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Electrohydrodynamic jet process for pore-structurecontrolled 3D fibrous architecture as a tissue regenerative material: Fabrication and cellular activities Minseong Kim, and GeunHyung Kim Langmuir, Just Accepted Manuscript • DOI: 10.1021/la501080c • Publication Date (Web): 27 Jun 2014 Downloaded from http://pubs.acs.org on July 2, 2014
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Electrohydrodynamic jet process for pore-structure-controlled 3D fibrous architecture as a tissue regenerative material: Fabrication and cellular activities
Min Seong Kim and GeunHyung Kim*
Department of Biomechatronic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Republic of Korea 440-746
*
Corresponding author:
Prof. GeunHyung Kim, Tel.: +82-31-290-7828, E-mail address:
[email protected] Postal address: 2066 Sebu-Ro, Jangan-Gu, Suwon-Si Gyeonggi-Do, South Korea
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Abstract In this study, we propose a new scaffold fabrication method, “direct electro-hydrodynamic jet process,” using the initial jet of an electrospinning process and ethanol media as a target. The fabricated three-dimensional (3D) fibrous structure was configured with multi-layered micro-sized struts consisting of randomly entangled micro/nanofibrous architecture, similar to that of native extracellular matrixes (ECMs). The fabrication of the structure was highly dependent on various processing parameters, such as the surface tension of the target media, and the flow rate and weight fraction of the polymer solution. As a tissue regenerative material, the 3D fibrous scaffold was cultured with pre-osteoblasts to observe the initial cellular activities in comparison with a solidfreeform fabricated 3D scaffold sharing a similar structural geometry. The cell-culture results showed that the newly developed scaffold provided outstanding micro-cellular environmental conditions to the seeded cells (about 3.5-fold better initial cell attachment and 2.1-fold better cell proliferation).
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1. Introduction Micro/nanofibers have been used widely in various biotechnologies, such as biosensors, artificial organ components, tissue engineering scaffolds, and drug delivery.1,2 In particular, the use of micro/nanofibers in biomedical scaffolds has been very extensive because the fibers are topographically and spatially very similar to native extracellular matrixes (ECMs), which can support the proliferation and differentiation of cells into configurations that are analogous to those of native tissues and organs.3-7 As a biomedical scaffold, electrospun micro/nanofibers can be fabricated mainly into twodimensional (2D) flat surfaces. However, compared to 3D fibrous structures, the 2D nanofibers cannot induce fully 3D cellular morphology and differentiation under physiological conditions8; moreover, the small pore sizes, which are very difficult to control during the electrospinning process, has been a serious limitation due to the low cell infiltration into the thickness of the scaffold, resulting in spreading only on the fiber surface or limited infiltration within the fiber mat.9,10 Recently, to achieve a 3D fibrous structure with controllable pore structure, various spinning techniques have been proposed, such as wet electrospinning,11,12,13 low-temperature electrospinning,14 electrospinning with a blowing agent,15 electrospinning using electrostatic repulsion,16 and centrifugation.17 The proposed methods have provided outstanding developments to achieve 3D fibrous structures, but there are still some limitations, such as insufficient 3D construction and low controllability of pore size, to obtaining a realistic biomedical scaffold for various tissue engineering applications. Also, Kumar et al18 reported that 2D electrospun poly(ε-caprolactone) (PCL) nanofibers provoke human bone marrow stromal cells (hBMSCs) into an extended, branched morphology, which may be associated with osteogenic differentiation outcomes in nanofiber scaffolds, but provide a low cell proliferation rate. In comparison, a 3D layer-by-layer PCL scaffold fabricated by the solidfreeform method provides enhanced hBMSC proliferation, but low osteogenic differentiation, due to the high surface area of the 3D structure, which can result in a large area for proliferation. Based on their findings, Kumar et al18 proposed a new concept for an ideal scaffold for bone tissue regeneration that involves a structurally 3D porous shape, like a solid-freeform fabricated 3D scaffold, but the struts can consist of micro/nanofibers, which can provide a large number of cell attachment sites to facilitate cell proliferation with simultaneous support of osteogenic differentiation. Based on this excellent concept, we aimed to realize a highly potential 3D scaffold consisting of micro/nanofibers. To achieve this kind of scaffold, an innovative direct electrohydrodynamic jet process (EJP) using ethanol as a target media was proposed. Through the process, a 3D structure like a micro-sized mesh was composed with randomly and evenly distributed micro/nanofibers, which could mimic the fibrous structure of ECMs and induce stereoscopic cellular activities. A fabrication process using ethanol media as a target bath was validated with various
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processing parameters. Also, to observe the potential of the scaffold as a tissue regenerative material, the initial attachment and proliferation of pre-osteoblasts were evaluated.
2. Experimental Section Materials: Poly(ε-caprolactone) (density: 1.135 g/cm3; molecular weight (Mn): 90 000 g/mol; melting point: 60°C) was obtained from Sigma–Aldrich Co. (St. Louis, MO, USA). To fabricate the scaffold, various weight fractions (8 – 16 wt%) of PCL in a 20:80 solvent mixture of methylene chloride (MC; surface tension: 28.1 mN/m) and dimethyl formamide (DMF; surface tension: 37.1 mN/m) (Junsei Chemical Co., Tokyo, Japan) were used with a 16-G electrospinning nozzle and a 20-mL glass syringe. As a target media in the bath, 99% ethanol (EtOH; surface tension: 22.1 mN/m; Duksan, Korea) and water (surface tension: 72.9 mN/m) were used.
Fabrication of a 3D scaffold consisting of micro/nanofibers: Various electric fields (1.2 – 2.6 kV/cm) and flow rates (0.1 – 0.4 ml/h) were used. For the fabrication of the fibrous scaffolds, an electrospinning system was connected to a three-axis robot (DTR3-2210-T-SG, DASA Robot, South Korea). The machine moved automatically according to the path designed by a CAD model. The moving speed of the nozzle was set to 10 mm s-1. As a target, an EtOH media was used with a grounded copper plate immersed in the bath. After dispensing the 3D fibrous structures, the EtOH was removed with water. For drying, the 3D fibrous structures were vacuum freeze-dried using a freeze dryer (SFDSM06; Samwon, Busan, South Korea) at –76°C for 1 day. The flow rate of the PCL solution was controlled using a syringe pump (KDS 230; KD Scientific, Holliston, MA, USA). A power supply (SHV300RD-50K; Convertech, Seoul, South Korea) was used to produce the electric field. The temperature during the electrospinning was 28°C and the humidity was 36 ± 3%. To fabricate a control consisted of microsized struts, PCL powder was moved to the heating barrel (110 oC) of the plotting system. To stably attach between the struts of the layers, the extruding struts over the previous layer were faintly pressed, so that the gap between the nozzle tip (diameter = 250 µm) and the previous layer was set about 92% compared to the diameter of extruded struts. The moving speed of the plotter and extruding pneumatic pressure were set as 3 mm s-1 and 673 ± 31 kPa, respectively.
Characterization: SEM (SNE-3000M, SEC, Inc., South Korea) was used to characterize the surface morphology of micro/nanofibrous struts. Before observation, the 3D fibrous scaffolds were completely dried using the freeze dryer at –76°C for 1 day and then sputter-coated with gold. Sample preparation and measurements were performed according to the manufacturer’s instructions. Using the SEM images, the morphology of single cells was evaluated after culturing for 4 h and 1 day. Also, Image J software (National Institutes of Health, Bethesda, MD, USA) was used to measure the area of
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F-actin and the cell number from DAPI/phalloidin result. The porosity of the scaffolds was calculated using the equation, 1 − M / (ρV), where M represents the mass of the scaffold, ρ is the density of PCL (1.135 g/cm3), and V is the volume of the structure (assumed to be rectangular). The ability for protein absorption was measured using a bicinchoninic acid (BCA) protein assay (Pierce Kit; Thermo Scientific, Waltham, MA, USA). Samples (diameter = 8 mm and weight = 3 mg ± 1.5 mg) were placed in 24-well plates containing Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone) and incubated at 37°C for 1, 4, 6, and 12 h. Specimens were washed with PBS and lysed with 0.1% Triton X-100. An aliquot of the lysate (25 µL) was added to 200 µL of BCA working reagent, and the mixture was incubated for 30 min at 37°C. The absorbance at 562 nm was determined using a plate reader. Samples incubated in serum-free medium were used as blanks. The protein adsorption was calculated as the mean ± the standard deviation (n = 5). In vitro cell culture: Scaffolds (5 mm × 5 mm) were sterilized with 70% ethanol and ultraviolet (UV) light, and then placed in culture medium overnight. Mouse pre-osteoblast cells (MC3T3-E1; ATCC, Manassas, VA, USA) were used to evaluate the behavior of cells cultured on the scaffolds. The cells were cultured for up to four passages in 24-well plates containing α-minimum essential medium (Life Sciences, USA) containing 10% fetal bovine serum (Gemini Bio-Products, USA) and 1% antibiotic/antimycotic (Cellgro, USA). The cells were collected by trypsin–ethylenediaminetetraacetic acid (EDTA) treatment, seeded onto the scaffolds at a density of 1 × 105 per sample, and incubated at 37°C in an atmosphere of 5% CO2. After 4 h, 1 day, and 3 days of cell culture, the scaffolds were subjected to diamidino-2-phenylindole (DAPI) fluorescent staining to detect cell nuclei. Phalloidin (Invitrogen, Carlsbad, CA, USA) staining was performed to visualize the actin cytoskeleton of proliferated cells. The cell number identified by DAPI staining was measured from the surface and cross-section fluorescence images of the scaffolds using ImageJ. The stained scaffolds were analyzed under a microscope (TE2000-S; Nikon, Tokyo, Japan) equipped with an epifluorescence attachment and a SPOT RT digital camera (SPOT Imaging Solutions, Sterling Heights, MI, USA). Cell proliferation was determined by MTT measurement (Cell Proliferation Kit I; Boehringer Mannheim, Mannheim, Germany). The MTT-assay was established on cleavage of the yellow tetrazolium salt MTT by mitochondrial dehydrogenases in viable cells to generate purple formazan crystals. Cell/scaffolds were put in incubation with 0.5 mg mL-1 MTT for 4 h at 37 oC. The absorbance at 570 nm was measured using a microplate reader (EL800; Bio-Tek Instruments, Winooski, VT). Five samples were used for each incubation period, and each test was measured in triplicate.
Statistical analyses: All data are presented as the mean ± SD. Statistical analyses were performed
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using SPSS software (ver. 20.0; SPSS, Inc.), and consisted of single-factor analyses of variance (ANOVAs). In all analyses, p*
