Preparation and bioactive properties of novel bone-repair bionanocomposites based on hydroxyapatite and bioactive glass nanoparticles

Preparation and bioactive properties of novel bone-repairbionanocomposites based on hydroxyapatite and bioactive glassnanoparticles

Francisco Valenzuela,1 Cristian Covarrubias,1 Constanza Martı́nez,2 Patricio Smith,2

Mario Dı́az-Dosque,1 Mehrdad Yazdani-Pedram3

1Departamento de Ciencias B�asicas, Facultad de Odontologı́a, Universidad de Chile, Sergio Livingstone 943, Independencia,

Santiago, Chile2Facultad de Medicina, Pontificia Universidad Cat�olica de Chile, Santiago, Chile3Departamento de Quı́mica Org�anica y Fisicoquı́mica, Facultad de Ciencias Quı́micas y Farmac�euticas, Universidad de Chile,

Santiago, Chile

Received 6 December 2011; revised 18 April 2012; accepted 24 April 2012

Published online 16 June 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.32736

Abstract: Bionanocomposites based on ceramic nanopar-

ticles and a biodegradable porous matrix represent a prom-

ising strategy for bone repair applications. The preparation

and bioactive properties of bionanocomposites based on hy-

droxyapatite (nHA) and bioactive glass (nBG) nanoparticles

were presented. nHA and nBG were synthesized with nano-

metric particle size using sol–gel/precipitation methods.

Composite scaffolds were prepared by incorporating nHA

and nBG into a porous alginate (ALG) matrix at different

particle loads. The ability of the bionanocomposites to

induce the crystallization of the apatite phase from simu-

lated body fluid (SBF) was systematically evaluated using X-

ray diffraction (XRD), scanning electron microscopy with

energy dispersive X-ray analysis, and Fourier transform

infrared spectroscopy. Both nHA/ALG and nBG/ALG compo-

sites were shown to notably accelerate the process of crys-

tallization and growth of the apatite phase on the scaffold

surfaces. For short immersion times in SBF, nBG (25%)-

based nanocomposites induced a higher degree of apatite

crystallization than nHA (25%)-based nanocomposites, prob-

ably due to the more reactive nature of the BG particles.

Through a reinforcement effect, the nanoparticles also

improve the mechanical properties and stability in SBF of

the polymer scaffold matrix. In addition, in vitro biocompati-

bility tests demonstrated that osteoblast cells are viable and

adhere well on the surface of the bionanocomposites. These

results indicate that nHA- and nBG-based bionanocompo-

sites present potential properties for bone repair applica-

tions, particularly oriented to accelerate the bone

mineralization process. VC 2012 Wiley Periodicals, Inc. J Biomed

Mater Res Part B: Appl Biomater 100B: 1672–1682, 2012.

Key Words: bioactive nanoparticles, bionanocomposites, bio-

active glass, hydroxyapatite, bone regeneration

How to cite this article: Valenzuela F, Covarrubias C, Martı́nez C, Smith P, Dı́az-Dosque M, Yazdani-Pedram M. 2012. Preparationand bioactive properties of novel bone-repair bionanocomposites based on hydroxyapatite and bioactive glass nanoparticles.J Biomed Mater Res Part B 2012:100B:1672–1682.

INTRODUCTION

Bioactive glasses (BGs) and hydroxyapatite (HA) are prob-ably the most attractive ceramic materials for bone tissuerepair. Although the bioactivity mechanism of BGs is not yetfully understood, this resorbable and osteoproductive mate-rial promotes bone-tissue formation on its surface and itbonds to surrounding living tissue when implanted in thebody. HA has also been shown to have excellent biocompati-bility and bioactivity properties with respect to bone cellsand tissues, probably due to its similarity with the hard tis-sues of the body. Bioactive composites1,2 prepared by incor-porating bioactive ceramic particles into biodegradable poly-

meric matrices are interesting materials that combine thebioactivity of the inorganic particles with the supportingproperties of the three-dimensional polymeric matrix, whichpromote cellular attachment, and growth, and mineral ma-trix deposition. Bionanocomposites are a promising class ofnew hybrid nanomaterials that use a combination of nano-scale bone graft materials with biodegradable polymer mat-rices. These nanocomposites offer larger surface area, highsurface reactivity, relatively strong interfacial bonding,design flexibility, and enhanced mechanical properties com-pared to conventional bulk composites.3 Most of the studiesreport the preparation of bioactive composites using BG or

Correspondence to: C. Covarrubias; e-mail: [email protected]

Contract grant sponsor: CONICYT; contract grant number: FONDECYT Project No. 11100495

Contract grant sponsor: Vicerrectorı́a de Investigaci�on y Desarrollo (VID)—Universidad de Chile (Faculty Travel Grant Program ‘‘U-Apoya: Lı́nea

Ayuda de Viaje’’)

1672 VC 2012 WILEY PERIODICALS, INC.

HA particles with sizes in the micrometer range obtainedthrough a high-temperature preparation process. Nowadays,the advances in the synthesis of nanomaterials by the sol–gel technique offer the possibility of synthesizing BG andHA particles with controlled nanometric particle size usingrelatively low processing temperatures. Sanosh et al.4

reported the synthesis of nano HA by a simple sol–gel pre-cipitation method. HA nanoparticles presented morphology,size and crystallinity comparable to those of HA constitutinghuman hard tissue. Despite the promising characteristics ofthese HA nanoparticles, their bioactive properties such astheir ability to induce apatite mineralization in simulatedbody fluid (SBF) has not yet been evaluated. The synthesisof BG particles with nanometric size was reported by Honget al.5 The BG nanoparticles prepared by the sol–gel methodand then incorporated into a poly(L-lactide) (PLLA) matrixshowed a greater capability to induce the formation of anapatite layer in SBF media compared to pure PLLA scaffold.Some investigations have established that BG has a higherbioactivity than HA6 probably due to the biologically activesilica-rich layer produced by ion leaching from the BG struc-ture, which strongly promotes bone growth. These compara-tive studies have been carried out using HA and BG par-ticles with traditional micrometric size. Biocompositesprepared with nanosized HA or BG particles are expected tohave improved bioactive properties for bone tissue repair,due to the higher aspect ratio exhibited by the nanodimen-sional materials.6 Smaller crystals will dissolve more rapidlythan larger crystals of the same composition, due to thehigher surface area exposed to the biological environmentand to the larger number of lattice defects.8,9 Thus, the useof nanosized HA or BG should accelerate the rate of forma-tion and growth of the biologically active apatite layer,10

allowing a chemical link between the materials and thenewly formed bone, as well as the later attachment and dif-ferentiation of stem cells.

Bionanocomposites should be also fabricated by choos-ing an adequate polymer matrix. Strict requirements forscaffold materials are biocompatibility, a three-dimensionalporous structure, an appropriate surface chemistry for celladhesion and mineralization, sufficient mechanical strengthto withstand in vivo stress and physiological loading, and anadequate biodegradation rate with nontoxic byproducts. Al-ginate (ALG) is a naturally occurring and biocompatiblepolysaccharide widely used for cell transplantation,11 regen-eration of skin,12 cartilage,13 bone,14 liver,15 and cardiac tis-sue.16,17 As a hydrophilic polymer, the ALG sponge is easilywettable, allowing more efficient penetration of cells intothe matrix.18 ALG forms a stable gel by cross linkage withimmunologically inert calcium ions and it is also known tobreakdown to simpler glucose type residues, which aretotally absorbable.19 In spite of these valuable properties,the preparation of ALG-based bionanocomposites has beenscantily studied. Turco et al.20 reported the preparation ofHA/ALG composites using commercial HA with averageparticle sizes of 0.15 lm and inducing ALG gelling with D-gluconic acid d-lactone. This composite efficiently sup-ported the adhesion and proliferation of cells, showing at

the same time adequate structural and physicochemicalproperties. The preparation of porous ALG scaffolds con-taining micrometric octacalcium phosphate has been alsoreported.21

In the present study, the bioactive properties of novelbionanocomposites prepared with nanosized HA and BGparticles using ALG as scaffold matrix are investigated.Bioactive nanoparticles were synthesized by sol–gel meth-ods and then incorporated into the biopolymer to produceporous bionanocomposite scaffolds. The work is focusedon comparing the ability of the bioactive nanomaterialsto accelerate the formation and growth of an apatitelayer from SBF. The viability of osteoblast bone-formingcells in the presence of the bionanocomposites is alsoevaluated.

MATERIALS AND METHODS

Synthesis of HA and BG nanoparticlesHA nanoparticles (nHA) were synthesized by the sol–gelprecipitation method reported by Sanosh et al.4: 50 mL of0.6M potassium dihydrogenphosphate (NH4H2PO4; May &Baker) solution were added dropwise to an equal volume of1M calcium nitrate (Ca(NO3)2�4H2O; Sigma-Aldrich) solutionunder constant stirring. Aqueous ammonia (NH3) was addeddropwise to the resulting solution until pH 11 was reached.The precipitated solution was stirred for 1 h and aged atroom temperature for 24 h. The white precipitate was sepa-rated and washed by repeated centrifugation and redisper-sion in fresh distilled water (three cycles). It was then driedat 40�C for 24 h, heated at a rate of 10�C/min to 200�C,and calcined at that temperature for 1 h. A calcination tem-perature of 200�C was chosen in order to obtain a less crys-talline and more reactive nHA particle.

Nanosized BG particles (nBG) were synthesized by thesol–gel method reported by Hong et al.5 However, in orderto obtain BG with better bioactivity the synthesis mixturewas prepared using the following molar composition:58SiO2:40CaO:5P2O5.

22 The synthesis of nBG was carriedout as follows: A calcium-based solution was prepared bydissolving 7.7 g of Ca(NO3)2�3H2O (Sigma-Aldrich) in 117mL of distilled water at room temperature. A second solu-tion was prepared by diluting 9.7 mL of tetraethylorthosili-cate (TEOS 98%; Aldrich) in 63.5 mL of ethanol, and it wasadded to the calcium nitrate solution, and the pH of theresulting solution was adjusted to 1–2 with citric acid.This transparent solution was slowly dropped under vig-orous stirring into a solution of 1.2 g of NH4H2PO4 (May& Baker) in 1500 mL of distilled water. During the drip-ping process the pH was kept at around 10 with aqueousammonia. The mixture was stirred for 48 h and aged for48 h at room temperature. The precipitate was separatedby centrifugation (12,000 rpm), and washed by three cen-trifugation-redispersion cycles with distilled water. Theseparated solid was dispersed in 200 mL of a 2% w/vaqueous solution of polyethylene glycol (PEG, 10,000), andstirred for 24 h. This suspension was freeze dried, andthen calcined at 700�C for 3 h to obtain a fine white nBGpowder.

ORIGINAL RESEARCH REPORT

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | AUG 2012 VOL 100B, ISSUE 6 1673

Preparation of bioactive nanocompositesnHA/ALG and nBG/ALG nanocomposites with 5, 25, and35% w/w nanoparticle content were prepared. A sodiumALG solution was prepared by dissolving 4 g of alginic acidsodium salt from brown algae (Medium viscosity, Sigma) in30 mL of distiller water. For each nanocomposite composi-tion, 0.04, 0.20, and 0.28 g of dried nanoparticle powderwas added to 20 mL of distilled water respectively, and dis-persed by sonication for 20 min. The nanoparticle disper-sion was then added to 30 mL of ALG solution with con-stant stirring, and the resulting mixture was furthersonicated for 20 min. In order to produce the ALG chaincrosslinking, 50 mL of a 0.4% w/v solution of calcium ni-trate was added dropwise under stirring. The resulting com-posite solution was 4% w/v sodium ALG. The composite gelformed was placed into 24-well plates, frozen at �80�C for12 h, and lyophilized for 48 h until dry. Pure ALG scaffoldswere also prepared following the procedure describedabove.

In vitro bioactivity assaysThe ability of the bionanocomposites to induce the forma-tion of apatite was assessed in acellular SBF, which hasinorganic ion concentrations similar to those of humanextracellular fluid. The SBF solution was prepared asdescribed by Kokubo et al.23 using the standard ion compo-sition (Naþ 142.0, Kþ 5.0, Mg2þ 1.5, Ca2þ2.5, Cl� 147.8,HCO�

3 4.2, HPO2�4 1.0, SO2�

4 0.5 mM). The fluid was bufferedat physiological pH 7.4 at 37�C with tri-(hydroxymethyl)aminomethane and hydrochloric acid. The cylindrical biona-nocomposite samples (1.0 cm � 0.5 cm) were individuallysoaked in 50 mL of SBF in polyethylene containers at36.5�C using a thermostatic bath. After incubation for a des-ignated time period, the scaffolds were removed from SBF,rinsed with distilled water, and dried at 60�C. The pH of theSBF medium was also recorded at different times.

Cell cultureHuman osteoblastic osteosarcoma cell line (SaOS-2) wasused for cell culture analyses. The viability of the osteo-

blast-like cells on the bionanocomposites was assessedusing the 3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assayaccording to the protocol provided by the manufacturer(CellTiter Aqueous One Solution cell proliferation assay kitfrom Promega). Approximately 50 � 103 cells in 1 mL Dul-becco’s modified Eagle medium (D-MEM) (Invitrogen LifeTechnologies) were seeded on the scaffold. The mediumcontained 10% fetal bovine serum (FBS GIBCO), 50 IU/mLpenicillin, and 50 mg/mL streptomycin. The cell suspensionwas seeded on sterilized scaffolds, incubated at 37�C in ahumidified air atmosphere containing 5% CO2, and MTSassays were performed in quadruplicate 2 days after cellseeding. Briefly, after 2 h of incubation with the MTS rea-gent in a humidified 5% CO2 atmosphere, the medium wascollected from the scaffolds and absorbance was measuredat a wavelength of 490 nm. Cells adhered on the surface ofsome nanocomposites after 2 h of incubation were exam-ined by Scanning Electron Microscopy (SEM) (Zeiss, DMS940). For this purpose, adherent cells were fixed in 2.5%glutaraldehyde, then progressively dehydrated in ethanol,

FIGURE 2. XRD pattern of (a) nBG and (b) nHA bioactive nanopar-

ticles synthesized by sol–gel/precipitation methods.

FIGURE 1. TEM image of (a) nBG and (b) nHA bioactive nanoparticles synthesized by sol–gel/precipitation methods.

1674 VALENZUELA ET AL. PREPARATION AND BIOACTIVE PROPERTIES OF NOVEL BONE-REPAIR BIONANOCOMPOSITES

dried in super-critical CO2, and finally coated with gold forSEM observation.

Material characterizationBioactive nanoparticles, bionanocomposites, and apatite for-mation were analyzed by X-ray diffraction (XRD). XRD pat-terns were measured on a Siemens D 5000 diffractometerusing CuKa radiation within a 2y range of 5�–50� at a scan-ning speed of 1.2�/min. For XRD analysis, the scaffolds sam-ples were removed from SBF solution, then rinsed with dis-tilled water and dried at 60�C for 24 h. Bioactivenanoparticles were also examined by Transmission Elec-tron Microscopy (TEM) in a Philips Tecnai 12 Bio Twinmicroscope. Specimens were prepared by transferring asmall drop of sample-ethanol suspension to carbon-film-coated copper grids. Apatite formation on the bionano-composites was also analyzed by SEM and Fourier Trans-form Infrared Spectroscopy (FTIR). SEM images were

taken with a Jeol JSM 5410 microscope equipped withenergy-dispersive X-ray spectroscopy (EDX). FTIR analysiswas carried out on a Bruker Vector 22 FTIR spectrometer.SBF-treated bionanocomposites were crushed, homoge-neously mixed with KBr (1 wt.%), and pressed toobtain circular wafers. FTIR transmission spectra wererecorded in the 4000–400 cm�1 range with a resolutionof 1 cm�1.

The compressive mechanical strength and modulus ofscaffolds were tested with an Instron 4505 mechanical tes-ter with a 100N load cell. The exact dimensions of each cy-lindrical scaffold were measured right before the test (typi-cally 15 mm in height � 5 mm in diameter). The crossheadspeed was set at 2 mm/min and load was applied until thespecimens were compressed to approximately 50% of theiroriginal height. The slope of the initial linear section of thecompressive stress–strain curve was used to estimateYoung’s modulus.

FIGURE 3. SEM images of (a,d) neat ALG scaffold, (b,e) nHA(25%)/ALG, and (c,f) nBG(25%)/ALG nananocomposite scaffolds. (g) Photography of

nBG(25%)/ALG.



RESULTS AND DISCUSSION

Preparation of bioactive nanocompositesHA and BG were synthesized with nanometric particle size(Figure 1). TEM observations revealed that nHA and nBGpresent an estimated particle size of around 40 and 70 nm,respectively. The XRD pattern of nHA exhibits all reflections

corresponding to the HA crystal structure (JCPD 09-0432)(Figure 2), whereas the nBG pattern presents broader peakscharacteristic of the less-crystalline BG structure. Thesenanosized particles were incorporated into a crosslinkedALG matrix to prepare nHA/ALG and nBG/ALG nanocompo-site scaffold materials. Typical SEM images of the porousscaffold structures are shown in Figure 3. Neat ALG scaffoldpresented a relatively uniform macroporous structure withpore size in the 100–500 lm range. Incorporation of thenanoparticles into the ALG matrix tended to increase the

FIGURE 4. XRD analysis of (a) nHA(35%)/ALG and (b) nBG(35%)/ALG

nanocomposites, and samples after 7 days immersion in SBF: (c) ALG

scaffold, (d) nHA(5%)/ALG, (e) nHA(25%)/ALG, (f) nHA(35%)/ALG, (g)

nBG(5%)/ALG, (h) nBG(25%)/ALG, and (i) nBG(35%)/ALG.

FIGURE 5. SEM images of nanocomposites after 7 days immersion in SBF: (a) nHA(5%)/ALG, (b) nHA(25%)/ALG, (c) nHA(35%)/ALG, (d)

nBG(5%)/ALG, (e) nBG(25%)/ALG, and (f) nBG(35%)/ALG.

FIGURE 6. XRD analysis of nanocomposites after different immersion

times in SBF: (a–c) neat ALG scaffold, (d–f) nHA(25%)/ALG, and (g–i)

nBG(25%)/ALG.


macropore size of the resulting nanocomposite scaffolds[Figure 3(b,c)]. This is an indication that the HA and BGnanoparticles probably affect the mechanism of pore forma-tion from ice crystal templates. It has been found that dif-ferent sizes and types of nanoparticles can induce differentinstability in the ice front, nanoparticle surface energy canalso cause different ice-nanoparticle segregation behav-ior24,25 or induce different ice structures26; affecting thusthe size and shape of the pores. Nanocomposites preparedwith 25 wt% nanoparticle content exhibited more distortedand asymmetrical pores about 250–700 lm in size. Thismacroporous size range has been suggested as suitable forcell adhesion, ingrowth and reorganization, and would pro-vide the necessary space for neovascularization in vivo.27–30

Some microsized HA and BG clusters were also observed onpore walls of the scaffolds, which is an indication that nano-

particles undergo some degree of agglomeration duringnanocomposite formation. Polymer nanocomposite structurewas examined at higher SEM magnification on fracturednanocomposite samples [Figure 3(d–f)]. Some nanoparticlescan be observed as small microsized cluster dispersed intothe ALG polymer matrix, which is an indication that nano-particles undergo some degree of agglomeration duringnanocomposite formation. On the other hand, the nanocom-posite preparation procedure used in this work, offeredeasy processability of the materials in different shapes andsizes [Figure 3(g)].

In vitro bioactivity assaysThe bioactivity of nanocomposite scaffolds prepared withdifferent nanoparticle loads was initially evaluated in SBFfor 7 days. The ability of the nanocomposites to induce the

FIGURE 7. SEM images of nanocomposite surfaces after different immersion times in SBF: (a) neat ALG scaffold, (b) nHA(25%)/ALG and (c)

nBG(25%)/ALG.



formation of apatite phase was analyzed by XRD (Figure 4).It was found that the degree of apatite crystallizationincreases with nanoparticle content, as judged by the inten-sity of the most characteristic apatite peak at 31.7�, corre-sponding to the 211 reflection of the apatite crystal (JCPD250166). The crystallization of the apatite phase on the bio-materials strongly depends on the concentration of Ca2þ

and PO3�4 ions in the surrounding fluid.8,31 Dissolution of

BG particles produces free ions, which in combination withother ions from SBF form the apatite phase. In the case ofHA, these particles act as nanosized nucleation centers forapatite growth, eliminating thus the nuclei induction phase.It can also be noted that at the same particle load, thedegree of apatite crystallization is higher on nBG/ALG com-posites than on nHA/ALG composites. This effect can beattributed to the higher solubility and reactivity of the BGstructure, as consequence of its siliceous and less crystallinenature.32 These differences in the ability of the nanocompo-sites to induce mineralization of apatite were also confirmedby SEM (Figure 5). nHA/ALG nanocomposites with 5–25 wt% particle content exhibited small apatite clusters on theirsurface, which did not densely cover the entire surface[Figure 5(a,b)]. In contrast, nBG/ALG nanocompositesinduced the formation of a more continuous apatite layer

FIGURE 8. EDX elemental analysis of nanocomposite surfaces after different immersion times in SBF: (a) neat ALG scaffold, (b) nHA(25%)/ALG

and (c) nBG(25%)/ALG.

FIGURE 9. FTIR spectra of nanocomposites after different immersion

times in SBF: (a–c) neat ALG scaffold, (d–f) nHA(25%)/ALG, and (g–i)

nBG(25%)/ALG.


made of larger sized apatite clusters [Figure 5(e)]. Whenthe nHA/ALG nanocomposite was prepared with 35 wt %HA, an increase in apatite crystallization was observed interms of both the extent of the mineralization and the clus-ter size deposited on the scaffold surface [Figure 5(c)]. Thiseffect was much more marked for the case of nBG(35%)/ALG nanocomposite [Figure 5(f)], which led to the forma-tion of a dense apatite material with bone-like appearance.Although nanocomposites prepared with 35 wt % nanopar-ticles induce a high degree of mineralization, this particlecontent could not be compatible with the desired porosityof the scaffold.

Bioactive nanoparticles are expected to considerablyaccelerate the formation of apatite during the early stagesof the bone repair process. In order to evaluate this prop-erty, apatite crystallization was analyzed on the neat ALGscaffold and on the 25 wt % filled nanocomposites aftershort immersion times in SBF. Figure 6 shows the gradualincrease of apatite crystallization on the nanocompositesurfaces with increasing time in SBF. The formation of crys-talline apatite on both the unfilled scaffold and the nano-composites was detected by XRD at 24 h of incubation in

SBF. Beyond this time, the intensity of the XRD apatite peakis considerably higher on the nanocomposite samples com-pared to that of the neat biopolymer matrix, confirming ahigher degree of apatite crystallization in the presence ofthe nanoparticles. Among the nanocomposites studied, nBG/ALG presented a higher rate of apatite crystallization thannHA/ALG for short immersion times in SBF. As already com-mented, siliceous and more amorphous BG structure is gen-erally more soluble and reactive than crystalline HA, therebyaccelerating the apatite formation process through differentionic dissolution products. Si is known to be an essentialelement for metabolic processes associated with the forma-tion and calcification of bone tissue.33,34 High Si contentshave been detected in early stages of bone matrix calcifica-tion,31 whereas aqueous Si was shown to be able to induceprecipitation of HA.35 The evolution of apatite mineraliza-tion was also examined by SEM (Figure 7). Although, noXRD apatite peaks were detected at 12 h of immersion inSBF, SEM observations revealed the presence of mineralizedclusters on different regions of the nanocomposite surfaces,particularly on the nBG(25%)/ALG surface. EDX chemicalcomposition analysis (Figure 8) showed high Ca and P con-tent in the precipitates deposited on the nanocomposites,whereas only Na and Cl were detected on the neat ALG sur-face. These results indicate that clusters formed on the

FIGURE 10. SEM images showing the macroporous scaffold structure after 7 days immersion in SBF: (a) neat ALG scaffold, (b) nHA(25%)/ALG

and (c) nBG(25%)/ALG.

FIGURE 11. pH variations of SBF medium versus immersion time for

the neat ALG scaffold and the bionanocomposites.

FIGURE 12. Mechanical properties of neat ALG scaffold and

bionanocomposites.



nanocomposites at 12 h may correspond to an amorphouscalcium phosphate phase (ACP), which is a precursor ofcrystalline apatite. After 24 h of incubation in SBF, nearly allthe area of the surfaces of the bionanocomposites was cov-ered with apatite crystals. Among the nanocomposites stud-ied, significant differences in terms of cluster sizes and den-sity of the apatite layer formed on their surface weredetected. In the case of the nHA(25%)/ALG nanocomposite,relatively small and discrete apatite precipitates wereformed on its surface, contrasting with the larger clustersand denser apatite layer induced on the surface of thenBG(25%)/ALG nanocomposite [Figure 7(b,c)]. Calcium andphosphorous contents (Ca þ P) measured by EDX on thenBG-based nanocomposite were higher than those found onthe nHA-based nanocomposite (Figure 8), confirming thegreater presence of apatite on the nanocomposite filled withnBG, as already detected by XRD and SEM. The formation ofapatite on the nanocomposites was also verified by FTIRanalysis (Figure 9), mainly from the presence of two bandsaround 600 cm�1, attributed to the PO4 bending vibrationand from another strong band in the 1000–1100 cm�1 area,attributed to the PO4 symmetric stretching vibration in crys-

talline apatite.36 Although the ACAOACA antisymmetricstretching mode of the ALG structure also appears in the1027–1081 cm�1 range, the intensity of the apatite PO4

stretching band is predominant. As the apatite layer beginsto develop on the nanocomposite’s surface, the PO4 stretch-ing peak becomes more intense and sharper. At 12 h ofimmersion in SBF, nanocomposite filled with nBG exhibits amore intense PO4 apatite band than that observed on thenHA-based nanocomposite. These observations confirm thebetter ability of the nBG-based nanocomposite compared tothe nHA-nanocomposite to accelerate the mineralization ofapatite after short immersion times in SBF.

Stability and mechanical propertiesChemical stability and mechanical properties of bionano-composites are also important aspects for their use in tissuerepair applications. The stability of the porous structure ofthe bionanocomposites was evaluated in SBF medium. Fig-ure 10 shows SEM images of the neat ALG scaffold and ofthe bionanocomposites after 3 days of immersion in SBF. Itcan be seen that the porous structure of neat ALG is consid-erably altered after the soaking period in SBF, whereas thenanoparticle-filled scaffolds retain to a large extent theiroriginal macroporous structure. This result can be attrib-uted to a reinforcement effect produced by the incorpora-tion of the nanoparticles into the biopolymer matrix. Thiseffect must result either from a restriction of the molecularmotion in the amorphous regions of the polymer or fromsimple mechanical reinforcement by the dispersed nanopar-ticles. The pH variation of the SBF media was also moni-tored during the scaffold immersion period (Figure 11). Allthe scaffolds produced an abrupt pH decrease after a fewhours of immersion in SBF. After that, pH of the SBF me-dium increased, reaching a plateau around 7.1. The pHdecrease can be attributed to the release of uronic acidmonomers into the SBF medium, as a consequence of poly-saccharide depolymerization via cleavage of the glycosidicbonds.37 In despite of the observed pH decrease, ALGweight loss was not detected in SBF due to the apatite pre-cipitation effect on the biopolymer surface. Although there

FIGURE 13. Osteoblast cell viability (MTS) on neat ALG scaffold and

bionanocomposites.

FIGURE 14. SEM image of osteoblastic cell attached on the nBG(25%)/ALG bionanocomposite surface (a) and amplification showing filopodia-

biomaterial interaction (b).


are few studies about the exact mechanism of ALG degrada-tion in SBF, it has been found that ALG has a faster degrada-tion rate in SBF than in deionized water at 37�C.38 The pHincrease was slightly higher in the case of the nBG(25%)/ALG composite, and it can be attributed to the bufferingeffect of neutralizing basic anions produced from nBG disso-lution. Thus, nBG nanoparticles contribute to compensatethe pH decrease produced by the degradation of the ALGmatrix.

In tissue engineering, porous scaffolds act as a temporalsupport for tissue regeneration through cell adhesion, prolif-eration and differentiation. They have to possess sufficientstrength and stiffness that will bear in vivo loads so that thescaffolds can function before the growing tissue replacesthe gradually degrading scaffolds matrix. Compression testsof the bionanocomposites were also carried out, and thecompressive strength and Young’s modulus are shown inFigure 12. A significant improvement in the mechanicalproperties of the neat ALG scaffold is achieved with theincorporation of the nanoparticles. Compressive strengthand Young’s modulus were increased by �40 and 22%,respectively. Young’s modulus of nHA(25%)/ALG wasslightly higher than that of nBG(25%)/ALG, probably due toa higher reinforcement effect of the more crystalline HAnanoparticles. Thus, the presence of HA and BG nanopar-ticles in the ALG matrix also contributes to improve thescaffold’s stiffness (Young’s modulus) needed for bonerepair applications.

Cell viabilityQuantitative MTS assays were performed to determine themetabolic activity of osteoblast-like cells (SaOS-2) associatedwith each type of scaffold after 48 h of incubation (Figure13). Although absorbance values of the bionanocompositestended to be higher compared to those of the neat ALG andcontrol (cells cultured in the absence of the materials), nostatistical differences were found. These results indicatethat the viability and proliferation of the bone-forming cellsis not altered in contact with the bionanocomposites pre-pared in this work. In addition to the cytocompatibilityexhibited by the nanocomposites, osetoblastic cells adheredwell on the surface of these materials (Figure 14). The cellsdeveloped extended filopodia intimately adhered to thebionanocomposite surface. These cell culture assays furthersupport the potential of the nBG and nHA-based bionano-composites for their possible use in bone repairapplications.

CONCLUSIONS

HA and BG nanoparticles synthesized by the sol–gel methodcan be used for the preparation of bioactive nanocompositematerials for bone repair. Bionanocomposite scaffolds pre-pared by incorporating the nanoparticles into a macropo-rous ALG matrix, exhibited outstanding bioactive propertiesin SBF medium. The nanoparticles notably accelerate thecrystallization process of the apatite phase on the scaffoldsurfaces. For short immersion times in SBF, nBG-basednanocomposites induce a higher degree of apatite crystalli-

zation than nHA-based nanocomposites, as a consequence ofthe more reactive nature of the BG particles. Through areinforcement effect HA and BG nanoparticles also improvethe mechanical properties and stability of the polymer scaf-fold matrix in SBF. In addition, in vitro biocompatibility testsshow that osteoblast cells are viable and adhere well on thesurface of the bionanocomposites. Hence, the nHA and nBG-based bionanocomposites present potential properties forbone repair applications, particularly oriented to acceleratethe bone mineralization process.

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Preparation and bioactive properties of novel bone-repair bionanocomposites based on hydroxyapatite and bioactive glass nanoparticles