Surface Chemical Functionalization of Wrinkled Thiol–ene Elastomers for Promoting Cellular Alignment

Abstract

Wrinkled polymer surfaces find broad applicability; however, the polymer substrates are often limited to poly(dimethylsiloxane) (PDMS), which limits spatial control over wrinkle features and surface chemistry. An approach to surface functionalization of wrinkled elastomer substrates is demonstrated through versatile, multistep thiol–ene click chemistry. The elastomer is formed using a thiol-Michael reaction of tetrathiol with excess diacrylates while wrinkle formation is induced through a second free radical UV polymerization of the acrylates on the surface of the elastomer. Due to oxygen inhibition of the free radical polymerization, pendant acrylates at the surface remain unreacted and are subsequently functionalized with a multi-functional thiol, which can be further reacted through a number of thiol-X ‘click’ reactions. As a demonstration, these thiol surfaces are further modified to either promote cell adhesion of human mesenchymal stem cells (hMSCs) through coupling with RGDS-containing peptides or surface passivation through reaction with hydrophilic hydroxyl ethyl acrylate moieties. Through engineering a combination of surface chemistry and surface topography, hMSCs exhibited increased spreading and cell density on RGDS-functionalized surfaces and a two-fold increase in cell alignment when cultured on wrinkled substrates. Gradient functionalized surfaces created by tuning the wrinkle wavelength with UV irradiation enabled rapid screening of the effect of topography on the hMSCs. Further, this novel application of click chemistry enables simultaneous tuning of wrinkle topology and surface chemistry towards targeted material applications.

Introduction

Wrinkling or buckling on polymer surfaces presents a fast, cost-effective approach to generate surface topography. One route to form wrinkles is to mount a high modulus thin film on a thicker lower modulus substrate that is mechanically strained. Upon release of the strain, the contraction of the thicker substrate produces a lateral compression of the thin film top layer, which is relaxed through out-of-plane deformation (into and out of the low modulus substrate) to produce sinusoidal wrinkles of wavelength, λ.¹ The wavelength is a function of the film thickness (h) and the film and substrate modulus (E_f and E_s, respectively), described by λ~h(E_f/E_s)^(1/3), which is independent of strain in the linear viscoelastic region. Through this approach, topography can be rapidly generated over large surface areas and has led to numerous applications, including optical and photovoltaic coatings,^{2, 3} switchable wetting surfaces,^4-6 tunable diffraction gratings,⁷ antifouling coatings,⁸ substrates for directed cell-alignment,^9-12 and flexible optoelectronics,^{13, 14} among many others. Despite the versatility of wrinkling, the majority of studies have been limited to poly(dimethylsiloxane) (PDMS) substrates,^{8, 15-24} which lack facile methods to confine and align wrinkle features. Moreover, control over surface chemistry, an important handle for enhancing material performance, is limited.

Functionalization of PDMS is typically performed by first transforming the naturally hydrophobic surface to a nanometer-thick hydrophilic SiO_x surface using plasma or ultraviolet oxidation (UVO).^25-32 When performed on PDMS that is mechanically strained, the newly formed silicate layer acts as the high modulus thin film that enables wrinkle formation upon removal of the deformation. The silicate layer is then readily functionalized, typically via a functional silane agent. One drawback of this functionalization strategy is that PDMS undergoes short-term hydrophobic recovery, a process which restores the hydrophobic state of the PDMS surface over hours to days.^{25, 26, 29} Hydrophobic recovery occurs due to inversion of polar groups from the surface into the bulk, diffusion of low molecular weight PDMS chains to the surface, and condensation of any free silanol groups.^{25-27, 33} Covalently-attached surface functional groups on PDMS are stable.^{28, 31, 32, 34-36} However, these protocols require long surface chemical treatment times, lack spatial control,³⁷ and necessitate the use of plasma treatments - all of which are not scalable, thereby limiting the commercial viability of wrinkling technology. Extended plasma treatments have also been shown to induce crack formation in the PDMS surface.³⁸ On wrinkled PDMS substrates, functionalization has been introduced through physical adsorption of surface moieties, which also lack spatiotemporal control over chemical modification and results in a less mechanically robust coating.³⁹

To address these limitations, we demonstrate a two-tiered polymerization approach to generate wrinkled materials with surface functionality suitable for further chemical modification (Scheme 1).⁴⁰ The two-tiered polymerization scheme employs a thiol-Michael polymerization reaction between multifunctional thiol and acrylate monomers, where the formulation contains excess acrylate monomer. The resultant elastomer substrate thus contains excess pendant acrylates within the polymer network. In addition to the thiol and acrylate monomers, a photoinitiator and a photoabsorber are included in the elastomer formulation. The photoinitiator enables subsequent free-radical polymerization of the acrylates initiated using light, while the photoabsorber attenuates light, thereby isolating the polymerization to the thin skin layer of the elastomer. Upon irradiation of a strained elastomer, a higher modulus skin layer is formed that results in wrinkle formation upon release of the deformation. This approach affords simple control of the wrinkle wavelength by controlling the light intensity and irradiation time, as well as control of the wrinkle confinement and alignment through photopatterning.

Scheme 1. Two-tiered polymerization for wrinkle formation.

Tetrathiol, PETMP, and excess diacrylate, TEGDA, were mixed with photoinitiators (I651 and I819), photoabsorber (T479), and triphenyl phosphine (TPP) to generate an acrylate-rich elastomeric substrate. Straining and irradiating the elastomer with 365nm UV light triggers radical polymerization of the pendant acrylates. The photoabsorber, which absorbs 365 nm light, prevents light penetration into the bulk, thus confining the second polymerization to the surface. Upon destraining the material, wrinkles are generated due to the elastic moduli mismatch between the film and substrate.

Subsequent functionalization of the elastomer formed using this two-tiered approach is enabled by leveraging oxygen inhibition of the free-radical polymerization. Owing to the presence of oxygen at the air-elastomer interface, the high modulus thin layer contains unreacted acrylate moieties available for functionalization via a thiol-Michael click reaction. Moreover, by first reacting with excess multifunctional thiols, the acrylate-rich surface can be transformed into a thiol-rich surface capable of further functionalization by thiol-X reactions⁴¹ to yield a broad range of possible surface chemistries.

As a demonstration of the functionalization efficacy, we create chemically-modified elastomers to i) prevent cell adherence, through surface passivation, or ii) to promote attachment and direct alignment of human mesenchymal stem cells (hMSCs), a model primary cell type that exhibits alignment in the presence of wrinkled topography.^{11, 42-45} We establish that the material can not only be modified with surface chemical cues (i.e., RGDS) to promote cell adhesion and spreading on the surface, but that controlling surface topology induces cellular response by alignment of the cells within the troughs of the wrinkled structures.

Experimental

Materials:

Pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) was donated by Bruno Bock. Tetraethylene glycol diacrylate (TEGDA, >90%), 1,3,5-trimethylbenzene (TMB), 1,8-diazabicyclo[5.4.0]undec-7ene (DBU), quinine, 3-allyloxy-1,2-propanediol (APD), and methanesulfonic acid (MsOH, >99.0%) were purchased from TCI America. Photoinitiator, Irgacure 651 (I651) (2,2-dimethyoxy-1,2-diphenylethan-1-one) and Irgacure 819 (I819) (Bis(2,4,6-trimethylbenzoyl)-phenylphosphineoxide), and photoabsorber, Tinuvin 479 (T479) were donated by BASF. Triphenylphosphine (TPP, 99%) was purchased from Acros Organics. Diisopropylethylamine (DIPEA), hydroxy ethyl acrylate (HEA), 1H-1H-2H-2H-perfluorodecane thiol (PFDT) and dimethylphenylphosphine (DMPP) were purchased from Sigma Aldrich. Acetonitrile (ACS Certified), dimethylformamide (DMF), methanol, and tetrahydrofuran (THF) were purchased from Fisher Scientific. Deuterated acetonitrile, methanol, and water were also purchased from Fisher Scientific. Amino acids, including allyloxycarbonyl-protected lysine (K(alloc)), used in peptide synthesis were purchased from Chem-Impex or Chempep. Fluorescent label, Alexafluor 647 (AF647) carboxylic acid succinimidyl ester, was purchased from Life Technologies Corporation. Trifluoroacetic acid (TFA) and triisopropylsilane (TIPS) was purchased from Chem-Impex. Human mesenchymal stem cells (hMSCs) were purchased from Lonza, basic fibroblast growth factor from Peprotech, phenol red-free DMEM from Corning, and all other cell culture reagents from Life Technologies Corporation. Bovine serum albumin (BSA) and phalloidin tetramethylrhodamine B isothiocyanate (Phalloidin-TRITC) were purchased from Sigma Aldrich, and Dulbecco’s phosphate-buffered saline (DPBS), paraformaldehyde, Triton-X, and DAPI from Fisher Scientific. For LAP synthesis, 2,4,6-trimethylbenzoylchloride, dimethylphenylphosphonite, lithium bromide and 2-butonone were all purchased from Sigma Aldrich. All chemicals were used without further purification.

Generation of wrinkled elastomers:

Photoinitiator I651 (0.5wt%), I819 (0.1wt%) and photoabsorber T479 (1.0wt%) were added to TEGDA in a scintillation vial and stirred at 1000 RPM until all components were fully dissolved. TPP (1.0 wt%) and MsOH (0.3 wt%) were then added to the mixture and stirred for 15 minutes. PETMP was then added to the mixture in a 1:2 thiol:acrylate stoichiometry and stirred for 3 additional minutes. The mixture was then cast between two glass slides with 1/16 inch spacer and allowed to cure overnight. All films were irradiated and wrinkled roughly 14.5 hours after casting.

Elastomer samples were placed on a custom-made stretching apparatus and strained 20% uniaxially (Supporting Information, Figure S1). A glass coverslip was placed atop the elastomer, which was then irradiated with 365 UV light (1 mW/cm²) (Omnicure S2000, equipped with a liquid light guide and collimating lens) for a preset amount of time. Samples were held in the strained position for five minutes after irradiation was complete and then destrained to yield wrinkles.

Gradient samples were created by covering elastomers with an opaque mask, which was slowly withdrawn over the irradiation duration.⁴⁶ After 365 nm irradiation, samples were held in the strained position for 5 minutes. All samples were then destrained, placed on a glass slide, and irradiated with 405 nm light (2 mW/cm², 10 minutes) without a coverslip to react the remaining free acrylates in the bulk. Samples were cut into 6mm x 6mm squares for functionalization. Wrinkle wavelengths were measured on a Nikon Eclipse (LV100, 5MP CCD) Optical Microscope). Wavelengths were recorded as an average wavelength over at least nine periods.

Peptide Synthesis:

Pendant RGDS-containing peptides were synthesized as a functional handle to promote cell adhesion on the elastomer surface. Briefly, a sequence AhxWGRGDSK(alloc)G (RGDS) was synthesized using standard automated Fmoc solid phase peptide synthesis techniques (PS3, Protein Tech).^47-49 After final deprotection of the Ahx monomer, 2 mg of the AF647 carboxylic acid succinimidyl ester was dissolved in 8 mL of DMF with 0.5 mmoles of peptide and 100 μL of DIPEA overnight. Following this reaction, the peptide was cleaved from the resin using a solution of TFA/TIPS/deionized water (95/2.5/2.5) for three hours and precipitated with excess diethyl ether. The crude peptide was purified via HPLC (85:15 to 77:23 Water:Acetonitrile over 9 minutes, XBridge C18 OBD 5 μm column, Waters Corporation), lyophilized, and analyzed by mass spectrometry (Supporting Information, Figure S2).

LAP Synthesis:

The LAP photoinitiator was synthesized according to previously established protocols.^{49, 50} Briefly, 2,4,6-trimethylbenzoyl chloride (1.6 g, 0.009 mol) was reacted with an equimolar amount of dimethylphenylphosphonite (1.5 g, 0.009 mol). The solution was stirred for 18 hours at room temperature under argon, and subsequently lithium bromide in 2-butanone was added to the solution at a 4x molar excess and heated to 50 °C for 10 minutes. After cooling, the solid precipitate was filtered and rinsed 3 times with 2-butanone to remove unreacted reagents. The remaining product was dried and analyzed using ¹H NMR (Supporting Information, Figure S3).

Functionalization:

Solutions containing PETMP in acetonitrile (15 mg/mL) were prepared in 20 mL scintillation vials. Catalyst, DMPP, was then added (0.15mol%) and the mixture was vortexed for 30 seconds. Elastomer squares were submerged into the thiol-containing solution for 60 seconds to functionalize the surface. After functionalization, the elastomer squares were removed and sonicated in 4 mL of acetonitrile for an additional 60 seconds.

HEA solutions were prepared by adding 0.5 mol% DBU to 2 mL of pure HEA. Freshly prepared thiol-functionalized elastomers were submerged into the HEA solution for 60 seconds. Samples were then removed and sonicated in 4 mL of deionized water for 60 seconds.

To prepare RGDS-functionalized samples, 25 μL of RGDS solution (40 mg/mL in deionized water, 22 mM LAP) were added to the surface of thiol-functionalized elastomers placed between two 1/16” Teflon spacers. A glass coverslip was immediately placed on top of the sample to ensure even coverage of the RGDS on the surface. Samples were irradiated with 365 nm UV light (10 mW/cm², 60 seconds). Samples were subsequently sonicated in 3 mL of deionized water for one minute. All samples were sonicated in an additional 4 mL of DI water for 60 minutes and dried under high vacuum at room temperature overnight.

Contact Angle:

Static contact angle measurements were performed by depositing 2 mL of water (purified with a Milli-Q water purification system) on the various functionalized samples (native, thiol, PETMP/HEA, and PETMP/RGDS) and analyzed using a KrüssGmbH FM41 Easy Drop goniometer under ambient conditions. Images were taken 1 second after droplet deposition and analyzed using the accompanying Drop Shape Analysis software. All measurements were performed in triplicate.

X-ray photoelectron spectroscopy (XPS):

To determine the appropriate operating solution concentrations for surface functionalization, XPS experiments were conducted using a Thermo Fisher K-Alpha+ XPS on PFDT-functionalized elastomer substrates, at varying PFDT solution concentrations between 0 mg/mL to 70 mg/mL. PFDT was selected due to the high fluorine signals observed in XPS. Surveys were conducted using 100 eV pass energy, 1 eV step size and 10 ms dwell time. For high resolution fluorine data, 20 eV pass energy, 0.1 eV step size and 50 ms dwell time were used. All experiments were conducted using the flood gun for charge compensation.

Fourier Transform Infrared Spectroscopy (FTIR):

Kinetic data was obtained using a Nicolet iS350 FTIR in transmission mode. All experiments were performed in near-IR. Time-dependent conversions, ρ(t), were taken as 1 – Peak Area(t)/Peak Area (t=0) for both thiols (2509-2612 cm⁻¹) and the C=C ‘ene’ bond (6131-6265 cm⁻¹). Formulations were casted between two glass slides, separated by 0.635 mm PVC shims. Long-time experiments (4 hours) for the thiol-Michael reaction were conducted using a room temperature DTGS detector, with a spectral resolution of 4 cm⁻¹, averaging over 32 spectra. To characterize the free radical polymerization, which is significantly faster, FTIR spectra was obtained using the MCT detector, averaging over 8 spectra with a spectral resolution of 4 cm⁻¹. Conversion data were collected for 3 minutes, with the irradiation of the films starting 30 seconds after FTIR data collection began, in order to obtain a baseline.

Nuclear Magnetic Resonance (NMR) spectroscopy:

¹H-NMR studies were performed using a Bruker AVIII 400 or AVIII 600 NMR spectrometer.

Human mesenchymal stem cell culture and seeding:

Bone marrow-derived human mesenchymal stem cells (hMSCs, Lonza) were expanded on tissue culture treated polystyrene in growth medium (low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 50 U/mL penicillin, 50 μg/mL streptomycin, 0.2% v/v Amphotericin B, 10% v/v fetal bovine serum, and 1 ng/mL basic fibroblast growth factor (bFGF)). Cells were fed every 2-3 days and were passaged at approximately 85% confluency.

Prior to cell seeding, all material samples were sterilized via germicidal UV light irradiation (254nm) for 30 minutes and placed into non-tissue culture treated 48 well plates. Cells were trypsinized from the tissue culture treated plates, centrifuged, and counted with a hemocytometer using standard techniques. Cells were then suspended in phenol red- and serum-free high glucose DMEM (supplemented with 50 U/mL penicillin, 50 μg/mL streptomycin, 0.2% Amphotericin B) and were seeded on top of the materials at 15,000 cells cm⁻². After 12 hours of culture, the media was replaced with phenol red-free high glucose DMEM containing 10% fetal bovine serum (in addition to 50 U/mL penicillin, 50 μg/mL streptomycin, 0.2% Amphotericin B).

Confocal Microscopy:

Cells were washed with DPBS after 3 days of culture on the substrates and fixed with 4% paraformaldehyde for 15 minutes at room temperature on a rocker. After washing (1x 5 min with DPBS and 2x 5 min with 3% w/v bovine serum albumin (BSA) + 0.05% v/v Triton-X in DPBS), cells were blocked and permeabilized (5% w/v BSA + 0.1% v/v Triton-X in DPBS) at room temperature for 1 hour. Cells were then incubated with Phalloidin-TRITC solution (1:250 dilution, 5% w/v BSA, 0.1% v/v Triton-X in DPBS) for 3 hours at room temperature, followed by washing (1 x 5 min DPBS) and staining with DAPI (700 nM in DPBS, 30 minutes at room temperature). The substrates were washed 3x in DPBS prior to imaging. Samples were kept on a rocker during all incubation and wash steps.

Samples were imaged on a Zeiss LSM 810 confocal microscope (z-stacks, 80 images per stack, 2 μm spacing). For quantification of nuclear orientation relative to wrinkles, average cell area, and cell density, fluorescent images (nuclei and F-actin) were processed and analyzed using NIH ImageJ software (Supporting Information, Figure S11). The materials autofluoresce in the blue-green region, and thus we were able to capture images of the material surface using the DAPI channel. Wrinkles were viewed in Ortho view in Zen Blue, and wavelength and amplitude of the wrinkled materials treated with PETMP and RGDS were measured using NIH ImageJ software (Supporting Information, Figure S8).

Results and Discussion

An orthogonal, two-tiered polymerization approach was developed to form the elastomer and the wrinkles independently. Tetrathiol and excess diacrylate were mixed with photoinitiators (Irgacure 651 and Irgacure 819), photoabsorber (Tinuvin 479) and a catalytic amount of triphenylphosphine to form a homogeneous base elastomer (Scheme 1). Methanesulfonic acid (MsOH) was added to create a reaction inhibition time that allowed the thiol and ene components to be mixed and casted as films before polymerization occurs.⁵¹ Because of the self-limiting nature of the thiol-Michael reaction, the stoichiometric excess of acrylates remained unreacted and attached to the crosslinked network backbone. Consistent with the 1:2 thiol:ene stoichiometry used to form the elastomer, in situ FTIR spectra of conversion vs. time for the thiol-Michael reaction (Supporting Information, Figure S4) confirms quantitative thiol conversion and roughly 50% acrylate conversion.

The elastomer was then strained above the critical strain necessary for wrinkle formation,^{1, 52, 53} with a coverslip placed directly on top, to create an oxygen barrier at the surface. The degree of additional crosslinking is controlled through timed irradiation with 365 nm UV light, while the penetration depth of the light is controlled by the concentration of the UV photoabsorber, which confines the acrylate homopolymerization to the surface of the material. Samples were held in the strained state using clamps for over a minute after irradiation, owing to the observation of additional dark polymerization after the light is ceased. The acrylate conversion continues to increase for roughly 30 seconds after the initial UV irradiation (Supporting Information, Figure S5). This dark polymerization is most likely due to limited diffusion of radical species in the elastomer network generated during the photoirradiation, which hinders termination of radicals. The coverslip was subsequently removed, and the strained elastomer was released from the clamps to yield wrinkles. Wrinkle features were controlled through irradiation time and intensity (Figure 1), with larger wavelengths observed at higher intensity and longer irradiation times.

Figure 1. Wrinkle wavelength vs. Time and Intensity.

As light controls the film formation necessary to generate wrinkles, both light intensity and irradiation time dictate the size of wrinkle features. Increasing both the intensity and time will lead to increasing wrinkle wavelengths. Adapted from ref 62.

The surface of the wrinkled elastomer was chemically functionalized using a procedure shown in Scheme 2. The wrinkled polymer was subjected to 405 nm irradiation (without coverslip), which is outside the wavelength absorption of the photoabsorber, but still capable of triggering polymerization of the remaining acrylates within the bulk of the elastomer. This final photocure procedure increases the crosslink density and helps mitigate diffusion of solvent into the network during the next surface functionalization step. It should also be noted that this irradiation step was carried out in the presence of oxygen (i.e., without the cover slip) so that the free radical polymerization was inhibited at the air/polymer interface, ensuring pendant acrylates were available for post-functionalization.

Scheme 2. Chemical modification of the acrylate-rich elastomer surface.

Wrinkled surfaces are irradiated with 405 nm light to induce bulk radical polymerization of the remaining acrylates. This post-processed material is then submerged into a solution of tetrathiol, PETMP, which converts the acrylate-rich surface into a thiol-rich surface via the thiol-Michael reaction. In generating the thiol-rich surface, a second modification can be carried out. In the presence of a nucleophile or base catalyst, the pendant thiols will undergo a second thiol-Michael reaction with electron-poor enes (i.e., hydroxyethyl acrylate). Alternatively, thiols can react with electron-rich enes (i.e., allyl) through a photoinitiated radical thiol–ene mechanism. Because this latter reaction is light-triggered, spatial control over chemical functionalization is possible. An RGDS-containing peptide was synthesized with an allyloxycarbonyl(alloc)-protected lysine amino acid residue, which contains the electron-rich ene that can be coupled to the thiol-rich elastomer. Adapted from ref 62.

While different functionalization schemes can be implemented to modify the pendant acrylates at the wrinkled surface, the surface was first reacted with a large excess of a tetrathiol momomer, PETMP, to produce a thiol-rich interface. By transforming the acrylate-rich to a thiol-rich surface, it is possible to employ the diverse thiol-X ‘click’ reaction toolbox to further modify the surface with specialized molecules for material applications. Wrinkled elastomers were placed into PETMP solutions, reacted for one minute and rinsed. High thiol solution concentration ensures excess thiols are present after reaction at the surface. Thiol coupling to the acrylate-rich surface was confirmed using x-ray photoelectron spectroscopy (XPS) (See Supporting Information, Figure S6).

The thiol-rich surface can be further functionalized through additional thiol-X reactions, including the thiol-Michael and the radical mediated thiol–ene reactions. Thiol-Michael reactions, which were used to generate the base elastomer and create the thiol-rich wrinkled surface, are initiated in the presence of an electron-deficient vinyl functional group (e.g., an acrylate) and a base or nucleophile catalyst(e.g., a phosphine). As hydrophilic surfaces often exhibit antifouling properties,^54-56 hydroxyethyl acrylate (HEA) was selected for the second thiol-Michael functionalization to impart hydrophilicity to the surface of the elastomer via a hydroxyl functional group. Alternatively, with an electron-rich vinyl functional group (e.g., allyl), the reaction will proceed through a radical-mediated reaction in the presence of a radical initiator. Using a photoinitiator and photopatterned light, this latter thiol–ene reaction introduces spatial control over chemical modification of the polymer surface. Allyl-containing peptide, AF647-AhxWGRGDSK(alloc)G (AF647-RGDS), was synthesized (ESI MS in Supporting Information, Figure S2) to promote cell adhesion of human mesenchymal stem cells (hMSCs) through integrin binding of the RGDS sequence.⁵⁷

The chemical modification of the surfaces was assessed using contact angle goniometry (Supporting Information, Figure S7). Native (acrylate-rich) samples exhibited a contact angle of 32.1 ± 1°. The thiol-rich surfaces, created by reacting a hydrophobic PETMP to the acrylate-rich surface showed a commensurate increase in contact angle (75.5 ± 4°). With further coupling of the excess surface thiols to hydrophilic monomers (i.e., hydroxyethyl acrylate, AF647-RGDS), the surfaces are rendered more hydrophilic, thus lowering the contact angle (37.5 ± 1.2°).

The polymer materials were modified with the various functional handles for cell culture experiments using hMSCs. hMSCs are multipotent progenitor cells that can differentiate into a variety of cell types that comprise different tissues, including bone and connective tissues, that require cellular alignment for proper morphology and function.^{58, 59, 60, 42} Non-strained elastomers were irradiated with 365 and 405 nm light under the same processing conditions described in Scheme 2 (i.e., 365 nm irradiation with coverslip for high modulus film formation followed by 405 nm irradiation bulk cure of bulk free acrylates) and subsequently functionalized with just PETMP, PETMP/HEA, or PETMP/RGDS (Figure 2a). ‘Native’, unfunctionalized samples (i.e., free acrylate only) were used as a control. hMSCs were seeded on these flat, chemically modified surfaces to assess surface chemistry effects on cell density and spreading (Figure 2b and c). As expected, PETMP/RGDS functionalized elastomers significantly promoted both cell attachment and spreading (Figure 2b and c) as compared to all other conditions. Thiol-rich surfaces also facilitated some hMSC attachment and spreading but to a much lesser extent. It is worth noting that acrylate-rich elastomers phototreated with just the AF647-RGDS (with LAP) also facilitated similar cell density and spreading as PETMP surfaces. We suspect this is due to some radical polymerization occurring between the acrylate surface and the allyl group on the RGDS peptide in the presence of photoinitiator, both in solution (LAP) as well as in the polymer (Irgacure 651, Irgacure 819).

Figure 2. Effects of flat functionalized surfaces on hMSC attachment.

(a) Representative confocal microscopy images of hMSCs seeded on various functionalized surfaces (Native, PETMP (thiol-rich), PETMP/HEA (hydrophilic), PETMP/RGDS, and RGDS only). Scale bar represents 100 μm. Cell nuclei are stained blue, whereas the cytoskeleton is shown in red. hMSCs seeded on PETMP/RGDS-functionalized surfaces exhibited significant improvements in cell spreading (b) and cell density (c) as compared to other functionalization conditions. For PETMP-functionalized samples, hMSCs attached and spread to a lesser degree, possibly due to nonspecific interactions with the free thiol moieties. Homopolymerization of the allyl moiety in the AF647-RGDS sequence (RGDS only) to the free acrylates may also promote hMSC attachment. The data illustrates the mean with error bars representing the standard error, determined by a two-sided Student’s t-test after evaluation of variance for each population. Roman numerals are statistically the same; the full list of p-values is given in Supporting Information table, Table S1). Adapted from ref 62.

hMSCs were seeded on PETMP/RGDS-functionalized wrinkled elastomers prepared using three different 365 nm irradiation times (5.5, 7.0 and 9.0 seconds) corresponding to three distinct wrinkle wavelengths (λ~ 190, 345, 480 μm, respectively; confirmed via confocal microscopy, Supporting Information, Figure S8). Cell spreading increases with increasing wrinkle wavelengths (Figure 3a and b), due to the cells’ preference to settle in the troughs of the wrinkle structures. As wrinkle wavelengths increase, the troughs of the structures widen, and the curvature near the trough begins to flatten, increasing the surface area of the troughs for cells to settle and spread. Inversely, as wavelengths decrease, steepness between trough and peak increases and the troughs become narrower. This forces cells to spread primarily in the direction parallel to the trough and limits perpendicular spread toward the peaks, leading to a decrease in cell area. Consistent with this trend, flat samples, which can be thought of as an infinitely large wrinkle, had the largest degree of cell spreading. This is consistent with flat samples having the largest degree of accessible surface for cell deposition.

Figure 3. hMSCs seeded on PETMP/RGDS-functionalized wrinkled substrates with increasing wrinkle wavelength.

(a) Representative confocal microscopy images of hMSCs seeded on wrinkled substrates reveal alignment of cells with the troughs of wrinkled structures (vertical in images) versus a flat PETMP/RGDS-functionalized substrate. Scale bar represents 100 μm. Cell nuclei are stained in blue, whereas the cytoskeleton is stained red. (b) As the cells tend to settle and spread along the troughs of the wrinkle structures, increasing the wrinkle wavelengths increases the accessible surface area in the troughs for the cells to spread. (c) Cell density decreases with increasing 365 nm irradiation time. The data illustrate the mean with error bars representing the standard error, determined by a two-sided Student’s t-test after evaluation of variance for each population. Roman numerals are statistically the same; the full list of p-values is given in Supporting Information table, Table S2). Adapted from ref 62.

In contrast to cell spreading, the cell density of hMSCs decreases with increasing wrinkle wavelength (Figure 3c). One reason for this may be variations in PETMP/RGDS density. As the wrinkle formation process is carried out with a coverslip (i.e., negligible O₂ inhibition), some acrylate conversion is expected at the very surface of the elastomer. As wrinkle wavelength increases with increasing 365 nm irradiation, the amount of remaining pendant acrylates available for post modification should decrease, thus lowering the final RGDS surface concentration.

Cell nuclear alignment was also analyzed based on previously established protocols³⁹ with aligned nuclei taken within ±15° off the axis parallel to the wrinkle wavelength direction (Figure 4). All nuclei (>30 nuclei per image) not in contact with the edge of the image were analyzed. In contrast to previous cell alignment studies performed on wrinkle substrates, in which topographic features smaller than the size of a cell were used to induce alignment,³⁹ in our system, the wrinkle wavelengths (λ~190-480 μm) were larger than the size of an hMSC (~10-100 μm). Wrinkle amplitudes (λ~27-41 μm, Supporting Information, Figure S8) were also much larger than the height of seeded hMSCs (on the order of a single micrometer). Wrinkled samples exhibit a two-fold increase in nuclear alignment as compared to flat PETMP/RGDS samples, demonstrating that cells can align with topographical features much larger than the size of a single cell. However, no statistical difference in nuclear alignment was observed between the three wrinkle wavelength conditions. This is likely due to the fact that even the smallest wrinkle wavelength analyzed (~150 μm) was still larger than the hMSC, and the variation in wavelengths probed was limited. This effect is explored further by creating a gradient wrinkled surface.

Figure 4. Nuclear Alignment vs. wrinkle wavelength on PETMP/RGDS surfaces.

Cell alignment measurements, taken as ±15° parallel to the wrinkle wavelength, demonstrated a two-fold increase in nuclear alignment of hMSCs on PETMP/RGDS-functionalized wrinkle substrates as compared to a flat substrate. The data illustrate the mean with error bars representing the standard error, determined by a two-sided Student’s t-test after evaluation of variance for each population. Roman numerals are statistically the same; the full list of p-values is given in Supporting Information table, Table S3). Adapted from ref 62.

This “photowrinkle” system enables spatiotemporal control over wrinkle formation.^{40, 61} This feature expedites the creation of gradient wrinkle systems, which allows rapid screening of surface interactions over a continuum of topographical length scales while only utilizing a single sample (Figure 5). To produce a gradient wrinkle sample, a photomask was placed on top of a strained elastomer. The masked elastomer was then irradiated with 365 nm light as the photomask was withdrawn at constant velocity over the surface of the elastomer to create a linear gradient in the UV light exposure as a function of time (illustrated in Figure 5a). Because the irradiation dose across the surface varies as a function of irradiation time, a modulus (crosslink density) gradient forms in the surface of the elastomer along the stretch direction. Upon releasing the strain, gradient wrinkle formation occurs due to the gradient in modulus mismatch variation across the surface.

Figure 5. hMSC seeding on wrinkled gradient samples.

(a) Elastomer samples are strained and irradiated with a linearly increasing 365 nm irradiation dose producing a crosslink density gradient across the XY planar surface of the material. This gradient modulus alters the modulus mismatch required to produce wrinkle features, thus enabling rapid production of gradient wrinkles. (b) A gradient sample is functionalized with PETMP/RGDS and employed in an hMSCs seeding experiment. As anticipated, hMSCs (F-actin stained with Phalloidin-TRITC – red) seeded on smaller wrinkle wavelengths exhibit more aligned cell bodies than at larger wrinkle wavelengths. The ability to produce gradient wrinkles facilitates probing surface interactions over a continuum of length scales while only utilizing one sample. Wrinkle wavelength ranges from ~100 μm (left end) to ~430 μm (right end). Scale bar represents 1 mm. (c) Enlarged sections corresponding to the gradient image in Figure 5b. Scale bar represent 500 μm.

hMSC cells are introduced and found to interact with this gradient in surface topology in a systematic manner, as seen in Figure 5b. The hMSCs (stained in red) seeded on PETMP/RGDS functionalized gradient samples exhibit visually different cell body alignment across the gradient wrinkled surface. At small wrinkle wavelengths (~100 μm), single hMSCs align with the troughs of the wrinkle structures because the cell size is commensurate with the wrinkle features. As the wrinkle wavelength increases to hundreds of microns, however, the cells exhibit less alignment. It should be noted that the wavelength and amplitude of these materials are coupled, and thus we cannot make specific conclusions about whether the wavelength or amplitude is the dominating factor in governing cell alignment. Ultimately, the degree of cell alignment appears to be a result of the slope between the peak and trough, which is determined by both wavelength and amplitude. The use of this wrinkle wavelength gradient may be particularly interesting as a tool to promote various cell differentiation pathways in response to degree of alignment. This may be especially useful in tissue regeneration applications focusing on interfaces between tissue types.

Conclusions

Thiol–ene photowrinkle systems present a powerful tool for controlling surface interactions of polymer materials. Through stoichiometrically controlled thiol–ene formulations, two-tiered polymerization schemes can be implemented towards rapid generation of wrinkles. Importantly, this topography can be further enhanced through post-chemical functionalization, by leveraging oxygen inhibition associated with the free radical photopolymerization. By employing thiol–ene chemistry to chemically modify wrinkle surfaces, it is possible to independently tailor the topography and the surface chemistry towards a targeted application. While in this study we implemented a two-step functionalization protocol to adapt the elastomer for cell culture experiments, due to the modular nature of thiol–ene ‘click’ chemistry, many commercially available and synthesizable multifunctional thiols and enes can be utilized. In conjunction with the ability to rapidly form spatially aligned and confined topography, the versatility of this approach to controlling surface properties will enable the development of next generation functional coating materials.