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carboxymethyl chitosan nanofibrous scaffolds for bone tissue engineering application
Accepted Manuscript Polycaprolactone/carboxymethyl chitosan nanofibrous scaffolds for bone tissue engineering application
Fereshteh Sharifi, Seyed Mohammad Atyabi, Dariush Norouzian, Mojgan Zandi, Shiva Irani, Hadi Bakhshi PII: DOI: Reference:
S0141-8130(18)30638-X doi:10.1016/j.ijbiomac.2018.04.045 BIOMAC 9453
To appear in: Received date: Revised date: Accepted date:
9 February 2018 5 April 2018 9 April 2018
Please cite this article as: Fereshteh Sharifi, Seyed Mohammad Atyabi, Dariush Norouzian, Mojgan Zandi, Shiva Irani, Hadi Bakhshi , Polycaprolactone/carboxymethyl chitosan nanofibrous scaffolds for bone tissue engineering application. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), doi:10.1016/j.ijbiomac.2018.04.045
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ACCEPTED MANUSCRIPT Polycaprolactone/Carboxymethyl Chitosan Nanofibrous Scaffolds for Bone Tissue Engineering Application Fereshteh Sharifi1, Seyed Mohammad Atyabi*2, Dariush Norouzian2, Mojgan Zandi3, Shiva Irani1, Hadi Bakhshi4
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran,
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Department of Biomaterial, Iran Polymer and Petrochemical Institute, Tehran, Iran.,
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Macromolecular Chemistry II, University of Bayreuth, Universitätsstraße 30, Bayreuth,
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Germany
*Corresponding author. Seyed Mohammad Atyabi, Email: [email protected],
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[email protected], Tel: +98-21-66953311
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Department of NanoBiotechnology, Pasteur Institute of Iran, Tehran, Iran
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Iran
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ACCEPTED MANUSCRIPT Abstract: This research focused on the physical properties and cell compatibility of nanofibrous scaffolds based on polycaprolactone/chitosan (PCL/CTS) and PCL/carboxymethyl chitosan (PCL/CMC) blends for bone tissue engineering application. Scaffolds were fabricated by electrospinning technique. SEM images showed that the undesirable ultrafine and splitting
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fibers in PCL/CTS scaffolds are eliminated by replacing CTS with CMC. PCL/CMC
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scaffolds exposed significantly improved surface hydrophilicity improvement comparing to
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PCL/CTS ones. The water contact angle of PCL scaffold was reduced on the addition of 15% CMC from 123±1° to 51±3° in high concentration of CMC scaffold. The average diameter of
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fibers in PCL/CTS 15% and PCL/CMC 15% were 439 and 356 nm, respectively, which demonstrated higher concentrations of CMC resulted in decrease fibers diameter than other FTIR spectroscopy confirmed the composition of PCL/CTS and
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blended scaffolds.
PCL/CMC scaffolds. The culturing of human osteoblast cells (MG63) on the scaffolds
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showed that all scaffolds are biocompatible. The PCL/CMC nanofibers exhibited promoting
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proliferation trend, compared to the PCL and PCL/CTS ones, especially at maximum concentrations of CMC. The results demonstrate that the PCL/CMC electrospun scaffolds can
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be an excellent candidate for bone tissue engineering application.
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Keywords: carboxymethyl chitosan; nanofibers; osteoblast cells; electrospinning; bone tissue engineering.
1. Introduction The biological and physical agents within the extracellular matrix (ECM) guide the cells to organize bone tissue (1). Thus, designing a scaffold that can imitate the native ECM for cell fate determination is a major challenge in bone tissue engineering (2, 3). Manufacturing a
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ACCEPTED MANUSCRIPT scaffold for bone regeneration as a hard tissue with high toughness and tensile strength entails a biomaterial with adequate mechanical stability as well as appropriate porosity and surface hydrophilicity to support the cell attachment and growth (2). Polycaprolactone (PCL) is one of the most common synthetic polymers in bone tissue engineering with suitable properties such as biocompatibility, biodegradability, and higher toughness (4-6). However, the
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hydrophobic nature of PCL, which influences the cell attachment and consequently their
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proliferation and differentiation (7, 8), is the most important challenge in the initial step of
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cell culturing (6, 9).
Chitosan (CTS) as natural biopolymer has been extensively used in tissue engineering,
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biomedicine implants, and drug delivery devices (10, 11). It has great advantages such as biocompatibility, biodegradability as well as antifungal, antibacterial and anticancer activities
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(10, 12). However, it suffers from drawbacks such as insoluble at physiological pH and deficient mechanical stability (13, 14). Carboxymethylation of the amine and hydroxyl
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groups of chitosan can improve its water solubility and processability for tissue engineering
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application (11, 15, 16). Incorporation of CTS or CMC within other hydrophilic polymers promoted the cell attachment and proliferation (11, 17). Therefore, a mixture of PCL with
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CTS or CMC can lead to blended scaffolds with more robust properties for bone tissue engineering. Scaffolds based on PCL and CTS or CMC can be fabricated via various methods
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such as salt-leaching (18), freeze-drying (19), wet-spinning (20) and electrospinning (11, 17, 21). Among these methods, electrospinning process has many preferences including similarity to the natural ECM, large surface area to volume ratio, and highly porosity compared to other techniques (22). In this study, PCL scaffolds containing different amounts of CTS or CMC were fabricated via the electrospinning process. We hypothesized that carboxymethylation of CTS can create a structure similar to the functional factors of native polysaccharide within the natural ECM.
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ACCEPTED MANUSCRIPT The physical and morphological properties of the fabricated scaffolds were characterized and the effects of incorporated CTS or CMC on their cellular interaction were evaluated.
2. Experimental 2.1. Materials
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CTS extracted from crab shell with a degree of deacetylation of >90% was purchased
diphenyltetrazolium
bromide
(MTT),
4’,6-diamidino-2-
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dimethylthiazol-2-yl]-2,5
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from Bio Basic (Canada). PCL with an average molecular weight (Mn) of 80,000, 3-[4,5-
phenylindole (DAPI) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich
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(Germany). Acetic acid, formic acid, sodium hydroxide, isopropanol, monochloroacetic acid, and ethanol were purchased from Merck (Germany). The human osteoblast cells (MG63)
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were obtained from the Cell Bank Stem Cell Technology Institute (Iran). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum, and trypsin/EDTA solution (0.25%)
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were obtained from Gibco (Canada). Home-lab phosphate buffered saline (PBS, pH=7.4) was
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prepared based on NaCl, KCl, Na2HPO4, and KH2PO4 reagents.
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2.2. Synthesis of carboxymethyl chitosan (CMC) CTS (1 g) was first dissolved in acetic acid/deionized water mixture (1/9, v/v) at room
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temperature with continuous stirring overnight. Sodium hydroxide was then added to precipitate out the CTS. The precipitated CTS was isolated and washed with deionized water and isopropanol. The purified CTS was dissolved in isopropanol and sodium hydroxide and stirred for 5 hours. Afterward, a mixture of monochloroacetic acid and isopropanol was added dropwise and stirred at room temperature for 8 hours (Figure 1a) (23). Finally, the precipitate was filtered, washed with ethanol and dried in a vacuumed oven (Memmert, Germany).
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2.3. Electrospinning of Scaffolds Solutions of PCL (12.5 wt%) containing CTS or CMC (5%, 10% or 15 wt%) were prepared in a mixture of acetic acid/formic acid (2/3, v/v) at room temperature (Table 1). The prepared solutions were filled into a 5 mL plastic syringe and placed in the electrospinning
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apparatus (Nano-Azma, Iran). The electrospinning process was carried out at a flow rate
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range of 0.1-0.7 mL/h, a voltage of +18-30 kV and a needle-to-collector distance of 16-20
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cm. All scaffolds were collected on aluminum sheets rolled over the collector.
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2.4. Instruments
FTIR spectra were recorded on a Burker spectrometer (Equinox 55, Germany) from 400
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to 4000 cm-1 with a 4 cm-1 resolution. The viscosity of the electrospinning solutions was measured by a Brookfield viscometer (DV-11+Pro) at ambient temperature using 3 mL of
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each well-mixed solution. The morphology of fibrous scaffolds was studied by scanning
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electron spectroscopy (SEM, VEGA//TESCAN). All specimens were coated with a gold layer before imaging. Fibers diameter was determined by Image J. software using SEM
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images.
The surface hydrophilicity of scaffolds was investigated by water contact angle
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measurement. For this purpose, 5 µl of ultrapure water was placed on the surface of the scaffolds in ambient condition and the contact angle was measured. All measurements were repeated four times on different sites of scaffolds.
2.5. Biocompatibility assays MG63 were maintained in T25 tissue culture flasks at 37°C in a 5% humidified atmosphere using Dulbecco’s modified Eagle’s medium (DEME) as the culture media
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ACCEPTED MANUSCRIPT containing 20% fetal bovine serum (FBS, Gibco, Germany). The cells were passaged depending on the cell proliferation and confluence every 3-4 days. Scaffolds (0.5×0.5 cm2) were sterilized under UV lamp for 40 min (both sides) and placed in 96-well plates, where MG63 cells were seeded on them (104 cells/well). After incubating about 2 h, the culture medium was added to each well and incubated for 24, 48 and 72 h. The cell viability was
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evaluated by an inverted microscope (Bell, INV-100FL) qualitatively. The cell viability was
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also measured by MTT assay. For this purpose, the culture medium was replaced with 200
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µL of MTT solution (5 mg/mL) and incubated for 3 h. The formed formazan crystals were dissolved in DMSO and the absorption was measured at 570 nm. Plate wells without
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scaffolds were used as a control. The reported values are an average of three repetitions. The morphology of cells cultured on the scaffold was studied by SEM. The cell-seeded
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scaffolds were rinsed with PBS twice and fixed via immersing in glutaraldehyde solution (4.5%) for 3 h. Dehydration of scaffolds was done using a series of ethanol solutions (from
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60 to 100%).
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For DAPI staining, the cell-seeded scaffolds were washed with PBS, fixed with paraformaldehyde solution (4%), immersed in Triton 100 solution (0.1%) to permeabilize the
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cells, and washed with PBS twice. Finally, cells were stained with DAPI and kept in dark
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before fluorescent microscopy.
2.6. Statistical analysis Data were evaluated by a Dunnet one-way analysis of variance (ANOVA) using SPSS software (version 16.0). The a priori alpha value was set at 0.05 with the level of significance for all statistical analyses (p ≤ 0.05).
3. Results and Discussion
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peak for stretching vibration of aliphatic C–H bonds appeared at 2924 cm-1 (24, 25). The
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broad peak at 1604 cm-1 is due to the bending vibrations of N–H bonds of amine groups (24, 26). The bending vibrations of –CH2– and –CH3 groups resulted in a peak at 1411 cm-1 (24).
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The peak at 1254 cm-1 is related to the stretching vibration of C–N bonds of amine groups
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(24). The peak for asymmetric stretching vibration of C–O bonds of ether bridges appeared at 1163 cm-1 (24, 26). Carboxymethylation of CTS resulted in a new peak at 1728 cm-1
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attributing to the stretching vibrations of carbonyl (C=O) bonds of carboxylic acid groups (27). Meanwhile, the intensity of peaks for the bending vibrations of N–H bonds of amine
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groups at 1601 cm-1 was decreased due to their consumption in the course of
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carboxymethylation reaction. These results confirmed the successful synthesis of CMC.
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4000350030002500200015001000 500
1604-
1411-
29243444-
4000
3500
3000
2500
2000
1500
11201076-
29252864-
172816601601146613831286-
CMC
3444-
Absorbance (%)
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125411531070-
(b)
1000
500
Wavenumber (cm-1)
Figure 1. (a) Synthesis of CMC. (b) FTIR spectra of CTS and CMC.
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3.2. Fabrication of electrospun fibrous scaffolds Various techniques such as salt-leaching (18), freeze-drying (19), nanotopography modulating (28) and electrospinning (5, 6, 29) are being used for manufacturing substrates with drastically enlarge the surface area as scaffolds for tissue engineering. Meanwhile,
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electrospinning process can produce scaffolds with highly porosity and similarity to the
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natural ECM. Fibrous scaffolds based on PCL containing CTS or CMC (5%, 10% or 15%)
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was fabricated through electrospinning process. Results showed that the viscosity of electrospinning solutions was significantly improved by increasing the concentration of CTS
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or CMC from 5% to 15%, at a constant PCL concentration of 12.5% (Table 1), which is related to the polyelectrolyte behavior of CTS and its derivative (30). Meanwhile, the
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viscosity of PCL/CMC solutions was lower than corresponding PCL/CTS ones. Table 1. Formulation and viscosity of electrospinning solutions.
PCL (%)
CTS (%)
CMC (%)
12.5
0
0
12.5
5
0
534
12.5
10
0
599
12.5
15
0
1215
PCL/CMC 5%
12.5
0
12.5
153
PCL/CMC 10%
12.5
0
12.5
164
PCL/CMC 15%
12.5
0
12.5
590
PCL PCL/CTS 5% PCL/CTS 10%
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PCL/CTS 15%
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Scaffold
Viscosity (cp)
The morphology of fibrous scaffolds was evaluated by SEM (Figure 2). SEM image of PCL scaffold showed homogenous fibers with a smooth surface and an average diameter of 201±27 nm. Both PCL/CTS and PCL/CMC scaffolds displayed higher average fiber diameters comparing to PCL one (Figure 3a). This phenomenon is attributed to the higher viscosity of the corresponding electrospinning solutions that expose higher resistance against 8
ACCEPTED MANUSCRIPT jet and faster solidification of CTS or CMC that both lead to thicker fibers (11, 30). The homogeneity of PCL fibers was decreased via incorporating CTS or CMC, as higher relative standard deviation (RSD) values were observed for average diameter of fibers (Figure 3a). Meanwhile, PCL/CMC 10% and PCL/CMC 15% fibers showed lower RSD values (more homogeneity) than other blended ones. SEM images of PCL/CTS scaffolds displayed some
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ultrafine fibers (<20 nm) between the main fibers (Figure 2). This phenomenon was
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intensified at higher concentrations of CTS. In fact, CTS is a polycationic biopolymer and
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increased the charge intensity of the electrospinning solution. When this higher-charge solution injected, more elongation and branching forces are imposed on it leading to the
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splitting of jet and formation of ultrafine fibers (31). On the other hand, SEM images of PCL/CMC scaffolds showed homogenous fibers with a smooth surface and no undesirable
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ultrafine and splitting fibers (Figure 2). It is worth to mention that, due to lower solution viscosity, the diameter of PCL/CMC 10% and PCL/CMC 15% fibers was less than
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corresponding PCL/CTS ones (Figure 3a).
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Figure 2. SEM images of electrospun fibrous scaffolds.
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500 400 300 453
200 100
471
439
434
361
356
201
0 PCL/CTS PCL/CTS PCL/CTS PCL/CMC PCL/CMC PCL/CMC 5% 10% 15% 5% 10% 15%
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PCL
120
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100 80 60
123
112
96
40
92
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Water Contact Angle (°)
(b)140
58
20
51
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0
56
PCL
PCL/CTS PCL/CTS PCL/CTS PCL/CMC PCL/CMC PCL/CMC 5% 10% 15% 5% 10% 15%
4000350030002500200015001000 500
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136612411185104716001366124213851047-
1728-
29442865-
1366124118651047-
16041729-
29462865-
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3435-
PCL/CMC 15%
4000
1730-
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3358-
PCL/CTS 15%
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Absorbance (%)
29452866-
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(c) PCL
3500
3000
2500
2000
1500
Wavenumber (cm-1)
1000
500
Figure 3. (a) Average fiber diameter of scaffolds. (b) Water contact angle of scaffolds. (c) FTIR spectra of scaffolds.
Surface hydrophilic is one of most important property for scaffolds used in tissue engineering application (3, 5). The surface hydrophilicity of the fabricated scaffolds was studied through measuring the water contact angle (Figure 3b). PCL scaffold showed a hydrophobic surface with a contact angle of 123±1°. The incorporation of CTS and especially
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ACCEPTED MANUSCRIPT CMC improved the surface hydrophilicity of scaffolds, whereas a contact angle of 51±3° was observed for PCL/CMC 15% scaffold (Figure 3b). The difference between PCL/CTS and PCL/CMC scaffolds is related to the presence of additional hydrophilic carboxylic acid groups within the backbone of CMC (32, 33). The FTIR spectroscopy was applied to confirm the presence of CTS and CMC in the
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blended scaffolds (Figure 3c). The FTIR spectrum of PCL scaffold showed two peaks at 2945
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and 2866 cm-1 attributing to the stretching vibrations of aliphatic C–H bonds. The peak for stretching vibration of carbonyl bond of ester groups appeared at 1730 cm -1. The peaks at
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1185 and 1047 cm-1 are due to stretching vibration of ether (C–O) bonds of ester groups (11,
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34). The blended scaffolds showed similar characteristic peaks for PCL accompanied with some extra peaks (Figure 3c). The peaks at 3358 cm-1 (O–H and N–H stretching vibrations)
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and 1600 cm-1 (N–H bending vibration) demonstrated the presence of CTS in PCL/CTS 15% scaffold was by. The existence of CMC in PCL/CMC 15% scaffold was confirmed with the
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peak at 3435 cm-1 (stretching vibrations for residue O–H and N–H bonds) and 1604 cm-1
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3.3. Biocompatibility
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(bending vibration of N–H bonds) (24, 27).
The biological response of the fabricated scaffolds was evaluated through the cytotoxicity
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and cell attachment. In vitro cytotoxicity of the scaffolds was carried out through direct contact of MG63 cells with them for 72 hours. Optical microscopy images showed good biocompatibility for all scaffolds up to 72 hours, whereas MG63 cells displayed the original spindle morphology and the affinity to grow to confluence on the samples (Figure 4).
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Figure 4. Inverted microscopy images of MG63 cells on the scaffolds after 24-72 hours of incubation.
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Plate wells without scaffold were used as control. S shows the scaffolds. The magnifications of images are 100x.
The suitability of scaffolds for tissue engineering application is investigated via studying
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the viability of cells cultured on their surfaces (35). For this purpose, MG63 cells were cultured on the scaffolds for 24, 48 and 72 hours and their viability was determined using
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MTT assay (Figure 5a). Results showed that of the proliferation of seeded cells depended on the concentrations of CTS and CMC. The attachment of cells on the scaffolds was appropriate for all concentrations of CTS and CMC during the initial 24 hours. The cell viability for all scaffolds exhibited a progressive trend in the second day compared to the previous day. After 48 hours, the cell viability for blended scaffolds containing 10% of CTS or CMC was higher than other concentrations. In the third day, a decrease in the number of cells for all scaffolds and control was observed. This decrease is related to ongoing
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ACCEPTED MANUSCRIPT proliferation rates, shortage in cell culture space on the scaffolds and cell-cell interaction led to the inhabitation of the continuous cells proliferation. After 72 hours, the number of cells on the blended scaffolds was significantly higher than the PCL one. Meanwhile, PCL/CMC 10% or PCL/CTS 10% scaffolds demonstrated a moderately better cellular promotion trend compared to the others (Figure 5a). Substitution CMC instead of chitosan blended with PCL
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was developed a novel scaffold for stimulating the recruitment of human osteoblast. The 24h
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MG63 cells attachment results also declared that the CMC scaffolds were a suitable for
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osteoblast cells attachment. The suitability of the CMC scaffolds for supporting and protecting attached osteoblast cells was not surprising, due to the presence of CMC may have
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chelating calcium and enhance MG63 proliferation. Among the PCL/CMC scaffolds, the PCL/CMC10% was the best scaffold for osteoblast cell attachment and proliferation (36, 37).
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Shalumon et al. fabricated chitosan solution with a concentration of 4 wt% with PCL, while in the present study electrospun scaffolds constructed form CTS and CMC solution with
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concentration of 5- 15 wt % with PCL. Its noteworthy that, the cell viability of PCL- chitosan
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was significantly lower than the presence of hydroxyapatite and bioglass (21). While in the present study, addition CMC in the PCL electrospun solution without additive anything has
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undeniable affected on cell proliferation. One of the limitation of PCL biopolymer is absence of cells recognized site. According to this, increment contain of natural derivative
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biomaterials in fabricated scaffolds could significantly improve its limitation.
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24 h
48 h
72 h
0.6
OD
0.5 0.4 0.3
*
0.2
*
0.1 0.0 PCL
PCL/CTS PCL/CTS PCL/CTS PCL/CMC PCL/CMC PCL/CMC 5% 10% 15% 5% 10% 15%
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Control
Figure 5. (a) Viability of MG63 cells on the scaffolds after 24-72 hours of incubation determined by
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MTT assay. Plate wells without scaffold were used as control. Data are subjected to Dun-net one-way
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analysis of variance (ANOVA, n = 3, *: p ≤0.05). (b) SEM images of MG63 cells attached on the scaffolds. (c) Fluorescent microscopy images for the seeded scaffolds after staining by DAPI. The magnifications of images are 100x.
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The shape of cells, their spreading and orientation on the scaffolds were studied by SEM (Figure 5b). The seeded MG63 cells on PCL scaffold remained in a more or less rounded
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morphology and focal adhesion after 24 hours of incubation. But, the cells on PCL/CMC 10% were started to spreading with a filopodial extension morphology during the first day (Figure 5b). After 48 hours, the morphology of the cells became flat and they spread well on the PCL/CMC 10% scaffold. Suitable cell-cell and cell- environment interactions were observed for the cells on the PCL/CMC 10% scaffold. DAPI staining is used to evaluate the healthiness of cells’ nucleases. The DAPI images confirmed the viability and complete healthiness of cells cultured on the surface of all
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4. Conclusion Scaffolds based on PCL/CTS and PCL/CMC were fabricated using electrospinning
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process. Using CMC instead of CTS could adjust the viscosity and charge density of the
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electrospinning solution and led to uniform fibers without formation of any undesirable
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ultrafine and splitting fibers, especially at high concentrations. PCL/CMC scaffolds exposed more hydrophilic surfaces comparing to PCL and PCL/CTS ones. The biological
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performance of the scaffolds was studied through direct contact with MG63 cells. Although all blended scaffolds exhibited excellent initial cells attachment only PCL/CMC 10%
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displayed an acceptable trend of cell proliferation. The results showed that the incorporation of CMC to PCL fibers provided a more promising scaffold for bone tissue engineering
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applications.
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optimization of degree of folate grafted on chitosan and carboxymethyl-chitosan. Progress in Biomaterials. 2016;5:1-8.
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Shalumon KT, Anulekha KH, Girish CM, Prasanth R, Nair SV, Jayakumar R. Single step
electrospinning of chitosan/poly (caprolactone) nanofibers using formic acid/acetone
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Graphical abstract
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Fereshteh Sharifi, Seyed Mohammad Atyabi, Dariush Norouzian, Mojgan Zandi, Shiva Irani, Hadi Bakhshi PII: DOI: Reference:
S0141-8130(18)30638-X doi:10.1016/j.ijbiomac.2018.04.045 BIOMAC 9453
To appear in: Received date: Revised date: Accepted date:
9 February 2018 5 April 2018 9 April 2018
Please cite this article as: Fereshteh Sharifi, Seyed Mohammad Atyabi, Dariush Norouzian, Mojgan Zandi, Shiva Irani, Hadi Bakhshi , Polycaprolactone/carboxymethyl chitosan nanofibrous scaffolds for bone tissue engineering application. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), doi:10.1016/j.ijbiomac.2018.04.045
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ACCEPTED MANUSCRIPT Polycaprolactone/Carboxymethyl Chitosan Nanofibrous Scaffolds for Bone Tissue Engineering Application Fereshteh Sharifi1, Seyed Mohammad Atyabi*2, Dariush Norouzian2, Mojgan Zandi3, Shiva Irani1, Hadi Bakhshi4
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran,
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Department of Biomaterial, Iran Polymer and Petrochemical Institute, Tehran, Iran.,
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Macromolecular Chemistry II, University of Bayreuth, Universitätsstraße 30, Bayreuth,
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Germany
*Corresponding author. Seyed Mohammad Atyabi, Email: [email protected],
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[email protected], Tel: +98-21-66953311
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Department of NanoBiotechnology, Pasteur Institute of Iran, Tehran, Iran
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Iran
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ACCEPTED MANUSCRIPT Abstract: This research focused on the physical properties and cell compatibility of nanofibrous scaffolds based on polycaprolactone/chitosan (PCL/CTS) and PCL/carboxymethyl chitosan (PCL/CMC) blends for bone tissue engineering application. Scaffolds were fabricated by electrospinning technique. SEM images showed that the undesirable ultrafine and splitting
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fibers in PCL/CTS scaffolds are eliminated by replacing CTS with CMC. PCL/CMC
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scaffolds exposed significantly improved surface hydrophilicity improvement comparing to
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PCL/CTS ones. The water contact angle of PCL scaffold was reduced on the addition of 15% CMC from 123±1° to 51±3° in high concentration of CMC scaffold. The average diameter of
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fibers in PCL/CTS 15% and PCL/CMC 15% were 439 and 356 nm, respectively, which demonstrated higher concentrations of CMC resulted in decrease fibers diameter than other FTIR spectroscopy confirmed the composition of PCL/CTS and
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blended scaffolds.
PCL/CMC scaffolds. The culturing of human osteoblast cells (MG63) on the scaffolds
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showed that all scaffolds are biocompatible. The PCL/CMC nanofibers exhibited promoting
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proliferation trend, compared to the PCL and PCL/CTS ones, especially at maximum concentrations of CMC. The results demonstrate that the PCL/CMC electrospun scaffolds can
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be an excellent candidate for bone tissue engineering application.
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Keywords: carboxymethyl chitosan; nanofibers; osteoblast cells; electrospinning; bone tissue engineering.
1. Introduction The biological and physical agents within the extracellular matrix (ECM) guide the cells to organize bone tissue (1). Thus, designing a scaffold that can imitate the native ECM for cell fate determination is a major challenge in bone tissue engineering (2, 3). Manufacturing a
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ACCEPTED MANUSCRIPT scaffold for bone regeneration as a hard tissue with high toughness and tensile strength entails a biomaterial with adequate mechanical stability as well as appropriate porosity and surface hydrophilicity to support the cell attachment and growth (2). Polycaprolactone (PCL) is one of the most common synthetic polymers in bone tissue engineering with suitable properties such as biocompatibility, biodegradability, and higher toughness (4-6). However, the
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hydrophobic nature of PCL, which influences the cell attachment and consequently their
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proliferation and differentiation (7, 8), is the most important challenge in the initial step of
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cell culturing (6, 9).
Chitosan (CTS) as natural biopolymer has been extensively used in tissue engineering,
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biomedicine implants, and drug delivery devices (10, 11). It has great advantages such as biocompatibility, biodegradability as well as antifungal, antibacterial and anticancer activities
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(10, 12). However, it suffers from drawbacks such as insoluble at physiological pH and deficient mechanical stability (13, 14). Carboxymethylation of the amine and hydroxyl
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groups of chitosan can improve its water solubility and processability for tissue engineering
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application (11, 15, 16). Incorporation of CTS or CMC within other hydrophilic polymers promoted the cell attachment and proliferation (11, 17). Therefore, a mixture of PCL with
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CTS or CMC can lead to blended scaffolds with more robust properties for bone tissue engineering. Scaffolds based on PCL and CTS or CMC can be fabricated via various methods
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such as salt-leaching (18), freeze-drying (19), wet-spinning (20) and electrospinning (11, 17, 21). Among these methods, electrospinning process has many preferences including similarity to the natural ECM, large surface area to volume ratio, and highly porosity compared to other techniques (22). In this study, PCL scaffolds containing different amounts of CTS or CMC were fabricated via the electrospinning process. We hypothesized that carboxymethylation of CTS can create a structure similar to the functional factors of native polysaccharide within the natural ECM.
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ACCEPTED MANUSCRIPT The physical and morphological properties of the fabricated scaffolds were characterized and the effects of incorporated CTS or CMC on their cellular interaction were evaluated.
2. Experimental 2.1. Materials
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CTS extracted from crab shell with a degree of deacetylation of >90% was purchased
diphenyltetrazolium
bromide
(MTT),
4’,6-diamidino-2-
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dimethylthiazol-2-yl]-2,5
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from Bio Basic (Canada). PCL with an average molecular weight (Mn) of 80,000, 3-[4,5-
phenylindole (DAPI) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich
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(Germany). Acetic acid, formic acid, sodium hydroxide, isopropanol, monochloroacetic acid, and ethanol were purchased from Merck (Germany). The human osteoblast cells (MG63)
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were obtained from the Cell Bank Stem Cell Technology Institute (Iran). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum, and trypsin/EDTA solution (0.25%)
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were obtained from Gibco (Canada). Home-lab phosphate buffered saline (PBS, pH=7.4) was
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prepared based on NaCl, KCl, Na2HPO4, and KH2PO4 reagents.
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2.2. Synthesis of carboxymethyl chitosan (CMC) CTS (1 g) was first dissolved in acetic acid/deionized water mixture (1/9, v/v) at room
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temperature with continuous stirring overnight. Sodium hydroxide was then added to precipitate out the CTS. The precipitated CTS was isolated and washed with deionized water and isopropanol. The purified CTS was dissolved in isopropanol and sodium hydroxide and stirred for 5 hours. Afterward, a mixture of monochloroacetic acid and isopropanol was added dropwise and stirred at room temperature for 8 hours (Figure 1a) (23). Finally, the precipitate was filtered, washed with ethanol and dried in a vacuumed oven (Memmert, Germany).
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2.3. Electrospinning of Scaffolds Solutions of PCL (12.5 wt%) containing CTS or CMC (5%, 10% or 15 wt%) were prepared in a mixture of acetic acid/formic acid (2/3, v/v) at room temperature (Table 1). The prepared solutions were filled into a 5 mL plastic syringe and placed in the electrospinning
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apparatus (Nano-Azma, Iran). The electrospinning process was carried out at a flow rate
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range of 0.1-0.7 mL/h, a voltage of +18-30 kV and a needle-to-collector distance of 16-20
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cm. All scaffolds were collected on aluminum sheets rolled over the collector.
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2.4. Instruments
FTIR spectra were recorded on a Burker spectrometer (Equinox 55, Germany) from 400
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to 4000 cm-1 with a 4 cm-1 resolution. The viscosity of the electrospinning solutions was measured by a Brookfield viscometer (DV-11+Pro) at ambient temperature using 3 mL of
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each well-mixed solution. The morphology of fibrous scaffolds was studied by scanning
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electron spectroscopy (SEM, VEGA//TESCAN). All specimens were coated with a gold layer before imaging. Fibers diameter was determined by Image J. software using SEM
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images.
The surface hydrophilicity of scaffolds was investigated by water contact angle
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measurement. For this purpose, 5 µl of ultrapure water was placed on the surface of the scaffolds in ambient condition and the contact angle was measured. All measurements were repeated four times on different sites of scaffolds.
2.5. Biocompatibility assays MG63 were maintained in T25 tissue culture flasks at 37°C in a 5% humidified atmosphere using Dulbecco’s modified Eagle’s medium (DEME) as the culture media
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ACCEPTED MANUSCRIPT containing 20% fetal bovine serum (FBS, Gibco, Germany). The cells were passaged depending on the cell proliferation and confluence every 3-4 days. Scaffolds (0.5×0.5 cm2) were sterilized under UV lamp for 40 min (both sides) and placed in 96-well plates, where MG63 cells were seeded on them (104 cells/well). After incubating about 2 h, the culture medium was added to each well and incubated for 24, 48 and 72 h. The cell viability was
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evaluated by an inverted microscope (Bell, INV-100FL) qualitatively. The cell viability was
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also measured by MTT assay. For this purpose, the culture medium was replaced with 200
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µL of MTT solution (5 mg/mL) and incubated for 3 h. The formed formazan crystals were dissolved in DMSO and the absorption was measured at 570 nm. Plate wells without
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scaffolds were used as a control. The reported values are an average of three repetitions. The morphology of cells cultured on the scaffold was studied by SEM. The cell-seeded
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scaffolds were rinsed with PBS twice and fixed via immersing in glutaraldehyde solution (4.5%) for 3 h. Dehydration of scaffolds was done using a series of ethanol solutions (from
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60 to 100%).
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For DAPI staining, the cell-seeded scaffolds were washed with PBS, fixed with paraformaldehyde solution (4%), immersed in Triton 100 solution (0.1%) to permeabilize the
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cells, and washed with PBS twice. Finally, cells were stained with DAPI and kept in dark
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before fluorescent microscopy.
2.6. Statistical analysis Data were evaluated by a Dunnet one-way analysis of variance (ANOVA) using SPSS software (version 16.0). The a priori alpha value was set at 0.05 with the level of significance for all statistical analyses (p ≤ 0.05).
3. Results and Discussion
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peak for stretching vibration of aliphatic C–H bonds appeared at 2924 cm-1 (24, 25). The
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broad peak at 1604 cm-1 is due to the bending vibrations of N–H bonds of amine groups (24, 26). The bending vibrations of –CH2– and –CH3 groups resulted in a peak at 1411 cm-1 (24).
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The peak at 1254 cm-1 is related to the stretching vibration of C–N bonds of amine groups
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(24). The peak for asymmetric stretching vibration of C–O bonds of ether bridges appeared at 1163 cm-1 (24, 26). Carboxymethylation of CTS resulted in a new peak at 1728 cm-1
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attributing to the stretching vibrations of carbonyl (C=O) bonds of carboxylic acid groups (27). Meanwhile, the intensity of peaks for the bending vibrations of N–H bonds of amine
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groups at 1601 cm-1 was decreased due to their consumption in the course of
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carboxymethylation reaction. These results confirmed the successful synthesis of CMC.
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4000350030002500200015001000 500
1604-
1411-
29243444-
4000
3500
3000
2500
2000
1500
11201076-
29252864-
172816601601146613831286-
CMC
3444-
Absorbance (%)
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125411531070-
(b)
1000
500
Wavenumber (cm-1)
Figure 1. (a) Synthesis of CMC. (b) FTIR spectra of CTS and CMC.
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3.2. Fabrication of electrospun fibrous scaffolds Various techniques such as salt-leaching (18), freeze-drying (19), nanotopography modulating (28) and electrospinning (5, 6, 29) are being used for manufacturing substrates with drastically enlarge the surface area as scaffolds for tissue engineering. Meanwhile,
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electrospinning process can produce scaffolds with highly porosity and similarity to the
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natural ECM. Fibrous scaffolds based on PCL containing CTS or CMC (5%, 10% or 15%)
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was fabricated through electrospinning process. Results showed that the viscosity of electrospinning solutions was significantly improved by increasing the concentration of CTS
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or CMC from 5% to 15%, at a constant PCL concentration of 12.5% (Table 1), which is related to the polyelectrolyte behavior of CTS and its derivative (30). Meanwhile, the
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viscosity of PCL/CMC solutions was lower than corresponding PCL/CTS ones. Table 1. Formulation and viscosity of electrospinning solutions.
PCL (%)
CTS (%)
CMC (%)
12.5
0
0
12.5
5
0
534
12.5
10
0
599
12.5
15
0
1215
PCL/CMC 5%
12.5
0
12.5
153
PCL/CMC 10%
12.5
0
12.5
164
PCL/CMC 15%
12.5
0
12.5
590
PCL PCL/CTS 5% PCL/CTS 10%
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PCL/CTS 15%
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Scaffold
Viscosity (cp)
The morphology of fibrous scaffolds was evaluated by SEM (Figure 2). SEM image of PCL scaffold showed homogenous fibers with a smooth surface and an average diameter of 201±27 nm. Both PCL/CTS and PCL/CMC scaffolds displayed higher average fiber diameters comparing to PCL one (Figure 3a). This phenomenon is attributed to the higher viscosity of the corresponding electrospinning solutions that expose higher resistance against 8
ACCEPTED MANUSCRIPT jet and faster solidification of CTS or CMC that both lead to thicker fibers (11, 30). The homogeneity of PCL fibers was decreased via incorporating CTS or CMC, as higher relative standard deviation (RSD) values were observed for average diameter of fibers (Figure 3a). Meanwhile, PCL/CMC 10% and PCL/CMC 15% fibers showed lower RSD values (more homogeneity) than other blended ones. SEM images of PCL/CTS scaffolds displayed some
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ultrafine fibers (<20 nm) between the main fibers (Figure 2). This phenomenon was
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intensified at higher concentrations of CTS. In fact, CTS is a polycationic biopolymer and
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increased the charge intensity of the electrospinning solution. When this higher-charge solution injected, more elongation and branching forces are imposed on it leading to the
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splitting of jet and formation of ultrafine fibers (31). On the other hand, SEM images of PCL/CMC scaffolds showed homogenous fibers with a smooth surface and no undesirable
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ultrafine and splitting fibers (Figure 2). It is worth to mention that, due to lower solution viscosity, the diameter of PCL/CMC 10% and PCL/CMC 15% fibers was less than
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corresponding PCL/CTS ones (Figure 3a).
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Figure 2. SEM images of electrospun fibrous scaffolds.
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500 400 300 453
200 100
471
439
434
361
356
201
0 PCL/CTS PCL/CTS PCL/CTS PCL/CMC PCL/CMC PCL/CMC 5% 10% 15% 5% 10% 15%
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PCL
120
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100 80 60
123
112
96
40
92
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Water Contact Angle (°)
(b)140
58
20
51
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0
56
PCL
PCL/CTS PCL/CTS PCL/CTS PCL/CMC PCL/CMC PCL/CMC 5% 10% 15% 5% 10% 15%
4000350030002500200015001000 500
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136612411185104716001366124213851047-
1728-
29442865-
1366124118651047-
16041729-
29462865-
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3435-
PCL/CMC 15%
4000
1730-
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3358-
PCL/CTS 15%
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Absorbance (%)
29452866-
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(c) PCL
3500
3000
2500
2000
1500
Wavenumber (cm-1)
1000
500
Figure 3. (a) Average fiber diameter of scaffolds. (b) Water contact angle of scaffolds. (c) FTIR spectra of scaffolds.
Surface hydrophilic is one of most important property for scaffolds used in tissue engineering application (3, 5). The surface hydrophilicity of the fabricated scaffolds was studied through measuring the water contact angle (Figure 3b). PCL scaffold showed a hydrophobic surface with a contact angle of 123±1°. The incorporation of CTS and especially
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ACCEPTED MANUSCRIPT CMC improved the surface hydrophilicity of scaffolds, whereas a contact angle of 51±3° was observed for PCL/CMC 15% scaffold (Figure 3b). The difference between PCL/CTS and PCL/CMC scaffolds is related to the presence of additional hydrophilic carboxylic acid groups within the backbone of CMC (32, 33). The FTIR spectroscopy was applied to confirm the presence of CTS and CMC in the
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blended scaffolds (Figure 3c). The FTIR spectrum of PCL scaffold showed two peaks at 2945
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and 2866 cm-1 attributing to the stretching vibrations of aliphatic C–H bonds. The peak for stretching vibration of carbonyl bond of ester groups appeared at 1730 cm -1. The peaks at
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1185 and 1047 cm-1 are due to stretching vibration of ether (C–O) bonds of ester groups (11,
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34). The blended scaffolds showed similar characteristic peaks for PCL accompanied with some extra peaks (Figure 3c). The peaks at 3358 cm-1 (O–H and N–H stretching vibrations)
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and 1600 cm-1 (N–H bending vibration) demonstrated the presence of CTS in PCL/CTS 15% scaffold was by. The existence of CMC in PCL/CMC 15% scaffold was confirmed with the
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peak at 3435 cm-1 (stretching vibrations for residue O–H and N–H bonds) and 1604 cm-1
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3.3. Biocompatibility
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(bending vibration of N–H bonds) (24, 27).
The biological response of the fabricated scaffolds was evaluated through the cytotoxicity
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and cell attachment. In vitro cytotoxicity of the scaffolds was carried out through direct contact of MG63 cells with them for 72 hours. Optical microscopy images showed good biocompatibility for all scaffolds up to 72 hours, whereas MG63 cells displayed the original spindle morphology and the affinity to grow to confluence on the samples (Figure 4).
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Figure 4. Inverted microscopy images of MG63 cells on the scaffolds after 24-72 hours of incubation.
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Plate wells without scaffold were used as control. S shows the scaffolds. The magnifications of images are 100x.
The suitability of scaffolds for tissue engineering application is investigated via studying
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the viability of cells cultured on their surfaces (35). For this purpose, MG63 cells were cultured on the scaffolds for 24, 48 and 72 hours and their viability was determined using
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MTT assay (Figure 5a). Results showed that of the proliferation of seeded cells depended on the concentrations of CTS and CMC. The attachment of cells on the scaffolds was appropriate for all concentrations of CTS and CMC during the initial 24 hours. The cell viability for all scaffolds exhibited a progressive trend in the second day compared to the previous day. After 48 hours, the cell viability for blended scaffolds containing 10% of CTS or CMC was higher than other concentrations. In the third day, a decrease in the number of cells for all scaffolds and control was observed. This decrease is related to ongoing
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ACCEPTED MANUSCRIPT proliferation rates, shortage in cell culture space on the scaffolds and cell-cell interaction led to the inhabitation of the continuous cells proliferation. After 72 hours, the number of cells on the blended scaffolds was significantly higher than the PCL one. Meanwhile, PCL/CMC 10% or PCL/CTS 10% scaffolds demonstrated a moderately better cellular promotion trend compared to the others (Figure 5a). Substitution CMC instead of chitosan blended with PCL
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was developed a novel scaffold for stimulating the recruitment of human osteoblast. The 24h
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MG63 cells attachment results also declared that the CMC scaffolds were a suitable for
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osteoblast cells attachment. The suitability of the CMC scaffolds for supporting and protecting attached osteoblast cells was not surprising, due to the presence of CMC may have
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chelating calcium and enhance MG63 proliferation. Among the PCL/CMC scaffolds, the PCL/CMC10% was the best scaffold for osteoblast cell attachment and proliferation (36, 37).
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Shalumon et al. fabricated chitosan solution with a concentration of 4 wt% with PCL, while in the present study electrospun scaffolds constructed form CTS and CMC solution with
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concentration of 5- 15 wt % with PCL. Its noteworthy that, the cell viability of PCL- chitosan
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was significantly lower than the presence of hydroxyapatite and bioglass (21). While in the present study, addition CMC in the PCL electrospun solution without additive anything has
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undeniable affected on cell proliferation. One of the limitation of PCL biopolymer is absence of cells recognized site. According to this, increment contain of natural derivative
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biomaterials in fabricated scaffolds could significantly improve its limitation.
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24 h
48 h
72 h
0.6
OD
0.5 0.4 0.3
*
0.2
*
0.1 0.0 PCL
PCL/CTS PCL/CTS PCL/CTS PCL/CMC PCL/CMC PCL/CMC 5% 10% 15% 5% 10% 15%
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Control
Figure 5. (a) Viability of MG63 cells on the scaffolds after 24-72 hours of incubation determined by
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MTT assay. Plate wells without scaffold were used as control. Data are subjected to Dun-net one-way
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analysis of variance (ANOVA, n = 3, *: p ≤0.05). (b) SEM images of MG63 cells attached on the scaffolds. (c) Fluorescent microscopy images for the seeded scaffolds after staining by DAPI. The magnifications of images are 100x.
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The shape of cells, their spreading and orientation on the scaffolds were studied by SEM (Figure 5b). The seeded MG63 cells on PCL scaffold remained in a more or less rounded
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morphology and focal adhesion after 24 hours of incubation. But, the cells on PCL/CMC 10% were started to spreading with a filopodial extension morphology during the first day (Figure 5b). After 48 hours, the morphology of the cells became flat and they spread well on the PCL/CMC 10% scaffold. Suitable cell-cell and cell- environment interactions were observed for the cells on the PCL/CMC 10% scaffold. DAPI staining is used to evaluate the healthiness of cells’ nucleases. The DAPI images confirmed the viability and complete healthiness of cells cultured on the surface of all
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4. Conclusion Scaffolds based on PCL/CTS and PCL/CMC were fabricated using electrospinning
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process. Using CMC instead of CTS could adjust the viscosity and charge density of the
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electrospinning solution and led to uniform fibers without formation of any undesirable
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ultrafine and splitting fibers, especially at high concentrations. PCL/CMC scaffolds exposed more hydrophilic surfaces comparing to PCL and PCL/CTS ones. The biological
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performance of the scaffolds was studied through direct contact with MG63 cells. Although all blended scaffolds exhibited excellent initial cells attachment only PCL/CMC 10%
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displayed an acceptable trend of cell proliferation. The results showed that the incorporation of CMC to PCL fibers provided a more promising scaffold for bone tissue engineering
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applications.
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