IOP PUBLISHING Biomed. Mater.

2 (2007) 181188

BIOMEDICAL MATERIALS

doi:10.1088/1748-6041/2/3/003

Preparation of electrospun silk broin ber mats as bone scaffolds: a preliminary study
Chidchanok Meechaisue1, Patcharaporn Wutticharoenmongkol2, Rujira Waraput1, Thanapol Huangjing1, Nantana Ketbumrung1, Prasit Pavasant3 and Pitt Supaphol2
1 Department of Materials Technology, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand 2 Technological Center for Electrospun Fibers and The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330, Thailand 3 Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand

E-mail: pitt.s@chula.ac.th

Received 16 March 2007 Accepted for publication 4 June 2007 Published 23 August 2007 Online at stacks.iop.org/BMM/2/181 Abstract In the present contribution, electrospinning (e-spinning) was used to fabricate ultra-ne bers of silk broin (SF) from cocoons of indigenous Thai silkworms (Nang-Lai) and Chinese/Japanese hybrid silkworms (DOAE-7). The effects of solution concentration (i.e., 1040% (w/v) in 85% (v/v) formic acid) and applied electrostatic eld strength (EFS; 10, 15 and 20 kV/10 cm) on morphology and size of the electrospun (e-spun) SF products were investigated by scanning electron microscopy. The average diameter of the resulting e-spun SF bers was found to increase with an increase in both the solution concentration and the EFS value. Specically, the average diameter of the e-spun SF bers from Nang-Lai SF solutions ranged between 217 and 610 nm, while that of the bers from DOAE-7 SF solutions ranged between 183 and 810 nm. The potential for use of the e-spun SF ber mats as bone scaffolds was assessed with mouse osteoblast-like cells (MC3T3-E1) in which the cells appeared to adhere and proliferate well on their surface.

1. Introduction
Electrospinning (e-spinning) is a process by which ultrane bers with diameters in the sub-micrometer down to nanometer range can be fabricated. This process deals with the application of a high electrical potential from an emitting electrode of a high-voltage power supply to a polymer liquid (i.e., solution or melt) across a nite distance between a conductive capillary and a grounded collecting device [1, 2]. At a critical electrostatic eld strength (EFS), charges of the same polarity on the surface of a partially spherical droplet destabilize the shape of the droplet to resemble that of a cone (i.e., Taylors cone). Further increase in the EFS causes the Coulombic repulsion to exceed the surface tension, leading to an ejection of a charged stream of the polymer liquid (i.e., the
1748-6041/07/030181+08$30.00

charged jet). The charged jet travels in a straight trajectory for a certain distance before undergoing a bending instability, resulting in the formation of a looping trajectory [3]. During its ight to the collector, the charged jet thins down and, simultaneously, dries out or solidies to leave ultra-ne bers on the collector. Ultra-ne bers obtained from the e-spinning process exhibit numerous interesting characteristics, e.g., high surface area to mass or volume ratio, high density of pores in sub-micrometer or nanometer length scale of the obtained non-woven ber mat and vast possibilities for surface functionalization. These unique properties render e-spun ultra-ne bers as excellent candidates for various biomedical applications, e.g., carriers for topical or transdermal drug delivery, wound-dressing materials and scaffolding materials 181

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for tissue and cell culture [4]. The challenge in tissue engineering is the design of scaffolds that mimic the structure and biological functions of the natural extra-cellular matrix (ECM). E-spun ber mats are uniquely suitable for use as scaffolds because of their three-dimensional structure with high porosity, which is similar to brous collagen in the natural ECM. In spite of the interesting structure of the e-spun ber mats, the selection of materials for fabrication into scaffolds is also very important. The natural silk bers from domesticated silkworms, Bombyx mori, have been used as high-quality textile bers and sutures for a long time. The natural silk bers consist of two types of proteins: broin and sericin. Fibroin is the protein that forms the laments of silkworm silk, whereas sericin is a group of gummy proteins that bind the broin laments. Silk broin (SF) has been used in various forms, such as gels, powders, bers or membranes, depending on the applications. Silk bers or silk membranes have been explored as scaffolding materials in tissue engineering [513]. This is because SF exhibits several useful properties that are suitable for scaffolding applications, including good biocompatibility, biodegradability, good oxygen and water vapor permeability, and minimal inammatory reaction [14]. In recent years, many reports were focused on the e-spinning of SF [1521] and the use of the e-spun SF ber mats as scaffolds for tissue and cell culture [2224]. In the present contribution, silk bers from two types of silkworms, i.e., a Thai and a hybrid race, were used as raw materials for the preparation of SF. The SF sponges were dissolved in formic acid and then e-spun into ultra-ne ber mats. The effects of solution concentration and EFS on morphology and size of the e-spun SF bers were investigated using scanning electron microscopy (SEM). The potential for use of the e-spun SF ber mats as scaffolding materials for bone cell culture was evaluated in vitro with mouse osteoblast-like cells (MC3T3E1), in which the attachment and proliferation of the cells were analyzed.

bers were de-gummed three times with 0.5% (w/v) Na2CO3 solution at 100 C for 30 min and then rinsed with warm water. De-gummed silk was dissolved in a ternary solvent system of CaCl2/ethanol/H2O in a 1:2:8 mol ratio at 70 C. This solution was then dialyzed in water using a cellulose tubular membrane (SigmaAldrich, USA) for 3 days. The obtained SF solution was ltered and lyophilized to obtain SF sponges. The SF solutions for e-spinning were nally prepared by dissolving weighed amount of SF sponges in 85% formic acid at various concentrations ranging from 10 to 40% (w/v) with 5% (w/v) increment. Prior to e-spinning, these solutions were measured for their shear viscosity by a Brookeld DV-III programmable rheometer. 2.3. Fabrication and characterization of e-spun SF ber mats Each of the as-prepared SF spinning solutions was contained in a 5 ml glass syringe connected with a blunt gauge-26 stainlesssteel hypodermic needle, used as the nozzle. The emitting electrode of positive polarity from a Gamma High-Voltage Research D-ES30PN/M692 power supply was used to charge the spinning solution by being connected with the needle, while the grounding one was connected to a collecting device. To investigate the effects of spinning solution concentration and electrostatic eld strength on morphology and/or size of the e-spun products, a piece of aluminum sheet on a rigid plastic backing was used as the stationary collecting device. The distance between the tip of the needle and the screen collector dened a collection distance, which was xed at 10 cm, while the collection time was xed at 10 min. It should be noted that both the syringe and the needle were tilted 45 from a horizontal baseline to maintain a constant presence of a solution droplet at the tip of the needle. Morphology and size of the individual bers in the espun SF ber mats were observed by a JEOL JSM-6400 scanning electron microscope (SEM). The specimens for SEM observation were prepared by cutting an Al sheet covered with the e-spun ber mats and the cut sections were carefully afxed on copper stubs. Each specimen was gold-coated using a JEOL JFC-1100 sputtering device prior to observation under SEM. Diameters of the e-spun bers were determined from SEM images by means of SemAphore 4.0 software. To prepare SF brous scaffolds for bone cell culture, the SF solution from the cocoons of Nang-Lai silkworms at the concentration of 35% (w/v) in 85% formic acid was e-spun into ber mats under a xed EFS of 20 kV/10 cm and a xed collection time of 8 h. In this case, an aluminum sheet on a rotating cylindrical drum (OD 15 cm, rotation speed 50 rpm) was used as the collecting device. Again, both the syringe and the needle were tilted 45 from a horizontal baseline to maintain a constant presence of a solution droplet at the tip of the needle. The thickness of the e-spun ber mats was about 200 m. The e-spun ber mats were immersed in a 90/10 (v/v) methanol/water mixture for 10 min to induce a conformational change of the SF molecules from amorphous to -sheet [22] and later dried in vacuo at 35 C overnight. The necessity for inducing the amorphous to sheet conformational transition was to reduce solubility of the

2. Experimental details
2.1. Materials Cocoons from two different types of Bombyx mori silkworms were used as raw materials for the preparation of silk broin (SF). Nang-Lai is an indigenous Thai silkworm race having yellow cocoons, whereas DOAE-7 is a Chinese/Japanese hybrid silkworm race having white cocoons. It should be noted that DOAE-7 is a hybrid silkworm race developed by Department of Agricultural Extension, Ministry of Agriculture and Co-operatives, Thailand. Both of these races are well known for their high quality yarns. The chemicals used for the preparation of SF and its spinning solutions were sodium carbonate (Na2CO3), calcium chloride (CaCl2), ethanol and 85% formic acid (all reagent grade). These chemicals were purchased from Carlo Erba (Italy) and used as-received. 2.2. Preparation of SF and SF spinning solutions Both types of cocoons were rst boiled in water and then dried at 60 C for 24 h in an oven to obtain raw silk bers. These 182

Preparation of electrospun silk broin ber mats as bone scaffolds

Table 1. Shear viscosity of two different types of SF solutions at various concentrations and diameters of e-spun bers from these solutions. Solution concentration (% w/v) 10 15 20 25 30 35 40 Shear viscosity (mPa s) Nang-Lai 16 38 88 216 389 888 2144 DOAE-7 12 31 76 178 467 1233 3323 Fiber diameters (nm) Nang-Lai 217 13 223 17 250 25 495 30 610 61 DOAE-7 183 13 222 20 290 29 655 35 810 67

obtained SF brous scaffolds in the aqueous medium used during bone cell culture. 2.4. Cell culture and cell seeding Mouse osteoblast-like cells (MC3T3-E1; clonal osteogenic cell line derived from newborn C57BL/6 inbred mouse) were cultured as a monolayer in Minimum Essential Medium (with Earles Balanced Salts) (MEM; Hyclone, USA), supplemented by 10% fetal bovine serum (FBS; BIOCHROM AG, Germany), 1% l-glutamine (Invitrogen Corp., USA) and 1% antibiotic/antimycotic formulation (containing penicillin G sodium, streptomycin sulfate and amphopericin B (Invitrogen Corp., USA)). The medium was replaced once in every 3 days and the cultures were maintained at 37 C in a humidied atmosphere containing 5% CO2. Each scaffold was cut into circular disks (15 mm in diameter) and the disk specimens were placed in wells of a 24-well tissue-culture polystyrene plate (TCPS; Biokom Systems, Poland) and later sterilized in 70% ethanol for 30 min. The specimens were then washed with autoclaved de-ionized water and later immersed in MEM overnight. To ensure a complete contact between the specimens and the wells, the specimens were pressed with a metal ring (12 mm in diameter). MC3T3-E1 from the cultures were trypsinized (0.25% trypsin containing 1 mM EDTA (Invitrogen Corp., USA)), counted by a hemacytometer (Hausser Scientic, USA) and seeded at a density of about 36 000 cells cm2 on the scaffold specimens. 2.4.1. Cell attachment and cell proliferation. For attachment study, MC3T3-E1 were allowed to attach to the scaffold specimens for 2, 4 and 8 h. At each time point, the number of the attached cells was quantied by a 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; SigmaAldrich, USA) assay. Each specimen was rinsed with phosphate buffer saline (PBS; SigmaAldrich, USA) to remove unattached cells prior to the MTT assay. For the proliferation study, the cells were rst allowed to attach on the specimens for 8 h. The proliferation of cells on the specimens was determined at day 1, 3 and 5 after culture. At 8 h of the attachment period, the cells were starved with MEM supplemented by 2% FBS, 1% l-glutamine and 1% antibiotic/antimycotic formulation. The number of cells was, again, quantied by the MTT assay.

2.4.2. Quantication of viable cells. The MTT assay is based on the reduction of the yellow tetrazolium salt to purple formazan crystals by dehydrogenase enzymes secreted from the mitochondria of metabolically active cells. The amount of purple formazan crystals is proportional to the number of viable cells. First, each sample was incubated at 37 C for 1 h with 250 l/well of MTT solution at 0.5 mg ml1 without phenol red. After incubation, MTT solution was removed. A buffer solution containing dimethylsulfoxide (DMSO; Carlo Erba, Italy) (900 l/well) and glycine buffer (pH = 10) (125 l/well) was added to the wells to dissolve the formazan crystals. After 10 min of rotary agitation, the solutions were transferred into a cuvette and placed in a Thermospectronic Genesis10 UVvisible spectrophotometer, from which the absorbance at 540 nm representing the number of viable cells was measured. 2.4.3. Morphological observation of cultured cells. After removal of the culture medium, the cell-cultured scaffold specimens were rinsed with PBS twice and the cells were then xed with 3% glutaraldehyde solution (diluted from 50% glutaraldehyde solution (Electron Microscopy Science, USA) with PBS) at 500 l/well. After 30 min, they were rinsed again with PBS. After cell xation, the specimens were dehydrated in an ethanol solution of varying concentration (i.e., 30, 50, 70, 90 and 100%, respectively) for about 2 min at each concentration. The specimens were then dried in 100% hexamethyldisilazane (HMDS; Sigma, USA) for 5 min and later dried in air after removal of HMDS. Finally, the specimens were mounted on copper stubs, coated with gold and observed by SEM.

3. Results and discussion

3.1. Morphology and size of e-spun SF bers 3.1.1. Effect of SF solution concentration. Solutions of SF prepared from the cocoons of Nang-Lai and DOAE-7 silkworms in 85% (v/v) formic acid were prepared at various concentrations (i.e., 10, 15, 20, 25, 30, 35 and 40% (w/v)). These solutions were measured for their shear viscosity prior to e-spinning and the results are summarized in table 1. For each type of the SF solutions, the shear viscosity increased monotonically with the increase in the solution concentration. Specically, the SF solutions from the cocoons of Nang-Lai silkworms (hereafter, Nang-Lai SF solutions) showed the shear viscosity values in the range of 162144 mPa s, while those 183

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(a)

(e)

(b)

(f )

(c)

(g)

(d )

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Figure 1. SEM images (magnication = 3000, scale bar = 10 m) of e-spun bers from two types of SF solutions in 85% (v/v) formic acid at four different concentrations: (a, e) 10%, (b, f ) 20%, (c, g) 30% and (d, h) 40% (w/v). The two types of SF solutions were those prepared from cocoons of (ad ) Nang-Lai and (eh) DOAE-7 silkworms. The applied EFS was 20 kV/10 cm.

from the cocoons of DOAE-7 silkworms (hereafter, DOAE7 SF solutions) showed the property values in the range of 123323 mPa s. The signicant increase in the viscosity of the solutions with the increase in the solution concentration is obviously due to the increased molecular entanglements. E-spinning of these solutions was carried out at a xed EFS of 20 kV/10 cm. Figure 1 shows selected SEM images of the e-spun bers from both types of the SF solutions at various concentrations (i.e., 10, 20, 30 and 40% (w/v)). 184

Figures 1(a)(d ) show the SEM images of the bers from the Nang-Lai SF solutions, while gures 1(e)(h) show those of the bers from the DOAE-7 SF ones. Evidently, the morphology of the e-spun products at equivalent concentrations for both types of SF solutions was similar. At low concentrations (i.e., 10 and 15% (w/v)), a large number of discrete beads were observed (see, for examples, gures 1(a) and (e)). At 20 and 25% (w/v), a mixture of beaded and smooth bers was obtained (see, for example, gures 1(b)

Preparation of electrospun silk broin ber mats as bone scaffolds

(a)

(a)

(b)

( b)

Figure 2. SEM images (magnication = 3000, scale bar = 10 m) of e-spun bers from 40% (w/v) SF solution prepared from cocoons of DOAE-7 silkworms at two values of applied EFS of (a) 10 and (b) 20 kV/10 cm, respectively.

and (f )). At 30% (w/v) and greater, the beads disappeared, leaving only smooth bers on the screen collector (see, for example, gures 1(c), (d ), (g) and (h)). Since either beaded or smooth bers could only be prepared from the SF solutions with concentrations in the range of 2040%, the e-spun bers from these solutions were then measured for their diameters and the results are also summarized in table 1. The average diameter of the bers, regardless of the types of the SF solutions, increased monotonically with the increase in the solution concentration, with the average diameter of the bers from Nang-Lai SF solutions being in the range of 217610 nm, while that of the bers from DOAE-7 SF solutions being in the range of 183810 nm. The observed increase in the solution viscosity was responsible for the monotonic increase in the ber diameters [25]. 3.1.2. Effect of EFS. To investigate the effect of the applied EFS on morphology and size of the obtained e-spun SF bers, 20% and 40% (w/v) solutions of SF from the cocoons of both Nang-Lai and DOAE-7 silkworms in 85% (v/v) formic acid were prepared. The applied EFS was 10, 15 or 20 kV/10 cm. At 20% (w/v), e-spinning of both types of SF solutions produced beaded bers (results not shown). The number of beads was found to decrease, and their shape appeared to be more elongated, with the increase in the applied EFS. Figure 2 shows selected SEM images of the e-spun bers from 40% (w/v) DOAE-7 SF solution prepared at the EFS values of 10 and 20 kV/10 cm. Evidently, smooth bers were obtained from these spinning conditions and the size of these bers was observed to increase with the increase in the applied EFS. A similar behavior was also observed for the e-spun bers from

( c)

Figure 3. Low-magnication SEM images (magnication = 500, scale bar = 50 m) of MC3T3-E1 on e-spun brous scaffolds from 35% (w/v) SF solution prepared from cocoons of Nang-Lai silkworms at (a) 2 h, (b) 4 h and (c) 8 h after cell seeding.

40% (w/v) Nang-Lai SF solution. Specically, the average diameter of the e-spun bers from 40% Nang-Lai SF solution increased from about 440 nm at the EFS of 10 kV/10 cm to 610 nm at 20 kV/10 cm, while that of the e-spun bers from 40% (w/v) DOAE-7 SF solution increased from about 480 nm at the EFS of 10 kV/10 cm to 810 nm at 20 kV/10 cm. The observed increase in the average ber diameter with the increase in applied EFS was postulated to be a result of the increase in the mass throughput in response to the increase in the electrostatic force [26]. 3.2. Cell attachment and cell proliferation on SF brous scaffolds Both the attachment and the proliferation of cells on a given substrate are prerequisites for a functional scaffold. For the particular studies, the e-spun ber mats from 35% (w/v) NangLai SF solution were prepared and the potential for use of these ber mats as scaffolds for bone tissue culture was assessed using mouse osteoblast-like cells (MC3T3-E1) as the reference 185

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(a)

(a )

(b)

(b)

(c )

(c)

Figure 4. High-magnication SEM images (magnication = 3500, scale bar = 5 m) of MC3T3-E1 on e-spun brous scaffolds from 35% (w/v) SF solution prepared from cocoons of Nang-Lai silkworms at (a) 2 h, (b) 4 h and (c) 8 h after cell seeding.

Figure 5. Low-magnication SEM images (magnication = 500, scale bar = 50 m) of MC3T3-E1 on e-spun brous scaffolds from 35% (w/v) SF solution prepared from cocoons of Nang-Lai silkworms at day (a) 1, (b) 3 and (c) 5 after cell culture.

bone cells. For the attachment study, MC3T3-E1 were seeded on the scaffold specimens and were allowed to attach on the surface of these scaffolds for 2, 4 and 8 h. Quantication of the viable cells at each seeding time point was based on the UV absorbance from the MTT assay. It was found that the viability of the attached cells on the SF brous scaffolds at every time point investigated was similar. Specically, the absorbance values at 2, 4 and 8 h after cell seeding were 0.338 0.032, 0.346 0.037 and 0.334 0.028, respectively. For the proliferation study, MC3T3-E1 were rst allowed to attach to the SF brous scaffolds for 8 h. The proliferation of the cells on these scaffold specimens was then determined at day 1, 3 and 5. Apparently, the viability of the proliferated cells on the SF brous scaffolds was found to increase from that of the attachment period of 8 h to 0.391 0.038 at day 1 to 0.554 0.023 at day 3 and nally to 0.634 0.033 at day 5. However, in comparison with the viability of the cells 186

on TCPS, used as the positive control, the values observed at every time point were lower. The morphology of MC3T3-E1 that were either seeded or cultured on the surfaces of the SF brous scaffolds was further investigated by SEM. The images in two different magnications were taken, with the low-magnication images showing the behavior of cells on a large area of the scaffold surfaces, while the high-magnication ones providing a closer examination of individual cells. Figures 3 and 4 show selected low- and high-magnication SEM images (i.e., 500 and 3500, respectively) of MC3T3-E1 during the attachment period on the SF brous scaffolds at different time points. Since the size of pores on the SF brous scaffolds was much smaller than that of the cells, cells could only attach and propagate on the surface of the scaffolds. In a slight contradiction suggested by the MTT assay during the attachment period that showed similar absorbance values at all three different time points, the number of cells on the surfaces of the SF brous scaffolds was found to increase with the increase in the cell-seeding time (see gure 3). At 2 h after

Preparation of electrospun silk broin ber mats as bone scaffolds

(a)

increase in the cell-culturing time, with the cells appeared to fully cover the surface of the scaffold at day 5 after cell culture (see gure 5), and all of the cells assumed their fully expanded state. Interestingly, since it was proposed that growth factors stimulate the formation of lopodia in broblasts to direct broblast division [27, 28], the highmagnication SEM images taken at days 1 and 5 may reveal a snapshot of the role of lopodia during an on-going cell division of MC3T3-E1 as the cells tried to ll the empty surface of the SF brous scaffolds (see gures 6(a), (c)). The facts that the coverage of the cells on the SF brous scaffolds appeared in monolayer and that the cells assumed their fully expanded state with evidence of lopodia around the edge of the cells conrm the potential for use of the e-spun SF ber mats as scaffolds for bone tissue culture.

4. Conclusion
Silk broin (SF) from cocoons of indigenous Thai silkworms (Nang-Lai) and Chinese/Japanese hybrid silkworms (DOAE-7) was fabricated into ultra-ne bers by electrospinning (e-spinning). The effects of solution concentration (i.e., 1040% (w/v) in 85% (v/v) formic acid) and applied electrostatic eld strength (EFS; 10, 15 and 20 kV/10 cm) on morphology and size of the electrospun (e-spun) SF products were investigated by scanning electron microscopy (SEM). At low solution concentrations (i.e., 1025% (w/v)), e-spinning of both types of SF solutions produced either discrete beads or beaded bers, while, at high solution concentrations (i.e., 30% w/v), only smooth bers were observed. The average diameter of the e-spun bers from both types of SF solutions was found to increase monotonically with the increase in the solution concentration, with the value of the resulting e-spun SF bers from Nang-Lai SF solutions being in the range of 217610 nm, while that of the bers from DOAE-7 SF solutions being in the range of 183810 nm. For beaded bers, an increase in the EFS value caused the number of beads to decrease and the shape of beads to be more elongated, while, for smooth bers, it was responsible for the observed increase in the ber diameters. The potential for use of the e-spun SF ber mats as scaffolds for bone tissue culture was assessed with mouse osteoblast-like cells (MC3T3-E1) that were either seeded or cultured on the surface of SF ber mats prepared from 35% (w/v) Nang-Lai SF solution. It appeared that the e-spun SF ber mats could be used as scaffolding materials for bone cell culture, as the cells adhered and proliferated well on their surfaces.

(b)

(c)

Figure 6. High-magnication SEM images (magnication = 2000, scale bar = 10 m) of MC3T3-E1 on e-spun brous scaffolds from 35% (w/v) SF solution prepared from cocoons of Nang-Lai silkworms at day (a) 1, (b) 3 and (c) 5 after cell culture.

cell seeding, the majority of the cells on the surface of the scaffold were still in round shape (see gure 3(a)), but a closer examination around the edge of the cells revealed evidence of lopodia (i.e., slender cytoplasmic projections extending from the leading edge of migrating cells [27]) that helps the cells during their migration over the surface of the scaffold (see gure 4(a)). At 4 h after cell seeding, the majority of the cells started to extend their cytoplasm over the surface of the scaffold, evidence of the ability of the cells to attach on the surface (see gures 3(b) and 4(b)). At 8 h after cell seeding, expansion of the cytoplasm of the majority of the cells became more evident (see gures 3(c) and 4(c)). Figures 5 and 6 show selected low- and highmagnication SEM images (i.e., 500 and 2000, respectively) of MC3T3-E1 during the proliferation period on the SF brous scaffolds at different time points. In line with the MTT assay during the proliferation period that showed a monotonic increase in the absorbance values with increase in the time in culture, the number of cells on the surface of the SF brous scaffolds was found to increase with the

Acknowledgments
The authors acknowledge partial support received from the Ramkhamhaeng University, National Research Council of Thailand and Chulalongkorn University (through invention and research grants from the Ratchadapesek Somphot Endowment Fund). 187

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