Materials Letters 65 (2011) 2404–2406

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Materials Letters

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Preparation of fluoride-containing gelatin nanofiber scaffold

Jia Xu a,⁎, Jing Yan b, Qiang Gu b, Junfeng Li b, Hongyan Wang b,⁎a School of Chemistry and Environmental Engineering, Changchun University of Science and Technology, Changchun 130022, Chinab College of Chemistry, Jilin University, Changchun 130012, China

⁎ Corresponding authors. Tel.: +86 431 85583008.E-mail addresses: xujia@cust.edu.cn (J. Xu), wang_h

0167-577X/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.matlet.2011.04.080

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 October 2010Accepted 24 April 2011Available online 29 April 2011

Keywords:ElectrospinningFluorideNanofiberGelatinBiomaterialsNanocomposites

A novel material, fluoride-containing gelatin nanofiber scaffold, was prepared by electrospinning processsuccessfully. Scanning electron microscopy (SEM) image showed that the morphology of nanofibers wasuniform and smooth, and the average diameter was about 200 nm. An even distribution in the fiber matrix ofsome nanoparticles which is about 20 nm was also observed in transmission electron microscopy (TEM)images. X-ray diffraction (XRD) demonstrated that the nanoparticles dispersed in the gelatin fiber matrixwere CaF2 crystals. This scaffold was crosslinked with glutaraldehyde (GTA) vapor at room temperature for4 days in order to improve the water-resistant ability. After soaking in Dulbecco's Modified Eagle's Medium(DMEM) solution (37 °C) for 4 weeks the crosslinked scaffold stillmaintained a good appearance andmorphology.All the properties of this novel material show a good potential to be a bone tissue engineering scaffold.

y@jlu.edu.cn (H. Wang).

ll rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Fluoride is one of the few inorganic ions which are able to stimulateosteoblasts (OB). In 1983, Fareley et al. [1] confirmed that fluoride coulddirectly stimulate osteoblastic proliferation, increase the activity ofalkaline phosphatase (ALP) and enhance collagen synthesis. Dequeker[2] thought that fluoride could increase the deposition rate and thenumberofOBby stimulatingOBmitosis. Therefore, the researchof addingfluoride into scaffoldmaterialwas considered bymore andmore scholarsin the field of bone tissue engineering. Harrison et al. [3] preparedhydroxyfluorapatite (FHA) using fluoride to replace the hydroxyl grouppartly and testified the active effect of fluoride for culturing embryonicstem cells. Kim et al. [4] introduced fluorine ions into the HA–PCLbioactive composites through a novel processing technique. After thefluoridation, the MG63 (human osteoblast) cells on the compositesexhibitedahigher expression level of bone-relatedgenes (specificallyALPand osteocalcin).Moreover, the intracellular levels of ALP and osteocalcinproduced by the cells on the FHA–PCL composites were significantlyhigher than those on the HA–PCL without fluoridation. Cooper et al. [5]found that thegrit-blasted surfaceoffluoride-modificationTiO2enhancedosteoblastic differentiation in vitro and interfacial bone formation in vivo.

With the development of electrospinning [6], nanofiber productwas increasingly used as the scaffold of bone tissue engineeringgradually. Many materials, including PLA [7], PLGA [8], PCL [9],chitosan [10], silk [11], collagen [12], gelatin [13] and their mixtureshad been electrospun into nanofiber scaffold in bone tissue engineer-

ing. Badami et al. [7] prepared PLA nanofiber scaffold and studied theeffect of fiber diameter on spreading, proliferation and differentiationof osteoblastic cells. In order to improve the properties of scaffold inbone tissue engineering, hydroxyapatite was introduced into nano-fibers using electrospinning by Zhang et al. [10] and Kim et al. [13] andthe material had obtained active effect. However, the preparation offluoride-containing nanofiber scaffold was rarely reported.

In this study, fluoride has been introduced into the gelatinnanofiber scaffold by electrospinning. The distribution of fluorideparticles in the fiber was observed by TEM. Glutaraldehyde was usedas a crosslinking agent to improve its water-resistance. This scaffoldwill be used to culture the osteoblasts of nude rat to find the bestcontent of fluoride in our future work.

2. Experimental

2.1. Materials

Gelatin type A from porcine skin (approximately 300 Bloom) waspurchased from Sigma-Aldrich. 2,2,2-trifluoroethanol (TFE) wasobtained from Rhodia Company Ltd. Aqueous glutaraldehyde (GTA)solution (50%) was purchased from Tianjin Fuchen Chemical ReagentsFactory. Sodium fluoride (NaF) was purchased from Beijing TongguangFine Chemicals Company. Calcium chloride (CaCl2) was obtained fromBeijing Chemicals Company.

2.2. Preparation

0.1 g CaCl2 and 0.0757 g NaF were dissolved in 2 ml and 1 mldistilled water, respectively, and stirred vigorously for 30 min. Then,

2405J. Xu et al. / Materials Letters 65 (2011) 2404–2406

9 ml TFE was added into the CaCl2 aqueous solution and stirredvigorously for 30 min. 2.55 g gelatinwas dissolved in the above solutionand stirred vigorously for 6 h at 35 °C. NaF aqueous solution was addedinto the above blended solution dropwise with vigorous stirring. Themixture was then stirred at room temperature for 6 h. The spinningliquid had been prepared. The apparatus for the electrospinningexperiments was similar to previous report [7]. At room temperature,the spinning liquid was placed into a glass syringe with the tip of innerdiameter of 1 mm. A clamp connectedwith high voltage power supplierwas attached to the glass syringe. As grounded collector, a piece ofaluminum foil was placed towards the tip at a distance of 20 cm. Themembrane of fluoride-containing gelatin nanofibers was formed on thealuminum foil at 16 kV. The membrane was then stored in a vacuumoven for 24 h at 40 °C to remove residual solvent.

2.3. Crosslinking

The fluoride-containing gelatin nanofiber membrane was sealed ina dessicator and was crosslinked in the glutaraldehyde (GTA) vaporfor 4 days at room temperature. The crosslinked membrane was driedin a vacuum oven for 24 h at 45 °C to remove residual GTA.

2.4. Characterization

The morphology of the electrospun fibers was observed under aSEM (SHIMADZU SSX-550) at an accelerating voltage of 15 kV. TheTEM images were recorded on a JEM-2000EX microscopy. XRDpatterns were obtained with a Siemens D5005 diffractometer usingCu Kα radiation. FTIR spectra were recorded on a Nicolet InstrumentsResearch series 5PC Fourier Transform Infrared spectrometer.

3. Results and discussion

Gelatin could dissolve in water easily, but its aqueous solutioncould not be electrospun into fiber. Huang et al.[14] had successfullyelectrospun gelatin into nanofibers using TFE as solvent in 2004. Inthis study, calcium fluoride was introduced into gelatin nanofiberssuccessfully.

Fig. 1 outlined the procedure of the preparation of the fluoride-containing spinning liquid and the nanofibers. Firstly, CaCl2 wasdissolved in the hybrid solvent of TFE and water, and then gelatin wasadded into the above solution (Fig. 1(A)). Fig. 1(B) was the key stepfor producing spinning liquid. Calcium fluoride was producedhomogeneously without accumulation in the system when NaF wasdropped slowly. This phenomenon indicates that the colloidalsolution of gelatin hindered the accumulation of calcium fluoride,thus calcium fluoride could be dispersed in spinning liquid homoge-neously. A homogeneous spinning liquid was prepared (Fig. 1(C)).

Fig. 1. The preparation of fluoride-contai

Fig. 2(A) showed the SEM image of the fluoride-containing gelatinscaffold. We can see that the morphology of fluoride-containinggelatin fibers was uniform and the average diameter of fibers is about200 nm. Fig. 2(B and C) showed TEM images of the fluoride-containing gelatin nanofibers. It could be seen that some nanoparti-cles with the average diameter of about 20 nmwere well dispersed inthe polymer fiber matrix. These nanoparticles should be CaF2 fromthe reaction between NaF and CaCl2, which had been validated byFig. 2(D). Fig. 2(D) showed an X-ray diffraction pattern of the fluoride-containing gelatin nanofiber scaffold. A peak at 2θ=21.8° wascharacteristic of gelatin [15]. Three weak peaks were observed at28.3°, 47.5° and 55.7°, which were in agreement with the standardXRD peaks of crystalline CaF2 and showed that the CaF2 crystals werein the form of (111), (220) and (311) [16]. The result of XRD provedconvincingly that the nanoparticles dispersed in the gelatin fibermatrix were CaF2 crystals.

Fig. 3(A) showed the wide scan FTIR spectra for gelatin (a) andfluoride-containing gelatin (b). It could be obviously seen that the FTIRspectra of the fluoride-containing gelatinwere different from that of thegelatin. From Fig. 3(A (a)), we could observe the characteristicabsorption band of gelatin: the amide I band (antisymmetric stretchingvibration of carboxyl group or C_O vibration) at 1636.64 cm−1 and theamide II band (N\Hbending vibration) at 1525.10 cm−1. But the amideI band shifted to 1650.70 cm−1 and the amide II band shifted to1538.79 cm−1 in Fig. 3(A (b)). It was concluded that there was aninteraction between CaF2 and carboxyl group of gelatin. This interactionmight be the reason that gelatin could prevent the aggregation of CaF2nanoparticles.

Gelatin could dissolve in water easily and the nanofiber scaffoldhad a particularly high surface area, so the scaffold would disappearimmediately after putting it into water which made it impossible toculture cells in aqueous environment. To reduce the water solubilityof the scaffold, GTA vapor was used to crosslink fluoride-containinggelatin scaffold according to the work from Zhang et al. [17]. Themembrane crosslinked became visibly yellowish. The color changewas due to the establishment of aldimine linkages (CH_N) betweenthe free amine groups of gelatin and glutaraldehyde [18]. The fiberscaffold crosslinked for 4 days in GTA vapor was immersed into theDMEM solution (37 °C) which mimic the real physiological environ-ment to test its dissolvability. After immersing for 4 weeks, thescaffold still kept an intact appearance in macroscopic view. FromFig. 3(B), it was obvious that the fiber still kept a good morphology.The water-resistance was greatly improved compared to the non-crosslinked scaffold. It is indicated that the crosslinked fluoride-containing gelatin nanofiber membranes could be applicable to thescaffold of culturing cells. More studies on the growth of theosteoblasts on the scaffold and the application of the scaffold in thebone tissue engineering will be in our future publications.

ning spinning liquid and nanofibers.

Fig. 2. SEM image (A) and TEM image (B, C) of fluoride-containing gelatine fibers; X-ray diffraction pattern of the fluoride-containing gelatin nanofiber scaffold (D).

Fig. 3. FTIR spectra of gelatin nanofibers (A (a)) and fluoride-containing gelatin nanofibers (A (b)); SEM image of fluoride-containing gelatin scaffold after immersing (B).

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4. Conclusions

Under the colloid protection, fluoride was successfully introducedinto gelatin nanofibers with a uniform and smooth morphology byelectrospinning process. CaF2 nanoparticles with average diameter ofabout 20 nm were well dispersed in the gelatin nanofiber matrix. Thewater resistant character of the fibers resulted from crosslinking withGTAvapormade it a very potentmaterial for the bone tissue engineering.

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