Ultrasound-assisted fabrication of a biocompatible magnetic hydroxyapatite Gang Zhou,1,2* Wei Song,1,2* Yongzhao Hou,1 Qing Li,1,3 Xuliang Deng,4 Yubo Fan1,2 1

Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing 100191, People’s Republic of China 2 Research Institute of Beihang University in Shenzhen, Shenzhen 518057, People’s Republic of China 3 The Second Dental Center, Peking University School and Hospital of Stomatology, Beijing 100101, People’s Republic of China 4 Department of Geriatric Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081, People’s Republic of China Received 25 September 2013; revised 30 October 2013; accepted 18 November 2013 Published online 12 December 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35043 Abstract: This work describes the fabrication and characterization of a biocompatible magnetic hydroxyapatite (HA) using an ultrasound-assisted co-precipitation method. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and transmission electron microscopy (TEM) were used to characterize the structure and chemical composition of the produced samples. The M–H loops of synthesized materials were traced using a vibrating sample magnetometer (VSM) and the biocompatibility was evaluated by cell culture and MTT (3-(4,5dimethylthiazol-2-yl)22,5-diphenyltetrazolium bromide) assay. Furthermore, in vivo histopathological examinations were used to evaluate the potential toxicological effects of Fe3O4-HA composites on kidney of SD rats injected intraperitoneally with Fe3O4-HA particles. The results showed that magnetic iron oxide particles first replace OH ions of HA, which are parallel to the c axis, and then enter the HA crystal lattice which pro-

duces changes in the crystal surface of HA. Chemical bond interaction was observed between PO432 groups of HA and iron ions of Fe3O4. The saturation magnetization (MS) of Fe3O4HA composites was 46.36 emu/g obtained from VSM data. Cell culture and MTT assays indicated that HA could affect the growth and proliferation of HEK-293 cells. This Fe3O4-HA composite produced no negative effects on cell morphology, viability, and proliferation and exhibited remarkable biocompatibility. Moreover, no inflammatory cell infiltration was observed in kidney histopathology slices. Therefore, this study succeeds to develop a Fe3O4-HA composite as a prospective C biomagnetic material for future applications. V 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3704–3712, 2014.

Key Words: hydroxyapatite, Fe3O4, composite, magnetic, biocompatibility

How to cite this article: Zhou G, Song W, Hou Y, Li Q, Deng X, Fan Y. 2014. Ultrasound-assisted fabrication of a biocompatible magnetic hydroxyapatite. J Biomed Mater Res Part A 2014:102A:3704–3712.

INTRODUCTION

Magnetic materials have attracted considerable attention for applications in medical devices and pharmaceuticals,1 where they are commonly used for bioseparation,2 drug delivery,3 cell sorting,4 and diagnostic analysis.5 There has been a recent surge in the use of Fe3O4 nanoparticles6 for biological applications7,8 due to the tremendous work achieved in the synthesis and functionalization of such materials9–11 Recently, Fe3O4-based nanocomposites have been developed into a new magnetically responsive nanodevice suitable for intravenous drug administration, which has been shown to

be advantageous for intracellular drug delivery.12 For magnetic resonance imaging (MRI) applications, Fe3O4 nanoparticles have been utilized as probes.13,14 However, it has been reported that Fe3O4 nanoparticles could induce cell death in malignant tissues.15,16 This would inherently limit its use in biomedical science. Non-toxicity is the most essential requirement for Fe3O4 for biomedical applications. Thus, an ideal magnetic particle intended for therapeutic treatment should be clinically safe and have a low toxicity, simultaneously minimizing undesired side effects.17–19 Also, any material intended for use

*These authors contributed equally to this work. Correspondence to: Y. Fan; e-mail: [email protected] Contract grant sponsor: National Basic Research Program of China; contract grant number: 2011CB710901 Contract grant sponsor: NSFC; contract grant numbers: 11002016, 61227902, 10925208, 11120101001 Contract grant sponsor: National Basic Research Program of China; contract grant number: 2011CB710901 Contract grant sponsor: National Key Technology R&D Program; contract grant numbers: 2012BAI18B06, 2012BAI18B05 Contract grant sponsor: 111 Project of China; contract grant number: B13003 Contract grant sponsors: International Joint Research Center of Aerospace Biotechnology and Medical Engineering, Ministry of Science and Technology of China, and the Fundamental Research Funds for the Central Universities of China

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within the body should be biocompatible and, ideally, not produce any undesired biological response. The design of a biomagnetic composite for biomedical applications is therefore challenging. The composite should be excellent magnetization and biocompatibility. Various polymers have been investigated for coating magnetic particles,20 such as polyvinyl alcohol (PVA), polylactic acid (PLA), and polyglycolic acid (PGA) and typically made of lipids, proteins, gelatin, alginate, and so on.21 The polymer is implemented as a shell around the iron oxide core without molecular shell iron oxides nanoparticles which will rapidly aggregate through interactions between themselves. But the core-shell structure coating results in a sharp decrease in the magnetic properties of Fe3O4. This modification also affects the colloidal and functional stability of superparamagnetic iron oxide nanoparticles.22 Recently, second phase reinforcement of composites has been used for improving material biocompatibility. The intrinsic biological properties of hydroxyapatite (HA: Ca10(PO4)6(OH)2), such as superior biocompatibility and bioactivity,23,24 make it very suitable as a second phase. Especially HA interacts better with divalent metals25 by ion exchange.26,27 Xie et al.28,29 demonstrated that the phosphonate functional groups were stable and strong anchors on the Fe3O4 nanoparticle surface. Therefore, to develop nontoxic and biocompatible Fe3O4-based compounds, introducing HA is often highly desirable. However, obtaining magnetic biomaterials which combine bioactivity and magnetic properties is not an easy task; the inclusion of iron (Fe) seems to diminish the bioactivity. Traditionally, the sol–gel and coating methods11,30 are inadequate methods for obtaining Fe3O4-HA composites because the iron becomes segregated, forming nonmagnetic precipitates. Besides, the conventional preparation of Fe3O4-HA composites is carried out into the aqueous solution. The precipitates31,32 tend to aggregate and cannot be dispersed sufficiently because the HA nanoparticles decrease their high surface energy, leading to serious agglomeration of nanosized composites. And ultrasound irradiation33 can create a special reaction environment to deagglomerate nanoparticles and improve the reaction greatly owing to the strong chemical and mechanical effects of acoustic cavitation. This research aimed to design and synthesize a novel magnetic hydroxyapatite composite based on the ultrasound-assisted co-precipitation method. This method being presented for magnetic nanoparticle preparation allows for quicker formation of composite nanoparticles. Due to all the materials used in the experiments being environmentally benign and renewable, this method allows for the production of ecofriendly and biocompatible materials. The properties of the developed composite were characterized by XRD, FT-IR, TEM, and VSM. Besides the present study investigated the in vitro biocompatibility and bioactivity of Fe3O4-HA nanoparticles in cultures of cells. These studies were also followed subsequently by a pilot study to verify in vivo biocompatiability of these innovative materials.

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MATERIALS AND METHODS

All the chemical reagents used in this work were of analytical grade. Synthesis of Fe3O4 FeCl36H2O (Beijing Chemical Reagents, 97 wt% pure, 0.5 mol/L) and FeCl24H2O (Beijing Chemical Reagents, 99 wt% pure, 0.25 mol/L) were contemporarily added to the basic suspension as sources of Fe21 and Fe31 ions. NH4•OH (Beijing Chemical Reagents, 25 wt% pure, 1.5 mol/L) was then titrated into solution with vigorous stirring and the pH was adjusted to 14 at 25 C. After titration and aging for 24 h, the precipitates were collected by centrifuging the suspension at 3000 r/min and washing in distilled water. Finally, the Fe3O4 powders were dried and sieved at 150 lm. Synthesis of HA HA slurry was synthesized by a precipitation method.34 Briefly, trisodium phosphate (Na3PO412H2O) (Beijing Chemical Reagents, 85 wt% pure, 0.3 mol/L) solution was added dropwise into calcium nitrate (Ca(NO3)24H2O, Beijing Chemical Reagents, 95 wt% pure, 0.5 mol/L) according to a Ca/P molar ratio of 5:3. The reaction was subjected to vigorous stirring and the pH was adjusted to 14 by adding an ammonia solution (NH4•OH, Beijing Chemical Reagents, 25 wt% pure) at 25 C. After titration and aging for 24 h, the precipitates were collected by centrifuging the suspension at 4 g and washing in distilled water. Finally, the solution was then dried to obtain the HA powder. Synthesis of Fe3O4-HA Ca(NO3)24H2O(0.5mol/L), FeCl36H2O (0.5 mol/L) and FeCl24H2O (0.25 mol/L) were dissolved in water as solution [A]. Na3PO412H2O (0.3 mol/L) and NH3H2O (1.5 mol/L) were also dissolved in water as solution [B]. Solution [B] was then titrated into solution [A] with ultrasound-assisted stirring. After ultrasound-assisted dispersion at 25 C for 5 h and then ageing for 24 h at room temperature without further stirring, the precipitates were separated from mother liquor by centrifugation, then washed with distilled water and centrifuged three times. Finally, the Fe3O4-HA powder was collected after drying. Characterization of materials The crystal structure of the synthesized materials was determined by X-ray diffractometry (XRD, X’Pert pro-MPD PANalytical B.V.) with Cu/Ka radiation (k 5 0.15405 nm) (40 kV, 40 mA). Chemical analysis of the composite was carried out by infrared spectroscopy (IR, Thermo Nicolet 170SX FT-IR Spectrometer) from 4000 cm21 to 400 cm21 at a resolution of 2 cm21, averaging 100 scans. The analysis of powder morphology was carried out by transmission electron microscopy (TEM) analyses, performed by a JEOL JEM 1200EX (120 kV). The magnetic properties were characterized using the Vibrating Sample Magnetometer 7307 at 25 C.

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Cell culture and cell viability assays HEK-293 cells were cultured and maintained in a logarithmic growth phase in DMEM (Gibco, USA) medium containing 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin at 37 C and 5% CO2. Cell morphology was assessed using an inverted phase contrast microscope (Olympus IX71). Cell viability was measured using a 3-(4,5dimethylthiazol-2-yl)22,5-diphenyltetrazolium bromide (MTT) assay. The dye MTT is taken and metabolized to purple by viable mitochondria. Cells were counted and plated in 96-well plates at a rate of 1 3 104 cells per well and incubated in 200 mL cell culture medium. After 12 h, when cells had adhered to the wells, the medium was removed and replaced by 200 mg/mL HA, Fe3O4, or Fe3O4-HA solutions. Prior to dilution with the culture medium, the HA, Fe3O4, or Fe3O4-HA powder was sterilized by spreading it in a thin layer and then exposing it to UV light for 12 h. After incubation for 7 days, a 20 mL MTT solution (5 mg/mL, prepared with PBS, pH 7.4) was added to each well and incubated at 37 C and 5% CO2 for an additional 4 h. The purple MTT was dissolved in 150 mL dimethyl sulfoxide solution (DMSO) (Sigma, USA). The activity of the mitochondria, reflecting cellular viability, was evaluated by measuring the optical density at 490 nm using an ELISA microplate reader (Bio-Rad Instruments, Inc.). The cell viability (%) of the treated cells was calculated in relation to the negative controls (100%). F-actin specific cytoskeleton staining HEK-293 cells were cultured on a glass microscope slide at 5 3 105. After 12 h in culture, when cells had adhered to the slides, the medium was removed and replaced by sterilized 200 mg/mL HA, Fe3O4, or Fe3O4-HA solutions. After 7 days, these slides were washed twice in PBS and fixed with 4% paraformaldehyde for 30 minutes. After fixation, the cells were washed twice with PBS and then permeabilized in PBS containing 0.1% Triton X-100 for 5 min. After that, cells were blocked with 1% BSA/PBS at 25 C for 30 min. The slides were then washed twice with PBS and incubated in rhodamin conjugated phalloidin at 25 C for 30 min. The slides were washed again in PBS and incubated in 1 mg/mL DAPI for 3 min. The staining signal of cell was recorded by a fluorescence microscope. In vivo experiments Male Sprague Dawley rats (200–220 g body weight, Vital River Laboratory Animal Technology Co. Ltd.) were housed in polycarbonate cages placed in a ventilated, temperaturecontrolled room that was maintained at 20 6 2 C and 60 6 10% relative humidity. A 12 h light/12 h dark cycle was used. Standard food and distilled water were available ad libitum. All procedures used in this animal study were in compliance with approved protocols by the School of Biological Science and Medical Engineering Committee on the use of Laboratory Animals. HA, Fe3O4, Fe3O4-HA nanoparticles were suspended in freshly sterilized saline solution (8.3 mg/mL) and ultrasonicated for 15 min to ensure complete dispersion, respectively.

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FIGURE 1. XRD patterns of materials (a: HA; b: Fe3O4; c: Fe3O4-HA). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

In each group, eight rats were randomly assigned and injected intraperitoneally with Fe3O4-HA solution (8.3 mg/ mL) three times per week for four weeks. Control group was injected with an equal volume of physiologic saline. At the end of the fourth week, all animals were killed and their kidneys were excised and kept in 10% formalin for histopathological examination. Sections (5–6 mm) of kidney tissues embedded in paraffin blocks were stained with hematoxylin-eosin (HE) and then observed using an optical microscope (Nikon U-III Multipoint Sensor System, USA). Ethics statement All experiments involving the use of animals were in compliance with Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation and were approved by Beijing Municipal Science & Technology Commission (Permit Number: SCXK (Beijing) 20060008 and SYXK (Beijing) 2006-0025). Statistical analysis All data were expressed as means 6 standard deviation (SD). A one-way analysis of variance (ANOVA) and L.S.D. test were performed with SPSS software (version 11.5). Significance was determined at p  0.05. RESULT AND DISCUSSION

XRD measurement X-ray diffraction of the synthesized materials yielded characteristic peaks (shown in Fig.1). They were broadening diffraction peaks, which mean a poor crystalline structure, typical of nonstoichiometric HA (Referred from JCPDS 09– 0432). According to Jade 9.0 software, the mean particle size of synthesized HA was calculated to be 28.47 6 0.4 nm in the Fe3O4-HA system; the characteristic peaks of Fe3O4 also appeared in Figure 1(c). The diffraction peaks at

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TABLE I. Lattice Parameters and Cell Volumes of Different Samples with Standard Deviation Lattice Parameters

HA

Fe3O4-HA

A 5 b (nm) c (nm) a5b g Cell volume (nm3)

0.9409 0.6879 90 C 120 C 0.5242

0.9428 0.6880 90 C 120 C 0.5281

SCHEME 1. Mechanism of preparation.

36.51 C, 47.09 C, 57.08 C, and 62.96 C corresponding to planes (311), (400), (333), and (440) provided clear evidence for the formation of spinal structures on the ferrite.35 This observation corresponds to previous publications of similar phenomena.36,37 However, the samples showed these changes in crystal structure. HA, Ca10(PO4)6(OH)2, belongs to the hexagonal system with a space group of P63/m, a 5 b 5 0.9360.946 nm, c 5 0.6780.690 nm.38 From I, there are evident changes in the lattice parameters and cell volumes of different samples. Rietveld analysis showed that an increase of a axis from 0.9409 to 0.9428 and the increase of c axis from 0.6879 to 0.6880 was attributed to the Ca substitution with ion species that is a lower radius. Compared with the HA group, a, b, and c axes all increased in the Fe3O4-HA composite. With the addition of Fe3O4, the composite’s cellular structure increased to 0.5281 nm3. These changes correspond to distortion of the HA. The distortion may be attributed to the magnetic iron oxide particles replacing the –OH, which is parallel to the c axis, and then entering the HA crystal lattice, which causes these changes to the crystal surface of HA (210) and (212). The Fe ion in Fe3O4 coordinates with PO432 of HA, thus acting as a bridge to PO432 groups (Fig. 2), which is another reason for the changes taking place in the HA unit cell. According to Scheme 1, the obtained Fe3O4-HA composites are highly stable and may be separated as a powder and readily redispersed in water. The redispersibility of the Fe3O4-HA composite indicates that Fe3O4 are capped by the PO432 groups of HA.

FT-IR analysis FT-IR spectra of the synthesized HA, Fe3O4, and Fe3O4-HA composite powder were shown in Figure 3(a,b,c), respectively. The major HA bonds, which were associated with PO432 (1037, 961, and 606 cm21) and OH2 (3572 cm21), appeared in Figure 2(a). The IR spectra indicated that there were some changes in the frequencies of PO432 and OH2, which all shifted to the low wave numbers in the composite. This is especially true for the band attributed to OH2 (3572 cm21), which cannot be found in Figure 2(c). This means Fe-O affected the OH2 molecules of the HA crystal. The 580 cm21 band is characteristic of Fe3O412,39 and also shifted significantly [Fig. 3(c)]. Thus, the iron via phosphate groups of HA is strongly bound to the Fe3O4-HA composite via an easy synthetic route by recognizing the facts which favor HA crystal growth on iron oxide nanoparticle surface. Iron oxide nanoparticles favor the nucleation of Ca21 ions during the first step in HA nucleation over their surface. The further nucleation of PO432 ions results in the formation of nanoparticle-dispersed HA crystals. Eventually, the change in bond lengths of the ion interactions resulted in a change in vibrational frequencies. This trend is well appreciated for XRD conclusion. While in the traditionally coating method the polymer lacks a well-defined high affinity anchor, the poor polymer-Fe3O4 interface affects protein adsorption and drastically decreases blood circulation time.22 This synthesized magnetic hydroxyapatite composites overcome the attractive Van der Waals between two phases. n-HA is successfully bound to the Fe3O4 surface. Meanwhile, the ultrasound method plays a very important role in the prepartion. The ultrasonic waves promote the fast migration of the newly formed Fe3O4 nanoparticles to the HA surface. Ultrasound irradiation results in

FIGURE 2. Schematic diagram showing the mode of binding of HA and Fe3O4.

FIGURE 3. FT-IR spectra of synthesized materials (a: HA; b: Fe3O4; c: Fe3O4-HA). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 4. TEM of Fe3O4-HA composite.

increasing of solubility, and thus a reduced supersaturation of growth species in the solution. The small size particles become unstable and dissolve back into the solution which may be the reason why the particles strongly stick to the HA, which also increased high-velocity interparticle collisions among the particles, in turn preventing the formation of larger particles. These two effects are important advantages of using ultrasonic irradiation for the preparation of nano-materials. TEM observation Fe3O4-HA composite nanopowder morphology was investigated using TEM analysis that showed a very low concentration of dark spots (2–5 nm in size) corresponding to inclusions of iron rich phases (Fig. 4). While TEM investigation also confirmed that HA particles had needle-like morphology, the quasi-amorphous HA matrix contains uniformly distributed iron. The Fe3O4-HA composite was rather heterogeneous in size, 1–5 nm in width, and up to 15–40 nm in length (Fig. 4). Ultrasound-assisted frabrication prevents the formation of larger particles due to the high-velocity interparticle collision. VSM study To understand the magnetic properties of nanoferrite, it was characterized using VSM.40 The hysteresis loop of the sample is shown in Figure 5. The saturation magnetization (Ms) of the Fe3O4-HA composite was calculated as 46.36 emu/g. It was calculated that Ms of Fe3O4 was 55.63 emu/g, which was smaller than the synthesized Fe3O4 (Ms 5 70.65 emu/g). This was attributed to the new interface formed between Fe3O4 and HA. The difference in the value of Ms could be explained in the second phase distribution. In the present work, the proposal of HA formed in the composite changed these values of magnetic moment and lattice constant. Numerous materials possess a geometrically frustrated arrangement of magnetic atoms.41 The decrease in Ms value may be attributed to the site occupancies of metal cations between HA and Fe3O4. According to XRD data, the altered

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FIGURE 5. Magnetization hysteresis loops of materials at 300 K (a: Fe3O4; b: Fe3O4-HA composite). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

HA cell possesses a geometrically frustrated arrangement of magnetic atoms. Meanwhile, ultrasonic treatment affected the size and crystallinity of the ferrite phase, causing a reduction in magnetization of Fe3O4-HA. And superparamagnetic iron oxide used for clinical applications are primarily coated with polymer. The magnetic response of Fe3O4 is directly related to the core size and the shell thickness. However, the radius of the polymer is many times larger than that of magnetic core, which decreases the magnetic property sharply. From this VSM study, the magnetization of Fe3O4-HA was much higher than that of core-shell samples.20,21 It was speculated that HA can act as assistants, which were assumed to be the reason for this exceptionally strong magnetic property.

Cell culture Under an inverted microscope, the morphologies of living cells indicated that HEK-293 cells were fibriform and fusiform [shown in Fig. 6(a)]. Some cells transformed from spindle-like to cube-like or polygonal and aggregated on the seventh day of being induced to HA [Fig. 6(b)]. The results suggested that the HEK-293 cells can attach, spread, and proliferate when co-cultured with HA. There was little cell growth in the Fe3O4 group during the seven days. This was attributed to the toxicity of iron ions in Fe3O4. With the HA introduced into the composite, some phenomena were changed in Figure 6(d). At the third day, only a few HEK-293 cells presented an elongated fusiform shape. On the fifth day, a large amount of cells proliferated and formed a cell colony. By the seventh day, the population of cells increased and fully attached to the disc. Obviously, the Fe3O4-HA composite produced no negative effect on the cell morphology, viability, and proliferation. Compared with positively charged iron particles, the Fe3O4-HA composite with neutral surface resulted in increased cell growth. It also

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FIGURE 6. Cell morphological changes at different days (3rd day, 5th day, 7th day). Scale bar: 100 mm. (a) Control group; (b) HA stimulation groups; (c) Fe3O4 stimulation groups; (d) Fe3O4-HA stimulation groups. HEK-293 cell treated with medium containing 200 lg/mL materials. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

demonstrated the potential applicability of this biomagnetic material to tissue engineering. Effects of materials on cell viability The cellular behavior on biomaterials is an important factor for the evaluation of biocompatibility. Cell growth is the first sequential reaction upon contact with a material surface, which is crucial for cell survival.42 To evaluate relative cell proliferation of HEK-293 cells co-cultured with different materials (HA, Fe3O4, and Fe3O4-HA), the MTT assay was employed in the present study. Figure 7 showed relative cell proliferation after culturing for seven days. The absorbance (490 nm) of groups was detected and cell viability was calculated by contrast with control. An obvious decrease in cell viability was noticed upon contact with Fe3O4. Fe3O4 powders, i.e., it significantly inhibited the growth of HEK-293 cells, which was attributed to the cytotoxic effects of Fe3O4 particles; iron ions are released from Fe3O4 and caged proteins of HEK-293 cells. While the Fe3O4-HA composite showed excellent biocompatibility, there was no significant

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FIGURE 7. MTT assays for proliferation of HEK-293 cells co-cultured with materials cultured for 7 days (a: control; b: HA; c: Fe3O4; d: Fe3O4-HA). Results were shown as the mean SD values (n 5 16) *denotes p < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 8. Fluorescence images of the cytoskeletal organization of HEK-293 cells co-cultured with materials (a: Control without materials; b: HA; c: Fe3O4; d: Fe3O4-HA) after 7 days. Cells stained for F-actin (red) and nucleus (blue). Scale bar 50 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

difference compared with the HA group, clarifying the important role played by the addition of HA to the composite. Some bonds formed between PO432 and iron ions, which restricted the release of iron ions and decreased the cytotoxicity of the composite. Effects of materials on cytoskeleton organization The cytoskeleton is a highly dynamic system, which is made up of a large variety of specialized proteins. It plays diverse roles in human disease.43,44 To investigate the growth behavior of HEK-293 cells with three different materials, cells were visualized with a double staining of actin (cytoskeleton) and nucleus. Fluorescence microscopy was used to observe cytoskeletal reorganization after seven days of incubation (Fig. 8). F-actins and nucleolus were stained by rhodamin-conjugated phalloidin and DAPI, respectively. Cells treated by HA were linear and stretched arrays of actin microfilaments [Fig. 8(b)], and cells exhibited the features of HEK-293 cells, such as a large and clear nucleus, visible nucleolus, polygonal or spindle appearance, and typical cobblestone-like arrangement. However, there were limited cells on the Fe3O4 group. Compared with the control [Fig. 8(a)], some cells were shrunken and the cell density decreased significantly, as shown in Figure 8(c). F-actin in the cytoplasm was also concentrated in specific regions of cells after Fe3O4 stimulation. This meant that Fe3O4 powders inhibited the expression of F-actin due to the release of iron ions. These ions also induce oxidative injury to phospholipids in cell organelle membranes. In addition, with the introduction of HA as the second phase, the Fe3O4-HA

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composite presented excellent cytoskeletal reorganization. There was no significant difference in cell density compared with the HA group. HA enhanced biocompatibility of Fe3O4 through the coupling strategy process of the composite buildup. This analysis deduced that Fe3O4-HA composites were not cytotoxic to HEK-293 cells and possessed excellent biocompatibility. In vivo analysis Magnetic nanoparticles contain unique magnetic properties, making them attractive candidates for cell labeling imaging and tracking. The biocompatibility of magnetic nanoparticles is the most important role for bioapplication. In vivo analysis is a critical method to characterize the biocompatibility. After administration of treatments for four weeks, the kidneys were excised from each rat for histological analysis. Cells were observed in the kidney tissues of the Fe3O4-HA group under a light microscope [Fig. 9(d)]. No inflammatory cell infiltration was observed in the kidney slices. This showed excellent biocompatibility of Fe3O4-HA particles in kidney tissues, without inflammation or injury. The vast majority of iron oxide nanoparticles were targeted toward the kidney, liver, or lymph nodes—locations they naturally end up during clearance.22 The kidney physiologically filters and captures nondegradable particles. A pathological response induced by nanoparticles was observed in the kidney. This evidence can be used to characterize the biocompatibility of a material.45 From Figure 9, it may be speculated that Fe3O4-HA composite materials are less toxic than the Fe3O4 group. HA acts as a second phase bonding

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FIGURE 9. Histopathological observation of the tissue of kidney for administration accumulation of materials for 4 weeks: (a) Control group; (b) HA group; (c) Fe3O4 group; (d) Fe3O4-HA. Scale bar 100 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

with Fe3O4. The physical characteristics of Fe3O4-HA composite affect their in vivo performance. This surface modification gives the magnetic particles their wide-ranging potential in the biomedical arena. This ultrasound-assisted co-precipitation method could develop novel biocompatible superparamagnetic nanoparticles, which avoid the use of poorly tolerated magnetite based nanoparticles, extending the applications. Therefore, future goals will be to explore composite materials that have better biocompatibility with perfect biological functionality. CONCLUSIONS

In this study, a magnetic hydroxyapatite composite was prepared through ultrasound-assisted co-precipitation method. XRD, FT-IR, TEM, and VSM were applied to investigate the physical and chemical properties of the magnetic materials. The PO432 groups of HA can interact with iron ions of Fe3O4. The lattice parameters a, b, and c of HA changed after forming a compound and the Fe3O4-HA composite showed good saturation magnetization. The cell culture data were used to evaluate the biocompatibility of the Fe3O4-HA composite, showing no cytotoxic effects on HEK-293 cells. Histological analysis revealed that no significant inflammation or injury was induced in kidney tissue by the Fe3O4-HA composite. This approach produced a composite with excellent magnetic properties without sacrificing biocompatibility. The current study suggested that these novel Fe3O4-HA composites may be particularly relevant for strategies of

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ULTRASOUND-ASSISTED BIOCOMPATIBLE MAGNETIC HYDROXYAPATITE

Ultrasound-assisted fabrication of a biocompatible magnetic hydroxyapatite.

This work describes the fabrication and characterization of a biocompatible magnetic hydroxyapatite (HA) using an ultrasound-assisted co-precipitation...
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