SCENCENEWS m-journal de magnetic field can readily mimic the diurnal shifting magne- Table 1. Number of adsorbed residues of FN-l17-10 on membranes with toelectric environment. I4 Altogether, these studies point to different CFO contents(MD simulations) promising applications of magnetoelectric materials in tissue regeneration. In this study, we fabricated the CoFe2O4 (CFO)/ 5w:% ASN13,ASP15, THR16,CLY17,VAL18, LEU19,GLY46, ASN47,SER48, 44 poly(vinylidene fuoridetrifluoroethylene)[P(VDF-TrFE)) mag U49, GLU50, GLU51, VAL52, VAL53, HIS54 ASP56, GLN57, CYS60 netoelectric nanocomposite membranes, which can be regu THR61, PHE62, ASP63, ASN64,SER66, LEU69, ASP81 ASP122 lated by application of a remote DC magnetic field to generate LEU123, THR124, ASN125, GLU141, SER145, PRO146, SER147, ASP148 a built-in magnetoelectric microenvironment. Molecula TYR170, GLUI7I, GLN172, PRO289, PRO338, GLY339, ASP341 TYR366.ARG367.THR368 content of CFO nanoparticles for attaining the greatest argi- 10 wt% GLUT, ASN13, PRO14 ASPI5, HR16, GLYT/, VALIS, LEU19, THR20 nylglycylaspartic acid(RGD) sites exposure. The magneto- EU49, GLU51, VAL52, CAL53, HIS54, ALA55, ASP56, GLN57, SER59 electric microenvironment provided by the magnetoelectric CYS60, THR61, PHE62, ASP63, ASN64, LEU65, SER66, PRO67-ASP122 membranes can enhance osteogenesis and bone regenera GLU141SER145SER147ASP196ILE197.GLU223.GLY249THR250 tion within the bone defect area. The bone regeneration can GLU251, GLN271, SER273, THR274, VAL275, SER276ASP277, VAL278 PRO289, GLU312, THR313, GLY314, GLY315, ASN316, SER317, LYS337 not only be attributed to the direct osteogenic effect of bone RO338, GLY339, VAL340, ASP341, TYR366, ARG367, THR368 marrow mesenchymal stem cells(BM-MSCs), but also to the improved osteoimmunomodulatory microenvironment. 15 wt% ASP15, GLYI, VAL18, THR32, LEU49, GLU50,GLU51,VAL52,VAL53,31 HIS54, ALA55, ASP56, GLN57, SER58, SER59, CYS60, THR61, PHE62 The interaction between BM-MSCs and macrophages enhance ASP63, ASN64, ASP80, GLU137, GLU141, ASP277, PRO289, GLU312, bone repair by accelerating the transition from inflammatory immune response to regenerative immune response within the 20 wt% ASP56, CLN57 ASP63 ASP122, LU137, ASP138 GLU141 SER147, 19 bone defect areas( Figure 1b) GLLUI71, PRO289, THR313, GLY314, ASN316, PRO338, GLY33 VAL340, ASP341, ARG367, THR368 2. Results and Discussion RGD peptide is an archetypical ligand in the 10th type Ill 2.1. MD Simulation of RGD Exposure on CFO/P(VDF-TrFE) domain of FN, which mediates cell adhesion through specific anes interactions with various integrin receptors. [I5I The simulation predicted that the RGD sequence on 10 wt% CFO content mem- To predict the biological properties of the built-in magneto- branes were exposed towards the solution phase(Figure 2a) electric microenvironment provided by our fabricated magne- which are conducive for integrin binding. The root mean toelectric nanocomposite membrane, MD simulations were square deviation values!(0. 193 for 5 wt%, 0. 211 for 10 wt%, performed. The CFO/P(VDF-TrFE) magnetoelectric nanocom- 0.160 for 15 wt%, 0.148 for 20 wt%)of RGD indicated the high posite membrane was composed of ferromagnetic CFO nano- interdomain elasticity and flexibility of the RgD configuration particles embedded within a ferroelectric P(VDF-TrFE) matrix in the 10 wt% CFO content membranes. The enhanced RGD that provide excellent flexibility for the membrane. In this elasticity and flexibility could in turn facilitate RGD-integrin study, nanocomposite membranes with 5-20 wt% CFO content binding. RGD peptide was reported to facilitate cell spreading were selected for the MD simulations(Figure Sla, Supporting and motility, I stem cell differentiation, IS) and nanoparticle Information). Within the simulation models(Figure S1b, Sup- internalization, ib, 19 possibly through the assembly of clus porting Information), 10 wt% CFO content membranes exhib- ters of adhesion proteins. 120 Overall, the MD simulations indi ited the minimum distance between the FN-Ill7-10 protein cated that the 10 wt% CFO content membranes could enhance and nanocomposite membrane(Figure SIc, Supporting Infor- FN-I1I7-10 adsorption and optimize RGD domain exposure to mation). Additionally, the 10 wt% CFO content membranes promote cell adhesion. 21) According to previous studies, the exhibited strong interaction between the fibronectin (FN) FN adsorption capacity is an important extracellular environ nodule and the surface of the membrane, which possessed mental factor, which directly effects initial cell attachment and the greatest numbers of adsorbed FN-lll7-10 residues within proliferation, 2 Cells adhesion and spreading area was also 0.35 nm(Table 1). The enhanced FN-1ll7-10 protein adsorption positively correlated with increased surface RGD density. 2) The by 10 wt% CFO content membranes was also supported by the strongest van der Waal interactions and the highest value of Table 2. The interac interaction energy(Table 2). Under the influence of long-range different CFO contents MD By between FN-I117-10 and membranes with electrostatic interactions, the protein gradually moved to the surface. As the protein gets closer to the surface, the short. dw)(k)me ange van der Waals interaction also starts to affect the protein o 702725 481.366 221.359 Therefore, the protein continuously adjusts its conformation and finally adsorbs stably on the surface, under the synergistic effects of van der waals and electrostatic interactions, There. 15%6 701.053 488.772 212.281 fore in our simulation, 10 wt% CFo content membranes were 20 wt% -543.027 134935 predicted to have the most favorable surface for FN-1117-10 pro- E interaction energy: bEee electrostatic interaction energy, E van de tein adsorption Adv Funct. Mater. 2020. 2006226 2006226日3of1) o 2020 Wiley-VCH GmbHwww.advancedsciencenews.com www.afm-journal.de 2006226 (3 of 11) © 2020 Wiley-VCH GmbH magnetic field can readily mimic the diurnal shifting magne￾toelectric environment.[14] Altogether, these studies point to promising applications of magnetoelectric materials in tissue regeneration. In this study, we fabricated the CoFe2O4 (CFO)/ poly(vinylidene fluoridetrifluoroethylene) [P(VDF-TrFE)] mag￾netoelectric nanocomposite membranes, which can be regu￾lated by application of a remote DC magnetic field to generate a built-in magnetoelectric microenvironment. Molecular dynamics (MD) simulation was used to predict the optimal content of CFO nanoparticles for attaining the greatest argi￾nylglycylaspartic acid (RGD) sites exposure. The magneto￾electric microenvironment provided by the magnetoelectric membranes can enhance osteogenesis and bone regenera￾tion within the bone defect area. The bone regeneration can not only be attributed to the direct osteogenic effect of bone marrow mesenchymal stem cells (BM-MSCs), but also to the improved osteoimmunomodulatory microenvironment. The interaction between BM-MSCs and macrophages enhance bone repair by accelerating the transition from inflammatory immune response to regenerative immune response within the bone defect areas. (Figure 1b) 2. Results and Discussion 2.1. MD Simulation of RGD Exposure on CFO/P(VDF-TrFE) Composite Membranes To predict the biological properties of the built-in magneto￾electric microenvironment provided by our fabricated magne￾toelectric nanocomposite membrane, MD simulations were performed. The CFO/P(VDF-TrFE) magnetoelectric nanocom￾posite membrane was composed of ferromagnetic CFO nano￾particles embedded within a ferroelectric P(VDF-TrFE) matrix that provide excellent flexibility for the membrane. In this study, nanocomposite membranes with 5–20 wt% CFO content were selected for the MD simulations (Figure S1a, Supporting Information). Within the simulation models (Figure S1b, Sup￾porting Information), 10 wt% CFO content membranes exhib￾ited the minimum distance between the FN-III7-10 protein and nanocomposite membrane (Figure S1c, Supporting Infor￾mation). Additionally, the 10 wt% CFO content membranes exhibited strong interaction between the fibronectin (FN) module and the surface of the membrane, which possessed the greatest numbers of adsorbed FN-III7-10 residues within 0.35 nm (Table 1). The enhanced FN-III7-10 protein adsorption by 10 wt% CFO content membranes was also supported by the strongest van der Waal interactions and the highest value of interaction energy (Table 2). Under the influence of long-range electrostatic interactions, the protein gradually moved to the surface. As the protein gets closer to the surface, the short￾range van der Waals interaction also starts to affect the protein. Therefore, the protein continuously adjusts its conformation and finally adsorbs stably on the surface, under the synergistic effects of van der Waals and electrostatic interactions. There￾fore in our simulation, 10 wt% CFO content membranes were predicted to have the most favorable surface for FN-III7-10 pro￾tein adsorption. RGD peptide is an archetypical ligand in the 10th type III domain of FN, which mediates cell adhesion through specific interactions with various integrin receptors.[15] The simulation predicted that the RGD sequence on 10 wt% CFO content mem￾branes were exposed towards the solution phase (Figure 2a), which are conducive for integrin binding. The root mean square deviation values[16] (0.193 for 5 wt%, 0.211 for 10 wt%, 0.160 for 15 wt%, 0.148 for 20 wt%) of RGD indicated the high interdomain elasticity and flexibility of the RGD configuration in the 10 wt% CFO content membranes. The enhanced RGD elasticity and flexibility could in turn facilitate RGD-integrin binding. RGD peptide was reported to facilitate cell spreading and motility,[17] stem cell differentiation,[18] and nanoparticle internalization,[17b,19] possibly through the assembly of clus￾ters of adhesion proteins.[20] Overall, the MD simulations indi￾cated that the 10 wt% CFO content membranes could enhance FN-III7-10 adsorption and optimize RGD domain exposure to promote cell adhesion.[21] According to previous studies, the FN adsorption capacity is an important extracellular environ￾mental factor, which directly effects initial cell attachment and proliferation.[22] Cells adhesion and spreading area was also positively correlated with increased surface RGD density.[23] The Table 1. Number of adsorbed residues of FN-III7-10 on membranes with different CFO contents (MD simulations). Surface Adsorbed residues Total 5 wt% ASN13,ASP15,THR16,GLY17,VAL18,LEU19,GLY46,ASN47,SER48, LEU49,GLU50,GLU51,VAL52,VAL53,HIS54,ASP56,GLN57,CYS60, THR61,PHE62,ASP63,ASN64,SER66,LEU69,ASP81,ASP122, LEU123,THR124,ASN125,GLU141,SER145,PRO146,SER147,ASP148, TYR170,GLU171,GLN172,PRO289,PRO338,GLY339,ASP341, TYR366,ARG367,THR368 44 10 wt% GLU11,ASN13,PRO14,ASP15,THR16,GLY17,VAL18,LEU19,THR20, LEU49,GLU51,VAL52,CAL53,HIS54,ALA55,ASP56,GLN57,SER59, CYS60,THR61,PHE62,ASP63,ASN64,LEU65,SER66,PRO67,ASP122, GLU141,SER145,SER147,ASP196,ILE197,GLU223,GLY249,THR250, GLU251,GLN271,SER273,THR274,VAL275,SER276,ASP277,VAL278, PRO289,GLU312,THR313,GLY314,GLY315,ASN316,SER317,LYS337, PRO338,GLY339,VAL340,ASP341,TYR366,ARG367,THR368 58 15 wt% ASP15,GLY17,VAL18,THR32,LEU49,GLU50,GLU51,VAL52,VAL53, HIS54,ALA55,ASP56,GLN57,SER58,SER59,CYS60,THR61,PHE62, ASP63,ASN64,ASP80,GLU137,GLU141,ASP277,PRO289,GLU312, PRO338,GLY339,ASP341,ARG367,THR368 31 20 wt% ASP56,GLN57,ASP63,ASP122,GLU137,ASP138,GLU141,SER147, GLLU171,PRO289,THR313,GLY314,ASN316,PRO338,GLY339, VAL340,ASP341,ARG367,THR368 19 Table 2. The interaction energy between FN-III7-10 and membranes with different CFO contents (MD simulations). Surface Einta) [kJ mol−1 ] Eeleb) [kJ mol−1 ] Evdwc) [kJ mol−1 ] 5 wt% −702.725 −481.366 −221.359 10 wt% −773.174 −431.619 −341.555 15 wt% −701.053 −488.772 −212.281 20 wt% −543.027 −408.092 −134.935 a)Eint, interaction energy; b)Eele, electrostatic interaction energy; c)Evdw, van der Waals interaction energy. Adv. Funct. Mater. 2020, 2006226