Date of Award

June 2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Chemical Engineering

Advisor(s)

Jeremy L. Gilbert

Second Advisor

Michelle M. Blum

Keywords

Cytotoxicity, Galvanic corrosion, Magnesium and magnesium alloys, Oxidation and reduction reactions, Reactive oxygen species

Subject Categories

Engineering

Abstract

This dissertation characterized how galvanic coupling of magnesium (Mg) with titanium (Ti) via sputtering enhanced the cytotoxicity of Mg particles due to increased corrosion rate. Mg and Mg-Ti particles were characterized using scanning electron microscopy (SEM), which showed that Mg and Mg-Ti particles have the average diameter of 50 µm, which makes them slightly larger than an average mammalian cell. Therefore, the cytotoxicity of the particles is not due to the ingestion of the particles by cells via phagocytosis, but solely due to electrochemical effects. Sputtering of Ti to partially cover Mg particles results in a very thin layer, in the range of nm. The surface area of Ti covering the Mg particles matters more than the thickness of the Ti layer, as the Ti layer serves as a catalyst for the reduction reactions, spatially separating the oxidation reaction of Mg and the reduction reaction of water/oxygen.

The first part of the dissertation focuses on the in vitro cell viability experiments to show that Mg and Mg-Ti particles kill cells in a dosage-dependent manner, where Mg-Ti particles kill cells more effectively than Mg alone. Cytotoxicity of Mg and Mg-Ti was measured on different types of cells, such as murine pre-osteoblast cells, human osteosarcoma cells, and bacterial Escherichia coli cells. For murine pre-osteoblast cells, Mg killed cells completely at 1500 µg/ml (less than 1%), while Mg-Ti killed cells completely at 750 µg/ml, which is half of the concentration of Mg. Doubling and tripling the pre-osteoblast cell density did not increase the particle concentration that required to kill cells completely, but did decrease the rate of cell viability drop over particle concentration. Mg and Mg-Ti killed pre-osteoblast cells within 18-24 hours, and if many cells were viable, cells proliferated and recovered after 24 hours, while if more than 50% cells died, remaining cells stayed alive but did not proliferate. Cytotoxicity of Mg and Mg-Ti also depended on proximity, where only cells less than 2 mm away from the cluster of particles were killed. For osteosarcoma cells, Mg could not kill cells completely even at 2500 µg/ml, while Mg-Ti killed cells completely at 1250 µg/ml by 24 hours. By 72 hours, Mg killed cells completely at 2500 µg/ml, and Mg-Ti killed cells completely at 1000 µg/ml. Higher concentrations and longer periods of time were needed to kill osteosarcoma cells, compared to murine pre-osteoblast cells. Osteosarcoma cells also showed the same trend as pre-osteoblast cells, where at low particle concentrations, the cells were able to proliferate and recover, at medium particle concentrations, the remaining live cells did not proliferate, and at high particle concentrations, the cells died and did not recover. The remaining live cells and their daughter cells did not gain resistance to the particle treatment as they were killed again without having to increase the particle concentration. 24-hour grown E. coli biofilms and planktonic cells were also treated with high concentrations of particles, and compared to conventional antibiotics, such as ofloxacin, Mg and Mg-Ti were able to kill biofilm more significantly, although not completely (down to 2%). However, Mg and Mg-Ti showed great promise in killing and preventing biofilms.

The second part of the dissertation focuses on investigating whether pH is the main cause of Mg cytotoxicity as many studies have claimed, and if the pH is the major or sole cause of enhanced cytotoxicity of Mg-Ti. This study showed that the pH levels of Mg and Mg-Ti were the same at the same particle concentration, which does not explain the increased killing of Mg-Ti. Also, Mg and Mg-Ti did not just kill cells directly, but also affected the components, such as the proteins, in the cell culture medium.

Overall, this study provides better understanding of cytotoxicity of Mg and Mg-Ti, so that not only Mg and Mg-Ti can be used to target and kill cancer and bacterial infections for the therapeutic effect, but to show that Mg and Mg-based alloys may not be as biocompatible, which is still the common conception of this biomaterial.

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Open Access

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