Limit lines indicate standard error (n = 20) for each bar. Significant reductions in the NFs with cells group was apparent at 12 weeks. Immunostaining for CD8 was performed (D). The thickness of the capsule was visualized using Masson trichrome–stained sections on nanofibers (NFs) (A) and NFs with cells (B) (original magnification ×40). F, G, and H, NF + C at 4, 8, and 12 weeks, respectively. C, D, and E, NFs at 4, 8, and 12 weeks, respectively. Hematoxylin-eosin–stained slides show little inflammation and cellular residence as well as host extracellular matrix (ECM). A slight offset was used to allow individual data points to be resolved. The bar indicates the median value, with individual data points shown by open circles, which are all integer numbers. Histological score of blood vessel penetration (bottom graph) (0 indicates sparse 1, on the periphery 2, scattered 3, well distributed and 4, very dense). Limit lines indicate standard error (n = 30). Cell seeding increased blood vessels early, but NFs without cells sustained blood vessels longer. B, Area (shown as a percentage) of blood vessels outside the implant border (top graph). Host-tissue deposition between the nanofiber (NF) layers was variable but not statistically significant ( P > .10 for all comparisons of NFs vs NFs + cells ). Limit lines indicate the standard deviation of the means of all the layers in 6 implants. Radiographic density became more punctate following implant.ī P < .05 with a connecting line shows the indicated comparison.Ī, Thickness of host tissue (arrows) was measured on cross sections of the implants (original magnification ×40). D, Renderings of thresholded reconstruction of implants from micro–computed tomographic scans before (left) and after (right) weeks in a subcutaneous pocket. At 4 weeks, 10 samples were available for all implants at 8 weeks, 8, 9, 9, and 8 samples were available for NFs, NFs with cells, human ECM, and porcine ECM, respectively and at 12 weeks, 10 samples were available for all implants. The seeding of cells did not have a significant effect until 12 weeks after implantation. The porcine extracellular matrix (ECM) was significantly more stable. Each implant was referenced to that individual implant prior to surgery. C, Percent change in volume at 4, 8, and 12 weeks of implantation based on micro–computed tomographic scan numerical evaluation. Calcein also reveals a spindlelike cell footprint on the nanofiber substrate compared with the TCPS. Notice in the overlay that calcein-stained cells were not stained by PI, showing the accuracy of the stains. Calcein AM is converted to the fluorescent calcein in the cytoplasm of viable cells, while propidium iodide (PI) stains the nuclear material in cells that do not have a patent cell membrane. B, Representative images of cell viability. Data represent the mean of at least 3 samples in each group at each time point. C indicates cells (in part A, C indicates concentration), d, distance between needle and collector ECM, extracellular matrix eGFP, enhanced green fluorescent protein f, flow rate H, human N (in part E) or NF, nanofiber P, porcine V +, positive applied voltage and WD, working distance.Ī, Quantification of cell (C) viability on nanofibers (NFs) vs tissue culture polystyrene (TCPS). Histologic specimens are shown with antibody (F) and without antibody (G). Fibroblasts were identified using P4HB antibody. Postexplant scanning (3) was completed after fixation in paraformaldehyde therefore, the samples were soaked in 70% ethanol at the second scanning, while implants for the first scan were soaked in aqueous media. Implants were scanned (1), implanted (2), explanted, scanned a final time (3), and processed for histologic analysis (4). Each animal received 1 of each of the 4 implant types in distinct subcutaneous pockets on the dorsum. The cell-preseeded scaffolds were compared with unseeded scaffolds and decellularized dermis as controls in a single rat (E). A cross section of a scaffold shown below. After cells were allowed to attach and culture on the fibers, the scaffold was folded and cut (D). Primary fibroblasts were harvested (C) and seeded on the scaffold in a monolayer. An example 10 × 10-cm prepared construct is pictured next to a ruler measuring about 16 cm long (B). The constructs were lifted from the collector and sterilized. Electrospun nanofibers made of polycaprolactone (PCL)/collagen (A) were imaged under a scanning electron microscope.
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