Fibroblast Culture on Poly(L-lactide-co-ε-caprolactone) an Electrospun Nanofiber Sheet

Transcription

1 Macromolecular Research, Vol. 20, No. 12, pp (2012) DOI /s pissn eissn Fibroblast Culture on Poly(L-lactide-co-ε-caprolactone) an Electrospun Nanofiber Sheet Bong Seok Jang 1,2, Youngmee Jung 1, Il Keun Kwon 2, Cho Hay Mun 1, and Soo Hyun Kim*,1 1 Center for Biomaterials, Korea Institute of Science and Technology, Seoul , Korea 2 Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul , Korea Received November 1, 2011; Revised April 3, 2012; Accepted April 3, 2012 Abstract: Electrospinning has been used to make a nanofibrous matrix for vascular tissue engineering applications. The poly(l-lactide-co-ε-caprolactone) (PLCL) copolymer (50:50), which is biodegradable and elastic, was used to fabricate electrospun nanofiber sheets with a thickness of µm. The objective of this study was to investigate the behavior of fibroblast cells on the PLCL electrospun sheet. The cell proliferation on the PLCL electrospun sheet was evaluated. The cell morphology was observed using scanning electron microscopy. Several coating materials were evaluated to increase cell adhesion, including fibronectin, Type-I collagen, and gelatin. Among the coating materials tested, Type-I collagen gave the best result. Cell proliferation at all cell densities was tested steadily increase up to 3 weeks. Single side cell seeding and double side cell seeding were compared. During cell proliferation for 3 and 7 days, the single side cell seeding slowly increased, whereas rapid cell growth was observed for the double side seeding. We evaluated the mechanical properties of electrospun nanofiber scaffolds cultured with different cell volumes. In these experiments, a higher cell volume resulted in higher tensile strength and Young s modulus. Further studies are being conducted to design a functional tubular vascular scaffold with adequate mechanical properties and architecture to promote cell growth. Keywords: electrospinning, PLCL, fibroblast cells, cell matrix engineering. Introduction The search of ideal vascular substitute materials for cardiovascular applications as a bypass or replacement of obstructed blood vessels due to diseases or trauma has thus far been a half-century endeavor. Large-diameter (>6 mm) vascular grafts such as Polyethylene terephthalate (PET, Dacron) and expanded polytetrafluoroethylene (eptfe) are the standard materials currently used, but no ideal method for using autologous vein grafts is currently available for small diameter (<5 mm) applications due its high failure rates from thrombosis, stenosis, and occlusion. Therefore, finding a solution for small-diameter vascular grafting has recently become a major focus of attention. Integrating the principles of tissue engineering with innovations in biomaterial technology holds promise for use in the development of a new generation of vascular substitutes. The extracellular matrix (ECM), which is composed of a basement membrane and a cross-linked network of proteins, glycosaminoglycans and collagen and elastin fibers nanoscale structures, plays an important role in controlling cell *Corresponding Author. soohkim@kist.re.kr behavior in living systems. Fabrication of nanofibers that mimic the ECM is one of the essential components for the development of an ideal scaffold for vascular grafts. 1-6 During the last two decades, significant advances have been made in the development of biocompatible and biodegradable materials for biomedical applications. The biodegradable polymer must be biocompatible and meet other criteria to be qualified as a biomaterial, i.e., easily processable, sterilizable, and capable of controlled stability or degradation in response to biological conditions. Poly(L-lactic acid) (P L LA), poly(d-lactic acid) (P D LA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL) and poly(l-lactic-co-ε-caprolactone) (PLCL) are biodegradable materials. PGA has been widely used and has a degradation rate of around 6~8 weeks. However, this rate is too fast for vascular graft tissue engineering applications because cell culture usually requires longer periods for inducement of tissue regeneration. 7 Recently, other biodegradable polymers such as P L LA and PCL have been studied because they have slower degradation rates. 8 For the regeneration of blood vessels, the elasticity of the polymer is as important as its biodegradable properties. The Polymer Society of Korea 1234

2 Fibroblast Culture on Poly(L-lactide-co-ε-caprolactone) an Electrospun Nanofiber Sheet Recently, our group had evaluated using PLCL as biomaterial. This material is suitable for making artificial blood vessels and also for tissue engineering due to its excellent in elasticity and biodegradable property. In this study, PLCL was synthesized with same mole ratio of L-lactide (LA) and ε-caprolactone (CL). 9,10 PLCL is basically a soft copolymer that consists of PCL as the soft matrix and PLA as the hard domains and exhibits a complete rubberlike elasticity, good biocompatibility to SMCs and proper biodegradability Therefore, PLCL holds great promise for use in vascular tissue engineering and tissue culture under cyclic mechanical stimulation. Electrospun scaffolds have been widely used in tissue engineering due to their similar structure to native extracellular matrices (ECM). Their unique fibrous structures would promote cell attachment and proliferation on the scaffolds. Electrospinning technique, which enables the production of continuous polymer micro- or nanofibers with diameters in the range of 50-5,000 nm from polymer solutions or melts under high electric field, has been proposed as a promising alternative for fabricating vascular scaffolds because of its simple setup, low cost productivity and facile control of fiber diameter and porosity Fibroblasts have important function in the healing process, tissue repair and synthesizing interstitial collagens, fibronectins, and other matrix components. 18 As such, fibroblasts can provide a favorable support and maintain the integrity of the collagen matrix. Fibroblasts can also produce their own matrix and thus can balance the synthesis and degradation of the naturally produced scaffold. Moreover, fibroblasts are capable for producing and secreting Figure 1. Schematic diagram of cell matrix engineering. angiogenic factors, 19 growth factors including vascular endothelial growth factors (VEGFs) and fibroblast growth factors (FGFs), 20 and other factors 21,22 which can promote cell viability and function with no need for genetic manipulation. Fibroblasts can produce essential growth factors. In addition syngeneic or autologous fibroblasts are currently available, which will make clinical transplantation more feasible. In this study, we cultured fibroblasts on PLCL electrospun sheets with the goal of evaluating the potential of using this system in tissue engineering applications. The sheet was rolled to produce a tubular structure that was similar structure to native blood vessel (Figure 1). In this paper, we evaluated the production parameter of PLCL electrospun scaffold and cell adhesion and proliferation on electrospun sheets containing different coating materials. We also compared the tensile properties of electrospun sheet at various cell densities. Experimental Materials and Methods. Electrospinning Apparatus: A custom-made electrospinning apparatus was constructed using a high-voltage supplier, an infusion pump, a syringe with a stainless steel blunt-ended needle (22 G x 1 ) and a custom-made rotating mandreltype collector. The syringe was horizontally fixed on the infusion pump and the polymer solution was led through a teflon tube. The polymer solution was electrostatically sprayed from the tip of the needle by applying a high voltage between the electrode and the collector. Preparation of PLCL Copolymer Sheet. The poly(llactide-co-ε-caprolactone) (PLCL 50:50) was synthesized by using ring opening polymerization method and polymers with a molecular weight of approximately 418,000 were used to fabricate electrospun sheet (Figure 2). PLCL was synthesized using previous described procedures (Jeong, Kim et al. 2004; Kim, Kwon et al. 2006). Briefly the polymerization of PLCL (50:50) was conducted in a 50 ml glass ampule containing L-lactide (100 mm), ε-caprolactone (100 mm) and 1,6-hexanediol (0.5 mm) using stannous octoate (1.0 mm) as a catalyst. The ampule was sealed under vacuum after purging three times with nitrogen at 90 o C and heating to 150 o C for 24 h with stirring Figure 2. Schematic diagram of chemical structure of the PLCL 50:50 copolymer. Macromol. Res., Vol. 20, No. 12,

3 B. S. Jang et al. in an oil bath. After the reaction completed, the obtained polymer was dissolved in chloroform and filtered through a 4.5 µm pore membrane filter. The polymer was precipitated into an excess of methanol, filtered, and dried under vacuum. Nanofiber Fabrication. The Polymers Used PLCL 50:50: The fabricated electrospun nanofiber sheets had a length of 28 cm and width of 20 cm with a thickness of 50 µm. scanning electron microscopy (SEM) images of the resulting fibers showed nanoscale fiber diameters and a random orientation of fibers. The fibers of the electrospun PLCL sheet were produced from 10% (wt/vol) PLCL solution in several kinds of organic solvent such as tetrahydrofuran (THF): dimethylformamide (DMF) (6:4), methylene chloride, chloroform and HFIP (1,1,1,3,3,3-hexafluoroisopropanol). The 10 wt% PLCL solution was delivered by a syringe pump (ESP200D, NanoNC) at a constant flow rate (1 ml/h). The blunt-ended 22-gauge needle was clamped to the positive electrode of a high voltage power supply (ESP200D, NanoNC) generating 15 kv of electric field, and the negative electrode was connected to an Al foil collector with an air gap distance of 15 cm. The mandrel was rotated at 400 r/min. The electrospun PLCL sheet was vacuum dried for 3 days at room temperature and stored in desicators until subsequent uses. Scanning Electron Microscopy (SEM). The morphology and surface topography of nanofiber sheets and cell-seeded sheets were examined using a scanning electron microscope (NOVA NanoSEM200, Fei, Netherlands) operated at 20 kv. For SEM examination, sheets were seeded with cells fixed in 4% (v/v) formaldehyde for 24 h, and then dehydrated using a graded ethanol series (50%, 70%, 90%, and 100%) and dried. Small amounts of nanofiber sheets or cell-seeded sheets samples were attached to SEM stubs using carbon tabs and the stub surfaces were then coated with platinum using a sputter-coater (E1030, Hitachi, Japan). Cell Isolation and Culture. Fibroblasts were isolated from the back skin of 3 weeks male Newzealand white rabbits weighing 3-4 kg. Cells were dissected and incubated in a sterile conical tube containing an enzymatic dissociation buffer under agitation on an orbital shaker (60 rpm) for 90 min at 37 o C. This buffer contains mg/ml elastase (Sigma), 3.0 mg/ml collagenase (CLS Type-I, 204 units/mg, Worthington Biochemical, USA), mg/ml soybean trypsin inhibitor (Type-I-S, Sigma) and 2.0 mg/ml crystallized bovine serum albumin (BSA, Gibco). Following the complete dissolution of the matrix, the sample was recentrifuged at 200 g for 5 min. The pellet was resuspended in growth medium consisting of Dulbecco s modified Eagle s medium (DMEM, Sigma Chemical, st.louis, MO, USA) supplemented with 10% (v/v) FBS, 2 mm L-glutamine (Gibco), 100 units/ ml penicillin (Gibco) and 0.1 mg/ml streptomycin (Gibco). Isolated fibroblasts were allowed to adhere for 1 day in tissue-culture flasks (25 cm 2 ) under a humidified atmosphere of 5% CO 2 and 95% air at 37 o C. Adhered fibroblasts were further cultured in the medium, which was changed at 2-day intervals. The morphologies of adhered fibroblasts were observed under a phase-contrast microscope (TE 2000-U, Nikon, Japan). Fibroblasts at passage 4 were used in this study. Coating on the Nanofiber Sheets. The electrospun PLCL nanofiber sheet was punched round type (10 mm Diameter), washed with ethanol and deionized water and then dried at room temperature before use. The nanofiber sheets were coated with fibronectin, Type-I collagen, and gelatin. The control groups were tissue culture polystyrene (TCP) and uncoated nanofibers. Phosphate buffer saline (PBS)-prewetted nanofibers were immersed in Type-I collagen solution in 1% acetic acid at a concentration of 1 mg/ml at 4 o C overnight. The sheets were also immersed in fibronectin solution in 1x PBS at a concentration of 25 µg/ml and gelatin solution in 1% acetic acid with a concentration of 40 mg/ml at 4 o C for overnight. Subsequently, the coated nanofiber sheets were rinsed with PBS three times to remove residual coating solution before seeding cells. Cell Viability Assay. Cell viability was evaluated using a water-soluble tetrazolium salt (WST) assay (Dojindo Laboratories, Japan), which is based on the ability of living cells to reduce a tetrazolium salt into a soluble coloured formazan product. The culture medium was washed after 2 days and 50 µl of WST solution was added to each well containing 450 µl of DMEM. Cells were then incubated in the dark at 37 o C for 4 h. For each sample solution, 100 µl was transferred to a 96-well plate to measure the absorbance of the formazan product at an optical density (OD) of 450 nm. A blank value from media exposed to cell-free scaffolds was subtracted from each of the experimental values. Cell Morphology and Phenotype Study. After certain time points, the cultured fibroblasts were rinsed with a PBS solution three times and fixed with 4% formaldehyde at room temperature for over 1 h. After thorough washing with DW (deionized water), cells were dehydrated using a graded series of ethanol (50%, 70%, 90%, and 100%) and lyophilized overnight. Completely dried samples were sputtercoated with platinum before viewing under a SEM. Different Density of Cell Seeding. The electrospun PLCL nanofiber sheet was punched round type (10 mm diameter), washed with ethanol and deionized water and then dried at room temperature before use. The fibroblasts were seeded onto the coating material coated nanofiber sheets at each density of cells/0.8 cm 2, cells/0.8 cm 2, cells/0.8 cm 2, and cells/0.8 cm 2. After culturing the cells for 1, 3, 7, 14, and 21 days, the unattached cells were washed and the cells attached on the sheets were further cultured in the medium, which was changed at 2-day intervals. Different Side of Cell Seeding. To assess the effect of the sides on cell growth, fibroblasts were seeded onto the coated 1236 Macromol. Res., Vol. 20, No. 12, 2012

4 Fibroblast Culture on Poly(L-lactide-co-ε-caprolactone) an Electrospun Nanofiber Sheet nanofiber sheets at a density of cells/0.8 cm 2. One group consisted of cells that were seeded on to upper side of the nanofiber sheet at a density of cells/0.8 cm 2 and the other group consisted of cells seeded on to the upper and lower side of the nanofiber sheet each at a density of cells/0.8 cm 2. The total cell volumes of both groups were exactly same ( cells/0.8 cm 2 ). Mechanical Properties Test. Tensile strength, strain, and elastic modulus of the scaffolds were determined by tensiometry (Instron Model 5567 Instron Corp). Unidirectional stress was applied to determine the longitudinal tensile strengths of the grafts. Samples with a length of 10 mm and width of 5 mm width were used to determine the strength. Samples were fixed into the jaws of the tensile machine using grips and measurements were conducted at a crosshead speed of 10 mm/min and analyzed using Blue hill software. E-modulus was calculated by the slope of the initial values of the stress-strain curves using origin software. Statistical Analysis. All the quantitative results were obtained from triplicate samples. Data are expressed as means ± SD. Statistical analysis was carried out using the unpaired Student s t-test. A value of ρ<0.05 was considered to be statistically significant. Results and Discussion Fabrication of PLCL Electrospun Nanofiber Sheet. Biodegradable PLCL electrospun nanofiber sheets were fabricated using a horizontal type electrospinning device. The important electrospinning parameters for the fiber morphology and diameter were polymer concentration, viscosity, applied voltage, needle diameter, the delivery rate of polymer solution, and solvent. 23,24 Several different conditions were used to optimize fabrication of the nanofiber sheets. To determine the best solvent for electrospinning, we used a fixed PLCL concentration (10% w/v) and several kinds of solvents (Figure 3). The effect of solvent type on the electrospun sheets was evaluated and the results are shown in Figure 3. The average of diameter of fibers is 0.9, 1.2, 1.3, and 2.5 µm in THF:DMF (6:4), chloroform, HFIP, and methylene chloride. The fiber size in the solvent THF:DMF (6:4), chloroform and methylene chloride was not homogeneous. Some fusion between the fibers occurred when the sheets were formed. The fibers fabricated in the THF:DMF (6:4) solvent were also not connected. During electrospinning in several time, the fiber fabricated in the chloroform solvent was made bead formation (data not shown). The fibers fabricated in the chloro- Figure 3. SEM images of PLCL electrospun sheets. PLCL 10 wt% solution in THF:DMF (6:4) (A,E,I), chloroform (B,F,J), HFIP (C,G,K), and methylene chloride (D,H,L). Macromol. Res., Vol. 20, No. 12,

5 B. S. Jang et al. Figure 4. SEM images of PLCL electrospun sheets. PLCL 6 wt% solution (A-C), 9 wt% solution (D-F), and 10 wt% solution (G,H) in HFIP. form and methylene chloride solvent were occurred fusion due to residual solvent between fiber and fiber. And also, the fiber size in the methylene chloride solvent was too big at the same conditions. On the other hand, the HFIP solvent produced good result. Under these conditions, the fiber size was homogeneous and no fusion was observed. In addition, the fiber density from the HFIP solvent was higher than the methylene chloride solvents, which expected would increase the physical properties. To determine the best PLCL concentration, we used the HFIP solvent and different PLCL concentrations ranging from 6% to 10% (w/v) (Figure 4). By increasing the concentration, the fiber size also increased. In addition, the 6% mixture had the lowest viscosity, which would result in increased fusions and the creation of nodes. These attributes are not good for cell growth. For the 9% and 10% samples, the fiber size was bigger and no fusion was observed. In addition, the 9% concentration produced a higher fiber density than the 10% concentration because the fiber diameter for the 10% was bigger than 9%. The morphologies of electrospun PLCL sheets were determined by SEM. As shown in Figure 4(D-F), the fiber diameter ranged from 500 nm-1 µm. In addition, the fiber size was homogeneous and no fusion was observed. From the results shown in Figures 3 and 4, 9 wt% PLCL solution in HFIP could produce 5 µm thickness per hour. Based on this result, we made sheets with different thickness (Figure 5). Coating Material for Cell Adhesion. Collagen is the most common protein in animals and there are over 10 kinds of different types. Among them, collagen type I, II, and III are the most common collagen in the humans. These collagens are known as fibrous collagen, because they form long strands. All together, collagen types I, II, and III are 80% to 90% of the collagen in people. Because they are structurally similar, the processes for producing collagen types I, II, and III are in tissues also are similar. 25,26 As shown in Figures 6 and 7, Type-I collagen coating resulted in better cell adhesion compared to the other coating at 1 day and adhesion gradually increased at 3 and 7 days. The same level of cell adhesion was observed for the fibronectin coating as the Type-I collagen coating at 1 day; however, the cell count for the Type-I collagen coating (180,000±3,000) was significantly higher than the fibronectin coating group Figure 6. Cell adhesion test for electrospun PLCL sheets coated with various materials (ELSP: Electrospun without any coating, COL: Type-I collagen, FN: Fibronectin, and GLT: Gelatin). TCP (tissue culture plate) as commercial product was used for positive control. ELSP was also used for negative control. Cell count was determined by WST assay. Values are means±sd, n=3. Figure 5. SEM images of PLCL 9 wt% solution in HFIP electrospun sheets. Cross-section images of scaffolds that were 50 µm thick (A), 30 µm thick (B), and 20 µm thick (C) Macromol. Res., Vol. 20, No. 12, 2012

6 Fibroblast Culture on Poly(L-lactide-co-ε-caprolactone) an Electrospun Nanofiber Sheet Figure 7. DAPI staining of fibroblasts grown on electrospun PLCL sheets that had been coated with various materials for 1 week (blue color is nucleus). The scale bars indicate 500 µm (large square) and 100 µm (small square). Table I. Contact Angles of the Electrospun Sheet Coated with Different Materials a PLCL Type-I Collagen Fibronectin Gelatin Contact Angle (deg.) o ±1.10 o o ±1.97 o o ±1.31 o o ± 2.23 o a Values are means±sd, n=3. (150,000±5,000). Table I shows the contact-angle data of the different materials coated onto the electrospun sheets measured with a surface-analysis instrument. As expected, Type-I collagen showed much better wettability than the other groups because of its hydrophilic attributes. This property will be extremely useful for increasing initial cell adhesion and attachment and explains why the Type-I collagen coated group showed better cell adhesion and proliferation compared to the other groups. The main aim of using collagen as coating material, the Type-I collagen was chosen due to major component of blood vessel for vascular tissue engineering. And also, compared to other ECM proteins such as fibronectin and gelatin, the collagen is associated dynamic tissue such as blood vessel due to much effect on the mechanical strength. 25,26 Cell Density for Cell Proliferation. Figure 8 shows the cell proliferation profiles at different cell densities on electrospun PLCL sheets as a function of culture duration. The Figure 8. Cell proliferation test for Type-I collagen coated onto electrospun PLCL sheets having various cell density vs. time. Cell count was determined using the WST assay. Values are means±sd, n=3. Macromol. Res., Vol. 20, No. 12,

7 B. S. Jang et al. Figure 9. SEM images of Type-I collagen coated electrospun PLCL sheets at various cell densities at 7 days after cell seeding; (A) cells/0.8 cm 2, (B) cells/0.8 cm 2, (C) cells/0.8 cm 2, (D) cells/0.8 cm 2. Images were acquired at a magnification of 500 (left), 1000 (center), and 2000 (right). cell viability was consistently higher in all groups over time. At 1 day after cell seeding, the cell density for the cells/0.8 cm 2 group increased by a factor of 10 relative to the initial cell seeding density. Similar results were also observed when 4'-6-diamidino-2-phenylindole (DAPI) staining was used (Figure 9). Cell growth at 7 days after cell seeding was observed in the SEM analysis. The cell density of A, B, C, and D were cells/0.8 cm 2, cells/0.8 cm 2, cells/0.8 cm 2, and cells/0.8 cm 2, respectively. As shown in Figure 9(C) and (D), cell densities of cells/0.8 cm 2 and cells/0.8 cm 2 were too high. The cells covered the entire surface of the electrospun sheet and blocked the pores, which prevented mass transfer. However, as shown in Figure 9(A) and (B), cell densities of cells/0.8 cm 2 and cells/0.8 cm 2 were sufficient for mass transfer. Under these conditions, the pores were not covered by the cells so mass transfer was still possible. The cell density in Figure 9(B) was higher than Figure 9(A), which is appropriate for a longer cell proliferation period. Cell Seeding of Single Side and Double Side. The initial cell density on the Type-I collagen coated electrospun PLCL sheets were cells/0.8 cm 2 and cells/0.8 cm 2 on the single side and double side, respectively. From Figure 11, we can see that for the single side cell seeding, the cell density only slightly increased and during the cell proliferation period between 3 and 7 days, the single side cell density did not increase significantly. However, the cell density on the double side cell seeding group increased rapidly. This difference may be caused by limited cell-cell interactions due to a lack of free volume on the sheet surface. This will also decrease the possibility for cell spreading. For vessel engineering applications, the 2D electrospun sheet must be rolled to create a 3D shape that mimics native vessels. Because the 3D-type rolled scaffold made from our system will have a larger surface area than the 3D-type tubular scaffold, we confirmed that double side cell seeding is better than single side cell seeding. Mechanical Properties. Uniaxial Tensile Tests: Tensile properties of the Type-I collagen coated electrospun PLCL sheets at various cells density were measured. The average tensile strength, tensile strain, Young s modulus, and failure elongation are summarized in Table II. The combination of cells and Type-I col Macromol. Res., Vol. 20, No. 12, 2012

8 Fibroblast Culture on Poly(L-lactide-co-ε-caprolactone) an Electrospun Nanofiber Sheet Figure 10. DAPI staining of Type-I collagen coated electrospun PLCL sheets at various cell densities vs. time (blue color is nucleus). The scale bars indicate 500 µm (large square) and 100 µm (small square). coated electrospun PLCL sheets without cell seeding and pure electrospun PLCL sheet without any treatment. It is worth noting that a higher elongation and energy at the tension break were observed for the combination of cells and Type-I collagen coated electrospun PLCL sheet, which indicates good deformability and flexibility. Thus, the increase in cell volume resulted in higher tensile strength and Young s modulus due to the increased density of the materials. Conclusions Figure 11. Comparison of cell proliferation on Type-I collagen coated electrospun PLCL sheets with different cell seeding side. Cell count was determined using the WST assay. Values are means ± SD, n = 3. lagen coated onto electrospun PLCL sheet gave rise to a higher modulus when compared to only Type-I collagen Macromol. Res., Vol. 20, No. 12, 2012 In this study, we evaluated the behavior of cells on the PLCL electrospun sheet. Fibroblasts actively attached and proliferated when cultured on the electrospun sheet coated with Type-I Collagen. In addition, when the fibroblast seed density on the electrospun sheet was increased, cell proliferation also increased with time. When cells were seeded on both sides of the electrospun sheet, better cell attachment and proliferation were observed compared to seeding on a single side at the same cell volume and time. In addition, the electrospun sheet cultured with fibroblast showed good mechan1241

9 B. S. Jang et al. Table II. Tensile Properties of Electrospun PLCL Sheets at Various Cell Densities Tensile Stress (MPa) Tensile Strain (mm/mm) Young s Modulus (MPa) Elongation at Break (mm/mm) Electrospun Sheet Collagen Coated Electrospun Sheet cells/collagen Coated Electrospun Sheet after 7 days cells/collagen Coated Electrospun Sheet after 7 days cells/collagen Coated Electrospun Sheet after 7 days cells/collagen Coated Electrospun Sheet after 7 days ical properties. Based on these findings, further studies are being conducted to design a functional tubular vascular scaffold with adequate mechanical properties and architecture to promote cell growth. Acknowledgement. This study was supported by a grant from the Korea Healthcare technology R&D Project, Ministry of Health & Welfare (MOHW), Republic of Korea (A and A110328). References (1) F. Hass, Microsurgery, 6, 59 (1985). (2) L. Xue and H. P. Greisler, J. Vasc. Surg., 37, 472 (2003). (3) T. Gumpenberger, J. Heitz, D. Bauerle, H. Kahr, I. Graz, C. Romanin, V. Svorcik, and F. Leisch, Biomaterials, 24, 5139 (2003). (4) S. J. Lee, J. J. Yoo, G. J. Lim, A. Atala, and J. Stitze, J. Biomed. Mater. Res. A, 83A, 999 (2007). (5) R. D. Sayers, S. Raptis, M. Berce, and J. H. Miller, Br. J. Surg., 85, 934 (1998). (6) T. A. Telemeco, C. Ayres, G. L. Bowlin, G. E. Wnek, E. D. Boland, N. Cohen, C. M. Baumgarten, J. Mathews, and D. G. Simpson, Acta Biomater., 1, 377 (2005). (7) S. H. Kim, S. H. Kim, and Y. H. Kim, Tissue Eng. Reg. Med., 3, 13 (2006). (8) S. N. Lakshmi and C. T. Laurencin, Prog. Polym. Sci., 32, 762 (2007). (9) S. I. Jeong, B. S. Kim, S. W. Kang, J. H. Kwon, Y. M. Lee, S. H. Kim, and Y. H. Kim, Biomaterials, 25, 5939 (2004). (10) S. I. Jeong, B. S. Kim, Y. M. Lee, K. J. Ihn, S. H. Kim, and Y. H. Kim, Biomacromolecules, 5, 1303 (2004). (11) S. I. Jeong, J. H. Kwon, J. I. Lim, S. W. Cho, Y. M. Jung, W. J. Sung, S. H. Kim, Y. H. Kim, Y. M. Lee, B. S. Kim, C. Y. Choi, and S. J. Kim, Biomaterials, 26, 1405 (2005). (12) A. Baji, Y. W. Mai, S. C. Wong, M. Abtahi, and P. Chen, Compos. Sci. Technol., 70, 703 (2010). (13) H. Wu, J. Fan, C. C. Chu, and J. Wu, J. Mater. Sci. Mater. Med., 21, 3207 (2010). (14) Q. P. Pham, U. Sharma, and A. G. Mikos, Biomacromolecules, 7, 2796 (2006). (15) J. M. Lee, G. Y. Tea, Y. H. Kim, I. S. Park, S. H. Kim, and S. H. Kim, Biomaterials, 29, 1872 (2008). (16) H. Liu, X. Li, G. Zhou, H. Fan, and Y. Fan, Biomaterials, 32, 3784 (2011). (17) M. F. Leong, K. S. Chian, P. S. Mhaisalkar, W. F. Ong, and B. D. Ratner, J. Biomed. Mater. Res. A, 89, 1040 (2009). (18) T. Krieg, D. Abraham, and R. Lafyatis, Arthritis Res. Ther., 9, S4 (2007). (19) H. Liu, B. Chen, and B. Lilly, Angiogenesis, 11, 223 (2008). (20) V. R. lyer, M. B. Eisen, D. T. Ross, G. Schuler, T. Moore, J. C. F. Lee, J. M. Trent, L. M. Staudt, J. H. Jr., M. S. Boguski, D. Lashkari, D. Shalon, D. Botstein, and P. O. Brown, Science, 283, 83 (1999). (21) A. Rabinovitch, T. Russell, and D. H. Mintz, Diabetes, 28, 1108 (1979). (22) K. Kobayashi, Y. Imanishi, A. Miyauchi, N. Onoda, T. Kawata, H. Tahara, H. Goto, T. Miki, E. Ishimura, T. Sugimoto, T. Ishikawa, M. Inaba, and Y. Nishizawa, Eur. J. Endocrinol., 154, 93 (2006). (23) Q. P. Pham, U. Sharma, and A. G. Mikos, Tissue Eng., 12, 1197 (2006). (24) Y. Zhang, H. Ouyang, C. T. Lim, S. Ramakrishna, and Z. M. Huang, J. Biomed. Mater. Res. B: Appl. Biomater., 72, 156 (2005). (25) L. Harvey, B. Arnold, Z. S. Lawrence, M. Paul, B. David, and D. James, in Molecular Cell Biology, 4th ed., W. H. Freeman, New York, 2000, Section (26) G. David, Collagen, Protein Data Bank Rutgers University and University of California San Diego, San Diego, 2000, PDB Macromol. Res., Vol. 20, No. 12, 2012