Preparation and characterization of bioactive calciumsilicate and poly(e-caprolactone) nanocomposite forbone tissue regeneration

Jie Wei,1,2,3 S. J. Heo,1,4 Changsheng Liu,2 D. H. Kim,1 S. E. Kim,4 Y. T. Hyun,4

Ji-Wang Shin,1 Jung-Woog Shin1,3

1Team of BK21, First Project Team, Department of Biomedical Engineering, Inje University, Gimhae,Gyeongnam 621-749, Republic of Korea2Key Laboratory for Ultrafine Materials of Ministry of Education, Engineering Research Center for Biomedical Materialsof Ministry of Education, East China University of Science and Technology, Shanghai 200237,People’s Republic of China3R & D Department, Taesan Solutions Ltd, Seoul 135-080, Republic of Korea4Future Technology Center, Korea Institute of Materials Science, Changwon, Gyeongnam 641-831, Republic of Korea

Received 27 April 2007; revised 13 October 2007; accepted 29 April 2008Published online 18 June 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32139

Abstract: A novel biocomposite of nanosized calcium sili-cate (n-CS) and poly(e-caprolactone) (PCL) was successfullyfabricated directly using n-CS slurry, not dried n-CS pow-der, in a solvent-casting method. The in vitro bioactivity ofthe composite was evaluated by investigating the apatite-forming ability in simulated body fluid. A proliferationassay with mouse L929 fibroblasts was used to test thein vitro biocompatibility. The composition, hydrophilicity,and mechanical properties were also evaluated. Results sug-gest that the incorporation of n-CS could significantlyimprove the hydrophilicity, compressive strength, and elas-tic modulus of n-CS/PCL composites, with the enhance-ments mainly dependent on n-CS content. The n-CS/PCLcomposites exhibit excellent in vitro bioactivity, with surfaceapatite formation for 40% (w/w) n-CS (C40) exceeding that

of 20% (w/w) n-CS (C20) at 7 and 14 days. The Ca/P ratiosof apatite formed on C20 and C40 surfaces were 1.58 and1.61, respectively, indicating nonstoichiometric apatite withdefective structure. Composites demonstrated significantlybetter cell attachment and proliferation than that of PCLalone, with C40 demonstrating the best bioactivity. The apa-tite layers that formed on the composite surfaces facilitatedcell attachment (4 h) and proliferation during the earlystages (1 and 4 days). Collectively, these results suggest thatthe incorporation of n-CS produces biocomposites withenhanced bioactivity and biocompatibility. � 2008 WileyPeriodicals, Inc. J Biomed Mater Res 90A: 702–712, 2009

Key words: calcium silicate; poly(e-caprolactone); nano-composite; bioactivity; cell proliferation

INTRODUCTION

Many studies have shown that silicon-containingbioactive materials such as bioactive glasses and sili-con-doped calcium phosphate materials exhibitedexcellent ability to induce bone-like apatite formingin vitro and in vivo.1,2 Furthermore, solutions with ahigh silicon concentration show the potential to acti-

vate bone-related gene expression, stimulate osteo-blast proliferation, and promote new bone forma-tion.3,4 Therefore, silicon-containing bioactive materi-als may open new possibilities in the field of boneregeneration and replacement. Calcium silicate(CaSiO3) bioceramics exhibit excellent in vitro bioac-tivity, and the formation rate of surface apatite isfaster than those of other biocompatible glasses andglass-ceramics in simulated body fluid (SBF) solu-tion.5,6 In addition, wollastonite (natural CaSiO3)composites plasma sprayed on titanium alloy sub-strates and wollastonite/polymer composites showgood mechanical properties and bioactivity, provid-ing favorable implants for bone tissue regenera-tion.7,8 Although existing bioactive CaSiO3 bioceram-ics possess high bioactivity, they are unfortunatelyvery brittle and have inherently poor tensile proper-ties and difficulty for processing.9

Correspondence to: J.-W. Shin; e-mail: sjw@bme.inje.ac.kror biomater2006@yahoo.com.cnContract grant sponsor: Fundamental Research Program

of Korea Institute of Materials ScienceContract grant sponsor: Ph.D. Programs Foundation of

Ministry of Education of China; contract grant number:20070251007

� 2008 Wiley Periodicals, Inc.

Poly(e-caprolactone) (PCL), one of the most com-mercially available biodegradable polymers, iswidely used in biomedical fields for its biodegrad-ability, biocompatibility, and formability.10 Theunique nature of PCL is its mechanism of biodegra-dation through hydrolysis of the ester linkage,resulting in decomposition products that are normalintermediates of cell metabolism.11 However, a num-ber of problems have been encountered regardingthe use of PCL for bone regeneration. The primarylimiting factor is the lack of bioactivity; the hydro-phobic nature of PCL prevents new tissue fromattaching to the polymer surface.12,13 In balancedconsideration of the limitations and advantages ofpolymers and bioactive ceramics materials, combin-ing these materials should provide bioactive inor-ganic–organic composites with optimized proper-ties.14 To date, a variety of bioactive composites ofbiodegradable polymers, bioceramics, and bioactiveglasses have shown varying degrees of success asbone tissue engineering scaffolds.15,16 Because boneis a nanocomposite of inorganic minerals and or-ganic biopolymers, nanobiocomposites of bioactiveceramics and biodegradable polymers have recentlygained interest and are perceived to be beneficialover conventional methods in the field of bone graft-ing.17,18 Specifically, emphasis has been placed onbioactive particles dispersed in suitable polymericmatrices.19,20

We investigated a novel, bioactive, hydrophilic,polymer-based biocomposite that incorporates nano-sized calcium silicate (n-CS) in PCL, with the goal ofproducing flexible bone implants, enhanced mechan-ical properties, and favorable bioactivity and bio-compatibility. Because hydrothermally synthesizedn-CS slurries dried in air agglomerate, reducing ho-mogeneity, we used the n-CS/DMF (dimethyl form-amide) slurry directly to prepare PCL composites,using a simple solvent-casting method. The in vitrobioactivity of the n-CS/PCL composite was eval-uated by investigating the apatite-formation abilityin SBF. The in vitro biocompatibility of the compositewas evaluated using mouse L929 fibroblasts in aproliferation assay.

MATERIALS AND METHODS

Preparation of nano-CaSiO3

n-CSs were synthesized using a chemical precipitationmethod according to the following chemical reaction:

Na2SiO3 þ CaðNO3Þ2 ! CaSiO3 þ 2NaNO3

Ca(NO3)2�4H2O and Na2SiO3�9H2O were used as the start-ing materials in this reaction. The two reagents in 1:1 stoi-

chiometric proportion were dissolved in deionized waterin beakers and the concentration was adjusted to 0.5 mol/L; 0.2% (w/w) polyethylene glycol was added to 300 mLof Ca(NO3)2 solution at ambient temperature as a dispers-ant. The Ca(NO3)2 solution was stirred while 300 mL ofNa2SiO3 solution was added. After the precipitation wascomplete, CaSiO3 precipitate was obtained and fullywashed with deionized water. CaSiO3 precipitate was thenmixed in a solid–solution ratio of 1% (w/w) with DMF ina three-necked flask; the mixture was stirred while thetemperature was gradually increased to 100–1208C toevaporate the water and kept at 1208C for 5 h, and thencooled at room temperature. The obtained n-CS/DMFslurry was stored in a beaker until use (DMF as dispers-ant). A portion of the n-CS slurry was vacuum-dried atroom temperature to obtain n-CS powder samples thatwere then characterized for phase composition and surfacemorphology using X-ray diffraction (XRD, Geigerflex,Rigaku, Japan) and scanning electron microscopy (FE-SEM,Hitachi, S-4300SE, JPN).

Preparation of n-CS/PCL composites

The n-CS content (w/v) in the slurry (DFM) was testedso that we could know how much slurry we need to takewhen we made 20% and 40% (w/w) n-CS in the compos-ite. Composites were prepared by solvent-casting method.To uniformly disperse the n-CS into the composite andprevent the n-CS from aggregation in the PCL, we madethe composite using co-solution method that mixed the n-CS/DFM slurry with PCL/chloroform solution. Briefly,poly(e-caprolactone) (PCL) pellets were used as supplied(Sigma-Aldrich Corporation, St. Louis; MW 5 65,000). PCLpellets were dissolved in chloroform at 20% (w/v) accord-ing to the weight ratio of n-CS to PCL. A prescribedamount of n-CS/DFM slurry, according to the desired n-CS content, was then added to the PCL/chloroform solu-tion under continuous stirring for 2 h to obtain uniformdispersion. The mixture was then heat-dried at 808C for2 h to evaporate the chloroform, resulting in an n-CS/PCL/DMF mixture. Subsequently, the mixture temperaturewas gradually increased to 100–1208C under constant stir-ring and held for 2 h. The obtained mixture was cast in a100 3 100 3 3 mm3 Teflon mold and air-dried under thefume hood for 24 h to evaporate the DMF; remaining sol-vent was removed by vacuum-drying at 508C for 48 h. Af-ter washing the samples in deionized water and ethanol,samples were dried at room temperature in a fume hoodfor 48 h. The obtained samples were cut to 10 3 10 3

3 mm3 and stored in a desiccator until use. For hydrophi-licity, phase composition, surface morphology, and me-chanical property analyses, the composite samples werefabricated by solvent-casting in different Teflon molds. Thephase composition and surface morphology of the compos-ite specimens were examined by XRD (Geigerflex, Rigaku,Japan) and SEM (FE-SEM, Hitachi, S-4300SE, JPN).

Mechanical properties test

The compressive strength of n-CS/PCL composites (103 10 3 10 mm3) was measured at room temperature at a

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constant displacement rate of 2 mm/min (MTS 858.20Bionix, MTS System Corp., MN). The jigs were speciallydesigned to provide a uniform load on the composite sam-ples. The compressive strength and elastic modulus wascalculated within the linear range of the stress–straincurve. The results represent the average of four or fivespecimens for each composition.

Hydrophilicity

Surface wettability of the n-CS/PCL composite speci-mens was determined by measuring the contact angle ofwater droplets on the surfaces using a contact angle mea-surement system (SEO 300A, Surface and Electro-Optics,Ansan, Korea). The water contact angle method was usedto determine the polar interactions at the material-waterinterface. Water droplets of 10 lL were made at three dif-ferent points on each sample using a microsyringe (Per-fektim, Popper & Sons, Japan), and the contact angleswere measured after 30 s. The results represent the mean6 standard deviation (SD) of four or five contact anglesper sample.

Bioactivity in SBF

The bioactivity of n-CS/PCL composite specimens wasevaluated by examining the apatite formation on their sur-faces in SBF. SBF was prepared by dissolving reagentgrade NaCl, NaHCO3, KCl, K2HPO4�3H2O, MgCl2�6H2O,CaCl2, and Na2SO4 in deionized water; the ion concentra-tions were similar to those in human blood plasma (TableI).21 The solution was buffered at pH 7.4 with tris(hydrox-ymethyl) aminomethane ((CH2OH)3CNH2) and 1M hydro-chloric acid (HCl) at 378C.22

Nonsterilized, disk-shaped samples with the size of3 mm in thickness, 10 mm in diameter were cut, polishedwith diamond paste, washed in distilled water, and driedin a vacuum at room temperature. Samples were thensoaked in 30 mL of SBF at 378C for 1, 3, 5, 7, and 14 dayswithout shaking and change the solution during soakingfor each sample. After soaking, specimens were removedfrom the SBF solution, gently rinsed with deionized water,and dried at room temperature. Scanning electron micros-copy (FE-SEM, Hitachi, S-4300SE, JPN) and energy disper-sive X-ray spectrometer (EDX) were used to monitor themorphology and composition of apatite formed on thecomposite surfaces.

Ionic concentration measurement

At each sample time during the bioactivity assessment,samples of SBF were removed to measure calcium (Ca),phosphorous (P), and silicon (Si) ion concentrations in theSBF solution using inductively coupled plasma atomicemission spectroscopy (ICP-AES).

Cell attachment and proliferation assay

L929 fibroblast cell was used to evaluate the preliminarybiocompatibility of n-CS/PCL composite. For attachmentand proliferation studies, disc-shaped samples of 10 mmin diameter and 3 mm in thickness were sonicated in etha-nol and sterilized using ultra-violet light. For cell adhesionexperiments, L929 fibroblast cells were seeded on the sam-ples at a density of 2 3 103 cells/sample. Cells wereallowed to adhere for 1 h before each well was gentlyflooded with 1 mL of medium. Cell attachment was deter-mined after incubation for 4 h. Cell proliferation was eval-uated after seeding cells at a density of 2 3 103 cells/sam-ple, followed by incubation for 1, 4, and 7 days, with themedium replaced every second day. Viable cells on thesubstrates were assessed quantitatively using the MTTassay. In brief, sample-cell constructs were placed in cul-ture medium containing MTT and incubated in a humidi-fied atmosphere at 378C for 4 h. After the supernatantswere removed, dimethyl sulfoxide (Sinopharm, Shanghai,China) was added to each well to completely dissolve theMTT reagent. The optical density (OD) of each well wasmeasured at 590 nm in a microplate reader (ELX 800, Bio-Tek, USA) using a reference wavelength of 620 nm. Sixspecimens of each sample were tested for each incubationperiod, and each test was performed in triplicate. Resultsare reported as OD units.

Statistical analysis

Statistical analysis was performed using one-wayANOVA with post hoc tests. All results are expressed asthe mean 6 SD. Differences were considered statisticallysignificant at p < 0.05.

RESULTS

SEM analysis

In scanning electron micrographs, the synthesizedn-CS was uniformly needle-like, with powderagglomerating among the particles (Fig. 1). This maybe due to the small particle size, with high surfaceenergy resulting in their aggregation. High magnifi-cation shows that the n-CS aggregations were com-posed of needle-like grains 50–90 nm in diameterand 100–250 nm in length.

TABLE IIon Concentrations in Simulated Body Fluid (SBF) and

Human Blood Plasma

Specimen

Ion Concentration (mM)

Naþ Kþ Mg2þ Ca2þ Cl2 HCO32 HPO422 SO4

22

SBF 142.0 5.0 1.5 2.5 147.8 4.2 1.0 0.5Blood

plasma 142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5

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XRD analysis

In the XRD patterns of PCL, the n-CS/PCL 40%composite, and n-CS, the CaSiO3 powder tracedepicts only a single peak and low background,indicating the occurrence of a low-crystallized state(Fig. 2).

Hydrophilicity determination

Water contact angles on the composite specimenswere significantly reduced with increasing n-CS con-tent (p < 0.01; Table II), indicating improved hydro-philicity.

Mechanical properties

Composite samples were not completely densedue to the solvent-casting method. The compressive

strength and elastic modulus of compositesincreased with the increasing n-CS content (p < 0.05;Table III). The 40% (w/w) n-CS sample resulted inthe highest compressive strength (54 MPa) and elas-tic modulus (537 MPa), indicating that the additionof n-CS to PCL improves the mechanical propertiesto a point. The highest n-CS content (60%) yielded alower compressive strength (37 MPa) and elasticmodulus (316 MPa) than did C40 (p < 0.05; TableIII), indicating that there is a critical concentrationbeyond which additional n-CS diminishes the me-chanical properties of the composite.

Apatite formation in SBF

In the surface morphology of C20 and C40 compo-sites, the n-CS was entrapped by the PCL polymermatrix, and it was difficult to distinguish n-CS par-ticles and PCL (Fig. 3). The surface of both speci-mens was coarse, but the surface roughness of C40was higher than that of C20.

After soaking in SBF for 7 days, typical sphericalapatite granules were partly formed on the surfaceof C20 [Fig. 4(a)], whereas spherical apatite granuleswere fully formed on the surface of C40 [Fig. 5(a)].After soaking in SBF for 14 days, the apatite depositson both C40 and C20 showed typical spherical gran-ules in densely packed apatite layers [Figs. 4(b) and5(b)]. Higher magnifications [Figs. 4(c,d) and 5(c–f)]showed that the spherical deposits contained manyflake-like crystals. Granules on the C20 specimenhad a loosely packed apatite layer, whereas those on

Figure 1. Scanning electron micrographs of nano calcium silicate with different modification. (a) 330 K, (b) 360 K.

Figure 2. X-ray diffraction pattern of (a) poly(e-caprolac-tone) (PCL), (b) calcium silicate (CS)/PCL composite with40% (w/w) nano-sized CS (n-CS), and (c) n-CS.

TABLE IIWater Contact Angles on Poly(e-caprolactone) (PCL) and

Composite Specimens (C20–C60; mean ± SD, n 5 5)

Sample Water Contact Angle (8)

PCL 77.5 6 0.8C20 59.2 6 1.2C40 26.7 6 0.9C60 0

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the C40 specimen showed a densely packed apatitelayer. The flake-like apatite crystals that formed onthe surfaces of both C40 and C20 were 100–200 nmin length and 20–60 nm in width.

The initial size and number of apatite crystals onthe surface of C40 were larger than those on the sur-face of C20; this was maintained throughout the 14days of the test period. The sphere-like apatite gran-ules on these two composites became flake-like andmore compact from 7 to 14 days. As well, the apatiteformation rate increased with increasing n-CS con-tent. We compared the surface morphology of purePCL specimens before and after soaking in SBF for14 days (Fig. 6). No apatite deposits formed on thePCL surface, indicating that pure PCL does not havethe ability to form apatite within this timeframe.

EDX analysis of formed apatite

The EDX spectra were examined for the apatitelayers that formed on the surfaces of compositespecimens after soaking in SBF for 14 days (Fig. 7).The Ca and P peaks were detected and the Ca/Pratios of the C40 and C20 specimens were 1.61 and1.58 (data not shown), respectively, which is lessthan the 1.67 Ca/P ratio in hydroxyapatite. Thisindicates that the apatite that formed on both com-posite samples was nonstoichiometric, with defectivestructure. Apatite did not develop on the surface ofPCL after soaking in SBF for 14 days (data notshown).

Changes in SBF solution ion concentrations

We examined the changes in the elemental concen-trations of calcium, phosphorous, and silicon in theSBF solutions after soaking the specimens for vari-ous periods (Fig. 8). Calcium and silicon concentra-tions increased quickly up to 3 days for all speci-mens, and thereafter, continued to increase at aslower rate up to 14 days. In addition, the compositewith higher n-CS content (i.e., C40) showed a moreintensive release of both Ca and Si ions compared tothe composite with lower n-CS content (i.e., C20). Incontrast to the increasing Ca and Si concentrations, Pion concentrations decreased gradually throughoutthe soaking period, and the decrease was more pro-nounced with the C40 than the C20 specimen.

Effect of composite on fibroblasts

In the MTT assay of L929 fibroblasts, the OD val-ues for composites were not significantly differentwhen compared to those of PCL samples after 1 day(Fig. 9). However, the OD values for both compo-sites were higher than for the PCL control at 4 and 7days (p < 0.05). In addition, the OD values for C40were significantly higher than those for C20 at 4 and7 days (p < 0.05).

In the MTT assay of L929 fibroblasts cultured onC40, the OD values for the composite after soakingin SBF were significantly higher than those of thestarting composite at 4 h, 1 day, and 4 days (p <0.05); there was no significant difference at 7 days(Fig. 10). After soaking in SBF for 14 days, C40improved fibroblast attachment (4 h) and prolifera-tion (1 and 4 days) relative to the starting composite,suggesting that apatite formation on composite sur-faces facilitates cell adhesion and proliferation.

DISCUSSION

Ideally, biomaterials need to interact actively withcells and tissues, and stimulate tissue regeneration.

TABLE IIIMechanical Properties of Poly(e-caprolactone) (PCL) and

Composite Specimens (C20–C60; mean ± SD, n 5 5)

SamplesCompressive

Strength (MPa)Elastic

Modulus (MPa)

PCL 37 6 2 191 6 8C20 n-composite 43 6 5 345 6 21

C40 54 6 6 537 6 24C60 37 6 5 316 6 12

Figure 3. Scanning electron micrographs of (a) C20 and (b) C40 composites.

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With the increase in orthopedic implants in clinicalapplications, efforts have continued to develop newbiocompatible implanted materials.23,24 Inorganic–or-ganic biocomposite containing bioactive ceramicsand biocompatible polymers may result in implantedmaterials that possess improved comprehensiveproperties relative to individual materials, possiblyovercoming the use limitations of bioactive ceramics,for example, brittleness and processing difficulty,and of polymer biomaterials, for example, bioactivityand hydrophobicity.25

We successfully designed and fabricated a com-posite consisting of PCL and n-CS using a nano cal-cium silicate slurry. The compressive strength andelastic modulus of the n-CS/PCL composites weresignificantly higher than those of PCL alone. Thus,the prepared composites possessed improved me-chanical properties compared to those of the purepolymer due to the incorporation of n-CS in PCL. Inthe composite system, the compressive strength andelastic modulus of C40 were significantly higherthan those of C20 and C60, suggesting that a weightratio of about 40% (w/w) would be optimal. Compo-sites containing high n-CS content, for example, 60%,may result in a dramatic reduction in the structuralintegrity and mechanical strength of the composite.In addition to mechanical properties, surface proper-ties greatly affect the performance of an implantedmaterial. The hydrophilicity of biomaterials is an im-portant factor for cell adhesion and growth, with

improved surface hydrophilicity improving interac-tions between the material and cells to elicit con-trolled cellular adhesion and to maintain differenti-ated phenotypic expression.26–28 The water contactangles of the n-CS/PCL composites showed remark-able improvement in hydrophilicity through theincorporation of n-CS, suggesting that the incorpora-tion of hydrophilic inorganic materials into hydro-phobic polymers is a viable method of improvingthe hydrophilicity of polymers.

A common feature among bioactive materials isthat they can bond to living bone through bone-likeapatite layers formed on their surfaces when in con-tact with SBF.29 Bone-like apatite plays an essentialrole in the formation, growth, and maintenance of thetissue-biomaterial interface.30 The n-CS was com-pounded as bioactive filler in PCL to form novel bio-active n-CS/PCL composites; these showed the abilityto form apatite layers on the composite surfaces uponsoaking in SBF. Because the apatite formation rate onC40 specimens was higher than that on C20 speci-mens, we conclude that apatite formation on n-CS/PCL was significantly dependent on the CS content.We suspect that the resulting higher concentration ofn-CS at the composite surface increased the formationof apatite. Thus, it may be desirable to control the n-CS surface content in the composite according to itsapplication. Bone-like apatite could not form on thesurface of pure PCL after soaking in SBF for 14 days,indicating that pure PCL was not bioactive.

Figure 4. C20 composite specimens immersed in simulated body fluid for (a) 7 days and (b–d) 14 days at differentmagnifications.

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The Ca and Si ion concentrations in the SBFincreased with time during the soaking period, andincreased more rapidly with the C40 than with the

C20 specimens. This increase was attributed to thedissolution of Ca and Si ions from the composite.Although the formation of the bone-like apatite layer

Figure 5. C40 composite specimens immersed in simulated body fluid for (a) 7 days and (b–f) 14 days at differentmagnifications.

Figure 6. Scanning electron micrographs of poly(e-caprolactone) (a) before and (b) after immersion in simulated bodyfluid for 14 days.

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consumed some Ca ions, Ca ion dissolution from thecomposite exceeded its consumption.31,32 The P ionconcentration in the SBF decreased graduallythroughout the soaking period, with the decrease forthe C40 specimens exceeding that for the C20 speci-mens. This indicates that C40 formed more apatitethan C20. The decrease in P concentration was attrib-uted to the formation of amorphous calcium phos-phate and the subsequent formation of apatite byincorporating OH2 ions from the SBF,33 providingan indirect indication that a precipitation reactionhad occurred.

The EDX results showed that the Ca/P molarratios of the apatite that formed on the C20 and C40specimens were 1.58 and 1.61, respectively. Accord-ing to the formula for nonstoichiometric apatite:

Ca10�xðOHÞ2�xðHPO4ÞxðPO4Þ6�xð0 � x � 1Þ

When x 5 0, the apatite is hydroxyapatite (Ca/P 5

1.67); when x 5 1, the apatite is tricalcium phosphate(Ca/P 5 1.5), which is a special nonstoichiometricapatite called apatite-TCP.34 When the Ca/P molarratio is between 1.67 and 1.5 (0 < x < 1), the apatiteis expressed using the above formula and callednonstoichiometric apatite. This apatite has defective

structure relative to stoichiometric hydroxyapatite;nonstoichiometric apatite is similar to the bioapatitein human bone and is usually called bone-like apa-tite.35 Therefore, the results suggest that the apatitethat formed on the surfaces of the n-CS/PCL compo-sites was nonstoichiometric apatite.

The mechanism of apatite formation on wollaston-ite in SBF is Ca2þ release and ionic interchange ofCa2þ for 2Hþ, resulting in the formation of an amor-phous silica layer on the surface of wollastonite andproviding a favorable site for apatite nucleation.36,37

The dissolution of wollastonite increases the ionic ac-tivity product of apatite in SBF, thereby promotingthe nucleation of apatite.38 We found that althoughn-CS was imbedded in the PCL matrix, the micropo-rous structure (pore size less than 1 lm) of the com-posites allows the release of Ca and Si ions to sup-port apatite deposition. Furthermore, the profile ofchanges in Ca, Si, and P ion concentrations in SBFwere similar to those of bioactive wollastoniteceramics. These results suggest that the mechanismof bone-like apatite formation on the surfaces of n-CS/PCL composites is similar to that of bioactivewollastonite ceramics.

There are some reports that ionic dissolution prod-ucts containing Ca and Si from bioactive glasses can

Figure 7. Energy dispersive X-ray spectrum of a C40 specimen soaked in simulated body fluid for 14 days. [Color figurecan be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 8. Changes in Ca, Si, and P concentrations in the simulated body fluid with (a) C20 and (b) C40. [Color figure canbe viewed in the online issue, which is available at www.interscience.wiley.com.]

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stimulate osteoblast proliferation and gene expres-sion.39 Xynos et al.40 showed that ionic products, Siand Ca in particular, could stimulate osteoblast pro-liferation. Chou et al.41 reported that sol–gel bio-glass1 has a significant osteogenic effect by releasinga high level of Si. The inductively coupled plasmaatomic emission spectroscopy results showed that Siand Ca ions could be released from the compositesin SBF and that Ca and Si ion concentrationsincreased more quickly from C40 than from C20 toprovide a higher basic ion concentration in SBF solu-tion. MTT tests showed that the C20 and C40 com-posites significantly stimulated L929 fibroblast prolif-eration over that on PCL alone, with proliferationfor C40 being significantly higher than for C20.Thus, the promotion of cell proliferation by thecomposites depended largely on the CS content.Therefore, the dissolution associated with the com-posites produces a calcium and silicon rich environ-ment that may be responsible for stimulating cellproliferation.

Bone-like apatite layers may provide a suitablesubstrate for osteoblast-like cell adhesion, prolifera-tion, and function, which would provide a strongbond between the implanted material and the sur-rounding bone tissue.42 Loty et al.43 found that a bio-logical apatite layer on A-W glass-ceramic promotedosteoblast-like cell differentiation and subsequentapposition of bone matrix in rats. Olmo et al.44 sug-gested that the biocompatibility of bioactive sol–gelglasses with apatite layers were greatly enhancedcompared to those without an apatite layer. TheMTT tests showed that L929 fibroblasts adhered bet-ter to C40 after soaking in SBF for 14 days than tothe starting C40 composite within the first 4 h of cul-ture. The cell proliferation levels on C40 after soak-

ing in SBF for 14 days were significantly higher thanthat of the starting composite after 1 and 4 days ofculturing (p < 0.05). Apatite formation on the com-posite surface promoted cell attachment and prolifer-ation during the early stages of cell culture. Thus,the apatite that formed on the composite surfacesprovides a good environment for the attachment andproliferation of cells.

CONCLUSION

Novel bioactive composites containing n-CS andPCL were successfully synthesized directly using n-CS slurry, not dried n-CS powder, in a solvent-cast-ing method. The addition of n-CS to PCL resulted ina structure with better mechanical properties andhydrophilicity than PCL alone, with the enhance-ment largely dependent on the n-CS content. The n-CS/PCL composites were bioactive; apatite layersformed on their surfaces in an n-CS content depend-ent manner after immersion in SBF. The Ca/P ratiosof the apatite formed on C20 and C40 were 1.58 and1.61, respectively, representing nonstoichiometricbone-like apatite with defective structure. Apatitewas formed through the release of Ca and Si ionsfrom the composites, similar to the mechanism withCaO-SiO2-based bioactive ceramics. The n-CS/PCLcomposite significantly promoted fibroblast cell pro-liferation over pure PCL, with the enhancementincreasing with increased n-CS content. The apatitelayers that formed on the composite surfaces facili-tated cell attachment (4 h) and proliferation duringthe early stages (1 and 4 days), although there wasno difference at 7 days. The effects of n-CS contenton mechanical properties, hydrophilicity, and the

Figure 9. MTT assay of L929 fibroblasts at different cul-ture times with (a) poly(e-caprolactone) (PCL), (b) compos-ite specimen C20, and (c) composite specimen C40. Datarepresent the mean 6 SD, n 5 6. [Color figure can beviewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 10. MTT assay of L929 fibroblasts cultured on C40(a) before and (b) after soaking in simulated body fluid for14 days. Data represent the mean 6 SD, n 5 6. [Color fig-ure can be viewed in the online issue, which is available atwww.interscience.wiley.com.]

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apatite formation rate, as well as on promoting cellproliferation, varied with the level of n-CS content.Thus, it is desirable to control the CS content in thecomposite according to the intended application.Our results demonstrate initial in vitro cell compati-bility and bioactivity of n-CS/PCL biocompositesand their potential applications for bone repair.

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