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Diffusion-Induced Hydrophilic Conversion of Polydimethylsiloxane/ Block-Type Phospholipid Polymer Hybrid Substrate for Temporal Cell-Adhesive Surface Ji-Hun Seo†,‡ and Kazuhiko Ishihara*,† †
Department of Materials Engineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Department of Materials Science and Engineering, School of Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea
‡
ABSTRACT: In this study, diffusion-induced hydrophobic−hydrophilic conversion of the surface of the cross-linked polydimethylsiloxane (PDMS) substrate was realized by employing a simple swelling−deswelling process of PDMS substrate in a block-type polymer solution with the aim of development of a temporal celladhesive substrate. The ABA block-type polymer composed of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) segment and PDMS segment with over 70% of dimethylsiloxane unit composition could be successfully incorporated in the PDMS substrate during the swelling−deswelling process to prepare the PDMS/phospholipid block copolymer hybrid substrates. During the aging process of the PDMS substrate for 4 days in aqueous medium, its surface property changed gradually from hydrophobic to hydrophilic. X-ray photoelectron spectroscopy and atomic force microscopy data provided strong evidence that the timedependent hydrophilic conversion of the PDMS/block-type phospholipid polymer hybrid substrate was induced by the diffusion of the hydrophilic PMPC segment in the block-type polymer to be tethered on the substrate. During the hydrophilic conversion process, surface-adsorbed fibronectin was gradually desorbed from the substrate surface, and this resulted in successful detachment of two-dimensional connected cell crowds. KEYWORDS: poly(2-methacryloyloxyethyl phosphorylcholine), polydimethylsiloxane, swelling−deswelling process, hybrid material, cell adhesion
1. INTRODUCTION A cross-linked polydimethylsiloxane (PDMS) possesses several attractive properties, such as good transparency, high oxygen permeability, good formability, high flexibility, and controllable stiffness, suitable to prepare many engineering devices.1,2 In recent years, PDMS-based cell culture devices also have been gaining much attention in the biomedical field, in addition to an increased interest in their use for mechanotransduction in the fields of cell biology and stem cell engineering.3 Because aspects of adhering cells such as the cell morphology, intracellular signaling pathway, and downstream stem cell lineage are known to depend strongly on the stiffness of material surfaces,4 the need for development of cell-adhesive surfaces with controllable stiffness has increased remarkably in the past decade.5 For this reason, cross-linked PDMS substrate, whose stiffness can be easily changed by controlling cross-linking density, has been broadly used to regulate the functions of adhering cells.6,7 Meanwhile, detached confluent cell crowds as a sheet-form are highly beneficial for being transplanted onto damaged tissue for the purpose of tissue regeneration.8−10 From this point of view, a PDMS-based cell culturing device capable of detaching confluent cell crowds as a two-dimensional sheet structure is © XXXX American Chemical Society
anticipated as a useful tool for tissue engineering, because the physiological activity of withdrawn cell crowds can be also regulated by changing the stiffness of PDMS substrates. On this account, introduction of temperature-responsive poly(Nisopropylacrylamide) (PNIPAAm) on the PDMS surface has been attempted with the aim of developing a substrate surface capable of controlling cell functions and withdrawing confluent cell crowds as a sheet-form.11,12 Although this is a very smart method to develop temporal cell-adhesive PDMS substrate, it is also valuable to examine other possible methods from the point of view of simplicity of process or without providing stimulation such as temperature change for in situ application of cell sheet. The slightly cross-linked PDMS substrate, when immersed in an appropriate organic solvent such as chloroform, hexane, or dichloromethane, swells up; then, as the swollen PDMS substrate dries up naturally, its original dimensions are restored.13 On the basis of this property, we make a hypothesis Received: June 19, 2016 Accepted: August 4, 2016
A
DOI: 10.1021/acsami.6b07414 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
solvent, the samples were dried at 60 °C for 6 h in vacuo. The deswollen substrates were then placed in 5 mL of water at room temperature for 3 days and subsequently subjected to thorough rinsing with water and drying in a vacuum oven for 1 day. During the aging process, surface characterizations of the aged PDMS substrates were conducted every 24 h after their drying in vacuo. 2.4. Contact Angle Measurement in Water. The static water contact angles were measured using a goniometer (Kyowa Interface Science Co., Tokyo, Japan) at room temperature. PDMS was immersed and fixed in water; then, air bubbles were brought into contact with the PDMS surface for 3 s; subsequently, the contact angles were measured using photographic images. For each sample, measurements were performed at more than three positions. 2.5. X-ray Photoelectron Spectroscopy (XPS) Measurement. The surface composition of the PDMS substrates was investigated by XPS using Mg Kα sources. A takeoff angle of photoelectron was fixed as 90° (Kratos/Shimadzu, Kanagawa, Japan). All of the samples were vacuum-dried for 1 day before the measurement. The P/Si ratio was obtained from the integration values of the XPS spectra of the P 2p and Si 2p peaks referenced to the C 1s peak at 285.0 eV. 2.6. Atomic Force Microscopy (AFM) Observations. AFM analysis under the wet condition was conducted using NanoScope IIIa (Nihon Veeco, Tokyo, Japan). The excitation frequency range was 7.8−9 kHz, and the scan rate and scan scales were 0.5 Hz and 100 nm, respectively. 2.7. Protein Adsorption Test. Before and after being subjected to water aging, the PDMS substrates were immersed in a mixture of 0.030 g/dL bovine plasma fibrinogen and 0.045 g/dL BSA in PBS (pH 7.4, ion strength 0.15 M) for 60 min at 37 °C. The substrates were then rinsed with fresh PBS twice by the stirring method (300 rpm for 5 min). The adsorbed protein was detached in sodium dodecyl sulfate (SDS, 1 wt % in water) by sonication for 20 min, and the protein concentration in the SDS solution was determined using the MicroBCA kit. 2.8. Surface Density of Fibronectin. The surface density of adsorbed fibronectin was monitored by the enzyme-linked immunosorbent assay (ELISA). Deswollen PDMS substrates were immersed in 10 μg/mL fibronectin solution in PBS for 1 h at 37 °C. After being gently washed with fresh PBS, PDMS substrates were immersed in distilled water and aged for 4 days. The PDMS substrates (aged for 1, 2, 3, and 4 days) were then gently washed with fresh PBS and brought into contact with 2 mg/mL of the mouse antifibronectin antibody solution for 1 h at room temperature. After being rinsed with PBS three times, the substrates were allowed to react with 8 mg/mL of the antimouse IgG (HRP-conjugated) in BSA-pretreated 24-well plates for 2 h. After washing of the substrates with PBS six times, 0.50 mL of a solution (mixture of 10 mL of guanylic acid buffer (pH 3.5), 0.125 mL of 3,3′,5,5′-tetramethylbenzidine (44 mM), and 0.018 mL of H2O2) was added to each substrate in the BSA-pretreated well. After the reaction was quenched with 2.0 N sulfuric acid, the absorbance at 450 nm in each solution was measured using a microplate reader (Multiskan FC; Thermo Fisher Scientific, St. Herblain, France). 2.9. L929 Cell Adhesion Test. Deswollen PDMS substrates were immersed in 10 μg/mL fibronectin solution (PBS) for 1 h at 37 °C. After being gently washed with fresh PBS, PDMS substrates were placed in the 24-well plate, and 1.0 mL of NIH3T3 mouse fibroblast cell suspension (2.0 × 104 cells/mL, 10% FBS, 50 μg/mL penicillin, minimum essential medium) was added to the wells. All of the samples were stored in a 100% humidified incubator at 37 °C with 5% CO2 for 4 days. After being washed with fresh medium, all of the PDMS substrates were observed under an optical microscope.
that when an ABA block-type polymer containing a hydrophilic segment (A block) and a hydrophobic PDMS segment (B block) is dissolved in PDMS-swellable solvent, the block copolymer might get incorporated in the PDMS substrate during the swelling−deswelling process14 and then diffuse out when immersed in aqueous medium. Subsequently, the hydrophobic cell-adhesive PDMS substrate surface would gradually change to a noncell-adhesive hydrophilic surface, thereby enabling the use of this phospholipid block copolymerincorporated PDMS substrate as a temporal cell-adhesive surface. To prove and optimize this hypothesis, block copolymers with different compositions were synthesized given that the efficiency of hydrophilic conversion is believed to be strongly dependent on the compositions of the A and B segments. Various compositions of ABA block-type polymers composed of poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) (PMPC) segments as the A block and PDMS segment as the B block were synthesized and applied for inducing a time-dependent hydrophilic conversion of the PDMS substrate surface that would be sufficient for detaching adhering cell crowds as a sheet-form. PMPC is a very well-known antibiofouling, hydrophilic polymer that is able to suppress protein adsorption and the following cell adhesion.15−17 Therefore, if PMPC segment could be tethered on the PDMS substrate by a diffusion process, cell-adhesive PDMS substrate is anticipated to be converted to an antiadhesive one. The purpose of this research was to confirm the feasibility of the simple swelling−deswelling process for bringing about the diffusion-induced hydrophilic conversion of the PDMS substrate surface with the aim of developing a temporal celladhesive PDMS substrate.
2. MATERIALS AND METHODS 2.1. Materials. MPC was purchased from NOF Co., Ltd. (Tokyo, Japan), which was synthesized by a previously reported method.15 The preparation kit of Sylgard 184 was purchased from Dow Corning (Midland, MI), and the PDMS substrate (10 wt % cross-linker) was prepared as per the manufacturer’s instructions. Bovine serum albumin (BSA), human plasma fibronectin, and mouse monoclonal antihuman fibronectin antibody were purchased from Sigma-Aldrich (St. Louis, MO). A goat polyclonal antibody to mouse IgG (horseradish peroxidase (HRP)-conjugated) was purchased from Abcam (Cambridge, UK). The Micro-BCA protein-assay kit was purchased from Pierce Chemical (Rockford, IL); other chemical reagents were purchased from Tokyo Kasei Co. (Tokyo, Japan). 2.2. Preparation of ABA Block-Type Polymers. Three different compositions of block copolymers were synthesized by a previously reported method.18 Briefly, 1,1,3,3-tetramethyldisiloxane (TMS, 1.36 mmol) and octamethylcyclotetrasiloxane (D4, 0.068 mol) were placed in a round-bottomed flask and degassed with dry Ar. Next, after 0.13 mL of sulfonic acid was injected in the flask, it was sealed immediately; the mixture was allowed to react in a 55 °C oil bath for 3 days. The reaction mixture was then dissolved in diethyl ether and thoroughly washed with water until it was neutralized. The organic layer was then stirred with magnesium sulfate, which was followed by filtration and drying in vacuo. Three different molecular weights of silylhydrated end-functional PDMS were prepared by controlling the D4/TMS ratios,19 and the end-functional groups were substituted into the atom transfer radical polymerization (ATRP) initiator. Subsequently, ATRP was conducted with the MPC to synthesize the ABA block-type polymers by the previously reported method.18 2.3. Swelling−Deswelling Process of PDMS. The synthesized block-type copolymers were dissolved in ethanol/chloroform (30/70 vol %) mixed solvent at 30 mg/mL. Next, the PDMS substrates (1 cm × 1 cm × 0.2 cm) were immersed in 1 mL of each polymer solution for 5 days at room temperature. After being rinsed with a fresh mixed
3. RESULTS AND DISCUSSION To develop the temporal cell-adhesive substrate, protein adsorption must be initially induced on the PDMS substrate and the protein must be spontaneously desorbed with cell crowds after the desired period, because cell adhesion behavior is dominated by adsorbed proteins. Protein adsorption is known to depend strongly on the physicochemical properties of B
DOI: 10.1021/acsami.6b07414 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 1. Reaction scheme of PMPC−PDMS−PMPC block copolymer and overall experimental concept for the development of temporal celladhesive PDMS substrate by swelling−deswelling process.
Table 1. Molecular Profiles and Solubilities of Synthesized Block Copolymersa composition (unit %, 1H NMR) symbol PDMS1 PDMS2 PDMS3 B-1 B-2 B-3
solubility (30 mg/mL)
MPC
DMS
ethanol
chloroform
ethanol/chloroform (30/70)
Mn (×104 g/mol, SEC)
Mw/Mn
89 53 24
100 100 100 11 47 76
− − − ++ ++ +
++ ++ ++ − − −
++ ++ ++ ++ ++ ++
0.092 0.42 1.5 2.6 2.3 3.4b
1.4 1.9 1.7 1.2 1.4 b
Solubility (++, soluble; +, partially soluble; −, insoluble), DMS: dimethylsiloxane. bMn was determined by 1H NMR because of its solubility problem in SEC solvent. a
Figure 2. Surface hydrophilicity of PDMS substrates treated with block copolymers with three different compositions. (a) Water contact angle under dry condition and (b) air bubble contact angle under wet condition. All samples were aged in water for 3 days before the contact angle measurement (*p < 0.05).
study. Figure 1 shows the overall reaction scheme of the ABA block-type polymer composed of PMPC and PDMS, as well as the concept of developing a temporal cell-adhesive PDMS substrate. We demonstrated in a previous study that an ABA block-type polymer composed of PMPC and PDMS can be easily incorporated into the PDMS substrate during the swelling process in the polymer solution and that the hydrophilic PMPC segment diffuses out and tethers to the
a surface, such as the surface roughness, electrical charge, degree of hydrogen bonding, and hydrophobicity.20,21 Among these properties, control of the property of hydrophobicity of a material surface is known as an effective approach for regulating the adsorption and desorption behaviors of proteins.22 Therefore, the development of a PDMS substrate capable of spontaneously changing the nature of its surface from hydrophobic to hydrophilic was the primary goal of the present C
DOI: 10.1021/acsami.6b07414 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 3. Surface morphology of PDMS substrates observed by wet-mode AFM. All samples were aged in water for 3 days before the AFM observation.
block copolymer is also considerably variable, possibly affecting the efficiency of hydrophilic conversion because the molecular weight of B-3 block copolymer, which shows the best efficiency for hydrophilic conversion, is largest among the synthesized block copolymers. However, due to the limited types of block copolymers, the relevant discussion is thought to be excluded in the present study and should be included in a future study. A similar result was obtained when the hydrophilicity was measured using air bubbles under the wet condition. As shown in Figure 2b, B-2- and B-3-treated PDMS substrates showed higher hydrophilicity than did the B-1-treated PDMS substrate, which had the lowest DMS composition. These results indicate that the B-3 block polymer has the optimum composition for inducing effective conversion of the hydrophobic surface of the PDMS substrate to a hydrophilic surface after aging in water. The swelling of the PDMS substrate surface induced by hydrophilic conversion was observed by AFM. Figure 3 shows the wet-mode AFM images of the PDMS substrate captured after 3 days of aging in water. In contrast to the flat, unswollen state of the untreated PDMS surface, a heterogeneous swollen morphology was observed for the block-type polymercontaining PDMS substrate, and the degree of surface swelling increased gradually from B-1 to B-3. This result is in good agreement with that of the contact angle measurement. Because the surface swelling of PDMS substrate is thought to be induced by the hydration of the tethered hydrophilic PMPC segment near the surface, B-3-treated PDMS was expected to show the most swollen morphology after hydrophilic conversion. The effect of block copolymer composition on the hydrophilic conversion of the PDMS substrate was also confirmed by investigating the amount of proteins on the pretreated PDMS substrate surfaces after aging in water. All samples were immersed in water for 3 days, and a protein adsorption test was conducted by using a mixture of BSA and fibrinogen. As a result, the block-type polymer-treated PDMS substrate showed a considerably smaller amount of adsorbed proteins, whereas the untreated PDMS substrate showed a large amount of adsorbed proteins even after 3 days of aging (Figure 4). Among the various block-type polymer-treated PDMS substrates, the B-3-treated PDMS substrate showed the smallest amount of adsorbed proteins, which is identical to the result of the background level (∼0.3 μg/cm2) of the micro-BCA experimental method. Interestingly, when the B-3-treated PDMS substrate was aged in water for 1 day, the antifouling property did not increase significantly in comparison to that when the B-3-treated PDMS substrate was aged for 3 days. This result indicates that the hydrophilic conversion of the PDMS substrate is a time-dependent process, and this time-dependent hydrophilic conversion is thought to be useful for developing a
substrate surface when it is aged in water. However, PMPC, which does not contain a PDMS segment, was not able to be included in the substrate during the swelling process because of its extremely different polarity between PMPC and PDMS substrate. This previous study indicates that the efficiency of hydrophilic conversion of the PDMS substrate is possibly dependent on the compositions of the block copolymer dissolved in the solvent that facilitates swelling of PDMS. Therefore, in the present study, block copolymers with different compositions of MPC units and DMS units were synthesized to optimize the hydrophilic conversion of the PDMS substrate. Initially, PDMS ATRP macroinitiators with three different molecular weights, 0.92 × 103, 4.12 × 103, and 15.1 × 103, were synthesized and used to prepare the block copolymers. Table 1 lists the resulting compositions, solubilities, and molecular weights of the three synthesized block copolymers. Each copolymer exhibited limited solubility in chloroform, which is a suitable solvent for inducing swelling of PDMS. However, the three block copolymers were completely soluble in ethanol/ chloroform mixed solvent (30/70), in which a swelling ratio of 150% for the PDMS substrate can be achieved. To confirm the optimum composition for the hydrophilic conversion of the PDMS substrate after its aging in aqueous medium, separate PDMS substrates including the three different block copolymers were aged in water for 3 days after the completion of the swelling−deswelling process in each block-type polymer solution. Figure 2 shows the results of contact angle measurements after 3 days of aging using water droplets and air bubbles. The water contact angle measured after surface drying decreased gradually as the DMS unit composition in the block copolymer increased. Two factors are expected to govern the efficiency of hydrophilic conversion of the PDMS substrate by the swelling−deswelling process. The first factor is the amount of block copolymer chain incorporated in the substrate after the swelling−deswelling process, and the second factor is the amount of tethered PMPC segment after the aging process. Because PMPC, without any PDMS segment, could not be incorporated into the PDMS substrate during the swelling process, it is anticipated that a block polymer with a higher DMS composition will be incorporated more effectively into the PDMS substrate. On the other hand, a block copolymer with a higher MPC unit composition is expected to result in a larger amount of tethered PMPC segment after aging once it has been incorporated into the PDMS substrate. Although the exact mechanism of the composition dependency of hydrophilicity is yet to be clearly understood, it can be hypothesized that a block copolymer with a higher DMS unit composition is more effective in inducing the hydrophilic conversion of the PDMS substrate because it can be incorporated more effectively into the substrate during the swelling process. The molecular weight of the incorporated D
DOI: 10.1021/acsami.6b07414 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
The surface swelling of the PDMS substrate induced by hydrophilic conversion with an increase in the aging time was also investigated by AFM observations (Figure 6). As is clearly seen from this figure, the surface swelling that occurred after 1 day of aging was almost insignificant. However, a rough surface began to be observed after 2 days of aging, and the surface changed more significantly after 3 and 4 days of aging. Because the changes in the surface morphology were induced by the swelling of the tethered PMPC segment, this result reconfirms the diffusion-induced hydrophilic conversion of the B-3-treated PDMS substrate and is highly consistent with the results of the contact angles and P/Si ratio. To develop a temporal cell-adhesive substrate, the adsorption behavior of the cell-adhesive protein on the substrate surface must primarily be controlled because cell adhesion is induced by surface proteins.23 The physiological activity of cells on material surfaces is known to be affected by the type, density, and orientation of adsorbed proteins called the ECM.24 Various types of cell-adhesive proteins are included in the ECM when the cells are cultured in serum-containing medium; examples of such proteins include fibronectin, collagen, laminin, and vitronectin.25 Among these, fibronectin is a widely used celladhesive protein that influences cell adhesion, differentiation, proliferation, and other cell cycle progressions because of its two main active sites: the PHSRN and RGD sequences.26,27 These two active sequences can specifically bind to α5β1 integrin molecules on eukaryotic cells and activate small GTPase proteins via focal adhesion kinase for regulating the physiological activity of cells.28 In the present study, fibronectin was coated on the PDMS substrate to induce cell adhesion, and its density change with an increase in the aging time was evaluated by ELISA. Figure 7 shows the intensity of the surface fibronectin relative to that of the antifouling PDMS substrate, which was passivated by a previously reported method.29 In the case of the bare PDMS substrate, the density of the adsorbed fibronectin did not change significantly over the aging time (4 days). This indicates that the fibronectin molecules were irreversibly adsorbed on the PDMS substrate surface because of the strong hydrophobic interactions that occurred between the fibronectin molecules and the substrate surface. In contrast, the surface density of fibronectin on the B-3-treated PDMS substrate surface decreased gradually as the aging time increased, and most of the adsorbed fibronectin was desorbed when the aging time reached 4 days. Okano et al. proposed a useful technique for developing a temporal cell-adhesive surface by temperature-responsive PNIPAAm grafting on cell culture
Figure 4. Amount of protein adsorbed on block copolymer-treated PDMS substrates. All samples were aged in water for 3 days before the protein adsorption tests (*p < 0.05, **p < 0.005).
temporal cell-adhesive substrate, which was the goal of this study. The time-dependent hydrophilic conversion of the B-3treated PDMS substrate was evaluated by confirming the changes in the air bubble contact angle and the ratio of the surface elements with aging. Figure 5 shows the results of the changes in the air bubble contact angle and P/Si ratio on the B3-treated PDMS substrate with aging in water. In the case of the air bubble contact angle, no significant change was observed on the bare PDMS substrate surface even after 4 days of aging in water. Contrary to this, the hydrophobic B-3-treated substrate surface gradually changed to a hydrophilic one, and the contact angle was saturated when the aging time reached 4 days. The ratio of surface elements, that is, the P/Si ratio, was evaluated by XPS analysis. As shown in Figure 5b, almost no P atoms were present on the B-3-treated substrate surface after 1 day of aging. However, the number of P atoms increased gradually as the aging time increased; in contrast, no P atoms were observed on the bare PDMS substrate surface even after 4 days of aging. Interestingly, the increasing tendency of the P/Si ratio with aging is rather similar to the trend of the change in the air bubble contact angle. This result indicates that the hydrophilic conversion of the PDMS substrate is a diffusioninduced process of the incorporated block copolymer chains, and thus some time is required for the diffusion of the chains to enable them to satisfactorily reach the outermost surface, which could result in a change from the hydrophobic nature of the surface to a hydrophilic one.
Figure 5. Changes in (a) air bubble contact angle in water and (b) P/Si surface element ratio of B-3-treated PDMS substrate with aging in water (*p < 0.05). E
DOI: 10.1021/acsami.6b07414 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 6. Morphological changes of B-3-treated PDMS substrate with aging in water. AFM analysis was performed under the wet condition.
phenomenon was not observed. In contrast, temporal celladhesive behavior was observed on the B-3-treated PDMS substrate surface. As shown in Figure 8, cells adhered stably to and proliferated on the B-3-treated PDMS substrate surface until 3 days of culturing. However, the beginning of spontaneous cell peeling was also observed on a part of the substrate surface after 3 days, and most cells detached easily from the surface after 4 days. As a result, spontaneously or easily detached two-dimensional (2D) cell aggregates were frequently observed at the edge of the substrate surface, as shown in Figure 9. Because of the weakened interaction Figure 7. Intensity of surface fibronectin measured from absorbance at 450 nm after quenching of reaction of HRP-conjugated antifibronectin antibody and substrate relative to that of passivated PDMS (*p < 0.05, **p < 0.005, ***p < 0.001).
plate.30 This technique is based on the concept of converting a hydrophobic cell culturing surface to a hydrophilic one to induce desorption of ECM proteins. On the basis of the results of the contact angle measurements and XPS and AFM analyses, a similar effect is thought to be applicable in the case of the present results. That is, a weakened hydrophobic interaction between the fibronectin molecules and the substrate surface via hydrophilic conversion is responsible for the desorption of the adsorbed fibronectin. The temporal cell adhesion property of the prepared PDMS substrate was examined by the culturing of NIH3T3 mouse fibroblasts after fibronectin coating. Figure 8 shows the optical microscopy images of the cells adhering onto the bare and B-3treated PDMS substrate surfaces. In the case of the bare PDMS substrate, cells were found to adhere stably to the substrate surface even after 4 days, and a spontaneous peeling
Figure 9. Detached cell crowds on B-3 block copolymer-incorporated PDMS substrate surface after 4 days (bar = 500 μm).
between the fibronectin layer and the hydrophilically converted PDMS substrate surface, cell aggregates are thought to detach easily from the surface as a sheet-form. Development of 2D connected cell crowds is a crucial technique for repair of damaged tissue in the field of regenerative medicine.31 Because the stiffness of cell-contacting
Figure 8. Optical microscopy images of NIH3T3 fibroblasts on bare and B-3-treated PDMS substrate surfaces at different culturing times (bar = 500 μm). F
DOI: 10.1021/acsami.6b07414 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
(7) Evans, N. D.; Minelli, C.; Gentleman, E.; LaPointe, V.; Patankar, S. N.; Kallivretaki, M.; Chen, X. Y.; Roberts, C. J.; Stevens, M. M. Substrate Stiffness Affects Early Differentiation Events in Embryonic Stem Cells. Eur. Cells Mater. 2009, 18, 1−14. (8) Shimizu, T. Fabrication of Pulsatile Cardiac Tissue Grafts Using a Novel 3-Dimensional Cell Sheet Manipulation Technique and Temperature-Responsive Cell Culture Surfaces. Circ. Res. 2002, 90 (3), 40e−48. (9) Shimizu, T.; Yamato, M.; Kikuchi, A.; Okano, T. Cell Sheet Engineering for Myocardial Tissue Reconstruction. Biomaterials 2003, 24 (13), 2309−2316. (10) Yang, J.; Yamato, M.; Kohno, C.; Nishimoto, A.; Sekine, H.; Fukai, F.; Okano, T. Cell Sheet Engineering: Recreating Tissues without Biodegradable Scaffolds. Biomaterials 2005, 26 (33), 6415− 6422. (11) Rayatpisheh, S.; Heath, D. E.; Shakouri, A.; Rujitanaroj, P. O.; Chew, S. Y.; Chan-Park, M. B. Combining Cell Sheet Technology and Electrospun Scaffolding for Engineered Tubular, Aligned, and Contractile Blood Vessels. Biomaterials 2014, 35 (9), 2713−2719. (12) Rayatpisheh, S.; Li, P.; Chan-Park, M. B. Argon-Plasma-Induced Ultrathin Thermal Grafting of Thermoresponsive PNIPAm Coating for Contractile Patterned Human SMC Sheet Engineering. Macromol. Biosci. 2012, 12 (7), 937−945. (13) Lee, J. N.; Park, C.; Whitesides, G. M. Solvent Compatibility of Poly(dimethylsiloxane)-Based Microfluidic Devices. Anal. Chem. 2003, 75 (23), 6544−6554. (14) Seo, J.-H.; Matsuno, R.; Konno, T.; Takai, M.; Ishihara, K. Surface Tethering of Phosphorylcholine Groups onto Poly(dimethylsiloxane) Through Swelling-Deswelling Methods with Phospholipids Moiety Containing ABA Block-Type Polymers. Biomaterials 2008, 29 (10), 1367−1376. (15) Ishihara, K.; Ueda, T.; Nakabayashi, N. Preparation of Phospholipid Polylners and Their Properties as Polymer Hydrogel Membranes. Polym. J. 1990, 22 (5), 355−360. (16) Inoue, Y.; Onodera, Y.; Ishihara, K. Preparation of a Thick Polymer Brush Layer Composed of Poly(2-Methacryloyloxyethyl Phosphorylcholine) by Surface-Initiated Atom Transfer Radical Polymerization and Analysis of Protein Adsorption Resistance. Colloids Surf., B 2016, 141 (1), 507−512. (17) Ishihara, K.; Kitagawa, T.; Inoue, Y. Initial Cell Adhesion on Well-Defined Surface by Polymer Brush Layers with Varying Chemical Structures. ACS Biomater. Sci. Eng. 2015, 1 (2), 103−109. (18) Seo, J.-H.; Matsuno, R.; Takai, M.; Ishihara, K. Cell Adhesion on Phase-Separated Surface of Block Copolymer Composed of Poly(2methacryloyloxyethyl phosphorylcholine) and Poly(dimethylsiloxane). Biomaterials 2009, 30 (29), 5330−5340. (19) Miller, P. J.; Matyjaszewski, K. Atom Transfer Radical Polymerization of (Meth)acrylates from Poly(dimethylsiloxane) Macroinitiators. Macromolecules 1999, 32 (26), 8760−8767. (20) Gessner, A.; Lieske, A.; Paulke, B. R.; Muller, R. H. Influence of Surface Charge Density on Protein Adsorption on Polymeric Nanoparticles: Analysis by Two-Dimensional Electrophoresis. Eur. J. Pharm. Biopharm. 2002, 54 (2), 165−170. (21) Cole, M. A.; Voelcker, N. H.; Thissen, H.; Griesser, H. J. Stimuli-Responsive Interfaces and Systems for the Control of ProteinSurface and Cell-Surface Interactions. Biomaterials 2009, 30 (9), 1827−1850. (22) Wu, Y.; Simonovsky, F. I.; Ratner, B. D.; Horbett, T. A. The Role of Adsorbed Fibrinogen in Platelet Adhesion to Polyurethane Surfaces: A Comparison of Surface Hydrophobicity, Protein Adsorption, Monoclonal Antibody Binding, and Platelet Adhesion. J. Biomed. Mater. Res., Part A 2005, 74 (4), 722−738. (23) Arima, Y.; Iwata, H. Effect of Wettability and Surface Functional Groups on Protein Adsorption and Cell Adhesion Using Well-Defined Mixed Self-Assembled Monolayers. Biomaterials 2007, 28 (20), 3074− 3082. (24) Gumbiner, B. M. Cell adhesion: The Molecular Basis of Tissue Architecture and Morphogenesis. Cell 1996, 84 (3), 345−357.
materials is known to be an important factor in the modulation of functions of adhering cells by mechanostransduction,32 a temporal cell-adhesive substrate capable of changing its surface stiffness has a great potential utility in the field of biomedical engineering for tissue regeneration. To this end, a simple and convenient method for developing a temporal cell-adhesive PDMS substrate surface has been proposed in the present study. The hydrophilic conversion of the PDMS substrate requires at least 3 days, and this time lag provides an opportunity to induce fibronectin adsorption and subsequent cell adhesion and proliferation. Although further study is required to perform complete control the period, degree of hydrophilicity, and uniformity of cell detachment, the feasibility of using the swelling−deswelling process for developing a temporal cell-adhesive PDMS substrate has been successfully confirmed in the present study.
4. CONCLUSIONS The feasibility of diffusion-induced hydrophilic conversion of PDMS substrate by hybridization with an amphiphilic ABAtype block polymer composed of PMPC and PDMS segments for the development of a temporal cell-adhesive substrate was verified. Time-dependent hydrophilic conversion of the block polymer-treated PDMS substrate provides a useful time lag for sufficiently cultivating 2D connected cell crowds. After the surface nature of the PDMS substrate is converted to hydrophilic, connected cell crowds spontaneously detach from the substrate surface because of the spontaneous removal of surface fibronectin. Although several optimization processes are required to enable control of the diffusion time, degree of hydrophilicity, and uniformity of cell peeling, it is suggested that the swelling−deswelling process of PDMS in the blocktype polymer solution is a simple and useful method for the development of a temporal cell-adhesive substrate, which has potential applicability in the development of 2D connected tissue.
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[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Schneider, F.; Fellner, T.; Wilde, J.; Wallrabe, U. Mechanical Properties of Silicones for MEMS. J. Micromech. Microeng. 2008, 18 (6), 065008−065017. (2) Park, J. H.; Park, K. D.; Bae, Y. H. PDMS-Based Polyurethanes with MPEG Grafts: Synthesis, Characterization and Platelet Adhesion Study. Biomaterials 1999, 20 (10), 943−953. (3) McBeath, R.; Pirone, D. M.; Nelson, C. M.; Bhadriraju, K.; Chen, C. S. Cell Shape, Cytoskeletal Tension, and RhoA Regulate Stem Cell Lineage Commitment. Dev. Cell 2004, 6 (4), 483−495. (4) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 2006, 126 (4), 677−689. (5) Weng, S.; Fu, J. Synergistic Regulation of Cell Function by Matrix Rigidity and Adhesive Pattern. Biomaterials 2011, 32 (36), 9584− 9593. (6) Guilak, F.; Cohen, D. M.; Estes, B. T.; Gimble, J. M.; Liedtke, W.; Chen, C. S. Control of Stem Cell Fate by Physical Interactions with the Extracellular Matrix. Cell stem cell 2009, 5 (1), 17−26. G
DOI: 10.1021/acsami.6b07414 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces (25) Juliano, R. L.; Haskill, S. Signal Transduction from the Extracellular-Matrix. J. Cell Biol. 1993, 120 (3), 577−585. (26) Aota, S.; Nomizu, M.; Yamada, K. M. The Short Amino Acid Sequence Pro-His-Ser-Arg-Asn in Human Fibronectin Enhances CellAdhesive Function. J. Biol. Chem. 1994, 269, 24756−24761. (27) Cutler, S. M.; Garcia, A. J. Engineering Cell Adhesive Surfaces That Direct Integrin α5β1 Binding Using a Recombinant Fragment of Fibronectin. Biomaterials 2003, 24 (10), 1759−1770. (28) Hotchin, N. A.; Kidd, A. G.; Altroff, H.; Mardon, H. J. Differential Activation of Focal Adhesion Kinase, Rho and Rac by the Ninth and Tenth FIII Domains of Fibronectin. J. Cell Sci. 1999, 112, 2937−2946. (29) Seo, J.-H.; Shibayama, T.; Takai, M.; Ishihara, K. Quick and Simple Modification of a Poly(dimethylsiloxane) Surface by Optimized Molecular Design of the Anti-Biofouling Phospholipid Copolymer. Soft Matter 2011, 7 (6), 2968−2976. (30) Yang, J.; Yamato, M.; Shimizu, T.; Sekine, H.; Ohashi, K.; Kanzaki, M.; Ohki, T.; Nishida, K.; Okano, T. Reconstruction of Functional Tissues with Cell Sheet Engineering. Biomaterials 2007, 28 (34), 5033−5043. (31) Yamato, M.; Okano, T. Cell Sheet Engineering. Mater. Today 2004, 7 (5), 42−47. (32) Even-Ram, S.; Artym, V.; Yamada, K. M. Matrix Control of Stem Cell Fate. Cell 2006, 126 (4), 645−647.
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DOI: 10.1021/acsami.6b07414 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
