In this study, we engineered a vascularized 3D dermis by layering multiple highly aligned prevascularized sheets with human mesenchymal stem cells to mimic the capillary loops observed in native dermis tissue. Monocyte-derived macrophages were seeded onto these prevascularized constructs to mimic the innate immune-response in vitro. To evaluate macrophage responses to these scaffolds, we used multiphoton microscopy to detect the metabolic phenotypes of macrophages on engineered scaffolds over time. NADH and FAD are coenzymes that emit endogenous fluorescence and participate in various metabolic pathways, and their fluorescence lifetime is associated with different protein binding states and microenvironments. Fluorescence lifetime images of macrophages were obtained on day 3 and day 7 after seeding, and significant differences in NADH and FAD fluorescence lifetime features can be observed in macrophages on different scaffolds.
The human skin dermis is vascularized soft tissue containing highly organized extracellular matrix (ECM) architecture. Macrophages mediate wound healing within the skin but technologies for detection of macrophage function simultaneously with ECM properties are limited. In this study, we use multiphoton microscopy to evaluate the metabolic function of macrophages and quantify collagen alignment. Multiphoton microscopy is label-free and non-damaging allowing time-course studies of turbid samples and in vivo measurements. The fluorescence lifetime imaging technique detects the time fluorophores remain in an excited state before relaxing to the ground state. Reduced nicotinamide dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) are auto-fluorescence coenzymes that participate in various metabolic pathways including glycolysis, oxidative phosphorylation, fatty acid synthesis, and fatty acid oxidation, and label-free fluorescence lifetime imaging is a functional method to track cellular metabolism. In this paper, macrophages were seeded on anisotropic and isotropic ECM. Fluorescence lifetime images of macrophages and second harmonic generation (SHG) images of collagen in the ECM were obtained with a two-photon fluorescence lifetime microscope every two days for one week. Significant changes in macrophage size and fluorescence lifetime features were observed with time. The free fraction of NAD(P)H and bound fraction of FAD are both higher for macrophages on random ECM compared with aligned collagen. In summary, multiphoton microscopy provides a label-free method to track metabolic variation of macrophages concurrently with imaging of the ECM.
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