In vitro cyto-biocompability and cell detachment of temperature-sensitive dextran hydrogel

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1 In vitro cyto-biocompability and cell detachment of temperature-sensitive dextran hydrogel Fei Xiao 1, Li Chen 1, Rui-Feng Xing 1, Yi-Ping Zhao 1, Jing Dong 1, Gang Guo 2, and Rui Zhang 2 1 Tianjin Key Laboratory of Fiber Modification and Functional Fiber, School of Materials Science and Chemical Engineering, Tianjin Polytechnic University, Tianjin , China 2 Institute of Endocrinology, Tianjin Medical University, Tianjin , China chenlis@tjpu.edu.cn Abstract A series of poly(nipaam-co-gma-dex) hydrogels were synthesized from glycidyl methacrylate-derivatized dextran (GMA-Dex) and N-isopropylacrylamide(NIPAAm) in aqueous solution. The swelling properties, cell culture and detachment behaviors of these hydrogels were investigated. It has found that poly(nipaam-co-gma-dex) hydrogels are not only sensitive to temperature in deionized water and Dulbecco s modified eagle medium (DMEM) (10%FBS), but also exhibit good biocompatibility. In addition, L929 cells were found to detach from the hydrogel surface naturally without affecting the function of L929 by controlling environmental temperature. Keywords: Hydrogels; N-isopropylacrylamide; Dextran; Temperature-sensitivity; Cell culture 1 Introduction Poly(N-isopropylacrylamide) (PNIPAAm), a well-known thermo-responsive polymer which exhibits a coil-to-globule transition in aqueous solution at a lower critical solution temperature (LCST) of around 32, has been used in controlled cell adhesion[1-2]. Okano et al[3] found that PNIPAAm-grafted surface of polystyrene cell culture dishes can exhibit hydrophobicity and hydrophilicity at temperature above and below the LCST, respectively. As a result, endothelial cells and hepatocyte attached and proliferated on the PNIPAAm-grafted surfaces at 37, could be readily detached from the surface by lowering the incubation temperature without the usual damage due to trypsinization. However, PNIPAAm has a poor biocompatibility which restricts the cell attach application. Therefore, introducing other polymer with good biocompatibility into PNIPAAm was used to improve its biocompatibility. Furthermore, spontaneous cell sheet detachment from surfaces of NIPAAm-grafted tissue culture polystyrene (TCPS) is a relatively slow process, occurring gradually from the sheet periphery toward the interior. Thus, rapid detachment of cultured cell sheets is a very important recovery method that permits facile manipulation of the sheets. Hydrogel is a three-dimensional network of hydrophilic polymer, which can absorb tremendous amounts of water from the enviorment. Due to the low interfacial free energy of the hydrophilic surface, proteins and cells are hard to be adhered to the surface. However, hydrophobic molecule chains on the hydrogel surface can improve the cell adhesion[4-7]. An 1

2 important advantage of hydrogels is that the nature of soft and rubbery state minimizes any potential irritant reaction with surroundings tissue, and exhibits a good bio-compatibility. Moreover, the porous conformation of hydrogels can accelerate the hydration of the material. Dextran, consisting of α-1,6-linked D-glucopypyranose residues, is a bacterial-derived polysaccharide generally produced by enzymes from certain strains of Leuconostoc or Streptococcus. Dextran and its derivatives have been examined extensively in biomedical, pharmaceutical, and biotechnological applications due to their biocompatible, biodegradable, and non-toxic properties[8 12]. Crosslinked dextran hydrogel beads have been widely used as low protein-binding matrices for column chromatography applications and microcarrier for cell culture technology [4,13]. We have previously synthesized dextran hydrogels with NIPAAm (Poly(NIPAAm-co-GMA-Dex)), and its swelling properties, mechanical properties and bioseparation behaviors were investigated as well[14,15]. It was found that Poly(NIPAAm-co-GMA-Dex) hydrogels are temperature-sensitive while exhibit much better swelling and mechanical properties than the PNIPAAm gel at the same time. In this paper, a series of poly(nipaam-co-gma-dex) hydrogels were synthesized by copolymerization of GMA-Dex with NIPAAm in aqueous solution. The cyto-biocompability and cell detachment behaviors on the surfaces of poly(nipaam-co-gma-dex) hydrogels were investigated. It is expected that the hydrogel will show good cell attachment and growth ability, and a rapid cell sheet detachment. 2 Materials and methods 2.1 Materials NIPAAm (Kohjin Co. Ltd., Japan) was used after recrystallization from n-hexane. 4-Dimethylaminopyridine (DMAP) and glycidyl methacylate (GMA) (Yuanji Chemical Industry Co. Ltd.)were used as received. Dextran and dimethtyl sulfoxide (DMSO) (Shanghai Chemical Reagents Co., China). Ammonium persulfate (APS), N,N,N,N -tetra-methylenediamine (TEMED), and N,N -methylenebis(acrylamide) (MBAA) were analytical grade made in China. Phosphate-buffered saline (PBS, ph 7.4) was used for hydrogel sterilization. Dulbecco s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (Minhai biotechnologies), penicillin and streptomycin, Trypsin and Ethylenediaminetetraacetic acid (EDTA) were obtained Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.. Water was deionized by Millipore Direct-Q apparatus. 2.2 Synthesis of poly(nipaam-co-gma-dex) hydrogels GMA-Dex monomer with double bonds was obtained by the transesterification of GMA with Dextran at room temperature as previously reported[14]. Poly(NIPAAm-co-GMA-Dex) hydrogel was prepared by the free radical copolymerization of NIPAAm and GMA-Dex in aqueous solution. NIPAAm and GMA-Dex were dissolved in deionized water according to a weight ratio, r, in the feed ((GMA-Dex)/(GMA-Dex+NIPAAm)) of 0.2, 0.4, 0.5, 0.6, and 0.8 respectively, and then initiator APS (0.2wt%) and accelerant TEMED (0.5wt%) were added. After bubbling with nitrogen gas to get rid of oxygen while stirred by vortex for 10 min, the mixture was injected into the glass mold and let it react at room temperature for 48 h. The formed hydrogels were cut into discs with a diameter of approximately 9mm and immersed in an excess of 2

3 distilled water for 1 week with daily water exchange to remove the unreacted monomer. The obtained hydrogels were dehydrated for 2 days by freeze-drying before characterization. The reaction procedure is shown in Scheme 1. Pure PNIPAAm hydrogel was also prepared as control sample in the present of cross-linker MBAA (0.5wt%). Scheme 1: Schematic representation of poly(nipaam-co-gma-dex) hydrogel. 2.3 Swelling characterization of the hydrogels The dried hydrogels were immersed in deionized water to swell for 24 h over a range of temperature from 20 to 50. Equilibrium swelling ratio is defined as W s /W d, where W s and W d are the weight of equilibrium swollen gels and dried gels, respectively. The LCST is defined as the temperature where the volume of hydrogel compacts dramatically. 2.4 L929 cell culture The pre-measurement hydrogels were divided into 3 groups and placed into 6-well tissue culture plates. 2 ml ethanol was added to sterilize the gel for 5 min, and irradiation was carried out for 30min using 15w ultraviolet light. Then 2-3 ml DMEM with 100U/mL of penicillin and 100U/mL of streptomycin were added into 6-well to pre-incubate overnight. After the old DMEM was decanted, 4 mlof trypsin was added to digest L929 cell for 3-5 min. The antenna vanished as the shape of L929 cell turned into round. Removed the assimilation solution mixture and then 4mL fresh DMEM containing double amount of antibiotic was added. The bottom of flask was blown carefully by sucker. The suspended cell solution was transferred into a scale centrifuged tube and diluted consecutively to the density of cells/ml. 2.5 L929 cell attachment The incubated L929 cell medium was decanted, and the obtained suspension was injected into the well. The cell was incubated at 37 in a humid atmosphere containing 5% CO 2, the medium was exchanged every two days. 6-well TCPS was taken out after culturing 2, 4, 6, 8 days, respectively, and one group was digested with trypsin solution for counting the amount of cells by using haemacytometer. The morphological changes of L929 cell attached on transparent hydrogels surfaces were directly observed by phase-contrast inverted light microscopy (OLYMPUS ckx31, Japan). L929 cell attached on the opaque hydrogels surface was fixed with 2% glutaraldehyde, and then the 6-well was incubated at 4 overnight. The fixed cells were lyophilized by freeze-dryer (Boyikang experimental instrument 3

4 Co. Beijing). The samples were observed using a scanning electron microscopy (SEM, Quanta 200, FEI Co.) by sputtering with gold. 2.6 L929 cell detachment L929 cells were seeded onto hydrogels discs at a density of cells/ml and cultured at 37 under a humid atmosphere of 5% CO 2 in air. After 2 days, the temperature of the cell system was decreased from 37 to 20 by cooling the culture dishes in bio-chemical incubator. The change image of L929 detachment morphology was captured by phase-contrast microscopy. Detached cells from hydrogels were collected and the amount of cell was estimated by heamacytometer. 2.7 L929 Cell viability assay The cell viability was estimated by the MTT method[16]. The L929 cells during exponent growth period were first diluted into suspension at a density of cells/ml. After seeding onto the hydrogel discs, the culture systems were incubated at 37 in a humidatmosphere containing 5% CO 2 for 8 days, and the DMEM was exchanged every 2 days. At each time point (2, 4, 6 and 8 days), the samples were taken out of the incubator and about 100 µl MTT solution (5mg/mL) was added to each well to incubate for 4h. After decanting the liquid at top of DMEM, 300 µl DMSO was added to each well. In order to dissolve the formazan crystal, the culture plates were shaked on a rocker platform at a low speed for 10 min. Then, 100 µl solution was injected into 96-well TCPS each time. Each well OD value were determined by Auto Microplate Reader (Σ960, Metertech Co., America) at a wavelength of 570 nm. Data of the cell viability were collected from triplicate samples and expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA with a Scheffé test. 3 Results and discussion 3.1 Effect of temperature on swelling behaviors of hydorgels The effect of temperature on swelling behaviors of poly(nipaam-co-gma-dex) hydrogels with different ratio r, were investigated and shown in Figure 1. It is clear that poly(nipaam-co-gma-dex) hydrogels still exhibit temperature-sensitivity, suggesting NIPAAm component plays a key role in the tuning the volume phase transition behavior of hydrogels. Figure 1b showed the relationship between LCST and r. It is seen that the LCST of poly(nipaam-co-gma-dex) hydrogels is more obvious than that of PNIPAAm hydrogels and increases with increasing r, that is with increasing amount of GMA-Dex. It might be because that the introduction of hydrophilic group (-OH) of dextran into PNIPAAm hydrogels improves its hydrophilicity and increases the amount of hydrogen bonds. Therefore, more energy is needed in order to break the hydrogen bonds, and the LCST value of hydrogels increased at the same time. The swelling behaviors of poly(nipaam-co-gma-dex) hydrogels in cell culture medium were also studied for further cell culture, and the similar temperature-sensitive property was observed. Figure 1b shows the swelling curves of poly(nipaam-co-gma-dex) hydrogel with r=0.4 in deionized water and DMEM, respectively. It is seen that the LCST of the hydrogel in DMEM is about 28, which is lower than that of hydrogel in deionized water. It might be attributed to a great amount of NaCl and NaHCO 3 in the DMEM which inhibits the forming of the hydrogen bonds. 4

5 Swelling ratio (a) PNIPAAm r=0.2 r=0.4 r=0.5 r=0.6 r=0.8 LCST( o C) (b) Temperature( o C) r 20 (c) Swelling ratio H 2 O DMEM Temperature( o C) Figure 1: Temperature dependence of the Swelling ratio of (a) poly(nipaam-co-gma-dex) and PNIPAAm hydrogels in deionized water (b) poly(nipaam-co-gma-dex) hydrogel with r=0.4 in deionized water and DMEM. In order to understand the reversibility and speed of the response of poly(nipaam-co-gma-dex) gels to the change of temperature, oscillatory swelling experiments were conducted at different temperatures. As shown in Figure 2, poly(nipa-co-gma-dex) (r=0.4) gels could swell and de-swell over a period of time when the temperature was varied periodically between 20 and 50 in distilled water and cell culture medium Water retaining(%) H 2 O DMEM Time(h) Figure 2: Swelling reversibility of poly(nipaam-co-gma-dex) hydrogels in deionized water and DMEM 5

6 3.2 Cell attachment and growth The cell compatibility of the poly(nipaam-co-gma-dex) hydrogels were examined by morphology study and adhesion assessing of the cell. L929 cells exhibits different morphologies for different cultured time on hydrogels discs. Optical microscope images at 2, 4, 8 and 10 days after cell seeding are shown in Figure 3. Two days after cell seeding, attached L929 was scarce and remained rounded, which is similar to the morphologies of L929 before seeding. However, poly(nipaam-co-gma-dex) hydrogels with at both r=0.4 and r=0.8 groups showed a closer packing of cells on the hydrogel discs after 4 days. After 8 and 10 days, the amount of L929 on hydrogel discs was increased continually. It is obvious that the density of cell on hydrogel (r=0.4) is higher than that of hydrogel (r=0.8). The images clearly showed the cell adhered to and grew well on the surface of hydrogels, indicating that poly(nipaam-co-gma-dex) hydrogels have good cell compatibility. Figure 3: Phase contrast micrographs (100 ) of L929 cell attached on poly(nipaam-co-gma-dex) hydrogels with r=0.4(a, b, c and d)and r=0.8(e, f, g and h) after culturing 2, 4, 8 and 10 days respectively Due to the opacity of PNIPAAm hydrogel disc, SEM measurement was further employed to investigate the cell morphology. As shown in Figure 4, the cell amount increased on both poly(nipaam-co-gma-dex) hydrogel and PNIPAAm hydrogel with increasing cultured time. However, L929 cells began to spread in all directions on poly(nipaam-co-gma-dex) hydrogel discs at 4th day, while at 6th day in the case of PNIPAAm hydrogel, suggesting that the cell adhesion and spreading was better on poly(nipaam-co-gma-dex) hydrogels compared with PNIPAAm hydrogel. 6

7 Figure 4: SEM micrographs (400 ) of L929 cell attached on poly(nipaam-co-gma-dex) hydrogel with r=0.4(a, b, c and d) and PNIPAAm hydrogel(e, f, g and h) after culturing 2, 4, 6, 8 days respectively Proliferation curves of L929 cell on poly(nipaam-co-gma-dex) hydrogels (r=0.4 and 0.8) and PNIPAAm hydrogel were shown in Figure 5. Although no cell amount changes in first 4 days, an abruptly increase were found in 4-6 days for all hydrogels, where the amount of cell on poly(nipaam-co-gma-dex) hydrogels started to increase greatly at 4 th day after seeding while that on PNIPAAm started at 6 th day. These results suggest that poly(nipaam-co-gma-dex) hydrogel surfaces supported more cells than PNIPAAm hydrogel, and poly(nipaam-co-gma-dex) hydrogels showed better cell compatibility due to the introduction of dextran. It was also found from Figure 5 that the cell adhesion on the surface of poly(nipaam-co-gma-dex) hydrogel with r=0.8 was lower than that on poly(nipaam-co-gma-dex) with r=0.4. It might be speculated that dextran could limit immobilization of L929 cells, which is consist with the mechanism of immobilization theory[17-19]. Furthermore, cell movement and aggregation seemed to be slightly restricted by the uneven surface topography of hydrogel discs. A similar result was obtained by the cell viability assay. As shown in Figure 6, at all time points measured, the cell adhesion to poly(nipaam-co-gma-dex) hydrogel with r=0.4 surface was better than that on the PNIPAAm surface, but worse than that on the TCPS control surface. Among them cells on poly(nipaam-co-gma-dex) surface with r=0.4 showed significantly higher viability (p<0.01) than those on the PNIPAAm surface at 2nd, 4th and 8th day. Cell number( 10 5 cells/ml) PNIPAAm r=0.4 r= Culture time(days) Figure 5: Proliferation curves of L929 cell on poly(nipaam-co-gma-dex) hydrogels with r=0.4 and 0.8 and PNIPAAm hydrogels 7

8 Tcps PNIPAAm r=0.4 r=0.8 ** Absorption(570nm) ** ** Culture time(days) Figure 6: Viability of L929 cell after culturing 2, 4, 6, 8 days for different hydrogels In order to evaluate substrates cytotoxicity, the cell proliferation inhibition index (CPII) was determined using the following equation: CPII=100%-(test culture OD 570nm /control culture OD 570nm 100%) It is obvious that the higher the CPII value, the larger the cytotoxicity. Figure 7 shows the CPII value of different hydrogel discs after culturing 2, 4, 6, 8 days. It is found that the CPII value of PNIPAAm hydrogel is higher than that of poly(nipaam-co-gma-dex) hydrogels during 6 days culture time. Among all hydrogel discs, poly(nipaam-co-gma-dex) hydrogel with r=0.4 group showed the smallest CPII value during all culturing days, that is, the lowest cytotoxcity. Cultrue time(days) r=0.8 r=0.4 PNIPAAm Cell proliferation inhibit index(%) Figure 7: Cell proliferation inhibition index after culturing 2, 4, 6, 8 days for different hydrogels 3.3 Influence of temperature on cell detachment Since poly(nipaam-co-gma-dex) hydrogels exhibit well-defined temperature-sensitivity, it is 8

9 expected that cells cultured on poly(nipaam-co-gma-dex) hydrogels could be detached simply by decreasing the temperature from 37 o C (hydrophobic) to 20 o C (hydrophilic). L929 cells were cultured as previously, after culturing for 2 days at 37, the temperature of the cell culture systems was then decreased from 37 to 20 by cooling the culture dishes. The morphology of L929 cells was observed by optical microscope after 60 min incubation at 20. The cells started to change shape from spread to round, and finally cells were observed to detach completely from the surface. It is most likely that hydration change of polymer chain at the cell-substrate interface is an important stimulus to induce cell detachment. Figure 8 shows the correlation between the percentage of detached cells and incubation time. It can be seen that all groups showed similar detachment tendency, L929 cells detached around 60% after 15 min incubation at 20, and cells detached over 85% after 60 min incubation. Thus, the cells could detach from these thermoresponsive hydrogels successfully by reducing the temperature from 37 to 20. This cell detachment is attributed to the formation of an expanded, swollen, and hydrophilic surface, which weakening cellular adhesion and resulting in spontaneous cell detachment due to the hydration of PNIPAAm below its LCST[20]. In contrast, in the case of conventional TCPS dishes, the cell could not be detached using the same treatment because of no surface property alteration with temperture Detached cells(100%) PNIPAAm r=0.4 r= Incubation time at 20 (min) Figure 8: Percentage of detached L929cell from poly(nipaam-co-gma-dex) hydrogels (r=4, r=8) and PNIPAAm hydrogel as a function of incubation time in culture medium at 20 It is noted that the spread L929 cells cultured on PNIPAAm and poly(nipaam-co-gma-dex) hydrogels detached around 60% at 15 min incubation after cooling cell culture systems from 37 to 20, while that on PNIPAAm-grafted TCPS dish detached only 30% at 15 min incubation as reported by Okano et al[20], suggesting the cell detachment on the hydrogels is more rapidly than those on PNIPAAm-TCPS dish. In addition, hydrogel has porous conformation, which could accelerate the hydration of the material. When the temperature was decreased from 37 to 20, the hydrogels reswelled due to the hydration of PNIPAAm. The reswelling of hydrogel supplied water molecules to the cells not only from the periphery of each cell but also through pores underneath the adherent cells. The spread cells on poly(nipaam-co-gma-dex) hydrogels showed little weaker detachment behavior than that on PNIPAAm hydrogel, which might be relating to the property of materials surface, 9

10 since it is usually believed that the hydration on substrate results in hydrophilic/hydrophobic changes. Okano et al [3] considered that cell detachment process was affected by the cell metabolic processes as well as the hydration of the culture surface where cell detachment is implicated on. Thus, after cells contact surfaces (passive adhesion), cells are always dynamically altering their cell membrane and morphology to optimize interactions and to stabilize the cell-material surface interface (active adhesion), both physicochemical and biological. 4 Conclusion Poly(NIPAAm-co-GMA-Dex) hydrogels were prepared in this study, which are temperature-sensitive and exhibit much better swelling ability in both distilled water and cell culture medium. In vitro test was conducted successfully on dextran-based hydrogels with low cytotoxicity and good biocompatibility. It has found that cells can naturally detach from the hydrogel disc without trypsin digest, indicating that poly(nipaam-co-gma-dex) hydrogels are biocompatible and promising biomaterials for improving specific cell interactions. Acknowledgements Gratefully thanks for expert technical assistance from Institute of Endocrinology. This research was financially supported by the National Nature Science Foundation of China (Contract grant number: ),and the Specialized Research Fund for the Doctoral Program of Higher Education (Contract grant number: ) References [1] H. A. Von-Recum, Kim-SW, A. Kikuchi, et al. J. Biomed. Mater Res. 40 (1998) 631. [2] H. A. von-recum, A. Kikuchi, M. Okuhara, et al. J. Biomater. Sci. Polym. Ed. 9 (1998) [3] T. Okano, N. Yamada, M. Okuhara, et al. Biomaterials 16 (1995) 297. [4] C. J. De Groot, M. J. A. Van Luyn, W. N. E. Van Dijk-Wolthuis, et al. Biomaterials 22 (2001) [5] H. Park, K. Park. Pharm. Res. 13(1996) [6] D. L. Coleman, D. E. Gregonis, J. D. Andrade. J. Biomed. Mater. Res. 16 (1982) 381. [7] K. Jr. Smetana. Biomaterials 14 (1993) [8] L. Hovgaard, H. Brondsted. J. Control. Rel. 36(1995) 159. [9] P. Edman, B. Ekman, I. Sjoholm. J. Pharm. Sci. 69 (1980) 838. [10] H. Brondsted, C. Anderson. J. Controlled Release 53 (1998) 7. [11] K. M. Huh, Y. Kumashiro, T. Ooya, et al. Polym. J. 33 (2001) 108. [12] M. Kurisawa, N. Yui. Macromol. Chem. Phys. 199 (1998) [13] S. G. Levesque, M. S. Shoichet. Biomaterials 27(2006) [14] L. Chen, J. Dong, Y. M. Ding, et al. J. Appl. Polym. Sci. 96(2005) [15] J. Dong, L. Chen, Y. M. Ding, et al. Macromol.Chem.Phys. 206(2005) [16] J. Sun, L. Wang, M. A. Waring, et al. Artif. Organs 21 (1997) 408. [17] S. P. Massia, J. Stark, D. S. Letbetter. Biomaterials 21(2000) [18] F. Maury, J. Honiger, D. Pelaprat, et al. J. Mater. Sci. - Mater. Med. 8 (1997) 571. [19] K. N. Chua, W. S. Lim, P. C. Zhang, et al. Biomaterials 26 (2005) [20] O.H. Kwon, A. Kikuchi, M. Yamato, et al. Biomaterials 24 (2003)