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Wioleta Pawlak (Przeradzka) - Polsko-Japoński Program Doktorancki

Wioleta Pawlak (Przeradzka)

Name: Wioleta Przeradzka

Internship Term: From 04.07.2011 to 27.08.2011

Host Research Laboratory / Group: MANA, Biometal Group

Supervisor NIMS: Akiko Yamamoto, Ph.D

Supervisor WUT: prof. dr hab. inż. Małgorzata Lewandowska

 

Internship Subject: Effect of pure magnesium surface treatment on degradation behavior and cell growth.

Description of research:

Magnesium (Mg) and its alloys were first applied to orthopedic implants in early 20’s century. Recent attempts on stent application open a new stage as a bioabsorbable metal.

Mg generates H2 and OH along the progress of its corrosion reaction with water, the pH of the fluid around Mg surface will increase, which may affect surrounding cellular and tissue function.

In this internship, I examined degradation behavior and cytocompatibility of Mg surface. Using pure Mg, surface treatment with silane reagents was applied to control the surface hydrophilicity. It was examined cytocompatibility and degradation behavior in cell culture condition to find the effect of the hydrophilicity on their degradation behavior and cell growth.

The cells which were used in this project were murine fibroblast L929.

Cells were keeping in E-MEM + 10%FBS. It is physiological solutions similating the body fluids. Samples of pure magnesium after polishing and washing in ultrasonic cleaner were coating by silane reagents: ethyltriethoxysilane LS2410 and octadecyltriethoxysilane LS6970. Some samples were no coating. Next samples have been sterilized by ethylene oxide gas(EOG).

The first measurement was contact angle of water. I have done it to check hydrophobicity. When hydrophobicity is improved it is expected to retard initial rapid corrosion of magnesium.

A contact angle can be measured by producing a drop of liquid on a solid. The angle formed between the solid/liquid interface and the liquid/vapor interface is referred to as a contact angle. The most common method for measurement involves looking at the profile of the drop and measuring two-dimensionally the angle formed between the solid and the drop profile with the vertex at the three-phase line.

A drop with a contact angle over 90° is hydrophobic. This condition is exemplified by poor wetting, poor adhesiveness and the solid surface free energy is low. A drop with a small contact angle is hydrophilic. This condition reflects better wetting, better adhesiveness, and higher surface energy.

WST-1 assay is performed to measure number of cells. The samples are put on dishes with culture medium solutions and kept in CO2 incubator and remove after 7days incubation.

WST-1 assay after day 1, day 4, day 7 incubation is relying on measuring light absorbance at 450 nm in culture medium solution. Bigger absorption shows the more cells living on sample.

Mg2+ Quantification is measurement to check corrosion rate. It is Xylidyl blue colorimetric method. It was done after day 1, day 4, day 7 incubation. It is relying on measuring light absorbance at 520 nm and 650 nm in culture medium solution from dishes which in were samples.

ThepH measurement was doing upto 24 hours to check corrosion rate and cultural condition. It used special electrode and a z-stage placed in incubator to control distance from sample surface to be 1 mm. It takes 24 hours and measured 720 data for one sample. One data is for two minutes.

Immersion test without cells is similar to WST-1 but this was done without cells on mirror polishing surface. The samples were put on to E-MEM+10%FBS without phenol red. Next I was doing Mg2+ Quantification to check corrosion condition.

 

Summary of reaserch:

The most hydrophobic surface is LS6970 but it is unstable. It could be caused by not good concentration of the reagent and not perfect coating. LS2410 is hydrophobic and stable.

The biggest amount of cells is observed on pMg, LS6970 have similar amount.

The most corroded is LS2410. It could be caused by surface morphology produced by polishing. On microscopic observation of samples, we can see many polishing marks that means corrosion progressed along polishing marks.

The biggest pH was measured for LS2410, suggesting that it has the highest corrosion rate. The sample corroded severely, not many cells were observed on it.

Since the polishing marks influence the corrosion behavior of surface coated samples, I prepared mirror polished sample with LS2410 and LS6970 coating. In case of LS6970, coating condition is also changed. Immersion test without cells is performed in a similar manner to cytocompatibility evaluation.

 

After mirror polishing:

The most hydrophobic surface is LS6970, the least is LS2410. All samples increased hydrophobicity and have stable contact angle to water.

The most corroded is LS2410, the least corroded is pMg. One of the possible causes is buff polishing solution which reacts with silane reagent. On microscopic observation, pMg surface is homogenious, but coating samples have marks of air bubbles. This is because of the increase of hydrophobicity by surface coating since it contributes to stabilize air bubble on sample surface. It should be minded that water contact angel would be different from that of culture medium since it contains lipids and proteins.

For further investigation, following can be performed:

  • Change silane reagents on hydrophobicity
  • Improve mirror polishing – change buff polishing solution
  • Check cell culture reaction on mirror polishing surface

 

Overall impression:

I liked very much student internship in Japan. I learned many new things there. During the week I worked in a laboratory in the group of biomaterials. I met many interesting people. And on the weekends I visited Tsukuba, Tokyo and the surrounding Japanese towns. I would recommend this trip to younger colleagues.

 

Group from Poland, NIMS 2011