Article pubs.acs.org/Langmuir
Fabrication of a Nanofibrous Mat with a Human Skin Pattern Jeong Hwa Kim,† Jinah Jang,‡ Young Hun Jeong,*,§ Tae Jo Ko,*,∥ and Dong-Woo Cho⊥ †
Department of Mechanical Engineering, Korea Polytechnic University, Gyeonggi 429-793, South Korea Division of Integrative Biosciences and Biotechnology, POSTECH, Gyeongbuk 790-784, South Korea § School of Mechanical Engineering, Kyungpook National University, Daegu 702-701, South Korea ∥ School of Mechanical Engineering, Yeungnam University, Gyeongbuk 712-749, South Korea ⊥ Department of Mechanical Engineering, POSTECH, Gyeongbuk 790-784, South Korea ‡
ABSTRACT: A number of studies on skin tissue regeneration and wound healing have been conducted. Electrospun nanofibers have numerous advantages for use in wound healing dressings. Here, we present an electrospinning method for alteration of the surface morphological properties of electrospun mats because most previous studies focused on the materials used or the introduction of bioactive healing agents. In this study, a micromachined human skin pattern mold was used as a collector in an electrospinning setup to replicate the pattern onto the surface of the electrospun mat. We demonstrated the successful fabrication of a nanofibrous mat with a human skin pattern. To verify its suitability for wound healing, a 14-day in vitro cell culture was carried out. The results indicated that the fabricated mat not only induces equivalent cell viability to the conventional electrospun mat, but also exhibits guidance of cells along the skin pattern without significant deterioration of pattern geometry. such dressings.17,18 The dressings can be classified according to their function, material, and physical form.17 With regard to function, for example, debridement, occlusive, antibacterial, absorbent, and adherent dressings have been developed. From the viewpoint of physical form, wound dressings can be divided into ointments, films, sheets, and gels. Moreover, dressings can be considered vehicles for delivery of bioactive materials or therapeutic agents to the wound surface. Electrospun nanofiber webs are among the most suitable materials for wound dressing because they allow excellent drainage of wound exudate and permeation of air into the wound, due to their highly porous nature, high surface-area-tovolume ratio, and mechanical compliance.19 Moreover, their ability to incorporate various additional materials enables delivery of bioactive or therapeutic agents,20 which can provide an enhanced chemical environment in the wound to improve healing. Various types of electrospun biopolymer nanofibers with advanced properties have been developed.19,21−23 However, most studies focused on the functionality and healing properties of the mat with regard to materials and bioactive molecular guidance. The introduction of surface morphological properties to nanofibrous wound dressings has not been considered despite the considerable potential of nanofibers for wound healing.19 There have been many studies regarding the morphological properties of electrospun nanofibrous mats. The standard
1. INTRODUCTION There have been significant advances in biomedical engineering over the last several years with improvements in various technological areas. Tissue engineering1 applications are attracting a great deal of attention because they provide opportunities to address incurable diseases and to minimize invasive therapy and pain in patients with various conditions and wounds.2 Therefore, much research regarding the regeneration of various tissues has been conducted,3,4 of which a key component is the preparation of suitable biomaterials for the target tissues; i.e., scaffolds.5 In this regard, many fabrication methods, ranging from traditional methods such as phase separation6,7 and solvent casting,2 to advanced techniques, such as solid free-form fabrication,8 have been developed. Some of the greatest advances in the development of biomaterials for tissue engineering have been made in the areas of skin tissue regeneration and wound healing.4,9 There are two typical approaches, i.e., permanent and temporary skin replacements.10 The former (permanent dermal replacement) has been developed by two prominent groups. Burke and Yannas devised artificial skin as a tissue regenerative matrix, consisting of a thin silastic layer and collagen-chodroitin-6-sulfate.11−14 Bell et al. developed another promising type of artificial skin known as a preengineered skin-equivalent graft.15,16 Temporary skin replacement is typically known as wound dressing, and plays a role in temporary wound closure to protect the tissue underlying the wound bed against bacterial inflammation or microbial contamination and to maintain an environment conducive to healing. There have been many studies of wound healing with the aid of © 2014 American Chemical Society
Received: July 31, 2014 Revised: November 24, 2014 Published: December 5, 2014 424
dx.doi.org/10.1021/la503064r | Langmuir 2015, 31, 424−431
Langmuir
Article
approach to morphological control in electrospinning involves unidirectional alignment of nanofibers using specialized collectors, such as a drum and a pair of parallel plates.24,25 The aligned nanofibers fabricated using these methods exhibited various functionalities in terms of cell guidance.26,27 Direct-write patterning is a useful alternative because it facilitates introduction of the desired morphological properties by scanning.28−30 Another common method is use of a collector with micropatterned electrodes prepared using lithographic technology.31,32 Moffa et al.33 and Rogers et al.34 used collectors with microgrooves to produce nanofibrous mats with three-dimensional patterns that could affect cell culture. In this study, an advanced electrospinning method capable of introducing a specific morphology into the nanofibrous mat was developed. This method consists of a combination of electrospinning and pattern replication; thus nanofibers are electrospun onto a conductive mold with a human skin pattern to fabricate a human-skin-patterned nanofibrous mat for wound healing applications. The mold, which was used as a collector in electrospinning, was composed of an aluminum alloy, and the pattern on the top plane was machined using a micromilling process. In addition, we used direct-write electrospinning, which is an improved method that uses a focused electric field and two-dimensional collector motion for scanning, to fabricate mats of uniform thickness and fiber density. The results demonstrated that a nanofibrous mat with a human skin pattern could be fabricated using the proposed method with no loss of the intrinsic properties of nanofibers. To demonstrate the applicability of the fabricated mat, 14-day in vitro cell tests using a mouse embryonic fibroblast cell line (NIH-3T3) were carried out. The fabricated mat provided excellent morphological guidance of cells along the skin pattern, and, as expected, cells cultured thereon exhibited a level of viability equivalent to those cultured on conventional electrospun fibers.
Figure 1. Experimental setup for micromilling of the human-skinpatterned mold. (a) Vertical-type three-axis micromilling machine used in mold preparation; (b) micromilling cutter.
2. MATERIALS AND METHODS 2.1. Mold Preparation. A mold with a human skin pattern was produced using a micromilling surface-texturing process, which was developed in our previous study.35 First, an image of actual human skin was converted to grayscale. Then, simplified three-dimensional geometric information on the skin pattern was reconstructed using pixel-intensity-based height mapping after several morphological image operations.35 Finally, a tool path for micromilling was generated using commercial computer-aided manufacturing (CAM) software (NX5; Unigraphics) from a three-dimensional solid model that included the reconstructed skin surface. In this study, a laboratory-built micromilling machine, consisting of a vertical-type 3-axis CNC machine with an ultraprecision air spindle as shown in Figure 1, was used in mold machining. The cutting tool was a titanium aluminum nitride (TiAlN)-coated carbide flat-end mill with two flutes and a tool diameter of 177.8 μm (equivalent to 0.007″). The surface speed of the flute was 45.72 m/min, which corresponded to the cutting conditions on feed rate and spindle speed of 30 mm/min and 80000 rpm, respectively. An aluminum alloy (Al 6061) was selected as the mold material for easy machining, and the mold was of approximate dimensions 15 × 15 × 6 mm3. The machining method was 2.5-dimensional cavity milling according to the generated tool path, in which the depth of cut and number of layers of cut were 10 μm and 10, respectively. The resulting pattern height was ∼100 μm. Figure 2 shows the prepared Al mold with a human skin pattern. 2.2. Electrospinning. The polymer for electrospinning was polycaprolactone (PCL) with an average molecular weight (Mw) of 70000−90000 (440744; Sigma-Aldrich), and the solvent for the polymer was 99.5% chloroform (C0584; Samchun Pure Chemical Co.).
Figure 2. Actual human-skin-patterned mold (AL6061). PCL was dissolved in the solvent to a concentration of 8.8 wt % and stirred for 120 min to obtain a homogeneous solution. The electrospinning setup was constructed by modifying the directwrite electrospinning apparatus used in our previous study,29 and consisted of a high-voltage power supply, a syringe pump, a nozzle spinneret with an inner diameter of 200 μm, a cylindrical sidewall electrode with an inner diameter of 150 mm, and a grounded human skin-patterned mold on a thick polyoxymethylene (POM) plate (Figure 3). The human-skin-patterned mold acted as a collector plate. A flat-surface mold of the same dimensions was also prepared as a control for comparison. Two-dimensional planar motion governed by a commercial motion controller (Turbo Clipper; Delta Tau Data Systems, Inc.) was introduced to the POM plate carrying the mold to improve the uniformity in thickness and fiber density of the 425
dx.doi.org/10.1021/la503064r | Langmuir 2015, 31, 424−431
Langmuir
Article
Table 1. Experimental Conditions for Electrospinning parameter (unit)
value
temperature (°C) humidity (%) voltage (kV) tip-to-collector distance (mm) scan speed (mm/s) flow rate (ml/h) number of layers (−)
19−20 50−55 21 60 40 0.1 20
Figure 4. SEM images of a nanofibrous line pattern on the mold. The nanofibrous mat electrospun onto the patterned surface of the mold was heat-treated using an oven to improve physical interconnectivity among the nanofibers. The treatment temperature and time were 60 °C and 5 min, respectively. However, natural cooling after heat treatment resulted in significant warping of the mat because of residual stress caused by the temperature difference between its top and bottom surfaces during cooling. In detail, the top surface facing the air had a lower temperature, while the bottom surface was in contact with the hot mold. Therefore, the top surface showed significantly greater shrinkage than the bottom surface. To avoid this warping, the mold with the electrospun mat was cooled rapidly for 3 min on a metal plate with a temperature of ca. −30 °C. 2.3. Morphological Observation. The morphologies of the fabricated mats were investigated by high-resolution scanning electron microscopy (NOVA NANO200; FEI) and field-emission scanning electron microscopy (FE-SEM) (S4700; Hitachi). Figures 1b, 5c, and 6b were obtained by high-resolution scanning electron microscopy, while the others were obtained by FE-SEM. The thickness was measured using an ultraprecision micrometer (No. 227-221; Mitutoyo) with constant compression of 0.5 N. The dimensions were measured on the obtained SEM images using image-processing software (ImageJ; National Institutes of Health). 2.4. Cell Culture. The biocompatibility and viability of cells cultured on the fabricated human skin-patterned mat were assessed to evaluate its suitability for biomedical engineering. The assessments were performed using the mouse embryonic fibroblast cell line NIH3T3 cultured in alpha-MEM medium (Thermo Scientific−Hyclone) supplemented with 10% fetal bovine serum (FBS; Invitrogen GIBCO) and 1% penicillin−streptomycin (P/S; Invitrogen GIBCO). Cell cultures were maintained in a humidified incubator under 5% CO2 at 37 °C. The fabricated mats were sterilized in 70% ethanol for 2 h, and then irradiated with ultraviolet (UV) light for 2 h. Before cell seeding, they were prewetted in culture medium for 3 h after washing three times with phosphate-buffered saline (PBS; Invitrogen GIBCO). The cell suspension was seeded directly onto a prewetted mat in a 24-well plate. The density of the cell suspension was ∼1 × 104 cells/sample. Cells were allowed to attach to the nanofibrous mats for 24 h,
Figure 3. Electrospinning setup using a human-skin-patterned mold for fabrication of a human-skin-patterned nanofibrous mat. (a) Schematic diagram; (b) actual setup. electrospun mat. Planar motion following a raster scan path with a pitch size of 200 μm was applied to the POM plate carrying the mold. The scan area was ∼30 × 30 mm2, which was double that of the patterned surface of the mold; most of the nanofibers electrospun out from the nozzle spinneret were deposited on the mold surface because of the focusing effect of the electric field. As a result, nanofibers could be deposited evenly over the patterned surface of the mold. Moreover, the thickness of the mat could be controlled by repeating the planar motion along the same path in a layer-by-layer process. The nozzle spinneret tip-to-collector (i.e., patterned surface of the mold) distance was maintained at 60.0 mm. The electrical potential between the nozzle spinneret and the pattern mold was ∼21 kV. The sidewall electrode had the same electrical potential as the nozzle. The flow rate of PCL solution was controlled by a syringe pump at 0.1 mL/h. The electrospinning setup was isolated from the environmental conditions by means of being housed in an acrylic chamber. During the experiments, the temperature was maintained at 19−20 °C, and relative humidity at 50−55%. The scan speed of the mold was ∼40 mm/s and the number of layers was 20. Table 1 summarizes the electrospinning conditions used. 426
dx.doi.org/10.1021/la503064r | Langmuir 2015, 31, 424−431
Langmuir
Article
Figure 6. Photograph (a) and SEM image (b) of a human-skinpatterned nanofibrous mat. and histological analyses. Cell proliferation was evaluated using a Cell Counting Kit-8 (CCK-8; Dojindo), while cell morphology was assessed using SEM images and F-actin/DAPI staining. Gene expression was quantified by real-time RT-PCR. For histological analysis, we used hematoxylin and eosin (H&E) staining. Each experimental group consisted of at least three samples to facilitate statistical analysis. At 1, 3, 5, and 7 days, the samples were carefully washed twice with PBS to remove detached cells; the wells were then refilled with a mixture of CCK-8 reagent and fresh medium according to the manufacturer’s protocol. After incubation for 4 h under 5% CO2 at 37 °C, the absorbance at 450 nm was measured using a microplate reader (Sunrise Monochrom Control TW; TECAN). Cells seeded on skin-patterned and flat-surfaced mats after 7 and 14 days of culture were rinsed with PBS, and then fixed with 3.7% formaldehyde-PBS for 30 min. The samples were dried in air for 72 h after washing with PBS. Then, the samples were sputtered with palladium under vacuum, and their surface morphology visualized using SEM. Also, cell morphology on the mat after 7 days of culture were observed using F-actin and DAPI. Cultured cells on the mat were stained with phalloidin-fluorescein isothiocyante (PhalloidinFITC, Sigma-Aldrich) solution after fixing and permiabilizing. Then cell nuclei were counterstained using Bisbenzimide H 33258 (DAPI, Sigma-Aldrich) solution. After 14 days of cell culture, total RNA was extracted using guanidinium isothiocyanate-phenol-chloroform (TRIzol; Life Technologies) according to the manufacturer’s protocol. RNA concentration was measured by spectrophotometric analysis (Nanodrop; Thermo Scientific). Reverse transcription was carried out using a cDNA synthesis kit (Maxima First Strand cDNA synthesis Kit; Thermo Scientific). Then, gene expression was quantified using SYBRgreen with an RT-PCR system (Applied Biosystems 7500 Real-Time PCR system; Life Technologies). The collagen type I (COL1), collagen type III (COL3), elastin (ELN), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) genes were amplified using the following primers: COL1: 5′-GCATGGCCAAGAAGACATCC-3′ and
Figure 5. SEM images of nanofibers. (a) Flat-surfaced mold; (b) skinpatterned surface mold; (c) enlarged image of nanofibers; (d) distribution of nanofiber diameter. following which the medium in each chamber was replaced with fresh medium every other day (48 h). 2.5. Assays (Biological Analysis). Cell behavior on the mats was analyzed by assaying proliferation, morphology, gene expression, 427
dx.doi.org/10.1021/la503064r | Langmuir 2015, 31, 424−431
Langmuir
Article
2.7. Statistical Analysis. Statistical analyses were performed by one-way analysis of variance (ANOVA) using Microsoft Excel 2013. In all analyses, P < 0.05 was taken to indicate statistical significance.
5′-CCTCGGGTTTCCACGTCTC-3′; COL3:5′-TTCCTGAAGATGTCGTTGATGTG-3′ and 5′-TTTTTGCAGTGGTATGTAATGTTCTG-3′; ELN: 5′-GTGTATACCCAGGCGGAGTG-3′ and 5′-GCTTTGACTCCTGTGCCAGT-3′. COL1, COL3, and ELN expression levels were normalized relative to that of GAPDH and investigated using the 2-ΔΔCT method. Each sample was tested in triplicate. 2.6. Histological Analysis. Samples cultured for 14 days were harvested and fixed with 3.7% formaldehyde. The constructs were embedded in optimal cutting temperature (OCT) compound and cut into frozen sections 10 μm thick. The sections were then rinsed with PBS, fixed again, and stained with H&E.
3. RESULTS AND DISCUSSION 3.1. Geometric Analysis of the Fabricated Human Skin-Patterned Mat. Figure 4 shows a single line pattern of nanofibers on the human-skin-patterned mold. As shown in the figure, a line pattern with straight-line geometry was fabricated successfully. Based on the 50 measurements of the width of the line pattern fabricated under the same conditions as shown in Table 1, the actual line pattern width was 199.5 ± 47.8 μm. In addition, there was no significant difference in line patterns between flat and wrinkled regions of the mold. From the measurements, we set the pitch size of the raster scan path to 200 μm to introduce sufficient overlap between line patterns after focusing by the electric field. The thickness of the mat was ca. 364.25 ± 55.03 μm, which was close to the minimum thickness of the commercialized wound dressings.36 Morphological differences between nanofibers on the flat mold (i.e., no pattern) and skin-patterned mold composed of the same material were investigated. Nanofibers were electrospun onto both molds under the conditions shown in Table 1. Figure 5a,b shows the fabricated nanofibrous mats on both flatand patterned-surface molds. The nanofibers were efficiently electrospun onto both molds. In addition, there were no significant differences in fiber-scale morphology (Figure 5a,b). However, nanofibers on the patterned mold successfully replicated the skin pattern present on the surface of the mold, while nanofibers on the flat surface showed no patterning or wrinkles. The mats had an almost identical fiber morphology (Figure 5c), which shows the nanofibers after heat treatment. The fiber diameter distribution is shown in Figure 5d; the fiber diameter ranged from 250 to 3000 nm. Figure 6a,b shows a photograph and SEM image of a fabricated human-skin-patterned mat. The size of the fabricated mat was ∼15 × 15 mm2, which was trimmed to 10 × 10 mm2 to fit the 24-well culture plates. 3.2. Cell Proliferation and Gene Expression Analyses. As mentioned above, the fabricated skin-patterned mat had similar properties to the conventional electrospun mat (plain mat), with the exception of the presence of skin patterns consisting of complex wrinkled geometry, which seemed to have little effect on the biocompatibility of the mat. Moreover, the electrospun nanofibrous structure is known to have excellent biocompatibility.19 Therefore, the biocompatibility of the skin-patterned mat was compared with that of the plain mat using a CCK-8 assay to evaluate cell proliferation and realtime RT-PCR to assay gene expression levels.
Figure 7. CCK-8 assay results of NIH-3T3 cells on skin-patterned and conventional nanofibrous mats after 1, 3, 5, and 7 days in culture.
Figure 8. Gene expression levels of ECM-secreting cells cultured on the skin-patterned and conventional nanofibrous mats for 14 days as determined by real-time RT-PCR.
Figure 9. Cell morphology on a skin-patterned mat after 7 days of culture. (a) F-actin and DAPI staining image; (b) SEM image. 428
dx.doi.org/10.1021/la503064r | Langmuir 2015, 31, 424−431
Langmuir
Article
Figure 11. Histological analysis.
the two groups throughout the test period, although the number of cells on the plain mat was slightly greater than that on the patterned mat after 5 days of culture. Cell proliferation did not change markedly in the two groups over the first 3 days after cell seeding, but increased significantly up to 7 days in both groups to a similar level. Therefore, both mats provided an environment conducive to cell proliferation. Similarly, the levels of expression of the genes such as COL1, COL3 and ELN, which are well-known major extracellular matrix (ECM) proteins secreted from fibroblasts, were investigated. Figure 8 shows the COL1, COL3, and ELN gene expression levels on both the plain and skin-patterned mats up to 14 days of cell culture. As expected, there were no significant differences in COL1, COL3, or ELN gene expression between the two mats because p-values of each gene were not less than 0.05. In addition, the fabricated skin-patterned mats supported cellular functions to a level comparable to the plain mats. Therefore, the fabricated skin-patterned mat exhibited a biocompatibility similar to that of the conventional plain electrospun mat. 3.3. Morphological and Histological Analyses. F-actin staining enables one to observe the actin filaments in the cytosol; therefore, we examined the stretched cell morphology on both a patterned and a conventional mat by using F-actin staining. There was little difference in cell morphology between two cases. As shown in Figure 9a, it could be seen that fibroblasts had the spindle-like shape, which was the natural morphology of the fibroblast on the two-dimensional surface. Moreover, SEM image showed the identical result with the F-actin staining result (Figure 9b). The morphology of NIH-3T3 cells and their secreted ECM after 7 and 14 days in cell culture were examined by SEM. Figure 10a,b shows cells on skin-patterned and plain mats. In both cases, cells were spread evenly over the mats. As shown in Figure 10a, the skin pattern on the mat remained visible even though its surface was covered with cells. Similarly, the flat surface of the cell-seeded plain mat showed no deterioration. However, the cells and secreted ECM did not cover the surface of either of the mats completely. With regard to the cytoskeleton, the cells cultured on both types of mat stretched and showed attachment. The surface morphologies of both cell-seeded mats after 14 days in cell culture are shown in Figure 10c,d. In both cases, the cells spread over the whole of the mats, and synthesized ECM was distributed evenly over the surface. The pattern of the skin-patterned mat remained visible up to 14 days, although the cell-secreted ECM layer cracked upon drying. All such cracks appeared in the patterned mat, and particularly in wrinkled regions due to surface tension caused by residual stress upon drying. Nanofibers beneath the cellECM layer were visible through the cracks, which indicated that cell migration into the mat was negligible.
Figure 10. SEM images of the ECM-secreting cells cultured on the mats. (a) Skin-patterned mat after 7 days; (b) plain mat after 7 days; (c) skin-patterned mat after 14 days; (d) plain mat after 14 days.
Figure 7 shows the relative cell proliferation levels evaluated after 3, 5, and 7 days in culture. Two groups cultured on the patterned mat and the plain mat were compared. There was no significant difference (P > 0.05) in the number of cells between 429
dx.doi.org/10.1021/la503064r | Langmuir 2015, 31, 424−431
Langmuir
Article
(5) Kim, B.-S.; Mooney, D. J. Development of biocompatible synthetic extracellular matrices for tissue engineering. Trends Biotechnol. 1998, 16 (5), 224−230. (6) Mao, J.; Zhao, L.; de Yao, K.; Shang, Q.; Yang, G.; Cao, Y. Study of novel chitosan−gelatin artificial skin in vitro. J. Biomed. Mater. Res., Part A 2003, 64 (2), 301−308. (7) Choi, Y. S.; Hong, S. R.; Lee, Y. M.; Song, K. W.; Park, M. H.; Nam, Y. S. Study on gelatin-containing artificial skin: I. Preparation and characteristics of novel gelatin−alginate sponge. Biomaterials 1999, 20 (5), 409−417. (8) Hollister, S. J. Porous scaffold design for tissue engineering. Nat. Mater. 2005, 4 (7), 518−524. (9) Yang, E. K.; Seo, Y. K.; Youn, H. H.; Lee, D. H.; Park, S. N.; Park, J. K. Tissue engineered artificial skin composed of dermis and epidermis. Artif. Organs 2000, 24 (1), 7−17. (10) Schulz Iii, J.; Tompkins, R.; Burke, J. Artificial skin. Annu. Rev. Med. 2000, 51 (1), 231−244. (11) Yannas, I.; Burke, J. F. Design of an artificial skin. I. Basic design principles. J. Biomed. Mater. Res. 1980, 14 (1), 65−81. (12) Yannas, I.; Burke, J.; Gordon, P.; Huang, C.; Rubenstein, R. Design of an artificial skin. II. Control of chemical composition. J. Biomed. Mater. Res. 1980, 14 (2), 107−132. (13) Dagalakis, N.; Flink, J.; Stasikelis, P.; Burke, J.; Yannas, I. Design of an artificial skin. Part III. Control of pore structure. J. Biomed. Mater. Res. 1980, 14 (4), 511−528. (14) Yannas, J. B. I.; Quinby, W., Jr.; Bondoc, C.; Jung, W. Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury. Ann. Surg. 1981, 194 (4), 413. (15) Bell, E.; Ivarsson, B.; Merrill, C. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc. Natl. Acad. Sci. U. S. A. 1979, 76 (3), 1274−1278. (16) Bell, E.; Ehrlich, H. P.; Buttle, D. J.; Nakatsuji, T. Living tissue formed in vitro and accepted as skin-equivalent tissue of full thickness. Science 1981, 211 (4486), 1052−1054. (17) Boateng, J. S.; Matthews, K. H.; Stevens, H. N. E.; Eccleston, G. M. Wound healing dressings and drug delivery systems: A review. J. Pharm. Sci. 2008, 97 (8), 2892−2923. (18) Chaby, G.; Senet, P.; Vaneau, M.; Martel, P.; Guillaume, J. C.; Meaume, S.; Teot, L.; Debure, C.; Dompmartin, A.; Bachelet, H.; Carsin, H.; Matz, V.; Richard, J. L.; Rochet, J. M.; Sales-Aussias, N.; Zagnoli, A.; Denis, C.; Guillot, B.; Chosidow, O. Dressings for acute and chronic wounds - A systematic review. Arch. Dermatol. 2007, 143 (10), 1297−1304. (19) Zahedi, P.; Rezaeian, I.; Ranaei-Siadat, S. O.; Jafari, S. H.; Supaphol, P. A review on wound dressings with an emphasis on electrospun nanofibrous polymeric bandages. Polym. Adv. Technol. 2010, 21 (2), 77−95. (20) Ji, W.; Sun, Y.; Yang, F.; van den Beucken, J. J.; Fan, M.; Chen, Z.; Jansen, J. A. Bioactive electrospun scaffolds delivering growth factors and genes for tissue engineering applications. Pharm. Res. 2011, 28 (6), 1259−1272. (21) Schneider, A.; Wang, X.; Kaplan, D.; Garlick, J.; Egles, C. Biofunctionalized electrospun silk mats as a topical bioactive dressing for accelerated wound healing. Acta Biomaterial. 2009, 5 (7), 2570− 2578. (22) Rujitanaroj, P. O.; Pimpha, N.; Supaphol, P. Wound-dressing materials with antibacterial activity from electrospun gelatin fiber mats containing silver nanoparticles. Polymer 2008, 49 (21), 4723−4732. (23) Thakur, R. A.; Florek, C. A.; Kohn, J.; Michniak, B. B. Electrospun nanofibrous polymeric scaffold with targeted drug release profiles for potential application as wound dressing. Int. J. Pharm. 2008, 364 (1), 87−93. (24) Katta, P.; Alessandro, M.; Ramsier, R.; Chase, G. Continuous electrospinning of aligned polymer nanofibers onto a wire drum collector. Nano Lett. 2004, 4 (11), 2215−2218. (25) Li, D.; Wang, Y.; Xia, Y. Electrospinning of polymeric and ceramic nanofibers as uniaxially aligned arrays. Nano Lett. 2003, 3 (8), 1167−1171.
The cross-sectional morphology of cells on the patterned mat after 14 days in cell culture was investigated. Figure 11 shows an H&E-stained cross-sectional image of a mat with cultured cells. The whole surface was covered by an organized sheath consisting of cells and secreted ECM, which corresponded to Figure 10c. In addition, most of the fibroblasts attached to the top surface of the mat, but some infiltrated the mat to a depth of up to 100 μm. Finally, the mat was capable of guiding tissue regeneration along the skin pattern.
4. CONCLUSIONS This study showed that an electrospun nanofibrous mat with a human skin pattern could be fabricated using direct-write electrospinning with a human-skin-patterned mold as a collector. The skin pattern for texturing was obtained from actual human skin by morphological imaging. Then, the patterned mold was machined using a 2.5-dimensional micromilling-based texturing process. The mold was successfully implemented in the electrospinning process and yielded excellent replication of the human skin pattern on the electrospun nanofibrous mat. To investigate the feasibility of this fabricated human skinpatterned mat for wound healing, 14-day in vitro tests were carried out. The cells seeded on the patterned mats showed vigorous proliferation for 7 days after seeding and sufficient ECM protein secretion, to levels almost identical to those on the plain mat fabricated using conventional electrospinning. In particular, the skin pattern remained for up to 14 days in cell culture with excellent guidance of cell growth along the pattern. Therefore, cells cultured on the fabricated human skinpatterned mat exhibited a level of viability similar to those cultured on the conventional nanofibrous mat. Moreover, the pattern provided morphological guidance for cell growth, which could play a crucial role in the esthetic outcomes of skin tissue regeneration and wound healing.
■
AUTHOR INFORMATION
Corresponding Authors
*(Y.H.J.) Tel: 82-53-950-5577; Fax: 82-53-950-6550; E-mail:
[email protected]. *(T.J.K.) Tel: 82-53-810-2576; Fax: 82-53-810-4627; E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This research was supported by the Pioneer Research Center Program (NRF-2012-0009665) and Creative Research Initiative (CRI) Program (No. 2010-0018294) through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning, Republic of Korea.
■
REFERENCES
(1) Langer, R.; Vacanti, J. P. Tissue engineering. Science 1993, 260 (5110), 920−926. (2) Shin, H.; Jo, S.; Mikos, A. G. Biomimetic materials for tissue engineering. Biomaterials 2003, 24 (24), 4353−4364. (3) Griffith, L. G.; Naughton, G. Tissue engineeringCurrent challenges and expanding opportunities. Science 2002, 295 (5557), 1009−1014. (4) Lanza, R.; Langer, R.; Vacanti, J. P. Principles of Tissue Engineering; Academic Press: New York, 2011. 430
dx.doi.org/10.1021/la503064r | Langmuir 2015, 31, 424−431
Langmuir
Article
(26) Xu, C. Y.; Inai, R.; Kotaki, M.; Ramakrishna, S. Aligned biodegradable nanotibrous structure: A potential scaffold for blood vessel engineering. Biomaterials 2004, 25 (5), 877−886. (27) Nain, A. S.; Phillippi, J. A.; Sitti, M.; MacKrell, J.; Campbell, P. G.; Amon, C. Control of cell behavior by aligned micro/nanofibrous biomaterial scaffolds fabricated by spinneret-based tunable engineered parameters (STEP) technique. Small 2008, 4 (8), 1153−1159. (28) Sun, D.; Chang, C.; Li, S.; Lin, L. Near-field electrospinning. Nano Lett. 2006, 6 (4), 839−842. (29) Lee, J.; Lee, S. Y.; Jang, J.; Jeong, Y. H.; Cho, D.-W. Fabrication of patterned nanofibrous mats using direct-write electrospinning. Langmuir 2012, 28 (18), 7267−7275. (30) Dalton, P. D.; Joergensen, N. T.; Groll, J.; Moeller, M. Patterned melt electrospun substrates for tissue engineering. Biomed. Mater. 2008, 3, 034109. (31) Zhang, D.; Chang, J. Patterning of electrospun fibers using electroconductive templates. Adv. Mater. 2007, 19 (21), 3664−3667. (32) Liu, Y.; Zhang, L.; Li, H.; Yan, S.; Yu, J.; Weng, J.; Li, X. Electrospun fibrous mats on lithographically micropatterned collectors to control cellular behaviors. Langmuir 2012, 28 (49), 17134−17142. (33) Moffa, M.; Sciancalepore, A. G.; Passione, L. G.; Pisignano, D. Combined nano- and micro-scale topographic cues for engineered vascular constructs by electrospinning and imprinted micro-patterns. Small 2014, 10 (12), 2439−2450. (34) Rogers, C. M.; Morris, G. E.; Gould, T. W.; Bail, R.; Toumpaniari, S.; Harrington, H.; Dixon, J. E.; Shakesheff, K. M.; Segal, J.; Rose, F. R. A novel technique for the production of electrospun scaffolds with tailored three-dimensional micro-patterns employing additive manufacturing. Biofabrication 2014, 6 (3), 035003. (35) Syahputra, H. P.; Ko, T. J. Application of image processing to micro-milling process for surface texturing. Int. J. Precis. Eng. Manuf. 2013, 14 (9), 1507−1512. (36) Thomas, S.; Loveless, P. A comparative study of the properties of twelve hydrocolloid dressings. World Wide Wounds 1997, 1−14.
431
dx.doi.org/10.1021/la503064r | Langmuir 2015, 31, 424−431
