Thermoresponsive and bioactive poly(vinyl ether)-based hydrogels synthesized by radiation copolymerization and photochemical immobilization

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Radiation Physics and Chemistry 77 (2008) 154–161 www.elsevier.com/locate/radphyschem

Thermoresponsive and bioactive poly(vinyl ether)-based hydrogels synthesized by radiation copolymerization and photochemical immobilization Ays- e Go¨nen Karakec- ˙ili˙a, Cristina Satrianob,c, Menems- e Gu¨mu¨s-dereli˙og˘lua,, Giovanni Marlettab,c b

a Chemical Engineering Department, Hacettepe University, 06800 Beytepe, Ankara, Turkey Laboratory for Molecular Surfaces and Nanotechnology (LAMSUN), Dipartimento di Scienze Chimiche, University of Catania, Italy c Consorzio Interuniversitario per i Sistemi a Grande Interfaccia (CSGI), V. le A. Doria 6, 95125 Catania, Italy

Received 25 February 2007; accepted 21 April 2007

Abstract A thermoresponsive hydrogel was synthesized by radiation copolymerization of ethylene glycol vinyl ether (EGVE) and butyl vinyl ether (BVE) in the presence of cross-linking agent diethylene glycol divinyl ether. The gel was modified by a cell adhesion factor RGD by photochemical immobilization technique. While the unmodified hydrogel shows fully hydrated form at low temperatures (+4 1C) and it extensively dehydrates at 37 1C, the biomodified hydrogel still kept its thermoresponsive character after immobilization. The effectiveness of immobilization was checked with FTIR-ATR and XPS. The use of bioactive thermoresponsive hydrogels in cell culture applications was investigated. For this purpose, cell culture experiments were realized by L929 mouse fibroblasts. Cell attachment experiments revealed the effect of immobilized RGD with higher values of cell attachment (85%), which were obtained especially in the absence of serum. The thermoresponsive character of the hydrogel was useful for the application of low-temperature treatment in order to recover the attached viable cells from the surface of the hydrogel without using trypsin. When the culture temperature was decreased from 37 to 10 1C for 30 min 80% of the cells were detached from the hydrogel surface. r 2007 Elsevier Ltd. All rights reserved. Keywords: Thermoresponsive hydrogel; Biomodification; Photochemical immobilization; Cell culture; Cell detachment

1. Introduction The temperature-sensitive hydrogels, which display volume transitions in response to temperature changes in the environment, are gaining much attention for their possible use in tissue engineering applications as cell support materials. There are various studies reporting the control of cell-surface adhesion by exploiting cell culture temperature on thermoresponsive polymers (Ebara et al., 2004; Akiyama et al., 2004; Shimizu et al., 2003; Okano et al., 1993). Poly(N-isopropylacrylamide) (PIPAAm) is one of the most extensively used thermoresponsive polymer Corresponding author. Tel.: +90 312 2977447; fax: +90 312 2992124.

E-mail address: [email protected] (Menemse Gu¨mu¨s-dereli˙og˘lu). 0969-806X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2007.04.014

for temperature controlled cell attachment/detachment. Cells adhered on PIPAAm surfaces at 37 1C were spontaneously lifted by reducing culture temperature below the lower critical solution temperature (LCST, 32 1C) without any need for trypsin or EDTA. PIPAAm-grafted tissue culture grade polystyrene dishes were used for the application of cell-sheet engineering in which the cells were harvested from the thermoresponsive surfaces without damaging the extracellular matrix proteins and cell-to-cell connections (Ebara et al., 2004; Akiyama et al., 2004; Shimizu et al., 2003; Okano et al., 1993). Recently, vinyl ether based monomers were polymerized by g-irradiation to produce thermoresponsive gels with any desired temperature transition (Gu¨mu¨s- dereli˙og˘lu et al., 2004; Mun et al., 1999) and were used to control the cell attachment/detachment behaviour by temperature changes

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(Gu¨mu¨s- dereli˙og˘lu and Karakec- ˙ili˙, 2003). The wettability of water-swellable copolymers of vinyl ethers significantly changes with temperature and this change initiates the cell detachment from the polymeric surface (Gu¨mu¨s- dereli˙og˘lu and Karakec- ˙ili˙, 2003). Moreover, the specific interactions between biosignal molecules and cell receptors play an important role in mediating cellular functions like cell attachment and spreading. Among these biosignal molecules, the arginine-glycine-aspartic acid (RGD) sequence of fibronectin has been widely reported to enhance the integrin-mediated cell adhesion (Park et al., 2005; Hersel et al., 2003; Gu¨mu¨s-dereli˙og˘lu and Tu¨rkog˘lu, 2002; Drumheller and Hubbell, 1994; Hirano et al., 1993). In our previous study, vinyl ether based hydrogels were synthesized by using a hydrophilic monomer ethylene glycol vinyl ether (EGVE) and hydrophobic monomer butyl vinyl ether (BVE) by g-irradiation. The synthesized hydrogel with thermoresponsive character was biomodified with RGD by using a four-step immobilization method in which the surface hydroxyl groups were isocyanated and subsequently converted to activated ester (Kondoh et al., 1993). Unfortunately, the hydrogel lost its thermoresponsive character after the immobilization process (Gu¨mu¨s- dereli˙og˘lu et al., 2004; Karakeci˙li˙, 2006). In this study, it is aimed to synthesize a bioactive thermoresponsive hydrogel and investigate its possible use as a cell support material. For this purpose, EGVE– BVE hydrogel was synthesized as reported previously (Gu¨mu¨s- dereli˙og˘lu et al., 2004; Mun et al., 1999). Photochemical immobilization process (Chung et al., 2002, 2003) was used for the biomodification of the synthesized thermoresponsive hydrogel by RGD. While the bioactive property obtained after RGD immobilization will support the cell adhesion, the thermoresponsive character will lead to the efficient recovery of viable cells by lowering the temperature without using proteolytic enzyme. Here, (i) the effect of biomodification process on thermoresponsive character of the gel, (ii) the effect of RGD biosignal molecule on cell attachment and (iii) the effect of thermoresponsive character on cell attachment/detachment process by low-temperature treatment were reported. 2. Materials and methods 2.1. Materials Comonomers ethylene glycol vinyl ether (EGVE), butyl vinyl ether (BVE) and cross-linking agent diethylene glycol divinyl ether (DEGDVE) were obtained from Aldrich (Germany). Arginine-glycine-aspartic acid (Arg-Gly-Asp) sequence of fibronectin (RGD; FW ¼ 346.3 g/g mol) was purchased from Sigma (Germany). Photochemical cross-linker sulfosuccinimidyl-6-(40 -azido-20 -nitrophenylamino)hexanoate (sulfo-SANPAH, FW ¼ 492.4 g/g mol) was obtained from Pierce (USA). All the chemicals were used without further purification. Phosphate buffer saline (PBS) tablets were purchased from Sigma (Germany).

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Tissue culture flasks and plates were purchased from Nunc (Germany). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 0.01% trypsin/10 mM EDTA were obtained from Sigma (Germany). 2.2. Synthesis of hydrogels The crosslinked copolymer was synthesized according to the procedure described in our previous study (Gu¨mu¨s-dereli˙og˘lu et al., 2004). In brief, the mixture consisting of comonomers in 60:40 mole ratio (EGVE:BVE) and crosslinker (DEGDVE) (4.0 mol%) were placed into the Pyrex test tube, frozen in nitrogen, degassed and sealed in vacuum. The irradiation copolymerization was carried out on a 60Co source at an irradiation dose rate of 3.51 kGy/h during 30 h. After the polymerization, the gels were removed, cut into cylinders (diameter: 13 mm, height: 1.1 mm) and washed out in distilled water during 3 weeks. 2.3. Biomodification of hydrogels Photochemical immobilization technique was used to modify the synthesized temperature-sensitive hydrogel by the RGD sequence of cell-adhesion protein fibronectin (Chung et al., 2002, 2003). In the first step, RGD was coupled with the photoreactive molecule sulfo-SANPAH in order to prepare photoreactive RGD molecules. For this purpose, RGD (5 mM in PBS pH: 7.4) and sulfo-SANPAH (5 mM in PBS pH: 7.4) were reacted in dark at room temperature for 2 h. The resulting phenyl-azido derivatized RGD (500 ml) was then poured onto the hydrogel. The gel was removed from the solution after 1 h and air-dried at room temperature. The dried gel was irradiated by UV light for 4 min to induce the photochemical fixation of RGD on the thermoresponsive hydrogel surface. The gel was fully rinsed with distilled water after immobilization process in order to remove the un-reacted reagents. The amount of immobilized RGD was estimated indirectly by ninhydrin method (Ito et al., 1993). For this purpose the immobilization procedure was performed at two stages. At the first stage, the hydrogel was activated by using the photochemical cross-linker sulfo-SANPAH by embedding the hydrogel in 5 mM sulfo-SANPAH solution at room temperature for 1 h. The hydrogel was then removed from the solution and air-dried. UV light was applied for 4 min to induce the photochemical activation of hydrogel. After photoactivation, the hydrogel was washed thoroughly in PBS (pH: 7.4) to remove the unreacted reagents. Subsequently, 5 mM RGD was pipetted on to the hydrogel. RGD was allowed to react in dark at room temperature for 2 h. After that the hydrogel was removed and the amount of RGD remaining in the solution was determined by ninhydrin method (Ito et al., 1993). A calibration curve was used to determine the amount of RGD. This amount was then subtracted from the initial amount (5 mM) and the immobilized RGD amount was determined indirectly.

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2.4. Characterization of hydrogels 2.4.1. FTIR-ATR analysis Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectra for the unmodified (EGVE–BVE) and RGD-modified (EGVE–BVE–RGD) hydrogels were obtained by using a Perkin-Elmer Spectrum One IR spectrometer (USA). The spectra of samples were taken at 400–2000 cm1 wavelength and analyzed with a standard software package, Perkin-Elmer, Spectrum One. 2.4.2. X-ray photoelectron spectroscopy (XPS) XPS analysis was performed by using a PHI 5600 Multi Technique Spectrometer equipped with dual Al/Mg anode, hemispherical analyser and electrostatic lens system (Omni Focus III). The electron take-off angle was typically 451, corresponding to a sampling depth of about 6 nm. The analyser was operated in FAT mode by using the Al Ka1,2 radiation with pass energy of 11.75 eV for the wide scans. The pressure in the main vacuum chamber during the analysis was kept below 3  108 mbar. The binding energy (BE) of 285.0 eV of the main C1s component (assigned to C–C and C–H bondings) was used as a reference to calibrate the energy position of the various peaks. 2.4.3. Temperature sensitivity of hydrogels In order to determine the temperature sensitivity of hydrogels, dynamic swelling measurements were done by gravimetric means at four different temperatures, i.e., 4, 10, 25 and 37 1C. The cylindrical samples were dried to the constant weight in vacuum and then immersed in a constant temperature bath filled with PBS (50 ml, pH: 7.4). The samples were removed from the bath at appropriate intervals, blotted with filter paper and weighed by using an analytical balance (Precisa 205 A SCS; sensitivity70.0001 g; Switzerland) and then returned to PBS. The measurements were repeated until the equilibrium was reached. All the experiments were carried out in an incubator (NUVE, ES 500, Turkey). The percent water content was calculated on dry weight basis by using the following equation: Water contentð%Þ ¼ ðW s  W d Þ 

100 , Wd

(1)

where Ws is the swollen weight and Wd is the dry weight of polymer gels. All measurements were repeated at least three times and data were expressed as means. 2.5. Cell culture studies 2.5.1. Cell line and maintenance In order to investigate the cell attachment on EGVE–BVE and EGVE–BVE–RGD hydrogels, cell culture studies were carried out with L929 mouse fibroblasts. L929 cell line was obtained from HUKUK Cell Line Collection (no: 92123004; Foot and Mouth Disease Institute, Ankara, Turkey). The cells were subcultured in flasks using

DMEM, supplemented with 10% (v/v) FBS and maintained at 37 1C in a humidified CO2 (5%) atmosphere (Heraus Instruments, Germany). Cells were dissociated with 0.01% trypsin/10 mM EDTA, centrifuged and resuspended in medium. The culture medium was replaced every 2 days. The specific growth rate and doubling time for L929 cell line were determined as 0.0204 h1 and 32 h, respectively, when they are cultivated in PS Petri dishes containing DMEM and 10% (v/v) FBS. Prior to cell culture experiments, 24-well tissue culture plates (TCPS) were precoated with parafilm and were soaked in 96% ethanol and placed under UV light for 30 min for sterilization. Hydrogels of EGVE–BVE and EGVE– BVE–RGD with 13 mm diameter were sterilized with 70% ethanol (30 min, 2 times), rinsed with sterile Dulbecco’s PBS (pH ¼ 7.4, 30 min, 2 times) and suspended in conditioning medium (15 min) prior to cell seeding. 2.5.2. Cell attachment For cell attachment experiments, the cell density was adjusted to 5  104 cells/ml in DMEM containing 10% FBS. A 1 ml of cell suspension was added to each well of the 24-well TCPS containing EGVE–BVE and EGVE– BVE–RGD hydrogels. In order to determine the number of adhesive cells haemocytometric counting was performed. At short culture times within 24 h, the medium was aspirated and the non-adhered cells were counted in the medium with a Neubauer haemocytometer by using trypan blue exclusion method. Cell attachment experiments were conducted in DMEM without serum also. 2.5.3. Cell detachment by low-temperature treatment After a 30 h incubation period, temperature was decreased from 37 to 10 1C for 30 min by taking into account the results of our previous study (Gu¨mu¨s-dereli˙og˘lu and Karakec- ˙ili˙, 2003). The detached cells from each well were collected and haemocytometric counting was performed by using trypan blue exclusion method. The number of cells detached from the hydrogel surface was expressed as a percentage of the detached cells to the total cells cultured on each disc at the end of 30 h. 2.5.4. Cell detachment by trypsin treatment At the end of the 30 h cultivation period, the cells were harvested from the hydrogels by incubation at 37 1C for 15 min with 0.01% trypsin/10 mM EDTA solution and haemocytometric counting was performed by using trypan blue exclusion method to determine the number of detached cells. 2.6. Statistical analysis All data were expressed as means7standard deviations of a representative of three similar experiments carried out in triplicate. Statistical analysis was performed by using the Statistical Package for the Social Sciences (SPSS) version 9.0 software. Statistical comparisons were made by analysis

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of variance (ANOVA). Bonferroni tests were used for post hoc evaluations of differences between groups. In all statistical evaluations po0.05 was considered as statistically significant. 3. Results and discussion 3.1. Synthesis of EGVE–BVE hydrogels Linear and crosslinked copolymers of vinyl ethers can be synthesized by radiation polymerization as they belong to hard polymerization monomers, owing to their low reactivity (Mun et al., 1999). Copolymers of a vinyl ether of ethylene glycol and butyl vinyl ether containing both hydrophilic and hydrophobic groups exhibit a thermoresponsive character, owing to the strengthening of the hydrophobic interactions with increasing temperature (Mun et al., 1999). In this study, a vinyl ether based thermoresponsive hydrogel of EGVE–BVE was synthesized by g-irradiation copolymerization and the total dose for gel formation was 105.6 kGy. The synthesized gel consisting of comonomers in 60:40 mole ratio (EGVE: BVE) in feed, corresponding to 70:30 (EGVE:BVE) in copolymer composition was determined to have a thermoresponsive character (Gu¨mu¨s- dereli˙og˘lu et al., 2004). The thermoresponsive hydrogel was modified by the RGD sequence of fibronectin and the uses of these hydrogels in cell culture were investigated. 3.2. Biomodification of EGVE–BVE hydrogels Recent studies have revealed that specific interactions through molecular recognition between immobilized ligand biomolecules and cell membrane receptors play an important role for inducing cellular functions like cell attachment, spreading and proliferation on biomaterials. The RGD peptide sequence of fibronectin has been widely studied as an immobilized specific ligand for cell adhesion (Park et al., 2005; Hersel et al., 2003; Gu¨mu¨s-dereli˙og˘lu and Tu¨rkog˘lu, 2002; Drumheller and Hubbell, 1994; Hirano et al., 1993). In this study, RGD was immobilized on thermoresponsive EGVE–BVE hydrogel to construct a thermoresponsive bioactive cell support material. The presence of RGD molecules should enhance the cell adhesion on the hydrogel surface and the thermoresponsive character should be useful for detachment of cells by a temperature change. In our previous study, the biomodification of EGVE–BVE hydrogel was realized in a fourstep process starting with isocyanation of hydroxyl groups (Gu¨mu¨s- dereli˙og˘lu et al., 2004). The surface isocyanate group was then hydrolyzed and RGD was immobilized through the activated surface amino groups. However, the swelling experiments revealed that the synthesized hydrogel lost its thermoresponsive character after biomodification, mostly owing to the complexity of this modification process (Gu¨mu¨s-dereli˙og˘lu et al., 2004). In this study, photochemical immobilization technique which does not

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require specific activated groups on the surfaces (Chung et al., 2003) was used for the modification of EGVE–BVE hydrogel. Phenyl-azido derivatized RGD molecules were immobilized on the hydrogel surface upon UV radiation (290–370 nm) according to the method previously described by Chung et al. (2002, 2003). The phenyl-azido group is photolyzed in the presence of UV radiation and generates highly reactive nitrene group, which induces reactions with the neighbouring compounds on the hydrogel (Fig. 1) (Matsuda and Sugawara, 1995). The immobilized RGD amount was calculated on the order of 106 mol/g using the ninhydrin method and it would be sufficient to affect the cell attachment behaviour (Drumheller and Hubbell, 1994). Since the diffusion of RGD biomolecules should be taken into account, the immobilized amount is given on mol basis for unit weight of dry hydrogel (the average weight of a hydrogel is 0.12670.0085 g) as an overall estimation of RGD molecules introduced in surface and bulk structure of the hydrogel after the immobilization process. 3.3. Characterization of hydrogels 3.3.1. FTIR-ATR analysis The effect of immobilization process on the surface of EGVE–BVE hydrogel was investigated by FTIR-ATR spectra. These spectra are shown in Fig. 2a for the unmodified hydrogel (EGVE–BVE) and in Fig. 2b for the RGD-modified hydrogel (EGVE–BVE–RGD). After immobilization process, there was an increase in the absorption at 1650 cm1, which is the characteristic band of Amid I present in RGD molecule. Also a broad shoulder was observed at 1520 cm1, which belongs to the Amid II absorption in RGD molecule. 3.3.2. X-ray photoelectron spectroscopy (XPS) Table 1 reports the surface composition and the amount of C1s, N1s and O1s of EGVE–BVE and EGVE– BVE–RGD hydrogels obtained from the wide scan XPS spectra. N1s atomic concentration was detected after the biomodification process as 2.2%. The presence of N1s and the increase in O1s can be attributed to the RGD molecules O N3

NH

(CH2)5

C

NH

RGD

UV . N .

N3

+

N2

nitrene Fig. 1. Production of nitrene groups in phenyl-azido derivatized peptide (RGD) by UV irradiation.

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94.8 90 85 1517

80

a

75 70 b

65 %T

60 55 50 45 40 35 30 25 20 14.4 2000.0 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900

800

650.0

-1

cm

Fig. 2. FTIR-ATR spectra of unmodified (a) and RGD-modified (b) EGVE–BVE hydrogel.

Table 1 Average surface atomic compositiona and corresponding atomic ratios for EGVE–BVE hydrogels Hydrogels

O1s (%)

C1s (%)

N1s (%)

C/O

N/C

EGVE–BE EGVE–BE–RGD

23.8 24.1

76.2 73.7

– 2.2

3.20 3.06

0 0.03

a

Typical error ¼ 71%.

present on the surface of the hydrogel after immobilization as previously reported (Satriano et al., 2005). The detailed analysis of the high resolution C1s spectra, reported in Fig. 3, shows the signals for –C–C– and –C–H– (at 285.070.2 eV) and –C–O– (at 286.470.2 eV) for the unmodified EGVE–BVE hydrogel and the appearance of new {CQO groups at 288.170.2 eV of BE for the RGD-modified EGVE–BVE hydrogel. N1s peak for the RGD-modified hydrogel appears at 400.170.2 eV of BE for the new –N–CQO– groups. Correspondingly in the O1s peak –O–C– groups (at 532.670.2 eV) and –O–H– groups (at 533.670.2 eV) for the unmodified hydrogel are detected. Upon RGD immobilization, the contribution of {CQO groups at 532.070.2 eV of BE in the O1s peak takes place for the EGVE–BVE–RGD hydrogel.

in our previous study (Gu¨mu¨s- dereli˙og˘lu et al., 2004), the synthesized amphiphilic hydrogel shows a volume-phase transition in the temperature range of 10–25 1C. The temperature decrease favours the uptake of PBS into the gel and EGVE–BVE hydrogel is hydrophilic and holds water molecules at 4 1C. However, the hydrogel expel water molecules and is relatively hydrophobic resulting in a reduced polymer volume at 37 1C. These results reveal that the synthesized hydrogel changes from hydrophobic to hydrophilic by decreasing temperature and can be used for cell detachment in cell cultures similar to PNIPAAm gels (Gu¨mu¨s-dereli˙og˘lu et al., 2004). The swelling kinetic data obtained for unmodified and RGD-modified hydrogel were compared in Fig. 4. The equilibrium water contents for hydrogels do not change after the photochemical immobilization process and all hydrogels have similar swelling behaviour. The equilibrium water content (EWC) values are also given in Table 2. All hydrogels have approximately the same EWC at each temperature. These results indicate that the biomodification by using photochemical immobilization does not change the thermoresponsive behaviour of EGVE–BVE hydrogels unlike the biomodification process realized in our previous study (Gu¨mu¨s-dereli˙og˘lu et al., 2004). 3.4. Cell culture studies

3.3.3. Temperature sensitivity of hydrogels The temperature sensitivity of unmodified and RGDmodified hydrogels was determined by equilibrium swelling experiments performed in PBS having a pH of 7.4 at four different temperatures (4, 10, 25 and 37 1C). As explained

3.4.1. Cell attachment It has been reported that RGD is a cell adhesion promoting peptide and improve the affinity of cells when immobilized on surfaces (Park et al., 2005;

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EGVE-BVE

159

EGVE-BVE-RGD

C1 C1

C2

C2

C3 295

290

285

280 300

295

290

285

415

280

N1

Intensity (a.u.)

300

410

405

400

395 415

410

405

400

395

O1 O1

O2

545

540

O2

535 BE (eV)

530

525 545

540

535 BE (eV)

O3

530

525

Fig. 3. XPS photoelectronic peaks of C1s, N1s and O1s for unmodified (EGVE–BVE) and RGD-modified (EGVE–BVE–RGD) thermoresponsive hydrogels.

140 120

T=4°C T=10°C

water content %

100 80

T=25°C

60

T=37°C

40 20

EGVE-BVE EGVE-BVE-RGD

0 0

500

1000 time (min)

1500

2000

Fig. 4. Comparison of swelling kinetics of unmodified and RGD-modified EGVE–BVE hydrogels at different temperatures.

Gu¨mu¨s-dereli˙og˘lu and Tu¨rkog˘lu, 2002; Hirano et al., 1993). In this study, L929 mouse fibroblasts were used and the time-dependent cell attachment onto the unmodified and

RGD-modified EGVE–BVE thermoresponsive hydrogels was studied both in serum-free and 10% (v/v) FBS containing cultures. The effects of RGD on fibroblast attachment are shown in Fig. 5. The results show that the cell attachment on all surfaces was nearly completed in 4-h culture period and the increase in culture time up to 24 h did not considerably affect the number of attached cells. In 10% (v/v) FBS containing culture, at the end of 4 h, 84% of the cells were attached on the unmodified EGVE–BVE hydrogel. The percentage of attached cells at the end of 4 h was 90% in the presence of RGD molecules on the biomodified EGVE–BVE hydrogel which is very close to the unmodified EGVE–BVE hydrogel. In the absence of serum, the presence of RGD accelerated the cell attachment and also increased the number of cells attached to the hydrogel. At the end of 4 h culture period, the results obtained from haemocytometric countings showed that 85% of cells were attached on RGD-modified EGVE–BVE hydrogel whereas this amount was only 55% on the unmodified EGVE–BVE hydrogel. These results indicate that L929 mouse fibroblast attachment occurs as a result of the specific interaction between the integrin receptors in the cell membrane and tripeptide, RGD. In the absence of immobilized RGD molecules, the

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Table 2 The equilibrium swelling values of the unmodified and RGD-modified EGVE–BVE hydrogels at different temperatures (PBS, pH ¼ 7.4)

Table 3 Low-temperature treatment and trypsinization for the cell recovery from thermoresponsive hydrogels

Hydrogels

Hydrogels

EWC (%)

EGVE–BVE EGVE–BVE–RGD

number of attached cells / cm2 x(10-4)

3.5 3.0

4 1C

10 1C

25 1C

37 1C

11773 11672

10272 10373

6172 6272

4472 4573

EGVE-BVE (10% FBS) EGVE-BVE-RGD (10% FBS) EGVE-BVE (without serum) • EGVE-BVE-RGD (without ∗ serum)

EGVE–BVE EGVE–BVE–RGD

∗

•

∗

•

∗ •

2.5 2.0 1.5 1.0 0.5 0.0 1

4

2

24

time (h) Fig. 5. L929 fibroblast attachment on unmodified and RGD-modified EGVE–BVE hydrogels in 10% FBS and in the absence of serum. Initial cell concentration: 5  104 cells/ml, n ¼ 3, po0.01: control group is EGVE–BVE (with 10% FBS) and Kpo0.01: control group is EGVE–BVE (without serum).

attachment can be explained by the moderately wettable surface of the hydrogel at 37 1C (water contact angle: 501 at 37 1C) which is similar to that of TCPS (water contact angle: 47.91) (Gu¨mu¨s- dereli˙og˘lu and Karakec- ˙ili˙, 2003). 3.4.2. Cell detachment The detachment of L929 fibroblasts from the EGVE– BVE and EGVE–BVE–RGD hydrogel surfaces was realized by low-temperature treatment and trypsinization. An optimum route selected in a previous study was followed during the low-temperature treatment process (Gu¨mu¨s- dereli˙og˘lu and Karakec- ˙ili˙, 2003). According to this, the culture medium (at 37 1C) was replaced with fresh medium at 10 1C and the culture dishes were cooled to 10 1C. The number of detached cells was determined at the end of 30 min. At lower temperatures, the thermoresponsive polymer chains maintain expanded conformations so the interactions between the cell and the polymeric surfaces are reduced. There is a loss of cell tension on hydrated polymer resulting in cell detachment. However, it should be considered that there is an optimum process temperature as the cell metabolism can be suppressed at very low

Attached cells (%) (10% FBS containing medium)

8471 9072

Detached cells (%) Lowtemperature treatment; 37 1C-10 1C, 30 min

Trypsinization

8373 8072

91 91

temperatures resulting in a decreased number of detached cells (Okano et al., 1995). In our previous study, the effect of cell metabolism was evident as the number of detached cells in case the temperature was lowered to 4 1C was smaller when compared to the low temperature treatment at 10 1C (Gu¨mu¨s-dereli˙og˘lu and Karakec- ˙ili˙, 2003). The cell recovery from EGVE–BVE and EGVE– BVE–RGD hydrogels was realized by trypsinization also. The number of detached cells obtained from both procedures is given in Table 3. The comparative results showed that while the trypsinization procedure is leading to 90% cell recovery, the low-temperature treatment causes 80% cell recovery. This is why, the lowtemperature treatment process with the hydration change of the surface can be considered as an effective procedure for the recovery of L929 mouse fibroblasts. In the presence of immobilized RGD molecules for EGVE–BVE–RGD hydrogel, the detachment is also explained by the loss of cell tension and surface anchoring because of surface hydration. The hydrated and softened polymer surface promotes the dissociation between the ligand biomolecule and cell receptor. There is a loss of cell tension by swelling and the association between cell integrins and RGD is decreased. It should be noted that low-temperature treatment does not directly cause cell receptor–ligand biomolecule dissociation in case there is no surface hydration as the cell-matrix binding is known to be retained even at 4 1C (Grinnell et al., 1982). There was a slight decrease in the percentage of the detached cells in the presence of immobilized RGD molecules, which shall not be considered as a significant difference. Although not presented in this study, it is likely that the immobilized RGD content may affect the detachment of fibroblasts. 4. Conclusion The thermoresponsive copolymer of ethylene glycol vinyl ether and butyl vinyl ether was modified by using photochemical immobilization of cell adhesive molecule RGD. The results demonstrated that the immobilization process did not change the thermoresponsive behaviour of synthesized hydrogel. The cell culture studies showed that the presence of RGD molecules triggered cell attachment

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especially in the absence of serum. The thermoresponsive characteristics and bioactive property were combined making EGVE–BVE hydrogels possible to use in cell cultures without using proteolytic enzyme. Our ongoing research studies are directed to the proliferation of specific cells on bioactive thermoresponsive EGVE–BVE hydrogels and the applicability of these hydrogels in cell sheet engineering studies. Acknowledgements This study was financially supported by Grant No. 03 K 120 570-5 by Turkish Prime Ministry State Planning Organization (DPT) and by FIS-CNR (Rome) and FIRB RBNE01458S grants. A. Karakec- ˙ili˙ acknowledges the grant for the period spent at LAMSUN at University of Catania provided by the Consorzio Interuniversitario per i Sistemi a Grande Interfaccia (CSGI; Florence, Italy). References Akiyama, Y., Kikuchi, A., Yamato, M., Okano, T., 2004. Ultrathin poly(N-isopropylacrylamide) grafted layer on polystyrene surfaces for cell adhesion/detachment control. Langmuir 20, 5506–5511. Chung, W.T., Lu, Y.F., Wang, S.S., Lin, Y., Chu, S., 2002. Growth of human endothelial cells on photochemically grafted GRGD chitosans. Biomaterials 23, 4803–4809. Chung, W.T., Liu, D.Z., Wang, S.Y., Wang, S.S., 2003. Enhancement of the growth of human endothelial cells by surface roughness at nanometer scale. Biomaterials 24, 4655–4661. Drumheller, P.D., Hubbell, J.A., 1994. Polymer networks with grafted cell adhesion peptides for highly biospecific cell adhesive substrates. Anal. Biochem. 222, 380–388. Ebara, M., Yamato, M., Aoyagi, T., Kikuchi, A., Sakai, K., Okano, T., 2004. Temperature-responsive cell culture surfaces and RGDS ligands. Biomacromolecules 5, 505–510. Grinnell, F., Lang, B.R., Phan, T.V., 1982. Binding of plasma fibronectin to the surfaces of BHK cells in suspension at 4 1C. Exp. Cell Res. 142, 499–504. Gu¨mu¨s-dereli˙og˘lu, M., Karakec- ˙ili˙, A., 2003. Uses of thermoresponsive and RGD/insulin-modified poly(vinyl ether)-based hydrogels in cell cultures. J. Biomat. Sci. Polym. E 14, 199–211.

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