Preparation and in vitro bioactivities of calcium silicate nanophase materials

Materials Science and Engineering C 25 (2005) 455 – 461 www.elsevier.com/locate/msec

Preparation and in vitro bioactivities of calcium silicate nanophase materials Xianghui WanT, Chengkang Chang, Dali Mao, Ling Jiang, Ming Li State Key Laboratory of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiaotong University, 1954 Huashan Road, Shanghai 200030, PR China Received 14 May 2004; received in revised form 28 September 2004; accepted 18 December 2004 Available online 2 February 2005

Abstract Nano-sized amorphous CaSiO3 (A-CS) and h-wollastonite (h-CS) powder were produced by chemical precipitation method and subsequent heat treatment, using Ca(NO3)2!4H2O and Na2SiO3!9H2O as the starting materials, polyethylene glycol as the dispersant. Bulk materials were produced using the two powders as precursors. Rough, porous, and elongated particles occurred in A-CS material and the grains of h-CS were smooth, dense, and spheric. The particle sizes of A-CS and h-CS were about 40 nm and 90 nm in diameter respectively. The bioactivities of A-CS and h-CS materials were investigated by soaking the two materials in SBF solution at 36.5 8C for 6 h to 15 days. Experiment results revealed that CaCO3 crystallized accompanied with HAP on the A-CS surfaces. HAP crystallized constantly on the surfaces of h-CS. The HAP layer formed on h-CS was composed of uniformly sized particles. A layer of irregular particle size was formed on the A-CS owing to the co-existence of CaCO3. The A-CS showed a faster formation of HAP due to the high release rate of Ca ion. D 2005 Elsevier B.V. All rights reserved. Keywords: Calcium silicate; Nanophase; Chemical precipitation method; In vitro test; Hydroxyapatite

1. Introduction During last several decades, various kinds of biomaterials known as bbioactive materialsQ such as 45S5bioglass [1], sintered hydroxyapatite [2], A/W glass–ceramic containing apatite and wollastonite [3], have been synthesized and developed for hard tissue repair and replacement. A common feature for all bioactive materials is that they can bond to living bone through a hydroxyapatite (HAP) layer formed on their surfaces when in contact with Simulated Body Fluid (SBF) [4]. The results from the previous studies implied that the element P plays a very important role in the material composition. However, new researches shown that the presence of phosphorus in the material composition was not an essential requirement for the development of the HAP layer [5]. The CaO and SiO2 could serve as the main

T Corresponding author. Tel.: +86 21 62933344; fax: +86 21 62932522. E-mail address: [email protected] (X. Wan). 0928-4931/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2004.12.003

components for biomaterials to promote the bonding between implants and hard tissue [6–8]. So, studies have been focused on the CaO–SiO2 binary system. Calcium silicate (CaSiO3) has the chemical composition of 48.3% CaO and 51.7% SiO2. It exhibited excellent in vitro bioactivity [7,8] and the formation rate of HAP on its surface is faster than those of the other biocompatible glasses and glass–ceramics in SBF solution [9]. In addition, wollastonite composites as plasma sprayed coating on titanium alloy substrates [10,11] and wollastonite/TiO2 [12] or wollastonite/polymer [13] composites showed good mechanical properties as well as bioactivity. So CaSiO3 ceramics will be a candidate material for artificial bone and dental root. The calcium silicate materials prepared in previous studies were in micrometer scale. However, CaSiO3 nanophase materials have not been reported. The present work prepared nano-sized amorphous CaSiO3 and h-wollastonite powders by chemical precipitation method and subsequent heat treatment. The two powders were pressed and sintered

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Table 1 Ionic concentration of simulated body fluid and human plasma (in mmol/l) Ion +

Na K+ Mg2+ Ca2+ Cl HCO 3 HPO2 4 2 SO4

SBF

Human plasma

142.0 5.0 1.5 2.5 147.8 4.2 1.0 0.5

142.0 5.0 1.5 2.5 103.8 27.0 1.0 0.5

to form bulk materials. In vitro tests were performed to evaluate their bioactivities. Significant differences of the in vitro bioactivities of the two materials were observed and studied in detail.

2. Experimental procedure 2.1. Preparation of CaSiO3 nanophase materials Theoretically, amorphous CaSiO3 (A-CS) powder in nano-scale were synthesized by precipitation method according to the following chemical reaction: Na2 SiO3 þ CaðNO3 Þ2 ¼ CaSiO3 A þ 2NaNO3 : Ca(NO3)2 4H2O and Na2SiO3 9H2O were used as the starting materials in this reaction. The two reagents in stoichiometric proportion were dissolved in deionized water in two beakers; the concentrations were adjusted to 0.5 mol/l 0.5% (wt.%) polyethylene glycol as the dispersant was added to 200 ml Ca(NO3)2 solution. The Ca(NO3)2 solution was stirred while dropping another 200 ml Na2SiO3 solution into the Ca(NO3)2 solution. After the precipitation was completed, the obtained precipitate was washed three times with de-ionized water, filtered, washed again with ethanol, dispersed in ethanol and dried at 80 8C, finally obtaining the nano-sized CaSiO3 powder. After drying, the amorphous powder was submitted to differential thermal analysis (DTA) and thermogravimetric analysis (TG). 6 mg of powder was heated to 1000 8C at a constant heating rate of 10 8C min1 under air atmosphere. DTA traces were used to determine the temperature at which crystallization of the relevant crystalline phases occurred. On the basis of the DTA results, heat treatment was performed to obtain the crystal phase. The two powders were ground and sieved before compacted. An amount of 1 g of each powder was pressed into disks of 20 mm in diameter and 2 mm in thickness at 50 MP uniaxial pressures and sintered at 600 8C to form bulk materials for in vitro test. The phase compositions of the powders before and after thermal treatment were identified using X-ray diffractometer at 40 Kv and 25 mA. Scans were run from 108 to 708 2h at a speed of 28 min1 and a step of 0.028 using Cu Ka X-rays of

d

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wavelength of 1.5406 2. Observations of the morphologies of two compacts were conducted by field emission scanning electron microscope (JSM6700F). 2.2. In vitro test In order to evaluate the bioactivity of the CaSiO3 materials, the compact disks were put into SBF, proposed by Kokubo et al. [14], at 36.5 8C in sterile polyethylene containers. The SBF solution has a composition and concentration similar to those of the inorganic part of human plasma. The ionic concentrations of SBF solution and human plasma were listed in Table 1. The reagents used and the order of dissolving reagents are NaCl, NaHCO3, KCl, K2HPO4 3H2O, MgCl2 6H2O, 1N–HCl, CaCl2, Na2SO4, NH2(CH2OH)3, otherwise precipitation will be occurred. The soaking times were 6, 12, and 24 h, 3, 7, and 15 days. After being soaked, the disks were rinsed with deionized water and dried at room temperature. The in vitro bioactivities of the two CaSiO3 materials were evaluated by studying the changes in the morphologies of the crystalline phases formed on the surfaces of the disks and the variation of ionic concentrations in SBF solution with the time. XRD and SEM were employed to characterize the formation of new layer on the surfaces of two materials after different soaking time in SBF. The concentrations of Ca, P, and Si in SBF solution were measured by inductively coupled plasma atomic emission spectroscopy (ICP).

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3. Results and discussion 3.1. Characterization of powders Fig. 1 shows the TG/DTA curves of the powder after drying at 80 8C. There are an exothermic peak and several endothermic peaks. The endothermic process appeared at 89 8C was attributed to loss of residue water and ethanol, corresponding to a weight loss of about 22%. The second

Fig. 1. DTA and TG curves of the powder after being dried at 80 8C.

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smooth, dense, and spheric. The different morphologies resulted from the different CaSiO3 phase of the starting powders. Before heat treatment, the powder was amorphous and contained a great deal of water. After being heated to 800 8C, h-wollastonite crystallized and the water was removed from the powder, causing the particles to densify. It is clear from the results described above that the obtained amorphous and crystal materials were composed of particles in nano-scale. Since the nano-sized particles have large surface areas and higher surface energy, the materials exhibited higher reactivity when immerged in SBF. 3.2. Formation of new layer on the surfaces of CaSiO3 compacts Fig. 2. XRD patterns of powder before and after heat treatment.

endothermic process occurred at a temperature range from 150 to 210 8C, which was caused by the evaporation of dispersant, the polyethylene glycol. Third, the remove of crystal water formed a endothermic process at 513 8C. Finally, an exothermic process was detected at 781 8C, due to the crystallization of CaSiO3. These results let us to determine that the optimal heat treatment temperature for the crystallization of CaSiO3 is 800 8C, at which h-wollastonite crystallizes completely, on the other hand, heat treatment at this temperature avoided the grains growing to bigger. The XRD patterns of the two CaSiO3 powders are shown in Fig. 2. It is clear to see that the XRD patterns of as-precipitated CaSiO3 powder showed just flat baseline, while the powder after heat treatment at 800 8C for 2 h showed numerous sharp peaks, which confirmed that the powder was consisted of pure crystal phase h-wollastonite. The two powders were pressed into disks at 50 MP uniaxial pressures and sintered at 600 8C to form bulk materials. Fig. 3a and b present the SEM micrographs of ACS and h-CS materials respectively. A-CS was rough, porous, and composed of elongated particles. The elongated particles had a size of 40 nm in diameter and about 300 nm in length. The h-CS grains with 90 nm in diameter were

Fig. 4 gives the XRD patterns of A-CS (a) and h-CS (b) before and after soaking in SBF solution for various time periods. In the A-CS, crystalline XRD peaks appeared after soaking for 6 h (Fig. 4a) were identified as calcite and aragonite (CaCO3). The FTIR C–O absorption band corresponding to this amorphous CaCO3 was detected at about 1520 cm1 in the spectrum of the A-CS (Fig. 5a). HAP formation was however detected in the FTIR spectrum of the 6 h soaked sample (Fig. 5a) as a composite absorption band at about 1189 cm1, composed of the P–O stretch of HAP and the Si–O vibration of the silica rich phase and at 634 cm1, the P–O bending of HAP. A crystalline peak of HAP at 31.88 2h corresponding to the 211 reflection was clearly observed in the XRD pattern after 1 day soaking. After prolonged soaking the HAP peaks became the main constituent of the XRD patterns as the HAP content increased and completely covered the surfaces of the compacts, but strong peaks of calcite were also observed. It was considered that the peaks of CaCO3 arose from the growth of small CaCO3 nuclei on the initial A-CS material [15], since the precipitation reaction was carried out in aqueous medium. In addition, the FTIR spectra show the characteristic bands of HAP at about 1149, 630, and 586 cm1 and also show the absorption bands at about 1414– 1509 cm1 of carbonate.

Fig. 3. SEM micrographs of A-CS (a) and h-CS (b).

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Fig. 4. XRD patterns of A-CS (a) and h-CS (b) after soaking for various times.

In the h-CS, the 211 peak of HAP started to appear in the XRD pattern (Fig. 4b) after 1 day soaking although the hwollastonite peak at 308 2h was still observed even after prolonged soaking and there were no other crystalline phases. The presence of calcite was not observed in h-CS because the carbonate was completely removed from the hCS during firing. The intensities of the HAP peaks in the hCS increased up to 15 days, indicating that the HAP crystallized constantly on the surfaces. The FTIR spectra of Fig. 5b show a combination of the P–O absorption bands of the HAP and the Si–O absorption bands of the silica-rich phase, which occur in almost the same positions. The C–O absorption bands of the carbonate group are thought to be incorporated into the HAP. These vibrational modes of carbonate are typical of carbonate groups substituting for the phosphate (PO43) or hydroxide (OH) groups in the HAP structure. Fig. 6 displayed the SEM micrographs of the surfaces of A-CS and h-CS compacts after soaking in SBF solution for various time periods. It is obvious that both compacts could

develop a HAP layer on their surfaces after 15 days of soaking, indicating that the two CaSiO3 materials exhibited excellent in vitro bioactivities. In the A-CS, the surface was almost covered with a layer of clusters after 12 h of soaking, whereas only a little particles appeared on the h-CS surface. This finding implied that the A-CS had the higher reactivity with SBF. The high-resolution graphics of two compacts after being soaked in SBF for 12 h and 15 days were shown in Fig. 7. It can be seen from the micrographs that uniformly sized particles like worm were formed on the surfaces of hCS (Fig. 7c,d). The round particles, thought to be CaCO3, were formed on A-CS for 12 h of soaking (Fig. 7a) because CaCO3 was the main crystallization phase at that time. After 15 days of soaking, the particles became irregular (Fig. 7b) due to the co-existence of HAP and CaCO3. The different crystallization behaviors of two compacts could explain the differences in microstructures. Since only HAP crystallized on the surfaces of h-CS, the precipitated particles were simple and regular. While for A-CS, HAP and CaCO3 crystallized together on the surfaces.

Fig. 5. FTIR spectra of A-CS (a) and h-CS (b) after soaking.

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Fig. 6. SEM micrographs of the surfaces of A-CS and h-CS soaked in SBF for various time periods (a), (b), (c): A-CS; (d), (e), (f ): h-CS.

3.3. Concentration changes of the SBF solution Fig. 8 shows changes of concentrations of Ca, P, and Si of SBF solution measured by ICP after soaking for various time periods. At early stages the Ca and Si concentrations in SBF increased rapidly, corresponding to the steep

decrease of P concentration in the first 6 h of soaking. Since the surface area of the soaked samples is 1.5 cm2 and the SBF volume is 20 ml (S/V=0.075), obviously most of the phosphate were absorbed on the surface of compacts and only a little were assumed to form amorphous HAP as shown in the FTIR spectra of A-CS. The A-CS has a

Fig. 7. High resolution graphic of the surfaces of two compacts after soaking 12 h and 15 days (a), (b): A-CS; (c), (d): h-CS.

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P ion concentration 40

600

30

ppm

ppm

Ca ion concentration 800

400 200

20 10

0 0h

6h

12h

24h

3d

7d

0

15d

0h

6h

Soaking time

ppm

50 40 30 20 10 0

24h

3d

7d

15d

Soaking time

β–CS

A–CS

12h

A–CS

β–CS

Si ion concentration

0h

6h

12h

24h

3d

7d

15d

Soaking time A–CS

β–CS

Fig. 8. Changes of Ca, P, and Si concentrations of the SBF solution.

higher reactivity with SBF solution than the h-CS material because of the higher release rate of Ca into the SBF solution. This can be explained by the differences in the microstructure and crystalline phase. A-CS has a smaller particle size than the h-CS, which will produce a larger specific surface areas when immersed into SBF solution and finally promote the ion release. Second, the Ca–O bond of A-CS is not as strong as that in h-CS lattice, since amorphous materials are not as stable as the crystalline materials. Therefore, the ions in A-CS are easier to be released into the SBF solution. After a long period of soaking, the Ca, P, and Si concentration became almost constant, indicating that an apparent equilibrium between the dissolution of the powder surface and the formation of HAP.

4. Conclusions Two forms of CaSiO3 nanophase materials were prepared with different amorphous and crystalline phases using relevant nano-sized powders as precursors produced by chemical precipitation method. A-CS compact was rough, porous and composed of elongated particles, while the h-CS crystallites were smooth, dense and spheric and some crystallites formed agglomerates. The results of in vitro test revealed that only HAP crystallized on h-CS surfaces. A-CS was however covered with CaCO3, which crystallized from the amorphous CaCO3 nuclei present in the starting powder. Uniformly sized particles like a worm were formed on the h-CS. The particles formed on the A-CS were irregular, due to the co-existence of CaCO3 and HAP. The A-CS showed a faster formation of HAP because of the high release rate of Ca. The results suggest that both A-CS and h-CS have good

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