ARTICLE pubs.acs.org/JPCC
Dehydration-Induced Structural Changes in the CollagenHydroxyapatite Interface in Bone by High-Resolution Solid-State NMR Spectroscopy Ratan Kumar Rai and Neeraj Sinha* Centre of Biomedical Magnetic Resonance, SGPGIMS Campus, Raibarelly Road, Lucknow 226014 India
bS Supporting Information ABSTRACT: Study of interactions among different components of amorphous biomaterial such as bone is important to understand the mechanism of its formation and biomineralization process and for its unique mechanical properties. In this article, we present dehydration-induced structural changes at the interface of the collagen protein and hydroxyapatite interface in intact mammalian bones by high-resolution solid-state NMR (SSNMR) spectroscopy. With recent advances in SSNMR methodologies, this is the only spectroscopic technique which can provide atomic piercing structural details on amorphous systems such as bone. We performed three SSNMR experiments on bone samples with different degrees of hydration level to probe comparative structural changes. One of the SSNMR experiments is a 13C{31P} Rotational Echo Double Resonance (REDOR) SSNMR experiment which gives an estimate of distances between collagen protein side chain residues and the hydroxyapatite surface in bone. Other SSNMR experiments such as relaxation measurement (T2 measurement) of 13C resonances of collagen protein along with 1H chemical shift measurement give structural changes in the bone matrix due to dehydration. These experiments were performed on bone samples with different degree of water content and also on bone samples in which water was exchanged with D2O, thereby reducing the strength of the hydrogen bonding network. We find that when water molecules from the bone matrix are removed the distance of collagen with the hydroxyapatite surface decreases significantly. The present study explains the role of water in stabilizing the structural properties of amorphous biomaterial like bone and will help in synthesizing bone implant materials with desired properties.
1. INTRODUCTION With new advances in spectroscopic techniques, the structural study of amorphous biomaterial such as bone is an active area of research.1 The compositions of bones have been well established consisting of inorganic phosphates such as hydroxyapatite (HAP), organic macromolecules such as proteins, lipids, polysaccharides, and water molecules. Ultrastructural arrangement of inorganic mineral, water, and organic components provides unique strength and elastic property of bone. Any alteration in bone compositions or structural arrangement gives rise to diseases related to bone weakening such as osteoporosis, osteomalacia, etc. Among various organic components in the bone matrix, collagen is the most abundant protein and consists 90% of organic components.2 There are approximately 5% proteins other than collagen, and the rest of the organic components consist of various lipids35 and polysaccharides. Collagen is responsible for bone strength,6 and other proteins such as statherin or glycoprotein perform other important functions.7 The inorganic part of the bone matrix is mainly hydroxyapatite (Ca10(PO4)6(OH)2), and water is 20% of the bone weight.8 In such a complex system, the knowledge of different types of interaction between organic components and the inorganic r 2011 American Chemical Society
mineral surface is very crucial to understanding the ultrastructural property of bone. Various proteins in bones have been characterized, and its short-range interaction with inorganic surface has been studied.9,10 Different experimental approaches have been applied to understand the interaction of organic components with inorganic mineral to understand bone structure. Solid-state NMR (SSNMR) experiments such as 13C{31P} Rotational Echo Double Resonance (REDOR)11 have been applied previously to measure distances between organic and inorganic surfaces in bone samples and in bone-like model systems where distances up to 6 Å were measured.9,10,1219 It has been shown that the organic mineral interface consists of mainly polysaccharide10 for distances less than 5 Å. However, polysaccharide consists of less than 5% of organic matter in the bone matrix, much less than collagen protein. Recently, it has also been shown that citrate forms close linking with the hydroxyapatite surface in bones.20 These studies were carried out to measure distances of Received: March 18, 2011 Revised: May 7, 2011 Published: May 18, 2011 14219
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The Journal of Physical Chemistry C glycosaminoglycan (GAGS), citrate, and protein statherin from the inorganic surface in bone minerals and bone-like model systems. In the work reported by Jaeger et al.,9 they have shown that the organicmineral interface is mainly through polysaccharide and very recently shown that it is mainly citrate.20 This was concluded based on distance measurement by 13C{31P} REDOR for 13C resonance of GAGS and citrate. The exclusive measurement of long-range interaction which predominantly involves interaction of collagen with the mineral interface will be difficult by other spectroscopic techniques due to the amorphous nature of the bone matrix. In this direction, solid-state NMR (ssNMR) can provide useful structural information for such systems. Water is another most studied component by several spectroscopic techniques such as FTIR,21 Magnetic Resonance Imaging (MRI),2224 and SSNMR.8,2528 These studies showed two types of water in bone matrix, which are mobile water (free water) in the Harversian and Lacuna-canalicular system and bound water, associated with inorganic components and protein collagens.29 A recent study by Zu et al. has utilized intact bone for SSNMR studies and showed that dehydration-induced structural changes in collagen can be measured.26 Fernandez et al. have shown that mechanical properties of bone reduce by dehydration in the bone matrix.30 Robinson et al. have shown that as mineralization in bone proceeds water is getting displaced in the Osteiod part of bone.31 Nyman et al. in their work have shown that age-related changes significantly reduce the bound water content of bone, whereas there is no change in free water content.32 Few other recent age-related studies by various groups have shown that cross-linking between bone minerals and collagen proteins increases as age progresses, resulting in bone weakening.33,34 Wilson et al. proposed the organized water layer as a component in the ultrastructure of bone, existing at the interface between the inorganic surface and collagen.25 This water is described as being in the spaces near bone mineral and collagen. Zhu et al. studied dehydration-induced time-dependent structural changes in intact bone by the 13C NMR spectrum of organic components.26 On the basis of the 13C NMR spectrum of dehydration bone and bone with water exchanged with D2O, they concluded that a hydrogen bonding network exists between collagen and the surrounding environment through water molecules.26 Natural abundance 43Ca NMR studies performed by Xu et al. provided atomic level piercing information about the inorganic matrix of bone and bone protein interaction.35 Hence, it is important to study water-dependent interaction among different components to understand the ultrastructure of bone. Such an understanding will help in designing bone implants with desired properties. We present here the study of interaction of collagen protein with the inorganic surface in the bone matrix through water molecules. Our study involves measurement of distance between the collagen protein and inorganic surface in bone samples with different levels of bound water content by 13C {31P} REDOR. We also measure the same distance in the bone matrix when the hydrogen bonding network is weaken by the exchange of water with D2O. This has given an understanding of the hydrogen bonding network through water molecules in the organicmineral interface. Other SSNMR experiments on the measurement of the local motional order parameter of collagen and 1H chemical shifts by 13C/1H Heteronuclear Correlation (HetCor) experiment in bone samples with different water networks have given insight into structural changes due
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to dehydration and H/D exchange. Such information by other spectroscopic techniques is not possible on amorphous systems such as bone.
2. MATERIAL AND METHODS 2.1. Sample Preparation. For SSNMR experiments, Indian Goat (Capra hircus, 23 years old) femora bone was taken from a local slaughter house. Intact bone was cylindrical cut (8.0 mm long with the radius of 1.0 mm) so that it could be fixed into a 3.2 mm Zirconium rotor (Supporting Information Figure S1). Various degrees of dehydration of bone samples were achieved by placing it in a lyophilizer for 24 and 72 h, respectively. For deuterated bone, it was dipped into D2O (Sigma Aldrich, USA) for 48 h to allow maximum exchange of water present in the bone with D2O. We have chosen intact bone for our study since it has been shown earlier that grinding of bone for SSNMR experiments changes water content as well as homogeneity of the bone matrix.26 2.2. NMR Experimental Parameters. All SSNMR spectra were recorded on a 600 MHz NMR spectrometer (Avance III, Bruker Biospin, Switzerland) operating at 600.154 MHz for 1H, 242.94 MHz for 31P, and 150.154 MHz for 13C frequencies with a Bruker 3.2 mm DVT probe. Magic Angle Spinning (MAS) frequency was 10.0 kHz for all experiments. The spinning speed was controlled by a Bruker MAS pneumatic unit within an accuracy of (2 Hz. Pulse lengths for the Rotation Echo Dipolar Recoupling (REDOR)11 experiment (see Supporting Information Figure S2) were 1.8 μs for 1H π/2 pulse, 6.35 μs for 31P π pulse, and 14 μs for 13C π pulse. Recycle delay used for all experiments was 5.0 s. For the REDOR experiments with dephasing time of 4.0, 8.0, 20.0, and 40.0 ms, signal averaging of 5k, 10k, 17k, and 26k number of transients was used. Total signal averaging times were 6.8, 13.8, 23.6, and 36.1 h, respectively. The sample and probe stability for such long signal averaging were checked before (Supporting Information). For long signal averaging experiments, small sets of REDOR experiments (with (S) and without dephasing pulses (S0)) with 512 scans were recorded and were added later for better signal-tonoise ratio. Total acquisition times for each REDOR experiment were 11 ms with 1k data points. For the 13C 1D spectrum ramp cross-polarization sequence with SPINAL-6436 spin, 1H decoupling (100 kHz 1H r.f. field) and 1.0 ms contact time were used. For the 13C {31P} REDOR experiment, a sequence with alternating π pulses on the 13C observed channel and 31P on the dephasing channel was utilized (Supporting Information Figure S2). XY-8 phase cycling on observed and dephasing channels was used to compensate pulse imperfections.37 The 1H decoupling during the REDOR dephasing period was 100 kHz with a SPINAL-64 decoupling sequence. The details of REDOR pulse sequence used for experiment are given in the Supporting Information. The REDOR experiments with (S) and without (S0) π pulses on the 31P channel were acquired for different dephasing times. The ratio of signal intensity (S/S0) for different dephasing times gives a REDOR curve. For 1H/ 13C and 1H/ 31P Heteronuclear correlation (HetCor)38 experiments (Supporting Information Figure S3), effective fields during the 1H homonuclear decoupling period (FFLG)39,40 were 80 and 50 kHz, respectively. High power 1H decoupling (100 kHz) was applied during the t2 period. 2.3. Simulation and Data Fitting. REDOR simulation curves for different spin pairs were generated by a SIMPSON simulation environment.41 The simulation program is given at the end of the 14220
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Table 1. Various Distances and 13C T2 Measured in Different Bone Samples three-spin system without
three-spin system with homonuclear
two-spin system (Å)
homonuclear coupling (Å)
coupling (600 Hz) (Å)
fresh bone
9.0 ( 0.6
9.8 ( 0.9
9.7 ( 0.7
75.4 ( 7.0
one day dehydration
8.5 ( 0.4
8.6 ( 0.5
9.0 ( 0.5
39.7 ( 4.0
residue hydroxyproline Cγ
glycine CR
proline Cδ
13
C T2 values (ms)
three day dehydration
7.2 ( 0.5
7.9 ( 0.6
8.1 ( 0.5
23.4 ( 2.0
H/D exchanged bone
8.5 ( 0.6
8.5 ( 0.6
8.6 ( 0.6
26.6 ( 1.5
fresh bone
9.1 ( 0.7
9.8 ( 0.6
9.9 ( 0.7
32.6 ( 2.2
one day dehydration
8.5 ( 0.4
9.1 ( 0.5
9.1 ( 0.5
24.39 ( 1.1
three day dehydration H/D exchanged bone
7.2 ( 0.6 8.6 ( 0.7
7.9 ( 0.6 8.6 ( 0.5
8.1 ( 0.6 8.5 ( 0.5
16 ( 1.3 15.8 ( 1.4
fresh bone
8.9 ( 0.7
9.8 ( 1.0
9.6 ( 0.8
32.48 ( 2.5
one day dehydration
8.6 ( 0.8
9.3 ( 0.7
9.1 ( 0.7
18.53 ( 2.0
three day dehydration
-
-
-
15.0 ( 1.2
H/D exchanged bone
7.2 ( 0.7
7.9 ( 0.5
7.8 ( 0.7
16.6 ( 1.4
fresh bone
8.7 ( 0.6
9.4 ( 0.8
9.2 ( 1
43.5 ( 3.2
one day dehydration
8.6 ( 0.7
9.1 ( 0.7
8.9 ( 0.7
20.9 ( 2.1
three day dehydration H/D exchanged bone
7.3 ( 0.8 7.6 ( 0.4
7.9 ( 0.8 8.5 ( 0.5
8.0 ( 0.7 8.5 ( 0.5
17.2 ( 2.3 19.3 ( 2.1
pro Cβ
fresh Bone
8.8 ( 0.8
9.3 ( 0.6
9.5 ( 0.7
-
proline Cγ
fresh bone
9.6 ( 0.6
10.2 ( 0.8
10.4 ( 1.0
-
alanine CR
Supporting Information. REDOR curves for two spin systems with 13C and 31P nuclei and for three spin systems with one 13C nuclei dipolar coupled with two 31P nuclei were considered (Supporting Information Figure S2 (B)). For both types of spin systems, dipolar coupling values corresponding to different distances between 13C and 31P nuclei were considered, and REDOR curves were generated. These spin systems have been shown earlier to accurately represent the dipolar coupling network for the study of the organic mineral interface in bones.14,42 Distances between different 13C nuclei of collagen and 31P nuclei of hydroxyapatite were found in intact bone at different levels of hydration and in H/D exchange conditions (Table 1). T2 values of the 13C signal of collagen in intact bone at different levels of hydration and H/D exchange were calculated by recording the 13 C spectrum as a function of dephasing time in the S0 REDOR experiment. Best fits to the experimental data were calculated by the MATLAB (The Mathworks Inc.) program (Supporting Information Figure S5). T2 values corresponding to different resonances in bone samples are given in Table 1.
3. RESULTS Two types of bones samples were used earlier for the SSNMR experiments in the literature. These samples were cryogenically ground bone and intact bone. Cryogenic grinding changes the ultrastructural properties of bone,26,43 and water content reduces during the course of the SSNMR experiment due to Magic Angle Spinning (MAS). Recently, it has been shown that time-dependent dehydration studies can be performed on bones by putting a small hole on top of the NMR rotor to allow water molecules to escape.26 We performed SSNMR experiments on intact Indian Goat femora bone by cutting it into a small piece to fit inside the MAS rotor (Supporting Information Figure S1). The bone sample was sealed with Teflon tape to avoid any escape of water molecules during the NMR experiment. We find that the water content was almost the same even with seven days of MAS (Supporting Information Figure S4). This was necessary to check
since our experiment for measuring the long-range distance between collagen and the inorganic surface requires signal averaging for a long time period. Water content was verified with 1H NMR spectra recorded each day in seven days of continuous MAS. Natural abundance 13C chemical shift of collagen is a very sensitive indicator of any change in the water content.26 The 13C NMR spectra with 1H decoupling of bone were recorded each day during seven days of MAS. We did not find any significant change in the 13C chemical shift and line width during MAS for seven days. These results suggest no significant change in the water content of bone by MAS. In our study, we used four bone samples with different strength of the hydrogen bonding network and water content. Reduction in the strength of the hydrogen bonding network by exchange of 1H with 2H is well-known.26 This is due to the fact of electronegativity differences in HO and DO bonds. The one-dimensional (1D) 13 C NMR spectrum along with two-dimensional (2D) 1H31P HetCor experiments of all four bone samples are shown in Figure 1. The bound water level content in these samples can be seen in the 2D 1H31P spectrum which shows resolved peaks from OH and bound water. In samples with one day and three days of dehydration, bound water peaks are significantly low in intensity compared to fresh bone sample (Figure 1). Also in the H/D exchange bone sample, the bound water peak intensity is somewhere between fresh and dehydrated bone. The corresponding natural abundance 13C spectra are shown for all four bone samples. Various resonances in the 13C spectrum corresponding to organic components can be identified and assigned.26,28,4446 Most of the resonances correspond to Type 1 collagen residues and citrate which resonate at 76 ppm. The carbonyl carbons of collagen resonate around 175 ppm. As bound water contents are reduced, the carbonyl resonances merge, and the line width of aliphatic peaks increases slightly. It should be noted that dehydration-induced changes observed in the 13C spectrum are consistent with the observations of Zhu et al.26 To see structural changes due to different levels of bound water content and the hydrogen bonding network, we performed 14221
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Figure 1. 13C NMR spectra (ad) of intact bone recorded with cross-polarization with magic angle spinning and 1H decoupling. (a) Fresh intact bone, (b) bone dehydrated for one day, (c) bone dehydrated for three days, and (d) fresh intact bone H/D exchanged for 48 h. Two-dimensional 1H/31P correlation NMR spectra of bones (eh) at various stages. (e) Fresh intact bone, (f) bone dehydrated for one day, (g) bone dehydrated for three days, and (h) fresh intact bone deuterated for 48 h. Various 13C signals corresponding to collagen are shown at the top of the spectrum. In 2D 1H/31P NMR (eh) spectra, the water peak (H2O) and hydroxyl ion (OH) peaks are marked in the spectrum.
SSNMR experiments to measure distance between collagen residues and 31P of inorganic components (HAP). We performed 13C{31P} Rotational Echo Double Resonance (REDOR) NMR experiments to measure distances between the collagen side chain and inorganic part.11 In the present method of using intact bone in sealed conditions, we could observe 13C transverse relaxation time (T2) of the order of 50.0 ms, making large distance measurement possible. The signal intensity of REDOR with and without dephasing pulses was recorded for different dephasing times. REDOR experiments have been recorded for dephasing times of 4.0, 8.0, 20.0, and 40.0 ms. For 8.0 ms, significant dephasing is only observed for citrate resonance at 76 ppm, and a slight change in intensity is observed in the aliphatic region (2070 ppm). It was shown earlier that in the 13C spectrum of bone the concentration of other phosphorylated compounds is too small to be observed in the NMR spectrum. Hence, whatever dephasing we observe in 13C{31P}
REDOR is from the inorganic surface.9 This observation is consistent with earlier reported studies.9 It can be seen that significant dephasing was observed for 20 and 40 ms dephasing times (Figure 2). The amount of dephasing observed is inversely proportional to the distance between two nuclei. For 20.0 and 40.0 ms dephasing time, significant amounts of dephasing were observed in the aliphatic region of the collagen protein spectrum. One such spectrum for a dephasing time of 20.0 and 40.0 ms is shown in Figure 2. The amount of dephasing corresponding to different residues can be measured, and this makes it possible to estimate the long-range distance between the collagen side chain and the inorganic mineral interface. REDOR measured S/S0 for different dephasing times corresponding to the hydroxy-proline Cγ residue along with a simulated curve for different distances, which are shown in Figure 3. The REDOR curve for different distances can be simulated, and the best fit to experimental data can be measured. It can be seen from Figure 3 that changes in 14222
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Figure 2. 13C spectrum of fresh bone showing dephasing at (a) 20 ms and (b) 40 ms. The red spectrum was acquired without 31P dephasing pulse and the green one with 31P pulses on.
Figure 3. 13C REDOR data of fresh intact bone, bone dehydrated for three days, and fresh intact bone with H/D exchanged for two days. The data show a plot of S/S0 (for hydroxyproline Cγ residue) as a function of dephasing time and various best fit curves corresponding to different 13C31P distances. The best fit REDOR data for the (a) two-spin system (CP), (b) three-spin system (CP2), and (c) three-spin system (CP2) with PP dipolar coupling of 600 Hz are shown in the figure. Blue color corresponds to fresh intact bone, red for three days dehydrated bone sample, and green for the H/D exchanged bone.
distance for bone sample with different degrees of water content and hydrogen bonding network are beyond the experimental error limit. For most of the side chain residues of collagen, the distances with inorganic phosphorus were measured, and these are shown in Table 1. The distances were measured in all four bone samples with different degrees of hydration and H/D exchange bone samples. The corresponding errors in the estimation of various distances are also shown in Table 1. It is interesting to note that 1H decoupling efficiency during the REDOR period will be crucial for this type of experiment. We have used high-power 100 kHz SPINAL-64 1H decoupling during the REDOR dephasing period. This is very large decoupling power and will be sufficient. Also, the decoupling efficiency will be the same for the reference (S0) as well as dephased (S)
REDOR spectrum. Hence, when we take the ratio of S/S0, it will be independent of decoupling efficiency and will depend entirely on collagen hydroxyapatite distance. Comparative changes observed in distance for all four bone samples will give direct evidence to the change in the interaction of collagen with the inorganic surface due to a change in water content and reduction in hydrogen bonding network strength. We can see that the distance between collagen and the inorganic surface reduces by reduction in bound water content. The distance measured in H/D exchange bone is somewhere between distances measured in fresh and completely dry bone. This trend was observed for all residues of collagen. It has been shown earlier that a mechanical property of bone reduces due to H/D exchange indicating a change in internal structure.30 Figure 4 represents changes in 14223
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Figure 4. REDOR dephasing S/S0 observed for resonances corresponding to (a) hydroxyproline Cγ, (b) glycine CR, (c) proline Cβ, and (d) alanine CR side chain of collagen in bones with different dehydration and H/D exchange level. The square box represents dephasing S/S0 observed at 20 ms, and the round curve corresponds to 40 ms.
S/S0 values for 20 and 40 ms dephasing time for a few residues of collagen for bone samples with different hydration level. The corresponding change in S/S0 values will give direct evidence of change in distance between collagen and the inorganic surface as a function of hydration level in bones. It can be seen that as water content in bone reduces there is a significant reduction in S/S0 values for all four side chain residues of collagen. This clearly indicates that collagen is coming closer to the inorganic surface when water content reduces in the bone matrix. Further, for bone samples with H/D exchange, S/S0 values are in between those corresponding to fresh bone and completely dry bone. In the bone matrix statherine, other lipids (25%) and polysaccharides along with other proteins (5%) constitute less than 10% of organic components. Collagen is 90% of the organic components of the bone matrix. Hence, it is difficult to observe the natural abundance 13C NMR spectrum from other organic components due to dynamic range problems. The natural abundance 13C spectrum of bone will be predominantly from collagen. However, the contribution in REDOR dephasing due to other organic components will be very small and can be neglected. Further, our present study is mainly focused on measuring dehydration-induced structural changes in the collagen hydroxyapatite surface. Hence, we neglect contribution from other organic components since it will only reduce marginally our estimate of distances between collagen and the HAP surface.
The change in the local environment of collagen due to different bound water content can also be measured by 1 H13C HetCor and T2 measurement of different 13C resonances. For the same bone samples with different degrees of hydration level, 1H13C HetCor experiments and T2 measurements were performed. Figure 5 shows the 1H13C HetCor spectrum for bone samples with different levels of water content and hydrogen bonding network. The resolution in the 2D 1 H13C HetCor spectrum is good enough to resolve the 1H chemical shift of various side chain residues of collagen. The corresponding assignments along with the 1D 13C spectrum are shown in Figure 5. We observe that in bones with different levels of bound water content there is a significant low-field shift in 1H chemical shift, although the 13C chemical shift does not show a significant change. This confirms no significant change in collagen structure due to the difference in hydration level, although there is a significant change in its local environment. Here also, the 1H chemical shifts corresponding to H/D exchange bone are somewhere in between fresh and completely dehydrated bone. The low-field change in 1H chemical shifts observed in samples with different degrees of hydration and H/D exchange is attributed to the change in hydrogen bonding network.47,48 The 13C transverse relaxation rate T2 is an indicator of local motion and environment. Measured T2 values corresponding to different side chains of collagen are shown in Table 1. As bone gets dehydrated, T2 values reduce, corresponding to the lesser 14224
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The Journal of Physical Chemistry C side chain motional order parameter. The T2 values of H/D exchanged bone sample are in between fresh and completely dehydrated bone.
4. DISCUSSION Our experimental results indicate the bone mineral, protein collagen, and water molecules corresponding to a model shown in Figure 6. As bound water level in the bone matrix reduces, cross-linking between collagen and the inorganic surface increases due to which collagen comes closer to the inorganic surface. In fresh bone, the distance between the collagen side chain and inorganic phosphorus is mostly around 9.0 Å. In our study, such large distance measurements became possible due to the large T2 we observed in our intact bone samples. Earlier heteronuclear distances of this order were measured by REDOR in different studies too. For example, by 13C{15N} REDOR, typically a distance of 5.5 Å was detected in amyloid fibrils.49 Such distance can be translated to an 15N13C dipolar coupling constant of 18.4 Hz, which is quite comparable to the 13C31P coupling constant for a distance corresponding to 9 Å. The previous bone study by the Drobny group has illustrated that the statherin protein binds to the inorganic surface via a specific
Figure 5. 2D 1H/13C heteronuclear correlation NMR spectra of intact bone at various stages of hydration and H/D exchange. Blue curve shows 2D correlation spectra of fresh intact bone, green curve corresponds to H/D exchanged bone, and red corresponds to dehydrated bone.
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residue, and each specific residue distance from inorganic phosphate has been reported around 4.55 Å.14,1619 Duer et al. in their study showed that polysaccharide GAGS is within 5 Å distance from the inorganic surface.9,10 These earlier studies were focused toward measuring the distance of statherin protein and polysaccharide such as GAGS in bone and bone-like model systems. For fresh bones, and with different degrees of dehydration, the distance between carbons of the collagen side chain and phosphorus of hydroxyapatite is of the order of 9.0 ( 0.5, 8.4 ( 0.5, and 7.6 ( 0.5 Å, respectively. The same trend is observed when we exchange water with D2O which has reduced hydrogen bonding strength. We observe distances of the order 8.2 ( 0.6 Å in H/D exchanged bone samples. Our measured distances have error bars (Table 1). The error bars were calculated based on detailed error analysis which takes into account signal-to-noise ratio as well as spread in the REDOR simulation curves for various experimental measurements. This is the reason for some large error bars in distance measurement. Hence, we report only average distances of various collagen residues from phosphorus of the inorganic surface. These reported average distance changes with water content in the bone matrix. This can be explained only when water forms a hydrogen bonding network with the inorganic surface and collagen. The systematic weakening of the hydrogen bonding network as dehydration level increases results in a decrease in the distance between collagen and the inorganic surface. The change in 1H chemical shifts and T2 values of 13C resonances also confirm that collagen is coming closer to the inorganic surface due to dehydration as well as H/D exchange. Hence, interaction strength between collagen and the inorganic surface increases due to dehydration and H/D exchange. Increases in this interaction strength cause bone to be more susceptible to fracture due to the restricted motion of water molecules which provides tensile strength against any external pressure.32 It has been shown earlier that water content in the bone matrix is directly related to the mechanical properties of bone.50 Hence, we can conclude that water forms a hydrogen bonding network with inorganic phosphorus and collagen. Such a network stabilizes the bone matrix and is responsible for mechanical properties of bone.
5. BONE MODEL Various proteins in bone minerals have been identified earlier. One of the proteins in the bone matrix is statherin which is responsible for initiating mineralization. Various studies have been carried out to understand the structure of statherin in the bone matrix.13,14,16,17,5153 It is widely accepted that this protein is within a distance of 4 Å from the inorganic surface.14,54 Other
Figure 6. Model to show the effect of dehydration between the inorganic surface and collagen of bone. After dehydration, the distance between the collagen and hydroxyapatite surface decreases. 14225
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The Journal of Physical Chemistry C studies confirm that polysaccharide GAGS is within 5 Å distance from the inorganic surface.9,10 A recent study confirms that citrate is closer to the inorganic surface.20 There are various other molecules responsible for cross-linking between collagen and the inorganic surface.7 Water plays a crucial role in the hydrogen bonding network between collagen and the inorganic surface. Recent studies by Klaus Schmidt-Rohr et al. measure the 3 nm thickness of the inorganic surface in the bone matrix.55 Assuming collagen to be 3 nm wide, this model corresponds to approximately 10% of water content in the bone matrix. If we take into account CH bond length and distance of phosphorus from the inorganic surface, water layer thickness will be around 7 Å in completely hydrated bone. This distance will change due to dehydration and H/D exchange. Earlier studies on estimation of water content by various other groups estimate similar water content in fresh bone.8 The hydrogen bonded network of water molecules acts as a lubricant for the relative motion of collagen with the inorganic surface due to external stress.8,32 Water movement allows bone to withstand external stress with less deformation and acts as a sacrificial layer, protecting collagen from shear under uniaxial stress. This makes bones flexible and less susceptible to fracture in the event of external stress. As water content reduces in the bone matrix, collagen comes closer to the inorganic surface, and its cross-linking with collagen increases. This makes bone more susceptible to fracture. The mechanical properties measured in bones with different water content are consistent with this model.32,50
6. CONCLUSION In the present study, we studies the measurement of long-range distance of collagen with the inorganic surface in intact bone. The study is based on a high-resolution solid-state NMR experiment to measure the distance between collagen 13C and 31P of the inorganic surface by a REDOR experiment. A reduction in bound water content results in a decrease of this distance. A further SSNMR experiment to measure the local order parameter of collagen and 1H chemical shift confirms that collagen is coming closer to the inorganic surface as water content reduces. Our finding gives new structural insight into the role of water in bone strength. This type of information on dehydration-induced structural changes in intact bone is not possible by other spectroscopic techniques. This may have possible implications in understanding the bone weakening mechanism due to disease conditions and designing suitable materials of bone implants. ’ ASSOCIATED CONTENT
bS
Supporting Information. Figures S1S7 and the SIMPSON program used for simulating REDOR curves corresponding to different 13C and 31P distances. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Fax: þ91-522-2668215.
’ ACKNOWLEDGMENT Financial support from the Department of Biotechnology, India (Grant number BT/PR12700/BRB/10/719/2009), is gratefully acknowledged.
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