Alizarin Red Mineralization Assay.

<p>Only small, diffusely distributed mineral deposits are detected in phase contrast images of stained cultures that are more prominent in hMSCs in osteogenic differentiation than in propagation media. Quantitative analysis by absorption at 405 nm of eluted alizarin red stain reveals significantly more staining of NO cells than HO (denoted by asterisk). N = 6.</p

SHG evaluation of fibrous collagen.

<p>The gradual deposition of fibrous collagens is apparent in SHG images acquired on days 4, 8,12 and 16 from hMSCs in osteogenic medium at (A) 5% oxygen and (B) 20% oxygen. Quantitative evaluation of pixel density in SHG images reveals that collagen deposition evolves earlier for HO conditions than for NO cells. N = 4. (Bar = 50 µm).</p

TPEF analysis of hMSCs following KCN or FCCP treatment.

<p>The inverse fluctuations of NAD(P)H and FP fluorescence in response to KCN or FCCP treatment were detectable via either TPEF emission spectra excited at 755 nm, or TPEF images excited at 755 nm and 860 nm. For each method, means are displayed relative to values before treatment with standard error bars. Spectral data from multiple microscopic fields (N = 9) were used for the analysis of the TPEF emission spectra while the results from TPEF images were calculated on a cell by cell basis from multiple image sets (N = 42).</p

RT-PCR.

<p>Transcript levels of COL 1a1, COL 2a1, BSP and LPL were assessed for each imaging time point relative to GAPDH. Statistically significant differences in mRNA transcript levels are denoted by a single asterisk (in comparison to the propagation medium at the same oxygen tension) or by a double asterisk (significance in comparison to cells of the same medium condition, at 20% oxygen tension). N = 4.</p

Lysosomal localization of lipofuscin.

<p>Lipofuscin autofluorescence (green channel) is co-localized with (A) Lysotracker red (red channel) and not (B) Mitotracker Orange (red channel). Co-localization is indicated by the yellow color in panel A. Note that not all lysosomes contain lipofuscin. Bar  = 30 µm.</p

Oil Red - O Staining for lipid droplets.

<p>hMSCs maintained in adipogenic differentiation medium were stained with Oil Red - O to highlight the lipid contents of cells (top images, bar  = 100 µm). Higher magnification transmission images of HA and NA (bottom images, bar = 50 µm) before staining reveal larger, more numerous lipid droplets in hMSCs cultured in adipogenic differentiation medium at 20% oxygen. Quantification of eluted Oil Red - O by absorption of 490 nm reveals an increase in total Oil Red - O staining amongst HA or NA cells, but the differences are not statistically significant. N = 6.</p

Components of hMSC TPEF emission spectra.

<p>(A) hMSC TPEF emission spectra collected at either 755 nm or 860 nm excitation with corresponding fits by the ALS script. (B) linear unmixing methods reveal the contributing cellular fluorophores to be NAD(P)H, Lipofuscin and FP. When components of intrinsic cellular fluorescence are used to produce the fits displayed in panel A the NAD(P)H component does not contribute to cellular spectra excited at 860 nm.</p

Changes in cell redox ratio and density with time.

<p>A) Average cellular redox ratio at 0, 1, and 2 wks in perfusion compared to previously published static culture experiments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055696#pone.0055696-Quinn1" target="_blank">[9]</a>. In both static and perfusion culture, no change in redox was observed between 0 and 1 wks, but the redox ratio decreased (p<0.001) from 1 to 2 wks. Although a significant decrease was observed in both cultures at wk 2, the redox ratio was signficantly higher in the perfusion cultures. B) Cell population measurements per image stack (238×238×40 µm volume) at 0, 1, and 2 wks in perfusion flow demonstrate an increase over time, but substantial sample-to-sample variability.</p

Experimental timeline describing cell seeding, induction and imaging time points.

<p>The total number of cells seeded on each scaffold was scaled up by scaffold volume to maintain a consistent density with previous work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055696#pone.0055696-Kang1" target="_blank">[15]</a>.</p

False colored redox maps of adipose tissues <i>in vitro</i> after 0, 1, and 2 wks within the perfusion environment.

<p>The grayscale structures in the images are the silk biomaterial 3D scaffolds, while the sparsely distributed colored objects are cells.</p