Standard curve of GFP fluorescent signal from lysed GFP-MSCs.

<p>GFP-MSCs were counted using a hemocytometer and set numbers of cells were spun down in a centrifuge. Cell pellets were lysed and solution fluorescence was measured by a fluorometer. The coefficient of determination for the linear regression was 0.999, showing a very strong linear correlation between GFP-MSC number and solution fluorescence.</p

Chemotactic responses of MSCs.

<p>GFP-expressing MSCs (4×10⁴) were seeded onto the top of transwell chambers, with various cytokines/chemokines placed in the bottom of the chambers; some wells contained serum-free media (SFM) as a negative control. After a 20 hr incubation at 37°C, the GFP-MSCs that had migrated across the transwell membrane were lysed and quantitated by measuring fluorescence intensity of GFP. The following chemoattractants were evaluated: <b><i>A)</i></b> recombinant human PDGF-BB, PDGF-AB, or a mixture of SDF-1α, CXCL16, MIP-1α, MIP-1β, and RANTES, each at the indicated concentrations (ng/mL) (representative of 3 independent runs) <b><i>B)</i></b> PDGF-BB and BMP-2 (representative of 3 independent runs) and <b><i>C),</i></b> varying concentrations of PDGF-BB showing dose response.</p

PDGF-BB released from PCL/col/HA scaffolds stimulates MSC chemotaxis.

<p>The lower wells of transwell chambers were filled with either purified PDGF-BB (10 ng/mL), serum-free medium (SFM) or PDGF-BB-containing conditioned media collected from PDGF-BB-coated PCL/col/HA scaffolds after 72 hrs. GFP-MSCs were seeded in the upper chambers and allowed to migrate for 20 hrs. After this interval, MSCs adherent to the underside of the transwells were visualized by fluorescent microscopy (top panel, representative images). In addition, MSC migration to the underside of the filter was quantified by lysing cells and measuring solution fluorescence (lower panel). Three independent experiments were performed for solution fluorescence. Analysis of variance with Tukey’s HSD post-hoc was used to establish significance (* denotes <i>p</i><.01).</p

Released PDGF-BB induces chemotaxis of MSCs in a stringent migration assay. <i>A)</i>

<p>Schematic showing experimental set-up (not drawn to scale). GFP-MSCs were seeded in 8-well rectangular plates. After cell confluence was established, cells were completely removed from the top half of the well by scraping along a pre-drawn central line. Subsequently, a PDGF-BB-adsorbed PCL/col/HA/scaffold, placed on a steel wire mesh, was placed 1.5 cm away from the cell front. As a control, some chambers were set up with PCL/col/HA scaffolds lacking PDGF-BB. <b><i>B)</i></b> After a 72 hr-incubation in the chambers described, MSCs were stained with DAPI and visualized by fluorescence microscopy. The original cell front created is denoted by a white line. <b><i>C)</i></b> GFP-images showing change in cell morphology of MSCs exposed to PDGF-BB. <b><i>D)</i></b> Significantly greater cell number was observed migrating toward PDGF-BB coated scaffolds compared to uncoated scaffolds. <b><i>E)</i></b> DAPI-stained images were further analyzed by counting the number of cells in three defined regions of distance beyond the original cell front. The distribution of cells in the wells with PDGF-BB-coated scaffolds showed that a greater percentage of the total cells that had migrated beyond the cell front had localized to the region beyond 400 µm. In comparison, the greatest percentage of cells in the control wells localized to the region below 150 µm. A total of six samples were analyzed for each condition. An asterisk (*) denotes significant differences observed with <i>p</i><.01, whereas (**) denotes <i>p</i><.0001.</p

Adsorption and release of PDGF-BB from scaffolds. <i>A)</i>

<p>Scaffolds were incubated in PBS containing 1.5 µg PDGF-BB for 24 h at 4°C. ELISA assays were used to measure the unbound PDGF-BB in the supernatants. Adsorption of PDGF-BB to the scaffolds was determined by subtracting the unbound PDGF-BB from the 1.5 µg of PDGF-BB initially added. Data are from three independent experiments (* denotes p<0.01). <b><i>B)</i></b> ELISAs were used to measure the amounts of PDGF-BB in conditioned PBS solution collected from the scaffolds at the indicated time intervals over a period of 8 weeks (for many of the data points, error bars are too small to be visualized).</p

Contraction of porous 70:30 col/PCL scaffolds containing seeded fibroblasts.

<p>Scaffold diameters were measured at each time interval to quantify contraction (plotted as percent decrease in scaffold diameter). Values represent means and standard deviation for five scaffolds per time point. Two independent experiments were performed. A one way Anova was performed to compare the percent change in scaffold contraction of the various groups. *Represents significant difference (p<0.01) relative to all other groups.</p

Scanning electron microscopy (SEM) images of scaffolds with 160 μm pores.

<p>A) Magnification of 150x. B) Magnification of 1834x.</p

Fibroblast viability on 70:30 col/PCL scaffolds.

<p><b>(A)</b> F344 fibroblasts grown on scaffolds for 1, 4, 7, and 14 days were stained for either living (green) or dead (red) cells. Scale bar = 40 μm. <b>(B)</b> Values represent means and standard error of the mean for live and dead cells measured from three distinct fields per scaffold, with multiple scaffolds evaluated. * represents significant difference in live cell number compared to dead cell number at each time point (p<0.05); # represents difference in live cell number relative to live cell number at day 1 (p<0.05).</p

Images (10X) of wound healing over a 21-day time period show the structure of the matrix in each treatment group.

<p>The matrix in wounds containing scaffolds pre-seeded with fibroblasts appears more normal to skin tissue with loose, wavy basket-weave matrix and the formation of hair follicles. Scale bar = 10 μm.</p

Scaffolds pre-seeded with fibroblasts promote formation of ECM with a high degree of basket-weave structure, resembling unwounded skin tissue.

<p><b>(A)</b> Representative image of a cross-section of a wound bed harvested from a rat implanted with a cell-seeded microporous scaffold. White dashed line designates the area of basket-weave matrix. Scale bar = 40 μm. <b>(B)</b> Graph depicts the average basket-weave area of harvested tissues containing scaffolds seeded for 4 days with fibroblasts (4d), scaffolds seeded for 1 day (1d), acellular porous scaffolds, and sham wounds (<i>n</i> = 5 rats per group, with multiple microscopic fields examined per specimen). A repeated measure Anova was performed to compare significance between treatment groups. *Represents p<0.01 for all treatment groups that are significantly different from each other within the same time point. ^# Represents treatment groups significantly different than their respective groups at the 7 day time point (p<0.05).</p