Preparation and in vitro characterization of electrospun 45S5 bioactive glass nanofibers

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CERAMICS INTERNATIONAL

Ceramics International 41 (2015) 417–425 www.elsevier.com/locate/ceramint

Preparation and in vitro characterization of electrospun 45S5 bioactive glass nanofibers Aylin M. Deliormanlın Celal Bayar University, Department of Materials Engineering, Manisa, Turkey Received 4 July 2014; received in revised form 20 August 2014; accepted 23 August 2014 Available online 1 September 2014

Abstract Bioactive glasses are widely used in biomedical applications due to their ability to bond to bone and even to soft tissues. In this study, 45S5 bioactive glass fibers were prepared through sol–gel processing and electrospinning technique. A precursor solution containing poly vinyl alcohol and bioactive glass sol was used to produce fibers. The mixture was electrospun at a voltage of 20 kV by maintaining tip to a collector distance of 8 cm. The fibers with an average diameter of 337 7 81 nm (before calcination) were successfully obtained. Results showed that the crystalline phase of the fibers was largely influenced by the calcination temperature. Hydroxyapatite formation on calcined 45S5 fibers was investigated in simulated body fluid (SBF) using different fiber/SBF (F/S) ratios (0.5, 1, 2 and 10 mg/ml) at 37 1C. When immersed in SBF, conversion to a calcium phosphate material showed a strong dependence on the F/S ratio. At high solid concentration (10 mg/ml), surface of the fibers could not be converted to the HA-like material in SBF after 30 days. At lower solid concentrations (2, 1 and 0.5 mg/ml) an amorphous calcium phosphate layer formation was observed followed by the conversion to hydroxyapatite. & 2014 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

Keywords: Bioactive glass; Electrospinning; Fibers; Tissue engineering applications

1. Introduction Electrospinning is a unique approach using electrostatic forces to produce fine fibers from solutions or melts. This method has emerged a widespread technology to produce nanofibrous structures for tissue engineering applications [1]. These structures have morphologies and fiber diameters in a range comparable with those found in the extracellular matrix of human tissues [1,2]. Electrospun fibers are found to possess features such as high porosity and effective mechanical properties [1–3]. In this process, electric field is subjected to the end of a capillary tube that contains the polymer fluid held by its surface tension. This induces a charge on the surface of the liquid. Mutual charge repulsion causes a force directly opposite to the surface tension [2,4]. The entanglements in the polymer macromolecules provide necessary extensional viscosity for the stabilization of the n

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http://dx.doi.org/10.1016/j.ceramint.2014.08.086 0272-8842/& 2014 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

solution jet. The jet moves toward a ground plate or rotating cylinder which acts as a counter electrode. Then, nanofibers deposit randomly in the form of a thin sheet on this counter electrode [4,5]. One of the attractive properties of this approach is that the obtained nanofiber mats possess high surface area [6]. It has been used successfully to spin a number of synthetic and natural polymers into fibers with diameters varying from 3 nm to 5 mm [2,5,7]. Although electrospinning is used mainly for polymeric systems, it can also be applied to ceramic systems. A number of ceramic fibrous structures have been produced including ferroelectric materials, magnetic materials and biomaterials such as hydroxyapatite, alumina, silica and titania using this approach [5,6,8–11]. Bioactive glasses are promising scaffold materials for bone and soft tissue regeneration [12–14]. The silicate bioactive glass designated 45S5 (Bioglass) has been the most widely used glass for biomedical applications [12]. To date, clinical applications of bioactive glasses are limited to those materials synthesized by melting processes. Silicate based glasses prepared by sol–gel

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processing have also been investigated for tissue engineering applications [15]. Recently, some sodium-containing bioactive glass–ceramic compositions including 45S5 Bioglass was synthesized by the sol–gel method [16–18]. Cacciotti et al. [18] studied the synthesis and thermal behavior of 45S5. Similarly, Pirayesh and Nychka synthesized the 45S5 using the sol–gel method followed by heat treatment to produce semi-crystalline structure and compared the bioactivity against amorphous melt cast 45S5 powder [16]. Additionally, the method for making inorganic bioactive glass nanofibers by combining the sol–gel processing with electrospinning technique has been utilized by different research groups [19–21]. Kim et al. [19] first fabricated bioactive glass nanofibers using the electrospining process. Results showed that fibers possessed high bioactivity and osteogenic potential in vitro. Similarly, Xia et al. [20] studied the effect of electrospinning parameters on the diameter and morphology of bioactive glass bioactive glass nanofibers and their in vitro biomineralization. Although there are some studies in literature on the preparation of elecrospun bioactive glass nanofibers, preparation of sol–gel derived 45S5 bioactive glass as a nanoscale fiber by means of electrospinning technique has not been reported yet. Therefore, the aim of this study was to prepare 45S5 bioactive glass electrospun nanofibers for tissue engineering applications. 2. Material and methods 2.1. Fiber preparation The composition of the 45S5 bioactive glass is (in mol%): 46.1% SiO2, 24.4% Na2O, 26.9% CaO and 2.6% P2O5 [12]. Following chemicals were used as precursors for the sol preparation: tetraethyl orthosilicate (TEOS), triethyl phosphate (TEP), calcium nitrate tetrahydrate (Ca(NO3)2  4H2O), and sodium nitrate (NaNO3) (all from Sigma-Aldrich, USA). For the preparation of the nanofibers, initially 8.375 ml TEOS was added into 0.1 M HNO3 aqueous solution at room temperature. To achieve a clear sol, the molar ratio between water and TEOS was set at 18 [16]. The mixture was allowed to stir for at least 60 min for hydrolysis. Then, each compound (0.725 ml TEP, 3.38 g NaNO3 and 5.0325 g CaNO3) was added (in the sequence) only when the previous solution became clear and was stirred for 1 h. Prior to electrospining aqueous poly (vinyl alcohol) (PVA) ( Mw:88,000–97,000, 88% hydrolyzed, Alfa Aesar) solution (10 wt%) containing 0.5 vol% surfactant (Surfynol SE, Air Products Inc. USA) and 1 vol% ethyl alcohol (Sigma-Aldrich, USA) were added to the glass sol at 1/1/ vol ratio to adjust the viscosity and the surface tension. The mixture was allowed to stir overnight for homogenization at 25 1C prior to electrospinning process. Further, experiments were also performed to study the effect of PVA concentration (4.5–6 vol % in overall solution) on the electrospinning process. Electrospinning was performed using a laboratory scale nanospinner (NE-300, Inovenso, TR). In order to produce the nanofibers, homogeneous, transparent solutions were loaded (10 ml) into a plastic syringe. The solution was injected at a rate of 1 ml/h to a stainless steel nozzle with a diameter of 0.8 mm. A grounded stainless steel cylinder, covered with an aluminum foil served as

the collector. The experiments were carried out by maintaining a distance of 8 cm between the nozzle and the rotating collector. A voltage of 20 kV was applied to the solution and a mat of PVA/ 45S5 sol composite fibers was collected on the aluminum foil. A schematic drawing of the electrospinning set up is shown in Fig. 1. The elecrospun fibers were aged at 60 1C and then dried at 120 1C for 1 day in air atmosphere. Fibers were subsequently heat treated first at 300 1C for 4 h by a heating rate of 1 1C/min followed by a treatment at 600, 650, 680 and 700 1C for 2 h (heating rate 1 1C/min). 2.2. Characterizations The microstructure of the fabricated bioactive glass fibers were examined using SEM (Philips XL-30 S FEG) at an accelerating voltage of 5 kV and a working distance of 10 mm. About 30 randomly selected fibers taken from the SEM micrographs were utilized to obtain the fiber diameter distribution and determine the average fiber diameter. X-ray diffraction, XRD (Philips X'Pert Pro) was used to analyze the presence of any crystalline phase formation in the prepared fibers; XRD was performed using Cu Kα radiation at a scanning rate of 0.011/min in the range of 10–901 2θ. The glass transition and crystallization properties of the fibers were analyzed using a differential thermal analyzer (Perkin Elmer SII 7300) combined with a thermogravimetric analyzer as a function of temperature. Samples were heated to 900 1C in N2 atmosphere at 10 1C/min. In vitro bioactivity of the fibers was investigated in a simulated body fluid (SBF) under static conditions. SBF was prepared in compliance with the protocol of Kokubo et al. [22], by dissolving reagent-grade chemicals of NaCl, NaHCO3, KCl, K2HPO4  3H2O, MgCl2  6H2O, CaCl2 and Na2SO4 (Sigma Aldrich, USA) in deionized water and buffering at a pH of 7.40 with tris (hydroxymethyl) aminomethane ((CH2OH)3CNH2) and 1 M hydrochloric acid (Fisher Scientific Inc., USA) at 37 1C. Different fiber/SBF (F/S) ratios (0.5, 1, 2 and 10 mg/ml) were utilized in the experiments. Each sample (1 g of fiber) was immersed in a polyethylene bottle containing the SBF solution having different

Fig. 1. Schematics view of the electrospining set up.

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Exothermic peaks represent the phase transformation and crystal formation. In Fig. 2a DTA curve the peak at 350 1C may be due to removal of the water because of the condensation of the precursor materials. The peak around 800 1C is due to the crystallization of the 45S5 fibers. XRD diagram of the as-prepared 45S5 nanofiber is demonstrated in Fig. 2b. Accordingly, sodium nitrate (PDF #036-1474) is presented in the sample before heat treatment. After heat treatment at 600 1C, crystalline combeite Na5.27Ca3Si6O18 (PDF# 01-078-1650) formed in the sample (Fig. 3). It is known that sodium nitrate may convert to combeite phase above 600 1C [16]. Additionally, some peaks of clinophosinate Na3CaPSiO7 (PDF# 00-035-0485) were also observed at the same temperature. Starting from 680 1C all the major peaks of Na3CaPSiO7 were detected. At 700 1C, a crystalline sodium calcium silicate phosphate Na3Ca(SiO3)(PO4) (PDF# 01-084-0151) started to form in the structure. Sodium nitrate peak disappeared at 700 1C which means that all the nitrates were removed from the structure at this temperature. These results revealed that the crystalline phase of the fibers was largely influenced by the calcination temperature.

θ ο ΔΔ

Fig. 2. (a) Differential thermal analysis and thermogravimetric analysis curves of non-stabilized 45S5 fibers and (b) XRD pattern of the as-prepared 45S5 nanofibers. NaNO3 (*).

F/S ratios, and kept for up to 30 days without shaking in an incubator at 37 1C. SEM was utilized to analyze the structure of the reacted fibers, using the conditions described previously. Fourier transform infrared spectroscopy (FTIR-ATR, Agilent Cary 660) was also used to characterize HA-like layer formed on the surfaces of the glass. FTIR analysis was performed in the wavenumber range 400–4000 cm  1.

Intensity (a.u)

θ Δο

Δ

θ Δ

Δ Δθ ο Δ

Δ

* Δ

Δ Δ

Δ

Δθ

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ο θ

θ

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Δ

680 °C

Δ

650 °C

Δ

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Δ

* *

Δ

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40 50 60 2 theta (degree)

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Fig. 3. XRD patterns of the electrospun 45S5 naofibers calcined at various temperatures. NaNO3 (*), combeite Na5.27Ca3Si6O18 (Δ), clinophosinate Na3CaPSiO7 (θ), Na3Ca(SiO3)(PO4) (ο).

3. Results and discussions 3.1. Electro-spun Fibers P-O bending

Si-O-2NBO (a)

Absorbance

Through Section 3 unless stated otherwise 45S5 fibers refers to the fibers prepared at (1/1):(45S5 sol/PVA) ratio (total PVA concentration in solution 5 vol%). The DTA/TG curves of the asprepared 45S5 nanofibers is shown in Fig. 2a. Accordingly, weight loss below 100 1C is due to the removal of traces of adsorbed water molecules. The weight loss between 350 and 450 1C is due to the decomposition of PVA in the fibers. The TGA curve shows high amount of weight loss around this range of temperature. The weight loss up to 800 1C may be attributed to the decomposition of nitrates in the structure and conversion to oxides. The total weight loss was about 70% at 800 1C. The DTA curve was found to contain both endothermic and exothermic peaks. The endothermic peaks correspond to the removal of sodium nitrate and other nitrogen containing compositions.

Si-O stretching

400

(b)

600

800

1000

1200

1400

1600

Wavenumber (1/cm) Fig. 4. FTIR spectra of the 45S5 bioactive glass samples (a) fibers calcined at 700 1C and (b) as-prepared fibers before calcination.

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Fig. 4 shows the FTIR spectra of the 45S5 fibers. The peaks ranging from 950 to 1100 cm  1 were attributed to the Si–O stretching. The primary resonances in the spectrum consisted of the vibrational modes of the Si–O–Si bond in the glass network such

as the stretching vibration at 1030 and 1055 [23]. According to the literature, the main absorption bands of amorphous 45S5 bioactive glass are Si–O bending peak at 460 cm  1 and Si–O stretching at 926 and 1024 cm  1 [16]. In the current study, the broad resonance 8

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Fiber diameter (nm) Fig. 5. SEM micrographs of the 45S5 fibers having different PVA concentrations and their fiber diameter distributions. (a) 4.5%, (b) 5.0%, (c) 5.5%, and (d) 6.0%.

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in the range of 925–950 cm  1 corresponded to Si–O–2NBO (nonbridging oxygen) vibrational modes associated with the Ca þ 2 and Na þ 1 ions in the glass network [23]. This implies the occurrence of crystallization. Since combeite has more NBO bonding compared to amorphous glass, this peak confirms that combeite is present in the structure. The peak at 1010 cm  1 refers to stretch Si–O which is attributed to the silicon bond of phosphorous rich phase [16]. This peak appears in the sample after treatment at 700 1C. It is known that the use of polymer solutions with inorganic precursors is an effective way for producing ceramic fibers. Polymers such as poly (vinyl alcohol), poly (ethylene oxide) and poly (vinyl pyrolidone) are the most common polymers used for these applications [2]. The addition of the sol to the polymer lowers the solution viscosity and can affect the fiber morphology during electrospinning [9,11]. Fig. 5 shows the SEM micrographs of the as-prepared 45S5 fibers having different PVA concentrations. Nanofibers deposited randomly in the form of a thin sheet on the counter electrode. At 4.5% and 5% PVA concentrations a bead free, stable fibrous structure was produced. At higher PVA concentrations some overlapping in fibers and difficulties were noticed during electrospinning. Lowest average fiber diameter (28074.6 nm) was obtained at 4.5% PVA concentration. As the PVA concentration was increased from 4.5% to 6%, a slight 350

Fiber diameter (nm)

340 330 320 310 300 290 280 270

4.5

5

5.5

6

PVA Concentration (%) Fig. 6. Graph showing the effect of PVA concentration on the average fiber diameter.

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increase was observed in fiber diameter (33574.6 nm) (Fig. 6). Based on the previous studies, it is known that addition of the precursor sols to the polymer solutions lowers the viscosity of the mixture and thereby leads to a reduction of fiber diameter [5,9,11]. However, in the current study, the difference in fiber diameter depending on the PVA concentration was not so significant presumably due to the narrow range of polymer concentration (between 4 and 6%). To see the effect of PVA content on the fiber diameter, some further experiments should be performed at higher PVA concentrations. However, this may also cause some difficulties at calcination step since removal of organics at high concentrations requires special heat treatment regimes. Fig. 7 shows the SEM micrographs of the 45S5 fibers after heat treatment at 700 1C. Fibers retained their fibrous nature after calcination and grain formation was observed due to crystallization. Average fiber diameter was measured to be 2207 12 nm after calcination at 700 1C for 2 h. Based on the EDS analysis (Fig. 8), some combeite phase was detected in fibers calcined at 700 1C. On the other hand, formation of NaNO3 crystals was observed by EDS analysis in as-prepared fibers before calcination process (Fig. 8b). The bioactivity of the calcined fibers was investigated in vitro in a simulated body fluid (SBF) under static conditions. When immersed in an aqueous phosphate solution, such as the body fluid, bioactive glasses convert to an amorphous calcium phosphate or hydroxyapatite (HA)-like material, which is responsible for their strong bonding with surrounding tissue [12–14,23,24]. Fig. 9 shows the SEM micrographs of the 45S5 fibers after treatment in SBF for 1 day (F/S ratio:1 mg/ml and 0.5 mg/ml). After one day of immersion in SBF, a second phase material presumably a calcium phosphate or crystalline HA formation occurred on the surface of the fibers at both F/S ratios. Similarly, Fig. 10 demonstrates the SEM micrographs of the calcined 45S5 fibers after treatment in SBF at different concentrations for 30 days. Accordingly, at F/S ratio (10 mg/ml) HA formation was not observed on the surface of the fibers after 30 days. SEM and EDS analysis (not shown) revealed the formation of some calcite crystals on the surface of the fibers treated in SBF at that F/S ratio (Fig. 10(a)). As the SBF amount increased (F/S ratio: 2 mg/ml), crystalline HA formation occurred after 30 days of immersion. Similarly, even

10 μm

10 μm

Fig. 7. SEM micrographs of the electrospun 45S5 nanofibers; (a), (b) before calcination and (c) after calcination at 700 1C.

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Fig. 8. EDS spectrum of the 45S5 nanofibers (a) calcined at 700 1C and (b) as-prepared.

at higher SBF amount (F/S ratio: 1 mg/ml) SEM analysis showed again the formation of HA-like material after 30 days. The converted layer on the fibers was composed of plate like particles. This morphology is typical of HA-like material formed by the conversion of silicate based bioactive glasses in an aqueous phosphate solution [23–25]. Jones et al. [26] investigated the reactivity of 45S5 by changing the powder/ solution volume ratio in SBF solution. It was reported that, higher concentration of the material in solution caused larger increase in pH, and this implied that calcium carbonate formed at the expense of HA [26]. Effect of solid/solution ratio on the hydroxyapatite and calcite formation from 70SiO2–30CaO bioactive glasses in simulated body fluid was also studied by Lukito and co-workers [27]. When the samples were soaked in SBF for 1 day, calcite crystalline phases formed and covered up the pre-formed HA phases on the surfaces of the bioactive glasses with S/S ratios of 10, 5, 3.3 and 2.5 mg/ml. FTIR spectroscopy of the SBF treated fibers (Fig. 11a) showed resonances at 1000–1100 cm  1 and at 570 cm  1 corresponding to a calcium phosphate [22,25]. The two P–O bending peaks at 560 and 604 cm  1 are the main peaks for characterizing the HA formation [16,23]. A crystalline Ca–P

layer, as indicated by the divided P–O bending vibration band between 500 and 600 cm  1, formed after 30 days for the sample immersed in SBF at a 1 mg/ml F/S ratio. Additionally, these two peaks were detectable after 7 days of immersion and became more intense after 30 days. A C–O stretching vibration band also appeared between 890 and 800 cm  1 indicating the formation of carbonated calcium phosphate [28]. The resonances at 1390 cm  1 were attributed to C–O in the (CO3)2– group. Results revealed that a crystalline Ca–P layer, as indicated by the divided P–O bending vibration band between 500 and 600 cm  1, formed only after 1 day for the sample immersed in SBF at 0.5 mg/ml F/S ratio (Fig. 11b). Higher intensity in P–O bending peaks in this sample suggests a faster HA formation rate compared to the samples treated in SBF at 1 mg/ml. SEM image of the same type of sample also provided information for the existence of HA like material on the surface. Additionally, the resonance at 800 cm  1 assigned to the tetragonal Si–O–Si group was present in the spectrum for the fibers immersed for 1 day at 0.5 mg/ml F/S ratio revealing the polymerization of silanol groups. Based on the previous studies, crystallized bioactive glass 45S5 has been shown to be less bioactive than its amorphous counter

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10 μm

2 μm

10 μm

2 μm

Fig. 9. SEM micrographs of calcined 45S5 nanofibers after immersion in SBF for 1 day at different F/S ratios; (a), (b) 1 mg/ml; (c), (d) 0.5 mg/ml; magnifications  10,000 and  50,000.

10 μm

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Fig. 10. SEM micrographs of calcined 45S5 nanofibers after immersion in SBF for 30 days at different F/S ratios; (a), (d) 10 mg/ml; (b), (e) 2 mg/ml; and (c), (f) 1 mg/ml.

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4. Conclusions

PO4 Si-O-Si

PO4

Absorbance

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7 days

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Wavenumber (1/cm) 0.35

PO4

Absorbance

0.30

The 45S5 bioactive glass fibers with an average diameter of 337781 nm (when 45S5 sol/PVA ratio was 1/1) were successfully fabricated using electrospinning technique. The use of inorganic precursors of 45S5 glass with aqueous PVA solution was an effective way for producing bioactive glass fibers. Results showed that the crystalline phase formation of the fibers was largely influenced by the calcination temperature. A heat treatment at 700 1C led to the formation of combeite as the main crystalline phase and fiber diameter reduced to 220712 nm after heat treatment. Below 700 1C nitrates could have not removed from the structure. In spite of the crystalline nature, fabricated 45S5 nanofibers showed high bioactivity in SBF. A crystalline Ca–P layer, as indicated by the divided P–O bending vibration band between 500 and 600 cm  1, formed only after 1 day for the sample immersed in SBF at 0.5 mg/ml F/S ratio.

0.25

Acknowledgments

0.20 0.15 0.10

PO4

Si-O-Si

PO4 CO3

0.05

CO3

0.00 600

800

1000

1200

1400

Wavenumber (1/cm) Fig. 11. (a) FTIR spectra of the calcined 45S5 fibers after treatment in SBF (F/S ratio: 2 mg/ml) for different time periods and (b) FTIR spectrum of the calcined 45S5 fibers after treatment in SBF (F/S ratio: 0.5 mg/ml) for 1 day.

parts as measured by time to form HA [29,30]. However, study of Pirayesh et al. [16] revealed that in sol–gel derived and melt cast 45S5 bioactive glass powders P–O bending peaks were detectable after 3 days of immersion and became more intense with increasing immersion time for both powder types. In that study glass concentration in SBF was 2 mg/ml and average median particle size was 11.8 mm and 8 mm for the sol–gel derived and melt cast 45S5 powders, respectively. Their results showed that crystalline gel-derived 45S5 glass showed a similar reaction rate and time to form HA as compared with melt-cast 45S5. Similarly, Liu et al. [23] showed that in melt derived amorphous 45S5 bioactive glass nanofibers (diameter of the fibers ranged from 500 nm to 5 mm) the splitting of the (PO4)3 v4 resonance at 605 and 560 cm  1 were evident after 3 days of immersion in SBF (F/S ratio 2 mg/ml). High resolution TEM also revealed that isolated nanocrystalline regions in the surface region of silicate 45S5 glass fibers that were immersed for 3 days in SBF, as observed from the presence of lattice fringes [23]. On the other hand, the results of the present work showed a conversion of 45S5 glass fibers to crystalline HA after only 1 day of immersion in SBF at 0.5 mg/ml F/S ratio. Therefore, it is possible to conclude that, in spite of their semi crystalline nature, 45S5 fibers fabricated by elecrospinning showed high bioactive response in SBF due to their high surface area.

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