COMPARATIVE STUDY ON PREPARATION AND ...excellent mechanical strength, chemical stability and biocompatibility, however, they are considered as bioinert materials [1-4]. To improve

25Comparative study on preparation and characterization of bioactive coatings for biomedical...

© 2017 Advanced Study Center Co. Ltd.

Rev. Adv. Mater. Sci. 48 (2017) 25-51

Corresponding author: Csaba Balázsi, e-mail: [email protected]

COMPARATIVE STUDY ON PREPARATION ANDCHARACTERIZATION OF BIOACTIVE COATINGS FOR

BIOMEDICAL APPLICATIONS – A REVIEW ON RECENTPATENTS AND LITERATURE

Monika Furkó, Katalin Balázsi and Csaba Balázsi

Hungarian Academy of Sciences, Centre for Energy Research, Budapest Konkoly-Thege M. 29-33. H–1121,Hungary

Received: December 16, 2016

Abstract. Up to now, there are enormous efforts to develop such coatings that can enhance thebiocompatibility properties of metallic implant materials (Ti, Ti6Al4V, CoCrMo, stainless steel)and even provide antimicrobial effect. The most interesting candidates for these coatings arecalcium phosphate (CaP) or specifically hydroxyapatite (HAp) phases due to their biocompatibility,osteoconductive properties, and chemical similarity to the inorganic phase of human bone. Theionic modification of CaP phases can improve the biocompatibility property of materials evenmore. This review provides an overview of the recent results achieved on the preparation meth-ods of pure and ion-substituted calcium phosphates as well as their characterization. In addition,we summarize the effect of ion modification (Si, Sr, Mg, Zn, Fe, Cu, and Ag) on the crystal structureand biological properties of coatings as well as the biological role of ion-substituents.

1. INTRODUCTION

Ti6Al4V, CoCrMo, and stainless steel are widelyused as orthopedic prostheses. This is due to theirexcellent mechanical strength, chemical stabilityand biocompatibility, however, they are consideredas bioinert materials [1-4]. To improve theirbiocompatibility and enhance the bone growth ap-propriate coating is needed [5]. Pure HAp is a sto-ichiometric apatite phase with a Ca/P molar ratio of1.67 and the most stable calcium phosphate salt atnormal temperatures and pH between 4 and 12 [6].The chemical composition, crystallinity, size, andmorphology of the HAp crystals and their aggre-

gates play critical roles in determining their proper-ties and potential applications [7-9]. Nano sized HApcrystals have high surface energy [10], andbioceramics with enhanced mechanical propertiescan be fabricated using HAp nanopowders as rawmaterials. In addition, nano HAp bioceramics ex-hibit better bioactivity and higher resorbability thanthose in microscale sizes [11]. There are severaltechniques to prepare coatings such as plasmaspraying [12-15], sol–gel technique [16-20], chemi-cal vapor deposition (CVD) [21], plasma enhancedMOCVD [22,23], electrodeposition [24-26], pulsedlaser deposition (PLD) [27-29] and so on. The prepa-ration methods can also be regarded as wet chemi-

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26 M. Furkó, K. Balázsi and C. Balázsi

cal and solid state reactions. Depending on thecoating process and preparation parameters, crys-tallinity and phase composition of the HAp can vary.The presence of amorphous phase accelerates thecoating dissolution, which can affect the the long-term influence of the coating. The faster dissolutionrate enhances bone growth through biological apa-tite formation but may lead to the faster degrada-tion of the coating [30]. It is well known that HAp’schemistry can be made even closer to bone min-eral with the substitution of other ions into the lat-tice, including carbonate, silicon, magnesium, stron-tium and zinc.

It is also possible to tailor the apatite to specificneeds and a lot of work has been carried out in thefield of substituted apatites [31]. These ionic sub-stitutions can also modify the biodegradation prop-erties and biosorption kinetics of different calciumphosphate phases such as amorphous calciumphosphates (ACP), dicalcium phosphates (DCP),tricalcium phosphates (TCP) and hydroxyapatite(HAp). Ionic substitutions for the component ions(Ca2+, PO

43- and OH-) of the HAp lattice or -TCP

lattice are effective to control the biodegradation orbioresorption of the CaP ceramics. For example Mgsubstitution in the Ca-site decreases the extent ofdissolution of -TCP [32]. Magnesium [32], stron-tium [33], zinc [34,35], fluoride [36], silicon [37] andcarbonate [38] ions have been substituted in theHAp lattice or the TCP lattice so far. Among them,F- substitution decreases, while Mg, Sr, or carbon-ate substitutions increase the extent of dissolutionof synthetic HAp [39]. The Mg2+, Sr2+, Zn2+, Si4+,and F- ions released due to the dissolution of CaPceramic materials containing these ions in the lat-tice may affect osteoblast and/or osteoclast activi-ties and bone formation [40,41].

In addition, these ionic substitutions remarkablyaffect the HAp lattice parameters, crystallinity, solu-bility, thermal stability, and dissolution rate, enhanc-ing the in vitro proliferation of human osteoblast-likecells and consequently encouragingosseointegration. Among the several ions, siliconhas attracted a lot of attention, as it has an impor-tant role in bone tissue metabolism and is able tostimulate osteoblast action and, consequently, newbone formation [42].

The aim of this review is to summarize the effectof different preparation methods on the structure,stability and biological activity of pure and ionicmodified CaP phases as well as describe the mainadvantages and disadvantages of the current prepa-ration techniques.

2. DESCRIPTION OF SYNTHESISMETHODS

2.1. Plasma spraying (PS)

The plasma spraying technique is based on the useof plasma flames at very high temperatures and highvelocities that lead the HAp particles onto the sur-face of substrates. The CaP coating obtained in thiscase mainly comprises HAp, but other crystallineand amorphous calcium phosphate (ACP) phasescan be also present. The amounts of these co-pro-duced phases depend on the conditions of spray-ing, such as gas flow, plasma temperature, natureof the gas, cooling conditions and the distance be-tween the substrate and the flame as well as thesize of the HAp particles [43-46]. Wu et al. [14]described the method for preparing porous HAp lay-ers using suspension. They added pore-formingagent into the hydroxyapatite suspension with asolid content of 16%-45%. After full stirring, the feed-stock materials for plasma spraying were transferredinto the injection system, and injected into the hightemperature area of the central plasma flame. Thethus achieved porous structure could improve thethe biological properties of the coatings, such asbioactivity and capability for bone ingrowth. Gan etal. [15] disclosed an invention related to preparingantimicrobial silver containing HAp coatings byplasma spraying technique. In their patent, onemethod of incorporating a silver derivative into HApincludes the steps of sequentially applying layersof stable silver oxide and HAp powder to an implant.However, this method did not form a homogeneoussilver-doped HAp coating. Consequently, coveragewas not uniform, and ion release was not steadilymaintained after implantation. Even if the silver ox-ide was mixed with the HAp powder prior to plasma-spraying, the oxide cannot chemically react andcombine with conventional HAp powder to form ahomogenous formulation. In the other method, theysoaked an implant having a hydroxyapatite coatingin a silver nitrate solution for approximately 24 hoursand then dried the implant in air or an inert environ-ment. The thickness of the plasma sprayed coatingon a medical implant can typically be from about 1micron, and typically is in the range of 10 or a fewtens to a few hundreds of microns.

2.1.1. Molecular plasma deposition(MPD)

The MPD process was originally developed by Cha-meleon Scientific (Longmont, CO, USA) and en-


ables the deposition of HAp coatings onto metals,polymers, or other ceramics at thickness levelsranging from nanometers to tens of microns. TheMPD process utilizes a corona discharge at a highvoltage to allow coatings to be applied uniformly atlow temperatures onto a wide range of substrates[47]. Balasundaram et al. [47] described nano-hy-droxyapatite deposition onto titanium surface viaMPD for the first time. In their work, the HAp con-taining solution was ionized via corona discharge,and HAp was then deposited on the anodizednanotubular Ti by introducing the ionized solutioninto the vacuum chamber. This research group fileda patent also on this topic [48]. They found that thenano-HAp coating deposited by MPD is highly ad-herent and free of microparticles.

2.2. Pulsed laser deposition (PLD)

Generally, in PLD technique, a pulsed laser beamis focused on a rotating HAp target in a vacuumchamber under a controlled water vapour atmo-sphere. The material that is vaporized is depositedconsequently onto a parallel substrate, which canbe heated. Several CaP phases can be obtained byvarying the deposition parameters, such as thefluence of the beam laser, the water vapour pres-sure and substrate temperature [49]. This methodenables to generate adherent and crystalline filmsand has been proven to be efficient and reliable [50].A comparative study on the adhesion of differentnatural-derived HAp composites and commercialHAp thin films was carried out by Duta et al. [51].They observed the adherence values recorded forthe HAp films were generally similar to the onesoften reported in the literature for this type of PLDfilms. Komath et al. [52] studied the possibility ofobtaining adherent and crystalline HAp on titaniumsubstrates at 200 °C through PLD and subsequenthydrothermal treatment in an alkaline medium. Theyfound a remarkable increase in adhesion with thesubstrate as a result of the treatment. Man et al.[53] investigated the effect of different pre-treatmentmethod for enhancing the adhesion strength of HApand they observed in general the adhesion strengthincreased with surface roughness.

2.3. Chemical vapor deposition (CVD)

The process of producing coatings and films withCVD involves the chemical reactions of gaseousreactants on or near the vicinity of a heated sub-strate surface. CVD can be employed to manufac-ture single-layer, multilayer, composite,

nanostructured, and functionally graded coatingmaterials with well-controlled dimension and uniquestructure at low processing temperatures.

This atomistic deposition method can offer highlypure materials with structural control at atomic ornanoscale levels in addition to the coating of com-plex-shaped biomedical prostheses and the fabri-cation of nanodevices and composites [21]. Themain CVD process parameters such as tempera-ture, pressure, reactant gas concentration and to-tal gas flow require accurate control and monitor-ing. The chemical reactions during CVD processinclude pyrolysis, oxidation, reduction, hyrolysis ora combination of these, and can be catalysed bythe substrate. The chemical reactions determine therequired operating temperature range. The tempera-ture at which the coating is deposited is critical asit controls both the thermodynamics and the kinet-ics of the coating process. The deposition tempera-ture must be achieved and maintained in order forthe reaction to occur on the substrate and not inthe gas phase, and with an appropriate microstruc-ture (grain size and shape). Small changes in thetemperature may change the reaction, and/or itskinetics, resulting in an inferior coating [21]. Theability of the reactant gases to reach the substratesurface and the temperature at which the reactionis gas diffusion limited, are important in determin-ing the uniformity of the coating [54,55].

2.4. Sol gel technique (SG)

In a sol-gel system, colloidal particles in the size of1-1000 nm are dispersed. The stability of sol par-ticles can be modified by reducing their surfacecharge. If it is reduced sufficiently, gelation is in-duced and the resultant gel is able to keep its shapewithout using any mould. Gels can be regarded ascomposites since they consist of solid skeleton thatenclose the liquid phase or solvent [16]. The sol-gelmethod can be performed by dip coating process(for large samples with complex shape) or by spincoating (for smaller samples with flat surface). Gen-erally, it can be said that this technique offers arelatively easy, convenient and cheap solution toprovide bioceramic coatings compared to the abovediscussed high-temperature techniques that requirehigh energy. Moreover, sol-gel process providessuperior chemical and physical homogeneity of thefinal ceramic product compared to other routes, suchas solid-state synthesis, wet precipitation or hydro-thermal formation. In the SG coating process, sub-strate metal degradation due to thermally-inducedphase transformations, microstructure modification


or oxidation can also be avoided. The ceramic coat-ings prepared by SG process has better structuralintegrity, purity and phase composition than otherconventional methods, such as thermal spraying.and this process provides significantly milder con-ditions of the synthesis of calcium phosphate films.This results in a much better structural integritywhereas the defects originated from plasma spray-ing can be largely avoided. Generally a sol-gel pro-cess for preparing a crystallized hydroxyapatitecomprises hydrolysing a phosphor precursor in awater based medium, adding a calcium salt precur-sor to the medium after the phosphite has beenhydrolysed to obtain a hydroxyapatite gel and sub-sequent calcination of the crystallized hydroxyapa-tite at a suitable elevated temperature. Existing sol-gel hydroxyapatite (HAp) synthesis methods requirecalcination temperatures higher than 500 °C to de-velop a well-crystallized HAp phase. A high degreeof HAp crystallinity is required for bioactive applica-tions, because partially crystalline, or amorphouscalcium phosphate coatings are rapidly resorbedby living tissue. Metal alkoxides such as calciumdiethoxide and phosphorus esters (for example,trialkyl phosphites and trialkyl phosphates), are usedas Ca and P precursors, respectively, in SG syn-thesis of CaP [18].

2.5. Wet chemical precipitation /Hydrothermal synthesis

Wet chemical precipitation reactions are the mostwidely used methods for preparing ceramic coat-ings or powders. In wet-chemical coating proceduressolutions, suspensions, colloids of dissolved or sus-pended precursors are used in order to coat boneimplant surfaces. The main advantage of this tech-nique is that ambient temperature and pressurescan be applied using relatively simple equipment.In addition, objects with complicated shapes canbe completely covered with the coating and the pro-cess can easily be upscaled. However its main draw-back is that heat treatment is necessary after depo-sition to enhance the adherence of coatings [56].

CaP coatings have already been intensively in-vestigated for several decades owing to their prop-erties to enhance the integration of implants intobone [57]. Numerous reviews have been publisheddealing with the deposition of biologically active (in-organic) CaP layer on titanium implants [57,58]. Thehydrothermal synthesis is similar to wet precipita-tion method, however, in this case, treatment isperformed at elevated (>80 °C) temperatures duringa relatively prolonged period of time (>1.5 h), the

CaPO4 deposits are usually crystalline and they offer

good control on morphology and chemical stoichi-ometry [59-63].

2.6. Electrochemical deposition (ED)

Electrochemical deposition is a versatile coatingtechnique that has been used widely in biomedicalapplications. Up to now, a large amount of work hasbeen devoted to the electrochemical HAp coatingon implant surfaces [64,65]. Using electrochemicalprocess to deposit CaP and modified CaP coatingsis more cost effective compared to other physicaldeposition methods (CVD, PLD). The processingtemperature is relatively low compared to plasmaspraying. Furthermore, the properties of the coat-ing can be easily controlled. The other advantage ofelectrochemical methods that homogenous layer canbe deposited on 3D materials with complex geom-etry. In a conventional electrochemical depositionprocess, a two-electrode electrochemical cell is usedwhere the cathode is the substrate material to becoated and the anode is some inert metal such asplatinum. During electrochemical deposition, the pHat the cathode/electrolyte interface can be con-trolled. In an aqueous electrolyte, the following re-actions occur at the surface of the cathode (reduc-tion of water, proton discharge, reduction of dis-solved oxygen):

2H2O +2e– H

2 + 2OH–, (1)

2H3O+ + 2e– H

2 + 2H

2O, (2)

O2 + H

3O+ + 4e– 3OH–. (3)

This results in the formation of hydroxyl ions, andhence increasing pH close to the surface.

The main electrochemical reactions at the elec-trode surfaces might be as follows:

Ca2+ + HPO4

2- CaHPO4, (4)

Ca2+ + HPO4

2- + 2H2O CaHPO

4.2H

2O, (5)

10CaHPO4 + 12OH– Ca

10(PO

4)

6(OH)

2 +

4PO4

3- + 10H2O. (6)

The electrochemical deposition can be performedby direct and pulse current. The main advantage ofpulse current deposition over the direct current isthat more homogeneous and dense layer can beachieved. In the pulse current deposition the time ofcurrent impulses applied are in the order of milli-second which is followed by a relaxation time. Thisrelaxation time allows the ions-depleted region inthe vicinity of electrode to regenerate. Moreover,


since the short current pulses prefer nucleigenetarion over growth, coatings even smaller par-ticles can be deposited which enhance the corro-sion properties of samples [66].

2.7. Electrophoretic deposition (EPD)

The electrophoretic deposition (EPD) process em-ploys electric fields to deposit charged nanoparticlesfrom a solution onto a substrate. Earlier industrialuse of the EPD process used organic solvent solu-tions and therefore typically generated hazardouswaste as a by-product of the process [67]. In addi-tion, the shapes, compositions, densities, and mi-crostructures of materials formed through EPD pro-cesses have typically been difficult to control. It isextremely difficult to form structures from more thanone material. That is to say, typical EPD processesare limited in that they are only capable of formingplanar, homogenous structures [68,69].

2.8. Electrospraying (ES)

Electrospraying is a relatively new technique to de-posit coatings by generating droplets, which canthen later be collected as films or particulates, un-der the maintenance of an electric field. Severalworks have recently developed CaP suspension orcoatings on substrates by electrospraying method[70-72]. In addition, using polymeric additives theparticulate form of CaPs can also be produced byadjusting the solution properties or collector design.In practice, a range of polymeric suspensions hasbeen generated into spherical particulates [73,74].Eltohamy et al. [73] developed TCP bioceramicmicrospheres through the electrospraying methodand utilized these spherical microparticulates as drugdelivery systems. They claimed that the generationand form of particles highly depend on the proper-ties of the initial solution, showing a switch fromcontinuous fibers (electrospinning) into discontinu-ous particles (electrospraying). In particular, duringthe electrospraying, the shape turned into a spheri-cal cup. Lagaron Cabello et al. filed an invention ondevelopment of bioactive coatings by electrospinningfor biomedical application [75]. In their invention, theydescribed that the electrospinning technique makesit possible to obtain coatings generally based onfibers of submicrometric size from a wide range ofbiocompatible and biodegradable polymers. Due tothe fiber size, the electrospun coatings can mimicthe real structure of live tissues and organs, whichfavours adhesion, growth, migration and cell differ-entiation. Furthermore, the materials of greatest in-

terest as the base of implants are also polymeric,and can be both temporary and permanent.Electrospinning shares the characteristics of bothelectrospraying and the conventional solution of thedry spinning of fibers [75].

Summary of advantages and disadvantages of dif-ferent deposition techniques is presented in Table 1.

3. CHARACTERIZATION OF PUREAND ION-SUBSTITUTED CALCIUMPHOSPHATE COATINGS

In this section, the physical and chemical proper-ties of calcium phosphate coatings prepared by dif-ferent methods are discussed.

3.1. Properties of CaP coatingsprepared by plasma spraying (PS)

Malshe et al. [13] investigated the mechanical prop-erties of PS deposited HAp in their patent. Theyhave succesfully fabricated HAp coating with a grainsize from 50 to 300 nm and a gradient of nano-to-micron pore sizes. The nano-HAp coating had a Ca/P ratio of about 1.6, very close to natural bone, andthus favorable for bone cell growth. Moreover, thenano-HAp coating was crystalline after sintering.They performed adhesion tests and microscratchtests according to ASTM C1624. The microscratchtest results showed that the critical load of coatingdelamination reached as high as 10 N. Gan et al.[15] carried out mechanically tests (pull-out tests)to determine the shear strength and interface stiff-ness of the implant-bone interface zone in case ofsilver modified HAp coatings. They found that themodified coatings with the lower or higher Ag+ addi-tions performed similarly. Moreover, the pull-out forcefor implant removal increased with increasing im-plantation period with significantly higher pull-outforces being recorded for the 16-day implantedsamples compared with the 9-day samples. Thevalues of interface zone stiffness were not signifi-cantly different for the 9-day and 16-day implantedsamples although the mean values were higher forthe 16-day implants. Wang et al. [76] fabricated HApcoatings by micro-plasma spraying method. Theyfound that the coating phase distribution significantlychanged throughout the coating depth. CrystallineHAp, ACP and ß-TCP were observed near the coat-ing-substrate interface. On the other hand, HApcontent increased with increasing distance from in-terface, and it was ~90% at top surface of the coat-ing ( > 40 mm) according to micro-Raman analysis.Columnar structure was found in such region, which


Low cost,Low temperature,Parameters are easy to control.

Table 1. Summary of advantages and disadvantages of different deposition techniques [12-29].

Method Advantage Disadvantage

Plasma Spray

Molecular PlasmaDeposition

Pulsed Laser Deposition

Chemical VaporDeposition

Sol/Gel Technique

Wet Chemical synthesis/Hydrothermal

Electrodeposition

Electrophoretic deposition

Electrospraying

Homogeneous , dense layer,High deposition rate,Coating thickness and depositionparameters are easy to control,Layer adherence is good,Improved wear and corrosion resistance.

Expensive equipment,High temperature,Possible thermal decomposition,Materials with complex shape aredifficult to coat (Line-of-sighttechnique),Rapid cooling produces cracksPoor control of the physicochemi-cal parameters of the coating.

Homogeneous, dense layer,High deposition rate,Coating thickness and depositionparameters are easy to control,High adhesive strength,Improved wear and corrosion resistance.

Expensive equipment,Line-of-sight technique,High temperature,Thermal decomposition.

Homogeneous, dense layer,Coating thickness and depositionparameters are easy to control,Layer adherence is good,Improved wear and corrosion resistance.

Expensive equipment,Line-of-sight technique,Low deposition rate.

Homogeneous, dense layer,highly pure materials,coating thickness and depositionparameters are easy to control,layer adherence is good,low processing temperatures.

Expensive equipment,Line-of-sight technique,Low deposition rate.

High purity,Homogeneous, thin coating withoutresidual stresses,Non-line-of-sight technique,Low temperature.

Edge cracking,Post-treatment needed (heattreatment to enhance the adher-ence of coatings)Layer adherence is poor,Controlled atmosphere processingrequirement,Expensive raw materials.

Homogeneous coating,Non-line-of-sight technique,Low temperature.

Post-treatment needed (heat treat-ment to enhance the adherence ofcoatings)Layer adherence is poor.

Low cost,Low temperature,Non-line-of-sight technique,Parameters are easy to control.

Inhomogeneous layer,Coating adherence is relatively poor.

Low cost,Low temperature,Non-line-of-sight technique,Parameters are easy to control.

Inhomogeneous layer,Coating adherence is relativelypoor,Difficult to produce crack-free coatings.Inhomogeneous layer,Coating adherence is relatively poor,Line-of-sight technique.


was related to a strong (002) crystallographic tex-ture. They also investigated the dissolution behaviourof the coating by immersing the material in balancedsalt solution for 14 days. The study revealed thatHAp coating exhibited superior chemical stability,and the columnar structure emerged on the surfaceof the coating during immersion experiment.Gligorijevic et al. [77] studied the surface structuralheterogeneity of high power plasma-sprayed hy-droxyapatite coatings. In their work they examinedthe dependence of the local surface structure, thesurface structural heterogeneity on the local thick-ness (thickness uniformity) and structure proper-ties along the thickness of hydroxyapatite coatings(HAp) deposited by using the high power (52 kW)laminar plasma jet. The results showed heteroge-neous phase distribution on the surface of each HApanalyzed and non-uniform thickness of HAp coat-ings. A correlation existed between the local sur-face structure and the local thickness of HAp coat-ing. The microstructural analyses along the thick-ness of HAp coatings suggested that different localstructure in the different surface locations of thesame HAp was caused by the occurrence of differ-ent recrystallization along the thickness of HAparound the corresponding surface locations duringthe plasma deposition process. Tsui et al. [78]sprayed HAp coatings onto substrates of Ti6Al4V,mild steel and tungsten, using a range of input powerlevels and plasma gas mixtures. They found thatthe HAp coating had low porosity, high cohesivestrength, good adhesion to the substrate, a highdegree of crystallinity and high chemical purity aswell as phase stability. They also discovered thatas the plasma power level increased, the crystallin-ity and OH- ion content of the coatings decreased,while the amount of non-HAp calcium phosphatecompounds present increased. These changes wereattributable to an increased degree of particle melt-ing. The melted material freezed to an amorphousphase. As the plasma power level increased, theporosity level and extent of microcracking in thecoatings decreased. These are associated increasesin the Young’s modulus, quenching stress and re-sidual stress in the coating and an increase in in-terfacial adhesion.

3.2. Properties of CaP coatingsprepared by pulsed laserdeposition (PLD)

There are several studies on investigating the prop-erties of pulse laser deposited HAp coatings [27-29,51,77-80]. Popescu-Pelin et al. [79] grown HAp

thin films onto titanium substrates and studied theirmechanical, physico–chemical and biological prop-erties. They evidenced rough and irregular morpholo-gies with specific micrometric droplets on the filmsurface. In addition, granular surface topographieswith micro- and nano-cavities were found for coat-ings deposited by PLD. Ca/P ratios were within therange of 1.73 ± 0.3 which indicates the presence ofHAp with a moderately excess of calcium. Theyobserved that the crystallinity of coatings improvedby PLD deposition. The micrographs of the filmsshowed a uniform distribution of spheroidal particu-lates with a mean diameter of around 2 mm. Pull-off measurements demonstrated excellent bondingstrength values between the hydroxyapatite filmsand the titanium substrates. Nishikawa et al. [80]studied the variation of the Ca/P ratio in hydroxya-patite thin films in relation to the spot size of theablation laser for two different spatial energy distri-butions in pulsed laser deposition. They found thatthe more uniform spatial energy distribution of theablation laser improved the Ca/P ratio. Duta et al.[51] prepared doped hydroxyapatite thin films byPLD. The deposited hydroxyapatite powder and filmsproved to be polycrystalline and the Fourier trans-form infrared spectroscopy evidenced the vibrationalbands characteristic to a hydroxyapatite materialslightly carbonated.

3.3. Properties of CaP coatingsprepared by chemical vapordeposition (CVD)

Krumdiek et al. [22] used pulsed-pressure (PP)metal-organic (MO)CVD to deposit layers of calciumphosphate onto flat biomedical grade titanium(Ti6Al4V) substrates. In their researches, they usedcalcium lactate and trimethyl phosphate (TMP),combined in a methanol solution as precursor. Theyfound that the films were continuous and the size ofsurface structures increases at higher temperaturesand precursor concentration. The films consisted ofamorphous calcium phosphate in elemental Ca/Pratios similar to the standard bioceramic, hydroxya-patite (1.66).

A qualitative micro-indentation method was alsoemployed to examine the adhesion of CVD depos-ited calcium phosphate coatings. They noticed thatthe adhesion was relatively consistent, and none ofthe coatings deposited at temperatures above 500°C, or at any concentration, were observed to crackor delaminate upon cooling.

Gao et al. [23] filed a patent on a method inwhich they coated the substrate with a calcium


phosphate compound using plasma enhancedMOCVD. They claimed that the ideal substrates fordeposition were solid materials that can be porousor non-porous, including but not limited to metal,ceramic, glass and their combinations. After thecoating process the samples were placed into asolution with an agent selected from an approriategroup of protein, antibiotic, antimicrobial, growthfactor and their combinations that can be adsorbedinto the coating before implantation.

3.4. Properties of CaP coatingsprepared by Sol/Gel method (SG)

Preparing CaP coatings by sol-gel methods is awidely applied and investigated procedure.Takahashi et al. [81] developed a gel route prepara-tion method using calcium nitrate andphosphonoacetic acid (HOOCCH

2PO(OH)

2) in an

aqueous solution and obtained pure HAp powder at700 °C. The crystallinity of HAp increased with tem-perature up to 1100 °C. Chai et al. [82] comparedtwo calcium precursors, namely calcium diethoxideand calcium propionate in reaction with triethyl phos-phite as phosphorus precursor to form HAp coat-ing. They observed that HAp phase appeared at 500°C for calcium propionate solution, but no HAp formedwhen calcium ethoxide was used. However, theydid not explain the influence of chemical nature ofthe precursors on phase formation. Qiu et al. [83]used calcium nitrate and ammonium dihydrogenphosphate (NH

4H

2PO

4) to synthesize HAp in highly

basic solution. They obtained HAp at calcinationtemperatures of 400 -1100 °C and indicated that thecrystallinity of the HAp improved with increasingtemperature.

Haddow et al. [84] used calcium acetate with anumber of phosphorus precursors, i.e. phosphoricacid (H

3PO

4), phosphorus pentoxide (P

2O

5), and tri-

ethyl phosphite for HAp coating preparation. Theyfound that the films prepared from triethyl phosphiteand calcium acetate showed the best wetting char-acteristic and the temperature required to form anapatitic phase was greater than 600 °C. Troczynskiet al. [19] disclosed an invention to manufacturebiofunctional HAp coatings and microspheres fordrug encapsulation. They designed the coatings andmicrospheres to perform a defined biological func-tion related to drug delivery. In this process theyused calcium phosphate cement (CPC) slurry andincubated it to precipitate HAp phase within themicrosphere or the coating. They claimed that byadding drugs and proteins into colloidal suspension(CPC slurry) of the microsphere or coating precur-

sors, a direct in-situ encapsulation and subsequentcontrolled release of the therapeutically active agentsfrom the apatite microspheres can be achieved. Thevarying degree of crystallinity of the microsphereswas used to control and customize their resorptionprocess in body fluids, and thus the rate of drugrelease. Usinskas et al. [85] deposited porous andhydrophilic calcium hydroxyapatite coating ontomodified titanium substrate by sol-gel method com-bined with dip-coating technique. In their researchwork they modified the surface of titanium substratewith calcium titanate (CaTiO

3) sublayer or additional

preheating at 650 °C to achieve a better quality ofHAp coatings. During the preparation process cal-cium acetate monohydrate was used as precursor.Ca(CH

3COO)

2 was added to the aqueous solution

of the 1,2-ethandiol and the obtained mixture wasthen stirred for 30 minutes at 65 °C. Afterward,ethylenediaminetetraacetic acid (EDTA) was added.They applied triethanolamine (TEA) also as acomplexing agent. Then diluted orthophosphoricacid (H

3PO

4 85%) was added into the solution to

obtain the Ca/P ratio of 1.67. Finally, this solutionwas mixed with PVA dissolved in distilled water. Theyfound that the surface modification of Ti substratedid not have any influence on the morphology of theHAp thin films.

Malakauskaite-Petrulevicienen et al. [86] dem-onstrated in their research work that sol–gel pro-cessing route is suitable for the fabrication of hy-droxyapatite thin films on Si substrate. The sub-strate was spin-coated by precursor sol solution 1,5, 15, and 30 times. The samples were annealedafter each spin-coating procedure at 1000 °C for 5hin air. In the sol–gel process ethylendiamintetra-acetic acid and 1,2-ethandiol as well as triethanola-mine and polyvinyl alcohol were used as complexingagents and as gel network forming agents, respec-tively. They claimed that the properties of hydroxya-patite thin coatings depend on spinning and anneal-ing times.

3.5. Properties of CaP coatingsprepared using wet chemicalprecipitation and hydrothermalmethods

In wet chemical preparation methods most researchwork are using calcium nitrate as calcium precur-sor [87,88].

The nitrate method is based on Eq. (7) and avoidsionic contamination because the ammonium nitrateby-product does not incorporate into the HAp lat-tice.


10Ca(NO3)

2.4H

2O + 6(NH

4)

2HPO

4 + 8NH

4OH

Ca10

(PO4)

6(OH)

2 + 20NH

4NO

3 + 20H

2O (7)

In this case extensive washing after precipitation isrequired to remove this residual ammonium nitrateand ammonia.

Thus, an alternative method was employed byAkao and Osaka [89,90] which focused on a cal-cium hydroxide method intended for large-scale in-dustrial use. The reaction process in Eq. (8) showesthat in this case the only by-product is water.

10Ca(OH)2+6H

3PO

4 Ca

10(PO

4)6(OH)

2 + 18H

2O (8)

There are numerous other research works usingsame precursors. Nörenberg et al. [91] also usedcalcium hydroxide as Ca precursor and phosphoricacid as P precursor to produce rod shaped apatitecrystals with a specific length-to-width ratio wherethe length-to-width-ratio of the crystal was at least 5. Saita et al. [92] filed an invention on methodpreparing HAp coating films using coating liquor com-prising a colloidal dispersion of hydroxyapatite hav-ing a fine particle size of 0.5 ìm or less. Troczynskiet al. [93] in their invention described a novel room-temperature process for obtaining calcium phos-phate, in particular hydroxyapatite, coatings andmicrospheres that encapsulate drugs, proteins,genes, DNA for therapeutical use.

However, the main problem with wet chemicalprecipitation reactions is a lack of reproducibilitydue to the formation of non stoichiometric CaPphases or variations in crystallinity and morphol-ogy. An important factor is the pH control in order toavoid the formation of TCP or other phases. Theaqueous calcium source must be buffered at a highpH in order to ensure the presence of orthophos-phate ions in solution produced from aquous H

3PO

4

[94]. Other finding that the reaction temperaturesbetween 25 and 37 °C have produced particle sizessimilar to those in bone, whereas 90 °C had particlesizes similar to dentin [95]. The ionic substitutioncan also be problematic for the Ca/P ratio and thesubsequent phase purity. Also, when producingsome substituted apatites, it is necessary to per-form the reaction in an inert gas atmosphere in or-der to avoid excessive carbonate substitution[96,97]. Bonfield et al. [98] filed a patent on prepa-ration of a single phase magnesium- and carbon-ate-substituted hydroxyapatite composition. Theprocess comprises the steps of preparing an aque-ous solution containing CO

32- and PO

43- ions in the

substantial absence of cations other than H+ ionsand mixing the solutions with an aqueous calcium-and magnesium-containing solution or suspension.

The precipitate formed were collected and dried. Theratio of (Ca+Mg/P) in the calcium- and magnesium-containing solution or suspension and the phospho-rus-containing solution, when mixed together, wasmaintained at 1.67. The product contained up to0.5% magnesium and up to 1% of carbonate sub-stituted into the hydroxyapatite structure and didnot contained Na+ or NH

4+ ions and the ratio of

(Ca+Mg/P) was greater than 1.67.Bingöl et al. [99] prepared hydroxyapatite from

calcium sulfate hemihydrate and ammonium phos-phate solution under mild hydrothermal conditions.In their research they found that the formation ofHAp at a reaction temperature lower than 50 °Coccurs at very limited extend while at 25 C HApformation starts after 7 days. Extending the reac-tion time for low temperature reactions improves theHAp formation efficiency, meanwhile this also pro-motes precipitation of another calcium phosphatesuch as monetite together with HAp. Saita et al.[100] invented a method for forming a hydroxyapa-tite coating film on substrates. The method com-prises coating a dispersion of flocculated colloidsof hydroxyapatite on a substrate and subsequentdrying. The coating method of this invention did notrequire heating of the coated substrate to high tem-perature and hence can also be applied to substrateswhich are easily deteriorated with heat. They claimedthat the coated substrate had excellent strength andadhesion force and was useful in a variety of fields,particularly as an implant [63]. The source of Ca2+

for electrodeposition can be not only syntheticCa(NO

3)2 but there are several reseach works where

the authors used so-called organic calcium compo-nents derived from either seashell [101,102] or egg-shell [103-107]. Narayanan and co-workers [101]used commercially available calcium nitrate andcalcium nitrate produced from calcined seashell assources of calcium in the electrolysis. In their re-search work the seashell was powdered and cal-cined for 1 h at 900 °C. One part of this powder wastreated with two parts of HNO

3 and the product was

washed in water then dried. They found that thecoatings prepared using organic Ca source werethin, containing lower amounts of carbonate andshowed better resistance to corrosion in SBF me-dium compared to coatings with inorganic Ca(NO

3)2.

These coatings had low roughness values and theycontained some amount of sodium and bioactivehydroxyl groups which is suitable for body implantapplication showing better biological performance.While Balázsi et al. [104] prepared hydroxyapatitepowder by ball milling and attrition milling from egg-


shell. They found that attrition milling resulted innanosized grains and after the heat treatment at900 °C, the particle size was around 100 nm. In thecase of ball milling the grains sticked together. Co-agulated samples with smooth surfaces wereachieved after heat treatment at 900 C and smallerparticle size with homogeneous size distribution wasachieved with attritor milling compared to ball mill-ing.

Fig. 1 shows the SEM images of HAp partcilesof different origin (commercial HAp purchased fromSigma Aldrich (sHAp) and HAp from eggshell (eHAp)prepared by wet chemical method and ball milling).It is visible that sHA had smaller size of constituentgranules than eHA. Low-magnification images (in-set in Figs. 1a and 1b) showed that eHAp hadsmaller particle size than sHAp. It might be due tothe weak aggregation tendency between the con-stituent granules in eHAp.

3.6. Properties of CaP coatingsprepared by electrochemicaldeposition (ECD)

The number of research work and patents is abun-dant in scientific databases since this techniqueoffers a cheap and versatile opportunity for prepar-ing bioceramic coatings on metallic implant materi-als. Benhayoune et al. [108] filed an invention on aprocess for the electrodeposition of calcium-phos-phorus coatings onto a rough metallic support. Theyapplied the electric current in pulsed mode betweenthe support that forms the cathode and a counterelectrode and the electrolyte comprised a solutionof Ca(NO

3)

2.4H

2O and of NH

4H

2PO

4. This coating

can be optionally doped with metallic ions such asstrontium. The strontium was introduced to replacepart of the calcium atom in the calcium phosphate

coating in proportions such that (Ca + Sr) / P =1.67. The pH value was adjusted to 4.4 by addingNaOH solution. They claimed that the depositedHAp had homogeneous microscopic structure andcrystallographic structure similar to that of the sto-ichiometric HAps The percent of crystallinity wasgreater than 90% or even 95%, and the coatingshad good mechanical strength and adhesion to thesupport. The crystal structure of HAp was hexago-nal and close-packed. The preparation was per-formed at near room temperature. They stated thatmetal dopant can be incorporated into the calciumphosphate biomaterials by adding precursors in theelectrolytic solution. The dopants can be selectedfrom zinc, magnesium, manganese, silicon, stron-tium, sodium, silver, titanium and fluorine, and thecorresponding precursors capable of releasing insolution e.g. Zn2+ ions , Mg2+, Mn2+, Si4+, Sr2+, Na +,

Ag+, Ti4+ and F– mixtures. Preferably, the dopantswere selected from Zn2+, Mg2+, Sr2+ and its mixtures.LeGeros et al. [26] described a method in theirpatent in which they prepared highly adherent coat-ing of a desired calcium phosphate phase on tita-nium-based substrates by electrochemical method.They found that the deposited layer had homogenousmorphology, composition and thickness. They alsoinvestigated the dissolution properties of CaP ac-cording to ASTM F1926 specifications (“Test methodfor evaluation of the environmental stability of cal-cium phosphate coating”). They found that the coat-ing composition (mainly the HAp/ACP ratio) signifi-cantly affects in vitro dissolution properties of thecoating: the lower the ratio, the more soluble thecoating. Gupta et al. [109] invented a method toprepare discrete region of CaP coatings onto medi-cal implant by electrochemical deposition. Theshapes of the deposited HAp crystals were mainlyneedle-like. Thanh et al. [110] investigated the ef-

Fig. 1. SEM measurements on (a) hydroxyapatite from eggshells (eHA) (b) synthetic hydroxyapatite (sHA).Magnification 50000X. Low magnification (1000X) images of eHAp (a) and sHAp (b) in the inset of Figure.


fect of electrolyte/precursor concentrations, the tem-perature and the H

2O

2 content on the morphology,

structure and composition of the coating. They foundthat H

2O

2 addition leads to smoother HAp coatings,

when intervening the reaction mechanism and elec-trolyte composition and its concentration signifi-cantly affect the deposition kinetics as well as theHAp coating thickness. Blackwood et al. [111] dis-covered that the cathodic deposition of CaP leadsto a mixture of poorly crystalline HAp and amor-phous calcium phosphates that were weakly ad-hered to the Ti substrate. They found that both thecrystallinity of the HAp and its fraction in the de-posited layer can be increased by post-treatmentwith NaOH and increased adhesion and crystallineHAp can be obtained by either pre-treating the Tisubstrate with NaOH followed by a heat treatmentat 600 °C or the addition of H

2O

2 to the electrolyte.

In addition, both the crystallinity of the HAp and itsadhesion to the Ti substrate increased at elevatedtemperatures. Rotating the substrate at low revolu-tions was also beneficial for adhesion to the sub-strate as the centrifugal forces both pump more Ca2+

and PO4

2- ions towards the substrate and removeshydrogen bubbles. But, at higher rotation rates OH-

ions were pushed away from the substrate so thatbulk precipitation was favoured over film formation.

It was also discussed in some research works[112-115] that the main phase of as-deposited CaPlayer is monetite which can be transformed to HApby either heat treatment at 900 °C or by immersingthe samples into 1M NaOH solution at 70 °C for 2hours.

The characteristic morphology and EDX elemen-tal analysis of as deposited Monetite phase are vis-ible in Fig. 2. It is visible that the Ca/P ratio in thiscase is 0.97. The morphology of hyrdoxyapatite af-ter phase transformation by alkali-treatment is vis-ible in Fig. 3 (SEM, TEM and EDX analysis). Theelemental analysis in this case revealed the Ca/Patomic ratio to be 1.74, which is closer to the el-emental ratio in hydroxyapatite (1.67).

The electrochemical deposition can also be per-formed by pulse current instead of direct current.This method provide coatings with even better char-acteristic with regard to mechanical and corrosionproperties. With pulse current, more dense, homo-geneous layers can be deposited with smaller grainsize compared with direct current deposition. A typi-cal current wave form can be seen in Fig. 4.

Gopi et al. [116] described a comparative studyon direct and pulse current deposition of hydroxya-patite. Their reseach revealed that the pulsed cur-rent more positively influences the crystallinity andadhesion of HAp films deposited on substrate thanthe one obtained from direct continuous currentmethod. They claimed that the off part of the cyclein pulsed electrodeposition method gives Ca2+ andPO

43- ions in the bulk solution sufficient time to dif-

fuse to the vicinity of the substrate maintaining morefavorable conditions for HAp deposition. Microscopicstudies of the as-deposited HAp coating by pulsedmethod demonstrated that the most compact uni-form layer was obtained at low current density withlonger off time. Therefore the coatings obtained bypulsed method were better adherent.

Blackwood et al. [117] have also deposited hy-droxyapatite onto titanium substrate and investi-gated the adhesion properties of coatings. Theyfound that pulsed deposition at low current densi-ties increased the thickness, crystallinity and ad-hesion of HAp coatings, making them more suit-able for biomedical applications than their DC coun-terparts. However, they also discovered that HApcoating deposited with high AC current density for ashort time had similar properties as the one coated

Fig. 2. SEM image of Monetite coating on titaniumalloy deposited by pulse current as well as the cor-responding EDX elemntal analysis.


Fig. 3. SEM (a) and TEM (b) images on HAp coating prepared by pulse current electrodeposition andsubsequent surface treatment in 1M NaOH solution as well as the corresponding EDX elemntal analysis.

with a low DC current density over a long time tothe same charge density.

The other possibility to manipulate the mechani-cal, corrosion and biological properties of calciumphosphate coatings is ion doping. It is well discussedthat the ion-modification can interfere with the hy-droxyapatite crystal lattice influencing its param-eters.

According to the thorough literature survey, themost common doping elements are silver and zinc.The role of silver incorporation is to provide antibac-terial properties while the zinc content can enhancethe biocompatible properties of coatings and canpromote wound healing process. In some researchworks [118-122] the XRD results showed phase

Fig. 4. Schematic pulse-current waveform.

shifting to higher 2q value due to the replacement oflarger sized Ca2+ (0.099 Å) ions with smaller sizedZn2+ ions (0.77 Å) or even with Mg2+ ions (0.69 Å)[120-127].

In other research work, Ziani et al. [121] foundbroadening of the reflections due to the reduction inthe crystallite size and increase in the lattice disor-der, which they attributed to the Mg2+ substitutionin the HAp lattice. On the other hand, the substititionof strontium and silver can cause phase shifting tolower 2 indicating an increase in the lattice param-eters, which can be attributed to the higher ionicradius of Sr (1.13 Å) and Ag (1.15 Å), as comparedto Ca2+ [122]. Moreover, the incorporation of Zn ionsinto CaP crystal structure can also result in a newphase, Parascholzite CaZn

2(PO

4)2.2(H

2O), formation

[123-128].It is visible in Fig. 5 that the ion-modified CaP

coating had mainly a mixture of non-uniform smallneedle-like particles in nanometer size and largerthin, laminated plate-like particles in micrometersize. Moreover, when silver and/or zinc particles wereincorporated into the layer, flake-like aggregateswere also formed. The Ca/P ratio in modified CaPlayer varied from 1.11 to 1.68 which confirmed thedifferent calcium phosphate phases present in thelayer.


Fig. 5. SEM-EDX investigation on AgCaP (a) on ZnCaP (b) and AgZnCap coated Ti6Al4V (c).

3.7. Properties of CaP coatingsprepared by electrophoreticdeposition (EPD)

Theoretically, in EPD deposition direct current (DC)or high voltage is applied to suspended powder par-ticles in a liquid medium to charge the particles anddeposit them onto a conductive substrate of oppo-site charge [67-75]. Rose et al. [68] in their patentdemonstrated that electrophoretic deposition (EPD)is capable of, at small length scales, being performedusing aqueous (water-based) solutions. In addition,EPD can be performed using a wide variety ofcharged nanoparticles, such as oxides, metals,polymers, semiconductors, diamond and so on.Rojaee et al. [129] prepared nanostructured HApcoatings onto magnesium alloy implants via elec-trophoretic deposition. The obtained nHAp coatingsconsisted of an outer course layer with the meanthicknesses of 87.31 ± 4.52 mm and the nHAp coat-ing showed an average roughness of 5.05 ± 0.72mm.

Zhong et al. [130] deposited biomimetic zincsubstituted hydroxyapatite coatings with chitosanand multiwalled carbon nanotubes onto titaniumsubstrate with electrophoretic method. In their re-search, they discovered that the use of silk fibroinas template and zinc as substitution led to changesin HAp crystal morphology and the silk fibroin actedas template for nucleation and growth of HAp. Theresultant HAp nanoparticles were rod-like and ZnHApnano-particles were in wrinkled sheet-like shape.Furthermore, the ZnHAp composite coating hadbetter corrosion resistance compared to HAp coat-ings.

3.8. Properties of CaP coatingsprepared by electrospraying (ES)

Various bioceramic coatings can be deposited byelectrospaying technique. Leeuwenburgh et al. [70]deposited thin calcium phosphate layers onto com-mercially pure cp-Ti substrates using electrostaticspray deposition. The results showed that the coat-


ing structure and morphology could be tailored bychoosing the appropriate combination of depositionparameters. Moreover, with this technique, varioussurface morphologies, ranging from dense to veryporous coatings, can be obtained. Particularly in-teresting was a unique reticular coating morphol-ogy characterized by a three-dimensionally inter-connected pore network. The X-ray diffraction (XRD)and Fourier transform infrared spectrometry (FTIR)analyses showed that crystalline carbonate apatitecoatings were formed after heat treatment of as-deposited ESD coatings. Huang et al. [72] preparednano-sized HAp coatings by electrospraying. In theirresearch work the nHA was synthesized by a pre-cipitation reaction between calcium hydroxide(Ca(OH)

2) and orthophosphoric acid (H

3PO

4) with a

Ca/P ratio of 1.67. The prepared nHA droplets werethen sprayed on glass substrate for morphologicalexamination. They claimed that the electrosprayingoffers the potential to create both micro- and nano-scale surface topography and appropriate coveragefor a favorable cell response. Eltohamy et al. [73]investigated the electrosprayed tricalcium phos-phate (TCP) spherical cups. The prepared particleswere of a few micrometers in size (average=2.74mm). They mixed sol–gel precursors, containingcalcium and phosphate (Ca/P=1.5) with polyvi-nylpyrrolidone in ethanol at varying concentrations,and then sprayed under a controlled electrostaticfield. Singh et al. [131] coated nano hydroxyapatite(n-HA) onto orthopaedic implant material. The syn-thesis of n-HA was performed using wet chemicalmethod and the n-HA was further coated on tita-nium alloy (Ti6Al4V), using the electrostatic spraydeposition. In they research they found that thesurface roughness of Ti alloy increased from 2.34mm to 2.77 mm after coating with n-HAp and corro-sion resistance improved drastically. XRD patternof n-HAp coated Ti alloyh, owever, showed broad-ening of HAp associated peak, reflecting changefrom crystalline to amorphous phase of n-HA.

Schouten et al. [132] prepared electrosprayedCaP coatings and investigated their mechanicalproperties. The prepared coatings showed a rough,porous network of densely packed nano-CaP par-ticles distributed over the entire implant surface.They also carried out Torque test to evaluate themechanical properties of coated implants. The re-sults showed that the mean torque-in values were32.1 N cm-1, and the mean torque-out values after 6weeks implantation were 42.8 N cm-1 for nano-CaP-coated implants.

4. BIOLOGICAL PERFORMANCE OFPURE AND ION-SUBSTITUTEDHYDROXYAPATITE COATINGS

For biomedical application, the developed HAp coat-ings must meet strict requirements. The criticalquality specifications for HAp coatings include thick-ness, phase composition, crystallinity, Ca/P ratio,microstructure, surface roughness, porosity, implanttype and surface texture, which influence the re-sulting mechanical properties of the implant, suchas cohesive and bond strength, tensile strength,shear strength, Young’s modulus, residual stressand fatigue life [133-141]. Changes to these vari-ables can produce coatings with varied bioactivityand durability. It has been suggested that a coatingfor orthopaedic implants should have low porosity,strong cohesive strength, good adhesion to the sub-strate, a high degree of crystallinity, and high chemi-cal and phase stability [142]. Generally, it can bestated that after implantation into the human body,the implant surface will immediately contact thephysiological fluid, which contains numerous ionsand proteins that guide the adhesion of particularcell types to the surface. There are numerous bio-logical fluids that can simulate the real biologicalconditions within the body. These solutions are SBF[143,144], Hanks’ solution [145,146], fetal bovineserum solution [147], buffering fluid [148], salinesolution [149, 150], phosphate-buffered saline [151],and fast calcification solution [152].

4.1. Biocompatibility investigations onpure HAp layers

Up to now, the mechanical strength of pure HApbioceramics obtained by various technologies is stilllower than that of natural bones. Since the mechani-cal strength of HAp ceramics is still too poor forapplication of implant materials in load bearing place,therefore HAp ceramics are often coated as a thinlayer on metal materials to increase thebiocompatibility and osteoconductivity of the dentaland orthopedic implants [153]. Furthermore, thechemistry and surface topography of HAp or othercalcium phosphate crystals deposited as thin filmon implants are known to accelerate early bone for-mation and increase the strength of the bond be-tween implant and bone.

Pugh et al. [17] invented a method to prepareartifical stabilized calcium phosphate phases withunique morphology capable of supporting bone cellactivity. The morphology of prepared coating had acharacteristic form of loosely interconnected globu-


lar structure resembling coral. The size of the gran-ules varied from approximately 0.5-1 mm in lateraldimension. The coating was porous in the directionperpendicular to the substrate with a larger planardensity closer to the surface than near to the sub-strate. This morphology allowed for the percolationof liquid. Hontsu et al. [29] examined the in vitroand in vivo biocompatibility of a thin hydroxyapatite(HAp) deposited by pulsed laser ablation. Thin HApfilm was deposited on titanium discs using pulsedlaser operating at a repletion rate of 10 Hz and an-nealed by heating at 380 °C for 1 h. They used actinstaining to test the bioactive properties of coating.The staining with rhodamine phalloidin revealed thatthe spread of clonal stromal cells was markedlygreater on HAp coating than on untreated titanium.When infused with clonal stromal cells, the HAp-coated titanium could indeed generate bone forma-tion in the backs of nude mice. However, the ap-pearance of new bone was not enhanced by theHAp coating, likely because air in the porous scaf-fold during cell loading reduces the cell-scaffoldcontact. Wu et al. [14] in their invention investigatedthe biocompatible properties of porous HAp coat-ing. They added ammonium carbonate, ammoniumbicarbonate, ethanol, hydrogen peroxide as poreforming agent into the CaP suspension, and themixture was extensively stirred to be used as thefeedstock materials for plasma spraying. The po-rosity of the porous hydroxyapatite prepared by thisinvention was 2%-50%, and the pore size was 0.1-200 microns. They discovered that its bioactivity wasbetter than the coatings prepared without the pore-forming agent, which can promote the proliferationand growth of the osteoblast cells. It is obvious sincethe porous hydroxyapatite coatings are beneficialto the delivery of oxygen and nutrients and the ex-cretion of metabolites. They also investigated theproliferation and growth rate of MG 63 cells by MTTassay. The results indicated that the coatings werebeneficial to the proliferation and growth of the cells,in addition to the lack of cytotoxicity. Balasundaramet al. [47,48] in their invention discovered that thenano-HAp coated nanotubular Ti surface promotesosteoblast cell adhesion and the coatings were par-ticularly suitable for orthopedic and dental implantswhere deposition of osteoblasts and other proteinsis important in bone formation. The HAp coating wasstrongly adhered to the Ti surface. Anchorage-de-pendent cells, including osteoblasts, exhibited en-hanced adhesion to the nanoparticulate HAp com-pared to microparticulate HAp surfaces, thus effec-tively promoting accumulation of calcium-contain-

ing minerals required for new bone formation fromthe extracellular matrix. The described nano HApcoated nanotubular titanium surfaces promoted thecell adhesion to a greater extent than to nanotubulartitanium surfaces without the HAp coating. The in-ventors claimed that the greater density and adher-ence of osteoblast cells to the nano-particulate HApsurfaces provided significant advantage over currentlyused coatings in orthopaedic implants.

Knabe et al. [154] investigated the biocompatibleproperties of titanium and hydroxyapatite dentalimplant surfaces using a rat bone marrow stromalcell culture system. They found that after 14-dayincubation, multiple RBM cell layers and elabora-tion of a multilayered extracellular matrix (ECM)occurred on all of the test substrates, however, onthe Ti-HAp specimens, cell layers and the extracel-lular matrix seemed to be less dense and stratifiedwith fewer fibrillar components. Sewing et al. [24]filed a patent on method of electrodepositing cal-cium phosphate layers onto Ti6Al4V alloy by meansof galvanostatic polarization using cathodic currentflow at –10 mA/cm3. The as-deposited layer con-sisted of amorphous calcium phosphate(Ca

9H(PO

4)

6.nH

2O), hydroxyapatite (Ca

10(PO

4)

6

(OH)2), octacalcium phosphate (Ca

8H

2(PO

4)6.5H

2O),

brushite (CaHPO4·2H

2O). After 30 minutes of depo-

sition they immersed the samples in a collagensolution for 10 minutes and subsequent partial min-eralization of the collagen was carried out undercathodic current flow at –10 mA/cm3 for 15 min-utes. The deposited coating was whitish and thethickness was 0.04-150 mm. They investigated thecell proliferation of MC3T3 mouse osteoblasts onsamples using WST-1 tests. The developed coat-ings showed increased cell adherence and cell pro-liferation began after shorter time. Thian et al. [155]studied the biocompatibility of electrosprayed HApcoatings. To evaluate the bioactive capacity of thesenanoHAp (nHA) coatings in vitro, an acellular simu-lated body fluid soaking experiment and a humanosteoblast (HOB) cell culture work were conducted.Under these physiological conditions, more accel-erated apatite precipitation process occurred on thenHA-coated titanium surfaces as compared to theuncoated titanium surfaces. HOB cells developedmature cytoskeletons with distinct evidence of ac-tin stress fibres and vinculin adhesion plaques onthese nHA coatings. Leeuwenburgh et al. [156] in-vestigated the in vitro and in vivo reactivity of po-rous, electrosprayed calcium phosphate coatings.They found that all apatitic electrosprayed coatingsinduced the formation of homogeneous and adher-


ent CaP precipitation layers. Amorphous CaP dis-played a delayed precipitation of poorly adherentCaP layers, whereas heterogeneous calcificationwas observed on top of b-TCP-coated substrates,indicating that b-TCP and amorphous CaP coatingsexhibit a poor ability of inducing calcification in SBFas compared to crystalline apatitic coatings. The invivo tests showed no adverse tissue reactions (toxiceffects/inflammatory cells) according to light micros-copy investigations, and all coatings became sur-rounded by a dense, fibrous tissue capsule afterimplantation. All coatings degraded gradually at adissolution rate depending on the chemical phase,thereby enabling synthesis of CaP coatings with atailored degradation rate. Jonge et al. [157] devel-oped a coating to mimic the biphasicbiomineralization process, where both the enzymealkaline phosphatase (ALP) and calcium phosphate(CaP) were coated onto Ti discs. In this case, theywere able to trigger enzymatically and physico-chemically controlled biomineralization pathways.In vitro soaking studies in cell culture medium re-vealed that crystal growth initially proceeded at afaster rate on CaP-coated Ti than on ALP-contain-ing coatings, but mineral deposition onto ALP-coatedTi caught up with the calcification behaviour of CaPcoatings upon long-term soaking. Cell culture ex-periments with osteoblast-like cells, however, dem-onstrated the opposite effect in mineral depositionon the electrosprayed CaP and ALP coatings. TheALP–CaP composite coatings showed delayed pro-liferation as well as accelerated mineralization incomparison to cells cultured on the CaP-coated anduncoated Ti. In conclusion, these in vitro resultsshowed that the osteogenic potential of Ti can bestimulated by ALP-containing coatings. This researchgroup also investigated the osteogenic effect ofelectrosprayed nanoscale collagen/calcium phos-phate coatings in their other manuscript [158]. Theycharacterized the mechanical and biological coat-ing properties using tape tests (ASTM D-3359) andin vitro cell culture experiments. The results revealedthat co-deposition of collagen significantly improvedcoating adhesive and cohesive strength, resultingin a remarkably high coating retention of up to 97%for coating thicknesses below 100 nm. In vitro cellculture experiments showed that electrosprayedCaP and coll-CaP composite coatings enhancedosteoblast differentiation, leading to improved min-eral deposition [158].

In addition, some clinical case study results onHAp coated implants can also be found in the sci-entific literature. Aebli et al. [159] carried out a his-

tological study of a proximally hydroxyapatite (HAp)-coated femoral component, retrieved after 9.5 yearsof good function. They found that the HAp coatingbecame completely degraded. Bone was in directcontact with the titanium surface in all the areaswhich had been coated, with no interposing fibroustissue. There were no signs of particles, third-bodywear, adverse tissue reactions or osteolysis. Boneremodelling was evident in this case by the pres-ence of resorption lacunae; tetracycline labellingshowed bone laid down six years after implanta-tion. They also discovered that the loss of the HAp-coating had no negative effect on the osseo-inte-gration of the stem. They concluded that the HApcoating contributed to the fixation of the implant andits degradation did not adversely affect the long termfixation. Delaunay et al. [160] investigated the ef-fect of hydroxyapatite coating on the radio-clinicalresults of a grit-blasted titanium alloy femoral taper.This work was a case-control study of 198cementless primary total hip arthroplasty with theAlloclassicTM system. They found that long-termcementless press-fit (“flat wedge-shaped”) fixationwas reliable and HAp coating improved the radio-logical results of the proximal bone-prosthesis in-terface. Results were excellent or good in 184 hips(95%) with no significant difference between the 2groups (without HAp compared to with HAp coated;p = 0.59) and radiographic signs of stableosseointegration were observed in 173 hips (90% ofthe cases). Nodzo et al. [161] investigated the shortterm effect of HAp coated metal backed patellas. Intheir work, they evaluated the records and radio-graphs of 101 knees with a hydroxyapatite coatedmetal backed patella (HAp) and 50 knees with acemented polyethylene patella (CP) with minimumtwo year clinical follow up. They discovered thatpatients in both the HAp and CP groups had similarclinical outcomes at final follow-up. Forty-five per-cent of patients in the HAp group had 1–2 mm ar-eas of decreased trabecular bone density aroundthe pegs, which were not observed in the CP group,and may represent stress shielding. Thisuncemented HAp component had satisfactory earlyclinical outcomes, but long-term follow up is neces-sary to determine the durability of this implant. Satoet al. [162] compared the survival of cups in totalhip arthroplasty with and without HAp coating dur-ing a minimum follow-up of 18 years and found nosignificant difference in cup survival rates betweenthe control and HAp coated groups based on theirexperiments. In their work, 163 patients were ana-lyzed, including 73 cups with HAp coating (HAp+


group) and 110 without HAp coating (HAp- group);otherwise, the cups had identical titanium-sprayedrough surfaces and were fixed with screws. The re-sults suggested that HAp coating did not have ei-ther beneficial or adverse effects on the long-termcup survival in primary cementless total hip arthro-plasty.

4.2. Biocompatibility investigations onion-modified HAp layers

It is widely discussed that the biocompatibility and/or antimicrobial properties of HAp coatings can beenhanced even more by doping elements or com-ponents. The doping elements can be cationic, suchas Ag+ [15,122,126-128,163-170], Zn2+ [34,35,119,123-129,171-175], Cu2+ [186], Mg2+ [34,40,98,118,120,121], Sr2+ [8,9,40,122], Si4+ [37,96], Fe2+ [102]and anionic such as fluorine [120,196], chlorine [196]and carbonate [194, 195] ions.

4.2.1. Substitution with cations

There are numerous research work on doping theHAp coating with silver ions to provide the layerantimicrobial properties. The silver express its anti-microbial properties in cationic form by forming astrong bond with electron donor groups in biologicalmolecules. Kose et al. [163] prepared silver iondoped calcium phosphate-based ceramicnanopowder-coated prosthesis and found that theyincreased the infection resistance in New ZealandWhite rabbit modells. Lu et al. [164] prepared nano-Ag-loaded hydroxyapatite coatings on titanium sur-faces by electrochemical deposition. They performedantibacterial and cell culture tests to evaluate theantibacterial properties and biocompatibility of HAp/Ag composite coatings. The results indicated thatthe as-prepared coatings had good antibacterial prop-erties and biocompatibility. However, an appropri-ate silver content should be chosen to balance thebiocompatibility and antibacterial properties. Thesilver-loaded HAp had shown antibacterial effect, andalso good osteoconduction. The biocompatibilitytests were carried out by Alamar Blue assay onosteoblast cells grown on different samples. Theyfound that pure HAp coatings had the highest prolif-eration rate, followed by 0.5 HAp/Ag and 1 HAp/Agafter 3 days of culture. Erakovic et al. [165] pre-pared silver doped hydroxyapatite thin films bypulsed laser deposition. They tested the cytotoxicactivity with HEp2 cells against controls. The anti-fungal efficiency of the deposited layers was testedagainst the Candida albicans and Aspergillus niger

strains. Their studies on cytotoxicity showed thatthe tested biomaterials did not influence the cellu-lar adhesion, viability, morphology and proliferationrate. Their development onto the tested materialsurfaces was similar to the glass control.

Song et al. [166] developed three kinds of anti-bacterial ingredients loaded hydroxyapatite (HAp)coating: antibiotic (ampicillin sodium salt), silver ionsand water soluble chitosan. They prepared two dif-ferent kinds of HAp coatings with dense and porousstructures by the air plasma spraying (APS) andliquid precursor plasma spraying (LPPS) processes.They found that the antibiotic loaded HAp coatingshad the best antibacterial activity, while the silverion loaded HAp coatings showed the worst bacte-rial inhibitory effects among the three kinds of anti-bacterial materials. They found that the HAp coat-ing with porous structure had increased loading ofantibacterial materials and enhanced antibacterialproperties. Such observations suggested that bothantibacterial material and coating structure havestrong influences on the antibacterial properties ofthe HAp coatings. Gan et al. [15] described amethod for incorporating a silver derivative into HApin their invention. They used WST-1 reagent to quan-tify the metabolic activity of the MC3T3-E1 cellsgrown on different samples and exposed to the con-ditioned media. Their results showed that the1%AgHAp and 2%AgHAp groups increased themetabolic activity of cells compared to the positivecytotoxic control group (PVC), and the negativecontrol group (HDPE). The metabolic activity of cellsexposed to pure HAp was however higher than ei-ther of the two Ag containing groups. The metabolicactivity of cells exposed to the Ti6A14V group wasbelow that of all HAp containing groups and thenegative control HDPE. However, Ti6Al4V stimulatedthe mineralization of cells to a level that was twicethat of the positive control.

The other most commonly applied doping ele-ment is zinc. It is well known that the zinc is animportant mineral in the normal growth and devel-opment of the skeletal system and its deficiency isassociated with a decrease in bone density [171].Moreover, Zn inhibits osteoclast differentiation, andpromotes osteoblast activity thus promoting boneformation [172]. Li et al. [173] studied the osteo-clast responses of tricalcium phosphate and zinc-containing tricalcium phosphate in culture mediumsupplemented with zinc. They found that the cul-ture medium supplemented with zinc within therange between 0.3 and 6.8 ppm induced an increasein osteoclast apoptosis and a decrease in actin ring


formation. ZnTCP sample releasing comparable zincion inside the resorption lacuna did not influenceosteoclast apoptosis and actin ring formation. Otherresearchers found that ZnTCP ceramic with 0.5 and1.0 mol.% zinc suppressed resorption by matureosteoclasts in vitro compared with TCP [174]. Ortizet al. [175] studied the in vitro performance of zincapatite coatings on titanium surfaces. The percent-age of zinc incorporated in the coatings was 7 at%in this case and the Ca/P ratio was 1.61 for thepure and Zn substituted apatite, suggesting that Znwas incorporated substitutionally, replacing Ca at-oms in the HAp structure. They found that the in-corporation of Zn in the HAp structure changed thecrystal morphology, reduced crystal size and de-creased the deposition rate showing that zinc is aninhibitor of the growth of HAp crystal. They performedscratch tests to evaluate the coating adhesionstrength and no significant difference was foundbetween the two tested groups, indicating the goodadhesion of ZnHAp to Ti substrates. The in vitroresponse of human osteoblasts (HOB) exposed tothe surface of HAp and ZnHAp coatings were alsostudied. The results showed that ZnHAp sampleshad better adhered and spread cells compared withHAp. ZnHAp coatings presented cells with elongatedor polygonal shapes and clearly more spreaded thanHAp. The quantitative analysis also showed thatthere was a significantly higher number of cells ad-hered to ZnHAp coatings compared to HAp, indi-cating the zinc in corporation stimulates osteoblastproliferation.

There are some research works on preparing andcharacterizing strontium substituted calcium phos-phate coatings. The role of strontium in these sub-stituted coating is to decrease bone resorption byinhibition of osteoclast-resorbing activity and osteo-clastic differentiation. In addition, it promotes boneformation by enhancing pre-osteoblastic cell repli-cation and osteoblastic differentiation. There are alsosome positive results obtained in clinical studieson long-term Sr treatment [176]. Li et al. [177] in-vestigated the effect of strontium-substituted hy-droxyapatite coating on implant fixation in ovariec-tomized rats. In their study, they prepared stron-tium-substituted hydroxyapatite coatings with 10mol.% Ca2+ replaced by Sr2+ (10% SrHAp) usingsol-gel dip methods. The results showed that the10% SrHA coated implants improved theosseointegration compared to HAp, with the bonearea ratio and bone-to-implant contact increased by70.9% and 49.9% in histomorphometry, the bonevolume ratio and percent osseointegration by 73.7%

and 45.2% in micro-CT evaluation, and the maxi-mal push-out force and ultimate shear strength by107.2% and 132.9% in push out test. These resultsdemonstrated that 10% SrHAp coatings could en-hance implant osseointegration in OVX rats, andsuggested the feasibility of using SrHAp coatingsto improve implant fixation in osteoporotic bone.Chung et al. [178] prepared Sr-HAp coatings ontotitanium by micro-arc treatment (MAT). They claimedthat this method can replace calcium (Ca) with dif-ferent percentages of strontium (Sr). They also in-vestigated how Sr content affects the microstruc-ture of and osteoblast/osteoclast growth on the coat-ings. The experimental results indicated that an in-crease in the Sr content in the electrolyte bath re-sulted in a greater degree of Sr substitution at Casites within the HAp phase, facilitating the forma-tion of Sr-HAp coatings with Sr fully solid soluble inthe HAp phase. Irrespective of the Sr content, mostcoatings were similar in porous morphology and poresize. Additionally, the Sr-HAp coating showed higherosteoblast compatibility than raw titanium metal andthe HAp coating. Moreover, cell adhesion and prolif-eration after 48 h was greater than that after 4 h,indicating that Sr can stimulate osteoblast adhe-sion and proliferation. Further, Sr significantly inhib-ited osteoclast differentiation when the Sr-HAp coat-ings exceeded 38.9 at.% Sr. Boyd et al. [179] pre-pared strontium-substituted hydroxyapatite coatingsby co-deposition sputter technique. They revealedthat Sr could be successfully incorporated into theHAp lattice to form SrHAp coatings. It was observedthat as the number of SrHAp sputtering targets wereincreased (increasing Sr content), the depositionrate decreased. Malshe et al. [13] filed a patent onpreparing coating that contained HAp and ZnO par-ticles and investigated their biocompatible and anti-microbial properties. They claimed that ZnOnanoparticles can be incorporated in the HApnanoparticles to form a composite coating mate-rial. The role of ZnO was to provide infection resis-tance due to its inherent antimicrobial properties.They discovered that HAp-ZnO nanocomposite couldalso promote cell organization through contact guid-ance, topology, and its unique cell interactions.

There are research works on preparing and char-acterization of silicon substituted HAp coatings. Itcan be stated that silicon is an essential trace ele-ment for bone development. It was reported that sili-con was only located in the active areas of youngbone and was involved in the early stages of bonecalcification [180,181]. A relative high concentrationof silicon is present in metabolically active osteo-


blasts and is considered to be essential in the for-mation of extracellular matrix in bone and cartilage[182]. Qiu et al. [183] electrodeposited novelbioactive silicon doped calcium phosphate coatingon AZ31 magnesium alloy. They prepared dual-layerstructure with a porous lamellar-like and an outer,block-like apatite layer. In vitro immersion tests werecarried out in simulated body fluid within 28 days ofimmersion. In addition, further formation of an apa-tite-like layer on the surface after immersion provedbetter integrity and biomineralization performanceof the coating. Biological characterization was car-ried out for viability, proliferation and differentiationof MG63 osteoblast-like cells. The coatings showedgood cell growth and an enhanced cell proliferation.Moreover, an increased activity of osteogenic markerALP was found. All the results demonstrated thatthe Si-doped calcium phosphate can enhance thebioactivity, which would facilitate the rapidity of bonetissue repair.

The magnesium is also an important trace ele-ment in cell functions at biological, chemical andmolecular levels. It plays a key role in bone me-tabolism, since it influences osteoblast and osteo-clast activity, and thereby bone growth [184].Kheradmandfard et al. [185] studied the effect ofMg content on the bioactivity and biocompatibilityof Mg-substituted fluorapatite nano-powders pre-pared by mechanical activation. They found that bio-activity depended on the amount of Mg substitutionand cell culture suggested that the addition of Mg2+

had no adverse effect and Mg-apatite samples hadgood biocompatibility.

The HAp coatings can be modified by more thanone cation by co-deposition. There are researchworks on electrodeposited silver and zinc co-dopedhydroxyapatite powders and coatings [126-128].Iqbal et al. [126] prepared Zn–Ag–HAp nanoparticlesby using microwave-assisted wet precipitation pro-cess. In their research work, the antibacterial testsof the nanoparticles revealed their antibacterial ac-tivity against Staphylococcus aureus and Escheri-chia coli. By using simulated body fluid (SBF), anapatite layer formation was observed in 28 days. Invitro cell adhesion assay confirmed the cell attach-ment of normal human osteoblast (NHOst) cells tothe disc surface. MTT [(3(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] assay indicatedthat the cells were viable, and the cells proliferatedfaster on the disks than on the control surface dueto the presence of metal ions. Huang et al. [186]electrodeposited strontium/copper substituted hy-droxyapatite (SrCuHA) coating onto commercially

pure titanium (CP-Ti). They studied the antibacte-rial and in vitro cytocompatible properties of coat-ings. Cu was incorporated into HAp in order to im-prove its antimicrobial properties while the Sr wasadded as a second binary element to improve thebiocompatibility. They found that the presence ofSr2+ and Cu2+ in solution led to reduced roughnessof the coating and that finer nucleus size was formed.The results highlighted that Sr2+ and Cu2+ werehomogenously incorporated into HAp lattice to formSrCuHAp coatings. The antimicrobial test resultsrevealed that SrCuHAp coatings can kill Escheri-chia coli to a certain extent during the first few days,which might be due to the Cu substitution in thecoating. An enhancement of in vitro osteoblast ad-hesion, proliferation and alkaline phosphatase ac-tivity was also observed. There are other discus-sions on the role of Cu ions that prove its vital parteither in osteogenesis and/or in angiogenesis. Cop-per is an essential trace element with its highestabundance in the liver tissue is known for its stimu-latory effect on endothelial cells towards angiogen-esis. Moreover, Cu functions as a cofactor and isan important component for structural and catalyticproperties of many enzymes. Although copper canexist in its oxidized cupric (Cu2+) or reduced cu-prous (Cu+) state, the bivalent Cu2+ has attractedmuch attention for its role in angiogenic processes[187].

Gopi et al. [188] developed a bilayer coating byelectropolymerisation of poly(3,4-ethylenedioxythiophene) (PEDOT) on surgical gradestainless steel (316L SS) and subsequent elec-trodeposition of strontium (Sr) and magnesium (Mg)substituted porous hydroxyapatite (SrMg-HAp). Theresults showed that the PEDOT/Sr,MgHAp bilayerexhibited greater adhesion strength than the SrMg-HAp coated 316L SS. In vitro cell adhesion test ofthe SrMg-HAp coating on PEDOT coated specimenwas found to be more bioactive compared to that ofthe single substituted hydroxyapatite (Sr or Mg-HA)on the PEDOT coated 316L SS. As a conclusion,the PEDOT/SrMg-HAp bilayer coated 316L SS canserve as a prospective implant material for biomedi-cal applications. This research group in other work[189] deposited minerals (Sr, Mg, and Zn) substi-tuted HAp by pulse current. They also investigatedthe biological performance of the prepared coatings.The in vitro cell adhesion test results revealed thatthe M-HAP coating was appropriate for the forma-tion of new cell growth which proved the enhancedbiocompatible properties of the coatings. Yuan etal. [190] filed a patent on a method providing the


calcium phosphate material with an effective amountof trace elements. Trace elements used in this in-vention are selected from the group consisting of F,Si, Cu, Li, Al, Mg, Mn, Zn, Ge, Sr and their combi-nations. The prepared calcium phosphate particleshad a diameter in the range of 200-300 mm. Suchprepared calcium phosphate material was capableof release of the trace elements comprised in it.The inventors found that the osteoinductivity of cal-cium phosphate materials could be considerablyimproved by providing the calcium phosphate mate-rial with an effective amount of trace elements, whichtrace elements are then released over time from thematerial. The results also showed that these coat-ings enhanced the bone formation, stimulated pro-liferation and differentiation of collagen-synthesiz-ing cells.

4.2.2. Substitution with anions

It is well discussed that the hydroxyapatite crystalcan also be modified by anionic substitutions. Thebiologically most relevant anionic substitutions in-volve F-; Cl-, CO

32-, SO

42-, for the OH- group. The

fluorinated HAp increased stability and crystallinityof materials. The partially F-substituted HAp haslower solubility compared to HAp or fluoroapatiteitself [191]. CO

32- can inhibit HAp crystal growth,

and the incorporation may result in poorly crystal-line structures with increased solubility [192]. Theanionic modification can be used to perpare apa-tites with tunable solubility. In addition, a biomimeticHAp should also contain significant amount of CO

32-

ions. On this basis, enormous efforts have beenmade to synthesize biomimetic nanocrystallineapatite containing carbonate as raw material for themanufacture of biomaterials [193]. These anionicsubstituted apatites can be prepared by wet chemi-cal methods [191-196] or via electrochemical depo-sition [64] and are mainly used in tissue engineer-ing. Zhou et al. [194] prepared carbonated hydroxya-patite (CHAp) nanospheres through nanoemulsion.The synthesis was performed by mixing an acetonesolution of Ca(NO

3)

2.4H

2O with an aqueous solution

of (NH4)2HPO

4 and NH

4HCO

3. Four reaction tempera-

tures, namely, 4, 25, 37, and 55 °C, were investi-gated and no surfactant was added in allnanoemulsion processes. Wet slurries of CHAp fromthe nanoemulsions were then freeze-dried to obtaindry powders. Xiao et al. [195] developed hollow HApmicrospheres with controllable amounts of carbon-ate substitution (0–12 wt.%) using a novel glassconversion route and evaluated the powder in vitroand in vivo. Hollow HAp microspheres with ~12 wt.%

of carbonate (CHAp) showed higher surface areathan conventional hollow HAp microspheres andfaster degradation rate in a potassium acetate buffersolution. They found that after implantation for 12weeks in rat calvarial defects, the CHAp and HApmicrospheres showed a limited capacity to regen-erate bone but the CHAp microspheres resorbedfaster than the HA microspheres.

Wang et al. [64] deposited fluoridated hydroxya-patite (FHAp) coatings on titanium substrates us-ing an electrochemical technique. In their work, dif-ferent concentrations of F- ions were incorporatedinto the apatite structure by adding NaF into theelectrolyte. Typical apatite structures were obtainedfor all the coatings after electrodeposition and sub-sequent post-treatment, including alkaline immer-sion and vacuum calcination. The coatings wereuniform and dense, with a thickness of 5 m. Theyfound that when the F- concentration was higher than0.012 M in the electrolyte, a saturation of fluor intothe coating occurred and the F/Ca ratio in the coat-ings became almost constant (F/Ca ratio = 0.125).They observed that the FHA coatings had higherbonding strength and lower dissolution rate than HApcoating, particularly for those with a fluoridation levelof 0.5–0.625. This coating also exhibited higher bio-logical affinity like cell proliferation and alkaline phos-phatase activity. For clinical application, it is sug-gested that a moderate content of fluor, such asCa

5(PO

4)

3(OH)

0.375-0.5F

0.5-0.625, is most suitable as a

compromise among cell attachment, cell prolifera-tion, apatite deposition and dissolution resistance.

Fahami et al. [196] prepared chlorine and fluo-rine substituted hydroxyapatite and investigated theircharacteristics and bioactivity properties. Theymeasured the zeta potential of the prepared sus-pension. The obtained zeta potential values werenegative which was described to favorosseointegration, apatite nucleation, and bone re-generation. The bioactivity of samples was investi-gated on sintered pellets soaked in simulated bodyfluid (SBF) solution and apatite crystals formed onthe surface of the pellets after being incubated for14 days.

5. USE OF PURE AND ION-SUBSTITUTED HYDROXYAPATITECOATINGS IN BIOMEDICAL FIELDS

Nano-hydroxyapatite bioceramics have a wide vari-ety of applications. The most important ones areapplications in bone tissue engineering scaffolds[107,197-200,216] in bone void f illers[105,106,201,202], in orthopaedic and dental implant


coatings [5-18], and in drug and gene delivery sys-tems [19,73,93,198,203-206]. Moreover, HAp canalso be used as a compound to mimic thebiomineralization processes [205-208]. Lee et al.[105] performed a comparative study on synthetic(sHAp) and eggshell-derived hydroxyapatite (eHAp)as a material for bone regeneration. They investi-gated the regenerative ability of bone using the twotypes of HAp in a rabbit calvarial defect model. Theydiscovered that both types of HAp showed higherbone formation than the unfilled control. However,eHAp had significantly higher bone formation thanthe unfilled control at 8 weeks after operation.

Concerning current clinical applications of HApcoatings, the plasma spraying technology is themost widely used over the last twenty years, withseveral companies (APS Materials Inc, Inc.,Fluidinova, SA, Premier biomaterials Ltd, HiMed,Surface Dynamics) manufacturing commersiallyavailable HAp coated implants. For application inorthopaedic surgery, coating degradation is one ofthe major concerns because of the potential latefailure of the implant caused by the HAp particles’migration into the joint space. So far, abundant num-ber of researches and clinical case studies havebeen performed on HAp coated implants [159-162,210-214]. The results of these clinical trials arecontroversary. While Aebli [159], Delaunay [160],D’Antonio [210], McNally [211] and Furlong [212]reported positive effect of HAp coatings on osteo-genesis induced by the coating leading to perma-nent and better fixation of the implant over non-coated implants within a few days to weeks. Evenso, there are some cases where clinical studiesreported no additional benefits of HAp coating[213,214]. However, in these cases, thick HAp coat-ings (~155 mm) were used, which might be the rea-son of the lack of positive effect, owing to poorermechanical properties leading to coating delamina-tion. Despite the comprehensive investigation of pureHAp coatings, there are a very few example of clini-cal applications of ionic modified or particles dopedHAp coatings in the available scientific literature.One clinical study was performed by Eto et al. [215]investigating the performance of silver oxide con-taining hydroxyapatite (Ag-HAp) coating preparedby thermal spraying. They evaluated the potentialissues of total hip arthroplasty (THA) with an Ag-HAp coated implant since prosthetic joint infectionis a serious complication of implant therapy. Theimplant contained silver at a maximum quantity of2.9 mg / implant. The results showed that the Ag-HAp coated implants markedly improved patients’

activities of daily living without causing any adversereactions attributable to silver in the human body.Ag-HAp is expected to reduce postoperative infec-tions and prevent decreased quality of life in pa-tients undergoing prosthetic arthroplasty, thus lead-ing to more favorable outcomes.

6. CONCLUSION & FUTUREDIRECTIONS

The aim of this comprehensive literature survey wasto provide a widespread overview of state of the artin the area of preparing and developing biomedicalimplant coatings.

The available research studies and clinical trialshighlighted that preparing and manufacturing of pureHAp coatings are well elaborated and it is a work-ing technology. However, there is still high demandto develop ionic doped HAp coatings onto implantmaterials for clinical application since applying dop-ing elements into the HAp crystals can improve thebiocompatibility of implants and can mimic thenanostructure of bone matrix even more. It is obvi-ous that there are numerous parameters that canaffect the performance of HAp coatings such as pre-paring methods, preparing parameters, quantity andquality of doping elements. It means that there arestill need to perform thorough research to optimizethe above mentioned parameters to enhance thebiocompatibility performance of HAp coatings anddecrease the number of post-operative failures, in-fections in orthopaedic surgery.

REFERENCES

[1] J.R. Davis, In: Handbook of Materials forMedical devices, ed. by I.R. Davis (ASMInternational (2003) ISBN: 0-87170-790-X).

[2] S. Shrivastava, In: Medical Device Materials,Proceedings of the Materials & Processes forMedical Devices Conference, ed. byShrivastava (ASM International (2003) ISBN:978-0-87170-798-7).

[3] C. Fleck and D. Eifler // Intl. J. Fatigue 32(2010) 929.

[4] Y.S. Han and S.H. Hong // Mater. Sci. Eng.A 266 (1999) 276.

[5] P. Habibovic, F. Barrčre, C.A.V Blitterswijk,K.D. Groot and P. Layrolle // J. Am. Ceram.Soc. 83 (2002) 517.

[6] S. Koutsopoulos // J. Biomed. Mater. Res. 62(2002) 600.


[7] L. Xia, K. Lin, X. Jiang, Y. Xu, M. Zhang,J. Chang et al. // J. Mater. Chem. B 1 (2013)5403

[8] K. Lin, L. Xia, H. Li, X. Jiang, H. Pan, Y. Xu etal. // Biomaterials 34 (2013) 10028.

[9] Y. Shen, J. Liu, K. Lin and W. Zhang // Mater.Lett. 70 (2012) 76.

[10] K. Lin, L. Chen and J. Chang // Int. J. Appl.Ceram. Technol. 9 (2012) 479.

[11] M. Sadat-Shojai, M.T. Khorasani,E. Dinpanah-Khoshdargi and A. Jamshidi //Acta Biomater. 9 (2013) 7591.

[12] K.A. Gross and S. Saber-Samandari // Surf.Coat. Technol. 203 (2009) 2995.

[13] A.P. Malshe and W. Jiang, Nanostructuredhydroxyapatite coating for dental andorthopedic implants (Patent:WO2011022642A1, 2011).

[14] F. Wu, Y. Huang, L. Song et al. Method forpreparing porous hydroxyapatite coatings bysuspension plasma spraying (Patent:US8,877,283 B2, 2014).

[15] L. Gan, M.L. Scott and S.C. Jani, Coatingand coating method (Patent:WO 2009111307A2, 2009)

[16] B. Ben-Nissan and A.H. Choi //Nanomedicine 1 (2006) 311.

[17] S.M. Pugh, T.J.N. Smith, M. Sayer andD. Langstaff, Artificial stabilized compositionof calcium phosphate phases particularlyadapted for supporting bone cell activity(Patent:US 2007/0184035A1, 2007).

[18] T. Troczynski and D.M. Liu, Sol-gel calciumphosphate ceramic coatings and method ofmaking same (Patent:US 6,426,114 B1,2002).

[19] T. Troczynsky, D.M. Liu and Y. Quanzu,Process for manufacture of biofunctionalhydroxyapatite coatings and microspheres forin situ drug encapsulation (Patent:EP 1 383481 B1, 2004).

[20] L. Gan, M.L. Scott and S.C. Jani, Gradientcoating for biomedical applications(Patent:US 9011965 B2, 2015).

[21] K.L. Choy // Prog. Mater. Sci. 48 (2003) 57.[22] S.P. Krumdieck, B.P. Reyngoud, A.D.

Barnett, D.J. Clearwater, R.M. Hartshorn,C.M. Bishop, B.P. Redwood and E.L. Palmer// Chem. Vap. Deposition 16 (2010) 55.

[23] Y. Gao and A.A. Campbell, Method of coatinga substrate with a calcium phosphatecompound (Patent:US006113993A, 2000).

[24] A. Sewing, M. Dard, S. Rossler,D. Scharnweber and H. Worch, Coating formetallic implant materials (Patent:US 2002/0018798 A1, 2002).

[25] M. Shirkanzadeh, Method for depositingbioactive coatings on conductive substrates(Patent:US005205912A, 1993).

[26] R.Z. LeGeros, J.P. LeGeros and S. Lin,Methods for producing adherent coatings ofcalcium phosphate phases on titanium andtitanium alloy substrates by electrochemicaldeposition (Patent:US 2005/0000819 A1,2005).

[27] Y. Hashimoto, M. Kusunoki, R. Hatanaka,K. Hamano, H. Nishikawa, Y. Hosoi,S. Hontsu and M. Nakamura // Adv. Sci.Technol. 49 (2006) 282.

[28] Y. Yang, S.R. Paital and N.B. Dahotre //J. Mater. Sci. Mater. Med. 21 (2010) 2511.

[29] S. Hontsu, Y. Hashimoto, Y. Yoshikawa,M. Kusunoki, H. Nishikawa and A. Ametani //J. Hard Tissue Biol. 21 (2012) 181.

[30] L. Sun, C.C. Berndt, K.A. Gross andA. Kucuk // J. Biomed. Mater. Res. 58 (2001)570.

[31] J. Shepherd, D. Shepherd and S. Best //J. Mater. Sci. Mater. Med. 23 (2012) 2335.

[32] C. Tardei, F. Grigore, I. Pasuk and S. Stoleriu// J. Optoelectron. Adv. Mater. 8(2) (2006)568.

[33] S. Kannan, A.F. Lemos, J.H.G. Rocha andJ.M.F. Ferreira // J. Am. Ceram. Soc. 89(9)(2006) 2757.

[34] S.J. Kalita and H.A. Bhatt // Mater. Sci. Eng.C 27(4) (2007) 837.

[35] A. Ito, K. Senda, Y. Sogo, A. Oyane,A. Yamazaki and R.Z. LeGeros // Biomed.Mater. 1(3) (2006) 134.

[36] L.J. Jha, S.M. Best, J.C. Knowles,I. Rehman, J.D. Santos and W. Bonfield //J. Mater. Sci. Mater. Med. 8(4) (1997) 185.

[37] I.R. Gibson, S.M. Best and W Bonfield //J. Biomed. Mater. Res. 44(4) (1999) 422.

[38] D.G.A. Nelson, J.D.B. Featherstone, J.F.Duncan and T.W. Cutress // Caries. Res.17(3) (1983) 200.

[39] R.Z. LeGeros // Clin. Mater. 14(1) (1993) 65.[40] M. Roy and S. Bose // J. Biomed. Mater.

Res. A 100A(9) (2012) 2450.[41] Y. Wang, S. Zhang, X. Zeng, L. Ma,

W. Weng, W. Yan and M. Qian // ActaBiomater. 3(2) (2007) 191.


[42] M. Vallet-Regi and J.M. Gonzalez-Calbet //Prog. Solid State Chem. 32 (2004) 1.

[43] J. Weng, X.G. Liu, X.D. Li and X.D. Zhang //Biomaterials 16 (1995) 39.

[44] W. Tong, J. Chen, X. Li, Y. Cao, Z. Yang,J. Feng et al. // Biomaterials 17 (1996) 1507.

[45] K.A. Gross, C.C. Berndt and H. Herman //J. Biomed. Mater. Res. 39 (1998) 407.

[46] S.W.K. Kweh, K.A. Khor and P. Cheang //Biomaterials 23 (2002) 381.

[47] G. Balasundaram, D.M. Storey and T.J.Webster // Internat. J. Nanomed. 10 (2015)527.

[48] G. Balasundaram, T.M. Shimpi and D.M.Storey, Hydroxyapatite coated nanostructuredtitanium surfaces (Patent:US 2009/0035722A1, 2009).

[49] L. Cléries, J.M. Fernandez-Pradas and J.L.Morenza // J. Biomed. Mater. Res. 49 (2000)43.

[50] V. Nelea, C. Morosanu, M. Iliescu and I.N.Mihailescu // Appl. Surf. Sci. 228 (2004) 346.

[51] L. Duta, F.N. Oktar, G.E. Stan, G. Popescu-Pelin, N. Serban, C. Luculescu and I.N.Mihailescu // Appl. Surf. Sci. 265 (2013) 41.

[52] M. Komath, P. Rajesh, C.V. Muraleedharan,H.K. Varma, R. Reshmi and M.K. Jayaraj //Bull. Mater. Sci. 34 (2011) 389.

[53] H.C. Man, K.Y. Chiu, F.T. Cheng and K.H.Wong // Thin Solid Films 517 (2009) 5496.

[54] V. Kosma, T. Tsoufis, T. Koliou, A. Kazantzis,K. Beltsios et al. // Mat. Sci. Eng. B 178(2013) 457.

[55] H. Li, L. Wang, C. Liang, Z. Wang andW. Zhao // Mat. Sci. Eng. B 166 (2010) 19.

[56] A.W.G. Nijhuis, S.C.G. Leeuwenburgh andJ.A. Jansen // Macromol. Biosci. 10 (2010)1316.

[57] L.T. de Jonge, S.C.G. Leeuwenburgh, J.C.G.Wolke and J.A. Jansen // Pharm. Res. 25(2008) 2357.

[58] T. Hanawa // Jpn. Dent. Sci. Rev. 46 (2010)93.

[59] J.S. Earl, D.J. Wood and S.J. Milne //Journal of Physics: Conference Series 26(2006) 268.

[60] Y. Wang et al. // Mater. Lett. 60(12) (2006)1484.

[61] Y. Fujishiro et al. // J. Chem. Tech. Biotech.57(4) (1993) 349.

[62] D.M. Roy and S.K. Linnehan // Nature 247(1974) 220.

[63] S.K.F. Asl, S. Nemeth and M.J. Tan // Surf.Coat. Techn. 270 (2015) 197.

[64] J. Wang, Y.L. Chao, Q.B. Wan, Z.M. Zhu andH.Y. Yu // Acta Biomater. 5 (2009) 1798.

[65] N. Eliaz, O. Ritman-Hertz, D. Aronov,E. Weinberg, Y. Shenhar, G. Rosenman,M. Weinreb and E. Ron // J. Mater. Sci.Mater. Med. 22 (2011) 1741.

[66] M.S. Chandrasekar and M. Pushpavanam //Electrochim. Acta 53 (2008) 3313.

[67] M.A. Worsley, T.F. Baumann, J.H. Satcher,T.Y. Olson, J.D. Kuntz and K.A. Rose,Methods of electrophoretic deposition forfunctionally graded porous nanostructuresand systems thereof (Patent:US 8968865B2, 2015).

[68] K.A. Rose, J.D. Kuntz and M.A. Worsley,Methods of three-dimensional electrophoreticdeposition for ceramic and cermetapplications and systems thereof (Patent:US9453289 B2, 2016).

[69] M.A. Worsley, J.D. Kuntz and K.A. Rose,Stabilization of green bodies via sacrificialgelling agent during electrophoreticdeposition (Patent:US 20120269218 A1,2012).

[70] S. Leeuwenburgh, J. Wolke, J. Schoonmanand J. Jansen // J. Biomed. Mater. Res. A66(2) (2003) 330.

[71] A. Muthutantri, J. Huang and M. Edirisinghe// J. Mater. Sci. Mater. Med. 19(4) (2008)1485.

[72] J. Huang, S.N. Jayasinghe, S.M. Best, M.J.Edirisinghe, R.A. Brooks and W. Bonfield //J. Mater. 39 (2004) 1029.

[73] M. Eltohamy, U. Sang Shin, J.E. Won et al.// Mater. Lett. 65 (2011) 2043.

[74] Y. Wu, S.J. Kennedy and R.L. Clark //J. Biomed. Mater. Res. B 91(1) (2009) 381.

[75] J.M. Lagaron Cabello, S.T. Giner, J.V.Gimeno Alcaniz and M.J. Zapata,Development of bioactive electrospuncoatings for biomedical applications(Patent:US 2012/0301514 A1, 2012).

[76] Y. Wang, T. Fan, Z. Zhou and D. He // Mater.Lett. 185 (2016) 484.

[77] B.R. Gligorijevic, M. Vilotijevic,M. Scepanovic, D. Vidovic and N.A. Radovi //J. Alloy Compd. 687 (2016) 421.

[78] Y.C. Tsui, C. Doyle and T.W. Clyne //Biomaterials 19 (1998) 2015.

[79] G. Popescu-Pelin, F. Sima, L.E. Sima, C.N.Mihailescu, C. Luculescu, I. Iordache,


M. Socold, G. Socol and I.N. Mihailescu //Appl. Surf. Sci. (2016) DOI 10.1016/j.apsusc.2016.10.043.

[80] H. Nishikawa, T. Hasegawa, A. Miyake,Y. Tashiro et al. // Mater. Lett. 165 (2016) 95.

[81] H. Takahashi, M. Yashima, M. Kakihana andM. Yoshimura // E. J. Sol. St. Inorg. Chem.32 (1995) 829.

[82] C.S. Chai, B. Ben-Nissan, S. Pyke andL. Evans // Mater. Manuf. Processes 10(1995) 205.

[83] Q. Qiu, P. Vincent, B. LoWenberg, M. Sayerand J.E. Davies // Thin Films in Vitro Cellsand Mater. 3(4) (1993) 351.

[84] D.B. Haddow, P.F. James and R. Van Noort //J. Mater. Sci. Mater. Med. 7 (1996) 255.

[85] P. Usinskas, Z. Stankeviciute,A. Beganskiene and A. Kareiva // Surf. Coat.Techn. (2016) doi: 10.1016/j.surfcoat.2016.10.032.

[86] M. Malakauskaite-Petrulevicienen,Z. Stankeviciute and G. Niaura // Ceram.Internat. 41 (2015) 7421.

[87] M. Jarcho, C.H. Bolen, M.B. Thomas,J. Bobick, J.F. Kay and R.H. Doremus //J. Mater. Sci. 11 (1976) 2027.

[88] H. Scheicher and E. Wendler, Bonereplacement material on the basis ofcarbonate and alkali containing calciumphosphate apatites (PatentNr: 4,917,702,1990).

[89] M. Akao, H. Aoki and K. Kato // J. Mater.Sci. 16 (1981) 809.

[90] A. Osaka, Y. Miura, K. Takeuchi, M. Asadaand K. Takahashi // J. Mater. Sci. Mater.Med. 2 (1991) 51.

[91] N. Nörenberg, H.J. Sterzel and V. Koch, Rodshaped apatite crystals with a specificlength-to-width ratio (Patent:WO 2003000588A1, 2003).

[92] K. Saita and S. Fujiwara, Method for forminghydroxyapatite coating film using coatingliquor containing hydroxyapatite (Patent Nr.5,030,474, 1991).

[93] T. Troczynski, D.M. Liu and Q. Yang,Biofunctional hydroxyapatite coatings andmicrospheres for in situ drug encapsulation(Patent:US 6,730,324 B2, 2004).

[94] A.K. Lynn and W. Bonfield // Acc. Chem.Res. 38 (2005) 202.

[95] M. Vallet-Regí and J.M. Gonzalez-Calbet //Prog. Solid State Chem. 32 (2004) 1.

[96] S. Gomes, J.M. Nedelec, E. Jallot,D. Sheptyakov and G. Renaudin // Cryst.Growth Des. 11 (2011) 4017.

[97] S.R. Kim, J.H. Lee, Y.T. Kim, D.H. Riu, S.J.Jung, Y.J. Lee, S.C. Chung and Y.H. Kim //Biomaterials 24 (2003) 1389.

[98] W. Bonfield and I.R. Gibson, Process for thepreparation of magnesium and carbonatesubstituted hydroxyapatite (Patent:WO1999032400 A1, 1999).

[99] O.R. Bingöl and C. Durucan // Am. J.Biomed. Sci. 4(1) (2012) 50.

[100] K. Saita and S. Fujiwara, Method forforming hydroxyapatite coating film(Patent:US 5128169 A, 1992).

[101] R. Narayanan, S. Dutta and S.K. Seshadri// Surf. Coat. Techn. 200 (2006) 4720.

[102] J. Trinkunaite-Felsen, A. Prichodko,M. Semasko and R. Skaudzius // Adv.Powd. Techn. 26 (2015) 1287.

[103] B. Hosseini, S.M. Mirhadi, M. Mehrazin,M. Yazdanian et al. // Trauma Monthly DOI:10.5812/traumamon.36139.

[104] C. Balázsi, F. Wéber, Z. Kövér, E. Horváthand C. Németh // J. Eur. Cer. Soc. 27(2007) 1601.

[105] S.W. Lee, S.G. Kim, C. Balázsi, W.S. Chaeand H.O. Lee // Oral Surg. Oral Med. OralPathol. Oral Radiol. Endod. 113(3) (2012)348.

[106] K. Balázsi, G. Gergely, C.H. Chae, H.Y.Sim, J.Y. Choi, S.G. Kim and C. Balázsi //Cells and Materials 24 (2012) 18.

[107] H.Y. Kweon, K.G. Lee, C.H. Chae,C. Balázsi, S.K. Min, J.Y. Kim, J.Y Choiand S.G. Kim // J. Oral Maxillofac. Surg.69(6) (2011) 1578.

[108] H. Benhayoune, N.J. Dumelie andR. Drevet, Process for the electrodepositionof calcium-phosphorus coatings ontometallic substrates(Patent:WO2010055231A2, 2010).

[109] G. Gupta and A. Sewing, Methods ofdepositing discrete hydroxyapatite regionson medical implants(Patent:US20110089041A1, 2011).

[110] D.T. Thanh, P.T. Nam, N.T. Phuong and L.X.Que et al. // Mater. Sci. Eng. C 33 (2013)2037.

[111] D.J. Blackwood and K.H.W. Seah // Mater.Sci. Eng. C 29 (2009) 1233.

[112] W.J. Shih, Y.H. Chen, S.H. Wang, W.L. Liet al. // J. Cryst. Growth 285 (2005) 633.


[113] W.J. Shih, M.C. Wang, K.M. Chang, C.L.Wang, S.H. Wang, W.L. Li and H.H. Huang// Metall. Mater. Trans. A 41(3) (2010) 3509.

[114] C. Huang and P. Cao // Mater. Chem. Phys.181 (2016) 159.

[115] M.H. Prado Da Silva, J.C.H. Lima, G.A.Soares, C.N. Elias, M.C. de Andrade, S.M.Best and I.R. Gibson // Surf. Coat. Techn.137 (2001) 270.

[116] D. Gopi, J. Indira and L. Kavitha // Surf.Coat. Techn. 206 (2012) 2859.

[117] D.J. Blackwood and K.H.W. Seah // Mater.Sci. Eng. C 30 (2010) 561.

[118] A. Bigi, G. Falini, E. Foresti, M. Gazzano,A. Ripamonti and N. Roveri // ActaCrystallogr. 52 (1996) 87.

[119] F.Z. Ren, R.L. Xin, X. Ge and Y. Leng // ActaBiomater. 5 (2009) 3141.

[120] Y. Cai, S. Zhang, X. Zeng, Y. Wang, M. Qianand W. Weng // Thin Solid Films 517(17)(2009) 5347.

[121] S. Ziani, S. Meski and H. Khireddine // Int.J. Appl. Ceram. Tec. 11(1) (2014) 83.

[122] Z. Geng, Z. Cui, Z. Li, S. Zhu et al. // Mat.Sci. Eng. C 58 (2016) 467.

[123] M.H. Prado da Silva, F.N. Moura, D. Navarroda Rocha et al. // Surf. Coat. Tech. 249(2014) 109.

[124] F. Miyaji, Y. Kono and Y. Suyama // Mater.Res. Bull. 40 (2005) 209.

[125] Y. Tang, H.F. Chappell, M.T. Dove et al. //Biomaterials 30 (2009) 2864.

[126] N. Iqbal, M. Rafiq, A. Kadir, N.H. Mahmoodet al. // Ceram. Int. 40 (2014) 4507.

[127] M. Furko, Y. Jiang, T.A. Wilkins and C.Balázsi // Mater. Sci. Eng. C 62 (2016) 249.

[128] M. Furko, Y. Jiang, T.A. Wilkins and C.Balázsi // Ceram. Internat. 42 (2016) 4924.

[129] R. Rojaee, M. Fathi and K. Raeissi // Appl.Surf. Sci. 285P (2013) 664.

[130] Z. Zhong, J. Qin and J. Ma // Ceram.Internat. 41 (2015) 8878.

[131] S. Singh, V.K. Meena, M. Sharma andH. Singh // Mater. Design 88 (2015) 278.

[132] C. Schouten, G.J. Meijer, J.J.J.P. van denBeucken and S.C.G. Leeuwenburgh // ActaBiomater. 6 (2010) 2227.

[133] L.T. de Jonge, S.C.G. Leeuwenburgh,J.G.C. Wolke and J.A. Jansen // Pharm.Res. 25 (2008) 2357.

[134] S.V. Dorozhkin // Biomaterials 31 (2010)1465.

[135] R.B. Parekh, O. Shetty and R. Tabassum //Int. J. Oral Implantol. Clin. Res.3 (2012)116.

[136] L. Sun, C.C. Berndt, K.A. Gross andA. Kucuk // J. Biomed. Mater. Res. 58(2001) 570.

[137] C.C.G. Moura, M.A. Souza, P. Dechichi,D. Zanetta-Barbosa, C.C. Teixeira and P.G.Coelho // J. Biomed. Mater. Res. A 94(2010) 103.

[138] R. Bosco, J. Van Den Beucken,S. Leeuwenburgh and J. Jansen // Coatings2 (2012) 95.

[139] A. Oyane, X. Wang, Y. Sogo, A. Ito andH. Tsurushima // Acta Biomater. 8 (2012)2034.

[140] R. Bosco, E.R.U. Edreira, J.G.C. Wolke,S.C.G. Leeuwenburgh, J.J.J.P. van denBeucken et al // Surf. Coat. Technol. 233(2013) 91.

[141] R.A. Surmenev // Surf. Coat. Technol. 206(2012) 2035.

[142] M. Epple and R.A. Surmenev // RSC Adv. 3(2013) 11057.

[143] C. Aparicio, D. Rodriguez and F.J. Gil //Mater. Sci. Eng. C 31 (2010) 320.

[144] T.P. Ntsoane, M. Topic and R. Bucher //Powder Diffr. 26 (2011) 138.

[145] G.L. Zhao, G. Wen, Y. Song and K. Wu //Mater. Sci. Eng. C 31 (2011) 106.

[146] H.C. Man, K.Y. Chiu, F.T. Cheng and K.H.Wong // Thin Solid Films 517 (2009) 5496.

[147] A.R. Boyd, G.A. Burke, H. Duffy,M. Holmberg, C. O’ Kane, B.J. Meenan etal. // J. Mater. Sci: Mater. Med. 22 (2011)71.

[148] Y. Chen, X. Zheng, Y. Xie, H. Ji, C. Ding,H. Li et al. // Surf. Coat. Technol. 205(2010) 1892.

[149] R.A. Surmenev, M.A. Surmeneva, E.V.Shesterikov, V.F. Pichugin, T. Peitsch andM. Epple // J. Mater. Sci: Mater. Med. 21(2010) 1233.

[150] K. Ueda, T. Kawasaki, T. Goto, J. Kurihara,H. Nakagawa, H. Kawamura et al. // J.Biomechan. Sci. Eng. 4 (2009) 392.

[151] P.G. Coelho, S.L. de Assis, I. Costa andV.P. Thompson // J. Mater. Sci. Mater. Med.20 (2009) 215.

[152] Y. Huang, Y. Qu, B. Yang, W. Li, B. Zhangand X. Zhang // Mater. Sci. Eng. C 29(2009) 2411.


[153] R.A. Surmenev, M.A. Surmeneva and A.A.Ivanova // Acta Biomater. 10 (2014) 557.

[154] C. Knabe, F. Klar, R. Fitzner, R.J. Radlanskiand U. Gross // Biomaterials 23 (2002)3235.

[155] E.S. Thian, X. Li, J. Huang, M.J.Edirisinghe, W. Bonfield and S.M. Best //Thin Solid Films 519 (2011) 2328.

[156] S.C.G. Leeuwenburgh, J.G.C. Wolke, M.C.Siebers, J. Schoonman and J.A. Jansen //Biomaterials 27 (2006) 3368.

[157] L.T. de Jonge, J.J.J.P. van den Beucken,S.C.G. Leeuwenburgh et al.// ActaBiomater. 5 (2009) 2773.

[158] L.T. de Jonge, S.C.G. Leeuwenburgh,J.J.J.P. van den Beucken, J. te Riet et al. //Biomaterials 30 (2010) 2461.

[159] N. Aebli, J. Krebs, D. Schwenke, H. Stich,P. Schawalder and J.C. Theis // J. BoneJoint Surg. [Br] 85-B (2003) 499.

[160] C. Delaunay // Orthop. Traumatol. Surg.Res. 100 (2014) 739.

[161] S.R. Nodzo, D.W. Hohman, A.S. Hoy, M.Bayers-Therin et al. // J. Arthroplasty 30(2015) 1339.

[162] T. Sato, Y. Nakashima and K. Komiyama //J. Arthroplasty 31(6) (2016) 1228.

[163] N. Kose, A. Otuzbir, C. Peks et al. // Clin.Orthop. Relat. Res. 471 (2013) 2532.

[164] X. Lu, B. Zhang, Y. Wang, X. Zhou et al. //J. R. Soc. Interface 8 (2011) 529.

[165] S. Erakovic, A. Jankovic, C. Ristoscu,L. Duta, N. Serban, A. Visan, I.N.Mihailescu et al. // Appl. Surf. Sci. 293(2014) 37.

[166] L. Song, Y.F. Xiao, L. Gan, Y. Wu, F. Wuand Z.W. Gu // Surf. Coat. Techn. 206(2012) 2986.

[167] S. Erakovic, A. Jankovic, I.Z. Matic, Z.D.Jurani et al. // Mater. Chem. Phys. 142(2013) 521.

[168] C. Shi, J. Gao, M. Wang, J. Fu, D. Wangand Y. Zhu // Mater. Sci. Eng. C Mater. Biol.Appl. 55 (2015) 497.

[169] X. Liu, Y. Mou, S. Wu and H.C. Man // Appl.Surf. Sci. 273 (2013) 748.

[170] C.S. Ciobanu, S.L. Iconaru, P. LeCoustumer et al. // Nanoscale Res. Lett. 7(2012) 324.

[171] M. Yamaguchi // J. Trace Elem. Exp. Med.11 (1998) 119.

[172] B.S. Moonga and D.W. Dempster // J. BoneMiner. Res. 10 (1995) 453.

[173] X. Xia Li, K. Senda, A. Ito, Y. Sogo andA. Yamazaki // Bioceramics Developmentand Applications 1 (2011) 1-4. doi:10.4303/bda/D101106.

[174] Y. Yamada, A. Ito, H. Kojima, M. Sakane,S. Miyakawa, T. Uemura et al. // J. Biomed.Mater. Res. A 84 (2008) 344.

[175] I.Y. Ortiz, A.R. dos Santos, A.M. Costa etal. // Ceram. Internat. 42 (2016) 15502.

[176] J.Y. Reginster, O. Bruyere, A. Sawicki,A. Roces-Varela, P. Fardellone, A. Robertset al. // Bone (2009) doi: 10.1016/j.bone.2009.08.004.

[177] Y. Li, Q. Li, S. Zhu, E. Luo, J. Li, G. Feng,Y. Liao and J. Hu // Biomaterials 31 (2010)9006.

[178] C. J. Chung and H.Y. Long // Acta Biomater.7 (2011) 4081.

[179] A.R. Boyd, L. Rutledge, L.D. Randolph andB.J. Meenan // Mater. Sci. Eng. C 46 (2015)290.

[180] E.M. Carlisle // Science 167 (1970) 2879.[181] S. Klaus and B.M. Divid // Nature 239 (1972)

333.[182] E.M. Carlisle // J. Nutr. 106 (1976) 478.[183] X. Qiu, P. Wan, L. Tan, X. Fan and K. Yang

// Mater. Sci. Eng. C 36 (2014) 65.[184] M. Percival // Appl. Nutr. Sci. Rep. 5 (1999)

1.[185] M. Kheradmandfard, M.H. Fathi, F. Ansari

and T. Ahmadi // Mater. Sci. Eng. C 68(2016) 136.

[186] Y. Huang, M. Hao, X. Nian, H. Qiao andX. Zhang // Ceram. Internat. 42 (2016)11876.

[187] S. Bose, G. Fielding, S. Tarafder andA. Bandyopadhyay // Trends Biotechnol.31(10) (2013) 594.

[188] D. Gopi, S. Ramy, D. Rajeswari,M. Surendiran and L. Kavitha // ColloidSurface B 114 (2014) 234.

[189] D. Gopi, A. Karthika, S. Nithiya andL. Kavitha // Mater. Chem. Phys. 144 (2014)75.

[190] H. Yuan, B.J.D. De and G.K. De, Method ofimproving the osteoinductivity of calciumphosphate (Patent:WO 2006115398 A1,2006).

[191] E.C. Moreno, M. Kresak and R.T. Zahradnik// Caries Res. 11 (1977) 142.

[192] J.D.B. Featherstone, I. Mayer, F.C.M.Driessens, R.M.H. Verbeeck andM. Heijligers // Calcif. Tissue Int. 35 (1983)169.


[193] D. Tadic, F. Peters and M. Epple //Biomaterials 23 (2002) 2553.

[194] W.Y. Zhou, M. Wang, W.L. Cheung, B.C.Guo and D.M. Jia // J. Mater. Sci: Mater.Med. 19 (2008) 103.

[195] W. Xiao, B.S. Bal and M.N. Rahaman //Mater. Sci. Eng. C 60 (2016) 324.

[196] A. Fahami, G.W. Beall and T. Betancourt //Mater. Sci. Eng. C 59 (2016) 78.

[197] S. Dorozhkin // Materials 2 (2009) 1975.[198] S. Bose and S. Tarafder // Acta Biomater. 8

(2012) 1401.[199] S.S. Kim, S.M. Park, O. Jeon, C.Y. Choi

and B.S. Kim // Biomaterials 27 (2006)1399.

[200] R. Detsch and A.R. Boccaccini // J. TissueEng. Regen. Med. 9 (2015) 1133.

[201] L. Xia, K. Lin, X. Jiang, Y. Xu, M. Zhang,J. Chang et al. // J. Mater. Chem. B 1(2013) 5403.

[202] M. Valletregi // Prog. Solid State Chem. 32(2004) 1.

[203] S. Dorozhkin // Materials 2 (2009) 221.[204] V. Uskokovic and D.P. Uskokovic //

J. Biomed. Mater. Res. B Appl. Biomater.96 (2011) 152.

[205] K. Lin, L. Chen, P. Liu, Z. Zou, M. Zhang,Y. Shen et al. // Cryst. Eng. Comm. 15(2013) 2999.

[206] K. Lin, P. Liu, L. Wei, Z. Zou, W. Zhang,Y. Qian et al. // Chem. Eng. J. 222 (2013)49.

[207] M. Sadat-Shojai, M.T. Khorasani,E. Dinpanah-Khoshdargi and A. Jamshidi //Acta Biomater. 9 (2013) 7591.

[208] Y. Cai and R. Tang // J. Mater. Chem. 18(2008) 3775.

[209] L. Li, H. Pan, J. Tao, X. Xu, C. Mao, X. Guet al. // J. Mater. Chem. 18 (2008) 4079.

[210] J.A. D’Antonio, W.N. Capello, O.D.Crothers, W.L. Jaffe and M.T. Manley //J. Bone Joint Surg. 74 (1992) 995.

[211] S.A. McNally, J.A.N. Shepperd, C.V. Mannand J.P. Walczak // J. Bone Joint Surg. Br.82B (2000) 378.

[212] R.J. Furlong and J.F. Osborn // J. BoneJoint Surg. Br. 73B (1991) 741.

[213] C.H. Rorabeck // J. Arthroplasty 21 (2006)85.

[214] Y.H. Kim, J.S. Kim, S.H. Oh and J.M. Kim// J. Bone Joint Surg. 85 (2003) 1682.

[215] S. Eto, S. Kawano, S. Someya,H. Miyamoto, M. Sonohata andM. Mawatari // The Journal of Arthroplasty(2016) 1-6. http://dx.doi.org/10.1016/j.arth.2015.12.034.

[216] P. Gouma, R. Xue, C.P. Goldbeck,P. Perrotta and C. Balázsi // J.Mat. Sci.Eng. C 32(3) (2012) 607.

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