Perforation Does Not Compromise Patterned Two-DimensionalSubstrates for Cell Attachment and Aligned SpreadingStephen B. Bandini,⊥,† Joshua A. Spechler,⊥,‡ Patrick E. Donnelly,⊥,† Kelly Lim,⊥,† Craig B. Arnold,‡

Jean E. Schwarzbauer,§ and Jeffrey Schwartz*,†

†Department of Chemistry, ‡Department of Mechanical and Aerospace Engineering, §Department of Molecular Biology, PrincetonUniversity, Princeton, New Jersey 08544, United States

*S Supporting Information

ABSTRACT: Polymeric sheets were perforated by laser ablation and were uncompromised by a debris field when first treatedwith a thin layer of photoresist. Polymer sheets perforated with holes comprising 5, 10, and 20% of the nominal surface area werethen patterned in stripes by photolithography, which was followed by synthesis in exposed regions of a cell-attractive zirconiumoxide-1,4-butanediphosphonic acid interface. Microscopic and scanning electron microscopy analyses following removal ofunexposed photoresist show well-aligned stripes for all levels of these perforations. NIH 3T3 fibroblasts plated on each of theseperforated surfaces attached to the interface and spread in alignment with pattern fidelity in every case that is as high as thatmeasured on a nonperforated, patterned substrate.

KEYWORDS: polymer laser ablation, perforated substrate, two-dimensional patterned cell alignment

A challenge for tissue engineering is to direct cellorganization in a three-dimensional (3D) device;1 an

ideal device should induce cells to adopt the specific, native-likeorganization characteristic of a particular tissue type that isrequired for proper function.2−5Among such scaffold modelsare 3D printed structures, hydrogels, porous foams, andexogenous extracellular matrix (ECM).6−8 These scaffoldclasses have been constructed artfully using synthetic or naturalmaterials to incorporate, for example, growth factors or cell-attachment peptides to improve bioactivity; others have beendesigned to incorporate microfluidic channels to mimicvascularization.9,10 Several of these have shown clinical promisefor tissue repair.11 Templating spatially aligned cell growth in a3D construct that leads to similarly aligned ECM might providethe basis for a scaffold model to recapitulate native tissueproperties.12 3D printing occurs, effectively, through layer-by-layer two-dimensional (2D) printing;13−16 3D constructs canthus be envisaged as a stack of 2D patterned sheets in whichspatially controlling cell spreading on their surfaces is key.17−21

Our method to accomplish aligned cell spreading and ECMassembly was demonstrated on a range of biomaterial polymers.It involves patterning with a cell-attractive, two-componentinterface consisting of micrometer-dimension stripes of azirconium oxide (ZrO2) layer that is terminated with a self-assembled monolayer of an α,ω-diphosphonate (SAMP). Thisnanometer-thin construct templates attachment and prolifer-

ation of plated cells in register with the chemical pattern; cellproliferation is unconstrained and remains aligned over theentire surface,22 and cell-assembled ECM is also in register withthe ZrO2−phosphonate pattern.23 Stacking such sheets in a 3Dconstruct could, however, be unfavorable for cell viability in thedepths of the device: lateral transport of oxygen and nutrientsfrom the device periphery to cells growing toward its center,and removal of waste products from it, could be limiting.24,25 Itis well-established that perforation facilitates transport inbiological26−33 and engineered34−38 systems. Therefore, wereperforations to be present in each 2D-patterned layer of a 3Dconstruct, transport of nutrients and waste products throughoutthe device would be facilitated, yet these very holes couldcompromise our cell templating approach. Here, we show thatfilms of polyetherether ketone (PEEK) can be perforatedsystematically to 20% of their nominal surface areas by laserablation and that such perforations do not compromisecontrolled patterning with a cell-adhesive ZrO2−phosphonateinterface.39 Furthermore, we show that these surfaces templatecell spreading with spatial alignment control as strong as thatachieved using nonperforated surfaces.23

Received: May 30, 2017Accepted: September 28, 2017Published: September 28, 2017

Letter

pubs.acs.org/journal/abseba

© 2017 American Chemical Society 3123 DOI: 10.1021/acsbiomaterials.7b00339ACS Biomater. Sci. Eng. 2017, 3, 3123−3127

In a typical experiment, a 355 nm diode pumped, solid statepulsed Nd:YVO4 laser was used to perforate 50 μm-thick PEEKfilms. The laser beam was focused to a diameter ca. 8 μm at anenergy ca. 40 μJ, yielding a fluence of ca. 80 J/cm2, which is inaccord with published values for the ablation threshold andhole depth for PEEK films ablated by a 308 nm XeCl eximerpulsed laser;40,41 it was adequate to cut through the PEEK withone pass. The laser pulsed every 1 μm while tracing out theperforations, and the stages moved at a speed of 10 mm/s,giving a laser repetition rate of 10 kHz. Initial attempts atablating clean 1 × 1 cm PEEK substrates showed that theperforation process yielded a debris field that could bevisualized by optical microscopy as large, dark areassurrounding the ablated holes (Figure 1A); sonication inethanol did not remove the debris. This problem was obviatedthrough the simple expedient of first coating the polymercoupons with the same photoresist that was used subsequentlyfor surface photolithographic patterning (Figure 1B). Thus,

when photoresist AZ-5214-E was spin-cast on both sides of thePEEK coupons and then heated (95 °C) to cure, debris fromablation procedure landed on top of the photoresist; removalby sonication in ethanol showed the formation of well-definedperforations that were surrounded by clean polymer (Figure1B).Perforations 130 × 30 μm were chosen to be large enough to

prevent blockage by a single cell;42 perforations on PEEKsurfaces were introduced in several patterns such that thedistance from an attached cell to any hole would be no morethan 200 μm, the estimated maximum distance that oxygendiffuses from a capillary to support metabolically active tissue.43

We prepared substrates in which 5, 10, and 20% of theirnominal surface areas consisted of holes to test the limits of ourpatterning procedures. Doubling the number of holes toconvert 5% nominal surface coverage to 10% was done either“head to tail” (Figure 2B) or “side to side” (Figure 2C);surfaces with 20% nominal hole coverage were created by

Figure 1. Protection scheme for laser ablation of PEEK films. (A) Perforations are introduced by laser ablation without a photoresist cover layer;after sonication in ethanol, a large debris field remains around the ablated hole. (B) With photoresist protection, debris lands on the photoresistlayer, and sonication in ethanol yields a well-defined hole with minimal debris surrounding it. Optical images; scale bar = 40 μm.

Figure 2. Optical images of perforated PEEK substrates with (A) 5% surface coverage by holes, (B) 10% coverage via “head-to-tail” ablation, (C)10% coverage by “side-to-side” ablation, and (D) 20% coverage. Each surface was functionalized with the ZrO2−phosphonate interface patterned in30 × 30 μm stripes; scale bar = 100 μm. A SEM image shows well-defined patterning around the laser-ablated perforation (E), and the EDX maps ofZrLα1 (F) and PKα1 (G) confirm that the ZrO2−phosphonate modification conforms to the pattern.

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doubling these numbers of ablations. Photolithography by spin-coating photoresist on heavily perforated surfaces has not beenreported, and a minor complication was that perforated PEEKdid not attach to the spin coater chuck as is necessary todeposit photoresist: the holes obviate a vacuum seal between itand the chuck of the spin coater. This was easily addressed byfirst adhering the perforated polymer to a glass slide usingseveral drops of uncured photoresist; the PEEK/glass ensemblewas then spin-coated with additional photoresist at 4000 rpmfor 40 s, then baked at 95 °C for 45 s. The ensembles wereexposed to UV (365 nm, 4W) through a photomask for 40 s tocreate 30 μm wide stripes separated by 30 μm (30 × 30 μm); ineach case patterned stripes of the photomask were orientedparallel to the perforated holes. Treating with AZ MIF 300developer for 35 s gave oriented, striated patterns on theperforated PEEK (Figure 2). The ensembles were then exposedto vapor of zirconium tetra(tert-butoxide) (1) for 5 min at 10−3

Torr; oxygen-containing functionalities of the PEEK enable itscoordinative bonding to 1.39 Heating to 65 °C gave a surface-bound, cross-linked, mixed zirconium oxide/tert-butoxidepattern, which was converted to patterned ZrO2−phosphonateby immersion in a solution of 1,4-butanediphosphonic acid inethanol (0.25 mg/mL);22 this ethanol treatment also removedresidual photoresist. Optical microscopy showed the patternedregions to be parallel with the ablated holes; both holes andpatterns were uniform over the entire PEEK surface, with holecoverage of 5, 10, and 20% of the substrates nominal areas(Figures 2A−E). Apparently, even when 20% of the surface hasbeen ablated away, the substrate could still be spin-coated withan adequate layer of photoresist in both uniformity andthickness for photolithographic patterning. EDX analysis(Figure 2F−G) confirmed the composition of these patternsto be the desired ZrO2−phosphonate. Because the ZrO2interface is only about 1 nm-thick and its phosphonatetermination is a monolayer,39 EDX signals for Zr and P areweak and difficult to distinguish from background; line scans of

EDX data are supportive of the assignments made here(Supporting Information, Figure 1).NIH 3T3 fibroblasts were plated on ZrO2−phosphonate-

patterned PEEK substrates with 5, 10, and 20% nominal surfacehole coverages to determine if perforation adversely affectedcell attachment and spreading in alignment with the pattern.Cells were plated at 30 000 cells/1 cm2 of nominal substratesurface in DMEM with 10% bovine calf serum19 and weregrown for 3 days before being fixed with 3.7% formaldehyde inphosphate-buffered saline, permeabilized with NP-40, andstained with rhodamine−phalloidin to visualize actin filaments.Analysis showed that the cells had spread around and between,but not over, the holes and were in alignment with the ZrO2−phosphonate pattern (Figure 3). Actin was found to be alignedover the surfaces in register with the patterns after cells hadreached confluence (Supporting Figure 1).Quantitatively comparing cell alignment on patterned,

nonperforated versus patterned, perforated surfaces was doneby measuring aspect ratios of Fast Fourier Transform (FFT)outputs from stained actin images of adhered cells (Figure 3).22

FFT analysis was done on four 10× images each a perfectsquare of 1024 × 1024 pixels. Image contrast was normalizedwith pixel saturation set at 0.4%; FFT was determined usingImageJ software in which grayscale output was colorized usingthe “spectrum” table. We define the aspect ratio by dividing thewidth of the FFT output oval by its length; thus, the smaller theaspect ratio, the better the alignment. This ratio was 0.69 ±0.09 for cells on patterned, nonperforated PEEK (Figure 3A);patterned, perforated substrates had ratios of 0.53 ± 0.05, 0.59± 0.09, 0.62 ± 0.09, and 0.59 ± 0.03 for cells on surfaces with5% hole coverage (Figure 3B), 10% hole coverage via head-to-tail ablation (Figure 3C), 10% hole coverage via side-to-sideablation (Figure 3D), and 20% hole coverage (Figure 3E),respectively. These data show that the cells are at least as well-aligned on patterned, perforated PEEK as they are onpatterned, nonperforated PEEK; this conclusion is further

Figure 3. Top row: Representative images of actin stained NIH 3T3 fibroblasts grown on patterns of the ZrO2−phosphonate on perforated PEEKsubstrates after 3 days: (A) nonperforated PEEK, reproduced with permission from ref 22, copyright 2013, the Royal Society of Chemistry; (B) 5%nominal surface coverage by holes; (C) 10% coverage via the head-to-tail ablation scheme or (D) by the side-to-side ablation scheme; (E) 20%coverage. Pattern direction is in the direction of the red arrow on each substrate; magnification 10×, scale bar = 100 μm. Bottom row: RepresentativeFFT outputs of actin images for each of the above surfaces. FFT outputs are shown with red lines though each oval to measure the aspect ratio,which is indicated at the bottom as average ±1 standard deviation; the lower the number, the better the alignment. See also Supporting Information,Figure 2 for an image taken at 7 days on a substrate with 5% nominal surface coverage by holes.

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supported by ANOVA (α = 0.05), where no statisticaldifference was found for the aspect ratio among all of thePEEK surfaces (p = 0.114). It is not known to what extent, ifany, the presence of holes affects the FFT output, althoughthere is no apparent correlation between hole number oralignment with measured aspect ratios.We showed that clean perforation of a surface-protected

polymer film can be accomplished by laser ablation and thatthis material can be patterned to template spatially controlledcell spreading in a way that is as effective as on unperforatedpolymer substrates. Protecting PEEK with a thin layer ofphotoresist prior to laser treatment causes all debris fromablation to be deposited on top of this layer, which is thenremoved by sonication in ethanol. Ablation-perforated PEEK iseasily patterned by photolithography to prepare cell-adhesiveZrO2−phosphonate striations; patterned PEEK films templatecell spreading in alignment with these striations, even whenperforations account for 20% of the nominal surface area. Giventhe ease of implementing our methods, our procedures formaterial transformation might provide an effective approach toenvisaged 3D constructs based on stacked 2D-patterned,perforated polymer sheets that would be designed also toobviate layer-to-layer compression. Coupled with demonstratedexcellence in spatially determined surface chemical modificationof polymer films and of ECM assembly on them,23 thesemethods may help inform new routes for fabricating tissuescaffolds comprising cell-assembled matrix44 or organ-on-a-chiptechnologies45 that effectively recapitulate native tissuearchitectures in which biodegradable polymeric substrates canbe accommodated.

■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsbiomater-ials.7b00339.

Image of confluent layer of cells on a 5% perforatedsubstrate at 7 days (PDF)

■ AUTHOR INFORMATION

Corresponding Author*E-mail: jschwart@princeton.edu.

ORCID

Jeffrey Schwartz: 0000-0001-9873-4499Author Contributions⊥The manuscript was written through contributions of allauthors. All authors have given approval to the final version ofthe manuscript. S.B.B., J.A.S., P.E.D., and K.L. contributedequally.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The authors thank the National Science Foundation PrincetonMRSEC (Grant DMR-1420541), the Princeton Institute forthe Science and Technology of Materials, and the NationalInstitutes of Health (Grant R01 CA160611 to J.E.S.).

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