International Biodeterioration & Biodegradation 64 (2010) 81e85

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International Biodeterioration & Biodegradation

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Short communication

Biodegradation of metallic surgical implants investigated using an

ultrasound-assisted process combined with ICP-OES and XRD
Anna Lesniewicz, Lukasz Gackiewcz, Wieslaw Zyrnicki*
Wroclaw University of Technology, Chemistry Department, Analytical Chemistry Division, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Biodegradation of metallic implant materials was investigated in solutions to simulate conditions in the
Received 19 August 2008 human body. Saliva and body fluids were represented by phosphate-buffered saline solution, Hank's
Received in revised form solution, and fluoridated saliva solution. Alloys of titanium, niobium, nickel, and two austenitic stainless
16 September 2009
steels were examined as the implant materials. In vitro metals released from the alloys by ultrasonically
Accepted 20 September 2009
Available online 3 December 2009
assisted leaching and conventional static immersion tests were compared to evaluate the application of
ultrasound in a long-term implant biodegradation study. Atomic emission spectroscopy (ICP-OES) was
employed to determine the metals liberated during immersion of the implant materials. Corrosion of the
Keywords:
Metals and alloys materials and morphological changes in the surface structure were investigated using an X-ray powder
Implant materials diffraction method.
Biodegradation Ó 2009 Elsevier Ltd. All rights reserved.
Ultrasonically assisted elemental release
ICP-OES
XRD

1. Introduction
Metal alloys used in surgical implants must demonstrate unique
properties such as good mechanical strength and biocompatibility,
high corrosion resistance, safety, ductility, and wear resistance.
The most frequently used alloys-stainless steel (Gebert et al.,
2007), cobaltechromium (Sudhakar, 2005), and titanium alloys
(Meticos-Hukovic et al., 2006)dhave been used in medicine and
dentistry for fracture fixation plates, bone screws, joint replacement
devices, and dental implants. Also, the noble alloys (Wataha et al.,
2001), NiTi memory alloys (Li and Zheng, 2006), NiCr-based alloys
(Huang, 2003), and several polymers, including biodegradable
materials (Shum and Mak, 2003), have been thoroughly examined
and are commonly employed in biomedical and dental applications.
Risk assessment in the clinical use of implants and other
materials is concerned mainly with decomposition and degrada-
tion of the materialsdthe processes leading to implant failure.
Most often, electrochemical corrosion studies have been performed
using potentiometric and voltammetric techniques in order to
determine the corrosion resistance of alloys in different media
simulating body fluids (Sudhakar, 2005; Meticos-Hukovi c et al.,
2006; Oliviera et al., 2006; Chieh et al., 2003; Khan et al., 1999; Li
* Corresponding author. Tel./fax: þ48 71 320 24 94.
E-mail address: wieslaw.zyrnicki@pwr.wroc.pl (W. Zyrnicki).
et al., 2006). The influence of conditions inside the human body on
corrosion of implant materials has been investigated using solu-
tions reproducing saliva and body fluids, for example, blood serum
and tissue extracts (Cheng et al., 2007). A saliva substitute solution
called Fusayama or FusayamaeMeyer artificial saliva, containing
NaCl, KCl, CaCl2, NaH2PO4, Na2S, and urea, has commonly been
prepared (Huang, 2003).
Phosphate-buffered saline (PBS) solution has been prepared by
dissolution of NaCl, KCl, Na2HPO4, and KH2PO4, with the addition of
NaN3 to prevent bacterial growth for studies investigating a rela-
tionship between degradation and time (Shum and Mak, 2003).
The corrosion resistance of different alloys has also been verified
in Hank's solution, a nutritive salt solution for culturing animal cells
containing CaCl2, MgSO4, KCl, KH2PO4, NaHCO3, NaCl, and Na2HPO4,
usually used in combination with naturally occurring bodily
substances (e.g., glucose, blood, serum and tissue extracts) (Meticos-
Hukovi c et al., 2006).
Implant failure mechanisms have also been studied previously
in alkaline (NaOH) (Yaniv et al., 1997), neutral (phosphate buffer
(Oliviera et al., 2006), NaCl (Meticos-Hukovi c et al., 2006)), and
acidic solutions (e.g., HCl, HNO3, H2SO4, HClO4, or H3PO4) (Chieh
et al., 2003).
The influence of the degradation environment on the corrosion
and wear behaviour characteristics of these materials has been
examined for various periods of time, ranging from 10 min
(Khan et al., 1999) up to 200 days (Gebert et al., 2007). These studies

0964-8305/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2009.09.012
82 A. Lesniewicz et al. / International Biodeterioration & Biodegradation 64 (2010) 81e85

have used changes in weight (Chieh et al., 2003) and material Table 2
hardness (Khan et al., 1999) as outcomes and have included surveys Chemical composition and properties of PBS, and Hank's and saliva solutions.

of material surfaces performed using techniques such as surface Extractant Chemical composition Properties
profilometry (Khan et al., 1999), atomic force microscopy (Chieh Constituent Concentration
et al., 2003), thermal analysis (Shum and Mak, 2003), and X-ray [g l1]
diffraction (Li et al., 2006). Since biodegradation can result in the Phosphate Buffer NaCl 8.00 Isoosmolar phosphate
dissolution of the metallic implants, elemental release from these Saline (PBS) KCl 0.20 buffer taken as model
alloys should be investigated as a reason for clinical implant failure solution; Na2HPO4  12H2O 0.63 body fluid
pH ¼ 7.4 KH2PO4 0.63
and for allergic, inflammatory, toxic, and even carcinogenic effects.
NaN3a 0.20
Chemical analysis of extracts obtained after immersion of the
metallic materials in solutions simulating body fluids has usually Hank's solution; NaCl 8.00 Solution taken as
pH ¼ 7.4 KCl 0.40 simulated body fluid
been performed with the use of spectroscopic methods, for
NaHCO3 0.35
example AAS, ICP-OES, and ICP-MS (Meticos-Hukovic et al., 2006; CaCl2 0.14
Wataha et al., 2001). MgCl2  6H2O 0.10
The purpose of the present study was to evaluate the applicability Na2HPO4  12H2O 0.06
of ultrasound assistance in investigating the corrosion of selected KH2PO4 0.06
MgSO4  7H2O 0.06
metals and alloys (considered as potential implants), and the use of Glucose 1.00
X-ray diffraction to study surface degradation and changes of the NaN3a 0.20
implants in solutions reproducing human body fluids.
Fluoridated NaCl 0.40 Artificial saliva enriched
saliva KCl 0.40 with fluoride ions
2. Materials and methods solution; CaCl2 0.90
pH ¼ 5.3 NaH2PO4  2H2O 0.70
Na2S  9H2O 0.01
2.1. Implant materials and artificial body fluids Urea 1.00
NaF 1.00
Nickel, niobium, and titanium and their alloys, as well as two NaN3a 0.20
kinds of austenitic stainless steel were used for investigation and a
NaN3 addition prevents bacterial growth.
employed in the form of planar pieces of metal. Table 1 shows the
nominal composition of the examined materials.
Before the experiments, the metal surfaces were cleaned for days of alloy immersion. The flasks were agitated gently every day.
15 min in 10 M HCl in the ultrasonic bath, then rinsed once with In the ultrasound-assisted tests for metal release investigation, the
acetone and several times with deionised water, and finally dried as metal concentrations in the solutions were measured after 30, 50,
described by Takeuchi et al. (2003). 100, and 150 h. The sonication was carried out in a 50-Hz ultrasonic
All reagents used in this study were of analytical grade or bath (Ultrasons-H model, 930 W, J.P. Selecta).
better. Extractants, i.e., the PBS, Hank's, and saliva solutions, were
prepared by dissolution and/or dilution of appropriate commer-
cially available salts and solutions. Detailed information about the 2.3. Measurement methods
chemical compositions of the solutions reproducing human body
fluids is given in Table 2. Aqueous standard solutions used for For studying release of the metals into solution, their concen-
calibration in the optical emission spectrometry were prepared via trations were measured in the extracts by inductively coupled
dilution of the ICP multi-element standards obtained from Merck plasma-optical emission spectrometry (ICP-OES) using a Jobin-
KGaA. All dissolutions of salts and dilutions were performed with Yvon 38S spectrometer equipped with a V-groove nebuliser and
deionised water obtained from an EASYpureÔ unit (Barnstead). Scott-type spray chamber. The operating parameters and analytical
line wavelengths chosen for the measurements were typical. To
2.2. Static and ultrasound-assisted immersion tests ensure high-quality trace metal determination, with each set of
samples a blank sample was prepared through the complete
Precisely weighed metal planar forms were placed in conical analytical procedure and used for correction of analytical signals
flasks containing 100 or 250 ml of the PBS, Hank's solution, and measured by ICP-OES.
artificial saliva solution. The flasks were protected with laboratory An X-ray powder diffraction method (XRD) was applied to
sealing film and placed in a water bath at 37  C (human body investigate morphological changes in the surface structures of the
temperature). In the conventional static tests, the released element examined metallic implant materials. Measurements were done in
concentrations were measured in samples collected after 45 and 84 the symmetric q/2q Bragg-Brentano geometry using a Philips

Table 1
The nominal chemical composition (in weight percentage) of the studied materials.

Material Elements concentration [%]

Al Cr Fe Mo Mn Nb Ni Ti Others
Niobium <0.003 <0.005 bal <0.003 C, H, N, O, Si, Ta, W, Zr
Niobium alloy 0.005 0.005 0.02 0.02 0.005 bal 0.02 0.005 Zr: 0.8e1.2, C, H, N, O, Si, Ta, W
Nickel <0.2 <0.3 bal <0.1 C, Cu,
Nickel alloy 22.5 0.5 9.5 1 bal C, Ce, Si
Titanium 0.005 0.095 bal C, N, O
Titanium alloy 6.12 3.87 bal C, N, O
Austenitic stainless steel 19e19 bal <2 8e10 <0.15% C, <1.0% Si
Austenitic stainless steel SUS 316L 0.94 16e18.5 bal 2e3 <2 10e14 <0.03% C, <0.045% P, <0.03% S, <1% Si
 A. Lesniewicz et al. / International Biodeterioration & Biodegradation 64 (2010) 81e85 83

Saliva solution 3. Results and discussion

3,5
Fe concentration [µg/g]

3 3.1. Conventional and ultrasound-assisted metal release

2,5
The fundamental requirements addressed by metallic implant
2
CE materials are high chemical and physical resistance to biodegra-
1,5 UE dation in the human body over a very long time. However, this
1 means that any evaluation of the results of biodegradation should
0,5 also be carried out over a long time period. Therefore, a search for
0
methods allowing the acceleration of such processes is of high
SUS 316 L steel Austenitic steel Ti alloy importance. It is well known that sonication accelerates digestion
and leaching processes. Thus, it seemed worthwhile to compare
Hank's solution the efficiencies of conventional leaching during a long-lasting
0,7 static immersion and an ultrasonically accelerated extraction.
Metal releases from nickel, niobium, and titanium metals and their
Fe concentration [µg/g]

0,6

0,5
alloys and austenitic stainless steels into the solutions simulating
human body conditions were investigated and the concentration of
0,4 CE
a number of elements was measured. The elemental releases evidently
0,3 UE
depended upon the composition of the extraction solution. The
0,2 leaching efficiencies of the static process and sonication also differed
0,1
considerably for the alloys examined. The precision of the measure-
ments was investigated via the analysis of five sub-samples in each of
0
SUS 316 L steel Austenitic steel Ti alloy
two runs; relative standard deviations varied from 5% to 15%.
As a rule, the release was the highest for phosphate-buffered
Fig. 1. Comparison of the conventional extraction (CE) and ultrasonically assisted (UE) saline solution (PBS), at an intermediate level for artificial saliva,
leaching efficiencies (static immersion: 84 days; sonication: 150 h). and the lowest for Hank's solution.
A comparison of the metal extraction efficiencies for the
conventional (84 days) and the ultrasound-assisted leaching
(150 h) techniques was conducted. At such a choice of the leaching
X'PERT system. The diffractometer was equipped with a CuKa times the metal concentrations in solutions were comparable and
(l ¼ 0.154 nm) source. Identification of the phases was performed easily measurable. Fig. 1 shows a compilation of the selected
by comparison of the experimental powder diffraction patterns elemental concentration determinations in extracts. As can be
with reference patterns collected in the Powder Diffraction Files deduced from these results, the use of ultrasound energy caused
(International Centre for Diffraction Data PDF-2 base). a remarkable shortening of the leaching time necessary to observe

Titanium alloy Titanium alloy - leaching in the saliva solution

2500
4500
2250
4000
2000
3500 28.3
1750
Counts

3000
Counts

1500
2500
1250
2000
1000
1500 35.5
750
1000 10.4 25.6
500 31.0
500
250
0 0
5 15 25 35 45 55 65 5 15 25 35 45 55 65

2 Theta angle range 2 Theta angle range

Titanium alloy - leaching in the Hank's solution Titanium alloy - leaching in the PBS solution
5000 31.6 4000
4500 31.8
3500
4000
3000
3500
Counts
Counts

3000 2500

2500 2000
10.6 28.7
2000 1500
1500 35.5
28.5 1000 9.6
35.2
1000 45.2
500
500 56.3
0
0
5 15 25 35 45 55 65
5 15 25 35 45 55 65
2 Theta angle range 2 Theta angle range

Fig. 2. Ti-alloy X-ray diffraction patterns.

84 A. Lesniewicz et al. / International Biodeterioration & Biodegradation 64 (2010) 81e85

Niobium alloy - leaching in the Hank's solution Nickiel alloy - leaching in the PBS solution

2000 6000
1800
5000
1600
1400 4000

Counts
Counts

1200 31.6
1000 31.4 3000
28.5
800
2000 27.2
600 28.3 13.3
400
27.8 1000
200 10.4
0 0
5 15 25 35 45 55 65 5 15 25 35 45 55 65

2 Theta angle range 2 Theta angle range

SUS 316L steel - leaching in the Hank's solution SUS 316L steel - leaching in the PBS solution
31.7
12000 10000
11000
9000
10000
8000
9000 28.6 31.6

Counts
Counts

8000 7000

7000 6000
6000 5000
5000
4000 10.4
4000
3000
3000
2000 2000
5 15 25 35 45 55 65 5 15 25 35 45 55 65

2 Theta angle range 2 Theta angle range

Fig. 3. X-ray diffraction patterns niobium and nickel alloys and austenitic steels.

comparable metal releases. This means that ultrasound-assisted
leaching is unquestionably a useful technique in biomaterial
degradation investigations.
To evaluate the influence of the extraction time, examined
material samples were immersed in the solutions simulating
human body fluids for 45 and 84 days (the conventional extraction)
and for 50, 100, and 150 h (sonication).
The metal releases from the alloys into the leaching solutions
increased over time, at observed rates that were comparable to
those found elsewhere in the literature. However, in the case of
steel materials, the continuous increase in leaching versus time was
observed in sonication only up to a time of 100 h, after which the Cr,
Fe, Mn, and Ni concentrations in the solutions stabilised. This effect
could be explained by changes in alloy surface structures, mainly
formation and deposition of any metal salts on the alloy surface.
Therefore, surface investigations by X-ray diffraction spectrometry
were deemed necessary.
3.2. Phase changes in metal surfaces
Although the X-ray powder diffraction method has been used
quite often to study the biodegradation of metallic implant
surfaces, to the best of our knowledge its application to an inves-
tigation and comparison of alloy degradation due to various bio-
logical fluids and liquids has not been reported to date.
The solutions used here differed both in chemical composition
and in pH value. The results for the Ti alloy are presented in Fig. 2;
Fig. 3 shows the results for the niobium alloy, nickel alloy, and SUS
316L austenitic stainless steel. The X-ray diffraction data showed
the formation of new compounds on the alloy surfaces.
The XRD analysis of the titanium alloy (performed before the
degradation process) revealed the presence of seven peaks
distinctly associated with the Ti-alloy phases. The X-ray diffraction
pattern of the titanium alloy after 84 days of degradation in the
saliva, Hank's solution, and PBS solution showed the presence of
several additional peaks (see Fig. 2, peaks marked with B). The
peak positions and intensities depended on the composition of the
extractant solution used. The peaks were identified as Na2HPO4
(main peaks position according to the ICDD PDF-2 base:
2q ¼ 25,530 and 35,508), NaH2PO4 (for hydrate 2q ¼ 25,531), KCl
(2q ¼ 28,345), NaCl (2q ¼ 31,691 and 45,447), and Na2CO3
(2q ¼ 35,051). This means that one can observe, in addition to
corrosion, the process of deposition on the implant surface of salts
from ions existing in the solution used.
Similarly, X-ray diffractograms obtained for the niobium alloy,
nickel alloy, and austenitic steels after immersion also show the
appearance of peaks indicating the formation of metal salts during
degradation occurring in Hank's and PBS solutions (peaks marked
with B, Fig. 3).
The dependence of this process on the chemical composition of
the degradation environment could be explained by the stabilisa-
tion of ionic concentrations during the long-term immersion
studies. The new peaks found by XRD on the surface of the alloys
immersed in Hank's solution were identified as belonging to KCl
(2q ¼ 28,345) and NaCl (2q ¼ 31,691), while the peaks occurring in
PBS solution were determined to be NaH2PO4 (2q ¼ 10,324), K3PO4
(2q ¼ 13,438), NaCl (2q ¼ 31,691), and KCl (2q ¼ 28,345).
4. Conclusions
The ICP-OES and XRD methods were found here to be very suit-
able and mutually complementary for studying the biodegradation of
metal alloys in artificial body fluids. As such, a combination of these
two methods was developed and applied for the first time here for
the alloys of titanium, niobium, and nickel, as well as for both
austenitic stainless steels when in contact with phosphate-buffered
saline solution, Hank's solution, and fluoridated saliva solution.
It was demonstrated that the application of ultrasound energy
accelerated the implant material biodegradation process by 20e40
times, allowing for the relatively rapid evaluation of normally long-
term processes occurring when metal implants are affected by
body fluids.
 A. Lesniewicz et al. / International Biodeterioration & Biodegradation 64 (2010) 81e85
The XRD study showed that several salts that are normal compo-
nents of body fluids were deposited on the implant surfaces, which
might protect the implants against further corrosion to some degree.
More extended studies on the corrosion and degradation
processes of implants under conditions simulating human body
conditions are currently being undertaken.
References
Cheng, F.T., Lo, K.H., Man, H.C., 2007. An electrochemical study of the crevice
corrosion resistance of NiTi in Hank's solution. Journal of Alloys and
Compounds 437, 322e328.
Chieh, T.C., Chu, J., Liu, C.T., Wu, J.K., 2003. Corrosion of Zr52.5Cu17.9Ni14.6Al10Ti5 bulk
metallic glasses in aqueous solutions. Materials Letters 57, 3022e3025.
Gebert, A., Roth, S., Gopalan, R., Kündig, A.A., Schultz, L., 2007. Corrosion behavior of
FePt-based bulk magnets in artificial saliva solution. Journal of Alloys and
Compounds 436, 309e312.
Huang, H.-H., 2003. Surface characterization of passive film on NiCr based dental
casting alloys. Biomaterials 24, 1575e1582.
Khan, M.A., Williams, R.L., Williams, D.F., 1999. Conjoint corrosion and wear in
titanium alloys. Biomaterials 20, 765e772.
85
Li, C., Zheng, Y.F., 2006. The electrochemical behavior of a Ti50Ni47Fe3 shape
memory alloy. Materials Letters 60, 1646e1650.
Li, M.G., Sun, D.Q., Qiu, X.M., Yin, S.Q., 2006. Corrosion behavior of the laser-brazed
joint of TiNi shape memory alloy and stainless steel in artificial saliva. Materials
Science and Engineering A 441, 271e277.
Meticos-Hukovi c, M., Pili
c, Z., Babi
c, R., Omanovi c, D., 2006. Influence of alloying
elements on the corrosion stability of CoCrMo implant alloy in Hank's solution.
Acta Biomaterialia 2, 693e700.
Oliviera, N.T.C., Ferreira, E.A., Duarte, L.T., Biaggio, S.R., Rocha-Filho, R.C., Bocchi, N.,
2006. Corrosion resistance of anodic oxides on the Ti-50Zr and Ti-13Nbe13Zr
alloys. Electrochimica Acta 51, 2068e2075.
Shum, A.W.T., Mak, A.F.T., 2003. Morphological and biomechanical characterization
of poly(glycolic acid) scraffolds after in vitro degradation. Polymer Degradation
and Stability 81, 141e149.
Sudhakar, K.V., 2005. Investigation of failure mechanism in vitallium 2000 implant.
Engineering Failure Analysis 12, 257e262.
Takeuchi, M., Abe, Y., Yoshida, Y., Nakayama, Y., Okazaki, M., Akagawa, Y., 2003. Acid
pre-treatment of titanium implants. Biomaterials 24, 1821e1827.
Wataha, J.C., Nelson, S.K., Lockwood, P.E., 2001. Elemental release from dental
casting alloys into biological media with and without protein. Dental Materials
17, 409e414.
Yaniv, A.E., Lumsden, J.B., Staehle, R.W., 1997. The composition of passive films
on ferritic stainless steels. Journal of the Electrochemical Society 124 (4),
490e496.