Y. Nakagawaa,
S. Ohtab,
M. Nakamurac and
T. Ito*ab
aDepartment of Chemical System Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
bCenter for Disease Biology and Integrative Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: taichi@m.u-tokyo.ac.jp
cGraduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan
First published on 7th December 2017
Novel precursor polymers for three-dimensional (3D) inkjet printing of hydrogels are required for various biomedical applications. We have, for the first time, investigated 3D inkjet printing of ionically cross-linked star block copolymer hydrogels, for which the precursor polymer has a dendritic polyester core, a poly(oligo(ethylene glycol) methyl ether acrylate) inner layer, and a poly(acrylic acid) outer layer. The star block copolymer solution (8.0 wt%) showed a viscosity of 7–8 mPa s, which is suitable for inkjet printing. This solution formed a homogeneous hydrogel upon the addition of metallic ions, such as the zinc, copper(II), aluminum, and ferric ion. The elasticity of the resulting hydrogels was dependent on the ion species. The rapid sol–gel transition induced by the metallic ions enabled 3D inkjet printing of the star block copolymer hydrogels, through ejection of the star block copolymer solution and subsequent ionic cross-linking to achieve layer-by-layer deposition of the gelled droplets.
While various hydrogels can be 3D-printed through robotic dispensing,5,15,16 only a few materials, such as alginate (or its combination with other materials)5 and fibrin,17,18 have been reported as useful for 3D inkjet printing. Since the suitable viscosity range for the ink-jetting process is very low, usually 3.5–12 mPa s,15 the 3D inkjet printing of hydrogels has been achieved mainly through an in-process cross-linking approach,19 where the ejection of precursor solution and subsequent cross-linking of the droplets are repeatedly conducted to achieve layer-by-layer deposition of the hydrogel. In this process, extremely fast gelation, as well as low viscosity of the precursor solution, is required to avoid the droplet spreading upon impact onto the already-gelled surface. These strict requirements have precluded use of hydrogels other than alginate or fibrin. Therefore, further investigation of 3D-inkjet-printable hydrogels that allow a facile molecular design, as well as large-scale synthesis in an affordable manner, is required.
We have previously reported a star block copolymer that can form a homogeneous hydrogel without polymer precipitation through mixing with calcium ion (Ca2+)20 or ferric ion (Fe3+).21 This star block copolymer has a dendritic polyester (DPE) core, a poly(oligo(ethylene glycol) methyl ether acrylate) (polyOEGA) inner layer, and a poly(acrylic acid) (PAA) outer layer (Fig. 1a). In the current study, 3D inkjet printing of this synthetic, star block copolymer hydrogel was investigated. Since the star block copolymer solution shows fast gelation upon the addition of metallic ions, this material can be used in 3D inkjet printing through an in-process cross-linking approach (Fig. 1b).
The honey-comb-shaped star block copolymer hydrogels were 3D-printed at room temperature using layer-by-layer printing of 20 images onto the gel substrate mentioned above using the star block copolymer solution as an ink. According to the previous report published by our group,24 process parameters were set as follows. Distance from the printer to the substrate was fixed at 4 mm. Head-speed and dot-pitch were 32000 μm s−1 and 48 μm, respectively. The 3D-printed hydrogels were removed from the substrate through the gentle addition of saline onto the substrate to allow the hydrogels to float.
With a view to using the star block copolymer solution as a precursor ink, the printability of the material through an inkjet nozzle was examined. We measured the solution viscosity of the star block copolymer at varying shear rate (Fig. 2), which is an important measurement to determine printability. The solution viscosity depended on the polymer concentration and varied from 3–4 mPa s at 5 wt% to 54–56 mPa s at 20 wt%. From this result, considering that the viscosity range required for inkjet-printable materials is reported to be 3.5–12 mPa s,15 a star block copolymer solution at a concentration below 10 wt% would be preferable for inkjet-printing. Therefore, for the following experiments, we fixed the polymer concentration at 8.0 wt%, where the solution viscosity was 7–8 mPa s.
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Fig. 2 Viscosity of the star block copolymer solution at different concentrations as a function of shear rate. Saline was used as a solvent. |
Compared with conventional precursor polymers for the 3D inkjet printing of hydrogels, such as alginate, the star block copolymer showed a much lower solution viscosity. The viscosity of the 5.0 wt% star block copolymer solution was 3–4 mPa s, whereas that of a 5.0 wt% alginate solution was 500–1500 mPa s (data not shown). Alginate shows high solution viscosity because of the rigidity of the polymer chain, especially at GG blocks, and electrostatic repulsion arising from the carboxylic moieties. As a result of this high solution viscosity, the concentration of an inkjet-printable alginate solution is reported to be at most ∼2 wt%.25 On the other hand, the solution viscosity of the star block copolymer was within the viscosity range of inkjet-printable materials (3.5–12 mPa s)15 even at 10 wt%. The star-shaped structure would contribute to the decrease of the solution viscosity; star-shaped polymers have been reported to show lower solution viscosity compared with linear polymers with the same molecular weights.26 This low solution viscosity of the star block copolymer is one of the advantages when considering its application as an ink material.
We then evaluated the gelation ability of the star block copolymer solution through cross-linking using several metallic ions, such as zinc (Zn2+), copper(II) (Cu2+), aluminum (Al3+), and Fe3+. While use of Ca2+ or Fe3+ as a cross-linker to generate the star block copolymer hydrogel has been previously reported by our group,20,21 that of other metallic ions has not been investigated. We simply added 20 μL of the metallic ion solution in saline to 20 μL of the 16 wt% star block copolymer solution in saline (Fig. S1†). The star block copolymer solution showed gelation upon addition of Zn2+, Cu2+, Al3+, or Fe3+ aqueous solution at a concentration above a certain threshold. Interestingly, the amount of the metallic ion required to induce gelation was different depending on the ion species in the order of Zn2+ > Cu2+ > Al3+ ∼ Fe3+.
We further evaluated the viscoelasticity of the obtained hydrogels. We fixed the ratio of the concentration of the metallic ion to that of the carboxylate ([Mn+]/[COO−]) at 1.0. We measured the frequency dependencies of the storage modulus (G′) and loss modulus (G′′) of the hydrogels using a rheometer (Fig. 3). The measured G′ and G′′ of the star block copolymer hydrogels cross-linked with Zn2+, Cu2+, Al3+, or Fe3+ were frequency-independent, and the G′ was larger than G′′. These results indicate that all the obtained hydrogels behaved as viscoelastic solids, regardless of the ion species. In addition, the hydrogels showed a different viscoelasticity depending on the ion species used. The loss tangent (tanδ), which is a ratio of G′′ to G′, of the hydrogels cross-linked using Zn2+, Cu2+, Al3+, or Fe3+ was 0.554, 0.415, 0.195, and 0.112, respectively (Fig. S2†). These results indicate that the elasticity of the hydrogels increases with the ion species, in the following order: Fe3+ > Al3+ > Cu2+ > Zn2+. To evaluate the gelation time, the time dependence of G′ and G′′ of these materials was also measured at a frequency of 1 Hz (Fig. S3†). As a result, in case of the star block copolymer containing Cu2+, Al3+, or Fe3+, G′ was already larger than G′′ from the beginning of the measurement, indicating that the gelation time was extremely short. In case of the star block copolymer containing Zn2+, on the other hand, G′ exceeded G′′ 16 min after the addition of Zn2+, suggesting that gelation time of this sample was relatively longer compared to the others.
The observed difference in the threshold concentration of the metallic ions for gelation and the viscoelasticity of the resulting hydrogels is considered to result from the variation in the stability constant of the metal–carboxylate complex depending on the ion species. Henderson et al.27 reported that the life-time and strength of the ionic cross-links are determined mainly by the bond stability of the metal–ligand complex, which has a linear relationship with the thermodynamic equilibrium constant of the complex formation. The life-time and strength of the cross-links are considered to affect the cross-linking density, which would further determine the gelation threshold and stiffness of the resulting hydrogel.
Finally, we examined 3D inkjet printing of the star block copolymer hydrogels cross-linked using metallic ions. We used a custom-made inkjet printer (Fig. 4a), as reported previously.24 The hydrogels were 3D-printed via layer-by-layer printing of 20 images, all of which have the same honey-comb-shaped design. To achieve 3D hydrogel printing through an in-process cross-linking approach, the 8.0 wt% star block copolymer solution was printed onto the agarose gel substrate containing Zn2+, Cu2+, Al3+, or Fe3+. Upon impact, the ejected droplets were gelled through the up-ward diffusion of the metallic ion (Fig. 4b). By repeating this process layer by layer, the star block copolymer hydrogel was 3D-printed. After printing, patterned 3D hydrogels cross-linked using Cu2+, Al3+, or Fe3+ were successfully formed and were transferred from the substrate, while that cross-linked using Zn2+ was just spread on the substrate and was unable to maintain its structure when transferred from the substrate (Fig. 4c). The poor 3D-printability of the hydrogel cross-linked using Zn2+ would be due to the long gelation time as mentioned above. The appearance of the 3D-printed hydrogels was different depending on the ion species used as a cross-linker; the 3D-printed hydrogels cross-linked using Fe3+ or Al3+ showed a clear edge, while that crosslinked using Cu2+ showed a rather blurred edge (Fig. 4d). These results suggest that the printing resolution was different depending on the ion species used.
The printing resolution is dependent on the droplet spreading on the substrate. Generally, the ejected droplet spreads after its impact on a substrate, leading to a lower printing resolution.28 In the 3D hydrogel printing process, droplet spreading is inhibited by gelation. Therefore, the gelation rate predominantly determines the printing resolution. In the above experiments, the gelation rate is considered to be affected by the quantity of metallic ions required to induce gelation; if more ions are required, more time is needed to allow enough ions to diffuse into the droplet. This is the reason that higher resolution was achieved with Fe3+ or Al3+, which requires less ions for gelation.
Further improvement in the printing resolution is also expected to be achieved through optimization of printing process parameters. For example, we previously found that different pattern of driving waveform generated droplets with different shapes, which would result in difference in the printing resolution. Further optimization of distance from the printer to the substrate might ameliorate the printing resolution, considering the fact that drag from air currents in the printing environment affects drop placement accuracy.28 In the current system, number of printing layers is also an important parameter to control the printing resolution, because more time is required to achieve sufficient supply of ions into the droplets deposited onto the higher layers, which would deteriorate the printing resolution of the upper layers. Since substrate surface topography is reported to affect the printing resolution for 2D printing,15 this effect should also be considered when conducting 3D inkjet printing to improve the printing resolution.
These results represent the successful 3D inkjet printing of ionically cross-linked star block copolymer hydrogels. The 3D-printable hydrogels must satisfy some criteria; they need to be self-supporting, and must gel extremely rapidly.15 In the case of the inkjet systems, in particular, the low droplet viscosity can further accelerate droplet spreading on the substrate, and therefore even faster gelation is required. As a result, 3D-inkjet-printable hydrogels have been less investigated, and alginate gels or gels involving the combination of alginate with other materials have been used in most cases.5 Other than alginate gels, a few studies have examined fibrin gels, resulting in rather blurred structures.17,18 In the current study, the star block copolymer hydrogels allowed 3D inkjet printing with high resolution comparable to alginate gels. The fast formation of homogeneous hydrogels induced by complexation with free metallic ion is considered to be a key feature, which is achieved by prevention of intra-molecular cross-linking through a strategic molecular design of the precursor polymer.20,21
Since the material presented here is composed of a synthetic polymer, it potentially allows engineering of the material by introducing biodegradable star arms or other chelating ligands instead of the carboxylates used in the current polymer, which might decrease the quantity of metallic ions required for gelation to ameliorate the printing resolution. In addition, our material has the potential for scaled-up synthesis in an affordable manner, which is important for future applications. Although a further investigation on the biocompatibility of our materials is required for future biomedical application, our previous research20,21 has suggested a high biocompatibility of the star block copolymer hydrogel cross-linked using Ca2+ or Fe3+, as well as low cytotoxicity of the precursor polymer itself. The fast gelation rate of our material would also enable 3D inkjet printing of cell-loaded hydrogels in culture media. This system represents a promising platform technology to expand the design flexibility of 3D-inkjet-printable hydrogels, which have potential for a variety of applications.
Footnote |
† Electronic supplementary information (ESI) available: Additional pictures of the hydrogels, loss tangent data of the hydrogels, and gelation kinetics. See DOI: 10.1039/c7ra11509a |
This journal is © The Royal Society of Chemistry 2017 |