Influence of fluorine substitution on the morphology and structure of hydroxyapatite nanocrystals prepared by hydrothermal method

Materials Chemistry and Physics 137 (2013) 967e976

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Influence of fluorine substitution on the morphology and structure of hydroxyapatite nanocrystals prepared by hydrothermal method A. Joseph Nathanael a, b, D. Mangalaraj c, *, S.I. Hong b, **, Y. Masuda d, Y.H. Rhee e, H.W. Kim e a

Department of Nanomaterials Engineering, Chungnam National University, Daejeon 305-764, South Korea Thin Film and Nanomaterials Laboratory, Department of Physics, Bharathiar University, Coimbatore 641 046, India c Department of Nanoscience and Technology, Bharathiar University, Coimbatore 641 046, India d National Institute of Advanced Industrial Science and Technology (AIST), 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan e Department of Microbiology, Chungnam National University, Daejeon 305-764, South Korea b

h i g h l i g h t s < Fluorapatite nanorods were produced hydrothermally with different fluorine content. < Fluorine substitution was found to alter the morphology of crystals appreciably. < It enhances the crystallinity, orientation dependent growth and hence aspect ratio. < In vitro cellular analysis shows excellent cell viability of the fluorapatite.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 January 2012 Received in revised form 2 July 2012 Accepted 1 November 2012

Hydroxyapatite (HAp) nanocrystals with different levels of fluorine substitution (P/F ¼ 0, 6, 4 and 2) on the OH sites were produced via hydrothermal method. The fluorine substitution was found to alter the morphology of crystals appreciably. The aspect ratio and the crystallinity of HAp crystals increased with increasing fluorine substitution. The presence of broad ring and hallow ring patterns in electron diffraction suggests the low-crystalline nature of HAp crystals. With increasing fluorine substitution, the diffraction patterns exhibited discrete rings and numerous diffraction spots, implying the increased crystallinity. Raman spectra from the HAp nanoparticles also support the less-crystalline nature of the pristine HAp and the enhanced crystallization by fluorine substitution. In HAp crystals processed with no fluorine substitution, surface energy and planar Ca2þ density are less sensitive to the crystallographic orientation because of its low-crystalline nature, favoring equi-axed or slightly elongated particles. The addition of fluorine apparently increased the crystallinity, enhancing the orientation dependent growth and accordingly the aspect ratio. Osteoblast proliferation was observed to be enhanced by fluorine substitution in HAp. In vitro biological data support that the excellent osteoblastic cell viability and functional activity of the fluoridated apatite. Ó 2012 Elsevier B.V. All rights reserved.

Keywords: Biomaterials Nanostructures Crystal growth Electron microscopy Mechanical testing

1. Introduction Hydroxyapatite (HAp, Ca10 (PO4)6 (OH)2) has attracted much interest as an implant material for teeth and bones, due to the similarity of its crystallography and chemical composition to the mineral of human hard tissues. Nanocrystalline HAp is found in natural bone, where nanoscale apatite-like crystals mineralize * Corresponding author. Tel.: þ91 422 2425458; fax: þ91 422 2425706. ** Corresponding author. Tel.: þ91 82 42 8216595; fax: þ91 82 42 8225850. E-mail addresses: [email protected] (A. Joseph Nathanael), dmraj800@ yahoo.com (D. Mangalaraj), [email protected] (S.I. Hong). 0254-0584/$ e see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matchemphys.2012.11.010

collagen fibers [1e3]. Along with pristine HAp, HAp with the addition of other inorganic elements such as fluorine [4,5], strontium [6e8], europium [9], yttrium [10] etc., has been used for some specific application such as drug delivery [7,9] and light emitting [5] and luminescence applications [2,8]. Fluor-hydroxyapatite [FHAp; Ca10 (PO4)6 (OH,F)2] its fluoridated form, has gained its importance in the area of dental restoration because of its specific biological benefits including the resistance of apatite to resorption by body fluids [11]. Incorporation of fluorine is known to protect the teeth from caries formation [4]. The bioactivity, osteoconductivity and therapeutic effects of HAp are dependent on the solubility in biological environment.

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Pure fluorapatite (FA; Ca10 (PO4)6 (F)2) is known to have a much lower solubility in biological fluids than HAp, because FA possesses a greater stability than HAp, both chemically and structurally. The partial incorporation of fluorine in order to elicit its biological function is one of the main themes in dental restoration research. Dissolution of HAp is influenced by many issues like: crystallinity, chemical composition and its stoichiometry. Substitution of OH groups in HAp by F ions is one of the appropriate procedures for decreasing the dissolution rate of apatite, which leads to the formation of fluorine-substituted HAp (FHAp). As a result, increase in crystallinity, a decrease in crystal strain, and an increase in thermal and chemical stability are obtained [12,13]. The existence of large amount of fluorine on the outer layer of the teeth enamel is known to protect against the formation of dental caries and to stimulate the bone cell response and matrix synthesis. This is due to the formation of fluoridated HAp in teeth which has a higher acid resistance than pristine HAp [14]. Other than above-mentioned applications, since fluoride incorporation in the bioactive glasses results in fluoride release for caries prevention, fluoride-containing bioactive glasses find application in toothpastes [15]. Additionally, fluorine-substituted bioactive glasses have been shown to form fluorapatite in physiological solutions, which has high chemical stability than HAp [16,17]. The FA forms fluor-hydroxyapatite solid solution with HAp through the replacement of OH by F. Hence the modification of the extent of fluoride substitution provides an effective way of controlling the stability of the apatite [12]. It is generally expected that the introduction of F will retard the decomposition of nanoscale HAp crystals. However, the effects of fluorine substitution on the morphology, structure and chemistry of nanocrystalline apatite are not well reported and understood. Prior to utilization of the nanocrystalline fluoridated apatite as hard tissue replacements and/or a bio-ceramics suitable for long-term implant fixation [12], their mechanical reliability and structural stability need to be studied. Nanocomposites of apatite and synthetic polymer provide a combination of favorable mechanical performance and bioactivity and, therefore, have been developed as bone analogue composite mimicking the properties and the structure of bone. Polyethylene (PE) is established as a biocompatible material and is extensively used in orthopedics applications [18] while HAp strongly resembles bone minerals. Extensive work has been carried out on HMWPE/ HAp composite as a substitute for cortical bone, the major load bearing type of bone. The mechanical performance of HMWPE/HAp composites was found to be strongly influenced by the structure and shape of nanoscale HAp crystals [1,19,20]. It would be, therefore, of great interest to study the mechanical performance of HMWPE matrix composites reinforced with nanoscale HAp crystals modified by fluorine substitution. This paper deals with the formation of fluoridated hydroxyapatite nanocrystals by means of hydrothermal synthesis and their morphological, phase and structural changes as a function of fluorine substitution. Their mechanical properties and in vitro cellular assay were also studied. 2. Experimental section 2.1. Synthesis In a typical synthesis process, calcium nitrate (Ca(NO3)2$4H2O) and diammonium hydrogen phosphate ((NH4)2HPO4) were used as calcium (Ca) and phosphate (P) sources, respectively. The HAp was prepared by taking calcium nitrate and diammonium hydrogen phosphate separately and mixed with de-ionized water with the molar ratio of 1:0.6 in order to maintain the Ca/P ratio 1.67 which is the stoichiometric molar ratio of hydroxyapatite. The pH of the

phosphate containing solution was increased to 9 by adding ammonium hydroxide (30%). Three sets of P solutions containing different concentrations of fluorine ion were also prepared by dissolving ammonium fluoride (NH4F) in water. The molar ratio of P to F (P/F)in fluorine-substituted HAp (FHAp) were chosen to be 6, 4 and 2 and designated as FHAp1 (P/F ratio ¼ 6), FHAp2 (P/F ratio ¼ 4) and FHAp3 (P/F ratio ¼ 2), respectively, afterwards. During the reaction the phosphate containing solution was added drop wise into the calcium containing solution with vigorous stirring for 1 h. The mixed solution was transferred to the Teflon beaker of the stainless steel autoclave and placed in the oven at 180  C for 12 h for reaction. After the reaction the autoclave was cooled down to room temperature automatically. The final precipitate were washed several times with distilled water and dried at 100  C overnight. The dried powders were crushed with mortar and pestle and calcined at 600  C for 2 h. 2.2. Preparation of polymer-nanoparticle composite for mechanical analysis For mechanical strength analysis, a high molecular weight polyethylene (HMWPE) was reinforced with HAp for fabricating HAp/polymer composite. HAp and FHAp nanorods prepared by hydrothermal method were mixed with HMWPE. Before mixing, HMWPE and hydroxyapatite nanoparticles were dried in an oven at 120  C for 1 h and cooled down to room temperature to remove the moisture contents. 10% of nanorods prepared by hydrothermal method were mixed with the HMWPE and the blending was carried out in a micro compounder (HAAKE MiniLab II) with the mixing temperature of 180  C. The rotor speed at 80 rpm for a mixing time of 20 min was used for all the sample preparation. A piston injection moulding system (HAAKE MiniJet 557-2270) was used for preparing composite specimen for the mechanical performance analysis. 2.3. In vitro cellular assay To investigate the cellular response to the F doped hydroxyapatite nanorods, CHO model animal cells were cultured on. In vitro cellular assay was carried out by the following procedure: CHO cells (CHO-K1, Korean Collection for Type Cultures), the model animal cell, were used to study F doped HAp nanopowders. The powder samples were coated on the 3 M adhesion tape. The prepared films were washed with PBS for 24 h and were then placed at the bottom of the wells of a multi-well tissue culture plate. After removing the PBS solution from the multiwall tissue culture plate by pipetting, the CHO cells (4  104 cm2) were seeded to the film surfaces. Ham’s F-12 nutrient mixture (Gibco Laboratories) containing 5% fetal bovine serum, 100 U/mL penicillin and 100 mg mL1 gentamycin was used as the culture medium. The cells were cultured in an incubator at 37  C under a 5% CO2 atmosphere. At the end of each incubation period, the supernatant was withdrawn and each well was washed with PBS and treated with trypsin (0.05% trypsin/0.02% ethylene-diamine-tetra-acetic acid, Gibco). The morphology of the cultured cells, which were fixed in 2.5% glutaraldehyde solution, was observed using a JEOL JSM-7000F scanning electron microscope (SEM). 2.4. Characterization The prepared samples were structurally characterized by X-ray diffraction (XRD) analysis using a Cu-Ka1 radiation (Rigaku X-ray diffractometer D/max-2200). The Raman spectra of the present study were recorded using a laser Raman spectrometer (Renishawin Via Raman Microscope) at an output power of 10 mW of

A. Joseph Nathanael et al. / Materials Chemistry and Physics 137 (2013) 967e976

a 514 nm Arþ laser. Spectra were corrected using LO-phonon mode of Si (100) substrates observed at 520.5 eV. Particles on carbon tapes were evaluated with an X-ray photoelectron spectroscopy (XPS). For this work, XPS, Kratos analytical, ESCA-3400, Shimadzu equipment was utilized. The X-ray source (MgKa, 1253.6 eV) was operated at 10 kV and 20 mA. Resolutions for survey analyses or narrow analyses were 1.15 eV or 0.95 eV, respectively. Step size (eV) or dwell time (s) for survey analyses or narrow analyses were 1 eV, 150 s or 0.1 eV, 300 s, respectively. Spectra were corrected by 1.25 eV using standard binding energy of CeC bonds (C 1s, 284.6 eV) in surface contaminations. The morphology, particle size and size distribution of particles were investigated by a field emission scanning electron microscope (FESEM JEOL JSM-6500) at 10 kV after Sputter coated platinum for conduction. To gain further insight into the microstructures, transmission electron microscopic (TEM) investigations were performed using JEOL JEM-2100. Samples for TEM analysis were prepared by air-drying a drop of a sonicated suspension of the dried precipitate in ethanol onto copper grids. Selected area electron diffraction (SAED) pattern was also taken to know the crystallinity of the samples. Nitrogen adsorptionedesorption isotherms of the present study were obtained using Autosorb-1 (Quantachrome Instruments). Samples were out gassed at 110  C under 102 mmHg for more than 6 h prior to measurement. Specific surface area was calculated by BET (BrunauereEmmetteTeller) method using adsorption isotherms. Pore size distribution was calculated by BJH (Barrette JoynereHalenda) method using desorption isotherms. The tensile experiment was performed using a universal testing machine (810 Material Test Systems) at a crosshead speed of 10 mm min1. A 100 kN load was used for tensile tests. The yield strength was determined from the upper yield point and the fracture strain was the elongation at break point in the tensile curve. The reported mechanical properties were calculated by averaging the measurements from five specimens.

969

3. Result and discussion 3.1. Morphological analyses The microstructure of the hydrothermally synthesized FHAp nanoparticles in the present study was observed by FESEM. Fig. 1aed shows the morphology of pristine HAp (1(a)), FHAp1 (1(b)), FHAp2 (1(c)) and FHAp3 (1(d)), respectively. Some elongated crystals along with mostly spherical ones were seen for pristine HAp in Fig. 1a. The fluorine substitution was found to alter the morphology of crystals appreciably as shown in Fig. 1aed. The rod-shaped crystals increased with the increasing fluorine substation. It appears that the aspect ratio of crystals increased with increasing fluorine substitution as will be discussed below. TEM images (Fig. 2) provide a further insight into the nanostructure and morphology of HAp and FHAp. Fig. 2aeh exhibits the TEM images (2aed) and diffraction patterns (2eeh) of pristine HAp (2(a) and (e)), FHAp1 (2(b) and (f)), FHAp2 (2(c) and (g)) and FHAp3 (2(d) and (h)), respectively. Consistent with the SEM observation, the morphology of crystals was found to be modified appreciably by the fluorine substitution as shown in Fig. 2aed. It is obvious that not only the population of rod-shaped crystals increased but also the aspect ratio of crystals increased with increasing fluorine substitution. The shape of the HAp crystallite observed by TEM is observed to be mostly equi-axed and some slightly elongated with a mean particle size of 45  5 nm in diameter. For pristine HAp the aspect ratio is 1.2  0.4, supporting the formation of slightly elongated nanoparticles. The average diameter and length of FHAp1 crystals were found to be 30  5 and 80  15 nm, respectively. TEM images of FHAp2 and FHAp3 supports that the aspect ratio of FHAp particle increased appreciably with increasing fluorine substitution. For initial fluorine addition (FHAp1) the aspect ratio is 2.5  0.5. The aspect ratio of the nanorods increased upon further increasing the F

Fig. 1. FESEM images of (a) HAp, (b) FHAp1, (c) FHAp2, and (d) FHAp3. Inset: higher magnification of corresponding images (scale bar is 100 nm).

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Fig. 2. TEM images of (a) pure HAp, (b) FHAp1, (c) FHAp2, (d) FHAp3, and (eef) SAED patterns of (aee), respectively.

concentration (for FHAp2 aspect ratio is 3.2  0.2; for FHAp3 it is 4.5  0.3). The corresponding electron diffraction pattern analyses exhibited broad ring patterns along with some diffraction sports, suggesting low-crystalline nature of apatite crystals. With increasing fluorine substitution, the diffraction patterns exhibited discrete rings and more diffraction spots, suggesting the enhanced crystallinity. For FHAp3, the diffraction patterns show distinct numerous diffraction spots (Fig. 2h), implying the highly crystalline nature of crystals.

3.2. Structural analyses The structural characterization of the hydrothermally prepared HAp and FHAp is shown in Fig. 3. The HAp and FHAp nanocrystals exhibited typical peaks associated with apatite although there is some modification of the sharpness of the peak associated with the crystallinity of nanocrystals. A close examination revealed the peak shifted slightly to the right with the addition of fluorine. The peak shifted steadily with increasing fluorine concentration. The shifts in

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a (Ao)

(210)

(102)

(002)

9.45 9.42 9.36

c (Ao)

Intensity (a.u)

b

6.88

c/a ratio

30

FHA3

FHA2

0.734

c

0.732 0.730

Aspect ratio Cryst. deg. (%)

29

(004)

(222) (213)

27 28 2θ (d eg )

(310)

26

Intensity (arb.units)

(002) (102) (210) (211) (300) (220)

6.89 6.87

100 75 50 25 0 6 4

d

e

2 0

HAp FHA1

HAp

20

a

9.39

6.90

25

25

30

971

35 40 45 2θ (degrees)

50

55

FHAp1

FHAp2

FHAp3

Samples Fig. 4. Change in (a) a (b) c lattice parameters and (c) c/a ratio (d) degree of crystallinity and (e) aspect ratio, respectively, as a function of different fluorine substitution.

60

Fig. 3. XRD patterns of (a) pure HAp, (b) FHAp1, (c) FHAp2, and (d) FHAp3. Inset: Enlarged view of the specific part of the XRD patterns shows the shift in 2q with increase in F concentration. Dotted lines show the peak position of pure HAp.

the peaks were shown in the inset of Fig. 3. These peak shifts reflect the change in lattice parameters of the apatite structures resulting from the fluorine incorporation. It should be also noted that the (211) peak become much sharper and the peak gradually split into two peaks as the fluorine concentration increased, implying the enhanced crystallinity with increased fluorine substitution. Fluorine substitution was also found to significantly affect the lattice parameters of the apatite structure. The lattice parameters were calculated from XRD analysis and it supports that fluorine substitution notably affects the lattice parameters. The changes in the lattice parameters are presented in Fig. 4. The lattice parameters of the HAp nanocrystals were calculated to be 9.417  A (a-axis) and 6.882  A (c-axis) whereas those of FHAp1were 9.398  A (a-axis) and 6.881  A (c-axis). These values for HAp nanorods are in good agreement with the theoretical values of: 9.417  A (a-axis) and 6.881  A (c-axis). In the case of FHAp, there was an apparent decrease in the a-axis with little change in c-axis (Fig. 4(a, b)). As fluorine ion substituted the hydroxyl ion in apatite, the lattice contracts along the a-axis with little change in the c-axis. When the molar ratio of P to F changed from 6 to 2, the lattice constant also changed accordingly. When the P/F ratio is 2, the apatite structure is thought to be close to that of FA structure, and the a-axis value decreased to 9.379  A, which is close to the reported value for pure FA (a-axis 9.368  A). Also the 2q value also changed from 25.8 to 26.4 for (002) plane as shown in inset of Fig. 3. The a-axis decreased almost linearly with increasing fluorine substitution [21e23]. As a result, the c/a ratio increased with the increasing fluorine substitution (Fig. 4(c)). The fluorine substitution in nanoscale particles produced by the hydrothermal method in the present study has the similar influence on the lattice constants as in micro-scale apatite crystals [23].

The changes in the lattice constant can be attributed to the replacement of OH by F [12]. The 10 calcium atoms in HAp structure can be considered to consist of Ca (I) (four atoms) and Ca (II) (six atoms). Le Geros [24] suggested that OH groups shown as a single unit in this arrangement (Fig. 5) are positioned between the imaginary calcium triangles in a linear OeHeOeH arrangement parallel to c-axis as exhibited in Fig. 5. The apatite structures are known to accommodate readily various anionic or cationic substitutions [25]. The negatively charged F ions added at the time of synthesis have a tendency to replace the OH ions placed in the A) disordered positions (Fig. 5). Since the ionic radius of F ion (1.32 A), F ions are likely to fit better in the is smaller than OH ion (1.68  imaginary Ca triangles than OH ions [25]. It appears that the decrease of lattice constant along a-axis in FA with the fluorine substitution can be explained by this simple geometrical consideration. A special note in the XRD data is the variation in the intensity and width of the apatite peaks for the HAp and FHAp nanocrystals and this variation is known to be closely related to the crystallinity and the crystallite size of the hydrothermally prepared nanocrystals. Addition of fluorine content notably increased the crystallinity as well as the aspect ratio of HAp crystals as exhibited in Fig. 2aed. To estimate the average crystallite size, L, of the nanoparticles we employed DebeyeScherer formula:

Fluoride Calcium Oxygen Hydrogen Fig. 5. The relative positions of the OH and F atoms at the centre of the Ca (II) imaginary triangle [20]. Courtesy of R.Z. Le Geros.

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L ¼ kl=bcos q

(1)

where k ¼ 0.94 (shape factor), l is the wavelength of the radiation used (1.54056  A), b is the full width at half maximum of the peak in radians and q is the Bragg angle. The equation was applied to the major peaks in the corresponding powder diffraction patterns shown in Fig. 3. The crystallite size increases from 10  5 nm to 30  7 with the increase of fluorine addition. It is reported that the existence of fluorine within the apatite structure promotes the crystallization of the apatite structure along the c-axis direction. The increase of fluorine addition was observed to influence not only the crystallite size but also the crystallinity. Degree of crystallinity increasing with increasing F concentration can be demonstrated by the increasing sharpness of XRD peaks in Fig. 3. The degree of crystallinity, corresponding to the fraction of crystalline phase present in the examined volume, was evaluated by the relation [26]:

  Xc z1  Vð112=300Þ =Ið300Þ

(2)

1049 1080

588

435

Intensity(arb.units)

where I300 is the intensity of (300) reflection and V112/300 is the intensity of the valley among (112) and (300) planes. As the fluorine ion concentration increases V112/300 is visible whereas for pristine HAp it was completely disappears since the crystallinity is very low (Fig. 4(d)). It is plausible to assume that the crystallinity and the aspect ratio are inter related. It has been reported that the crystalline HAp grows along the c-axis because of the high surface energy and low planar density of Ca2þ ions on (001) plane [27]. In HAp crystals processed with no fluorine substitution, surface energy and planar Ca2þ density is less sensitive to the crystallographic orientation because of its low-crystalline nature, favoring equi-axed or slightly elongated particles. The addition of fluorine apparently increased the crystallinity, enhancing the orientation dependent growth into rod-shaped crystals and accordingly the aspect ratio (Fig. 4(e)). The reason why the fluorine substation increases the crystallinity will be discussed at the end of this section. Fig. 6 shows the Raman spectrum obtained from the HAp and FHAp nanoparticles. Peaks are found at 435 cm1, 588 cm1, 1049 cm1, and 1080 cm1. The characteristic peak at 958e 965 cm1 is due to symmetric stretching vibrations of the

(d) (c) (b) (a)

200

400

600

800

1000

1200

-1

Wave number (cm ) Fig. 6. Raman spectra of (a) HAp, (b) FHAp1, (c) FHAp2, and (d) FHAp3 nanoparticles. Inset: Enlarged view of the specific peak which shows the shift in the wave number with the increase in F concentration.

phosphate group (PO3 4 ). The position of this peak is a good indicator of the degree of crystallinity of the material: it shifts to w955 cm1 in more amorphous apatite, but is found at w965 cm1 in more ordered, non-carbonated apatite [28]. It is clearly observed from inset figure in Fig. 6 that, initially the pristine HAp shows the value at 958 cm1 which supports the less-crystalline nature of apatite. But as the fluoride content is increased the shift of the peak toward the crystalline nature of the apatite was observed along with the increase in the peak intensity. The 965 cm1 peak of the FHAp3 nanoparticles is very sharp, indicating it is well crystallized. The weak peak at 1080 cm1 can be assigned to the carbonate group (B-type carbonate, n3) [28,29]. This suggests that the improvement in crystallinity is caused by the increase of the F content, consistent with the results from the XRD analyses. The F concentration in the prepared FHAp is determined by XPS as shown in Fig. 7(aec). The dotted line (Fig. 7d) shows the ideal fluorine content where all F in the solution completely incorporated into the HAp lattice. The dashed line with the data points measure F concentrations in the synthesized FHAp nanoparticles. A discrepancy is observed between the ideal F incorporation and the measured F incorporation, especially at high concentrations, which is attributed to the loss of fluorine in the form of n-CaF2 during the reaction process [30]. XPS narrow scan reveals only one peak located at 684.2 eV belonging to F1s which is the fingerprint of F in the FHAp or FA structure [31,32], indicating that fluoride ions have been successfully incorporated into the HAp lattice structure. In Fig. 7(c), a trace of F1s in CaF2 was found at 686.7 eV, which is due to reaction between Ca2þ and F ions at a higher F concentration [22,30]. Fig. 8 shows the variation in P/F, Ca/F and F/Ca ratios with the increase of F ion concentration. The P/F was set at 6, 4 and 2 in the preparation process and the measured ratios were found to be similar to these values. Only small variations are observed from the initial value. The Ca/P ratio varies from 1.56 to 1.66 on increasing the F ion concentration. For pure HAp, it was 1.67 which is close to the stoichiometric Ca/P ratio of apatite. As reported earlier, this can be related to the smaller fluorine ion size in comparison with the OH ions in pristine HAp. Higher F ion concentration possibly promotes faster incorporation of tiny F ions into the larger OH ions sites of apatite structure [21]. In a pristine HAp, F/Ca ¼ 0 since there is no fluorine. In an FHAp, the F/Ca ratio varies from 0 to 0.2. In a pure FA, F/Ca ¼ 2:10 ¼ 0.2 which is the stoichiometric limit [30] for fluorapatite. In our case, the F/Ca varies from 0.11 to 0.19 by increasing the F concentration. In the higher concentration also, F is not sufficient enough to form fluorapatite. Further increase in the F may result in the formation of FA. This is compatible with the XRD analyses of the present study in which the lattice parameters increased with fluorine substitution, but have not reached the exact value of stoichiometric FA. This is also supported by the presence of F1s trace in CaF2 observed at 686.7 eV in Fig. 7(c), which is due to the reaction between Ca2þ and F ions at a higher F concentration [22,30]. The increase of the crystallinity with increasing fluorine substitution was confirmed by electron diffraction analyses (Fig. 2), XRD analyses (Figs. 3 and 4) and Raman spectra analyses (Fig. 6). The enhanced crystallinity of HAp crystal by fluorine substitution can be explained in terms of the substitution of OH by F in its structure. Usually the HAp structure can be viewed as the isolated 2þ ions parallel to the a-axis, PO3 4 tetrahedral oriented toward Ca     along with the X (OH or F or Cl ) anions of the electronegative elements placed around Ca2þ ions in the direction perpendicular to the c-axis [33,34]. Once the OH are partially substituted by the F, the existing hydrogen atoms of the OH group are tightly bound to the nearby F anions because of the higher affinity of the fluorine with respect to the oxygen, producing a quite well-ordered apatite

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a

b

973

684.2 eV

Intensity (arb.units)

Intensity (arb.units)

684.2 eV

687

686

685

683 687

684

686

684

683

682

681

Binding energy (eV)

Binding energy (eV)

c 686.7

Measured F- concentration (y)

684.2

0.3

Intensity (arb.units)

685

d

0.2

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0.0 689

688

687

686

685

684

683

682

0.0

0.1

0.2

0.3

-

Binding energy (eV)

Intented F concentration (x)

Fig. 7. XPS F1s spectrum of (a) FHAp1, (b) FHAp2, (c) FHAp3, and (d) a discrepancy observed between the intended (x value) and the measured F incorporation (y value).

P/F ratio

6

structure, which leads to increasing crystallinity [33,34]. From the XPS analysis it is found that, initially there was a decrease in the Ca/ P ratio, that is, a decrease in the stoichiometry of the apatite. When lower amount of F is added to HAp, which was not sufficient to replace the considerable amount of OH in HAp resulting in lower stoichiometry (Fig. 7). By increasing the F concentration, increase in crystallinity as well as stoichiometry was observed.

5 4 3 2 1

Ca/P ratio

1.70

3.3. Specific surface area

1.65

The determination of the specific surface area (SSA) is often a prerequisite for quantization study and interpretation of adsorption properties. In the study of bioactive materials, the evolution of SSA has seldom been considered a suitable monitor of surface activity/reactivity. In this work, the specific surface area and pore size distribution for the prepared nanoparticles were measured by the nitrogen adsorptionedesorption isotherms using the BET and BJH methods. The particle size (DBET) was estimated by assuming the primary particles to be spherical

1.60 1.55

F/Ca ratio

1.50

0.20

DBET ¼ 6=rs

0.15

0.10

FHAp1

FHAp2

FHAp3

Samples Fig. 8. Variation in P/F, Ca/F and F/Ca ratios with the increase of F ion concentration.

(3)

where r is the theoretical density of the sample (3.156 g cm3 for HAp and 3.13 g cm3 for FHAp) and s is the SSA. The adsorptione desorption isotherms and the pore size distribution are shown in Fig. 9. The FHAp shows a higher SSA with respect to the corresponding pure HAp and it is in good agreement with the reported data [35,36]. The distinct hysteresis loop for all the samples can be

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40

150

Stress (MPa)

Volume Adsorbed (cc/g)

200

50

a

Adsorption Desorption

100

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30

(b)

(c)

20

10

0 0.0

0.2

0.4

0.6

0.8

0

1.0

0

100

Relative Pressure (P/P0)

200

300

Elongation (%)

b

Adsorption Desorption

250

Fig. 10. Stressestrain curve of HMWPE reinforced with (a) pristine HAp, (b) FHAp1, (c) FHAp2, and (d) FHAp3.

200

bonding between HWMPE and HAp crystals can be enhanced with increase of SSA. SSA establishes the amount of surface contact between the polymer matrix and the filler surfaces. More surface contact and bonding between the filler and the polymer matrix is achieved by the fillers with higher specific surface areas and this increases the mechanical properties of the polymer/ nanocomposite materials [20].

150

100

50

3.5. In vitro cellular analysis

0 0.0

0.2

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0.6

0.8

1.0

Relative Pressure (P/P 0) Fig. 9. Adsorptionedesorption isotherms of (a) pristine HAp and (b) FHAp3. Inset: corresponding pore size distribution.

categorized as type III isotherms. All HAp, FHAp1, FHAp2 and FHAp3 nanocrystals exhibited mesoporous nature with an SSA of 63.28, 66.39, 71.21 and 75.57 m2 g1, respectively. 3.4. Mechanical property The effect of the morphology of HAp and FHAp nanocrystals on the mechanical performance of the HMWPE/nanoparticle composite was analyzed and stress-strain responses are plotted in Fig. 10. As explained earlier, nanocomposites of apatite and synthetic polymers provide a combination of favorable mechanical performance and can been developed as bone analogue material to mimic the properties and the structure of bone. Hence, it would be, of immense significance to study the mechanical performance of HMWPE matrix composites reinforced with different kind of nanoscale HAp crystals. The reinforcement of polymer with HAp crystals is expected to enhance the bioactivity, but reduce ductility of the polymer. It is observed that the ductility of FHAp/HMWPE nanocomposite was improved compared to that of pristine HAp (Fig. 10(bed)). The increased ductility may be associated with the enhanced interfacial bonding between HWMPE and HAp crystals. In order to explore the relationship between the mechanical performance of HWMPE/HAp composites and surface properties of HAp crystals, the ductility of HWMPE/HAp composites is plotted against the specific surface area of the HAp and FHAp nanocrystals in Fig. 11. The fracture strain increased with the specific surface area of the HAp and FHAp nanocrystals, suggesting the interfacial

It was observed that osteoblast proliferation increased on all nanophase fluorine substituted HAp tested in the present study (Fig. 12) compared to conventionally formed HAp. Especially, the proliferation of osteoblasts increased with increase of F concentration. The degree of osteoblast proliferation was found to be similar for pure HAp and FHAp1 (Fig. 11a and b), but increased with further increasing the F concentration (FHAp2 and FHAp3) (Fig. 11c and d). Based on the in vitro biological data, it is deduced that the cell viability and activity for the fluoridated apatite are better than those of pure HAp. This observation supports the potential use of the fluorine-substituted apatite, because HAp has already proven its excellent biocompatibility both in vitro and in vivo. The improved cell viability of FHAp can be attributed to the

325 FHAp3

300 FHAp2

Ductility (%)

Volume Adsorbed (cc/g)

(d)

(a)

275 FHAp1 250 HAp 225

200 60

65

70

75

80

2

Specific surface area (m /g) Fig. 11. Relationship between the specific surface area and the ductility of the different polymer nanocomposites.

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Fig. 12. SEM image of the CHO cells grown on the HAp and FHAp nanoparticles: (a) pure HAp, (b) FHAp1, (c) FHAp2, and (d) FHAp3.

high specific surface area [37e40] compared to pure HAp. Many reports revealed that the higher specific surface area allows improved cell adhesion, protein and drugs [41e45]. Some researchers suggested that the porous structure and high surface area of the fibers were capable of facilitating the transportation of oxygen, nutrients and the metabolic waste of cells, the migration of cells and communication between them [20,44,45], all of which contribute to better cell growth. In the literature, higher cellular responses for the FHAps were also associated with the effect of fluorine itself [24,46]. It is well documented that fluorine promotes bone formation in vivo and enhances re-mineralization (repair) and calcification in vitro [24,46]. Also it was explained that [47], when F replaces the OH groups in the HAp, the negative charges on the surface of the material increase. It has been revealed that the OH groups in the HAp supply binding sites for cell absorption. More fluorine ions might be released into the cell culture medium with increase of the cell culture time, and thereby stimulates cell attachment [47]. These results collectively suggested that not only hydroxyl groups, but also fluorine ions released from the FHAp nanocrystals improve the cell attachment and subsequent cell activities. The fluorine ion is known to enhance the cellular responses when administrated within an appropriate range of concentration (105e107) [4]. In the present study, the fluorine released from the FHAps is observed to be below the detection limit (<107) under the test conditions in vitro. Therefore, the cellular response might not be affected appreciably by the compositional difference of fluorine in this study. However, the long-term use of the fluorine-substituted apatite in vivo may reveal the fluorine effect. This issue, raised by

the in vitro tests, requires further in vivo animal study in order to confirm the biological feasibility of the FHAps. 4. Conclusion The synthesis of fluorine-substituted hydroxyapatite with varied concentration of substituted anions has been achieved by hydrothermal method. The substitution of fluorine ions remarkably alters the morphology, aspect ratio, degree of crystallinity. With increasing fluorine substitution, the diffraction patterns exhibited discrete rings and numerous diffraction spots, implying the increased crystallinity of HAp crystals with increase of fluorine content. In HAp crystals processed with no fluorine substitution, surface energy and planar Ca2þ density is less sensitive to the crystallographic orientation because of low crystallinity, favoring equi-axed or slightly elongated particles. The addition of fluorine apparently increased the crystallinity, enhancing the orientation dependent growth and accordingly the aspect ratio. The increase of the crystallinity with increasing fluorine substitution was confirmed by electron diffraction analyses, XRD analyses and Raman spectra analyses. The enhanced crystallinity of HAp crystal by fluorine substitution can be associated with the substitution of OH by F in its structure. The ductility of HMWPE/FHAp nanocomposite improved compared to that of pristine HAp, which is associated with the enhanced interfacial bonding due to the increased specific surface area in FHAp nanocrystals. The enhanced osteoblast proliferation by fluorine substitution in HAp can also be attributed to the high specific surface area of FHAp nanocrystals. The mechanical and cell compatibility analysis revealed that the F

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substituted HAp nanorods are the promising candidate for the bone analogue materials for biomedical application.

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