Enhanced cell viability of hydroxyapatite nanowires by surfactant mediated synthesis and its growth mechanism

Abstract

Hydroxyapatite (HA) nanowires were grown by a simple hydrothermal method using cetyl-trimethyl-ammonium-bromide (CTAB) as a surfactant. The growth of preferentially oriented nanowires were suggested to be formed by the orientation-dependent electrostatic interaction between the CTAB ions and HA crystals. We prepared HA nanowires with a diameter of 30 ± 7 nm and the length of few micrometers. The prolonged hydrothermal treatment with the ideal concentration of CTAB induce the preferential growth of HA nanowires in the c-axis direction. The pronounced peak broadening of mainly (002) planes with the increase of the length of nanowires can be attributed to the natural bending of nanowires with an extremely high aspect ratio. The in vitro cell cultural studies of the HA nanowires showed that the osteoblast cell response enhanced with the uniformity and the specific surface area of HA nanowires.

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1. Introduction

One dimensional (1-D) nanostructured materials exhibit interesting and novel size- and morphology-dependent properties. Different inorganic 1-D nanostructures characterize the diameter dependence of nanostructures for several physical and chemical properties and have accordingly been synthesized for applications in numerous fields. 1D nanostructures will form by the way in which atoms are assembled into a structure with a nanometer thickness but with a much larger length¹ due to the highly orientation dependent preferential growth. They exhibit attractive and unique properties in the nanoscale which are very different from that of bulk materials.^2–4 Substantial development has been found in the formation of one and two dimensional (1D, and 2D) nanostructures such as nanoparticles,⁵ rods⁶ (wires, tubes, ribbons),^7–9 and sheets.¹⁰

One dimensional nanowires (NWs) are of increasing attention, because of their physico-chemical properties such as photoluminescence,¹¹ transparent conducting films,¹² field emission,¹³ and ferromagnetism.¹⁴ Usual methodologies for the synthesis of nanowires require irksome procedures and distinct conditions such as raising temperature, addition of catalysts or modification of templates. It will be much more complicated with the introduction of templates in the reaction system which in some cases raises the impurity concentration in the product. Hence, a simpler and more efficient approach for the synthesis of 1D nanowires would be much favored. The alternative and attractive approach for 1D nanostructure synthesis is a low temperature solution phase synthesis.¹⁵

Hydroxyapatite (HA; Ca₁₀(PO₄)₆(OH)₂) is a mineral component, well-known for its presence in the bone, dentine, and calcified cartilage of the mineralized tissues of vertebrates.^16,17 But each one has a different organic matrix composition. HA is of significance in the field of biomedical for the production of artificial bone grafts and teeth joints.¹⁸ Also it is a favorable alternate as reinforcing filler for composites and insulating agents.¹⁸ Because of its versatility, HA is one of the most favorable materials for biomedical application. The high aspect ratio of HA nanoparticles enriched various useful and desirable properties by reducing the grain boundary problems and increasing the mechanical flexibility and toughness. Widespread studies on HA revealed that it possess good osteoconductive and biocompatible properties on human cells.^19–21

HA nanoparticles were synthesized in many different methods such as sol–gel,²² hydrothermal,⁶ solvothermal,²³ co-precipitation,²⁴ reflex condensation²⁵ etc. Among this, hydrothermal method was considered as one of the most promising methods to prepare HA nanostructures due to highly crystalline products, higher efficiency, large scale synthesis, controllable morphology and Ca/P ratio close to stoichiometric HA.^26–28 Many different nanostructures like nanorods,⁶ plates,²⁹ rings¹⁹ and hierarchical³⁰ structures were reported for HA by hydrothermal method. Surfactant/soft template assisted syntheses of nanoparticles were growing due to the possible formation of tunable nanostructures with high aspect ratio, higher surface area.^31–33 Some important surfactants/soft templates are cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulphate (SDS), polyvinyl pyrrolidone (PVP). These surfactants/templates were used in synthesis to form a rod like structures.²⁷ Surfactant assisted synthesis is considered as an effective, convenient and mild synthetic methodology.²⁶ Many researchers were used CTAB in HA synthesis to control the morphology and the crystalline size.^26,34,35 It is found that CTAB induce the c-axis oriented growth in HA to form rods or wire like nanostructure. Many reports were available for the CTAB assisted HA nanorods. But high aspect ratio nanowire like structure in HA was restricted due to its unique structural features. It is reported that, the HA nanowires with higher lengths will have improved properties and hence they will broaden and enable their applications in numerous fields such as drug delivery, bone tissue engineering and adsorbent for organic pollutants. Even though some advancement has been made, the synthesis of longer HA nanowires is extremely challengeable and has been hardly reported. Recently some reports are available for the formation of HA nanowires by solvothermal,³⁶ hydrothermal³⁷ and microwave assisted techniques³⁸ using different precursors. Jiang et al. used solovthermal method to prepare HA nanowire using calcium oleate precursor.³⁶ Zhao et al. prepared HA nanowires using hydrothermal method and applied it for protein adsorption studies.³⁷ Microwave assisted hydrothermal synthesis of HA nanowire was reported by Qi et al.³⁸ Lu et al. made flexible and non-flammable paper from HA nanowires;³⁹ sol–gel hydrothermal method for the preparation one and three dimension of HA nanowires were reported by Costa et al.⁴⁰

In this paper, we demonstrate an extensive parametric analysis on CTAB assisted hydrothermally induced freestanding HA nanowires. Earlier, reverse micelle and surfactants in hydrothermal/solvothermal processes were used to produce nanowires and nanorods of varying aspect ratios.^41–43 It is reported that, surfactants offer controlled organic templates that lead the growth of inorganic nanomaterials through geometric, electrostatic and stereochemical interactions.⁴⁰ Freestanding HA nanowires in an aqueous solution was prepared by optimizing the solution and surfactant concentration, hydrothermal reaction time and temperature and calcination temperature. By carefully increasing the CTAB concentration we determine the growth stages of HA from nanorods to uniform nanowires. As discussed earlier, even though there are many reports on CTAB assisted HA nanorods, reports on nanowire formation were limited. Some report suggested the improved performance of nanowires compared to nanoparticles.⁴⁴ In energy harvesting field, some studies found that the nanowires achieves two to 20-fold efficiency.^44–46 Hence in this study, we are particularly focused on the uniform growth of HA nanowires, rather than nanorods, by the addition CTAB. The growth mechanism of the surfactant assisted nanowires is suggested on the basis of the CTAB concentration and processing temperature during the synthesis and morphological development thereof. The functional property was analyzed over in vitro osteoblast cell response.

2. Experimental methods

2.1. Reagents

Calcium nitrate (Ca(NO₃)₂·4H₂O), diammonium hydrogen phosphate ((NH₄)₂HPO₄), cetyl-trimethyl-ammonium-bromide (CTAB), ammonium hydroxide (NH₄ OH) were purchased from Aldrich and used without further purification.

2.2. Synthesis of HA nanowire

The HA nanowires were synthesized by surfactant assisted hydrothermal method. Ca(NO₃)₂·4H₂O and (NH₄)₂HPO₄ were individually mixed with double distilled water with the Ca/P ratio of 1.67 to maintain the stoichiometric ratio of HA. In the calcium containing solution, 0.5, 0.8 and 1 mmol (0.2, 0.3 and 0.4 gm respectively) of CTAB was added as a surfactant. NH₄OH solution was added drop-wise to the phosphate containing solution to increase the pH to 8. After continuous stirring for some time, the phosphate solution was added drop wise into the calcium solution and the stirring was continued for further 30 min. Then the mixed solution was then transferred to Teflon container of the stainless steel autoclave. The autoclave was placed in the furnace and the temperature of the furnace was maintained at 180 °C for 6 and 12 h. The autoclave was remaining in the furnace until it reaches down to room temperature. After that the precipitate in the autoclave was washed several times with ethanol and distilled water. The precipitates were dried at 80 °C and finally calcined at 600 °C for 2 h.

2.3. Characterization

FESEM images were recorded by Helios 600 system. Analytical TEM and HRTEM analysis were done by using FE-TEM, Tecnai F30 S-Twin with operating voltages of 160 and 200 kV respectively. Powder X-ray diffraction (XRD) study was executed with a Rigaku, D/MAX 2500H diffractometer using Cu-Kα radiation (λ = 1.5418 Å).

2.4. Osteoblast metabolic activity: MTT assay

Checking cellular behavior towards an artificial matrix was performed by different biochemical assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (tetrazolium dye MTT) assay is simple colorimetric method for indirectly determining the cellular proliferation since MTT reagent is broken-down into insoluble formazan crystals by the mitochondrial enzymes of metabolically active cells, hence high the change in MTT color (yellow to purple) in indicative of better rate of cell proliferation. Briefly, MTT was weighed and dissolved in serum free-DMEM (5 mg ml⁻¹) and 100 μl of the solution was used for test and control well. Medium from the samples was discarded and incubated with MTT solution for 90 min at 37 °C. After, incubation MTT solution was carefully discarded and DMSO was added to the samples to dissolve the formazin crystal formed during the MTT break down process. DMSO was collected and read using spectrophotometer at 490 nm. Samples were set up in duplicate to derive significance of data. Furthermore, cell attachment to synthesized nanomaterials was evaluated using well known nuclear stain 4′,6-diamidino-2-phenylindole (DAPI, Sigma). Medium was removed from the test samples and washed thrice with PBS (0.01 M, pH 7.4) and incubated with DAPI solution for 10 min at 37 °C. Samples tested on Day 1, 5 and 15 were visualized using inverted fluorescent microscope (NiKon ECLIPSE Ti, Japan).

2.5. Analysis of alkaline phosphatase (ALP) activity

The ALP activity was observed biochemically by Fast Violet B salt (Sigma) and naphthol AS-MX phosphate (Sigma). Firstly, the test samples consisting growing cells were gently washed with phosphate buffer saline (PBS, pH 7.4, temp 37 °C) then treated with ALP staining followed by three repetitive washing with PBS. Five images were taken at random locations in each sample for ALP staining using inverted fluorescent microscope (NiKon ECLIPSE Ti, Japan). Images were analyzed by Image J software (NIH) for counting of cells in a localized area by DAPI staining and total image area for percentage ALP stain.

2.6. Live-dead viability assay

Staining to find the live/dead was done to check the biocompatibility of the nanowire. 20 μl of ethidium homodimer-1 stock solution was dissolved in 10 ml PBS and along with 5 μl calcein am stock solution, to prepare the working solution. This working solution (100 μl) was used for staining system. Samples were incubated for 30 min at room temperature followed by washing with PBS and observed under fluorescent microscope (TI-DH, NIKON, Japan).

3. Results and discussion

3.1. General morphology

The morphological and structural developments of the nanowires produced by hydrothermal treatment were studied by FESEM and XRD analyses. FESEM images of the nanostructures of HA from the initial stage (pristine) up to final bamboo like nanowire structure due to the addition of CTAB was shown in Fig. 1. The pristine HA prepared by hydrothermal method have a clear nanorod morphology with the length up to 600 nm and diameter of 100–150 nm (Fig. 1). The development of nanowire is clearly visible with the addition of CTAB in the consecutive figures (Fig. 1(b–h)). With the addition of 0.5 mmol of CTAB, the length of the nanorod was increased with a slight reduction in the diameter. One interesting observation is that nanowires tended to bend slightly as the aspect ratio increases. It is natural that long nanowires with an extremely high aspect ratio (long wires with small diameter) bends because of the high bending moment due to its high self-weigh and small cross-sectional area.

Fig. 1 FESEM micrographs of HA nanorods and nanowires hydrothermally treated at 180 °C for 6 (a, c, e and g) and 12 (b, d, f and h) h: (a and b) pristine (c and d) 0.5 (e and f) 0.8 and (g and h) 1 mmol of CTAB, (i and j) magnified image and EDX pattern of (h).

The crystal structure and the phase purity of the HA nanowires prepared by hydrothermal method in the presence of CTAB were examined by XRD. Samples were annealed at 600 °C to increase the crystallinity of the sample. Fig. 2 shows the XRD pattern of the nanorods/nanowires with different concentration of CTAB and pristine HA. The diffraction peaks close to 26 and 31° can be assigned to HA (001), (211) diffractions, respectively, which represents the typical character of a crystalline hexagonal HA phase with an estimated lattice constant, a = 9.418 Å and c = 6.884 Å.⁶

Fig. 2 (i) XRD pattern of HA nanorods and nanowires hydrothermally treated at 180 °C for 12 h (a) pristine (b) 0.5 (c) 0.8 and (d) 1 mmol of CTAB. (ii) Blow-up image of (i) to show the minor peak broadening with the addition of CTAB.

Obviously, there is no other distinct diffraction peaks in all spectra than those of the peaks mentioned above, indicating that all HA nanowires have prevailing hexagonal HA crystal structure irrespective of the addition of CTAB as a surfactant. It is clear that the prepared HA is highly crystalline. The increased relative intensity of the (002) indicates that the HA nanowires grown preferentially along the [001] direction. Compare with the (211) peak, (002) peak became stronger and stronger from pristine to 1 mmol of CTAB. As the CTAB concentration increases the peak width increases. The diffraction patterns are matched with hexagonal HA structure and calculated lattice parameters are similar with the standard value (JCPDS # 09-0432). From the XRD results, it can be concluded that highly pure hexagonal HA was achieved by the current method with and without the addition of CTAB.

The blow-up images of XRD in Fig. 2(ii) exhibit the change in the FWHM of the pristine and CTAB added nanorods and wires. The broadening of (002) peaks appear to be much more pronounced than those of any other peaks. The peak broadening of (002) peak intensified when the diameter decreased and the length increased drastically with the addition of CTAB ≥ 0.5 mmol, suggesting (002) peak broadening can be attributed to the morphological change of crystals. The effect of morphological change on (002) peak broadening can be attributed to the natural bending of nanowires with an extremely high aspect ratio because of the high bending moment due to its high self-weigh and small cross-sectional area. It is not clear that these nanowires were bent elastically or plastically. Irrespective of whether bending of HA nanowires is elastic or plastic, the tensile and compressive stress develops depending on the sign of curvature,^47,48 which would results in the peak broadening. The d-spacing of the (002) peak was calculated at three different 2θ values (lower, main peak and higher angle) to understand the bending effect of the nanowire. There was a considerable change in the d-spacing over the (002) peak range. The actual d spacing value at the peak is 0.343 nm; at lower angle and higher angle side it has the value of 0.348 and 0.339 respectively. Even though it is a small value, it correlates the peak broadening with bending of the nanowire. The nature of bending and its effect will be discussed in relation to TEM images in the next section.

3.2. Nanostructural analysis

Transmission electron microscopy (TEM), high resolution TEM (HRTEM) and selected area electron diffraction pattern (SAED) analyses were further used to explore the morphology and crystal structure of the pristine and CTAB added HA nanowires. Fig. 3 shows the TEM and high resolution TEM (HRTEM) images of the HA nanocrystals. The results well coincide with the FESEM analysis which clearly displays the progression of uniform nanowires with the addition of CTAB. From the TEM analysis, it is found that HA nanowires have an average diameter of 30 nm and few micrometers in length.

Fig. 3 TEM micrographs of HA nanorods and nanowires hydrothermally treated at 180 °C for 6 (a, c, e and g) and 12 (b, d, f and h) h: (a and b) pristine (c and d) 0.5 (e and f) 0.8 and (g and h) 1 mmol of CTAB.

The pristine HA revealed nanorods with the diameter of 100–150 nm. There is not much change in the diameter of the nanorods for the pristine HA with the different reaction time. With the addition of CTAB content, the morphological development of HA crystals was modified greatly and the shape, size and aspect ratio vary appreciably depending on the concentration of CTAB. 0.5 mmol of CTAB addition considerably increases the length of the nanorods as shown in the morphological analysis but with the small change in the diameter of the nanorods. For 6 h hydrothermal treated sample we found wide distribution in the diameter. That is, these samples show non-uniform distribution in diameter. With the increase of the reaction time, the uniformity was achieved significantly. With further increase of the CTAB content, it is observed that the 12 h treatment always provided better uniformity in shape and size of nanowires than 6 h heat treatment.

Sample with 0.8 mmol of CTAB showed further reduction in the diameter and the pronounced increase of the length. Similarly for 1 mmol of CTAB addition, the 12 h hydrothermally treated sample exhibited uniform nanowires with significantly reduced diameter. The average measured diameter of this nanowires is 30 ± 4 nm. These results revealed the ability to produce fine nanowires by adding optimized amount of CTAB and controlling simple processing parameters such as reaction time, temperature, pH and concentration of the precursor. Again, nanowires tended to bend slightly as the aspect ratio increases. The average rod/wire diameter was further calculated and confirmed from the average of four field of view and 50 measurements per field of view (Fig. 4).

Fig. 4 Diameter distribution of HA nanorods and nanowires hydrothermally treated at 180 °C for 6 h (a, c, e and g) and 12 h (b, d, f and h): (a and b) pristine (c and d) 0.5 (e and f) 0.8 and (g and h) 1 mmol of CTAB.

The crystal structure of the HA nanowire was examined by high resolution TEM (Fig. 5). The crystal lattice of HA indicates that the single crystalline HA nanowires were grown in the [001] direction with the d-spacing of 0.34 nm. This value corresponds to the (002) peak in the XRD pattern (Fig. 2). It is also confirmed that the (001) direction aligned with the nanowire axis and it is confirmed that the HA nanowires were grown along the preferred c-axis direction. The d-spacing of the (100) direction is 0.82 nm which is in good agreement with the reported value. Through examination of the HRTEM images did not reveal any defects such as dislocations in the nanowires which indicate that the nanowires are of high quality. The presence of dislocations in nanowire is energetically unfavorable because of their high elastic energy and the image force which attracts the dislocations out of the surface if there are any.¹⁹ In some materials, the critical size for the instability of lattice dislocations⁴⁹ was suggested to be 15–50 nm depending on the surface properties, elastic modulus, size of Burgers vector, and the lattice friction. In the HA nanowires with the average diameter of 30 nm, mobile dislocations are not likely to be present. The corresponding SAED pattern of the nanowire is shown in Fig. 4e and the diffraction spots in the pattern is indexed. It is also confirmed the single crystalline nature of the final HA nanowires.

Fig. 5 (a–c) Higher magnification images of HA nanowires, (d and e) respective HRTEM and SAED pattern of selected part from (c).

In order to examine the nature of bending in long nanowires and its effect on the XRD peak broadening, the bent region of wires were imaged using a HRTEM (ESI†). It is natural that long nanowires with an extremely high aspect ratio (long wires with small diameter) bends because of the high bending moment due to its high self-weigh and small cross-sectional area. Because of bending, convex side of wire is in tension and the concave side is in compression. A careful examination of the enlarged images from the tension and compression side of the nanowire revealed the absence of dislocations in both sides (ESI†). Bending could take place either elastically or plastically by introducing geometrically necessary dislocations.^50,51 Careful and through examination of HRTEM images the tension and compression side of the nanowire did not reveal the geometrically necessary dislocations in bending, suggesting that the bending occurs elastically after growth and drying of HA nanowires. During the growth of nanowires in solution, the bending force due to the self-weight of nanowires is not likely as high because of buoyancy in solution and no defect was introduced. The maximum elastic strain in the bent nanowires can be calculated using an equation ε = ±d/2ρ, where d is the average width of nanowire and ρ is the radius of curvature of the bent nanowire. The radius of curvature of the bent wire was measured to be 750 nm and the diameter was measured to be 30 nm. The maximum strain imposed on the bent nanowires is calculated to be ±0.02. The d-spacing of (002) planes with 0.02 elastic strain would be 0.34 nm ± 0.0068 nm (0.336–0.350 nm), which is close to that (0.339–0.348 nm) calculated from the width of (002) peak in Fig. 2. The slight difference between the (002) spacing calculated based on the bent curvature and that from (002) peak width may be attributed to the difference in the cross section shape of rod (square cross section vs. circular cross section).⁴⁷

It is well known that when a material is strained, the crystal lattice experienced contraction and elongation, which change the inter-planar spacing of the lattice plane. This change resulted change in d-spacing cause change in the diffraction pattern.⁴⁸ Bending of nanowires (tension on the right-hand side surface and contraction at the left-hand side surface) after growth and drying produce non-uniform lattice strain through the thickness, and would cause the broadening of the XRD peak. Very careful analysis of the lattice spacing of the elongated side (0.348 nm) and contracted side (0.339 nm) revealed that the lattice strain is developed in bending, which would result in XRD peak broadening.^52,53 The reason why (002) peak broadening is more pronounced than any other peaks can also be explained nicely by the bending nature and preferential growth direction of nanowires. As shown in Fig. 4, HA nanowires grew along the preferred c-axis direction, suggesting (002) planes are perpendicular to the long axis of nanowires. If the nanowires are bent elastically with respect to the long axis, (002) planes are most significantly affected by bending. The d spacing of (002) planes would increase in the tension side and decrease in the compression side, resulting the more dramatic XRD peak broadening.

While further increasing the CTAB concentration to 1.3 mmol showed destruction in the nanostructure (Fig. 6(a)) and many beads like structures were observed in the nanowire after 12 h of hydrothermal treatment. While increasing the hydrothermal treatment time to 24 h revealed the no uniformity with beads like structure throughout the nanowires (Fig. 6(b)). But for 1 mmol sample, after treating the sample for 24 h there was no prominent difference observed in the uniformity as well as morphology. Hence we concluded that the optimum concentration of CTAB and the hydrothermal treatment time is 1 mmol and 12 h respectively (additional FESEM and TEM images of HA nanowires are available in ESI†). For comparison, the FESEM and XRD pattern of bulk HA was analyzed and presented in Fig. 6(c and d) respectively. Any specific nanostructures were not found from the FESEM analysis. The XRD pattern revealed that the material is highly crystalline and well coincide with the HA peaks with standard JCPDS data (JCPDS # 09-0432). The peak intensity is higher than the nanostructured materials presented in Fig. 1. It shows typical XRD pattern of the bulk material and broadening of the peak is hardly observed.

Fig. 6 HA nanoparticles with 1.3 mmol of CTAB concentration (a) 12 (b) 24 h hydrothermal treatment. (c and d) FESEM and XRD pattern of the bulk HA particles.

3.3. Possible growth mechanism of HA nanowires

CTAB concentration and hydrothermal reaction time with constant reaction temperature (180 °C) on the nanoparticles were thoroughly examined to understand the growth mechanism of the HA nanowires. The reaction temperature of 180 °C was optimized with previous experiments.⁶ The parameters were optimized by repeating the experiments several times for the formation of fine HA nanowires. From the TEM and SEM results, we propose a possible growth mechanism of the HA nanowires by hydrothermal method.

Many reports are available for the formation of various materials in nanowire form by different preparation methods.^7,12,53–57 However, in this work very fine, uniform nanowires with the diameter of approximately 30 nm and few micrometer length have been obtained with the addition of CTAB in a simple hydrothermal method. In the present study, we identified that CTAB has been playing an important role in the formation of ultrafine HA nanowires apart from hydrothermal reaction time. Our strategy for making freestanding HA nanowire is to control the interaction between CTAB and HA by carefully optimizing the CTAB concentration. In the pristine HA synthesis by hydrothermal reaction produces nanorods with the length of 600 nm with 120 nm diameter. The reduction in the diameter of the nanorods/wires due to the addition of CTAB was reported previously.⁵⁸ It has been reported that, CTAB acted as soft template for the synthesis of nanorods and nanowires and the diameter of the rodlike micellar could be changed by adjusting the concentration of CTAB. We found that as the concentration of the CTAB increased the diameter of the nanowire was reduced considerably. By keeping the reaction temperature (180 °C) and pH (8) constant, the change in the diameter and length was quite clear for the change in the CTAB concentration. Different hydrothermal treatment time also analyzed. As the CTAB concentration increased from 0.5 to 0.8 and 1 mmol, the average diameter of the nanowire decreased from 82 to 54 and 33 nm. The uniformity in the diameter of the nanowires also increased considerably while increasing the CTAB concentration.

Different mechanisms have been proposed for the formation of nanorods in the presence of CTAB for various materials including HA.^26,34,35 It is reported that, CTAB would ionize entirely in an aqueous solution and become a cation with tetrahedral structure.⁵⁹ The phosphate anion for HA is also a tetrahedral structure and the structure complementarity endows CTAB with the capability to control the crystallization.⁵⁹ We propose here that the formation of nanorods and nano needles with extremely high aspect ratio is promoted by the stereochemistry and orientation dependent anisotropic charge distribution of HA. The interaction of positively charged CTAB to negatively charged phosphate anions is suggested to play an important role in the formation of nanowires with extremely high aspect ratio observed in the present study. The formation of HA nanowires is promoted by the anisotropic charge distribution on the different faces of the HA. CTAB cations are supposed to attach preferentially to certain planes with higher negative charge distribution of HA than those with positive charge distribution. Rulis et al.⁶⁰ calculated electron charge density by ab initio density functional calculation and reported that the (001) surface is more positive than negative. On balance, the (100) surface is more negative. Hong et al.⁶¹ further suggested that a consequence of the anisotropic distribution of atoms is the orientation dependence of surface energy and surface charge, which is dependent on the orientation dependence of the bonding nature of crystalline apatite.

Osteoblast cell response on nanowires. Cell viability and proliferation analysis of biomaterials are a prerequisite for clinical applications. Human osteoblast cells were seeded to evaluate the in vitro cellular response on HA nanowires. HA nanowires were pre-equilibrated with cell-culture media and 1 × 10⁴ cells per well per nanowire was seeded on to a 5 mm section.

Changes in cellular metabolic activity due the presence of nanoparticles were analyzed with control (2D). HA nanorods and nanowires exhibit more cell viability and more metabolic activity than control (2D) samples. In the presence of HA nanowires, the metabolic action of the cells improved with increasing time signifying the biocompatible nature of HA nanowires as shown in Fig. 7 and 8. HA nanowires are supposed to have numerous advantages over HA nanoparticles as well as its bulk counterparts. From our studies, we found that compared with HA nanoparticles (spheres, rods and bulk), the agglomeration is very minimum and it was reported that the nanowires may also functionalized with large molecules like fibronectin.⁶² Hence, HA nanowires might be an effective material than nanoparticle of other morphology. Many reports suggest that size of the nanoparticles determine the cell viability of that particular material. The diameter of <50 nm might be ideal for efficient nanomaterial uptake into cells due to combinations of physicochemical and biological interactions at the bio-nano interface.⁶² The diameter of HA nanowire is around 30 nm and hence we found that the cell viability is higher than nanorods with the diameter more than 80 nm. In order to find the actual effect of the nanoparticle, the bulk HA were analyzed for osteoblast cell response. The response was very low compared to HA nanorods and nanowires. HA nanowires shows excellent and improved cell response compared to all the other forms of HA especially over bulk HA.

Fig. 7 MTT assay of different nanoparticles which indicate total metabolic cells at any given time interval.

Fig. 8 DAPI nuclei staining fluorescent image of osteoblast interaction shows cell attachment on HA nanorods and nanowires with different days (scale bar: 100 μm).

ALP and live/dead analysis. MTT assay and ALP levels were monitored to analyze the metabolic activity and functional activities, respectively, for up to 30 days with control (2D) sample. ALP is known as marker for differentiation of osteoblast cells at a relatively early bone forming stage during mineralization and matrix protein formation.⁶³ The ALP expression was noted only from day 5 in both HA-NR and NW indicating that both these materials were potentially osteoconductive. The ALP expression is observed on 15^th day onwards which could also explain the decrease in the proliferation rate of the cells since its known phenomena for cells to stop proliferating once they enter the differentiation cycle (Fig. 9). Depending upon the materials used for the material fabrication, the interaction between the matrix and cells can be modulated. It is noted that natural ECM based materials enhances osteoblast attachment as it interacts with specific cell surface receptors which in turn results in activation of precise intracellular signaling cascade. Depending upon the protein receptors present on the surface of biomaterials that cell identifies it triggers a specific cellular response. For example, tissue culture plated coated with coll I is shown to increase osteogenesis in mesenchymal stem cells (MSCs) by activating Akt and ERK (extracellular-signal-regulated kinases) pathways and this is noted by increase in ALP activity of these cells. Along with other transcription factor such as SPP1 (osteopontin marker), ALP is known to be maturation marker. Experiments performed by Hansen et al.,⁶⁴ has shown increase in osteoblast stiffness when cultured in nano-islands of 11 and 38 nm and result of nanotopography of the materials. Besides the effect on the cellular phenotypes, the mechanic-sensitivity of cells can be altered by nano-patterning techniques which can be observed in study performed by Salvi et al.,⁶⁵ nano-island led to increased intracellular calcium level in MSCs as response to flow rate of fluid on smaller islands compared to cells cultured on larger islands. Müller et al.⁶⁶ reported that the cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts by increased ALP activity and expression of osteogenic genes. By monitoring alkaline phosphatase (ALP) activity Birmingham et al.⁶⁷ quantified osteogenic differentiation of MSCs. They reported that the intracellular ALP was found to peak earlier and there was greater calcium deposition when MSCs were co-cultured with osteocytes rather than osteoblasts. Over last few years it has been established that topography along with other mechanical cues presented by the nanomaterials can downstream signaling cascade thereby modulating osteoblast proliferation and differentiation cascade and most often used marker to identify this change is ALP. Similar to earlier published results we found that osteoblasts exhibited different phenotypes and ALP activity as response to HA nanowires. The dimensions and depth of the nano-features of the material directly effects cell–surface interaction. Cells cultured in nano-pits are found to be more homogenous in size than nano-islands cells.

Fig. 9 Osteoblasts cultured on HA-NR and HA-NW were stained for ALP expression at different time point (scale bar: 100 μm).

Further live/dead staining assay was used to examine cell viability. Live and dead cells emitted green fluorescence and red fluorescence, respectively. After 25 days of incubation, the cell viability in the presence of HA nanowire remains high, and dead cells were barely visible as presented in Fig. 10. This confirms that the synthesized HA nanowires have very low toxicity toward fibroblast cells. When the cells come into contact with the adjacent cell it will stop growing.⁶⁸ Since control (2D) sample has limited space and hence dead cells are observed due to growth constrain. HA nanowires provide larger surface area and high aspect ratio which permits the cell to proliferate for a longer period of time.

Fig. 10 Live and dead imaging of osteoblast on control (2D) and HA-NR and HA-NW at different time point (scale bar: 100 μm).

4. Conclusions

HA nanowires were synthesized by simple hydrothermal method with the addition of CTAB as a surfactant. Many parameters such as CTAB concentration, reaction time and temperature have been optimized to obtain fine nanowire. The more significant peak broadening of (002) planes with the increase of length of nanowires can be attributed to natural bending of nanowires with an extremely high aspect ratio due to its high self-weigh and small cross-sectional area. This theory was further confirmed based on our findings in the HRTEM images on the bended part of the nanowire. Detailed analysis revealed that the nanowires were grown on c-axis direction of HA. We proposed possible formation mechanism based on our findings. The mechanism for the growth of preferentially oriented nanowires was proposed based on the orientation-dependent electrostatic interaction between the CTAB ions and HA crystals with anisotropic charge distribution. Finally, in vitro cellular analysis revealed that the prepared HA nanowires shown better cell response compared to other nanostructures and its bulk counterpart. Hence the present study revealed that the prepared HA nanowire has a potential application in the biomedical field.

Conflict of interest

The authors declare no competing financial interest.

Acknowledgements

We acknowledge the supports from Yeungnam University research grants in 2014 and Korea National Research Foundation (0077110).

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Footnote

† Electronic supplementary information (ESI) available: Additional FESEM and HRTEM images. See DOI: 10.1039/c6ra01155a

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