Carrageenan Hydrogels Constructed through an

Subscriber access provided by UNIV OF DURHAM

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

Rubbery chitosan/carrageenan hydrogels

2

constructed through electroneutrality system and

3

their potential application as cartilage scaffold

4

Xichao Liang1, ‡, Xiaolan Wang2,‡, Qi Xu1, Yao Lu2, Yu Zhang2, Hong Xia2, Ang Lu1*, and

5

Lina Zhang1,3*

6

1

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, China

7

2

Department of Orthopedics, Guangdong Key Lab of Orthopedic Technology and Implant

8

Materials, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou

9

510010, China

10

3

11

China

School of Chemistry and Chemical Engineering, Guangxi University, Nanning, 530004,

12 13 14 15 16

1

ACS Paragon Plus Environment

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

17

KEYWORDS: chitosan, carrageenan, polyelectrolyte hydrogels, electroneutral system, cell

18

differentiation, cartilage scaffold.

19

ABSTRACT: In the present work, the bulk and homogeneous composite hydrogels were

20

successfully constructed from positively charged chitosan (CS) and negatively charged

21

carrageenan (CG) in alkali/urea aqueous solution via a simple one-step approach, for the first

22

time. An electroneutral CS solution was achieved in alkali/urea, leading to a homogeneous

23

solution blended by CS and CG, which could not be realized in acidic medium, because of the

24

agglomeration caused by polycation and polyanion. Subsequently, the CS/CG composite

25

hydrogels with multiple crosslinked networks were prepared from blend solution by using

26

epichlorohydrin (ECH) as the crosslinking agent. The composite hydrogels exhibited

27

hierarchically porous architecture, excellent mechanical properties as well as pH- and

28

salt-responsiveness. Importantly, the composite hydrogels were successfully applied for

29

spreading ATDC5 cells, showing high attachment and proliferation of cells. The results of

30

fluorescent micrographs and scanning electronic microscope images revealed that the CS/CG

31

composite hydrogels enhanced the adhesion and viability of ATDC5 cells. The alcian blue

32

staining, glycosaminoglycan quantification and real-time PCR analysis proved that the

33

CS/CG composite hydrogels could induce chondrogenic differentiation of ATDC5 cells in

34

vitro, exhibiting great potential for the application in cartilage repair. This work opened up a

35

facile and fast fabrication pathway for the construction of ampholytic hydrogel from

36

polycation and polyanion in electroneutrality system.

37 2


Page 2 of 40

Page 3 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

38 39 40 41

INTRODUCTION

42

Damage of cartilage have a great impact on the quality of life, as a result of the functional

43

and the metabolic properties of the original tissue will hardly ever be restored spontaneously.1,

44

2

45

the articular cartilage is an avascular tissue with a highly complex structure, consisting of

46

relatively few cells, proteoglycans and proteins, which has limited intrinsic capacity for

47

self-repair due to the lack of neural, lymphatic networks, as well as progenitor cells.3-5 Thus,

48

the repair of articular cartilage injury presents great challenges in clinical and experimental

49

research. Recently, significant interest has been focused on tissue engineering approaches for

50

the regeneration of cartilage defects, and the strategy of repairing osteochondral defects with

51

cell-seeded scaffold materials by implantation surgery has become prevalent.6-9 This approach

52

credited to its cost affordable nature and high efficiency is considered quite promising in

53

cartilage regeneration.10-14

Therefore, cartilage repair materials are urgently needed in clinical practice. It is noted that

54

Ocean is continuously producing new forms of life,15 and the marine biomass is considered

55

an excellent candidate for biomedical materials due to its inherent biocompatibility and

56

activity. The polysaccharides based hydrogels have displayed soft and rubbery consistency,

57

low interfacial tension and three-dimensional network structures, which mimic the natural

58

extracellular matrix (ECM) in native cartilaginous tissue.16-20 Moreover, their high tissue-like

59

water content not only enable efficient transportation of nutrients and wastes, but also

60

provides important feature for cell functionality.21,

61

abundant polysaccharides among marine biomass. Chitosan (CS), derived from the

62

deacetylation of chitin, exhibits intrinsic biocompatibility, biodegradability, non-toxicity,

22

Chitin and carrageenan (CG) are

3



Page 4 of 40

63

antimicrobial activity as well as low immunogenicity, thus has been widely applied in drug

64

delivery, gene therapy and tissue engineering.23-29 It is composed of N-glucosamine and

65

N-acetyl-glucosamine

66

glycosaminoglycan (GAG) present in articular cartilage which not only maintains the

67

structural integrity of the cartilage, but also plays pivotal role in cell adhesion, proliferation

68

and differentiation.30-32 On the other hand, CG derived from red seaweeds is a sulfated and

69

linear polysaccharide with negative charge which consists of sulfate esters of galactose and

70

3,6-anhydrogalactose copolymers, linked by alternating α-1,3 and β-1,4 glycosidic linkages.

71

Recently, it has attracted much attentions in biomedical applications due to the roles of

72

sulphate groups in metabolism and biodistribution, and its resemblance to natural GAGs

73

owing to their backbone composition of sulphated disaccharides.33-35 Moreover, its

74

advantageous properties for cartilage tissue repair and improving cell attachment and viability

75

have been demonstrated.36-38 Furthermore, articular cartilage defects and degeneration is

76

associated with loss of GAGs.39 Thus, a worthwhile endeavor would be to construct CS/CG

77

composite hydrogels to achieve better effectiveness in articular cartilage regeneration and

78

repairing.

units,

and

possesses

structural

characteristics

similar

to

79

CS is usually dissolved in acidic medium, and possesses positive charges, whereas CG has

80

negative charge. Due to the strong electrostatic interaction, polycation and polyanion will

81

rapidly aggregate in aqueous system to form agglomeration, precipitation or polyion

82

complexes, rather than homogeneous solution. Our strategy is that CS can be dissolved in

83

alkaline/urea system by destroying the intermolecular hydrogen bonding rather than

84

protonation,40 to gain electroneutrality, on which basis a homogeneous solution can be formed.

85

Thus, composite hydrogel with multiple crosslinked networks can be prepared by crosslinking.

86

In the present work, CS and CG were dissolved separately in alkali/urea followed by blending,

87

resulting in a transparent and homogeneous CS/CG blend solution. Subsequently, bulk and 4


Page 5 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

88

homogeneous CS/CG composite hydrogels were prepared by using the epichlorohydrin (ECH)

89

as crosslinking agent. Their structure and properties were studied, and the adhesion and

90

proliferation of chondrogenic ATDC5 cells on CS/CG composite hydrogels were evaluated in

91

vitro. Furthermore, the differentiation behavior of ATDC5 cells cultured on composite

92

hydrogels were also examined. This work points out that the CS/CG composite hydrogels

93

exhibited uniform multiple crosslinked network structure, excellent mechanical properties,

94

pH- and salt-responsive behavior, and excellent biocompatibilities. This is important in the

95

biological applications in terms of cartilage repair.

96

EXPERIMENTAL SECTION

97

Materials. Commercial grade chitosan powder (with a deacetylation degree of 89%) was

98

purchased from Ruji Biotechnology Co., Ltd (Shanghai, China). Carrageenan powder was

99

purchased from Aladdin (Shanghai, China). Other chemical reagents were of analytical grade

100

supplied by Sinopharm Chemical Reagent Co., Ltd (Beijing, China) and used without further

101

purification. Ultrapure water (15MΩ cm) used in all experiments was obtained with a Milli-Q

102

apparatus (Millipore, Bedford, MA, USA).

103

Preparation of CS/CG Composite Hydrogels. The CS/CG composite hydrogel was

104

constructed via an environment-friendly method by a sol-gel transition method as follows. 8 g

105

of CS powder was dispersed and stirred in 192 g of mixture of LiOH, KOH, urea, and

106

ultrapure water (4.5:7:8:80.5, by weight) to obtain a suspension. Subsequently, the suspension

107

was frozen at -30 oC for 4 h, followed by thawing and stirring at room temperature. The

108

freezing-thawing cycle was repeated twice to obtain a transparent chitosan solution, with a

109

chitosan concentration of 4 wt %. CG powder was dissolved in the same solvent through the

110

same way to obtain a transparent solution with the concentration of 4 wt %. Then the resultant

111

CS and CG solutions were blended by changing the weight ratio of CS to CG by w/w% of 9:1,

112

8:2, 7:3, 6:4 and 5:5, which was coded as C-CGEL1 to C-CGEL5 respectively. ECH (2 mL) 5



Page 6 of 40

113

as a cross-linker was added to the CS/CG mixture (100 g) and was stirred at -25 oC for 2 h to

114

obtain a homogeneous pre-gel solution. After removing air bubbles by centrifugation at 8000

115

rpm for 10 min at 0 oC, the transparent CS/CG pre-gel solution was poured into mold and kept

116

at room temperature for 5 h. Thus, the CS/CG composite hydrogels were fabricated, and

117

immersed in distilled water to remove the residual reagent for 3 days. During this period,

118

distilled water was replaced five times a day. The physical composite hydrogels prepared by

119

heating at 60 oC without cross-linker were also fabricated, and the composite hydrogels

120

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

121

coded as P-CGEL1 to P-CGEL5, respectively. Moreover, the pure CS hydrogel without CG as

122

a contrast in experiment was also prepared in the same way, and coded as CSGEL.

123

Characterization. Scanning electron micrograph (SEM) measurements were carried out on

124

a field emission scanning electron microscopy (FESEM, Zeiss, SIGMA, Germany) at an

125

accelerating voltage of 5 kV. The wet hydrogels were frozen in liquid nitrogen and snapped

126

immediately, and then freeze-dried. The surface and fracture sections were sputtered with gold

127

for SEM observation. The structures of the hydrogels were characterized by Fourier transform

128

infrared spectroscopy (FT-IR, NICOLET 5700, Thermo Nicolet, U.S.A), X-ray photoelectron

129

spectrometer (XPS, Kratos XSAM800, England), and thermogravimetric Analysis (TG, STA

130

449C, NETZSCH, German). The samples were cut into particle-like size after being

131

freeze-dried and then vacuum-dried for 24 h at 60 oC before measurements. Solid-state

132

NMR spectra with cross polarization/ magic angle spinning (CP/MAS) of the samples were

133

carried out on a 300 MHz NMR spectrometer (Bruker Advance III, German) at ambient

134

temperature. The light transmittance of the pristine CS hydrogel and CS/CG composite

135

hydrogels were determined by ultraviolet-visible (UV-vis) spectroscopy (UV-6, Mapada,

136

China) at wavelengths ranging from 200 to 800 nm. The freeze-dried hydrogel samples were

137

cut into powders and dispersed in ultrapure water, and their zeta potential were measured by a 6


13

C

Page 7 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

138

Zetasizer (Nano ZS, Malvern Instruments). The concentration of all the dispersions were 5 mg

139

mL-1.

140

Mechanical Performance and Swelling Studies. The mechanical properties of the

141

hydrogels were characterized by compression and tension tests, which were measured on a

142

universal tensile-compressive tester (CMT 6503, Shenzhen MTS/SANS, China). A cylindrical

143

hydrogel with a diameter of 10 mm and a height of 10 mm was placed on the lower plate and

144

compressed by the upper plate at a strain rate of 1 mm min–1. For the stretching measurements,

145

a hydrogel sheet 50 mm long and 5 mm wide was stretched at a tension speed of 2 mm min–1.

146

For each group, the data were obtained under the same surroundings. For each sample, at least

147

five parallel experiments were conducted.

148

The gravimetric method was employed to measure the swelling ratios of the hydrogels with

149

different feed ratios in ultrapure water, NaCl solutions with different concentrations, and

150

different pH solutions. The swelling temperature was set as 37 oC to simulate body

151

temperature for exploring potential biomedical application. The pH values were adjusted by

152

HCl and NaOH solution, ranging from 1 to 13, which were determined by using a FE-20K pH

153

meter (Mettler-Toledo, Ohio, U.S.A). The ionic strength of the pH solution was controlled to

154

be 0.1 M by adding an appropriate amount of NaCl solutions. The swelling ratio (SR) was

155

calculated as

SR=

Ws -Wd ×100% Wd

(1)

156

where Ws and Wd are the weight of the swollen and dried hydrogel at 37 oC respectively. For

157

each test, at least three parallel samples were measured and an average result was reported.

158

The deswelling kinetics of the swollen hydrogels in phosphate buffered saline (PBS) (pH =

159

7.2) was measured gravimetrically at 37 oC. At selected time intervals, the hydrogels samples

160

were taken out from PBS and weight after removing the excess on the surfaces with wet filter

161

paper. Water retention (WR) in the hydrogel was defined as 7



WR=

Wt -Wd ×100% Ws

Page 8 of 40

(2)

162

where Wt is the weight of wet hydrogel at time t and Ws is the weight of swollen hydrogel in

163

ultrapure water while Wd is the weight of dried hydrogel. For each test, at least three parallel

164

samples were measured and an average result was reported.

165

The hydrophilicity of the CS/CG composite hydrogels was characterized by the water

166

contact angle measurement to investigate the effect of addition of CG on the hydrophilicity of

167

the composite hydrogels. The water contact angle, which is an indicator of the hydrophilicity

168

of surfaces, were measured at room temperature by DSA100 (Krüss, Germany). Water was

169

used as the liquid phase. The contact angle was measured 5 times on each hydrogel and an

170

average value was calculated by statistical method.

171

Hemolysis Tests. The hemolytic activity of the CS/CG composite hydrogels was

172

investigated with healthy New Zealand white rabbit blood in heparinized-tubes. The blood

173

was diluted with physiological saline in a ratio of 4:5 by volume. Then, CS/CG composite

174

hydrogels were dipped in 1 mL of physiological saline and incubated at 37 oC for 30 min. 1

175

mL of deionized water was used as a positive control and 1 mL of physiological saline as a

176

negative control. Then 0.2 mL of diluted blood was added into each sample and the mixtures

177

were incubated at 37 oC for 60 min. After that, all samples were centrifuged for 5 min at 3000

178

rpm and the supernatant was collected. ODs of the supernatants were determined at 545 nm

179

using a microplate reader (Thermo, Multiskn Go, U.S.A). The hemolysis rate was calculated

180

by using the following equation41: Hemolysis rate %=

Asample -Acontrol(-) ×100% Acontrol(+) -Acontrol(-)

(3)

181

where Asample, Acontrol(-) and Acontrol(+) represent the absorbance of the samples, negative and

182

positive controls, respectively.

183

In Vitro Cell Culture. Pre-chondrogenic ATDC5 cells obtained from RIKEN cell bank 8


Page 9 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

184

(Japan) were seeded on the surface of the hydrogels to evaluate the cytocompatibility. The

185

cells were cultured at 37 oC in a 5% CO2 incubator in 25 cm3 flasks (beaver, cyagen, China)

186

containing DMEM/F12 medium (Gibco, life technology, U.S.A) supplement with 5% fetal

187

bovine serum (FBS, Gibco, life technology, U.S.A), 1% antimicrobial of penicillin and

188

streptomycin (Hyclone, U.S.A). The culture medium was changed every 2 days.

189

Cell Viability and Cell Proliferation. The pure CS and CS/CG composite hydrogels with

190

10 mm diameter and a thickness of about 0.1 cm were sterilized by 70% alcohol for 30 min,

191

and then were exposed to ultraviolet light for 30 min. The samples were then transferred to

192

the bottom of 24-well plastic culture plates and washed with sterilized physiological saline for

193

30 min. Cell viability was determined by the live/dead staining. ATDC5 cells suspension at a

194

density of 5×104 cells/well were seeded on each sample for 24 h. Then, the samples on the 24

195

wells were washed by physiological saline and were stained with 1 mM calcein AM and 2

196

mM ethidium homodimer-1 solutions (Sigma, U.S.A) at 37 oC for 30 min, which were

197

visualized with a fluorescence microscope (Olympus BX51, Olympus Corporation, Japan).

198

3-(4, 5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT, sigma, U.S.A) assay was

199

used to evaluate the proliferation rate of cells growing on the surface of the samples.42 ATDC5

200

cells suspension at a density of 2×104 cells/well were seeded on each sample for 1, 4 and 7

201

days. After culturing for 1, 4 and 7 days, the medium was aspirated, and an aliquot of 50 µL

202

MTT dissolved in 450 µL DMEM/F12 medium was added to each sample. The plates were

203

then incubated at 37 oC for 4 h. After the culture medium was removed, the formazan reaction

204

products were dissolved in 500 µL of dimethyl sulfoxide (DMSO, Aladdin, China) for 20 min.

205

The formazan solution was transferred to a 96-well plate, and the absorbance at 490 nm was

206

measured using a microplate reader (Thermo, Multiskan Go, U.S.A).

207

Cell Morphology. The cell morphology was analyzed by SEM (FESEM, Zeiss, Germany).

208

For morphological analysis of the ATDC5 cells at a density of 5×104 cells/well after being 9



Page 10 of 40

209

cultured on different samples for 48 h, the samples were washed with PBS thrice and fixed

210

with 3% glutaraldehyde for 4 h at 4 oC. The cells were dehydrated through graded

211

concentrations of ethanol (20%, 40%, 60%, 80%, and 100%) and then tertiary butanol, twice

212

for 30 min each. The resultant hydrogels were lyophilized and sputtered with gold for SEM

213

observation.

214

In Vitro Degradation Study. The in vitro degradation behavior of the hydrogels was

215

studied by incubating certain weights of the pristine CS hydrogel and CS/CG composite

216

hydrogels (10 mm diameter) in 4 mg/mL three-times-recrystallized egg white lysozyme in 0.1

217

M PBS at pH 7.4 and 37 oC.43, 44 After pre-defined time intervals, hydrogel samples were

218

taken out from the lysozyme solution and washed with ultrapure water three times. The

219

hydrogel samples were frozen at - 20 oC overnight and freeze-dried for 2 days. The extent of

220

the in vitro degradation was characterized by the percentage of the weight of the dried

221

hydrogels before and after the lysozyme treatment by the following equation: Weight Remaining %=

WT ×100% W0

(4)

222

where WT and W0were the dry weight of the hydrogel sample at certain degradation time and

223

initial dry weight of the hydrogel respectively.

224

Alcian Blue Staining. ATDC5 cells on the samples at 7 and 14 days were rinsed twice with

225

phosphate-buffered saline and fixed in 4% paraformaldehyde solution for 30 min, after which

226

cells were stained with 0.1% alcian blue 8GX (sigma-Aldrich, U. S. A) in 0.1 M HCl

227

overnight. After overnight staining, the plates were washed twice with 0.1 M HCl for 5 min to

228

remove any nonspecific staining of the plates, followed by a 30 s rinse with 3% acetic acid

229

three times, and then visualized with an inverted microscope (Leica, Germany).

230

Glycosaminoglycan (GAG) Quantification. GAG content of the ATDC5 cells seeded

231

samples was evaluated by GAG assay using 1, 9-Dimethyl-Methylene Blue (DMMB, sigma,

232

U.S.A.). DMMB dye solution (0.016 mg/mL of DMMB, 0.01 M HCl, 2.37 mg/mL of NaCl, 10


Page 11 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

233

3.4 mg/mL of Glycine) was prepared and stored at room temperature shielded from light.

234

After 7 and 14 days, GAG was harvested from the cells by digestion with papain solution

235

containing (125 µg/mL of papain, 5 mM L-cystein,100 mM Na2HPO4, 5 mM EDTA)

236

maintaining a pH of 6.8 at 60°C for 16 h. The dye solution was transferred to a 96-well plate,

237

and the absorbance at 525 nm was measured using a microplate reader (Thermo, Multiskan

238

Go, U.S.A.). A standard curve was established using chondroitin-4-sulfate (sigma, U.S.A.),

239

and the total GAG amounts were determined from the OD value which correlated to the

240

corresponding GAG amount in the standard curve.45 The total protein was determined using

241

BCA Protein Quantitation Kit (Thermo, U.S.A.), and the absorbance at 562 nm was measured

242

using a microplate reader. GAG content was normalized according to the protein contents (µg

243

GAG/mg protein).

244

Quantitative Real Time PCR (RT-PCR) Analysis. The expressions of cartilage-related

245

genes, namely aggrecan, SOX9, collagen type II (COL2) and collagen type X (COL10) were

246

investigated to evaluate the ability of ATDC5 cells on samples using RT-PCR analysis. The

247

ATDC5 cells at a density of 2×104 cell/well were cultured with DMEM/F12 medium

248

supplement with 5 % fetal bovine serum and 1 % antimicrobial of penicillin and streptomycin.

249

For chondrogennic induction, the medium was replaced with medium containing 10 µg/mL

250

bovine insulin, 5.5 µg/mL human transferrin and 0.005 µg/mL sodium selenite (ITS Liquid

251

Media Supplement, Sigma, USA). After 7 and 14 days, total RNA was extracted from

252

cell-hydrogel constructs by 750 µL/well trizol reagent (Life technology, USA). 750 µL/well of

253

chloroform was added followed by vigorous shaking for 20 s, and centrifuging at 12,000 rpm

254

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

256

12,000 rpm for 10 min at 4 oC. The supernatant was removed and the RNA pellet was washed

257

with 1 mL of 75% ethanol for 5 min at 4 oC. The concentration and purity of the RNA was 11



258

assessed using a Nanodrop 2000 (Thermo, U.S.A). Afterwards, the RNA was converted to

259

cDNA using a Prime Script RT Master Mix reagent kit (Takara, Japan). Then the analysis was

260

performed with the Rotor-Gene 6000 (QIAGEN, German) with SYBR Green Real time PCR

261

Master Mix (DBI Bioscience, China). The qRT-PCR results were analyzed using the

262

Rotor-Gene Real-Time analysis software 6.0. And the relative gene expression was calculated

263

using the 2-∆∆Ct method. The relative expression levels of the genes evaluated were

264

normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

265

The synthesized primers used in this study were listed in Table 1.

266

Real-time RT-PCR thermal cycles correspond to the following periods: 2 min at 95 oC,

267

followed by 40 cycles of 95 oC for 10 s corresponding annealing temperature of 55 oC for 30 s

268

and an extension step at 72 oC for 30 s.

269

Statistical Analysis. All of the data were expressed as means ± standard deviations (SD;

270

n = 3). The statistical analysis was performed with OriginPro 9.0. The statistical significance

271

of the difference was measured using one-way analysis of variance, and values of p < 0.05

272

were considered statistically significant. Values of p < 0.01 and < 0.001 are marked as **

273

and *** respectively.

274

RESULTS AND DISCUSSION

275

Construction and Structure of CS/CG Composite Hydrogels. In order to avoid the

276

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

278

alkali/urea with a concentration of 4 wt %, and then were directly blended with the weight

279

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

281

and 1b, the CS/CG blend solution in alkali/urea was transparent and homogeneous, and no

282

obvious aggregates were observed. It was attributed to the fact that CS was dissolved in this 12


Page 12 of 40

Page 13 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

283

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


13

C NMR


308

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


Page 14 of 40

Page 15 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

333

Biomacromolecules

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



358

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


Page 16 of 40

Page 17 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

383

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



408

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


Page 18 of 40

Page 19 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

433

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



458

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


Page 20 of 40

Page 21 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

483

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



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


Page 22 of 40

Page 23 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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



558

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


Page 24 of 40

Page 25 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

582

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

(1) Deng, J.; She, R.; Huang, W.; Dong, Z.; Mo, G.; Liu, B. J. Mater. Sci. Mater. Med. 2013, 24, 2037-2046. (2) van Haaften, E. E.; Ito, K.; van Donkelaar, C. C. J. Orthop. Res. 2017, 35, 1265-1273. (3) Vilela, C. A.; Correia, C.; Oliveira, J. M.; Sousa, R. A.; Espregueira-Mendes, J.; Reis, R. L. ACS Biomater. Sci. Eng. 2015, 1, 726-739. (4) Correa, D.; Lietman, S. A. Semin. Cell Dev. Biol. 2017, 62, 67-77. (5) Duda, G. N.; Sittinger, M.; Eniwumide, J. O.; Lippens, E. In Regenerative Medicine from Protocol to Patient; Springer: Cham, 2016; p 305. (6) Ren, K.; He, C.; Xiao, C.; Li, G.; Chen, X. Biomaterials 2015, 51, 238-249. (7) Lam, J.; Clark, E. C.; Fong, E. L. S.; Lee, E. J.; Lu, S.; Tabata, Y.; Mikos, A. G. Biomaterials 2016, 83, 332-346. (8) Kim, M.; Hong, B.; Lee, J.; Kim, S. E.; Kang, S. S.; Kim, Y. H.; Tae, G. Biomacromolecules 2012, 13, 2287-2298. 25



611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654

(9) Markstedt, K.; Mantas, A.; Tournier, I.; Martínez Ávila, H.; Hägg, D.; Gatenholm, P. Biomacromolecules 2015, 16, 1489-1496. (10) Eslahi, N.; Abdorahim, M.; Simchi, A. Biomacromolecules 2016, 17, 3441-3463. (11) Maturavongsadit, P.; Luckanagul, J. A.; Metavarayuth, K.; Zhao, X.; Chen, L.; Lin, Y.; Wang, Q. Biomacromolecules 2016, 17, 1930-1938. (12)Rodrigues, M. N.; Oliveira, M. B.; Costa, R. R.; Mano, J. F. Biomacromolecules 2016, 17, 2178-2188. (13) Balakrishnan, B.; Banerjee, R. Chem. Rev. 2011, 111, 4453-4474. (14) Torres-Rendon, J. G.; Femmer, T.; De Laporte, L.; Tigges, T.; Rahimi, K.; Gremse, F.; Zafarnia, S.; Lederle, W.; Ifuku, S.; Wessling, M.; Hardy, J. G.; Walther, A. Adv. Mater. 2015, 27, 2989-2995. (15) Bork, P.; Bowler, C.; de Vargas, C.; Gorsky, G.; Karsenti, E.; Wincker, P. Science 2015, 348, 873. (16) Giammanco, G. E.; Carrion, B.; Coleman, R. M.; Ostrowski, A. D. ACS Appl. Mater. Interfaces 2016, 8, 14423-14429. (17)Chen, F.; Yu, S.; Liu, B.; Ni, Y.; Yu, C.; Su, Y.; Zhu, X.; Yu, X.; Zhou, Y.; Yan, D. Sci. Rep. 2016, 6, 20014. (18) Radhakrishnan, J.; Subramanian, A.; Krishnan, U. M.; Sethuraman, S. Biomacromolecules 2017, 18, 1-26. (19) Naderi-Meshkin, H.; Andreas, K.; Matin, M. M.; Sittinger, M.; Bidkhori, H. R.; Ahmadiankia, N.; Bahrami, A. R.; Ringe, J. Cell Biol. Int. 2014, 38, 72-84. (20) Van Vlierberghe, S.; Dubruel, P.; Schacht, E. Biomacromolecules 2011, 12, 1387-1408. (21) Park, H.; Choi, B.; Hu, J.; Lee, M. Acta Biomater. 2013, 9, 4779-4786. (22) Fan, M.; Ma, Y.; Mao, J.; Zhang, Z.; Tan, H. Acta Biomater. 2015, 20, 60-68. (23) Prabaharan, M. 2015, 72, 1313-1322. (24) Saravanan, S.; Leena, R. S.; Selvamurugan, N. Int. J. Biol. Macromol. 2016, 93, 1354-1365. (25) Andrey, S. K.; Stanislav, A.; Yury, A. S. Russ. Chem. Rev. 2017, 86, 231. (26) Choi, B.; Kim, S.; Lin, B.; Wu, B. M.; Lee, M. ACS Appl. Mater. Interfaces 2014, 6, 20110-20121. (27) Sivashankari, P. R.; Prabaharan, M. Int. J. Biol. Macromol. 2016, 93, 1382-1389. (28)Konovalova, M. V.; Markov, P. A.; Durnev, E. A.; Kurek, D. V.; Popov, S. V.; Varlamov, V. P. J. Biomed. Mater. Res. Part A 2017, 105, 547-556. (29) Zhang, Y.; Choi, S.-W.; Xia, Y. Macromol. Rapid Commun. 2012, 33, 296-301. (30) Fang, J.; Zhang, Y.; Yan, S.; Liu, Z.; He, S.; Cui, L.; Yin, J. Acta Biomater. 2014, 10, 276-288. (31) Tan, H.; Chu, C. R.; Payne, K. A.; Marra, K. G. Biomaterials 2009, 30, 2499-2506. (32) Dash, M.; Chiellini, F.; Ottenbrite, R. M.; Chiellini, E. Prog. Polym. Sci. 2011, 36, 981-1014. (33) Mihaila, S. M.; Popa, E. G.; Reis, R. L.; Marques, A. P.; Gomes, M. E. Biomacromolecules 2014, 15, 2849-2860. (34) Popa, E.; Reis, R.; Gomes, M. Biotechnol. Appl. Biochem. 2012, 59, 132-141. (35) Popa, E. G.; Gomes, M. E.; Reis, R. L. Biomacromolecules 2011, 12, 3952-3961. (36) Popa, E. G.; Carvalho, P. P.; Dias, A. F.; Santos, T. C.; Santo, V. E.; Marques, A. P.; 26


Page 26 of 40

Page 27 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681

Biomacromolecules

Viegas, C. A.; Dias, I. R.; Gomes, M. E.; Reis, R. L. J. Biomed. Mater. Res. Part A 2014, 102, 4087-4097. (37) Popa, E. G.; Caridade, S. G.; Mano, J. F.; Reis, R. L.; Gomes, M. E. J. Tissue Eng. Regener. Med. 2015, 9, 550-563. (38) Santo, V. E.; Frias, A. M.; Carida, M.; Cancedda, R.; Gomes, M. E.; Mano, J. F.; Reis, R. L. Biomacromolecules 2009, 10, 1392-1401. (39) Koller, U.; Apprich, S.; Schmitt, B.; Windhager, R.; Trattnig, S. Int. Orthop. 2017, 41, 969-974. (40) Duan, J.; Liang, X.; Cao, Y.; Wang, S.; Zhang, L. Macromolecules 2015, 48, 2706-2714. (41) Huang, Y.; Ding, X.; Qi, Y.; Yu, B.; Xu, F.-J. Biomaterials 2016, 106, 134-143. (42) He, M.; Wang, X.; Wang, Z.; Chen, L.; Lu, Y.; Zhang, X.; Li, M.; Liu, Z.; Zhang, Y.; Xia, H.; Zhang, L. ACS Sustainable Chem. Eng. 2017, 5, 9126-9135. (43) Ding, B.; Gao, H.; Song, J.; Li, Y.; Zhang, L.; Cao, X.; Xu, M.; Cai, J. ACS Appl. Mater. Interfaces 2016, 8, 19739-19746. (44) Wu, S.; Duan, B.; Lu, A.; Wang, Y.; Ye, Q.; Zhang, L. Carbohydr. Polym. 2017, 174, 830-840. (45) Arslan, E.; Guler, M. O.; Tekinay, A. B. Biomacromolecules 2016, 17, 1280-1291. (46)Zhou, Y.; Yang, B.; Ren, X.; Liu, Z.; Deng, Z.; Chen, L.; Deng, Y.; Zhang, L.-M.; Yang, L. Biomaterials 2012, 33, 4731-4740. (47) Lee, J. H.; Lee, J. W.; Khang, G.; Lee, H. B. Biomaterials 1997, 18, 351-358. (48) Mihaila, S. M.; Gaharwar, A. K.; Reis, R. L.; Marques, A. P.; Gomes, M. E.; Khademhosseini, A. Adv. Healthcare Mater. 2013, 2, 895-907. (49) Bi, W.; Deng, J. M.; Zhang, Z.; Behringer, R. R.; de Crombrugghe, B. Nat. Genet. 1999, 22, 85. (50) Goldring, M. B.; Tsuchimochi, K.; Ijiri, K. J. Cell. Biochem. 2006, 97, 33-44. (51) Hardingham, T.; Tew, S.; Murdoch, A. Arthritis Res. Ther. 2002, 4, S63. (52) Pearle, A. D.; Warren, R. F.; Rodeo, S. A. Clin. Sports Med. 2005, 24, 1-12.

682 683 684 685 686 687 688 689 690 27



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


Page 28 of 40

Page 29 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

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



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


Page 30 of 40

Page 31 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

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



764 765

766 767

Figure 3. Schema to describe the formation and structure of CS/CG composite hydrogels.

768 32


Page 32 of 40

Page 33 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

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



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

34


Page 34 of 40

Page 35 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

781 782

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

35



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.

36


Page 36 of 40

Page 37 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

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

37



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

***).

823 824 825 826 827 38


Page 38 of 40

Page 39 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

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



835 836

Table of Contents Graphic (TOC)

837

40


Page 40 of 40

Carrageenan Hydrogels Constructed through an

Recommend Documents