Preparation, characterization and in vitro testing of poly(lactic-co-glycolic) acid/barium titanate...

Preparation, characterization and in vitro testing of poly(lactic-co-glycolic) acid/barium titanate nanoparticlecomposites for enhanced cellular proliferation

Gianni Ciofani & Leonardo Ricotti &Arianna Menciassi & Virgilio Mattoli

Published online: 28 October 2010# Springer Science+Business Media, LLC 2010

Abstract The recent advancements in tissue engineeringand, more in general, in cell-based applications, has led toan ever increasing interest toward new materials forsustained cell proliferation and differentiation. Here, thepreparation and the characterization of scaffolds based onpoly(lactic-co-glycolic) acid / barium titanate nanoparticlecomposites are presented. In vitro testing on H9C2 cell linedemonstrates how the presence of the nanoparticlespositively affects both the proliferation and the differenti-ation of this muscle-like cell line. Finally, the possibility toobtain porous scaffolds and, therefore, an actual 3D culturesystem, is introduced.

Keywords Polymer/ceramic composites . Barium titanatenanoparticles . Poly(lactic-co-glycolic) acid .Muscle cells .

Tissue engineering

1 Introduction

The development of innovative materials useful as scaffoldsfor the sustained growth of cells is of particular interest inregenerative medicine and tissue engineering, because theycan be potentially tailored to mimic the natural extracellularmatrix (ECM) in terms of structure, chemical composition,

and mechanical properties (Shin et al. 2003; Dankars et al.2005; Brandl et al. 2007).

Among the biodegradable polymers, poly(α-hydroxyesters) such as poly(L-lactic acid) (PLLA) and poly(lactic-co-glycolic acid) (PLGA) are widely being investigated asmaterials to regenerate several kind of tissues (Lin et al.2002). These biodegradable materials are not only biocom-patible with host tissues, but also aid in the differentiationand proliferation of the desired cell types. They can beeasily and reproducibly fabricated into intended geometriesof porous and/or bulk scaffolds that can degrade to non-toxic low-molecular-weight compounds (Wang et al. 1990;Mikos et al. 1994).

On the other hand, for several applications, strongermechanical features are often desired respect to those onesoffered by the bare copolymers. Researchers have usedseveral reinforcements for improving the mechanicalproperties of different classes of copolymers. Thesereinforcements include, among others, hydroxyapatite(Causa et al. 006; Cerrai et al. 999; Russias et al. 2006),tricalcium phosphate (Yang et al. 2006; Loher et al. 2006),glasses (Ranne et al. 2007; Meretoja et al. 2006), andcarbon nanotubes (CNT) (Harrison and Atala 2007; Lahiriet al. 2009; Lee et al. 2009; Edwards et al. 2009). Thebiocompatibility of the reinforcement is a major concern.Given that reinforcement may remain within the livingtissues for a longer time after the biodegradable matrix isabsorbed, highly compatible materials should be employed.Motivated by this necessity, we propose in this paper theapplication of an innovative nanomaterial, barium titanatenanoparticles (BTNPs), as the second phase reinforcementfor the PLGA copolymer.

Nanostructured materials have been extensively exploredin many biological applications because of their intriguingphysical and chemical properties (Gao and Xu 2009). In

G. Ciofani (*) : L. Ricotti :A. Menciassi :V. MattoliItalian Institute of Technology, Smart Materials Lab,Center for Micro-BioRobotics c/o Scuola Superiore Sant’Anna,Viale Rinaldo Piaggio, 34,56025 Pontedera, Pisa, Italye-mail: [email protected]

G. Ciofanie-mail: [email protected]

Biomed Microdevices (2011) 13:255–266DOI 10.1007/s10544-010-9490-6

particular, the intrinsic optical, magnetic, and electricalproperties owned by nanomaterials can offer remarkableopportunities of interaction with complex biological pro-cesses for several biomedical applications.

Ceramic materials based on perovskite-like oxides are ofintense interest because of their applications in electricaland electronic devices (Chung et al. 2004). Due to its highdielectric constant, barium titanate (BaTiO3) is probablyone of the most studied compounds of this family and stillrepresents the basis for the preparation of multilayerceramic capacitors and thermistors with positive tempera-ture coefficient of resistivity (Hennings et al. 2001;Buscaglia et al. 2000).

We already proposed nanomedicine applications of adifferent type of BTNPs (100 nm in size, cubic structure).They have been proven to be non-toxic even at highconcentrations, and can be efficiently exploited as proteincarriers (Ciofani et al. 2010a) and as enhancers of the up-take of low molecular weight drugs such as doxorubicin(Ciofani et al. 2010b). Problems related to Ba2+ ionleaching (Yoon et al. 2003) are minimized thanks both pHphysiological values and polymer and/or surfactant stabili-zation; however, in vivo long-term biocompatibility experi-ments will be needed before any realistic clinicalapplications. Notwithstanding, our results have been prom-ising and, very recently, other applications of BTNPs in thenano-bio field have started to emerge: Hsieh et al., forexample, have proposed their use as second harmonicradiation imaging probes upon conjugation with specificantibody (Hsieh et al. 2010).

Here we show, for the first time, preparation and in vitrocharacterization of PLGA/BTNPs composites for possibleapplications in tissue engineering and regeneration. Afteran accurate characterization of the nanoparticles used in thisstudy, we present the preparation of bulk and porousPLGA/BTNPs scaffolds exploited as substrates for cellculturing. Two doping ratios (10 and 30 wt% BTNPs/PLGA) have been investigated and compared to analogousstructures made of plain PLGA (0 wt%). After the analysisof the mechanical properties with appropriate tensile tests,and of the surface features via electron microscopy, wehave carried out experiments with H9C2 cells, a widelyused cardiac-like cell line. We have shown that the presenceof nanoparticles does not affect the cell viability, but, on thecontrary, a huge increment of the cell proliferation wasnoticed in all the tested doped structures. Finally, we havedemonstrated that also the differentiation of H9C2 cells ispositively affected by the presence of the BTNPs in thePLGA matrix, being the myotube development highlyincreased on the doped scaffolds.

These interesting results do not seem to be related to the,even strong, improvement of the mechanical properties, butrather to the presence of the nanoparticles, and some

hypotheses have been withdrawn in order to explain thisphenomenon.

2 Materials and methods

2.1 Barium titanate nanoparticle characterizationand preparation of the samples for biocompatibility testing

BTNPs were purchased by Nanostructured & AmorphousMaterials, Inc. (Houston, TX). Details of sample purity andcomposition, as provided by the supplier, include purity:99.9%; APS: 200 nm; SSA: 5.0–5.6 m2/g; color: white;morphology: spherical. After purchasing, BTNP sampleswere further analyzed in order to obtain additionalcharacterization.

Energy-dispersive X-ray (EDS) microanalysis was per-formed on BTNP samples using a scanning electronmicroscope (SEM, Jeol JSM-5600 LV). Afterwards, sam-ples were sputter coated and observed with the SEM for amorphological evaluation at an accelerating voltage of20 kV.

X ray diffraction (XRD) patterns were recorded using anX-ray powder diffractometer (Kristalloflex 810, Siemens)using Cu Kα radiation (λ=1.5406 A) at a scanning rate of0.016°s−1 with 2θ ranging in 10°–80° and a temperature of25°C.

For a preliminary testing of their cytocompatibility,nanoparticles were dispersed in cell culture medium thanksto a non-covalent wrapping with glycol-chitosan (GC,G7753 from Sigma) as already described in (Ciofani et al.2010b).

2.2 Polymer/ceramic composites preparationand characterization

Composites of poly(lactic-co-glycolic acid) (PLGA;50:50 wt%; P2191 from Sigma) were prepared using asolvent casting process. Briefly, 500 mg of PLGA wasadded to 5 ml of chloroform; the beaker was placed in awater bath and allowed to heat at about 55°C until allPLGA pellets had completely dissolved. Thereafter, BTNPwere added to the PLGA solution at a concentration of 0(control), 10 and 30 wt%. After a sonication of about20 min in order to disperse the nanoparticles, the mixturewas poured into a Teflon-mold, and allowed to dryovernight at room temperature, and for further 48 h into avacuum oven at room temperature.

A similar procedure was adopted in order to obtainporous scaffolds, with a salt leaching approach: in thiscase, 3.75 g of NaCl (S7653 from Sigma, crystalsdiameter up to 250 μm) were added to the PLGA/BTNPs dispersions. The final products, after having

256 Biomed Microdevices (2011) 13:255–266

dried in the vacuum oven, were soaked in deionizedwater for 72 h; the water being changed every 12 h inorder to allow the salt to leach out.

Mechanical properties of the samples were evaluatedwith an INSTRON 4464 Mechanical Testing System, usinga ±10 N load cell, and performing traction tests. Sampleswere cut into 20×5×2 mm slices and allocated betweentwo aluminium clamps. All samples were pulled at theconstant speed of 5 mm/min, until reaching sample failure.Data were recorded at a frequency of 100 Hz, the stress wascalculated as the load divided by the cross-section area oftensile specimens, while the strain was calculated as ratiobetween the extension and the initial length of tensilespecimens. The tensile modulus for each tested sample wascalculated starting from the stress/strain curve, according toa standard procedure (Callister et al. 2003). Mechanicaltests were repeated on three different samples, for each kindof sample.

Microphotographs of the constructs were obtained with afocused ion beam FEI 200 FIB microscope.

2.3 Cell culture and WST-1 testing on barium titanatenanoparticles

H9C2 rat cardiomyocites (ATCC CRL-1772) were culturedin expansion medium, composed of Dulbecco’s modifiedEagle’s medium (DMEM) supplemented with 10% fetalbovine serum (FBS), 100 IU/ml penicillin, 100 μg/mlstreptomycin and 2 mML-glutamine. Cells were maintainedin normal culture conditions (37°C, saturated humidityatmosphere at 95% air / 5% CO2).

H9C2 is a subclone of the original clonal cell linederived from embryonic BD1X rat heart tissue. Myoblasticcells in this line fuse to form multinucleated myotubes andrespond to acetylcholine stimulation (Kimes and Brandt1976). In our studies, differentiation of H9C2 cells intomyotubes was induced by switching, in cell specimens at80%–90% confluence, the culture medium from expansionto differentiation medium, composed of DMEM supple-mented with 2 mML-glutamine, 100 IU/ml penicillin,100 μg/ml streptomycin, 1% Insulin-Transferrin-Selenium(ITS, I3146 from Sigma) and 1% FBS.

For preliminary testing of nanoparticle cytocompatibil-ity, WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoilium monosodium salt, providedin a pre-mix electro-coupling solution, BioVision) cellproliferation assays were carried out. After trypsinizationand counting with a hemocytometer, 5,000 cells wereseeded in 96-well plate chambers. Once adhesion wasverified (after about 12 h since seeding), cells wereincubated with 0, 5, 10, 20, 50 and 100 μg/ml of glycol-chitosan coated BTNPs for 48 h and, at the endpoint,further incubated with 100 μl of culture medium +10 μl of

the pre-mix solution for 2 h. Finally, absorbance at 450 nmwas read with a microplate reader (Victor3, Perkin Elmer).

2.4 Proliferating cultures: viability staining and DNAquantifications

Proliferation tests were performed seeding 20,000 cells onscaffolds (dimensions about 1×1 cm) obtained from thePLGA/BTNPs composites, both bulk and porous. After a72 h incubation, viability was investigated with the LIVE/DEAD® viability/cytotoxicity Kit (Molecular Probes). Thekit contains calcein AM (4 mM in anhydrous DMSO) andethidium homodimer-1 [EthD-1, 2 mM in DMSO/H2O 1:4(v/v)]. Live cells are distinguished by the presence ofubiquitous intracellular esterase activity, determined by theenzymatic conversion of the non-fluorescent cell-permeantagent, calcein AM, to an intensely fluorescent molecule,calcein. Calcein is well retained within live cells, producingan intense uniform green fluorescence. Conversely, EthD-1enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleicacids, thereby producing a bright red fluorescence in deadcells. EthD-1 is excluded by the intact plasma membrane oflive cells.

At the endpoint, scaffolds (n=3) were rinsed with PBSand treated for 10 min at 37°C with 2 μM calcein AM and4 μM EthD-1 in PBS, and finally observed with an invertedfluorescent microscope (Eclipse TI, Nikon) equipped with acooled CCD camera (DS-5MC USB2, Nikon) and with NISElements AR imaging software.

Proliferation was quantitatively evaluated assessing theds-DNA content in each type of scaffolds (doped with 0, 10and 30 wt% of nanoparticles, both bulk and porous). Allassays (n=3) were performed following 72 h of cultures. Atthe endpoint, scaffolds were removed from the cell culturesand treated with appropriate volumes of double distilled(dd)-H2O. Cell lysates were thus obtained by 2 freeze/thawcycles of the samples: overnight freezing at −80°C, 15 minthawing at 37°C in sonication bath to enable the ds-DNA togo into solution.

Ds-DNA content in cell lysates was measured using thePicoGreen kit (Molecular Probes; Singer et al. 1997). ThePicoGreen dye binds to ds-DNA and the resulting fluores-cence intensity is directly proportional to the ds-DNAconcentration in solution. Standard solutions of DNA inddH2O at concentrations ranging from 0–6 μg/ml wereprepared and 50 μl of standard or sample was loaded forquantification in a 96-well black microplate. Workingbuffer and PicoGreen dye solution were prepared accordingto the manufacturer’s instructions and 100 and 150 μl/welladded, respectively. After a 10 min incubation in the dark atroom temperature, fluorescence intensity was measured ona microplate reader (Victor3, PerkinElmer) using an

Biomed Microdevices (2011) 13:255–266 257

excitation wavelength of 485 nm and an emission wave-length of 535 nm.

2.5 Differentiating cultures: viability and f-actin staining

For differentiation testing, cells were seeded at confluenceon scaffolds (about 60,000 cells/cm²) as described in theprevious Section. After 24 h incubation, culture mediumwas switched from the proliferating one to the differentia-tion one, allowing a further incubation of 72 h. Thereafter,cultures underwent viability/cytotoxicity testing as previ-ously described for the proliferation experiments. Differen-tiation status was monitored evaluating myotubes width andlength, as reported in the literature (Huang et al. 2006),after calcein or f-actin staining.

The latter was obtained with a treatment with labelledphalloidin, according to the following procedure. Cellswere rinsed with PBS 1X, then fixed using paraphormal-deyde (158127 from Sigma) 4% in PBS for 20 minfollowed by other three PBS washing steps (5 min each).Thereafter, cultures were treated with Triton X-100 (78787from Sigma), 0.1% in PBS for 15 min, for membranepermeabilisation; saturation was allowed for 10 min, using0.1% porcine gelatin (271616 from Sigma) in PBS. Finally,cells underwent a 30 min incubation with a 100 μMsolution of rhodamine-labelled phalloidin (R415 fromInvitrogen). After incubation, cells were rinsed in PBShigh-salt (0.45 M NaCl in PBS) for 1 min, followed by PBSrinsing (three times, 5 min each).

2.6 Statistical analysis

Analysis of the data was performed by analysis of variance(ANOVA) followed by Student’s t-test to test for signifi-cance, which was set at 5%. WST-1 assays and DNAquantification assays were performed in exaplicate; all theother assays in triplicate if not otherwise specified.Myotube analyses were performed on, at least, 20 cellsper sample. In all cases, three independent experimentswere carried out. Results are presented as mean value ±standard error of the mean (SEM).

3 Results and discussion

3.1 Nanoparticle characterization

Figure 1(a) shows a SEM image of the BTNPs used in thiswork, as purchased form the supplier. EDS analysis (Fig. 1(b)) performed on the samples confirmed the data providedby the supplier: no significant impurities were detected bythe micro-analysis, with a quantitative composition inweight as following: Ba~63%, Ti~20% and O~17%.

From XRD analysis, BTNPs resulted to have aperovskite-like crystallographyc structure. Tetragonal phaseof BaTiO3 was detected (Fig. 1(c)) with two close peaks at2θ=44.85° and 45.38° (Tao et al. 2008).

Preliminary toxicity testing denoted an excellent cyto-compatibility, as provided by the WST-1 assay (Fig. 1(d)):only at a concentration of 100 μg/ml of BTNPs in theculture medium a moderate (about 20%) but significant (p<0.05) decrement of cell proliferation can be detected. Theseresults are in agreement to the data already reported forother type of BTNPs (Ciofani et al. 2010b).

3.2 Composite characterization: imaging and mechanicalproperties

Figure 2(a) shows FIB images of bulk (non porous) PLGA/BTNPs composites. Low magnification (1,000×, uppermicropictures) show an homogeneous surface, withoutqualitative differences among the samples (0, 10, and30 wt% content of nanoparticles). Increased magnification(30,000×, lower micropictures) enable nanoparticles to bedetected, uniformly dispersed in the PLGA matrix.

Mechanical properties, assessed with a traction test,dramatically change in the doped scaffolds. Figure 2(b)shows stress/strain plots representative of the three testedsamples; in the inlet a zoom of the trend for ε<1 isreported, in order to better appreciate the differences amongthe samples. All the data discussed in the following areextracted from the stress/strain plots obtained as reported inthe previous Section, with n=3 for each case.

Tensile modulus increases non-linearly with the presenceof BTNPs, being 2 MPa for the plain PLGA, but reaching avalue of about 55 MPa for the samples with 30 wt% ofnanoparticles (p<0.01) and and 4 MPa for the 10 wt%sample (p<0.05). Also the tensile strength at the yieldconsiderably increases along with the content of BTNPs,being 0.6, 0.9 and 2 MPa for a concentration of 0, 10, and30 wt% of BTNPs, respectively (in both case p<0.05 respectto the plain PLGA). On the other hand, the elongation at thebreak is significantly reduced in the presence of the nano-particles, being about 12.5 for the plain PLGA, 4.5 in the10 wt% samples and 1.35 in the 30 wt% sample.

FIB imaging of scaffold obtained with salt-leachingtechnique well evidences highly porous structures, in all theconsidered samples (Fig. 3(a)). Once again, high magnifi-cation micropictures of the surfaces show the presence ofuniformly dispersed nanoparticles in the PLGA matrix.

Mechanical properties, in this case, are independent onthe concentration of the nanoparticles. An average tensilemodulus of about 2 MPa (p<0.05) was found for all thetested structures (doped with 0, 10, and 30 wt% of BTNPs).Both tensile strength at yield (about 0.25 MPa) andelongation at the break (about 0.45) are statistically non-


different (p>0.05) in the doped samples respect to the plainPLGA.

3.3 Enhanced cellular activity on composites

Cellular interactions with the prepared structures wasevaluated both qualitatively, with viability/cytotoxicityassays, and quantitatively with the assessment of the DNAcontent, a direct indication of the number of cells grown onthe scaffolds (Martins et al. 2009).

In Fig. 4(a) the results of the live/dead assay is reportedfor the non-porous, bulk scaffolds, for increasing concen-trations of doping BTNPs. No appreciable evidence of celldeath (red fluorescence) was noticed in all the testedstructures, but high viability was confirmed in each case.An apparent higher cellularity was detected in the BTNPdoped scaffolds. This qualitative observation was confirmedby the evaluation of the DNA content after a 3-day culture:proliferation doubled in the 10 wt% and 30 wt% dopedscaffolds (DNA concentration about 0.3 μg/ml) respect to

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Fig. 1 Barium titanate nanoparticle characterization: (a) SEM imaging, (b) EDS analysis, (c) XRD spectrum, and (d) cell viability expressed bythe WST-1 assay following incubation of H9C2 cells in culture media modified with increasing concentrations of nanoparticles (* p<0.05)


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Fig. 2 Bulk PLGA/BTNPs composite characterization: (a) FIB imaging (upper images low magnification, lower images high magnification); (b)stress/strain plot (the inlet shows a zoom of the plots for ε<1)


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Fig. 3 Porous PLGA/BTNPs composite characterization: (a) FIB imaging (upper images low magnification, lower images high magnification);(b) stress/strain plot


the plain PLGA (DNA concentration about 0.15 μg/ml; n=6; p<0.05; Fig. 4(b)).

This strong increment in cell proliferation on the dopedscaffold does not seem to be dependent on the mechanicalproperties of the structures. As discussed in the previousSection, in fact, tensile modulus of the 30 wt% BTNPdoped scaffolds is considerably higher than the 10 wt%structures, but cell proliferation is similar (p>0.05), thussuggesting that the enhanced cellular activity is mainly dueto an effect of the nano-roughness determined by thepresence of the nanoparticles in the PLGA matrix, ratherthan to an improvement of the mechanical features.

This hypothesis is confirmed when results of cellinteractions with the porous structures are considered,where the mechanical properties present non-substantialdifferences among the different doping ratios. Figure 5(a)depicts the results of the viability/cytotoxicity assays onthese structures: also in this case the cell viability isoptimal, with a negligible presence of red-fluorescent,necrotic cells. In Fig. 5(b) a 3D rendering of an area ofthe three structures is presented (Z=15 μm): cells have wellcolonized pores of the scaffolds and, also in this case, a

strong increment of the proliferation is evident in the caseof doped scaffolds. Once again, DNA quantificationconfirm these qualitative results: DNA concentration wasfound to be about 0.4 μg/ml in the cultures on the 10 wt%and 30 wt% BTNP doped structures, while about 0.2 μg/mlon the plain PLGA.

As final test to prove the better cellular functionalityupon cultivation on PLGA/BTNPs composites, H9C2 cellswere seeded at confluence on bulk scaffolds and induced todifferentiate, forming characteristic myotubes. After a 3-days incubation in differentiating conditions, myotubeassessment was carried out as outlined in Section 2.5.Figure 6(a) shows myotube organization in each testedscaffold, as evidenced both by the calcein staining and bythe TRITC-phalloidin labelling of f-actin. Most interesting-ly, myotube length is affected by the presence of thenanoparticles in the PLGA matrix: about 60 μm in the plainPLGA, but more than 100 μm in the 10 wt% and 30 wt%composites (p<0.05; Fig. 6(b)). The BTNP doping seemsto influence also the myotube width, being about 9 μm onthe plain PLGA, and reaching about 14 μm in the case ofcultures on the 30 wt% doped scaffolds.

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Fig. 4 Cell proliferation investigation on bulk PLGA/BTNPs scaffolds: (a) viability/cytotoxicity testing and (b) DNA concentrationquantification


Summarizing, we have showed as the preparation ofPLGA scaffolds, both porous or not, doped with differentconcentrations of BTNPs, positively affects the interactionsbetween the structures and the cells, fostering an incrementof cell proliferation and differentiation.

4 Conclusion

In the past decades, an urgent necessity of innovative,“smart” materials for tissue engineering raised, in order toobtain constructs able to sustain and promote cell prolifer-

ation and tissue regeneration (Rosso et al. 2005), andparticular attention was dedicated to polymer/ceramicscomposites, in order to take advantage by the physco-chemical properties of these two classes of materials (Yunoset al. 2008).

Here, we have proposed an innovative approach basedon PLGA and barium titanate nanoparticles. We havedemonstrated that doped scaffolds are not only able toguarantee proliferation and differentiation of the tested cellline, but also to enhance both phenomena. Obviously,further investigations are needed in order to clarify thisaspect: we have demonstrated how the improved culture

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Fig. 6 Cell differentiation on bulk PLGA/BTNPs scaffolds: (a) calcein staining, (b) f-actin staining and quantitative evaluation of myotubeslength (c) and width (d). (* p<0.05)


conditions are independent on the mechanical properties ofthe constructs, so a realistic hypothesis could be a favorableinfluence due to the nanostructured surfaces of the dopedconstructs, as already documented in the literature (Richertet al. 2008). However, we cannot exclude a positive effectto an interaction of the nanoparticles with serum proteins,as documented by Wang et al. (Wang et al. 2010) in thecase of PLGA/TiO2 nanoparticle composites. In this case,osteoblasts cultured on the composite scaffolds withdifferent TiO2 content displayed increased cell proliferationcompared with pure PLGA scaffold, jointly to an enhancedalkaline phosphatase activity and higher calcium secretion.TiO2 particles have already attracted considerable attentionin the biomedical field. TiO2 powders are effective inapatite formation thanks to their ability to enhance cellattachment and proliferation, to improve the osteoconduc-tivity in vitro and in vivo, and to promote protein absorptionand osteoblast adhesion (Liu et al. 2006).

However, an intriguing feature of BaTiO3 is its piezo-electric nature, that could positively influence cellularactivity (Baxter et al. 2010). Thanks to piezoelectricity,BaTiO3 has the distinct potential to act as “smart” andactive material in tissue engineering following appropriatestimulation: mechanical activation via ultrasounds ofPLGA/BTNPs are for example under investigation, andfurther benefits are expected for cellular proliferation andactivity (Ciofani et al. on line).

Acknowledgments The authors gratefully thank Mr. Carlo Filippe-schi (CRIM Lab, Scuola Superiore Sant’Anna, Pisa, Italy), and Mr.Piero Narducci (Department of Chemical Engineering, University ofPisa, Pisa, Italy) for their valuable technical contributions.

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