Surface chemical immobilization of bioactive peptides on synthetic polymers for cardiac tissue engineering.

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Surface chemical immobilization of bioactive peptides on synthetic polymers for cardiac tissue engineering a

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Elisabetta Rosellini , Caterina Cristallini , Giulio D. Guerra & Niccoletta Barbani

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Department of Civil and Industrial Engineering, University of Pisa, Largo Lucio Lazzarino, 56126 Pisa, Italy b

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Institute for Composite and Biomedical Materials, C.N.R., o.u. Pisa, Pisa, Italy Accepted author version posted online: 19 Mar 2015.Published online: 21 Apr 2015.

To cite this article: Elisabetta Rosellini, Caterina Cristallini, Giulio D. Guerra & Niccoletta Barbani (2015): Surface chemical immobilization of bioactive peptides on synthetic polymers for cardiac tissue engineering, Journal of Biomaterials Science, Polymer Edition, DOI: 10.1080/09205063.2015.1030991 To link to this article: http://dx.doi.org/10.1080/09205063.2015.1030991

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Journal of Biomaterials Science, Polymer Edition, 2015 http://dx.doi.org/10.1080/09205063.2015.1030991

Surface chemical immobilization of bioactive peptides on synthetic polymers for cardiac tissue engineering


Elisabetta Rosellinia*, Caterina Cristallinib, Giulio D. Guerrab and Niccoletta Barbania a Department of Civil and Industrial Engineering, University of Pisa, Largo Lucio Lazzarino, 56126 Pisa, Italy; bInstitute for Composite and Biomedical Materials, C.N.R., o.u. Pisa, Pisa, Italy

(Received 24 November 2014; accepted 26 February 2015) The aim of this work was the development of new synthetic polymeric systems, functionalized by surface chemical modification with bioactive peptides, for myocardial tissue engineering. Polycaprolactone and a poly(ester-ether-ester) block copolymer synthesized in our lab, polycaprolactone–poly(ethylene oxide)–polycaprolactone (PCL–PEO–PCL), were used as the substrates to be modified. Two pentapeptides, H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS) from fibronectin and H-Tyr-Ile-Gly-Ser-ArgOH (YIGSR) from laminin, were used for the functionalization. Polymeric membranes were obtained by casting from solutions and then functionalized by means of alkaline hydrolysis and subsequent coupling of the bioactive molecules through 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride/N-hydroxysuccinimide chemistry. The hydrolysis conditions, in terms of hydrolysis time, temperature, and sodium hydroxide concentration, were optimized for the two materials. The occurrence of the coupling reaction was demonstrated by infrared spectroscopy, as the presence on the functionalized materials of the absorption peaks typical of the two peptides. The peptide surface density was determined by chromatographic analysis and the distribution was studied by infrared chemical imaging. The results showed a nearly homogeneous peptide distribution, with a density above the minimum value necessary to promote cell adhesion. Preliminary in vitro cell culture studies demonstrated that the introduction of the bioactive molecules had a positive effect on improving C2C12 myoblasts growth on the synthetic materials. Keywords: functionalization; fibronectin; laminin; bioactive scaffold

Introduction In recent years, a large number of synthetic polymers, having suitable mechanical stability and biodegradability, were suggested to make scaffolds for cardiac tissue regeneration.[1–3] One important remaining problem is inadequate interaction between polymer and cells. In vivo, tissue development and functionality are regulated by highly precise cell– cell and cell–extracellular matrix (ECM) interactions.[4] For this reason, over the past years, the attention was focused on the development of bioactive scaffolds, containing specific chemical and structural information that control tissue formation, similarly to cell–ECM communications during embryological development.[5] Approaches to improve biomaterials include material modification by immobilization of cell recognition *Corresponding author. Email: [email protected] © 2015 Taylor & Francis


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motifs to obtain controlled interaction between cells and synthetic substrates.[6] Initially, these materials were coated with cell adhesive proteins such as fibronectin, collagen, or laminin.[7,8] The use of entire proteins, however, bears some disadvantages in view of medical applications, such as undesirable immune responses, increased infection risks, proteolytic degradation, and unsuitable orientation on the surface. One strategy to solve most of these problems is to use, instead of entire proteins, cell recognition motifs as small immobilized peptides.[9,10] Peptides present several advantages, such as higher stability toward sterilization conditions, heat treatment and pH variation, storage and conformational shifting, as well as easier characterization and cost effectiveness. Furthermore, thanks to lower space requirement, they can be packed with higher density on surfaces. In multicellular organisms, contacts of cells with neighboring cells and the surrounding ECM are mediated by cell adhesion receptors. Integrin family represents the most numerous and versatile group.[11] Integrins play a major role as anchoring molecules, but they are also involved in important processes like embryogenesis, cell differentiation, immune response, wound healing, and hemostasis. It is well established that integrin-mediated cell spreading and focal adhesion formation trigger survival and proliferation of anchorage-dependent cells. In contrast, loss of attachment causes apoptosis in many cell types. Apoptosis can even be induced by the presence of immobilized ECM molecules when non-immobilized soluble ligands are added. In order to provide a stable linking, peptides should be covalently attached to the polymer, through functional groups like hydroxyl-, amino-, or carboxyl groups. Since many synthetic polymers do not have functional groups on their surface, these need to be introduced by blending, co-polymerization, chemical or physical treatment.[12–17] Different strategies were also examined to covalently bind peptides or proteins to polymer surfaces: the most common one involves the reaction between substrate carboxylic groups and amino groups of the bioactive molecule, to generate an amide bond. An additional point to take in consideration is the surface density of the bioactive molecules. It is well known that the number of attached cells is clearly related to peptide surface density and in particular cell attachment, as a function of peptide concentration, has a sigmoid increase. With reference to this, Massia and Hubbell demonstrated that a density of 1 fmol peptide/cm2 is sufficient for cell spreading, while 10 fmol/cm2 for focal contact formation, on a Arg-Gly-Asp (RGD) functionalized glycophase glass surface.[18] The aim of our work was the surface modification of synthetic polymers, via hydrolysis in sodium hydroxide solution and subsequent covalent attachment of biologically active peptide sequences. On the basis of the results obtained in a previous screening work, in which we identified polymeric materials with suitable properties for cardiac tissue engineering application,[19] the materials used as substrates to be modified were commercial polycaprolactone (PCL) and a synthetic polymer synthesized in our lab, a tri-block poly(ester-ether-ester) copolymer. The tri-block poly(ester-ether-ester) copolymer, polycaprolactone–poly(ethylene oxide)–polycaprolactone (PCL–PEO–PCL), was obtained by reaction of preformed poly(ethylene glycol) with ε-caprolactone.[20] The good biocompatibility and hemocompatibility of the copolymer were widely demonstrated.[21] In terms of degradability, bulk hydrolysis of the copolymer and the biocompatibility of degradation byproducts were reported.[22–24] In agreement with the definitions provided by Vert et al. [25], PCL–PEO–PCL can be considered a hydrolytically degradable polymer.


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The bioactive molecules chosen for the functionalization were the two pentapeptides: H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS), from fibronectin, and H-Tyr-Ile-Gly-SerArg-OH (YIGSR), from laminin. The oligopeptide RGD is the most commonly used cell adhesion peptide because it is found in several adhesion proteins, such as fibronectin, vitronectin, laminin, and collagen type I. Many cell types demonstrated good adhesion on synthetic scaffolds functionalized with RGD-containing peptides.[6] In particular, the GRGDS peptide was shown to promote the adhesion of several cell types, including myoblasts, and to stimulate integrins relevant in early cardiac development (α5β1, αvβ3).[26,27] YIGSR has also been studied extensively as an adhesion peptide.[28,29] In addition, laminin has been demonstrated to increase the ability of stem cells to differentiate into beating cardiomyocytes [30] and therefore, the YIGSR laminin sequence could be involved not only in adhesion, but also in differentiation processes. The goal of this work was the setup of the functionalization protocol, employing a four-step procedure: (i) alkaline hydrolysis for the formation of COO− groups on the polymeric surfaces; (ii) protonation with HCl; (iii) activation of carboxyl groups using the coupling reagents 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS); and (iv) coupling of the peptide sequences. In particular, the hydrolysis conditions were optimized for the two polymers. Then, the functionalized materials underwent a complete morphological and physicochemical characterization to assess the occurrence of the coupling reaction and to study the effect of the functionalization procedure on materials properties. A preliminary biological investigation, carried out by in vitro cell culture tests, finally verified the ability of the functionalized materials to improve cell–material interactions. Materials and methods Materials PCL–PEO–PCL [21] had an ester to ether units 66:34 M ratio, a central polyether chain with a mean Mn value of 35.0 KDa and a total mean Mn value of 203.7 KDa; PCL (Sigma Aldrich, St. Louis, MO) had a mean Mn value of 80.0 KDa. PCL–PEO–PCL and PCL were used as the substrates to be modified. The hydrolyzed protein (HP) used for the optimization of the coupling reaction (gelatin type B from bovine skin) was purchased from Sigma Aldrich. The cell-recognizing peptides having the sequence GRGDS (>97.4%) and YIGSR (>98%) were from Bachem (Bubendorf, Switzerland) and used as supplied. Hydrochloride solution was diluted from stock solution (Carlo Erba Reagenti, Milan, Italy). Sodium hydroxide pellets were from Carlo Erba Reagenti. EDC and NHS were from Fluka (St. Louis, MO). Phosphate buffer saline (PBS, Sigma Aldrich) was newly prepared prior to use. The water used was deionized and sterilized from a Millipore (Billerica, MA) system. Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, fetal bovine serum, glutamine, penicillin, and streptomycin were from Cambrex (East Rutherford, NJ). Formaldehyde and 4′,6-diamidino-2phenylindole (DAPI) were from Sigma Aldrich. Other reagents were all commercially available and used as received. Preparation of PCL and PCL–PEO–PCL membranes The first step of the work was the preparation of membranes based on the two synthetic polymers by casting technique.

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PCL and PCL–PEO–PCL 2% (w/v) chloroform solutions were prepared and they were cast on glass Petri dishes by solvent evaporation at room temperature, obtaining homogeneous membranes with a thickness of 156 ± 24 μm.


Alkaline hydrolysis For the alkaline hydrolysis treatment, the polymeric membranes, cut into squares of 1 cm × 1 cm, were immersed in sodium hydroxide solutions of appropriate concentrations and reacted for a predetermined period of time, at different temperatures. During the reaction, the samples were maintained suspended inside the solution, in order to treat both surfaces. The conditions of hydrolysis tested for the two materials are summarized in Table 1. Hydrolysis conditions were optimized for each material with regard to hydrolysis time, temperature, and concentration of the NaOH solution. After hydrolysis, the membranes were immersed in 0.01 mol/L HCl to yield polymer surfaces bearing carboxylic groups. Attachment of peptide Polymeric samples bearing carboxylic groups were immersed into a buffer solution (pH 5) containing an EDC/NHS mixture, with a 3:1 M ratio, at 4 °C for 3 h. After several washing in deionized water to remove unreacted compounds, the samples were reacted with a peptide solution of 0.5 mg/ml in PBS (pH 7.4) at 4 °C for 12 h. Finally, the samples were washed again for three times in deionized water to remove the non-immobilized biomolecules. Wash waters were collected for subsequent chromatographic analysis. Before performing peptide immobilization, the same procedure was applied to verify the efficacy of the coupling reaction, using as ligand the HP. After activation with EDC/NHS, samples were reacted with a 0.1% w/v solution of the HP, with pH 8, at 4 °C for 12 h. Fourier transform infrared (FTIR) spectroscopy analysis The efficacy of the hydrolysis treatment performed on sample surface, as well as the occurrence of the coupling reaction, was verified through infrared spectroscopy, using a Spectrum One Fourier transform infrared (FTIR) spectrometer (Perkin Elmer, Waltham,

Table 1. Hydrolysis conditions in terms of hydrolysis time, temperature, and alkaline concentration, tested for the two materials.

PCL PCL–PEO–PCL

NaOH (M)

Temperature (°C)

Time (h)

0.6 1 1 0.05 0.1 0.5 1 1

25 25 30 25 25 25 25 30

1, 2, 6, 16 1 1, 24, 48, 72 1 0.5, 1, 3 1 1 1, 2, 3, 6, 16, 24


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MA) equipped with ATR objective lens with a penetration depth of 2–3 μm. All spectra were obtained at 4 cm−1 and represented the average of 16 scans.


Chromatographic analysis The peptide surface density was determined measuring the corresponding concentration in the coupling solution (Cf ) after coupling reaction, as well as in the wash waters (Cw), by high-performance liquid chromatography (HPLC, 200 Series HPLC system, Perkin Elmer, with a UV/VIS detector). An HP Prosphere C4 300A 5u column (250 mm length × 4.5 mm internal diameter, Alltech Associates, Deerfield, IL) was used. The mobile phase was: A = 0.085% trifluoroacetic acid (w/v) in acetonitrile; B = 0.1% trifluoroacetic acid (w/v) in water. The elution condition was a linear binary gradient at a flow rate of 1 ml/min and the gradient was from 30% A and 70% B to 60% A and 40% B in 15 min. The injection volume was 50 μl. The detector wavelength was set at λ = 280 nm. Knowing the concentration of the coupling solution before the reaction (Ci), the volume of the coupling solution and of the wash waters (V) and the area (A) of the treated surface, the peptide surface density was calculated according to the following equation: Density ¼

V ðCi Cf Cw Þ A

FTIR chemical imaging The distribution of carboxyl groups on hydrolyzed materials, as well as of the biomolecules on the functionalized surfaces, was investigated through FTIR chemical imaging. Spectral images of functionalized polymeric membranes were acquired with an infrared imaging system (Spotlight 300, PerkinElmer). The spectral resolution was 4 cm−1. The spatial resolution was 100 × 100 μm. Background scans were obtained from a region of no sample. IR images were acquired with a liquid nitrogen-cooled mercury cadmium telluride line detector composed of 16 pixel elements. Each absorbance spectrum composing the IR images and resulting from 16 scans was recorded for each pixel in the μATR mode with the Spotlight software. We collected the spectra by touching the ATR objective on the sample and recording the spectrum generated from the surface layer of the sample. The Spotlight software used for the acquisition was also used to preprocess the spectra. IR spectral images were produced with the absorbance in a given frequency range, 4000–720 cm−1. Spectra contained in the spectral images were analyzed with a compare correlation image. The obtained correlation map indicated the areas of an image where the spectra were most similar to a reference spectrum. In these analyses, the reference spectrum used was the most frequent one of the chemical map (the medium spectrum). Morphological analysis The morphological analysis of the functionalized surfaces was performed through a scanning electron microscope JSM 5600 (Joel Ltd, Tokyo, Japan). Before analysis, the samples were mounted on metal stubs and coated with gold to a thickness of 200–500 Å with a gold splutter.

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Thermal analysis


Differential scanning calorimetry (DSC) was used to study the effect of the functionalization procedure on the crystallinity of the treated materials. The thermal behavior was studied in the range of 30–200 °C by a DSC7 Perkin Elmer, at a rate of 10 °C/min under a nitrogen flow, using aluminum opened pans. The endothermic peaks were measured with the DSC7 software. Cell culture protocol For a preliminary evaluation of the ability of the functionalized materials to promote tissue regeneration, in vitro cell adhesion and proliferation tests were performed with C2C12 myoblasts, as a model of a possible cell source for cardiac tissue engineering.[31] C2C12 myoblasts were obtained from European Collection of Cell Culture (London, UK). Polymeric membranes were prepared for cell culture according to the following procedure. Dry samples, cut into squares of 1 cm2, were sterilized by washing with 70% (v/v) ethanol solution in sterile water, followed by UV exposure for 15 min on each sample side. Two-dimensional scaffolds were placed in a 24-well plate and seeded with C2C12 (about 105 cells/ml). Cells were also cultured directly on the wells of the tissue culture plates (TCPs) and on untreated polymeric materials, as control. Growth medium contained DMEM with high glucose, 10% fetal bovine serum, 2 mM glutamine, penicillin 100 U/ml, and streptomycin 100 μg/ml. Culture was maintained in an incubator equilibrated with 5% CO2 at 37 °C. The duration of the whole experiment was seven days; the medium was completely removed every two days and replaced with fresh medium. The cell number on the functionalized materials was evaluated at different times after seeding (1, 3 and 7 days) labeling cells with DAPI, a fluorescent dye that binds to cell nuclei. At appointed times, the culture medium was removed and substrates with attached cells were rinsed two times with PBS for 10 min. Attached cells were fixed in 4% (v/v) paraformaldehyde (PFA) for 30 min at 4 °C and washed more times, to remove PFA. The fixed cells were subsequently incubated for 2 min in the dark with DAPI and rinsed three times with PBS. Images were collected using a fluorescent microscope TE2000U (Nikon Co., Tokyo, Japan). Results and discussion Surface modification Polymeric samples were hydrolyzed in sodium hydroxide solution in order to functionalize their surfaces. After hydrolysis, the samples were treated with 0.01 M HCl to obtain surfaces with carboxylic acid groups. GRGDS and YIGSR peptides reacted via the N-terminus with carboxyl groups, preactivated with a carbodiimide and NHS to generate an active ester. The peptides were used since they have been proved to be biologically important for enhancing cell interaction with biomaterial surfaces. The experimental conditions of alkaline hydrolysis tested for the two materials are reported in Table 1. The optimal conditions were identified through infrared analysis of the treated materials, as detailed in the next section. It is important to underline that all the characterization tests performed to evaluate the efficacy of the functionalization procedure were performed on both sample sides.


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Table 2. C=O/CH band ratio, before and after hydrolysis, for the selected hydrolysis treatments. The percentage variation of the band ratio was calculated according to the formula: (Rbh − Rah/ Rbh ) × 100; where Rbh is the band ratio before hydrolysis and Rah is the band ratio after hydrolysis.

C=O/CH band ratio


PCL PCL–PEO–PCL

Before hydrolysis

After hydrolysis

Percentage variation of the band ratio

2.80 1.20

1.74 1.09

38 9

Hydrolysis conditions NaOH (M)

T (°C)

Time (h)

1 1

30 30

24 2

Infrared spectroscopy studies Band ratio technique is commonly used for quantitation in infrared spectroscopy. This technique involves the measurement of the band intensity or area of an internal reference, with respect to that of a band of interest. This method requires to choose an appropriate band measure, an appropriate reference band and a good base line. In order to evaluate the base hydrolysis reaction, ATR spectra of hydrolyzed samples, prepared according to the conditions reported in Table 1, were acquired to calculate the ratio among the area of the band among 1800–1600 cm−1, due to C=O groups, and that among 3000–2500 cm−1, due to CH groups, since this last band does not undergo a significant modification after the hydrolysis treatment. The calculated ratios were compared with those of the untreated samples. A reduction in the ratio indicates a decrease in the C=O groups due to ester bonds, as a consequence of their hydrolysis. For values of NaOH concentration below 1 M and for temperatures below 30 °C, we did not observe significant variations of the ratio for both polymers. For both polymers, the optimal hydrolysis conditions were identified as a concentration of NaOH equal to 1 M and a temperature of 30 °C, for a reaction period of 24 h in the case of PCL and of 2 h for PCL–PEO–PCL: for times longer than these, no further reduction in the ratios was observed. In Table 2, the minimum ratios obtained for the two polymers and the conditions of hydrolysis used are reported. These conditions were chosen for performing the functionalization procedure. Spectra supporting the results collected in Table 2 are reported in Figure 1.

Figure 1. FTIR spectra of (a) untreated PCL and hydrolyzed PCL, treated according to the optimized hydrolysis conditions detailed in Table 2; (b) untreated PCL–PEO–PCL and hydrolyzed PCL–PEO–PCL, treated according to the optimized hydrolysis conditions detailed in Table 2.

Figure 2. FTIR chemical imaging analysis of hydrolyzed PCL (a–c) and PCL–PEO–PCL (d–f). (a and d): Chemical maps; (b and e) medium spectra; (c and f) correlation maps.


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As shown by the band ratio values, the degree of hydrolysis was higher for PCL than for PCL–PEO–PCL, as a consequence of the higher number of hydrolyzed ester bonds on the surface of PCL. In Figure 2, the medium spectra of PCL (Figure 2(b)) and PCL–PEO–PCL (Figure 2(e)) samples, treated according to the chosen hydrolysis conditions, are reported. The presence of the adsorption peak at 1,560 cm−1 is a clear spectrophotometric evidence for the formation of carboxylate groups in the hydrolyzed materials.

Figure 3. Infrared spectra of PCL and PCL–PEO–PCL before and after the coupling reaction with the (HP).


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Figure 4. Infrared spectra showing the typical adsorption peaks of the biomolecules used for the functionalization: HP, GRGDS, and YIGSR.

In Figure 3, the ATR spectra of the PCL and PCL–PEO–PCL samples, before and after coupling with the HP, used for the optimization of the coupling reaction, are reported. Spectra of untreated PCL and PCL–PEO–PCL showed the adsorption peaks (νas,CH2 = 2,944 cm−1; νs,CH2 = 2,866 cm−1; νC=O=1,722 cm−1; νC–C(=O)–O = 1,240 cm−1; νC–O–C = 1,160 cm−1) which are typical of polyesters. After the coupling reaction, both functionalized materials also showed the typical adsorption peaks of the protein material used for functionalization (νO–H = 4,000–3,000 cm−1; νN–H = 3,285 cm−1; νC=O = 1,645 cm−1; δN–H = 1,558 cm−1, Figure 4), demonstrating the occurrence of the coupling reaction. The same technique of analysis was used to evaluate the coupling reaction with the two peptides, comparing the spectra of functionalized materials (Figure 5) with those of the biomolecules used for functionalization (Figure 4). The attachment of GRGDS and YIGSR peptides was demonstrated by the presence, in all the acquired spectra, of a band among 3,700–3,000 cm−1, due to the stretching of NH and OH groups. The enlargement of this adsorption band, particularly evident in the case of GRGDS, was explained as a consequence of the increase in hydrophilicity of the samples, for the presence of the peptide. Such enlargement was less evident for YIGSR-functionalized polymers, due to the lower hydrophilicity of the amino acid residues contained in this peptide. The adsorption peak due to amide I (1,645 cm−1, νC=O) was observed as a shoulder on the high adsorption band of the ester C=O group. The adsorption peak due to Amide II (1,558 cm−1, νN-H) had a low intensity, but it was however detectable on all the analyzed functionalized samples.

Figure 5. On the left: infrared spectra of GRGDS- and YIGSR-modified PCL–PEO–PCL. On the right: infrared spectra of GRGDS- and YIGSR-modified PCL. The occurrence of the coupling reaction was demonstrated by the presence on functionalized materials of the typical absorption peaks of the bioactive peptides.


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E. Rosellini et al. Peptide surface density on functionalized polymers.

PCL PCL–PEO–PCL

GRGDS (mg/cm2)

YIGSR (mg/cm2)

0.75 0.18

0.80 0.25


Chromatographic analysis The peptide surface density on functionalized polymers was determined by HPLC. The results, expressed as mg of peptide/cm2, are shown in Table 3. Considering the molecular weight of the peptide sequences (490.47 for GRGDS; 594.67 for YIGSR), the immobilized peptide density of the surface was on the order of 10−7 mol/cm2. On the basis of the reference values reported in literature,[18] the amount of immobilized peptide should be sufficient to affect cell adhesion and proliferation. The peptide superficial density was higher on PCL because of the higher number of hydrolizable ester bonds (PCL–PEO–PCL contains 70% of PCL). Moreover, in the case of PCL–PEO–PCL, it can be supposed that an unhomogeneous distribution of constituent blocks or the higher crystallinity of the polymeric matrix, demonstrated by the higher melting enthalpy ΔH (ΔHPCL–PEO–PCL = 100–89 J/g; ΔHPCL = 84–65 J/g), made the alkaline hydrolysis more difficult.[32] FTIR chemical imaging Hydrolyzed samples of PCL and PCL–PEO–PCL were characterized using the chemical imaging apparatus, in order to investigate the homogeneity of the alkaline hydrolysis treatment. Chemical maps of the two samples were acquired (in Figures 2(a) and (d), for PCL and PCL–PEO–PCL, respectively) and from them, the medium spectra were collected, showing the typical adsorption peak of the carboxylate groups (as underlined by the arrows in Figures 2(b) and (e)). Then, the correlation maps between the chemical maps and the medium spectra were elaborated in the range of adsorption of the carboxylate groups (1,700–1,500 cm−1), as reported in Figures 2(c) and (f). Being the correlation index close to one on all the analyzed surfaces, it was demonstrated that carboxylate groups were nearly homogeneously distributed on the treated samples. The distribution of the biomolecules on the functionalized surfaces was also investigated by FTIR chemical imaging. The use of the instrument in μATR mode permits the acquisition of the surface chemical map of the sample. The chemical maps of the PCL and PCL–PEO–PCL samples functionalized with GRGDS and YIGSR were recorded. From the chemical map, the medium spectrum was acquired, where it was possible to identify the typical absorption peak of the two bioactive peptides used for the surface chemical modification, among 3,700–3,000 cm−1. The correlation maps among the chemical map and the adsorption peaks in the interval of interest were elaborated. For all the analyzed samples, most of the area of the correlation maps showed high values of the correlation index, demonstrating that peptide distribution on sample surface was enough homogeneous. As an example, the chemical imaging investigation performed on GRGDS-modified samples is reported in Figure 6.

Figure 6. On the top: correlation map (c) among the chemical map of GRGDS-modified PCL (a) and the relative medium spectrum (b). On the bottom: correlation map (f) among the chemical map of GRGDS-modified PCL–PEO–PCL (d) and the relative medium spectrum (e).


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Morphological analysis The morphological analysis was performed to evaluate possible morphological variations after the functionalization treatment. The results of the analysis, carried out by SEM, are reported in Figure 7. SEM micrographs were acquired for untreated and treated polymeric membranes, after hydrolysis and after coupling. The surface of untreated PCL membrane surface showed the presence of spherulitic structures immersed inside an amorphous matrix, due to high polymer crystallinity [33]; after the hydrolysis treatment, the dimension of the globular structures appeared reduced and the amorphous matrix increased, confirming the results obtained in the DSC analysis. After the functionalization, the globular structures appeared less evident. The surface of PCL–PEO–PCL was characterized by the presence of polygonal structures, strictly in contact among them; after the hydrolysis treatment, such structures appeared smaller and more separated. After the coupling reaction, the crystalline structures were less evident. Morphological modifications occurring after the coupling reaction could be attributed to a thin peptide film covering the sample surface. Thermal analysis The thermal analysis, carried out by DSC on untreated and hydrolyzed polymers, showed only one thermal event, due to polymer melting. The calorimetric data collected in Table 4 show a significant reduction in the enthalpy value for the hydrolyzed samples, linked to the crystallinity degree of both polymers. This result was in agreement with the observations made on SEM micrographs. The hydrolysis procedure produced a reduction in surface crystallinity for both polymers. This reduction was advantageous, since it is well known that surfaces with a high degree of crystallinity do not promote cell adhesion.[34] The thermal analysis was performed also on both polymers after the coupling reaction, but in this case, no variation of melting temperature or enthalpy was observed in comparison to hydrolyzed samples.

Figure 7.

SEM analysis of untreated and treated substrates.

Journal of Biomaterials Science, Polymer Edition Table 4.

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Calorimetric data of PCL and PCL–PEO–PCL, before and after hydrolysis. ΔH (J/g)

63.4 ± 3.1 60.7 ± 2.9 62.2 ± 5.6 55.8 ± 4.8

106.7 ± 5.0 94.6 ± 2.1 74.5 ± 2.5 67.0 ± 2.0


PCL–PEO–PCL untreated PCL–PEO–PCL after hydrolysis PCL untreated PCL after hydrolysis

Tm (°C)

Figure 8. C2C12 myoblast proliferation test on untreated polymers, GRGDS- and YIGSR-modified polymers, and TCPs.

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Figure 9. Appearance of multinucleated myotubes on YIGSR-modified polymers in 7 days after seeding, without differentiation medium. (a and b) TCPs control; (c) untreated PCL; (d) untreated PCL–PEO–PCL; (e) GRGDS-modified PCL; (f) GRGDS-modified PCL–PEO–PCL; (g) YIGSRmodified PCL; (h) YIGSR-modified PCL–PEO–PCL.


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Cell culture test C2C12 myoblasts seeded on functionalized polymers were used as a preliminary assessment of cellular responses to the surface chemical modification with bioactive peptides. Cells were examined at 1, 3, and 7 days post-seeding, using DAPI staining and fluorescent microscopy. The results of C2C12 myoblast proliferation test are collected in Figure 8. As shown by the graphs, functionalized substrates were able to promote cellular adhesion and proliferation, better than the untreated materials. The increase in proliferation was particularly evident starting from the third day after seeding: at this time, the number of cells on functionalized materials exhibited at least a twofold increase with respect to the untreated samples and became even higher than the TCPs control. Comparing the results obtained for the two different peptide sequences used for polymer functionalization, even if both were able to promote myoblasts adhesion and proliferation, cell number was higher on the fibronectin pentapeptide than on the laminin sequence, suggesting a higher efficiency of GRGDS than YIGSR in promoting C2C12 myoblasts growth. For YIGSR-modified polymers, a decrease in cell number was observed between day 3 and 7. This result was explained as a consequence of the fusion of myoblasts into multinucleated myofibrils, as a step toward myoblasts differentiation.[35] In fact, as shown in Figure 9, the appearance of multinucleated myotubes in 7 days after seeding was observed on polymers functionalized with YIGSR in the absence of the differentiation medium. This result suggested also a possible action of the laminin sequence in promoting myoblasts differentiation. Conclusions Alkaline hydrolysis and subsequent aqueous carbodiimide chemistry have been used to covalently graft GRGDS and YIGSR peptides to synthetic polymers containing ester groups. The hydrolysis condition was optimized with regards to hydrolysis time, temperature, and sodium hydroxide concentration for two synthetic polymers. The chemical imaging analysis provided information about the chemical modifications produced on material surface by the functionalization procedure and confirmed a nearly homogeneous distribution on both sample sides of the carboxylate groups (after the hydrolysis step) and of the peptides (after the coupling step). The introduction of the bioactive molecules had a positive effect on improving C2C12 myoblast proliferation on the synthetic materials. In particular, the fibronectin GRGDS sequence showed a higher ability of promoting cell proliferation, while the laminin YIGSR sequence appeared able to promote cell differentiation, as shown by the appearance of multinucleated myotubes in the absence of differentiation medium. This work can be considered a first step in the development of a synthetic ECM substitute, in which ligand type and density may be easily varied, in order to guide and control cardiac tissue formation from stem cells. As future developments, it would be interesting to perform the surface modification on preformed three-dimensional scaffolds or injectable materials and to make a deeper biological characterization, using also mesenchymal and cardiac stem cells. Disclosure statement No potential conflict of interest was reported by the authors.

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