Materials Science and Engineering C 29 (2009) 1207–1215

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Materials Science and Engineering C

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Surface characterization and biocompatibility of micro- andnano-hydroxyapatite/chitosan-gelatin network films

Junjie Li a,b, Yan Dou c, Jun Yang d, Yuji Yin b, Hong Zhang a, Fanglian Yao a,⁎, Haibin Wang c, Kangde Yao b,⁎a School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, Chinab Research Institute of Polymeric Materials, Tianjin University, Tianjin, 300072, Chinac Shanxi Medical University, Taiyuan, 030001, Chinad Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China

⁎ Corresponding authors. Fanglian Yao is to be contacKangde Yao, Tel.: +86 22 27404983.

E-mail addresses: yaofanglian@tju.edu.cn (F. Yao), ri

0928-4931/$ – see front matter © 2008 Elsevier B.V. Adoi:10.1016/j.msec.2008.09.038

a b s t r a c t

a r t i c l e i n f o

Article history:

Hydroxyapatite (HA)/polym Received 30 July 2008Received in revised form 11 September 2008Accepted 17 September 2008Available online 7 October 2008

Keywords:Surface characterizationNano-hydroxyapatiteMicro-hydroxyapatiteBiocompatibilityTopographyMesenchymal stem cells

er composites have been widely used in bone tissue engineering due to theirchemical similarity to natural bone. And the surface characters of the composites are crucial to influencetheir biological properties. Here, nano-hydroxyapatite/chitosan-gelatin (nHCG) films were prepared viabiomineralization of chitosan-gelatin (CG) network films in Ca(NO3)2-Na3PO4 Tris buffer solution at alkalinecondition. And the micro-hydroxyapatite/chitosan-gelatin (mHCG) films were formed through immersingthe CG network films into the HA crystal (with average size 5 μm) suspensions. The surface chemicalcharacteristics of nHCG and mHCG were evaluated by Fourier transformed infrared (ATR-FTIR) and X-rayphotoelectron spectroscopy (XPS). Surface topographies of the samples were observed by atomic forcemicroscopy (AFM) and scanning electron microscope (SEM). Results suggest that the ion/polar interactionsare the main drive forces for nHCG formation via biomineralization. And the hydrogen bonds between COOH,OH, -NH2 of CG films and OH groups of HA crystals take the important role in the formation process of mHCG.A comparative study of mesenchymal stem cells (MSCs) behaviors on the nHCG and mHCG surface layer wascarried out. Both nHCG and mHCG have excellent biocompatibility, moreover, the MSCs on nHCG presenthigher osteogenic differentiation activity than on mHCG. The nHCG is a potential biomaterial in bone tissueengineering.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Hydroxyapatite (HA, Ca10(PO4)6(OH)2) is the major inorganiccomponent in mammalian hard tissues and has been studiedextensively for medical applications because of its excellent biocom-patibility, bioactivity and osteoconductivity [1,2]. For example, it hasbeen shown that HA and its composites are suitable for attachment,proliferation and differentiation of mesenchymal stem cells (MSCs),owing to their structure and chemical compositions. However, HA alsohas some disadvantages, instability of the particulate HA is oftenencountered when the particles are mixed with saline or patient'sblood and hence migrate from the implanted site into surroundingtissues to causing damage to health tissue [3]. In order to obtainintelligent biomaterials, a great attention has been focused on thecomposites of HA in conjugation with some synthesized and naturalpolymers via different methods, such as biocomposites [4], biominer-alization [5], HA crystalline surface patterning [6], surface modifica-tion [7] and biological self-assembly [8]. The chemical composition

ted at Tel.: +86 22 27402893.

pm@tju.edu.cn (K. Yao).

ll rights reserved.

and topography structure of HA/polymers composites can becontrolled through different ways. Moreover, topographies of extra-cellular microenvironments can influence cellular response fromattachment and migration to differentiation of new tissue [9,10].Previous research has elucidated the true effect of the particle size ofHA granules response to the MSCs, results suggested that HA crystalsin nano scale is more beneficial to promote differentiation of MSCs invitro than that at micrometer size level [11]. Although, cells in theirnatural environment interact with extracellular matrix (ECM)components at the nanometer scale [12], but whether nanometerscale is necessary to produce significant effect on stem cells behaviorshas no definite conclusion till now. Ecans et al. [13] pointed out thatstructures containing both micro- and nano-patterned should bedesigned to enhance the cell functions. Yim et al. [14] found thatnanopatterns (350 nm×350 nm×700 nm) could have a moresignificant effect on the differentiation and proliferation of stem-cells compared to micro terms in neuronal induction media.

In our previous research, micro-hydroxyapatite/chitosan-gelatinnetwork scaffolds were prepared via biocomposites and the rat caldariaosteobalsts could attach and proliferate in the scaffolds well [15,16]. Thenano-hydroxyapatite (nHA) crystals were formed on the surface ofchitosan-gelatin network films via biomineralization [17]. Based on

Fig. 1. ATR-FTIR spectra for chitosan (a), gelatin (b), nHCG (cycle 5) (c) and mHCG(d) network films.

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these results, here, chitosan-gelatin network films modified with mHA(mHCG, the average sizeofHAwasabout5 μm)ornHA(nHCG,17–25nm)were prepared. The relationships between the surface performances(physical and chemical) of nHCG or mHCG and the MSCs behaviors wereinvestigated. The results suggested that the surface properties of nHCGplay more important roles in regulating MSCs adhesion and proliferationthan that of mHCG. Moreover, the nHCG takes more important effects onosteogenic differentiation of MSCs.

2. Materials and methods

2.1. Materials

Chitosan (mean molecular weight 2.0×105 Da and deacetylationdegree 85%) was supplied by the Qingdao Medical Institute (Qingdao,China), and purified as follows. The chitosan was dissolved in a 2%acetic acid aqueous solution until a homogeneous 1% chitosan solutionwas obtained. The pH of this solution was adjusted to 9.0 in order toprecipitate chitosan with a 10% NaOH aqueous solution, and thenwashed with deionized distilled water and air-dried. The gelatinpowder (bovine) was purchased from the Sigma Chemical Co. HAwasproduced by Biomedical Center of Sichuan University (China). Allother reagents were of analytic grade.

2.2. Preparation of nHCG and mHCG composites

nHCG network films were prepared as previously reported [17].Briefly, first, chitosan-gelatin (CG) network films were prepared bycasting the mixed solution of chitosan (30 ml 2% (wt.%) in a 1% aceticacid solution) and gelatin (15 ml aqueous, 4% (wt.%)) after cross-linking by glutaraldehyde (0.25(wt.%)). Keep drying at room tem-perature until the water evaporated, a film with ca. 0.1 mm thicknesswas formed. The films were treated with NaOH solution (1% (wt.%)) toneutralize the acetic acid, washed with deionized distilled water topH=7.0 and retreated with sodium borohydride (NaBH4) solution toeliminate the residual glutaraldehyde. Samples were repeatedlywashed with deionized water and dried at room temperature. Then,the CG network films were immersed in 0.1 M Ca (NO3)2 Tris buffersolution for 12 h. After being washed with deionized water, the CGnetwork films were soaked for another 12 h in 0.1 M Na3PO4 Trisbuffer solution with a pH of 11–14 adjusted using NaOH solution. Theabove nHA formation stepwas repeated several times according to thesediment amount of nHA. In this article, the samples were denoted byusing nHCG (cycle X), where X is the repeated times of nHA sediment.

The CG network films were immersed into a suspension ofhydroxyapatite with a mean size of 5 μm in deionized water for12 h. The mHCG was formed after drying.

2.3. Characterization of nHCG and mHCG composites.

Surface chemical compositions of sampleswere investigated byX-rayphotoelectron spectroscopy (XPS, Rigaku D/max 2500v/pc) andreflectance FTIR (MAGNA-560, Nicolet, USA), surface topographies ofsamples were examined by Atomic Force microscopy (AFM) (Digitalinstruments Inc., Santa Barbara, CA) in contact method. Three dimen-sional images and surface topography parameters (Ra and Rt) datawereacquired using nanoscope image software. The surface morphologicalchanges of the samples were studied by a scanning electron microscope(SEM, XL30, PHILIPS, Holand)with an accelerating voltage of 20 kV aftersputtering gold.

2.4. Solubility analysis of HA from nHCG and mHCG composites

nHCG and mHCG films with a diameter of 14 mm were immersedinto the 2.0 ml α-MEM culture media and maintained the tempera-ture at 37 °C, after 2, 4 and 7 days, 1 ml of culture media was diluted

using 5 ml deionized water, 100 μl NH4Cl solution (5% (wt.%)) wasadded and the [Ca2+] of the culture media was measured usingMETTLER TOLEDO.

2.5. Cell experiments

MSCs were isolated from the bone shaft of femurs of 3-week-oldmale mouse according to the technique reported by Lennon et al. [18]and expression with CD44+ and CD71+. These cells were expandedusingα-MEM complete media and grown at 37 °C and 5% CO2. The 4thpassage cells were used in the subsequent cultures.

2.5.1. Cell morphologyA 500 μl cell suspension with approximately 3.0×104 cells was

seeded on the top of mHCG and nHCG composite film in 48-well tissueculture plates. After the MSCs were cultured for 2–7 days at 37 °C and5% CO2, the cell-grown film samples were fixed with 2.5% glutar-aldehyde in 0.1 M PBS (pH 7.2), followed the specimens weredehydrated throw a graded series of ethanol, vacuum dried and goldcoated for scanning electronmicroscope (SEM, XL30, PHILIPS, Holand)observation.

2.5.2. Cell attachmentCell attachment on the surface of nHCG andmHCG film at different

time intervals was studied. Culture medium (500 μl) containing about6.0×104 MSCs were added to wells covered with nHCG and mHCGfilms. The cells were allowed to attach to the films undisturbed in ahumidified incubator (37 °C, 5% CO2) for 0.5, 1, 2, 4 and 6 h,respectively. At each time interval, unattached cells were removed bythoroughly washing with PBS, and attached cells were fixed withformalin (4% in PBS) at 4 °C for 30min. The adherent cells stainedwith0.5% crystal violet for 30 min and excess dye was washed with PBSthree times. The dye bound to the nucleus of the cells was recoveredwith 2% SDS solution, and then the number of attached cells wasmeasured bymonitoring optical absorbance by the solution at 570 nmusing an enzyme-linked immunosorbent assay (ELISA) plate reader(TECAN SPECTRA III).

2.5.3. MTT assayThe MTT assay was used as a measure of relative cell viability [19].

After the MSCs were cultured on nHCG and mHCG films in 48-welltissue culture plates for 2, 4, and 7 days at 37 °C and 5% CO2, and themedia were changed every other day. The cell viability was evaluatedusing the MTT assay, in which 50 μl of MTT (Sigma, 5 mg/ml inDulbecco's PBS)was added to eachwell and incubated at 37 °C, 5% CO2

Fig. 2. The interactions between the CG network films and HA crystals in nHCG composites (A) and mHCG composites (B) ( hydrogen bonds interaction,coordination interaction, ionic interaction and polar interaction).

Fig. 3. XPS survey spectra of (a) mHCG, (b) nHCG (cycle 3), and (c) nHCG(cycle 5).

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for 4 h. After removal of the medium, the converted dye was dissolvedwith acidic isopropanol (0.05MHCl in absolute iospropanol). Solution(100 μl) of each samplewas transferred to a 96-well plate. Absorbanceof converted dye is measured at a wavelength of 570 nm using anenzyme-linked immunosorbent assay (ELISA) plate reader (TECANSPECTRA III).

2.5.4. Alkaline phosphatase (ALP) activity stainingThe MSCs were seeded on nHCG andmHCG at 37 °C in a humidified

atmosphere of a 5% CO2. In order to induce osteogenic differentiation,50 μg/mL L-ascorbic acid, 10 mmol/L β-(magnesium phosphate)pentahydrate and 10 nmol/L dexamethasone (Dex) were also addedto the medium. The alkaline phosphatase (ALP) activity was measuredusing alkaline phosphatase reagent kit (Zhongsheng Beikong Bio-Technology Science Inc., China) at day 4 and day 7. Briefly, cell lysatesand culture supernatant were combined with 50 ml of ALP reagent andthe activity measured in a 96-well plate following 30 min incubation at37 °C. The data was read at 405 nm using an enzyme-linkedimmunosorbent assay (ELISA) plate reader (TECAN SPECTRA III).

2.6. Statistical analysis

Statistical analysis was performed with the Orginpro Software forWindows. The results of cells viability were reported as meanstandard deviation for n=4, and the differences between the sampleswere tested by Student's t-test with a significance level of pb0.05 orpb0.01.

3. Results

3.1. Surface chemical characters of mHCG and nHCG network films

In our previous work [20], it has been found that the chemicalcomponent of hydroxyapatite in nHCG and mHCG is similar. The HAcrystalline size in nHCG is about 15–27 nm, which is far smaller than

that in mHCG (5 μm). In order to investigate the differences of theinteractions between HA and CG network in nHCG and mHCG, here,ATR-FTIR spectroscopy was performed (as shown in Fig. 1). The ATR-FITR spectra of all these samples showed the typical peaks of phosphatevibration at 1027 cm−1, which implied the existence of HA on the surfaceof nHCGandmHCG.Moreover, peaks forCO3

2−vibrationmode is appearedat the position of 876 cm−1 in nHCG composites, this observation meansthat the PO4

3− sites of nHA structure were partially substituted by CO32−

groups in nHCG composites. While there is no CO32− group in mHCG

composites. For mHCG composites, which formed via biocomposites, theabsorption at 1653 cm−1 can be attributed to COOH groups of gelatin inmHCG. It shifted to the direction of higher wavenumber compared with

Fig. 4. Surface composition of nHCG (cycle 5) and mHCG detected by XPS.

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COOHgroups (1639 cm−1) of gelatin itself. The OH absorption of chitosanandgelatin inmHCGcanbeobservedat 3367–3312 cm−1,whichbecomeswider than that of chitosan and gelatin themselves. So the characteristicabsorptions of COOH and OH in mHCG suggested the formation ofhydrogen bond between HA crystalline and CG network (as shown inFig. 2(A)). Compared with the IR spectra of pure chitosan and gelatin, theabsorptions of COOH at 1639 cm−1 and OH at 3367–3312 cm−1 of nHCGbecomemuchweaker andnearly disappear.Meanwhile, a newabsorptionpeak at 1410 cm−1 appeared. That means the existence of much strongerinteraction between HA and CG networks in nHCG. According to thebiomineralization process of HA crystals on CG network, the pH value ofthe medium is higher than isoelectric point of gelatin. So COOH of gelatinin nHCG exists in the form of COO− and the ionic or polar interactionbetween COO− and Ca2+ could be formed [17]. Li et al. [21] found that thehydrogen bonds may also be existed between -NH2 and HA. On the otherhand, two crystalographically different calciumsites exist in theHAcrystalstructure. Ca (2) appears on the terminated surface of HA crystals. The Ca(2)has acoordinationnumberof7, and is strictlyheld in the structure [22].On the surface, 2 coordinates of Ca (2) are lost, therefore, which allows tothe coordination interactions of Ca (2)with COO−, -COOHandNH2 on thesurface of CG network (as shown in Fig. 2(B)).

Fig. 3 shows the XPS analysis of nHCG and mHCG. Ca, P, O and Cwere detected on the surface layer. Photoelectron signals at 355 eV

Fig. 5. A. The surface character of CG network films by AFM (column left) and SEM (column(column right). C. The surface character of mHCG network films by AFM (column left) and

(Ca2p) and 137 eV (P2p) were assigned to analyze the HA, and carbonC1s (284.8 eV) appeared in all samples. One can also find that thenHCG (cycle 3) and nHCG (cycle 5) have the same chemicalcompositions from Fig. 3(b) and (c) [23,24]. In addition, there aremore O1s on the surface of nHCG than that of mHCG, while more C1sappear on the surface of mHCG films. The content of Ca2p is 9.16% and3.769% on the surface of nHCG and mHCG films, respectively (asshown in Fig. 4). That means the surface of CG network was coveredwith HA particles and the covered ratio of nHCG is higher than mHCG.This may be caused by stronger interactions existing between HA andCG network in nHCG than that in mHCG.

3.2. Topography structure of nHCG and mHCG network films

The surface characteristics of nHCG and mHCG were observed byAFM and SEM, as shown in Fig. 5. The quantitative data of surfaceroughness parameters were listed in Table 1. One could find that CGnetwork films were almost smooth (Fig. 5A) and the value of Ra, Rtwas only 0.203 nm and 0.671 nm respectively (Table 1). With theincreasing of nHA content in the composites, the surface becameobviously rough and irregular.When the nHA crystals covered the film,the roughness increase to Ra=15.405 nm and Rt=82.932 nm (Fig. 5Band Table 1). With further enhancing the content of nHA, many largevalleys and peaks which being Ra=35.976 and Rt=115.14 nm wereproduced. Comparisonwith the nHCG surface, the roughness of mHCGis nearly one times higher (Ra=54.363 nm and Rt=227.39 nm), asshown in Fig. 5C and Table 1.

3.3. Dissolution analysis of HA from nHCG and mHCG network films

For bone regeneration, it is expected that the degradation rate of amaterial should match the process of tissue repair or regeneration.Moreover, dissolubility can be considered as one factor of biodegrada-tion. The dissolution behaviors of HA from nHCG and mHCG in culturemedia from 0 to 7 days were displayed in Fig. 6. It showed that allspecimens continue to dissolve after immersion. Fig. 6(A) suggestedthat there were higher [Ca2+] in nHCG (cycle 5) culture medium thanthat of mHCG at any time, and the [Ca2+] of mHCG culture mediumwas almost unchanged from 0 to 2 days, while in nHCG culturemedium it increased from 0.48 mmol/L to 4.76 mmol/L, whichindicated that nHA crystals have a more higher solubility anddissolution rate [25,26].

right). B. The surface character of nHCG network films by AFM (column left) and SEMSEM (column right).

Fig. 5 (continued).

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From Fig. 6(b), one can find that the dissolution behaviors of HAexhibited very different among nHCG (cycle 5), nHCG (cycle 3) andnHCG (cycle 1). Obviously, the solubility of nHA in nHCG (cycle 5) filmis higher than that in nHCG (cycle 3) and nHCG (cycle 1), and the

dissolution behaviors of nHA are similar in nHCG (cycle 3) and nHCG(cycle 1), which may be caused by the different interaction intensitybetween nHA crystals and CG templates. When there is a smallamount of nHA crystals (cf. Fig. 5B) precipitated on the CG films, the

Fig. 5 (continued).

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ionic and/or polar interactions between Ca2+, PO43− of nHA and COO−,

C=O, -NH2 of CG play important roles. However, the same kinds ofinteractions became weaker after cycle 5 because of the increase ofdistance between CG network films and nHA crystals layer (cf. Fig. 5B)[17].

3.4. Biocompatibility of nHCG and mHCG network films

Cell adhesion is an important previous process for sequent cellgrowth, cell migration and cell differentiation. Fig. 7(A) showed theattachment of the MSCs cultured on the surfaces of the test films andcontrol (TCPS) for a period of 6 h, and cell adhesion was recorded at0.5, 1, 2, 4 and 6 h. During the incubation period, cell's adhesion abilityincreased at early stage. The number of MSCs attached on the nHCGsurface is more than that on mHCG surface with pb0.05. However,there is no significant difference in comparison with the TCPS.Moreover, for the nHCG surface, the adhesion capability of MSCsenhanced with the increasing of the content of nHA crystals in thecomposites (as shown in Fig. 7(B)). Fig. 8(A) illustrated the MTT assayin term of formazan absorbance as a measure of MSCs viability seededonto nHCG films, mHCG and control (TCPS) after 7 days culture,respectively. The MTT assay absorbance of MSCs cultured on nHCGdisplays significant differences to mHCG (pb0.05) and the TCPS(pb0.01), and the proliferation capability of MSCs enhances with theincreasing of the content of nHA crystals in the composites (as shownin Fig. 8(B)). The morphology of MSCs cultured on the compositenetwork films was evaluated using SEM. Fig. 9(B) showed that thecells cultured on nHCG films were seen to be highly motile withfilopodia at day 2, which are better than that on the mHCG surface (cf.Fig. 9(A)). As shown in Fig. 9(C), cells have dramatically reproducedand aggregated with each other to form stratified cell layers at day 7and accompanying with filamentous fibers formed on the surface.

Table 1Surface parameters of nHCG and mHCG network films by AFM.

Surfaceparameter

Samples

CG nHCG(cycle 1) nHCG(cycle 3) nHCG(cycle 5) mHCG

Ra(nm)a 0.203 4.160 15.405 35.976 54.363Rt(nm)b 0.671 17.007 82.932 115.14 227.39

a Arithmetic mean deviation of the surface.b Maximum mean peak-to-valley height of the surface.

Obviously, the nHCG surface appeared to have no negative effect onthe cell morphology.

Fig. 10(A) shows the total ALP of MSCs cultured on the surfaces ofnHCG and mHCG. It indicated that the ALP activity of MSCs seeded on

Fig. 6. Calcium concentration changes in the a-MEM culture media during one weekimmersion of nHCG and mHCG, bars=means±SD (n=3).

Fig. 8. Proliferation levels of MSCs on the surface of (A) nHCG and mHCG network filmsand (B) nHCG network films(different precipitated times) over a 7-day period;bars=means±SD (n=6), Cells were seeded initially at a density of 6.0×104 cells/ml.(⁎pb0.05, ⁎⁎pb0.01).

Fig. 7. TheattachmentofMSCs to the surfaceof (A)nHCGandmHCGnetworkfilms and (B)nHCG network films (different precipitated times) over a 6 h period; bars=means±SD(n=3). Cells were seeded initially at a density of 6.0×104 cells/ml.

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nHCG network films was significant higher (pb0.01) than that onmHCG. On the nHCG surface, the ALP activity enhance with the nHAcrystals content increasing after 7 days culture in the presence ofosteogenic differentiation medium (cf. Fig. 10(b)).

4. Discussion

In the processing of tissue engineering, it is important to developnew biomaterials suitable for cell cultivation. The influence ofmaterials surfaces on cellular parameters, such as adhesion, prolifera-tion, migration and differentiation, has been extensively studied andshown to play an important role in the formation of tissues andorgans. Cells may interact with the substratum via chemical, physicaland topological surface parameters, which play an essential role inbiocompatibility of biomaterials [27,28]. These surface propertiescould affect not only cell morphology and cytoskeleton but also nuclei[29]. The HA coating has an excellent biocompatibility [30]. However,the effect of hydroxyapatite properties on cell response has not yetbeen fully understood. In fact, the cell responses depend upon thephysical and chemical characteristics of the substrates includingcrystalline particle size, surface structure and chemical composition. Itis important to understand the surface and interfacial chemistry of HAcomposites.

In order to study the relationship between surface characterizationand biocompaitibility, here the nHCG and mHCG composites wereprepared via biominerization and biocomposites, respectively. Resultssuggested that there are different interactions between HA crystalsand CG network in nHCG and mHCG. The size of HA in nHCG is 17–25 nm, which is smaller than that of mHCG (5 μm). And during thebiominerization process, some carbonate ions were incorporated intonHCG composites. In addition, the crystallinity of HA in nHCG is lowerthan that in mHCG [20]. These characterizations endow cells muchbetter adhesion capability on nHCG surface than that on mHCGsurface [31]. Moreover, nHA crystals have more amounts of atoms atthe surface compared to bulk. And there aremore surface defects suchas edge/corner sites and particle boundaries, and larger delocalizedregions on the surface of nHA than mHA crystals [32,33]. Theseproperties make the nHA crystals have stronger capability to absorbsome special peptide sequence (e.g. argininr-glycine-aspartic (RGD)sequence) and promote interactions of select serum protein(s) withHA crystals [34]. The adhesion ability of MSCs increases when the RGDwas absorbed to HA surface, whichmakes the MSCs have the ability todifferentiate along the osteoblast lineage [35,36]. Cell adhesionmolecules, such as fibronectin in serum or the molecules producedby the cells, could more easily support cellular adhesion andproliferation since many proteins are actively adsorbed on nHAsurfaces [37]. Attachment is implicated in the regulation of mostaspects of cellular activity and involves external structures of theplasma membrane such as filopodia, micropodia and fragile

Fig. 10. Alkaline phosphatase (ALP) activity of MSCs (A) on the surface of nHCG andmHCG network films, bars=mean±SD (n=6), pb0.01; and (B) on nHCG films(different precipitated times) after culturing for up to 4 days and 7 days. Cells wereattached initially at a density of 6.0×104 cells/ml. (⁎pb0.05 and ⁎⁎pb0.01).

Fig. 9. Cell morphology of mesenchymal stem cells seed on the mHCG cultured for2 days (A), nHCG (cycle 5) cultured for 2 days (B) and on the nHCG(cycle 5) cultured for7 days(C).

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microextensions [27], which are consist with our results (cf. Fig. 9).Cells utilize these structures for attachment andmigration. The qualityof this attachment will influence the cell's capacity to proliferate anddifferentiate.

Previous studies have shown that surface topography and particlegeometry/size are able to influence cellular behavior [38,39].Deligianni et al. [40] reported that cell attachment depends on thedegree of roughness of the material surface. They showed that as theroughness of the HA surface increased, the number of adherent cellsincreased. Tanaka et al. [41] also found that comparative roughnesssurface is more suitable for adhesion, differentiation of rat bonemarrow mesenchymal cells. While some literature reported adhesionand proliferation of cells decreased with increasing surface roughness[38,42]. In this research, the results indicated that it is appropriate topromote the cytocompatibility when the roughness is about35.976 nmbRab54.363 nm and 115.14 nmbRtb227.39 nm. Toosmooth surface is adverse for absorbing the protein and too rough

surface with deeper grooves influence the migration behavior of cell.These results suggest that nHA crystals exhibited good biocompat-ibility with respect to initial cell response to mHA crystals. As the cellsoccupied the grooves rather than the ridges and adhered to lowerparts of the patterned surface, relative roughness of mHCG surfacemade the MSCs migrate difficult [43].

Differentiation of MSCs is one of the key processes for boneregeneration. Despite diverse and ever-growing information concern-ing MSCs and their use in clinical strategies, the mechanisms thatgovern MSCs self-renewal and multilineage differentiation are notwell understood and remain an active area of investigation [44].Therefore, research efforts focused on identifying factors that regulateand control MSCs fate decisions. Previous researches [9,45,46] hadprovided compelling evidence that adult osteoprogenitor cells mayuse discrete changes in nanotopography (below 50 nm) as cues fordifferentiation or modulation of activity. In our vitro test, thedifferentiation of MSCs into osteoblast phenotype was quantitativelydetermined using ALP with the present of osteogenic differentiationmedium. The results of ALP activity indicated that nHCG can moreeffectively promote osteoblastic differentiation than mHCG. Thesefindings might due to the HA crystals in nHCG composites have highersolubility than that in mHCG, which made there be higher Ca2+

concentration in cell culture media of nHCG than that of mHCG.Moreover, additional Ca2+ concentration can enhance osteoblasticdifferentiation [47]. And more Ca atom exposed on the nHCG surface(as shown in Fig. 3) also improves this affectivity. As the same reason,the osteoblast differentiation capability improved with the increasing

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of nHA crystals content. As the MSCs occupied the grooves rather thanthe ridges and adhered to lower parts of the patterned surface, relativeroughness of mHCG surface made the MSCs migrate difficultly [43],which reduce the differentiation capacity. Of course, the exact reasonneed to be further studied.

5. Conclusions

In briefly, Chitosan-gelatin network films are common substratefor inducing formation of HA crystals in-situ and depositing sinteredHA crystals respectively. When nHA formed on the surface of CGnetwork via biomineralization, the corresponding ion interaction isthe main drive force. However, as the mHA crystals depositing on thesurface of CG network, the hydrogen bonds between COOH, OH, -NH2

of CG films and OH groups of HA crystals take the important role. BothnHCG and mHCG have excellent biocompatibility, but nHCG showedbetter biocompatibility due to their surface characters. It is revealedthat the nano size effect is more crucial in comparison to the othereffects for the MSCs behaviours cultured on nHCG or mHCGcomposites. Nano hydroxyapatite composites are potential biomater-ials in bone tissue engineering.

Acknowledgements

This work has been supported in part by the National NatureScience Foundation of China 30470482, 50773050, 30670572,50233020 and 50200300; the Tianjin Municipal National ScienceFoundation Key Project 043803211; Key Projects in the Tianjin Science& Technology Pillar Program via grant 06YFSZSF01000; Projectsupported by the International Cooperation from Ministry of Scienceand Technology of China (Grant No. 2008DFA51170) and theProgramme of Introducing Talents of Discipline to Universities, No.B06006.

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