International Journal of Biological Macromolecules 72 (2015) 403–409
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Mechanically tough biomacromolecular IPN hydrogel fibers by enzymatic and ionic crosslinking Xin Hu, Lingling Lu, Chen Xu, Xinsong Li ∗ School of Chemistry and Chemical Engineering, Southeast University, Nanjing 210018, China
a r t i c l e
i n f o
Article history: Received 4 April 2014 Received in revised form 9 August 2014 Accepted 10 August 2014 Available online 1 September 2014 Keywords: IPN hydrogel fiber Enzymatic crosslinking Mechanical strength
a b s t r a c t In this report, biological macromolecular full IPN hydrogel fibers composed of gelatin and alginate with an interpenetrating network (IPN) structure were prepared by wet spinning using a combination of enzymatic and calcium ions crosslinking. In the full IPN hydrogel fibers, mTG catalyzed the formation of one network of gelatin while calcium ions crosslinked another network of alginate intertwining with the former. The mechanical strength of the full IPN hydrogel fibers was measured by an electronic single fiber strength tester. The results showed that gelatin–alginate full IPN hydrogel fibers had a significant improvement of mechanical strength over gelatin–alginate semi-IPN gel fibers crosslinked only by calcium ions. The full IPN fiber has the highest tension of 62 cN and elongation of 739%, which are much higher than those of alginate hydrogel. Furthermore, biological evaluation indicated that gelatin–alginate full IPN hydrogel fibers enhance cell adhesion and proliferation significantly, illustrating the cyto-compatibility. A preliminary trial of hand weaving showed the knittablity of the mechanically tough full IPN hydrogel fibers. Because of their both excellent biocompatibility and mechanical strength, the biological macromolecular hydrogel fibers with full IPN structure may be desirable candidates for engineering tissue scaffolds. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hydrogel scaffolds have attracted much attention in the past decade in the field of tissue engineering scaffolds [1–3]. Their highly hydrated polymer networks may allow cells to adhere, proliferate and differentiate for the treatment of injured tissues and organs. However, poor mechanical properties of hydrogels limit their applications [4]. Fiber reinforcement within hydrogels is a promising approach for engineering the mechanical and physical properties of scaffolds [5]. Fibers can be arranged from monofilaments or multifilament yarns and made into textile substrates or superstructures that include woven, knit, braided, continuous randomly aligned (2D or 3D), staple fiber randomly aligned. They can be designed to have an extremely high porosity [6] for cellular infiltration, and exhibit good mechanical properties and suitable compliance for handling and conformability to varying tissue and organ shapes [7,8]. Apparently, both of biocompatible fibers and impregnated hydrogels or matrices are highly needed in order to fabricate fiber reinforced hydrogel scaffolds.
∗ Corresponding author. Tel.: +86 2583793456. E-mail address: [email protected] (X. Li). http://dx.doi.org/10.1016/j.ijbiomac.2014.08.043 0141-8130/© 2014 Elsevier B.V. All rights reserved.
Hydrogel fibers may be valuable candidates as building blocks for constructing 3D scaffolds [9]. Lin et al. [10] developed a calciumcrosslinked alginate fibrous scaffold with interconnected pores using a three-dimensional plotting system. Hu et al. [11] investigated the hydrodynamic spinning of hydrogel fibers by in situ cross-linking. They tried several biopolymer and synthetic polymer systems, but did not provide mechanical properties of the obtained hydrogels fibers in detail. Up to now, the development of hydrogel fibers with both excellent biocompatibility and mechanical strength remains a challenge. Biological macromolecular hydrogels composed of natural proteins or polysaccharides are widely applied in the development of multitude of tissue engineering scaffolds due to hydrophilicity, biocompatibility and biodegradability [12]. Unfortunately, most biomacromolecular hydrogels suffer from the lack of mechanical strength, and they are difficult to be handled to engineer 3D scaffolds with distinct geometries and physicochemical properties [13]. Chemical crosslinking is a common method to improve the mechanical properties of biomacromolecular hydrogels, the residues of crosslinkers such as glutaraldehyde, carbodiimide, and diphenylphosphoryl azide may bring cytotoxic side-effects even after thorough purification [14,15]. Therefore, hydrogels derived from biological macromolecular proteins and polysaccharides
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Table 1 Composition of gelatin (G)–alginate (A) bio-macromolecular hydrogel fibers. Sample
Gelatin (mg/mL)
0 A1 Semi-IPN 50 G5A1 G5A1.5 50 50 G5A2 G7.5A1 75 100 G10A1 IPN 50 G5A1 G5A1.5 50 50 G5A2 75 G7.5A1 100 G10A1
Alginate (mg/mL)
mTG (20 U/mL)
Ca2+ (1%)
10
–
Immerse
10 15 20 10 10
– – – – –
Immerse Immerse Immerse Immerse Immerse
10 15 20 10 10
Immerse Immerse Immerse Immerse Immerse
Immerse Immerse Immerse Immerse Immerse
prepared using biocompatible approaches with good mechanical properties are in high demand [16,17]. Utilizing two or more biomacromolecular proteins and polysaccharides to form an interpenetrating polymer network (IPN) hydrogel fibers by biocompatible approaches may enhance mechanical properties of the biopolymer hydrogels because of their highly entangled networks [18–20]. In this report, we present the preparation of gelatin–alginate hydrogel fibers with IPN structure using a combination of microbial transglutaminase (mTG) enzymatic and calcium ionic crosslinking [21]. The IPN hydrogel fibers were further characterized in terms of mechanical property, equilibrium water content, in vitro degradation and cytocompatibility. The results showed that the biomacromolecular hydrogel fibers prepared by biocompatible approaches may be applied in the development of tissue engineering scaffolds because of the combined biocompatibility and mechanical strength. 2. Materials and methods 2.1. Materials Sodium alginate (Laboratory Reagent Grade) was obtained from Huanghai Chemical Co. (Shandong, China), viscosity of 1% (w/v) solution in water is 94 mPa s. Gelatin (Type A, bloom strength at 11.1% moisture content is 250 g) was purchased from Aladdin Chemical Co., Ltd. (Shanghai, China). Anhydrous CaCl2 , dimethyl sulfoxide (DMSO) and papain (6000 U/mg) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Microbial transglutaminase (mTG, 1000 U/g) was a gift from Yiming Biological Products Co., Ltd. (Taizhou, China). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin, l-glutamine, trypsin and 3-(4,5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Invitrogen Co. (Carlsbad, CA). L929 mouse fibroblasts cells were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). 2.2. Preparation of IPN hydrogel fibers The gelatin (G)–alginate (A) IPN biohydrogel fibers were prepared with the composite as shown in Table 1 by wet spinning as illustrated in Fig. 1. Briefly, gelatin and alginate polymers were dissolved in distilled water and mixed, and a series of gelatin–alginate solutions with the concentration as showed in Table 1 were obtained. To prepare IPN hydrogel fibers, dope of gelatin–alginate solution was spun at a speed of 15 mL/h into aqueous coagulant bath with the concentration of CaCl2 1% and mTG 20 U/mL through a stainless steel spinneret with the diameter of 0.9 mm, and then the fibers were kept in oven at 55 ◦ C for 30 min to complete the enzymatically crosslinking. As a comparison, dope of polymer solution
was spun into aqueous coagulant bath with the concentration of CaCl2 1% in the absence of mTG, and semi interpenetrated network (semi-IPN) hydrogel fibers were prepared. Both semi-IPN and IPN hydrogel fibers were rinsed thoroughly with distilled water, and the diameter of these fibers was measured by a micrometer caliper, which is in the range of 1.0 ± 0.1 mm. 2.3. Equilibrium water content Gelatin-alginate IPN hydrogel fibers were incubated in distilled water at room temperature for 24 h to reach its equilibrium state. Then the weight (Ws ) of each hydrogel fiber at swollen state was measured after excess surface water was removed. After the sample was lyophilized, the weight (Wd ) of each sample at dried state was weighed. The equilibrium water content ratio was calculated using the following equation: Water content ratio =
Ws − Wd Ws
All the samples were measured in triplicates for each group. 2.4. Mechanical test Mechanical test for each gelatin–alginate hydrogel fiber was performed using an electronic single fiber strength tester (LLY-06B, Jinan, China) at room temperature. Hydrogel fiber with the length of 8 cm was subjected to tensile test at a strain rate of 20 mm/min. All samples were measured in triplicates. 2.5. In vitro enzymatic degradation To test enzymatic degradation of the IPN fibers, the samples were lyophilized and weighed the initial dry weight (W0 ), then immersed in PBS (0.01 M) containing papain (0.05 mg/ml) at 37 ◦ C under constant shaking at 80 rpm for 1, 3, 5, 7, 9 h. The fibers were washed carefully with deionized water and then freeze dried and weighed again (Wt ) at a predetermined time. The fractional mass remaining after in vitro degradation was calculated by the dry weight (Wt ) divided by the initial weight of the gel (W0 ) as follows:
Fractional mass remaining (%) =
Wt × 100% W0
Each group was tested in triplicates. 2.6. In vitro cytotoxicity The cytotoxicity of the gelatin–alginate IPN hydrogel fibers was evaluated by MTT assay. The fibers were steam sterilized at 121 ◦ C for 20 min, then immersed in DMEM solution at an extraction ratio of 0.75 cm2 /mL at 37 ◦ C for 24 h. The extract DMEM solutions were collected for MTT assay. Mouse fibroblast cells (L929) were allowed to seed at a density of 104 cells/well in 96-well plates and incubated in 100 L DMEM medium containing 10% FBS for 24 h in a humidified incubator (37 ◦ C, 5% CO2 ). Then the medium was replaced with 180 L DMEM medium (10% FBS in DMEM) and 20 L extract solution. After cultured for 1 day, 3 days and 5 days, 20 L of MTT solution (5 mg/mL in PBS) was added into each well for 4 h to allow formation of formazan crystal. Subsequently, supernatant was removed carefully and 150 L DMSO was added to each well. UV Absorbance was measured at 490 nm using the Bio-Rad Model 680 microplate reader (Philadelphia, PA). The cell viability was calculated as a percentage relative to the data obtained with blank control. All samples were tested for sextuplicates.
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Fig. 1. Schematic illustration of wet-spinning process: (1) metering pump; (2) spinning solution; (3) spinneret; (4) coagulation bath; (5, 6) take-up roller.
2.7. Cell seeding and imaging The gelatin–alginate IPN hydrogel fibers were incubated in DMEM containing 10% FBS at 37 ◦ C for 2 h. L929 cells were seeded onto the hydrogel scaffolds with a cell density of 104 /cm–2 and incubated in a humidified atmosphere (37 ◦ C, 5% CO2 ) for 5 days. Cell morphology was observed by microscope. After 5 days, the fibers were fixed with 2.5% glutaraldehyde solution at room temperature for 2 h, then rinsed three times with PBS and three times with deionized water. Subsequently, the samples were freeze-dried, and the cells morphology seeded on the fibers were observed under a scanning electron microscope (JSM6360LV, JEOL, Japan) after gold sputter-coating.
3. Results and discussion Gelatin-alginate IPN hydrogel fibers were prepared with the composite as shown in Table 1 by wet spinning (Fig. 1) using a mixture of calcium chloride and microbial transglutaminase (mTG) solution as a coagulantion bath. Transglutaminase is enzyme that catalyzes amide bond formation between glutamine and lysine side chains, with the loss of ammonia. So, gelatin can be crosslinked by the formation of covalent N--(␥-glutamyl) lysine amide bonds between individual gelatin molecules to form a permanent network catalyzed with mTG [22], while in the presence of calcium ions alginate G-blocks participate in gelation to form an “egg box”shaped structure network. As reported in our previous work [21], both gelatin and alginate formed crosslinked double networks in the presence of both mTG and calcium ions and hence, the obtained hydrogels can be considered as full IPN hydrogel fibers. When wet spinning of gelatin–alginate solution was conducted using a calcium chloride solution as a coagulation bath in the absence of mTG. Gelatin remained in its sol state in the obtained fibers. Therefore, the obtained hydrogels, in which alginate formed a crosslinked network in the presence of calcium ions while gelatin was entrapped within the alginate gel network, were considered as semi IPN hydrogels as shown in Scheme 1. Using semi IPN gel fibers as controls, the physicochemical and biological properties of
the gelatin–alginate IPN hydrogel fibers were investigated in detail as follows.
3.1. Equilibrium water content The equilibrium water content of the IPN hydrogel fibers was measured by gravimetric method as showed in Fig. 2. It was found that all of gelatin–alginate IPN hydrogel fibers and semi IPN hydrogel fibers have equilibrium water contents in the range from 85% to 95%, and the former has a lower water content than that of the latter. The reason is that uncrosslinked gelatin was entrapped into alginate network as in semi IPN case, resulting in a relatively loose structure which holds more water. Besides, gelatin–alginate IPN hydrogel fibers have a significant decreasing of water content with the increase of alginate content. It may be attributed to the increase of crosslinking degree and the formation of more compact alginate network. However, increasing the concentration of gelatin from 5% to 10%, the water content has a trend of slight increasing in IPN hydrogel fibers. This may be attributed to the loose crosslinking
Fig. 2. Equilibrium water content of gelatin–alginate full IPN hydrogel fibers after incubating in distilled water for 24 h, the composition of the fibers is shown in Table 1, A1 is alginate hydrogel crosslinked by calcium ions.
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Scheme 1. Formation of gelatin–alginate semi IPN and full IPN
network of gelatin because of the formation of amide bonds between glutamine and lysine side chains catalyzed by mTG. 3.2. Mechanical properties The mechanical properties of gelatin–alginate full IPN hydrogel fibers were measured by single fiber tensile tester at room temperature. As shown in Fig. 3, alginate hydrogel fiber (A1) crosslinked by calcium ions was easily fractured under a tension of 18 cN with an elongation about 150%. After the incorporation of gelatin with the absence of mTG, the tension of semi IPN hydrogel fibers decreased with the increase of gelatin content. But, the tension had a tendency of increase with elongation about 200% with the increase of alginate, resulting from more crosslinks of alginate. Most importantly, gelatin–alginate full IPN hydrogel fibers prepared in the presence of both mTG and calcium ions have much higher tension and elongation than those of alginate hydrogel and semi IPN hydrogel fibers crosslinked only by calcium ions. Increasing the concentration of alginate from 1% to 2%, the tension of full IPN fibers increased by more than 3-fold (18 cN versus 62 cN), and the elongation by more than 5-fold (145% versus 739%). With the increase of gelatin concentration from 0 to 10%, the tension of full IPN fiber G10A1 reached 29 cN, which was 1.6-fold higher than that of the alginate hydrogel fiber. Apparently, the gelatin–alginate full IPN fibers having the highest tension of 62 cN and elongation of 739% are so tough that they should withstand further handling and post processing. The obvious enhancement of mechanical strength with the increasing concentration of gelatin was only observed in the full IPN hydrogels but not in the semi IPN hydrogels. Actually, the
latter has a decrease trend of tension. Because the gelatin component remained in sol phase and did not participate in the formation of polymer network in semi IPN case. In the case of full IPN, the alginate network composed of ionic bonds between Ca2+ and guluronate blocks of alginate, formed a reinforced microstructure in the flexible gelatin network crosslinked by mTG [23]. The effect of crosslinking degree on tensile strength of gelatin–alginate full IPN hydrogel fibers was further investigated by changing the concentration of mTG. As indicated in Fig. 4, the full IPN hydrogel fibers showed increases of tension and elongation while mTG concentration was increased from 10 to 20 U/mL. That is to say, full IPN gels showed positive correlation between tension and mTG concentration. The elongation almost keeps constant with a slight increase of tension after mTG concentration is over than 20 U/ml. It means that the concentration of mTG has no significant effect on the mechanical strength of full IPN gel fibers because of the complete crosslinking. 3.3. Enzymatic degradation The degradation property of the gelatin–alginate full IPN hydrogel fibers was measured in the presence of papain by gravimetric method as showed in Fig. 5. We noticed that the fractional mass remaining of calcium-crosslinked alginate fibers decreased to zero after 3 h incubation. It may be attributed to the dissolution of the alginate fibers via Ca2+ exchanging with Na+ . All of the gelatin–alginate full IPN hydrogel fibers degraded completely after 7–9 h incubation. With the increase of gelatin concentration, the degradation time increases accordingly. It has been reported that it is uncontrollable and unpredictable for ionically crosslinked
X. Hu et al. / International Journal of Biological Macromolecules 72 (2015) 403–409
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Fig. 3. (A) Tension and (B) elongation of gelatin–alginate full IPN hydrogel fibers.
Fig. 4. Effect of mTG concentration on the tension (A) and elongation (B) of gelatin–alginate full IPN hydrogel fibers.
alginate hydrogels degrading into the surrounding medium [24]. Therefore, the combination of enzymatic proteolysis using papain with dissociation of calcium ions in PBS speeds up the degradation of full IPN fibers effectively.
quantitative assessment showed that all of full IPN fibers have L929 cell viability over 85%. Apparently, there was no cytotoxicity to L929 mouse fibroblast among all of the gelatin–alginate full IPN hydrogel fibers. The results indicated that the full IPN hydrogel fibers were non-toxic, and confirmed the potential for biomedical applications.
3.4. In vitro cytotoxicity 3.5. Cell seeding and imaging MTT assay was used to evaluate cell metabolic activity of L929 cells incubated with the extract solution of gelatin–alginate full IPN hydrogel fibers for 1, 3 and 5 days. As revealed in Fig. 6, the
Fig. 5. Weight change profiles of gelatin–alginate full IPN hydrogel fibers in PBS buffer solution containing 0.05 mg/mL papain at 37 ◦ C.
The ability of materials to support cell adhesion, spreading and proliferation is an important factor in tissue engineering. It is reported that unmodified alginate can promote minimal protein
Fig. 6. Cytotoxicity of extracts of gelatin–alginate full IPN hydrogel fibers.
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Fig. 7. Optical images (200×) of L929 mouse fibroblasts adhesion on gelatin–alginate full IPN hydrogel fibers. L929 seeded on hydrogel fibers at density of 1 × 104 cells/cm2 were cultured for 1, 3 and 5 days.
Fig. 8. SEM images of L929 mouse fibroblasts cultured on gelatin–alginate full IPN hydrogel fibers for 5 days (200×), (a) G5A1 without cells seeding; (b) G5A1 (insert image with magnification of 50), (c) G5A1.5, (d) G5A2 seeded with L929 cells.
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Fig. 9. Digital photograph of hand-woven braid made from G5A2 gelatin–alginate full IPN hydrogel fibers.
adsorption, leading to its disability for mammalian cells adhering or spreading on alginate polysaccharides [25,26]. To investigate the biological properties of the gelatin–alginate IPN hydrogel fibers, L929 fibroblasts were cultured on full IPN fibers for up to 5 days. As shown in Fig. 7, the addition of gelatin promoted cell interactivities. A large quantity of L929 cells adhesion and spreading were observed on the fibers prepared by enzymatic and ionic crosslinking approaches. Almost all the cells were found to grow well and there were no significant differences on the quantity of cells among the gelatin–alginate full IPN fibers with different alginate content. Fig. 8 shows SEM images of L929 cells morphology cultured on the hydrogel fibers after 5 days. It was found that there was a large amount holes with a diameter ca. 20 m distributed on the surface of the fiber before cell seeding. After seeding the cells on G5A1 were spindle-shaped and linked together, and similar morphologies were observed on G5A1.5 and G5A2 fibers. The results demonstrated that the gelatin–alginate full IPN hydrogel fibers prepared by enzymatic and ionic crosslinking approaches support cell attachment and proliferation and suitable to be a cytocompatible material. 3.6. Knittability of IPN hydrogel fibers Fibers should be suitable to control scaffold structure to engineer mechanical properties if they can be processed by machine or even hand. In order to test the knittability of the hydrogel fibers, a continuous filament of gelatin–alginate full IPN hydrogel was cut into shorter staple fibers of a finite length 20 cm. Braids of gelatin–alginate G5A2 full IPN hydrogel fibers were woven by hand with three or more threads as showed in Fig. 9. It is assumed that the gelatin–alginate semi IPN hydrogel fiber is strong enough to withstand the handling required for post knitting. 4. Conclusions The biological macromolecular gelatin–alginate IPN hydrogel fibers were prepared successfully by a combination of enzymatic and calcium ion crosslinking without using any extraneous crosslinking agents or toxic chemicals. mTG catalyzed the formation of gelatin network while calcium ions induced another network of alginate. Owing to the specific structure, the IPN hydrogel fibers showed a significant improvement of mechanical strength over
semi-IPN gel fibers. We also found that the tension of the IPN fibers increased with the increase of alginate or gelatin concentration. In addition, the effective cell adhesion and proliferation have shown the excellent biocompatibility of these fibers. The success in obtaining hand woven braid indicated their knittablity, suggesting considerable potential to exploit the wider research possibilities in the development of tissue engineering scaffolds. Acknowledgement Projects 51073036 and 51373034 were supported by the National Natural Science Foundation of China. References [1] K.Y. Lee, D.J. Mooney, Chem. Rev. 101 (2001) 1869–1880. [2] J.L. Drury, D.J. Mooney, Biomaterials 24 (2003) 4337–4351. [3] S. Varghese, J.H. Elisseeff, Hydrogels for musculoskeletal tissue engineering, in: Polymers for Regenerative Medicine, Springer, 2006, pp. 95–144. [4] J.Y. Sun, X. Zhao, W.R. Illeperuma, O. Chaudhuri, K.H. Oh, D.J. Mooney, J.J. Vlassak, Z. Suo, Nature 489 (2012) 133–136. [5] S.D. McCullen, C.M. Haslauer, E.G. Loboa, J. Mater. Chem. 20 (2010) 8776–8788. [6] V.J. Chen, P.X. Ma, Biomaterials 25 (2004) 2065–2073. [7] L.A. Smith, P.X. Ma, Colloids Surf. B: Biointerfaces 39 (2004) 125–131. [8] Z. Ma, M. Kotaki, R. Inai, S. Ramakrishna, Tissue Eng. 11 (2005) 101–109. [9] J.W. Nichol, A. Khademhosseini, Soft Matter 5 (2009) 1312–1319. [10] H.-Y. Lin, C.-W. Peng, W.-W. Wu, J. Mater. Sci.: Mater. Med. 25 (2014) 259–269. [11] M. Hu, R. Deng, K.M. Schumacher, M. Kurisawa, H. Ye, K. Purnamawati, J.Y. Ying, Biomaterials 31 (2010) 863–869. [12] J.-K. Francis Suh, H.W. Matthew, Biomaterials 21 (2000) 2589–2598. [13] D.L. Elbert, Curr. Opin. Biotechnol. 22 (2011) 674–680. [14] H.C. Liang, W.H. Chang, K.J. Lin, H.W. Sung, J. Biomed. Mater. Res. A 65 (2003) 271–282. [15] E. Gendler, S. Gendler, M. Nimni, J. Biomed. Mater. Res. 18 (1984) 727–736. [16] R. Meena, M. Chhatbar, K. Prasad, A.K. Siddhanta, Polym. Int. 57 (2008) 329–336. [17] A. Nakayama, A. Kakugo, J.P. Gong, Y. Osada, M. Takai, T. Erata, S. Kawano, Adv. Funct. Mater. 14 (2004) 1124–1128. [18] J. Picard, B. Doumèche, M. Panouillé, V. Larreta-Garde, Macromolecular Symposia, Wiley Online Library, 2010, pp. 337–344. [19] M.D. Brigham, A. Bick, E. Lo, A. Bendali, J.A. Burdick, A. Khademhosseini, Tissue Eng. A 15 (2008) 1645–1653. [20] S. Suri, C.E. Schmidt, Acta Biomater. 5 (2009) 2385–2397. [21] C. Wen, L. Lu, X. Li, Macromol. Mater. Eng. 299 (2014) 504–513. [22] C.W. Yung, L.Q. Wu, J.A. Tullman, G.F. Payne, W.E. Bentley, T.A. Barbari, J. Biomed. Mater. Res. A 83A (2007) 1039–1046. [23] K.Y. Lee, D.J. Mooney, Prog. Polym. Sci. 37 (2012) 106–126. [24] M.S. Shoichet, R.H. Li, M.L. White, S.R. Winn, Biotechnol. Bioeng. 50 (1996) 374–381. [25] J.A. Rowley, G. Madlambayan, D.J. Mooney, Biomaterials 20 (1999) 45–53. [26] E. Alsberg, K. Anderson, A. Albeiruti, R. Franceschi, D. Mooney, J. Dent. Res. 80 (2001) 2025–2029.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Mechanically tough biomacromolecular IPN hydrogel fibers by enzymatic and ionic crosslinking Xin Hu, Lingling Lu, Chen Xu, Xinsong Li ∗ School of Chemistry and Chemical Engineering, Southeast University, Nanjing 210018, China
a r t i c l e
i n f o
Article history: Received 4 April 2014 Received in revised form 9 August 2014 Accepted 10 August 2014 Available online 1 September 2014 Keywords: IPN hydrogel fiber Enzymatic crosslinking Mechanical strength
a b s t r a c t In this report, biological macromolecular full IPN hydrogel fibers composed of gelatin and alginate with an interpenetrating network (IPN) structure were prepared by wet spinning using a combination of enzymatic and calcium ions crosslinking. In the full IPN hydrogel fibers, mTG catalyzed the formation of one network of gelatin while calcium ions crosslinked another network of alginate intertwining with the former. The mechanical strength of the full IPN hydrogel fibers was measured by an electronic single fiber strength tester. The results showed that gelatin–alginate full IPN hydrogel fibers had a significant improvement of mechanical strength over gelatin–alginate semi-IPN gel fibers crosslinked only by calcium ions. The full IPN fiber has the highest tension of 62 cN and elongation of 739%, which are much higher than those of alginate hydrogel. Furthermore, biological evaluation indicated that gelatin–alginate full IPN hydrogel fibers enhance cell adhesion and proliferation significantly, illustrating the cyto-compatibility. A preliminary trial of hand weaving showed the knittablity of the mechanically tough full IPN hydrogel fibers. Because of their both excellent biocompatibility and mechanical strength, the biological macromolecular hydrogel fibers with full IPN structure may be desirable candidates for engineering tissue scaffolds. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hydrogel scaffolds have attracted much attention in the past decade in the field of tissue engineering scaffolds [1–3]. Their highly hydrated polymer networks may allow cells to adhere, proliferate and differentiate for the treatment of injured tissues and organs. However, poor mechanical properties of hydrogels limit their applications [4]. Fiber reinforcement within hydrogels is a promising approach for engineering the mechanical and physical properties of scaffolds [5]. Fibers can be arranged from monofilaments or multifilament yarns and made into textile substrates or superstructures that include woven, knit, braided, continuous randomly aligned (2D or 3D), staple fiber randomly aligned. They can be designed to have an extremely high porosity [6] for cellular infiltration, and exhibit good mechanical properties and suitable compliance for handling and conformability to varying tissue and organ shapes [7,8]. Apparently, both of biocompatible fibers and impregnated hydrogels or matrices are highly needed in order to fabricate fiber reinforced hydrogel scaffolds.
∗ Corresponding author. Tel.: +86 2583793456. E-mail address: [email protected] (X. Li). http://dx.doi.org/10.1016/j.ijbiomac.2014.08.043 0141-8130/© 2014 Elsevier B.V. All rights reserved.
Hydrogel fibers may be valuable candidates as building blocks for constructing 3D scaffolds [9]. Lin et al. [10] developed a calciumcrosslinked alginate fibrous scaffold with interconnected pores using a three-dimensional plotting system. Hu et al. [11] investigated the hydrodynamic spinning of hydrogel fibers by in situ cross-linking. They tried several biopolymer and synthetic polymer systems, but did not provide mechanical properties of the obtained hydrogels fibers in detail. Up to now, the development of hydrogel fibers with both excellent biocompatibility and mechanical strength remains a challenge. Biological macromolecular hydrogels composed of natural proteins or polysaccharides are widely applied in the development of multitude of tissue engineering scaffolds due to hydrophilicity, biocompatibility and biodegradability [12]. Unfortunately, most biomacromolecular hydrogels suffer from the lack of mechanical strength, and they are difficult to be handled to engineer 3D scaffolds with distinct geometries and physicochemical properties [13]. Chemical crosslinking is a common method to improve the mechanical properties of biomacromolecular hydrogels, the residues of crosslinkers such as glutaraldehyde, carbodiimide, and diphenylphosphoryl azide may bring cytotoxic side-effects even after thorough purification [14,15]. Therefore, hydrogels derived from biological macromolecular proteins and polysaccharides
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X. Hu et al. / International Journal of Biological Macromolecules 72 (2015) 403–409
Table 1 Composition of gelatin (G)–alginate (A) bio-macromolecular hydrogel fibers. Sample
Gelatin (mg/mL)
0 A1 Semi-IPN 50 G5A1 G5A1.5 50 50 G5A2 G7.5A1 75 100 G10A1 IPN 50 G5A1 G5A1.5 50 50 G5A2 75 G7.5A1 100 G10A1
Alginate (mg/mL)
mTG (20 U/mL)
Ca2+ (1%)
10
–
Immerse
10 15 20 10 10
– – – – –
Immerse Immerse Immerse Immerse Immerse
10 15 20 10 10
Immerse Immerse Immerse Immerse Immerse
Immerse Immerse Immerse Immerse Immerse
prepared using biocompatible approaches with good mechanical properties are in high demand [16,17]. Utilizing two or more biomacromolecular proteins and polysaccharides to form an interpenetrating polymer network (IPN) hydrogel fibers by biocompatible approaches may enhance mechanical properties of the biopolymer hydrogels because of their highly entangled networks [18–20]. In this report, we present the preparation of gelatin–alginate hydrogel fibers with IPN structure using a combination of microbial transglutaminase (mTG) enzymatic and calcium ionic crosslinking [21]. The IPN hydrogel fibers were further characterized in terms of mechanical property, equilibrium water content, in vitro degradation and cytocompatibility. The results showed that the biomacromolecular hydrogel fibers prepared by biocompatible approaches may be applied in the development of tissue engineering scaffolds because of the combined biocompatibility and mechanical strength. 2. Materials and methods 2.1. Materials Sodium alginate (Laboratory Reagent Grade) was obtained from Huanghai Chemical Co. (Shandong, China), viscosity of 1% (w/v) solution in water is 94 mPa s. Gelatin (Type A, bloom strength at 11.1% moisture content is 250 g) was purchased from Aladdin Chemical Co., Ltd. (Shanghai, China). Anhydrous CaCl2 , dimethyl sulfoxide (DMSO) and papain (6000 U/mg) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Microbial transglutaminase (mTG, 1000 U/g) was a gift from Yiming Biological Products Co., Ltd. (Taizhou, China). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin, l-glutamine, trypsin and 3-(4,5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Invitrogen Co. (Carlsbad, CA). L929 mouse fibroblasts cells were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). 2.2. Preparation of IPN hydrogel fibers The gelatin (G)–alginate (A) IPN biohydrogel fibers were prepared with the composite as shown in Table 1 by wet spinning as illustrated in Fig. 1. Briefly, gelatin and alginate polymers were dissolved in distilled water and mixed, and a series of gelatin–alginate solutions with the concentration as showed in Table 1 were obtained. To prepare IPN hydrogel fibers, dope of gelatin–alginate solution was spun at a speed of 15 mL/h into aqueous coagulant bath with the concentration of CaCl2 1% and mTG 20 U/mL through a stainless steel spinneret with the diameter of 0.9 mm, and then the fibers were kept in oven at 55 ◦ C for 30 min to complete the enzymatically crosslinking. As a comparison, dope of polymer solution
was spun into aqueous coagulant bath with the concentration of CaCl2 1% in the absence of mTG, and semi interpenetrated network (semi-IPN) hydrogel fibers were prepared. Both semi-IPN and IPN hydrogel fibers were rinsed thoroughly with distilled water, and the diameter of these fibers was measured by a micrometer caliper, which is in the range of 1.0 ± 0.1 mm. 2.3. Equilibrium water content Gelatin-alginate IPN hydrogel fibers were incubated in distilled water at room temperature for 24 h to reach its equilibrium state. Then the weight (Ws ) of each hydrogel fiber at swollen state was measured after excess surface water was removed. After the sample was lyophilized, the weight (Wd ) of each sample at dried state was weighed. The equilibrium water content ratio was calculated using the following equation: Water content ratio =
Ws − Wd Ws
All the samples were measured in triplicates for each group. 2.4. Mechanical test Mechanical test for each gelatin–alginate hydrogel fiber was performed using an electronic single fiber strength tester (LLY-06B, Jinan, China) at room temperature. Hydrogel fiber with the length of 8 cm was subjected to tensile test at a strain rate of 20 mm/min. All samples were measured in triplicates. 2.5. In vitro enzymatic degradation To test enzymatic degradation of the IPN fibers, the samples were lyophilized and weighed the initial dry weight (W0 ), then immersed in PBS (0.01 M) containing papain (0.05 mg/ml) at 37 ◦ C under constant shaking at 80 rpm for 1, 3, 5, 7, 9 h. The fibers were washed carefully with deionized water and then freeze dried and weighed again (Wt ) at a predetermined time. The fractional mass remaining after in vitro degradation was calculated by the dry weight (Wt ) divided by the initial weight of the gel (W0 ) as follows:
Fractional mass remaining (%) =
Wt × 100% W0
Each group was tested in triplicates. 2.6. In vitro cytotoxicity The cytotoxicity of the gelatin–alginate IPN hydrogel fibers was evaluated by MTT assay. The fibers were steam sterilized at 121 ◦ C for 20 min, then immersed in DMEM solution at an extraction ratio of 0.75 cm2 /mL at 37 ◦ C for 24 h. The extract DMEM solutions were collected for MTT assay. Mouse fibroblast cells (L929) were allowed to seed at a density of 104 cells/well in 96-well plates and incubated in 100 L DMEM medium containing 10% FBS for 24 h in a humidified incubator (37 ◦ C, 5% CO2 ). Then the medium was replaced with 180 L DMEM medium (10% FBS in DMEM) and 20 L extract solution. After cultured for 1 day, 3 days and 5 days, 20 L of MTT solution (5 mg/mL in PBS) was added into each well for 4 h to allow formation of formazan crystal. Subsequently, supernatant was removed carefully and 150 L DMSO was added to each well. UV Absorbance was measured at 490 nm using the Bio-Rad Model 680 microplate reader (Philadelphia, PA). The cell viability was calculated as a percentage relative to the data obtained with blank control. All samples were tested for sextuplicates.
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Fig. 1. Schematic illustration of wet-spinning process: (1) metering pump; (2) spinning solution; (3) spinneret; (4) coagulation bath; (5, 6) take-up roller.
2.7. Cell seeding and imaging The gelatin–alginate IPN hydrogel fibers were incubated in DMEM containing 10% FBS at 37 ◦ C for 2 h. L929 cells were seeded onto the hydrogel scaffolds with a cell density of 104 /cm–2 and incubated in a humidified atmosphere (37 ◦ C, 5% CO2 ) for 5 days. Cell morphology was observed by microscope. After 5 days, the fibers were fixed with 2.5% glutaraldehyde solution at room temperature for 2 h, then rinsed three times with PBS and three times with deionized water. Subsequently, the samples were freeze-dried, and the cells morphology seeded on the fibers were observed under a scanning electron microscope (JSM6360LV, JEOL, Japan) after gold sputter-coating.
3. Results and discussion Gelatin-alginate IPN hydrogel fibers were prepared with the composite as shown in Table 1 by wet spinning (Fig. 1) using a mixture of calcium chloride and microbial transglutaminase (mTG) solution as a coagulantion bath. Transglutaminase is enzyme that catalyzes amide bond formation between glutamine and lysine side chains, with the loss of ammonia. So, gelatin can be crosslinked by the formation of covalent N--(␥-glutamyl) lysine amide bonds between individual gelatin molecules to form a permanent network catalyzed with mTG [22], while in the presence of calcium ions alginate G-blocks participate in gelation to form an “egg box”shaped structure network. As reported in our previous work [21], both gelatin and alginate formed crosslinked double networks in the presence of both mTG and calcium ions and hence, the obtained hydrogels can be considered as full IPN hydrogel fibers. When wet spinning of gelatin–alginate solution was conducted using a calcium chloride solution as a coagulation bath in the absence of mTG. Gelatin remained in its sol state in the obtained fibers. Therefore, the obtained hydrogels, in which alginate formed a crosslinked network in the presence of calcium ions while gelatin was entrapped within the alginate gel network, were considered as semi IPN hydrogels as shown in Scheme 1. Using semi IPN gel fibers as controls, the physicochemical and biological properties of
the gelatin–alginate IPN hydrogel fibers were investigated in detail as follows.
3.1. Equilibrium water content The equilibrium water content of the IPN hydrogel fibers was measured by gravimetric method as showed in Fig. 2. It was found that all of gelatin–alginate IPN hydrogel fibers and semi IPN hydrogel fibers have equilibrium water contents in the range from 85% to 95%, and the former has a lower water content than that of the latter. The reason is that uncrosslinked gelatin was entrapped into alginate network as in semi IPN case, resulting in a relatively loose structure which holds more water. Besides, gelatin–alginate IPN hydrogel fibers have a significant decreasing of water content with the increase of alginate content. It may be attributed to the increase of crosslinking degree and the formation of more compact alginate network. However, increasing the concentration of gelatin from 5% to 10%, the water content has a trend of slight increasing in IPN hydrogel fibers. This may be attributed to the loose crosslinking
Fig. 2. Equilibrium water content of gelatin–alginate full IPN hydrogel fibers after incubating in distilled water for 24 h, the composition of the fibers is shown in Table 1, A1 is alginate hydrogel crosslinked by calcium ions.
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Scheme 1. Formation of gelatin–alginate semi IPN and full IPN
network of gelatin because of the formation of amide bonds between glutamine and lysine side chains catalyzed by mTG. 3.2. Mechanical properties The mechanical properties of gelatin–alginate full IPN hydrogel fibers were measured by single fiber tensile tester at room temperature. As shown in Fig. 3, alginate hydrogel fiber (A1) crosslinked by calcium ions was easily fractured under a tension of 18 cN with an elongation about 150%. After the incorporation of gelatin with the absence of mTG, the tension of semi IPN hydrogel fibers decreased with the increase of gelatin content. But, the tension had a tendency of increase with elongation about 200% with the increase of alginate, resulting from more crosslinks of alginate. Most importantly, gelatin–alginate full IPN hydrogel fibers prepared in the presence of both mTG and calcium ions have much higher tension and elongation than those of alginate hydrogel and semi IPN hydrogel fibers crosslinked only by calcium ions. Increasing the concentration of alginate from 1% to 2%, the tension of full IPN fibers increased by more than 3-fold (18 cN versus 62 cN), and the elongation by more than 5-fold (145% versus 739%). With the increase of gelatin concentration from 0 to 10%, the tension of full IPN fiber G10A1 reached 29 cN, which was 1.6-fold higher than that of the alginate hydrogel fiber. Apparently, the gelatin–alginate full IPN fibers having the highest tension of 62 cN and elongation of 739% are so tough that they should withstand further handling and post processing. The obvious enhancement of mechanical strength with the increasing concentration of gelatin was only observed in the full IPN hydrogels but not in the semi IPN hydrogels. Actually, the
latter has a decrease trend of tension. Because the gelatin component remained in sol phase and did not participate in the formation of polymer network in semi IPN case. In the case of full IPN, the alginate network composed of ionic bonds between Ca2+ and guluronate blocks of alginate, formed a reinforced microstructure in the flexible gelatin network crosslinked by mTG [23]. The effect of crosslinking degree on tensile strength of gelatin–alginate full IPN hydrogel fibers was further investigated by changing the concentration of mTG. As indicated in Fig. 4, the full IPN hydrogel fibers showed increases of tension and elongation while mTG concentration was increased from 10 to 20 U/mL. That is to say, full IPN gels showed positive correlation between tension and mTG concentration. The elongation almost keeps constant with a slight increase of tension after mTG concentration is over than 20 U/ml. It means that the concentration of mTG has no significant effect on the mechanical strength of full IPN gel fibers because of the complete crosslinking. 3.3. Enzymatic degradation The degradation property of the gelatin–alginate full IPN hydrogel fibers was measured in the presence of papain by gravimetric method as showed in Fig. 5. We noticed that the fractional mass remaining of calcium-crosslinked alginate fibers decreased to zero after 3 h incubation. It may be attributed to the dissolution of the alginate fibers via Ca2+ exchanging with Na+ . All of the gelatin–alginate full IPN hydrogel fibers degraded completely after 7–9 h incubation. With the increase of gelatin concentration, the degradation time increases accordingly. It has been reported that it is uncontrollable and unpredictable for ionically crosslinked
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Fig. 3. (A) Tension and (B) elongation of gelatin–alginate full IPN hydrogel fibers.
Fig. 4. Effect of mTG concentration on the tension (A) and elongation (B) of gelatin–alginate full IPN hydrogel fibers.
alginate hydrogels degrading into the surrounding medium [24]. Therefore, the combination of enzymatic proteolysis using papain with dissociation of calcium ions in PBS speeds up the degradation of full IPN fibers effectively.
quantitative assessment showed that all of full IPN fibers have L929 cell viability over 85%. Apparently, there was no cytotoxicity to L929 mouse fibroblast among all of the gelatin–alginate full IPN hydrogel fibers. The results indicated that the full IPN hydrogel fibers were non-toxic, and confirmed the potential for biomedical applications.
3.4. In vitro cytotoxicity 3.5. Cell seeding and imaging MTT assay was used to evaluate cell metabolic activity of L929 cells incubated with the extract solution of gelatin–alginate full IPN hydrogel fibers for 1, 3 and 5 days. As revealed in Fig. 6, the
Fig. 5. Weight change profiles of gelatin–alginate full IPN hydrogel fibers in PBS buffer solution containing 0.05 mg/mL papain at 37 ◦ C.
The ability of materials to support cell adhesion, spreading and proliferation is an important factor in tissue engineering. It is reported that unmodified alginate can promote minimal protein
Fig. 6. Cytotoxicity of extracts of gelatin–alginate full IPN hydrogel fibers.
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Fig. 7. Optical images (200×) of L929 mouse fibroblasts adhesion on gelatin–alginate full IPN hydrogel fibers. L929 seeded on hydrogel fibers at density of 1 × 104 cells/cm2 were cultured for 1, 3 and 5 days.
Fig. 8. SEM images of L929 mouse fibroblasts cultured on gelatin–alginate full IPN hydrogel fibers for 5 days (200×), (a) G5A1 without cells seeding; (b) G5A1 (insert image with magnification of 50), (c) G5A1.5, (d) G5A2 seeded with L929 cells.
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Fig. 9. Digital photograph of hand-woven braid made from G5A2 gelatin–alginate full IPN hydrogel fibers.
adsorption, leading to its disability for mammalian cells adhering or spreading on alginate polysaccharides [25,26]. To investigate the biological properties of the gelatin–alginate IPN hydrogel fibers, L929 fibroblasts were cultured on full IPN fibers for up to 5 days. As shown in Fig. 7, the addition of gelatin promoted cell interactivities. A large quantity of L929 cells adhesion and spreading were observed on the fibers prepared by enzymatic and ionic crosslinking approaches. Almost all the cells were found to grow well and there were no significant differences on the quantity of cells among the gelatin–alginate full IPN fibers with different alginate content. Fig. 8 shows SEM images of L929 cells morphology cultured on the hydrogel fibers after 5 days. It was found that there was a large amount holes with a diameter ca. 20 m distributed on the surface of the fiber before cell seeding. After seeding the cells on G5A1 were spindle-shaped and linked together, and similar morphologies were observed on G5A1.5 and G5A2 fibers. The results demonstrated that the gelatin–alginate full IPN hydrogel fibers prepared by enzymatic and ionic crosslinking approaches support cell attachment and proliferation and suitable to be a cytocompatible material. 3.6. Knittability of IPN hydrogel fibers Fibers should be suitable to control scaffold structure to engineer mechanical properties if they can be processed by machine or even hand. In order to test the knittability of the hydrogel fibers, a continuous filament of gelatin–alginate full IPN hydrogel was cut into shorter staple fibers of a finite length 20 cm. Braids of gelatin–alginate G5A2 full IPN hydrogel fibers were woven by hand with three or more threads as showed in Fig. 9. It is assumed that the gelatin–alginate semi IPN hydrogel fiber is strong enough to withstand the handling required for post knitting. 4. Conclusions The biological macromolecular gelatin–alginate IPN hydrogel fibers were prepared successfully by a combination of enzymatic and calcium ion crosslinking without using any extraneous crosslinking agents or toxic chemicals. mTG catalyzed the formation of gelatin network while calcium ions induced another network of alginate. Owing to the specific structure, the IPN hydrogel fibers showed a significant improvement of mechanical strength over
semi-IPN gel fibers. We also found that the tension of the IPN fibers increased with the increase of alginate or gelatin concentration. In addition, the effective cell adhesion and proliferation have shown the excellent biocompatibility of these fibers. The success in obtaining hand woven braid indicated their knittablity, suggesting considerable potential to exploit the wider research possibilities in the development of tissue engineering scaffolds. Acknowledgement Projects 51073036 and 51373034 were supported by the National Natural Science Foundation of China. References [1] K.Y. Lee, D.J. Mooney, Chem. Rev. 101 (2001) 1869–1880. [2] J.L. Drury, D.J. Mooney, Biomaterials 24 (2003) 4337–4351. [3] S. Varghese, J.H. Elisseeff, Hydrogels for musculoskeletal tissue engineering, in: Polymers for Regenerative Medicine, Springer, 2006, pp. 95–144. [4] J.Y. Sun, X. Zhao, W.R. Illeperuma, O. Chaudhuri, K.H. Oh, D.J. Mooney, J.J. Vlassak, Z. Suo, Nature 489 (2012) 133–136. [5] S.D. McCullen, C.M. Haslauer, E.G. Loboa, J. Mater. Chem. 20 (2010) 8776–8788. [6] V.J. Chen, P.X. Ma, Biomaterials 25 (2004) 2065–2073. [7] L.A. Smith, P.X. Ma, Colloids Surf. B: Biointerfaces 39 (2004) 125–131. [8] Z. Ma, M. Kotaki, R. Inai, S. Ramakrishna, Tissue Eng. 11 (2005) 101–109. [9] J.W. Nichol, A. Khademhosseini, Soft Matter 5 (2009) 1312–1319. [10] H.-Y. Lin, C.-W. Peng, W.-W. Wu, J. Mater. Sci.: Mater. Med. 25 (2014) 259–269. [11] M. Hu, R. Deng, K.M. Schumacher, M. Kurisawa, H. Ye, K. Purnamawati, J.Y. Ying, Biomaterials 31 (2010) 863–869. [12] J.-K. Francis Suh, H.W. Matthew, Biomaterials 21 (2000) 2589–2598. [13] D.L. Elbert, Curr. Opin. Biotechnol. 22 (2011) 674–680. [14] H.C. Liang, W.H. Chang, K.J. Lin, H.W. Sung, J. Biomed. Mater. Res. A 65 (2003) 271–282. [15] E. Gendler, S. Gendler, M. Nimni, J. Biomed. Mater. Res. 18 (1984) 727–736. [16] R. Meena, M. Chhatbar, K. Prasad, A.K. Siddhanta, Polym. Int. 57 (2008) 329–336. [17] A. Nakayama, A. Kakugo, J.P. Gong, Y. Osada, M. Takai, T. Erata, S. Kawano, Adv. Funct. Mater. 14 (2004) 1124–1128. [18] J. Picard, B. Doumèche, M. Panouillé, V. Larreta-Garde, Macromolecular Symposia, Wiley Online Library, 2010, pp. 337–344. [19] M.D. Brigham, A. Bick, E. Lo, A. Bendali, J.A. Burdick, A. Khademhosseini, Tissue Eng. A 15 (2008) 1645–1653. [20] S. Suri, C.E. Schmidt, Acta Biomater. 5 (2009) 2385–2397. [21] C. Wen, L. Lu, X. Li, Macromol. Mater. Eng. 299 (2014) 504–513. [22] C.W. Yung, L.Q. Wu, J.A. Tullman, G.F. Payne, W.E. Bentley, T.A. Barbari, J. Biomed. Mater. Res. A 83A (2007) 1039–1046. [23] K.Y. Lee, D.J. Mooney, Prog. Polym. Sci. 37 (2012) 106–126. [24] M.S. Shoichet, R.H. Li, M.L. White, S.R. Winn, Biotechnol. Bioeng. 50 (1996) 374–381. [25] J.A. Rowley, G. Madlambayan, D.J. Mooney, Biomaterials 20 (1999) 45–53. [26] E. Alsberg, K. Anderson, A. Albeiruti, R. Franceschi, D. Mooney, J. Dent. Res. 80 (2001) 2025–2029.
