Preparation and Characterization of a Novel Electrospun Spider Silk

In the paper, we successfully prepared spider silk fibroins (Ss)/poly(d,l-lactide) (PDLLA) ...... For a more comprehensive list of citations to this a...
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J. Phys. Chem. B 2008, 112, 11209–11216

11209

Preparation and Characterization of a Novel Electrospun Spider Silk Fibroin/ Poly(D,L-lactide) Composite Fiber Shaobing Zhou,*,† Hongsen Peng,† Xiongjun Yu,† Xiaotong Zheng,† Wenguo Cui,† Zairong Zhang,‡ Xiaohong Li,† Jianxin Wang,† Jie Weng,† Wenxiang Jia,‡ and Fei Li§ Key Laboratory of AdVanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong UniVersity, Chengdu 610031, People’s Republic of China, Department of Microbiology, Huaxi Basic Medicine and Forensic College, Sichuan UniVersity, Chengdu 610041, People’s Republic of China, and Nan Fang Spiders Breeding Research Institute, Nanning 530012, Guangxi, People’s Republic of China ReceiVed: January 29, 2008; ReVised Manuscript ReceiVed: June 7, 2008

In the paper, we successfully prepared spider silk fibroins (Ss)/poly(D,L-lactide) (PDLLA) composite fibrous nonwoven mats for the first time to the best of our knowledge. The morphology of the fibers was observed by a scanning electron microscope (SEM) and transmission electron microscope (TEM). The secondary structure change of the spidroin before and after electrospinning was characterized using Fourier transform infrared spectroscopy (FT-IR). Herein, a qualitative analysis of the conformational changes of the silk protein was performed by analyzing the FT-IR second-derivative spectra, from which quantitative information was obtained via the deconvolution of the amide I band. A mechanical test was carried out to investigate the tensile strength and the elongation at break. A water contact angle (CA) measurement was also performed to characterize surface properties of the fibers. The cytotoxicity of electrospun PDLLA and Ss-PDLLA nonwoven fibrous mats was evaluated based on a CCL 81(Vero) cells proliferation study. The results showed that the hydrophilic and mechanical property of the composite fiber were improved by introducing spidroin. 1. Introduction Over the past 2 decades, the field of tissue engineering has pursued a variety of materials and manufacturing processes to develop the engineering matrix/scaffold.1 Efforts to generate matrix construction have applied a variety of materials, including collagen, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), polycaprolactone, and other biocompatible polymers.2 In particular, PLA is biocompatible and undergoes scission in the body to lactic acids, which is induced by microbial fermentation of biomass. These characteristics make PLA [including PLLA and PDLLA (poly(D,L-lactide))] suitable for many biomedical applications.3–5 However, because it is strongly lipophilic and still lacks mechanical integrity, PLA often induces an inflammatory response.1 So recently, the research on the modification of PLA has attracted significant attention, but only limited success was reached.4,6 Spider silk has outstanding mechanical properties, such as high breaking energy and exceptional toughness, which makes it attractive for medical, military, and industrial applications.7,8 Most researchers have focused on studying spider silk genetics, the natural structure and function of silk protein, and artificial silk protein.9–13 However, the purification and preparation of silk protein for fiber spinning were very difficult due to its insolubility and unique properties, which limited its application disappointingly. But it was reported recently that some groups have obtained spin silk protein fibers from hexafluoro-2-propanol or from dilute protein solutions in concentrated formic acid.14,15 And recent successes in the identification, artificial synthesis, * Corresponding author. Tel.: 86-28-87634023. Fax: 86-28-87634649. E-mail: [email protected], [email protected]. † Southwest Jiaotong University. ‡ Sichuan University. § Nan Fang Spiders Breeding Research Institute.

the molecular structure, and expression of genes coding for spider silk protein have spurred interest in regenerating and processing fibers from dissolved natural spider silk.16–21 In this paper, we prepared electrospun fibers blending spider silk fibroins (Ss) with biodegradable PDLLA polymer in order to obtain a better biodegradable material by combining their respective advantages. Electrospinning is a fabrication technique to produce fibers of various materials with a diameter in range from nanometer to micrometer. In this study, to improve the mechanical properties and surface characteristics of pure PDLLA fibers, PDLLA solution in acetone was blended with different weight ratios of Ss solution in formic acid. By electrospinning, the mixed emulsion was deposited as a nonwoven fibrous mat on an aluminum foil. The fibers were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), mechanical testing, the contact angle (CA) measurement, and cytotoxicity evaluation. The secondary structural changes of spider silk before and after electrospinning were investigated in detail with Fourier transform infrared spectroscopy (FT-IR). Also the effect of the concentration of protein in the PDLLA polymer on the properties of fibers was studied. 2. Experimental Section 2.1. Materials. Poly(D,L-lactide) (MW 232 kDa) was synthesized by ring-opening polymerization of D,L-lactide monomer.22 The molecular weight and its distribution were determined by gel permeation chromatography (GPC, Waters 2695 and 2414, Milford, MA). Spider silk was produced from Agelena labyrinthica, which was purified five times in distilled water at room temperature.18 CCL 81(Vero) cells were provided by the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. All other chemical reagents were of reagent grade and used as received.

10.1021/jp800913k CCC: $40.75  2008 American Chemical Society Published on Web 08/19/2008

11210 J. Phys. Chem. B, Vol. 112, No. 36, 2008 SCHEME 1: Procedure for the Preparation of the Electrospun Solutions and Nonwoven Fiber Mat

2.2. Preparation of the Electrospun Solutions. The Ss were purified by heating in formic acid at 70 °C for 7 days. The insoluble fibers were separated by centrifuging (4000 rpm). After allowing evaporation of most of the formic acid at 60 °C, an 11% (w/w) solution of the silk fibroins was prepared. PDLLA was dissolved in acetone at room temperature. Then, an emulsion could be obtained by mixing polymer solution with the silk fibroins solution at different weight ratios for electrospinning. The process can be shown as the following Scheme 1. 2.3. Electrospinning. First, the mixed emulsion of PDLLA and Ss was added via a 5 mL syringe attached to a circularshaped metal capillary. The circular orifice of the capillary has an inner diameter of 0.6 mm. An oblong counter electrode is located about 15 cm from the capillary tip. The flow rate of the solution was controlled within 3.6-5.4 mL/h by a precision pump (Zhejiang University Medical Instrument Co., Hangzhou, China) to maintain a steady flow from the capillary outlet. The applied voltage was controlled within the range of 15-30 kV. Second, the pure PDLLA fiber and the synthetic fabric with diameter in the range of 200 nm and 1.2 µm was obtained with Ss weight ratios (Ss vs the fiber) of 5.0%, 10%, 15%, 20%, 25%, and 30%. Finally, the nonwoven fiber mats were dried under vacuum at room temperature for 3 days to completely remove solvent residue and stored at 4 °C. 2.4. Characterization of the Fibers. An SEM (FEI, Quanta 200, Philips, Netherlands) was used to record electron micrographs of the electrospun membranes to measure fiber diameter and investigate fiber morphology. The imaging condition was 20 kV, and the samples were imaged as-electrospun. Micrographs from the SEM analysis were digitized and analyzed with Image Tool 2.0 to determine the average fiber diameter of the mats produced. The samples for the TEM (Hitachi H-700H) observation were prepared by directly depositing the as-spun fibers onto the copper mesh. It was operated at 150 keV. The IR spectra were obtained with a Nicolet Magna 550 spectrometer equipped with a DTGS KBr detector. For dry samples, 0.8 mg of sample was mixed with 110-120 mg of KBr and annealed into disks. This process has previously been shown not to alter the IR spectra of dried proteins.23 The determination of the secondary structure of proteins was done with a method which combined the second derivative, the deconvolution, and band fitting. In the present work, a qualitative analysis of the conformation changes of the silk protein was

Zhou et al. performed by analyzing the FT-IR second-derivative spectra, whereas quantitative information was obtained from the deconvolution of the amide I band following a previously reported procedure.23–25 The data and positions of the peaks were taken from the derivative and the deconvoluted spectra. The compositions of the secondary structure could be obtained by fitting the deconvoluted spectrum with Gaussian curves. The area of the different fitting peaks can be associated with different types of the secondary structure. The nonwoven fibrous mats were subjected to stress-strain analysis using a universal testing machine Instron 5567, Instron Co., Massachusetts. For this mechanical testing, the samples were trimmed into a dog-bone shape specimen with the dimension of 75 mm × 4 mm and the gauge length of 25 mm. The mean thickness of each sample was 0.06 ( 0.01 mm, and the distance between the gripping point is 40 mm. The stress-strain analysis was conducted with the grips moving at a ratio of 10 mm/min. The data acquisition ratio was set to 20.0 Hz. The reported data of tensile strength and elongation represent the average results of five tests. The contact angle was measured using a sessile drop method at room temperature with the contact angle equipment (DSA 100, KRUSS, Germany). CA values of the right side and the left side of the distilled water droplet are both measured, and an average value is used. The contact angle was determined at 10 s after the droplet contacted on the surface of samples. All the CA data were an average of five measurements on different locations of the surface. The cytotoxicity of electrospun PDLLA and Ss-PDLLA nonwoven fiber mats was evaluated based on a CCL 81(Vero) cell proliferation study. The CCL 81 cells were grown in RPMI medium 1640 (Gibcos) with 10% fetal bovine serum (FBS). The cells were cultured in tissue culture flasks in the above medium and maintained at 37 °C in a humidified incubator with 5% CO2 and 95% air. After culturing for 2 days, the CCL 81 cells were removed from the culture flasks using trypsin (Sigma). Prior to cell seeding, the samples were minced into circular pieces of diameter 4 mm and sterilized by dipping in 75% ethanol for 15 min, followed by washing in phosphate-buffered saline (PBS) for five times. Then, the CCL 81 cells were seeded at a density of 1 × 103 cells/mL and 200 µL/well on the electrospun Ss-PDLLA nanofibrous mats in 96-well flatbottomed microassay plates (FMPS, Falcon Co., Becton Dickenson, Franklin Lakes, NJ). Nanofibrous scaffolds of PDLLA and FMPS substrate were used as controls. The cell proliferation was monitored for 1, 2, 3, and 4 days (n ) 6 for each time point per group). In order to monitor proliferations of cells in the well with different substrates, the attached well area of cells was determined by using an inverted microscope (DMIL, Leica, Germany). The cell viability (%) was calculated according to the following equation:

cell viability (%) ) OCA(sample)/WA × 100 where OCA(sample) represents the optical cell area of the wells treated with various nanofibrous mats and FMPS substrate only, and WA represents the well area. 3. Results and Discussion 3.1. Morphology of the Fibers. In the study, PDLLA concentration was found to be the most significant factor controlling the fiber diameter in the electrospun process. The PDLLA/acetone concentration (w/v) of 7.5% was selected as the optimized one. Figure 1 (i) shows the morphology of fibers obtained from pure PDLLA polymer. The PDLLA fibers with

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Figure 1. SEM micrograph of fibers electrospun from (i) PDLLA/acetone concentration of 7.5%: (a) 1000×, (b) 10 000×, and (ii) PDLLA solution/ formic acid with ratios (w/w) of (c) 5/5, (d) 6/4, and (e) 7/3, and (iii) the Ss/PDLLA emulsion with Ss ratios of (f) 5%, (g) 10%, (h) 15%, (i) 20%, (j) 25%, and (k) 30%.

TABLE 1: Diameter of the PDLLA and Ss-PDLLA Fibers with Different Ss Ratios fiber diameter (nm) samples

mean ( SD

max

min

PDLLA 5% 10% 15% 20% 25% 30%

236 ( 54 455 ( 115 411 ( 164 575 ( 217 231 ( 57 216 ( 54 494 ( 168

341 573 794 1050 364 302 893

162 142 125 237 161 89 217

average size of 236 nm were formed with random orientation and without beads, and they were uniform and not branched off. The mat produced was approximately 100 µm thick, which can be changed by simply adjusting the polymer concentration or by altering the electrospinning time. In the following stage,

the mixed emulsion consisting of PDLLA in acetone and Ss in formic acid would be used to electrospin. Taking this into consideration, our strategy had been to first investigate the effect of volume of formic acid on the morphology of fibers. The proportions (w/w) between PDLLA solution and formic acid at 5/5, 6/4, and 7/3 were investigated for electrospinning. Their SEM images are displayed in Figure 1 (i). When the amount of formic acid reached half in total volume, the fibers were formed with many beads (drops of polymer over the woven mat) as seen from Figure 1 (ii)c. With the decrease of the amount of formic acid, the amount of the beads was also decreased. And when the ratio of PDLLA solution to formic acid (w/w) arrived at 7/3, the beads were much fewer than those in other ratios. So on the basis of the above results, the PDLLA concentration and its ratio to formic acid could be fixed on in the following process. Figure 1 (iii) shows the morphology of fibers with Ss

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Figure 2. TEM images of the as-electrospun PDLLA (a) and Ss-PDLLA fibers with Ss ratios of (b) 5%, (c) 10%, (d) 15%, (e) 20%, (f) 25%, and (g) 30%.

ratios at 5%, 10%, 15%, 20%, 25%, and 30%. As seen from these images, the Ss ratio had a great effect on the diameter of fibers. When the ratios are lower than 15%, the electrospun fibers looked slightly uniform and were branched off. However, the average diameter of fibers with Ss ratios of 20% and 25% was uniform and was not branched off. The results showed that the more Ss in PDLLA solution, the better the electrospun fibers. Interestingly, the morphology of fibers containing larger amount of Ss looks much better than that of the fibers displayed in Figure 1 (ii). The reason may be that Ss with high concentration could be of great benefit to form fibers. Due to the limited solubility of Ss in formic acid at 25 °C, the highest ratio of Ss could reach only 30% in our experiment. However, even in this level, the average diameter of fibers was also not uniform and much larger than those in other ratios as shown in Table 1. To gain further information on the interior of the composite fibers, TEM recording had been carried out. From Figure 2, it could be seen that the electrospun PDLLA fibers had a smooth surface and a homogeneous structure, whereas the Ss-PDLLA fibers changed from the granular appearance to regular continuous fibrillar structure when Ss ratios increased from 5% to 30%. When the Ss ratio was lower than 15%, we could clearly see many irregular granules in the PDLLA fiber matrix, which consisted of spidroin. It suggested that the spidroin could not be electrospun to form fibers due to its low concentration. It seemed that the fiber with 15% ratio of Ss was the inflection between two extremes. Some cases

of irregular fibrillar structure began to appear within the fibers with lower Ss and on their surface, whereas beyond the 15% ratio, there was regular continuous fibrillar structure in the fiber matrix. The regular continuous fibrillar structure was also called a core-shell structure, in which the core was spidroin and the shell was PDLLA polymer. The reason to form sharp boundaries is probably associated with the immiscibility between the two components,26,27 since spidroin was hydrophilic and PDLLA was hydrophobic. The evaporating rate of acetone is faster than that of formic acid, which resulted in spidroin forming a core. Herein, apart from those core-shell fibers and others with the typical granular appearance, TEM images of the electrospun Ss-PDLLA fibers also showed that some fibers did not exhibit any core in the fiber and particle on its surface. 3.2. FT-IR Analysis. The amide group of proteins and polypeptides presents characteristic vibrational modes (amide modes) which are sensitive to the protein conformation. The amide I (1700-1600 cm-1 region) is primarily due to the CdO stretching vibration, amide II (1600-1480 cm-1 region) is due to the coupling of the N-H in-plane bending and C-N stretching modes, and amide III (1350-1190 cm-1 region) is due to the C-N stretching coupled to the in-plane N-H bending mode. Furthermore, vibrations of certain amino acid side chains have absorption bands within 1480-1350 and 1190-700 cm-1 regions and may give small contributions to the intensity of characteristic protein amide bands.28 Therefore, in order to study

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Figure 3. Original absorption spectra (left, note: (I) original spectra, (II) deconvoluted spectra, and (III) second-derivative spectra) and normalized spectra (right) of fibers in the range of 1700-1600 cm-1 compared with the spectra reconstructed by deconvolution method to produce the best fit; the individual Gaussian components are also shown: (a and b) original spider silk, (c and d) regenerated spider silk films, (e and f) 15%, (g and h) 20%, (i and j) 25%, and (k and l) 30%.

the change of the fibroins conformation, as-electrospun composite fibers with Ss ratios from 5% to 30%, original spidroin, and regenerated spidroin were all measured with FT-IR. In particular, the regenerated spidroin was gained by vaporizing the formic acid completely from the silk fibroins solution at room temperature. By applying the deconvolution procedure described in the literature25,26,28 to analyze the amide I region of protein from the FT-IR spectra, the spectral half-height bandwidth of the single Gaussian components (expressed in cm-1) and the percentage of each amide I component were obtained. Figure 3

shows comparison of FT-IR absorption spectra in the amide I region of all samples with the reconstructed spectra. Amide I bands centered between 1660 and 1652 cm-1 are generally considered to be characteristic of R-helical structures or other helical structures; for example, the polyglycine II 31-helix has an amide I absorption peak in the range of 1658-1663 cm-1, according to the structure for the glycine-rich region.28 Polypeptide fragments in a nonordered (random) conformation are usually associated with a broad infrared band at 1644-1648 cm-1. Infrared bands between 1610 and 1641 cm-1 are usually assigned to β-sheet.28,29 Theoretical calculations for β-turns also

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TABLE 2: Secondary Structure Analysis of Silk Proteins wavenumbers (cm-1) assignment original Ss regenerated Ss 15% β-sheets

random R-helix 31-helix turns

1610.6 1618.1 1625.4 1630.5 1637.9

1611.0 1618.3 1625.2 1633.8 1640.6

1647.6 1655.2 1662.1 1668.1 1674.11682.2 1690.0

1645.2 1652.4 1659.9 1667.9 1674.51682.1 1690.6

(vV) β-sheets 1697.2

1698.5

20%

25%

30%

1610.2 1610.1 1610.6 1610.4 1617.7 1618.1 1618.1 1617.6 1625.5 1629.1 1637.6 1647.1 1654.9 1662.5 1670.0 1676.8

1625.7 1629.1 1638.9 1647.1 1655.0 1662.2 1668.5 1675.1

1626.0 1631.0 1640.4 1647.9 1655.3 1662.6 1668.4 1673.3

1625.1 1636.1 1640.2 1647.0 1654.3 1661.5 1668.9 1675.8

1684.5 1682.0 1681.3 1683.3 1690.5 1689.7 1688.5 1689.3 1697.3 1694.5 1695.3

TABLE 3: Proportions of the Secondary Structures in the Silk Proteins secondary structure proportions (%) protein

β-sheets

random

R/31-helix

turns

original Ss regenerated Ss film 15% 20% 25% 30%

39 60 40 33 46 40

14 1 17 16 10 15

22 18 32 37 27 29

25 21 11 14 17 16

TABLE 4: Analysis of Water Contact Angle samples

water contact angle/deg ( SD

PDLLA film PDLLA fibrous mat 5% 10% 15% 20% 25% 30%

68.4 ( 1.5 137.4 ( 1.3 135.9 ( 1.5 131.4 ( 1.6 60.4 ( 1.6 0 0 0

predict an infrared-active mode between 1668 and 1691 cm-1; particularly, the simultaneous presence of the components at 1695 ( 4 cm-1 is typical of antiparallel (vV) β-sheets.28,29 The assignment of the negative peaks displayed in the secondderivative spectra, corresponding to the frequencies of amide I components obtained after deconvolution (Table 2), lays a foundation to correlate the percentage values of the amide I components recognized and the amounts of different secondary structures in the protein (Table 3). Quantitative analyses reveal that the secondary structure of regenerated spidroin contains 18% helix, 60% β-sheet (including antiparallel), 21% turns, and 1% random structures. In comparison to the secondary structure of original spidroin, the β-sheet structure is increased by 21% with concomitant decrease in the helical structure by 4%, in β-turn by 4%, and in random structures by 13%. Thus, it is clear that the regeneracy of spidroin should be accompanied by increasing of helical to the β-sheet structures, as reported previously,20 where the solvent evaporation normally resulted in β-sheet structure. By comparing the spectra of regenerated spidroin and the electrospun Ss-PDLLA fibrous mats with different weight ratios of Ss, significant differences in the secondary structure of the protein (Table 3) could be found out. The spectra of the electrospun Ss-PDLLA fibrous mats showed a higher percentage of helix (32%, 37%, 27%, and 29%) and random (17%,

Figure 4. Strain-stress curves for electrospun PDLLA and Ss-PDLLA fiber mats with Ss ratio of (a) 5%, (b) 10%, (c) 15%, (d) 20%, (e) 25%, and (f) 30%.

16%, 10%, and 15%) but a smaller percentage of β-sheet (40%, 33%, 46%, and 40%) and turns (11%, 14%, 17%, and 16%). The results suggest that three-dimensional folding and supersecondary structures in the two samples are different. Therefore, the spectral changes observed after electrospinning are probably a result of the electrospinning process, as has been described previously where the electric field applied may inhibit β-sheet formation and produce a protein fiber that adopts a predominantly helical conformation.20 The appearance of a predominantly helical conformation in the electrospun membrane could result from the elongational strain applied during the processing. However, in comparison with the secondary structure of original Ss, the β-sheet almost kept stable after electrospinning, whereas the helix increased obviously due to the turns turning into it. 3.3. Stress-Strain Measurements. It is well-known that spider silk has outstanding mechanical properties, such as high breaking energy and exceptional toughness. In order to investigate its effect on mechanical properties of Ss-PDLLA, the mechanical test was employed. Figure 4 displays the strain-stress curves for electrospun PDLLA and Ss-PDLLA fiber mats. As seen from the Figure 4, the mechanical properties of Ss-PDLLA had a great increase compared with those of PDLLA fiber. The tensile strength and the elongation at break of PDLLA fiber were 1.8 ( 0.3 MPa and 20.0% ( 4.8%, respectively, whereas the tensile strength and the elongation at break of Ss-PDLLA fiber with Ss ratio of 30% increased to 3.7 ( 0.4 MPa and 61.9% ( 6.2%, respectively. By increasing Ss ratio, the mechanical properties increased, which showed that the mechanical properties of PDLLA were improved by blending with Ss. Consensus ensemble repeat motifs of these proteins comprise a number of segments and contain polyalanine regions linked by glycine-rich segments. Glycine-rich regions are thought to contribute to the elasticity of the silk fiber, whereas polyalanine regions adopt a β-sheet-like conformation and are believed to endow the silks with strength and stiffness.30 Comparative FTIR studies have indicated that the electrospun Ss-PDLLA fibrous mats appear to have a same proportion of β-conformations (mean, 40%) with original Ss but more helix (mean, 31%), which can also explain why the mechanical properties of Ss-PDLLA fibers could be improved after electrospinning by introducing Ss into the polymer matrix. 3.4. Water Contact Angle Analysis. Although both surface energy and surface roughness are the dominant factors for wettability of materials, surface roughness is the key factor once the components of materials have been selected. To clarify the effect of the electrospinning process and Ss concentration on

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J. Phys. Chem. B, Vol. 112, No. 36, 2008 11215 4. Conclusion

Figure 5. Comparison of cell proliferation by culturing CCL 81 on the PDLLA electrospun fibrous mat and on the controls of FMPS substrate and the electrospun Ss-PDLLA fibrous mats with Ss ratios of 5%, 10%, 15%, 20%, 25%, and 30%.

the surface properties of electrospun fibers, water contact angles of electrospun fibers and polymer film gained by the polymer solution-cast method were measured and are summarized in Table 4. On one side, a more hydrophobic surface was observed for the PDLLA fiber mat than that of its films. The reason may be attributed to many pores among fibers, which bring a high water repellent property. On the other side, with the increase of Ss ratio in electrospun Ss-PDLLA fibers from 5% to 30%, CA values of the surfaces of the electrospun Ss-PDLLA fibrous mats decreased from 135.9° ( 1.5° to 0°, as shown in Table 4, which meant that the superhydrophobic property of PDLLA nanofibers could be turned into the superhydrophilic by the introducing of Ss. 3.5. Cytotoxicity Analysis. Although the electrospun SsPDLLA fibrous mats improved the hydrophilic ability and mechanical properties of the PDLLA, an adverse effect is the likely existence of the formic acid in the electrospun Ss-PDLLA fibrous mats, which is cytotoxic to cellular growth during in vitro or in vivo experiments. Moreover, the spider silk protein after such treatment could also bring some cytotoxicity. So it is very necessary to evaluate the cytotoxicity of electrospun Ss-PDLLA nonwoven fiber mats based on a CCL 81(Vero) proliferation study. Figure 5 shows that the OCA(sample) of the wells treated with both the Ss-PDLLA fibrous scaffolds and other control substrates continually increased from 1 to 4 day in a similar rate, which indicated that the cell took semblable expansions on the Ss-PDLLA fibrous mats to those on other control substrates. However, the slight cytotoxicity possibly from spider silk proteins could be observed by the fact that the cell expansions on the Ss-PDLLA fibrous scaffolds were inferior to control FMPS, though senior to the PDLLA fibrous scaffold. For a better cell proliferation, it is consequently suggested that the PDLLA should be compounded with spider silk protein of less cytotoxicity. Alternatively, we propose that electrospinning a complex of Ss and other biodegradable polymers in lacking of cell affinity should be appropriate in developing Ss-based composite nanofibrous scaffolds for favorable cell-scaffold interactions.

In the paper, the preparation and characterization of biodegradable composite fibers consisting of Ss and PDLLA were investigated in detail. The result from SEM images showed that a better morphology of the fiber could be obtained by increasing the Ss ratio into polymer matrix. The quantitative analyses of the secondary structure of spidroin indicated the increase of the helix structure and the retention of the β-sheet after electrospinning and showed how mechanical properties of Ss-PDLLA fibers could be improved by increasing the Ss ratio. It might be the reason that polyalanine regions adopt a β-sheet-like conformation and endow the silks with strength and stiffness. The results of water contact angle testing displayed that the superhydrophobic property of PDLLA fibers could be turned into the superhydrophilic property by introducing Ss. Very interestingly, the composite fibers showed excellent biocompatibility according to the results from the evaluation of cytotoxicity. Therefore, the electrospun nonwoven fabrics would find wide applications as scaffolds for tissue engineering, tissue repair substitutes, wound dressing materials, and carriers for drug delivery due to their high hydrophilicity, specific surface area, and porous structure. Acknowledgment. This work was supported by Project 50773065 supported by the National Natural Science Foundation of China and Programs for New Century Excellent Talents in University NCET-07-0719 and Sichuan Youth Science and Technology Foundation (08ZQ026-040). References and Notes (1) Wnek, G. E.; Carr, M. E.; Simpson, D. G.; Bowlin, G. L. Nano Lett. 2003, 3, 213. (2) Gruber, H. E.; Ingram, J. A.; Leslie, K.; Norton, H. J.; Hanley, E. N. Biotechnol. Histochem. 2003, 78, 109. (3) Liu, X. B.; Zou, Y. B.; Li, W. T.; Cao, G. P.; Chen, W. J. Polym. Degrad. Stab. 2006, 91, 3259. (4) Gao, Y.; Kong, L. J.; Zhang, L.; Gong, Y. D.; Chen, G. Q.; Zhao, N. M.; Zhang, X. F. Eur. Polym. J. 2006, 42, 764. (5) Cui, W. G.; Li, X. H.; Zhu, X. L.; Yu, G.; Zhou, S. B.; Weng, J. Biomacromolecules 2006, 7, 1623. (6) Gao, C. L.; Xia, Y. Z.; Ji, Q.; Kong, Q. S.; Li, Q. Y. Mater. ReV. 2006, 20, 372. (7) Vollrath, F.; Knight, D. Nature 2001, 410, 541. (8) Dai, L. R.; Zhang, Y.; Ou-Yang, Z. C. Thin Solid Films 2003, 382, 438–439. (9) Kaplan, D.L.; Adams, W.W.; Farmer, B.; Viney, C. ACS Symp. Ser. 1994, 544, 2. (10) Guerette, P. A.; Ginzinger, D. G.; Weber, B. H.; Gosline, J. M. Science 1996, 272, 112. (11) Cappello, J. The biological production of protein polymers and their use. Trends Biotechnol. 1990, 8, 3091. (12) Xu, M.; Lewis, R. V. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 7120. (13) Gosline, J. M.; Denny, M. W.; DeMont, M. E. Nature 1984, 309, 551. (14) Fahnestock, S. R. International Patent Application No. WO 94/ 29450, 1994. (15) Lewis, R. V.; Hinman, M.; Kothakota, S.; Fournier, M. Protein Expression Purif. 1996, 7, 400. (16) Ko, F. K.; Jovicic, J. Biomacromolecules 2004, 5, 780. (17) Seidel, A.; Liivak, O.; Calve, S.; Adaska, J.; Ji, G. D.; Yang, Z. T.; Grubb, D.; Zax, D. B.; Jelinski, L. W. Macromolecules 2000, 33, 775. (18) Gellynck, K.; Verdonk, P.; Almqvist, K. F.; Van Nimmenl, E.; Gheysens, T.; Mertens, J.; Van Langenhove, L.; Kiekens, P.; Verbruggen, A. Eur. Cells Mater. 2005, 10, 45. (19) Trancik, J. E.; Czernuszka, J. T.; Cockayne, D. J.; Viney, C. Polymer 2005, 46, 5225. (20) Stephens, J. S.; Fahnestock, S. R.; Farmer, R. S.; Kiick, K. L.; Chase, D. B.; Rabolt, J. F. Biomacromolecules 2005, 6, 1405. (21) Putthanarat, S.; Tapadia, P.; Zarkoob, S.; Miller, L. D.; Eby, R. K.; Adams, W. W. Polymer 2004, 45, 1933. (22) Deng, X. M.; Zhou, S. B.; Li, X. H.; Yuan, M. L.; Zhao, J. J. Controlled Release 2001, 71, 165. (23) Yang, T. H.; Dong, A.; Meye, J.; Johnson, O. L.; Cleland, J. L.; Carpenter, J. F. J. Pharm. Sci. 1999, 88, 161.

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