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Effects of Chitin Whiskers on Physical Properties and Osteoblast Culture of Alginate Based Nanocomposite Hydrogels Yao Huang, Mengyu Yao, Xing Zheng, Xichao Liang, Xiaojuan Su, Yu Zhang, Ang Lu, and Lina Zhang Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 22 Sep 2015 Downloaded from http://pubs.acs.org on September 26, 2015

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Effects of Chitin Whiskers on Physical Properties and Osteoblast Culture of Alginate Based Nanocomposite Hydrogels

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Yao Huang †, Mengyu Yao ‡, Xing Zheng †, Xichao Liang†, Xiaojuan Su†, Yu Zhang‡,

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Ang Lu†*, Lina Zhang†*

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†

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China

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‡

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Command of Chinese PLA, Guangzhou 510010, China

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

Department of Orthopedics, General Hospital of Guangzhou Military Area

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Keywords: chitin whisker, sodium alginate, nanocomposite hydrogel, proliferation

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of osteoblast cells, bone tissue engineering

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Abstract: Novel nanocomposite hydrogels composed of polyelectrolytes alginate and

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chitin whiskers with biocompatibility were successfully fabricated based on the pH

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induced charge shifting behavior of chitin. The chitin whiskers with mean length and

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width of 300 and 20 nm were uniformly dispersed in negatively charged sodium

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alginate aqueous solution, leading to the formation of the homogeneous

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nanocomposite hydrogels. The experimental results indicated that their mechanical

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properties were significantly improved compared to alginate hydrogel and the

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swelling trends were inhibited, as a result of the strong electrostatic interactions

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between the chitin whiskers and alginate. The nanocomposite hydrogels exhibited 1

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certain crystallinity and hierarchical structure with nanoscale chitin whiskers, similar

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to the structure of the native extracellular matrix. Moreover, the nanocomposite

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hydrogels were successfully applied as bone scaffolds for MC3T3-E1 osteoblast cells,

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showing their excellent biocompatibility and low cytotoxicity. The results of

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fluorescent micrographs and scanning electronic microscope (SEM) images revealed

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that the addition of chitin whiskers into the nanocomposite hydrogels markedly

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promoted the cell adhesion and proliferation of the osteoblast cells. The

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biocompatible nanocomposite hydrogels have potential application in bone tissue

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engineering.

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1. Introduction

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It has been estimated that over 200 million people worldwide suffer from

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osteoporosis, fracture and other bone defects1. Bone tissue regeneration, which aims

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to use synthetic scaffolds or graft materials to aid the natural healing of bone defects,

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remains as a clinical challenge2, 3. An ideal bone scaffold is required to be nontoxic,

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biocompatible and mechanically tough, with a three dimensional structure that mimics

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the bone extracellular matrix (ECM) secreted by the osteoblasts4. ECM is well

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acknowledged to exhibit

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structures5 and cells are inherently sensitive to its surface properties such as roughness,

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topography, and chemistry6. However, it is almost impossible to meet all the

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requirements by single component polymer materials. As a result, nanocomposites

a hierarchical organization from nano- to macro-scale

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involving biodegradable polymeric matrices and bioactive nanofillers have attracted

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intensive attention in bone tissue engineering and regeneration7, 8.

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The exploitation and application of marine biomass as biomedical materials has

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not only conformed to the ideas of sustainable development but also promoted the

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blue economic growth. Chitin is a natural polysaccharides with important biological

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function9 and widely exists as the skeletal material of crustaceans, insects, algae, etc.

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As an original component of living organisms, chitin has been generally recognized to

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be biocompatible, biodegradable, and thus a good candidate for biomaterials10 due to

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the diverse biofunctions, such as enhancing the biological self-defense function in

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animals,11 accelerating wound healing,12 hemostatic and antimicrobial properties,13

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etc. Currently, the chitin and its deacetylated product, chitosan, are used in only a few

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niche areas of industrial chemistry, however, their potential are much greater.14 Chitin

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whiskers retain the intrinsic structure and bioactivities of α-chitin, so they possess

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excellent biocompatibility, as well as high aspect ratios, large surface areas and

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longitudinal modulus as high as 150 GPa. Therefore, the chitin whiskers are expected

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to be promising candidates as load-bearing components for biomedical applications,

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especially when compared with the non-biocompatible reinforcing nanofillers15-17.

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Specifically, chitin and chitosan have already been acknowledged to be highly

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osteoconductive and promote the differentiation of the rat calvarial osteoblast18-21.

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Moreover, the formation of apatite layer achieved by deposition of calcium phosphate

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on chitin22-24 can further improve the mechanical performance and osteo-inductive

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properties.25 3


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Alginates are also natural marine resources, which are anionic polysaccharides

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extracted from seaweeds, consisting of β-D-mannuronic acid and α-L-guluronic.

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Alginate hydrogels cross-linked with Ca2+ have been widely employed in drug

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delivery, cell encapsulation and tissue engineering applications due to their excellent

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biocompatibility, 3-D structure and biodegradability in vivo.26, 27 However, there were

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few work regarding sole alginate gel as bone scaffolds, since the mammalian cells

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cannot interact with unmodified alginate.28 On the other hand, alginate cross-linked

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with Ca2+ degrades rapidly in cell culture medium due to the exchange of the Na+ or

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K+ ions with Ca2+ ions.29 It is worth noting that polyion complex (PIC) or

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polyelectrolyte complex (PEC) composed of oppositely charged polymers appear as

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an option to reduce the tendency of swelling and enhance the mechanical properties of

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the composite materials.30-32 Chitin whiskers are positively charged due to the

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protonation of amino groups facilitated by acid.33 In view of the above case, PEC

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composed of negatively charged alginate and positively charged chitin whiskers

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should be feasible and would be potential in bone tissue engineering. However, few

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works have been done as a result of the easily flocculation of chitin whiskers in

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negatively charged matrix.34 In the present work, nanocomposite hydrogels composed

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of chitin whiskers and alginate were successfully fabricated. Our strategy to overcome

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this problem is the utilization of chitin whiskers with pH dependent charge shifting

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properties. In basic solution, the chitin whiskers were negatively charged and

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homogeneously blended with sodium alginate. After neutralization, the electrostatic

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interaction occurred between the chitin whiskers and alginate macromolecules, 4


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contributing to enhanced mechanical properties of the composite hydrogels. The

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effects of chitin whiskers on the structure and properties of the nanocomposite were

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studied. Furthermore, the promoted adhesion and proliferation of osteoblast by

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addition of chitin whiskers were investigated and evaluated. This work has potential

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applications in bone tissue engineering.

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2. Experimental

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2.1 Materials

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Chitin powder with a degree of acetylation (DA) of 90% was purchased from

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Golden-Shell Biochemical Co. Ltd (Zhejiang, China). The weight-average molecular

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weight (Mw) of the chitin powder was determined to be 59.6 × 105 in 5% (w/v)

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LiCl–DMAc by dynamic light scattering (DLS, ALV/GGS-8F, ALV, Germany). The

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DA values of raw chitin and chitin whiskers were measured by conventional

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potentiometric titration method35. Sodium alginate (SA) and other chemical reagents

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were purchased from Sinopharm Chemical Reagent Co., Ltd and used without further

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purification. Ultra-pure water (Millipore) was used for all experiments.

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2.2 Preparation of composite hydrogels

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The chitin whiskers were prepared by acid hydrolysis, according to our previous

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work36. Briefly, the chitin powder were subjected to hydrolysis by 3 M H2SO4 (30 mL

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per gram of chitin) at 90 °C for 6 h with vigorous stirring. Then the resultant

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suspension was diluted with water and centrifuged to remove excessive acid in the

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supernatant, followed by dialysis until the pH value no longer changed. For better 5


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dispersion of the chitin whiskers, the suspension was subjected to a further ultrasonic

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treatment (800 W) on an ultrasonic cell disruptor (JY92-IID, Ningbo Scientz

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Biotechnology Co., Ltd., China). Centrifugation of the chitin whisker suspension at

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7200 rpm was performed to remove any visible precipitate, since most of the whiskers

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were converted to nanoscale. Finally, the chitin whisker suspension with a solid

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concentration of 3 wt. % was stored at 4 oC for use, denoted as CW.

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The CW suspension were diluted to various concentration as 0.5 wt. %, 1 wt. %,

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2 wt. % and 2.5 wt. %. Subsequently, 0.1M NaOH was added drop wisely into the

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diluted suspension to control the pH in range of 11.0-11.5. Ultrasonication was

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applied to the suspension for 5 min before calculated amount of sodium alginate was

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added and dissolved by stirring overnight. The concentration of sodium alginate was 2

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wt. % for all samples. The composite solution was centrifuged at 6000 rpm for 10 min.

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Subsequently, the solution was cast in a mold and then cross-linked with 3 wt. %

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CaCl2 solution overnight. The cross-linked hydrogels were rinsed with 0.01M HCl

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and then ultrapure water to remove the excess NaOH and salt. The hydrogels were

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decoded as SCa-b, where a/b referred to the weight ratio of alginate/chitin whisker.

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Pure alginate hydrogel without addition of the chitin whiskers was also prepared in

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the same procedure as a control and denoted as SA.

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2.3 Characterization

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X-ray photoelectron spectra (XPS) were recorded on a Thermo Fisher

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ESCALAB 250Xi X-ray photoelectron spectrometer, using Al Kα radiation as the

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excitation source. Infrared spectroscopy of the chitin whiskers and the composite 6


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hydrogels was recorded on a NICOLET 5700 FTIR spectrometer at room temperature.

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X-ray diffraction (XRD) measurement was carried out on a wide angle X-ray

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diffraction (WXRD) diffractometer (D8-Advance, Bruker, U.S.A.) with Cu Kα

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radiation (λ = 0.154 nm). The XRD patterns were recorded in the range 2θ = 5−40° at

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a scanning speed of 2° /min. The samples were lyophilized and trimmed into small

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particles and dried in vacuum oven at 40 oC for 48 h before testing. Scanning electron

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microscopy (SEM) was carried out on a field emission scanning electron microscopy

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(FESEM, Zeiss, SIGMA). The wet hydrogels were frozen in liquid nitrogen, snapped

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immediately, and then freeze-dried. The surfaces and fracture sections were sputtered

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with gold for the SEM observation. Transmission electron microscopy (TEM) images

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of chitin whisker suspension and ultrathin slice of composite hydrogel SC4-4 were

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observed on a JEOL JEM-2010 (HT) electron microscope, with an accelerating

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voltage of 200 kV. The compression strength of the gels was measured on a universal

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tensile tester (CMT 6503, Shenzhen SANS Test machine Co. Ltd., Shenzhen, China)

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according to ISO527−3−1995 (E) at a speed of 1 mm/min. For each group, the data

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were obtained under the same surroundings. For each sample, at least 5 parallel

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experiments were conducted and an average data was reported.

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

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hydrogels37 in 0.1M NaCl solution with different pH. The hydrogels were oven-dried

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at 60 oC before immersing in swelling medium. The swelling temperature was set at

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37 oC. The pH values were adjusted by HCl and NaOH solutions, ranging from 1.01

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to 10.13, which were determined by using a FE-20K pH-meter (Mettler-Toledo, Ohio, 7


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USA). The ionic strength of the pH solution remained 0.1 M by adding an appropriate

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amount of NaCl. The equilibrium swelling ratio (ESR) was calculated as ESR = Ws/Wd

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(1)

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Where Ws is the weight of the swollen gel at 37 oC and Wd is the weight of the oven

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dried gel. For each test, three parallel samples were measured and an average result

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was reported.

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2.4. Cell Adhesion and Cytotoxicity Assay

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The alginate and composite gels were cut into cylindrical slices with a thickness

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of about 0.1 cm, sterilized by autoclaving, soaked in the 0.1 mg/mL physiological

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saline for 0.5 h, and then transferred to the bottom of 24-well plastic culture plates.

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The preosteoblast extracted from the calvaria of Mus musculus MC3T3-E1

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(ATCC, CRL-2592, USA) were cultured in α-minimal essential medium (α-MEM,

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Gibco, Life technologies, Carlsbad, USA) supplemented with 10% fetal bovine serum,

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containing 100U/mL penicillin and 100 mg/mL streptomycin. The cells were

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incubated at 37 oC in 5% CO2 and 95% air and passaged every 2 days.

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The proliferation of MC3T3-E1 cells on different substrates were characterized

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by using 3-(4, 5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay

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after the cells were cultured for 1, 3 and 7 days. Cells were seeded onto the substrates

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at a density of 4 ×104 cells/mL. For MTT assay, at each of the designated time points

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(1, 3 and 7 days), 40 mL of MTT (Sigma, USA) solution with concentration of 5

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mg/mL in phosphate buffered saline (PBS) was added to 300 mL of culture medium

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and incubated for 4 h at 37oC. After the culture medium was removed, the formazan 8


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reaction products were dissolved in 400 μL dimethylsulfoxide (DMSO) for 20 min.

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The optical density of the formazan solution was read using a microplate reader

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(Thermo, Multiskn Go) at 490 nm.

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To characterize the cell viability by live/dead fluorescent staining, the

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MC3T3-E1 cells seeded at a density of 2 ×104 cells/ml were incubated on the

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substrates at 37 oC in a humidified atmosphere of 5% v/v CO2 for 24 h and 48 h,

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respectively. After incubation was completed, the media was removed and the

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adherent cells were subjected to live/dead staining following the manufacturer's

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protocol (Sigma, USA). Briefly, 1 mM calcein AM and 2 mM ethidium homodimer-1

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solutions were prepared in PBS. After the culture media were removed, the cells were

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rinsed once in PBS, followed by addition of 300 μL of 1 mM calcein AM and 2 mM

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ethidium homodimer-1 solution. The cells were stained at 37 oC for 30 min, and then

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visualized with a fluorescence microscope (Olympus BX51, Olympus Corporation,

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Japan).

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The cell morphology and adhesion were analyzed by SEM (FESEM, Zeiss,

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SIGMA). For morphological analysis of the MC3T3-E1 cells after being cultured with

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different substrates for 48 h, the samples were washed with PBS for three times and

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

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graded concentrations of ethanol (30%, 50%, 70%, 90%, and 100%) and then tertiary

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butanol, twice for 10 min each. The resultant hydrogels were lyophilized and

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sputtered with gold for SEM observation.

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3. Results and discussion

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3.1 Structure and intermolecular interaction of the nanocomposite hydrogels

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The surface amino groups on chitin whiskers act as weak polyelectrolyte with

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pKa of approximately 6.3 at room temperature.38 When the positively charged chitin

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whiskers were directly mixed with alginate sodium solution, serious flocculation

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occurred (as shown in Figure 1b). In our findings, the charge properties of chitin

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whiskers were pH dependent, as shown in Figure 1. Since the raw chitin powder

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exhibited partial deacetylation with a degree of deacetylation of 10%, the resultant

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chitin whiskers were positively charged under lower pH, as a result of the pronation

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of –NH2 group in acidic solution. When pH reached 10, however, the chitin whiskers

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turned to be negatively charged. It can be ascribed to the complete deprotonation of

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–NH2, leading to the appearance of negative charge. This could be explained that the

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–OSO3- groups induced by sulfuric acid hydrolysis occurred on the chitin whiskers,

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and the existence of –OSO3H was confirmed by XPS spectra (Figure S1). The

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appearance of negative charge of the chitin whiskers in alkaline solution

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similar to that of the cellulose nanocrystal.39, 40 Such pH-responsive charge shifting

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behavior has also been reported for sulfated cellulose nanocrystal decorated with

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amino based group.41 Therefore, the chitin whiskers could be well dispersed in both

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acidic and basic solutions, as a result of the electrical repulsion. When the pH was

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above 13, however, the chitin whiskers flocculated, due to the possible desulfation in

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concentrated NaOH42 or the charge effects suppressed by the screening effect of

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increased ionic strength.43 Thus, we controlled the pH of chitin whisker suspension in 10


is

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the range between 11 and 12. Subsequently, the alginate sodium was dissolved in the

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above chitin whisker suspension with vigorous stirring, and their homogeneous

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mixture could be obtained (Figure 1a). In such mixtures, the negatively charged chitin

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whiskers were evenly distributed in the sodium alginate matrix, which returned to be

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positively charged after crosslinking with CaCl2 and HCl neutralization, leading to the

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formation of the nanocomposite hydrogels where strong electrostatic interaction

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occurred between the chitin whiskers and alginate.

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Figure 1. Zeta potential of chitin whiskers in aqueous suspensions with different pH

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values. Insert: the mixture of chitin whiskers and sodium alginate in (a) NaOH

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solution of pH 11 and (b) neutral aqueous solution at a weight ratio of 1:1 for alginate

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/ chitin.

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Solid-state

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C NMR spectra of the chitin whisker, alginate hydrogel and

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composite hydrogel are presented in Figure S2. The assignment of the observed

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signals to various types of carbons was also demonstrated. Both the signals of chitin 11


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whiskers and alginate were presented in the composite hydrogels (Figure S2c),

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indicating the coexistence of the tow components with intact chemical structure.

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FT-IR spectra of the nanocomposite hydrogels are shown in Figure 2. With an

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increase of the CW content, the intensity of the characteristic peaks for chitin (amide I

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at 1661 and 1622 cm-1 and amide II at 1559 cm-1

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peaks for alginate appeared at 1738 and 1628 cm-1 were ascribed to the carboxylic

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acids (–COOH) and carboxylate ions (–COO-), respectively, indicating that besides

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electrostatic interaction of NH3+ and COO-, hydrogen bonding might also existed

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between the –COOH of alginate and –OH of chitin 45. The enhanced band from 1620

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to 1660 cm-1 that arose from overlapping bands of amide I and –COO- further

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confirmed the electrostatic interaction between alginate and chitin whisker,46 which is

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beneficial to the formation of homogeneous architecture between SA and CW.

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) increased. The characteristic

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Figure 2. FT-IR spectra of chitin whisker (CW), alginate hydrogel (SA) and the

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composite hydrogels.

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The XRD patterns of alginate hydrogel, chitin whiskers and composite hydrogels

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are shown in Figure 3. The pure alginate calcium hydrogel displayed no distinct peaks,

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indicating its amorphous structure.47 All the other samples exhibited diffraction peaks

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at 2θ = 9.3 o, 12.8 o, 19.2 o, 20.7 o, 23.4 o and 26.4 o, ascribed to the (020), (021), (110),

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(120), (130) and (013) crystal plane of α-chitin.44 When the crystal plane [110] was

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regarded as the crystal components, the crystallinity could be calculated as follows. 48 Cr I110 (%) = (I110 - Iam) × 100/ I110

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(2)

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Where I110 and Iam refer to the intensity at 19.2 o and the baseline height at 16.0°as

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amorphous part, respectively. The results indicated that with an increase of the CW

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content, the crystallinity increased from 24.99% (SC4-1) and 37.18% (SC4-2) to

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65.06% (SC4-4). It is seeing that CW exhibited high crystallinity as 91.19%. The

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enhanced crystallinity is beneficial for a tissue scaffold as it may lead to enhanced

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mechanical strength and comply with tissue structures, since the natural ECM

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components (fibrous proteins and proteoglycans) exhibit a certain degree of

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crystallinity.49

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Figure 3. XRD patterns of chitin whisker (CW), alginate hydrogel (SA) and the

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composite hydrogels.

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Figure 4 shows TEM images of the chitin whisker dispersed in water and the

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ultrathin section of SC4-4 hydrogel. The rod-like shape of chitin whiskers with

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diameters of ~20 nm and lengths of 200-500 nm were observed. Namely, the chitin

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whiskers exhibited nanosize and aspect ratio larger than 10, indicating the tendency to

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form entangled network structure when well dispersed 50. The result of TEM images

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of the ultrathin section of the SC4-4 hydrogel (Figure 4b) confirmed the existence and

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relatively evenly distribution of chitin whiskers in the alginate calcium matrix. It was

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noted that the chitin whiskers were easily aggregated even in aqueous solution

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without sufficient ultrasonication (Figure S3). However, in the composite hydrogels

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with CW content as high as 50 wt%, the chitin whiskers just entangled with each

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other but no obvious aggregation was observed, indicating good compatibility

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between the chitin whiskers and the alginate matrix. 14


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Figure 4. TEM images of the chitin whisker dispersed in water (a) and the ultrathin

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section of SC4-4 hydrogel (b).

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On the basis of the above results, when the pH was in the range of 11 to 12, the

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chitin whiskers could be well dispersed in the alginate matrix to form homogeneous

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architecture, and after crosslinking and neutralization, the electrostatic interaction

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occurred between chitin whiskers and alginate in the composite hydrogels. Herein, the

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chitin whiskers retained nano-sized distribution and formed entangled structure in the

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alginate matrix.

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3.2 Effects of chitin whiskers on properties of the composite hydrogel

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Figure 5 shows the compressive stress-strain curves for the nanocomposite hydrogels

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and SA. As mentioned above, the chitin whiskers exhibited a network structure in the

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alginate calcium matrix, which has been proven to be efficient for stress transfer.51

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The compressive strength of pure alginate hydrogel was 1.39 MPa, lower than that of

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the composite hydrogels. With an increase of the chitin whisker ratio from SC4-1 to

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SC4-4 the compressive strength of the composite hydrogels increased from 2.04 to

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3.18 MPa, without obvious decrease in strain. When the ratio increased to SC4-5,

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however, the compressive strength decreased to 1.97 MPa, possibly due to the phase

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separation caused by excessive whisker aggregation. Moreover, the introduction of 15


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the chitin whisker also enhanced the compressive modulus of the composite hydrogels

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from 5.08 MPa (for SA) to 13.72 MPa (for SC4-4). Furthermore, the hydrogels were

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tough enough to be twisted and recovered without fracture afterwards (Figure S4). It

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has been reported that the acetylamino group in chitin contribute to its binding and

5

chelating capacity with calcium ions, and thus can accelerate the deposition of

6

hydroxyapatite on the surface of chitin derivates52. Moreover, the primary amide

7

group in chitin might also interact with calcium ion.20 In our findings, the positively

8

charged chitin whiskers were able to bind negatively charged alginate as well as

9

electroneutral CaCl2. To illustrate this interaction, the CaCl2 solution and sodium

10

alginate solution was added respectively to the top of the CW suspension without

11

agitation, and gelation occurred in both situations (Figure S5 bottom). Moreover, the

12

shape of the hydrogel consisted of CW and CaCl2 maintained better when compared

13

with the others, confirming the interaction between chitin whisker and CaCl2.

14

Therefore, CaCl2 as cross-linker acted an important role for the binding of both

15

components in the hydrogels, leading to enhanced strength. On the other hand, the

16

strong electrostatic interaction between the chitin whiskers and alginate also

17

contributed to the mechanical enhancement53. Compared to the generally reported

18

compressive strength of alginate calcium hydrogel (less than 1 MPa),54,

19

composite hydrogels prepared in this work exhibited excellent mechanical properties.

20

It is well-known that the mechanical strength is one of the priorities of the scaffolds

21

for bone tissue engineering, to support the bone tissue regeneration at the site of

22

implantation and maintain sufficient integrity during both in vitro and in vivo cell 16


55

the

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1

growth56. Therefore, the high mechanical strength of the composite hydrogels (SC4-4

2

with compressive strength of 3.18 MPa and modulus of 13.72 MPa) assured its

3

application as bone replacement or graft.

4 5

Figure 5. Compressive stress-strain curves of the composite hydrogels and SA

6

hydrogel. Insert: photographs of SA, SC4-2 and SC4-4.

7

Apart from mechanical performance, the topological structure also played an

8

important role in the cell behavior such as adhesion and proliferation. As shown in

9

Figure 6, the surface of the SA hydrogels displayed groove-like morphology due to

10

the inhomogeneity of ionic crosslinking.55 However, the surfaces became compact

11

with the introduction of chitin whiskers (Figure 6 a-c), suggesting that a denser

12

network was formed by chitin whiskers inside the alginate matrix. The conclusion

13

was consistent with the results of the above IR, NMR, XRD and TEM. Moreover, the

14

cross section of the composite hydrogel displayed laminated porous structure,

15

allowing the diffusion of oxygen, nutrients and metabolic products to and from

16

encapsulated cells57, endowing the composite hydrogels potentials as scaffolds for 17


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1

bone tissue engineering. The phase separation of SC4-5 was further confirmed in

2

Figure 6 (d, h), which was ascribed to the dramatically increased viscosity of the

3

chitin whisker suspension with high concentrations (2.5 wt %), resulting in difficulty

4

of the homogeneous blending with sodium alginate. Therefore, this sample (SC4-5)

5

was excluded from the following cell culture experiment.

6 7

Figure 6. Surface (top) and cross section (bottom) of SA (a, e), SCW4-2 (b, f),

8

SCW4-4 (c, g) and SCW4-5 (d, h). Scale bar: 1 μm (top) and 200 nm (bottom).

9

Figure 7 shows the swelling behavior of the composite hydrogels and SA.

10

Compared with SA hydrogels, the composite hydrogels exhibited relatively low

11

swelling ratios, due to the enhancement effect through the electrostatic interaction

12

between the chitin whiskers and alginate.46 With the increase of CW content, the

13

swelling ratios of the composite hydrogel was significantly decreased from 34.6 (SA)

14

to 17.0 (SC4-4). The electrostatic affinity between alginate and chitin whisker

15

reduced the chance of Ca2+ exchange with soluble ions in physical fluids such as Na+

16

and K+. Thus, the dimension stability of the composite hydrogels in saline solution

17

was remarkably enhanced, and the main disadvantage of SA (poor stability) in 18


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biomedical application was improved by introducing chitin whiskers. Therefore, the

2

nanocomposite hydrogels became more suitable as scaffolds in bone tissue

3

engineering. Furthermore, the composite hydrogels displayed pH responsive swelling

4

behavior (Figure S6). Namely, the swelling ratio of the composite hydrogel increased

5

with an increase of pH. As the chitin whiskers were negatively charged in the basic

6

solution, the electrostatic repulsion between them and –COO- led in swelling. While

7

in acidic medium, the chitin whiskers were positively charged and their electrostatic

8

attraction with –COO- resulted in shrinkage. Especially, when the pH was below the

9

pKa of –COOH (about 3.8),58 the protonation of –COO- facilitated the hydrogen

10

bonding formation59 between alginate and chitin whiskers, leading to the dramatic

11

contraction.

12 13

Figure 7. Swelling ratio of the composite hydrogels and SA hydrogel in 0.1 M NaCl

14

solution at 37 oC.

15

3.3 Effects of chitin whiskers on the osteoblast adhesion and proliferation

16

To evaluate whether the composite hydrogels met the fundamental requirement as

17

scaffolds in bone tissue engineering, the MC3T3-E1 cells were cultured on the 19


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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

1

hydrogels, and cell attachment and proliferation were evaluated. Figure 8 shows the

2

results of MTT assay for different hydrogels. Compared to the SA hydrogel, the cell

3

viability increased obviously with increasing CW content, which is in accordance

4

with the reports that chitin is biocompatible and suitable for osteoblast culture.11, 24

5

The enhanced osteoblast proliferation was further confirmed by the florescent

6

photographs of cells cultured the composite hydrogels for different time scales, as

7

shown in Figure 9. In this case, the live cells were stained by calcein AM and appear

8

in green with dead ones in red. After 24 h culturing, the osteoblast started to attach as

9

isolated cells onto the surface of the hydrogel. After 48 h, the cells proliferated on

10

both SA hydrogel and composite hydrogels, and the proliferation significantly

11

increased with the introduction of chitin whiskers. It was evidently displayed that the

12

osteoblasts cultured on the composite hydrogels connected with each other by discrete

13

filopodia and formed an entangled network. This observation was consistent with the

14

MTT results. It could be concluded that the addition of chitin whiskers significantly

15

improved the cytocompatibility of the composite hydrogels as well as the osteoblast

16

adhesion and proliferation on the hydrogels.

20


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1 2

Figure 8. MTT results of MC3T3-E1 cells seeded on SA hydrogel and the composite

3

hydrogels with culture time of 1, 3 and 7 days.

4 5

Figure 9. Fluorescent micrographs of MC3T3-E1 cells after 24 (top) and 48 h (bottom)

6

postseeding onto SA hydrogel (a, e) and the PEC composite hydrogels SC4-1 (b, f),

7

SC4-2 (c, g) and SC4-4 (d, h). Scale bar is 100 μm.

8

The morphology of the MC3T3-E1 cells on the scaffolds was examined by SEM.

9

Figure 10 shows the SEM micrographs of osteoblasts on the composite hydrogels

10

after 48 h of culture. The osteoblast cells spread on the surface of the hydrogels and 21


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

1

the amount of cells increased as the chitin whisker contents increased. From the

2

enlarged view in Figure 10 (c) and (d), the osteoblast exhibited an elongated shape,

3

and were anchored into the surface of the hydrogels by discrete filopodia, indicating

4

the excellent adherent performance of the composite hydrogels. No obvious cell

5

spreading was observed on the SA hydrogel, probably due to the drastic swelling of

6

SA hydrogel in salty culture medium29 and the resultant shrinkage during drying,

7

along with deformation or detachment of the adherent cells. Moreover, it has been

8

reported that the MC3T3-E1 cells have showed little adherence to unmodified

9

alginate hydrogels.60 In contrast, chitin and chitosan have been confirmed to support

10

the initial attachment and spreading of osteoblasts preferentially over fibroblasts.24, 61

11

On the other hand, the chitin whiskers could increase the surface roughness of the

12

composite hydrogels, as indicated by Figure 6. Namely, the chitin whiskers were

13

capable of promoting the cell adhesion.62 Moreover, the hydrophilicity of chitin and

14

functional groups like -NH2 was supposed to facilitate the effective calcium

15

phosphate deposition63 and formation of apatite layer, which can further promote the

16

osteo-conductivity.64 Therefore, the introduction of chitin whiskers significantly

17

improved the osteoblast cell affinity, and thus enhanced the cytocompatibility of the

18

composite hydrogels, leading to the great potentials of the chitin whisker–alginate

19

composite hydrogels as a natural polymer-based scaffolds for bone tissue engineering.

22


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1 2

Figure 10. Top: SEM images of the osteoblasts spreading on the composite hydrogels

3

SC4-1 (a) and SC4-4 (b) after 48h of culture. Scale bar = 20 μm; Bottom: Enlarged

4

view of single cell on SC4-1 (c) and SC4-4 (d). Scale bar = 2 μm.

5 6

Conclusion

7

The nanocomposite hydrogels from chitin whiskers and alginate were

8

successfully constructed on the basis of the pH-dependent charge behavior of the

9

chitin whiskers. By controlling pH at 11-12, the chitin whiskers were negatively

10

charged, and could be homogeneously dispersed in sodium alginate matrix.

11

Subsequently, after crosslinking with CaCl2 and neutralization, the chitin whiskers

12

returned to be positively charged, leading to the formation of the hydrogels. The

13

strong electrostatic interaction between the chitin whiskers and alginate led to the

14

excellent mechanical performance and the decreased swelling behavior. Furthermore,

15

the introduction of chitin whiskers greatly improved the cytocompatibility of the 23


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

1

composite hydrogel. With an increase of the chitin whisker content, the adhesion,

2

spreading and proliferation of osteoblast MC3T3-E1 on the composite hydrogels were

3

significantly enhanced. The composite hydrogels would be important as potential

4

scaffolds in bone tissue engineering. It is worth noting that as a skeletal material in

5

living organism, chitin has intrinsic biocompatibility and biofunctions. Therefore, the

6

materials fabricated directly from chitin through physical process are expected to be

7

perfect candidate in bone repair and tissue engineering. This work opened up a new

8

avenue to fabricate bone-repairing scaffolds from ocean resources via physical

9

process.

10 11

Supporting Information.

12

Additional figures about XPS measurements, solid state 13C NMR spectra,

13

photographs and swelling performance are reported in the supporting information file.

14

This material is available free of charge via the Internet at htpp://pubs.acs.org.

15

Corresponding Author

16

* Lina Zhang, email: [email protected]; Ang Lu, email: [email protected]

17

Author Contributions

18

The manuscript was written through contributions of all authors.

19

Funding Sources

20

This work was supported by the Major Program of National Natural Science

21

Foundation of China (21334005), the National Natural Science Foundation of China 24


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1

(20874079, 51203122 and 81271957), the Fundamental Research Funds for the

2

Central Universities (2014203020202), the National Basic Research Program of

3

China (2012CB619106) and Guangdong Key Lab of Orthopedic Technology and

4

Implant Materials Construction Grant (2011233-32).

5

25


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Polyelectrolyte complex hydrogels based on alginate and chitin whiskers were successfully fabricated, showing excellent mechanical properties and cytocompatibility to osteoblast cells.

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Polyelectrolyte complex hydrogels based on alginate and chitin whiskers were successfully fabricated, showing excellent mechanical properties and cytocompatibility to osteoblast cells. 41x30mm (300 x 300 DPI)


Effects of Chitin Whiskers on Physical Properties ... - ACS Publications

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