3D electrospun silk fibroin nanofibers for fabrication of artificial skin Faheem A. Sheikh PhD, Hyung Woo Ju MS, Jung Min Lee MS, Bo Mi Moon MS, Hyun Jung Park MS, Ok Joo Lee PhD, Jung-Ho Kim MS, Dong-Kyu Kim MD, Chan Hum Park MD, PhD PII: DOI: Reference:

S1549-9634(14)00571-1 doi: 10.1016/j.nano.2014.11.007 NANO 1029

To appear in:

Nanomedicine: Nanotechnology, Biology, and Medicine

Received date: Revised date: Accepted date:

11 June 2014 29 August 2014 17 November 2014

Please cite this article as: Sheikh Faheem A., Ju Hyung Woo, Lee Jung Min, Moon Bo Mi, Park Hyun Jung, Lee Ok Joo, Kim Jung-Ho, Kim Dong-Kyu, Park Chan Hum, 3D electrospun silk fibroin nanofibers for fabrication of artificial skin, Nanomedicine: Nanotechnology, Biology, and Medicine (2014), doi: 10.1016/j.nano.2014.11.007

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3D electrospun silk fibroin nanofibers for

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fabrication of artificial skin

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Faheem A Sheikh, PhDa Hyung Woo Ju, MSa Jung Min Lee, MSa Bo Mi Moon, MSa Hyun Jung Park, MSa Ok Joo Lee, PhDa Jung-Ho Kim, MSa Dong-Kyu Kim, MDb Chan Hum Park MD,

a

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PhDa,b*

Nano-Bio Regenerative Medical Institute, College of Medicine, Hallym University, Chuncheon,

*b

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200-702, South Korea

Department of Otorhinolaryngology-Head and Neck Surgery, School of Medicine, Hallym

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University, Chuncheon, 200-704, South Korea First two authors have contributed equally in this work.

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Corresponding Author: Chan Hum Park M.D., Ph.D., E-mail : [email protected], Tel: +82-33-

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240-5262, Fax: +82-33-241-2909

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Short title: 3D nanofibers for artificial dermis The word count for abstract: 148

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A complete manuscript word count (including body text and figure legends): 5380 Number of references: 43 Total figures: 11 (+ 1 Figure in Supplementary Materials) Acknowledgments: This work was supported by the Hallym University Research Fund and grant from Bio-industry Technology Development (112007-05-3-SB010), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. Authors are thankful to Korea basic science institute (KBSI) branch at Chuncheon for providing facilities to perform VP-FE-SEM. Conflict of interest: All authors confirm that have no conflict of interest.

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ACCEPTED MANUSCRIPT Abstract Tissue-engineered skin substitutes such as nanofibers from traditional electrospinning

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may offer an effective therapeutic option for the treatment of patients suffering from skin damages such as burns and diabetic ulcers. However, it is generally difficult for cells to infiltrate

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the nanofibers due to its small pore size and sheets-like appearance. In the present study, a facile and efficient strategy have successfully been introduced that can produce 3D silk fibroin

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nanofibers, obviating an intrinsic limitation of traditional and salt-leaching electrospinning by

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introducing cold-plate electrospinning. The cell attachment and infiltration studies indicated the use of 3D nanofiber scaffolds by cold-plate electrospinning as a potential candidate to overcome

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intrinsic barriers of electrospinning techniques. The 3D nanofiber scaffolds using this technique presented a high porosity with controlled full-thickness and an easy way to deposit on facial

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curvature; the resultant nanofibers scaffolds laid best attributes is the ideal candidate for artificial

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skin reconstruction.

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Keywords: Cold-plate electrospinning; Full-thickness scaffolds; Artificial skin; 3D nanofibers

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ACCEPTED MANUSCRIPT Introduction Recently, nanofibers have emerged as multifunctional materials that are suitable for

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biomedical applications because of their superior porous, mechanical, and chemical properties.1

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The nanofibers produced for various applications are mainly produced by one of three techniques: self-assembly,2 phase separation,3-4 and electrospinning.5 Electrospinning is

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considered to be the most widely used and preferred technique for fabricating the nanofibers

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used in tissue-engineering. The fibers may be of polymer, ceramic, and/or composite in nature, and all these types have been successfully prepared via electrospinning.5-7 The electrospinning

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technique had a distinct advantage to develop as skin substitution compared with other methods. Because the structural and physical properties of electrospun nanofibers resembles with those of

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extra cellular matrix, the electrospun nanofibers from synthetic or regenerated natural polymers

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showed an increased cellular proliferation and enhanced a transport of nutrient/waste.8-9 However, because the traditional electrospinning (TE) process produces uncontrolled and

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densely packed fibers, it often results in compact 2D nanofibers in a membrane-like structure,

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which constitutes a superficial porous network of nanofibers.10 The membrane-like nanofiber sheets produced by TE are limited in the diversity of tissue reconstruction, and these limitations are difficult to overcome because of the electrostatic repulsion between incoming and alreadydeposited fibers. To address this issue, various improvisations have been proposed to increase the porosity of these sheets at a fundamental level; however, the effort to achieve full-thickness is often omitted in the literature, because it is extremely challenging. For instance, the use of air impedance on the collecting mandrel to create deep pores via air pressure can produce porous capillary tubes.11 The co-electrospinning of PCL and water-soluble poly(ethylene oxide) (PEO) and/or gelatin, in which the “sacrificial” PEO and/or gelatin was removed during post-treatment,

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ACCEPTED MANUSCRIPT has also successfully improvised the porosity, but only with an uncontrolled and irregular pore structure.12-13 Additionally, sonic waves can aid in loosening individual nanofibers, thereby

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resulting in the perforation of electrospun mats, but at the cost of destabilizing the fiber integrity,

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which can result in a loss of mechanical strength.14 The use of cryogenic electrospinning as a fabrication technique to enhance porosity, has been developed both by spinning nanofibers onto

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liquid nitrogen and by introducing ice crystals onto the rotating mandrel, resulting in the creation

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of porous nanofibers; however, these approaches suffer from low overall yields and irregular porous structures, the nanofibers deposited cannot retain any shape using mold as collector, and

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the final thickness was not discussed in these studies.15-16 Interestingly, Nam et al. claim to have produced 3D nanofiber scaffolds by introducing salt leaching electrospinning (SLE) for creating

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highly porous PCL nanofibers, in which salt crystals were then removed during post-processing

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to create void areas that allow appropriate cell infiltration.17 However, this technique produced an irregular internal pore structure, had a low thickness of nanofibers, which may be dissolved or

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experience collapse of their porous structure during the post-treatment process. Moreover, our

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previous experiences with porous PCL and water-soluble silk fibroin nanofibers created by SLE, indicate that this technique results in a reduction of the thickness and the pores created after the removal of the porogen were prone to collapse.17-19 However, a very small number of reports do exist that consider the fabrication of thick scaffolds in the form of uncontrolled cotton-like sponges. Unfortunately, most of these reports concern the use of water-insoluble polymers, e.g., poly(-caprolactone) (PCL)20-21 and polystyrene.22 However, cell-culture and in vivo tests might result in the collapse of these loosely arranged nanofibers.

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ACCEPTED MANUSCRIPT Until now, the progress of biotechnology suggests that the electrospinning of polymeric nanofibers is an interesting approach to counteract organ deficit. Especially, natural polymers

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have been known to one of the most effective biomaterials.23 Therefore, in the past several

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decades, numerous studies have been performed to develop artificial skin using natural polymers, including collagen sponge with silicone film,24 chitosan film and sponge25 and chitosan with

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gelatin.26 These natural polymers have been exploited as substitute scaffold for skin regeneration

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because of their inherent biocompatibility than those of synthetic polymers which often result in toxicity to tissues.27 In this connection, silk fibroin is considered as natural protein produced by

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Bombyx mori, and is highly biocompatible, minimally immunogenic, non-toxic, noncarcinogenic and biodegradable, therefore is considered as ideal candidate for skin

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regeneration.28-31 Moreover, there is a urgent need to develop silk-based scaffolds for the

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reconstruction of artificial skin used in tissue engineering applications, having excellent intrinsic properties, biocompatibility, biodegradability and mechanical strength.32-33 Currently, there are

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several methods to fabricate the porous silk fibroin scaffolds for tissue engineering applications.

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For example, freeze-drying, salt-leaching, and traditional electrospinning are commonly employed techniques.34-38 However, all these approaches either lead to a superficially porous or a thin sheet-like nanofibers resulting in small and irregular pore structures, which drastically inhibit the further development of these improvements to the next levels. In this study, we demonstrate the use of cold-plate electrospinning (CPE) technology for a water-soluble polymer (i.e., silk fibroin) to overcome the aforementioned shortcomings of electrospinning technique. Efficient system, avoiding the use of toxic solvents and using only aqueous solutions as solvents were used to electrospin of silk fibroin nanofibers. The nanofibers obtained using this technique were studied in comparison with 2D nanofiber sheets produced via TE and 3D nanofibers

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ACCEPTED MANUSCRIPT scaffolds produced using SLE techniques. Furthermore, the aim of the present study was to develop the skin substitute using the 3D nanofibers by CPE.

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Materials and methods

Cocoons from Bombyx mori were kindly gifted by Rural Development Administration

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(Suwon, Republic of Korea). Sodium chloride (NaCl), particle sizes 200 m was purchased from (Dae Jung, Siheung, Gyeonggi, Republic of Korea). Dulbecco’s modified Eagle medium

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(DMEM) supplemented with 10% fatal bovine serum, cocktail of 1% penicillin-streptomycin and

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Trypsin, were obtained from Welgene, Fresh Media™ (Dalseogu, Daegu, Republic of Korea). Phosphate Buffer Saline (PBS) pH 7.4 (1X) and Trypan Blue Stain 0.4% was obtained from

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Gibco® (Life Technologies Corporation, Gaithersburg, MD, USA). Tissue culture flasks and microplates for cell seeding and growth were purchased from BD Falcon™, (Winston-Salem,

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NC, USA) and SPL Life Sciences, (Pocheon-si, Gyeonggi-do, Republic of Korea).

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Fabrication of nanofibers by TE, SLE and CPE The electrospinning setup for the fabrication of the 2D nanofibers (i.e., using the TE

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technique) is similar to one that we used previously to electrospin silk fibroin nanofibers.39-40 A water-based solution containing nominally 8 wt% of silk fibroin was isolated from cocoons of Bombyx mori following our previously established procedures.39-40 In a typical procedure, we prepared silk fibroin nanofibers by loading the blend of silk fibroin and PEO solution into a plastic syringe equipped with a 22-gauge stainless steel needle (2.41 mm OD × 1.80 mm ID). To improve the processibility of silk solutions for electrospinning, while maintaining biocompatibility, 30 wt% of PEO in aqueous solution was added to silk fibroin solution. Finally, the blend solutions of silk fibroin and PEO in the ratio of (5:1) were loaded in syringes and used for electrospinning to prepare nanofibers.41 The needle was connected to a high-voltage power 6

ACCEPTED MANUSCRIPT supply with a continuous supply of solution using a syringe pump at a rate of 0.2 mL/h. The voltages used for electrospinning were +20 kV and −1 kV, and the collecting distance from the

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needle tip to the collector was maintained at 15 cm. The electrospinning setup for creating

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nanofibers using salt as a porogen was inspired by the work of Nam et al. and by our previous experiences in producing porous PCL and silk fibroin nanofibers using SLE techniques (Figure

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S1).17-19 The 3D nanofibers scaffolds prepared by CPE technique was performed using a custom-

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designed immersion chiller (IMC-90, Operon, Gyeonggi-do, Gimpo City, Korea) capable of cooling to temperatures of −90°C (Figure 1A). A flexible cooling transfer pipe leading from the

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chiller was packed inside an insulated box with orifices for cold plate (aluminum). The collector was connected to an ice chiller capable of cooling to −90°C, as depicted in (Figure 1A). The

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same electrospinning conditions (with regard to voltage and tip to collector distance) used for the

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fabrication of the 2D and 3D nanofibers via the TE and SLE techniques were applied. Same time the electrospinning was allowed to begin when an cold plate had sensed the below 0°C, which

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resulted in the simultaneous accumulation of ice crystal layers and deposition of nanofibers on

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the collector. Furthermore, the layer-by-layer accumulation of ice particles continued to enhance the electrical conductivity on cold plate, which resulted in an increased likelihood of nanofibers being deposited with full-thickness (Figure 1A). The humidity was strictly controlled by using custom designed humidity controller installed in the electrospinning chamber by NanoNC (ESR200R2D, eS-robot®, Geumcheon-gu, Seoul, Republic of Korea). Finally, all the nanofibers fabricated by TE, SLE and CPE, were immediately freeze-dried, immersed in 95% ethanol for crystallization and then removal of “sacrificial” PEO in de-ionized water39 as indicated in (Figure 1B).

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ACCEPTED MANUSCRIPT Results The gross findings of the nanofibers produced via CPE were excellent in terms of full-

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thickness (Figure 1C). We observed that the average thickness of the 2D nanofiber sheets

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produced using the TE technique was 0.033 ± 0.01 mm and that the average thickness of the nanofiber scaffolds produced using the SLE technique was 0.14 ± 0.25 mm. In contrast, the

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nanofibers scaffolds produced using the CPE technique possessed an average thickness of 8.53

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± 1.82 mm. Moreover, we could successfully achieve in fabricating nanofbers about 10 mm with maximum thickness via CPE technique (Figure 1D). Also, it was observed that CPE can

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easily be helpful to deposit nanofibers on mold surfaces with typical curvature surfaces of face

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and ear (Figure 1D).

The methods used to calculate average swelling, water up-take and liquid porosity

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percentages are mentioned in (supplementary materials) and its results are presented in (Figure

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1E). In case of 2D nanofiber sheets and 3D nanofiber scaffolds obtained using the TE and SLE process had average swelling ratio of (15.5 ± 1.1 and 19.5 ± 3.2%), and swelling percentage of

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nanofibers scaffolds prepared by CPE had average percentage of 29.2 ± 4.9, which is higher than the former techniques. The average water up-take percentages of the nanofibers obtained using the TE and SLE techniques were found to be (93.9 ± 2.4 and 95.12 ± 7.2%), respectively, and the nanofibers produced using the CPE technique had an average water up-take of (96.6 ± 2.5%), which higher than former techniques. The liquid porosities of nanofibers prepared by TE and SLE had average percentage of 69.3 ± 4.5 and 71.3 ± 1.7, respectively. Meanwhile, the nanofiber scaffolds obtained via CPE had an average liquid porosity of (75.1 ± 3.5%), which is considerably higher than those achieved using the other techniques. All these physical properties (i.e., swelling, water up-take and liquid porosity results) indicate that nanofiber scaffolds 8

ACCEPTED MANUSCRIPT prepared by CPE had excellent properties than that of nanofibers prepared by TE and SLE techniques.

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Figure 2 presents the FE-SEM results for the nanofibers obtained using the three different

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techniques. In case of the fibers produced via the TE technique (Figure 2A), the samples exhibited a smooth morphology of the nanofibers, which is consistent with the typical nanofiber

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morphology observed after any electrospinning event. Moreover, the nanofibers were arranged in

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a compact manner, which resulted in the formation of a small pore size between the fibers (Figure 2A). The average diameter of the nanofibers were calculated from FE-SEM images,

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using the software (Innerview 2.0, Dong, Bundang Daeduk Plaza, Republic of Korea), after measuring (10-20 diameters per sample). These investigations revealed that nanofibers prepared

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by TE technique were having average fiber diameter of 508 ± 178 nm, while as, the average

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diameter for fibers prepared by SLE technique were 577 ± 275 nm, and the average diameter for fibers prepared by CPE technique was 812 ± 208 nm, respectively. The fiber morphology in

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terms smoothness remained the same for all fabrication techniques. However, a difference was

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observed in the fiber diameter and pore size of the nanofibers produced by SLE and CPE techniques (Figures 2A, C and E). The average pore size of the nanofibers was measured from the FE-SEM images, after measuring (20-30 pore diameters per sample). The average pore diameters of the nanofibers were strongly affected by the different techniques. For the 2D nanofiber sheets, the average pore diameter was 0.86 ± 1.14 m, and the average pore diameter of the 3D nanofibers produced using the SLE technique was calculated to be 18.56 ± 0.1 m, which was greater than the pore diameter of the 2D nanofibers. However, the nanofibers produced using the CPE technique had an average pore diameter of 41.42 ± 0.8 m, which is considerably higher than the pore diameter observed for the nanofibers produced using either of

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ACCEPTED MANUSCRIPT the former techniques. The cross-sectional images (Figures 2B, D and F) of the nanofibers provide greater insight into the internal structures with regard to the pore architecture and

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thickness of the nanofibers. The nanofibers produced using the CPE technique had the loose

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arrangement of fibers and highest thickness among all samples, loosely arranged fibers by CPE can be beneficial for anchoring cell attachments and penetration. Moreover, the results revealed

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that a regular pore structure resembling a honeycomb-like architecture was present beneath the

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surfaces of the nanofiber scaffolds produced via CPE (Figure 2F). In comparison, the nanofiber sheets obtained using the TE technique exhibited a small pore size and small thickness (Figures

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2A and B), and the nanofiber scaffolds obtained using the SLE technique (Figures 2C and D) had moderate porosity and physical appearance of these nanofibers indicated lost in integrity after

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salt leaching process.

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The methods used to carry out cell culture studies are explained in (supplementary materials). The results of cell-attachment and cell infiltration on nanofiber combinations after

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culturing NIH 3T3 fibroblasts for 2 weeks are presented in (Figures 3A, C and E). The average

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thickness of the nanofiber scaffolds used for all cell culture studies were 0.033 ± 0.01 mm for TE, 0.14 ± 0.25 mm for SLE, and 8.53 ± 1.82 mm for CPE nanofibers, respectively. In all cases, cells were attached in the usual pattern as observed for any other electrospun sheet. However, the cross-sections of the nanofibers after the culturing of the fibroblasts proved to be more informative with respect to the cell infiltration (Figures 3B, D and F). A different degree of cell penetration was observed for each nanofiber configuration. The infiltration of the 2D nanofiber sheets prepared via the TE technique by the cells was clearly difficult. However, the nanofibers produced using the SLE technique showed a moderate level of infiltration with collapsed pore architecture after cell culturing. The nanofibers produced using the CPE technique showed

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ACCEPTED MANUSCRIPT intense cell infiltration and retention of the pores after cell culturing (Figure 3F), which suggests the superiority of this technique. To have more insight about the cell infiltration, NIH 3T3

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fibroblasts cells were cultured in presence of nanofibers for 4 weeks of time; samples were cross-

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sectioned and viewed under confocal fluorescent microscopy. In this regard, the (Figure 4) shows images of cross-sectioned nanofibers samples under confocal microscopy. It can be

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observed, that cells are peripherally located on the surface of nanofibers and are not completely

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infiltrated into nanofibers due to small pore size of nanofibers prepared by TE technique (Figures 4A and B). The nanofibers prepared by SLE technique shows mild cell infiltration (Figures 4C

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and D). Comparatively, the nanofibers prepared by CPE technique shows maximum level of cell infiltration than that of nanofibers prepared by either of the former techniques (Figures 4E and

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F).

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The abundant formation of ice particles in and around the nanofibers depends on the humidity present in the environment during CPE; this phenomenon enhances the porosity and

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thickness of the fibers obtained via the CPE technique. Furthermore, this improvisation in CPE

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technique can presumably achieve greater porosity if the ambient humidity during electrospinning is increased. To investigate this possibility, we tested several different humidity levels (i.e., 30, 50, and 90%) in the electrospinning chamber during the collection of the fibers on the cold plate. The surface morphology and cross-sectional results at controlled humidity after electrospinning and freeze-drying are presented as FE-SEM images (Figure 5). The surface morphology shows a loose arrangement of the nanofibers due to increased humidity, resulting increase in pore diameters (Figures 5A, C and E). From the cross-sectional images, an increase in the humidity during electrospinning following the CPE technique is concluded to be accompanied by an increase in the internal pore size and thickness of the resultant nanofiber

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ACCEPTED MANUSCRIPT scaffolds (Figures 5B, D and F). The humidity also influenced the swelling ratio, liquid porosity and water up-take capacity of the nanofiber scaffolds prepared CPE. The (Figure S2) presents the

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results of these properties under the influence of (30, 50, and 90%) humidity during CPE

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technique. The swelling percentages were calculated to be 16.97 ± 2.7, 29.2 ± 4.9 and 31.22 ± 4.7, the water up-take percentages were 94.38 ± 7.6, 96.63 ± 2.5 and 96.85 ± 2.4, and the liquid

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porosity were found to be 72.69 ± 1.4, 75.14 ± 3.5 and 95.46 ± 6.5% for nanofibers fabricated in

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the presence of (30, 50, and 90%) humidity, respectively. The higher values in these measurements indicate that swelling, liquid porosity and water up-take properties can be

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improved by increasing the humidity during the fabrication of nanofibers using the CPE technique.

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While achieving this goal the 3D nanofiber scaffolds fabricated under the influence of

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50% humidity during CPE were selected to construct artificial dermis by co-culturing two different cell lines in air-liquid culture system. In this connection, methods used to co-culture

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fibroblasts and keratinocytes is mentioned in (supplementary files). The (Figure 6A) shows the

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schematic illustration using air-liquid culture system, consisting of keratinocytes co-cultured with fibroblasts in presence of 3D nanofiber scaffolds produced by 50% of humidity during CPE. The method to explain H&E and MT staining is explained at (supplementary files) results after culturing 6, 7 and 8 weeks in presence of 3D nanofibers fabricated by 50% of humidity CPE are presented in (Figures 6 (b, c and d)). As the time of incubation pass, we can observe that number of fibroblasts and keratinocytes are infiltrating into 3D nanofiber scaffolds. Fibroblasts can be seen in deep layers and more differentiation of keratinocytes at superficial layer, giving it appearance of artificial dermis. Figure 6(e), represents the results of MT staining, after coculturing for 8 weeks, from this image we can clearly observe influx of collagen-like ECM is

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ACCEPTED MANUSCRIPT predominately present in deeper layer of 3D nanofiber scaffolds. The data from this study suggests that the 3D nanofibers prepared by CPE might be a suitable for skin tissue-engineering.

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Discussions

To construct an artificial dermis with 2D nanofiber sheets prepared by TE is straight

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forward impossible. Also, the 3D nanofiber scaffolds prepared by SLE had some drawbacks like;

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collapsing of pores, loss of physical integrity, having less thickness, compared to that of nanofibers prepared by CPE technique. Before assigning the nanofibers for constructing artificial

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skin we have to look on basic architect of skin. Which consists of outer epidermis layer that contains keratinocytes and serving as a barrier to infection. Whereas, the dermis is a relatively

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thick and an inner layer that contains fibroblasts and extra cellular matrix mainly comprising collagen. These extra cellular matrix proteins provides a strength, flexibility and networks

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systems for body homeostasis.42 Till this date, there were several studies for bilayer skin

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substitutes which were developed with two different biomaterials. However, the previous studied

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fabricating a bilayered structure using two different biomaterials had some issues.24-26, 43 Thus, fabricating the bilayer skin substitutes with one biomaterial (i.e., silk fibroin at present) may be helpful for addressing this issue. To our knowledge, although our histologic findings shows a primitive result; however, this is the first study to develop the bilayer skin substitutes, using single nanofibers materials to form artificial dermis. The thickness of achieved nanofibers by CPE is higher than any thickness reported in the literatures.11-12,

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It is noteworthy in

mentioning that fibers coming out of needle of TE apparatus find it hard to deposit on curvature surfaces. Therefore, the nanofibers prepared by TE technique can barely deposit on typical collector surface with a full-thickness. Also, SLE techniques could help to improve the porosity and thickness to some extent; however, this technique also fails to deposit nanofibers on 13

ACCEPTED MANUSCRIPT curvature surfaces and with full-thickness nanofiber mats is hard to obtain. However, in case of CPE, we could successfully deposit the nanofibers with full-thickness on minute curvature

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surfaces of skin (e.g., on face and ear) which can apply to use these nanofibers in facial

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highly functional dermal substitute using CPE technique.

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reconstruction surgery (Figure 3B). Moreover, the further in vivo studies are needed to create

It has been well known that the electrospun nanofibers are considered as an excellent

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scaffold for tissue engineering due to extra cellular matrix mimicking factor. However, a full-

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thickness and large-pore 3D nanofiber scaffold is strongly recommended for skin reconstruction. Skin defect is one of the oldest and very difficult problems in regenerative medicine. Recently,

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several skin substitutes are commercially available for clinical applications and fundamental research. However, each type of skin substitutes still has its limitations and inconvenient aspects.

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The current study investigated that CPE technique results in a controllable highly porous and

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full-thickness 3D nanofibrous form, which provides an improvement in overall yield and better cell infiltration. Electrospinning using a cold-plate can produce nanofibers with higher porosity

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than those achieved using the TE and SLE techniques. The resultant nanofibers have a more regular pore architecture, higher porosity and water-binding abilities compared with other 2D and 3D nanofibers produced using the TE and SLE techniques that have been reported previously. The thorough porosity of the nanofibers obtained using the CPE technique leads to an improved cell infiltration compared to the nanofibers obtained using the previously developed techniques. Moreover, it is possible to achieve a full-thickness 3D nanofiber scaffold of greater than 10 mm using CPE technique because of the enhancement in electrical conductivity caused by the deposition of ice crystals and enhanced humidity facilitates accumulation of more ice particles resulting in deposition of nanofibers with full-thickness on the cold plate. This 14

ACCEPTED MANUSCRIPT technique can be directly applied to facilitate the fabrication of biomaterials with exact shapes and morphologies; in particular, such nanofibers are highly desirable for creating structures that

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resemble the dermis, nose, or ear or for repairing other facial defects.

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ACCEPTED MANUSCRIPT References 1. Huang Z-M, Zhang Y-Z, Kotaki M, Ramakrishna S. A review on polymer nanofibers by

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ACCEPTED MANUSCRIPT 10. Rajesh V, Dhirendra S K, Nanofibers and their applications in tissue engineering. Int J Nanomedicine. 2006; 1:15-30.

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removal of sacrificial fibers. Biomaterials. 2008; 29:2348-58. 13. Zhang Y, Ouyang H, Lim CT, Ramakrishna S, Huang Z-M, Electrospinning of gelatin fibers

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ACCEPTED MANUSCRIPT Figure legends: Figure 1. (A) Schematic illustration of the cold-plate electrospinning technique used to produce

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highly porous nanofibers. A high-voltage power supply generator was used to provide

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electric charge to the needle tip of syringe and at flat-bed collector (a). Silk fibroin solution was pumped using a syringe pump and the metallic needle was connected to

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the positive electrode (b). 3D electrospun silk fibroin nanofiber matrix (c). A

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conductive cold plate (aluminum) collector (160 mm in length, 100 mm in breadth and 10 mm in height) (d). A cooling transfer pipe (e). An illustration of the

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simultaneous accumulation of ice crystals and deposition of nanofibers on cold plate (f). The ultra-low temperature chiller (g). (B) Schematic illustration for crystallization

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method of 3D electrospun silk fibroin nanofibers. The nanofibers were freeze dried

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and immersed in 95% ethanol for 30 minutes and further placed in deionized water to allow removal of PEO for one day. The nanofibers were then lyophilized in a freeze-

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dryer for 48 h. (C) Gross finding of the TE, SLE and CPE techniques. (D)

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Photographs of the full-thickness 3D bionic face and ear fabricated via the CPE technique. (E) The results of the swelling ratio, water up-take and liquid porosity of the nanofibers fabricated via TE, SLE and CPE techniques.

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ACCEPTED MANUSCRIPT Figure 2. Field-emission scanning electron microscopy (FE-SEM) images of the nanofibers produced using the three different techniques. The surface morphology of the 2D

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nanofiber sheets obtained using the TE technique (A) and the corresponding cross-

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sectional image (B). The surface morphology of the nanofiber scaffolds obtained using the SLE technique (C) and the corresponding cross-sectional image (D).

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Controlled surface morphology of the nanofiber scaffolds obtained using the CPE

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technique (E) and the corresponding cross-sectional image (F). Figure 3. Cell-attachment and cell infiltration results using NIH 3T3 fibroblasts on the 2D

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nanofibers sheet obtained via the TE technique (A) and a cross-sectional image indicating cell infiltration (B). Cell attachment on the nanofibers scaffolds obtained

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via the SLE technique (C) and a cross-sectional image indicating cell infiltration (D).

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Cell attachment on the nanofibers scaffolds fabricated via the CPE technique (E) and a cross-sectional image indicating cell infiltration (F).

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Figure 4. Cross-sectional confocal fluorescent microscopy images of NIH 3T3 fibroblast after 4

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weeks of culture. The 2D nanofibers sheets obtained via the TE technique (A, B). The 3D nanofiber scaffolds obtained via the SLE technique (C, D). The nanofibers scaffolds fabricated via the CPE technique (E, F).

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Figure 5. Field-emission scanning electron microscopy images of the nanofiber scaffolds

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produced at controlled humidity during fabrication via controlled CPE. The surface

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morphology of the nanofiber scaffolds obtained at 30% humidity (A) and the corresponding cross-sectional image (B). The surface morphology of the nanofiber

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scaffolds obtained at 50% humidity (C) and the corresponding cross-sectional image

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(D). The surface morphology of the nanofiber scaffolds obtained at 90% humidity (E) and the corresponding cross-sectional image (F).

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Figure 6. Schematic illustration for co-cultured method (fibroblasts and keratinocytes) using air-liquid culture system (A). Histological appearance after co-culturing of 3D

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nanofibers scaffolds prepared via 50% humidity by CPE technique. The H&E

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staining results after co-culturing for 6 weeks (b), H&E staining results after coculturing 7 weeks (c) and H&E staining results after co-culturing 8 weeks (d). The

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(e).

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results of MT staining after co-culturing of fibroblasts and keratinocytes for 8 weeks

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Graphical Abstract

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2D nanofibers produced using the traditional electrospinning technique play an important role in

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a variety of applications. Nevertheless, a full-thickness, large-pore 3D nanofiber scaffold is strongly desired for use as biomaterial in tissue engineering. In this work, we have successfully introduced a new technique that can produce 3D nanofiber scaffolds with high porosity and, most importantly, with full thickness for tissue regeneration using cold-plate electrospinning.

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