Materials Science and Engineering C 53 (2015) 262–271
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Preparation, in vitro mineralization and osteoblast cell response of electrospun 13–93 bioactive glass nanofibers Aylin M. Deliormanlı Celal Bayar University, Department of Materials Engineering, Manisa, Turkey
a r t i c l e
i n f o
Available online 22 April 2015 Keywords: Bioactive glass Electrospinning Fibers Tissue engineering applications
a b s t r a c t In this study, silicate based 13–93 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 10 cm. The amorphous glass fibers with an average diameter of 464 ± 95 nm were successfully obtained after calcination at 625 °C. Hydroxyapatite formation on calcined 13–93 fibers was investigated in simulated body fluid (SBF) using two different fiber concentrations (0.5 and 1 mg/ml) at 37 °C. When immersed in SBF, conversion to a calcium phosphate material showed a strong dependence on the fiber concentration. At 1 mg/ml, the surface of the fibers converted to the hydroxyapatite-like material in SBF only after 30 days. At lower solid concentrations (0.5 mg/ml), an amorphous calcium phosphate layer formation was observed followed by the conversion to hydroxyapatite phase after 7 days of immersion. The XTT (2,3-Bis-(2-Methoxy-4-Nitro-5Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay was conducted to evaluate the osteoblast cell response to the bioactive glass fibers. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Fibrous scaffolds obtained from biomaterials are very important in biomedical engineering as the biomaterials mimic the main components of tissues. Biomimetic architectures should include a tailored structural hierarchy, a certain fiber orientation, and a certain pore size so that large cells may be able to migrate, adhere and spread over them [1]. These structures have morphologies and fiber diameters in a range comparable with those found in the extracellular matrix of human tissues [2,3]. They have high surface area, porosity and effective mechanical properties [2–4]. They have been used to engineer various soft connective tissues such as the skin, ligament, muscle, and tendon, as well as bone tissue regeneration [5]. In recent years, electrospinning has gained widespread interest as a potential tissue-engineering scaffolding technique [2,5]. In electrospinning process, electric field is subjected to the end of a capillary tube that contains the solution. This induces a charge on the surface of the liquid [5,6]. The solution undergoes extensional flow and deposits on to a target by the application of an external electrostatic field. If the intensity of the electric field produces enough stress in the droplet to overcome surface tension a solution jet can be created moving towards the ground collector. But before reaching the collector the solvent partially evaporates and the solution jet is subjected to extensional strain leading to formation of nanofibers. Non-woven, aligned, layered and
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http://dx.doi.org/10.1016/j.msec.2015.04.037 0928-4931/© 2015 Elsevier B.V. All rights reserved.
coaxial nanofibers with a high surface area can be produced by controlling the electrospinning parameters [4–8]. It has been used successfully to spin a number of synthetic and natural polymers such as PLLA [9], PCL [9–12], silk [13], chitosan [14], collagen [11] and gelatine [15] into fibers with diameters varying from 3 nm to 5 μm [16]. It can also be applied to glass and ceramic systems [17–19]. Bioactive glasses can be utilized for bone and soft tissue regeneration [20–23]. The silicate bioactive glass designated as 45S5 (Bioglass) has been the most widely used glass for biomedical applications [20]. Another silicate bioactive glass designated as 13–93, with the composition (wt.%): 53 SiO2, 6 Na2O, 12 K2O, 5 MgO, 20 CaO, and 4 P2O5, has been receiving interest for biomedical applications [22]. 45S5 glass is approved for in vivo use by the U.S. Food and Drug Administration and products of 13-93 glass have been approved for in vivo use in Europe [23]. In vitro cell culture experiments showed no marked difference in the proliferation and differentiated function of osteoblastic MC3T3-E1 or MLO-A5 cells between dense disks of 45S5 and 13–93 glass [22,23]. However, 13–93 glass converts to a hydroxyapatite-like material more slowly than 45S5 glass [23]. To date, clinical applications of these bioactive glasses are limited to those materials synthesized by melting processes. A sol–gel process, involving the foaming of a sol with the aid of a surfactant, has been used to prepare porous scaffolds of a few bioactive glasses and bioactive glass powders [23–25]. Recently, Pirayesh and Nychka synthesized the 45S5 glass using the sol–gel method followed by heat treatment to produce semi-crystalline structure [24]. Similarly, sol–gel processing combined with electrospinning technique has been utilized by different research groups to produce bioactive glass fibers [26–29].
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Table 1 Surface tension and the viscosity of the solutions prepared in the study.
13–93 sol 13–93 sol/10 wt.% PVA mixture (1/1) 13–93 sol/PVA mixture including 1 vol.% ethanol 13–93 sol/PVA mixture including 1 vol.% ethanol and Surfynol SE 13–93 sol/PVA mixture including 1% ethanol in the presence of F-68 (1 vol.%)
Surface tension (mN/m)
Viscocity (mPa·s) @ 3 rpm
30.67 ± 0.28 37.25 ± 0.45 34.45 ± 0.31
2.5 55.2 50.7
27.37 ± 0.25
50.5
25.21 ± 0.17
48.1
2
Heat flow (W/g)
1 0 -1 -2 -3 200
400
600
800
Temperature (C) Fig. 3. Differential thermal analysis curve of the as-prepared 13–93 fibers.
Fig. 1. Flowchart of the solution preparation procedure for electrospinning of 13–93 glass.
Because of their high surface area, bioactive glass nanofibers degrade rapidly and convert to HA. As in the case of sol–gel-derived bioactive glasses, the bioactivity of these glass nanofibers is maintained over a larger SiO2 compositional range when compared to melt-derived glasses. Kim et al. [26] fabricated bioactive glass nanofiber by electrospinning of bioactive glass sol-poly(vinyl-butyral) mixture followed by heat treatment to remove the organic component. They also showed in vitro attachment and differentiation of bone marrowderived stem cells on the bioactive nanofibrous scaffold and compared it with bioactive glass films of the same composition. Allo et al. [28] studied the synthesis and electrospinning of ε-polycaprolactone-bioactive glass (composed of silica, calcium oxide and phosphate) hybrid biomaterials via a sol–gel process. 45S5 bioactive glass–ceramic fibers were synthesized previously through electrospinning method by the author and in vitro bioactivity of the fibers was investigated in simulated body fluid. Results revealed that 45S5 bioactive glass fibers can be fabricated successfully by electrospinning method however crystallization occurs during calcination at 700 °C [29]. In the current study, preparation of 13–93 bioactive glass as a nanoscale fiber by means of electrospinning technique was reported. The effects of the solution parameters on the morphology of electrospun glass nanofibres were studied to produce uniform, bead free fibers with a narrow diameter distribution. In vitro bioactivity of the fibers in SBF was elucidated as a function of immersion time and fiber concentration. Osteoblast cell response of electrospun fibers was investigated using the XTT method. 2. Experimental studies 2.1. Solution preparation
Fig. 2. Schematic view of the electrospinning set up. A similar (not the same) schematic was utilized in the previous paper of the author (Ref. 29, Ceram Int. Vol 41, 2015, 417425 Figure 1).
The composition of the 13–93 bioactive glass is (in mol%): 54.6% SiO2, 22.1% CaO, 7.9% K2O, 7.7% MgO, 6.0% Na2O, and 1.7% P2O5 [22].
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(b) Intensity
Absorbance
(a)
400
600
800 1000 1200 1400 1600 1800 2000
10
20
30
40
50
60
70
80
2 theta (degree)
Wave number (1/cm)
Fig. 4. (a) Fourier transform infrared spectroscopy (FTIR) spectra; (b) XRD diagram of as-prepared 13–93 fibers (◊ denotes sodium nitrate).
* * *
Intensity
725 oC
700 oC 680 oC 625 oC
20
40
60
80
2 theta (degree) Fig. 5. XRD diagram of the 13–93 fibers calcined at various temperatures (* denotes HA).
The method utilized in solution preparation was modified from the previous study of the author [29]. Precursors utilized for the sol preparation were as follows: tetraethyl orthosilicate (TEOS), triethyl phosphate (TEP), calcium nitrate tetrahydrate (Ca(NO3)2·4H2O, potassium nitrate
(a)
(b)
KNO3, magnesium nitrate hexahydrate (Mg(NO3)2·6H2O), and sodium nitrate NaNO3 (all from Sigma-Aldrich, USA). For the preparation of the nanofibers, initially 39.676 ml of TEOS was added into 0.1 M HNO3 aqueous solution at room temperature. The molar ratio of H2O/TEOS was maintained at 18 [24]. The mixture was allowed to stir for at least 60 min for hydrolysis. Then, each compound (1.90 ml TEP, 3.340 g NaNO3, 16.538 g Ca(NO3)2·4H2O, 6.317 g Mg(NO3)2·6H2O and 5.230 g KNO3) was added successively only when the previous solution became clear and was stirred for 1 h. Prior to electrospinning, aqueous poly (vinyl alcohol) (PVA) (Mw: 88,000–97,000, 88% hydrolyzed, Alfa Aesar) solution (10 vol.%) (13–93/PVA: 1/1 ratio), 1 vol.% ethyl alcohol (absolute, Sigma-Aldrich, USA) and 0.5 vol.% Surfynol SE (Air Products, USA) were added to the sol to adjust the viscosity and the surface tension. The mixture was allowed to stir overnight for homogenization at 25 °C prior to electrospinning process. Additionally, the effect of a non-ionic surfactant on the fiber diameter and morphology was determined. For this purpose, solutions were prepared in the presence of Pluronic F-68 (Sigma Aldrich, USA) at two different concentrations (0.25 vol.% and 1 vol.% F-68) using the procedure described above. Pluronic F-68 is a block copolymer with a molecular weight of 8350 g/mol. It contains two terminal polyethylene oxide and one central polypropylene oxide groups. The number of ethylene oxide units in the structure is reported to be 151 by the manufacturer company.
(c)
Fig. 6. Optical images of the 13–93 bioactive glass nonwoven mats (a) before and (b) and (c) after calcination at 625 °C.
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(d)
40 µm
(b)
20 µm
(e)
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10 µm
8
12
680 ±142 nm
(c)
10
464 ±95 nm
(f)
6
Count
Count
8
4
6 4
2 2 0
0 400
500
600
700
800
900
1000
Fiber diameter (nm)
300
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Fiber diameter (nm)
Fig. 7. SEM micrographs of the 13–93 fibers and their fiber diameter distributions (a), (b) and (c) before heat treatment (d), (e) and (f) after heat treatment at 625 °C.
Fig. 1 summarizes the solution preparation procedure utilized in the study. Ultra-pure water with resistivity of 18.2 MΩ- cm was used for solution preparation through the experiments.
1 day in air atmosphere. Fibers were heat treated at 250 °C for 4 h by a heating rate of 1 °C/min followed by a treatment at 625 °C for 4 h (heating rate 1 °C/min) to remove the nitrates and organic materials in the structure.
2.2. Electrospinning 2.3. Characterizations In order to produce the 13–93 bioactive glass nanofibers, a laboratory scale nanospinner device (NE-300, Inovenso, TR) was utilized. The device comprises an illuminator, an exhaust, a high precision pump, a drum stroke and a rotating drum. Schematic view of the set up is given in Fig. 2. For electrospinning, a homogeneous, transparent 13– 93/PVA solution containing ethanol and surfactant was loaded 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 aluminium foil served as the collector. The solution was electrospun under an applied DC voltage of 20 kV using a distance of 10 cm between the nozzle and the rotating collector. The electrospun fibers were first aged at 60 °C and then dried at 120 °C for
Surface tensions of the electrospinning solutions were measured using a tensiometer (Attension, Sigma 702) by the Du Nouy ring method. The measurements were performed at 25 °C and the platinum ring was flamed until it glowed red before each measurement to avoid organic contamination. The Huh and Mason model was used in correction calculations made in the device. For each sample three different measurements were done and the results were averaged. Before each measurement the device was tested using deionized water at room temperature. The viscosity of the prepared solutions was measured at 25 °C by a rotational viscometer (Brookfield, DV-II+) using a small sample UL adaptor.
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(a)
(b)
10 µm
Fiber diameter (nm)
1000
10 µm
(c)
800 600 400 200 0 0,0
0,2
0,4
0,6
0,8
1,0
1,2
F-68 concentration (vol%) Fig. 8. SEM micrographs of the as-prepared 13–93 bioactive glass fibers in the presence of F-68 (a) 0.25 vol.%, (b) 1 vol.%, and (c) the graph showing the effect of F-68 on the average fiber diameter.
The microstructure of the fabricated bioactive glass fibers was examined using scanning electron microscopy, SEM (Philips XL-30S FEG) at 5 kV. Samples were gold coated to achieve conductivity and lessen charging prior to analysis. About 40 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 technique, XRD (Philips X'Pert Pro) was used to analyze the formation of crystalline phases in the prepared fibers. The diffractometer was operated using Cu Kα radiation with a step size of 0.01°/min and at a 2θ range of 10–90°. As-prepared fibers were also analyzed using a differential thermal analyzer (Perkin Elmer SII 7300) as a function of temperature. Samples were heated to 900 °C in N2 atmosphere at 10 °C/min for this purpose. 2.4. In vitro bioactivity test Simulated body fluid (SBF) was used to evaluate the in vitro bioactivity of the fibers. SBF was prepared in compliance with the protocol of Kokubo et al. [30]. For this purpose required amount of NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, CaCl2 and Na2SO4 (all reagent grade, from Sigma Aldrich, USA) was dissolved in ultra-pure water and buffered at a pH of 7.40 with tris (hydroxymethyl)aminomethane ((CH2OH)3CNH2) and 1 M hydrochloric acid (Fisher Scientific Inc., USA) at 37 °C. Two different fiber/SBF (F/S) ratios (0.5 and 1 mg/ml) were utilized in the experiments. Each sample (1 g of fiber) was immersed in a polyethylene bottle containing the SBF solution, and kept for up to 30 days without shaking in an incubator at 37 °C. At the end of immersion periods, fibers were removed from the bottle, washed
with deionized water to prevent further reaction and dried in an oven at 60 °C for 24 h. SEM was utilized to analyze the structure of the reacted fibers. Fourier transform infrared spectroscopy (FTIR-ATR, Agilent Cary 660) was also used to characterize HA forming ability of the glass fibers. 2.5. Cell culture studies Fifty milligrams of 13–93 glass fibers was placed inside a cylindrical ceramic (alumina) mold. The molds holding the randomly oriented glass fibers were placed into an oven preheated to 650 °C, and then heated for 60 min. After heating, the molds were removed from the oven and cooled to room temperature. The disk-shaped scaffolds 4 ± 0.1 mm in diameter and 2 ± 0.2 mm in thickness were prepared and stored in a desiccator. Same type of scaffolds was also prepared using amorphous 13–93 glass powders (d50 = 2.1 μm) synthesized by the melt-cast method and these scaffolds designated as the control group. The in vitro cytotoxicity of the 13–93 fiber scaffolds was determined by XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium5-Carboxanilide) assay. Cell culture experiments were performed using the mouse bone/calvaria preosteoblastic MC3T3-E1 (Subclone 4) cells (ATCC CRL-2593). Prior to experiments, scaffolds were rinsed 3 times with 70% ethanol and dry heat sterilized at 350 °C overnight. The cells were cultured in a modified minimum essential medium (aMEM; GIBCO, Invitrogen Corporation, Grand Island, N.Y.), supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO, Invitrogen Corporation, Grand Island, N.Y.), 100 U/ml penicillin and 100 U/ml streptomycin. The cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Tested scaffolds were placed into each
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(b)
2 µm
10 µm
(c)
(d)
10 µm
(e)
2 µm
(f)
2 µm
10 µm
(g)
(h)
10 µm
2 µm
Fig. 9. SEM micrographs of the 13–93 fibers immersed in SBF (1 mg/ml) for (a) and (b) 1 day; (c) and (d) 5 days; (e) and (f) 7 days and (g) and (h) 30 days.
well of 96-well plates (NUNC; Thermo Fisher Scientific) before cell seeding. Scaffolds were seeded with cells by adding an MC3T3-E1 cell suspension onto the scaffolds (5 · 104 cells in 100 μl of medium per
well). The cell suspension was fully absorbed, thereby allowing the cells to be distributed within the scaffolds. The cell-seeded scaffolds were incubated for 4 h to allow the cells to adhere to the scaffolds.
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(a)
(c)
2 µm
10 µm
(d)
(b)
2 µm
10 µm
Fig. 10. SEM micrographs of the 13–93 fibers immersed in SBF (0.5 mg/ml) for (a) and (c) 1 day; (b) and (d) 7 days.
Samples were cultured for 48 h and then yellow XTT solution was added. The absorbance was monitored at 450 nm using an Elisa Plate Reader. XTT is a colorless or slightly yellow compound that when reduced becomes brightly orange. This color change is accomplished by the breakup of positively-charged quaternary tetrazole ring. The formazan product of XTT reduction is soluble and can be used in realtime assays. Cell Proliferation Kit II (XTT) (Roche) was used in the experiments according to the manufacturer's protocol. Three replicate samples were tested for each condition. For morphological observation of cultured cells after incubation for 48 h, each scaffold was removed, rinsed three times with PBS, and the cells were fixed with 2.5% glutaraldehyde solution (Sigma Aldrich, USA). After an overnight soak in PBS at 4 °C, the scaffolds were washed three times with PBS (10 min at each) and dehydrated through a graded series of ethanol (50, 60, 70, 80, 90 and 100%) for 15 min at each concentration. After drying for 48 h at room temperature, the samples were sputter-coated with gold and observed in an SEM using the conditions described previously. The morphology of cells at culture time was also determined by optical microscopy. 2.6. Statistical analysis Three samples in each group were used for cell culture experiments. The data are presented as the mean standard deviation. Statistical analysis was carried out using one-way analysis of variance (ANOVA) and Tukey's post hoc test, with the level of significance set at p b 0.05. 3. Results and discussion 3.1. Solution properties Surface tension and viscosity are crucial factors in electrospinning and they are functions of solvent compositions of the solution. By reducing the surface tension of the solution, beaded fibers can be converted into smooth fibers [31]. On the other hand, it has been proven that
continuous and smooth fibers cannot be obtained in very low viscosity, whereas very high viscosity results in the hard ejection of jets from the solution [31–33]. Therefore, it is necessary to adjust properly these parameters to achieve a successful electrospinning operation. The surface tension and viscosity values of the as-prepared solutions are listed in Table 1. Accordingly, the surface tension of 13–93 sol alone was measured to be 30.67 mN/m. In the presence of PVA, the surface tension increased to 37.25 mN/m. Results also revealed that fibers cannot be fabricated from pure 13–93 glass solution, because Si–O network of glass was not sufficient to create intermolecular interactions. Therefore, it was essential to add a polymeric material with long chains into the glass such as PVA to enhance the formation of fibers. To reduce the surface tension and increase the volatility, 1% of ethanol was added to the aforementioned solution. By this way, a decrease was observed in the surface tension of the solution to 34.45 mN/m. Introduction of a surfactant to this system led to a further decrease in the surface tension. Results also revealed that viscosity of the electrospinning solution was 50.5 mPa·s at 3 rpm and a decrease in viscosity to 10 mPa·s was observed as the shear rate increased to 50 rpm (results not shown). Therefore it is possible to conclude that the solution has a shear thinning flow behavior. 3.2. Bioactive glass fibers The DTA curve of the as-prepared 13–93 nanofibers is shown in Fig. 3. The 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. Exothermic peaks represent the phase transformation and crystal formation. In Fig. 3, the peak below 100 °C is due to the removal of traces of adsorbed water molecules and the peak at 250–300 °C is may be due to removal of the water because of the condensation of the precursor materials. The peak in the range of 350–400 °C can be attributed to the decomposition of PVA in the fibers. The peak between 700-800 °C is due to the crystallization of the 13–93 fibers.
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P-O bending
Absorbance
30 d
7d
1d
(a) 600
800
1000
1200
Wavenumber (1/cm)
Absorbance
P-O bending
7d
1d
(b) 600
800
1000
1200
Wavenumber (1/cm) Fig. 11. FTIR spectra of the 13–93 fibers treated in SBF for different times (a) F/S ratio: 1 mg/ml; (b) F/S ratio: 0.5 mg/ml.
Fig. 4-a shows the FTIR spectra of the as-prepared 1393 fibers. The peaks ranging from 920 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 cm−1 [25]. According to the literature, the main absorption bands of amorphous silicate based glass are Si– O bending peak at 460 cm−1 and Si–O stretching at 926 and 1024 cm−1 [24]. XRD diagram of the as-prepared 13–93 nanofibers was demonstrated in Fig. 4-b. Accordingly, sodium nitrate (PDF# 036-1474) was presented in the sample before heat treatment. Sodium nitrate peak disappeared in samples calcined at 625 °C, which means that all the nitrates were removed from the structure at this temperature. Results revealed that the heat treatment temperature (625 °C) was high enough to eliminate the nitrates in the structure of as-spun fibers and it did not cause crystallization of the glass. On the other hand, after heat treatment at 680 °C, crystalline hydroxyapatite Ca10(PO4)6OH2 (PDF# 01-072-1243) formed in the sample (Fig. 5). It is known that 13–93 has better processing characteristics compared to 45S5 Bioglass by viscous flow sintering (larger window between Tg and the onset of crystallization), the glass phase in scaffolds can be sintered to high density without crystallization [23]. In the current study, a crystalline phase formation was observed only in samples calcined at 680 °C for 4 h. Fig. 6 shows the images of the as-prepared nonwoven mats before and after calcination at 625 °C. During electrospinning, nanofibers deposited randomly in the form of a thin sheet on the counter electrode. As a result, homogeneous and flexible bioactive glass mats were manufactured. After calcination, nonwoven mats retained their flexible character as shown in Fig. 6(b) and (c). Fig. 7 demonstrates the SEM micrographs of the as-prepared and heat treated fibers. Accordingly,
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when 13–93 glass sol/PVA (1/1 ratio) mixture was utilized for electrospinning a bead free, stable fibrous structure was produced. At higher PVA concentrations some overlapping in fibers was noticed during electrospinning (results not shown). The average fiber diameter was calculated to be 680 ± 142 nm before heat treatment. Fibers retained their fibrous nature after calcination and grain formation were not observed due to amorphous nature of the glass at that temperature. Average fiber diameter was measured to be 464 ± 95 nm after calcination at 625 °C for 4 h. The effect of surfactant addition on the fiber morphology was also investigated in the study. In the presence of F-68 at 0.25 vol.% and 1 vol.%, a decrease was observed in average fiber diameter to 175.56 ± 42 nm and 126.6 ± 31 nm, respectively (Fig. 8). It is known that the surfactants can be introduced to decrease the surface tension and increase the net charge density [31–33]. Lin et al. [32] used cationic surfactants to increase the jet charge density; then instability motion was enhanced and the beaded nanofibers were also overcome. The instability motion is an important factor to stretch and thin the charged jet. Jia and Qin [33] used surfactants to change the surface tension of electrospinning solution; then the thermal performance and inner structure of nanofibers were adjusted. Similarly, Zheng and coworkers [31] studied the effect of ionic and non-ionic surfactants on the morphology of the electrospun fibers. The nanofiber diameter and diameter distribution range decreased with the increase of surfactant concentration in the solution. Attributed to the larger net charge density, the cationic surfactant provided a better way to prevent forming beaded structures [31]. In the current study, the decrease observed in fiber diameter may be presumably due to the reduction of the surface tension of the solution since net charge density insignificantly changes in the presence of a non-ionic surfactant. 3.3. In vitro bioactivity The bioactivity of the calcined fibers was investigated in vitro in a simulated body fluid (SBF) under static conditions. The prominent feature of bioactive glass is the formation of amorphous calcium phosphate or hydroxyapatite like layer on their surface when they are immersed in physiological fluid such as simulated body fluid and phosphate solution. The composition of this hydroxyapatite (HA) layer is almost similar to bone mineral and it provides adhesion with the surrounding tissues [34–36]. Fig. 9 shows the SEM micrographs of the 1393 fibers after treatment in SBF for different time intervals (F/S ratio: 1 mg/ml). After 1 day of immersion in SBF, a second phase material, presumably a calcium phosphate or crystalline HA formation, occurred on the surface of the fibers. The amount of this second phase material increased as the immersion time in SBF increased. The morphology of the calcium phosphate based material that formed on the surface of the fibers immersed for 1 day was significantly different compared to those treated for longer times such as 5 days. Similarly, Fig. 10 demonstrates the SEM micrographs of the calcined 13–93 fibers after treatment in SBF (F/S ratio: 0.5 mg/ml) for 1 and 7 days. Accordingly, SEM analysis showed the formation of HA like material after 1 day of immersion in SBF. The amount of HA-like material formed on the surface of the fibers was higher compared to the samples treated in SBF at 1 mg/ml. 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 [23,34–36]. The effect of glass concentration on the dissolution behavior and bioactivity of silicate bioactive glasses was studied by Jones et al. [37]. Results revealed that as the glass concentration in the solution increased the intensity of the peaks that identify HA formation decreased, which means reduction in bioactivity. 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 [37]. Lukito et al. [38] investigated the bioactivity of 70SiO2–30CaO bioactive glass using different ratios of bioactive glass to SBF solution (2 mg to 10 mg/ml). They reported that the initial 6 h of soaking resulted in the formation of HA
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Absorbance (at 450 nm)
(a)
13-93 fiber 13-93 control group
3
2
1
0
(b)
(c)
(d)
(e)
Fig. 12. Graph showing the result of XTT assay (absorbance at 450 nm) (a); optical microscope images showing the morphology of the cells at culture time,13–93 control group (b), 13–93 fibers (c); SEM micrographs of the MC3T3-E1 cells grown on the 13–93 fiber scaffolds at culture time (d) and (e).
phase regardless of the amount of glass to SBF ratio. After 6 h of soaking except for the 2 mg/ml ratio SBF solutions showed tendencies to form calcite. For the evaluation of carbonated HA formation, phosphate and carbonate bonds are of interest. Phosphate groups show four modes that are active in the infrared region: (i) bending vibration of PO4 at 560–610 and 430–460 cm− 1 and (ii) asymmetric stretching: broad band at 1000–1150 and at 960 cm−1 [39,40]. FTIR spectroscopy of the SBF treated 13–93 bioactive glass fibers (Fig. 11-a) showed resonances at 1000–1100 cm−1 and at 570 cm−1 corresponding to calcium phosphate. The two P–O bending peaks at 560 and 604 cm−1 are the main peaks for characterizing the HA formation [24,25,41]. 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 [39,40,42]. The resonances at 1390 cm−1 were attributed to C–O in the (CO3)2− group [42]. Results also revealed that a crystalline Ca–P layer, as indicated by the divided P–O bending vibration band between 500 and 600 cm−1, appeared only after 1 day and higher intensity peaks formed after 7 days 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
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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. It is known that 13–93 glass degrades (and converts to an HA-like material) more slowly than 45S5 glass. A previous study [34] showed that 45S5 electrospun fibers can covert to HA after 1 day of immersion under the same conditions. On the other hand, a crystalline Ca–P layer, as indicated by the divided P–O bending vibration band between 500 and 600 cm−1, formed only after 7 days for the 13–93 sample immersed in SBF at 0.5 mg/ml F/S ratio under the same conditions. Similarly, in the previous study of Fu et al. [41], in vitro bioactivity of the 13–93 bioactive glass scaffolds was observed by the conversion of the glass surface to a nanostructured hydroxyapatite layer within 7 days in simulated body fluid at 37 °C. To summarize, the results of the current study revealed that 13–93 bioactive glass nanofibers can be manufactured through electrospinning technique and the resultant fibers show high bioactive response in SBF (at 0.5 mg/ml F/S ratio). 3.4. Cell viability and morphology In order to evaluate the biocompatibility of the 13–93 glass fibers, an XTT assay was performed. The tetrazolium salt XTT is cleaved to formazan by a complex cellular mechanism. This bioreduction occurs in viable cells only, and is related to NAD(P)H production through glycolysis. Therefore, the amount of formazan dye formed directly correlates to the number of metabolically active cells in the culture. The assay is based on the cleavage of the tetrazolium salt XTT in the presence of an electron-coupling reagent, producing a soluble formazan salt [43,44]. This conversion only occurs in viable cells. The measured absorbance directly correlates to the number of viable cells [43]. Fig. 12-a shows the cell viability of MC3T3-E1 pre-osteoblast cells incubated with the sample. The difference in cell viabilities with control group samples after 48 h of incubation was negligibly small, and there was no cytotoxicity. This result suggests that electrospun 13–93 glass fibers have good biocompatibility. The microscopic examination of cultures confirmed that cells remained viable in the presence of the XTTformazan. Fig. 12-b and c shows the optical morphology of cells grown in culture wells for 2 days, including wells that contained the glass scaffolds. The cells reached near confluence and had an elongated and well-spreading morphology similar to that of the control. The cell morphology on the 13–93 glass fibers was observed with SEM (Fig. 12-d, e). After 2 days of culture, the cells were found to actively grow on the surface of glass fibers. MC3T3-E1 cells spread well and attached firmly on the surfaces, indicating good biocompatibility of the scaffolds. The cells also appeared to penetrate inside the pore structure. Furthermore, the cells had flattened bodies and many cytoskeletal extensions that were in intimate contact with the glass fiber substrate. 4. Conclusions The 13–93 bioactive glass fibers with an average diameter of 680 ± 142 nm were successfully fabricated using electrospinning technique. The use of inorganic precursors of 13–93 glass with aqueous PVA solution was an effective way for producing bioactive glass fibers. Results showed that the fibers kept their amorphous nature after a heat treatment at 625 °C and fiber diameter reduced to 464 ± 95 nm after heat treatment. XRD analysis showed that at 625 °C all the nitrates were removed from the structure. Fabricated 13–93 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 7 days for the sample immersed in SBF at 0.5 mg/ml F/S ratio. The biocompatibility tests on MC3T3-E1 pre-osteoblast cells using XTT assay revealed no cytotoxicity of the fibers.
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Acknowledgments The financial support for this research was provided by the Scientific and Technical Research Council of Turkey (TUBITAK), 1001 grant program for Scientific And Technological Research Projects; grant no: 111M766. The author would like to thank Dr. Harika Atmaca (Celal Bayar University, DEFAM) for her help in cell culture experiments.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Preparation, in vitro mineralization and osteoblast cell response of electrospun 13–93 bioactive glass nanofibers Aylin M. Deliormanlı Celal Bayar University, Department of Materials Engineering, Manisa, Turkey
a r t i c l e
i n f o
Available online 22 April 2015 Keywords: Bioactive glass Electrospinning Fibers Tissue engineering applications
a b s t r a c t In this study, silicate based 13–93 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 10 cm. The amorphous glass fibers with an average diameter of 464 ± 95 nm were successfully obtained after calcination at 625 °C. Hydroxyapatite formation on calcined 13–93 fibers was investigated in simulated body fluid (SBF) using two different fiber concentrations (0.5 and 1 mg/ml) at 37 °C. When immersed in SBF, conversion to a calcium phosphate material showed a strong dependence on the fiber concentration. At 1 mg/ml, the surface of the fibers converted to the hydroxyapatite-like material in SBF only after 30 days. At lower solid concentrations (0.5 mg/ml), an amorphous calcium phosphate layer formation was observed followed by the conversion to hydroxyapatite phase after 7 days of immersion. The XTT (2,3-Bis-(2-Methoxy-4-Nitro-5Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay was conducted to evaluate the osteoblast cell response to the bioactive glass fibers. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Fibrous scaffolds obtained from biomaterials are very important in biomedical engineering as the biomaterials mimic the main components of tissues. Biomimetic architectures should include a tailored structural hierarchy, a certain fiber orientation, and a certain pore size so that large cells may be able to migrate, adhere and spread over them [1]. These structures have morphologies and fiber diameters in a range comparable with those found in the extracellular matrix of human tissues [2,3]. They have high surface area, porosity and effective mechanical properties [2–4]. They have been used to engineer various soft connective tissues such as the skin, ligament, muscle, and tendon, as well as bone tissue regeneration [5]. In recent years, electrospinning has gained widespread interest as a potential tissue-engineering scaffolding technique [2,5]. In electrospinning process, electric field is subjected to the end of a capillary tube that contains the solution. This induces a charge on the surface of the liquid [5,6]. The solution undergoes extensional flow and deposits on to a target by the application of an external electrostatic field. If the intensity of the electric field produces enough stress in the droplet to overcome surface tension a solution jet can be created moving towards the ground collector. But before reaching the collector the solvent partially evaporates and the solution jet is subjected to extensional strain leading to formation of nanofibers. Non-woven, aligned, layered and
E-mail address: [email protected].
http://dx.doi.org/10.1016/j.msec.2015.04.037 0928-4931/© 2015 Elsevier B.V. All rights reserved.
coaxial nanofibers with a high surface area can be produced by controlling the electrospinning parameters [4–8]. It has been used successfully to spin a number of synthetic and natural polymers such as PLLA [9], PCL [9–12], silk [13], chitosan [14], collagen [11] and gelatine [15] into fibers with diameters varying from 3 nm to 5 μm [16]. It can also be applied to glass and ceramic systems [17–19]. Bioactive glasses can be utilized for bone and soft tissue regeneration [20–23]. The silicate bioactive glass designated as 45S5 (Bioglass) has been the most widely used glass for biomedical applications [20]. Another silicate bioactive glass designated as 13–93, with the composition (wt.%): 53 SiO2, 6 Na2O, 12 K2O, 5 MgO, 20 CaO, and 4 P2O5, has been receiving interest for biomedical applications [22]. 45S5 glass is approved for in vivo use by the U.S. Food and Drug Administration and products of 13-93 glass have been approved for in vivo use in Europe [23]. In vitro cell culture experiments showed no marked difference in the proliferation and differentiated function of osteoblastic MC3T3-E1 or MLO-A5 cells between dense disks of 45S5 and 13–93 glass [22,23]. However, 13–93 glass converts to a hydroxyapatite-like material more slowly than 45S5 glass [23]. To date, clinical applications of these bioactive glasses are limited to those materials synthesized by melting processes. A sol–gel process, involving the foaming of a sol with the aid of a surfactant, has been used to prepare porous scaffolds of a few bioactive glasses and bioactive glass powders [23–25]. Recently, Pirayesh and Nychka synthesized the 45S5 glass using the sol–gel method followed by heat treatment to produce semi-crystalline structure [24]. Similarly, sol–gel processing combined with electrospinning technique has been utilized by different research groups to produce bioactive glass fibers [26–29].
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Table 1 Surface tension and the viscosity of the solutions prepared in the study.
13–93 sol 13–93 sol/10 wt.% PVA mixture (1/1) 13–93 sol/PVA mixture including 1 vol.% ethanol 13–93 sol/PVA mixture including 1 vol.% ethanol and Surfynol SE 13–93 sol/PVA mixture including 1% ethanol in the presence of F-68 (1 vol.%)
Surface tension (mN/m)
Viscocity (mPa·s) @ 3 rpm
30.67 ± 0.28 37.25 ± 0.45 34.45 ± 0.31
2.5 55.2 50.7
27.37 ± 0.25
50.5
25.21 ± 0.17
48.1
2
Heat flow (W/g)
1 0 -1 -2 -3 200
400
600
800
Temperature (C) Fig. 3. Differential thermal analysis curve of the as-prepared 13–93 fibers.
Fig. 1. Flowchart of the solution preparation procedure for electrospinning of 13–93 glass.
Because of their high surface area, bioactive glass nanofibers degrade rapidly and convert to HA. As in the case of sol–gel-derived bioactive glasses, the bioactivity of these glass nanofibers is maintained over a larger SiO2 compositional range when compared to melt-derived glasses. Kim et al. [26] fabricated bioactive glass nanofiber by electrospinning of bioactive glass sol-poly(vinyl-butyral) mixture followed by heat treatment to remove the organic component. They also showed in vitro attachment and differentiation of bone marrowderived stem cells on the bioactive nanofibrous scaffold and compared it with bioactive glass films of the same composition. Allo et al. [28] studied the synthesis and electrospinning of ε-polycaprolactone-bioactive glass (composed of silica, calcium oxide and phosphate) hybrid biomaterials via a sol–gel process. 45S5 bioactive glass–ceramic fibers were synthesized previously through electrospinning method by the author and in vitro bioactivity of the fibers was investigated in simulated body fluid. Results revealed that 45S5 bioactive glass fibers can be fabricated successfully by electrospinning method however crystallization occurs during calcination at 700 °C [29]. In the current study, preparation of 13–93 bioactive glass as a nanoscale fiber by means of electrospinning technique was reported. The effects of the solution parameters on the morphology of electrospun glass nanofibres were studied to produce uniform, bead free fibers with a narrow diameter distribution. In vitro bioactivity of the fibers in SBF was elucidated as a function of immersion time and fiber concentration. Osteoblast cell response of electrospun fibers was investigated using the XTT method. 2. Experimental studies 2.1. Solution preparation
Fig. 2. Schematic view of the electrospinning set up. A similar (not the same) schematic was utilized in the previous paper of the author (Ref. 29, Ceram Int. Vol 41, 2015, 417425 Figure 1).
The composition of the 13–93 bioactive glass is (in mol%): 54.6% SiO2, 22.1% CaO, 7.9% K2O, 7.7% MgO, 6.0% Na2O, and 1.7% P2O5 [22].
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(b) Intensity
Absorbance
(a)
400
600
800 1000 1200 1400 1600 1800 2000
10
20
30
40
50
60
70
80
2 theta (degree)
Wave number (1/cm)
Fig. 4. (a) Fourier transform infrared spectroscopy (FTIR) spectra; (b) XRD diagram of as-prepared 13–93 fibers (◊ denotes sodium nitrate).
* * *
Intensity
725 oC
700 oC 680 oC 625 oC
20
40
60
80
2 theta (degree) Fig. 5. XRD diagram of the 13–93 fibers calcined at various temperatures (* denotes HA).
The method utilized in solution preparation was modified from the previous study of the author [29]. Precursors utilized for the sol preparation were as follows: tetraethyl orthosilicate (TEOS), triethyl phosphate (TEP), calcium nitrate tetrahydrate (Ca(NO3)2·4H2O, potassium nitrate
(a)
(b)
KNO3, magnesium nitrate hexahydrate (Mg(NO3)2·6H2O), and sodium nitrate NaNO3 (all from Sigma-Aldrich, USA). For the preparation of the nanofibers, initially 39.676 ml of TEOS was added into 0.1 M HNO3 aqueous solution at room temperature. The molar ratio of H2O/TEOS was maintained at 18 [24]. The mixture was allowed to stir for at least 60 min for hydrolysis. Then, each compound (1.90 ml TEP, 3.340 g NaNO3, 16.538 g Ca(NO3)2·4H2O, 6.317 g Mg(NO3)2·6H2O and 5.230 g KNO3) was added successively only when the previous solution became clear and was stirred for 1 h. Prior to electrospinning, aqueous poly (vinyl alcohol) (PVA) (Mw: 88,000–97,000, 88% hydrolyzed, Alfa Aesar) solution (10 vol.%) (13–93/PVA: 1/1 ratio), 1 vol.% ethyl alcohol (absolute, Sigma-Aldrich, USA) and 0.5 vol.% Surfynol SE (Air Products, USA) were added to the sol to adjust the viscosity and the surface tension. The mixture was allowed to stir overnight for homogenization at 25 °C prior to electrospinning process. Additionally, the effect of a non-ionic surfactant on the fiber diameter and morphology was determined. For this purpose, solutions were prepared in the presence of Pluronic F-68 (Sigma Aldrich, USA) at two different concentrations (0.25 vol.% and 1 vol.% F-68) using the procedure described above. Pluronic F-68 is a block copolymer with a molecular weight of 8350 g/mol. It contains two terminal polyethylene oxide and one central polypropylene oxide groups. The number of ethylene oxide units in the structure is reported to be 151 by the manufacturer company.
(c)
Fig. 6. Optical images of the 13–93 bioactive glass nonwoven mats (a) before and (b) and (c) after calcination at 625 °C.
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265
(d)
40 µm
(b)
20 µm
(e)
10 µm
10 µm
8
12
680 ±142 nm
(c)
10
464 ±95 nm
(f)
6
Count
Count
8
4
6 4
2 2 0
0 400
500
600
700
800
900
1000
Fiber diameter (nm)
300
400
500
600
700
Fiber diameter (nm)
Fig. 7. SEM micrographs of the 13–93 fibers and their fiber diameter distributions (a), (b) and (c) before heat treatment (d), (e) and (f) after heat treatment at 625 °C.
Fig. 1 summarizes the solution preparation procedure utilized in the study. Ultra-pure water with resistivity of 18.2 MΩ- cm was used for solution preparation through the experiments.
1 day in air atmosphere. Fibers were heat treated at 250 °C for 4 h by a heating rate of 1 °C/min followed by a treatment at 625 °C for 4 h (heating rate 1 °C/min) to remove the nitrates and organic materials in the structure.
2.2. Electrospinning 2.3. Characterizations In order to produce the 13–93 bioactive glass nanofibers, a laboratory scale nanospinner device (NE-300, Inovenso, TR) was utilized. The device comprises an illuminator, an exhaust, a high precision pump, a drum stroke and a rotating drum. Schematic view of the set up is given in Fig. 2. For electrospinning, a homogeneous, transparent 13– 93/PVA solution containing ethanol and surfactant was loaded 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 aluminium foil served as the collector. The solution was electrospun under an applied DC voltage of 20 kV using a distance of 10 cm between the nozzle and the rotating collector. The electrospun fibers were first aged at 60 °C and then dried at 120 °C for
Surface tensions of the electrospinning solutions were measured using a tensiometer (Attension, Sigma 702) by the Du Nouy ring method. The measurements were performed at 25 °C and the platinum ring was flamed until it glowed red before each measurement to avoid organic contamination. The Huh and Mason model was used in correction calculations made in the device. For each sample three different measurements were done and the results were averaged. Before each measurement the device was tested using deionized water at room temperature. The viscosity of the prepared solutions was measured at 25 °C by a rotational viscometer (Brookfield, DV-II+) using a small sample UL adaptor.
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(a)
(b)
10 µm
Fiber diameter (nm)
1000
10 µm
(c)
800 600 400 200 0 0,0
0,2
0,4
0,6
0,8
1,0
1,2
F-68 concentration (vol%) Fig. 8. SEM micrographs of the as-prepared 13–93 bioactive glass fibers in the presence of F-68 (a) 0.25 vol.%, (b) 1 vol.%, and (c) the graph showing the effect of F-68 on the average fiber diameter.
The microstructure of the fabricated bioactive glass fibers was examined using scanning electron microscopy, SEM (Philips XL-30S FEG) at 5 kV. Samples were gold coated to achieve conductivity and lessen charging prior to analysis. About 40 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 technique, XRD (Philips X'Pert Pro) was used to analyze the formation of crystalline phases in the prepared fibers. The diffractometer was operated using Cu Kα radiation with a step size of 0.01°/min and at a 2θ range of 10–90°. As-prepared fibers were also analyzed using a differential thermal analyzer (Perkin Elmer SII 7300) as a function of temperature. Samples were heated to 900 °C in N2 atmosphere at 10 °C/min for this purpose. 2.4. In vitro bioactivity test Simulated body fluid (SBF) was used to evaluate the in vitro bioactivity of the fibers. SBF was prepared in compliance with the protocol of Kokubo et al. [30]. For this purpose required amount of NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, CaCl2 and Na2SO4 (all reagent grade, from Sigma Aldrich, USA) was dissolved in ultra-pure water and buffered at a pH of 7.40 with tris (hydroxymethyl)aminomethane ((CH2OH)3CNH2) and 1 M hydrochloric acid (Fisher Scientific Inc., USA) at 37 °C. Two different fiber/SBF (F/S) ratios (0.5 and 1 mg/ml) were utilized in the experiments. Each sample (1 g of fiber) was immersed in a polyethylene bottle containing the SBF solution, and kept for up to 30 days without shaking in an incubator at 37 °C. At the end of immersion periods, fibers were removed from the bottle, washed
with deionized water to prevent further reaction and dried in an oven at 60 °C for 24 h. SEM was utilized to analyze the structure of the reacted fibers. Fourier transform infrared spectroscopy (FTIR-ATR, Agilent Cary 660) was also used to characterize HA forming ability of the glass fibers. 2.5. Cell culture studies Fifty milligrams of 13–93 glass fibers was placed inside a cylindrical ceramic (alumina) mold. The molds holding the randomly oriented glass fibers were placed into an oven preheated to 650 °C, and then heated for 60 min. After heating, the molds were removed from the oven and cooled to room temperature. The disk-shaped scaffolds 4 ± 0.1 mm in diameter and 2 ± 0.2 mm in thickness were prepared and stored in a desiccator. Same type of scaffolds was also prepared using amorphous 13–93 glass powders (d50 = 2.1 μm) synthesized by the melt-cast method and these scaffolds designated as the control group. The in vitro cytotoxicity of the 13–93 fiber scaffolds was determined by XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium5-Carboxanilide) assay. Cell culture experiments were performed using the mouse bone/calvaria preosteoblastic MC3T3-E1 (Subclone 4) cells (ATCC CRL-2593). Prior to experiments, scaffolds were rinsed 3 times with 70% ethanol and dry heat sterilized at 350 °C overnight. The cells were cultured in a modified minimum essential medium (aMEM; GIBCO, Invitrogen Corporation, Grand Island, N.Y.), supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO, Invitrogen Corporation, Grand Island, N.Y.), 100 U/ml penicillin and 100 U/ml streptomycin. The cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Tested scaffolds were placed into each
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2 µm
10 µm
(c)
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(e)
2 µm
(f)
2 µm
10 µm
(g)
(h)
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Fig. 9. SEM micrographs of the 13–93 fibers immersed in SBF (1 mg/ml) for (a) and (b) 1 day; (c) and (d) 5 days; (e) and (f) 7 days and (g) and (h) 30 days.
well of 96-well plates (NUNC; Thermo Fisher Scientific) before cell seeding. Scaffolds were seeded with cells by adding an MC3T3-E1 cell suspension onto the scaffolds (5 · 104 cells in 100 μl of medium per
well). The cell suspension was fully absorbed, thereby allowing the cells to be distributed within the scaffolds. The cell-seeded scaffolds were incubated for 4 h to allow the cells to adhere to the scaffolds.
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(a)
(c)
2 µm
10 µm
(d)
(b)
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Fig. 10. SEM micrographs of the 13–93 fibers immersed in SBF (0.5 mg/ml) for (a) and (c) 1 day; (b) and (d) 7 days.
Samples were cultured for 48 h and then yellow XTT solution was added. The absorbance was monitored at 450 nm using an Elisa Plate Reader. XTT is a colorless or slightly yellow compound that when reduced becomes brightly orange. This color change is accomplished by the breakup of positively-charged quaternary tetrazole ring. The formazan product of XTT reduction is soluble and can be used in realtime assays. Cell Proliferation Kit II (XTT) (Roche) was used in the experiments according to the manufacturer's protocol. Three replicate samples were tested for each condition. For morphological observation of cultured cells after incubation for 48 h, each scaffold was removed, rinsed three times with PBS, and the cells were fixed with 2.5% glutaraldehyde solution (Sigma Aldrich, USA). After an overnight soak in PBS at 4 °C, the scaffolds were washed three times with PBS (10 min at each) and dehydrated through a graded series of ethanol (50, 60, 70, 80, 90 and 100%) for 15 min at each concentration. After drying for 48 h at room temperature, the samples were sputter-coated with gold and observed in an SEM using the conditions described previously. The morphology of cells at culture time was also determined by optical microscopy. 2.6. Statistical analysis Three samples in each group were used for cell culture experiments. The data are presented as the mean standard deviation. Statistical analysis was carried out using one-way analysis of variance (ANOVA) and Tukey's post hoc test, with the level of significance set at p b 0.05. 3. Results and discussion 3.1. Solution properties Surface tension and viscosity are crucial factors in electrospinning and they are functions of solvent compositions of the solution. By reducing the surface tension of the solution, beaded fibers can be converted into smooth fibers [31]. On the other hand, it has been proven that
continuous and smooth fibers cannot be obtained in very low viscosity, whereas very high viscosity results in the hard ejection of jets from the solution [31–33]. Therefore, it is necessary to adjust properly these parameters to achieve a successful electrospinning operation. The surface tension and viscosity values of the as-prepared solutions are listed in Table 1. Accordingly, the surface tension of 13–93 sol alone was measured to be 30.67 mN/m. In the presence of PVA, the surface tension increased to 37.25 mN/m. Results also revealed that fibers cannot be fabricated from pure 13–93 glass solution, because Si–O network of glass was not sufficient to create intermolecular interactions. Therefore, it was essential to add a polymeric material with long chains into the glass such as PVA to enhance the formation of fibers. To reduce the surface tension and increase the volatility, 1% of ethanol was added to the aforementioned solution. By this way, a decrease was observed in the surface tension of the solution to 34.45 mN/m. Introduction of a surfactant to this system led to a further decrease in the surface tension. Results also revealed that viscosity of the electrospinning solution was 50.5 mPa·s at 3 rpm and a decrease in viscosity to 10 mPa·s was observed as the shear rate increased to 50 rpm (results not shown). Therefore it is possible to conclude that the solution has a shear thinning flow behavior. 3.2. Bioactive glass fibers The DTA curve of the as-prepared 13–93 nanofibers is shown in Fig. 3. The 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. Exothermic peaks represent the phase transformation and crystal formation. In Fig. 3, the peak below 100 °C is due to the removal of traces of adsorbed water molecules and the peak at 250–300 °C is may be due to removal of the water because of the condensation of the precursor materials. The peak in the range of 350–400 °C can be attributed to the decomposition of PVA in the fibers. The peak between 700-800 °C is due to the crystallization of the 13–93 fibers.
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P-O bending
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Fig. 4-a shows the FTIR spectra of the as-prepared 1393 fibers. The peaks ranging from 920 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 cm−1 [25]. According to the literature, the main absorption bands of amorphous silicate based glass are Si– O bending peak at 460 cm−1 and Si–O stretching at 926 and 1024 cm−1 [24]. XRD diagram of the as-prepared 13–93 nanofibers was demonstrated in Fig. 4-b. Accordingly, sodium nitrate (PDF# 036-1474) was presented in the sample before heat treatment. Sodium nitrate peak disappeared in samples calcined at 625 °C, which means that all the nitrates were removed from the structure at this temperature. Results revealed that the heat treatment temperature (625 °C) was high enough to eliminate the nitrates in the structure of as-spun fibers and it did not cause crystallization of the glass. On the other hand, after heat treatment at 680 °C, crystalline hydroxyapatite Ca10(PO4)6OH2 (PDF# 01-072-1243) formed in the sample (Fig. 5). It is known that 13–93 has better processing characteristics compared to 45S5 Bioglass by viscous flow sintering (larger window between Tg and the onset of crystallization), the glass phase in scaffolds can be sintered to high density without crystallization [23]. In the current study, a crystalline phase formation was observed only in samples calcined at 680 °C for 4 h. Fig. 6 shows the images of the as-prepared nonwoven mats before and after calcination at 625 °C. During electrospinning, nanofibers deposited randomly in the form of a thin sheet on the counter electrode. As a result, homogeneous and flexible bioactive glass mats were manufactured. After calcination, nonwoven mats retained their flexible character as shown in Fig. 6(b) and (c). Fig. 7 demonstrates the SEM micrographs of the as-prepared and heat treated fibers. Accordingly,
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when 13–93 glass sol/PVA (1/1 ratio) mixture was utilized for electrospinning a bead free, stable fibrous structure was produced. At higher PVA concentrations some overlapping in fibers was noticed during electrospinning (results not shown). The average fiber diameter was calculated to be 680 ± 142 nm before heat treatment. Fibers retained their fibrous nature after calcination and grain formation were not observed due to amorphous nature of the glass at that temperature. Average fiber diameter was measured to be 464 ± 95 nm after calcination at 625 °C for 4 h. The effect of surfactant addition on the fiber morphology was also investigated in the study. In the presence of F-68 at 0.25 vol.% and 1 vol.%, a decrease was observed in average fiber diameter to 175.56 ± 42 nm and 126.6 ± 31 nm, respectively (Fig. 8). It is known that the surfactants can be introduced to decrease the surface tension and increase the net charge density [31–33]. Lin et al. [32] used cationic surfactants to increase the jet charge density; then instability motion was enhanced and the beaded nanofibers were also overcome. The instability motion is an important factor to stretch and thin the charged jet. Jia and Qin [33] used surfactants to change the surface tension of electrospinning solution; then the thermal performance and inner structure of nanofibers were adjusted. Similarly, Zheng and coworkers [31] studied the effect of ionic and non-ionic surfactants on the morphology of the electrospun fibers. The nanofiber diameter and diameter distribution range decreased with the increase of surfactant concentration in the solution. Attributed to the larger net charge density, the cationic surfactant provided a better way to prevent forming beaded structures [31]. In the current study, the decrease observed in fiber diameter may be presumably due to the reduction of the surface tension of the solution since net charge density insignificantly changes in the presence of a non-ionic surfactant. 3.3. In vitro bioactivity The bioactivity of the calcined fibers was investigated in vitro in a simulated body fluid (SBF) under static conditions. The prominent feature of bioactive glass is the formation of amorphous calcium phosphate or hydroxyapatite like layer on their surface when they are immersed in physiological fluid such as simulated body fluid and phosphate solution. The composition of this hydroxyapatite (HA) layer is almost similar to bone mineral and it provides adhesion with the surrounding tissues [34–36]. Fig. 9 shows the SEM micrographs of the 1393 fibers after treatment in SBF for different time intervals (F/S ratio: 1 mg/ml). After 1 day of immersion in SBF, a second phase material, presumably a calcium phosphate or crystalline HA formation, occurred on the surface of the fibers. The amount of this second phase material increased as the immersion time in SBF increased. The morphology of the calcium phosphate based material that formed on the surface of the fibers immersed for 1 day was significantly different compared to those treated for longer times such as 5 days. Similarly, Fig. 10 demonstrates the SEM micrographs of the calcined 13–93 fibers after treatment in SBF (F/S ratio: 0.5 mg/ml) for 1 and 7 days. Accordingly, SEM analysis showed the formation of HA like material after 1 day of immersion in SBF. The amount of HA-like material formed on the surface of the fibers was higher compared to the samples treated in SBF at 1 mg/ml. 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 [23,34–36]. The effect of glass concentration on the dissolution behavior and bioactivity of silicate bioactive glasses was studied by Jones et al. [37]. Results revealed that as the glass concentration in the solution increased the intensity of the peaks that identify HA formation decreased, which means reduction in bioactivity. 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 [37]. Lukito et al. [38] investigated the bioactivity of 70SiO2–30CaO bioactive glass using different ratios of bioactive glass to SBF solution (2 mg to 10 mg/ml). They reported that the initial 6 h of soaking resulted in the formation of HA
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Fig. 12. Graph showing the result of XTT assay (absorbance at 450 nm) (a); optical microscope images showing the morphology of the cells at culture time,13–93 control group (b), 13–93 fibers (c); SEM micrographs of the MC3T3-E1 cells grown on the 13–93 fiber scaffolds at culture time (d) and (e).
phase regardless of the amount of glass to SBF ratio. After 6 h of soaking except for the 2 mg/ml ratio SBF solutions showed tendencies to form calcite. For the evaluation of carbonated HA formation, phosphate and carbonate bonds are of interest. Phosphate groups show four modes that are active in the infrared region: (i) bending vibration of PO4 at 560–610 and 430–460 cm− 1 and (ii) asymmetric stretching: broad band at 1000–1150 and at 960 cm−1 [39,40]. FTIR spectroscopy of the SBF treated 13–93 bioactive glass fibers (Fig. 11-a) showed resonances at 1000–1100 cm−1 and at 570 cm−1 corresponding to calcium phosphate. The two P–O bending peaks at 560 and 604 cm−1 are the main peaks for characterizing the HA formation [24,25,41]. 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 [39,40,42]. The resonances at 1390 cm−1 were attributed to C–O in the (CO3)2− group [42]. Results also revealed that a crystalline Ca–P layer, as indicated by the divided P–O bending vibration band between 500 and 600 cm−1, appeared only after 1 day and higher intensity peaks formed after 7 days 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
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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. It is known that 13–93 glass degrades (and converts to an HA-like material) more slowly than 45S5 glass. A previous study [34] showed that 45S5 electrospun fibers can covert to HA after 1 day of immersion under the same conditions. On the other hand, a crystalline Ca–P layer, as indicated by the divided P–O bending vibration band between 500 and 600 cm−1, formed only after 7 days for the 13–93 sample immersed in SBF at 0.5 mg/ml F/S ratio under the same conditions. Similarly, in the previous study of Fu et al. [41], in vitro bioactivity of the 13–93 bioactive glass scaffolds was observed by the conversion of the glass surface to a nanostructured hydroxyapatite layer within 7 days in simulated body fluid at 37 °C. To summarize, the results of the current study revealed that 13–93 bioactive glass nanofibers can be manufactured through electrospinning technique and the resultant fibers show high bioactive response in SBF (at 0.5 mg/ml F/S ratio). 3.4. Cell viability and morphology In order to evaluate the biocompatibility of the 13–93 glass fibers, an XTT assay was performed. The tetrazolium salt XTT is cleaved to formazan by a complex cellular mechanism. This bioreduction occurs in viable cells only, and is related to NAD(P)H production through glycolysis. Therefore, the amount of formazan dye formed directly correlates to the number of metabolically active cells in the culture. The assay is based on the cleavage of the tetrazolium salt XTT in the presence of an electron-coupling reagent, producing a soluble formazan salt [43,44]. This conversion only occurs in viable cells. The measured absorbance directly correlates to the number of viable cells [43]. Fig. 12-a shows the cell viability of MC3T3-E1 pre-osteoblast cells incubated with the sample. The difference in cell viabilities with control group samples after 48 h of incubation was negligibly small, and there was no cytotoxicity. This result suggests that electrospun 13–93 glass fibers have good biocompatibility. The microscopic examination of cultures confirmed that cells remained viable in the presence of the XTTformazan. Fig. 12-b and c shows the optical morphology of cells grown in culture wells for 2 days, including wells that contained the glass scaffolds. The cells reached near confluence and had an elongated and well-spreading morphology similar to that of the control. The cell morphology on the 13–93 glass fibers was observed with SEM (Fig. 12-d, e). After 2 days of culture, the cells were found to actively grow on the surface of glass fibers. MC3T3-E1 cells spread well and attached firmly on the surfaces, indicating good biocompatibility of the scaffolds. The cells also appeared to penetrate inside the pore structure. Furthermore, the cells had flattened bodies and many cytoskeletal extensions that were in intimate contact with the glass fiber substrate. 4. Conclusions The 13–93 bioactive glass fibers with an average diameter of 680 ± 142 nm were successfully fabricated using electrospinning technique. The use of inorganic precursors of 13–93 glass with aqueous PVA solution was an effective way for producing bioactive glass fibers. Results showed that the fibers kept their amorphous nature after a heat treatment at 625 °C and fiber diameter reduced to 464 ± 95 nm after heat treatment. XRD analysis showed that at 625 °C all the nitrates were removed from the structure. Fabricated 13–93 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 7 days for the sample immersed in SBF at 0.5 mg/ml F/S ratio. The biocompatibility tests on MC3T3-E1 pre-osteoblast cells using XTT assay revealed no cytotoxicity of the fibers.
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Acknowledgments The financial support for this research was provided by the Scientific and Technical Research Council of Turkey (TUBITAK), 1001 grant program for Scientific And Technological Research Projects; grant no: 111M766. The author would like to thank Dr. Harika Atmaca (Celal Bayar University, DEFAM) for her help in cell culture experiments.
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