Template-Free Growth of Novel Hydroxyapatite Nanorings - American

Article pubs.acs.org/crystal

Template-Free Growth of Novel Hydroxyapatite Nanorings: Formation Mechanism and Their Enhanced Functional Properties A. Joseph Nathanael,†,‡ Sun Ig Hong,*,‡ D. Mangalaraj,*,§ N. Ponpandian,§ and Pao Chi Chen⊥ †

Department of Physics, Bharathiar University, Coimbatore 641 046, India Department of Nanomaterials Engineering, Chungnam National University, Daejeon 305-764, South Korea § Department of Nanoscience and Technology, Bharathiar University, Coimbatore 641 046, India ⊥ Department of Chemical and Materials Engineering, Lunghwa University of Science and Technology, Taoyuan, Taiwan ‡

S Supporting Information *

ABSTRACT: Template-free, single crystalline novel hydroxyapatite (HAp) nanorings with an inner diameter of 70 nm were grown by a combined high gravity and hydrothermal approach. Nanodisks were suggested to be formed by oriented aggregation and Ostwald ripening of mostly calcium pyrophosphate nanospheres prepared initially by the high gravity method with a stepwise increase of flow rate of phosphate solution. The prolonged hydrothermal treatment of nanodisks appeared to induce the nanoring formation via acid penetration along the dislocations in HAp nanodisks. The presence of edge dislocations in the central region of nanodisks was confirmed by high resolution transmission electron microscopy. The mechanical evaluation of high molecular weight polyethylene (HMWPE) composite with various shaped HAp nanocrystals and in vitro cellular analysis of HAp nanocrystals revealed that mechanical and bioactive performances improved with an increase of the specific surface area of HAp nanocrystals. The enhanced mechanical performance of HMWPE/ HAp nanoring composite and the excellent cell viability for HAp nanorings are attributed to the superior interface bonding and cell activity, respectively, both of which are enhanced by the high specific surface area.

1. INTRODUCTION One of the most challenging processes in materials engineering is the controlled fabrication of materials with user-defined shapes to provide an increasingly precise control over the structures and hence their properties. Significant progress has been made in the fabrication of one- and two-dimensional (1D and 2D) nanomaterials such as nanoparticles, rods (wires, cables, tubes, ribbons, and helixes), and sheets.1−7 Among those, hollow nanostructures such as nanocages, nanospheres, and nanorings have attracted much interest because these hollow cavities improve the functionality of these nanomaterials to be used in various applications.8−12 However, it is still a challenge to develop such functional hollow ring-like shapes in exact circular, oval, or polygonal morphologies with large yield. It should be noted that in various studies, the ringlike nanostructures were either grown directly onto templates followed by template removal or rearranged from a particular part of raw material.7,13−17 Development of free-standing ringlike nanostructures using self-organized tiny building blocks has been a challenging task until now. Recently, a few successes were made in the synthesis of freestanding rings (e.g., CdS, ZnO, Au, Cd(OH)2, and Ag2V4O11) by solution-based processes based on different synthesis mechanisms, such as self-assembly of primary nanoparticles, central-etching of disks, and self-coiling of nanobelts.7,18−22 © 2012 American Chemical Society

Since nanorings have a unique structural texture, they exhibit novel properties caused by the presence of cavities. This cavities may greatly enhance the functionality of nanomaterials.14−17,23 For example,24 the cavities of gold nanorings influence an extremely uniform field enhancement effect. It has been suggested that in sensing and spectroscopy applications they could act as a resonant nanocavities for probing or carrying tiny nanostructures such as quantum dots, biomolecules etc.24 Similarly α-Fe2O3 nanorings exhibit excellent characteristics as a sensor. It has shown long-term stability, good reproducibility, and function as a highly sensitive electro-catalyst mainly due to the high surface-to-volume ratio and the unique network of interconnected pores in the nanorings (Fe2O3).12 Therefore, research for new synthesis methods to achieve such hollow ring-like morphologies is imperative. The mineralized tissues of vertebrates include bone, dentine, and calcified cartilage. All these tissues have hydroxyapatite (HAp; Ca10(PO4)6(OH)2) as their mineral component but with some modification of their organic matrix composition. The HAp has been studied extensively for cell cultures and has been found to possess good osteoconductive properties.25,26 Received: March 26, 2012 Revised: May 27, 2012 Published: May 29, 2012 3565

dx.doi.org/10.1021/cg3003959 | Cryst. Growth Des. 2012, 12, 3565−3574

Crystal Growth & Design

Article

phosphate flow rate was increased gradually from 300 mL/min to 500 mL/min (condition II); or (3) flow rate of the phosphate solution was increased stepwise from the beginning of the experiment (condition III). In all three cases, the solutions were mixed thoroughly with an rpm of 3000 and reacted to form the product. After the solution was mixed by the high gravity method, the products were dried and heat treated at 600 °C for 2 h. After that the products were hydrothermally treated at 180 °C at different time intervals of 12, 24, and 48 h. Finally, obtained nanoparticles were dried at 150 °C for 24 h before further characterization. 2.2. Preparation of HAp/Polymer Nanocomposites. In order to investigate the effect of HAp nanoparticles on the mechanical performance of high molecular weight polyethylene (HMWPE)/HAp composites, the mechanical properties of HMWPE matrix (with an average molecular weight of 1 × 106 g/mol) composites reinforced with 10 wt % of the different HAp nanoparticles were analyzed. Before mixing, HMWPE and the nanoparticles were dried in an oven at 120 °C for 1 h and cooled down to room temperature to remove the moisture contents. The blending was carried out by melt extrusion method using a high speed rotating micro compounder (HAAKE MiniLab II) with a mixing temperature of 180 °C. Initially polymer granules were loaded in the extruder. After the melting started, nanoparticles were fed into the extruder in order to avoid the deposition of nanoparticles in the bottom of the extruder. A mixing time of 20 min was optimized and fixed for all the samples with a rotor speed of 80 rpm. A piston injection molding system (HAAKE MiniJet 557−2270) was used for preparing the specimen to study the mechanical behavior. Pure HMWPE was also tested to compare the effect of nanoparticle reinforcement. 2.3. Characterization. The powder X-ray diffraction (XRD) analysis was performed with a Rigaku D/MAX 2200 diffractometer using Cu-Kα radiation (λ = 1.5418 Å). Field emission scanning electron microscopy (FESEM) images were recorded by the JEOL JSM 6500 system. Analytical transmission electron microsopy (TEM) and high resolution TEM (HRTEM) investigations were performed by using JEOL JEM-2100 with operating voltages of 160 and 200 kV, respectively. Particle size distribution analysis was carried out using an HORIBA LB-550 particle size analyzer. The Raman spectra of the present study were recorded using a laser Raman spectrometer (Renishaw inVia Raman Microscope) at an output power of 10 mW of a 514 nm Ar+ laser. Spectra were corrected using LO-phonon mode of Si(100) substrates observed at 520.5 eV. The tensile strength experiment was performed using a universal testing machine (810 Material Test Systems) at a crosshead speed of 10 mm/min with a load of 100 kN. 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. 2.4. In Vitro Cellular Analysis. The in vitro cellular analysis was carried out using CHO cells (CHO-K1, Korean Collection for Type Cultures), the model animal cell. These cells were used to study the effects of nanostructures on the cell compatibility of various HAp nanoparticles obtained in this work. The detailed experimental details are given elsewhere.31 The morphology of the CHO cell growth was analyzed using FESEM analysis. Additionally, the human osteosarcoma cells (MG-63 cells) were cultured to determine the effect of morphology on osteoblast cell viability. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 in air containing cell culture medium (DMEM media (phenol red free) Welgene), 10% fetal bovine serum (FBS, Welgene), 2 mM L-glutamine, 1% antimicrobial of penicillin, and streptomycin (Antibiotic, Gibco,). Then the cells were seeded in 96-well microassay plates at a concentration of 1 × 104 cells/ well and cultured for 24 h. After that the sterilized HAp nanoparticle samples were added into the wells at the concentration of 100 μg/ mL−1 and were cocultured with cells for different time periods (1, 2, and 3 days). Cell proliferation or viability of cells was determined using CCK-8 kit (Dojindo, CK-04-13). Five samples were tested for each culturing time period and the mean value was reported.

Several studies have shown that the mechanical properties of the ceramics and composites could be improved remarkably by utilizing nanoscale building blocks, such as nanorods, nanofibers, and nanotubes.27−29 Furthermore, the central hollow space or area in the nanoscale materials may enhance the functionality and structural effectiveness or structural performance of those materials.1−7 Studies are needed to explore the possible fabrication and application of nanoscale HAp crystals with a central cavity. In the literature, there are few reports which suggest the possible formation of hollow space in HAp. Jongebloed et al.30 reported the acid penetration into rod-type HAp single crystals of the millimeter scale parallel to the c-axis. The dissolution of HAp in acids is an important issue because it is likely to have a significant influence on bone resorption and the progression of carious process. It was suggested by Jongebloed et al.30 that the acids attacked the dislocation site (which they presumed to exist in HAp crystals) and formed an etch pit, which extended into the crystal in the c-axis direction. A cavity was formed in a later stage. To the knowledge of the authors, no effort has been made on the synthesis of nanoscale crystalline HAp with central cavities (so-called “nanotube” or “nanoring”) taking advantage of the acid penetration process on HAp nanocrystals. HAp nanocrystals with central cavities may have some advantages over regular crystals because they are more bioactive due to the high surface area to volume ratio and central cavities can be used to store and carry some bio- or electrofunctional materials. In this paper, an effort to process HAp nanorings was made through a deliberate chemically induced morphological modification process, and the formation of freestanding novel HAp nanorings in an aqueous solution prepared by a chemical method is reported for the first time. The growth mechanism of the nanodisks and rings are proposed on the basis of the synthesis and morphological modification process. The functional/structural properties were also studied through in vitro cellular analysis and mechanical performance evaluation.

2. EXPERIMENTAL METHODS 2.1. Synthesis of HAp Nanorings. The HAp nanorings were synthesized by a combined high gravity and hydrothermal method using calcium nitrate (Ca(NO3)2·4H2O) and diammonium hydrogen phosphate ((NH4)2HPO4) as calcium and phosphorus sources, respectively. The detailed synthesis of hydroxyapatite nanoparticles using a high gravity method by varying different parameters such as flow rates of the solutions, rpm of the rotating packed bed (RPB)m etc., was given in detail in our previous report.31 Briefly, calcium nitrate and diammonium hydrogen phosphate were separately mixed with deionized water with a molar ratio of 1:0.6 in order to maintain the Ca/P ratio of 1.67, which is the stoichiometric molar ratio of hydroxyapatite. The pH of the phosphate containing solution was increased to 9 by adding aqueous solution of NH3 (30%). The prepared Ca and P solutions were pumped through the two different solution inlets into the RPB of the high gravity setup where both solutions were mixed and reacted with each other to form a product. In this work we have controlled only the flow rate of the phosphate containing solution in order to control the reaction kinetics. Other parameters such as pH, concentration of the solutions, rotating speed of the RPB, etc. were maintained constant as optimized earlier. The flow rate of Ca and P solutions was controlled by using the liquid flow meter. In the present experiment, three different phosphate flow rates were analyzed. Flow rates of calcium and phosphate solutions were varied as follows: (1) maintained constantly at 300 mL/ min throughout the experiment (condition I); (2) initially the flow rates of the calcium and phosphate solutions were maintained constantly at 300 mL/min, and at the final stage of the process, 3566



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3. RESULTS AND DISCUSSION 3.1. Phase Analysis. The phase purity and crystal structure of the HAp nanoparticles prepared by the high gravity method were examined by XRD. Figure 1 shows the XRD pattern of the

Figure 2. XRD pattern of HAp nanoparticles formed after hydrothermal treatment at 180 °C for (a) 12, (b) 24, and (c) 48 h. Figure 1. XRD pattern of HAp nanoparticles with different phosphate flow rates (a) constant, (b) stepwise, and (c) continuously increasing flow rates.

phase in the hydrothermal process. This can be described by the following reaction paths: 4Ca(NO3)2 ·4H 2O + 4(NH4)2 HPO4 + 110NH4OH

as-prepared nanoparticles with different flow rates before the hydrothermal treatment. For the constant flow rates of both solutions (condition I), hexagonal HAp crystal phase was observed as exhibited in Figure 1. For stepwise increase of flow rates (condition II), calcium pyrophosphate (Ca2P2O7) crystal phase were mostly observed along with minor HAp phase. For continuously increasing flow rate (condition III), various forms of calcium phosphate phases, mostly Ca3(PO4)2, were observed (Figure 1). This shows that the flow rates of the solution have a great influence on the composition, stoichiometry, crystal structure, and morphology of the nanoparticles. Since there are many forms of calcium phosphate compounds with different free energy and activation energy barrier for reaction/transformation, controlled reaction conditions are suggested to be most important to control the stability and volume fraction of each phase.32 Hence the formation of various calcium phosphate compounds is sensitive to the flow rate of solutions; therefore it is highly likely that different forms of calcium phosphate compounds formed, when the flow rates of calcium and phosphates were not balanced to produce Ca/P ratio of 1.67. In condition II, during the initial stage of the reaction, the flow rates of both calcium and phosphate containing solutions were uniform (300 mL/min), whereas at the final stage the flow rate of phosphate solution was increased (500 mL/min). Spherical nanoparticles produced in condition II developed into nanostructures with different morphologies (disks, etching and rings) subject to different hydrothermal reaction times (12, 24, and 48 h). Figure 2 shows the XRD pattern of the nanoparticles prepared under condition II and subsequently treated by a hydrothermal process with various times (12, 24, and 48 h). The presence of few Ca2P2O7 peaks was obvious after hydrothermal treatment for 12 and 24 h. Hydrothermal treatment for 48 h yielded a highly crystalline pure HAp, and no secondary phase was observed. It is observed from the XRD results in Figures 1 and 2 that the intermediate phase Ca2P2O7 was primarily formed when the stepwise flow rate was used in the high gravity method but gradually transformed to the HAp

→ 2Ca 2P2O7 + 107NH4NO3 + 83H 2O

(1)

5Ca 2P2O7 + 19H 2O → Ca10(PO4 )6 (OH)2 + 4PO4 −3 + 12H3O+

(2)

It was found that all Ca2P2O7 had been converted into HAp during the prolonged hydrothermal treatment (48 h). Formation of HAp from Ca2P2O7 after the hydrothermal reaction is given by eq 2.32,33 The average crystallite size calculated from the XRD pattern for the nanorings using the Scherrer formula was 25 nm. All the diffraction peaks and lattice parameters calculated from the XRD patterns match well with the pure hexagonal HAp phase (JCPDS # 09-0432). 3.2. Nanostructural Analysis. The morphological and nanostructural evolution of the nanocrystals produced under condition II and subsequent hydrothermal treatment were studied by electron microscopic analysis. Figure 3 presents the typical FESEM images of the nanostructures in various morphologies such as nanospheres, nanodisks, and donutshaped nanorings (Supporting Information (Figure s1) provides the FESEM images for all other conditions). The samples prepared by the high gravity method (before hydrothermal processing) have a spherical morphology with a uniform size distribution (Figure 3a). The HAp crystals obtained with a hydrothermal treatment for 12 h show a disklike morphology as seen in Figure 4b. HAp crystals hydrothermally treated for 24 h have a disklike morphology (Figure 3c) with a hole or a cavity partially formed at the center of the disk. The prolonged hydrothermal reaction for 48 h changes the morphology to nanorings (Figure 3d,e) with clearly defined central holes. Most of the nanorings were observed to have a hexagonal or semihexagonal morphology. The low magnification SEM images, especially Figure 3c,d, show that large-scale production of nanodisks and nanorings with relatively uniform size and shape distribution can be produced by the combined high gravity and hydrothermal method used in this study. 3567



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developed nanorings (Figure 3d) appears smoother than those of the nanodisks (Figure 3b) and partially formed nanorings (Figure 4c). This indicates that the surface smoothening and rearrangement took place driven by the reduction of surface energy during the hydrothermal treatment.34 The chemical composition of the HAp nanoparticles were evaluated by energy dispersive spectroscopy (EDS), and the typical EDS spectrum of HAp nanorings is presented in Figure 3f. It shows the presence of Ca, P, and O in HAp nanorings. The Raman spectra of the nanorings show a very strong PO4 υ1 peak at 963 cm−1 as can be seen in Figure 4. This characteristic peak at 963 cm−1 was due to a totally symmetric stretching mode (υ1) of the PO4 group (P−O bond). Apart from this peak, other weak peaks were found at 435 cm−1 (double degenerate bending mode (υ2) of the PO4 group (O− P−O bond)) and 588, 610 cm−1 ((triply degenerate bending mode (υ4) of the PO4 group (O−P−O bond)). The position of this peak is a good indicator of the degree of crystallinity of the material: it is found at 963 cm−1 in more ordered, highly crystalline noncarbonated apatite and at ∼958 cm−1 in polycrystalline apatite.35 The presence of the peak at 963 cm−1 for nanorings in Figure 4 suggests that the nanorings were highly crystalline. On the other hand, the presence of the peak at 959 cm−1 for nanodisks suggests that the nanodisks were not highly crystalline at this stage and may contain some defects. Also a medium peak is observed at 1045 cm−1 which is the triply degenerate asymmetric stretching mode (υ2) of the PO4 group (P−O bond). This kind of peak is mainly observed in calcium pyrophosphate compounds which confirm the presence of Ca2P2O7 in the nanodisks.36 Further insight into the microstructural evolution toward the donut-shaped nanorings can be obtained through the TEM study. Figure 5 presents the TEM and HRTEM images of the HAp nanocrystals with various shapes. The image for the HAp nanospheres prepared by high gravity method without hydrothermal treatment is presented in Figure 5a. These spheres were found to be the basic building blocks for the nanorings during the hydrothermal treatment. Figure 5b presents the TEM micrograph of the HAp disks after 12 h hydrothermal reaction. This image shows the disks that could be formed by the aggregation of small particles (indicated by an arrow) during the hydrothermal reaction. In the micrograph shown in Figure 5c, it appears that hole formation or thinning took place at the center of the HAp nanodisks during the hydrothermal reaction of 24 h. The increase in the hydrothermal reaction time (48 h) leads to the formation of perfect nanorings with a hole at the center as seen in Figure 5d. The shape of the well-developed nanorings is hexagonal-shaped, and this may be due to the surface relaxation of the rings parallel to the prism planes with a low surface energy.37 Figure 5e presents the SAED pattern acquired from HAp nanorings and it shows the hexagonal symmetry. This SAED was acquired with the [001] zone axis and the diffraction spots in the pattern is indexed. Figure 5, panels f and g are the HRTEM images for the HAp acquired from different regions of the rim of nanorings. In both images, (110) planes are marked demonstrating the single crystalline nature of each ring. The top plane of the nanorings is the (001) and side planes are (110) planes. It is apparent that the nanorings and nanodisks have a hexagonal crystal structure with the c-axis perpendicular to the flat plane. Detailed TEM analysis of the HAp nanorings gives an average outer diameter of 200 ± 15 nm an inner

Figure 3. FESEM micrographs of (a) nanospheres before hydrothermal treatment, (b) nanodisks after 12 h, (c) nanodisks with slight etch at the center after 24 h, (d) nanorings after 48 h hydrothermal treatment, (e) higher magnification image of nanorings (scale bar in the inset is 100 nm and values are in nm), and (f) EDS spectrum acquired from the HAp nanorings.

Figure 4. Raman spectra of nanodisks (a) and nanorings (b). Inset shows the deviation of the specific peak.

Hu et al.12 prepared hematite nanorings by enlarging the hole at the center of nanodisks using a microwave-assisted hydrothermal method. They suggested that phosphate ion enrichment at the center induced the hole formation and the inner part of the disk was etched during the formation of nanorings, but the dissolution was found to be not uniform from disk to disk or even within a single disk. In contrast, the size of nanorings prepared in this study was observed to be quite uniform. The surface of the finally obtained well3568



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increased at the final stage nanoparticles exhibited mostly calcium pyrophosphate (Ca2P2O7) peaks along with few HAp peaks. Stoichiometric HAp (Ca/P = 1.67) production needs well controlled mixing of Ca and P sources with respective molar ratios. If the molar ratio and the mixing speed were modified, other calcium phosphate compounds were observed to be produced. When the flow rate was increased at the final stage, mostly calcium pyrophosphate (Ca2P2O7) was produced. From the literature, it is found that calcium pyrophosphate has a spherical geometry and tends to form a netlike matrix which may induce the formation of disk-like morphology by aligned aggregation.41 During the hydrothermal treatment, joining of aggregated spheres to form disks (Figure 5b) and phase transformation from calcium pyrophosphate to hydroxyapatite (Figure 2) as described in eqs 1 and 2 appeared to have occurred. From Raman and XRD analysis, the presence of calcium pyrophosphate was observed. Prolongation of the hydrothermal reaction to 48 h completely transformed the product to hydroxyapatite with an inappreciable modification of the disklike morphology. A combined high gravity and hydrothermal method induced the nucleation, coarsening, and partial dissolution for the formation of HAp nanorings. Nanocrystals grown in solution typically involve the fast nucleation of primary particles followed by subsequent growth by two primary mechanisms: oriented aggregation and Oswald coarsening. In the oriented aggregation, the crystalline lattice planes may be almost perfectly aligned or dislocations at the contact areas between the adjacent particles lead to defects in the finally formed bulk crystals.42 The reorientation of two contacting nanoparticles could be driven by Brownian motion.43Oriented aggregation of nanocrystals leads to single crystalline solids with defects such as edge and screw dislocations to accommodate the slight mismatch between oriented adjoin particles.44 Single crystalline aggregation is energetically favorable over polycrystalline aggregation because of the energy reduction in the absence of grain boundaries. Ostwald ripening is a mechanism driven by the fact that the chemical potential of a particle increases with a decrease in particle size. Larger crystals grow at the expense of smaller crystals. 3.5. Formation of Nanorings. The dissolution process in apatites due to the effect of acid treatment was well reported with different acids and time periods.30,45,46 Concerning the anisotropic and preferential dissolution process, the dissolution at preferred orientation or spots such as defects and chemical in-homogeneities were suggested. It is well-known that dislocations, linear defects at an atomic scale, are preferential sites for dissolution. It was reported that the acid attack or penetration starts with one etch pit in the HAp basal plane which is obviously the preferred dissolution spot or active site.30 Although the prism faces are attacked slightly by the acid, the main effect of the acid is the formation of a longitudinal hole parallel to the c-axis. In the present work, the pH of the initial phosphate solution was adjusted to 9. However, after the hydrothermal treatment at 180 °C it was observed that the solution’s pH was decreased to below 3. It is apparent from eq 2 that the increase of hydronium ion [H3O+] causes the decrease in pH during the hydrothermal treatment47 as the reaction proceeds and it creates an acidic environment which may promote etching of the basal plane to form HAp nanorings. The microstructural observation indicates that the protective environment against the etch-pit or hole formation maintained up to 12 h hydrothermal treatment and the acidic

Figure 5. TEM micrographs of the HAp nanostructures (a) nanospheres without hydrothermal treatment, (b) nanodisks after 12 h, (c) single nanodisk with slight etch at the center after 24 h, (d) single nanoring after 48 h hydrothermal treatment, (e) SAED pattern of the HAp nanorings, and (f, g) HRTEM images at different places of the HAp nanorings.

diameter of 70 ± 10 nm, and a periphery thickness of 60 ± 7 nm. 3.3. Formation Mechanism. Time-dependent morphological evolution was examined in order to understand the formation mechanism of hollow HAp nanorings during the hydrothermal treatment at 180 °C. The experiment was repeated several times to optimize the parameters for the formation of HAp disks and rings. The detailed possible formation mechanism is given below. 3.4. Formation of Nanodisks. Many reports are available for the formation of HAp nanorods by different preparation methods, especially by the hydrothermal method.38−40 However, surprisingly in this work we have obtained a disklike structure after initial hydrothermal treatment for 12 h (see Figures 3b and 5b) when the flow rate was increased stepwise during the initial high gravity process (condition II). The formation of disks rather than rods may be explained as follows: As explained in the structural analysis, the XRD pattern from nanospheres prepared under flow rate condition I exhibited peaks from the hexagonal HAp phase. However, under flow rate condition II, as the flow rate of the phosphate solution was 3569



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instability is likely to increase by an increase of strain energy of dislocations and thermal activation at the processing temperature of nanorings (180 °C). The absence of dislocations at the rim of nanoring/nanodiks as displayed in Figure 5f,g is supported by the increasing importance of Ostwald ripening in the late stage of nanodisk formation and the large image force for dislocation in HAp crystals. It was suggested that hollow−core type dislocations can be formed in materials with a large Burger vector (like apatites).62 The strain energy reduction around the dislocation site was compensated by creating a tube or hollow surface around the dislocation line. This may be the possible reason for the presence of a “hollow-core” dislocation, and it does not indicate that there is no material in the hollow-core type dislocation. This dislocation core most possibly consists of loosely bound, stained, and highly reactive atoms.63 It was confirmed from the HRTEM images Figure 6 that edge-type dislocations with the dislocation line parallel to the c-axis exist in the central region of nanodisks. In the acidic environment, etch pits can be created at the dislocation site, which can be extended into the crystal in the direction perpendicular to the (001) plane. In the later stage of the reaction, a cavity can be formed parallel to the caxis with hexagonal symmetry. To investigate whether the formation of holes along the dislocation lines are energetically favorable, the energy criterion was evaluated by assuming that the stable hole is created in the nanorings if the reduction of strain energy associated with the removal of a dislocation outweighs the increase of the surface energy associated with the creation a hollow tube. Here, both the nanorings and the central holes were assumed to take the cylindrical shapes with a limited height (thickness), l. The loss of total strain energy associated with dislocation is given as

environment favoring the hole formation prevailed and the cavity or hole was formed at 24 h hydrothermal treatment. The HRTEM image of the central region of nanodisks is shown in Figure 6. The dislocations were mostly observed in

Figure 6. HRTEM images of the nanodisks showing the edge type dislocations. Inset shows the schematic representation of edge dislocation.

the central region of the disk, and no dislocations were observed at the rim of nanodisk/nanorings as exhibited in Figure 5f. The dislocations in Figure 6 are edge-type. The screw dislocation which is likely to be grown during the growth of crystals was not observed. The absence of screw dislocation in Figure 6 does not necessarily mean that there is no screw dislocation in HAp crystals. In the disk, a screw dislocation with the Burgers vector perpendicular to the disk plane cannot be imaged. The presence of edge-type dislocation, however, suggests that the oriented attachment-based growth occur dominantly over Ostwald ripening in the early stage of coarsening.48,49 During the nanodisks formation by aggregation and oriented attachment, a slight misorientation between adjoining particles would result in the trapping of dislocations in the nanodisk.50 It is observed from the HRTEM images that only the center of the disks has the dislocation sites (Figures 5 and 6). The absence of dislocations at the rim of the nanoring/nanodisk suggests that the Oswald ripening process took over the oriented attachment process in the late stage of nanodisk formation. It has been reported that pits and defects are likely to be absent in particles formed by Ostwald ripening (point defects may be present).43As the particle size increased, oriented attachment process becomes less important because of the lower mobility of large particles,48 and the Oswald ripening process may take over in the late stage of disk growth, which favors the absence of edge dislocations at the rim. It is also well accepted that dislocations near a free surface are attracted to and removed from the surface,51,52 and therefore no dislocations were observed in nanostructured crystals with a high surface area such as metallic/ceramic fibers and whiskers.53 The dislocations close to the surface, if there are any, are thought to be unstable because of its image force and move out of the surface. Many investigators54−56 attempted to confirm the critical size of crystals below which the instability of dislocation prevails. In metals, the critical size was suggested to be 15−50 nm57−61 depending on the surface properties, elastic modulus, size of Burgers vector, and the lattice friction. The thickness of the nanoring rim in Figure 5f,g is thicker than the critical size suggested for metals. Since the image force which impels dislocations out of a free surface increases with an increase of Burgers vector, the image force is likely to be large in HAp crystals. Furthermore, the critical size of dislocation

Est =

Gb2l r ln 4π r0

(3)

where G is shear modulus of apatite which is given as 41.8 GPa;64 b is the Burgers vector of dislocation parallel to c-axis (9.4 Å in case of edge dislocations);65 l is the thickness of the nanorings; and r and ro are the radius from the dislocation center and the dislocation core radius, respectively. If the cylindrical hole with the radius r was formed perpendicular to the nanodisks plane, the creation of surface energy is given as Esurf = 2πrl(100)

(4)

where γ(100) is the surface energies of the prismatic plane (30 mJ/m2).66 The total energy change is given as ΔEtotal = Est + Esurf

(5)

The change of the total energy in eq 5 is plotted against the radius of the cylindrical hole along with the changes of strain energy associated with dislocation and the surface energy due to hole formation in Figure 7. The radius of the stable radius with the minimum total energy assuming the presence of a single edge dislocation was found to be 15 nm. The predicted diameter of the cylindrical hole (30 nm) is smaller than that of the observed hole (70 nm). The presence of extra dislocations (more than a single dislocation) would increase the predicted diameter. The presence of several dislocations as observed in Figure 6 are suggested to be enough to induce as cylindrical hole as large as 70 nm even if the stress field of each dislocation is appreciably overlapped. The careful observation of the 3570



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nanospheres, coarsening and coalescing with the oriented aggregation and Ostwald ripening. Dissolution of the central region of nanodisks driven mostly by the reduction of strain energy associated with dislocations to produce the perfect hollow rings. 3.6. Mechanical Analysis. 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 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.67 It would be, therefore, of great interest to study the mechanical performance of HMWPE matrix composites reinforced with nanoscale HAp crystals with various shapes. The effect of shape of HAp crystals on the mechanical performance of HMWPE/HAp composite was investigated and the obtained stress strain curves are shown in Figure 9. For

Figure 7. The change of total energy as a function of the radius of the central hole.

nanorings revealed that the thickness of the rim of the nanoring is quite uniform indicating the expansion of the hole was limited by the stable dislocation-free structure of the rim as exhibited in Figure 5f,g. On the basis of the above results and explanation, the possible formation mechanism of the HAp hollow nanorings could be proposed. The ultrafine nanoparticles (Figures 3a and 5a) formed by the high gravity method are unstable due to their high chemical potential and high surface energy. The coarsening process leads to the formation of monodispersed calcium pyrophosphate nanodisks initially. The prolonged hydrothermal treatment induces calcium pyrophosphate to transform to HAp with inappreciable modification of the initial disklike morphology. The dissolution along the dislocations perpendicular to the basal plane as the pH decreases with hydrothermal reaction induces nanostructured ring-shaped crystals. The schematic illustration of the formation mechanism of hollow HAp nanorings is presented in Figure 8. We conclude that the possible mechanism for the formation of the hollow HAp nanorings can be depicted as nucleation and growth of

Figure 9. Typical stress−strain curve of HMWPE reinforced with (a) HAp nanodisks, (b) nanospheres, (c) partially formed nanorings, (d) HAp nanorings, (e) pure HMWPE.

comparison, mechanical performance of the pure HMWPE is given in the figure (curve “e” in Figure 9). Reinforcement with HAp crystals is expected to enhance the bioactivity of HMWPE, but at the expense of ductility. The ductility of HMWPE reinforced with HAp nanodisks and nanospheres decreased significantly as shown in Figure 9. However, surprisingly enough nanoring-reinforced composite exhibited the excellent mechanical performance compared to the composites with other reinforcements. The tensile fracture strain of the HAp nanoring-reinfoced HMWPE composites showed the highest value than other nanoparticle reinforced composites, comparable to that of pure HMWPE. This demonstrated the advantages of HAp nanorings as the filler which may increase the biocompatibility of HMWPE with negligible loss of the ductility and strength. It was also noted that the mechanical property of composite with the partially formed HAp nanorings exhibited better mechanical performance compared to that reinforced with the nanodisks. The tensile fracture strain reached over 300% with the addition of

Figure 8. Schematic illustration of HAp nanorings formation. 3571



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partially formed nanodisks while it decreased drastically (∼190%) with the addition of nanodisks. Ring-like and tube-like structures with the central holes were suggested to possess higher surface area68−71 than other solid structures and exhibit enhanced characteristic properties such as catalytic property, biological performance, surface enhanced Raman scattering, etc. In nanorings and nanotubes, their hollow interior structure allows external agents to be encapsulated inside,69 which promotes more frequent and active interaction between nanocrystals and the surrounding matrix. In order to correlate the mechanical performance of HMWPE/HAp composites and the interface characteristics between HMWPE and HAp crystals, the specific surface areas (SSA) and pore size distributions of various shaped HAp nanocrystals were studied via BET analysis by the N2 adsorption/desorption method, which are given in Supporting Information. We have found that the specific surface area of the nanorings (89.62 m2/ g) is higher than those of nanodisks (46.32 m2/g) and partially etched nanodisks (58.24 m2/g). Apparently, the specific surface area increased with the formation of central holes in the nanodisks (See Supporting Information Figure s2 and Table s1). The fracture strain of HMWPE matrix composites reinforced with various shaped HAp crystals was plotted against the specific surface area (Supporting Information Figure s4) and the fracture strain increased with the specific surface area (SSA) of HAp nanocrystals. The pore size distributions and pore volumes of various shaped HAp nanocrystals are also provided in Supporting Information (Figure s3 and Table s1). The pore volume of nanorings is significantly greater than those of their counterparts. The increase of pore volume may be associated with the center holes in the nanorings which helped to create more channels on the surface, resulting in an increase of porosity and pore volume.70 It is well documented that72,73 the physical characteristics (shape, size, size distribution, etc.) of the filler are very important in determining mechanical properties of polymer nanocomposites. Apart from that, the specific surface area is one of the most important parameter of the fillers in the polymer composite formation.73 SSA is known to determine the amount of surface contact between the polymer matrix and the filler. Fillers with higher surface areas will contribute to more surface contact and bonding between the filler and matrix, thus increasing the mechanical properties of the composite. The shape of the filler also plays a major role in mechanical performance of composites. Compared to smooth spherical particles, the irregular shaped tiny particles are more preferable since the molten polymer can penetrate into troughs on the particle surface during high temperature processing route resulting in the mechanical interlock72−74 between HAp and HMWPE, in addition to any chemical bonding. The mechanical interlocking of HAp particles by the matrix developed upon the shrinkage of the polymer during cooling from the processing temperature.72−74 The same interlocking mechanism is believed to take place in HMWPE/nanorings composite, resulting in the enhanced mechanical performance. 3.7. In Vitro Cellular Assay. The SEM image in Figure 10 shows the CHO cells grown on the different HAp nanostructures. The cells are proliferated actively on the HAp nanorings compared to other nanostructured HAp crystals. Cell proliferation is also known to be dependent on the surface properties including the surface area.75−79 The cells were shown to grow and spread well, being in intimate contact with

Figure 10. Typical morphology of the CHO cells grown on HAp (a) nanospheres, (b) nanodisks, (c) nanodisks with slight etch at the center, and (d) nanorings.

the surface and the cell compatibility property also improved notably in HAp nanorings. The cell proliferation of the MG-63 human osteosarcoma cell on different morphologies of HAp nanocrystals as a function of days are also shown in Figure 11. It is observed that the cell

Figure 11. Cell viability of MG-63 cells on different nanostructures as a function of days.

viability varied with the change in morphology of HAp crystals. The cell proliferation rate reveals that there was inappreciable cytotoxicity on the prepared HAp nanoparticles on MG-63 cells. Cell viability in various shaped HAp nanocrystals were also plotted against the specific surface area (Supporting Information Figure s5), and the graph revealed that cell viability also increased with increase of the specific surface area, resulting in the superior cell viability in HAp nanorings with the higher specific surface area. Nanospheres and nanorods show almost similar cell viability property. Nanodisks and nanoparticle obtained by continuous flow rates (condition 3) which possess irregular shape with some different calcium phosphate compounds (Figure 1c) exhibited poor cell viability compared to other morphologies. 3572



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The excellent cell viability in nanorings is also attributed to the high specific surface area.68−71 Many reports suggest that the larger surface area allows enhanced adhesion of cell, protein, and drugs.75−79 Along with their larger surface areas, their hollow interior structure allows external agents to be encapsulated inside,69 enhancing the biological cell activity. 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, all of which contribute to better cell growth,78−81 supporting the excellent cell viability in nanorings in the present study.

ASSOCIATED CONTENT

S Supporting Information *

Additional FESEM images for all conditions, BET specific surface area, and pore size distribution related data are available. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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4. CONCLUSIONS The large-scale production of nanorings with relatively uniform size and shape distribution was produced by a combined high gravity and hydrothermal method in this study. The ringshaped HAp nanostructured crystals were obtained after optimizing several preparation conditions without using any templates for the first time. The mechanism for the formation of the hollow HAp nanorings can be depicted as the nucleation and growth of nanospheres, coarsening by the oriented aggregation and Ostwald ripening, and the acid penetration along the dislocations. Dissolution of the central region of nanodisks was presumed to be driven mostly by the reduction of strain energy associated with dislocations to produce the perfect hollow rings. The presence of edge dislocations in the central region of nanodisks was confirmed by TEM. The energy criterion based on the strain energy associated with the dislocations and the surface energy associated with the hole formation supported that the formation of holes along the dislocation lines are energetically favorable. The predicted diameter of the cylindrical hole based on the energy criterion assuming the presence of several dislocations are supposed to be close to the observed diameter of the hole in the nanodisks. The enhanced biocompatibility of nanorings and good mechanical performance of HMWPE/HAp nanoring composite are attributed to the enhanced interface bonding and cell activity associated with the high specific surface area of nanorings.

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AUTHOR INFORMATION

Corresponding Author

*(S.I.H.): [email protected]. Phone: +82-42- 8216595. Fax: + 82-42-822-5850. (D. M.): [email protected]. Phone: +91422- 2425458. Fax: + 91-422-2425706. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.J.N. gratefully acknowledges the financial support from the Ministry of Higher Education, Taiwan, through Taiwan−India collaborative research project. Also this work was partially supported by Korea National Research Foundation (20090077110). 3573



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