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Biological and Medical Applications of Materials and Interfaces

Cooperative Assembly of a Peptide Gelator and Silk Fibroin Afford an Injectable Hydrogel for Tissue Engineering Baochang Cheng, Yufei Yan, Jingjing Qi, Lianfu Deng, ZengWu Shao, Ke-Qin Zhang, Bin Li, Ziling Sun, and Xinming Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01725 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 27, 2018

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ACS Applied Materials & Interfaces

Cooperative Assembly of a Peptide Gelator and Silk Fibroin Afford an Injectable Hydrogel for Tissue Engineering Baochang Cheng,†,¶ Yufei Yan,

‡,¶

Jingjing Qi,† Lianfu Deng,‡ Zeng-Wu Shao,§ Ke-Qin Zhang,∥

Bin Li, *,⊥ Ziling Sun,*,# Xinming Li*,† †

College of Chemistry, Chemical Engineering and Materials Science, Soochow University,

Suzhou, 215123, China. ‡

Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai Institute of Orthopaedics and

Traumatology, Shanghai Ruijin Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai, 200025, China. §

Department of Orthopaedics, Union Hospital, Tongji Medical School, Huazhong University of

Science and Technology, Wuhan, 430022, China. ∥

National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering,

Soochow University, Suzhou, 215123, China. ⊥

Department of Orthopaedics, The First Affiliated Hospital, Orthopaedic Institute, Soochow

University, Suzhou, 215006, China. #

School of Biology and Basic Medical Science, Soochow University, Suzhou, 215123, China.

KEYWORDS: hydrogel, peptide, silk fibroin, self-assembly, tissue engineering.

ACS Paragon Plus Environment

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ABSTRACT: Silk fibroin (SF) from Bombyx mori has received increasing interest in biomedical fields, because of its slow biodegradability, good biocompatibility, and low immunogenicity. Although SF based hydrogels have been studied intensively as potential matrix for tissue engineering, weak gelation performance and low mechanical strength are major limitations that hamper their widespread applicability. Therefore, searching for new strategies to improve SF gelation property is highly desirable in tissue engineering research. Herein, we report a facile approach to induce rapid gelation of SF by a small peptide gelator (e.g., NapFF). Following simply mixing SF and NapFF in water, a stable hydrogel of SF was obtained in a short time period at physiological pH, and the minimum gelation concentration of SF can reach as low as 0.1%. In this process of gelation, NapFF can not only behave itself as a gelator for supramolecular self-assembly, but also can trigger the conformational transition of SF molecule from random coil to β-sheet structure via hydrophobic and hydrogen bonding interactions. More importantly, in order to generate a scaffold with favorable cell-surface interactions, a new peptide gelator (NapFFRGD) with RGD domain was applied to functionalize SF hydrogel with improved bioactivity for cell adhesion and growth. Following encapsulating vascular endothelial growth factor (VEGF), the SF gel was subcutaneously injected in mice, and served as an effective matrix to trigger the generation of new blood capillaries in vivo.

1. INTRODUCTION Silk fibroin (SF) is a fibrous protein isolated from the cocoons of domestic silkworm Bombyx mori,1 and has been widely recognized as an effective scaffold for preparing various forms of biomaterials, such as fibers, particles, films, sponges, hydrogels and electrospun mats.2-6 Among these materials formats, SF hydrogel has emerged as one of promising candidates for biomedical applications ranging from tissue engineering to drug delivery,7-8 because of its slow


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biodegradability, good biocompatibility, and low immunogenicity.9-11 But the gelation process of a SF solution is quite slow under physiological conditions, which usually takes a relatively long time (>5 days) at a high fibroin concentration (> 4%),12-13 greatly limiting its applications in biomedical fields. Recent studies revealed that the slow dynamics of gelation for a pure SF solution is mainly relevant with conformational transition from random coil to β-sheet structure, which then gradually self-assembles into close-packed β-sheet crystals, acting as physical crosslink to stabilize SF hydrogel.8, 14-15 Although many physical treatment methods (e.g., pH, vortexing, sonication, and electrical field),16-19 or the addition of certain organic molecules (e.g., ethanol, surfactants or hydrophilic polymers) are able to increase the gelation rate of SF by adjusting protein chain-chain interactions,20-29 the gelation process induced by instrumental parameters may not be compatible with certain clinical environments, and potential toxicity of certain organic molecules can raise a significant concern for biomedical applications. Thus, searching for a simple and biocompatible way to trigger the gelation of SF is highly desirable. Peptide self-assembly has become an effective approach for producing varied biocompatible hydrogel from spontaneous association of molecular gelators via differential non-covalent interactions.30-35 In addition, the integration of peptide self-assembling system with certain biopolymers (e.g., hyaluronic acid, alginate, detran, bovine serum albumin, or β-lactoglobulin) could feasibly modulate the self-assembling behaviors of peptide gelators and generate hybrid nanomaterials or hydrogels.36-41 Encouraged by these studies, we decided to investigate the selfassembling behaviors of peptide gelators within the system of SF, examine its potential to induce the gelation of SF and generate a novel biocompatible hydrogel from SF.


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In this study, we chose a small synthetic peptide which contains a naphthyl group and a Phe– Phe dipeptide as a standard molecular gelator (namely, NapFF), and examine its potential to trigger the gelation of SF. NapFF was a small peptide-based gelator and could self-assemble efficiently to form well-defined one dimensional nanostructures and hydrogel in water via noncovalent hydrophobic and hydrogen-bonding interactions.42 Following simply mixing SF and NapFF in water, a stable hydrogel was obtained at physiological pH, and the minimum gelation concentration of SF can reach as low as 0.1%, which is much lower than the threshold concentration of gelation achieved by other physical approaches. The dynamics of gelation and the mechanical strength of resulting hydrogel could be effectively regulated by the concentrations of both SF and NapFF. More importantly, in order to generate a favorable microenvironment for tissue development, both a new peptide gelator (NapFFRGD) with RGD domain and VEGF can be easily applied to modify SF hydrogel with improved bioactivity, such as cell adhesion, cell growth and angiogenesis. After subcutaneous injection in mice, the selfassembled SF gel can serve as an effective matrix to trigger the generation of new blood capillaries in vivo. 2. RESULTS AND DISCUSSION NapFF was prepared by following a typical procedure of solid-phase synthesis with the application of Fmoc-Phe-OH and 2- naphthylacetic acid (Figure S1-S3). NapFF exhibited good solubility in water, and self-assembled to form well-defined nanofibers at a low concentration of 0.4 wt% (Figure S6). Although a pure SF solution (> 2%) was capable to form a physically cross-linked hydrogel, its gelation time ranged from days to weeks (Figure S7), depending on its concentration in solution. In order to test the potential of NapFF to improve the gelation property of SF, we added different amounts of NapFF to SF, and prepared a series of solutions at pH=7.4,


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in which the concentrations of NapFF ranged from 0.1 to 0.4 wt% (Table 1), and the concentration of SF was fixed at 0.1%. For the sample containing 0.1 wt% of NapFF and 0.1% of SF, no gelation occurred in 24 h (Sol 1 in Table 1). But when the concentration of NapFF in SF solution was increased to 0.2%, a sol-gel transition was observed in 1.5 h (Figure 1A-1C, Gel 1 in Table 1). Further increasing NapFF concentration to 0.4 wt% can greatly shorten the gelation time to 30 min (Gel 2 in Table 1). These results indicated that NapFF molecule present in the solution of SF can significantly improve the gelation property of SF by decreasing gelation concentration and shortening gelation time, and the efficiency was dependent on the concentration of NapFF. The transmission electron microscopy (TEM) images revealed the formation of nanostructures in different morphologies by NapFF and SF during the process of supramolecular gelation. For example, before mixing these two solutions together, we identified the presence of both onedimensional nanostructures with diameter around 12 nm in the blank solution of NapFF (Figure S6C), and a large amount of thin single fibers (diameter=7 nm) and nanobundles in the solution of SF (Figure S7C). After mixing them together for gelation, we observed the formation of highly cross-linking nanofibrous networks from both NapFF and SF (Figure 1D). And the selfassembled nanofibers in 12 nm from NapFF were well-distributed and entangled with both thin single fibers (7 nm) and the untwisted nanobundles from SF. Rheological test confirmed that the self-assembled gel containing NapFF (0.2 wt%) and SF (0.1%) exhibited typical solid-like rheological behavior with the storage moduli (G’) greater than the loss moduli (G’’) within the investigated oscillating frequency limit (0.1−100 Hz) (Figure 1E). These results indicated that NapFF can perform supramolecular self-assembling properly in the solution of SF by forming


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well-defined one-dimensional nanostructures, and these nanostructures interpenetrated with SF to form three-dimensional nanofibril networks. Circular dichroism spectroscopy (CD) and Fourier transform infrared spectroscopy (FTIR) studies provided information about the molecular conformations of NapFF and SF during the process of supramolecular gelation. As shown in Figure 2A, CD spectra of the blank solutions of NapFF and SF showed negative peaks at 214 and 196 nm and positive troughs around 193 and 184 nm, respectively, implying their less-ordered structures in their solution states.13,

17, 43

However, after mixing them together for supramolecular gelation, a positive band at 188 nm and a negative band at 208 nm in CD spectrum appeared, which was a characteristic of β-sheet conformation.13, 17, 43 Additional Cotton effects near 230 and 285 nm could be attributed to the ordered arrangement of aliphatic residues and phenyl groups within β-sheet structures.44 Fourier transform infrared (FTIR) spectroscopy analysis provided further information about the conformation changes of SF during supramolecular gelation (Figure 2B). For example, the spectrum of blank solution of NapFF showed a slight band at 1625 cm-1 in its spectrum, which was a characteristic peak of β-sheet conformation. The blank solution of SF revealed the presence of two peaks at 1648 and 1580 cm-1, respectively, which were located within the amide I and amide II regions of a random coil conformation.13-14, 17 In comparison, the mixed hydrogel from NapFF and SF displayed a sharp peak at 1621 cm-1 and two small shoulders at 1636 and 1686 cm-1 in its spectrum, implying the coexistence of β-sheet and random coil structures of SF in a gel state. The results suggested that the sol-gel transition of SF induced by NapFF was consistent with the conformational transformation of SF from a random coil to a β-sheet structure, and NapFF molecules in SF solution exhibited high potentials to promote this process of conformational transition of SF.


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In order to understand the process of cooperative self-assembling between NapFF and SF, we used fluorescence quenching titration method to examine the potentials of intermolecular interactions between them.45-46 In this experiment, a NapFF solution with a fixed concentration at 500 μM was prepared, which is much lower than the concentration required for self-assembly. Followed by the titration with SF at varied concentrations, the fluorescence emission at 332 nm from the naphthyl group of NapFF was quenched gradually, due to the physical binding between NapFF and SF clusters (Figure 2C). Based on the linear plot between fluorescence intensities of NapFF and SF concentrations in solutions (Figure S12 and S13), the binding constant of NapFF and SF protein was estimated to be 6.40×105 M-1. This result indicated that there existed intermolecular interactions between NapFF and SF via hydrophobic and hydrogen bonding, which may play an important role in the molecular conformational transformation of SF and its rapid gelation triggered by NapFF. After confirming the cooperative self-assembling property of NapFF and SF, we further investigate whether these effects can be exploited to tune the gelation rate and mechanical strength of resulting hydrogel. As shown in Figure 2D, the solution with 0.1 wt% NapFF and 0.1% SF showed a slow rate of gelation and its G’ value still remained around 22 Pa after 4500 s. However, when the concentrations of NapFF were increased to 0.2 and 0.4 wt%, rapid gelation of SF solutions was observed within 1.5 h and 30 min, respectively. Besides, the resulting Gel 1 and Gel 2 both exhibited dominant elastic properties, as exemplified by the high storage modulus (Figure 2D and Table 1). In addition, when the concentration of NapFF was fixed at 0.4 wt%, increasing the concentration of SF to 0.5, 1.0 and 2.0%, respectively, can afford the formation of Gel 3-5 (Table 1 and Figure S8-S10), which displayed further decreased gelation time and enhanced mechanical strength, in comparison with Gel 2 (Figure 2E and 2F).


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Easy control of the gelation dynamics and mechanical strength of the hydrogel from NapFF and SF encouraged us to examine the potential of preparing injectable hydrogels from them. Firstly, we used Gel 5 as an example to evaluate its injectability, because of its relatively high gelation rate and mechanical strength. After extrusion through a needle (0.7 mm in diameter), the extruded solution collected in a vial can quickly transform into a stable gel within 10 min (Figure 3A-3D). Scanning electron micrograph (SEM) image revealed the presence of a threedimensional porous network with mesh size around from 4.3 μm to 26.6 μm (Figure 3E). In order to prove shear-shinning and recovery properties, we performed a simple time-dependent step-strain experiment. As shown in Figure 3F, when the applied angular frequency was fixed at 1 Hz, and 1% strain was applied on this hydrogel, the G’ value was much higher than its G’’ value, indicating a gel-like feature. After the strain increased to 100%, both G’ and G’’ values decreased dramatically to 1245 Pa and 3022 Pa, respectively, due to the collapse of the hydrogel network. However, when the strain returned to its original value (1%), both G’ and G’’ can recover about 75% and 83% of their original values, indicating the thixotropic property of Gel 5 at relatively high concentrations of NapFF (0.4 wt%) and SF (2.0%). Because of high water content, good biocompatibility and stabilities of the hydrogel composed of NapFF and SF (Figure S14-S15), we examined its suitability to work as a new scaffold for cell adhesion and growth. On the basis of live−dead assays shown in Figure 4A, human umbilical vein endothelial cells (HUVEC) showed relatively low binding affinity to the surface of Gel 5, as exemplified by the rounded morphology of cells and disordered F-actin inside cells at day 1 (Figure 4C), due to the biological inertness of both SF and NapFF. But these cells can keep growing and form cell colonies over the course of a 5-day culture (Figure 4B), indicating the high biocompatibility of both NapFF and SF molecules. In order to improve cell adhesion


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properties of SF hydrogel, we designed and synthesized a new gelator (e.g., NapFFRGD) from NapFF by attaching with a RGD segment (Figure S4 and S5), which is an effective cell binding ligand derived from native extracellular matrices (ECM).47 By mixing SF with NapFFRGD, we obtained a new and bioactive SF hydrogel (namely, Gel RGD) containing multiple RGD ligands for cell adhesion (Figure S11). After being seeded on the surface of Gel RGD, HUVEC exhibited improved cell attachment and spreading properties on Gel RGD in comparison to Gel 5, as exemplified by the appearance of a typical polyhedral-like morphology after 1 day incubation, and the formation of highly elongated and well-defined stress actin filaments (green) inside cell (Figure 4D). Cell density quantification revealed that Gel RGD can induce a steady increase of cell densities of HUVEC (>4.5-fold) over the course of a 5-day culture (Figure 4B). These results confirmed that the supramolecular hydrogel prepared from SF protein and NapFF exhibited good cytocompatibility, and application of a peptide gelator with RGD ligands can further improve its bioactivity for cell adhesion and growth. Vascularization plays an important role in tissue regeneration by supplying adequate oxygen and nutrients to ensure the survival and function of engineered tissue constructs.48 The formation of new blood vessel in vivo is a complex process, achieved by the coordinated interplay between different components, such as tissue, cells, growth factors and ECM.49-50 Particularly, physical attachment of endothelial cell on ECM is fundamentally important to the process of vascular formation.51 Considering high similarity with ECM in structures and good cell adhesion property towards HUVEC, we decided to examine its potentials of Gel RGD to work as a new matrix for supporting microvasculature formation with the assistance of VEGF. VEGF is a major regulator of vascularization in nature under both physiological and pathological conditions, but its low biostability in vivo can significantly affect its efficiency for inducing neovascularization.52-53


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Encapsulation of VEGF in hydrogels proved to be a feasible approach to control its stability and maintain its bioactivity for an extended period of time through sustained release (Figure S17 and S18).54-57 The angiogenic activity of VEGF encapsulated in the gel matrix of Gel RGD was firstly examined by an in vitro tube formation assay (Figure 5A). After HUVECs were planted on the surface of Gel RGD containing VEGF (200 ng/mL), cell morphogenesis was examined over the course of 3-day culture (e.g., 24, 48 and 72 h). HUVECs spread well on the surface of gel matrix, and formed cell–cell contacts and short capillary-like sprouts in 24 h (Figure 5A). With the extension of incubation time, the cells started to undergo tubulogenesis and formed well-recognized capillary tubular network structures in 48 h. When the incubation time was prolonged to 72 h, a more intense tubule network was observed, as exemplified by the increasing numbers of closed meshes and junctions, as well as total tubule length per unit of area (Figure 5B-5D). In comparison, no formation of obvious short cord or capillary structures was observed in the hydrogel without encapsulating VEGF within 72 h, confirming the crucial role of VEGF in the process of endothelial cell morphogenesis. In vivo tissue response to the VEGF-loaded hydrogel (Gel RGD) for microvascularization was further evaluated after subcutaneous implantation into the dorsal side of mice for three weeks, and the corresponding hydrogel without VEGF loading was used as a control. Before subcutaneous injection, we firstly checked the in vitro gelation properties of Gel RGD at 37 °C. Based on the results shown in Figure S19, we found that a much shorter gelation time was observed at 37 °C for Gel RGD, and a stable gel of Gel RGD could be obtained in 10 min. This result was consistent with the studies that elevated gelation temperatures can increase the gelation rate of silk fibroin.17 Besides, TEM images and rheological results of Gel RGD formed at both room temperature and 37 °C revealed the presence of similar morphologies of nanofibers


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with width around 9 nm, and the formation of stable hydrogels with storage moduli (G’) around 5500 Pa for both of them. Therefore there was no obvious difference in the gel properties of Gel RGD which were formed at room temperature and 37 °C, besides a much shorter gelation time of Gel RGD at 37 °C. After subcutaneous injection, the implanted hydrogel remained localized in the injection site and was identifiable through 21 days, but was completely resorbed after 28 days, because of slow bioresorption and degradation in vivo (Figure S20). H&E staining confirmed that the bulk gel remained intact at day 3, but slowly degraded into gel fragments together with cells invasion and tissue ingrowth (Figure S21). In addition, during the course of implantation through 28 days, we did not observe significant inflammatory reaction induced by the implanted gel matrix to the surrounding tissues (Figure S21), indicating the good biocompatibility of our gel matrix for biomedical applications. More importantly, based on H&E staining images shown in Figure 6, the sample group containing VEGF showed the presence of more numbers of blood capillaries at day 3, compared with the group without VEGF. And the density of blood capillary was increased with time within the course of implantation, due to the long-term bioavailability of VEGF from Gel RGD. Immunohistochemical staining with CD31 antibodies further confirmed that the blood vessels observed on the margin of the implanted gel matrix were composed of endothelial cells (Figure 7). Quantitative analyses revealed that the average area of blood capillaries for the VEGF-loading group was around 1.11 % per observed field at day 3, and gradually increased to 1.39 % and 1.96 % at day 10 and day 21, respectively (Figure S22). In comparison, a much lower capillary density (e.g., 0.13 % at day 3, 0.31 % at day 10 and 1.07 % at day 21) was observed in the blank gel without VEGF over the course of 21-day implantation (Figure S22). These results suggested that the supramolecular hydrogel of Gel RGD can work as an effective matrix for encapsulation and delivery of VEGF and supporting


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microvasculature formation through the coordinated interactions with HUVEC, gel matrix and VEGF. 3. CONCLUSIONS In summary, we report a facile approach for inducing rapid gelation of SF by a small peptide gelator. Because of the independent self-assembling property of NapFF in SF solution and its intermolecular interaction with SF protein, the incorporation of NapFF with SF solution can effectively improve the gelation process of SF by lowering its threshold gelation concentration to 0.1%, and shortening gelation time to seconds. More importantly, in order to generate a favorable microenvironment for biomedical applications, a bioactive peptide gelator (NapFFRGD) or protein (VEGF) can be easily applied to functionalize SF gel for improved biofunctionalities, such as cell adhesion, cell growth and angiogenesis. Compared with other silk fibrion hydrogel triggered by organic molecules, our developed approach exhibit numerous advantages: (i) lower gelation concentration; (ii) higher biocompatibility; (iii) easy modification with bioactive peptide gelators or proteins; (iv) and simple and facile preparation method of peptide gelators from solid-phase synthesis. Therefore, we expected that the incorporation of peptide gelator with SF could provide a new approach for preparing biocompatible and biofunctional SF hydrogels, and the feasibility of modifying SF with other bioactive peptide gelators and its potential applicability in different biomedical engineering fields worth studying further. 4. EXPERIMENTAL SECTION Synthesis of peptide hydrogelator. Peptide gelator was synthesized by following typical solidphase synthesis procedures. Generally, 2-chlorotrityl chloride resin (1.0 g, 1.3−1.8 mmol/g) was swelled in dry dichloromethane (DCM) with N2 bubbling for 30 min, and washed by dry DMF


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five times. Afterward, the solution containing N,N-diisopropylethylamine and Fmoc-Phe-OH in DMF was added. After reaction for 1 h, the solvent was removed and the resin was washed by dry DMF and quenched by blocking solution. Then, the resin was treated with 20% piperidine for 30 min, and washed thoroughly with DMF. The peptide chain was prolonged by following standard Fmoc solid-phase synthesis protocols and using O-benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate as a coupling reagent. Finally the synthetic peptide was removed from the resin by using trifluoroacetic acid. The resulting peptide molecule was purified by column chromatography to afford NapFF in a yield of 78%. 1H NMR (400 MHz, DMSO-d6): δ12.80 (s, 1H), 8.30-8.24 (dd, 2H), 7.83-7.43 (m, 6H), 7.17 (m, 10H), 4.55(m, 1H), 4.42 (m, 1H), 3.53-3.47 (dd, 2H), 3.02-2.70 (m, 4H). MS: calcd M=480.2, obsd (M+H)+=481.2. Preparation of silk solution. Degummed SF solution was prepared by following a previously reported method.58 Briefly, B. mori cocoons were boiled in a Na2CO3 solution (0.02 M) for 1 h and then washed by deionized water to remove sericin proteins and other impurities. After drying at room temperature overnight, the extracted SF was dissolved in 9.3 M LiBr solution at 60 °C for 4 h, and the solution was dialyzed against distilled water using dialysis tube (molecular weight cut off: 3500) for 3 days. Then the solution was enriched by PEG20000, and centrifuged at 4000 rpm for 40 min at 4 °C to remove silk aggregates formed during these processes. The final concentration of SF solution was approximately 10%. SF solutions at lower concentrations were prepared by diluting stock SF solution (10%) with deionized water or PBS buffer (10 mM, pH=7.4). Gelation of silk fibroin/NapFF solution. In order to investigate the potentials of NapFF to induce the gelation of SF, we used the solution of NapFF with concentration at 0.4 wt% and pH=7.4 as stock solution. SF solutions at varied concentrations (0.1, 0.5, 1.0 and 2.0%) were


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prepared from its stock solution (10%) by successive dilution. After adding desired volumes of NapFF to SF solutions, followed by vortex and gentle heating, we obtained mixed samples containing both NapFF and SF with final concentrations from 0.1 to 0.2% and 0.1 to 2.0%, respectively. The sample containing higher concentration of NapFF at 0.4 wt% was prepared by dissolving a desired amount of NapFF in the solution of SF. Then, 300 μL of each sample was transferred to a 2 mL flat-bottomed vial and stored at room temperature for gelation. The state of the sample in tube together with its gelation time was examined by turning the tube upside down. Rheological tests. Rheological study was taken on a Thermo Scientific HAAKE RheoStress 6000 rheometer at 25 °C. Approximately, 300 μL of hydrogel was loaded on a parallel plate (diameter=20 mm). i) The dynamic strain sweep was performed from 0.1% to 10% strain with frequency at 6.282 rad/s; ii) the dynamic frequency sweep test was conducted from 0.628 to 628 rad/s with strain at 1.0%; iii) the dynamic time sweep was performed at an angular frequency of 6.282 rad/s and strain of 1.0%; iv) for shear-recovery test, the sample was oscillated firstly at low strain amplitude (1.0%) to reach equilibrium, followed by the application of a strong deformation (strain at 100%). After 30 s, the deformation at high strain was stopped and a time sweep measurement was carried out at constant strain (1.0%) and frequency (6.28 rad/s). Circular dichroism and FTIR characterization. Approximately, 20 μL of each sample was placed evenly on a quartz cuvette of 1 mm thickness and scanned from 180 to 400 nm by using a JASCO J-810 spectrometer under N2 atmosphere. The solutions and hydrogels for FTIR characterization were prepared by using deuterium oxide (D2O) as a solvent. The FTIR spectra were collected on a PerkinElmer spectrophotometer by loading the samples into a KBr cuvette. TEM and SEM characterizations. 10 μL of each hydrogel sample was loaded on a carbon films on copper grid, and stained with phosphotungstic acid (2.0% (w/v)). Then the copper grid


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was observed on a Tecnai G220 transmission electron microscope. The silicon wafers coated with hydrogel sample were frozen in liquid nitrogen and dried in vacuum for 24 h. After sputtercoating with a thin layer of gold, the samples were visualized on a scanning electron microscope (Hitachi S-4800). Determination of intermolecular interactions between SF and NapFF. The intermolecular interactions between SF and NapFF were determined by fluorometric experiments as a previously reported method.39 SF solutions at varied concentrations (0.0025-0.2%) were prepared from its stock solution (10%) by successive dilution with 10 mM PBS buffer (pH=7.4). NapFF solution with concentration at 500 μM was made in 10 mM PBS buffer (pH=7.4). The fluorescent intensities of these mixed solutions at 332 nm were examined and recorded. The binding constant (Ka) between SF and NapFF was calculated, according to literature reported procedures. In vitro cytocompatibility tests. Cytocompatibility tests were performed by following CCK-8 (Cell Counting Kit-8) protocols. Human umbilical vein endothelial cells (HUVEC) obtained from ATCC (American Type Culture Collection), were suspended in a 96-well flat bottomed microplate with a final concentration of 4×103 cells/well and cultured at 37 °C under 5% of CO2 atmosphere for 24 h. The medium was replaced with complete culture medium supplemented with different concentrations of molecular gelators (e.g., 0, 10, 20, 50, 100 and 200 μM), or SF (0, 0.1, 0.2, 0.5, 1.0 and 2.0%). After culturing for 24 h, fresh medium with 10% CCK-8 solution was added and incubated for 2 h. The optical absorbance at 450 nm was recorded on a Thermo Scientific Varioskan Flash spectral scanning multimode reader. Cell viability was expressed as a percentage of the quantity of control (untreated) cells, and viability in the control group was designated as 100%. All tests were performed in quintuplicate.


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Cell viability in vitro. Viability of the cells seeded on the hydrogels was tested according to manufacturer’s instruction for live/dead assay. Approximately, 100 μL of hydrogel was coated on a 48-well plate and covered with 100 μL of DMEM (Dulbecco’s modified Eagle’s medium) containing 10% FBS and 1% antibiotics for buffer exchange. The medium in each well was then replaced with fresh medium after 24 h of incubation. Then 200 μL of cell suspension (8.0×103 cells) in complete medium was pipetted into each well, and incubated at 37 °C under 5% of CO2 atmosphere. The medium was changed every other day and the cells were stained by calcein AM/propidium iodide and imaged by an OLYMPUS IX71 fluorescence microscope. The cell number was quantified by Image J software. Microfilament staining. Morphologies and cytoskeleton structures of cell cultured on gel sample were examined with FITC-phalloidin assays according to manufacturer’s instruction. After incubation for 24 h, cells were washed by PBS buffer (pH=7.4) twice, followed by the addition of 4% paraformaldehyde for cell fixation. Then the cell-seeded hydrogels were washed by 0.1% triton X-100 in PBS buffer four times (5 min for each time), and treated with FITCphalloidin (40 μg/mL) and Hoechst (1 μg/mL) for 1 h. The stained samples were visualized by a confocal laser scanning microscope with excitation filters at 488 nm (green, FITC) and 346 nm (blue, Hoechst). Determination of VEGF165 release in vitro using ELISA. 100 μL of SF hydrogel was prepared in triplicate with the presence of human VEGF165 (200 ng/mL). Then the hydrogel was covered with 1 mL of PBS containing 0.1% BSA, and incubated in the incubator at 37 °C and 5% CO 2 for VEGF165 release. 50 μL of PBS buffer was taken out at different time-scale (12 h, 1d, 2, 3, 4, 5, 6, 7, 10, 14 and 21 days), followed by the supplement of the same volume of PBS buffer on


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the top of hydrogels. The amount of VEGF165 in PBS buffer was determined by ELISA assays according to manufacturer’s protocols. Examination of the bioactivity of VEGF released from SF hydrogel. The bioactivity of VEGF165 released from hydrogel was collected and assessed by culturing HUVEC on tissue culture plates. HUVEC were seeded in 96-well plates with density at 4000 cells per well and maintained with DMEM containing 10% FBS, 1% antibiotics and VEGF165 (500 pg/mL) released from SF hydrogel. After incubation for 24 h, a CCK-8 assay was performed to quantify cell viability. The sample without VEGF165 was used as a control. Vascularization in vitro. 50 μL of SF hydrogel containing 200 ng/mL human VEGF165 was coated on a 48-well plate, then 200 μL of cell suspension (8.0×103 cells) in DMEM containing 10% FBS and 1% antibiotics was pipetted into each well, and incubated at 37 °C under 5% of CO2 atmosphere. The cell status was recorded by an OLYMPUS IX71 fluorescence microscope at each designed time point. The formations of the capillary networks, the number of meshes and junctions, as well as the total tubule length per unit of area were quantified by a computer image analysis (ImageJ). In vivo animal experiment. This experiment was performed by complying with the institutional regulations established and approved by the Animal Research Committee at the laboratory animal center of Soochow University. Forty male Balb/c inbred mice (8-week-old) were purchased from the laboratory animal center of Soochow University, and they all meet safety specifications. Under chloral hydrate anesthesia (4%), 100 μL of gel sample encapsulating 1 μg of VEGF was subcutaneously injected into the dorsal side of mice using a 1 mL syringe. At each designed time point, the mice were sacrificed, and their subcutaneous tissues containing gel scaffold were excised, fixed in 4% formaldehyde solution, paraffin embedded, sectioned with a


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microtome (5 μm) and stained with either H&E or anti-CD31 antibody. Blood vessel number, size and area were determined by using ImageJ software from three captured images of H&E and immunohistochemical staining slides from each animal. Statistical analysis. Statistical analysis was carried out by using the GraphPad Prism V.6.00 software. One-way ANOVA with a Bonferroni post-test was used to analyze results from tube formation tests. Two-way ANOVA with a Bonferroni post-test was used to analyze results of cell density and average blood vessel areas from CD31 staining experiments. The varied levels of significance were also explained in the legend corresponding to each figure.

ASSOCIATED CONTENT Supporting Information. Materials and methods, synthesis and characterizations of peptide gelator NapFF and NapFFRGD, determination of the binding constant between SF and NapFF, sustained release behavior of VEGF from Gel RGD. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected]; [email protected] Author Contributions ¶

These two authors contribute equally.

Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT We thank the financial support by the National Key R&D Program of China (Ministry of Science and Technology of China, 2016YFC1100100) to ZW. Shao, KQ. Zhang and X. Li, the National Natural Science Foundation of China (51673142) to X. Li; the Natural Science Foundation of Jiangsu Province (BK20151218) to X. Li; and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). REFERENCES 1. Jin, H. J.; Kaplan, D. L., Mechanism of silk processing in insects and spiders. Nature 2003, 424, 1057-1061. 2. Altman, G. H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R. L.; Chen, J. S.; Lu, H.; Richmond, J.; Kaplan, D. L., Silk-based biomaterials. Biomaterials 2003, 24, 401-416. 3. Koh, L.-D.; Cheng, Y.; Teng, C.-P.; Khin, Y.-W.; Loh, X.-J.; Tee, S.-Y.; Low, M.; Ye, E.; Yu, H.-D.; Zhang, Y.-W.; Han, M.-Y., Structures, mechanical properties and applications of silk fibroin materials. Prog. Polym. Sci. 2015, 46, 86-110. 4. Maghdouri-White, Y.; Bowlin, G. L.; Lemmon, C. A.; Dreau, D., Bioengineered silk scaffolds in 3D tissue modeling with focus on mammary tissues. Mater. Sci. Eng. C 2016, 59, 1168-1180. 5. Manchineella, S.; Thrivikraman, G.; Basu, B.; Govindaraju, T., Surface-Functionalized Silk Fibroin Films as a Platform To Guide Neuron-like Differentiation of Human Mesenchymal Stem Cells. ACS Appl. Mater. Interfaces 2016, 8, 22849-22859.


19


Page 20 of 36

6. Manchineella, S.; Thrivikraman, G.; Khanum, K. K.; Ramamurthy, P. C.; Basu, B.; Govindaraju, T., Pigmented Silk Nanofibrous Composite for Skeletal Muscle Tissue Engineering. Adv. Healthcare Mater. 2016, 5, 1222-1232. 7. Kapoor, S.; Kundu, S. C., Silk protein-based hydrogels: Promising advanced materials for biomedical applications. Acta Biomater. 2016, 31, 17-32. 8. Kim, U. J.; Park, J. Y.; Li, C. M.; Jin, H. J.; Valluzzi, R.; Kaplan, D. L., Structure and properties of silk hydrogels. Biomacromolecules 2004, 5, 786-792. 9. MacIntosh, A. C.; Kearns, V. R.; Crawford, A.; Hatton, P. V., Skeletal tissue engineering using silk biomaterials. J. Tissue Eng. Regen. Med. 2008, 2, 71-80. 10. Meinel, L.; Betz, O.; Fajardo, R.; Hofmann, S.; Nazarian, A.; Cory, E.; Hilbe, M.; McCool, J.; Langer, R.; Vunjak-Novakovic, G.; Merkle, H. P.; Rechenberg, B.; Kaplan, D. L.; Kirker-Head, C., Silk based biomaterials to heal critical sized femur defects. Bone 2006, 39, 922-931. 11. Wang, X.; Zhang, X.; Castellot, J.; Herman, I.; Iafrati, M.; Kaplan, D. L., Controlled release from multilayer silk biomaterial coatings to modulate vascular cell responses. Biomaterials 2008, 29, 894-903. 12. Hanawa, T.; Watanabe, A.; Tsuchiya, T.; Ikoma, R.; Hidaka, M.; Sugihara, M., New oral dosage form for elderly patients: preparation and characterization of silk fibroin gel. Chem. Pharm. Bull. 1995, 43, 284-288. 13. Matsumoto, A.; Chen, J.; Collette, A. L.; Kim, U.-J.; Altman, G. H.; Cebe, P.; Kaplan, D. L., Mechanisms of silk fibroin sol-gel transitions. J. Phys. Chem. B 2006, 110, 21630-21638.


20

Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


14. Ayub, Z. H.; Arai, M.; Hirabayashi, K., Mechanism of the Gelation of Fibroin Solution. Biosci., Biotechnol., Biochem. 1993, 57, 1910-1912. 15. Numata, K.; Katashima, T.; Sakai, T., State of Water, Molecular Structure, and Cytotoxicity of Silk Hydrogels. Biomacromolecules 2011, 12, 2137-2144. 16. Kojic, N.; Panzer, M. J.; Leisk, G. G.; Raja, W. K.; Kojic, M.; Kaplan, D. L., Ion electrodiffusion governs silk electrogelation. Soft Matter 2012, 8, 6897-6905. 17. Nagarkar, S.; Nicolai, T.; Chassenieux, C.; Lele, A., Structure and gelation mechanism of silk hydrogels. Phys. Chem. Chem. Phys. 2010, 12, 3834-3844. 18. Wang, X.; Kluge, J. A.; Leisk, G. G.; Kaplan, D. L., Sonication-induced gelation of silk fibroin for cell encapsulation. Biomaterials 2008, 29, 1054-1064. 19. Yucel, T.; Cebe, P.; Kaplan, D. L., Vortex-Induced Injectable Silk Fibroin Hydrogels. Biophys. J. 2009, 97, 2044-2050. 20. Jang, M. J.; Um, I. C., Effect of sericin concentration and ethanol content on gelation behavior, rheological properties, and sponge characteristics of silk sericin. Eur. Polym. J. 2017, 93, 761-774. 21. Lu, S.; Sun, S.; Zhang, F.; Li, J.; Xing, T.; Jin, J., Cationic Surfactant-Induced Instantaneous Gelation of Silk Fibroin Solution. Asian J. Chem. 2014, 26, 5667-5672. 22. Luo, K.; Yang, Y.; Shao, Z., Physically Crosslinked Biocompatible Silk-Fibroin-Based Hydrogels with High Mechanical Performance. Adv. Funct. Mater. 2016, 26, 872-880.


21


Page 22 of 36

23. Numata, K.; Yamazaki, S.; Katashima, T.; Chuah, J.-A.; Naga, N.; Sakai, T., Silk-Pectin Hydrogel with Superior Mechanical Properties, Biodegradability, and Biocompatibility. Macromol. Biosci. 2014, 14, 799-806. 24. Park, J. H.; Kim, M. H.; Jeong, L.; Cho, D.; Kwon, O. H.; Park, W. H., Effect of surfactants on sol-gel transition of silk fibroin. J. Sol-Gel Sci. Technol. 2014, 71, 364-371. 25. Silva, S. S.; Popa, E. G.; Gomes, M. E.; Oliveira, M. B.; Nayak, S.; Subia, B.; Mano, J. F.; Kundu, S. C.; Reis, R. L., Silk hydrogels from non-mulberry and mulberry silkworm cocoons processed with ionic liquids. Acta Biomater. 2013, 9, 8972-8982. 26. Wu, X.; Hou, J.; Li, M.; Wang, J.; Kaplan, D. L.; Lu, S., Sodium dodecyl sulfate-induced rapid gelation of silk fibroin. Acta Biomater. 2012, 8, 2185-2192. 27. Zhong, T.; Xie, Z.; Deng, C.; Chen, M.; Gao, Y.; Zuo, B., Copolymer-induced silk-based hydrogel with porous and nanofibrous structure. J. Appl. Polym. Sci. 2013, 127, 2019-2024. 28. Lin, C.; Xu, M.; Zhang, W.; Yang, L.; Xiang, Z.; Hong, J.; Liu, X., A Multiple Controllable Sol-Gel Preparation of 2D Silica Lamellar Composites Based on Azoimidazolium Surfactants. Adv. Mater. Interfaces 2017, 4, 1601249. 29. Dubey, P.; Kumar, S.; Aswal, V. K.; Ravindranathan, S.; Rajamohanan, P. R.; Prabhune, A.; Nisal, A., Silk Fibroin-Sophorolipid Gelation: Deciphering the Underlying Mechanism. Biomacromolecules 2016, 17, 3318-3327. 30. Boekhoven, J.; Stupp, S. I., 25th Anniversary Article: Supramolecular Materials for Regenerative Medicine. Adv. Mater. 2014, 26, 1642-1659.


22

Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


31. Du, X.; Zhou, J.; Shi, J.; Xu, B., Supramolecular Hydrogelators and Hydrogels: From Soft Matter to Molecular Biomaterials. Chem. Rev. 2015, 115, 13165-13307. 32. Rajagopal, K.; Schneider, J. P., Self-assembling peptides and proteins for nanotechnological applications. Curr. Opin. Struc. Biol. 2004, 14, 480-486. 33. Ulijn, R. V.; Smith, A. M., Designing peptide based nanomaterials. Chem. Soc. Rev. 2008, 37, 664-675. 34. Zhang, S. G., Fabrication of novel biomaterials through molecular self-assembly. Nat. Biotechnol. 2003, 21, 1171-1178. 35. Manchineella, S.; Murugan, N. A.; Govindaraju, T., Cyclic Dipeptide-Based Ambidextrous Supergelators: Minimalistic Rational Design, Structure-Gelation Studies, and In Situ Hydrogelation. Biomacromolecules 2017, 18, 3581-3590. 36. Brizard, A.; Stuart, M.; van Bommel, K.; Friggeri, A.; de Jong, M.; van Esch, J., Preparation of nanostructures by orthogonal self-assembly of hydrogelators and surfactants. Angew. Chem. Int. Ed. 2008, 47, 2063-2066. 37. Capito, R. M.; Azevedo, H. S.; Velichko, Y. S.; Mata, A.; Stupp, S. I., Self-assembly of large and small molecules into hierarchically ordered sacs and membranes. Science 2008, 319, 18121816. 38. Chen, L.; Revel, S.; Morris, K.; Spiller, D. G.; Serpell, L. C.; Adams, D. J., Low molecular weight gelator-dextran composites. Chem. Commun. 2010, 46, 6738-6740. 39. Javid, N.; Roy, S.; Zelzer, M.; Yang, Z.; Sefcik, J.; Ulijn, R. V., Cooperative Self-Assembly of Peptide Gelators and Proteins. Biomacromolecules 2013, 14, 4368-4376.


23


Page 24 of 36

40. Rozkiewicz, D. I.; Myers, B. D.; Stupp, S. I., Interfacial Self-Assembly of Cell-like Filamentous Microcapsules. Angew. Chem. Int. Ed. 2011, 50, 6324-6327. 41. Zhang, X.; Chu, X.; Wang, L.; Wang, H.; Liang, G.; Zhang, J.; Long, J.; Yang, Z., Rational Design of a Tetrameric Protein to Enhance Interactions between Self-Assembled Fibers Gives Molecular Hydrogels. Angew. Chem. Int. Ed. 2012, 51, 4388-4392. 42. Zhang, Y.; Kuang, Y.; Gao, Y.; Xu, B., Versatile Small-Molecule Motifs for Self-Assembly in Water and the Formation of Biofunctional Supramolecular Hydrogels. Langmuir 2011, 27, 529-537. 43. Iizuka, E.; Yang, J. T., Optical rotatory dispersion and circular dichroism of the beta-form of silk fibroin in solution. Proc. Nat. Acad. Sci. U.S.A. 1966, 55, 1175-1182. 44. Zhang, Y.; Gu, H. W.; Yang, Z. M.; Xu, B., Supramolecular hydrogels respond to ligandreceptor interaction. J. Am. Chem. Soc. 2003, 125, 13680-13681. 45. Bourassa, P.; Dubeau, S.; Maharvi, G. M.; Fauq, A. H.; Thomas, T. J.; Tajmir-Riahi, H. A., Locating the binding sites of anticancer tamoxifen and its metabolites 4-hydroxytamoxifen and endoxifen on bovine serum albumin. Eur. J. Med. Chem. 2011, 46, 4344-4353. 46. van de Weert, M.; Stella, L., Fluorescence quenching and ligand binding: A critical discussion of a popular methodology. J. Mol. Struct. 2011, 998, 144-150. 47. Ruoslahti, E.; Pierschbacher, M. D., New perspectives in cell adhesion: RGD and integrins. Science 1987, 238, 491-497. 48. Novosel, E. C.; Kleinhans, C.; Kluger, P. J., Vascularization is the key challenge in tissue engineering. Adv. Drug Deliv. Rev. 2011, 63, 300-311.


24

Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


49. Phelps, E. A.; Garcia, A. J., Engineering more than a cell: vascularization strategies in tissue engineering. Curr. Opin. Biotechnol. 2010, 21, 704-709. 50. Rouwkema, J.; Khademhosseini, A., Vascularization and Angiogenesis in Tissue Engineering: Beyond Creating Static Networks. Trends Biotechnol. 2016, 34, 733-745. 51. Stromblad, S.; Cheresh, D. A., Cell adhesion and angiogenesis. Trends Cell Biol. 1996, 6, 462-468. 52. Ferrara, N., Vascular endothelial growth factor: Basic science and clinical progress. Endocr. Rev. 2004, 25, 581-611. 53. Kumar, V. A.; Taylor, N. L.; Shi, S.; Wang, B. K.; Jalan, A. A.; Kang, M. K.; Wickremasinghe, N. C.; Hartgerink, J. D., Highly Angiogenic Peptide Nanofibers. Acs Nano 2015, 9, 860-868. 54. Chiu, Y.-C.; Cheng, M.-H.; Engel, H.; Kao, S.-W.; Larson, J. C.; Gupta, S.; Brey, E. M., The role of pore size on vascularization and tissue remodeling in PEG hydrogels. Biomaterials 2011, 32, 6045-6051. 55. Li, Z.; Qu, T.; Ding, C.; Ma, C.; Sun, H.; Li, S.; Liu, X., Injectable gelatin derivative hydrogels with sustained vascular endothelial growth factor release for induced angiogenesis. Acta Biomater. 2015, 13, 88-100. 56. Silva, E. A.; Mooney, D. J., Effects of VEGF temporal and spatial presentation on angiogenesis. Biomaterials 2010, 31, 1235-1241. 57. Tabata, Y.; Miyao, M.; Ozeki, M.; Ikada, Y., Controlled release of vascular endothelial growth factor by use of collagen hydrogels. J. Biomater. Sci., Polym. Ed. 2000, 11, 915-930.


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58. Yamada, H.; Nakao, H.; Takasu, Y.; Tsubouchi, K., Preparation of undegraded native molecular fibroin solution from silkworm cocoons. Mater. Sci. Eng., C 2001, 14, 41-46.


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Scheme 1. Illustration of the process of supramolecular gelation from SF and peptide gelator and its potential biofunctionality for inducing angiogenesis in vivo.


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Figure 1. Optical images of (A) the blank solution of NapFF (0.4 wt%, pH=7.4), (B) the blank solution of SF (0.2%, pH=7.4), and (C) the mixed hydrogel containing NapFF (0.2 wt%) and SF (0.1%) at room temperature. (D) TEM image of the nanostructures from SF gel shown in Figure 1C. (E) Frequency dependence of the dynamic storage moduli (G’) and the loss moduli (G’’) of SF gel shown in Figure 1C.


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Figure 2. (A) Circular dichroism and (B) FTIR spectra analysis of the blank solution of NapFF, SF and the mixed gel in deuterium oxide (D2O) (pH=7.4) shown in Figure 1. (C) Fluorescence spectra of NapFF in the presence of varied concentrations of SF in 10 mM PBS buffer (pH=7.4). (D) Dynamic time sweep of the mixed hydrogels composed of varied concentrations of NapFF (Sol 1: 0.1 wt%, Gel 1: 0.2 wt%, and Gel 2: 0.4 wt%) and 0.1% of SF. (E) Gelation time of different hydrogels containing 0.4 wt% of NapFF and varied concentrations of SF. (F) Storage modulus of different hydrogels containing 0.4 wt% of NapFF and varied concentrations of SF.


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Figure 3. Optical images of (A) the mixed hydrogel containing NapFF (0.4 wt%) and SF (2.0%), (B) the process of extruding the prepared hydrogel through a 0.7 mm needle, (C) the sol state of sample collected from the procedure shown in Figure 3B, (D) the recovered gel from the solution shown in Figure 3C in 10 min. (E) SEM image of the recovered gel shown in Figure 3D. (F) Time-dependent of repetitive cycles of the step-strain analysis of the hydrogel shown in Figure 3A.


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Figure 4. (A) Fluorescence images of live/dead assays of HUVEC cultured on the hydrogels of Gel 5 and Gel RGD over the course of 5 days. (B) Cell density changes of HUVEC cultured on the hydrogels of Gel 5 and Gel RGD shown in Figure 4A over the course of 5 days. Morphologies and cytoskeleton structures of HUVEC cultured on the hydrogels of (C) Gel 5 and (D) Gel RGD shown in Figure 4A.


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Figure 5. (A) Light microscopy images of HUVEC morphogenesis on the blank hydrogel of Gel RGD or Gel RGD loading with VEGF (200 ng/mL) over 72 h. Quantification of (B) the number of meshes per unit of area, (C) the number of junction per unit of area, and (D) the total tube length per unit of area on Gel RGD loading with VEGF (200 ng/mL) over 72 h. Values are shown as mean ± standard deviation (n=3). *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar = 50 μm.


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Figure 6. Histological images of blood vessels in implanted hydrogel with/without VEGF loading. (A-C) H&E stained sections of the implanted Gel RGD hydrogel without VEGF loading on day 3, day 10 and day 21, respectively; (D-F) high magnification images of the boxed areas shown in Figure 6A, 6B and 6C. (G-I) H&E stained sections of the implanted Gel RGD hydrogel loading with VEGF on day 3, day 10 and day 21, respectively; (J-L) high magnification images of the boxed areas shown in Figure 6G, 6H and 6I. Black arrows indicate blood vessels. Scale bar = 100 μm.


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Figure 7. (A-C) Fluorescent images of sections of implanted hydrogel without VEGF loading at day 3, day 10 and day 21, respectively. The samples were stained by using CD31 antibodies (red) and DAPI (blue); (D-F) high magnification images of the boxed areas shown in Figure 7A, 7B and 7C, individually. (G-I) Fluorescent images of sections of implanted hydrogel loading with VEGF at day 3, day 10 and day 21, respectively; (J-L) high magnification images of the boxed areas shown in Figure 7G, 7H and 7I, individually. Scale bar = 50 μm.


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Table 1. Summary of the conditions and properties of hydrogels formed by NapFF and SF.

Sample

Sol 1

Gel 1

Gel 2

Gel 3

Gel 4

Gel 5

NapFF (wt%)

0.1

0.2

0.4

0.4

0.4

0.4

SF (%)

0.1

0.1

0.1

0.5

1.0

2.0

pH

7.4

7.4

7.4

7.4

7.4

7.4

-

0.17

0.24

1.13

1.64

2.39

-

659

1289

7051

9902

18560

-a

1.5 h

30 min

483 s

314 s

97 s

Gel images Critical (%) G’ (Pa)

strain

Gelation time [a]

The gelation process did not occur in 24 hr. Bar=0.5 cm.


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TOC Graphic


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Cooperative Assembly of a Peptide Gelator and ... - ACS Publications

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