Home

Search

Collections

Journals

About

Contact us

My IOPscience

Electrospun silk-elastin-like fibre mats for tissue engineering applications

This content has been downloaded from IOPscience. Please scroll down to see the full text. 2013 Biomed. Mater. 8 065009 (http://iopscience.iop.org/1748-605X/8/6/065009) View the table of contents for this issue, or go to the journal homepage for more

Download details: IP Address: 194.27.18.18 This content was downloaded on 16/08/2014 at 10:38

Please note that terms and conditions apply.

IOP PUBLISHING

BIOMEDICAL MATERIALS

doi:10.1088/1748-6041/8/6/065009

Biomed. Mater. 8 (2013) 065009 (13pp)

Electrospun silk-elastin-like fibre mats for tissue engineering applications Raul Machado 1,6 , Andr´e da Costa 1 , Vitor Sencadas 2,3 , Carmen Garcia-Ar´evalo 4,5 , Carlos M Costa 2 , Jorge Padr˜ao 1 , Andreia Gomes 1 , Senentxu Lanceros-M´endez 2 , Jos´e Carlos Rodr´ıguez-Cabello 4,5 and Margarida Casal 1,6 1

CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal 2 Centro/Departamento de F´ısica, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal 3 School of Technology, Polytechnic Institute of C´avado and Ave., 4750-810 Barcelos, Portugal 4 Bioforge (Group for Advanced Materials and Nanobiotechnology), Centro I+D, Universidad de Valladolid, Valladolid, Spain 5 Networking Research Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), E-47011 Valladolid, Spain E-mail: [email protected] and [email protected]

Received 22 August 2013 Accepted for publication 9 October 2013 Published 28 November 2013 Online at stacks.iop.org/BMM/8/065009 Abstract Protein-based polymers are present in a wide variety of organisms fulfilling structural and mechanical roles. Advances in protein engineering and recombinant DNA technology allow the design and production of recombinant protein-based polymers (rPBPs) with an absolute control of its composition. Although the application of recombinant proteins as biomaterials is still an emerging technology, the possibilities are limitless and far superior to natural or synthetic materials, as the complexity of the structural design can be fully customized. In this work, we report the electrospinning of two new genetically engineered silk-elastin-like proteins (SELPs) consisting of alternate silk- and elastin-like blocks. Electrospinning was performed with formic acid and aqueous solutions at different concentrations without addition of further agents. The size and morphology of the electrospun structures was characterized by scanning electron microscopy showing its dependence on the concentration and solvent used. Treatment with methanol-saturated air was employed to stabilize the structure and promote water insolubility through a time-dependent conversion of random coils into β-sheets (FTIR). The resultant methanol-treated electrospun mats were characterized for swelling degree (570–720%), water vapour transmission rate (1083 g/m2/day) and mechanical properties (modulus of elasticity ∼126 MPa). Furthermore, the methanol-treated SELP fibre mats showed no cytotoxicity and were able to support adhesion and proliferation of normal human skin fibroblasts. Adhesion was characterized by a filopodia-mediated mechanism. These results demonstrate that SELP fibre mats can provide promising solutions for the development of novel biomaterials suitable for tissue engineering applications. S Online supplementary data available from stacks.iop.org/BMM/8/065009/mmedia (Some figures may appear in colour only in the online journal)

6

Author to whom any correspondence should be addressed.

1748-6041/13/065009+13$33.00

1

© 2013 IOP Publishing Ltd

Printed in the UK & the USA

Biomed. Mater. 8 (2013) 065009

R Machado et al

Typically, SELPs are composed of silk-like and elastinlike repeating units with amino acid sequences GAGAGS (G: glycine, A: L-alanine, S: L-serine) and V1PG1V2G2 (V: L-valine, P: L-proline), respectively [20, 21]. However, modifications involving point substitutions of V2 for L-glutamic acid to create pH stimuli-sensitive materials [22, 23], or L-lysine to provide points for covalent crosslinking [24, 25], are reported. Several SELP combinations, with different size and silk to elastin block ratio have been reported in the literature [26, 27] and, depending on their specific sequence and composition, are able to display an irreversible sol-to-gel transition at body temperature or even at room temperature. This temperature-mediated gelation process allowed its exploitation for a variety of applications including matrix-mediated delivery systems for gene therapy [28–32], drug delivery systems [21, 33], stimuli (pH, temperature and ionic strength) sensitive materials [22, 23] and as scaffolds for encapsulation and chondrogenesis of human mesenchymal stem cells [34]. Additionally, SELPs in the form of gels subcutaneously implanted in guinea pigs displayed excellent in vivo biocompatibility [21]. SELPs have also been processed into free standing films by solvent evaporation at room temperature [35–37]. These were shown to be optically transparent, displaying mechanical properties resembling those of native aortic elastin and able to encapsulate and release drugs, demonstrating its potential as ophthalmic drug delivery systems [37]. Electrospun fibre mats of SELP copolymers can be interesting candidates for applications in tissue regeneration and wound dressing applications as the design of SELPs combines the tensile strength of silk and the resilience of elastin in a single molecule. To date, only a few reports describe the spinning of SELP copolymers. Nagarajan et al produced fibres of 200–300 nm diameter by electrospinning a SELP-67K aqueous solution with the addition of a PEO–SDS complex [38]. Addition of PEO and SDS showed to have simultaneously changed the solution viscosity, electrical conductivity and surface tension, facilitating the process of fibre formation from aqueous solution. In the same work, the authors also describe the production of fibres within the same range (200–300 nm) with a SELP-67K 15 wt% formic acid solution [38]. Ner et al described the electrospinning of nanoribbons from a SELP-47K aqueous solution [39] and spun microdiameter polydisperse fibres (less than 10 μm to over 60 μm) were obtained by wet-spinning a solution of SELP-47K in formic acid [40]. More recently, the fabrication of a biocompatible tissue scaffold by electrospinning a solution of 15 wt% SELP-47K in formic acid producing a polydisperse population of fibres with diameters ranging from 50 to 600 nm was reported [41]. However, to our knowledge, there is no report comparing the influence of the silk-block size, the type of solvent and SELP concentration in the morphology of the electrospun structures. Previously, we have reported the synthesis and biological production [42, 43] of a new class of SELPs consisting of multiple blocks of a silk-like sequence (GAGAGS) combined with an elastin-like analogue sequence in which the most commonly used elastin pentamer VPGVG was altered to

1. Introduction Protein-based polymers (PBPs) are macromolecules present in a wide variety of organisms fulfilling critical structural and mechanical roles. These specialized polymeric molecules are based on repetitive blocks of amino acid sequences that propagate through the natural protein encoding a basic structural property that is, ultimately, responsible for the physical and mechanical properties of the polymeric material. Due to its remarkable mechanical properties but also to their improved biocompatibility, natural PBPs have drawn much attention in the development of nanostructured scaffolds directed for tissue engineering applications [1]. In the process of creating nanofibrous scaffolds, electrospinning is a method of fibre fabrication that has received increasing attention as a technique to produce sub-micro and nanofibre mats due to its easy implementation on a lab scale and high versatility. When the diameter of the fibre is reduced from the macroscale to the sub-microscale or nanoscale, several new characteristics appear, such as flexibility in surface functionalities and superior mechanical performance [2]. Furthermore, the porous structure of electrospun scaffolds contains microscale interconnected pores that are essential to transport the oxygen and nutrient supply to support cell growth [3]. Elastin or its soluble precursor tropoelastin [4, 5], silk fibroin [3, 6], collagen [7, 8], gelatin [4, 9] and fibrinogen [10, 11] are amongst the most used protein-based biomaterials for the development of nanofibre scaffolds produced by electrospinning. The advance of protein engineering, emerging from the increase of knowledge in structural and molecular biology, combined with the use of recombinant DNA technology made possible the advent of a new class of artificial biomacromolecules, the recombinant protein-based polymers (rPBPs). This new class of protein-based materials, with precisely controlled polypeptide sequences, mimic the properties of their natural counterparts but can also display functions and properties that are not found in nature. Indeed, with the aid of recombinant DNA technology, it is possible to combine in the same polypeptide chain the properties of two or more different proteins, creating copolymers with distinct properties from their native equivalents [12–14]. One example of such artificial macromolecules is the silk-elastinlike proteins (SELPs). These artificial PBPs are inspired by nature and composed of multiple repeats of silk- and elastinlike blocks founded on the conservative amino acid motifs present in the natural proteins, silk fibroin and elastin. By combining the high tensile strength of silk fibroin [15] with the elasticity and resiliency of elastin [16], it is possible to create copolymers that, in principle, will combine the properties of both proteins. The silk-like units spontaneously self-assemble into packed antiparallel β-sheet structures stabilized by hydrogen bonding [17], providing crystallinity and mechanical strength. The elastin-like unit, on the other hand, has been reported as displaying a highly flexible conformation [18, 19]. The periodic introduction of elastomeric sequences will therefore reduce the overall crystallinity of the system by disrupting the silk-like blocks and consequently, increasing its flexibility and water solubility. 2

Biomed. Mater. 8 (2013) 065009

R Machado et al

VPAVG. This simple substitution of the central glycine in the VPGVG sequence by an L-alanine has demonstrated to dramatically change the properties of elastin-like polypeptides (ELPs) resulting in singular behaviour. Although displaying a reversible phase-transition, a feature commonly shared by ELPs, this was found to be characterized by an acute hysteresis [44, 45]. Moreover, it was suggested that this substitution results in a change of the ELP mechanical response from elastic to plastic [19]. Therefore, preparation of SELPs with the VPAVG pentapeptide should give rise to a new set of copolymers with distinct properties, pushing forward and enhancing the application potential of these recombinant proteins. With the aim of developing high performance PBPs directed for tissue engineering applications, the present study explores the potential of two novel silk-elastin-like proteins (SELP-1020-A and SELP-59-A), based on silk fibroin (GAGAGS) crystalline blocks and elastin-like (VPAVG) blocks, for the fabrication of fully protein-based fibrous mats produced by electrospinning. The effect of concentration and solvent type in the morphology of the electrospun structures was conducted using formic acid or water as solvents. Changes in the secondary structure of the SELP fibre mats were analysed by Fourier transform infrared (FTIR) and correlated with the transition from water-soluble to water-insoluble state. The mechanical properties of a representative electrospun fibre membrane were evaluated, along with its wettability, degree of swelling and water vapour permeability. As the aim of this study is to develop and evaluate the potential of SELP fibre mats as biomaterials directed for tissue regeneration applications, cell adhesion and proliferation of normal human skin fibroblasts was also investigated.

2.2. Electrospinning and post-processing treatment A custom-build electrospinning apparatus was used for the fabrication of the non-woven mats. The polymer solution was placed in a commercial plastic syringe fitted with a steel 18 gauge (inner diameter of 0.84 mm) blunt tip needle. Electrospinning was conducted by applying an electric field (Glassman model PS/FC30P04) between 1.7 and 2.4 kV cm−1 (table S1, supplementary data available from stacks.iop.org/BMM/8/065009/mmedia) and a collecting distance of 15 cm. A syringe pump fed the polymer solution into the tip at a rate of 0.25 mL h−1. During processing, temperature and moisture inside the electrospinning chamber were kept at 25 ± 1 ◦ C and 40 ± 7%, respectively. The generated electrospun mats were collected on a static grounded metallic collector. For post-processing treatment, fibres produced from 17 wt% SELP-59-A/FA (FA, formic acid) were chosen as representative sample. The as-spun fibre mats were immersed in a methanol solution for 20 and 60 min or exposed to methanol-saturated air at 25 ◦ C for 1, 6, 18 and 48 h in a vapour chamber. For the latter, a Petri dish containing 15 mL of liquid methanol (99.8%, Panreac) was placed at the bottom of the desiccator. The electrospun SELP mats were placed on a perforated ceramic plate, separating the bottom chamber with methanol from the top chamber. After the treatment, the fibre mats were air-dried at room temperature for at least 48 h, before any characterization. Throughout the text, fibre mats treated with methanol-saturated air will be denoted as methanol-treated samples. 2.3. Characterization of electrospun fibre mats Electrospun samples were coated with a thin gold layer using a sputter coating (Polaron model SC502) and their morphology was analysed by scanning electron microscopy (SEM, Leica Cambridge) with an accelerating voltage of 20 kV. The average diameter and distribution of the fibres was calculated with approximately 100 randomly selected fibres with ImageJ image processing software [46]. Contact angle measurements (sessile drop in dynamic mode) were performed at room temperature in a Data Physics OCA20 device using ultrapure water as test liquid. The contact angles were measured by depositing ultrapure water drops (3 μL) on the sample surface and analysed with SCA20 software. At least five measurements in each sample were performed in different membrane locations and the average contact angle was taken as the result for each sample. Infrared (FTIR) spectra were acquired at room temperature with a Bruker Tensor 27 spectrometer from Bruker Optics in attenuated total reflection mode (ATR) from 4000 to 600 cm−1. FTIR spectra were collected after 64 scans with a resolution of 4 cm−1. Baseline subtraction, second derivative and band fitting were performed with OriginPro 8.1 (OriginLab, Northampton, MA). For component analysis, a linear baseline was first applied to the amide I region (1705–1595 cm−1) and second derivative with a nine-point Savitsky–Golay smoothing function was applied. Frequencies detected by second derivative were used as peak position

2. Materials and methods 2.1. SELP production and preparation SELP-1020-A and SELP-59-A copolymers were designed to have a similar silk to elastin block ratio of 1:2, and molecular weight of ∼55 kDa, with different block lengths (supplementary data, figure S1, available from stacks.iop.org/BMM/8/065009/mmedia). SELP-1020-A consists in four tandem repetitions of S10E20 and SELP-59-A in nine tandem repetitions of S5E9, where S is the silk block with sequence GAGAGS and E the elastin block with sequence VPAVG. Genetic constructions were obtained by seamless cloning using previously described standard molecular biology procedures [12, 42, 45]. Both recombinant SELPs were produced by means of auto-induction and purified by acidbased cell lysis followed by ammonium sulphate purification, according to previously described methodology [42]. Pure lyophilized SELP copolymers were completely dissolved in either deionized water (H2O) or formic acid (FA, 98–100%, MERCK) at different concentrations of 5, 9, 13, 17 and 21 wt%. The solutions were maintained at ice-cold temperature, dissolved with the help of a magnetic stirrer until complete dissolution and allowed to set at room temperature for 30 min before electrospinning. 3

Biomed. Mater. 8 (2013) 065009

R Machado et al

guides for curve fitting iteratively with a Gaussian function (R2 > 0.999), while fixing the peak centres. The contribution of each fitted component to the amide I band was determined by integrating the area under the curve and normalizing for the total area of the amide I band. The stress–strain mechanical measurements were carried out at room temperature (∼25 ◦ C) with a TST350 tensile testing system (Linkam Scientific Instruments) at a strain rate of 2 mm min−1 in rectangular samples of 35 × 7 mm2 and 30–35 μm thickness. The modulus of elasticity (E), ultimate tensile strength (UTS) and strain-to-failure (ε) were calculated as average values from at least five test samples. The modulus of elasticity was calculated as the slope of the stress–strain curves in the linear zone of elasticity within 2% of elongation.

methanol-saturated air for 48 h. The membranes were then air-dried at room temperature for at least 48 h and sterilized by UV exposure (254 nm) for 20 min. All the experiments were performed in triplicate. 2.6.1. Cell culture. The BJ-5ta cell line (telomeraseimmortalized normal human skin fibroblasts) was obtained from the American Type Culture Collection (ATCC) through LGC standards and cultured at 37 ◦ C, 5% CO2, in humidified environment and according to ATCC recommendations (BJ-5ta medium—4 parts of Dulbecco’s modified Eagle’s medium containing 4 mM L-glutamine, 4.5 g L−1 glucose, 1.5 g L−1 sodium bicarbonate, and 1 part of Medium 199, supplemented with 10% (v/v) of fetal bovine serum, 1% (v/v) penicillin/streptomycin solution and 10 μg mL−1 hygromycin B).

2.4. Degree of swelling The degree of swelling was assessed gravimetrically in methanol-treated 17 wt% SELP-59-A electrospun mats by measuring the difference in weight between dry and swollen samples. The methanol-treated electrospun fibre mats were immersed in deionized water for different time periods at room temperature (∼25 ◦ C). After each time interval, the sample was taken out and the excess of water removed by gently soaking up the samples with filter paper. The degree of swelling was calculated as follows and expressed in terms of percentage relatively to the dry sample: Degree of Swelling (%) = [(Ws − Wd )/Wd ] × 100

2.6.2. Cell viability. Short term cell viability in response to methanol-treated SELP-59-A/FA electrospun fibre mats R Aqueous was performed by the MTS assay (CellTiter 96 One Solution Cell Proliferation, Promega) according to manufacturer’s instructions. MTS is a viability/proliferation test, and an inverse correlation between toxicity and colour conversion can be assumed. The cytotoxicity of the electrospun fibre mats in BJ-5ta cell line was assessed by indirect contact in surface treated 96-well cell culture plates (Nunclon polystyrene 96-well MicroWell, Thermo Scientific). One hundred microlitres of BJ-5ta cell suspension (6.6 × 104 cells mL−1) were seeded and cultured for 24 h according to the cell culture conditions described above. In parallel, UV sterilized methanol-treated fibres were incubated with 1 mL of cell culture medium for 24 h at 37 ◦ C, 5% CO2, in humidified environment. After 24 h of cell growth, the cell culture medium was removed and replaced by the same volume of the medium conditioned by contact with the SELP-59-A electrospun fibres. Cells were incubated for an additional 24, 48 and 72 h at 37 ◦ C, 5% CO2, in humidified environment after which cell viability was measured using the MTS proliferation assay. Briefly, cells in 1x sterile phosphate buffered saline (PBS, 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 per litre, at pH 7.4) were incubated with MTS solution for 2 h at 37 ◦ C followed by reading the absorbance at 490 nm with standard PBS used as blank measurement. Live cells react with tetrazolium salt in the MTS reagent producing a soluble formazan dye, which has absorbance at a wavelength of 490 nm. In the linear range of the absorbance curve, the absorbance intensity is proportional to the cell number. DMSO 30% and cells cultured in standard culture medium at the conditions tested were used as negative and positive controls for cell viability, respectively. Results were expressed as percentage of viability related to the positive control (set as 100% viability).

(1)

where Ws is the mass of the swollen sample and Wd is the initial dry mass. Each value was averaged from three independent measurements. 2.5. Water vapour transmission rate Water vapour transmission rate (WVTR) of methanoltreated 17 wt% SELP-59-A electrospun mats was determined gravimetrically based on the standard ASTM E96-95 method [47] with slight modifications. Glass vials containing 1/3 of ultrapure water were covered with electrospun membranes, fixed with parafilm and equilibrated overnight at room temperature. The vials were placed on a desiccator containing silica gel and maintained in temperature-controlled incubators at 25 ◦ C. In this way, the gradient in relative humidity (RH) between the outside and inside of the glass vials was 0:100 (RH outside: RH inside). The samples were weighted (Mettler Toledo AG245, ± 0.1 mg) at regular time intervals over a period of 10 h and a weight loss versus time plot was constructed. From the slope of the plot, the WVTR was calculated by WVTR(g/m2 /day) = (slope∗24)/A

(2)

where A is the area exposed to the permeant (m2). Experiments were performed with at least six replicas.

2.6.3. Cell adhesion and proliferation. SELP-59-A/FA methanol-treated fibre mats were cut in 13 mm diameter circles and used to coat sterile, surface-treated, non-pyrogenic and non-cytotoxic polyethylene cell culture coverslips (Sarstedt).

2.6. In vitro cell culture studies Cell culture studies were performed in electrospun membranes produced from 17 wt% SELP-59-A/FA and subjected to 4

Biomed. Mater. 8 (2013) 065009

R Machado et al

Non fibre-coated cell culture coverslips and 30% DMSO solution were used as positive and negative controls for cell proliferation, respectively. Both the fibre-coated and the non fibre-coated coverslips were previously sterilized by UV exposure. Before incubation with cells, the coverslips were equilibrated with 100 μL of BJ-5ta medium added to each well, followed by incubation at 37 ◦ C, 5% CO2 for 4 h, in humidified environment. The medium was then removed and 100 μL of BJ-5ta cell suspension (300 cells/μL) was added to each well followed by incubation for 24 h at 37 ◦ C, 5% CO2 to allow cell adhesion. Loosely adherent or non-adhered cells were removed by aspiration and washing the wells twice with PBS. Adhered cells were observed by scanning electron microscopy (SEM). For SEM analysis, the samples were soaked in a fixation solution (1 mL of 2.5% glutaraldehyde in PBS) for 1 h at room temperature, rinsed with distilled water (1 mL) and dehydrated through immersion for 30 min in a series of successive ethanol–water solutions (0.5 mL of 55%, 70%, 80%, 90%, 95% and 100% v/v of ethanol). The samples were then dried at room temperature and coated with gold prior to SEM analysis. To monitor the effect of the methanoltreated electrospun fibre mats on skin fibroblasts by direct contact, cell proliferation was assessed by the MTS assay as described before. Measurements were performed after three and seven days of cell culture. Culture medium was renovated every 24 h. After each time interval, cells were washed twice with PBS to remove non-adherent cells. All measurements were performed in triplicate. Mycoplasm contamination was monitored by sequence-specific PCR.

producing a clear solution without any evidence of gelation or aggregation. The optimal electrospinning conditions were obtained for both SELPs and are summarized in table S1 (supplementary data, available from stacks.iop.org/BMM/8/065009/mmedia). The optimum applied field was in the range of 2.2–2.4 kV cm−1, with a feed rate of 0.25 mL h−1 and a copolymer concentration between 13 and 17 wt% for SELP-1020-A and up to 21 wt% for SELP-59-A. By changing the concentration of SELP-1020-A/FA and SELP-59-A/FA, an assortment of different electrospun structures was obtained, ranging from particles/spheres characteristic of an electrospray process where no polymer entanglement occurs to nano and sub-micro fibres and ribbons (figures 1 and 2). The electrospun structures showed the same trend for both polymers, with the concentration of 13 wt% representing the critical threshold for the formation of non-defective fibres. At concentrations below 13 wt%, the formation of either beads (5 wt%) or beaded fibres (9 wt%) was observed, while an increment in the concentration to 13 wt% and above resulted in the absence of such defects. This can be explained by the increasing viscosity of the solution and polymer entanglement, allowing the formation of smooth and bead-free fibre mats randomly distributed in the flat static collector. The solution viscosity is proportional to the polymer concentration; thus, for fibre formation during electrospinning, the solution must consist of enough polymer content to promote adequate viscosity. The effect of concentration in fibre diameter is graphically represented in figure 3. Under the experimental conditions and for SELP-1020-A, the average fibre diameter was calculated as 100 ± 12 nm for 13 wt% and 270 ± 155 nm for 17 wt%; however, the broader diameter distribution found in the latter case is noticeable. Regarding SELP-59-A, the mean fibre diameter was found to be 150 ± 22 nm for 13 wt%, 183 ± 36 nm for 17 wt% and 330 ± 40 nm for 21 wt%, with similar distribution of fibre diameters. A comparison between the as-spun structures, obtained for both copolymers, demonstrates that besides concentration, the silk- and elastin-block content also influenced the morphology of the electrospun fibres. Indeed, for the same concentration of 13 wt%, the fibres obtained with SELP-1020-A are thinner and distributed over narrower diameter dispersion than those obtained with SELP-59-A. This disparity in fibre morphology is greatly emphasized when comparing the electrospun structures obtained with a concentration of 17 wt%. Regarding SELP-1020-A, the formation of a mix population of fibres and ribbons, contributing to the wide dispersion of diameters observed in figure 1, contrasts with the regular fibres obtained with SELP-59-A. This ribbon morphology is a consequence of the formation of a thin skin around the liquid jet due to rapid evaporation of the solvent at the jet surface. Before all the solvent diffuses through the skin, it solidifies resulting in a thin layer of solid skin with liquid core. As the solvent inside evaporates, the skin remains as a hollow tube, collapsing into ribbon-like structures [50, 51]. This ribbon morphology was also observed in other electrospun polymeric materials such as poly(ether imide) [51], elastin [52] and silk fibroin [53]. No fibres were

2.6.4. Statistics and data analysis. One-way analysis of variance (ANOVA) with Bonferroni’s post-test was carried out to compare the means of different data sets within each experiment in GraphPad Prism 5 software. A value of P < 0.05 was considered to be statistically significant.

3. Results and discussion 3.1. Fabrication of SELP fibre mats by electrospinning In this work, electrospinning of SELP-1020-A and SELP-59A was performed using formic acid (FA) or water (H2O) as solvents, in order to study the influence of the solvent used in fibre formation. The selection of formic acid was based on its ability to dissolve silk fibroin [3, 48, 49] and a SELP analogue [40, 41] at adequate concentrations for electrospinning. A stable Taylor cone is required for the formation of uniform fibres, so preliminary electrospinning experiments (data not shown) were conducted in order to evaluate the best conditions while maintaining its stability. A collecting distance of 150 mm, a flow rate of 0.25 mL h−1 and a needle of 18 gauge have shown the optimum conditions and therefore were kept constant in all the experiments while the influence of the applied electrical field and concentration on fibre diameter and its distribution were characterized. 3.2. Electrospinning formic acid SELP solutions Both copolymers (SELP-1020-A and SELP-59-A) were completely dissolved in FA at different concentrations, 5

Biomed. Mater. 8 (2013) 065009

R Machado et al

(A)

(B)

(C)

(D)

Figure 1. SEM images of the electrospun structures obtained with SELP-1020-A/FA at different concentrations: (A) 5 wt%, (B) 9 wt%, (C) 13 wt%, (D) 17 wt%. Inset figures represent the diameter distribution for each corresponding concentration. A normal distribution curve was applied for 13 wt% whereas none was applied for 17 wt% due to the high dispersion of diameters. Scale bars: 5 μm.

(A)

(B)

(C)

(D)

Figure 2. SEM images of the electrospun structures obtained with SELP-59-A/FA at different concentrations: (A) 5 wt%, (B) 9wt%, (C) 13 wt%, (D) 17 wt%, (E) 21 wt%. Inset figures represent the diameter distribution for each corresponding concentration. A normal distribution curve was applied for all histograms. Scale bars: 5 μm. 6

(E)

Biomed. Mater. 8 (2013) 065009

R Machado et al

images of the electrospun structures obtained for SELP-59A/H2O at concentrations of 5, 9 and 13 wt%. As expected, an increase in the concentration led to the deposition of wider fibres and ultimately to the deposition of ribbons. In a concentration-dependent way, electrospinning of SELP-59A resulted in a low density of fibres with short lengths (5 wt%, figure 4(A)), in a high density of branched fibres with triangle-shaped bifurcations (9 wt%, figure 4(B)) or in a mixed population of flat ribbons and branched fibres with triangle-shaped bifurcations (13 wt%, figure 4(C)). The triangle-shaped bifurcation is a consequence of fibre splitting resulting from the ejection of smaller jets from the primary one. When the primary jet from the charged droplet travels towards the collector, it undergoes changes in both the local charge units and local polymer concentration leading to instabilities in jet flow; these instabilities can be minimized by ejection of smaller jets or by splitting into two smaller jets, resulting in branched fibres [51]. The dramatic differences between both copolymers can be attributed to the high density of hydrogen bonding structures in SELP-1020-A than in SELP-59-A. As the formation of hydrogen bonds between the silk-like blocks is thought to be the primary driving force for SELP gelation, serving as contact points between the polymer chains [21, 25], the gelation rate of SELPs is strongly dependent on the number of silk-like blocks in the polymer chain. Although each of the bonds by itself is rather weak, the cooperative interaction of all the bonds results in a highly stable structure. Therefore, higher density of silk blocks leads to an increase in interlocking points for physical chain entanglement, decreasing the amount of polymer necessary to form a crosslinked network, explaining the higher gelation rate of SELP-1020-A when compared to SELP-59-A.

Figure 3. Graphical representation displaying the critical concentration threshold (represented by dashed line) for the production of defective and non-defective fibres.

obtained for SELP-1020-A at a concentration of 21 wt% as the solution was too viscous to be processed and clogging at the needle tip was observed. In this case, the high cohesiveness of the solution leads to instability in the fluid flow, hampering the electrospinning. On the other hand, SELP-59-A at the same concentration produced fibres with diameters between 243 and 433 nm. Given the similar molecular weights and the same silk to elastin block ratio in both copolymers, the difference in fibre diameter and morphology must be attributed to the doubled number of tandem silk-like blocks present in SELP-1020-A. Although with an overall lower number of silk-like blocks per polymeric chain (40 in SELP-1020-A and 45 in SELP-59-A), the higher number of tandem silk blocks in SELP-1020-A (10 in SELP-1020-A and 5 in SELP-59-A) contributes to a large extent to the viscosity of the polymer solution. 3.3. Electrospinning SELP aqueous solutions

3.4. Post-processing treatment with methanol

Both SELPs were dissolved in water at the same concentrations used for formic acid. However, as opposed to the formic acid mixtures, even at a concentration as low as 5 wt%, the SELP-1020-A/H2O solution was highly viscous, showing observable signs of gelation, therefore not spinnable. In fact, after some time, the solution turned into a soft gel matrix by a process accelerated by increasing the incubation temperature to 37 ◦ C (data not shown). As for SELP-59-A, pieces of evidence of the sol-to-gel transition were observed only with higher concentrations of 17 and 21 wt%, leading to non-spinnable solutions. Figure 4 shows representative

The electrospun mats are highly soluble in water, completely dissolving when in contact with an aqueous solution. In order to extend their use for applications that require contact with aqueous environments or body fluids there is a need to stabilize the structure. To make the dry fibres stable when in contact with water, the secondary structure of the silk-blocks can be modified in order to induce formation of a higher fraction of hydrogen-bonded beta-sheets. The use of methanol has been described as a method to promote such conformation by inducing crystallization of the

(A)

(B)

(C)

Figure 4. SEM images of electrospun fibres obtained by SELP-59-A/H2O at 5 wt% (A), 9 wt% (B) and 13 wt% (C). Scale bars: 5 μm. 7

Biomed. Mater. 8 (2013) 065009

R Machado et al

(A)

(B)

(C)

(A)

Figure 5. Representative images of the fibre mats obtained after treatment with liquid methanol for (A) 20 and (B) 60 min, and (C) after treatment with methanol-saturated air. Scale bars: 5 μm.

silk blocks and consequently making the fibres more stable in contact with water [54, 55]. In the present work, the electrospun mats obtained with 17 t% SELP-59-A/FA were chosen as representative samples to study the structural changes induced by methanol. Treatment with liquid methanol was shown to compromise the fibre morphology, transforming the individualized fibres into a fibrous film (figures 5(A) and (B)). In order to overcome this issue, dry electrospun mats were exposed to air saturated with methanol for periods of time up to 48 h, shown to maintain morphological integrity with minor differences in fibre width (174 ± 44 nm and 183 ± 36 nm for the methanol-treated and non-treated fibre mats, respectively) (figure 5(C)). Surface wettability is an important property for material applications in tissue and biomedical engineering, playing an important role in cell proliferation and attachment [56–59]. The average water contact angle of the methanol-treated SELP59-A/FA membranes was calculated as 69◦ ± 3◦ indicating that wetting is favourable on the surface of the fibre mats and within the optimal conditions for fibroblast cell adhesion and growth. Regarding the non-treated membranes, the water contact angle was calculated as 28◦ ± 2◦ within the first minute of contact, quickly reaching negligible values and leading to fibre dissolution. To understand the changes induced by methanol in the secondary structure of the electrospun mats, SELP-59A fibres were exposed to air saturated with methanol at different time-scale points and analysed by ATR-FTIR. After methanol treatment, the amide I peak (C=O stretching) progressively shifted to lower wavenumbers (figure 6), reflecting a conversion from the unordered, random coil structure to an ordered, beta-sheet conformation. The analysis of the infrared spectra suggests that this conversion is a gradual event involving a transition from random coil to beta-sheet. In fact, the initial peak, corresponding to the non-treated sample centred at 1647 cm−1, shifts to values of 1632, 1630 and 1628 cm−1 after 6, 18 and 48 h methanol treatment, respectively, while after 1 h with methanol, the peak centred at 1647 cm−1 is attributed to a random coil conformation, the peaks located in the range of 1628–1637 cm−1 are attributed to antiparallel beta-sheet structures [41, 54]. The infrared spectra of non-treated electrospun samples from SELP-59-A/H2O and 13 wt% SELP-59-A/FA and H2O solutions, exhibited an amide I band centred at 1646 cm−1 (data not shown) showing that both the concentration and the solvent

Figure 6. Experimentally determined ATR–FTIR spectra for the non-treated (a) and treated samples with methanol-saturated air for 1, 6, 18 and 48 h (spectra b–e, respectively). Image on the left shows the full spectra in the range of wavenumber 600–4000 cm−1. Image on the right highlights the amide I region (1600–1700 cm−1) which is associated with C=O stretching and related to backbone conformation.

do not affect the unordered secondary structure of the spun fibres. Regarding the samples immersed in liquid methanol, the peak from amide I was found to be centred at 1627 cm−1 (data not shown), indicating the presence of an antiparallel betasheet conformation and thus demonstrating the crystallization effect of methanol. To study this transition further in detail from random coil to beta-sheet structure upon methanol treatment, the amide I region was curve fitted and the fraction of fitted components was calculated. The number of components and peak positions used as starting parameters for curve fitting were obtained from the second derivative of the original spectra. For each sample, a total of eight component bands were fitted to the amide I band. Figures 7(A) and (B) show examples of the Gaussian curve fitting in the amide I region for the untreated and the 48 h methanol-treated samples. For each fitted band, a structural conformation was assigned (table S2, supplementary data available from stacks.iop.org/BMM/8/065009/mmedia), except for aggregated strands that were considered as contributions to the beta-sheet content. The relative content of each assigned structural conformation is summarized in figure 7(C). While the content of turns and bends is maintained almost constant all over the study, representing approximately 27 + 2% of the total, the relative percentage of beta-sheet conformation is balanced by the number of extended chains. 8

Biomed. Mater. 8 (2013) 065009

(A)

R Machado et al

(B)

(C)

Figure 8. Degree of swelling determined for methanol-treated SELP-59-A/FA fibre mats during 240 min of immersion in deionized water at room temperature. Inset represents the degree of swelling obtained during the first 5 min of immersion. Measurements were performed with three independent samples.

142 ± 48 MPa, an average ultimate tensile strength of 14 ± 2 MPa and an average strain-to-failure of 22 ± 6%. These values contrast with the mechanical properties previously determined for nanoribbons of a SELP analogue (SELP47K) where the modulus of elasticity, ultimate tensile strength and strain-to-failure were found to be 880 MPa, 31 MPa and 8%, respectively [39]. Although losing tensile strength, the strain-to-failure of the SELP-59-A nanofibres showed a two-fold increase in relation to SELP47K nanoribbons.

Figure 7. Curve-fitted spectra (top) of non-treated (A) and 48 h methanol-treated (B) samples. Eight Gaussian bands (green line) were fitted iteratively to the amide I band (black line) using the peak positions obtained from the second derivative spectra. (C) represents the fractional distribution of each component to the amide I absorbance spectrum of non-treated and methanol-treated samples.

3.6. Degree of swelling and water vapour transmission rate

In the case of the non-treated sample, the content of beta-sheet, extended chains and turns 30, 44 and 26%, respectively. After treatment with methanol-saturated air these values change to 39, 35 and 26%, reflecting an increase in the beta-sheet content and loss of extended chains conformation. With longer methanol curing times, the content in ordered structures, characterized by an enrichment of beta-sheet structures, reaches values of 47 and 51% (6 h and 18 h treatment, respectively), with a subsequent loss in the extended chains content, where the relative amount decreases to 25 and 21%. A steady state is reached after 18 h treatment as no significant changes were observed in a further increase in curing time (48 h treatment). In light of this quantitative analysis, the conversion to the beta-sheet structure occurs due mostly to the random coil → beta-sheet conformation transition, whereas the fraction of turns and bends changes only slightly.

The water uptake capability (degree of swelling) of the methanol-treated electrospun membranes was evaluated by immersing the fibres in deionized water for different time intervals while measuring changes in weight (figure 8). Upon immersion, the methanol-treated membranes rapidly swelled, reaching its maximum water uptake capacity within the first 2 min. After the time of the experiment (4 h) the degree of swelling for the methanol-treated samples was found to be in the range of 578 ± 168%. According to the British Pharmacopeia [60] and ASTM standards [47], the WVTR measures the steady water vapour flow in unit time through unit area under specific conditions of temperature and humidity. Considering the potential application of SELP fibre mats as wound dressing for skin regeneration, this parameter thus characterizes the rate at which the water is transmitted from the wound bed to the external environment through the graft. The WVTR of a skin graft should be adequate in order to prevent both excessive dehydration due to high WVTR and wound maceration and contamination due to low WVTR. The WVTR of the methanol-treated SELP-59-A/FA fibre mats was determined to be 1083 g/m2/day (figure S3, supplementary data available from stacks.iop.org/BMM/8/065009/mmedia). According to Lamke et al [61] the WVTR of normal skin is 204 ± 12 g/m2/day, while in injured skin this value can range

3.5. Mechanical properties of electrospun fibres The mechanical properties of 17 wt% SELP-59-A/FA electrospun fibre mats were analysed in the dry state under uniaxial tensile analysis. A characteristic uniaxial stress–strain curve of methanol-treated fibres is shown in the supplementary data, figure S2 available from stacks.iop.org/BMM/8/065009/mmedia. The electrospun membranes revealed an average modulus of elasticity of 9

Biomed. Mater. 8 (2013) 065009

R Machado et al

Figure 9. Cytotoxicity test of SELP-59-A/FA fibre mats (indirect contact) on normal human skin fibroblasts (BJ-5ta cell line) L with positive (control) and negative controls (DMSO 30%) for cell viability. Bars represent means ± SD. Cell viability in the SELP fibres was similar or higher than that of the control during the time of the experiment (24, 48 and 72 h of cell culture), ∗ p < 0.05 was considered to be statistically significant using ANOVA followed by Bonferroni’s post hoc tests (ns—non significant, ∗ p  0.05, ∗∗∗∗ p  0.0001; ANOVA).

Figure 10. Proliferation of normal human skin fibroblasts (BJ-5ta) seeded on SELP-59-A/FA fibre mats in relation to the control after three days and seven days of cell culture. Data are expressed as mean ± SD. No significant differences were detected between the control and SELP fibres after three days of cell culture. After seven days of culturing, a slight decrease in cell proliferation was observed for cells seeded in the electrospun fibre mats, ∗ p < 0.05 was considered to be statistically significant using ANOVA followed by Bonferroni’s post hoc tests (ns—non significant, ∗∗∗∗ p  0.0001; ANOVA).

from 278 ± 26 g/m2/day for first degree burns to 5138 ± 202 g/m2/day for a granulating wound. The results obtained for the WVTR indicate that SELP-59-A fibre mats are more adequate to moderate exuding wounds.

In the determination of the biocompatibility of a biomaterial, the cellular behaviour is an important factor as cells undergo specific morphological changes in order to stabilize the cell–material interface. In this process of surface recognition involving cell adhesion and spreading, cells undergo several sequential changes namely cell attachment, filopodial growth, cytoplasmic webbing, flattening of the cell mass and ruffling of peripheral cytoplasm [62]. Fibroblasts are a major component of the connective tissue that composes the human body, namely in the skin, being major producers of the extracellular matrix. To evaluate the adhesion of the fibroblasts onto the SELP-59-A/FA fibre mats, normal human skin fibroblasts were cultured on the fibre mats for 24 h and the resulting cellular morphologies were examined by electron microscopy for more detailed cell–material interactions. Figure 11 shows micrographs of representative BJ-5ta cells adhered onto SELP-59-A/FA fibre mats after 24 h of incubation. The micrographs showed cells spherical or roughly spherical in shape, with smooth rounded surface and adhered on the surface with lamellipodia and filopodia extensions. In general, the morphology of the adhered cells was dominated by cells in advanced stage of adhesion (figure 11(A)) and cells apparently in early stages of spreading (figure 11(B)). The filopodia extensions have been reported as being associated to substrate-exploring functions with various cell types expressing long transient filopodia in their spherical state prior spreading [63]. On flat surfaces, these transient filopodia quickly disappear during spreading in favour of lamellipodiamediated spreading mechanism. In fact, Albuschies and Vogel [64], demonstrated that fibroblasts cultured on a micropatterned silicon nanowire surface spread through a filopodia-mediated mechanism. In contrast, the authors demonstrated that fibroblasts cultured on flat surfaces for the

3.7. Fibroblast viability and proliferation In this work, viability of normal human skin fibroblasts in SELP-59-A/FA fibre mats was evaluated in vitro by using the MTS assay which gives an indication of cell metabolic activity. The results of the indirect contact study (exposure to leachables in pre-conditioned cell culture medium) after fibroblast incubation with material extracts showed no cytotoxicity regardless of incubation time. Figure 9 represents the viability results for BJ-5ta cells in contact with undiluted pre-conditioned media after 24, 48 and 72 h of incubation. The metabolic activity of cells in contact with the leachables was similar to or higher than that obtained with the positive control (culture medium). The high values of cell viability obtained with indirect contact showed that the electrospun fibre mats were stable when in contact with cell culture medium; with possibly very few leachables and definitely nothing toxic was liberated from the mats to the medium. To evaluate the ability of the SELP-59-A/FA fibre mats to directly support cell proliferation, skin fibroblasts were seeded on the fibre mats and grown for three and seven days after which proliferation was assessed by measuring the metabolic activity of the cells with MTS assay. Figure 10 shows the proliferation profile of BJ-5ta seeded on the surface of methanol-treated electrospun SELP-59-A/FA fibre mats, after three and seven days of cell culturing. After three days of incubation, the fibroblasts cultured on the fibre mats displayed a proliferation profile similar to that obtained with the control, where cells were cultured on standard cell culture coverslips. After seven days of incubation, proliferation of the cells grown on the SELP fibre mats was slightly lower when compared to the control. 10

Biomed. Mater. 8 (2013) 065009

(A)

R Machado et al

(B)

Figure 11. SEM micrographs of BJ-5ta fibroblasts cultured on the SELP-59-A/FA fibre mats for 24 h showing cells in advanced stage of adhesion (A) and cells apparently in early stage of spreading (B). Cells adhered to the surface with short filopodia (black arrows) extending into long conical protrusions (dendritic extensions, white arrows) that interpenetrated into the SELP fibre mats.

microscopy demonstrated that skin fibroblasts adhered and spread on the fibre mats by a filopodia-mediated mechanism. In this work, characterization of the electrospun membranes in terms of its mechanical properties, wettability, degree of swelling and WVTR suggest that SELP fibre mats meet the requirements for application as materials suitable for wound dressing for skin regeneration. One of the foreseen limitations is the poor biodegradability of these materials though this characteristic can be adjusted by modifying the molecular composition of SELPs, which also influences the physical– chemical properties. Cell–material interaction can also be improved in future studies by inclusion of cell adhesion recognition sites or bioactive molecules. Further developments may include the fabrication of active biological scaffolds using cell electrospinning [65]. This technique can be employed for the development of three-dimensional active biological structures using, for example, a coaxial needle arrangement [66]. In summary, the extreme versatility of rPBPs opens a wide, new window for the development of application tailored materials.

same time period are almost depleted of filopodia with the occurrence of circumferential lamellipodium. In the present work, the morphology of the fibroblasts displayed short radial filopodia extending into long filopodia and conical protrusions with lengths reaching near the diameter of the spherical cell mass (figure 11). Apparently, the fibroblasts interacted and integrated well with the surrounding SELP fibres penetrating into the mats, with the ends of the dendritic extensions from the SELP fibres becoming difficult to differentiate by SEM. Together with the viability and proliferation assays, these results further show that SELP-59-A fibre mats are biocompatible, non-cytotoxic and able to support proliferation of normal human fibroblasts.

4. Conclusions Fibres of SELP copolymers can be interesting candidates for several biomedical applications including three-dimensional scaffolds, skin grafts and drug delivery materials. In this work, two new genetically engineered silk-elastin-like protein copolymers, SELP-1020-A and SELP-59-A, were electrospun from formic acid without addition of any external agents into an assortment of structures with different morphologies that can be explored for such applications. With both SELPs a relative monodisperse population of non-defective fibres was produced, with diameters much smaller than previously reported. The tunable morphology, merely by changing concentration and by the number of silk blocks, confers a unique flexibility in the development of tailored scaffolds for tissue engineering applications, where the topography is of interest. As the use of organic solvents can limit the range of applications for a certain material, we have shown that SELP-59-A can be electrospun from aqueous solution, producing nanoribbons to be further explored for encapsulation/incorporation of enzymes or drugs. Structure stabilization and water insolubility were rendered by treatment with methanol, leading to an increase in the β-sheet content due to a time-dependent transition from random coil to betasheet conformation. Electrospun mats of SELP-59-A/FA were explored as biomaterial for the culture of normal human skin fibroblasts. Indirect cytotoxicity assessment revealed good cytocompatibility and promotion of cell proliferation. SEM

Acknowledgments This work was financially supported by the European Commission via the 7th Framework Programme project EcoPlast (FP7-NMP-2009-SME-3, collaborative project number 246176), by Portuguese funding from FEDER through POFC-COMPETE and PEst project C/BIA/UI4050/2011 (Portugal), PEST-C/FIS/UI607/2011 and PEST-C/QUI/UIO686/2011. By MICINN (MAT 200914195-C03-03, IT2009-0089, ACI2009-0890, MAT201015310 and MAT2010-15982), the JCyL (VA034A09, VA030A08 and VA049A11-2) and CIBER-BBN. EC (Spain). The authors also thank funding from Matepro-Optimizing Materials and Processes , ref. NORTE-07-0124-FEDER000037 , co-funded by the ‘Programa Operacional Regional do Norte’ (ON.2—O Novo Norte), under the ‘Quadro de Referˆencia Estrat´egico Nacional’ (QREN), through the ‘Fundo Europeu de Desenvolvimento Regional’ (FEDER). RM, AC, CMC and VS acknowledge FCT for SFRH-BPD/86470/2012, SFRH/BD/75882/2011, SFRH/ BD/68499/2010 and SFRH/BPD/63148/2009 grants, respectively. 11

Biomed. Mater. 8 (2013) 065009

R Machado et al

References

[21] Cappello J et al 1998 In-situ self-assembling protein polymer gel systems for administration, delivery, and release of drugs J. Control. Release 53 105–17 [22] Nagarsekar A, Crissman J, Crissman M, Ferrari F, Cappello J and Ghandehari H 2002 Genetic synthesis and characterization of pH- and temperature-sensitive silk-elastinlike protein block copolymers J. Biomed. Mater. Res. 62 195–203 [23] Nagarsekar A, Crissman J, Crissman M, Ferrari F, Cappello J and Ghandehari H 2003 Genetic engineering of stimuli-sensitive silk elastin-like protein block copolymers Biomacromolecules 4 602–7 [24] Haider M et al 2005 Molecular engineering of silk-elastinlike polymers for matrix-mediated gene delivery: biosynthesis and characterization Mol. Pharm. 2 139–50 [25] Dandu R, Von Cresce A, Briber R, Dowell P, Cappello J and Ghandehari H 2009 Silk-elastinlike protein polymer hydrogels: influence of monomer sequence on physicochemical properties Polymer 50 366–74 [26] Megeed Z, Cappello J and Ghandehari H 2002 Genetically engineered silk-elastinlike protein polymers for controlled drug delivery Adv. Drug Deliv. Rev. 54 1075–91 [27] Gustafson J A and Ghandehari H 2010 Silk-elastinlike protein polymers for matrix-mediated cancer gene therapy Adv. Drug Deliv. Rev. 62 1509–23 [28] Megeed Z, Cappello J and Ghandehari H 2002 Controlled release of plasmid DNA from a genetically engineered silk-elastinlike hydrogel Pharm. Res. 19 954–9 [29] Greish K et al 2009 Silk-elastinlike protein polymer hydrogels for localized adenoviral gene therapy of head and neck tumors Biomacromolecules 10 2183–8 [30] Gustafson J, Greish K, Frandsen J, Cappello J and Ghandehari H 2009 Silk-elastinlike recombinant polymers for gene therapy of head and neck cancer: from molecular definition to controlled gene expression J. Control. Release 140 256–61 [31] Gustafson J A, Price R A, Greish K, Cappello J and Ghandehari H 2010 Silk-elastin-like hydrogel improves the safety of adenovirus-mediated gene-directed enzyme-prodrug therapy Mol. Pharm. 7 1050–6 [32] Price R, Gustafson J, Greish K, Cappello J, McGill L and Ghandehari H 2012 Comparison of silk-elastinlike protein polymer hydrogel and poloxamer in matrix-mediated gene delivery Int. J. Pharm. 427 97–104 [33] Dinerman A A, Cappello J, Ghandehari H and Hoag S W 2002 Swelling behavior of a genetically engineered silk-elastinlike protein polymer hydrogel Biomaterials 23 4203–10 [34] Haider M, Cappello J, Ghandehari H and Leong K W 2008 In vitro chondrogenesis of mesenchymal stem cells in recombinant silk-elastinlike hydrogels Pharm. Res. 25 692–9 [35] Teng W, Huang Y, Cappello J and Wu X 2011 Optically transparent recombinant silk-elastinlike protein polymer films J. Phys. Chem. B 115 1608–15 [36] Teng W, Cappello J and Wu X 2009 Recombinant silk-elastinlike protein polymer displays elasticity comparable to elastin Biomacromolecules 10 3028–36 [37] Teng W, Cappello J and Wu X 2011 Physical crosslinking modulates sustained drug release from recombinant silk-elastinlike protein polymer for ophthalmic applications J. Control. Release 156 186–94 [38] Nagarajan R, Drew C and Mello C M 2007 Polymer–micelle complex as an aid to electrospinning nanofibers from aqueous solutions J. Phys. Chem. C 111 16105–8 [39] Ner Y, Stuart J A, Whited G and Sotzing G A 2009 Electrospinning nanoribbons of a bioengineered silk-elastin-like protein (SELP) from water Polymer 50 5828–36

[1] Bhardwaj N and Kundu S C 2010 Electrospinning: a fascinating fiber fabrication technique Biotechnol. Adv. 28 325–47 [2] Huang Z M, Zhang Y Z, Kotaki M and Ramakrishna S 2003 A review on polymer nanofibers by electrospinning and their applications in nanocomposites Compos. Sci. Technol. 63 2223–53 [3] Min B M, Lee G, Kim S H, Nam Y S, Lee T S and Park W H 2004 Electrospinning of silk fibroin nanofibers and its effect on the adhesion and spreading of normal human keratinocytes and fibroblasts in vitro Biomaterials 25 1289–97 [4] Li M Y, Mondrinos M J, Gandhi M R, Ko F K, Weiss A S and Lelkes P I 2005 Electrospun protein fibers as matrices for tissue engineering Biomaterials 26 5999–6008 [5] Nivison-Smith L, Rnjak J and Weiss A S 2010 Synthetic human elastin microfibers: stable cross-linked tropoelastin and cell interactive constructs for tissue engineering applications Acta Biomater. 6 354–9 [6] Zhou J, Cao C and Ma X 2009 A novel three-dimensional tubular scaffold prepared from silk fibroin by electrospinning Int. J. Biol. Macromol. 45 504–10 [7] Buttafoco L et al 2006 Electrospinning of collagen and elastin for tissue engineering applications Biomaterials 27 724–34 [8] Jha B S et al 2011 Electrospun collagen: a tissue engineering scaffold with unique functional properties in a wide variety of applications J. Nanomater. 2011 348268 [9] Powell H M and Boyce S T 2008 Fiber density of electrospun gelatin scaffolds regulates morphogenesis of dermal–epidermal skin substitutes J. Biomed. Mater. Res. A 84A 1078–86 [10] Carlisle C R, Coulais C, Namboothiry M, Carroll D L, Hantgan R R and Guthold M 2009 The mechanical properties of individual, electrospun fibrinogen fibers Biomaterials 30 1205–13 [11] Baker S et al 2012 The mechanical properties of dry, electrospun fibrinogen fibers Mater. Sci. Eng. C 32 215–21 [12] Rodr´ıguez-Cabello J, Girotti A, Ribeiro A and Arias F 2012 Synthesis of genetically engineered protein polymers (recombinamers) as an example of advanced self-assembled smart materials Nanotechnology in Regenerative Medicine ed M Navarro and J A Planell (Totowa, NJ: Humana Press) p 17–38 [13] Rabotyagova O S, Cebe P and Kaplan D L 2011 Protein-based block copolymers Biomacromolecules 12 269–89 [14] van Hest J C M and Tirrell D A 2001 Protein-based materials, toward a new level of structural control Chem. Commun. 2001 1897–904 [15] Altman G H et al 2003 Silk-based biomaterials Biomaterials 24 401–16 [16] Urry D W et al 2002 Elastin: a representative ideal protein elastomer Phil. Trans. R. Soc. Lond. B 357 169–84 [17] Fossey S A, N´emethy G, Gibson K D and Scheraga H A 1991 Conformational energy studies of β-sheets of model silk fibroin peptides: I. Sheets of poly(Ala-Gly) chains Biopolymers 31 1529–41 [18] Rodr´ıguez-Cabello J C, Alonso M, D´ıez M I, Caballero M I and Herguedas M M 1999 Structural investigation of the poly(pentapeptide) of elastin, poly(GVGVP), in the solid state Macromol. Chem. Phys. 200 1831–8 [19] Urry D W 2006 What Sustains Life? Consilient Mechanisms for Protein-Based Machines and Materials (New York: Springer) [20] Cappello J et al 1990 Genetic engineering of structural protein polymers Biotechnol. Prog. 6 198–202 12

Biomed. Mater. 8 (2013) 065009

R Machado et al

[40] Qiu W, Teng W, Cappello J and Wu X 2009 Wet-spinning of recombinant silk-elastin-like protein polymer fibers with high tensile strength and high deformability Biomacromolecules 10 602–8 [41] Qiu W, Huang Y, Teng W, Cohn C M, Cappello J and Wu X 2010 Complete recombinant silk-elastinlike protein-based tissue scaffold Biomacromolecules 11 3219–27 [42] Machado R et al 2013 High level expression and facile purification of recombinant silk-elastin-like polymers in auto induction shake flask cultures AMB Express 3 11 [43] Collins T, Azevedo-Silva J, da Costa A, Branca F, Machado R and Casal M 2013 Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation Microb. Cell Fact. 12 21 [44] Reguera J, Lagaron J M, Alonso M, Reboto V, Calvo B and Rodriguez-Cabello J C 2003 Thermal behavior and kinetic analysis of the chain unfolding and refolding and of the concomitant nonpolar solvation and desolvation of two elastin-like polymers Macromolecules 36 8470–6 [45] Machado R et al 2009 Exploiting the sequence of naturally occurring elastin: construction, production and characterization of a recombinant thermoplastic protein-based polymer J. Nano Res. 6 133–45 [46] Rasband W S Image J, US National Institutes of Health, Bethesda, MD, USA http://imagej.nih.gov/ij/ [47] ASTM 1995 Standard test methods for water vapour transmission of materials ASTM E96-95 [48] Marelli B, Alessandrino A, Fare S, Freddi G, Mantovani D and Tanzi M C 2010 Compliant electrospun silk fibroin tubes for small vessel bypass grafting Acta Biomater. 6 4019–26 [49] Sukigara S, Gandhi M, Ayutsede J, Micklus M and Ko F 2003 Regeneration of Bombyx mori silk by electrospinning–part 1: processing parameters and geometric properties Polymer 44 5721–7 [50] Reneker D H and Yarin A L 2008 Electrospinning jets and polymer nanofibers Polymer 49 2387–425 [51] Koombhongse S, Liu W and Reneker D H 2001 Flat polymer ribbons and other shapes by electrospinning J. Polym. Sci. B 39 2598–606 [52] Rnjak-Kovacina J et al 2012 Electrospun synthetic human elastin:collagen composite scaffolds for dermal tissue engineering Acta Biomater. 8 3714–22 [53] Cao H, Chen X, Huang L and Shao Z 2009 Electrospinnning of reconstituted silk fiber from aqueous silk fibroin solution Mater. Sci. Eng. C 29 2270–4

[54] Hu X, Kaplan D and Cebe P 2006 Determining beta-sheet crystallinity in fibrous proteins by thermal analysis and infrared spectroscopy Macromolecules 39 6161–70 [55] Wilson D, Valluzzi R and Kaplan D 2000 Conformational transitions in model silk peptides Biophys. J. 78 2690–701 [56] Dowling D P, Miller I S, Ardhaoui M and Gallagher W M 2011 Effect of surface wettability and topography on the adhesion of osteosarcoma cells on plasma-modified polystyrene J. Biomater. Appl. 26 327–47 [57] Lampin M, WarocquierClerout R, Legris C, Degrange M and SigotLuizard M F 1997 Correlation between substratum roughness and wettability, cell adhesion, and cell migration J. Biomed. Mater. Res. 36 99–108 [58] Lourenco B N et al 2012 Wettability influences cell behavior on superhydrophobic surfaces with different topographies Biointerphases 7 46 [59] Kim S H et al 2007 Correlation of proliferation, morphology and biological responses of fibroblasts on LDPE with different surface wettability J. Biomater. Sci Polym. Ed. 18 609–22 [60] BSBE 2002 Test methods for primary wound dressings. Part 1: aspects of absorbency. Fluid handling capacity BSBE 13726-1 [61] Lamke L O, Nilsson G E and Reithner H L 1977 The evaporative water loss from burns and the water-vapour permeability of grafts and artificial membranes used in the treatment of burns Burns 3 159–65 [62] Rajaraman R, Rounds D E, Yen S P and Rembaum A 1974 A scanning electron microscope study of cell adhesion and spreading in vitro Exp. Cell. Res. 88 327–39 [63] Partridge M A and Marcantonio E E 2006 Initiation of attachment and generation of mature focal adhesions by integrin-containing filopodia in cell spreading Mol. Biol. Cell. 17 4237–48 [64] Albuschies J and Vogel V 2013 The role of filopodia in the recognition of nanotopographies Sci. Rep. 3 1658 [65] Jayasinghe S N 2013 Cell electrospinning: a novel tool for functionalising fibres, scaffolds and membranes with living cells and other advanced materials for regenerative biology and medicine Analyst 138 2215–23 [66] Townsend-Nicholson A and Jayasinghe S N 2006 Cell electrospinning: a unique biotechnique for encapsulating living organisms for generating active biological microthreads/scaffolds Biomacromolecules 7 3364–9

13