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Fabrication and design of bioactive agent coated, highly-aligned electrospun matrices for nerve tissue engineering: Preparation, characterization and application
Accepted Manuscript Title: Fabrication and Design of Bioactive Agent Coated, Highly-Aligned Electrospun Matrices for Nerve Tissue Engineering: Preparation, Characterization and Application Author: Sang Jin Lee Min Heo Donghyun Lee Dong Nyoung Heo Ho-Nam Lim Il Keun Kwon PII: DOI: Reference:
S0169-4332(17)30552-4 http://dx.doi.org/doi:10.1016/j.apsusc.2017.02.179 APSUSC 35288
To appear in:
APSUSC
Received date: Revised date: Accepted date:
13-12-2016 16-2-2017 19-2-2017
Please cite this article as: S.J. Lee, M. Heo, D. Lee, D.N. Heo, H.-N. Lim, I.K. Kwon, Fabrication and Design of Bioactive Agent Coated, Highly-Aligned Electrospun Matrices for Nerve Tissue Engineering: Preparation, Characterization and Application, Applied Surface Science (2017), http://dx.doi.org/10.1016/j.apsusc.2017.02.179 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fabrication and Design of Bioactive Agent Coated, Highly-Aligned Electrospun Matrices for Nerve Tissue Engineering: Preparation,
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Characterization and Application
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Sang Jin Lee1, Min Heo1, Donghyun Lee1, Dong Nyoung Heo2, Ho-Nam Lim2, Il Keun
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Kwon2,∗
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Department of Dentistry, Graduate School, Kyung Hee University, 26 Kyungheedae-ro,
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Dongdaemun-gu, Seoul 02447, Republic of Korea 2
Department of Dental Materials, School of Dentistry, Kyung Hee University, 26 Kyungheedae-ro,
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Dongdaemun-gu, Seoul 02447, Republic of Korea
* Correspondence to Il Keun Kwon, Ph. D. Department of Dental Materials, School of Dentistry, Kyung Hee University, 26
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Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea Tel.: +82-2-961-0350
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E-mail address: [email protected] (Il Keun Kwon).
Abstract
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In this study, we designed highly-aligned thermoplastic polycarbonate urethane (PCU)
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fibrous scaffolds coated with bioactive compounds, such as Poly-L-Lysine (PLL) and Poly-LOrnithine (PLO), to enhance cellular adhesion and directivity. These products were
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characterized by scanning electron microscope (SEM) analysis which demonstrated that highly aligned fiber strands were formed without beads when coated onto a mandrel rotating
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at 1800 rpm. During in vitro cell test, PLO-coated, aligned PCU scaffolds were found to have significantly higher proliferation rates than PLL coated and bare PCU scaffolds. Interestingly,
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dental pulp stem cells (DPSCs) were observed to stretch along the longitudinal axis parallel to the cell direction on highly aligned scaffolds. These results clearly confirm that our
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strategy may suggest a useful paradigm by inducing neural tissue repair as a means to
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remodeling and healing of tissue for restorative procedures in neural tissue engineering.
Keywords: electrospinning, aligned nanofiber, dental pulp stem cell, neural tissue engineering
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1. Introduction
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For a long time, human nerve diseases such as peripheral nerve injury (PNI) have been a significant concern to humanity all over the world. PNI commonly occurs with physical
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injuries suffered during construction/transportation accidents, natural disasters, military
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service injuries, and other trauma [1, 2]. Approximately 360,000 patients suffer from upper extremity paralytic syndromes annually in the Unites States, and over 300,000 people suffer
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peripheral nerve injuries in Europe, annually [1]. To solve this issue, many clinical and bioengineering researchers have been working towards repairing damaged neural tissue using
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polymeric scaffolds [3-6].
Tissue engineering strategies have received a great deal of attention for use in reconstruction
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of damaged nerves by providing a cell-growth promoting environment [7]. Amongst the
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many bioengineered approaches, aligned electrospun nanofibrous scaffold made by electrospinning (ELSP) is a promising tactic because the fibrous structure mimics that of native neural extracellular matrix [8-10]. This scaffold is also able to regulate cellular behaviors for cell attachment or migration, nutrient transportation, and signal transduction [11]. In general, synthetic polymeric fiber scaffolds have been useful for nerve tissue repair on account of their potent mechanical properties [12, 13]. However, their capabilities for supporting cell proliferation, migration, and viability are limited due to their crystalline and hydrophobic properties [14-16]. Thus, hydrophobic platforms should be converted to hydrophilic substrates to promote cellular activity. In this case, hydrophobic scaffolds can be readily adapted to provide for hydrophilic surfaces via coating of bioactive agents [16-18].
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In this study, we designed a highly aligned thermoplastic polycarbonate urethane (BionateⓇ II, PCU) fibrous scaffolds coated with bioactive compounds such as Poly-L-Lysine (PLL) and Poly-L-Ornithine (PLO) to enhance cellular behaviors such as adhesion and proliferation. The PLL and PLO are cationic bioactive compounds used in neuroscience [19, 20]. These are
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hydrophilic agents that alter the surface properties of hydrophobic scaffolds to promote
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cellular adhesion, migration, and proliferation. This supports neurite growth to form a network for nerve cells [21, 22]. Furthermore, These are also able to promote neural cell
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differentiation [23, 24]. As for the substrate, PCU is a Food and Drug Administration (FDA)
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approved commercially-available, medical-grade polymer that has been extensively used in long-term implants due to its bio-stability, biocompatibility, and appropriate mechanical
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properties [14, 25, 26].
The major aim of this study is to determine not only the effectiveness of the scaffold for
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supporting cellular adhesion and proliferation behavior, but also to determine the interaction of the scaffold with dental pulp stem cell (DPSCs). The fabricated scaffolds were
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characterized by scanning electron microscopy (SEM), water contact angle, X-ray
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photoelectron spectroscopy (XPS) analysis. Finally, in vitro cell proliferation was investigated. Additionally, directional cell morphology was visualized by confocal laser scanning microscopy (CLSM). This preliminary study will pave the way for future investigations.
2. Materials and methods 2.1 Materials Poly-L-Lysine hydrobromide (Mw range 500-2,000), FITC labeled Poly-L-Lysine (Mw range 30,000-70,000), and Poly-L-Ornithine hydrobromide (Mw > 100,000) were purchased from Sigma-Aldrich (St. Louis, MO). N,N-Dimethylformamide (99.5%), Tetrahydrofuran
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(99.5%) were purchased from JUNSEI (JUNSEI CHEMICAL Co. Ltd., Japan). Commercially available thermoplastic polycarbonate polyurethane (Bionate II®, 80A) was synthesized by DSM Biomedical (Berkeley, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate buffered saline (PBS), fetal bovine serum (FBS),
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trypsin–EDTA, and penicillin streptomycin were purchased from Gibco BRL (Invitrogen Co.
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Ltd, Carlsbad, CA, USA). Deionized-distilled water (DDW) was produced by an ultrapure
analytical grade and used without further purification.
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water system (Puris-Ro800; Bio Lab Tech., Korea). All other reagents and solvents were of
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2.2 Preparation of highly aligned thermoplastic polycarbonate urethane scaffolds
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The fibrous scaffolds were prepared using ELSP according to our previously described with some modification [27-30]. Briefly, PCU compounds were dissolved in mixed DMF/THF
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(5:5) solvent to produce an 8 w/v % solution. For ELSP, the dissolved solution was loaded into a luer-lock syringe attached to a 20 G metal blunt needle and electrospun on an
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aluminum foil covered rotating mandrel at 20 kV using a high-voltage DC power supply (Nano NC, Korea) with a 1 ml/h feed rate (KDS-200, KD Scientific Inc.) and a 15 cm needle
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tip-to-collector distance. For different conditions of alignment, the mandrel speed was varied between 100, 900, and 1800 rpm, respectively. Finally, the resultant PCU nanofiber was dried overnight under vacuum to remove any residual solvent.
2.3 Poly-L-lysine, FITC labeled Poly-L-lysine, and Poly-L-ornithine coating on fibrous scaffolds For physical adsorption of bioactive agents on substrates, we applied using the previously described methods with some modification [31]. Cover glasses and PCU fibrous scaffolds (48 well size) were submerged in 1 mL of PBS solutions containing either 500 ug/mL of PLL,
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FITC labeled PLL, and PLO for 24 h at room temperature with gentle shaking. These were then washed three times in 10 mL of PBS.
2.4 In vitro DPSCs adhesion and proliferation test on scaffolds
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Dental pulp stem cells (DPSCs) were donated by the Department of Oral and Maxillofacial
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Surgery of Kyung Hee University Dental Hospital. Prior to cell culture on scaffolds, specimens were sterilized with UV lamp on a clean bench. DPSCs were cultured in DMEM
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supplemented with 10% FBS and 1% penicillin–streptomycin in a 5% CO2 incubator at 37 °C. The DPSCs were drop seeded onto the scaffolds at a density of 2 x 104 cells per well. After 2
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h, medium was filled onto the scaffolds in each culture plate, respectively. Cell adhesion was
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determined after 2 days of culture and proliferation was determined at 1, 3, and 7 days by using the cell counting kit (CCK-8) assay (n = 4). The absorbance of the medium was
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measured at 450 nm using a microplate reader (ELISA, Bio-Rad, Hercules, CA, USA). These experiments were repeated in triplicate. For F-actin staining, the proliferated cells on scaffold
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groups were fixed with 3.9% formaldehyde for 1 h. After that, these were washed with PBS followed by staining with 1 mL of Oregon green 514 phalloidin (1:200) and 4′,6-diamidino-
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2-phenylindole (DAPI) (1:1000) solution.
2.5 Analysis equipment
The surface morphologies of PCU nanofibers were characterized using a scanning electron microscope (SEM, Hitachi S-2300, Japan) at an acceleration voltage of 15 kV. All samples were dried at room temperature, and then sputter-coated with gold by an IB-3 (Eiko, Japan) sputter coater for 10 min. The fibrous angle characterization was carried out using image analysis software (Eyeview-Analyzer (Digiplus, Korea)). Surface water contact angle measurements were performed using a contact angle meter (Phoenix 150, SEO, Korea) using
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8-µL of distilled water at room temperature. X-ray photoelectron spectroscopy (XPS) was performed with a K-Alpha instrument (Thermo Electron, UK) to evaluate the surface elements of the scaffolds. The visualization of the FITC labeled PLL coating on PCU
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CLSM (Eclipse E600W, Nikon, Tokyo, Japan) after staining.
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scaffolds and the morphology of the cells growing on the scaffolds were analyzed using
2.6 Statistical analysis
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All experiments were carried out in triplicate. All values are expressed as mean ± standard deviation. Multiple comparisons of groups were analyzed using two-way analysis of variance
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(ANOVA) followed by Dunnett's T3 post hoc paired comparison test. Statistical analysis was
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performed using PASW Statistics 18 software (SPSS, Inc., Chicago, IL). A value of P < 0.05 is considered to be statistically significant denoting “*”. The statistical analyses are presented
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3. Results
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within figure legends.
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3.1 Characterization of fabricated PCU scaffolds (SEM) For neural tissue regeneration, aligned PCU scaffolds were fabricated by ELSP procedure. The resultant scaffolds were then characterized by SEM analysis (Fig. 2a). To obtain a highly-aligned fiber surface, we controlled the rotating mandrel speed at 100, 900, and 1800 rpm, respectively. The fiber strands obtained from the 100 and 900 rpm mandrel speeds showed poor alignment. On the other hand, unidirectional fiber strands were obtained from the 1800 rpm condition. The average angle of each fabricated scaffold relative to the vertical axis was measured based on the rotating mandrel speed (Fig. 2b). This measurement indicated that fiber strands were more highly aligned when the speed increased. From these results, we selected the 1800 rpm condition for fabrication of PCU scaffolds used for in vitro
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assessments. We hypothesized that highly aligned fibrous scaffolds may influence the directional stretch of neuro-potential cells.
3.2 Surface characterization of PLL and PLO coated PCU fibrous scaffolds
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To confirm the different surface characteristics of PLL and PLO coated fibrous scaffolds,
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water contact angle and XPS analysis was carried out. Fig. 3 shows the results of water contact analysis. The non-coated random and aligned PCU fibers did not absorbed any water.
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This indicates that the PCU scaffold is highly hydrophobic. However, the surface treated PCU scaffolds absorbed water within 30 sec. The random PCU fiber provided for more rapid
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water absorption than the aligned PCU fiber. This result is due to the decrease in pore size
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because the surface topography of these two scaffolds is different due to the different fabrication processes. This may have a direct influence on the surface property of the PCU
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scaffolds [32, 33]. Based on the water contact experiment, we analyzed surface elements of manufactured fibrous scaffolds by XPS. As shown in Fig. 4, both random and aligned PCU
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nanofibers did not exhibit N1s peaks. However, the PLL and PLO coated PCU, both random and aligned nanofibers, presented characteristic N1s peaks. This phenomenon is due to the
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presence of the amine groups of PLL and PLO which were well introduced onto bare PCU scaffolds. This indicates that the physical coating of bioactive agents was successfully performed in this study. Interestingly, the N1s were slightly increased for the PLO coating as compared to the PLL coating. From these results, we hypothesized that the PLO coated PCU scaffolds may be more suitable for cell proliferation than PLL coated PCU scaffolds.
3.3 Visualization of FITC labeled PLL coated on PCU fibrous scaffolds In order to confirm the formation of a uniform coating of the bioactive agent, we visualized FITC-labeled PLL coated random and aligned PCU scaffolds by confocal laser scanning
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microscopy. As shown in Fig. 5a, the entangled fiber morphology displayed in random PCU scaffold was well observed. On the other hand, the fibrous strands were observed to be well aligned in highly aligned PCU scaffolds. This result indicates that the bioactive agent is uniformly coated on both random and aligned PCU scaffolds as well as alignment may affect
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directional cell behavior on scaffolds [7, 11].
3.4 Characterization of DPSCs adhesion on cover glass with and without coating
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To investigate the effectiveness of PLL and PLO coatings for promoting cellular adhesion, cell viability was confirmed using the CCK-8 assay both with and without bioactive agent
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coatings on glass coverslips after 2 days of culture (Fig. 6a and b). As shown in Fig. 6a, the
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absorbance was higher in surface coated cover glass as compared with bare cover glass. This means that PLL and PLO promote cell adhesion. Interestingly, DPSCs had significantly
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higher adhesion on PLO coated cover glass as compared to PLL coating. This result implies that DPSCs can be well adhered and may proliferate more effectively on the PLO coated
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group as compared to the PLL coated group.
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3.5 Characterization of DPSCs proliferation on aligned PCU scaffolds Based on the cell adhesion test, the cell proliferation rate of DPSCs was verified on aligned PCU scaffolds both with and without bioactive agents. As shown in Fig. 7b, after 1 day in culture, all groups exhibited a similar proliferation rate. However, after 4 and 7 days of culture, PLL and PLO coated scaffolds showed a significantly higher proliferation rate than bare scaffolds. This is due to PLL and PLO interactions with the DPSCs. Similar to the cell adhesion test results (Fig. 6), PLO coated PCU scaffolds had higher cell proliferation than PLL coated PCU scaffolds (Fig. 7b). This demonstrates that PLO provides for superior DPSCs attachment than PLL in this condition.
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3.6 Characterization of cellular behavior of DPSCs on bioactive agent coated random and aligned PCU scaffolds The shape and morphology of DPSCs on random and aligned PCU nanofiber scaffold both
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with and without bioactive agents was examined after 1 and 7 days of culture, respectively.
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Fig. 7a and S1 showed different cell morphologies for random and aligned PCU nanofibers. CLSM images show the actin cytoskeleton morphologies of DPSCs. The cytoskeleton
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displays random entanglement on random PCU nanofibers from 1 to 7 days of cell culture (Fig. S1). However, DPSCs presented a stretched, spreading morphology when grown on
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aligned PCU nanofiber groups at all culture time-points (Fig. 7a, white arrow). This
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phenomenon indicates that aligned fibers provide a guide for cell growth by presenting an elongated environment as compared to random PCU nanofibers [34, 35]. Interestingly, a
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greater number of DPSCs were observed for PLL and PLO coated nanofiber scaffolds than bare scaffolds regardless of alignment. This result corresponds with the cell proliferation test
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results (Fig. 7b). These results suggest that cell adhesion and cell morphology were affected by the substrates environments both with and without bioactive coatings. This may help
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neural potential cells with elongation and stretching along the longitudinal axis, parallel to the cell direction, which is the morphology observed in neural tissue [34, 36, 37].
4. Discussion
In the field of neural tissue engineering, implantable and biocompatible scaffolds are highly desirable for maintaining a biomimetic environment for neural cell migration [38]. In this case, electrospun, aligned, fibrous scaffolds are well suited for this application because of their highly-oriented fibers, which can induce directional cell migration. These also provide for uniform cell alignment, highly striated extracellular matrix, and guided cell outgrowth
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during neural tissue formation [11, 39]. Thus, in this study, we devised a highly aligned fibrous scaffold and coated it with bioactive agents to promote cell adhesion and proliferation (Fig. 1). During ELSP, highly-aligned fiber scaffolds were formed when fiber strands were collected on a rotating mandrel at a controlled speed [12]. By testing different mandrel speeds,
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we obtained highly aligned PCU fiber scaffolds at 1800 rpm which provided for cell
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extension (Fig. 2). To manufacture fiber mats, we used DMF/THF blended solvent for preparation of PCU ELSP solution. This allows for the generation of a uniformly formulated
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fibrous morphology. DMF/THF solvents are able to clearly dissolve PCU compounds. This mixture has been well employed in our previous studies [28-30].
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The topography guidance approach, by use of aligned fibers, can control cellular migration
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and promoting directional orientation [40]. Alignment of the fibers greatly influences the cellular growth behavior and outstandingly controls cell directional morphology. Additionally,
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the cells grown on the aligned scaffolds have increased proliferation rates with reinforced cell-matrix interactions as compared with cells grown on random substrates [41]. To promote
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nervous tissue regeneration, a topographical strategy can play a pivotal role for promoting cell adhesion, alignment, spreading, and morphological changes [42]. However, the exact
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mechanisms of topography-dependent neural cell responses are still unclear [43]. Nevertheless, many bio-engineering researchers have made an effort to illuminate the cellular mechanisms of this alignment effect which occurs on oriented fibrous scaffolds. Chew et al. reported that the culturing of cells on aligned fibers lead to significant up-regulation of the myelin-specific gene as compared to cells cultured on randomly oriented fibers. This suggests that the alignment and elongation of human neural cells on aligned fibers can promote their maturation as compared to those grown on randomly oriented fibers. They concluded that integrin may interact with the cytoskeleton or mediate myelin-related gene expressions [37]. In another study, Wang et al. demonstrated that neural progenitor cells expanded faster to
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promote proliferation on aligned nanofibers as compared with those grown on randomly oriented nanofibers [36]. They revealed that there was a higher level of cyclin D1 and CDK2, two downstream genes of ß1 integrin/MAPK signaling, on aligned nanofibers as compared to those on randomly oriented nanofibers. The ß1 integrin is well known to transmit cell
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signaling to mediate cell proliferation, differentiation, and migration [44]. Based on these
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findings, we determined that our highly-aligned nanofibers are selective for nerve tissue regeneration (Fig. 2).
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In the present study, we used PCU as it is a commercially available polymer with excellent mechanical properties for use in the biomedical engineering field [14]. In neural tissue
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engineering, neuro-potential materials are required to remain for long periods of time. These
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should be also manufactured from bio-stable materials. For these reasons, we choose PCU polymer as an adoptable biomaterial for neural scaffold generation. However, PCU is a
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hydrophobic synthetic polymer which may impede cell proliferation, migration, and viability. This is due to its crystallinity and hydrophobic properties. Thus, surface treatment of these
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scaffolds should be carried out for encouraging a cell favorable environment [16-18]. This issue can surmount through attachment of bioactive molecules to potentially guide and
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enhance cell adhesion and proliferation on the scaffolds [13, 15]. For this reason, we utilized PLL and PLO coatings to provide for favorable sites with improved adhesion and proliferation of neuro-potential DPSCs [19, 21, 22, 24, 45-48]. Although the two substances have a similar property, we anticipated that their bioactivity and cellular activity may be different. We verified the coatings and the different characteristics of the formed scaffold surfaces via water contact angle experiments and XPS analysis (Figs. 3 and 4). As expected, the bioactive agent coated PCU scaffolds showed higher hydrophilicity which provides for a favorable cellular environment (Fig. 3) [19, 45]. PLO coated random and aligned PCU scaffolds exhibited a more rapid water absorption as compared to PLL coated random and
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aligned PCU scaffolds. Although the same quantity of PLO and PLL were coated on the PCU scaffolds, a higher amount of PLO may be immobilize on the PCU scaffolds because the PLO used in this study had a higher molecular weight than the PLL. The XPS analysis clearly correlated with the contact angle outcomes (Fig. 3). The PLO coated scaffolds had slightly
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increased amine groups as compared to the PLL coated scaffolds. This indicates that there
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were more amino groups introduced on the bare PCU scaffolds for the PLO coated scaffolds. Our previous report, Lee et al. also demonstrated that immobilization of amine molecules on
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polyester scaffolds significantly increased water absorption [49, 50]. In another study, Zheng et al. established that increasing the amount of PLL and PLO coating on scaffolds showed
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decreasing water contact angles with increasing N1s peak from XPS analysis [46]. Through
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water contact angle and XPS analysis, we clearly demonstrated that our developed scaffolds have hydrophilic activity. We also visualized FITC-labeled PLL coating on random and
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alignment PCU scaffolds. After coating, our developed mats maintained their existing morphology because PCU has excellent mechanical properties and moderately good bio-
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stability [51]. Khan et al. revealed that four different commercial polyurethane scaffolds did not present any significant changes in mechanical properties over a 3-year incubation period
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in PBS at 37°. This was established by UTS and strain experiments [14]. In our study, PCU fibrous scaffolds were coated by submerging them in 1 mL PBS solutions containing bioactive agents for 24 h at room temperature with gentle shaking. Thus, we determined that our developed fiber mats might have mechanical stability after coating process. In tissue engineering, hydrophilic scaffolds are crucial to provide for more effective cell interaction. The substrate properties determine several parameters including the degree of cell migration, proliferation, and adhesion [28, 50, 52, 53]. From in vitro assessments, we obtained good cell proliferation results. The PLL and PLO coated cover glass exhibited significantly higher cell proliferation of DPSCs (p < 0.05) as compared with bare cover glass,
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meaning that PLL and PLO are useful for enhancing cell proliferation (Fig. 6). Likewise, the PLL and PLO coated electrospun PCU fibrous scaffolds exhibited higher cell proliferation as compared to bare PCU scaffolds (Fig. 7a and b). At time points of 4 and 7 days, the PLL and PLO coated scaffolds had statistically significant different cell proliferation as compared to
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the 1 day culture time point. This indicates that PLL and PLO are able to promote cell
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proliferation as time goes on. Our topography approach also provided for elongated DPSCs morphology (Fig. 7a compared to Fig. S1). Interestingly, the PLO coated scaffolds had
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significantly increased DPSCs proliferation as compared to PLL coated substrates in the cell proliferation test. In the comparison between PLO and PLL, Ge et al. determined that PLO
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significantly increased neural stem progenitor cells proliferation and induced higher degrees
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of differentiation, as compared to PLL [54]. Additionally, Haile et al. previously indicated that PLO-laminin coated hydrogels significantly improved the adhesion and viability of neuro
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potential cells due to the molecule’s physical and chemical properties better than PLL [55]. They asserted that the improved surface qualities of bio-substrates may play an important role
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in determining the fate and morphologies of cells. Based on these aspects and our results, we established that PLO coating is a more suitable bioactive agent as compared to PLL coating
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for neural cell proliferation in nerve tissue engineering. For in vitro evaluations in the present study, we used DPSCs, primarily derived from pulp tissues of primary incisors, exfoliated deciduous, and permanent human third molar teeth, are a heterogeneous stem cell population, which has neurogenic potential for neuronal differentiation [56, 57]. In previous reports, Gervois et al. demonstrated that DPSCs are capable of neuronal commitment with distinct features of neuronal differentiation during PLO treatment [58]. In a further study, Király et al. indicated that PLL coated plastic surfaces showed a higher mRNA expression of neuronal marker genes as they act to induce differentiation of DPSCs into distinct neuronal phenotypes [59]. In this case, our developed scaffolds are attractive and may be able to play an important
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role in neural tissue engineering and clinical application.
5. Summary
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In summary, in this study, a highly-aligned PCU fibrous scaffold was manufactured by ELSP process followed by PLL and PLO coating. This scaffold allowed for enhanced DPSCs
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adhesion and proliferation. After ELSP at 1800 rpm, nearly parallel fiber strands were observed. During cell culture studies, DPSCs grown on PLL and PLO coated scaffolds were
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observed to present a parallel morphology along the longitudinal axis. Finally, PLO coated
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PCU scaffolds showed a significant increase in cell proliferation as compared to PLL coated and bare substrates. In the present study, we suggest that physical guidance of DPSCs is an
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important aspect in their growth and proliferation. This preliminary study will allow for more detailed future investigations. This may include investigating the chemical binding of
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bioactive agents to the substrates for better attachment. Our study implies the tremendous potential of these scaffolds for application to neural tissue engineering. In the near future,
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based on current research, we will develop a highly-aligned neuro-potential fibrous scaffold
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with sustain release of bioactive molecules and proteins using heparin chemistry for clinical neural tissue engineering applications.
Acknowledgement
This study was supported by a grant from The Korean Health Technology R&D Project (HI13C1527), Ministry of Health & Welfare, and by Bio-industry Technology Development Program (312062-5) of iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
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Figure Legends
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Fig. 1. Schematic illustration of the preparation method for generating thermoplastic polycarbonate polyurethane fibrous scaffolds with bioactive agents for neural tissue regeneration. Fig. 2. Surface characterization of fiber substrates. Scanning electron microscopy images of aligned PCU fibrous scaffolds with rotation at 100, 900, and 1800 rpm, respectively (a). Angle counts of aligned PCU fibrous scaffolds (b).Fig. 3. Water contact angle analysis of bare and coated fiber substrates for 30s. Fig. 4. X-ray photoelectron spectroscopy characterization of fiber substrates. XPS Spectrum of O, C, N, peaks (a and b), and surface chemical composition (c and d) on fiber substrates. Fig. 5. Imaging of FITC labeled PLL coating on random (a) and aligned (b) fiber substrates by confocal laser scanning microscopy. Fig. 6. Evaluation of DPSCs adhesion test using CCK-8 assay on bare cover glass, PLL, and PLO coated PCU fibrous scaffolds (a), confocal laser scanning microscopy images of bare cover glass, PLL and PLO coated PCU fibrous scaffolds after 2 days in cell culture (n=4, *p < 0.05 compared to cover glass). Fig. 7. Confocal laser scanning microscopy images of aligned bare PCU, PLL and PLO coated PCU scaffolds after 1 and 7 days of culture (a), Cell proliferation of DPSCs grown on bare, aligned PCU fibrous scaffolds, PLL and PLO coated aligned PCU fibrous scaffolds over 7 days (b). (n=4, *p < 0.05 as compared to 1 day of culture)
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:: Highlights ::
● Highly aligned thermoplastic polycarbonate urethane fibrous scaffolds were well fabricated
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using electrospinning process under a 1800 rpm rotating condition on collector. ● Poly-L-Ornithine coated fiber scaffolds showed a significant increase in cell proliferation as
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compared to Poly-L-Lysine coated and bare substrates. ● dental pulp stem cells were observed to present a parallel morphology along the longitudinal axis on highly aligned Poly-L-Lysine and Poly-L-Ornithine coated scaffolds.
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Graphic abstract
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S0169-4332(17)30552-4 http://dx.doi.org/doi:10.1016/j.apsusc.2017.02.179 APSUSC 35288
To appear in:
APSUSC
Received date: Revised date: Accepted date:
13-12-2016 16-2-2017 19-2-2017
Please cite this article as: S.J. Lee, M. Heo, D. Lee, D.N. Heo, H.-N. Lim, I.K. Kwon, Fabrication and Design of Bioactive Agent Coated, Highly-Aligned Electrospun Matrices for Nerve Tissue Engineering: Preparation, Characterization and Application, Applied Surface Science (2017), http://dx.doi.org/10.1016/j.apsusc.2017.02.179 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fabrication and Design of Bioactive Agent Coated, Highly-Aligned Electrospun Matrices for Nerve Tissue Engineering: Preparation,
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Characterization and Application
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Sang Jin Lee1, Min Heo1, Donghyun Lee1, Dong Nyoung Heo2, Ho-Nam Lim2, Il Keun
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Kwon2,∗
1
Department of Dentistry, Graduate School, Kyung Hee University, 26 Kyungheedae-ro,
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Dongdaemun-gu, Seoul 02447, Republic of Korea 2
Department of Dental Materials, School of Dentistry, Kyung Hee University, 26 Kyungheedae-ro,
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Dongdaemun-gu, Seoul 02447, Republic of Korea
* Correspondence to Il Keun Kwon, Ph. D. Department of Dental Materials, School of Dentistry, Kyung Hee University, 26
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Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea Tel.: +82-2-961-0350
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E-mail address: [email protected] (Il Keun Kwon).
Abstract
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In this study, we designed highly-aligned thermoplastic polycarbonate urethane (PCU)
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fibrous scaffolds coated with bioactive compounds, such as Poly-L-Lysine (PLL) and Poly-LOrnithine (PLO), to enhance cellular adhesion and directivity. These products were
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characterized by scanning electron microscope (SEM) analysis which demonstrated that highly aligned fiber strands were formed without beads when coated onto a mandrel rotating
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at 1800 rpm. During in vitro cell test, PLO-coated, aligned PCU scaffolds were found to have significantly higher proliferation rates than PLL coated and bare PCU scaffolds. Interestingly,
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dental pulp stem cells (DPSCs) were observed to stretch along the longitudinal axis parallel to the cell direction on highly aligned scaffolds. These results clearly confirm that our
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strategy may suggest a useful paradigm by inducing neural tissue repair as a means to
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remodeling and healing of tissue for restorative procedures in neural tissue engineering.
Keywords: electrospinning, aligned nanofiber, dental pulp stem cell, neural tissue engineering
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1. Introduction
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For a long time, human nerve diseases such as peripheral nerve injury (PNI) have been a significant concern to humanity all over the world. PNI commonly occurs with physical
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injuries suffered during construction/transportation accidents, natural disasters, military
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service injuries, and other trauma [1, 2]. Approximately 360,000 patients suffer from upper extremity paralytic syndromes annually in the Unites States, and over 300,000 people suffer
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peripheral nerve injuries in Europe, annually [1]. To solve this issue, many clinical and bioengineering researchers have been working towards repairing damaged neural tissue using
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polymeric scaffolds [3-6].
Tissue engineering strategies have received a great deal of attention for use in reconstruction
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of damaged nerves by providing a cell-growth promoting environment [7]. Amongst the
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many bioengineered approaches, aligned electrospun nanofibrous scaffold made by electrospinning (ELSP) is a promising tactic because the fibrous structure mimics that of native neural extracellular matrix [8-10]. This scaffold is also able to regulate cellular behaviors for cell attachment or migration, nutrient transportation, and signal transduction [11]. In general, synthetic polymeric fiber scaffolds have been useful for nerve tissue repair on account of their potent mechanical properties [12, 13]. However, their capabilities for supporting cell proliferation, migration, and viability are limited due to their crystalline and hydrophobic properties [14-16]. Thus, hydrophobic platforms should be converted to hydrophilic substrates to promote cellular activity. In this case, hydrophobic scaffolds can be readily adapted to provide for hydrophilic surfaces via coating of bioactive agents [16-18].
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In this study, we designed a highly aligned thermoplastic polycarbonate urethane (BionateⓇ II, PCU) fibrous scaffolds coated with bioactive compounds such as Poly-L-Lysine (PLL) and Poly-L-Ornithine (PLO) to enhance cellular behaviors such as adhesion and proliferation. The PLL and PLO are cationic bioactive compounds used in neuroscience [19, 20]. These are
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hydrophilic agents that alter the surface properties of hydrophobic scaffolds to promote
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cellular adhesion, migration, and proliferation. This supports neurite growth to form a network for nerve cells [21, 22]. Furthermore, These are also able to promote neural cell
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differentiation [23, 24]. As for the substrate, PCU is a Food and Drug Administration (FDA)
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approved commercially-available, medical-grade polymer that has been extensively used in long-term implants due to its bio-stability, biocompatibility, and appropriate mechanical
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properties [14, 25, 26].
The major aim of this study is to determine not only the effectiveness of the scaffold for
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supporting cellular adhesion and proliferation behavior, but also to determine the interaction of the scaffold with dental pulp stem cell (DPSCs). The fabricated scaffolds were
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characterized by scanning electron microscopy (SEM), water contact angle, X-ray
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photoelectron spectroscopy (XPS) analysis. Finally, in vitro cell proliferation was investigated. Additionally, directional cell morphology was visualized by confocal laser scanning microscopy (CLSM). This preliminary study will pave the way for future investigations.
2. Materials and methods 2.1 Materials Poly-L-Lysine hydrobromide (Mw range 500-2,000), FITC labeled Poly-L-Lysine (Mw range 30,000-70,000), and Poly-L-Ornithine hydrobromide (Mw > 100,000) were purchased from Sigma-Aldrich (St. Louis, MO). N,N-Dimethylformamide (99.5%), Tetrahydrofuran
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(99.5%) were purchased from JUNSEI (JUNSEI CHEMICAL Co. Ltd., Japan). Commercially available thermoplastic polycarbonate polyurethane (Bionate II®, 80A) was synthesized by DSM Biomedical (Berkeley, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate buffered saline (PBS), fetal bovine serum (FBS),
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trypsin–EDTA, and penicillin streptomycin were purchased from Gibco BRL (Invitrogen Co.
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Ltd, Carlsbad, CA, USA). Deionized-distilled water (DDW) was produced by an ultrapure
analytical grade and used without further purification.
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water system (Puris-Ro800; Bio Lab Tech., Korea). All other reagents and solvents were of
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2.2 Preparation of highly aligned thermoplastic polycarbonate urethane scaffolds
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The fibrous scaffolds were prepared using ELSP according to our previously described with some modification [27-30]. Briefly, PCU compounds were dissolved in mixed DMF/THF
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(5:5) solvent to produce an 8 w/v % solution. For ELSP, the dissolved solution was loaded into a luer-lock syringe attached to a 20 G metal blunt needle and electrospun on an
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aluminum foil covered rotating mandrel at 20 kV using a high-voltage DC power supply (Nano NC, Korea) with a 1 ml/h feed rate (KDS-200, KD Scientific Inc.) and a 15 cm needle
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tip-to-collector distance. For different conditions of alignment, the mandrel speed was varied between 100, 900, and 1800 rpm, respectively. Finally, the resultant PCU nanofiber was dried overnight under vacuum to remove any residual solvent.
2.3 Poly-L-lysine, FITC labeled Poly-L-lysine, and Poly-L-ornithine coating on fibrous scaffolds For physical adsorption of bioactive agents on substrates, we applied using the previously described methods with some modification [31]. Cover glasses and PCU fibrous scaffolds (48 well size) were submerged in 1 mL of PBS solutions containing either 500 ug/mL of PLL,
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FITC labeled PLL, and PLO for 24 h at room temperature with gentle shaking. These were then washed three times in 10 mL of PBS.
2.4 In vitro DPSCs adhesion and proliferation test on scaffolds
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Dental pulp stem cells (DPSCs) were donated by the Department of Oral and Maxillofacial
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Surgery of Kyung Hee University Dental Hospital. Prior to cell culture on scaffolds, specimens were sterilized with UV lamp on a clean bench. DPSCs were cultured in DMEM
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supplemented with 10% FBS and 1% penicillin–streptomycin in a 5% CO2 incubator at 37 °C. The DPSCs were drop seeded onto the scaffolds at a density of 2 x 104 cells per well. After 2
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h, medium was filled onto the scaffolds in each culture plate, respectively. Cell adhesion was
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determined after 2 days of culture and proliferation was determined at 1, 3, and 7 days by using the cell counting kit (CCK-8) assay (n = 4). The absorbance of the medium was
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measured at 450 nm using a microplate reader (ELISA, Bio-Rad, Hercules, CA, USA). These experiments were repeated in triplicate. For F-actin staining, the proliferated cells on scaffold
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groups were fixed with 3.9% formaldehyde for 1 h. After that, these were washed with PBS followed by staining with 1 mL of Oregon green 514 phalloidin (1:200) and 4′,6-diamidino-
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2-phenylindole (DAPI) (1:1000) solution.
2.5 Analysis equipment
The surface morphologies of PCU nanofibers were characterized using a scanning electron microscope (SEM, Hitachi S-2300, Japan) at an acceleration voltage of 15 kV. All samples were dried at room temperature, and then sputter-coated with gold by an IB-3 (Eiko, Japan) sputter coater for 10 min. The fibrous angle characterization was carried out using image analysis software (Eyeview-Analyzer (Digiplus, Korea)). Surface water contact angle measurements were performed using a contact angle meter (Phoenix 150, SEO, Korea) using
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8-µL of distilled water at room temperature. X-ray photoelectron spectroscopy (XPS) was performed with a K-Alpha instrument (Thermo Electron, UK) to evaluate the surface elements of the scaffolds. The visualization of the FITC labeled PLL coating on PCU
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CLSM (Eclipse E600W, Nikon, Tokyo, Japan) after staining.
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scaffolds and the morphology of the cells growing on the scaffolds were analyzed using
2.6 Statistical analysis
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All experiments were carried out in triplicate. All values are expressed as mean ± standard deviation. Multiple comparisons of groups were analyzed using two-way analysis of variance
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(ANOVA) followed by Dunnett's T3 post hoc paired comparison test. Statistical analysis was
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performed using PASW Statistics 18 software (SPSS, Inc., Chicago, IL). A value of P < 0.05 is considered to be statistically significant denoting “*”. The statistical analyses are presented
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3. Results
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within figure legends.
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3.1 Characterization of fabricated PCU scaffolds (SEM) For neural tissue regeneration, aligned PCU scaffolds were fabricated by ELSP procedure. The resultant scaffolds were then characterized by SEM analysis (Fig. 2a). To obtain a highly-aligned fiber surface, we controlled the rotating mandrel speed at 100, 900, and 1800 rpm, respectively. The fiber strands obtained from the 100 and 900 rpm mandrel speeds showed poor alignment. On the other hand, unidirectional fiber strands were obtained from the 1800 rpm condition. The average angle of each fabricated scaffold relative to the vertical axis was measured based on the rotating mandrel speed (Fig. 2b). This measurement indicated that fiber strands were more highly aligned when the speed increased. From these results, we selected the 1800 rpm condition for fabrication of PCU scaffolds used for in vitro
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assessments. We hypothesized that highly aligned fibrous scaffolds may influence the directional stretch of neuro-potential cells.
3.2 Surface characterization of PLL and PLO coated PCU fibrous scaffolds
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To confirm the different surface characteristics of PLL and PLO coated fibrous scaffolds,
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water contact angle and XPS analysis was carried out. Fig. 3 shows the results of water contact analysis. The non-coated random and aligned PCU fibers did not absorbed any water.
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This indicates that the PCU scaffold is highly hydrophobic. However, the surface treated PCU scaffolds absorbed water within 30 sec. The random PCU fiber provided for more rapid
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water absorption than the aligned PCU fiber. This result is due to the decrease in pore size
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because the surface topography of these two scaffolds is different due to the different fabrication processes. This may have a direct influence on the surface property of the PCU
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scaffolds [32, 33]. Based on the water contact experiment, we analyzed surface elements of manufactured fibrous scaffolds by XPS. As shown in Fig. 4, both random and aligned PCU
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nanofibers did not exhibit N1s peaks. However, the PLL and PLO coated PCU, both random and aligned nanofibers, presented characteristic N1s peaks. This phenomenon is due to the
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presence of the amine groups of PLL and PLO which were well introduced onto bare PCU scaffolds. This indicates that the physical coating of bioactive agents was successfully performed in this study. Interestingly, the N1s were slightly increased for the PLO coating as compared to the PLL coating. From these results, we hypothesized that the PLO coated PCU scaffolds may be more suitable for cell proliferation than PLL coated PCU scaffolds.
3.3 Visualization of FITC labeled PLL coated on PCU fibrous scaffolds In order to confirm the formation of a uniform coating of the bioactive agent, we visualized FITC-labeled PLL coated random and aligned PCU scaffolds by confocal laser scanning
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microscopy. As shown in Fig. 5a, the entangled fiber morphology displayed in random PCU scaffold was well observed. On the other hand, the fibrous strands were observed to be well aligned in highly aligned PCU scaffolds. This result indicates that the bioactive agent is uniformly coated on both random and aligned PCU scaffolds as well as alignment may affect
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directional cell behavior on scaffolds [7, 11].
3.4 Characterization of DPSCs adhesion on cover glass with and without coating
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To investigate the effectiveness of PLL and PLO coatings for promoting cellular adhesion, cell viability was confirmed using the CCK-8 assay both with and without bioactive agent
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coatings on glass coverslips after 2 days of culture (Fig. 6a and b). As shown in Fig. 6a, the
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absorbance was higher in surface coated cover glass as compared with bare cover glass. This means that PLL and PLO promote cell adhesion. Interestingly, DPSCs had significantly
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higher adhesion on PLO coated cover glass as compared to PLL coating. This result implies that DPSCs can be well adhered and may proliferate more effectively on the PLO coated
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group as compared to the PLL coated group.
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3.5 Characterization of DPSCs proliferation on aligned PCU scaffolds Based on the cell adhesion test, the cell proliferation rate of DPSCs was verified on aligned PCU scaffolds both with and without bioactive agents. As shown in Fig. 7b, after 1 day in culture, all groups exhibited a similar proliferation rate. However, after 4 and 7 days of culture, PLL and PLO coated scaffolds showed a significantly higher proliferation rate than bare scaffolds. This is due to PLL and PLO interactions with the DPSCs. Similar to the cell adhesion test results (Fig. 6), PLO coated PCU scaffolds had higher cell proliferation than PLL coated PCU scaffolds (Fig. 7b). This demonstrates that PLO provides for superior DPSCs attachment than PLL in this condition.
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3.6 Characterization of cellular behavior of DPSCs on bioactive agent coated random and aligned PCU scaffolds The shape and morphology of DPSCs on random and aligned PCU nanofiber scaffold both
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with and without bioactive agents was examined after 1 and 7 days of culture, respectively.
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Fig. 7a and S1 showed different cell morphologies for random and aligned PCU nanofibers. CLSM images show the actin cytoskeleton morphologies of DPSCs. The cytoskeleton
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displays random entanglement on random PCU nanofibers from 1 to 7 days of cell culture (Fig. S1). However, DPSCs presented a stretched, spreading morphology when grown on
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aligned PCU nanofiber groups at all culture time-points (Fig. 7a, white arrow). This
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phenomenon indicates that aligned fibers provide a guide for cell growth by presenting an elongated environment as compared to random PCU nanofibers [34, 35]. Interestingly, a
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greater number of DPSCs were observed for PLL and PLO coated nanofiber scaffolds than bare scaffolds regardless of alignment. This result corresponds with the cell proliferation test
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results (Fig. 7b). These results suggest that cell adhesion and cell morphology were affected by the substrates environments both with and without bioactive coatings. This may help
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neural potential cells with elongation and stretching along the longitudinal axis, parallel to the cell direction, which is the morphology observed in neural tissue [34, 36, 37].
4. Discussion
In the field of neural tissue engineering, implantable and biocompatible scaffolds are highly desirable for maintaining a biomimetic environment for neural cell migration [38]. In this case, electrospun, aligned, fibrous scaffolds are well suited for this application because of their highly-oriented fibers, which can induce directional cell migration. These also provide for uniform cell alignment, highly striated extracellular matrix, and guided cell outgrowth
Page 10 of 33
during neural tissue formation [11, 39]. Thus, in this study, we devised a highly aligned fibrous scaffold and coated it with bioactive agents to promote cell adhesion and proliferation (Fig. 1). During ELSP, highly-aligned fiber scaffolds were formed when fiber strands were collected on a rotating mandrel at a controlled speed [12]. By testing different mandrel speeds,
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we obtained highly aligned PCU fiber scaffolds at 1800 rpm which provided for cell
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extension (Fig. 2). To manufacture fiber mats, we used DMF/THF blended solvent for preparation of PCU ELSP solution. This allows for the generation of a uniformly formulated
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fibrous morphology. DMF/THF solvents are able to clearly dissolve PCU compounds. This mixture has been well employed in our previous studies [28-30].
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The topography guidance approach, by use of aligned fibers, can control cellular migration
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and promoting directional orientation [40]. Alignment of the fibers greatly influences the cellular growth behavior and outstandingly controls cell directional morphology. Additionally,
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the cells grown on the aligned scaffolds have increased proliferation rates with reinforced cell-matrix interactions as compared with cells grown on random substrates [41]. To promote
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nervous tissue regeneration, a topographical strategy can play a pivotal role for promoting cell adhesion, alignment, spreading, and morphological changes [42]. However, the exact
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mechanisms of topography-dependent neural cell responses are still unclear [43]. Nevertheless, many bio-engineering researchers have made an effort to illuminate the cellular mechanisms of this alignment effect which occurs on oriented fibrous scaffolds. Chew et al. reported that the culturing of cells on aligned fibers lead to significant up-regulation of the myelin-specific gene as compared to cells cultured on randomly oriented fibers. This suggests that the alignment and elongation of human neural cells on aligned fibers can promote their maturation as compared to those grown on randomly oriented fibers. They concluded that integrin may interact with the cytoskeleton or mediate myelin-related gene expressions [37]. In another study, Wang et al. demonstrated that neural progenitor cells expanded faster to
Page 11 of 33
promote proliferation on aligned nanofibers as compared with those grown on randomly oriented nanofibers [36]. They revealed that there was a higher level of cyclin D1 and CDK2, two downstream genes of ß1 integrin/MAPK signaling, on aligned nanofibers as compared to those on randomly oriented nanofibers. The ß1 integrin is well known to transmit cell
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signaling to mediate cell proliferation, differentiation, and migration [44]. Based on these
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findings, we determined that our highly-aligned nanofibers are selective for nerve tissue regeneration (Fig. 2).
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In the present study, we used PCU as it is a commercially available polymer with excellent mechanical properties for use in the biomedical engineering field [14]. In neural tissue
an
engineering, neuro-potential materials are required to remain for long periods of time. These
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should be also manufactured from bio-stable materials. For these reasons, we choose PCU polymer as an adoptable biomaterial for neural scaffold generation. However, PCU is a
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hydrophobic synthetic polymer which may impede cell proliferation, migration, and viability. This is due to its crystallinity and hydrophobic properties. Thus, surface treatment of these
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scaffolds should be carried out for encouraging a cell favorable environment [16-18]. This issue can surmount through attachment of bioactive molecules to potentially guide and
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enhance cell adhesion and proliferation on the scaffolds [13, 15]. For this reason, we utilized PLL and PLO coatings to provide for favorable sites with improved adhesion and proliferation of neuro-potential DPSCs [19, 21, 22, 24, 45-48]. Although the two substances have a similar property, we anticipated that their bioactivity and cellular activity may be different. We verified the coatings and the different characteristics of the formed scaffold surfaces via water contact angle experiments and XPS analysis (Figs. 3 and 4). As expected, the bioactive agent coated PCU scaffolds showed higher hydrophilicity which provides for a favorable cellular environment (Fig. 3) [19, 45]. PLO coated random and aligned PCU scaffolds exhibited a more rapid water absorption as compared to PLL coated random and
Page 12 of 33
aligned PCU scaffolds. Although the same quantity of PLO and PLL were coated on the PCU scaffolds, a higher amount of PLO may be immobilize on the PCU scaffolds because the PLO used in this study had a higher molecular weight than the PLL. The XPS analysis clearly correlated with the contact angle outcomes (Fig. 3). The PLO coated scaffolds had slightly
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increased amine groups as compared to the PLL coated scaffolds. This indicates that there
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were more amino groups introduced on the bare PCU scaffolds for the PLO coated scaffolds. Our previous report, Lee et al. also demonstrated that immobilization of amine molecules on
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polyester scaffolds significantly increased water absorption [49, 50]. In another study, Zheng et al. established that increasing the amount of PLL and PLO coating on scaffolds showed
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decreasing water contact angles with increasing N1s peak from XPS analysis [46]. Through
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water contact angle and XPS analysis, we clearly demonstrated that our developed scaffolds have hydrophilic activity. We also visualized FITC-labeled PLL coating on random and
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alignment PCU scaffolds. After coating, our developed mats maintained their existing morphology because PCU has excellent mechanical properties and moderately good bio-
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stability [51]. Khan et al. revealed that four different commercial polyurethane scaffolds did not present any significant changes in mechanical properties over a 3-year incubation period
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in PBS at 37°. This was established by UTS and strain experiments [14]. In our study, PCU fibrous scaffolds were coated by submerging them in 1 mL PBS solutions containing bioactive agents for 24 h at room temperature with gentle shaking. Thus, we determined that our developed fiber mats might have mechanical stability after coating process. In tissue engineering, hydrophilic scaffolds are crucial to provide for more effective cell interaction. The substrate properties determine several parameters including the degree of cell migration, proliferation, and adhesion [28, 50, 52, 53]. From in vitro assessments, we obtained good cell proliferation results. The PLL and PLO coated cover glass exhibited significantly higher cell proliferation of DPSCs (p < 0.05) as compared with bare cover glass,
Page 13 of 33
meaning that PLL and PLO are useful for enhancing cell proliferation (Fig. 6). Likewise, the PLL and PLO coated electrospun PCU fibrous scaffolds exhibited higher cell proliferation as compared to bare PCU scaffolds (Fig. 7a and b). At time points of 4 and 7 days, the PLL and PLO coated scaffolds had statistically significant different cell proliferation as compared to
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the 1 day culture time point. This indicates that PLL and PLO are able to promote cell
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proliferation as time goes on. Our topography approach also provided for elongated DPSCs morphology (Fig. 7a compared to Fig. S1). Interestingly, the PLO coated scaffolds had
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significantly increased DPSCs proliferation as compared to PLL coated substrates in the cell proliferation test. In the comparison between PLO and PLL, Ge et al. determined that PLO
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significantly increased neural stem progenitor cells proliferation and induced higher degrees
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of differentiation, as compared to PLL [54]. Additionally, Haile et al. previously indicated that PLO-laminin coated hydrogels significantly improved the adhesion and viability of neuro
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potential cells due to the molecule’s physical and chemical properties better than PLL [55]. They asserted that the improved surface qualities of bio-substrates may play an important role
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in determining the fate and morphologies of cells. Based on these aspects and our results, we established that PLO coating is a more suitable bioactive agent as compared to PLL coating
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for neural cell proliferation in nerve tissue engineering. For in vitro evaluations in the present study, we used DPSCs, primarily derived from pulp tissues of primary incisors, exfoliated deciduous, and permanent human third molar teeth, are a heterogeneous stem cell population, which has neurogenic potential for neuronal differentiation [56, 57]. In previous reports, Gervois et al. demonstrated that DPSCs are capable of neuronal commitment with distinct features of neuronal differentiation during PLO treatment [58]. In a further study, Király et al. indicated that PLL coated plastic surfaces showed a higher mRNA expression of neuronal marker genes as they act to induce differentiation of DPSCs into distinct neuronal phenotypes [59]. In this case, our developed scaffolds are attractive and may be able to play an important
Page 14 of 33
role in neural tissue engineering and clinical application.
5. Summary
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In summary, in this study, a highly-aligned PCU fibrous scaffold was manufactured by ELSP process followed by PLL and PLO coating. This scaffold allowed for enhanced DPSCs
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adhesion and proliferation. After ELSP at 1800 rpm, nearly parallel fiber strands were observed. During cell culture studies, DPSCs grown on PLL and PLO coated scaffolds were
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observed to present a parallel morphology along the longitudinal axis. Finally, PLO coated
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PCU scaffolds showed a significant increase in cell proliferation as compared to PLL coated and bare substrates. In the present study, we suggest that physical guidance of DPSCs is an
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important aspect in their growth and proliferation. This preliminary study will allow for more detailed future investigations. This may include investigating the chemical binding of
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bioactive agents to the substrates for better attachment. Our study implies the tremendous potential of these scaffolds for application to neural tissue engineering. In the near future,
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based on current research, we will develop a highly-aligned neuro-potential fibrous scaffold
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with sustain release of bioactive molecules and proteins using heparin chemistry for clinical neural tissue engineering applications.
Acknowledgement
This study was supported by a grant from The Korean Health Technology R&D Project (HI13C1527), Ministry of Health & Welfare, and by Bio-industry Technology Development Program (312062-5) of iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
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Figure Legends
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Fig. 1. Schematic illustration of the preparation method for generating thermoplastic polycarbonate polyurethane fibrous scaffolds with bioactive agents for neural tissue regeneration. Fig. 2. Surface characterization of fiber substrates. Scanning electron microscopy images of aligned PCU fibrous scaffolds with rotation at 100, 900, and 1800 rpm, respectively (a). Angle counts of aligned PCU fibrous scaffolds (b).Fig. 3. Water contact angle analysis of bare and coated fiber substrates for 30s. Fig. 4. X-ray photoelectron spectroscopy characterization of fiber substrates. XPS Spectrum of O, C, N, peaks (a and b), and surface chemical composition (c and d) on fiber substrates. Fig. 5. Imaging of FITC labeled PLL coating on random (a) and aligned (b) fiber substrates by confocal laser scanning microscopy. Fig. 6. Evaluation of DPSCs adhesion test using CCK-8 assay on bare cover glass, PLL, and PLO coated PCU fibrous scaffolds (a), confocal laser scanning microscopy images of bare cover glass, PLL and PLO coated PCU fibrous scaffolds after 2 days in cell culture (n=4, *p < 0.05 compared to cover glass). Fig. 7. Confocal laser scanning microscopy images of aligned bare PCU, PLL and PLO coated PCU scaffolds after 1 and 7 days of culture (a), Cell proliferation of DPSCs grown on bare, aligned PCU fibrous scaffolds, PLL and PLO coated aligned PCU fibrous scaffolds over 7 days (b). (n=4, *p < 0.05 as compared to 1 day of culture)
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:: Highlights ::
● Highly aligned thermoplastic polycarbonate urethane fibrous scaffolds were well fabricated
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using electrospinning process under a 1800 rpm rotating condition on collector. ● Poly-L-Ornithine coated fiber scaffolds showed a significant increase in cell proliferation as
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compared to Poly-L-Lysine coated and bare substrates. ● dental pulp stem cells were observed to present a parallel morphology along the longitudinal axis on highly aligned Poly-L-Lysine and Poly-L-Ornithine coated scaffolds.
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M
cr
ip t
Fig. 1
-1.0 kv
M an
us
+20.0 kv
i
High voltage
cr
Figure Click here to download Figure: 06 Figure.pptx
Rotating collector
Ac
ce pt
ed
Syringe pump
Random PCU fiber (100 rpm)
Align PCU fiber (1800 rpm)
Page 26 of 33 Dental pulp stem cell
i us
cr
Fig. 2
ce pt
ed
M an
(a)
50
40
Ac
Counts (n) (n) Counts
(b)
Random 100 rpm 900 rpm 1800 rpm
30
20
10
0 0
30
60
90
Angle (˚)
120
150
180
Page 27 of 33
Ac
PCU / PLO PCU / PLL PCU
ed
91°
44°
47°
0°
0° 0°
93°
93° 93°
90° 74° 0°
49° 15° 0°
M an
0 sec 93°
i
6 sec
30 sec
93°
cr
us
PCU
Random nanofibers PCU / PLO PCU / PLL
ce pt
Aligned nanofibers
Fig. 3 93°
Page 28 of 33
i us
cr
Fig. 4 XPS
Aligned nanofibers
(a)
M an
Random nanofibers
(b)
PCU PCU / PLL PCU / PLO
O1s
O1s
ed
C1s
PCU PCU / PLL PCU / PLO
1000
800
600
Ac
1200
ce pt
N1s
400
200
N1s
0
1200
1000
Binding energy (eV)
(c)
Groups
C1s
800
600
400
200
0
Binding energy (eV)
C
N
O
PCU
77.85
0
PCU / PLL
77.23
PCU / PLO
75.92
(d)
Groups
C
N
O
22.15
PCU
76.42
0
23.58
4.26
18.51
PCU / PLL
75.48
4.91
19.61
5.20
18.88
PCU / PLO
75.95
5.25
18.80 Page 29 of 33
i M an
us
cr
Fig. 5
Random PCU / FITC labeled PLL
(b)
Ac
ce pt
ed
(a)
Align PCU / FITC labeled PLL
Page 30 of 33
i (b)
Cover glass
M an
us
cr
Fig. 6
PLL coating
Ac
ce pt
ed
(a)
PLO coating
Page 31 of 33
i ce pt
ed
M an
us
cr
(a)
(b)
Ac
Fig. 7
Page 32 of 33
Graphic abstract
-1.0 kv
M an
us
+20.0 kv
i
High voltage
cr
Graphical Abstract (for review)
Rotating collector
Ac
ce pt
ed
Syringe pump
Random PCU fiber (100 rpm)
Align PCU fiber (1800 rpm)
Page 33 of 33 Dental pulp stem cell