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148
7. DEVELOPMENT OF LAYERWISE
HYDROXYAPATITE COATING: PHOSPHATE INTER
LAYERED COATING
7.1. INTRODUCTION
Metallic implants are generally used for load bearing applications [1]. A
thin hydroxyapatite (HA) coating, having the composition Ca10(P04)6(0H)2
similar to bone apatite, can prevent the release of metal ions from the substrate into
the biological environment [2]. Commonly developed HA coatings on metallic
substrates suffer from many problems like cracking and peeling off [3], which
results in the release of hannful metal ions to the body environment. Also the
adherence of the HA coating to the substrate is very poor. In order to overcome
inferior adhesion an interlayer coating can be provided in between the metal
substrate and the HA outer layer [ 4]. The present study had the approach of
providing a zinc phosphate interlayer on metallic substrate in order to improve the
adherence of HA to the substrate.
Zinc is an essential trace element in human body and has stimulatory effect
on bone fonnation in-vitro and in-vivo. The zinc content of human bone ranges
from 0.0126-0.0217 wt%. Zinc containing tricalcium phosphate(ZnTCP) has
pharmaceutical effect in bone formation [ 5]. Zinc releasing calcium phosphates
favour human marrow cell culture [6]. Great advances have been achieved in
combining hydroxyapatite with zinc phosphate (7, 8, 9]. ZnTCP/HA ceramics are
149
superior in terms of supporting osteogenic differentiation [10]. Zn3(P04)2.4H20
(Hopeite) gets converted into hydroxyapatite when immersed in an aqueous
sol�tion containing 0.1 mol/L Ca(N03)z at 60°
C for 3 h [11].
In the present study a zinc phosphate (ZnP) inter-layer was provided on
316L SS by conversion coating after hot-dip galvanization of the substrate.
Stainless steel is much cheaper when compared with titanium; the only demerit is
the extent of biocompatibility. In the present case, the substrate was ensured that it
act merely as an interior mass of the system because it has no possibility of getting
its surface exposed into the environment. Hence the focus point became cheaper
substrate. The hot-dip galvanization process resulted in the formation of alloying
layers between iron and zinc. These layers could control the corrosion of the
substrate and prevent the release of metal ions from the substrate. Conversion
coati ngs on metals generally enhance paint or lacquer adhesion and prevent
corrosion [12]. In the present work a conversion coating of ZnP layer acted as a
barrier preventing probable metal ion release from the substrate and increased the
adhesion of a HA superficial layer formed on it. The ZnP layer facilitated efficient
re-growth of HA when there was an artificial destruction in aggressive biological
environments followed by immersion in Simulated Body Fluid (S. B. F.). The HA
layer was evaluated by different techniques. The detailed results are discussed in
this chapter.
150
7.2. EXPERIMENT AL
7.2.1. The hot-dip galvanization process
Commercially available stainless steel (3 l 6L SS) was selected as the
substrate. The pre-treatment was carried out as discussed in Sec. 3.3.2. and in
3.3.2.2. The hot - dip galvanization was carried out as discussed in Sec. 3.3.2.2.
The phosphating was also discussed in detail in that section.
7.2.2. HA coating by electrodeposition
The conversion coated coupons were rinsed with acetone, dried in air and
subjected to electrochemical deposition of calcium phosphate at 60°C in
galvanostatic mode at a current density ranging from 0.6-1.4 mA/cm2 and a voltage
of -l.40V vs SCE. The electrolyte used for the electrodeposition contained 0.084M
Ca (N03)2 and 0.050 M NHtH2P04 [12]. The pH of the bath was adjusted to 4.60
by adding dilute ammonia water. The zinc phosphate coated substrate was kept as
the cathode against a platinum foil anode. After electrodeposition, the calcium
phosphate ceramic coating was treated in 0.1 M NaOH at 60°C for 2 days.
7.2.3. Physico-chemical characterization of the coating
The surface morphology of the HA coating developed on the ZnP coated
substrate was analyzed by scanning electron microscopy (SEM, HITACHI, S-
2400). The HA coated coupons were rinsed with acetone, dried in air and its
surface was gold coated in order to make it conductive during the SEM analysis.
Porosity of the coating was analyzed by using modified ferroxyl reagent test [13,
14]. The normal ferroxyl test was not suitable for zinc coating since the exposed
portion of iron was cathodically protected by sacrificial action of zinc. In the
151
present case an external anodic potential of 0.400 V was impressed on the coupons
to overcome the galvanic action. The ferroxyl reagent, consisting of a solution
containing potassium ferricyanide, sodium chloride and agar-agar in hot water was
applied on the surface of the coupons for 30 minutes and inspected for any
prussian blue colouration. The adherence of the HA coating was evaluated by
scratching the surface using a brush followed by washing in distilled water. The
surface of the HA coated coupons were scratched with a nylon brush and it was
immersed in distilled water for 1 hour. The brush was selected in such a way that it
would not get any deterioration during the whole test duration. The extent of
adherence was evaluated only relatively as a qualitative parameter. This test was
conducted as there was no conventional specific test available for evaluation of
adherence of HA coated implants. Also the surface of the HA coatings was
scratched with graphite pencils (H, HB and 2B) and then immersed in distilled
water for 1 hour for any HA dissolution. The composition of the conversion
coating was determined by X-ray diffraction analysis using Cu-Ka radiation (X'
pert Pro analyzer), after rinsing the coupons with distilled water and drying at
100°c.
7.2.4. Evaluation of bioactivity of the coating
Bio activity of the ZnP coating was evaluated by soaking in S. B. F. as
discussed in Sec. 3.6.5. The release of zinc ions into the biomimetic bath was also
evaluated by AAS analysis. The absorbance measurements of the sample solutions
were performed using a GBC-A VENT A atomic absorption spectrophotometer
(AAS).
152
7.2.5, Electrochemical evaluation of the coating
The corrosion characteristics of the HA coated ZnP coating was
evaluated in 0.9% physiological saline solution. The trend of variation in open
circuit potential (O.C.P.) was recorded for a period of two weeks at a temperature
of 37 ± 1 °C. A saturated calomel electrode (SCE) was used as the reference
electrode.
Electrochemical impedance spectroscopic (EIS) studies were carried out
using an Autolab PGST AT 30 plus FRA 2 corrosion measurement system. The
impedance analysis was carried out using Ringer's physiological solution as the
electrolyte. Ag/ AgCl, Pt and the coupon having 1 cm2 exposed area was used as
reference, counter and working electrodes respectively. Impedance analysis at the
frequency range of 1 MHz to 10 Hz was carried out with reference to O.C.P. after
30 minutes of exposure of the coupons in the electrolyte.
7.2.6. Evaluation under special conditions
The stability of the HA coating developed biomimetically on ZnP substrate
was analyzed in aggressive physiological solution. In order to make aggressive
physiological condition the pH of Ringer's solution was changed to acidic one. The
initial pH of the Ringer's solution was 6.74, which is converted to an acidic pH
(4.5) by adding a drop of 0.1 M HCL The HA coated substrates were immersed in
the modified Ringer's solution for 2, 4, 6 & 8 days. Then the re-growth
characteristics of the HA superficial coating developed biomimetically on the ZnP
interlayer was studied in S. B. F. The biomimetic HA coating was initially
evaluated for its stability in modified Ringer's solution (pH 4.5) for 2 to 8 days, at
153
a temperature of 37 ± 0.5°C. The open circuit potential (0. C. P) variation during
this process was evaluated by using a Saturated Calomel Electrode (SCE) as the
reference electrode. The surface morphology of the coatings was then evaluated
using an optical microscope (Olympus SZ 61, magnification x40). The re-growth
characteristics of the destructed coupons were then analyzed in S. B. F. similar to
the process as mentioned in Sec. 2.6. The OCP of the coupons were monitored
during the biomimetic growth. The re-growth morphology was also evaluated
using an optical micrograph (Olympus SZ61, magnification x40).
7.3. SELECTION AND STANDARDIZATION OF THE
PROCESS
316L SS having the composition mentioned in Sec. 3.3.2 were chosen as
the substrate for the present study. The hot-dip galvanization process conditions
were previously practiced and standardized in our laboratory [15, 16]. Generally
hot-dip galvanization process facilitates the alloying reaction between iron and
zinc. In the present work, prior to conversion coating of the galvanized surface, the
zinc rich outer layer was etched with 2% H2S04 for one minute to drain off the
excess zinc present on the surface, i.e., the pure zinc layer was almost completely
etched out. Then the surface zinc layer was completely converted into zinc oxide
that involved in the phosphating reaction. In order to achieve a coating with high
bond strength and good accommodation character to bone, a gradient structure
should be preferred [17]. In this context, the present work had the approach of
providing an inter phosphate layer on the Fe-Zn alloy layer that was gradient in
15 4
zinc content. The conversion coated layer enhanced the deposition of calcium
phosphate onto its surface. The calcium phosphate coating was then subjected to
an alkaline treatment (Sec. 7.2.2) as HA is the predominant phase at pH greater
than 6.9 [18]. After standardization of the process in terms of reproducibility and
reliability with optimum values, the experimental parameters were kept constant
throughout the entire study.
7.4. COMPOSITION OF THE COATING
The composition of both ZnP and ZnP/HA coatings were characterized
based on XRD analysis [Fig. 7.l(a, b& c)]. The peaks [Fig. 7.l(a)] corresponding
to (211), (11 2) and (300) plane at 31.77°, 32.19° and 32.90° revealed the presence
of HA as dominant phases in the coating. Eventhough traces of other calcium
phases were also seen in the XRD pattern, only the predominant phase was
considered in the present case. It should be noted that the development of refined
HA was not the only prime objective of the present work but to develop an
adherent calcium phosphate coating with high bio activity was the ultimate. The
conversion coating of the ZnP layer was found to be hopeite, Zn3(P04)2.4H20. The
strong peak at 31.35° was a clear evidence of the presence of hopeite phase in the
coating (Fig. 7. l(c). Other peaks that appear at 32-34° (broad peak), 39°, 4 4°, 53°,
60° and 65° (Fig 7 .1 (b)) could be probably due to the presence of some other
phases than hopeite, which are not identified in the present study. In the present
study only the. major hopeite phase was considered and proceeded with
characterization of its bio activity. Hopeite is known to decompose by losing two
155
water molecules at I 00°C. But heating at 50-60°C as in the present case could not
cause �y such lose of water from hopeite leading to any structural change. The
hopeite structure of zinc phosphate has high osteogenic property [19] and could
facilitate better bone growth.
!jll]J • 800
:i 600 (.1111)
400 tJ
200
0 30 40 60 60
2 Theta
2000 b
11600
1000 tJ
600
0 20 30 40 60 60
2 Theta
100K
BOK
&OK
40K:
20K
20 30 40
156
C
60 60
2 Theta
Fig. 7.1. The XRD patterns of the (a) the HA coating, (b) the
conversion coated layer and (c) that with high count Y-axis.
7.5. POROSITY AND ADHERENCE OF THE COATING
The porous nature of the galvanized coatings was analyzed based on
modified ferroxyl reagent test. This analysis was carried out in order to assess the
exposure of stainless steel surface towards the environment. As per the modified
ferroxyl reagent test (Sec. 7.2.3), the galvanized coating was found to be non
porous; not showing the characteristic blue spots upon reaction with the substrate
iron and ferroxyl reagent. The ZnP layer formed by conversion coating was found
to be some what porous. This porous nature facilitated the calcium phosphate
coating via electrodeposition as there is an electrical contact between the
electrolyte and the substrate surface.
The HA coating was developed on the ZnP surface with the aim to develop
adherent coatings which is stable under in vivo condition. The adherence property
of the HA coatings to the ZnP substrate was evaluated by scratch test followed by
157
dissolution in distilled water. The HA coating was found to be very much adherent
to the substrate as revealed by scratch test and its dissolution in distilled water.
This adherence property of the HA coating revealed that the coating is suitable for
in vivo applications. In the present study, the ferroxyl reagent test, scratching test
and dissolution test were conducted only qualitatively. Also ferroxyl reagent test is
conducted only to know whether any substrate surface was exposed to the
environment.
7.6. SURFACE MORPHOLOGY OF THE COATING
7.6.1. Surface morphology
In order to evaluate the nature of HA layer formed on the surface of ZnP,
the morphology of the coating was analyzed by SEM. The surface morphology of
the electrodeposited HA coating is shown in Fig. 7.2 (i & ii). The ZnP conversion
coated layer was covered with HA, leaving uniform micro spots of uncovered area
of less than IO µm length. HA coating exhibited segregated growth. The porous
nature of the ZnP layer favoured the growth of HA during electrodeposition, by
exposing Zn2+
ions through the pores of the ZnP layer in the cathode, as ZnP is not
electrically conductive.
158
Fig.7.2. The surf: ce morphology [Scanning Electron Micrographs] of
the electrodeposited HA co ting on ZnP [magnification (i) x 600 and
(ii) xl.Ok].
7.6.2. Layerwise structure
159
IHA layer IIZnP layer I
Alloy layer ISubstrate
Specimen holder
Fig. 7.3 The surface morphology [Scanning Electron Micrographs] of
the electrodeposited HA coating on ZnP (the cross sectional view)
[magnification (i) xSO, (ii) x600, enlarged view of the alloy layer and
(iii) x60).
The cross sectional morphology of the coating showed the layering nature
of the Zn-ZnP-HA coating [Fig. 7.3 (iii)]. The cross sectional view of the HA
coated substrate revealed that the steel surface was entirely protected by the inner
alloy layers and the conversion coated layer, over which HA coating was present.
Actually the zinc phosphate layer and the HA outer layer together had porosity.
The steel surface was efficiently protected by the inner alloy layers over which the
porous conversion coated layer and HA coating was present. The steel surface was
entirely protected by the zinc phosphate and calcium phosphate layers (HA). The
gradient nature of the alloy layers could enhance the protection of steel from
getting exposed to the environment.
160
7.7. CORROSION RESISTANCE OF THE COATING
7.7.1. Variation of open circuit potential
w O.Ci
0.3
0.1
> -0.1
ii -0.3
-0.Ci
-0.7
-0.9
-1.1
0 Ci 10 15
Immersion time, days
Fig. 7.4. The variation in O.C.P. of the coupons during long term
immersion test in stagnant 0.9% NaCl solution at 37 ± 0.5°C [0- bare
SS, A- ZnP, D - ZnP /HA coating].
The alloy layer formed during hot-dip galvanization is crucial for
preventing corrosion, as it does not allow the release of hannful metal ions into
biological solutions. The corrosion resistance tendency was evaluated by the
measurement of open circuit potential (O.C.P). The ZnP layer behaved almost inert
in physiological saline solution as evidenced in Fig. 7.4. There was very less
potential shift of the ZnP coating in physiological solution. Both the ZnP and .
ZnPIHA coating exhibited the similar potential shift, which indicated that the
mechanism of action of Cl- ion on both the phosphates is same, same protective
nature for ZnP and HA in normal saline solution.
161
7.7.2. Electrochemical impedance analysis
The impedance analysis of the pure zinc coating, phosphate zinc coating
and HA coating revealed the protective nature of the ZnP coated substrates in
Ringer's physiological solution. The Solution resistance (Rs) values were - 72.76, -
75.54, - 77.29 Ohm, Polarization resistance (Rp) values were 82.03, 87.34, 77.57
Ohm and constant phase element (CPE/F) values were 1.621 x 10-8, 1.303 x 10-8
,
1.252 x 10-3 for pure zinc coating, ZnP coating and HA coating respectively. The
Rp values of ZnP coating exhibited protective nature than the Rp values of HA. The
HA coating may easily get attacked by er ions and hence has low protective
nature than ZnP coating.
7.8. BIO GROWTH- BIOMIMETIC EVALUATION
The biocompatibility of the ZnP coating was evaluated by soaking the
substrate in S. B. F. as mentioned in Sec. 3.6.5. The surface morphology of the
biomimetically developed HA coating (Fig. 7.5.) was the clear evidence for the
biocompatibility of the ZnP layer.
162
Fig.7.S. The surface morphology [Scanning Electron Micrographs] of
the biomimetically developed HA coating on ZnP layer [magnification
(i) x600 and (ii) xl.Ok].
Both the extent of electrodeposit ion and biomimetic deposition of HA
were compared in order to eval uate the bio activity of ZoP under in vitro
conditions. Unlike the electrodeposited HA coating, the biomimetically developed
coating exhibited less exposed sites. The biomimetic deposition facilitated the
natural growth of HA from S. B. F. The ZnP layer acts as nucleation sites for
further HA growth. In this thin layer coating the phosphate component in the layer
enhanced the adhesion of Ca2+ easily, by acting as a negatively charged layer. This
Ca2+ adsorption from S. B. F. enhanced further apatite growth by adsorbing the
corresponding phosphate moiety. During the initial period of immersion zinc ions
were released into the biomimetic bath. But the biocompatibility of the coating was
not affected as the extent of zinc ions released falls within the allowed level [5]. In
the present case it was thought about the inhibitory effect of Zn. But, in the present
case only HA coated substrate was immersed. Secondly, the zinc concentration
became less than 1 ppm within 24 hours and less than 0.2 ppm within 48 hours.
These test results revealed to rule out the possibility of any inhibitory effect of HA.
163
Also zinc ions could favour the osteogenic activity. As the thin HA layer got
attached to ZnP it facilitated further growth of HA. Similar to the ZnP bone
cements (20] the developed ZnP thin layer had better biocompatibility. The zinc
ion concentration in S. B. F. under a sufficiently acidic condition, pH 4.5 was also
measured during initial days. There was no significant change in the zinc
concentration in the S. B. F. revealing that there was no formation of hopeite and
amorphous zinc calcium phosphate phases because only HA coated specimens
were immersed in S. B. F. The bio activity of the developed biocompatible
coatings was further evaluated with a new approach.
7.9. BIOACTIVITY
7.9.1. Destruction
In order to evaluate the stability and efficiency of the HA coating
developed biornimetically on the ZnP substrate, it was subjected to a new type of
evaluation technique. The coating was initially subjected to destructive analysis in
physiological media mentioned in Sec. 2.8. For obtaining aggressive physiological
solution, the pH of Ringer's physiological solution was changed from 6.0 to 4.5 by
adding dil. HCl drop wise. The pH of 5 mimics the acidic body environment
during the early inflammation reactions [21]. In the actual body condition some
adverse effect of proteins is found, here only the pH variation is considered. The
aggressive environment had a higher er concentration. The HA coating should
peel off from the surface due to er ion attack.
164
w -0.4
-0.5
-0.6
-0.7
-D.B
D 2 4 6 8
Tim•, days
Fig. 7.6. The variation in O.C.P. of the ZnP/HA coated coupon during
long term immersion test in modified Ringer's solution (pH 4.5), at a
temperature of 37 ± o.5°c.
The evaluation was carried out for a period of 2 to 8 days to assess the
stability of the coating. The O.C.P. variation monitored during the analysis (Fig.
7.6) exhibited the potential shift in less active region during the initial period. As
the immersion continued the potential value shifted to active region. On the eighth
day of immersion the HA coated coupons exhibited a potential variation of -
0.25V with respect to the SCE, in modified Ringer's solution.
Even after 8 days of exposure to aggressive environment, the coating did
not get destructed considerately [Fig. 7.8(a)]. Only the topmost HA layer got
slightly destructed. A thin porous layer of HA coating was found to be adherent to
the substrate even after 8 days of immersion. The pH of the Ringer's solution
which was initially at 6. 74 was reduced to 4.5 and then the HA coated coupon was
immersed. The pH increased to 7 .22 within 24 hours due to the dissolution of HA
resulting in release of OH·. This observation revealed that there was no destruction
165
in the HA coating. The initial dissolution of HA prevented further HA dissolution
by increasing the pH.
7.9.2, Re growth
w -Q4
-Q5
-Q&
-Q7
-QBa.
0 5 10 15
lmm•nlan tlm•, days
Fig. 7.7. The variation in O.C.P. of the ZnP/HA coated coupon during
re-growth of HA in simulated body fluid, after destruction for various
periods in modified Ringer's solution (pH 4.5) at a temperature of
37±0.s'C [ o-2 days, •-4days, .6. -6days, • -8 days]
The coatings after subjected to destructive analysis were then analyzed for
its re-growth characteristics in S. B. F. fo r a period of 14 days. Each of the coupons
destructed for different periods from 2 to 8 days were immersed in S. B. F. at a
temperature of 37 ± 0.5°C. The variation of potential of the coupons during the
biomimetic deposition exhibited almost same initial O.C.P. for the coupons
subjected to destructive analysis for 2 and 4 days. The coupons that were subjected
to the destructive analysis for 6 and 8 days exhibited almost similar initial O.C.P.
(Fig. 7.7). As the biomimetic deposition continued the O.C.P. of the coupons
exhibited a shift in potential towards the less active region. Irrespective of the
extent of destru�tive analysis period, the coatings regained similar bio-growth after
14 days.
166
Fig. 7.8. (a) The surface morphology (optical micrograph,
magnification x40) of the biomimetic HA coating after subjecting to
immersion in modified Ringer's solution (at a pH 4.5 and at a
temperature of 37 ± O.SoC) (i) for 2 days & (ii) for 8 days. (b) The
surface morphology (optical micrograph, magnification x40) of the
destructed HA coating after re-growth of HA by immersing in S. B. F.
for a period of two weeks, at a temperature of 37 ± O.50C and at a pH
of 7.4 (i) HA re-growth of (a (i) ) (ii) HA re-growth of (a (ii) )
As per visual observation, the destructed coatings had a uniform growth of
HA soon after the immersion in S. B. F. It showed a uniform growth ofHA as that
of the fresh ZnP layer. The surface morphology (optical micrograph,
magnification x40) of the two extreme cases, i.e., the coatings subjected to
167
destruction for 2 and 8 days and their re-growth after immersing in S. B. F. for 14
days was 'shown in Fig. 7.8 (a & b) for comparison. The coating destructed for 2
and 8 days exhibited almost the same HA re-growth after immersion in S. B. F. for
a period of 2 weeks.
7.10. SUITABILITY OF ZINC PHOSPHATE FOR FURTHER
COATING
The ZnP layer was very much adherent to the substrate and was of porous
in nature. Even if, any zinc ions present in the porous ZnP layer, it would not cause
any harmful effects to the biological activity of the developed coating. The zinc
releasing calcium phosphates could favour osteogenic differentiation if the level of
zinc ions released will fall in between the allowed level [5]. In the present case,
the zinc ions released into the S. B. F. during the first day of immersion was only
1.004 ppm and it decreased and reached almost zero value on the seventh day of
immersion as evidenced in Fig. 7.9. Thereafter the release of zinc ions got ceased
because the substrate surface was fully covered with hydroxyapatite coating. Based
on the reliability and efficiency of the developed coating its applicability and
feasibility in the biological environment was analyzed.
168
E 1.2 A. A.
1 C
0.8 0
·o
0.6
0.4
C 0.2 0
0
0 2 4 6 8
Immersion time, days
Fig.7.9. The plot showing the amount of Zinc released into the bath
during the biomimetic deposition.
316 L SS implants had adverse effect due to harmful metal ion release [22].
In this context the minimization of harmful metal ions is beneficial. Zinc metal is
somewhat toxic to human body, even though it is a dietary supplement in permitted
levels. In the present study, the zinc layer formed is etched with H2S04 and the
surface layer is completely transformed into a phosphate layer. A. Ito et. al
described ZnP as an "intelligent material" with self release regulating ability [5].
Also a large number of literature support the biological application of zinc
phosphate [ 5-1 O]. In the present study the biological application of zinc phosphate
is only marginal. The main aim of zinc phosphate layer is to act as an adhesive
layer for hydroxyapatite growth. As the zinc phosphate surface has Po/· species, it
can facilitate the adsorption of Ca2+
from the electrolytic solution. Once the HA
coating was developed on the ZnP layer, the question of bio activity diminishes.
The destruction of the HA coating may be an adverse factor. Even though the HA
coating get destructed the underlying ZnP layer has esteogenic property and it will
facilitate further HA growth.
169
7.11. CONCLUSIONS
In the present study 316L SS was modified as a biocompatible substrate for
hydroxyapatite growth. A unique layering system of pure substrate, thick Fe-Zn
alloy layers, thin pure zinc layer, thin ZnP layer on which a HA layer was
developed. The hot-dip glavanization process adopted for the formation of zinc
coating on SS substrate facilitated the formation of alloy layers on the metallic
substrate. The conversion coating of ZnP on the galvanized substrate acted as a
biocompatible layer for hydroxyapatite growth. The ZnP conversion coating also
facilitated hydroxyapatite growth from simulated body fluid even after subjecting
to aggressive physiological conditions. The ZnP/HA coating exhibited stability
during the immersion in aggressive physiological condition simulated to early
inflammation period. Subsequently it exhibited effective bio-growth of HA during
biomimetic deposition i.e., immersion in simulated body fluid for a period of two
weeks.
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