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Rubbery chitosan/carrageenan hydrogels constructed through electroneutrality system and their potential application as cartilage scaffold Xichao Liang, Xiaolan Wang, Qi Xu, Yao Lu, Yu Zhang, Hong Xia, Ang Lu, and Lina Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01456 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017
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Biomacromolecules
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Rubbery chitosan/carrageenan hydrogels
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constructed through electroneutrality system and
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their potential application as cartilage scaffold
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Xichao Liang1, ‡, Xiaolan Wang2,‡, Qi Xu1, Yao Lu2, Yu Zhang2, Hong Xia2, Ang Lu1*, and
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Lina Zhang1,3*
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1
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, China
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2
Department of Orthopedics, Guangdong Key Lab of Orthopedic Technology and Implant
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Materials, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou
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510010, China
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3
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China
School of Chemistry and Chemical Engineering, Guangxi University, Nanning, 530004,
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KEYWORDS: chitosan, carrageenan, polyelectrolyte hydrogels, electroneutral system, cell
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differentiation, cartilage scaffold.
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ABSTRACT: In the present work, the bulk and homogeneous composite hydrogels were
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successfully constructed from positively charged chitosan (CS) and negatively charged
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carrageenan (CG) in alkali/urea aqueous solution via a simple one-step approach, for the first
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time. An electroneutral CS solution was achieved in alkali/urea, leading to a homogeneous
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solution blended by CS and CG, which could not be realized in acidic medium, because of the
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agglomeration caused by polycation and polyanion. Subsequently, the CS/CG composite
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hydrogels with multiple crosslinked networks were prepared from blend solution by using
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epichlorohydrin (ECH) as the crosslinking agent. The composite hydrogels exhibited
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hierarchically porous architecture, excellent mechanical properties as well as pH- and
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salt-responsiveness. Importantly, the composite hydrogels were successfully applied for
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spreading ATDC5 cells, showing high attachment and proliferation of cells. The results of
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fluorescent micrographs and scanning electronic microscope images revealed that the CS/CG
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composite hydrogels enhanced the adhesion and viability of ATDC5 cells. The alcian blue
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staining, glycosaminoglycan quantification and real-time PCR analysis proved that the
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CS/CG composite hydrogels could induce chondrogenic differentiation of ATDC5 cells in
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vitro, exhibiting great potential for the application in cartilage repair. This work opened up a
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facile and fast fabrication pathway for the construction of ampholytic hydrogel from
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polycation and polyanion in electroneutrality system.
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INTRODUCTION
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Damage of cartilage have a great impact on the quality of life, as a result of the functional
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and the metabolic properties of the original tissue will hardly ever be restored spontaneously.1,
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2
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the articular cartilage is an avascular tissue with a highly complex structure, consisting of
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relatively few cells, proteoglycans and proteins, which has limited intrinsic capacity for
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self-repair due to the lack of neural, lymphatic networks, as well as progenitor cells.3-5 Thus,
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the repair of articular cartilage injury presents great challenges in clinical and experimental
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research. Recently, significant interest has been focused on tissue engineering approaches for
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the regeneration of cartilage defects, and the strategy of repairing osteochondral defects with
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cell-seeded scaffold materials by implantation surgery has become prevalent.6-9 This approach
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credited to its cost affordable nature and high efficiency is considered quite promising in
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cartilage regeneration.10-14
Therefore, cartilage repair materials are urgently needed in clinical practice. It is noted that
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Ocean is continuously producing new forms of life,15 and the marine biomass is considered
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an excellent candidate for biomedical materials due to its inherent biocompatibility and
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activity. The polysaccharides based hydrogels have displayed soft and rubbery consistency,
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low interfacial tension and three-dimensional network structures, which mimic the natural
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extracellular matrix (ECM) in native cartilaginous tissue.16-20 Moreover, their high tissue-like
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water content not only enable efficient transportation of nutrients and wastes, but also
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provides important feature for cell functionality.21,
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abundant polysaccharides among marine biomass. Chitosan (CS), derived from the
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deacetylation of chitin, exhibits intrinsic biocompatibility, biodegradability, non-toxicity,
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Chitin and carrageenan (CG) are
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antimicrobial activity as well as low immunogenicity, thus has been widely applied in drug
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delivery, gene therapy and tissue engineering.23-29 It is composed of N-glucosamine and
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N-acetyl-glucosamine
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glycosaminoglycan (GAG) present in articular cartilage which not only maintains the
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structural integrity of the cartilage, but also plays pivotal role in cell adhesion, proliferation
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and differentiation.30-32 On the other hand, CG derived from red seaweeds is a sulfated and
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linear polysaccharide with negative charge which consists of sulfate esters of galactose and
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3,6-anhydrogalactose copolymers, linked by alternating α-1,3 and β-1,4 glycosidic linkages.
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Recently, it has attracted much attentions in biomedical applications due to the roles of
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sulphate groups in metabolism and biodistribution, and its resemblance to natural GAGs
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owing to their backbone composition of sulphated disaccharides.33-35 Moreover, its
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advantageous properties for cartilage tissue repair and improving cell attachment and viability
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have been demonstrated.36-38 Furthermore, articular cartilage defects and degeneration is
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associated with loss of GAGs.39 Thus, a worthwhile endeavor would be to construct CS/CG
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composite hydrogels to achieve better effectiveness in articular cartilage regeneration and
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repairing.
units,
and
possesses
structural
characteristics
similar
to
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CS is usually dissolved in acidic medium, and possesses positive charges, whereas CG has
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negative charge. Due to the strong electrostatic interaction, polycation and polyanion will
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rapidly aggregate in aqueous system to form agglomeration, precipitation or polyion
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complexes, rather than homogeneous solution. Our strategy is that CS can be dissolved in
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alkaline/urea system by destroying the intermolecular hydrogen bonding rather than
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protonation,40 to gain electroneutrality, on which basis a homogeneous solution can be formed.
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Thus, composite hydrogel with multiple crosslinked networks can be prepared by crosslinking.
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In the present work, CS and CG were dissolved separately in alkali/urea followed by blending,
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resulting in a transparent and homogeneous CS/CG blend solution. Subsequently, bulk and 4
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homogeneous CS/CG composite hydrogels were prepared by using the epichlorohydrin (ECH)
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as crosslinking agent. Their structure and properties were studied, and the adhesion and
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proliferation of chondrogenic ATDC5 cells on CS/CG composite hydrogels were evaluated in
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vitro. Furthermore, the differentiation behavior of ATDC5 cells cultured on composite
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hydrogels were also examined. This work points out that the CS/CG composite hydrogels
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exhibited uniform multiple crosslinked network structure, excellent mechanical properties,
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pH- and salt-responsive behavior, and excellent biocompatibilities. This is important in the
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biological applications in terms of cartilage repair.
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EXPERIMENTAL SECTION
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Materials. Commercial grade chitosan powder (with a deacetylation degree of 89%) was
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purchased from Ruji Biotechnology Co., Ltd (Shanghai, China). Carrageenan powder was
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purchased from Aladdin (Shanghai, China). Other chemical reagents were of analytical grade
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supplied by Sinopharm Chemical Reagent Co., Ltd (Beijing, China) and used without further
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purification. Ultrapure water (15MΩ cm) used in all experiments was obtained with a Milli-Q
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apparatus (Millipore, Bedford, MA, USA).
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Preparation of CS/CG Composite Hydrogels. The CS/CG composite hydrogel was
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constructed via an environment-friendly method by a sol-gel transition method as follows. 8 g
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of CS powder was dispersed and stirred in 192 g of mixture of LiOH, KOH, urea, and
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ultrapure water (4.5:7:8:80.5, by weight) to obtain a suspension. Subsequently, the suspension
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was frozen at -30 oC for 4 h, followed by thawing and stirring at room temperature. The
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freezing-thawing cycle was repeated twice to obtain a transparent chitosan solution, with a
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chitosan concentration of 4 wt %. CG powder was dissolved in the same solvent through the
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same way to obtain a transparent solution with the concentration of 4 wt %. Then the resultant
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CS and CG solutions were blended by changing the weight ratio of CS to CG by w/w% of 9:1,
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8:2, 7:3, 6:4 and 5:5, which was coded as C-CGEL1 to C-CGEL5 respectively. ECH (2 mL) 5
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as a cross-linker was added to the CS/CG mixture (100 g) and was stirred at -25 oC for 2 h to
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obtain a homogeneous pre-gel solution. After removing air bubbles by centrifugation at 8000
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rpm for 10 min at 0 oC, the transparent CS/CG pre-gel solution was poured into mold and kept
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at room temperature for 5 h. Thus, the CS/CG composite hydrogels were fabricated, and
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immersed in distilled water to remove the residual reagent for 3 days. During this period,
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distilled water was replaced five times a day. The physical composite hydrogels prepared by
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heating at 60 oC without cross-linker were also fabricated, and the composite hydrogels
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prepared by heating with the weight ratio of CS to CG by w/w% of 9:1, 8:2, 7:3, 6:4; 5:5 were
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coded as P-CGEL1 to P-CGEL5, respectively. Moreover, the pure CS hydrogel without CG as
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a contrast in experiment was also prepared in the same way, and coded as CSGEL.
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Characterization. Scanning electron micrograph (SEM) measurements were carried out on
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a field emission scanning electron microscopy (FESEM, Zeiss, SIGMA, Germany) at an
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accelerating voltage of 5 kV. The wet hydrogels were frozen in liquid nitrogen and snapped
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immediately, and then freeze-dried. The surface and fracture sections were sputtered with gold
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for SEM observation. The structures of the hydrogels were characterized by Fourier transform
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infrared spectroscopy (FT-IR, NICOLET 5700, Thermo Nicolet, U.S.A), X-ray photoelectron
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spectrometer (XPS, Kratos XSAM800, England), and thermogravimetric Analysis (TG, STA
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449C, NETZSCH, German). The samples were cut into particle-like size after being
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freeze-dried and then vacuum-dried for 24 h at 60 oC before measurements. Solid-state
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NMR spectra with cross polarization/ magic angle spinning (CP/MAS) of the samples were
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carried out on a 300 MHz NMR spectrometer (Bruker Advance III, German) at ambient
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temperature. The light transmittance of the pristine CS hydrogel and CS/CG composite
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hydrogels were determined by ultraviolet-visible (UV-vis) spectroscopy (UV-6, Mapada,
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China) at wavelengths ranging from 200 to 800 nm. The freeze-dried hydrogel samples were
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cut into powders and dispersed in ultrapure water, and their zeta potential were measured by a 6
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C
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Zetasizer (Nano ZS, Malvern Instruments). The concentration of all the dispersions were 5 mg
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mL-1.
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Mechanical Performance and Swelling Studies. The mechanical properties of the
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hydrogels were characterized by compression and tension tests, which were measured on a
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universal tensile-compressive tester (CMT 6503, Shenzhen MTS/SANS, China). A cylindrical
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hydrogel with a diameter of 10 mm and a height of 10 mm was placed on the lower plate and
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compressed by the upper plate at a strain rate of 1 mm min–1. For the stretching measurements,
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a hydrogel sheet 50 mm long and 5 mm wide was stretched at a tension speed of 2 mm min–1.
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For each group, the data were obtained under the same surroundings. For each sample, at least
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five parallel experiments were conducted.
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The gravimetric method was employed to measure the swelling ratios of the hydrogels with
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different feed ratios in ultrapure water, NaCl solutions with different concentrations, and
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different pH solutions. The swelling temperature was set as 37 oC to simulate body
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temperature for exploring potential biomedical application. The pH values were adjusted by
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HCl and NaOH solution, ranging from 1 to 13, which were determined by using a FE-20K pH
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meter (Mettler-Toledo, Ohio, U.S.A). The ionic strength of the pH solution was controlled to
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be 0.1 M by adding an appropriate amount of NaCl solutions. The swelling ratio (SR) was
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calculated as
SR=
Ws -Wd ×100% Wd
(1)
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where Ws and Wd are the weight of the swollen and dried hydrogel at 37 oC respectively. For
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each test, at least three parallel samples were measured and an average result was reported.
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The deswelling kinetics of the swollen hydrogels in phosphate buffered saline (PBS) (pH =
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7.2) was measured gravimetrically at 37 oC. At selected time intervals, the hydrogels samples
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were taken out from PBS and weight after removing the excess on the surfaces with wet filter
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paper. Water retention (WR) in the hydrogel was defined as 7
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Wt -Wd ×100% Ws
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(2)
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where Wt is the weight of wet hydrogel at time t and Ws is the weight of swollen hydrogel in
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ultrapure water while Wd is the weight of dried hydrogel. For each test, at least three parallel
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samples were measured and an average result was reported.
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The hydrophilicity of the CS/CG composite hydrogels was characterized by the water
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contact angle measurement to investigate the effect of addition of CG on the hydrophilicity of
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the composite hydrogels. The water contact angle, which is an indicator of the hydrophilicity
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of surfaces, were measured at room temperature by DSA100 (Krüss, Germany). Water was
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used as the liquid phase. The contact angle was measured 5 times on each hydrogel and an
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average value was calculated by statistical method.
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Hemolysis Tests. The hemolytic activity of the CS/CG composite hydrogels was
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investigated with healthy New Zealand white rabbit blood in heparinized-tubes. The blood
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was diluted with physiological saline in a ratio of 4:5 by volume. Then, CS/CG composite
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hydrogels were dipped in 1 mL of physiological saline and incubated at 37 oC for 30 min. 1
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mL of deionized water was used as a positive control and 1 mL of physiological saline as a
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negative control. Then 0.2 mL of diluted blood was added into each sample and the mixtures
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were incubated at 37 oC for 60 min. After that, all samples were centrifuged for 5 min at 3000
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rpm and the supernatant was collected. ODs of the supernatants were determined at 545 nm
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using a microplate reader (Thermo, Multiskn Go, U.S.A). The hemolysis rate was calculated
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by using the following equation41: Hemolysis rate %=
Asample -Acontrol(-) ×100% Acontrol(+) -Acontrol(-)
(3)
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where Asample, Acontrol(-) and Acontrol(+) represent the absorbance of the samples, negative and
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positive controls, respectively.
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In Vitro Cell Culture. Pre-chondrogenic ATDC5 cells obtained from RIKEN cell bank 8
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(Japan) were seeded on the surface of the hydrogels to evaluate the cytocompatibility. The
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cells were cultured at 37 oC in a 5% CO2 incubator in 25 cm3 flasks (beaver, cyagen, China)
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containing DMEM/F12 medium (Gibco, life technology, U.S.A) supplement with 5% fetal
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bovine serum (FBS, Gibco, life technology, U.S.A), 1% antimicrobial of penicillin and
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streptomycin (Hyclone, U.S.A). The culture medium was changed every 2 days.
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Cell Viability and Cell Proliferation. The pure CS and CS/CG composite hydrogels with
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10 mm diameter and a thickness of about 0.1 cm were sterilized by 70% alcohol for 30 min,
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and then were exposed to ultraviolet light for 30 min. The samples were then transferred to
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the bottom of 24-well plastic culture plates and washed with sterilized physiological saline for
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30 min. Cell viability was determined by the live/dead staining. ATDC5 cells suspension at a
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density of 5×104 cells/well were seeded on each sample for 24 h. Then, the samples on the 24
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wells were washed by physiological saline and were stained with 1 mM calcein AM and 2
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mM ethidium homodimer-1 solutions (Sigma, U.S.A) at 37 oC for 30 min, which were
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visualized with a fluorescence microscope (Olympus BX51, Olympus Corporation, Japan).
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3-(4, 5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT, sigma, U.S.A) assay was
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used to evaluate the proliferation rate of cells growing on the surface of the samples.42 ATDC5
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cells suspension at a density of 2×104 cells/well were seeded on each sample for 1, 4 and 7
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days. After culturing for 1, 4 and 7 days, the medium was aspirated, and an aliquot of 50 µL
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MTT dissolved in 450 µL DMEM/F12 medium was added to each sample. The plates were
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then incubated at 37 oC for 4 h. After the culture medium was removed, the formazan reaction
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products were dissolved in 500 µL of dimethyl sulfoxide (DMSO, Aladdin, China) for 20 min.
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The formazan solution was transferred to a 96-well plate, and the absorbance at 490 nm was
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measured using a microplate reader (Thermo, Multiskan Go, U.S.A).
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Cell Morphology. The cell morphology was analyzed by SEM (FESEM, Zeiss, Germany).
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For morphological analysis of the ATDC5 cells at a density of 5×104 cells/well after being 9
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cultured on different samples for 48 h, the samples were washed with PBS thrice and fixed
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with 3% glutaraldehyde for 4 h at 4 oC. The cells were dehydrated through graded
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concentrations of ethanol (20%, 40%, 60%, 80%, and 100%) and then tertiary butanol, twice
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for 30 min each. The resultant hydrogels were lyophilized and sputtered with gold for SEM
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observation.
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In Vitro Degradation Study. The in vitro degradation behavior of the hydrogels was
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studied by incubating certain weights of the pristine CS hydrogel and CS/CG composite
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hydrogels (10 mm diameter) in 4 mg/mL three-times-recrystallized egg white lysozyme in 0.1
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M PBS at pH 7.4 and 37 oC.43, 44 After pre-defined time intervals, hydrogel samples were
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taken out from the lysozyme solution and washed with ultrapure water three times. The
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hydrogel samples were frozen at - 20 oC overnight and freeze-dried for 2 days. The extent of
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the in vitro degradation was characterized by the percentage of the weight of the dried
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hydrogels before and after the lysozyme treatment by the following equation: Weight Remaining %=
WT ×100% W0
(4)
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where WT and W0were the dry weight of the hydrogel sample at certain degradation time and
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initial dry weight of the hydrogel respectively.
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Alcian Blue Staining. ATDC5 cells on the samples at 7 and 14 days were rinsed twice with
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phosphate-buffered saline and fixed in 4% paraformaldehyde solution for 30 min, after which
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cells were stained with 0.1% alcian blue 8GX (sigma-Aldrich, U. S. A) in 0.1 M HCl
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overnight. After overnight staining, the plates were washed twice with 0.1 M HCl for 5 min to
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remove any nonspecific staining of the plates, followed by a 30 s rinse with 3% acetic acid
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three times, and then visualized with an inverted microscope (Leica, Germany).
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Glycosaminoglycan (GAG) Quantification. GAG content of the ATDC5 cells seeded
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samples was evaluated by GAG assay using 1, 9-Dimethyl-Methylene Blue (DMMB, sigma,
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U.S.A.). DMMB dye solution (0.016 mg/mL of DMMB, 0.01 M HCl, 2.37 mg/mL of NaCl, 10
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3.4 mg/mL of Glycine) was prepared and stored at room temperature shielded from light.
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After 7 and 14 days, GAG was harvested from the cells by digestion with papain solution
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containing (125 µg/mL of papain, 5 mM L-cystein,100 mM Na2HPO4, 5 mM EDTA)
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maintaining a pH of 6.8 at 60°C for 16 h. The dye solution was transferred to a 96-well plate,
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and the absorbance at 525 nm was measured using a microplate reader (Thermo, Multiskan
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Go, U.S.A.). A standard curve was established using chondroitin-4-sulfate (sigma, U.S.A.),
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and the total GAG amounts were determined from the OD value which correlated to the
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corresponding GAG amount in the standard curve.45 The total protein was determined using
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BCA Protein Quantitation Kit (Thermo, U.S.A.), and the absorbance at 562 nm was measured
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using a microplate reader. GAG content was normalized according to the protein contents (µg
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GAG/mg protein).
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Quantitative Real Time PCR (RT-PCR) Analysis. The expressions of cartilage-related
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genes, namely aggrecan, SOX9, collagen type II (COL2) and collagen type X (COL10) were
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investigated to evaluate the ability of ATDC5 cells on samples using RT-PCR analysis. The
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ATDC5 cells at a density of 2×104 cell/well were cultured with DMEM/F12 medium
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supplement with 5 % fetal bovine serum and 1 % antimicrobial of penicillin and streptomycin.
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For chondrogennic induction, the medium was replaced with medium containing 10 µg/mL
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bovine insulin, 5.5 µg/mL human transferrin and 0.005 µg/mL sodium selenite (ITS Liquid
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Media Supplement, Sigma, USA). After 7 and 14 days, total RNA was extracted from
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cell-hydrogel constructs by 750 µL/well trizol reagent (Life technology, USA). 750 µL/well of
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chloroform was added followed by vigorous shaking for 20 s, and centrifuging at 12,000 rpm
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for 20 min at 4 oC. The aqueous phase was collected and put in a fresh 1.5 mL tube, and 0.5
255
mL of isopropanol (100%) was added to the collected aqueous phase and centrifuged at
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12,000 rpm for 10 min at 4 oC. The supernatant was removed and the RNA pellet was washed
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with 1 mL of 75% ethanol for 5 min at 4 oC. The concentration and purity of the RNA was 11
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assessed using a Nanodrop 2000 (Thermo, U.S.A). Afterwards, the RNA was converted to
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cDNA using a Prime Script RT Master Mix reagent kit (Takara, Japan). Then the analysis was
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performed with the Rotor-Gene 6000 (QIAGEN, German) with SYBR Green Real time PCR
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Master Mix (DBI Bioscience, China). The qRT-PCR results were analyzed using the
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Rotor-Gene Real-Time analysis software 6.0. And the relative gene expression was calculated
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using the 2-∆∆Ct method. The relative expression levels of the genes evaluated were
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normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
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The synthesized primers used in this study were listed in Table 1.
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Real-time RT-PCR thermal cycles correspond to the following periods: 2 min at 95 oC,
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followed by 40 cycles of 95 oC for 10 s corresponding annealing temperature of 55 oC for 30 s
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and an extension step at 72 oC for 30 s.
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Statistical Analysis. All of the data were expressed as means ± standard deviations (SD;
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n = 3). The statistical analysis was performed with OriginPro 9.0. The statistical significance
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of the difference was measured using one-way analysis of variance, and values of p < 0.05
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were considered statistically significant. Values of p < 0.01 and < 0.001 are marked as **
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and *** respectively.
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RESULTS AND DISCUSSION
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Construction and Structure of CS/CG Composite Hydrogels. In order to avoid the
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strong electrostatic interaction, which leads to the heterogeneous flocculation, the alkali/urea
277
aqueous solution was utilized as solvent to dissolve CS and CG. CS and CG were dissolved in
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alkali/urea with a concentration of 4 wt %, and then were directly blended with the weight
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ratio of 1:1. For comparison, CS and CG were also dissolved in 2 wt % acetic acid with the
280
same concentration and blended with the same weight ratio, as control. As shown in Figure 1a
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and 1b, the CS/CG blend solution in alkali/urea was transparent and homogeneous, and no
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obvious aggregates were observed. It was attributed to the fact that CS was dissolved in this 12
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solvent, as a result of the destruction of the intermolecular hydrogel bonds of CS in alkali/urea
284
which led to the neutral feature of CS. However, small heterogeneous lamellar membrane
285
appeared in the acidic system (Figure 1c and 1d), due to the strong electrostatic adsorption
286
interaction between cationic CS and anionic CG. This could be explained that CS was
287
dissolved in acidic system due to the protonation of the amino groups, thus CS is positive
288
charged and easily formed heterogeneous flocculation with negatively charged CG via
289
electrostatic interaction, which led to hindrance to fabricate bulk materials.
290
To investigate the effect of the ratio of two components on the structure and properties of
291
the composite hydrogels, CS/CG blend solutions in alkali/urea with different weight ratio
292
from 9:1 to 5:5 were prepared here. To evaluate the stability, the rheological properties and
293
gelation behavior of the blend solution were investigated with dynamic viscoelastic
294
measurements. Figure 1e shows the effects of the weight ratio on storage modulus G′ and loss
295
modulus G″. The CS/CG blend solutions displayed higher gelation temperature than the
296
pristine CS solution, and the gelation temperature increased with an increase of the CG
297
content, suggesting that the stability of the blend solution in alkali/urea was improved by the
298
addition of CG, favoring the construction of bulk CS/CG composite materials.
299
CS/CG composite hydrogels were then successfully fabricated with epichlorhydrine (ECH)
300
as a cross-linking agent (Figure S1a). The photographs of pristine CS hydrogel and CS/CG
301
composite hydrogels are shown in Figure S1b. Compared with the pristine CS hydrogel, the
302
transparency of the composite hydrogels was improved, with an increase of CG content
303
(Figure S1c). The CS/CG composite hydrogels exhibited the optical transmittance, indicating
304
good miscibility between chitosan and carrageenan. The FT-IR spectra and solid
305
spectra were applied to investigate their structure and composition. As shown in Figure S2,
306
the FT-IR spectra of CS displayed two amide peaks at 1655 and 1598 cm-1, ascribed to
307
vibrations of amide I and amide II band, respectively. The peak at 1081 cm-1 was ascribed to 13
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the vibration of glycosidic bonds. The FT-IR spectra of CG exhibited characteristic peaks at
309
1263, 1069, 928 and 846 cm-1, corresponding to the sulfate groups, the glycosidic linkage, the
310
3, 6-anhydrogalactose and galactose-4-sulfate, respectively. The amide peaks of CS in
311
C-CGEL2 and C-CGEL4 were identified and nearly converted to a single band, indicating the
312
interaction between the amino groups of CS with CG. On the other hand, the typical bands of
313
CG in C-CGEL2 and C-CGEL4 were also identified, revealing that the CS/CG composite
314
hydrogels were successfully constructed from CS and CG. Figure 2 displays the solid-state
315
13
316
were presented in C-CGEL4, indicating the coexistence of CS and CG in the CS/CG
317
composite hydrogels. It is noted that the peaks at 61.2 ppm for C6 of CS (Figure 2a) and 63.1
318
ppm for G6 of CG (Figure 2c) exhibited a large loss resolution in C-CGEL4 (Figure 2b), and
319
the two peaks appeared at 60.9 and 62.7 ppm, respectively. The results indicated that
320
cross-linking reaction occurred in the alkaline condition, and both CS and CG were involved
321
in the etherification reaction, forming hydrogel networks. Moreover, in the TGA curves of the
322
composite hydrogels (Figure S3), decomposition was observed between 240 and 300 oC,
323
demonstrating sufficient thermal stability for application as biomaterials.
C NMR spectra of CS, C-CGEL4 and CG. Apparently, all the typical signals of CS and CG
324
To ascertain the chemical bonding in the CS/CG composite hydrogels, XPS was applied.
325
The results (Figure S4a) indicated that C, N, O and S existed in C-CGEL2, whereas only C, O
326
and N was presented in CS, and C, O and S appeared in CG, suggesting the co-existence of
327
CS and CG in the composite hydrogels. The typical N 1s XPS spectra of CS and C-CGEL2
328
and XPS spectra in the S 2p region of CG and C-CGEL2 (Figure S4b and S4c) indicated that
329
both the N 1s and S 2p spectra of C-CGEL2 shifted to higher binding energies as compared to
330
CS and CG, confirming the existence of strong electrostatic interaction between the amino
331
groups of CS and sulfate groups of CG. Therefore, the CS/CG composite hydrogels were
332
successfully constructed from CS and CG through hydrogen bonding, chemical crosslinking 14
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and electrostatic interactions.
334
Figure 4 shows the SEM images of the inner part of the pristine CS hydrogel (Figure 4a1)
335
and CS/CG composite hydrogels (Figure 4b1-f1). The composite hydrogels all displayed
336
well-defined, interconnected, three-dimensional porous network architecture and evenly pore
337
distribution, suggesting good compatibility between CS and CG. The average pore size and its
338
distribution showed that the pore diameter of the composite hydrogels increased with an
339
increase of CG content (Figure 4a2-f2), due to the high water absorption of sulfate ester
340
groups on CG. Compared with the pristine CS hydrogel, the structure of the composite
341
hydrogels became loose with an increase of the CG content, consistent with changes of pore
342
size. Furthermore, the pore size distribution of the composite hydrogels became wide,
343
indicating the multiple porous structure, supported by the SEM results. The porous structure
344
of the hydrogels was conductive to the diffusion of oxygen, nutrients and metabolic products
345
to and from encapsulated cells, endowing the CS/CG composite hydrogels potentials as
346
scaffolds for articular cartilage repair.
347
In view of these results, a schematic diagram to describe the formation and architecture of
348
CS/CG composite hydrogels is proposed in Figure 3. The CS contributed to support the
349
hydrogel matrix, and the CG was bonded with chitosan through hydrogen bonding and ionic
350
bonding. The CS/CG composite hydrogels were formed by chemical cross-linking and
351
physical cross-linking to form multiple network structures, which was supported by the
352
results in Figure 1, 2, 4, S1, S2 and S4. In our findings, CG acted as an expanding to enhance
353
the pore size, whereas CS as a backbone reinforced the hydrogel.
354
Mechanical Properties and Swelling Behavior of CS/CG Composite Hydrogels.
355
Mechanical properties are one of the most important factors of the scaffolds for cartilage
356
tissue engineering, which allow the scaffolds to support the articular cartilage regeneration at
357
the site of implantation and maintain sufficient integrity during both in vitro and in vivo cell 15
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growth. As shown in Figure 5, the CS/CG hydrogels exhibited good compressive and tensile
359
strength, and the C-CGEL2 could be severely knotted, twisted and pressed without
360
deformation, suggesting good mechanical properties. In particular, as shown in Figure 5c, the
361
composite hydrogels could be recovered after compression, exhibiting excellent elasticity.
362
Figure 5d and 5e shows the typical compressive and tensile stress-strain curves of CS/CG
363
composite hydrogels at room temperature, and the detailed mechanical parameters are
364
summarized in Table 2. These results strongly demonstrate that CS/CG composite hydrogels
365
have excellent mechanical properties. With an increase of the CG content, the compressive
366
and tensile fracture stress and strain of the composite hydrogels decreased, whereas the water
367
content increased, further validating that CS contributed to enhance the mechanical properties,
368
and CG contributed to increase the water content. Therefore, the mechanical properties of
369
CS/CG hydrogels can be controlled by adjusting the content of CG to meet the different
370
requirements. Moreover, mechanical stability is another important performance of the
371
scaffolds for cartilage scaffold materials. The representative loading-unloading curves
372
including cyclic compression and tension of C-CGEL3 (Figure 5f and 5g) exhibited efficient
373
recovery of CS/CG composite hydrogels after loading. The results of the loading-unloading
374
cycles for 50 times demonstrated that the composite hydrogels had good elasticity and
375
mechanical stability, as a result of the multiple network structure. To highlight the important
376
role of chemical cross-linking on the composite hydrogels, the physical composite hydrogels
377
without ECH were prepared by heating the blend solution. Clearly, the physical hydrogels had
378
poor mechanical properties (Figure S5a) and was unstable in acids (Figure S5b). Therefore,
379
the CS/CG composite hydrogels with high strength and elasticity due to multiple network
380
structure were obtained by chemical cross-linking in the present work.
381
The swelling behavior of hydrogels is also an essential feature. The swelling ratio of the
382
CS/CG hydrogels was investigated at 37 oC, as shown in Figure 6a. As the feed ratio of CS to 16
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CG in the hydrogels decreased from 9:1 to 5:5, swelling ratio of the hydrogels increased from
384
13.81 to 128.87 g/g, owing to high water absorbability of CG. To clarify the effect of CG on
385
the hydrophilicity of the composite hydrogels, the contact angle measurement was carried out
386
and shown in Figure S6. The hydrophilicity of the CS/CG composite hydrogels was
387
significantly improved by adding CG, compared with pristine CS hydrogel. Moreover, the
388
higher the CG content, the better the hydrophilicity of the CS/CG composite hydrogels,
389
indicating the strong water absorbability and hydrophilicity of sulfate ester groups. The
390
hydrophilicity of composite hydrogels is of great importance to biological application.
391
As is well known, the swelling behavior of hydrogels is usually sensitive to pH condition.
392
Figure 6b shows the effects of pH on the swelling behaviors of the composite hydrogels. All
393
composite hydrogels exhibited lower swelling ratio in buffers with 0.1 M ionic strength at
394
pH=7 in comparison with that in ultrapure water, as a result of the high ionic strength. The
395
CS/CG composite hydrogels with different feed ratio exhibited different pH sensitive behavior.
396
C-CGEL1 displayed the maximum swelling ratio at pH=1, due to the protonation of the
397
amino groups on CS, leading to the strong electrostatic repulsion in acidic medium. However,
398
the composite hydrogels from C-CGEL2 to C-CGEL5 obtained relatively low swelling ratio
399
at the same pH condition (pH=1), owing to the positive charges of CS being neutralized by
400
sulfate ester groups on CG. On the contrary, as the feed ratio of CS to CG in the hydrogels
401
decreased from 9:1 to 5:5, the swelling ratio of the hydrogels at pH=13 increased as
402
increasing the CG content, due to the CS was electroneutral and the electrostatic repulsion
403
between the sulfate ester groups on CG increased with an increase of CG content. The results
404
indicated that the feed ratio of CS to CG played an important role in controlling the pH
405
responsiveness through regulating the electrostatic interaction between CS and CG at different
406
pH values.
407
The swelling behavior of the composite hydrogels in salt was also evaluated. Figure 6c 17
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shows the swelling ratio of the composite hydrogels in NaCl solutions with different
409
concentration. The swelling ratio of all the hydrogel samples decreased with the increase of
410
the salt concentration, and the shrinking behaviors of hydrogels in salt solution could be
411
controlled by the ionic interactions between mobile ions and the fixed charges, which made
412
great contributions to the osmotic pressure between the interior hydrogel and the external
413
solution. Moreover, the deswelling kinetic of CS/CG hydrogels in PBS was shown in Figure
414
6d. All the swollen hydrogels displayed fast responsive properties and revealed shrinking
415
behavior with different water retention. The water retention values of the hydrogels were 70%,
416
60%, 31%, 27% and 23% for C-CGEL1, C-CGEL2, C-CGEL3, C-CGEL4 and C-CGEL5,
417
respectively. From the above results, the CS/CG hydrogels exhibited pH- and salt-sensitive
418
properties. This stimulus response behavior is important in biomedical materials.
419
Acute Hemolysis Test of CS/CG Composite Hydrogels. The blood compatibilities of the
420
CS/CG composite hydrogels and pristine CS hydrogel were studied by acute hemolysis
421
experiments. The release of hemoglobin was used to quantify the damaging properties of the
422
hydrogels. The hemolysis index is regarded as safe when it is less than 5%, according to ISO
423
document 10 993-5 1992.46 The degree of hemolysis of CS/CG composite hydrogels and
424
pristine CS hydrogel (Figure S7a) indicated that C-CGEL1-4 and CSGEL displayed a low
425
degree of hemolysis (< 2%), whereas that of C-CGEL5 was higher than 5%. The results
426
confirmed good blood compatibility of the CS/CG composite hydrogels on the whole. The
427
visual observation of the hemolytic phenomenon (Figure S7b) showed that positive control
428
displayed a red solution in the tube due to the presence of released hemoglobin. Negative
429
control showed no red color in the tube, indicating no hemolysis occurred. There was no
430
indication of the hemolysis phenomenon after treatment with C-CGEL1 to C-CGEL4 and
431
CSGEL, suggesting that the CS/CG composite hydrogels except C-CGEL5 were not
432
hemolytic to rabbit red blood cells. Moreover, C-CGEL5 had poor mechanical properties and 18
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high swelling ratio, which was not suitable as implant material, thus C-CGEL5 was excluded
434
from the following cell culture experiments.
435
Chondrocyte Adhesion, Proliferation and Viability on CS/CG Composite Hydrogels.
436
To investigate whether the CS/CG composite hydrogels meet the fundamental requirement in
437
articular cartilage repair, the ATDC5 cells were cultured on the hydrogels, and cell attachment,
438
viability and proliferation were evaluated in vitro. Figure 7f shows the results of MTT assay
439
for CS/CG composite hydrogels (C-CGEL1 to C-CGEL4) and pristine CS hydrogel (CSGEL)
440
as a contrast. For the culture time of 1 d, the optical density (OD) values of C-CGEL1 to
441
C-CGEL 4 were similar to that of CSGEL, and there was no obvious difference. When the
442
culture time reached 4 d, the OD values of C-CGEL2 and C-CGEL4 increased obviously.
443
From the MTT results for 7 d, the cells viability of C-CGEL1 to C-CGEL4 was all higher
444
than that of CSGEL, suggesting that the CS/CG composite hydrogels were favorable for the
445
culture of ATDC5 cells. This might probably because that the composite hydrogels provided a
446
weakly charged surface (Table S1) which is suitable for attached cell growth.30, 47 And the
447
cells viability of the different hydrogels could be also related to the mechanical properties and
448
structural morphology of the hydrogels. Moreover, the MTT results also indicated that the
449
addition of CG in composite hydrogel could enhance the ability of proliferation of the ATDC5
450
cells. The enhanced viability of chondrocyte was further confirmed by the live/dead staining
451
florescent photographs of ATDC5 cells cultured on the CS/CG composite hydrogels and
452
pristine CS hydrogel, as shown in Figure 7a-e. Green and red staining indicated viable and
453
dead cells, respectively. The ATDC5 cells cultured on the CS/CG composite hydrogels were
454
dense and connected with each other to form an entangled network, indicating that the
455
composite hydrogels were beneficial to the adhesion and growth of chondrocytes. Moreover,
456
after 24 h culturing, the cell viability values on CSGEL and CS/CG composite hydrogels
457
(Figure S8) all displayed a high degree (> 90%), confirming good biocompatibility of both CS 19
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and CG. And the cells viabilities of C-CGEL3 and C-CGEL4 were significantly higher than
459
CSGEL, suggesting that the composite hydrogels could improve the cell viability. This can be
460
attributed to the fact that the sulfated group in CG is an important group of chondroitin sulfate
461
in cartilage, which has an intrinsic biological activity on cartilage growth.48 This is important
462
for the application of articular cartilage repairing and regeneration.
463
The morphology of the ATDC5 cells on the CS/CG hydrogels and pristine CS hydrogel
464
were observed by SEM. Figure 8 displays the SEM micrographs of chondrocytes spreading
465
on the CSGEL, C-CGEL1, C-CGEL2, C-CGEL3 and C-CGEL4 after 48 h of culture.
466
C-CGEL1 to C-CGEL4 all exhibited high attachment efficiency of cells (Figure 8b1-e1),
467
compared with CSGEL (Figure 8a1). And the ATDC5 cells spread well on the surface of the
468
composite hydrogels, whereas there were only scattered chondrocytes on the CSGEL,
469
indicating the composite hydrogels were favorable for the culture of ATDC5 cells. The
470
chondrocyte was anchored onto the surface of composite hydrogels by filopodia (Figure
471
8b2-e2), and weaved together to form an entangled network, indicating the excellent adherent
472
performance on the CS/CG composite hydrogels, compared with CSGEL (Figure 8a2). The
473
SEM results further confirmed that the CS/CG composite hydrogels improved the
474
chondrocyte cell affinity and the cytocompatibility, owing to the inherent biocompatibility of
475
CS and CG. Moreover, to mimic the in vivo degradation performance, the in vitro degradation
476
behavior of hydrogels has been investigated by using egg white lysozyme. The CS/CG
477
composite hydrogels displayed a lower degradation rate than the pristine CS hydrogel (Figure
478
S9) due to the existence of the interaction between CS and CG, indicating the composite
479
hydrogels had feasible biodegradation rate in the presence of lysozyme in vivo. This is
480
beneficial to the retention of the morphological and mechanical performance of hydrogels
481
during the earlier period of the cartilage regeneration. Therefore, the CS/CG composite
482
hydrogels exhibited great potentials for articular cartilage repair. 20
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Evaluation of Cell Differentiation by Alcian Blue Staining and GAG Quantification.
484
GAGs are critical components of cartilaginous ECM, and the presence of GAG secretion is an
485
important index to chondrogenic differentiation. To investigate the deposition of GAG during
486
chondrogenic differentiation, the GAG deposition in the pure CS hydrogel (CSGEL) as well
487
as CS/CG composite hydrogels with low CG content (C-CGEL1) and high CG content
488
(C-CGEL4) laden with ATDC5 cells was evaluated by alcian blue staining. Figure 9 depicts
489
the positive staining of ATDC5 cells encapsulated in CSGEL, C-CGEL1 and C-CGEL4 for
490
alcian blue staining after 7 and 14 days of culture, indicating starting deposition of GAGs,
491
which is commonly found in native articular cartilage ECM. The intensity of alcian blue
492
staining increased with an increase of the CG content, suggesting that the addition of CG in
493
the composite hydrogels could promote the deposition of GAG during chondrogenic
494
differentiation. The ATDC5 cells cultured on C-CGEL1 and C-CGEL4 both promoted the
495
GAG deposition as indicated by alcian blue staining after 7 and 14 days of culture, especially
496
the C-CGEL4 had better stimulating effects (Figure 9c1 and 9c2). This could be explained
497
that the composite hydrogels with high CG content was more beneficial to cartilage repair,
498
indicating that CG played an important role in chondrogenic differentiation.
499
Cell differentiation is very important in biomedicine. To further prove the important role of
500
CG in chondrogenic differentiation, GAG content/total protein in the CSGEL, C-CGEL1 and
501
C-CGEL4 was evaluated, which was used to indicate ECM deposition and synthetic activity,
502
as shown in Figure 9d. The amount of GAG/total protein secreted by ATDC5 cells seeded on
503
the C-CGEL1 and C-CGEL4 was higher than CSGEL at 7 days, and both of them had
504
statistical difference (***p < 0.001), indicating that the addition of CG in the composite
505
hydrogels was beneficial to chondrogenic differentiation. The amount of GAG/total protein
506
secreted by ATDC5 cells seeded on the CSGEL, C-CGEL1 and C-CGEL4 were all improved
507
at 14 days, and the GAG/total protein production secreted by ATDC5 cells seeded on the 21
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508
C-CGEL1 and C-CGEL4 hydrogels were both significantly higher compared with
509
CSGEL(***p < 0.001). Importantly, the amount of GAG/total protein in C-CGEL4 was much
510
higher than that of in C-CGEL1 at 7 and 14 days, suggesting that the composite hydrogel with
511
high CG content could be a potential cartilage repair material.
512
Gene Expression Analyzed with Quantitative Real-Time PCR (qRT-PCR). To verify
513
whether the CS/CG composite hydrogels could promote the chondrogenic differentiation of
514
ATDC5 cells, the expressions of cartilage-related genes including aggrecan, SOX9, COL2 and
515
COL10 on the CSGEL, C-CGEL1 and C-CGEL4 after 7 and 14 days of culture were
516
quantified by real-time PCR. As shown in Figure 10a-d, after 7 days of culture, the gene
517
expression of aggrecan, SOX9, COL2 and COL10 in cells grown on the C-CGEL1 and
518
C-CGEL4 were all greater than that seen on the CSGEL (Figure10a-d). Obviously, CS/CG
519
composite hydrogels had a promoting effect on cartilage differentiation compared with
520
pristine CS hydrogel. The gene expression of aggrecan, SOX9, COL2 and COL10 in cells
521
grown on C-CGEL4 were significantly greater than that on CSGEL, whereas only COL10 (*P
522
< 0.05) gene expression in cells grown on the C-CGEL1 were significantly higher than
523
CSGEL at 7 days (Figure10d), suggesting that the composite hydrogels with high CG content
524
resulted in greater chondrogenic gene expression. Additionally, SOX9 is known to be
525
essential for cartilage formation and is often considered an early chondrogenic marker.49, 50
526
The results (Figure 10b) indicated that the addition of CG in the composite hydrogels
527
primarily stimulated the expression of chondrogenic gene markers at early time. After 14 days
528
of culture, the chondrocytes in the composite hydrogels exhibited an increase in the gene
529
expression of cartilage-related genes, and the gene expression level of aggrecan and COL2
530
increased markedly during the incubation period compared to that at 7 days (Figure 10a and
531
10c). Aggrecan and COL2 are marker genes for normal chondrocytic differentiation, and are
532
considered to be the two major and most important constituents of hyaline cartilage ECM.51, 52 22
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Biomacromolecules
533
These results suggested that the composite hydrogels facilitated the cartilaginous specific
534
matrix production and differentiation of chondrocytes in vitro. Moreover, the gene expression
535
of aggrecan, SOX9, COL2 and COL10 in cells grown on C-CGEL4 at 14 days were
536
significantly greater than CSGEL, whereas only aggrecan (*P < 0.05), COL2 (*P < 0.05) and
537
COL10 (**P < 0.01) gene expression in cells grown on the C-CGEL1 had statistically
538
significant difference compared with CSGEL (Figure 10a, 10c and 10d). These results further
539
demonstrated that the introduction of CG in the composite hydrogels promoted the
540
chondrogenic differentiation of ATDC5 cells, and particularly the composite hydrogels with a
541
relatively high content of CG significantly enhanced the chondrogenic differentiation
542
phenomenon. The gene expression of aggrecan in cells grown on C-CGEL4 was obviously up
543
regulated both in 7 and 14 days (Figure 10b), in agreement with the GAG quantification
544
(Figure 9c1 and 9c2), indicating the CS/CG composite hydrogels indeed promoted the
545
formation of cartilaginous nodules. In summary, the results from RT-PCR analysis
546
demonstrated that the CS/CG composite hydrogels could improve the chondrocyte
547
differentiation of pre-chondrogenic ATDC5 cells compared with pristine CS hydrogel, and the
548
composite hydrogels with high CG content significantly enhanced the chondrogenic
549
differentiation of ATDC5 cells. Clearly, the introduction of CG in the composite hydrogels
550
was able to stimulate specific cellular responses at the molecular level, leading to the
551
increased expression of cartilage genes. Therefore, the CS/CG composite hydrogels enabled
552
the adhesion, proliferation and viability of ATDC5 cells and showed their efficiency to
553
support the production of GAG and other chondrogenic features, as well as their ability to
554
enhance the chondrogenic differentiation of ATDC5 cells. It further demonstrated a great
555
potential for articular cartilage repair.
556
CONCLUSION
557
Novel polyelectrolyte composite hydrogels were successfully constructed from CS and CG 23
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in alkali/urea aqueous systems via a simple one-step approach, based on the electroneutrality
559
of the CS in blend solutions. The composite hydrogels exhibited homogeneous multi-scale
560
porous architecture, excellent mechanical properties as well as pH- and salt-sensitive
561
behaviors. The CS/CG composite hydrogels with multiple networks were constructed by
562
chemical cross-linked CS acts as polycation and CG acts as polyanion, in which physical
563
cross-linking formed through hydrogen bonds and ionic bonds between the two
564
polyelectrolytes also existed. The hydrophilicity and transparency of the composite hydrogels
565
increased with an increase of the CG content. The in vitro studies indicated that the CS/CG
566
composite hydrogels promoted the adhesion, viability and proliferation of ATDC5 cells.
567
Moreover, compared with pristine CS hydrogel, the CS/CG composite hydrogels could
568
promote the chondrogenic differentiation of ATDC5 cells in vitro, and with an increase of CG
569
content, the expression of cartilage markers significantly enhanced, indicating that CG played
570
an important role in the chondrogenic differentiation of ATDC5 cells. Therefore, the CS/CG
571
composite hydrogels are expected to be promising cell-carrier materials for cell delivery, with
572
application in the articular cartilage scaffold.
573
ASSOCIATED CONTENT
574
Supporting Information
575
Preparation process of CS/CG composite hydrogel; FT-IR spectra of CS, C-CGEL2,
576
C-CGEL4 and CG; TG and DTG curves of CS/CG composite hydrogels; XPS spectra of CS,
577
CG and C-CGEL2; compressive stress-strain curves of composite hydrogels prepared by
578
heating; water contact angle measure and hemolytic activity of the different hydrogels; cell
579
viability of ATDC5 cells on the CS hydrogel and CS/CG composite hydrogels after 24 h
580
culturing; in vitro degradation of the CS hydrogel and CS/CG composite hydrogels; zeta
581
potential values of the dispersions of different hydrogel powders (PDF).
24
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AUTHOR INFORMATION
583
Corresponding Authors
584
* E-mail:
[email protected].
585
* E-mail:
[email protected].
586
ORCID
587
Lina Zhang: 0000-0003-3890-8690
588
Author Contributions
589
‡Xichao Liang and Xiaolan Wang contributed equally to this work.
590
Notes
591
The authors declare no competing financial interest.
592
ACKNOWLEDGMENTS
593
This work was supported by the National Key Research and Development Program of
594
China (2016YFB0700803), the Major Program of National Natural Science Foundation of
595
China (21334005), the Major International (Regional) Joint Research Project (21620102004)
596
and the National Natural Science Foundation of China (51573143, 31700880 and 31771038).
597
REFERENCES
598 599 600 601 602 603 604 605 606 607 608 609 610
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691 692 693 694 695 696 697 698 699 700 701
Figure Caption
702
Figure 1. Photographs of the CS/CG blend in alkali/urea aqueous solvent (a) and in acid (c);
703
optical photomicrograph of the CS/CG blend in alkali/urea aqueous solvent (b) and SEM
704
image of the small lamellar membrane appeared in the CS/CG blend in acid (d); temperature
705
dependence of the storage modulus (G′) and loss modulus (G″) for CS/CG blend solution in
706
alkali/urea aqueous solvent with different weight ratio of CS to CG by w/w% of 9:1, 8:2, 7:3,
707
6:4 and 5:5 (e).
708
Figure 2. Solid state 13C NMR spectra of chitosan (a), C-CGEL4 (b), and κ-carrageenan (c).
709
Figure 3. Schema to describe the formation and structure of CS/CG composite hydrogels.
710
Figure 4. SEM images of cross-sectional structures of CSGEL (a1), C-CGEL1 (b1),
711
C-CGEL2 (c1), C-CGEL 3 (d1), C-CGEL4 (e1) and C-CGEL5 (f1); pore size distribution of
712
CSGEL (a2), C-CGEL1 (b2), C-CGEL2 (c2), C-CGEL 3 (d2), C-CGEL4 (e2) and C-CGEL5 28
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713
(f2).
714
Figure 5. The photographs of C-CGEL3 under knotting (a), twisting (b) and compression (c).
715
Typical compressive (d) and tensile (e) stress-strain curves of CS/CG amphiprotic composite
716
hydrogels. Representative loading-unloading compression (f) and tension (g) curves (50 runs)
717
of C-CGEL3.
718
Figure 6. The swelling ratio of the CS/CG composite hydrogels in ultrapure water as a
719
function of the composition of CS and CG at 37 oC (a). Effects of pH on the swelling
720
behaviors of CS/CG composite hydrogels (b). Swelling ratio of CS/CG composite hydrogels
721
in NaCl solution (c) and water retention of CS/CG composite hydrogels in PBS at 37 oC (d).
722
Figure 7. Live/dead staining florescent photographs of ATDC-5 cells after 24 h post seeding
723
onto CSGEL (a), C-CGEL1 (b), C-CGEL2 (c), C-CGEL3 (d) and C-CGEL4 (e), the scale bar
724
is 200 µm. MTT results of ATDC-5 cells seeded on CSGEL and CS/CG composite hydrogels
725
with culture time of 1, 4 and 7 days. Stars indicate statistically significant differences
726
regarding control of CSGEL (p < 0.05 = *; p < 0.01 = **; p < 0.001 = ***).
727
Figure 8. (Top) SEM images of the chondrocytes spreading on the CSGEL (a1), C-CGEL1
728
(b1), C-CGEL2 (c1), C-CGEL3 (d1) and C-CGEL4 (e1) after 48 h of culture; scale bar = 100
729
µm. (Bottom) Enlarged view of single cell on CSGEL (a2), C-CGEL1 (b2), C-CGEL2 (c2),
730
C-CGEL3 (d2) and C-CGEL4 (e2); scale bar = 5 µm.
731
Figure 9. Optical microscopy images of histological section obtained from CSGEL (a1, a2),
732
C-CGEL1 (b1, b2) and C-CGEL4 (c1, c2), collected after 7 and 14 days of culture and stained
733
with alcian blue. GAG/protein content of CSGEL, C-CGEL1 and C-CGEL4 seeded with
734
ATDC5 cells at 7 and 14 days of culture (d). Stars indicate statistically significant differences 29
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735
regarding control of CSGEL (p < 0.001 = ***).
736
Figure 10. Gene expression of aggrecan, SOX9, type-2 collagen (COL2) and type-10
737
collagen (COL10) in ATDC5 cells cultured on the CSGEL, C-CGEL1 and C-CGEL4 at 7 and
738
14 days of culture (a-d). The expression of these genes was normalized against the
739
housekeeping gene GAPDH and calculated by the ∆CT method. Stars indicate statistically
740
significant differences regarding control of CSGEL (p < 0.05 = *; p < 0.01 = **; p < 0.001 =
741
***).
742
Table 1. Primers sequences used for quantitative RT-PCR.
743
Table 2. Physical and mechanical properties of CS/CG amphiprotic hydrogels.
744 745 746 747
748 749
Figure 1. Photographs of the CS/CG blend in alkali/urea aqueous solvent (a) and in acid (c);
750
optical photomicrograph of the CS/CG blend in alkali/urea aqueous solvent (b) and SEM 30
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751
image of the small lamellar membrane appeared in the CS/CG blend in acid (d); temperature
752
dependence of the storage modulus (G′) and loss modulus (G″) for CS/CG blend solution in
753
alkali/urea aqueous solvent with different weight ratio of CS to CG by w/w% of 9:1, 8:2, 7:3,
754
6:4 and 5:5 (e).
755
756 757
Figure 2. Solid state 13C NMR spectra of chitosan (a), C-CGEL4 (b), and κ-carrageenan (c).
758 759 760 761 762 763 31
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764 765
766 767
Figure 3. Schema to describe the formation and structure of CS/CG composite hydrogels.
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769 770
Figure 4. SEM images of cross-sectional structures of CSGEL (a1), C-CGEL1 (b1),
771
C-CGEL2 (c1), C-CGEL 3 (d1), C-CGEL4 (e1) and C-CGEL5 (f1); pore size distribution of
772
CSGEL (a2), C-CGEL1 (b2), C-CGEL2 (c2), C-CGEL 3 (d2), C-CGEL4 (e2) and C-CGEL5
773
(f2).
33
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774 775
Figure 5. The photographs of C-CGEL3 under knotting (a), twisting (b) and compression (c).
776
Typical compressive (d) and tensile (e) stress-strain curves of CS/CG composite hydrogels.
777
Representative loading-unloading compression (f) and tension (g) curves (50 runs) of
778
C-CGEL3.
779 780
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Figure 6. The swelling ratio of the CS/CG composite hydrogels in ultrapure water as a
783
function of the composition of CS and CG at 37 oC (a). Effects of pH on the swelling
784
behaviors of CS/CG composite hydrogels (b). Swelling ratio of CS/CG composite hydrogels
785
in NaCl solution (c) and water retention of CS/CG composite hydrogels in PBS at 37 oC (d).
786 787 788 789 790 791
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792 793
Figure 7. Live/dead staining florescent photographs of ATDC-5 cells after 24 h post seeding
794
onto CSGEL (a), C-CGEL1 (b), C-CGEL2 (c), C-CGEL3 (d) and C-CGEL4 (e), the scale bar
795
is 200 µm. MTT results of ATDC-5 cells seeded on CSGEL and CS/CG composite hydrogels
796
with culture time of 1, 4 and 7 day. Stars indicate statistically significant differences regarding
797
control of CSGEL (p < 0.05 = *; p < 0.01 = **; p < 0.001 = ***).
798
799 800
Figure 8. (Top) SEM images of the chondrocytes spreading on the CSGEL (a1), C-CGEL1
801
(b1), C-CGEL2 (c1), C-CGEL3 (d1) and C-CGEL4 (e1) after 48 h of culture; scale bar = 100
802
µm. (Bottom) Enlarged view of single cell on CSGEL (a2), C-CGEL1 (b2), C-CGEL2 (c2),
803
C-CGEL3 (d2) and C-CGEL4 (e2); scale bar = 5 µm.
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804 805
Figure 9. Optical microscopy images of histological section obtained from CSGEL (a1, a2),
806
C-CGEL1 (b1, b2) and C-CGEL4 (c1, c2), collected after 7 and 14 days of culture and stained
807
with alcian blue. GAG/protein content of CSGEL, C-CGEL1 and C-CGEL4 seeded with
808
ATDC5 cells at 7 and 14 days of culture (d). Stars indicate statistically significant differences
809
regarding control of CSGEL (p < 0.001 = ***).
810 811 812 813 814 815
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816 817
Figure 10. Gene expression of aggrecan, SOX9, type-2 collagen (COL2) and type-10
818
collagen (COL10) in ATDC5 cells cultured on the CSGEL, C-CGEL1 and C-CGEL4 at 7 and
819
14 days of culture (a-d). The expression of these genes was normalized against the
820
housekeeping gene GAPDH and calculated by the ∆CT method. Stars indicate statistically
821
significant differences regarding control of CSGEL (p < 0.05 = *; p < 0.01 = **; p < 0.001 =
822
***).
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828 829
Table 1. Primers sequences used for quantitative RT-PCR. Gene
Forward primer sequence(5′-3′)
Reverse primer sequence(5′-3′)
GAPDH
CATGGCCTTCCGTGTTCCTA
CCTGCTTCACCACCTTCTTGAT
Aggrecan
TCGAATCCCCAAATCCCTCAT
ACATTGCTCCTGGTCTGCAA
SOX9
GCCACGGAACAGACTCACAT
GGACCCTGAGATTGCCCAGA
COL2
GCCAGGATGCCCGAAAA
TTGTCACCACGATCACCTCTG
COL10
CATCTCCCAGCACCAGAATC
GCTAGCAAGTGGGCCCTTTA
830 831
Table 2. Physical and mechanical properties of CS/CG composite hydrogels. Water Sample
content
Compressive properties
Tensile properties
σb (MPa)
εb (%)
E (MPa)
σb (MPa)
εb (%)
E (MPa)
(%) C-CGEL1
93.25
3.94
88.28
0.38
0.53
109.74
0.73
C-CGEL2
95.54
2.99
86.79
0.37
0.44
101.77
0.52
C-CGEL3
96.74
1.16
81.37
0.18
0.38
98.71
0.45
C-CGEL4
98.07
0.25
66.26
0.12
0.09
61.53
0.16
C-CGEL5
99.23
0.16
63.54
0.09
0.03
44.52
0.07
832 833 834 39
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Table of Contents Graphic (TOC)
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