Biospinning by silkworms: Silk fiber matrices for tissue engineering applications

Biospinning by silkworms: Silk fiber matrices for tissue engineering applications

Acta Biomaterialia 6 (2010) 360–371 Contents lists available at ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabioma...

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Acta Biomaterialia 6 (2010) 360–371

Contents lists available at ScienceDirect

Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat

Biospinning by silkworms: Silk fiber matrices for tissue engineering applications Biman B. Mandal, Subhas C. Kundu * Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, India

a r t i c l e

i n f o

Article history: Received 25 November 2008 Received in revised form 24 August 2009 Accepted 25 August 2009 Available online 27 August 2009 Keywords: Silk Biospinning Biocompatibility Scaffolds Cell culture

a b s t r a c t The mechanism of biospinning of natural silk fibers has been an open issue for decades. In this report a natural bio-polymeric matrix based on biospun silk fibers obtained from Antheraea mylitta, a wild nonmulberry tropical tasar silkworm, is put forward for potential applications. This report deals with the conformational transitions of silk fibroin during the biospinning process and its potential to support cell adherence and proliferation. The silk fibers obtained were aligned into linear, mixed or random patterns forming interconnected, macroporous three-dimensional matrices. The matrices were morphologically and functionally characterized with respect to fiber diameter, crystallinity, mechanical strength and biocompatibility using feline fibroblast cells. Drawn silk fibers showed enhanced stability to protease treatment in comparison with naturally occurring native gland fibroin protein. A viability assay suggested biocompatibility of these matrices in vitro. Fluorescence and confocal microscopy indicated normal cell attachment, spreading and proliferation on these biospun silk matrices. The results provided evidence for the use of biospun silk matrices as natural, inexpensive and alternative substrata for tissue engineering applications. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Tissue engineering techniques are being constantly explored to replace diseased and damaged tissues and organs. In this connection, reconstructed bio-engineered scaffolds have gained prime importance of late in the field of tissue engineering, to be used as grafts, matrices for immobilization and drug delivery vehicles [1–3]. Scaffolds are being specifically designed and modified for applications intended to support cell adhesion, migration and proliferation by mimicking the extracellular matrix under in vivo conditions [4,5]. Of primary need in most in vivo applications scaffolds must satisfy structural parameters such as easy fabrication, biodegradability, biocompatibility, mechanical strength and interconnectivity, depending on the application [1,6,7]. A wide range of materials, both biologically derived and synthetically prepared, have been tested for three-dimensional (3D) scaffold fabrication [3,6,8–11]. However, there is a constant search for materials (both synthetic and natural) that satisfy these parameters and fulfill the needs of tissue engineering. Compared with solid 3D matrices, microporous fibrous matrices with a high surface to volume ratio are always desirable for cell adherence and proliferation [12]. Electrospinning is one such attractive fiber fabrication technique to produce fibers of the de-

* Corresponding author. Address: Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal 721302, India. Tel.: +91 3222 283764; fax: +91 3222 278433. E-mail address: [email protected] (S.C. Kundu).

sired diameter for tissue engineering applications, such as producing fibrous matrices, but this requires instrumental set-up [13,14]. In comparison, silkworms may not produce fibers of varied diameter but at the same time do not require any costly apparatus set-up for silk fiber biofabrication. This highly specialized natural fiber spinning process by silkworms is appropriately termed ‘‘biospinning” and the silk fibers obtained through this natural process as ‘‘bio-engineered biospun silk”. It is expected that highly porous fibrous constructs fabricated utilizing biospinning will facilitate not only easy passage of migrating cells but also transport of nutrients and toxic waste to and from the proliferating cells. Silk is a natural polymer that has been used as sutures for decades [7,15]. The reported low inflammatory response to silk fibroin protein both in vivo and in vitro makes it an ideal choice for matrix fabrication of materials to be used for biomedical applications [16,17]. However, little is known about the wild non-mulberry silk spun by Antheraea mylitta, a member of the Saturniidae family, compared with its counterpart, the extensively studied silk of the mulberry silkmoth Bombyx mori, of the Bombycidae family [1,3,6,7]. This led us to explore the possibilities of producing non-mulberry silk as a natural biomaterial. The wild saturniid silkworm A. mylitta is a holometabolous insect that completes its life cycle in four stages, namely egg, larva, pupa and adult [6,18–20]. The silkworms pass through five larval intermoults (stages) called instars. The final (fourth) moult produces a matured fifth instar larva, which pupates a by spinning a cocoon of silk thread, a ‘‘bio-engineered fiber”. The silk produced in epithelial cells is stored in the lumen of specialized silk glands

1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.08.035

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of mature fifth instars before they commence biospinning. Several methods of artificially spinning regenerated silk proteins into silklike fibers have been tried. It has been reported that artificial silk fibers have inferior mechanical properties compared with naturally spun silk, giving an edge to this natural spinning method over others [21–23]. The non-mulberry silkworms comprise the tropical tasar (A. mylitta), the temperate oak tasar (Antheraea pernyi), the muga (Antheraea assamensis) and the eri (Philosamia ricini) [19,20,24,25]. A. mylitta produces the largest known cocoons, which have peduncles/stalks, spun before cocoon formation for the purpose of attachment to twigs. These peduncles are the source of high molecular weight sericin, which has antioxidant and anti-apoptotic potential [26–28]. A fibrous protein (fibroin) core and a glue protein (sericin) surrounding the fiber makes it very strong [26,27]. The fibroin protein of the tasar silkworm is a homodimer, each monomer of which has a molecular weight of 197 kDa [18,29]. Previous reports on silk have emphasized regenerating silk fibroin protein solutions from both cocoons and the silk glands of B. mori and A. mylitta using various protocols for its dissolution [29–31]. In this study we report, for the first time, the use of biospun silk fibroin protein fibers directly drawn from silkworms as a natural matrix, without undergoing the process of fibroin dissolution and regeneration. Usage of biospun silk fibers circumvents the use of organic and inorganic chaotropic salts needed during the regeneration process. Silk fibroin protein fibers obtained from silkworms, both naturally and drawn manually, were effectively transformed into 3D functional scaffolds for cell culture. This study is an attempt to characterize these natural matrices for future potential applications in tissue engineering and related biomedical applications.

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for 30 min to remove sericin from the core fibroin protein fibers [27]. 3D biospun silk fiber matrices were fabricated by placing layers of mats on top of one another to form a porous scaffold measuring 20  25 mm, similar to those of the linear and random samples in terms of dimensions (Fig. 2f). 2.3. Fourier transform infrared (FTIR) spectroscopy The infrared spectrum of A. mylitta gland fibroin protein was analyzed using a FTIR spectrophotometer (Nicolet NEXUS-870 FTIR) equipped with a TE-cooled DTGS detector with a KBr window. The spectra had a resolution of 4 cm1. The equipment was purged with dry air before data collection to eliminate errors due to water vapour absorption. Background measurements were recorded and subtracted from the sample readings. 2.4. X-ray diffraction X-ray diffraction was used to determine the degree of crystallinity of A. mylitta silk fibroin fiber samples. X-ray scans of the samples were performed with a Philips PW 1710 diffractometer. All scans extended from 10° to 50° in 2h at a speed of 3.0° min1. Ni-filtered CuKa was the X-ray source. 2.5. Scanning electron microscopy (SEM)

2. Materials and methods

Fiber diameter, surface morphology and interconnectivity of matrices were examined using a JEOL JSM-5800 scanning electron microscope at an operating voltage of 20 keV. The specimens were sputter coated with gold before analysis. The pore sizes were determined by measuring 50 random pores from SEM images using Corel software. Biospun matrices seeded with cells were first fixed in formaldehyde, followed by dehydration in graded alcohol. Critical point drying was performed followed by gold sputter coating.

2.1. Materials

2.6. In vitro enzymatic degradation

Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum, trypsin and penicillin–streptomycin antibiotic mixture (Gibco BRL), protease XIV from Streptomyces griseus and thiazoyl blue (Sigma), calcein-AM (Fluka, Switzerland), rhodamine–phalloidin and Hoechst 33342 (Invitrogen), Live Dead Kit (Molecular Probes), Alamar assay kit (AbD Serotec), tissue culture grade polystyrene plastic flasks and plates (Tarsons) and other chemicals (Sigma or Aldrich) were used for the present experiments. All chemicals were used without further purification.

Enzymatic degradation of biospun silk matrices and gland fibroin protein was evaluated in vitro using protease XIV from S. griseus with an activity of 4.5 U mg1. Biospun silk fiber matrices of 5  5  3 mm and fabricated silk gland fibroin protein films (prepared by spreading fibroin protein evenly on Teflon-coated plates followed by drying) measuring 15 mm in diameter were immersed in 2 ml of phosphate-buffered saline (pH 7.4) (PBS) containing 2 U protease enzyme and incubated at 37 °C to obtain uniform results. Every 3 days this solution was replaced with freshly prepared solution. Samples immersed in PBS without enzyme under similar conditions were used as a control. Weight loss was calculated using the formula:

2.2. Fabrication of linear, mixed and random aligned biospun matrices Fifth instar larvae of A. mylitta were collected from local tasar silk farms and fed with host plant leaves (Termanalia arjuna) at our departmental farm until they began spinning fibers. Matrices (linear and mixed orientations) were fabricated on coverslip surfaces measuring 20  25 mm. The linear orientation of fibers was carried out manually on a glass coverslip (Fig. 1A). The linearly aligned fibers were unidirectional, running parallel to each other, while the mixed alignment fibers ran perpendicularly to each other. The fabricated silk fiber matrices were highly porous, having an average pore size ranging between 50 and 200 lm, determined using Corel software. The randomly aligned fibers were obtained using a different strategy. The silkworms were placed on Teflon-coated glass plates. The silk fibers (matrices), spun layer by layer on these plates, were later peeled off the glass surface (Fig. 1B). The naturally fabricated biospun matrices were degummed by boiling with 0.02 M Na2CO3

S ¼ ½ðW 0  W 1 Þ=W 0   100 where S is percentage solubility, W0 is the initial weight of the sample (mg) and W1 is the final weight of the sample (mg). The experiment was repeated three times and the average value was obtained. 2.7. Tensile properties Tensile strength was measured using a Housfield-H25KS universal testing machine equipped with a 0.1 kN load cell at ambient room temperature. Five individually drawn silk fibers were wound together to form a single set for testing. Fibers measuring 0.01 cm in diameter and 10 mm in gauge length were tested at 25 °C and 70% relative humidity. The samples fiber sets were degummed by boiling for 30 min in 0.02 M Na2CO3 to remove sericin and were

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Fig. 1. Procedures followed for the fabrication of biospun matrices from silk fibroin protein obtained from fully mature fifth instar A. mylitta silkworms. (A) Images showing fabrication of linear and mixed alignment fiber matrices. Scanning electron micrographs showing fiber alignment and cell growth on degummed (without sericin) aligned silk fibers (day 1). (B) Representative images showing fabrication of randomly aligned biospun matrices. (a) Fully mature fifth instar silkworm; (b) biospinning of silk fibers by a silkworm using its spinneret; (c) biospinning of randomly aligned fibers on Teflon-coated glass plates; (d) spun fiber matrices on a flat surface; (e) AH 927 fibroblast cells seeded on fiber matrices showing profuse growth (day 5); (f) micrographs of degummed matrices showing the porous structure.

examined at a crosshead speed of 2 mm min1. The tensile strength was determined for n = 3 sets of fibers. 2.8. Culture of a feline fibroblast cell line for biocompatibility assessment AH 927 cells are adherent feline fibroblasts that require a substratum to adhere to and proliferate. The cells were cultured in

DMEM medium supplemented with 10% fetal bovine serum and 1% UI ml1 penicillin–streptomycin in tissue culture flasks at 37 °C with 5% CO2. The culture medium was changed twice a week. Subculturing was done on a weekly basis by detaching cells from the flasks using 0.05% trypsin–0.1% EDTA solution followed by splitting at a 1:4 ratio. Before each experiment Trypan blue staining was used to check cell viability. Cells with >95% viability were used for further experiments.

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Fig. 2. Fiber morphology and cell culture on A. mylitta silk protein fibers. (a and b) Silk fibers as observed on the cocoon surface; (c and d) biospun silk fibers drawn directly from silkworms. Schematic representation of the cell culture system specifically devised for culture on randomly aligned biospun silk fiber mats. (e) Cells were seeded on top of the matrices hanging between coverslip clamps submerged in medium. (f) Schematic representation of biospun scaffold fabrication from naturally spun silk fibers.

2.9. Cell proliferation and mitochondrial activity assessment Biospun silk fibroin 3D matrices (linear, mixed and random aligned) were degummed by boiling for 30 min in 0.02 M Na2CO3 solution to remove sericin. This was followed by repeated washing with sterilized PBS and drying. The samples were presterilized by steam autoclaving before cell culturing. Matrices were conditioned with DMEM for 4 h before cell seeding and kept in culture dishes.

The cells were seeded directly on top of the fiber meshes in the case of linear and mixed alignment fibers. A special cell culturing procedure was devised for seeding cells on randomly aligned fiber scaffolds. Glass slides with coverslips at the side were stacked together to support the matrix on top without touching the base (Fig. 2e). Cells seeded on the top of the matrix were allowed to adhere and proliferate. The entire set-up was kept in a sterile culture dish with medium. Equal numbers of cells (104) were seeded on

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each matrix and polystyrene culture plate controls. In brief, cells were suspended in 50 ll medium and seeded on presterilized biospun matrices for efficient seeding without cell spillage. The coverslips having linear and mixed fibers were previously blocked with 0.5 wt.% bovine serum albumin to prevent cell attachment to the glass slides. After 4 h of initial cell attachment to the fiber matrix surface the matrices were transferred to fresh culture plates containing medium. The medium was replaced every other day. The cultures were incubated for 1, 3, 5 or 7 days in a humidified atmosphere containing 5% CO2 at 37 °C. Mitochondrial activity assessment was carried out using the MTT assay. For the MTT substrate (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrasodium bromide) to be reduced by living cells to a dark blue formazan requires active mitochondria. This provides an accurate measurement of mitochondrial activity of cells in culture. An aliquot of 100 ll of MTT (5 mg ml1) diluted 1:10 in PBS was added to each well, followed by incubation for 4 h at 37 °C. At the end of the assay the blue formazan product was dissolved in 1 ml of dimethyl sulfoxide. Absorbance was measured at 595 nm in a Bio-Rad microplate reader. Cell proliferation on biospun matrices was monitored using the Alamar blue dye reduction assay (AbD Serotec) at specified intervals of 1, 3, 5 and 7 days. Cell proliferation was plotted in terms of number of cells using a standard graph of known cell number under similar incubation conditions. Absorbance was measured at 570 and 600 nm using a multiplate reader and proliferation calculated according to the manufacturer’s protocol. 2.10. Viability assay on biospun matrices Cell viability was assessed using a Live/Dead viability/cytotoxicity kit (Molecular Probes) using the manufacturer’s protocol. Equal numbers of fibroblast cells (105) were seeded on each matrix and the polystyrene culture plate controls using a similar technique to that mentioned earlier. The cultures were incubated for 1, 3, 5 or 7 days in a humidified atmosphere containing 5% CO2 at 37 °C. On the requisite days the cells were trypsinized and isolated from the matrices. The cell suspensions were centrifuged at 1000 rpm and resuspended in 1 ml of fresh medium. To this was added 2 ll of 50 lM calcein-AM solution and 4 ll of 2 mM ethidium homodimer I per milliliter of cells. After incubation for 30 min in the dark the samples were analyzed by flow cytometry (FACS Calibur B–D using Cell Quest Pro software) with 488 nm excitation, measuring green fluorescence emission for calcein (live cells) and red fluorescence emission for ethidium homodimer I (dead cells). Cells were gated (to exclude debris) and the voltage and compensation adjusted before final images were taken. 2.11. Confocal microscopy Attachment of AH 927 fibroblast cells to biospun silk fibroin constructs was revealed using confocal microscopy. The silk matrices were seeded with fibroblast cells (104) and cultured for 3, 5 or 7 days in complete medium. After harvesting the matrices were washed three times with PBS (pH 7.4), followed by incubation in 3.7% formaldehyde in PBS for 10 min. The samples were further washed with PBS and preincubated with 1% BSA for 30 min. The constructs were then permeabilized using 0.1% Triton X-100 for 5 min. Incubation with rhodamine–phalloidin for 20 min at room temperature, followed by washing with PBS and staining with 5 lg ml1 Hoechst 33342 for 30 min. Fluorescence images from stained constructs were obtained using a confocal laser scanning microscope (Olympus FV 1000 attached to a IX 81 inverted microscope) equipped with argon (488 nm) and helium–neon (534 nm) lasers. Two-dimensional multichannel image processing was carried out using FV 1000 Advance software v. 4.1 (Olympus).

2.12. Fluorescence microscopy Attachment and viability of seeded cells on biospun silk fibroin matrices was verified using fluorescence microscopy. After an incubation period of 3, 5 or 7 days cells seeded on biospun silk fiber nets (linear, mixed and random aligned) were placed into a medium containing 0.1 lM calcein acetoxymethyl ester (calcein-AM) and incubated for 10–20 min at 37 °C. Calcein-AM becomes fluorescent when absorbed by viable cells. The stained matrices were then placed on a microscope slide and examined by fluorescence microscopy (Leica TCS NT). Image acquisition and processing was done using Qwin software. Figures show one representative sample per group. 2.13. Statistical analysis All quantitative experiments were run in triplicate and the results are expressed as means ± standard deviation for n = 3. The Alamar test and MTT test results are expressed as means ± standard deviation for n = 6. Statistical analysis of the data was performed by one-way analysis of variance (ANOVA). Differences between groups at P 6 0.05 were considered statistically significant and at P < 0.01 highly significant. 3. Results and discussion 3.1. Biospun silk fibroin matrices: natural spinning versus forced drawing Insects, such as silkworm larvae, and spiders produce fine silk fibers naturally. The exact mechanism of fiber spinning by silkworms still remains a mystery. The silk protein fibroin exists as a concentrated solution in a random coil conformation in the silk gland [29,32]. During the highly specialized process of biospinning the silkworm extrudes the fluid through a spinneret (a video clip of the A. mylitta silk biospinning process is provided as a supplement). The silk fibers are drawn by pulling the spinneret away from the point of attachment. The coiled fibroin protein is converted into crystalline b-sheets on exposure to air. During the process the extruded fibers are coated with a layer of the silk protein sericin, which acts as a glue. Most researchers agree that the natural biospinning procedure involves the transition of fibroin from a random coiled or helical structure to b-sheet under shear and/or rapid elongational flow. Other factors like K+ ion concentration are also known to affect this process [32,33]. In B. mori heavy chain fibroin is largely composed of repeats of the motif (Gly-Ala-Gly-Ala-Gly-Ser)n [34]. Heavy chain fibroin is thought to be present as a silk I-like conformation within the lumen of the posterior and middle divisions of the gland, lacking ordered secondary structure [35,36]. The amino acid composition of A. mylitta fibroin indicates that it is rich in glycine and alanine as the major amino acids [18]. During natural spinning the silk I-like conformation is thought to be converted into silk II, which is predominantly formed of crystalline b-sheets [37,38]. These b-sheets are orientated along the axis of the fibers, giving it strength and stiffness comparable with or superior to that of high performance synthetic materials [39,40]. The silk fibers comprise a fibrous protein (fibroin) core with a glue protein (sericin) surrounding it [26,27]. A silk fiber is composed of two individual fibroin filaments bonded by sericin. Differences in fibroin fiber diameter have been observed by SEM during forced fiber drawing. When silk threads are manually drawn by hand the diameter ranges between 25 and 30 lm compared with naturally spun cocoon fibers, which are in the range 65–70 lm (Fig. 2b and d). The diameter of individual biospun fibroin fibers re-

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duced from 30–35 to 12–15 lm when drawn manually, in comparison with cocoon fibers. The external force exerted during manual drawing of fibers may be one of the factors responsible for the decrease in fiber diameter. It may be possible to fabricate thinner fibers than those we report by manipulating the drawing force using a motorized set-up at a specific speed. This method could be of immense potential for the fabrication of fibers for specific biomedical applications requiring lesser diameter fibers of high surface to volume ratio. A change in terms of shape of the fiber was also observed through SEM observations. Manually drawn fibers were observed to be round in shape due to the external stretching force compared with cocoon fibers, which have a flat, ribbon-like appearance (Fig. 2a–d). 3D matrices of biospun fibers showed high porosity with a wide range of pore sizes due to the randomness of the process. Pore sizes in the 3D matrices ranging from 100 to 500 lm were observed by SEM (Fig. 1). The obvious question of feasibility of the process still remains. In particular, manual alignment to obtain linear and mixed aligned matrices, while time consuming, is possible (a similar problem has been reported for electrospinning to obtain aligned fibers). We noticed that silkworms may fabricate large random biospun matrices up to a size of 14  10 cm (depending on available surface area and time allowed for spinning) within 3–4 h, which would allow the fabrication of 4–5 scaffolds (Fig. 1d). To scale up the process a greater number of silkworms may be individually placed on different coated plates at the same time to obtain a sufficient number of randomly oriented fiber matrices. Furthermore, one can acquire a sufficient number of mature fifth instar larvae (one female moth usually lays 130 eggs) and a crop can be obtained three times a year, as this silkworm has a trivoltine life cycle. 3.2. Mechanical strength of biospun silk fibers Antheraea mylitta degummed silk fibers showed remarkable mechanical strength. Five individual fibers taken together as a single interwoven set gave an average value of 467 ± 160 MPa for each individual fiber, comparable with many well-known fiber materials used today. The mechanical strength of this bio-engineered fiber (containing b-sheets) was much higher than that of fibroin films (mainly helices and coils) prepared earlier by us from A. mylitta regenerated gland fibroin [6,30]. A comparison of the mechanical properties of common silks (silkworm and spider dragline) with several types of biomaterial fibers and tissues are listed in Table 1. The mechanical strength of these fibers would presumably be higher if measured without removing the sericin glue. One report has suggested a decrease in fiber strength upon degumming due to the harsh treatment conditions [41]. Being a well-studied natural polymer, silk has an advantage over other materials where high strength is required for day to day engineering needs. 3.3. Protein conformational analysis by FTIR Conformational transitions of regenerated silk gland fibroin were investigated by FTIR spectroscopy and X-ray diffraction. Due to the presence of amide groups in the protein, the characteristic vibration bands between 1630 and 1650 cm1 for amide I (C@O stretching) and 1540 and 1520 cm1 for amide II (secondary NH bending) were observed. These vibrations have specific frequencies which relate to the b-sheet and random coil conformations in silk protein molecules. In Fig. 3c the gland silk protein fibroin shows a typical random coil conformation and a-chains with peaks at 1550 and 1650 cm1, corresponding to amide II and amide I, respectively [42]. Treatment with 70 vol.% ethanol for 30 min led to the induction of a b-sheet transition in silk fibroin with signature peaks at 1630 and 1530 cm1, both related to b-sheets, for amide I and amide II, respectively [9,16]. This confirms that a transition in the fibroin molecules

Table 1 A comparison study of mechanical properties of common silks (silkworms and dragline) to several types of biomaterial fibers and tissues. Materials

Tensile strength (MPa)

References

B. mori silk (with sericin)a B. mori silkb Spider silkc A. mylitta (fibers without sericin) PLAd Bone Tendon Kevlar

500 740 875–972 467 ± 160

Perez-Rigueiro et al. [62] Cunniff et al. [63] Cunniff et al. [63] This study

28–50 160 150 3600

Engelberg and Kohn [64] Gosline et al. [65] Gosline et al. [65] Gosline et al. [65]

a Bombyx mori silkworm silk—determined from bave (multithread fibers naturally produced from the silkworm coated with sericin). b Bombyx mori silkworm silk—average calculated from data in Ref. [63]. c Nephila clavipes silk produced naturally and through controlled silking. d Polylactic acid with molecular weights ranging from 50,000 to 300,000.

due to rearrangement of hydrogen bonds in the protein moiety led to the band shifts [43,37,38]. 3.4. Crystallinity determination by XRD X-ray diffraction was used to further confirm conformational transitions in and crystallinity of fibroin fiber matrices and protein. Diffractograms of silk fibers and gland fibroin samples are presented in Fig. 3b. Untreated fibroin protein isolated directly from silk glands showed two broad peaks at around 2h = 12° and 23°, representing the amorphous state of the protein due to abundant random coils. The h values on 70 vol.% ethanol treatment shifted to 2h = 21° and 24°, with a hump at 2h = 30.5°, suggesting induction of crystallinity as a result of its transition from random coil to b-sheet [44–46]. Similar observations were noted in the case of native non-mulberry silk fibers drawn from cocoons, which exhibited two strong peaks around 2h = 20° and 22.3° characteristic of b-sheet crystals of silk fibroin [47]. The results suggest a conformational transition of fibroin protein from an amorphous to a crystalline state, similar to naturally existing fibroin fibers. However, the b-sheet content is less compared with native silk fibers. The double peak curves observed in the case of A. mylitta fibroin reflect a-helix crystals, as previously reported in the case of A. pernyi fibroin, a member of the non-mulberry silkworm family [6,30,47]. To further elucidate conformational transitions and the degree of crystallinity of fibroin fibers of different spin lengths spun fiber matrix samples were collected at various stages of biospinning (Fig. 3a). The spun fiber matrices were arranged in the order top, middle and bottom layers for crystallinity determination. Silk fibers spun by silkworms during spinning initiation formed the top layer, while the middle and bottom layers were spun during the latter stages of spinning. Interestingly, it can be observed that all three layers had strong peaks at 2h = 17°, 20° and 22° characteristic of b-sheet crystals of silk fibroin, similar to natural reeled cocoon fibers [44,45,47]. The results suggest that silk fiber drawing is the dominant factor in crystallinity induction [48]. Also, throughout the spinning process the degree of crystallinity remained the same in all the fiber layers and throughout the fiber length. This observation gives an insight into the spinning process whereby naturally existing amorphous gland fibroin is transformed to a highly crystalline conformation during the highly specialized biospinning process [32]. 3.5. Cytocompatibility of biospun silk fiber matrices Cytocompatibility of biospun fibers in terms of cell proliferation and mitochondrial activity was analyzed using AH 927 feline fibro-

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Fig. 3. Protein conformation and degradation behavior. (a) X-ray diffractogram of A. mylitta silk fiber matrices at various stages of biospinning. (a) Bottom layer; (b) middle layer; (c) top layer. (Inset) Diffractogram of native silk fiber drawn from a cocoon. (b) X-ray diffractogram of ethanol treated and non-treated silk gland fibroin protein. (c) FTIR spectra of ethanol treated and non-treated silk gland fibroin protein films. (d) Degradation patterns of biospun silk fiber mats and fibroin protein in both enzyme and PBS. Data are plotted as means ± standard deviation.*P < 0.01, n = 3.

blast cells on fabricated 3D silk biospun matrices. An Alamar blue cell proliferation assay showed that randomly aligned matrices better supported cell growth and proliferation compared with linear and mixed fiber matrices and were comparable with control polystyrene plates (Fig. 4a). On day 7 total cells present on randomly oriented matrices (54,000 cells) exceeded the control condition (49,000 cells). It was also observed that cell proliferation on the control plates was hampered from day 5 to day 7, probably due to contact inhibition (confluence) with the formation of only 3000 new cells. In comparison, on randomly oriented fibers 9000 new cells were formed over the same time period. This was probably due to the 3D mesh-like structure of the randomly oriented fibers providing a larger surface area for cells to grow and proliferate compared with the limited surface of flat polystyrene plates. In contrast, linearly arranged and mixed fibers showed less cell proliferation compared with the controls. It can be assumed from the proliferation graphs that during the initial phase (day 1) of cell proliferation both the linear and mixed matrices showed fewer cell numbers compared with the controls and random fibers. These differences in cell number were probably due to the arrangement of the fibers within the matrices. The controls showed the highest cell number on day 1 (9200 cells) as they had a flat surface without pores and thus all the viable cells seeded onto it attached. In the

case of random fibers only 5400 cells became attached on day 1. The rest must have penetrated the macroporous matrix through its pores, into the medium. In contrast, only 1800 and 2900 cells were attached to the linear and mixed fibers on day 1. These account for their lower cell proliferation in comparison with the random and control groups. We know that an adhesive force is a must when an adherent cell encounters a surface, allowing it to attach firmly. Non-specific interactions like Van der Waal’s forces, electrostatic forces and other specific bond forces between cell adhesion molecules and their receptors play a role in cell attachment. Similar reports of fibroblastic cell culture on regenerated fibroin and non-woven nets of silk fibroin protein from cocoons of B. mori using conventional methods have already been reported [12,49]. Non-mulberry silk fibroins from A. pernyi and A. mylitta have sequences similar to RGD, which help in cell attachment [6,50,51]. However, due to large fiber diameters of 25–30 lm, which is similar in size to a single cell, the cells have to rest on the fibers to adhere to the surface and proliferate, without passing through. Thus, for cell adherence to fibrous matrices the mesh-like network is an important parameter for cell support in the initial phase of attachment, holding the cells onto it. Once cells attach to the fibers they proliferate normally, as can be seen from the Alamar assay values from day 1 to day 7 of culture. The higher cell numbers observed in

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Fig. 4. (a) Alamar blue cell proliferation assay showing fibroblast cell growth on biospun silk fibers. (b) MTT assay for mitochondrial activity of fibroblast cells on biospun silk matrices. (c) Calcein-AM stained fluorescence micrographs showing cell attachment and viability on biospun non-mulberry silkworm silk matrices. Data are plotted as means ± standard deviation. *P < 0.05, n = 6.

the mixed fiber matrices compared with the linear matrices were due to the close resemblance to random fiber mats in morphology, leading to higher cell attachment to their fibers in the initial phase. To further elucidate the mitochondrial activity of the cells growing on these matrices MTT assays were performed. MTT reagent is reduced to formazan when incubated with viable cells. Thus, the absorbance of formazan directly reflects mitochondrial activity and cellular metabolism of the attached cells on these fibroin matrices. Among the 3D constructs the matrices fabricated using random biospun fiber mats showed higher mitochondrial activity compared with the linear and mixed fibers throughout the culture period (Fig. 4b). A similar trend was observed in the case of the Alamar cell proliferation assay. Mitochondrial activity observed in the case of random fibers was comparable with cells grown on control polystyrene plates until day 5 (P < 0.05). On day 7 the cells grown on random fibers showed higher activity compared with the controls. This was due to the presence of RGD sequences and the higher surface area provided by the randomly aligned fibers, for better cell attachment and proliferation compared with control plates lacking RGD and having restricted space for cell proliferation. The highly porous structure of the biospun matrices allows cells to migrate and proliferate within the interconnected macroporous 3D matrices. The MTT findings directly reflect the Alamar blue assay results in terms of mitochondrial activity. Thus it may now be safely assumed that differences in activity on different silk matrices were due to differences in initial cell attachment and not due to other external factors. The cells proliferating on these matrices showed normal mitochondrial activity, comparable with that on polystyrene plates, and any differences were solely due to differences in

the level of initial cell attachment. From the results obtained it may be assumed that silk fiber matrices are cytocompatible and that fiber orientation in the resultant matrices to form a mesh-like network are important for cell attachment and proliferation in the initial phase. As observed by SEM, random fibers had the maximum surface area for cell adhesion and growth, compared with linear and mixed fibers. The cells were observed to form a sheath on the matrices, a sign of a high level of proliferation (Fig. 1). Further, the presence of RGD sequences within non-mulberry silk fibroin may promote faster cell attachment and spreading on the silk fibers. It is expected that RGD sequences will in turn act as chemical cues for the seeded cells, allowing migration within porous silk fiber scaffolds to facilitate functional construct fabrication for potential tissue engineering applications. It is assumed that the highly porous nature of the silk fibroin matrices will not only facilitate easy passage of nutrients to the proliferating cells but also aid in faster diffusion of waste roducts from the inner core. In our study cell adherence and proliferation in all three matrices were evaluated and cells were observed to be growing normally in comparison with the controls. Looking at practical applications, randomly oriented fibers would seem to be a better choice than the other two due to its less cumbersome method of fabrication. Large volumes of these randomly oriented meshes can be fabricated in a relatively shorter time to meet the demands of the tissue engineering industry (Fig. 1). Still, all three aligned fiber meshes could be effectively used for different cell-based needs, depending on the desired application, with directed and aligned fibers being required for directed cell growth, e.g. in the case of myofibroblast and neural cells [13,14].

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3.6. Cell viability assay To assess the biocompatibility of the matrices for use as potential biomaterials it is important to determine cell responses on them and evaluate living and dead cells. Flow cytometric analysis was done on these matrices using a Live/Dead Kit from Molecular Probes (Fig. 5). On day 1 nearly 97% of the cells were alive on the controls, and similar results were observed for the case of random and mixed oriented fibers, with 94% and 93% viable cells. Linear fiber matrices showed 89% viable cells on the initial day of cell attachment. On day 3 similar results were observed for all cases, with cell viabilities of 90%. Cell viability decreased on days 5 and 7 for the case of the linear and mixed fibers, compared with the random and control sets. This may be due to the limited porosity of these matrices and the surface area for attachment of proliferating cells. As already observed by SEM, the porosity of these two matrices was limited, with smaller pores compared with the macropores of the randomly oriented fiber matrices (Fig. 1). This might have restricted the entry of growing cells into the inner cores of the matrices. Thus, due to the lack of a surface to adhere to a greater

number of cells died at the end of days 5 and 7 on the linear and mixed matrices. In comparison, the randomly aligned fiber matrices continued to support cells and at the end of day 7 the percentage of viable cells was higher than that on the controls, with nearly 88% live cells, compared with 79% in the case of control plates. Lack of free substratum and contact inhibition due to cell confluence might have resulted in this decrease in cell viability in the case of control plates at the end of day 7. The results further support the earlier findings of the Alamar and MTT assays, which showed that random matrices supported better cell attachment and viability. The results were comparable, and better than the controls on day 7. This result further emphasizes that these matrices are best suited for cell culture applications as they allow nutrients and growing cells to pass through and reach the innermost core for growth and proliferation.

3.7. Confocal and fluorescence microscopy Acetoxymethyl (AM) ester derivatives of calcein are used to study live cells. Non-fluorescent cells permeable to derivatives

Fig. 5. Flow cytometric data showing cell viability on biospun A. mylitta silk matrices and a polystyrene plate as a control. The viable cells were stained with calcein-AM (green) and dead cells with ethidium homodimer I (red). The percentage of viable and dead cells are shown in the inset in each representative figure (L, live; D, dead). (For interpretation of colour mentioned in this figure, the reader is referred to the web version of this article.)

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of calcein becomes fluorescent on hydrolysis. AH 927 fibroblast cells seeded on fibroin fiber matrices showed intense fluorescence on incubation with calcein, suggesting their viability on these silk fibers [12,52]. Cells were firmly attached to the silk fibers of all matrices, i.e. linear, mixed and randomly aligned fibers, as observed by fluorescence microscopy (Fig. 4c). Initially, cell attachment was lower to the linearly aligned fibers compared with the mixed and randomly aligned fibers. This was assumed to be due to the linear arrangement of fibers. The fluorescence observations complemented the results obtained earlier from the Alamar and MTT tests. Compared with day 3 profuse cell growth with cells attached to all three different matrices was observed on days 5 and 7. Confocal studies of cells seeded on biospun fibroin matrices complemented earlier SEM and fluorescence studies. Cells were observed to attach firmly on linearly, mixed and randomly arranged fibers, suggesting normal attachment, growth and proliferation. Distinct rounded nuclei were observed throughout the fiber matrices with well spread out actin filaments, suggesting biocompatibility of the fibroin fiber matrices (Fig. 6) [6]. 3.8. Response of biospun silk fibers to enzymatic degradation The degradation rate of a matrix is an important parameter for a biomaterial designed to be used for tissue engineering applications. On incubation with proteases biospun A. mylitta 3D silk fibroin matrices and two-dimensional (2D) gland protein fibroin

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films show controlled degradation patterns over a period of 28 days (Fig. 3d). The fibroin films were fabricated by air drying extracted gland fibroin protein, whereas biospun silk fiber matrices were naturally spun by silkworms, with a highly random orientation. The biospun fiber matrices kept in PBS devoid of enzymes showed little or no degradation after 28 days incubation [6]. On incubation with protease (2 U) silk fibers showed a slow pattern of degradation. The remaining mass was 84, 70 and 46 wt.% after 7, 14, 21 and 28 days incubation, respectively (P < 0.01). In comparison, fabricated gland fibroin films showed a higher degradation rate, with remaining masses of 38 and 6 wt.% in PBS after 7 and 14 days incubation, respectively (P < 0.01). The films have been completely dissolved by day 18 of incubation in PBS. Enhanced degradation was observed for 2D fibroin films on incubation with enzymes, with 10 wt.% mass remaining after 7 days incubation (P < 0.01). In the presence of enzymes total degradation was achieved within 8 days of incubation. The enhanced degradation rate of gland fibroin 2D films compared with biospun silk fibers was due to the amorphous nature of the gland fibroin protein, as revealed by FTIR spectroscopy and XRD. Random coil and a-helical structures accelerate the process of degradation in both PBS and enzyme solutions in comparison with that of biospun fibroin fibers, formed of b-sheets [6,32,42]. The crystalline structure of silk fiber threads, due to a conformational transition to b-sheet during biospinning, resulted in enhanced stability in both PBS and enzyme solution [32,48]. The results suggest that functional matrices can be fabricated from these silk fibroin fibers, which allow cells to grow and

Fig. 6. Confocal micrographs showing cell attachment on biospun silk matrices made using silk from A. mylitta non-mulberry silkworms. Cells were grown for 5 days on random, mixed and linearly aligned fibers. A single biospun fibroin thread (showing two distinct fibroin fibers bound to each other) with attached cells. Samples were stained with rhodamine–phalloidin (red) for actin filaments and Hoechst 33342 (blue) for nuclei. (For interpretation of colour mentioned in this figure, the reader is referred to the web version of this article.)

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proliferate while undergoing slow degradation at the implanted/ grafted site with ingress of new cells. 3.9. Potential uses and future scope of biospun silk matrices In recent years immense stress has been laid on the production and use of fibrous materials for biomedical applications. Of all the technologies developed for the production of fibrous matrices the most acceptable one is electrospinning. In contrast, in this study we have looked into and discussed in detail a natural biospinning process for the efficient production of silk fibers in the micron range. These fibers exhibited tremendous potential due to their unique characteristics that closely resemble manmade electrospun fibers. The low inflammatory response they invoked makes them a promising candidate for future biomedical applications [17]. Their importance is further evident as many researchers have reported that nanometer scale surface features and topography play an important role in regulating cell behavior in terms of cell adhesion, activation, proliferation, alignment and orientation [53,54]. That polymer nanofibers already exist and are being considered for use as scaffolds to engineer tissues such as cartilage, bones, arterial blood vessels, heart tissue, nerves and vascular grafts further add to their advantages [5,55,56]. Silk fiber matrices may provide an alternative route, taking advantage of their macroporous structure to promote osteogenesis as in earlier reports, in which researchers successfully engineered bone cells on 3D fibroin structures [57,58]. Silk nets can be used to grow mesenchymal stem cells for regeneration of cartilage, tendons and ligaments [1,2,59,60]. Successful delivery of protein-based drugs and growth factors can be achieved by both physical adsorption and chemical bonding, due to the high surface area to volume ratio of these matrices [57,61]. Biospun fibers may be effectively used for biosensor development. A larger surface area will improve the sensitivity of conductometric sensors. The present and earlier studies have clearly suggested the potential of naturally biospun matrices for various biomedical applications in the future.

4. Conclusions This preliminary study has demonstrated the potential of tropical non-mulberry tasar silkworm fibroin protein-based biospun fibers as 3D matrices for cell growth and proliferation in vitro. Our method provides a new and alternative route using silk protein fibers directly from silkworms produced through the natural spinning process without any chemical processing. Slow degradability, high porosity, high tensile strength and easy biofabrication with high cell attachment and viability indicate immense potential as a natural bio-polymeric material. This study is a step towards the versatile usage of biospun silk fibroin protein fibers for potential future biomedical and tissue engineering applications. Acknowledgements We are grateful to Dr. Laura A. Poole-Warren and Dr. Penny J. Martins, University of New South Wales, Sydney, Australia, for their critical reading and meaningful suggestions on the manuscript. The Department of Biotechnology and its bioinformatics facility (BT/PR6035/MED/14/733/2005), the Indo-Australia Biotechnology Fund (BT/PR9552/ICD/16/755/2006), the Council of Scientific Industrial Research (a fellowship to B.B.M.), the Government of India and the Indo-US Science and Technology Forum, New Delhi, are greatly acknowledged for funding our project.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2009.08.035.

Appendix B. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1–6, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi: 10.1016/j.actbio.2009.08.035.

References [1] Wang Y, Kim HJ, Vunjak-Novakovic G, Kaplan DL. Stem cell-based tissue engineering with silk biomaterials. Biomaterials 2006;27:6064–82. [2] Wang Y, Blasioli DJ, Kim HJ, Kim HS, Kaplan DL. Cartilage tissue engineering with silk scaffolds and human articular chondrocytes. Biomaterials 2006;27:4434–42. [3] Vepari C, Kaplan DL. Silk as a biomaterial. Prog Pol Sci 2007;32:991–1007. [4] Lutolf MP, Weber FE, Schmoekel HG, Schense JC, Kohler T, Muller R, et al. Repair of bone defects using synthetic mimetics of collagenous extracellular matrices. Nat Biotechnol 2003;21:513–8. [5] Stitzel JD, Pawlowski K, Wnek GE, Simpson DG, Bowlin GL. Arterial smooth muscle cell proliferation on a novel biomimicking vascular graft scaffold. J Biomater Appl 2001;16:22–33. [6] Mandal BB, Kundu SC. Non-bioengineered silk fibroin protein 3-D scaffolds for potential biotechnological and tissue engineering applications. Macromol Biosci 2008;8:807–18. [7] Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen J, et al. Silk-based biomaterials. Biomaterials 2003;24:401–16. [8] Mauney JR, Nguyen T, Gillen K, Kirker-Head C, Gimble JM, Kaplan DL. Engineering adipose-like tissue in vitro and in vivo utilizing human bone marrow and adipose-derived mesenchymal stem cells with silk fibroin 3-D scaffolds. Biomaterials 2007;28:5280–90. [9] Kim UJ, Park J, Kim HJ, Wada M, Kaplan DL. Three-dimensional aqueousderived biomaterial scaffolds from silk fibroin. Biomaterials 2005;26:2775–85. [10] Adelow C, Segura T, Hubbell JA, Frey P. The effect of enzymatically degradable poly(ethylene glycol) hydrogels on smooth muscle cell phenotype. Biomaterials 2008;29:314–26. [11] Lutolf MP, Hubbell JA. Synthesis and physicochemical characterization of endlinked poly(ethylene glycol)-co-peptide hydrogels formed by Michael-type addition. Biomacromolecules 2003;4:713–22. [12] Unger RE, Wolf M, Peters K, Motta A, Migliaresi C, Kirkpatrick CJ. Growth of human cells on a non-woven silk fibroin net: a potential for use in tissue engineering. Biomaterials 2004;25:1069–75. [13] He W, Yong T, Ma ZW, Inai R, Teo WE, Ramakrishna S. Biodegradable polymer nanofiber mesh to maintain functions of endothelial cells. Tissue Eng 2006;12:2457–66. [14] Yang F, Murugan R, Wang S, Ramakrishna S. Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering. Biomaterials 2005;26:2603–10. [15] Moy RL, Lee A, Zalka A. Commonly used suture materials in skin surgery. Am Fam Physician 1991;44:2123–8. [16] Meinel L, Hofmann S, Karageorgiou V, Kirker-Head C, McCool J, Gronowicz G, et al. The inflammatory responses to silk films in vitro and in vivo. Biomaterials 2005;26:147–55. [17] Acharya C, Ghosh SK, Kundu SC. Silk fibroin protein from mulberry and nonmulberry silkworms: cytotoxicity, biocompatibility and kinetics of L929 murine fibroblast adhesion. J Mater Sci Mater Med 2008;19:2827–36. [18] Datta A, Ghosh AK, Kundu SC. Purification and characterization of fibroin from the tropical saturniid silkworm, Antheraea mylitta. Insect Biochem Mol Biol 2001;31:1013–8. [19] Mahendran B, Acharya C, Dash R, Ghosh SK, Kundu SC. Repetitive DNA in tropical tasar silkworm Antheraea mylitta. Gene 2006;370:51–7. [20] Mahendran B, Ghosh SK, Kundu SC. Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA and cytochrome oxidase subunit I genes. J Genet 2006;85:31–8. [21] Liivak O, Blye A, Shah N, Jelinski LW. A microfabricated wet-spinning apparatus to spin fibers of silk proteins. Structure–property correlations. Macromolecules 1998;31:2947–51. [22] Seidel A, Liivak O, Calve S, Adaska J, Ji GD, Yang ZT, et al. Regenerated spider silk: processing, properties, and structure. Macromolecules 2000;33:775–80. [23] Shao ZZ, Vollrath F, Yang Y, Thgersen HC. Structure and behaviour of regenerated spider silk. Macromolecules 2003;36:1157–61. [24] Saha M, Kundu SC. Molecular identification of tropical tasar silkworm (Antheraea mylitta) ecoraces with RAPD and SCAR merkers. Biochem Genet 2006;44:72–85. [25] Mahendran B, Ghosh SK, Kundu SC. Molecular phylogeny of silk producing insects based on internal transcribed spacer DNA1. J Biochem Mol Biol 2006;39:522–9.

B.B. Mandal, S.C. Kundu / Acta Biomaterialia 6 (2010) 360–371 [26] Dash R, Mukherjee S, Kundu SC. Isolation, purification and characterization of silk protein sericin from cocoon peduncles of tropical tasar silkworm, Antheraea mylitta. Int J Biol Macromol 2006;38:255–8. [27] Dash R, Ghosh SK, Kaplan DL, Kundu SC. Purification and biochemical characterization of a 70 kDa sericin from tropical tasar silkworm, Antheraea mylitta. Comp Biochem Physiol B: Biochem Mol Biol 2007;147:129–34. [28] Dash R, Acharya C, Bindu PC, Kundu SC. Antioxidant potential of silk protein sericin against hydrogen peroxide-induced oxidative stress in skin fibroblasts. Biochem Mol Biol Rep 2008;41:236–41. [29] Mandal BB, Kundu SC. A novel method for dissolution and stabilization of nonmulberry silk gland protein fibroin using anionic surfactant sodium dodecyl sulfate. Biotechnol Bioeng 2008;99:1482–9. [30] Mandal BB, Kundu SC. Non-bioengineered silk gland fibroin protein: characterization and evaluation of matrices for potential tissue engineering applications. Biotechnol Bioeng 2008;100:1237–50. [31] Tsukada M, Freddi G, Gotoh Y, Kasai N. Physical and chemical properties of tussah silk fibroin films. J Polym Sci B: Polym Phys 1994;32:1407–12. [32] Calvert P. Silk and sequence. Nature 1998;393:309–10. [33] Magoshi J, Magoshi Y, Nakamura S. Mechanism of fiber formation of silkworm. ACS Symp Ser 1994;544:292–310. [34] Zhou CZ, Confalonieri F, Medina N, Zivanovic Y, Esnault C, Yang T, et al. Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acids Res 2000;28:2413–9. [35] Asakura T, Suzuki H, Watanabe Y. Conformational characterization of silk fibroin in intact Bombyx mori and Philosamia cynthia ricini silkworms by 13C NMR spectroscopy. Macromolecules 1983;16:1024–6. [36] Jones NA, Sikorski P, Atkins EET, Hill MJ. Nature and structure of once-folded nylon 6 monodisperse oligoamides in lamellar crystals. Macromolecules 2000;33:4146–54. [37] Asakura T, Kuzuhara A. Conformation characterization of Bombyx mori silk fibroin in the solid state by high-frequency 13C cross polarization–magic angle spinning NMR, X-ray diffraction, and infrared spectroscopy. Macromolecules 1984;18:1841. [38] Asakura T, Watanabe Y, Itoh T. NMR of silk fibroin. 3. Assignment of carbonyl carbon resonances and their dependence on sequence and conformation in Bombyx mori silk fibroin using selective isotopic labeling. Macromolecules 1984;17:2421–6. [39] Gosline JM, DeMont ME, Denny MW. The structure and properties of spider silk. EndeaVour 1986;10:37–43. [40] Shao ZZ, Vollrath F. The surprising strength of silkworm silk. Nature 2002;418:41. [41] Pérez-Rigueiro J, Elices M, Llorca J, Viney C. Effect of degumming on the tensile properties of silkworm (Bombyx mori) silk fiber. J Appl Polym Sci 2002;84:1431–7. [42] Miyazawa T, Blout ER. The infrared spectra of polypeptides in various conformations: amide I and II bands. J Am Chem Soc 1961;83:712–9. [43] Mingzhong L, Shenzhou L, Zhengyu W, Haojing Y, Jingyu M, Lihong W. Studies on porous silk fibroin materials. I. Fine structure of freeze dried silk fibroin. J Appl Polym Sci 1999;79:2185. [44] Li M, Tao W, Lu S, Kuga S. Compliant film of regenerated Antheraea pernyi silk fibroin by chemical crosslinking. Int J Biol Macromol 2003;32:159–63. [45] Li M, Tao W, Kuga S, Nishiyama Y. Controlling molecular conformation of regenerated wild silk fibroin by aqueous ethanol treatment. Polym Adv Technol 2003;14:694–8. [46] Kweon H, Um IC, Park YH. Thermal behavior of regenerated Antheraea pernyi silk fibroin film treated with aqueous methanol. Polymer 2000;41:7361–7.

371

[47] Tao W, Li M, Zhao C. Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution. Int J Biol Macromol 2007;40: 472–8. [48] Trabbic KA, Yager P. Comparative structural characterization of naturally- and synthetically-spun fibers of Bombyx mori fibroin. Macromolecules 1998;31:462–71. [49] Tamada Y. New process to form a silk fibroin porous 3-D structure. Biomacromolecules 2005;6:3100–6. [50] Datta A, Ghosh AK, Kundu SC. Differential expression of the fibroin gene in developmental stages of silkworm, Antheraea mylitta (Saturniidae). Comp Biochem Physiol B 2001;129:197–204. [51] Minoura N, Aiba AI, Higuchi M, Gotoh Y, Tsukada M, Imai Y. Attachment and growth of fibroblast cells on silk fibroin. Biochem Biophys Res Commun 1995;208:511–6. [52] Bondar B, Fuchs S, Motta A, MigliaresI C, Kirkpatrick CJ. Functionality of endothelial cells on silk fibroin nets: comparative study of micro- and nanometric fibre size. Biomaterials 2008;29:561–72. [53] Flemming RG, Murphy CJ, Abrams GA, Goodman SL, Nealey PF. Effects of synthetic micro- and nano-structured surfaces on cell behavior. Biomaterials 1999;20:573–88. [54] Craighead HG, James CD, Turner AMP. Chemical and topographical patterning for directed cell attachment. Curr Opin Solid State Mater Sci 2001;5:177–84. [55] Mo XM, Xu CY, Kotaki M, Ramakrishna S. Electrospun P(LLA-CL) nanofiber: a biomimetic extracellular matrix for smooth muscle cell and endothelial cell proliferation. Biomaterials 2004;25:1883–90. [56] Silva GA, Czeisler C, Niece KL, Beniash E, Harrington DA, Kessler JA, et al. Selective differentiation of neural progenitor cells by high-epitope density nanofibers. Science 2004;303:1352–5. [57] Meinel L, Hofmann S, Oliver B, Fajardo R, Merkle HP, Langer R, et al. Osteogenesis by human mesenchymal stem cells cultured on silk biomaterials: comparison of adenovirus mediated gene transfer and protein delivery of BMP-2. Biomaterials 2006;27:4993–5002. [58] Kim KH, Jeong L, Park HN, Shin SY, Park WH, Lee SC, et al. Biological efficacy of silk fibroin nanofiber membranes for guided bone regeneration. J Biotechnol 2005;120:327–39. [59] Altman GH, Horan RL, Lu HH, Moreau J, Martin I, Richmond JC, et al. Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials 2002;23: 4131–41. [60] Meinel L, Hofmann S, Karageorgiou V, Zichner L, Langer R, Kaplan DL, et al. Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds. Biotechnol Bioeng 2004;88:379–91. [61] Karageorgiou V, Tomkins M, Fajardo R, Meinel L, Snyder B, Wade K, et al. Porous silk fibroin 3-D scaffolds for delivery of bone morphogenetic protein-2 in vitro and in vivo. J Biomed Mater Res 2006;78A:324–34. [62] Perez-Rigueiro J, Viney C, Llorca J, Elices M. Mechanical properties of singlebrin silkworm silk. J Appl Polym Sci 2000;75:1270–7. [63] Cunniff PM, Fossey SA, Auerbach MA, Song JW, Kaplan DL, Adams WW, et al. Mechanical and thermal properties of dragline silk from the spider, Nephila clavipes. Polym Adv Technol 1994;5:401–10. [64] Engelberg I, Kohn J. Physicomechanical properties of degradable polymers used in medical applications: a comparative study. Biomaterials 1991;12:292–304. [65] Gosline JM, Guerette PA, Ortlepp CS, Savage KN. The mechanical design of spider silks: from fibroin sequence to mechanical function. J Exp Biol 1999;202:3295–303.