The Heart Valve Society

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Macrophage Polarization State Influences Synthetic Heart Valve Degradation: Insights In The Fragile Balance Between Tissue Formation And Biomaterial Degradation
Tamar Wissing1, Marieke Brugmans2, Anthal Smits3, Carlijn Bouten3.
1Technical University Eindhoven, Eindhoven, Netherlands, 2Xeltis B.V., & Technical University Eindhoven, Eindhoven, Netherlands, 3Institute for Complex Molecular Systems & Technical University Eindhoven, Eindhoven, Netherlands.

Background
Novel immunomodulatory, resorbable synthetic heart valve substitutes are being developed that once implanted elicit an inflammatory response succeeded by a regenerative response in situ. For long-term functionality of these load-bearing substitutes, mechanical integrity should be maintained throughout the regenerative process. The mechanical integrity is highly dependent on the delicate balance between tissue formation and biomaterial degradation.
The versatile macrophage has been shown to play a decisive role in this balance, as macrophages contribute to tissue formation and remodelling, as well as biomaterial degradation. Therefore, the goal of this study is to quantify the degradative products (e.g. esterases, reactive oxygen species (ROS)) produced by different macrophage phenotypes.
Methods
Macrophages were cultured in 2D for four days and meanwhile polarized into the pro-inflammatory (M1) (20ng/ml IFN-ɤ, 100 ng/ml LPS), the anti-inflammatory (M2a) (20 ng/ml IL-4, 20ng/ml IL-13) or the regulatory (M2b) (50 ng/ml IL-10) phenotype. Unpolarized macrophages (M0) served as the control group. Medium samples were collected at day 2 and 4 to quantify the esterase production in time. At day 4, cells were sacrificed and processed for further analyses to 1) visualize active ROS production (CellROX reagent, Life Technologies), 2) quantify lipid peroxidation as a measure for oxidative stress (Lipid Peroxidation (MDA) assay, Sigma Aldrich), 3) determine the DNA content and 4) confirm macrophage polarization on gene and protein level.
Results
The unpolarised (M0), pro-inflammatory (M1) and regulatory (M2b) macrophages showed pronounced active ROS production at day 4, where limited active ROS production was detected in the anti-inflammatory (M2a) macrophage group (fig 1A). Furthermore, MDA analysis revealed increased percentages of lipid peroxidation (a measure for oxidative stress) for the M2a (39.1 6.5), M2b (25.9 1.6) and M1 (96.7 3,0 ) macrophages in comparison to the unpolarised control (M0) (fig. 1B).
Conclusions
The current results illustrate that the pro-inflammatory macrophage phenotype (M1) may accelerate oxidative biomaterial degradation. Follow-up studies will focus on the ROS and esterase production of polarized macrophages when cultured on 3D electrospun synthetic substitutes used for in situ tissue regeneration. Eventually, these insights can be translated towards immunomodulatory design strategies to tailor biomaterial degradation.


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