Surface & Coatings Technology 201 (2006) 1902–1906www.elsevier.com/locate/surfcoat

Short communication

Preparation of bone-like composite coating using a modified simulated bodyfluid with high Ca and P concentrations

Kai Hu, Xian-Jin Yang ⁎, Yan-Li Cai, Zhen-Duo Cui, Qiang Wei

School of Materials Science and Engineering, Tianjin University, Tianjin 300072, PR China

Received 31 December 2005; accepted in revised form 22 February 2006Available online 29 March 2006

Abstract

A carbonate hydroxyapatite (HA)–collagen (Col) composite coating on NiTi shape memory alloy (SMA) was synthesized by biomimeticgrowth in modified simulated body fluid (MSBF), which containing high Ca and P ions concentrations. The morphology of composite coating wasuniform and porous. The major phase of coating, identified by Infrared Spectroscopy (IR) and X-ray diffraction (XRD), was HA with B-typecarbonate substitution. It was also proved by IR results that the collagen was present in the coating. Transmission Electron Microscopy (TEM) andhigh resolution TEM analysis showed that the c-axis of HA crystals aligned parallel to the longitudinal direction of the collagen fibrils. Therelative orientation maintained in the composite coating may benefit the NiTi SMA to achieve better properties in hard tissue replacement.© 2006 Elsevier B.V. All rights reserved.

Keywords: Hydroxyapatite; Collagen; Composite coating; Nickel titanium; Modified simulated body fluid

1. Introduction

Bone tissue consists mainly of hydroxyapatite and collagen;since the organic and inorganic phases organize themselves invivo in a so-called multi-level hierarchical structure [1]. Boneis considered a chemically bonded composite between apatiteand type I collagen [2]. This definition inspired earlier studiesof hydroxyapatite–collagen composite [3–6]. However withthe evolution of the researches and experiments, it becameobvious that only mimicking the composition of bone is notsufficient to obtain composites with properties similar tonatural bone [7]. Some researchers have employed the methodof co-precipitation to form bulk calcium phosphate–collagencomposites by self-assembly of collagen and the precipitationof calcium phosphate [8–14]. In vivo evaluation did provedthat the composite favorably enhanced new bone growth[15,16]. However, the composite was still limited in practicalapplication because of its poor mechanical properties.Glutaraldehyde cross-linked hydroxyapatite–collagen compos-

⁎ Corresponding author. Tel.: +86 22 2740 1524; fax: +86 22 2740 5874.E-mail address: xjyang@tju.edu.cn (X.-J. Yang).

0257-8972/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.surfcoat.2006.02.036

ite showed improved mechanical behavior even though themicrostructural characteristics of hydroxyapatite and collagendisappeared [17].

Applying calcium phosphate–collagen composite coatingto the surface of metallic implant is without doubt a novelmethod to achieve an excellent combination of the advan-tages of the coating and substrate. The metallic substrateprovides strong mechanical support to the implant, whereasthe bioactive coating promotes growth and healing of newbone tissue. Some studies of calcium phosphate–collagencomposite coating indicated a problem with the homogeneityof collagen absorption [18,19]. However, electrolytic depo-sition of a calcium phosphate–collagen coating yielded auniform distribution of composite coating on the workingelectrode [20]. To the best of our knowledge, the structuralrelationship between hydroxyapatite and collagen during thecomposite coating formation on the substrate has not beenreported yet.

The aim of this work is to produce a bone-like apatite–collagen composite coating on NiTi shape memory alloy by themethod of biomimetic growth. The biomimetic process consistsof soaking implants under moderate conditions of pH andtemperature into simulated body fluid (SBF) solutions that have

Table 1Ionic concentration of SBF and MSBF solutions, in comparison with those ofhuman blood plasma (HBP) (mM)

Na+ K+ Ca2+ Mg2+ HCO3− Cl− HPO42− SO4

2−

HBP 142.0 5.0 2.5 1.5 27.0 103.0 1.0 0.5SBF 142.0 5.0 2.5 1.5 4.2 148.0 1.0 0.5MSBF 142.0 5.0 12.5 1.5 4.2 159.8 5.0 0.5

Fig. 1. ESEM micrographs of composite coating grown biomimetically for3days in MSBF, (a) and (b) are at low and high magnifications respectively.

1903K. Hu et al. / Surface & Coatings Technology 201 (2006) 1902–1906

a similar inorganic content as human blood plasma [21]. Toshorten the immersion periods while maintaining the properconcentrations and proportions of apatite and collagen, wemodified the SBF by increasing the Ca and P ion concentra-tions. We will demonstrate that the biomimetic growthtechnique in modified simulated body fluid (MSBF) results information of carbonate hydroxyapatite–collagen compositecoatings on NiTi alloy substrate, and that the characteristicorientation between hydroxyapatite and collagen is beingmaintained in the composite coating.

2. Materials and methods

NiTi shape memory alloy blocks (50.8 at.% Ni; ShenyangTianhe New Materials Development Co., Ltd.) of 10×3×3mm dimensions were used as substrates. All chemicalregents were purchased from Sigma-Aldrich. Water-solubletype I collagen powder was purchased from Mingrang Bio-tech Co., Ltd.

The NiTi substrates were abraded with no. 360, 600, 800sand papers in succession, and then cleaned ultrasonicallyin deionized water for 3min. The cleaned samples weredipped in 30% HNO3 aqueous solution at 60°C for 30min,boiled in 1.2M NaOH aqueous solution for 2h, and cleanedultrasonically before and after the alkali treatment. Thesamples were finally soaked in the MSBF. The ionconcentrations of MSBF were identical to Kokubo's SBF[21] but the [Ca2+] and [HPO4

2−] concentration wereincreased fivefold (see Table 1). The pH of MSBF wascontrolled at 6.13 with 1M HCl. The water-soluble type Icollagen powder was dissolved at a concentration of0.556g/L in MSBF. In the process of biomimetic growth,the pH of MSBF was adjusted by tris-hydroxymethylaminomethane[(CH2OH)3CNH3] to rise slowly to 7.15within 24h. The samples were kept in a water bath at50°C, and the MSBF was renewed everyday. After soakingin MSBF for 3 and 4days respectively, the samples weretaken out, cleaned ultrasonically in deionized water, andthen dried in air.

The surfaces of the specimens were examined by environ-mental scanning electron microscopy (ESEM, XL30), X-raydiffraction meter (XRD, RIGAKUD-MAX) and X-ray photo-emission spectroscopy (XPS, PHL1600ESCA system). Thecomposite coating was scraped off the NiTi substrates andground into a fine powder. The powder was tested with infraredspectroscopy (IR, BIO-RAD FTS3000), field emission guntransmission electron microscopy (TEM, TECNAI G2 F20) andconventional transmission electron microscopy (TEM, JEOL-100CX-II).

3. Results and discussion

3.1. ESEM

Fig. 1 shows the morphology of the surface of NiTi SMAafter biomimetic growth in MSBF for 3days. The coatingformed on the surface of the substrate was uniform (Fig. 1a). Itcould be seen that the coating was composed by many smallspherical particles, forming a loose arrangement. The averagediameter of the single particle was about 1μm. There werecolloid-like connections among the small particles (Fig. 1b).

The stability of the MSBF containing high calcium andphosphate concentration is due to the acidic condition(pH=6.13) adjusted by HCl. It is known that the relationshipbetween Ca–P precipitation in solution and Ca–P coatinggrowth is competitive [22]. If the calcifying solutions reach theirsupersaturation point rapidly, it will lead to Ca–P precipitateinto the solution and hold Ca–P coating back from growing onthe substrate. We gradually controlled the release of Tris-hydroxymethyl aminomethane into MSBF within 24h. Thisdelayed the Ca–P precipitation in solution and ensured that aCa–P coating formed simultaneously. On the other hand, theisoelectric point of collagen used in the experiment is pH 5.9–6.4. Accompanying the rising pH, collagen molecules assem-bled themselves into collagen fibrils and became negativelycharged ions whose terminal groups were mostly –COO− that

Fig. 2. IR spectra of composite coating (a), a mechanical mixture of pure HA andcollagen (b) and H2O (c).

1904 K. Hu et al. / Surface & Coatings Technology 201 (2006) 1902–1906

might absorb Ca2+ [23]. Therefore a calcium phosphate–collagen composite coating formed on the substrate.

3.2. IR

IR spectrometry was carried out on the coating to confirm thepresence of collagen and determine the inorganic groups. TheIR spectra of the coating and a mechanical mixture of pure HAand collagen are shown in Figs. 2 and 3.

Fig. 2a and b exhibit the typical peaks of collagen. In thespectrum of mechanically mixed powder (Fig. 2b), weobserved the typical bands such as the CfO stretchingvibration at 1654cm−1 of the amide group I, the N–Hdeformation vibration at 1521cm−1 of the amide group II andN–H deformation vibration at 1284cm−1 of the amide groupIII band. Normally, the amide I band is strong, the amide IIband is weak and the amide III band is moderately intense.

Fig. 3. IR spectra of composite coating (a), and a mechanical mixture of pure HAand collagen (b).

The band at 1638cm−1, which combined with the peak1654cm−1 of the amide I to form a shoulder structure, wasthe typical peak of H2O (Fig. 2c). The presence of H2O mightbe due to the fact that the specimen was affected by moisturein the experiment. In the spectrum of the coating (Fig. 2a), itwas noticed that there was also a similar shoulder between1600 and 1700cm−1. Besides the band at 1638cm−1 for H2O,the other band at 1651cm−1 could be confirmed as being thetypical peak of amide I of collagen. It is believed that the “redshift” of amide I was caused by a covalent bond formationwith Ca2+ ions of HA crystals [24]. The band of amide II at1521cm−1 was very weak and the amide III band almostdisappeared. It was implied that HA crystals grew on the self-assembled collagen fibrils and blocked the vibration oforganic groups such as carboxyl and carbonyl groups [25].The amide II and III bands not only corresponded to the CfOstretching vibration, but also included the N–H bendingvibration. For this reason, the effects of mineralization on theamides II and III were greater than for amide I.

Fig. 3a shows the typical peaks of inorganic groups in thecomposite coating. Therewere PO4

3− v1mode at 964cm−1, PO43−

v2 mode at 474cm−1, PO43− v4 mode at 562 and 604cm−1, and

–OH band at 632cm−1. These vibrational bands could also beseen in the mechanical mixture of pure HA and collagen (Fig.3b). The 1037cm−1 and 1096cm−1 bands in Fig. 3b were PO4

3−

v3 mode and asymmetric HA respectively. However, both werevery weak in the IR spectrum of composite coating (Fig. 3a).Furthermore, we could see the CO3

2− v2 mode at 865cm−1 andthe CO3

2− v3 mode at 1461cm−1 in the spectrum of compositecoating. According to Chang and Tanaka [26], the CO3

2− v2mode at 865cm−1 indicated the formation of carbonate apatitewith B-type substitution in the HA–Col composite coating.This substitution leads to a distortion of the crystallographiclattice, i.e. a-axis contraction and c-axis extension [27].

Fig. 4. XRD pattern of coatings after biomimetic growth in MSBF for 4days (a),and SBF for 7days (b). (♦) Hydroxyapatite, (■) NiTi.

1905K. Hu et al. / Surface & Coatings Technology 201 (2006) 1902–1906

3.3. XRD

The crystallographic structure of the coating was investigat-ed by X-ray diffraction. Fig. 4 shows the XRD pattern of thecoatings after biomimetic growth in MSBF for 4days as well asin SBF for 7days. Both solutions contain identical concentra-tions of collagen as mentioned above. The phases of the coatingobtained in MSBF had broader diffraction peaks, indicating thatthe coatings were poorly crystallized with small particle size.The main diffraction peaks could be assigned to HA as shown inFig. 4a. An acute diffraction peak at 26.2° possibly corre-sponded to the most intense diffraction line of CaCO3, whichmight be caused by carbonate substitution in HA–Colcomposite coating proved by IR results above. So it could bespeculated that the phases of the coating after biomimeticgrowth in MSBF were bone-like apatite. Compared with thecoating formed in SBF after 7days (Fig. 4b), the coating grown

Fig. 5. TEM micrographs of the powder of composite coating showing HAnuclei formed on the collagen fibril in the initial stage of mineralization at low(a) and high (b) magnifications.

Fig. 6. TEMmicrographs of the mineralized collagen fibril in composite coating.The inset shows a selected area electron diffraction (SAED) pattern of themineralized collagen fibril.

in MSBF for 4days possessed a higher degree of crystallinity,while high Ca and P concentrations caused the coatings to formmore quickly in the solution.

3.4. TEM

TEM was employed to study the micromorphology andmicrostructure between HA and collagen fibrils. A plate-likeparticle grew in the middle of a collagen fibril of 800nm inlength and 20nm in diameter (Fig. 5a). At high magnification(Fig. 5b), the particle showed a typical crystallographicstructure, compared with the amorphous structure of uncalcifiedcollagen fibril. This suggests that the method we used couldfavor the calcium phosphate crystal particles to nucleate on thefibril in the initial stage of mineralization.

Fig. 7. High resolution TEM image of fully mineralized collagen fibril.

1906 K. Hu et al. / Surface & Coatings Technology 201 (2006) 1902–1906

To investigate the relative orientation between HA crystalsand collagen fibril, we selected a mineralized collagen fibril foranalysis by electron diffraction (Fig. 6). In the electrondiffraction pattern, the ring shaped diffraction was ascribed to(211) of HA, and the two pairs of bright diffraction points wereascribed to (002) and (004) of HA. This means that the c-axis ofHA crystals aligned from above-right to bottom-left, i.e. the c-axis was the preferential orientation and oriented parallel to thelongitudinal direction of the collagen fibril.

This special orientation could be observed more directlyfrom high resolution TEM images. As shown in Fig. 7, HAcrystals grown on the surface of a fully mineralized collagenfibril exhibited overall uniform lattice plane distribution. Byvirtue of the calculation of lattice plane spacing, the lattice planeof 0.2638nm corresponded to the HA lattice plane 202(0.2630nm) and the plane of 0.2734nm corresponded to theHA lattice plane 300 (0.2720nm). The (202) and (300) planes ofHA crystal are ascribed to the ⟨010⟩ zone axis, thus the ⟨010⟩direction is perpendicular to the paper plane. Because the ⟨001⟩direction of the HA crystal is perpendicular to ⟨010⟩ directionand parallel to (300) lattice plane, we could get the sameconclusion that the c-axis of HA crystal aligned parallel to thelongitudinal direction of the collagen fibril. Moreover, we alsomeasured the lattice plane spacing in Fig. 5b as shown. Bycomparison, the (202) plane of HAwas basically perpendicularto the longitudinal direction of collagen fibril, which wasdifferent from the situation of Fig. 7. It meant that there was anangle between the c-axis of HA and the longitudinal direction ofcollagen fibril. We believed that the individual size of crystallitewas smaller during onset of mineralization, and the calciumphosphate crystals nucleated on the collagen fibril oriented atrandom (Fig. 5b). With the development of mineralization, thecrystallites became larger and the c-axis of HA exhibited thepreferential orientation (Fig. 7). Gradually, the crystallite sizealong the c-axis of HA even grew beyond the size of thediameter of the collagen fibril. Therefore, the c-axis orientedparallel to the longitudinal direction of the fibril in the laterperiod of mineralization by means of structural self-organiza-tion between HA and collagen fibril [9].

The TEM results confirm that the relative orientationbetween HA crystals and collagen fibrils, similar to thenanostructure of calcified natural tissue [28,29], could beobtained in the composite coating by biomimetic growth inMSBF.

4. Conclusion

A carbonate hydroxyapatite–collagen composite coatingwas obtained on NiTi SMA by biomimetic growth in MSBFcontaining high Ca and P ions concentrations. The morphologyof the composite coating was uniform. The main phase of thecoating was HA with B-type carbonate substitution. In theformation process of the composite coating, the c-axis of HAcrystals aligned themselves parallel to the longitudinal directionof the collagen fibrils. This meant that the composite coatingcontained the microstructure similar to bone. Therefore, the

coating is likely to possess bone-like properties and reaction atthe implant site. The relative orientation maintained in thecomposite coating may benefit the surface-modified NiTi SMAto achieve better properties in hard tissue replacement.

Acknowledgements

This work was supported by the National Natural ScienceFoundation of China (Project No. 50471048), SpecializedResearch Fund for the Doctoral Program of Higher Education(Project No. 20040056016), and Scientific and TechnologicalProject of Tianjin (Project No. 043186311).

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