Hydrogel Assembly with Hierarchical Alignment by Balancing Electrostatic Forces

Hydrogels with hierarchical alignment are described by balancing electronic repulsion and attraction among silk protein nanofibers. The nanofiber alignment is cruicial to the formation of these hydrogels, dependent on electric fields and the inherent repulsion from negatively charged silk, which induces alignment layer separation. Aligned cell growth on the hydrogels implies promising medical applications. Anisotropy or alignment of materials is a theme of interest due to its fundamental importance in structures generated by biological organisms.1, 2 In nature, tissues such as heart, brain, and muscle exhibit highly organized hierarchical structures,3, 4 assembled from molecules to generate long-range alignment of extracellular fibrils and cells which are crucial for biological functions. Inspired by these natural systems, generating anisotropic materials has been highlighted for biomaterials to tune or optimize mechanical properties as well as to provide new systems for tissue engineering and regenerative medicine. Although successful fabrication of aligned structures has been achieved in solid materials and liquid crystals,5, 6 the introduction of anisotropy to hydrogels remains a challenge.1, 7 A few processes were studied to induce anisotropy in hydrogels, including electric fields,8 enzymatic hydrogelation,9 and mechanical stretching,10 however, the inherent limitations of these processes limit the formation of aligned structures using synthetic polymers. Hierarchical aligned structures, typical features of functional materials in living organisms, remain to be exploited. In light of anisotropic hydrogels and their critical functions in biological systems,11, 12 reproducing these features via self-assembly is a main approach.1, 9, 13 Silk, produced by Bombyx mori, is a suitable model protein material for the fabrication of anisotropic materials due to its unique sequence-specific hierarchical self-assembly.14 Silk hydrogels composed of nanofibers were achieved via several controllable self-assembly processes.15-17 However, like natural biomaterials, these nanofibers remain as random entanglements forming isotropic hydrogels, but fail to further assemble into aligned hierarchical structures. Recently, several exceptional examples of supramolecular hydrogels offered hints for generating anisotropic hydrogels, in which molecules could assemble into higher order arrays of aligned nanofibers in the presence of external forces.18-22 Isotropic silk hydrogels (E-gel) were prepared under electric fields and aggregated near the anode.23 Recently, a novel solution-hydrogel system composed of silk nanofibers was generated by tuning silk self-assembly in aqueous solution.16 In this system, the nanofibers contained a significantly higher negative charge than silk materials previously reported,24 making them dispersible in aqueous solution. Under the effect of the external electric field, the charged silk nanofibers will migrate toward the anode, the same as what happens in electrophoresis.25 We hypothesize that the charged silk nanofibers would also align due to the electrostatic interactions between charged silk nanofibers and the electric field,26 resulting in aligned structures. 2 wt% aqueous solutions of silks that self-assemble into nanofibers with high beta-sheet content were prepared. The silk nanofibers uniformly dispersed in aqueous solutions due to the repulsive forces between the nanofibers.16 The hydrogel formation was triggered by low direct current voltage (50 V) that induced the negative charged silk nanofibers migrate toward the anode (Figure S1, Supporting Information). Within seconds of the application of the voltage, a visible hydrogel (E-N-Gel) began to appear, gradually grew near the anode and finally formed stable structures after 20 min (Figure S1A (Supporting Information) and Figure 1A,B), since the negatively charged silk nanofibers were repelled from the cathode and concentrated near the anode. Rhodamine-labeled silk was used to facilitate observations during the gelation processes and suggested that most of the silk nanofibers moved to the gelation area (Figure S1A,B, Supporting Information). This observation prompted further investigation into the response of the solution under different voltages and silk concentrations. At higher voltage or lower silk concentration the E-N-Gel formed more quickly. E-N-Gels became thicker with an increase of initial silk concentration, but maintained same size when they were prepared at same concentration but at different voltages. Unlike E-gels previously reported that could dissipate near the new cathode and form again near the new anode if the electrodes were reversed after e-gel formation,23 E-N-Gels remained unchanged without a transition back to the solution state at higher temperature or through the reversed electric field, suggesting the formation of a more stable structure (Figure S1C, Supporting Information). Birefringence is a material property derived from molecular anisotropy, thus polarized microscopy was used to study the birefringence of the hydrogels. The E-N-Gels displayed birefringence, suggesting uniform alignment of the nanofibers into organized structures (Figure 1C). In contrast, nanofibers with random orientations did not show this level of birefringence (Figure 1C). To further verify the alignment of nanofibers in the E-N-Gel, hydrogels in liquid nitrogen were flash frozen and then freeze-dried for scanning electron microscopy (SEM) (Figure 1D,E). The E-N-Gels were composed of aligned lamellae with thickness of several microns and inter-lamellar distances of about 100–200 μm. Although the morphology of the nanofibers on the lamellae looked mesh-like, higher magnifications and Fourier transform analysis (Figure 1E,G and Figure S2, Supporting Information) indicated that the lamellae consisted of bundles of nanofibers with oriented structures, implying the formation of hierarchically anisotropic features. Confocal microscopy and transmission electron microscopy (TEM) confirmed the hierarchical orientation of the nanofibers in E-N-Gels (Figure 1F,G). A uniform layered structure of hydrogel appeared in confocal images while TEM of the layers revealed parallel aligned nanofibers. Some nanofibers entangled the oriented nanofibers to form aligned bundles that were also found in the SEM images (Figure 2). In view of the hierarchical anisotropic features embedded within E-N-Gels, the gels were further investigated during the gelation process. Considering the gradual growth of the hydrogel resulted in different degrees of gelation, thus samples were obtained immediately near the anode at designed gelation time points. Without voltage, silk nanofibers were randomly distributed with entangled structures (Figure 2A(a)). When the electric field was applied, silk nanofibers migrated toward the anode due to isoelectric focusing26 and rotated to orient parallel to the longer axis of the fibers (Figure 2A(b)). Similar to the response of collagen molecules in an electric field,26 the rotation of the silk nanofibers could be explained by electrostatic interactions between charged silk nanofibers and the electric field. As an amphiphilic molecule, charges on silk nanofibers are pH dependent. Given the pH change of the silk solution between the electrodes, the end terminus of the nanofibers closer to the cathode would be relatively more negatively charged than the end terminus closer to the anode, creating a pH-induced electric dipole. Therefore, the end terminus closer to the cathode will be electrostatically repelled more intensely by the cathode than the end which is further away, resulting in rotational electrostatic interactions and finally orientation. Under the effect of the external electric field, the negatively charged silk fibers migrated and accumulated near the anode due to the lower local pH as a result of electrolysis of water, generating H+ at the anode.27-29 Considering similar nanofiber structures and charged groups between silk and collagen, it is reasonable to form the oriented structures for the silk nanofibers, similar to that happened for collagen in an electric field.26 The nanofibers formed oriented bundles and then developed into thin sheets with a depth of several hundred nanometers (Figure 2A(c)), and further assembled to form thicker layers of several microns (Figure 2A(d–f)), which could be explained by the electrostatic forces and a steep oH gradient.26 This assembly process was confirmed by TEM. Following the gelation process, the width of the entangled nanofiber bundles with aligned structures gradually increased from tens of nanometers to about 1 μm (Figures 1G and 2B), similar to that found by SEM. Several phenomena remain unexplained in the gelation process, including the separation of the layers and the homogeneous distance between the layers inside the hydrogels (Figure S3, Supporting Information). A recent study of anisotropic hydrogels illustrated how electrostatic repulsion could be harnessed to achieve functional efficiency.18 Taking into account that in our hydrogel silk nanofibers are also negatively charged, we consider that the likely explanation for the separate layered structures with homogeneous spacing is the electrostatic repulsion among the cofacially oriented silk nanofiber layers. That is, the electrostatic interaction between the silk nanofibers and the electric field caused the orientation and movement of the silk nanofibers while the electrostatic repulsion from the nanofibers resulted in the separation of the layers. This interpretation was supported by the structural changes of the hydrogels under different voltages and silk concentrations (Figure S4, Supporting Information). We noted that E-N-Gels derived from the same nanofiber solution had similar layer depths and similar spacing between the layers under different voltages although the stable gels formed more quickly at higher voltages. It is suggested that the layer depth and the spacing between layers in the oriented structures were independent of the electric field. Subsequently the layer depth decreased following the decrease of silk nanofiber concentration, might be because the thickness of the layers was depended on the degree of entanglement of the nanofibers. Unlike conventional hydrogels and scaffolds,30, 31 the narrower rather than wider spacing appears in the hydrogels with lower concentrations. This result is reasonable since the less negative charges on the thinner layer provided weaker repulsion, yet the underlying mechanism remains to be elucidated. Therefore, both higher charge density and nanofiber structure are critical factors in reducing the hierarchical anisotropy of the hydrogels (Figure 3A). To gain deeper insight in the role of the nanofiber structure and charge density, silk fibroin solutions composed of nanofibers with low charge density (zeta potential −5 mV), and nanoparticles with high charge density (zeta potential −50 mV) were prepared respectively (Figure S5, Supporting Information). The nanofibers with low charge density remained unchanged in the electric field without hydrogel formation near the anode due to the deficiency in the electrostatic interactions. When a silk nanoparticle solution was placed under an electric field, a hydrogel appeared near the anode but it had significantly inferior anisotropic features since the nanoparticles lack the capacity for alignment (Figure S5, Supporting Information). It is worth noting that this strategy for anisotropic hydrogel fabrication is based on the regulation of nanostructure and charge density, thus offers universal options. Peptide nanofibers containing negative charge were treated under the electric fields and also formed the anisotropic hydrogels, confirming this universality (Figure S6, Supporting Information). Although more functional materials with specific nanostructures and charge densities need to be designed to control the hierarchical structures, this general mechanism should provide a roadmap towards such goals. The anisotropic nature of the hydrogels also resulted in anisotropic mechanical properties. In rheological measurements of the silk hydrogels (1 and 2 wt%), shearing parallel to the silk layers gave a significantly higher storage modulus than when shearing the same hydrogel sample orthogonal to the silk layers (Figure 3B). Similar anisotropy for the modulus has been reported in other hydrogels with aligned structures.32-35 These studies confirmed that mechanical anisotropy was derived from the aligned structures in the hydrogels.32-35 Many applications for this process can be envisioned. For example, more complex anisotropic structures can be designed by adjusting the shape of the positive electrode due to the aligned directions of the layers of silk hydrogel parallel to the electrode surface (Figure S7, Supporting Information). Mesenchymal stem cells showed oriented structures on the hydrogels, suggesting future applications in different tissue regeneration strategies where isotropy is a key, such as in nerve and muscle regeneration (Figure 3C). The stem cells were cultured on the freeze-dried hydrogels with aligned structures for 12 d. The cells grew along the aligned layers and showed better cytocompatibility than on the freeze-dried hydrogels without aligned structure (Figure S8, Supporting Information). These results suggested that the orientated structure had no negative influence on cell compatibility. Further, silk has been well-documented as a biodegradable material, with the degradation process mediated by proteases.36-38 Many previous studies have confirmed that the aligned microstructures of various biomaterials had a significant influence on cell fate, such as cell orientation, differentiation and growth.39-42 Our initial study here revealed that the cells could be orientated on the aligned hydrogels, suggesting the feasibility in regulating cell behavior. Further studies will be needed to clarify whether the cell behavior could be tuned by changing layer thickness, the spacing between layers as well as the stiffness of the aligned hydrogels. In conclusion, a rational design is demonstrated that utilizes electrostatic interactions to drive the hierarchical alignment of nanofibers to generate anisotropic hydrogels. The alignment of nanofibers is initially triggered by the electrostatic interaction of pH induced electric dipole of silk nanofiber with electric field. The layers composed of the aligned nanofibers form because of nanofiber entanglement and the inherent repulsion from negative charge results in the separation of the aligned layers, forming hierarchical anisotropic structures. This approach represents a new strategy for designing aligned hydrogels with broad implications. When combined with the biocompatibility of silk, these aligned hydrogel systems could have broad utility in biological systems. Detailed experimental protocols describing the preparation of silk solutions, nanofiber formation, preparation of peptide hydrogels, SEM, TEM, polarized optical microscopy, confocal microscopy, ultrasonic treatment, zeta potential, cell culture, dynamic oscillatory rheology, and atomic force microscopy characterization can be found in the Supporting Information. The authors thank the National Basic Research Program of China (973 Program 2013CB934400), NSFC (21174097, 21574024), and the NIH (R01 DE017207) for support of this work. The authors also thank the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Excellent Youth Foundation of Jiangsu Province (BK2012009), and the Natural Science Foundation of Jiangsu Province (Grants BK20140397) for support of this work. As a service to our authors and readers, this journal provides supporting information supplied by the authors. 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