SEM and EDX studies of bioactive hydroxyapatite coatings

on titanium implants

Gabriela Ciobanu a,*, Gabriela Carja a, Octavian Ciobanu b, Ion Sandu c, Andrei Sandu d

a ‘‘Gh. Asachi’’ Technical University of Iasi, Faculty of Chemical Engineering, D. Mangeron Boulevard,

No. 71, Iasi 700050, Romaniab ‘‘Gr. T. Popa’’ Medicine and Pharmacy University of Iasi, Faculty of Medical Bioengineering, Universitatii Street,

No. 16, Iasi 700115, Romaniac ‘‘Al.I. Cuza’’ University of Iasi, Department of Cultural Heritage, Closca Street, No. 9, Iasi 700066, Romania

d Romanian Inventors Forum, Sf. P. Movila Street, No. 3, Et. 3, Iasi 700089, Romania

Received 23 October 2007; received in revised form 28 November 2007; accepted 30 November 2007

Abstract

This work presents a study on an alternative coating method based on biomimetic techniques which are designed to form a

crystalline hydroxyapatite layer very similar to the process corresponding to the formation of natural bone. The HA formation on the

surface of titanium alloy pretreated with NaOH solution is investigated. Two types of solutions such as supersaturated calcification solution

(SCS) and modified SCS (M-SCS) were used to investigate bone-like apatite formation on alkali-treated titanium. The hydroxyapatite

deposits are investigated by means of scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX). The data suggest

that the method utilized in this work can be successfully applied to obtain deposition of uniform coatings of crystalline hydroxyapatite on

titanium substrates.

# 2007 Elsevier Ltd. All rights reserved.

Keywords: Hydroxyapatite; Titanium support; SEM-EDX structural characterization

www.elsevier.com/locate/micron

Available online at www.sciencedirect.com

Micron 40 (2009) 143–146

1. Introduction

Titanium and its alloys are the best metallic materials for

biomedical applications, such as dental, orthopedic implants

and osteosynthesis applications. This is due to high mechanical

resistance, low modulus of elasticity, high corrosion resistance,

and excellent general biocompatibility and atoxicity (Niinomi,

2003; Boehlert et al., 2005).

Hydroxyapatite (HA) is widely used as a bioactive ceramic

since it forms a chemical bonding to bone. Applications

include coatings of orthopedic and dental implants and

scaffolds for bone growth (Chern Lin et al., 2001; Bourgeois

et al., 2003).

The biomimetic methods, applied to produce HA coatings,

have attracted considerable research attention in last decades

(Kokubo, 1996; Ryu et al., 2005; Wu et al., 2006). These

* Corresponding author. Tel.: +40 332 417468; fax: +40 232 271311.

E-mail address: gciobanu03@yahoo.co.uk (G. Ciobanu).

0968-4328/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.micron.2007.11.011

methods produce HA coatings by immersing metal implants in

an aqueous solution containing calcium and phosphate ions at

pH and physiological temperatures.

The aim of this paper is to present a study on an alternative

coating method based on biomimetic techniques which are

designed to form a crystalline HA layer in a way similar to

the process of natural bone formation.

2. Experimental

2.1. Preparation of Ti6Al4V strips

Ti6Al4V alloy bar was cut into rectangular strips with

typical dimensions of 10 mm � 10 mm � 3 mm. Strips were

cleaned with acetone, ethanol and de-ionized water. Samples

were then treated in 0.6 M NaOH solution at 160 8C in a

pressure chamber for 72 h, with heating rates of 5 8C /min.

After alkaline pre-treatment, samples were washed in

deionised water for 5 min and were finally heat-treated at

60 8C for 4 h.

Fig. 1. The SEM micrograph of the Ti6Al4V sample after alkaline/heat

treatment.

Fig. 2. The SEM photographs of the surfaces of Ti6Al4V samples a

G. Ciobanu et al. / Micron 40 (2009) 143–146144

2.2. Coating solutions

SCS solution was prepared by dissolving CaCl2�2 H2O,

NaH2PO4�H2O and NaHCO3 in 1 l of deionized water. The ion

concentrations of SCS solution are 4.0 Mmol/l Na+, 5.0 Mmol/l

Ca+, 10.0 Mmol/l Cl�, 2.5 Mmol/l H2PO4�, and 1.5 Mmol/l

HCO3�. A modified SCS (M-SCS) treatment was used to

deposit HA coating on Ti substrate. M-SCS was prepared by

adding at original SCS quantities of vitamin A (A) and vitamin

D2 (D), with respect A/D rapport of 4.545.

In order to simulate the in vivo process, as-treated titanium

plate was directly immersed into 200 ml SCS solution contained

in a glass beaker, which was kept at 37 8C in a shaking water bath.

The SCS was refreshed every 2 days in order to keep the ion

concentration stable. The titanium samples were taken out of the

solutions after 144 h immersion, rinsed with deionized water,

followed by drying in air at 60 8C for 1 h.

2.3. Samples characterization

Scanning electron microscopy (SEM) coupled with energy

dispersive X-ray spectroscopy (EDX) (VEGA//TESCAN

fter 144 h soaking in: SCS solution (a); M-SCS solution (b–d).

Fig. 3. SEM-EDX spectrum of the biomimetic apatite layer deposited in M-

SCS solution.

G. Ciobanu et al. / Micron 40 (2009) 143–146 145

instrument) was used to observe the morphology and

chemical composition of samples. Silver sputtering was used

to make the coating surfaces conductive for the SEM

investigations.

3. Results and discussion

Biomimetic treatment in SCS solutions used in this study

consisted in two main steps:

� T

reatment in alkaline solutions of NaOH: in this step the

metallic samples are oxidizing in NaOH diluted solutions at

160 8C, and finally heat-treated at 60 8C for 4 h. The

crystalline sodium titanate Na2Ti5O11 (Fig. 1) was formed on

the titanium surface. The structural characteristics of the

coatings followed by XRD (figure not shown) indicate the

presence of Na2Ti5O11 on the titanium surface.

� T

reatment in SCS solution: in this step the apatite layer is

deposited on the surface of metallic samples, in SCS solution.

Two types of solutions such as SCS and modified SCS (M-

SCS) were used to investigate bone-like apatite formation on

alkali-treated titanium. M-SCS was prepared by adding at

original SCS appropriate quantities of vitamin A – retinol (A)

and vitamin D2 (D) to modify the physical structure of the

final product and to enhance the osteoinductive and

biochemical properties of coatings.

For some clinical applications, it may be favourable to

include additional components into the SCS solution during

the formation of the HA. Vitamin A (retinol) plays an

essential role in normal bone and tooth development.

Vitamin D2 (ergocalciferol) promotes bone formation and

mineralization and is essential in the development of

skeleton and tooth.

Fig. 2 shows a comparison of the SCS and M-SCS

treatments of Ti samples and the resulting HA layers. Sample

immersed in SCS had exhibited acicular shaped HA crystals

and many areas of noncoverage. After 144 h soaking titanium

sample in SCS, inhomogeneously distributed HA precipitates

with a diameter of approximately 2–5 mm were formed on the

Ti surface (Fig. 2a).

Sample immersed in M-SCS had exhibited a completely

covered layer on Ti surface (Fig. 2b); the uniformity of the

coatings is revealed by the results presented in Fig. 2b–d (at

different magnitudes). It can be observed that the apatite layer

exhibits a petal rose-like morphology.

The previous published literature data, point out that the

conventional SCS solution is buffered with Hepes or Tris–

hydroxymethyl aminomethane (TRIS) to adjust the pH value

to 7.2 or 6.2 in order to maintain the chemical stability of the

solution (Ryu et al., 2005; Wu et al., 2006). We have not used

a buffer in the SCS or M-SCS (modified SCS) treatments for

obtaining a more economic and simpler method. This

explains the fact that the HA growth is poor, as presented in

Fig. 2a. But, in M-SCS, Fig. 2b–d indicates a very good HA

growth. This is due, probably, to the vitamins added. The

structural characteristics of the coatings followed by XRD

(figure not shown) indicate the HA presence on the titanium

surface.

SEM-EDX analysis showed that the petal rose-like apatite

crystallites (in Fig. 2b) are composed mainly of hydroxyapatite

and exhibits a molar Ca/P ratio of 1.67 (Fig. 3).

The samples were seeded with osteoblastic cells and

cultured for 36 h in a CO2 incubator at 37 8C. After culturing,

alkaline phosphatase (ALP) activity was assayed. These

coatings are biocompatible, but this is subject of other studies.

This study is only regarding to characterize the HA coatings in

SCS and M-SCS solutions.

4. Conclusions

The goal of this study was to develop an improved method

for depositing HA coatings, which promotes biological

activity, on the surface of Ti implant materials. The proposed

biomimetic method is a simple way to grow HA coatings on

titanium substrates at room temperature using a technique

more effective than those reported previously. A modified

SCS (M-SCS) treatment was used in the present study to

deposit HA coating on titanium substrate after alkaline/heat

treatment. M-SCS was prepared by added at original SCS

appropriate quantities of vitamin A (A) and vitamin D2 (D),

with respect A/D rapport of 4.545. The vitamin A (A) and

vitamin D2 (D) are included in minor amounts in our M-SCS

solution to modify the physical structure of the final product

and to enhance the osteoinductive and biochemical properties

of coatings. SEM-EDX studies confirm the formation of HA

on the Ti substrate. Experimental results suggested that this

modified biomimetic method is very simple and highly

effective and it can be successfully applied to obtain

deposition of uniform coatings of crystalline hydroxyapatite

on titanium substrates.

G. Ciobanu et al. / Micron 40 (2009) 143–146146

Acknowledgement

The authors gratefully acknowledge the financial assistance

from the Ministry of Education and Research, Romania (Grant

al Academiei Romane, No. 62/04.09.2007).

References

Boehlert, C.J., Cowen, C.J., Jaeger, C.R., Niinomi, M., Akahori, T., 2005.

Tensile and fatigue evaluation of Ti–15Al–33Nb (at.%) and Ti–21Al–29Nb

(at.%) alloys for biomedical applications. Mater. Sci. Eng. C 25/3, 263–275.

Bourgeois, B., Laboux, O., Obadia, L., Gauthier, O., Betti, E., Aguado, E.,

Daculsi, G., Bouler, J.-M., 2003. Calcium-deficient apatite: a first in vivo

study concerning bone ingrowth. J. Biomed. Mater. Res. A. 65 (3), 402–408.

Chern Lin, J.H., Kuo, K.H., Ding, S.J., Ju, C.P., 2001. Surface reaction of

stoichiometric and calcium-deficient hydroxyapatite in simulated body

fluid. J. Mater. Sci.: Mater. Med. 12, 731–741.

Kokubo, T., 1996. Formation of biologically active bone-like apatite on metals

and polymers by a biomimetic process. Thermochim. Acta 280/281, 479–

490.

Niinomi, M., 2003. Recent research and development in titanium alloys for

biomedical applications and healthcare goods. Sci. Technol. Adv. Mater. 4,

445–454.

Ryu, H.S., Song, W.-H., Hong, S.-H., 2005. Biomimetic apatite induction on

Ca-containing titania. Curr. Appl. Phys. 5/5, 512–515.

Wu, J.-M., Zhang, S.-C., Li, Y.-W., Zhao, F.-D., Wang, M., Osaka, A., 2006.

Influence of film thickness on in vitro bioactivity of thin anatase films

produced through direct deposition from an aqueous titanium tetrafluoride

solution. Surf. Coat. Technol. 201/6, 3181–3187.