Tuned Morphological Electrospun Hydroxyapatite Nanofibers via pH

Journal of Bionic Engineering 9 (2012) 478–483

Tuned Morphological Electrospun Hydroxyapatite Nanofibers via pH Xiaofeng Song1,2, Fengguang Ling1,2, Haotian Li1, Zhantuan Gao2, Xuesi Chen2 1. School of Chemical Engineering, Changchun University of Technology, Changchun 130012, P. R. China 2. Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China

Abstract The concept of biocompatible, osteoconductive and noninflammatory material mimicking the structure of natural bone has generated a considerable interest in recent decades. Hydroxyapatite (HA) is an important bionic material that is used for bone grafting in osseous defects and drug carriers. HA with various morphologies and surface properties have been widely investigated. In this paper, HA nanofibers are produced through a combination of electrospinning and sol-gel technique. The morphologies, composition and structure are investigated by Scanning Electron Microscopy (SEM), Thermogravimetic Analysis (TGA), Fourier Transform Infrared (FTIR), X-ray Diffraction (XRD) patterns, Transmission Electron Microscopy (TEM). The results show that HA nanofibers are even and well-crystallized, and pH is crucial for producing HA nanofibers. With the change of pH from 4 to 9, nanofibers grow densely along (210) plane and become compact while surface area, pore volume and pore size decrease correspondingly. The synthesized HA nanofibers are nontoxic and safe. Zn can be also incorporated into HA nanofibers, which will endow them with more perfect function. Keywords: electrospinning, sol, hydroxyapatite, pH Copyright © 2012, Jilin University. Published by Elsevier Limited and Science Press. All rights reserved. doi: 10.1016/S1672-6529(11)60143-1

1 Introduction Synthetic hydroxyapatite [HA, Ca10(PO4)6(OH)2] is a significant biomaterial in the health care industry because its composition is similar to the natural calcium phosphate minerals in human biological hard tissues, such as bones and teeth[1–3]. Previously reported approaches of HA synthesis include solid-state reaction[4], co-precipitation[5,6], hydrothermal method[7], and sol-gel route[8–12]. Ceramic materials synthesized by sol-gel route exhibit many advantages over the others such as high product purity, homogeneous composition, and low synthesis temperature. Micron-sized HA shows poor bioresorbability and brittle characteristic[13,14], then nano-HA is becoming more interests. Also, the chemical and biological properties of HA are closely related to their nanoscale dimensions[15,16]. For example, Yang prepared mesoporous europium-doped HA nanorod as drug storage through a simple one-step route using cationic surfactant as template. He found that drug release could be easily Corresponding author: Xiaofeng Song E-mail: [email protected]

tracked and monitored by the change of the luminescence intensity[17]. One dimensional (1D) inorganic nanomaterials, such as nanotubes, nanowires, nanobelts, and nanorods, have been a subject of intensive research due to their intriguing properties like large surface-to-volume ratio. They have the potential application in many areas such as materials science, chemistry, physics, biology and engineering[18,19]. So far, a number of successful techniques have been developed to fabricate 1D nanostructures such as template-directed[20], vapor–liquid–solid (VLS)[21], solution–liquid–solid[22], hydrothermal[23], solvothermal[24], selfassembly[25], and electrospinning[26]. Among these methods, electrospinning has proven to be a simple and versatile method for producing 1D nanostructure. A wide range of 1D polymeric, composite, and inorganic materials have been successfully synthesized by this technique with the characteristics of easy control of fiber morphology, low cost and safety[27–30]. In this work, HA nanofibers are synthesized successfully using a combination of electrospinning and

Song et al.: Tuned Morphological Electrospun Hydroxyapatite Nanofibers via pH

sol-gel technique. The grown crystal and surface structure of HA nanofibers can be conveniently tailored through increasing or decreasing pH of sol. The other content such as Zn can be also incorporated into HA nanofibers by this combination method.

2 Experiment 2.1 Chemicals and synthesis of HA fibers The follows are the abbreviations of the chemicals and the corresponding manufacturers: Polyvinylpyrrolidone (PVP, Mn=1300000, Boai NKY Pharmaceuticals Ltd, China, analytical grade); Triethyl phosphate ((C2H5O3)3PO, TEP, Sinopharm Chemical Reagent Co. Ltd, China, chemically pure); Calcium nitrate (Ca(NO3)2·4H2O, Beijing Chemical Works, China, analytical grade); ethanol (Beijing Chemical Works, China, analytical grade). PVP was selected for fibrous template due to its dissolution in water or ethanol. In a typical procedure, 1.2 g of Ca(NO3)2·4H2O was mixed with distilled water and ethanol under vigorous stirring for 1h. Subsequently, 0.138 g of TEP and a suitable amount of ammonia were added. This solution was closely capped and stirred at 80 C – 90 C for 20 h. After it was cooled down to room temperature, a required amount of PVP was added to the above solution, followed by magnetic stirring for 24 h for electrospinning. Then the precursor solution was loaded into a 30 ml glass syringe equipped with a metallic needle (inner diameter: 0.8 mm). The needle was connected to a high-voltages supply that is capable of generating DC voltages up to 30 kV. In our experiment a voltage of 3 kV was applied for electrospinning. A piece of flat aluminum foil was placed 25 cm under the tip of the needle to collect the fibers. The electrospinning was conducted in air. Calcinations of the dried electrospinning fibers film in air at 600 C for 1 h at 100 C·h1 were used to prepare HA nanofibers. Then the obtained fibers were filtered and washed with distilled water and dried at 80 C. 2.2 Characterization Environmental Scanning Electron Microscopy (ESEM) images were recorded on a Model XL30 ESEM FEG instrument equipped with Energy Dispersive X-ray (EDX). Thermogravimetic Analysis (TGA) was measured using a Perkin Elmer thermal analyzer from room temperature to 600 C under an air flux of 100 ml·min1

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ramping at 10 C·min1. Fourier Transform Infrared (FT-IR) spectroscopy analysis were carried out on a Bruker IFS 66V/S spectrophotometer. The X-Ray powder Diffraction (XRD) data were collected on a Rigaku D/max 2500V PC diffractometer (Cu K X-radiation at 40 kV and 200 mA). Transmission Electron Micrographs (TEM) were performed on a JEM-2010 JEOL equipment. The fiber surface structure was analyzed by nitrogen adsorption-desorption data collected in a Quantachrome Autosorb-1 gas adsorption analyzer at 77 K.

3 Results and discussions Fig. 1a shows ESEM images of these collected fibers by electrospinning PVP/ sol-gel precursor at initial pH = 9. These fibers were randomly distributed on the substrate to form a thin mat and their lengths could reach several centimeters. The diameter of these fibers was found in the range of 700 nm to 1 m. After annealing the sample in air at 600 C, the PVP was removed, and a well-defined fiber texture kept unchangeable, but the fibers exhibited shrinkage and diameter was reduced (Fig. 1b). The inset TEM image depicts that each fiber took on a rough surface structure. Their average diameter was found to be 500 nm. Moreover, as shown in low magnification Fig. 1c, the self-supporting mat made of HA nanofibers showed non-woven cloth-like characteristic which should be particularly useful in modifying surface features for site specific delivery like drug, protein, polypeptides or gene.

Fig. 1 (a) ESEM images of precursor fibers that were electrospun from the sol containing 0.07 g·ml1 PVP at initial pH = 9; (b) ESEM images of HA fibers after being calcined in air at 600 C for 1 h; (c) low-magnification ESEM images of a self-supporting mat made of sub- micrometer HA fibers. The inset of (b) shows TEM of the HA after calcinations.

Journal of Bionic Engineering (2012) Vol.9 No.4

To figure out the role of pH in HA synthesis, we produced HA nanofibers under different initial pH values. Fig. 3 shows XRD patterns of two samples synthesized at pH = 4 and 9, respectively, which are consistent with the standard database (1997, JCPDS 73-1731). Interestingly, with the increase in initial pH value, diffraction peak of (210) crystal plane was enhanced notably, suggesting that crystal epitaxy changed. The orientation index (p) of HA crystal could be calculated from the formulae: R = I(210) / I(211) and p = R/R0, where I is the relative intensity of crystal plane, R and R0 are the ratios of crystal plane intensity with and without preferential orientation. The index p at pH = 9 is three times higher than that at pH = 4, showing crystal grain preferentially grown is dense along (210) plane at high pH value.

Deriv.weight (%/min)

Weight (%)

TGA curves of Fig. 2a suggest that weight-loss of precursor fibers kept little changeable at near 600 C, showing that the volatile substance such as hydrate, ethanol and the remaining TEP as well as PVP had been removed completely, and inorganic HA nanofibers were formed. FTIR spectra further show that the characteristic peaks of the PVP had been disappeared after heating at 600 C for 1 hour, which indicat that the polymer had been degraded (see Fig. 2b). The new peaks at ca. 1094 cm1, 1029 cm1, 959 cm1, and 602 cm1 are assigned to phosphate group[31]. The absorption bands at 634 cm1 and 3600 cm1 associated with hydroxide group, were typically observed in a HA FT-IR spectrum. The spectrum also shows carbonate group bands at 868 cm1 and 1420 cm1–1480 cm1, which could be attributed to take in carbon dioxide from air during preparation. Carbonate was substituted for phosphate or hydroxide sites in the structure of HA, tending to increase its solubility in comparison with stoichiometric HA[32]. Then it can be more easily resorbed by the living cells and leads to faster bone regeneration.

Intensity (counts)

480

Reflectance (a.u.)

Fig. 3 XRD patterns of HA fiber samples made of the sol at initial pH = 9 (1) and 4 (2), respectively.

Fig. 2 (a) TGA curve of the precursor fibers that were electrospun from the sol containing 0.07 g·ml1 PVP; (b) FT-IR spectrum of PVP fiber (1) and HA fibers (2) before calcination and (3) after calcination at 600 C.

Fig. 4 gives the effect of pH on surface structure of HA nanofibers. In Fig. 4a, HA fiber showed well-crystallized dense structure, while pH was reduced from 9 to 4, HA fiber turned into porous structure and fiber diameter decreased from 500 nm to 400 nm, as observed in Fig. 4b. Moreover, the textural properties of two samples are summarized in Table 1. The surface areas were calculated according to the Barrett-Emmett-Teller (BET) equation. The pore parameters were calculated from the adsorption branches of the isotherm from the Barrett-Joyner-Halanda (BJH) method. The specific areas of the samples obtained at pH = 9 was 48.5 m2·g1 while that of the sample at pH = 4 increased to 97.3 m2·g1. The pore volume and average pore size of the samples also showed the same trend.

Song et al.: Tuned Morphological Electrospun Hydroxyapatite Nanofibers via pH

Fig. 4 TEM image of HA fiber samples made of the sol containing 0.07 g·ml1 PVP at pH = 9 (a) and pH =4 (b), respectively.

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Fig. 5 The effect of pH on textural properties of HA nanofibers: (a) pH = 4; (b) pH = 9.

Table 1 Textural properties of the samples made from the sol with different pH pH

Surface area (m2·g1)

Pore volume (cm3·g1)

Average pore diameter (nm)

4

97.3

0.12

23

9

48.5

0.03

12

Organic phosphorus derivatives of various ester functional groups exhibit characteristics of hydrolysis upon exposure to water molecules[33,34]. The hydrolyzed phosphate may react with Ca precursor in aqueous environment through Eq. (1), bringing about a lot of H+. Then hydrolysis of TEP is very slow at PH <7, which reduces the P-O-Ca linkage formation. As expected in Fig. 5a, gel precursor domains were fewer and more dispersed as sphere in electrospun fibers, resulting in more pores in HA nanofibers after removing organic content. With initial pH increased to 9 in sol, the morphological structure of HA nanofiber also varies correspondingly (Fig. 5b). The cytotoxicity test for 3T3 mouse embryonic fibroblast cells suggested the obtained HA fibers were fairly non-toxic and safety. Exhilaratingly, we also found in the experiment that Zn incorporated HA fibers could be produced by tuning this combinatorial process and following Eq. (2), as seen in Fig. 6, and the incorporating amount of Zn might be tailored between 0 and 10 mol.%. HPO(OC2H5)3x(OH)x +Ca2+ + 2NO3P-O-Ca intermediate + H+,

4 Conclusion We demonstrate a method combining electrospinning with sol-gel to synthesize hydroxapatite nanofibers. After annealing in air at 600 C for 1 hour, electrospun PVP/sol-gel fibers turn into pristine HA nanofibers. We can control not only grown crystal but also morphological structure of HA fiber by tuning pH value. This novel and versatile strategy for the formation of 1D HA fiber also exhibits a key feature: the convenient introduction of dopants, such as Zn, Eu and Mg, into products through adjusting the precursor composition, which offers an opportunity to generate more perfect function.

Acknowledgments (1)

(10–x)Ca2++xZn2++ 6 PO43 +2OH Ca10–xZnx(PO4)6(OH)2 (0x 1).

Fig. 6 ESEM images of 5 mol% Zn-doped HA fibers. The inset shows the fibrous EDX spectrum.

(2)

We gratefully acknowledge the support from the National Natural Science Foundation of China (Project No: 20574066), the Natural Science Foundation of Jilin province (201115147), and project of Education Department of Jilin (2012115).

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References [1]

[14] Murugan R, Ramakrishna S. Aqueous mediated synthesis of

Zhao Y F, Ma J. Triblock co-polymer templating synthesis of

mesostructured

hydroxyapatite.

Microporous

and

Mesoporous Materials, 2005, 87, 110–117. [2]

Chen F, Wang Z C, Lin C J. Preparation and characterization of nano-sized hydroxyapatite particles and hydroxyapatite/chitosan nano-composite for use in biomedical materials.

[3]

orthopaedic/dental implant efficacy. Bioceramics, 2000, 13,

Bioactive, luminescent and mesoporous europium-doped

Ramachandra R R, Roopa H N, Kannan T S. Solid state

hydroxyapatite as a drug carrier. Biomaterials, 2008, 29,

G T. Photoresponse of sol-gel-synthesized ZnO nanorods.

Cuney T A, Korkusuz F, Timicin M, Akkas N. An investi-

Appied Physics Letters, 2004, 84, 5022–5024.

gation of the chemical synthesis and high-temperature sin-

[19] Sadasivan S, Khushalani D, Mann S. Synthesis of calcium

tering behavior of calcium hydroxyapatite (HA) and trical-

phosphate nanofilaments in reverse micells. Chemistry of

Science: Materials in Medicine, 1997, 8, 91–96.

Materials, 2005, 17, 2765–2770. [20] HanY J, Kim J, Stucky G D. Preparation of noble metal

Rhee S H, Tanaka J. Hydroxyapatite coating on a collagen

nanowires using hexagonal mesoporous silica SBA-15.

membrane by a miomimetic method. Journal of American

Chemistry of Materials, 2000, 12, 2068–2069.

Ceramic Society, 1998, 81, 3029–3031.

[21] Hu S J, Odom T W, Lieber C M. Chemistry and physics in

Liu H S, Chin T S, Lai L S, Chiu S Y, Chung K H, Chang C

one dimension: Synthesis and properties of nanowires and

S, Lui M T. Hydroxyapatite synthesized by a simplified

nanotubes. Accounts of Chemical Research, 1999, 32,

hydrothermal method. Ceramics International, 1997, 23,

435–445.

19–25. Layrolle P, Ito A, Tateishi T. Sol-gel synthesis of amorphous calcium phosphate and sintering into microporous hydroxyapatite bioceramics. Journal of American Ceramic [9]

4341–4347. [18] Ahn S E, Lee J S, Kim H, Kim S, Kang B H, Kim K H, Kim

Materials in Medicine, 1997, 8, 511–518.

cium phosphate (TCP) bioceramics. Journal of Materials

[8]

321–324. [17] Yang P P, Quan Z W, Li C X, Kang X J, Lian H Z, Lin J.

Bionic Engineering, 2012, 9, 224–233.

composite ceramic powders. Journal of Materials Science:

[7]

Biomaterials, 2000, 21, 1803–1810. [16] Webster T J, Siegel R W, Bizios R. Enhanced surface and

Wu X D, Song X F, Li D S, Liu J G, Zhang P B, Chen X S.

synthesis and thermal stability of HAP and HAP- -TCP

[6]

Enhanced functionsof osteoblasts on nanophase ceramics.

mechanical properties of nanophase ceramics to achieve

surfactant template method for protein delivery. Journal of

[5]

[15] Webster T J, Ergun C, Doremus R H, Siegel R W, Bizios R.

Materials Letters, 2002, 57, 858–861. Preparation of mesoporous nano-hydroxyapatite using a

[4]

bioresorbable nanocrystalline hydroxyapatite. Journal of Crystal Growth, 2005, 274, 209–213.

[22] Chen C C, Chao C Y, Lang Z H. Simple solution-phase synthesis of soluble CdS and CdSe nanorods, Chemistry of Materials, 2000, 12, 1516–1518. [23] Zhou Z H, Zhou P L, Yang S P, Yu X B, Yang L Z. Con-

Society, 1998, 81, 1421–1428.

trollable synthesis of hydroxyapatite nanocrystals via a

Jillavenkatesa A, Condrate Sr R A. Sol-gel processing of

dendrimer-assisted hydrothermal process. Materials Re-

hydroxyapatite. Journal of Materials Science, 1998, 33, 4111–4119. [10] Lopatin C M, Pizziconi V, Alford T L, Laursen T. Hydroxyapatite powders and thin films prepared by a sol-gel technique. Thin Solid Films, 1998, 326, 227–232.

search Bulletin, 2007, 42, 1611–1618. [24] Ma M G, Zhu J F. Solvothermal synthesis and characterization

of

hierarchically

nanostructured

hydroxyapatite hollow spheres. European Journal of Inorganic Chemistry, 2009, 2009, 5522–5526.

[11] Varma H K, Kalkura S N, Sivakumar R. Polymeric precur-

[25] Mohamed M B, Ismail K Z, Link S, El-Sayer M A. Thermal

sor route for the preparation of calcium phosphate com-

reshaping of gold nanorods in micelles. Journal of Physical

pounds. Ceramics International, 1998, 24, 467–470.

Chemistry B, 1998, 102, 9370–9374.

[12] Kordas G, Trapalis C C. Fourier transform and multi di-

[26] Li D, Xia Y N. Electrospinning of nanofibers: Reinventing

mensional EPR spectroscopy for the characterization of

the wheel?. Advanced Materials, 2004, 16, 1151–1170.

sol-gel derived hydroxyapatite. Journal of Sol-Gel Science

[27] Fong H, Chun I, Reneker D H. Beaded nanofibers formed

Technology, 1997, 9, 17–24.

during electrospinning. Polymer, 1999, 40, 4585–4592.

[13] Cengiz B, Gokce Y, Yildiz N, Aktas Z, Calimli A. Synthesis

[28] Song X F, Wang Z J, Liu Y B, Wang C, Li L J. A highly

and characterization of hydroxyapatite nanoparticles.

sensitive ethanol sensor based on mesoporous ZnO–SnO2

Song et al.: Tuned Morphological Electrospun Hydroxyapatite Nanofibers via pH nanofibers. Nanotechnology, 2009, 20, 075501. [29] Song X F, Qi Q, Zhang T, Wang C. A humidity sensor based on KCl-doped SnO2 nanofibers. Sensors and Actuators B, 2009, 138, 368–373.

483

mal processing. Journal of American Ceramic Society, 1996, 79, 837–842. [32] Pan H, Darvell B W. Effect of carbonate on hydroxyapatite solubility. Crystal Growth & Design, 2010, 10, 845–850.

[30] Lee K H, Kim H Y, Khil M S, Ra Y M, Lee D R. Charac-

[33] Schrotter J C, Cardenas A, Smaihi M, Hovnanian N. Silicon

terization of nano-structured poly( -caprolactone) non-

and phosphorus alkoxide mixture: Sol-gel study by spec-

woven mats via electrospinning. Polymer, 2003, 44,

troscopic techniques. Journal of Sol-Gel Science Technol-

1287–1294.

ogy, 1995, 4, 195–204.

[31] Russel S W, Luptak K A, Suchicital C T A, Alford T L,

[34] Tian F, Pan L, Wu X. The NMR studies of the P2O5-SiO2 sol

Pizzicoui V B. Chemical and structural evolution of

and gel chemistry. Journal of Non-Crystalline Solids, 1988,

sol–gel-derived hydroxyapatite thin films under rapid ther-

104, 129–134.