Nanostructural Surfaces with Different Elastic Moduli Regulate the

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Nanostructural Surfaces with Different Elastic ModulI Regulate the Immune Response by Stretching Macrophages Lan Chen, Donghui Wang, Feng Peng, Jiajun Qiu, Liping Ouyang, Yuqin Qiao, and Xuanyong Liu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b00237 • Publication Date (Web): 15 May 2019 Downloaded from http://pubs.acs.org on May 15, 2019

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Nanostructural Surfaces with Different Elastic Moduli Regulate the Immune Response by Stretching Macrophages

Lan Chena,b, Donghui Wanga, Feng Penga,b, Jiajun Qiua,b, Liping Ouyanga,b, Yuqin Qiaoa, Xuanyong Liua,b*

a

State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai

Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China b

Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy

of Sciences, Beijing 100049, China

*Corresponding Authors: Prof. Xuanyong Liu: E-mail: [email protected]; Tel.: +86 21 52412409; Fax: +86 21 52412409.

ACS Paragon Plus Environment

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Abstract A proper immune response is key for the successful implantation of biomaterials, and designing and fabricating biomaterials to regulate immune responses is the future trend. In this work, three different nanostructures were constructed on the surface of titanium using a hydrothermal method, and through a series of in vitro and in vivo experiments, we found that the aspect ratio of nanostructures can affect the elastic modulus of a material surface and further regulate immune cell behaviors. This work demonstrates that nanostructures with a higher aspect ratio can endow a material surface with a lower elastic modulus, which was confirmed by experiments and theoretical analyses. The deflection of nanostructures under the cell adsorption force is a substantial factor in stretching macrophages to enhance cell adhesion and spreading, further inducing macrophage polarization toward the M1 phenotype and leading to intense immune responses. In contrast, a nanostructure with a lower aspect ratio on a material surface leads to a higher surface elastic modulus, making deflection of the material difficult and creating a surface that is not conducive to macrophage adhesion and spreading, thus reducing the immune response. Moreover, molecular biology experiments indicated that regulation of the immune response by the elastic modulus is primarily related to the NF-κB signaling pathway. These findings suggest that the immune response can be regulated by constructing nanostructural surfaces with the proper elastic modulus through their influence on cell adhesion and spreading, which provides new insights into the surface design of biomaterials.

Key words: surface elastic modulus, macrophage polarization, immune response, implants, molecular mechanism


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At present, there are enormous demands for biomedical implants, and a proper immune response is key for successful implantation of biomaterials.1 After implantation, the proteins in body fluids are adsorbed onto the surface of the material within a very short time (nanosecond level).2 Then, the body forms blood clots around the material through activation of the complement system, and the intrinsic and exogenous coagulation pathway, meanwhile, recruits immune cells through secreting chemokines to enhance the immune response and osteoblasts to form new bone.3, 4

Thus, the immune response is a necessary step in the process of implant-bone binding.5, 6 Macrophages play an essential role in the innate immune response, and can be polarized into

two major phenotypes under the stimulus of implanted materials, the classical activated M1 phenotype and alternatively activated M2 phenotype.7-10 M1 phenotype inflammatory macrophages are activated by Th1 cytokines and secrete a large number of pro-inflammatory cytokines, such as interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), which can kill bacteria and other pathogens. Moreover, they can participate in the Th1 type inflammatory response as initiator and effector cells.11, 12 M2 phenotype macrophages participate in Th2 type inflammation by secreting a massive number of anti-inflammatory cytokines, such as interleukin4 (IL-4) and interleukin-10 (IL-10), and can promote extracellular matrix (ECM) reconstruction and new bone formation by releasing various growth factors including vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB) and transforming growth factorbeta (TGF-β).13-15 Thus, the M1/M2 macrophage proportion is very important for obtaining a proper immune response.16-18 Recent studies have indicated that the surface structure of materials, especially the nanostructure, is an essential factor in regulating immune cell behaviors.19-22 Chen et al. demonstrated that the adhesion, migration and cytokine secretion of macrophages on the surface


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of materials with a parallel groove striatum width of 250 nm to 2 μm were significantly different; notably, the immune response of cells on the surface of materials with nanoscale furrows, was significantly decreased.23 Wang et al. found that pro-inflammatory factor secretion by macrophages on the material surface of nanotube arrays was significantly decreased.24 Ma et al. showed that samples with small nanotubes were more likely to induce macrophage polarization into the M2 phenotype than those with large nanotubes.25 To date, many studies have focused on the mechanisms underlying the cellular behaviorregulation abilities of nanostructures and found that they may be affected by the following three factors:26, 27 (1) Wettability, construction of nanostructures will change the surface wettability, which will influence protein adsorption and affect cell adhesion, migration and differentiation.28, 29

(2) Size and spacing of the nanostructures, large surface asperity can block the movement of the

filopodial tip, delay its extension and push cell elongation.30,

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(3) Matrix elastic modulus or

stiffness, the surface modulus of a material can be affected by the nanostructure density and shape. It has been reported that a stiffer material surface will lead to more focal adhesion complex activation and stronger cell tension, which will further affect cell adhesion, migration, proliferation, and differentiation.32,

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However, there is still no general agreement on the

mechanisms underlying “nanostructure controlled cell behavior”, and there is a lack of studies focused on immune-related cells, which limits development of a new generation of biomaterials with the ability to control immune responses. In this regard, examining how biomaterials with different topographies mediate cell adhesion and the immune response of macrophages is of great scientific and engineering importance. Titanium-based materials have been extensively used in biomedical fields, especially orthopedic implants, because of their excellent biocompatibility.34 At present, there are many


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methods for constructing nanostructures on titanium surfaces, such as microarc oxidation treatment and anodic oxidation treatment, among others.34, 35 In this work, different nanostructures were constructed using a hydrothermal method with adjustable parameters and a stable and manageable procedure. The effects of nanostructures with different sizes on the behavior of mouse macrophages were evaluated through in vitro and in vivo experiments using RAW264.7 cells and C57BL/6 mice.36 The results revealed that nanostructures of various sizes possess significantly different surface elastic moduli and different deformabilities. When immune cells adhere to their surface, the nanostructures with various surface elastic moduli bend and stretch the macrophages to a different extent, which further affects the polarization of macrophages through the FAK-NFκB signaling pathway. The surface characterization of Ti samples before and after hydrothermal treatment, including morphology, roughness, crystal phase, element types and surface elastic modulus results, is shown in Figure 1. After mixed acid treatment, the oxide layer on the titanium surface is removed, and the surface of the titanium is flat with micron-scale ridge-like structures under an electron microscope (Figure 1a). After hydrothermal treatment, the original microstructure on the titanium surface is destroyed, and uniform nanoleaf-, nanosponge- and nanowire-like structures can be observed on different samples, with the obtained specimens denoted as “NL”, “NS” and “NW”, respectively (Figure 1a and Figure S1). The diameter and height of a single nanoleaf on the NL sample were 102.4 ± 8.1 and 305.4 ± 12.9 nm, respectively; those of a nanosponge on the NS sample were 29.0 ± 0.9 and 474.0 ± 25.3 nm; and for the NW sample, these values were 38.0 ± 1.6 and 3.5 ± 0.2 μm (Figure S1-2 and Table S1-3). The aspect ratio of the nanostructures showed the following trend: NL < NS < NW. Furthermore, it can be observed from the low-magnification SEM images that the nanostructures were clustered into bundles, and there was a significant


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difference in the density of the bundles. The NL sample possessed the largest bundle density (27.3 ± 1.7 μm-2), whereas, the density of the nanobundles was significantly decreased on the NS sample, and the density of nanowire bundles on the NW samples was the lowest (5.9 ± 0.8 vs. 0.0077 ± 0.0021 μm-2, respectively) (Figure S3 and Table S4). The NW sample exhibited the largest mean roughness (Ra) and root-mean-square height data (Rq) values compared with the other three samples, while the NS sample had the smallest Ra and Rq values (Figure 1b). The red area represents the concave region of the material surface, while the purple area represents the convex region of the material surface. The maximum fluctuation heights of the Ti, NL, NS, and NW surfaces were 136.7, 171.5, 192.5, and 288.7 nm, respectively, indicating that the surface roughness of the samples exhibited the following trend: NW > NS > NL > Ti. XRD patterns of the various samples are shown in Figure 1c. A diffraction peak centered at 2θ = 27.4°, which corresponds to the characteristic peak of anatase TiO2,37 was detected in the XRD patterns of all the samples with nanostructures, but no peak was present at 2θ = 27.4° in the XRD pattern of Ti. This finding indicates that the nanostructures on the surface of the NL, NS and NW samples are composed of anatase TiO2. XPS was used to detect the element types and the corresponding chemical states of the various samples (Table S5). There was no difference in the Ti or O high-resolution spectra of the NL, NS, and NW samples (Figure 1d). The Ti 2p peaks at 464.25 eV (Ti 2p1/2) and 458.55 eV (Ti 2p3/2) correspond to the binding energy of Ti4+ in the Ti (OH)62- group. The O 1s peak at 529.80 eV corresponds to the O2- binding energy of (Ti-OH) groups.38 The XRD and XPS results indicate that the nanostructures on the surfaces of the NL, NS, and NW samples have the same composition. The wettability of the sample surfaces is displayed in Figure 1e. The water contact angle of the Ti sample is approximately 60°, decreases to 18° for the NL and NS samples and further decreases to 0° for the NW sample. The superhydrophilicity


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of the NW samples may be the result of their special fibrous structures, which have a relatively high specific surface area and more crystal defects, endowing them with higher surface energy. Thus, more water molecules can be adsorbed onto the sample surface, consequently improving the sample hydrophilicity.19 The average surface elastic moduli of the different nanostructures as a function of indentation depths from 100 nm to 500 nm are depicted in Figure 1f, and the values for the NL, NS, and NW samples were 72.6 ± 8.01, 19.1 ± 2.06, and 0.8 ± 0.12 GPa, respectively. It was found that the long and flexible TiO2 nanowire structures endow the NW sample surface with the lowest elastic modulus, while the short and inflexible TiO2 nanoleaves give the NL sample surface the highest elastic modulus (Figure 1f). The trend of the average surface elastic modulus of these samples was Ti > NL > NS > NW. The elastic modulus represents the ratio of stress to strain in the elastic deformation stage, and is a stable index of mechanical properties and related to the chemical composition of materials and how the material is produced and processed.39-43 In this work, nanostructures with the same composition and crystal phase (TiO2) were constructed by a similar hydrothermal treatment (Figure 1c and d). Thus, the intrinsic elastic modulus (ETiO2) of different samples should remain constant. The huge variation in the tested elastic modulus (E′) is the result of significant differences in the structures of the various samples. In general, nanostructures with a higher aspect ratio are more easily bent under pressure and show a lower surface elastic modulus. Theoretically, E′ and ETiO2 have the following relation (See supplementary Equation S1-4 for the formula derivation process): 𝑑 3

𝐸′ = 𝐴 × 𝑛 × (ℎ) × 𝐸𝑇𝑖𝑂2 ,

(1)


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where A is a parameter related to testing conditions and the basic properties of samples, n is the number of nanostructures below the probe, and d and h are the diameter and height, respectively, of the TiO2 nanostructures. Therefore, the average elastic modulus of a nanostructured surface is inversely proportional to the third power of the nanostructure aspect ratio (h/d) and proportional to the density (n) of nanostructure bundles. Thus, nanostructures with denser bundles and a lower aspect ratio would endow the material surface with a larger surface elastic modulus, and nanostructures would be less likely to deform under external forces than nanostructures with a high aspect ratio and sparse bundles. The morphology and cell attachment area of macrophages on different samples are shown in Figure 2a and b. Although cell proliferation on the nanostructure surface was not inhibited compared with that on the Ti sample (shown in Figure S4a-c), the cell adhesion and spreading behaviors observed by SEM on various nanostructures were significantly different (Figure 2a). After cultured for 4 hours, most of the macrophages had already adhered to the samples; the cells on the Ti samples resembled polygonal stars, and the cells on the NL samples with nanoleaf structures were round, but the cells on the NS and NW samples were found to be well spread with longer filopodia. Especially, the cells on the NW samples with nanowire structures were entirely spread. Quantification of these results based on SEM examination after cultured for 4 hours is shown in Figure 2b, Figure S5 and Table S6. The mean values and standard deviations of the cell attachment areas were obtained by calculating all the cell areas on the sample surface under two randomly selected low-magnification visual fields (500 times magnification by SEM), and the results revealed that the average macrophage spreading area on the NW sample (162.9 ± 12.2 μm2) was approximately 3 times and 1.6 times greater than that on the NL and NS sample (48.4 ± 5.5 and 100.4 ± 7.6 μm2), respectively, while that on the Ti sample was 73.7 ± 9.9 μm2, and the cell


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spreading area on the NL sample was the lowest among the four groups. These results indicate that the nanostructure can affect cell adhesion and spreading. Previous studies have suggested that the material surface wettability, roughness or zeta potential may result in differences in cell adhesion and spreading. However, in the present study, the surface roughness (Figure 1b) among the three samples with various nanostructures was not significantly different, and although the water contact angles of the NL and NS samples are the same (Figure 1e), the responses of cells to these samples differed, indicating that the above factors are not the main causes of the different behavior of cells cultured on various samples. Note that there is a significant difference in the elastic modulus of the surface of samples with different nanostructures (Figure 1f); thus, it is conceivable that the regulation of macrophages behaviors by nanostructures may be primarily due to the differences in their elastic modulus. It has been reported that the elastic modulus of a material surface is an important factor that can affect cell adhesion and spreading.44, 45 In this work, to illustrate the relationship between deformation and the surface elastic modulus of nanostructures after contact with macrophages, the Euler-Bernoulli beam theory was applied. In this theory, a fixed load P acts on the tip of the nanostructures during interactions between macrophages and the nanostructures. The force is decomposed into forces along the nanostructure and forces perpendicular to the nanostructure, and only movement perpendicular to the nanostructure will affect adhesion of the cells. The relationship between deflection δ in the horizontal direction and the force P can be determined as shown in Equation 2; calculation details can be seen in Supplementary data Equation S5-8 and Figure S6. P = A′ × n ×

3

(𝑑ℎ)

(2)

× E′ × δ

where A' is a parameter related to test conditions and the basic properties of samples. Based on Equation 2, the relation between deflection δ and force P is shown in Figure 2c. The force P


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generated by cell adsorption onto the nanostructure surface is constant. Under the same force P, as the surface elastic modulus decreases and the aspect ratio increases, the deflection of nanostructures increases. In other words, when cells adhere to the surface of samples, the nanostructures with different sizes will bend to different extents, which is primarily related to differences in their surface elastic modulus. The nanowire structures with the highest aspect ratio on the NW sample, which had the lowest surface elastic modulus, exhibited the maximum deformation among all the samples, causing adhered cells to be stretched and consequently increasing the cell spreading area (Figure 2a and b). In contrast, the nanoleaf structures with the lowest aspect ratio on the NL sample give the NL sample surface the highest surface elastic modulus and exert minimal deformation, making it difficult for cells to adhere and spread on the sample surface. A schematic diagram is shown in Figure 2d. It is also worth noting that cell adhesion and spreading on the NW sample can be further improved by the fact that nanowires produce fewer barriers for cell adhesion and migration compared with other nanostructures. As shown in Figures S7-S8 and Table S7, the distance between two tips of nanostructure bundles on the samples was 27.05 ± 1.68, 5.85 ± 0.75, and 0.0077 ± 0.0021 μm (Figure S7 and Table S7), and the number of nanobundles below each cell on the sample was 15.16 ± 0.72, 6.86 ± 0.34, and 1.27 ± 0.25, respectively (Figure S8). In the case of the NL samples, denser bundles of nanoleaf structures were present, and the spaces between the adjacent bundles were the smallest among the surfaces (Figures S3 and S7), providing a large number of barriers that limit filopodia extension and further inhibit cell adhesion, while for the NW samples, there were fewer bundles of nanowire structures, and the spaces between the nanowire bundles were the largest (Figures S3 and S7) and were larger than the diameters of the


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macrophages, producing fewer barriers and allowing cells to adhere on the side of deflected nanowires and exhibit flat and extensive spreading. C-C chemokine receptor type 7 (CCR7), which is highly expressed on the surface of M1 macrophages, and mannose receptor CD206, which is highly expressed on M2 macrophages, were selected to evaluate the polarization of macrophages on samples toward the M1 and M2 phenotypes via flow cytometry.46-48 As shown in Figure 3a, the proportion of M2 macrophages showed the following trend: NL > NS = NW > Ti; conversely, the proportion of M1 macrophages exhibited the following trend: NS > NW > NL > Ti. However, the ratio of M2 and M1 macrophages on the NL samples was the highest compared with that on the other samples (Table S8). This result indicates that the NL sample significantly induced macrophage polarization to the M2 phenotype and decreased macrophage polarization toward the M1 phenotype, evidenced by the high proportion of M2 macrophages expressing the surface marker CD206 and the low proportion of M1 macrophages expressing the surface marker CCR7 on the NL surface compared with the other nanostructure samples. By contrast, on the NS and NW samples, the proportion of M1 macrophages was increased. Since M1 macrophages exhibit cytotoxic and pro-inflammatory functions, while M2 macrophages are associated with tuning of inflammatory responses, tissue remodeling and repair, this result indicates that the inflammatory response of cells on the NL sample would be the weakest.49 iNOS is primarily located in the cytoplasm, and CD206 is a transmembrane protein localized both intracellularly and at the cell surface; therefore, iNOS and CD206 were selected for immunofluorescence staining test because they are easily distinguished after staining in macrophages.48, 50, 51 Images and the mean optical density of iNOS (green; M1 macrophages) and CD206 (red; M2 macrophages) staining of macrophages on samples are shown in Figures 3b and


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S9. After incubation for 4 hours, a small number of macrophages were adhered to the surface of the samples, and the green fluorescence intensity of cells cultured on all the samples is strong. One day later, the red fluorescence intensity of macrophages on the NL sample was the strongest (p < 0.001) among all the groups. After incubation for 4 days, a large number of macrophages were growing on the sample surfaces, and the green fluorescence intensity of cells cultured on all the samples was stronger than the red fluorescence intensity, while the red fluorescence intensity of macrophages on the NL sample was the strongest (p < 0.001) among all the samples. The fluorescence intensity of samples indicates the following trend in the proportion of M2 macrophages on different samples: NL > Ti > NW > NS. These results suggest that macrophages on NL samples express the highest level of the M2 macrophage marker CD206, which is consistent with the results of the flow cytometry experiment. Based on the fact that M1 macrophages generate pro-inflammatory cytokines, such as IL-1β, IL-6 and TNF-α, to increase inflammation, while M2 macrophages suppress inflammation to enhance fibrosis and wound healing by increasing levels of anti-inflammatory cytokines, including IL-4, IL-10 and TGF-β, ELISAs were used to detect the release of IL-4, IL-6, IL-10, and TNF-α by macrophages (Figure 3c).52-59 The amounts of IL-4 and IL-10, which inhibit inflammation, released by cells on the NL sample were the highest and those released by cells on the NW sample were the lowest, and the general trend was NL > Ti> NS > NW. In contrast, the amounts of IL-6 and TNF-α, which promote inflammation, released by cells on the NL sample were the lowest compared with those released by cells on the other surfaces, and the amounts of IL-6 and TNF-α released by cells on the NW sample were the largest; the trend was NW > NS ≈ Ti > NL. These results can be explained by the fact that the NL sample induced macrophage polarization toward the M2 phenotype (Figure 3a and b), which resulted in increased IL-4 and IL-10 production and


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decreased IL-6 and TNF-α production, decreasing ROS generation (Figure S10) and the inflammation response.60, 61 The expression levels of immune-related gene were detected by RT-PCR, and the results are shown in Figure 3d. The expression level of the CCR7 gene in cells cultured on the nanostructure samples was significantly (p < 0.01) lower than that of cells on Ti, while the expression level of the CD206 gene in cells cultured on nanostructure samples was significantly (p < 0.01) higher than that of cells on Ti, but there was no obvious difference in CCR7 and CD206 gene expression levels in cells grown on the different nanostructure samples. The gene expression levels of the antiinflammatory cytokines IL-4 and IL-10 were the highest and those of the inflammatory cytokines IL-6 and TNF-α were the lowest in the four groups, which is consistent with the ELISA results. The general trend of the expression level of the inflammatory-related genes was NS ≈ Ti > NW > NL, and the general trend of the expression level of the anti-inflammatory-related genes was NL > Ti > NS and NW. However, these trends do not totally coincide with the ELISA results because PCR was used to detect the expression level of mRNA in macrophages on the fourth day after seeding, while ELISAs were used to detect the expression levels of proteins in cells at 4 days. The PCR results do not represent the same gene expression levels. Furthermore, the transcription and translation processes are affected by many factors, and thus, the expression levels of proteins detected by ELISA are not only determined by mRNA but also affected by other factors, such as the protein half-life and the speed of synthesis.62-64 The expression levels of FAK-NF-κB-related genes were measured by RT-PCR, and the results are shown in Figure 3e. The expression levels of FAK, Integrin αv, and Integrin β3, which are related to cell adhesion, were consistent with the cell adhesion and proliferation behaviors and presented the following trend: NS > NW > Ti > NL. Moreover, the cell adhesion-related gene


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expression levels affect the activation of downstream NF-κB-related genes. The NF-κB-related gene expression levels in cells on the NL sample were the lowest compared with those in the other samples, and the low NF-κB-related gene expression levels would likely decrease the expression levels of inflammatory-related genes. To reveal the molecular mechanism of the immune response induced by different nanostructures, the expression levels of typical signaling proteins were measured (Figure 3f). There were similar expression trends among FAK, NF-κB p65, and phosphorylated-NF-κB p65 (Ser536). Cells on the NW samples had the highest (p < 0.01) protein level compared with the other cells, while cells on the NL samples possess the lowest (p < 0.01) protein level among the four groups. These results are consistent with the RT-PCR analysis, and the general trend of the signaling protein expression levels was as follows: NW > NS > Ti > NL. PCR and WB (Figure 3e and f) results indicated that the immune responses of cells on the nanostructures with different surface elastic moduli could be regulated through the NF-κB signaling pathway (Figure 4). The expression levels of adhesion-related genes, including FAK, integrin αv, and integrin β3, in cells on the NL samples (with a high surface elastic modulus) were the lowest among the four groups, which would lead to inhibition of PI3K gene expression and further suppress the expression levels of downstream genes (including PDK1, AKT, and IKKα) in the NF-κB inflammatory signaling pathway.65, 66 A decrease in the IKKα expression level will directly reduce the catalytic activity of IκBs, which is the catalytic subunit of kinase IKK. IκBs combines with NF-κB dimers and maintains NF-κB in an inactive state. After IκBs is catalyzed by IKK, two conservative serine residues in IκBs are phosphorylated, leading to polyubiquitination under the catalytic activity of the SCF-E3 ubiquitin enzyme complex and further degradation by a protease to release NF-κB. Activated NF-κB is translocated into the nucleus and binds to the associated DNA sequence to induce transcription of target genes.67-69 Therefore, the reduction in


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the catalytic activity of IKKα in cells on NL samples will result in abnormal release of NF-κB from the inhibitory complex and a reduction in the number of NF-κB molecules transported into the nucleus, which further decreases the expression levels of downstream genes related to inflammation. According to the above results, it is believed that when the surface of a sample is not conducive to adhesion and spreading of macrophages, the inflammation response of macrophages is inhibited to a certain extent. The immune response of various samples in vivo was studied using a the mouse air-pouch model.70-72 Histological observation was performed at postoperative days 1 and 4, and air-pouch tissue sections were stained with hematoxylin-eosin (HE) and Masson’s trichrome (Figure 5a). A fibrous layer formed around the implanted samples, indicated by the arrow. HE and Masson’s trichrome staining of sections revealed that both the Ti and nanostructure implants were completely surrounded by a fibrous layer, predominantly composed of neutrophils, macrophages, and monocytes, at 1 and 4 days after implanted.73 The thickness of the fibrous layer and the total number of infiltrating cells throughout the Ti and NW implant site at day 4 were increased from that at day 1, while the number of infiltrating cells around the NL and NS samples decreased at day 4, and the thickness of the fibrous layer around the NL sample reduced by half, while that around the NS sample exhibited no obvious change. Quantitative analysis demonstrated that the fibrous capsule around the NL sample was the thinnest, and the thickness of the fibrous layer around the Ti, NL, NS, and NW samples at day 1 was 42.08 ± 4.08, 27.18 ± 1.28, 56.93 ± 2.78 and 74.93 ± 1.19 μm, respectively. In addition, at day 4, the thickness of the fibrous layer around the Ti, NL, NS, and NW samples was 61.63 ± 5.31, 15.67 ± 1.06, 62.73 ± 2.79 and 74.27 ± 5.43 μm, respectively. This is in agreement with the trend in the number of infiltrating inflammatory cells: NW > Ti > NS > NL. These findings may be attributed to the ability of the NL sample (with


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nanoleaf structures) to significantly promote M2 macrophage polarization, which could inhibit the inflammatory response to the biomaterial, including reducing inflammatory cell infiltration and inhibiting fibrous capsule formation.74 The implanted samples were also subjected to immunofluorescence staining for iNOS (green; M1 macrophages) and CD206 (red; M2 macrophages), as shown in Figure 5b, which also shows the mean optical densities (MODs). The iNOS-positive area was the largest (p < 0.01) in the NW group and the smallest (p < 0.01) in the NL group, while the CD206-positive area was the largest (p < 0.01) in the NL group at days 1 and 4. The trend in the MOD of CD206-positive cells was NL > Ti > NS > NW, but the trend in the MOD of iNOS-positive cells was the opposite: NW > NS ≈ Ti > NL. These results are consistent with the in vitro experiments and indicate that the implanted nanostructure samples induced macrophage polarization to the M1 or M2 phenotype in vivo. Moreover, the findings show that the NL sample in particular promoted M2 macrophage polarization the most effectively compared with the other samples. In conclusion, in this study, the elastic modulus of a material surface was found to be adjusted by construction of nanostructures on the material surface. The results show that nanostructures with dense bundles and a low aspect ratio on a material surface imbue the surface with a large elastic modulus, while nanostructures with a high aspect ratio and sparse bundles give the material surface a low elastic modulus. Furthermore, the elastic modulus of the material surface was found to be a primary factor in regulating immune cell behavior. For material surfaces with a low elastic modulus, the nanostructures easily deform, causing stretching of cells adhered to the surface, increasing cell spreading area, and promoting macrophages to polarize into the M1 phenotype to boost inflammation. The molecular mechanism was verified to be related to the FAK-NF-κB


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signaling pathway. Our work demonstrates that adjusting the aspect ratio of nanostructures can affect the elastic modulus of a material surface and regulate immune cell behaviors.

Associated Content

Author Information Corresponding Authors E-mail: [email protected]

Author Contributions The manuscript was written by Lan Chen, Donghui Wang and Xuanyong Liu; the experiments were performed by all the authors. All authors have approved the final version of the manuscript.

Notes The authors declare no competing financial interest.

Supporting Information The authors declare that data supporting the findings of this study are available within the paper and the Supplementary Information or are available from the authors upon request. The


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supporting information includes quantitative data, quantitative descriptions of some results, elastic modulus and force analyses, materials, and methods.

Acknowledgments Financial support from the National Natural Science Foundation for Distinguished Young Scholars of China (51525207), National Natural Science Foundation of China (51831011, 31870944), Science and Technology Commission of Shanghai Municipality (18410760600, 18YF1426900), International Partnership Program of Chinese Academy of Sciences Grant No.GJHZ1850 are acknowledged.

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Figure 1. Characterization of samples with different nanostructures. (a) SEM images of the surface morphology of various samples at low and high magnification (i, ii, iii, and iv present Ti, NL, NS, and NW, respectively). (b) AFM images of (i, iv, vii, x) three-dimensional morphology, (ii, v, viii, xi) two-dimensional morphology, and (iii, vi, ix, xii) the depth profile of different samples (i, ii, and iii present Ti; iv, v, and vi present NL; vii, viii, and ix present NS; x, xi, and xii present NW). (c) XRD patterns of the Ti, NL, NS, and NW samples. (d) XPS spectra of the NL, NS, and NW samples. (e) Water contact angles of the Ti, NL, NS, and NW samples. (f) Elastic modulus values of the Ti, NL, NS, and NW samples as a function of indentation depths from 100 nm to 500 nm. 177x188mm (300 x 300 DPI)


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Figure 2. Morphology and cell attachment area of macrophages on various samples in vitro. (a) SEM morphology of macrophages cultured on samples at low and high magnification (i and v present Ti; ii and vi present NL; iii and vii present NS; iv and viii present NW). (b) The cell attachment area of macrophages on Ti, NL, NS, and NW samples; two low-magnification fields (500 times magnification via SEM) were randomly selected to calculate the mean values and standard deviations. (c) Force P in N vs. tip deflection δ in μm for nanostructures with different elastic moduli. As the elastic modulus decreases, the same force leads to larger tip deflection. Similarly, a nanostructure with a high elastic modulus undergoes less bending under the same force. (d) Schematic diagram of macrophages adherence and spreading on nanostructures with different elastic moduli. 177x177mm (300 x 300 DPI)


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Figure 3. Immune response of macrophages on various samples in vitro. (a) Flow cytometry analyses of cellsurface markers on macrophages (i and v present Ti; ii and vi present NL; iii and vii present NS; iv and viii present NW). (b) Mean optical density for iNOS (M1 marker, stained green) and CD206 (M2 marker, stained red) in macrophages on Ti, NL, NS, and NW samples in immunofluorescence images; nuclei were stained with DAPI (blue). (c) ELISA determination of cytokines secreted from macrophages after incubation on samples for 4 days. (d) Relative mRNA expression levels of the immune-related genes CCR7, CD206, IL-4, IL-6, IL-10, and TNF-α in macrophages at day 4 after culture on sample surfaces. (e) PCR results showing FAK-NF-κB-related gene levels in macrophages at day 4 after culture on the Ti, NL, NS, and NW samples. (f) Western blotting images showing FAK (D2R2E), NF-κB p65 (D14E12), and phosphorylated-NF-κB p65 (Ser536) protein levels in cells cultured on samples, and the corresponding gray values of the three proteins. 177x199mm (300 x 300 DPI)


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Figure 4. The FAK-NF-κB signaling pathway mediates cell adhesion and the immune response of macrophages cultured on the surface of the nanostructure materials. 83x63mm (600 x 600 DPI)


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Figure 5. Immune response of macrophages on various samples in vivo. Images of hematoxylin eosin (HE) staining and Masson’s trichrome staining of air-pouch tissues adjacent to different samples at 1 and 4 days after implantation. (a) Images of HE staining of tissues showing the entire structure and layers of the airpouch tissues, and images of Masson’s trichrome-stained tissues obviously displaying the fibrous tissues and the infiltrating cells at days 1 and 4. The corresponding thickness of the fibrous layer and the number of infiltrated cells at days 1 and 4. (b) Immunofluorescence images and mean optical density of iNOS (M1 marker, stained green) and CD206 (M2 marker, stained red) in macrophages cultured on the Ti, NL, NS, and NW samples at 1 and 4 days after implantation; nuclei were stained with DAPI (blue). 177x151mm (300 x 300 DPI)


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Table of Content: Nanostructures of different sizes were constructed via hydrothermal treatment of a titanium surface, and the results showed that changes in nanostructure can affect the elastic modulus of a material surface. Biological experiments showed that the nanostructures endowed the material surface with different elastic moduli and could regulate the immune response by stretching macrophages to influence the cell adhesion and spreading. 83x35mm (600 x 600 DPI)


Nanostructural Surfaces with Different Elastic Moduli Regulate the

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