Fig 1.
Scanning electron microscopy (SEM) images of scaffolds with 160 μm pores.
A) Magnification of 150x. B) Magnification of 1834x.
Fig 2.
Fibroblast viability on 70:30 col/PCL scaffolds.
(A) 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) 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).
Fig 3.
Contraction of porous 70:30 col/PCL scaffolds containing seeded fibroblasts.
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.
Fig 4.
Extracellular matrix is deposited into the 160 μm pores of 70:30 col/PCL scaffolds.
Top-down phase-contrast images of pores at 3, 7, 10, and 14 days following cell seeding reveal fibrous matrix deposition over time. Scale bar = 40 μm.
Fig 5.
Fibroblast infiltration into scaffolds with or without 160 μm pores.
Fibroblasts were pre-loaded with red nanocrystals and then seeded onto: (A) standard electrospun 70:30 col/PCL scaffolds (lacking micropores), or (B) 70:30 col/PCL scaffolds with introduced 160 μm micropores. After 10 days of cell culture, scaffolds were OCT-embedded, cross-sectioned, labeled with Hoescht, and imaged. Fibroblasts can be seen migrating into the micropores.
Fig 6.
Collagen and fibronectin are deposited into 160 μm pores within scaffolds.
(A) A picrosirius red stain was used to detect fibrillar collagen in 70:30 col/PCL scaffolds. Scale bar = 40 μm. (B) Immunoblotting for collagen I and fibronectin was performed on homogenates prepared from microporous scaffolds with adherent fibroblasts grown for 1, 7, and 14 days. The no cell negative control was prepared from scaffolds that were not seeded with fibroblasts.
Fig 7.
Images of skin wounds implanted with cell-seeded or acellular microporous scaffolds over a 21-day interval.
Four full-thickness defects were created in the backskin of each rat, with 5 rats examined per time point. One wound was implanted with microporous scaffolds pre-seeded with fibroblasts for 4 days (4D), another was implanted with microporous scaffolds pre-seeded with fibroblasts for 1 day (1D), a third was implanted with an acellular microporous scaffold (acellular), and the final wound was covered with gauze only (no implant, “Sham”). Representative images are shown for each time point.
Fig 8.
Pre-seeded scaffolds promote more effective tissue regeneration.
(A) A cross-section of rat skin tissue undergoing the wound-healing process. Black dashed lines designate junctions between abnormal tissue and normal skin morphology. Scale bar = 40 μm. (B) Graph depicts average abnormal tissue areas (n = 5 rats per group) of harvested tissues containing porous scaffolds seeded for 4 days with fibroblasts (4d), porous scaffolds seeded for 1 day (1d), acellular porous scaffolds, and sham wounds. 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. Also, all treatment groups significantly decreased over time (p<0.001).
Fig 9.
Scaffolds pre-seeded with fibroblasts promote formation of ECM with a high degree of basket-weave structure, resembling unwounded skin tissue.
(A) 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) 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 (n = 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).
Fig 10.
Images (10X) of wound healing over a 21-day time period show the structure of the matrix in each treatment group.
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.