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European Polymer Journal 42 (2006) 3171–3179

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Macromolecular Nanotechnology

A study on the bioactivity of chitosan/nano-hydroxyapatitecomposite scaffolds for bone tissue engineering

Lijun Kong, Yuan Gao, Guangyuan Lu, Yandao Gong,Nanming Zhao, Xiufang Zhang *

Department of Biological Sciences and Biotechnology, State Key Lab of Biomembrane and Membrane Biotechnology,

Tsinghua University, Beijing 100084, China

Received 17 May 2006; received in revised form 25 July 2006; accepted 4 August 2006Available online 2 October 2006

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Abstract

In order to increase the biocompatibility and bioactivity of chitosan, hydroxyapatite had been in situ combined intochitosan scaffolds. The bioactivity of the composite scaffolds was studied by examining the apatite formed on the scaffoldsby incubating in simulated body fluid and the activity of preosteoblasts cultured on them. The apatite layer was assessedusing scanning electronic microscope (SEM), X-ray diffraction (XRD), Fourier-Transformed Infrared spectroscopy(FTIR) and weight measurement. Composite analysis showed that after incubation in simulated body fluid on both ofthe scaffolds carbonate hydroxyapatite was formed. With increasing nano-hydroxyapatite content in the composite, thequantity of the apatite formed on the scaffolds increased. Compared with pure chitosan, the composite with nano-hydroxy-apatite could form apatite more readily during the biomimetic process, which suggests that the composite possessed bettermineralization activity. Furthermore, preosteoblast cells cultured on the apatite-coated scaffolds showed different behav-ior. On the apatite-coated composite scaffolds cells presented better proliferation than on apatite-coated chitosan scaffolds.In addition, alkaline phosphatase activities of cells cultured on the scaffolds in conditioned medium were assessed. The cellson composite scaffolds showed a higher alkaline phosphatase activity which suggested a higher differentiation level. Theresults indicated that the addition of nano-hydroxyapatite improved the bioactivity of chitosan/nano-hydroxyapatite com-posite scaffolds. On the other hand, that is to say composition of substrates could affect the apatite formation on them, andpre-loaded hydroxyapatite can enhance the apatite-coating. It will also be significant in preparation of apatite-coatingpolymer scaffolds for bone tissue engineering.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Bioactivity; Apatite; Mineralization; Simulated body fluid (SBF); Tissue engineering

0014-3057/$ - see front matter � 2006 Elsevier Ltd. All rights reserved

doi:10.1016/j.eurpolymj.2006.08.009

* Corresponding author. Tel.: +86 10 6278 3261; fax: +86 106279 4214.

E-mail address: zxf-dbs@mail.tsinghua.edu.cn (X. Zhang).

1. Introduction

Chitosan is one of the most widely-used naturalpolymers in tissue engineering research. It canbe obtained by partially deacetylating of chitinwhich can be extracted from crustacean. It is a

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polysaccharide composed of glucosamine andN-acetyl glucosamine linked with a b 1-4 glucosidiclinkage. Chitosan is biocompatible and can bedegraded by enzymes in human body, and the deg-radation product is nontoxic [1]. It has been studiedin many biomedical fields, including tissue engineer-ing for bone [2–5], blood vessel [6] and nerve [7–9].However, it is not the ideal material for tissue engi-neering: its bioactivity need to be improved for spe-cific tissue like most of polymers. For theimprovement of the bioactivity on chitosan, it isoften combined to other bioactive materials. As amajor inorganic component of natural bone,hydroxyapatite (HA) is a biomimetic material withgood biocompatibility and bioactivity in bone tissueengineering. Nevertheless, its brittleness makes ithard to shape. Chitosan can be molded in variousforms and can form porous structure with lyophili-zation [10]. In order to improve the bioactivity ofchitosan in bone tissue engineering and get shape-able HA-contained materials, the preparation of achitosan/nano-HA composite scaffolds was reportedelsewhere [2].

For the increase of bioactivity and mechanicalproperty, some composites of polymer and bioac-tive ceramics has been developed for bone tissueengineering [2,11–15]. It was reported that the addi-tion of hydroxyapatite (HA) in the polymer/HAcomposites could improve the activity and viabilityof cells cultured on them [13], or improve both themechanical and cell-attachment properties of thealginate scaffolds [15]. Ma and his coworkers dem-onstrated that HA could impart osteoconductivityinto the highly porous PLLA/HA composite scaf-folds for bone tissue engineering [11] and improvethe protein adsorption capacity [14]. Besides theaforementioned properties, for the bioactive ceram-ics, one of their most important characteristic is atime-dependent kinetic modification of the surfacethat occurs upon implantation: a biological activecarbonate hydroxyapatite layer can form on theirsurface, which is chemically and structurally equiv-alent to the mineral phase in bone and providesan interfacial bonding between materials and tissues[12,16,17]. Consequently, the bioactivity of theseartificial materials can be attributed to the forma-tion of a biologically active bone-like, carbonate-containing apatite layer [18,19].

The process of apatite formation on the bioactivematerials in living body could be reproduced in sim-ulated body fluid (SBF), which means that in vivo

bone bioactivity of a material can be predicted by

assessing apatite formation on its surface in SBF[19,20]. Recently, several studies have made effortsto accelerate the coating process by using higherion concentrations than 2 · SBF. It has beenreported that apatite was formed on metal sub-strates in one day using supersaturated SBF(5 · SBF) [21–23]. As a result, to evaluate thein vitro bioactivity of biomaterials, a concentratedsimulated body fluid was used to accelerate the pro-cess [13,24,25].

In addition, apatite-coating is another method toprepare a surface-active scaffold for bone tissueengineering. It is quick and easy, hence it causesmuch attention [12]. The biomimetic method usingSBF can prepare an apatite layer on a substratewithout using special equipment or extremely highprocessing temperatures. It has numerous advanta-ges compared with other apatite-coating methods[26], including plasma spraying and ion-assisteddeposition. The formation of an apatite coating onmany types of substrates using biomimetic processeshas been reported [21,22,24,25,27]. The propertiesof the apatite layer can affect cell viability and pro-liferation. Even subtle changes in the apatite micro-environment can cause varied cell responses [28].Therefore, re-searching the influence of the sub-strate on the forming of the apatite layer is mean-ingful for the apatite-coating biomaterials study.

In this study, the bioactivity of chitosan/nano-HA and chitosan scaffolds was examined in anin vitro biomimetic process. Influence of nano-HAon bioactivity of chitosan scaffolds was investigated.The influence of substrate component on theformation of apatite was evaluated. Furthermore,MC 3T3-E1 cells were inoculated on the apatitelayer formed on the two kinds of scaffolds and theimpact of apatite layer formation on different sub-strates on cell proliferation and differentiation wasinvestigated.

2. Materials and methods

2.1. Materials

Chitosan was obtained from Haisheng Co.(Qingdao, China). The degree of deacetylation wasestimated to be 93.5% using the 1H-NMR methodand the viscosity-average relative molecular weightwas 1.8 · 106 Da. Ca(NO3)2, (NH4)2HPO4 and allthe inorganic salts for SBF were of analytical gradeand were purchased from Beijing Chemical Engi-neering Factory (Beijing, China).

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2.2. Methods

2.2.1. Preparation of chitosan/nano-HA composite

scaffoldsPorous chitosan/nano-HA composite scaffolds

were made as previously described [2]. Briefly,Ca(NO3)2 solution and (NH4)2HPO4 solution wereslowly added into a 2% chitosan acetic acid solu-tion, mixed thoroughly and then frozen at �20 �C.After lyophilization and neutralization in NaOHsolution, the scaffolds were rinsed with deionizedwater in order to remove any remaining NaOH.Chitosan scaffolds were made with similar processexcept for the addition of those inorganic salt solu-tions. The porous structures of the two kinds ofscaffolds were similar (Fig. 1) and their porositieswere nearly 95% and pore diameter was mainlyabout 20–60 lm [2]. Nano-HA particles of about70–100 nm could be observed in the microscopicmorphologies with original magnification of 5000·(Fig. 3). Since the similarity of their porous struc-

Fig. 1. Porous structure of chitosan and chitosan/nano-HAcomposite scaffolds. (a) chitosan scaffold; (b) chitosan/nano-HAcomposite scaffold with 12% of HA. Original magnification: 80·.

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ture and HA particles size, 12% HA content com-posite scaffold was selected as a representativesample for the further analysis and comparison withchitosan scaffold.

2.2.2. Preparation of 5 · SBFSBF containing nearly five times the inorganic ion

concentration of human blood plasma was preparedby dissolving NaCl, NaHCO3, KCl, K2HPO4 Æ3H2O, MgCl2 Æ 6H2O, Na2SO4, and CaCl2 in deion-ized water. Solution pH value was adjusted withHCl before the addition of CaCl2 to prevent a precip-itate from forming. The final pH of the solution was6.5. The concentration of SBF were 710.0 mM Na+,12.7 mM Ca2+, 7.7 mM Mg2+, 25.0 mM K+,739.7 mM Cl�, 5.0 mM HPO2�

4 , 21.0 mM HCO�3and 2.5 mM SO2�

4 [27].

2.2.3. In vitro mineralization process

Disc-like chitosan/nano-HA composite scaffoldsspecimens with a 14 mm diameter and 2 mm inthickness were immersed in 50 ml of 5 · SBF in aplastic bottle in a shaker bath set at 60 rpm at37 �C. After incubation for 36 h, the specimens wereremoved from the 5 · SBF solution, washed care-fully with deionized water to remove soluble inor-ganic ions, and then lyophilized. The chitosanscaffolds (14 mm diameter, 2 mm thickness) weretreated as the composite scaffolds.

2.2.4. Weight measurement

The mass change of the samples before and afterincubation in 5 · SBF for 36 h was measured byusing an analytical balance (accuracy: 0.1 mg). Allthe samples were freeze-dried before weighing. Sixspecimens were measured for each kind of scaffoldsto obtain an averaged result.

2.2.5. Scanning electron microscopy (SEM)

examination

After incubation in 5 · SBF for 36 h and washingwith deionized water, the samples were lyophilizedand cut with a razor blade to expose the inner sur-faces. After being coated with gold in a sputteringdevice, the scaffolds were examined with a scanningelectron microscope (KYKY-2800, Apparatus Fac-tory, Chinese Academy of Sciences, Beijing, China)with an accelerating voltage of 20 kV.

2.2.6. X-ray diffraction (XRD)

To investigate the components of the apatitelayer, the samples were analyzed using an X-ray

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powder diffractometer (D8 Advance, Bruker,Germany) with monochromatic Cu Ka radiation.

2.2.7. Fourier-transformed infrared spectroscopy

(FTIR)

A PerkinElmer system 2000 FTIR spectrometer(PerkinElmer, Norwalk, CT) was used for FTIRanalysis. The spectra were collected over the rangeof 4000–400 cm�1.

2.2.8. Cell culture

MC 3T3-E1 cells were cultured in a-MEM(Gibco, Grand Island, NY) supplemented with10% fetal bovine serum (FBS) (National HyClone(Lanzhou) Bio-Engineering Co., China), 100 U/mlpenicillin (Sigma, St. Louis, MO), and 100 lg/mlstreptomycin (Sigma). Cells were incubated at37 �C in a 5% CO2 incubator and the medium waschanged every 2 days. After the cells reached conflu-ence, they were harvested by trypsinization followedby the addition of fresh culture medium to create acell suspension. All the scaffolds after incubation in5 · SBF were put in 24 well culture dishes. 500 ll ofcell suspension containing 6 · 104 cells was seededon the scaffolds. Another 500 ll fresh culture med-ium was then added to each scaffold after about6 h. Culture medium was changed every two days.

2.2.9. Cell morphology

Acridine orange (AO) was used to stain the cellson the apatite-coated scaffolds. AO is a cationicfluorescent dye that interacts with DNA by interca-lation or electrostatic attraction [29]. It is commonlyused in cell morphology examination [29–31]. Thesamples were stained in 0.01% AO solution for10 min in the dark. After being rinsed with phos-phate buffered saline solution (PBS) to remove thedye adsorbed on the scaffolds, the samples wereexamined using a confocal laser scanning micro-scope (CLSM) (Olympus, Japan) to investigate thecells morphologies. The emission wavelength ofthe AO used in this research was 488 nm [31].

2.2.10. Cell proliferationA modified MTT (3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide) assay was appliedto quantitatively assess the number of viable cellsattached and grown on the scaffolds [2]. Briefly, allthe cell-loaded scaffolds were placed in a new 24-well flat-bottom culture plate after cultured for 12days, and 1 ml of serum-free medium with 100 llof MTT (Sigma) solution (5 mg/ml in PBS) was

added to each sample. After the samples were incu-bated at 37 �C for 4 h to allow MTT formazan for-mation, the upper solvent was removed and 1 ml of10% sodium dodecyl sulfate (Sigma) in 0.01 N HClwas added to dissolve the formazan crystals for 6 hat 37 �C. During the dissolving period, the spongyscaffolds were squeezed every 30 min to ensure thecomplete extraction of formazan crystals. The opti-cal density (OD) at 570 nm was determined againsta sodium dodecyl sulfate solution blank. Three par-allel replicates were read for each sample and t-testwas used for the statistical analysis.

2.2.11. Cell differentiation

Scaffolds with cells were cultured in conditionmedium consisting of 90% a-MEM containing a50 lg/ml concentration of L-Ascobric acid 2-phos-phate, 10 mM b-glycerophosphate disodium saltand 10% FBS, and culture medium was changedevery 2 days. After cultured for 21 days, alkalinephosphatase (ALP) activities of cells cultured inun-conditioned medium (a-MEM medium with10% FBS) and cells cultured in conditioned mediumwere evaluated with ALP assay kit (ZhongshengBeikong Bio-Technology and Science Inc. (China)).

3. Results and discussion

3.1. Quantity assessment

For the reason of mechanical properties, the HAcontent of the scaffolds in this study was controlledin the range of 0–14%. The higher HA content in thescaffold, the less elastic property was found (dataare not published). The dry weight of the scaffoldswas measured before and after the incubation andthe percentage of weight increase was calculated asfollows:

P ¼ ðW a � W bÞ=W b � 100% ð1Þ

P percentage of weight increase,Wa weight of sample after incubation in

5 · SBF,

Wb weight of sample before incubation in5 · SBF.

The weight increase of the composite scaffoldsafter incubation in 5 · SBF was significantly higherthan that of chitosan scaffolds. The percent ofweight increase exhibited a trend of increasingalong with the nano-HA content in the scaffolds(Fig. 2). On the composite scaffolds, more apatite

Fig. 2. Percentage of weight increase with increasing HA contentin the substrate scaffolds. The scaffolds were incubated in 5 · SBFat 60 rpm, 37 �C for 36 h and the dry weight of the samples beforeand after incubation was evaluated. Data represent the mean± SD of six samples. One-way ANOVA was used for thestatistical analysis. * p < 0.01 compared with apatite on chitosanscaffolds.

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was formed and the amount of apatite formedincreased with the increasing of HA content intoit. The results indicated that the addition ofnano-HA enhanced the mineralization bioactivityof the scaffolds. The mechanism of apatite forma-tion in SBF has been described by many research-ers [16,32]. It has been reported that the formationof apatite on artificial materials is induced by func-tional groups which could reveal negative chargeand further induce apatite via the formation ofamorphous calcium phosphate. In this researchthe major reason of enhancement of apatite forma-tion on the composite scaffolds might be the nano-HA particles acted as nucleation initiation sites.The more nano-HA content in the composite scaf-folds, the more nucleation initiation sites existed,as a result the more apatite was formed faster.Once the apatite nuclei are formed, they can growspontaneously by consuming the calcium andphosphate ions present in the surrounding fluid[33].

3.2. Morphology examination

The morphology of chitosan and composite scaf-folds with 12% of nano-HA before and after incuba-tion in SBF was evaluated using SEM. On both thetwo kinds of scaffolds which were incubated in SBF,

apatite was formed. The size of the apatite particlesformed on the chitosan scaffolds was larger thanthat of the particles on the composite scaffolds. Nev-ertheless, the amount of apatite formed on thechitosan scaffolds was less than that of apatite onthe composite scaffold. This result is also supportedby the results of the weight increase testing. On thecomposite scaffolds the apatite particles were den-ser, as shown in Fig. 3.

SBF is a metastable calcium phosphate solutionsupersaturated with respect to apatite. It is reportedthat the barrier for homogeneous nucleation of apa-tite is too high and a stimulus is required to inducethe heterogeneous nucleation of apatite from theSBF. On the chitosan/nano-HA composite scaffoldsthere were many nano-HA particles which could actas nucleation sites. As a result, apatite could beformed more efficiently on the composite scaffoldsthan on the chitosan scaffolds. Therefore, in thesame interval of time, more apatite was depositedon the composite than on the chitosan alone. Witha limited amount of inorganic ions in the local min-eralization microenvironment, the more nuclei thatwere formed, the less amount of minerals that couldbe aggregated on each nucleus. Consequently eachparticle formed on the composite scaffold was smal-ler than on the chitosan scaffold (Fig. 3).

3.3. Compositional analysis

The composition of the apatite layer on the twokinds of scaffolds was analyzed with XRD andFTIR. Composite scaffold with 12% nano-HA wasselected as a representative sample of the composite.The XRD patterns of the two kinds of scaffoldsafter incubation in 5 · SBF are shown in Fig. 4.After incubation in 5 · SBF, the scaffolds showedboth the characteristic peaks of chitosan (20.305�,29.96�) and the characteristic peaks (25.8�, 31.7�and 48.5�) of carbonate hydroxyapatite (Fig. 4).From Fig. 4 it can be concluded that on both ofthe scaffolds an apatite layer was formed. The peaksof the chitosan/nano-HA composite after incuba-tion were sharper and stronger, which suggestedthat, on chitosan/nano-HA composite scaffolds,apatite with higher levels of crystallinity wasformed. The FTIR spectrum of the chitosan andchitosan/nano-HA composite after incubation in5 · SBF showed the presence of PO3�

4 and hydroxylalong with a CO2�

3 absorption band (Fig. 5). Com-bined with the results of XRD, it can be deducedthat the induced apatite was a carbonated HA.

Fig. 3. Morphology of scaffolds before and after incubation in 5 · SBF at 37 �C, 60 rpm for 36 h, examined with SEM. Originalmagnification 5000· (a) chitosan scaffold before incubated in 5 · SBF; (b) chitosan scaffold after incubated in 5 · SBF; (c) compositescaffold with 12% nano-HA content before incubated in 5 · SBF; (d) composite scaffold with 12% nano-HA content after incubated in5 · SBF.

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3.4. Biocompatibility examination

Impacts of apatite layer formed on the two kindsof scaffolds on cell viability were investigated. Thedifference of substrate scaffolds caused differenceof apatite coating on quantity and topology, andfurther affected the cell growth and proliferation.In this study, the compatibility between bone-forming cells and the chitosan/nano-HA compositescaffolds was evaluated with MC 3T3-E1, a preos-teoblast cell line derived from newborn mouse cal-varia. The cell morphology was observed withCLSM after staining the cells with AO (Fig. 6). Itcan be seen that the cells on the apatite layer formedon composite scaffolds were distributed in clustershaving more cells than those on the apatite layer

formed on chitosan scaffolds. Cell proliferationwas examined with an MTT assay. Compared withthe apatite-coated chitosan scaffolds, the viability ofcells cultured on the apatite-coated composite scaf-folds was higher (Fig. 7). This can also be seen fromthe results of AO staining. It has been reported thatsurface roughness, topography in micro-scale [34–37] and nano-scale [38,39] can influence cell mor-phology and proliferation. Wu et al. have alsoreported the influence of biomimetic apatite struc-ture on osteoblast viability and proliferation [28].In that study the coating process influenced thestructure of apatite formed and further caused dif-ferent cell responses. In this study, the componentsof the substrates affected the structure of the biomi-metic apatite layer formed on them: topography and

Fig. 5. FTIR spectrum of chitosan and composite scaffolds afterincubated in 5 · SBF; Composite-SBF: composite scaffolds with12% of nano-HA after incubated in 5 · SBF; chitosan-SBF:chitosan scaffold after incubated in 5 · SBF.

Fig. 6. AO staining of MC 3T3-E1 cultured on scaffolds afterincubated in 5 · SBF, examined with CLSM. (a) chitosanscaffold; (b) chitosan/nano-HA composite scaffold with 12% ofnano-HA.

Fig. 4. XRD patterns of chitosan scaffolds and compositescaffolds after incubation in 5 · SBF. composite-SBF: compositescaffolds with 12% of nano-HA after incubated in 5 · SBF;chitosan-SBF: chitosan scaffold after incubated in 5 · SBF. *:characteristic peaks of carbonate hydroxyapatite; #: characteris-tic peaks of chitosan.

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quantity, which further affected the viability andproliferation of cells seeded on the apatite layer.The results indicated that not only the coating pro-cess [28] but also the component of substrate couldhave an effect on the apatite structure and furtheraffect the cells responses, which would be instructivein research of preparation of biomimetic apatite-coating polymer scaffolds.

ALP activity is one of the characteristic parame-ter of osteoblast cells differentiation. ALP activitiesof cells cultured in un-conditioned or conditionedmedium were examined respectively. After cultured

in conditioned medium, the ALP activities of cellson composite scaffolds increased significantly com-pared with cultured in un-conditioned medium.And the ALP activity of cells on composite scaffoldswas much higher than that of cells on chitosan scaf-folds after cultured in conditioned medium. TheALP activity assessment indicated that cells on thecomposite scaffolds showed higher differentiationlevel than chitosan scaffolds (Fig. 8). It was sug-gested that the composite scaffolds with nano-HAparticles had better bone bioactivity. The additionof nano-HA could develop the properties of chito-san to fit bone tissue engineering.

Fig. 7. The MTT assay of cells cultured on chitosan andchitosan/nano-HA composite scaffolds with 12% of nano-HAcontent after incubated in 5 · SBF. All the cells were cultured onthe scaffolds for 12 days. Data represent the mean ±SD of threesamples. * p < 0.05: compared with chitosan scaffolds afterincubation at the same culture time.

Fig. 8. ALP activity of cells cultured on the scaffolds for 21 days.Un-chi: cells cultured on chitosan scaffolds in un-conditionedmedium; Un-com: cells cultured on composite scaffolds with 12%of nano-HA content in un-conditioned medium; chi: cellscultured on chitosan scaffolds in conditioned medium; com: cellscultured on composite scaffolds with 12% of nano-HA content inconditioned medium. Data represents the mean ±SD for threesamples. * p < 0.05.

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4. Conclusion

An accelerated process of biomineralization withconcentrated SBF was used in the assessment of bio-activity of biomaterials and studying the influencesof substrate composition on apatite coating whichwas used in preparation of biomimetic materials.The chitosan/nano-HA composite scaffolds showed

better biomineral activity than chitosan scaffolds. Inthe composite scaffolds, nano-HA particles pro-vided nuclei in the mineralization process. As aresult more apatite formed on the composite scaf-folds than on the chitosan scaffolds. The additionof nano-HA influenced the composition of the apa-tite layer. The results also suggested that nano-HAcould enhance the coating of apatite layer on bio-materials, which could be used to produce apatite-polymer composite scaffolds. Furthermore, it wasshown that the structure of the apatite layer alsoinfluenced the viability and differentiation of preos-teoblast cells. The apatite formation and cells viabil-ity reflected the properties of the substrate: thechitosan/nano-HA composite showed a higher bio-activity for bone tissue engineering. The existenceof nano-HA particles could increase the amountof apatite coating and influence the topography,and further increase the proliferation and differenti-ation of cells. It could be concluded that the addi-tion of nano-HA to chitosan scaffold improved itsbone bioactivity, which could develop the use ofchitosan in bone tissue engineering.

Acknowledgements

This research was supported by the NationalBasic Research Program (also called ‘‘973’’ Project)of China (No. 2005CB623905) and the Tsinghua-Yue-Yuen Medical Science Fund. The authors alsoacknowledge the support provided by the professorsin the Analysis Center Tsinghua University and allthe colleagues in our laboratory.

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