Chapter 4 Result
4.1 Microstructure analysis of 316L alloy with and without treatment51
A. XRD
Putting the treated and untreated 316L stainless steel through the X-ray Diffraction Analysis, the results are shown as below [fig. 4-1]. The γ-phase of the untreated 316L stainless steel in the control group showed in the color black, compared with the other electrochemistry treated 316L have no obvious changes. This means that in the electrochemistry treated surfaces, the surface properties of the 316L have not changed drastically. The treated 316L have the same surface properties of untreated 316L and the crystallization still existed in the ET-316L.
B. AFM, SEM
By using the Atomic Force Microscope (AFM), we can see in the control group [fig. 4-2], the surface of each grain is quite smooth and with few shallow indents. The average roughness (Ra) measured is 112.84 nm, and the mean roughness (Rm) is -23.096nm. After the surface treatment by electrochemistry
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with the parameter of 3V-20min [fig. 4-3], many pebbles can be seen within few µm, and the value for Ra 66.53nm and Rm 110.91pm. With further treatment of electrochemistry 5V-20min [fig. 4-4], many pores were formed and created many groves with fused grain boundaries, the Ra value is 99.959nm and the Rm value is 45.423nm.
Under the Scanning Electron Microscopy (SEM), the control group [fig.
4-5] shows clear and definite grain boundaries. Each grain is larger in size and with the smoother surfaces. As in the 3V-20min [fig. 4-6] the grain boundaries are not as clear. Also the sizes of each grain are smaller and with groves and pores with created rougher surfaces. With the further treatment 5V-20min [fig.
4-7], the boundaries are even less definite and the numerous pores were seen in the diagram. Nevertheless, there are little pores exists in the big pores which greatly increases its roughness and the surface area.
C. Wettability and Surface Energy
We then look at the 316L to see how well a material can adhere to the surface through its contact angle and its surface energy. Glass having the lowest contact angle, the untreated and treated 316L have no significance difference in contact angle. The contact angle results are taken with CCD camera as shown
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[fig. 4-8].
Testing the surface energy of both the treated and untreated 316L stainless steel. Acting as positive control, glass still has the highest amount of the surface energy. Both treated and untreated 316L have no significance differences in the surface energy [fig. 4-9].
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D. Cross-section
In the cross-section view of Scanning Electron Microscopy (SEM), the control group of untreated 316L has shown a very thin and unorganized (fragile) outer oxide layer over a smooth inner layer [fig. 4-10]. Conversely, the treated 316L resulted a thicker and more uniformed oxide layer, with a porous inner layer throughout these surfaces [fig. 4-11].
Micro-implants are increasingly popular in clinical orthodontics to have impact on skeletal anchorage. However, biofilm formation on their surfaces and subsequent infection of peri-implant tissues can result in either exfoliation or surgical removal of these devices.
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4.2 Hemocapatibility of 316L alloy with and without treatment
A. Clotting Time
We have demonstrated the effect of clotting time to either treated or untreated 316L, and each sample is being measured by optical density versus time. The data results are formulated into graphs shown as below [fig. 4-12].
When the uncoagulated red blood cells are immersed into the distilled water with the samples, the bilirubin inside the red blood cell burst out of the cells due to the osmotic diffusion between the distilled water and the cell. It was observed that the more of the unattached and uncoagulated blood, the redder the distilled water was seem. As shown in the data, the glass grabbed the most coagulated blood cell within the first 20 minutes compares to the other 316L alloys. After 20 minutes, the control group grabbed more red blood cells than glass; only the treated 316L especially the samples treated with 5V still maintain lesser cells adhered than glass.
B. Plasma Protein Assay (Fibrinogen)
Fibrin (also called Factor Ia) is a fibrous protein involved in the clotting of blood. It is a fibrillar protein that is polymerized to form a mesh, that forms a
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haemostatic plug or clot (in conjunction with platelets) over a wounded site.
Fibrin is made from fibrinogen, a soluble plasma glycoprotein that is synthesized by the liver. Processes in the coagulation cascade activate the zymogen prothrombin to the serine protease thrombin, which is responsible for converting fibrinogen into fibrin. Fibrin is then cross linked by factor XIII to form a clot. Therefore the data are obtained and as shown in fig. 4-13. The treatment with 2V and 5V have the highest amount of fibrinogen analyzed, the others are about the same as the non-treated stainless steel.
C. Platelet adhesion assays (CD 61)
Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61), also known as ITGB3, is a human gene. CD61 is a cluster of differentiation found on thrombocytes. This protein product is the integrin beta chain beta 3. As we know, integrins are integral cell-surface proteins composed of an alpha chain and a beta chain. Any given chain may combine with multiple partners resulting in different integrins. Integrin beta 3 is found along with the alpha IIb chain in platelets. Integrins are known to participate in cell adhesion as well as cell-surface mediated signaling. So we used CD 61 to test how well the platelets can adhere to the samples we prepared, and the result is shown in fig. 4-14.
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Glass having the least amount of platelets adhere to the surfaces and the samples with 1V and 5V have the highest amount of the platelets adhered to the surface. The other samples are only slight lower than the non-treated stainless steel, in terms of platelet adherence.
D. Platelet activation assay (p-selectin)
P-selectin is a cell adhesion molecule found in granules in endothelial cells (cells lining blood vessels) and activated platelets. Other names for P-selectin include CD62P, Granule Membrane Protein 140 (GMP-140), and Platelet Activation-Dependent Granule to External Membrane Protein (PADGEM).
P-selectin plays an essential role in the initial recruitment of leukocytes (white blood cells) to the site of injury during inflammation. The endothelial cells are activated by the molecules such as histamine or thrombin during inflammation, then the P-selectin moves from an internal cell location to the endothelial cell surface.
Thrombin is the one that triggers and stimulates endothelial-cell release of P-selectin, and recent studies suggest an additional Ca2+ independent pathway involved in release of P-selectin. Ligands for P-selectin on eosinophils and neutrophils are similar sialylated, protease-sensitive, endo-beta-galactosidase
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-resistant structures, clearly different than those reported for E-selectin, and suggest disparate roles for P-selectin and E-selectin during recruitment during inflammatory responses.
The result of the P- selectin test is shown in fig. 4-15. The glass and the sample with 3V have the highest P-selectin in the data, which means the inflammation occurs more rapidly than the other samples, the lower voltage samples have lower inflammation than the non-treated, it may due to the etching treatment that causes the grooves to be more deeper. The 5V samples have only slight higher inflammation than the non-treated stainless steel.
E. Clotting Time Samples under SEM
XPure Human Blood
Under the SEM, in the Control group (Non-treated stainless steel) in fig.
4-16, the red blood cell is still in its unique shape without any deformation.
Very little fibrin can be seen stretched out to other red blood cells or adhere on to the stainless steel surface. As shown in the treated sample 5V in fig.
4-17, the pores are obvious in the picture. Noted the smaller pores inside the big pores, with the red blood cells shoot out multiple fibrins to withhold its self from falling into the porous hole. This orientation allowed the trapped
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cells to grab more cells nearby creating a huge cluster of cells. It is quite similar to the geometry of spider webs we often seen in the nature used to trap its prey on to the web.
Y Pure Human Blood Clotted After 10 mins
In the sample 1V [fig. 4-18], the surface of the stainless steel only formed little shallow and very wide pores compare to the 5V. Only two of the cells in the picture extended the fibrins to grab each other, the other cells are “stopped” in the crater with no fibrin extension nor cluster together. The sample 3V [fig. 4-19] have narrower and deeper grooves. Nearly all cells have their fibrin extended and one cell was burst and deformed. Compare to the 1V sample, the cell aggregation is more obvious. In the sample 5V [fig.
4-20], notice that the surface was very rough with pores. The fibroblast can be seen with fibrin extended out to grab onto the surface and also the red blood cells.
ZPure Human Blood Clotted After 40 mins
As shown in the 1V sample [fig. 4-21], compare to 10 mins, there were more fibroblasts and adhered more firmly to the stainless steel surface with more fibrin extension. In the 3V sample [fig. 4-22], more red blood cells were trapped by many fibroblasts. The shape of the red blood cells
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maintain in a contant stable form. The adherence ability of the 3V sample is more obvious than the 1V sample. The 5V sample [fig. 4-23] were seen very few red blood cells attached, though a big cluster of fibroblasts were noted in the picture. As a result, it was demonstrated that 3V sample would give good adherence ability than the other voltage samples.
[ Platelet rich plasma (PRP) and Platelet poor plasma (PPP) with treated and non-treated stainless steel
In the control group with PRP [fig. 4-24] and PPP [fig. 4-25], the amount of cells noted in the PPP were lesser than the PRP, though the fibrin can still been seen in the picture. Comparing the 1 V sample with PPP [fig. 4-26] and with PRP [fig. 4-27], still more cells were seen in the PRP sample than those with PPP. However, there were more platelets cells than the red blood cells in the PRP. As shown in the 3V sample treated with either PRP [fig. 4-28]
and PPP [fig. 4-29], more platelets cells but no obvious fibrin were shown in the picture taken using PPP sample. In the other hand, less fibrin were noted in the PPP [fig. 4-30] of 5V sample picture compare to PRP [fig. 4-31].
Moreover, more platelets and red blood cells were observed in the 5V PRP picture, though most of the platelets had no fibrin extension except for the red blood cells.
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Fig. 4-1 XRD
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Fig. 4-2 AFM analysis (Control Group)
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Fig. 4-3 AFM analysis (3V-20min)
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Fig. 4-4 AFM analysis (5V-20min)
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10 μm 10 μm
Fig. 4-5 SEM (Control)
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10 μm
Fig. 4-6 SEM 3V-20min
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10 μm
Fig. 4-7 SEM 5V-20min
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Fig. 4-8 Contact Angle CCD Camera
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Fig. 4-9 Surface Energy and CCD Camera
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Fig. 4-10 Cross section View of Control Group
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(a)
(b)
Fig. 4-11 Treated 316L cross-section view (a) 3V-20 min (b) 5V-20 min
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Fig. 4-12 Clotting time with various condition
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Fig. 4-13 Plasma Protein Assay (Fibrinogen)
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Fig. 4-14 Platelet adhesion assays (CD 61)
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Fig. 4-15 Platelet activation assay (p-selectin)
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Fig. 4-16 Clotting Time Test (control at 0 min) in SEM, with pure human
blood
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Fig. 4-17 Clotting Time Test (5V at 0 min) in SEM, with pure human blood
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Fig. 4-18 Clotting Time Test (1V at 10 min) in SEM, with pure human blood
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Fig. 4-19 Clotting Time Test (3V after 10 min) in SEM, with pure human
blood
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Fig. 4-20 Clotting Time Test (5V after 10 min) in SEM, with pure human
blood
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Fig. 4-21 Clotting Time Test (1V after 40 min) in SEM, with pure human
blood
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Fig. 4-22 Clotting Time Test (3V after 40 min) in SEM, with pure human
blood
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Fig. 4-23 Clotting Time Test (5V after 40 min) in SEM, with pure human
blood
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Fig. 4-24 Clotting Time Test (Control) in SEM, with PRP
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Fig. 4-25 Clotting Time Test (Control) in SEM, with PPP
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Fig. 4-26 Clotting Time Test (1V sample) in SEM, with PPP
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Fig. 4-27 Clotting Time Test (1V sample) in SEM, with PRP
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Fig. 4-28 Clotting Time Test (3V sample) in SEM, with PRP
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Fig. 4-29 Clotting Time Test (3V sample) in SEM, with PPP
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Fig. 4-30 Clotting Time Test (5V sample) in SEM, with PPP
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Fig. 4-31 Clotting Time Test (5V sample) in SEM, with PRP
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