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Biomaterials 30 (2009) 1080–1088

Contents lists avai

Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

Preparation and characterization of bioactive mesoporouswollastonite – Polycaprolactone composite scaffold

Jie Wei a,b, Fangping Chen a, Jung-Woog Shin b, Hua Hong a, Chenglong Dai a, Jiancan Su c,Changsheng Liu a,*

a Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology,Shanghai 200237, PR Chinab Department of Biomedical Engineering, Inje University, Gimhae, Gyeongnam 621-749, Republic of Koreac Department of Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai 200433, PR China

a r t i c l e i n f o

Article history:Received 8 August 2008Accepted 23 October 2008Available online 18 November 2008

Keywords:Mesoporous wollastoniteComposite scaffoldHydrophilicityBioactivityCell attachment and proliferation

* Corresponding author.E-mail addresses: biomater2006@yahoo.com.cn, n

0142-9612/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.biomaterials.2008.10.046

a b s t r a c t

A well-defined mesoporous structure of wollastonite with high specific surface area was synthesizedusing surfactant P123 (triblock copolymer) as template, and its composite scaffolds with poly(3-capro-lactone) (PCL) were fabricated by a simple method of solvent casting-particulate leaching. Themeasurements of the water contact angles suggest that the incorporation of either mesoporouswollastonite (m-WS) or conventional wollastonite (c-WS) into PCL could improve the hydrophilicity ofthe composites, and the former was more effective than the later. The bioactivity of the compositescaffold was evaluated by soaking the scaffolds in a simulated body fluid (SBF) and the results show thatthe m-WS/PCL composite (m-WPC) scaffolds can induce a dense and continuous layer of apatite aftersoaking for 1 week, as compared with the scattered and discrete apatite particles on the c-WS/PCLcomposite (c-WPC) scaffolds. The m-WPC had a significantly enhanced apatite-forming bioactivitycompared with the c-WPC owing to the high specific surface area and pore volume of m-WS. In addition,attachment and proliferation of MG63 cells on m-WPC scaffolds were significantly higher than that ofc-WPC, revealing that m-WPC scaffolds had excellent biocompatibility. Such improved properties of m-WPC should be helpful for developing new biomaterials and may have potential use in hard tissue repair.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

It is clear that a significant characteristic of bioactive materials istheir ability to bond with living bone through the formation of anapatite interface layer. Various types of biomaterials containingCaO–SiO2, such as bioactive glasses, AW glass–ceramics, andwollastonite (WS) have been investigated as bioactive biomaterialsfor tissue repair and replacement [1–3]. Recently, some studieshave shown that WS has excellent bioactivity and is a potentialcandidate of new biomaterials for tissue repair [4,5]. Nevertheless,the extensive use of WS is still limited by their brittle nature.Biodegradable polymers and their copolymers are widely used inbone regeneration, dental repair, orthopedic fixation devices, aswell as many other biomedical applications [6,7]. Yet, there exist nopure polymers that can effectively bond to bone in vivo, and thecomposites of biodegradable polymers with bioactive inorganicphases are still a promising approach. Biodegradable polymer,polycaprolactone (PCL) has attracted much interest because of its

ic7505@263.net (C. Liu).

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cost-efficiency, high toughness, and processability resulting fromits relatively low melting temperature, and a combination of PCLwith bioactive inorganic materials could bring the advantages ofboth materials [8].

Over the past few years, a family of mesoporous materials withhigh specific surface area has received much interest because oftheir potential applications in catalysts, optical devices, etc [9,10]. Inbiomaterials research, mesoporous materials have been mainlyinvestigated for use in drug delivery for controlled drug release[11]. Up to now, there were only a few reports about the applicationof mesoporous materials in biomaterial research for bone regen-eration, though there are a number of papers focusing on develop-ment of micro- and macroporous biomaterials in order toimprove their dissolution, cell incorporation, resorption, and boneregeneration [12,13].

Studies showed that increasing the specific surface area andpore volume of biomaterials for tissue repair may greatly acceleratethe kinetic process of apatite deposition and therefore, enhance thebone-forming bioactivity [14]. More importantly, precise controlover porosity, pore size, and internal pore architecture of bioma-terials on different length scales is essential for the understandingof the structure–bioactivity relationship and the rational design of

J. Wei et al. / Biomaterials 30 (2009) 1080–1088 1081

better bone-forming biomaterials [15]. As the mesoporous mate-rials have unique structural characteristics, it is reasonable toassume that mesoporous materials may possess different physical–chemical and biological properties from non-mesoporous bioma-terials. Recently, the study of mesoporous Si–Ca–P glasses provedthat the mesoporous glasses had better in vitro bioactivity thantraditional bioactive glasses, and mesoporous silicate materialshave been proposed for biomedical application as drug deliverysystems and bone tissue regeneration materials [16,17].

There have been a number of studies in the literature focusingon the composites created by combining biodegradable polymersand c-WS [18–20]. However, the bioactivities of these compositeswere dependent on the specific surface area of WS, and still notsatisfactory because of the low surface area of c-WS as comparedwith mesoporous materials. To the best of our knowledge, therewas no previous report about the preparation of m-WS/PCLcomposite scaffolds for use as bone repair material. It is expectedthat if the synthesis of highly ordered mesoporous bioactive WSwith high specific surface area and pore volume, was used insteadof c-WS, the bioactivity of the composites scaffold should beimproved. Therefore, in this study, m-WS/PCL composite scaffoldswere prepared, and the effects of m-WS on hydrophilicity,degradability, in vitro bioactivity, and cell attachment and prolif-eration on the composite scaffolds were investigated.

2. Materials and methods

2.1. Preparation and characterization of m-WS

The mesoporous wollastonite (m-WS) powders were prepared by sol–gelmethod. Briefly, 3.0 g of surfactant EO20PO70EO20 ((polyethylene oxide)20(polypro-pylene oxide)70(polyethylene oxide)20, P123) was dissolved in 120 mL of 2 M HCland 30 mL of distilled water solution while stirring at 37 �C in water bath untilthe solution became clear. 8.50 g of tetraethyl orthosilicate (TEOS) and 9.64 g ofCa(NO3)2$4H2O were then added into the solution. The mixture was stirred at 37 �Cfor 24 h, and the resulting precipitate was dried at 110 �C for 12 h in air without anyfiltering and washing. After that, the m-WS was calcined at 700 �C for 3 h to removethe surfactant template and obtain the final m-WS products, then the m-WS wereground into powders and sifted through 400 mesh sieve for future use. Theconventional wollastonite (c-WS) was also synthesized by an almost identicalprocess but without using surfactant (P123) as control. The morphology of m-WSpowders was analyzed using TEM (JEM2010, Japan) working at 200 kV N2 adsorp-tion–desorption isotherms were obtained using a Micromeritics porosimeter(Tristar 3000, USA) at 77 K under a continuous adsorption condition. Brunauer–Emmet–Teller (BET) and Barrett–Joyner–Halenda (BJH) analyses were used todetermine the surface area, pore size, and pore volume.

2.2. Preparation of m-WS/PCL composite scaffolds

The m-WS/PCL composite scaffolds were prepared by a solvent casting-partic-ulate leaching method using NaCl particles as the porogen. PCL, supplied as pellets(Mw ¼ 80,000; Sigma–Aldrich, St. Louis, MO, USA) were dispersed in 10 mLchloroform and then a certain amount of m-WS (or c-WS) powder was added intothe solution and continuously stirred for 24 h to disperse m-WS (or c-WS) uniformly.The ratio of the PCL and m-WS (or c-WS) mixture to chloroform was fixed at 10%(wt/v). Sodium chloride (NaCl) particles with diameters 300–500 mm were thenincorporated into the suspension, and the dispersion was cast into a B60 mm mold.The samples were air-dried under flowing air for 24 h to allow the solvent toevaporate and subsequently vacuum-dried at 40 �C for 48 h to remove anyremaining solvent. The samples were then immersed in deionized water for 72 h,and during the period the water was replaced approximately every 6 h with freshwater at room temperature to leach the salt particulates out. Then the samples weretaken out, air-dried for 24 h, and vacuum-dried overnight to obtain the sponge-likescaffolds. The composite scaffolds with the addition of 20 and 40 wt% m-WS (or c-WS) were prepared, respectively. Pure PCL scaffolds were also prepared as control.

2.3. Mechanical properties, hydrophilicity and degradability

The mechanical strength of m-WPC and c-WPC samples (10� 10� 10 mm3) wasmeasured at room temperature using a compression test at a constant displacementrate of 2 mm/min. The jigs were specially designed to provide a uniform load on thecomposite samples, and a Bionix858.20 (MTS Systems Corp., Eden Prairie, MN, USA)was used to conduct tests. The compressive strength and elastic modulus werecalculated within the linear range of the stress–strain curve. For each composition,four to five specimens were tested and the results were averaged.

The hydrophilicity of m-WPC and c-WPC with desired ratio of WS was evaluatedby measuring the water contact angles of the nonporous discs with the size of10 � 10 � 3 mm3 (contact angle system, OCA15, dataphysics Co., Germany), and PCLas control. The water contact angle method was employed to determine the polarinteractions across the material–water interface. The sample was mounted on theplatform and a goniometer (Rame Hart, NJ, USA) was aligned and focused on thematerial–air interface. Water droplets of 10 mL formed by a micro-syringe(Perfektim, Popper & Sons Inc., Japan) were dropped onto three different points ofeach sample surfaces and water contact angles were measured after 30 s. Thepresent data represent the mean � SD of four to five contact angles per sample.

The weight loss of the m-WPC, c-WPC and PCL specimens with the size of10 � 10 � 3 mm3 during degradation in phosphate buffered saline (PBS) (pH 7.4)solution with refreshing daily was measured by the changes in dry weight afterincubation for specified time periods, and the buffer was refreshed every 3 days.After soaking, the specimens were removed from the liquid, rinsed with distilledwater, and dried in a vacuum oven for 12 h. All the values presented are the averageof three specimens. Percent weight loss was computed according to the followingequation:

Weight lossð%Þ ¼ 100ðW0 �WtÞ=W0

where W0 is the starting dry weight and Wt is the dry weight at time t.

2.4. Scaffolds soaking in simulated body fluid

The SBF was prepared and buffered at pH 7.4 with tris-(hydroxymethyl)-aminomethane [(CH2OH)3CNH2] and hydrochloric acid (HCl). The bioactivity ofm-WS (or c-WS)/PCL composite scaffolds in vitro was tested by immersing scaffoldsin SBF at a concentration of 1 mg/mL at 37 �C to monitor the formation of HA on thesurface of scaffolds with time. Scaffold samples were immersed in simulated bodyfluid (SBF) for 1 and 2 weeks. Soaking was carried out at 37 �C with stirring andrefreshing the SBF solution at every 2 days. After soaking, samples were removedfrom SBF, gently washed with deionized water, and dried at room temperature.

The surface morphology and composition of the composite scaffolds before andafter soaking in the SBF solutions were characterized by scanning electron micros-copy (SEM; JSM-6700F, JEOL, Tokyo, Japan), and energy-dispersive X-ray spectro-meter (EDS, INCA Energy, Oxford Instruments, UK). Average porosity of thesescaffolds was measured using the Archimedes’ principle. The phase composition ofthe m-WS, m-WPC and m-WPC immersed in SBF for 1 week was examined by X-raydiffraction (XRD, Geigerflex, Rigaku Co., Japan). The SBF solutions after soaking for 1,3, 5 and 7 days were collected for determining the ion concentrations of Ca, P, and Siby inductively coupled plasma atomic emission spectroscopy (ICP-AES; Varian,USA).

2.5. Cell attachment and proliferation

For attachment and proliferation studies, m-WPC and c-WPC samples with thesize of 10 mm in diameter and 3 mm thick were sonicated in ethanol and sterilizedusing ultra-violet light. For cell adhesion experiments, MG63 cells were seeded onthe discs at a density of 2�103 cells/disc. Cells were allowed to adhere for 1 h beforeeach well was gently flooded with 1 mL of medium. Cell attachment was determinedusing the MTT assay after incubation for 4 h. Cell proliferation was evaluated afterseeding cells at a density of 2 � 103 cells/disc, followed by incubation for 1, 4, and 7days, with the medium replaced every 2 days. Adhesion and viable cells on thesubstrates were assessed quantitatively using the MTT assay. In brief, composite-cellconstructs were placed in culture medium containing MTT and incubated ina humidified atmosphere at 37 �C for 4 h. Cell growth was determined by an MTTassay (MTT Kit, Roche Diagnosis Corp., IN, USA). The absorbance value was measuredat 570 nm with microplate reader. Six specimens were tested for each incubationperiod, and each test was performed in triplicate. Results are reported as OD units.

2.6. Statistical analysis

Statistical analysis was performed using one-way ANOVA with post hoc tests. Allresults are expressed as the mean � standard deviation (SD). Differences wereconsidered statistically significant at p < 0.05.

3. Results

3.1. Characterization of the m-WS powders

Fig. 1 illustrates the transmission electron micrographs of thecalcined m-WS with different magnifications. The TEM imagesshow that the m-WS with a mesoporous pore diameter of about4 nm has the highly ordered 1-D cylindrical channels of hexagonalarrangement. It can be obtained that the prepared m-WS hasa uniform mesoporous structure.

Fig. 1. Transmission electron micrographs of mesoporous wollastonite with different magnifications, (a) �50k and (b) �400k.

J. Wei et al. / Biomaterials 30 (2009) 1080–10881082

N2 sorption isotherms of m-WS and c-WS are shown in Fig. 2.The BET surface area, pore volume, and pore size were calculated tobe 405 m2/g1, 0.55 cm3/g, and 3.55 nm, respectively. For compar-ison, calcined c-WS synthesized without surfactant P123 hasa much lower BET surface area and pore volume of 47 m2/g and0.04 cm3/g, respectively. Nitrogen sorption isotherms furtherconfirm the existence of mesoporous structure, which gives thepore size distribution curve calculated from the adsorption branchby the BJH model. The pore size distribution of m-WS is narrow,indicating the m-WS has a uniform mesoporous structure and thepores were homogeneous. The nitrogen sorption result is consis-tent with the TEM image of the m-WS.

3.2. Characterization of the composite scaffolds

The mechanical strength of compressive strength of thecomposite scaffolds decreased with increasing porosity as shown inTable 1. The porosity was significantly affected by the amount of theporogens (NaCl particulates) used in making the scaffolds.

The surface morphology and microstructure of the as-fabricatedm-WS/PCL composite porous scaffolds are shown in Fig. 3. The

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00

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Relative Presure (P/P0)

AdsorptionDesorption

3

Vo

lu

me A

dso

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ed

cm

3/g

S

TP

a

Fig. 2. (a) N2 sorption isotherms and (b) the pore

scaffold exhibited a macroporous structure with completely inter-connected open pores. The high magnification SEM micrographshows that the pores were almost spherical in shape and the poresize varied from 300 to 500 mm. Some small-sized pores, generallyin the sizes of 1–10 mm, were easily observed distributed across themacropore walls, and the thickness of the wall was about 50–100 mm. The porosity of composite scaffolds prepared by thismethod could reach about 80%.

3.3. Hydrophilicity and degradability of the m-WS/PCL composite

Table 2 shows the results of the measurements of water contactangles of the sample surfaces. It can be seen that water contactangles on both the m-WPC and the c-WPC specimens weresignificantly reduced with increasing WS content (p < 0.01; Table2), indicating improved hydrophilicity. Moreover, the water contactangles of the m-WPC were lower than c-WPC both 20 wt% and40 wt% c-WS content in the composites, which indicated that thehydrophilicity of m-WPC was higher than c-WPC.

Fig. 4 presents weight loss in both (a) m-WPC and (b) c-WPCsamples as a function of incubation time. The figure reveals that

10 1000.00

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re V

olu

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b

size distribution of mesoporous wollastonite.

Table 1Mechanical properties of the m-WPC scaffolds with different porosities.

Samples NaCl/PCL(wt/wt)

Porosity(%)

Compressive strength(MPa)

1 8:1 80 � 3.1 3 � 0.52 7:1 74 � 2.3 5 � 0.73 6:1 63 � 1.7 10 � 1.24 5:1 57 � 2.1 12 � 1.1

Table 2Water contact angle of m-WPC and c-WPC with different WS content (wt%), poly-(3-caprolactone) (PCL) as control.

Samples Water contact angles (�)

m-WPC c-WPC

PCL 77.5 þ 0.8WS (20%) 41.8 � 1.2 53.4 � 1.0WS (40%) 0 22.6 � 1.3WS (50%) 0 0

J. Wei et al. / Biomaterials 30 (2009) 1080–1088 1083

weight loss in both types of samples increased with incubationtime, 37 and 28 wt% for m-WPC and c-WPC containing 40 wt% WScontent after 12 weeks, respectively. Weight loss in m-WPCsamples was significantly greater than c-WPC at 12 weeks. Pure PCLsamples exhibited no significant weight loss, losing only 2.1% of itsinitial weight at 12 weeks.

3.4. Apatite formation on the composite scaffolds

SEM images of the m-WPC and c-WPC scaffolds before and aftersoaking in SBF for 1 week are shown in Figs. 5–7. For the compositescaffolds, m-WS and c-WS particles attached on the pore surfaceand/or embedded in PCL shown in Fig. 5. After soaking for 1 week,the surface morphology of the composite scaffold has changed anda continuous layer of dense deposits formed on the surface of them-WPC (Fig. 6a,b). A higher magnification examination shows thatthe sizes of the elongated deposit grains were 200–400 nm inlength (Fig. 6c). For the c-WPC scaffold, the morphological char-acteristics before soaking in SBF are similar to m-WPC shown inFig. 5. However, after soaking for 1 week, there were some scatteredand discrete deposits on the surface of the composite scaffold, andthe grain sizes of the deposits were also about 200–400 nm inlength (Fig. 7c).

The prerequisite for biomaterial to bond to living bone is theformation of a bioactive apatite layer on the surface of implant inthe body. Therefore, in this experiment, the bone-forming activityof the m-WS based composite in vitro was tested in SBF to monitorthe formation of apatite on its surface. The EDS spectra wereexamined for the apatite layers that formed on the compositesurfaces after soaking in SBF for 1 week. Both Ca and P peaks weredetected as shown in Fig. 6d and Fig. 7d; Ca/P ratios for m-WPC andc-WPC specimens were 1.63 and 1.75, respectively, almost similarto the 1.67 Ca/P ratio of hydroxyapatite.

The XRD patterns of m-WS (a), m-WPC (b) and m-WPC (c)immersed in SBF for 1 week as shown in Fig. 8. It can be shown thatthe apatite phase presents on the scaffold surfaces and the peak ofm-WS disappears after m-WPC immersed in SBF for 1 week(Fig. 8c). The analysis of XRD results is consistent with the resultsfrom SEM and EDX.

Fig. 3. SEM images of the m-WPC scaffolds at d

3.5. Ion concentration of the SBF solution

Fig. 9 shows the changes in ion concentrations of Ca, P, and Si inSBF solutions for the m-WPC and c-WPC scaffolds measured by ICPafter soaking for various time periods. The Ca and Si concentrationsin SBF increase while P concentration decreases. It is obvious thatthe m-WPC scaffold shows a more intensive release of Ca and Siions as compared with the c-WPC scaffold, and the P concentrationfor the m-WPC scaffold decreases more quickly than c-WPC. Inaddition, the P concentration for the m-WPC scaffold is slightlylower than that of c-WPC at the end of the experiment.

3.6. Cell attachment

The relative number of cells adherent to the different materialswas assessed using MTT assay because optical density absorbancevalues can provide an indicator of the relative number of cells onthe composites. Cell attachment was investigated using MTT assayof MG63 cells cultured on the m-WPC, c-WPC and pure PCL, andtissue culture plate as a control. The results of cell attachment areprofiled in Fig. 10. In a period of 4 h, the absorbance value of the m-WPC is significantly higher than that of c-WPC, pure PCL andcontrol (p < 0.05). The results show that improved cell attachmentrelative to the m-WPC specimens than that of c-WPC, suggestingthat the m-WPC specimens facilitate cell adhesion on their surfacesand can promote cell growth.

3.7. Cell proliferation

The proliferation of MG63 osteoblast-like cells cultured on bothm-WPC and c-WPC was assessed using MTT assay because ODvalues can provide an indicator of the cells growth and proliferationon the different materials, and tissue culture plate as a control. It canbe seen from Fig. 11 that the OD values for m-WPC specimens aresignificantly higher those of c-WPC at 4 days, and 7 days (p < 0.05);there is no significant difference at 1 day. The results show thatimproved cell growth and proliferation (4 and 7 days) relative to them-WPC specimens than c-WPC, suggesting that the m-WPC speci-mens facilitate cells growth and can promote cell proliferation.

ifferent magnificent, (a) �70 and (b) �250.

0

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1 2 3 5 7 9 12Time (week)

We

ig

ht lo

ss

(w

t %

)

abc

Fig. 4. Change of weight loss of (a) m-WPC and (b) c-WPC with 40 wt% WS content inSBF with time, (c) pure PCL as a control. Data represent the mean � standard deviation,n ¼ 5.

J. Wei et al. / Biomaterials 30 (2009) 1080–10881084

4. Discussions

A biocompatible and biodegradable scaffold with high levels ofporosity, suitable pore size, and highly interconnected pore struc-ture is required to offer sufficient space for tissue growth and toincrease the volume of the invasion of surrounding tissue [21]. Ithas been found that pores between 50 and 150 mm determineosteoid growth and pores larger than 150 mm facilitate proliferationof cells, vascular ingrowth, and internal mineralized bone forma-tion [22]. In this study, about 80% porosity of the m-WPC scaffoldwith the addition of 40 wt% m-WS content can be achieved. Thesecomposite scaffolds exhibit open macropores and pore sizes mainlyin the range of 300–500 mm, which is in accord with the size ofthe salt particles, and the porosity of scaffold increases with theincrease of porogen (NaCl particles) used in the experiments. Theporosity of the composite scaffold has obvious effect on itscompressive strength. The higher the porosity, the lower thecompressive strength of the scaffold has, and the scaffolds exhibitthe maximal porosity of 80%, with minimal compressive strength ofis 3 MPa.

The surface characteristics would greatly affect the performanceof a biomaterial in a biological environment. For example, thehydrophilicity of materials is an important factor for cell adhesionand growth, and improved surface hydrophilicity of materials is

Fig. 5. SEM images of (a) m-WPC and (b) c

beneficial to the interactions between materials and cells througheliciting controlled cellular adhesion and maintaining differenti-ated phenotypic expression [23]. The results of the present studyshow that a significant improvement of the hydrophilicity of twokinds of composites with the addition of WS into PCL. The watercontact angle of the m-WPC decreases from 77.5 to 0 while c-WPCdecreases from 77.5 to 22.6 with the WS content increases from 0 to40 wt% in the composite. The different texture of m-WS and c-WSaccount for this different result as m-WS has high surface area andwell-defined pore channels. In addition, the m-WS has a muchhigher pore volume and therefore lower apparent density than c-WS, the addition of m-WS and c-WS with the same weight ratiointo PCL would result in higher apparent volume fraction of m-WSphase in m-WPC than that c-WS in c-WPC, which may be respon-sible for the quickly decrease of the contact angle for the m-WPCthan that of c-WPC. For the higher loadings of WS (m-WPC� 40% orc-WPC � 50%) in the composites, water contact angles of thecomposites were 0 (contact angle was determinate as 0� whenwater droplet was absorbed into composite). The water dropletabsorbed into the composite was due to the capillary effect(adsorption water filling up the pores of the m-WPC). Therefore, thecapillarity also affected the contact angle of the composites.

The weight loss of both m-WPC and c-WPC in PBS increasedwith the incubation time and weight loss in m-WPC samples wassignificantly higher than m-WPC after 2 weeks. 37 wt% of the m-WPC while 28 wt% of c-WPC of their initial weight were lost at theend of the degradation experiment (12 weeks). In addition, thepure PCL did not show significant weight loss during incubationand lost only 2.1% of its initial weight after 12 weeks, indicating thatthe increased weight loss of the composites might be mainly theresult of the degradation of WS. The results suggest that m-WPChas a much faster degradation than c-WPC because of its micro-structure, such as, huge specific surface area and pore volume.

It has been proposed by Kokubo that the examination of apatiteformation on a material in SBF is useful for predicting the in vivobone bioactivity of a biomaterial, and the formation of the apatitelayer shows the ability of a given implanted material to forminterfacial bonds with tissues when in contact with physiologicalfluid [24]. There are several reports about the potential ofcombining the bioactive materials and polymers to form bioactivecomposite materials for bone repair biomaterials [25,26]. In thisstudy, m-WS or c-WS, as a bioactive and degradable inorganiccomponent, was incorporated into PCL to form novel compositeswith good bioactivity, and an apatite layer was formed on the poresurface of the composite after soaking in SBF solution.

ICP measurement results suggest that the Ca and Si concentra-tions in SBF increased while P concentration decreased due to theformation of apatite by consuming P ions from SBF solutions during

-WPC scaffolds before soaking in SBF.

Fig. 7. SEM images (a–c) and EDX (d) of c-WPC scaffolds after soaking in SBF for 1 week at different magnifications. (a) �8k, (b) �30k, (c) �60k.

Fig. 6. SEM images (a–c) and EDX (d) of m-WPC scaffolds after soaking in SBF for 1 week at different magnifications. (a) �8k, (b) �30k, (c) �70k.

J. Wei et al. / Biomaterials 30 (2009) 1080–1088 1085

0

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In

te

ns

ity

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.u

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Fig. 8. XRD patterns of m-WS (a), m-WPC (b) and m-WPC (c) immersed in SBF for 1week, in which :, ;, C represent m-WS, PCL and apatite phase, respectively.

J. Wei et al. / Biomaterials 30 (2009) 1080–10881086

soaking. The m-WPC scaffold shows a more intensive release of Caand Si ions as compared with the c-WPC, and the P concentrationfor the m-WPC decreased more quickly than that for the c-WPCbecause of the consumption of P species in SBF for the formation ofapatite. These suggest that much shorter induction time for theapatite formation and, subsequently, significantly higher deposi-tion rate of apatite was obtained for the m-WPC in the presentinvestigation than c-WPC scaffold. In addition, the P concentrationfor the m-WPC scaffold was slightly lower than that of c-WPC at theend of the experiment. It could be concluded that the m-WPCexhibits significantly improved bioactivity as compared with the c-WPC scaffold. The most probable reason for the enhanced bioac-tivity of m-WPC is that the well-defined mesoporous structure ofthe m-WS has suitable pore size, high specific surface area and porevolume while c-WS does not.

The formation mechanism of apatite on bioactive biomaterials(such as bioglass) was proposed by Hench et al. [27]. The exchangeof hydrogen H3Oþ from the SBF solution with Ca in WS gives rise tothe formation of Si-OH groups and leaves behind a silica-richleached layer. Si-OH groups had been proposed to be a catalyzingagent and could provide specific favorable sites for the apatitenucleation [28,29]. Therefore, the formation of apatite may beclosely related to the amount of Si-OH groups present on thesurface of bioactive materials. The amount of Si-OH groups isrelated to the surface area and pore volume, which could accountfor the different bioactivities between the m-WPC and c-WPCscaffold in this study.

The BET surface area of the m-WS was calculated to be405 m2/g, which was almost 4 times of that of c-WS (47 m2/g).

0123456

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Elem

en

tal

co

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tra

tio

n (m

M)

Ca P Si

a

Fig. 9. Changes of Ca, Si and P concentrations of SBF solution after soak

The high specific surface area, together with the presence ofamounts of pores (pore volume, 0.55 cm3/g), facilitates theexchange of Ca2þ in the m-WS with H3Oþ from the SBF solutionand lead to higher density of Si-OH groups on the m-WS surface,which results in the higher apatite nucleation rate. Moreover,after the nucleation of apatite, the high specific area and highpore volume of the m-WS facilitate the transport of Ca2þ, HPO4

2�,OH�, and CO3

2- species from the SBF solution to the nuclei and thefast deposition of apatite.

The results show that the surface of the m-WPC scaffold wascovered with a dense apatite layer, which is apparently differentfrom the loose texture of apatite on the c-WPC surface. It isconsidered that the large but uniform pores about 4 nm of the m-WS should be responsible for the dense texture of apatite layer. Asthe superposition of the surface potentials inside the pores increasethe ionic concentration and degree of supersaturation of the Ca andP ions. It is believed that the uniform pore structure facilitates theformation of dense apatite, and the m-WS with a large enoughsurface area and pore volume can facilitate the transport of dis-solved Ca2þ and the subsequent dense deposition of apatite.However, c-WS does not show well-defined pore structure, andhigh specific surface area and pore volume; therefore, the formedapatite layer on c-WPC show loose structure.

In summary, there are two reasons for the difference in bioac-tivity between m-WPC and c-WPC scaffold. One is that the m-WPCscaffold with 40 wt% mesoporous wollastonite content has a lowercontact angle (0 for m-WPC) than that of c-WPC scaffold (22.6 for c-WPC). A lower contact angle facilitates the entrance of the SBFsolution into the composite and the improvement of the bioactivity.The other is that m-WS in the composite with high specific surfacearea and pore volume has a greatly enhanced apatite-formingbioactivity than conventional c-WS.

Cellular response and behavior can be influenced by the char-acteristics of material surfaces in vitro. Attachment belongs to thefirst phase of cell/material interactions and the quality of this firstphase will influence the cell’s capacity to growth, morphology andproliferate upon contact with the implant materials [30]. In thepresent study, the cell attachment experimental results shown thatMG63 osteoblast-like cells adhered better to m-WPC than that of c-WPC, PCL and tissue culture plate within the first 4 h of culture. Theenhancement of the MG63 attachment on m-WPC than on c-WPC islikely to be associated with material surface features, such asthe pore size, surface adsorbability, and protein adsorption of them-WPC and c-WPC in cell culture medium. The m-WS may havespecial surface properties due to its huge specific surface area thatpromote cells attachment, and the higher hydrophilic surface of m-WPC may be useful for cell adhesion than c-WPC.

It is known that the cellular responses to a material, such asattachment, growth and proliferation, depend not only on

Ca P Si

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0 1 3 5 7Time (days)

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tal

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tio

n (m

M)

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ing (a) m-WPC and (b) c-WPC with 40 wt% WS content with time.

0

0.05

0.1

0.15

0.2

0.25

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Control PCL m-WPC c-WPCSpecimens

O.D

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Fig. 10. MG63 attached on the surfaces of (a) m-WPC and (b) c-WPC at 4 h. Tissueculture plate as a control, data represent the mean � SD, n ¼ 5.

J. Wei et al. / Biomaterials 30 (2009) 1080–1088 1087

physical status (surface morphology of implant) but also onchemical compositions of the material [31,32]. The differentchemical compositions also play a crucial role in determining thecell responses, which was relevant to the level of ions releasedfrom the material and a consequent cell–material interaction.There are some reports that ionic dissolution products containingCa and Si from bioactive glasses can stimulate osteoblast prolif-eration and gene expression. Xynos et al. showed that ionicproducts, Si and Ca in particular, could stimulate osteoblastproliferation [33–37].

In our study, the inductively coupled plasma atomic emissionspectroscopy results show that Si and Ca ions could be releasedfrom the composites in SBF, and that Ca and Si ion concentrationsincreased more quickly from m-WPC than from c-WPC to providea higher basic ion concentration in SBF solution. Moreover, MTTtests showed that both the m-WPC and thec-WPC significantlystimulated MG63 osteoblast-like cells growth and proliferationwith the time, with cell growth and proliferation for m-WPCbeing significantly higher than for c-WPC. The continuous disso-lution of mesoporous wollastonite associated with the compositesproduces a calcium- and silicon-rich environment that might beresponsible for stimulating cell growth, and subsequentproliferation.

0

0.1

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0.3

0.4

0.5

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1 4 7Time (day)

O.D

m-WPCc-WPCControl

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Fig. 11. Proliferation of MG63 cells cultivated on the m-WPC and c-WPC for 1, 4, and 7days, data were represented as mean � standard deviation; n ¼ 5. Asterisks indicatethat the proliferation rate of cells seeded on m-WPC was significantly higher than thatof c-WPC at 4 and 7 days (p < 0.05).

5. Conclusions

Bioactive composite scaffold of poly(3-caprolactone) (PCL) andmesoporous wollastonite (m-WS) was fabricated in this study. Theincorporation of WS into PCL could improve the hydrophilicity ofthe composites, which was inorganic content dependent, and m-WS could improve the hydrophilicity of the composites moreeffectively than c-WS at the same content. The degradation of m-WPC in SBF was significantly higher than c-WPC during 12 weeks,which might be caused by the differences in the microstructure anddissolution between m-WS and c-WS in the composites becausethe m-WS has a higher specific surface and pore volume than c-WS.In vitro bioactivity studies of the m-WPC scaffolds shows that itcould induce a dense layer of apatite after soaking in SBF for 1 weekcompared with scattered and discrete apatite particles on c-WPCsurfaces. Owing to the well-defined mesoporous structure, suitablepore size, high specific surface area, and high pore volume, the m-WPC had an apparently enhanced apatite-forming bioactivitycompared with the c-WPC. MTT results show that the cellcompatibility of both m-WPC and c-WPC, and the attachment andproliferation of MG63 cells were significantly higher on the surfaceof the m-WPC than c-WPC. In conclusion, the m-WPC had thehydrophilicity, degradability, in vitro bioactivity and cell behaviorbetter than that of c-WPC. These findings suggest that the m-WPCscaffold was promising in terms of its mesoporous structure andbioactivity, and may find potential applications for bone repair.

Acknowledgements

The authors appreciate financial support from the NationalScience Foundation for Distinguished Young Scholars of China(Grant No.20425621), the Major State Basic Research Program ofChina (No. 2005CCA01000), Shanghai Pujiang Program (2008),Shanghai Leading Academic Discipline Project (B502) and Nanospecial program of Science and Technology Development ofShanghai (No. 0852nm02700).

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