Article pubs.acs.org/journal/abseba
Corona Discharge: A Novel Approach To Fabricate ThreeDimensional Electrospun Nanofibers for Bone Tissue Engineering Wei Song,† Liang Chen,† Joseph Seta,† David C. Markel,†,‡ Xiaowei Yu,§ and Weiping Ren*,†,‡ †
Department of Biomedical Engineering, Wayne State University, Detroit, Michigan 48201, United States Department of Orthopaedic Surgery, Providence Hospital, Southfield, Michigan 48075, United States § Department of Orthopaedics, Shanghai Sixth People’s Hospital, Shanghai 200231, China ‡
S Supporting Information *
ABSTRACT: The electrospinning process produces dense two-dimensional (2D) nanofiber (NF) sheets with small pore size that limits cell infiltration and proliferation. This study aims to fabricate three-dimensional (3D) NF sheets by designing a NF collector mounted with multiple movable needles. The corona discharge effect leads to continuous deposition of 3D polycaprolactone (PCL) NF matrices on the surface of the NF collector. The increase of the pore size, pore volume, and pore interconnectivity of the formed 3D NF sheet was confirmed by scanning electron microscopy, 3D confocal laser scanning microscopy, and micro-computerized tomography, respectively. An increased crystallinity of 3D NFs was observed by thermal and rheological analysis. Furthermore, cell growth on the 3D NF matrices was evaluated using murine pre-osteoblastic MC3T3 cells. When compared with 2D NF matrices, 3D NF matrices demonstrated enhanced cell infiltration, proliferation, and differentiation. We believe that a corona dischargebased NF collector design represents a promising approach to fabricate 3D NF matrices with desirable geometry, and microstructure. This simple, controllable, one-step process may help move forward the clinical translation of electrospun NFs in regenerative medicine. KEYWORDS: three-dimensional nanofiber, electrospinning, corona discharge, porous structure, cellular activity
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INTRODUCTION Bone is an organ with a complex hierarchical structure composed of organic, and inorganic constituents.1 Various types of synthetic bone substitutes have been developed over the past decades to treat bone defects.2 Although these bone substitutes possess sufficient mechanical strength, they often show poor osteoinduction and osteoconduction to engage bone ingrowth.3 In order to augment bone tissue regeneration with enhanced interaction between bone cells and implant materials, numerous attempts have been made to recreate a fibrous structure mimicking the fibrous collagen network of native extracellular matrix (ECM) of the bone.2,4 Electrospun nanofibers (NFs) provide an ideal scaffold for bone tissue engineering due to the nanoscale topography assisting critically in osteointegration.4b,5 Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; aligned NFs form with a rapidly rotating collector and randomly oriented fiber mats with a stationary collector.6 Traditional electrospinning process usually produces dense two-dimensional (2D) fibrous mats over time leading to a compact structure with small pore size and poor interconnectivity that inhibit cell infiltration and proliferation.7 A three-dimensional (3D) NF scaffold with controlled geometry and larger pore sizes is more desirable to facilitate the bone © XXXX American Chemical Society
regeneration and repair with enhanced cell infiltration, proliferation, and differentiation. Several methods to increase the pore size and/or electrospun NF layer thickness have been recently reported.8 Some focused on varying the electrospinning process parameters such as electric charge, external force on spinneret jet, and magnetic field,8a to create 3D fibrous structures. Systems combining insoluble fibers and sacrificial co-fibers,9 salt leaching,7a ice crystals,10 and photopatterning11 have been used to increase the pore size of prepared NFs. Alternatively, the use of a patterned collector provides a promising approach to increase the pore size.12 Advanced NF collection techniques including wet electrospinning,13 rolling or stacking collectors,14 and yarn15 were demonstrated to be suitable for NF mass production. NF scaffolds with larger pore size can be generated using collectors with diversified patterned surface textures.16 However, most of these NFs are randomly wrapped lacking controlled porosity, pore size, pore shape, and scaffold geometry that are essential for bone tissue engineering. Received: January 26, 2017 Accepted: April 17, 2017 Published: April 17, 2017 A
DOI: 10.1021/acsbiomaterials.7b00061 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
tivity) by programmed control of collector movement speed using micro stepper motors.
The working mechanism of the electrospinning process is similar to the plasma discharge phenomenon, in which the NF formation is driven by high static voltage to repulsively deposit onto low potential targets in the form of a solidified jet.6b Under normal circumstances, a charged polymer jet will evenly deposit on a flat surface with equipotential density. However, if a spike projects from the surface, a nonluminous electrical discharge, or point discharge (PD), would favorably build up at the tip of the spike. As a result, the local electric field close to a pointed conductor extends above surrounding objects and maintains strength many-fold higher than that of any surrounding conductors, leading to acceleration of free electrons to a sufficiently high velocity, ionizing neutral air molecules. Therefore, during electrospinning, the charged polymeric jet favorably deposits onto the extended sharp points, as shown in Figure 1a. An ordered NF pattern is
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EXPERIMENTAL METHODS
Materials. Polycaprolactone (PCL, Mw = 70 000−90 ,000), dimethyl sulfoxide (DMSO), calcein, chloroform, and dimethylformamide (DMF) were purchased from Sigma-Aldrich (St. Louis, MO). Alpha-modified Minimum Essential Medium, trypsin, Dulbecco’s phosphate-buffered saline (DPBS buffer), and DiI cell-label solution were purchased from Invitrogen (Grand Island, NY). MC3T3-E1 pre-osteoblast cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA). Quant-iT PicoGreen dsDNA Assay Kit was purchased from Thermo Fisher Scientific (Waltham, MA). Alkaline Phosphatase Activity Colorimetric Assay Kit was purchased from BioVision (Milpitas, CA). Electrospinning of PCL Nanofibers. A polycaprolactone mixture (0.2 mg/mL) was prepared by dissolving PCL into chloroform overnight to homogenize. Additionally, 10 mL of DMF was added to the mixture and stirred for 3 h until it was evenly homogenized. For imaging purposes, Calcein fluorescent labeling dye (8 μg/mL) was mixed with the PCL solution to finalize as PCL−calcein solution for electrospinning. The solution was then loaded into a syringe (5 mL, BD Scientific, Franklin Lakes, NJ) and connected with high-purity tubing (IDEX Health & Science, Oak Harbor, WA) to a blunt tipped (B-D Scientific, San Jose, CA) 26 G1/2 needle (0.6 mm i.d.). The entire syringe, tubing, and needle system was attached to a syringe pump (R100E, Razel Scientific Instruments, St. Albans, VT) with a set flow rate Q. The needle tip was connected using alligator clips to a high voltage supply (ES40P, Gamma High Voltage Research Inc., Ormond Beach, FL). The electrospinning process was performed with the following settings: flow rate (Q) = 1 mL/h, voltage (V) = 19−20 kV, and a needle tip-to-collector plate distance of 10 cm. A static immobile collector plate was utilized as opposed to a spinning collector. The collector was a 3 × 3 × 1 cm foam plate wrapped in aluminum foil and pierced by multiple forward−reverse movable needles. The needles were placed in concentric squares, evenly distributed along the plate. Eleven rounds of electrospinning were conducted at 20 min/round for construction of the 3D scaffold. Embedded needles were moved forward and backward at every round as needed to focus fiber distribution on certain locations. Collected 3D NF scaffolds were placed in aluminum-covered Petri dishes and stored in the dark. Scanning Electron Microscopy (SEM). The PCL 3D NF scaffold was first gold-coated (Gold Sputter, EFFA Coater, Redding, CA), and the morphology of the NFs was then characterized by SEM (JSM6510LV-LGS, Peabody, MA) at 25 kV accelerating voltage. 3D Confocal Laser Scanning Microscopy (CLSM). Samples were analyzed by a color 3D confocal laser scanning microscope (Keyence VK-9700, Itasca, IL), which combines optical, confocal, and scanning electron microscopy. Each measurement forms three types of images: light intensity, color height, and 3D images. The pore volume distribution and surface roughness were measured using VK-Analyzer software (Keyence). For pore volume measurements (color height image, 15000× magnification), about 80 pores at the same height along the z-axis were selected and their volumes measured individually in each scaffold (n = 3). The pore volume distribution was calculated as the percent of a range of pore volumes versus the total pore volume. The surface roughness was represented by arithmetic mean roughness (Ra), which is automatically calculated using eq 1 according to JIS B 0601-1994 surface texture parameter:
Figure 1. Fabrication of polycaprolactone 3D nanofiber scaffold. (a) Electrospinning setup with self-designed needle-pierced collector. (b) Illustration of a cross-sectional view of electrospun fibers built-up between the spinneret and needle-collector. (c) Photograph of collected fibers deposited along needles and platform during electrospinning. (d) Multiple rounds (5, 10, and 20) to form 3D nanofibers on needle collectors.
achieved by electrospinning onto numerous projections, as shown in Figure 1b. Our results demonstrate the feasibility of fabricating more sophisticated 3D NF scaffolds with tailored structures using this novel PD collector with grids of projections. In this study, we designed a NF collector composed of numerous movable needles where NFs would favorably deposit according to the PD mechanism. By gradually pushing the needles forward, NFs continually build on the top of the needle grid layer by layer. NFs constantly deposit along the direction of the moving needle, allowing a much thicker 3D NF scaffold to be produced. The influence of 3D NF scaffolds with higher porosity and interconnected structure on cell infiltration and proliferation was assessed in vitro. We chose polycaprolactone (PCL) in this study to fabricate electrospun NF scaffolds. PCL is a biodegradable polyester that has been approved by the U.S. Food and Drug Administration for many tissue engineering applications because of its excellent biocompatibility. We have also demonstrated the use of PCL as a NF material in our previous report.17 Our novel PD collecting method is a simple approach to prepare 3D NF matrices with controlled pore size and thickness. This technology has great potential for the preparation of NF scaffolds with defined geometry and microstructures (pore size, pore shape, and pore interconnec-
Ra =
1 S
∫0
S
|f (x)| dx
(1)
where S indicates the whole measuring length and x indicates the distance from measuring point to point 0. Differential Scanning Calorimetry (DSC). The calorimetric measurement was performed on a Q2000 differential scanning calorimeter (TA Instruments Inc., New Castle, DE) in a dry nitrogen atmosphere. The instrument was calibrated with standard Indium. All samples (about 8 mg) were heated to the elevated temperature of 80 B
DOI: 10.1021/acsbiomaterials.7b00061 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering °C, held for 5 min, and then quenched to room temperature. The DSC thermograms were recorded at a heating and cooling rate of 5 °C/min. The crystallization temperatures (Tc) and the melting temperatures (Tm) were taken as the temperatures at the maximum and the minimum of both endothermic and exothermic peaks, respectively. Macro-Tensile Testing. NF scaffolds were cut into 3.8 × 1.3 cm rectangular dumbbell-shaped tensile testing samples with cross sectional width of 0.9 cm for testing region. Sample thickness was measured with an iGAGING caliper (iGAGING, San Clemente, CA). Macro-tensile measurements were performed using an electromechanical universal tester (Instron, Elancourt, France). All samples were mounted between holders at a distance of 1 cm. Tensile testing was conducted at a rate of 0.1 mm/s. A photograph of the experimental setup is shown in Figure 2Sa. Rheology Characterization of Electrospun Nanofibers. An AR-G2 rheometer (TA Instruments Inc., New Castle, DE) with an environmental temperature chamber and torsion clamp geometry was used for the rheological characterization of NF scaffolds. Oscillatory stress sweep and frequency sweep were employed. The stress sweep was performed while holding the temperature (25 °C) and frequency (2π rad/s) constant and increasing the stress level. The linear viscoelastic region (LVR) from 100 to 10 000 Pa was determined as the safe-region without structural breakage from oscillatory force. Samples were subjected to an oscillatory stress sweep and the corresponding storage modulus (G′) and loss modulus (G″) measured. The frequency sweep was performed at a fixed force corresponding to a point within the LVR as frequency increased from 0.1 to 100 rad/s, and the plots of G′ and G″ toward frequency were obtained. An oscillatory temperature ramp was performed to study the thermal behavior of NFs and determine the heat distortion temperature (HDT). The samples were scanned in a temperature ramp in oscillatory mode from 25 to 80 °C at a rate of approximately 2 °C/min with a constant oscillatory stress of 1000 Pa and a constant frequency of 2π rad/s. G′ and G″ were recorded at an interval of 30 s. Micro-Computerized Tomography (μ-CT). Each scaffold was scanned with the VivaCT 40 (SCANCO Medical AG, Fabrikweg, Brüttisellen, Switzerland) using a voltage of 45 kVp and a current of 177 μA at 10 μm resolution. A cylindrical volume of interest with 785 slices was selected for the 3D scaffold, and 400 slices were selected for the homogeneous scaffold. The morphology of the scaffolds was determined using software from the manufacturer to measure the average wall thickness (Tb.Th), average pore size (Tb.Sp), porosity, pore size distribution, and interconnectivity. Validation of μ-CT in measuring the porosity of NF scaffolds is achieved by 3D CLSM as described previously, showing
