Materials Science and Engineering C 23 (2003) 551 – 560

www.elsevier.com/locate/msec

Relationship between surface properties (roughness, wettability)

of titanium and titanium alloys and cell behaviour
L. Ponsonnet a,*, K. Reybier a, N. Jaffrezic a, V. Comte b, C. Lagneau b,
M. Lissac b, C. Martelet a
a
Laboratoire d’Ingénierie et Fonctionnalisation des Surfaces (IFoS), Ecole Centrale de Lyon, UMR 5513, Bâtiment D5,
BP 163, Ecully Cedex F-69134, France
b
Laboratoire d’Etudes des Interfaces et des Biofilms en Odontologie, Université Claude Bernard-Lyon I, Lyon Cedex 69372, France
Received 23 January 2003; accepted 18 March 2003

Abstract

Cell attachment and spreading to titanium-based alloy surfaces is a major parameter in implant technology. In this paper, substratum
surface hydrophobicity, surface free energy, interfacial free energy and surface roughness were investigated to ascertain which of these
parameters is predominant in human fibroblast spreading. Two methods for contact angle measurement were compared: the sessile drop
method and the captive bubble two-probe method. The relationship between surface roughness and the sessile drop contact angles of various
engineered titanium surfaces such as commercial pure titanium (cp-Ti), titanium – aluminium – vanadium alloy (Ti – 6Al – 4V), and titanium –
nickel (NiTi), was shown. Surface free energy (SFE) calculations were performed from contact angles obtained on smooth samples based on
the same alloys in order to eliminate the roughness effect. SFE of the surfaces have been calculated using the Owens – Wendt (OW) and Van
Oss (VO) approaches with the sessile drop method. The OW calculations are used to obtain the dispersive (cd) and polar (cp) component of
SFE, and the VO approach allows to reach the apolar (cLW) and the polar acid – base component (cab) of the surface. From captive bubble
contact angle experiments (air or octane bubble under water), the interfacial free energy of the different surfaces in water was obtained. A
relationship between cell spreading and the polar component of SFE was found. Interfacial free energy values were low for all the
investigated surfaces indicating good biocompatibility for such alloys.
D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Contact angle; Sessile drop method; Captive bubble method; Cell proliferation; Surface roughness; Human fibroblasts; Commercial pure titanium;
Ti6Al4V; NiTi

1. Introduction
Cell adhesion and cell spreading is an important param-
eter for implant engineering. Low biomaterial efficiency is
often due to poor integration of the implant with surround-
ing tissue [1]. Tissue integration is conditioned by the
adhesion and spreading ability of cells (fibroblasts) on
implant surfaces. Cell behaviour on biomaterial surfaces
depends upon implant – cell interactions, correlated with
surface properties. Surface hydrophilicity, roughness, tex-
ture, chemical composition, charge and morphology
strongly affect cellular responses in contact with the
* Corresponding author. Tel.: +33-4-72-18-65-14; fax: +33-4-78-43-
11-40.
E-mail address: laurence.ponsonnet@ec-lyon.fr (L. Ponsonnet).
implants [2]. Many studies have shown that implant success
is dependent not only on the physico-chemical properties of
the implant surface such as surface free energy or interfacial
free energy, but also on its roughness [3– 11].
Hallab et al. [4] demonstrated that surface free energy
(SFE) was a more important surface characteristic than sur-
face roughness for cellular adhesion strength and prolifer-
ation, and that the surface energy components of the various
tested material were shown to be related to cellular adhesion
strength. Schakenraad et al. [5] found that, despite the great
number of parameters interfering with cellular adhesion and
spreading, the solid surface free energy apparently is a
dominant factor in cellular attachment to a polymer surface
and remains so, even if the solid surface has been covered by a
protein layer. In Ruardy et al.’s study [6], it was shown that
the spread area of human fibroblasts increased with wett-
ability when going from the hydrophobic to the hydrophilic

0928-4931/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0928-4931(03)00033-X
552 L. Ponsonnet et al. / Materials Science and Engineering C 23 (2003) 551–560
end of a surface composed of a wettability gradient. Georgi et
al. [7] demonstrated increased cell proliferation with increas-
ing material surface wettability (water sessile drop method).
The relative importance of surface wettability on fibroblast
spreading was studied by Webb et al. [8]. It was shown that
cell attachment and spreading were significantly greater on
hydrophilic surfaces than on hydrophobic surfaces, and that
moderately hydrophilic surfaces promoted the highest level
of cell attachment. Groth and Altankov [9] studied the role of
tyrosine phosphorylation during fibroblast spreading on
surfaces with different values of wettability. The authors
concluded that the effect of hydrophobic substrata on cells
with respect to adhesion and proliferation is due to a transfer
of signals via integrins from the substratum to the cell interior.
The pre-adsorption of fibronectin on hydrophobic substrata
may provide better initial conditions, thus improving the
tissue compatibility of the material. Redey et al. [10] studied
osteoclast adhesion and activity on synthetic hydroxyapatite,
carbonated hydroxyapatite, and natural calcium carbonate
and the relationship to surface free energies. Surface energy
was found to play an essential role in osteoclast adhesion,
whereas osteoclast spreading was found to depend on surface
chemistry, especially on protein adsorption and newly formed
apatite layers. Den Braber et al. [11] evaluated the effect of
parallel surface microgrooves and surface energy on cell
growth. The most significant conclusion was that physico-
chemical parameters such as wettability and surface free
energy influence cell growth but play no measurable role in
the shape and orientation of cells on microtextured surfaces.
Previously, we had pointed out a correlation between
human gingival fibroblasts spreading on titanium based
alloys and surface chemical composition and roughness
[12]. In this paper, we studied the effect of surface energy
and interfacial free energy on cell spreading on metallic
biomaterials, taking into account surface roughness effects.
2. Material and methods
2.1. Substrate preparation
The present study was performed on nickel –titanium
alloy (NiTi) (AMF, Reuilly, France) whose bulk composi-
tion was 55.4 wt.% Ni and 44.6 wt.% Ti. Alloy plates 3 mm
in thickness were cut into square samples (0.55 cm2). The
latter were prepared in order to obtain three different surface
roughnesses. The NiTi samples were at first heated in order
to obtain at room temperature the high-temperature auste-
nitic phase: 30 min at 680 jC then 1 h at 400 jC. The
treatment was performed under a primary vacuum. X-ray
diffraction was used to verify the efficiency of the treatment.
NiTi plates were mechanically polished with wetted metal-
lographic polishing (grades 80, 400, and 2400) SiC papers
(ESCIL, Chassieu, France). Depending on the paper grade
used, the samples will be referred to as NiTi 80, NiTi 400,
NiTi 2400.
The other titanium alloy materials (industrial polishing)
used as substrates were commercially pure titanium grade 2
(cp-Ti) and a titanium alloy (Ti6Al4V) (SERF, Decines,
France). Both biomaterials were in the form of discs (1.13
cm2 in surface area and 2 mm thick). After each surface
treatment, all the samples were ultrasonically cleaned for 20
min, firstly in acetone, secondly in 70% ethanol (in volume)
before a final rinsing with distilled water. Prior to cell culture,
the samples were sterilised by g-irradiation (Groupe-Lépine,
Bron, France/Ionisos, Dagneux, France). The g-source used
was cobalt 60 (25 000 Gy).
2.2. Surface characterisation
2.2.1. Scanning electron microscopy (SEM) for surface
observation
To determine the surface topography of the sub-
strates, SEM images were taken using a Philips XL
20 scanning electron microscope. Five different areas
per sample were examined under different magnifications
(
100,
400,
800).
2.2.2. Profilometry-atomic force microscopy
The Perthometer C5D (Perthen) instrument was used to
assess substrate surface roughness. Four specimens of each
sample (NiTi 80, NiTi 400, NiTi 2400, Ti6Al4V, cp-Ti) were
randomly selected for recordings (five measurements per
specimen). Average roughness (Ra), peak-to-valley rough-
ness (Rz), maximum roughness (Rmax) and finally the
highest difference between the maximum and the average
surface heights (Rp) were measured. Details of such measure-
ments were presented in a previous paper [12].
The atomic force microscope (AFM) was used to
characterize the roughness of samples after a smooth
mechanical polishing and to verify that the roughness is
lower than 100 nm for SFE calculations. The AFM
apparatus was a Park Scientific Instrument microscope
and the measurements were performed in the contact mode
(microlever tip).
2.3. Wettability
2.3.1. Sessile drop method and surface free energy (SFE)
determination
Contact angles were obtained using the sessile drop
method with a GBX Scientific Instrument (Romans, France).
The drop image was stored by a video camera and an image
analysis system calculated the contact angle (H) from the
shape of the drop. Three liquids were used as a probe for
surface free energy calculations: diiodomethane (Sigma, St.
Louis, MO, USA), formamide (Sigma) and distilled water.
For each sample three drops were analysed (three different
samples per surface type). The final contact angle used for
calculation or for comparison of different samples was the
average of left and right angles of each drop. The surface free
energies of the different alloys were calculated using two
 L. Ponsonnet et al. / Materials Science and Engineering C 23 (2003) 551–560
theoretical models, Owens Wendt (OW) and Van Oss (VO)
models. The OW model gives the long-range dispersion
(Lifshitz – van der Waals) (cd) and the short-range polar
(hydrogen bonding) (cp) components of surface free energy
[13] and the VO approach brings the dispersive (cLW) and the
polar acid – base (cab) components, the latter divided into two
parts, acidic (c+) and basic (c) [14] according to the follow-
ing equations:
1 þ cosh ¼ 2ðcds Þ1=2 ððcdL Þ1=2 =cL Þ þ 2ðcps Þ1=2 ððcPL Þ1=2 =cL Þ
h i
LW 1=2 þ 1=2 þ 1=2
ð1 þ coshÞcL ¼ 2 ðcLW s cL Þ þ ðcL cs Þ þ ðc s cL Þ
where cs is the SFE of the surface, cL the SFE of the liquid and
 + 1/2
cab
s =(cs cL ) .
For both methods, the spreading pressure was not taken
into account. This pressure gives the contribution to SFE of
the adsorption of an external layer from the atmosphere and
has to be calculated for SFE calculation correction if SFE is
higher than 60 mJ/m2 [15]. In the present study, SFE values
are lower than this limit and the spreading pressure can be
neglected.
Contact angles for SFE calculations were measured at
room temperature and the drop was put on the surface after
the same cleaning procedures as before culture: all the
samples were ultrasonically cleaned for 20 min, firstly in
acetone, secondly in 70% V/V ethanol before a final rinsing
with distilled water before air drying.
Fig. 1. Scanning electron microscopy of the different surfaces at various roughnesses.
553
The thermodynamical equilibrium was determined
from kinetic storage of dynamic angle evolution with
time. The angle was generally measured during the first
second. The surface was polished by mechanical polish-
ing until the roughness was less than 100 nm in order to
minimise the effect of surface roughness on contact angle
values. Water contact angle measurements were performed,
as a function of time, for various roughnesses on NiTi
surfaces. Contact angle values were retained at 1 s and
at 20 s after water drop deposition. Four NiTi samples
were studied for each grade of roughness (NiTi 80, 400
and 2400). The contact angles were measured in two
directions with respect to surface strips (camera parallel
to the strips or perpendicular). The evolution and the
decrease during time of the contact angle values were
studied as a function of roughness.
2.3.2. Captive bubble two-probe method and interfacial free
energy
Samples were placed in an environmental chamber to
obtain contact angles using the captive bubble two-probe
method [16]. Two microliter bubbles of either n-octane
(99.9% Gold Label, Aldrich Chemical) or room air were
introduced with a syringe. The contact angles (/ for air,
h for octane) for each material were the average of a
total of three bubble measurements made on three differ-
ent samples. These data were used to calculate the polar
p
(csv ) and dispersive (cdsv) components of SFE, and the
554 L. Ponsonnet et al. / Materials Science and Engineering C 23 (2003) 551–560

Fig. 2. Relationship between cell proliferation and surface roughness. The proliferation is higher if the roughness is lower than 1 Am.

interfacial free energy (csw) according to the following in equilibrium with vapour; csv = surface tension of the solid
Andrade et al. [16] equations: in equilibrium with vapour; csw = interfacial free energy of the
solid in water.
Isw ¼ 50:5ð1  cosHoct Þ
ð72cosUair þ 72:1  Isw Þ2 2.4. Cell culture
cdsv ¼
86:4
2 2.4.1. Tissue culture
I
cpsv ¼ sw Explant cultures were obtained from biopsies of clini-
202
cally healthy human gingiva tissues obtained during extrac-
csv ¼ cpsv þ cdsv tion of teeth for orthodontic treatment, and used with the
csw ¼ csv  ð72:1cosUair Þ informed consent of the patient. Routine histological exami-
nation of randomly selected specimens showed no sign of
with s = solid; w = water; v = gas vapour (air); Isw = non-dis- inflammation. Immediately following removal, gingiva tis-
persive interaction at the solid– water interface; cos Hoct = sues were stored in a Dulbecco’s modified Eagle’s medium
cosine of the octane contact angle; cos Uair = cosine of the air (DMEM) (EUROBIO: Les Ulis, France) supplemented with
contact angle; cdsv = dispersive component of the solid in 100 U/ml penicillin (Boehringer Mannheim, Germany), 50
p
equilibrium with vapour; csv = polar component of the solid Ag/ml streptomycin (Boehringer Mannheim, Ottweiler, Ger-

Fig. 3. Cell proliferation as a function of the surfaces. Effect of chemical composition and of roughness at 2, 4 and 7 days of culture.
 L. Ponsonnet et al. / Materials Science and Engineering C 23 (2003) 551–560 555

many), and 250 Ag/ml fungizone (GIBCO, Paisley, UK),

then cut into small pieces of about 2 mm3 and put on plastic
cultured dishes. The cultures were suspended in DMEM
supplemented with 10% foetal bovine serum (Boehringer
Mannheim), penicillin/streptomycin and fungizone. They
were placed in a humidified incubator at 37 jC in an
atmosphere of 5% CO2 in air. After confluence, cells were
trypsinized and seeded on culture dishes. Confluent cultures
of gingiva fibroblasts were seeded at a concentration of 104
cells per cm2 onto substrates, which had been placed in 24-
well dishes.

2.4.2. Proliferative activity: MTT assay

The proliferative activity of cultured cells was determined
with the MTT colorimetric assay as described by Eirich [21].
Two, four and seven days after seeding, cells were incubated
with a tetrazolium salt solution (MTT), 3-[4,5-dimethylthio-
zol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma, Saint
Louis, Missouri, USA) for 4 h at 37 jC. The MTT was
reduced to an insoluble formazan precipitate by mitochon-
drial succinic dehydrogenase of viable cells. After removal of
the medium, ethanol-dimethyl sulfoxide (DMSO) (Amresco,
Solon, Ohio, USA) solution (1:1) was added to each well.
After complete solubilisation of the dark-blue crystal of MTT
formazan, the absorbance of the content of each well was
measured at 570 nm with a 96-well microplate reader on a
spectrophotometer. The blank reference was taken from wells
without cells, also incubated with the MTT solution.
3. Results
3.1. Surface characterisation
3.1.1. SEM and roughness
Fig. 1 shows SEM micrographs for all tested samples
(magnification
400), NiTi, Ti6Al4V and cp-Ti. The differ-
Fig. 5. Water contact angle measurements on NiTi grade 80 with strips
parallel to the camera axis.
ent NiTi surfaces are ranked from the roughest to the smooth-
est. The difference in density and size of grooves between all
the samples is clearly observed. The lowest roughness is seen
on NiTi 2400 (few strips compared to all the other surfaces
and minimum average roughness Ra (0.057 Am)).
3.1.2. Roughness determination and cell proliferation
Peak-to-valley roughness (Rz) values for all the sub-
strates are correlated to cell proliferation in Fig. 2. The same
type of correlation is observed for the other roughness
parameters [12].
3.2. Cell proliferation
The behaviour of cultured fibroblasts on the previously
studied surfaces is shown in Fig. 3. The cell density was
measured over different periods (2, 4 and 7 days) with the
MTT assay. Cells cultured on the NiTi 2400 show high rates

Fig. 4. Water contact angle measurements on NiTi grade 80 perpendicular Fig. 6. Water contact angle measurements on NiTi grade 400 perpendicular
to the camera axis. to the camera axis.
556 L. Ponsonnet et al. / Materials Science and Engineering C 23 (2003) 551–560

Table 2
Sessile drop measurements on different surfaces after polishing leading to
roughness lower than 0.015 Am
Contact angle NiTi cp-Ti Ti6Al4V
Water 60.4 F 5.8 53.9 F 5.1 50.0 F 3.1
Formamide 46.5 F 5.5 35.2 F 0.7 31 F 3.1
Diiodomethane 34.7 F 2.1 35.4 F 3.5 35.1 F 4.2

Fig. 7. Water contact angle measurements on NiTi grade 400 with strips
parallel to the camera axis.
of proliferation and therefore a high cell density compared
to those obtained on the other NiTi substrates characterised
by a higher roughness (NiTi 400 and NiTi 80). The higher
the roughness, the lower the cell proliferation. Ti6AL4V
behaves in the same manner as cp-Ti, with a lower pro-
liferation rate than that of NiTi 2400, even if the roughness
is similar for these three surfaces.
3.3. Wettability
3.3.1. Sessile drop method
3.3.1.1. Water contact angle measurements on rough
surfaces. Fig. 4 shows the decrease of water contact angle
over time on NiTi 80, storing the image of the drop shape on
surfaces with strips perpendicular to the axis of the camera. It
can be seen that contact angle values are different from one
sample to another, ranging from 66j to 78j in the first second
(average 73j). Twenty seconds after, the contact angle is
strongly decreased (  7j). The drop is guided by the top-
ography of the surface and its shape is modified. When the
strips are placed parallel to the camera axis (Fig. 5), the angles
at 1 s range from 57j to 80j (average 69j) and the decrease
after 20 s is equal to 4j. The roughness is not homogeneous
and the contact angle measurements constitute an indicator of
the homogeneity of the surface. The same type of experiment
has been conducted on NiTi 400 (Figs. 6 and 7). The angles at
1 s range from 47j to 57j (average 50j) and the contact angle
decrease is equal to 2.5 in the perpendicular view (axis of the
camera perpendicular to the strips of the surface). In the
parallel view (axis of the camera parallel to the strips of the
surface), the angles at 1 s range from 40j to 58j (average 51j)
and the decrease after 20 s is equal to 2.5j. Table 1
summarises these results.
Table 2 indicates that the results with water and forma-
mide are similar for Ti6Al4V and cp-Ti and different than
that of NiTi, whereas the results with diiodomethane are the
same for each material.
3.3.1.2. Contact angle measurements on smooth surfaces
and SFE calculation. Table 2 gives the contact angles
obtained by the sessile drop method on the different surfaces
after mechanical polishing to reach a minimum roughness.
The roughness obtained through atomic force microscopy
measurements is 0.015 F 0.002 Am. Cp-Ti and Ti6Al4V
show similar angles for the three liquids (water, formamide
and diiodomethane), whereas NiTi is similar to the other
alloys for diiodomethane. The different components of SFE
are expressed. In order to compare the results more easily,
histograms are drawn for the overall results. Fig. 8 clearly
indicates that two groups of samples are visible for cs and
for the polar (cp) and dispersive (cd) components of SFE
(OW method). NiTi is on the one hand and cp-Ti and
Ti6Al4V are on the other hand. The total SFE is of the
same order of magnitude for all the samples. The small
differences are explained by the difference in the polar
component, the dispersive component being exactly the
same for all the samples. These results are in agreement

Table 1
Water contact angle on NiTi of various grades (80, 400 and 2400) and
measured by the camera with strips parallel to the camera axis or
perpendicular
Water contact 80 80 400 400
angle perpendicular parallel perpendicular parallel
1s 73 F 5 69 F 11 50 F 5 51 F 8 Fig. 8. Total surface free energy (c), polar (cp) and dispersive (cd)
20 s 65 F 5 66 F 10 47 F 5 48 F 9 components of surface free energy calculated using the Owens Wendt
D (20 – 1) s 7 F 1.4 4F2 2.8 F 1.0 2.5 F 0.6 approach.
 L. Ponsonnet et al. / Materials Science and Engineering C 23 (2003) 551–560 557

Table 3
Captive bubble measurements
Bubble captive NiTi cp-Ti Ti6Al4V
measurements
and calculations
Uair (deg) 48.57 F 1.61 49.56 F 0.78 48.46 F 1.42
Hoct (deg) 125.82 F 2.71 125.07 F 0.26 127.35 F 1.37
cSV (mJ/m2) 50.05 F 1.64 49.17 F 0.71 49.97 F 1.29
cD (mJ/m2) 18.33 F 3.17 17.88 F 0.86 17.38 F 2.06
cP (mJ/m2) 31.72 F 1.53 31.30 F 0.15 32.59 F 0.77
cSW (mJ/m2) 2.36 F 0.15 2.40 F 0.05 2.17 F 0.07

nents. It can be seen that no significant difference is

observed for csv between all the samples (50 mJ/m2) and
that the interfacial free energy is extremely small (less than 3
Fig. 9. Total (c) and dispersive (cd) component of surface free energy
mJ/m2).
calculated using the Van Oss approach.

with those obtained by the VO method. Fig. 9 confirms that
the dispersive component (cd) is the same for all the samples
and the total SFE values are similar for both methods (45
mJ/m2 for NiTi and 51 mJ/m2 for cp-Ti and Ti6Al4V). Fig.
10 indicates that the acido – basic component is different for
all the samples. Nevertheless, again, cp-Ti and Ti6Al4V
exhibit similar basic SFE components, the acidic component
being very low. All the surfaces have a low electron
acceptor (acidic) character (ca) and a greater electron donor
(basic) character (cb). The polar component of SFE (cp)
determined by the OW method is always greater than the
acidic – basic component (cab ) calculated by the VO
approach.
3.3.2. Captive bubble method
In the captive bubble method, we used two probe bubbles
(air and octane) so that we were able to measure both the
polar (cp) and dispersive (cd) forces that are present on the
surface. These two forces together contribute to the inter-
facial free energy. Table 3 summarises the contact angle
values and the surface and interfacial free energy compo-
Fig. 10. Polar acid – basic (cab), acid (ca) and basic (cb) components of
surface free energy calculated using the Van Oss approach.
4. Discussion
The objective of this study was to tend to a better
understanding of the relationships between material surface
properties (roughness or surface and interfacial free energy)
and cell spreading. Surfaces were prepared with different
roughnesses and surface physicochemical properties. The
relation between roughness and proliferation is clearly
demonstrated in Fig. 2 as it has already been seen in a
previous study [12]. What had not yet been analysed was the
effect of wettability and surface or interfacial free energy on
cell spreading.
4.1. Water contact angle measurements on NiTi rough
surfaces
First of all, water contact angle measurements were
performed directly on rough NiTi samples (Niti 80, 400
and 2400). According to Figs. 4– 7, the water drop spreads
continuously along the strips and the angle decreases with
time, the greater the roughness, the higher the decrease. In
Table 1, one can see that the decrease is higher in the
perpendicular view (axis of the camera perpendicular to the
strips) than in the parallel view (axis of the camera parallel
to the strips). The order of magnitude of the water contact
angles as a function of roughness is in agreement with the
work of Young et al. [17] on cp-Ti with angles ranging from
45j to 75j and roughness from 0.3 to 3 Am. The roughest
surface has the highest water contact angle.
In conclusion, the shape of the water drop is modified
by the surface strips over time, and measuring a contact
angle on such rough surfaces cannot give correct values of
surface hydrophobicity. Roughness strongly modifies wett-
ability, and if one wants to characterise the surfaces in
terms of surface free energy (SFE), smooth surfaces must
be used, because SFE is not a measurement of the topo-
graphic morphology of the surface but an indication of the
surface tension of the solid surface. Thus, SFE calculations
cannot be performed from measurements using rough
558 L. Ponsonnet et al. / Materials Science and Engineering C 23 (2003) 551–560
surfaces, that is why we used smooth ones for SFE
determination.
4.2. Contact angle measurements on smooth surfaces
4.2.1. Water contact angle measurements on smooth
samples
In order to separate the effect of roughness from that of
wettability, the surfaces were polished to obtain smooth
surfaces (Ra < 0.015 Am) for contact angle measurements
and for SFE calculations. Table 2 indicates that the water
contact angles remain in the middle range (50j to 65j) for
all the surfaces corresponding in general to good adhesion
and cell proliferation [24]. If we want to correlate cell
proliferation (Fig. 3) and sessile drop SFE calculations,
we must only discuss the smoothest samples exhibiting a
similar low roughness (Fig. 1), i.e. NiTi 2400, cp-Ti and
Ti6Al4V (Ra < 0.07 Am).
Water contact angle measurements (Table 2) indicate that
all the surfaces are moderately wettable surfaces (60 F 6j
for NiTi, 54 F 5j for cp-Ti and 50 F 3j for TiA6Al4V) and
are all hydrophilic surfaces with similar water contact angles
(55 F 5j). Proliferation is higher for NiTi than for cp-Ti and
Ti6Al4V (Fig. 3). Webb et al. [18] have stated that, when
material surfaces are exposed to dilute serum, fibroblast
attachment and fibroblast spreading are greater on hydro-
philic surfaces compared to hydrophobic surfaces. Many
authors have also observed this effect [19], in the presence
or absence of adsorbed proteins [9]. However, differences
between the hydrophilic surfaces in water wettability influ-
enced cell attachment but not spreading or cytosquelette
organisation [18].
Thus, our results are in agreement with such published
results. Indeed, fibroblast proliferation cannot be directly
correlated to the water contact angles.
4.2.2. Surface free energy calculations using sessile drop
method on smooth samples
The surface energy is a sum of the polar and dispersive
components of surface tension. According to the Owens
Wendt calculations (Fig. 8), one can conclude that for NiTi a
higher proliferation can be associated with a low total SFE
(NiTi: 46 F 3 mJ/m2 and cp-Ti/Ti6Al4V: 52 F 2 mJ/m2).
Because the dispersive component is the same for all the
surfaces (31 F 1 mJ/m2), the difference is due to the Owens
Wendt polar component of the SFE (NiTi: 14.3 mJ/m2 and
cp-Ti/Ti6Al4V: 21.2 mJ/m2). This is confirmed by the Van
Oss approach (Fig. 10) with the polar acid –base component
(NiTi: 2.5 F 2 mJ/m2 and cp-Ti/Ti6Al4V: 8 F 2 mJ/m2) and
a similar dispersive component (42 F 1 mJ/m2). The total
SFE values are close to those obtained by the OW approach:
45 F 2 mJ/m2 for NiTi and 50 F 1 mJ/m2 for cp-Ti/Ti6Al4V.
The influence of the polar component of SFE on fibro-
blast adhesion, which represents the interaction energy of
the surface with water, has been previously published [20]
and it has been shown that cellular adhesion is maximal at a
fractional polarity, FP (cp/(cp + cd)), equal to 0.3. If we
calculate this FP parameter in our study with the Owens
Wendt approach, we obtain a FP value of 0.3 for NiTi and
0.4 for cp-Ti and Ti6Al4V. Thus, the region of fractional
polarity at 0.3 might be expected to have optimal fibroblast
compatibility. These results are in agreement with the Van
Oss approach where cell proliferation is increased by a low
FP (0.05 for NiTi and 0.15 and 0.19 for cp-Ti and Ti6Al4V,
respectively). One can conclude that a low polar component
(or a low fractional polarity) is the major parameter for good
fibroblast proliferation.
The same type of correlation has been published for
cellular adhesion, whereas poor correlation is observed
between the dispersion component of SFE and adhesion
strength, compared to the polar component of SFE [4,10].
This is the same information we obtained, because here the
dispersive component for fibroblast proliferation is the same
for all the surfaces and for both approaches (OW and VO).
Moreover, it has been demonstrated that, if cellular adhesion
on metals is correlated with surface energy, this parameter
may be a more important determiner of cell adhesion and
proliferation than surface roughness. In the present study,
one cannot give such a conclusion because roughness (in the
micron range) seems to be the major parameter for cell
proliferation. Indeed, only smooth surfaces (roughness less
than 1 Am) show significant proliferation, and this is true
whatever the wettability. Thus, there may exist a roughness
threshold (between 0.08 and 1 Am) over which cell prolif-
eration becomes difficult and one can see that the higher the
roughness, the lower the proliferation (Fig. 2).
Another approach has been published on the thermody-
namic aspects of fibroblast spreading on solid substrata [5].
This study shows poor cell spreading of various cell types
on low surface free energy substrata and good cell spreading
on high surface free energy substrata. The inflection point of
the curves, indicating the change from poor cell spreading to
good cell spreading, is around 57 mJ/m2. This limit is higher
than the SFE values obtained in the present study and the
curve corresponding to fibroblast proliferation indicates
that, before the inflection point in the 30 – 50 mJ/m2 range,
the relationship between SFE and proliferation can be
reversed: proliferation can be higher for lower SFE. If we
include the SFE results of the present study in such a curve,
one can see that no clear correlation is observed in this SFE
range.
4.2.3. Interfacial and surface free energy calculations using
captive bubble method
Table 3 summarises the captive bubble results. All the
surfaces exhibit a similar interfacial free energy. This energy
corresponds to the energy of the interface between the
surface and water. It is well-known that a high energy metal
surface in contact with air is covered with various layers,
each leading to a decrease of the real surface free energy of
the surface [21]. The first layer may be an oxide or a
sulphide on the metal, followed by strong polar bonds, such
 L. Ponsonnet et al. / Materials Science and Engineering C 23 (2003) 551–560
as water or OH – adsorption (water or alcohol interacts
strongly with the –OH groups), the final layer having a
relatively low surface energy as found in this study. Minimal
interfacial free energy may be correlated with optimum
biocompatibility for surfaces such that their water interfacial
energies are less than 5 mJ/m2. A strong hydrophilic surface
with a high water concentration, such as hydrogel (three-
dimensional networks formed by polymers in aqueous
solution), could constitute such a surface. If this hypo-
thetical hydrogel contained about 90% water, the interfacial
free energy would probably be less than 3 mJ/m2. Such gels
have been elaborated and are biocompatible biomedical
materials [22,23]. Andrade [24] shows that interfacial free
energy is a much more representative parameter to attempt
to correlate with bio-compatibility than work of adhesion
between the surface and the water.
Thus, in the present study, and in agreement with
Electron Spectroscopy for Chemical Analysis in a previous
study on the same surfaces [12], there could be a hydrogel
on all the surfaces because interfacial free energy values,
which represents unsatisfied bonding of the substratum in
the water, are between 2 and 3 mJ/m2 for all the surfaces
with an error in measurement corresponding to an uncer-
tainty of about 0.15 mJ/m2. This is confirmed by the fact
that the hydrogel is measured to be a flexible material by
comparing the sessile drop method and the captive bubble
one. Indeed, all the surfaces have different air bubble
contact angles in water (180 – 50 = 130j) than water sessile
drop in air angles (50j to 60j) as already demonstrated by
Cunanan et al. [25] for polymers. Like these authors
propose, the case in hand might be one where in water,
the polar parts of the surface will interact with water and this
interaction will contribute to the decrease in the interfacial
free energy, even though the material itself will have a high
surface free energy. Thus, the small differences in SFE
observed between all the surfaces in the present study by
the sessile drop method might be suppressed by this effect
with the captive bubble method. However, the captive
bubble method is quite an interesting approach because
the global system is close to the real one when cell culture
is performed.
5. Conclusion
A strong correlation between surface roughness and cell
proliferation has been demonstrated in a previous study
[12]. In that study, the effect of roughness on water contact
angle measurements was shown. A correlation with the
surface free energy for smooth samples was noted, and it
was concluded that a low polar component (or low frac-
tional polarity) was the major parameter for good fibroblast
proliferation. Taking roughness into account, it was dem-
onstrated that there might exist a roughness threshold
(between 0.08 and 1 Am) over which cell proliferation
became difficult and that the higher the roughness, the
559
lower the proliferation. Captive bubble measurements indi-
cated that all the surfaces were covered by a hydrogel,
helping to decrease the theoretical high surface free energy
of the metallic surfaces analysed. In conclusion, despite the
high number of parameters conditioning cell adhesion and
proliferation, surface free energy appears to be a dominant
factor, but roughness can strongly disturb the relationships
between surface free energy and cell proliferation.
For further studies, one may recall that the adsorption of
the culture medium proteins may be influenced by the
surface free energy components of the various surfaces.
Initial fibroblasts contact with these proteins could be a
major parameter for cell adhesion and proliferation. Various
hypotheses can be proposed to study protein adsorption as a
mediator in cell response [20]. One of them is that composi-
tional differences in the first adsorbed proteins could affect
cell behaviour. Immunolabelling for specific adhesive pro-
teins such as fibronectin could be performed and we plan on
further study of these proteins in future work.
Acknowledgements
The authors wish to thank B. Beaugiraud and C. Pernier
for AFM measurements.
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