Construction of Magnetic Nanochains to Achieve Magnetic Energy Coupling in Scaffold

doi:10.21203/rs.3.rs-1448231/v1

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Research Article

Construction of Magnetic Nanochains to Achieve Magnetic Energy Coupling in Scaffold

https://doi.org/10.21203/rs.3.rs-1448231/v1

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Background: Fe₃O₄ nanoparticles are highly desired for constructing endogenous magnetic microenvironment in scaffold to accelerate bone regeneration due to their superior magnetism. However, their random arrangement easily leads to mutual consumption of magnetic poles, thereby weakening the magnetic stimulation effect.

Methods: In this study, magnetic nanochains are synthesized by magnetic-field-guided interface co-assembly of Fe₃O₄ nanoparticles. In detail, multiple Fe₃O₄ nanoparticles are aligned along the direction of magnetic force lines and are connected in series to form nanochain structures under an external magnetic field. Subsequently, the nanochain structures are covered and fixed by depositing a thin layer of silica (SiO₂), and consequently forming linear magnetic nanochains (Fe₃O₄@SiO₂). The Fe₃O₄@SiO₂ nanochains are then incorporated into poly l-lactic acid (PLLA) scaffold prepared by selective laser sintering technology.

Results: The results show that the Fe₃O₄@SiO₂ nanochains with unique core-shell structure are successfully constructed. Meanwhile, the orderly assembly of nanoparticles in the Fe₃O₄@SiO₂ nanochains enable to form magnetic energy coupling and obtain a highly magnetic micro-field. The in vitro tests indicate that the PLLA/Fe₃O₄@SiO₂ scaffolds exhibit superior capacity in enhancing cell activity, improving osteogenesis-related gene expressions, and inducing cell mineralization compared with PLLA and PLLA/Fe₃O₄ scaffolds.

Conclusion: In short, the Fe₃O₄@SiO₂ nanochains endow scaffolds with good magnetism and cytocompatibility, which have great potential in accelerating bone repair.

Magnetic microenvironment

Fe3O4 nanoparticles

Magnetic nanochains

Magnetic energy coupling

Bone scaffold

Recent scaffolds lack the capacity to effectively modulate cell growth or tissue reconstruction, resulting in slow bone regeneration and even failure of bone implantation [1–3]. It is well known that cells are magnetically sensitive due to the diamagnetism of cell membranes [4], and exposure to magnetic fields served to alter membrane flux and regulate ion channels and biochemical pathways [5, 6]. In this case, a series of cell behaviors will be mediated. Inspired by this, it is significant to construct an endogenous magnetic microenvironment in bone scaffold to mediate cell behaviors and tissue regeneration via magnetic stimulation.

As a typical magnetic material, Fe₃O₄ nanoparticles have been widely used in tissue engineering field due to their superior magnetic properties and good biocompatibility [7]. In addition, Fe₃O₄ nanoparticles enable to decompose into oxygen and iron in the body and can be easily removed from the body after degradation by oxygen transport and metabolism [8, 9]. The introduction of Fe₃O₄ nanoparticles into bone scaffolds is expected to construct an endogenous magnetic microenvironment to enhance cell activity and accelerate cell growth. It is worth noting that the random arrangement of Fe₃O₄ nanoparticles in scaffold easily leads to mutual repulsive of magnetic poles between adjacent nanoparticles, thereby weakening the magnetic strength. In this case, the scaffold cannot fully exert its regulation on cell behaviors.

The directional assembly of Fe₃O₄ nanoparticles into ordered nanochain structures is expected to solve the above problem. Under external magnetic field, the internal magnetic dipole moment of Fe₃O₄ nanoparticles enable to be rapidly deflected to the direction of magnetic field [10, 11]. Moreover, the attractive magnetic dipole interaction drives multiple Fe₃O₄ nanoparticles to assemble into ordered magnetic nanochain structures along with magnetic force lines [12, 13]. Compared with randomly arranged Fe₃O₄ nanoparticles, the magnetic nanochain structure can realize the magnetic energy coupling between the nanoparticles, thereby enhancing the magnetic strength [14–16]. Li et al. synthesized uniform linear cobalt nanochains with a coating layer of polyvinylpyrrolidone in aqueous solution under an external magnetic field [17]. Zhang et al. synthesized one-dimensional nickel nanochains by dropping method [18].

In this study, Fe₃O₄@SiO₂ magnetic nanochains were synthesized utilizing a magnetic-field-guided interface co-assembly of nanoparticles. In short, Fe₃O₄ nanoparticles were firstly coated with a layer of protective SiO₂ film. Then, the above products were aligned and assembled into nanochains under an external magnetic field. Thereafter, the nanochains were fixated by further depositing with a layer of SiO₂ to permanent keep their structure. The microscopic morphology, chemical composition, and physicochemical properties of Fe₃O₄@SiO₂ were analyzed. Then, the Fe₃O₄@SiO₂ were loaded into PLLA scaffold which prepared using selective laser sintering (SLS). The cell activity, osteogenic differentiation and mineralization abilities induced by the scaffolds were investigated and analyzed in detail. Moreover, the osteogenesis-related gene expressions of runt-related transcription factor-2 (Runx2), osteopontin (OPN), osteocalcin (OCN) and osterix (OSX) of cells were assessed. Additionally, the stimulation mechanism of the magnetic scaffold on cells was also discussed in depth.

Materials

Medical-grade PLLA powders were obtained from Shenzhen Polymtek Biomate-rial Co., Ltd. (Shenzhen, China). Fe₃O₄ nanoparticles with an average diameter of 100 nm, tetraothorsilicate (TEOS), sodium citrate dihydrate (C₆H₅Na₃O₇·2H₂O), sodium hydroxide (NaOH) and concentrated ammonia solution (NH₃·H₂O, 28 wt.%) were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). The above chemicals were applied as received without further purification.

Synthesis of one-dimensional Fe₃O₄@SiO₂ nanochains

The Fe₃O₄@SiO₂ nanochains were prepared through the magnetic-field-guided interface co-assembly of Fe₃O₄ nanoparticles, as shown in Fig. 1. The synthesis procedure was partly based on A. Saha et al [19–21]. First, 20 mg of Fe3O4 nanoparticles was dispersed in 60 mL ethanol-aqueous solution by ultrasonication for 20 min. Subsequently, 5 mL of ammonia solution (28 wt.%) was dropped into the resulting solution with mechanical agitating (800 rpm) for 30 min. Then, the agitating speed was reduced to 300 rpm, and 10 mL of ethanol solution with TEOS (V_ethanol:V_TEOS=9:1) were dropped in the solution to coat Fe₃O₄ nanoparticles with a layer of protective SiO₂ film in the early stage of the sol-gel reaction of TEOS. After reaction for 15 min, the above solution was exposed in a static magnetic field (55 mT-65 mT) for 2 min to induce the linear arrangement of Fe₃O₄ nanoparticles without stirring, forming nanochain structures. After standing for another 10 min, the nanochain structures were further coated by SiO₂. Finally, the fixated core-shell Fe₃O₄@SiO₂ magnetic nanochains were obtained after magnetically collection, cleaning, and drying.

Preparation of scaffolds

For comparison, PLLA, PLLA/Fe₃O₄ and PLLA/Fe₃O₄@SiO₂ scaffolds were respectively fabricated. Prior to fabricating, PLLA/Fe₃O₄@SiO₂ and PLLA/Fe₃O₄ powders were prepared as follows. 4.6 g of PLLA powders and 0.4 g of Fe₃O₄@SiO₂ nanochains were added to a beaker containing 30 mL of ethanol and then ultrasound for 30 min, following by vigorously stirring 1 h to obtain dispersed suspensions. Subsequently, the suspensions were vacuum dried for 24 h at 65 ℃, obtaining PLLA/Fe₃O₄@SiO₂ powders. Meanwhile, PLLA/Fe₃O₄ powders were also prepared in the same way.

The representative PLLA/Fe₃O₄@SiO₂ scaffold with honeycomb structure was fabricated via SLS technology. In detail, the PLLA/Fe₃O₄@SiO₂ powders were paved on the powder bed and selectively laser scanned according to the designed three-dimensional model, with laser power at 2.5 W, scanning speed at 100 mm/s and scanning distance at 0.24 mm [22, 23]. The modeling platform gradually descended with each layer of powders was sintered until the scaffold was completely formed.

Measurements and characterization

The morphologies of the Fe₃O₄@SiO₂ nanochains were observed by transmission electron microscope (TEM, TALOS F200X, USA). The chemical structure, compositions and crystal structures of the Fe₃O₄@SiO₂ or/and Fe₃O₄ powders were detected by X-ray photoelectron spectrometer (XPS, EscaLab 250Xi, USA), Fourier transform infra-red spectrometer (FTIR, FTIR 850, China) and X-ray diffractometer (XRD, D/MAX-RA, Japan). The magnetic properties of Fe₃O₄@SiO₂ and Fe₃O₄ powders were evaluated using a vibrating sample magnetometer (VSM, LakeShore7404, USA). Water contact angle on PLLA, PLLA/Fe₃O₄ and PLLA/Fe₃O₄@SiO₂ scaffolds were assessed using an optical contact angle meter (DM-501, Japan).

Cytocompatibility

The PLLA, PLLA/Fe₃O₄, PLLA/Fe₃O₄@SiO₂ scaffolds (φ 8 × 2 mm³) were sterilized by immersing in 70% ethanol solution for 2 h and irradiating with UV for 12 h. Then, the sterilized scaffolds were individually placed in 48-well culture plates. MG-63 cells (Sigma, Shanghai, China) were selected to investigate the cytocompatibility of scaffolds. MG-63 cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) were washed with D-Hanks solution three times and were digested with trypsin. The resulting solution were centrifuged at 1000 r/min for 5 min. The cell suspension was diluted to 8 × 10³ cells/mL. All the cells were incubated in a humidified condition with 5% CO₂ at 37°C.

The cells at a density of 4 × 10³ cells/well were incubated on the surface of sterilized scaffolds in 48-well plates containing DMEM, in which the DMEM was updated daily. After incubating for 3 and 7 days, each cell-scaffold sample was washed with PBS three times, and then immersed in 5% glutaraldehyde for 30 min to fix cells. Thereafter, the cell-scaffold samples were dehydrated with 30%, 50%, 70%, 80%, 90%, 95% and absolute ethyl alcohol in sequence and dried for 12 h at room temperature. After coated with gold, the cell morphology on each scaffold was observed by scanning electron microscope (SEM).

Cell viabilities on the scaffolds were studied by a live/dead staining kit (Beyotime, China). At the specified time (3 and 7 days), the culture medium was removed, and the cells were detached from scaffolds and rinsed using PBS three times. Subsequently, the cells were stained with 2 µM Calcein-AM for 30 min at 37 ℃. Finally, the stained cells were visualized using a fluorescence microscope (Olympus, Japan).

Cell proliferation was quantitatively assessed using Cell Counting Kit-8 (CCK-8, Beyotime, China) assay. The cell-scaffold samples were harvested from culture medium after 1, 4 and 7 days of cultivating. Then, the samples were washed with PBS and transferred into 96-well plates containing 100 µL CCK-8 reagent. After incubating for 2 h, the absorbance of the solution was detected utilizing a microplate reader (Thermal, USA) at 450 nm. Each group was carried out three parallel experiments.

The alkaline phosphatase (ALP) activity of the cells on scaffolds was determined to assess the osteogenic differentiation. After incubating for 7 days, the harvested cells from samples were washed with PBS 3 times and fixed with 4% paraformaldehyde for 15 min. Then, ALP staining kit (Beyotime, China) was dropped to stain the cells, and the stained cells were monitored using an inverted microscope (TE2000U, Japan).

The mineralization nodules of MG-63 cells cocultured with scaffolds were qualitatively investigated using Alizarin Red staining. The cells were seeded on scaffolds in 6-well plates for 7 days at a density of 1×10⁴ cells/mL. After that, the cell-scaffold samples were fixed using 4% paraformaldehyde and rinsed with PBS. Subsequently, the samples were stained with 0.04 M Alizarin Red for 10 min. After rinsing, the samples were observed under a light microscope.

Scaffold-mediated cell differentiation and osteogenesis were further studied. For this, the expression of several relative genes containing runt-related transcription factor-2 (Runx2), osteopontin (OPN), osteocalcin (OCN) and osterix (OSX) were detected with quantitative real-time polymerase chain reactions (RT-PCR). After incubating for 3 and 7 days, the RNA isolation of cells was employed using TRIzox reagent, and then the RNA was reverse transcribed to cDNA using PrimeScript 1st strand cDNA synthesis kit. Finally, the levels of Runx2, OPN, OCN and OSX were calculated using the 2^−ΔΔCt method. Each sample was analyzed three times.

Statistical analysis

All data were conducted by Student’s t-test for independent samples and presented as means ± standard deviation, where **p < 0.01 and *p < 0.05 represented significant difference.

Fe₃O₄@SiO₂ nanochains

The representative one-dimensional structure of the Fe₃O₄@SiO₂ nanochains were observed using TEM. As shown in Figs. 2a and 2b, the Fe₃O₄@SiO₂ nanochains presented a unique core-shell structure, in which Fe₃O₄ nanoparticles served as cores with a diameter of about 100 nm and SiO₂ layers served as shells with a thickness of about 25 nm. Based on Fast Fourier transform (FFT, Figs. 2c and 2d), the interplanar distance between adjacent lattice fringes was 0.25 nm, which was in great agreement with the (311) lattice planes of Fe₃O₄. Moreover, the diffraction rings depicted in the selected area electron diffraction (SAED) patterns respectively corresponded to the (220), (311), (400), (440) and (422) lattice planes of cubic Fe₃O₄ (Fig. 2e). The Si, Fe and O elements were clearly exhibited in the elemental mapping images (Figs. 2f-i). Particularly, the distribution of Si and Fe elements further confirmed the shell-core structure of the nanochains. The results confirmed that the Fe₃O₄ nanoparticles could be induced to align in a nanochain though magnetic dipolar interaction under external magnetic field.

Magnetic field distribution around single Fe₃O₄ nanoparticle and nanochain in the same direction of external magnetic field was analyzed using finite element method (COMSOL Multiphysics), as shown in Figs. 3a-c. It could be seen that the magnetic dipole moment of a Fe₃O₄ nanoparticle reached a saturated value under the adequately strong magnetic field (Fig. 3a). When the centerline between two adjacent nanoparticles was aligned with the direction of the external magnetic field, the dipole-dipole interaction was attractive (Fig. 3b). When the interaction energy was large enough to overcome thermal fluctuations, the magnetic dipole-dipole force drove the self-assembly of nanoparticles into nanochains along the dipole moment (Fig. 3c). In this case, the dipole-dipole coupling reached the maximum. Therefore, the magnetism of nanochains can be regarded as the magnetic energy coupling between nanoparticles.

The magnetic properties of Fe₃O₄ nanoparticles and Fe₃O₄@SiO₂ nanochains were detected and presented in Fig. 3d. Magnetization curves confirmed the superparamagnetism of nanoparticles and nanochains due to the absence of hysteresis. The nanochains preserved their superparamagnetism during the synthetic processing from superparamagnetic nanoparticles, which was an important advantage for their applicability in biomedicine [24, 25]. The saturation magnetization of Fe₃O₄ was 52.2 emu/g. The relatively low saturation magnetization of Fe₃O₄@SiO₂ was due to the introduction of non-magnetic SiO₂ shells decreased the weight ratio of Fe₃O₄ in Fe₃O₄@SiO₂ [26]. This phenomenon was also discovered by M. Tarhini and A. Bitar et al [20, 21].

XPS spectra of Fe₃O₄@SiO₂ nanochains were presented in Fig. 3e. The typical Fe2p, O1s and Si2p peaks were clearly observed (Fig. 3e₁). In detail, the peaks centered at 711.4, 723.0 and 726.0 eV were respectively corresponded to Fe²⁺2p_3/2, Fe²⁺2p_1/2 and Fe³⁺2p_1/2 of Fe₃O₄ (Fig. 3e₂), while the peaks centered at 102.7 and 104.2 eV were assigned to Si-OH and Si-O-Si of SiO₂ (Fig. 3e₃) [27]. It was worth noting that the binding energy of O1s was 533.1 eV (Fig. 3e₄), which was higher than that of Fe₃O₄ (529.6 eV) by 3.5 eV. This was mainly due to the formation of Fe-O-Si chemical bond (530.2 eV) decreased the electronic density of O binding Fe, resulting in the chemical shift of binding energy of O1s. Moreover, the coexistence of Si-O-Si and Fe-O-Si verified the coating of SiO₂ on the nanochains.

The XRD patterns of Fe₃O₄ nanoparticles and Fe₃O₄@SiO₂ nanochains were exhibited in Fig. 3f. There were typical diffraction peaks of (200), (311), (400), (422), (511) and (440) planes, which corresponded to Fe₃O₄ presented in both patterns, confirming that the crystal structure of Fe₃O₄ nanoparticles were completely preserved during the synthesis of Fe₃O₄@SiO₂. Compared with Fe₃O₄, a new broad diffraction at around 20° appeared in Fe₃O₄@SiO₂, which was attributed to the amorphous SiO₂ shell [28]. In the FTIR spectrum (Fig. 3g), the absorption peak at 1076 cm^− 1 in Fe₃O₄@SiO₂ was adscripted to Si-O bond while the peak at 592 cm^− 1 was attributed to Fe-O bond [29–31], which further confirmed that the core-shell structure of Fe₃O₄@SiO₂ nanochains.

Physical and chemical properties

The porous scaffold with honeycomb structure was shown in Fig. 4a. The pore size of the scaffold was 800 ± 50 µm, which was proven to be beneficial to cell adhesion and climbing growth [32]. The magnetic behaviors of the PLLA, PLLA/Fe₃O₄ and PLLA/Fe₃O₄@SiO₂ scaffolds were shown in Figs. 4b and 4c. It was clearly seen that the introduction of Fe₃O₄ and Fe₃O₄@SiO₂ endowed the non-magnetic PLLA scaffold favorable magnetic properties (Figs. 4b). Although the magnetic saturation intensity of the PLLA/Fe₃O₄@SiO₂ scaffold was slightly lower than that of the PLLA/Fe₃O₄ scaffold, the low remanence (Mr) similar to Fe₃O₄ proved that the PLLA/Fe₃O₄@SiO₂ scaffold still possessed good superparamagnetism (Fig. 4c). This was conducive to construct magnetic microenvironment in scaffold, which was expected to enhance cell viability and promote cell growth through magnetic stimulation.

The phase composition of the scaffolds was assessed using XRD (Fig. 4d). It can be clearly observed that the diffraction peaks belonged to (010), (110), (203) and (205) planes of PLLA [33]. By contrast, the new diffraction peaks corresponding to (311), (400), (551) and (440) crystal planes confirmed the spinel structure of Fe₃O₄ in PLLA/Fe₃O₄ and PLLA/Fe₃O₄@SiO₂ scaffolds [34].

Generally, a scaffold with favorable hydrophilicity is more conducive to cell adhesion [35]. The hydrophilicity of PLLA, PLLA/Fe₃O₄ and PLLA/Fe₃O₄@SiO₂ scaffolds was investigated via water contact angle test. As shown in Fig. 4e, the contact angle on the PLLA scaffold was 86.2 ± 2.8°. By contrast, the contact angle decreased after the adding Fe₃O₄ nanoparticles, indicating the improvement of hydrophilicity. This could be attributed to the presence of hydroxyl groups on the Fe₃O₄ nanoparticles in aqueous environment. Moreover, the PLLA/Fe₃O₄@SiO₂ scaffolds exhibited the best hydrophilicity, which was mainly due to the silanol groups of SiO₂ absorbed water molecule via hydrogen bonding.

Cytocompatibility

The cytocompatibility of scaffolds is a necessary and crucial element in the bone repair process because it determines whether cells can adhere, grow, and proliferate on the scaffold [36]. Herein, the cytocompatibility of the PLLA, PLLA/Fe₃O₄ and PLLA/Fe₃O₄@SiO₂ scaffolds were assessed. As shown in Fig. 5a, cells adhered well on all scaffolds, indicating that the PLLA, PLLA/Fe₃O₄ and PLLA/Fe₃O₄@SiO₂ scaffolds had good biocompatibility. Particularly, better cell adhesion morphology displayed on PLLA/Fe₃O₄@SiO₂ scaffold than that on PLLA and PLLA/Fe₃O₄ scaffolds at the same time point. Moreover, the cells completely expanded and essentially presented normal topological configuration on PLLA/Fe₃O₄@SiO₂ scaffold after 7 days of cultivation, indicating that the Fe₃O₄@SiO₂ nanochains in scaffold were more conducive to cell adhesion and expansion.

Cell viability is also an important indicator for evaluating the cytocompatibility of the scaffold [37–39]. To investigate the cells viability induced by the PLLA, PLLA/Fe₃O₄ and PLLA/Fe₃O₄@SiO₂ scaffolds, the cells were strained with calcein AM. Normally, calcein AM only stains living cells, because calcein AM as a dye can be transformed into a membrane impermeable fluorescent analogue by the cell esterases, and the fluorescence will leak out when the cell membrane is completely damaged [40, 41]. As shown in Fig. 5b, the density of living cells in PLLA, PLLA/Fe₃O₄ and PLLA/Fe₃O₄@SiO₂ scaffold groups was significantly enhanced with time, confirming that all scaffolds possessed the ability to enhance cell activity. Notably, the cells increased exponentially from 3 to 7 days with the highest density observing in the cells which cocultured with PLLA/Fe₃O₄@SiO₂ scaffold, indicating that the Fe₃O₄@SiO₂ nanochains in scaffold significantly enhanced cell viability and promoted cell proliferation.

Cell proliferation is one of the important physiological functions of living cells[42]. To quantitatively study the cell proliferation capacity on the PLLA, PLLA/Fe₃O₄ and PLLA/Fe₃O₄@SiO₂ scaffolds, the CCK-8 assay was carried out (Fig. 5c). It could be clearly seen that the optical density (OD) value of cells on all the scaffolds increased significantly with incubating time. Compared with the PLLA scaffold, higher OD value of cells presented on the PLLA/Fe₃O₄ scaffold, indicating that the Fe₃O₄ nanoparticles in the scaffold promoted cell proliferation. Especially, the OD value of cells on PLLA/Fe₃O₄@SiO₂ scaffold was markedly higher than that on PLLA/Fe₃O₄ scaffold, indicating that the Fe₃O₄@SiO₂ nanochains further promoted cell proliferation.

As one of the early indicators of osteogenic differentiation [43], ALP activity of cells cultured on the scaffolds for 7 days was qualitatively analyzed (Fig. 6a). It could be clearly seen that the cells cocultured with the PLLA/Fe₃O₄@SiO₂ scaffold samples exhibited higher ALP activity than PLLA and PLLA/Fe₃O₄ samples. As one of the late markers of osteogenic differentiation [44], the Alizarin Red staining was performed to assess the extracellular matrix mineralization of cells cocultured with the scaffolds for 7 days (Fig. 6b). As expected, there were obvious red precipitates in all scaffold groups. It was worth noting that the mineral deposition was significantly enhanced in PLLA/Fe₃O₄@SiO₂ samples compared to other groups, mainly due to the stronger magnetic stimulation effect of Fe₃O₄@SiO₂ nanochains. The above results demonstrated that the Fe₃O₄@SiO₂ nanochains in scaffold markedly enhanced cell activity and promoted cell proliferation, differentiation, and mineralization.

The bone-related gene expressions including RUNX2, OPN, OCN and OSX on PLLA, PLLA/Fe₃O₄ and PLLA/Fe₃O₄@SiO₂ scaffolds were investigated (Fig. 7). From an overall perspective, the expression levels of RUNX2, OPN, OCN and OSX on day 7 were greatly higher than on day 3. Especially, the expression level of them on the PLLA/Fe₃O₄@SiO₂ scaffold were markedly higher than that on PLLA/Fe₃O₄ and PLLA scaffolds at any time. The results showed that the Fe₃O₄@SiO₂ nanochains provided a more favorable magnetic microenvironment for cell differentiation than Fe₃O₄ nanoparticles in scaffolds, confirming that the superior capability of Fe₃O₄@SiO₂ nanochains to promote cell differentiation.

It is well known that various cells, such as mesenchymal stem cells, osteoblasts, and endothelial cells are magnetically sensitive due to the diamagnetism of cell membranes [45]. Inspired by these, researchers have applied different external magnetic fields to study the roles of magnetic stimulation in bone repair in recent years [46, 47]. It was found that the external magnetic fields could induce a series of cell behaviors by regulating cell surface receptors and signaling pathways via magnetic stimulations, thereby accelerating new bone regeneration or inhibiting osteoclast resorption. However, the need of magnetic field generators limits the clinical application of magnetic stimulation to a certain extent.

To solve the above problem, it would be an effective means to construct an endogenous magnetic microenvironment in bone scaffolds by introducing magnetic materials. As a highly biocompatible and magnetic materials, Fe₃O₄ nanoparticles have received clinical approval from the Food and Drug Administration. The scaffolds loaded with Fe₃O₄ nanoparticles indeed effectively enhanced cell viability and promoted cell proliferation [48, 49]. However, the random arrangement of Fe₃O₄ nanoparticles in the scaffolds greatly compromised their positive magnetic stimulation effects, due to the mutual repulsive between adjacent magnetic dipoles.

In present study, we constructed Fe₃O₄@SiO₂ nanochains with uniform shell-core structure by magnetic-field-guided interface co-assembly of Fe₃O₄ nanoparticles (Fig. 2). The simulation analysis results of magnetic field distribution proved the orderly assembly of Fe₃O₄ nanoparticles in the Fe₃O₄@SiO₂ nanochains formed magnetic energy coupling and obtained a highly magnetic micro-field (Figs. 3a-c). The results are consistent with the analysis of Yin Yadong's team [50–52]. From the results of magnetic tests, the Fe₃O₄@SiO₂ nanochains still preserved the superior superparamagnetism of Fe₃O₄ nanoparticles. The good magnetism and high surface areas endowed Fe₃O₄@SiO₂ nanochains with great potential for use in biomedicine.

To better understand the biological advantages of Fe₃O₄@SiO₂ nanochains in scaffolds, a series of in vitro cell experiments were performed. Compared to PLLA/Fe₃O₄ scaffolds, PLLA/Fe₃O₄@SiO₂ scaffolds are more conducive to cell adhesion and expansion, especially further enhancing cell viability, proliferation, differentiation, mineralization, and bone-related gene expressions (Figs. 5–7). It could be attributed to the stronger magnetic stimulation effect of Fe₃O₄@SiO₂ nanochains. In terms of mechanism, the orderly assembly of Fe₃O₄ nanoparticles obtained magnetic energy coupling, resulting in a highly micro-field that stimulated the surrounding cells to respond (Fig. 8). In this case, the membrane flux of the diamagnetic cell membrane would be modified. Moreover, the strong magnetic singles would activate receptors on the cell membrane, thereby modulating a series of signaling pathways including Ca²⁺ channels, mitogen-activated protein kinase (MAPK), bone morphogenetic protein-2 (BMP-2) and integrins [5, 9, 43, 53]. Then, the corresponding downstream transcription factors were regulated, and consequently osteogenesis-related gene expressions of RUNX2, OPN, OCN and OSX were up-regulated. Hence, the Fe₃O₄@SiO₂ nanochains in scaffold possessed great potential in accelerating bone repair.

A magnetic-field-guided interface co-assembly of Fe₃O₄ nanoparticles had been demonstrated to rationally synthesis unique Fe₃O₄@SiO₂ nanochains. The obtained Fe₃O₄@SiO₂ nanochains exhibited high magnetic susceptibility and excellent magnetic induction intensity. Importantly, the superior magnetic properties of nanochains enhanced the interaction between PLLA/Fe₃O₄@SiO₂ scaffold and cells. As a result, the nanochains in scaffold effectively enhanced cell activity, proliferation, differentiation, and mineralization as well as bone-related gene expressions. These findings confirmed the superparamagnetic scaffold incorporated with Fe₃O₄@SiO₂ nanochains could accelerate the repair of bone defect.

PLLA

Poly l-lactic acid

SiO₂

Silica

SLS

Selective laser sintering

TEOS

Tetraothorsilicate

TEM

Transmission electron microscope

XPS

X-ray photoelectron spectrometer

FTIR

Fourier transform infra-red spectrometer

XRD

X-ray diffractometer

VSM

Vibrating sample magnetometer

DMEM

Dulbecco's Modified Eagle's Medium

SEM

Scanning electron microscope

CCK-8

Cell Counting Kit-8

ALP

Alkaline phosphatase

Runx2

Runt-related transcription factor-2

OPN

Osteopontin

OCN

Osteocalcin

OSX

Osterix.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by the following funds: (1) The Natural Science Foundation of China (52105352, 51935014, 52165043, 82072084, 81871498); (2) The Project of Jiangxi University of Science and Technology (205200100535); (3) JiangXi Provincial Natural Science Foundation of China (2020ACB214004, 20202BAB214011); (4) The Provincial Key R&D Projects of Jiangxi (20201BBE51012); (5) The Project of State Key Laboratory of High Performance Complex Manufacturing.

Authors’ contributions

Shuai CJ and Chen X contributed to acquisition, analysis of data, drafted and revised the manuscript. He CX, Qian GW and Shuai Y contributed to analysis and interpretation of the data. Yao J, Xu WD and Peng SP contributed to performing the experiments. Deng YW and Yang WJ conceptualized the experimental research, acquired the financial support/experimental resources, supervised the research activities, validated the data and reviewed the paper. All authors read and approved the final manuscript.

Acknowledgments

Not applicable.

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You are reading this latest preprint version

Construction of Magnetic Nanochains to Achieve Magnetic Energy Coupling in Scaffold

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Version 1

Abstract

Figures

Introduction

Experimental Sections

Materials

Synthesis of one-dimensional Fe₃O₄@SiO₂ nanochains

Preparation of scaffolds

Measurements and characterization

Cytocompatibility

Statistical analysis

Results

Fe₃O₄@SiO₂ nanochains

Physical and chemical properties

Cytocompatibility

Discussion

Conclusions

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors’ contributions

Acknowledgments

References

Status:

Version 1

Construction of Magnetic Nanochains to Achieve Magnetic Energy Coupling in Scaffold

Status:

Version 1

Abstract

Figures

Introduction

Experimental Sections

Materials

Synthesis of one-dimensional Fe3O4@SiO2 nanochains

Preparation of scaffolds

Measurements and characterization

Cytocompatibility

Statistical analysis

Results

Fe3O4@SiO2 nanochains

Physical and chemical properties

Cytocompatibility

Discussion

Conclusions

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors’ contributions

Acknowledgments

References

Status:

Version 1

Synthesis of one-dimensional Fe₃O₄@SiO₂ nanochains

Fe₃O₄@SiO₂ nanochains