Cytocompatibility of pure metals and experimentalbinary titanium alloys for implant materials

Yeong-Joon Park a,*, Yo-Han Song a, Ji-Hae Ana, Ho-Jun Song a,Kenneth J. Anusavice b

aDepartment of Dental Materials and MRC for Biomineralization Disorders, School of Dentistry,

Chonnam National University, Gwangju 500-757, South KoreabDepartment of Restorative Dental Sciences, College of Dentistry, University of Florida, Gainesville,

FL 32610-0415, USA

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 1 2 5 1 – 1 2 5 8

a r t i c l e i n f o

Article history:

Received 25 July 2013

Received in revised form

11 September 2013

Accepted 13 September 2013

Keywords:

Cytocompatibility

Titanium alloy

Implant

WST-1 test

Agar overlay test

a b s t r a c t

Objective: This study was performed to evaluate the biocompatibility of nine types of pure

metal ingots (Ag, Al, Cr, Cu, Mn, Mo, Nb, V, Zr) and 36 experimental titanium (Ti) alloys

containing 5, 10, 15, and 20 wt% of each alloying element.

Methods: The cell viabilities for each test group were compared with that of CP-Ti using the

WST-1 test and agar overlay test.

Results: The ranking of pure metal cytotoxicity from most potent to least potent was as

follows: Cu > Al > Ag > V > Mn > Cr > Zr > Nb > Mo > CP-Ti. The mean cell viabilities for

pure Cu, Al, Ag, V, and Mn were 21.6%, 25.3%, 31.7%, 31.7%, and 32.7%, respectively, which

were significantly lower than that for the control group ( p < 0.05). The mean cell viabilities

for pure Zr and Cr were 74.1% and 60.6%, respectively ( p < 0.05). Pure Mo and Nb demon-

strated good biocompatibility with mean cell viabilities of 93.3% and 93.0%, respectively. The

mean cell viabilities for all the Ti-based alloy groups were higher than 80% except for Ti–

20Nb (79.6%) and Ti–10V (66.9%). The Ti–10Nb alloy exhibited the highest cell viability

(124.8%), which was higher than that of CP-Ti. Based on agar overlay test, pure Ag, Cr,

Cu, Mn, and V were ranked as ‘moderately cytotoxic’, whereas the rest of the tested pure

metals and all Ti alloys, except Ti–10V (mild cytotoxicity), were ranked as ‘noncytotoxic’.

Significance: The results obtained in this study can serve as a guide for the development of

new Ti-based alloy implant systems.

# 2013 Elsevier LtdElsevier B.V. All rights reserved.

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1. Introduction

Recently, commercially pure titanium (CP-Ti) has become

widely used as a biomaterial for dental implants, orthopaedic

implants, cardiovascular appliances, and implant-supported

dental crowns because of outstanding characteristics that

* Corresponding author at: Department of Dental Materials, School of Dgu, Gwangju 500 757, South Korea. Tel.: +82 62 530 4871; fax: +82 62 5

E-mail addresses: yjpark@jnu.ac.kr, parkyj61@hanmail.net (Y.-J. Pa

0300-5712/$ – see front matter # 2013 Elsevier LtdElsevier B.V. All righttp://dx.doi.org/10.1016/j.jdent.2013.09.003

include high specific strength, high resistance to corrosion,

greater biocompatibility, low modulus of elasticity, and high

capacity to be osseointegrated with bone.1–3 However, the use

of unalloyed CP-Ti requires further improvement to overcome

its limitations including strength, hardness, wear resistance,

fatigue strength, and poor grindability. It is desirable also to

decrease its elastic modulus as close as possible to that of bone

entistry, Chonnam National University, 300 Yongbong dong, Buk30 4875.rk).

hts reserved.

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 1 2 5 1 – 1 2 5 81252

tissue.4–11 Reports have focused on improved properties of CP-

Ti for use as implant materials. In a study of Ti–Ag and Ti–Cu

alloys,12 Ti–20% Ag and Ti–5% Cu alloys exhibited better

grindability than pure Ti, which is a desirable property for

dental CAD/CAM alloys. The results from a previous study

utilizing experimental Ti–Au (5–20 wt% Au), Ti–Ag (5–20 wt%

Ag), and Ti–Cu (2–10 wt% Cu) alloys suggested that their

hardness and tensile strength were higher than these

properties for pure Ti.13 In another study, Ti–Cr alloys

exhibited greater flexure strength than CP-Ti, which was

associated with the strengthening effect of the v phase.5 The

flexure strength of the Ti–20Cr alloy was about 80% greater

than that for CP-Ti, and the elastic recovery capability was

460% greater than that of CP-Ti. The lower elastic modulus of

CP-Ti and Ti alloys, compared with 316L stainless steel and Cr–

Co alloys, is potentially advantageous for preserving the

surrounding bone by minimizing the reduction in physiologi-

cal stress within bone and a reduction in bone density. This

occurs because of a more favourable match of elastic modulus

between the implants and bone.14–18 The elastic moduli of

recently developed b-Ti alloys range from 55 to 85 GPa, which

are much lower than those of 316L stainless steel, Cr–Co alloys,

and CP-Ti.2 Manganese (Mn), molybdenum (Mo), and niobium

(Nb) have been investigated as b stabilizers for Ti alloys and

studies have suggested that they can decrease the elastic

modulus and increase other important mechanical properties

of Ti-based alloys.19–27 Biocompatibility tests for dental alloys

including Ti alloys have been performed in various stud-

ies.19,28–31 When metallic materials are implanted inside a

body, they may corrode and/or wear. The released metal ions

and/or debris can be toxic or irritating to surrounding tissues.

The release of metallic ions during the destruction of the

passive film can cause side effects in the body. Even though

the Ti–6Al–4V alloy is an established implant material in

orthopaedics, it is reported that ions associated with Ti–6Al–

4V alloy inhibit the normal differentiation of bone marrow

stromal cells to mature osteoblasts in vitro.32 If biologically

relevant molecules are released from the metallic biomaterial

and interact with biologically relevant molecules, biologically

active organo-metallic and metallic salts can be formed.30,33–40

Thus, the biocompatibility of the metallic materials used for

implant treatment should be evaluated during the develop-

ment of Ti alloys.

Cytotoxicity of a biomaterial can be examined using either

a monolith of the material,41 a particulate form,42–44, or

Table 1 – Materials used in the study.

Raw material Specification

CP-Ti (Grade 2) Rod, 10 mm dia. ASTM B265

Titanium Sponge 3 mm and down 99.9%

Aluminium Foil, 1.0 mm thick, annealed, 99.99%

Chromium Pieces, 2–3 mm thick, 99.995%

Copper Shot, 13 mm dia., 99.99%, oxygen free

Manganese Granules, 0.8–10 mm, 99.98%

Molybdenum Foil, 1.0 mm thick, 99.95%

Niobium Sheet, 99.9%

Silver Silver shot, 1–5 mm, Premion1, 99.99%

Vanadium Pieces, 99.7%

Zirconium Foil, 0.02 mm thick Annealed, 99.8%

extracted solutions, which may contain several types of

released metallic compounds.45–48 Most of the biocompatibili-

ty reports for metallic elements have been based on the use of

metal salts or on extracted solutions containing the metal

cations.45,49,50 Because the cytotoxicity evaluations of metallic

compounds are performed at different places by various

methods using several kinds of cell lines, the results cannot be

compared directly to each other. Moreover, the mass release of

a particular element is not expected to be proportional to its

atomic percentage in the alloy, and the lability of an element

can be altered by other elements in the alloy.51,52 Thus, when

developing new Ti alloys, cytotoxicity assays for the fabricated

alloys and the constituent pure metals are useful to study the

effect of each component on the behaviour of cells. The in vitro

cytotoxicity tests are relatively fast and they can be standard-

ized relatively easily. Thus, the in vitro tests can provide highly

reliable data and reproducible measurements even though

their relevance to clinical practice is not always consistent.53

The evaluation of the bulk alloy cytotoxicity in a biological

environment should be performed initially. However, to date,

there is scant information about the cytotoxicity of Ti-alloy

ingots.

The aim of this study was to evaluate the biocompatibility

of candidate Ti-alloys using well-characterized fibroblast-like

cell lines. Candidate alloying elements with Ti (Ag, Al, Cr, Cu,

Mn, Mo, Nb, V, and Zr) were evaluated. Titanium alloys with

varying elemental contents of alloying elements were evalu-

ated for their cytotoxicity, consistent with the aim of

developing new Ti-alloy systems as dental implant materials.

2. Materials and methods

2.1. Sample preparation

Binary Ti–A alloys that varied in the concentrations of element

A (where A was Ag, Al, Cr, Cu, Mn, Mo, Nb, V, and Zr), in

concentrations of 5, 10, 15 or 20 wt% in the Ti alloys, were

fabricated using vacuum arc melting under a high purity argon

atmosphere on a water-cooled hearth (Table 1). To homoge-

nize the alloys, the prepared ingots were melted seven times,

and the alloy specimens were treated for 4 h at temperatures

150 8C below the respective solidus temperatures and furnace

cooled at an approximate rate of 10 8C/min to 600 8C in a high

purity argon atmosphere followed by air cooling to room

Lot no. Manufacturer

04RB-10 Daido Steel Co. Ltd., Nagoya, Japan

129Q001 Alfa Aesar, Ward Hill, USA

40762 Alfa Aesar, Ward Hill, USA

38494 Alfa Aesar, Ward Hill, USA

36686 Alfa Aesar, Ward Hill, USA

K21T034 Alfa Aesar, Ward Hill, USA

36215 Alfa Aesar, Ward Hill, USA

SH-NB04 GMH Stachow-Metall GmbH, Goslar, Germany

12186 Alfa Aesar, Ward Hill, USA

42775 Alfa Aesar, Ward Hill, USA

44752 Alfa Aesar, Ward Hill, USA

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 1 2 5 1 – 1 2 5 8 1253

temperature. Prepared alloy buttons were cut into disks with a

diameter of 10 mm and a thickness of 1.2 mm. The disks were

polished by a sequence of coarser to finer abrasives succes-

sively through 2000 grit SiC abrasive. They were ultrasonically

cleaned in acetone, ethanol, and distilled water. CP-Ti disks

were used as a control group.

2.2. Cell viability test

The viability of cells was analysed using a colorimetric assay to

quantify cleavage of the tetrazolium salt WST-1 (Roche,

Germany) by mitochondrial dehydrogenases. The resulting

dye was quantified spectrophotometrically and directly

correlated to the number of metabolically active cells in the

culture.54 For the test, L-929 mouse fibroblast cells were grown

on a 100-mm-diameter Petri dish containing 10 mL of

RPMI1640 supplemented with 10% foetal bovine serum

(FBS). When sufficient number of cells had proliferated,

100 mL of the cell suspension at a cell density of

5 � 104 cells/mL were seeded on the test samples and

incubated at 37 8C under 5% CO2. After 24 h of incubation,

the cells were detached with 10 mL of 0.05% Trypsin–EDTA

solution (Gibco, Canada) for 3 min, and 100 mL of RPMI1640

were added to each well. Then, 100 mL of the mixture were

transferred into a 96-well culture plate, and 10 mL of WST-1

solution were added to each well. The cells were incubated at

37 8C in 5% CO2 for 4 h, shaken for 1 min, and the absorbance

was measured at 450 nm by a microplate reader (Bio-Rad,

USA). The cell viability on each pure metal and their alloy

samples was compared with that of CP-Ti, and this result was

expressed as the cell viability ratio (%) vs. CP-Ti. Analyses were

performed for seven samples per test group.

2.3. Agar overlay test

Ten microlitre aliquots of L-929 cell suspensions at a cell

density of 3 � 105 cells/mL were seeded in 100-mm-diameter

cell culture dishes (Corning, USA) and incubated to confluence

at 37 8C in 5% CO2. After 24 h of incubation, the medium was

replaced with 10 mL of freshly prepared agar/nutrient medium

containing RPMI1640, 5% FBS, and a 3% agarose mixture

(Sigma–Aldrich, USA). Ten millilitres of neutral red solution

(0.01% in phosphate-buffered saline; Sigma–Aldrich, USA)

were added and the cells were incubated for 15 min at room

temperature. Excess dye was removed and the test specimens

were placed on the agar surface. A 0.25% zinc dibutyldithio-

carbamate polyurethane film was used as a positive control

and a polyethylene sheet was used as negative control. The

dishes were incubated for 24 h at 37 8C in 5% CO2. Thereafter,

the cultures were examined under a microscope. Neutral red is

a weak cationic dye that readily diffuses across plasma and

organelle membranes, and accumulated in the lysosomes.

Loss of membrane integrity induced by toxic substances

results in decreased retention of neutral red. Thus, damaged

or dead cells appear decolorized compared with healthy

control cells.50,55

The decolorized zones and cell lysis around and/or under

the specimens were evaluated according to ISO 7405.56 Each

test was repeated five times. The decolorized zones were

scored as follows: 0 for no decolorization detectable; 1 for

decolorization only under the specimen; 2 for a decolorization

zone not greater than 5 mm from the specimen; 3 for a

decolorization zone not greater than 10 mm from the speci-

men; 4 for a decolorization zone greater than 10 mm from the

specimen; and 5 when the total culture was decolorized. Cell

lysis was defined as a loss of cell membrane integrity that was

visible by light microscopy. Cell lysis was scored as follows: 0

when no cell lysis was detectable; 1 for less than 20% cell lysis;

2 for 20–40% cell lysis; 3 for >40% to <60% cell lysis; 4 for 60% to

80% cell lysis; and 5 for more than 80% cell lysis. Analyses were

performed on five samples per test group. For each specimen,

one score was given, and the median score from each

specimen was calculated for both the decolorization zone

index and the lysis index. The cell response was classified as

follows: 0/0 as noncytotoxic; 1/1 as mildly cytotoxic; 2/2 to 3/3

as moderately cytotoxic; and 4/4 to 5/5 as markedly cytotoxic.

The median values were calculated to describe the central

tendency of the scores since the results were expressed as an

index in a ranking scale.56,57

2.4. Statistical analyses

The software, Statistical Package for the Social Sciences (SPSS,

version 19.0, SPSS, Inc., an IBM Company, Chicago, Illinois,

USA), was used to analyse the data from the WST-1 test based

on a Kruskal–Wallis one-way analysis of variance and

Duncan’s multiple range test. The results were expressed as

the mean � standard deviation (SD) for seven separate

experiments. A p-value < 0.05 was considered statistically

significant.58

3. Results

3.1. Cell viability

Compared with the cell viability of cells seeded into culture

wells containing only media, the cell viability for the control

group (cells seeded on CP-Ti) was 89.8 � 19.0%. Pure metals

may be considered biocompatible if the cell viability on the

metals is equivalent to or greater than that of the CP-Ti

control. After a cell culture period of 24 h, the pure metals

exhibited the following decreasing order of cell viability: Mo,

Nb, Zr, Cr, Mn, V, Ag, Al, and Cu (Fig. 1). The cell viabilities for

pure Mn, V, Ag, Al, and Cu, were 32.7 � 12.8%, 31.7 � 8.9%,

31.7 � 16.3%, 25.3 � 7.3%, and 21.6 � 10.5%, respectively,

which were significantly lower than that for the corresponding

control group ( p � 0.001, Table 2). These results demonstrate

the cytotoxicity of these five pure metals.

The cell viability for pure Zr and Cr were 74.1 � 23.3% and

60.6 � 20.9%, respectively, compared with that of CP-Ti. The

pure metals, Mo and Nb, demonstrated good biocompatibility

as evidenced by cell viabilities of 93.3 � 14.4% and 93.0 � 7.6%,

respectively, compared with that of the control group.

The cell viability for CP-Ti was 100%, which is the

comparison scale limit (Fig. 2). The cell viability for all Ti-

based alloy groups exceeded 80%, except for Ti–20Nb

(79.6 � 7.8%) and Ti–10V (66.9 � 22.0%). When Ag, Al, Cu,

Mn, and V were alloyed with Ti, the cell viability for Ti-based

alloys increased markedly, i.e., by a factor of more than three.

Fig. 1 – Mean W SD percent cell viability on pure metal

specimens of Ag, Al, Cr, Cu, Mn, Mo, Nb, V, and Zr versus

on the CP-Ti control after cell culture for 24 h. N = 7;

*Significantly different from the control, p < 0.05.

Fig. 2 – Mean W SD cell viability on pure metals and on Ti

based alloys containing these elements versus the CP-Ti

control after cell culture for 24 h. N = 7; *Significantly

different from the control, p < 0.05.

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 1 2 5 1 – 1 2 5 81254

Interestingly, among the tested Ti alloys, Ti–10Nb alloy

showed the highest cell viability of 124.8 � 16.9%, which

was greater than that of CP-Ti. However, as the concentration

of Nb in the Ti alloy increased above 10 wt%, the cell viability

decreased gradually to 79.6 � 7.8% for the Ti–20Nb alloy group

( p > 0.05 vs. CP-Ti).

3.2. Cytotoxicity based on the agar overlay test

The agar overlay test results are listed in Table 3. Decoloriza-

tion of the stained agar layer was observed around the positive

control and pure Ag, Cu, Mn, and V. For pure Cr, even though

the decolorization zone appeared just under the sample, many

cells in the decolorization zone were lysed, producing a cell

response of 1/3. Pure Ag, Cr, Cu, Mn, and V were ranked as

Table 2 – Percent cell viability (%) on pure metal ingot vs.on CP-Ti after cell culture for 24 h based on the WST-1test.

Samples Mean (SD)

CP-Ti 100.0a (19.0)

Pure Ag 31.7d (16.3)

Pure Al 25.3d (7.3)

Pure Cr 60.6c (20.9)

Pure Cu 21.6d (10.5)

Pure Mn 32.7d (12.8)

Pure Mo 93.3a,b (14.4)

Pure Nb 93.0a,b (7.6)

Pure V 31.7d (8.9)

Pure Zr 74.1b,c (23.3)

N = 7; SD = standard deviation.

By Kruskal–Wallis statistics: K = 54.916, p � 0.001.

Means with the same letter are not significantly different

( p > 0.05).

Duncan post hoc grouping: a > b > c > d.

‘moderately cytotoxic’, with cell responses of 2/4, 1/3, 3/3, 3/4,

and 3/2, respectively (Table 3). Pure Zr was ‘mildly cytotoxic’,

with a cell response of 1/1. Pure Mo and Nb did not decolorize

the neutral-red-stained cells, and all tested Ti alloys, except

for Ti–10V (Table 3, cell response of 1/1), were ranked as

‘noncytotoxic’. The results from the agar overlay test were

consistent with those from the WST-1 test.

4. Discussion

The release of metal ions from metallic implants affects implant

biocompatibility and may cause various complications, such as

Table 3 – Cytotoxicity of pure metals and Ti-based alloysevaluated by the agar overlay test using L-929 cells.

Samples DI LI Cytotoxicity

Positive control (polyurethane) 3 3 Moderate

Negative control (polyethylene) 0 0 None

CP-Ti 0 0 None

Pure Ag 2 4 Moderate

Pure Al 0 0 None

Pure Cr 1 3 Moderate

Pure Cu 3 3 Moderate

Pure Mn 3 4 Moderate

Pure Mo 0 0 None

Pure Nb 0 0 None

Pure V 3 2 Moderate

Pure Zr 1 1 Mild

Ti–10V 1 1 Mild

Ti–(5,10,15,20)wt% Aa 0 0 None

N = 5; DI = decolorization index; LI = lysis index.a All the tested Ti–(5, 10, 15, and 20)wt% A alloys, where A = Ag, Al,

Cr, Cu, Mn, Mo, Nb, V, and Zr, alloys (except Ti–10V alloy) showed a

cell response of ‘0/00, demonstrating noncytotoxicity in the agar

overlay test.56

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 1 2 5 1 – 1 2 5 8 1255

osteolysis, allergic reactions, remote site accumulation, and,

eventually, implant failure.59–62 The frequently observed

unwanted biological effects of different metals require biologi-

cal tests of metallic implants before they are used in humans.

The cytotoxicity test serves as an efficient screening test to

identify the behaviour of cells in the presence of biomaterials.37

Cytotoxicity tests for metallic materials have been per-

formed mainly with metallic compounds, which are formed

from the alloying elements.45–49 Sometimes they are exam-

ined in particulate form,42–44 which react directly with cells. In

rare cases such as in the present study, the cytotoxicity tests

were performed using metallic ingots.25,41 Even with the same

types of component metals, their mechanical, physico-

chemical, and corrosion properties are significantly affected

depending on the compositional variations of the alloys.51,52,5

Therefore, besides testing with a related metallic salt solution,

biocompatibility testing of the metal ingot itself is necessary

for the development of new metallic biomaterials.

Because the cytotoxicity evaluation of metallic materials

has been performed with various metallic forms in several

labs by several methods using several types of cell lines, the

results cannot be compared directly with each other.30,34–40

Even though there are various test methods for evaluating the

cytotoxicity of biomaterials, the agar overlay and MTT tests

are well established for assessing the cytocompatibility of

biomaterials.25,31,38 The tests are accurate and easy to perform,

and the detailed procedure is well described in ISO stan-

dards.56

In the present study, we investigated the reaction of

fibroblast-like cells to nine types of pure metals and 36 types of

Ti alloys (5, 10, 15, or 20 wt% of each alloying element) using

the WST-1 test. The WST-1 assay has advantages over the MTT

assay; it is simple to perform, it provides a more effective

signal, and the toxicity of the assay procedure itself to cells is

decreased.54

In the WST-1 cell proliferation assay, the degree of cleavage

of a tetrazolium salt by mitochondrial dehydrogenases to form

formazan in viable cells was evaluated. The greater the

number of metabolically active cells, the greater was the

amount of formazan produced. By detection of the formazan

level in the cells, the cytotoxicity was measured. The

generation of the dark-yellow formazan was colorimetrically

measured at 450 nm and was directly correlated to the cell

number.

The pure metals tested in this study using L-929 cells for

metal ingots demonstrated different degrees of cytotoxicity.

The ranking of pure metal cytotoxicity, from most to

least potent, was as follows: Cu > Al > Ag > V > Mn > Cr >

Zr > Nb > Mo > CP-Ti (Table 2, Fig. 2). This result differs to

some extent from the ranking reported from other studies.63

Yamamoto et al.63 evaluated the cytotoxicity of 43 metal salts

using two kinds of culture cells, and reported that the intensity

of the metal salts’ cytotoxicity were quite similar between

MC3T3-E1 and L-929 cells. Based on their results using L-929

cells, they reported that the cytotoxicity order from most

to least toxic was as follows: V3+ > Ag+ > Cr3+ > Cu2+ > Mn2+ >

Nb5+ > Al3+ > Mo5+. The differences in rankings between the

present and previous study are related to the difference in the

form of the tested samples, i.e., metal ingots vs. dissolved

metal ions.

In the agar overlay test, pure Mn, V, Ag, and Cu showed a

cell response of 3/4, 3/2, 2/4, and 3/3, respectively, which

means that they were moderately cytotoxic to fibloblasts

(Table 3). In the cell morphological study of Cortizo et al.,64

osteoblast-like cells exposed to Cu or Ag progressively died

after cell division was arrested. The authors reported that

apoptosis was caused by Cu and Ag, as evidenced by the

observation of membrane blebs and apoptotic bodies. After a

longer metal exposure, signs of necrosis became more

prevalent. In our agar overlay test results, the cells contacting

pure Mn, V, Ag, and Cu through agar overlay for 1 d changed to

a globular shape and most of the cells showed blebbing at 200�magnification, demonstrating their apoptosis, while cells

under negative control and against CP-Ti maintained their

cell morphology. However, decolorization at the perimeter of

the samples was weaker than that of the positive control,

which demonstrates that some of the cells were damaged, and

cells experiencing necrosis were scarcely seen.

In the WST-1 test, when Ag, Cu, Mn, and V were alloyed

with Ti, the cell viability for Ti-based alloys increased

drastically by a factor of three or more (Fig. 2). As shown in

Table 3, apoptosis of L929 cells was evident for pure Ag, Cr, Cu,

Mn, and V, whereas the viability of L929 cells was maintained

for all tested Ti alloys, except for Ti–10V (cell response 1/1).

Based on a study of the MG-63 osteoblast cell viability (%) of

pure Ti and Ti–Mn, the cell viabilities of Ti–5Mn, Ti–8Mn, Ti–

12Mn, and pure Mn were 89%, 86%, 72%, and 50%, respectively,

demonstrating that only a very high concentration of Mn

(12 wt% in that study) inhibits cell proliferation.25 In our study,

the cell viabilities of Ti–Mn alloys using L929 cells were

99.9 � 20.8%, 83.3 � 10.7%, 109.3 � 28.3%, 97.8 � 19.9%, and

32.7 � 12.8% for Ti–5Mn, –10Mn, –15Mn, –20Mn, and pure Mn,

respectively. We hypothesize that this fluctuation in cell

viability was derived from altered electrochemical character-

istics that correspond to the variation in the microstructural

phase characteristics that was associated with the composi-

tional change. To verify the influence of these factors on

biocompatibility, a further investigation of the corrosion rate

and dissolved metal ion species and microstructural phase

analysis are required.

It has been reported that Al3+ can significantly suppress the

expression of alkaline phosphatase, osteocalcin, and osteo-

pontin genes of the osteoblastic cell line, whereas Ti4+, V3+,

and Cr3+ display few inhibitory effects in cytocompatibility

tests.65 In our WST-1 tests, the cell viability of pure Al was

25.3 � 7.3% compared with that of the control group. This was

similar in cytotoxicity level compared with those of pure Ag,

Cu, Mn, and V (Fig. 1). However, neither the decolorized zone

nor cell lysis was observed for pure Al in the agar overlay test

(Table 3). When analysing these data, one must consider that

the agar overlay test requires substances to diffuse through

the agar layer before they can be detected as toxic, and there is

no direct contact of the cells with the material.66 Thus, the

differentiation of the cytotoxicity between tested material

groups can be possible only by use of a series of materials with

widely different toxic properties. In our study, the WST-1 test

seemed to be more sensitive for detecting the cytocompat-

ibility of materials compared with the agar overlay test, and

pure Al seemed to have a detrimental influence on the

metabolic function of the cells, even though the cell

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 1 2 5 1 – 1 2 5 81256

membranes remained intact when they contacted the pure Al

ingot.

Li et al.30 reported that a 72-h extracted solution of Zr metal

(99.8% pure) in the form of powder or bulk was noncytotoxic.

Several reports have demonstrated the favourable cell

viability property of Zr.67,68 However, in our study, the cell

viability for pure Zr and Cr (purity of 99.8% and 99.995%,

respectively) displayed moderate cell viabilities of

74.1 � 23.3% and 60.6 � 20.9%, respectively, which demon-

strates a significant difference when compared with that of

CP-Ti ( p < 0.05, Table 2). Based on the results of our agar

overlay test, pure Zr was mildly cytotoxic (cell response 1/1).

However, pure Cr showed a moderate cytotoxicity (cell

response 1/3), as demonstrated by the appearance of the

decolorization zone just under the sample and the presence of

numerous lysed cells in the decolorization zone (Table 3). The

differences between the present and previous results may be

associated with the experimental form of the metal, i.e., metal

ingots vs. metallic salts or extracts. Based on our results in

which a Cr ingot was used as well as on previous results using

metallic salts,69 the cytotoxicity of pure Cr prompted consid-

eration of its use. However, when pure Cr or Zr were alloyed

with Ti, cell viability increased up to a level similar to that of

CP-Ti ( p > 0.05), and those Ti-alloys were ranked as ‘non-

cytotoxic’ based on the agar overlay test (Table 3, Fig. 2).

A previous investigation of the effects of Al, Cr, Cu, Mo, Nb,

V, and Zr ions at concentrations ranging from 0.05 to 5.0 mM

on human T lymphocytes demonstrated that Nb was the most

toxic metal, inducing <50% viability at a concentration of

approximately 0.5 mM.70 On the other hand, pure Mo and Nb

ingots in our study demonstrated good biocompatibility, as

evidenced by cell viabilities for pure Mo and Nb of 93.3 � 14.4%

and 93.0 � 7.6%, respectively, compared with that of the

control group (Fig. 1). In the agar overlay test, pure Mo and Nb

were noncytotoxic (Table 3). These contradictory results may

have occurred because the cytotoxicity of metallic implant

materials was influenced by the difference in the released

metal ionic forms and their concentrations.34,48,49,63,68–74 Even

though the extent of dissolution for a toxic element deter-

mines the severity of cytotoxicity, when the cytotoxic metal is

alloyed with another metal, the mass release of a particular

element is generally not independent of its atomic concentra-

tion in the alloy.64,75 The cytotoxicity of a metal ingot is

dependent on the concentration of released ions, on the

exposure time of the metal to the cells, and on the sensitivity

of the specific cells or tissues to the released metal.75

Among the tested Ti alloys, the Ti–10Nb alloy exhibited the

highest cell viability (124.8 � 17%), which was even greater

than that of CP-Ti (Fig. 2). However, as the concentration of Nb

in the Ti alloy increased above 10 wt%, the cell viability

decreased gradually to 79.6 � 7.8% for the Ti–20Nb alloy group

( p > 0.05). Also, there was no linear relationship between

alloying element concentration and its cytocompatibility

(Fig. 2). As the concentration of the alloying elements in the

fabricated Ti alloys increased, the crystallographic phase

change and, for some compositions, an intermetallic com-

pound could be formed, and it can influence the physical and

corrosion properties of the alloys along with their resultant

cytocompatibility behaviours.12,25,51,5 This phenomenon indi-

cates that cytotoxicity of the metal alloys is related not only to

the type of component metallic elements but also to their

stability in the biological environment that may vary

considerably because of differences in their microstructures

and electrochemical properties.

5. Conclusions

This study employed WST-1 and agar overlay tests to analyse

the reaction of fibroblast-like cells to nine types of pure metal

ingots and 36 experimental titanium alloys that contained 5,

10, 15, and 20 wt% of alloying elements. The ranking of pure

metal cytotoxicity from most potent to least potent was as

follows: Cu > Al > Ag > V > Mn > Cr > Zr > Nb > Mo > CP-Ti.

In the agar overlay test, pure Mn, V, Ag, and Cu were

moderately cytotoxic. However, the other pure metals and all

tested Ti alloys, except Ti–10V, were noncytotoxic. The Ti–10V

alloy exhibited mild cytotoxicity. The cell viabilities for all of

the Ti-based alloy groups were higher than 80%, except for Ti–

20Nb (79.6 � 7.8%) and Ti–10V (66.9 � 22.0%). Among the tested

Ti alloys, the Ti–10Nb alloy demonstrated the highest cell

viability (124.8 � 16.9%), which was even greater than that of

CP-Ti. Even though Ti and the Ti alloys tested herein, except

for the Ti–10V group, were biocompatible, our cytotoxicity test

results indicate that Ti alloys containing Ag, Al, Cr, Cu, Mn, V,

and Zr need to be investigated further for their long-term

safety in a biological corrosive environment. The results

obtained in this study can serve as a guide for the development

of new Ti-based alloy systems.

Acknowledgements

This study was financially supported by Special Research

Program of Chonnam National University and by the National

Research Foundation of Korea (NRF) grant funded by the Korea

government (MEST) (No. 2012-0009424).

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