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of Plasma Enhanced Chemical Vapor Deposited Poly(2-hydroxyethyl methacrylate) Films for Biomimetic Replication of the Intestinal Basement Membrane...
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Biomacromolecules 2010, 11, 1579–1584

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Biocompatibility of Plasma Enhanced Chemical Vapor Deposited Poly(2-hydroxyethyl methacrylate) Films for Biomimetic Replication of the Intestinal Basement Membrane Courtney A. Pfluger, Daniel D. Burkey, Lin Wang, Bing Sun, Katherine S. Ziemer, and Rebecca L. Carrier* Department of Chemical Engineering, Northeastern University, Boston, Massachusetts 02115 Received February 24, 2010; Revised Manuscript Received April 13, 2010

It is recognized that topographical features such as ridges and grooves can dramatically influence cell phenotype, motivating the development of substrates with precisely biomimetic topography for study of the influence on cultured cells. Intestinal basement membrane topography has been precisely replicated using plasma enhanced chemical vapor deposition (CVD) of poly(2-hydroxyethyl methacrylate) (pHEMA) on native tissue. The ability for CVD pHEMA to coat and retain the complex architecture of the intestinal basement membrane at the micrometer scale was demonstrated using electron microscopy and surface chemical analysis (XPS). The suitability of CVD pHEMA as a cell culture substrate was assessed. Caco-2 cells maintained a high (>85%) viability on CVD pHEMA. Cell attachment and proliferation on CVD pHEMA were similar to those observed on materials traditionally used for cell culture and microfabrication purposes. Results indicate that CVD pHEMA is useful for development of precise (micrometer-scale) topographically biomimetic substrates for cell culture.

1. Introduction Topographical cues have been shown to be important to cell function, affecting cell phenotype and protein expression.1-5 Pins et al. developed microfabricated analogs of the basal lamina with polydimethylsiloxane (PDMS) and collagen and used these analogs to elucidate the influence of topography on epithelial cell proliferation and differentiation.6 Most studies of substrate topography on cell phenotype have involved simple topographical features (e.g., grooves) of a single scale (e.g., nanoscale features), raising the question of how more irregular, multiscale complex topographical features of native tissue influence cell function. While traditional lithographic techniques are suitable for fabrication of regular topographical features, a different methodology is required to replicate irregular, complex topography. One approach is to use native tissue as a template. The ability to form thin layers (a micrometer or smaller) on the surface of native tissue structure could be very useful in mimicking these complex topographies. Plasma enhanced chemical vapor deposition (PECVD) can deposit conformal films as thin as 15 nm and ranging up to several micrometers in an enclosed, one-step process.7 Poly(2-hydroxyethyl methacrylate) (pHEMA) has been shown to be a suitable polymer for use in biomedical applications because of its biocompatibility and Hydro-gel forming properties.8-11 PHEMA can be cross-linked to strengthen the polymer matrix and control its swelling and degradation properties for potential drug delivery and tissue scaffold applications.12,13 PHEMA films produced by solvent based polymerization techniques have been used as artificial skin,10 and articular cartilage replacements.11 While pHEMA has generally not been thought to be an optimal material for cell attachment due to its highly hydrophilic nature, it has been demonstrated to be a suitable culture substrate for some cell types.14 Horak et al. used pHEMA as a substrate to culture mouse embryonic stem cells; * To whom correspondence should be addressed. E-mail: r.carrier@ neu.edu.

they cross-linked pHEMA with N,O-dimethacryloylhydroxyamine (DMHA) to control degradation and utilize phase separation and salt-leaching to impart porosity.15 Stem cells were only able to grow on the cross-linked (but not noncross-linked) pHEMA. Mabilleau et al. demonstrated that J774.2 macrophage cells cultured on pHEMA microbeads proliferated and maintained high viability.16 Also, Merrett and co-workers cultured corneal epithelial cells on peptide modified pHEMA.17 The pHEMA films utilized for cell culture applications have been produced by traditional solution based chemistry; these wet polymerization methods are time intensive procedures, involve handling of harmful reagents and solvents, and are bulk deposition methods. CVD pHEMA films have been characterized and shown to have similar properties as solution phase pHEMA films. Tarducci et al. studied pulsed and continuous plasma polymerization of HEMA via X-ray photoelectron, infrared, and nuclear magnetic resonance spectroscopies and confirmed deposition of structurally well-defined pHEMA films.18 Lopez et al. demonstrated that these plasma-deposited thin films were biocompatible through a series of protein adsorption studies and demonstrated that these films are favorably similar to conventional pHEMA surfaces (spin-cast pHEMA, radiation grafted pHEMA, and bulk pHEMA gels).19 Chan and Gleason conducted extensive characterization of initiated chemical vapor deposition (iCVD) pHEMA and studied how hydrogel formation can be controlled via cross-linking.20 In this work, we explore the application of plasma enhanced CVD pHEMA for development of topographically biomimetic cell culture substrates. The fabrication and cross-linking of thin pHEMA films using a plasma enhanced CVD technique is described. As pHEMA is generally regarded as being a poor substrate for cell attachment, utility of CVD pHEMA for intestinal cell culture is tested. Low and high cross-linked pHEMA thin films were evaluated for their biocompatibility and suitability for cell culture by seeding Caco-2 intestinal cells on the surface and comparing attachment, cell growth, and

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viability with those observed on substrates commonly used for cell culture and microfabrication. A thin coating of cross-linked pHEMA was then deposited on the surface of a porcine intestinal basement membrane and was analyzed by SEM and XPS to confirm a uniform coating with comparable chemical makeup to traditional solution cast pHEMA. The ultimate goal will be to produce a thin, biocompatible pHEMA film with appropriate biomimetic topography that accurately represents the in vivo environment, improving the relevance of in vitro cell culture scaffolds.

2. Materials and Methods 2.1. Materials. 2-Hydroxyethyl methacrylate (HEMA; Aldrich, 99%), tert-butyl peroxide (TBPO; Aldrich, 98%), and ethylene glycol diacrylate (EGDA; Aldrich, 90%) were used with no further purification or modifications. Copper(II) chloride crystals (Aldrich, 97%) were added to both liquid monomer HEMA and EGDA to discourage autopolymerization when heating. 2.2. CVD Reactor. Thin film depositions were performed in a custom-built stainless steel vacuum chamber with a heated precursor delivery manifold. Precursor molecules were contained in Pyrex jars wrapped in heating tape and volatilized for flow into the reactor. HEMA was heated to 68 °C, while TBPO had sufficient vapor pressure at ambient temperature to not require heating for volatilization. EGDA was heated to between 40 and 60 °C, depending upon the desired EGDA flow rate. HEMA and TBPO vapors were admitted to the reactor through a shower head assembly above the deposition stage. EGDA vapors were admitted through a side port. Vacuum was achieved using an Edwards E2M40 rotary vane pump, and chamber pressure was controlled using a butterfly valve connected to an MKS model 252-A exhaust valve controller and an MKS Baratron capacitance manometer. Deposition pressure was constant at 350 mTorr. Plasma activation was achieved using a Comdel CPS-500 radio frequency power source operating at 13.56 MHz attached to a HeathKit SA-2060 matching network. Deposition power was held constant at 20 W for all films. Films were deposited onto 550 µm thick silicon wafers from Montco Silicon (Lot# S4988) or intestinal tissue prepared as described below. 2.3. Deposition Conditions. PHEMA films were produced using 1 sccm of TBPO as an initiator, 4 sccm of HEMA, and 5 sccm of Argon, used to help strike the plasma. Cross-linked pHEMA films were deposited by keeping the HEMA flow rate into the reactor constant at 6 sccm and varying the EGDA container temperature to change the EGDA flow rate into the reactor and, therefore, the amount of crosslinking of the pHEMA matrix. Films with low and high cross-link densities were deposited to enable assessment of the influence of crosslinking density on the degradation characteristics of the pHEMA films. Low cross-linked pHEMA films were deposited using EGDA heated to 42 °C and high cross-linked films used EGDA heated to 48 °C. While depositing on porcine intestine, a silicon wafer was also placed in the chamber to shadow the deposition for determining film composition with FT-IR and deposition thickness with ellipsometry. 2.4. Tissue Preparation. A porcine intestine stored on ice and received shortly after slaughter from a local abbatoir was used for preparation of an intestinal basement substrate for CVD deposition. A segment of jejunal tissue 63 in. from the pyloric stomach-duodenum junction was removed from the intestine and cut open in the direction of the flow axis. Any bulk material was removed; the tissue was washed with Hanks balanced salt solution (HBSS) and halved along the flow axis. The samples were then washed again with fresh HBSS, blotted on filter paper to remove bulk mucus, and immersed in a mixture of 1% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer solution (PBS, pH 7.3) at 4 °C overnight. The next morning, the samples were cut into smaller pieces (∼1 cm2) in preparation for critical point drying and rinsed one final time in PBS for 1 h. To remove cellular material, samples underwent secondary fixation and maceration/

Pfluger et al. osmication; these samples were further rinsed twice in PBS and then immersed in 0.1% osmium tetroxide (OsO4) buffer in 0.1 M PBS in six-well plates at 20 °C for 48 h. Note that solid osmium tetroxide is highly volatile and toxic and should be handled very carefully in a chemical fume hood at all times. Following this, the six-well plates were agitated vigorously to remove all cellular material, and samples were rinsed three times in distilled water.21 Samples were then dehydrated in a graded ethanol series (30, 50, and 70% ethanol, 10 min each). Samples were stored in 70% ethanol at 4 °C until critical point drying. For final preparation, samples were washed (85 and 95% ethanol for 15 min each, 100% ethanol three times for 15 min each) and critical point dried using supercritical CO2. Following critical point drying, the intestinal samples were ready either for SEM analysis or pHEMA coating. 2.5. Characterization. 2.5.1. FTIR. Structural analysis of the thin films deposited on silicon wafers was done using a Perkin-Elmer Spectrum GX-2000 Fourier Transform infrared spectrometer (FTIR) running the Spectrum software suite (version 5.3.1). Spectra represent the average of 32 scans over the range between 4000 to 400 cm-1 at a resolution of 4 cm-1. Measurements were done in absorbance mode, and spectra were thickness normalized for quantitative comparison and baseline corrected using the Spectrum software. 2.5.2. Ellipsometry. Thicknesses of thin films deposited on silicon wafers were measured using a J.A. Woollam Co., Inc. M-2000 spectroscopic ellipsometer at angles of 65, 70, and 75°. The Cauchy equation was used to model the system and determine thicknesses.22 2.5.3. X-ray Photoelectron Spectroscopy (XPS). XPS analysis was performed using a PHI 10-360-4-015 hemispherical analyzer with a PHI 04-173-0-077 Mg/Al dual anode nonmonochromatic X-ray source in an ultrahigh vacuum chamber with a base pressure of ∼2 × 10-9 Torr. All XPS data reported was collected with the Al anode operating at 15 kV and 300 W with a spot size of 1.1 mm2. It is noted that the chamber pressure could not be pumped below 2 × 10-8 Torr for the intestinal substrate samples, even after more than 4 h of pumping time. The minimum full-width at half-maximum (fwhm) value of 1.4 eV was determined by a gold standard, and gold and copper standards were used for energy and scale calibration. A pass energy of 89.45 and 35.75 eV was used for survey spectra and high resolution spectra, respectively. An energy step of 1.0 eV was used for survey spectra, and 0.05 eV was used for the high resolution spectra. Peak deconvolution and quantification of the elements was accomplished using AugerScan software and Phi sensitivity factors. XPS peak curve fitting was performed with an 80% Gaussian and 20% Lorenzian peak shape with Shirley background subtraction. 2.5.4. SEM. Following critical point drying, either before or after pHEMA deposition, samples to be imaged via SEM were sputter coated with palladium. They were then imaged using a Hitachi S-4800 UHR field emission scanning electron microscope. 2.6. Biocompatibility Study. 2.6.1. Cell Culture. Caco-2 cells were seeded at 7.6 × 104 cells/mL using Eagle’s minimum essential medium (Invitrogen) with 20% fetal bovine serum (American Type Culture Collection, Manassas, VA) and 1% antibiotic-antimycotic solution (10000 units penicillin, 10 mg streptomycin, and 25 µg amphotericin B per mL, Sigma-Aldrich). Caco-2 cells were maintained at 37 °C in a humidified 10% CO2 incubator in T-75 flasks and were split 1:2 after 5 to 7 days when the cells reached confluency. Confluent cell layers were treated with 0.25% (w/v) Trypsin in 0.53 mM EDTA solution and incubated at 37 °C until cells fully detached. Cells were then resuspended in cell culture medium at a density of 6.3 × 104 cells/mL. A 3 mL suspension was added to each well of a six-well plate containing the substrates. 2.6.2. Test Material Preparation. Cell culture wells in 12-well culture plates were coated with PECVD deposited pHEMA films at 1 µm thickness. These films were cross-linked with EGDA at a low or a high cross-linking density, as described above. PHEMA surfaces were sterilized by treatment with 70% w/v ethanol solution and washing with sterile phosphate buffered saline (PBS). Uncoated wells of

Biomimetic Replication of the Intestinal Membrane polystyrene were used as controls. The cell culture medium was exchanged every 3 days. For comparison to pHEMA, cells were grown on tissue culture plastic and PDMS. For preparation of PDMS surfaces, Sylgard 184 (Dow Corning, Midland, MI) was used. A 10:1 w/w ratio of base to curing agent was utilized; cross-linking occurred via a hydrosilylation reaction. A 1.5 mL aliquot of PDMS mixture was added into each well of a six-well plate. The mixture was degassed in a vacuum desiccator for 30 min, and then baked in an oven at 70 °C for 2 h. Prepared PDMS surfaces were sterilized by treatment with 70% w/v ethanol solution overnight and washing with sterile PBS. 2.6.3. Cell Counting and Viability Determination. Cell counts and viability determination were conducted on days 1, 2, 3, 6, 9, and 12 of culture for low and high cross-linked PHEMA as well as polystyrene. At the appropriate time, the supernatant was decanted and the cells that were attached to the surface were quickly rinsed with 1 mL of PBS and then incubated in 200 µL of Trypsin (Invitrogen) per well at 37 °C for 30 min to detach cells from the substrate surface. An aliquot of 800 µL of fresh medium was added to each well to stop the reaction. Cell counts were performed using a hemacytometer, where a 1:5 ratio of cells to Trypan Blue (Invitrogen) was used to determine cell viability.23 Trypan blue was used for viability assessment, as the autofluorescence of pHEMA precluded the use of some other viability assays. On days 3, 6, and 9, the supernatant was removed and fresh medium was added to the wells. Two cell counts were performed for each well, and two wells were analyzed for each group. The entire experiment was completed twice, giving eight data points for each surface on each sampling day. Percent cell attachments on polystyrene, PDMS, low and high cross-linked pHEMA were calculated at 1, 2, and 3 days after seeding by dividing the amount of cells attached to the surface by the total number of cells seeded. Relative cell attachment was calculated by dividing the number of attached cells on each surface by that on polystyrene on day 1. 2.7. Statistical Significance. Statistical significance was calculated by applying one way ANOVA analysis with Tukey’s post hoc comparison. A P value of less than 0.05 was used to indicate statistical significance.

3. Results 3.1. Fourier Transform Infrared Spectroscopy of pHEMA and Cross-Linking Properties. FTIR characterization of CVD deposited pHEMA films indicated that the main monomer functional groups remained intact after the deposition process. The O-H stretching vibrations between 3600 and 3300 cm-1, and the CdO band between 1750 and 1650 cm-1 strongly indicated that the side chain functionality of the monomer, containing the hydroxyl and carbonyl groups, had been retained in the polymer (data not shown). Increasing the flow rate of EGDA into the CVD reactor resulted in an increase in the amount of cross-linking in the pHEMA films. EGDA contains two CdO groups and therefore increases the overall number of CdO bonds in the film. Cross-linking was confirmed by FTIR analysis of the films, which indicated an increase in the carbonyl peak intensity between 1650 and 1750 cm-1 with an increase in flow rate of EGDA, as well as by degradation rate results reported as part of a separate study.24 Low and high cross-linked films were cross-linked at 10 and 37%, respectively, as determined by XPS as described in this same study. Ellipsometry indicated that deposited films were 1 µm thick. 3.2. Biocompatibility. Hydrogel formation and degradation properties of plasma deposited pHEMA films, together with how cross-linking affects these properties, have been previously reported.24 It was demonstrated that plasma deposited low crosslinked and high cross-linked pHEMA films form hydrogels

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Figure 1. Relative cell attachment on cross-linked pHEMA and PDMS compared to polystyrene on day 1 over the first three days of culture: (*) indicates statistical significance compared to polystyrene (p < 0.02) and (+) indicates statistical significance compared to low cross-linked pHEMA and PDMS (p < 0.05).

which undergo 27 and 12% degradation (as determined by a decrease in dry film thickness), respectively over 21 days in an aqueous environment. In the present work, a biocompatibility study was conducted to examine how cross-linked polymer degradation affects the attachment, proliferation, and viability of cells. Note that non-cross-linked pHEMA was not used in this study, as it was found to completely degrade after 7 days in a culture environment and would not be useful as a culture substrate. Caco-2 intestinal cells were cultured on low and high cross-linked pHEMA as well as polystyrene (positive control), and total attached cells as well as cell viability were examined over time in culture. Cell attachment on PDMS was also tested for comparison since it is a substance that has been utilized widely to create complex structures and topographies for microfabricated microfluidic and cell culture substrate surfaces. 3.2.1. RelatiVe Cell Attachment on Polystyrene Compared to Cross-Linked pHEMA and PDMS. Comparison of cell attachment over the first three days of culture on polystyrene relative to attachment on cross-linked pHEMA and PDMS generally indicated that PECVD pHEMA provides a suitable environment for cell attachment (Figure 1). One day after cell seeding, percent attachment ranged from approximately 43% on PDMS and just above 50% on low cross-linked pHEMA to greater than 90% on high cross-linked pHEMA and polystyrene. Over the three day period, attachment on high cross-linked pHEMA was generally similar to that on tissue culture plastic but significantly greater than that on low cross-linked pHEMA and PDMS (p < 0.02). The lowest cell attachment was consistently observed on PDMS and low cross-linked pHEMA. 3.2.2. Cell Growth and Viability on Cross-Linked pHEMA Coated Surfaces. Caco-2 cells grew continuously on the surfaces of low and high cross-linked pHEMA as well as polystyrene over 12 days in culture (Figure 2). After 3 days in culture, there

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Figure 3. SEM images of (a, b) bare porcine intestinal basement membrane and (c, d) high cross-linked pHEMA 1 µm thick coated pig intestinal membrane surface. The closed arrow heads point to crypt structures, open arrows indicate villi and black arrows indicate pores.

Figure 2. Total attached Caco-2 cells on high cross-linked and low cross-linked pHEMA compared to polystyrene.

was no significant difference in cell number on the three substrates. In general, cell growth results indicate that crosslinked PECVD deposited pHEMA can support cell attachment and proliferation to a similar extent as traditional cell culture plastic. Cell viability did not drop below 85% over 12 days of culture (data not shown). There was no significant difference between cell viability on high cross-linked pHEMA and on polystyrene. Thus, the high viability of cells cultured on pHEMA, and its similarity to that observed on widely used tissue culture substrates, reinforce the suitability of cross-linked pHEMA as a culture substrate for intestinal cells. 3.3. Deposition of High Cross-Linked pHEMA on Intestine. As our ultimate goal is to utilize pHEMA as a cell culture substrate with biomimetic topography created via CVD on actual basement membrane, the ability of pHEMA to conformally coat actual intestinal basement membrane was tested. Figure 3a,b shows a scanning electron micrograph (SEM) of a porcine intestinal basement membrane that has the epithelium removed at a low and high magnification. The villi and crypts are evident, and the porous, rough surface is shown. While the complete removal of intestinal epithelium, particularly in crypts, is difficult to assess via SEM, investigation of multiple SEM images of crypt-villus structures postremoval of epithelium has demonstrated a basement membrane surface which appears continuous from villi into crypts, supporting complete removal of epithelium (data not shown). Figure 3c,d depicts SEM images of the same porcine intestinal basement membrane with highly cross-linked pHEMA deposited on the surface using plasma enhanced CVD. The 1 µm thick films of both low and high cross-linked pHEMA conformally coated the entire surface of the intestine, preserving the structure of the column-like villi and deep invaginated crypts at the bases of the villi. Porcine villi are approximately 50-150 µm wide and 100-200 µm tall, and the crypts are approximately 20-50 µm in diameter. Also, the micrometer-scale porous surface structure of the intestinal basement membrane is still visible after polymer deposition.

Figure 4. XPS survey scans of bare intestine (top) and pHEMA on intestine (bottom), displaying sodium, nitrogen, and osmium peaks on the bare intestine and the disappearance of those peaks after coating with pHEMA.

3.3.1. XPS Analysis to Confirm Intestine Coating. XPS surface scan analysis of high cross-linked pHEMA on a silicon wafer and on fixed and critically point dried bare intestinal basement membrane was conducted to confirm pHEMA coating of the intestinal surface. Figure 4 shows the survey scan comparison of the fixed and dried porcine intestine and the highly cross-linked pHEMA coated intestine. Elemental sodium 1s (1072 eV), oxygen 1s (532 eV), nitrogen 1s (399 eV), carbon 1s (285 eV), and osmium 4f7 (52 eV) peaks were observed in the bare intestine. Oxygen satellite peaks in the 1000-970 eV range were also present. Carbon, oxygen, nitrogen, and sodium are expected elements of the intestine. Osmium and sodium are believed to be residue from the intestine preparation. In the survey scan of the pHEMA-coated intestine sample, osmium and sodium peaks disappeared, and the intensity of the nitrogen peak decreased by 73%. This indicates that the pHEMA, which

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pHEMA deposited on the silicon wafer than to the bare intestine. This further suggests that the pHEMA deposited by CVD is successfully coating the intestine.

4. Discussion

Figure 5. XPS atomic oxygen analysis on (a) bare intestine, (b) pHEMA on silicon wafer, and (c) pHEMA on intestine. The bare intestine and pHEMA coated on intestine were multiplied by factors of 3 and 4.5, respectively.

is not expected to contain these elements, has successfully coated the intestine to a thickness greater than the ∼19.6 nm sampling depth. The oxygen peak tight scans for bare intestine, high crosslinked pHEMA on a silicon wafer, and high cross-linked pHEMA on the intestine were also compared (Figure 5). Under the same instrument conditions and acquiring the scans for the same amount of time, the total counts from the O1s tight scan for bare and coated intestine samples were at least three times less than the O1s total counts for the pHEMA-coated silicon wafer, used as a control. This decrease in counts is believed to be due to the topography of the intestine samples.25 For illustrative purposes, the counts of the O1s peaks in Figure 5 for the bare intestine (Figure 5a) and the polymer-coated intestine (Figure 5c) were multiplied by factors of 3 and 4.5, respectively. The peak positions were corrected for differences in sample charging. The multiple spectra scanned from different spots on the coated intestine sample were consistent, suggesting a uniform coating on the intestine down to the 1 mm spatial resolution of the XPS analysis. The oxygen peak is composed of three oxygen bonding states corresponding to the carbonyl, hydroxyl, and ester functional groups. The groups are identified by their difference in peak energy location with a 0.80 eV shift between the oxygen bound in the carbonyl and hydroxyl groups and a 1.48 eV shift between the oxygen bound in the carbonyl and ester groups. This is consistent with other published XPS analysis of pHEMA films deposited on silicon by CVD.20 The high cross-linked pHEMA deposited on silicon has an approximate intensity ratio of 1:1.00: 1.08 for the carbonyl, hydroxyl, and ester groups, and this is also consistent with other published work.20 The bare intestine also contains carbonyl, hydroxyl, and ester functional groups, but the ratio between them is 1:2.32:2.61, which is significantly different from the ratio of oxygen bonding states in pHEMA on silicon. The pHEMA-coated intestine shows a 1:1.06:1.10 ratio, indicating a chemical makeup much more similar to the

4.1. Suitability of PECVD as a Cell Culture Scaffold Material. FTIR analysis of the deposited pHEMA films demonstrated that low and high levels of cross-linking were achieved, and the level of cross-linking was shown to correlate with the degradation rate;24 these results indicate that PECVD pHEMA will enable tuning of degradation properties for tissue scaffold applications. In addition, cell attachment on high crosslinked pHEMA was higher than on PDMS, which is a material commonly used for developing substrates with complex topographies (Figure 1). Also, there was no statistical difference between percent cell attachment on high cross-linked pHEMA and polystyrene over the first two days of culture. Over 12 days of culture, cells on low and high cross-linked pHEMA had the same growth trends as cells cultured on polystyrene but at slightly lower cell concentrations, possibly due to the initial difference in cell attachment (Figures 1 and 2). Greater cell attachment on high cross-linked pHEMA compared to low cross-linked pHEMA and PDMS (Figure 1) may be due to relative surface hydrophilicity. It has been shown that cells prefer attachment to surfaces with a certain range of hydrophilicity and do not attach well to highly hydrophobic or hydrophilic surfaces.26 By increasing the cross-linking of pHEMA with EGDA, the hydrophilicity of the film is decreased due to loss of hydroxyl groups.24 A related possible explanation for the differences in cell attachment is variable amounts of protein adsorption from the cell culture medium on the varying degrees of cross-linked pHEMA. Protein adsorption happens more readily on hydrophobic surfaces.27 The lower percent cell attachment on low cross-linked pHEMA compared to high cross-linked pHEMA could also be related to the degradation of polymer. Fast degradation of a polymer substrate has been shown to negatively affect cell attachment.28 It is noted that pHEMA does not contain labile bonds other than those created by cross-linking with EGDA, and it is thus believed that the degradation observed is a result of low molecular weight pHEMA diffusion from the polymer matrix. However, cell proliferation levels on the pHEMA materials were comparable to those on polystyrene, indicating that cell function was not significantly inhibited by the degradation of the cross-linked pHEMA over 12 days of culture. Taken together, the results support the suitability of CVD cross-linked pHEMA (in particular with high levels of cross-linking) as a cell culture substrate or scaffold offering a significant advantage for creating complex topographies, as PECVD of pHEMA is a dry, one-step process, in comparison to multistep solution phase polymer substrate fabrication. 4.2. Deposition of High Cross-Linked pHEMA on Intestine. XPS and microscopic analyses indicated that pHEMA indeed was coated onto the surface of the porcine intestinal basement membrane and that the structure of the intestine at the micrometer to submicrometer scale was kept intact. These results indicate that plasma CVD is a prime technique for replicating the structure of the intestine for use as a tissue engineering scaffold. While complex biological topography in deposited pHEMA films is promising, it will be extremely important in future studies to assess the preservation of this topography upon removal of the pHEMA from underlying substrate. A major challenge is the removal of the pHEMA substrate from the

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intestinal basement membrane. To overcome this barrier, we are investigating the precoating of intestinal basement membrane with silica, removal of tissue by degradation in bleach, and deposition of pHEMA membranes onto silica. It has been demonstrated that CVD of silica can precisely replicate biological structures. Cook et al. demonstrated the replication of the complex nano- and microscale structures of butterfly and housefly wings with depositions of 100-150 nm coating of silica.29 We intend to expand on this idea and apply it to the development of an inorganic, rigid mold of the intestinal basement membrane for pHEMA deposition. Once the method for separation of pHEMA from a rigid replica is achieved, the cellular response to precisely biomimetic topography and potentially the host response to implanted material can be assessed. It is not anticipated that culturing cells on the complex topographical features of natural intestinal basement membrane will present problems with respect to culturing a confluent cell monolayer, as observed in native epithelium. It was previously demonstrated that cells seeded on surfaces with approximately biomimetic cryptlike microwells initially settle mainly within the microwells, but eventually cover the entire surface, including vertical side-walls of microwells.30

5. Conclusion As the field of tissue engineering grows, scaffolds facilitating tissue growth and development using chemical and topographical cues will be increasingly in demand. Here, PECVD technique has been utilized to produce biocompatible and biodegradable polymer thin films conformally coating the complex biological geometries of the intestinal basement membrane. Topographical details at the micrometer to submicrometer scale were preserved, and the suitability of pHEMA with varying degrees of crosslinking as a cell culture substrate was demonstrated. These results support further investigation of PECVD of pHEMA for development of cell culture substrates and tissue engineering scaffolds with precise biomimetic topography. Acknowledgment. The authors would like to acknowledge NSF CMMI MDSE funding of this work under Award CMMI#0727984, NSF CBET BBBE funding for this work under Award CBET-#0700764, and the Chemical Engineering department at Northeastern University for additional funding.

References and Notes (1) Teixeira, A. I.; Abrams, G. A.; Bertics, P. J.; Murphy, C. J.; Nealey, P. F. J. Cell Sci. 2003, 116, 1881–1892.

Pfluger et al. (2) Clark, P.; Connolly, P.; Curtis, A. S.; Dow, J. A.; Wilkinson, C. D. DeVelopment 1987, 99, 439–448. (3) Clark, P.; Connolly, P.; Curtis, A. S.; Dow, J. A. DeVelopment 1990, 108, 635–644. (4) Meyle, J.; Gultig, K.; Nisch, W. J. Biomed. Mater. Res. 1995, 29, 81–88. (5) Kemkemer, R.; Jungbauer, S.; Kaufmann, D.; Gruler, H. Biophys. J. 2006, 90, 4701–4711. (6) Pins, G.; Toner, M.; Morgan, J. FASEB J. 2000, 14, 593–603. (7) Karches, M.; Morstein, M.; von Rohr, P. R.; Pozzo, R. L.; Giombi, J. L.; Baltanas, M. A. Catal. Today 2002, 72, 267–279. (8) Peppas, N. A.; Moynihan, H. J.; Lucht, L. M. J. Biomed. Mater. Res. 1985, 19, 397–411. (9) Hoffman, A. S. AdV. Drug DeliVery ReV. 2002, 43, 3–12. (10) Young, C.; Wu, J.; Tsou, T. Biomaterials 1998, 19, 1745–1752. (11) Kon, M.; de Visser, A. Plast. Reconstr. Surg. 1981, 67, 288–294. (12) Montheard, J. P.; Chatzopoulos, M.; Chappard, D. J. Macromol. Sci. ReV. Macromol. Chem. Phys. 1992, C32, 1–34. (13) Mabilleau, G.; Stancu, I.; Honore, T.; Legeay, G.; Cincu, C.; Basle, M.; Chappard, D. J. Biomed. Mater. Res., Part A 2005, 77A, 35–42. (14) Slaughter, B. V.; Khurshid, S. S.; Fisher, O. Z.; Khademhosseini, A.; Peppas, N. A. AdV. Mater. 2009, 21, 3307–3329. (15) Horak, D.; Kroupovia, J.; Slouf, M.; Dvorak, P. Biomaterials 2004, 25, 5249–5260. (16) Mabilleau, G.; Moreau, M. F.; Filmon, R.; Basle, M. F.; Chappard, D. Biomaterials 2004, 25, 5155–5162. (17) Merrett, K.; Griffith, C.; Deslandes, Y.; Pleizier, G.; Sheardown, H. J. Biomater. Sci., Polym. Ed. 2001, 12, 647–671. (18) Tarducci, C.; Schofield, W.; Badyal, J. Chem. Mater. 2002, 14, 2541– 2545. (19) Lopez, G.; Ratner, B.; Rapoza, R.; Horbett, T. Macromolecules 1993, 26, 3247–3253. (20) Chan, K.; Gleason, K. Langmuir 2005, 21, 8930–8939. (21) Takahashi-Iwanaga, H.; Iwanaga, T.; Isayama, H. Arch. Histol. Cytol. 1999, 62, 471–481. (22) Bosch, S.; Perez, J.; Canillas, A. Appl. Opt. 1998, 37, 1177–1179. (23) Shapiro, H. Practical Flow Cytometry, 3rd ed.; John Wiley & Sons: New York, NY, 1995; pp 111-113. (24) Pfluger, C.; Carrier, R.; Sun, B.; Ziemer, K.; Burkey, D. Macromol. Rapid Commun. 2009, 30, 126–132. (25) Chatelier, R. C.; St. John, H. A.; Gengenbach, T. R.; Kingshott, P.; Griesser, H. J. Surf. Interface Anal. 1997, 25, 741–746. (26) Fredriksson, C.; Khilman, S.; Kasemo, B.; Steel, D. M. J. Mater. Sci.: Mater. Med. 1998, 9, 785–788. (27) Grinnell, F.; Feld, M. J. Biol. Chem. 1982, 257, 4888–4893. (28) Zislis, T.; Mark, D. E.; Cerbas, E. L.; Hollinger, J. O. J. Oral Implantol. 1989, 15, 160–167. (29) Cook, G.; Timms, P. L.; Goltner-Spickermann, C. Angew. Chem., Int. Ed. 2003, 42, 557–559. (30) Wang, L.; Murthy, S. K.; Fowle, W. H.; Barabino, G. A.; Carrier, R. L. Biomaterials 2009, 30, 6825–6834.

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