Biomaterials 23 (2002) 3733 3740
Effect of metal alloy surface stresses on the viability of ROS-17/2.8
osteoblastic cells
. . .
Anita Kapanena,*, Anatoli Danilova,c, Petri Lehenkarib, Jorma Ryhanenb, Timo Jamsac,
Juha Tuukkanena
a
Biocenter Oulu and Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000 FIN-90014 Oulu, Finland
b
Department of Surgery, University of Oulu, P.O. Box 5000 FIN-90014 Oulu, Finland
c
Department of Medical Technology, University of Oulu, P.O. Box 5000 FIN-90014 Oulu, Finland
Received 2 July 2001; accepted 14 March 2002
Abstract
In this study we compared the effect of structural stresses and surface roughness on biocompatibility of NiTi- and Ti-alloy for
ROS-17/2.8 osteoblastic cells. We suggest here that cell viability and cell attachment are linear functions of internal (structural)
stress and subgrain size of the implant alloy. However, this is not the case with surface roughness. The two-phase state in these
materials is characterized by different mean values of structural stresses (s) in a-martensite and b-phase. We found a straight
correlation between cell viability and sb=sa ratio. Atomic force microscopy revealed that, even after equal surface polishing
treatments, roughness varied significantly between the different alloys. The effect of the surface structure of the alloy on the
osteoblastic ROS-17/2.8 cell survival rate was studied with combined calcein-ethidium-homodimer fluorescence labeling. The
possible effects on cell attachment to substrate were studied by staining the focal contacts with paxillin antibody. All the NiTi
surfaces were tolerated well and the cells attached most abundantly to the roughest NiTi surface but the smoothest Ti-alloy surface.
However, other parameters of the material state, such as the surface stresses created by hot rolling seem to be responsible for some
of the attachment and cell survival features observed in this study. r 2002 Elsevier Science Ltd. All rights reserved.
Keywords: NiTi; Titanium alloys; Surface stresses; AFM; ROS-17/2.8
1. Introduction (2000) with three different cell lines showed that surface
roughness promotes osteogenic differentiation of less
A material consisting of nearly equiatomic parts of mature cells. More mature cells exhibit a reduced
nickel and titanium (Nitinol) has superelasticity, ther- sensitivity to their substrate, but are still affected by
mal shape memory and good damping properties, which changes in surface roughness [8]. However, there are in
make it a promising surgical implant material, especially vitro studies that indicate attachment favoring smooth
in orthopedics [1 3]. Studies done with human fibro- surfaces [9 14]. Some in vivo studies done with dogs,
blasts and human osteoblasts show good in vitro rabbits and pigs indicate that increasing surface rough-
biocompatibility of NiTi [4,5]. ness is associated with enhanced bone formation at
There are several papers convincing that surface implant surfaces [15 17]. But surface roughness, being a
roughness contributes to osteoblast attachment and characteristic of surface topography, could not describe
spreading. Rat calvaria-derived osteoblast attachment the whole spectrum of subtle differences in material
increased as a function of surface roughness [6]. A study surface state caused by the differences in its structures
on chemically pure Ti and Ti-alloy surfaces further and phase composition. At the same time, it is well
indicated increased attachment as a function of surface known that the internal or structural stresses are very
roughness [7]. The recent study by Lohmann et al. sensitive characteristics of the state of the material. The
structural stresses reflect such structural parameters as
density and distribution of dislocations, twins and
*Corresponding author. Tel.: +358-8-537-5188; fax: +358-8-537-
stacking faults. The stresses react on phase composition
5172.
E-mail address: anita.kapanen@oulu.fi (A. Kapanen). changes. In addition, the ratios of stresses in different
0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 14 2 - 9 6 12 ( 0 2 ) 0 0 10 7 - 2
3734 A. Kapanen et al. / Biomaterials 23 (2002) 3733 3740
phases inform about probable consequences of phase cloth with chromium oxide paste (group 1200, n ź 6).
transformations on corrosion and electrochemical prop- The rubber wheel was used for NiTi samples to achieve
erties. Among the important causes of internal stresses the high polished state, which could not be achieved
we may mention mechanical, chemical, heat and only by polishing with cloth because of the high wear-
radiation treatments. Therefore, we find it relevant to proofness of NiTi. The test disks were then washed in an
apply definition of structural stress to biocompatibility ultrasonic vibrobath, degreased with 70% ethanol for
investigations. 10 min and autoclaved at 1201C for 20 min before use.
The aim of the present study was to assess the effect of
structural stresses in NiTi- and two-phase (a þ b) Ti-
2.2. Surface roughness measurement
alloys on the viability of ROS-17/2.8 osteoblastic cells in
comparison with the effect of surface roughness.
AFM measurements were performed with Explorer
Different NiTi surfaces were compared with those of a
system (Thermomicroscopes, Sunnyvale, USA) and
Ti-alloy with two different hot-rolling treatments. An
SPMLabNT software ver. 5.01 Explorer AFM (Ther-
atomic force microscope (AFM) and X-ray structural
momicroscopes, Sunnyvale, USA). The sizes of scanned
analysis was used to examine the surface characteristics.
area were 100 100 mm2.
The following roughness parameters were measured:
Ra is the average roughness is the arithmetic average
deviation from the mean line. Rp the maximum peak is
2. Materials and methods
the maximum height or the highest peak of the
roughness profile above the mean line. Rt the maximum
2.1. Test materials
peak to valley is the sum total of the maximum peak and
maximum valley measurements of roughness within the
The tested materials were binary NiTi shape memory
length assessed. Rtm is the more representative mean
alloy (Ti 44 wt%, Ni 56 wt%, Unitec) and two phase
value of the entire profile.
(a þ b) Ti-based alloy (Ti 90.5 wt%, Al 6 wt%, Mo
Three disks in each test group were analyzed on three
2.2 wt%, Cr 1.3 wt%, Institute of Light Metals, Mos-
randomly chosen lines, and the means of the parameters
cow, Russia) vacuum-melted and hot-rolled. The choice
were calculated. Table 1 displays the surface roughness
of materials was governed by their good perspectives for
parameters of the different alloys.
biomedical application: NiTi because of its well-known
biomechanical and corrosion properties, Ti6Al2.2-
Mo1.3Cr alloy seems to be a competitor to wide-spread 2.3. X-ray structural analysis
Ti6Al4V-alloy because of toxic vanadium substitution
for less toxic elements as Mo and Cr. To initiate X-ray structural analysis of phase composition,
different phase composition and thus different structural internal stresses and subgrain sizes (in NiTi and TiI
stresses, two rolling temperatures for two groups of Ti- samples) was performed on the X-ray diffractometer
alloy were used: 8501C (TiI) and 10501C (TiII). The DRON-3.0 (X-ray equipment Co   Burevestnik  , Saint-
rolling temperature of NiTi was 9501C. Petersburg, Russia) with filtered Cu Ka-radiation. The
To initiate further surface stress grades, test disks of mean values of lattice strains (e) that were used for stress
5 mm in diameter and 3 mm in thickness were ground by calculation, and the mean size of subgrains were
grinding stone N80 (group 80, n ź 6) followed by determined in accordance with the procedure described
carbon silicon paper of decreasing coarseness 240, 320, in [18]. The necessity to separate the effect of stresses
400, 600 (group 600, n ź 6), 800, 1200 and finally and subgrain sizes on diffraction line broadening was
polished by rubber wheel (only NiTi-alloy samples) and the initial reason to analyze both of these structural
Table 1
Roughness parameters of the tested alloys
Alloy Ra (nm) Rp (nm) Rt (nm) Rtm (nm)
NiTi 80 362.27209.2 636.67152.6 1434.37501.1 448.4754.8
NiTi 600 156.0720.5 632.77195.2 633.27443.5 506.9741.4
NiTi 1200 95.2741.4 158.0738.9 425.2784.1198.4721.5
TiI 80 1479.5738.9 3120.07583.4 6847.07301.9 2568.0712.7
TiI 600 1428.57173.2 1850.071064.2 5823.07707.1 1198.07142.8
TiI 1200 304.8731.4 846.07548.7 1353.071061.2 539.87161.7
TiII 80 732.1749.12285.07304.13740.0772.1948.1749.9
TiII 600 436.3710.4 1098.07210.2 2244.07318.9 669.0756.1
TiII 1200 339.2739.9 579.8785.8 1381.0711.3 418.2721.4
A. Kapanen et al. / Biomaterials 23 (2002) 3733 3740 3735
parameters. The structural stresses s were calculated (ZYMED Laboratories, Inc., San Francisco, CA,
from formula s ź Ee; where Young s modulus E was USA) at 1:100 in PBS for 45 min on ice. Staining was
7 104 MPa for NiTi, 10.3 104 MPa for b-phase and carried out with rhodamine-conjugated rabbit anti-
11.3 104 MPa for a-martensite in titanium alloy. The mouse immunoglobulin secondary antibodies (DAKO,
values of structural stresses and subgrain sizes in Glostrup, Denmark) for 30 min on ice. To visualize the
martensitic phase of NiTi were not studied because of nuclei, the cells were incubated with the DNA-binding
the small amounts of martensite (weak diffraction lines). fluorochrome Hoechst 33258 (1:1000) for 10 min at
The analysis of internal stresses in TiII group samples room temperature. The focal contacts were studied
was not performed in the present study. under a confocal microscope LSM 510 equipped with an
inverted microscope Axiovert 100M and 63 objective
2.4. Cell culture (NA 1.2/water, Zeiss, Germany). From each sample
disk, 6 frames were scanned with 1024 1024 frame size
Rat osteosarcoma cell line ROS-17/2.8 (a generous (pixel size 0.14 0.14 mm2). The number of focal
gift from G.A. Rodan, Merck Research Laboratories, contacts was measured with a digital image analyzer
West Point, PA, USA) cultures were carried out in (MCID M4 v.3.0.re.1.1, Imaging Research Inc., Cana-
minimal essential medium (MEM, Gibco) supplemented da). The measured region of interest was
with 10% fetal calf serum (Bioclear), antibiotics (100 U 146.2 146.2 mm2. The confocal microscope images
of penicillin/ml, 100 mg of streptomycin/ml) and l- were segmented based on red color intensity. The
glutamine (2 mm) at +371C (5% CO2, 95% air). The interactively defined paxillin-containing focal contacts
cultures were allowed to reach confluency before were automatically counted from the region of interest.
subculturing onto metal alloy disks. The cells were
washed with 371C phosphate-buffered saline (PBS), and 2.7. Statistical analysis
adherent cells were detached by using trypsin-EDTA.
Five thousand cells were seeded per disk (n ź 6) and Mean values and standard deviations were computed.
allowed to attach for 3 h. The cells were allowed to grow Analysis of variance (ANOVA) and Student s t-test
for 48 h before staining with a cytotoxicity test kit or were utilized to assess the level of significance of the
fixation with 4% paraformaldehyde (PFA). differences between the experimental groups. Bonferro-
Because cells grown on disks cannot be seen in normal nis corrections were applied to the t-tests. All statistical
light microscopy, the cells were also cultured on glass analyses were performed with commercial software
cover slips 10 mm in diameter to assess the time of (Origin 5.0, Microcal Software, Inc., USA).
subculture confluency.
2.5. Cytotoxicity test 3. Results
The cells on the disks were washed twice with a warm 3.1. X-ray structural analysis
PBS solution and stained with a LIVE/DEADsVia-
bility/Cytotoxicity kit (Molecular Probes, Oregon, The results of X-ray experiments showed that the
USA). The optimal concentration of the ethidium above treatments of tested alloys resulted in different
homodimer-1 (EthD-1) dye was 0.1 mm and that of the phase state of samples. The initial hot-rolled state of
calcein dye 1 mm. The samples were incubated for 15 min NiTi sample was austenite. The grinding by stone N80
at 371C and viewed under a fluorescence microscope. did not change the phase composition but gave rise to
Dead cells (stained red) and live cells (stained green) diffraction line broadening that is typical for cold-
counted from six randomly chosen areas (0.849 mm2) on worked metals because of increase in structural stresses.
each disk were viewed. The cells were counted visually Further grinding and polishing were accompanied first
under a fluorescence microscope (Nikon Eclipse E600, by remarkable decrease of structural stresses (group
Nikon, Japan) with a 10 objective, NA 0.25 (Nikon, 600) and then by their negligible increase (group 1200).
Japan), and the ratio of dead to live cells was computed. Another structural parameter that was parallel stu-
Approximately 350 cells were seen in each area. The died mean size of subgrains changed in opposite
number of dead cells per image was expressed as per manner: group 80 was characterized by the smallest
1000 cells. sizes, in group 600 samples subgrain sizes were the
biggest and in group 1200 this parameter again became
2.6. Immunofluoresence microscopy of focal contacts smaller (Table 2). Simultaneously, small amounts of
martensitic phase appeared in the samples of groups 600
The PFA-fixed cells were permeabilized with 0.1% and 1200.
Triton-X-100 in PBS for 10 min on ice. The cells were The same behavior of structural stresses was observed
stained by using monoclonal paxillin antibody in the basic martensitic a-phase of titanium alloy. The
3736 A. Kapanen et al. / Biomaterials 23 (2002) 3733 3740
Table 2
The structural stresses and mean values of subgrain size in austenite of NiTi, b-phase and a-martensite of titanium alloy and Ti-alloys sb=sa ratio
Sample group NiTi Structural stresses (MPa) Subgrain size (nm)
80 25971935
600 1437988
1200 1627943
Sample groupTiI b-phase a-martensite
Structural stresses (MPa) Subgrain size (nm) Structural stresses (MPa) Subgrain size (nm) sb=sa
80 1647628 12477 62 1.32
600 22871339 4573 100 5.07
1200 306722 110 7975 235 3.87
highest values of sa were in samples of group 80, in
group 600 they were the smallest and then rose again in
group 1200. As a result of the described behavior, the
ratio sb=sa had the minimum value in group 80,
maximum in group 600, and again decreased in group
1200. Nevertheless, the mean values of stresses in b-
phase (sb) steadily increased as well as the subgrain sizes
in both b-phase and a-martensite (Table 2). The
mechanical treatment of titanium alloy was not accom-
panied by changes in phase composition, and initial b-
phase content (10 12 vol%) were kept in the samples
within investigated groups.
3.2. Cytotoxicity test
The cells cultured on sandpapered surfaces appeared
larger in a visual examination. On the roughest disks in
the TiII group, the cultures did not reach complete
confluency in 48 h, as did the cultures on NiTi. The
cytotoxicity test showed that the roughest NiTi and TiI
surfaces were significantly better in view of cell viability
than the other surfaces in the test groups. When we
compared the different alloys within the same roughness
Fig. 1. (A) Results of the cytotoxicity test. NiTi nickel titanium
alloy, TiI titanium alloy with 8501C hot rolling, TiII titanium alloy
group, it turned out that, in group 80, NiTi and TiI had
with 10501C hot rolling. źpp0:05; ź pp0:01; źpp0:001:
significantly fewer dead cells (12721 and 15721,
(B) Number of focal adhesions. źpp0:05; ź pp0:01; ź
respectively) than TiII (34729), (pp0:001). The 600
pp0:001:
and 1200 groups did not differ significantly. Within the
NiTi test group, the number of dead cells on the
roughest (80) surface (12721) was significantly lower
compared to the 600 (23728), (pp0:05) and 1200 the roughest surface, but was also observed on the other
(22726), (pp0:05) surfaces. The results in the TiI group surfaces.
were similar, with the 80 surface (15721), showing a We determined the number of focal adhesions based
significantly lower number of dead cells compared to the on the paxillin staining of the cells. The results showed
600 (34737), (pp0:01) and 1200 surfaces (27727), that NiTi 80 strongly stimulated the formation of focal
(pp0.05). The TiII group showed no significant adhesions formation. Only TiI 1200 was equally efficient
differences between the three roughness groups as NiTi 80 as a promoter of cell attachment. Overall, TiI
(34729, 23729 and 25743, respectively) (Fig. 1A). was a better matrix for osteoblast attachment than TiII.
The different surface roughness grades of NiTi did not
3.3. Attachment of cells significantly differ in the number of focal adhesions
(6117325, 4607272 and 4857343). In the TiI group,
The focal adhesions of the cells grown on the test the number of focal adhesions was significantly lower on
materials seemed to locate parallel to the grinding the roughest surface (2697177) compared to the 600
grooves (Fig. 2). This phenomenon was clearly seen on (4237222), (pp0:01) and 1200 (6587355), (pp0:001)
A. Kapanen et al. / Biomaterials 23 (2002) 3733 3740 3737
Fig. 2. Confocal microscope image of ROS-17/2.8 cultures on surfaces of different roughness. The small white arrows show the direction of the
grinding grooves, to which the focal contacts are parallel. The white arrowheads point out the diffusively stained cells without clear focal contacts.
Scale 20 mm.
surfaces. The TiI 1200 group had a significantly increased the cell attachment but was in reverse
higher number of focal adhesions than the 600 group association to cell death rate (Fig. 3A). Interesting
(pp0:01). The TiII 1200 surface had a significantly finding was that in the b-phase of TiI alloy both the
higher number of focal adhesions (3517194) than the cell attachment and the cell death rate increased with
surfaces of the other two roughness grades (2237151 in increasing structural stress (Fig. 3B). In the a-martensite
group 80 and 2487156 in group 600), (pp0:01) phase of TiI alloy the effect of structural stress was
(Fig. 1B). opposite (Fig. 3C).
3.4. Effect of surface stress on biological parameters
4. Discussion
The analysis of the results obtained from biological
tests and X-ray experiments demonstrate that mean To detect common regularities of biocompatibility
values of both biocompatibility parameters, cell survival relative to the surface of the implant, our study suggests
and cell attachment, seem to be linear functions of that it would be better to characterize surface stress than
internal stresses and subgrain sizes, respectively. In roughness. A cyclic recovery effect (structural stress
the NiTi group, the increase in structural stress decrease) during permanent deformation of pure metals
3738 A. Kapanen et al. / Biomaterials 23 (2002) 3733 3740
24
28 700
NiTi
TiI(²)
600
22
26
600
20
24
550
500
18
22
400
16
500
20
14
300
18
450
12
16 200
140 160 180 200 220 240 260 160 180 200 220 240 260 280 300 320
(a) Structural stress, MPa (b)
Structural stress, MPa
700
Ti I(Ä…)
35
600
30
500
25
400
20
300
15 200
40 60 80 100 120 140
(c) Structural stress, MPa
Fig. 3. (A) Biocompatibility parameters as functions of structural stresses in NiTi. (B) Biocompatibility parameters as functions of structural stresses
of TiI-alloy b-phase. (C) Biocompatibility parameters as functions of structural stresses of TiI-alloy a-martensite.
has been long known [18] and is a result of dislocation cobalt chromium, titanium and hydroxyapatite
redistribution, which is testified by subgrain sizes [19,20,14]. However, our results on Ti-alloy are in line
changes. Further investigations showed that the same with these studies. In both Ti-alloy groups, focal
effect takes place as a result of strain-induced phase contacts were less numerous on rough surfaces than
reactions. Obviously, both of these mechanisms deter- on smooth ones.
mine stress behavior in NiTi alloy, but only the In addition, we found that focal contacts seemed to
dislocation redistribution is responsible for stress align with the grinding grooves of the rough NiTi and
behavior in titanium alloy, though its details in b-phase TiI surfaces. In cell culture studies, Anselme et al. (2000)
and martensite are different because of their different observed that the rougher the surface, the more
crystal lattices. disorganized was the cell layer [19]. In our experiments,
The initial softness or hardness of the metal alloy no such correlation was seen.
affects surface roughness. Therefore, in our study, the We further found that there was a change in cell size
roughness of the sandpaper used did not correlate with related to surface roughness. Larger cells were more
the measured surface roughness parameters in Table 1. numerous on the roughest surface, especially on NiTi
Our results showed the roughest NiTi surface to be disks. Large cells were also seen on the second roughest
favorable for osteoblastic cells. Both the low number of surface (600). However, only few of them were noticed
dead cells and the high number of focal contacts showed on the smoothest surface (1200) and none on any of the
that a rough NiTi surface is well tolerated by ROS-17/ TiII alloy specimens. A study done with human
2.8 cells. Despite the fact that attachment number does osteoblastic MG-63 cells showed that the cells cultured
not give data about attachment strength, inverse on the roughest surfaces had more cuboidal morphology
correlation between attachment site number and cell and were more differentiated [12].
death rate proves that cells do not tolerate different The regularities of biocompatibility parameters as
surfaces on the same manner. Our finding of the effect of functions of structural stresses in NiTi-alloy reveal
NiTi surface roughness is contradictory to some earlier that higher stresses promote better biocompatibility
studies done with other metal alloys, such as Ti6Al4V, parameters. This result is in contradiction with the
Focal adhesions
Focal adhesions
Focal adhesions
Dead cells /1000 cells
Dead cells/1000 cells
Dead cells/1000 cells
A. Kapanen et al. / Biomaterials 23 (2002) 3733 3740 3739
well-known negative effect of stresses on the corrosion 5. Conclusion
properties of a material and the straight correlation
between these properties and biocompatibility [18]. Our results indicate low cytotoxicity of NiTi, even
Small number of experiments does not allow discussion after very rough surface treatment. NiTi disks were well
of the structural reasons of this phenomenon. tolerated by osteoblastic ROS-17/2.8 cells. Because the
Results obtained for biocompatibility parameters in initial softness or hardness of metal alloys has an impact
titanium alloy b-phase point out the existence of on surface roughness, the characterization of surface
common structural causes for such behavior in stresses could be a better method for assessing the
homogeneous materials with b-phase structure. How- surface state of the implant after equal surface
ever, it is possible that unusual NiTi-alloys proper- manipulation. The results of the present study showed
ties provide different biochemical interaction with that definition of structural stresses might be a sensitive
cells. instrument in biocompatibility investigations.
If taken into account that biocompatibility para-
meters are the characteristics of whole sample and that
the basic phase in titanium alloys sample was a-
Acknowledgements
martensite, the obtained regularities of biocompatibility
parameters behavior in a þ b titanium alloy reflect the
This study was supported by Technology Develop-
same regularities for basic a-martensite. But the
ment Center of Finland (TEKES).
behavior of cell viability as function of structural
stresses in titanium alloy b-phase remains unexplained.
To explain this experimental fact the effect of sb=sa
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