PLoS One. 2017 Aug 10;12(8):e0182914.

Biodegradability and platelets adhesion assessment of magnesium-based alloys using a microfluidic system

Lumei Liu1,2, Youngmi Koo1,2, Boyce Collins1, Zhigang Xu1, Jagannathan Sankar1, Yeoheung Yun1, 2∗

1 National Science Foundation-Engineering Research Center for Revolutionizing Metallic Biomaterials, North Carolina Agricultural and Technical State University, Greensboro, North Carolina, USA

2 FIT BEST Laboratory, Department of Chemical, Biological, and Bioengineering, North Carolina Agricultural and Technical State University, Greensboro, North Carolina, USA

∗ Corresponding author

E-mail: yyun@ncat.edu (YY)

 

Abstract

Magnesium (Mg)-based stents are extensively explored to alleviate atherosclerosis due to their biodegradability and relative hemocompatibility. To ensure the quality, safety and cost-efficacy of bioresorbable scaffolds and full utilization of the material tunability afforded by alloying, it is critical to access degradability and thrombosis potential of Mg-based alloys using improved in vitro models that mimic as closely as possible the in vivo microenvironment. In this study, we investigated biodegradation and initial thrombogenic behavior of Mg-based alloys at the interface between Mg alloys’ surface and simulated physiological environment using a microfluidic system. The degradation properties of Mg-based alloys WE43, AZ31, ZWEK-L, and ZWEK-C were evaluated in complete culture medium and their thrombosis potentials in platelet rich plasma, respectively. The results show that 1) physiological shear stress increased the corrosion rate and decreased platelets adhesion rate as compared to static immersion; 2) secondary phases and impurities in material composition induced galvanic corrosion, resulting in higher corrosion resistance and platelet adhesion rate; 3) Mg-based alloys with higher corrosion rate showed higher platelets adhesion rate. We conclude that a microfluidic-based in vitro system allows evaluation of biodegradation behaviors and platelets responses of Mg-based alloys under specific shear stress, and degradability is related to platelets adhesion.

https://doi.org/10.1371/journal.pone.0182914

 

Supplement:

Cardiovascular diseases (CVDs) are disorders of heart and the blood vessels (1). Biodegradable metallic stents, especially those made of magnesium (Mg)-based alloys, provide significant advantages that reduce restenosis, long-term endothelial dysfunction compared with permanent polymers (2,3), and provide better mechanical properties (tensile, compressive and shearing loadings) than bioasorbable polymers (4). Magnesium (Mg)-based alloys are degradable and biocompatible. The degradability means the main component, Mg, reacts with aqueous environment and then dissolves completely (5). Their degradability of makes these biomaterials a great choice for clinical devices, especially for orthopedic and cardiovascular applications. The biocompatibility of Mg-based alloys refers to their ability to interact with the body organic tissues without causing an unacceptable degree of harm.

Magnesium-based alloys are promising biodegradable and biocompatible materials for next generation cardiovascular stents. However, current ASTM standard in vitro corrosion tests (e.g. ASTM-G31-72 (6) and ISO 10993 series (7)) are not applicable to predict corrosion behavior of Mg alloys in vivo, because the degradation rates of magnesium-based scaffolds tested in vitro do not match those observed in clinical trials. In vivo and in vitro test results of Mg and Mg alloy corrosion typically show differences and even opposite outcomes (8). For example, the average corrosion rate of pure Mg (>99 wt.%) varies from 0.15 to 1.68 mm/year with different immersion solutions (8). Pure Mg (>99 wt.%), LAE442 and AZ91D showed different corrosion rates achieved from corrosion tests in vitro (ASTM standard immersion) and in vivo, (9,10). These facts revealed the unreliability of conventional degradation tests.

Microfluidic systems allow the detailed analysis of alloys under conditions found in vivo, such as blood-induced shear stress and blood cells for a variety of location and applications. The reported application facilitates the controlled and reliable assessment of different biodegradable alloys and aids the development of the optimal degradation behavior and biocompatibility. The quality and safety of Mg-based alloys can be evaluated in terms of degradability (corrosion behavior, type and product) and thrombogenic potential, including nidus thrombosis indicated by platelets adhesion (11) and downstream thrombosis indicated by particulate analysis(12).

A microfluidic system (Figure 1) was used to evaluate the degradation rates and thrombogenic potential of Mg-based alloys in culture medium (Figure 1, A-a) and platelets rich-plasma (Figure1, B-a), respectively. The shear stress was 6.81 dyne/cm2 to mimic in vivo coronary artery mean wall shear stress (13). The static condition was also tested by immersing the alloys in culture medium and platelets rich plasma (PRP). The tested Mg-based alloys were WE43, AZ31, ZWEK-L and ZWEK-C. WE43 stent has been studied in vivo (14-16); AZ31 has been tested in vitro (17); ZWEK-L and ZWEK-C are same material but cut from the longitudinal and cross-sectional directions from the extruded rod, respectively. Micro-X-ray computed tomography (CT, GE Phoenix Nanotom-M™, GE Sensing & Inspection Technologies GmbH) was conducted on samples before and after testing in microfluidic system, as well as after removing corrosion product on alloys surface by chromic acid dip. Degradation rates were calculated. Scanning electron micrographs (SEMs) was used to observe the platelet adhesion and the adhesion rate was calculated.

The degradation rate from low to high is WE43≈ZWEK-C<AZ31<ZWEK-L (P<0.05). This property is closely related to alloys’ design (element composition), fabrication quality (secondary phases and impurities), and extrusion direction facing to flow for extruded alloys. Mg-based alloy will have improved corrosion resistance if element composition and secondary phases are optimized, impurities-induced galvanic corrosion minimalized, and angel between extrusion direction and flow adjusted.

Platelets adhesion rate form low to high in dynamic condition is WE43≈ZWEK-C<AZ31<ZWEK-L (P<0.05). It reveals the thrombosis potential of WE43 and ZWEK-C is the lowest which is more biocompatible than the other two alloys. It is found that the degradation rate and platelets adhesion rates had the same trend of the alloys at dynamic condition. So a relation map was plotted to show their relation (Figure 1, C). It shows that with higher corrosion rate, the alloy is also with higher platelets adhesion rate. This phenomenon indicates that the fierce degradation behavior is a stimulus for platelet adhesion thus thrombosis and provides clue for future study on Mg-based alloys and their prospective clinical application.

 

 

Figure 1. Work flow of evaluation of degradability and biocompatibility of Mg-based alloys. The corrosion rate and the platelet adhesion rate at dynamic condition were WE43≈ZWEK-C<AZ31<ZWEK-L (P<0.05). The mild positive relation between degradability and thrombogenic potential was preliminarily revealed with the microfluidic system.

 

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