Reorientation of Cellulose Nanowhiskers in Agarose Hydrogels under

Article pubs.acs.org/Biomac

Reorientation of Cellulose Nanowhiskers in Agarose Hydrogels under Tensile Loading Anayancy Osorio-Madrazo,*,†,‡,# Michaela Eder,† Markus Rueggeberg,†,§,∥ Jitendra Kumar Pandey,† Matthew James Harrington,† Yoshiharu Nishiyama,⊥ Jean-Luc Putaux,⊥ Cyrille Rochas,⊥ and Ingo Burgert†,§,∥ †

Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Wissenschafspark Golm, D-14424 Potsdam, Germany Centre de Recherches sur les Macromolécules Végétales (CERMAV-CNRS; affiliated with Université Joseph Fourier and member of the Institut de Chimie Moléculaire de Grenoble), F-38041 Grenoble cedex 9, France § Institute for Building Materials, ETH - Swiss Federal Institute of Technology Zurich, CH-8093 Zurich, Switzerland ∥ Applied Wood Materials Laboratory, EMPASwiss Federal Laboratories for Materials Testing and Research, CH-8600 Dübendorf, Switzerland ‡ Institut für Forstbenutzung und Forstliche Arbeitswissenschaft, Albert-Ludwigs University of Freiburg, D-79085 Freiburg, Germany # Freiburger Materialforschungszentrum−FMF, Albert-Ludwigs University of Freiburg, D-79104 Freiburg, Germany ⊥

ABSTRACT: Agarose hydrogels filled with cellulose nanowhiskers were strained in uniaxial stretching under different humidity conditions. The orientation of the cellulose whiskers was examined before and after testing with an X-ray laboratory source and monitored in situ during loading by synchrotron Xray diffraction. The aim of this approach was to determine the process parameters for reorienting the cellulose nanowhiskers toward a preferential direction. Results show that a controlled drying of the hydrogel is essential to establish interactions between the matrix and the cellulose nanowhiskers which allow for a stress transfer during stretching and thereby promote their alignment. Rewetting of the sample after reorientation of the cellulose nanowhiskers circumvents a critical increase of stress. This improves the extensibility of the hydrogel and is accompanied by a further moderate alignment of the cellulose nanowhiskers. Following this protocol, cellulose nanowhiskers with an initial random distribution can be reoriented toward a preferential direction, creating anisotropic nanocomposites.

■

INTRODUCTION Composite materials made of reinforced hydrogels are found in many biological systems such as plant tissues or the extracellular matrix of mammal conjunctive tissues. Specific functions and desirable behaviors often arise from the ability to anisotropically align the stiff reinforcing phase within the hydrogel matrix. A better understanding of the underlying processes is required before such concepts can be applied to technical applications. In this study, we aimed at aligning cellulose nanowhiskers (CNWs) within a bulk agarose hydrogel matrix by applying a mechanical stress, akin to the deformation of the primary cell wall of plants during cell growth. Agarose is an alternating copolymer found in some seaweeds consisting of 1,4-linked 3,6-anhydro-α-L-galactose and 1,3linked β-D-galactose derivatives. Agarose can form physical hydrogels, which have found use in many applications,1,2 for instance, as a model system to understand thermoreversible gelation mechanisms of polysaccharides.3−6 Agarose hydrogels possess a homogeneous and rigid fibrillar network morphology,5,6 which can show a linear stress−strain behavior and high extensibility depending on the intrinsic viscosity.3 A specific © 2012 American Chemical Society

feature of agarose gels is their high rigidity, which originates from the gelation of rigid polymer chains in the sol state and results in peculiar mechanical and thermal properties.4,6−8 Cellulose nanofibers have gained increasing interest as stiff and fibrous reinforcing elements in the form of CNWs in composites made up of both synthetic9−14 and natural polymer matrices,15−17 and in the form of the micro/nanofibrillated cellulose type.14,18−24 As a result of their exceptionally high stiffness (Young’s modulus: 114−140 GPa),25−29 a significant reinforcing capacity has been reported, even at low CNW content.15,30 Producing an anisotropic structure would allow one to tune the mechanical properties or to design, for instance, actuable membranes for targeted movements.31−33 However, due to difficulties in processing the materials, the high expectations concerning CNW reinforcement have remained largely unrealized.31 To exploit their remarkable mechanical properties, Received: December 11, 2011 Revised: January 31, 2012 Published: February 1, 2012 850

dx.doi.org/10.1021/bm201764y | Biomacromolecules 2012, 13, 850−856

Biomacromolecules

Article

composed of a P-1000 pump (Thermo Fisher Scientific) connected to the polyvinyl alcohol GRAL-LIN pre- and main-columns (Polymer Standards Service (PSS), Germany). The columns were thermostated at 70 °C. A total of 100 μL of filtered agarose solutions were injected and eluted with DMSO at a flow rate of 1 mL·min−1. A UV-1000 detector (Thermo Fisher Scientific) operating at 300 nm was coupled online with a Shodex RI-71 refractive index detector (MZAnalysentechnik, Germany). Pullulan (PSS, Germany) was used as the calibration standard for molecular weight determination. By using this method, we measured number-average (Mn) and weight-average (Mw) molecular weights of 9.04 × 104 and 2.34 × 105 g/mol, respectively, in agreement with values previously reported for agarose.7,8 Transmission Electron Microscopy (TEM). Drops of a 0.001% (w/v) CNW suspension were deposited on glow-discharged carboncoated copper grids. The samples were negatively stained with 2% (w/ v) uranyl acetate after removal of the liquid in excess and before complete drying. The specimens were observed using a Philips CM200 microscope operating at 80 kV. Microtensile Straining. For the tensile straining experiments, ∼2 mm wide and ∼7 mm long strips were cut out from the prepared hydrogel nanocomposite with a razorblade. Care was taken to not damage the sample edges. The samples were glued onto a foliar frame with a test span of about 7 mm by using cyanoacrylate glue (Loctite 454). Then the frame with the sample was mounted onto a tensile tester surrounded by a sealed sample chamber which allowed to control relative humidity (RH) during the experiment. The humidity generator (Wetsys, Setaram Instrumentation, France) mixes dry air and water-saturated air in a controlled way to supply a gaseous flow with a stable RH that can be tuned between 5 and 95% (±0.3%) at a given temperature. The hydrogel nanocomposite was uniaxially stretched at a constant speed of 0.2 μm/s. RH was kept constant during the whole experiment or alternated between 23 and 93%, which had an influence on the water content of the sample itself. However, it should be emphasized that it was not possible to measure the exact moisture content within the hydrogel in situ as the humidity was changed. The applied force F was measured by a load cell with a maximum capacity of 50 N. The displacement of the motorized linear stage was recorded. Nominal stress σ = F/(w0t0) and nominal strain ε = (l − l0)/l0 values were estimated with w0 being the initial width, t0 the initial thickness, l the actual length, and l0 the initial length of the cut hydrogel strip. At least three different strips of a given sample were tested under similar experimental conditions. It is worth noticing that the true stress of the sample should be different from the calculated one (which is shown in the manuscript) because of a reduction of the sample cross-sectional area (wt) caused by straining and/or partial drying. Wide- and Small-Angle X-ray Scattering (WAXS and SAXS). Nanostar Laboratory Set Up. Transmission mode measurements were performed by using a Nanostar instrument from Bruker AXS (Germany) operating at 40 kV and 35 mA using Cu Kα radiation (λ = 1.542 Å) and a two-dimensional detector (Hi-Star). The diameter of the beam was approximately 400 μm and the sample to detector distance was 70 mm to analyze the WAXS range. The sample was placed in a sample holder sealed with Kapton foil windows to avoid water evaporation. Silver behenate and corundum (Al2O3) were used to calibrate the q-range. At least nine measurements were made at different points on the hydrogel composites with acquisition times of 2400 s for the nonstretched sample and 1200 s after stretching and a mean value of the degree of orientation was calculated. For data processing, background subtraction, and transmission corrections were made. Synchrotron Set Up. Simultaneous SAXS and WAXS experiments in transmission mode during tensile loading were performed using synchrotron X-rays at the microfocus (μSpot) beamline at BESSY II (Berlin). Data were collected at a wavelength λ = 0.8267 Å using a two-dimensional MARCCD detector placed at a distance of 300 mm from the sample, to simultaneously measure SAXS and WAXS. The synchrotron X-ray beam (diameter ≈ 10 μm) passed through the

it is essential to favor molecular interactions between the CNWs and the matrix as well as to regulate the spatial orientation of the CNWs. This is quite difficult to achieve in bulk structures and typical processing results in a random distribution of CNWs, which leads to an isotropic material response. Up to this point, several methods have been used to align fibrous reinforcements to fabricate anisotropic nanocomposites.34,35 Dynamic mechanical analysis (DMA) of nanocomposites oriented by magnetic fields showed that the storage modulus in the axial direction was higher than in the transverse direction.23,24 In addition to magnetic fields,35−38 both electric fields39 and shearing forces40 have been shown to orient CNWs. Another approach for orienting CNWs in hydrogels is the use of fiber-drawing techniques. Here, it was demonstrated that CNWs could be aligned after immersion in an aqueous sodium alginate solution and wet spinning into a CaCl2 bath41 and after extrusion of PVA-CNWs suspensions into cold methanol.42 Due to the orientation of the nanocrystals, enhanced mechanical properties of the nanocomposite fibers were reported.41−43 A suitable biological example from which we can draw inspiration for the fabrication of anisotropic composites is the primary cell wall of plants.44−47 During cell growth, the cell wall expands due to internal pressure of the cell and the stiff cellulose microfibrils are oriented from a perpendicular to a parallel orientation with respect to the cell axis.48,49 Along these lines, a pioneering study showed that it is possible to align βchitin crystalline microfibrils suspended in a hydrated fibrin clot by mechanical stretching.50 A water-activated shape-memory effect was shown for initially dry CNW/polyurethane nanocomposites after swelling in water and a further stretching, drying, and stress-release.51 The fixation of the temporary sample shape was partly explained by the formation of a moderately aligned CNW network. Taking a cue from these deformation principles, we applied an external load to produce anisotropic CNW-reinforced agarose hydrogels. The hydrogels were uniaxially stretched in a microtensile tester under controlled humidity conditions and the orientation of the CNWs was monitored by X-ray scattering. By this in situ approach the role of various process parameters influencing the reorientation of the CNWs toward a preferential direction could be reported.

■

MATERIAL AND METHODS

CNW/Agarose Hydrogel Nanocomposites. As described in detail by Nishiyama et al.,38,52 CNWs were prepared by heterogeneously acid-hydrolyzing the cell wall of the green algae Cladophora sp. collected at the Japanese coast (Chikura, Chiba). Agarose supplied by Hispanagar (Spain, Batch: D1_LE) was added to an aqueous colloidal suspension of CNWs to prepare mixtures with 5% (w/v) agarose and 1% (w/v) CNWs. The mixture was sonicated twice for 1 min, by using a Sonoplus HD 200 ultrasonic homogenizer (Bandelin, Germany) with titanium alloy MS 73 microtip at 30% of amplitude, to ensure a homogeneous dispersion of CNWs and agarose powder in the sample. Then, the mixture was boiled for 20 min at 90 °C in a silicon oil bath while refluxing (to avoid water evaporation) and under mechanical stirring. The hot viscous agarose-CNW solution was transferred to a Petri dish and allowed to cool at room temperature in a mold to obtain a rigid hydrogel with flat and smooth surface. Hydrogel nanocomposites with a thickness between 1 and 2 mm were prepared. Molecular Weight. Weight- and number-average molecular weights (MWs) of agarose and MW distribution were evaluated by size exclusion chromatography (SEC). The 0.1% (w/v) agarose solutions were prepared in DMSO and filtered through 0.45 μm pore size membranes (Sartorius). The chromatographic equipment was 851


Biomacromolecules

Article

hydrogel nanocomposite clamped in the microtensile tester with Kapton foil windows. Acquisition times of 3 s were used to measure SAXS and WAXS at different strain levels of the sample. Silver behenate and quartz were used to calibrate the q-range and transmission corrections and background subtraction were made. Like for laboratory experiments, the orientation of the CNWs was measured at nine points (in a grid like pattern with distances between the points of 200 μm in the horizontal and 400 μm in the vertical direction) on the hydrogel and a mean value of the degree of orientation was calculated. The degree of orientation of the CNWs was evaluated by measuring the azimuthal intensity of the (110) reflection of the Iα allomorph of cellulose (q = 1.61 Å−1). A fit of the curve of azimuthal intensity vs azimuthal angle with two Gaussians allowed calculating the ρparameter which represents the degree of orientation of the CNWs. The ratio of the Gaussian peak areas (Apeak1 + Apeak2) to the total area beneath the curve (Atotal) yields the ρ-parameter according to the relation:

ρ(%) =

(A peak1 + A peak2) A total

× 100

(1)

The crystalline structure of pure CNWs was studied by synchrotron X-ray analysis on a 1 mm thick pellet which was prepared by pressing the CNWs after freeze-drying of the original colloidal suspension.

■

RESULTS AND DISCUSSION CNW Crystal Structure. As seen from TEM images (Figure 1a,b), the Cladophora CNWs are very long needleshaped single crystals with a high aspect ratio. They have an average width of 30 ± 12 nm and a length ranging from 200 nm to 4 μm. A synchrotron 2D X-ray diffraction pattern of a pellet of Cladophora cellulose nanocrystals was recorded and radially averaged to obtain a profile (Figure 1c) which contains diffraction peaks indexed as the (100), (010), (002), (110), and (1̅1̅4) reflections of triclinic Iα cellulose allomorph.53 The relative intensities reveal a significant uniplanarity of the sample most likely due to the compression of the CNW powder during the pellet preparation. Orientation of the CNWs by Uniaxial Stretching. Uniaxial stretching of the hydrogel nanocomposite strips was performed to reorient the CNWs from a random distribution toward a preferential orientation within the agarose matrix. Figure 2a shows a representative stress−strain curve for a sample loaded at 93% RH. The sample broke at ∼13% maximum strain and a maximum stress of 0.08 MPa. The rather low stress level could be related to the high water content, which limited the strength of the agarose matrix and, probably, the stress transfer from the matrix to the CNWs.54 The determined ρ-parameters (eq 1) before and after testing revealed that the CNWs did not reorient toward the straining direction. Presumably, the high water content promotes the competition between the water molecules and agarose to establish hydrogen-bonds with the CNWs which should result in weak interactions between the CNWs and the agarose matrix. Figure 2b shows a stress−strain curve of a sample that was stretched at low humidity conditions (23% RH) to reduce the water content of the initially wet sample. The stress−strain curve shows a superimposition of stresses coming from stretching and drying (which leads to shrinkage). After stretching, an increase of the ρ-parameter was observed, which indicates a reorientation of the CNWs toward a preferential direction. It is plausible that the interaction between the soft agarose matrix and the stiff CNWs is

Figure 1. (a,b) TEM images of negatively stained Cladophora cellulose whiskers; (c) wide-angle synchrotron X-ray diffraction profile from a pellet of Cladophora cellulose whiskers. The indexation of the main peaks corresponds to that of triclinic cellulose Iα allomorph.37.

augmented through the drying process via increased hydrogen bonding, permitting stress transfer at the interface between the agarose and the CNWs. The stress transfer would be expected to promote alignment of the CNWs toward the stretching direction. In contrast to the work of Bica et al.,55,56 a free rotation of the CNWs in the hydrogel was not observed because the aspect ratio of the CNWs was much higher than the pore diameters in 5% (w/v) agarose hydrogels. The formation of pores in the agarose physical hydrogel is linked to an equilibrium state. The pore size mostly depends on the agarose concentration and this size will barely increase even under changing humidity conditions. Due to the fact that the CNWs were longer than the agarose network pore size diameter (cryo-scanning electron microscopy images not shown),57 we assume that reorientation during straining is 852


Biomacromolecules

Article

Figure 3. () Stress−strain curves (left Y-axis) of cellulose nanowhiskers filled agarose hydrogel composite stretched under alternating humidity conditions: 93% RH (gray background), 23% RH (white background). (a) Comparison between the agarose hydrogel alone and filled with cellulose whiskers; (b) Composite stretched under eight steps of alternating humidity with determination of the degree of orientation of the cellulose whiskers (▲: ρ-parameter) before stretching and after sample fracture by XRD analysis at the Nanostar under laboratory conditions.

Figure 2. () Stress−strain curves (left Y-axis) of cellulose nanowhiskers filled agarose hydrogel composite stretched at (a) 93% RH (gray background); (b) 23% RH (white background). (▲) ρParameter (degree of orientation of the cellulose nanowhiskers) determined before stretching and after sample fracture by XRD analysis at the Nanostar under laboratory conditions.

due to the entrapment of the CNWs which permits stress transfer. In this experiment at 23% RH, the ongoing drying process led to a large increase in stress until the sample fractured at about 7 MPa at a maximum strain of 13% (similar to the wet sample tested at 93% RH in Figure 2a). To achieve higher sample strains, stress levels were kept below a critical threshold by straining under alternating phases of low (23% RH) and high (93% RH) relative humidity. Before critical stress levels caused by drying and stretching were achieved, the sample was rewetted by increasing the RH to 93% for ∼20 min. Afterward, the RH was reduced again to 23%. Figure 3a shows stress−strain curves of the CNW/agarose hydrogel nanocomposite and of the pure 5% (w/v) agarose hydrogel under alternating humidity conditions. At 23% RH, the slope was similar for both samples and although the drying of the pure agarose hydrogel took slightly longer, the stresses were comparable for both samples. Increasing RH to 93% resulted in a higher stress decrease in the pure agarose hydrogel, which indicates a higher sensitivity to the humidity. A subsequent decrease in RH to 23% led to a fast increase in stress in the nanocomposite, but only to a moderate increase in the pure agarose hydrogel. This suggests an important reinforcing capacity of the CNWs as soon as a critical drying state has been reached. Figure 3b shows a stress−strain curve of a composite sample that was subjected to eight steps of alternating humidity during stretching. This procedure maintained a stress level below 1.5 MPa and resulted in a maximum strain of approximately 25%. The ρ-parameter, which was measured after sample rupture, had increased up to a value

of about 28%, indicating a pronounced alignment of the CNWs along the stretching direction (Figure 3b). Under laboratory conditions, measurements of the ρparameter of the CNWs could only be performed before and after a given straining (usually after sample fracture). In order to follow the change of the ρ-parameter during the stretching of the sample under varying humidity conditions, in situ measurements were performed at the BESSY microfocus beamline. Figure 4 shows scattering diagrams, calculated ρparameters, and the corresponding stress−strain diagram of a stretching experiment with two humidity steps. The straining was interrupted for 10 min at the point of humidity change, to allow a rewetting of the sample without additional straining. The 2D synchrotron X-ray scattering diagrams shown in Figure 4a correspond to the recording before stretching and at a strainlevel of 12%, just before sample fracture. The SAXS signal of the 2D scattering patterns reveals the preferential orientation of the CNWs after stretching. Nevertheless, the evaluation of the orientation is underestimated when just plotting the azimuthal intensity averages of the SAXS q-range. In this range, the anisotropic scattering of the cellulose nanocrystals is partially obscured by the more isotropic scattering of the agarose microfibrils constituting the hydrogel network. The analyzed WAXS data of the unstrained sample (Figure 4a) shows that the reflections of cellulose Iα appear as diffraction rings (see peaks assignment in Figure 1c), indicating a random orientation of the CNWs. The WAXS signal of water at q = 2.0 Å−1 is strong in the pattern before stretching and less significant after 853


Biomacromolecules

Article

reveals that via straining of the hydrogel, the peak areas of the curves increase, which correspond to the proportion of oriented CNWs, whereas the area beneath the baseline (defined as passing through the curve minima) decreases, which corresponds to the proportion of randomly distributed CNWs. Figure 4c shows the corresponding stress−strain diagram and the evolution of the CNWs orientation degree estimated through the ρ-parameter. The most significant change of the ρparameter was observed at ∼9% strain where the stress value increased. Then, the ρ-parameter continued to moderately increase, finally reaching a value of ρ = 21.5 ± 2.1% (Figure 4a) after 12.5% of strain. The results reported in Figures 2−4 show that a preferential orientation of CNWs can be achieved by uniaxial tests under adjusted humidity conditions. However, one cannot conclude from these observations which experimental parameter (strain, stress or moisture) is crucial for the alignment of the CNWs. Therefore, further experiments with in situ analysis were performed to discern the influence of each experimental parameter, in particular which conditions facilitate the orientation of the CNWs at relatively low stress levels. Figure 5 shows two further in situ synchrotron experiments that serve to elucidate the role of the hydration level and the

Figure 4. In situ tensile straining of agarose hydrogel filled with cellulose nanowhiskers under varying humidity conditions at the synchrotron beamline (a) 2D synchrotron X-ray scattering before stretching and after 12% straining; (b) Azimuthal intensity average evolution of the (110) reflection of cellulose Iα for different strain values: each curve with standard deviations (gray bars) represents the average data of nine points measured on the sample for each strain value; (c) () Stress−strain curve (left Y-axis) and ρ-parameter evolution (▲) determined by synchrotron X-ray analysis during the stretching, initially under 23% RH (white background), then under 93% RH (gray background). Strain was interrupted for ten minutes at the point of humidity change.

Figure 5. () Stress−strain curves (left Y-axis) and ρ-parameter evolution (▲) determined by synchrotron X-ray analysis for samples stretched at alternating humidity conditions: 23% RH (white background); 93% RH (gray background).

12% of strain due to drying of the sample. After stretching, the appearance of (100), (010), and (110) diffraction arcs in the equatorial sector of the diagram (perpendicular to the stretching direction) reveals the preferential orientation of the CNWs along the stretching direction. The change of the azimuthal profile at q = 1.6 Å−1 of the (110) reflection for different strain values under the stretching conditions can be followed in situ in Figure 4b. Each curve with standard deviations (gray bars) represents the average data of nine points measured on the sample at a given strain value. Figure 4b

stress level in inducing CNW alignment. In these experiments, hydrogels at 23% RH were stretched and rewetted (93% RH) after reaching two different stress levels. In the first experiment (Figure 5a), hydrogels stretched at low humidity (23% RH) were only permitted a moderate increase in stress before rehydration. Under these conditions, an increase in the ρparameter was simultaneously observed. During the wetting phase at 93% RH, a further increase of the ρ-parameter was observed before it reached a first plateau (Figure 5a). The following humidity cycle, in which stress was kept below a 854


Biomacromolecules critical level, resulted in a further but modest increase of the ρparameter. In the second experiment (Figure 5b), the hydrogel was allowed to reach a higher stress level prior to rehydration than in the previous experiment. However, a comparable increase of the ρ-parameter was still observed even in the absence of rewetting. Again, during the following humidity cycles, the degree of orientation remained high. In both experiments (Figure 5a,b), the jump of the ρparameter occurred at a strain level of about 12.5%, regardless of whether the humidity was increased or not. This strain corresponds to a stress level of about 0.25 MPa in the first experiment and about 0.45 MPa in the second. The most important result from these two experiments (Figure 5) is that the pronounced increase of the ρ-parameter is achieved under different moisture conditions. This indicates that during a tensile test which maintains a relatively low stress level, a rewetting of the hydrogel is not required to reorient the CNWs, but allows to continue the stretching and thereby contribute to increase the strain at failure and the reorientation of CNWs.

■

CONCLUSIONS

■

AUTHOR INFORMATION

■

ACKNOWLEDGMENTS

■

REFERENCES

Article

We thank the German Research Foundation DFG (Project BU 2132/1-1) and the Max-Planck Society for supporting this study. The support of I. Zenke, A. Martins, M. Gräwert, M.-F. Marais, F. Saxe, and L. Bertinetti in the laboratory was highly appreciated. We are grateful to Hispanagar Company (Spain) for the gift of the agarose sample and also to L. David for helpful discussions. We thank S. Siegel, C. Li, and B. Aichmayer for assistance at the microfocus beamline at BESSY II, Berlin.

(1) Liang, S.; Xu, J.; Weng, L.; Dai, H.; Zhang, X.; Zhang, L. J. Controlled Release 2006, 115, 189−196. (2) Toussaint, J.-F.; Dubois, A.; Dispas, M.; Paquet, D.; Letellier, C.; Kerkhofs, P. Vaccine 2007, 25, 1167−1174. (3) Watase, M.; Nishinari, K. Rheol. Acta 1983, 22, 580−587. (4) Guenet, J.-M.; Brûlet, A.; Rochas, C. Int. J. Biol. Macromol. 1993, 15, 131−132. (5) Ramzi, M.; Rochas, C.; Guenet, J.-M. Macromolecules 1998, 31, 6106−6111. (6) Guenet, J.-M.; Rochas, C. Macromol. Symp. 2006, 242, 65−70. (7) Rochas, C.; Lahaye, M. Carbohydr. Polym. 1989, 10, 289−298. (8) Mitsuiki, M.; Mizuno, A.; Motoki, M. J. Agric. Food Chem. 1999, 47, 473−478. (9) Favier, V.; Canova, G. R.; Cavaillé, J.-Y.; Chanzy, H.; Dufresne, A.; Gauthier, C. Polym. Adv. Technol. 1995, 6, 351−355. (10) Favier, V.; Chanzy, H.; Cavaillé, J. Y. Macromolecules 1995, 28, 6365−6367. (11) Helbert, W.; Cavaillé, J.-Y.; Dufresne, A. Polym. Compos. 1996, 17, 604−611. (12) Dufresne, A.; Cavaillé, J. Y.; Helbert, W. Polym. Compos. 1997, 18, 198−210. (13) Liu, H.; Laborie, M.-P. Cellulose 2011, 18, 619−630. (14) Siqueira, G.; Fraschini, C.; Bras, J.; Dufresne, A.; Prud’homme, R.; Laborie, M.-P. Eur. Polym. J. 2011, 47, 2216−2227. (15) Dubief, D.; Samain, E.; Dufresne, A. Macromolecules 1999, 32, 5765−5771. (16) Dufresne, A. Compos. Interfaces 2000, 7, 53−67. (17) Dufresne, A.; Kellerhals, M. B.; Witholt, B. Macromolecules 1999, 32, 7396−7401. (18) Svagan, A. J.; Samir, M. A. S. A.; Berglund, L. A. Biomacromolecules 2007, 8, 2556−2563. (19) Svagan, A. J.; Samir, M. A. S. A.; Berglund, L. A. Adv. Mater. 2008, 20, 1263−1269. (20) Zimmermann, T.; Bordeanu, N.; Strub, E. Carbohydr. Polym. 2010, 79, 1086−1093. (21) Zimmermann, T.; Pöhler, E.; Schwaller, P. Adv. Eng. Mater. 2005, 7, 1156−1161. (22) Eyholzer, C.; Borges de Couraça, A.; Duc, F.; Bourban, P. E.; Tingaut, P.; Zimmermann, T.; Månson, J. A. E.; Oksman, K. Biomacromolecules 2011, 12, 1419−1427. (23) Nakagaito, A. N.; Fujimura, A.; Sakai, T.; Hama, Y.; Yano, H. Compos. Sci. Technol. 2009, 69, 1293−1297. (24) Suryanegara, L.; Nakagaito, A. N.; Yano, H. Compos. Sci. Technol. 2009, 69, 1187−1192. (25) Hepworth, D. G.; Bruce, D. M. J. Mater. Sci. 2000, 35, 5861− 5865. (26) Hsieh, Y. C.; Yano, H.; Nogi, M.; Eichhorn, S. Cellulose 2008, 15, 507−513. (27) Sakurada, I.; Nukushina, Y.; Ito, T. J. Polym. Sci. 1962, 57, 651− 660. (28) Sturcova, A.; Davies, G. R.; Eichhorn, S. J. Biomacromolecules 2005, 6, 1055−1061. (29) Nishino, T.; Takano, K.; Nakamae, K. J. Polym. Sci., Part B: Polym. Phys. 1995, 33, 1647−1651. (30) Angles, M. N.; Dufresne, A. Macromolecules 2001, 34, 2921− 2931.

CNW-reinforced agarose hydrogels were strained under varying and controlled humidity conditions. In situ investigations by Xray scattering during straining showed that a preferential orientation of the CNWs was achieved within the agarose matrix. Based on these studies, the following can be concluded on the influence of specific parameters (i.e., strain, stress, and moisture) on the degree of orientation: (1) Uniaxial straining was crucial for the reorientation of the nanocrystals, because free sample drying without any fixation and straining did not result in a change of the ρparameter (data not shown). However, the results did not allow for determination of critical thresholds for the strain-level and the stress-level, since the ρ-parameter started to increase at different strain and stress levels in the in situ experiments. (2) Rehydration did not seem to be crucial for an increase of the ρ-parameter (Figure 5a,b). However, repeated rehydration helped to keep the stress level below a critical value and thereby allowed for increased straining and further moderate increase of the ρ-parameter. In light of these observations, we conclude that a combination of uniaxial tensile straining and hydrogel drying results in a reorientation of the CNWs. One probable explanation is that the drying process establishes the required molecular interaction between agarose and CNWs that mediates load transfer from the matrix to the fibres inducing a preferential alignment of the latter. Finally, via this approach CNWs can be reoriented from an initial random distribution toward the straining direction, resulting in anisotropic hydrogel nanocomposite.

Corresponding Author

*Tel.: +49 761 2039242. Fax: +49 761 2033763. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 855


Biomacromolecules

Article

(31) Burgert, I.; Fratzl, P. Philos. Trans. R. Soc., A 2009, 367, 1541− 1557. (32) Fratzl, P.; Elbaum, R.; Burgert, I. Faraday Discuss. 2008, 139, 275−282. (33) Elbaum, R.; Zaltzman, L.; Burgert, I.; Fratzl, P. Science 2007, 316, 884−886. (34) Li, D.; Liu, Z.; Al-Haik, M.; Tehrani, M.; Murray, F.; Tannenbaum, R.; Garmestani, H. Polym. Bull. 2010, 65, 635−642. (35) Kvien, I.; Oksman, K. Appl. Phys. A: Mater. Sci. Process. 2007, 87, 641−643. (36) Sugiyama, J.; Chanzy, H.; Maret, G. Macromolecules 1992, 25, 4232−4234. (37) Kimura, F.; Kimura, T.; Tamura, M.; Hirai, A.; Ikuno, M.; Horii, F. Langmuir 2005, 21, 2034−2037. (38) Nishiyama, Y.; Kuga, S.; Wada, M.; Okano, T. Macromolecules 1997, 30, 6395−6397. (39) Bordel, D.; Putaux, J.-L.; Heux, L. Langmuir 2006, 22, 4899− 4901. (40) Ebeling, T.; Paillet, M.; Borsali, R.; Diat, O.; Dufresne, A.; Cavaille, J.-Y.; Chanzy, H. Langmuir 1999, 15, 6123−6126. (41) Ureña-Benavides, E. E.; Brown, P. J.; Kitchens, C. L. Langmuir 2010, 26, 14263−14270. (42) Jalal Uddin, A.; Araki, J.; Gotoh, Y. Biomacromolecules 2011, 12, 617−624. (43) Ureña-Benavides, E. E.; Kitchens, C. L. Macromolecules 2011, 44, 3478−3484. (44) Cosgrove, D. J. Annu. Rev. Cell Dev. Biol. 1997, 13, 171−201. (45) Carpita, N. C.; Gibeaut, D. M. Plant J. 1993, 3, 1−30. (46) McCann, M. C.; Roberts, K. In The Cytoskeletal Basis of Plant Growth and Form; Lloyd, C. W., Ed.; Academic Press: London, 1991; pp 109−129. (47) Cosgrove, D. J. Nat. Rev. Mol. Cell Biol. 2005, 6, 850−861. (48) Wolters-Arts, A. M. C.; Sassen, M. M. A. Planta 1991, 185, 179−189. (49) Baskin, T. I. Annu. Rev. Cell Dev. Biol. 2005, 21, 203−222. (50) Blackwell, J. Biopolymers 1969, 7, 281−298. (51) Mendez, J.; Annamalai, P. K.; Eichhorn, S. J.; Rusli, R.; Rowan, S. J.; Foster, E. J.; Weder, C. Macromolecules 2011, 44, 6827−6835. (52) Marchessault, R. H.; Morehead, F. F.; Walter, N. M. Nature 1959, 184, 632−633. (53) Sugiyama, J.; Vuong, R.; Chanzy, H. Macromolecules 1991, 24, 4168−4175. (54) Rusli, R.; Shanmuganathan, K.; Rowan, S. J.; Weder, C.; Eichhorn, S. J. Biomacromolecules 2010, 11, 762−768. (55) Bica, C. I. D.; Borsali, R.; Geissler, E.; Rochas, C. Macromolecules 2001, 34, 5275−5279. (56) Bica, C. I. D.; Borsali, R.; Rochas, C.; Geissler, E. Macromolecules 2006, 39, 3622−3627. (57) Rochas, C.; Hecht, A.-M.; Geissler, E. Macromol. Symp. 1999, 138, 157−163.

856


Reorientation of Cellulose Nanowhiskers in Agarose Hydrogels under

Recommend Documents