Promotion of Adipogenesis of 3T3-L1 Cells on Protein Adsorption

Nov 3, 2016 - Stem cell differentiation is an important issue in regenerative medicine and tissue engineering. It has been reported that cell shape is...
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Promotion of Adipogenesis of 3T3-L1 Cells on Protein AdsorptionSuppressing Poly(2-methoxyethyl acrylate) Analogs Takashi Hoshiba,*,†,‡,§ Eri Nemoto,§,∥ Kazuhiro Sato,∥ Hiroka Maruyama,∥ Chiho Endo,∥ and Masaru Tanaka*,†,⊥ †

Frontier Center for Organic Materials and ∥Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510, Japan ‡ International Center for Materials Nanoarchitectonics, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan ⊥ Institute for Materials Chemistry and Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, Fukuoka 819-0395, Japan S Supporting Information *

ABSTRACT: Stem cell differentiation is an important issue in regenerative medicine and tissue engineering. It has been reported that cell shape is one of the factors that determine the lineage commitment of mesenchymal stem cells (MSCs). Therefore, the substrates have been developed to control their shapes. Recently, we found that poly(2-methoxyethyl acrylate) (PMEA) analogs can control tumor cell shape through the alteration of protein adsorption. Here, the adipogenesis of an adipocyte-progenitor cell, 3T3-L1 cells, was attempted; adipogenesis was to be regulated by surfaces coated with PMEA analogs through the control of their shape. The adipogenesis of 3T3-L1 cells was promoted on the surfaces coated with PMEA and its analogs, PMe3A and PMe2A. Evident focal adhesions were hardly observed on these surfaces, suggesting that integrin signal activation was suppressed. Additionally, actin assembly and cell spreading were suppressed on these surfaces. Therefore, the surfaces coated with PMEA analogs are expected to be suitable surfaces to regulate adipogenesis through the suppression of cell spreading. Additionally, we found that protein adsorption correlated with actin assembly and adipogenesis.

1. INTRODUCTION Control of stem cell functions is one of the most important issues in tissue engineering and regenerative medicine.1 In particular, the control of stem cell differentiation is crucial to reconstruct functional tissues and organs in vitro and in vivo. Numerous efforts have been made to control stem cell differentiation, focusing on soluble factors (e.g., growth factors, hormones, and small compounds),2,3 culture substrates (e.g., synthetic polymers and extracellular matrix proteins),3,4 and mechanical stimulation (e.g., tensile stimulation and elastic substrates).3,5−8 Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into osteoblasts, chondrocytes, and adipocytes.9 There have been many efforts to control the osteogenesis and adipogenesis of MSCs. These differentiation processes exhibit a contrary relationship.10,11 For example, fibronectin exhibited promotive effects on the osteogenesis of MSCs and suppressive effects on their adipogenesis via the regulation of actin fiber assembly.12,13 It has also been reported that osteogenesis is promoted in spreading shaped cells, whereas adipogenesis is promoted in round-shaped cells.13,14 These reports indicated that the adipogenesis and osteogenesis of MSCs can be regulated via the control of cell shapes. The control of cell shape is usually achieved using the substrate © 2016 American Chemical Society

surfaces where the adhesion area of isolated cells is restricted by patterning technique with polyethylene glycol (PEG) and poly(vinyl alcohol) (PVA).14,15 The patterned substrate surfaces are useful to regulate the shape of the cells as desired and have substantially progressed to reveal the relationship between cell shape and MSC differentiation.14−16 However, the patterned substrate surfaces are unfavorable for obtaining a large number of cells because the restricted adhesion area will limit the capacity of cultured cells. Developing substrate surfaces that can regulate cell shape without the patterning technique is desired. Serum proteins are adsorbed onto the surface of synthetic polymer substrates, and adsorbed proteins allow cells to adhere and spread on the substrate surfaces via the interaction between adsorbed proteins and integrin on the cells.17 Therefore, it is expected that suppression of protein adsorption prohibits the cells to spread on the substrate surfaces. Indeed, the cells did not spread on the poly(2-hydroxyethyl methacrylate) (PHEMA)-coated surface, which suppresses protein adsorption.18 In addition to PHEMA-coated surface, PEG- and Received: September 8, 2016 Revised: October 24, 2016 Published: November 3, 2016 3808

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2.2. 3T3-L1 Cell Culture. 3T3-L1 cells were purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank, Osaka, Japan). The cells were subcultured in DMEM/F-12 medium containing 10% fetal bovine serum (FBS, Equitech-Bio, Kerrville, TX) on TCPS. Before starting the experiments, the cells were harvested from the TCPS with a 0.25% trypsin/EDTA solution (Gibco, Carlsbad, CA). 2.3. Cell Adhesion Assay. The polymer-coated surfaces were incubated in DMEM/F-12 medium containing 10% FBS or serum-free DMEM/F-12 medium for 1 h at 37 °C prior to cell adhesion assay. The cells were plated onto the polymer-coated surfaces at a density of 1 × 104 cells/cm2. The cells were allowed to adhere to the surfaces in DMEM/F-12 medium containing 10% FBS or in serum-free DMEM/ F-12 medium for 1 and 3 h at 37 °C. The nonadherent cells were removed by washing the plates with PBS twice, and the adherent cells were fixed with 0.1% glutaraldehyde overnight at room temperature. The cells were stained with 0.2% crystal violet (Wako, Osaka, Japan).23 After the staining, the adherent cells were counted in three randomly selected fields using an optical microscope. For the inhibition assay, the cells were incubated with 5 mM ethylenediaminetetraacetic acid (EDTA) for 10 min at 37 °C prior to plating. The cell adhesion assay was performed after EDTA treatment, as described above. 2.4. Cell Growth Assay. A polymer-coated 96-well plate was incubated in DMEM/F-12 medium containing 10% FBS for 1 h at 37 °C prior to cell growth assay. The 3T3-L1 cells were plated on a polymer-coated 96-well plate at a density of 1 × 104 cells/cm2, and the cells were cultured for 1, 2, and 4 days. After the culture, the grown cell numbers were quantified with a standard curve by colorimetric WST-8 assay (Dojindo Laboratories, Kumamoto, Japan). Also, viable and dead cells were stained with live/dead staining after the culture of 4 days. The viable cells were stained by the incubation with 2 μM of calcein-AM (Dojindo Laboratories) for 15 min at 37 °C. After the incubation, the dead cells were stained by the incubation with 4 μM of propidium iodide (PI) for 5 min at 37 °C. The labeled cells were observed with a fluorescent microscope. 2.5. Adipogenic Culture of 3T3-L1 Cells. The polymer-coated surfaces were incubated in DMEM/F-12 medium containing 10% FBS at 37 °C for 1 h prior to the adipogenic culture. The 3T3-L1 cells were plated on the surfaces at a density of 1 × 104 cells/cm2 and were cultured for 4 days in DMEM/F-12 medium containing 10% FBS, 1 μM dexamethasone, 0.5 mM methyl-isobutylxanthine, and 10 μg/mL insulin (adipogenic medium). Half of the medium was changed to fresh adipogenic medium after 2 days. After 6 days of adipogenic culture, the cells were stained with 100 ng/mL nile red (Sigma, St. Louis, MO), following the fixation with 4% paraformaldehyde in PBS (Wako) for 10 min at 37 °C. Also, cell nuclei were counterstained with 10 μg/mL of Hoechst 33258 (Dojindo Laboratories). The labeled cells were observed with a fluorescent microscope. 2.6. Adipogenic Gene Expression Assay. Total RNA was extracted from the cells after 4 days of adipogenic culture using a Sepasol-RNA I Super G reagent (Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s instructions. The total RNA (1 μg) was used as a first strand and mixed with random hexamer primers and ReverTra Ace-α reverse transcriptase (Toyobo, Osaka, Japan). Realtime polymerase chain reaction (PCR) was amplified for genes coding glyceraldehyde 3-phosphate dehydrogenase (Gapdh), fatty acid synthase (Fasn), glycerol-3-phosphate dehydrogenase 2 (Gpd2), and fatty acid binding protein 4 (Fabp4) using TaqMan Gene Expression Assays (Gapdh: Mm99999915_g1, Fasn: Mm00662319_m1, Gpd2: Mm00439082, Fabp4: Mm00662319_m1, Thermo Fisher Scientific, Waltham, MA). The reaction was performed with 10 ng of cDNA, TaqMan Expression Assays, and Premix Ex Taq (Probe qPCR; TaKaRa, Shiga, Japan), according to the manufacturer’s instructions. The gene expression levels relative to Gapdh were calculated using the comparative Ct method. 2.7. Immunocytochemical Analysis. The polymer-coated surfaces were incubated in DMEM/F-12 medium containing 10% FBS at 37 °C for 1 h prior to the culture. The cells were plated on the surfaces at a density of 1 × 104 cells/cm2 and were cultured for 3 and 24 h in DMEM/F-12 containing 10% FBS. After the culture, the cells

poly(2-methacryloxyethyl phosphoryl choline) (MPC)-coated surfaces can also suppress the adsorption of proteins.17,19−21 However, the cells hardly adhered to the surfaces coated with these polymers, which are unsuitable for cell adhesive culture.17,19−21 We have developed poly(2-methoxyethyl acrylate) (PMEA) and its analogs as blood compatible polymers.22 Recently, we reported that cancer cells could adhere to the surfaces coated with PMEA and its analogs although protein adsorption was suppressed on these surfaces.23−26 In particular, cell shapes were altered from a spreading shape to a round shape according to the type of PMEA analogs.23,24 Therefore, it is expected that the adipogenesis and osteogenesis of MSCs can be regulated on the surfaces coated with PMEA analogs. In this study, we examined whether the surfaces coated with PMEA and its analogs can regulate adipogenesis. For this purpose, 3T3-L1 cells, a mouse adipocyte-progenitor cells, were cultured on the surfaces coated with PMEA analogs. The growth and adipogenesis of 3T3-L1 cells were examined. Additionally, we clarified the regulation mechanism of adipogenesis on these surfaces, focusing on cell adhesion mechanisms, actin assembly, and cell spreading. Finally, we compared the correlation between 3T3-L1 cell functions and protein adsorption on the surfaces coated with PMEA analogs.

2. MATERIALS AND METHODS 2.1. Preparation of Polymer-Coated Surfaces. PMEA (Mw: 62000 g/mol) and poly(tetrahydrofurfuryl acrylate) (PTHFA) (Mw: 150000 g/mol) were synthesized as described in previous reports.22,27 Poly(2-(2-methoxyethoxy) ethyl acrylate-co-butyl acrylate) (30:70 mol %, PMe2A, Mw: 93400 g/mol) and poly(2-(2-methoxyethoxy) ethoxy ethyl acrylate-co-butyl-acrylate) (30:70 mol %, PMe3A, Mw: 94400 g/ mol) were also synthesized as previously described.28 Poly(2methacryloyloxyethyl phosphorylcholine-co-butyl methacrylate) (30:70 mol %, PMPC, Mw: 600000 g/mol) was kindly gifted from the NOF Corporation (Tokyo, Japan). The chemical structures of these polymers are shown in Supporting Information, Figure S1. PMEA, PMe2A, PMe3A, and PMPC were dissolved in methanol at a concentration of 0.2 and 1.0% (w/v). PTHFA was dissolved in methanol/chloroform (5:1) at a concentration of 0.2 and 1.0% (w/v). The polymer-coated surfaces were prepared on polyethylene terephthalate (PET) discs (ϕ = 14 mm, thickness = 125 μm, Mitsubishi Plastics, Tokyo, Japan). Each polymer solution (40 μL, 0.2% (w/v)) was cast on the PET disc and spin-coated twice under the following conditions: 500 rpm for 5 s, 2000 rpm for 10 s, 2000−4000 (slope) for 5 s, 4000 rpm for 10 s, and 4000 to 0 rpm (slope) for 5 s.23 For adipogenic culture of 3T3-L1 cells, PET discs (ϕ = 51 mm) were also spin-coated to prepare polymer-coated surfaces. Each polymer solution (224 μL, 1.0% (w/v)) was used for the spin-coating. The spin-coating was performed twice under the following conditions: 1000 rpm for 7 s, 2000 rpm for 5 s, 2000−4000 rpm (slope) for 5 s, 4000 rpm for 10 s, and 4000 to 0 rpm (slope) for 4 s. Additionally, the back side, which is not for cell culture, was spin-coated with PMPC as described above. The sterilization of polymer-coated surfaces was performed by UV exposure for 1 h. To prepare a fibronectin (FN)-coated surface as a positive control of integrin-dependent cell adhesive surface, a 24-well tissue culture polystyrene (TCPS) plate (IWAKI, Tokyo, Japan) was coated by the incubation with 300 μL/well of FN solution (10 μg/mL, Calbiochem, Darmstadt, Germany) at 37 °C for 1 h. After incubation, the plate was washed with phosphate buffered saline (PBS) twice before being used for the following experiment. For the cell growth assay, each polymer was cast on a 96-well TCPS plate (IWAKI). Briefly, 12 μL of each polymer solution (0.2% (w/v)) was casted to a 96-well TCPS plate, and the plate was air-dried for 1 week.23 3809

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Biomacromolecules were fixed with 4% paraformaldehyde in PBS for 10 min at 37 °C and the cells were permeabilized three times with 1% Triton X-100 in PBS for 10 min at room temperature. Permeabilized cells were incubated with an antivinculin antibody (Ab; Millipore, Billerica, MA) for 2 h at 37 °C, followed by treatment with Alexa Fluor 488-conjugated phalloidin (Invitrogen, Carlsbad, CA), and Alexa Fluor 568-conjugated antimouse IgG Ab (Invitrogen) for 1 h at 37 °C. Can Get Signal (ToYoBo) was used for the labeling. ProLong Gold Antifade Reagent with DAPI (Invitrogen) was added on the samples. The samples were observed by confocal laser scanning microscopy (Olympus, Tokyo, Japan). The actin fibers were quantified as a positive area of actin staining with Photoshop CS4.0 and ImageJ.29 2.8. Statistical Analysis. All of the data are presented as the means ± SD. The significance of the differences between two samples was analyzed through an unpaired Student’s t test using Microsoft Excel 2010. The statistical analyses used to analyze the differences between three or more samples were performed using R, a language and environment for statistical computing. The significance of the differences was analyzed using analysis of variance (ANOVA). Tukey’s multiple comparison test was applied as a posthoc test. Differences with P values less than 0.05 were considered statistically significant.

3. RESULTS 3.1. 3T3-L1 Cell Functions on the Surfaces Coated with PMEA Analogs. First, it was confirmed whether 3T3-L1 cells can adhere to the surfaces coated with PMEA analogs by cell adhesion assay after 1 and 3 h of incubation (Figure 1A). The cells started to adhere to polymer-coated surfaces except to the PMPC-coated surface within 1 h. Over 80% of the cells adhered to TCPS, PTHFA-, PMEA-, and PMe3A-coated surfaces after 3 h. Approximately 67% of the cells adhered to the PMe2A-coated surface after 3 h. Few cells adhered to the PMPC-coated surface even after 3 h. This result indicated that the surfaces coated with PMEA analogs allowed the adhesion of 3T3-L1 cells. The growth of 3T3-L1 cells was also compared among the surfaces coated with PMEA analogs by WST-8 assay (Figure 1B). The cells can grow on PTHFA-, PMEA-, PMe3A-, and PMe2A-coated surfaces. The grown cell numbers were in the order of TCPS > PTHFA > PMEA > PMe3A > PMe2A after 4 days of the culture. Particularly, the growth of the cells was significantly suppressed on PMe3A- and PMe2A-coated surfaces compared with TCPS after 4 days. The cell numbers on PMe3A- and PMe2A-coated surfaces were approximately 68 and 61% of the numbers on TCPS after 4 days. Also, the cytotoxicity of PMEA analogs was examined by live/dead staining (Figure 1C). Almost all cells were calceinAM positive, whereas PI-positive cells were hardly observed on the surfaces coated with PMEA analogs, suggested that the surfaces coated with PMEA analogs exhibited little cytotoxicity on 3T3-L1 cells. These results indicated that the surfaces coated with PMEA analogs could support the growth of 3T3L1 cells but exhibited different cell growth rates with little cytotoxicity. Additionally, the adipogenesis of 3T3-L1 cells was compared among the surfaces coated with PMEA analogs. The lipid accumulation was started within 6 days of adipogenic culture on TCPS and PTHFA-, PMEA-, PMe3A-, and PMe2A-coated surfaces, suggesting that the adipogenesis can occur on the surfaces coated with PMEA analogs (Supporting Information, Figure S2). To further compare the adipogenesis on these surfaces, adipogenic gene expression (Fasn, Gpd2, and Fabp4) was compared by real-time PCR analysis after the 4 days of adipogenic culture (Figure 2). The expression levels of Fasn on PMEA-, PMe3A-, and PMe2A-coated surfaces were 1.7−2.1×

Figure 1. Cell adhesion (A) and growth (B) on the surfaces coated with PMEA analogs. The data presented are the means ± SD (n = 3). *P < 0.05 vs TCPS. (C) Live/Dead staining of the cells on the surfaces coated with PMEA analogs. Green: calcein-AM-positive cells (viable cells); Red: PI-positive cells (dead cells).

Figure 2. Adipogenesis of 3T3-L1 cells on the surfaces coated with PMEA analogs. The expression levels of adipogenic genes, Fasn (A), Gpd2 (B), and Fabp4 (C) were measured after 4 days of adipogenic culture by real-time PCR analysis. The data presented are the means ± SD (n = 3). *P < 0.05, **P < 0.01 vs TCPS, †P < 0.05 vs PTHFA. 3810

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Biomacromolecules higher than that on TCPS (Figure 2A). The expression levels of Gpd2 on PMEA-, PMe3A-, and PMe2A-coated surfaces were 2.1−3.3× higher than that on TCPS (Figure 2B). The expression levels of Fabp4 on PMEA-, PMe3A-, and PMe2Acoated surfaces were 3.1−3.8× higher than that on TCPS (Figure 2C). The highest expression of these adipogenic genes was observed in the cells cultured on the PMe2A-coated surface. These results suggested that the adipogenesis of 3T3L1 cells was promoted on PMEA-, PMe3A-, and PMe2A-coated surfaces compared to PTHFA-coated surface and TCPS, a conventional cell culture substrate. 3.2. Characterization of 3T3-L1 Cell Adhesion Mechanisms. As a next step, adhesion mechanisms of 3T3-L1 cells were examined to clarify the regulation mechanisms of 3T3-L1 cell functions. It has already been reported that protein adsorption is suppressed on the surfaces coated with PMEA analogs, leading to the suppression of integrin-dependent adhesion on the surfaces.23−25 Therefore, integrin-dependency for 3T3-L1 cell adhesion was examined by the cell adhesion assay with EDTA, an inhibitor of integrin-dependent adhesion (Figure 3A).30 Few cells adhered to the TCPS and PTHFAcoated surface under the condition with EDTA after 1 h of incubation, as well as on fibronectin (FN)-coated surface, which is a natural extracellular matrix (ECM) protein to lead integrin-dependent adhesion. In contrast to the TCPS- and PTHFA-coated surface, the cells can adhere to the PMEA-, PMe3A-, and PMe2A-coated surfaces even under the condition with EDTA. The inhibitory percentages of EDTA on cell adhesion were 10−29% on these surfaces, suggesting that integrin-dependency for 3T3-L1 cell adhesion was low on PMEA-, PMe3A-, and PMe2A-coated surfaces. To further examine integrin-dependency for the adhesion, vinculin localization was observed as an indicator of focal adhesions on these surfaces after 1 h of incubation by immunocytochemistry (Figure 3B).31 Many focal adhesions were evidently observed in 3T3-L1 cells on the TCPS, PTHFA-, and FN-coated surfaces, whereas only a few focal adhesions were found on the PMEA-, PMe3A-, and PMe2Acoated surfaces. Therefore, these results indicated that 3T3-L1 cells adhered to PMEA-, PMe3A-, and PMe2A-coated surfaces via both integrin-dependent and−independent mechanisms. Additionally, it is indicated that the surfaces coated with PMEA analogs can alter the integrin-dependency of 3T3-L1 cell adhesion to the surfaces. In addition to the condition in the presence of serum-derived adsorbed proteins, we also examined whether 3T3-L1 cells can adhere to the surfaces coated with PMEA analogs without adsorbed proteins. A cell adhesion assay was performed in serum-free medium. The cells can adhere to TCPS and the surfaces coated with PMEA analogs even in serum-free medium as well as in serum-containing medium within 1 h of incubation (Figure 3C). This result indicated that the cells can directly interact with the substrate surfaces to give rise to integrinindependent cell adhesion when protein adsorption was suppressed. 3.3. 3T3-L1 Cell Shapes on the Surfaces Coated with PMEA Analogs. Finally, it was examined whether 3T3-L1 cell shapes altered on the surfaces coated with PMEA analogs. The cells shapes were observed using a confocal laser microscope after the staining of actin fibers (Figures 3B and 4A). The cells started to spread on TCPS, PTHFA-, and FN-coated surfaces within 1 h (Figure 3B) and were completely spread on these surfaces after 1 day of the culture (Figure 4A). In contrast to

Figure 3. Adhesion mechanisms of 3T3-L1 cells on the surfaces coated with PMEA analogs. (A) Inhibitory effect of EDTA on cell adhesion within 1 h. The data presented are the means ± SD (n = 3). *P < 0.05 vs PMPC (−). (B) Focal adhesion formation after 1 h of culture. Blue, green, and red indicate cell nuclei, actin fibers, and vinculin, respectively; * indicates focal adhesions. (C) Cell adhesion in the absence of serum after 1 h. The data presented are the means ± SD (n = 3).

these surfaces, the cells kept a round shape on PMEA-, PMe3A-, and PMe2A-coated surfaces after 1 h of the culture (Figure 3B). The cells formed thin pseudopodia on PMEA-, PMe3A-, and PMe2A-coated surfaces after 1 day (Figure 4A). These results indicated that the cell spreading was suppressed and was delayed on PMEA-, PMe3A-, and PMe2A-coated surfaces. It has been reported that adipogenesis is suppressed by the assembly of actin fibers.13,14 Long and thick actin fibers were found on the TCPS, PTHFA-, and FN-coated surfaces whereas short and thin fibers were mainly observed on the PMEA-, PMe3A-, and PMe2A-coated surfaces (Figure 4A). For further analysis of the assembly of actin fibers, visualized actin areas were quantified using ImageJ in a single cell on the surfaces coated with PMEA analogs after 1 day of culture (Figure 4B). Actin areas were 3.4−6.2× larger on the TCPS and PTHFAcoated surfaces than the PMEA-, PMe3A-, and PMe2A-coated surfaces. These results indicated that actin fibers were more 3811

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Additionally, the formation of focal adhesions, which is a complex to activate the downstream pathways of integrin signaling, was delayed on PMEA-, PMe3A-, and PMe2A-coated surfaces even after 1 day (Figure 3B and Figure 4A). Focal adhesions are generally formed by the interaction between integrin and ECM proteins.31,32 On PMEA-, PMe3A-, and PMe2A-coated surfaces, protein adsorption was suppressed compared with TCPS and PTHFA-coated surface.25 It has been reported that low adsorbed ECM proteins suppress the formation of focal adhesions and integrin signaling activation.18,33 Therefore, it is also speculated that the formation of focal adhesions is suppressed due to low adsorbed ECM proteins on PMEA-, PMe3A-, and PMe2A-coated surfaces, leading the suppression of integrin signaling activation. Additionally, it might be possible that the integrin-independent adhesion mechanism inhibited the formation of focal adhesions. It is well-known that actin assembly followed by cell spreading requires the activation of integrin signaling.32 As discussed above, the activation of integrin signaling was suppressed on PMEA-, PMe3A-, and PMe2A-coated surfaces. Therefore, 3T3-L1 cells exhibited low actin assembly and a round shape on these surfaces (Figure 4A,B). Our results are consistent with a previous report by Horbett et al. that showed the suppression of protein adsorption inhibits 3T3 cell spreading on the surface coated with poly(hydroxyethyl methacrylate-co-ethyl methacrylate).18 4.2. Different Growth and Adipogenesis of 3T3-L1 Cells on the Surfaces Coated with PMEA Analogs. Similar to actin assembly, cell growth also requires integrin signaling.34 We found that the growth of 3T3-L1 cells was significantly suppressed on PMEA-, PMe3A-, and PMe2A-coated surfaces compared to TCPS (Figure 1B). As discussed above, integrin signaling is suppressed on PMEA-, PMe3A-, and PMe2Acoated surfaces. Therefore, cell growth was suppressed on PMEA-, PMe3A-, and PMe2A-coated surfaces due to weak integrin signaling. The adipogenesis of 3T3-L1 cells was promoted on PMEA-, PMe3A-, and PMe2A-coated surfaces (Figure 2). It has been reported that adipogenesis is promoted when the cells were round in shape.13,14 It is also reported that actin assembly inhibits the adipogenesis of MSCs through the decrease of cellular tension.14 To promote the adipogenesis, proliferativeactivated receptor-γ (PPARγ) should be activated.35 PPARγ activity is regulated by the molecules which are relating to cell shape, such as Rho-associated kinase (ROCK) and TAZ. When ROCK is activated for cell spreading, ROCK can inhibit PPARγ activity through the activation of phosphatidylinositol-4phosphate 5-kinase.36 The suppression of ROCK activity can lead to increase the PPARγ activity.36 Also, TAZ can interact with PPARγ to suppress PPARγ activity when TAZ is localized in cell nucleus.37,38 It is known that nuclear localization of TAZ is inhibited when actin assembly is suppressed.29,39 Thus, the suppression of actin assembly might lead to increase PPARγ activity. Active PPARγ can increase CCAAT/enhancer-binding protein α (C/EBPα) expression. And C/EBPα expression positively regulates PPARγ expression to promote the adipogenesis.40 We showed that the cells exhibited a round shape and suppressed actin fiber formation on PMEA-, PMe3A-, and PMe2A-coated surfaces (Figure 3B, Figure 4A,B). On these polymer surfaces, ROCK activity and nuclear localization of TAZ might be suppressed. Due to above reasons, it is speculated that the adipogenesis of 3T3-L1 cells was suppressed on these surfaces (Figure 5A). We cannot exclude

Figure 4. Cell shape and actin assembly on the surfaces coated with PMEA analogs after 1 day of culture. (A) The formation of actin fibers and focal adhesions. Blue, green, and red indicate cell nuclei, actin fibers, and vinculin, respectively; * indicates focal adhesions. (B) Quantification analysis of actin assembly. The data presented are the means ± SD (n = 5−9).

highly assembled on the TCPS and PTHFA-coated surfaces compared to the PMEA-, PMe3A-, and PMe2A-coated surfaces.

4. DISCUSSION 4.1. Adhesion Mechanisms of 3T3-L1 Cells on the Surfaces Coated with PMEA Analogs. 3T3-L1 cells adhered to PMEA-, PMe3A-, and PMe2A-coated surfaces via both integrin-dependent and -independent mechanisms (Figure 3A,B). It has already been shown that approximately 40−55% of protein adsorption is suppressed on PMEA-, PMe3A-, and PMe2A-coated surfaces compared with TCPS.25 It is expected that these surfaces are not covered completely with adsorbed proteins, which may expose the surfaces and may allow the cells to interact to the substrate surface directly. Indeed, 3T3-L1 cells could adhere to the surfaces coated with PMEA analogs without adsorbed proteins (Figure 3C). Therefore, it is possible that 3T3-L1 cells adhered to PMEA-, PMe3A-, and PMe2Acoated surfaces via integrin-adsorbed protein interaction and direct interaction to the surfaces. Similar results were obtained with several tumor cells (HT-1080, MDA-MB-231, and MCF-7 cells) and a hepatocyte model (HepG2), as we reported previously.23−25 3812

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Biomacromolecules other possible reasons. Further detailed mechanisms should be examined in the future.

Figure 5. Putative regulation mechanisms of adipogenesis (A) and osteogenesis (B) of 3T3-L1 cells/MSCs on the surafaces coated with PMEA analogs.

4.3. Correlations between 3T3-L1 Cell Functions and Protein Adsorption on the Surfaces Coated with PMEA Analogs. The correlations between 3T3-L1 cell functions and protein adsorption on the surfaces were compared (Figure 6). The percentage of cell adhesion inhibition by EDTA was approximately 100% when protein adsorption amounts relative to TCPS were over 85% (Figure 6A). When protein adsorption amounts relative to TCPS were less than 85%, the percentage of cell adhesion inhibition dramatically decreased. Similar to the percentage of cell adhesion inhibition, actin assembly dramatically decreased below 85% of adsorbed proteins relative to TCPS (Figure 6B). In contrast to cell adhesion inhibition and actin assembly, adipogenic gene expression exhibits an increased tendency with the decrease of adsorbed protein amount (Figure 6C−E). Horbett et al. reported that protein adsorption onto the surfaces is a critical factor to control cell spreading.18 Similar to the correlation between protein adsorption and cell spreading, it is expected that protein adsorption can be a factor to control the adipogenesis of 3T3L1 cells. There are several factors to control protein adsorption onto the surfaces (e.g., hydrophilicity and surface free energy).41,42 It is also reported that several factors (e.g., mobility factor and surface molecular chirality) can regulate stem cell differentiation.43,44 We have focused on the water structure in hydrated polymers. We have suggested that a unique water structure, called “intermediate water”, might play an important role in the expression of blood compatibility.45 Recently, we have proposed that intermediate water in PMEA analogs can be a critical factor to control protein adsorption.25 It is shown that protein adsorption is suppressed when the contents of intermediate water increased. Additionally, we have reported that the contents of intermediate water in PMEA analogs can be tuned by the alteration of primary structure of PMEA analogs.28,46 Therefore, it is expected that adipogenesis can be

Figure 6. Correlations of FBS protein adsorption with (A) cell adhesion inhibition with EDTA, (B) actin assembly, (C) Fasn expression, (D) Gpd2 expression, and (E) Fabp4 expression. The data presented are means + SD (A: n = 3, B: n = 5−9, C−E: n = 3). Protein adsorption amounts relative to TCPS have been already reported in our previous report.22

tuned by coating with PMEA analogs designed to possess various intermediate water contents. 4.4. Possibility of the Regulation of MSC Differentiation on the Surfaces Coated with PMEA Analogs. 3T3-L1 cells were used as a model of adipocyte-progenitor cells in this study. In the case of MSC adipogenesis, cell shape also plays a pivotal role in the decision of lineage commitment of MSC. McBeath et al. reported that the adipogenesis of MSCs was promoted by the round shape of the cells.14 Therefore, it is also expected that the adipogenesis of MSCs would be promoted on PMEA-, PMe3A-, and PMe2A-coated surfaces, which can suppress protein adsorption (Figure 5A). Additionally, MSCs can undergo osteogenesis, which exhibits a contrary relationship to adipogenesis.12,14 It is reported that osteogenesis is promoted by the spreading form of the cells in contrast to adipogenesis.14 Additionally, osteogenesis requires focal adhesion kinase (FAK) activation and following integrin signaling.47 On PTHFA-coated surface, many types of cells exhibit a spreading form and evident focal adhesions were formed.23,24 MSCs are likely to undergo osteogenesis rather than adipogenesis on PTHFA-coated surface (Figure 5B). Therefore, it is expected that the differentiation of MSCs can be switched between adipogenesis and osteogenesis on the surfaces coated with PMEA analogs, which should be confirmed in future studies. 4.5. Possible Applications of PMEA Analogs in Stem Cell Engineering. The regulation of stem cell differentiation is an important problem to be solved in regenerative medicine 3813

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Biomacromolecules

supported by the Center of Innovation (COI) Program from the Japan Science and Technology Agency (JST).

and tissue engineering. We showed the possibility to switch between adipogenesis and osteogenesis of MSCs through the control of protein adsorption, which determines the activation of integrin signal and cell shapes. Cell shape often determines stem cell differentiation such as chondrogenesis and myogenesis.48 We have already reported that the surfaces coated with PMEA analogs can control the shape in several cells as well as 3T3-L1 cells.23,24 Therefore, the surfaces coated with PMEA analogs can be utilized as the surfaces to promote stem cell differentiation, which is also determined by the cell shape for regenerative medicine such as osteochondral tissue regeneration. Previously, we have reported the composition of culture media that can simultaneously induce both osteogenesis and adipogenesis.49 It is expected that MSCs can dominantly differentiate into osteoblasts and adipocytes on the surface patterned-coated with PTHFA and PMe3A under simultaneous differentiation conditions, respectively. In osteoporosis patients, it is known that the balance between the osteogenesis and adipogenesis of MSCs is disrupted.50 Such patterned surfaces to induce patterned MSC differentiation might provide a useful culture system to develop new drugs for osteoporosis by the analysis of drugs’ effects on the balance of MSC differentiation.

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5. CONCLUSION In this study, we showed that integrin-dependent cell adhesion followed by the integrin signal activation, actin assembly, and cell spreading can be regulated by surfaces coated with PMEA analogs which can control protein adsorption. Additionally, the adipogenesis of 3T3-L1 cells was promoted on PMEA-, PMe3A-, and PMe2A-coated surfaces via the suppression of integrin signal activation, followed by actin assembly and cell spreading. Therefore, the surfaces coated with PMEA analogs are expected to regulate stem cell differentiation, which is also determined by the cell shape for regenerative medicine and pharmaceutical applications.

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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/acs.biomac.6b01340. Chemical structure of the polymers used in this study; Nile red staining of 3T3-L1 cells on the surfaces coated with PMEA analogs (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +81-238-26-3585. E-mail: [email protected]. *Tel.: +81-92-802-6235. E-mail: [email protected]. kyushu-u.ac.jp. Author Contributions §

These authors contributed equally (T.H. and E.N.).

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Rumi Sawada of the National Institute of Health Sciences for fruitful discussion. This work was supported by a Grant-in-Aid for Young Scientists (A) (26702016), funded by MEXT, Japan. In addition, T.H. and M.T. were partially 3814

DOI: 10.1021/acs.biomac.6b01340 Biomacromolecules 2016, 17, 3808−3815

Article

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DOI: 10.1021/acs.biomac.6b01340 Biomacromolecules 2016, 17, 3808−3815