The role of dialysis and freezing on structural conformation, thermal properties and morphology of silk fibroin hydrogels

Abstract

Silk fibroin has been widely explored for many biomedical applications, due to its biocompatibility and biodegradability. The aim of this work was to study the role of dialysis and freezing on structural conformation, thermal properties and morphology of silk fibroin hydrogels. Hydrogels were prepared after 3 and 7 days of dialysis and the effect of freezing was analyzed. For that purpose, a part of the fibroin hydrogels underwent freezing at -20 °C for 24 h, followed by lyophilization and the rest of the hydrogels were kept at 8 °C for 24 h, with further lyophilization. The fibroin hydrogels were characterized by X-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). Measurements by XRD and FTIR indicated that silk I and silk II structures were present in the fibroin hydrogels and that the secondary structure of fibroin is transformed mostly to β-sheet during the gelation process. Thermal analysis indicated that fibroin hydrogels are thermally stable with the degradation peak at around 330–340 °C. SEM micrographs showed porous structures and the fibroin hydrogels subjected to freezing presented a much larger pore size. Results indicate that the dialysis time and freezing did not alter the material crystallinity, conformation or thermal behavior; however, hydrogel microstructure was strongly affected by dialysis time and freezing, showing controlled pores size. This study provides fundamental knowledge on silk fibroin hydrogels preparation and properties and the studied hydrogels are promising to be used in the biomaterial field.

Introduction

Replacement of functional tissues requires the development of three-dimensional (3D) scaffolds that can provide an optimum microenvironment for tissue growth and regeneration.^1,2 Pore architecture in 3D polymeric scaffolds is known to play a critical role in tissue engineering as it provides the growth, adhesion, migration and proliferation of cells. Therefore, the scaffolds should have suitable properties such as interconnecting network of pores for the effective migration, growth and attachment of cells, sufficient porosity for the effective transport of nutrients and waste, biocompatibility, and biodegradation properties, for being exploited in tissue engineering applications.³

Silk fibroin (SF) derived from Bombyx mori silkworm is a structural protein which possesses many important properties for tissue engineering and regenerative medicine, such as versatile processing, slow biodegradation, good biocompatibility, low immunogenicity, low inflammatory response, adjustable mechanical properties, high permeability to oxygen and water vapor and resistance to enzymatic degradation.^4-6

The cocoon of the silkworm is mainly composed of two protein components, fibroin and sericin. Fibroin is the water insoluble structural protein component of silk fibers whereas sericin is the water-soluble glue-like protein that holds SF fibers together.⁴ Since the sericin contamination was identified to be the main source of problems, such as unwanted immunological reactions in vivo,^7,8 purified SF has become a novel, promising biomaterial and has found increasing numbers of applications in clinical devices including in tissue engineering of cartilage, bone, muscle, ligament and tendon tissues.^9-13 Fibroin is the core protein which accounts for 70% of the cocoon and is a hydrophobic glycoprotein.⁴ SF consists mainly of the amino acids glycine, alanine and serine which form antiparallel β-sheets in the spun fibers and provide stability and interesting mechanical properties to the fibers.^4,14 SF can assume distinct conformations; α-helix and random-coil conformations (also called silk I) and the β-sheet conformation (silk II). Silk I is a metastable form that is soluble in water and silk II is insoluble in water and is the most stable structure, where SF chains are connected by hydrogen bonds between the adjacent segments of polypeptide chains.^5,15-17

SF fibers, extracted from B. mori cocoons, are insoluble in water and in the majority of organic solvents. SF dissolves in concentrated acid solutions and in concentrated aqueous, organic and aqueous-organic salts solutions.¹⁸ These solutions contain highly concentrated salts that need to be removed by dialysis in order to prepare SF-based materials, such as hydrogels,¹⁹ films¹⁷ and nanoparticles.²⁰ SF solution after dialysis is metastable (predominance of silk I) and it can easily convert to a more stable form, by a conformational transition to silk II, forming stable hydrogels. SF gelation from aqueous solution is a kinetic process and depends on factors such as SF concentration, temperature, pH²¹ and presence of other materials, such as poly(N-isopropylacrylamide)²² and poly(ethylene oxide) polymers²³. SF gelation process is also influenced by dialysis parameters, since the rate of salt removal will directly influence the conformational transition of SF. The rate of salt removal can influence SF conformational transition, since the salt is responsible for SF solvation and stabilization in solution. When salt is removed, a metastable solution is obtained and at this point gelation is just a kinetic process. If the necessary amount of salt is not removed during dialysis, SF-based materials cannot be prepared due to the strong salt solvation. On the contrary, the more salt is removed, the more rapid is the gelation process.¹⁹ Thus, dialysis is a key parameter on SF gelation and the study of dialysis time is important to define SF hydrogels properties.

Cryogenic processes, based on freezing of hydrogels, and subsequent lyophilization, are widely used for scaffold preparation because of the biocompatibility of both the template and the process for template removal.^24-26 During the freezing step, solvent crystals grow at a controllable rate and solute molecules are excluded from the frozen solvent until the sample is completely frozen. The freezing step is very important in order to produce desirable porous structures, with control over the pores size and quantity, depending on parameters such as freezing temperature and rate.²⁷

The purpose of the present study was to study the role of dialysis and freezing on structural conformation, thermal properties and morphology of silk fibroin hydrogels. Hydrogels were prepared after 3 and 7 d of dialysis and the effect of freezing was analyzed. The structure and properties of SF hydrogels were determined by X-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM).

Results

Crystallinity

Figure 1 shows the X-ray diffractograms of SF hydrogels prepared from SF solution subjected to different dialysis time and freezing. The halos at 2θ = 20.4°, 2θ = 20.2°, 2θ = 20.7° and 2θ = 20.6° appearing in the XRD spectra of SF hydrogels formed at 37 °C after 3 d of dialysis subjected to freezing (Fig. 1A), SF hydrogels formed at 37 °C after 3 d of dialysis kept at 8 °C (Fig. 1B), SF hydrogels formed at 37 °C after 7 d of dialysis subjected to freezing (Fig. 1C) and SF hydrogels formed at 37 °C after 7 d of dialysis kept at 8 °C (Fig. 1D), respectively, are attributed to the silk II (β-sheet) conformation.^14,20,28-30

Figure 1. X-ray diffractograms of SF hydrogels formed at 37 °C after 3 d of dialysis subjected to freezing (A) or kept at 8 °C (B) and SF hydrogels formed after 7 d of dialysis subjected to freezing (C) or kept at 8 °C (D).

Structural conformation

By analyzing the infrared spectrum, the structural conformation of SF can be determined, depending on the wavenumber location of the absorption bands of amides I, II and III. Amide I, II, and III bands are attributed to C = O stretching, N-H deformation, and O-C-N bending, respectively.³¹

The infrared spectra of the SF hydrogels are shown in Figure 2. The SF hydrogel formed at 37 °C after 3 d of dialysis and subjected to freezing (Fig. 2A) presented absorption bands at 1625 cm⁻¹ (amide I) and 1525 cm⁻¹ (amide II), corresponding to the silk II structural conformation.^32-34 Other adsorption band was observed at 1234 cm⁻¹ (amide III), which is characteristic of the silk I conformation.³⁵

Figure 2. FTIR-ATR spectra of SF hydrogels formed at 37 °C after 3 d of dialysis subjected to freezing (A) or kept at 8 °C (B) and SF hydrogels formed after 7 d of dialysis subjected to freezing (C) or kept at 8 °C (D).

The SF hydrogel formed at 37 °C after 3 d of dialysis and kept at 8 °C (Fig. 2B) showed adsorption bands at 1631 cm⁻¹ (amide I) and 1533 cm⁻¹ (amide II), indicating the existence of the silk II structural conformation.^32-34 The adsorption band at 1240 cm⁻¹ (amide III) is characteristic of the silk I conformation.³⁶

The SF hydrogels formed after 7 d of dialysis subjected to freezing or not (Fig. 2C and D, respectively) presented similar spectra, with adsorption band at 1635 cm⁻¹ (amide I) corresponding to the SF silk II structural conformation.^32-34 The adsorption bands at 1525 and 1527 cm⁻¹ (amide II), for SF hydrogels formed after 7 d of dialysis subjected to freezing or not (Fig. 2C and D, respectively), is also attributed to the SF silk II structural conformation.^32-34 The adsorption band at 1234 cm⁻¹ (amide III), for both SF hydrogels formed after 7 d of dialysis is ascribed as silk I structural conformation.³⁵

In all the analyzed hydrogels the co-existence of silk I and silk II conformations was observed, which is typical of SF hydrogels.

Thermal analysis

Thermogravimetric curves of SF hydrogels are shown in Figure 3. The thermograms of all samples showed similar trend. The initial weight loss of SF hydrogels at around 100 °C is due to loss of water. The second weight loss took place within the temperature range between 270 and 380 °C and is associated with the breakdown of side chain groups of amino acid residues as well as the cleavage of peptide bonds.^37,38

Figure 3. TGA curves of SF hydrogels formed at 37 °C after 3 d of dialysis subjected to freezing (A) or kept at 8 °C (B) and SF hydrogels formed after 7 d of dialysis subjected to freezing (C) or kept at 8 °C (D).

For observation of SF hydrogels degradation peaks, derivate of the thermogravimetric curves were analyzed, as shown in Figure 4. It is possible to observe that all samples presented similar behavior, with a peak at around 330–340 °C, which is attributed to the thermal degradation of SF hydrogels.

Figure 4. DTGA wide scan curves of SF hydrogels and detected degradation peaks (inset) in all hydrogels. SF hydrogels formed at 37 °C after 3 d of dialysis subjected to freezing (A) or kept at 8 °C (B) and SF hydrogels formed after 7 d of dialysis subjected to freezing (C) or kept at 8 °C (D).

Morphology

Figure 5 shows the micrographs of the SF hydrogels porous structure obtained by SEM. These revealed that all samples had 3D morphologies with uniform porous structures and they showed the effect of freezing on SF hydrogels formed at 37 °C after 3 d of dialysis (Fig. 5A) and SF hydrogels formed after 7 d of dialysis (Fig. 5C), which were frozen at -20 °C for 24 h, compared with the SF hydrogels kept at 8 °C (Fig. 5B and D). The SF hydrogels subjected to freezing presented a pore size much larger than the hydrogels kept at 8 °C. Moreover, it is possible to see that the SF hydrogels formed after 7 d of dialysis kept at 8 °C showed a thicker fibroin network with smaller pores (Fig. 5D) than SF hydrogels formed after 3 d of dialysis (Fig. 5B).

Figure 5. SEM micrographs of the porous structure SF hydrogels formed at 37 °C after 3 d of dialysis subjected to freezing (A) or kept at 8 °C (B) and SF hydrogels formed after 7 d of dialysis subjected to freezing (C) or kept at 8 °C (D).

Discussion

Significant work has been performed over the last decade to develop natural polymers for biomedical applications, particularly for biocompatible implantable materials.³⁹ Among natural polymers, silk is an interesting candidate component for tissue engineering. The goal of this research was to study the role of dialysis and freezing on structural conformation, thermal properties and morphology of silk fibroin hydrogels.

The SF solution, before dialysis, is supersaturated and can be stored without undergoing gelation for several months. This happens due to the high ionic strength that promotes SF fibers solvation. During the dialysis, the salt ions diffuse from SF solution into the dialysis water and the ionic force decreases. This allows more interaction between SF molecules and the solution may undergo a sol-gel transition.¹⁹

Through X-ray diffraction measurement, some information about SF crystalline structure and its conformations of silk I and silk II can be obtained from its crystal structure transition. The distinct halos found in the X-ray diffractograms at 2θ = 20.4°, 2θ = 20.2°, 2θ = 20.7° and 2θ = 20.6° for SF hydrogels presented typical patterns of β-sheet structure (silk II). Previous studies by Kim et al. presented that all hydrogels prepared in your work showed a distinct halo at 2θ = 20.6°, which is attributed to β-sheet crystalline structure of SF.¹⁴ Kundu et al. observed a halo at 2θ = 20.2° denoting the silk II structure appeared in the XRD pattern of the SF nanoparticles made from B. mori protein.²⁰ Kim et al. observed a halo at 2θ = 20.4° for SF scaffolds indicating that is a β-sheet crystalline structure (silk II).²⁸ Lv et al. showed the presence of the diffraction halo at 2θ = 20.7° in silk fibroin films, corresponding to β-sheet structure.³⁰ This β-sheet conformation is formed during SF solution gelation, as a result of SF molecular chain dehydration and their intra and intermolecular hydrogen bond formations. In solution, SF molecules are predominantly organized into the α-helix and random coil structure. However, this structure tends to convert to the β-sheet upon hydrogel formation, which is thermodynamically more stable.⁴⁰ No remarkable differences were observed in the XRD spectra of the SF hydrogels formed under different conditions within this study.

FTIR spectroscopy is very sensitive to conformational modifications of silk fibroin. Each SF crystalline form shows a specific absorption band in distinct vibrational regions associated with the amide groups in proteins. The more interesting infrared bands to analyze proteins are the amide bands: amide I, amide II and amide III.³¹ Results of FTIR investigation showed that there were slight shifts of the adsorption bands of amides but without conformational changes. This indicates that SF presented in the hydrogel is highly stable and does not change its conformation due to changes in external factors.

Thermal decomposition of SF is mainly influenced by the intrinsic morphological and physical properties of the protein.^41,42 The results obtained by TGA suggest that SF hydrogels thermal degradation occurs at 330–340 °C, which according to the literature is related to SF materials with crystalline β-sheet (silk II) conformation.^42,43 This result is in agreement with the XRD result, which determined that the secondary structure of SF hydrogels is predominantly β-sheet (silk II). By FTIR analysis was possible to verify the presence of silk I conformation on the amide III absorption band, indicating that this structure may occur in the fibroin hydrogels, but in a lower proportion when compared with the silk II.

Morphology of SF hydrogels, reveled by SEM micrographs, indicates the presence of a porous structure with three-dimensional interconnectivity.^14,43-47 The SF hydrogels subjected to freezing (–20 °C) showed larger pore size when compared with SF hydrogels kept at 8 °C. The SF hydrogels kept at 8 °C were frozen with liquid nitrogen before the lyophilization. In the lyophilization procedure, pore size of the material is controlled by the size of the ice crystals formed during freezing. The freezing process with liquid nitrogen is particularly rapid and, ideally, the water presented within the pores of the hydrogel turns into ice crystals that sublimate during lyophilization, maintaining the same shape and size of the pores of the hydrogel. However, when the freezing process is performed at a higher temperature, such as freezing at –20 °C, ice nucleation is slow and the nuclei tend to grow into larger ice crystals, which leads to the generation of materials with large and random pores.⁴⁸ The porous features resulting from freezing are in good agreement with precise results. Guan et al. showed that when polyurethane scaffolds were frozen in liquid nitrogen small pores were observed and most of the pores were less than 10 μm in size.⁴⁹ Chun et al. observed that the pore size of porous poly(D,L-lactic-co-glycolic acid) scaffolds frozen at liquid nitrogen temperature was smaller than 5 μm, but the pore size was enlarged up to 30–50 μm when frozen at –20 °C for 24 h.⁵⁰ Similarly, Ma et al. observed that when the temperature was decreased from –20 °C to –196 °C (liquid nitrogen) the pore size of poly(L -lactic acid) scaffolds was greatly decreased from 115–140 µm to 20–40 µm.⁵¹ Chung et al. also showed that the pores of alginate/galactosylated chitosan scaffolds formed after liquid nitrogen treatment were small than the pores formed after freezing at –20 °C and –70 °C.⁵²

Other processes for the preparation of porous materials include: solvent casting/particulate leaching,³⁸ emulsion templating³⁹ and phase separation.⁴⁰ In general, these methods require the use of large amounts of organic solvents and a lengthy washing or etching procedure.^38,42,43 A freezing process can provide certain advantages over the traditionally used techniques cited above.⁴⁴ Water is an environment-friendly solvent and the use of ice crystals as porogens is green and sustainable. This is particularly beneficial for biological applications. When removing the solvent, the freezing process does not bring impurities into the samples and a further purifying process is therefore not necessary.

Materials and Methods

Preparation of silk fibroin solution

Cocoons of Bombyx mori silkworm were supplied by Bratac. To obtain pure SF fibers from silkworm cocoons it was necessary to perform a degumming process of silk cocoons to remove the sericin coating. This process was achieved by initially washing the cocoons with distilled water to remove any impurity, followed by the immersion of cocoons in 1 g/L of Na₂CO₃ solution at 85 °C for 1 h and 30 min, with solution change every 30 min. A final wash in distilled water was done to remove Na₂CO₃ residues. The SF fibers were dried and dissolved in a ternary solvent of CaCl₂:CH₃CH₂OH:H₂O, in a molar ratio of 1:2:8, at 85 °C until total dissolution, to a SF salt solution of 10% (w/v).¹⁷

Preparation of fibroin hydrogels

We studied two different methods for the preparation of fibroin hydrogels. In the first method, SF solution at a concentration of 10% (w/v) was dialyzed (cellulose membrane, Viscofan 22 EU – 20 USA) against distilled water for 3 d, at 8 °C, with water change every 24 h. After dialysis, the solution was filtered by gauze to remove small amounts of aggregates. The final concentration of the silk fibroin aqueous solution was 4% (w/v), which was determined by weighing the remaining solid after drying. The dialyzed solution was placed on specific molds (diameter: 25 mm) in a thermostatic bath at 37 °C until the formation of gels (3 d). Hydrogel formation was observed when the sample presented an opaque white color and did not fall when the mold was inverted for 30s.^14,53 Cylinder-shaped samples sized 25 mm in diameter and 10 mm in thickness were used. The SF hydrogels were then subjected to a freezing at -20 °C for 24 h. To analyze the effect of freezing, control samples of SF hydrogels were kept at 8 °C for 24 h.

In the second method, SF salt solution in a concentration of 10% (w/v) was dialyzed (cellulose membrane, Viscofan 22 EU – 20 USA) against distilled water at 8 °C, with water change every 24 h, until the formation of hydrogels inside the dialysis tube (7 d). The influence of freezing on this hydrogels was also analyzed by freezing SF hydrogels at -20 °C for 24 h, while the control samples (non-frozen hydrogels) were kept at 8 °C for 24 h.

Four different types of samples were prepared: 1) dialysis for 3 d followed by hydrogel formation at 37 °C and freezing by 24h at -20 °C; 2) dialysis for 3 d followed by hydrogel formation at 37 °C and kept at 8 °C for 24 h; 3) dialysis for 7 d until hydrogel formation inside the tube and freezing by 24h at -20 °C and 4) dialysis for 7 d until hydrogel formation inside the tube and kept at 8 °C for 24h.

All hydrogels were characterized after freezing by liquid nitrogen and lyophilization (Liobras, L101, Brazil) within the first 24 h to prevent structural changes.

Characterization

X-ray Diffraction

Changes in the crystallinity of hydrogels were followed with X-ray diffraction (XRD), performed by a X'Pert-MPD diffractometer (Philips Analytical X Ray) with Cu-Kα radiation, with a wavelength of 1.54 Å. The X-ray source was operated at 40 kV and 40 pA. The scanning speed was 0.06°/s, step size of 0.02°, and the measurement range was 2θ = 10–40°. Three samples were used for each type of material.

Fourier Transformed Infrared Spectroscopy

The structural changes of silk fibroin hydrogels were analyzed in the range of 675–4000 cm⁻¹, with a resolution of 4 cm⁻¹, using Fourier transformed infrared spectroscopy with attenuated total reflection apparatus (FTIR-ATR) (Nicolet 6700, Thermo Scientific). FTIR was used to identify the secondary structure of the protein samples through the location of the peaks of amide I, II and III. Three samples were used for each type of material.

Thermogravimetric Analysis

Thermogravimetric analysis (TGA) measurements were performed on the hydrogels using a thermogravimetric analyzer (TGA-50, Shimadzu) in the temperature range of 30–600 °C with a slope of 10 °C/min and a N₂ flow of 50 mL/min, in order to analyze the influence of dialysis and freezing on the weight loss of the samples with respect to the temperature. Three samples were used per each type of material.

Scanning Electron Microscopy

The cross-section morphology of fibroin hydrogels was observed by scanning electron microscopy (SEM). The analysis was performed on the lyophized samples coated with a gold layer and then examined with an EVO MA15 scanning electron microscope (Zeiss), at 200× and 5000× magnification, with an accelerating voltage of 10 kV.

Conclusions

The present work describes the formation and characterization of silk fibroin hydrogels. Three-dimensional silk fibroin hydrogels were successfully prepared with an interconnected porous structure. From the results of X-ray diffraction and FTIR-ATR analysis, it is concluded that SF transforms into β-sheet after gelation, and both silk I and silk II structures exist simultaneously in all hydrogels. Thermal analysis showed the degradation peak for all SF hydrogels at around 330–340 °C, indicating high thermal resistance for these hydrogels. Longer dialysis time (7 d) resulted in a thicker fibroin network after lyophilization, showing that the dialysis time has an effect on the material porous structure and morphology. The effect of freezing was also visible on the porous structure of SF hydrogels, considerably increasing pore sizes. These scaffolds may hold promise in bone tissue engineering, and the degradation rate of the SF porous structures and in vitro citocompatibility tests are subject of future work.

10.4161/biom.28536