Use of Raman Microscopy and Multivariate Data Analysis to Observe

Jan 26, 2009 - In the current study, an advanced multivariate data analysis approach was implemented. The collected Raman mapping data were subjected ...
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Anal. Chem. 2009, 81, 1442–1449

Use of Raman Microscopy and Multivariate Data Analysis to Observe the Biomimetic Growth of Carbonated Hydroxyapatite on Bioactive Glass Regina K. H. Seah,† Marc Garland,‡ Joachim S. C. Loo,*,† and Effendi Widjaja*,‡ School of Materials Engineering, Nanyang Technological University, Nanyang Avenue, Singapore 639798, and Institute of Chemical and Engineering Sciences, Agency for Science, Technology and Research (ASTAR), 1 Pesek Rd, Jurong Island, Singapore 627833 In the present contribution, the biomimetic growth of carbonated hydroxyapatite (HA) on bioactive glass were investigated by Raman microscopy. Bioactive glass samples were immersed in simulated body fluid (SBF) buffered solution at pH 7.40 up to 17 days at 37 °C. Raman microscopy mapping was performed on the bioglass samples immersed in SBF solution for different periods of time. The collected data was then analyzed using the band-target entropy minimization technique to extract the observable pure component Raman spectral information. In this study, the pure component Raman spectra of the precursor amorphous calcium phosphate, transient octacalcium phosphate, and matured HA were all recovered. In addition, pure component Raman spectra of calcite, silica glass, and some organic impurities were also recovered. The resolved pure component spectra were fit to the normalized measured Raman data to provide the spatial distribution of these species on the sample surfaces. The current results show that Raman microscopy and multivariate data analysis provide a sensitive and accurate tool to characterize the surface morphology, as well as to give more specific information on the chemical species present and the phase transformation of phosphate species during the formation of HA on bioactive glass. In the past decade, there has been an increasing interest in the concept of biological fixation of prostheses, where the interface between implant and tissues develop a type of biological bond.1 Bioactive glasses, ceramics, and composites have been developed as implants and put into clinical use as bone-regenerative material in dental and orthopedic applications. When these bioimplants are immersed in a simulated body fluid (SBF), a layer of bioactive coatings such as carbonated hydroxyapatite (HA) is formed on the surface that has chemical composition similar to the mineral phase of bones.2 The first well-characterized bioactive glass is the melt-derived 45S5 Bioglass with the weight composition of 45% SiO2, 24.5% * To whom correspondence should be addressed. E-mail: joachimloo@ ntu.edu.sg (J.S.C.L.), [email protected] (E.W.). † Nanyang Technological University. ‡ Institute of Chemical and Engineering Sciences. (1) Jones, J. R.; Hench, L. L. Mater. Sci. Technol. 2001, 17, 891–900. (2) Cao, W. P.; Hench, L. L. Ceram. Int. 1996, 22, 493–507.

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Na2O, 24.5% CaO, 4% P2O5 developed by Hench et al.3 This bioactive glass is considered as having the closest mechanical properties to cortical bone, as well as giving a quick biochemical response when it is immersed in physiological fluid.1,3,4 An extensive study on the mechanism of reactions occurring at the surface of this bioactive glass has been also carried out by Clark et al.5 As reported, the formation of carbonated HA required the presence of hydrated silica, that is, Si-OH groups. The presence of the specific functional group Si-OH reacts with the OH- in the SBF to give Si-O-. The negatively charged Si-O- then attracts the positively charged calcium ions present in the body fluid, forming calcium silicate, Si-O-Ca+. The calcium ions attached to the Si-O- then further attract the phosphate ions, (HPO42-), forming an amorphous calcium phosphate, Cax(PO4)y, which is the precursor for the carbonated HA crystallites. Numerous in vitro spectroscopic studies have been performed to characterize and to understand the formation of HA on the surface of bioactive glasses. These included the use of spectroscopic techniques, such as Fourier Transform Infrared (FT-IR) Spectroscopy,6-8 Raman,8-11 X-ray Photoelectron Spectroscopy,12-15 and Nuclear Magnetic Resonance.16-19 In a recent (3) Hench, L. L.; Splinter, R. J.; Allen, W. C.; Greenlee, T. K., Jr. J. Biomed. Mater. Res 1971, 2, 117–141. (4) Hench, L. L. Biomaterials 1998, 19, 1419–1423. (5) Clark, A. E.; Hench, L. L.; Paschall, H. A. J. Biomed. Mater. Res. 1976, 10, 161–174. (6) Jones, J. R.; Sepulveda, P.; Hench, L. L. J. Biomed. Mater. Res., Part B Appl. Biomater. 2001, 58, 720–726. (7) Sepulveda, P.; Jones, J. R.; Hench, L. L. J. Biomed. Mater. Res. 2002, 61, 301–311. (8) Rehman, I.; Karsh, M.; Hench, L. L.; Bonfield, W. J. Biomed. Mater. Res. 2000, 50, 97–100. (9) Notingher, I.; Boccaccini, A. R.; Jones, J.; Maquet, V.; Hench, L. L. Mater. Charact. 2003, 49, 255–260. (10) Gonzalez, P.; Serra, J.; Liste, S.; Chiussi, S.; Leon, B.; Perez-Amor, M. J. Non-Cryst. Solids 2003, 320, 92–99. (11) Rehman, I.; Hench, L. L.; Bonfield, W.; Smith, R. Biomaterials 1994, 15, 865–870. (12) Serra, J.; Gonza´lez, P.; Liste, S.; Serra, C.; Chiussi, S.; Leo´n, B.; Pe´rezAmor, M.; Yla¨nen, H. O.; Hupa, M. J. Non-Cryst. Solids 2003, 332, 20–27. (13) Vallet-Regı´, M.; Pe´rez-Pariente, J.; Izquierdo-Barba, I.; Salinas, A. J. Chem. Mater. 2000, 12, 3770–3775. (14) Takadama, H.; Kim, H. M.; Kokubo, T.; Nakamura, T. J. Am. Ceram. Soc. 2002, 85, 1933–1936. (15) Skipper, L. J.; Sowrey, F. E.; Pickup, D. M.; Fitzgerald, V.; Rashid, R.; Drake, K. O.; Lin, Z. J.; Saravanapavan, P.; Hench, L. L.; Smith, M. E.; Newport, R. J. J. Biomed. Mater. Res. 2004, 70A, 354–360. (16) Hayakawa, S.; Tsuru, K.; Ohtsuki, C.; Osaka, A. J. Am. Ceram. Soc. 1999, 82, 2155–2160. 10.1021/ac802234t CCC: $40.75  2009 American Chemical Society Published on Web 01/26/2009

report by Bonino et al., an in situ Raman microscopy approach was used to observe the reactivity of bioactive glass when it was in direct contact with TRIS (tris(hydroxymethyl)aminomethane) buffered solution.20 The use of Raman microscopy to study the integration of HA-coated metallic implant into bone has also been reported.21-24 However, in these spectroscopic studies, measurements were only performed on single or multiple points of the samples. In the present contribution, more extensive investigations were conducted using a Raman microscope in mapping mode. Raman microscopy mapping was carried out on the bioglass samples, which had been made into disk form and were immersed in SBF solution for different periods of time. For each sample, two mappings were performed. One was on the edge and the other was on the center of the disk. The mapping measurements were performed by scanning in a grid pattern over the surface of the sample via programmed movements of the microscope stage. Therefore, using this approach, spatially resolved spectral information for each sample being investigated was collected. The homogeneity of the HA growth on the surface of bioglass samples could also be examined. In the current study, an advanced multivariate data analysis approach was implemented. The collected Raman mapping data were subjected to a chemometric technique, namely, self-modeling curve resolution (SMCR), to extract the pure component spectra of the observable constituents present in the system. There are many well-known SMCR techniques that can be used, such as SIMPLISMA,25 orthogonal projection approach,26 multivariate curve resolution-alternating least-squares,27 and so forth. However, in the current analysis, we use band-target entropy minimization (BTEM)28-30 that has proven capable of recovering pure component spectra of minor components having weak infrared and/ or Raman signals at better signal-to-noise (S/N) ratios than those mentioned above.31,32 BTEM was initially developed to analyze in situ FT-IR spectra collected from organometallic reactive29,31 and non-reactive systems.30,33 However, in the past few years, (17) Hayakawa, S.; Tsuru, K.; Iida, H.; Ohtsuki, C.; Osaka, A. Phys. Chem. Glasses 1996, 37, 188–192. (18) Eckert, H. Prog. Nucl. Magn. Reson. Spectrosc. 1992, 24, 159–293. (19) Lin, K. S. K.; Tseng, Y. H.; Mou, Y.; Hsu, Y. C.; Yang, C. M.; Chan, J. C. C. Chem. Mater. 2005, 17, 4493–4501. (20) Bonino, F.; Damin, A.; Aina, V.; Miola, M.; Verne, E.; Bretcanu, O.; Bordiga, S.; Zecchina, A.; Morterra, C. J. Raman Spectrosc. 2008, 39, 260–264. (21) Darimont, G. L.; Gilbert, B.; Cloots, R. Mater. Lett. 2003, 58, 71–73. (22) Taddei, P.; Tinti, A.; Reggiani, M.; Monti, P.; Fagnano, C. J. Mol. Struct. 2003, 651-653, 427–431. (23) Frauchiger, V. M.; Schlottig, F.; Gasser, B.; Textor, M. Biomaterials 2004, 25, 593–606. (24) Dopner, S.; Muller, F.; Hildebrandt, P.; Muller, R. T. Biomaterials 2002, 23, 1337–1345. (25) Windig, W.; Guilment, J. Anal. Chem. 1991, 63, 1425–1432. (26) Sanchez, F. C.; Toft, J.; Van den Bogaert, B.; Massart, D. L. Anal. Chem. 1996, 68, 79–85. (27) Tauler, R.; Kowalski, B. R.; Flemming, S. Anal. Chem. 1993, 65, 2040– 2047. (28) Widjaja E. Development of band-target entropy minimization (BTEM) and associated software tools, PhD Thesis, National University of Singapore, Singapore, 2002. (29) Widjaja, E.; Li, C. Z.; Garland, M. Organometallics 2002, 21, 1991–1997. (30) Chew, W.; Widjaja, E.; Garland, M. Organometallics 2002, 21, 1982–1990. (31) Li, C. Z.; Widjaja, E.; Chew, W.; Garland, M. Angew. Chem. 2002, 41, 3784– 3789. (32) Widjaja, E.; Crane, N.; Chen, T. S.; Morris, M. D.; Ignelzi, M. A., Jr.; McCreadie, B. R. Appl. Spectrosc. 2003, 57, 1353–1362. (33) Widjaja, E.; Li, C. Z.; Chew, W.; Garland, M. Anal. Chem. 2003, 75, 4499– 4507.

because of its generality, BTEM has been applied to many other applications, including the analysis of hyperspectral image data collected from biomedical,32 pharmaceutical,34 and commercial samples.35 Since most chemical compounds have their own unique pure component vibration spectra, the pure component spectral estimates obtained from BTEM can be used for component identification including previously unknown chemical components. In the present study, BTEM was applied to recover the underlying pure component spectra from samples collected at different contact times with the SBF solution. Accordingly, information on the phase transformations leading to carbonated HA formation can be obtained, and the time-dependent spatial distribution of HA on the surface can also be investigated. Comparison of the results obtained from point measurements and Raman mapping measurements is also presented. MATERIALS AND METHODS All materials used to prepare and to synthesize bioglass (Ca(NO3)2 · 4H2O, tetraethyl orthosilicate, triethyl phosphate, HCl, and ethanol) and SBF (NaCl, NaHCO3, KCl, K2HPO4 · 3H2O, MgCl2 · 6H2O, HCl, CaCl2, Na2SO4, and (CH2OH)3CNH2) were purchased from Sigma-Aldrich (Singapore). In the present study, bioglass was prepared through a sol-gel processing method to control the ultrastructure, texture, and compositions of the glasses. This method has been shown to be able to produce bioglasses that give regenerative bioactive behavior in vivo.36 The typical synthesis of highly ordered and partially crystalline bioglass involved the use of 3.3 g of Ca(NO3)2 · 4H2O, 5.0 g of tetraethyl orthosilicate, 0.73 g of triethyl phosphate, and 1.0 g of 0.5 M HCl. All these materials were then dissolved in 60 g of ethanol and were stirred at room temperature for 1 day. The resulting sol was introduced into a Petri dish to undergo evaporation induced self-assembly. The dried gel was calcined at the desired temperature of 500 °C for 5 h. The final product after calcination was then ground into powder and pressed into disks of 13 mm diameter using a die press. Each disk contained about 150 mg of product. The protocol for preparing simulated body fluid (SBF) solution was proposed by Kokubo.37 This solution has ion concentrations almost identical to those of human blood plasma. SBF was prepared by dissolving the following reagent grade chemicals one by one into ion-exchanged and distilled water, giving 1 L of final solution. The salts used were NaCl (7.996 g), KCl (0.224 g), NaHCO3 (0.350 g), K2HPO4 · 3H2O (0.228 g), MgCl2 · 6H2O (0.305 g), CaCl2 (0.278 g), and Na2SO4 (0.071). Afterward, the solution was buffered at pH 7.40 with tris-hydroxymethylaminomethane [(CH2OH)3CNH2] and 1 M hydrochloric acid (HCl) at 36.5 °C. The bioglass disk samples were placed in cell culture wells and were immersed into the prepared SBF solution for a predetermined time duration ranging from 0 to 17 days at a temperature of about 37 °C, which is similar to human body temperature. The SBF solution was replaced every 2-3 days during the course of the experiment, and the samples were placed (34) Widjaja, E.; Seah, R. K. H. J. Pharm. Biomed. Anal. 2008, 46, 274–281. (35) Widjaja, E.; Garland, M. Anal. Chem. 2008, 80, 729–733. (36) Yan, X. X.; Deng, H. X.; Huang, X. H.; Lu, G. Q.; Qiao, S. Z.; Zhao, D. Y.; Yu, C. Z. J. Non-Cryst. Solids 2005, 351, 3209–3217. (37) Kokubo, T. J. Non-Cryst. Solids 1990, 120, 138–151.

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Figure 1. SEM images of the surfaces of the bioglasses before (a,b) and after immersion in SBF for 17 days (c,d).

again in clean cell culture wells after each SBF replacement. After each immersion period (0, 3, 5, 7, 10, 12, 14, and 17 days), the samples were taken out of the incubation solution, rinsed with distilled water, dried at room temperature for 1 week, and stored in desiccator before surface analysis. The first surface analysis was performed to observe the microstructure of the bioglass disk before and after it was immersed for 17 days in SBF solution. This analysis was carried out by scanning electron microscopy (SEM, Jeol, JSM-6700F), which was operated at 2.0 to 5.0 kV depending on the sample being analyzed. The second analysis was carried out by Raman microscopy (InVia Reflex, Renishaw) equipped with near-infrared enhanced deep-depleted thermoelectrically Peltier cooled CCD array detector (576 × 384 pixels) and a high grade Leica microscope. The Raman scattering was excited with a 785 nm near-infrared diode laser, and a 20 × objective lens was used to collect the backscattered light. Scans were performed in an extended spectral window from 300 to 1800 cm-1. The acquisition time for each Raman spectrum was around 30 s. Two types of Raman measurements were performed for each bioglass disk, (i) Raman point measurements at three different points, and (2) Raman mapping measurements at two different locations. Raman mapping was performed in an area of 200 µm × 200 µm with a step size of 20 µm in both the x and y directions. The first map was performed at the edge of disk, and the second map was performed at the center of disk. For each Raman map, a total of 121 Raman spectra were collected. Spectral preprocessing that includes spectral smoothing and spike removal due to cosmic rays were carried out first before the Raman data was further analyzed using the BTEM algorithm. For the first type of Raman measurements (pointwise collection), the collected spectra at three different points was not subjected to BTEM analysis, but instead, they were only averaged. 1444

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COMPUTATIONAL ASPECTS Unlike most SMCR techniques, BTEM was specially developed to reconstruct one pure component spectrum at a time from a set of mixture spectra without using any spectral libraries or any a priori knowledge. The first step in the analysis is the use of singular value decomposition38 to extract the singular vectors that describe the variance of the original mixture data set. With the present data set, to extract an estimate of a pure Raman scattering coefficient, a selected band in the first few non-noise right singular vectors is targeted. The BTEM algorithm then retains this feature and, at the same time, returns an entire full-length spectrum containing this band and all correlated bands, which have minimum entropy. This routine is repeated for all important observable physical features in the selected right singular vectors. Criterion for selecting the band targets is simple. A user can first set a tolerance for accepting/rejecting a feature. Then, all the observed spectral features in the first few right singular vectors with intensity above this threshold are selected as band-targets for BTEM. After applying BTEM to all of these band-targets, a superset of reconstructed pure component Raman scattering coefficients is obtained, and this set can be reduced to eliminate redundancies. This results in an enumeration of all observable pure component spectra. As mentioned, the targeted bands are retained during the reconstruction. As part of the process, the resulting pure spectral patterns are returned in a normalized form. When all normalized observable pure component spectra have been reconstructed, relative contributions of these signals can be calculated by projecting them onto the original data set. For a detailed description of the BTEM algorithm, readers are referred to refs 28-30. (38) Golub, G. H.; Van Loan, C. F. Matrix Computations; The John Hopkins University Press: Baltimore, 1996.

Figure 2. Raman spectra of bioactive glass as received (a), after 3 days (b), after 5 days (c), after 7 days (d), after 10 days (e), after 12 days (f), after 14 days (g), and after 17 days (h) of immersion in SBF buffer solution.

RESULTS Scanning Electron Microscope (SEM). Panels a and b of Figure 1 show the generally rough and uneven surface morphology of a bioglass disk before it was immersed in SBF solution. At higher magnification, it can be seen that many round particles fused together to give a continuous layer. In panels c and d of Figure 1, the growth of HA particles in the form of agglomerates can be clearly observed after 17 days of immersion in SBF solution. Raman Point-Wise Measurements. A series of Raman spectra of bioactive glass as synthesized and after immersion in SBF buffer solution as a function of immersion time are shown in Figure 2. As can be seen in Figure 2a, the Raman spectrum of bioactive glass as synthesized (before reaction) has well-defined vibrational bands at 713, 742, 961, and 1086 cm-1 and a broad and unresolved band centered at 1064 cm-1. Previous studies suggested that the peak at 961 cm-1 may be ascribed to the presence of phosphate, one of the constituents used to form bioactive glass,39 or it may be associated with the bending mode of Si-O-Si.40 The band assignment for the Raman peak at 1086 cm-1 was also inconclusive. This peak was suggested to belong to both the asymmetric stretching vibration of PO43groups and a νCO mode of carbonates.39 In panels b-h of Figure 2, it can also be seen that during reaction in SBF buffer solution, the Raman peak intensity at 961 cm-1 progressively increases and the broadband at 1051-1068 cm-1 gradually decreases until it can not be detected after 14 days immersion. The Raman peak at 713 cm-1 was still found after 17 days immersion. However, the Raman peak at 742 cm-1 disappeared after 5 days immersion. The increase in the ratio of the peak intensities of 961 cm-1 (symmetric stretching mode of PO43-) to 1087 cm-1 (calcite) is an indicator for the (39) Cerruti, M.; Bianchi, C. L.; Bonino, F.; Damin, A.; Perardi, A.; Morterra, C. J. Phys. Chem. B 2005, 109, 14496–14505. (40) Balamurugan, A.; Sockalingum, G.; Michel, J.; Faure, J.; Banchet, V.; Wortham, L.; Bouthors, S.; Laurent-Maquin, D.; Balossier, G. Mater. Lett. 2006, 60, 3752–3757.

Figure 3. Pre-processed Raman mapping spectra measured at the edge of a sample as synthesized (before immersed into SBF solution). Note that about three spectra have dramatically different signals in the range about 1200-1600 cm-1 (marked with numbers 1-3).

progressive growth of carbonated HA on the surface of the bioactive glass. Although Raman point-wise measurements may be useful to monitor changes on the bioglass surface, it has some limitations with respect to identifying the chemical species involved and does not allow an accurate determination of their spatial distributions. Further complications arise when some species in the system have highly overlapping Raman signatures. Raman Mapping Measurements. As mentioned above, Raman mapping measurements were performed on two different locations (center and edge of the disk) for each sample, before and after immersing into SBF solution. Raman Mapping Data Analysis of a Sample As Synthesized before Immersed into SBF Solution. Edge Location Before Immersion. Figure 3 shows the despiked and smoothed Raman data taken from mapping measurements (11 pixel by 11 pixel) on the edge of a sample as synthesized (before immersed into SBF solution). These 121 Raman spectra show that many similar band maxima are seen in most spectra, but that large variation in backgrounds can occur (baseline changes). Also, about three spectra show dramatically different features in the region about 1200-1600 cm-1. The pre-processed Raman mapping data were then subjected to BTEM analysis, and four pure spectral patterns were reconstructed. The relative contributions from each constituent were then obtained by projecting these spectral estimates onto the preprocessed normalized mixture Raman spectra. The mixture spectra were normalized to the band at 1086 cm-1 (L∞ norm) to overcome the problem of pixel-to-pixel Raman intensity variation during mapping measurements. The resulting relative contributions provide a map or spatial distribution for each component. The four pure spectral patterns obtained via BTEM analysis and their spatial distributions are shown in Figure 4. Note that the axes for the spatial distributions are in pixel number, which can be directly converted to distance by multiplying by 20 µm for each pixel. It is immediately apparent that the first 2 patterns (a and b) are widely distributed across the sample, and that the last 2 patterns (c and d) are only detectable in 3 or 4 pixels. The reconstructed pure spectral estimates via BTEM analysis were then compared to known spectral libraries associated with Analytical Chemistry, Vol. 81, No. 4, February 15, 2009

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Figure 5. BTEM pure spectral pattern estimates and their spatial distribution obtained from the center of a sample as synthesized (before immersed into SBF solution): (a) calcite, (b) silica glass.

Figure 4. BTEM pure spectral pattern estimates and their score images that represent their spatial distribution obtained from the edge of a sample as synthesized (before immersed into SBF solution): (a) calcite, (b) silica glass, (c) organic impurity 1, (d) organic impurity 2.

bioactive glass. It was found that the first spectral estimate corresponds to the Raman spectrum of calcite. The strong and symmetric peak centered at 1086 cm-1 is due to the stretching vibration (ν1 mode) of CO3, and the weaker peak at 713 cm-1 is due to the ν4 mode of symmetric CO3 deformation.41 The second spectral estimate has a strong peak at 1066 cm-1 and two visibly weaker peaks at 741 and 960 cm-1. This pure constituent spectral estimate may be associated with dry silica glass before any reaction with SBF solution. The strong and weak peaks observed may correspond to the Si-O-Si stretching and bending modes of the crystalline phase of the sample, respectively. In addition, the peak at 960 cm-1 (δSi-O-Si) can be used to determine the amount of silica present in the sample.40 The reconstructed peaks at 741 and 1066 cm-1 are higher in wavenumber compared to those of a previous study (720 and 1060 cm-1).40 As discussed before, the Raman bands of bioactive silica glass are very sensitive to composition changes and the addition of alkali and alkali earth oxides to the silica network. As such, Raman peak shifting and intensity variations are commonly observed because of the increase or decrease of the local symmetry of the silica network.40 The third and fourth spectral estimates obtained via BTEM analysis show good signal-to-noise ratios, and therefore, should not be artifacts. The resolved bands are typically broad and have rather clear Lorenzian shapes. They also occur in the typical fingerprint area of organic compounds. Also, as mentioned above, they only occur in about 3-4 pixels. Taken together, these results suggest that these two spectral estimates are due to some sort of organic impurity in the samples, possibly particulate. These impurities might be derived from handling or even storage of the samples. Center Location Before Immersion. Similar analysis was carried out, and again BTEM analysis reconstructed four pure (41) Degen, I. A.; Newman, G. A. Spectrochim. Acta 1993, 49A, 859–887.

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Figure 6. New BTEM pure spectral pattern estimates from samples immersed in SBF solution for 3, 5, and 7 days: (a) another form of silica glass, (b) amorphous calcium phosphate-like phase, and (c) octacalcium phosphate-like phase.

spectral patterns. Figure 5 shows the pure component Raman spectra of calcite and silica glass (the major contributions to the measurements) with their corresponding spatial distributions. Comparison of Figures 4 and 5 shows that the reconstructed pure component spectra from both the edge and center of the disk are similar, and the two main identified components (associated with the bioglass structure) are well distributed on the areas being mapped. Raman Mapping Data Analysis for Samples after Immersed in SBF Solution for 3, 5, and 7 days. BTEM analysis of the Raman mapping data obtained from samples immersed in SBF solution for 3, 5, and 7 days provided similar pure component spectra as shown in Figures 4 and 5, that is, calcite, dry silica glass, and some organic impurities. In addition, BTEM analysis also revealed some new spectral pattern estimates, which are not found in sample before reaction. These three new spectral pattern estimates are shown in Figure 6. The first spectral estimate was reconstructed from all three samples (3, 5, and 7 days), and it is assigned to another form of silica glass. Three well-defined bands at 741, 962, and 1052 cm-1

were recovered. The first two peaks at 741 and 962 cm-1 have similar peak positions to the Raman spectrum of dry silica glass before reaction, and the third peak is shifted from 1066 to 1052 cm-1. Since the Raman spectrum of silica glass is very sensitive to Si-O-Si environment, this shift could be possibly due to the effect of silica compositional changes during reaction with SBF solutions. This change has also led to changes in the relative Raman intensities of the bands. The second spectral estimate was only recovered from samples immersed in SBF solution for 3 and 5 days. It has well defined and broader peaks at 952 and 1055 cm-1, which are possibly associated with ν1PO4 and ν3PO4 of amorphous calcium phosphate (ACP)-like mineral, respectively. It has been previously determined that ACP may be a precursor phase in bone formation.42 In a recent study, Bonino et al.20 has also suggested that a phase transition of the surface phosphate component will occur when bioactive glass reacts with TRISbuffered solution. The phosphate component starts as an amorphous phase and gradually changes to a crystalline phase. The third spectral estimate was only recovered from a sample after immersion in SBF solution for 7 days. It has a sharper peak at 955 cm-1 compared to the second estimate and a small peak at 1015 cm-1. This spectral estimate can be assigned to octacalcium phosphate (OCP), which is the transient intermediate species during bone formation. Crane et al. observed this transient species (OCP or OCP-like phase) in living tissue during intramembranous mineralization.43 Kazanci et al. have also observed this species during in vitro conversion of ACP to HA.44 The two resolved peaks, 955 and 1015 cm-1, can be attributed to ν1PO4 and P-O stretching of monohydrogen phosphate, respectively. Raman Mapping Data Analysis for Samples after Immersed in SBF Solution for 10, 12, 14, and 17 days. Longer contact times between the bioglass and the SBF were also conducted. BTEM analysis of the Raman mapping data obtained from all samples immersed in SBF solution for 10, 12, 14, and 17 days provided spectral estimates of calcite, carbonated HA, and once again some organic impurities. In contrast, none of the samples immersed in SBF solution for 10, 12, 14, and 17 days showed the presence of ACP and dry silica glass. Only the sample associated with 10 days immersion showed OCP. In addition BTEM analysis of the Raman mapping data indicated the presence of another form of silica glass (i.e., that previously shown in Figure 6a) only for the sample immersed for 10 days, and not those samples immersed for 12, 14, and 17 days. These results indicate that the phase transformations from OCP to HA are essentially completed after 12 days of immersion. Consequently, after 12 days immersion, only calcite and carbonated HA were the observables on the surface of the bioactive glass. Figure 7 shows the BTEM pure component spectral estimates of carbonated HA recovered from samples with different immersion time from 10 to 17 days. As seen in this Figure, all spectral estimates have similar features with a sharp and strong vibration at 961-962 cm-1, which can be attributed to ν1PO4. Other (42) Termine, J. D.; Posner, A. S. Calc. Tissue Int. 1967, 1, 8–23. (43) Crane, N. J.; Popescu, V.; Morris, M. D.; Steenhuis, P.; Ignelzi, M., Jr. Bone 2006, 39, 434–442. (44) Kazanci, M.; Fratzl, P.; Klaushofer, K.; Paschalis, E. P. Calc. Tissue Int. 2006, 79, 354–359.

Figure 7. BTEM pure spectral estimates of carbonated HA obtained from samples with various immersion time: (a) 10 days, (b) 12 days, (c) 14 days, and (d) 17 days.

distinct peaks are also observed in these estimates. These peaks include 430-450, 588-592, 1050-1052, and 1074-1085 cm-1, which can be associated with ν2PO4, ν4PO4, ν3PO4, and ν1CO3, respectively. Whereas the positions of the ν1PO4 and ν3PO4 peaks are quite stable, the ν2PO4 and ν1CO3 peaks are shifted to lower wavenumbers, and the ν4PO4 peak is shifted to higher wavenumbers as a function of immersion time. These vibrational shifts have been observed by Kazanci et al.44 during in vitro conversion of ACP to HA and were attributed to the maturation of mineral crystallites of carbonated HA. From Figure 7, it can also be observed that the signal-to-noise ratio of the spectral estimates improved considerably as a function of immersion time, particularly from 10 to 14 days. Since the signal-to-noise ratio of pure component spectral estimates correlate with concentration or amount of a species present in a system, it can be used to indicate if a spectrum corresponds to a major or a minor species. The present analyses show that carbonated HA was only a minor component after 10 days immersion; however, its concentration increased as a function of immersion time and stabilized after 14 days. The signal-to-noise ratio of the carbonated HA spectral estimates are comparable for the 14 and 17 day samples. Progressive Changes in the Spatial Distributions of Calcite and Carbonated HA. The Raman score images were generated by projecting the BTEM pure component spectral estimates back onto the normalized and baseline-corrected Raman mapping data. The distribution of the calcite and carbonated HA components over the same areas are shown in Figures 8 to 9. Areas on both the center of the disk and the edge of the disk were used, and the corresponding sizes of these images were 200 × 200 µm2. The distribution of calcite appears in Figure 8. Calcite is initially homogenously distributed, and most domains have high concentration of calcite after 3 and 7 days exposure to SBF. However, the distribution of calcite becomes more random at longer exposure time to the SBF solutions. This is because with Analytical Chemistry, Vol. 81, No. 4, February 15, 2009

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Figure 8. Raman score images showing the distribution of calcite at (a) the edge and (b) the center of the bioactive glass disk for samples exposed to SBF for (i) 3 days, (ii) 7 days, (iii) 14 days, and (iv) 17 days, respectively. The axes of score images are in pixels and can be directly correlated to distance by multiplying each pixel with 20 µm.

Figure 9. Raman score images showing the distribution of HA at (a) the edge and (b) at the center of bioactive glass surface for samples exposed to SBF for (a) 10 days, (b) 12 days, (c) 14 days, and (d) 17 days, respectively. The axes of score images are in pixels and can be directly correlated to distance by multiplying each pixel with 20 µm.

prolonged exposure to SBF, the calcium ions can be leached out into the SBF solution. The leaching of Ca2+ contributes to the formation of calcium phosphate in the liquid phase. After 14 and 17 days exposure, the concentration of calcite is clearly reduced, on both the edge and disk center, and the distributions are very inhomogeneous. For the edge samples after 17 days (Figure 8a-iv), some pixels still show about 80% of the original intensity of the calcite signal, while other pixels show only about 20% of the original signal intensity. For the disk center samples after 17 days (Figure 8b-iv), numerous pixels still show about 80-100% of the original intensity, while other pixels show a minimum of about 40% of the original signal intensity. Taken together, the results of panels a and b of Figure 8 suggest that the reactivity is not identical for surfaces on the edge and disk center. The spatial distribution of carbonated HA on the bioactive glass surface as a function of immersion time appears in Figure 9. Since the samples before 10 days (3, 5, and 7 days samples) 1448

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did not show any carbonated HA, Figure 9 only show the Raman score images of HA for 10, 12, 14, and 17 days samples. As can be seen from both Figure 9a-i and Figure 9b-i, there is a small signal for carbonated HA for virtually all pixels after 10 days exposure. After 12 days exposure, the distribution of carbonated HA is very heterogeneous. Some pixels still have only about 10-20% of the maximum observed signal, while some pixels are already exhibiting about 100% signal intensity. Again, comparison of the edge data Figure 9a-ii and the disk center data Figure 9b-ii indicates that the edge surface is more reactive. However, as immersion period in SBF increases to 14 and 17 days, the spatial distribution of HA on both the edge and the disk center has become nearly uniform, and maximum signal intensity (red) is observed for most pixels. The presence of a few light red regions appears to be due, at least in part, to the presence of some organic impurities. Indeed, the spectra from some of these pixels exhibited a pattern similar to those observed in Figure 4c,d.

DISCUSSION Comparison of Mapping and Classic Point-Wise Analysis. The present results have shown that a Raman mapping measurements approach certainly improves the spectral and chemical information available, in comparison to Raman point-wise measurements. Although the time needed for mapping measurements and subsequent analysis is much higher, more accurate species identification and component spatial distribution information can be obtained. Combining these measurements with a multivariate data analysis technique, such as BTEM, has also allowed the reconstruction of the underlying pure component spectra of species involved in the HA formation. This greatly facilitates the subsequent positive identification of the species present. Indeed, pure component spectra of calcite, dry silica glass, another form of silica, ACP, OCP, carbonated HA, and some organic impurities were all identified in this study. Having identified the constituents present, the temporal evolution of surface phosphate species during HA formation could be determined. From 3 and 5 day samples, the pure component spectrum of ACP was resolved with Raman characteristic peak at 952 cm-1, whereas from the 7 and 10 day samples, the pure component spectrum of OCP was resolved with Raman characteristic peak at 955 cm-1. Also from 10 days onward, the pure component spectrum of matured carbonated HA with Raman characteristic peak at 961-962 cm-1 was recovered. Accordingly, it appears that the formation of mature carbonated HA starts from ACP followed by OCP. Such details would certainly be difficult to achieve if only classical point-wise measurements alone, as shown in Figure 2, were used. High spectral overlapping among observable species in the present system makes classical pointwise measurements more difficult to interpret. However, with the current analysis, high spectral overlap is no longer an issue. As an example, small peak shifting due to mineral crystallite maturation as shown in Figure 7 can also be detected and investigated. The phosphate phase transformation observed in the current study

is in agreement with results shown by Crane et al.43 and Kazanci et al.44 However, it is also worthwhile to note that this transformation sequence (ACP to OCP and OCP to matured HA) has not been universally accepted. Competing interpretation based on NMR studies also exists, which conclude that there is no intermediate phase during bone apatite formation.45,46 In the current study, using multivariate analysis, the spatial distribution and homogeneity of certain components can also be studied. Changes in the spatial distribution of carbonated HA formation on the bioactive glass surface as a function of time may be important to understand in detail when it is in contact with physiological fluid. CONCLUSIONS The present contribution addressed the combined use of Raman microscopy mapping coupled with the novel multivariate data analysis algorithm, BTEM, for elucidating the phase transformations of surface phosphate species when bioactive glass reacts with SBF solution. The transformation starts from amorphous calcium phosphate followed by octacalcium phosphate, and then finally full transformation to carbonated HA. With the reconstruction of pure component spectra of all observable species via BTEM, detailed information is more directly obtained. The present spectroscopic and multivariate approach is rather general and could possibly be extended to investigate the reaction between bioactive glass and more complex physiological solutions containing amino acids and proteins.

Received for review October 22, 2008. Accepted January 8, 2009. AC802234T (45) Jager, C.; Welzel, T.; Meyer-Zaika, W.; Epple, M. Magn. Reson. Chem. 2006, 44, 573–580. (46) Grynpas, M. D.; Omelon, S. Bone 2007, 41, 162–164.

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