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In vitro behavior of fluoridated hydroxyapatite coatings inorganic-containing simulated body fluid

Yongsheng Wang a, Sam Zhang a,⁎, Xianting Zeng b, Kui Cheng a, Min Qian b, Wenjian Weng c

a School of Mechanical and Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798b Singapore Institute of Manufacturing Technology, 71 Nanyang Drive, Singapore 638075

c Department of Materials Science and Engineering, Zhejiang University, Hangzhou, Zhejiang, 310027, PR China

Received 8 July 2005; accepted 3 March 2006Available online 7 July 2006

Abstract

A dense and pure hydroxyapatite [HA, Ca10(PO4)6(OH)2] coating and a fluoridated HA [Ca10(PO4)6(OH)0.67F1.33] are deposited on Ti6Al4Vsubstrates by sol-gel dip coating method. Glucose and bovine serum albumin have been added in standard simulated body fluid (SBF) to formorganic-containing SBF in simulation of the physiological blood plasma. The HA and the fluoridated HA coatings are immersed in the standardand modified SBF for time periods of 2, 4, 7, 14 and 28 days at 37±0.1 °C. After soaking, the coating surface is examined for nucleation andgrowth of apatite using SEM morphological observation. The post-soaking SBF solutions are analyzed via Inductively Coupled Plasmaspectroscopy for calcium ion concentration. The results show that at concentration of 40 g/L, bovine serum albumin has significant retardationeffect on apatite precipitation from SBF onto pure or fluoridated HA coatings; Fluorine-incorporation in HA has positive bio-activation effect inboth standard SBF and organic-containing SBF. However, glucose addition in SBF does not generate significant influence on the bioactivity ofHA and fluoridated HA.© 2006 Elsevier B.V. All rights reserved.

Keywords: Fluoridated Hydroxyapatite (FHA); In vitro; Simulated body fluid; Glucose; Bovine serum albumin; Sol-gel

1. Introduction

Hydroxyapatite [HA, Ca10(PO4)6(OH)2] has been devel-oped as coatings on metallic implants in the field oforthopedics and dentistry due to its chemical and biologicalsimilarity to human hard tissues and also direct bondingcapability to the surrounding tissues [1,2]. It has beenestablished that HA coatings promote early bone appositionand fixation for HA-coated implants by encouraging chemicalbonding between new bone and the surface of HA [3]. HAcoating is also believed to protect the metallic substrate formcorrosion in the biological environment, as well as serving asan effective barrier against the release of toxic metal ionsfrom the metallic substrates into the living body [4].However, the high dissolution rate of HA renders itquestionable long term stability because dissolution of HA

leads to disintegration of the coatings thus hinders the fixationof implants to the surrounding host tissues. Recently,fluoridated hydroxyapatite (fluoridated HA, Ca10(PO4)6(OH)2−xFx, x is the degree of fluoridation) has been developedthat possesses lower solubility than pure HA while maintain-ing the comparable bioactivity and biocompatibility [5–7].

In vitro tests that employ cell cultures are performed toevaluate the biological response of the related cells, whileinvestigations in cell-free solutions with compositions that aresimilar to human body fluid allow the determination of chemicaland mineralogical changes of the implants under a simulatephysiological environments [8]. Simulated body fluid (SBF)with inorganic ion concentrations nearly to those of humanblood plasma, which was first introduced by Kokubo et al., isthe most popular simulating solution used in in vitro tests [9].However, actual body fluid contains not only the inorganiccomponents but also various kinds of organic components (suchas carbohydrates and proteins) and the organic componentswould exert noticeable influence on the implants. Jaou et al.[10] and Balint et al. [11] reported that although sugar and/or

Materials Science and Engineering C 27 (2007) 244–250www.elsevier.com/locate/msec

⁎ Corresponding author. Tel.: +65 6790 4400; fax: +65 6791 1859.E-mail address: msyzhang@ntu.edu.sg (Sam Zhang).

0928-4931/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.msec.2006.03.012

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glucose have a minor influence on crystallization of HA, theysignificantly inhibit the crystallization process of FluoridatedApatite (FA). This effect was attributed to the formation ofnonstoichiometric apatite in the presence of sugar. Theinhibition effects of carbohydrates on the bone mineralizationalso reported by other researchers [11,12]. Dorozhkin et al.[13,14]concluded that glucose exhibited negligible influence oncrystallization of calcium phosphate based on in vitro tests withthe glucose modified SBF solution. On effects of proteins,extensively investigations have been done on CaP biomaterials,especially on HA [15,16]. It has been reported that plasma

proteins would adsorb immediately on the surface of HA after itwas implanted in vivo, and the initial cellular response waspartly dependent on the proteins adsorbed by the implantsurfaces [17]. The first protein layer adsorbed on the implantsurface affects the cellular adhesion [18,19], differentiation andproduction of extracellular matrix production. It also affectsdissolution [17], nucleation and crystal growth of HA [15] aswell as the final fixation between the implant and surroundingtissues. Albumin is usually selected for this kind of study due toits high concentration in blood plasma, favorable diffusioncoefficient and ability to bind other ions and molecules [20].Therefore, it is unwise to neglect the influences of organiccomponents in the in vitro tests. The objective of the currentwork is to study the effect of proteins and glucose on the apatitedeposition on an HA and an FHA coating.

Table 1Chemical composition of human blood plasma compared to the ion concentration of Kokubo's SBF

Inorganic ion concentration (mM) Organic composition (mg/dL)

Ca2+ HPO42− Na+ Cl− Mg2+ K+ HCO3

− SO42− Albumin Globulin Fibrinogen Glucose

Blood plasma 2.5 1 142 103 1.5 5 27 0.5 3300–4000 880–3530 340–430 100Kokubo's SBF 2.5 1 142 148 1.5 5 4.2 0.5 / / / /

Fig. 1. XRD patterns of the coating fired at 600 °C: a) HA, b) fluoridated HA.

Fig. 2. XPS patterns of the fluoridated HA coating fired at 600 °C.Fig. 3. Surface morphology of the sol-gel derived a) HA coating and b)fluoridated HA coating.

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2. Experimental

2.1. Coating preparation and characterization

The process of preparation for dip-sols and deposition ofFHA coatings are detailed in our previous work [6,7]. Based onour previous work, Ca10(PO4)6(OH)0.67F1.33 has much betterbioactivity than other composition FHA coatings [21]. There-fore, current work chose to focus on this specific FHA for itsbioactivity in organic-containing SBF. An HA coating was usedas control. Titanium alloy (Ti6Al4V) of 20×30×1.2 mm with afinal finish of polishing by silicon carbide sandpaper (1200#)was used as substrates. The deposition run was repeated 4 timesfor a final coating thickness of ∼1.5 μm. The phasecharacterization of the prepared coatings was conducted by X-ray diffraction analysis (XRD, Philips PW 1830) usingmonochromatic CuKα radiation with a step size of 0.02°. Thefluorine concentrations were determined by X-ray Photoelec-tron Spectroscopy (XPS, Kratos-Axis Ultra System) usingmonochromatic Al Kα X-ray source (1486.7 eV). The surfacemorphology of FHA coatings was characterized using scanningelectron microscopy (SEM, LEICA S360).

2.2. In vitro tests in standard SBF and modified SBF solutions

Three kinds of solutions were used in the immersion tests:(1) the standard SBF solution, (2) glucose modified SBF

solution (G-SBF) and (3) bovine serum albumin modified SBFsolution (A-SBF). The standard SBF solution was preparedaccording to Kokubo's protocol by dissolving appropriatequantities of the relevant reagent-grade chemicals in deionizedwater [9]: NaCl, NaHCO3, KCl, K2HPO4U3H2O, MgCl2U6H2O,CaCl2, HCl (1 M), Na2SO4 and NH2C(CH2OH)3. After all thereagents were dissolved, the solution was then heated to 37 °Cand maintained at this temperature while titrating the solution toa pH of 7.4 with 1 M HCl or NH2C(CH2OH)3. The inorganicion concentrations in the standard SBF solution are almost thesame in human blood plasma Table 1. However, as shown in thetable, besides the inorganic components, there are many organiccompounds in human blood plasma, which are not included inthe Kukobo's SBF solution. Bovine serum albumin (BSA,Merck) and glucose (D(+)-Glucose anhydrous, Merck) wereselected respectively for the preparation of protein andcarbohydrate-containing SBF solutions. The glucose modifiedSBF solution (G-SBF) and BSA modified SBF solution (A-SBF) were obtained by dissolving 1 g/L for glucose and 40 g/Lfor BSA respectively in the prepared SBF solution.

The coatings were placed in sterilized bottle containingsolution with a liquid/area ratio of 50 ml/cm2. Before soaking inthe solution, the samples were washed ultrasonically in acetonefor 10 min, and then sterilized in ethanol. The soaking tookplace in a temperature-controlled shaking water bath for variousperiods of 2, 4, 7, 14, 21 and 28 days at 37 °C±0.1 °C. After thedesired immersion, the samples were taken out, gently washed

Fig. 4. SEMmicrographs of the fluoridated HA coating after the in vitro tests in SBF andG-SBF: a) 2 days in SBF, b) 7 days in SBF, c) 2 days in G-SBF, d) 7 day in G-SBF.

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with deionized water and dried at room temperature before theeffect characterization.

Scanning electron microscopy (SEM, LEICA S360) wasused to examine the chemical and surface morphology changesthat occurred after the immersion in simulated solutions. TheCa2+ ion concentration of the solutions were measured withinductively coupled plasma atomic emission spectrometer (ICP-AES, PerkinElmer Optima 2000). An average of threemeasurements was taken for each sample.

3. Results and discussion

3.1. Phase, composition and surface morphologies of theprepared coatings

The XRD profiles of the Fluoridated HA coating and theHA coating are shown in Fig 1. The coatings have similardiffraction profile. The peaks (002), (211), (112), (300) arethose of the HA structure (JCPDS file card #9-432). Notricalcium phosphate (TCP), CaO, CaF2 or other phases aredetected in these coatings.

The concentration of Ca, F, and P in the coating is determinedby ratio of the area under the respective elemental peak in theXPS narrow scan spectrum [7]. As such, the Ca/P molar ratio inthe coating is 1.65 in HA and 1.63 in Fluoridated HA, which isvery close to the stoichiometric value of 1.67.

Fig 2 shows the XPS spectra of fluoridated HA. F1s peak isevident in the wide scan. The narrow scan analysis around684 eV (the inset) reveals only one peak at ∼684.3 eVbelonging to F1s. That peak is the fingerprint for fluorine inFHA structures [22]. This indicates that the fluorine ions havebeen successfully incorporated into the coating.

Fig 3 shows the surface morphologies of the sol-gel derivedHA and fluoridated HA coatings. Two observations are noticed:(1). both coatings have complete coverage of the substrate; thecoatings look uniform and generally smooth. (2). the fluoridatedcoating appears rougher (Fig 3b) since the Fluorine agent(HPF6) promotes gelation in FHA deposition process [23].

3.2. Effect of glucose

Fig 4 shows the characteristic surface morphologies on thefluoridated HA coating after in vitro tests in SBF and G-SBF.After 2 days in standard SBF (Fig 4a), a new apatite layerprecipitated from the solution completely covers the surfaceof the FHA coating deposited from the sol-gel process. After7 days, the new apatite layer becomes smooth and uniform(unlike the rough appearance after 2 days), as is seen in Fig4b. At low magnification, the surface appears dense andcontains spherical apatite particles. At higher magnification(Fig 4b inset), however, porosity can still be seen in thesurface. Further increase of soaking time has no influence onthe morphology of the newly grown apatite layer exceptincrease in thickness. Fig. 4c and d are the same coating inglucose-modified SBF for 2 days and 7 days respectively.Comparing Fig 4a with Fig 4c and b with Fig 4d, one doesnot find too much difference except that more pin-holes are

observed after 2 days in G-SBF, but after 7 days the newapatite seems to be smoother and without the apatite granules(Fig 4d). These results indicate that glucose does not seem tohave a significant influence on the nucleation and growth ofapatite on fluoridated HA coating. This is also observed onpure HA coating [13,14].

3.3. Effect of albumin

After soaking in the protein-containing SBF or A-SBF for2 days (Fig 5a), there is only sporadic nucleation of new apatiteon the Fluoridated HA surface. Compare Fig. 5a with Fig 4a,one can easily see that albumin addition drastically sloweddown nucleation of apatite from SBF. This retardation effect islikely carried to the growth stage of the newly nucleated apatitecrystals. Only after 28 days can the new apatite layer becomescontinuous (Fig 5b, the cracks are drying artifacts), which onlyneeds 7 days in standard SBF (Fig 4b) or glucose modified SBF.The newly deposited apatite layer is usually considered gel-like.After drying, the gel-like structure shrinks and thus cracks form[24]. Higher magnification (inset in Fig 5b) also revealsporosity in the newly precipitated apatite layer. Fig 6 comparesthe bioactivity of pure HA coatings in SBF and A-SBF. After2 days in standard SBF (Fig 6a), obvious apatite nucleation is

Fig. 5. SEM micrographs of the fluoridated HA coating after soaking in A-SBFfor: a) 2 days, b) 28 days.

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pyobserved, and after 14 days, a continuous layer is formed (Fig.6b). But 2 days in A-SBF (Fig 6c) results in much less newapatite than that in SBF. Even after 28 days, the apatiteformation is still sporadic (Fig. 6c). Therefore, protein additionin SBF adversely affect apatite nucleation and growth on bothHA and FHA coatings. According to Combes and Rey [25], theslow deposition and growth comes from the fact that theadsorption of albumin reduces the interfacial energy of apatitenuclei with the solution. That deprives the driving force for thegrowth of the new apatite.

3.4. Effect of fluorine

The effect of fluorine inclusion in HA has been extensivelystudied recently [7], and it has been reported that the degree offluoridation of x∼1.33 improves biomineralization in HA [21].Comparing this fluoridated HA (where fluoridation extent is1.33) with pure HA in standard SBF for 2 days (Fig 4a vs Fig6a), one sees a drastic increase in nucleation rate as fluorine isincorporated in HA structure. To have a continuous apatite layerformed, it requires 14 days on a pure HA coating (Fig 6b) butonly 7 days on fluoridated HA (Fig 4b). This bio-activationeffect of fluorine is also effective in the presence of protein as ismanifested in the in vitro tests in A-SBF: without fluorine in thecoating, 2 days soaking in A-SBF results in almost no apatitenucleation (Fig. 6c), but with fluorine, obvious nucleation isobserved (Fig. 5a). In the case of pure HA, 28 days in A-SBFgives rise to only sporadic apatite (Fig. 6d), however, on thefluoridated HA, a continuous thick layer of apatite is formed(Fig. 5b).

3.5. Variation of Ca2+ concentration during in vitro tests

The variation of Ca2+ concentration in SBF, G-SBF and A-SBF solutions are shown in Fig 7 as a function of soaking time.The ups and downs of the Ca2+ concentration respectivelyindicate dissolution and precipitation of apatite from the solutionto the surface of the coatings. In Fig 7 a), during the first 4 daysthe pure HA coating experienced more dissolution thanreprecipitation that resulted in the increase of Ca2+ concentrationin the solution. The Ca2+ released from the coating may result ina supersaturation of Ca2+, a situation more favorable fornucleation [15,26]. Therefore, spontaneous growth of apatitelayer takes place which consumes calcium and phosphate ions inSBF causing the gradually decrease of Ca2+ concentration. Withthe incorporation of F− ion into the HA lattice, no obviousdissolution process is observed. It seems that during the wholetest process, the Ca2+ ion concentration maintains a gradualreduction, indicating a continuous precipitation of apatite. Thisagrees well with SEM morphological observations.

Fig 7b describes the Ca2+ concentration in G-SBF. Asglucose has no obvious influence on the precipitation process oneither HA or FHA coating, it is not surprising that the Ca ionconcentration profile does looks very much the same as that inFig. 7a.

Fig 7c shows the Ca2+ concentration in A-SBF after the invitro test. It is interesting to note that the curves show asudden drop in the first 2 days for both HA and FluoridatedHA coatings. After that, calcium concentration increases andthen slowly decreases. The sudden drop of Ca2+ concentrationis resulted from the adsorption of BSA on the coating surface,

Fig. 6. SEM micrographs of pure HA coating after soaking for: a) 2 days in SBF, b) 14 days in SBF, c) 2 days in A-SBF, d) 28 days in A-SBF.

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which captures large amount of Ca2+ from the solution. Theinteractions between BSA and the coatings are complex.Generally, after the coating is immersed into the A-SBFsolution, BSA will be immediately adsorbed onto the surfaceand thus inhibit both the dissolution of the coating into thesolution and the precipitation of apatite from the solution onto the coating surface [27]. It has been suggested that theproteins compete with ions, e.g. Ca2+ and PO4

3− etc., in thesolution for the same surface binding sites [24]. Therefore theadsorption of BSA to the surface of coating surface reducesthe number of nucleation and growth sites for apatite. On the

other hand, the isoelectric point of BSA is 4.7, therefore,BSA will be negatively charged in the physiological solutionwith a pH of 7.4, thus tends to bind positive ions like Ca2+ inthe solution [28]. This strongly affects the available Ca2+ ionsfor nucleation and growth of apatite.

4. Conclusions

This work has examined the biological response ofhydroxyapatite (HA) and a fluoridated HA with a fluoridationdegree of 1.33, i.e., Ca10(PO4)6(OH)0.67F1.33, in standardSimulated Body Fluid (SBF) and organic-containing, i.e.,glucose and protein-modified, SBF. This work concludes that

1. At concentration of 40 g/L, bovine serum albumin hassignificant retardation effect on apatite precipitation fromSBF onto pure or the fluoridated HA coatings;

2. Fluorine-incorporation in HA has positive bio-activationeffect in both standard SBF and organic-containing SBF.

3. Glucose has negligible influence on the bioactivity of HAand the fluoridated HA.

Acknowledgements

This work is supported by the Agency for ScienceTechnology and Research, Singapore (A⁎Star) through project032101 0005 and the SIMTech-NTU collaboration projectU03-S-389B.

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