Hybrid mesoporous nanostructured scaffolds as dielectric biosimilar restorative materials

Abstract

BACKGROUND:

The intricate structure of natural materials is in correspondence with its highly complex functional behaviour. The health of teeth depends, in a complex way, on a heterogeneous arrangement of soft and hard porous tissues that allow for an adequate flow of minerals and oxygen to provide continuous restoration. Although restorative materials, used in clinics, have been evolving from the silver amalgams to actual inorganic fillers, their structural and textural properties are scarcely biomimetic, hindering the functional recovery of the tissue.

OBJECTIVE:

The objective of this work is to compare and test the hybrid mesoporous silica-based scaffolds as candidates for dentine restoration applications.

METHODS:

In this work, we present the development and the physical properties study of biocompatible hybrid mesoporous nanostructured scaffolds with a chemically versatile surface and biosimilar architecture. We test their textural (BET) and dielectric permittivity (ac impedance) properties.

RESULTS:

These materials, with textural and dielectric properties similar to dentine and large availability for the payload of therapeutic agents, are promising candidates as functional restorative materials, suitable for impedance characterization techniques in dental studies.

CONCLUSIONS:

Structural, textural, morphological characterization and electrical properties of hybrid mesoporous show a large degree of similarity to natural dentin samples.

Keywords

Mesoporous scaffolds restorative materials textural properties dielectric properties

1. Introduction

Some of the most promising areas in medical research, like the regenerative/restorative materials field, include nowadays nanostructured phases in their compositions benefiting from enhanced therapeutic performance and architecture control at the nanometre range.

Dentine is a multiscale hierarchic porous dental material, capped on top by a crown made of highly mineralized and protective enamel, and covered in the root, by a cement layer involved in the attachment of the teeth to the bony socket. The central part is filled up by a non-mineralized soft tissue, pulp, that contains nerves and a vascular network connected with surrounding tissues, the periodontal ligament and the bony socket. This tubular arrangement, with interconnected channels, facilitates the flow of biological fluids inside dentine, and is responsible for its remarkable dielectric response: an extremely high dielectric constant at low frequencies followed by a falling off as the excitation frequency is increased [1]. This specific signature makes dentine recognizable when using diagnostic impedance techniques and allows monitoring its biological functionality when it is damaged.

Nowadays, mainly all dental restorations use light cured composite materials due to their aesthetic appearance, handling advantages and resistance to wear [2]. Conventionally used polymer composites present good mechanical properties when resin matrices are filled with silane treated nonporous inorganic particles (e.g. zirconium oxide, barium glass). However, although silane treatment facilitates the interfacial bond between fillers and the resin matrix, in the oral environment [3] silane coupling agents hydrolyse and reduce the average lifetime of dental composites [4] provoking particles' leaching and the degradation of the matrix inside the teeth structure. Therefore, together with a compromised chemical stability, that may affect the neighbouring healthy tissues, the absence of porosity hinders the flow of dentine fluids and prevents the functional recovery of the damaged piece. These materials, with a scarce biologic performance, porosity or chemical stability and a dielectric response that is far from natural tissues, do not meet the biomimetic paradigm, highly sought in the tissue engineering field.

Restorative materials with biosimilar dielectric permittivity signature and structural porosity are crucial for the biological recovery of the treated teeth, to ensure an adequate flow of biological fluids and undistorted dielectric response to facilitate diagnosis procedures, such as bioimpedance characterizations [5].

In order to allow for a high degree of integration and the recovery of biological functions, a new paradigm in restorative materials is emerging under the premise of providing a high degree of similarity with the properties of the natural tissue. Biocompatible mesoporous silica nanostructures are a new generation of materials for tissue engineering applications due to their biocompatibility, osteointegration, high stability under strong pH/temperature variations, chemical versatility for surface functionalization or tailorable structural/textural properties or large availability for drug loading [6], that fulfil many of the requirements for dental tissue restoration. The objective of this work is to compare and test the hybrid mesoporous silica-based scaffolds as candidates for dentine restoration applications.

2. Materials and methods

2.1. Synthesis of SBA-15 mesoporous silica (S15H)

Hexagonally ordered SBA-15 mesoporous silica nanostructures can be synthesized by a soft template method [7], by solving Pluronic 123 (P123, PEO20-PEO70-PEO-20), a triblock copolymer that works as structure direct agent, in a mixture of deionised water with hydrochloric acid (HCl, 37%) under magnetic stirring at 35 °C until its complete dissolution. Afterwards, tetraethyl orthosilicate (TEOS, 98%), working as the silica precursor, is added with a final molar composition of 1.0SiO₂/0.017P123/3.4HCl/208H₂O. The reaction continues under vigorous magnetic stirring during 24 h, followed by an aging step at 100 °C for 24 h. The final product, mesoporous silica nanostructures (S15H), are afterwards filtered under vacuum and dried at 60 °C for 12 h, and submitted to several washing cycles with different mixtures of organic solvents (isooctane/ethanol, acetone/deionised water and deionised water), to eliminate the remaining surfactant from the inner channels.

2.2. Synthesis of a hybrid mesoporous silica-based scaffold (Sc1)

With the aim of providing mechanical and biological support, a three-dimensional biocompatible scaffold was synthesized by integrating mesoporous silica nanostructures in a mixture of chitosan/kappa-carrageenan. Typically, 300 mg of chitosan were dissolved in 5 mL of deionised water, together with 340 mg of the S15H ceramic, while 100 mg of kappa-carrageenan were dissolved in 5 mL of deionised water at 70 °C, for its subsequent addition under vigorous manual stirring into the chitosan solution. The mixture was blended at 24000 rpm followed by the addition of 0.4 mL of 1,4-butanediol diglycidyl ether (BDDE) and, afterwards transferred into a 10 mL plastic syringe and kept at −20 °C. Once frozen, the scaffold was immersed in a basic solution of potassium chloride at 60 °C for 24 h to stabilize its cylindrical shape for its demolding and washed with distilled water and lyophilized. The so prepared hybrid materials, show large porosity, large biocompatibility and sustained drug delivery tested for in vitro conditions [6].

2.3. Characterization of silica materials

Structural characterization was performed by low-angle X-ray powder diffraction (XRPD) using a PANalytical X’Pert Powder Empyrean (Panalytical, Callo End, UK) with Cu Kα radiation (𝜆 = 1.4506 Å), with a step size of 0.01° and counting time of 5 s per step from 0.25° to 6° (2𝜃).

Morphological characterization was realized by scanning electron microscopy (SEM) using a Zeiss FE-SEM ULTRA Plus microscope operating at 30 kV (Zeiss, Oberkochen, Germany) and transmission electron microscopy (TEM) using a JEOL JEM-1011 microscope (JEOL, Tokyo, Japan) operating at 100 kV.

Textural characterization was performed by the N₂ adsorption isotherms of a degassed sample using a Quantachrome Autosorb IQ2 (Quantachrome Instruments, Florida, USA), from which pore size distributions and specific surface area were estimated.

In addition, micro-computer tomography scans were performed, at room temperature, on a dried sample specimen, for a 3D visualization of the scaffolds. For the measurements at 35 kV no filter was used. The specimen was measured at 0.15° rotational step with 180° rotation, 5 micron pixel size and an exposure time of 1062 ms. The data were reconstructed using NRecon software (Bruker, Kontich, Belgium) and analysed using a CTan software (Bruker, Kontich, Belgium). The image rendering was performed on the reconstructed images in CTvox (Bruker, Kontich, Belgium).

Thermogravimetric analysis (TGA) was performed on heating, from room temperature to 850 °C, at 10 °C/min under nitrogen flow (20 mL/min) using a Perkin Elmer Pyris 7 thermal gravimetric analyzer (Perkin, Waltham, MA, USA).

2.4. Dentine sample preparation

Sound human premolar teeth were extracted by orthodontic prescription, and immediately after, they were placed in physiological solution and stored at 5–6 °C. Soft tissues were removed applying ultrasounds, prior to the subsequent sectioning perpendicularly to the long axis using a diamond-blade saw. Sections, with an averaged thickness of 1 mm, were obtained from the dentinal region between the occlusal surface and the pulp camera, with a commercial cutter model 650A (South Bay Technology).

2.5. Electrical impedance spectroscopy

Complex dielectric permittivity, 𝜀^∗(𝜔), of dentine, mesoporous silica and hybrid scaffold samples, were measured at frequencies from 10 to 1 × 10⁶ Hz with a parallel-plate capacitor coupled to a Solartron 1260 Impedance/Gain-Phase Analyzer (Solartron Analytical, Hampshire, UK). The capacitor was mounted in a Janis SVT200T cryostat (Leica Biosystems, Barcelona, Spain) refrigerated with liquid nitrogen and with a Lakeshore 332 (Lakeshore, Carson, USA) incorporated to control the temperature, which allowed fixing a temperature at 37 °C. The material surfaces were painted with silver paint to ensure a good electrical contact with the electrodes. The measurements were performed for dry and wet materials. In the case of wet materials, the measurements were performed immediately after taking out the specimens from physiological solution and externally drying with tissue paper for the teeth samples and Sc1 scaffold or filtering for SH15 mesoporous silica matrix. The sample-holder was pre-heated before measurement to avoid delays that could cause significant variations of water content in the tested materials. A maximum of five permittivity measurements per specimen were performed. The impedance analysis software SMART (Solartron Analytical, Hampshire, UK) was used for data acquisition and processing.

3. Results

The hybrid mesoporous silica-based scaffolds are a mixture of highly ordered mesoporous ceramic embedded inside a scaffold matrix of non-ordered porous biopolymers composed of chitosan and k-carrageen. Therefore, the hybrid 3D scaffold shows diffraction patterns at angles in the range 0.5° ≤ 2𝜃 ≤ 6° when the Bragg conditions are fulfilled for the order phase of mesoporous silica. According to this, Fig. 1A shows the low-angle XRPD pattern of the S15H mesoporous silica matrix, where the three well-resolved peaks characteristic of the SBA-15 materials are observed, corresponding to (100), (110) and (200) reflections of the P6mm hexagonal symmetry group. From these characteristic reflections of a highly ordered 2D hexagonal SBA-15 structure the unit cell parameter, a ₀, and interplanar distance, d, the distance between the crystallographic planes, have been obtained to be 12.43 Å and 10.77 Å, respectively. These values are in accordance with previously reported works [8–10]. In summary, although a 3D amorphous microstructure was obtained, its order at mesoscale allows having a better control over drug loading and release as was tested before by our group [6].

Fig. 1.

A: Low-angle XRPD pattern of the S15H mesoporous silica matrix; B: TGA/DTG curves of S15H, where NW and W correspond to the sample before and after removing the surfactant; C: TGA curves of the main components of the Sc1 scaffold, including it.

To guaranty an available surface area of the mesoporous silica matrix, it was necessary to remove the template by submitting it to several washing cycles with different mixtures of organic solvents. To quantify the residual amount of surfactant, the samples were characterized by TGA/DTG analysis. Figure 1B shows an initial weight loss of 5 and 9% between 25 and 120 °C for the sample before and after removal surfactant, respectively, which is related to the thermodesorption of physically adsorbed water. The well-defined peaks obtained by DTG analysis displayed the temperature ranges in which the Pluronic P123 molecules are degraded. The most intense peak (110–450 °C) is associated with the S15H material before it has been subjected to the surfactant removal process, and the smallest one (170–450 °C) is associated to the S15H material obtained after this process, demonstrating an important surfactant weight loss. However, around 11% of Pluronic P123 still remains within the mesopores after the surfactant removal process. On the other hand, the TGA curves of the main materials involved in the synthesis of Sc1 scaffold, including it, are shown in Fig. 1C. The TGA analysis of the Sc1 scaffold is consistent with the expected result based on the amount employed of each component as well as their degradation behaviour (Table 1). At 850 °C, the chitosan and kappa-carrageenan kept around 25.3% of their weight, while the S15H ceramic 76.5%. Thus, the Sc1 scaffold displayed a remnant weight of 33.4%. Based on the expected weight calculation, the residual weight percentage of the Sc1 scaffold should be 29.8% because the BDDE was completely removed at this temperature. However, the crosslinking reaction mediated by BDDE improved the degradation behaviour of the scaffold. Therefore, a higher weight percentage is observed. Although the scaffold starts to degrade at a temperature higher than 𝜅-carrageenan but lower than chitosan, its negative slope is less abrupt than those polysaccharides, which indicates that its degradation process is gradual and occurs in several steps.

Table 1

Remnant mass after TGA analysis of the materials involved in the synthesis of Sc1 scaffold including it

Component	Weight (mg)	Remnant weight (mg) at 850 °C
𝜅-Carrageenan	100	25.3
Chitosan	300	75.9
S15H ceramic	340	260.1
BDDE	440	0.0
Sc1 scaffold^∗	1180	351.3

^∗without considering additional components as acetic acid and water.

The SEM images of the Sc1 scaffold (Figs 2A and B) show a highly porous structure with a remarkable roughness surface. The three-dimensional matrix exhibits randomly oriented pores, ranging from macropores to micropores based on IUPAC definition [11]. In this regard, the scaffold not only provides a place to cells adhesion and proliferation but also the ability to load and release a wide variety of molecules of biological interest. This ability is given by the presence of the S15H mesoporous silica matrix that has long filament morphology with particle size between 500 nm and 1 μm (Figs 2C and D).

Fig. 2.

A and B: SEM micrographs at different magnifications and locations of the Sc1 scaffold; C and D: SEM micrographs at different magnifications; E and F: TEM micrographs at different magnifications and orientation of the pore channels system of the S15H mesoporous silica matrix; G and H: SEM micrographs at different magnifications of dentin surface.

By TEM microscopy and when the incident electron beam is parallel to the main axis of the mesopores a hexagonal disposition of uniform pores was observed in S15H matrix (Fig. 2E). Furthermore, when the electron beam is perpendicular to the channel axis unidirectional cylindrical channels are observed (Fig. 2F).

The scaffolds of mesoporous materials have porous nature and their tubular structure is reminiscent of the dentine tubules (Figs 2G and H). Dentine is a mineralized tissue that contains numerous tubules filled with fluid that extend from the pulp chamber in the centre of the tooth to it outer surface [12]. This dental tissue is composed of 50% inorganic matter, 30% organic matter and 20% fluids by volume. The inorganic components are mainly hydroxyapatite crystals randomly distributed, and the organic material is predominantly type I collagen (90%). Both materials delimit the dentinal tubules through which the dentinal fluids circulate, that are responsible for the dentin permeability.

A 3D structure of a scaffold is shown in Fig. 3. The highly radiopaque silica particles are uniformly distributed through the whole volume however; they tend to form aggregates. The analysis performed in CT a revealed a porosity of 90%. As seen in Fig. 3, the orientation and shape of the pores is relatively random which may lead to more isotropic mechanical properties. The estimated average structure thickness was 50 μm and the average structure separation (distance between the closest walls) revealed 410 μm (Fig. 4).

Fig. 3.

A and B: 3D rendering of the micro CT data showing the structure of the scaffolds. The red particles are areas with higher radiopacity, supposedly with higher concentration of the S15H particles; C and D: cross-section of a specimen showing the connectivity of the internal channels. The size of CT-image is 791X791 pixels, and scale length of 20,16 (μm/pixel).

Fig. 4.

Distribution of (A) the structure thickness and (B) structure separation of the Sc1 mesoporous scaffold.

3.1. Textural properties

Nitrogen adsorption isotherm (Fig. 5A) shows a type IV N₂ adsorption isotherm that are similar to those reported previously [13,14] and characteristic of good-quality SBA-15 mesoporous materials. Similarly, to the nitrogen adsorption isotherms for large-pore MCM-41 [15–17] the isotherm of SBA-15 featured hysteresis loops if of H1-type with sharp adsorption and desorption branches. The sharpness of the adsorption branches is indicative of a narrow mesopore size distribution. The pore size distributions (PSDs) were analyzed and calculated by Non-Local Density Functional Theory (NLDFT) and Barrett–Joyner–Halenda (BJH) methods and are presented in Fig. 5B.

Fig. 5.

A: N₂ sorption isotherm; B: pore size distributions of the S15H mesoporous silica matrix and Sc1 mesoporous scaffold.

Fig. 6.

A: Real and B: imaginary part of the relative dielectric permittivity for dry materials; C: real and D: imaginary part of the dielectric permittivity for wet materials, measured as a function of frequency at 37 °C.

The compiled results are displayed in Table 2. The PSDs of both the S15H ceramic and Sc1 scaffold, where the rod-like S15H particles are covered by a high amount of polysaccharides mixture, display values ranging between 8 and 9 nm. As is expected, a decrease in Sc1 surface area is observed since the SBA-15 mesoporous silica is partially blocked by the polymeric matrix. However, in general the pore sizes, BET specific surface areas, and total pore volumes were in good agreement with those reported previously [7,18].

Table 2

Textural and structural values of SBA-15 and Sc1 materials. Surface area (S_BET), pore volume (V_p), pore size by NLDFT method (PS_DFT), pore size by BJH method (PS_BJH), unit cell parameter (a ₀) obtained from low-angle XR diffraction, wall thickness of the mesopores (t _wall = a ₀ − PS_DFT)

Sample	S_BET (m²/g)	V_p (cm³/g)	PS_DFT (nm)	PS_BJH (nm)	a ₀ (nm)	t _wall (nm)
S15H	639.29	0.954	8.15	8.98	12.43	4.285
Sc1	145.68	0.308	—	8.46	10.77	—

3.2. Dielectric properties

The permittivity of a material describes the polarization response of a dielectric medium exposed to an electric field [19]. Generally, polarization of materials in an alternating field does not respond instantaneously [20], leading to a phase delay between polarization and an external oscillating electric field. This time lag is due to various processes: ionic and dipolar relaxation, and atomic and electronic resonances at higher frequencies. The dielectric permittivity spectrum over a wide range of frequencies can help to identify the specific polarization mechanisms and absorption losses, which are important for opening up new applications in the materials engineering [21].

The linear permittivity of a material is usually given relative to the permittivity of free space (𝜀₀ = 8.85 × 10⁻¹² F/m) as a relative permittivity, 𝜀_r, and the actual permittivity is then given by the following equation: $\begin{eqnarray}\displaystyle {\varepsilon}={\varepsilon}_{r}{\varepsilon}_{0}. & & \displaystyle \nonumber\end{eqnarray}$

In addition to this, the response of materials to at external fields depends on the frequency of the field and the dielectric permittivity can be expressed as a complex magnitude (𝜀^∗) [22]: $\begin{eqnarray} \displaystyle {\varepsilon}\rightarrow {\varepsilon}^{\ast }({\omega})={\varepsilon}^{\prime }({\omega})-j{\varepsilon}^{\prime \prime }({\omega})={\varepsilon}^{\prime }({\omega})-j{\sigma}/{\omega}. & & \displaystyle \nonumber \end{eqnarray}$ Where, the real part, 𝜀^′, of the permittivity accounts for dielectric constant and the imaginary part, 𝜀^′′, for losses; j, is the imaginary number; 𝜔 =2𝜋𝜈, is the angular frequency and 𝜎, is the conductivity.

Figures 6A and B display the real part of the relative dielectric permittivity of dentine, mesoporous S15H matrix and Sc1 scaffold measured as a function of frequency. As expected in ordered porous materials, the permittivity of the dry samples is practically independent of the frequency and shows a low dielectric constant (Fig. 6A). Silica bulk materials have a low dielectric constant around 4, but the presence of nanoporous or mesoporous structures in the silica, decreases substantially these values from 4 close to the unity. These mesoporous materials as ultra-low dielectric constant materials are of interest for applications in electronic devices because they reduce cross talk noise in modern digital circuits [23]. On the other hand, in the case of wet samples of dentine and mesoporous materials (Figs 6C and D), the dielectric permittivity shows a significant frequency-dependent dispersion. The changes in the values of the dielectric permittivity are larger at lower frequencies and smaller at higher frequencies, so the dielectric response is greatly affected by the frequency. At about 1000 Hz the curves split up, showing a larger relaxation for dentine. Also, at high frequencies an almost flat dielectric response can be observed, indicating the poor conductive behaviour of these materials and how the relaxation process changes from ionic to dipolar at these frequencies.

The permittivity values measured for dentine agree with those reported previously in the literature [24]. Also, the mesoporous S15H and Sc1 scaffold show a similar dielectric behaviour with dentine. We have to note that the S15H matrix and Sc1 scaffolds were measured after being immersed in physiological solution, to simulate the in vivo state of the dentine [25], taking into account that the aqueous solution inside the dentinal tubules dramatically enhances the real and imaginary part of the dielectric permittivity, since a high water content produces an increase of dipolar polarization as well as a faster mobility of the ions in the sample.

These results can be explained by the presence of numerous discontinuities separating liquids, containing dissolved salts as in the case of the dental fluids and solid phases, as mesoporous silica, which give rise to Maxwell–Wagner polarization and deeply affect the dielectric permittivity behaviour. The electromagnetic complex interplays of this type of wet porous structures with liquid additives shaping the response of low-frequency dielectric permittivity which depends on different ionic charges present in the porous structure [26]. In the framework of the Maxwell–Wagner model for interfacial polarization [27] a capacitor formed by two dielectric materials with conductivities 𝜀₁ and 𝜀₂ and dielectric permittivity 𝜀₁ and 𝜀₂ gives a relaxation spectrum ( $\varepsilon_r$ vs. 𝜈 = 𝜔∕2𝜋) indistinguishable from that expected for a single-phase dielectric which obeys the Debye model. However, the relaxation process occurs at a characteristic frequency that depends on the properties of both components. Meanwhile, in the ${\varepsilon}_{\text{r}}^{\prime \prime }$ vs. 𝜈 curve differences with Debye model are found. This is due to the fact, that the imaginary part of the dielectric permittivity contains a conductivity term owing to both phases, which modifies the curve shape especially at low frequencies. To better visualize the influence of the conductivity on the dielectric behaviour the Cole- Cole plots ( $\varepsilon^{\prime\prime}_r$ vs. ${\varepsilon}_{r}^{\prime }$ ) were employed. If a system is composed by a conductive phase (𝜎₁≠ 0) embedded in an insulting phase (𝜎₂ = 0), the observed response agrees with the ideal Debye model, in which the Cole-Cole plot results in a semicircular arc. As 𝜀₂ grows, the ideal semicircular shape disappears, showing a steep rise in ${\varepsilon}_{r}^{\prime \prime }$ .

The Cole-Cole plots obtained for dentine and mesoporous S15H matrix and Sc1 scaffold are shown in Fig. 7. A different behaviour from that predicted by Debye model can be observed in these samples, which is consequence of the high conductivity produced by the free carriers existing in these specimens.

Fig. 7.

Cole-Cole plot of wet dentine, mesoporous S15H and Sc1 scaffold measured at 37 °C.

4. Discussion

The advantage of using a hybrid mesoporous scaffold is to combine in one material the bioactive properties of a mixture of natural polysaccharides as chitosan and kappa-carrageenan with the mechanical resistant and chemical versatility of the SBA-15 mesoporous silica. In this regard, the scaffold not only provides a place to cells adhesion and proliferation but also the ability to load and release a wide variety of biological substances of interest. This ability is given by the presence of the S15H mesoporous silica matrix which has porosity higher than that of mature dentin [28]. Also, the good connected pores provide a sufficient nutritional supply and interchange between a living tissue and a scaffold and show many similarities with dentine tubular structure in which flow occurs through a variety of channels that vary in diameter and shape. On the other side, this high porosity may worsen the mechanical stability and resistance to stress. The degradation characteristics of Pluronic together with the bioactive potential of silica make it interesting for materials supporting a self-regeneration [29,30]. The structure separation defines indirectly the average pore diameter and is therefore important for the capillary forces and absorption characteristics. The estimated structure thickness of 50 μm is larger in comparison with Pluronic scaffolds found in literature [29,31]. This can be affected by the micro CT resolution, additional artefacts and low contrast of air with this organic substance. Another reason may be the preparation procedure and incorporation of the mesoporous silica.

From an electromagnetic point of view, the properties of dentine and mesoporous materials are very similar. Both wet materials shown high dielectric permittivity and conductivity values at low frequencies. The high porosity of these synthetic mesoporous materials is what explains the observed dielectric properties that are very similar to natural dentine, which is also a porous material.

These porous materials are very different from other materials that are currently used to repair tooth decay, as is the case of silver amalgams or new nanocomposite materials, which do not present any porosity.

All these results show the suitability of this material, which offers excellent potential as scaffolds for dental tissue engineering due to it similar response to the dentine in nature.

5. Conclusions

In this work, hybrid mesoporous silica-based scaffolds are proposed as good candidates for dentine restoration applications, due to their textural (BET) and dielectric permittivity (ac impedance) properties similar to the native tissue. Structural, textural, morphological characterization and electrical properties of hybrid mesoporous are showing a large degree of similarity to natural dentin samples.

Footnotes

Conflict of interest

None to report.

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