FIG. 5.

Fragment viability, and sprouting in hypoxic conditions. E- and E+I-circulated DIP chamber–conditioned constructs were sectioned and stained for HIF1α (brown indicates positive staining). HIF1α-positive fragments were found throughout E-circulated constructs, but where highly concentrated in ROI3 (a and b). In E+I-circulated constructs, few HIF1α fragments were present within examined sections. Example E+I-circulated ROI3 images are presented (c, d). Gray boxes in (a) and (c) illustrate magnified fields of view in (b) and (d). About 250 μL MVF constructs were cultured in standard (typical cell culture conditions, 5% CO₂ and 37°C) and hypoxic conditions (1% O₂ and 37°C). Day 5 standard and hypoxic constructs were stained with a fluorescently labeled anti-HIF1α followed by three-dimensional stack generation via confocal microscopy. Stained standard constructs (e) presented less fluorescent signal than hypoxic constructs (f ). MVFs were costained with a viability marker (green represents viable fragments [g, h]). Three time-points were selected for evaluating sprouting within cultured constructs (i). After 4 days of hypoxic conditioning, standard constructs (average sprouting index [ASI] of 1.35 ± 0.07) displayed significantly more sprouting than hypoxic constructs (ASI of 0.1 ± 0.04) (Pearson Chi-square test, n = 96, df = 2, χ² = 78.78, *p < 0.0001). After 7 days, standard constructs exhibited more sprouting than previously hypoxic constructs, respectively, ASI of 2.0 ± 0 and 0.1 ± 0.04 (Pearson Chi-square test, n = 96, df = 2, χ² = 96.0, *p < 0.0001). Similarly, day 10 standard samples (ASI of 1.56 ± 0.08) exhibited more sprouting than previously hypoxic specimens (ASI of 0.19 ± 0.06) (Pearson Chi-square test, n = 96, df = 2, χ² = 67.67, *p < 0.0001). After 6 days of culture (j), MVF viability was significantly less in hypoxic constructs than standard constructs, 80 ± 2.0% and 90 ± 1.3% respectively (two-tailed Student's t-test, n = 40, df = 37.54, *p < 0.0001). Hypoxic cultures were then returned to standard conditions, but viability did not significantly change after 1 day of standard culturing. All calculated values are presented as mean ± s.e.m. HIF1α, hypoxia inducible factor-1α. Color images available online at www.liebertonline.com/ten.