To observe the early adsorption of extracellular matrix and blood plasma
proteins to magnesium-incorporated titanium oxide surfaces, which has shown
superior bone response in animal models.
Material and Methods
Commercially pure titanium discs were blasted with titanium dioxide
(TiO2) particles (control), and for the test group,
TiO2 blasted discs were further processed with a micro-arc
oxidation method (test). Surface morphology was investigated by scanning
electron microscopy, surface topography by optic interferometry,
characterization by X-ray photoelectron spectroscopy (XPS), and by X-ray
diffraction (XRD) analysis. The adsorption of 3 different proteins
(fibronectin, albumin, and collagen type I) was investigated by an
immunoblotting technique.
Results
The test surface showed a porous structure, whereas the control surface
showed a typical TiO2 blasted structure. XPS data revealed
magnesium-incorporation to the anodic oxide film of the surface. There was
no difference in surface roughness between the control and test surfaces.
For the protein adsorption test, the amount of albumin was significantly
higher on the control surface whereas the amount of fibronectin was
significantly higher on the test surface. Although there was no significant
difference, the test surface had a tendency to adsorb more collagen type
I.
Conclusions
The magnesium-incorporated anodized surface showed significantly higher
fibronectin adsorption and lower albumin adsorption than the blasted
surface. These results may be one of the reasons for the excellent bone
response previously observed in animal studies.
titanium dioxidemagnesiumimmunoblottingfibronectinsalbuminscollagen type I.INTRODUCTION
In living systems, blood (plasma) is the first component to come in contact with
biomaterials such as a titanium implant during surgery [1]. It is known that immediately after contact with plasma,
rapid adsorption of plasma proteins onto the biomaterial takes place [2], which influences subsequent cell attachment,
spreading, proliferation, and differentiation [3]. In bone-to-implant binding, i.e. osseointegration, the attachment of
blood-derived proteins such as plasma fibronectin to the implant surface enhances
the chemotaxis [4] and focal adhesion of
osteogenic cells [5]. Fibronectin is a
high-molecular weight extracellular matrix (ECM) protein (approximately 440 kDa)
that binds to integrins, which are membrane-spanning receptor proteins [6]. One form of fibronectin, the plasma
fibronectin, is produced by hepatocytes in the liver and circulates in the blood
[7]. It activates signaling pathways that
direct cell-cycle progression, gene expression, matrix mineralization [8], and the regulation of the survival of
osteoblasts [9]. In addition, plasma
fibronectin is known to be a regulator for bone density, and bone biomechanical
properties [7], and it has been reported that
plasma fibronectin interacts with bone morphogenetic protein 1, indicating that it
has an important role in osteogenesis [10].
Ever since the importance of a moderately roughened surface was proposed, the rate of
osseointegration has been enhanced by surface modification [11], and further, in recent times, surface modification is
being carried out even at the nano-level. It is therefore reasonable that implant
surfaces should be modified at this level, given that it has been proven that cells
react sensitively to nano-topographies [12].
Several studies have investigated the effect of the modified surfaces on osteogenic
cell reactions, and surface modifications aimed at enhancing cell responses have
been carried out [13,14]. However, few modifications have actually focused on the
protein reactions underlying such cell reactions.
We have used electrochemical oxidation incorporating protein "bindable"
ions such as sulfate, phosphate [15], and
calcium [16] to create surface modifications
aimed to theoretically attract ECM proteins or bone matrix proteins with high
bonding properties. The concept behind these novel modifications is the hypothetical
biochemical bonding between bone and the implant [16]. These so-called "bioactive" implants showed improved bone
responses compared to machined implants or other surfaces available on the market. A
recent study by Sul et al. [17] validated the
presence of a biochemical bond of surface chemistry modified smooth surface implants
in bone but also measured the relative quantity of their biochemical bond strength
at the bone-to-implant interface. Magnesium (Mg)-incorporated oxidized implants
showed stronger and faster bone integration as compared to commercially available
oxidized or dual acid-etched implants [18].
The enhanced bone response of the Mg implants was most likely due to the Mg titanate
chemistry effects, possibly attracting ECM proteins or bone matrix proteins. It has
been reported that Mg supplementation to young mice influenced bone formation,
resorption, and mineralization [19]. It has
also been shown that Mg deficiency may disturb bone metabolism and lead to
osteoporosis [20].
The objective of this in vitro study was to investigate if
adsorption of three different proteins, which are purportedly involved in bone
apposition to implant surfaces is altered in Mg-modified surfaces, which would
substantiate the in vivo data.
MATERIAL AND METHODS
Disc sample preparation
The disc samples (commercially pure titanium, CpTi, ASTM grade 4, 10 mm × 5 mm) were
manufactured using a CNC (computer numerical control) machine and then blasted with
TiO2 particles in the range of 100 - 150 μm. The implants were
degreased by sonication in an aqueous solution of phosphate-free Extran® MA 03
(Merck, Darmstadt, Germany)/deionized water (1 : 100) and absolute ethanol for 2 ×
15 min. Next, they were rinsed with deionized water, and then dried in an oven at 60
°C for 24 h. The samples were divided into 2 groups, the blasted control group and
the oxidized test group. The test group was fabricated in an electrolyte mixture
containing Mg ions. They were fabricated using a micro arc oxidation (MAO) process
as previously described [21,22]. In brief, the MAO process was conducted in
galvanostatic mode, with the anodic forming voltage increased at a rate of dV/dt and
controlled at 0.5 V/s by a combination of electrochemical parameters. The
electrolyte mixture was stirred with a magnetic stirring bar at 300 rpm. Currents
and voltages were recorded at 1-s intervals using a computer that was interfaced
with the power supply. The samples were rinsed with deionized water and then dried
in an oven at 60 °C for 24 h.
Surface properties and analysis techniques
The morphologies of the samples were observed using SEM (LV-SEM, JSM-6380LV; JEOL,
Sollentuna, Sweden). The chemical composition of the samples was measured by X-ray
photoelectron spectroscopy (XPS, ESCALAB 250, Thermo-VG, England) using a
monochromatic Al Kα X-ray source (1486.7 eV, 300 W; beam size, 400 µm
diameter). The electron take-off angle was fixed at 45° and the vacuum pressure
was maintained below 10-9 torr during spectral data acquisition. XPS data
were acquired before and after sputtering. In order to remove the superficial
contaminant (2 monolayers), Ar sputter cleaning was carried out for 3 s (beam
energy, 2 KeV; primary current, 2 µA; raster area, 3.14 mm2). The binding
energy of the target elements was determined with a resolution of 0.1 eV, using the
binding energy of carbon (C 1s: 284.8 eV) as a reference.
The crystal structure was determined by low-angle XRD with a thin film collimator
(X`Pert PRO-MRD, Philips Ltd, Netherlands) on a plate-type sample prepared with the
same electrochemical parameters as the test screw-shaped implants. The step size
used in the scan was 0.02° over the range of 15° to 70°. The spectra
were recorded using Cu Ka radiation (0.154056 Å) generated at an acceleration
voltage of 35 kV and a current of 25 mA.
Surface roughness was measured using an optical profilometer (MicroXamTM,
Phase-Shift, Arizona, USA). Three discs each from the test group and from the
control group were measured at 3 areas to give a total of 9 measurements for each
group. The measuring area was 230 µm × 230 µm for each group. A Gaussian filter, 50
µm × 50 µm, was used to separate the roughness from errors of form and waviness.
Attachment of purified albumin to disc surfaces
Relative amounts of attached albumin was analyzed by electrophoresis and Coomassie
blue staining of the gels after solubilization of the disc-associated albumin. Discs
in triplicate were immersed in 40 mg/ml purified human serum albumin (Sigma, St
Louis, MO, USA) for 16 h at 37 °C to saturate binding, then washed in
phosphate-buffered saline (PBS). Attached albumin was solubilized by boiling discs
for 5 min in detergent buffer (0.1% sodium dodecyl sulfate [SDS], 1% Igepal CA-630
and 0.5% sodium deoxycholate in PBS), and samples were analyzed by 5%
SDS-polyarylamide gel electrophoresis followed by Coomassie blue staining of the
gels. Evaluation of staining intensities was performed by analysing images using
Sigma Gel software (SPSS Science Software GmbH, Erkrath, Germany).
Attachment of collagen and fibronectin to disc surfaces
A total of 100 µl with 0.2 mg/ml bovine plasma fibronectin (Sigma, St Louis, MO, USA)
or bovine collagen type I (Nutacon BV, Leimuden, Netherlands) in PBS was added to
the tops of the discs in triplicate and incubated for 16 h at 37 °C. The discs
were washed 3 times in PBS and transferred to microcentrifuge tubes containing 500
µl 1% Igepal CA-630, 0.5% sodium deoxycholate, and 0.1% SDS. The discs were boiled
in this solution for 5 min, then chilled on ice, and the solutions were stored at -
80 °C. The samples were thawed on ice, and 25 µl of each sample was mixed with
25 µl 2XLaemmli sample buffer (BioRad, Hercules, CA, USA) and a final concentration
of 8% β-mercaptoethanol. The samples were boiled for 5 min, chilled on ice, and
run on a 5% SDS polyacrylamide gel electrophoresis. Proteins were
electro-transferred to polyvinylidene difluoride membranes (BioRad), and plasma
fibronectin and collagen type I were detected by immunoblotting with specific
antibodies. Rabbit anti-human fibronectin antibodies F-3648 were obtained from Sigma
(Sigma, St Louis, MO, USA), and rabbit anti-human collagen type I antibodies ab292
were from Abcam plc, Cambridge UK. Blots were developed using horseradish
peroxidase-coupled secondary antibodies and an Advance Western Blotting Detection
Kit (GE Health Care, Buckinghamshire, UK). Evaluation of staining intensities was
performed by analysing images using Sigma Gel software.
Statistical analysis
All statistical analyses in the present study were performed with the KaleidaGraph
software (Synergy Software, Essex Junction, VT, USA). The mean and standard
deviation values for the in vitro parameters were calculated. The average values
were compared by paired Student's t-test and analysis of variance (ANOVA) followed
by a post hoc Tukey-Kramer test with the value of statistical significance set at
the 0.05 level.
RESULTS
Surface characterization
Figure 1 shows SEM micrographs that
characterize blasted pits and facets in the control surface and homogeneous porous
structure with an average pore size of 1 - 2 µm in the test surface. The surface
roughness after filtering showed an Sa value (arithmetic average height deviation,
μm) of 0.81 (± 0.31) for the control and 0.75 (± 0.14) for the test group. No
significant differences regarding surface roughness were observed. Figure 2 shows
high resolution XPS spectra of the major elements extracted from the Ti 2p3/2 (458.9
± 0.1 eV), O 1 s (531 ± 0.5 eV) (Figure 2A),
and Mg 2p (50.4 ± 0.1 eV) core-level energy regions of the electron orbitals before
and after argon ion (Ar+) sputter cleaning (Figure
2B). Table 1 shows the quantitative differences between the chemical
compositions of the samples. The test sample showed the major doublet peaks of the O
1 s at 530.8 eV and 531.7 eV, which may be attributed to the Mg titanate and –OH
functional groups. The blasted implants consisted mainly of TiO2. Figure 3 shows the XRD patterns of the
amorphous structure in the control group and a mixture of anatase and rutile phase
in the test group (Figure 3).
Scanning electron microscopy image of the surface blasted with titanium
particles (control), and Mg-incorporated anodized surface (test) (Scale bar:
5 µm).
Ti 2p and O 1 s spectra of control and test surface. The dashed line
indicates the binding energy of peak position at Ti 2p and O 1 s for the
control surface.
Mg cation incorporation during the MAO process, characterizing the binding
energy at the Mg 2p of as-received and Ar + sputter-cleaned surfaces. The
dashed line indicates the binding energy of peak position at Mg 2p of
as-received surface.
X-ray diffraction patterns of control and test surface. Amorphous, anatase
and rutile phase of TiO2 were detected on the control and test
groups. Ti = titanium; A = anatase; R = rutile.
Binding energiesa and atom concentration rateb of
elements at as-received and sputter cleaned surface in XPS analysis.
Atom
Control
Test
Beforec
Afterd
Beforec
Afterd
at. %
BE
at. %
BE
at. %
BE
at. %
BE
Ti
12.5
458.8
19.3
458.8
10.0
458.8
17.9
458.9
O
55.8
530.2
67.9
530.3
53.1
531.5
60.7
530.5
Mg
-
-
-
-
6.9
50.4
9.2
50.5
C
31.3
284.8
12.8
284.8
27.9
284.8
9.8
284.8
N
0.4
400.2
-
-
2.1
400.3
1.6
400.4
aBinding energy value in eV
bAtom concentration rate in at.%
cAs-received surface
dSputter cleaned surface
BE = binding energies
Adsorption of purified albumin, fibronectin, and collagen type I to control and
test surfaces
The intensity of the protein stainings for each surface is shown in Figure 4A. The results of this study showed
that significantly more albumin adhered to control surfaces than to test surfaces,
and significantly less purified fibronectin adhered to control surfaces than to test
surfaces (P ≤ 0.05) (Figure 4B and 4C).
For collagen type I, no significant difference was detected, although a binding
tendency was found in favour of the test surface (P = 0.09) (Figure 4D).
Attachment of extracellular matrix and blood plasma proteins to control and
test surfaces (n = 3 in each group, performed in triplicate). Purified
albumin, fibronectin, or collagen type I were allowed to attach to surfaces
for 16 h at 37 ˚C. Relative binding was measured by electrophoresis
followed by Coomassie blue staining or immunoblotting of solubilized
proteins (A). Significant differences by densitometry (B - D) using paired
Student's t-test (P ≤ 0.05) are indicated (*).
DISCUSSION
This study focused on the effect of titanium surface property changes with particular
attention to the initial protein behaviour. Surface characterization determined
major differences of surface chemistry and crystal structure but minor differences
of surface roughness between the control and test surfaces.
In the test group, the characteristic element of Mg, 7 - 9 at.%, was incorporated
into the oxide layer through the field-associated ion incorporation during the MAO
process [22,23]. The finding of the hydroxyl group in the test surface is consistent
with the findings of previous studies [22,24].
It has been reported that enhanced osteogenic cell responses in vitro, and bone
apposition in vivo, have been observed in surfaces possessing an external layer of
anatase and rutile phases [25]. The present
results of surface characterization are congruent with those of the implants used
for the previous in vivo studies. This was an important aspect of our study, since
its aim was to validate in vivo initial protein interactions with in vitro data,
possibly correlating the latter to the enhanced bone apposition seen in the animal
studies [21,22,24,26].
The individual protein adsorption test showed that the amount of albumin adsorbed
onto the test surface was significantly lower than that adsorbed onto the control
surface. Moreover, the amount of fibronectin adsorbed to the test surface was
significantly higher than that adsorbed to the control surface. The amount of
collagen type I adsorbed onto the test surface was also higher, although the
difference was not statistically significant. The results of the study clearly
showed the characteristics of each surface with regard to specific protein binding.
It has been reported that osteoblasts grown on Mg-incorporated surfaces show higher
expression of β1, and α5β1 integrin receptors than do
non-Mg-incorporated surfaces [27]. Since the
β1, and α5β1 integrin receptors are known to be fibronectin
receptors, these results suggest that Mg attracts more fibronectin to the surface
than occurs with non-Mg-incorporated surfaces.
In a preliminary experiment, we used human plasma obtained from healthy blood donors
and incubated this on control and test samples for 16 h. The precipitated proteins
were run on SDS-polyacrylamide gel-electrophoresis, and protein bands detected by
Commassie blue staining were cut out of the gel and sent for protein identification
by mass spectrometry (Pick'n Post Service, VWR International AB, Stockholm, Sweden).
The results showed that at 16 h, the proteins above detection level were plasma
albumin, and the amount of adsorption showed different results than that from
purified albumin. Similarly, analysis of fibronectin by immunoblotting after
incubation of human plasma on discs showed different results compared to
corresponding purified protein demonstrated in the current study. It would be
interesting to study more thoroughly the protein adsorption using human plasma.
However, this is a difficult task because the amount and type of protein adsorption
changes rapidly due to competitive protein adsorption [28] (Vroman effect). The Vroman effect is the competitive
nature of protein adsorption onto the surface depending on the molecular weight of
the protein [29]. In future studies, it will
be interesting to observe different time points and clarify the mechanisms of this
phenomenon.
Collagen type I is the major constituent of bone matrix protein [30], which is assembled in the presence of
plasma fibronectin [31]. It is an essential
protein in osteogenesis [32], which occurs
later in the biological process. The reason for observing collagen type I adsorption
in the individual protein adsorption test was to investigate its reaction to the Mg
surface, because of its central role as structural component in bone, and the lack
or abnormality of both collagen type I and Mg causes osteogenesis imperfecta [33]. Although there was no significant
difference, the test surface tended to have higher amounts of collagen type I
adsorption, which may be one of the factors for the enhanced bone apposition seen in
animal studies.
Albumin is a major protein included in plasma (approx. 60%, molecular weight 65 kD)
which is also a well-known blocking protein used in laboratory experiments. It has
been reported that albumin has characteristics that prevent other protein adsorption
and cell adhesion on its coated surface [34].
The relationship between plasma fibronectin and albumin has been investigated by
Grainger and colleagues [35], who stated that
albumin "masks" adsorbed plasma fibronectin and lowers the amount of cell
attachment, and that on specific hydrophobic surfaces, albumin out-competes with
other ECM proteins, including plasma fibronectin, even if the concentration of the
plasma fibronectin is comparatively high. It is well known that plasma fibronectin
binds more to hydrophilic surfaces [36],
whereas albumin binds more to hydrophobic surfaces [37]. Anodic oxidized Ti surfaces have been reported to present
hydrophilicity [38,39]. It has also been reported that anodic oxidized Ti surfaces
have high surface energy [40,41], which is essential for maintaining surface
hydrophilicity [42]. This suggests that the
enhanced adsorption of fibronectin and reduction of albumin may be a result of
surface energy-related hydrophilicity as well as Mg incorporation. Since surface
roughness showed no significant differences, our study results strongly suggest the
involvement of theses abovementioned factors.
CONCLUSIONS
In this study, the effect of titanium property changes on the amount of fibronectin,
albumin, and collagen type I adsorption was investigated. Mg-incorporated titanium
oxide surfaces showed major differences of surface chemistry and crystal structure,
albeit similar surface roughness values compared to the control TiO2
blasted surface. In the protein adsorption investigation, the test surface
significantly reduced the adsorption of albumin and significantly enhanced
fibronectin adsorption as compared to the control. The presence of Mg, the high
surface energy, and hydrophilicity most likely influenced the enhancement of protein
adsorption. This may be a reason for the enhanced bone apposition observed in
previous animal studies.
ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS
This research was supported by the research grant from the Swedish Research
Council, Project no: 621-2005-3402, and from the Biotechnology development
project (2009-0084195) from the Ministry of Education, Science and Technology of
Korea.