Cellular Response to Non-contacting Nanoscale Sublayer: Cells

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Cellular Response to Non-contacting Nanoscale Sublayer: Cells Sense Several Nanometer Mechanical Property Tomoyuki Azuma,† Yuji Teramura,†,‡ and Madoka Takai*,† †

Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Department of Immunology, Genetics and Pathology (IGP), Rudbeck Laboratory C5:3, Uppsala University, SE-751 85 Uppsala, Sweden

‡

S Supporting Information *

ABSTRACT: Cell adhesion is influenced not only from the surface property of materials but also from the mechanical properties of the nanometer sublayer just below the surface. In this study, we fabricated a well-defined diblock polymer brush composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) and 2-aminoethyl methacrylate (AEMA). The underlying layer of poly(MPC) is a highly viscous polymer, and the surface layer of poly(AEMA) is a cell-adhesive cationic polymer. The adhesion of L929 mouse fibroblasts was examined on the diblock polymer brush to see the effect of a noncontacting underlying polymer layer on the cell-adhesion behavior. Cells could sense the viscoelasticity of the underlying layers at the nanometer level, although the various fabricated diblock polymer brushes had the same surface property and the functional group. Thus, we found a new factor which could control cell spread at the nanometer level, and this insight would be important to design nanoscale biomaterials and interfaces. KEYWORDS: block polymer brush, cell adhesion, non-contacting layer, XRR, QCM-D

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the substrate might influence the cell-adhesion behavior.9,10 They showed that fibroblasts adhered while maintaining their round morphology on a bilayered polymer membrane made of polystyrene and polyisoprene where the thickness of the upper polystyrene layer was less than 25 nm. This study implied that the cell adhesion on polymer bilayers could be influenced from the second underlying polymer layer where cells could not directly contact. Therefore, it is important to evaluate the indirect effect of the non-contacting sublayer of the materials. In the previous study, although they could make a doublelayered polymer membrane, those two layers were sequentially coated with each polymer and not chemically bonded. It is not obvious for us whether the interaction between layers is necessary or not for the cell adhesion because the interlayer interaction might be different when the thickness of each layer is different in the case of a multilayer polymer system. A bilayer film with well-controlled and chemically bonded layers could be ideal to resolve previous issues and clarify the influence of the non-contacting layer. Polymer brushes, which have welldefined, chemically bonded, and controllable structures at the nanometer level,11 should be useful for this objective. In this study, we used such a well-defined diblock polymer brush and examined the influence of the non-contacting

INTRODUCTION Controlling the surface property of biomaterials is important for designing biocompatible materials because protein adsorption and cellular adhesion are known to be largely influenced by the surface property of such artificial materials. In particular, the influence of the different surface properties on cell adhesion has been studied in detail, including the wettability,1 the charge density,2 or the chemical functionality.3 In general, firm cell adhesion was observed on moderately hydrophilic surfaces with water contact angles between 40° and 60°1 and on charged surfaces with high absolute ζ potentials.2 In addition to these surface properties of the interface, the density of immobilized RGD is also a critical factor for the celladhesion process.4 The cell adhesion and spreading take place on the packed RGD surfaces where the distance between RGD motifs is less than 58−73 nm.4 Yet another critical factor for cell adhesion is the elasticity of the biomaterials.5−8 Engler et al. reported that stem cell lineage is actually affected by the material stiffness. Mesenchymal stem cells (MSCs) differentiated into neurocytes on soft matrices that mimic the brain (0.1−1 kPa), into myocytes on stiffer matrices that mimic the muscle (8−17 kPa), and into osteocytes on comparatively rigid matrices that mimic the collagenous bone (25−40 kPa). Thus, the cell adhesion is largely affected by the direct interaction between cellular surfaces and material surfaces where they come directly in contact. On the other hand, there were also interesting reports that the non-contacting sublayer of © 2016 American Chemical Society

Received: January 29, 2016 Accepted: April 11, 2016 Published: April 11, 2016 10710

DOI: 10.1021/acsami.6b01213 ACS Appl. Mater. Interfaces 2016, 8, 10710−10716

Research Article

ACS Applied Materials & Interfaces underlying polymer layer on the cell adhesion. We used homopolymer brushes (poly(MPC) and poly(AEMA)) as well as copolymer brushes (PMbA15 and PMbA50). Our diblock polymer brush was composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer as the lower layer and 2aminoethyl methacrylate (AEMA) as the upper layer (poly(MPC-block-AEMA) (PMbA)). MPC polymer is one of the bioinert materials and interacts with water.12 Also, MPC polymer shows high viscosity since it is in a hydrogel state in water due to the high interaction with water.13 Our hypothesis is that the highly viscous sublayer of poly(MPC) might influence the cell adhesion. Poly(AEMA) is selected as the surface layer since the polycationic surface is strongly interactive with the cell surface. The diblock polymer brush of MPC and AEMA has already been reported, and the property of this bilayer film was well-studied in our group.14,15 Here, we changed the unit number of the poly(AEMA) layer, which was located at the surface of the bilayer film, and examined the influence of the non-contacting poly(MPC) layer on the cell adhesion of L929 mouse fibroblasts.

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Figure 1. Chemical structures of polymers prepared in this study. was fabricated as previously reported.14 The concentrations of AEMA monomer, CuBr, bpy, and ethyl-2-bromoisobutyrate were 0.5, 0.01, 0.02, and 0.01 M, respectively where the mixture of H2O/2-propanol = 1/1 (v/v) was used as the solvent. After the resulting mixture was bubbled with Ar for 10 min at room temperature, ethyl 2bromoisobutyrate and the initiator-immobilized substrates were simultaneously added in the mixture and reacted for 4 h at room temperature. The reaction was stopped by adding O2, and then the substrates were collected. The collected substrates were washed with H2O and 2-propanol for 3 min by sonication and dried in vacuo overnight. In the case of PMbA50, 50-unit-long poly(MPC) brushes were fabricated and 50-unit-long poly(AEMA) brushes were then grown on top of the poly(MPC) brushes. Specifically, after poly(MPC) brushes were fabricated onto the initiator-immobilized substrates as described previously, the substrate was added into the mixture of CuBr/bpy/AEMA (0.01, 0.02, and 0.5M, respectively). The initiator (0.01 M) was finally added into the solution, and then they were reacted for 4 h at room temperature. In the case of PMbA15, 50unit-long poly(MPC) brushes were fabricated and 15-unit-long poly(AEMA) brushes were fabricated on top of poly(MPC) brushes. After poly(MPC) brushes were fabricated onto the initiatorimmobilized substrates as described previously, the substrate was added into the mixture of CuBr/bpy/AEMA (0.03, 0.07, and 0.5 M, respectively). The initiator (0.03 M) was finally added into the solution, and then they were reacted for 1 h at room temperature. The resultant reaction mixtures were collected and evaluated by 1H NMR (JNM-GX 270, JEOL, Tokyo, Japan) to calculate the conversion ratio. Methanol-d4 was used as the deuterated solvent for poly(MPC), and D2O was used for poly(AEMA). The graft density of poly(MPC) was changed as previously reported to examine the relationship between thickness and graft density.17 In order to change the graft density, the mixed-SAM of BrC10TCS and dodecyltrichlorosilane was used. BrC10TCS and dodecyltrichlorosilane were mixed at molar ratio of 10/90, 20/80, or 50/50, and the mixture solution was used for the coating of the surface. Then, poly(MPC) brush was grown on the mixed-SAM surface. Static Contact angle. The static contact angles of air bubbles in water on the polymer brush surfaces were measured by a contact angle meter (CA-W, Kyowa Interface Science Co., Tokyo, Japan) at room temperature. Substrates were immersed in water, and 10 μL of air bubbles were placed onto the substrates. ζ Potential. The ζ potentials of the polymer brush surfaces were measured by an electrophoretic light-scattering spectrophotometer (ELSZ1000Z, Otsuka Electron, Osaka, Japan) at room temperature. Polymer brushes were fabricated on the glass substrates, and their surface potentials were evaluated. Measurements were performed in 10 mM NaCl aqueous solution. Spectroscopic Ellipsometry. The thickness of polymer brushes was measured by a spectroscopic ellipsometry (alpha-SE, J. A. Woollam, Lincoln, NE, USA) in air at room temperature. A He−Ne

EXPERIMENTAL SECTION

Materials. 2-Methacryloyloxyethyl phosphorylcholine was purchased from NOF Co. (Tokyo, Japan). 2-Aminoethyl methacrylate hydrochloride, copper(I) bromide (CuBr), 2,2′-bipyridyl (bpy), ethyl2-bromoisobutyrate, dodecyltrichlorosilane, methanol-d4, deuterium oxide (D2O), bovine serum albumin (BSA), and fluorescein isothiocyanate (FITC)-labeled BSA were purchased from SigmaAldrich Co. (St. Louis, MO, USA). MPC and AEMA were used as received. Hexane, ethanol, methanol, acetone, 2-propanol, and toluene were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). These solvents were extra-pure grade and used without further purification. Dulbecco’s phosphate buffered saline (PBS, without calcium chloride and magnesium chloride), Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from Invitrogen Co. (Carlsbad, CA, USA). L929 cells were purchased from Riken Cell Bank (Ibaraki, Japan). Fabrication of Polymer Brush Surfaces. (11-(2-Bromo-2methyl)propionyloxy)undecyltrichlorosilane (BrC10TCS) was synthesized and used as the initiator for subsequent reactions, as previously reported.15 Here, we used a Si wafer with 10 nm thick SiO2, a SiO2coated QCM sensor chip, and slide glass. Those surfaces were washed in hexane, ethanol, and acetone for 3 min by sonication (2510-DTH ultrasonic cleaner, BRANSON, Kanagawa, Japan) and treated with O2 plasma for 5 min (300 W; 100 mL/min gas flow; PR500, Yamato Scientific Co., Ltd., Tokyo, Japan). Those treated surfaces were exposed to 2 mM BrC10TCS solution in toluene at room temperature overnight. Those surfaces were washed with toluene by sonication and dried in vacuo overnight. In order to fabricate polymer brushes of poly(MPC), poly(AEMA), and poly(MPC-block-AEMA) (PMbA) on the initiator-immobilized substrates, surface-initiated atom transfer radical polymerization (SI-ATRP) was used (Figure 1).15 In this study, we aimed to fabricate 50 units of poly(MPC) and poly(AEMA), the surface properties of which have already been well-characterized and reported by our group.14 Also 15 or 50 units of poly(AEMA) were polymerized on 50 units of poly(MPC) (PMbA15 and PMbA50, respectively). Poly(MPC) brush was fabricated as previously reported.16 Briefly, CuBr, bpy, and MPC monomers were dissolved in degassed methanol where the concentrations of MPC monomers, bpy, and CuBr were 0.5, 0.02, and 0.01 M, respectively. After the resulting mixture was bubbled with Ar for 10 min at room temperature, ethyl-2-bromoisobutyrate (0.01 M), used as the sacrificial initiator, and the initiator-immobilized substrates were simultaneously added in the mixture and reacted for 24 h at room temperature. The reaction was stopped by adding O2, and then the substrates were collected. The collected substrates were washed with methanol for 3 min by sonication and dried in vacuo overnight. Poly(AEMA) brush 10711

DOI: 10.1021/acsami.6b01213 ACS Appl. Mater. Interfaces 2016, 8, 10710−10716

Research Article

ACS Applied Materials & Interfaces Table 1. Properties (SCA, ζ Potential, Thickness, and Density) of Polymer Brushes Fabricated in This Study SCA of air in water (deg) ζ potential (mV) thicknessa (nm) thicknessb (nm) roughnessa (nm) graft densitya (chains/nm2) a

poly(MPC)

poly(AEMA)

PMbA15

PMbA50

168 ± 2 −3 ± 4 6.6 ± 0.6 5.9 ± 0.6 0.6 0.38 ± 0.02

131 ± 4 22 ± 3 6.0 ± 0.1 5.0 ± 0.1 1.5 0.56 ± 0.01

129 ± 6 15 ± 6 7.5 ± 0.1 6.3 ± 0.3 0.6 0.41 ± 0.02 (MPC layer) 0.34 ± 0.01 (AEMA layer)

138 ± 7 7±5 9.0 ± 0.5 8.5 ± 0.7 1.0 0.30 ± 0.02 (MPC layer) 0.19 ± 0.01 (AEMA layer)

Obtained by XRR. bObtained by spectroscopic ellipsometry.

Figure 2. (a) Raw XRR data of polymer brushes (where, for example, 1.E-01 represents 1 × 10−1). (b) Fitting data of XRR spectrum of poly(MPC). (c) Relationship between density and thickness of poly(MPC) fabricated on mixed-SAM, BrC10TCS and dodecyltrichlorosilane. (d) Relationship between graft density of poly(AEMA) layer and ζ potential. laser (632.8 nm) was used, and the incident angle was 70°. The obtained data were fitted with the Cauchy layer model, and the refractive index was assumed to be 1.488.18 X-ray Reflectivity Measurement. The thickness and the density of fabricated polymer brushes were evaluated by X-ray refectivity (XRR) measurements (SmartLab (9 kW), Rigaku Co., Tokyo, Japan) in air at room temperature, as previously reported.19 X-ray radiated from a Cu Kα source was focused on the substrates using a collimating mirror. The incident angle of X-ray was horizontal, and the detector was rotated by 2θ (0° < 2θ < 10°) while the substrates were rotated by θ during the measurements. The data fitting was conducted by the software Global Fit (Rigaku). In the cases of poly(MPC) and poly(AEMA), fitting was performed with a three-layer model (SiO2, BrC10TCS, and each polymer). In the case of PMbA, fitting was performed with a four-layer model (SiO2, BrC10TCS, poly(MPC), and poly(AEMA)). AFM Measurement. The surface topography of PMbA15 and PMbA50 was evaluated by AFM (NanoNavi II, SII Nano technology, Chiba, Japan). Measurement was operated in tapping mode under ambient conditions with the commercially available cantilever (RTESP

MPP-11100-10, Bruker, Kanagawa, Japan). The root-mean-square value was calculated from obtained contrast images. Amount of BSA Adsorption. The amount of adsorbed BSA was evaluated by the fluorescence intensity of adsorbed FITC-labeled BSA. Fabricated substrates were incubated in 1.0 mg/mL FITC-BSA/BSA solution (FITC-BSA/BSA = 1/9 (by molar ratio)) in PBS for 1 h at 37 °C. Then, the substrates were rinsed by PBS 3 times and observed by a fluorescence microscope (Axioskop2 plus, Carl Zeiss, Jena, Germany) at the exposure time of 1/5 s. Analyses of Interaction between BSA and Polymer Brush by QCM-D. The interaction between BSA and the polymer brushes surface was evaluated by quartz crystal microbalance with energy dissipation (QCM-D E4, Q-Sense, Gothenburg, Sweden). The measurement was conducted in a flow chamber at 25 °C. The ATcut quartz crystal sensors with SiO2 coating (fundamental resonance frequency was 4.95 MHz) were used, and polymer brushes were fabricated on these surfaces, as described earlier. The flow chamber was filled with PBS, and the signal was collected after the baseline was stabilized. After the surface was exposed to 1 mg/mL BSA solution in PBS for 30 min by injection at a flow rate of 0.25 mL/min, PBS was 10712

DOI: 10.1021/acsami.6b01213 ACS Appl. Mater. Interfaces 2016, 8, 10710−10716

Research Article

ACS Applied Materials & Interfaces

Figure 3. (a) Fluorescence intensity of adsorbed FITC-labeled BSA. (b) QCM charts of Δf about BSA adsorption on poly(MPC), poly(AEMA), PMbA15, and PMbA50. (c) QCM charts of ΔD about BSA adsorption on poly(MPC), poly(AEMA), PMbA15, and PMbA50. injected into the flow cell to rinse the unbound protein. The resonance frequency change (Δf) was calculated. The energy dissipation change (ΔD) was also calculated, and the adsorbed protein was evaluated. Cell Experiments. L929 cells were cultured in DMEM supplemented with 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin. Cells were cultured at 37 °C in 5% CO2 and 95% air. Polymer brushes were fabricated on the slide glasses (1 cm × 1 cm), and those plates were placed on tissue culture polystyrene dishes (TCPSs). After L929 cells were collected by trypsinization, cells were seeded on the polymer brush surfaces in the culture medium (2.0 × 104 cells in 1.0 mL) and cultured for 3 days. Cells were observed by a phase-contrast microscopy after 5, 24, 48, and 72 h postseeding (IX73, Olympus Co., Tokyo, Japan). The numbers of the adherent cells in microscopic images were manually counted, and five to six images were analyzed for each substrate.

poly(AEMA), PMbA15, and PMbA50 were 22 ± 3, 15 ± 6, and 7 ± 5 mV, respectively. Since the surface ζ potential was positive, the surface property is cationic on poly(AEMA). It was suggested that the block copolymerization of 15 units of AEMA onto poly(MPC) was enough to cover the whole surface. Next, those polymer brush surfaces were analyzed by XRR. Obtained spectra are shown in Figure 2a,b. The three-layer model (SiO2/BrC10TCS/MPC or AEMA) was used for the fitting of the spectra for poly(MPC) and poly(AEMA). The four-layer model (SiO2/BrC10TCS/MPC/AEMA) was used for the fitting of the spectra for PMbA15 and PMbA50. Figure 2c shows the relationship between graft density and the thickness of poly(MPC). The thickness was relatively constant in the low-graf t-density region, whereas the thickness exponentially increased as the graft density increased in the high-graf t-density region, showing that the poly(MPC) was in a mushroom state or a brush state, respectively.20 The boundary value was estimated to be approximately 0.1 chains/nm2, which is close to the previously reported value.21 Based on the XRR analyses, all of the polymer brushes were in a brush state since the graft densities of our polymers were more than 0.1 chains/ nm2. Based on the fitting, the graft density of the poly(AEMA) layer of PMbA15 was 0.34 chains/nm2, and 83% of the lower poly(MPC) layer (0.41 chain/nm2) was occupied with the upper poly(AEMA) layer. For the PMbA50, the graft density of the poly(AEMA) layer was 0.19 chains/nm2, indicating that the occupation ratio onto poly(MPC) (0.30 chain/nm2) was 63%. The conversion ratio of poly(AEMA) from poly(MPC) was lower than we anticipated because it is difficult to fabricate a block polymer brush by SI-ATRP.22 The catalyst-based side reaction occurs in normal SI-ATRP, which resulted in the loss

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RESULTS AND DISCUSSION Characterization of Polymer Brush Surfaces. Fabricated polymer brushes of poly(AEMA), poly(MPC), PMbA15, and PMbA50 were analyzed by measuring the static contact angle, the ζ potential, and the surface density. Table 1 summarizes the static contact angle of air in water, the surface ζ potential, the thickness, and the density of those surfaces. The static contact angles of air in water of poly(AEMA), PMbA15, and PMbA50 were 131 ± 4°, 129 ± 6°, and 138 ± 7°, respectively, which indicates that there are no differences in wettability among those three surfaces. Also, the static contact angle of air in water of poly(MPC) was 168 ± 2°. These results indicate that the surface properties of PMbA15 and PMbA50 reflect only poly(AEMA), and no influence of poly(MPC) located at the bottom layer was seen on the wettability. The ζ potential of poly(MPC) was neutral (−3 ± 4 mV), whereas those of 10713

DOI: 10.1021/acsami.6b01213 ACS Appl. Mater. Interfaces 2016, 8, 10710−10716

Research Article

ACS Applied Materials & Interfaces

Figure 4. Microscopic images of adhered L929 on TCPS (upper left), poly(MPC) (upper middle), poly(AEMA) (upper right), PMbA50 (lower left), and PMbA15 (lower middle) after 5 (a), 24 (b), and 72 h (c) from seeding. (d) Number of adhered L929 on poly(AEMA), PMbA50, and PMbA15 after 24, 48, and 72 h from seeding.

0.6, 6.0 ± 0.1, 7.5 ± 0.1, and 9.0 ± 0.5 nm, respectively. These results are in agreement with the ellipsometric analyses. For the analysis of the influence of the upper poly(AEMA) layer on the ζ potential, the ζ potential was plotted to the graft density

of chain-end functionality. In this case, bromine chain-end functionality was lost while polymerization of MPC and the conversion of poly(AEMA) remained low. The thicknesses of poly(MPC), poly(AEMA), PMbA15, and PMbA50 were 6.6 ± 10714

DOI: 10.1021/acsami.6b01213 ACS Appl. Mater. Interfaces 2016, 8, 10710−10716

Research Article

ACS Applied Materials & Interfaces (Figure 2d). The ζ potential was high on the surface with high graft density of poly(AEMA), suggesting that the ζ potential depends on the graft density of poly(AEMA). Therefore, the lower layer of poly(MPC) was fully covered with the upper layer of poly(AEMA) and no effect of poly(MPC) on the surface property was seen. Additionally, the roughness of PMbA15 and PMbA50 was evaluated by AFM. The root-meansquare values of PMbA15 and PMbA50 were 0.15 and 0.79 nm, respectively. This result was in agreement with that of XRR and also indicates that the lower layer of poly(MPC) was fully covered with the upper layer of poly(AEMA). Thus, the block polymer brush was successfully fabricated in order to examine the indirect influence of the lower poly(MPC) layer on protein adsorption and cell adhesion. BSA Adsorption onto Polymer Brush Surfaces. We performed the adsorption tests using FITC-labeled BSA on our polymer brush surfaces (Figure 3a). There was indeed BSA binding onto the surface, and no significant differences were seen in the amount of adsorbed BSA among poly(AEMA), PMbA15, and PMbA50 surfaces. Also, the BSA adsorption on the surfaces was analyzed by QCM-D (Figure 3b,c). Figure 3b shows the frequency change, and this value relates with the change in mass on the surface. There was no BSA adsorption on the poly(MPC) surface while BSA adsorption was observed on the poly(AEMA) surface, which is consistent with previous studies.23 On the other hand, substantially larger frequency changes were observed on PMbA15 and PMbA50 than that on poly(AEMA). We expected that the QCM results would be similar to that of the BSA adsorption tests using FITC-labeled BSA; however, the results were different among the poly(AEMA), PMbA15, and PMbA50. In addition, the data of the energy dissipation changes were confounding (Figure 3c), where the energy dissipation change relates with the change in viscoelasticity of the surface. Although the energy dissipation changes were understandable on poly(MPC) and poly(AEMA) surfaces, the changes on PMbA15 and PMbA50 were unexpected since a negative energy dissipation change was seen on PMbA15, while a large positive change was seen on PMbA50 upon BSA binding. Presumably, the lower poly(MPC) layers of PMbA15 and PMbA50 exhibit high viscosity.24 Previous reports showed that the QCM-D measurement on surfaces with high viscosity results in the divergence from the ideal mass change (obtained by the Sauerbrey equation).25,26 Due to the high viscosity of the poly(MPC) layer, inaccurate values for both the frequency and the energy dissipation changes may have been observed upon BSA adsorption. However, this phenomenon implies that the lower layer of poly(MPC) significantly affects the viscoelasticity during the protein adsorption onto the upper layer of poly(AEMA). Cell Adhesion onto Polymer Brush Surfaces. Cell adhesion was then examined on our polymer brush surfaces using mouse L929 fibroblasts (Figure 4a−c). No cell adhesion was observed on poly(MPC) for 72 h as previously reported.27 On the other hand, the adhesion of L929 was observed on poly(AEMA), PMbA15, and PMbA50 surfaces. On poly(AEMA) and PMbA50 surfaces, L929 immediately adhered and spread on the surface over time to an extent similar to that of the TCPS surface. However, on the PMbA15 surface, L929 did not spread well for 72 h and the morphology of the adhered cells remained round although those cells adhered immediately. The growth rates of adhered cells were the same among poly(AEMA), PMbA50, and PMbA15 surfaces (Figure 4d).

Interestingly, the adherent cells on PMbA15 proliferated while maintaining their round morphology at almost the same rate as those adhered on poly(AEMA) and PMbA50. Since there were no significant differences among the surface properties of poly(AEMA), PMbA50, and PMbA15, the differences in cell-adhesion behavior cannot be explained directly by the interactions between cell surfaces and polymer brush surfaces. Our hypothesis is that the viscoelasticity of the non-contacting lower poly(MPC) layer of PMbA15 could have influenced the cell adhesion. So far, it has been well-known that cell adhesion is influenced by the surface property of culture dishes; however, there is hardly information on the effect of this non-contacting layer. Shimomura et al. reported that cell adhesion of fibroblast depends on the mechanical instability of the underlying layer, where this sublayer was not a cellcontacting layer.10 They made thin polystyrene films on the bulk polyisoprene by spin-coating. The morphology of adhered fibroblasts was round when the polystyrene film was thinner than approximately 25 nm. On the other hand, adhered cells spread well on polystyrene films with thickness of more than 25 nm. Although the adhesion mechanism is not fully understand, the cell adhesion is influenced by the non-contacting underlying layer. The adhered cells may have sensed the mechanical instability of the underlying polyisoprene layer. Because the polystyrene film was not chemically bonded with the underlying polyisoprene in their study, the response of the adhered cells may have been lower than that observed in our study. However, their results are in agreement with our hypothesis. The cell adhesion on the PMbA15 surface clearly showed the round state of adhered cells, which was different from PMbA50 and poly(AEMA) surfaces. The underlying layer of poly(MPC) with high viscosity actually influenced the cell adhesion. Although PMbA50 is a block copolymer of 50 units of poly(MPC) and 50 units of poly(AEMA), the adhered cells spread well, which was different from PMbA15. Since the 50 units of the poly(AEMA) layer were long enough to compromise the effect of the poly(MPC) layer, cells presumably did not sense the mechanical property. Additionally, we changed the polymerization degree of poly(MPC) from 50 to 100 units to evaluate the influence of the underlying layer of PMbA50 and examined the cell-adhesion behavior (Supporting Information Figure S1). L929 firmly adhered and well-spread on the surface, indicating that 50 units of the poly(AEMA) layer was long enough to compromise the effect of the underlying poly(MPC) layer. Also the mechanical property of fabricated diblock polymer brush was evaluated in PBS by AFM (Cypher ES AFM, Asylum Research, Santa Barbara, CA, USA; Figure S.2). The force curve of PMbA15, which is related to the mechanical property, was not the same with PMbA50 and close to the poly(MPC). Thus, our results suggested that cells could sense the mechanical property of the underlying layer, even at the nanometer level. And it was suggested that cells recognize not only the outermost surfaces where cells adhere but also a few nanometer region from the outermost surface as the scaffold.

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CONCLUSION We found that the influence of the mechanical property originating from the nanoscale underlying layer can influence the cell adhesion and spreading. Cells could sense the mechanical property of the underlying layer and proliferate normally while keeping their rounded morphology if the surface layer was within a few nanometers in which region the 10715

DOI: 10.1021/acsami.6b01213 ACS Appl. Mater. Interfaces 2016, 8, 10710−10716

Research Article

ACS Applied Materials & Interfaces

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cells may be able to recognize as their scaffold. Thus, we found a new factor, which could control cell spread at the nanometer sublayer. This nanoscale effect on mechanical and surface properties would be an important prospect for designing new biomaterials.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b01213. Effect of underlying poly(MPC) layer and force curve measurement by AFM (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +81-3-5841-7125. Fax: +81-3-5841-0621. E-mail: takai@ bis.t.u-tokyo.ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas ‘‘Molecular Soft-Interface Science’’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan (20106001). A part of this work was conducted in the Research Hub for Advanced Nano Characterization. We thank Dr. K. Kushiro from the University of Tokyo for helpful discussions and comments. We also thank Mr. H. Sugasawa from the Oxford Instruments for the technical support of AFM (Cypher ES AFM, Asylum Research, Santa Barbara, CA, USA).

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REFERENCES

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DOI: 10.1021/acsami.6b01213 ACS Appl. Mater. Interfaces 2016, 8, 10710−10716