Surface Modification of Polyimide Sheets for Regenerative Medicine

Biomacromolecules 2010, 11, 2731–2739

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Surface Modification of Polyimide Sheets for Regenerative Medicine Applications S. Van Vlierberghe,† M. Sirova,‡ P. Rossmann,‡ H. Thielecke,§ V. Boterberg,† B. Rihova,‡ E. Schacht,† and P. Dubruel*,† Polymer Chemistry and Biomaterials Research Group, Ghent University, Krijgslaan 281 S4-bis, 9000 Ghent, Belgium, Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic, and Biohybrid Systems, Fraunhofer Institute for Biomedical Engineering, Ensheimerstr. 48, 66386 St. Ingbert, Germany Received July 13, 2010; Revised Manuscript Received August 30, 2010

In the present work, two strategies were elaborated to surface-functionalize implantable polyimide sheets. In the first methodology, cross-linkable vinyl groups were introduced on the polyimide surface using aminopropylmethacrylamide. In the second approach, a reactive succinimidyl ester was introduced on the surface of PI. Using the former approach, the aim is to apply a vinyl functionalized biopolymer coating. In the latter approach, any amine containing biopolymer can be immobilized. The foils developed were characterized in depth using a variety of characterization techniques including atomic force microscopy, static contact angle measurements, and X-ray photoelectron spectroscopy. The results indicated that both modification strategies were successful. The subcutaneous implantation in mice indicated that both modification strategies resulted in biocompatible materials, inducing only limited cellular infiltration to the surrounding tissue. Table 1. Overview of the Properties of Nonmodified, Thermally Cured PI19

1. Introduction Surface modification of biomaterials to improve the final biocompatibility or the cell-interactive character of implants has gained increasing interest over the last decade.1-4 In the present work, the modification of polyimide (PI) membranes to be applied as implants for ocular diseases, including age-related macular degeneration, (AMD) is studied. PI has already found widespread application in industry for insulation of electrical machines,5,6 as optical materials,7 for flexible print circuits,8 and so on. In addition, PI-based materials have also been applied as electrodes in biomedical microdevices.9-11 However, for nonsensor-related applications, its use in the biomedical field is virtually unexplored. One rare example includes the application of PI to function in hollow fiber membrane oxygenerators for intrathoracic and intravenous devices to support failing lungs.12 Some PI are thus perfectly biocompatible but lack the cell interactive properties required for tissue repair applications. To improve this property, PI-based materials have to be surface modified. Several methods have already been described to enable PI surface modification. The most frequently applied technique is plasma treatment.13-16 In addition, several alternative procedures have also been explored including sulphuric acid and hydrogenperoxidetreatment,alkalinehydrolysis,andaminolysis.5,6 Massia et al. previously modified PI with bioactive peptides, including RGD and IKVAV, to improve the performance of neural prosthetic devices.17 In the present work, two strategies are elaborated to enable surface modification of PI membranes to function as cell carriers. In the first strategy, methacrylamide groups will be incorporated to enable subsequent covalent grafting of bioactive molecules possessing double bonds. In the second approach, reactive succinimidyl esters will be introduced * To whom correspondence should be addressed. Tel.: 003292644466. Fax: 003292644972. E-mail: [email protected]. † Ghent University. ‡ Academy of Sciences of the Czech Republic. § Fraunhofer Institute for Biomedical Engineering.

stress (10 µm film) tensile strength elongation density glass transition temperature (Tg) decomposition temperature moisture uptake weight loss (500 °C in air, 2 h)

2 MPa 35 kg/mm2 25% 1.4 g/cm >400 °C 620 °C 0.5% 1%

on the PI surface, enabling coupling of any amine containing bioactive molecules in a subsequent step. The present paper focuses on an in-depth comparison of both surface functionalization methods, including both a chemical and a biological analysis.

2. Materials and Methods 2.1. Development of Polyimide Foils. For the fabrication of the polyimide (PI) foils, semiconductor process technology was applied. First, a 5 µm thick layer of polyimide resin (Pyralin PI 2611, HD Microsystems, Bad Homburg, Germany) was spun onto a polished silicon wafer that served as a support structure during the whole process. Pyralin PI 2611 is based on biphenylanhydride and 1,4-phenylenediamine and is supplied as a polyamic acid precursor dissolved in N-methyl-2-pyrrolidone. PI 2611 is known to be highly anisotropic because of its rigid-rod nature.18 To obtain foils with a thickness of 10 µm, the spinning process was repeated twice. The polyimide was thermally cured in an oven (PB 6-2, YES) under a nitrogen atmosphere at 350 °C into a fully aromatic polyimide film. The wafer was cleaned with isopropanol and deionized water in an ultrasonic bath. Finally, the foil was mechanically stripped from the wafer using tweezers. Table 1 shows an overview of the properties of thermally cured, nonmodified PI.19 2.2. Development of Methacrylamide-Modified Polyimide Foils. The PI foil (1 × 1 cm, 10 µm thick; Pyralin PI2611, HD MicroSystems) was incubated overnight at ambient temperature in 2 mL of methanol

10.1021/bm100783h  2010 American Chemical Society Published on Web 09/10/2010

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Van Vlierberghe et al.

Figure 1. Reaction scheme for the introduction of cross-linkable groups on PI using aminopropylmethacrylamide.

Figure 2. Static contact angle measurements of blank and modified PI (PI-// and PI-AFB). The values obtained are an average of three separate measurements.

(Chromasolv, Sigma-Aldrich) in the presence of 20 mg (0.11 mmol) aminopropylmethacrylamide · HCl (Polysciences) and 0.5 mL (2 mmol) of distilled tributylamine (Acros) in the absence of light. Next, the methacrylamide-modified PI (PI-//) membrane was washed twice with MeOH and double distilled water, followed by an overnight drying step at ambient temperature. 2.3. Development of Succinimidyl Ester-Modified Polyimide Foils. A polyimide (PI) foil (1 × 1 cm, 10 µm thick) was incubated at ambient temperature in 1 mL of ethyl acetate (Acros) for 4 h in the presence of the succinimidyl ester of 4-azido-2,3,5,6-tetrafluorobenzoic acid (AFB; 5 mg/mL, Invitrogen) in the dark. Next, the PI foil was irradiated for 15 min with UV-C lamps (250 nm, TUV 15W/G15 T8, Philips), followed by two washing steps with ethyl acetate and phosphate buffer (pH 8), yielding succinimidyl ester-modified PI foils (PI-AFB). 2.4. Characterization of Surface-Modified Polyimide Foils. 2.4.1. Static Contact Angle Measurements. For each static contact angle measurement (SCA), 1 µL of double distilled water was placed on the polymer sheet. The spreading of the droplet was imaged using a video camera using 25 frames/s. The contact angle was determined on the

screen using the imaging software provided by the supplier (SCA 20, version 2.1.5 build 16). 2.4.2. Atomic Force Microscopy. Atomic force microscopy studies were performed with a Nanoscope IIIa Multimode equipped with 4.43r8 software (Digital Instruments, Santa Barbara, CA) applying “tapping mode” in air. Changes in surface morphology are quantified by rootmean-square (rms) roughness values (Rq). The rms roughness is defined as follows:

Rq )

∑ (Z - Z

2

avg)

i

N

(1)

where Zavg is the average Z height value within a given area, Zi is the current Z value, and N is the number of points within the given area. 2.4.3. X-ray Photoelectron Spectroscopy. The chemical composition of the polyimide sheets was determined using FISONS S-PROBE, a dedicated X-ray photoelectron microscopy (XPS) instrument designed to give the ultimate in performance while providing a high sample

Polyimide Sheets for Regenerative Medicine

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Figure 3. AFM images of (A) unmodified PI, (B) PI modified with aminopropylmethacrylamide (PI-//), and (C) PI modified with the succinimidyl ester of AFB (PI-AFB).

throughput. The fine focus Al-Ka source with a quartz monochromator, developed by Fisons Instruments Surface Science ensures lower background and higher sensitivity than conventional twin anode sources. All measurements were performed in a vacuum of at least 10-9 Pa wide and a narrow-scan spectra was acquired at a pass energy of 158 and 56 eV, respectively. The binding energy was calibrated by the C 1s peak at 284.6 eV. The spot size used was 250 µm on 1 mm. Data analysis was performed using S-PROBE software. The measured spectrum was displayed as a plot of the number of electrons (electron counts) versus electron binding energy in a fixed, small energy interval. Peak area and peak height sensitivity factors were used for the quantifications. All data are expressed as atomic %. 2.5. In Vivo Biological Evaluation. Male C57BL/6 and female BALB/c mice were obtained from the breeding colony of the Inst. of Physiology ASCR, v.v.i.. The mice were housed and treated in accordance with approved guidelines and provided with food and water ad libitum. Table 2. Surface Roughness Values (rms) of PI, PI-//, and PI-AFB Obtained Using AFM Analysis rms (Rq; nm) PI PI-// PI-AFB

0.54 3.52 3.26

Blank PI sheets, PI-//, and PI-AFB (3 mm Ø) were sterilized using a cold cycle ethylene oxide, and subcutaneously implanted in mice. The skin on the left flank was shaved one day before the implantation. For the operation, the mice were anaesthetized by intraperitoneal injection of 0.3 mL of 4% chloral hydrate, and the skin was treated with surface disinfectant. The implants were aseptically placed into pockets made in the left dorsal region cranially to the incision by blunt dissection. The incision was then sealed by a tissue glue (Histoacryl, B. Braun, Germany). The mice were sacrificed 6 days later, and samples of sera were collected and stored at -20 °C for cytokine analysis. The tissue adjacent to the implants was excised, fixed in 5% formalin, and embedded in paraffin, after which the 5 µm cut sections were stained with hematoxylin/eosin. Sham-operated mice were included to distinguish the histological findings resulting from the operation itself from those generated by the implants. Table 3. Atomic Composition of PI, PI-//, and PI-AFB Acquired by Means of XPS

C N O F

PI (%)

PI-// (%)

PI-AFB (%)

79 5 16

74 7 19

72 6 21 1

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Figure 4. Nitrogen peak of unmodified PI (part A) and PI-// (part B) obtained using XPS.

Figure 5. Reaction scheme for the introduction of amine reactive functional groups on PI using the succinimidyl ester of AFB.

Cytokine levels in sera of mice were determined using multiple analyte detection FlowCytomix (Bender MedSystems, Austria). The assays were performed according to the manufacturer’s protocol, and the measurements were done on LSR II flow cytometer (Becton Dickinson, U.S.A.). Bender MedSystems provided software for the data analysis. 2.6. Polymer Immobilization on Functionalized PI Foils: Gelatin As a Case Study. Methacrylamide-modified gelatin (MW 155 kDa, polydispersity 2.5) was prepared as described previously.20 Briefly, the amine side groups in gelatin were chemically modified with methacrylamide moieties, resulting in a degree of substitution (DS) of 60% relative to the original content of the primary amines.20 Next, PI-// was incubated in an aqueous solution of modified gelatin (5 w/v % gel-MOD), containing the photoinitiator Irgacure 2959 (2 mol % relative to the methacrylamide side chains) for 2 h. Subsequently, the PI foil was exposed to UV light (279 nm, 10 mW/cm2) on both sides for 30 min. In a final step, the foil was incubated in double-distilled water at 40 °C for 3 h to remove the physically adsorbed (i.e., non covalently bound) gelatin. ATR-IR spectroscopic analyses were performed to obtain qualitative proof of the immobilization of gelatin onto the PI foil. Transmittance spectra were acquired using a Biorad FT-IR spectrometer FTS 575C equipped with a “Golden Gate” ATR accessory. The latter was fitted with a diamond crystal. The spectra were recorded over the range 4000-600 cm-1 and averaged over 16 scans.

3. Results and Discussion In the present work, two strategies were elaborated and compared with the aim to functionalize polyimide (PI) membranes with moieties, enabling the subsequent introduction of bioactive molecules including proteins, glycosaminoglycans, peptides, and so on.

The PI foils applied were already evaluated before during 20 months in vitro.21 Rubehn et al. incubated the samples in PBS and performed gravimetric analysis, infrared spectroscopy, X-ray diffractometry (XRD), and X-ray photoelectron spectroscopy.21 No change in material behavior was observed after incubation at 37 and 60 °C. This was a good indication of the long-term stability of PI with respect to PBS.21 The XRD measurement indicated that the PI applied was semicrystalline, with a lattice spacing of 4.7 and 3.4 Å.21 3.1. Development and Characterization of Methacrylamide-Modified Polyimide Foils. In a first approach, aminopropylmethacrylamide was applied to introduce double bonds on PI surfaces (see Figure 1). A nucleophilic attack of the amines of aminopropylmethacrylamide on the imide groups of PI results in the formation of covalent amide linkages between PI and the methacrylamide moieties. The modified surfaces (PI-//) were first characterized using static contact angle (SCA) measurements. Figure 2 indicates that the SCA increased significantly (P < 0.05) from 59 to 70° upon grafting methacrylamide moieties. The latter was anticipated based on the hydrophobic character of the grafted functionality. In addition to SCA measurements, atomic force microscopy (AFM) analysis was also performed to evaluate the morphological alterations induced on the PI surface after reaction with aminopropylmethacrylamide. As indicated in Figure 3, the chemical modification significantly alters the PI surface morphology. The nonmodified PI membrane possesses a smooth surface with no distinguishable features (see figure 3A), while the surface of PI-// shows a large number of nanoscale depressions (see figure 3B). Differences in morphology between nontreated and surface-modified PI membranes can be quantified using root-mean-square roughness values (Rrms). Blank PI has



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Figure 6. (A) Generation of nitrene from azide and (B) reaction of nitrene with aromatic CdC and CsH bonds.

Figure 7. Histological analysis of tissues 6 days after subcutaneous implantation of PI-// and PI-AFB in C57BL/6 mice, H/E staining. (A) Introductory canal with solitary mononuclear cells (left). Inflammatory edema and scattered mononuclear infiltration in the adjacent tissue. The implanted foil (PI-//) was lost during the specimen preparation. (B) General picture representing the implantation canal for the PI-AFB foil. The canal with collateral edema and scarce, chiefly mononuclear infiltration. Solitary cells (mainly macrophages) adhere to the implant. No giant cells are present. Muscle plate (right) does not show any damage. At the top, a part of the insertion canal with denser inflammatory infiltrate chiefly consisting of macrophages is shown. (C) Detail of the transversely sectioned PI-AFB foil adhering to the wall of the insertion slit. Narrow layer of hemorrhage and edema with sporadic infiltration, chiefly of mononuclear (macrophage) cells in the adjacent connective tissue. Early stage of fibroplasia close to the implant is seen. Giant cells were not observed. The optically empty space (bottom right) originates from artificial detachment of the lower wall of the insertion slit from the implant.

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Table 4. Cytokine Levels (pg/mL) Detected in Sera of C57BL/6 Mice with Blank PI, PI-//, and PI-AFB Implantsa untreated IL-1R IL-6 TNFR GM-CSF IL-1R IL-6 TNFR GM-CSF

28; 17 0; 0 0; 0 13; 0

PI

PI-//

PI-AFB

22 0 0 12

6h 26 0 0 14

6h 24 0 0 13

27 0 0 15

6 days 29 0 0 21

6 days 31 0 0 18

LPS-injected 6h 39 11045 1009 49

a The implants were transplanted subcutaneously. Blood samples were collected after 6 h or 6 days and pooled from three individual mice. As controls, sera of normal mice (two separate pools, three mice each) and mice injected with lipopolysaccharide (LPS; from Salmonella typhimurium; 10 mg per dose, injected intraperitoneally in sterile PBS; sera pooled from three mice) were explored. Cytokine levels were detected using FlowCytomix assay.

an Rrms value of 0.54 nm, while PI-// possesses an Rrms value of 3.52 nm (see Table 2). The root-mean-square surface roughness value of blank PI is in the range of the value obtained by Xu et al. (i.e., 0.7 nm).22 Finally, X-ray photoelectron spectroscopy (XPS) was applied to analyze possible differences in the chemical composition between non- and surface-modified PI. Table 3 indicates an increase in nitrogen content (i.e., from 5 to 7%) after reaction with aminopropylmethacrylamide due to the presence of nitrogen in the grafted methacrylamide moieties. To further confirm the successful functionalization, peak deconvolution was performed on the nitrogen signal (see Figure 4). Blank PI resulted in one nitrogen peak (Figure 4A), while PI-// possessed two nitrogen signals (peak deconvolution in Figure 4B). The technique thus enables to distinguish between nitrogen atoms from different functional groups. Unmodified PI only contains imide nitrogens, while PI-// possesses both imide as well as amide nitrogens.

The different characterization techniques applied thus enables us to conclude that the modification of PI with methacrylamide functionalities was successful. 3.2. Development and Characterization of Succinimidyl Ester-Modified Polyimide Foils. As an alternative strategy, reactive ester groups were incorporated on the PI surface. This approach would enable the direct coupling of amine-containing biomolecules including peptides and proteins. Figure 5 illustrates the grafting of the succinimidyl ester of AFB via the azide moieties. The heterobifunctional derivative was already applied earlier as a successful method to immobilize reactive ester groups on polymer surfaces.23,24 In addition, azide-containing compounds were already immobilized before onto Kapton films (i.e., poly(oxidiphenylene-pyromellitimide)) by Harmer.25 He proposed the establishment of a covalent linkage between the azide compound and PI. UV light is applied to enable conversion of the tetrafluorophenylazido group to phenylnitrene (see Figure 6A). This highly reactive compound could form covalent linkages with both the PI as well as neighboring AFB molecules leading to the formation of a polymeric multilayer. The latter phenomenon was already observed before by Roger et al. when grafting azide-containing molecules onto poly(ethylene terephtalate) surfaces.26 In addition, the nitrene intermediates can also result in the formation of amines, nitro, or nitroso compounds and even ring expansion is possible.26 However, these side products are removed during the washing step.26 Low et al. proposed a reaction mechanism of nitrene and aromatic CdC and C-H bonds (see Figure 6B), which could be applicable for PI.27 However, due to the stability of the aromatic ring, the C-H bonds of the phenyl group are not very reactive toward the nitrene radicals. Therefore, the reaction between nitrene and alkyl C-H bonds from neighboring AFB molecules is anticipated to occur more frequently.27 This corresponds to the polymerization of the azide on the PI surface, as already indicated higher. The cycloaddition of nitrenes to the CdC bonds of the phenyl ring is not anticipated based on previous research from Low et al.27

Figure 8. (A) Blank PI, 2 days: Detail of early perifocal inflammatory cellularity consisting chiefly of lymphocytes and macrophages, with rare polymorphonuclear leukocytes. A thin layer of fibrin adheres to the surface of implantation canal. (B) PI-AFB, 2 days: The membrane shows some adherent mononuclear cells. Sporadic similar cells appear inside the cavity. Edema and scattered mononuclear nonsuppurative infiltrate are seen in the adjacent connective tissue. The epidermis, corium, and superficial muscular layer are intact. (C) PI-//, 2 days: Detail of early periphocal inflammatory cellularity consisting of lymphocytes and macrophages, with rare polymorphonuclear leukocytes. A thin layer of exudate covers the surface of implantation canal. (D) Blank PI, 6 days: Minimal response in the tissue on both sides of the implant and focal adhesion of cells to the surface of the PI foil were observed. Some scattered chiefly mononuclear infiltrate in the surrounding tissue. (E) PI-AFB 6 days: Six days after implantation, multiple mononuclear cells (chiefly macrophages) appear on the surface of membrane and on the wall of canal, with several giant elements. Similar infiltration and early perifocal fibroplasias are seen on the top. (F) PI-//, 6 days: Several mononuclear, partly multinuclear cells adhere to the membrane and wall of the canal. Early collateral scarring.


Figure 9. Copolymerization of double bonds of methacrylamidemodified gelatin and PI-// in the presence of a photoinitiator upon applying UV irradiation.

First, the modified membranes (PI-AFB) were characterized using SCA measurements. Figure 2 indicates that the SCA of PI-AFB did not significantly differ from that of blank PI. Next, AFM analysis was performed to evaluate possible morphological differences between non- and surface modified PI (see Figure 3). Similarly, as for the methacrylamide-modified PI, nanoscale depressions were observed on the surface of PI-AFB (Figure 3C). The differences in surface morphology before and after modification were confirmed by the increase in Rrms value from 0.54 to 3.26 nm. It can thus be concluded that the obtained surface roughness was similar for both modification approaches. Finally, XPS was also applied to analyze possible differences in the chemical composition between non- and AFB-modified PI. Table 3 indicates the presence of 1% F after reaction with the azide of AFB due to the presence of fluorine in the grafted AFB moieties. The different characterization techniques applied thus enables us to conclude that the modification of PI with succinimidyl ester functionalities was successful. 3.3. In Vivo Biological Evaluation. PI is often used for biosensor encapsulation and more recently as a substrate for neural or ocular implants. In vitro assays indicated that PI implants are biocompatible, eliciting only a moderate coagulation, negligible cytotoxicity, and hemolysis.28 In addition, PI supports fibroblast adhesion and growth.29 In vivo, PI implants exhibited good tissue integration and induced a very mild foreign body reaction, limited to a small area around the implants after a prolonged time period.30,31 In addition, Klinge et al and Rodriguez et al have demonstrated earlier the in vivo biocompatibility of the PI starting material we have selected in the present work (i.e., PI2611).32,33 We anticipated that the chemical modification of PI should not affect significantly its biocompatibility. To detect possible alterations in the biocompatibility of the modified PI implants (i.e., PI-// vs PI-AFB), we focused on the search for an acute response upon subcutaneous implantation.


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The test performed on the functionalized PI in C57BL/6 mice showed that both PI-// as well as PI-AFB are biocompatible materials. In the short interval after the implantation (6 h), the microscopic lesion was mainly due to the operation itself, as evidenced by the comparison with the sham-operated mice (data not shown). We have not observed any massive infiltration of polymorphonuclear cells that could be attributable to the inflammation-inducing or stimulatory capacity of the implanted foils. Six days after implantation it was evident that the foils induced only scattered cellular infiltration in the adjacent tissue. The infiltrating cells represented a mixed population, consisting of monocytes/macrophages and a small number of polymorphonuclear leukocytes. In addition, mild tissue response to the implant was observed, which was localized at the interface between the implant and the surrounding tissue in the tela subcutanea. The onset of fibroplasia indicated tissue recovery process (see Figure 7). Presence of giant cells characteristic of granuloma formation was not found. No significant differences between the two membrane types (i.e., PI-// vs PI-AFB) were observed. Summarizing, the tissue response to both implants can be evaluated as mild, permitting good integration into the tissue at later times post-implantation. In addition to the histological examination, we also measured pro-inflammatory cytokines in the sera of the implanted mice either immediately after the implantation (6 h) or upon collection of the histological samples (i.e., after 6 days; Table 4). No cytokine response related to the chemical modifications of the polyimide foils was observed. The cytokines that are typically produced in response to pro-inflammatory stimuli such as LPS (see last column of Table 4), that is, TNFR or IL-6, were not detected in the sera of the mice 6 days post PI-// and PI-AFB implantation. In addition, levels of IL-1R and GM-CSF were within the range found in normal untreated mice. Thus, the results were in excellent correlation with the histological analysis, showing overall mild and localized response to the implanted material. Cells of macrophage lineage play a central role in the orchestration and resolution of acute and chronic responses to an implanted artificial device. Individuals with a particular phenotype can mount different responses to such a material. The BALB/c inbred strain of mice was used in the next series of evaluations as it is known to develop responses skewed to an alternative (M-2) type of macrophage activation in contrast to C57BL/6 strain that mounts to a classic (M-1) functional program.34 M-1 macrophages tend to produce higher amounts

Figure 10. ATR-IR spectrum of unmodified PI, methacrylamide-modified gelatin, and gelatin-coated PI.

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of pro-inflammatory cytokines than M-2 ones that are able to tune inflammatory responses, effectively scavenge tissue debris, promote tissue remodeling, repair, and wound healing.35 Both types of the modified PI foils were compared with blank (unmodified) PI foils. The histological samples were collected 2 and 6 days following the subcutaneous implantation. After 2 days, mild edema and weak cellular infiltration in the tissue adjacent to the implanted foils were observed. The cellular infiltrates were mixed, consisting of mononuclear cells (macrophages and lymphocytes) and polymorphonuclear cells. The foils were not well integrated in the tissue, and some were lost during the sample preparation (see Figure 8A,C). Six days after the implantation, the first signs of fibroplasia were observed suggestive of tissue repair and resolution of the overall mild acute response. Few multinucleated giant cells were observed on the surface of PI-AFB (Figure 8E) and also PI-//. The unmodified PI induced similar response in the tissue as the two PI modifications. Again, no significant differences were found between the AFB- and PI-//-modified foils. The cytokine detection in sera collected 2 and 6 days after the implantation showed levels comparable with sera of normal BALB/c mice, and no increase of pro-inflammatory cytokines in either type of the PI foil tested. In conclusion, the results of the in vivo evaluation unequivocally showed very good bio- and immunocompatibility of the two PI modifications studied in the present work. 3.4. Polymer Immobilization on Functionalized PI Foils: Gelatin As a Case Study. Because both surface modification schemes did not lead to noticeable changes in immunocompatibility, we investigated as a proof of concept the immobilization of methacrylamide containing gelatin on the PI-// foils. The biopolymer immobilization is thus realized by copolymerization of the double bonds of gelatin with those on the PI surface (see Figure 9). The successful anchoring was confirmed using ATRIR spectroscopy (see Figure 10). The ATR-IR spectrum of gelatin shows three important characteristic regions (i.e., amide A, amide I, and amide II bands). The amide A mode consists of a band at 3274 cm-1 that corresponds to an NH stretch, coupled with hydrogen bonding.36,37 The amide I band, which is due primarily to CdO stretching of the peptide groups, is extremely sensitive to changes in the gelatin chain conformation. The wavenumber of the characteristic band of these carbonyl groups is determined by the local chemical environment and the degree of dipole-dipole interaction between neighboring carbonyl groups.38 The band at 1665 cm-1 is related to the presence of triple helices and β-turns.38 The amide II region consists of a band at 1556 cm-1 (i.e., NH bend coupled with CN stretch).39 The three characteristic protein peaks at 1556, 1665, and 3274 cm-1 are also present in the IR-spectrum of gelatin-modified PI. The imide stretches at 1353, 1703, and 1771 cm-1 (see IR spectrum of PI) are not present in the spectrum of gelatin-coated PI because uncoated PI was recorded as a blank (i.e., reference spectrum).

4. Conclusions In the present work, two strategies were elaborated and compared to functionalize implantable polyimide (PI) foils with reactive moieties. Various characterization techniques including atomic force microscopy, static contact angle measurements, and X-ray photoelectron spectroscopy indicated that the methodologies developed were successful. The in vivo biocompatibility of the modified PI implants was demonstrated using a subcutaneous murine model. As a proof of concept, we studied


the immobilization of methacrylamide modified gelatin on the PI-//. The present work is highly relevant for applications in which implantation of PI is required. Acknowledgment. The authors would like to acknowledge the PolExGene consortium. PolExGene is a STREP project (Contract No. 019114) funded under the EU sixth framework programme. Sandra Van Vlierberghe is postdoctoral fellow of the Research Foundation-Flanders (FWO, Belgium). V. Pakanova is greatly acknowledged for the in vivo biological evaluation. The authors would like to thank V. Vermeersch and E. Vanderleyden for the sample preparation and the XPS measurements, respectively.

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Surface Modification of Polyimide Sheets for Regenerative Medicine

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