Influence of physical cues from the degrading magnesium implants on human cells

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Chapter 2.1, Migration assay:
To compare the migration behaviour of human gingival fibroblasts (HGF) on magnesium membranes (Botiss biomaterials GmbH, Berlin, Germany, Purity: 99.95%, 13 x10 mm, thickness: 140 µm) with them on current used non-resorbable GBR-membranes, the established migration assay, which is described in my paper [4] was performed on gamma-sterilized titanium discs (Goodfellow, Friedberg, Germany, Purity: 99.6%, Ø15 mm, thickness: 140 µm). The migration curve, obtained from the evaluated images using the software Tscratch, represents the averaged cell free area of six replica as a function of the time.
2.2, Surface characterization:
The roughness and topography of uncorroded and 72 h pre-corroded gamma-sterilized magnesium membranes, which were used for the migration assay, were already determined during my previous research [4]. The analysis was completed by roughness measurements with the atomic force microscopy (AFM) of titanium discs and tissue culture plastic (TCP, 24-well plate, Sarstedt, Nürnbrecht, Germany) and topography images of titanium discs using the scanning electron microscopy (SEM). Additionally, the wettability was determined for magnesium (uncorroded and pre-corroded), titanium discs and TCP. The utilized surface methods are described in the following sections.
2.2.1, Topography - Scanning electron microscopy (SEM):
In contrast to light microscope, the scanning electron microscope (SEM) uses electrons for imaging, whereby a magnification of up to 105 x and a resolution of 1-5 nm can be achieved [13]. The high depth of sharpness and the high magnifications of the SEM allow capturing of high-resolution topography images. Samples need to be electrically conductive and resistant to vacuum. Non-conductive sample can be made electrically conductive by coating with a thin gold layer in a sputtering process [14]. But, there are already modern SEM working with low vacuum to investigate non-conductive samples without sputtering [15].
An electron gun generates a beam of electrons by thermal emission (Fig.2) [14]. The electrons dissolve from the cathode and were accelerated to the anode under vacuum. The acceleration voltage determines the energy of the electrons leaving the anode, which is between 2-30 keV [14]. Operating under vacuum avoids electrons to collide with gas molecules and allows using the wave properties of electrons [16]. Electrons were focused by the condenser lens to a small electron beam, which is scanned in a raster pattern over the sample surface [14]. Scanning is achieved by the beam deflector containing scanning coils. When electrons hit the sample, they interact elastically or inelastically with the sample atoms [17]. During elastically interaction, the electron is scattered by the positive atom nucleus. The generated backscattered electrons (BSE) are characterized by a low energy loss and were detected with a semiconductor detector [17]. Inelastically interaction occurs when the impinging electron knocks another electron from its atomic shell, which is called secondary electron (SE) [17]. Secondary electrons (SE) show a high energy loss with a high deflection angle and are detected with the Everhart-Thornley detector, a type of scintillation-photomultiplier system [17]. The detector signal is amplified and converted in an indirect image on the screen. Every voltage signal is assigned to a brightness value. Topography images are principally captured using SE-signals, whereas BSE signals provide information about the chemical composition of the sample [14]. Although SE are generated in the whole sample, the generated SE in deeper layers cannot escape the sample due to their low energy and are thus absorbed. Therefore, only the SE in direct vicinity to the interface form the signal (escape depth for metals: 5 nm and for isolators: 50 nm), which are 1% of all formed SE [18].