DOI: 10.1002/cphc.201200080

Recent Advances in Research Applications of NanophaseHydroxyapatiteKate Fox,*[a] Phong A. Tran,[b] and Nhiem Tran*[c]

1. Introduction

What separates a biomaterial from all others is its ability toremain in a harsh biological environment without elicitingdamage either to its surroundings or to itself. Accordingly, sig-nificant research has been undertaken in the last century toidentify and optimise the “best” biomaterial for medical appli-cations. For orthopaedic use, calcium phosphate based materi-als are widely represented as an optimal material to aid bonerepair, replacement and regeneration. The use of calcium phos-phates as a medical implant material has been extensivelystudied since the 1950s; however, the first use of calciumphosphates as a bone repair material can be traced back toAlbee et al. in 1920[1] whilst Monroe et al.[2] and Hubbard[3] in1971 and 1974, respectively, are linked with the first indicationsof hydroxyapatite use as an implant material.[4] It was, however,not until the later end of the century that calcium phosphatesin the form of hydroxyapatite gained significant attention.

1.1. Bone: Structure and its Process of Regeneration

Bone is a porous composite material made up of an organiccomponent (90–95 % collagen) and an inorganic component(mainly hydroxyapatite, a calcium phosphate material in theform of crystalline Ca10(PO4)6(OH)2).[5] The composite nature issufficient to compensate for any perceived weaknesses of eachcomponent. In fact, in addition to the aforementioned collagenfibres and calcium phosphate material, bone also comprisestwo additional phases, water and organic substances such asnon-collagenous proteins (often chemically bound to glycos-aminoglycans), whereby the fractions of the four phases differdepending on the use of the bone, patient characteristics suchas age or health, and the location of the bone within the body.

The mechanical strength of the composite comes from theinorganic hydroxyapatite, which, combined with type 1 colla-

gen fibres and the extracellular matrix, is able to provide a sup-port scaffold.[5a, 6] The inorganic hydroxyapatite, however, de-spite its good compressive strength is torsionally weak witha high modulus of elasticity. To compensate, the organic com-ponent in the form of collagen offers a high tensile strength.Accordingly, bone is a composite device in which the collagenframework is interspersed with nanoscale hydroxyapatite parti-cles (20–40 nm) between collagen fibres (see Figure 1).[4] Thus,the two components of bone enable it to be strong yet allowsome deformation. Having said that, it remains unknownwhere mineralisation first occurs, that is, within this gap be-tween the collagen fibres or within the collagen fibre overlapregion.[7] In fact, biologically, carbonated apatite crystals arethe smallest crystals known with average sizes of 50 � 25 �2–4 nm in bone and 100 � 50 � 50 nm in tooth enamel.[8]

Further to the mineral component, bone is also populatedby cells, macromolecules, proteins and blood vessels. To main-tain a healthy regenerative bony matrix, there are three celltypes which combine to build and remove bone. Osteoblasts

Hydroxyapatite, the main inorganic material in natural bone,has been used widely for orthopaedic applications. Due to sizeeffects and surface phenomena at the nanoscale, nanophasehydroxyapatite possesses unique properties compared to itsbulk-phase counterpart. The high surface-to-volume ratio, reac-tivities, and biomimetic morphologies make nano-hydroxyapa-tite more favourable in applications such as orthopaedic im-plant coating or bone substitute filler. Recently, more effortshave been focused on the possibility of combining hydroxy-apatite with other drugs and materials for multipurpose appli-

cations, such as antimicrobial treatments, osteoporosis treat-ments and magnetic manipulation. To build more effectivenano-hydroxyapatite and composite systems, the particle syn-thesis processes, chemistry, and toxicity have to be thoroughlyinvestigated. In this Minireview, we report the recent advancesin research regarding nano-hydroxyapatite. Synthesis routesand a wide range of applications of hydroxyapatite nanoparti-cles will be discussed. The Minireview also addresses severalchallenges concerning the biosafety of the nanoparticles.

[a] Dr. K. FoxSchool of PhysicsUniversity of MelbourneParkville, Victoria 3010 (Australia)Fax: (+ 61) 3 9347 4783E-mail : kfox@unimelb.edu.au

[b] Dr. P. A. TranDepartment of Chemical and Biomolecular EngineeringUniversity of Melbourne, Parkville, Victoria 3010 (Australia)

[c] Dr. N. TranDepartment OrthopaedicsRhode Island Hospital and Alpert Medical SchoolBrown UniversityProvidence, RI 02903 (USA)E-mail : nhiem_tran@brown.edu

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and osteoclasts build and remove bone, respectively. This isdone by the secretion of collagenous proteins in the case ofosteoblasts and the secretion of acids and proteases in thecase of osteoclasts to build or degrade hydroxyapatite.[9] Inmore detail, the inorganic hydroxyapatite component of boneenables bone remodelling through cellular signalling via thethird cell type, osteocytes. This is achieved by adsorption of ex-tracellular matrix proteins which when adsorbed promote os-teoblast proliferation, differentiation and adhesion about thehydroxyapatite material.[9] This is essential as net bone lossoccurs with imbalances in the bone remodelling system. Thebone remodelling system is a cycle consisting of five phases:

activation, resorption, reversal, formation and quiescence de-pendent on the interactions of osteoclasts and osteoblasts.[9, 10]

The activation phase involves the interaction of precursorcells (osteoblasts and osteoclasts) which allows the differentia-tion and commencement of resorptive activity by osteoclasts.[9]

After the resorption phase there is a period of reversal inwhich growth factors are deposited to attract osteoblast cellsto the interface. The osteoblasts then start the deposition ofthe bony matrix that later becomes mineralised in the forma-tion phase. The cycle will then continue, consistently deposit-ing matrix and then mineralised bone and removing damagedbone for constant structural remodelling. Net bone loss occurswhen osteoclastic bone resorptive activity is increased by anincrease in either the number or activity of osteoclast cells. Inconjunction, the reduction of osteoblast cells and a subsequentdecrease in bone formation will result in a net imbalance.

Accordingly, synthetic hydroxyapatite has been effective inreducing aseptic loosening associated with post-implant sur-gery by enhancing the early mechanical stability of the bone/implant interface. This is due to an increased bone appositionand osteoconductivity as the hydroxyapatite facilitates chemi-cal bonding with surrounding hard tissue.[6, 12]

1.2. Synthetic Equivalents of Hydroxyapatite

Synthetic hydroxyapatite must be as close to the natural hy-droxyapatite component of bone as possible. Accordingly, syn-thetic versions must be both crystallographically and chemical-ly comparable to natural hydroxyapatite.[13]

Natural hydroxyapatite is normally found in the form ofCa10(PO4)6(OH)2 with a stoichiometric Ca/P ratio of 1.67(Table 1). However, calcium phosphates can also exist in otherchemical phases predominantly in the forms of octacalciumphosphate (OCP, Ca8H2 (PO4)6, Ca/P = 1.3), dicalcium phosphate(Ca2P2O7, Ca/P = 1) and b-tricalcium phosphate (b-TCP,Ca3(PO4)2, Ca/P = 1.5). Due to calcium depletion, b-TCP is moresoluble than crystalline hydroxyapatite.[14] Generally, all calciumphosphates are osteoconductive in vivo.[15] However, hydroxya-patite(Ca/P=1.67) is the most thermodynamically stable calciumphosphate in the physical environment (pH�5.4) and hence isthe most common calcium phosphate material used.[16]

Figure 1. Representation of the elements that comprise bone. It is evidentthat bone consists of macro, micro and nano components.[11]

Table 1. The various forms of calcium phosphate. Adopted from ref. [18].

Ca/P ratio Apatite type Comments

0.5 monocalcium phosphate monohydrate (MCPM) Ca(H2PO4)2·H2O0.5 monocalcium phosphate anhydrous (MCPA) Ca(HPO4)2

1.0 dicalcium phosphate dihydrate (DCPD), mineral brushite CaHPO4·2H2O1.0 dicalcium phosphate anhydrous (DCPA), mineral monetite CaHPO4

1.33 octacalcium phosphate (OCP) Ca8(HPO4)2(PO4)4·5H2O1.5 a-tricalcium phosphate (a-TCP) a-Ca3(PO4)2

1.5 b-tricalcium phosphate (b-TCP) b-Ca3(PO4)2

1.2–2.2 amorphous calcium phosphate (ACP) CaxHy(PO4)z·nH2O, n = 3–4.5; 15–20 % H2O1.5–1.67 calcium-deficient hydroxyapatite (CDHA) Ca10�x(HPO4)x(PO4)6�x(OH)2�x (0<x<1)1.67 hydroxyapatite (HA or OHAp) Ca10(PO4)6(OH)2

1.67 fluorapatite (FA or FAp) Ca10(PO4)6F2

2.0 tetracalcium phosphate (TTCP or TetCP), mineral hilgenstockite

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Hydroxyapatite is a hexagonally crystalline structure witha P63/m space group.[17] The crystallographic, physical andchemical properties are changed by ion substitution (Ca2+ ,PO4

3� or the OH� groups). Accordingly, it is not uncommon tosee ions such as CO3

2�, HPO42� and F� replacing the stoichio-

metric components of the hydroxyapatite lattice. Replacedions are known to change the inherent properties of thematrix. Substitutions such as OH� to Cl� , PO4

3� to CO32� and

Ca2 + to Mg2+ are recognised to weaken the hydroxyapatitestructure by increasing solubility, whilst OH� to F� willstrengthen the structure and decrease solubility.[7]

Synthetic hydroxyapatite is a bioactive ceramic having an in-terfacial response that results in bone bonding.[5a] Consequent-ly synthetic hydroxyapatite is commonly used as an implantcoating material to stimulate bone growth around the implantleading to strong bone–hydroxyapatite–prosthesis bonds.[19]

This can be seen in tests where the shear strength of hydrox-yapatite-coated titanium is 5–8 times stronger and bone in-growth is improved in comparison to non-hydroxyapatite-coated titanium.[20] This is of great importance because al-though titanium is commonly used in orthopaedics, its surfaceoxide prevents true interaction with bone (in fact, the attach-ment between bone and uncoated titanium implants has beenconsidered a “tight apposition” rather than true bonding[4]).

The true benefits of hydroxyapatite possibly lie in the gener-ation of an improved bone/implant interface during the post-surgery healing phase. Recent studies[21] have suggested thatin hip implants, at least, no clinical advantage can be linked tohydroxyapatite use on the femoral component. Each study ex-planted hip implants after a minimum implantation time of2 years. At this period the interfacial bone bonding did notdiffer between hydroxyapatite-coated and uncoated implants.However, the true benefit of hydroxyapatite is most likely ob-scured by this stage since other studies[12, 22] suggest that a hy-droxyapatite coating will improve bone apposition at the im-plant/bone interface, at least within the first month of implan-tation.

2. Synthesis of Hydroxyapatite and Hydroxy-apatite Composite Nanomaterials

Hydroxyapatite can be synthesised using a wide variety ofroutes (see Table 2). In general, it can be considered that thereare two main pathways: wet chemistry and high-temperatureprocessing. Methods include precipitation, sol–gel, hydrother-

mal, metathesis, plasma spraying, particle sintering and combi-nations thereof.

2.1. Wet Chemistry

The wet chemistry or precipitation method relies on a supersa-turated solution of calcium and phosphorus ions to precipitateonto an exposed surface.[12c, 24] It is a wet method in whichsamples are held within the solution to enable calcium phos-phate deposition. As seen in Figure 2, the saturated solutioncan be controlled to generate crystalline or amorphous sub-strates depending on the deposition rate. Heterogeneous nu-

cleation is the preferred adhesion and accordingly, samples areoften vertically suspended. The wet chemical methods are sig-nificantly cheaper and easier than other methods of hydroxy-apatite formation (refer to Table 2). No specialised equipmentis required and chemical concentrations are scalable to pro-duce bulk solutions; however, the major disadvantage of thetechnology is the unreliable nature of morphology and crystal-linity plus the time taken for a decent deposition.[25]

The size of hydroxyapatite particles in the wet chemicalmethods varies significantly. It is, however, considered for themost part that synthesis by precipitation will result in averageparticle sizes of less than approximately 1 mm but not evenlydispersed.[23] Other methods can therefore be used in conjunc-tion to ensure the desired particle size.

One common wet chemistry method to generate hydroxy-apatite is to combine calcium nitrate tetrahydrate with an am-monium phosphate. This can be performed over a range of

Table 2. Generation of apatite by different processing methods. Summarised with updates from refs. [18, 23] .

Method Average particle size Comments

wet chemical 0.07–0.64 mm cheap, simple and slow; difficult to regulate particle sizesolid-state process 0.1–1 mm difficult to regulate particle size and multiple phases seen in crystallisationhydrothermal 1 mm difficult to regulate particle sizemechanical–chemical synthesis 0.13–0.14 mm simple but process requires strict adherence to recipesol–gel nanosized subject to impuritieschemical synthesis 50 nm subject to impuritiessputter coating 0.5–3 mm cheap but line of sight technique and high temperatures introduce additional phasesplasma spraying 30–200 mm uniform coating but surface coating amorphous

Figure 2. Effect of supersaturation in wet precipitation processes, whereinKsp represents the solubility constant of the solution, S(a) is the solubilityproduct of the amorphous phase, S(c) is the solubility product of the crystal-line phase and Q represents the reaction coefficient.[7]

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temperatures (1 to 90 8C) whereby calcium nitrate tetrahydrateCa(NO3)2·4H2O is added to ammonium dihydrogen phosphate,NH4H2PO4. Ammonium hydroxide is added to control the pH.This method is reasonably quick but time may be sunk by anadditional washing step in which the nitrates and ammoniumhydroxide are removed from the precipitate.

Alternatively, drops of orthophosphoric acid can be addedto a solution of calcium hydroxide under stirring. Precipitationof apatite is slow and crystallinity is controlled by temperature(higher temperature leads to higher crystallinity).

The method is characterised by the following reactions:Reaction of calcium and phosphate salts :

10Ca(NO3)2 + 6(NH4)2HPO4 + 8NH4OH!Ca10(PO4)6(OH)2 +

20NH4NO3 + 6H2ONeutral reaction of acidic and alkaline solutions:

10Ca(OH)2 + 6H3PO4!Ca10(PO4)6(OH)2 + 18H2OAlternatively, hydroxyapatite nanoparticles may be generat-

ed by complexing calcium chloride with tartaric acid under theinfluence of orthophosphoric acid and ammonium hydroxide:10Ca2 + + 6PO4

3�+ 2OH�!Ca10(PO4)6(OH)2

Wet chemistry also enables hydroxyapatite growth usingmore unusual sources. For example, research has shown thathighly sinterable, nanosized hydroxyapatite powders can besynthesised using recycled eggshell (due to its CaCO3 phase)and phosphoric acid as calcium and phosphorus sources, re-spectively.[26] The eggshell was shown to be converted to calci-um oxide by calcination and the resultant nano-hydroxyapatite(particle size, 70 nm) was fully densified when sintered for 1 hat 1300 8C.[26a]

The calcium phosphate phase and morphology can be con-trolled by adjusting the pH value of the solution the samplesare exposed to. For example, amorphous calcium phosphatesare considered to form at pH values between 5 and 12 whilstoctacalcium phosphates are stable within a narrow windowbetween pH 5.5 and 7.[27] Hydroxyapatite is considered to de-velop best at pH values around that of the body (i.e. , 7.4).[24a, 28]

Another method to generate hydroxyapatite is to use a su-persaturated ionic solution similar to that of human bloodplasma. Simulated body fluid was developed by Kokuboet al. ,[12c] who demonstrated that a glass–ceramic apatite–wol-lastonite implanted in vivo and growing a calcium phosphatefilm can be replicated in an acellular simulated body fluid withion concentrations comparable to those of human bloodplasma. Reagent-grade chemicals (NaCl, NaHCO3, KCl,K2HPO3·3H2O, MgCl2·6H2O, CaCl2, Na2SO4) are systematically dis-solved in purified water and the solution brought to physio-logical pH using hydrochloric acid. The precipitation reactionoccurs at 37 8C. Precipitation of thermodynamically stable apa-tite onto samples is thought to occur through the processes ofdissolution, re-nucleation and crystal growth and eventualtransformation to hydroxyapatite.[27] Accordingly, the techniquefollows the classical theory of precipitation, that is, nucleationof calcium phosphate on an exposed substrate activatesa spontaneous precipitation of a solid phase from a supersatu-rated solution.[29]

2.2. Sol–Gel Synthesis of Hydroxyapatite

The second hydroxyapatite synthesis technique is sol–gel, inwhich a colloidal solution is converted to an amorphous gelwhich is then solidified through drying.[30] The sol–gel tech-nique is a two-phase process (solid–liquid) whereby colloidalparticles are suspended in solution. The process is dominatedby the intrasolution forces (van de Waals, intrasolution particlecharging). Accordingly, the stability of the sol is controlled bydecreasing surface charge of nanoparticles to form a gel. Forapatites, the process is based on polymerising organometallicprecursors of the M(OR)n alkoxide type.[5a, 31] The synthesisedhydroxyapatite particle size is in the nanometre range.[32] How-ever, Masuda et al. found that by using the sol–gel methodand nanopowders as the calcium phosphate precursor andmetal alkoxide as the starting material, the powder precursorwill form larger agglomerates.[33]

The sol–gel sample is then dried to remove excess solvents.It is considered, however, that films less than 500 nm in thick-ness cannot be dried without film cracking.[25b] This is becausethe rate of shrinkage is somewhat proportional to the moisturecontent of the film. Finally, the film is heated at around 600 8Cto ensure that remnant organic material is combusted. Al-though the firing is not essential, it eliminates apatite contami-nation.

The sol–gel technique is particularly conducive to synthesisefluoro-hydroxyapatite (FHA). FHA is formed via the followingreaction: 3Ca3(PO4)2 + CaF2!Ca10(PO4)6F2 (at 900 8C). FHA, in itspurest form, is more soluble than hydroxyapatite due to thenegative fluorine ions reportedly enhancing mineralisation andre-crystallisation in vivo.[34] Using the sol–gel method, nano-FHA is formed using calcium nitrate, triethyl phosphate (TEP)and ammonium fluoride as the precursors for calcium, phos-phorus and fluorine, respectively. FHA is preferred in somecases to hydroxyapatite because of its known superior me-chanical properties under high-temperature sintering due toan improved thermal stability compared to hydroxyapatite.[35]

Figure 2 shows that, using the same deposition technique,FHA can be produced as a much more consistent nanoparticlethan “standard” hydroxyapatite.

2.3. Hydrothermal

The hydrothermal method of hydroxyapatite synthesis isa high-pressure, high-temperature process used to transformslurries into a crystalline phase. The particle size is approxi-mately 1 mm but can be produced in needle-like form withlengths less than 200 nm.[23] Jiang et al. explored the formationof nano-hydroxyapatite at temperatures between 130 and160 8C, finding that the temperature did not change particlesize as expected, instead temperature increase led to increasedhydroxyapatite crystallinity (Figure 3).[36]

For instance, hydroxyapatite particles are generated fromthe reaction of calcium carbonate and diammonium hydrogenphosphate at re-crystallisation temperatures of 200–300 8C andpressures around 1–2 kbar.[25b, 37]

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2.4. Metathesis

The metathesis method is a variety of the above methodolo-gies. First, precipitation of hydroxyapatite onto a substrate isfacilitated via a chemical reaction within an aqueous solu-tion.[38] The precipitate is then migrated using electrophoreticdeposition whereby charged particles migrate towards a coun-ter-charged electrode.[38b,c, 39] The dimensions of the hydroxy-apatite particles within the coating are less than 50 nm. Thetechnique does not rely on a line of sight and thus can beused to generate conformal coatings. The chemical reaction is :

10Ca(NO3)2 + 6NH4H2PO4 + 8NH4OH!Ca10(PO4)6(OH)2 +

20NH4NO3 + 6H2O

2.5. Plasma Spraying Synthesis and Coating ofHydroxyapatite

The most common commercially utilised method for apatiteformation is plasma spraying. The technique uses a direct cur-rent arc to produce a gas plasma. More specifically, the directcurrent coverts argon to plasma, which heats the powder andpropels it onto a substrate. Plasma spaying is difficult to use togenerate hydroxyapatite nanoparticles due to inconsistencieswithin the plasma.[40]

Although plasma-sprayed hydroxyapatite is produced withhigh reproducibility and economic efficiency for fast bone in-growth,[41] it can often have poor mechanical stability.[41a, 42] Inparticular, the high-temperature process associated withplasma-sprayed hydroxyapatite can lead to problems withcoating integrity due primarily to variations in crystallinity andmorphology within the sample.[40, 42, 43] The plasma-sprayedcoating comprises highly amorphous hydroxyapatite contentat the top of the coating (due to the cooling phase of the

spray process) and subsequently, the top often varies signifi-cantly from the underlying crystalline hydroxyapatite.[44] Fur-ther, the high temperature can cause dissociation of the hy-droxyapatite during plasma coating and the introduction ofa number of calcium phosphate phases, which, although notaffecting the mechanical stability of the coating, influences thesolubility of the coating within a physiological environ-ment.[40, 42, 43, 45] More importantly, plasma-sprayed hydroxyapa-tite does not have any true bond with the substrate.[14d] Due tothe high-speed, high-temperature process, no covalent or ionicbonding may occur.

2.6. Extra Processing

2.6.1. Sintering

In many of the above methods of ascertaining hydroxyapatiteor calcium phosphate material, sintering is a crucial procedureto acquire tailored materials. Assessments on the required sin-tering temperature have been exhaustively studied.[17, 18, 38a, 46] Itis well established that sintering hardens the hydroxyapatitebut is also responsible for the introduction of multiple phases.However, high-temperature sintering is important to removeorganic materials and water vapour, reduce shrinkage, reducecrystal size and eliminate acidic phases.[18] The requisite sinter-ing temperature is often more than 1000 8C which can lead tocracking in the case of hydroxyapatite coatings on implantsdue to the differences in their mechanical properties.[18, 25b] Fineparticles are usually generated by the chemical route withouthigh-temperature processing. Nanosized hydroxyapatite parti-cles, however, require abrupt precipitation where overlap ofthe nucleation and crystal growth phases can be prevented.[7]

By sintering hydroxyapatite after chemical growth, it is possi-ble to still obtain nanoparticles without unwieldy chemicalprocessing.

2.6.2. Ultrasound

Recent attention has been given to the use of ultrasound irra-diation to assist nano-hydroxyapatite formation. It is well es-tablished that nanopowders aggregate to produce microme-tre-sized clumps which determine the final ceramic microstruc-ture.[33] Ultrasound is used to reduce such particle agglomera-tion and produce superfine slurries, which increase chemicalreaction time and yield (Figure 4).[25a] Researchers have foundthat ultrasonic power can effectively control hydroxyapatiteparticle size and morphology when used in conjunction withwet chemical formation methods.[25a, 47] For example, Poinernet al.[25a] found that nano-hydroxyapatite in the range of 30 nmcan be produced with ultrasonic irradiation power of 50 W anda temperature of 400 8C. It was considered that the particlesize was limited by microjetting and microbubble dispersionthrough the solution.[25a] Further improvements in agglomera-tion have also been achieved by adding a surfactant duringprecipitation and ultrasound treatment.[48]

Figure 3. TEM images of hydroxyapatite powder hydrothermally treated at130, 150 and 160 8C. It is evident that each sample has uniform rod-shapednanoparticles.[36]

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3. Applications of Nano-Hydroxyapatite andComposites

3.1. Advantages of Nano-Hydroxyapatite

Nanosized hydroxyapatite particles are attracting interest dueto links between small particle size and inhibition of negativecellular activities.[49] Sun et al.[49a,b] showed that an inhibitoryeffect on cellular activity occurred in an in vitro osteoblast cul-ture treated with particles of size between 500 and 3000 nm.Since this research was performed, fabrication techniques haveimproved and nanoscale hydroxyapatite powders are routinelygenerated. Cai et al. ,[50] for example, used nano-hydroxyapatiteat approximately 20, 40 and 80 nm in diameter to compare theeffects on bone marrow mesenchymal stem cells and showedimproved cell vitality and proliferation when exposed to nano-particles relative to those exposed to the more conventionalsub-micrometre hydroxyapatite particles.

The importance of synthetic nanosized particles relates tothe natural hydroxyapatite crystals seen in biology. As previ-ously discussed, in vivo the hydroxyapatite crystals are nano-scopic. This enables the constant regeneration of bone as partof remodelling. Accordingly, when we seek to replace this with

a synthetic alternative, it is not surprising that the body ex-pects to see the same. It is beneficial if the surface roughnessof synthetic hydroxyapatite is in the nanoscale. In particular,nanoscale topography appears to have a positive effect on os-teoblastic differentiation and proliferation.[49d, 51] For example,Webster et al. published a series of reports showing that whenmaintaining the same material profile (phase, chemistry, crys-tallinity), a nanoroughened hydroxyapatite surface (grain sizeless than 100 nm) was superior to a less roughened one forpositive bone cell responses and therefore better osseointegra-tion.[52] More specifically, osteoblast adhesion, proliferation andcell differentiation markers, such as alkaline phosphatase syn-thesis and calcium deposition, were significantly greater onnanoscale topographical surfaces than on a standard hydroxy-apatite sample surface with roughness greater than100 nm.[52b] The same group showed that primary osteoblastactivity was supported by the nanophase ceramic whereby tar-trate-resistant acid phosphatase synthesis (TRAcP, a phenotypicmarker for osteoclasts which express this enzyme) and resorp-tion pitting was seen to be significantly greater for cells ex-posed to nanostructured surfaces (compared to a hydroxyapa-tite sample with surface area greater than 100 nm).[52c]

The reasons behind such improvements are still largelyunder investigation. However, a common hypothesis is thatprotein adsorption and bioactivity on surfaces or particles withnanometre features is different from that on conventionalnanometre-smooth surfaces.[52a, 53] A cell interacts with a bioma-terial via receptors on the cell surface and a layer of proteinsadsorbed onto material surfaces. The receptors can only recog-nise certain types of proteins. Previously, Webster et al.showed that of all the proteins contained in serum, vitronectinadsorbed in the highest concentration to nanophase surfacesof alumina while albumin adsorbed in the greatest amount onconventional alumina.[53a] Vitronectin is known to be crucial forosteoblast adhesion, since surfaces treated with vitronectingreatly enhance osteoblast adhesion compared to other pro-teins contained in serum.[52b]

3.2. Hydroxyapatite for Hard Tissue Repair

Synthetic hydroxyapatite has long been preferred as the mate-rial used in hard tissue repair over autografts and allografts.This is due to problems in locating bone grafts from autograftand allograph sources and particularly limitations in allograftusage because of the fear of disease transmission and graft re-jection. As discussed in Section 1.2, synthetic hydroxyapatitehas been shown to reduce aseptic loosening when used as animplant coating material in the immediate phase after surgeryby improving bone apposition and osteoconductivity at theimplant/bone interface.[12, 22a] Accordingly, nano-hydroxyapatitehas found use in areas such as bone repair, bone augmenta-tion and coating of implants as well as acting as fillers in boneor teeth.[18, 51, 54]

Nano-hydroxyapatite appears capable of rectifying the prob-lems of standard hydroxyapatite. At present, the use of hydrox-yapatite is somewhat restricted to non-load-bearing applica-tions or as a coating to much stronger materials such as stain-

Figure 4. Ultrasonically treated hydroxyapatite (d–g) compared to sampleswithout ultrasonic treatment (a–c). Reproduced with permission fromref. [25a] .

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less steel and titanium. This is due to the perceived weaknessin compressive strength.[55] Nano-hydroxyapatite, however, hasimproved strength through increased surface roughness, areaand densification, which may improve its mechanical proper-ties under load.[18, 51]

Nano-hydroxyapatite compressed into cylinders has beenimplanted into the spongy bone of rabbit and the integrationwith surrounding bone assessed.[56] After 28 days, it was clearthat the nano-hydroxyapatite was incorporated into the pre-existing spongy bone.[56] Similar results were seen when elec-tropolished titanium cylinders were coated with nano-hydrox-yapatite and implanted.[57] The integration of nano-hydroxya-patite may be associated with the increased topographicalroughness; however, integration also appears to be dependenton bone type (roughness may be less important in corticalbone than in trabecular bone, primarily due to the pre-existingprimary stability of implants with cortical bone).[58] The in vivoanimal results have been supported by human trials[59] andnano-hydroxyapatite is now integrated into commercial prod-ucts such as nanOss�, Ostim�, Cerabone� and BoneSave�.[18]

3.3. Hydroxyapatite as a Drug Carrier

In the mid-1970s, it was found that proteins could reversiblyadsorb to hydroxyapatite depending upon surface charge, pro-tein charge and protein structure.[60] Adsorption of proteins,drugs and the like to hydroxyapatite is very attractive particu-larly where targeted drug delivery is required. Whilst adsorp-tion of drugs such as bisphosphonates onto the surface of hy-droxyapatite has been achieved,[61] it is questionable whethersurface-based adsorption will be effective over the long term.Problems such as burst release and rapid dissolution of hy-droxyapatite in vivo, for example, hinder its use.

Hydroxyapatite drug-delivery systems have been limited dueto the difficulty in loading drugs that are insoluble in water.[62]

Therefore, hydroxyapatite nanoparticles are considered to bebeneficial as a nanocarrier. Nanoparticulate carriers are desira-ble for both systemic and localised delivery due to the in-creased circulation time and the capacity to transport hydro-phobic materials.[7] Nano-hydroxyapatite in particular is there-fore attractive due to its solubility in vivo and its capacity topenetrate cell membranes.[7]

Accordingly, Ijntema et al.[60a] used bovine serum albumin(BSA), an acidic protein known not to desorb either from hy-droxyapatite at phosphate concentrations less than 0.5 m or inthe presence of CaCl2 adsorbed to hydroxyapatite, to mimica drug microcarrier.[60a,c] The flaw in the experiment was thatthe BSA bound so tightly to the hydroxyapatite that the onlymechanism for release was not BSA desorption but hydroxya-patite dissolution. For example, in water after 3 weeks only0.01 % of BSA had desorbed from the hydroxyapatite.

Chitosan and hydroxyapatite have together formed a formi-dable partnership as drug-delivery vehicles. This is due to thenegatively charged hydroxyapatite core (�13.9 mV) forminga tight attraction to the positively charged chitosan.[63] For ex-ample, Venkatesan et al.[63] recently showed that hydroxyapa-tite–chitosan nanoparticles used to encapsulate Celecoxib en-

abled sustained release of the drug due to the strong hydrox-yapatite–chitosan bond which restrained the loaded drug.After 24 h, less than 15 % of the drug was released by the“burst effect” common to adsorbed drugs and after 15 daysover 10 % of the drug remained. The combination of Celecoxib,hydroxyapatite and chitosan was shown to shrink colon tu-mours in mice (Figure 5).

3.4. Hydroxyapatite Nanocomposites as AntibacterialMaterials

In addition to drug delivery, the use of ceramics comprisingantibacterial properties is highly appealing. Given that infec-tion and macrophagic cell responses can result in the removaland replacement of a medical device, it is essential that anynegative effects of implantation are ameliorated. For example,silver ions are known to have an antibacterial effect. Accord-ingly, silver ions have been incorporated into the hydroxyapa-tite matrix during production. Silver ions have been shown togive hydroxyapatite excellent antibacterial properties, whichwere most likely due to the interaction of silver ions with thethiol groups in bacterial proteins.[64] Recently, Li et al. testedthe effect of silver–hydroxyapatite on bacteria by comparingthe inhibition of Escherichia coli on hydroxyapatite–titania tocomposite hydroxyapatite–titania–silver. It was shown that thesilver composite material had a 100 % bacterial reduction aftera 24 h incubation period.[64e] Ewald et al. also supported thesefindings by showing that, by doping apatite with silver, materi-als (in the form of brushite) were produced with antimicrobialactivity comparable to antibiotic treatments.[64d] These types ofcomposites are particularly interesting due to the increasingresistance of bacteria to antibiotics.

Hydroxyapatite is also coupled with antibiotic to form dis-solvable antibacterial microspheres.[65] For example, co-deposi-tion of drugs or antibiotics with hydroxyapatite to generatehidden or deeper pockets has been preliminarily investiga-ted.[61b, 65a] Another mechanism that appears promising is the

Figure 5. Effect of drug delivery on colon tumours injected subcutaneouslyinto the right flank of nude mice. Shown below the mice are the excised tu-mours post treatment. Hap = hydroxyapatite, Cht = chitosan. (Adopted fromref. [63] .)

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use of calcium phosphate cements (see Figure 6) to generatecohesive drug–apatite or antibiotic–apatite formulations.[54a]

One reason is the capacity for controlled drug release via diffu-sion from the cement rather than via dissolution of the apatitematerial, as the cement has less in vitro solubility than typicalblock apatites.[66] However, release is different for each drugtype and highly soluble drugs will still undergo burst relea-se.[6, 22a]

3.5. Magnetic Hydroxyapatite Composites

A recent interesting method to utilise the nano-hydroxyapatitehas been to couple it with magnetic particles or to polarise it.First, polarisation of hydroxyapatite has been shown to prefer-entially attract cells that will establish bone growth around theimplant. Polarisation in a dc field (1 kV cm�1 at 400 8C) hasbeen shown to modify cellular activity around the implant.[67]

This is most likely due to the generation of positive and nega-tive charges on the implant which mimic streaming potentialsseen within natural bone.[68] In animal models, bone penetra-tion into polarised hydroxyapatite implants has been found tooccur as early as 3 weeks after surgery, half the time of a “stan-dard” hydroxyapatite implant (6 weeks).[67a,e] The preferred po-larisation, positive or negative, is inconclusive. For example, invitro study by Dekhtyar et al. showed that a negativelycharged hydroxyapatite surface had 10 times more cells thana neutral surface and that bone regrowth was favoured ona negatively polarised surface.[69] However, another in vitrostudy by Kizuki et al. indicated favoured cell adhesion on posi-tively polarised surfaces.[70]

Coupling with magnetic particles such as Co, Fe and mag-netite (Fe3O4) has been shown to be possible.[7, 71] In a recentreview by Uskokovic,[7] it was suggested that the magneticcoupling could be utilised for external control of particle deliv-ery by using a magnetic field. The identified use for this was todetoxify bloodborne toxins and remove these by drawing thehydroxyapatite–toxin away from the blood vessel. Additionally,researchers such as Wu et al.[71d] successfully showed that mag-netic hydroxyapatite could be formed by adding iron(II) chlo-ride tetrahydrate to a calcium hydroxide orthophosphate sus-pension during ageing, which could be used to treat tumours.The particle size was around 20–50 nm with a shape of short

rod or sphere with a magnetisation of around 3.42–20.92 emu g�1.[71d] In vivo, when injected into murine colorectalcancer cells, the magnetic hydroxyapatite nanoparticles causedsignificantly greater shrinkage of a murine colorectal tumourthan non-magnetised hydroxyapatite.[71c] In addition, the mag-netic hydroxyapatite showed little toxicity when subcutaneous-ly injected.[71c]

More recently, Tran et al. suggested that hydroxyapatite-coated magnetic nanoparticles could be used to reverse osteo-porosis.[72] Briefly, osteoblasts were cultured in vitro in the pres-ence of hydroxyapatite-coated Fe3O4 nanoparticles. The resultsshowed that in the presence of 200 mg mL�1 hydroxyapatite-coated Fe3O4 magnetic nanoparticles, osteoblast density andosteoblast differentiation markers, such as alkaline phospha-tase, type I collagen and calcium deposition, were significantlyincreased.[72b] Assessment of osteoblast cells demonstrated at-tachment of the hydroxyapatite-coated Fe3O4 nanoparticles tothe surface of the cell within 24 h (Figure 7). The authors spec-ulate that matrix proteins, such as vitronectin and fibronectin,may have adsorbed in greater amounts to the hydroxyapatite-coated iron oxide nanoparticles. Subsequent interactions be-tween the hydroxyapatite-coated iron oxide nanoparticles andosteoblast membranes may have enhanced osteoblast prolifer-ation and differentiation by triggering key integrin receptors.The data indicated that magnetic hydroxyapatite could be di-

Figure 6. Nanostructure of apatite crystals in a bone cement.[54a]

Figure 7. TEM images showing hydroxyapatite-coated Fe3O4 nanoparticleuptake by two different osteoblast cells (a) and (b).[72b] The majority of thenanoparticles attached to the surface of the cells.

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rected by an external magnetic field to porous sites, improvebone-forming cell growth and reverse osteoporosis. Sucha method can enhance the drug effectiveness of the treat-ment.

3.6. Nano-Hydroxyapatite for Gene Therapy

The use of calcium phosphate based materials for in vitrotransfection is well established.[73] However, difficulty lies withinthe capacity of the research to translate off the bench and intoanimal models. Subsequently, the beneficial advantage ofnanosized particles appears to be in the field of gene therapy.As discussed in Section 3.3, nanocarriers are desirable thera-peutic delivery vehicles due to their increased circulation time,adherence to hydrophobic materials and capacity to penetratecell membranes.[7] Nucleic acids such as DNA and RNA are gen-erally incapable of penetrating cell membranes.[18] Further-more, nano-hydroxyapatite appears to be highly capable ofco-precipitating with DNA and RNA. For example, Zhu et al.[74]

reported that using DNA–nano-hydroxyapatite complexes, thetransfection efficiency of plasmid DNA (pDNA) into cells (SGC-7901) could be up to 80 % without any clear toxicity within thefirst 2 weeks after in vivo injection. Advancing this, Wu et al.[75]

used superparamagnetic nano-hydroxyapatite particles for tar-geted gene therapy, with the nanoparticles displaying a highbinding affinity for pDNA (average of 97 %).

Similarly Hossain et al.[76] recently demonstrated that nano-hydroxyapatite provides a good delivery mechanism for silenc-ing RNA (siRNA or small-interfering RNA). Typically, like DNA,siRNA use in gene therapy (to silence target genes at the tran-scriptional level) is hindered by the lack of suitable targeteddelivery systems as the naked siRNA cannot passively pene-trate the cell membranes.[76] Accordingly, carbonate apatite–siRNA complex (average size of 274.6 nm) was used to deliversiRNA to HeLa cells. The results indicated that the carbonateapatite provided delivery of siRNA with efficient endocytosisand endosomal escape of the siRNA. In fact, 10 nm releasedsiRNA led to significant inhibition of cancer cells by silencingthe cyclin B1 gene.

4. Challenges

The biosafety of hydroxyapatite nanoparticles remains contro-versial. As a drug-delivery or protein-coupling agent, there islittle literature to suggest that the combination is biotoxic.[77]

Accordingly, the commercial medical use of hydroxyapatite inthis manner remains under debate. At present, however, regu-latory approval remains limited to the use of nanoparticles asbone matrix fillers.[7, 77]

It is currently considered that the disadvantages of nanopar-ticles are the potential for wear particle generation and thesubsequent osteolytic potential of these. Nanoparticles are sus-ceptible to degradation by hydrolysis and oxidation and thusare readily releasable.[49d] Wear particles can cause damage viatwo mechanisms. First, hydroxyapatite wear particles, especial-ly when used in joint replacement products, can become en-trapped in the joint and degrade the remaining materials as

well as cause cellular inflammation. Subsequent implant loos-ening will occur after net bone loss due to this inflammatoryresponse. Second, wear particles can migrate away from thesource of implantation, for example via the synovial fluid. Mi-gration can be particularly damaging where hydroxyapatitedebris is taken up into the bloodstream from the body fluidthrough intercellular space between endothelial cells.[78]

Several researchers have shown that nano-hydroxyapatite isbiocompatible.[25b, 26a, 27, 49c–e, 51, 52, 71d, 79] The studies by Websteret al. showed that nanophase hydroxyapatite initiated a betterosteoblast or osteoclast cellular response than larger particlesizes.[52a–c] However, the proposition was that cellular responseswere improved due to three factors: 1) the increased surfaceroughness; 2) the enhanced surface wettability ; and 3) the in-creased surface solubility.[52b,c] Accordingly, it is likely that nano-particles of calcium phosphate material are readily releasedinto surrounding tissues.[18] A recent study by Lahiri et al.looked into whether wear particle size has any effect of cyto-toxicity.[80] Interestingly, due to the inherent agglomeration ofhydroxyapatite nanoparticles, wear debris ranged in size be-tween 300 nm and 9.5 mm (average 3 mm). Particle size was re-duced when the nano-hydroxyapatite was combined withcarbon nanotubes (100 nm to 3 mm, average 600 nm). Thestudy found that nano-hydroxyapatite wear particles wereindeed more cytotoxic to osteoblast activity. This was consid-ered to be due to the difficulties with phagocytosis of largersized particles.

This result was also supported by Shi et al. ,[81] who used twosizes of nano-hydroxyapatite (20 and 80 nm) to treat osteosar-coma (malignant bone tumour cells). Both types of hydroxy-apatite nanoparticles trended towards apoptotic cell death butincreased apoptosis was observed with the larger particles(80 nm). However, other researchers suspected that since bothnano- and microsized hydroxyapatite particles could be dis-solved progressively in lysosomes, the reported increased cyto-toxicity toward macrophages might be due to intracellular cal-cium level rather than particle size.[77, 82]

The concept of biocompatibility of nanoparticles is perhapsno longer the appropriate term, in particular when nanoparti-cles are used as a delivery mechanism. Accordingly, buzz termssuch as biostability and biodegradation have slipped into thecommon vernacular, particularly when referring to materialssuch as hydroxyapatite where a stable equilibrium is not nec-essarily desired but rather it is expected to degrade.[83] The bio-compatibility and biostability of nano-hydroxyapatite havebeen reported in a variety of papers.[52a,c, 72a, 82a, 84]

Biostability, for example, has been tested by immersingnano-hydroxyapatite in highly acidic solutions and testing forCa ion depletion.[51] As an example, in work by Lim et al. ,[85] hy-droxyapatite discs and hydroxyapatite-coated teeth wereplaced in a highly acidic solution (pH 3.26) for up to 3 months.Although both samples showed an increase of calcium withinthe solution over time, it was considered that both substrateshad remarkable chemical resistance and stability. Furthermore,Vickers hardness testing of substrates before and after acidicexposure showed minimal change, thus suggesting that hy-droxyapatite hardness is not unduly affected by calcium deple-

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tion. Alternatively, nanoparticles may be left to soak in synthet-ic body fluids and tested for colloidal change.[82a] As a colloidalsolution, nano-hydroxyapatite can be unstable due to the cat-ionic and anionic dual charges within the hydroxyapatitewhich can easily lead to flocculation and the aforementionedparticle agglomeration.[86]

The discussion about safety concerns associated with ultra-small particles is ongoing and it is fair to say that the healthconsequences are not well understood. It is likely that nano-particles will interact with DNA and RNA as particle size de-creases and transport across cell membranes such as theblood–brain barrier becomes probable.[79a] There is, however,no clear evidence that nanoparticulate hydroxyapatite is eithercytotoxic or genotoxic.[18, 77] Recently, Scheel and Hermann[77]

undertook a risk assessment of the use of hydroxyapatite asa dental material, particularly where hydroxyapatite nanoparti-cles were used. The study concluded that no carcinogenic find-ings, adverse subchronic or chronic effects, due to hydroxy-apatite exposure could be discovered in the literature. Current-ly, hydroxyapatite nanoparticles are used in commercial prod-ucts and have gained the requisite regulatory approval. Ostim�(Osartis, Obernburg, Germany), for example, is a nanoparticle-sized precipitated hydroxyapatite paste comprising about 40 %water, which is used as an injectable bone filling material totreat bone defects.[54b] To sell its product in Europe (CE mark)and the US (FDA approval), comprehensive preclinical evalua-tion including biocompatibility and genotoxicity testing hasshown that it is safe for the market.[77] This supports the find-ings of other researchers which have shown that it takes largedoses of micro- or nanoparticulate hydroxyapatite as a sol insaline solution injected directly into the bloodstream to betoxic (medium dose 200 mg kg�1 nanoparticles in a rabbitmodel and 160 mg kg�1 in a rat and dog model).[87]

Another doubt over nano-hydroxyapatite, however, relatesto possible particle aggregation within blood vessels. It isknown that artery calcification as part of atherosclerosis is dueto calcium hydroxyapatite.[88] Although this is yet to be fullyunderstood, it is possible that nano-hydroxyapatite may accu-mulate in blood vessels in a manner similar to cholesterol.Given the similar crystal lattices of hydroxyapatite and choles-terol, it is proposed that atherosclerotic complications mayoccur with ongoing hydroxyapatite nanoparticle use.[7]

5. Outlook

It is well established that synthetic hydroxyapatite is a benefi-cial material for medical applications. By sharing a good por-tion of its chemistry with bone, hydroxyapatite opens up ave-nues for applications throughout the body. Nano-hydroxyapa-tite, in particular, appears to hold many advantages over mi-crometre-sized particulate hydroxyapatite. The traditional hy-droxyapatite deposition processes, such as wet chemistry, sol–gel and electrophoresis, appear to be well suited to make hy-droxyapatite particle sizes well below 1 mm; however, it ap-pears that reproducibility of nanosized particles is an issue. Ad-ditional processing such as ultrasonics appears to at least

ameliorate the problem and de-cluster agglomerated particle-s.[25a, 48]

Nano-hydroxyapatite is desirable due to its ability to crosscellular membranes. Particularly in drug-delivery applications,encapsulation of drugs within a nano-hydroxyapatite scaffoldor coupled to a nano-hydroxyapatite particle will enable tar-geted delivery. The known dissolution rate of apatite materials,such as hydroxyapatite in particular, may prove vital to over-coming the issue of burst release of drugs as well as uptake ofhydrophobic materials into cells. Tran et al. , for example,showed that nano-hydroxyapatite is readily absorbed into anosteoblast cell within 24 h.[49a] This shows that nano-hydroxya-patite can be used effectively to treat bone diseases such asosteoporosis. As a coupling agent, hydroxyapatite can be usedwith bone drugs such as bisphosphonates to treat bone dis-eases.[61]

Interestingly, however, the medical benefits of using nano-hydroxyapatite are not limited to bone disorders. Rather thereis growing evidence to show that nano-hydroxyapatite aloneor as a coupled agent is highly efficient at treating tu-mours.[63, 81] Further, coupling to magnetic agents to producemagnetic nanoparticles is possibly the solution to targetedtreatment.[71a,d, 72c]

Although there are some concerns about the biosafety ofnanoparticles,[89] there is no clear evidence that suggests thatnano-hydroxyapatite is causing, or will cause, any future medi-cal damage to users.[77] In vitro and in vivo trials have found nocytotoxic effects when compared to micrometre-sized particle-s.[82a] In fact, research has shown that nano-hydroxyapatite isbiocompatible.[25b, 26a, 27, 49c–e, 51, 52, 71d, 79]

Nano-hydroxyapatite is shaping up as a new material totreat bone disorders. Its advantages build from the knownbenefits of synthetic hydroxyapatite as a bone filler, scaffoldand coating material. When produced at the nanoscale, theparticles have the added benefit of an increased surface areaproviding an ideal material for cellular processing. The use ofnano-hydroxyapatite as a targeted drug-delivery vehicle is a rel-atively new field and accordingly the true long-term benefitsof nano-hydroxyaptite are sure to be revealed in the nextdecade. At present, however, it is certainly looking promising.

Acknowledgements

P.A.T. acknowledges funding from the McKenzie Fellowship, Uni-versity of Melbourne, Australia. N.T. is supported by the Stein/Bellet Foundation and Vietnam Education Foundation, USA.

Keywords: biotechnology · hydroxyapatite · nanomaterials ·nanoparticles · orthopaedics

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Received: January 30, 2012Published online on March 29, 2012

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