New Nanocomposite System with Nanocrystalline ApatiteEmbedded into Mesoporous Bioactive GlassMonica Cicuendez,†,‡ María Teresa Portoles,§ Isabel Izquierdo-Barba,*,†,‡ and María Vallet-Regí*,†,‡
†Departamento de Química Inorganica y Bioinorganica, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramon yCajal s/n, 28040 Madrid, Spain‡Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain§Department of Biochemistry and Molecular Biology I, Faculty of Chemistry, Universidad Complutense de Madrid, CiudadUniversitaria s/n, 28040-Madrid, Spain
*S Supporting Information
ABSTRACT: Crystalline nanoparticles are very attractive building blocks forthe preparation of nanostructured materials. These particles can be dispersed indifferent noncrystalline mesostructured matrixes in order to obtain nano-composite systems which combine the properties of both componentsbroadening their functionality. In the present study, a novel nanocompositebioceramic formed by nanocrystalline apatite particles uniformly embedded intoa mesostructured SiO2−CaO−P2O5 glass wall has been synthesized through theevaporation-induced self-assembly (EISA) method, commonly used formesoporous bioactive glass synthesis, but accelerating the sol−gel apatitecrystallization rate by strong acidification. Moreover, the use of F127 surfactantas a structure directing agent in this synthesis has allowed the homogeneousnanocrystalline apatite particles incorporation inside of the amorphousmesoporous glass. In vitro bioactive assays have shown a fast apatite-likephase formation similar to that exhibited by mesoporous bioactive glasses.Furthermore, the response of L929 fibroblasts and Saos-2 osteoblasts to this new nanocomposite has indicated a significantimprovement in its biocompatibility compared with conventional mesoporous bioactive glasses.
1. INTRODUCTIONA great challenge for the scientific community in the field ofbiomaterials is the design of novel and advanced thirdgeneration bioceramics which promote bone tissue regener-ation, accelerating the healing processes.1,2 The rapid growth ofnanotechnology has allowed numerous advances in this fieldsince nanosized and nanocrystalline bioceramics clearlyrepresent a promising class of orthopedic and dental implantformulations with improved biological and biomechanicalproperties.3−5 In this sense, nanocrystalline hydroxyapatiteconstitutes a revolutionary bioceramic with better propertiesthan coarser crystals.6 Furthermore, due to its close similaritieswith bone mineral component, bioactivity, exceptionalbiocompatibility, and osteoconduction, this nanobioceramicreceives considerable attention in hard tissue replacement andreconstruction applications.6,7
On the other hand, mesostructured bioactive glasses (MBGs)have recently been suggested as excellent candidates for bonetissue regeneration.2,8−10 Their structural and textural proper-ties offer higher surface/volume ratios and a chemicalsynergistic effect which provoke the most accelerated bioactivekinetics shown up to date. Moreover, their mesoporous
arrangement and chemical composition confer them the abilityto act as excellent matrixes for local drug delivery.11−14
“Nanocomposite systems” in which nanocrystals are dispersedin different noncrystalline matrixes are currently the subject ofintensive research activity because they combine the propertiesof both components, thus broadening their functionalitybeyond the pure materials.15,16 To guarantee such functionality,the synthesis process of such systems requires a homogeneousdistribution of both components, preserving the intrinsiccharacteristics of each constituent. In this sense, the design ofa nanocomposite material formed by nanocrystalline apatiteparticles embedded into an MBG mesoporous wall would allowto obtain a novel third generation bioceramic with an enhancedfunctionality. Several routes toward the preparation of silicamesoporous−apatite composite have been explored.17−21 Themost common methods are based in grafting apatite precursorson preformed silica mesoporous material or coating apatiteparticles with mesoporous silica films. However, such routes donot satisfy the requirements for preserving the properties of
Received: November 16, 2011Revised: February 21, 2012Published: February 22, 2012
both components, and the materials obtained in the processpresent very heterogeneous distribution and phase segrega-tion.19−21
Exploring new routes to obtain this new nanocomposite andtaking into account that during the synthesis of evaporation-induced self-assembly (EISA)-derived MBGs, clusters ofamorphous calcium phosphate (ACP) are formed into themesoporous wall;22,23 a modification of such process in order toincrease the crystallinity of this ACP to apatite could be analternative. However, the incorporation of nanocrystallineparticles into noncrystalline matrixes is a challenging taskwhere the surfactant plays an important role. Thus, the use ofF127 surfactant, whose molecules are larger in the hydrophilicregion than Pluronic P123, produces a homogeneousdistribution of the nanocrystalline particles into amorphousmesoporous matrixes, avoiding phase segregation, as it has beendemonstrated in other nanocomposite systems.24
Therefore, in the present study, we report the synthesis of ananocomposite material (MG-HA) formed by nanocrystallineapatite particles embedded into SiO2−CaO−P2O5 mesoporousglass. The synthesis has been carried out through EISA methodusing F127 as surfactant in very acidic conditions, which willlead to acceleratation of the crystallization rate of sol−gelapatite. A deep physicochemical characterization of MG-HAmaterial has been performed to determine its final properties.On the other hand, its in vitro bioactive response as well as itsbiocompatibility on both murine L929 fibroblasts and humanSaos-2 osteoblasts have been evaluated.
2. EXPERIMENTAL SECTION2.1. Synthesis of MG-HA Nanocomposite. Highly mesostruc-
tured nanocomposite (MG-HA) material formed by nanocrystallineapatite particles embedded into SiO2−CaO−P2O5 mesporous glasswall has been synthesized through the evaporation-induced self-assembly (EISA) method25 using a nonionic surfactant, Pluronic F127(BASF), and tetraethyl orthosilicate (TEOS, 98%, Sigma−Aldrich),triethyl phosphate (TEP, 99.8%, Sigma−Aldrich), and calciumchloride26 (CaCl2‑4H2O, 99%, Sigma−Aldrich) as SiO2, P2O5, andCaO sources, respectively. The synthesis of the nanocomposite MG-HAmaterial is based mainly on the synthesis of MBGs in the SiO2−CaO−P2O5 system9 but with specific modifications: (i) by an increase ofmolar ratio of [HCl/TEOS + TEP] from 0.013 to 0.287, which willlead to an acceleration in the hydrolysis of different alkoxides as well asthe sol−gel apatite crystallization28−30 and (ii) the use of surfactantPluronic F127 instead of Pluronic P123 to facilitate the incorporationand homogeneous distribution of nanocrystalline apatite nanoparticleswithin the amorphous mesostructured glassy matrix. In this synthesis,the evaporation self-assembly starts with a homogeneous solution ofnanocomposite precursors and surfactant prepared in ethanol/watersolvent with c0 ≪ cmc, with cmc being the critical micellarconcentration. The concentration of the system is progressivelyincreasing by ethanol evaporation which drives to self-assembly ofsilica−surfactant micelles and further organization into liquidcrystalline mesophase. This process goes at 30 °C in air atmosphere.25
A mesoporous material containing only silica and denoted Si100sample has also been synthesized in the same conditions in order tocompare with MG-HA (see summary characterization in SupportingInformation, S1). Briefly, 19.5 g of F127 was dissolved in 168.6 mL ofabsolute ethanol (99.5%, Panreac) with 12.8 mL of 1.0 M HCl(prepared from 37% HCl, Panreac) solution and 19.4 mL of Milli-Qwater. Afterward, the appropriate amounts of TEOS, TEP, and CaCl2were added in 1 h intervals under continuous stirring during 4 h at 40°C and subsequently maintained in static conditions at the sametemperature overnight. Note that, to prepare Si100 sample, onlyTEOS was added in the synthesis process. The resulting sols were castin Petri dishes (9 cm diameter) to undergo the EISA method at 30 °C.
The gelation process occurred after 3 days, and the gels were aged for7 days in the Petri dishes at 30 °C. Finally, the dried gels wereremoved as homogeneous and transparent membranes (severalhundreds of micrometers thick) and calcined at 700 °C during 6 hto remove the surfactant, organics residue, and chloride ions. Table 1displays the amount of each reactive as well as the final composition ofMG-HA material determined by X-ray fluorescence (XRF) in a PhilipsPANalytical AXIOS spectrometer (Philips Electronics NV) where theX-rays were generated using an Rh Kα source at k = 0.614 Å andCHNS elemental chemical analysis in a Perkin-Elmer 2400CHNSthermo analyzer. The obtained results show a composition in verygood agreement with the nominal composition and the totalelimination of organic material and chlorides.
A deep physic-chemistry characterization of the synthesized materialhas been carried out by X-ray diffraction (XRD) and transmissionelectron microscopy (TEM), solid-state nuclear magnetic resonance(NMR), N2 adsorption, and Fourier transform infrared spectroscopy(FTIR). Moreover, the in vitro response concerning bioactivity and invitro biocompatibility in the presence of murine L929 fibroblasts orhuman Saos-2 osteoblasts of this synthesized material has been alsoperformed. The details of such characterization are shown inSupporting Information.
3. RESULTS AND DISCUSSIONFigure 1A displays a small-angle XRD pattern corresponding toMG-HA material demonstrating the presence of mesoporous
arrangement. XRD diffractogram shows a well-defineddiffraction maximum at 2θ = 0.71 degree and wide maximaaround 2θ = 1.36 and 1.56 degree, which can be indexed as 10,11, and 20 reflections of a 2D-hexagonal structure,31 accordingto TEM results. Wide angle XRD pattern (Figure 1B) revealsthe presence of nanocrystalline apatite phase exhibiting verybroad (002), (211), (310), and (113) reflections.32,33 Thesedata indicate that the inorganic framework may exhibit
Table 1. Amounts of Reactants Used in the Synthesis of MG-HA Materiala
Reactants
TEOS (mL) TEP (mL) CaCl2 (g)
37.2 2.6 2.1Final composition
χSib χCa
b χPb stoichiometric formulac Cl (wt%)d C (wt%)e
0.86 0.08 0.05 Ca0.09SiP0.06O2.24 0.00 0.17aFinal composition analyzed by XRF and elemental chemical analysis.bFinal composition analyzed by XRF where χE corresponds to theatomic fraction of the E element. cStoichiometric formula normalizedto Si from the composition analyzed by XRF. dPercentage of chlorineanalyzed by XRF. ePercentage of carbon analyzed by elementalchemical analysis.
Figure 1. (A) Small-angle and (B) low-angle XRD patterns of calcinedMG-HA material.
nanocrystalline apatite domains. However, on the basis of thisevidence alone, the possibility that these nanocrystallinedomains are associated with a phase separation cannot bestrictly excluded. An important technique to resolve thisquestion and with particular value for the characterization ofmesoscopically ordered semicrystalline inorganic framework isTEM. Figure 2A shows a typical image of a 2D-hexagonal
structure taken with the electron beam perpendicular to thechannels, revealing the presence of dark contrast ovoid particlesaround 12 × 9 nm which are homogeneously distributedthroughout the mesoporous wall. High resolution TEM imagesof two of these particles (Figure 2B,C) show lattice spacings at0.34 and 0.28 nm, corresponding to the reflections (002) and(211) of apatite phase, according to the results by XRD (Figure1B). Moreover, selected-area electron diffraction pattern (ED)recorded on MG-HA material, showing characteristic diffuseelectron diffraction rings (Figure 2D), confirms that the wall ofthis material is made by nanocrystalline apatite particlesuniformly embedded in a continuous amorphous glassyinorganic matrix.In order to determine at the atomic level the chemical
environment of the various constituents of the MG-HA sample,a study by NMR solid state has been carried out. Figure 3shows the 31P and 29Si NMR spectra obtained by single pulse(SP) and cross-polarization (CP) under MAS conditions.Moreover, Table 2 shows the relative populations (expressed aspercentages) of Qn for 29Si units, which have been obtained bydeconvolution of the experimental spectra. Concerning 31PNMR, SP spectrum shows an intense signal at 2.1 ppm, havinga full width at half-maximum height (fwhm) around 4.02 ppm.This chemical shift could be assigned to q0 enviromental oftypical nanocrystalline apatite as it has been previously reportedby Lin et al.34 Although this assignment could have some
controversy compared to Jager et al., who attributed the samechemical shift with lower fwhm value (around 2.3 ppm) tonanocrystalline apatite,35 XRD and TEM results reveal thenanocrystalline nature of these particles. Moreover, the smallparticle size of nanocrystalline apatite observed by TEM couldexplain that the signal found in MG-HA is broader than thatobserved by Jager et al., where a nanocrystalline apatite with aparticle size of 10 nm of diameter and 30−50 nm of length wasstudied.Furthermore, a less intense second signal around −8 ppm
was observed. This signal falls within the range typically foundfor 31P in q1 tetrahedral and may as such conform to either ofthe P−O−X (X = P, Si) scenarios.36 Similar 31P signals havebeen reported as P−O−Si in mesoporous bioactiveglasses.22,23,37 Moreover, the spectrum displays a very weaksignal around −1.7 ppm, which could be assigned to dicalciumphosphate dehydrate.38 The obtained results in the presentstudy reveal that the joint presence of Ca2+ and PO4
3− provokesthe formation nanocrystalline apatite particles located at themesopore wall as it has been evidenced by TEM and XRDtechniques. In this case, 56% of phosphorus present in thesample is in the form of nanocrystalline apatite particles, while32% remaining is making up the silica network as a P−O−Sibond. These results differ from the obtained in MBGs with thesame silica content, since MBGs exhibited ACP clusters and noevidence of phosphorus incorporation into the silica net-work.22,23 Such differences could be explained by theconditions used for MG-HA nanocomposite synthesis. For thismaterial, the use of higher [HCl/TEOS + TEP] molar ratioprovokes an acceleration of TEP hydrolysis,39 increasing thephosphorus incorporation into the silica network and thecrystallization rate of sol−gel apatite.19−21Moreover, the use of F127 surfactant in the present study
plays a decisive role in the mesostructure formation as well as inthe homogeneous distribution of nanocrystalline apatite
Figure 2. TEM study corresponding to MG-HA material. (A) Lowmagnification TEM image and its FT pattern with the electron beamperpendicular to the pore arrangement of a 2D hexagonal structure.(B, C) Higher resolution TEM images corresponding to the darkercontrasts, showing lattice spacing at 0.28 and 0.34 nm apatite phase.(D) The selected area ED pattern of MG-HA material.
Figure 3. (A) Solid-state 31P single pulse and (B, C) 29Si single-pulseand cross-polarization MAS NMR spectra (with their qn and Qn
phosphorus and silicon environments, respectively). The areas for theQn and qn units were calculated by Gaussian line-shape deconvolu-tions.
particles into amorphous matrixes.24 In fact, when the MG-HAmaterial was synthesized with Pluronic P123 instead of F127,the glass mesostructure formation was greatly disturbed due tothe presence of agglomerates located outside of mesoporousarrangement (Supporting Information, S2).
29Si NMR spectroscopy was used to evaluate the networkconnectivity of MG-HA material. Q2, Q3, and Q4 represent thesilicon atoms (denoted Si*) in (NBO)2 Si*-(OSi)2, (NBO)Si*-(OSi)3, and Si*(OSi)4 (NBO = nonbonding oxygen),respectively. In the case of the SP spectrum, two signals,centered at −112 and −102 ppm, are observed, correspondingto the Q4 and Q3
H environments of silicon, respectively. Incontrast, in the spectrum obtained by CP, 4 signals centered at−112, −102, −93, and −88 ppm are observed, correspondingto the Q4 environments, Q3
H, Q2H, and Q2
Ca, respectively. Thepresence of a signal in CP spectrum corresponding to Q2
Caenvironment reveals that a small amount of free CaO (notentrapped as apatite particles) is forming part of the glassystructure as network modifier. Moreover, the nonexistence ofthis signal in SP spectrum demonstrates that this specie is closeto the protons sited at the material surface.22 Therefore, fromour results, we can consider the apatite crystallization restrictsthe amount of Ca ion available for silica matrix, obtaining amesoporous glassy matrix poor in Ca and richer in Si and Pcations. Moreover, MG-HA also shows a similar disruptionsilica network grade in the surface, in comparison to MBGswith the same silica content,22,23 due to that the apatitenanoparticles have a similar effect on the silica networkdisruption that the ACP clusters in the MBGs.Textural properties of MG-HA nanocomposite were quanti-
tative studied by N2 adsorption isotherms. Figure 4A showstype IV isotherm with H1-type hysteresis identified ascylindrical mesopores. This material exhibits a BET surfacearea value of 235 m2/g and a pore volume value of 0.4 cm3/g. Itis also important to remark on the presence of micropores (<2nm), with an area of 9.25 m2/g, which could act to interconnectthe mesoporous channels, as it has been demonstrated in other2D-hexagonal mesoporous structures as SBA-15.40 The study of
pore size distribution reveals the existence of a largemesoporous size of 8 nm (Figure 4B). This nanocompositealso exhibits lower BET surface area and pore volume values incomparison with MBGs in the SiO2−CaO−P2O5 system. Thisdecrease could be attributed to the presence of largermesopores sizes (8 nm instead of 4.4 nm). To determine ifthe existence of the nanocrystalline apatite particles intomesoporous wall affects the MG-HA textural properties, suchmaterial was compared with Si100 sample (Figure 4A, B). Theresults evidence higher BET surface area and pore volume(Table 2) in the MG-HA material, exhibiting similar pore sizediameter (around 8 nm). These results could be directly relatedto the presence of micropores, which are not evidenced inSi100 sample (Figure 4). As it has been commented above, thedisrupting effect of apatite nanoparticles on silica frameworkprovokes (Q4 unit of 29Si CP NMR, Table 2) a notable increasein the microporosity, in a similar way that titania particlesbehave on silica framework in titania−silica mesoporousnanocomposites films.24 To assess the functionality of theMG-HA nanocomposite, its in vitro bioactivity in simulated bodyfluid (SBF) and its biocompatibility on L929 fibroblasts andSaos-2 osteosblasts have been evaluated.In order to compare the bioactive behavior of MG-HA with
MBGs, the bioactivity has been analyzed in the similarconditions by keeping the same external surface/volumeratio.9 In this case, FTIR spectroscopy cannot be used todetermine the changes in the MG-HA surface after SBFexposure, because the starting material exhibits the character-istic doublet at 560 and 600 cm−1 corresponding to crystallinephosphate (see Supporting Information, S3).41 Therefore,SEM-EDS and TEM-ED studies were carried out to determinethe changes in the MG-HA surface as function of soaking time.Figure 5 displays SEM micrographs and their correspondingEDS spectra, showing a thin layer composed of needle-shapedcrystallites after 8 h in SBF. The morphological evolution ismore evident after 24 h, when MG-HA surface is fully coveredby this new layer composed of hemispherical particles formedby needle-shaped crystallites, typical of biomimetic processes of
Table 2. Textural Properties Obtained by N2 Adsorption of the Calcined MG-HA and Si100 Materialsa
aRelative populations, expressed as percentages, corresponding to the Qn 29Si units obtained by deconvolution of the experimental spectra NMRsolid state obtained by single pulse and cross-polarization, respectively.
Figure 4. (A) Nitrogen sorption isotherms and (B) pore size distribution of calcined MG-HA and Si100 materials.
bioactive surfaces.42 EDS analyses reveal a notable increase inphosphorus and calcium content in the MG-HA surface after 8and 24 h of incubation. To confirm the new formed phase onthe MG-HA material, TEM study has been performed. Resultsshow that in the first 8 h the surface is plenty covered byneedle-like particles, which correspond to apatite phase, as ithas been evidenced by ED study. A higher number of theseneedle-like apatite particles are observed in TEM image after 24h. Moreover, it is important to note that the mesoporousarrangement is kept after 8 and 24 h, as it can be evidenced inTEM images.Figure 6 shows the calcium, pH, and silicon variations in the
SBF over soaking time. In the first 8 h of incubation, calciumconcentration displays a slight increase around 10 ppm,
followed by a gradual decrease until 24 h. Finally, a suddendecrease until almost the total calcium depletion at 48 h isobserved. Concerning the pH variation, this parameter followsthe same initial trend of calcium variation values, being constantalong the time of the experiment. In the case of silicon, agradual lixiviation over time is observed.The obtained results reveal the highly bioactive behavior of
MG-HA material when it is soaked in SBF. The initial slightincrease of Ca2+ content and pH evolution in SBF suggests thatan ionic exchange between Ca2+of MG-HA material (in thiscase, calcium of glass network as network modifier) and H+
from SBF takes place. In agreement with Hench theory,43 thisprocess leads to silanol formation on its surface which is amandatory step for the subsequently apatite precipitation. Theobtained TEM/SEM results show that MG-HA and MBGsmaterials have similar bioactive behaviors.9,44
With the purpose of evaluating the cell response to the MG-HA nanocomposite, in vitro biocompatibility studies were carriedout with cultured murine L929 fibroblasts and human Saos-2osteoblasts. Figure 7 shows the effect of MG-HA material oncell proliferation of both cell types after 4 days of treatment.The cell number obtained in the presence of different MG-HAdoses was referred as percentage in relation to the controls(100%) carried out in the absence of biomaterial. As it can beobserved in all cases, high proliferation values were obtained
Figure 5. In vitro bioactivity in SBF: study of the nanocomposite MG-HA surface by SEM/EDS and TEM/ED before and after differenttimes in SBF.
Figure 6. (A) Calcium, pH, and (B) silicon variations in the SBF solution over time.
Figure 7. Effect of MG-HA bioceramic nanocomposite on proliferationof cultured murine L929 fibroblasts and human Saos-2 osteoblastsafter 4 days of treatment. The cell number obtained in the presence ofdifferent MG-HA doses was referred as percentage in relation to thecontrols (100%) carried out in the absence of biomaterial.
and no significant differences exist between MG-HA treatedcells and controls (p > 0.05). Previous studies using MBGs withthe same silica content also showed satisfactory results.However, a cytostatic effect was observed through thereduction of cell proliferation with MBGs.45 The absence ofthis cytostatic effect when MG-HA was added to the cellcultures can be due to the presence of nanocrystalline apatiteparticles embedded into the mesoporous glass walls, whichimproves the biocompatibility of MG-HA with respect toMBGs with ACP clusters. It has been demonstrated that cellsare sufficiently sensitive to alterations at the nanoscale levelwhich can elicit diverse cell behaviors.6 In this sense, previousstudies have shown that the nanocrytalline apatites comparedto ACP enhance notably the adhesion, proliferation, anddifferentiation of osteogenic cells.46,47
Figure 8 shows the cell morphology of murine L929fibroblasts (A) and human Saos-2 osteoblasts (B) cultured
for 4 days in the presence of 5 and 2.5 mg/mL of MG-HAbioceramic nanocomposite, respectively. This biomaterial doesnot produce morphological alterations in these cell types, whichproliferate in the presence of MG-HA, showing their normalcharacteristics as it was observed by optical microscopy. Nochanges of cell size and complexity were detected by flowcytometry after MG-HA treatment (data not shown).High values of cell viability (around 90%), evaluated by
propidium iodide exclusion and flow cytometry, were obtainedafter 4 days of treatment of cultured murine L929 fibroblastsand human Saos-2 osteoblasts with different doses of MG-HA(Table 3). In order to know if this biomaterial inducesapoptosis in these cell types, the cell percentage in each cellcycle phase was analyzed by flow cytometry and the SubG1
fraction (cells with fragmented DNA) was used as an indicationof apoptosis. Very low values of apoptosis (lower than 1%) inboth murine L929 fibroblasts and human Saos-2 osteoblastswere obtained after 4 days of treatment with different doses ofMG-HA (Table 3). All these results indicate the excellentbiocompatibility of this novel MG-HA bioceramic nano-
composite where the nanocrystalline apatite nanoparticlesimprove the cell response.
4. CONCLUSIONSIn this manuscript, the synthesis of a novel mesostructurednanocomposite formed by nanocrystalline apatite particlesuniformly embedded into mesoporous SiO2−CaO−P2O5matrix is shown. This nanocomposite presents a mesoporousarrangement with a well-defined 2D-hexagonal structureexhibiting high surface area and pore volume values withlarge pore size of 8 nm.The in vitro bioactivity assays have shown a fast apatite-like
phase formation on the surface of this nanocomposite material.The fibroblast and osteoblast response to this material reveals asignificant biocompatibility improvement compared with themesoporous bioactive glasses which could be due to thepresence of nanocrystalline apatite units distributed intomesoporous glass architecture. The obtained results indicatethat this novel nanocomposite offers a synergy of its twoconstituents, enhancing its potential for bone tissue regener-ation purposes.
■ ASSOCIATED CONTENT*S Supporting InformationDetailed description of Material and Methods. Figure S1showing the structural and textural characterization correspond-ing to Si100 material. Figure S2 displaying a TEM imagecorresponding to MG-HA material using P123 surfactant asstructure directing agent instead of F127. Figure S3 showingFTIR spectrum corresponding to calcined MG-HA material(PDF). This material is available free of charge via the Internetat http://pubs.acs.org.
NotesThe authors declare no competing financial interest.
■ ACKNOWLEDGMENTSIn memoriam of Prof. Purificacion Escribano Lopez. We thankthe following for funding this work: the Spanish CICYTthrough project MAT2008-00736, the Comunidad Autonomade Madrid via the S2009MAT-1472 program grant, and theMinisterio de Ciencia e Innovacion through project CS02010-11384-E. We also thank Fernando Conde (CAI X-ray
Figure 8. Cell morphology of murine L929 fibroblasts (A) and humanSaos-2 osteoblasts (B) cultured for 4 days in the presence of 5 and 2.5mg/mL of MG-HA nanocomposite respectively.
Table 3. Effect of MG-HA Nanocomposite Different Doses onCell Viability and Apoptosis of Cultured Murine L929Fibroblasts and Human Saos-2 Osteoblasts after 4 Days ofTreatment
Diffraction), CAI Elemental analysis, CAI NMR, ICTS CentroNacional de Microscopia Electrónica of Universidad Complu-tense de Madrid. M.C. is grateful to MICINN for the financialsupport through FPI fellowship.
■ REFERENCES(1) Hench, L. L.; Polak, J. M. Science 2002, 295, 1014.(2) Vallet-Regí, M. J. Int. Med. 2010, 267, 22.(3) Willians, D. Biomaterials 2008, 29, 1737.(4) Vallet-Regí, M; Ruiz-Hernandez, E. Adv. Mater. 2011, 23, 5177.(5) Thomas, V.; Dean, D. R.; Vohra, Y. K. Curr. Nanosci. 2006, 2,155.(6) Dorozhkin., S. V. Acta Biomater. 2010, 6, 715.(7) Vallet-Regí, M; Gonzalez-Calbet, J. M. Prog. Solid State Chem.2004, 32, 1.(8) Yan, X.; Yu, C.; Zhou, X.; Tang, J.; Zhao, D. Angew. Chem., Int.Ed. 2004, 43, 5980.(9) Lopez-Noriega, A.; Arcos, D.; Izquierdo-Barba, I.; Sakamoto, Y.;Terasaki, O.; Vallet-Regí, M. Chem. Mater. 2006, 18, 3137−3144.(10) Shi, Q.; Wang, J.; Zhang, J.; Fan, J.; Stucky, G. D. Adv. Mater.2006, 18, 1038.(11) Xia, W.; Chang, J. J. Controlled Release 2006, 110, 522.(12) Arcos, D.; Lopez-Noriega, A.; Ruiz-Hernandez, E.; Terasaki, O.;Vallet-Regí, M. Chem. Mater. 2009, 21, 1000.(13) Lopez-Noriega, A.; Arcos, D.; Vallet-Regí, M. Chem.−Eur. J.2010, 16, 10879.(14) Lin, H.-M.; Wang, W.-K.; Hsiung, P.-A.; Shyu, S.-G. ActaBiomater. 2010, 6, 3256.(15) Yang, P.; Zhao, D.; Margolesem, D. I.; Chemelka, B. F.; Stucky,G. D. Nature 1998, 396, 152.(16) Pinna, N.; Niederberger, M. Angew. Chem., Int. Ed. 2008, 47,5292.(17) Andersson, J.; Areva, S.; Spliethoff, B.; Linden, M. Biomaterials2005, 26, 6827.(18) Sousa, A.; Souza, K. C.; Sousa, E. M. B. Acta Biomater. 2008, 4,671.(19) Diaz, A.; Lopez, T.; Manjarrez, J.; Basaldella, E.; Martínez-Blanes, J. M.; Odriozola, J. A. Acta Biomater. 2006, 2, 173.(20) Borwoka, A.; Szczes, A. Mater. Lett. 2011, 65, 175.(21) Jagadeesan, D.; Deepak, C.; Siva, K.; Inamdar, M. S.; Eswara-Moorthy, M. J. Phys. Chem. C 2008, 112, 7379.(22) Leonova, E.; Izquierdo-Barba, I.; Arcos, D.; Lopez-Noriega, A.;Heddin, N.; Vallet-Regí, M.; Eden, M. J. Phys. Chem. C 2008, 112,5552.(23) García, A.; Cicuendez, M.; Izquierdo-Barba, I.; Arcos, D.; Vallet-Regí, M. Chem. Mater. 2009, 21, 5474.(24) Fattakhova-Rohlfing, D.; Szeifert, J. M.; Yu, Q.; Kalousek, V.;Rathousky, J.; Bein, T. Chem. Mater. 2009, 21, 2410.(25) Brinker, C. J.; Lu, Y. F.; Sellinger, A.; Fan, H. Y. Adv. Mater.1999, 11, 579.(26) Yu, B.; Poologasundarampillai, T.; Turdean-Ionescu, C.; Smith,M. E.; Jones, J. R. Biocer. Develop. Apps. 2011, 1, ID D110178.(27) Westheimer, F. H.; Huang, S.; Coritz, F. J. Am. Chem. Soc. 1988,110, 181.(28) Chai, C. S.; Gross, K. A.; Ben Nissan, B. Biomaterials 1998, 19,2291.(29) Jillavenkatesa, A.; Condrate, R. A. J. Mater. Sci. 1998, 33, 4111.(30) Liu, D. M.; Yang, Q.; Troczynski, T.; Tseng, W. J. Biomaterials2002, 23, 1679.(31) Zhao, D.; Feng, J. P.; Huo, Q.; Melosh, N.; Fredrickson, G. H.;Chmelka, B. F.; Stucky, G. D. Science 1998, 279, 548.(32) Panda, R. N.; Hsieh, M. F.; Chung, R. J.; Chin, T. S. J. Phys.Chem. Solids 2003, 64, 193.(33) Padilla, S.; Izquierdo-Barba, I.; Vallet-Regí, M. Chem. Mater.2008, 20, 5942.(34) Lin, K. S. K.; Tseng, Y. H.; Mou, Y.; Hsu, Y. C.; Yang, C. M.;Chan, J. C. C. Chem. Mater. 2005, 17, 4493.
(35) Jager, C.; Welzel, T.; Meyer-Zaika, W.; Epple, M. Magn. Reson.Chem. 2006, 44, 573.(36) Mankenzie, K. J. D.; Smith, M. E.Multinuclear solid-state NMR ofinorganic materials; Pergamon Press: Amsterdam, 2002.(37) Gunawidjaja, P. N.; Lo, A. Y. H.; Izquierdo-Barba, I.; García, A.;Arcos, D.; Stevensson, B.; Grins, J.; Vallet-Regí, M.; Eden, M. J. Phys.Chem. C 2010, 114, 19345.(38) Zhongqi, H.; Wayne Honeycutt, C.; Baoshan, X.; McDowel, R.W.; Pellechia, P. J.; Tiequan, Z. Soil Sci. 2007, 172, 7.(39) Tilocca, A.; Cormack, A. N. J. Phys. Chem. B 2007, 111, 14256.(40) Liu, Z.; Terasaki, O.; Ohsuna, T.; Hiraga, K.; Shin, H. J.; Ryoo,R. Chem. Phys Chem. 2001, 2, 229.(41) Vallet-Regí, M.; Romero, A. M.; Ragel, C. V.; LeGeros, R. Z. J.Biomed. Mater. Res. 1999, 44, 416.(42) Vallet-Regi, M.; Ragel, C. V.; Salinas., A. J. Eur. J. Inorg. Chem.2003, 1029.(43) Hench, L. L.; Andersson, O. In Hench, L.L.; Wilson, J., Eds.;Bioactive Glasses. An Introduction to Bioceramics; Elsevier Science: NewYork, 1995; p 477.(44) Izquierdo-Barba, I.; Arcos, D.; Sakamoto, Y.; Terasaki, O.;Lopez-Noriega, A.; Vallet-Regí, M. Chem. Mater. 2008, 20, 3191.(45) Alcaide, M.; Portoles, P.; Lopez-Noriega, A.; Arcos, D.; Vallet-Regí, M.; Portoles, M. T. Acta Biomater. 2010, 6, 892.(46) Ginebra, M. P.; Espanol, M.; Montufar, E. B.; Perez, R. A.;Mestres, G. Acta Biomater. 2010, 6, 2863.(47) Hu, Q.; Tan, Z.; Liu, Y.; Tao, J.; Cai, Y.; Zhang, M.; Pan, H.; Xu,X.; Tang, R. J. Mater. Chem. 2007, 17, 4690.
