Calcium phosphate-coated magnetic nanoparticles for treating bone diseases

6 Calcium phosphate-coated magnetic nanoparticles for treating bone diseases R. A. PARETA, Wake Forest Institute for Regenerative Medicine, USA and S. SIRIVISOOT, King Mongkut’s University of Technology Thonburi, Thailand

Abstract: Bone diseases like osteoporosis, osteoarthritis and bone cancer affect a significant population. It has been previously shown that magnetic nanoparticles (MNPs) can be directed in the presence of a magnetic field to any part of the body, allowing for site-specific drug delivery and possibly an immediate increase in bone density. In this chapter, we will discuss an MNP-based approach to treat bone diseases. Based on our own and a few other research groups, results are very encouraging and indicate that MNPs and calcium phosphate (CaP) coatings on MNPs, specifically (γ-Fe2O3), significantly increased osteoblast density. Thus, CaP-coated MNPs can be used to treat bone diseases like osteporosis. Key words: iron oxide magnetic nanoparticles, calcium phosphate, osteoporosis, surfactant, osteoblasts.

6.1

Introduction

Recent advances in nanotechnology have enabled researchers to discover new processes and phenomena which can be applied to medical and biomedical applications. In particular, the capability to characterize and analyze interactions of nanomaterials and biological entities at the cellular and molecular level has resulted in novel applications like biosensor, molecular imaging and nanomedicine.

6.1.1 Magnetic nanoparticles Magnetic nanoparticles (MNPs) have attractive properties such as superparamagnetism, high field irreversibility and high saturation fields. These phenomena arise from size and surface effects dominated by the magnetic behavior of individual nanoparticles. Besides applications in information storage and electromagnetic devices, MNPs have found potential applications for medical diagnostics and therapeutics. MNPs are of great interest owing to their potential biomedical applications, such as protein or cell 131 © Woodhead Publishing Limited, 2012

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separation, drug delivery vehicles, magnetic resonance imaging (MRI), signal enhancement agents and cancer hyperthermia treatment (Fig. 6.1), but there is hardly any literature on tissue repair using MNPs. When their diameter is less than 20 nm, MNPs will become superparamagnetic – that is, they are non-magnetic on a macroscale without an exterior magnetic field, and seem like common materials. However, each particle can be considered as a single magnetic domain: a magnetic field in a nanoscale (Yao, 2010). There are various types of MNPs, ranging from well-known iron oxide-based to metallic such as Mn, Fe, Co, Ni, Zn, Mg and their oxides (Kumar and Mohammad, 2011). Here we will only focus on iron oxide MNPs.

6.1.2 Osteoporosis Osteoporosis is characterized by the structural deterioration of bone and subsequent low bone mass. It is commonly attributed as causing fractures in the hip, spine, wrists and ribs. In the USA today, 10 million individuals are estimated to already have the disorder and almost 34 million more are estimated to have low bone density, placing them at increased risk for

Hyperthermia based therapy Polymegle Magnatic particle nanoparticle Controlled release

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6.1 A schematic representation of some of the unique advantages of magnetic nanomaterials for hyperthermia-based therapy and controlled drug delivery (Kumar and Mohammad, 2011).

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osteoporosis and broken bones (National Osteoporosis Foundation). The desirability of reversing low bone density is not, however, limited to cases of osteoporosis. Other conditions such as bone cancer, arthritis and bone fractures would also benefit from highly effective bone-building strategies. In all of these cases, the site-specific anabolic behavior of a drug delivery system would be advantageous.

6.1.3 Limitations of present osteoporosis treatments At present, some drugs are available to treat low bone density such as bisand di-phosphonates, estrogen modulators, etc. Anti-resorptive bone agents are primarily used to prevent further breakdown of bone (Hughes, 1995; Hughes et al., 1989). But non-specific action of bis-phosphonates on other cell lines has been shown, including decreasing the proliferation of osteoblasts (bone forming cells) and inducing apoptosis in macrophages (Adami and Zamberlan, 1996; Reinholz, 2000). Another limitation is the inability of these agents to increase bone growth, instead only working to prevent further loss. Anabolic treatments are the most promising in terms of building bone mass, resulting in increased osteoblast activity and a reduction of 65–70% of vertebral and about 50% of non-vertebral fractures. However, some anabolic treatments (like the use of bone morphogenetic proteins (BMPs)), while potent, are not selective inducers of bone mass and, thus, generate calcium deposits in areas not desirable (like the liver). Thus, an effective drug delivery system (such as the use of iron oxide MNPs) could be engineered to increase site-specific anabolic effects of bone-building agents.

6.1.4 Iron oxide magnetic nanoparticles Iron oxide particles are known to be non-toxic and are eventually broken down and incorporated into hemoglobin (Berry and Curtis, 2003). For the above reasons, superparamagnetic iron oxide nanoparticles have been recognized as a promising tool for the site-specific delivery of drugs and of diagnostic agents (Weissleder et al., 1995). The application of small iron oxide MNPs during in vitro diagnostics has been practiced for nearly 40 years (Gilchrist et al., 1957). Specifically, studies have included mostly maghemite, γ-Fe2O3, or magnetite (Fe3O4) single particles about 5–20 nm in diameter, as these are very promising candidates due to their biocompatibility and relative ease to functionalize for a wide range of applications (Oliveira et al., 2006; Schwertmann and Cornell, 2000; Thunemann et al., 2006). In particular, the application of these iron oxide nanoparticles in drug delivery has been well addressed for non-bone applications (Xu and

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Sun, 2007). Briefly, superparamagnetic iron oxide MNPs with tailored surface chemistry have been widely used experimentally for numerous in vivo applications such as MRI contrast enhancements, tissue repair, immunoassays, detoxification of biological fluids, hyperthermia, drug delivery and in cell separation (Chouly et al., 1996; Gupta and Gupta, 2005; Gupta and Hung, 1989; Reimer and Weissleder, 1996). However, the use of iron oxide nanoparticles in orthopedic applications remains largely neglected. This is despite the promise of iron and iron oxide to increase bone health. For example, it has previously been shown that iron restriction can have an inhibitory effect on the mineralization of osteoblasts in vitro (Harris, 2003). The beneficial link between iron and bone density has been demonstrated in vivo by the association of dietary iron and a healthy bone mineral density (Abraham, 2006; Parelman et al., 2006). Moreover, experimental evidence also suggests that there may be some positive association between iron metabolism and the in vitro proliferation of non-bone cell lines such as fibroblasts (Le and Richardson, 2004). Iron oxide nanoparticles can also aid in the site-specific generation of bone remodeling, which can be accomplished by directing such MNPs to diseased bone under an external magnetic field. Our group was the first to investigate the potential of CaP-coated iron oxide MNPs to treat bone diseases. We determined the cytocompatibility and proliferation of osteoblasts with magnetite and maghemite. CaP was coated on the iron oxide nanoparticles in the presence of bovine serum albumin (BSA) and citric acid (CA) to decrease particle agglomeration. Our results provided evidence that maghemite MNPs enhanced osteoblast density compared with controls (Pareta et al., 2008).

6.2

Iron oxide magnetic nanoparticle synthesis

Two iron oxide MNPs (magnetite and maghemite) were synthesized. These MNPs are known to have uniform sizes and can be easily synthesized in lab with reproducible properties.

6.2.1 Magnetite nanoparticle synthesis Monodispersed magnetite (Fe3O4) nanoparticles were synthesized by Sun et al. through a high-temperature solution phase reaction of Fe(acac)3 and 1,2-hexadecanediol in the presence of oleic acid and oleylamine as surfactants. They tuned the particle diameter from 3 to 20 nm by varying reaction conditions or by seed-mediated growth. For example, 4 nm Fe3O4 nanoparticle seeds were synthesized by refluxing a reaction mixture composed of

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Fe(acac)3, diphenyl ether, 1,2-hexadecanediol, oleic acid and oleylamine. By controlling the quantity of the nanoparticle seeds and reaction conditions, they generated various larger-sized Fe3O4 nanoparticles. Magnetic measurements on all those Fe3O4 nanoparticles indicate that the nanoparticles were superparamagnetic at room temperature (no hysteresis; Sun et al., 2003; Sun and Zeng, 2002). Recently, iron oxide MNPs with high chemical stability and good dispersion were synthesized by Sun’s group via controlled oxidation of the iron surface to crystalline Fe3O4 shell (Peng et al., 2006). The crystalline Fe3O4 shell offered more robust protection to the metallic core of iron, and the core/shell-structured nanoparticles were stable in hexane or water dispersion. It led to the possibility to make small Fe nanoparticles (<10 nm in radius) with desired stability for highly efficient biological separation, drug delivery and high-sensitivity biological detection. To make these core–shell nanoparticles more stable, Sun et al. generated the crystalline Fe3O4 shells by controlled oxidation of the as-synthesized iron nanoparticles using an oxygen transferring agent (CH3)3NO. The thickness of the shell was tunable by controlling the amount of (CH3)3NO added into the reaction mixture. Magnetic measurements of the 2.5/5 nm Fe/ Fe3O4 nanoparticles showed that they were superparamagnetic with a magnetic moment reaching 61.6 emu/g. After their exposure to air for 8 h, the nanoparticles still possessed a reasonably high magnetic moment at 56.2 emu/g. In our lab, we wanted to utilize a really simple and reliable method to synthesize magnetite nanoparticles. They were synthesized by a wet chemistry method under high pH as previously reported (Kang et al., 1996). Briefly, iron(II) chloride and iron(III) chloride were dissolved in 25 mL of deoxygenated water in the presence of 0.4 M hydrochloric acid. This solution was added drop-wise at 0.5 mL/min to a 250 mL solution of 1.5 M sodium hydroxide under nitrogen gas flow. This solution was centrifuged at 5000 rpm and the supernatant decanted. The particles were dispersed in double deionized water while vortexing was followed by sonication. This was repeated three times whereby a black solution of magnetite was obtained. The particles were sterilized through dispersion in ethanol once, centrifuged at 5000 rpm, and decanted before suspension in double deionized water (Pareta et al., 2008).

6.2.2 Maghemite nanoparticle synthesis Hyeon et al. synthesized monodispersed and highly crystalline maghemite (γ-Fe2O3) nanoparticles via oxidation. Monodispersed iron nanoparticles were first synthesized, and then they were transformed to monodispersed γ-Fe2O3 nanocrystals by controlled oxidation using trimethylamine N-oxide ((CH3)3NO) as a mild oxidant (Hyeon et al., 2001). Based on the thermal

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decomposition of a metal–surfactant complex, Hyeon et al. further developed the ultra-large-scale synthesis of monodispersed iron oxide nanocrystals. Instead of using toxic and expensive organometallic compounds such as Fe(CO)5, they prepared the iron–oleate complex by reacting inexpensive and environmentally friendly compounds, namely metal chlorides and sodium oleate. They generated monodispersed iron oxide nanocrystals by heating a 1-octadecene (ODE) solution with iron−oleate complex to 320°C and keeping that temperature for 30 min (Park et al., 2004). To synthesize maghemite nanoparticles, we also oxidized magnetite (synthesized earlier). The oxidation of the cleaned magnetite nanoparticles was carried out under low pH and high temperature. Magnetite nanoparticles were sonicated and vortexed. The pH of the nanoparticle solution was adjusted to 3.5 using 0.1 M hydrochloric acid. This solution was then heated to 100°C in oxygenated water while vigorously magnetically stirring for 30 min. The resulting solution was a reddish-brown suspension of maghemite. This solution was centrifuged and decanted, whereby it was sterilized in ethanol as described above. The cleaned solution was then resuspended in double deionized water (Pareta et al., 2008).

6.3

Surface modification of iron oxide magnetic nanoparticles

Iron oxide-based nanoparticles and nanostructures promise broad applications in biomedicine and biology due to their inherent biocompatibility. For sitespecific targeting of such applications, the size of nanoparticles is very critical and needs to be engineered for the applications. MNPs need to have a proper size (i.e., 8–20 nm), as well as appropriate surface functionalization to satisfy other prerequisites, including aqueous dispersibility, stability, biocompatibility and biodegradability, to be regarded as biofunctional MNPs. For in vivo applications, biofunctional MNPs must meet all four of these prerequisites. Generally, there are three ways to connect biofunctional molecules to the surface of a nanoparticle. The most commonly used method is to coat the nanoparticle with polymers, and then form covalent bonds between the polymeric coating and the biofunctional molecules. Although widely used to functionalize nanoparticles that have large sizes (≥ 50 nm in diameter), polymeric coatings often lead to non-specific absorption of other substances than the desired targets, which decreases selectivity. In a second route, a monolayer of molecules that bear a reactive group attaches on the nanoparticles; then the biofunctional molecules react with the monolayer to yield the biofunctional nanoparticles. In the latter method, the surface anchor is conjugated with the biofunctional molecule before the conjugate reacts with the nanoparticle to give the desired product. The second route is simple and versatile, but may leave unconsumed reactive groups, while

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the last route produces a well-defined monolayer, but it requires a considerable effort to engineer a biofunctional molecule that will bear a surface anchoring group.

6.3.1 Calcium phosphate coating Surfactants have been shown to have a stabilizing effect on MNPs (Gupta and Gupta, 2005). Various ligands to stabilize magnetite nanoparticles are dextran, cationic liposomes, polyvinyl alcohol, hydrogel and lauric acid, while maghemite nanoparticles (γ-Fe2O3) are stabilized by ligands such as dextran. We coated the iron oxide MNPs with CaP in order to treat bone diseases where increased CaP at that location would help bone density and help recovery. CaP coatings were produced on MNPs in the presence of BSA or CA capping agents. BSA and CA were used, the intention being to disperse the MNPs so that an effective CaP coating could be created. Calcium nitrate and monopotassium phosphate were used at a molar ratio of 1.67, which is the ratio of calcium to phosphate for the natural hydroxyapatite phase of bone (Suchanek et al., 1997). 60 mmol of calcium nitrate was added to 150 mL solution of magnetically stirred double deionized water, and the pH was adjusted to 10 with ammonium hydroxide. Iron oxide nanoparticles at 13.33 mg/mL and respective capping agents (BSA and CA) at 1.9 mg/mL were added to the calcium solution. Separately, a 150 mL solution was prepared using 36 mmol of monopotassium phosphate whereby the pH was adjusted to 10 using ammonium hydroxide. This solution was added drop-wise to the calcium solution. Then, the mixture was moved to a polytetrafluoroethylene (PTFE) hydrothermal unit and heated for 9 h at 70°C. After cooling, particles were centrifuged (5000 rpm) and the aqueous component was decanted, followed by dispersion in ethanol for sterilization purposes. After subsequent sonication, the coated MNPs were again centrifuged and the solvent decanted. This was repeated a total of three times before their dispersion in double deionized water (Pareta et al., 2008).

6.4

Characterization of iron oxide magnetic nanoparticles

MNPs were characterized to investigate their composition, morphology and magnetic properties.

6.4.1 Composition characterization: X-ray diffraction We characterized both uncoated and CaP-coated MNPs with X-ray diffraction (XRD) to verify the various phases present using Cu Kα radiation (Siemens Diffractometer D5000 Kristalloflex; Bruker AXS Inc; Chicago, IL, USA). The

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6.2 XRD patterns for (a) magnetite (Fe3O4) nanoparticles, (b) magnetite nanoparticles coated with CaP in the presence of BSA and (c) magnetite nanoparticles coated with CaP in the presence of CA. H = hydroxyapatite peak; M = magnetite peak.

2θ angle was varied from 20° to 70° at 10° min−1. Diffraction signal intensity was recorded and processed using DiffracPlus: TexEval (Bruker AXS, Inc., Palo Alto, CA, USA) software. Figure 6.2 shows the XRD pattern for the uncoated and CaP-coated magnetite (Fe3O4) nanoparticles. The peaks for the nanoparticle samples matched with the pattern for magnetite and hydroxyapatite standards. The magnetite nanoparticle XRD peaks matched those of the magnetite standard and so did the CaP-coated magnetite nanoparticles formulated in the presence of CA, but the peaks for the CaP-coated magnetite nanoparticles formulated in the presence of BSA did not match those of magnetite. Also, only CaP-coated magnetite nanoparticles formulated in the presence of BSA showed an exact match for the hydroxyapatite standard while CaP-coated magnetite nanoparticles formulated in the presence of CA displayed other phases of CaP. Uncoated and CaP-coated maghemite (γ-Fe2O3) nanoparticle XRD patterns are shown in Fig. 6.3. All three samples matched the maghemite standard and both CaP-coated nanoparticles also matched the hydroxyapatite standard (Pareta et al., 2008).

6.4.2 Characterization of morphology: transmission electron microscopy We characterized the morphology of magnetite and maghemite nanoparticles (both uncoated and coated) with transmission electron microscopy (TEM). The dispersions of nanoparticles in deionized water were allowed to slowly dry on formvar-coated copper grids. All imaging was carried out using a Philips Joel 140 kV TEM (New York, NY, USA). The morphologies of the various nanoparticles are shown in Fig. 6.4. Prepared magnetite

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6.3 XRD patterns for (a) maghemite (γ -Fe2O3) nanoparticles, (b) maghemite nanoparticles coated with CaP in the presence of BSA and (c) maghemite nanoparticles coated with CaP in the presence of CA. H = hydroxyapatite peak; G = maghemite peak.

nanoparticles displayed a cubic structure with only one phase visible and diameters less than 10 nm. Maghemite nanoparticles were very similar in shape and size (around 15 nm individually) but seemed more agglomerated, possibly due to higher emu/g as shown in Table 6.1 without the presence of any surfactant. Table 6.1 gives an estimation of MNP size as observed by TEM micrographs. The maghemite nanoparticles seemed finer than magnetite nanoparticles. With the CaP-coated MNPs, two different phases were observed. The iron particles were seen as dark cubes and spheres and the CaP coating as an amorphous surrounding. The iron particles could be seen encapsulated within the needle-like arrays of the CaP sheath. It does seem that formulating nanoparticles in the presence of both BSA and CA helped to disperse the nanoparticles.

6.4.3 Characterization of magnetic properties: vibrating sample magnetometry (VSM) The magnetic properties (hysteresis loop) of the dried nanoparticles were assessed using vibrating sample magnetometry (VSM; LakeShore 7400; Chicago, IL, USA) at 300 K. VSM measurements showed that all MNPs possessed magnetic properties. The maximum electromagnetic unit per gram (emu/g) for each sample is shown in Table 6.2. Maghemite nanoparticles had higher magnetic saturation values compared with magnetite nanoparticles (this was also true for the CaP-coated maghemite). As expected, the CaP-coated nanoparticles had a decreased magnetic saturation per gram compared to uncoated MNPs. Between CaP-coated nanoparticles, those

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6.4 TEM images of (a) magnetite (Fe3O4) nanoparticles, (b) maghemite (γ-Fe2O3) nanoparticles, (c) CaP-coated magnetite (Fe3O4) nanoparticles dispersed in BSA, (d) CaP-coated maghemite (γ-Fe2O3) nanoparticles dispersed in BSA, (e) CaP-coated magnetite nanoparticles dispersed in CA and (f) CaP-coated maghemite (γ-Fe2O3) nanoparticles dispersed in CA.

synthesized in the presence of CA had higher magnetic saturations compared with those synthesized in the presence of BSA. Figure 6.5 compares the normalized magnetization curves (hysteresis loop) of the uncoated and CaP-coated Fe3O4 (magnetite) nanoparticles formulated in the presence of BSA and CA. Figure 6.6 compares the normalized magnetization curves (hysteresis loop) of uncoated and CaP-coated γ-Fe2O3 (maghemite) nanoparticles formulated in the presence of BSA and CA.

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Table 6.1 Particle size analysis of MNPs, uncoated and coated Sample

Size (nm) ± standard deviation

Fe3O4 Fe3O4-CaP(BSA) Fe3O4-CaP(CA) γ-Fe2O3 γ-Fe2O3-CaP(BSA) γ-Fe2O3-CaP(CA)

11.5 ± 3.5 125 ± 25 long, 50 ± 10 wide 125 ± 25 long, 50 ± 10 wide 12.5 ± 2.5 125 ± 25 long, 50 ± 10 wide 175 ± 25 long, 65 ± 15 wide

Note: BSA = bovine serum albumin; CA = citric acid.

Table 6.2 VSM readings for the MNPs Sample

Emu/g

Fe3O4 Fe3O4-CaP(BSA) Fe3O4-CaP(CA) γ-Fe2O3 γ-Fe2O3-CaP(BSA) γ-Fe2O3-CaP(CA)

29.77 3.47 12.67 33.58 8.48 9.12

Note: BSA = bovine serum albumin; CA = citric acid.

The reduction of the saturation magnetization of the coated iron oxide nanoparticles could be largely due to the weight contribution from the CaP and surfactants (Xu, 2007). But the hysteresis loop from the present study showed that the CaP-coated nanoparticles were still superparamagnetic. Clearly, this property could be utilized to direct such MNPs to a desirable bone site in the body to allow the MNPs to treat local bone disease. For example, after such a magnetic field is removed, a functionalized version of the CaP-coated nanoparticles can attach themselves to osteoporotic bone, immediately building bone mass. Moreover, maghemite nanoparticles coated with hydroxyapatite in the presence of BSA were shown in this study to significantly increase the proliferation of bone cells. Thus, after such maghemite-coated nanoparticles attach, they may also increase bone formation. This study also showed that the magnetite nanoparticles were better dispersed after early time points as compared with maghemite due to lower emu/g values. In the absence of any surface coating, magnetic iron oxide particles have relatively hydrophobic surfaces (compared with the cell culture media) with a large surface area to volume ratio. Due to hydrophobic interactions between the particles, these particles agglomerated and formed large clusters, resulting in increased particle size. When nanoparticles agglomerate, they decrease the effective surface area for protein, and subsequently cell interactions. This is important as previous studies have

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6.6 Normalized magnetization curves of maghemite (Fe3O4) nanoparticles: (a) uncoated, (b) CaP-coated in the presence of BSA and (c) CaP-coated in the presence of CA.

demonstrated greater vitronectin adsorption (a protein important for promoting osteoblast adhesion) on individual nanoparticles than when nanoparticles agglomerated into micron-sized particles (Webster et al., 2001). In this manner, greater vitronectin adsorption on nanoparticles that do not agglomerate is a key design parameter for promoting osteoblast functions. Most importantly, this study provided the first evidence of greater osteoblast densities in the presence of maghemite nanoparticles than magnetite nanoparticles and controls after five and eight days.

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Biological applications of magnetic nanoparticles

MNPs offer exciting opportunities for biomedical applications. First, their size could be controlled; ranging from a few nanometers up to tens of nanometers, so the optimal size of MNPs can be easily achieved to match a specific biological entity of interest. MNPs can be coated with biofunctional molecules to make them interact with or bind to an entity with high affinity, thereby providing a controllable means of ‘tagging’. Second, since they are magnetic, they obey Gauss’s law, and could be manipulated by an external magnetic force. This ‘action at a distance’ opens up many applications, including the biological separation, purification, detection and drug delivery. Third, the MNPs can be made to resonantly respond to an alternating magnetic field, with advantageous results related to the transfer of energy from the exciting field to the nanoparticles, which leads to their biological application as hyperthermia agents. Forth, MNPs play an important role as MRI contrast agents because the signal of magnetic moment of a proton can be captured by resonant absorption, which occurs in the interaction between MNPs and a time-varying magnetic field.

6.5.1 Iron oxide magnetic nanoparticles biocompatibility and potential to treat osteoporosis Magnetic therapy has been considered a promising alternative to treat various bone diseases. Bassett first pointed out that pulsed electromagnetic fields (PEMF) were very helpful in osteonecrosis and orthodontics (Bassett et al., 1974, 1989). Bone defects were improved to increase bone density and calcium content, and decrease or reverse osteoporosis when exposed to magnetic fields (Chang and Chang, 2003; Taniguchi and Kanai, 2007). For osteoarthritis, the magnetic field was shown to even relieve pain and increase mobility (Sutbeyaz et al., 2006). It was also used for the treatment of fractures, spine fusion, distraction osteogenesis, delayed union, pseudoarthrosis and so on with some positive effects (Glazer et al., 1997; Grace et al., 1998; Kesemenli et al., 2003; Kort et al., 1982). Meanwhile, many researchers also addressed the mechanism of magnetic therapy. They indicated that the magnetic field could stimulate the proliferation and differentiation of osteoblasts, promote the expression of BMP to increase osteointegration and, finally, accelerate new bone formation (Ohkubo and Xu, 1997; Yan et al., 1998; Yamamoto et al., 2003). In our studies, osteoblasts were grown in complete DMEM (Dulbecco’s Modified Eagle Medium, supplemented with 10% FBS (fetal bovine serum) and 1% P/S (penicillin/streptomycin)) on the bottom of a 96-well polystyrene plate at a seeding density of 3000 cell/cm2 with or without magnetite (Fe3O4)

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or maghemite (γ-Fe2O3) uncoated nanoparticles for one, five and eight days. For CaP-coated MNPs, culture was done for one day. Nanoparticles were dispersed by sonication and vortexed before their immediate addition (at a constant volume of 20 µL and at a concentration of 4.25 mg/mL for both magnetite and maghemite nanoparticles) to the cells in 200 µL complete DMEM. The cell culture medium was not changed throughout the experiment. Cell counts were performed using the MTS (CellTiter 96® aqueous non-radioactive cell proliferation) assay. Cell proliferation assays after one day indicated that osteoblast density was similar in the presence of magnetite (Fe3O4) nanoparticles and the controls (i.e., osteoblasts grown on a polystyrene cell culture plate without nanoparticles). Osteoblast density was also similar in the presence of CaP-coated magnetite nanoparticles (synthesized in the presence of both BSA and CA) compared with the controls as shown in Fig. 6.7. Osteoblast density in the presence of the uncoated and CaP-coated maghemite (γ-Fe2O3) synthesized with CA was similar to the controls, while for the CaP-coated maghemite synthesized with BSA, osteoblast density after 24 h was significantly higher (p < 0.05) than the controls as shown in Fig. 6.8. Osteoblast density was similar in the presence of magnetite and maghemite after one day. In contrast, after five and eight days, osteoblast density in the presence of maghemite nanoparticles was significantly higher than controls while magnetite was similar to the controls (Fig. 6.9). In this work, the motivation was the possibility of applying MNPs to the treatment of osteoporosis. The promise of these particles (particularly maghemite) to immediately increase bone density at the site of disease was emphasized. Also, drug molecules can be attached and the magnetic properties of these nanoparticles can be utilized as they are guided under a magnetic field to desirable bone sites. Thus, the success of such a treatment requires that the particles are cytocompatible when in contact with osteoblasts. It is therefore beneficial that the results 9000 8000

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6.7 Similar osteoblast density in the presence of magnetite (Fe3O4) nanoparticles coated with CaP after 1 day of culture.

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6.9 Increased osteoblast density in the presence of maghemite (γ-Fe2O3) nanoparticles after 5 and 8 days of culture.

of the present study demonstrated that no iron oxide nanoparticle solution had antiproliferative effects on osteoblasts; they were all better or at least comparable to controls in which no particles were added.

6.6

Conclusions

Magnetite and maghemite nanoparticles have the potential to treat bonerelated diseases. The CaP-coated maghemite synthesized in the presence of BSA showed significantly higher osteoblast densities compared with controls and CaP-coated maghemite synthesized in the presence of CA after one day. For longer time durations (five and eight days), maghemite nanoparticles performed best, with significantly higher osteoblast densities compared with magnetite and controls. MNPs are very attractive for bone

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disease treatment. Their biocompatibility and drug delivery potential, with the added bonus of ability to control their movement in vivo through external magnetic fields, can be utilized to increase local bone density and as well as bone regeneration. Similarly, MNPs are being used for protein/cell separation, MRI signal enhancement and cancer hyperthermia treatment.

6.7

Future trends

CaP-coated MNPs provide a novel field where local bone density can be increased very fast and presence of osteo-inductive agents like CaP can further aid in rapid bone regeneration. Our work lays down the foundation for more deliberate attempts to treat osteoporosis and other bone-related diseases. Future studies should investigate the potential of these nanoparticles in vivo to monitor their transport properties and efficacy in treating bone diseases.

6.8

References

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