The polyimide (PI) foils (Pyralin PI 2611, HD Microsystems, Bad Homburg, Germany) were developed as described earlier [27]. In brief, a polyimide (PI) foil (1 cm × 1 cm, 10 μm thick) was incubated at ambient temperature in 1 mL ethyl acetate (Acros) for 4 hours in the presence of the succinimidyl ester of 4-azido-2,3,5,6-tetrafluorobenzoic acid (AFB) (5 mg/mL, Invitrogen) protected from light. Next, the PI foil was irradiated for 15 minutes 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).

Next, the modified foils were incubated in an aqueous gelatin (type B, MW 155 kDa, polydispersity 2.5) solution (5 w/v%, pH 8) at 40 °C for 3 hours. In a final step, the foils were incubated in double distilled water at 40 °C for three hours to remove the physically adsorbed (i.e., non covalently bound) gelatin.

Methacrylamide-modified gelatin (type B, MW 155 kDa, polydispersity 2.5) (Gel-mod) was prepared as described previously [34]. Briefly, the amine groups in gelatin were chemically modified with methacrylamide moieties, resulting in a degree of substitution (DS) of 78% relative to the original content of primary amines [34].

The pre-treatment and silanisation of the Ti samples was performed as described previously [28]. In brief, the Ti samples were degreased and the native oxide layer was etched before treatment with an oxidizing agent. Next, the oxidized Ti samples were silanized using a 3-(trimethoxysilyl)propyl methacrylate (TMSPMA) solution (10 v/v% in pentane). The samples were then dipcoated in a Gel-mod solution (10 w/v%, in milliQ-water), containing the photoinitiator Irgacure® 2959 (2 mol% relative to the amount of crosslinkable groups) at 40 °C. In a final step, the samples were irradiated with UV-C lamps (250 nm, TUV 15W/G15 T8, Philips) for 30 min. In a final step, the gelatin-coated Ti samples were incubated in double distilled water at 40 °C for three hours to remove the physically adsorbed (i.e., non covalently bound) gelatin.

In a first part of the present work, the development and characterization of gelatin-coated polyimide (PI) foils was studied. In order to surface-activate PI, reactive ester groups were introduced on the PI surface using the succinimidyl ester of 4-azido-2,3,5,6-tetrafluorobenzoic acid (AFB) as heterobifunctional reagent (see Figure 1). Via its azide group, AFB can be coupled to the PI surface under UV illumination. This approach enables the subsequent direct coupling of any amine-containing biomolecule such as gelatin using the reactive ester moieties introduced in the first step. The heterobifunctional derivative selected was already applied successfully to immobilize reactive ester groups on polymer surfaces [27,35,36]. In addition, azide-containing compounds were previously immobilized on Kapton films (i.e., poly-oxydiphenylene-pyromellitimide) by Harmer [37]. The UV light is required to enable conversion of the tetrafluorophenylazido group to phenylnitrene. This highly reactive compound forms covalent linkages with the PI as well as neighboring AFB molecules leading to the formation of a polymeric multilayer. In addition, the nitrene intermediates can also lead to the formation of amines, nitro or nitroso compounds and even ring expansion is possible [38]. However, these side products are removed during the washing step [38]. Therefore, the reaction between nitrene and alkyl C-H bonds from neighboring AFB molecules is anticipated to occur more frequently [39]. The mechanism was already elucidated before by our research group [27].

The modified foils were characterized using a variety of techniques. Static contact angle (SCA) measurements (Figure 2) indicate that the SCA of PI-AFB did not significantly differ from that of blank PI (P > 0.05). However, after applying a gelatin coating, the SCA decreased from 59° to 49° (P < 0.05), as anticipated based on the hydrophilicity increase of the surface after immobilizing gelatin. In addition to SCA measurements, atomic force microscopy (AFM) analysis was performed to study possible morphological alterations induced on the PI surface after reaction with the succinimidyl ester of AFB and/or the subsequent gelatin immobilization. As indicated in Figure 3, the chemical modification significantly alters the PI surface morphology. The non-modified PI foil possesses a smooth surface with no distinguishable features (see Figure 3(a)), while the surface of PI-AFB shows a large number of nano-scale depressions (see Figure 3(b)). After applying a gelatin coating, the size of the nano-scale depressions decreases to a certain extent (see Figure 3(c)), which is also illustrated by the root-mean-square roughness values (R_rms). Blank PI has an R_rms value of 0.5 nm, while PI-AFB and gelatin-coated PI possess an R_rms value of 3.3 nm and 1.9 nm respectively (see Table 1). We hypothesize that gelatin fills up the nano-scale depressions on the surface-activated PI to a certain extent. Therefore, the final surface roughness is determined by both the gelatin coating as well as by the surface-activation on PI using the succinimidyl ester of AFB. Furthermore, the surface plot of the gelatin-coated PI (see Figure 3(c)) clearly indicates the absence of globular domains, which is atypical for proteins deposited on a surface. We therefore now aim at elucidating the effect of this protein conformation on the subsequent cell adhesion, spreading and proliferation using in vitro biocompatibility assays. The results of this study will be the topic of a forthcoming paper. This is highly relevant since, as an example, Guerra et al. already reported on the influence of fibronectin conformations on the subsequent cell attachment [40]. We thus anticipate that the gelatin conformation will also significantly affect the subsequent cell adhesion.

In a subsequent part of the material surface analysis, X-ray photoelectron spectroscopy (XPS) was applied to analyze the obtained differences in chemical composition between non- and surface-modified PI. Table 2 indicates the presence of 1% F after reaction with AFB, due to the presence of fluorine in the grafted AFB moieties. In addition, a nitrogen increase from 6% to 11% was observed when comparing PI-AFB and gelatin-coated PI respectively, which proves the successful protein deposition on the PI surface.

The successful gelatin grafting was also further confirmed using ATR-IR mapping (see Figure 4). The ATR-IR spectrum of gelatin is mainly characterized by the presence of two important characteristic regions (i.e., the amide I and the amide II band) [41]. The amide I band, which is due primarily to C=O 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 [42]. The band at 1,664 cm⁻¹ is related to the presence of both triple helices and β-turns [42]. The amide II region consists of a band at 1,556 cm⁻¹ (i.e., NH bend coupled with CN stretch) [43]. The two characteristic protein peaks at 1,556 and 1,664 cm⁻¹ are also present in the IR-spectrum of gelatin-modified PI (data not shown). In order to study whether the PI surface was completely biofunctionalized with gelatin, ATR-IR maps (100 μm × 100 μm) were recorded at 1,556 cm⁻¹ (Figure 4(a)) and 1,664 cm⁻¹ (Figure 4(b)). The IR maps clearly indicate that gelatin is indeed present throughout the entire PI surface. As a final surface analysis technique, confocal fluorescence microscopy (CFM) measurements were performed to visualize surface immobilized texas red-labeled gelatin. The results (see Figure 5) indicate that without PI activation, texas red-labeled gelatin is not grafted on the PI surface (Figure 5(a)). When applying the labeled gelatin on PI-AFB, the CFM analysis clearly showed the complete coverage of the PI surface with gelatin (Figure 5(b)).

The different characterization techniques applied thus enabled us to safely conclude that the PI modification with succinimidyl ester functionalities and the subsequent gelatin immobilization was successful.

In a second part of the present work, we aimed at applying a gelatin coating on a Ti surface in order to improve the final in vitro and in vivo cell-interactive properties of Ti implants. The procedure comprised two steps, now also involving a polymer modification step. In a first step, gelatin was modified using methacrylic anhydride in order to introduce polymerizable methacrylamide moieties, as described earlier [34]. Next, the Ti samples were cleaned, oxidized and silanized using TMSPMA in order to surface-functionalize the samples with polymerizable methacrylate groups. The gelatin immobilization was performed by dipcoating the silanized Ti samples in a Gel-mod solution containing Irgacure 2959, under UV-irradiation. The grafting strategy elaborated in the present work is illustrated in Figure 6. As with the modified PI foils, the modified surfaces were characterized in depth using a variety of surface (imaging) techniques.

SCA measurements (see Figure 7) indicated a contact angle increase from 67° for a commercially pure (cp) Ti surface to 101° for a TMSPMA silanized Ti surface (Sil Ti) (P < 0.05). The latter was anticipated based on the hydrophobic nature of the propyl and the methacrylate functionality of TMSPMA. After gelatin immobilization, a contact angle of 54° was obtained confirming the presence of the more hydrophilic protein layer on the Ti surface.

In order to study possible morphological changes of the Ti surface upon surface modification, AFM measurements were performed (see Figure 8). The cleaning, the oxidation and the subsequent silanization procedure influenced the Ti surface morphology to a great extent. An overall increase in surface roughness was observed from 66 nm for cp Ti to 190 nm for silanized Ti (see Table 3). This is mainly due to the formation of TMSPMA aggregates during the silanization reaction (Figure 8(b)). After gelatin immobilization, the surface roughness decreased to 95 nm (Table 3, Figure 8(c)). We anticipate that the final surface roughness is determined by the gelatin layer. As a result, cell attachment will mainly depend on both the surface roughness as well as on the gelatin conformation. Similar to the PI samples, in vitro biocompatibility studies will be performed to further study the effect of these phenomena. We thus anticipate that the tissue engineering field would benefit significantly from fine-tuning the coating thickness in such a way that, in this case, the gelatin degradation profile matches the formation of new bone within the porous Ti scaffold.

Next, the influence of the modification on the chemical composition of the Ti surface was determined using XPS measurements. Table 4 summarizes the atomic surface compositions of a cp Ti surface, a silanized Ti surface and a gelatin-coated Ti surface. After silanization, 9% Si was detected on the surface while the Ti signal disappeared. The latter indicated a complete coverage of the Ti surface by TMSPMA. The subsequent gelatin immobilization was confirmed by the presence of a N signal. The atomic composition of the modified Ti surface resembled the composition of a pure Gel-mod hydrogel.

Very interestingly, and in contrast to the PI foils included in this work (§ 3.1), ATR-IR could not be applied on the silanized Ti samples. Our current working hypothesis is that the siloxane layer is thinner than the penetration depth of the laser (see Figure 9). After applying the gelatin coating, the spectrum obtained resembles the one of Gel-mod (Figure 9). In this case, the thickness of the gelatin coating exceeds the penetration depth of the IR laser and reflection is avoided.

In addition to AFM, the gelatin coating was also visualized by means of CFM after staining the gelatin coating using Oregon Green (see Figure 10). Figure 10(b) illustrates the presence of a homogeneous gelatin coating on the Ti surface, in contrast to the control sample (i.e., an Oregon Green-stained cp Ti sample (Figure 10(a)).

As with the modified PI samples (§ 3.1), it can again be concluded that the different characterization techniques clearly indicated a successful modification of Ti with methacrylate moieties and its subsequent functionalization using gelatin with polymerizable side groups.