Investigation of human urothelial cells in response to magnesium-zinc-strontium alloys in exposure culture

摘要

Introduction: Magnesium (Mg) alloys have promising potential for bioabsorbable medical device application, e.g. urological device application. Current materials for ureteral stents present major problems, such as infection by bacterial accumulation and blockage by encrustation. Mg alloys showed antibacterial properties in our previous study, and Mg degrades in physiological environment which eliminates painful device removal. In this study, we investigated the cytocompatibility of Mg-4Zn-xSr alloys with human urothelial cells (HUCs), as well as how the main degradation products of Mg affect the viability of HUCs. Materials and Methods: HUCs were cultured in the plates that were coated with 2 μg/cm2 poly-L-lysine. Phase-contrast images were taken for each well to obtain the initial cell density, before inducing the substrates. ZSr41_A and ZSr41_B which had 0.15 wt% and 0.5 wt% Sr respectively, and 4 wt% Zn in both, together with Mg, PU and glass were weighed and disinfected under UV light The substrates were placed in the Transwell® inserts with pore size of 0.45 μm, and kept a distance of 0.9 mm from the cells. After the exposure culture for 24 or 48 hours, substrates were collected from the media and air dried overnight. The substrates were weighed and characterized using scanning electron microscope and energy dispersive X-ray spectroscopy for surface morphology and elemental composition. The media were collected for pH and Mg2+ ion concentration evaluation. Adhered cells were fixed and stained for fluorescence imaging to evaluate the final cell density and morphology after culture. HUCs were also cultured with media of various pH and Mg2+ ion concentrations for 24 hours to evaluate the effects of Mg degradation products, and the HUCs density after culture were quantified. Every group is prepared in triplicate. ANOVA analysis were performed, p＜0.05 was considered as statistically significant. Results and Discussion: The cell density ratio (final/initial) after exposure culture for both 24 and 48 hours were summarized in Figure 1. The ZSr41_A resulted in the highest cell density ratio in average. Both ZSr41_A and ZSr41_B showed no statistically significant difference compared with glass and cells only controls. As shown in Figure 2a, as the pH of the media increased, the amount of live cells decreased after a 24-hour culture. The HUC density cultured with pH of 7.4 and 8.0 was statistically significant higher than those of 8.6,9.5 and 10. The HUC density after 24 hours of culture with varying Mg2+ ion concentrations showed no statistically significant difference (Figure 2b). Conclusion: The Mg-4Zn-xSr alloys showed good cytocompatibility with HUCs in exposure culture. The HUCs showed no significant difference in cell density when cultured with pH less than 8 and Mg2+ ion concentration less than 40 mM. The results indicated that high alkalinity might be the dominate cytotoxic factor of Mg alloy with HUCs, Mg alloy with slower degradation rate is preferred for the future in vivo studies.