Materials Science and Engineering C 37 (2014) 363–368
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Formation of nanostructured fluorapatite via microwave assisted solution combustion synthesis Maryam Nabiyouni a, Huan Zhou b,c,⁎, Timothy J.F. Luchini d, Sarit B. Bhaduri c,e a
Department of Bioengineering, The University of Toledo, Toledo, OH, USA Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, Jiangsu, China Department of Mechanical, Industrial and Manufacturing Engineering, The University of Toledo, Toledo, OH, USA d Composite Vehicle Research Center, Michigan State University, East Lansing, MI, USA e Division of Dentistry, The University of Toledo, Toledo, OH, USA b c
a r t i c l e
i n f o
Article history: Received 23 September 2013 Received in revised form 24 December 2013 Accepted 5 January 2014 Available online 11 January 2014 Keywords: Fluorapatite Microwave assisted solution combustion synthesis Nanotubes
a b s t r a c t Fluorapatite (FA) has potential applications in dentistry and orthopedics, but its synthesis procedures are time consuming. The goal of the present study is to develop a quick microwave assisted solution combustion synthesis method (MASCS) for the production of FA particles. With this new processing, FA particles were successfully synthesized in minutes. Additionally, unique structures including nanotubes, hexagonal crystals, nanowhiskers, and plate agglomerates were prepared by controlling the solution composition and reaction time. In particular, the as-synthesized FA nanotubes presented a “Y” shape inner channel along the crystal axis. It is supposed that the channel formation is caused by the crystal growth and removal of water soluble salts during processing. The as-synthesized FA nanotubes showed good cytocompatibility, the cells cultured with a higher FA concentration demonstrated greater growth rate. With this new and easily applied MASCS processing application, FA nanoparticles have increased potential in dental and orthopedic applications. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Apatitic mineral phase makes up 95–96 wt.% of dental enamel along with water (3 wt.%), and organic matter (1 wt.%) [1]. Though, calcium phosphate salts like hexagonal hydroxyapatite (Ca10(PO4)6(OH)2, HA) account for the majority of the apatite forms in human enamel, natural teeth always contain some fluoride (F), in the form of fluorapatite (Ca10(PO4)6 F2, FA) or fluor-hydroxyapatite (Ca10(PO4)6(OH,F)2, FHA) [2,3]. Both HA and FA crystals are composed of six PO34 − groups surrounded by ten Ca2+ ions with two OH− or F− ions located along the c axis. The distinguishing features of two apatites are the presence of either ion along the c axis, the position and orientation of the ion with respect to the nearby calcium atoms, and the cell volume [4]. The F− ion has a smaller size than OH−, 1.28 Å compared to 1.37 Å. As a result, F− ions can pack more closely resulting in smaller cell volume [5]. Synthetic FAs are harder and have slightly better thermal stability than the original apatite phase of human teeth [6,7]. Great stiffness, ⁎ Corresponding author at: Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, Jiangsu, China. Tel.: +86 1 419 530 8223, fax: +86 1 419 530 8206. E-mail address: [email protected] (H. Zhou). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2014.01.018
significant resistance to acid damage, and suitable biocompatibility of FAs make them potential candidates in restorative dentistry. The application of FA could come in different forms like crowns, inlays, dentin simulators, coatings and cements [7–10]. In addition to dentistry, it is also worth noting that FA is important in orthopedics to promote bone regeneration [11–15]. In reviewing the literature, it is reported that FA can be synthesized via different techniques including the sol–gel process [16–18], wetchemical processing [19,20], solid-state reaction [21,22], and the hydrothermal process [9,19,23]. Details of all synthesis techniques and resulting features of the as-synthesized FA are summarized in Table 1. However, the common limitation of all reported technologies is that they are time consuming. Our group has made great strides in synthesizing calcium phosphate whiskers via a rapid microwave-assisted solution combustion synthesis (MASCS) process [24–26]. In the process, aqueous solutions containing NaNO3, HNO3, Ca(NO3)2.4H2O, KH2PO4 and different additives were irradiated in a household microwave oven for 5 min. The solidified substances were then simply stirred in water at room temperature to obtain the whiskers of the desired CaP phase including HA, tricalcium phosphate (Ca3(PO4)2, TCP), biphasic calcium phosphate (BCP), and chlorapatite (Ca10(PO4)6Cl2, CA) [24–26]. In the present work, MASCS was used to efficiently produce FA nanoparticles with unique features.
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Table 1 Summary of FA synthesis technologies. Method
Reactants
Time
Structure
Ref.
Sol–gel Sol–gel Sol–gel Sol–gel Wet-chemical Wet-chemical Solid-state Hydrothermal Hydrothermal Hydrothermal
(C2H5O)3P, NH4F, Ca(NO3)2.4H2O, ethanol (NH4)2HPO4, NH4F, Ca(NO3)2.4H2O, H2O CaCl2.6H2O, Na2HPO4.12H2O, KF, H2O, octane, surfactant CaCl2.6H2O, Na2HPO4.12H2O, KF, H2O, diethyleneglycol Ca10(PO4)6(OH)2, NaF, H2O, HNO3, NH3.H2O Ca(NO3)2, KH2PO4, KF, acrylamide, polyethylene oxide, polyacrylamide, N,N,N′N′-tetramethylenediamine CaF2, Ca3(PO4)2 CaHPO4, Na2HPO4, NaH2PO4, KF Ca10(PO4)6(OH)2, NaF, H2O, HNO3, EDTA-Na4.4H2O Ca(NO3)2, NaF, NaH2PO4, poly(acrylic acid)
N8 days 21 h 52 h N28 h N5 days N4 days N2 h N84 h N6 h 1h
100 nm spherical particles N/A 20–180 nm particles 100–300 nm particles 20–50 nm nanorods/nanowires 300–1000 nm hexagonal crystals plate-like irregular nano particles N/A 1–5 μm nanorods/nanowires 152 (24) × 38 (6) nm nanospindles
[16] [17] [18] [18] [19] [20] [21,22] [9] [19] [23]
Some of these features include FA nanoparticles with high aspect ratio and crystallinity and the nanoparticles can be synthesized using a simple process, not previously offered by other reported techniques. 2. Experiments 2.1. Synthesis Reagents for whisker synthesis such as NaNO3, NaF, Ca(NO3)2.4H2O, and KH2PO4 were purchased from Fisher Scientific, USA. In addition, CaF2 was purchased from Sigma–Aldrich, USA. Different experimental conditions, such as compositions and durations, were studied in order to find the best composition and optimum condition to produce FA whiskers (Table 2). CaF2 and NaF were both used as sources for F− in reaction. The difference is NaF is water-soluble and CaF2 is insoluble. The reaction solution was prepared by adding chemicals one by one to 10 mL de-ionized water in a 30 mL beaker with 200 rpm stirring. The final mixture was a clear solution with salts in suspension, although excess CaF2 particles were visible. After 10 min of stirring, beakers containing the mixture were placed in a household microwave oven (700 W, Kenmore, USA) and were covered with a 250 mL inverted Pyrex beaker. The assembly was then heated at 100% power for 3, 5, or 7 min separately. Resulting substances were left to air-dry for 15 min. The 30 mL beakers were placed in a 250 mL Pyrex beaker containing 100 mL of water to cool and solidify completely. Air-dried substances were magnetically stirred at 400 rpm in 500 mL of de-ionized water to suspend formed particles and dissolve water-soluble salts. Finally, the solution was washed with approximately 2 L of de-ionized water and filtered using a filter paper (Whatman Grade 5, Fisher Scientific, USA). The filtrate was then placed in an 80 °C oven overnight for further characterization and evaluation. 2.2. Characterization The as-synthesized particles were first visualized by scanning electron microscope (SEM, S4800, Hitachi, USA) to determine which composition resulted in the desired one-dimensional structure. Inner structures of FA particles were investigated using transmission electron microscopy (TEM, HD-2300, Hitachi, USA) with a voltage of 200 kV. The phase compositions of particles were characterized by X-ray diffraction (XRD, Ultima III, Rigaku, USA) with monochromated Cu Kα radiation, setting the operating conditions at a voltage of 40 kV and a current of
44 mA. Particles were examined at 2θ angles from 10° to 60° at a scanning speed of 1° per minute. To identify the phases present in the sample, the XRD pattern of the sample was compared with every calculated pattern in a Powder Diffraction File (PDF) database from the International Center for Diffraction Data (ICDD). Using the search-match capabilities of the JADE (MDI, USA) XRD software and the ICDD-PDF database, all phases present in the samples were identified. Fourier transform infrared spectroscopy (FTIR, UMA-600 Microscope, Varian Excalibur Series, USA) was applied for chemical analysis of FA particles. The transmittance of samples was recorded with 256 scans with resolution of 4 cm−1 between 4000 and 400 cm−1. Zeta potential measurements were performed using a Zeta Potential/Particle Sizer (Nicomp 380ZLS, Particle Sizing Systems, USA) to measure the surface charge of samples. The measurements were performed using the multi-angle square cell setup and the photon source was set on photomultiplier tubes only. The zeta potentials were determined by measuring the electrophoretic movements of charged particles under the applied electric field. In order to examine stability of FA particles in physiological conditions, the particles were incubated in simulated body fluid (SBF) at 37 °C environmental conditions. Composition of the as-prepared SBF is shown in Table 3 [27,28]. The solution was replenished every other day. After 7 days, the incubated FA particles were dried and characterized using SEM coupled with energy dispersive spectrometry (EDS). 2.3. Preosteoblast culture Preosteoblast cells (MC3T3-E1, CRL-2593™, ATCC, USA) were used to study the indirect effect of FA on preosteoblast proliferation and differentiation. Centrifuge tubes containing 25, 50, 100, and 150 mg/mL FA in alpha minimum essential medium (α-MEM, Thermo Scientific HyClone) were incubated overnight prior to cell seeding. Cells were initially grown at 37 °C and 5% CO2 in α-MEM, augmented by 10% fetal bovine serum (FBS, Thermo Scientific HyClone, USA). The culture medium was replenished every other day until the cells reached 90% confluency. To study cell proliferation, MC3T3-E1 cells were seeded to wells (Flacon™ 12 wells cell culture plates, BD Biosciences, USA) at a density of 10,000 cells/well. Immediately after seeding cells, 50 μL of FA containing α-MEM with respective FA concentration was added to the wells together with 500 μL cell culture medium. Cell density was measured after 24 h, and 7 days using CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega, USA). For statistical analysis, all
Table 2 Reaction solution compositions and microwave irradiation time for FA synthesis. Experimental conditions
H2O (mL)
NaNO3 (g)
CaF2 (g)
NaF (g)
Ca(NO3)2.4H2O (g)
KH2PO4 (g)
HNO3 (mL)
MW time (min)
FA-1 FA-2 FA-3 FA-4 FA-5
10 10 10 10 10
5.00 5.00 5.00 5.00 5.00
1.00 – 1.00 1.00 0.70
– 0.53 – – –
1.00 1.00 1.00 1.00 1.00
0.384 0.384 0.384 0.384 0.384
1.20 1.20 1.20 1.20 1.20
5 5 7 3 3
M. Nabiyouni et al. / Materials Science and Engineering C 37 (2014) 363–368 Table 3 Composition of 1 L SBF. Order
Reagent
SBF
1 2 3 4 5 6 7 8 9 10
NaCl NaHCO3 KCl Na2HPO4 MgCl2.6H2O 1 M HCl CaCl2.2H2O Na2SO4 Tris-Base 1 M HCl
6.5456 g 2.2682 g 0.3727 g 0.1419 g 0.3045 g 10 mL 0.3881 g 0.072 g 6.063 g 33.3 mL
experiments were performed three or more times, and one-way ANOVA analysis was applied for data analysis. 3. Results and discussion 3.1. SEM & TEM The different morphological features of as-synthesized FA nanoparticles are shown in Fig. 1. As illustrated in (Fig. 1a&b), FA-1 experimental conditions resulted in the formation of hexagonal FA nanotubes. In contrast, the replacement of CaF2 using NaF (FA-2) inhibited the formation of FA nanotubes (Fig. 1c). In this case, larger FA hexagonal crystals with over 2 μm length were observed. The effects of heating times are reflected in FA-3 and FA-4. As shown in (Fig. 1d), the increase in heating time from 5 min to 7 min results in hexagonal FA nanotubes with reduced inner channels while reduction of heating time to 3 min forms FA nanowhiskers with dense cores (Fig. 1e). Finally, to confirm the necessity of CaF2 presence on formation and morphology of FA nanotubes, lower amounts of CaF2 were added. As presented in Fig. 1f, reducing the CaF2 content resulted into non-uniform plate-like particles. These particles lack both the hexagonal geometry as well as the tube-like structure. Therefore, FA particles in the form of nanotubes, hexagonal crystals, nanowhiskers, and plate-like crystals can be synthesized using MASCS process with controlled reaction solution compositions and operating
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parameters. The as-prepared FA crystals in the present work are quite different from those known in the literature (Table 1). Thus, MASCS can be a powerful tool to produce FA materials with a variety of unique features [29,30]. TEM imaging of the FA-1 showed the presence of inner channels in the crystal structure (Fig. 2). The channel presents a “Y” shape along the crystal axis. It is also noted that the channels in FA-1 nanotubes were not continuous or uniform along the axis. The particles become highly crystallized as they are formed at high temperatures. The formation of FA nanotubes is an important achievement of this work. There are several signatures of this structure: 1) the channel can provide a site for target material loading and sustained release, especially considering the poor dissolution rate of FA in saline; 2) the release rate of loaded materials can be adjusted with respect to the environmental changes since the dissolution rate of FA can be greatly improved once local pH decreases [31] due to bacterial activity; 3) the unique “Y” shaped structure along the crystal axis with a sealed oneend reduces the possibility of content (e.g. drug) leakage at the time of delivery; 4) the positively charged nanotube surface makes them ideal for DNA/plasmid loading and enhances their specific cellular uptake [32]; and 5) if applied as a filler in composites, the interaction between the tube structure and the matrix can work as a “locking” mechanism to improve the bonding between FA and matrix. In our previous work, we argued that the growth of apatite in MASCS probably follows a “dissolution–crystallization–whisker-growth” process [24–26]. The molten NaNO3 and the high energy generated during microwave ignited combustion dissolve all the initially formed apatite precipitates to form a uniform liquid phase under continuing microwave irradiation. Solid apatite phases are formed through the nucleation and growth processes during the cooling of the molten salt bath. During cooling, rapid crystallization occurs along the preferred growth axes of the apatite phase. In the case of CA synthesis, we observed that under the same experimental conditions, chloride to hydroxyl substitution can lead to an expansion in the a-axis and a decrease in the c-axis, enhancing the crystal growth of the as-synthesized particles to elongated structure. Therefore, in this work, we suggest that the presence of F− ions in the system determines the hexagonal structure of formed apatite phase. Indeed, the previous papers suggested that hexagonal structure was first formed in the nascent crystals during the biomimetic FA formation [33]. Since the substitution of OH− by F−
Fig. 1. SEM images of synthesized FA particles using different parameters: a) and (b) FA-1; c) FA-2; d) FA-3; e) FA-4; and (f) FA-5.
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Fig. 2. TEM images of synthesized FA-1 nanotubes. Red arrows specify the presence of nanochannels. The structure of “Y” shape is composed of a wide open mouth and a narrowed channel along the crystal axis.
can pack the atoms in a cell, it is not surprising that the aspect ratio (of length and base) of the as-formed FA-2 (NaF as F− source) is much smaller than the previously synthesized CA whiskers (NaCl as Cl− source) [25]. In thermal properties, CaF2 has a much higher melting point (1418 ˚C) than Pyrex glass (softening point of 820 ˚C), which acted as the container for the reactants. Therefore, it is impossible to melt CaF2 to induce the dissolution–crystallization–whisker-growth process since glass would have already melted before the melting temperature of CaF2 is reached. A reasonable explanation is that CaF2 can be incorporated in the structure of FA when it is exposed to molten salts and microwave irradiation. Chander et al. [34] reported that CaF2 can be converted to FA in phosphate solutions at various temperatures ranging between 25 and 75 ˚C in the pH range of 6.5 to 8.5. Hence, we suggest that in the present method, CaF2 provides the F− ion source and acts as a template to form elongated hexagonal FA via diffusion through molten calcium and phosphate sources. This process is different from the conventional solution mediated role of CaF2 in production of FA. Additionally, it is noted that excessive amounts of CaF2 is necessary to assist the formation of an elongated hexagonal structure instead of an irregular plate-like structure (FA-5). The cooling process consists of two steps: slow cooling under microwave irradiation, and fast cooling after microwave shutdown. As addressed above, the high temperature of the molten system is mainly due to the combustion. Though microwave irradiation can provide thermal energy to the reactants, this process is less energetic than the combustion process. Therefore, after combustion the temperature of the molten system can decrease to a certain level in which the absorbed
heat from microwave irradiation equals to the heat loss following the air exposure. The temperature drops significantly after microwave heating due to the heat loss. Based on the results of conditions FA-1, FA-3 and FA-4, it is believed that the “Y” shape channel formation and size differences are related to microwave irradiation time and temperature. Under longer microwave irradiation, the size of FA crystals is expected to increase via reaction with molten calcium and phosphate, thus, resulting in the size differences achieved under FA-1, FA-3 and FA-4. The ionic diffusion derived from the whisker core and the newly formed outer shell is expected to assist the FA growth. Consequently, the inner core of formed FA becomes hollow and molten salts fuse into the channel. Later, the entire system temperature drops to room temperature during cooling. This could cause two phenomena: 1) the ionic flow rate gradually decreases to zero, stopping the crystal growth of FA; 2) the fused molten salts form apatite inside the hollow structure to seal the cavity. At this point, phase separation occurs between apatite and NaNO3 salts. The FA-1 condition provided longer microwave incubation, resulting in more apatite formation in the channels, and caused the creation of different channel sizes between FA-1 and FA-3. Additionally, newly formed apatite has better epitaxial relationship to the already existing apatite instead of NaNO3. Therefore, NaNO3 is slowly pushed out from the shrinking FA channel by the solidified apatite. In fast cooling, the molten salt movement is stopped, and the residual NaNO3 is removed by H2O leaving the “Y” channel along the FA crystals. This may also result in the slight decrease in surface charge seen in FA-1 when compared to pure FA in literature [35]. The schematic of the process is summarized in Fig. 3.
Fig. 3. Schematic of the formation kinetics of FA-1.
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3.2. XRD & FTIR The XRD pattern of the FA-1 is shown in Fig. 4a. The pattern matches the FA (JCPDS PDF#97-024-0629) data indicating that the assynthesized particles were FA. Of note, the resulting FA is highly crystallized because of the significant temperature increase in combustion. In Fig. 4b, the FTIR spectrum of FA-1 is shown. Broad PO−3 peaks 4 were observed at 968, 1025, 1050, and 1086 cm− 1, and sharp peaks were seen at 580, and 805 cm−1. This is in agreement with the previously reported FA FTIR spectrum [36]. Additionally, a CO2− band was 3 observed in the 1400 cm− 1 range of sample FA-1. During cooling, CO2 in air can react with FA causing impurities. This phenomenon is expected to slightly improve the dissolution rate of as-synthesized FA as compared to the pure FA [37].
3.3. Zeta potential The zeta potential analysis shows the surface charge of FA-1 to be 2.37 ± 0.45 mV. The value is slightly lower than the reported FA surface charge, which is higher than 4 mV [35].
Fig. 5. SEM images of FA-1 after 7 days in SBF and related EDS result.
3.5. Preosteoblast culture 3.4. In vitro SBF soaking After 7 days SBF incubation, the hexagonal geometry and inner channels of FA-1 particles were preserved and no apatite coating was observed to FA-1 surface (Fig. 5). Related EDS scans showed FA-1 particles containing Na and F elements even after SBF incubation, where Na comes from the doping of Na+ during FA formation. This phenomenon indicates the high stability of FA-1 in physiological solution. The growth of apatite on bioactive surfaces is attributed to the surface nucleation sites, such as sodium titanate of NaOH etched titanium [38], Si–OH sites of silica [39], and active Ca2+ sites of calcium phosphates [24]. FA is believed to require much more time to create enough active Ca2 + sites for apatite deposition as compared to HA, therefore FA is more resistant to chemical attack.
Fig. 4. XRD (a) and FTIR (b) results of as-synthesized FA-1.
Results of preosteoblast culture are presented in Fig. 6. Day 1 results show no statistical cell number variations in all samples (p N 0.05). However, the 7 day cell count demonstrates the significant effect of FA-1 on preosteoblast cell proliferation. Although 25 mg/mL FA-1 extract did not significantly change the preosteoblast proliferation rate, groups (100, and 150 mg/mL) showed much higher cell growth than the FA-free control group (p b 0.05). The average cell number increased relative to the FA-1 concentration in medium. Biocompatibility is one of the crucial criteria for dental substitutes. Cytotoxicity of the examined biomaterials can be tested via direct or indirect contact. The direct contact assay can indicate toxicity for different reasons such as the release of toxic leachable species, and lack of cell adhesion. Meanwhile, the indirect method only indicates the cytotoxicity of the studied biomaterials [40]. Therefore, in order to test the capability of FA-1 particles, the preosteoblasts were cultured with different concentrations of FA-1 extracts using the indirect method. The in vitro cell study using extracts from different FA-1 concentrations showed that released ions from FA-1 could promote the proliferation of preosteoblast cells. A similar study on fluoride doped hydroxyapatite (F-HA) coated Mg–Zn alloys demonstrated the ability of F-HA in releasing F− ions and consequently enhancing the viability of human bone marrow stromal cells [41]. This cell culture result indicates that FA synthesized via MASCS is cytocompatible, which is in agreement with other work in literature [8,11,42].
Fig. 6. Effects of the extracts of FA-1 with different concentrations on MC3T3 preosteoblast cells after culture for 24 h and 7 days.
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4. Conclusion In summary, unique structures of FA including nanotubes, hexagonal crystals, nanowhiskers, and plate-like crystals can be synthesized by changing the processing parameters of the MASCS method. The crystal features are controlled by reaction solution compositions and operation parameters. The as-synthesized FA nanotubes were shown to be stable in physiological conditions and were cytocompatible. The in vitro study showed that cell proliferation rates were parallel to increasing the concentration of FA extract. It is believed that these FA particles have potential for numerous dental and biomedical applications. Acknowledgment This work was partially supported by NSF CMMI 0753479. References [1] J.L. Cuy, A.B. Mann, K.J. Livi, M.F. Teaford, T.P. Weihs, Arch. Oral Biol. 47 (2002) 281–291. [2] X. Chatzistavrou, S. Papagerakis, P.X. Ma, P. Papagerakis, Int. J. Dent. 2012 (2012) 5. [3] E.I.F. Pearce, G.E. Coote, M.J. Larsen, J. Dent. Res. 74 (1995) 1775–1782. [4] P. Rulis, L. Ouyang, W.Y. Ching, Phys. Rev. B 70 (2004) 155104. [5] J.M. Hughes, M. Cameron, K.D. Crowley, Am. Mineral. 74 (1989) 870–876. [6] E. Menéndez-Proupin, S. Cervantes-Rodríguez, R. Osorio-Pulgar, M. Franco-Cisterna, H. Camacho-Montes, M.E. Fuentes, J. Mech. Behav. Biomed. Mater. 4 (2011) 1011–1020. [7] Y. Chen, X. Miao, Biomaterials 26 (2005) 1205–1210. [8] J. Wei, J. Wang, W. Shan, X. Liu, J. Ma, C. Liu, J. Fang, S. Wei, J. Mater. Sci.: Mater. Med. 22 (2011) 1607–1614. [9] M.J. Sladek, Pat. WO1994023944, (1994). [10] H.W. Denissen, H.M. Nieuport, W. Kalk, H.G. Schaeken, A. Hooff, Biocer. Hum. Body, 1992, pp. 130–140. [11] B.-H. Yoon, H.-W. Kim, S.-H. Lee, C.-J. Bae, Y.-H. Koh, Y.-M. Kong, H.-E. Kim, Biomaterials 26 (2005) 2957–2963. [12] W.J.A. Dhert, P. Thomsen, C.P.A.T. Klein, K. Groot, P.M. Rozing, L.E. Ericson, J. Mater. Sci.: Mater. Med. 5 (1994) 59–66. [13] L.C. Chow, S. Takagi, Pat. WO2012003438A1, (2012). [14] S. Takagi, S. Frukhtbeyn, L.C. Chow, J. Res. Inst. Stand. Tech. 2010 (2012) 267–276.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Formation of nanostructured fluorapatite via microwave assisted solution combustion synthesis Maryam Nabiyouni a, Huan Zhou b,c,⁎, Timothy J.F. Luchini d, Sarit B. Bhaduri c,e a
Department of Bioengineering, The University of Toledo, Toledo, OH, USA Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, Jiangsu, China Department of Mechanical, Industrial and Manufacturing Engineering, The University of Toledo, Toledo, OH, USA d Composite Vehicle Research Center, Michigan State University, East Lansing, MI, USA e Division of Dentistry, The University of Toledo, Toledo, OH, USA b c
a r t i c l e
i n f o
Article history: Received 23 September 2013 Received in revised form 24 December 2013 Accepted 5 January 2014 Available online 11 January 2014 Keywords: Fluorapatite Microwave assisted solution combustion synthesis Nanotubes
a b s t r a c t Fluorapatite (FA) has potential applications in dentistry and orthopedics, but its synthesis procedures are time consuming. The goal of the present study is to develop a quick microwave assisted solution combustion synthesis method (MASCS) for the production of FA particles. With this new processing, FA particles were successfully synthesized in minutes. Additionally, unique structures including nanotubes, hexagonal crystals, nanowhiskers, and plate agglomerates were prepared by controlling the solution composition and reaction time. In particular, the as-synthesized FA nanotubes presented a “Y” shape inner channel along the crystal axis. It is supposed that the channel formation is caused by the crystal growth and removal of water soluble salts during processing. The as-synthesized FA nanotubes showed good cytocompatibility, the cells cultured with a higher FA concentration demonstrated greater growth rate. With this new and easily applied MASCS processing application, FA nanoparticles have increased potential in dental and orthopedic applications. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Apatitic mineral phase makes up 95–96 wt.% of dental enamel along with water (3 wt.%), and organic matter (1 wt.%) [1]. Though, calcium phosphate salts like hexagonal hydroxyapatite (Ca10(PO4)6(OH)2, HA) account for the majority of the apatite forms in human enamel, natural teeth always contain some fluoride (F), in the form of fluorapatite (Ca10(PO4)6 F2, FA) or fluor-hydroxyapatite (Ca10(PO4)6(OH,F)2, FHA) [2,3]. Both HA and FA crystals are composed of six PO34 − groups surrounded by ten Ca2+ ions with two OH− or F− ions located along the c axis. The distinguishing features of two apatites are the presence of either ion along the c axis, the position and orientation of the ion with respect to the nearby calcium atoms, and the cell volume [4]. The F− ion has a smaller size than OH−, 1.28 Å compared to 1.37 Å. As a result, F− ions can pack more closely resulting in smaller cell volume [5]. Synthetic FAs are harder and have slightly better thermal stability than the original apatite phase of human teeth [6,7]. Great stiffness, ⁎ Corresponding author at: Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, Jiangsu, China. Tel.: +86 1 419 530 8223, fax: +86 1 419 530 8206. E-mail address: [email protected] (H. Zhou). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2014.01.018
significant resistance to acid damage, and suitable biocompatibility of FAs make them potential candidates in restorative dentistry. The application of FA could come in different forms like crowns, inlays, dentin simulators, coatings and cements [7–10]. In addition to dentistry, it is also worth noting that FA is important in orthopedics to promote bone regeneration [11–15]. In reviewing the literature, it is reported that FA can be synthesized via different techniques including the sol–gel process [16–18], wetchemical processing [19,20], solid-state reaction [21,22], and the hydrothermal process [9,19,23]. Details of all synthesis techniques and resulting features of the as-synthesized FA are summarized in Table 1. However, the common limitation of all reported technologies is that they are time consuming. Our group has made great strides in synthesizing calcium phosphate whiskers via a rapid microwave-assisted solution combustion synthesis (MASCS) process [24–26]. In the process, aqueous solutions containing NaNO3, HNO3, Ca(NO3)2.4H2O, KH2PO4 and different additives were irradiated in a household microwave oven for 5 min. The solidified substances were then simply stirred in water at room temperature to obtain the whiskers of the desired CaP phase including HA, tricalcium phosphate (Ca3(PO4)2, TCP), biphasic calcium phosphate (BCP), and chlorapatite (Ca10(PO4)6Cl2, CA) [24–26]. In the present work, MASCS was used to efficiently produce FA nanoparticles with unique features.
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Table 1 Summary of FA synthesis technologies. Method
Reactants
Time
Structure
Ref.
Sol–gel Sol–gel Sol–gel Sol–gel Wet-chemical Wet-chemical Solid-state Hydrothermal Hydrothermal Hydrothermal
(C2H5O)3P, NH4F, Ca(NO3)2.4H2O, ethanol (NH4)2HPO4, NH4F, Ca(NO3)2.4H2O, H2O CaCl2.6H2O, Na2HPO4.12H2O, KF, H2O, octane, surfactant CaCl2.6H2O, Na2HPO4.12H2O, KF, H2O, diethyleneglycol Ca10(PO4)6(OH)2, NaF, H2O, HNO3, NH3.H2O Ca(NO3)2, KH2PO4, KF, acrylamide, polyethylene oxide, polyacrylamide, N,N,N′N′-tetramethylenediamine CaF2, Ca3(PO4)2 CaHPO4, Na2HPO4, NaH2PO4, KF Ca10(PO4)6(OH)2, NaF, H2O, HNO3, EDTA-Na4.4H2O Ca(NO3)2, NaF, NaH2PO4, poly(acrylic acid)
N8 days 21 h 52 h N28 h N5 days N4 days N2 h N84 h N6 h 1h
100 nm spherical particles N/A 20–180 nm particles 100–300 nm particles 20–50 nm nanorods/nanowires 300–1000 nm hexagonal crystals plate-like irregular nano particles N/A 1–5 μm nanorods/nanowires 152 (24) × 38 (6) nm nanospindles
[16] [17] [18] [18] [19] [20] [21,22] [9] [19] [23]
Some of these features include FA nanoparticles with high aspect ratio and crystallinity and the nanoparticles can be synthesized using a simple process, not previously offered by other reported techniques. 2. Experiments 2.1. Synthesis Reagents for whisker synthesis such as NaNO3, NaF, Ca(NO3)2.4H2O, and KH2PO4 were purchased from Fisher Scientific, USA. In addition, CaF2 was purchased from Sigma–Aldrich, USA. Different experimental conditions, such as compositions and durations, were studied in order to find the best composition and optimum condition to produce FA whiskers (Table 2). CaF2 and NaF were both used as sources for F− in reaction. The difference is NaF is water-soluble and CaF2 is insoluble. The reaction solution was prepared by adding chemicals one by one to 10 mL de-ionized water in a 30 mL beaker with 200 rpm stirring. The final mixture was a clear solution with salts in suspension, although excess CaF2 particles were visible. After 10 min of stirring, beakers containing the mixture were placed in a household microwave oven (700 W, Kenmore, USA) and were covered with a 250 mL inverted Pyrex beaker. The assembly was then heated at 100% power for 3, 5, or 7 min separately. Resulting substances were left to air-dry for 15 min. The 30 mL beakers were placed in a 250 mL Pyrex beaker containing 100 mL of water to cool and solidify completely. Air-dried substances were magnetically stirred at 400 rpm in 500 mL of de-ionized water to suspend formed particles and dissolve water-soluble salts. Finally, the solution was washed with approximately 2 L of de-ionized water and filtered using a filter paper (Whatman Grade 5, Fisher Scientific, USA). The filtrate was then placed in an 80 °C oven overnight for further characterization and evaluation. 2.2. Characterization The as-synthesized particles were first visualized by scanning electron microscope (SEM, S4800, Hitachi, USA) to determine which composition resulted in the desired one-dimensional structure. Inner structures of FA particles were investigated using transmission electron microscopy (TEM, HD-2300, Hitachi, USA) with a voltage of 200 kV. The phase compositions of particles were characterized by X-ray diffraction (XRD, Ultima III, Rigaku, USA) with monochromated Cu Kα radiation, setting the operating conditions at a voltage of 40 kV and a current of
44 mA. Particles were examined at 2θ angles from 10° to 60° at a scanning speed of 1° per minute. To identify the phases present in the sample, the XRD pattern of the sample was compared with every calculated pattern in a Powder Diffraction File (PDF) database from the International Center for Diffraction Data (ICDD). Using the search-match capabilities of the JADE (MDI, USA) XRD software and the ICDD-PDF database, all phases present in the samples were identified. Fourier transform infrared spectroscopy (FTIR, UMA-600 Microscope, Varian Excalibur Series, USA) was applied for chemical analysis of FA particles. The transmittance of samples was recorded with 256 scans with resolution of 4 cm−1 between 4000 and 400 cm−1. Zeta potential measurements were performed using a Zeta Potential/Particle Sizer (Nicomp 380ZLS, Particle Sizing Systems, USA) to measure the surface charge of samples. The measurements were performed using the multi-angle square cell setup and the photon source was set on photomultiplier tubes only. The zeta potentials were determined by measuring the electrophoretic movements of charged particles under the applied electric field. In order to examine stability of FA particles in physiological conditions, the particles were incubated in simulated body fluid (SBF) at 37 °C environmental conditions. Composition of the as-prepared SBF is shown in Table 3 [27,28]. The solution was replenished every other day. After 7 days, the incubated FA particles were dried and characterized using SEM coupled with energy dispersive spectrometry (EDS). 2.3. Preosteoblast culture Preosteoblast cells (MC3T3-E1, CRL-2593™, ATCC, USA) were used to study the indirect effect of FA on preosteoblast proliferation and differentiation. Centrifuge tubes containing 25, 50, 100, and 150 mg/mL FA in alpha minimum essential medium (α-MEM, Thermo Scientific HyClone) were incubated overnight prior to cell seeding. Cells were initially grown at 37 °C and 5% CO2 in α-MEM, augmented by 10% fetal bovine serum (FBS, Thermo Scientific HyClone, USA). The culture medium was replenished every other day until the cells reached 90% confluency. To study cell proliferation, MC3T3-E1 cells were seeded to wells (Flacon™ 12 wells cell culture plates, BD Biosciences, USA) at a density of 10,000 cells/well. Immediately after seeding cells, 50 μL of FA containing α-MEM with respective FA concentration was added to the wells together with 500 μL cell culture medium. Cell density was measured after 24 h, and 7 days using CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega, USA). For statistical analysis, all
Table 2 Reaction solution compositions and microwave irradiation time for FA synthesis. Experimental conditions
H2O (mL)
NaNO3 (g)
CaF2 (g)
NaF (g)
Ca(NO3)2.4H2O (g)
KH2PO4 (g)
HNO3 (mL)
MW time (min)
FA-1 FA-2 FA-3 FA-4 FA-5
10 10 10 10 10
5.00 5.00 5.00 5.00 5.00
1.00 – 1.00 1.00 0.70
– 0.53 – – –
1.00 1.00 1.00 1.00 1.00
0.384 0.384 0.384 0.384 0.384
1.20 1.20 1.20 1.20 1.20
5 5 7 3 3
M. Nabiyouni et al. / Materials Science and Engineering C 37 (2014) 363–368 Table 3 Composition of 1 L SBF. Order
Reagent
SBF
1 2 3 4 5 6 7 8 9 10
NaCl NaHCO3 KCl Na2HPO4 MgCl2.6H2O 1 M HCl CaCl2.2H2O Na2SO4 Tris-Base 1 M HCl
6.5456 g 2.2682 g 0.3727 g 0.1419 g 0.3045 g 10 mL 0.3881 g 0.072 g 6.063 g 33.3 mL
experiments were performed three or more times, and one-way ANOVA analysis was applied for data analysis. 3. Results and discussion 3.1. SEM & TEM The different morphological features of as-synthesized FA nanoparticles are shown in Fig. 1. As illustrated in (Fig. 1a&b), FA-1 experimental conditions resulted in the formation of hexagonal FA nanotubes. In contrast, the replacement of CaF2 using NaF (FA-2) inhibited the formation of FA nanotubes (Fig. 1c). In this case, larger FA hexagonal crystals with over 2 μm length were observed. The effects of heating times are reflected in FA-3 and FA-4. As shown in (Fig. 1d), the increase in heating time from 5 min to 7 min results in hexagonal FA nanotubes with reduced inner channels while reduction of heating time to 3 min forms FA nanowhiskers with dense cores (Fig. 1e). Finally, to confirm the necessity of CaF2 presence on formation and morphology of FA nanotubes, lower amounts of CaF2 were added. As presented in Fig. 1f, reducing the CaF2 content resulted into non-uniform plate-like particles. These particles lack both the hexagonal geometry as well as the tube-like structure. Therefore, FA particles in the form of nanotubes, hexagonal crystals, nanowhiskers, and plate-like crystals can be synthesized using MASCS process with controlled reaction solution compositions and operating
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parameters. The as-prepared FA crystals in the present work are quite different from those known in the literature (Table 1). Thus, MASCS can be a powerful tool to produce FA materials with a variety of unique features [29,30]. TEM imaging of the FA-1 showed the presence of inner channels in the crystal structure (Fig. 2). The channel presents a “Y” shape along the crystal axis. It is also noted that the channels in FA-1 nanotubes were not continuous or uniform along the axis. The particles become highly crystallized as they are formed at high temperatures. The formation of FA nanotubes is an important achievement of this work. There are several signatures of this structure: 1) the channel can provide a site for target material loading and sustained release, especially considering the poor dissolution rate of FA in saline; 2) the release rate of loaded materials can be adjusted with respect to the environmental changes since the dissolution rate of FA can be greatly improved once local pH decreases [31] due to bacterial activity; 3) the unique “Y” shaped structure along the crystal axis with a sealed oneend reduces the possibility of content (e.g. drug) leakage at the time of delivery; 4) the positively charged nanotube surface makes them ideal for DNA/plasmid loading and enhances their specific cellular uptake [32]; and 5) if applied as a filler in composites, the interaction between the tube structure and the matrix can work as a “locking” mechanism to improve the bonding between FA and matrix. In our previous work, we argued that the growth of apatite in MASCS probably follows a “dissolution–crystallization–whisker-growth” process [24–26]. The molten NaNO3 and the high energy generated during microwave ignited combustion dissolve all the initially formed apatite precipitates to form a uniform liquid phase under continuing microwave irradiation. Solid apatite phases are formed through the nucleation and growth processes during the cooling of the molten salt bath. During cooling, rapid crystallization occurs along the preferred growth axes of the apatite phase. In the case of CA synthesis, we observed that under the same experimental conditions, chloride to hydroxyl substitution can lead to an expansion in the a-axis and a decrease in the c-axis, enhancing the crystal growth of the as-synthesized particles to elongated structure. Therefore, in this work, we suggest that the presence of F− ions in the system determines the hexagonal structure of formed apatite phase. Indeed, the previous papers suggested that hexagonal structure was first formed in the nascent crystals during the biomimetic FA formation [33]. Since the substitution of OH− by F−
Fig. 1. SEM images of synthesized FA particles using different parameters: a) and (b) FA-1; c) FA-2; d) FA-3; e) FA-4; and (f) FA-5.
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Fig. 2. TEM images of synthesized FA-1 nanotubes. Red arrows specify the presence of nanochannels. The structure of “Y” shape is composed of a wide open mouth and a narrowed channel along the crystal axis.
can pack the atoms in a cell, it is not surprising that the aspect ratio (of length and base) of the as-formed FA-2 (NaF as F− source) is much smaller than the previously synthesized CA whiskers (NaCl as Cl− source) [25]. In thermal properties, CaF2 has a much higher melting point (1418 ˚C) than Pyrex glass (softening point of 820 ˚C), which acted as the container for the reactants. Therefore, it is impossible to melt CaF2 to induce the dissolution–crystallization–whisker-growth process since glass would have already melted before the melting temperature of CaF2 is reached. A reasonable explanation is that CaF2 can be incorporated in the structure of FA when it is exposed to molten salts and microwave irradiation. Chander et al. [34] reported that CaF2 can be converted to FA in phosphate solutions at various temperatures ranging between 25 and 75 ˚C in the pH range of 6.5 to 8.5. Hence, we suggest that in the present method, CaF2 provides the F− ion source and acts as a template to form elongated hexagonal FA via diffusion through molten calcium and phosphate sources. This process is different from the conventional solution mediated role of CaF2 in production of FA. Additionally, it is noted that excessive amounts of CaF2 is necessary to assist the formation of an elongated hexagonal structure instead of an irregular plate-like structure (FA-5). The cooling process consists of two steps: slow cooling under microwave irradiation, and fast cooling after microwave shutdown. As addressed above, the high temperature of the molten system is mainly due to the combustion. Though microwave irradiation can provide thermal energy to the reactants, this process is less energetic than the combustion process. Therefore, after combustion the temperature of the molten system can decrease to a certain level in which the absorbed
heat from microwave irradiation equals to the heat loss following the air exposure. The temperature drops significantly after microwave heating due to the heat loss. Based on the results of conditions FA-1, FA-3 and FA-4, it is believed that the “Y” shape channel formation and size differences are related to microwave irradiation time and temperature. Under longer microwave irradiation, the size of FA crystals is expected to increase via reaction with molten calcium and phosphate, thus, resulting in the size differences achieved under FA-1, FA-3 and FA-4. The ionic diffusion derived from the whisker core and the newly formed outer shell is expected to assist the FA growth. Consequently, the inner core of formed FA becomes hollow and molten salts fuse into the channel. Later, the entire system temperature drops to room temperature during cooling. This could cause two phenomena: 1) the ionic flow rate gradually decreases to zero, stopping the crystal growth of FA; 2) the fused molten salts form apatite inside the hollow structure to seal the cavity. At this point, phase separation occurs between apatite and NaNO3 salts. The FA-1 condition provided longer microwave incubation, resulting in more apatite formation in the channels, and caused the creation of different channel sizes between FA-1 and FA-3. Additionally, newly formed apatite has better epitaxial relationship to the already existing apatite instead of NaNO3. Therefore, NaNO3 is slowly pushed out from the shrinking FA channel by the solidified apatite. In fast cooling, the molten salt movement is stopped, and the residual NaNO3 is removed by H2O leaving the “Y” channel along the FA crystals. This may also result in the slight decrease in surface charge seen in FA-1 when compared to pure FA in literature [35]. The schematic of the process is summarized in Fig. 3.
Fig. 3. Schematic of the formation kinetics of FA-1.
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3.2. XRD & FTIR The XRD pattern of the FA-1 is shown in Fig. 4a. The pattern matches the FA (JCPDS PDF#97-024-0629) data indicating that the assynthesized particles were FA. Of note, the resulting FA is highly crystallized because of the significant temperature increase in combustion. In Fig. 4b, the FTIR spectrum of FA-1 is shown. Broad PO−3 peaks 4 were observed at 968, 1025, 1050, and 1086 cm− 1, and sharp peaks were seen at 580, and 805 cm−1. This is in agreement with the previously reported FA FTIR spectrum [36]. Additionally, a CO2− band was 3 observed in the 1400 cm− 1 range of sample FA-1. During cooling, CO2 in air can react with FA causing impurities. This phenomenon is expected to slightly improve the dissolution rate of as-synthesized FA as compared to the pure FA [37].
3.3. Zeta potential The zeta potential analysis shows the surface charge of FA-1 to be 2.37 ± 0.45 mV. The value is slightly lower than the reported FA surface charge, which is higher than 4 mV [35].
Fig. 5. SEM images of FA-1 after 7 days in SBF and related EDS result.
3.5. Preosteoblast culture 3.4. In vitro SBF soaking After 7 days SBF incubation, the hexagonal geometry and inner channels of FA-1 particles were preserved and no apatite coating was observed to FA-1 surface (Fig. 5). Related EDS scans showed FA-1 particles containing Na and F elements even after SBF incubation, where Na comes from the doping of Na+ during FA formation. This phenomenon indicates the high stability of FA-1 in physiological solution. The growth of apatite on bioactive surfaces is attributed to the surface nucleation sites, such as sodium titanate of NaOH etched titanium [38], Si–OH sites of silica [39], and active Ca2+ sites of calcium phosphates [24]. FA is believed to require much more time to create enough active Ca2 + sites for apatite deposition as compared to HA, therefore FA is more resistant to chemical attack.
Fig. 4. XRD (a) and FTIR (b) results of as-synthesized FA-1.
Results of preosteoblast culture are presented in Fig. 6. Day 1 results show no statistical cell number variations in all samples (p N 0.05). However, the 7 day cell count demonstrates the significant effect of FA-1 on preosteoblast cell proliferation. Although 25 mg/mL FA-1 extract did not significantly change the preosteoblast proliferation rate, groups (100, and 150 mg/mL) showed much higher cell growth than the FA-free control group (p b 0.05). The average cell number increased relative to the FA-1 concentration in medium. Biocompatibility is one of the crucial criteria for dental substitutes. Cytotoxicity of the examined biomaterials can be tested via direct or indirect contact. The direct contact assay can indicate toxicity for different reasons such as the release of toxic leachable species, and lack of cell adhesion. Meanwhile, the indirect method only indicates the cytotoxicity of the studied biomaterials [40]. Therefore, in order to test the capability of FA-1 particles, the preosteoblasts were cultured with different concentrations of FA-1 extracts using the indirect method. The in vitro cell study using extracts from different FA-1 concentrations showed that released ions from FA-1 could promote the proliferation of preosteoblast cells. A similar study on fluoride doped hydroxyapatite (F-HA) coated Mg–Zn alloys demonstrated the ability of F-HA in releasing F− ions and consequently enhancing the viability of human bone marrow stromal cells [41]. This cell culture result indicates that FA synthesized via MASCS is cytocompatible, which is in agreement with other work in literature [8,11,42].
Fig. 6. Effects of the extracts of FA-1 with different concentrations on MC3T3 preosteoblast cells after culture for 24 h and 7 days.
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4. Conclusion In summary, unique structures of FA including nanotubes, hexagonal crystals, nanowhiskers, and plate-like crystals can be synthesized by changing the processing parameters of the MASCS method. The crystal features are controlled by reaction solution compositions and operation parameters. The as-synthesized FA nanotubes were shown to be stable in physiological conditions and were cytocompatible. The in vitro study showed that cell proliferation rates were parallel to increasing the concentration of FA extract. It is believed that these FA particles have potential for numerous dental and biomedical applications. Acknowledgment This work was partially supported by NSF CMMI 0753479. References [1] J.L. Cuy, A.B. Mann, K.J. Livi, M.F. Teaford, T.P. Weihs, Arch. Oral Biol. 47 (2002) 281–291. [2] X. Chatzistavrou, S. Papagerakis, P.X. Ma, P. Papagerakis, Int. J. Dent. 2012 (2012) 5. [3] E.I.F. Pearce, G.E. Coote, M.J. Larsen, J. Dent. Res. 74 (1995) 1775–1782. [4] P. Rulis, L. Ouyang, W.Y. Ching, Phys. Rev. B 70 (2004) 155104. [5] J.M. Hughes, M. Cameron, K.D. Crowley, Am. Mineral. 74 (1989) 870–876. [6] E. Menéndez-Proupin, S. Cervantes-Rodríguez, R. Osorio-Pulgar, M. Franco-Cisterna, H. Camacho-Montes, M.E. Fuentes, J. Mech. Behav. Biomed. Mater. 4 (2011) 1011–1020. [7] Y. Chen, X. Miao, Biomaterials 26 (2005) 1205–1210. [8] J. Wei, J. Wang, W. Shan, X. Liu, J. Ma, C. Liu, J. Fang, S. Wei, J. Mater. Sci.: Mater. Med. 22 (2011) 1607–1614. [9] M.J. Sladek, Pat. WO1994023944, (1994). [10] H.W. Denissen, H.M. Nieuport, W. Kalk, H.G. Schaeken, A. Hooff, Biocer. Hum. Body, 1992, pp. 130–140. [11] B.-H. Yoon, H.-W. Kim, S.-H. Lee, C.-J. Bae, Y.-H. Koh, Y.-M. Kong, H.-E. Kim, Biomaterials 26 (2005) 2957–2963. [12] W.J.A. Dhert, P. Thomsen, C.P.A.T. Klein, K. Groot, P.M. Rozing, L.E. Ericson, J. Mater. Sci.: Mater. Med. 5 (1994) 59–66. [13] L.C. Chow, S. Takagi, Pat. WO2012003438A1, (2012). [14] S. Takagi, S. Frukhtbeyn, L.C. Chow, J. Res. Inst. Stand. Tech. 2010 (2012) 267–276.
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