Hydroxyapatite Nanocrystals for Biomedical Applications - American

Oct 11, 2010 - fluorescence (x axis, FL1-H) vs PI fluorescence (y axis, FL2-H) shows logarithmic intensity. Quadrants: lower left (FITC-annexin V-/PI-...
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J. Phys. Chem. C 2010, 114, 18352–18357

Hydroxyapatite Nanocrystals for Biomedical Applications Huaqiang Cao,*,† Lu Zhang,‡ He Zheng,† and Zhao Wang*,‡ Department of Chemistry and Protein Science Key Laboratory of the Ministry of Education, School of Medicine, Tsinghua UniVersity, Beijing 100084, People’s Republic of China ReceiVed: July 1, 2010; ReVised Manuscript ReceiVed: September 21, 2010

This paper reports a biomolecule-assisted hydrothermal method to synthesize hydroxyapatite (HAP) nanocrystals. The as-synthesized samples were characterized by X-ray diffraction (XRD), Raman, transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), and Fourier transform infrared (FT-IR) spectroscopy. The data showed that the growth process of the HAP nanocrystals was composed of nucleation and growth processes. HAP nanocrystals can inhibit the proliferation of human HeLa cells in dose- and time-dependent manners, suggesting the promising potential of HAP nanocrystals in cancer therapy. Also, HAP nanocrystals own excellent mechanical properties, suggesting promising applications in dental restoration. This approach opens up vast opportunities for the synthesis and potential applications of nanostructures in the biomedical field. Introduction Nature is full of biomineral materials, such as shell, ivory, teeth, magnetic crystals in bacteria, and so on. Many complex biomaterials consist of inorganic minerals and macromolecular compounds, which have many fascinating functions. These inorganic compositions of biomineral materials can be found everywhere in nature and show excellent strength, crack roughness, highly smooth finish of surface, as well as other special functions. The special functions of biomineral materials are attributed to the self-assembly process and fine microstructures obtained under special bioprocesses,1 which inspires us to develop novel synthesis methods to generate inorganic nanocrystals via controlling the nucleation and growth processes of inorganic materials. Inorganic nanocrystals are a class of novel materials whose properties are defined by their submicrometer dimensions. The physicochemical properties of nanocrystals are different significantly from those of the corresponding bulk materials. However, the controlled synthesis of nanocrystals with novel physicochemical properties is still a great challenge. The synthesis, bioactivity, and application of hydroxyapatite [HAP, Ca10(PO4)6(OH)2] have been studied since 1970. HAP, being the major component of inorganics of bone textures in the human body, shows good biocompatibility and compression strength, which can be used as the infilling materials of the damaged site of bone underdevelopment, man-made ear bones, false eyeballs, medico-carriers, etc.2 Recently, great interest has been shown for synthesizing HAP crystals. Various techniques have been developed for generating nanostructured HAP, such as constructing complex HAP-based composites with stiffness (10 GPa), strength (150 MPa), and work of fracture (220 J/m2) that match those of compact bone;3 fabricating peptide-amphiphile nanofibers via pH-induced self-assembly and mineralization of HAP;4 generating HAP nanorods through liquid-solid-solution * Corresponding authors: (H.C.) tel +86 10 62794233, fax +86 10 62794233, e-mail [email protected]; (Z.W.) tel +86-1062772240, fax +86-10-6277675, e-mail [email protected]. † Department of Chemistry. ‡ Protein Science Key Laboratory of the Ministry of Education, School of Medicine.

technique;5 preparing platelike nanosized HAP via a homogeneous precipitation method in an ultrasound field;6 forming rodlike HAP particles via a biomimetic route;7 synthesizing HAP crystals via urea-assisted hydrothermal method;8 and others. Cuisinierk believed that the growth of bone was conducted step by step. First, ions were adsorbed on organic matrix and then HAP nanoparticles were nucleated and generated, accompanied by reorganization of matrix out of cells with interface molecules and molecule adsorption between organic ligands and inorganic crystals. Second, HAP nanoparticles extended to form needlelike crystals.9 The nucleotide ATP (adenosine triphosphate) is the cell’s most common energy carrier in all of the biological systemssthat is, the chemical energy reservoir of the cells and has been acquired by the remnant mitochondria of Encephalitozoon cuniculi.10 Recently, ATP has been employed as a building block for the self-assembly of nanowires. It is demonstrated that small biomolecules such as ATP and cyanine dyes can self-assemble into functional nanoarchitectures in the noncovalent combinatorial manner.11 ATP is used as a template to synthesize inorganic nanoparticles involved in the biomineralization process.12 ATP-triggered release of semiconductor CdS nanoparticles is also reported.13 Herein, we report a simple hydrothermal method to generate rodlike HAP nanocrystals by using ATPNa2 [C10H8N4O2NH2(OH)2(PO3H)3H] molecule as reactant reagent and to provide the possible growth mechanism. The anticancer activities and mechanical properties of the as-synthesized HAP nanocrystals are studied.

Experimental Section Synthesis. Generation of HAP nanocrystals was carried out via an ATP-assisted hydrothermal synthesis route. In a typical

10.1021/jp106078b  2010 American Chemical Society Published on Web 10/11/2010

Hydroxyapatite Nanocrystals procedure, 1.5 mmol of CaCl2 (analytic reagent, AR) was added into 20 mL of deionized water with stirring for 10 min to form solution A, while ATPNa2 (Beijing Kebio Biotechnology Co., Ltd., >98% purity; molar ratio of Ca2+/ATPNa2 ) 5:3) was dissolved into 19 mL of deionized water with stirring for 10 min to form solution B. Solution B was then was added dropwise into solution A, followed by addition of 1 mL of NH3 · H2O with stirring for 30 min at room temperature. The mixture was sealed into a 50 mL Teflon-lined autoclave, heated to 110 °C, and maintained at this temperature for a selected time (ranging from 1 to 120 h). After the autoclave was cooled down to room temperature naturally, the products were collected and washed via centrifugal method by using deionized water and then absolute alcohol. The cycle was repeated three times, followed by drying at 80 °C for 3 h. Characterization. Products were characterized by powder X-ray diffraction (XRD) on a Bruker D8 Advance diffractometer using Cu KR radiation (λ ) 1.5418 Å). Transmission electron microscopy (TEM) was carried out on a JEOL TEM-1200 operating at 120 kV and Hitachi H-800 operating at 200 kV. High-resolution transmission electron microscopy (HRTEM) was obtained by use of a JEM 2010 high-resolution transmission electron microscope using an accelerating voltage of 200 kV. The Fourier transform infrared (FT-IR) spectra were measured on a Nicolet 560 Fourier transform infrared spectrophotometer. Resonance Raman spectra (Renishaw, RM 1000) were measured with excitation from the 514.5 nm line of an Ar-ion laser. Cell Culture and Treatment. The human HeLa cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units/mL penicillin, and 100 µg/mL streptomycin and maintained at 37 °C in the air with mixture of 5% CO2. MTT (Thiazolyl Blue) Assay. A 100 µL suspension of HeLa cells per well was seeded in a 96-well plate at a density of 5 × 104 cells/mL. Cells grew for 12 h after seeding and were then treated with HAP nanocrystals or Ca3(PO4)2 at the designated concentrations (50, 100, 150, and 200 µg/mL) for different times (12, 24, and 48 h). Then, the cells were incubated in 200 µL of culture medium with 20 µL of MTT (5 mg/mL) for each well for 4 h. Finally, all media were removed, 150 µL of dimethyl sulfoxide (DMSO) was added to each well, and the plate was shaken for 10 min. The absorbance was read at a wavelength of 490 nm by use of a Benchmark microplate reader (Bio-Rad Corp.). Fluorescence Observation of HeLa Cells Stained by Hoechst 33342. To distinguish living cells from apoptotic and necrotic cells, HeLa cells were stained with fluorescent dye. A 500 µL suspension of HeLa cells per well was seeded in a 24well plate at a density of 5 × 104 cells/mL. Cells grew for 12 h after seeding and then were treated with HAP nanocrystals or Ca3(PO4)2 at the designated concentrations (100 and 200 µg/ mL) for different times (12, 24, and 48 h). After being washed with ice-cold PBS twice, cells were fixed with 200 µL of methanol for 10 min at room temperature. Cells were washed with PBS twice. Then, 20 µL of Hoechst 33342 (100 µg/mL) was added to 80 µL of PBS for each well. Cells were stained at 37 °C for 10 min. After being washed with PBS twice, the cells were observed, and pictures were taken with a DMIRB inverted fluorescence microscope (Leica Corp.). Flow Cytometer Measurement. The percentage of apoptotic and necrotic cells was assayed with an Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit (AP101, BioVision). A 1 mL suspension of HeLa cells per well was seeded in a 6-well plate at a density of 1 × 105 cells/mL.

J. Phys. Chem. C, Vol. 114, No. 43, 2010 18353 TABLE 1: Experimental Parameters of As-Synthesized Nanorod Products sample

Ca2+/ATP (molar ratio)

n(Ca2+) (mmol)

temp (°C)

time (h)

HAP-1 HAP-2 HAP-3 HAP-4 HAP-5 HAP-6 HAP-7 HAP-8 HAP-9 HAP-10

5:3 5:3 5:3 5:3 5:3 5:3 5:3 5:3 5:3 5:3

3.0 3.0 1.5 1.5 1.5 1.5 3.0 3.0 3.0 0.75

110 110 110 110 110 110 110 110 110 110

1 5 10 15 48 120 10 48 120 48

Cells grew for 12 h after seeding and were treated with HAP nanocrystals or Ca3(PO4)2 at the designated concentrations (50, 100, and 200 µg/mL) for different times (12, 24, and 48 h). The cells were gently trypsinized (0.25% tryspin with 0.02% EDTA) and washed with serum-containing medium two times. After being resuspended in 500 µL of annexinbinding buffer, the cells were coincubated with 5 µL of annexin V FITC and 10 µL of PI in the absence of light for 5 min at room temperature. Finally, fluorescence intensities of cells stained were analyzed by flow cytometer (CellLab Quanta SC, Beckman). Nanoindentation Test. Nanoindentation measurements were performed by use of an MTS XP nanoindenter (MTS Systems Co., Nano Instruments) equipped with a Berkovich (three-sided pyramidal) diamond tip (100 nm radius). The load and displacement resolution of the instrument were 50 nN and 0.01 nm, respectively. The continuous stiffness measurement (CSM) technique14 was used to determine the stiffness of the sample continuously as a function of depth with constant strain rate of 0.05 s-1. Results and Discussion A series of as-synthesized products with different reaction times, concentrations of reactants, and molar ratios of reactants are listed in Table 1. The effect of reaction time is investigated by changing the reaction time from 1 h (denoted as HAP-1, Figure S1a in Supporting Information) to 5 h (denoted as HAP2, Figure S1b in Supporting Information), 10 h (denoted as HAP-7, Figure S1g in Supporting Information), 48 h (denoted as HAP-8, Figure S1h in Supporting Information), and 120 h (denoted as HAP-9, Figure S1i in Supporting Information) with other conditions remaining constantsidentical concentrations (Ca2+ concentration ) 3.0 mmol/40 mL, ATP concentration ) 1.8 mmol/40 mL) and identical reaction temperature of 110 °C. We found that HAP-1 and HAP-2 are particles of 5-15 nm (HAP-1) and 10-25 nm (HAP-2), while HAP-7, HAP-8, and HAP-9 are rods 10-20 nm in diameter/20-50 nm in length (HAP-7), 10-20 nm in diameter/100-300 nm in length (HAP8), and 10-20 nm in diameter/30-100 nm in length (HAP-9). Also, we worked another group experiment with different reaction times, changing the reaction time from 10 h (denoted as HAP-3, Figure S1c in Supporting Information) to 15 h (denoted as HAP-4, Figure S1d in Supporting Information), 48 h (denoted as HAP-5, Figure S1e in Supporting Information), and 120 h (denoted as HAP-6, Figure S1f in Supporting Information) with other conditions remaining constantsidentical concentrations (Ca2+ concentration ) 1.5 mmol/40 mL, ATP concentration ) 0.9 mmol/40 mL) and identical reaction temperature of 110 °C. The sizes are ∼10 nm in diameter/10-30 nm in length (HAP-3), 10-20 nm in diameter/10-50 nm in length (HAP-

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Figure 1. (a) XRD pattern and (b) Raman spectrum of a typical as-synthesized sample (HAP-9).

Figure 2. (a) TEM image, (b) corresponding SAED pattern, and (c) HRTEM image of the as-synthesized sample (HAP-9).

4), 10-20 nm in diameter/20-50 nm in length (HAP-5), and 10-30 nm in diameter/30-90 nm in length (HAP-6). The longer the reaction time, the larger the size of the HAP nanocrystals. The concentration effect is also investigated by changing the reaction concentration of Ca2+ from 0.75 mmol/40 mL (denoted as HAP-10, Figure S1j in Supporting Information) to 1.5 mmol/ 40 mL (denoted as HAP-5, Figure S1e in Supporting Information) to 3.0 mmol/40 mL (denoted as HAP-8, Figure S1h in Supporting Information) with other conditions remaining constant, at the identical reactant molar ratio (Ca2+/ATPNa) of 5:3, with the identical reaction temperature of 110 °C and the identical reaction time of 48 h. The sizes are 10-20 nm in diameter/20-50 nm in length (HAP-10), 10-20 nm in diameter/ 20-50 nm in length (HAP-5), and 10-20 nm in diameter/ 100-300 nm in length (HAP-8). The larger the Ca2+ concentration, the larger the size of HAP nanocrystals. The phase structure and purity of the obtained products are investigated by powder XRD and Raman analysis (Figure 1). All the diffraction peaks can be attributed to pure HAP (JCPDS card file 76-0694). The broad XRD peaks (Figure 1a) are attributed to the very small particle size, which is also demonstrated by TEM observation. Typically, the Raman spectrum of the as-synthesized sample (Figure 1b) shows a very strong characteristic peak at ∼961 cm-1, which is attributed to the symmetric stretching mode ν1(PO43-).15 Both XRD and Raman analysis demonstrate that the as-synthesized sample belongs to pure HAP phase. More information about the microstructure can be obtained via TEM and HRTEM observation (Figure 2). Figure 2a shows a representative TEM image of monodisperse rodlike HAP nanocrystals. The average size of the HAP nanocrystals is ∼17 nm in diameter and 104 nm in length. The rings of the selected area electron diffraction (SAED) pattern (Figure 2b) confirm the HAP nanocrystals to be multicrystalline. More information about the crystal can be derived from the HRTEM image (Figure 2c). The lattice fringes observed in the HRTEM image agree well with the separation between the (002), (242), and (343) lattice planes, respectively, which also demonstrates the HAP nanocrystals to be multicrystalline.

Figure 3. FT-IR spectrum of a typical as-synthesized sample (HAP7).

FT-IR spectrum (Figure 3) recorded from the HAP nanorods shows absorption bands at 3570, 3440, 1640, 1460, 1030, 962, 877, 634, 604, 565, and 472 cm-1. The bands at 3570 and 634 cm-1 are attributed to ν(OH) stretching vibration and ν(OH) librational vibration, respectively, due to hydroxyl group O-H.15 Usually, the O-H bands occur at higher frequency of about 3600 cm-1 and have a sharp absorption peak. However, the formation of hydrogen bonds results in a shift to lower wavelengths, from 3500 to 2900 cm-1, accompanied by a broadening of O-H stretching bands of FT-IR data.16 The broad band at 3440 cm-1 is assigned to the absorbed water.17 The band at 1640 cm-1 is assigned to the ν2 bending mode of the H2O molecules.18 The bands at 1110, 1030, 962, 604, 565, and 472 cm-1 are assigned to vibrations of the phosphate group, PO43-. Among these bands, the band at 962 cm-1 is due to the symmetric stretching mode ν1(PO43-), 472 cm-1 to the bending mode ν2(PO43-), 1110 and 1030 cm-1 to the vibration mode ν3(PO43-), and 604 and 565 cm-1 to bending modes ν4(PO43-).15,18 The weak bands at 877 and 1455 cm-1 are attributed to vibration modes of ν2(CO32-) and ν3(CO32-), respectively.18 These data demonstrate incorporation of carbonate groups into the crystal structure of HAP. Carbonate ions are a common impurity in HAP.18,19 It is worth pointing out that pure ATP does not show any peak in this FT-IR zone of as-prepared HAP nanostructures

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Figure 4. Schematic pattern of the growth mechanism of HAP nanocrystals.

Figure 5. Effects of HAP nanocrystals on cell viability of HeLa cells. (a) Control, control group; Ca200, 200 µg/mL Ca3(PO4)2 group; HAP50, 50 µg/mL HAP group; HAP100, 100 µg/mL HAP group; HAP200, 200 µg/mL HAP group. In comparison to the control and negative control [200 µg/mL Ca3(PO4)2 group], HAP-treated groups had significant inhibition of HeLa cell proliferation (p < 0.001). (b) HAP groups with designated concentrations (50, 100, and 200 µg/mL) for different times (12, 24, and 48 h), respectively. HAP nanocrystals produced a decrease of proliferative activity of HeLa cells, with significant changes from 12 to 48 h of cell culture (*p < 0.001).

(The FT-IR spectrum of ATPNa2 is shown in Supporting Information, Figure S2.) The crystal morphology is controlled by the thermodynamic energy and kinetic factors in the crystal growth process. Crystal formation, that is, a stable solid-state equilibrium phase generated via nucleation and growth, is usually attributed to the result of minimum interface energy. In the view of dynamics, the crystal planes with the lowest growth rate will form the largest crystal surface, while the crystal plane with the largest growth rate will have a small crystal surface or even disappear in the growth process. On the basis of the experimental data and analysis, we believe that the crystal plane with low interface energy is the crystal plane with a low growth rate.20 The possible mechanism behind the formation of the crystalline HAP nanorods is presented in Figure 4. It is composed of two stages: nucleation and orientational growth. The first stage is the nucleation process, that is, the initial reaction between Ca2+ and ATP2- ions, which is too fast to generate the HAP [Ca10(PO4)6(OH)2] nuclei. The nucleation process is due to the surface stabilization of nanoparticles, which is driven by thermodynamic parameters that are related to the particle size.21 It is believed that small seed particles are initially generated by the decomposition of highly reactively precursors, thereby explaining the fast growth rate, shown as follows:

10Ca2+ + 2ATP2- f Ca10(PO4)6(OH)2

(1)

Figure 6. Fluorescent images of HeLa cells apoptosis induced by HAP with different concentrations and different times (× 400). (a) Control; (b-d) 100 µg/mL for 12, 24, and 48 h, respectively; (e-g) 200 µg/ mL for 12, 24, and 48 h, respectively. Several apoptotic cells are marked with white arrowheads.

Usually, the nucleation occurs over some time with constant monomer concentrationsthe nucleation ends when the monomer concentration falls below the critical level for nucleation (critical supersaturation level).22,23 During the second stage, nanorods are generated from the growth of nuclei. The nanorods can be regarded as quasi-one-dimensional nanoparticles. The formation of nanorods via the oriented attachment mechanism24 is determined by the surface energy, which must be minimal for

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Figure 7. Flow cytometric analysis of HeLa cells treated with HAP nanocrystals for 12, 24, or 48 h and then double-labeled with FITClinked annexin V/PI. Dual-parameter dot plot of FITC-annexin V fluorescence (x axis, FL1-H) vs PI fluorescence (y axis, FL2-H) shows logarithmic intensity. Quadrants: lower left (FITC-annexin V-/PI-), viable cells; lower right (FITC-annexin V+/PI-), early apoptotic cells; upper right (FITC-annexin V+/PI+), necrotic or late apoptotic cells; upper left (FITC-annexin V-/PI+), damaged cells. (a) Control; (b) negative control of HeLa cells treated with 200 µg/mL Ca3(PO4)2 for 48 h; (c-e) cells treated with HAP nanocrystals at 50, 100, and 200 µg/mL, respectively, for 12 h; (f-h) cells treated with HAP nanocrystals at 50, 100, and 200 µg/mL, respectively, for 24 h; (i-k) cells treated with HAP nanocrystals at 50, 100, and 200 µg/mL, respectively, for 48 h.

a given volume for a crystal in equilibrium with its surroundings.21 It is well-known that when a large solid piece of material is broken into smaller parts, it needs to cut the bonds between the neighboring atoms and generates new surfaces, which needs energy (i.e., surface energy) to overcome the bonds.25 Obviously, the surface atoms have fewer nearest-neighboring atoms, compared with bulk atoms, which will raise the energy and therefore lower the stability of those atoms as well as the surfaces themselves. Minimizing surface area is the driving force that exists in materials. The smaller the surface-to-volume ratio, the lower the energy state of the material.21 In order to minimize the surface energy, directed bonds in anisotropic lattices raise crystallization in rods or platelets, which leads to the formation of one- or two-dimensional particles, such as rods or plates. To assess the anticancer activity of the HAP nanocrystals, the live human cervical cancer cells (HeLa) are used as a cell model in vitro. The inhibition of HeLa cell proliferation by HAP

nanocrystals is measured by MTT colorimetric analysis,25,26 and the results are shown in Figure 5. After HeLa cells are incubated with HAP nanocrystals or Ca3(PO4)2 at the designated concentrations (50, 100, and 200 µg/mL), a time-dependent decrease of cell viability from 84.43% to 65.35% or from 81.00% to 56.61% compared with the control is observed while time is prolonged from 12 to 48 h, respectively. For a negative control experiment, we select Ca3(PO4)2 as a negative control reagent, which has similar physical properties to HAP and shows no inhibition for HeLa cell proliferation. Also, the dose-dependent decrease of cell viability from 90.03% to 81.00%, 82.71% to 71.56%, and 68.69% to 56.61%, accompanying the HAP concentrations changing from 50 to 100 to 200 µg/mL at designated times of 12, 24, and 48 h, respectively, is observed. Obviously, HAP nanocrystals have effectively inhibited HeLa cell proliferation. We also determine the apoptosis induced by HAP nanocrystals using the fluorescent microscope and flow cytometer.25 In the control group, as shown in Figure 6a, the nucleus is round and free of condensation and fragmentation, with a smooth nuclear membrane. However, increased nuclear fragmentation and apoptotic body formation are identified in HAP nanocrystaltreated groups (Figure 6). The higher the concentration and the longer the treatment time, the more apoptotic cells there are. These results are further confirmed by flow cytometric analysis. Only a few apoptotic cells can be observed when HeLa cells are incubated with normal culture medium or Ca3(PO4)2 at the designated times and concentrations. When the incubation time is prolonged from 12 to 24 or 48 h, increases in the percentages of apoptotic cells are observed in HAPsfrom 5.23% to 8.32% and 37.03% for 50 µg/mL HAP, from 8.23% to 13.05% and 36.04% for 100 µg/mL HAP, and from 9.06% to 14.49% and 43.77% for 200 µg/mL HAP, respectively (Figure 7). However, the apoptotic rates of the control and Ca3(PO4)2 (concentration ) 200 µg/mL) experiments are 0.08% and 2.24% (Figure 7a,b). These results suggest that HAP nanocrystals can induce apoptosis or necrosis of HeLa cells. And the apoptotic rates increase with prolonged treatment time and increasing HAP nanocrystal concentrations, which is in agreement with the observation from fluorescence microscopy. Teeth basically consist of three different calcified tissues, namely, enamel, dentin, and cementum. It is known that dental hard tissues are considered as complex hydrated biological composites composed of inorganic materials (HAP), organic materials, and water.27 Enamel rod is composed of carbonated HAP nanocrystals, each separated from the other by a tiny intercrystalline space composed of organic materials and water, while cementum also consists of an inorganic component (65 wt %) mainly composed of HAP. The mechanical properties of as-synthesized HAP nanocrystals are studied by using nanoindentation technique. It is popular to evaluate the me-

TABLE 2: Comparison of the Results Obtained for Hardness and Elastic Modulus of Disc-Shaped HAP Nanocrystal Specimens with Related Values Given by the Sample and Published Data (HAP-9) ref this work 29 29 30 31 31 31 32 32

sample

type

load (mN)

hardness (mean ( SD) (GPa)

elastic modulus (mean ( SD) (GPa)

HAP nanocrystals enamel dentin dentin cements cements cements cements cements

disc-shaped primary molars primary molars permanent molars self-curing cements light-curing cements dual-curing cements biocements biocements

43-45 50, 150 50, 150 5 100 100 100 100 100

0.88 ( 0.02 0.48 ( 0.35 0.92 ( 0.11 0.49 0.21 ( 0.01 0.31 ( 0.03 0.31 ( 0.01 0.406 0.331

27.91 ( 0.83 80.35 ( 7.71 19.89 ( 1.92 19.26 4.9 ( 0.2 8.8 ( 0.5 7.4 ( 0.2 10.9 9.7

Hydroxyapatite Nanocrystals chanical properties of dental materials on a small scale by using nanoindentation tests changed from traditional microhardness tests, due to its ability to test vary small quantities of material, as well as providing simultaneous data on hardness and elastic modulus.28 Herein, we carried out the measurement of the mechanical properties of as-synthesized HAP by nanoindention techniques, including hardness and elastic modulus. The hardness and elastic modulus data provided by the manufactures is checked by testing seven different positions of the as-synthesized HAP disk-shaped specimens of size of 13 mm in diameter and 1 mm in thickness via the pressed disk technique. Hardness is the ability of a material to resist a permanent indentation, while elastic modulus is the ratio of stress to corresponding strain below the proportional limit.29 The hardness and elastic modulus data for enamel based on nanoindentation from various publications and our results are shown in Table 2. According to the results obtained, our specimen presents excellent hardness and elastic modulus compared with other data. This suggests that the as-synthesized HAP nanocrystals could find applications as a filling material of dental restoration for their excellent mechanical properties. Conclusion In summary, we provide a simple hydrothermal route to synthesize HAP nanocrystals and investigate their primary biomedical applications. The characterization and analysis, including TEM, HRTEM, XRD, Raman, and FT-IR analysis, allows us to propose a nucleation-growth mechanism for the generation of HAP nanocrystals. The as-synthesized HAP nanocrystals have obvious anticancer properties in HeLa cells via an apoptosis mechanism assessed by fluorescence observation and flow cytometric measurement. This research indicates the as-synthesized HAP nanocrystals effectively suppress the proliferation of HeLa cells, as well as being a material used for the restoration of lost tooth structure. Our findings open up a new biomedical application of inorganic nanocrystals in the future. Acknowledgment. We gratefully acknowledge the financial support from the National Natural Science Foundation of China (20921001, 20535020), the Innovation Method Fund of China (20081885189), the National High Technology Research and Development Program of China (2009AA03Z321), and the Tsinghua-Yue-Yuen Medical Sciences Fund (THYY 20070008). Supporting Information Available: Two figures showing TEM images of the as-synthesized HAP nanocrystals and the FTIR spectrum of ATPNa2. This material is available free of charge via the Internet at http://pubs.acs.org.

J. Phys. Chem. C, Vol. 114, No. 43, 2010 18357 References and Notes (1) Biomaterials Science, 2nd ed.; Cui, F., Fen, Q., Eds.; Tsinghua University: Beijing, China, 2004. (2) Inorganic Biomaterials; Tang, S., Mao, X., Eds.; South of China University of Technology Press, Guangzhou, China, 2008. (3) Deville, S.; Saiz, E.; Nalla, R. K.; Tomsia, A. P. Science 2006, 311, 515. (4) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Science 2001, 294, 1684. (5) Wang, X.; Zhuang, J.; Peng, Q.; Li, Y. AdV. Mater. 2006, 18, 2031. (6) Jevtic´, M.; Sˇkapin, S.; Jancˇar, B.; Ignjatovic´, N.; Uskokovic´, D. Cryst. Growth Des. 2008, 8, 2217. (7) Zhang, Y.; Lu, J. Cryst. Growth Des. 2008, 8, 2101. (8) Neira, I.; Kolen’ko, Y. V.; Lebedev, O. I.; Tendeloo, G. V.; Gupta, H. S.; Guitia´n, F.; Yoshimura, M. Cryst. Growth Des. 2009, 9, 466. (9) Controlling Matrix of Biomineralization and Biomimc Applications; Ouyang, J., Ed.; Chemical Industry Press: Beijing, China, 2006. (10) Tsaousis, A. D.; Kunji, E. R. S.; Goldberg, A. V.; Lucocq, J. M.; Hirt, R. P.; Embley, T. M. Nature 2008, 453, 533. (11) Morikawa, M.-A.; Yoshihara, M.; Endo, T.; Kimizuka, N. J. Am. Chem. Soc. 2005, 127, 1358. (12) Berti, L.; Burley, G. Nat. Nanotechnol. 2008, 3, 81. (13) Ishii, D.; Kinbara, K.; Ishida, Y.; Ishii, N.; Okochi, M.; Yohda, M.; Aida, T. Nature 2003, 423, 628. (14) Oliver, W. C.; Pharr, G. M. J. Mater. Res. 2004, 19, 3. (15) Koutsopoulos, K. J. Biomed. Mater. Res. 2002, 62, 600. (16) Organic Chemistry; Clayden, J., Greeves, N., Warren, S., Wothers, P., Eds.; Oxford University Press: Oxford, U.K., 2001. (17) Zhang, Y.; Lu, J. Cryst. Growth Des. 2008, 8, 2101. (18) Jevtic, M.; Mitric, M.; Sˇkapin, S.; Jancˇar, B.; Ignjatovic´, N.; Uskokovic´, D. Cryst. Growth Des. 2008, 8, 2217. (19) Markovı´c, M.; Flower, B. O.; Tung, M. S. J. Res. Natl. Inst. Stand. Technol. 2004, 109, 553. (20) Biomineralization; Cui, F. Z.; Ed.; Tsinghua University Press: Beijing, China, 2006. (21) Introduction to Nanoscience; Hornyak, G. L., Dutta, J., Tibbals, H. F., Rao, A. K., Eds.; CRC Press: Boca Raton, FL, 2008. (22) Xiao, Y.; Cao, H.; Liu, K.; Zhang, S.; Chernow, V. Nanotechnology 2010, 21, 145601. (23) Viswanatha, R.; Sarma, D. Growth of Nanocrystals in Solution. In Nanomaterials Chemistry; Rao, C. N. R., Ed.; Wiley-VCH: Weinheim, Germany, 2007. (24) Banfield, J. F.; Welch, S. A.; Zhang, H. Z.; Ebert, T. T.; Penn, R. L. Science 2000, 289, 751. (25) Nanomaterials: An introduction to synthesis, properties and applications; Vollath, D., Ed.; Wiley-VCH: Weinheim, Germany, 2008. (26) Wu, Q. Z.; Cao, H. Q.; Luan, Q. Y.; Zhang, J. Y.; Wang, Z.; Warner, J. H.; Watt, A. A. R. Inorg. Chem. 2008, 47, 5882. (27) Angker, L.; Swain, M. V. J. Mater. Res. 2006, 21, 1893. (28) Nanoindentation; Fischer-Cripps, A. C., Ed.; Springer Verlag: New York, 2002. (29) Mahoney, E.; Holt, A.; Swain, M.; Kilpatrick, N. J. Dent. 2000, 28, 589. (30) Van Meerbeek, B.; Willems, G.; Celis, J. P.; Roos, J. R.; Braem, M.; Lambrechts, P.; Vanherle, G. J. Dent. Res. 1993, 72, 1434. (31) Ceballos, L.; Garrido, M. A.; Fuentes, V.; Rodrı´guez, J. Dent. Mater. 2007, 23, 100. (32) Peluccio, M. S.; Bignardi, C.; Lombardo, S.; Montevecchi, F. M.; Carossa, S. J. Phys.: Condens. Matter. 2007, 19, 395003.

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