Scholarly article on topic 'Calcium phosphate-forming ability of magnetite and related materials in a solution mimicking in vivo conditions'

Calcium phosphate-forming ability of magnetite and related materials in a solution mimicking in vivo conditions Academic research paper on "Nano-technology"

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{"Calcium phosphate" / "Iron oxide" / "Iron oxyhydroxide" / "Simulated body fluid"}

Abstract of research paper on Nano-technology, author of scientific article — Yasuyuki Kato, Taishi Yokoi, Euisup Shin, Ill Yong Kim, Masakazu Kawashita, et al.

Abstract Iron-based compounds, especially magnetite (Fe3O4), can be a candidate of thermoseeds for hyperthermia therapy. When iron-based compounds are applied for bone tumor treatment, they should have a heat-generating property and a bone-bonding property. However, the bone-bonding property of iron-based compounds is still unclear. The bone-bonding property of materials is estimated by their bone-like apatite formation property in simulated body fluid (SBF). The method to estimate apatite forming ability of materials by utilizing SBF was introduced by Kokubo et al. We thus report fundamental research into the behavior of iron oxides and an iron oxyhydroxide namely: FeO, Fe3O4, α-Fe2O3, γ-Fe2O3, and α-FeOOH, in SBF. Calcium phosphate precipitation was found in Fe3O4 and α-Fe2O3 within 7 and 28 days after soaking in SBF, respectively, while FeO, γ-Fe2O3, and α-FeOOH did not. Our results indicate that Fe3O4 and α-Fe2O3 have a better potential bone-bonding property than FeO, γ-Fe2O3, and α-FeOOH. The induction of apatite precipitation in SBF can be attributed to the specific structure of FeOH groups on the surface of Fe3O4 and α-Fe2O3.

Academic research paper on topic "Calcium phosphate-forming ability of magnetite and related materials in a solution mimicking in vivo conditions"

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Journal of Asian Ceramic Societies

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Calcium phosphate-forming ability of magnetite and related materials in a solution mimicking in vivo conditions ^

Yasuyuki Katoa, Taishi Yokoib *, Euisup Shina, 111 Yong Kim Koichi Kikutaa, Chikara Ohtsukia

Masakazu Kawashita1

a Department of Crystalline Materials Science, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan b Graduate School of Environmental Studies, Tohoku University, 6-6-20 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan c Department of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, 6-6-12-208 Aramaki-Aoba, Aoba-ku, Sendai 980-8579, Japan

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ARTICLE INFO

Article history: Received 24 July 2014 Received in revised form 21 September 2014 Accepted 20 October 2014 Available online 8 November 2014

Keywords: Calcium phosphate Iron oxide Iron oxyhydroxide Simulated body fluid

ABSTRACT

Iron-based compounds, especially magnetite (Fe3 O4), can be a candidate of thermoseeds for hyperthermia therapy. When iron-based compounds are applied for bone tumor treatment, they should have a heat-generating property and a bone-bonding property. However, the bone-bonding property of iron-based compounds is still unclear. The bone-bonding property of materials is estimated by their bone-like apatite formation property in simulated body fluid (SBF). The method to estimate apatite forming ability of materials by utilizing SBF was introduced by Kokubo et al. We thus report fundamental research into the behavior of iron oxides and an iron oxyhydroxide namely: FeO, Fe3 O4, a-Fe2 O3,7-Fe2 O3, and a-FeOOH, in SBF. Calcium phosphate precipitation was found in Fe3 O4 and a-Fe2 O3 within 7 and 28 days after soaking in SBF, respectively, while FeO, 7-Fe2O3, and a-FeOOH did not. Our results indicate that Fe3O4 and a-Fe2O3 have a better potential bone-bonding property than FeO, 7-Fe2O3, and a-FeOOH. The induction of apatite precipitation in SBF can be attributed to the specific structure of F^OH groups on the surface of Fe3O4 and a-Fe2O3.

© 2014 The Ceramic Society of Japan and the Korean Ceramic Society. Production and hosting by

Elsevier B.V. All rights reserved.

1. Introduction

Hyperthermia therapy has attracted much attention because it is a low-invasive cancer treatment. Magnetic hyperthermia has attracted special attention as a safe method that can be used on tumors deep within the body. Some iron oxides can be used as a thermoseed in hyperthermia treatment [1,2]. A type of magnetic fluid containing iron oxide nanoparticles has been approved for the treatment of glioblastoma [3]. Magnetite (Fe3O4) is a suitable candidate for this treatment because of its magnetic properties and low toxicity. Researches have been conducted into the fabrication of magnetite microspheres [4], magnetite nanoparticles [5], magnetite/mesoporous silica hybrids [6], and magnetite/carboxymethyldextran hybrids [7] for tumor treatment.

For the hyperthermic treatment of bone tumors, the materials require biological affinity to surrounding bone tissue in addition to thermoseed properties. Previously, composite materials that

* Corresponding author. Tel.: +81 22 795 4274; fax: +81 22 795 4274. E-mail address: yokoi@mail.kankyo.tohoku.ac.jp (T. Yokoi). Peer review under responsibility ofThe Ceramic Society ofJapan and the Korean Ceramic Society.

consist of magnetite and hydroxyapatite (HAp, Cai0(PO4)6(OH)2) were developed for bone tumor treatment [8,9] because HAp has a bone-bonding property, i.e., osteoconductivity. The design of magnetite/HAp composites is based on the heat-generating property of Fe3O4 and the osteoconduction property of HAp.

Based on a fundamental understanding of the osteoconduction of ceramic biomaterials, the bone-bonding property is known to depend on the formation of a bone-like apatite layer on surfaces after exposure to body fluids. This means that a surface design capable of inducing bone-like apatite formation results in Fe3O4 having a high affinity for living bone. Our previous report indicated that iron-based materials can possibly induce bone-like apatite deposition in an aqueous solution that mimics physiological conditions [10]. Therefore, Fe3O4 and its related compounds potentially have an excellent bone-bonding property, and they can be used as substrates to form the apatite layer. However, the required surface characteristics of Fe3O4 for the formation of bone-like apatite in a body environment have not been fully determined.

In this study, the behavior of iron-based materials was investigated in a solution that mimics body fluid to determine the fundamental requirements for bone-like apatite formation on the surface of Fe3O4 and related materials in bony defects. Bone-like apatite formation by iron oxides and an iron oxyhydroxide was

2187-0764 © 2014 The Ceramic Society ofJapan and the Korean Ceramic Society. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.Org/10.1016/j.jascer.2014.10.007

Table 1

Reagents forthe preparation of 1000 cm3 simulated body fluid.

Order Reagent Amount

1 NaCl 7.996g

2 NaHCO3 0.350g

3 KCl 0.224g

4 K2HPO43H2O 0.228 g

5 MgCl2-6H2O 0.305 g

6 1.0 mol dm-3 HCl 40 cm3

7 CaCl2 0.278 g

8 Na2SO4 0.071 g

9 (CH2OH)3CNH2 6.057g

investigated in the simulated body fluid (SBF) suggested by Kokubo et al. They reported that the bone-bonding properties of materials can be estimated by bone-like apatite formation in SBF [11-13]. The inorganic ion concentrations in SBF are similar to those in human blood plasma.

2. Experimental procedures

in the order given in Table 1. Each reagent was allowed to completely dissolve before the addition of the next reagent. The solution was kept at 36.5 °C and the pH was adjusted to 7.25 by adding a 1.0moldm-3 (M) HCl solution. After the pH adjustment, the solution was transferred to a volumetric flask and ultra-pure water was added to adjust the total volume of the solution to 1000 cm3. All the reagents shown in Table 1, with the exception of the 1.0 M HCl solution, were purchased from Nacalai Tesque, Inc., Kyoto, Japan. The 1.0 M HCl solution was prepared by diluting a 35mass% HCl solution (Wako Pure Chemical Industries Ltd., Osaka, Japan).

An aliquot of SBF was placed in polystyrene bottle. Wustite (FeO), Fe3O4, hematite (a-Fe2O3), maghemite (^-Fe2O3), and goethite (a-FeOOH) were statically soaked in SBF. The polystyrene bottle was covered tightly and maintained at 36.5 °C for up to 28 days. The ratio of (weight of the powder):(volume of SBF) was 20mg:10cm3. These powders were purchased from Kojundo Chemical Laboratory Co., Ltd., Saitama, Japan. The soaked powder was removed after 1, 3, 7, 14, and 28 days by filtration and the extracted powders were rinsed with ultra-pure water and ethanol and dried at 40 °C.

2.1. Soaking of samples in SBF

SBF was prepared as follows: 700 cm3 of ultra-pure water was added to a 1000 cm3 glass beaker, stirred with a magnetic stirrer, and the reagent-grade chemicals listed in Table 1 were dissolved

2.2. Characterization

The specific surface areas of the samples were measured using the Brunauer-Emmett-Teller (BET) method with N2 gas as the

10 20 30 40 50 60 70 20/ degree

10 20 30 40 50 60 70 20/ degree

a-Fe2O Y : a-Fe2O3

28 days ▼ .. . 1 ▼ ITt TTT^ i 1 T 11

0 day ▼ 1 h 1 IT ik

10 20 30 40 50 60 70 20 / degree

10 20 30 40 50 60 70 20 / degree

w c et c

10 20 30 40 50 60 70 20 / degree

a-FeOOH ■ : a-FeOOH

Jjf! 28 days

1 ■ 0 day m m

. 1 . 1 . 1 . 1 . 1 .

Fig. 1. Powder X-ray diffraction patterns of the studied samples before and after soaking in SBF for 28 days. "0 day" indicates the sample before soaking in SBF.

adsorbate (NOVA 1000e; Yuasa Ionics Co. Ltd., Osaka, Japan). The samples were heat-treated at 200 °C for 2h under vacuum as a pretreatment for the specific surface area measurements.

The pH of the SBF was measured after soaking the sample powders using a glass-electrode type pH meter (D-51; Horiba Ltd, Kyoto, Japan). The concentrations of calcium, phosphate, and iron ions in the SBF were measured using inductively coupled plasma atomic emission spectroscopy (Optima 2000DV; PerkinElmer Japan, Kanagawa, Japan) to determine the time-dependent changes of these ions concentrations in the SBF.

The morphology of the samples was observed under a scanning electron microscope (SEM; JSM5600; JEOL Ltd., Tokyo, Japan), after the application of a thin gold coating. Sample compositions were characterized qualitatively by energy dispersive spectroscopy (EDS; EX-54140 MSK; JEOL Ltd.).

The crystalline phases of the extracted powder samples were characterized using powder X-ray diffraction (XRD; RINT PC2100; Rigaku Co., Tokyo, Japan) using Cu Ka radiation (k = 0.154056 nm) in the range of 29 = 10-70° with a scanning rate of 2.0° min-1.

3. Results

Table 2 shows the specific surface areas of the samples. The specific surface areas of FeO, Fe3O4, a-Fe2O3, ^-Fe2O3, and a-FeOOH were 0.14, 7.02, 3.98, 21.9, and 17.5 m2 g-1, respectively.

Table 2

Specific surface areas of the samples measured by the BET method.

Sample Specific surface area (m2 g-1)

FeO 0.14

Fe3O4 7.02

a-Fe2O3 3.98

7-Fe2 O3 21.9

a-FeOOH 17.5

Fig. 1 shows XRD patterns of the samples before and after soaking in SBF for 28 days. Hereafter, "0 day" indicates the sample before soaking in SBF. Crystalline phase change was not detected in all samples upon soaking the FeO, Fe3O4, a-Fe2O3, ^-Fe2O3, and a-FeOOH in SBF. No crystalline substance formation was detected upon soaking these samples in SBF.

Fig. 2 shows SEM micrographs of the samples before and after soaking in SBF for 0, 7, and 28 days. Granules of several micrometers in size were observed for the FeO sample before soaking in SBF. No morphological change was observed for these FeO granules upon soaking the FeO granules in SBF. Particles of several hundred nanometers in size were observed for the Fe3O4 particles before soaking in SBF. After soaking in SBF for 7 and 28 days, spherical particles approximately 10 |im in diameter with a network-like surface structure were observed in addition to the Fe3O4 particles. Particles several hundred nanometers in size were observed for

Fig. 2. Scanning electron microscope images of the samples soaked in SBF for 0, 7, and 28 days. "0 day" indicates the sample before soaking in SBF.

Fig. 3. Scanning electron microscope images and energy dispersive spectroscopic spectra of Fe3O4 soaked in SBF for0 and 7 days. "0 day" indicates the sample before soaking in SBF.

a-Fe2O3 before soaking in SBF. Only a-Fe2O3 particles were found after soaking in SBF for 7 days, while spherical particles approximately 10 |im in diameter with a network-like surface structure in addition to the a-Fe2O3 particles were observed after soaking in SBF for 28 days. Aggregates of needle-shaped particles that were several hundred nanometers in size were observed for ^-Fe2O3 before soaking in SBF. No morphological change in the ^-Fe2O3 granules was observed upon soaking the ^-Fe2O3 granules in SBF. Needle-shaped particles were observed for a-FeOOH before soaking in SBF. No significant morphological changes were observed for the needle-shaped a-FeOOH particles that were soaked in SBF for 7 and 28 days.

SEM images and EDS spectra of Fe3O4 before and after soaking in SBF for 7 days are shown in Fig. 3. According to these SEM images, Fe3O4 particles of several hundred nanometers in size were present before soaking in SBF. After soaking in SBF for 7 days, Fe3 O4 particles and spherical particles of approximately 10 |im in diameter with a network-like surface structure were present. Fe was detected in the EDS spectrum of Fe3 O4 before soaking in SBF. Fe, Ca, and P were detected in Fe3O4 after soaking in SBF. These findings indicate that the spherical particles with a network-like surface structure contained Ca and P. Cu, Zn, and Au were detected in the EDS analysis. Cu and Zn are derived from the SEM sample holder, which is made of brass. Au was derived from the gold coating that was applied before SEM observation.

Time-dependent changes in the concentrations of calcium, phosphate, and iron ions in the SBF upon soaking FeO, Fe3O4, a-Fe2O3, 7-Fe2O3, and a-FeOOH are shown in Fig. 4. In the case of FeO, no significant decrease in the concentrations of calcium and phosphate ions was detected. When a-FeOOH was soaked in SBF, the concentrations of calcium and phosphate ions in the SBF decreased gradually as the soaking period increased. For Fe3O4, the concentrations of calcium and phosphate ions decreased at 7 days significantly and they then decreased gradually with an increase in the soaking period. Significant decreases in the concentrations of calcium and phosphate ions were found at 14 days and 28 days for a-Fe2O3 and ^-Fe2O3, respectively. The iron ion concentrations in the SBF upon soaking FeO, Fe3O4, a-Fe2O3, ^-Fe2O3, and a-FeOOH were less than the detection limit.

Fig. 5 shows time-dependent changes in the pH of the SBF containing FeO, Fe3O4, a-Fe2O3, ^-Fe2O3, and a-FeOOH. The pH of the SBF solutions was maintained at around 7.2 for up to 28 days. No

Fig. 4. Time dependence of the calcium, phosphate, and iron ion concentrations in SBF while soaking the studied samples. Initial calcium, phosphate, and iron ion concentrations in SBF are 2.5,1.0, and 0 mol m-3, respectively.

Fig. 5. Time dependence of the SBF pH while soaking the studied samples. Initial pH of SBF was 7.25.

significant changes in the pH of the SBF solutions were apparent in these samples.

4. Discussion

4.1. Calcium phosphate-forming ability of the samples

Aggregates with a network-like structure were observed in the Fe3O4 and a-Fe2O3 samples after soaking in SBF (Fig. 2), although

the formation of a crystalline compound was not detected in these samples by XRD (Fig. 1). According to Fig. 3, the aggregates that formed in Fe3O4 after soaking in SBF for 7 days contained Ca and P. Additionally, the concentrations of calcium and phosphate ions in SBF decreased upon Fe3O4 soaking (Fig. 4). These experimental results indicate that the aggregates with a network-like structure that formed in the Fe3 O4 sample were calcium phosphate. The morphology of the aggregates that formed in the a-Fe2O3 sample was similar to that of the Fe3O4 sample. Moreover, decreases in the concentrations of calcium and phosphate ions in SBF were detected in the a-Fe2O3 sample. Therefore, the aggregates that formed in the a-Fe2O3 sample were likely calcium phosphate. For the soaking of 7-Fe2O3 and a-FeOOH, we found that the calcium and phosphate ion concentrations in SBF at 28 days were almost equal to those of the SBF upon Fe3O4 and a-Fe2O3 soaking, although calcium phosphate formation was not observed in 7-Fe2O3 and a-FeOOH after soaking in SBF (Fig. 2). Decreases in the calcium and phosphate ion concentrations in SBFupon 7-Fe2O3 and a-FeOOH samples soaking can be attributed to the adsorption of these ions onto the surface of 7-Fe2O3 and a-FeOOH particles.

The formation of calcium phosphate was observed in the Fe3O4 and a-Fe2 O3 samples after soaking in SBF (Fig. 2), but calcium phosphate phase was not detected in these samples by XRD (Fig. 1 ). The morphology of the calcium phosphate precipitates in the Fe3O4 and a-Fe2O3 samples after soaking in SBF was similar to that of bone-like apatite that precipitated from SBF [14]. Additionally, the formation of defective structure and/or small crystallite HAp, which has been referred to as bone-like apatite, in SBF has been reported [15]. Therefore, the calcium phosphate precipitates that were observed in the Fe3O4 and a-Fe2O3 samples after soaking in SBF were likely bone-like apatite. However, no HAp diffraction peaks were detected by XRD in the Fe3O4 and a-Fe2O3 samples after soaking in SBF. This can be attributed to defective structure and/or small crystallites. Additionally, insufficient quantities of HAp can also result in XRD detection difficulties.

4.2. Dominant factor in calcium phosphate formation

Specific functional groups induce the heterogeneous nucleation of HAp in SBF. Typical functional groups are Si—OH [16] and— COOH [17].Titanium [18], zirconium [19], tantalum [20], and niobium [21] oxide gels induce bone-like apatite formation in SBF by Ti—OH, Zr—OH, Ta—OH, and Nb—OH, respectively. We propose that an Fe—OH group with a specific structure on the particle surfaces of Fe3O4 and a-Fe2O3 induces the formation of calcium phosphate in SBF based on the following discussion. The following three factors are controlling factors for calcium phosphate formation on the materials in SBF: crystal structure of the substrate material, formation of Fe—OH groups on material surfaces, and the amount and structure of the Fe—OH groups. We will discuss each factor separately.

4.2.1. Crystal structure of the substrate material

The calcium phosphate-forming ability of a titanium oxide (TiO2) substrate depends on its crystal structure, namely rutile or anatase [22]. Information about the calcium phosphate-forming ability of TiO2 is useful in understanding differences in the calcium phosphate-forming ability of a-Fe2O3 and 7-Fe2O3, as they have the same chemical composition. As shown in Fig. 2, a-Fe2O3 induces calcium phosphate formation and 7-Fe2O3 does not. This implies that a-Fe2O3 has an appropriate crystal structure for the induction of calcium phosphate formation. However, it is unlikely that a-Fe2O3 has a crystal structure that is exclusively responsible for the promotion of calcium phosphate formation because both a-Fe2O3 and Fe3O4 formed calcium phosphate. We believe that the induction of calcium phosphate formation by lattice matching

between the substrate materials and precipitated calcium phosphate is a result of an epitaxial growth-like phenomenon. Epitaxial growth occurs under limited conditions where the lattice mismatch is within several percent [23].Therefore, this phenomenon does not occur easily. Fe3O4 and a-Fe2O3 have different crystal structures and hence it is reasonable to consider the other factors that lead to the induction of calcium phosphate formation, rather than the crystal structure of substrate materials.

4.2.2. Formation ofFe—OH groups on the material surface

For TiO2, Ti—OH groups induce calcium phosphate formation [18]. The Fe—OH group is thus more important for calcium phosphate formation than the crystal structure of the substrate materials. According to Fig. 2, calcium phosphate formation occurred after 7 days for Fe3O4 and 14 days for a-Fe2O3. These results suggest the existence of induction periods for calcium phosphate formation in these samples. During the induction periods, it is presumed that the surfaces of Fe3O4 and a-Fe2O3 are hydrolyzed to form Fe—OH groups and the Fe—OH groups induce calcium phosphate formation.

Fig. 2 shows that the morphologies of the FeO, Fe3O4, a-Fe2O3, 7-Fe2O3, and a-FeOOH particles did not change up to 28 days. Fig. 4 indicates that Fe was not detected in SBF after soaking the FeO, Fe3O4, a-Fe2O3, 7-Fe2O3, and a-FeOOH materials for up to 28 days. The pH of the SBF solution was maintained at around 7.2, which is a very mild condition for these compounds (Fig. 5). These results imply that FeO, Fe3O4, a-Fe2O3, 7-Fe2O3, and a-FeOOH hardly dissolve in SBF and that the hydrolysis of these materials only occurs on their surfaces. Although the surfaces of all the investigated samples would have been hydrolyzed to form Fe—OH groups in SBF, only Fe3O4 and a-Fe2O3 induced calcium phosphate formation and the other samples did not. These findings imply that the formation ofFe-OH groups and the amount as well as structure of the Fe—OH groups on the iron-based material surfaces should be considered.

4.2.3. Amount and structure of the Fe—OH groups

a-FeOOH particles will contain many Fe—OH groups on their surface because of their component hydroxide ions. However, a-FeOOH did not induce calcium phosphate formation. If 7-Fe2O3 has the same reactivity as a-Fe2O3 in SBF, the number of Fe—OH groups on 7-Fe2O3 that were formed by the hydrolysis of the particle surface would be larger than that of a-Fe2O3, because the specific surface area of 7-Fe2O3 is approximately 5.5 times larger than that of a-Fe2O3 (Table 2). However, a-Fe2O3 induced calcium phosphate formation and 7-Fe2O3 did not. Materials of higher solubility should form more Fe—OH groups in aqueous solutions. The relevant order of solubility is 7-Fe2O3> a-FeOOH > a-Fe2O3 [24]; however, a-Fe2O3 induced calcium phosphate formation and 7-Fe2O3 did not. The absence of calcium phosphate formation by the a-FeOOH and 7-Fe2O3 samples implies that the amount of Fe—OH groups on the material surface is not important for the induction of calcium phosphate formation.

From research into TiO2, basic and acidic Ti—OH groups have a different calcium phosphate-forming ability [25,26]. This indicates that the structure of the Ti—OH groups is important for the induction of calcium phosphate formation in SBF. This suggests that Fe—OH groups with a specific structure will be the dominant factor for the induction of calcium phosphate formation in SBF. In future work, the surface structures of the materials before and after soaking in SBF will be carefully investigated by X-ray photo-electron spectroscopy, Fourier transform infrared spectroscopy, and zeta potentiometry to determine the calcium phosphate formation mechanism on these material surfaces. Additionally the time-dependence of calcium phosphate formation should be investigated by transmission electron microscopy to obtain the evidence of heterogeneous nucleation of calcium phosphate on Fe3O4 and

a-Fe2O3 particle surface. However, it is noteworthy that Fe3O4 and [5 related materials have the potential to form calcium phosphate in a body-like environment. This finding can benefit potential biomed-

ical applications of Fe3O4 and related materials. [7

[8 [9 [10

5. Conclusions

We investigated the behavior of iron oxides and an iron oxy-hydroxide in SBF. Upon soaking Fe3O4 and a-Fe2O3 in SBF, Fe3O4 was found to induce the formation of calcium phosphate with a network-like structure in the SBF within 7 days and a-Fe2 O3 did the same within 28 days. FeO, a-FeOOH, and ^-Fe2O3 did not induce calcium phosphate formation. Fe—OH groups with a specific structure on the particle surfaces of Fe3 O4 and a-Fe2 O3 likely induce the heterogeneous nucleation of calcium phosphate in SBF.

Acknowledgment

This work was partially supported by a Grant-in-Aid for Scientific Research (No. 22107007) on the Innovative Areas of "Fusion Materials: Creative Development of Materials and Exploration of Their Function through Molecular Control" (No. 2206) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT).

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