Introduction

Recent scaffolds lack the capacity to effectively modulate cell growth or tissue reconstruction, resulting in slow bone regeneration and even failure of bone implantation [1,2,3,4]. It is well known that cells are magnetically sensitive due to the diamagnetism of cell membranes [5], and exposure to magnetic fields served to alter membrane flux and regulate ion channels and biochemical pathways [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.

Fig. 2
figure 2

a-c TEM images, (d) the inverse-FFT image, (e) SAED patterns and (f-i) element map** images of Fe3O4@SiO2 nanochains

Full size image

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 Fig. 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 centerlines of two adjacent nanoparticles were aligned with the direction of the external magnetic field, the dipole–dipole interaction was attractive (Fig. 3b). In the case of the interaction energy was large enough to overcome thermal fluctuations, the magnetic dipole–dipole force drove the self-assembly of nanoparticles into nanochain along the dipole moment (Fig. 3c). In this condition, the dipole–dipole coupling reached the maximum, and the magnetism of nanochain could be regarded as the magnetic energy coupling among multiple nanoparticles. The magnetic strength of nanochain was higher than that of randomly arranged Fe3O4 nanoparticles.

Fig. 3
figure 3

a Magnetic field distribution around a Fe3O4 nanoparticle. The attractive (b) dipole–dipole forces between two adjacent nanoparticles drive the formation of nanochains along the magnetic field (c). d Magnetization curves, (e) XPS spectra of Fe3O4@SiO2 nanochains. (e1) XPS survey spectrum along with the spectra of (e2) Fe2p, (e3) Si2p and (e4) O1s. (f) XRD patterns, (g) FTIR spectra

Full size image

The magnetic properties of Fe3O4 nanoparticles and Fe3O4@SiO2 nanochains were detected and presented in Fig. 3d. It could be seen that both nanoparticles and nanochains exhibited superior magnetism, which was an important advantage for their applicability in biomedicine [22, 23]. 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. This phenomenon was also discovered by M. Tarhini and A. Bitar et al. [24, 25].

XPS spectra of Fe3O4@SiO2 nanochains were presented in Fig. 3e. The typical Fe2p, O1s and Si2p peaks were clearly observed (Fig. 3 e1). In detail, the peaks centered at 711.4, 723.0 and 726.0 eV were respectively corresponded to Fe2+2p3/2, Fe2+2p1/2 and Fe3+2p1/2 of Fe3O4 (Fig. 3 e2), while the peaks centered at 102.7 and 104.2 eV were assigned to Si–OH and Si–O-Si of SiO2 (Fig. 3 e3) [26]. It was worth noting that the binding energy of O1s was 533.1 eV (Fig. 3 e4), 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 [27]. 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 [28,29,30], 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 [31, 32]. The phase composition of the scaffolds was assessed using XRD (Fig. 4b). 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].

Fig. 4
figure 4

a Optical graphs of SLS prepared honeycomb scaffold. b XRD patterns. c Magnetization curves. d Magnetization behaviors at magnetic field from -90 to 90 Oe. e Compressive strength and compressive modulus (n = 5, *p < 0.05), (f) tensile stress–strain curves, (g) TGA curves and (h) hydrophilicity of PLLA, PLLA/Fe3O4 and PLLA/Fe3O4@SiO2 scaffolds

Full size image

The magnetic behaviors of the PLLA, PLLA/Fe3O4 and PLLA/Fe3O4@SiO2 scaffolds were shown in Fig. 4c and d. It was clearly seen that the introduction of Fe3O4 and Fe3O4@SiO2 endowed the non-magnetic PLLA scaffold favorable magnetic properties. This was conducive to construct magnetic microenvironment in scaffold, which was expected to enhance cell viability and promote cell growth through magnetic stimulation.

Mechanical properties of the scaffolds were evaluated via compressive and tensile tests, with the results shown in Fig. 4e and f. The compressive strength and modulus of PLLA scaffolds were only 22.3 ± 0.9 MPa and 78.4 ± 6.2 MPa. Encouragingly, the compressive strength and modulus of the PLLA/Fe3O4@SiO2 scaffolds were 41.8 ± 1.6 MPa and 142.6 ± 8.5 MPa, which were increased by 87.4% and 80.6% compared with PLLA scaffolds. Moreover, the PLLA/Fe3O4@SiO2 scaffolds also exhibited much higher the tensile strength and strain than those of PLLA and PLLA/Fe3O4 scaffolds, which were 13.34 ± 1.2 MPa and 2.13%, respectively. The high mechanical properties of PLLA/Fe3O4@SiO2 scaffolds were attributed to that the Fe3O4@SiO2 nanochains acted as rigid reinforcement to enhance the stress transfer efficiency in the matrix.

The TGA measurement were performed to analyze the thermal decomposition and the corresponding residual weight of scaffolds (Fig. 4g). The slight weight loss below 300 °C of the scaffolds was related to the evaporation of adsorbed water molecules. Obviously, the thermal decomposition temperature of PLLA scaffolds was about 300 ~ 400 °C, while the range of decomposition temperature leftward shifted and narrowed after adding Fe3O4 and Fe3O4@SiO2. This confirmed that the addition of Fe3O4 and Fe3O4@SiO2 catalyzed the thermal decomposition of PLLA. Additionally, the residual weight of the PLLA/Fe3O4 and PLLA/Fe3O4@SiO2 scaffolds was 7.4 wt.% and 7.6 wt.%, respectively, which was close to the nominal content (8 wt.%) of Fe3O4 and Fe3O4@SiO2 introduced into PLLA matrix.

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. 4h, 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, 37]. 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.

Fig. 5
figure 5

a Adhesion morphologies of MG-63 cells on PLLA, PLLA/Fe3O4 and PLLA/Fe3O4@SiO2 scaffolds. b Fluorescence images of cells cultured after 3 and 7 days on the scaffolds. c Cell proliferation on the scaffolds (n = 5, *p < 0.05, **p < 0.01)

Full size image

Cell viability is also an important indicator for evaluating the cytocompatibility of the scaffold [38]. 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 [39, 40]. 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. 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 [41], 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 [42], 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.

Fig. 6
figure 6

a The ALP and (b) Alizarin Red staining of cells on PLLA, PLLA/Fe3O4 and PLLA/Fe3O4@SiO2 scaffolds after 7 days of cultivation

Full size image

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.

Fig. 7
figure 7

The expressions of (a) RUNX2, (b) OPN, (c) OCN and (d) OSX on the scaffolds after 3 and 7 days of culture (n = 5, *p < 0.05, **p < 0.01)

Full size image

Discussion

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 [43]. Inspired by these, researchers have applied different external magnetic fields to study the roles of magnetic stimulation in bone repair in recent years [44, 45]. 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 [46, 47]. 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 (Fig. 3a-c). The results are consistent with the analysis of Yin Yadong's team [48,49,50]. 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, 6 and 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 [6, 10, 41, 51]. 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.

Fig. 8
figure 8

Schematic illustration of Fe3O4@SiO2 magnetic nanochains in up-regulating osteogenesis-related gene expressions

Full size image

Conclusions

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.