The behavior of aged regenerated Bombyx mori silk fibroin solutions studied by 1H NMR and rheology

Biomaterials 29 (2008) 4268–4274

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The behavior of aged regenerated Bombyx mori silk fibroin solutions studied by 1H NMR and rheology Zainuddin a, b, c, *, Tri T. Le d, Yoosup Park e, f, Traian V. Chirila a, b, e, g, Peter J. Halley e, f, Andrew K. Whittaker c, e a

Queensland Eye Institute, Brisbane, Qld, Australia School of Medicine, Faculty of Health Sciences, University of Queensland, Brisbane, Qld, Australia Centre for Magnetic Resonance, University of Queensland, Brisbane, Qld, Australia d Department of Chemistry, University of Queensland, Brisbane, Qld, Australia e Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Qld, Australia f Department of Chemical Engineering, University of Queensland, Brisbane, Qld, Australia g School of Physical and Chemical Sciences, Queensland University of Technology, Brisbane, Qld, Australia b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 June 2008 Accepted 28 July 2008 Available online 19 August 2008

As part of a project to utilize the regenerated silk fibroin (RSF) membranes as a supporting matrix for the attachment and growth of corneal stem/progenitor cells in the development of tissue engineered constructs for the surgical restoration of the ocular surface, the behavior of the aged RSF solutions has been investigated. The solutions were produced from domesticated silkworm (Bombyx mori) cocoons according to a protocol involving successive dissolution steps, filtration and dialysis. The solutions were kept at 4  C in a refrigerator for a certain period of time until near the gelation time. The changes in molecular conformation were studied by solution-state 1H NMR, while the flow of the solutions was characterized by rheological method. Upon ageing turbidity developed in solutions and the viscosity continuously decreased prior to a drastic increased near the gelation time. The 1H resonances of aged solutions showed a consistent downfield shift as compared to the 1H resonances of the fresh solution. Shear thinning with anomalous short recovery within a certain range of low shear rates occurred in both fresh and aged solutions. While the solutions behave as pseudo-plastic materials, the chain conformation in aged solutions adopted all secondary configurations with b-strand being predominant. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Silk fibroin Ageing 1 H NMR Rheology Chain conformation Pseudo-plastic flow

1. Introduction It has been known that the ocular surface disorders (OSDs) caused by various chronic conditions such as thermal or chemical burns, Stevens–Johnson syndrome, cicatricial pemphigoid and chronic contact lens wear can damage the progenitor cells, leading to deficiency of limbal epithelial stem cells. A severe implication of prolonged limbal stem cell deficiency is blindness due to progressive ingrowth of fibrous tissue and opacification of the cornea [1]. Clinical studies have shown that the surgical treatment to restore OSDs either by autografts or allografts has many limitations. Autografting is restricted by the amount of tissue that can be removed from the other eye and may cause complications to the healthy eye, whilst allografting is limited by the availability of

* Corresponding author Queensland Eye Institute, 41 Annerley Road, South Brisbane, Qld 4101, Australia. Tel.: þ61 7 3010 3381; fax: þ61 7 3010 3390. E-mail address: [email protected] (Zainuddin). 0142-9612/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2008.07.041

donor tissue, possible rejection and biosafety concerns. In a relatively new approach, limbal stem cells are isolated through biopsy, expanded in vitro on a suitable matrix and then transplanted to injured/diseased corneal surface [2–5]. Providing that the supporting material is capable of promoting cell attachment, growth and spreading, the latter technique offers a viable alternative for restoring the ocular surface. Currently, denuded human amniotic membrane (AM) is the most widely used substrate for ocular surface repair [2,6–10]. However, as the AM is a human-derived tissue, it is a potential vector for transferring infectious disease [11]. Other drawbacks of using this biological material have also been reported [12–14]. Therefore, the search for alternative materials is increasingly important. To date, several alternative matrices have been evaluated including collagen and its derivatives, fibrin gel, MatrigelÒ and some synthetic polymers [4,5], but only few of them have reached animal experimentation or clinical trials and the results were unsatisfactory. Hence, in our previous reports [15,16], we have proposed and evaluated the feasibility of the regenerated silk fibroin (RSF)

Zainuddin et al. / Biomaterials 29 (2008) 4268–4274

membranes as a supporting matrix for cultivation of human limbal epithelial (HLE) cells. We demonstrated that the RSF membranes were able to support the growth of HLE cells at a level comparable to that on tissue culture plastic. A comprehensive study on cell behavior and its long-term differentiation is currently undergoing in our laboratories and the results will be published in due course. It is known that the cell-adhesive properties of the RSF membranes are also influenced by the protocol for preparation of the membranes [17–22], a phenomenon which probably relates to the conformational change in solution that occurs during processing and storage [23–25] and by solvent treatment of the membranes [17–19,26–28], leading to the formation of, and an interplay between crystallizable forms known as silks I, II and III, where b-forms and a-helix structures predominate, and amorphous forms containing mainly random coil structures [29–36]. It was Coleman and Howitt who, in their seminal paper on fibroin [37], showed probably for the first time that different conformations of the silk fibroin can be reciprocally converted. They noticed that if water-soluble fibroin (‘‘denatured’’), which we know now as silk II, was dissolved in aqueous solution of copper ethylenediamine and then dialyzed against water, the resulting water-soluble fibroin (renatured) does not differ essentially from native fibroin (as found in the silk gland), which we call now silk I. However, at that time, they could not refer to silk I or silk II as these terms were to be coined a few years later by Kratky and coworkers [38,39], who were also the first to give a correct interpretation to this observation by showing that silk II can be converted into silk I after dissolution of the former in an appropriate solvent followed by dialysis against water [40,41]. In the last decades, there have been considerable efforts to understand the transformation of the viscous silk I in the middle section of domesticated silkworm’s glands (about 25% of concentration) to crystalline fibrous silk II spun through the spinneret [42–47]. As a result, many factors have been identified to affect the transition of silk I to silk II, including pH, removal of calcium ions and water molecules from the ducts and the presence of external forces during the spinning process [28,42–47]. Based on the experiments developed to mimic the natural spinning process of silkworm silk it was revealed that the external force played an important role in the transition of silk I to silk II [42–47]. Within this context, the rheometer has also been utilized to generate a similar structural transformation by applying shear force to the silk fibroin solutions [42–55]. Although limited studies on the rheology of the RSF aqueous solutions have been reported [47–50,53,55], it appeared that shear thinning dominated the process and the formation of aggregates was occasionally observed at considerably high shear rate [47,53,55]. Another factor which also contributes to the transformation of silk I to silk II is the structural nature of the silk fibroin. It is well known that the silk fibroin is constituted by highly repeated hydrophobic and crystallisable molecules with the primary sequences of amino acid residues of Gly–Ala–Gly–Ala–Gly–Ser, [Gly–Ala]n–Gly–Tyr and [Gly–Val]n–Gly–Ala (n ¼ 1–8) separated by 11 amorphous region of mainly Gly–Ala–Gly–Ser and Gly–Ala–Gly– Ala–Gly–Ser sequences [51]. Therefore, upon storage, the RSF solution tends to undergo self-transformation to adopt a more stable conformation by forming intra- and/or intermolecular hydrogen bonds [23,56–58]. In this study, to shed more light on the conformational change during storage and to determine the useful life time of the RSF solutions prior to gelation, the behavior of RSF solutions was monitored by using rheological and proton NMR methods. This knowledge is essential, particularly when the RSF membranes are intended to be used as substrates for tissue engineering applications.

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2. Materials and methods 2.1. Materials The Bombyx mori cocoons were purchased from Tajima Shoji Co. Ltd. (Yokohama, Japan). Sodium carbonate (Na2CO3) and lithium bromide (LiBr) were obtained from Sigma-Aldrich. Methanol and deionized water (18.2 MUcm) were used as solvents. 2.2. Preparation and ageing of RSF aqueous solutions The Bombyx mori cocoons were cut in smaller pieces, vacuum dried, weighed, and placed in 1 L boiling solution of Na2CO3 (0.02 M) for 1 h to remove sericins. Subsequently the silk fibers were rinsed three times in 1 L hot water (w70  C) for 20 min each and then let to dry in a fume hood overnight. After sericin-free silk fibers were dried under vacuum for few hours, they were dissolved in an aqueous lithium bromide solution (9.3 M) at 60  C for 4 h to obtain a silk concentration of about 10%. The solution was pre-filtered through a syringe filter with a pore size of 0.8 mm (MinisartÒ-GF, Sartorius) and followed by a 0.20-mm pore size filter (MinisartÒHigh-Flow, Sartorius). About 10 mL of the filtrate was injected into a 3–12 mL dialysis cassette with a molecular weight cut-off of 3500 (Slide-A-LyzerÒ, Pierce) to be dialysed against water. Following six changes of water within 3 days of dialysis, the resulting solution was collected and filtered through a 0.20-mm pore size filter (MinisartÒHigh-Flow). The resulting aqueous solution with a concentration in silk fibroin of about 3.8% was kept at 4  C for 1 to 4 months. 2.3.

1

H NMR measurements

The samples were diluted to four times of the initial volume of the RSF aqueous solutions with deuterated water (D2O). 1H NMR spectra were obtained on a 500 MHz Avance Bruker spectrometer operating at 500.13 MHz with a 5 mm probe. Water (HDO) resonance was used as an internal standard for determination the 1H chemical shifts. The water signal was suppressed by a thousand fold or more using watergate pulse sequence with gradient double echo. Phasing and baseline corrections were completed manually. The software used for these procedures was TOPSPIN version 1.3. 2.4. Rheological measurements The viscosity of the RSF aqueous solutions was measured using an AR-G2 Rheometers (TA Instrument Ltd., USA). The shear viscosity was acquired by linearly increasing the shear rate without oscillation from 0.05 to 500 s1. Meanwhile, the measurement of storage (G0 ) and loss (G00 ) modulus as a function of frequency (u) was performed within the linear viscoelastic region using 40-mm diameter cone plate with 57 mm gap. All rheological measurements were carried out at room temperature (25  0.5  C). A solvent trap was used throughout the experiment to avoid the possible loss of water.

3. Results and discussion 3.1.

1

H NMR analysis

Though the proton (1H) resonances of many proteins and peptides in the solution-state have been well documented, little data are available on the solutions of silk fibrous proteins, including the regenerated Bombyx mori silk fibroin [59]. The fact that the silk fibroin does not readily dissolve in most organic solvents and it is structurally unstable in aqueous environment due to conformational change. Our observation on the effect of ageing on the behavior of the aqueous silk fibroin solution, however, has prompted us to carry out the 1H NMR measurement in aqueous system. Under this condition we are aware that the interference of the water 1H resonance is imminent. However, because all of the samples contain the same amount of water and this study was to compare the 1H resonances of the aged and fresh silk fibroin solutions, the same magnitude of interference would apply to all samples, thus the changes in the molecular structure due to ageing effect would directly correspond to the changes in chemical shift and line shape. Fig. 1 shows the 1H NMR spectra of the fresh and 4 month old silk fibroin solutions with and without water peak suppression. As expected, the major peaks were mainly contributed by the primary structure of the silk fibroin (i.e. alanine, glycine, serine, tyrosine and valine). Close inspection of the spectra (see Fig. 2a–f) reveals that while the global chemical shift of the aged solutions consistently moved downfield (relative to the fresh silk

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Zainuddin et al. / Biomaterials 29 (2008) 4268–4274

A

4

the aromatic side-chain 1H resonances of Tyr and Phe can be seen at 6.50–7.50 ppm (Fig. 2e), the amide protons of all amino acid residues expand at 7.85–8.45 ppm (Fig. 2f). The appearance of few very narrow peaks in all spectra (1.76/1.81, 2.12, 3.25 and 8.29/8.34 ppm) suggests that both fresh and aged silk solutions contained small portion of low molecular weight peptides which were free from hydrogen bonding [67].

HDO

0 3.2. Rheological study

Gly α Ser β

B

Tyr 4H

Ala, β Tyr β

4

Val, γ

0 9

8

7

6

5

4 (ppm)

3

2

1

0

Fig. 1. 1H NMR spectra of fresh and aged RSF aqueous solutions without (A) and with (B) water peak suppression measured at 298 K. 0 denotes fresh solution and 4 denotes 4 months old solution.

solution) the shape of the lines remains unchanged. This behavior suggests that both fresh and aged solutions contained all of the three secondary structures of the silk fibroin proteins (random coil, a-helix and b-strand) with b-strands content increased with increasing ageing time. The presence of b-strands in the fresh RSF solutions is not unexpected, particularly in the solutions that are not clarified after dialysis against water [24]. Additionally, the transformation of silk fibroin from random coil/a-helix to b-strands and/or b-sheets (adjacent b-strands link together via hydrogen bonds) during storage has been reported elsewhere [23–25]. Accordingly, the appearance (even if it is weak and only observed in the 4 month old sample) of new 1H resonances at 5.60 ppm and possibly at around 5.30 ppm (Fig. 1) which are characteristic of the b forms [60–62] was anticipated. It seems that even though b forms is structurally more packed than the random coil the molecular chain remains flexible, as reflected by small downfield shift (about 0.06 ppm). Hence, our hypothesis is that ageing the RSF solution up to near the gelation time would result in a transformation of random coil to mainly b-strand, while formation of b-sheet primarily occurred at the onset of gelation. Therefore, the 1H resonances of the RSF aqueous solution prior to gelation have been assigned by closely examining the expanded 1 H NMR spectra (Fig. 2a–f) and matching their values with the values of all amino acid residues available in the literature [60–66], as summarized in Table 1. Taking into account that both fresh and aged silk solution strongly demonstrated the presence of mixed conformations, so the 1H resonances were determined as an average of all resonances emerged from random coil, a-helix and b-strand. The peaks appearing at 0.6–0.9 ppm were attributed to Ile (Hg,d) and Leu (Hd), with a small contribution of Hg of Val (Fig. 2a). The multi-peaks at 1.1–1.5 ppm were originated mainly from Hg of Ile overlapped with Hb of Ala, Hg of Lys and Hg of Thr residues (Fig. 2a,b). As shown in Fig. 2b, at least eight amino acid residues were detected under the peaks which stretch from 1.5 to 2.5 ppm, namely Leu (Hb,g), Arg (Hb,g), Lys (Hb,d), Met (Hb,g,3), Ile (Hb), Pro (Hb,g), Val (Hb) and Glu (Hb,g). The main peak appearing at 2.65–3.05 ppm was contributed by Hb of Tyr, Phe, Asp, Cys and Hb,3 of Lys residues (Fig. 2c). A significant Ha resonances emerge at 3.5–4.5 ppm was mainly due to Ha of Gly as major component of the silk fibroin, overlapping with Hb of Ser and Hd of Pro residues (Fig. 2d). Other Ha resonances which should appear at 4.4–5.7 ppm were not observable (n/a) due to water peak suppression. Whilst

Izuka [48] reported that the viscosity of the RSF aqueous solutions measured at 14  C was extremely low, even at a fibroin concentration of about 10% (x0.09 Pa s). The authors suggested that the silk fibroin solution might be considered as liquid crystalline. To further elaborate on this behavior the shear viscosity of the freshly prepared and aged silk solutions has been measured and reported in this study. Fig. 3 shows the dependence of viscosity on the shear rate for fresh and aged silk fibroin solutions. It can be seen that all solutions demonstrated a similar trend of viscosity behavior. Initially, the viscosity is almost independent of shear rate, approaching Newtonian flow, particularly for silk solutions of 1–3 month old and then follows plastic flow behavior with continuous shear thinning. More interestingly, there was a sensitive region in which a drastic drop of viscosity followed by a short recovery occurred before a continuous shear thinning. This behavior suggests that under shear forces the silk fibroin underwent structural transformations. Having the viscosity continuously decreasing with increasing shear rate the model of non-Newtonian pseudoplastic flow can be applied. Typically, the profile of this model shows three zones, i.e. a low shear rate plateau followed by a near power-law decrease that ends finally at high shear rate plateau [68]. There numerous mathematical models have been proposed to explain this behavior [69], however, the one proposed by Cross [70] was found to be more practical in the analyses of our experimental results. The model is expressed in the following equation.

h ¼ hN þ ðh0  hN Þ= 1 þ lym




(1)

Parameters h0 and hN are the limiting viscosity values at shear  / 0 and y  / N, respectively. l is a constant with units of rate y time and m > 0 is a dimensionless constant that measure the severity of shear thinning, typically ranges from 2/3 to 1. As shown in Fig. 3, irrespective of anomalous (a drastic drop and short recovery) region, the congruency of fitting curves and experimental data is sufficient to estimate the zero-shear viscosity for each of the solutions, and the results are presented in Fig. 4. It can be seen that the viscosity of the solution gradually decreased with extension of ageing time up to 3 months, and then it increased significantly in the following month. This behavior is consistent with our early hypothesis, i.e. following b-strand transformation which led to lower viscosity, b-sheet transformation occurred, causing an increase in the viscosity. Noting that the solution gelled just a few days after 4 months timepoint, the viscosity of the solution must have sharply increased and reached an infinite value at the onset of gelation. Clearly, the macroscopic effect of the conformational changes can be visually observed from the development of turbidity in the solutions (see inserted images in Fig. 4). The more b-strand and/or b-sheet was formed the more light would be diffracted, thus increasing the turbidity. Based on the above observation, it is unambiguous that the viscosity behavior shown in Fig. 3 was dictated by both shear rate and the propensity of the silk fibroin to undergo self-transformation. Under shear, the molecules aligned into more uniform orientation and promoted the formation of either folded or unfolded chain, depending on the shear rate. At low shear rate

Zainuddin et al. / Biomaterials 29 (2008) 4268–4274

a

b

Ala Thr IIe Val IIe Leu IIe

4

Glu 4 Glu

3

3

2

2

1

1

0

0

1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5

c

Pro

4 His

Tyr Phe Arg His Cys

Asp Phe Lys His Cys Tyr

d

Asp

2

2

1

1

0

0

3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4

Tyr

4

Ser Thr Pro Leu Arg Lys 4 Met 3

e

Tyr Arg

Val Glu Pro Met

Leu IIe Leu Pro Arg Leu Lys Lys Arg Lys Met Met

Gly Ala Val Glu Thr IIe

Gly Ser Pro

Ser

4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5

f NH of all residues

Tyr Phe His Tyr 4

3

3

2

2

1

1

0

0

7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5

Glu Pro Arg Lys Met

2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3

3

Phe Arg Lys

4271

Phe Lys His

8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 (ppm)

Fig. 2. Expanded 1H NMR spectra for fresh and aged RSF aqueous solutions with water peak suppression. 0 denotes fresh solution and 1–4 denote aged solution to 1, 2, 3 and 4 months, respectively.

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Zainuddin et al. / Biomaterials 29 (2008) 4268–4274

Table 1 1 H NMR chemical shifts of the RSF aqueous solutions measured at 298 K Residue (%)

Fresh solution

Aged solution

Averaged chemical shift values for all conformations

Averaged chemical shift values for all conformations

Ha

Hb

NH

Others

Ha

Hb

NH

Others

Gly (46.2) Ala (29.7) Ser (10.8) Tyr (4.9)

4.15, 3.65 4.18 4.31 n/a

– 1.24 3.95, 3.81 3.08, 2.76

8.35 8.10 8.29 8.26

4.20, 3.70 4.24 4.37 n/a

– 1.28 4.00, 3.86 3.12, 2.81

8.39 8.16 8.34 8.30

Val (2.1) Asp (1.5) Glu (0.9) Thr (0.8) Phe (0.6) Ile (0.5)

4.10 n/a 4.22 n/a n/a 4.12

2.05 2.89, 2.56 2.10, 1.98 4.20 3.12, 2.84 1.77

8.20 8.36 8.37 8.16 7.99 8.05

4.16 n/a 4.26 n/a n/a 4.16

2.11 2.94, 2.62 2.11, 1.99 4.24 3.16, 2.90 1.81

8.25 8.40 8.35 8.20 8.02 8.10

Pro (0.4)

4.30

2.18, 1.84

–

4.36

2.22, 1.90

–

Leu (0.6)

4.32

1.74, 1.57

8.23

4.38

1.80, 1.61

8.27

Arg (0.4)

4.33

1.86, 1.71

8.27

4.37

1.92, 1.77

8.32

Lys (0.3)

4.28

1.90, 1.73

8.29

4.34

1.97, 1.78

8.35

His (0.2) Met (0.1)

n/a 4.29

3.28, 2.86 1.84, 1.58

8.28 8.22

n/a 4.35

3.34, 2.90 1.92, 1.62

8.33 8.32

Cys (0.1)

n/a

3.17, 2.80

8.17

– – – d-CH (7.15, 7.04) 3-CH (6.91, 6.64) g-CH3 (n/a, 0.72) – g-CH2 (2.36, 2.28) g-CH3 (1.19) 3-CH (7.38, 7.26) g-CH2 (1.23, 1.04) g-CH3 (0.76) d-CH3 (0.71) g-CH2 (1.89, 1.74) d-CH2 (3.67, 3.48) g-CH (1.53) d-CH3 (0.78, 0.67) g-CH2 (1.60, 1.52) d-CH2 (3.12, 3.08) NH (7.36, 6.62) g-CH2 (1.40, 1.36) d-CH2 (1.61, 1.57) 3-CH3 (2.96, 2.93) 3-NH3 (7.44) d-CH (7.70, 7.14) g-CH3 (2.20, 1.87) 3-CH3 (1.50) –

n/a

3.21, 2.84

8.23

– – – d-CH (7.21, 7.10) 3-CH (6.97, 6.69) g-CH3 (n/a, 0.78) – g-CH2 (2.39, 2.32) g-CH3 (1.24) 3-CH (7.44, 7.30) g-CH2 (1.26, 1.10) g-CH3 (0.82) d-CH3 (0.77) g-CH2 (1.94, 1.78) d-CH2 (3.69, 3.54) g-CH (1.57) d-CH3 (0.84, 0.72) g-CH2 (1.65, 1.56) d-CH2 (3.16, 3.13) NH (7.41, 6.67) g-CH2 (1.45, 1.41) d-CH2 (1.66, 1.63) 3-CH3 (3.02, 2.99) 3-NH3 (7.49) d-CH (7.76, 7.20) g-CH3 (2.26, 1.98) 3-CH3 (1.55) –

(<0.15 s1), the viscosity of the fresh solution was slightly reduced with increasing the shear rate, suggesting that the alignment of molecules which are consisted of mainly random coil improved the molecular flow and promoted the formation of b-strand. On the other hand, in aged solutions, the viscosity was independent of shear rate up to 3 months and then slightly reduced at 4 months time. This behavior might be attributed to the b-strand conformation formed by self-transformation during storage. As b-strand is a folded conformation, it is more packed and more easily tumbling, thus alignment of the molecules by shear would not significantly alter the molecular flow and so as the viscosity. In the 4 months old solution, there was an indication that the b-sheet has also been formed, as reflected by an increase in the zero-shear viscosity (see Fig. 4) and further shifting of the chemical shift to downfield

(Fig. 2a–f). However, it seemed that this infant b-sheet structure was easily disrupted, even by low shear rate, resulting in a gradual decrease of the viscosity. At shear rates of 0.2–1.0 s1, the viscosity of all solutions appeared to be highly sensitive with a drastic drop of viscosity followed by a short recovery. We suggest that the shear force at this sensitive region was able to induce a massive breakage of transient crosslinked points (H-bonds), leading to the unfolding of the folded chains. Clearly, after a short recovery, the viscosity continuously decreased and eventually reached plateau at end shear rate of 500 s1. This suggests that once all of the unfolded chains were aligned and straighten by the flow field the adjacent molecules started to aggregate. Episodically, the macroscopic effect of this aggregation was observed as white material surrounded by a clear

1.0 Fresh, m=0.97 1 month, m=0.80 2 months, m=0.77 3 months, m=0.70 4 months, m=0.93 Curve fitting

0.1

0.01

0.001 0.01

Zero-shear viscosity (Pa.s)

Viscosity (Pa.s)

1

Infinity 0.8 Gel

0.6

Solution

0.4 0.2 0.0

0.1

1

10

100

1000

Shear rate (s-1) Fig. 3. Viscosity behavior of the fresh and aged RSF aqueous solutions (3.8% w/v) under shear force.

0

1

2 3 Time (month)

4

5

Fig. 4. Effect of ageing time on zero-shear viscosity of the RSF aqueous solutions (3.8% w/v). Turbidity (inserted images) developed as the ageing time increased.

Zainuddin et al. / Biomaterials 29 (2008) 4268–4274

G′ and G′′ (Pa)

10 1

Fresh 1 month 2 months 3 months 4 months

0.1

(G′′/G′)

10 1 0.1 0.01 0.01

0.1

1

10

100

1000

(rad s-1) Fig. 6. Tan delta (d) of fresh and aged RSF aqueous solutions (3.8% w/v).

significantly higher than the fresh solution. This again confirms that at 1 and 4 months the solutions behave like a network-structure, while at 2 and 3 months they behave like liquid crystals. Also, the tendency of the tan d to approach unity at u above 10 rad s1 suggests that the aggregation of the b-strands is likely to occur at high frequency. 4. Conclusions The tendency of 1H NMR chemical shifts and the shape of spectral line suggested that all three molecular conformations, i.e. random coil, a-helix and b-strand were present in both fresh and aged RSF aqueous solutions. As anticipated, the transformation from random coil/a-helix to b-strand increased upon ageing and this transformation eventually formed b-sheet structure at the onset of gelation. The flow of RSF aqueous solutions showed characteristic of non-ideal pseudoplastic behavior with the viscosity gradually decreased from approximately 0.30 Pa s to as low as 0.012 Pa s, which is nearly the same as the viscosity of water (z0.01 Pa s) at 3 months time. Our observation revealed that the useful life-time of RSF aqueous solution (3.8% w/v) in which the resultant membranes were still easy to handle with forceps in a similar fashion to pieces of cellophane was 3 months. Beyond this period, the membranes were very brittle. Acknowledgements

References

0.01 0.001 0.0001 0.01

100

This work was sponsored by an unrestricted grant from Prevent Blindness Foundation, Brisbane, Australia, through Viertels Vision. We would like to thank Professor Kaplan and Dr Matsumoto, both at Tufts University, Medford, MA, USA, for expert advice. We also acknowledge the support from the group of Professor Justin Cooper-White, allowing us to use the rheometer at the Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Australia.

100 G′ G′′

1000

tan

liquid at the end of shear experiments, a phenomenon which has been previously noticed [52–54,71]. More evidences for the hybrid behavior of the RSF solutions were obtained from the storage (G0 ) and loss (G00 ) modulus measurements. As shown in Fig. 5, within the oscillatory frequency (u) range tested (0.1–100 rad s1), initially, both G0 and G00 were almost independent of u. However, after reaching certain u values, the solutions seemed to follow the typical plastic flow behavior with G0 and G00 increased with increasing u. The value of G0 for the fresh solution was higher than the G00 , in agreement with the trend reported by Ochi et al. [51] for the natural silk solution of similar concentration. For the aged solutions, the above trend fluctuated against the ageing time. At 1 and 4 months, it followed the fresh solution – indicating a network-like structure, but at 2 and 3 months the trend reversed – suggesting a liquid crystal-like structure. Whilst the G0 at 4 month was higher than the G0 of fresh solution, the G0 at 1–3 months was lower. This variation is interpreted as the result of conformational change during storage. As early mentioned that upon ageing the silk fibroin underwent structural transformation from random coil/a-helix to b-strand, then b-sheet and eventually formed three dimensional networks through self-assembly. It seemed that in early transition (at 1 month) the formed b-strand was still not yet aligned and probably entangled to some extent; consequently, the elastic modulus G0 dominated the loss modulus G00 and the G0 value became slightly lower than the G0 of the fresh solution. In contrast, at 2 and 3 months the b-strand has relatively well aligned and separated; accordingly, the solutions might be considered as a liquid crystal. In this condition, the modulus values, particularly the G0 , would appreciably drop below the modulus values of the fresh solution and the G00 dominated the G0 . While at 4 months, the adjacent b-strands started to link together through formation of hydrogen bonds between carbonyl and amine groups of the amino acid residues. As expected, the latter association would shift both modulus values to a higher level with G0 being predominant. Another phenomenon which also reflects the complexity of the RSF solutions is an appreciably high increase of G0 and G00 at u above 10 rad s1. This increase implies that at higher frequency the silk fibroin tends to form aggregates, regardless of the ageing time. Moreover, since there was no crossover point between G0 and G00 observed in all samples the solutions behave entirely like a liquid (no gelation). By plotting tan d (¼G00 /G0 ), it is immediately seen that the values of tan d for aged solutions were all above the fresh solution (Fig. 6). As seen, the tan d at 1 and 4 months was just slightly higher, while the tan d at 2 and 3 months it was

4273

0.1

1

10 (rad

100

1000

s-1)

Fig. 5. Storage (G0 ) and loss (G00 ) modulus of fresh and aged RSF aqueous solutions (3.8% w/v)

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