Magnetic cell-scaffold interface constructed by superparamagnetic

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Biological and Medical Applications of Materials and Interfaces

Magnetic cell-scaffold interface constructed by superparamagnetic IONPs enhanced osteogenesis of adipose-derived stem cells Huimin Chen, Jianfei Sun, Zibin Wang, Yi Zhou, Zhichao Lou, Bo Chen, Peng Wang, Zhirui Guo, Hui Tang, Junqing Ma, Yang Xia, Ning Gu, and FeiMin Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b17427 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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ACS Applied Materials & Interfaces

Magnetic cell-scaffold interface constructed by superparamagnetic IONPs enhanced osteogenesis of adipose-derived stem cells Huimin Chen

a, ¶,

Jianfei Sun

b,¶,

Zibin Wang c, Yi Zhou d, Zhichao Lou

e,b,

Bo Chen

f,b,

Peng

Wang g, Zhirui Guo h, Hui Tang a, Junqing Ma a, Yang Xia a,b*, Ning Gu b,i*, Feimin Zhang a,i*

a.

Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu

210029, China b.

Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and

Medical Engineering, Southeast University, Nanjing, Jiangsu 210096, China c.

Analysis and Test Center, Nanjing Medical University Nanjing, Jiangsu 211166, China

d. Yixing e.

People’s Hospital, Yixing Jiangsu 214200, China

College of Materials Science and Engineering, Nanjing Forestry University, Nanjing, Jiangsu

210037, China f.

Materials Science and Devices Institute, Suzhou University of Science and Technology,

Suzhou, Jiangsu 215009, China g.

Department of Sports Medicine and Adult Reconstructive Surgery, Drum Tower Hospital

affiliated to Medical School of Nanjing University, Nanjing, Jiangsu 210008, China

ACS Paragon Plus Environment

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ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

h.

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Department of Geriatrics, The Second Affiliated Hospital, Key Laboratory for Aging &

Disease, Nanjing Medical University, Nanjing, Jiangsu 210011, China i.

Collaborative Innovation Center of Suzhou Nano Science and Technology, Suzhou, Jiangsu

215123, China

¶ These authors contributed equally to this paper.

ABSTRACT One of the key factors in tissue engineering and regenerative medicine is to optimize the interaction between seed cells and scaffolds, such that the cells can grow in naturally biomimetic conditions. Their similarity to macromolecules and many unique properties mean that functional nanoparticles have promising potential for the modification and improvement of traditional scaffolds to obtain excellent biocompatibility, tunable stiffness, physical sensing, and stimulusresponse capabilities. In the present study, we report magnetic poly(lactic-co-glycolic acid)/polycaprolactone (PLGA/PCL) scaffolds that were fabricated using a combination of the electrospinning technique and layer-by-layer assembly of superparamagnetic iron oxide nanoparticles (IONPs). PLGA/PCL scaffolds assembled with gold nanoparticles were prepared using the same method for comparison. The results showed that the assembled film of nanoparticles on the surface greatly enhanced the hydrophilicity and increased the elastic modulus of the scaffold, which subsequently improved the osteogenesis of the stem cells. Furthermore, the magnetic property of the IONPs was proved to be the key factor in enhancing osteogenic differentiation, which explained the superior osteogenic capacity of the magnetic scaffolds compared with that of the gold nanoparticles-assembled scaffold. These results


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demonstrated the importance of magnetic nanomaterials as a bioactive interface between cells and scaffolds, and will promote the design of biomaterials to improve tissue engineering and regenerative medicine efficacy.

Keywords: Iron oxide nanoparticles; Ferumoxytol; electrospinning; layer-by-layer assembly; bone tissue engineering.

1. Introduction Bone disorders, such as bone infections, bone tumors, and bone loss, remain a challenge for worldwide public health.1 This issue becomes increasingly important with aging populations because some degenerative diseases, such as osteoporosis, can lead to a higher incidence in bone fracture and more difficult bone repair.2 For cases of weak osteogenic activity in synthetic bone grafts, the scaffold should be more suitable for cell adherence, capable of enhancing cellular growth, and should promote osteogenic differentiation.3 Commonly, stem cells are used as the seed cells because of their pluripotent differentiation into multiple tissue cells.4 Moreover, degradable polymer scaffolds, such as the Food and Drug Administration (FDA)-approved polylactic acid (PLA) or poly(lactic-co-glycolic acid) (PLGA), are considered as promising scaffolds in clinic because of their biocompatibility and tunable properties.5 Amounts of studies have demonstrated the important role of mechanic stimuli in regulation of cellular fate, which resulted in the emerging field of mechanobiology.6 Thus, the studies on clinical scaffolds for bone repair have focused on the improvement of cellular adhesion, mechanic strength and active stimulation in local niche. Some biological molecules, such as the arginine-glycine-aspartate (RGD) peptide, were often used to modify the surface of scaffold to enhance the cellular


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adhesion.7,8 However, the uniform and compact control of surface properties using molecular modification remains a challenging task. On the premise of ensuring biological activity, it is hard for the molecular capping layer to cover a large scale on bulk scaffold and escape from dissociation in vivo.9,10 Moreover, the surface modification with molecules is unable to regulate the mechanical property of the bulk scaffold. Currently available reinforcing methods for the mechanic strength of scaffold include the regulation of micro-structure of bulk materials, incorporation of inorganic nanomaterials and application of novel monomer.10-13 However, these strategies are complex, complicated and costly. Moreover, the reagents or the nanomaterials in these processes are often incompatible with the clinical standards. Also due to the strict clinical standards for implantable medical appliances, most studies on the stiffness-based stimuli for the regulation of cellular functions have been in vitro studies.11–14 Presently, the mechanic property of PLA/PLGA scaffolds is determined by the crosslinking degree and molecular weight of the polymer. And there have been few studies about the stiffness-based stimuli to regulate cells after implantation in vivo. This compels us to look for other strategy to enhance the stimulation for cellular growth. Magnetic fields have been discovered to have a significant positive effect on bone growth and fracture healing.15 Moreover, the magnetic effect could also be tuned remotely by the external field after implantation in vivo.16 Thus, the magnetic scaffold would play a special role in clinical bone repair by integration of multiple functions. Superparamagnetic iron oxide nanoparticles (IONPs) can be facilely modified onto the scaffold and magnetized by external magnetic field. In addition, IONPs have some common features with biological macromolecules, such as adhesion loci for cells.17 Thus, we proposed that the IONPs can be used as the capping layer on the scaffold instead of biological molecules. We previously studied the in vivo effects of


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FDA-approved magnetic iron oxide (γ-Fe2O3) nanoparticles (IONPs; brand name Ferumoxytol) 18,19

loaded onto a gelatin sponge scaffold (IONPs-GS) using incisor socket model in Sprague-

Dawley rats, and compared them with empty incisor sockets and those filled with GS alone.20 Micro-computed tomography and histological observations showed that the IONPs-GS group had better bone regeneration than both control groups 4 weeks after surgery. These results clearly indicated that IONP-modified scaffolds induced active osteogenesis and angiogenesis.20 Therefore, it is important to determine whether the effects came from the magnetic or the mechanical cues. In clinic, adipose-derived stem cells (ADSCs) and PLGA/Polycaprolactone (PCL) scaffold were popular for bone repair because of clinical availability and safety.21,22 In this study, IONPs were employed to compactly and uniformly modify the surface of PLGA/PCL electrospun scaffold using layer-by-layer (LBL) assembly. The IONPs were synthesized by following procedures of Ferumoxytol which was approved by Food & Drug Administration (FDA) for clinical use.18,19 This nanomaterial was composed of a 10 nm γ-Fe2O3 spherical core and a 30 nm capping layer of polyglucose sorbitol carboxymethyether (PSC) molecules,18,19 which has also been approved by Chinese FDA for clinical application. In addition, to elucidate the role of magnetism, gold nanoparticles (GNPs) were also assembled on the scaffold surface using an identical process. Then, the cellular effect of the magnetic scaffold on ADSCs was investigated. It was discovered that the surface modification with the assembled film of nanoparticles significantly reinforced the mechanical properties and greatly promoted the osteogenic differentiation of ADSCs. Furthermore, the enhancement of magnetic scaffolds was much more significant than that of the GNPs-modified scaffolds, which resulted from the magnetic effect.


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We believe that these results will be beneficial to design of the next-generation scaffolds with magnetic stimuli for bone tissue engineering.

2. Experimental Section Preparation of electrospun scaffolds: 0.45g PLGA (75/25, 12 kDa average Mw), 0.45g PCL (11 kDa average Mw) were both from Jinan Daigang Biomaterial (Jinan, China), and 0.1g gelatin (Aladdin, Shanghai, China) were dissolved in TFEA (7.3g 2,2,2-Trifluoroethanol, Aladdin, Shanghai, China) with 10 μL of glacial acetic acid by overnight stirring. A syringe pump with a metal needle (internal diameter = 0.4 mm; Jianpai, Jintan, China) was filled with the above polymer solution and connected to a high-voltage power supply (16 kV), injected at 0.8 mL/h. A nanofibrous structure was formed on the aluminized collecting plate. The obtained scaffolds were dried for 72 h before use (Huaxing Technology, Develop Co Ltd, Beijing, China). LBL assembly of the nanoparticles: The IONPs and GNPs were prepared by our group as previously reported.18,23 The detailed preparation procedures are shown in the Supporting Information. The LBL approach was used to assemble the IONPs and GNPs onto electrospun scaffolds, resulting in three groups. (1) ES (control): untreated electrospun scaffold; (2) IO-ES: IONPs-assembled electrospun scaffold, using IONP solution (10 mg/mL); (3) G-ES: GNPs-assembled electrospun scaffold, using GNP solution (0.5 mg/mL). The detailed coating procedure comprised: First, the electrospun scaffolds were treated with nitrogen plasma for 30s at 220 V and 1.5~1.8A. The distance between the two electrodes was 55


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mm. This was to achieve a negative charge on the surface. Then, the charged film was immersed into 2 mg/mL poly(Diallyldimethylammonium chloride) (PDDA) solution for 30 min, then thoroughly rinsed with pure water. Afterward, the positively charged electrospun scaffolds were dipped in negatively charged nano-solution (IONP and GNP solution) for 30 min at RT, and thoroughly rinsed again. The last step was freeze-drying, which was to ensure the firm adhesion of the nanoparticles. The whole procedure was repeated four times to obtain a dense and uniform nano-assembly. Scaffold morphology and mechanical properties characterization: After being thoroughly dried and sputter-coated with gold, the samples from the three groups (ES, IO-ES, and G-ES) were examined using SEM (scanning electron microscopy; S-3400N II, Hitachi, Japan). Ultra-thin sections were prepared for TEM (transmission electron microscopy; Tecnai G2 Spirit Bio TWIN, FEI, Hillsboro, OR, USA) observation. The hysteresis loops, representing the magnetic properties of the scaffolds, were measured using a vibrating sample magnetometer (VSM, Lakeshore 7470, Lake Shore Cryotronics, Inc., Lake Shore, CA, USA). The content of the nanoparticles on the electrospun fibers was measured using thermogravimetric analysis (TGA, Pyris 1 DSC, PerkinElmer, Waltham, MA, USA). Atomic force microscopy (AFM) imaging and force spectroscopy were performed using an Agilent 5500 (Agilent, Chandler, AZ, USA) “closed loop” AFM system, with a piezoelectric sensor (Agilent). AFM probes with Au coating on the reflective side of the pre-magnetically coated cantilever were used; the tip was sphere-shaped with a curvature radius of 8 nm. The spring constant of the cantilever was tested as 0.1 N/m. The images and the force curves were obtained in air, and were acquired using Agilent AAC mode AFM. The obtained AFM images were processed using the WSxM software (http://www.wsxm.es/download.html). Resonance


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frequency changes induced by a small mass24 was used to calibrate the AFM cantilever spring constant. The Young’s moduli were calculated using Hertz model:25

(1)

where F represents the loading force, v represents Poisson’s ratio (assumed to be 0.5), δ represents the indentation depth, E represents the elastic modulus, and R represents the radius of the tip. Surface Properties: Attenuated total reflectance Fourier transform infrared spectroscopy (ATRFTIR; NICOLET6700FT-IR, Thermo Scientific, Texas, USA) was used to analyze the prepared samples. Water contact angles were measured using a contact angle meter (JC2000C2, Shanghai Zhongchen Powereach Company, China) 26 to indicate the surface energy of the samples. Protein adsorption: The effect of nanocoating on protein adsorption onto the scaffolds was determined as previously reported.27 Samples (6 mm in diameter) were pre-immersed for 2 h in 1×phosphate-buffered saline (PBS). Then, they were transferred to a 4.5 g/L protein solution (bovine serum albumin, BSA; Sigma-Aldrich, St. Louis, MO, USA) for 12 h at 37 °C. Afterward, they were rinsed, transferred to 1% SDS (sodium dodecyl sulfate) /PBS solution, and treated for 20 min by sonication to fully uncouple the protein from the scaffolds. The amount of protein adsorbed onto the sample was assessed (BCA Protein Assay Kit, Leagene Biotechnology Co., Ltd, Beijing, China). In vitro cell assay: OriCell Sprague-Dawley (SD) rat ADSCs (Cyagen Biosciences, Guangzhou, China). Cells at passage 3–5 were used. Dexamethasone (10-7 M), 50 μM ascorbate-2-phosphate,


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and 10 mM β-glycerol phosphate (Sigma-Aldrich) were supplemented into the culture media to form osteogenic medium. The morphology of the ADSCs attached to the scaffolds (6 h and 24 h) was investigated using CLSM (confocal laser scanning microscopy; Zeiss-LSM510, Carl Zeiss, Oberkochen, Germany). For CLSM, the scaffolds were washed, fixed, permeabilized, and incubated with rhodamine phalloidin from Cytoskeleton, Inc (Denver, CO, USA.) at 1:200 for 30 min. After rinsing with 1 × PBS, the samples were incubated with DAPI (4′,6-diamidino-2-phenylindole) from Beyotime (Shanghai, China) at 1:2000 for 30s. For each sample, three photos were obtained randomly. Ten representative images of each group were analyzed (Image-Pro Plus 6.0 software, www.mediacy.com/imagepro). We calculated the cell spreading area by:

(2)

In the above formula, Stotal refers to total cell spreading area, and Ncell refers to cell number. The adhered cells aspect ratio (at 6 h) was calculated using the formula below:

(3)

The ratio of cell adhesion at 6 h was determined by CCK-8 (cell counting kit-8) from Beyotime (Shanghai, China). And the results were analyzed according to previous reports,28 using the formula below:


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(4)

At 1, 4, 7, and 10 days, the cell number was calculated using the same CCK-8 kit. After 2 h incubation in the dark, the OD (optical density) at 450nm was measured (Titertek, Helsinki, Finland). The average result was taken from five replications of each sample. For SEM observation, two-day-old cell-scaffolds were ﬁxed, dehydrated, freeze-dried and sputter-coated with platinum. Cell morphology on the scaffolds was observed using SEM. The alkaline phosphatase (ALP) activity of the cells (4 days, 7 days, 14 days) on the scaffolds were assayed using an ALP Assay kit from JianCheng Bioengineering Institute (NanJing, China) and determined according to previous reports. 29 At 7 and 14 days after cell seeding, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract the total RNA. Genes, including ALP, COL1 (collagen type 1), RUNX2 (runtrelated transcription factor 2), OCN (osteocalcin), and ISCA1 (iron-sulfur cluster assembly protein 1) were then assayed using RT-PCR (reverse transcription polymerase chain reaction). The housekeeping gene was glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequences of the primers are listed in Table S1. The RNA concentration and purity was detected using a NanoVue Plus spectrophotometer (GE Healthcare, Piscataway, NJ, USA). A PrimeScript RT reagent kit (Takara Bio Co., Ltd., Otsu, Japan) was used in the reverse transcription reactions. Quantitative real-time RT-PCR (qRT-PCR) reactions were done by a RT-PCR System from Applied Biosystems (ABI 7300, Foster City, CA, USA): a 5 min denaturation step at 95 °C was followed by 40 cycles of 10 s at 95 °C and 31 s at 60 °C. 2-∆∆Ct method was used to evaluate the relative gene expressions which were normalized by the cycle threshold (Ct) of the


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housekeeping gene GAPDH in triplicate. The calibrator was the Ct of the genes from ADSCs cocultured with ES.30 2% ARS (Alizarin Red S) from Millipore (Billerica, MA, USA) was used to stain calciumrich deposits on the scaffolds. The mineralization rate was quantified according to previous report 31 using the absorbance of ES at 550 nm as calibrator. Interaction between nanoscaffolds and co-cultured ADSCs: To detect the interaction between nanomaterials and ADSCs, scaffold samples with cells were prepared after seeding for 7 days. Then, the samples were examined using a Tecnai™ G2 Spirit Twin system from FEI (Hillsboro, OR, USA). Statistics: Average ± SD were used to quantify all results. The Statistical Product and Service Solutions (version 22.0) from IBM Corp (Armonk, New York, USA) was utilized. Group comparisons were done by one-way-analysis of variance with Bonferroni post-hoc tests. Results were considered significant (p < 0.05), and highly significant (p < 0.01).

3. Results and Discussion The synthesized IONPs and GNPs are shown in Supporting Information, Figure S1, from which it can be seen that both nanomaterials were spherical and homogeneously distributed. The nanoparticle concentrations were chosen based on requirements of LBL assembly and our experimental experience. The reasons have been explained and added in Supporting Information. The coating process using the LBL method is illustrated in Figure 1a. Sample images of ES, IOES, and G-ES are shown in Figure 1b. The scaffolds appeared to be completely covered by a


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film of nanoparticles, which was further confirmed by SEM and TEM characterization (Figure 1c-e). The nanoparticles formed a compact capping layer around the electrospun fibers. Indeed, the LBL technique can control the surface capping of nanoparticles. It was found experimentally that the magnetization intensity, the coating thickness, and the iron content of the scaffold increased as the number of layers increased (Supporting Information, Figure S2 and Table S2). However, the increment became non-significant between the 3-layer and the 4-layer assembly. Thus, the nanoparticles were assembled into 4 layers in our experiments. All scaffolds comprised randomly oriented fibers with diameters ranging from 0.7–1.4 μm and had a three-dimensional (3D) macroporous structure. After the surface was coated with nanoparticles, the smooth surface of the electrospun fibers became rough. As shown by the TEM cross-sectional images, the coating of nanoparticles was nearly continuous, dense, and homogenous around the fibers, with a thickness of about 50–100 nm. After the surface was coated with IONPs, the scaffold exhibited an obvious magnetic response in the presence of a magnet. The magnetization property was also measured using VSM. From the hysteresis loop, the magnetic scaffold showed an obvious superparamagnetism and the saturation magnetization was about 3.56 emu/g (Figure 1f). AFM was used to further characterize the 3D surface morphology of the scaffolds (Figure 2). The ES (blank scaffold) presented a relatively flat and smooth surface, with stripe-like and wavy patterns, which should result from the structure of the fibers. The G-ES (gold nanoparticles-modified scaffold) showed a similar surface morphology to the ES, except that the surface was coated by a layer of small nanospheres. However, the layer of IONPs covering the surface of scaffold was thicker than that of the GNPs. Thus, the stripe-like surface pattern was not observed for the IO-ES (iron oxide nanoparticles-modified scaffold), which reflected the thicker stabilizer of polysaccharide molecules on the surfaces of IONPs than the citrate


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molecules on GNPs. The content of the nanoparticles on the scaffolds was determined using TGA (Supporting Information, Figure S3). TGA can be used to quantify the nanomaterials assembled on the scaffolds. All samples were presented similar behaviors under heat treatment. Water removal below 250 C caused first slight weight loss, while the decomposition of PLGA at approximately 280–350 C resulted in the second weight loss, and the decomposition of PCL at approximately 350–400 C presented as the third weight loss. The final weight remaining at temperature higher than 410 C represented the amount of assembled nanoparticles. The portions of remnant weight were 16.4% and 4.9% for IO-ES and G-ES, respectively. This result confirmed the TEM observations quantitatively. Mechanical properties are vital for the practical application of polymer scaffolds. Representative Deflection-Distance curves of the electrospun fibers are shown in Supporting Information, Figure S4. Based on these data, the elasticity moduli of the surfaces of different scaffolds were calculated (Figure 3a). The elasticity modulus was significantly enhanced after surface modification by the "hard" nanoparticles (p < 0.01). Interestingly, the value for the IOES scaffold was lower than that for G-ES scaffold (p < 0.01), although the assembled film of IONPs was thicker than that of GNPs. The thicker stabilizing layer for IONPs could account for this phenomenon. The stabilizing layer of polysaccharide molecules was soft, which reduced the elasticity modulus. The mechanical measurement using a nanoindentor showed that the mechanical property of the interface could be reinforced by surface coating with inorganic nanoparticles between the scaffold and the cells. ATR-FTIR spectra for the three samples are shown in Supporting Information, Figure S5, presenting the influence of the nanoparticles on the chemical radicals of the scaffold surface. It appeared that there was little alteration on the surfaces; however, a new peak at around 1600 cm-1 (marked by arrows) appeared for the IO-ES


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surface, indicating the presence of carboxyls from PSC. Wettability was also investigated by measuring the water contact angle (Figure 3b). The ES group exhibited a hydrophobic surface, whose contact angle was about 105.8°. After the surface coating by nanoparticles, the contact angle was significantly decreased to approximating 0°. This indicated remarkably improved wettability resulting from the presence of hydrophilic nanoparticles. Moreover, the presence of nanoparticles is beneficial for cell adhesion because of the augmented surface roughness and extended surface area.32,33 Thus, the exposure of nanoparticles on the surface should improve the protein adsorption ability of the scaffolds. The protein adsorption quantities for the nanoparticlemodified scaffolds are shown in Figure 3c. The values for IO-ES and G-ES were significantly increased compared with that for ES (p < 0.01). However, there was little difference between the two nanoparticle-coated scaffolds (p > 0.05). The difference between IONPs and GNPs was possibly eliminated because the scaffold surface was covered by a compact, uniform, and continuous film of assembled nanoparticles rather than the individual units. Next, we cultured ADSCs on the scaffolds. The scaffold thickness plays a critical role in tissue engineering and regeneration.34,35 The thickness of the scaffold before and after the LBL process was calculated (Supporting Information, Figure S6a-d). For IO-ES, the thickness before and after LBL was 165.26 ± 3.52 μm and 167.66 ± 4.69 μm (p > 0.05), respectively. For G-ES, the thickness before and after LBL was 87.24 ± 4.16 μm and 87.28 ± 2.84 μm (p > 0.05), respectively. No significant differences were found in the thickness of the scaffold before and after LBL process, in both IO-ES and G-ES groups. This was because that the coating of nanoparticles was too thin to affect the bulk thickness of the scaffold. Therefore, the process of LBL assembly scarcely changed the thickness of the scaffolds.


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The behaviors including adhesion and spreading of ADSCs were observed using CLSM (Figure 4a-f). For the ES group, cellular spreading was not obvious at 6 h (Figure 4a), but was clearly observed at 24 h (Figure 4d). Compared with the ES group, the cells cultured on IO-ES and G-ES showed substantial spreading with active cytoskeletal processes as early as 6 h (p < 0.01). In addition, the cells on IO-ES presented the highest aspect ratio after 6 h of culture (p < 0.01). This meant that these cells were more stretched and spindle-like compared with those on ES and G-ES. The quantitative data for the cellular spreading area and aspect ratio are shown in Figure 4g, h. However, the difference disappeared after 24 h of culture. After 2 days of culture, the cells and the scaffolds were observed using SEM, which showing similar results for all three groups of no significant differences in cell shape (Supporting Information, Figure S7). These results indicated that the magnetic nanoparticles-modified scaffolds affected cellular behavior at an early stage. The cell shape itself is an inherent cue to regulate stem cell differentiation; therefore, we were believed that the yield of osteogenesis should be dependent on the aspect ratio.11 The adherent ADSCs on the scaffolds after 6 h of culture was assessed using a CCK-8 kit to indicate the condition of initial cell adhesion. Significantly more cells adhered to IO-ES (p < 0.01, Figure 4i) than to the other scaffolds. Characterization of the scaffolds and co-cultured ADSCs using TEM further confirmed this point (Supporting Information, Figure S8). Thus, the surface of IO-ES provided better sites for cellular adhesion. This result may be related to the advantages of Ferumoxytol, which is a clinically approved inorganic nanodrug. It was reported that the assemblies using these IONPs were better for cellular adhesion.17,36 However, in the present study, the proliferation of ADSCs showed non-significant variations among the different scaffolds (p > 0.05, Supporting Information, Figure S9). This result indicated that the surface


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coating of nanoparticles influenced the cellular adhesion at the early stage rather than during the whole growth process. The osteoinduction capacities of the scaffolds were then tested. Scaffold stiffness has been recognized as an influencing factor for the osteogenic differentiation of stem cells.12–14 To detect the stiffness of bulk scaffolds, a hardness test was carried out using a Vickers diamond indenter on an automated hardness tester (Supporting Information, Figure S10). No differences were found among ES, IO-ES, and G-ES (p > 0.05). Therefore, the LBL process did not change the overall stiffness of the scaffolds. Furthermore, the IONPs and GNPs, with the same size and surface capping, were used to modify the polymer scaffold to eliminate the influence resulting from the elemental units. The scaffolds were modified with a compact and uniform layer of both nanoparticles using the LBL assembly. The resultant scaffolds, IO-ES-2 and G-ES-2, were used as controls. The detailed preparation procedures are shown in the Supporting Information. The characterization of IONPs2, GNPs-2, IO-ES-2, and G-ES-2 is shown in Figure S11. ALP activity was chosen as an indicator to evaluate osteogenic differentiation of the ADSCs (Figure 5a). The cells cultured on IO-ES showed the highest ALP activity among the three groups after 7 days of culture (p < 0.01) and was maintained until 14 days of culture (p < 0.01). Comparatively, the cells cultured on G-ES also showed increased ALP activity, which was significantly higher than that in the cells cultured on the ES (p < 0.01). However, the cellular ALP activity of G-ES was always significantly lower than that of IO-ES (p < 0.01) and IO-ES-2 (p < 0.01). Interestingly, there was no difference between IO-ES and IO-ES-2 (p > 0.05), and GES and G-ES-2 (p > 0.05). This result may indicate that the particle size and surface modifiers of


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the nanoparticles were not influencing factors in determining the osteoinduction capacities of the scaffolds. Transcription of some important biomarkers, including ALP, RUNX2, COL1, and OCN, which are associated with the osteogenic differentiation of ADSCs, were measured using qRT-PCR (Figure 5b-e). At 7 and 14 days the expressions levels of these genes in cells on the nanoparticle-modified scaffolds were markedly upregulated and this effect was increasingly significant with increasing time (p < 0.05). Therefore, qRT-PCR results indicated that the nanoparticle-modified scaffolds could greatly enhance the osteogenic differentiation of ADSCs. G-ES upregulated the expression of COL1 to a greater extent than IO-ES at 7 days and 14 days (p < 0.01). However, it should be noted that the expression levels of ALP and RUNX2 at 7 and 14 days and OCN at 14 days in IO-ES were much higher than those for G-ES (p < 0.01). RUNX2 is the earliest and the most specific marker for bone formation.37 ALP and OCN are markers of osteogenic differentiation in the early and late stage, respectively, and both are essential for bone mineralization and bone formation.38 COL1 is the most abundant extracellular protein in bone and provides the structural framework for bone formation. Thus, COL1 is highly sensitive to the mechanical property of the extracellular matrix.11 According to the obtained results, although the bulk hardness of the two matrices was similar, the rigidity of G-ES surface was more than that of IO-ES surface, which led to higher upregulation of COL1. However, G-ES did not result in higher expressions of three osteogenic marker genes (OCN, ALP, and RUNX2). Therefore, magnetic effect was considered to play a greater role in the enhancement of osteogenesis than the mechanic effect. Moreover, it was found that there were no differences in the expressions of all four biomarkers between IO-ES and IO-ES-2 (p > 0.05), and between G-ES and G-ES-2 (p > 0.05). Based on the results of the overall osteogenic differentiation performance of the cells, IO-


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ES was superior to G-ES. In addition, the particle size and surface modifiers of the nanoparticles were not influencing factors in determining the osteoinduction capacities of the scaffolds. Quantitative measurements of synthesized bone mineral matrix for three scaffolds after 21 days of osteogenic induction were plotted and shown in Figure 5f. Both nanoparticle-modified scaffolds showed greatly enhanced (more than 2-fold) bone mineral synthesis compared with that induced by the blank scaffold. Furthermore, the enhancement of bone mineral synthesis in the IO-ES group was significantly higher that of the G-ES group. Therefore, this result confirmed the conclusion that the osteogenesis capacities of IO-ES were better than those of G-ES. More importantly, very few nanoparticles entered into the cells (Supporting Information, Figure S12), meaning that the osteogenic enhancement phenomenon could not have resulted from the transmembrane effect of the nanoparticles. Therefore, we hypothesized that the magnetic effect resulting from the assembly of magnetic nanoparticles would contribute to this phenomenon. Gene expression of an exogenous magnetoreceptor, ISCA1, at 14 days was examined to confirm this hypothesis. A remarkable upregulated of ISCA1 expression would be found if magnetism had an obvious effect.39 As expected, there were obvious differences in ISCA1 expression, which was significantly higher in the IO-ES group than that in the G-ES and ES control groups (p < 0.01, Figure 6a). Meanwhile the expression of this biomarker was relatively low in the G-ES and the ES control groups. Furthermore, the correlation coefficients of the expression of ISCA1 with those of ALP, COL1, RUNX2, and OCN were calculated (Figure 6b). The results showed that the expression levels of ISCA1/ALP and ISCA1/RUNX2 were highly correlated (absolute value > 0.9), while the expression levels of ISCA1/COL1 were not correlated (absolute value < 0.1). Therefore, magnetism should be a significant influencing factor, even though an external magnetic field was


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absent. It has been observed that a magnetic field can improve the differentiation of stem cells.40 In our experiments, the cells were actually cultured on a layer of magnetic nanoparticles. Indeed, the aggregation of magnetic nanoparticles can lead to phase transition of the magnetism, from the superparamagnetism to weak ferromagnetism.39 In the present study, we believed that a similar effect was involved. Neighboring magnetic moments are inclined to form a concordant alignment to reduce the systematic energy; therefore, the compact, uniform, and large-scaled layer of IONPs could be regarded as comprising a number of tiny magnets. Although the film of nanoparticles exhibited little net magnetic effect because of counteractions between the number of magnets with a random orientation, the aggregates of magnetic nanoparticles could have the significant, but highly localized, magnetic effect on the cells. Thus, the IO-ES showed superior performance in terms of osteogenic differentiation of ADSCs compared with that of the G-ES (Figure 6c). Finally, a molecular effect on cellular behavior from the capping layer of nanoparticles was excluded. The IONPs were stabilized by large PSC molecules and the GNPs were stabilized by small citrate molecules; these two molecules were also used to modify the surfaces of blank electrospun scaffolds, separately, in the absence of the nanoparticles. The cellular ALP activities for the PSC-modified scaffold and the citrate-modified scaffold after 14 days of culture were measured using the blank scaffold as the control (Supporting Information, Figure S13). There was no significant difference between the ALP activities of the two scaffolds and the control (p > 0.05). Thus, we concluded that the cellular effects resulted from the nanoparticles rather than the surface molecules, and the nanoparticles truly played a beneficial role in the construction of scaffolds for tissue repair, which will be complementary to the surface modification with molecules.


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Concerning the fabrication method, LBL assembly of nanoparticles can finely tune the surface morphology and roughness through assembly cycles, while maintaining the original structure and porosity of the scaffolds, which makes it quite different from conventional magnetic scaffold fabricating methods, such as dip-coating and co-electrospinning. 16,41-43 This technique has been used to immobilize bioactive substances on scaffold surfaces.44 More recently, it was used to assemble silica nanoparticles on the fiber surface of a fibrous scaffold and enhanced the cellular responses of osteoblast cells.45 This is also consistent with the aim of interface tissue engineering (ITE), which is to stimulate the repair or regeneration the functions of diseased or damaged zones at the interface of different tissue types.46 Lu et al implanted tantalum (Ta) ions into polyetheretherketone (PEEK) using plasma immersion ion implantation (PIII) to form Ta2O5 nanoparticles in the near surface.47 The Ta-PIII PEEK surfaces induced enhanced osteogenic differentiation of rat BMSCs in vitro, and faster and better osteointegration in vivo.47 According to the reported results, the biological effects were not significant in the absence of an external magnetic field under conditions of dispersed magnetic nanoparticles in the matrix of the scaffolds.16 However, our work assembled the nanoparticles into a dense and uniform granular layer. For non-magnetic nanoparticles, the assemblies can stimulate cells by changing the mechanical properties of the interface, which is consistent with the results of our G-ES group. While for the magnetic nanoparticles, the assemblies could not only change the mechanical properties of the interface, but also promoted the osteogenic differentiation of cells via a magnetic effect. Thus, the assembled film of IONPs provided additional capabilities to the composites, such as providing better cell adhesion sites, modulating matrix rigidity, and


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mediating local magnetic effects, which agreed with the results of our previous studies.36,49-51 Therefore, magnetic IONPs presented a much stronger effect than GNPs. Currently, novel strategies using magnetic methods to enhance the function of seed cells, the properties of synthetic scaffolds, and the delivery of growth factor are a hot research topic in bone tissue engineering.52 Due to the assistance of magnetic force, magnetic scaffolds can increase the osteogenic differentiation and angiogenesis of stem cells, and bone regeneration compared with those of the controls.53 The underlying mechanisms were identified as involving the activation of signaling pathways including mitogen-activated protein kinase (MAPK), integrin, bone morphogenetic protein (BMP), and nuclear factor kappa B (NF-κB).53,54 Researchers have also attributed the improvements to the involvement of magnetism.16,43,54–58 A magnetic field can promote the differentiation of stem cells into osteoblasts, thereby enhancing bone growth and promoting bone repair.40 In the present study, magnetic nanoparticles were used to modify scaffolds and construct magnetic cell-scaffold interfaces. Although no external magnetic field was applied, the magnetic scaffolds could still be magnetized because of the existence of a geomagnetic field. Therefore, a weak local magnetic field can be produced at the interface. This endogenous magnetic field could affect the stem cells by promoting their osteogenic differentiation and osteogenesis. The recently identified magnetosensing protein, ISCA1, which can respond to magnetic fields, could be used to indicate the role of magnetic effects.33,39 However, how this magnetosensing protein induces the subsequent signaling cascades inside cells remains unknown. Nevertheless, the current study demonstrated the importance of magnetic nanomaterials as a bioactive interface between cells and scaffolds, and will promote the design of biomaterials to improve tissue engineering and regenerative medicine efficacy. Further studies will be focused


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on the in vivo performance of this magnetic scaffold and the underlying mechanisms by which magnetism enhances the osteogenic differentiation of stem cells including the effects from enhanced cell adhesion, and controlled scaffold degradation which could match the rate of tissue ingrowth.

4. Conclusion The present study demonstrated for the first time that an assembled layer of functional nanoparticles was suitable for the surface modification of tissue engineering scaffolds, which substantially enhanced osteogenesis using stem cells. We chose the PSC-capped γ-Fe2O3 nanoparticles to cover the PLGA/PCL electrospun scaffold with a compact, uniform, and tailorable film using the Layer-by-Layer assembly technique. Surface modification with nanoparticles greatly improved the hydrophilicity and elasticity of the interface and its affinity for stem cells. More importantly, the magnetism of γ-Fe2O3 nanoparticles significantly enhanced the osteogenic differentiation of ADSCs, which provided a novel and convenient strategy to fabricate scaffolds with active stimulation and remote-control functions. These results reveal a promising approach for tissue engineering and regenerative medicine applications.

ASSOCIATED CONTENT Supporting Information. Preparation and characterization of nanoparticles; The reasons for choosing 10 mg/mL IONP solution and 0.5 mg/mL GNP solution; TEM images and magnetic properties of IONPs and GNPs; Magnetic hysteresis loops, water contact angles, TEM images and SEM with EDS of IOES samples with zero layer (0), one layer (1), two layers (2), three layers (3) and four layers (4)


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of IONPs assemblies; Thermogravimetric Analysis of the samples weight loss during thermal treatment; Representative Deflection-Distance curves of the electrospun scaffolds; Attenuated Total Reflection Flourier Transformed Infrared Spectroscopy spectra for ES, IO-ES, and G-ES; The thickness of IO-ES and G-ES scaffolds before and after LBL assembly; Cell morphology on the scaffolds, as assessed by scanning electron microscope at 2 days after seeding; Low magnification Transmission electron microscope images presenting the condition of ADSCs with the scaffolds after 7 days of co-culture; Cell proliferation on ES, IO-ES, and G-ES scaffolds; Hardness of ES, IO-ES, and G-ES scaffolds; Characterization of IONPs-2, GNPs-2, IO-ES-2, and G-ES-2; High magnification TEM images of ADSCs cultured on the scaffolds after 7 days, presenting the endocytic condition of the nanomaterials; The cellular ALP activity for the PSCmodified scaffold and the citrate-modified scaffold; Real-time polymerase chain reaction primers used in this study; Iron content detection of IO-ES with different coating layers using EDS.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected](Y.X.). Fax: +86-25-86516414. *E-mail: [email protected] (N.G.). Fax: +86-25-83272460. *E-mail: [email protected] (F.Z.). Fax: +86-25-86516414. ORCID Yang Xia: 0000-0001-7962-8930 Ning Gu: 0000-0003-0047-337X Feimin Zhang: 0000-0003-3833-8923 ⊥ Huimin Chen and Jianfei Sun contributed equally to this work. Author Contributions


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Jianfei Sun and Yang Xia both conceived the project and wrote the manuscript. Feimin Zhang, Ning Gu, Jianfei Sun, and Yang Xia designed the experiments. Feimin Zhang and Ning Gu gave instructions. Huimin Chen, Yi Zhou, and Hui Tang performed the fabrication and characterization of the scaffolds. Bo Chen, Zhirui Guo, and Peng Wang prepared the nanoparticles. Huimin Chen and Yi Zhou carried out the cell culture, cell activity tests, and captured the CLSM images. Huimin Chen and Yi Zhou carried out the ALP and RT-PCR experiments. Junqing Ma gave some instructions on cell culturing, sterilization, and RT-PCR. Yang Xia performed the statistical analysis. Zibin Wang performed the TEM. Zhichao Lou performed the AFM. All authors reviewed the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation (NSF) of China (81771044 and

61601227),

the

National

Key

R&D

Program

of

China

(2017YFA0104301,

2016YFA0201704/2016YFA0201700), the Southeast University-Nanjing Medical University Cooperative Research Project (2242018K3DN16), the Qing Lan Project, the Jiangsu Medical Youth Talent (QNRC2016853), the NSF of Jiangsu Province (BK20160939), the Collaborative Innovation Centre of Suzhou Nano Science and Technology, and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions （ PAPD , 2018-87）.


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Figure Captions Figure 1. (a) Schematic illustration of the Layer-by-Layer assembly process on the scaffolds. (b) As fabricated two scaffolds with assembled nanoparticles and untreated ES control. SEM and TEM images of the three groups: ES Control (c); IO-ES (d); G-ES (e). The images present the morphology of the nano-assemblies on the surface of the electrospun scaffolds. (f) Hysteresis loops of the three scaffolds, indicating the superparamagnetism of IO-ES. ES, electrospun scaffold; SEM, scanning electron microscopy; TEM, transmission electron microscopy; IO-ES electrospun scaffold-iron oxide (γ-Fe2O3) nanoparticles; G-ES, electrospun scaffold with gold nanoparticles. Figure 2. AFM images of ES, IO-ES, and G-ES. Two-dimensional AFM images of ES (a), IOES (d) and G-ES (g). Three-dimensional AFM reconstruction of ES (b), IO-ES (e), and G-ES (h). Low magnification SEM images of ES (c), IO-ES (f), and G-ES (i). AFM, atomic force microscopy ES, electrospun scaffold; IO-ES electrospun scaffold-iron oxide (γ-Fe2O3) nanoparticles; G-ES, electrospun scaffold with gold nanoparticles; SEM, scanning electron microscopy. Figure 3. (a) Young’s modulus of ES, IO-ES, and G-ES by nanoindentation. (b) Water contact angles of the three groups. (c) Measurements of protein adsorption (n = 9). In each plot, bars indicated by different letters are significantly different from each other (p < 0.05). ES, electrospun scaffold; IO-ES electrospun scaffold-iron oxide (γ-Fe2O3) nanoparticles; G-ES, electrospun scaffold with gold nanoparticles. Figure 4. Confocal laser scanning microscopy images (×400) of the morphology of ADSCs cultured on the four scaffolds (ES, control, IO-ES, and G-ES) at 6 h (a, b, c) and 24 h (d, e, f). Cells were stained using F-actin (red) and nucleus (blue). (g) Quantitative analysis of the cell spreading area at 6h. (h) Quantitative analysis of the adhered cell aspect ratio at 6h. (i) Quantitative analysis of the adhered cell ratio at 6h after seeding (n = 4). In each plot, bars indicated by different letters are significantly different from each other (p < 0.05). ADSC, adipose-derived stem cell; ES, electrospun scaffold; IO-ES electrospun scaffold-iron oxide (γFe2O3) nanoparticles; G-ES, electrospun scaffold with gold nanoparticles.


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Page 33 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. (a) ALP activities of seeded cells at 7 and 14 days after seeding (n = 4). (b) (c) (d) and (e) The expressions of ALP, COL1, RUNX2, and OCN on the scaffolds after 7 and 14 days of culture (n = 4). (f) Quantitative analysis of mineral synthesis by the cells (n = 6). In each plot, bars indicated by different letters are significantly different from each other (p < 0.05). ALP, alkaline phosphatase; COL1, collagen type 1, RUNX2, runt-related transcription factor 2; OCN, osteocalcin. Figure 6. (a) The expressions of ISCA1 on the scaffolds after 14 days of culture (n = 4). (b) Correlation coefficients of ISCA1/ALP, ISCA1/COL1 ISCA1/RUNX2, and ISCA1/OCN, respectively. The results showed that the expression of ISCA1/ALP and ISCA1/COL1 were highly correlated. (c) Schematic showing of the mechanism for effect of the IONPs-assembled electrospun scaffold on the cells. The key point lies in the local magnetic field. The magnetization could enhance the osteogenic differentiation of stem cells. ISCA1, iron-sulfur cluster assembly protein 1; ALP, alkaline phosphatase; COL1, collagen type 1, RUNX2, runtrelated transcription factor 2; OCN, osteocalcin; IONP, iron oxide (γ-Fe2O3) nanoparticle.


33


ES

1(a) Nitrogen plasma 2 (c) 3 Electrospun 4 5 Scaffold (-) (-) 6 7 8 9 10 11 12 PDDA Nano Deionized Deionized 13 Solution solution water water 14 (+) (-) 15 16 (d) 17 18 Electrospun 19 Scaffold 20 Electrospun 21 Scaffold 22 23 Repeated 24 25 Electrospun Scaffold 26 Nanoparticles 27 assembled with 28 nanoparticles 29 (e) 30 (f) 31 (b) IO-ES 32 33 34 ES 35 36 G-ES 37 38 39 IO-ES ES G-ES 40 41 42 43 ACS Paragon Plus Environment 44 45 46

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+- +- +- +- +-

1μm

200nm

1μm

200nm

G-ES

Magnetization ( emu/g)

IO-ES

-

+-- +-- +-- +- +--

1μm

200nm Fig. 1

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(a)

(b)

ES

(c)

1μm

(d)

20μm

(e) IO-ES

(f)

1μm

(g)

20μm

(h)

(i)

G-ES

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


1μm

20μm


Fig. 2


c

1.5

b

1

0

(b)

IO-ES

0.5

G-ES

a

ES

Nanoindentation

Young’s Modulus (GPa)

2

(a)

ES

G-ES

IO-ES

(c) 6

a

G-ES

2

b

IO-ES

4

b

ES

Protein adsorption (μg / mm2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Electrospun scaffold

Nano assembly

Protein

Fig. 3 ACS Paragon Plus Environment

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0

4

(i)

90

b a

a

70 60

G-ES

80

ES

20μm

Cell adhesion rate ( % control well )

100

20μm

G-ES

2 0

(f)

a

a IO-ES

20μm

b

6

IO-ES

(c)

300

ES

(e)

20μm

a

(h)

IO-ES

(b)

20μm

b

G-ES

ES

20μm

600

b

IO-ES

(d)

(a)

900

ES

24h

Aspect ratio of adhered cells at 6h

6h

Cell spreading area at 6h(μm2)

(g)

G-ES

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



a

b

7 days b b a c c

14 days

b

b

c

(f)

14 daysACS Paragon Plus Environment b

1

0

b

7 days

a

c

c

7 days

4

3

a

a

(d) 12

b b

c

G-ES-2

14 days

c

G-ES

IO-ES-2

IO-ES

ACS Applied Materials (b)& Interfaces

G-ES-2

c

ES

G-ES-2

c

IO-ES-2

G-ES-2

G-ES

b

IO-ES

0

IO-ES-2

IO-ES

b

G-ES

8 G-ES

b

IO-ES-2

a

ES

4 IO-ES

ES

ALP Expression (folds)

0

G-ES

a

ES

14 days

4

IO-ES

b

COL1 Expression (folds)

G-ES-2

f

ES

c G-ES-2

G-ES

e

Cell-synthesized bone minerals (%)

b G-ES

IO-ES-2 f

G-ES-2

b IO-ES-2

d

G-ES

7 days IO-ES

e

IO-ES-2

c IO-ES

7 days

IO-ES

ES

G-ES-2

IO-ES-2

IO-ES

c

ES

c

G-ES-2

ES

c

ES

G-ES-2

b

G-ES

b

G-ES

b

IO-ES-2

b

G-ES

IO-ES-2

a

IO-ES

ES

ALP activity (U / g protein)

a

IO-ES

ES

RUNX2 Expression (folds)

20 1 2 3 4 15 5 6 7 10 8 9 10 11 5 12 13 14 0 15 16 17 18(c) 9 19 20 21 22 23 6 24 25 26 27 28 3 29 30 31 32 33 0 34 35 36(e) 37 15 38 39 40 41 42 10 43 44 45 46 47 5 48 49 50 51 52 53 0 54 55 56 OCN Expression (folds)

(a) Page 38 of 39

12 b

8

c c

14 days

b c

2

a

Culture time (21 days) Fig. 5

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b

3 2

Correlation Coefficient

(b)

(a)

1

0.938 0.924 0.732

0.6

0

IO-ES

1

G-ES

a

a ES

ISCA1 Expression (folds)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.2 0

-0.0876

(c)

Electrospun scaffold

Magnetic effect

IONPs assembly

Stem cell


Magnetic cell-scaffold interface constructed by superparamagnetic

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