Mesoporous bioactive scaffolds prepared with cerium-, gallium- and zinc-containing glasses

Acta Biomaterialia 9 (2013) 4836–4844

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Acta Biomaterialia

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Mesoporous bioactive scaffolds prepared with cerium-, gallium- andzinc-containing glasses

Shruti Shruti a, Antonio J. Salinas b,c,⇑, Gigliola Lusvardi a, Gianluca Malavasi a,⇑, Ledi Menabue a,M. Vallet-Regi b,c

a Department of Chemical and Geological Sciences, University of Modena and Reggio Emilia, Via G. Campi 183, 41125 Modena, Italyb Departamento de Quimica Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spainc Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain

a r t i c l e i n f o

Article history:Received 27 May 2012Received in revised form 12 September 2012Accepted 19 September 2012Available online 28 September 2012

Keywords:Scaffolds with hierarchical porosityMesoporous glasses with Ce2O3, Ga2O3 or ZnORapid prototypingIn vitro bioactivityBone tissue engineering

1742-7061/$ - see front matter � 2012 Acta Materialhttp://dx.doi.org/10.1016/j.actbio.2012.09.024

⇑ Corresponding authors. Addresses: DepartamenBioinorgánica, Facultad de Farmacia, Universidad CoMadrid, Spain. Tel.: +34 91 394 1861; fax: +34Department of Chemical and Geological Sciences, UniEmilia, Via G. Campi 183, 41125 Modena, Italy. Tel.059373543 (G. Malavasi).

E-mail addresses: [email protected] (A.J. Sunimore.it (G. Malavasi).

a b s t r a c t

Mesoporous bioactive glass scaffolds (MBG_Scs), based on 80% SiO2–15% CaO–5% P2O5 (in mol.%) meso-porous sol–gel glasses substituted with Ce2O3, Ga2O3 (both 0.2% or 1.0%) and ZnO (0.4% or 2.0%), weresynthesized by combination of evaporation-induced self-assembly and rapid prototyping techniques.Cerium, gallium and zinc trace elements were selected because of their inherent beneficial biologicalproperties. Fabricated scaffolds were characterized and compared with unsubstituted scaffold (B_Sc).All of them contained well interconnected ultralarge pores (pores >400 lm) ideal for vascular ingrowthand proliferation of cells. Macropores of size 100–400 lm were present inside the scaffolds. In addition,low-angle X-ray diffraction showed that B_Sc and scaffolds with substituent contents up to 0.4% exhib-ited ordered mesoporosity useful for hosting molecules with biological activity. The textural properties ofB_Sc were a surface area of 398 m2 g�1, a pore diameter of 4.3 nm and a pore volume of 0.43 cm3 g�1. Aslight decrease in surface area and pore volume was observed upon substitution with no distinct effect onpore diameter. In addition, all the MBG_Scs except 2.0% ZnO_Sc showed quite quick in vitro bioactiveresponse. Hence, the present study is a positive addition to ongoing research into preparing bone tissueengineering scaffolds from bioceramics containing elements of therapeutic significance.

� 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Bone regeneration is a natural phenomenon by which new boneis formed during the normal remodeling process, as well as afterinjury. However, there are certain clinical situations where bonecannot heal itself because the defect is too large or the bone haslost its regenerative capabilities. Bone tissue engineering hasemerged as a promising technique in such situations, stimulatingregeneration of host bone without posing constraints found in goldstandard bone grafting methods [1,2]. It utilizes three-dimensionalporous biomaterial scaffolds which act as a temporary frameworkproviding a suitable environment for normal cell growth, andhence helps in tissue regeneration [3].

The success of a synthetic scaffold depends on whether it satis-fies requirements similar to those found in nature for normal bone

ia Inc. Published by Elsevier Ltd. A

to de Quimica Inorgánica ymplutense de Madrid, 2804091 394 1786 (A.J. Salinas),

versity of Modena and Reggio: +39 0592055041; fax: +39

alinas), gianluca.malavasi@

development. Natural bones possess hierarchical porosity in therange of 1–3500 lm, which is necessary for several physiologicalfunctions [4]. Ideally the template must consist of an intercon-nected porous structure with �90% porosity. Pore sizes greaterthan 100 lm enable cell seeding, tissue ingrowths and vascularisa-tion. Pores in the microporous (<2 nm) or mesoporous (2–50 nm)range promote cell adhesion, adsorption of biological metabolitesand resorbability at rates controlled to match that of tissue repair[5–8].

Mesoporous bioactive glass scaffolds have set a mark in the fieldof bone tissue engineering for exhibiting a well-interconnectedmacroporous network along with mesopores, enabling them tocarry therapeutic drugs [9–12]. Recently, efforts have being madeto incorporate elements that have a relevant function and biologi-cal significance in the glass matrix [13–16]. This approach is con-sidered to be economical and stable, as such elements do notpose a risk of decomposition during scaffold manufacture [17].

Recently, the beneficial biological features of cerium, galliumand zinc have prompted scientists to study them in different glasssystems [18–22]. Studies have shown that cerium has a positive ef-fect on primary mouse osteoblasts in vitro and cerium oxide nano-particles act as neuroprotective agents [23,24]. In addition, galliumincreases bone calcium content, inhibits osteoclast activity and

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shows antimicrobial activity [25–27]. Zinc has a stimulatory effecton bone formation, and also shows antimicrobial activity [28,29].

These studies motivated us to incorporate xCe2O3, xGa2O3 orxZnO into sol–gel (BGs) and mesoporous bioactive glasses (MBGs)with the composition (80 � x)% SiO2–15% CaO–5% P2O5 (in mol.%)[30,31]. Structural characterization of Ce2O3-, Ga2O3- or ZnO-substituted BGs by 29Si magic angle spinning nuclear magneticresonance showed that the glass network connectivity of unsubsti-tuted BGs (75% of Q4 content) reduced upon the addition of Ce ions,which thus acts mainly as a network modifier, while gallium andzinc ions behave as intermediate ions. On the other hand, experi-mental results confirmed that MBGs substituted with a low con-centration of Ce2O3, Ga2O3 or ZnO maintained their mesoporousorder, high textural properties such as surface area of 462 m2 g�1,pore size of 4.4 nm, pore volume of 0.49 cm3 g�1 and the abilityto form apatite rapidly in vitro (except xZnO over 2.0%), and thatthe cerium, gallium and zinc were distributed homogeneously inthe glass network. On the other hand, xCe2O3, xGa2O3 and xZnO-BGs took �7–15 days for hydroxycarbonate apatite (HCA) forma-tion, except xZnO over 4.0%.

Preliminary experimental results indicate that Ce-, Ga- and Zn-MBGs possess the optimum properties required for a material to beused for fabrication of scaffolds. Thus, in the present study we tookthe further step of preparing three-dimensional (3-D) scaffoldsfrom quaternary MBGs substituted with cerium, gallium or zincelements, which possessed valuable features (see Fig. 1). The focusof the study was to determine if the fabricated scaffolds exhibithierarchical porosity and maintain the textural properties andin vitro response of MBG powder with the view to recommendingthem for bone tissue engineering.

2. Materials and methods

2.1. Synthesis of MBG powders

Cerium, gallium and zinc-containing (80 � x)% SiO2–15% CaO–5% P2O5 (in mol.%) mesoporous sol–gel glasses, the compositionsof which are given in Table 1, were synthesized according to amethod described in a previous publication [32] . In the presentstudy, glasses were prepared by replacing a small part of SiO2 withCe2O3, Ga2O3 or ZnO, whereas the amounts of CaO and P2O5 werekept constant in order to maintain the optimal Ca/P ratio required

Fig. 1. Schematic representation of possible biological properties possessed byCe3+-, Ga3+- and Zn2+-substituted MBG_Scs prepared by rapid prototyping: 3-Dprinting.

for fast bioactive response, as reported in another study on SiO2–CaO–P2O5 sol–gel glasses [33]. During synthesis, Pluronic P123,tetraethyl orthosilicate, triethyl phosphate, calcium nitrate(Ca(NO3)2.4H2O) and cerium nitrate, gallium nitrate or zinc nitratewere dissolved in ethanol containing 0.5 N HNO3. The sol obtainedwas added to a Petri dish to undergo evaporation-induced self-assembly (EISA) [34] for 7 days. The dried gels were then calcinedat 700 �C for 3 h in order to remove surfactant. The glasses thus ob-tained were subjected to milling to obtain grains of size below32 lm for mesoporous bioactive glass scaffold (MBG_Sc)preparation.

2.2. Preparation of MBG scaffolds by rapid prototyping: 3-D printing

The rapid prototyping technique [35] consists of reproducing aprevious computer-aided design format by injecting a paste using arobot injector.

2.2.1. Preparation of paste for robot injectionMBG powder (6 g) was suspended in dichloromethane with the

aid of ultrasound. Simultaneously, polycaprolactone (PCL) granules(4 g) were dissolved in the same quantity of dichloromethane bymagnetic stirring at room temperature. The dichloromethane con-taining the MBG powder was then added to the completely dis-solved PCL–dichloromethane solution. This mixture was allowedto evaporate by continuous magnetic stirring at room temperatureuntil it had formed a paste with the right consistency for injection.

2.2.2. Rapid prototyping: 3-D printing layer by layerMBG_Scs were prepared by introducing paste into a polyethyl-

ene injection cartridge that was fixed in an EnvisionTEC GmbH 3-DBioplotter™ printing device. The injection parameters were deter-mined using the computer program PRIMCAM version 2.98, whichdirects the injector robot. The speed of the tip was set at320 mm min�1 in the horizontal plane and 50 mm min�1 in thevertical plane. The orifice of the polyethylene conical tip usedwas 0.58 mm. The dimensions of the MBG_Scs were8 mm � 8 mm � 4 mm, and the scaffolds consisted of 10 layers.The MBG_Scs obtained were dried in an oven at 70 �C for 2 h toevaporate the dichloromethane and then calcined at 500 �C for3 h to remove the PCL.

2.3. Characterization of scaffolds

Powder X-ray diffraction (XRD) experiments were performedwith a Philips X’Pert diffractometer equipped with Cu Ka radiation(wavelength = 1.5418 Å). XRD patterns were collected in the 2hrange between 0.6� and 8�, with a step size of 0.02� and a countingtime of 5 s per step.

Nitrogen adsorption–desorption at 77.35 K was used to deter-mine the textural properties using a Micromeritics ASAP 2020porosimeter. Before adsorption measurement, the MBG_Scs weredegassed under a vacuum for 24 h at 120 �C. The surface areawas obtained by applying the Brunauer–Emmett–Teller (BET)method. The pore size distribution was determined by the Bar-ret–Joyner–Halenda method from the adsorption branch of theisotherm.

The macroporosity of the MBG_Scs was examined by means ofenvironmental scanning electron microscopy (FEI Quanta 200, FeiCompany, The Netherlands).

Thermogravimetry (TG) and differential thermal analyses (DTA)were carried out in a Pyris Diamond TG/DTA thermal analyzerusing an air flow of 200 ml min�1 and heating from 35 to 1000 �Cat 5 �C min�1.

Table 1Molar % composition of synthesized MBG scaffolds.

Sample code Samples SiO2 CaO P2O5 Substituent x

B_Sc 80SiO2–15CaO–5P2O5 80 15 5 – –xCe2O3_Sc (80 � x)% SiO2–15CaO–5P2O5–xCe2O3 0.2 1.0xGa2O3_Sc (80 � x)% SiO2–15CaO–5P2O5–xGa2O3 0.2 1.0xZnO_Sc (80 � x)% SiO2–15CaO–5P2O5–xZnO 0.4 2.0

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2.4. In vitro bioactivity test

In vitro bioactivity test was carried out by mounting the MBG_Scvertically in a platinum scaffold in filtered simulated body fluid(SBF) [36]. The SBF was filtered with a 0.22 lm Milipore system fil-ter to avoid bacterial contamination. Each sample was soaked forfour different time intervals (8 h and 1, 3 and 7 days) in 20 ml ofSBF at 37 �C. The scaffolds were kept completely submerged inSBF. All operations were performed in a laminar air flow to avoidcontamination with microorganisms. After soaking, the MBG_Scswere taken out and gently rinsed, first in distilled water and thenin ethanol, before being dried for 24 h in a laminar air flow.

The chemical composition of the SBF was analyzed with aninductively coupled plasma (ICP) spectrometer (ICP Optima4200DV, Perkin Elmer) to evaluate the changes in the concentra-tions of calcium, phosphorus, silicon, cerium, gallium and zinc dur-ing the in vitro bioactivity test. The pH level of the SBF wasdetermined using an Ilyte Na+ K+ Ca2+ pH system.

2.4.1. Characterization of surface of scaffolds after in vitro bioactivitytest

Scanning electron microscopy (SEM) and Fourier-transforminfrared spectroscopy (FTIR) were used to study the evolution of

Fig. 2. SEM images of (A) xCe2O3_Sc, (B) xGa2O3_Sc, (C) xZnO_Sc and (D) B_Sc showinMBG_Scs showing homogeneous distribution of Si, Ca, P, Ce, Ga and Zn elements.

the MBG_Sc surface. The SEM analysis was performed in a JEOL6400 microscope and the FTIR was carried out with a NicoletMagna IR 550 spectrometer. Energy-dispersive X-ray (EDX) analy-sis was also performed with the JEOL 6400 microscope to study theamounts of silicon, calcium, phosphorus, cerium, gallium and zincbefore and after in vitro assay.

3. Results and discussion

Fig. 2 represents SEM images of unsubstituted scaffold (B_Sc),xCe2O3_Sc, xGa2O3_Sc and xZnO_Sc, consisting of a lattice of rodsstacked one above the other in the form of layers. The top-viewimage of each MBG_Sc showed ultralarge pores (pores > 400 lm)present between two adjacent rods formed by 3-D rapid prototyp-ing (RP). Moreover, the digital image of the MBG_Sc in Fig. 3Ashowed a quite uniform pore structure from top to bottom. Thebottom pores were also open and did not deform due to the weightof the layers above. The cross-section SEM image (see Fig. 2C)showed well-defined gaps between adjacent layers, as seen inthe case of xZnO_Sc. The same was observed in the transverse sec-tion of MBG_Sc presented in Fig. 3C, suggesting that well-intercon-nected ultralarge pores formed by RP.

g giant macropores (top view) and macropores (cross-section). (E) EDX spectra of

Fig. 3. Digital images of xCe2O3-, xGa2O3- and xZnO-substituted MBG_Scs (A) beforeand (B) after dye test (top view of scaffold surface); (C) transverse section of eachscaffold. ( Green circle = ultralarge pores; small yellow dots = pores on thesurface; blue square box = macropores; vertical arrow = gap between twoadjacent layers.)

Fig. 4. XRD patterns of xCe2O3-, xGa2O3- and xZnO-substituted MBG_Scs along withB_Sc.

S. Shruti et al. / Acta Biomaterialia 9 (2013) 4836–4844 4839

The top-view SEM image (Fig. 2) of all MBG_Scs showed rodswith a rough surface without any visible pores. However, macrop-ores ranging in size between 100 and 400 lm appeared in thecross-section SEM micrograph of each MBG_Sc (see Fig. 2). Thesemacropores were formed due to the removal of PCL during the heattreatment of the MBG_Sc.

A preliminary test was performed to determine the presence ofpores on the surface and the interconnections between the pores.Each scaffold was subjected to an ethanol-based red-colored dye,as reported in a previous publication [9]. The test gave a positiveresult, as all the samples changed color from white to red(Fig. 3B) due to the flow of fluid through the pores which werenot visible on the surface. In addition, the transverse sections ofthe samples confirmed that the dye could access to the cores ofthe scaffolds (Fig. 3C). Hence, the results indicate that each scaffoldhas some interconnection between the pores present on the sur-face and the macropores present inside, though they do not con-firm whether the interconnections are suitable for cell ingrowth.It is thus necessary to carry out an in vitro cell test in the futureto confirm this assumption.

Generally scaffolds should possess large enough macropores forefficient regeneration of bone tissue. Most studied have shown thatpores between 50 and 150 lm are required for osteoid growth andpores larger than 150 lm facilitate the proliferation of cells, vascu-lar ingrowth and internal mineralized bone formation [9]. There-fore the ultralarge pores and macroporosity showed by cerium-,gallium- and zinc-substituted MBG_Scs are within the parametersof the porosity required for bone tissue engineering.

The EDX spectra of substituted MBG_Scs (see Fig. 2E) show thehomogeneous distribution of Si, Ca, P, Ce, Ga and Zn, and signals ofcerium, gallium and zinc, indicating their presence in the glassmatrix.

Fig. 4 collects the low-angle XRD patterns of B_Sc, xCe2O3_Sc,xGa2O3_Sc and xZnO_Sc after calcination. B_Sc and the MBG_Scssubstituted with 0.2% Ce2O3, 0.2%Ga2O3 and 0.4% ZnO show almostidentical diffraction patterns to those observed in our previouswork on Ce-, Ga- and Zn-MBG powders. The sharp diffraction max-ima at a 2h of 1.3–1.4�, with two poorly resolved peaks in the 2h

range 2.16–2.70 �C, can be assigned to reflections (10), (11) and(20) of an ordered 2-D hexagonal structure [37]. However, onincreasing the amount of cerium and gallium no diffractionmaxima was observed, except a weak maxima in case ofzinc -substituted MBG_Sc, suggesting a defective arrangement ofmesopores. The results suggest that mesoporous order is not al-tered in B_Sc and MBG_Scs substituted with the smallest amountsof cerium, gallium and zinc during the manufacturing process.However, in 1.0% Ce2O3_Sc, 1.0%Ga2O3_Sc and 2.0% ZnO_Sc, themesostructure could not withstand milling or a second thermaltreatment during scaffold fabrication. Thus, MBG_Scs containinglarger amounts of xCe2O3/Ga2O3 (where x P 2.0 mol.%) or xZnO(where x P 4.0 mol.%) were not prepared.

Fig. 5. Nitrogen adsorption–desorption isotherm plots of cerium-, gallium- and zinc-substituted MBG_Scs along with B_Sc. Inset: pore size distribution curves and texturalfeatures – SBET, surface area (m2 g�1); DP, pore diameter (nm); VP, pore volume (cm3 g�1).

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Fig. 5 shows the nitrogen adsorption isotherms and pores sizedistribution along with the derived textural properties (surfacearea (SBET), pore diameter (DP) and pore volume (VP)) of B_Sc andcerium-, gallium- and zinc-substituted MBG_Scs. All curves canbe identified as type IV isotherms characteristic of mesoporousmaterials. The hysteresis loops obtained are of type H1 in the mes-opore range, which is characteristic of cylindrical pores. All sam-ples show a single-mode pore size distribution centered between3.7 and 4.3 nm. The SBET and VP of B_Sc and the 0.2% Ce2O3,0.2%Ga2O3 and 0.4% ZnO MBG_Scs were approximately the same(means of 390 m2 g�1 and 0.395 cm3 g�1, respectively). In contrast,the disordered arrangement of mesopores observed by the XRDpatterns of 1.0% Ce2O3_Sc, 1.0%Ga2O3_Sc and 2.0% ZnO_Sc had animpact on their SBET and VP values, reducing them to as little as344 m2 g�1 and 0.327 cm3 g�1, respectively.

TG/DTA analysis of MBG_SC (not shown for the sake of brevity)showed an approximately 40% loss of weight in the TG curve below450 �C. In addition, two exothermic peaks were observed (at 360and 420 �C) in the DTA curve, corresponding to initial and finaldecompositions of PCL [38]. The results indicate that, in total,40 wt.% PCL was initially present in a scaffold – in agreement withthe experimental procedure – which is completely eliminated by450 �C. This helped us to decide the calcination temperature (i.e.500 �C) for the removal of PCL from the as-prepared MBG_Scs.

3.1. In vitro bioactivity test

A significant characteristic of MBG scaffolds is their ability toform an apatite layer on their surface, which, it has been claimed,is responsible for bond formation with living bone. An in vitro bio-activity assay can be performed by soaking material in fluid mim-icking human plasma. During the assay, rapid exchange in cationslike Ca2+and H+ occurs, leading to an initial increase in the Ca2+

concentration of the solution. Later, Ca2+ and PO�34 precipitate from

the solution onto the SiO2-rich layer formed on the surface ofmaterials, resulting in an amorphous CaP layer. This layer crystal-lizes into an apatite layer via the incorporation of OH� and CO3

2�

anions from the solution. In the present study, the formation ofsuch an apatite layer was monitored by two ways: directly, by ana-lyzing the apatite formation on the surface of the MBG_Sc by FTIRand SEM–EDX; and indirectly, by evaluating changes in the pH andthe calcium, phosphorus, silicon, cerium, gallium and zinc concen-trations of the SBF.

Fig. 6 shows the FTIR spectra of B_Sc and Ce-, Ga- and Zn-MBG_Scs before and after soaking in SBF for three different timeintervals (8 h, 1 day and 7 days). Before soaking, all MBG_Scsshowed intense silicate group absorption bands. The intense bandsat 1035 and 442 cm�1 are assigned to the Si–O–Si asymmetricstretch, and the band at 800 cm�1 to the Si–O symmetric stretch.

Fig. 6. FTIR spectra of B_Sc, 1.0% Ce2O3_Sc, 1.0%Ga2O3_Sc and 2.0% ZnO_Sc, before and during SBF treatment.

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On soaking B_Sc in SBF, an amorphous phosphate band ap-peared at 558 cm�1 after 8 h. However, by 1 day the FTIR spectrumof B_Sc was similar to that of carbonate hydroxyapatite, as clearphosphate group bands at 1026, 950, 600 and 560 cm�1 were vis-ible, along with carbonate bands at 1489, 1419 and 871 cm�1,which increased in intensity with time [39].

No significant change in the in vitro response was observed onsubstituting B_Sc with cerium, gallium or zinc up to 0.4 mol.%. Onthe other hand, the evolution of the FTIR spectra of 1.0% Ce2O3_Scand 1.0%Ga2O3_Sc followed same pattern as B_Sc, though withslightly less intense phosphate and carbonate bands. However, adrastic decrease was observed in the in vitro bioactivity of 2.0%ZnO_Sc. An amorphous phosphate band at 559 cm�1 appearedafter 8 h, though doublet formation at 561 and 600 cm�1, corre-sponding to crystalline phosphate, was observed only after 7 days,along with low-intensity carbonate bands [40]. This clearly indi-cates that all samples except 2.0% ZnO_Sc show a quick in vitrobioactive response.

Fig. 7 shows SEM images of B_Sc, 1.0% Ce2O3_Sc, 1.0%Ga2O3_Scand 2.0% ZnO_Sc before and during SBF treatment. At 0 h, the het-erogeneous surface of a typical glass was observed in all the sam-ples. After soaking for 8 h, the B_Sc micrograph shows that there isa surface layer with an irregular distribution, although no clear evi-dence of surface layer formation was observed in 1.0% Ce2O3_Sc,1.0%Ga2O3_Sc and 2.0% ZnO_Sc. However, by 1 day, the surfacemorphology of B_Sc, 1.0% Ce2O3_Sc and 1.0%Ga2O3_Sc had changedcompletely, as a flaked layer of thin-edged crystals forming pseu-do-spherical agglomerates covered the entire surface of the scaf-folds. In contrast, in 2.0% ZnO_Sc only an initial coating of acalcium phosphate layer was observed, which turned into a layerof spherical particles covering the entire glass surface by 7 days.In the B_Sc, 1.0% Ce2O3_Sc and 1.0%Ga2O3_Sc the formed layer be-comes denser, forming spherical aggregates of elongated particlesafter 7 days. The evolution of the surface layers of MBG_Scs substi-tuted with the smallest amounts of cerium, gallium and zinc wasfound to be similar to that of B_Sc (not shown for the sake ofbrevity).

The EDX spectra of B_Sc recorded a steady increase in calciumand phosphorus concentrations during SBF immersion. By 7 days,

the silicon content was found to be less than calcium and phospho-rus, with the Ca/P molar ratio of 1.64 corresponding to the value ofbiological apatite [31]. The EDX spectra of 1.0% Ce2O3_Sc and1.0%Ga2O3_Sc were analogous to B_Sc. However, in 2.0% ZnO_Scthe levels of Ca and P show very slow increases up to 1 day.However, by 7 days the Ca/P ratio was equal to 1.54, indicatingthe formation of an apatite layer on the surface.

Fig. 8 shows the changes in Ca, P and Si contents and pH value ofSBF during in vitro bioactivity of B_Sc and cerium-, gallium- andzinc-substituted MBG_Scs. In B_Sc, Ca concentration increased dur-ing the first 24 h (to 2.7 mM), then showed a diminishing trend,reaching 2.2 mM after 168 h. The Ca variations in xCe2O3_Sc, xGa2-

O3_Sc and xZnO_Sc (except 2.0% ZnO_Sc) followed similar patternsto that of B_Sc. However, the increment during the first 24 h wasmuch higher (3.5–4.0 mM), before later decreasing, although thelevel was still found to be higher than B_Sc after 168 h. On theother hand, in 2.0% ZnO_Sc the level of Ca only increased, confirm-ing the FTIR and SEM–EDX results, which showed the formation ofan HCA layer on its surface only after 7 days.

In all the samples, P and Si ions exhibited contrasting variations,with the P concentration showing only a decreasing trend, fallingbelow 0.2 mM after 8 h; the opposite was observed in the case ofSi release. In addition, MBG_Scs showed much higher Si releasethan MBG powders, indicating a greater rate of dissolutionin vitro. The probable reason for this is that the in vitro bioactivityof the MBG powders was measured using pellets, which have acompact structure, unlike the open framework of scaffolds. Thisis an advantage of the fabricated MBG_Scs, as released Si acts asan important stimulator of osteogenic genes.

During in vitro bioactivity, the Ce, Ga and Zn concentrationswere always found to be lower than the lowest standard prepared(not shown). The ion exchange during the in vitro assay caused thepH of the SBF to increase, reaching a maximum value of 7.8. How-ever, after 24 h it remained fairly constant, at around 7.6–7.8 in allthe cases.

Thus, all the Ce-, Ga- and Zn-MBG_Scs except 2.0% ZnO_Sc exhi-bit a large in vitro response. These results are in accordance withour previous study on MBG powders [31], indicating: (i) a fasterin vitro response as compared with conventional BGs [30]; and

Fig. 7. SEM images of B_Sc, 1.0% Ce2O3_Sc and 1.0%Ga2O3_Sc, showing a quick in vitro response, and 2.0% ZnO_Sc, showing a low in vitro response. Inset: EDX results.

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(ii) no change in in vitro response due to the scaffold preparationprocedure. During ICP analysis, the Ca ion concentration increasedin first few hours, whereas the P ion concentration only showed adecreasing trend, probably due to the large surface area exhibitedby the cerium-, gallium- and zinc-containing MBG_Scs. This notonly causes leaching and reprecipitation of Ca and P ions to occursimultaneously, but also exposes many silanol groups, which act asnucleation centers for HCA formation [41,42]. On the other hand,the cause of the low in vitro response of 2.0% ZnO is due to theinvolvement of zinc ion in the formation of zinc phosphate. Thisis clearly depicted in the ICP results of 2.0% ZnO_Sc, where theCa concentration shows continuous incrementation up to 168 h,with a drastic decrease in P concentration and the absence of zincion in the SBF. The solubility product constant of Zn3(PO4)2 is4.55 � 10�4 times lower than that of Ca3(PO4)2. Therefore, in aque-ous solution PO�3

4 ions combine more easily with Zn2+ ions thanwith Ca2+. It is thus presumed that, during in vitro assay, Ca2+

and Zn2+ ions are released from 2.0% ZnO_Sc, but that the Zn2+ ionscombine with PO�3

4 , because of which some of the PO�34 is used in

Zn3(PO4)2 formation rather than HCA formation, leading to lowbioactivity. However, xCe2O3_Sc and xGa2O3_Sc do not show such

behavior, as Ce and Ga ions are not detected in SBF, which maythus consume phosphate and retard HCA formation.

Cerium-, gallium- and zinc-substituted MBG_Scs exhibitedquite similar structural and textural properties along with thesame in vitro response as found earlier in the parent MBG [31].These satisfactory results indicate that 3-D porous MBG scaffoldssubstituted with cerium, gallium and zinc can be prepared by acombination of EISA and RP without hampering basic propertiesof MBG powder.

4. Conclusions

In the present study, 3-D scaffolds were prepared from quater-nary MBG with composition (80 � x)% SiO2–15% CaO–5% P2O5 con-taining biologically potent cerium, gallium and zinc. Hierarchicalporosity was obtained by use of the non-ionic surfactant P123 asa mesostructure directing agent, PCL as a macroporous templateand RP for ultralarge macropores. The obtained scaffolds containedinterconnected ultralarge pores suitable for vascularization, nutri-ent supply and normal cell growth. In addition, macropores were

Fig. 8. Variation of Ca, P and Si concentrations and pH values of SBF during in vitro study of B_Sc and cerium-, gallium- and zinc-substituted MBG_Scs.

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found inside the scaffolds, though their interconnection with thepores on the surface needs to be confirmed by in vitro cell tests.All scaffolds maintained optimum textural properties and in vitrobehavior of MBG powder. Despite the severe preparation condi-tions, mesopore order was maintained by B_Sc and the lowestsubstituted Ce2O3, Ga2O3 and ZnO MBG scaffolds. Hence, 3-D mac-romesoporous MBG scaffolds containing Ce, Ga and Zn therapeuticions are promising candidates for bone tissue engineering.

Acknowledgements

Financial support thought Comisión Interministerial de Cienciay Tecnología (CICYT, Spain) (MAT2008-736), Comunidad Autónomade Madrid (S2009/MAT-1472) and the Network of Excellence ofSpanish MICINN (CSO2010-11384-E) is acknowledged.

Appendix A. Figures with essential colour discrimination

Certain figures in this article, particularly Figs. 1, 3 and 8, aredifficult to interpret in black and white. The full colour imagescan be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2012.09.024.

References

[1] Porter JR, Ruckh TT, Popat KC. Bone tissue engineering: a review in bonebiomimetics and drug delivery strategies. Biotechnol Prog 2009;25:1539–60.

[2] Griffith LG, Naughton G. Tissue engineering: current challenges and expandingopportunities. Science 2002;295:1009–14.

[3] Vallet-Regí M. Evolution of bioceramics within the field of biomaterials. CRChim 2010;13:174–85.

[4] Arcos D, Vallet-Regí M. Sol–gel silica based biomaterials and bone tissueregeneration. Acta Biomater 2011;6:2874–88.

[5] Gerhardt L, Boccaccini AR. Bioactive glass and glass ceramics scaffolds for bonetissue engineering. Materials 2010;3:3867–910.

[6] Vallet-Regí M, Ruiz-Hernández E. Bioceramics: from bone regeneration tocancer nanomedicine. Adv Mater 2011;23:5177–218.

[7] Mouriño V, Boccaccini AR. Bone tissue engineering therapeutics: controlleddrug delivery in three-dimensional scaffolds. J R Soc Interf 2012;7:209–27.

[8] Manzano M, Vallet-Regí M. New developments in ordered mesoporousmaterials for drug delivery. J Mater Chem 2010;20:5593–604.

[9] García A, Izquierdo-Barba I, Colilla M, López de Laorden C, Vallet-RegíLópez deLaorden M. Preparation of 3-D scaffolds in the SiO2–P2O5 system with tailoredhierarchical meso-macroporosity. Acta Biomater 2011;7:1265–73.

[10] Salinas AJ, Vallet-Regí M. Evolution of ceramics with medical application. ZAnorg Allg Chem 2007;633:1762–73.

[11] Wu C, Luo Y, Cuniberti G, Xiao Y, Gelinsky M. Three dimensional printing ofhierarchical and tough mesoporous bioactive glass scaffolds with acontrollable pore architecture, excellent mechanical strength andmineralization ability. Acta Biomater 2011;7:2644–50.

[12] Vallet-Regí M, Izquierdo-Barba I, Colilla Montserrat. Structure andfunctionalization of mesoporous bioceramics for bone tissue regenerationand local drug delivery. Philos Trans R Soc A: Math Phys Eng Sci2012;370:1400–21.

[13] Zhu Y, Li X, Yang J, Wang S, Gao H, Hanagata N. Composition–structure–property relationships of the CaO–MxOy–SiO2–P2O5 (M = Zr, Mg, Sr)mesoporous bioactive glass (MBG) scaffolds. J Mater Chem 2011;21:9208–18.

[14] Wu C, Miron R, Sculean A, Kaskel S, Doert T, Schulze R, et al. Proliferation,differentiation and gene expression of osteoblasts in boron-containingassociated with dexamethasone deliver from mesoporous bioactive glassscaffolds. Biomaterials 2011;32:7068–78.

[15] Zhu Y, Zhang Y, Wu C, Fang Y, Yang J, Wang S. The effect of zirconiumincorporation on the physiochemical and biological properties of mesoporousbioactive glass scaffolds. Micropor Mesopor Mater 2011;143:311–9.

[16] Wang X, Li X, Ito A, Sogo Y. Synthesis and characterization of hierarchicallymacroporous and mesoporous CaO–MO–SiO2–P2O5 (M = Mg, Zn, Sr) bioactiveglass scaffolds. Acta Biomater 2011;7:3638–44.

[17] Mouriño V, Cattalini JP, Boccaccini AR. Metallic ions as therapeutic agents intissue engineering scaffolds: an overview of their biological applications andstrategies for new development. J R Soc Interf 2012;9:401–19.

[18] Leonelli C, Lusvardi G, Malavasi G, Menabue L, Tonelli M. Synthesis andcharacterization of cerium-doped glasses and in vitro evaluation of bioactivity.J Non-Cryst Solids 2003;316:198–216.

[19] Valappil SP, Ready D, Abou Neel EA, Pickup DM, Ó Dell LA, Chrzanoweki W,et al. Controlled delivery of antimicrobial gallium ions from phosphate-basedglasses. Acta Biomater 2009;5:1198–210.

[20] Valappil SP, Ready D, Abou Neel EA, Pickup DM, Chrzanoweki W, Ó Dell LA,et al. Antimicrobial gallium-doped phosphate based glasses. Adv Funct Mater2008;18:732–42.

[21] Neel EA, O’Dell LA, Chrzanowski W, Smith ME, Knowles JC. Control of surfacefree energy in titanium doped phosphate based glasses by co-doping with zinc.J Biomed Mater Res B 2009;89:392–407.

[22] Balamurugan A, Balossier G, Kannan S, Michel J, Rebelo AH, Ferrerira JM.Development and in vitro characterization of sol–gel derived CaO–P2O5–SiO2–ZnO bioglass. Acta Biomater 2007;3:255–62.

[23] Jinchao Z, Cuilian L, Yaping L, Jing S, Peng W, Keqian D, et al. Effect of ceriumion on the proliferation, differentiation and mineralization function of primarymouse osteoblasts in vitro. J Rare Earth 2010;28:138–42.

[24] Schubert D, Dargusch R, Raitano J, Chan SW. Cerium and yttrium oxidenannoparticles are neuroprotective. Biochem Biophys Res Commun 2006;342:86–91.

4844 S. Shruti et al. / Acta Biomaterialia 9 (2013) 4836–4844

[25] Bockman RS, Boskey AL, Blumenthal NC, Alcock NW, Warrell Jr RP. Galliumincreases bone calcium and crystallite perfection of hydroxyapatite. CalcifTissue Int 1986;39:376–81.

[26] Hall TJ, Chambers TJ. Gallium inhibits bone resorption by a direct effect onosteoclast. Bone Miner 1990;8:211–6.

[27] Harrington JR, Martens RJ, Cohen ND, Bernstein LR. Antimicrobial activity ofgallium against virulent Rhodococcus equi in vitro and in vivio. J Vet PharmacolTher 2006;29:121–7.

[28] Ito A, Kawamura H, Otsuka M, Ikeuchi M, Ohgushi H, Ishikawa K, et al. Zinc-releasing calcium phosphate for stimulating bone formation. Mater Sci Eng C2002;22:21–5.

[29] Aydin SB, Hanley L. Antimicrobial activity of dental composites containingzinc oxide nanoparticles. J Biomed Mater Res B: Appl Biomater 2010;94:22–31.

[30] Shruti S, Salinas AJ, Malavasi G, Lusvardi G, Menabue L, Ferrara C, et al.Structural and in vitro study of cerium, gallium and zinc containing sol–gelbioactive glasses. J Mater Chem 2012;22:13698–706.

[31] Salinas AJ, Shruti S, Malavasi G, Menabue L, Vallet-Regi M. Substitution ofcerium, gallium and zinc in ordered mesoporous bioactive glasses. ActaBiomater 2011;7:3452–8.

[32] López-Noriega A, Arcos D, Izquierdo-Barba I, Sakamoto Y, Terasaki O, Vallet-Regí M. Ordered mesoporous bioactive glasses for bone tissue regeneration.Chem Mater 2006;18:3137–44.

[33] Malavasi G, Manubue L, Menziani MC, Pedone A, Salinas AJ, Vellet-Regi M. Newinsight in the bioactivity of SiO2–CaO and SiO2–CaO–P2O5 sol–gel glasses by

molecular dynamics simulation. J Sol–gel. J Sol–Gel Sci Technol 2011. http://dx.doi.org/10.1007/ S10971-011-2453-4.

[34] Brinker CJ, Lu Y, Sellinger A, Fan H. Evaporation-induced self assembly:nanostructures made easy. Adv Mater 1999;11:579–85.

[35] Yang SF, Leong KF, Du ZH, Chua CK. The design of scaffolds for use in tissueengineering. Part II. Rapid prototyping techniques. Tissue Eng 2002;8:1–11.

[36] Kokubo T, Takadama H. How useful is SBF in predicting in vivo bonebioactivity. Biomaterials 2006;27:2907–15.

[37] Garcia A, Cicuendez M, Izquierdo-Barba I, Arcos D, Vallet-Regi M. Essential roleof calcium phosphate heterogeneities in 2D-hexagonal and 3D-cubic SiO2–CaO–P2O5 mesoporous bioactive glasses. Chem Mater 2009;21:5474–84.

[38] Lee B, Quang DV, Youn M, Song H. Fabrication of biphasic calcium phosphates/polycaprolactone composites by melt infiltration process. J Mater Sci: MaterMed 2008;19:2223–9.

[39] Salinas AJ, Vallet-Regi M, Izquierdo-Barba I. Biomimetic apatite deposition oncalcium silicate gel glasses. J Sol–Gel Sci Technol 2001;21:13–25.

[40] Salinas AJ, Martin AI, Vallet-Regi M. Bioactivity of three CaO–P2O5–SiO2 sol–gelglasses. J Biomed Mater Res 2002;61:524–32.

[41] Jones JR, Lin S, Yue S, Lee PD, Hanna JV, Smith ME, et al. Bioactive glassscaffolds for bone regeneration and their hierarchical characterization. ProcInst Mech Eng Pt H: J Eng Med 2010;224:1373–87.

[42] Lusvardi G, Malavasi G, Aina V, Bertinetti L, Cerrato G, Magnacca G, et al.Bioactive glasses containing Au nanoparticles. Effect of calcinationstemperature on structure, morphology and surface properties. Langmuir2012;26:10303–14.