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Chapter 5

Study of bone through solid state NMR

98

5.1. Introduction

With new advances in spectroscopic techniques, the structural studies of amorphous bio-

material such as bone is an active area of research1. 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 (Figure 5.1(a)

and (b)). Ultra-structural arrangement of inorganic mineral, water and organic components

provide unique strength and elastic property of bone. Any alteration in bone compositions or

structural arrangement give rise to diseases related to bone weakening such as osteoporosis,

osteomalacia, etc. Among various organic components in bone matrix, collagen is the most

abundant protein and consists 90% of organic components2. There are approximately 5%

proteins other than collagen and rest of organic components consists of various lipids 3-5

and

polysaccharides.

Figure 5.1.(a)Intact cortical bone (b) Diagram depicting the Composition of bone

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Collagen is responsible for bone strength6 and other proteins such as statherin, glycoprotein

perform other important functions7. The inorganic part of bone matrix is mainly

hydroxyapatite(HA) (Ca10(PO4)6(OH)2) and water consist 20% of bone weight8. Whereas HA as

has a Ca:P ratio of 5:3 (1.67), bone mineral itself has Ca:P ratios ranging from 1.37 - 1.87. This

is because the composition of bone mineral is much more complex and contains additional ions

such as silicon, carbonate and zinc. In such a complex system, the knowledge of different type

of interaction between organic components and inorganic mineral surface are very crucial to

understand the ultra-structural property of bone (Figure 5.2).

Figure 5.2. Hierarchical collagen structure from tropocollagen molecule to fiber level along with hydroxyapatite (HA).

100

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 13

C{31

P} Rotational Echo Double

Resonance (REDOR)11

has been applied earlier to measure distances between organic and

inorganic surfaces in bone samples and in bone like model systems where distances up to 6Ǻ

were measured9; 10; 12-19

. It has been shown that organic mineral interface consists of mainly

polysaccharide10

for distance less than 5Å. However, polysaccharide consists of less than 5% of

organic matter in bone matrix, much less than collagen protein. Recently it has also been shown

that citrate forms close linking with hydroxyapatite surface in bones20

. These studies were

carried out to measure distances of glycosaminoglycan (GAGS), citrate and protein statherin

from inorganic surface in bone minerals and bone like model systems. In the work reported by

Jaeger et al.9, they have shown that organic mineral interface is mainly through polysaccharide

and very recently shown that it is mainly citrate20

. This was concluded based on distance

measurement by 13

C{31

P} REDOR for 13

C resonance of GAGS and citrate. The exclusive

measurement of long range interaction which predominantly involves interaction of collagen

with mineral interface will be difficult by other spectroscopic techniques due to amorphous

nature of 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(Fourier transform infrared spectroscopy)21

, Magnetic Resonance Imaging (MRI)22-24

and

Solid State Nuclear Magnetic Resonance (ssNMR)8; 25-28

. These studies showed two types of

water in bone matrix, which are mobile water (free water) in Harversian and Lacuna-canalicular

system and bound water, associated with inorganic components and protein collagens29

.

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Fernandez et al. have showed that mechanical properties of bone reduce by dehydration in bone

matrix30

. Robinson et al. have showed that as mineralization in bone proceeds, water is getting

displaced in osteiod part of bone31

. Nyman et al. in their work have shown that age related

changes significantly reduces bound water content of bone whereas there is no change in free

water content32

. Few other recent age related studies by various groups have shown that cross

linking between bone minerals and collagen proteins increases as age progress resulting in bone

weakening 33; 34

. Wilson et al. proposed that organized water layer as a component in the ultra-

structure of bone, existing at the interface between inorganic surface and collagen25

. This water

is described as being in the spaces near bone mineral and collagen. Zhu et al. studied de-

hydration induced time dependent structural changes in intact bone by 13

C NMR spectrum of

organic components26

. Based on 13

C NMR spectrum of dehydration bone and bone with water

exchanged with D2O, they concluded hydrogen bonding network exist between collagen and

surrounding environment through water molecules26

.

Although bone structural and mechanical properties have been well-studied, the knowledge

about how collagen fibrils and HAP crystals interact at the molecular scale and how they deform

as an integrated system under external stress are not well understood. Developing a deeper

understanding of the properties of bone from the level of its building blocks requires a thorough

investigation of the interplay of the organic protein molecules with the mineral crystals. This, in

turn, requires an atomistic-level investigation of the properties of the organic–inorganic

interfaces and its correlation with the overall mechanical behavior.

5.2. Aim and objective

The aim of this chapter is to study water dependent interaction among different components to

understand the ultra – structure of bone. Such an understanding will help in designing bone

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implants with desired properties. In this chapter, we present here the study of interaction of

collagen protein with inorganic surface in bone matrix through water molecules. Our study

involves measurement of distance between collagen protein and inorganic surface in bone

samples with different level of bound water content by 13

C {31

PF REDOR. We also measure

same distance in bone matrix when hydrogen bonding network is weaken by the exchange of

water with D2O. This has given understanding on hydrogen bonding network through water

molecules in organic – mineral interface. Other ssNMR experiment on measurement of local

motional order parameter of collagen and 1H chemical shifts by

13C/

1H Heteronuclear

Correlation (HetCor) experiment in bone samples with different water network has given insight

on structural changes due to de-hydration and H/D exchange.

5.3. Material and Methods

5.3.1 Sample preparation

For ssNMR experiments, Indian Goat (Capra hircus, 2-3 year old) femora bone was taken

from local slaughter house. Intact bone was cylindrical cut (8.0 mm long with the radius of 1.0

mm) so that it can fix into 3.2 mm Zirconium rotor (Figure 5.3). Various degree of dehydration

of bone samples was achieved by placing it in lyophilizer for 24 hour and 72 hours respectively.

For deuterated bone, it was dipped into D2O (Sigma Aldrich USA) for 48 hour to allow

maximum exchange of water present in bone with D2O. We have chosen intact bone for our

study since it has been shown earlier that grinding of bone for ssNMR experiment changes

water content as well as homogeneity of bone matrix26

.

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Figure 5.3. Picture of intact bone along with piece cut to fit into solid state NMR rotor.

5.3.2 NMR experimental Parameters

All ssNMR spectra were recorded on 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 Bruker 3.2 mm DVT probe. Magic Angle Spinning (MAS) frequency was 10.0

kHz for all experiments. The spinning speed was controlled by Bruker MAS pneumatic unit

within accuracy of 2Hz. Pulse length for the Rotation Echo Dipolar Recoupling (REDOR)11

experiment (Figure 5.4) were 1.8 μsec for 1H π/2 pulse , 6.35 μsec for

31P π pulse and 14 μsec

for 13

C π pulse. Recycle delay used for all experiments were 5.0 s. For the REDOR experiments

with de-phasing time of 4.0, 8.0, 20.0, 40.0 ms signal averaging of 5k, 10k, 17k and 26K

number of transients were used. Total signal averaging times were 6.8 hour, 13.8 hour, 23.6

hour and 36.1 hour respectively. The sample and probe stability for such long signal averaging

was checked before. For long signal averaging experiments, small sets of REDOR experiments

(with (S) and without dephasing pulses (S0)) with 512 scans were recorded and was added later

for better signal to noise ratio. Total acquisition time for each REDOR experiments were 11 ms

with 1K data points. For 13

C 1D spectrum ramp cross polarization sequence with SPINAL-6435

spin 1H decoupling (100 kHz

1H r.f. field) and 1.0 ms contact time was used. For

13C {

31P}

REDOR experiment, a sequence with alternating π pulses on 13

C observed channel and 31

P on

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Figure 5.4. (a) 1D pulse sequence (b) 1H/

13C Cross polarization(CP) (c) Pulse sequence used

for recording 1H-

31P and

1H-

13C Hetero-nuclear correlation experiment for intact bone at

different hydration and deuteration levels. The pulse sequence consists of first /2 pulse on 1H

followed by magic angle m pulse to put 1H magnetization perpendicular to the effective field

during t1 period. Frequency shift Lee-Goldburg decoupling was used for homomuclear decoupling during t1 period. After the t1 period, chemical shift evolved

1H magnetization is spin

locked for cross polarization to 31

P or 13

C nuclei. A short cross polarization (CP) time 1ms and

70s was used for one bond correlation for1H-

31P and

1H-

13C Hetero-nuclear correlation

experiment respectively. Finally 31

P or 13

C signal is recorded in presence of SPINAL-64 decoupling on

1H channel. (d)

13C {

31P} REDOR pulse sequence with XY8 phase cycling and

alternating π pulses.

105

dephasing channel was utilized (Figure 5.4). XY-8 phase cycling on observed and dephasing

channel was used to compensate pulse imperfections36

. The 1H decoupling during REDOR

dephasing period were 100 kHz with SPINAL-64 decoupling sequence. The details of REDOR

pulse sequence used for experiment are given in Figure 5.4. The REDOR experiment with (S)

and without (S0) π pulses on 31

P channel were acquired for different dephasing time. The ratio of

signal intensity (S/S0) for different dephasing time gives REDOR curve. For 1H/

13C and

1H/

31P

Hetronuclear correlation (HETCOR)37

experiments (Figure 5.4) effective field during 1H

homonuclear decoupling period (Frequency Switched Lee- Goldburg, FSLG)38

were 80 kHz and

50 kHz respectively. High power 1H decoupling (100 kHz) was applied during t2 period.

5.3.3 Simulation and data fitting:

REDOR simulation curves for different spin pairs were generated by SIMPSON simulation

environment39

. The simulation program is given in Appendix II. REDOR curves for two spin

systems with 13

C and 31

P nuclei and for three spin systems with one 13

C nuclei dipolar coupled

with two 31

P nuclei were considered (Figure 5.5). For both type of spin system, dipolar

coupling values corresponding to different distances between 13

C and 31

P nuclei were

considered and REDOR curves were generated. These spin systems have been shown earlier to

accurately represent dipolar coupling network for the study of organic mineral interface in

bones14; 40

. Distance between different 13

C nuclei of collagen and 31

P nuclei of hydroxyapatite

were found in intact bone at different level of hydration and in H/D exchange conditions (Table

5.1). T2 values of 13

C signal of collagen in intact bone at different level of hydration and H/D

exchange were calculated by recording 13

C spectrum as a function of de-phasing time in S0

REDOR experiment. Best fit to the experimental data were calculated by MATLAB

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Figure 5.5. Two spin system and three spin system spin topologies used in REDOR simulation. Arrows direction in the figure shows the dipolar coupling that is varied in the REDOR simulation. For the 31P spin pair 600 Hz homonuclear dipolar coupling (fixed) was considered as reported in previous studies. (The Mathworks Inc.) program. T2 values corresponding to different resonances in bone

samples are given in Table 5.1.

5.3.4 Stability of water content in bone under Magic Angle Spinning: To check the change in hydration level of intact bone under Magic Angle Spinning,

13C

spectrum of bone were recorded after it undergoes MAS continuously for one week. The

resulting spectra recorded at different days are shown in Figure 5.6 (A-F). The spectra were

recorded with 512 signal averaging scans. It can be seen that spectrum recorded after one week

of continuous MAS (Figure 5.6) is similar to the initial spectrum shown in Figure 5.6(A).

Three distinct carbonyl peaks shown around 178 ppm remains same even after one week of

MAS. This shows that hydration level of intact bone under MAS in sealed condition remains

constant. This has been achieved due to the fact that we used intact bone sealed with Teflon tape

to prevent any escape of water molecule from MAS rotor.

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Figure 5.6. To check the stability of the bone sample a quick 13

C spectra with 1H decoupling

were recorded under Magic Angle Spinning (MAS) for 7 days. (A) 13

C spectra of fresh bone (B) 13

C spectra of same bone after MAS for two days (C) 13

C spectra of same bone after three days of MAS. (D), (E) and (F) shows the spectra recorded after four, five and seven days of MAS respectively. It can be seen that the effect of MAS on dehydration of intact bone is negligible.

5.4. Results

Two types of bones samples were used earlier for the ssNMR experiments in literature. These

samples were cryogenically ground bone and intact bone. Cryogenic grinding changes the ultra

– structural properties of bone26; 41

as well as water content reduces during the course of 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

NMR rotor to allow water molecules to escape26

. We performed ssNMR experiments on intact

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Indian Goat femora bone by cutting in a small piece to fit inside MAS rotor (figure 5.3). The

bone sample was sealed with Teflon tape to avoid any escape of water molecules during NMR

experiment. We find that water content was almost same even with seven days of MAS (figure

3.6). This was necessary to check since our experiment for measuring long – range distance

between collagen and inorganic surface requires signal averaging for long time period. Water

content was verified with 1H NMR spectra recorded each day in seven days of continuous MAS.

Natural abundance 13

C chemical shift of collagen is very sensitive indicator of any change in the

water content26

. The 13

C NMR spectrum with 1H decoupling of bone were recorded each day

during seven days of MAS. We didn’t find any significant change in the 13

C 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 hydrogen bonding

network and water content. Reduction in strength of hydrogen bonding network by exchange of

1H with

2H is well known

26. This is due to the fact of electro negativity differences in H-O and

D-O bonds. The one dimensional (1D) 13

C NMR spectrum along with two – dimensional (2D)

1H –

31P HetCor experiments of all four bone samples are shown in Figure 5.7. The bound

water level content in these samples can be seen in 2D 1H –

31P spectrum which shows resolved

peaks from OH- and bound water. In samples with one day and three days of dehydration, bound

water peaks is significantly low in intensity compared to fresh bone sample (Figure 5.7). Also

in H/D exchange bone sample, bound water peak intensity is somewhere in between fresh and

dehydrated bone. The corresponding natural abundance 13

C spectra are shown for all four bone

samples. Various resonances in 13

C spectrum corresponding to organic components can be

identified and assigned 26; 28; 42-44

. Most of the resonances correspond to Type 1 collagen

residues and citrate which resonates at 76 ppm. The carbonyl carbons of collagen resonate

109

around 175 ppm. As bound water contents are reduced, the carbonyl resonances merge and line

width of aliphatic peaks increases slightly. It should be noted that dehydration induced changes

observed in 13

C spectrum is consistent with observation of Zhu et al26

.

In order to see structural changes due to different level of bound water content and

hydrogen bonding network, we measured performed ssNMR experiment to measure distance

between collagen residues and 31

P of inorganic components (HAP). We performed 13

C{31

P}

Rotational Echo Double Resonance (REDOR) NMR experiment to measure distances between

collagen side chain and inorganic part11

. In present method of using intact bone in sealed

conditions, we could observe 13

C transverse relaxation time (T2) of the order of 50.0 ms making

large distance measurement possible. Signal intensity of REDOR with and without de-phasing

pulses was recorded for different de-phasing time. REDOR experiment have been recorded for

dephasing time of 4.0 ms, 8.0 ms, 20.0 ms and 40.0 ms. For the 8.0 ms significant dephasing is

only observed for citrate resonance at 76 ppm and slight change in intensity is observed in

aliphatic region (20-70 ppm). It was shown earlier that in the 13

C spectrum of bone,

concentration of other phosphorylated compound is too small to be observed in NMR spectrum.

Hence whatever dephasing we observe in 13

C{31

P} REDOR is from inorganic surface9. This

observation is consistent with earlier reported studies9. It can be seen that significant dephasing

was observed for 20ms and 40 ms dephasing time (Figure 5.8). The amount of and 40.0 ms

dephasing time, significant amount of dephasing were observed in aliphatic region of collagen

protein spectrum. One such spectrum for dephasing time of 20.0 ms and 40.0 ms is shown in

Figure 5.8. Amount of dephasing corresponding to different residues can be measured and this

makes possible to estimate the long range distance between the collagen side chain and

inorganic mineral interface. REDOR measured S/S0 for different dephasing times corresponding

to hydroxy-proline C residue along with simulated curve for different distances is shown in

110

Figure 5.7: 13

C NMR spectra (a-d) 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 hours. Two dimensional

1H /

31P correlation NMR spectra of bones (e-h) at various stages. (e) Fresh intact

bone, (f) bone dehydrated for one day, (g) bone dehydrated for three days and (h) fresh intact bone deutarted for 48 hours. Various

13C signals corresponding to collagen are shown at top of

large distance measurement possible. Signal intensity of REDOR with and without de-phasing pulses was recorded for different de-phasing time. REDOR experiment have been recorded for dephasing time of 4.0 ms, 8.0 ms, 20.0spectrum. In 2D

1H/

31P NMR (E-H) spectra, water peak

(H2O) and hydroxyl ion (OH) peaks are marked in the spectrum.

111

Figure 5.8. 13

C spectrum of fresh bone showing de-phasing at (a) 20 ms and (b) 40ms. The red spectrum was acquired without

31P de-phasing pulse and green one with

31P pulses on.

dephasing observed is inversely proportional to the distance between two nuclei.

Figure 5.9. The REDOR curve for different distances can be simulated and best fit to

experimental data can be measured. For most of the side chain residues of collagen, the

distances with inorganic phosphorus were measured and these are shown in Table 5.1. The

distances were measured in all four bone samples with different degree of hydration and H/D

exchange bone samples. The corresponding errors in the estimation of various distances are also

shown in Table 5.1. It is interesting to note that 1H decoupling efficiency during REDOR period

will be crucial for this type of experiment. We have used high power 100 kHz SPINAL-64 1H

112

decoupling during REDOR dephasing period. This is very large decoupling power and will be

sufficient. Also, the decoupling efficiency will be same for reference (S0) as well as dephased

Figure 5.9. 13

C REDOR data of bone fresh intact bone, bone dehydrated for one day, bone dehydrated for three days, fresh intact bone H/D exchanged for two days. The data shows plot of S/S0 as a function of dephasing time and various best fit curves corresponding to different hetronuclear distances. Above figure solid lines are for best fit distances of hydroxyapatite

surface and collagen amino acid hydroxyproline Cresidue. The best fit REDOR data for (a) two spin system (C-P) (b) three spin system (C-P2) and (c) three spin system (C-P2) with P-P dipolar coupling of 600 Hz is shown in figure. Blue color corresponds to fresh intact bone, red for three days dehydrated bone sample and green for the H/D exchanged bone.

(S) REDOR spectrum. Hence, when we take 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 in the change in

interaction of collagen with inorganic surface due to change in water content and reduction in

hydrogen bonding network strength. We can see that the distance between collagen and

inorganic surface reduces by reduction in bound water content. The distance measured in H/D

exchange bone is somewhere in 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 change in internal structure30

. Figure

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5.10 represent change in S/S0 values for 20 ms and 40 ms dephasing time for few residues of

collagen for bone samples with different hydration level.

Table 5.1 Various distances and 13

C T2 measured in different bone samples.

Residue Two spin system

(Å)

Three spin system Without Homonuclear

coupling

(Å)

Three spin system With

Homonuclear coupling(600Hz)

(Å)

13C T2 Values

(ms)

Hydroxyproline

C

Fresh bone 9.0±.06 9.8±.09 9.7±0.7 75.4 ±7.0

One day dehydration 8.5±0.4 8.6±.05 9.0±0.5 39.7±4.0

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±.06 8.5±.06 8.6±0.6 26.6±.1.5

Glycine C Fresh bone 9.1±0.7 9.8±.06 9.9±0.7 32.6 ±2.2

One day dehydration 8.5±0.4 9.1±.05 9.1±0.5 24.39± 1.1

Three day dehydration 7.2±0.6 7.9±.06 8.1±0.6 16 ± 1.3

H/D exchanged bone 8.6±0.7 8.6±.05 8.5±0.5 15.8±1.4

Proline C Fresh bone 8.9±.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±.07 16.6 ± 1.4

Alanine C 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 7.3±0.8 7.9±0.8 8.0±0.7 17.2 ± 2.3

H/D exchanged bone 7.6±0.4 8.5±0.5 8.5±0.5 19.3±2.1

Pro C Fresh Bone 8.8±0.8 9.3±0.6 9.5±.07 -

Proline C Fresh bone 9.6±0.6 10.2±0.8 10.4±1.0 -

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Figure 5.10. REDOR dephasing S/S0 observed for resonances corresponding to (a) Hydroxy –

proline C, (b) Glycine C, (c) Proline C and (d) Alanine C side chain of collagen in bones with different dehydration and H/D exchange level. Square box represents dephasing S/S0 observed at 20ms and round curve one corresponding to 40ms.

115

Figure 5.11. 13

C{P} REDOR data of Bone (Glycine C and Alanine C at different level of hydration and deuteration. Corresponding best fit with different distances and spin systems are shown inside each figure.

116

Figure 5.12. 13

C{P} REDOR data of Bone (Proline C at different level of hydration and H/D exchanged Corresponding best fit with different distances and spin systems are shown inside figure.

117

The corresponding change in S/S0 values will give direct evidence of change in distance

between collagen and inorganic surface as a function of hydration level in bones. It can be seen

that as water content in bone reduces, there is significant reduction in S/S0 values for all four

side chain residues of collagen. This clearly indicates that collagen is coming closer to inorganic

Figure 5.13. Curves showing variation of 13

C transverse magnetization with dephasing time for

T2 estimation of different resonances of collagen corresponding to Hydroxyproline C, Alanine

C, Proline C and Glycine C. . The estimation of T2 was carried out for bone under different level of hydration and deuteration. Corresponding T2 values are given in the figure.

118

surface when water content reduces in bone matrix. Further, for bone sample with H/D

exchange, S/S0 values are in between those corresponding to fresh bone and completely dry

bone.

Change in local environment of collagen due to different bound water content can also be

measured by 1H –

13C HetCor and T2 measurement of different

13C resonances. For same bone

samples with different degree of hydration level, 1H –

13C HetCor experiments and T2

measurements were performed (Figure 5.13). Figure 5.14 shows 1H –

13C HetCor spectrum for

bone samples with different level of water content and hydrogen bonding network. The

resolution in

Figure 5.14. 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.

119

2D 1H –

13C HetCor spectrum is good enough to resolve

1H chemical shift of various side chain

residues of collagen. The corresponding assignments along with 1D 13

C spectrum is shown in

Figure 5.14. We observe that bones with different level of bound water content, there is

significant low field shift in 1H chemical shift although

13C chemical shift does not show

significant change. This confirms no significant change in collagen structure due to difference in

hydration level although there is 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 13

C transverse relaxation rate T2 is an indicator of local

motion and environment. Measured T2 corresponding to different side chains of collagen are

shown in Table 5.1. As bone gets dehydrated, T2 values reduce corresponding to lesser side

chain motional order parameter. The T2 values of H/D exchanged bone sample are in between

fresh and completely dehydrated bone.

5.5. Discussion

Our experimental results indicate the bone mineral, protein collagen and water

molecules corresponding to model shown in Figure 5.15. As bound water level in bone matrix

reduces, cross linking between collagen and inorganic surface increases due to which collagen

comes closer to inorganic surface. In fresh bone, the distance between collagen side chain and

inorganic phosphorus is mostly around 9.0 Å. In our study, such large distance measurements

became possible due to 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

45. Such distance

can be translated to a 15

N-13

C dipolar coupling constant of 18.4 Hz, which is quite comparable

to the 13

C-31

P coupling constant for a distance corresponding to 9 Å. The previous bone study by

120

Drobny group has illustrated that the statherin protein bind to inorganic surface via specific

residue and each specific residue distance from inorganic phosphate has been reported around

4.5-5 Å14; 16-19

. Duer et. al. in their study showed that polysaccharide GAGS is within 5A

distance from inorganic surface9; 10

. These earlier study were focused towards measuring

distance statherin protein and polysaccharide such as GAGS in bone and bone like model

systems.

Figure 5.15. A 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. For fresh bones, and with different degree of dehydration, the distance between carbons

of collagen side chain and phosphorus of hydroxyapatite is of the order of 9.0±0.5 Å, 8.4±0.5 Å

and 7.6±0.5 Å respectively. Same trend is observed when we exchange water with D2O which

has reduced hydrogen bonding strength. We observe distance of the order 8.2±0.6 Å in H/D

exchanged bone samples. Our measured distances have error bars (Table 5.1). The error bars

were calculated based on detailed error analysis which takes into account of 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 inorganic surface. These reported

average distance changes with water content in bone matrix. This can be explained only when

121

water forms a hydrogen bonding network with inorganic surface and collagen. The systematic

weakening of the hydrogen-bonding network as dehydration level increases resulting in

decrease in the distance between collagen and inorganic surface. The change in 1H chemical

shifts and T2 values of 13

C 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 inorganic surface increases due to dehydration and H/D exchange.

Increase in this interaction strength cause bone to be more susceptible towards fracture due to

restricted motion of water molecules which provides tensile strength against any external

pressure32

. It has been shown earlier that water content in bone matrix is directly related to the

mechanical properties of bone46

. Hence, we can conclude that water forms a hydrogen bonding

network with inorganic phosphorus and collagen. Such network stabilizes bone matrix and is

responsible for mechanical properties of bone.

5.6. Bone Model

Various proteins in bone minerals have been identified earlier. One of the proteins in bone

matrix is statherin which is responsible for initiating mineralization. Various studies have been

carried out to understand the structure of statherin in bone matrix13; 14; 16; 17; 47-49

. It is widely

accepted that this protein is within a distance of 4 Ǻ from inorganic surface14; 50

. Other studies

confirm that polysaccharide GAGS is within 5 Ǻ distance from inorganic surface9; 10

. Recent

study confirms that citrate is closer to the inorganic surface20

. There are various other molecules

responsible for cross linking between collagen and inorganic surface7. Water plays a crucial role

in hydrogen bonding network between collagen and inorganic surface. Recent studies by Klaus

Schmidt-Rohr et al measure 3 nm thickness of inorganic surface in bone matrix51

. Assuming

collagen to be 3 nm wide, this model corresponds to approximately 10% of water content in

122

bone matrix. If we take into account on C-H bond length and distance of phosphorus from

inorganic surface, water layer thickness will be around 7Ǻ 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 bone8. The

hydrogen bonded network of water molecules act as lubricant for relative motion of collagen

with inorganic surface due to external stress8; 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 event of

external stress. As water content reduces in bone matrix, collagen comes closer to 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 model32; 46

.

5.7. Conclusion

In present study, we have given a study to measure long range distance of collagen with

inorganic surface in intact bone. The study is based on high resolution solid state NMR

experiment to measure distance between collagen 13

C and 31

P of inorganic surface by REDOR

experiment. Reduction in bound water content results in decrease of this distance. Further

ssNMR experiment to measure local order parameter of collagen and 1H chemical shift confirms

that collagen is coming closer to inorganic surface as water content reduces. Our finding gives

new structural insight into the role of water in bone strength. This may have possible

implications in understanding the bone weakening mechanism due to disease conditions and

designing suitable materials of bone implants.

123

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