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Bio-functionalized PCL nanofibrous scaffolds for nerve tissue engineering
Materials Science and Engineering C 30 (2010) 1129–1136
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Bio-functionalized PCL nanofibrous scaffolds for nerve tissue engineering Laleh Ghasemi-Mobarakeh a,b,c,d, Molamma P. Prabhakaran a,⁎, Mohammad Morshed c, Mohammad Hossein Nasr-Esfahani d, S. Ramakrishna a,e,⁎ a
Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore , 2 Engineering Drive 3, Singapore 117576, Singapore Islamic Azad University, Najafabad branch, Isfahan, Iran Department of Textile Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran d Department of Cell Sciences Research Center (Isfahan Campus), Royan Institute, ACECR, Tehran, Iran e King Saud University, 11451 Riyadh, Saudi Arabia b c
a r t i c l e
i n f o
Article history: Received 7 February 2010 Received in revised form 16 May 2010 Accepted 8 June 2010 Available online 19 June 2010 Keywords: Alkaline hydrolysis PCL Nanofibrous scaffolds Matrigel Nerve tissue engineering
a b s t r a c t Surface properties of scaffolds such as hydrophilicity and the presence of functional groups on the surface of scaffolds play a key role in cell adhesion, proliferation and migration. Different modification methods for hydrophilicity improvement and introduction of functional groups on the surface of scaffolds have been carried out on synthetic biodegradable polymers, for tissue engineering applications. In this study, alkaline hydrolysis of poly (ε-caprolactone) (PCL) nanofibrous scaffolds was carried out for different time periods (1 h, 4 h and 12 h) to increase the hydrophilicity of the scaffolds. The formation of reactive groups resulting from alkaline hydrolysis provides opportunities for further surface functionalization of PCL nanofibrous scaffolds. Matrigel was attached covalently on the surface of an optimized 4 h hydrolyzed PCL nanofibrous scaffolds and additionally the fabrication of blended PCL/matrigel nanofibrous scaffolds was carried out. Chemical and mechanical characterization of nanofibrous scaffolds were evaluated using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, contact angle, scanning electron microscopy (SEM) and tensile measurement. In vitro cell adhesion and proliferation study was carried out after seeding nerve precursor cells (NPCs) on different scaffolds. Results of cell proliferation assay and SEM studies showed that the covalently functionalized PCL/matrigel nanofibrous scaffolds promote the proliferation and neurite outgrowth of NPCs compared to PCL and hydrolyzed PCL nanofibrous scaffolds, providing suitable substrates for nerve tissue engineering. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nerve tissue engineering is a rapidly expanding area of research, providing new and promising approach to nerve repair and regeneration [1]. Design and fabrication of scaffolds suitable for neural tissue engineering is critical for the success of tissue regeneration [2]. Scaffold surfaces play a vital role for the success of tissue engineering as the interaction between cells and scaffolds occur at the surface of the scaffolds [3]. Synthetic biodegradable polymers are now widely used in tissue engineering due to their mechanical properties along with adjustable degradation rates [4]. However, cell affinity towards synthetic hydrophobic polymers is poor as a consequence of their low hydrophilicity and lack of surface cell recognition sites [5]. Recently nano-structured materials are receiving considerable attention for application in tissue engineering, essentially because the body consists of nanoscale structures of extra cellular matrix (ECM) providing a natural web of intricate nanofibers supporting cells and provides an instructive background guiding their ⁎ Corresponding authors. E-mail addresses: [email protected] (M.P. Prabhakaran), [email protected] (S. Ramakrishna). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.06.004
behavior [6]. Therefore improving the hydrophilicity of nanostructured scaffolds together with the incorporation of biological molecules on to the nanofibrous scaffolds could provide both physical and chemical signals required for cell growth. Poly (ε-caprolactone) (PCL) has been used for the reconstruction of various tissues such as bone, skin, nerve, retina and has several advantages including its biocompatibility, low cost and easy processability. However, being a synthetic biomaterial, PCL has a hydrophobic surface, lacks functional groups and hence it is not a good substrate for cell adhesion. To improve the hydrophilicity and biological properties of PCL nanofibrous scaffolds various techniques have been applied. Previous studies mostly modified the PCL nanofibrous scaffolds by incorporation of biologically active polymers such as gelatin and collagen or the hydrophilicity of PCL scaffolds was improved by methods such as laser and plasma treatment [1,7–13]. However, there are very few reports available on the functionalization of PCL scaffolds using biomolecules for tissue engineering [10–13]. Previous studies have shown that laminin, collagen IV, fibronectin and heparin sulphate proteoglycans support neurite outgrowth [14]. Our study is aimed at investigating a simple procedure such as alkaline hydrolysis of PCL nanofibrous scaffolds for improving its hydrophilicity and further attaching matrigel on them, to produce
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scaffolds suitable for nerve tissue engineering. Matrigel is a soluble and sterile extract of basement membrane proteins derived from the Engelberth–Holm–Swarm (EHS) mouse sarcoma and it is rich in extracellular proteins such as laminin, collagen IV, fibronectin and heparin sulphate proteoglycans [14–17]. Matrigel being an important component of the nervous system, functionalization of matrigel on the surface of an already modified PCL nanofibrous scaffold was rapidly achievable. For comparative evaluation studies, matrigel was also mixed with PCL solution and the electrospun ‘blended PCL/matrigel’ nanofibrous scaffolds were fabricated. The effect of incorporation of matrigel into PCL nanofibrous scaffolds towards the proliferation and differentiation of NPCs was further investigated.
2.4.2. Covalent attachment of matrigel on hydrolyzed PCL to obtain ‘functionalized PCL/matrigel’ scaffolds Hydrolyzed PCL (H-PCL) was used for the covalent attachment of matrigel on their surface. For covalent attachment of matrigel on PCL nanofibers, the H-PCL scaffolds were immersed in 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution (0.1 M, pH 5.0) of 5 mg/mL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) for 1 h at room temperature. Scaffolds were further rinsed with DI water and incubated with 20 μL of matrigel for 24 h and subsequently washed with PBS. Fig. 1 shows schematic representation of covalent attachment of matrigel on the surface of PCL nanofibrous scaffolds.
2. Materials and methods
2.5. Characterization of nanofibrous scaffolds
2.1. Materials
PCL (11% w/v) was dissolved in MC/DMF at a ratio of 80:20 and stirred for a period of 24 h at room temperature. The solution was electrospun from a 5 mL syringe with needle diameter of 0.4 mm and mass flow rate of 1 mL/h. A high voltage (13 kV) was applied to the tip of the needle attached to the syringe. The resulting fibers were collected on 15 mm cover slips placed on a flat aluminum plate collector kept at a distance of 12 cm from the needle tip.
The morphology of electrospun PCL, H-PCL, B-PCL and F-PCL nanofibers was studied by scanning electron microscopy (SEM) (JSM 5600, JEOL, Japan) at an accelerating voltage of 15 kV. Before observation, the scaffolds were coated with gold using a sputter coater (Jeol JFC-1200 fine coater, Japan). The diameter of the fibers was measured from the SEM micrographs using image analysis software (Image J, National Institutes of Health, USA). For determination of wettability (or hydrophilicity) of scaffolds, the contact-angle of electrospun nanofibers were measured by a video contact angle system (VCA Optima, AST Products). The droplet size was set at 0.5 μL. Five samples were used for each test and the average value was reported with standard deviation (±SD). ATR-FTIR is a powerful technique to understand the surface chemistry of a modified surface. ATR-FTIR spectroscopy of PCL, H-PCL, B-PCL and F-PCL nanofibrous scaffolds were performed over a range of 4000–400 cm− 1 at a resolution of 2 cm− 1 using a Nicolet spectrometer system. Mechanical properties of different scaffolds were determined using a table-top uniaxial testing machine (INSTRON 3345) using a 10-N load cell at a cross-head speed of 10 mm/min at ambient conditions. All samples were prepared in rectangular shapes with dimensions of 20 × 10 mm from the electrospun membranes. At least six samples were tested for each type of electrospun nanofibrous membrane and the average value was reported with standard deviation (±SD).
2.3. Modification of PCL nanofibrous scaffolds by alkaline hydrolysis
2.6. In vitro cell culture study
Electrospun PCL nanofibrous scaffolds were immersed in aqueous sodium hydroxide solution of 5% (w/v) for varying periods (1 h, 4 h and 12 h) at room temperature. After hydrolysis, the scaffolds were washed with de-ionized (DI) water repeatedly. For complete neutralization of NaOH, the samples were treated with 1 N HCL for 10 min at room temperature, washed with DI water and subsequently dried in a vacuum oven at room temperature for 24 h. The “Hydrolyzed PCL nanofibers” is termed as H-PCL, hereafter.
2.6.1. Neural precursor cell culture and seeding In vitro cell culture studies were carried out using Neonatal mouse cerebellum C17.2 stem cells. These cells can be used as neuron precursors since they are involved in the normal development of cerebellum, embryonic neocortex and other structures upon implantation into mouse germinal zones [18]. Neural precursor cells (NPCs) were cultured in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5% horse serum (HS) and 1% penicillin/streptomycin. After reaching 70% confluency, the cells were detached by trypsin/EDTA and viable cells were counted on a hemocytometer by trypan blue assay. Electrospun nanofibrous scaffolds were exposed to UV radiation for 2 h, washed 3 times with PBS and incubated with DMEM/F12 (1:1) mixture containing N2 supplement for 24 h before cell seeding. Cells were further seeded on nanofibrous scaffolds placed in a 24-well plate at a density of 15 × 103 cells/well and cultured with DMEM/F12 (1:1) mixture containing 1% N2 supplement at 37 °C, 5% CO2 and 95% humidity incubation conditions. Tissue cultured polystyrene cover glass (TCP) was used as the control.
PCL (Mw 80,000), 2-(N-morpholino)ethanesulfonic acid (MES), sodium hydroxide (NaOH), hydrochloric acid (HCL) and 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDAC) were obtained from Sigma-Aldrich (St. Louis, MO). Dimethyl formamide (DMF) and methylene chloride (MC) were purchased from Merck (Singapore) and hexamethyldisilazane (HMDS) was purchased from Fluka (USA). Dulbecco Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), trypsin-EDTA and horse serum (HS) were purchased from Gibco (Singapore). CellTiter 96® AQueous One solution reagent (MTS) used for cell viability measurement was purchased from Promega (Singapore) and matrigel was obtained from BD Biosciences (Singapore). 2.2. Fabrication of PCL nanofibrous scaffolds
2.4. Modification of PCL nanofibrous scaffolds by matrigel Matrigel was used for the modification of PCL nanofibrous scaffolds by two different methods: (i) matrigel was blended with PCL solution and the resultant solution was electrospun to obtain ‘blended PCL/matrigel’ nanofibrous scaffolds termed as B-PCL and (ii) matrigel was covalently attached on the surface of H-PCL nanofibrous scaffolds to obtain ‘functionalized PCL/matrigel’ scaffolds termed as F-PCL. 2.4.1. Electrospinning of ‘blended PCL/matrigel’ nanofibrous scaffolds Electrospun PCL/matrigel nanofibers were fabricated by blending PCL solution with matrigel at a ratio of 1000:1 (wt/wt). In short, matrigel was thawed at 4 ° C, mixed with PCL solution and stirred for 24 h at room temperature. The solution was electrospun at a high voltage of 13 kV and nanofibers were collected.
2.6.2. Cell proliferation study To study the cell proliferation on different substrates, cell proliferation was determined by the colorimetric MTS assay. MTS assay is based on the reduction of yellow tetrazolium salt in MTS to
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Fig. 1. Schematics of covalent attachment of matrigel on the surface of PCL nanofibrous scaffolds.
form purple formazan by dehydrogenase enzymes secreted from the mitochondria of metabolically active cells. The amount of formazan formed is directly proportional to the number of viable cells. After 2, 4 and 6 days of cell seeding on the scaffolds and TCP, the cells were washed with PBS and incubated with 20% MTS reagent containing serum free medium. After 3 h of incubation at 37 °C in 5% CO2, aliquots were pipetted into a 96-well plate and the absorbance of the content of each well was measured at 492 nm using a spectrophotometric plate reader (Fluostar Optima, BMG Lab Technologies, Germany). 2.6.3. Cell morphology study The morphology of nerve cells on nanofibrous scaffolds were observed under SEM. After 4 days of cell seeding, samples were fixed with 3% glutaraldehyde for 2 h, rinsed in water and dehydrated with graded concentrations (50, 70, 90 and 100% v/v) of ethanol. Subsequently the samples were treated with HMDS and kept in a fume hood for air drying. Finally the samples were coated with gold for observation of cell morphology under SEM. 2.6.4. Measurement of cells surface contact area Cell surface contact area was used as a parameter for indicating cell spreading on the different scaffolds and was assessed using image analysis software (Image J, National Institutes of Health, USA) on at least 50 randomly obtained SEM images of cells on different scaffolds. The total cell surface contact area was measured by summing the area covered by the cells over defined area of scaffold. The values of cell
surface contact area for each scaffold were obtained from the average of cell surface contact area values on 50 scaffolds. 2.6.5. Immunocytochemistry Immunocytochemistry was used for the investigation of differentiation of NPCs using antibodies that specifically target the peptides or protein antigens in the cells via specific epitopes. Immunofluorescent labeling for neurofilament 200 kDa (NF200) was performed to investigate the differentiation of NPCs seeded on the nanofibrous scaffolds using laser scanning confocal microscopy (LSCM, Fluoview FV300, Olympus). Briefly, after 4 days of culture, the samples were rinsed twice with PBS and fixed in 4% paraformaldehyde for 30 min at room temperature followed by permeation with 0.5% Triton X-100 for 5 min. The non-specific labeling was blocked by incubating the samples with 2% BSA and further the samples were incubated with anti-NF200 (diluted as 1:200, Sigma, USA) overnight. Subsequently the samples were washed with PBS and incubated in FITC conjugated rabbit anti-mouse secondary antibody (diluted at 1:50, Sigma, USA) for 1 h at 37 °C and finally the samples were mounted onto glass slides and viewed under LSCM. 2.7. Statistical analysis All data presented are expressed as mean ± standard deviation (SD). Statistical analysis was carried out using single-factor analysis of variance (ANOVA). A value of p ≤ 0.05 was considered statistically significant.
Fig. 2. Morphology of (A) H-PCL (4 h) nanofibers, (B) H-PCL (12 h) nanofibers.
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Table 1 Water contact angle and tensile strength of PCL and hydrolyzed PCL scaffolds. Scaffolds (hydrolyzed period)
Water contact angle (°)
Tensile strength (MPa)
PCL H-PCL (1 h) H-PCL (4 h) H-PCL (12 h)
118 ± 6 110 ± 2 83 ± 4 60 ± 5
3.76 ± 1.02 3.38 ± 0.9 3.00 ± 0.26 1.50 ± 0.22
3. Results
PCL (12 h) hydrolyzed scaffolds compared to PCL scaffolds. For determination of stability and integrity, the mechanical strength of scaffolds was assessed and the results revealed a significant decrease (p ≤ 0.05) in tensile strength of H-PCL (12 h) scaffolds compared to all other scaffolds of this study (Table 1), indicating the destructive effect of longer duration of alkaline hydrolysis. The optimized hydrolysis period for PCL was identified as 4 h during this study as it improved the hydrophilicity of PCL nanofibrous scaffolds without any morphology change or significant decrease in tensile strength, thus producing scaffolds with properties suitable for nerve regeneration.
3.1. Morphology and characterization of hydrolyzed PCL 3.2. Modification of PCL nanofibrous scaffolds with matrigel Alkaline hydrolysis of PCL was carried out for different periods of time (1 h, 4 h, and 12 h), in order to identify the best condition for process optimization of alkaline hydrolysis of electrospun PCL nanofibrous scaffolds. Analysis of SEM images showed no morphological changes of PCL nanofibrous scaffolds after 1 h and 4 h of alkaline hydrolysis while alkaline hydrolysis for 12 h resulted in nanofiber breakages (Fig. 2). Table 1 shows the results of contact angle measurement of PCL and H-PCL for 1 h, 4 h and 12 h. Contact angle of PCL nanofibrous scaffolds decreased with increasing duration of alkaline hydrolysis. Moreover the contact angle value decreased significantly for H-PCL (4 h) and H-
3.2.1. Characterization of electrospun PCL/matrigel (B-PCL) nanofibers A homogenous solution of PCL/matrigel was obtained using PCL/ matrigel at a ratio of 1000:1 (wt/wt) during this study. Higher concentrations of matrigel were attempted during this study. Addition of higher amounts of matrigel into PCL solution resulted in the separation of liquid and gel phase, that hindered the electrospinning process and continuous fibers were not obtained. Therefore the maximum ratio of PCL/matrigel was chosen for this study as 1000:1. Fig. 3 shows the SEM micrographs of PCL and B-PCL prepared by electrospinning process, along with their fiber diameter distributions.
Fig. 3. SEM micrographs of (A) PCL, (B) B-PCL and fiber diameter distributions of (C) PCL, (D) B-PCL.
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Fig. 4. SEM micrograph of (A) PCL and (B) F-PCL.
Comparison of fiber diameter distribution between PCL and B-PCL nanofibers revealed an increased fiber diameter distribution for B-PCL nanofibers compared to PCL nanofibers (135–1090 nm vs 214– 715 nm), suggesting the addition of matrigel into the electrospinning solution resulted in a wider fiber diameter distribution. Contact angle measurements showed that the incorporation of matrigel into PCL solution and electrospinning of resultant solution decreased the contact angle of PCL nanofibers from 118 ± 6° to 103 ± 4° for B-PCL nanofibers. Therefore the incorporation of matrigel into PCL nanofibers did not significantly change the hydrophilicity of nanofibers. 3.2.2. Characterization of matrigel functionalized PCL nanofibers (F-PCL) The SEM micrographs of PCL and F-PCL are shown in Fig. 4. Results of SEM analysis showed uniform nanofiber mat revealing a uniform distribution of matrigel on the surface of PCL nanofibrous scaffolds. Moreover, the fiber diameters of PCL and F-PCL nanofibrous scaffolds were found to be similar again revealing no formation of additional layer of matrigel around the nanofibers. The results of contact angle measurement showed that the water contact angle value decreased from 118±6° for PCL nanofibers to 44 ±6° for F-PCL indicating improved hydrophilicity of F-PCL nanofibrous scaffolds by covalent attachment of matrigel on the surface of PCL nanofibers. Fig. 5 shows the FTIR spectra of PCL and F-PCL nanofibers. The characteristic peaks in the FTIR spectra of PCL nanofibers include 2949 cm− 1 (asymmetric CH2 stretching), 2865 cm− 1 (symmetric CH2 stretching), 1727 cm− 1 (carbonyl stretching), 1293 cm− 1 (C–O and C–C stretching), 1240 cm− 1 (asymmetric COC stretching) and 1190 cm− 1 (OC–O stretching) [19–21]. The presence of extra peaks at 1650 cm− 1 and 1540 cm− 1 in the FTIR of F-PCL nanofibers confirms the presence of matrigel on the surface of PCL nanofibrous scaffold
Fig. 5. FTIR spectra of PCL and F-PCL nanofibrous scaffolds.
which are corresponding to the stretching vibrations of C O bond, and coupling of bending of N–H bond and stretching of C–N bonds, respectively [22,23].
3.3. In vitro cell culture study MTS assay was carried out to evaluate the proliferation of NPCs on PCL, H-PCL, B-PCL and F-PCL nanofibrous scaffolds. As shown in Fig. 6, cell proliferation increased significantly (p ≤ 0.05) on H-PCL compared to PCL nanofibrous scaffolds. Our results showed that the proliferation of NPCs on B-PCL was not significantly different (p N 0.05) compared to PCL nanofibrous scaffolds. However, the cell proliferation on F-PCL nanofibrous scaffolds was higher than that on PCL, H-PCL and B-PCL nanofibrous scaffolds indicating more effectiveness of this method for modification of PCL nanofibrous scaffolds compared to other methods used in this study. Fig. 7A–D shows the SEM micrographs of NPCs on PCL, H-PCL, B-PCL and F-PCL nanofibrous scaffolds. The morphology of nerve cells was retained on H-PCL than that on PCL nanofibrous scaffolds and H-PCL was found to be a better substrate for NPCs than PCL nanofibrous scaffolds. As can be seen in this figure, no obvious differences in morphology were observed between the cells grown on PCL and B-PCL nanofibrous scaffolds while nerve cells retained their original morphology on F-PCL nanofibrous scaffolds than PCL nanofibrous scaffolds and longer filapodial extensions and processes with more
Fig. 6. MTS results of nerve precursor cells on different nanofibrous scaffolds after 2, 4 and 6 days of cell seeding.
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Fig. 7. Morphology of cells on (A) PCL (B) B-PCL (C) H-PCL (D) F-PCL (magnification x3000) nanofibrous scaffolds after 4 days of cell culture.
spreading of NPCs were observed for cells seeded on F-PCL nanofibrous scaffolds compared to PCL and H-PCL nanofibrous scaffolds. The results of cell surface contact area measurements showed the cell surface contact area of 22 ± 3%, 40 ± 5% and 70 ± 5% for PCL, HPCL and F-PCL nanofibrous scaffolds respectively revealing more spreading of cells on F-PCL nanofibrous scaffolds followed by H-PCL nanofibrous scaffolds compared to cells spread on PCL nanofibrous scaffolds. Fig. 8A–C shows the LSCM micrographs of differentiated NPCs on PCL, H-PCL and F-PCL nanofibrous scaffolds indicating the differentiation of NPCs to nerve cells on the nanofibrous scaffolds. Longer neuritis was observed for cells grown on F-PCL nanofibrous scaffolds compared to the neurite length of cells grown on PCL and H-PCL nanofibrous scaffolds. 4. Discussion The fundamental approach in neural tissue engineering involves the fabrication of polymeric scaffolds with nerve cells to produce a three-dimensional functional tissue suitable for implantation [24] and fabrication of scaffolds with suitable chemical and mechanical properties is critical for the success of nerve tissue engineering. The hydrophilic–hydrophobic characteristic of scaffold is important in tissue culture and this can influence the initial cell adhesion and migration to a higher extent [25–28]. Extensive efforts have been devoted by various research groups towards increasing the hydro-
philicity of scaffolds by different methods [25–28]. Apart from hydrophilicity, the presence of functional groups on the surface of the scaffold is also important to aid cell adhesion. Coating of biomaterial surfaces with cell adhesive proteins such as fibronectin, vitronectin, collagen, laminin, or ECM resembling molecules such as chitosan and gelatin has been the most common and popular approaches for the development of improved biomaterial surfaces [3,9,11,13,29,30]. Although a simple physical coating of cell adhesive proteins can promote cell adhesion, this method is limited by poor stability of the cell adhesive proteins due to its weak interactive forces with the biomaterial surfaces [31]. Covalent binding of cell adhesive proteins to biomaterials could be a promising alternative approach that can be implemented for obtaining a stable layer on the surface of biomaterials. Yet another procedure is the blending of adhesive proteins with biomaterials to fabricate composite polymeric scaffolds. Blending results in a more uniform distribution of cell adhesive proteins in the biomaterial matrix and provides a more stable scaffold than that obtained by adsorption methods [31]. The major extracellular matrix proteins involved in contact of Schwann cells/axon units are the laminin and collagen IV [15]. Matrigel, in addition to TGF-beta, fibroblast growth factor, tissue plasminogen activator, is mainly composed of laminin, collagen IV, heparin sulfate proteoglycans, entactin and nidogen, and is a bioactive agent, used in numerous in vitro and in vivo studies for cell differentiations [16,17]. Qian et al coated tissue culture plate (TCP) with matrigel and investigated its impact on the expansion and neuronal differentiation
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Fig. 8. LSCM of nerve precursor cells after 4 day seeding on (A) PCL (B) H-PCL (C) F-PCL nanofibrous scaffolds.
of Mesenchymal stem cells and showed that matrigel enhanced neuronal differentiation and significantly improved cell expansion [32]. To the best of our knowledge, no studies have been performed on the functionalization of PCL nanofibrous scaffolds with matrigel that could be applied for effective nerve regeneration. In this study for the first time we functionalized the PCL nanofibrous scaffolds with matrigel and assessed its application towards nerve tissue engineering. In order to improve the hydrophilicity, alkaline hydrolysis was carried out for 1 h, 4 h and 12 h which overall revealed that the hydrolysis improves the hydrophilicity of PCL nanofibrous scaffolds (Table 1). Hydrophilicity was improved for 4 h and 12 h hydrolyzed scaffolds, but alkaline hydrolysis for 12 h period caused the breakage of
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nanofibers and significantly decreased the tensile strength of nanofiber mat. We identified the optimal duration for alkaline hydrolysis of PCL scaffolds as 4 h since it increased the hydrophilicity of PCL nanofibrous scaffolds without causing any adverse effects on tensile strength and fiber morphology. Tensile strength of H-PCL for 4 h was found to be 3.00± 0.26 MPa (Table 1) which is comparable to the tensile properties of fresh transected adult rat sciatic nerve (2.72± 0.97 MPa) reported by Broschel et al. [33]. The improved hydrophilicity of PCL nanofibrous scaffolds after alkaline hydrolysis is due to the formation of carboxyl and hydroxyl groups during alkaline hydrolysis and has been confirmed previously by some researchers [34–38]. Studies on the hydrolyzed degradation behavior of PCL and other aliphatic polyesters were also carried out by Pena et al. and Pamuła et al. [39,40]. To carry out any covalent attachment of biomolecules such as matrigel on the surface of scaffolds, a reactive group such as carboxyl, amino or hydroxyl must be present, which is usually not present in hydrophobic polymers such as PCL [41]. Although there are different approaches for introduction of reactive groups on the surface of polymers, including plasma treatment and subsequent exposing to air, treatment with hydrogen peroxide along with irradiation under UV for introduction of peroxide group (–OOH), alkaline hydrolysis used in this study appears to be a simpler and more effective approach for introduction of reactive groups on the surface of scaffolds. Our results showed that covalent attachment of matrigel on the surface of PCL nanofibrous scaffolds improved the hydrophilicity compared to H-PCL scaffolds. This appears to be due to the presence of more functional groups in the backbone of matrigel, thereby increasing the hydrophilicity of F-PCL nanofibrous scaffolds much more than the HPCL. In order to evaluate whether blending of PCL with matrigel and electrospinning of resultant solution results in a similar improvement, the matrigel was blended with PCL solution and the resultant solution was electrospun to nanofibers. The results revealed no significant improvement in hydrophilicity of the scaffolds. In addition, it induced a wider fiber diameter distribution. These observations are likely due to the non-homogeneity of the obtained solution and low concentrations of matrigel in the electrospinning solution. Results of MTS assay, SEM, LSCM observations and surface contact area of cells revealed higher proliferation of NPCs, longer neurite extension of cells and larger surface contact area of cells on H-PCL nanofibrous scaffolds than PCL nanofibrous scaffolds. The results of improved cell adherence and proliferation on H-PCL scaffolds are mainly due to the presence of reactive groups on the polymer surface and improved hydrophilicity after hydrolysis, similar to those reported by other researchers [7,36,38,42]. Incorporation of matrigel onto PCL electrospinning solution by physically mixing matrigel into PCL solution was found ineffective towards the cell proliferation, morphology and surface contact area of NPCs compared to PCL nanofibrous scaffolds. Therefore, it is not a suitable method for modification of PCL nanofibers. Cell proliferation, morphology and surface contact area of NPCs were further enhanced significantly on F-PCL than H-PCL and B-PCL nanofibrous scaffolds. Hydrophilicity/hydrophobicity may be the initial parameter affecting protein adsorption [43]. It can be concluded that hydrophilicity of a scaffold affects the surface energy of scaffolds, which might influence serum proteins that adhere to scaffolds, and in turn govern the biological response, such as cell adhesion and proliferation [44]. Additionally the presence of matrigel on the surface of PCL nanofibrous scaffolds allowed NPCs to preferentially attach to the proteins of their native environment and provide a bioactive scaffold. Hence the improvement observed on F-PCL nanofibrous scaffolds could be due to the concerted actions of its ECM components and inherent growth factors [32]. Moreover, previous studies have proved that terminal chemical groups could control cell growth and differentiation. Ren et al. showed that amino groups are more effective than hydroxyl and carboxyl groups towards nerve stem cell proliferation and migration [45]. It can be concluded that the presence of amino groups
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on the surface of F-PCL nanofibrous scaffolds and higher efficiency of amino groups than hydroxyl and carboxyl groups may account for more proliferation of NPCs and filopedia extension of nerve cells on F-PCL nanofibrous scaffolds than H-PCL scaffolds containing hydroxyl and carboxyl groups on their surface. Matrigel contain several different kinds of protein and the conjugation activity of individual proteins in matrigel with PCL might vary at different locations, though such investigations were not carried out during this study. A comparative study on the methods of surface modification using matrigel was only explored during this study and the F-PCL nanofibers were found more suitable for enhanced nerve regenerations. To the best of our knowledge, this is the first report which functionalized PCL nanofibrous scaffolds with biomolecules such as matrigel by making use of hydrolyzed PCL scaffolds. Our findings could encourage the development of novel biomaterials that mimic the chemical and physical microenvironment of neural tissues. The combined results of our study open new opportunities for further investigations on the modification of nanofibrous scaffolds using alkaline hydrolysis with different biomolecules. Moreover, the effect of matrigel on the proliferation and behavior of other cells for tissue engineering application can also be investigated. 5. Conclusion The hydrophilicity of PCL nanofibrous scaffolds was improved by the process of alkaline hydrolysis. Taking advantage of the available reactive groups on the surface of PCL nanofibrous scaffolds created by alkaline hydrolysis, we further carried out the covalent attachment of matrigel on PCL nanofibrous scaffolds. FTIR spectra confirmed the occurrence of covalent attachment of matrigel on the surface of PCL nanofibrous scaffolds. Blending of matrigel with PCL solution and electrospinning the resultant solution did not appear to have any significant effect on hydrophilicity of scaffolds, cell proliferation, morphology and surface contact area. Our results demonstrated that functionalized PCL nanofibrous scaffolds with matrigel promoted cell proliferation and cell surface contact area compared to PCL, H-PCL and B-PCL nanofibrous scaffolds. Acknowledgements We would like to express our thanks to National Medical Research Council (grant number: NMRC/1015/2005, R397000625213), Singapore for their financial support. References [1] M.P. Prabhakaran, J. Venugopal, C.K. Chan, S. Ramakrishna, Nanotechnology 19 (2008) 455102. [2] S. Blacher, V. Maquet, F. Schils, D. Martin, J. Schoenen, G. Moonen, R. Jérôme, J.P. Pirard, Biomaterials 24 (2003) 1033.
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Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Bio-functionalized PCL nanofibrous scaffolds for nerve tissue engineering Laleh Ghasemi-Mobarakeh a,b,c,d, Molamma P. Prabhakaran a,⁎, Mohammad Morshed c, Mohammad Hossein Nasr-Esfahani d, S. Ramakrishna a,e,⁎ a
Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore , 2 Engineering Drive 3, Singapore 117576, Singapore Islamic Azad University, Najafabad branch, Isfahan, Iran Department of Textile Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran d Department of Cell Sciences Research Center (Isfahan Campus), Royan Institute, ACECR, Tehran, Iran e King Saud University, 11451 Riyadh, Saudi Arabia b c
a r t i c l e
i n f o
Article history: Received 7 February 2010 Received in revised form 16 May 2010 Accepted 8 June 2010 Available online 19 June 2010 Keywords: Alkaline hydrolysis PCL Nanofibrous scaffolds Matrigel Nerve tissue engineering
a b s t r a c t Surface properties of scaffolds such as hydrophilicity and the presence of functional groups on the surface of scaffolds play a key role in cell adhesion, proliferation and migration. Different modification methods for hydrophilicity improvement and introduction of functional groups on the surface of scaffolds have been carried out on synthetic biodegradable polymers, for tissue engineering applications. In this study, alkaline hydrolysis of poly (ε-caprolactone) (PCL) nanofibrous scaffolds was carried out for different time periods (1 h, 4 h and 12 h) to increase the hydrophilicity of the scaffolds. The formation of reactive groups resulting from alkaline hydrolysis provides opportunities for further surface functionalization of PCL nanofibrous scaffolds. Matrigel was attached covalently on the surface of an optimized 4 h hydrolyzed PCL nanofibrous scaffolds and additionally the fabrication of blended PCL/matrigel nanofibrous scaffolds was carried out. Chemical and mechanical characterization of nanofibrous scaffolds were evaluated using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, contact angle, scanning electron microscopy (SEM) and tensile measurement. In vitro cell adhesion and proliferation study was carried out after seeding nerve precursor cells (NPCs) on different scaffolds. Results of cell proliferation assay and SEM studies showed that the covalently functionalized PCL/matrigel nanofibrous scaffolds promote the proliferation and neurite outgrowth of NPCs compared to PCL and hydrolyzed PCL nanofibrous scaffolds, providing suitable substrates for nerve tissue engineering. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nerve tissue engineering is a rapidly expanding area of research, providing new and promising approach to nerve repair and regeneration [1]. Design and fabrication of scaffolds suitable for neural tissue engineering is critical for the success of tissue regeneration [2]. Scaffold surfaces play a vital role for the success of tissue engineering as the interaction between cells and scaffolds occur at the surface of the scaffolds [3]. Synthetic biodegradable polymers are now widely used in tissue engineering due to their mechanical properties along with adjustable degradation rates [4]. However, cell affinity towards synthetic hydrophobic polymers is poor as a consequence of their low hydrophilicity and lack of surface cell recognition sites [5]. Recently nano-structured materials are receiving considerable attention for application in tissue engineering, essentially because the body consists of nanoscale structures of extra cellular matrix (ECM) providing a natural web of intricate nanofibers supporting cells and provides an instructive background guiding their ⁎ Corresponding authors. E-mail addresses: [email protected] (M.P. Prabhakaran), [email protected] (S. Ramakrishna). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.06.004
behavior [6]. Therefore improving the hydrophilicity of nanostructured scaffolds together with the incorporation of biological molecules on to the nanofibrous scaffolds could provide both physical and chemical signals required for cell growth. Poly (ε-caprolactone) (PCL) has been used for the reconstruction of various tissues such as bone, skin, nerve, retina and has several advantages including its biocompatibility, low cost and easy processability. However, being a synthetic biomaterial, PCL has a hydrophobic surface, lacks functional groups and hence it is not a good substrate for cell adhesion. To improve the hydrophilicity and biological properties of PCL nanofibrous scaffolds various techniques have been applied. Previous studies mostly modified the PCL nanofibrous scaffolds by incorporation of biologically active polymers such as gelatin and collagen or the hydrophilicity of PCL scaffolds was improved by methods such as laser and plasma treatment [1,7–13]. However, there are very few reports available on the functionalization of PCL scaffolds using biomolecules for tissue engineering [10–13]. Previous studies have shown that laminin, collagen IV, fibronectin and heparin sulphate proteoglycans support neurite outgrowth [14]. Our study is aimed at investigating a simple procedure such as alkaline hydrolysis of PCL nanofibrous scaffolds for improving its hydrophilicity and further attaching matrigel on them, to produce
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scaffolds suitable for nerve tissue engineering. Matrigel is a soluble and sterile extract of basement membrane proteins derived from the Engelberth–Holm–Swarm (EHS) mouse sarcoma and it is rich in extracellular proteins such as laminin, collagen IV, fibronectin and heparin sulphate proteoglycans [14–17]. Matrigel being an important component of the nervous system, functionalization of matrigel on the surface of an already modified PCL nanofibrous scaffold was rapidly achievable. For comparative evaluation studies, matrigel was also mixed with PCL solution and the electrospun ‘blended PCL/matrigel’ nanofibrous scaffolds were fabricated. The effect of incorporation of matrigel into PCL nanofibrous scaffolds towards the proliferation and differentiation of NPCs was further investigated.
2.4.2. Covalent attachment of matrigel on hydrolyzed PCL to obtain ‘functionalized PCL/matrigel’ scaffolds Hydrolyzed PCL (H-PCL) was used for the covalent attachment of matrigel on their surface. For covalent attachment of matrigel on PCL nanofibers, the H-PCL scaffolds were immersed in 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution (0.1 M, pH 5.0) of 5 mg/mL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) for 1 h at room temperature. Scaffolds were further rinsed with DI water and incubated with 20 μL of matrigel for 24 h and subsequently washed with PBS. Fig. 1 shows schematic representation of covalent attachment of matrigel on the surface of PCL nanofibrous scaffolds.
2. Materials and methods
2.5. Characterization of nanofibrous scaffolds
2.1. Materials
PCL (11% w/v) was dissolved in MC/DMF at a ratio of 80:20 and stirred for a period of 24 h at room temperature. The solution was electrospun from a 5 mL syringe with needle diameter of 0.4 mm and mass flow rate of 1 mL/h. A high voltage (13 kV) was applied to the tip of the needle attached to the syringe. The resulting fibers were collected on 15 mm cover slips placed on a flat aluminum plate collector kept at a distance of 12 cm from the needle tip.
The morphology of electrospun PCL, H-PCL, B-PCL and F-PCL nanofibers was studied by scanning electron microscopy (SEM) (JSM 5600, JEOL, Japan) at an accelerating voltage of 15 kV. Before observation, the scaffolds were coated with gold using a sputter coater (Jeol JFC-1200 fine coater, Japan). The diameter of the fibers was measured from the SEM micrographs using image analysis software (Image J, National Institutes of Health, USA). For determination of wettability (or hydrophilicity) of scaffolds, the contact-angle of electrospun nanofibers were measured by a video contact angle system (VCA Optima, AST Products). The droplet size was set at 0.5 μL. Five samples were used for each test and the average value was reported with standard deviation (±SD). ATR-FTIR is a powerful technique to understand the surface chemistry of a modified surface. ATR-FTIR spectroscopy of PCL, H-PCL, B-PCL and F-PCL nanofibrous scaffolds were performed over a range of 4000–400 cm− 1 at a resolution of 2 cm− 1 using a Nicolet spectrometer system. Mechanical properties of different scaffolds were determined using a table-top uniaxial testing machine (INSTRON 3345) using a 10-N load cell at a cross-head speed of 10 mm/min at ambient conditions. All samples were prepared in rectangular shapes with dimensions of 20 × 10 mm from the electrospun membranes. At least six samples were tested for each type of electrospun nanofibrous membrane and the average value was reported with standard deviation (±SD).
2.3. Modification of PCL nanofibrous scaffolds by alkaline hydrolysis
2.6. In vitro cell culture study
Electrospun PCL nanofibrous scaffolds were immersed in aqueous sodium hydroxide solution of 5% (w/v) for varying periods (1 h, 4 h and 12 h) at room temperature. After hydrolysis, the scaffolds were washed with de-ionized (DI) water repeatedly. For complete neutralization of NaOH, the samples were treated with 1 N HCL for 10 min at room temperature, washed with DI water and subsequently dried in a vacuum oven at room temperature for 24 h. The “Hydrolyzed PCL nanofibers” is termed as H-PCL, hereafter.
2.6.1. Neural precursor cell culture and seeding In vitro cell culture studies were carried out using Neonatal mouse cerebellum C17.2 stem cells. These cells can be used as neuron precursors since they are involved in the normal development of cerebellum, embryonic neocortex and other structures upon implantation into mouse germinal zones [18]. Neural precursor cells (NPCs) were cultured in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5% horse serum (HS) and 1% penicillin/streptomycin. After reaching 70% confluency, the cells were detached by trypsin/EDTA and viable cells were counted on a hemocytometer by trypan blue assay. Electrospun nanofibrous scaffolds were exposed to UV radiation for 2 h, washed 3 times with PBS and incubated with DMEM/F12 (1:1) mixture containing N2 supplement for 24 h before cell seeding. Cells were further seeded on nanofibrous scaffolds placed in a 24-well plate at a density of 15 × 103 cells/well and cultured with DMEM/F12 (1:1) mixture containing 1% N2 supplement at 37 °C, 5% CO2 and 95% humidity incubation conditions. Tissue cultured polystyrene cover glass (TCP) was used as the control.
PCL (Mw 80,000), 2-(N-morpholino)ethanesulfonic acid (MES), sodium hydroxide (NaOH), hydrochloric acid (HCL) and 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDAC) were obtained from Sigma-Aldrich (St. Louis, MO). Dimethyl formamide (DMF) and methylene chloride (MC) were purchased from Merck (Singapore) and hexamethyldisilazane (HMDS) was purchased from Fluka (USA). Dulbecco Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), trypsin-EDTA and horse serum (HS) were purchased from Gibco (Singapore). CellTiter 96® AQueous One solution reagent (MTS) used for cell viability measurement was purchased from Promega (Singapore) and matrigel was obtained from BD Biosciences (Singapore). 2.2. Fabrication of PCL nanofibrous scaffolds
2.4. Modification of PCL nanofibrous scaffolds by matrigel Matrigel was used for the modification of PCL nanofibrous scaffolds by two different methods: (i) matrigel was blended with PCL solution and the resultant solution was electrospun to obtain ‘blended PCL/matrigel’ nanofibrous scaffolds termed as B-PCL and (ii) matrigel was covalently attached on the surface of H-PCL nanofibrous scaffolds to obtain ‘functionalized PCL/matrigel’ scaffolds termed as F-PCL. 2.4.1. Electrospinning of ‘blended PCL/matrigel’ nanofibrous scaffolds Electrospun PCL/matrigel nanofibers were fabricated by blending PCL solution with matrigel at a ratio of 1000:1 (wt/wt). In short, matrigel was thawed at 4 ° C, mixed with PCL solution and stirred for 24 h at room temperature. The solution was electrospun at a high voltage of 13 kV and nanofibers were collected.
2.6.2. Cell proliferation study To study the cell proliferation on different substrates, cell proliferation was determined by the colorimetric MTS assay. MTS assay is based on the reduction of yellow tetrazolium salt in MTS to
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Fig. 1. Schematics of covalent attachment of matrigel on the surface of PCL nanofibrous scaffolds.
form purple formazan by dehydrogenase enzymes secreted from the mitochondria of metabolically active cells. The amount of formazan formed is directly proportional to the number of viable cells. After 2, 4 and 6 days of cell seeding on the scaffolds and TCP, the cells were washed with PBS and incubated with 20% MTS reagent containing serum free medium. After 3 h of incubation at 37 °C in 5% CO2, aliquots were pipetted into a 96-well plate and the absorbance of the content of each well was measured at 492 nm using a spectrophotometric plate reader (Fluostar Optima, BMG Lab Technologies, Germany). 2.6.3. Cell morphology study The morphology of nerve cells on nanofibrous scaffolds were observed under SEM. After 4 days of cell seeding, samples were fixed with 3% glutaraldehyde for 2 h, rinsed in water and dehydrated with graded concentrations (50, 70, 90 and 100% v/v) of ethanol. Subsequently the samples were treated with HMDS and kept in a fume hood for air drying. Finally the samples were coated with gold for observation of cell morphology under SEM. 2.6.4. Measurement of cells surface contact area Cell surface contact area was used as a parameter for indicating cell spreading on the different scaffolds and was assessed using image analysis software (Image J, National Institutes of Health, USA) on at least 50 randomly obtained SEM images of cells on different scaffolds. The total cell surface contact area was measured by summing the area covered by the cells over defined area of scaffold. The values of cell
surface contact area for each scaffold were obtained from the average of cell surface contact area values on 50 scaffolds. 2.6.5. Immunocytochemistry Immunocytochemistry was used for the investigation of differentiation of NPCs using antibodies that specifically target the peptides or protein antigens in the cells via specific epitopes. Immunofluorescent labeling for neurofilament 200 kDa (NF200) was performed to investigate the differentiation of NPCs seeded on the nanofibrous scaffolds using laser scanning confocal microscopy (LSCM, Fluoview FV300, Olympus). Briefly, after 4 days of culture, the samples were rinsed twice with PBS and fixed in 4% paraformaldehyde for 30 min at room temperature followed by permeation with 0.5% Triton X-100 for 5 min. The non-specific labeling was blocked by incubating the samples with 2% BSA and further the samples were incubated with anti-NF200 (diluted as 1:200, Sigma, USA) overnight. Subsequently the samples were washed with PBS and incubated in FITC conjugated rabbit anti-mouse secondary antibody (diluted at 1:50, Sigma, USA) for 1 h at 37 °C and finally the samples were mounted onto glass slides and viewed under LSCM. 2.7. Statistical analysis All data presented are expressed as mean ± standard deviation (SD). Statistical analysis was carried out using single-factor analysis of variance (ANOVA). A value of p ≤ 0.05 was considered statistically significant.
Fig. 2. Morphology of (A) H-PCL (4 h) nanofibers, (B) H-PCL (12 h) nanofibers.
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Table 1 Water contact angle and tensile strength of PCL and hydrolyzed PCL scaffolds. Scaffolds (hydrolyzed period)
Water contact angle (°)
Tensile strength (MPa)
PCL H-PCL (1 h) H-PCL (4 h) H-PCL (12 h)
118 ± 6 110 ± 2 83 ± 4 60 ± 5
3.76 ± 1.02 3.38 ± 0.9 3.00 ± 0.26 1.50 ± 0.22
3. Results
PCL (12 h) hydrolyzed scaffolds compared to PCL scaffolds. For determination of stability and integrity, the mechanical strength of scaffolds was assessed and the results revealed a significant decrease (p ≤ 0.05) in tensile strength of H-PCL (12 h) scaffolds compared to all other scaffolds of this study (Table 1), indicating the destructive effect of longer duration of alkaline hydrolysis. The optimized hydrolysis period for PCL was identified as 4 h during this study as it improved the hydrophilicity of PCL nanofibrous scaffolds without any morphology change or significant decrease in tensile strength, thus producing scaffolds with properties suitable for nerve regeneration.
3.1. Morphology and characterization of hydrolyzed PCL 3.2. Modification of PCL nanofibrous scaffolds with matrigel Alkaline hydrolysis of PCL was carried out for different periods of time (1 h, 4 h, and 12 h), in order to identify the best condition for process optimization of alkaline hydrolysis of electrospun PCL nanofibrous scaffolds. Analysis of SEM images showed no morphological changes of PCL nanofibrous scaffolds after 1 h and 4 h of alkaline hydrolysis while alkaline hydrolysis for 12 h resulted in nanofiber breakages (Fig. 2). Table 1 shows the results of contact angle measurement of PCL and H-PCL for 1 h, 4 h and 12 h. Contact angle of PCL nanofibrous scaffolds decreased with increasing duration of alkaline hydrolysis. Moreover the contact angle value decreased significantly for H-PCL (4 h) and H-
3.2.1. Characterization of electrospun PCL/matrigel (B-PCL) nanofibers A homogenous solution of PCL/matrigel was obtained using PCL/ matrigel at a ratio of 1000:1 (wt/wt) during this study. Higher concentrations of matrigel were attempted during this study. Addition of higher amounts of matrigel into PCL solution resulted in the separation of liquid and gel phase, that hindered the electrospinning process and continuous fibers were not obtained. Therefore the maximum ratio of PCL/matrigel was chosen for this study as 1000:1. Fig. 3 shows the SEM micrographs of PCL and B-PCL prepared by electrospinning process, along with their fiber diameter distributions.
Fig. 3. SEM micrographs of (A) PCL, (B) B-PCL and fiber diameter distributions of (C) PCL, (D) B-PCL.
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Fig. 4. SEM micrograph of (A) PCL and (B) F-PCL.
Comparison of fiber diameter distribution between PCL and B-PCL nanofibers revealed an increased fiber diameter distribution for B-PCL nanofibers compared to PCL nanofibers (135–1090 nm vs 214– 715 nm), suggesting the addition of matrigel into the electrospinning solution resulted in a wider fiber diameter distribution. Contact angle measurements showed that the incorporation of matrigel into PCL solution and electrospinning of resultant solution decreased the contact angle of PCL nanofibers from 118 ± 6° to 103 ± 4° for B-PCL nanofibers. Therefore the incorporation of matrigel into PCL nanofibers did not significantly change the hydrophilicity of nanofibers. 3.2.2. Characterization of matrigel functionalized PCL nanofibers (F-PCL) The SEM micrographs of PCL and F-PCL are shown in Fig. 4. Results of SEM analysis showed uniform nanofiber mat revealing a uniform distribution of matrigel on the surface of PCL nanofibrous scaffolds. Moreover, the fiber diameters of PCL and F-PCL nanofibrous scaffolds were found to be similar again revealing no formation of additional layer of matrigel around the nanofibers. The results of contact angle measurement showed that the water contact angle value decreased from 118±6° for PCL nanofibers to 44 ±6° for F-PCL indicating improved hydrophilicity of F-PCL nanofibrous scaffolds by covalent attachment of matrigel on the surface of PCL nanofibers. Fig. 5 shows the FTIR spectra of PCL and F-PCL nanofibers. The characteristic peaks in the FTIR spectra of PCL nanofibers include 2949 cm− 1 (asymmetric CH2 stretching), 2865 cm− 1 (symmetric CH2 stretching), 1727 cm− 1 (carbonyl stretching), 1293 cm− 1 (C–O and C–C stretching), 1240 cm− 1 (asymmetric COC stretching) and 1190 cm− 1 (OC–O stretching) [19–21]. The presence of extra peaks at 1650 cm− 1 and 1540 cm− 1 in the FTIR of F-PCL nanofibers confirms the presence of matrigel on the surface of PCL nanofibrous scaffold
Fig. 5. FTIR spectra of PCL and F-PCL nanofibrous scaffolds.
which are corresponding to the stretching vibrations of C O bond, and coupling of bending of N–H bond and stretching of C–N bonds, respectively [22,23].
3.3. In vitro cell culture study MTS assay was carried out to evaluate the proliferation of NPCs on PCL, H-PCL, B-PCL and F-PCL nanofibrous scaffolds. As shown in Fig. 6, cell proliferation increased significantly (p ≤ 0.05) on H-PCL compared to PCL nanofibrous scaffolds. Our results showed that the proliferation of NPCs on B-PCL was not significantly different (p N 0.05) compared to PCL nanofibrous scaffolds. However, the cell proliferation on F-PCL nanofibrous scaffolds was higher than that on PCL, H-PCL and B-PCL nanofibrous scaffolds indicating more effectiveness of this method for modification of PCL nanofibrous scaffolds compared to other methods used in this study. Fig. 7A–D shows the SEM micrographs of NPCs on PCL, H-PCL, B-PCL and F-PCL nanofibrous scaffolds. The morphology of nerve cells was retained on H-PCL than that on PCL nanofibrous scaffolds and H-PCL was found to be a better substrate for NPCs than PCL nanofibrous scaffolds. As can be seen in this figure, no obvious differences in morphology were observed between the cells grown on PCL and B-PCL nanofibrous scaffolds while nerve cells retained their original morphology on F-PCL nanofibrous scaffolds than PCL nanofibrous scaffolds and longer filapodial extensions and processes with more
Fig. 6. MTS results of nerve precursor cells on different nanofibrous scaffolds after 2, 4 and 6 days of cell seeding.
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Fig. 7. Morphology of cells on (A) PCL (B) B-PCL (C) H-PCL (D) F-PCL (magnification x3000) nanofibrous scaffolds after 4 days of cell culture.
spreading of NPCs were observed for cells seeded on F-PCL nanofibrous scaffolds compared to PCL and H-PCL nanofibrous scaffolds. The results of cell surface contact area measurements showed the cell surface contact area of 22 ± 3%, 40 ± 5% and 70 ± 5% for PCL, HPCL and F-PCL nanofibrous scaffolds respectively revealing more spreading of cells on F-PCL nanofibrous scaffolds followed by H-PCL nanofibrous scaffolds compared to cells spread on PCL nanofibrous scaffolds. Fig. 8A–C shows the LSCM micrographs of differentiated NPCs on PCL, H-PCL and F-PCL nanofibrous scaffolds indicating the differentiation of NPCs to nerve cells on the nanofibrous scaffolds. Longer neuritis was observed for cells grown on F-PCL nanofibrous scaffolds compared to the neurite length of cells grown on PCL and H-PCL nanofibrous scaffolds. 4. Discussion The fundamental approach in neural tissue engineering involves the fabrication of polymeric scaffolds with nerve cells to produce a three-dimensional functional tissue suitable for implantation [24] and fabrication of scaffolds with suitable chemical and mechanical properties is critical for the success of nerve tissue engineering. The hydrophilic–hydrophobic characteristic of scaffold is important in tissue culture and this can influence the initial cell adhesion and migration to a higher extent [25–28]. Extensive efforts have been devoted by various research groups towards increasing the hydro-
philicity of scaffolds by different methods [25–28]. Apart from hydrophilicity, the presence of functional groups on the surface of the scaffold is also important to aid cell adhesion. Coating of biomaterial surfaces with cell adhesive proteins such as fibronectin, vitronectin, collagen, laminin, or ECM resembling molecules such as chitosan and gelatin has been the most common and popular approaches for the development of improved biomaterial surfaces [3,9,11,13,29,30]. Although a simple physical coating of cell adhesive proteins can promote cell adhesion, this method is limited by poor stability of the cell adhesive proteins due to its weak interactive forces with the biomaterial surfaces [31]. Covalent binding of cell adhesive proteins to biomaterials could be a promising alternative approach that can be implemented for obtaining a stable layer on the surface of biomaterials. Yet another procedure is the blending of adhesive proteins with biomaterials to fabricate composite polymeric scaffolds. Blending results in a more uniform distribution of cell adhesive proteins in the biomaterial matrix and provides a more stable scaffold than that obtained by adsorption methods [31]. The major extracellular matrix proteins involved in contact of Schwann cells/axon units are the laminin and collagen IV [15]. Matrigel, in addition to TGF-beta, fibroblast growth factor, tissue plasminogen activator, is mainly composed of laminin, collagen IV, heparin sulfate proteoglycans, entactin and nidogen, and is a bioactive agent, used in numerous in vitro and in vivo studies for cell differentiations [16,17]. Qian et al coated tissue culture plate (TCP) with matrigel and investigated its impact on the expansion and neuronal differentiation
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Fig. 8. LSCM of nerve precursor cells after 4 day seeding on (A) PCL (B) H-PCL (C) F-PCL nanofibrous scaffolds.
of Mesenchymal stem cells and showed that matrigel enhanced neuronal differentiation and significantly improved cell expansion [32]. To the best of our knowledge, no studies have been performed on the functionalization of PCL nanofibrous scaffolds with matrigel that could be applied for effective nerve regeneration. In this study for the first time we functionalized the PCL nanofibrous scaffolds with matrigel and assessed its application towards nerve tissue engineering. In order to improve the hydrophilicity, alkaline hydrolysis was carried out for 1 h, 4 h and 12 h which overall revealed that the hydrolysis improves the hydrophilicity of PCL nanofibrous scaffolds (Table 1). Hydrophilicity was improved for 4 h and 12 h hydrolyzed scaffolds, but alkaline hydrolysis for 12 h period caused the breakage of
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nanofibers and significantly decreased the tensile strength of nanofiber mat. We identified the optimal duration for alkaline hydrolysis of PCL scaffolds as 4 h since it increased the hydrophilicity of PCL nanofibrous scaffolds without causing any adverse effects on tensile strength and fiber morphology. Tensile strength of H-PCL for 4 h was found to be 3.00± 0.26 MPa (Table 1) which is comparable to the tensile properties of fresh transected adult rat sciatic nerve (2.72± 0.97 MPa) reported by Broschel et al. [33]. The improved hydrophilicity of PCL nanofibrous scaffolds after alkaline hydrolysis is due to the formation of carboxyl and hydroxyl groups during alkaline hydrolysis and has been confirmed previously by some researchers [34–38]. Studies on the hydrolyzed degradation behavior of PCL and other aliphatic polyesters were also carried out by Pena et al. and Pamuła et al. [39,40]. To carry out any covalent attachment of biomolecules such as matrigel on the surface of scaffolds, a reactive group such as carboxyl, amino or hydroxyl must be present, which is usually not present in hydrophobic polymers such as PCL [41]. Although there are different approaches for introduction of reactive groups on the surface of polymers, including plasma treatment and subsequent exposing to air, treatment with hydrogen peroxide along with irradiation under UV for introduction of peroxide group (–OOH), alkaline hydrolysis used in this study appears to be a simpler and more effective approach for introduction of reactive groups on the surface of scaffolds. Our results showed that covalent attachment of matrigel on the surface of PCL nanofibrous scaffolds improved the hydrophilicity compared to H-PCL scaffolds. This appears to be due to the presence of more functional groups in the backbone of matrigel, thereby increasing the hydrophilicity of F-PCL nanofibrous scaffolds much more than the HPCL. In order to evaluate whether blending of PCL with matrigel and electrospinning of resultant solution results in a similar improvement, the matrigel was blended with PCL solution and the resultant solution was electrospun to nanofibers. The results revealed no significant improvement in hydrophilicity of the scaffolds. In addition, it induced a wider fiber diameter distribution. These observations are likely due to the non-homogeneity of the obtained solution and low concentrations of matrigel in the electrospinning solution. Results of MTS assay, SEM, LSCM observations and surface contact area of cells revealed higher proliferation of NPCs, longer neurite extension of cells and larger surface contact area of cells on H-PCL nanofibrous scaffolds than PCL nanofibrous scaffolds. The results of improved cell adherence and proliferation on H-PCL scaffolds are mainly due to the presence of reactive groups on the polymer surface and improved hydrophilicity after hydrolysis, similar to those reported by other researchers [7,36,38,42]. Incorporation of matrigel onto PCL electrospinning solution by physically mixing matrigel into PCL solution was found ineffective towards the cell proliferation, morphology and surface contact area of NPCs compared to PCL nanofibrous scaffolds. Therefore, it is not a suitable method for modification of PCL nanofibers. Cell proliferation, morphology and surface contact area of NPCs were further enhanced significantly on F-PCL than H-PCL and B-PCL nanofibrous scaffolds. Hydrophilicity/hydrophobicity may be the initial parameter affecting protein adsorption [43]. It can be concluded that hydrophilicity of a scaffold affects the surface energy of scaffolds, which might influence serum proteins that adhere to scaffolds, and in turn govern the biological response, such as cell adhesion and proliferation [44]. Additionally the presence of matrigel on the surface of PCL nanofibrous scaffolds allowed NPCs to preferentially attach to the proteins of their native environment and provide a bioactive scaffold. Hence the improvement observed on F-PCL nanofibrous scaffolds could be due to the concerted actions of its ECM components and inherent growth factors [32]. Moreover, previous studies have proved that terminal chemical groups could control cell growth and differentiation. Ren et al. showed that amino groups are more effective than hydroxyl and carboxyl groups towards nerve stem cell proliferation and migration [45]. It can be concluded that the presence of amino groups
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on the surface of F-PCL nanofibrous scaffolds and higher efficiency of amino groups than hydroxyl and carboxyl groups may account for more proliferation of NPCs and filopedia extension of nerve cells on F-PCL nanofibrous scaffolds than H-PCL scaffolds containing hydroxyl and carboxyl groups on their surface. Matrigel contain several different kinds of protein and the conjugation activity of individual proteins in matrigel with PCL might vary at different locations, though such investigations were not carried out during this study. A comparative study on the methods of surface modification using matrigel was only explored during this study and the F-PCL nanofibers were found more suitable for enhanced nerve regenerations. To the best of our knowledge, this is the first report which functionalized PCL nanofibrous scaffolds with biomolecules such as matrigel by making use of hydrolyzed PCL scaffolds. Our findings could encourage the development of novel biomaterials that mimic the chemical and physical microenvironment of neural tissues. The combined results of our study open new opportunities for further investigations on the modification of nanofibrous scaffolds using alkaline hydrolysis with different biomolecules. Moreover, the effect of matrigel on the proliferation and behavior of other cells for tissue engineering application can also be investigated. 5. Conclusion The hydrophilicity of PCL nanofibrous scaffolds was improved by the process of alkaline hydrolysis. Taking advantage of the available reactive groups on the surface of PCL nanofibrous scaffolds created by alkaline hydrolysis, we further carried out the covalent attachment of matrigel on PCL nanofibrous scaffolds. FTIR spectra confirmed the occurrence of covalent attachment of matrigel on the surface of PCL nanofibrous scaffolds. Blending of matrigel with PCL solution and electrospinning the resultant solution did not appear to have any significant effect on hydrophilicity of scaffolds, cell proliferation, morphology and surface contact area. Our results demonstrated that functionalized PCL nanofibrous scaffolds with matrigel promoted cell proliferation and cell surface contact area compared to PCL, H-PCL and B-PCL nanofibrous scaffolds. Acknowledgements We would like to express our thanks to National Medical Research Council (grant number: NMRC/1015/2005, R397000625213), Singapore for their financial support. References [1] M.P. Prabhakaran, J. Venugopal, C.K. Chan, S. Ramakrishna, Nanotechnology 19 (2008) 455102. [2] S. Blacher, V. Maquet, F. Schils, D. Martin, J. Schoenen, G. Moonen, R. Jérôme, J.P. Pirard, Biomaterials 24 (2003) 1033.
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