Micro Magnetic Field Produced by Fe3O4 Nanoparticles in Bone Scaffold for Enhancing Cellular Activity

The low cellular activity of poly-l-lactic acid (PLLA) limits its application in bone scaffold, although PLLA has advantages in terms of good biocompatibility and easy processing. In this study, superparamagnetic Fe3O4 nanoparticles were incorporated into the PLLA bone scaffold prepared by selective laser sintering (SLS) for continuously and steadily enhancing cellular activity. In the scaffold, each Fe3O4 nanoparticle was a single magnetic domain without a domain wall, providing a micro-magnetic source to generate a tiny magnetic field, thereby continuously and steadily generating magnetic stimulation to cells. The results showed that the magnetic scaffold exhibited superparamagnetism and its saturation magnetization reached a maximum value of 6.1 emu/g. It promoted the attachment, diffusion, and interaction of MG63 cells, and increased the activity of alkaline phosphatase, thus promoting the cell proliferation and differentiation. Meanwhile, the scaffold with 7% Fe3O4 presented increased compressive strength, modulus, and Vickers hardness by 63.4%, 78.9%, and 19.1% compared with the PLLA scaffold, respectively, due to the addition of Fe3O4 nanoparticles, which act as a nanoscale reinforcement in the polymer matrix. All these positive results suggested that the PLLA/Fe3O4 scaffold with good magnetic properties is of great potential for bone tissue engineering applications.


Introduction
Poly-l-lactic acid (PLLA) has become one of the main bone scaffold materials due to its advantages of good biocompatibility and easy processing [1][2][3]. Nevertheless, the low cellular activity limits its application in bone tissue engineering due to the lacking of active functional groups and weak cell affinity [4][5][6][7]. For enhancing cellular activity, researchers have added various cell growth factors, such as bone morphogenetic protein (BMP), transforming growth factor-beta (TGF-β), fibroblast growth factor, and so on [8][9][10][11]. Schofer et al. [8] incorporated BMP-2 into PLLA nanofibers and found that the BMP-2 improved the scaffold's cellular activity by increasing the expression of osteogenic marker proteins and osteogenesis. Zhu et al. [9] added TGF-β1 to nano-HA/PLLA composite scaffold and found that TGF-β1 released and promoted the adhesion, spreading, proliferation of mesenchymal stem cells (MSCs). Although the growth factors can improve cellular activity, they are very expensive and have a decay half-life [12][13][14]. The fast decay rate makes its biological activity decrease quickly and it cannot be continuously and steadily enhanced, which has greatly limited their wide range of clinical applications. was poured into a ball mill for 1 h for further dispersing. Finally, PLLA/Fe3O4 composite powders were obtained by filtering the mixed solution slowly using a filter paper with pore size of 0.45 µm (Millipore, HAWP01300) and drying in a drying box for 24 h at 50 °C. The magnetic composite scaffold was prepared by a SLS system with a 100 W CO2 laser (λ = 10.6 µm) and a galvanometric scanning system. In detail, the powder feeding cylinder piston rises, and then the powder spreading roller evenly lays a layer of powder on the sintering platform. Then under the control of the galvanometric scanning system, the powder layer was scanned and sintered by a laser beam followed the cross-sectional profiles of the model [32]. After sintering a layer, the piston of the forming cylinder was lowered by one layer thickness. Then, the powder spreading roller was controlled to lay a new layer of powder above the previously sintered layer, followed by the next sintering of the powder. The above operation was repeated in this way, and the sintered layers were stacked layer by layer until the whole scaffold was formed. The main process parameters were optimized as follows: scanning speed of 180 mm s −1 , scanning interval of 0.15 mm, and layer thickness of 0.1 mm. Six kinds of PLLA/Fe3O4 scaffolds with different contents of Fe3O4 (0, 1, 3, 5, 7, and 9 wt %) were fabricated by SLS, as shown in Figure 1. The optical color of the scaffolds gradually deepens with the increase of Fe3O4 content.

Characterization
The phase constituent of Fe3O4 nanoparticles and magnetic scaffolds was investigated via XRD (DMAX 2500, Japan Science Co., Tokyo, Japan) at a scan rate of 8°/min in the range of diffraction angle 2θ = 10° ~ 80°. The chemical group analysis was performed by FTIR (Nicolet 6700, Thermo Electron Scientific Instruments Co., Madison, WI, USA) with a test wavelength of 500 to 4000 cm −1 and a number of scans of 16 times. The TGA and DSC curves of the magnetic scaffolds at 30 to 600 °C were measured to evaluate the thermal stability, using a thermo gravimetric analyzer (TGA-105, Nanjing Dazhan Electromechanical Technology Research Institute, Nanjing, China) The magnetic composite scaffold was prepared by a SLS system with a 100 W CO 2 laser (λ = 10.6 µm) and a galvanometric scanning system. In detail, the powder feeding cylinder piston rises, and then the powder spreading roller evenly lays a layer of powder on the sintering platform. Then under the control of the galvanometric scanning system, the powder layer was scanned and sintered by a laser beam followed the cross-sectional profiles of the model [32]. After sintering a layer, the piston of the forming cylinder was lowered by one layer thickness. Then, the powder spreading roller was controlled to lay a new layer of powder above the previously sintered layer, followed by the next sintering of the powder. The above operation was repeated in this way, and the sintered layers were stacked layer by layer until the whole scaffold was formed. The main process parameters were optimized as follows: scanning speed of 180 mm s −1 , scanning interval of 0.15 mm, and layer thickness of 0.1 mm. Six kinds of PLLA/Fe 3 O 4 scaffolds with different contents of Fe 3 O 4 (0, 1, 3, 5, 7, and 9 wt %) were fabricated by SLS, as shown in Figure 1. The optical color of the scaffolds gradually deepens with the increase of Fe 3 O 4 content.

Characterization
The phase constituent of Fe 3 O 4 nanoparticles and magnetic scaffolds was investigated via XRD (DMAX 2500, Japan Science Co., Tokyo, Japan) at a scan rate of 8 • /min in the range of diffraction angle 2θ = 10 •~8 0 • . The chemical group analysis was performed by FTIR (Nicolet 6700, Thermo Electron Scientific Instruments Co., Madison, WI, USA) with a test wavelength of 500 to 4000 cm −1 and a number of scans of 16 times. The TGA and DSC curves of the magnetic scaffolds at 30 to 600 • C were measured to evaluate the thermal stability, using a thermo gravimetric analyzer (TGA-105, Nanjing Dazhan Electromechanical Technology Research Institute, Nanjing, China) under nitrogen at a temperature rise rate of 20 • C/min. Magnetic properties of the magnetic composite scaffolds were detected by a vibrating sample magnetometer (VSM7407, Lake Shore Cryotronics Inc., Westerville, OH, USA) and a permanent magnet. The hysteresis loop was measured in an applied magnetic field of ±20 kOe and the saturation magnetization was evaluated.
The compressive strength and modulus were evaluated using a universal testing machine (WD-D1, Shanghai Zhuoji Instrument Co., Ltd., Shanghai, China). The force-displacement curve was recorded automatically by a flat indenter with a slow loading speed of 0.5 mm/min. The compression strength and elastic modulus of the sample was calculated from the compression stress-strain curve. The hardness of composite scaffolds was assessed by a digital micro Vickers hardness tester (Micro Vickers Hardness Tester, HVS-1000C Shenzhen Shunhua Instrument Equipment Co., Ltd., Shenzhen, China) using an indentation test after polished. The Vickers hardness was calculated by the equation [33]: where F is the test force (N) and d is the diagonal length (mm). Each set of data was averaged and standard deviation from five replicate samples. The microscopic morphology of the surface pores and sections of the scaffold were characterized by scanning electron microscopy (SEM, Phenom ProX, Phenom-World BV, Eindhoven, Netherlands).

Cellular Compatibility
MG63 cells were cultured to evaluate the cell compatibility of scaffolds owning to their similar matrix synthesis and mineralization capabilities to osteoblasts. The cells were cultured in DMEM supplemented with sodium pyruvate, 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 • C in a humidified 5% CO 2 atmosphere. The magnetic scaffold was sterilized with an ultraviolet lamp for 2 h and then placed in a 24 well culture plate for evaluation of cell adhesion and proliferation. MG63 cells were seeded at a density of 4 × 10 5 cells per well and the cultured medium was changed daily. After 1, 3, and 7 days of culture, the cell-scaffold samples were taken out, rinsed with PBS, immobilized using 4% glutaraldehyde for 30 min, dehydrated with ethanol for 24 h, and completely dried. After being sputtered with gold, the morphology of the cells on the scaffolds was observed by SEM. At each evaluation period, after the cells were stained with 2 µM calcein acetoxymethyl ester for 30 min, the fluorescence microscope equipped with a digital camera was used for observation and analysis.
The CCK-8 method was used to evaluate the proliferation of cells planted on the scaffolds. After 1.0 × 10 4 MG63 cells were planted on the scaffold and cultured for different days, 40 µl of CCK-8 solution was added to each well and incubated for 4 h, and the absorbance at 450 nm was measured by a microplate reader. The biological activity of the magnetic scaffolds was evaluated by evaluating the degree of differentiation of the cells by detecting the activity of alkaline phosphatase in the medium solution of the scaffold and osteoblasts. After the induction of MG63 cells for 3, 5, and 7 days, the scaffolds were taken out, washed with PBS. The cells were separated by 0.25% trypsin, transferred to a new 24 well plate medium, and washed three times with PBS. After fixing with formalin for 30 s and washing twice with deionized water, they were stained with ALP reagent for 1 h, and finally photographed by a microscope (TE2000U, Nikon Co., Tokyo, Japan).

Statistical Analysis
The quantitative data were expressed as mean ± standard error. The statistical difference was analyzed using student's t-test and p < 0.05 was considered as the level of significance, which is expressed as *.

Physicochemical Properties and Thermal Properties
The phase composition of the scaffold was analyzed using XRD ( Figure 2a). PLLA shows two broad diffraction peaks at 16.5 • and 19.9 • , indicating that it was a semi-crystalline structure. were detected in the PLLA/Fe 3 O 4 composite scaffold, and their intensity increased with the increase of its content, which confirmed that Fe 3 O 4 was successfully introduced into the scaffold. Compared with the pure PLLA scaffold, the peak of PLLA in the composite scaffold was significantly weakened or even disappeared. This may be because the diffraction peak of Fe 3 O 4 was too strong and the relative peak intensity of PLLA was weakened. In addition, the positions of the diffraction peaks of PLLA and Fe 3 O 4 in the composite scaffold did not change, and no other peaks were observed, indicating that SLS preparation did not cause the formation of new phases or phase transformations.
Polymers 2020, 12, x FOR PEER REVIEW 5 of 15 planes (220), (311), (400), (422), (511), and (440) [34]. These characteristic peaks of Fe3O4 were detected in the PLLA/Fe3O4 composite scaffold, and their intensity increased with the increase of its content, which confirmed that Fe3O4 was successfully introduced into the scaffold. Compared with the pure PLLA scaffold, the peak of PLLA in the composite scaffold was significantly weakened or even disappeared. This may be because the diffraction peak of Fe3O4 was too strong and the relative peak intensity of PLLA was weakened. In addition, the positions of the diffraction peaks of PLLA and Fe3O4 in the composite scaffold did not change, and no other peaks were observed, indicating that SLS preparation did not cause the formation of new phases or phase transformations. The chemical functional groups of the scaffold were analyzed using FTIR ( Figure 2b). PLLA has characteristic absorption peaks at 3000, 1758, and 1500-1000 cm −1 , which correspond to the stretching vibration peaks of alkyl, carbonyl, and ether groups, respectively [35]. Fe3O4 has a characteristic absorption peak at 585 cm −1 , which corresponds to the stretching vibration peak of Fe-O [36]. This characteristic peak was also detected in the PLLA/7%Fe3O4 magnetic stent, which confirmed the successful introduction of Fe3O4 again. At the same time, several characteristic peaks of PLLA were clearly detected in the composite scaffold, which confirmed the existence of PLLA and made up for the results of XRD. The thermal stability of the composite scaffold was analyzed using DSC-TGA (Figure 2c,d). The magnetic scaffold exhibits significant thermal weight loss at 335 ~ 425 °C (Figure 2c), which was due to the thermal decomposition of PLLA [37]. After 425 °C, the residual weight of the scaffold hardly changed, which was due to the residual Fe3O4 with high thermal stability (melting point 1594.5 °C). The residual weight was about 0%, 1.2%, 3.4%, 5.2%, 7.6%, and 9.5%, respectively for PLLA/Fe3O4 scaffold with 0%, 1%, 3%, 5%, 7%, and 9% content, which was closed to the initial amount of Fe3O4 added. In the DSC curve, PLLA showed two endothermic peaks at 185.1 and 381.5 °C, which correspond to its melting temperature and decomposition temperature, respectively [30,38]. After Fe3O4 was added, the peak position at The chemical functional groups of the scaffold were analyzed using FTIR (Figure 2b). PLLA has characteristic absorption peaks at 3000, 1758, and 1500-1000 cm −1 , which correspond to the stretching vibration peaks of alkyl, carbonyl, and ether groups, respectively [35]. Fe 3 O 4 has a characteristic absorption peak at 585 cm −1 , which corresponds to the stretching vibration peak of Fe-O [36]. This characteristic peak was also detected in the PLLA/7%Fe 3 O 4 magnetic stent, which confirmed the successful introduction of Fe 3 O 4 again. At the same time, several characteristic peaks of PLLA were clearly detected in the composite scaffold, which confirmed the existence of PLLA and made up for the results of XRD. The thermal stability of the composite scaffold was analyzed using DSC-TGA (Figure 2c,d). The magnetic scaffold exhibits significant thermal weight loss at 335~425 • C (Figure 2c), which was due to the thermal decomposition of PLLA [37]. After 425 • C, the residual weight of the scaffold hardly changed, which was due to the residual Fe 3 O 4 with high thermal stability (melting point 1594.5 • C). The residual weight was about 0%, 1.2%, 3.4%, 5.2%, 7.6%, and 9.5%, respectively for PLLA/Fe 3 O 4 scaffold with 0%, 1%, 3%, 5%, 7%, and 9% content, which was closed to the initial amount of Fe 3 O 4 added. In the DSC curve, PLLA showed two endothermic peaks at 185.1 and 381.5 • C, which correspond to its melting temperature and decomposition temperature, respectively [30,38].
After Fe 3 O 4 was added, the peak position at 185.1 • C did not change, indicating that the melting point of the scaffold did not change, but the position of the peak at 381.5 • C shifted slightly to the left, which means the thermal decomposition point decreased slightly. This may be due to the addition of Fe 3 O 4 nanoparticles acting as a catalyst to accelerate the thermal decomposition of PLLA [39].

Magnetic Properties
The magnetic properties of the composite scaffolds at room temperature were qualitatively and quantitatively evaluated using permanent magnets and Vibrating Sample Magnetometer ( Figure 3). As can be seen from the illustration in the upper left corner, the PLLA/7%Fe 3 O 4 composite scaffold was firmly attracted by the permanent magnet from different sides, showing good magnetic properties. In the applied positive and negative magnetic fields (−20 kOe to +20 kOe), the magnetization curves of the scaffolds passed through the origin and were symmetrical at the origin without magnetization hysteresis, indicating that the scaffolds had good superparamagnetism [28]. The Ms is an extremely important parameter for magnetic performance evaluation, which refers to the maximum magnetization that can be achieved in a magnetic field [22,40]. The Ms of the composite scaffolds calculated from the magnetization curves are shown in the lower right corner of Figure 3. The value of Ms was positively related to the content of Fe 3 O 4 . In detail, the Ms of the 1%, 3%, 5%, 7%, and 9% Fe 3 O 4 composite scaffolds were 1.1, 1.8, 2.5, 4.0, and 6.1 emu/g. These showed that the composite scaffold had strong magnetism.
Polymers 2020, 12, x FOR PEER REVIEW 6 of 15 185.1 °C did not change, indicating that the melting point of the scaffold did not change, but the position of the peak at 381.5 °C shifted slightly to the left, which means the thermal decomposition point decreased slightly. This may be due to the addition of Fe3O4 nanoparticles acting as a catalyst to accelerate the thermal decomposition of PLLA [39].

Magnetic Properties
The magnetic properties of the composite scaffolds at room temperature were qualitatively and quantitatively evaluated using permanent magnets and Vibrating Sample Magnetometer ( Figure 3). As can be seen from the illustration in the upper left corner, the PLLA/7%Fe3O4 composite scaffold was firmly attracted by the permanent magnet from different sides, showing good magnetic properties. In the applied positive and negative magnetic fields (−20 kOe to +20 kOe), the magnetization curves of the scaffolds passed through the origin and were symmetrical at the origin without magnetization hysteresis, indicating that the scaffolds had good superparamagnetism [28]. The Ms is an extremely important parameter for magnetic performance evaluation, which refers to the maximum magnetization that can be achieved in a magnetic field [22,40]. The Ms of the composite scaffolds calculated from the magnetization curves are shown in the lower right corner of Figure 3. The value of Ms was positively related to the content of Fe3O4. In detail, the Ms of the 1%, 3%, 5%, 7%, and 9% Fe3O4 composite scaffolds were 1.1, 1.8, 2.5, 4.0, and 6.1 emu/g. These showed that the composite scaffold had strong magnetism.

Mechanical Properties
Mechanical properties were of great importance for use as a bone scaffold because they provided mechanical support in bone repair. The stress-strain curves after the compression test of the scaffolds are shown in Figure 4a. The stress of all the scaffolds increases almost linearly with the strain at the initial stage, and then continues to increase to the maximum peak and then appears an inflection point. The peak was defined as the intensity, and the slope of the initial linear phase was defined as the strength. Then the compressive strength ( Figure 4b) and compressive modulus

Mechanical Properties
Mechanical properties were of great importance for use as a bone scaffold because they provided mechanical support in bone repair. The stress-strain curves after the compression test of the scaffolds are shown in Figure 4a. The stress of all the scaffolds increases almost linearly with the strain at the initial stage, and then continues to increase to the maximum peak and then appears an inflection point. The peak was defined as the intensity, and the slope of the initial linear phase was defined as the strength. Then the compressive strength ( Figure 4b) and compressive modulus (Figure 4c) were calculated. The compressive strength and modulus in pure PLLA were 17.8 MPa and 1.6 GPa, respectively. After Fe 3 O 4 was added, the compressive strength of the scaffold was improved. When the content of Fe 3 O 4 was not more than 7%, the compressive strength increased with the content, and reached the maximum at 7%, which were 29.1 MPa and 2.9 GPa, increased by 63.4% and 78.9%, respectively. Then, when the Fe 3 O 4 content was further increased to 9%, the compressive strength and modulus decreased compared to 7%, to 26.4 MPa and 2.7 GPa, but were still higher than pure PLLA. The change trend of the Vickers hardness of the scaffolds as a function of with the content of Fe 3 O 4 was similar to the compression properties, and it reached the optimal value at 7%, which was 67.7 HV (Figure 4d).
Polymers 2020, 12, x FOR PEER REVIEW 7 of 15 ( Figure 4c) were calculated. The compressive strength and modulus in pure PLLA were 17.8 MPa and 1.6 GPa, respectively. After Fe3O4 was added, the compressive strength of the scaffold was improved. When the content of Fe3O4 was not more than 7%, the compressive strength increased with the content, and reached the maximum at 7%, which were 29.1 MPa and 2.9 GPa, increased by 63.4% and 78.9%, respectively. Then, when the Fe3O4 content was further increased to 9%, the compressive strength and modulus decreased compared to 7%, to 26.4 MPa and 2.7 GPa, but were still higher than pure PLLA. The change trend of the Vickers hardness of the scaffolds as a function of with the content of Fe3O4 was similar to the compression properties, and it reached the optimal value at 7%, which was 67.7 HV (Figure 4d). The distribution of nano reinforcing phases was closely related to the mechanical properties of polymer nanocomposites. Therefore, the dispersion of Fe3O4 in the PLLA matrix was analyzed using SEM ( Figure 5). The fracture surface of pure PLLA (Figure 5a) was relatively clean and smooth. After adding Fe3O4, the fracture surface became rough. Fe3O4 particles were randomly dispersed in the PLLA matrix. When the amount of Fe3O4 added was no more than 7% (Figure 5e), the number of Fe3O4 particles on the cross-section increased with the increase of the content of Fe3O4 added, and a good dispersion was maintained. However, when the Fe3O4 content was further increased to 9% (Figure 5f), obvious agglomeration began to appear in the scaffold. The distribution of nano reinforcing phases was closely related to the mechanical properties of polymer nanocomposites. Therefore, the dispersion of Fe 3 O 4 in the PLLA matrix was analyzed using SEM ( Figure 5). The fracture surface of pure PLLA (Figure 5a) was relatively clean and smooth. After adding Fe 3 O 4 , the fracture surface became rough. Fe 3 O 4 particles were randomly dispersed in the PLLA matrix. When the amount of Fe 3 O 4 added was no more than 7% (Figure 5e), the number of Fe 3 O 4 particles on the cross-section increased with the increase of the content of Fe 3 O 4 added, and a good dispersion was maintained. However, when the Fe 3 O 4 content was further increased to 9% (Figure 5f), obvious agglomeration began to appear in the scaffold. Usually, on the premise of uniform dispersion, the more the amount of nanoparticles added, the better the enhancing effect [41]. When the Fe3O4 content was less than or equal to 7%, the uniformly dispersed Fe3O4 nanoparticles acted as a nanoscale reinforcement in the polymer matrix and reached a peak at 7%. However, when the content of Fe3O4 was continuously added to 9%, the excess Fe3O4 was difficult to uniformly disperse in the matrix, forming more agglomerates, which reduced the enhancement efficiency [42]. Therefore, the mechanical properties of the PLLA/9%Fe3O4 scaffold no longer continue to increase compared with 7% Fe3O4.

Cell Responses
Biocompatibility is very critical for the application of bone scaffolds [43,44]. Based on the previous experimental results, the PLLA/7%Fe3O4 scaffold with the best comprehensive performance was selected for further culture experiments. The adhesion and morphology of MG63 cells on the scaffolds were characterized by SEM observation (Figure 6). After MG63 cells were cultured on the scaffolds for 1 day, they were spindle-shaped or ellipsoidal. After three days of incubation, the cells expanded on the scaffolds, and obvious filamentous pseudopodia appeared, which helped the cells to adhere tightly to the scaffold and continue to grow. After seven days, the number of cells increased. The cells completely spread out on the surface of the scaffold, and there was a fusion between the cells. On the PLLA/7%Fe3O4 scaffold, it can be seen that they have been connected to form a fusion layer (Figure 6f). More importantly, MG63 cells exhibited better adhesion morphology and proliferation levels on PLLA/7%Fe3O4 scaffolds than pure PLLA scaffolds at the same culture time. These showed that the magnetic scaffold had good cell compatibility. Usually, on the premise of uniform dispersion, the more the amount of nanoparticles added, the better the enhancing effect [41]. When the Fe 3 O 4 content was less than or equal to 7%, the uniformly dispersed Fe 3 O 4 nanoparticles acted as a nanoscale reinforcement in the polymer matrix and reached a peak at 7%. However, when the content of Fe 3 O 4 was continuously added to 9%, the excess Fe 3 O 4 was difficult to uniformly disperse in the matrix, forming more agglomerates, which reduced the enhancement efficiency [42]. Therefore, the mechanical properties of the PLLA/9%Fe 3 O 4 scaffold no longer continue to increase compared with 7% Fe 3 O 4 .

Cell Responses
Biocompatibility is very critical for the application of bone scaffolds [43,44]. Based on the previous experimental results, the PLLA/7%Fe 3 O 4 scaffold with the best comprehensive performance was selected for further culture experiments. The adhesion and morphology of MG63 cells on the scaffolds were characterized by SEM observation (Figure 6). After MG63 cells were cultured on the scaffolds for 1 day, they were spindle-shaped or ellipsoidal. After three days of incubation, the cells expanded on the scaffolds, and obvious filamentous pseudopodia appeared, which helped the cells to adhere tightly to the scaffold and continue to grow. After seven days, the number of cells increased. The cells completely spread out on the surface of the scaffold, and there was a fusion between the cells. On the PLLA/7%Fe 3 O 4 scaffold, it can be seen that they have been connected to form a fusion layer (Figure 6f). More importantly, MG63 cells exhibited better adhesion morphology and proliferation levels on PLLA/7%Fe 3 O 4 scaffolds than pure PLLA scaffolds at the same culture time. These showed that the magnetic scaffold had good cell compatibility. The behavior of MG63 cells on magnetic bone scaffolds was further studied using fluorescent staining. The results were shown in Figure 7. Obviously, compared with pure PLLA scaffold, there were more green fluorescent cells on magnetic bone scaffolds, and it was positively correlated with the culture time. Taking the number of cells on pure PLLA scaffolds after one day of culture as a 100% comparison, the statistical results are shown in Figure 7B. In order to further study the effect of magnetic scaffolds on the proliferation of MG63 cells, the CCK-8 test was used to evaluate the proliferation capacity of the scaffolds, and the results are shown in Figure 7C. After cell culture for three and seven days, the absorbance value (representing more living cells) on the PLLA/7%Fe3O4 scaffold was significantly higher than that of the pure PLLA scaffold (p < 0.05) indicating that the cell proliferation was enhanced [31]. It was shown that the Fe3O4 nanoparticles in the scaffolds could obviously promote the proliferation of MG63 cells. The behavior of MG63 cells on magnetic bone scaffolds was further studied using fluorescent staining. The results were shown in Figure 7. Obviously, compared with pure PLLA scaffold, there were more green fluorescent cells on magnetic bone scaffolds, and it was positively correlated with the culture time. Taking the number of cells on pure PLLA scaffolds after one day of culture as a 100% comparison, the statistical results are shown in Figure 7B. In order to further study the effect of magnetic scaffolds on the proliferation of MG63 cells, the CCK-8 test was used to evaluate the proliferation capacity of the scaffolds, and the results are shown in Figure 7C. After cell culture for three and seven days, the absorbance value (representing more living cells) on the PLLA/7%Fe 3 O 4 scaffold was significantly higher than that of the pure PLLA scaffold (p < 0.05) indicating that the cell proliferation was enhanced [31]. It was shown that the Fe 3 O 4 nanoparticles in the scaffolds could obviously promote the proliferation of MG63 cells.
ALP is a critical marker for the early differentiation of osteoblasts. Its activity was used to assess the level of differentiation of MG63 cells cultured on magnetic scaffolds ( Figure 8). It can be seen from the stained image that the ALP activity increased with increasing culture time. The ALP activity on the magnetic scaffold was higher than the ALP activity on the pure PLLA scaffold, indicating that osteogenic differentiation of MG63 cells was significantly up-regulated, which indicated that the magnetic scaffold had the ability to stimulate MG63 cell differentiation ability.
It is well known that the components contained in the scaffold have a significant effect on the cellular response. Among them, Fe 3 O 4 nanoparticles have strong magnetic features and unique superparamagnetic properties in nanometric dimensions. Meanwhile, the structure of cell membrane is complex. It not only contains charged lipid molecules, water, and protein, but also contains many ion channels such as K + , Na + , Ca 2+ and so on. There is also a large amount of Cl − , K + , Na + , and other anions and cations on the inner and outer surfaces of the membrane [45][46][47]. Therefore, Fe 3 O 4 nanoparticles can serve as a magnetic source for a single magnetic nanofield in a weak electromagnetic field formed by the cell due to the difference between internal and external ions and charges, thereby generating a biological effect of magnetic field on cells [48][49][50]. Maleki-Ghaleh. H et al. reported that in this weak electromagnetic field of cells, Fe 3 O 4 magnetic materials improved cell growth and activity by generating magnetic fields to enhance cell communication [24]. When it was combined with the matrix, a large number of tiny magnetic fields were generated on the pores or the surface of the scaffold. According to previous studies, magnetic fields may affect the nucleation of protein crystals in the culture medium and the distribution of proteins in the cell membrane, accelerating the specific recognition of integrin proteins on the cell surface and adsorbing to the extracellular matrix proteins on the surface of the scaffold, thereby promoting cell adhesion and spread [29,51]. Meanwhile, magnetic field stimulation can activate calcium ion (Ca 2+ ) channels on the cell membrane, which can increase the concentration of calcium ions in cells, thereby improving the function of Ca 2+ /calmodulin and the activity of cyclin-dependent kinase, promoting the proliferation of osteoblasts [52][53][54]. ALP is a critical marker for the early differentiation of osteoblasts. Its activity was used to assess the level of differentiation of MG63 cells cultured on magnetic scaffolds (Figure 8). It can be seen from the stained image that the ALP activity increased with increasing culture time. The ALP activity on the magnetic scaffold was higher than the ALP activity on the pure PLLA scaffold, indicating that osteogenic differentiation of MG63 cells was significantly up-regulated, which indicated that the magnetic scaffold had the ability to stimulate MG63 cell differentiation ability.  It is well known that the components contained in the scaffold have a significant effect on the cellular response. Among them, Fe3O4 nanoparticles have strong magnetic features and unique superparamagnetic properties in nanometric dimensions. Meanwhile, the structure of cell membrane is complex. It not only contains charged lipid molecules, water, and protein, but also contains many ion channels such as K + , Na + , Ca 2+ and so on. There is also a large amount of Cl − , K + , Na + , and other anions and cations on the inner and outer surfaces of the membrane [45][46][47]. Therefore, Fe3O4 nanoparticles can serve as a magnetic source for a single magnetic nanofield in a weak electromagnetic field formed by the cell due to the difference between internal and external ions and charges, thereby generating a biological effect of magnetic field on cells [48][49][50]. Maleki-Ghaleh. H et al. reported that in this weak electromagnetic field of cells, Fe3O4 magnetic materials improved cell growth and activity by generating magnetic fields to enhance cell communication [24]. When it was combined with the matrix, a large number of tiny magnetic fields were generated on the pores or the surface of the scaffold. According to previous studies, magnetic fields may affect the nucleation of protein crystals in the culture medium and the distribution of proteins in the cell membrane, accelerating the specific recognition of integrin proteins on the cell surface and adsorbing to the extracellular matrix proteins on the surface of the scaffold, thereby promoting cell adhesion and spread [29,51]. Meanwhile, magnetic field stimulation can activate calcium ion (Ca 2+ ) channels on the cell membrane, which can increase the concentration of calcium ions in cells, thereby improving the function of Ca 2+ /calmodulin and the activity of cyclin-dependent kinase, promoting the proliferation of osteoblasts [52][53][54].
In addition, the magnetic field can also activate various signal pathways of the cell, and collaboratively mediate the signal communication between them, such as the classic mitogen-activated protein kinase [55][56][57] and BMP signal pathway [17,58], thereby promoting the expression of growth factors, improving the activity of runt-related transcription factor 2 and ALP, accelerating the growth and differentiation of osteoblasts, and promoting bone repair finally. Of course, the micro magnetic force generated in the microenvironment of the magnetic scaffold can provide continuous dynamic mechanical stimulation to MG63 cells, which can also improve the cells adhere and migrate. In addition, the Fe3O4 nanoparticles in the scaffold have a large surface area to volume ratio. Uniform dispersion in the scaffold may show more contact surface area, thus providing more attachment sites for cell attachment [59]. However, the Fe3O4 nanoparticles are In addition, the magnetic field can also activate various signal pathways of the cell, and collaboratively mediate the signal communication between them, such as the classic mitogen-activated protein kinase [55][56][57] and BMP signal pathway [17,58], thereby promoting the expression of growth factors, improving the activity of runt-related transcription factor 2 and ALP, accelerating the growth and differentiation of osteoblasts, and promoting bone repair finally. Of course, the micro magnetic force generated in the microenvironment of the magnetic scaffold can provide continuous dynamic mechanical stimulation to MG63 cells, which can also improve the cells adhere and migrate. In addition, the Fe 3 O 4 nanoparticles in the scaffold have a large surface area to volume ratio. Uniform dispersion in the scaffold may show more contact surface area, thus providing more attachment sites for cell attachment [59]. However, the Fe 3 O 4 nanoparticles are nanomaterials, and previous studies have indicated that the nanomaterials may cause some potential adverse effects on cells and organs of the human body [60][61][62]. For example, Long et al. [60] investigated the effect of Fe 3 O 4 nanoparticles on cell activity of human hepatoma cell and lung adenocarcinoma cell, and their results indicated that the higher concentration of Fe 3 O 4 nanoparticles would cause cell death. PLLA is a biodegradable and biocompatibility bone scaffold material which has a relatively low degradation rate [63,64]. When the Fe 3 O 4 nanoparticles are incorporated into the PLLA scaffold, the slow degradation of scaffold can play the role of controlled release of Fe 3 O 4 nanoparticles, thereby continuously and steadily enhancing cellular activity without causing adverse effects to human body.

Conclusions
In this study, PLLA/Fe 3 O 4 scaffolds were successfully fabricated by SLS. The incorporated Fe 3 O 4 nanoparticles not only enhance the mechanical properties of the PLLA scaffold, but also effectively improve the biological activity of the scaffold. The results showed that the PLLA/7%Fe 3 O 4 composite scaffold exhibited increased compressive strength, modulus, and Vickers hardness, which were 29.1 MPa, 2.9 GPa, and 67.7 HV, respectively. Furthermore, the magnetic composite scaffold not only promoted cell attachment, diffusion, and interaction, but also significantly promoted MG63 cell proliferation and stimulated cell differentiation. All these positive results suggested that the SLS-processed PLLA/Fe 3 O 4 scaffold was of great potential for bone regeneration.