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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5370_Библиотеки_им_академика_М_И_Перельмана
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208 Surface Modification of Magnesium and its Alloys for Biomedical Applications
2010; Liu, Xin, Tian, & Chu, 2007; Liu et al., 2010; Yamamoto & Hiromoto, 2009;
Yang, Hort, Willumeit, & Feyerabend, 2012); however, the reason for this effect is
not always clear due to the complex nature of the interactions. For example, Mueller
et al. reported that corrosion rates of pure Mg in PBS increase when BSA is added to
0.1%, but then decrease as further BSA is added (up to 10%) (Mueller, De Mele,
Nascimento, & Zeddies, 2009). Further, this trend was dependent on alloy, with
AZ31 displaying very little change in corrosion rate across BSA concentrations, while
LAE442 steadily increased. Kirkland et al. found that the addition of 10% FBS slowed
the corrosion rate of a number of alloys as well as pure Mg in MEM (Kirkland &
Birbilis, 2013). However, increasing additions of BSA to MEM solutions from
20 to 60 g/L led to increasing corrosion rates for pure Mg, although all wer e ultimately
lower than in MEM alone (Kirkland & Birbilis, 2013). These varying trends suggest
the corrosion influence of proteins is more complicated than the simplistic adsorption
model suggests, because proteins in solution have a complicated relationship on the
passivation layers that form (Willumeit et al., 2011).
A number of tests that have been performed on Mg with the addition of proteins to a
base simulated body fluid are summarized in Table 6.1.
In the literature, the amount of protein added to solutions has varied widely, from
approximately 0.1 (Mueller et al., 2009) to 40 g/L (Rettig & Virtanen, 2009). It is important to consider that, when added in FBS form, the proteins are not solely BSA, and a
10% FBS solution would be equivalent to adding a total of w4 g/L of protein. Consequently, all FBS experiments had between 4 and 6 g/L of protein. Although none of the
experiments justified why this amount was chosen, it is a commonly used amount in cell
cultures, and FBS is expensive (Helgason & Miller, 2005).
Directly adding BSA proteins results in the same amount of protein in solution, as it
is in a pure form. Most experiments that used BSA additions also did not specify why a
certain amount was chosen. However, Mueller et al. chose three different amounts to
make comparisons between them (Mueller et al., 2009), and Rettig and Virtanen chose
to use 40 g/L in two separate experiments, the most realistic amount investigated to
date (Rettig & Virtanen, 2008, 2009). This was justified as it would equivalent
to the physi ological amount of HSA in the body.
Although the effect of proteins has been widely investigated for a number of
biomaterials, studies of their influence on Mg alloys has still been rather limited. Apart
from the tests mentioned above, very few studies have looked at how they might affect
the corrosion properties of coated alloys in vitro. As one of the key parameter s determining the success of an implant in the body, prote in interaction with the surface of
Mg alloys is still not well understood.
It is important to consider that the initial surface of the implant will be the surface
the proteins immediately come into contact with, and consequently it is this surface
that warrants the greatest study. Although CaP coatings do not chemically react
strongly with proteins, the changes in effective surface area, corrosion rate, local
pH, and diffusion will heavily affect the interaction with proteins, further altering
the corrosion behavior.
The biomimetic process uses solutions similar in ionic composition to physiological
fluids, with the aim of creating a coating that is similar in properties to the layer that
Effect of amino acids and proteins on the in vitro performance of coated magnesium 209
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Table 6.1 Magnesium in vitro experiments involving proteins
Primary author (reference) Base media Protein addition
Gu (Gu et al., 2009) MEM 10% FBS
Witte (Witte et al., 2007) MEM 10% FBS
Yamamoto (Yamamoto &
E-MEM 10% FBS
Hiromoto, 2009)
Pietak (Pietak, Mahoney,
MEM 15% FBS
Dias, & Staiger, 2007)
Liu (Liu et al., 2007) Basic SBF (not specified) 1 g/L BSA
Mueller (Mueller et al.,
PBS 1/10 g/L BSA
2007)
Mueller (Mueller et al.,
PBS 1/10 g/L BSA
2010)
Mueller (Mueller et al.,
PBS 0.1/1/10 g/L BSA
2009)
Rettig (Rettig & Virtanen,
Oyane m-SBF
a
40 g/L BSA
2008)
Eliezer (Eliezer & Witte,
0.9% NaCl, HBSS Not stated
2010)
Willumeit (Willumeit et al.,
DMEM, HBSS, Water 10/20% FBS
2011)
Salunke (Salunke et al.,
DMEM 10% FBS
2011)
Rettig (Rettig & Virtanen,
Oyane m-SBF
a
40 g/L BSA
2009)
Liu (Liu et al., 2010) Water, 0.9% NaCl 1/10 g/L BSA
Keim (Keim et al., 2011) “SBF,” DMEM, 100% FBS 10% FBS
a
Solution based on modified SBF by Oyane (Oyane et al., 2003).
would form in vivo (Barrere, 2002). The similarity of a biomimetic coating to natural
bone mineral can increase bioactivity of the surface (Baker et al., 2006). These coatings do not contain any elements not found in bone or body fluids and have been used
in various implant applications (Barrere, 2003; Kokubo, 1996; Lin & Li, 2006; Zhu &
Song, 2005). Biomimetic coatings can be created easily using the process described in
(Waterman et al., 2011). These coatings will be used to measure how an applied
coating can affect the corrosion response of amino acids and proteins in solution on
pure Mg. Due to its ubiquity, unique advantages, and ease of application, biomimetic
coatings were chosen in this work as the most ideal for further investigation.
210 Surface Modification of Magnesium and its Alloys for Biomedical Applications
6.3 Effect of amino acids on corrosion performance
of magnesium
6.3.1 Uncoated magnesium performance
Before proceeding to coated samples, it is first important to consider the impact of
amino acids on the corrosion of uncoated Mg. This provides a base for comparison
and further insight to the difference and protection coatings can offer.
The electrochemical behavior of pure Mg in a simple salt solution (Hanks Balanced
Salt Solution, HBSS) is shown in comparison to MEM, which contains amino acids.
Although the co mpositions of both media are similar, containing the same Mg
content, MEM has a lower Cl
as well as higher Ca2þand PO
3
concentrations
4
(Table 6.2). Theoretically, such a difference would suggest slightly decreased corrosion rates and increased CaP formation on the surface.
The polarization potential (E
in MEM than in HBSS (Figure 6.1). The corrosion current density (i
) of uncoated Mg was found to be higher for samples
Corr
) was also found
Corr
to be higher in MEM, which at a basic level is counterintuitive, given the aforementioned higher E
Table 6.2 Composition of corrosion media
and lower Clcontent in MEM. It is possible that this increased
Corr
2þ
ion
Hank’s
balanced
Human
Component
þ
Na
Cl
þ
K
2þ
Ca
2þ
Mg
2
HPO
4
2
SO
4
D-Glucose 5 5.5 5.5 5.5
Bicarbonate (HCO
plasma (HP)
142 145 117.4 117.4
103 144.6 123.5 123.5
5.0 5.8 5.4 5.4
2.5 1.3 1.8 1.8
1.5 0.4 0.4 0.4
1.0 0.8 1 1
0.5 0.4 0.4 0.4
)22e30 26.2 26.2 26.2
3
salt solution
a
(HBSS)
Minimum
essential
medium
b
(MEM)
HEPES e 25 25 25
Phenol red e 0.03 0.03 0.03
Albumin (g/L) 34e54 ee40
All concentrations in mmol/L unless otherwise stated.
Concentrations of inorganic blood contents given as in Warrel (2003).
a
H1641, Sigma-Aldrich.
b
56414C, Sigma-Aldrich.
c
MP Biomedical NZ Ltd.
MEM D bovine
serum albumin
(MEM D BSA)
c
Effect of amino acids and proteins on the in vitro performance of coated magnesium 211
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–1.45
–1.50
–1.55
–1.60
–1.65
–1.70
/ V vs SCE
–1.75
Corr
–1.80
E
–1.85
–1.90
–1.95
–2.00
MEM 30 min
MEM 8 h
MEM + BSA 30 min
MEM + BSA 8 h
Mg Biomimetic Mg Biomimetic
160
140
120
–2
100
cm
.
80
/ µA
Corr
i
60
40
20
0
MEM 30 min
MEM 8 h
MEM + BSA 30 min
MEM + BSA 8 h
Figure 6.1 Corrosion potential and current density for uncoated samples with and without
amino acids and proteins.
corrosion rate was observed due to the increased buffer capacity provided by amino
acids (Malda et al., 2008), as well as the proposed mechanism that amino acids chelate
with Mg
2þ
and inhibit passive layer formation (Yamamoto & Hiromoto, 2009).
Materials and methods
The pure Mg used for this work was a high-purity form (99.99%) obtained specifically for this
study (Timmenco Ltd., Toronto, Canada). The chemical compos itions were determined i ndepen dently via inductively coupled plasma atomic emission spectroscopy (results not shown herein).
Unless otherwise stated, all experiments presented in this chapter were performed using a threeelectrode flat-cell (K0235, Princeton Ap plied Research, TN, USA) with a volume of 300 mL of
media and an exposed working area of 1 cm
EC-Lab 10.2 software (BioLogic Inc., TN, USA).
2
. Results were recorded on a Biologic SP-150 using
Although MEM contains a similar composition of inorganic ions as HBSS, the
amino acids it contains may act as charge carriers, resulting in the possibility that
the higher current measurements were an artefact of solution conductivity. To investigate this, the conduct ivities of the solutions were measured, with MEM found to
possess a slightly lower conductivity than HBSS with HEPES, indicating that this
was not the case (Figure 6.2).
6.3.2 Effect on coated magnesium
Analyzing the polarization behavior of coated Mg samples in HBSS and MEM
revealed some interesting results (Figure 6.3). A similar trend was observed across
the samples, with an increase in both E
due to a large increase in the cathodic kinetics (i.e., increase in cathodic reactions
Corr
and i
in MEM over HBSS, primarily
Corr
212 Surface Modification of Magnesium and its Alloys for Biomedical Applications
12
11
10
9
8
7
6
5
4
3
Solution conductivity / mS / cm
2
1
0
HBSS MEM MEM + BSA
Figure 6.2 Solution conductivity at 37C with the HEPES buffer.
–1.4
–1.5
–1.6
–1.7
/ V vs SCE
WE
–1.8
E
–1.9
–2.0
–4 –3 –2 –1 0
Mg HBSS
Mg MEM
Biomimetic HBSS
Biomimetic MEM
log(|i / mA∙cm–2|)
Figure 6.3 PDP in HBSS versus MEM buffered with HEPES at 8 h immersion.
taking place). The nature of the corrosion layers can be determined through impedance
testing. Although it has been reported elsewhere that, for steel and aluminum, amino
acids increase polarization resistance (Ashassi-Sorkhabi et al., 2004, 2005), the opposite appears true for uncoated Mg in MEM (at least for the first few hours) (Figure 6.4).
From the Nyquist plots, it can be seen that, for each time point, both the film and EDL
resistance was decreased in the presence of the amino acids. Consequently, instead of
slowing short-term corrosion, amino acids are increasing the rate at which it proceeds.
This is supported by the findings of Yamamoto et al. (Yamamoto & Hiromoto, 2009).
Effect of amino acids and proteins on the in vitro performance of coated magnesium 213
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(a) 1 h
1.0
2
0.5
–Im(Z) / kΩ·cm
0.0
0.0 0.5 1.0 1.5
Re(Z) / kΩ·cm
Mg HBSS
Mg MEM
Biomimetic HBSS
Biomimetic MEM
2
(b) 7 h
1.0
2
0.5
–Im(Z) / kΩ·cm
0.0
0.0 0.5 1.0 1.5
Re(Z) / kΩ·cm
Figure 6.4 EIS of all samples in HBSS and MEM buffered with HEPES at 1 h (a) and 7 h (b)
immersion.
2
Coated samples exhibited similar behavior in the presence of amino acids, with the
polarization resistance remaining primarily cathodically controlled and increased in
MEM compared with HBSS. However, unlike uncoated Mg, this shift was accompanied by a decrease in the anodic kinetics, making the biomimetic-coated sample
display less corrosion in MEM than HBSS after 6 h. A similar behavior can be seen
when analyzing the polarization resistance, where the coated samples initially have
lower resistance in MEM, but become almost equal after 7 h immersion. Coupled
with the more noble potential, this leads to an overall reduction in the corrosion rate.
From these data, we can see that the effect of MEM is altered when the sample is
coated versus uncoated. For uncoated samples, the additional buffer capacity of the
MEM and/or the inhibition of the passive layers increased corrosion rates. But when
the coatings are applied and MEM is added, the effect of these passivation layers is
less important, because the bulk of the protection comes from the CaP coating.
214 Surface Modification of Magnesium and its Alloys for Biomedical Applications
Also, because the effective corrosion rate is reduced, pH rise due to corrosion drops,
and the buffering capacity of the amino acids becomes less important. The net result is
that the accelerating effects on corrosion one might expect amino acids to have is
reduced by the application of a coating.
6.4 Effect of proteins on magnesium biocorrosion
6.4.1 Uncoated magnesium
Uncoated Mg displayed highly consistent corrosion potential in both MEM and MEMcontaining 40 g/L BSA (MEM þ BSA) throughout the duration of the tests Figure
6.1(a). Proteins do not appear to directly interact with the corrosion of pure Mg in so-
lution. For uncoated Mg, the proteins decreased the cathodic and anodic kinetics for
scans at both 30 min (Figure 6.5) and 8 h immersion (Figure 6.6) without changing
the corrosion potential significantly. The effect of proteins is report ed to be focused
around the adsorption to form a protective layer (Liu et al., 2007, 2010). The total i
for Mg in MEM was initially high, more than 140 mA/cm2, but rapidly decreased over
the 8 h of investigation Figure 6.1(b). This behavior may be attributed to the formation
of the hydroxide layer, charge separation, and a rise in local pH, with longer immersion
times decreasing the corrosion rate. Looking at the polarization plots for uncoated samples, it can be seen that there was a slight drop in both anodic and cathodic branches,
consistent with the formation of a corrosion layer on Mg (Figure 6.7(a)). This is
confirmed via EIS (Figure 6.8(a) and (b)), as Mg in MEM displays the two-time
constant system that commonly characterizes semiprotective hydroxide layers. After
8 h immersion, the total impedance of both the oxide layer and the EDL has increased,
indicating the corrosion film became slightly more protective as corrosion progressed
and the layer formed, which can be seen later in Figure 6.9.
Corr
–1.4
–1.5
–1.6
–1.7
–1.8
/ V vs SCE
WE
E
–1.9
–2.0
–2.1
Figure 6.5 PDP of all samples in MEM and MEM þ BSA at 30 min.
Mg MEM
Mg MEM + BSA
Biomimetic MEM
Biomimetic MEM + BSA
–4 –3 –2 –1 0
log(|i / mA∙cm–2|)
Effect of amino acids and proteins on the in vitro performance of coated magnesium 215
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–1.4
–1.5
–1.6
–1.7
/ V vs SCE
WE
–1.8
E
–1.9
–2.0
Figure 6.6 PDP of all samples in MEM and MEM þ BSA at 8 h.
Mg MEM
Mg MEM + BSA
Biomimetic MEM
Biomimetic MEM + BSA
–4 –3 –2 –1 0
log(|i / mA∙cm–2|)
The addition of the protein albumin to the test s olution strongly affects the corrosion behavior of uncoated Mg. The corrosion potential, E
change with the addition of albumin; however, the i
Corr
, does not significantly
Corr
was found to drop considerably (Figure 6.1). BSA addition was found to decrease both the cathodic and
anodic reactions (Figure 6.7(a)). When corroding in BSA, the total corrosion current
density did not drop much from 30 min to 8 h , suggesting that the initial layers
that formed within 30 min did not change much over the duration of the test. The
Nyquist plots confirm this, showing similar impedance behavior for Mg in
BSA at both time points (Figure 6.8). The relative shape of the Nyquist plots
remain similar to MEM solutions alone. This is indicative of a greater film and
double-layer resistance due to proteins impeding corrosion by ads orbing to the
surface and forming a protective la yer (Liu et al., 2007, 2010; Rettig & Virtanen,
2008, 2009).
Previously, albumin has been shown to increase E
of Mg by decreasing the
Corr
anodic reaction rate on AZ91 (Liu et al., 2007), as well as increase the anodic reaction rate on WE43 and LAE442 (Mueller et al., 2010). Over the 8 h tests reported
here, both anodic and cathodic reactions decreased with the addition of BSA. This
matchesworkpreviouslyreportedby(Kirkland et al., 2010). Adsorption of proteins
to the Mg surface creates a layer that partially protects the underlying metal by
reducing the effective exposed surface area. While it has been reported that chelating
metal ions with the proteins would increase t he corrosion rate of other metals (Clark
& Williams, 1982), the data here support previous works that do not show this effect
on pure Mg (Willumeit et al., 2011). However, it is clear from the literature that
different alloys, surfaces, and corrosion conditions can affect the measured results.
To understand the effects of surface condi tio n and cor rosi on rat e on the amino
acid and protein modified solutions, the data presented above will be used as a baseline from which to compare coated samples.
216 Surface Modification of Magnesium and its Alloys for Biomedical Applications
(a) Uncoated Mg
–1.4
–1.5
–1.6
–1.7
–1.8
/ V vs SCE
–1.9
WE
E
–2.0
–2.1
–2.2
MEM 30 min
MEM + BSA 30 min
MEM 8 h
MEM + BSA 8 h
–4 –3 –2 –1 0
log(|i / mA∙cm–2|)
(b) Biomimetic
–1.4
–1.5
–1.6
–1.7
–1.8
/ V vs SCE
–1.9
WE
E
–2.0
–2.1
–2.2
Figure 6.7 PDP over time in MEM and MEM þ BSA for (a) uncoated Mg, (b) biomimetic
coated.
MEM 30 min
MEM + BSA 30 min
MEM 8 h
MEM + BSA 8 h
–4 –3 –2 –1 0
log(|i / mA∙cm–2|)
6.4.2 Proteins on biomimetically coated magnesium
The electrochemical behavior of biomimetically coated samples was tested under the
same conditions as the bare Mg substrates. The coatings have the effect of changing
the surface, reducing corrosion and diffusion rates from the corrosion sites to the
bulk solution. The coatings caused a decrease in the measured E
Although reduced in the short term, the corrosion potential of the biomimeticcoated Mg increased toward, but not reaching, the potential of the uncoated samples
in both solutions over the 8 h of testing (Figure 6.1). Although the addition of proteins
did not significantly affect the corrosion potential of uncoated Mg, when the coating is
for all samples.
Corr
Effect of amino acids and proteins on the in vitro performance of coated magnesium 217
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(a) 1 h
1.0
2
Mg MEM
Mg BSA
Biomimetic MEM
Biomimetic BSA
0.5
–Im(Z) / kΩ·cm
0.0
0.0 0.5 1.0 1.5 2.0
Re(Z) / kΩ·cm
2
(b) 7 h
1.0
2
0.5
–Im(Z) / kΩ·cm
0.0
0.0 0.5 1.0 1.5 2.0
Re(Z) / kΩ·cm
Figure 6.8 EIS of solutions with and without proteins at 1 h (a) and 7 h (b).
2
2.0
Mg MEM
Mg BSA
Biomimetic MEM
2
Biomimetic BSA
1.5
1.0
Resistance / kΩ·cm
0.5
0.0
012345678
Time / hours
Figure 6.9 EIS over time total polarization resistance.
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