4). In the dehydrated longitudinal sections, the half cylinders are packed closer together by contraction perpendicular to the lamellae, without a change in dimensions parallel to the lamellae. This would occur if, as demonstrated, part of the structure responsive to drying was also arranged in layers parallel to the lamellae. On the other hand, in the transverse sections, the internal forces arising from dehydration have induced the lamellae of the intact cylinders to come closer together. For this to happen, the lamellae would have to also change dimensions in the direction parallel to the lamellar boundaries. Based on the rotated plywood model of the lamella structure, we can deduce which half of the lamella is most likely to contract more during dehydration. In one half of a lamella most of the collagen fibrils have their grooves (inferred from the orientation of the crystal plane) aligned
Fig. 4. Schematic illustration showing the observed contraction directions in the transverse (left hand side) and longitudinal (right hand side) sections. Note that in the latter the cylindrically shaped lamellae are cut.
with the lamellar boundary to within about 30°. In the other half the grooves are orthogonal to or close to orthogonal to the lamellar boundary. If this part of the lamellae was less mineralized, then most of the contraction should occur parallel to the lamellar boundary plane and not perpendicular to the lamellar boundary plane, as was observed in longitudinal sections. From this we infer that it is most likely that the less mineralized part of a lamella is the part where the crystal layers and hence the grooves are approximately aligned with the lamellar boundary. Therefore, we propose (in the nomenclature of the rotated plywood model (Weiner et al., 1997, 1999)) that the more mineralized part of the lamellar unit corresponds to the 3rd and 4th sub-lamellae of the model and that 1st, 2nd and 5th sub-lamellae are less mineralized. In the transverse sections, the lamellae also contract during dehydration in the direction parallel to the lamellar boundary. This implies that the lamellar structure is not continuous along the lamella. We do not know what structural feature of the lamellae could enable this contraction behavior. We do note however that in the higher magnification BSE image in Fig. 4b, the darker and hence less mineralized portions are not continuous along the lamellae. The fact that this contraction does not occur when the osteons are disrupted in the longitudinal sections implies that in this direction the water content is not as easily removed and hence contraction does not occur independently.
5. Conclusions The water component of the lamellar structure clearly fulfills important structural and mechanical functions. About half of an individual lamella is less mineralized than the other half, and hence must have a higher proportion of channels within the collagen fibrils that are filled with water and not mineral. We suggest that it is the removal of the water from these channels that probably accounts for much
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of the observed contraction of lamellae upon dehydration in the ESEM. Acknowledgments We thank Dr. John Currey, University of York, Department of Biology, Dr. Ron Shahar, Hebrew University, Faculty of Veterinary Sciences and Dr. Meir Barak and Dr. Yurong Ma, The Weizmann Institute of Science and Dr. Sebnem Ozupek, Bogazici University, Department of Mechanical Engineering for their help and advice. This study has been supported by the Bogazici University Scientific Research Projects Grant No. 05X101D. Stephen Weiner is the incumbent of the Dr. Walter and Dr. Trude Burchardt Professorial Chair of Structural Biology. Support for this research was provided from grant RO1 DE006954 from the National Institute of Dental and Craniofacial Research to Dr. Stephen Weiner, The Weizmann Institute of Science. References Arsenault, A.L., 1989. A comparative electron microscopic study of apatite crystals in collagen fibrils of rat bone, dentin and calcified turkey leg tendons.. Bone and Mineral 6, 165–177. Boyde, A., Elliot, J.C., Jones, S.J., 1993. Stereology and histogram analysis of backscattered electron images: age changes in bone. Bone 14, 205–210. Bonar, L.C., Lees, S., Mook, H.A., 1985. Neutron diffraction studies of collagen in fully mineralized bone. J. Mol. Biol. 181, 265–270. Cowin, S.C., 1999. Bone poroelasticity. J. Biomech. 32, 218–238. Currey, J.D., 1984. Effects of differences in mineralization on the mechanical-properties of bone. Phil. Trans. R. Soc. B-Biol. Sci. 304, 509–518. Currey, J.D., 1990. Physical characteristics affecting the tensile failure properties of compact bone. J. Biomech. 23, 837–844. Currey, J.D., 2003. Role of collagen and other organics in the mechanical properties of bone. Osteo Int. 14, S29–S36. Currey, J.D., 2002. Bones, First ed. Structure and Mechanics, Princeton University Press, New Jersey. Eanes, E.D., Lundy, D.R., Martin, G.N., 1970. X-ray diffraction study of mineralization of turkey leg tendon. Calcif. Tis. Res. 6, 239–248. Eanes, E.D., Martin, G.N., Lundy, D.R., 1976. Distribution of water in calcified turkey leg tendon. Calcif. Tis. Res. 20, 313–316. Fernandez-Seara, M.A., Wehrli, S.L., Takahashi, M., Wehrli, F.W., 2004. Water content measured by proton–deuteron exchange NMR predicts bone mineral density and mechanical properties. J. Bone Min. Res. 19, 289–296. Fois, M., Lamure, A., Fauran, M.J., Lacabanne, C., 2001. Study of human cortical bone and demineralized human cortical bone viscoelasticity. J. App. Poul. Sci. 79, 2527–2533. Fratzl, P., Fratzl-Zelman, N., Klaushofer, K., 1993. Collagen packing and mineralization. An x-ray scattering investigation of turkey leg tendon. Biophys. J. 64, 260–266. Fratzl, P., 2003. Cellulose and collagen: from fibres to tissues. Curr. Opin. Coll. Interf. Sci. 8, 32–39. Handgraaf, J.W., Zerbetto, F., 2006. Molecular dynamics study of onset of water gelation around the collagen triple helix. Proteins: Struct. Funct. Bioinf. 64, 711–718.
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