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. 2024 Dec 5;28:0115. doi: 10.34133/bmr.0115

Fig. 3.

Fig. 3.

Recapitulation of cerebral-blood-vessel-specific cellular functionality and neuroinflammatory responses using 4 types of ECM biomaterial inks. (A) Validation of the cellular compatibility of different biomaterial inks. LIVE/DEAD (green/red) assay images of cells encapsulated in collagen, the BdECM, the VdECM, and the cerebrovascular-specific decellularized extracellular matrix (CBVdECM) after 14 d in culture (scale bars: 200 μm). (B) Overall morphological differences in human brain microvascular endothelial cells (HBMECs; 1 × 106 cells/ml) encapsulated in 1% BdECM or VdECM owing to proteomic differences (green: calcein-AM; scale bars: 100 μm). (C) Proliferation of cells encapsulated in ECM biomaterial inks. Data represent mean ± standard error of the mean (SEM, n = 4 per group; ***P < 0.001; ****P < 0.0001; 2-way ANOVA). (D) Schematic illustration of cerebral-blood-vessel-specific cellular functionality in the cerebrovascular (CBV) constructs. (E) Basement membrane-like structures (pink arrows) and endothelial sprouting morphologies (purple arrows) of endothelial junctions (vascular endothelial cadherin [VE-cadherin], green) formed in 1.5% collagen, BdECM, VdECM, or CBVdECM after 14 d in culture (HBMECs + human brain vascular pericytes [HBVPs], 2 × 106 cells/ml; scale bars: 100 μm). (F) Heatmap results of an inflammatory cytokine array after inducing neuroinflammation by tumor necrosis factor-α treatment. Each biomaterial showed different trends in their fold changes in inflammatory cytokines. (G) Quantification of the results presented in (E). Data represent mean ± SEM (n = 4 per group; *P < 0.05; **P < 0.01; ***P < 0.001; 2-way ANOVA). (H) Significant junctional down-regulation following intensive up-regulation of cytokines in the CBVdECM. Data represent mean ± SEM (n = 3 per group; *P < 0.05; **P < 0.01; ***P < 0.001; 2-way ANOVA). (I) Fold changes in 2 representative neuroinflammatory cytokines, namely, granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-X-C motif chemokine ligand 10 (CXCL10)/interferon-gamma-induced protein 10 (IP-10). Data represent mean ± SEM (n = 2 per group; ***P < 0.001; ****P < 0.0001; 2-way ANOVA).