4D Printing of Robust Hydrogels Consisted of Agarose Nanofibers and

Letter Cite This: ACS Macro Lett. 2018, 7, 442−446

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4D Printing of Robust Hydrogels Consisted of Agarose Nanofibers and Polyacrylamide Jinhua Guo, Rongrong Zhang, and Lina Zhang* College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China

Xiaodong Cao* Department of Biomedical Engineering, School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, People’s Republic of China National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, China S Supporting Information *

ABSTRACT: Hydrogels combined with complex 3D shapes and robust mechanical properties are extremely desired soft platforms in the fields of biomaterials, recently, 4D printing has been developed to be further shaped to form required patterns. On the basis of the excellent thixotropy of Laponite and the thermal-reversible sol−gel transition of agarose and easy formation of nanofibers below 35 °C, a 4D printing hydrogel (4D Gel) was fabricated by in situ polymerizing acrylamide in the agarose matrix containing Laponite. The experimental results demonstrated that Laponite played an important role in the improvement of 4D printing, such as endowing the ink with shear-thinning behavior to extrude easily and excellent shape stability after printing. The mechanical properties of 4D Gel were unexpectedly higher than those of both agarose and polyacrylamide hydrogels. The 4D Gel showed the ability to further transform its shapes, and was used successfully to construct a whalelike hydrogel, which opened mouth and cocked tail by treating with an external force and then cooling, as well as the octopus like hydrogel with waved tentacles to seem to “come alive”. This work opened a new avenue for creating more complex architectures than 3D with excellent properties, which is important in the macromolecule fields for the wide applications.

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product should be smart materials, which allow a printed 3D structure to change its form or function with time (the fourth dimension) in response to stimuli such as pressure, pH, light, and temperature.6 In the field of intelligent hydrogels, several strategies including specific structures actuated by bilayer gels,7 volume change of thermally sensitive hydrogel and anisotropic hydrogel enable the fabrication of 4D hydrogels. However, all of these methods suffer from low modulus (normally less than 100 kPa),5,8 limiting the applications of the hydrogels. In our previous work, a robust hydrogel with excellent biocompatibility was fabricated by in situ polymerizing acrylamide in the agarose matrix on the basis of the thermal-reversible sol−gel transition and the nanofiber formation of agarose below 35 °C.9 Meanwhile, the shear-thinning behaviors well-suited as inks for printing systems.10 Therefore, it is necessary to use internal scaffold material to modify the rheological behavior of the ink to meet the printing requirements. Laponite, a kind of nanocaly with excellent thixotropy after dispersing in water, which as a

he ability to create three-dimensional (3D) hydrogels on demand would enable a wide application in tissue engineering, such as organ repair,1 sensors, and biomedicine.2 The 3D bioprinting as an emerging technology is expected to meet the requirements of 3D structured hydrogels in the macromolecule field. However, in this printing, 3D structures are constructed through layer-by-layer positioning of 3D inks, which is often limited by lack of printable inks. Moreover, the most severe challenge of the current approaches are the printing of cantilever,2b hollow tubular structures,3 and the weak mechanical properties of the hydrogels. Recently, four dimensional (4D) printing has been developed to solve the ink insufficiency of 3D printing, and can be further shaped to form required patterns, leading to the construction of more complex architectures than 3D. 4D printing is an exciting emerging technology for creating dynamic devices that can change their shape and/or function on-demand over time.4 For example, a hollow tubular structures can be constructed through rolling a printed cuboid. However, most of the currently known 4D printing techniques rely on applying external stress to a printed 2D structure. After triggering, the stress is released and the 2D structure will transform into 3D.4b,5 Actually, 4D printed © XXXX American Chemical Society

Received: December 8, 2017 Accepted: March 20, 2018

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DOI: 10.1021/acsmacrolett.7b00957 ACS Macro Lett. 2018, 7, 442−446

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ACS Macro Letters

linkage of nanofibers through hydrogen bonding. It has been reported that the extended macromolecules in solution easily self-aggregated in parallel to form nanofibers.14 With further increase of the agarose concentration, the agarose nanofibers were physically cross-linked to form hydrogel. After the printing, a robust hydrogel containing agarose nanofibers, PAM and Laponite was created, supported by the results of in Figure 1d−f. The agarose hydrogels exhibited a nanofibers framework architecture, whereas 4D Gel displayed interpenetrated nanofibrous networks with the PAM micropores with pore wall. It was noted that the PAM hydrogel was consisted of low cross-linking density, leading to its soft and weak mechanical properties. The 4D Gel exhibited the good compatibility between the three components, in which the double networks containing the agarose nanofibers contributed to the reinforcing of the 4D Gel. Moreover, depending on the thixotropy of the Laponite, the ink could be easily extruded through a nozzle as a liquid and self-supported after extrusion as a solid. In view of the above results, a scheme to describe the formation and transform of 4D ink is proposed in Figure 1g. At 35 °C, the extended agarose chains easily self-aggregated in parallel to form nanofibers, and were highly crosslinked through hydrogen bonding interaction to generate three-dimensional networks, supported by the results in Figure 1a−c. After in situ polymerizing acrylamide, the agarose networks were interpenetrated with the PAM chemical networks having low crosslinking density to form homogeneous double networks, supported by the results in Figure 1d−f. The double networks containing nanofibers could significantly improve the strength of the composite hydrogels. At high temperature, the agarose nanofibers and their networks were disrupted into flexible chains, whereas the PAM chemical network still maintained its original state. Taking advantage of such thermal-reversible sol− gel transition behaviors of agarose, the 4D Gel was soften by heating to 95 °C and could be made into different shape, such a shape change could be hardened at ambient temperature. In our findings, to make the ink printable, Laponite was used as key component in the ink, which could endow the ink with shear-thinning behavior. As shown in Figure 2a, the rheological behavior at 95 °C of the 4D ink exhibited a high viscosity (η ≈ 104 Pa·s) at low shear rate (0.01 s−1), whereas the viscosity was declined rapidly with an increase of shear rate. When the shear rate was 100 s−1 the viscosity was only about 1 Pa·s. The high viscosity gave excellent shape stability after printing, whereas the low viscosity made the ink easy to be extruded. The result of strain amplitude sweep of the 4D ink is shown in Figure 2b. The storage modulus (G′) was about 5 kPa and exceeded the loss modulus (G″) at oscillation strain below 30%, indicating solid-like behavior up to this strain level; at oscillation strain higher than 30%, G″ exceeded G′, and the ink changed from a solid-like state to a liquid-like state. Meanwhile, on the base of the strain amplitude sweep results, the continuous step strain measurements were performed to study the rheological properties of the 4D ink. Dynamic strain in the range from 1 to 300% was taken to the 4D ink for 200 s (Figure 2c). The ink was in solid state at a given 1% oscillation strain; after the oscillation strain could rise to 300%, which changed rapidly to liquid state, so the sol−gel transition time was sufficiently short. These sol−gel and gel−sol transformations of 4D ink were repeatable. Such an efficient sol−gel transformation ensured the 4D printing application. As shown in Figure 2d, depending on the sol−gel transformation of the agarose, the ink could be

liquid when sustain shear force and self-supported after removing external force as a solid, has been investigated to serve as an internal scaffold material for the direct printing of hydrogel.11 Herein, we try to use the acrylamide, agarose and Laponite mixed solution as ink in direct inject printing. In the present work, we reported a 4D printing hydrogel (4D Gel) by in situ polymerization of acrylamide in the agarose matrix. By mixing Laponite with acrylamide and agarose precursors, the composites were endowed the thixotropy and could be printed directly into 3D structures in air. After printing, the ink was cross-linked through hydrogen bonds of agarose and chemical bonds of polyacrylamide (PAM). The thermal-reversible sol−gel transition of agarose12 led to the forth dimensional change of the hydrogel, and the double networks containing agarose nanofibers contributed to the reinforcement of the 4D Gel. Meanwhile, MTT test confirmed that 4D gel was nontoxic to the cells, the relative cell viability was approach 90% after 3 days culture.(Figure S1) Thus, the robust 4D Gel was able to transfer a 3D printed gel into a special structure with remarkable biocompatibility. Agarose/AM/Laponite ink (4D ink) was used successfully to fabricate agarose/PAM/Laponite hydrogels. As mentioned above, the ink was easy to be extruded at 95 °C, and formed continuous phase in suspended state (Figure S2). Figure 1a−c

Figure 1. AFM images of 4D ink: extremely diluted agarose solution of 1 × 10−7 g/mL at 25 °C (a), agarose solution of 1 × 10−4 g/mL at 25 °C (b) and concentrated solution 1 × 10−3 g/mL at 25 °C (c). SEM images of cross-section of hydrogels: Agarose (d), PAM (e), and 4D Gel (f). Schema to describe the network formation and transformations of 4D ink consisted of agarose, PAM, and Laponite (g).

shows AFM images of the agarose solution. Extended chain patterns with an average height (h) of 0.6 nm and average apparent length (l) of 500 nm were observed in the extremely diluted agarose solution at 25 °C.The direct evidence demonstrated that agarose existed as stiff chain conformation in the dilute aqueous solution at room temperature, consistent with its stiff double helix reported.13 Interestingly, a slight increase of the agarose concentration led to the formation of the numerous nanofibers with more height (h = 4 nm)and long nanofibers, as a result of the self-aggregation between the extended agarose chains in parallel as well as “head to tail” 443

DOI: 10.1021/acsmacrolett.7b00957 ACS Macro Lett. 2018, 7, 442−446

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ACS Macro Letters

to the 4D transition, the octopus waved its tentacle. As shown in Movie S1, based on the recovery process of the 4D Gel, the octopus seem to “come alive”. It was not hard to imagine that such a cantilever-like structure could not be directly printed by 3D printing technology without support. Furthermore, the 4D Gel could be capable to transform into different patterns repeatedly, and the 4D hydrogel could return to its original state by heating process. Furthermore, different complex shapes of human ear and nose like hydrogels with very fine details were also be printed (Figure S3). To evaluate the 4D transition process of the 4D Gel, the temperature-dependence storage (E′) modulus of the hydrogel was measured by rheology. As shown in Figure 3c, the storage modulus (E′) of 4D Gel decreased from 0.12 to 0.01 MPa with an increase of temperature, confirming that 4D Gel was softened at 95 °C, as a result of the disruption of the agarose nanofibers and physical networks. Meanwhile, E′ back to original levels at room temperature, indicating the hardening of 4D Gel, as a result of the reformation of highly physically cross-linked agarose networks containing nanofibers. Although many inks have been used in 3D printed hydrogel, including carrageenan,15 alginate,16 gelatin, and so on, these materials generally were unable to achieve high mechanical properties. In recent years, the double-network hydrogels have exhibited extraordinarily toughness, which was resulted from the sacrifice bond of the brittle network, dissipated large amounts of fracture energy.17 As for the 4D Gel, the typical tensile stress−strain curve was included in Figure 4a, the tensile

Figure 2. Rheological behaviors of the agarose/AM/Laponite 4D ink at 95 °C. Flow rheology pattern showing viscosity against shear rate (a). Oscillatory rheology pattern showing shear storage modulus (G′) and shear loss modulus (G″) evolution of inks used increasing shear strain (b). Oscillatory rheology pattern showing modulus evolution of inks between 1% and 300% strain (c). The storage modulus variation of 4D ink during cooling (d).

solidified rapidly at ambient temperature after printing, and got the products with accurate shape. The 4D printing technology is a universal way to construct cantilever and hollow tubular structures, which is a big challenge for direct 3D printing. Whale and octopus are used as models in the 4D printings. Here, the whalelike and octopus like hydrogels were printed through inkjet printing by a 3Dbioplotter. Figure 3 shows the examples of the 4D printing and

Figure 3. 4D printing product printed by 4D ink: a whale like hydrogel was used as a model to schematic the 4D transition process (a), the softening and hardening cycles of an octopus like gel (b), the storage modulus (E′) variation of 4D Gel during heating and cooling cycles measured by rheology (c).

the storage modulus variation process of the 4D gel. As shown in Figure 1g, after the UV photopolymerization, the chemically and physically cross-linked double networks containing the agarose nanofibers occurred in the agarose/PAM/Laponite system at low temperature to obtain ink. After the 3D printing process, as shown in Figure 3a, the whalelike hydrogel was generated, and then was remarkably softened after being heated in an oven at 95 °C for 15 min. Subsequently, the soft whalelike hydrogel was opened mouth and cocked tail by an external force, then hardened and fixed under cold water for 60 s to obtain its 4D pattern. Meanwhile, the octopus like hydrogel got its 4D pattern in the same way. As shown in Figure 3b, owing

Figure 4. Typical tensile stress−strain curves of 4D Gel (4 wt % agarose, 2 M acrylamide, 4.5 wt % Laponite), AgaL (4 wt % agarose, 4.5 wt % Laponite), and PAML (2 M acrylamide, 4.5 wt % Laponite) (a), tensile stress−strain curves of 4D Gel stored at 95 °C for different time after loading (b), the recovery (%) of elastic modulus and dissipated energy (e). Compression stress−strain curves of 4D Gel (c, d, f). 444

DOI: 10.1021/acsmacrolett.7b00957 ACS Macro Lett. 2018, 7, 442−446

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ACS Macro Letters

(21620102004), and the National Natural Science Foundation of China (20874079, 21574045).

strength was unexpectedly high, reaching 0.47 MPa at 1258%. More importantly, the 4D Gel achieved a toughness of 3.89 MPa, which was 16× and 864× higher than PAM/Laponite (PAML) single-network (SN) hydrogels (0.24 MPa) and agarose/Laponite (AgaL) SN hydrogels (0.0045 MPa). Moreover, 4D Gel also displayed much better compression strength than both PAML and AgaL (Figure 4c). The 4D Gel could achieve a compression toughness of 1.24 MPa. In contrast, the PAML were flabby, and the AgaL were very brittle. Usually, after the loading process, permanent damage would occur in the sacrificing highly cross-link network. However, in our findings, the mechanical properties of 4D Gel could recover by heating treatment. As shown in Figure 4b,d, the stress− strain curves of 4D Gel gradually closed to the original curve with an increase of heating time, and tended to be stable after 20 min. As seen from Figure 4e,f, two recovery rates, defined by a ratio of elastic modulus and dissipated energy, were over 80% at 95 °C after 20 min. This finding clearly indicated that the 4D Gel could be healed via the sol−gel transition of the agarose network. In summary, we demonstrated a route for 4D printing robust hydrogel by writing of agarose/AM/Laponite 4D hydrogel ink. In our findings, Laponite could make the ink suitable to be 4D printed, which played an important role in the improvement of the printing process, such as the endowing the ink with shearthinning behavior to extrude easily and excellent shape stability after printing. On the base of the sol−gel transformation behavior of agarose, the 4D Gel could reversibly soften by heating and harden by cooling, was able to make a 3D structure to transform its shape into different patterns. Moreover, the double network strategy of highly cross-linked agarose network was interpenetrated into lightly cross-linked PAM networks, leading to the high strength and toughness of the 4D Gel. This 4D ink was used successfully to construct a whalelike hydrogel, which was opened mouth and cocked tail by treating with an external force and then cooling, as well as the octopus like waved its tentacle and seem to “come alive”. Therefore, the 4D ink could meet the wide application in the macromolecule field, particularly, biomedical requirements such as scaffolds, sensors, soft robots, and medical devices on the basis of the biocompatibility of the 4D Gel.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00957. Experimental section (PDF). Movie S1 (MPG).

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Major Program of National Natural Science Foundation of China (21334005), the major Inter national (Regional) Joint Research Project 445

DOI: 10.1021/acsmacrolett.7b00957 ACS Macro Lett. 2018, 7, 442−446

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ACS Macro Letters (16) Bakarich, S. E.; Panhuis, M. i. h.; Beirne, S.; Wallace, G. G.; Spinks, G. M. Extrusion printing of ionic−covalent entanglement hydrogels with high toughness. J. Mater. Chem. B 2013, 1 (38), 4939. (17) Chen, Q.; Zhu, L.; Zhao, C.; Wang, Q.; Zheng, J. A robust, onepot synthesis of highly mechanical and recoverable double network hydrogels using thermoreversible sol-gel polysaccharide. Adv. Mater. 2013, 25 (30), 4171−6.

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DOI: 10.1021/acsmacrolett.7b00957 ACS Macro Lett. 2018, 7, 442−446