FIG. 3.

Cellular characteristics of cellularized PG-1. Images of epidermal component (keratinocytes) and dermal component (fibroblasts) of skin substitute are shown on the left side and right side respectively. Confocal microscopic images (20× magnification) of (A) top and (F) bottom of cellularized hydrogels (green—phalloidin, red—K14, blue—DAPI). Images taken 5 days post fibroblast seed and 3 days post keratinocyte seed show clusters of keratinocytes on the top surface of the hydrogel while elongated fibroblasts without keratinocytes are seen on the bottom of the hydrogel. Scale bar represents 50 μm. Live/dead viability (green—calcein AM [live cells], red—ethidium homodimer-1 [dead cells]) of (B) keratinocytes and (G) fibroblasts show 92.46% ± 2.26% (mean ± SEM) viable keratinocytes 3 days post seed and 97.74% ± 1.17% (mean ± SEM) viable fibroblasts 5 days post seed. Arrows indicate live cells while arrowheads indicate dead cells. Images taken at 20× magnification and scale bar represents 50 μm. BrdU proliferation assay (green—BrdU, blue—DAPI) of (C) keratinocytes and (H) fibroblasts show 18.27% ± 3.45% (mean ± SEM) BrdU-positive keratinocyte and 3.86% ± 1.42% BrdU-positive fibroblasts. Arrows indicate BrdU-positive cells while arrowheads indicate BrdU-negative cells. Images taken at 20× magnification and scale bars represent 50 μm. (D) Confocal microscopic image of different keratin expression of keratinocytes adhered to pullulan-gelatin hydrogel (green—phallodin, red—K10, blue—DAPI). Arrows indicate K14-positive keratinocytes while arrowheads indicate K10-positive keratinocytes. Images taken at 20× magnification and scale bars represent 50 μm. (E) Confocal microscopic image shows tight clustering of keratinocytes with adherens junction formation (green–phalloidin, red–e-cadherin, blue–DAPI). Image taken at 63× magnification and scale bar represents 20 μm. (I) Schematic showing total thickness of hydrogel and areas from which epidermal and dermal component images were taken. BrdU, 5-bromo-2′-deoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole. Color images available online at www.liebertpub.com/tea