Monodomain hydrogels prepared by shear-induced orientation and subsequent gelation

Abstract

Biomimetic soft materials with well-ordered structures have received increasing attention due to their potential applications in artificial tissues, soft actuators, and optical devices. We demonstrate here the synthesis of anisotropic hydrogels from concentrated liquid crystalline solution of hydroxypropylcellulose (HPC; content ≥ 45 wt%) with high viscosity. Monodomain precursor solution of HPC, in the presence of reactants, is obtained by mechanical shearing, which is immediately subjected to UV light irradiation to trigger the polymerization and thus to freeze the ordered structure of HPC before severe structural relaxation. The as-prepared HPC-containing poly(2-acrylamido-2-methylpropanesulfonic acid) gels are transparent and show uniform birefringence, Δn of 1.7 × 10⁻³, and pronounced anisotropic mechanical properties, with larger tensile modulus and breaking stress, yet smaller breaking strain when stretched parallel to the shear direction than those perpendicular to the shear direction. The gels also show anisotropic swelling ratio, with larger value in the direction perpendicular to the shear direction. The swelling anisotropy decreases from 1.22 in water to 1.09 in 1.5 M saline solution, which is related to the weakening of optical and mechanical anisotropy of swollen gels. This approach to prepare anisotropic gels should be suitable to other LC natural and synthetic macromolecules with high viscosity.

---

Introduction

Most soft bio-tissues are in a gel state and have hierarchically ordered structures at different scales, which are crucial for executing the functions of living organisms.¹ For example, Myosin assembles into liquid crystalline (LC) structures in sarcomere, contributing to the contraction and smooth motion of muscle fibers.² However, conventional synthetic hydrogels only have amorphous structures that limit their applications in optical devices, artificial tissues, etc. Inspired by the biological gels, there are many efforts to develop hydrogels with ordered structures at different scales. These ordered structures can be formed in hydrogels prior to or during the gelation process by molecular self-assembly, phase separation, and by applying electric or magnetic fields.^3–13 For example, Dobashi et al. have prepared physical LC hydrogels by dialyzing aqueous solutions of DNA or curdlan in a multivalent metallic ion solution, in which diffusion of cationic ion induces the molecular orientation and gelation of negatively charged, rigid biomacromolecules to form anisotropic structure.^14,15 Anisotropic graphene oxide (GO) gels are prepared by polymerization of GO-containing precursor solution under magnetic field.¹⁶ After reducing GO into reduced graphene oxide, the gels show anisotropic electroconductivity. In the above examples, the LC components usually have a low concentration, so that they are readily oriented under external fields in the precursor solutions with relatively low viscosity.

Shear-induced orientation is another facile approach to align the polydomain LC solution into monodomain.^17–19 After the cessation of shear, structural relaxation occurs due to the relaxation of chains. If the LC solution has relatively low viscosity, the monodomain alignment induced by shearing disappears within several seconds, which is not sufficient for arresting the preformed structure by forming permanent gel matrix. On the other hand, some biomacromolecules such as sodium alginate and hydroxypropylcellulose (HPC) only show LC phase in concentrated solutions, which usually have very high viscosity.^20–22 LC molecules in such viscous solutions are not easy to be aligned under electric or magnetic field due to the large resistance of molecular rotation. However, shear-induced orientation should be an ideal approach. The high viscosity results in slow structural relaxation after the cessation of shear, providing a “time window” to freeze the ordered structure by a gelation process. We attempt to use this method to align LC HPC solution and thus to prepare monodomain hydrogels.

HPC is a water soluble cellulose derivative, and its aqueous solutions show LC phase above a critical low concentration, , of ∼40 wt% and has a lower critical solution temperature (LCST) of ∼45 °C.^20,23 Polydomain LC HPC solutions can be oriented by mechanical shearing.^24–26 After the cessation of shear, the monodomain alignment gradually disorders due to the relaxation of HPC chains. To obtain anisotropic materials, the transient ordered structure of HPC induced by shearing should be frozen before the relaxation. Godinho et al. have fabricated anisotropic HPC film by blade coating and subsequent solvent evaporation under dry atmosphere.^27–29 Thus obtained films show anisotropic mechanical properties and reversible bending and unbending deformations by changing the environmental humidity. However, these films are not stable in highly humid conditions due to the absence of permanent polymer matrix. To broaden the application of HPC materials in aqueous environment, we aim to develop anisotropic HPC hydrogels.

In this paper, we investigate the shear-induced orientation and the structural relaxation of highly concentrated HPC solutions. Under polarizing optical microscope, the sheared HPC solutions, as well as HPC precursor solutions containing reactants, show strong and uniform birefringence. The birefringence gradually decreases after the cessation of shearing, yet it is still maintained to a considerable extent after 1000 s. Therefore, the monodomain alignment of HPC in the precursor solution can be frozen by subsequent polymerization and gelation process. The resultant hydrogels are transparent and present anisotropic optical, swelling, and mechanical properties, which should be an ideal candidate material for optical devices, soft actuators, etc. This approach should be applicable to other viscous LC solutions of natural and synthetic macromolecules.

Experimental

Materials

HPC purchased from Sigma-Aldrich has average weight molecular weight of 1.0 × 10⁵ and molar substitution of 3.5.²⁷ 2-Acrylamido-2-methylpropanesulfonic acid (AMPS; monomer), N,N′-methylenebis(acrylamide) (MBAA; crosslinker), and Irgacure 2959 (photoinitiator) were used as received from Sigma-Aldrich. The chemical structures of HPC, AMPS, and MBAA are shown in Fig. 1a. Milli-Q (18.2 MΩ) water was used in all experiments.

Fig. 1 (a) Chemical structures of HPC, AMPS, and MBAA. (b) Schematic for the preparation of anisotropic hydrogel by shear-induced orientation of LC HPC solution and subsequent gelation process. (c) POM images of LC HPC solution before (i) and after (ii) mechanical shearing, and the as-prepared gel (iii). Double arrow indicates the shear direction. Scale bar, 500 μm. A: analyzer; P: polarizer.

Shear-induced orientation and structural relaxation of HPC solution

Aqueous solutions of HPC with different concentrations were prepared by dissolving prescribed amount of HPC in water, which were kept in 4 °C refrigerator and stirred every day for at least 4 weeks, until used. The solutions were centrifuged (Velocity 18R Refrigerated Centrifuge) under 14 000 rpm at 15 °C for 30 min to remove the bubbles inside. These solutions showed LC mesophase when the concentration is above of ∼40 wt%. The viscosity change of HPC solutions with shear rate was studied using rheometer with parallel plate setup (ARG-2, TA). For the shear-induced orientation experiment, a certain amount of HPC solution was put atop a glass substrate and covered by another glass with a gap of 0.5 mm. Reciprocating shearing of HPC solution was performed at room temperature by using slide rail with controllable movement distance and speed. In this work, the distance was 1 cm, and the linear speed was in the range of 100–2000 mm min⁻¹, corresponding to shear rate of 3.2–64 s⁻¹. After 100 times reciprocating shear, the samples were immediately observed under polarizing optical microscope (POM) to monitor the structural relaxation and birefringence change with time. The optical retardation, R, was estimated by comparing the birefringence colors with a Michal-Levy chart.³⁰ The birefringence, Δn, is calculated by Δn = R/T, in which T is the thickness of the sample. The relaxation of HPC-containing precursor solutions was observed following a similar process.

Preparation of anisotropic HPC-containing hydrogels

The HPC-containing precursor solution was prepared by dissolving prescribed amount of AMPS, MBBA, and Irgacure 2959 in the homogeneous LC HPC (45 wt%) solution. After another 24 h, the precursor solution was poured into the reaction cells consisting of a pair of parallel glass plates with 0.5 mm spacing. After 100 times reciprocating shear, the sample was immediately exposed to UV light irradiation (365 nm, 5 mW cm⁻²; UVHAND100, Hönle) for 2.5 h. The photo-initiated polymerization and crosslinking froze the long-range ordered structure of HPC (Fig. 1b). To avoid the thermal induced phase separation of HPC, the sample was placed on cold plate with controlled temperature (15 °C) for polymerization. The as-prepared gel was swelled in water or saline solution with different concentration of NaCl, C_NaCl, for several days to achieve the equilibrium.

The precursor solutions and hydrogels are coded as xHPC–yAMPS and xHPC–yPAMPS, respectively, in which x and y are the concentration in wt% of HPC and AMPS. In the precursor solutions, the concentrations of MBAA and Irgacure 2959 were kept as 9 mol% and 4 mol% (relative to the monomer), respectively.

Polarizing optical microscope observation

The birefringence of LC HPC solution before and after mechanical shear was observed under polarizing optical microscope (POM; XS-402, Nanjing Xinxing Optical Instrument Co., Ltd.) with and without 530 nm tint plate. Images were taken by a camera coupled with the microscope at different time after the shearing. The birefringence of as-prepared and swollen gels was also observed under POM and calculated similar to that of sheared HPC solutions.

Swelling ratio of hydrogels

The as-prepared anisotropic gels were cut into rectangle or dumbbell shape and then swelled in a large amount of water or saline solutions with different C_NaCl. The swelling ratio in length, S, was measured by S = l/l₀, in which l and l₀ are the length (or width) of the as-prepared and swollen gel in the direction parallel or perpendicular to the shear direction; corresponding swelling ratios are noted as S_∥ and S_⊥. The anisotropy of swelling ratio, A, was calculated by A = S_⊥/S_∥. For comparison, the swelling ratio of PAMPS gel with identical composition of reagents was also measured.

Tensile test of hydrogels

The mechanical properties of the anisotropic gels in the as-prepared and swollen states were measured by tensile test (Reger Co., Ltd, RWT10) at room temperature. The gels were cut into a dumbbell shape standardized as the JIS-K6251-7 size (length: 35 mm; gauge length: 12 mm; width 2 mm) in the direction parallel or perpendicular to the previous shear direction.^31,32 The dumbbell shaped samples were swollen in saline solutions with different C_NaCl. The stress–strain curves were recorded while the samples were stretched at a constant velocity of 5 mm min⁻¹. The tensile modulus of gels was calculated based on the initial slope of the stress–strain curve (strain < 5%). The parameters of mechanical properties, including the tensile modulus E, breaking stress σ_b, and breaking strain ε_b, were the average of six parallel measurements.

Small-angle X-ray scattering

The anisotropic structure of as-prepared and swollen HPC/PAMPS gels was characterized by small-angle X-ray scattering (SAXS) performed at BL16B beamline of Shanghai Synchrotron Radiation Facility (SSRF). The wavelength of radiation source is 0.124 nm, and the sample-to-detector distance was 2 m. The sample was loaded in a sample cell covered by the X-ray-transparent polyimide thin film in X-ray transmission direction.

Scanning electron microscopy (SEM)

The structure of gels was observed by field emission scanning electron microscope (SEM; Hitachi S4800). The samples were prepared by freeze-drying and then cryogenically fractured in liquid nitrogen. Before SEM characterization, the fractured surface was coated with a thin layer of gold by the sputtering method.

Results and discussion

The HPC solutions with concentration above show LC mesophase (Fig. 1c(i)); however, the birefringence is not uniform, indicating the absence of long-range orientation. The polydomain LC solution can be readily oriented to monodomain one under mechanical shearing, which induces orientation of HPC along the shear direction. Strong and uniform birefringence can be observed during or after the shearing for a period of time (Fig. 1c(ii)). Because of the high viscosity, the structural relaxation of sheared HPC solution is slow.^33,34 The shear-induced monodomain alignment of HPC can be arrested by forming a permanent gel matrix. By shearing the precursor solution and subsequently irradiating it under UV light, monodomain HPC-containing poly(AMPS) hydrogel (HPC/PAMPS gel) is developed, which shows uniform birefringence (Fig. 1c(iii)).

The shear-induced orientation of HPC solution and its structural relaxation are crucial for the synthesis of anisotropic hydrogels. There should be a critical shear rate to induce the transformation of LC HPC solution from polydomain to monodomain. The variation of viscosity with the shear rate, [small gamma, Greek, dot above], is studied by rheology at room temperature (Fig. 2a). HPC solutions are non-Newtonian fluid; their viscosities at [small gamma, Greek, dot above] = 10⁻² s⁻¹ are higher than 1000 Pa s, which gradually decrease with the increase of shear rate in the range of 10⁻² to 10¹ s⁻¹.³⁵ In this range of shear rate, the viscosity of 45HPC–13AMPS and 45HPC–18AMPS solutions is higher than that of 45 wt% HPC solution. This is because AMPS is a strong acid, the addition of which decreases the content of free water and increases in the viscosity of HPC solution.³⁶ As [small gamma, Greek, dot above] increases further, the viscosity of HPC solutions rapidly decreases by two orders of magnitude at a narrow range of shear rate, 10–50 s⁻¹, corresponding to the transition from tumbling to flow-aligning of LC HPC.^35,37 Therefore, the critical shear rate, [small gamma, Greek, dot above]_c, to from monodomain nematic solution is ∼50 s⁻¹, after which the viscosity reaches a plateau. In the following experiments, the shear rate is set as 64 s⁻¹, the accessible maximum of the slide rail.

Fig. 2 (a) variation of viscosity of HPC and precursor solutions as a function of shear rate. (b, c) POM images of 45 wt% HPC solution (b) and 45HPC–13AMPS solution (c) taken at different time after shearing. The images in upper and bottom row were observed under crossed polarizers without and with 530 nm tint plate, respectively. Scale bar, 500 μm. A: analyzer; P: polarizer; X′: fast axis of the tint plate; Z′: slow axis of the tint plate. (d) Variation of birefringence as a function of relaxation time.

After the cessation of shearing, HPC solutions, as well as HPC-containing precursor solutions, show strong and uniform birefringence. However, the ordered structure in viscous solution is not stable and will gradually disorder and disappear with time due to the relaxation of HPC chains. The structural relaxation and birefringence variation of sheared solutions are observed in situ under POM. The birefringent color of sheared 45 wt% HPC gradually changes from blue to white-gray after 40 min, indicating that the monodomain alignment has relaxed to be globally isotropic (Fig. 2b). The HPC-containing precursor solutions show similar phenomena (Fig. 2c). The variation of birefringence, Δn, with the relaxation time, t, is shown in Fig. 2d. As expected, the birefringence quickly decreases with the time, especially within the first 1000 s. However, the sheared solutions still have considerable birefringence. Δn of 50 and 60 wt% HPC solutions at t = 1000 s is 1.4 × 10⁻³ and 7.4 × 10⁻⁴, respectively. In the HPC-containing precursor solution, the presence of reactants, especially the strongly acidic monomer, retards the relaxation process and thus enhances the birefringence. At t = 5 s, Δn of 45HPC–18AMPS solution is 2.5 × 10⁻³, two times that of 45 wt% HPC solution. At t = 1000 s, Δn of 45HPC–18AMPS and 45HPC–13AMPS solutions decreases to 1.4 × 10⁻³ and 8.6 × 10⁻⁴, respectively. The slow structural relaxation within 1000 s provides sufficient time to freeze the ordered structure by forming a permanent gel matrix.

After shearing, the HPC-containing precursor solution is subsequently exposed to UV light irradiation, which initiates the polymerization and crosslinking reactions. Thus obtained as-prepared gel of 45HPC–18PAMPS shows strong and uniform birefringence with Δn of 1.7 × 10⁻³, about 70% that of sheared precursor solution before UV light irradiation. Compared to the variation of solution birefringence with the relaxation time (Fig. 2d), the gelation time should be ∼8 min, after which the ordered structure of HPC is frozen in the gel matrix. This anisotropic structure of as-prepared HPC/PAMPS gel is also confirmed by SAXS measurement, which will be discussed in the following section.

After being swelled in water, the gel expands its volume due to the polyelectrolyte nature of PAMPS. Because of the anisotropic structure, the swelling of HPC/PAMPS gel is anisotropic; the swelling ratio in the direction parallel to the shear direction, S_∥ of 1.4, is smaller than that perpendicular to the shear direction, S_⊥ of 1.7 (Fig. 3a). The swelling ratio of PAMPS gel in water is 1.8 (Table 1). This result indicates that the incorporation of aligned HPC restrains the swelling of gel along the orientation of HPC, yet the swelling of gel perpendicular to the orientation of HPC is almost free. The swelling ratio of monodomain hydrogel decreases with the increase in C_NaCl of saline solutions where the gel is incubated (Fig. 3b). The anisotropy of swelling ratio, A = S_⊥/S_∥, also decreases with C_NaCl, as expected, from 1.22 in pure water to 1.09 in 1.5 M saline solution.

Fig. 3 (a) Photos of 45HPC–18PAMPS gel swollen in water (left) and 1.5 M saline solution (right). The samples numbered 1, 2, 3 are the as-prepared gel and swollen gels cut perpendicular and parallel to the shear direction, respectively. Scale bar, 1 cm. (b) Swelling ratio of the swollen gels in water and saline solutions measured in the direction parallel and perpendicular to the shear direction.

Table 1 Swelling ratio S, birefringence Δn, tensile modulus E, breaking strain, ε_b, and breaking stress σ_b of the as-prepared and swollen PAMPS gels and HPC/PAMPS gels.∥and ⊥ indicate the direction parallel and perpendicular to the shear direction, respectively

Hydrogels		S	Δn	E (kPa)	εb (%)	σb (kPa)
PAMPS	As-prepared	—	—	132 ± 33.1	46.2 ± 7.3	84.1 ± 13.8
	Water	1.78 ± 0.05	—	783 ± 35.8	9.5 ± 1.0	76.5 ± 1.3
	1 M NaCl	1.41 ± 0.07	—	158 ± 10.4	23.6 ± 5.0	48.1 ± 3.1
HPC/PAMPS	As-prepared ⊥	—	1.7 × 10^−3	285 ± 28.9	140.1 ± 10.1	446.8 ± 19.8
	As-prepared ∥	—		672 ± 73.9	91.8 ± 10.1	609.8 ± 16.8
	Water ⊥	1.66 ± 0.03	1.7 × 10^−5	262 ± 30.4	8.5 ± 0.4	22.6 ± 4.3
	Water ∥	1.36 ± 0.05		428 ± 23.2	7.5 ± 0.1	23.9 ± 4.4
	0.4 M NaCl ⊥	1.47 ± 0.03	2.5 × 10^−5	100 ± 8.7	9.9 ± 2.4	13.2 ± 0.7
	0.4 M NaCl ∥	1.25 ± 0.02		127 ± 16.2	12.4 ± 1.4	19.5 ± 0.9
	1 M NaCl ⊥	1.41 ± 0.02	4.4 × 10^−5	88 ± 16.2	19.6 ± 3.2	16.1 ± 1.3
	1 M NaCl ∥	1.25 ± 0.02		107 ± 26.9	16.6 ± 2.8	16.9 ± 1.4
	1.5 M NaCl ⊥	1.37 ± 0.02	9.2 × 10^−5	44.8 ± 2.0	24.3 ± 2.4	13.4 ± 0.1
	1.5 M NaCl ∥	1.26 ± 0.03		66.3 ± 2.6	23.3 ± 1.2	18.4 ± 0.1

---

The swelling of monodomain gel in water or saline solution results in variation of birefringence. As shown in Fig. 4a, the birefringent color of gel under crossed polarizers changes from orange in the as-prepared state to white-gray in the swollen state in 1.5 and 0.4 M saline solutions, respectively. The birefringence of gel decreases from 1.7 × 10⁻³ in the as-prepared state to 9.2 × 10⁻⁵ in the swollen state (1.5 M saline solution). The decrease in C_NaCl leads to further decrease in birefringence. This is because (i) the volume expansion of gel results in dilution of localized HPC concentration and (ii) the relatively larger swelling ratio in the direction perpendicular to the shear direction weakens the anisotropic structure of HPC. The later one is confirmed by SAXS measurements of the gels (Fig. 5). The as-prepared 45HPC–18PAMPS hydrogel shows an ellipsoid scattering pattern with a long axis in the meridian (ratio of major axis to minor axis, L₁/L₂, is 1.35). The elliptical scattering pattern indicates that the average distance between HPC molecules aligned parallel to the shear direction is slightly shorter than that aligned perpendicular to the shear direction. In contrast, the scattering pattern of swollen gel is almost isotropic (L₁/L₂, is 1.09). The decrease in structural anisotropy is associated with the anisotropic swelling. However, the anisotropic structure is still maintained to some extent in the swollen gel, as verified by SEM observation (Fig. 6). The 45HPC–18PAMPS swollen gel has anisotropic porous structure, whereas the conventional PAMPS gel only has isotropic one. We should note that the micron-scaled pore size of gel after freezing-dry is not the network mesh size of gel in the swollen state,³⁸ which is in the range of 1–10 nm.³⁹

Fig. 4 POM images (a) and birefringence (b) of the as-prepared and swollen 45HPC–18PAMPS gel in saline solutions. The upper and bottom images in (a) are taken under crossed polarizers without and with tint plate. Scale bar, 500 μm.

Fig. 5 SAXS measurements of the as-prepared (a) and swollen (b) HPC/PAMPS hydrogels. Schemes for scattering position on the specimen are shown atop the scattering pattern; the arrow shows the shear direction. The insets are the schematic of ellipse with the major axis L₁ and minor axis L₂ to show the anisotropy of scattering pattern.

Fig. 6 (a) Schematic to show the sample for SEM observation. (b, c) SEM images of the cross-section of swollen HPC/PAMPS gel (a) and neat PAMPS gel (b).

The anisotropic structure of gels also results in anisotropic mechanical properties. The tensile stress–strain curves of the as-prepared and swollen 45HPC–18PAMPS gels are shown in Fig. 7. Samples are cut from the bulk gels parallel or perpendicular to the shear direction. The as-prepared anisotropic gel has breaking stress σ_b of 610 kPa and tensile modulus E of 670 kPa, when the sample is stretched along the shear direction, higher than those of 450 kPa and 290 kPa, when stretched perpendicular to the shear direction (Fig. 7a). However, the as-prepared gel shows better extensibility (larger breaking strain, ε_b) in the direction perpendicular to the shear direction. When compared to the conventional PAMPS gel, the as-prepared HPC-containing PAMPS gel shows better mechanical properties (Table 1). After being swelled in water and saline solutions, the anisotropic gel become weak and brittle (Fig. 7b). σ_b and ε_b of swollen gel are smaller than those of as-prepared gel; however, the anisotropy of mechanical performances is still evident. The swollen gel has lager E and σ_b, yet smaller ε_b when stretched parallel to the shear direction, similar to the behaviors of as-prepared gel (Table 1). As C_NaCl increases, the swollen gel becomes softer and ductile. Meanwhile, the mechanical anisotropy weakens. This result is consistent with the change of optical and swelling anisotropy. The brittleness of swollen gels is due to the nature of PAMPS, which is a strong polyelectrolyte; the gel severely swells in water, making the matrix rigid yet fragile. In the presence of salt, the polyelectrolyte network contracts to some extent due to the electrostatic screening effect. This also merits tuning the anisotropic optical, swelling degree, and mechanical properties of gels by C_NaCl of the incubated solution. Other hydrogels can be used to develop such kind of hybrid anisotropic hydrogels, making them response to external stimulations such as temperature, pH, and light irradiation.

Fig. 7 Tensile stress–strain curves of as-prepared (a) and swollen (b) 45HPC–18PAMPS gels stretched parallel or perpendicular to the shear direction. The swollen gels were incubated in water and saline solution with different C_NaCl to achieve equilibrated state before measurements.

Conclusions

We have developed a transparent monodomain HPC-containing hydrogels based on shear-induced orientation of LC HPC and subsequent gelation process. Thus obtained gels show anisotropic optical, swelling capacity, and mechanical properties. The monodomain structure is formed by shear-aligning of a polydomain LC solution of HPC. After the cessation of shearing, the anisotropic structure is retained for a considerable period of time due to the high viscosity and slow relaxation of polymer chains, providing a “time window” to freeze the ordered structure by polymerization and crosslinking reactions. The as-prepared gel shows strong and uniform birefringence with Δn of 1.7 × 10⁻³, as well as evident mechanical anisotropy. After swelling the gel in water, the swelling ratio in length of gel perpendicular to the shear direction is 1.2 times that parallel to the shear direction, leading to a decrease in structural anisotropy. Therefore, the swollen gel shows weakened anisotropic optical and mechanical properties. The anisotropic properties of swollen gels are responsive to C_NaCl of the incubated solution due to the polyelectrolyte nature of gel matrix. This monodomain hydrogels should find applications as optical devices, artificial muscles, etc. The method presented here should be suitable to other natural and synthetic rigid macromolecules, of which LC solutions have high viscosity and are easily aligned by mechanical shearing.

Acknowledgements

This research was supported by Natural Science Foundation of Zhejiang Province, China (Y14E030021), National Natural Science Foundation of China (51403184), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.

Notes and references

1. L. P. Gartner and J. L. Hiatt, Color Textbook of Histology, Saunders, Philadelphia, 2nd edn, 2001.
2. C. M. Coppin and P. C. Leavis, Biophys. J., 1992, 63, 794.
3. Y. Kang, J. J. Walish, T. Gorishnyy and E. L. Thomas, Nat. Mater., 2007, 6, 957.
4. Z. L. Wu and J. P. Gong, NPG Asia Mater., 2011, 3, 57.
5. J. A. Kelly, A. M. Shukaliak, C. C. Y. Cheung, K. E. Shopsowiz, W. Y. Hamad and M. J. MacLachlan, Angew. Chem., Int. Ed., 2013, 52, 8912.
6. X. Pei, T. Zan, H. Li, Y. Chen, L. Shi and Z. Zhang, ACS Macro Lett., 2015, 4, 1215.
7. T. Pullawan, A. N. Wilkinson and S. J. Eichhorn, Biomacromolecules, 2012, 13, 2528.
8. Z. L. Wu, H. Furukawa, W. Yang and J. P. Gong, Adv. Mater., 2009, 21, 4696.
9. T. Inadomi, S. Ikeda, Y. Okumura, H. Kikuchi and N. Miyamoto, Macromol. Rapid Commun., 2014, 35, 1741.
10. F. Kleinschmidt, M. Hickl, K. Saalwachter, C. Schmidt and H. Finkelmann, Macromolecules, 2005, 38, 9772.
11. R. Takahashi, Z. L. Wu, M. Arifuzzaman, T. Nonoyama, T. Nakajima, T. Kurokawa and J. P. Gong, Nat. Commun., 2014, 5, 4490.
12. K. Amornwachirabodee, M. K. Okajima and T. Kanoko, Macromolecules, 2015, 48, 8615.
13. S. Wang, A. Lu and L. Zhang, Prog. Polym. Sci., 2016, 53, 169.
14. T. Dobashi, M. Nobe, H. Yoshihara and A. Konno, Langmuir, 2004, 20, 6530.
15. T. Dobashi, K. Furusawa, E. Kita, Y. Minamisawa and T. Yamamoto, Langmuir, 2007, 23, 1303.
16. L. Wu, M. Ohtani, M. Takata, A. Saeki, S. Seki, Y. Ishida and T. Aida, ACS Nano, 2014, 8, 4640.
17. M. Singh, V. Agarwal, D. D. Kee, G. McPherson, V. John and A. Bose, Langmuir, 2004, 20, 5693.
18. S. M. Zhang, M. A. Greenfield, A. Mata, L. C. Palmer, R. Bitton, J. R. Mantei, C. Aparicio, M. O. de la Cruz and S. I. Stupp, Nat. Mater., 2010, 9, 594.
19. M. A. Haque, G. Kamita, T. Kurokawa, K. Tsujii and J. P. Gong, Adv. Mater., 2010, 22, 5110.
20. R. S. Werbowyj and D. G. Gray, Macromolecules, 1980, 13, 69.
21. T. Nguyen, B. U. Peres, R. M. Carvalho and M. J. MacLachlan, Adv. Funct. Mater., 2016, 26, 2875.
22. I. W. Hamley, Soft Matter, 2010, 6, 1863.
23. S. Fortin and G. Charlet, Macromolecules, 1989, 22, 2286.
24. K. M. O. Hakansson, A. B. Fall, F. Lundell, S. Yu, C. Krywka, S. V. Roth, G. Santoro, M. Kvick, L. P. Wittberg, L. Wagberg and L. D. Soderberg, Nat. Commun., 2014, 5, 4018.
25. P. Navard, J. Polym. Sci., Polym. Phys. Ed., 1986, 24, 435.
26. R. Chiba and Y. Nishio, Biomacromolecules, 2006, 7, 3076.
27. Y. Geng, P. L. Almeida, S. N. Fernandes, C. Cheng, P. Palffy-Muhoray and M. H. Godinho, Sci. Rep., 2013, 3, 1028.
28. M. H. Godinho, J. G. Fonseca, A. C. Ribeiro, L. V. Melo and P. Brogueira, Macromolecules, 2002, 35, 5932.
29. C. Echevarria, L. E. Aguirre, E. G. Merino, P. L. Almeida and M. H. Godinho, ACS Appl. Mater. Interfaces, 2015, 7, 21005.
30. F. D. Bloss, An Introduction to the Methods of Optical Crystallography, Holt Rinehart and Winston, New York, 1961.
31. Z. L. Wu, T. Kurokawa, D. Sawada, J. Hu, H. Furukawa and J. P. Gong, Macromolecules, 2011, 44, 3535.
32. Z. L. Wu, D. Sawada, T. Kurokawa, A. Kakugo, W. Yang, H. Furukawa and J. P. Gong, Macromolecules, 2011, 44, 3542.
33. A. Patra, P. K. Verma and R. K. Mitra, J. Phys. Chem. B, 2012, 116, 1508.
34. M. Rubinstein and R. H. Colby, Polymer Physics, Oxford University Press, New York, 2003.
35. K. Hongladarom, V. Secakusuma and W. R. Burghardt, J. Rheol., 1994, 38, 1505.
36. S.-P. Rwei and M.-S. Lyu, Cellulose, 2012, 19, 1065.
37. R. G. Larson, The Structure and Rheology of Complex Fluids, Oxford University Press, New York, 1999.
38. The network structure of gel is influenced to some extent by the freeze-drying process. The micron-scaled porous structure in SEM image should be related to the micron-sized ice crystals formed during the “freezing” and removed during the “drying” process.
39. S. Oogaki, G. Kagata, T. Kurokawa, S. Kuroda, Y. Osada and J. P. Gong, Soft Matter, 2009, 5, 1879.

---