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Article Cite This: ACS Omega 2019, 4, 3114−3121
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Water-Rinsed Nonmulberry Silk Film for Potential Tissue Engineering Applications Feng Zhang,† Richen Yang,‡ Peng Zhang,§ Jianzhong Qin,*,§ Zhihai Fan,*,§ and Baoqi Zuo*,‡ †
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Department of Immunology, School of Biology and Basic Medical Sciences, Key Laboratory of Stem Cells and Biomedical Materials of Jiangsu Province and Chinese Ministry of Science and Technology and ‡National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China § Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215006, China ABSTRACT: The possibility of using nonmulberry silk fibroin as a biomaterial is a subject of broad interest due to its inherent RGD sequence. However, the extraction of silk fibroin from cocoons and silk glands remains a formidable task. In this work, we report a facile method to dissolve tussah silk using CaCl2− formic acid and a water-rinsing process for regeneration of tussah silk fibroin (TSF) film. The new technique is important and valuable because it can directly extract silk fibroin from cocoons and is advantageous over other previous reported methods using LiSCN and Ca(NO3)2. The morphology, chemical structure, and mechanical properties of regenerated film rinsed with water, ethanol, and methanol are characterized by scanning electron microscopy, Fourier transform infrared, and X-ray diffraction techniques in combination with mechanical testing. The results reveal that the regenerated TSF films possess a smooth surface and nanostructural cross section and exhibit predominantly β-sheet crystal structure and good mechanical properties in wet state. In addition, enhanced cell attachment and proliferation of bone marrow stem cells are observed on water-rinsed TSF film. The present study provides a novel way for dissolution and regeneration of tussah silk, and the resulting TSF films are a promising biomaterial for tissue engineering applications.
1. INTRODUCTION Silk is one of the most abundant biorenewable material, with a long and well-established technological base, and silk production existed in China from around 2500 B.C. Silk has been widely used for textile application due to its fineness and luster. Recently, silk has been transformed in just the past decade from the commodity textile world to a growing web of applications in high-technology directions, especially in use as biomaterials due to its superior mechanical properties comparable to those of spider silk, availability as large supply, good biocompatibility, and easy processing.1 Silks are a diverse family of natural materials, mainly classified as domestic ( Bombyx mori) and wild (tussah, etc.) silk, which have been extensively studied as the raw source of biomaterial preparation. Tussah silk fibroin (TSF) is mechanically superior to BSF and possesses RGD sequences.2,3 The noncytotoxic property and low level of inflammatory response make TSF an excellent material for tissue engineering.4,5 So, there is a great appeal of exploring regenerated TSF-based materials with desirable forms, structure, and property to meet the specific uses of biomaterials. A successful dissolution is the first step in the process of developing new and valuable silk-based materials for its further use in biomedical applications.6 In this aspect, B. mori silk has been found soluble easily in ionic liquids and concentrated chaotropic salts, such as calcium chloride and lithium bromide.6,7 However, these solvents have difficulty in © 2019 American Chemical Society
dissolving tussah silk because of its strong resistance to chemicals, which limits its further exploration and application compared with B. mori silk. Recently, We have explored a new solvent for B. mori silk dissolution, including CaCl2 and formic acid (FA).8,9 The ability of CaCl2−FA to disrupt hydrogen bonding makes it an attractive solvent for TSF fibers. In this article, we test the suitability of CaCl2−FA for solubilizing tussah silk and preparing regenerated films. The properties of regenerated silk films are very important from the practical viewpoint of application for nontextile materials, such as for drug delivery, tissue engineering scaffolds, cell culture, etc.10 Usually, the pure silk films, domestic and wild silk, tend to be brittle and stiff in the dry state, exhibiting high strength and low elongation.11 The mechanical properties of silk film can be significantly improved by blending with other materials or physical stretching.12,13 Most of the as-cast silk films must be stabilized by the induction of β-sheet formation through post-treatment with organic solvents, like methanol and ethanol.14 To avoid the use of organic solvent, water vapor annealing is explored to treat silk scaffolds.15 For our purpose, water instead of organic solvents is applied to rinse and promote the crystallization treatment of TSF and the Received: December 18, 2018 Accepted: January 25, 2019 Published: February 12, 2019 3114
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Figure 1. Dissolution and film formation of tussah silk: (a) degumming in sodium carbonate solution, (b) dissolving in CaCl2−FA solution, and (c) casting to form film. Raw silk fiber shows sericin coating and calcium oxalate crystals. Degummed silk shows nanofibril structure. Silk solution shows yellow color and lots of bubbles. Regenerated TSF film is transparent.
Figure 2. Optical polarizing microscopy photographs of tussah silk fibers in CaCl2−FA at different times. (a) 0 min, (b) 1 min, (c) 2 min, and (d) 5 min.
properties, biocompatibility of TSF films rinsed with water, ethanol, and methanol were characterized.
mechanical properties of TSF film in wet state are more adequate for taking into account its practical application. The present work was sought to address two goals: first is exploring a facile processing method for tussah silk dissolution and regeneration. To achieve this objective, CaCl2−FA was employed to dissolve tussah silk and the effect of CaCl2 concentration, dissolving temperature, and time on solubility was evaluated to determine its ability and optimum parameters to dissolve silk. Second, an all-aqueous treatment was sought to remove salts and acid and induce β-sheet formation, simultaneously avoiding unwanted chemical residuals with respect to biological uses of the materials when organic solvents were used. The chemical structure, mechanical
2. RESULTS AND DISCUSSION 2.1. Dissolution Behavior. Silk features exceptionally strong, extensible, and tough mechanical properties in spite of the simple protein building blocks, and the material’s protein structure endows silk with superior biocompatibility. Recently, the possibility of using tussah silk fibroin (TSF) as a biomaterial for biomedical and biotechnological applications has attracted a great deal of attention.16 However, only limited works had been reported on the regeneration of tussah silk due to its insolubility in the these solvents used for dissolving domestic silk.17 To date, Ca(NO3)2, LiSCN, and ionic liquid 3115
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helix structured.24 Therefore, CaCl2−FA dissolved silk through disrupting the hydrogen bonds in the crystalline region, meanwhile restraining the formation of stable β-sheet as well as metastable α-helix during the film formation. 2.2. Solubility. The solubility of TSF fibers (1 g in weight) in 10 mL CaCl2−FA under stirring was characterized to determine the optimum conditions. Figure 4a represented the solubility of TSF fibers in various concentrations of CaCl2 solution at room temperature for 2 h. TSF fibers were nearly insoluble in 2% CaCl2 concentration or less, about 20−80 wt % soluble in 4−8% CaCl2 concentration, and 100% soluble in 10% CaCl2 concentration. This suggested that 10% CaCl2 concentration was effective in dissolving 10 wt % silk fibers. Figure 4b shows the solubility of TSF fibers in 10 wt % CaCl2−FA at different temperatures for 2 h. The silk solubility was ∼30 wt % at 4 °C, about 80 wt % at 10 °C, and reaches 100 wt % when temperature ≥20 °C. Compared to the condition of CaCl2 concentration, the fiber solubility was less affected by dissolving temperature. Therefore, room temperature was enough for silk dissolution in CaCl2−FA. Figure 4c displayed the dissolution rate of TSF fibers in 10 wt % CaCl2−FA. Silk fibers with 80 wt % weight were dissolved within 0.5 h and completely dissolved in less than an hour. According to this result, it was suggested that 10 wt % CaCl2−FA was an efficient solvent for TSF silk dissolution. Taken together, the solubility increased with a higher CaCl2 concentration and dissolving temperature and increased dissolving time. The native silk was stabilized mainly by the presence of the strong inter-nanofibril interaction and β-sheet crystal structure. The ability of FA to disrupt hydrogen bonding made it an attractive solvent for polypeptides. However, native silk was insoluble in FA, which due to containing a small quantity of CaCl2 became a powerful solvent for silk. It was deduced that CaCl2−FA dissolved tussah silk by swelling the compact fibrous structure and breaking the β-sheet. During the dissolution process, the role played by FA was mainly swelling the silk,25 making the CaCl2 infiltration available. Then, the Cl− associated with the amino hydroxyl and Ca2+ linked with the ester oxygen. This interaction disrupted the β-sheet formed by hydrogen bonding in silk fibroin and led to silk dissolution. The dissolving process was also accompanied by a certain degree of degradation of fibroin molecular chains. Compared to aqueous solution of CaCl2 or LiBr, CaCl2−FA was more effective in dissolving silk due to the strong swelling ability of FA on silk. In the following study, 10 wt % TSF fibers were dissolved in 10 wt % CaCl2−FA at room temperature (∼20 °C) for 1 h. The resulting solution with yellow color was used to prepare regenerated TSF films. It was well known that as-cast films with amorphous structure were water soluble and water was infeasible for rinsing. But silk in CaCl2−FA featured a specific dissolution behavior, i.e., nanofibril preservation, approved in our previous reports.9,26 The existence of nanofibrils played an important role in maintaining water stability of the regenerated film just like their role in native silk. Therefore, we employed water, methanol, and ethanol to rinse the as-cast TSF film with the aim of removing CaCl2 and residual FA. After rinsing, a transparent and stable TSF film was formed, as shown in Figure 1. 2.3. Morphology. The morphology of TSF films rinsed with different solvents was observed by SEM, as shown in Figure 5. These films had similar morphology characteristics
were common used to extract TSF and silk gland was another way to prepare TSF-based materials.17−20 Recently, TSF with mesostructures was directly obtained from native tussah silk fibers for application in electronic and environmental fields.17 Here, we explored a new and facile way for tussah silk dissolution and regeneration, as shown in Figure 1. Silk fibers were composed of a fibroin core and gluelike sericin outside. The sericin component in silk should be removed completely to guarantee its biological properties. The raw tussah silk showed a fairly rough surface and microsized calcium oxalate crystals.21 Degumming in sodium carbonate solution generated sericin-free tussah silk that could be subsequently solubilized. A closer examination by scanning electron microscopy (SEM) indicated that these fibers were composed of fibrils that were ∼15 nm in diameter.22 The dissolution kinetics of silk in CaCl2−FA was studied by observing the changes in fiber birefringence with a polarizing microscope. As shown in Figure 2a, the native silk displayed birefringence, which was clearly referable to optical anisotropy.22 The silk fiber shrunk drastically, and part of its birefringence tended to be dark when it came in contact with CaCl2−FA. Then, it was found that with the increase of touch time, the fibers presented in CaCl2−FA disappeared as a consequence of silk dissolution. Meanwhile, some bubbles were observed in the silk dissolution position. It was known that the silk fiber has pore structure at a scale of nano to micro, which was formed during the silkworm spinning and water evaporation. As shown in Figure 1, a lot of bubbles generated in the dissolving solution confirmed that a massive amount of pores existed in TSF fibers.23 The porous structure was conducive to solvent penetration, thus promoting fiber swelling and dissolution. The resulting TSF solution was used to cast films on polystyrene Petri dishes. The crystal structure of TSF fiber and as-cast film was examined with X-ray diffraction (XRD). In Figure 3, raw and
Figure 3. XRD pattern of raw silk, degummed silk, and as-cast silk film.
degummed silks show a similar XRD spectrum, characterized by the β-sheet structure peaks at 16.7 and 20.5° and α-helix structure peak at 24.1°, suggesting the predominantly β-sheet crystalline structure but with a certain amount of α-helix, in agreement with a previous report.2 Compared to the crystalline fiber, the as-cast TSF film showed a broad amorphous halo around 16−30°, indicating mainly an amorphous structure.22 In previous reports, the conformations of TSF films regenerated from silk glands and cocoons dissolved in lithium thiocyanate/calcium nitrate solution were predominantly α3116
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Figure 4. Effect of concentration, temperature, and time on the solubility of tussah silk fiber in CaCl2−FA.
the air−solution interface, which restrained the evaporation of formic acid.27 In addition, CaCl2 had a strong adsorption ability for organic solvent, preventing the solvent evaporation. The residual solvent and CaCl2 leaching generated the pore structure at the nanoscale in the resulting films. 2.4. Structure. Changes in structure of silk films rinsed with water, ethanol, and methanol were characterized by Fourier transform infrared (FTIR) spectroscopy and XRD, as shown in Figure 6. The FTIR spectra of as-cast film exhibited absorption bands at 1637 cm−1 to amide I (C−O stretching), 1545 cm−1 to amide II (N−H in plane bending), and 1235 cm−1 to amide III (N−H deformation and C−N stretching), which were all attributed to amorphous structure.27 The amorphous structure of as-cast film was also confirmed by XRD pattern, as shown in Figure 3. This was different from previous results wherein the predominant α-helix instead of amorphous structure was found in the TSF films prepared from silk gland and cocoon dissolved in solvents of lithium thiocyanate or calcium nitrate.23 These results demonstrated the two roles played by CaCl2−FA in disintegrating β-sheet during silk dissolution and preventing structural transition to α-helix during film formation. After rinsing, the absorption peaks of films shifted to 1623, 1517, and 1265 cm−1, indicating the predominately β-sheet structure in the rinsed films.28 The β-sheet structure of solvent-rinsed films was further confirmed by XRD analysis, characterized by the diffraction peaks at 16.7, 20.1, and 24.1°; corresponding d-spacings were 0.530, 0.441, and 0.369 nm. Taken together, the secondary structure of the film transformed from random coil to β-sheet structure under three different rinse solvents, methanol, ethanol, and water. Methanol was often used as a coagulating solvent and was known to be effective in inducing the formation of β-sheet structure for silk films. Ethanol was another routinely used
Figure 5. SEM images of regenerated silk fibroin films rinsed using water, ethanol, and methanol.
featuring smooth surface and nanoporous structures. To further investigate the internal morphology structure, the film was fractured in liquid nitrogen. The nanoporous structure was also observed in the cross section. The smooth surface indicated the near-perfect packing of silk fibroin molecules with minimal surface defects and irregularities, and the nanoporous structure on the film surface and cross section was formed likely due to insufficient solvent evaporation. When the silk solution was cast, a thin layer or skin formed at
Figure 6. FTIR spectra and XRD pattern of regenerated silk fibroin films rinsed using water, ethanol, and methanol. 3117
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Figure 7. TG, differential thermogravimetric (DTG), and DSC curves of native silk and regenerated silk fibroin films rinsed using water, ethanol, and methanol.
solvent in inducing the structural transition to the crystalline βsheet of B. mori silk and usually used in a concentration of 75%. It was reported that ethanol with high concentration (≥80 wt %) was incapable of induction of the β-sheet formation of TSF film regenerated from lithium thiocyanate.28,29 However, the TSF film prepared from CaCl2−FA could be induced to form β-sheet when rinsed with pure ethanol, suggesting more sensitivity of this film to ethanol treatment. Usually, water could not be used as a coagulating solvent for film treatment and other material forms, such as porous scaffold and filament, because amorphous silk fibroin was soluble in water.7,30 One exception to this statement was that B. mori silk film ≥70 μm (films with thickness
