Biodegradable magnesium (Mg) and its alloys have attracted increasing attention recent years. Prior to approval mandated by the governmental regulatory body, Mg-based implants are required a series of testing for assurance of their biocompatibility. The preparation of extracts from implants is the critical step for both cytotoxicity and hemolysis tests in vitro. Currently, the preparation of the extracts from medical devices is performed following ISO 10993 Standards. However, the standards should be carefully reevaluated to predict potential health risks of Mg based implants due to their degradability.
Mg-based metals differ from non-degradable metals that they could react with water immediately and release Mg2+ accompanied with higher pH value and osmolality. In order to mimic in vivo environment, cell culture medium supplemented with serum is recommended as the extract medium for cytotoxicity test, and according to in vivo data it is suggested that extracts should be diluted 6-10 times by Wang[1].
For the hemocompatibility test, normal saline is commonly used as testing solution (extract medium), but the Cl - concentration is higher than in vivo, which accelerates the degradation rate and results in higher pH value and Mg2+ concentration of Mg-based implants in the test solution. According to our preliminary data, the Mg2+ concentration has not significant effect on the hemolysis ratio (Figure 1.A), while the pH value has (Figure 1.B). The composites of normal saline is so simple that there is not pH buffer system at all, which is not consistent with in vivo environment. It will lead to much higher pH value and then the false positive hemolysis test results. Furthermore, to avoid the interferences of amino acid and protein on hemoglobin, the phosphate-buffered simulated body fluid (SBF) is preferred to the cell culture medium or serum.
Figure 1 Hemolysis ratios of normal saline with different Mg2+ concentration and pH values.
In fact, both the cytotoxicity test and hemolysis test in vitro are based on the electrochemical process of Mg-based implants in extract medium, which is also called “degradation”. To search for the suitable extract medium of degradable behavior study for Mg-based implants in vitro, the SBF and DMEM were compared for pure-Mg degradation in our group. It was found that pure-Mg degrades faster in SBF than in DMEM (Figure 2). This reason may be that DMEM contains not only inorganic ion but also many kinds of amino acids, which are negatively charged with the pH value increasing caused by Mg degradation, bind with released Mg2+, deposite on the Mg surface, and ultimately prevent the degradation of Mg. It is also partially the reason for that Mg degrades slower in vivo, which has been demonstrated by Witte[2]. As a result, cell culture medium like DMEM is recommended as the extract medium for the degradable behavior study in vitro.
Figure 2 Mg2+ concentration and pH value of pure Mg degradation in SBF and DMEM at different time points.
All the above, the focus is on the extract medium component for how to mimic in vivo environment. In fact, the biggest difference between in vitro and in vivo conditions is that electrolyte is dynamic in vivo, while the extract medium is unrenewable in vitro. Therefore, how to re-design or modify the current protocols for biocompatibility evaluation and degradable behavior study in vitro for biodegradable Mg-based implants is critical and still needed further investigation.
National Basic Research Program of China (973 Program, 2012CB619102); Shenzhen Technology Innovation Plan (CXZZ20140731091722497 and CXZZ20140419114548507)
References:
[1] Wang J, Witte F, Xi T, Zheng Y, Yang K, Yang Y, et al. Recommendation for modifying current cytotoxicity testing standards for biodegradable magnesium-based materials. Acta Biomater 2015;21:237-49.
[2] Witte F, Fischer J, Nellesen J, Crostack HA, Kaese V, Pisch A, et al. In vitro and in vivo corrosion measurements of magnesium alloys. Biomaterials 2006;27(7):1013-8.