19
Synthesis, characterization, in vitro bioactivity and wettability of sol-gel derived SiO
2-CaO-P
2O
5and SiO
2-CaO-P
2O
5-Na
2O bioglasses
S. Bouhazma
(a), S. Chajri
(a), H. Barkai
(b), S. Elabed
(b), S. Ibnsouda Koraichi
(b), B. El Bali
(c)and M. Lachkar
(a)*(a)Engineering Laboratory of Organometallic and Molecular Materials (CNRST, URAC 19), Faculty of Sciences, University Sidi Mohamed Ben Abdellah, Po. Box 1796 (Atlas), 30000 Fez, Morocco.
(b)Laboratory of Microbial Biotechnology, Faculty of Sciences and Technology, University Sidi Mohamed Ben Abdellah, 30000 Fez, Morocco.
(c)Laboratory of Mineral Solid and Analytical Chemistry, Department of Chemistry, Faculty of Sciences, University Mohamed 1st, Po. Box 717, 60000 Oujda, Morocco.
*Corresponding author. E-mail : lachkar.mohammed@gmail.com Received 24 Sept 2014, Revised 24 Oct 2014, Accepted 10 Dec 2014
Abstract: Glasses in the ternary and quaternary system SiO2-CaO-P2O5 ; SiO2-CaO-P2O5-Na2O were prepared by means of a sol-gel route starting from tetraethyl orthosilicate, calcium nitrate, TEP, and sodium nitrate. The obtained glasses were characterized by X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). These materials were subjected to immersion studies in simulated body fluid (SBF), and have been subjected to water contact angle measurements. A comparative study on Na2O-containing (22Na) and Na2O-free bioactive glass ceramics (44C) indicated that Na2O could be an important constituent enabling achievement of an optimal combination of bioactivity and wettability.
Keywords: Sol-gel; bioactive glasses; sodium oxide; in vitro bioactivity; contact angle.
1.
Introduction
During the last decades, considerable attention has been directed towards the use of bioactive materials, where bioactivity is defined as interfacial bonding of an implant, or a bioactive scaffold to tissue by means of formation of a biologically active hydroxyapatite (HA) layer on the bioactive material surface [1,2].
Literature shows the importance of these materials because of their capacity to bond and integrate with bones in the living tissues [1-3]. This bonding to living bone tissue occurs upon a sequence of reactions on the material surface followed by cellular reactions [3]. The main applications of these bioactive glasses in the clinical field are mainly, if only to quote, the filling of bones (including teeth) cavities, the reconstruction of maxillofacial defects, and the production of dental devices. These Bioactive glasses are special systems which are generally composed of the oxides SiO2, CaO and P2O5. They can be synthesized by traditional melt quenching or by the versatile sol-gel process [4, 5]. Commercially produced bioactive glasses have been made by conventional glass powder manufacturing methods, i.e. melting and quenching. However,
20 calcium silicate glasses are tough to prepare via the traditional melt quenching method. This is because the compositions lie within the liquid–liquid immiscibility dome of the phase diagram, in the case of high CaO content, or have very high temperatures, in the case of low CaO content. Due to their potential advantages of higher purity and homogeneous materials that are produced at lower processing temperatures, sol-gel processings have been intensively studied as an alternative method for preparing ceramics and glasses for a wide variety of applications including the field of bioceramics. Li et al. showed that CaO-P2O5-SiO2
powders produced by this technique are more bio-active than the melt-derived glasses of the same composition [6]. Meanwhile, increasing research efforts are being invested in fabrication of bio-active glasses using the sol-gel technique [7]. A variety of sol-gel-derived bio-glasses materials, incorporating modifiers such as magnesium, cerium, boron, strontium and calcium have been reported with the aim of improving specific characteristics, namely the bioactivity, the compatibility and the mechanical strength [8, 9]. Chronologically dealing with the sol-gel route to synthesize bio-glasses materials, we remarked that Laczka et al. synthesized early in 2000 materials in the ternary CaO-P2O5-SiO2 system, adding a number of other elements, including sodium [10]. But in this case, the studied composition was quite different from the composition of the Bioglass® 45S5, which is commercially produced by the fusion and solidification process [11]. In 2005, Carta et al. proposed a synthesis method of a new glass of the P2O5-CaO-Na2O-SiO2
system; a phosphate glass with very low concentration of SiO2 (0-25% in mol) [12]. Later, Chen et al.
proposed a synthesis of similar bioactive glass 45S5 by the sol-gel method [13], which, in principle, shows several advantages: this method allowed for the production of bioactive glasses with higher purity and homogeneity, and also significantly expanded the ranges of compositions that exhibit bioactivity as compared to bioactive glasses prepared by the traditional method of melting/cooling [14-16], but a few disadvantages include the cost of the raw materials, shrinkage that accompanies drying and sintering, and processing times in comparison to the melting route [17]. Another point that should be highlighted is that, besides reducing the melting temperature for glass preparation by the traditional route, the addition of Na in these materials makes them more soluble in aqueous media, as previously mentioned [18]. These features of glass systems containing Na associated with the high intrinsic surface area of sol-gel glasses can result in a very interesting property that should be further explored, since the dissolution rate of materials designed for implants is a fundamental factor for their interaction with living tissues. Some properties of sol-gel glasses (without Na), such as dissolution rate and bioactivity, have been compared with melt derived glasses [19- 21]. The inclusion of Na2O in the fabrication of bioceramic materials would offer opportunities to improve mechanical strength without losing a satisfactory biodegradability. As to take part in the explorations of the bio-glasses, we report in the present work on the preparation, by a sol-gel method, of two glasses, i.e.
46%SiO2-44%CaO-10%P2O5 (44C) and 46%SiO2-22%CaO-10%P2O5-22%Na2O (22Na). We also study the effect of the composition on their in vitro bioactivity and their wetting properties.
2. Materials and methods
The chemicals used for synthesizing the bioactive glass were: tetraethyl orthosilicate (TEOS) (Aldrich, 99%), triethyl phosphate (TEP) (Eastman, 99.8%), sodium nitrate (Sigma–Aldrich, 99%) and calcium nitrate tetrahydrate (Sigma–Aldrich, 99%).
2.1. Glasses synthesis
21 Two glasses, with compositions presented in Table 1, were prepared by hydrolysis and polycondensation of TEOS, TEP and Ca(NO3)2.4H2O and/or NaNO3. The hydrolysis reaction, in a molar ratio of H2O/(TEOS + TEP) = 8, was catalyzed by adding few drops of HNO3 (2N). TEOS, deionized water, and nitric acid, were successively mixed and the miscellaneous was allowed to react for 1h under continuous stirring. Then appropriate amounts of a series of reagents were added in the following sequence: (TEP), Ca(NO3).4H2O, NaNO3, allowing 30 min for each reagent to react completely. Next, the solution was introduced in a hermetically sealed Teflon container, where it was allowed to gel at room temperature and then was aged at 70° C for 3d. Drying was carried out at 150°C for 4d; finally the stabilization treatment was carried out by heating the dried gel disks at 700°C for 3h. All glasses with compositions presented in Table 1 were prepared via a sol-gel technique and characterized by XRD, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM).
Table 1. Composition (mol %) of sol-gel glasses.
SiO2 CaO P2O5 Na2O
44C 46 44 10 0
22Na 46 22 10 22
2.2. XRD analysis
The characterization of the powders, resulting before and after immersion in SBF, was performed by X-ray diffraction. Measurements were performed with a Discover model equipped with a monochromatized Cu-Kα radiation (= 1.5418 Å) in the 2 range [20°-80°].
2.3. FTIR analysis
The monitoring of powder modification after in vitro bioactivity tests was performed by FTIR using an IR Bruker VERTEX 70 spectrometer. The FTIR spectra were recorded from 400 to 4400 cm−1 with the resolution of 4 cm−1.
2.4. Contact angle measurements
Contact angle measurements were performed by using a goniometer by the sessile drop method (GBX- France). Two polar liquids (water and formamide) and one non-polar liquid (diiodomethane) were used. The water contact angles of the different surfaces studied are under the theoretical limit of 65° between hydrophobic and hydrophilic character [18]. A drop of ultra-pure water, diiodomethane, formamide or SBF was placed on the glass disk mounted on a prepared substrate plate and the image was immediately sent by the camera to the computer for analysis. The contact angles of water were measured after 10s. The temperature and the humidity were constant during the experiment (22°C and 63% respectively). The measurements were repeated by depositing at least three drops on each substrate, and the final results were presented by the average of the measured drops.
2.5. SEM/EDX analysis
Morphological characterization of the pellets, regarding the surface modifications that occurred during the in vitro bioactivity tests, was performed by SEM. A set of samples was selected and analyzed before and after
22 soaking in SBF solution at different testing times. We used scanning electron microscopy coupled with energy-dispersive spectroscopy: ESEM Quanta 200 (FEI Company).
2.6. Evaluation of in vitro bioactivity
Assessment of in vitro bioactivity was carried out by soaking the powder, in 45 mL of simulated body fluid (SBF) in polyethylene containers maintained at 37 °C. In vitro tests were performed according to the method described by Kokubo and Takadama [22]. The solution used to conduct this study, known as SBF, is a cellular, protein-free and has a pH of 7.4. Composition of this solution is compared to human blood plasma [22] in Table 2. The SBF solution had a similar composition and concentration to those of human plasma.
No precipitation or changes of the content take place during the preparation or preservation of this solution.
It is important to mention that this solution is often used in the in vitro evaluation of the formation of a hydroxyapatite layer on the surface of materials designed for implants, according to ISO 23317, approved by the International Organization for Standardization [23].
Table 2. Composition of SBF and the inorganic part of human blood plasma (mmol/L).
Ion Simulate Body Fluid Blood plasma
Na+ 142.0 142.0
K+ 5.0 5.0
Mg2+ 1.5 1.5
Ca2+ 2.5 2.5
Cl- 148.8 103.0
HCO3-
4.2 27.0
HPO42- 1.0 1.0
SO42-
0.5 0.5
3. Results and Discussions
Figure 1 shows the XRD spectra of the sol-gel derived powders, from the two glasses 44C and 22Na, sintered at 700 °C. The results show that Na2Ca2Si3O9 is a major byproduct from the sintering of 22Na.
Chen et al. advanced the formation of sodium calcium silicates Na2Ca2Si3O9 and Na2Ca3Si6O16 upon treatment of all sample sets at 1000 °C [13]. Even prior to soaking in SBF, apatite peaks were evident in the XRD spectra of those two powders shown. However, this may not be considered surprising because the aqueous environment of the sol-gel process favors the precipitation of hydroxyapatite from the amorphous structure of silica-based bio-active glasses [24]. In 44C, the crystal form is identified to be -wollastonite CaSiO3. Worthy to notice that neither 22Na nor 44C bioactive glasses could be completely crystallized. In fact, Na2Ca2Si3O9 would require too much CaO to fully crystallize from bio-glass.
23 Figure 1. XRD spectra of sol-gel derived 44C and 22Na glass ceramics after sintering at 700°C for 3h.
In vitro bioactivity was evaluated by soaking the two bio-glasses 44C and 22Na powders in acellular SBF with a composition close to that of human blood plasma.
Figure 2. XRD spectra of the sol-gel derived bio-glass 44C (sintered at 700°C / 3h) before and after immersion in SBF for different times.
Figure 2 shows the XRD spectra of the sol-gel derived 44C glass crystallized after soaking in SBF for 10 days. The major diffraction peaks in the spectra of the soaked glass can be attributed to the formation of HA layer.
Figure 3: FTIR spectra of 44C bio-glass before and after soaking in SBF solution at different testing times.
24 The grinded sample 44C was examined by Fourier transform infrared spectroscopy (Fig. 3). Before soaking in SBF, the FTIR spectra of the sample exhibited Si–O–Si stretching and bending bands (Figure 3). In fact, the band around 1021 cm−1 corresponds to the vibrational mode of the asymmetric stretching of Si–O–Si, and the strong band at 449 cm−1 corresponds to the vibrational mode of the bending of Si–O–Si. After soaking at various times in SBF solution, additional peaks appeared in the FTIR spectra around 550-600 cm−1 corresponding to P–O bending (amorphous) [25]. Vibration bands at 795 cm−1 corresponding to antisymmetric vibration mode of P–O in amorphous calcium phosphate, and 548 cm−1 corresponds to carbonates observed for soaked glasses indicate apatite formation in SBF [26]. After 4 days, surfaces showed similar FTIR spectra as those observed for synthetic carbonated hydroxyapatite (CHA) [27]. In this work the pattern obtained for the glass 22Na after soaking for 6h, and 1d is similar to the one obtained before soaking in SBF. Patterns obtained for the glass 22Na after 4 d soaking showed one wide diffraction maxima at 32°, corresponding to the (211)-reflection of an apatite-like compound (Figure 4). After 10d, the (211)- and (210)-reflections of this apatite compound appeared owing to the increase in its crystallinity. The diffraction peaks of the Na2Ca2Si3O9 phase became shorter with increasing incubation time in SBF;
eventually disappearing after incubation for 4 days, leaving a broad halo pattern (indicating an amorphous structure) overlaid with small apatite peaks. Notice that our bio-glass 22Na is bio-active from 4d, more efficient than similar bio-active glasses synthesized by the same method which turned to be so from 15d [13].
Figure 4. XRD spectra of the sol-gel derived bioglass 22Na (sintered at 700°C / 3h) before and after immersion in SBF for different times.
After thirty days of immersion in simulated body fluid we can observe that the crystalline phase Na2Ca2Si3O9 disappeared completely and replaced by a typical strong (211)-peak of a hydroxyapatite layer (Figure 4). When the quantity of Silica is too low, the initial silicate network is more depolymerized, and the reaction requires a longer time in order to obtain a highly polymerized silica gel phase on which the amorphous calcium phosphate will precipitate. Rehman et al. [28] showed that when the rate of silica increases in glasses, the network takes longer time to release its CaP groups, leading to a slower HCA formation.
25 Figure 5. FTIR spectra of 22Na bioglass before and after soaking in SBF solution at different testing times.
The sol-gel process for producing ternary system (44C) is now well established [29]. Hence, the FTIR analysis in this work focused on the 22Na material produced with the sol-gel process (Figure 5). Among the bands appeared after soaking in SBF, we found those positioned at 1420 cm-1 corresponding to carbonates, 2272 and 2349 cm-1 corresponding to soluble CO2, and 1630, 3350 cm-1 to water, the band positioned in the region 777-790 cm-1 can be attributed to the crystalline phase which was confirmed by the XRD results.
During sintering at 700°C the formation of crystalline phase Na2Ca2Si3O9 is possible. After just 4 days we obtained the characteristics bands of CHA.
The surface properties of bioglasses materials are important criteria of biocompatibility. In fact, it was shown a relationship between hydrophobicity and surface tension with the cell/material interaction [30]. The wettability of a bio-active glass is an important factor, as it is considered to play an important role with respect to protein adsorption, cell attachment and spreading [30]. It involves the measurement of contact angles as the primary data, which indicates the degree of wetting when a solid and liquid interact. Small contact angles (90°) correspond to high wettability, while large contact angles (90°) correspond to low wettability.
Table 3. Contact angle of the glasses 44C and 22Na.
Samples
Contact angle (°)
Water SBF
44C 21.2 1.2 17.4 1.1
22Na 11.7 0.9 10.7 0.7
The results obtained for these two glasses 22Na and 44C are presented in Table 3. At least three measurements were carried out for each sample. We might thus conclude that both glasses are hydrophilic.
As matter of comparison 22Na presents a contact angle much smaller with only 11.7°, which means that this glass is more hydrophilic than the ternary glass 44C and 45S5 bioglass (52°) [31].
Accordingly, 44C is less suitable for cell attachment. This seems to have direct link with the composition. In fact, 22Na presents a higher percentage of Na2O, which means the release of a great number of Na+ ions in the medium, leading to a very hydrophilic surface [32]. With SBF solution, the contact angles of the
26 different surface studied are under the theoretical limit of 65°. Surfaces with a higher wettability, in a moderately hydrophilic range, are reported to be more adhesive for cells than those with lower one. It results then that 22Na presents more interest for cell proliferation than 44C and 45S5 glasses.
Figure 6. Scanning electron micrographs obtained from the glass 22Na before (a) and after soaking in SBF (b).
The formation of apatite was also confirmed by SEM/EDX examination (Figure 6 a&b), which identify the fine fibers of apatite crystals. EDX analysis on small particles of apatite formed at the sample surface is a useful tool to monitor the chemical mechanism of apatite formation [33]. The SEM observations provide evidence that two or more layers grew on the surface of the gel-glasses. The SEM micrograph of the 22Na gel glass after 4 days exposure (Figure 6b) reveals a difference in morphology within the formed CHA layer.
When phosphorus is added to the compositions, the two factors affecting the velocity of CHA formation are the Si and P percentage. The migration of these groups through the silica-rich layer improves the formation of the CaP phase at the surface of the material. The CHA formation time is also depending from the Si quantity. In order to yield apatite quickly, a bioactive glass must present a medium rate of SiO2 (between 44 and 50%). When the quantity of Si is too low, the initial silicate network is more depolymerized, and the reaction requires a longer time in order to obtain a highly polymerized silica gel phase on which the amorphous calcium phosphate will precipitate. If the Si rate is too high, the highly polymerized network slows down the migration of phosphate groups, and delays the formation of CHA at the surface [27]. The composition 22Na pointed out by the design reacts faster than Na bioglass obtained by Chen et al. [13]. The compositions are different, but the higher P2O5 content in 22Na allows a higher migration of CaP groups through the silica-rich layer, accelerating the apparition of CHA. Glass 22Na is a good compromise between network polymerization, solubility, quantity of orthophosphate groups in the matrix, and good wettability.
4. Conclusion
Bioactive glass powder was synthesized successfully via a sol-gel method that the processing is simple, low cost and highly efficient. Two bioactive glasses were performed in new formulation with the molar composition 46%SiO2-44%CaO-10%P2O5 and 46%SiO2-22%CaO-10%P2O5-22%Na2O as bioactive materials. These glasses are bioactive and form a hydrated carbonate apatite layer on their surface on exposure to SBF. The results indicate that bioglass 22Na is bioactive within the first 4 d of exposure to SBF.
Indeed, sample 22Na presents a good hydrophilic behavior permitting cell adhesion.
27 Acknowledgements-The authors would like to acknowledge the support and technical assistance of Interface Regional University Center (University Sidi Mohammed Ben Abdellah, Fez), and National Center for Scientific and Technical Research (CNRST-Rabat).
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