Page 68 - Magnesium-based supports for stem cell therapy of vascular disease - Mónica Echeverry Rendón

CHAPTER 4
l-glutamine (Lonza, NJ, USA) was maintained under culture until confluency. Next, cells were detached using trypsin at 37°C for 5min. About 50,000 cells were concentrated in 100μl of medium and this volume was loaded onto each sample and incubated for 30 min to allow cells to attach. After that, 1ml of fresh medium was gently added and cells were incubated 48 h at 37°C, 5% CO2 and 98% humidity. Then, cells were rinsed twice with PBS and fixed 30 min with 4% paraformaldehyde at room temperature. Next, cells were permeabilized with 0.5% triton X-100 for 15 min and blocked with 5% BSA-PBS. Finally, staining for actin was carried out by using 5μg/ml Phalloidin-TRITC (P1951, Sigma, Missouri, USA) while nuclei were stained with DAPI (D9542, Sigma, Missouri, USA). Cells were assessed using a fluorescence microscope (Nikon LABOPHOT-2) at a 10x objective magnification.
2.6.3 Hemolysis test
Citrated human blood was drawn and used for the hemolysis test. For this the citrated blood was diluted with saline solution in a ratio of 4:5. After that, material samples with and without coating were dipped in tubes containing 10 ml of saline solution and incubated at 37°C for 30 min. Next, 0.2 ml of diluted blood was added to each tube, mixed carefully and incubated at 37°C for 60 min. Then, tubes were centrifuged at 700 g for 5 min. Supernatants were transferred to a plate and absorbance was read at 545 nm. Deionized water and saline solution were used as positive and negative controls respectively. Hemolysis was calculated based on the Eq.2:
2.6.4 Thrombin generation assay (TGA)
Anodized and untreated Mg samples with dimensions of 0.5 cm x 0.5 cm and 1 mm of thickness were used for the thrombin generation assay. Low-density polyethylene (LDPE), polydimethylsiloxane (PDMS) and medical steel (MS) were used as reference materials for the test due to they are common materials used in biomedical applications. Reactions without material were used as negative control. Thrombin generation was determined by using a kit from Haemoscan (Groningen, The Netherlands) and processed according to the manufacturer’s protocol. First, samples were incubated in modified plasma in triplicate. Then, TGA reagent was added to initiate thrombin generation. After that, samples were collected every 2 minutes for 21 minutes. Then concentration of thrombin was measured by a colorimetric technique produced by an enzymatic reaction by using a thrombin-specific chromogenic substrate. Samples were read at an optical density of 405 nm with 540 nm as reference wavelength. After obtaining the data, thrombin concentration and rate of thrombin generation were calculated for each sample based on a calibration curve and compared with the reference materials and negative control.
3. Results
3.1 Characterization of the coatings
The curves of the anodizing process of c.p Mg using different electrolyte solutions are shown in Fig.1. The sample processed in NAF was operated under galvanostatic mode. The curve shows that the breakdown voltage was around 90V which was reached in 2s, this step is directly related to the onset of the generation of the oxide surface coating production of the barrier layer. Once this value was reached, the voltage oscillated around 150V during the rest of the time, while the thickness of the coating steadily increased. In contrast, HMT and MAN were operated at poten- tiostatic mode, once the process started, current density increased until it reached 170 mA.cm-2. Samples remained in this value for 35s for the HMT and 40 s for MAN. Then the current dropped down for 35 s for HMT and 80s for MAN until it reached 3 mA.cm-2 in both cases. For the two-steps anodic coatings, NAF¬HMT and NAF-MAN responded in a similar fashion: In both additives, a maximum current of 170 mA·cm-2 was reached that maintained for 22s for NAF HMT, and for 12s for NAF MAN. Next, in NAF-HMT the current decreased to 30 mA·cm-2 after 8s while in for NAF MAN it
66