reverse micelle-mediated synthesis and characterization of tricalcium phosphate nanopowder for bone...

Reverse Micelle-Mediated Synthesis and Characterization of TricalciumPhosphate Nanopowder for Bone Graft Applications

Sudip Dasgupta and Susmita Bosew

W. M. Keck Biomedical Materials Research Lab, School of Mechanical and Materials Engineering, Washington StateUniversity, Pullman, Washington 99164-2920

Nanocrystalline b-tricalcium phosphate (b-TCP) powder wassynthesized using reverse micelle as a template system. Cyclo-hexane was used as the oil phase, aqueous solutions of calciumnitrate and phosphoric acid as the aqueous phase, and poly(oxy-ethylene)5 nonylphenol ether (NP-5) and/or poly(oxyethylene)12nonylphenol ether (NP-12) as the surfactants. The powder weresynthesized at a fixed Ca/P molar ratio of 1.5 at a pH of 10.The synthesized powder were calcined at 8001C to obtain mono-phasic b-TCP. Particle size, morphology, and surface area ofthe synthesized powder were dependent on the chemistry of thesurfactant and composition of the microemulsion. The powderwere characterized using a BET surface area analyzer, powderX-ray diffraction, dynamic light scattering technique, and trans-mission electron microscopy. TCP nanoparticles had a particlesize between 32 and 135 nm, and a BET-specific average surfacearea between 57 and 103 m

2/g with controlled morphology.

The powder were consolidated and sintered at 12501C in a 3 kWmicrowave furnace in the form of a compact disk. Human osteo-precursor cells (osteoblastic precursor cell line 1 [OPC1]) wereused to assess the biocompatibility of TCP disks after 1, 5, and11 days in culture using scanning electron microscopy, MTTassay, and alkaline phosphatase expressions. Disk samples werebiocompatible and showed excellent OPC1 cell adhesion,growth, and proliferation. Biocompatible b-TCP nanopowderwere synthesized with controlled particle size, morphology, andsurface area using a reverse micelle-mediated template system.

I. Introduction

HYDROXYAPATITE (Ca10(PO4)6(OH)2, HA) and b-tricalciumphosphate (b-Ca3(PO4)2, b-TCP) are the two calcium

phosphate ceramics (CPCs) used in bone graft applicationsdue to their excellent biological responses to physiological envi-ronments.1–5 HA is a bioactive and osteoconductive ceramic,and closely chemically resembles the inorganic component ofhuman bone.3,4 b-TCP and HA implants become surrounded bynew bone within a few weeks after implantation at bony sites invivo.6 b-TCP is considered as a bioresorbable ceramic, as it isgradually replaced by new, natural, fully functional bone byvirtue of its extraordinary bioresorbability in biological envi-ronments.6

TCP powder can be synthesized by various processing routesthat can be divided into two broad categories, including solid-state processes7–10 and wet chemical methods.11–15 Powdersynthesized by solid-state processes suffer from stoichiometricalinhomogeneity, wide particle size distribution, and hard agglom-eration, which motivated researchers to study wet chemical pro-cesses. Destainville et al.16 have investigated the synthesis of

b-TCP nanopowder with an average specific surface area be-tween 8972 and 9572 m2/g by an aqueous precipitationmethod using a solution of calcium nitrate (Ca(NO3)2) and am-monium hydrogen phosphate ((NH4)2HPO4) at neutral andalkaline pH. Pena and Vallet-Regı17 studied the synthesis of b-TCP by a Pechini-based liquid-mix technique using calciumnitrate, ammonium hydrogen phosphate, and citric acid as pre-cursor materials. In these wet chemical synthesis routes, theprecipitate derived from aqueous solution is not of pure TCP,but apatitic TCP Ca9(HPO4)(PO4)5(OH), which requires furthercalcination at temperatures between 7001 and 8001C in order toproduce the b-TCP phase. Bow et al.18 used anhydrous 99.5%methanol, instead of water, to synthesize spherical b-TCP pow-der with an average particle size of 50 nm at 801C using calciumacetate and phosphoric acid. None of these aforementionedmethods showed precise control over particle morphology oragglomeration.

Calcium phosphate nanopowder synthesized using a micro-emulsion technique exhibit better control over nanoscale mor-phology compared with other wet chemical synthesis routes.Microemulsions are true dispersions of liquid droplets, water, oroil, in the size range between 10 and 100 nm, within anotherimmiscible liquid, oil, or water, stabilized by a surfactant. Thus,they are named as oil-in-water or water-in-oil microemulsions.In the case of water-in-oil (w/o) microemulsions, the heads ofsurfactant molecules/chains orient themselves into water and thetails into a continuous hydrocarbon phase, a phenomenoncalled reverse micellization. Cao et al.19 synthesized ultrahigh-aspect-ratio HA nanofibers using cetyltrimethylammoniumbromide (CTAB)/cyclohexane/n-pentanol/water in a reversemicelle-based template system. Sun et al.20 studied a reversemicroemulsion-directed synthesis of HA nanoparticles crystal-lized under hydrothermal conditions using surfactant. Theyfound a significant effect of surfactant on the morphology ofsynthesized HA nanoparticles. In our previous work, we havereported on both the micelle- and reverse micelle-basedmicroemulsion templated synthesis of HA nanopowder withcontrolled morphology.21,22

It is known that bone is a composite material consisting ofnanoscale minerals of biological apatite (length 50 nm, breadth25 nm, and thickness up to 4 nm) and a matrix of collagen fibers(50–70 nm in diameter).23 Although HA shows excellent osteo-conductivity, stoichiometric HA is not bioresorbable. b-TCP is abioresorbable material, and one of the most popular bone sub-stitutes for dental and orthopedic applications. However, appli-cations of b-TCP are restricted to relatively small bonedefects.24,25

Here we report the synthesis of b-TCP nanopowder withdifferent aspect ratios using a reverse micellization technique.This technique has better control over particle shape in the nano-scale compared with any other wet chemical synthesis route.Moreover, researchers have shown that nanoscale features im-prove cell–material interactions in different materials.26,27 Nano-grain materials have the potential to enhance strength andtoughness with regard to a micrometer scale material with thesame composition. b-TCP nanopowder with a different morphol-ogy can be used directly in drug or biomolecule delivery

T. Troczynski—contributing editor

This work was financially supported by the National Science Foundation (NSF) underthe Presidential CAREER Award for Scientists and Engineers (PECASE) to Dr. S. Bose(CTS # 0134476) and the National Institute of Health (Grant # NIH-RO1-EB- 007351).

wAuthor to whom correspondence should be addressed. e-mail: [email protected]

Manuscript No. 25569. Received November 30, 2008; approved June 8, 2009.

Journal

J. Am. Ceram. Soc., 92 [11] 2528–2536 (2009)

DOI: 10.1111/j.1551-2916.2009.03258.x

r 2009 The American Ceramic Society

2528

i:/BWUS/JACE/03258/[email protected]

applications or can be used for processing nanograin compactsfor bone grafts. Because b-TCP is a bioresorbable ceramic, itoffers immense opportunities in the field of tissue engineering.Bioresorbability of b-TCP nanopowder can be tailored by vary-ing particle size, morphology, surface area, and crystallinity.

The purpose of this research was to investigate the feasibilityof using a reverse microemulsion system in TCP nanoparticlesynthesis in which we would be able to control the particle shape.In our study, powder particle size and morphology were con-trolled by varying different synthesis parameters such as theaqueous to organic phase volume ratio, the nature of the sur-

factant, the pH of the reaction mixture, the aging or ripening timeof the reaction. Powder were characterized for their phase purity,particle size, shape, and distribution, and surface area using X-ray diffraction (XRD), Fourier transform infrared (FTIR) spec-troscopy, dynamic light scattering (DLS), transmission electronmicroscopy (TEM), and Brunauer, Emmett, and Teller (BET)surface area techniques. In vitro bone cell–material interactions ofTCP compacts were investigated for adhesion, proliferation, anddifferentiation of osteoblastic precursor cell line 1 (OPC1) humanosteoprecursor bone cells using scanning electron microscopy(SEM), MTT assay, and alkaline phosphatase (ALP).

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NP5

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Fig. 2. X-ray diffraction pattern of synthesized powder calcined at 80011C for 3 h using different surfactant systems.

November 2009 Synthesis and Characterization of Tricalcium Phosphate Nanopowder 2529

II. Experimental Procedure

(1) TCP Nanopowder Synthesis

Calcium nitrate tetrahydrate ((Ca(NO3)2 � 4H2O, Alfa Aeser,Ward Hill, MA), 90% orthophosphoric acid (H3PO4, Fisher,PA), ammonium hydroxide (NH4OH, Fisher, Pittsburgh, PA),cyclohexane (Fisher) as the oil phase, poly(oxyethylene)5 non-ylphenol ether (NP-5, Sigma-Aldrich, Milwaukee, WI) andpoly(oxyethylene)12 nonylphenol ether (NP-12, Sigma-Aldrich,WI, USA) as surfactants were used for the synthesis of b-TCPnanopowder. First, a 0.5M Ca21 solution was prepared by dis-solving the required amount of Ca(NO3)2 � 4H2O in deionizedwater. The mole ratio of Ca to P was maintained at 1.5:1.0 bythe dropwise addition of H3PO4. Ten volume percent of sur-factants, NP-5 and/or NP-12, were added to cyclohexane toprepare the organic phase. The aqueous phase was then mixedwith the organic phase in different aqueous to organic volumeratios (aq:org ratio) of 1:05, 1:10, and 1:15. Constant stirring for30 min on a hot plate produced a water-in-oil emulsion. The pHof the emulsion was adjusted to 10 by the slow addition of 90%concentrated NH4OH to the reaction mixture. The emulsionconverted into a transparent gel during mixing. The reactionmixture was aged at room temperature for 12 h and then dried

on a hot plate at 3001C followed by calcination at 8001C for 4 hin a muffle furnace to obtain nanocrystalline TCP powder.

(2) Powder Characterization

Phase analysis of calcined nanopowder was performed using aSiemens D500 Krystalloflex X-ray diffractometer with CuKaradiation (1.54018 A) with a Ni filter at room temperature overthe 2y range of 201–701 at a step size of 0.021 and a count time of0.5 s/step. The crystallite size was determined using the Scherrerformula t5 0.9l/b cos (y) from the (0210) peak at full-width athalf maxima (FWHM). The JCPDS files 09-169 for b-TCP and09-348 for a-TCP were used. The presence of characteristicchemical bonding in the synthesized powder was confirmed byFTIR analysis using a Nicolet 6700 FTIR spectrophotometer(Madison, WI). Powder particle size was measured by the DLStechnique using a NICOMPt 380 (Santa Barbara, CA) particlesize analyzer. A dilute aqueous suspension of powder at pH-10was ultrasonicated for 15 min to minimize the degree of ag-glomeration under the influence of strong electric double layerrepulsion around the negatively charged particle surface andwas used for particle size analysis. Particle size and morphologyof calcined TCP particles were studied using a TEM (JEM 120,JEOL, Peabody, MA). A 3 mL drop of a dilute aqueous sus-pension of powder was taken on a carbon-coated Cu grid andanalyzed. The aspect ratio of particles was calculated from TEMimages using a soft imaging system, Analysis 3.2 (Lakewood,CO). At least 10 TEM images were analyzed for each powder torepresent the aspect ratio data as mean7standard deviation (s)The aspect ratio data for all powder were analyzed with a SAS9.1 statistical analysis package (Cary, NC, USA)28 using one-way analysis of variance (ANOVA), and Tukey’s method forpairwise multiple comparison was carried out at a significancelevel of p5 0.05 in order to find out the differences in aspectratio of b-TCP nanopowder with variations in synthesis param-eters. The specific average surface area of powder was deter-mined using the BET method (five-point analyzer, TristarMicromeritics, Norcross, GA) after degassing samples overnightat 3501C with a continuous flow of nitrogen. Three samplesfrom each powder were used for BET surface area measurement,and data were represented as mean7standard deviation.

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Fig. 4. Fourier transform infrared spectroscopy of powder synthesized using surfactants at a fixed aqueous to organic phase ratio of 1:15 in the reactionmixture.

2530 Journal of the American Ceramic Society—Dasgupta and Bose Vol. 92, No. 11

(3) Powder Consolidation and Sintering

TCP nanopowder, synthesized using NP-5 surfactant at a fixedaqueous to organic composition of 1:15, was used to preparedisk compacts. Each compact was prepared by consolidatingapproximately 0.5 g of nanopowder by uniaxial pressing at 7.9MPa, followed by cold isostatic pressing at 345 MPa. The com-pact disks were sintered in a 3 kWmicrowave furnace at 12501Cfor 20 min. These compacts were used to study in vitro biocom-patibility evaluation.

(4) In Vitro Biocompatibility Evaluation

Sintered TCP disk samples were sterilized via one cycle of au-toclave at 1211C for 30 min. An immortalized, cloned, OPC1derived from human fetal bone tissue was obtained from OHSU(Portland, OR) and used in this study.29 OPC1 cells were seededonto the samples and then placed in 24-well plates with a celldensity of 2.0� 104 cells/well. OPC1 cells were cultured for 1, 5,and 11 days on sterilized TCP disks. TCP disks were replenishedwith fresh cell culture media every 2 days of the cell culture ex-periment. Three sintered TCP disks were used at each time pointfor cell culture studies. One milliliter of McCoy’s 5A mediumenriched with 5% fetal bovine serum and 5% bovine calf serum,containing 0.1 g/L penicillin and 0.1 g/L streptomycin, was

added to each well. Cultures were maintained at 371C underan atmosphere of 5% CO2.

(A) Morphology of OPC1 Cells: To investigate cell–ma-terial interactions by SEM, samples were fixed with 2% para-formaldehyde/2% glutaraldehyde in a 0.1M cacodylate bufferovernight at 41C after culturing them for 1, 5, and 11 days.Postfixation was performed with 2% osmium tetroxide (OsO4)for 2 h at room temperature. Fixed samples were then dehy-drated in an ethanol series (30%, 50%, 70%, 95%, and 100%three times), followed by 100% acetone, an equal mixture ofacetone and hexamethyldisilane (HMDS), and finally with pureHMDS.

(B) MTT Assay: The MTT assay (Sigma-Aldrich, St.Louis, MO) was performed to assess cell proliferation on sin-tered TCP disks. The MTT solution of 5 mg/mL was preparedby dissolving MTT in PBS, followed by filter sterilization. TheMTT was diluted (50 mL into 450 mL) in serum-free, phenol red-free Dulbeco’s minimum essential medium (DME). Five hun-dred microliters of diluted MTT solution was then added toeach sample in 24-well plates. After a 2-h incubation, 500 mL ofsolubilization solution made up of 10% Triton X-100, 0.1 NHCl, and isopropanol were added in order to dissolve the form-azan crystals. 100 mL of the resulting supernatant was trans-ferred into a 96-well plate, and read by a plate reader at 570 nm.Data are presented as mean7standard deviation.

(C) Immunocytochemistry and Confocal Micros-copy: Expression of ALP was assayed using confocal micros-copy. b-TCP substrates carrying osteoblasts were processedafter 1, 5, and 11 days of incubation. Three b-TCP disks ateach time point were used to study ALP expression from cul-tured osteoblast cells. Disks were washed in 20 mL scintillationvials containing 0.1M PBS for 15 min. PBS was removed andthe samples were fixed with 100% methanol at �101C for 15min, and 100% acetone at �101C for 15 min. Acetone was re-moved and the samples were stored at�101C for future use. Thesamples were rehydrated as required with 0.1M PBS for 10 min,and blocked with TBST/BSA (Tris-buffered saline with 1% bo-vine serum albumin, 250 mM NaCl, pH 8.3) for 1 h. Primaryantibody against ALP (Sigma-Aldrich, St. Louis, MO) wasadded at 1:100 dilution, and the samples were incubated atroom temperature overnight. The following day, the sampleswere rinsed with TBST/BSA three times for 10 min each. Thesecondary antibody, Oregon green goat antimouse IgG

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(Molecular Probes, Eugene, OR), was added at a 1:100 dilution,and the samples were incubated at room temperature for 1 h. Allthe samples were then rinsed with TBST/BSA three times for 10min each and mounted in Vectashield Mounting Medium (Vec-tor Labs, Burlingame, CA) containing propidium iodide (PI) ata concentration of 1.5 mg/mL. The samples were then observedusing a confocal microscope (BioRad 1024 MRC, Hercules,CA) with Laser Sharp 3.1 program software.

III. Results

(1) Phase Identification and Evolution

The XRD patterns (Fig. 1) of nanopowder synthesized usingNP-12 surfactant with an aqueous to organic ratio (aq:org ratio)of 1:15 and calcination at 6001 and 7001C mostly resembled thatof the calcium-deficient hydroxyapatite (CDHA) phase, which isconsistent with the earlier reports.30

All powder were calcined at 8001C, and their XRD patternshowed b-TCP as the major phase (JCPDS No. 9-169) (Fig. 2).The crystallite size was calculated using the (0210) peak of high-est intensity in the XRD patterns on the basis of the Debye–Scherrer equation. Irrespective of the nature or chemistry of thesurfactant used, such as NP-5 or NP-12 or NP-51NP-12, with adecrease in the aqueous content in the reaction mixture, the(0210) peak of the synthesized powder became broader. The

crystallite size of these synthesized b-TCP nanopowder de-creased with an increase in organic content in the reaction mix-ture (Fig. 3).

The chemistry of the surfactant did not have a significanteffect on the final chemical composition of calcined b-TCPnanopowder, which was also revealed in the FTIR spectroscopy(Fig. 4) of synthesized TCP nanopowder using different surfact-ants at a fixed aq:org ratio of 1:15 and calcination at 8001C.

(2) Particle Size, Morphology, and Surface Area Analysis

Synthesized b-TCP nanopowder showed a gradual decrease inparticle size (Fig. 5) and an increase in BET surface area (Fig. 6)with an increase in organic content in the reverse micelle. b-TCPnanopowder synthesized using NP-5 as the surfactant at a fixedaq:organic composition of 1:15 exhibited the lowest particle sizeof 32 nm and the highest BET surface area of 103 m2/g. Powdersynthesized with a fixed aq:org ratio of 1:05 using NP-5 surfact-ants showed the highest average particle size of 135 nm, but notnecessarily the lowest BET surface area. BET surface area wasthe lowest, B57 m2/g, for the powder with an average particlesize of B110 nm synthesized with NP-51NP-12,a mixed sur-factant system, at an aq:org ratio of 1:05.

Aspect ratios (Table I) of nanoparticles were calculated fromTEM micrographs (Figs. 7(a)–(c)) of b-TCP nanopowder syn-thesized at a fixed aq:org ratio of 1:15 using surfactants NP-5,NP-12 and a 1:1 mixture of NP-5 and NP-12 by volume. Nooutlier was found within 7s for any powder. Powder synthe-sized using NP-5 and a mixed surfactant of NP-51NP-12 gaveparticles with a lower aspect ratio of 1.170.15 and 1.370.2,respectively, which were not (p5 0.6714) significantly different.Elongated b-TCP particles with an aspect ratio of 3.970.4 wereobtained for powder synthesized using the NP-12 surfactant,which were significantly different in aspect ratio from b-TCPparticles synthesized using NP-5 (po0.0001) and NP-51NP-12(po0.0001).

TEM micrographs (Figs. 8(a)–(c)) of b-TCP nanopowdersynthesized using the NP-12 surfactant in a varying aq:org ra-tio in the microemulsion showed that with an increase in aque-

200 nm

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200 nm 200 nm

Fig. 7. Transmission electron micrographs of b-tricalcium phosphate particles synthesized at a fixed aqueous to organic phase composition usingdifferent surfactants: (a) NP-5, (b) NP-12, and (c) NP-51NP-12. NP-5, nonylphenol ether; NP-12, poly(oxyethylene)12 nonylphenol ether.

200 nm 200 nm

(a) (b) (c)

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Fig. 8. Transmission electron micrographs of b-tricalcium phosphate particles synthesized using the NP-12 surfactant with varying aqueous to organicphase ratios in the reaction mixture: (a) 1:15, (b) 1:10, and (c) 1:05. NP-12, poly(oxyethylene)12 nonylphenol ether.

Table I. Aspect Ratio of Synthesized TCP NanopowderUsing Different Surfactants at a Fixed Aq:Org Ratio of 1:15

in the Reaction Mixture

Surfactant Aspect ratio

NP-5 1.170.15NP-12 3.970.4NP-51NP-12 1.370.2

(a) po0.0001 for NP-5 vs NP-12; (b) po0.0001 for NP-12 vs NP-51NP-12;

and (c) p5 0.6714 for NP-5 vs NP-51NP-12. TCP, tricalcium phosphate; NP-5,

nonylphenol ether; NP-12, poly(oxyethylene)12 nonylphenol ether.


ous content in the reaction mixture, the particles became moreand more spherical. The aspect ratios (Table II) of b-TCP nano-powder synthesized at an aq:org ratio of 1:15, 1:10, and 1:05were 3.970.4, 2.470.5, and 1.570.4, respectively. In this case, asignificant variation in the aspect ratio of b-TCP nanopowderwas found as the aq:org ratio in the microemulsion was variedfrom 1:15 to 1:10 (p5 0.0002), 1:15 to 1:05 (po0.0001), and 1:10to 1:05 (p5 0.0044).

(3) In Vitro Biocompatibility Evaluation

b-TCP nanopowder synthesized using the NP-5 surfactant at afixed aqueous to organic composition of 1:15 were compactedand sintered for in vitro cell–material interaction study. The bulkdensity of sintered TCP disks was 89.5%71.5% of theoreticaldensity. The XRD pattern of the sintered sample (Fig. 9)showed the formation of a significant amount of the a-TCPphase. The SEM micrograph (Fig. 10) of the TCP compact sin-tered at 12501C showed some degree of porosity in the sinteredmicrostructure. SEMmicrographs (Figs. 11(a)–(c)) of bone cell–material interactions were analyzed for cell adhesion, growth,and differentiation after 1, 5, and 11 days, respectively. MTTassay (Fig. 12) was used to determine OPC1 cell proliferation onb-TCP substrates over the course of the experiment. After 11days of culture, the number of cells on the TCP substrate in-creased by approximately 10 times compared with day 1.Immunocytochemistry or ALP expression using OPC1 cellswas studied to determine bone cell differentiation towards anosteoblastic phenotype on these b-TCP samples. Confocal mi-

crographs (Fig. 13) for ALP expression of osteoblast cells onb-TCP compacts were analyzed after 5 and 11 days. ALP withinthe cells is identified by the expression of green fluorescence andnuclei counterstained with PI in the mounting medium ex-pressed red fluorescence. Although after 5 days the cells exhib-ited little amount of ALP expression, with an increase in culturetime up to 11 days, ALP activity increased significantly.

IV. Discussion

The objective of the present study was to synthesize b-TCPnanopowder with controlled size and morphology by varyingthe phase composition and hydrocarbon chain length of thesurfactant in a reverse micelle domain. Nanoscale CaP is muchmore bioresorbable, which attributes to its enhanced bioactivitycompared with micrometer-sized CaP. The precipitates obtainedfrom the aqueous solution were a nonstoichiometric apatite witha chemical formula of Ca10�x(HPO4)x(PO4)6�x(OH)2�x (0rxr1) where the Ca/P molar ratio could vary from 1.33 to

Table II. Aspect Ratio of TCP Nanopowder SynthesizedUsing NP-12 Surfactant with Variations in the Composition

of the Microemulsion

Aqueous to organic ratio Aspect ratio

1:15 3.970.41:10 2.470.51:05 1.570.4

(a) p5 0.0002 for 1:15 vs 1:10; (b) po0.0001 for 1:15 vs 1:05; and (c) p5 0.0044

for 1:10 vs 1:05. TCP, tricalcium phosphate; NP-12, poly(oxyethylene)12 nonyl-

phenol ether.

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Sintered at 1250 C

Fig. 9. X-ray diffraction pattern of samples calcined at 8001C/3h and sintered at 12501C/20 min in a microwave furnace.

Fig. 10. Microstructure of the tricalcium phosphate compact sinteredat 12501C.


1.65. The Ca:P molar ratio of 1.5:1 represents TCP. When x5 1,the above-mentioned general formula becomes Ca9(HPO4)(PO4)5(OH), which is called the calcium-deficient apatite(CDHA) phase. This CDHA transformed into b-TCP on heat-ing at and above 8001C, according to the following equation.30

Ca9ðHPO4ÞðPO4Þ5ðOHÞ ! 3Ca3ðPO4Þ2 þH2O (1)

For nanopowder calcined at 6001 and 7001C, the XRD pat-terns (Fig. 1) showed the formation of CDHA as the majorphase; however, after calcination at 8001C, b-TCP emerged asthe major phase (JCPDS No. 9-169), irrespective of the surfact-ant used during the synthesis process.

In a reverse micelle-based synthesis, powder size and mor-phology are directly related to the shape of nuclei formed duringthe reverse micellization and its change during synthesis. The

shape of the polar core in the reverse micelle domain is governedby two mutually opposing forces: (a) an attractive force due tothe hydrophobic attraction of the hydrocarbon chain units atthe hydrocarbon-water interface; and (b) a repulsive force be-tween the adjacent head groups with similar charge character-istics due to hydrophilic, steric, and ionic repulsion. The dropletsof water that were stabilized by surfactants act as nanoreactors,in which hydroxyl ions from NH4OH decreased the solubilityproduct of calcium-deficient apatite [Ca9(HPO4)(PO4)5(OH)],which gradually grew with aging. The chemical reaction, whichwas a simple precipitation reaction that occurred inside thenanodroplets of water, is shown in the following equation

9CaðNO3Þ2 þ 6H3PO4 þ 18NH4OH

¼ Ca9ðHPO4ÞðPO4Þ5ðOHÞ # þ18NH4NO3 þ 17H2O � � �(2)

With an increase in organic content in the microemulsion, thesize of the polar core in the reverse micelle domain decreased(Fig. 3), resulting in a decrease in the crystallite size as well as theparticle size of synthesized b-TCP powder. The BET surfacearea of any powder depends not only on particle size, but alsoon particle morphology and the inherent porosity of particles.This fact is reflected in the finding that synthesized b-TCP pow-der with the lowest particle size did not show the highest surfacearea (Figs. 5 and 6).

The chemistry of the surfactant did not have a significanteffect on the final chemical nature of calcined b-TCP powder.Irrespective of the chemistry of the surfactant, FTIR spectraof b-TCP phase (Fig. 4) are identified by the large band at900–1200 cm�1.31,32 The bands at 1022 and 1120 cm�1 are as-signed to the components of triply degenerate n antisymmetricP–O stretching mode. n, the nondegenerate P–O symmetricstretching mode, is detected at 972 cm�1. The bands at 606and 544 cm�1 are attributed to components of the triply degen-

(a) (b) (c)

Fig. 11. Scanning electron micrographs of osteoblast precursor cells on tricalcium phosphate after (a) 1 day, (b) 5 days, and (c) 11 days of cell culture.

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Fig. 12. MTT assays on tricalcium phosphate disks after 1, 5, and 11days of cell culture.

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Fig. 13. Confocal micrographs of alkaline phosphatase expression in osteoblast precursor cells cultured on tricalcium phosphate disks at(a) day 1, (b) day 5, and (c) day 11.


erate n O–P–O bending mode, and the doubly degenerate nO–P–O bending mode is evident at 438 and 498 cm�1. The ab-sence of characteristic bands at 631 and 3572 cm�1, correspond-ing to the hydroxyl group, indicates the absence of thehydroxyapatite phase in synthesized TCP nanopowder calcinedat 8001C. 32,33

Both the hydrocarbon chain length of the surfactant and theaq:org ratio in the reaction mixture had an effect on the mor-phology or aspect ratio of synthesized powder. The polar coresize in the reverse micelle domain was smaller for the lowestaqueous content in the reaction mixture. Separation distancebetween the hydrocarbon chain units was minimum when theyoriented themselves around the spherical hydrophilic core. Thiscaused an increased hydrophobic interaction between C5,C51C12, hydrocarbon tails of NP-5, and NP-51NP-12 sur-factants and dominated over the ionic or hydrophilic repulsionbetween the closely packed heads groups. Thus, the nucleiformed during the reverse micellization synthesis process usingNP-5 and NP-51NP-12 surfactants became more spherical withthe lowest water content in the microemulsion, resulting in b-TCP nanoparticles with a lower aspect ratio (Figs. 7(a) and (c)).In the case of the NP-51NP-12 system, NP-5 acted as a co-sur-factant. In the case of powder synthesized using only the NP-12surfactant, long C12 carbon chains needed a bigger surface areato orient them around the hydrophilic core of the reverse mi-celle. This may result in the formation of nuclei with a higheraspect ratio, and thus particle morphology could be elongatedfor powder synthesized using the NP-12 surfactant at the lowestaqueous content in the microemulsion (Fig. 7(b)). As water con-tent in the microemulsion increased, both steric hindrance fromC-12 hydrocarbon chains and repulsion between the hydrophilichead groups decreased, and by doing so, the reverse micelle do-main became more and more spherical. As a result, in the pres-ence of the NP-12 surfactant with a change in the aq:org ratiofrom 1:15 to 1:10 to 1:05 during synthesis, the aspect ratio of b-TCP nanopowder gradually decreased (Table II and Fig. 8).

The XRD pattern of the sintered compacts (Fig. 9) demon-strates the formation of a significant amount of the a-TCP phasedue to high-temperature phase transformation. Densificationof TCP compacts was hampered because of this b-a phasetransformation, as reflected in some degree of open pores inthe sintered microstructure of TCP compacts (Fig. 10) and thelow sintered density of 89.5%72%. During in vitro cell–mate-rial interaction study, these samples became porous, which wasdue to the faster dissolution of the a-TCP phase in the culturemedium. Cell attachment through a small micro extension ontothe sintered body surface was evident after 1 day of the OPC1cell culture (Fig. 11(a)). After day 5, cells proliferated on thesurface of the compacts (Fig. 11(b)). After day 11, the cellsdisplayed numerous lamellipodia and filopodia extensions toattach onto the sample surface and for cell migration as shownin Fig. 11(c). Optical density (Fig. 12) with respect to time pointsis directly related to the number of living cells in the culture.MTT solution is reduced to give a purple color in the presence ofmitochondrial dehydrogenase in living cells. The absorbance ofthis colored solution is quantified at 570 nm by a spectropho-tometer. The MTT assay and SEM observation showed thatTCP substrates were biocompatible, and promoted cell prolif-eration. A similar observation was reported by Cai et al.34 fromthe cell culture experiments conducted in order to study the sizeeffect of CaP nanoparticles on the proliferation of bone cells.CaP nanoparticles showed better cell attachment, proliferation,and differentiation compared with conventional CaP particles.Erisken et al.35 also showed that nanocomposites of TCP ex-hibited increased bone nodule formation and mineralization.

ALP expression appeared strongly positive in the TCP sub-strate cultured for 11 days, supporting the differentiationof osteoblastic cells (Fig. 13). ALP is a marker for osteoblastdifferentiation, and an increase in the specific activity of ALP ina population of bone cells indicates a corresponding shift to amore differentiated state.36 This study showed that TCP com-pacts made with synthesized nanopowder promoted bone cell

differentiation. Nanostructure processing of b-TCP compacts atlower sintering temperatures using a microwave sintering systemand relation between sintered density, microstructure and me-chanical strength of these compacts are currently underway.

V. Conclusion

Nanocrystalline b-TCP powder was synthesized using reversemicelle as a template system. Cyclohexane was used as the oilphase, aqueous solutions of calcium nitrate and phosphoric acidas the aqueous phase, and poly(oxyethylene)5 nonylphenol ether(NP-5) and/or poly(oxyethylene)12 nonylphenol ether (NP-12)as the surfactants. The composition of the microemulsion andthe surfactant chain length used in reverse micelle had an effecton the particle size, morphology, and surface area of synthesizedb-TCP nanopowder. Powder were prepared with a particle sizerange between 32 and 135 nm, a surface area varying from 57 to103 m2/g, with different aspect ratios. The particle size of thesynthesized powder increased with an increase in the aqueousphase content in the microemulsion. Cell–material interactionand immunocytochemistry studies using a OPC1 on sinteredTCP disks using the synthesized nanopowder showed excellentbiocompatibility.

Acknowledgment

The authors would like to acknowledge Ms. Brena Holman and Prof. HowardHosick for experimental support with the in vitro biocompatibility test.

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