Journal ofMaterials Chemistry B

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aKey Laboratory of Advanced Technologies o

of Materials Science and Engineering, S

610031, Sichuan, P. R. ChinabSchool of Life Science and Engineering,

610031, Sichuan, P. R. ChinacDepartment of Biochemistry and Molecula

Medicine, Sichuan University, Chengdu 610dSichuan Centre for disease control and pr

E-mail: Jwang@swjtu.edu.cn; J.wang63@gm

† Electronic supplementary informa10.1039/c3tb20552e

Cite this: J. Mater. Chem. B, 2013, 1,4484

Received 18th April 2013Accepted 2nd July 2013

DOI: 10.1039/c3tb20552e

www.rsc.org/MaterialsB

4484 | J. Mater. Chem. B, 2013, 1, 44

Synthesis of an RGD-grafted oxidized sodium alginate–N-succinyl chitosan hydrogel and an in vitro study ofendothelial and osteogenic differentiation†

Xia Liu,ab Wenzhen Peng,c Yingying Wang,a Minghua Zhu,d Tao Sun,d Qiang Peng,d

Yi Zeng,d Bo Feng,a Xiong Lu,a Jie Wenga and Jianxin Wang*ab

Hydrophilic surfaces for hydrogels as bone tissue engineering scaffolds are not beneficial for the adsorption of

protein and not conducive to the adhesion and growth of cells. In this study, we proposed to use an oxidized

sodium alginate–N-succinyl chitosan hydrogel as a bone tissue engineering scaffoldmaterial and to overcome

this issue by using RGD to modify this kind of hydrogel. The physicochemical properties of the obtained

hydrogels were characterized and an in vitro endothelial differentiation and osteogenic differentiation

study of bone-marrow-derived mesenchymal stem cells (BMSCs) was conducted to evaluate it. The results

showed that the RGD-grafted oxidized sodium alginate–N-succinyl chitosan hydrogel not only had a good

degradability but also enhanced cell adhesion and proliferation and promoted endothelial differentiation

and osteogenic differentiation of BMSCs. Based on the results, it can be expected that RGD-grafted

oxidized sodium alginate–N-succinyl chitosan hydrogel might be an optimal material for bone tissue

engineering scaffold whenever it is used alone, or composed with other materials in the future.

1 Introduction

In bone tissue engineering, 3D porous scaffolds canmaintain theoriginal shape of the organization, provide the places of board-ing, growth, proliferation and differentiation for cells, and guidethe regeneration of damaged tissue and control the structure ofthe regeneration of tissue.1–9 However, the research in this area isstill facing many problems such as material degradation, releaseof growth factors, formation of new bone and vascularization.10–15

Among them, the level of formation of new bone and vasculari-zation in bone tissue engineering scaffold implants is consideredas a critical factor for bone tissue engineering.16–19 The poorvascularization oen leads to necrosis of newly formed bone inthe central parts of the implants and thus to the failure inrepairing bone defect.19–21 Both synthetic and natural materialshave been explored as potential scaffolds for bone regeneration,among which, hydrogels, due to their unique properties such asgood biocompatibility, extracellular matrix-like structures, range

f Material, Minister of Education, School

outhwest Jiaotong University, Chengdu

Southwest Jiaotong University, Chengdu

r Biology, College of Basic and Forensic

041, P. R. China

evention, Chengdu 610041, P. R. China.

ail.com

tion (ESI) available. See DOI:

84–4492

of constituents and desirable degradability, have been consid-ered as good scaffoldmaterials that provide structural integrity totissue constructs, control drug and protein delivery to tissues andcultures.22–24 However, hydrophilic surfaces for hydrogels are notbenecial for the adsorption of protein and not conducive to theadhesion and growth of cells and in turn to formation of newbone and vascularization.25,26 In this study, we proposed anapproach using an RGD-graed oxidized sodium alginate–N-succinyl chitosan hydrogel as a bone tissue engineering scaffoldto overcome this issue. An in vitro endothelial and osteogenicdifferentiation study of bone-marrow-derivedmesenchymal stemcells (BMSCs) would be conducted to evaluate it.

2 Materials and methods2.1 Materials

Sodium alginate (SA,Mw: 612 kDa) and chitosan (CS,Mw: 209 kDa)were purchased from the Maichao Chemical Reagent (Shanghai,China) and Aokang Biological Technology Co., Ltd (Shandong,China), respectively. All the other chemicals and solvents werepurchased from the Kelong Chemical Reagent Factory (Chengdu,China) as reagent grade or better, and used without furtherpurication.

2.2 Preparation of oxidized sodium alginate (OSA)

Sodium alginate (5.0 g) was added to distilled water (250 mL)and stirred until it dissolved completely to form a 2% (w/v)solution. Then it was mixed with weighed sodium periodate

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according to the requiredmolar ratio of sodium periodate to thenumber of repetitive units of alginate (n(NaIO4)/n(SA) ¼ 0.2, 0.4,0.6, 0.8, 1.0) under stirring at room temperature in the dark. Theinvestigated oxidation reaction time points were 2 h, 4 h, 6 hand 8 h. The reaction was stopped by adding ethylene glycol(2 mL) over 0.5 h, followed by precipitation using an excessamount of ethyl alcohol (400 mL). The precipitates werecollected by centrifugation and then re-dissolved in distilledwater (50 mL) and subsequently dialyzed (MWCO 7000) againstdistilled water, ltered, and lyophilized.

The degree of oxidation (DO, %) of the OSA was dened as themolar ratio of the oxidized guluronate to the repetitive units ofalginate, and was determined by the method described byWu et al.27

2.3 Preparation of RGD-graed oxidized sodium alginate

Oxidized sodium alginate (OSA, 0.10 g) was dissolved indistilled water (1 mL) and then RGD (4mL) with a concentrationof 1 mg mL�1 was added under stirring. The reaction for theimmobilization of RGD was carried out at room temperature inthe dark for 36 h. The nal product was lyophilized.

2.4 Preparation of N-Succinyl chitosan (N-SC)

Chitosan (2.0 g) was dissolved in 80 mL HAc aqueous solutionwith a concentration of 1 wt% and then transferred into a reac-tion ask. Succinic anhydride (0.20 g) was dissolved in acetone(20 mL), and added drop-wise to the reaction ask under intensemechanical stirring for 30 min. Aer standing overnight at roomtemperature, the precipitates were collected by centrifugationand then soaked in ethyl alcohol. The pH value of the system wasadjusted to 10–12 using 5% w/v aq. NaOH and it was re-precipi-tated in an excess of acetone, ltered and then rinsed withethanol. Finally, the precipitate was dissolved in water and thesolution was subsequently dialyzed (MWCO 7000), ltered, andlyophilized. The degree of substitution (DS) which is the substi-tute number of hydroxyl groups or amino groups per glucos-amine unit in the N-succinyl chitosan was measured using thetitration method as described in ref. 28.

2.5 Preparation of hydrogels

The RGD-oxidized sodium alginate (RGD–OSA)–N-succinyl chi-tosan (N-SC) hydrogels were prepared using a Schiff base reactionunder aseptic condition. 1.0 g of sterilized N-succinyl chitosan(N-SC) was dissolved in 40 mL of a sterilized phosphate buffersolution (PBS, pH ¼ 7.4). Similarly, 2.0 g of sterilized RGD-oxidized sodium alginate (RGD-OSA) was dissolved in 20 mLsterilized phosphate buffer solution (PBS, pH ¼ 7.4). The steril-ized N-SC and RGD-OSA solutions were mixed under vigorousstirring. Subsequently, the mixed solution turned into a highlyviscous liquid, which nally formed a gel within a fewminutes at37 �C.

2.6 Characterization

The analysis of the material composition was performed using aFourier transform-infrared (FT-IR) spectrometer (Spectrum One,

This journal is ª The Royal Society of Chemistry 2013

Perkin Elmer, Norwalk, USA). 2 mg of each tested sample wascarefully mixed with 200 mg of KBr (infrared grade), ground andthen pressed into discs for the measurements. The spectra wererecorded from 500 to 4000 cm�1 wavenumber at a 4 cm�1 reso-lution, averaging 64 scans. The surface topographies of thelyophilized hydrogels were observed by scanning electronmicroscopy (SEM, Quanta 200, Philips, Netherlands). Thehydrogels were lyophilized at �80 �C and a gold layer was coatedon the hydrogel specimens' surface (1 cm � 1 cm). Uniaxialcompression was performed to measure the mechanical prop-erties of the hydrogels with an Instron 5567 mechanical tester(Instron Corporation, USA). Different ratios of the hydrogelsamples were moulded to be cylindrical, nominally 15 mm indiameter and 30 mm in height, to obtain at and parallelsurfaces for mechanical testing. The cylindrical probe (100 N) ofthe universal testing machine was placed on the surface of themeasured hydrogel, and then dropped down at a constantvelocity of 0.5 mm min�1. Three specimens were tested for eachsample. The averages and standard deviations were reported. Thedegradation of the hydrogels was performed in a conical asklled with 10 mL PBS (pH ¼ 7.4). The degradation experimentswere conducted by incubating the hydrogels in a thermostaticbath at 37 �C and by determining the weight loss aer recovery ofthe samples at predetermined time intervals. At the investigatedtime points, the samples were removed from the PBS mediumand then the medium on the surface of hydrogels was sucked upgently with a piece of lter paper before being weighed. Thedegradation was assessed by measuring the weight ratio (%),which was dened with the following expression:

Weight ratio (%) ¼ W2/W1 � 100%

here W1 and W2 are the weight of the gels before and aerdegradation, respectively.

2.7 Cell culture

The investigated hydrogels were coated on glass discs using aspinning coater. All the hydrogel-coated glass discs for cellculture were immersed in 75% ethanol for 2 h, followed byrinsing three times with PBS, before the cell culture. All thehydrogel-coated glass discs were put in 24-well plates for the cellculture.

2.7.1 Cell sourcing and seeding. Rat bone marrow mesen-chymal stem cells (rBMSCs) were obtained from 15 days oldnewborn rats using a whole bone marrow culture method. Thebone marrow cavities of the rats were ushed with a completemedium. Then, bone marrow cell suspensions from the bonemarrow cavities of rats were collected and cultured. Themedium was replaced with fresh medium every 3 days toremove the hematopoietic cells, and by controlling the passagetime, the rBMSCs were puried. The cells were planted on thehydrogel-coated glass discs at a density of 5 � 104 cells for eachwell in the 24-well plates, in a standard growth medium for upto 3 days.

2.7.2 Cell viability. The cell viability was determined bymeans of the Alamar blue assay as specied by the manufac-turer (Biosource, Nivelles, Belgium). The proliferation was

J. Mater. Chem. B, 2013, 1, 4484–4492 | 4485

Fig. 1 FT-IR spectra of (a) SA and oxidized SA, (b) oxidized SA and RGD-graftedoxidized SA; effect of the ratio of n(NaIO4)/n(SA) (c) and reaction time (d) on thedegree of oxidation of oxidized sodium alginate.

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indicated by measuring the reduction of resazurin to resorunas an indicator of the metabolic state of the cells. Cellulargrowth causes the reduction–oxidation indicator to changefrom an oxidized (non-uorescent, blue) to a reduced (uores-cent, red) form.29 Aer cell culture for 1, 3, 7 days, the mediumwas carefully removed and 300 mL of Alamar blue solution (10%Alamar blue, 80% media 199 (Gibcos) and 10% FBS; v/v) wasadded to each well and incubated for a further 3 h at 37 �C,under 5% CO2. A hydrogel without cells was used as the blankcontrol. A sample of 200 mL reduced Alamar blue solution waspipetted into a Costar opaque black bottom 96-well plate(Sigma) and read at 570 nm (excitation)/600 nm (emission) in anELISA microplate reader (Molecular Devices, Sunnyvale, CA).The results were presented as the mean � standard deviation,and the experiments were performed in triplicate.

Dual-uorescence viability, using acridine orange (AO) andpropidium iodide (PI), were employed for the accurate viabilityanalysis of the primary cells in our study. AO is permeable toboth live and dead cells and stains all nucleated cells togenerate a green uorescence, whilst PI enters dead cells withcompromised membranes and stains all dead nucleated cells togenerate a red uorescence. So, all live nucleated cells uorescegreen and all dead nucleated cell ones red.

Aer cell culture for 7 days, the cells were incubated withacridine orange (AO) and propidium iodide (PI) (both 5 mM) at37 �C for 10 min. The supernatant medium and excess dye wereremoved by washing three times with PBS, and then the cellswere photographed using an Olympus uorescencemicroscope,equipped with a digital camera.

2.7.3 In vitro osteogenic and endothelial differentiation ofrBMSCs. The cell culture was conducted in triplicate for eachsample. The hydrogel-coated glass discs were immersed in 75%alcohol for 2 h, and rinsed three times with PBS. The rBMSCswere digested, diluted into a cell suspension and then seededon the samples at a density of 5 � 104 cells for each well. Basisculture medium (L-DMEM + 10% FBS) (fatal bovine serum) wasadded aer the cells were seeded. The cells were cultured for24 h before replacing the medium.

Aer that, the medium was replaced with a different induc-tion media for each differentiation, for example, a boneinductionmedium containing H-DMEM + 10% FBS, 10 ngmL�1

BMP-2 (PeproTech, Rocky Hill, NJ, USA), 100 nM dexametha-sone (Sigma), 50 ng mL�1 ascorbic acid (Sigma), and 10 mMb-glycerophosphate (Sigma) was used for osteogenic differenti-ation, whilst a medium containing 10 ng mL�1 VEGF165(PeproTech, Rocky Hill, NJ, USA), 5 ng mL�1 bFGF (PeproTech,Rocky Hill, NJ, USA), and 2 mM L�1

L-glutamine (Sigma), 100 UmL�1 penicillin/streptomycin was used for endothelial differ-entiation. Control groups without BMP-2, VEGF165 and bFGFwere also included for the cell differentiation. The medium wasreplaced every 2 days. During the experiment, the system wasmaintained at 37 �C � 1 �C in humidied air, with 5% CO2.

On days 7, 14 and 21 aer being seeded, the examination ofthe osteogenic differentiation of the rBMSCs by monitoring theactivity of alkaline phosphatase (ALP) was conducted both byusing an alkaline phosphatase kit (Jiancheng BioengineeringInstitute, Nanjing, China) and by staining with a BCIP–NBT

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alkaline phosphatase kit (R&D Systems, Minneapolis, MN, USA)according to manufacturer's instruction. In the meanwhile, amineralization of osteogenic differentiation was analyzed byvon Kossa staining according to the protocol described previ-ously.30,31 Before staining, the cells were rinsed twice using PBSand then xed with 4% paraformaldehyde (Sinopharm Chem-ical Reagent Co., Ltd, Beijing) for 20 min at room temperature.

On day 21 aer seeding, the endothelial differentiation wasexamined by immunouorescence staining for the primaryantibodies: CD31 (platelet–endothelial cell adhesion molecule[PECAM]-1, 1 : 50, Sigma, USA), von Willebrand factor (vWF;1 : 100, Sigma, USA), uorescein isothiocyanate (FITC)-conju-gated goat-anti-rabbit immunoglobulin G (IgG) (1 : 100, BeijingZhongshan Glodenbridge Biotechnology Co., Ltd.) forsecondary antibody, and 2-(4-amidinophenyl)-6-indolecarba-midine dihydrochloride (DAPI) (Sigma, USA) for the nuclei.Finally, the cells were photographed using an Olympus uo-rescence microscope equipped with a digital camera and ImagePro-plus.

2.8 Statistical analyses

Three replicates were analyzed for each group. In the analysis ofthe quantitative data, a one way ANOVA combined with theStudent's t-test was used to detect statistically signicantdifferences at a signicance level (or p-value) of p < 0.05. Theresults are reported as the means � the standard deviations.

3 Results and discussion3.1 Characterization of RGD-graed oxidized sodiumalginate

3.1.1 Oxidation of sodium alginate. In order to understandthe oxidation of sodium alginate, FT-IR analysis was done.Fig. 1(a) shows the FT-IR spectra of the SA and oxidized SA.Compared with the SA, the oxidized SA presented a new char-acteristic peak at 1727 cm�1, which was assigned to an aldehydegroup C]O bond, indicating that the SA has been successfully

This journal is ª The Royal Society of Chemistry 2013

Fig. 2 (a) FT-IR spectra of chitosan (black line) and N-succinyl chitosan (red line);(b) effect of the anhydride/amino molar ratio on the degree of substitution of N-succinyl chitosan.

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oxidized by the NaIO4.32 The aldehyde groups formed in thealginate structure would be used for the immobilization of theRGD on the sodium alginate and for the Schiff base reactionbetween the oxidized sodium alginate and N-succinyl chitosanduring the formation of the hydrogels.33

3.1.2 Degree of oxidation. The degree of oxidation for theoxidized alginate was determined by the amount of sodiumperiodate and the reaction time. As shown in Fig. 1(c), duringthe same reaction time, the degree of oxidation of the sampleswas increased markedly with increasing the concentration ofthe NaIO4, however, when the ratio of n(NaIO4)/n(SA) was morethan 0.6, the degree of oxidation would never change signi-cantly. Moreover, increasing the reaction time could alsoincrease the degree of oxidation of the oxidized SA. The highestdegree of oxidation was obtained when the reaction time was 4hours. Aer that, a further increase in the reaction time wouldallow the degree of oxidation to drop down as shown inFig. 1(d). The reason may be due to a longer reaction timeleading to the degradation of the partially oxidized alginate insolution.

3.1.3 Preparation of RGD-graed oxidized sodium algi-nate. The aldehyde groups on the oxidized SA reacted withamino groups on the peptides, with the formation of C]N, sothat the RGD sequence could be immobilized on the oxidizedSA, aiming at enhancing the adhesion and function of the cellsseeded on the hydrogels.

Fig. 1(b) shows the FT-IR spectra of the oxidized SA and RGD-immobilized oxidized SA. For the oxidized SA (black line), apeak at 1727 cm�1 can be clearly seen, which corresponds to theprimary C]O characteristic peak whilst for the RGD-immobi-lized oxidized SA, the peak at 1727 cm�1 disappeared whilst anew peak at 1628 cm�1 appeared instead, indicating that theC]N bonds have been formed between the oxidized SA and theRGDs, thus suggesting that the RGD was successfully graedonto the oxidized SA.34

3.2 Characterization of N-succinyl chitosan

3.2.1 FT-IR spectra analysis. FT-IR spectra of chitosan(black line) and N-succinyl chitosan (red line) are shown inFig. 2(a). The FT-IR spectrum for chitosan showed the distinc-tive absorption bands at 3350–3500 cm�1 (the combination ofstretching of the –OH and –NH2), 1650 cm�1 (amide I), 1565cm�1 (–NH2 bending) and 1415 cm�1 (amide III). The absorp-tion bands at 1155 cm�1 (asymmetric stretching of the C–O–C),1075 and 1033 cm�1 (skeletal vibration involving the C–Ostretching) were characteristic of its saccharine structure.Compared with the FT-IR spectrum of chitosan, the absorptionbands of stretching in the IR spectrum, of the –OH and –NH2

(3434 cm�1) of N-succinyl chitosan became broader and shiedto a lower wavenumber. The intensity for the peaks at 3400 and1599 cm�1 (amino group characteristics) decrease greatly,whilst that for the peaks at 2924 cm�1 (stretching of –CH2–) andthe peak at 1650 cm�1 (amide I) and 1415 cm�1 (amide III)increased. A new absorption band at 1735 cm�1 correspondingto the symmetric stretching of the C]O in the –COOH groupcould be observed. The increased intensity of the peaks at

This journal is ª The Royal Society of Chemistry 2013

1650 cm�1 (amide I) and 1565 cm�1 (–NH2 bending) suggestedthat the amino groups of chitosan have been substituted,indicating that the succinyl introduction reaction took place atthe N-position and –NH –CO– groups have been formed.35,36

3.2.2 The degree of substitution of N-succinyl chitosan.The degree of substitution (DS) was the substituted number ofhydroxyl groups or amino groups per glucosamine unit in the N-succinyl chitosan. The anhydride/amino molar ratio, which canbe described as the molar ratio of succinic anhydride toN-succinyl chitosan, had a noticeable inuence on the DS of theN-succinyl chitosan. From Fig. 2(b), it can be seen that the DSincreased when the anhydride/amino molar ratio increasedfrom 0.5 to 3. The DS became at when the ratio was more than3, indicating that the number of hydroxyl groups or aminogroups which can be substituted has been depleted.

3.3 Characterization of RGD-graed oxidized sodiumalginate–N-succinyl chitosan composite hydrogels

3.3.1 FT-IR spectra analysis. Fig. 3(a) shows the FT-IRspectra of RGD-OSA, N-SC and the hydrogel (V(N-SC)/V(OSA) ¼ 8/2).It can be seen that the characteristic absorption peaks of RGD-oxidized alginate and N-succinyl chitosan at 1682 cm�1, 1735cm�1, 1650 cm�1, and 1565 cm�1 disappeared in the alginate–N-succinyl-chitosan composite hydrogel. A new absorptionband at 1640 cm�1 corresponding to the stretching vibration ofthe C]N groups could be observed,33,34 which indicated thatSchiff base reaction between the aldehyde group in RGD-oxidized alginate and amino group in N-succinyl chitosan hastaken place, thereby leading to cross-linked gels.

3.3.2 Morphology observation. Fig. 3(d–f) show the scan-ning electron microscope images of the hydrogels. It can beseen that pore structures with a uniform distribution of poresand dense walls could be observed in the composite hydrogelwith an 8/2 ratio of V(N-SC) to V(OSA), suggesting that an 8/2 ratioof V(N-SC) to V(OSA) might be a suitable ratio for the formation ofcross-linked gels.

3.3.3 Mechanical properties. Fig. 3(b) shows the compres-sive strength of gels with different ratios of V(N-SC) to V(OSA). Itcan be seen that the compressive strength for all the investi-gated gels with a ratio of V(N-SC) to V(OSA) from 2/8 to 9/1 was inthe range 12 kPa to 32.5 kPa. The highest compressive strengthof 32.5 kPa was found when the ratio of V(N-SC) to V(OSA) was 8/2,which was in good agreement with the scanning electron

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Fig. 3 (a) FT-IR spectra of RGD-immobilized oxidized SA (black line), N-succinyl chitosan (red line) and the composite hydrogel (blue line). (b) Compressive strength ofthe hydrogels as a function of the ratio of V(N-SC) to V(OSA). (c) Degradation behavior of the different components of the hydrogels in a PBS solution of pH 7.4 at 37 �C.(V(N-SC)/V(OSA)¼ 9/1, 8/2, 7/3, 5/5, 3/7, 2/8, 1/9). Scanning electronmicroscopy images of cross-sections of the hydrogels with different volume ratios: (d) 9 : 1, (e) 8 : 2and (f) 7 : 3, indicating pore size and interconnectivity (� 300).

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microscopy observations, as shown in Fig. 3(e), further sup-porting the fact that an 8/2 ratio of V(N-SC) to V(OSA) might be asuitable ratio for the formation of cross-linked gels. It can alsobe seen that the compressive strength increased with increasingthe ratio of the group V(N-SC) to V(OSA), which indicates that thelong-chain of N-succinyl chitosan might act as a skeleton forhydrogel networks. However, once the ratio of the group V(N-SC)to V(OSA) was less than 8/2, the decreased amount of the oxidizedsodium alginate might lead to a more weakly cross-linkedhydrogel molecular structure and thus to a lower compressivestrength.

3.3.4 In vitro degradation behavior of the hydrogels. Acritical requirement for polymeric matrices in tissue engi-neering applications is controllable biodegradation over time.In order to assess the degradation behavior of the hydrogelsformed from the oxidized alginate and N-succinyl chitosan, weincubated the gels in a PBS solution and monitored theirchange in weight. As shown in Fig. 3(c), it can be seen that thedegradation rate of the cross-linked hydrogel was greatlydependant on the volume ratio of N-SC to OSA under the sameexperimental conditions. A swelling process could be observedfor most cross-linked gels before degradation, especially forthose gels with a high ratio of V(N-SC)/V(OSA). This might bebecause the content of the strong suction group –COOHincreased with increasing the content of the N-succinyl chito-san. At the same time, the low cross-linking degree of the gelsmade the polymer network looser, thus absorbing water andswelling easily. For the gel with a 9/1 ratio of V(N-SC)/V(OSA), the

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swelling ratio could reach a maximum (175%) during the rst10 days. Aer that, these gels started to lose their mass becauseof the degradation of the gels. It can be observed that almosthalf of their mass was lost aer 60 days, due to the degradationof the gels. For the gels with the ratios of V(N-SC)/V(OSA) of 8/2, 7/3and 5/5, their degradation behavior was similar. They alldegraded slowly over time and totally degraded aer 60 days.When the volume ratio was less than 5/5, the lower the ratio, thehigher the rate of degradation was. They all degraded almostwithin about 30 days when the ratio of V(N-SC) to V(OSA) was 1/9.The dramatic decrease of the mass aer 30 days is likely due tothe lower molecular weight of the OSA. Aer oxidation, themolecular chain of the SA was partially broken, forming a smallmolecule oxidation alginate, which would be not conductive tothe formation of stable gels. Additionally, the higher the ratio ofOSA, the faster the degradation of the gels was. Thus, just bycontrolling the ratio of V(N-SC)/V(OSA), different degrees ofdegradation of the gels could be obtained.

3.4 In vitro cell culture

3.4.1 Cell proliferation. The cell viability was determinedby the OD values of the Alamar blue assay in our study. The ODvalues of the Alamer blue assay for all the groups exhibited asimilar increasing tendency over time, as shown in Fig. 4(a).From Fig. 4(a) and Table 1, it can be seen that the cell viabilitiesin the unmodied groups (ON and G-ON) were always lowerthan that in the control groups during the investigation period.

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Table 1 Increasing tendency of OD value of the Alamer blue assay for all thegroupsa

Samples Time (day) D-value D/BC (%) p

G-BC 1 0.012 7.8 0.0186 *

3 0.011 3.0 0.0313 *

7 0.016 3.2 0.1096ON 1 �0.023 �14.9 <0.01 **

3 �0.094 �25.6 <0.01 **

7 �0.078 �15.5 <0.01 **

G-ON 1 �0.006 �3.9 0.0287 *

3 �0.059 �16.1 <0.01 **

7 �0.057 �11.4 <0.01 **

RON 1 0.005 3.2 0.26083 0.020 5.4 0.0152 *

7 0.049 9.7 <0.01 **

G-RON 1 0.024 15.6 <0.01 **

3 0.026 7.1 0.07807 0.075 14.9 <0.01 **

a BC (Blank contrast), D-value (the difference value of OD between thesamples and the blank contrast).

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The main reason leading to this might be due to the fact thathydrophilic hydrogel surfaces were not benecial for theadsorption of protein and not conducive to the adhesion andgrowth of cells.25,26 In contrast, the cell viabilities in the RGD-modied groups (RON and G-RON) were higher than that in thecontrol groups. The cell viabilities in all the groups increased inthe order ON < G-ON < BC < G-BC < R-ON < G-RON. The RGDmodication enhanced the cell adhesion and proliferation,whilst the differentiation factor (BMP-2) had a similar function,but less powerful than RGD.

3.4.2 AO-PI uorescence staining. Dual-uorescenceviability using acridine orange (AO) and propidium iodide (PI)was also employed for the viability analysis of the primary cellsin our study. All the live nucleated cells would uoresce green,whilst all the dead nucleated cell would uoresce red.37 FromFig. 4(b), it can be seen that fewer dead cells existed and thegrowth of cells in the RGD-graed hydrogel group was betterthan that in the RGD-free hydrogel group. Similar results wereobtained when the cell growth factors were used. The investi-gation results show that both the RGD and the cell growthfactors can promote the growth and proliferation of cells.

3.4.3 Osteoblast differentiation3.4.3.1 Alkaline phosphatase (ALP) activity. The alkaline

phosphatase (ALP) activity was measured on days 7, 14 and 21,as shown in Fig. 5(a). The ALP activity for the RGD and BMP-2-free hydrogel groups was always not lower than the control, inthat way like the cell viability. By day 14, the ALP activity for theRGD and BMP-2-free hydrogel groups was higher than thecontrol, which means that the density of cells, althoughimportant, is not the only factor to inuence the ALP activity.The ALP activity for all the groups was the highest on day 14,among all the investigated points of time. The ALP activity forthe groups with RGD, BMP-2 and both was always higher thanthe control at any time point, which means that the RGD and

Fig. 4 (a) Cell proliferation determined by means of the Alamar blue assay on days 1performed in triplicate. * Stands for the data that has a significant difference (p < 0.0data that has a significant difference (p < 0.01, n ¼ 3) to the G-BC group at the samodified composite hydrogels were denoted as BC, ON and RON, respectively, whirespectively). (b) Images of the dual-fluorescence viability of the cells (after cell culturlive nucleated cells fluoresce green and all dead nucleated cell ones red. Original m

This journal is ª The Royal Society of Chemistry 2013

BMP-2 enhanced the up-regulation of the ALP expression.However, the expression of ALP for all groups decreased aer 14days of cultivation, this may be because further calciumdeposits inhibited the expression of the ALP.38 The assay ofalkaline phosphatase (ALP) activity show that a high density forrat bone marrow mesenchymal stem cells (rBMSCs), RGD andBMP-2 might be benecial for the differentiation of rBMSCsinto functional osteoblastic cells, which were responsible forthe mineralization of the osteoid matrix, characterized by Type Icollagen and ALP.

3.4.3.2 Alkaline phosphatase staining. Alkaline phosphataseis a marker for the osteogenic differentiation of rBMSCs at the

, 3 and 7. The results were presented as the mean� SD, and the experiments were5, n ¼ 3) to the blank control group (BC) at the same time point. ** Stands for theme time point. (Herein, the blank control group, composite hydrogels and RGDlst these groups with the growth factor were named as G-BC, G-ON and G-RON,e for 7 days) stained using both acridine orange (AO) and propidium iodide (PI): allagnification: 400�.

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Fig. 5 (a) Alkaline phosphatase activities of rBMSCs seeded on the substrates coated with different hydrogels for 7, 14 and 21 days. (Results were presented as themean� SD, and the experiments were performed in triplicate.) * Stands for the data that has a significant difference (p < 0.05, n¼ 3) to the blank control group (BC) atthe same time point. ** Stands for the data that has a significant difference (p < 0.01, n¼ 3) to the G-BC group at the same time point. (Herein, the blank control group,composite hydrogels and RGDmodified composite hydrogels were denoted as BC, ON and RON, respectively whilst these groups with the growth factor were named asG-BC, G-ON and G-RON, respectively). (b) Images of the alkaline phosphatase staining of the cells after osteogenic induction of 14 days. Original magnification: 100�.

Fig. 6 Images of cells stained with Alizarin red S after osteogenic induction of 21days. Original magnification: 100�.

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early stage. From Fig. 5(b), it can be seen that the cells in all thegroups showed the expression of alkaline phosphatase by day 14of cultivation, which in the groups with RGD and BMP-2 wasmore pronounced than that in the groups without RGD andBMP-2 and the control group, indicating the positive enhance-ment effect of RGD and BMP-2 on the osteogenic differentiationof rBMSCs.

3.4.3.3 Alizarin red staining. The ability of cells to form amineralized matrix is essential with regard to the developmentof materials for the formation of new bone, hence, the miner-alization of a matrix consisting of calcium salts can be used as aspecial marker for the formation of new bone. Alizarin red S(AR-S) as a dye can bind selectively to calcium salts and hence iswidely used for calciummineral histochemistry and the assay offormation of new bone.39 In our study, alizarin red staining wasused to examine the osteogenic differentiation of rBMSCs.Similar results to the alkaline phosphatase staining wereobserved. Calcium nodules in all groups were observed aer theculture of 21 days as shown in Fig. 6, further indicating thatrBMSCs have been induced into osteoblasts in vitro. However,the level of mineralization of the matrix was different for thedifferent groups. The highest level of mineralization of thematrix was detected in the group with RGD, when BMP-2 wasused and hence suggested the positive promoted effect of RGDand BMP-2 on the osteogenic differentiation of rBMSCs.

3.4.4 Endothelial differentiation. Some specic markersfor the expression of endothelial such as CD31 and vWF weredetected by using immunouorescence staining. The results ofthe immunouorescence staining (Fig. 7c) showed the positiveexpression of CD31 and vWF in cells in the group of G-RON. Aquantitative analysis of the uorescent intensity showed thatthe expression of CD31 (Fig. 7a) and vWF (Fig. 7b) in the cells onthe RGD-graed hydrogel group was better than that in theRGD-free hydrogel group. Moreover, the expression of CD31and vWF was remarkably enhanced by adding the cell growth

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factors (VEGF and bFGF) to the hydrogel. These data provide thedirect evidence for the endothelial differentiation of rBMSCs onthe RGD-graed hydrogel. Previous studies have illustrated thatgrowth factors (such as VEGF) could induce primary rBMSCsalong an endothelial cell lineage.40 VEGF was initially termed avascular permeability factor because of its ability to inducevascular leakage.41 The importance of VEGF in vascular devel-opment is highlighted by the fact that loss of a single VEGF-Aallele results in abnormal blood vessel development andembryonic death.42 Basic broblast growth factor (bFGF) is therst pro-angiogenic molecule to be identied.43 Both VEGF andbFGF are critical for the vasculogenesis of angioblasts inembryogenesis and for angiogenesis in physiological andpatho-physiological circumstances.44,45 In our preliminaryexperiment, the rBMSCs could not be effectively induced todifferentiate into endothelial cells by either VEGF or bFGF alone

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Fig. 7 A quantitative analysis of the fluorescence intensity of (a) CD31 and (b)vWF was performed and the values are expressed as percent fluorescent intensity.(The results are presented as the mean � SD, and the experiments were per-formed in triplicate. * Stands for the data that has a significant difference ( p <0.05, n ¼ 3) to the blank control group (BC) at the same time point. ** Stands forthe data that has a significant difference (p < 0.01, n ¼ 3) to the BC). (c) Images ofthe immunofluorescence staining of cells in the G-RON group after endothelialinduction of 21 days. The differentiated cells were detected for the expression ofendothelial cell markers CD31, and vWF and visualized by a FITC-conjugatedsecondary antibody. DAPI counter stained nuclei are in blue. DifferentiatedrBMSCs cells on the 21st day of induction using 10 ng mL�1 VEGF and 5 ng mL�1

bFGF showed a positive expression for both the CD31 and vWF. Originalmagnification: 200�.

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(data not shown). Furthermore, when the rBMSCs were exposedto VEGF alone, the cells grew more slowly. The addition of 5 ngmL�1 bFGF ensured a satisfactory cell expansion during theinduced differentiation, which is of practical signicance if alarge number of pre-treated cells are needed in tissue engi-neering. Unlike the previous study40 in which as high as 50 ngmL�1 VEGF was used, the endothelial differentiation of therBMSCs on the RGD-graed hydrogels was successfully inducedusing only 10 ng mL�1 VEGF plus 5 ng mL�1 bFGF, but withoutextending the time course of the differentiation. Above all, theseall provide the evidence for endothelial differentiation of therBMSCs on the RGD-graed hydrogels and the fact that thecombination of VEGF and bFGF was able to effectively inducethe endothelial differentiation of rBMSCs on the RGD-graedhydrogels.

4 Conclusions

Sodium alginate has been successfully oxidized with NaIO4 andthe obtained aldehyde groups in the alginate structure duringoxidation have been successfully used for the immobilization ofRGD on the sodium alginate and also for the formation ofhydrogels through a Schiff base reaction between the oxidizedsodium alginate and N-succinyl chitosan. The compressivestrength of the oxidized sodium alginate–N-succinyl chitosanhydrogels increased with increasing the ratio of V(N-SC) to V(OSA),

This journal is ª The Royal Society of Chemistry 2013

indicating that the long-chain of the N-succinyl chitosan mightact as a skeleton for hydrogel networks. The degradation rate ofcross-linked hydrogels was greatly dependant on the volumeratio of N-SC to OSA under the same experimental conditions.The time over which degradation to ½ of the weight for mostgels with different ratios of V(N-SC) to V(OSA) occurred was morethan 60 days. The results from the in vitro endothelial differ-entiation and osteogenic differentiation study showed that RGDmodication greatly enhanced cell adhesion and proliferationand promoted endothelial differentiation and osteogenicdifferentiation of the rBMSCs in the oxidized sodium alginate–N-succinyl chitosan hydrogels. Due to these properties, it maybe expected that an RGD-graed N-succinyl chitosan–oxidizedsodium alginate hydrogel might be an optimal material forbone tissue engineering scaffold whenever it is used alone orcomposed with other materials in the future.

Acknowledgements

This work was supported by the National Basic ResearchProgram of China (973 Program, 2012CB933600) and theNational Natural Science Foundation of China (no. 51072167).

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