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3 MATERIALS AND EXPERIMENTAL METHODS

3.1. EXPERIMENTAL MATERIALS

3.1.1. Titanium and Ti-6Al-4V

Commercially pure titanium (ASTM B 348 Gr. 1), hereafter called Ti 12 and Ti-

6Al-4V (ASTM B 348 Gr. 5), hereafter called Ti 31 were obtained from Mishra Dhatu

Nigam Ltd., Hyderabad in the form of rods of 5 mm and 8 mm diameter. These were

used as substrate metal to be coated with hydroxyapatite by plasma spray technique. The

microstructures of the as received Ti 12 and Ti 31 samples are shown in Fig. 3.1 and 3.2

respectively. The chemical compositions and mechanical properties as obtained from the

manufacturer are shown in Table 3.1 and 3.2.

3.1.2. Hydroxyapatite (HA)

In the present study hydroxyapatite was the surface coating material sprayed on Ti

12 and Ti 31 metal substrates. The hydroxyapatite powder was procured from Plasma

Biotal Limited, U.K. As reported by the manufacturer the powder contained HA with

minor traces of calcium oxide phosphate and alpha calcium orthophosphate with average

particle size of 30 micron and relative crystallinity 97%. The d-spacing of HA given by

the manufacturer is presented in Table 3.3. The morphology of feedstock HA powder

shown in Fig. 3.3 is characterized by a porous structure. The particles are irregular in

shape of varying size with particle mean size of 30 microns. Presence of large pores in

the HA feedstock means that the process of manufacture does not seem to be followed by

any kind of high temperature densification. This powder was coated on Ti 12 and Ti 31

samples of dimension 100 mm x 6 mm x 4 mm for further study.

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3.2. HEAT TREATMENT

The response of titanium and titanium alloys to heat treatment depends on the

composition of the metal and the effects of alloying elements on the structural

transformation of titanium. In the present study of heat treatment, Ti 12 and Ti 31 grade

alloys were subjected to annealing heat treatment at 600, 700, 800 and 900 C in an

electric furnace for 1 hour followed by air-cooling. Samples were protected from

oxidation by coating with borax and placing in airtight container during annealing.

3.2.1. Microstructural studies

On occasions it is necessary to examine the structural features of metals like grain

and phase that influence the properties of materials.

3.2.1.1. Optical microscopy

Optical microscopic examination is an extremely useful tool in the study and

characterization of materials. Microstructural examination helps to find association

between properties and structure. In the present study microstructures of all the heat

treated samples were studied qualitatively by optical microscopy. The sample surface was

polished by initially grinding with successively finer emery papers and finally polished

on a disc using lavigated alumina. After this the samples were etched with Kroll’s reagent

and viewed under optical microscope.

3.2.1.2. Grain size measurement

The grain size is often determined when properties of a polycrystalline material

are under consideration. In this regard there exist a number of techniques. Grain size in

the present study was measured by the linear intercept method using Filar eye piece

attached to the optical microscope. A known linear length calculated for particular

43

magnification was projected on the microstructure using Filar eye piece attached to the

optical microscope. The number of grains intersected was counted for the known

length. This was determined in four different directions and repeated in ten locations of

the microstructure. The grains intersected each time were counted. The line length

divided by the average of the number of grains intersected was determined to calculate

the average grain size.

3.3. TENSILE TEST

The effect of heat treatment on the tensile properties of Ti 12 and Ti 31 were

carried out on Instron machine of model 4206 at a cross head speed of 1 mm/min. The

dimensions of the samples are shown in Fig. 3.4 and Fig. 3.5. Ultimate tensile strength,

0.2% offset yield strength and percentage elongations were estimated from the load

versus displacement plot. Three samples were tested for each treatment condition and the

values reported are the average of these three tests.

3.4. PLASMA COATING

Plasma spray processing is a method of giving a material a protective coating

through the form of a plasma spray. In the present study plasma coating of

hydroxyapatite was given on Ti 12 and Ti 31 metal substrates. The metallic samples of

dimension 100 mm x 6 mm x 4 mm were prepared from the rods of Ti 12 and Ti 31

received from Mishra Dhatu Nigam Ltd., Hyderabad. Coating was done at Spraymet

Surface Technologies Pvt. Ltd., Bangalore. The surface of the substrate metal was

roughened before coating for better bonding between the metal substrate and HA coating.

Surface roughening was done by grit blasting with Al2O3 of grit size 24. The roughened

44

Ti 12 and Ti 31 samples were then washed with water followed by alcohol before plasma

coating.

Plasma spray process used a DC electric arc to generate a stream of high

temperature ionized plasma gas. The arc was struck between a tungsten cathode and a

copper anode within the torch. The torch was fed with a continuous flow of inert gas,

which was ionized by the DC arc, and was compressed and accelerated by the torch

nozzle so that it issues from the torch as a high velocity (in excess of 800 m/sec), high

temperature (12000 – 16000 K) plasma jet. The coating material in the powder form was

carried in an inert gas stream into the plasma jet where it was heated and propelled

towards the substrate. The coating was carried out separately using four gas atmospheres:

argon, argon/hydrogen, nitrogen and nitrogen/hydrogen. Here argon and nitrogen served

as primary gases and hydrogen acted as secondary gas. A pressure of 0.76 MPa for

primary gas (argon and nitrogen) and 0.069 MPa for secondary gas of 10% volume

(hydrogen) was applied to get desired plasma jet. The thickness of the coating was 100

micron and the standoff distance between the substrate and the jet was maintained at 8

cm during coating.

3.4.1. Optical microscopy

Microstructural examination is an extremely useful tool in the study and the

characterization of the materials. Microstructural examination helps in finding the

association between properties and structure. For the microstructural study by optical

microscopy, samples of 4 mm thickness were sectioned from the plasma coated specimen

and mounted using self-cure acrylic resin. The sample surface was polished by

45

initially grinding with successively finer emery papers and finally polished on a

disc using lavigated alumina. After this the samples were etched with Kroll’s reagent

and were viewed under optical microscope.

3.4.2. SEM study

The study was intended to understand the microstructure, phase, porosity,

solidification, crystallization mechanism, interfacial bonding and diffusion of coating and

metal constituents at interfacial region. For this purpose Analytical Scanning Electron

Microscope - JEOL JSM-6380LA model was used. The structure of HA surface of all the

samples coated in argon, argon/hydrogen, nitrogen and nitrogen/hydrogen atmosphere

were studied. EDAX analysis and the microstructure of cross sectioned samples mounted

in resin were examined. Before study the samples were sputter coated with platinum.

3.4.2.1. Porosity measurement

Argon, nitrogen, argon/hydrogen, and nitrogen/hydrogen atmosphere coated

samples were selected for quantitative analysis of porosity using Image Analyzer

software to understand the porosity formation in HA coated experimental materials.

For this purpose SEM photographs of cross sectioned sample were used in the Image

Analyzer.

3.4.2.2. EDAX analysis - Coating/Metal interface

To understand the nature of interfacial bonding between the metal and HA, the

coated samples were studied after sectioning. The sectioned samples were polished by

initially grinding with successively finer emery papers and finally polished on a disc

using lavigated alumina and investigated through SEM micrographs and EDAX.

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This was to determine the microstructural and compositional changes taking place during

the plasma coating. This would help us to find out the diffusion of elements from the

metal to the coating as well as from the coating to the metal at various depths.

3.4.3. X-ray diffraction studies

The qualitative and quantitative information of all the HA coated samples under

different gas atmospheres were obtained through X-ray diffraction to reveal the detailed

information about chemical composition, crystallography and structure. The XRD studies

were carried out on a JEOL-JDX-8P diffractometer using copper Kα radiation with nickel

filter. The tube voltage was 30 kV and tube current was 20 mA. A scan speed of 2 degree

per minute was employed over an angular 2θ range of 20-60°

3.4.4. Surface roughness study

Surface roughness is described by arithmetic mean value. The arithmetic mean

value, Ra, formerly identified as AA (for arithmetic average) or CLA (for center-line

average), is based on the schematic illustration of a rough surface, as shown in Fig. 3.6.

The arithmetic mean value is defined as,

Ra = dxyl

ynn

yyyy n

i

incba

1

01

11

where all ordinates ya, yb, yc, …..., are absolute values. The last term in the above

equation refers to the value Ra of a continuous surface or wave, as is commonly

encountered in signal processing. ℓ refers to the total profile length measured.

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The surface roughness profiles of coated samples were studied using Taylor Hobson

surface profilometer. Flat surface of the HA coating was scanned and Ra, surface

roughness value was determined.

3.4.5. Hardness measurement

Hardness of a material is the resistance to indentation or abrasion. It is an

important property as it measures the materials resistance to localized plastic

deformation. The hardness value in the study under consideration was measured using

Clemex Digital Micro hardness tester MMT X-7 with a load of 100 g for 10 seconds.

Knoop hardness values of hydroxyapatite coated samples in different atmospheres

were measured at 10 locations on each sample and the mean value is reported.

3.5. BIOLOGICAL EVALUATION

Before any biomaterial is placed inside the living body its biocompatibity evaluation

is very much essential to find the adverse effect on living body. This is done first

by in vitro study and then by in vivo study. In our present investigation the in vivo

study was not carried out due to some practical problems. Only the in vitro study

was carried out. It involved two parts:

1. The toxicity evaluation by MTT assay.

2. Platelet adhesion study.

3.5.1. Toxicity evaluation

In vitro evaluation of any biomaterial is very important in order to understand the

cell viability of the material before actual usage. In the present study of toxicity

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evaluation, all the coated and uncoated samples of dimension 6 mm x 4mm x 4 mm

(n = 6) were tested for cell viability by MTT assay (3-(4, 5-dimethyltiazole-2-yl)-2, 5-

diphenyl tetrazolium bromide) assay (Sigma Chemical Co.).

3.5.1.1. MTT Assay

The test was carried out according to the protocol described by Mossman89

. It is a

simple colorimetric test for cell proliferation and survival, which is used for the

measurement of cytotoxicity. The assay involves the ability of viable cells to convert a

soluble tetrazolium salt MTT into purple formazan end product by mitochondrial

dehydrogenase enzymes. The purple colour reaction is used as a measure of cell viability.

Exponentially growing V79 cells (104) were seeded into 96 well plates and

incubated for 24 hours at 37°C in CO2. Eight wells each served as control, blank and

rest of the wells were used for different test samples. The culture medium was removed

and substituted with fresh medium with different test samples labeled as 1, 2, 3, 4, 5, 6, 7

8, 9, 10 for Ti 12 argon, Ti 31 argon, Ti 12 argon/hydrogen, Ti 31 argon/hydrogen, Ti 12

nitrogen, Ti 31 nitrogen, Ti 12 nitrogen/hydrogen, Ti 31 nitrogen/hydrogen, Ti 12 and Ti

31 respectively for 24, 48 and 72 hours. After that 100 µl of the MTT stock (1mg/ml) was

added to each of the 96 wells followed by 4 hour incubation at 37oC in a 5% CO2

atmosphere. The test samples removed from the medium and purple colored precipitate

of formazan was solubilized by dissolving in a 100 µl buffer consisting of 23% sodium

dodecyl sulphate in 50% N, N-dimethyl formamide (pH 4.7). Greater magnitude of

optical density due to intense purple colouring is regarded as showing higher cell

viability. After 5-10 minutes of incubation at 37C, the optical densities (OD) were read

49

on multi-well spectrophotometer (Tecan, Austria) at 540 nm wavelength. Percent

viability was calculated as follows:

3.5.2. Platelet adhesion study

The activation of platelet in contact with any biomaterial affects the healing

process and it is a function of microtexture, composition, and other parameters. Platelet

adhesion is an initial, crucial and complex matter. Activation and adhesion of

platelet play a fundamental role in the development of thrombosis. For the platelet

adhesion study specimens of dimension 6 mm x 4 mm x 4 mm were cut from uncoated Ti

12 and Ti 31 and also from hydroxyapatite plasma coated samples of argon,

argon/hydrogen, nitrogen and nitrogen/hydrogen. The uncoated Ti 12 and Ti 31 samples

surface was polished by initially grinding with successively finer emery papers and

finally polished on a disc using lavigated alumina. All the test samples were cleaned

in acetone and ethyl alcohol solution for 5 min, and finally rinsed in distilled water

and dried.

3.5.2.1. Methodology of platelet adhesion82

Blood adhesion tests were conducted in a class of 10,000 clean room. The

samples were evenly distributed in two culture dishes. 200 ml blood (containing 3.8 ml

wt% citrate solution, blood/citrate acid = 9:1) extracted from a healthy adult

was centrifuged to form a platelet-rich-plasma (PRP) and erythrocyte. The PRP contained

Average of Test (OD) – Average of Blank

(OD) Average of Control (OD) – Average of Blank (OD)

Percent viability = ________________________________________ X 100

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about 30-35 x 107 platelets/ml. Then the blood was poured into culture dishes. After

culturing at 37◦ C for 30 minutes and 3 hours respectively, the PRP was taken out of the

wells. A phosphate buffer solution (PBS) was added to the wells and gently rinsed 2-3

times to get rid of platelets adsorbed nonspecifically on the surface. Then the samples

were soaked in 2% glutaraldehyde for 1 hour and 5% glutaradehyde for 12 hours to fix

the platelets, which adhered specifically on the surface. After rinsing with distilled water,

the samples were subsequently dehydrated through 50%, 75%, 90% and 100% ethanol

water solutions twice for 10 min each. After dehydrating, the samples were subsequently

dealcoholized through 50%, 75%, 90% and 100% isoamyl acetate water solution twice

for 10 min each. The samples were dried at a critical point over night. After spatter

coating with platinum, the samples were observed under SEM and photographs of the

platelets were randomly taken from the observation.

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Fig. 3.1 Microstructure of as received Ti 12

Fig. 3.2 Microstructure of as received Ti 31

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Table 3.1 Chemical analysis of Ti 12 and Ti 31 (in wt%)

Material C O N H Ti Fe Al V

Ti 12 0.037 0.111 0.008 0.0016 Bal 0.12 ---- ---

Ti 31 0.01 0.123 0.0072 0.0012 Bal 0.04 6.3 4

Table 3.2 Mechanical properties of Ti 12 and Ti 31

Material Diameter

(mm)

(0.2%) Y.S.

MPa

UTS

MPa

Elongation

%

RA

%

Ti 12 8 275 465 30 45

Ti 12 5 265 392 37 60

Ti 31 8 967 1027 16 47

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Table 3.3 d-spacing of as received HA

d-spacing Two theta d-spacing Two theta

4.057 21.91 2.804 31.92

3.869 22.99 2.777 32.24

3.492 25.51 2.709 33.07

3.440 25.90 2.626 34.15

3.168 28.17 2.520 35.62

3.069 29.11

2.998 29.80* 3.044 29.34*

2.905 30.78** 2.925 30.56**

Hydroxyapatite [Ca10 (PO4)6 (OH)2]

Calcium oxide phosphate*,

Alpha calcium orthophosphate**

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Fig. 3.3 SEM image of as received HA powder

Porosity

55

19.9

5r

Fig. 3.4 Dimensions of tensile specimen (in mm) for Ti 12

29

18

7

14

Fig. 3.5 Dimensions of tensile specimen (in mm) for Ti 31

Fig. 3.6 Surface roughness profile

3.5 5

5 8

6