Article pubs.acs.org/Langmuir
Wettability of Supramolecular Nanofibers for Controlled Cell Adhesion and Proliferation Xiao-Qiu Dou, Di Zhang, and Chuan-Liang Feng* State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China S Supporting Information *
ABSTRACT: By employing smart self-assembly of 1,4-benyldicarbonxamide-phenylalanine (C2) derived supramolecular gelators, a simple way to construct nanofibrous environments with the controllable wettability is developed. The fast cell adhesion and proliferation on the least wettable fibers indicates an efficient control over cells, which is proved to be mainly mediated by the interaction between protein and the fibers. One typical merit superior to other materials is that cell adhesion can be regulated not only on two-dimensional (2D) substrates but also in threedimensional (3D) microenvironments. This paves a novel way to deeply understand the influence of fiber wettability on cell behaviors in 3D environment.
1. INTRODUCTION
interaction to enhance the storage modulus of the resulting gels.22 Herein, a simple way to construct materials with the controllable wettability is developed by employing selfassembly of C2-benzene based supramolecular gelators. The cell adhesion on the self-assembled nanofibers can be efficiently tuned, which is mainly mediated by the interaction between protein (e.g., bovine serum albumin, BSA) and fibers. One typical merit superior to other materials is that cell adhesion can be regulated not only on two-dimensional (2D) substrates but also in three-dimensional (3D) microenvironments (Figure 1). This provides a novel way to gain insight into the influence of fiber wettability on cell behaviors in 3D environment, which is crucial for the design of a novel culture environment with desired wettability.23 Three types of C2-benzene based supramolecular gelators with ethylene glycol (EG) monomers as pendant groups (denoted as M1, M2, and M3) were synthesized (Figure 2). Through noncovalent interactions, the gelators can selfassemble into PEG-like nanofibrous hydrogels with different surface wetting properties,24,25 even though their other properties are similar. The influence of surface wettability on cell adhesion and proliferation was studied by using human hepatoma cell line 97H (MHCC-97H) and normal human skin fibroblast (NHSF). The fastest growth of metabolic activity was
Cell adhesion and growth on extracellular matrix (ECM) are greatly influenced by their properties including surface wettability,1−3 roughness,4 topography,5 and chemistry composition.6 Among these, surface wettability has been recognized as an important factor to control the dynamic interaction between an implanted surface and cells in vitro or in vivo.7−10 These surfaces with different wettabilities are usually achieved by introducing functional groups,11 incorporating amphiphilic moieties,12 creating charged materials,13 oxidization,14 and so on. Despite the achievements, most of these modifications still suffer from complex synthesis routes or long-term instability, which are not suitable for studying cell behaviors.15 Recently, the self-assembly of supramolecular gelators has been a classical example for tuning physical properties at the molecular level, since the assemblies arise from noncovalent interactions such as hydrogen bonds, hydrophobic interactions, π−π stacking, and electrostatic interactions, which are distinct from those formed by conventional polymers.16,17 This facilitates the easy control of the physical properties of the assembled aggregates by incorporating different groups into the supramolecules. For example, Stupp and co-workers studied the different self-assembled nanostructures including spheres, cylinders, twisted ribbons, belts, and tubes from peptide amphiphiles (PAs).18−20 Yang et al. reported the helicity of the supramolecular self-assemblies was facilely tuned by terminal chiral groups.21 Yang et al. designed a fusion protein with four binding sites and utilized the protein−peptide © 2013 American Chemical Society
Received: October 17, 2013 Revised: November 21, 2013 Published: November 22, 2013 15359
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2. MATERIALS AND METHODS 2.1. Synthesis of M1−M3. All chemicals were purchased from Aladdin and used without further purification. The gelators M1−M3 were synthesized according to ref 24. 1H nuclear magnetic resonance (1H NMR) experiments were carried out on a Bruker Advance III 400 instrument operating at 400 MHz. All spectra were recorded in dimethyl sulfoxide (DMSO). Mass spectra were recorded on a Waters Q-Tof mass instrument by positive mode electrospray ionization. Methanol was used as the solvent. M1. 1H NMR (400 MHz, DMSO-d6, δ): δ = 3.1 (dd, 4H, CH2), 3.4 (t, 2H, OH), 3.6 (m, 4H, CH2), 4.1 (t, 4H, CH2), 4.7 (dt, 2H, CH), 7.3 (m, 10H, Ph-H), 7.8 (s, 4H, Ph-H), 8.9 (d, 2H, NH) ppm. EI-MS for C30H32O8N2: calcd., 548.22; found, 549.22 [M+H]+. M2. 1H NMR (400 MHz, DMSO-d6, δ): δ = 3.1 (dd, 4H, CH2), 3.4 (m, 12H, CH2), 3.6 (t, 2H, OH), 4.2 (t, 4H, CH2), 4.7 (dt, 2H, CH), 7.2 (m, 10H, Ph-H), 7.8 (s, 4H, Ph-H), 8.9 (d, 2H, NH) ppm. EI-MS for C34H40O10N2: calcd., 636.71; found, 637.28 [M+H]+. M3. 1H NMR (400 MHz, DMSO-d6, δ): δ = 3.1 (dd, 4H, CH2), 3.4 (m, 20H, CH2), 3.6 (t, 2H, OH), 4.2 (t, 4H, CH2), 4.7 (dt, 2H, CH), 7.2 (m, 10H, Ph-H), 7.8 (s, 4H, Ph-H), 8.9 (d, 2H, NH) ppm. EI-MS for C38H48O12N2: calcd., 724.32; found, 725.33 [M+H]+. 2.2. Atomic Force Microscopy (AFM). The test concentrations of samples were all 0.01 wt %. The solution of sample (0.5 mL) was pipetted onto a freshly cleaved mica surface (1 cm × 1 cm) and dried under ambient conditions. AFM images were obtained by using a Vecco NanoScope IIIa atomic force microscope and MikroMasch NSC11 cantilevers/tips (radius of curvature less than 10 nm). AFM images were analyzed offline by using AFM software (Nanoscope 5.30r3sr3) provided by the company. 2.3. Rheological Measurement. Rheology was measured by using a TA Instruments AR G2 rheometer with a 20 mm diameter plate−plate steel geometry. The test concentrations of all hydrogels were 10 mg/mL. The measurements were performed by using a dynamic frequency sweep test in which a sinusoidal shear strain of constant peak amplitude (1%) was applied over a range of frequencies (0.1−10 Hz) at 25 °C. 2.4. Attenuated Total Internal Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. FTIR spectra were taken by using a Bruker EQUINOX55 instrument. The KBr disk technique was used for the solid-state measurement. The samples were scanned between the wavelengths of 4000 and 400 cm−1 at an interval of 1.9285 cm−1. The ATR spectra were measured by using a Nicolet In10MX instrument. The hydrogel was placed on the Ti substrate and dried for ATR test. 2.5. Ultraviolet−Visible (UV−vis) Spectroscopy. UV−vis absorption was recorded on a Tecan Infinite 200 Pro spectrometer. The UV per cell was constructed with 1 mm path length. The test concentrations for all samples were 0.02 mg/mL. 2.6. Circular Dichroism (CD) Spectroscopy. A JASCO J-815 CD spectrometer was used to collect CD data. Spectra were measured in a 10 mm path length cell at 25 °C. The data pitch was 0.2 nm. The scan speed was 100 nm/min. The test concentrations for all samples were 0.02 mg/mL. 2.7. Contact Angle Measurement. For determination of wettability of fibrous scaffolds, water contact angles of M1−M3 scaffolds were measured by using a Powereach JC2000D2 instrument. The images of water drops on the sample surface were recorded with a camera and then analyzed with software supplied by the manufacturer. Three different points were measured for each sample. The initial distilled water volume of 5 μL was used in each measurement. 2.8. Protein Adsorption. Drops of 100 μL of bovine serum albumin/fluoresceinisothiocyanate (BSA/FITC) conjugate (100 μg/ mL) on the glass (diameter: 1 cm) were covered by the fiber samples (0.02 mg). After a certain amount of time, the samples were washed with PBS (3 mL) and incubated in water (3 mL) twice for 2 min each. After drying, the fiber samples were dissolved in 5 mL of methanol. The fluorescence of BSA was measured with a Tecan Infinite 200 Pro spectrometer using λex = 473 nm and λem = 512 nm.
Figure 1. (a) Schematic diagram of cells cultured in 3D supramolecular hydrogels. The cells were encapsulated into 3D hydrogels by mixing Dulbecco’s modified Eagle’s medium (DMEM) and cells into gelators dimethyl sulfoxide (DMSO) solution. (b) Relative hydrophilic fibers with a low water contact angle (upper) induce less cell adhesion and slower proliferation than relative hydrophobic fibers with a high water contact angle (below).
Figure 2. (a) Molecular structure of C2-benzene based gelators M1− M3 and (b) their representative hydrogels.
observed on the least hydrophilic M1 surface. Quantification of cell proliferation in 3D nanofibrous environment revealed that the proliferation rates increased with the decrease of hydrophilicity of the fiber surface. As one of the key factors,26−28 protein adsorption on substrate plays a crucial role to determine the fate of adherent cells. The rate constants of BSA adsorption on M1, M2, and M3 surfaces were 0.23, 0.21, and 0.16 min−1, respectively. The final amount of adsorption on the least hydrophilic M1 surface was 1.5 times more than that on the most hydrophilic M3 surface. The result indicated that the more adsorbed protein from culture medium, the more adhered cells were on the substrate. This further proved that the cell adhesion on the wettable surface was mediated by the protein adsorption. The study makes it possible to establish a 3D environment to gain insight into the influence of surface wettability on cell adhesion, migration, and proliferation,29 rather than only on 2D surfaces.30−32 15360
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Figure 3. Tapping mode AFM scan for (a, b) M1, (d, e) M2, and (g, h) M3 dried gels. Height scans record of lines of (c) M1, (f) M2, and (j) M3 dried gels.
Figure 4. Transmission versus grazing angle ATR-FTIR spectra for xerogels of (a) M1 and (b) M3 (similar to M2; see the Supporting Information). (c) Proposed orientation of the hydrogen bonding interactions within the fibers.
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Figure 5. (a) UV−vis spectra of M1−M3. (b) CD spectra of M1−M3.
cm−1) is the out of plane bending of the amide’s N−H.34 Both ranges are characteristic of hydrogen-bonded amides. ATR grazing angle spectra showed that the carbonyl stretching band intensities reduced significantly when the samples were irradiated in grazing angle mode compared with that in transform mode. These results indicate a preferred orientation of the hydrogen bonding groups in a plane and the hydrogen bonding array is oriented along the axis of a nanofiber.35 Therefore, a model is proposed for M1−M3 self-assemblies in which the hydrogen bonding interactions within amide groups are along with the fiber axis (Figure 4c). Optical spectroscopy was utilized to probe the conformation of the conjugated moiety within the nanofibers. UV−vis spectra of all gels gave rise to absorbance maximum at 250 nm, indicative of π−π* transitions in the benzene groups (Figure 5a).36 CD spectra revealed the formation of random conformations (distinct trough near 198 nm) and β-turn-like structures (pronounced trough at 240 nm) within M1−M3 fibers (Figure 5b).37 Contact angle (CA) measurement was used to test the wettability of M1−M3 xerogels. The static angle on the surface reached balance state and did not significantly change after 3 s (Figure 6). With increasing the EG chain length, the average CA was 50°, 37°, and 30° for M1−M3, respectively. By changing terminal moieties of gelators, wettable properties of these gels are varied due to the inherent self-assembled ability
2.9. 2D/3D Cell Culture. For 2D cell culture, 100 μL of M1 (or M2, M3) solution (0.1 mg/mL) was added into one well of 96-well plates. Then 100 μL of cell suspension (1 × 105 cells) was added to one well of a 96-well plate coated with xerogels. In 3D culture, 20 μL of the mixed gelators in DMSO solution (300 mg/mL) was transferred to an insert and mixed with 980 μL of cell suspension (1 × 105 cells). The 100 μL of above cell suspension was then added to the well (96well plates) and incubated for 2 min under humidified atmosphere of 5% CO2 at 37 °C. Afterward, another 100 μL of fresh medium was added on the top of the gel surface. In both culture methods, the old medium is replaced every 48 h. 2.10. Live−Dead Staining. A fluorescent live−dead staining assay (Invitrogen) was used to visualize the proportion of viable and nonviable cells present after 24 h. A volume of 200 μL of the PBS assay solution containing 2 μM calcein AM and 4 μM propidium iodide (PI) was pipetted onto each cell−gel construct. After 15 min incubation under humidified atmosphere of 5% CO2 at 37 °C, the labeled cells were then viewed under a fluorescence microscope with excitation filters of 494 nm (green, calcein AM) and 545 nm (red, PI). 2.11. Cell Viability. A cell counting kit-8 (CCK-8) was employed to quantitatively evaluate the cell viability. A volume of 10 μL CCK-8 was added to one well of a 96-well plate, and three parallel replicates were prepared. After 2 h incubation at 37 °C, the absorbance at 450 nm was determined.
3. RESULTS AND DISCUSSION M1−M3 gelators were synthesized with high yields through conventional liquid phase reaction. Our previous study demonstrated that 1,4-diamide benzene core provided parallel interactions between hydrogen-bonding moieties. Hydrophobic phenylalanine side chains can provide hydrophobic and π−π interaction forces for self-assemblies.33 These noncovalent interactions were necessary to enforce self-assembly and hence allowed gelation to occur (Figure 2). The lowest concentrations of gelation were 0.2 wt % for M1, 0.07 wt % for M2, and 0.3 wt % for M3. AFM micrographs proved the nanostructured fibers in their self-assembled state (Figure 3) with diameters in the range of 10−500 nm. The root-mean-square roughness was almost the same for three dried gels: 68.6 ± 9.9 nm for M1, 56.6 ± 11.7 nm for M2, and 66.6 ± 10.8 nm for M3. Here, the mechanical property of the hydrogel was found to be slightly affected by the chain length of C-terminal EG monomer (storage modulus G′: ∼3.7 kPa for M1, ∼10.5 kPa for M2, and ∼8.1 kPa for M3).24 FTIR (Figure 4a and Supporting Information Figure S1) was used to unveil the interaction within the xerogels. The amide I band (between 1610 and 1690 cm−1) is primarily due to the CO stretch, and the amide II band (between 1510 and 1560
Figure 6. Relationship between contact angles on M1−M3 surfaces and the time of measurement. 15362
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of M1−M3. Subsequently, the effect of wettability on cell adhesion and proliferation were investigated by using two cell lines: MHCC-97H and NHSF. First, cytotoxicity of the M1−M3 surfaces was qualitatively tested by live−dead staining assay. In this assay, the live cells stained green and the dead cells stained red were observed after 24 h culture (Figure 7a). The overall survival rates for both cells
Figure 8. (a) Phase-contrast images of MHCC-97H cultured on 2D surfaces during 5 days. Scale bar represents 400 μm. CCK-8 assay results of (b) MHCC-97H and (c) NHSF cultured on M1−M3 surfaces.
The effect of M1−M3 gels on cell behaviors in 3D environment was further carried out. The cells were encapsulated into the hydrogels by mixing DMEM and cells with M1 (or M2, M3) of DMSO solutions (Figure 9a). The cells proliferated well in the hydrogel after long-term cell culture up to 8 days (Figure 9b, c). Phase-contrast microscope images showed that both cells displayed round morphology in the 3D matrix, which was distinct from the spread morphology on 2D culture (Figure 9d−i). This phenomenon was in agreement with the reported results by Feder-Mengus et al.39 Due to the diffusion-limited distribution of oxygen, nutrients, and metabolites, the cells proliferation rate in the 3D culture was relatively slow compared with that on the 2D culture.40 Quantification of both cell proliferations revealed that the proliferation rates also increased with the decrease of hydrophilicity of fiber surface. After 8 days of culture, OD tested by CCK-8 assay was 50% higher for MHCC-97H and 63% higher for NHSF in M1 cells construction than those in M3 cells construction. Varying interfacial wettability of scaffolds to tune cell growth was achieved not only on 2D substrates but also in 3D environments. In addition, the fastest cell proliferation in the M3 gels was partly influenced by the relatively fast degradation of the gels (degradation time of gel: 8 days for M1, 15 days for M2, and 9 days for M3).41 The results of cell growth in both 2D and 3D environments agree with the recent results that relatively less hydrophilicity and lower surface energy generally can promote cell adhesion and growth on biomaterials.42−44 A possible explanation to these phenomena is the difference of protein adsorption on the substrate. Indeed, the first stage in cell adhesion mechanisms is protein adsorption which appears to be a dominant player in dedicating cell response to the material.45 As one of the most important parameters to determine cell adhesion behaviors, protein adsorption on the gels was subsequently explored by using BSA since it plays a vital role during cell culture.46 After 25 min, BSA adsorption reached equilibrium state for all gels (Figure 10a). There was an obvious decrease in the amount of
Figure 7. (a) MHCC-97H and NHSF cells cultured on 2D surfaces after 24 h. Green staining indicates live cells, and red staining indicates dead cells. Scale bar represents 400 μm. (b) Dead cells were counted after live−dead staining. Note: values are averages counted by using Image J with at least three pictures.
were all above 95% on M1−M3 surfaces, similar to those on the control polystyrene (PS) surfaces (Figure 7b). This suggested that M1−M3 materials were biocompatible and permitted cell growth.38 Bright field optical microscopic images showed the highest density for cells cultured on the M1 surface (Figure 8a) after 24 h culture. Cell counting kit-8 (CCK-8) assays also proved the similar phenomena on the M1 surface with an optical density (OD) of ∼0.28 for MHCC-97H and ∼0.25 for NHSF. With cell culture durations up to 5 days, both MHCC-97H and NHSF adhered and proliferated on M1 substrate were at the highest rate (Figure 8b). The final MHCC-97H and NHSF cell density was 5.5 and 4.3 times higher than the original culture density, respectively. Compared with the M1 surface, the cell proliferation rate was lower on more hydrophilic M2 and M3 surfaces. This decrease in cell adhesion and proliferation with an increase in hydrophilicity was observed for the 2D gel coated substrates with water contact angles in the range of 30−50°. 15363
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Figure 9. (a) Photograph of cells encapsulated in 3D gels. CCK-8 assay results of (b) MHCC-97H and (c) NHSF cultured in M1−M3 gels. (d−i) Phase-contrast images of MHCC-97H and NHSF cultured in 3D M2 gels during 8 days. Scale bar represents 400 μm.
Figure 10. (a) BSA adsorption on surfaces of M1−M3. The adsorbed BSA on all surfaces reached equilibrium within 20 min. (b) Pseudo-first-order kinetic plots of BSA adsorbed on sample surfaces (blue line, M1; red line, M2; black line, M3). Point with cross and line: linear fit for the selected data.
BSA adsorbed on the fibrous networks from M1, M2, to M3. The maximum amount of the adsorbed BSA on M1 was 15.3 μg, while much less BSA adsorbed on more hydrophilic M2 (8.6 μg) and M3 (6.0 μg). Using the equation of a pseudo-first-order system by Lagergren, the rate constants of BSA adsorption were determined.47 It is described by the equation
M1, 435.98 mg/g for M2, and 306.44 mg/g for M3. The calculated k1 values of BSA adsorption on M1−M3 surfaces were 0.23, 0.21, 0.16 min−1, respectively. The results demonstrate that not only the most amount of BSA adsorb on the less hydrophilic M1 surface, but also BSA adsorption rate on M1 surface is highest among all the gel surfaces. Thus, it can be reasonably speculated that the wettability of fibers may be recognized by the cells through interaction between the fibers and protein, which may release different signals to the cells and result in different cell−fiber interaction.48,49 Though a greater level of biological understanding of how cells respond to different wettable supramolecular gel surfaces is currently missing, the investigation about the effect of supramolecular biomaterials’ wettability on the cell growth will increase our understanding further and develop supramolecular materials as 3D scaffolds.
ln(1 − qt /qe) = −k1t
where qt and qe are the mass of BSA adsorbed at time t (mg BSA/g material) and equilibrium mass of BSA adsorbed on material surface (mg BSA/g material), respectively. k1 is pseudo-first-order rate constant (min−1), and t is contact time (min). To obtain the rate constants, the straight line plots of ln(1 − qt/qe) against t for different substrates were tested (Figure 10b). The k1 values and correlation coefficients r12 were calculated from these plots and are given in Table S1 in the Supporting Information. The qe values were 769.94 mg/g for 15364
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with controlled surface chemistry combining minimal protein adsorption with specific bioactivation. Nat. Mater. 2011, 10, 67−73. (7) Park, J. H.; Wasilewski, C. E.; Almodovar, N.; Olivares-Navarrete, R.; Boyan, B. D.; Tannenbaum, R.; Schwartz, Z. The responses to surface wettability gradients induced by chitosan nanofilms on microtextured titanium mediated by specific integrin receptors. Biomaterials 2012, 33, 7386−7393. (8) Huang, L.; Yokoyama, Y.; Wu, W.; Liaw, P. K.; Pang, S. J.; Inoue, A.; Zhang, T.; He, W. Ni-free Zr-Cu-Al-Nb-Pd bulk metallic glasses with different Zr/Cu ratios for biomedical applications. J. Biomed. Mater. Res., Part B 2012, 100B, 1472−1482. (9) Van der Valk, P.; Pelt, A. W. J.; Busscher, H. J.; de Jong, H. P.; Wildevuur, C. R. H.; Arends, J. Interaction of fibroblasts and polymer surfaces: relationship between surface free energy and fibroblast spreading. J. Biomed. Mater. Res. 1983, 17, 807−817. (10) Faucheux, U.; Schweiss, R.; Lutzow, K.; Werner, C.; Groth, T. Self-assembled monolayers with different terminating groups as model substrates for cell adhesion studies. Biomaterials 2004, 25, 2721−2730. (11) Yang, J.; Wan, Y. Q.; Tu, C. F.; Cai, Q.; Bei, J. Z.; Wang, S. G. Enhancing the cell affinity of macroporous poly(L-lactide) cell scaffold by a convenient surface modification method. J. Polym. Int. 2003, 52, 1892−1899. (12) Klok, H. A.; Hwang, J. J.; Hartgerink, J. D.; Stupp, S. I. Selfassembling biomaterials: L-Lysine-dendron-substituted cholesteryl-(Llactic acid)n̅. Macromolecules 2002, 35, 6101−6111. (13) Zhu, H. G.; Ji, J.; Shen, J. C. Construction of multilayer coating onto poly-(DL-lactide) to promote cytocompatibility. Biomaterials 2004, 25, 109−117. (14) Yang, J.; Bei, J. Z.; Wang, S. G. Enhanced cell affinity of poly (D,L-lactide) by combining plasma treatment with collagen anchorage. Biomaterials 2002, 23, 2607−2614. (15) Fu, H. L.; Zou, T.; Cheng, S. X.; Zhang, X. Z.; Zhuo, R. X. Cholic acid functionalized star poly (DL-lactide) for promoting cell adhesion and proliferation. Tissue Eng. Regen. Med. 2007, 1, 368−376. (16) van Bommel, K. J. C.; van der Pol, C.; Muizebelt, I.; Friggeri, A.; Heeres, A.; Meetsma, A.; Feringa, B. L.; van Esch, J. Responsive cyclohexane-based low-molecular-weight hydrogelators with modular architecture. Angew. Chem., Int. Ed. 2004, 43, 1663−1667. (17) Lin, Y. Y.; Wang, A. D.; Qiao, Y.; Gao, C.; Drechsler, M.; Ye, J. P.; Yan, Y.; Huang, J. B. Rationally designed helical nanofibers via multiple non-covalent interactions: fabrication and modulation. Soft Matter 2010, 6, 2031−2036. (18) Matson, J. B.; Stupp, S. I. Self-assembling peptide scaffolds for regenerative medicine. Chem. Commun. 2012, 48, 26−33. (19) Sargeant, T. D.; Aparicio, C.; Goldberger, J. E.; Cui, H. G.; Stupp, S. I. Mineralization of peptide amphiphile nanofibers and its effect on the differentiation of human mesenchymal stem cells. Acta Biomater. 2012, 8, 2456−2465. (20) Moyer, T. J.; Cui, H. G.; Stupp, S. I. Tuning nanostructure dimensions with supramolecular twisting. J. Phys. Chem. B 2013, 117, 4604−4610. (21) Fu, Y. T.; Li, B. Z.; Huang, Z. B.; Li, Y.; Yang, Y. G. Terminal is important for the helicity of the self-assemblies of dipeptides derived from alanine. Langmuir 2013, 29, 6013−6017. (22) Zhang, X. L.; Chu, X. L.; Wang, L.; Wang, H. M.; Liang, G. L.; Zhang, J. X.; Long, J. F.; Yang, Z. M. Rational design of a tetrameric protein to enhance interactions between self-assembled fibers gives molecular hydrogels. Angew. Chem., Int. Ed. 2012, 51, 4388−4392. (23) Luo, Z. L.; Yue, Y. Y.; Zhang, Y. F.; Yuan, X.; Gong, J. P.; Wang, L. L.; He, B.; Liu, Z.; Sun, Y. L.; Liu, J.; Hu, M. F.; Zheng, J. Designer D-form self-assembling peptide nanofiber scaffold for 3-dimensional cell cultures. Biomaterials 2013, 34, 4902−4913. (24) Dou, X. Q.; Li, P.; Zhang, D.; Feng, C. L. RGD anchored C2benzene based PEG-like hydrogels as scaffolds for two and three dimensional cell cultures. J. Mater. Chem. B 2013, 1, 3562−3568. (25) Fan, X. W.; Lin, L. J.; Messersmith, P. B. Cell fouling resistance of polymer brushes grafted from Ti substrates by surface-initiated polymerization: Effect of ethylene glycol side chain length. Biomacromolecules 2006, 7, 2443−2448.
4. CONCLUSIONS In conclusion, supramolecular self-assembly can act as a facile and effective strategy to achieve wettability controlled cell responses in both 2D and 3D environments. For relatively hydrophilic M1−M3 with water contact angles from 30° to 50°, the adhesion and proliferation of cells were enhanced on less hydrophilic surfaces. The ability to vary supramolecular selfassembly wettability at the material−cell interface in a controlled manner (such as tuning the interfacial chemical groups) is suitable for a wide range of cell related applications, for example, culture systems with unique hydrodynamic properties for 2D/3D tissue culture.50 Further understanding of the relationship between the materials wettability and cell growth will be followed and may have instructive significance for biomaterial selection and design.
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ASSOCIATED CONTENT
S Supporting Information *
Figure showing transmission versus grazing angle ATR-FTIR spectra for M2 xerogels, and table with k1 and r12 values for M1−M3 surfaces. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Fax: +86 2154747651. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support came from the National Science Foundation of China (51173105, 51273111), the National Basic Research Program of China (973 Program2012CB933803), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. Research Fund for the Doctoral Program of Higher Education of China, SRF for ROCS, SEM. Shanghai Jiaotong Medical/Engineering Foundation (YG2012MS29).
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(26) Kolind, K.; Leong, K. W.; Besenbacher, F.; Foss, M. Guidance of stem cell fate on 2D patterned surfaces. Biomaterials 2012, 33, 6626− 6633. (27) Wang, X.; Gan, H.; Sun, T. L. Chiral design for polymeric biointerface: the influence of surface chirality on protein adsorption. Adv. Funct. Mater. 2011, 21, 3276−3281. (28) Cortese, B.; Riehle, M. O.; D́ Amone, S.; Gigli, G. Influence of variable substrate geometry on wettability and cellular responses. J. Colloid Interface Sci. 2013, 394, 582−589. (29) Zhang, S. G.; Gelain, F.; Zhao, X. J. Designer self-assembling peptide nanofiber scaffolds for 3D tissue cell cultures. Semin. Cancer Biol. 2005, 15, 413−420. (30) Zhuang, Z.; Fujimi, T. J.; Nakamura, M.; Konishi, T.; Yoshimura, H.; Aizawa, M. Development of a,b-plane-oriented hydroxyapatite ceramics as models for living bones and their cell adhesion behavior. Acta. Biomater. 2013, 9, 6732−6740. (31) Lampin, M.; WarocquierClerout, R.; Legris, C.; Degrange, M.; SigotLuizard, M. F. Correlation between substratum roughness and wettability, cell adhesion, and cell migration. J. Biomed. Mater. Res. 1997, 36, 99−108. (32) Moffa, M.; Polini, A.; Sciancalepore, A. G.; Persano, L.; Mele, E.; Passione, L. G.; Potente, G.; Pisignano, D. Microvascular endothelial cell spreading and proliferation on nanofibrous scaffolds by polymer blends with enhanced wettability. Soft Matter 2013, 9, 5529−5539. (33) Dou, X. Q.; Li, P.; Zhang, D.; Feng, C. L. C2-symmetric benzene-based hydrogels with unique layered structures for controllable organic dye adsorption. Soft Matter 2012, 8, 3231−3238. (34) Paramonov, S. E.; Jun, H. W.; Hartgerink, J. D. Self-assembly of peptide-amphiphile nanofibers: The roles of hydrogen bonding and amphiphilic packing. J. Am. Chem. Soc. 2006, 128, 7291−7298. (35) Rodriguez-Llansola, F.; Escuder, B.; Miravet, J. F.; HermidaMerino, D.; Hamley, I. W.; Cardin, C. J.; Hayes, W. Selective and highly efficient dye scavenging by a pH-responsive molecular hydrogelator. Chem. Commun. 2010, 46, 7960−7962. (36) Selvaraj, S. J.; Alphonse, I.; Britto, S. J. Isolation and characterization of novel esters from aerial parts of Tiliacora acuminata. Indian J. Chem., Sect. B 2009, 48, 1038−1040. (37) Ma, M. L.; Kuang, Y.; Gao, Y.; Zhang, Y.; Gao, P.; Xu, B. Aromatic−aromatic interactions induce the self-assembly of pentapeptidic derivatives in water to form nanofibers and supramolecular hydrogels. J. Am. Chem. Soc. 2010, 132, 2719−2728. (38) Panda, J. J.; Dua, R.; Mishra, A.; Mittra, B.; Chauhan, V. S. 3D cell growth and proliferation on a RGD functionalized nanofibrillar hydrogel based on a conformationally restricted residue containing dipeptide. ACS Appl. Mater. Interfaces 2010, 10, 2839−2848. (39) Feder-Mengus, C.; Ghosh, S.; Reschner, A.; Martin, I.; Spagnoli, G. C. New dimensions in tumor immunology: what does 3D culture reveal? Trends Mol. Med. 2008, 14, 333−340. (40) Derda, R.; Laromaine, A.; Mammoto, A.; Tang, S. K. Y.; Mammoto, T.; Ingber, D. E.; Whitesides, G. M. Paper-supported 3D cell culture for tissue-based bioassays. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 18457−18462. (41) Elowsson, L.; Kirsebon, H.; Carmignac, V.; Durbeej, M.; Mattiasson, B. Porous protein-based scaffolds prepared through freezing as potential scaffolds for tissue engineering. J. Mater. Sci.: Mater. Med. 2012, 23, 2489−2498. (42) Kennedy, S. B.; Washburn, N. R.; Simon, C. G.; Amis, E. J. Combinatorial screen of the effect of surface energy on fibronectinmediated osteoblast adhesion, spreading and proliferation. Biomaterials 2006, 27, 3817−3824. (43) Fu, H. L.; Zou, T.; Cheng, S. X.; Zhang, X. Z.; Zhuo, R. X. Cholic acid functionalized star poly (DL-lactide) for promoting cell adhesion and proliferation. J. Tissue Eng. Regener. Med. 2007, 1, 368− 376. (44) Hirata, E.; Akasaka, T.; Uo, M.; Takita, H.; Watari, F.; Yokoyama, A. Carbon nanotube-coating accelerated cell adhesion and proliferation on poly(L-lactide). Appl. Surf. Sci. 2012, 262, 24−27. (45) Anselme, K. Osteoblast adhesion on biomaterials. Biomaterials 2000, 21, 667−681. 15366
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Wettability of Supramolecular Nanofibers for Controlled Cell Adhesion and Proliferation Xiao-Qiu Dou, Di Zhang, and Chuan-Liang Feng* State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China S Supporting Information *
ABSTRACT: By employing smart self-assembly of 1,4-benyldicarbonxamide-phenylalanine (C2) derived supramolecular gelators, a simple way to construct nanofibrous environments with the controllable wettability is developed. The fast cell adhesion and proliferation on the least wettable fibers indicates an efficient control over cells, which is proved to be mainly mediated by the interaction between protein and the fibers. One typical merit superior to other materials is that cell adhesion can be regulated not only on two-dimensional (2D) substrates but also in threedimensional (3D) microenvironments. This paves a novel way to deeply understand the influence of fiber wettability on cell behaviors in 3D environment.
1. INTRODUCTION
interaction to enhance the storage modulus of the resulting gels.22 Herein, a simple way to construct materials with the controllable wettability is developed by employing selfassembly of C2-benzene based supramolecular gelators. The cell adhesion on the self-assembled nanofibers can be efficiently tuned, which is mainly mediated by the interaction between protein (e.g., bovine serum albumin, BSA) and fibers. One typical merit superior to other materials is that cell adhesion can be regulated not only on two-dimensional (2D) substrates but also in three-dimensional (3D) microenvironments (Figure 1). This provides a novel way to gain insight into the influence of fiber wettability on cell behaviors in 3D environment, which is crucial for the design of a novel culture environment with desired wettability.23 Three types of C2-benzene based supramolecular gelators with ethylene glycol (EG) monomers as pendant groups (denoted as M1, M2, and M3) were synthesized (Figure 2). Through noncovalent interactions, the gelators can selfassemble into PEG-like nanofibrous hydrogels with different surface wetting properties,24,25 even though their other properties are similar. The influence of surface wettability on cell adhesion and proliferation was studied by using human hepatoma cell line 97H (MHCC-97H) and normal human skin fibroblast (NHSF). The fastest growth of metabolic activity was
Cell adhesion and growth on extracellular matrix (ECM) are greatly influenced by their properties including surface wettability,1−3 roughness,4 topography,5 and chemistry composition.6 Among these, surface wettability has been recognized as an important factor to control the dynamic interaction between an implanted surface and cells in vitro or in vivo.7−10 These surfaces with different wettabilities are usually achieved by introducing functional groups,11 incorporating amphiphilic moieties,12 creating charged materials,13 oxidization,14 and so on. Despite the achievements, most of these modifications still suffer from complex synthesis routes or long-term instability, which are not suitable for studying cell behaviors.15 Recently, the self-assembly of supramolecular gelators has been a classical example for tuning physical properties at the molecular level, since the assemblies arise from noncovalent interactions such as hydrogen bonds, hydrophobic interactions, π−π stacking, and electrostatic interactions, which are distinct from those formed by conventional polymers.16,17 This facilitates the easy control of the physical properties of the assembled aggregates by incorporating different groups into the supramolecules. For example, Stupp and co-workers studied the different self-assembled nanostructures including spheres, cylinders, twisted ribbons, belts, and tubes from peptide amphiphiles (PAs).18−20 Yang et al. reported the helicity of the supramolecular self-assemblies was facilely tuned by terminal chiral groups.21 Yang et al. designed a fusion protein with four binding sites and utilized the protein−peptide © 2013 American Chemical Society
Received: October 17, 2013 Revised: November 21, 2013 Published: November 22, 2013 15359
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2. MATERIALS AND METHODS 2.1. Synthesis of M1−M3. All chemicals were purchased from Aladdin and used without further purification. The gelators M1−M3 were synthesized according to ref 24. 1H nuclear magnetic resonance (1H NMR) experiments were carried out on a Bruker Advance III 400 instrument operating at 400 MHz. All spectra were recorded in dimethyl sulfoxide (DMSO). Mass spectra were recorded on a Waters Q-Tof mass instrument by positive mode electrospray ionization. Methanol was used as the solvent. M1. 1H NMR (400 MHz, DMSO-d6, δ): δ = 3.1 (dd, 4H, CH2), 3.4 (t, 2H, OH), 3.6 (m, 4H, CH2), 4.1 (t, 4H, CH2), 4.7 (dt, 2H, CH), 7.3 (m, 10H, Ph-H), 7.8 (s, 4H, Ph-H), 8.9 (d, 2H, NH) ppm. EI-MS for C30H32O8N2: calcd., 548.22; found, 549.22 [M+H]+. M2. 1H NMR (400 MHz, DMSO-d6, δ): δ = 3.1 (dd, 4H, CH2), 3.4 (m, 12H, CH2), 3.6 (t, 2H, OH), 4.2 (t, 4H, CH2), 4.7 (dt, 2H, CH), 7.2 (m, 10H, Ph-H), 7.8 (s, 4H, Ph-H), 8.9 (d, 2H, NH) ppm. EI-MS for C34H40O10N2: calcd., 636.71; found, 637.28 [M+H]+. M3. 1H NMR (400 MHz, DMSO-d6, δ): δ = 3.1 (dd, 4H, CH2), 3.4 (m, 20H, CH2), 3.6 (t, 2H, OH), 4.2 (t, 4H, CH2), 4.7 (dt, 2H, CH), 7.2 (m, 10H, Ph-H), 7.8 (s, 4H, Ph-H), 8.9 (d, 2H, NH) ppm. EI-MS for C38H48O12N2: calcd., 724.32; found, 725.33 [M+H]+. 2.2. Atomic Force Microscopy (AFM). The test concentrations of samples were all 0.01 wt %. The solution of sample (0.5 mL) was pipetted onto a freshly cleaved mica surface (1 cm × 1 cm) and dried under ambient conditions. AFM images were obtained by using a Vecco NanoScope IIIa atomic force microscope and MikroMasch NSC11 cantilevers/tips (radius of curvature less than 10 nm). AFM images were analyzed offline by using AFM software (Nanoscope 5.30r3sr3) provided by the company. 2.3. Rheological Measurement. Rheology was measured by using a TA Instruments AR G2 rheometer with a 20 mm diameter plate−plate steel geometry. The test concentrations of all hydrogels were 10 mg/mL. The measurements were performed by using a dynamic frequency sweep test in which a sinusoidal shear strain of constant peak amplitude (1%) was applied over a range of frequencies (0.1−10 Hz) at 25 °C. 2.4. Attenuated Total Internal Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. FTIR spectra were taken by using a Bruker EQUINOX55 instrument. The KBr disk technique was used for the solid-state measurement. The samples were scanned between the wavelengths of 4000 and 400 cm−1 at an interval of 1.9285 cm−1. The ATR spectra were measured by using a Nicolet In10MX instrument. The hydrogel was placed on the Ti substrate and dried for ATR test. 2.5. Ultraviolet−Visible (UV−vis) Spectroscopy. UV−vis absorption was recorded on a Tecan Infinite 200 Pro spectrometer. The UV per cell was constructed with 1 mm path length. The test concentrations for all samples were 0.02 mg/mL. 2.6. Circular Dichroism (CD) Spectroscopy. A JASCO J-815 CD spectrometer was used to collect CD data. Spectra were measured in a 10 mm path length cell at 25 °C. The data pitch was 0.2 nm. The scan speed was 100 nm/min. The test concentrations for all samples were 0.02 mg/mL. 2.7. Contact Angle Measurement. For determination of wettability of fibrous scaffolds, water contact angles of M1−M3 scaffolds were measured by using a Powereach JC2000D2 instrument. The images of water drops on the sample surface were recorded with a camera and then analyzed with software supplied by the manufacturer. Three different points were measured for each sample. The initial distilled water volume of 5 μL was used in each measurement. 2.8. Protein Adsorption. Drops of 100 μL of bovine serum albumin/fluoresceinisothiocyanate (BSA/FITC) conjugate (100 μg/ mL) on the glass (diameter: 1 cm) were covered by the fiber samples (0.02 mg). After a certain amount of time, the samples were washed with PBS (3 mL) and incubated in water (3 mL) twice for 2 min each. After drying, the fiber samples were dissolved in 5 mL of methanol. The fluorescence of BSA was measured with a Tecan Infinite 200 Pro spectrometer using λex = 473 nm and λem = 512 nm.
Figure 1. (a) Schematic diagram of cells cultured in 3D supramolecular hydrogels. The cells were encapsulated into 3D hydrogels by mixing Dulbecco’s modified Eagle’s medium (DMEM) and cells into gelators dimethyl sulfoxide (DMSO) solution. (b) Relative hydrophilic fibers with a low water contact angle (upper) induce less cell adhesion and slower proliferation than relative hydrophobic fibers with a high water contact angle (below).
Figure 2. (a) Molecular structure of C2-benzene based gelators M1− M3 and (b) their representative hydrogels.
observed on the least hydrophilic M1 surface. Quantification of cell proliferation in 3D nanofibrous environment revealed that the proliferation rates increased with the decrease of hydrophilicity of the fiber surface. As one of the key factors,26−28 protein adsorption on substrate plays a crucial role to determine the fate of adherent cells. The rate constants of BSA adsorption on M1, M2, and M3 surfaces were 0.23, 0.21, and 0.16 min−1, respectively. The final amount of adsorption on the least hydrophilic M1 surface was 1.5 times more than that on the most hydrophilic M3 surface. The result indicated that the more adsorbed protein from culture medium, the more adhered cells were on the substrate. This further proved that the cell adhesion on the wettable surface was mediated by the protein adsorption. The study makes it possible to establish a 3D environment to gain insight into the influence of surface wettability on cell adhesion, migration, and proliferation,29 rather than only on 2D surfaces.30−32 15360
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Figure 3. Tapping mode AFM scan for (a, b) M1, (d, e) M2, and (g, h) M3 dried gels. Height scans record of lines of (c) M1, (f) M2, and (j) M3 dried gels.
Figure 4. Transmission versus grazing angle ATR-FTIR spectra for xerogels of (a) M1 and (b) M3 (similar to M2; see the Supporting Information). (c) Proposed orientation of the hydrogen bonding interactions within the fibers.
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Figure 5. (a) UV−vis spectra of M1−M3. (b) CD spectra of M1−M3.
cm−1) is the out of plane bending of the amide’s N−H.34 Both ranges are characteristic of hydrogen-bonded amides. ATR grazing angle spectra showed that the carbonyl stretching band intensities reduced significantly when the samples were irradiated in grazing angle mode compared with that in transform mode. These results indicate a preferred orientation of the hydrogen bonding groups in a plane and the hydrogen bonding array is oriented along the axis of a nanofiber.35 Therefore, a model is proposed for M1−M3 self-assemblies in which the hydrogen bonding interactions within amide groups are along with the fiber axis (Figure 4c). Optical spectroscopy was utilized to probe the conformation of the conjugated moiety within the nanofibers. UV−vis spectra of all gels gave rise to absorbance maximum at 250 nm, indicative of π−π* transitions in the benzene groups (Figure 5a).36 CD spectra revealed the formation of random conformations (distinct trough near 198 nm) and β-turn-like structures (pronounced trough at 240 nm) within M1−M3 fibers (Figure 5b).37 Contact angle (CA) measurement was used to test the wettability of M1−M3 xerogels. The static angle on the surface reached balance state and did not significantly change after 3 s (Figure 6). With increasing the EG chain length, the average CA was 50°, 37°, and 30° for M1−M3, respectively. By changing terminal moieties of gelators, wettable properties of these gels are varied due to the inherent self-assembled ability
2.9. 2D/3D Cell Culture. For 2D cell culture, 100 μL of M1 (or M2, M3) solution (0.1 mg/mL) was added into one well of 96-well plates. Then 100 μL of cell suspension (1 × 105 cells) was added to one well of a 96-well plate coated with xerogels. In 3D culture, 20 μL of the mixed gelators in DMSO solution (300 mg/mL) was transferred to an insert and mixed with 980 μL of cell suspension (1 × 105 cells). The 100 μL of above cell suspension was then added to the well (96well plates) and incubated for 2 min under humidified atmosphere of 5% CO2 at 37 °C. Afterward, another 100 μL of fresh medium was added on the top of the gel surface. In both culture methods, the old medium is replaced every 48 h. 2.10. Live−Dead Staining. A fluorescent live−dead staining assay (Invitrogen) was used to visualize the proportion of viable and nonviable cells present after 24 h. A volume of 200 μL of the PBS assay solution containing 2 μM calcein AM and 4 μM propidium iodide (PI) was pipetted onto each cell−gel construct. After 15 min incubation under humidified atmosphere of 5% CO2 at 37 °C, the labeled cells were then viewed under a fluorescence microscope with excitation filters of 494 nm (green, calcein AM) and 545 nm (red, PI). 2.11. Cell Viability. A cell counting kit-8 (CCK-8) was employed to quantitatively evaluate the cell viability. A volume of 10 μL CCK-8 was added to one well of a 96-well plate, and three parallel replicates were prepared. After 2 h incubation at 37 °C, the absorbance at 450 nm was determined.
3. RESULTS AND DISCUSSION M1−M3 gelators were synthesized with high yields through conventional liquid phase reaction. Our previous study demonstrated that 1,4-diamide benzene core provided parallel interactions between hydrogen-bonding moieties. Hydrophobic phenylalanine side chains can provide hydrophobic and π−π interaction forces for self-assemblies.33 These noncovalent interactions were necessary to enforce self-assembly and hence allowed gelation to occur (Figure 2). The lowest concentrations of gelation were 0.2 wt % for M1, 0.07 wt % for M2, and 0.3 wt % for M3. AFM micrographs proved the nanostructured fibers in their self-assembled state (Figure 3) with diameters in the range of 10−500 nm. The root-mean-square roughness was almost the same for three dried gels: 68.6 ± 9.9 nm for M1, 56.6 ± 11.7 nm for M2, and 66.6 ± 10.8 nm for M3. Here, the mechanical property of the hydrogel was found to be slightly affected by the chain length of C-terminal EG monomer (storage modulus G′: ∼3.7 kPa for M1, ∼10.5 kPa for M2, and ∼8.1 kPa for M3).24 FTIR (Figure 4a and Supporting Information Figure S1) was used to unveil the interaction within the xerogels. The amide I band (between 1610 and 1690 cm−1) is primarily due to the CO stretch, and the amide II band (between 1510 and 1560
Figure 6. Relationship between contact angles on M1−M3 surfaces and the time of measurement. 15362
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of M1−M3. Subsequently, the effect of wettability on cell adhesion and proliferation were investigated by using two cell lines: MHCC-97H and NHSF. First, cytotoxicity of the M1−M3 surfaces was qualitatively tested by live−dead staining assay. In this assay, the live cells stained green and the dead cells stained red were observed after 24 h culture (Figure 7a). The overall survival rates for both cells
Figure 8. (a) Phase-contrast images of MHCC-97H cultured on 2D surfaces during 5 days. Scale bar represents 400 μm. CCK-8 assay results of (b) MHCC-97H and (c) NHSF cultured on M1−M3 surfaces.
The effect of M1−M3 gels on cell behaviors in 3D environment was further carried out. The cells were encapsulated into the hydrogels by mixing DMEM and cells with M1 (or M2, M3) of DMSO solutions (Figure 9a). The cells proliferated well in the hydrogel after long-term cell culture up to 8 days (Figure 9b, c). Phase-contrast microscope images showed that both cells displayed round morphology in the 3D matrix, which was distinct from the spread morphology on 2D culture (Figure 9d−i). This phenomenon was in agreement with the reported results by Feder-Mengus et al.39 Due to the diffusion-limited distribution of oxygen, nutrients, and metabolites, the cells proliferation rate in the 3D culture was relatively slow compared with that on the 2D culture.40 Quantification of both cell proliferations revealed that the proliferation rates also increased with the decrease of hydrophilicity of fiber surface. After 8 days of culture, OD tested by CCK-8 assay was 50% higher for MHCC-97H and 63% higher for NHSF in M1 cells construction than those in M3 cells construction. Varying interfacial wettability of scaffolds to tune cell growth was achieved not only on 2D substrates but also in 3D environments. In addition, the fastest cell proliferation in the M3 gels was partly influenced by the relatively fast degradation of the gels (degradation time of gel: 8 days for M1, 15 days for M2, and 9 days for M3).41 The results of cell growth in both 2D and 3D environments agree with the recent results that relatively less hydrophilicity and lower surface energy generally can promote cell adhesion and growth on biomaterials.42−44 A possible explanation to these phenomena is the difference of protein adsorption on the substrate. Indeed, the first stage in cell adhesion mechanisms is protein adsorption which appears to be a dominant player in dedicating cell response to the material.45 As one of the most important parameters to determine cell adhesion behaviors, protein adsorption on the gels was subsequently explored by using BSA since it plays a vital role during cell culture.46 After 25 min, BSA adsorption reached equilibrium state for all gels (Figure 10a). There was an obvious decrease in the amount of
Figure 7. (a) MHCC-97H and NHSF cells cultured on 2D surfaces after 24 h. Green staining indicates live cells, and red staining indicates dead cells. Scale bar represents 400 μm. (b) Dead cells were counted after live−dead staining. Note: values are averages counted by using Image J with at least three pictures.
were all above 95% on M1−M3 surfaces, similar to those on the control polystyrene (PS) surfaces (Figure 7b). This suggested that M1−M3 materials were biocompatible and permitted cell growth.38 Bright field optical microscopic images showed the highest density for cells cultured on the M1 surface (Figure 8a) after 24 h culture. Cell counting kit-8 (CCK-8) assays also proved the similar phenomena on the M1 surface with an optical density (OD) of ∼0.28 for MHCC-97H and ∼0.25 for NHSF. With cell culture durations up to 5 days, both MHCC-97H and NHSF adhered and proliferated on M1 substrate were at the highest rate (Figure 8b). The final MHCC-97H and NHSF cell density was 5.5 and 4.3 times higher than the original culture density, respectively. Compared with the M1 surface, the cell proliferation rate was lower on more hydrophilic M2 and M3 surfaces. This decrease in cell adhesion and proliferation with an increase in hydrophilicity was observed for the 2D gel coated substrates with water contact angles in the range of 30−50°. 15363
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Figure 9. (a) Photograph of cells encapsulated in 3D gels. CCK-8 assay results of (b) MHCC-97H and (c) NHSF cultured in M1−M3 gels. (d−i) Phase-contrast images of MHCC-97H and NHSF cultured in 3D M2 gels during 8 days. Scale bar represents 400 μm.
Figure 10. (a) BSA adsorption on surfaces of M1−M3. The adsorbed BSA on all surfaces reached equilibrium within 20 min. (b) Pseudo-first-order kinetic plots of BSA adsorbed on sample surfaces (blue line, M1; red line, M2; black line, M3). Point with cross and line: linear fit for the selected data.
BSA adsorbed on the fibrous networks from M1, M2, to M3. The maximum amount of the adsorbed BSA on M1 was 15.3 μg, while much less BSA adsorbed on more hydrophilic M2 (8.6 μg) and M3 (6.0 μg). Using the equation of a pseudo-first-order system by Lagergren, the rate constants of BSA adsorption were determined.47 It is described by the equation
M1, 435.98 mg/g for M2, and 306.44 mg/g for M3. The calculated k1 values of BSA adsorption on M1−M3 surfaces were 0.23, 0.21, 0.16 min−1, respectively. The results demonstrate that not only the most amount of BSA adsorb on the less hydrophilic M1 surface, but also BSA adsorption rate on M1 surface is highest among all the gel surfaces. Thus, it can be reasonably speculated that the wettability of fibers may be recognized by the cells through interaction between the fibers and protein, which may release different signals to the cells and result in different cell−fiber interaction.48,49 Though a greater level of biological understanding of how cells respond to different wettable supramolecular gel surfaces is currently missing, the investigation about the effect of supramolecular biomaterials’ wettability on the cell growth will increase our understanding further and develop supramolecular materials as 3D scaffolds.
ln(1 − qt /qe) = −k1t
where qt and qe are the mass of BSA adsorbed at time t (mg BSA/g material) and equilibrium mass of BSA adsorbed on material surface (mg BSA/g material), respectively. k1 is pseudo-first-order rate constant (min−1), and t is contact time (min). To obtain the rate constants, the straight line plots of ln(1 − qt/qe) against t for different substrates were tested (Figure 10b). The k1 values and correlation coefficients r12 were calculated from these plots and are given in Table S1 in the Supporting Information. The qe values were 769.94 mg/g for 15364
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4. CONCLUSIONS In conclusion, supramolecular self-assembly can act as a facile and effective strategy to achieve wettability controlled cell responses in both 2D and 3D environments. For relatively hydrophilic M1−M3 with water contact angles from 30° to 50°, the adhesion and proliferation of cells were enhanced on less hydrophilic surfaces. The ability to vary supramolecular selfassembly wettability at the material−cell interface in a controlled manner (such as tuning the interfacial chemical groups) is suitable for a wide range of cell related applications, for example, culture systems with unique hydrodynamic properties for 2D/3D tissue culture.50 Further understanding of the relationship between the materials wettability and cell growth will be followed and may have instructive significance for biomaterial selection and design.
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ASSOCIATED CONTENT
S Supporting Information *
Figure showing transmission versus grazing angle ATR-FTIR spectra for M2 xerogels, and table with k1 and r12 values for M1−M3 surfaces. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Fax: +86 2154747651. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support came from the National Science Foundation of China (51173105, 51273111), the National Basic Research Program of China (973 Program2012CB933803), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. Research Fund for the Doctoral Program of Higher Education of China, SRF for ROCS, SEM. Shanghai Jiaotong Medical/Engineering Foundation (YG2012MS29).
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