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Efficiency of 1H→31P NMR cross-polarization in bone apatite and its mineral standards
ARTICLE IN PRESS
Solid State Nuclear Magnetic Resonance 29 (2006) 345–348 www.elsevier.com/locate/ssnmr
Efficiency of 1H-31P NMR cross-polarization in bone apatite and its mineral standards Agnieszka Kaflaka, Dariusz Chmielewskib, Andrzej Go´reckib, Anna Slosarczykc, Waclaw Kolodziejskia, a
Medical University of Warsaw, Faculty of Pharmacy, Department of Inorganic and Analytical Chemistry, ul. Banacha 1, 02-097 Warszawa, Poland b Medical University of Warsaw, Department of Orthopaedics, ul. Lindleya 4, 02-005 Warszawa, Poland c AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Al. Mickiewicza 30, 30-059 Krakow, Poland Received 7 November 2005 Available online 13 December 2005
Abstract Human bone mineral was studied using solid-state 31P NMR with cross-polarization (CP) from protons. The CP efficiency was determined for trabecular and cortical bone tissue from human adults and compared with synthetic mineral standards. The study shows the similarity between carbonatoapatite of type B and bone mineral as shown by their CP behaviour. The method can be used for the characterization of synthetic apatite-based implant materials. r 2005 Elsevier Inc. All rights reserved. Keywords: Bone; Mineral; Apatite;
31
P NMR; Cross-polarization
1. Introduction Bone is a complex and challenging material for NMR spectroscopy [1]. We have to differentiate between cortical (compact) and trabecular (sponge-like) tissue. They comprise ca. 80 and 20 wt%, respectively, of the human skeleton. The bone tissue cells (ca. 2 wt%) are spread throughout the bulk of the extracellular matter. This has mineral and organic constituents (ca. 65% and 25%, respectively), and contains water (ca. 10 wt%) [2]. The bone mineral is dominated by calcium apatite deposited on an organic matrix, which is ca. 90% collagen. Here, we focus our attention on the mineral part of bone. Unfortunately, all the complex structural information is contained in a single, featureless 31P line, appearing at 3.1 ppm from 85% H3PO4 [3]. It is often taken for granted that bone mineral is identical with calcium hydroxyapatite (HA), Ca10(OH)2(PO4)6, as first proposed by DeJong [4]. However, the HA crystal Corresponding author. Fax: +48 22 5720784.
E-mail address: [email protected] (W. Kolodziejski). 0926-2040/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ssnmr.2005.11.005
lattice (hexagonal space group P63/m [5]) can accommodate various extra ions, releasing in turn Ca2+, OH or PO34 ions, thus maintaining the electric charge balance [6]. In particular, carbonate ions CO23 can replace hydroxyl ions OH or ortophosphate ions PO34 , leading to carbonatoapatites of type A and B, respectively. Biological apatites are not pure HA and stoichiometric, and should rather be classified as carbonatoapatites [2,3,6–11]. Carbonate CO23 is the principal minor constituent of the bone mineral, estimated at 5–8 wt% [2,6,12–15]. Comparative infrared studies of synthetic carbonatoapatites and biological apatites indicate type B substitution in the latter materials [6–8,12–14,16–27]. Our earlier 1H-31P CP/MAS NMR results for human bone agree with this conclusion [3,10,11]. Cross-polarization (CP) was designed to enhance lowintensity solid-state NMR signals from dilute spins by polarization transfer from abundant spins, usually protons [28–30]. It can be also used for exposing protonated sites, for achieving 2D chemical shift correlation or for structural studies involving CP kinetics [31]. Since CP relies on heteronuclear dipolar couplings between the source and target spins, its efficiency is uniquely dependent on the
ARTICLE IN PRESS 346
A. Kaflak et al. / Solid State Nuclear Magnetic Resonance 29 (2006) 345–348
concentration, distribution and mobility of the nuclei involved. Therefore, the technique is remarkably sensitive to the composition, structure, properties and molecular dynamics in the solid state. The CP efficiency can be determined by comparison with the conventional Blochdecay (BD) experiment. However, we must remember that CP efficiency also strongly depends on experimental conditions, such as contact time, spin-lock power levels, decoupling power and the rate of magic-angle spinning (MAS). To make reliable comparisons, these experimental parameters must be rigorously kept identical and constant. CP also proceeds in systems where the source and target spins are similarly populated. This is the case for 1H-31P CP in apatites, where the CP signal is reduced in comparison with the BD signal of the same sample under optimized experimental conditions for both measurements. In this communication, the CP efficiency is presented for model compounds of bone mineral and compared with various samples of human bone. 2. Experimental Stoichiometric HA was synthesized by the wet chemical method (Ca/P ratio of 1.67) [32]. It was pre-dried in air to give as-synthesized sample HAh (h stands for hydrated). HAh was then calcined at 1073 K to give HAc (c stands for calcined). Carbonatoapatite of type B (CHA-B) with ca. 9 wt% of CO23 was synthesized according to Merry et al. [33]. Tricalcium phosphate Ca3(PO4)2 (TCP) was synthesized according to the standard procedure [34]. Brushite CaHPO4 2H2O (BRU) is a natural mineral from Japan. The purity and crystallinity of solid phosphates was confirmed by IR-microscopy (specular reflectance) and powder XRD. The samples of bone apatites (BHA) were gifts from the Glass and Ceramics Institute (Warsaw). The organic part of the bone was removed by chemical (leaching with NaHCO3) and thermal treatment (calcination at 1200 1C). Sample BHA1 was prepared from a piece of bone containing both trabecular and cortical tissue. Sample BHA2 was prepared exclusively from trabecular tissue. The bone samples were prepared at the Medical University of Warsaw. The cortical bone tissue was taken during autopsy from the tibia of a healthy 24-year old male killed in a car accident. The bone was not damaged and showed no pathological changes. The bone was powdered and divided into four samples, B1–B4, which were then treated in various ways (Table 1). The powdered bone
samples were examined with and without MAS. The trabecular bone was extracted from the femoral neck as small cylinders (samples B5 and B6), 6 mm in diameter and 20 mm long, to fit standard 7 mm Varian rotors. The bone cylinders were not capable of smooth spinning, so they were not examined with MAS. Sample B5 was taken during autopsy from a healthy 29-year old male, the victim of a car accident. The femur was not damaged and showed no pathological changes. Sample B6 was taken during surgical replacement of a hip joint with an alloplastic implant. The patient was a 69-year old female with osteopenia (low bone turnover), suffering from degenerative disease of the hip joints. Samples B5 and B6 were lyophylized, sterilized by gradiation (3.5 kGy) and stored at 277 K. The study was approved by the local research ethics committee. The HAh, HAc, CHA-B and BRU standards, and bone samples B1–B3 were the same as in our earlier study [3]. Solid-state 31P NMR spectra were acquired at 81 MHz on a Varian UNITY PLUS-200 spectrometer, using a Doty double-bearing probe and 7 mm zirconia rotors. All measurements were made at 298 K. The MAS spectra were acquired under rotation at 3 kHz. The rotors were spun using dry air. The intensities of the NMR lines from conventional pulse-acquire experiments, hereafter called BD experiments, and from CP experiments carried out from protons to phosphorus-31 (1H-31P CP), were compared. We used the proprietary Varian pulse program XPOLAR, capable of dealing with both BD and CP. The program executes the ordinary single-contact CP pulse sequence, with reversal of spin-temperature in the rotating frame. In each case, the BD and CP spectra were measured with XPOLAR in the arrayed (consecutive) mode within the same two-stage experiment. For CP, a contact time of 1 ms was chosen. BD and CP were both performed at the same high-power level for proton decoupling, with the same number of scans, the same proton p/2 pulses of 6.7 ms and the same recycle delays optimized for BD on a particular sample. Consequently, the same apodization was applied. The Hartmann–Hahn condition was carefully adjusted before each experiment, separately for MAS and static conditions. Signal intensities were measured at peak tops. 3. Results and discussion Apatite gives a single 31P line, with and without CP [1]. The 1H-31P polarization transfer in bone and synthetic apatites proceeds from protons of water and structural
Table 1 Preparation of cortical bone samples Sample
Preservation
Sterilization (radiation dose)
Storage temperature (K)
B1 B2 B3 B4
Lyophilization Freezing at 203 K Freezing at 203 K Lyophilization
— — b-radiation (35 kGy) b-radiation (35 kGy)
277 277 277 277
ARTICLE IN PRESS A. Kaflak et al. / Solid State Nuclear Magnetic Resonance 29 (2006) 345–348
hydroxyl groups [1,35]. The organic matter of bone does not participate in the 1H-31P CP process [36]. Water is mainly located on crystal surfaces and is loosely bound or strongly adsorbed. Therefore, CP is strongly dependent on the specific surface area of these materials and on the hydration of crystal surfaces. It follows that crystal dimensions and habits, and the state of surfaces (concentration and distribution of surface ions and water) are crucial for CP efficiency. The second CP contribution, that from structural hydroxyl groups, is strongly dependent on their concentration. It has been already established that bone apatite is deficient in hydroxyl groups [1,3,35,37,38]. Cho et al. [35] estimated the percentage of structural hydroxyl groups in bone mineral as 2171% of the stoichiometric content. All this assures us that the CP efficiency reflects many subtle factors determining the overall properties of our samples. The CP efficiency has to be adequately measured. Any definition will be arbitrary and we have to make reasonable decisions, considering both the conceptual and operational aspects. Note that the BD peak is from all the 31P nuclei of the material, while the CP peak is from the 31P nuclei located close to protons. Moreover, the BD intensity should be less sensitive to the experimental set up. It looks as if the BD experiment can provide us with some sort of reference intensity. For the sake of this argument, we define the CP efficiency as the percentage of the CP intensity in relation to the BD intensity, both assessed for the same sample on the basis of the signal amplitudes. Thus defined, CP efficiency can be measured for static samples or under MAS. We chose the MAS rate of 3 kHz, because for apatite it provides quite small spinning sidebands (Fig. 1) and it is sufficient to consider only the centreband. We decided to do CP with 1 ms contact time, because it roughly corresponds to the optimum intensity for various apatites (for bone mineral see [36]). The results (Table 2) fall between two outlying cases. TCP has a very small CP efficiency, because it does not contain structural hydrogen. BRU has very high CP
347
Table 2 1 H-31P CP efficiency for bone samples and various phosphates, determined as the intensity ratio in % of the CP and BD signal amplitudes Sample
HAh HAc BHA1 BHA2 CHA-B BRU TCP B1 B2 B3 B4 B5 B6 a
CP/BD (%) MAS at 3 kHz
Static
45 39 37 — 26 163 3 20 20 21 22 — —
37 44 40 28 26 129, 121, 130a 2 23 22 22 22 22 21
Split by dipolar coupling.
efficiency, because it contains proximate 1H and 31P spins located in the POH groups. BRU is interesting as a tractable model for predicting the NMR behaviour of HPO24 in bone apatite [1,39], although this has been questioned [40]. It is obvious that the HA and BHA samples show very different CP behaviour from the bone samples. In this respect, CHA-B is most similar to the bone mineral. There are practically no differences between the bone samples, although they were taken from three donors and were differently treated. In particular, the lyophilized samples B1 and B4 behave similarly to the hydrated samples B2 and B3. This means that lyophilization removes only loosely bound water, which does not take part in CP. At this stage of the study, we cannot make general statements on the bone samples, because we need to examine a larger and more diverse group. However, it is clear that the CP efficiency method is useful for the characterization and standarization of phosphate materials, and for matching their structure and composition. Application to composite, apatite-based prosthetic materials is straightforward.
4. Conclusions The conclusions of this study are summerized as follows:
Fig. 1. 31P BD NMR spectra of human bone tissue recorded with various MAS rates (sample B1).
(1) For 1H-31P CP efficiency, the CHA-B sample most resembles the bone samples B1–B6. This agrees with our former NMR studies [1,3,10,11], indicating that the bone mineral is closest in chemical structure to carbonatoapatite of type B. (2) CP efficiency is suitable for the characterization of phosphate materials.
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Acknowledgment The authors are grateful to the Medical University of Warsaw for financial support. References [1] W. Kolodziejski, Top. Cur. Chem. 246 (2005) 235. [2] R.Z. LeGeros, Prog. Cryst. Growth Charact. 4 (1981) 1. [3] A. Kaflak-Hachulska, A. Samoson, W. Kolodziejski, Calcif. Tissue Int. 73 (2003) 476. [4] W.F. DeJong, Res. Trav. Chim. 45 (1926) 445. [5] K. Sudarsanan, R.A. Young, Acta Crystallogr. B 25 (1969) 1534. [6] R.Z. LeGeros, in: P.W. Brown, B. Constanz (Eds.), Hydroxyapatite and Related Materials, CRC Press, Boca Raton, 1994, pp. 3–28. [7] R.Z. LeGeros, Crystallographic studies of the carbonate substitution in the apatite structure, Ph.D. Thesis, New York University, New York, 1967. [8] R.Z. LeGeros, Monographs in Oral Science, vol. 15. Calcium Phosphates in Oral Biology and Medicine, S. Karger, Basel, 1991. [9] J.D.B. Featherstone, I. Mayer, F.C. Driessens, R.M.H. Verbeeck, H.J.M. Heijligers, Calcif. Tissue Int. 35 (1983) 169. [10] A. Kaflak-Hachulska, D. Chmielewski, A. Go´recki, W. Kolodziejski, in: Materials of the Second Alpine Conference on Solid-State NMR. Chamonix–Mont Blanc, France, 2001. [11] W. Kolodziejski, A. Kaflak-Hachulska, in: Materials of the ISMAR 2001 Conference, Rhodes, Greece, 2001. [12] R. Legros, N. Balmain, G. Bonel, J. Chem. Res. (S) (1986) 8. [13] H. Aoki, Science and Medical Applications of Hydroxyapatite, Takayama Press, Tokyo, 1991. [14] F.C. Driessens, Z. Naturforsch. (C) 35 (1980) 357. [15] A.H. Roufosse, W.P. Aue, J.E. Roberts, M.J. Glimcher, R.G. Grffin, Biochemistry 23 (1984) 6115. [16] W.H. Emerson, E.E. Fisher, Arch. Oral Biol. 7 (1962) 671. [17] J.C. Elliott, in: M.V. Slack, R.W. Fearnhead (Eds.), Tooth Enamel, J. Wright & Sons, Bristol, 1965, pp. 20–22.
[18] R.Z. LeGeros, O.R. Trautz, J.P. LeGeros, E. Klein, Bull. Soc. Chim. Fr. 4 (1968) 1712. [19] R.Z. LeGeros, O.R. Trautz, E. Klein, J.P. LeGeros, Experientia 25 (1969) 5. [20] G. Bonel, Ann. Chim. 7 (1972) 127. [21] J.-C. Labarthe, G. Bonel, G. Montel, Ann. Chim. 8 (1973) 289. [22] R.M. Blitz, E.D. Pellegrino, Clin. Orthop. 129 (1977) 279. [23] M. Santos, P.F. Gonza´lez-Diaz, Inorg. Chem. 16 (1977) 2131. [24] J.D. Featherstone, S. Pearson, R.Z. LeGeros, Caries Res 18 (1984) 63. [25] M. Vignoles, G. Bonel, D.W. Holcomb, R.A. Young, Calcif. Tissue Int. 43 (1988) 33. [26] C. Rey, B. Collins, T. Goehl, I.R. Dickson, M.J. Glimcher, Calcif. Tissue Int. 45 (1989) 157. [27] C. Rey, V. Renugopalakrishnan, M. Shimuzu, B. Collins, M.J. Glimcher, Calcif. Tissue Int. 49 (1991) 259. [28] A. Pines, M.G. Gibby, J.S. Waugh, J. Chem. Phys. 59 (1973) 569. [29] C.A. Fyfe, Solid state NMR for Chemists, CFC Press, Guelph, 1983. [30] E.O. Stejskal, J.D. Memory, High resolution NMR in the solid state. Fundamentals of CP/MAS,, Oxford University Press, Oxford, 1994. [31] W. Kolodziejski, J. Klinowski, Chem. Rev. 102 (2002) 613. [32] A. Slo´sarczyk, E. Stobierska, Z. Paszkiewicz, M. Gawlicki, J. Am. Ceram. Soc. 79 (1996) 2539. [33] J.C. Merry, I.R. Gibson, S.M. Best, W. Bonfield, J. Mater. Sci. Mater. Med. 9 (1998) 779. [34] E. Salahi, J.G. Heinrich, Br. Ceram. Trans. 102 (2003) 79. [35] G. Cho, Y. Wu, J.L. Ackerman, Science 300 (5622) (2003) 1123. [36] A. Kaflak, D. Chmielewski, A. Go´recki, W. Kolodziejski, Solid State NMR 10 (1998) 191. [37] C. Rey, J.L. Miquel, L. Facchini, A.P. Legrand, M.J. Glimcher, Bone 16 (1995) 583. [38] C.-K. Loong, C. Rey, L.T. Kuhn, C. Combes, Y. Wu, S.-H. Chen, M.J. Glimcher, Bone 26 (2000) 599. [39] A. Kaflak-Hachulska, A. Slo´sarczyk, W. Kolodziejski, Solid State NMR 15 (2000) 237. [40] Y. Wu, M.J. Glimcher, C. Rey, J.L. Ackerman, J. Mol. Biol. 244 (1994) 423.
Solid State Nuclear Magnetic Resonance 29 (2006) 345–348 www.elsevier.com/locate/ssnmr
Efficiency of 1H-31P NMR cross-polarization in bone apatite and its mineral standards Agnieszka Kaflaka, Dariusz Chmielewskib, Andrzej Go´reckib, Anna Slosarczykc, Waclaw Kolodziejskia, a
Medical University of Warsaw, Faculty of Pharmacy, Department of Inorganic and Analytical Chemistry, ul. Banacha 1, 02-097 Warszawa, Poland b Medical University of Warsaw, Department of Orthopaedics, ul. Lindleya 4, 02-005 Warszawa, Poland c AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Al. Mickiewicza 30, 30-059 Krakow, Poland Received 7 November 2005 Available online 13 December 2005
Abstract Human bone mineral was studied using solid-state 31P NMR with cross-polarization (CP) from protons. The CP efficiency was determined for trabecular and cortical bone tissue from human adults and compared with synthetic mineral standards. The study shows the similarity between carbonatoapatite of type B and bone mineral as shown by their CP behaviour. The method can be used for the characterization of synthetic apatite-based implant materials. r 2005 Elsevier Inc. All rights reserved. Keywords: Bone; Mineral; Apatite;
31
P NMR; Cross-polarization
1. Introduction Bone is a complex and challenging material for NMR spectroscopy [1]. We have to differentiate between cortical (compact) and trabecular (sponge-like) tissue. They comprise ca. 80 and 20 wt%, respectively, of the human skeleton. The bone tissue cells (ca. 2 wt%) are spread throughout the bulk of the extracellular matter. This has mineral and organic constituents (ca. 65% and 25%, respectively), and contains water (ca. 10 wt%) [2]. The bone mineral is dominated by calcium apatite deposited on an organic matrix, which is ca. 90% collagen. Here, we focus our attention on the mineral part of bone. Unfortunately, all the complex structural information is contained in a single, featureless 31P line, appearing at 3.1 ppm from 85% H3PO4 [3]. It is often taken for granted that bone mineral is identical with calcium hydroxyapatite (HA), Ca10(OH)2(PO4)6, as first proposed by DeJong [4]. However, the HA crystal Corresponding author. Fax: +48 22 5720784.
E-mail address: [email protected] (W. Kolodziejski). 0926-2040/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ssnmr.2005.11.005
lattice (hexagonal space group P63/m [5]) can accommodate various extra ions, releasing in turn Ca2+, OH or PO34 ions, thus maintaining the electric charge balance [6]. In particular, carbonate ions CO23 can replace hydroxyl ions OH or ortophosphate ions PO34 , leading to carbonatoapatites of type A and B, respectively. Biological apatites are not pure HA and stoichiometric, and should rather be classified as carbonatoapatites [2,3,6–11]. Carbonate CO23 is the principal minor constituent of the bone mineral, estimated at 5–8 wt% [2,6,12–15]. Comparative infrared studies of synthetic carbonatoapatites and biological apatites indicate type B substitution in the latter materials [6–8,12–14,16–27]. Our earlier 1H-31P CP/MAS NMR results for human bone agree with this conclusion [3,10,11]. Cross-polarization (CP) was designed to enhance lowintensity solid-state NMR signals from dilute spins by polarization transfer from abundant spins, usually protons [28–30]. It can be also used for exposing protonated sites, for achieving 2D chemical shift correlation or for structural studies involving CP kinetics [31]. Since CP relies on heteronuclear dipolar couplings between the source and target spins, its efficiency is uniquely dependent on the
ARTICLE IN PRESS 346
A. Kaflak et al. / Solid State Nuclear Magnetic Resonance 29 (2006) 345–348
concentration, distribution and mobility of the nuclei involved. Therefore, the technique is remarkably sensitive to the composition, structure, properties and molecular dynamics in the solid state. The CP efficiency can be determined by comparison with the conventional Blochdecay (BD) experiment. However, we must remember that CP efficiency also strongly depends on experimental conditions, such as contact time, spin-lock power levels, decoupling power and the rate of magic-angle spinning (MAS). To make reliable comparisons, these experimental parameters must be rigorously kept identical and constant. CP also proceeds in systems where the source and target spins are similarly populated. This is the case for 1H-31P CP in apatites, where the CP signal is reduced in comparison with the BD signal of the same sample under optimized experimental conditions for both measurements. In this communication, the CP efficiency is presented for model compounds of bone mineral and compared with various samples of human bone. 2. Experimental Stoichiometric HA was synthesized by the wet chemical method (Ca/P ratio of 1.67) [32]. It was pre-dried in air to give as-synthesized sample HAh (h stands for hydrated). HAh was then calcined at 1073 K to give HAc (c stands for calcined). Carbonatoapatite of type B (CHA-B) with ca. 9 wt% of CO23 was synthesized according to Merry et al. [33]. Tricalcium phosphate Ca3(PO4)2 (TCP) was synthesized according to the standard procedure [34]. Brushite CaHPO4 2H2O (BRU) is a natural mineral from Japan. The purity and crystallinity of solid phosphates was confirmed by IR-microscopy (specular reflectance) and powder XRD. The samples of bone apatites (BHA) were gifts from the Glass and Ceramics Institute (Warsaw). The organic part of the bone was removed by chemical (leaching with NaHCO3) and thermal treatment (calcination at 1200 1C). Sample BHA1 was prepared from a piece of bone containing both trabecular and cortical tissue. Sample BHA2 was prepared exclusively from trabecular tissue. The bone samples were prepared at the Medical University of Warsaw. The cortical bone tissue was taken during autopsy from the tibia of a healthy 24-year old male killed in a car accident. The bone was not damaged and showed no pathological changes. The bone was powdered and divided into four samples, B1–B4, which were then treated in various ways (Table 1). The powdered bone
samples were examined with and without MAS. The trabecular bone was extracted from the femoral neck as small cylinders (samples B5 and B6), 6 mm in diameter and 20 mm long, to fit standard 7 mm Varian rotors. The bone cylinders were not capable of smooth spinning, so they were not examined with MAS. Sample B5 was taken during autopsy from a healthy 29-year old male, the victim of a car accident. The femur was not damaged and showed no pathological changes. Sample B6 was taken during surgical replacement of a hip joint with an alloplastic implant. The patient was a 69-year old female with osteopenia (low bone turnover), suffering from degenerative disease of the hip joints. Samples B5 and B6 were lyophylized, sterilized by gradiation (3.5 kGy) and stored at 277 K. The study was approved by the local research ethics committee. The HAh, HAc, CHA-B and BRU standards, and bone samples B1–B3 were the same as in our earlier study [3]. Solid-state 31P NMR spectra were acquired at 81 MHz on a Varian UNITY PLUS-200 spectrometer, using a Doty double-bearing probe and 7 mm zirconia rotors. All measurements were made at 298 K. The MAS spectra were acquired under rotation at 3 kHz. The rotors were spun using dry air. The intensities of the NMR lines from conventional pulse-acquire experiments, hereafter called BD experiments, and from CP experiments carried out from protons to phosphorus-31 (1H-31P CP), were compared. We used the proprietary Varian pulse program XPOLAR, capable of dealing with both BD and CP. The program executes the ordinary single-contact CP pulse sequence, with reversal of spin-temperature in the rotating frame. In each case, the BD and CP spectra were measured with XPOLAR in the arrayed (consecutive) mode within the same two-stage experiment. For CP, a contact time of 1 ms was chosen. BD and CP were both performed at the same high-power level for proton decoupling, with the same number of scans, the same proton p/2 pulses of 6.7 ms and the same recycle delays optimized for BD on a particular sample. Consequently, the same apodization was applied. The Hartmann–Hahn condition was carefully adjusted before each experiment, separately for MAS and static conditions. Signal intensities were measured at peak tops. 3. Results and discussion Apatite gives a single 31P line, with and without CP [1]. The 1H-31P polarization transfer in bone and synthetic apatites proceeds from protons of water and structural
Table 1 Preparation of cortical bone samples Sample
Preservation
Sterilization (radiation dose)
Storage temperature (K)
B1 B2 B3 B4
Lyophilization Freezing at 203 K Freezing at 203 K Lyophilization
— — b-radiation (35 kGy) b-radiation (35 kGy)
277 277 277 277
ARTICLE IN PRESS A. Kaflak et al. / Solid State Nuclear Magnetic Resonance 29 (2006) 345–348
hydroxyl groups [1,35]. The organic matter of bone does not participate in the 1H-31P CP process [36]. Water is mainly located on crystal surfaces and is loosely bound or strongly adsorbed. Therefore, CP is strongly dependent on the specific surface area of these materials and on the hydration of crystal surfaces. It follows that crystal dimensions and habits, and the state of surfaces (concentration and distribution of surface ions and water) are crucial for CP efficiency. The second CP contribution, that from structural hydroxyl groups, is strongly dependent on their concentration. It has been already established that bone apatite is deficient in hydroxyl groups [1,3,35,37,38]. Cho et al. [35] estimated the percentage of structural hydroxyl groups in bone mineral as 2171% of the stoichiometric content. All this assures us that the CP efficiency reflects many subtle factors determining the overall properties of our samples. The CP efficiency has to be adequately measured. Any definition will be arbitrary and we have to make reasonable decisions, considering both the conceptual and operational aspects. Note that the BD peak is from all the 31P nuclei of the material, while the CP peak is from the 31P nuclei located close to protons. Moreover, the BD intensity should be less sensitive to the experimental set up. It looks as if the BD experiment can provide us with some sort of reference intensity. For the sake of this argument, we define the CP efficiency as the percentage of the CP intensity in relation to the BD intensity, both assessed for the same sample on the basis of the signal amplitudes. Thus defined, CP efficiency can be measured for static samples or under MAS. We chose the MAS rate of 3 kHz, because for apatite it provides quite small spinning sidebands (Fig. 1) and it is sufficient to consider only the centreband. We decided to do CP with 1 ms contact time, because it roughly corresponds to the optimum intensity for various apatites (for bone mineral see [36]). The results (Table 2) fall between two outlying cases. TCP has a very small CP efficiency, because it does not contain structural hydrogen. BRU has very high CP
347
Table 2 1 H-31P CP efficiency for bone samples and various phosphates, determined as the intensity ratio in % of the CP and BD signal amplitudes Sample
HAh HAc BHA1 BHA2 CHA-B BRU TCP B1 B2 B3 B4 B5 B6 a
CP/BD (%) MAS at 3 kHz
Static
45 39 37 — 26 163 3 20 20 21 22 — —
37 44 40 28 26 129, 121, 130a 2 23 22 22 22 22 21
Split by dipolar coupling.
efficiency, because it contains proximate 1H and 31P spins located in the POH groups. BRU is interesting as a tractable model for predicting the NMR behaviour of HPO24 in bone apatite [1,39], although this has been questioned [40]. It is obvious that the HA and BHA samples show very different CP behaviour from the bone samples. In this respect, CHA-B is most similar to the bone mineral. There are practically no differences between the bone samples, although they were taken from three donors and were differently treated. In particular, the lyophilized samples B1 and B4 behave similarly to the hydrated samples B2 and B3. This means that lyophilization removes only loosely bound water, which does not take part in CP. At this stage of the study, we cannot make general statements on the bone samples, because we need to examine a larger and more diverse group. However, it is clear that the CP efficiency method is useful for the characterization and standarization of phosphate materials, and for matching their structure and composition. Application to composite, apatite-based prosthetic materials is straightforward.
4. Conclusions The conclusions of this study are summerized as follows:
Fig. 1. 31P BD NMR spectra of human bone tissue recorded with various MAS rates (sample B1).
(1) For 1H-31P CP efficiency, the CHA-B sample most resembles the bone samples B1–B6. This agrees with our former NMR studies [1,3,10,11], indicating that the bone mineral is closest in chemical structure to carbonatoapatite of type B. (2) CP efficiency is suitable for the characterization of phosphate materials.
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Acknowledgment The authors are grateful to the Medical University of Warsaw for financial support. References [1] W. Kolodziejski, Top. Cur. Chem. 246 (2005) 235. [2] R.Z. LeGeros, Prog. Cryst. Growth Charact. 4 (1981) 1. [3] A. Kaflak-Hachulska, A. Samoson, W. Kolodziejski, Calcif. Tissue Int. 73 (2003) 476. [4] W.F. DeJong, Res. Trav. Chim. 45 (1926) 445. [5] K. Sudarsanan, R.A. Young, Acta Crystallogr. B 25 (1969) 1534. [6] R.Z. LeGeros, in: P.W. Brown, B. Constanz (Eds.), Hydroxyapatite and Related Materials, CRC Press, Boca Raton, 1994, pp. 3–28. [7] R.Z. LeGeros, Crystallographic studies of the carbonate substitution in the apatite structure, Ph.D. Thesis, New York University, New York, 1967. [8] R.Z. LeGeros, Monographs in Oral Science, vol. 15. Calcium Phosphates in Oral Biology and Medicine, S. Karger, Basel, 1991. [9] J.D.B. Featherstone, I. Mayer, F.C. Driessens, R.M.H. Verbeeck, H.J.M. Heijligers, Calcif. Tissue Int. 35 (1983) 169. [10] A. Kaflak-Hachulska, D. Chmielewski, A. Go´recki, W. Kolodziejski, in: Materials of the Second Alpine Conference on Solid-State NMR. Chamonix–Mont Blanc, France, 2001. [11] W. Kolodziejski, A. Kaflak-Hachulska, in: Materials of the ISMAR 2001 Conference, Rhodes, Greece, 2001. [12] R. Legros, N. Balmain, G. Bonel, J. Chem. Res. (S) (1986) 8. [13] H. Aoki, Science and Medical Applications of Hydroxyapatite, Takayama Press, Tokyo, 1991. [14] F.C. Driessens, Z. Naturforsch. (C) 35 (1980) 357. [15] A.H. Roufosse, W.P. Aue, J.E. Roberts, M.J. Glimcher, R.G. Grffin, Biochemistry 23 (1984) 6115. [16] W.H. Emerson, E.E. Fisher, Arch. Oral Biol. 7 (1962) 671. [17] J.C. Elliott, in: M.V. Slack, R.W. Fearnhead (Eds.), Tooth Enamel, J. Wright & Sons, Bristol, 1965, pp. 20–22.
[18] R.Z. LeGeros, O.R. Trautz, J.P. LeGeros, E. Klein, Bull. Soc. Chim. Fr. 4 (1968) 1712. [19] R.Z. LeGeros, O.R. Trautz, E. Klein, J.P. LeGeros, Experientia 25 (1969) 5. [20] G. Bonel, Ann. Chim. 7 (1972) 127. [21] J.-C. Labarthe, G. Bonel, G. Montel, Ann. Chim. 8 (1973) 289. [22] R.M. Blitz, E.D. Pellegrino, Clin. Orthop. 129 (1977) 279. [23] M. Santos, P.F. Gonza´lez-Diaz, Inorg. Chem. 16 (1977) 2131. [24] J.D. Featherstone, S. Pearson, R.Z. LeGeros, Caries Res 18 (1984) 63. [25] M. Vignoles, G. Bonel, D.W. Holcomb, R.A. Young, Calcif. Tissue Int. 43 (1988) 33. [26] C. Rey, B. Collins, T. Goehl, I.R. Dickson, M.J. Glimcher, Calcif. Tissue Int. 45 (1989) 157. [27] C. Rey, V. Renugopalakrishnan, M. Shimuzu, B. Collins, M.J. Glimcher, Calcif. Tissue Int. 49 (1991) 259. [28] A. Pines, M.G. Gibby, J.S. Waugh, J. Chem. Phys. 59 (1973) 569. [29] C.A. Fyfe, Solid state NMR for Chemists, CFC Press, Guelph, 1983. [30] E.O. Stejskal, J.D. Memory, High resolution NMR in the solid state. Fundamentals of CP/MAS,, Oxford University Press, Oxford, 1994. [31] W. Kolodziejski, J. Klinowski, Chem. Rev. 102 (2002) 613. [32] A. Slo´sarczyk, E. Stobierska, Z. Paszkiewicz, M. Gawlicki, J. Am. Ceram. Soc. 79 (1996) 2539. [33] J.C. Merry, I.R. Gibson, S.M. Best, W. Bonfield, J. Mater. Sci. Mater. Med. 9 (1998) 779. [34] E. Salahi, J.G. Heinrich, Br. Ceram. Trans. 102 (2003) 79. [35] G. Cho, Y. Wu, J.L. Ackerman, Science 300 (5622) (2003) 1123. [36] A. Kaflak, D. Chmielewski, A. Go´recki, W. Kolodziejski, Solid State NMR 10 (1998) 191. [37] C. Rey, J.L. Miquel, L. Facchini, A.P. Legrand, M.J. Glimcher, Bone 16 (1995) 583. [38] C.-K. Loong, C. Rey, L.T. Kuhn, C. Combes, Y. Wu, S.-H. Chen, M.J. Glimcher, Bone 26 (2000) 599. [39] A. Kaflak-Hachulska, A. Slo´sarczyk, W. Kolodziejski, Solid State NMR 15 (2000) 237. [40] Y. Wu, M.J. Glimcher, C. Rey, J.L. Ackerman, J. Mol. Biol. 244 (1994) 423.