© Copyright 2001 American Chemical Society
JULY 24, 2001 VOLUME 17, NUMBER 15
Letters An Original Method to Assess Short-Term Physicochemical Reactions at the Periphery of Bioactive Glass Particles in Biological Fluids E. Jallot,* H. Benhayoune, L. Kilian, Y. Josset, and G. Balossier EA 2068 Biomate´ riaux: Laboratoire de Microscopie Electronique, 21 rue Cle´ ment Ader, 51685 REIMS BP 138 Cedex 02, France Received November 29, 2000. In Final Form: March 13, 2001 In this study, we analyze short-term physicochemical reactions at the interface between bioactive glass particles and biological fluids. The chemical analysis is performed under a micrometer scale by scanning transmission electron microscopy associated to energy-dispersive X-ray spectroscopy. However, microanalysis of diffusible ions such as sodium, potassium, or oxygen requires cryomethods for the specimen preparation and for its characterization. These techniques permit retention of all elements at their in vitro location. After 1 day of immersion in biological fluids, results demonstrate the formation of three surface layers (thickness of each layer ≈400 nm) at the bioactive glass periphery. The presence of a Si-Al rich layer permits the diffusion and the formation of a Ca-P-Mg rich layer. The third layer is composed of Na-O and can be considered as an exchanged layer between Na+ ions and H+ or H3O+ from the solution.
Introduction Bioceramics for hard tissue replacement can be divided into three categories: (a) bioinert ceramics, (b) surfacebioactive ceramics, and (c) resorbable bioactive ceramics. Bioinert ceramics are represented by alumina; surfacebioactive ceramics, by sintered hydroxyapatite, bioactive glasses, and glass ceramics; and resorbable bioactive ceramics, by low-crystalline hydroxyapatite, R or β-tricalcium phosphate, tetracalcium phosphate, and octocalcium phosphate.1-3 Since their discovery by Hench,4 new types of bioactive glasses have been developed and used in prosthetic * Corresponding author. Tel: 33 (0)3 26 05 75 64. Fax: 33 (0)3 26 05 19 00. E-mail:
[email protected]. (1) Oonishi, H.; Hench, L. L.; Wilson, J.; Sugihara, F.; Tsuji, E.; Kushitani, S.; Iwaki, H. J. Biomed. Mater. Res. 1999, 44, 31. (2) Jallot, E.; Benhayoune, H.; Weber, G.; Balossier, G.; Bonhomme, P. J. Phys. D: Appl. Phys. 2000, 33 (4), 321. (3) LeGeros, R. Z.; Daculsi, G. CRC Handbook of bioactive ceramics, Vol. II: Calcium Phosphate Ceramics; Yamamuro, N., Hench, L. L., Wilson, J., Eds.; CRC Press: Boca Raton, FL, 1990; p 17. (4) Hench, L. L.; Splinter, R. J.; Allen, W. C.; Greenlee, T. K. J. Biomed. Mater. Res. Symp. 1971, 2, 117.
applications and in repair of bone defects because of their well-documented biocompatibility as well as their osteoconductive and bioactive fixation properties.5 A bioactive fixation is defined as “an interfacial bonding of an implant to tissue by means of formation of a biologically active hydroxyapatite layer on the implant surface”.6 When exposed to body fluids, bioactive glasses undergo corrosion with a leaching of alkali ions resulting in the formation of a silica gel and a calcium phosphate layer on their surfaces. Successively, the Ca-P layer will recrystallize into hydroxycarbonate apatite.7 Bone-bonding properties of bioactive glasses are based on the formation of this layer.8 These bioactive materials can be used as coatings on metallic prostheses or as granules.9 But, the composition (5) Pajamaki, K. J. J.; Lindholm, T. S.; Andersson, O. H. J. Mater. Sci.: Mater. Med. 1995, 6, 14. (6) Hench, L. L. Biomaterials 1998, 19, 1419. (7) Oliva, A.; Salerno, A.; Locardi, B.; Riccio, V.; Della Ragione, F.; Iardino, P.; Zappia, V. Biomaterials 1998, 19, 1019. (8) Jallot, E.; Benhayoune, H.; Kilian, L.; Irigaray, J. L.; Oudadesse, H.; Balossier, G.; Bonhomme, P. Surf. Interface Anal. 2000, 29, 314.
10.1021/la001669m CCC: $20.00 © 2001 American Chemical Society Published on Web 06/22/2001
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has to be optimized to give a suitable compromise between bioactivity and solubility.10 For example, addition of Al2O3 may be used to control certain physical properties. By an increase of the silica content and by addition of alumina, the solubility of the bioactive glass could be minimized.9,11 Critical concentrations of elements such as magnesium and aluminum can play an important role in the regulation of physicochemical processes. Greenspan12 showed that 3% Al2O3 added to a bioactive glass inhibits bone bonding. In our case, 2% of Al2O3 was added and the bioactive glass is in the SiO2-Na2O-CaO-P2O5-K2O-Al2O3-MgO system. The aim of the present study is to analyze short-term physicochemical reactions at the interface between a bioactive glass and biological fluids. Many studies demonstrated a rapid alkali release and supposed an exchange with H+ or H3O+ for the formation of Si-OH bonds in the Si gel before the growth of the Ca-P layer.5,6 However, the formation of these layers is not explained in detail because of the complexity of short-term events that happen at the interface. Knowledge of the elemental distribution at the bioactive glass periphery is important to understand the physicalchemical mechanisms involved in forming the Si gel and the Ca-P layer. Structural and chemical evaluation of the bioactive glass particle periphery requires analysis under a micrometer scale. This study was performed by scanning transmission electron microscopy (STEM) associated to energy-dispersive X-ray spectroscopy (EDXS). On the other hand, microanalysis of elements such as oxygen, sodium, and potassium is a major problem.13,14 These elements are not very firmly bound in the glass which undergoes dissolution and becomes gel-like, or they can be solubilized during specimen preparation. Thin specimens for electron microscopy are generally prepared by a process involving fixation, postfixation, dehydration, embedding in resin, and sectioning. This procedure leads to marked changes in the elemental composition of the specimen. During usual chemical fixation, most of the diffusible ions are rapidly lost (within minutes) from the samples.14,15 To preserve the chemical identity of specimens, we adapted cryomethods for the preparation of bioactive glass particles immersed in a biological fluid. These methods permit retention of all elements of interest at their in vitro location and allow identification at the level of analytical resolution required. Materials and Methods Bioactive Glass Particle Characteristics. The bioactive glass composition is 50% SiO2, 20% Na2O, 16% CaO, 6% P2O5, 5% K2O, 2% Al2O3, and 1% MgO (% weight). The bioactive glass was obtained by melting the components at 1350 °C. Then, the glass was cast, crushed, and transformed into powder of grain size under 40 µm in diameter. Sample Treatment. The glass powder (2 mg) was immersed at 37 °C for 1 day in 1 mL of a standard Dulbecco’s Modified Eagle Medium (DMEM).16 DMEM contained the following (9) Kangasniemi, K.; Yli-Urpo, A. CRC Handbook of Bioactive Ceramics, Vol. 1: Bioactive Glasses and Glass-Ceramics; Yamamuro, N., Hench, L. L., Wilson, J., Eds.; CRC Press: Boston, 1990; p 97. (10) Andersson, O. H.; Liu, G.; Karlsson, K. H.; Niemi, L.; Miettinen, J.; Juhanoja, J. J. Mater. Sci.: Mater. Med. 1990, 1, 219. (11) Jallot, E.; Benhayoune, H.; Kilian, L.; Irigaray, J. L.; Barbotteau, Y.; Balossier, G.; Bonhomme, P. J. Colloid Interface Sci. 2001, 233, 83. (12) Greenspan, D. C.; Hench, L. L. J. Biomed. Mater. Res. 1976, 10, 503. (13) Zierold, K. J. Microsc. 1991, 161, 357. (14) Zierold, K.; Hentschel, H.; Wehner, F.; Wessing, A. Scanning Microsc., Suppl. 1994, 8, 117. (15) Roomans, G. M. J. Electron Microsc. Tech. 1988, 9, 3. (16) Xynos, I. D.; Edgar, A. J.; Buttery, L. D. K.; Hench, L. L.; Polak, J. M. Biochem. Biophys. Res. Commun. 2000, 276, 461.
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Figure 1. Al, Ca, P, and Na concentration profiles across the periphery of three bioactive glass particles in the original state (each point represents the mean value ( standard deviation). ingredients (mg/L): 6400 NaCl, 400 KCl, 200 CaCl2, 200 MgSO4‚ 7H2O, 124 NaH2PO4, and 3700 NaHCO3. Specimen Preparation and X-ray Microanalysis. After treatment, the glass powder was lying on the bottom of the box. The medium was carefully removed with a pipet. The bioactive glass particles were deposited on copper specimen holders (0.5 mm in diameter). Then, the particles were cryofixed by plunging into liquid ethane cooled by liquid nitrogen (-196 °C) with a velocity of 2 m s-1. The frozen specimens were stored in liquid nitrogen before cryosectioning. Cryosections were performed with a Leica Ultracut E ultramicrotome equipped with a cryosectioning attachment using a diamond knife. Sections were deposited onto a carbon coated collodion copper grid. The grid bearing cryosections was placed into a precooled GATAN cryospecimen holder and transferred into the microscope chamber. Sections were freeze-dried by warming up them slowly from -196 to -70 °C. The cryosections were studied at -170 °C with a scanning transmission electron microscope (Philips CM30) operating at a voltage of 100 kV. Elemental profiles from the center to the periphery of the particles were performed using an energydispersive X-ray spectrometer. The concentration profiles were made across three different particles. The elemental composition of dry specimens was determined by using the normalized Hall continuum method.17,18 In Hall’s method, the characteristic peaks to the continuum ratios of X-ray spectra are used for quantification of elements. The calibration procedure was performed with standards. Concentrations are expressed in mmol kg-1 of dry weight. A control measurement of the particles in the original state was performed.
Results Compositional variations near the interface were studied by electron probe X-ray microanalysis on the cryosections. Figures 1-3 show the distribution of Al, Ca, P, Na, Si, O, K, and Mg across the periphery of three bioactive glass particles in the original state. The distribution of these elements is homogeneous across the periphery of the bioactive glass particles. The STEM image showed a part of a bioactive glass particle after 1 day of immersion in standard culture medium. It revealed the formation of an “electron dense” layer (Figure 4). The thickness of the ion-exchange interface was several microns. Elemental profiles revealed four zones at the bioactive glass periphery (Figures 5-7): (17) Hall, T. A. Physical techniques in biological research, Vol. I Part A, 2nd ed.; Oster, G., Ed.; Academic Press: New York, 1971; Chapter 3. (18) Laquerriere, P.; Banchet, V.; Michel, J.; Zierold, K.; Balossier, G.; Bonhomme, P. Microsc. Res. Technol. 2001, 52, 231.
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Figure 2. Si and O concentration profiles across the periphery of three bioactive glass particles in the original state (each point represents the mean value ( standard deviation).
Figure 4. STEM micrograph of a freeze-dried cryosection of a bioactive glass particle after 1 day of immersion in standard culture medium (the dashed line is the scanning line; X is the start point of the scanning).
Figure 3. Mg and K concentration profiles across the periphery of three bioactive glass particles in the original state (each point represents the mean value ( standard deviation).
(a) The bioactive glass particles are in dissolution and are composed of Si, O, Ca, P, Na, K, Mg, and Al. (b) A layer of about 400 nm in thickness is enriched in Si and Al. In this layer, Ca, P, Na, and Mg concentrations were strongly decreased. On the other hand, an increase of oxygen was observed. (c) A second layer (≈400 nm in thickness) is enriched in Ca, P, and Mg. The Ca/P ratio was around unity. In this layer, an increase of Na was observed. Both Si and Al concentrations strongly decreased. Oxygen concentration slightly decreased. (d) A third layer is composed of sodium and oxygen without Al or Si. The evolution of sodium and oxygen concentrations appeared to be related. The Na decrease is related to the O increase. The concentrations of Ca, P, Mg, and K decreased. The distribution of Ca, P, and Mg concentrations was consistent across the interface. Discussion The study of ion distribution and ion release in the biological state is important to understand steps preceding the apatite layer formation at the bioactive glass periphery. This type of study required the development of cryotechniques for sample preparation. Conventional fixation and sectioning methods are therefore not the best choice to analyze diffusible ions. In the case of bioactive glass, only firmly bonded elements can be studied with these methods.
Figure 5. Al, Ca, P, and Na concentration profiles across the periphery of three bioactive glass particles after 1 day of immersion in standard culture medium (each point represents the mean value ( standard deviation).
Quantitative profiles show that the evolution of the bioactive glass particle periphery is characteristic for more than one particle. First, results demonstrate that bioactive glass particles immersed for 1 day in a biological fluid undergo dissolution. This phenomenon leads to physicochemical reactions and to the formation of four zones of different natures. As the glass matrix dissolves, various elements dispersed in the bioactive glass are free either to go into the solution or to combine with elements in the bioactive glass that make up surface layers. The first step in physicochemical reactions is the formation of a leaching layer (d) enriched in Na and O and depleted in Si, Al, Ca, P, K, and Mg. Na and O concentration gradients are opposited. This layer can be considered as an exchange layer between Na+ ions and H+ or H3O+ from the solution. But, during this time period, the exchange is still going on and should lead to a layer depleted in Na. The second step is the formation of a Si-Al rich layer (b) depleted in Ca, P, and Mg which permits the diffusion
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Figure 6. Si and O concentration profiles across the periphery of three bioactive glass particles after 1 day of immersion in standard culture medium (each point represents the mean value ( standard deviation).
Figure 7. Mg and K concentration profiles across the periphery of three bioactive glass particles after 1 day of immersion in standard culture medium (each point represents the mean value ( standard deviation).
and the formation of a Ca-P-Mg rich layer (c). The Si accumulation comes from breaking of Si-O-Si bonds. At this time, the siliceous layer is not a pure Si layer. This is due to the presence of aluminum in this type of bioactive glass. Al2O3 is firmly bonded to the Si and is not released together with Si. But, the oxygen increase can be related (19) Vallet-Regi, M.; Izquierdo-Barba, I.; Salinas, A. J. J. Biomed. Mater. Res. 1999, 46, 560.
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to the presence of SiO-+H3O+ and the beginning of the formation of a pure Si layer composed of SiOH groups. The Ca-P layer is not a stoichiometric apatite and is in majority composed of Ca, P, and Mg. The initial Ca/P ratio is around 1 for the Ca-P layer. Some results published report the formation of an hydroxyl carbonate apatite layer.7,19 However, the first step in the formation of this layer is the deposition of amorphous Ca, H2PO4, and CO3. Calcium might replace hydrogen in the hydroxyl group or bind with negatively charged oxygen species to the silica network. Then, the Ca ion is with one free valence and can bind with one phosphate group or another oxygen in the silica network. The Ca/P ratio is around unity. Only when further ions bind with the remaining OH group bonded to phosphate can the apatite crystal structure nucleate.20 In our case, the magnesium is incorporated in this layer, because this element can go into amorphous apatite with respect to Mg concentration in biological apatites.21 Conclusion The analysis of the interfacial zone, with fewer artifacts due to sample preparation and diffusion effects, needs special preparation/examination techniques. Cryomethods associated to electron probe microanalysis allow analysis of elemental distribution at the periphery of the bioactive glass particles in their in vitro state. We can investigate the chemical process of physicochemical reactions at a submicrometer scale. Physicochemical reactions occur in a relatively short time and lead to the formation of three surface layers. Our results demonstrate, for the first time to our knowledge, the presence of an enriched Na-O layer. The interactions between bioactive glass particles and biological fluids involve a preferential extraction of alkali with an exchange layer. Silicon is also removed from this layer at its interface with the solution. The formation of a SiAl rich layer leads to the formation of a Ca-P-Mg layer on top. The analysis of concentration gradients across the interface for different exposure times to biological solution will give information to better understand the mechanisms of the apatite formation at the bioactive glass periphery. Further investigations of the elemental concentration variations versus time would permit study of the evolution of the composition and thickness of these three layers. LA001669M (20) Kangasniemi, I. M. O.; Vedel, E.; De Blick-Hogerworst, J.; YliUrpo, A. U.; De Groot, K. J. Biomed. Mater. Res. 1993, 27, 1225. (21) Jallot, E.; Irigaray, J. L.; Oudadesse, H.; Brun, V.; Weber, G.; Frayssinet, P. Eur. Phys. J.: Appl. Phys. 1999, 6, 205.
