Label-free metabolic biomarkers for assessing valve interstitial cell calcific progression

Figures

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  Figure 1. Development of calcific nodules in osteogenic cultures. (a) Phase contrast images of VIC cultures for days 1 through 28 under quiescent or osteogenic conditions on compliant or stiff substrates. (b) Alizarin Red S stained 28-day VICs under quiescent or osteogenic conditions on compliant or stiff substrates. (c) Average number of nodules per coverslip. (d) Area covered by the nodules per coverslip normalized by the total area covered by the cells. N = 5–6, *p < 0.05, Scale bar = 500 µm.

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  Figure 2. Optical redox ratio and mitochondrial fractal dimension change with osteogenic differentiation of VICs. (a) Redox ratio maps of VIC cultures for days 1–28 under quiescent or osteogenic conditions on compliant or stiff substrates. (b) Average optical redox ratios and (c) Average fractal dimension for days 1 through 28 under quiescent or osteogenic conditions on compliant or stiff substrates. Scale bar = 100 µm. N = 5–6, *p < 0.05.

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  Figure 3. Optical redox ratio correlated with osteogenic markers. Optical redox ratio correlated with (a) ACTA2, (b) RHOA, (c) TGFβR1, (d) OPN, (e) OCN and (f) RUNX2 fold change for VIC cultures for days 1 through 28 under quiescent or osteogenic conditions on compliant or stiff substrates. Spearman’s Rank correlation coefficient is denoted as (Sp ρ).

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  Figure 4. Mitochondrial fractal dimension correlated with structural and osteogenic markers. Fractal Dimension correlated with (a) ACTA2, (b) RHOA, (c) TGFβR1, (d) OPN, (e) OCN and (f) RUNX2 fold change for VIC cultures for days 1 through 28 under quiescent or osteogenic conditions on compliant or stiff substrates. Spearman’s Rank correlation coefficient is denoted as (Sp ρ).

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  Figure 5. Nuclear morphology changes correlated with mitochondrial fractal dimension during VIC osteogenic differentiation. (a) Average nuclear area in pixels for VIC cultures for days 1 through 28 under quiescent or osteogenic conditions on compliant or stiff substrates. (b) Optical redox ratio correlated with average nuclear area. (c) Fractal dimension correlated with the average nuclear area. (d) Nuclear aspect ratio for VIC cultures for days 1 through 28 under quiescent or osteogenic conditions on compliant or stiff substrates. (e) Optical redox ratio correlated with nuclear aspect ratio. (f) Fractal dimension correlated with the nuclear aspect ratio. N = 5–6, *p < 0.05. Pearson’s correlation coefficient is denoted as (R).

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  Figure 6. Calcific nodules in osteogenic cultures were apoptotic in nature. (a) Representative Caspase immunostains (Caspase in red and nuclei in blue) of VIC cultures for days 1 through 28 under quiescent or osteogenic conditions on compliant or stiff substrates. (b) Average apoptosis index of nodules for days 1 through 28 under quiescent or osteogenic conditions on compliant or stiff substrates. N = 3, Scale bar = 500 µm. *p < 0.05 compared to corresponding quiescent media condition.

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  Figure 7. Cells and nodules in osteogenic cultures were proliferative in nature. (a) Representative Ki67 immunostains (Ki67 in red and nuclei in blue) of VIC cultures for days 1 through 28 under quiescent or osteogenic conditions on compliant or stiff substrates. Average proliferation index of (b) cells and (c) nodules for days 1 through 28 under quiescent and osteogenic conditions on compliant or stiff substrates. N = 3, Scale bar = 500 µm. *p < 0.05 compared to corresponding quiescent media condition.