**2.3.1 Evaluation of biocompatibility of TiNi SMA surfacely-modified**

Figure 2-1 shows the EDS results of Ca/P deposition on TiNi SMA before and after anodicoxidation respectively in SBF solution for 7d. It is seen from Figure 2-1a that there was small

Ti-O Film Cathodically-Electrodeposited on

concrete adsorption process is as following:

Ti4+(ox)HPO42- (ads) + OH-

substances in oxidation layer and ions in solution.

inflammation reaction caused due to fast degradation[25].

Ti (OH)3+(ox) + H2PO4-

And the OH-

further verification.

may mainly be HA.

the Surface of TiNi SMA and Its Bioactivity and Blood Compatibility 35

This is because of that Titanium dioxide's isoelectric point has a great influence on the behavior of Titanium in organism, the isoelectric point of TiO2 is about 6.2[19], which is somewhat lower than PH value (7.4) in physiological environment. This illustrates that under physiological environment, the Titanium surface may be with some weak negative charges. And this kind of surface with negative charges has a close relation to osseous integration between plant and its peripheral live bone[20-23]. In the body fluid, Ca2+ will combine with the negative charge on the TiO2 surface under the action of Coulombforce.

surface, thus it can introduce calcium phosphorus layer to deposit on the surface. The

Among them, (ads), (ox) and (aq) respectively stand for ions adsorbed on the alloy surface,

The self-produced oxidation film of TiNi SMA is too thin, not stable, but the TiO2 film produced by anodic-oxidation is thicker, so the capability to adsorb Ca/P layer is stronger. This illustrates that anodic-oxidized TiNi SMA may have some bioactivity, but it needs

Figure 2-2 shows an EDS result of Ca/P deposition in SBF solution of TiNi SMA before and after cathodic-electrodeposition. It can be seen from the figure that after dipping for 7d, there were higher contents of Ca and P elements deposited on the sample surface. Ca and P mole ratio reached to 1.5 : 1 in figure 2-2a; 2.4 : 1 in figure 2-2b; and 1.8 : 1 in figure 2-2c. The material's after cathodic-electrodeposition is the highest, the one's crystallized came second and the un-treated sample's was the lowest. This is because of, that the Ti-O film obtained after cathodic-electrodeposition was mainly Ti(OH)4, because of OHexistence, it was easy for apatite on the surface to nucleate. Li et al[24] found in research for bioactivity of titanium metals that the surface titanium oxide layer including Ti-OH group as well as being with electronegativity was one of the major factors to introduce HA nucleation. Because HPO42- and PO43- generally exist more easily under alkaline environment, crystallization treatment cause OH- loss, so nucleation is comparatively more difficult. There are researchs showing that the materials with high mole ratio are more stable than ones with low mole ratio under physiological environment. High Ca and P mole ratio is advantageous to prevent from host organization reaction of fast dissolution of small molecules after material planted and it can also prevent from the acute

At constant temperatures, solubility product of Ca10(PO4)(OH)2 (hydroxyapatite, HA) is far less than that of other phosphates, so HA oversaturation in calcification solution at the same concentration is higher than that of other phosphates. It can be inferred that surface crystal

on the surface will adsorb PO43- through hydrogen bond to gather forward to

Ti (OH)3+(ox) + HPO42- (aq) ——Ti4+(ox)PO43-(ads) + H2O (2.4)

——Ti4+(ox)HPO4 2-(ads) + H2O (2.2)

——Ti4+(ox)PO4 3-(ads) + H2O or (2.3)

amount of the elements of Ca, P and O on the sample before oxidation, this coincides with the Hanawa's investigation[18]. But there was a lot of the elements of Ca, P and O on the sample after oxidation, as shown in figure 2-1b.

Fig. 2-1. EDS result of Ca/P coating in SBF solution (PH7.40) (a TiNi SMA; b anodicoxidation)

amount of the elements of Ca, P and O on the sample before oxidation, this coincides with the Hanawa's investigation[18]. But there was a lot of the elements of Ca, P and O on the

a

b

Fig. 2-1. EDS result of Ca/P coating in SBF solution (PH7.40) (a TiNi SMA; b anodic-

oxidation)

sample after oxidation, as shown in figure 2-1b.

This is because of that Titanium dioxide's isoelectric point has a great influence on the behavior of Titanium in organism, the isoelectric point of TiO2 is about 6.2[19], which is somewhat lower than PH value (7.4) in physiological environment. This illustrates that under physiological environment, the Titanium surface may be with some weak negative charges. And this kind of surface with negative charges has a close relation to osseous integration between plant and its peripheral live bone[20-23]. In the body fluid, Ca2+ will combine with the negative charge on the TiO2 surface under the action of Coulombforce. And the OH on the surface will adsorb PO43- through hydrogen bond to gather forward to surface, thus it can introduce calcium phosphorus layer to deposit on the surface. The concrete adsorption process is as following:

$$\text{Ti (OH)}^{3\*}\text{(ox)} + \text{H}\_2\text{PO}\_4^{\cdot} - -\text{Ti^{4\*}}\_{\text{(ox)}}\text{HPO}\_4^{2^\*}\text{(ads)} + \text{H}\_2\text{O} \tag{2.2}$$

$$\mathrm{Ti^{4\*}\_{\mathrm{(ox)}}}\mathrm{HPO\_{4\*}^{2\*}}\mathrm{(ads)} + \mathrm{OH^{-}} - -\mathrm{Ti^{4\*}\_{\mathrm{(ox)}}}\mathrm{PO\_{4\*}^{3\*}}\mathrm{(ads)} + \mathrm{H\_2O} \text{ or } \tag{2.3}$$

$$\text{Ti(OH)}^{3\*}\text{(ox)} + \text{HPO4''}^{2\*}\text{(aq)} \longrightarrow \text{Ti4\*}^{4\*}\text{(ox)}\\\text{PO4''}^{3\*}\text{(ads)} + \text{H}\_2\text{O}\tag{2.4}$$

Among them, (ads), (ox) and (aq) respectively stand for ions adsorbed on the alloy surface, substances in oxidation layer and ions in solution.

The self-produced oxidation film of TiNi SMA is too thin, not stable, but the TiO2 film produced by anodic-oxidation is thicker, so the capability to adsorb Ca/P layer is stronger. This illustrates that anodic-oxidized TiNi SMA may have some bioactivity, but it needs further verification.

Figure 2-2 shows an EDS result of Ca/P deposition in SBF solution of TiNi SMA before and after cathodic-electrodeposition. It can be seen from the figure that after dipping for 7d, there were higher contents of Ca and P elements deposited on the sample surface. Ca and P mole ratio reached to 1.5 : 1 in figure 2-2a; 2.4 : 1 in figure 2-2b; and 1.8 : 1 in figure 2-2c. The material's after cathodic-electrodeposition is the highest, the one's crystallized came second and the un-treated sample's was the lowest. This is because of, that the Ti-O film obtained after cathodic-electrodeposition was mainly Ti(OH)4, because of OHexistence, it was easy for apatite on the surface to nucleate. Li et al[24] found in research for bioactivity of titanium metals that the surface titanium oxide layer including Ti-OH group as well as being with electronegativity was one of the major factors to introduce HA nucleation. Because HPO42- and PO43- generally exist more easily under alkaline environment, crystallization treatment cause OH- loss, so nucleation is comparatively more difficult. There are researchs showing that the materials with high mole ratio are more stable than ones with low mole ratio under physiological environment. High Ca and P mole ratio is advantageous to prevent from host organization reaction of fast dissolution of small molecules after material planted and it can also prevent from the acute inflammation reaction caused due to fast degradation[25].

At constant temperatures, solubility product of Ca10(PO4)(OH)2 (hydroxyapatite, HA) is far less than that of other phosphates, so HA oversaturation in calcification solution at the same concentration is higher than that of other phosphates. It can be inferred that surface crystal may mainly be HA.

Ti-O Film Cathodically-Electrodeposited on

electrodeposition; c annealed)

summarized with two stages[26].

Annealed

Unannealed

Intensity

the Surface of TiNi SMA and Its Bioactivity and Blood Compatibility 37

c

Figure 2-3 shows the surface XRD analysis results of cathodically-electrodeposited samples in SBF solution before and after crystallization respectively. It can be seen from the figure that HA existed on both samples. This illustrates when an amorphous Ti-O film riching in Ti-OH group was dipped in SBF solution for 7d, HA could form on the surface; this also illustrates that the obtained TiO2 after crystallization treatment still preserved some hydroxyl groups with bioactivity. The formation process of hydroxyapatite on a material surface is actually a formation and growth one of a new phase. The process can be

0 10 20 30 40 50 60 70 80

HA

Fig. 2-3. XRD result of HA on the surface of sample cathodically-electrodeposited

2 Theta

HA HA HA HA

TiNi(B2)

Fig. 2-2. EDS result of Ca/P coating in SBF solution (PH7.40) (a TiNi SMA; b

b

a

b

Fig. 2-2. EDS result of Ca/P coating in SBF solution (PH7.40) (a TiNi SMA; b electrodeposition; c annealed)

Figure 2-3 shows the surface XRD analysis results of cathodically-electrodeposited samples in SBF solution before and after crystallization respectively. It can be seen from the figure that HA existed on both samples. This illustrates when an amorphous Ti-O film riching in Ti-OH group was dipped in SBF solution for 7d, HA could form on the surface; this also illustrates that the obtained TiO2 after crystallization treatment still preserved some hydroxyl groups with bioactivity. The formation process of hydroxyapatite on a material surface is actually a formation and growth one of a new phase. The process can be summarized with two stages[26].

Fig. 2-3. XRD result of HA on the surface of sample cathodically-electrodeposited

Ti-O Film Cathodically-Electrodeposited on

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0

Fig. 2-4. Haemolysis rate of modified TiNi SMA

Hem

olysis r

ate/

%

the Surface of TiNi SMA and Its Bioactivity and Blood Compatibility 39

When blood connects with a foreign material surface some red cells will be destroyed to release hemoglobin, that is, haemolysis occurring. A material with good blood compatibility should have a lower haemolysis rate. Figure 2-4 shows a haemolysis rate tested result of TiNi SMA samples, which were untreated, anodically-oxidized and cathodically-electrodeposited respectively. It can be seen that the TiNi SMA and its surface-modified materials have met the medical requirement (<5%) for a biomaterial. Haemolysis rate of the modified material has decreased somewhat. And haemolysis rate of anodically-oxidized sample was lower than that of cathodically-electrodeposited one. This is because of that, TiO2 crystals have a good blood compatibility. Besides, the material surface smoothness and film quality have effects on haemolysis rate. Haemolysis rate of cathodically-electrodeposited sample is higher than that of anodically-oxidized one. This is relevant to the smoothness differences on sample surface.

a TiNi SMA

b TiNi anodic oxidation

c TiNi electrodeposition(annealed)

abc

The experiment on dynamic coagulation time is to test the degree of activating endogenous coagulation factor, the more smooth the dynamic coagulation curve is, the higher the absorbance at ordinate will be. This shows that, the longer the coagulation time, the lower the degree of activating coagulation factor. Generally, the time at O.D.=0.100 is often settled as the coagulation time, in order to comparing. Figure 2-5 shows the change curves of absorbance and blood connecting time of TiNi SMA, which were surface modified by anodically-oxidized and cathodically-electrodeposited respectively. It can be seen from the figure that the absorbance of the anodically-oxidized sample is highest, and the coagulation time is the longest. This illustrates that it has an excellent anti-coagulation property. The secondary is the cathodically-electrodeposited sample, and the last one is TiNi SMA sample (un-treated). Besides, material surface smoothness also has an effect on dynamic coagulation time. Generally the higher the surface smoothness, the longer the dynamic coagulation time.

**2.3.3 Dynamic coagulation time test of TiNi SMA after surface modification** 

**2.3.2 Haemolysis rate test of TiNi SMA after surface modification** 

### **2.3.1.1 Nucleation formation**

When a material connects to SBF solution, because of electrostatic attraction, Ca2+, HPO4 2 and PO43- directly acting on solid surface are adsorbed on the surface, to form calciumphosphorus compound, which interacts with HPO42- and CO3 2- in SBF solution, to form a new phase nucleus. According to two-dimension nucleation theory, as soon as a nucleus forms, the reactant ions will continuously nucleate on the deposited surface, and the crystal will continuously grow.

The factors to affect nucleation are those as following:① Calcium-phosphorus concentration at localization on surface plays a very key role to nucleation. For a sample cathodicallyelectrodeposited, the particle distribution on the surface film makes surface roughness of the sample increase, and surface area increase too. Thus the concentration at localization on the surface would be comparatively higher than that on a smooth surface. So, under the same conditions, it is easier for Ca2+ and HPO42- concentration on the roughness surface to reach the nucleation critical value. ② The interfacial energy of material surface. Apatite crystals form and directly grow on the material surface. It may be thought that is increasing ionic concentration in the saturated solution to form the low interfacial energy surface. According to Ostward's nucleation theory, the free energy to nucleate depends on solution's oversaturability (S), pure interfacial energy (σ), temperature (T) and particle surface area (A):

$$
\Delta G = -T \ln S + \sigma A \tag{2.5}
$$

This nucleation theory illustrates that: the increase of solution's oversaturability and decrease of pure interfacial energy are advantageous to interface heterogeneous nucleation. S increasing., will make nucleation free energy decrease. As long as S is high enough, even if a surface with low energy, which has not been subjected to any treatment, it can also introduce heterogeneous nucleation. Because ionic concentration is high, it can overcome the barrier of material's surface nucleation, to nucleate on the surface. ③ The geometric morphology of material surface. It is reported that a crystal nucleus firstly occurred at the roughness places on a surface. Because higher localization concentration can be kept in these regions, the critical value to nucleate would quickly be reached, at the same time these locations supply the nucleation spots.

### **2.3.1.2 Growth of a crystal nucleus**

In the process of hydroxyapatite formation, crystal nucleus formation is a homogeneous nucleation, that is, to form a nucleus on the certain substrate. Through the nucleation process, changes in crystal structure and composition accomplish simultaneously. A main role for grain boundaries to nucleate is to decrease boundaries' area and interface energy, thus to low the nucleation work. The decrease of interface energy drives crystal to grow. The deposition process of hydroxyapatite on sample surface in SBF solution is as following[27]:

$$\text{10Ca}^{2+} + 6\text{PO}\_4^{3-} + 2\text{OH} \cdot \longleftrightarrow \text{Ca}\_{10}\text{(PO4)}\_6\text{(OH)}\_2 \tag{2.6}$$

As a consequence, HA begins to deposit on sample, as long as apatite crystal nuclei form, the nuclei will consume Ca and P in solution, hydroxyapatite continuously deposit on sample surface.

When a material connects to SBF solution, because of electrostatic attraction, Ca2+, HPO42 and PO43- directly acting on solid surface are adsorbed on the surface, to form calciumphosphorus compound, which interacts with HPO42- and CO32- in SBF solution, to form a new phase nucleus. According to two-dimension nucleation theory, as soon as a nucleus forms, the reactant ions will continuously nucleate on the deposited surface, and the crystal

The factors to affect nucleation are those as following:① Calcium-phosphorus concentration at localization on surface plays a very key role to nucleation. For a sample cathodicallyelectrodeposited, the particle distribution on the surface film makes surface roughness of the sample increase, and surface area increase too. Thus the concentration at localization on the surface would be comparatively higher than that on a smooth surface. So, under the same conditions, it is easier for Ca2+ and HPO42- concentration on the roughness surface to reach the nucleation critical value. ② The interfacial energy of material surface. Apatite crystals form and directly grow on the material surface. It may be thought that is increasing ionic concentration in the saturated solution to form the low interfacial energy surface. According to Ostward's nucleation theory, the free energy to nucleate depends on solution's oversaturability (S), pure interfacial energy (σ), temperature (T) and particle surface area

This nucleation theory illustrates that: the increase of solution's oversaturability and decrease of pure interfacial energy are advantageous to interface heterogeneous nucleation. S increasing., will make nucleation free energy decrease. As long as S is high enough, even if a surface with low energy, which has not been subjected to any treatment, it can also introduce heterogeneous nucleation. Because ionic concentration is high, it can overcome the barrier of material's surface nucleation, to nucleate on the surface. ③ The geometric morphology of material surface. It is reported that a crystal nucleus firstly occurred at the roughness places on a surface. Because higher localization concentration can be kept in these regions, the critical value to nucleate would quickly be reached, at the same time these

In the process of hydroxyapatite formation, crystal nucleus formation is a homogeneous nucleation, that is, to form a nucleus on the certain substrate. Through the nucleation process, changes in crystal structure and composition accomplish simultaneously. A main role for grain boundaries to nucleate is to decrease boundaries' area and interface energy, thus to low the nucleation work. The decrease of interface energy drives crystal to grow. The deposition process of hydroxyapatite on sample surface in SBF solution is as following[27]:

 10Ca2+ + 6PO43- + 2OH- ←→ Ca10(PO4)6(OH)2 (2.6) As a consequence, HA begins to deposit on sample, as long as apatite crystal nuclei form, the nuclei will consume Ca and P in solution, hydroxyapatite continuously deposit on

(2.5)

**2.3.1.1 Nucleation formation** 

will continuously grow.

ln *G TS A*

locations supply the nucleation spots. **2.3.1.2 Growth of a crystal nucleus** 

sample surface.

(A):
