SCENCNNEWS A www.advancedsciencenews.com ww.afm-journalde of macrophages. 2 Hence, our data indicated that the RGD- 10-E/M membrane exhibited the strongest immunostaining integrin binding induced PI3K/Akt activation could be one intensity of CCR(Figure $15, Supporting Informa the potential mechanisms by which the CFO10-E/M group amongst all groups. The immunostaining signals of CD206 mediates M2 polarization of macrophages were weak in all groups(Figure S17, Supporting Information) To evaluate osteogenic differentiation in this co-culture After 4 days of implantation, cells from the interstitial fluid col- Sy'stem, ALP activity(Figure $13b, Supporting Information), lected at the defect region of the CFO 10-EM group showed zarin red staining(Figure 4c), immunofluorescence staining the highest ratio of CDllc-positive cells(MI macrophage ( Figure 4d), and osteogenic gene expression(Figure 4e, f) anal- marker) among all groups(Figure $18a, Supporting Informa is were performed. CFo 10-E/M exhibited the most BM-MSc tion). MI macrophages contribute to an initial acute inflamma osteogenic differentiation among all groups. Macrophages with tory stage in vivo s8 Meanwhile, the protein spectrum results M2 phenotype secrete cytokines, such as IL-10, BMP2, and demonstrated that complement components such as Clr, Cfh VEGF, which in turn modulate mesenchymal progenitor cell C5, Cfb, and C8b were upregulated in the CFo 10-E/M group recruitment, angiogenesis and bone regeneration. H Therefore, Enrichment of immune-related pathways and biological pro- M2 polarization of macrophages is one important mechanism cesses, such as endocytosis, complement, and coagulation cas- by which BM-MSC osteogenic differentiation is promoted by cade were also detected in the CFO 10-E/M group(Figure 5c, the magnetoelectric microenvironment. Figure S16b, Supporting Information). There is increasing evi- dence that the complement system, a crucial arm of the innate immune system, plays an important role in bone homeostasis 2.6. Biomimetic Magnetoelectric Microenvironment Accelerates regeneration, and inflammation. Fa The activated coagulation Bone Regeneration In Vivo and complement products can recruit immune cells to the injury site, which leads to a simultaneous early inflammatory The therapeutic efficacy of the magnetoelectric nanocomposite response. 42 Bone regeneration can be modulated by these membranes on bone defect repair were further investigated in inflammatory molecules. -I MI phenotype macrophages arrived vivo. The magnetoelectric nanocomposite membranes were at the injury site during the initial stage and are involved in mplanted to cover critical-sized (5 mm) calvarial defects in the early infammatory response. +4 In this study, the early M1 mature rats. I26 The P(VDF-TrFE) based membrane was not macrophage response in vivo could not be detected by in vitro sticky to the newly regenerated bone tissue, 1 which facilitates testing, and this might be because the complex osteoimmu- subsequent removal of the membrane and bone defect healing nomodulatory environment of the bone defect area is diffcult without residual materials. Since the removable membrane to mimic in vitro 37b These results thus imply that the CFO can be remotely tuned with an external DC magnetic field, the 10-E/M membrane could trigger the initial immune response removed membranes have the potential to be reusable. His. during the early stage of bone repair. logical analysis of Massons trichrome staining and H&E We next evaluated the of mi to m2 transition taining showed that the CFo 10-E/M led to complete healing, affected by built- in magnetoelectric microenvironment in vivo with flat and consecutive bone structures in 8 weeks. The After 4 days of implantation, assessment of adherent macro- mature osteoid tissue was present in the top center region of phages on the nanocomposite membranes showed that the the defect in the CFO 10-E/M group. These results revealed that immunostaining intensity of CD206 in the CFO 10-E/M group the CFO 10-E/M membrane promoted more new bone forma- was stronger than the other groups(Figure Se)and that the ion than the other groups( Figure S14a, b, Supporting Informa- immunostaining intensity of CCR7 in the CFO 10-E/M group on). In micro-CT tests, the CFO 10-E/M group demonstrated was weaker than the other groups( Figure S15, Supporting the most new bone formation at all-time points. After 4 and Information). After 14 days of implantation, adherent macro- 8 weeks post-surgery, the CFo 10-E/M group demonstrated phages on the nanocomposite membrane and cells from the homogeneous and contiguous regenerated mature bone tissue interstitial fluid collected at the defect regions of the CFO within the defect area. By contrast, in the other groups, new 10-E/M group exhibited the highest ratio of M2 macrophages bone tissue formed mostly at the marginal areas around the(Figures S17 and S18b, Supporting Information)Meanwhile, riginal bone defect( Figure 5a; Figure S14c, Supporting Infor- the adherent macrophages on nanocomposite membranes mation). Quantitative analysis revealed that the CFo 10-E/M demonstrated the weakest CCR7 immunostaining signals in membrane significantly increased regenerated bone volume the CFO 10-E/M group(Figure S16a, Supporting Informa- after 8 weeks of implantation(Figure 5b). These results thus tion). Notably, macrophage M2 polarization happened earlier confirmed that the magnetoelectric microenvironment pro- on the membrane than in the interstitial fuid of the CFO vided by the CFO 10-E/M membrane promotes enhanced bone 10-E/M group, which suggested that the cells adherent on the membrane can sense the magnetoelectric microenvironment directly and further accelerate the transition from the Mi to M2 phenotype. Cells within the membrane proximity will 7. Biomimetic Magnetoelectric Microenvironment Modulates sense the magnetoelectric microenvironment later than cells Osteoimmunomodulatory Responses In Vive that are in direct contact with the membrane since bone is a highly dynamic organ, the fracture healing is affected by the The osteoimmunomodulatory effects of the built-in magi surrounding fracture microenvironment, such as inflamma electric microenvironment were further investigated in tory processes. 4] The interstitial fluid provides the 3D env After 1 day of implantation, adherent macrophages on the ronment for inflammatory response, which is important for Adw. Funct Mater. 2020. 2006226 2006226(8of1) g 2020 Wiley-VCH GmbHwww.advancedsciencenews.com www.afm-journal.de 2006226 (8 of 11) © 2020 Wiley-VCH GmbH of macrophages.[32] Hence, our data indicated that the RGD￾integrin binding induced PI3K/Akt activation could be one of the potential mechanisms by which the CFO10-E/M group mediates M2 polarization of macrophages. To evaluate osteogenic differentiation in this co-culture system, ALP activity (Figure S13b, Supporting Information), alizarin red staining (Figure 4c), immunofluorescence staining (Figure 4d), and osteogenic gene expression (Figure 4e,f) anal￾ysis were performed. CFO 10-E/M exhibited the most BM-MSC osteogenic differentiation among all groups. Macrophages with M2 phenotype secrete cytokines, such as IL-10, BMP2, and VEGF, which in turn modulate mesenchymal progenitor cell recruitment, angiogenesis and bone regeneration.[41] Therefore, M2 polarization of macrophages is one important mechanism by which BM-MSC osteogenic differentiation is promoted by the magnetoelectric microenvironment. 2.6. Biomimetic Magnetoelectric Microenvironment Accelerates Bone Regeneration In Vivo The therapeutic efficacy of the magnetoelectric nanocomposite membranes on bone defect repair were further investigated in vivo. The magnetoelectric nanocomposite membranes were implanted to cover critical-sized (5  mm)  calvarial defects in mature rats.[26] The P(VDF-TrFE) based membrane was not sticky to the newly regenerated bone tissue,[13] which facilitates subsequent removal of the membrane and bone defect healing without residual materials. Since the removable membrane can be remotely tuned with an external DC magnetic field, the removed membranes have the potential to be reusable. His￾tological analysis of Masson’s trichrome staining and H&E staining showed that the CFO 10-E/M led to complete healing, with flat and consecutive bone structures in 8 weeks. The mature osteoid tissue was present in the top center region of the defect in the CFO 10-E/M group. These results revealed that the CFO 10-E/M membrane promoted more new bone forma￾tion than the other groups (Figure S14a,b, Supporting Informa￾tion). In micro-CT tests, the CFO 10-E/M group demonstrated the most new bone formation at all-time points. After 4 and 8 weeks post-surgery, the CFO 10-E/M group demonstrated homogeneous and contiguous regenerated mature bone tissue within the defect area. By contrast, in the other groups, new bone tissue formed mostly at the marginal areas around the original bone defect (Figure 5a; Figure S14c, Supporting Infor￾mation). Quantitative analysis revealed that the CFO 10-E/M membrane significantly increased regenerated bone volume after 8 weeks of implantation (Figure  5b). These results thus confirmed that the magnetoelectric microenvironment pro￾vided by the CFO 10-E/M membrane promotes enhanced bone regeneration in vivo. 2.7. Biomimetic Magnetoelectric Microenvironment Modulates Osteoimmunomodulatory Responses In Vivo The osteoimmunomodulatory effects of the built-in magneto￾electric microenvironment were further investigated in vivo. After 1 day of implantation, adherent macrophages on the CFO 10-E/M membrane exhibited the strongest immunostaining intensity of CCR7 (Figure S15, Supporting Information) amongst all groups. The immunostaining signals of CD206 were weak in all groups (Figure S17, Supporting Information). After 4 days of implantation, cells from the interstitial fluid col￾lected at the defect region of the CFO 10-E/M group showed the highest ratio of CD11c-positive cells (M1 macrophage marker) among all groups (Figure S18a, Supporting Informa￾tion). M1 macrophages contribute to an initial acute inflamma￾tory stage in vivo.[38] Meanwhile, the protein spectrum results demonstrated that complement components such as C1r, Cfh, C5, Cfb, and C8b were upregulated in the CFO 10-E/M group. Enrichment of immune-related pathways and biological pro￾cesses, such as endocytosis, complement, and coagulation cas￾cade were also detected in the CFO 10-E/M group (Figure 5c,d; Figure S16b, Supporting Information). There is increasing evi￾dence that the complement system, a crucial arm of the innate immune system, plays an important role in bone homeostasis, regeneration, and inflammation.[42a] The activated coagulation and complement products can recruit immune cells to the injury site, which leads to a simultaneous early inflammatory response.[42] Bone regeneration can be modulated by these inflammatory molecules.[43] M1 phenotype macrophages arrived at the injury site during the initial stage and are involved in the early inflammatory response.[44] In this study, the early M1 macrophage response in vivo could not be detected by in vitro testing, and this might be because the complex osteoimmu￾nomodulatory environment of the bone defect area is difficult to mimic in vitro.[37b] These results thus imply that the CFO 10-E/M membrane could trigger the initial immune response during the early stage of bone repair. We next evaluated the process of M1 to M2 transition affected by built-in magnetoelectric microenvironment in vivo. After 4 days of implantation, assessment of adherent macro￾phages on the nanocomposite membranes showed that the immunostaining intensity of CD206 in the CFO 10-E/M group was stronger than the other groups (Figure  5e) and that the immunostaining intensity of CCR7 in the CFO 10-E/M group was weaker than the other groups (Figure S15, Supporting Information). After 14 days of implantation, adherent macro￾phages on the nanocomposite membrane and cells from the interstitial fluid collected at the defect regions of the CFO 10-E/M group exhibited the highest ratio of M2 macrophages. (Figures S17 and S18b, Supporting Information) Meanwhile, the adherent macrophages on nanocomposite membranes demonstrated the weakest CCR7 immunostaining signals in the CFO 10-E/M group (Figure S16a, Supporting Informa￾tion). Notably, macrophage M2 polarization happened earlier on the membrane than in the interstitial fluid of the CFO 10-E/M group, which suggested that the cells adherent on the membrane can sense the magnetoelectric microenvironment directly and further accelerate the transition from the M1 to M2 phenotype. Cells within the membrane proximity will sense the magnetoelectric microenvironment later than cells that are in direct contact with the membrane. Since bone is a highly dynamic organ, the fracture healing is affected by the surrounding fracture microenvironment, such as inflamma￾tory processes.[45] The interstitial fluid provides the 3D envi￾ronment for inflammatory response, which is important for Adv. Funct. Mater. 2020, 2006226