Journal of Surface Engineered Materials and Advanced Technology, 2013, 3, 20-28 Published Online October 2013 (
Copyright © 2013 SciRes. JSEMAT
Characterization of Pectin Nanocoatings at Polystyrene
and Titanium Surfaces
Katarzyna Gurzawska1,2*, Kai Dirscherl3, Yu Yihua4, Inge Byg5, Bodil Jørgensen5, Rikke Svava5,
Martin W. Nielsen6, Niklas R. Jørgensen1, Klaus Gotfredsen2
1Research Center for Ageing and Osteoporosis, Departments of Medicine and Diagnostics, Copenhagen University Hospital Glostrup,
Glostrup, Denmark; 2Institute of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen,
Denmark; 3Dansk Fundamental Metrologi A/S, Lyngby, Denmark; 4Microtechnology and Surface Analysis, Danish Technological
Institute, Taastrup, Denmark; 5Department of Plant and Environment Sciences, Faculty of Science, University of Copenhagen,
Frederiksberg, Denmark; 6Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark.
Email: *
Received June 20th, 2013; revised July 19th, 2013; accepted August 6th, 2013
Copyright © 2013 Katarzyna Gurzawska et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
The titanium implant surface plays a crucial role for implant incorporation into bone. A new strategy to improve im-
plant integration in a bone is to develop surface nanocoatings with plant-derived polysaccharides able to increase adhe-
sion of bone cells to the implant surface. The aim of the present study was to physically characterize and compare poly-
styrene and titanium surfaces nanocoated with different Rhamnogalacturonan-Is (RG-I) and to visualize RG-I nano-
coatings. RG-Is from potato and apple were coated on aminated surfaces of polystyrene, titianium discs and titanium
implants. To characterize, compare and visualize the surface nanocoatings measurements of contact angle measure-
ments and surface roughness with atomic force microscopy, scanning electron microscopy, and confocal microscopy
was performed. We found that, both unmodified and enzymatic modified RG-Is influenced surface wettability, without
any major effect on surface roughness (Sa, Sdr). Furthermore, we demonstrated that it is possible to visualize the pectin
RG-Is molecules and even the nanocoatings on titanium surfaces, which have not been presented before. The compari-
son between polystyrene and titanium surface showed that the used material affected the physical properties of
non-coated and coated surfaces. RG-Is should be considered as a candidate for new materials as organic nanocoatings
for biomaterials in order to improve bone healing.
Keywords: Surface Properties; Titanium; Polystyrene; Rhamnogalacturonan-I; Osseointegration
1. Introduction
The implant surface plays a crucial role for implant in-
corporation into the bone and implant surface modifica-
tions which are continuously developed in attempts to
enhance and accelerate bone formation at the implant
surface [1-5]. The development has been approached by
chemically and physically modifications of the surface
[1,3-8]. The first concept focuses on incorporating inor-
ganic and/or organic molecules at the surface whereas
the second focuses on changing surface properties in-
cluding the surface topography [3,9]. The chemical and
physical surface modification can be performed at dif-
ferent levels [1,3,6]. From a biological point of view, the
osseointegration process takes place at the cellular level,
and therefore especially micro and nanoscale investiga-
tions have great importance for developing new surfaces
[4,6]. It has been demonstrated that nanoscale modifica-
tion of titanium implants affects surface properties, such
as hydrophilicity, biochemical bonding capacity and
roughness, which influence cell behaviour on the surface
such as adhesion, proliferation and differentiation of cells
as well as the mineralization of the extracellular matrix at
the implant surfaces [2,4-6,9-12].
The inorganic and organic nanocoatings are continu-
ously developed and tested in vitro and in vivo. For in
vitro examination, Tissue Culture Polystyrene Surfaces
(TCPS) or titanium discs (Grade 2 or 4) are most fre-
quently used [9], whereas for in vivo experiments tita-
nium implant surfaces of Grade 4 titanium are the most
frequently used [1,12]. To obtain the best conformity be-
*Corresponding author.
Characterization of Pectin Nanocoatings at Polystyrene and Titanium Surfaces
Copyright © 2013 SciRes. JSEMAT
tween in vitro and in vivo studies and thereby the best
prerequisite for interpretation of in vitro results, studies
characterizing and comparing how nanocoatings influ-
ence the surfaces are important.
It has been shown that both polystyrene and titanium
surfaces coated with Rhamnogalacturonan-I (RG-I) af-
fect osteoblast cell responses. By enhancing osteoblast
attachment, proliferation and mineralization compared to
uncoated surfaces [9-11,13-17]. The biological mecha-
nism when nanocoating RG-Is onto polystyrene and tita-
nium surfaces is however still not fully understood. The
positive biological effect might be connected to RG-Is’
structure, but also to the change of surface properties caus-
ed by RG-Is nanocoatings.
The aim of the presented study was to physically cha-
racterize and compare polystyrene and titanium surfaces
nanocoated with different Rhamnogalacturonan-Is (RG-
Is) and to visualize the RG-I nanocoatings.
2. Materials and Methods
In order to characterize the RG-Is pectin nanocoating,
three different types of material surfaces were used: 1)
Tissue Culture Polystyrene Plates (TCPS), (Nunc, Roskilde,
Denmark) with a diameter of 60 mm; 2) titanium discs
(Ti discs) with a diameter of 13 mm (Astra Tech, Möln-
dal, Sweden); 3) titanium implants (Ti implants) with a
diameter of 3.5 mm and a length of 8 mm (ANKYLOS,
Dentsply, Konstanz, Germany). We included 7 different
surfaces. Five were coated with RG-Is and two were non-
coated (Table 1).
2.1. Coating Procedure
To obtain a covalent bonding between the surfaces and
Table 1. Surfaces and nanocoatings used in the study.
Material of the surface
Polystyrene Titanium
TCPS Ti discs Ti implants
Untreated + + +
Aminated + + +
PU + + +
PA + + +
PG + +
PAG + +
AU + + +
+ used in study; not used in the study; TCPS: Tissue Culture Polystyrene
Surface Plates; Ti discs: Titanium discs, Ti implants: Titanium implants; PU:
Potato unmodified RG-I, PA: Potato dearabinanated RG-I; PG: Potato de-
galactanated RG-I; PAG: Potato dearabinanated degalactanated RG-I; AU:
Apple unmodified RG-I, RG-I: Rhamnogalacturonan-I.
the RG-I coatings all coated surfaces were aminated.
Surface amination of Tissue Culture Polystyrene Plates
(TCPS), titanium (Ti) discs and implants was performed
by plasma polymerization of allylamine following the
procedure described by Morra et al. [16] The RG-Is were
covalently coupled via reaction between the carboxyl
groups present in GalA of the RG-I backbone and the
primary amino groups on the surface [16].
2.2. Physical Characterization
Physical characterization of TCPS and titanium surfaces
was performed using contact angle measurements for
wettability, scanning electron microscopy (SEM) for vis-
ualization of the surface texture and atomic force mi-
croscope (AFM) for surface roughness measurements
and visualization of the nanocoating. The measurements
were performed on 3 samples (n = 3) of the TCPS, the Ti
discs and the Ti implants and at four different areas (m =
4) for each sample. The measurement areas of TCPS and
Ti discs were selected randomly on the surface and at the
Ti implants the measurements were performed at the top,
valley and flanks according to recommendation by Wen-
nerberg (2010) [18].
1) Contact angles (sessile angle) were measured using
a KRUSS Drop Shape Analysis System, DSA10-Mk2
(Kruss GmbH, Hamburg, Germany). A water droplet (2.5
µL on TCPS plates and Ti discs surface and 1 µL on crest
of Ti implants surface) was dropped on the surface and
recorded photographically. Contact angles were meas-
ured in the recorded image by using drop shape analysis
software, Scientific Drop Shape Analysis Software, DSA
1, Version 1.70 (Kruss GmbH, Hamburg, Germany) and
a curve fitting method (Tangent Method-1).
2) Scanning Electron Microscopy (SEM) was per-
formed with a ZEISS Ultra 55 scanning electron micro-
scope (Carl Zeiss NTS GmbH, Oberkochen, Germany)
operating at 3 kV and 20 kV in the secondary electron
imaging mode. Images were collected at 1 K, 5 K, 10 K,
20 K, 30 K, 35 K, 40 K or 50 K.
3) Surface roughness was measured with a metrologic
Atomic Force Microscope (AFM) DIM3100m (Bruker
AXS Inc., Fitchburg, WI, USA). The AFM had a scan
volume of 70 µm × 70 µm × 7 µm and intermittent scan
mode (tapping mode) was used to minimize interaction
force between tip and sample. The applied tip had a spe-
cified tip radius of 10 nm. The samples were scanned
with a scan rate of 0.1 Hz to minimize scan artefacts in
the image profiles. For the analysis of the roughness meas-
urements, the scanned images were pre-processed with a
first order lateral plane fit. This corrects for the residual
sample tilt and no further filtering was applied. The pa-
rameters selected for analysis of surface roughness were
Sa and Sdr using an area of 20 µm × 20 µm. [3] The
same conditions and equipment were used for visulaiza-
Characterization of Pectin Nanocoatings at Polystyrene and Titanium Surfaces
Copyright © 2013 SciRes. JSEMAT
tion of RG-I nanocoatings on titanium implant surfaces,
but the measurements were performed with 1 × 1 µm
scan area.
2.3. Visualization of RG-Is Nanocoatings
1) For visualization of RG-Is structures Atomic Force
Microscopy (AFM) imaging was performed using a mul-
timode AFM (Bruker AXS Inc., Fitchburg, WI, USA)
with a Nano V controller. RG-Is from potato unmodified
(PU), potato dearabinanated (PA), potato degalactanated
(PG), potato dearabinanated degalactanated (PAG) and
apple unmodified (AU) were dispersed into mili-Q water
to a concentration of 1 µg·mL1. Aliquots (10 mL1) of
the diluted RG-Is samples were deposited onto a freshly-
cleaved molecularly smooth surface [19-21] (Mica sur-
faces, Sa ~ 0 nm, Tedpella, Redding, CA, USA) and al-
lowed to dry under ambient conditions before imaging by
AFM in air [22]. The samples were scanned in inter-
mittent “tapping” mode with commercial tips “SSS-NCH”
from NanoSensors with a tip radius of 2 nm. Scannings
was performed in ambient conditions. The scan rate was
set to 0.5 Hz to limit the scan speed to 1 μm/s for a typi-
cal sized image of 1 µm × 1 µm. Using the image proc-
essing software SPIP (Image Metrology, Hørsholm, Den-
mark), the data was line-wise tilt-corrected with a first
order fit restricted to the data points of the Mica surface
only. This allows accurate height measurements relative
to the flat Mica reference surface with measurement un-
certainties below 1 nm.
2) Immunofluorescence labeling and confocal micros-
copy was performed on four implants, one non-coated
aminated Ti implant and three coated Ti implants with
(PU, PA, AU). The implants were placed in polystyrene
24-well plate (Nunc, Roskilde, Denmark) in separated
wells and blocked for 15 min with 1 ml/well of 5%
skimmed milk from Applichem (Darmstadt, Germany)
(5% solution of fat-free milk powder in phosphate buff-
ered saline (PBS), pH 7.2). Skimmed milk was removed
from the well and 1 ml/well of anti—(1 4)-β-galactan
LM5 (IgG2c) (PlantProbes, Leeds, UK) diluted 1:10 in
5% skimmed milk was added and placed on a shaker for
2 h. LM5 was removed from all wells and all implants
were washed with 5% skimmed milk 3 times (after add-
ing the milk to the wells, the plate was placed on a shaker
for 5 min). Secondary antibody, goat anti-rat IgG for
LM5 linked to FITC (fluorescein isothiocyanate) from
Sigma-Aldrich (Brøndby, Denmark) was diluted 1:200 in
5% of skimmed milk and applied 1ml/well. The plate
was covered by aluminum foil and placed on shaker for 2
hours. Subsequently. implants were washed three times.
1 ml of PBS was added to each well in order to store the
implants until examination by confocal microscopy at 4
degrees. Confocal images were done with a Leica TCS-
SP5 II confocal laser scanning microscope (Leica Mi-
crosystems, Exton, PA, USA) with PL Fluotar 10/x0.30
DRY objective with the same setting and conditions as
described in our previous work [23].
2.4. Osteoblast Cell Culture
SaOS-2 osteoblast-like cells were grown on Ti implants
under the same conditions as described in our previous
studies [23]. The cell morphology observations were
done with a Leica TCS-SP5 II confocal laser scanning
microscope (Leica Microsystems, Exton, PA, USA).
2.5. Statistical Analysis
Descriptive statistics were calculated as mean values and
standard errors of the mean. Results of surface analysis
experiments were analysed using ANOVA tests and Bon-
ferroni corrections for multiple comparisons using SPSS
11.5 software. A significance level of 0.05 was used
throughout the study. More sensitive statistics between
uncoated (untreated and aminated) samples and coated
(PU, PA, PG, PAG, AU) samples, as well as between
different type of surfaces (TCPS, Ti discs and Ti im-
plants) was applied.
3. Results
3.1. Physical Characterization
3.1.1. Contact Angle
When the contact angles of the 3 surfaces (TCPS, Ti
discs, Ti implants) were compared only significant dif-
ferences were found between TCPS and Ti disc surfaces
(p = 0.004). The highest SEM values were found for the
titanium implants (Figure 1).
When all 3 different surfaces were compiled the con-
Figure 1. The contact angle (sessile angle) measurements
results represent mean contact angle values and standard
error of the mean (mean ± SEM). Uncoated surfaces: un-
treated, aminated and coated surfaces: PU (potato unmodi-
fied), PA (potato dearabinanated), PG (potato degalactan-
ated), PAG (potato dearabinanated and degalactanated)
and AU (apple unmodified) of TCPS, Ti discs and Ti im-
plants surfaces. Ti: titanium.
Characterization of Pectin Nanocoatings at Polystyrene and Titanium Surfaces
Copyright © 2013 SciRes. JSEMAT
tact angle measurements demonstrated that nanocoating
with RG-Is (PU, PA, PG, PAG, AU), gave significantly
(p = 0.006) lower contact angles compared to the non-
coated control surfaces (untreated and aminated) (Figure
1). RG-I surfaces coated with PU (p = 0.02), PA (p =
0.01) and PG (p = 0.02) had significantly lower contact
angles compare to the untreated surfaces.
When analyzing differences between non-coated and
coated TCPS surfaces significant differences were found
for all coatings (p < 0.001). For the Ti discs significant
differences were found for all coatings compared to con-
trols, except for PAG compared to aminated surfaces.
For the titanium implants no significant differences be-
tween coated and non-coated surfaces (Figure 1).
3.1.2. Surface Roughness
The results for surface roughness (Sa, Sdr) measurements
are shown in Figures 2(a) and (b). In general the Ti im-
plants were significantly more rough than the Ti discs,
which were significantly more rough than the TCPS
Figure 2. Surface roughness measured with AFM and rep-
resented by amplitude parameter (Sa) (a) and hybrid pa-
rameter (Sdr); (b) (means ± SEM). Untreated, aminated,
PU (potato unmodified), PA (potato dearabinanated), PG
(potato degalactanated), PAG (potato dearabinanated and
degalactanated) and AU (apple unmodified) of TCPS, Ti
discs and Ti implants surfaces. Ti: titanium.
When the 3 different surfaces were compiled no sig-
nificant differences in Sa (p = 0.97) and Sdr (p = 0.86)
values were found between coated and non-coated sur-
When analyzing differences for the TCPS surfaces,
significant differences in Sa value were found for nano-
cating with PU (p = 0.02), PA (p = 0.003), PG (p =
0.001), PAG (p = 0.001) and AU (p = 0.001) compared
to untreated TCPS surface, but not to the aminated sur-
faces. When analyzing differences in Sa value of non-
coated and coated surfaces of Ti discs the only signifi-
cant difference (p = 0.011) was found between AU
coated and untreated Ti discs (p = 0.016) and aminated
Ti discs (p = 0.018). When analyzing differences in Sdr
values of uncoated and coated surfaces the only signifi-
cant difference was found on TCPS surfaces (p = 0.013),
between PU coated TCPS and non-coated TCPS surfaces
(p = 0.026). No significant differences in Sa and Sdr
values were found between coated and non-coated Ti
3.1.3. Scanning Electron Microscopy (SEM)
The SEM images of TCPS, Ti disc and Ti implants (Fig-
ure 3) showed differences in surface texture. On the sur-
face of titanium discs and implants, the texture pattern
(machined surface) from the manufacturing process
could be observed on the SEM images.
3.2. Visualization of Pectin Nanocoatings
3.2.1. RG-Is Structure Observed with AFM
The structure of potato RG-Is (scan area of 1 µm × 1 µm)
is demonstrated at Figure 4. The enzymatic modification
reduces the size of the pectin molecule. More than 70
measurements were conducted for each type for various
individual pectins. The height of the potato RG-Is are
shown in Figure 5. The results are sorted in decreasing
order for better visualization of the height differences.
After enzymatic modification of the arabinan and galac-
tan side chains, the height of PU decreased significantly
by approximately 3 nm on average (Figure 5).
3.2.2. RG-Is Nanocoating on Titanium Implant
Surfaces Visualized with AFM
A representative 3D image of PA RG-I nanocoating is
shown in Figure 6 with linear structures and a heteroge-
nous distribution of the RG-I molecules on the surface of
the Ti implants.
3.2.3. Immunofluorescence Labeling and Confocal
The confocal images showed presence of RG-Is nano-
coating on the coated titanium implant surface compared
to control aminated titanium implant surface (Figure 7).
Characterization of Pectin Nanocoatings at Polystyrene and Titanium Surfaces
Copyright © 2013 SciRes. JSEMAT
Figure 3. Representative images of untreated control sur-
face of TCPS, Ti disc and Ti implant performed with SEM
at 3 kV. TCPS: Tissue Polystyrene Plate, Ti: titanium, WD:
working distance, EHT: the high voltage, SE2: type of de-
3.3. Osteoblast Cell Culture
The confocal images from the Ti implants cultured with
SaO2 cells (Figure 8) showed spread morphology of the
osteoblast-like cells on RG-Is nanocoated titanium im-
plants as well as on the control aminated Ti implant.
4. Discussion
In this work, we assessed the effect on physical proper-
ties of nanocoatings with potato and apple RG-Is to
polystyrene and titanium surfaces. We found that native
(PU, AU) and modified RG-Is (PA, PG, PAG) influenced
surface wettability, without any major influence on sur-
face roughness (Sa, Sdr). Furthermore, we demonstrated
that it is possible to visualize the pectin molecules and
even the nanocoatings on titanium surfaces, which have
not been presented before. The comparison between poly-
styrene and titanium surfaces showed that both materials
became more hydrophilic after nanocoating and that the
RG-I molecules did not have any major effect on the
surface roughness.
In accordance with the present study, a number of
other studies have demonstrated that RG-Is nanocoatings
influenced the physical properties of polystyrene [11,13,
16,17] and titanium surfaces [10,14,15,23]. According to
findings by Morra et al. (2004), the difference in wet-
tability between RG-I coated and non-coated polystyrene
surfaces is caused by changes in the chemical composi-
tion after coating with RG-Is [16]. The same results were
presented in a work by Kokonnen et al. (2006), where
the authors proposed that the RG-Is’ side chains produce
a hydrated gel-like surface [11]. Our previous work also
demonstrated that by changing the chemical composition
of the surface with RG-Is nanocoatings the wettability of
the polystyrene surface was affected and the surface be-
came more hydrophilic compared to non-coated control
surfaces [14]. The same results were observed on tita-
nium surfaces showing that coating with RG-Is gave rise
to smaller contact angles compared to the controls i.e.
more hydrophilic surfaces [10,14,23]. In our study the
comparison of different surface TCPS, Ti discs and Ti
implants coated with RG-Is confirmed these findings.
However at the titanium implants there was a limited
access to measurements, which may explain the lack of
significant difference in the contact angle between coated
and non-coated surfaces. The increased hydrophilicity
obtained by nanocoating with RG-Is seemed to be similar
at the TCPS, Ti discs and titanium implants. The fact that
RG-I coatings created a more hydrophilic surface can
have a positive impact on osseointegration, as hydro-
philic surfaces are more suitable for interaction with bio-
logical fluids, cells and tissues than a more hydrophobic
surface [24]. In our study, a significant decrease in the
contact angle on the surface coated with PU, PA and PG
was shown, compared to the untreated control surface.
Therefore, these RG-Is should be considered for further
in vitro and in vivo studies.
The change in wettability of the surface has been re-
ported to be related not only to chemical modification but
Characterization of Pectin Nanocoatings at Polystyrene and Titanium Surfaces
Copyright © 2013 SciRes. JSEMAT
Figure 4. Representative images (2D and 3D) of RG-Is structure: potato unmodified (PU), potato dearabinanated (PA), po-
tato degalactanated (PG), potato dearabinanated degalactanated (PAG) on mica surface measured with a Multimode AFM 1
μm × 1 μm.
Characterization of Pectin Nanocoatings at Polystyrene and Titanium Surfaces
Copyright © 2013 SciRes. JSEMAT
Figure 5. Distribution plot of height measurements in nm of
RG-Is structure of PU, PA, PG, PAG performed with AFM
on Mica surface with 1 μm × 1 μm magnification. Potato
unmodified (PU), potato dearabinanated (PA) and potato
degalactanated (PG) and potato dearabinanated degalac-
tanated (PAG).
Figure 6. Representative image (3D) of RG-I nanocoating
on aminated titanium implant surface coated with potato
dearabinanated (PA aminated Ti implant), magnification 1 × 1
μm, measured using AFM with intermittent “Tapping mode”.
Figure 7. Representative confocal images of RG-Is nano-
coating visualised with immunofluorescence labeling by pri-
mary antibody, anti –(1 4)-β-galactan LM5 (IgG2c) and
secondary antibody, goat anti-rat IgG for LM5 linked to
FITC. Control: aminated Ti implant, Coated: PU Ti im-
plant; Ti: titanium, PU: potato unmodified.
also to the topographical changes of the surface [25,26].
Our surface roughness results showed that the RG-Is
used for nanocoating in general did not significantly af-
fect the roughness of the examined surfaces, which is in-
Figure 8. Representative confocal images of SaOS-2 cells
stained with Vybrant Cell-Labeling Solutions and cultured
on Ti implants surface nanocoated with RG-Is. Control:
aminated Ti implant, Coated: PU Ti implant; Ti: titanium,
PU: potato unmodified.
accordance with findings from Morra et al. (2004) [16].
The reason for different results of surface roughness (Sa,
Sdr) on polystyrene and titanium surface, when each of
the surface groups was compared, can be explained by
differences in surface texture, illustrated by the SEM im-
ages. It has also to be noticed that the surface roughness
was measured by AFM on a 20 µm × 20 µm square area
as recommended for a non quantitative overview of the
nanotopography [27]. Higher magnification would pro-
bably show difference between nanocoated and non-
coated surfaces as the RG-I molecules used for coating
are around 100 nm in size. Thus, our measurements per-
formed with AFM on a 1 µm × 1 µm area clearly visual-
ized the RG-Is nanocoating also on the titanium implant
surface. Nanocoatings of that size have not previously
been demonstrated. The visualization of the nanocoating
may be important for characterizing the physical proper-
ties of the nanocoated surface. The structure, as well as
the distribution and the topography of the nanocoating,
may play an important role in cell adhesion, as the nano-
coating can mimic the extracellular matrix (ECM) [28].
In the present study, we also visualized RG-Is nanocoat-
ing on titanium implants by immunofluorescence stain-
ing using the primary LM5 antibody, which specifically
binds to galactan side chains. By using the AFM tech-
nique with atomically smooth surfaces, we were able to
visualize and analyze the height and length of RG-Is
from potato. The length of the individual molecule was
in the range of 100 nm, which is in agreement with other
studies [22]. The height measurements showed a de-
crease in the height of modified RG-Is (PA, PG, PAG)
compared to unmodified RG-Is (PU), which showed that
enzymatic modification changed the RG-Is structure.
This corresponds to our previous findings, demonstrating
that enzymatic treatment of RG-Is decreases the amount
of galactan and arabinan in modified RG-Is compared to
unmodified RG-Is (PU). [14] Our height measurements
of modified RG-Is have not allowed us to distinguish
Characterization of Pectin Nanocoatings at Polystyrene and Titanium Surfaces
Copyright © 2013 SciRes. JSEMAT
between dearabinanated, degalactananated and debranch-
ed structures and therefore more detailed investigations
with AFM in “liquid cell” (AFM imaging in liquids) are
necessary [29].
The osteblast-like cells (SaOS2) grown on Ti implants
were spread on the surface, however the morphology,
cell viability and proliferation studies remain to be per-
formed to examine the osteoblast behavior on titanium
implant surface coated with RG-Is. On the other hand,
our previous studies showed a significant increase in cell
viability and matrix mineralization of the same type of
cells (SaOS2) on polystyrene and titanium discs surfaces
coated with RG-Is containing higher amounts of galac-
tose compared to controls [14]. Recent in vitro studies
[10,11,14-16] showed that the nanocoating of RG-I with
high amounts of galactose enhanced osteoblast spreading
and growth, in contrast to the nanocoating of RG-I with
high amounts of arabinose, which leads to aggregation
and decreased proliferation [11,13,14,16,17]. This find-
ing suggests that linear 1.4-linked galactans are impor-
tant for osteoblast adhesion, and that high content of ara-
binose can shield the galactans, and thus prohibiting their
interaction with osteoblasts [30]. It has previously been
shown that Galectin-3 binds specifically to galactose
residues [31]. As the osteoblastic cells contain Galectin-3,
it could therefore be speculated that osteoblast interaction
with the RG-I galactans is mediated through Galectin-3.
In addition, titanium surfaces coated with RG-Is have
been shown to positively influence cell adhesion, mor-
phology, proliferation and mineralization [10,15,23]. The
positive cell response on plant-derived molecules, espe-
cially RG-I with high amount of galactose, opens new
direction in the development of organic nanocoatings for
biomaterials in order to improve bone healing.
5. Acknowledgements
The authors thank Prof. Erik Fink Eriksen from Aarhus
Amtssygehus in Denmark for the gift of the SaOS-2 cell
line and Marco Morra for the aminated TCPS plates, Ti
discs and Ti implants.
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