7.DEVELOPMENT OF LAYERWISE …shodhganga.inflibnet.ac.in/bitstream/10603/92041/12/12...148 7.DEVELOPMENT OF LAYERWISE HYDROXYAPATITE COATING: PHOSPHATE INTER LAYERED COATING 7.1. INTRODUCTION

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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