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Kinetics of Short-Term Physicochemical Reactions at the Periphery of Bioactive Glass Particles. A Transmission Electron Microscopy Cryo-X-ray Microanalysis of Diffusible Ions Edouard Jallot,*,† Hicham Benhayoune,‡ Laurence Kilian,‡ and Yannick Josset‡ Laboratoire de Physique Corpusculaire de Clermont-Ferrand CNRS/IN2P3 UMR 6533, Universite´ Blaise Pascal, 24, avenue des Landais, 63177 Aubiere Cedex, France, and INSERM-ERM 0203, Laboratoire de Microscopie Electronique, UFR Sciences, 21, rue Cle´ ment Ader, BP 138, 51685 Reims, Cedex 2, France Received September 17, 2002. In Final Form: February 18, 2003 In this paper we analyze the kinetics of short-term physicochemical reactions at the interface between bioactive glass particles and biological fluids. Assessment of these reactions requires knowledge of elemental distribution under the micrometer scale at the interface between the bioactive glass and biological fluids. This analysis was performed by scanning transmission electron microscopy associated to energy-dispersive X-ray spectroscopy. But, microanalysis of diffusible ions such as oxygen, sodium, potassium, and magnesium is a major problem. To preserve the chemical identity of specimens, we used cryomethods for the preparation and the characterization of bioactive glass particles immersed in biological fluids. From 24 to 72 h, interactions between bioactive glass particles and biological fluids lead to a release of all elements and to the formation of three surface layers of the order of 300 nm in thickness. A first layer enriched in Si-Al, a second layer enriched in Ca-P-Mg (with a Ca/P ≈ 1), and a third layer enriched in Na-O. This paper demonstrates the formation of an exchange layer between Na+ ions from the glass and H3O+ from the solution. After 72 h, this layer disappears. The two other layers grow in thickness. The first layer becomes a pure Si layer with an increase of Si concentration. Ca, P, and Mg concentrations increase in the second layer, and the Ca/P ratio becomes near 1.7. The Si-rich layer permits the diffusion of Na, K, Ca, P, and Mg with a t-1/2 law and the precipitation of an apatite layer at the materials periphery. The apatite layer grows in thickness with time and is of the order of 3 µm after 672 h of immersion in biological fluids.

Introduction Bioactive glasses are under extensive investigation because of the peculiar reactivity they show when put in contact with human body fluids.1,2 These materials can stimulate a specific response in the surrounding tissues by means of a complex mechanism involving three main phases: ion leaching, partial dissolution of the glass surface, and precipitation of a bonelike apatite layer on the glass surface.3-5 Thanks to the precipitated apatite layer, these materials can provide a very strong chemical bond with hard tissues.6 Bioactive glasses can be proposed both as bulk and as coating for several kinds of applications, especially focused on orthopaedics, where load bearing is required.7,8 Several bioactive glasses can be used to prepare biocomposites, most of them belonging to the system SiO2-CaO-Na2O* To whom correspondence may be addressed: Tel, 33 (0)4 73 40 72 65; Fax, 33 (0)4 73 26 45 98; e-mail, [email protected]. † Laboratoire de Physique Corpusculaire de Clermont-Ferrand CNRS/IN2P3 UMR 6533, Universite´ Blaise Pascal. ‡ INSERM-ERM 0203, Laboratoire de Microscopie Electronique, UFR Sciences. (1) Hench, L. L. J Am. Ceram. Soc. 1998, 81, 1705. (2) Jallot, E.; Benhayoune, H.; Kilian, L.; Irigaray, J. L.; Oudadesse, H.; Balossier, G.; Bonhomme, P. Surf. Interface Anal. 2000, 29, 314. (3) Verne, E.; Vitale Brovarone, C.; Milanese, D. J. Biomed. Mater Res. 2000, 53, 408. (4) Jallot, E.; Benhayoune, H.; Kilian, L.; Irigaray, J. L.; Barbotteau, Y.; Balossier, G.; Bonhomme, P. J. Colloid Interface Sci. 2001, 233, 83. (5) Hench, L. L. Biomaterials 1998, 19, 1419. (6) Pie´trement, O.; Jallot, E. Nanotechnology 2002, 13, 18. (7) Oonishi, H.; Hench, L. L.; Wilson, J.; Sugihara, F.; Tsuji, E.; Kushitani, S.; Iwaki, H. J. Biomed. Mater Res. 1999, 44, 31. (8) Oliva, A.; Salerno, A.; Locardi, B.; Riccio, V.; Della Ragione, F.; Iardino, P.; Zappia, V. Biomaterials 1998, 19, 1019.

P2O5 with different amounts of other oxides (B2O3, Al2O3, MgO, Zr2O, etc.) which provided different properties in terms of surface reactivity.9,10 The following paper concerns a bioactive glass in the SiO2-Na2O-CaO-P2O5-K2OAl2O3-MgO system. Critical concentrations of magnesium can play an important role during physicochemical reactions at the material periphery. On the other hand, aluminum may inhibit apatite precipitation or reduces glass matrix dissolution.11 Together with the development of glasses, several efforts have been pursued in order to explain the mechanism responsible for their in vitro and in vivo behaviors.12-14 It has been observed that the apatite layer, especially in glasses contacting biological fluids, is preceded by formation of a silica gel layer. This layer acts as nucleation sites for Ca-P layer development. However, the formation of these layers is not explained in detail because of the complexity of short-term events occurring at the interface. Assessment of short-term physicochemical reactions requires knowledge of elemental distribution under the micrometer scale at the interface between the bioactive glass and biological fluids. We perform this analysis by scanning transmission electron microscopy (STEM) associated to energy-dispersive X-ray spectroscopy (EDXS). (9) Ferraris, M.; Verne´, E.; Ravaglioli, A.; Krajewski, A.; Parachini, L.; Vogel, J.; Carl, G.; Jana, C. Bioceramics; Sedel, L., Rey, C., Eds.; Paris, 1997; Vol. 10, p 195. (10) Kokubo, T.; Kushitani, H.; Ohtsuki, C.; Sakka, S.; Yamamuro, T. J. Mater. Sci. Mater. Med. 1992, 3, 79. (11) Greenspan, D. C.; Hench, L. L. J. Biomed. Mater. Res. 1976, 10, 503. (12) Hench, L. L. J Am. Ceram. Soc. 1991, 74, 1487. (13) Kokubo, T. J Non-Cryst. Solids 1990, 120, 138. (14) Andersson, O. H.; Karlsson, K. H. J Non-Cryst. Solids 1991, 129, 145.

10.1021/la0265629 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/01/2003

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Figure 1. Silicon concentrations (mmol‚kg-1 of dry weight) in zones I, II, III, IV, and V after 24, 72, 168, 336, and 672 h of immersion of bioactive glass particles in a standard culture medium.

Most of authors focused their investigations on Si, Ca, and P but other elements might play an important role during physicochemical reactions. The aim of this paper is to study all elements which are present in the bioactive glass in order to better understand physicochemical reactions. But, microanalysis of diffusible ions such as oxygen, sodium, potassium, and magnesium is a major problem.15,16 These elements are not very firmly bound in the glass matrix, which undergoes dissolution, or they can be solubilized during specimen preparation. Thin biological samples for transmission electron microscopy are usually prepared by a process involving fixation, postfixation, dehydration, embedding in resin, and sectioning. During this chemical fixation, most of the diffusible ions are rapidly lost (within minutes) from the samples.16,17 To preserve the chemical identity of specimens, we used cryomethods for the preparation of bioactive glass particles immersed in biological fluids. These methods permit retention of all elements of interest at their in vitro location and allow identification at the level of analytical resolution required.18 To advance in the knowledge of the short-term mechanisms responsible for bioactivity induction and to optimize the bioactive behavior of the bioactive glass, we studied the influence of exposure times to biological solution on the surface layers composition, size and on the growth rate of the apatite layer. 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 a mixture of raw materials in a platinum crucible at high temperature (2 h at 1200 °C and 3 h at 1350 °C). The mixture of raw materials consisted of sand Sibelco, aluminum oxide, anhydrous dibasic sodium phosphate, calcium carbonate, potassium carbonate, and basic magnesium carbonate. Then, the glass was cast, crushed, and transformed into powder of grain size under 40 µm in diameter. After production, the glass composition was determined by atomic emission spectrometry and its crystallinity was evaluated by X-ray diffraction. Sample Treatment. The glass powder (2 mg) was immersed at 37 °C for 24, 72, 168, 336, and 672 h in 1 mL of a standard Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Cergy Pontoise, France) (pH 7.3).19 DMEM contained the following ingredients: 6400 mg/L NaCl, 400 mg/L KCl, 200 mg/L CaCl2, 200 mg/L MgSO4‚7H2O, 124 mg/L NaH2PO4, and 3700 mg/L NaHCO3.

Specimens Preparation and X-ray Microanalysis. After treatment, the glass powder was lying in 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 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 energy dispersive 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.20 In Hall’s method the characteristic peak 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.

Results Concentration gradients of Si, Al, Na, O, Ca, P, Mg, and K across the periphery of bioactive glass particles were studied by electron probe X-ray microanalysis on the cryosections. Elemental profiles for different exposure times to biological solution revealed five zones from the center to the periphery of the bioactive glass (Figures 1-8, Table 1): From 24 to 72 h of Exposure to Biological Solution. Zone I: The bioactive glass particles are in dissolution (15) Zierold, K. J. Microscopy 1991, 161, 357. (16) Zierold, K.; Hentschel, H.; Wehner, F.; Wessing, A. Scanning Micros., Suppl. 1994, 8, 117. (17) Roomans, G. M. J. Electron Microsc. Tech. 1988, 9, 3. (18) Jallot, E.; Benhayoune, H.; Kilian, L.; Josset, Y.; Balossier, G. Langmuir 2001, 17, 4467. (19) Xynos, I. D.; Edgar, A. J.; Buttery, L. D. K.; Hench, L. L.; Polak, J. M. Biochem. Biophys. Res. Commun. 2000, 276, 461. (20) Hall, T. A. Physical techniques in biological research, 2nd ed.; Oster, G., Ed.; Academic Press: New York, 1971; Vol. I Part A, Chapter. 3.

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Figure 2. Aluminum concentrations (mmol‚kg-1 of dry weight) in zones I, II, III, IV, and V after 24, 72, 168, 336, and 672 h of immersion of bioactive glass particles in a standard culture medium.

Figure 3. Sodium concentrations (mmol‚kg-1 of dry weight) in zones I, II, III, IV, and V after 24, 72, 168, 336, and 672 h of immersion of bioactive glass particles in a standard culture medium. Table 1. Size (nm) of Zones I, II, III, IV, and V According to Time after Immersion of Bioactive Glass Particles in a Standard Culture Medium time, h

zone I

zone II

zone III

zone IV

zone V

24 72 168 336 672

>4800 >3600 >3500 >2900 >2900

900 ( 150 900 ( 150 1000 ( 200 1100 ( 200 1100 ( 200

300 ( 50 1300 ( 150 1400 ( 150 2200 ( 200 2400 ( 200

300 ( 50 1400 ( 150 1800 ( 150 2000 ( 200 3000 ( 200

300 ( 50 300 ( 50 0 0 0

and are composed of Si, O, Ca, P, Na, K, Mg, and Al. Si and Ca concentrations slowly decrease according to time of interaction between bioactive glass particles and fluids. Na, P, K, and Mg concentrations decrease rapidly. On the other hand, Al concentration slightly increases with time and O concentration stay constant. Zone II: All concentrations are lower than those in zone I and vary in the same way. Zone III: A layer enriched in Si, O, and Al. In this layer Ca, P, Na, K, and Mg concentrations were decrease. The size of this layer increases from 300 nm at 24 h after exposure to 1300 nm at 72 h.

Zone IV: A second layer (≈300 nm in thickness) enriched in Ca, P, O, and Mg. The Ca/P ratio was around unity after 24 h and increases near 1.3 at 72 h. In this layer, a high decrease of Na was observed. From 24 to 72 h Ca, P, and Mg concentrations increase. Both Si and Al concentrations decrease strongly. Oxygen concentration slightly decreases. The size of this layer increases from 300 nm at 24 h to 1400 nm at 72 h. Zone V: A third layer enriched in sodium and oxygen with low concentrations of Al and Si. The evolution of sodium and oxygen concentrations appeared to be related. The concentrations of Ca, P, and Mg increased with time, and high concentrations were found at 72 h. The size of this layer is of the order of 300 nm. After 72 h of Exposure to Biological Solution. Zone I: Si, O, Ca, P, Na, K, and Mg concentrations continue to decrease according to time of interaction. Al concentration increases with time. Zone II: Si, O, Ca, P, Mg, and Al concentrations are lower than those in zone I and decrease with time. Na and K concentrations are higher in this zone, and they decrease

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Figure 4. Oxygen concentrations (mmol‚kg-1 of dry weight) in zones I, II, III, IV, and V after 24, 72, 168, 336, and 672 h of immersion of bioactive glass particles in a standard culture medium.

Figure 5. Calcium concentrations (mmol‚kg-1 of dry weight) in zones I, II, III, IV, and V after 24, 72, 168, 336, and 672 h of immersion of bioactive glass particles in a standard culture medium.

with time to become similar to those in zone I after 672 h. The thickness of this layer is of the order of 1 µm. Zone III: This layer is now composed with high Si and O concentrations. Aluminum strongly decreases with time. The presence of Na and K is observed. Other elemental concentrations are very low. Its thickness increases from 1.4 µm at 168 h to 2.4 µm at 672 h. Zone IV: This layer is composed with high Ca, P, and O concentrations. An increase of Mg is observed in this layer. Ca, P, and Mg concentrations increase according to time after exposure. The presence of Na and K is observed. This layer grows in thickness from 1.4 to 3 µm. The Ca/P ratio is of the order of 1.7. Zone V: After 72 h, this layer disappears. At each time period, the distribution of Ca, P, and Mg concentrations was consistent across the interface. Analysis of the Diffusion Process for Na, K, Ca, P, and Mg. Variations of sodium, potassium, calcium, phosphorus, and magnesium concentrations in zone I

followed the law (eq 1) proposed by Philibert21 and Grauer22

CI,X ) atb

(1)

where CI,X is the mean concentration of the element X (Na, K, Ca, P, Mg) in zone I, t is the time period (day), and a and b are constants for the element X. When the concentrations are represented in logarithmic coordinates (Figures 9 and 10), the respective slope values (b) are defined with expression 2.22-24

log(CI,X) ) b log(at)

(2)

For the five considered elements, b is near -1/2 (Table 2), (21) Philibert, J. Atom movements, diffusion and mass transport in solids; De physique: Les Ulis, 1991; Chapter 10. (22) Grauer, R. EIR-Ber. 1985, 538, 83. (23) Hench, L. L.; West, J. K. Chem. Rev. 1990, 90, 33. (24) Bunker, B. C.; Tallant, D. R.; Headley, T. J.; Turner, G. L.; Kirkpatrick, R. J. Phys. Chem. Glasses 1988, 29, 3.

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Figure 6. Phosphorus concentrations (mmol‚kg-1 of dry weight) in zones I, II, III, IV, and V after 24, 72, 168, 336, and 672 h of immersion of bioactive glass particles in a standard culture medium.

Figure 7. Magnesium concentrations (mmol‚kg-1 of dry weight) in zones I, II, III, IV, and V after 24, 72, 168, 336, and 672 h of immersion of bioactive glass particles in a standard culture medium. Table 2. Slope Values (b) for Na, K, Ca, P, and Mg Determined When the Concentrations in Zone I According to Time after Implantation Are Represented in Logarithmic Coordinates slopes (b)

sodium

potassium

calcium

phosphorus

magnesium

-0.47 ( 0.11

-0.46 ( 0.10

-0.47 ( 0.09

-0.54 ( 0.09

-0.51 ( 0.11

which demonstrates a diffusion process. We only observe this phenomenon in zone I. Discussion The present work analyzes the interface changes of bioactive glass particles immersed into biological fluid. A physicochemical approach is adopted, namely, the analysis of the transformation kinetics of the glass composition by means of EDXS to understand mechanisms of interaction. Specimen preparation is an important step in X-ray microanalysis. The specimen can be irreparably mistreated in this step, with a consequence of making all further analytical work meaningless. The conventional preparation methods allow easy identification of the

ultrastructure of the specimen but have serious disadvantages from the analytical point of view. Cryomethods associated to electron probe microanalysis allow analysis of elemental distribution at the periphery of bioactive glass particles in their in vitro state. Analysis of concentration gradients across the interface for different exposure times to biological solution show that physicochemical reactions occur in a relatively short time. 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 distribution of silicon, aluminum, calcium, phosphorus, magnesium, sodium, and potassium differs between the glass particle center and

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Figure 8. Potassium concentrations (mmol‚kg-1 of dry weight) in zones I, II, III, IV, and V after 24, 72, 168, 336, and 672 h of immersion of bioactive glass particles in a standard culture medium.

Figure 9. Variation of sodium and potassium concentrations (mmol‚kg-1 of dry weight) in zone I according to time (day) after immersion of bioactive glass particles in a standard culture medium.

Figure 10. Variation of calcium, phosphorus, and magnesium concentrations (mmol‚kg-1 of dry weight) in zone I according to time (day) after immersion of bioactive glass particles in a standard culture medium.

the newly formed layers at the periphery (Figure 11). From 24 to 72 h after exposure, we observed the formation of five zones. The bioactive glass center is represented with zones I and II. The decrease of Si, Al, Ca, P, Mg, Na, and

K between these two zones shows the dissolution of the particles. A Si, Al, and O rich layer is observed (zone III). A second layer (zone IV) has a significantly higher concentration of Ca, P, and Mg and a decrease of Si and

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Figure 11. Schematic view of the five zones at the bioactive glass periphery after exposure to biological solution.

Al. A third layer (zone V) is enriched in Na and O with presence of K. From 72 to 672 h of exposure, physicochemical reactions continue. The Si, Ca, P, Mg, Na, and K decrease with time in zones I and II shows the particle dissolution. Al concentration increases in zone I because this element is firmly bound to Si and slowly released. Concentrations of Na and K are higher in zone II than those in zone I, which demonstrates a higher release of these elements. Then, a pure Si and O rich layer (zone III) of 2.4 µm in thickness is formed with low concentrations of Al. In zone IV, Ca, P, and Mg concentrations increase and the size of this layer increases. A Ca-P-Mg rich layer of about 3 µm in thickness is formed. After 72 h, zone V disappears. Formation of the five zones and their evolution with time of exposure to the biological solution represent important steps in bioactivity mechanisms: 1. Leaching Layer, Exchange Layer. The dissolution of the bioactive glass results from breakdown of the silica network and the associated release of all elements within it. The number of sites occupied by alkali ions in zones II, III, and IV is less than that inside the glass (zone I). The decomposition of the glass involves a preferential extraction of alkali so that there must be an alkali-deficient leached layer on the surface of the glass next to the solution. Then, a leaching layer is formed in zones II, III, and IV. Si, Al, Ca, and P may also be removed from this layer at its interface with the solution, and the interface will consequently move into the glass as the reaction proceeds. At the boundary between the leached layer and the solution, we observed an exchange layer in zones V, sites are available to cations which may be occupied preferentially by one of the ions in the solution. When the concentration of ions in solution is lowered, the second ion will begin to occupy the surface site. Occupancy of the surface sites will be determined by that equilibrium. The exchange process consists of a flow of H3O+ ions and an equivalent flow of sodium ions into the solution from the

glass represented as follows

SiO-Na+ + H3O+ f SiOH + H2O + Na+ Two reaction zones must therefore be identified, one at the leached layer-solution interface (zone V) where equilibrium is established between the surface sites and ions in solution, and the other at and near the leached layer-glass interface (zone II) consisting of counterion exchange. After 72 h of exposure, the Na concentration tends to zero in zone V. This also proves that all Na+ ions are mobile. 2. Formation of a Si Layer. Silicon passes into the solution as a result of the breaking of Si-O-Si bonds through attack by hydroxyl groups. Repolymerization of a hydrated Si-rich layer on the surface depleted in alkali and Ca, P, and Mg. At the beginning, this layer is enriched in Al because this element is firmly bound to Si. Extensive dissolution of the glass network is controlled by addition of Al2O3. In fact, Al is associated to Si (Si-O-Al) and enriched in the Si layer, thereby stabilizing it. When the glass dissolves, most of the Al2O3 is bonded by the Si but is not released together with other Si. Thus, the specific rate of Al released is smaller than the corresponding rate of Si released, because Al2O3 is a network former, not leached like Si. In the studied bioactive glass, addition of Al2O3 is used to control the solubility of the glass. 3. Migration of Calcium, Phosphate, and Magnesium from the Glass to the Surface of the Si Layer. Migration of Ca2+, PO43-, and Mg2+ ions from the bioactive glass particles through the Si gel layer into the biological solution further increases the concentrations. In the vicinity of the bioactive glass particles, concentrations of calcium, phosphorus, and magnesium follow a t-1/2 law which demonstrates a diffusion process through the Si layer. 4. Precipitation of a Ca-P-Mg-Rich Layer. Due to the diffusion of calcium, phosphate, and magnesium from

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the glass through the Si layer, a Ca-P-Mg film starts to build up on top of it. Ca increases degree of supersaturation of the biological fluid with respect to apatite, and silicon might play a role in providing favorable sites for nucleation of the apatite on the bioactive glass. The compositional profiles of Ca, P, and Mg follow each other through the periphery of the bioactive glass. Ca, P, and Mg amounts are low in the glass. The decrease in Ca, P, and Mg concentrations is attributed to the growth of the apatite nuclei induced on the Si layer. The apatite nuclei grow substantially by taking Ca and P from the materials and surrounding fluids. The induction period is of the order of 72 h. After 72 h, the Ca/P ratio is near 1.7, which is characteristics of apatite. Formation through precipitation of the apatite layer reduces the removal of alkali ions into biological fluids, but it does not stop it. The passage of sodium ions through the Si-rich layer and Ca-P-Mg-rich layer does not interfere with apatite precipitation and growth. Na diffuses through the siliceous layer and the apatite layer, but the presence of these two layers reduces its diffusion and leads to an accumulation of Na in zone II. Conclusion In the case of bioactive glasses that undergo dissolution due to ion diffusion, spatially resolved X-ray microanalyses associated to cryomethods are of great importance in evaluating short-term mechanisms of physicochemical reactions between material and biological fluids, because

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the phenomenon occurs under a micrometer scale. Cryomethods permit the preservation of the chemical identity of specimens and the microanalysis of diffusible ions of bioactive glass particles immersed in biological fluids. This paper is, to our knowledge, the first experimental study of the kinetics of short-term physicochemical reactions at the periphery of bioactive glass particles. These reactions can be separated into four steps. The first is rapid exchange of Na+ and K+ with H+ or H3O+ from solution through an exchange layer. The occupancy of the surface sites by either hydrogen ions or alkali ions which was thought to occur by clearly separable processes in fact occur simultaneously. The second reaction is a loss of soluble silica to the solution resulting from breaking of Si-O-Si bonds and repolymerization of a hydrated Sirich layer on the surface depleted in alkali and Ca, P, and Mg. The third reaction is diffusion of calcium, phosphate, and magnesium from the glass to the surface of the Si layer. The decomposition of glass can be described as a diffusion process through the siliceous layer. The fourth reaction is precipitation of a Ca-P-Mg-rich layer which grows in thickness with time of exposure to biological fluids. In our case, the buildup of an apatite layer occurs in vitro within a Si layer. Formation of the apatite layer represents bioactivity properties of the studied bioactive glass. Presence of 2% Al2O3 in the bioactive glass reduces its dissolution but does not inhibit its bioactivity. LA0265629