Colloids and Surfaces B: Biointerfaces 123 (2014) 742–746
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Alternating-current electrophoretic adhesion of biodegradable hydrogel utilizing intermediate polymers Taka-Aki Asoh 1 , Wataru Kawai, Akihiko Kikuchi ∗ Department of Materials Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan
a r t i c l e
i n f o
Article history: Received 20 August 2014 Received in revised form 21 September 2014 Accepted 8 October 2014 Available online 18 October 2014 Keywords: Hydrogel Adhesion Electrophoresis Scaffold
a b s t r a c t The adhesion of anionic charged biodegradable hydrogels each other utilizing oppositely charged watersoluble polymers as a binder has been achieved by applying alternating-current (AC) electric fields. The two gelatin based dextran sulfate gels (DS gels) were molecularly sutured together by AC electrophoretic adhesion when cationic charged quaternary ammonium chitosan (TMC) was applied between and held in contact with the two DS gels. The adhesive strength of the gels increased with increasing periodicity when a square wave was applied. Hydrogel constructs composed of DS microgels were prepared simply by AC electrophoretic adhesion utilizing intermediate TMC. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hydrogels are similar to the macromolecular-based constituents in the living body such as proteins, cells, and tissue, and therefore hydrogels have numerous applications in tissue engineering [1] and drug delivery systems [2]. For in situ hydrogel formation, several interaction as cross-linking points of the hydrogels have been used such as inclusion complexes [3], stereocomplexation [4], peptide interactions [5], hydrophobic interactions [6], and supramolecular complexes [7]. In particular, the preparation of hydrogel-like materials utilizing electrostatic interactions is a simple procedure in aqueous media at ambient temperature [8,9], and therefore the formation of polyion complex (PIC) provided positively and negatively charged particles and/or nano-gels has been used to form scaffolds by in situ aggregation [10–12]. However, control of the chemical and physical composition of self-assembling hydrogels was limited, because both cationic and anionic polymers or highly charged particles are needed for the formation of the PIC-driven hydrogel-like materials, and the gelation of oppositely charged polymer or particles occurs abruptly when they are mixed. On the other hand, if adhesion
∗ Corresponding author. Tel.: +81 3 5876 1415; fax: +81 3 5876 1639. E-mail address: [email protected] (A. Kikuchi). 1 Present address: Advanced Research Institute for Natural Science and Technology, Osaka City University, 3-3-138 Sugimoto Sumiyoshi-ku, Osaka-shi 558-8585, Japan. http://dx.doi.org/10.1016/j.colsurfb.2014.10.014 0927-7765/© 2014 Elsevier B.V. All rights reserved.
can be controlled, three-dimensional (3D) hydrogel constructs can be created. Therefore, adhesion of oppositely charged hydrogels via electrostatic interaction has been reported [13]. For hydrogel with the same cationic charge, adhesion using anionic polymer chains as binders has been reported [14,15]. There are adhesions based on surface modification of hydrogels with a polymer chain. Two cationic gels adhered to each other using anionic polymers, which changed the surface charge of the cationic gel from positive to negative. However, the spontaneous adhesion of hydrogels does not allow for the control of the configuration, shape, elasticity, or handling of hydrogels; therefore, a stimuli-responsive adhesion system is required for the preparation of 3D hydrogel constructs. Recently, we developed the electrophoretic adhesion of oppositely charged hydrogels [16–19]. The cationic and anionic hydrogels in contact adhered when a direct-current (DC) electric field was applied with a cationic and anionic hydrogel at the anode and the cathode, respectively, although two hydrogels did not adhere when in contact with each other without the electric field. Because the positively and negatively charged segments inside the hydrogels moved to the cathode and anode, respectively, a PIC was formed at the hydrogel-interface for adhesion of the two hydrogels through electrophoresis. Therefore, hydrogel constructs composed of oppositely charged microgels were simply and rapidly fabricated via the electrophoretic adhesion of microgel each other [18]. Electrophoretic adhesion as a preparation method for hydrogel constructs has advantages as compared with spontaneous gelation systems, because electrophoretic adhesion is able to control not only adhesion, but also gelation by manipulating
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Fig. 1. (A) Schematic illustration of the experimental procedure for the AC electrophoretic adhesion of anionic gels. The cationic polymers and anionic networks are shown in red and blue, respectively. On the bottom is a magnified image of the hydrogel interfaces during AC electrophoresis of the cationic polymers. (B) Images of the adhered DS30 gels, twisted 180-degrees. The DS gels were stained by the cationic dye methylene blue (MB). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the electric field. However, it is difficult to adhere hydrogels with the same charge to each other, and thus hydrogel constructs composed of both cationic and anionic gels are similar to spontaneous gelation systems [8–12]. In this study, we reported on the adhesion of biodegradable hydrogels with the same charge using an alternating-current (AC) electric field and oppositely charged water-soluble polymers as binders (Fig. 1). The adhesive strength was investigated as a function of voltage, frequency, and period when a square wave was applied for adhesion. Thus, we demonstrated the preparation of biodegradable hydrogel constructs by the 3D adhesion of microgels. 2. Materials and methods 2.1. Materials All reagents were used as purchased without further purification. Type A gelatin, sodium dextran sulfate (DS) (MW 500,000), and iodomethane were purchased from Wako (Japan), and chitosan (low molecular weight) was purchased from Aldrich (USA). 1-Ethyl3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was purchased from Dojindo (Japan). 2.2. Preparation of hydrogels (DS gel) The gelatin-based hydrogels were prepared by the chemical cross-linking of gelatin type A. To prepare the anionic gels, 500 mg of sodium dextran sulfate (DS) was added to 10 mL of the gelatin solution (100 mg mL−1 ) to yield DS gels. The aqueous pre-gel solution was injected into two glass plates sandwiched with 1-mm spacer, and then cured at 4 ◦ C for 1 h. The cured DS gels were immersed into 30 or 50 mM EDC solutions for chemical crosslinking. The swelling ratios (SRs) of the hydrogels were calculated from the ratio of the weight of the swelled gels (Ws ) to the weight of the dry gels (Wd ): SR = (Ws − Wd )/Wd . 2.3. Preparation of quaternary ammonium chitosan (N,N,N-trimethyl chitosan chloride: TMC) TMC with an 18% degree of substitution was synthesized according to the literature [20]. Briefly chitosan was reacted
with iodomethane in a 42-wt% aqueous ethanol solution at 50 ◦ C for 24 h. The product was isolated by acetone precipitation and subsequent centrifugation. The products were dissolved in NaCl containing aqueous solutions, re-precipitated using acetone, and then dissolved in water for the exchange of the counter ion iodide with chloride. The product was obtained as a yellow powder by lyophilization after the aqueous solution was dialyzed using a Spectra/Por regenerated cellulose membrane (molecular weight cut-off of 3500) against distilled water. The TMC was then characterized by 1 H NMR, and the quaternarization degree of TMC was found to be 18%.
2.4. Preparation of microgels Microgels were prepared based on a previous study [18]. To prepare the DS microgels, 500 mg of DS was added to 10 mL of the gelatin aqueous solution (100 mg mL−1 ), and to this viscous solution was added 40 mL paraffin oil with vigorous stirring at 45 ◦ C. Span 80 as a stabilizer was then added into the water/oil emulsion. The aqueous droplets were cured during this reverse suspension at 4 ◦ C for 30 min while stirring, and the paraffin oil was removed by washing with hexane. The cured DS microgels were immersed in 40 mL of the 30 mM EDC solutions for 24 h. The microgels were collected by lyophilization after washing with water, and then microgels with diameters from 100 to 125 m were collected using a sieving mesh.
2.5. Adhesion of DS gels by AC electric field impression The two DS gels with the same charge adhered to each other by AC electrophoretic adhesion. Cationic TMC solution was dropped onto one anionic DS gel surface, followed by placement of the other DS gels. Two DS gels with TMC were held between the two Pt electrodes, and then an AC electric field with a square wave was applied for adhesion of the hydrogels. The adhesive strength of the adhered DS gels was measured using a tensile tester as the lap shear adhesion force in ambient atmosphere [17]. The adhered DS gels were held at both ends of the supporting plates with two mechanical chucks. The failure strain was measured by a loading to failure in tensile mode at (1.0 mm s−1 ).
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Fig. 2. Chemical structure of (A) TMC and (B) DS.
3. Results and discussion 3.1. Electrophoretic adhesion of DS gels using AC voltage DS gels with 1 mm thickness were prepared by chemical crosslinking of gelatin type A with sodium dextran sulfate (DS). The gelatin was cured gels at 4 ◦ C for 1 h and then immersed in 30 and 50 mmol/L EDC solutions to form amide bonds as chemical cross-linking points between the primary amino and carboxyl groups of gelatin. These gels were designated as DS30 and DS50 gels, respectively. The swelling ratio (SR) and tensile strength (TS) were modulated by the EDC concentration. The TS and SR values were SR = 35.1 ± 3.0 and TS = 14.6 ± 1.0 kPa for the DS30 , and SR = 13.0 ± 1.2 and TS = 33.2 ± 6.2 kPa for the DS50 gels. A lower SR and higher TS was obtained by increasing the EDC concentration from 30 to 50 mmol/L, indicating increasing amide bond formation as cross-linking points [17]. For the adhesion experiment, we used DS gels and 18% quaternized TMC. Chemical structures of DS and TMC are shown in Fig. 2. At first, 5 L (2 mg mL−1 ) of TMC solution was dropped onto one DS gel surface, followed by placement of the other DS gels. The gels were cut in 5 mm × 15 mm strip specimens, and the adhesion area was 2 mm × 5 mm. Two DS gels did not adhere by pressure bonding in the present study, although adhesion of polyelectrolyte hydrogels utilizing oppositely charged polyelectrolyte as binders has been reported [14,15]. When the TMC in the solution contacted the DS gel, cationic TMC seemed to be adsorbed onto the anionic DS gel surfaces. Adhesion did not occur because the outermost surface of both DS gels became positively charged in the present study. Interestingly, they adhered when the AC
electric field was applied with a square wave (Fig. 1A). Adhesion of the two DS gels utilizing TMC binders was not observed when the DC electric field was applied. Therefore, the cationic TMC oscillated at the two anionic DS gel interface and then the polyion complex was formed at the interface of the two gels for hydrogel adhesion during AC electrophoresis of TMC. Gelatin gels without DS did not adhere using TMC and AC voltage, because polyion complex did not form due to the absence of anionic polymers. As shown in Fig. 1, adhered DS30 gels withstood stress of a 180◦ twist without detachment, indicating the formation of a strong and soft adhesive interface. 3.2. Adhesive strength of the DS gels A part of adhered DS30 gels (the adhesion area was 2 mm × 5 mm and the thickness of the gels was 1.0 mm) fractured during a tensile strength test while the adhesion interface remained intact. It is difficult to measure the adhesive strength of DS30 gels with reproducible results in this condition. Therefore, stronger DS50 gels with lower SRs (SR = 13.0 ± 1.2, TS = 33.2 ± 6.2 kPa) were used for measurement of the adhesive strength using the tensile test (Fig. 3A). The adhesive strength when using DS50 gels is shown in Fig. 3B. In the case of a 3.0 V mm−1 application (0.5 Hz, a square wave), the two DS50 gels did not adhere to each other (Fig. 3B; circle). On the other hand, adhesion was observed when 5.0 V mm−1 was applied (Fig. 3B; square), because the polymer mobility was proportionate to the applied voltage. These results were in good agreement with a previous study using DC voltage [17]. Interestingly, the adhesive strength of the gels increased with an increasing period
Fig. 3. (A) The experimental procedure for the lap shear tests on the adhered DS50 gels. (B) The adhesive strength of the adhered DS50 /DS50 gels as a function of the periodicity when a square wave with constant frequency (0.5 Hz) was applied between the two DS50 gels. (n = 5). The applied voltages were 3.0 (circle), 4.0 (triangle), and 5.0 (square) V mm−1 . The data are expressed as the mean ± standard deviation (SD) of 5 samples.
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Fig. 5. The oscillation mobility of TMC when a square wave was applied at the gel interface.
Fig. 4. (a) Adhesive strength of the DS50 /DS50 gels as a function of the frequency (n = 5). The applied voltage was 4.0 V mm−1 , and the periods were 5 (circle), 10 (triangle), and 20 (square). The data are expressed as the mean ± standard deviation (SD) of 5 samples.
(the period is the time required for one cycle of the waveform) when AC voltage was applied at 4.0 V mm−1 (Fig. 3B; triangle). In a previous study of electrophoretic adhesion using DC voltage and weak polyelectrolyte (e.g. primary amine salt and carboxylate)
hydrogels, the detachment of adhered hydrogels was observed when an inverse voltage was applied to the adhered hydrogels, because of the disintegration of the PIC due to the movement of the cationic and anionic polymers from the interface of the gels to the cathode and the anode, respectively [19]. Therefore, it is difficult to use an AC electric field for adhesion of hydrogels with the same charge because adhesive strength decreased due to intermediate binders overshot the gel-interface when electrophoresis time was increased [17]. However, in this study, we used a combination of strong polyelectrolyte (quaternary ammonium salt and sulfonate) which were stable in inverse voltage [18]. Therefore, the adhesive strength of the gels increased with an increasing period, indicating that the oscillated TMC interpenetrated quickly and stepwise at the interface of the DS gels during AC electrophoresis, and then PIC was formed for the adhesion of the DS gels. Next, we investigated adhesive strength as a function of frequency when the applied voltage was constant (4.0 V mm−1 ). As
Fig. 6. (A) Preparation of hydrogel constructs composed of DS30 microgels with TMC by AC voltage impression. The applied voltage and frequency were 5.0 V mm−1 and 0.25 Hz, respectively, and the electrophoresis time was 40 s in each electric field direction (x, y and z). (B) Macro and (C) microscopic images of the hydrogel constructs prepared by DS30 microgel adhesion. The DS30 microgels were stained by methylene blue.
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shown in Fig. 4, the adhesive strength decreased with increasing frequency, and adhesion of the hydrogels was not observed for 5 periods at frequency of 0.5 Hz (Fig. 4; circle). However, the adhesive strength increased with increasing periodicity; the adhesion strength was 5.57 ± 0.93 kPa for 10 cycles at 0.5 Hz (Fig. 4; triangle). When the frequency was 0.6 Hz, the hydrogels adhered to each other for 20 periods (Fig. 4; square), although adhesion was not observed for 10 periods (Fig. 4; triangle). These results indicate that the oscillation of the cationic polymers in response to an AC electric field was important for the adhesion of the hydrogels. It is well known that the migration length of the polymers depends on the applied voltage and/or electrophoresis time [21]. The polyion complex seemed to be formed between the hydrogel interface depending on the oscillation of TMC. The oscillations of TMC were lower when the frequency was higher and higher when the frequency was lower, at a constant electrophoresis periods (Fig. 5). When the frequency was lower (0.1–0.4 Hz), the hydrogel adhered early because of the large oscillations of TMC. On the other hand, adhesion of the hydrogels was observed when the cycle number was increased from 5 to 10 and/or 20 periods, although the hydrogel did not adhere when the frequency was higher (0.5–0.7 Hz). Therefore, the stepwise formation of the polyion complex with TMC oscillation at the interface of the two hydrogels was the driving force for the adhesion of two anionic hydrogels utilizing TMC as the binder. 3.3. Preparation of hydrogel constructs by microgels adhesion We also prepared hydrogel constructs by the adhesion of microgels. Anionic microgel powders (DS30 microgels) were swelled in water, and then cationic TMC droplets were added into the swelled microgels. Gelation was not observed before electrophoresis, which is important as spontaneous gelation can create unintended hydrogel shapes. After moulding a square shape, AC voltage was applied to a paste composed of DS30 microgels and TMC. The microgels adhered to each other and formed an elastic gel-like materials by AC electric field application on the x, y, and z axes as shown in Fig. 6A. Hydrogel constructs prepared by the adhesion of microgel were stable and with no microgel leakage into the water observed after the electric field was removed. The constructs wee also durable enough to be handled with forceps (Fig. 6B). Through the microscopic observations of the hydrogel constructs, we confirmed that the microgels had adhered to each other (Fig. 6C). Microgels formed 3D networks through the adhesion of the microgels by AC electrophoresis of TMC at the microgel interfaces.
4. Conclusions In conclusion, we report the AC electrophoretic adhesion of hydrogels with the same charge utilizing oppositely charged watersoluble polymers as a binder for building 3D hydrogel constructs. The two DS gels were adhered by AC electric field application when TMC was put between and held in contact with the two DS gels. When a square wave was applied, the adhesive strength of the gels increased with increasing periodicity. Hydrogel constructs composed of DS microgels were prepared by the AC electrophoretic adhesion of microgels utilizing intermediate TMC. The AC electrophoretic adhesion of biodegradable hydrogels will be strong tool for the fabrication of hydrogel-based 3D materials in the field of tissue engineering. Acknowledgment This study was supported by the Foundation for the Promotion of Ion Engineering and a Grant-in-Aid for Young Scientists (B) No. 22700497 from the Japan Society for the Promotion of Science. References [1] K.Y. Lee, D.J. Mooney, Chem. Rev. 101 (2001) 1869–1879. [2] T. Vermonden, R. Censi, W.E. Hennink, Chem. Rev. 112 (2012) 2853–2888. [3] F. van de Manakker, K. Braeckmans, N.E. Morabit, S.C. De Smedt, C.F. van Nostrum, W.E. Hennink, Adv. Funct. Mater. 19 (2009) 2992–3001. [4] H. Tsuji, F. Horii, S.H. Hyon, Y. Ikada, Macromolecules 24 (1991) 2719–2724. [5] C. Wang, R.J. Stewart, J. Kopeˇcek, Nature 397 (1999) 417–420. [6] B. Jeong, Y.H. Bae, D.S. Lee, S.W. Kim, Nature 388 (1997) 860–862. [7] N. Holten-Andersen, M.J. Harrington, H. Birkedal, B.P. Lee, P.B. Messersmith, K.Y.C. Lee, J.H. Waite, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 2651–2655. [8] M. Lemmers, J. Sprakel, J.K. Voets, J. van der Gucht, M.A. Cohen Stuart, Angew. Chem. Int. Ed. 49 (2010) 708–711. [9] J.N. Hunt, K.E. Feldman, N.A. Lynd, J. Deek, L.M. Campos, J.M. Spruell, B.M. Hernandez, E.J. Kramer, C.J. Hawker, Adv. Mater. 23 (2011) 2327–2331. [10] S.R. van Tomme, M.J. van Steenbergen, S.C. de Smedt, C.F. van Nostrum, W.E. Hennink, Biomaterials 26 (2005) 2129–2135. [11] Q. Wang, L. Wang, M.S. Detamore, C. Berkland, Adv. Mater. 20 (2008) 236–239. [12] H. Wang, M.B. Hansen, D.W.P.M. Löwik, J.C.M. van Hest, Y. Li, J.A. Jansen, S.C.G. Leeuwenburgh, Adv. Mater. 23 (2011) H119–H124. [13] H. Tamagawa, F. Nagato, S. Umemoto, N. Okui, S. Popovic, M. Taya, Bull. Chem. Soc. Jpn. 75 (2002) 383. [14] H. Tamagawa, Y. Takahashi, Mater. Chem. Phys. 107 (2008) 164–170. [15] H. Abe, Y. Hara, S. Maeda, S. Hashimoto, Chem. Lett. 43 (2014) 243–245. [16] T. Asoh, A. Kikuchi, Chem. Commun. 46 (2010) 7793–7795. [17] T. Asoh, W. Kawai, A. Kikuchi, Soft Matter 8 (2012) 1923–1927. [18] T. Asoh, A. Kikuchi, Chem. Commun. 48 (2012) 10019–10021. [19] T. Asoh, E. Kawamura, A. Kikuchi, RSC Adv. 3 (2013) 7947–7952. [20] T. Uragami, T. Katayama, T. Miyata, H. Tamura, T. Shiraiwa, A. Higuchi, Biomacromolecules 5 (2004) 1567–1574. [21] D.L. Smisek, D.A. Hoagland, Science 248 (1990) 1221–1223.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Alternating-current electrophoretic adhesion of biodegradable hydrogel utilizing intermediate polymers Taka-Aki Asoh 1 , Wataru Kawai, Akihiko Kikuchi ∗ Department of Materials Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan
a r t i c l e
i n f o
Article history: Received 20 August 2014 Received in revised form 21 September 2014 Accepted 8 October 2014 Available online 18 October 2014 Keywords: Hydrogel Adhesion Electrophoresis Scaffold
a b s t r a c t The adhesion of anionic charged biodegradable hydrogels each other utilizing oppositely charged watersoluble polymers as a binder has been achieved by applying alternating-current (AC) electric fields. The two gelatin based dextran sulfate gels (DS gels) were molecularly sutured together by AC electrophoretic adhesion when cationic charged quaternary ammonium chitosan (TMC) was applied between and held in contact with the two DS gels. The adhesive strength of the gels increased with increasing periodicity when a square wave was applied. Hydrogel constructs composed of DS microgels were prepared simply by AC electrophoretic adhesion utilizing intermediate TMC. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hydrogels are similar to the macromolecular-based constituents in the living body such as proteins, cells, and tissue, and therefore hydrogels have numerous applications in tissue engineering [1] and drug delivery systems [2]. For in situ hydrogel formation, several interaction as cross-linking points of the hydrogels have been used such as inclusion complexes [3], stereocomplexation [4], peptide interactions [5], hydrophobic interactions [6], and supramolecular complexes [7]. In particular, the preparation of hydrogel-like materials utilizing electrostatic interactions is a simple procedure in aqueous media at ambient temperature [8,9], and therefore the formation of polyion complex (PIC) provided positively and negatively charged particles and/or nano-gels has been used to form scaffolds by in situ aggregation [10–12]. However, control of the chemical and physical composition of self-assembling hydrogels was limited, because both cationic and anionic polymers or highly charged particles are needed for the formation of the PIC-driven hydrogel-like materials, and the gelation of oppositely charged polymer or particles occurs abruptly when they are mixed. On the other hand, if adhesion
∗ Corresponding author. Tel.: +81 3 5876 1415; fax: +81 3 5876 1639. E-mail address: [email protected] (A. Kikuchi). 1 Present address: Advanced Research Institute for Natural Science and Technology, Osaka City University, 3-3-138 Sugimoto Sumiyoshi-ku, Osaka-shi 558-8585, Japan. http://dx.doi.org/10.1016/j.colsurfb.2014.10.014 0927-7765/© 2014 Elsevier B.V. All rights reserved.
can be controlled, three-dimensional (3D) hydrogel constructs can be created. Therefore, adhesion of oppositely charged hydrogels via electrostatic interaction has been reported [13]. For hydrogel with the same cationic charge, adhesion using anionic polymer chains as binders has been reported [14,15]. There are adhesions based on surface modification of hydrogels with a polymer chain. Two cationic gels adhered to each other using anionic polymers, which changed the surface charge of the cationic gel from positive to negative. However, the spontaneous adhesion of hydrogels does not allow for the control of the configuration, shape, elasticity, or handling of hydrogels; therefore, a stimuli-responsive adhesion system is required for the preparation of 3D hydrogel constructs. Recently, we developed the electrophoretic adhesion of oppositely charged hydrogels [16–19]. The cationic and anionic hydrogels in contact adhered when a direct-current (DC) electric field was applied with a cationic and anionic hydrogel at the anode and the cathode, respectively, although two hydrogels did not adhere when in contact with each other without the electric field. Because the positively and negatively charged segments inside the hydrogels moved to the cathode and anode, respectively, a PIC was formed at the hydrogel-interface for adhesion of the two hydrogels through electrophoresis. Therefore, hydrogel constructs composed of oppositely charged microgels were simply and rapidly fabricated via the electrophoretic adhesion of microgel each other [18]. Electrophoretic adhesion as a preparation method for hydrogel constructs has advantages as compared with spontaneous gelation systems, because electrophoretic adhesion is able to control not only adhesion, but also gelation by manipulating
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Fig. 1. (A) Schematic illustration of the experimental procedure for the AC electrophoretic adhesion of anionic gels. The cationic polymers and anionic networks are shown in red and blue, respectively. On the bottom is a magnified image of the hydrogel interfaces during AC electrophoresis of the cationic polymers. (B) Images of the adhered DS30 gels, twisted 180-degrees. The DS gels were stained by the cationic dye methylene blue (MB). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the electric field. However, it is difficult to adhere hydrogels with the same charge to each other, and thus hydrogel constructs composed of both cationic and anionic gels are similar to spontaneous gelation systems [8–12]. In this study, we reported on the adhesion of biodegradable hydrogels with the same charge using an alternating-current (AC) electric field and oppositely charged water-soluble polymers as binders (Fig. 1). The adhesive strength was investigated as a function of voltage, frequency, and period when a square wave was applied for adhesion. Thus, we demonstrated the preparation of biodegradable hydrogel constructs by the 3D adhesion of microgels. 2. Materials and methods 2.1. Materials All reagents were used as purchased without further purification. Type A gelatin, sodium dextran sulfate (DS) (MW 500,000), and iodomethane were purchased from Wako (Japan), and chitosan (low molecular weight) was purchased from Aldrich (USA). 1-Ethyl3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was purchased from Dojindo (Japan). 2.2. Preparation of hydrogels (DS gel) The gelatin-based hydrogels were prepared by the chemical cross-linking of gelatin type A. To prepare the anionic gels, 500 mg of sodium dextran sulfate (DS) was added to 10 mL of the gelatin solution (100 mg mL−1 ) to yield DS gels. The aqueous pre-gel solution was injected into two glass plates sandwiched with 1-mm spacer, and then cured at 4 ◦ C for 1 h. The cured DS gels were immersed into 30 or 50 mM EDC solutions for chemical crosslinking. The swelling ratios (SRs) of the hydrogels were calculated from the ratio of the weight of the swelled gels (Ws ) to the weight of the dry gels (Wd ): SR = (Ws − Wd )/Wd . 2.3. Preparation of quaternary ammonium chitosan (N,N,N-trimethyl chitosan chloride: TMC) TMC with an 18% degree of substitution was synthesized according to the literature [20]. Briefly chitosan was reacted
with iodomethane in a 42-wt% aqueous ethanol solution at 50 ◦ C for 24 h. The product was isolated by acetone precipitation and subsequent centrifugation. The products were dissolved in NaCl containing aqueous solutions, re-precipitated using acetone, and then dissolved in water for the exchange of the counter ion iodide with chloride. The product was obtained as a yellow powder by lyophilization after the aqueous solution was dialyzed using a Spectra/Por regenerated cellulose membrane (molecular weight cut-off of 3500) against distilled water. The TMC was then characterized by 1 H NMR, and the quaternarization degree of TMC was found to be 18%.
2.4. Preparation of microgels Microgels were prepared based on a previous study [18]. To prepare the DS microgels, 500 mg of DS was added to 10 mL of the gelatin aqueous solution (100 mg mL−1 ), and to this viscous solution was added 40 mL paraffin oil with vigorous stirring at 45 ◦ C. Span 80 as a stabilizer was then added into the water/oil emulsion. The aqueous droplets were cured during this reverse suspension at 4 ◦ C for 30 min while stirring, and the paraffin oil was removed by washing with hexane. The cured DS microgels were immersed in 40 mL of the 30 mM EDC solutions for 24 h. The microgels were collected by lyophilization after washing with water, and then microgels with diameters from 100 to 125 m were collected using a sieving mesh.
2.5. Adhesion of DS gels by AC electric field impression The two DS gels with the same charge adhered to each other by AC electrophoretic adhesion. Cationic TMC solution was dropped onto one anionic DS gel surface, followed by placement of the other DS gels. Two DS gels with TMC were held between the two Pt electrodes, and then an AC electric field with a square wave was applied for adhesion of the hydrogels. The adhesive strength of the adhered DS gels was measured using a tensile tester as the lap shear adhesion force in ambient atmosphere [17]. The adhered DS gels were held at both ends of the supporting plates with two mechanical chucks. The failure strain was measured by a loading to failure in tensile mode at (1.0 mm s−1 ).
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Fig. 2. Chemical structure of (A) TMC and (B) DS.
3. Results and discussion 3.1. Electrophoretic adhesion of DS gels using AC voltage DS gels with 1 mm thickness were prepared by chemical crosslinking of gelatin type A with sodium dextran sulfate (DS). The gelatin was cured gels at 4 ◦ C for 1 h and then immersed in 30 and 50 mmol/L EDC solutions to form amide bonds as chemical cross-linking points between the primary amino and carboxyl groups of gelatin. These gels were designated as DS30 and DS50 gels, respectively. The swelling ratio (SR) and tensile strength (TS) were modulated by the EDC concentration. The TS and SR values were SR = 35.1 ± 3.0 and TS = 14.6 ± 1.0 kPa for the DS30 , and SR = 13.0 ± 1.2 and TS = 33.2 ± 6.2 kPa for the DS50 gels. A lower SR and higher TS was obtained by increasing the EDC concentration from 30 to 50 mmol/L, indicating increasing amide bond formation as cross-linking points [17]. For the adhesion experiment, we used DS gels and 18% quaternized TMC. Chemical structures of DS and TMC are shown in Fig. 2. At first, 5 L (2 mg mL−1 ) of TMC solution was dropped onto one DS gel surface, followed by placement of the other DS gels. The gels were cut in 5 mm × 15 mm strip specimens, and the adhesion area was 2 mm × 5 mm. Two DS gels did not adhere by pressure bonding in the present study, although adhesion of polyelectrolyte hydrogels utilizing oppositely charged polyelectrolyte as binders has been reported [14,15]. When the TMC in the solution contacted the DS gel, cationic TMC seemed to be adsorbed onto the anionic DS gel surfaces. Adhesion did not occur because the outermost surface of both DS gels became positively charged in the present study. Interestingly, they adhered when the AC
electric field was applied with a square wave (Fig. 1A). Adhesion of the two DS gels utilizing TMC binders was not observed when the DC electric field was applied. Therefore, the cationic TMC oscillated at the two anionic DS gel interface and then the polyion complex was formed at the interface of the two gels for hydrogel adhesion during AC electrophoresis of TMC. Gelatin gels without DS did not adhere using TMC and AC voltage, because polyion complex did not form due to the absence of anionic polymers. As shown in Fig. 1, adhered DS30 gels withstood stress of a 180◦ twist without detachment, indicating the formation of a strong and soft adhesive interface. 3.2. Adhesive strength of the DS gels A part of adhered DS30 gels (the adhesion area was 2 mm × 5 mm and the thickness of the gels was 1.0 mm) fractured during a tensile strength test while the adhesion interface remained intact. It is difficult to measure the adhesive strength of DS30 gels with reproducible results in this condition. Therefore, stronger DS50 gels with lower SRs (SR = 13.0 ± 1.2, TS = 33.2 ± 6.2 kPa) were used for measurement of the adhesive strength using the tensile test (Fig. 3A). The adhesive strength when using DS50 gels is shown in Fig. 3B. In the case of a 3.0 V mm−1 application (0.5 Hz, a square wave), the two DS50 gels did not adhere to each other (Fig. 3B; circle). On the other hand, adhesion was observed when 5.0 V mm−1 was applied (Fig. 3B; square), because the polymer mobility was proportionate to the applied voltage. These results were in good agreement with a previous study using DC voltage [17]. Interestingly, the adhesive strength of the gels increased with an increasing period
Fig. 3. (A) The experimental procedure for the lap shear tests on the adhered DS50 gels. (B) The adhesive strength of the adhered DS50 /DS50 gels as a function of the periodicity when a square wave with constant frequency (0.5 Hz) was applied between the two DS50 gels. (n = 5). The applied voltages were 3.0 (circle), 4.0 (triangle), and 5.0 (square) V mm−1 . The data are expressed as the mean ± standard deviation (SD) of 5 samples.
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Fig. 5. The oscillation mobility of TMC when a square wave was applied at the gel interface.
Fig. 4. (a) Adhesive strength of the DS50 /DS50 gels as a function of the frequency (n = 5). The applied voltage was 4.0 V mm−1 , and the periods were 5 (circle), 10 (triangle), and 20 (square). The data are expressed as the mean ± standard deviation (SD) of 5 samples.
(the period is the time required for one cycle of the waveform) when AC voltage was applied at 4.0 V mm−1 (Fig. 3B; triangle). In a previous study of electrophoretic adhesion using DC voltage and weak polyelectrolyte (e.g. primary amine salt and carboxylate)
hydrogels, the detachment of adhered hydrogels was observed when an inverse voltage was applied to the adhered hydrogels, because of the disintegration of the PIC due to the movement of the cationic and anionic polymers from the interface of the gels to the cathode and the anode, respectively [19]. Therefore, it is difficult to use an AC electric field for adhesion of hydrogels with the same charge because adhesive strength decreased due to intermediate binders overshot the gel-interface when electrophoresis time was increased [17]. However, in this study, we used a combination of strong polyelectrolyte (quaternary ammonium salt and sulfonate) which were stable in inverse voltage [18]. Therefore, the adhesive strength of the gels increased with an increasing period, indicating that the oscillated TMC interpenetrated quickly and stepwise at the interface of the DS gels during AC electrophoresis, and then PIC was formed for the adhesion of the DS gels. Next, we investigated adhesive strength as a function of frequency when the applied voltage was constant (4.0 V mm−1 ). As
Fig. 6. (A) Preparation of hydrogel constructs composed of DS30 microgels with TMC by AC voltage impression. The applied voltage and frequency were 5.0 V mm−1 and 0.25 Hz, respectively, and the electrophoresis time was 40 s in each electric field direction (x, y and z). (B) Macro and (C) microscopic images of the hydrogel constructs prepared by DS30 microgel adhesion. The DS30 microgels were stained by methylene blue.
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shown in Fig. 4, the adhesive strength decreased with increasing frequency, and adhesion of the hydrogels was not observed for 5 periods at frequency of 0.5 Hz (Fig. 4; circle). However, the adhesive strength increased with increasing periodicity; the adhesion strength was 5.57 ± 0.93 kPa for 10 cycles at 0.5 Hz (Fig. 4; triangle). When the frequency was 0.6 Hz, the hydrogels adhered to each other for 20 periods (Fig. 4; square), although adhesion was not observed for 10 periods (Fig. 4; triangle). These results indicate that the oscillation of the cationic polymers in response to an AC electric field was important for the adhesion of the hydrogels. It is well known that the migration length of the polymers depends on the applied voltage and/or electrophoresis time [21]. The polyion complex seemed to be formed between the hydrogel interface depending on the oscillation of TMC. The oscillations of TMC were lower when the frequency was higher and higher when the frequency was lower, at a constant electrophoresis periods (Fig. 5). When the frequency was lower (0.1–0.4 Hz), the hydrogel adhered early because of the large oscillations of TMC. On the other hand, adhesion of the hydrogels was observed when the cycle number was increased from 5 to 10 and/or 20 periods, although the hydrogel did not adhere when the frequency was higher (0.5–0.7 Hz). Therefore, the stepwise formation of the polyion complex with TMC oscillation at the interface of the two hydrogels was the driving force for the adhesion of two anionic hydrogels utilizing TMC as the binder. 3.3. Preparation of hydrogel constructs by microgels adhesion We also prepared hydrogel constructs by the adhesion of microgels. Anionic microgel powders (DS30 microgels) were swelled in water, and then cationic TMC droplets were added into the swelled microgels. Gelation was not observed before electrophoresis, which is important as spontaneous gelation can create unintended hydrogel shapes. After moulding a square shape, AC voltage was applied to a paste composed of DS30 microgels and TMC. The microgels adhered to each other and formed an elastic gel-like materials by AC electric field application on the x, y, and z axes as shown in Fig. 6A. Hydrogel constructs prepared by the adhesion of microgel were stable and with no microgel leakage into the water observed after the electric field was removed. The constructs wee also durable enough to be handled with forceps (Fig. 6B). Through the microscopic observations of the hydrogel constructs, we confirmed that the microgels had adhered to each other (Fig. 6C). Microgels formed 3D networks through the adhesion of the microgels by AC electrophoresis of TMC at the microgel interfaces.
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