Poly(N-isopropylacrylamide)-Grafted Polydimethylsiloxane Substrate

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Poly(N-isopropylacrylamide) grafted PDMS substrate for controlling cell adhesion and detachment by dual stimulation of temperature and mechanical stress Yoshikatsu Akiyama, Miki Matsuyama, Masayuki Yamato, Naoya Takeda, and Teruo Okano Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00992 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 6, 2018

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Biomacromolecules

Revised for Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Poly(N-isopropylacrylamide) grafted PDMS substrate for controlling cell adhesion and detachment by dual stimulation of temperature and mechanical stress

Yoshikatsu Akiyama1*, Miki Matsuyama1,2, Masayuki Yamato1, Naoya Takeda2*, Teruo Okano1* 1. Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8886, Japan. 2. Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University (TWIns), 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan

* Corresponding authors: Tel.: +81-3-3353-8112, ext. 43215; Fax: +81-3-3359-6046 (YA) E-mail: [email protected] (YA), [email protected] (NT), [email protected] (TO) Keywords

Mechanical stress, temperature-responsive cell culture surface, cell adhesion, polymer thin layer, PDMS

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Abstract

Stretchable

temperature-responsive

cell

culture

surfaces

composed

of

poly(N-

isopropylacrylamide (PIPAAm) gel-grafted polydimethylsiloxane (PIPAAm-PDMS) were prepared to demonstrate that dual stimulation of temperature and mechanical stress extensively altered graft polymer thickness, surface wettability, and cell detachment behavior. The PIPAAm-PDMS surface showed a hydrophilic/hydrophobic change with temperature lowering/raising across LCST, ascribing to phase transition of PIPAAm chains.

When

uniaxial stretching was applied, the grafted PIPAAm gel surface was modulated to be more hydrophobic as shown by increase in the contact angle.

Atomic force microscopy

observation revealed that uniaxial stretching made the grafted gel layer become thinner and deformed the nanoscale aggregates of the grafted PIPAAm gel, implying extension of the PIPAAm chains.

The stretched PIPAAm-PDMS became more cell adhesive than the

unstretched PIPAAm-PDMS at 37°C. Furthermore, dual stimulation ―shrinking the already stretched PIPAAm-PDMS and lowering the temperature― induced more rapid cell detachment than temperature change alone.

Similarly, when comparing with single

stimulation of temperature change or mechanical stress, dual stimulation accelerated cell sheets detachment and harvesting. This new stretchable and temperature-responsive culture surface can easily adjust the surface property to different cell adhesiveness by appropriately combining each stimulus, and enable the fabrication of cell sheets of various species.


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1. Introduction Cell sheet-based tissue engineering is a powerful technology for the treatment of damaged human tissues, and preclinical studies or clinical applications have already been achieved for regeneration of the corneal, heart, periodontal ligament tissue, esophagus, cartilage, and middle

ear.1

Temperature-responsive

cell

culture

surfaces,

such

as

poly(N-

isopropylacrylamide) (PIPAAm) gel immobilized in a nanometer-thick layer on a base substrate, are fundamental tool in this cell sheet engineering. Grafted PIPAAm chains show reversible hydration and dehydration by changing temperature.2-3 PIPAAm-grafted surfaces become hydrophobic due to dehydration above the lower critical solution temperature (LCST) of 32°C, enabling cell adhesion at 37°C, while the surface become hydrophilic below LCST, e.g., 20°C, leading to cell detachment. The cell adhesion and detachment properties strongly depend on the thickness of the PIPAAm layer, and the thickness is optimized at the nanoscale.3-4 Thicker PIPAAm layers never show cell adhesion property even at 37ºC, because the PIPAAm chains at the outermost surfaces remain to be hydrated. In contrast, thinner layers fail to detach cultured cells even below the LCST, because the polymer chains in close proximity to the base substrate are well dehydrated, and the temperature-induced transition hardly occur. In fact, thinner PIPAAm gel layers do not show significant changes in thickness in aqueous conditions even if the temperature is dynamically changed by crossing the LCST.4 Surface modification with a nanoscale layer of the PIPAAm gel was conventionally achieved by simultaneously polymerizing N-isopropylacrylamide monomer, crosslinking the polymer chains, and grafting the polymeric material to a polystyrene or aminated-glass substrate under electron beam (EB) exposure.1-3 Thickness of the PIPAAm gel depended on


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the amount of applied monomer, and it could not be changed once the polymeric material was fixed. Polydimethylsiloxane (PDMS) has been utilized as a stretchable culture surface to provide cyclic stretching stimulus to cells and to mimic in vivo conditions, such as the heartbeat,5-6 pulsatile movement of blood vessels and lungs,7-10 and dynamic movement of muscle tissue.11-12

This stretching affects the orientation,7,

cultured cells.14-15

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proliferation and differentiation of

Recently, a PDMS surface was used to dynamically modulate cellular

interactions with biomolecules.16-19

Enzymes embedded in a polyelectrolyte multilayer,

which was deposited on PDMS, were exposed and active at the outermost surface only under stretching. However, releasing the mechanical stress re-embedded the enzymes in the polyelectrolyte multilayer and prevented their enzymatic activity.16-19 Such implementation and release of mechanical stretching also enable modulation of the access of streptavidin or cells to biotin or RGD peptide and poly(ethylene glycol) (PEG) independently immobilized on PDMS.18

By contrast, a previous report demonstrated that the mechanical stretching

significantly affected grafting density of polyacrylamide (PAAm) brush on PDMS. When mechanical stretching was released from PAAm brush on stretched PDMS, grafting density of PAAm brush increased with PDMS shrinking.20

These studies demonstrate that

deformation of a PDMS substrate strongly affects the physical and chemical properties of the molecules thereon. Temperature-dependent volume variation of the PIPAAm gel deform cells embedded in the PIPAAm gel or in the patterned groove made of the PIPAAm gel.21 In this study, we combined temperature-responsive PIPAAm and elastic PDMS to develop a surface controllable by dual stimulation with temperature changes and mechanical stretching/shrinking.

The PIPAAm gel layer covalently immobilized on the PDMS

(PIPAAm-PDMS) base substrate dynamically changed its thickness via stretching and shrinking of the elastic substrate; the PIPAAm layer became thinner and more hydrophobic


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when stretched, while it became thicker and more hydrophilic via shrinking as schematically illustrated in Scheme 1. Namely, the stretching and shrinking concurrently changed the properties of the PIPAAm-PDMS surface. Furthermore, dual control with mechanical and temperature-stimuli was feasible to largely modulate the cell adhesion. In particular, cell detachment would be accelerated by making the PIPAAm layer thicker via PDMS shrinkage.


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2. Experimental section 2-1. Grafting of ultrathin PIPAAm gel on PDMS surfaces with EB irradiation A PDMS cell culture chamber (4-well type) was purchased from STREX (Osaka, Japan). Residual uncrosslinked PDMS and catalytic components in the PDMS were extracted by sequentially washing with triethylamine, ethyl acetate, and acetone solvents.22-23 Then, the PDMS surface was treated with O2 plasma (30 W, 80 mTorr O2, 10 seconds) to form silanol groups. The plasma treated PDMS surfaces were immediately immersed in a 1 wt% (v/v) aqueous solution of 3-aminopropyltrimethoxysilane (200 mL) and was reacted to immobilize aminopropyl groups at 70°C for 30 minutes (abbreviated as AP-PDMS) by referring a previous report.22 After the reaction, the AP-PDMS surface was washed with water, and completely dried with an air spray gun. Immediately after drying, 15 µL of a 40 wt% IPAAm monomer solution in a 2-propanol and methanol mixture (1:1 in volume ratio) was dropped and spread on each of the 4-well of the AP-PDMS chamber. The PDMS substrate with the IPAAm monomer solution was subjected to EB irradiation to graft an ultrathin PIPAAm gel layer on the surface. The substrate surface was washed with water after being immersed in cold water to remove unreacted monomer and ungrafted polymer. The PIPAAm gel grafted PDMS surface (PIPAAm-PDMS) was dried at 45°C for 6 hours.

2-2. ATR/FT-IR measurements of PIPAAm-PDMS graft density One well of the PIPAAm-PDMS chamber was cut out (approximately a 1.0 cm square of PDMS) and used for determining the polymer graft density by measuring FT-IR spectra in a ATR method (NICOLET 6700, Thermo Scientific, MA USA), by refereeing previous reports.3-4, 22, 24 The peak intensities at 1650 cm-1 and 1019 cm-1 (I1650, I1019) were measured,4,


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22, 24-27

and the ratio of I1650/I1019 was calculated to determine the grafted PIPAAm density by

referring to a calibration line (Fig. SI1). A calibration line was prepared as follows; appropriate amount of commercially available PIPAAm (SIGMA-Aldrich, US, MO) dissolved in 2-propanpol and methanol solution (1:1 volume ratio) was casted on an AP-PDMS surface (1.0 cm square (surface area = 1.0 cm2) to prepare PIPAAm coated PDMSs of known amounts. The PIPAAm samples were dried at room temperature for 12 hours, and peak intensities at 1650 cm-1 and 1019 cm-1 were measured by FT-IT/ATR (n=3). The ratio of I1650/I1019 was plotted against known amount of PIPAAm per square area of AP-PDMS surface (1.0 cm2) (Fig. SI1). The samples grafted at a density between 9.1 µg/cm2 and 14.7 µg/cm2 were selectively used as the PIPAAm-PDMS substrate.

2-3. Contact angle measurements Static contact angles were measured by the sessile drop method for the PDMS and PIPAAm-PDMS surfaces in both the unstrethced and stretched states. Stretched states were made by application of 20% of uniaxial stretching as described below.

Prior to the

measurements, the lateral wall of a PDMS chamber or the PIPAAm-PDMS chamber was partially punched out to make a hole for observing a 2 µL water droplet (yellow arrow in Fig. SI2 (A), (B) and (C)).

The elastic substrates were uniaxially stretched with a stretching

device (STREX, Osaka, Japan, Fig. SI2 (C)) in the direction of blue arrows. The stretching direction was indicated as blue colored arrows (Fig. SI2 (C)). The contact angle value of a water droplet was measured perpendicular to the stretching direction and was determined using the θ/2 method with a contact angle meter (DSA100, DAS3 software, KRÜSS GmbH,


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Hamburg, Germany). Contact angle measurement was conducted as soon as PDMS and PIPAAm-PDMS surfaces were dried. The humidity condition was maintained around 60% by humidity controller unit, when the static contact angle measurement conducted. The stretch ratio (SR) was defined according to a following equation; SR (%) = (Lx – L0) / L0 × 100 where Lx and L0 represent the lateral length of the uniaxially stretched and unstrethed cell culture chamber, respectively. samples.

The substrates with SR = 20% were used as the stretched

Three wells in a cell culture chamber were used for the contact angle

measurements. The contact angles of three different places on each well of the unstretched and stretched PIPAAm-PDMS surfaces were measured and averaged. Student’s t-test was used to determine differences in these contact angle values. These contact angles values were measured as soon as possible after removal unreacted components from PDMS (with organic solvent) and PIPAAm-PDMS (with washing) Contact angles of these PIPAAm-PDMS surfaces were also measured by the captive bubble technique in Milli-Q water at 37°C and 20°C by referring our previous report.24 PIPAAm-PDMS surfaces were immersed in the water at 37°C or 20°C for 1 day before the measurement.

2-4. XPS analysis XPS spectra were obtained using an X-ray photoelectron spectrometer (K-Alpha™, Thermo Fisher Scientific K.K., MA, USA) equipped with a monochromatized aluminum Kα X-ray source and a hemispherical energy analyzer. Compositional analyses (0–1100 eV) and high-resolution scans of the C1s, N 1s, O 1s and Si 1s regions were performed on all samples


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(take off angle = 15º). The binding energies of each atom and functional group were calibrated by referring to the C 1s peak at 285.0 eV. Atomic ratio was determined with the Avantage Data System software (Table SI1).

2-5. AFM observation and analysis Surface morphology observation and thickness measurements of the grafted PIPAAm gel layer were performed by AFM in tapping mode (NanoScope V, BRUKER AXS K. K., Tokyo, Japan). The spring constant and resonant frequency of cantilever (MPP-211000-10) were 3 N/m and 75 kHz, respectively. The PIPAAm-PDMS was stretched with a stretching device, and it was subsequently bonded to a glass coverslip (MATSUNAMI, No5, 5 mm × 5 mm in size, Osaka, Japan) with silicon glue (RTV type, Shin-Etsu Chemical Co., Ltd., Tokyo, Japan). A part of the stretched substrate bonded to the glass surface was cut out and put on a sample holder for AFM measurement. The surface morphology of the PIPAAm-PDMS and PDMS substrates in the unstretched and stretched states were observed, and the AFM images were subjected to 2nd order flattening with NanoScope V system software. Temperature was around 25°C and humidity condition was between 40% and 60% in each AFM measurement. To measure the thickness of the PIPAAm layer, a central part of the AP-PDMS surface was masked with 5-mm square glass coverslip, and only the surrounding area was grafted with PIPAAm. The PIPAAm grafted boundary was scanned in the stretched and untretched states by AFM (Fig. SI3). The images without digital flattening treatment were used to measure height difference between the (masked) ungrafted and grafted areas as the thickness of the PIPAAm gel layer (Fig. SI3 and SI4).


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A different type of the cantilever (Olympus Cooperation, Tokyo, Japan, OMCL-AC160TSW2), of which spring constant and resonant frequency were 42 N/m and 300 kHz, was used to obtain force-displacement curves of stretched and unstretched PIPAAm-PDMS.

The

loading rate of the cantilever probe was 1 µm/s. Three different places on each surface were measured.

The indentation depth and adhesion force values were determined from the

approaching and retracting processes of each force curves, respectively. Using a cover glass surface as a control surface, 10 nm depth was set as a trigger threshold value in the approaching process for safely measuring. Thus, the AFM cantilever automatically shifted to the retraction process, when the tip sensed that it had reached a depth of 10 nm from the surface. Three different places on each of PIPAAm-PDMS surfaces was measured, and the indentation depth and adhesion force values were represented as the mean values with standard deviation. For comparison, PDMS surface was also measured in a same way (Fig. SI5 and Table SI2). Student’s t-test was used to determine statistical significance.

2-6. Cell attachment/detachment control with temperature and mechanical stimuli Cells attachment/detachment assays were conducted using three cell culture wells of the PDMS or PIPAAm-PDMS chambers. The PIPAAm-PDMS chamber (SR = 20%) was stretched with the stretching device, and it was attached on a polystyrene cell culture dish (φ 150 mm, AGC technology, Shizuoka, Japan). An unstretched PIPAAm-PDMS chamber was also used for comparison. After 5 minutes of exposure to UV light for sterilization, BAECs (National Institute of Biomedical Innovation, Health and Nutrition, JCRB Cell Bank,Kobe, Japan, Passage number < 30) were seeded at 5.0 ×103 cells/cm2 and cultured at 37°C in a humidified atmosphere with 5% CO2 for initial 24 hours. The numbers of adhered cells were counted using phase-contrast microscopic images obtained at appropriate intervals up to 24


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hours. To evaluate the effect on cell detachment by lowering temperature and/or releasing the mechanical stretching (shrinking the stretched PIPAAm-PDMS), four different procedures as illustrated in Fig. SI6― were employed. Each procedure was as follows. Cells were seeded and cultured on unstretched PIPAAm-PDMS surfaces at 37°C for 24 hours as illustrated in top procedure of Fig. SI6. This procedure was denoted as UnSt-LoTemp, which indicated cells cultured on unstretched PIPAAm-PDMS were subjected to low temperature treatment. In order to evaluated an effect of single stimulus; the uniaxial mechanical shrinking or low temperature treatment, on cell adhesive properties of the stretched PIPAAmPDMS surfaces, cells were cultured on stretched PIPAAm-PDMS surfaces at 37°C. After 24 hours cell culture, second top and bottom procedures in Fig. SI6, were used for the evaluation of cell detachment properties. The second top procedure, which was denoted as St-LoTemp, indicated stretched PIPAAm-PDMS with low temperature treatment. In the bottom procedure, the stretched PIPAAm-PDMS substrates were shrunk to an unstretched state by unmounting from a stretching device without temperature change. The bottom protocol was denoted as St-UnSt-LoTemp, which indicated that stretched PIPAAm-PDMS was shrunk to unstretched state at 37°C. To evaluate effects of dual stimulus; the uniaxial mechanical stretching and low temperature treatment, on cell adhesive properties of PIPAAm-PDMS surfaces, cell detachment assay was conducted in a same way by using top three procedure in Fig. SI6. The top three procedure was denoted as St-UnSt-LoTemp, which indicated that stretched PIPAAm-PDMS was shrunk to unstretched state 37°C and was subjected to low temperature treatment at 20°C. In all protocols, after lowering of temperature and/or releasing the mechanical stretch, the microscopic images were captured from 0 minutes to 120 minutes at appropriate time-intervals, and the numbers and areas of adhered cells on three different places of each well were quantified using ImageJ software (National Institutes


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of Health, MD, USA) (Fig. SI7). These values are shown as the mean values with standard deviation and were analyzed by Student’s t-test.

2-7. Cell sheets detachment with mechanical and thermal stimuli To evaluate cell-sheet detachment profiles with dual stimuli, BAECs were seeded on the unstretched and stretched PIPAAm-PDMS surfaces (initial density: 1.0 × 105 cells/cm2) and were cultured at 37°C for 5 days. After the cells became confluent, cell-sheet detachment behaviors were investigated using UnSt-LoTemp, St-LoTemp, St-UnSt-LoTemp, and StUnSt as described above (Fig. SI6). Time required for complete cell-sheet detachment was measured on three wells and the values were averaged (Table SI3).


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3. Results and discussion 3-1. Spectroscopic analysis of ultrathin PIPAAm gel grafted PDMS surfaces with ATR/FT-IR and XPS Fig. 1 shows the attenuated total reflectance Fourier transform infrared spectroscopy (ATR/FT-IR) spectra of PDMS, aminated-PDMS (AP-PDMS) and PIPAAm grafted PDMS (PIPAAm-PDMS). PIPAAm gel was grafted on PDMS substrate by using electron beam (EB) irradiation. The graft polymer density was calculated from the ratio of peak intensities at 1650 cm-1 and 1019 cm-1, using a calibration line (Fig. SI1). Each sample preparations is described in the experimental section. The Pla-PDMS and AP-PDMS surfaces showed two broad signals and one sharp signal at 1019 cm-1, 1081 cm-1 and 1260 cm-1, which were assigned to the characteristic signals of Si-O-Si and the methyl groups of PDMS.25-26 After PIPAAm grafting, two new signals appeared at 1549 cm-1 and 1650cm-1, and these peaks were assigned to the C=O (amide I) and N-H (amide II) groups of grafted PIPAAm, respectively.27 X-ray photoelectron spectroscopy (XPS) analysis of PDMS, AP-PDMS and PIPAAmPDMS demonstrated that the nitrogen atom ratio gradually increased as the surface modification proceeded in this order, with 0.0% for PDMS, 2.7% for AP-PDMS, and 7.1% for PIPAAm-PDMS, while the silicon atom ratio decreased (Table SI1). These results demonstrate successful grafting of the PIPAAm gel onto the PDMS surface via amino groups (Fig. 1. and Table SI1). N-isopropylacrylamide (IPAAm) solution spread on AP-PDMS was exposed to EB irradiation for grafting PIPAAm gel on AP-PDMS. The PIPAAm gel hardly immobilized onto the PDMS without the amino propyl groups, corresponding to the results of our previous study,22 probably because, during EB exposure, these amino groups are necessary to provide radicals for subsequent polymerization and polymer grafting.22


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3-2. Temperature dependent contact angles change of PIPAAm grafted PDMS with and without mechanical stretching The effect of mechanical stretching on the wettability of the PDMS, AP-PDMS, and PIPAAm-PDMS surfaces was investigated by contact angle measurements as described in the experimental section (Fig. SI2). Uniaxial mechanical stretching (stretch ratio (SR) = 20%) was applied using a stretching device (Fig. SI2 (C)). There was no significant difference in contact angle between the unstrethced (109.8 ± 1.7° (mean ± SD), n = 3 and uniaxially stretched PDMS (111.1 ± 0.4°) surfaces at 37°C. These contact angles are in agreement with those reported in previous studies.25, 28 When the temperature was decreased to 20°C, the contact angle did not show large changes (unstretched and stretched PDMS surfaces showed contact angles of 111.8 ± 2.2° and 110.3 ± 0.6°, respectively). In the same manner, mechanical stretching did not affect the wettability of the AP-PDMS substrates (data not shown). In contrast, temperature change with mechanical stretching greatly changed the contact angle of the PIPAAm-PDMS surface (Fig. 2). Shape of the water droplet was shown in Fig. SI8. At 20°C, the contact angles was 72.8 ± 1.4° for unstretched PIPAAm-PDMS, and 78.7 ± 1.3° for stretched PIPAAm-PDMS. When the temperature was increased to 37°C, the contact angle of the unstretched and stretched PIPAAm-PDMS increased to 78.2 ± 3.5° and 84.4 ± 1.4°, respectively. Both the stretched and unstretched PIPAAm-PDMS surfaces showed temperature-dependent contact angle changes, demonstrating that the temperatureresponsive phase transition property is apparently maintained in PIPAAm-PDMS even under uniaxial stretching. Interestingly, mechanical stretching altered the PIPAAm-PDMS surface to be more hydrophobic at both 20°C and 37°C. Temperature dependent contact angle changes were also indicated by captive bubble method. The unstretched PIPAAm-PDMS


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surfaces indicated 29.9° ± 0.8° at 20°C and 39.7° ± 2.4° at 37°C, while the stretched PIPAAm-PDMS surfaces did 31.2° ± 2.6° at 20°C and 43.4° ± 5.3° at 37°C. To confirm whether an increase of this hydrophobicity attributed to decrease in the polymeric layer thickness by stretching, the grafted polymer thickness of both the unstrethced and stretched PIPAAm-PDMS surfaces was measured by atomic force microscopy (AFM) as described below.

3-3. Dynamic change of graft PIPAAm layer and its surface by mechanical-stretching The morphology of unstrethced and stretched PIPAAm-PDMS surfaces was observed by AFM (Fig. 3). The unstretched AP-PDMS surface showed a flat profile. In contrast, nanoscale granular aggregates (approximately 200 nm in size) were formed on the PIPAAmPDMS surface. (Fig. 3 (B)). The formation of these nanogranular aggregates is characteristic of the EB-grafted PIPAAm layer, as previously reported.27 Uniaxial mechanical stretching extended the substrate and flattened these nanoaggregates (Fig. 3 (C)). This morphological change was quantitatively shown by a decrease in the root-mean-square (RMS) value of the PIPAAm-PDMS, from 2.7 nm (unstretched) to 1.8 nm (stretched). For measurement of the thickness of grafted PIPAAm gel layer, the substrate, which partly lacked PIPAAm-grafting in a 5-mm square area, was prepared by the procedure described in the experimental section (Figs. SI3 and SI4). The thickness of the grafted PIPAAm gel layer in both the stretched and unstretched states was determined from the height difference of the section profile (Fig. 4). The red single-headed arrows show the boundaries between PIPAAm grafting and ungrafting area for the unstretched (arrow A) and stretched substrates (arrow B). The highest position of each profiles was indicated by red double-headed arrows, and was


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used to determine the grafted PIPAAm thickness. The thicknesses of the grafted PIPAAm layers was 700 nm and 460 nm for the unstrethced and stretched PIPAAm-PDMS, respectively. These results indicate that mechanical stretching decreased the thickness of the grafted PIPAAm layer. Thickness of the PIPAAm layer is a crucial factor for surface wettability and subsequent cell adhesion events,3-4, 24, 29 as have been demonstrated using the PIPAAm grafted tissue culture polystyrene (PIPAAm-TCPS). The thickness dependency is ascribed to marked dehydration of the grafted PIPAAm chains in the vicinity of basal hydrophobic TCPS. Dehydration of the PIPAAm chains at the outermost region is subsequently promoted. As a result, the thinner grafted PIPAAm layer provided a more hydrophobic PIPAAm-TCPS surface and enabled more cell adhesion.24, 29 Thickness of the PIPAAm layer is fixed in the PIPAAm-TCPS, whereas it can be modulated in the PIPAAm-PDMS surface with the external mechanical stretching, as shown by a decrease in the contact angle (Fig. 2) and deformation of nanoscale polymeric granular aggregates (Fig. 3). The mechanical stretching possibly strained the grafted PIPAAm chains and restricted their chain mobility. Such strain and restriction promoted dehydration of the PIPAAm chains,3 consequently enhancing hydrophobicity of PIPAAm-PDMS surface. Conversely, by releasing the mechanical stretching, the stretched PIPAAm-PDMS surface became hydrophilic with increasing thickness of the grafted polymeric layer. If PIPAAm grafted surfaces area were extended by uniaxial mechanical stretch to single direction at a SR of 20%, the grafted PIPAAm density of e.g. 12.3 µg/cm2 would decrease to 10.2 µg/cm2 by calculation. However, it actually decreased to 11.6 µg/cm2. This result could be ascribed to anisotropic deformation of the PIPAAm layer even by uniaxial mechanical stretching, suggesting that the substrate simultaneously shrank in the orthogonal direction.


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Thus, the change in the total area was probably small. By using a biaxial stretching device to enlarge the surface area,30 significant change in the graft density and polymer layer thickness could be obtained. Fig. 5 shows force-displacement curves measured by AFM for unstretched and stretched PIPAAm-PDMS surfaces under atmospheric condition (25°C), even where the grafted PIPAAm chains were relatively hydrated and loosely aggregated, as contact angles indicated (Fig. 2). In addition, the AFM silicon-probe is relatively hydrophilic, because of silanol groups formed on the probe.31-32 Accordingly, remarkable interaction was observed in the retraction process and the adhesion force was measured as 114 ± 24 nN (Fig. 5. (A)) for the unstretched PIPAAm-PDMS surface. Likewise, for the stretched PIPAAm-PDMS surface, an adhesion force was detected in the retraction profile, however, it became significantly smaller to be 46 ± 3 nN (Fig. 5. (B)). The larger adhesion forces of the unstretched PIPAAm-PDMS surface suggest that the hydrophilic AFM probe more strongly interacts with grafted PIPAAm chains through hydrogen-bonding, whereas the grafted polymer chains were strained to be rather dehydrated by mechanical stretching stress and their interaction with the hydrophilic AFM probe decreased (Fig. 3). In contrast, there was no significant difference adhesion forces between stretched and unstretched PDMS surfaces irrespective of mechanical stretch (34.0 ± 4 nN and 28.1 ± 1 nN, respectively, Fig. SI5). The surface stiffness of the unstretched and stretched PIPAAm-PDMS was also evaluated because the stiffness of an elastomer is a crucial factor for the adsorption of fibronectin and cell adhesion, migration, and differentiation behavior.33-34 The surface stiffness was compared using indentation depth values of the AFM cantilever determined from force curves during approach process (Table SI2). There was not significantly different in hardness between the stretched and unstretched PIPAAm-PDMS.


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3-4. Cell attachment and detachment assay Using PIPAAm-PDMS, a cell attachment and detachment assay was conducted. Bovine aortic endothelial cells (BAECs) were seeded and cultured on stretched or unstretched PIPAAm-PDMS surface according to the procedure in the experimental section. The assay procedure was illustrated in Fig. SI6. Fig. 6 shows BAEC adhesion profile on the unstretched and stretched PIPAAm-PDMS at 37°C. At 6 hours after seeding, the stretched surface showed a larger number of adhered cells (80%) than the unstretched surface (60%). This cell adhesive character of the stretched PIPAAm-PDMS surface was also demonstrated by the fact that the surface area of adhered cells on the stretched surface became larger than that on the unstretched surface (Fig. SI7). Fig. 7 shows microscopic images of BAECs on the PIPAAm-PDMS surfaces in four different procedures to evaluate the effect of single or dual stimulation with temperature and/or mechanical stretching (Fig. SI6). UnSt-LoTemp and St-LoTwmp procedures compare the cell detachment behaviors from the stretched and unstretched PIPAAm-PDMS surfaces with temperature stimulus. The unstretched PIPAAm-PDMS showed more rapid cell detachment, which completed within 60 minutes (Figs. 7 and 8), than the stretched substrate (completion at about 120 minutes), probably because the cells could not strongly adhered to the comparatively hydrophilic unstretched PIPAAm-PDMS surface. St-UnSt-LoTemp and St-UnSt procedures were employed to compare the cell detachment efficiency between the single mechanical stimulation and the dual stimulations with additional temperature-lowering. After culturing cells on the stretched PIPAAm-PDMS surfaces at 37°C for initial 24 hours, mechanical stretch was released in St-UnSt procedure


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without temperature change, while temperature-lowering to 20°C was accompanied by releasing mechanical stretch in St-UnSt-LoTemp procedures . In both protocols, the adhered and extended cells shrunk when releasing the mechanical stretch (Fig. SI9). This phenomenon demonstrate that the adhered cells efficiently responded to a change of mechanical stress. However, the single stimulus of mechanical-stress change with continuously culturing at 37°C in St-UnSt procedure was not enough to completely detach all the cells (Fig. 8). The number of detached cells was small during the initial 60 minutes (Fig. 8), and most of the cells still adhered, probably because the grafted PIPAAm chains only partially hydrated, as discussed above regarding the contact angles (Fig. 2), the AFM surface observations (Figs. 3 and 4) and the cell attachment profiles (Fig. 6).

By contrast, dual

stimulation (St-UnSt-LoTemp procedure) ―lowering of temperature and releasing the stretch― showed rapid cell detachment profile. Until 30 minutes, almost adhered cells detached. In comparison with St-LoTwmp procedure (single stimulation with low temperature) and Protocol D (single stimulation with releasing the stretch), dual stimulation in procedure St-UnSt-LoTempfor rapid cell detachment was remarkable. The release of mechanical stretching loosened strained PIPAAm chains and promoted hydration. Low temperature treatment also made PIPAAm chains hydrated by a transition to hydrophilic phase. Consequently, the dual stimulations induced remarkably higher hydration of grafted PIPAAm chains, and achieved an accelerated detachment of cells. The effect of single or dual stimulation with temperature and/or mechanical stretching on cell attachment and detachment properties were illustrated in Fig. 9.

3-5. Acceleration of harvesting the cell-sheets from PIPAAm-PDMS surfaces with dual stimuli.


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Cells were seeded at 1.0 × 105 cells/cm2 and cultured on unstretched or stretched PIPAAm-PDMS surface for 4 days. After the cells reached confluent, cell-sheets harvesting was attempted with four different procedures as did in the cell detachment experiments in Section 2.3 (Fig. SI6). Macroscopic images of confluent cells and harvested cell sheets are shown in Fig. 10. The time required for detaching the cell sheets was observed (Table SI3), when four different procedures; UnSt-LoTemp, St-LoTemp, St-UnSt-LoTemp, and St-UnSt illustrated in Fig. SI6., were used. With only a thermal stimulus (decreasing the temperature to 20°C), irrespective of the state of PIPAAm-PDMS surface, cell sheets were harvested after approximately 9 minutes in both the unstretched and stretched PIPAAm-PDMS substrates (UnSt-LoTemp and St-LoTemp procedures, Fig. 10 and Table SI3). When releasing mechanical stretching without decreasing the temperature (St-UnSt procedure), the cell sheets were recovered after more than 12 hours (Fig. 10). As described above, releasing mechanical stretching decreased the thickness of the grafted PIPAAm layer to become a more hydrophilic surface, which triggered detachment of the cell sheet even at 37°C. On the other hand, dual stimulation with a decrease in temperature and release of the mechanical stretching accelerated cell-sheet harvesting (St-UnSt-LoTemp procedure), and a detachment time was 6 ± 1 minutes (n = 3, Fig. 10). These results demonstrated that, as discussed in the cell-detachment experiments in Section 2.3 and indicated in Fig. 10, dual stimulation of releasing mechanical stretching with lowering the temperature synergistically worked to extensively hydrate the grafted PIPAAm chains, resulting in accelerated harvesting of the cell sheets.

4. Conclusions.


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The PIPAAm-PDMS surface properties, including surface wettability, grafted polymer thickness, and cell adhesion/detachment can be modulated by applying and releasing external mechanical stretching. To the best of our knowledge, this is the first report showing that mechanical stretching/shrinking enables modulation of the thickness and degree of hydration/dehydration of the graft polymer gels layer. This dynamic modulation will avoid optimization of the PIPAAm layer thickness in the preparation process, and enable the fabrication of various types of cell sheets, for which the best PIPAAm layer thickness is required on each cell species. Furthermore, dual stimulation of releasing the stretch and lowering the temperature of the PIPAAm-PDMS substrate is effective for cell-sheet harvesting as well as individual cell detachment. This new stretchable PIPAAm-PDMS would be applicable to fabrication of cell sheets with enhanced physiological function (e.g., angiogenesis,35 growth factor secretion36-37 and extracellular matrix deposition38-39) by cyclic stretching operation. Such functional cell sheets would work as useful grafts or model tissue in the tissue engineering therapies and studies.


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Supporting Information A calibration line for determination of the PIPAAm graft density, atomic ratio of PDMS and PIPAAm-PDMS surfaces, images of the PIPAAm-PDMS chamber and a stretching device, schematic diagrams illustrating the measurement of the thickness of grafted PIPAAm and the corresponding AFM images, force-displacement curves and indentation depth results measured with AFM, illustrative drawing of cell attachment and detachment assay, microscopic images of adhered cells with mechanical strething and/or shrinking applied, photographs of the water droplet on unstretched and stretched PIPAAm-PDMS surfaces, and time required for cell-sheet recovery.

Acknowledgments This work was partially supported by the Japan Society for the Promotion of Science (JSPS) under the A3 Foresight Program “Nano-Biomaterials and Delivery Strategies in Regenerative Medicine for Intractable Diseases”.

Grant-in-aid for Scientific Research

(JSPS KAKENHI Grant number, No. 26350531 and No. 18K12084) and Grant-in-aid for Scientific Research on Innovative Areas (MEXT KAKENHI Grant number, No. 23106009 and No. 23106719) are also acknowledged.


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Scheme 1. Conceptual scheme of modulating the thickness of the grafted PIPAAm layer and the properties of PIPAAm gel-grafted PDMS (PIPAAm-PDMS) surface with mechanical stretching and shrinking. (Left) PIPAAm-PDMS in an unstretched state. (Right) PIPAAmPDMS in a stretched state. The thickness of the grafted PIPAAm layer decreases, when mechanical stretching is applied to the PIPAAm-PDMS.

The decrease in the thickness

makes the PIPAAm-PDMS surface more hydrophobic.

By shrinking (releasing the

stretching) the stretched PIPAAm-PDMS substrate, the hydrophilicity of the PIPAAm-PDMS surface is enhanced with increasing the thickness of the grafted PIPAAm layer.

Scheme 1. Y. Akiyame et al.,


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Figure 1. ATR/FT-IR spectra of (A) PDMS, (B) AP-PDMS, and (C) PIPAAm-PDMS surfaces.

Fig. 1. Y. Akiyama et al.,


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Figure 2. Contact angle values of the unstretched and stretched PIPAAm-PDMS surfaces at (●) 20°C and (●) 37°C.

For comparison, the contact angles of the unstretched and stretched

PDMS surfaces were measured at (▴) 20°C and (▴) 37°C. The stretch ratio for stretched PIPAAm-PDMS was 20%. Statistical significance was analyzed with Student’s t-test in p

Poly(N-isopropylacrylamide)-Grafted Polydimethylsiloxane Substrate

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