In-Vitro Wound Contraction in the Horse: Differences Between Body and Limb Wounds
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Cell cultures were incubated at 37 degrees C in a five-percent carbon dioxide air environment. Readiness for subculturing was determined by the extent of fibroblast cell outgrowth (5 to 10 days). Cells were farmed successively in a 1:4 split ratio to passage 3 to 8 times for experimental use.
For seeding in the collagen matrix, NF from the six sites and GT from granulating limb wounds were harvested from tissue culture flasks with trypsin-EDTA washed and resuspended with DMEM. The cells were counted and resuspended at 1 x 106 cells/mL in DMEM medium.
Preparation and testing of collagen gels. The cells were then resuspended into type I collagen (Fred Baker Scientific, United Kingdom) at 2mg/mL and plated into six well tissue culture plates. Six gels of 1mL were made for each cell type (n = 18). Gelation occurred within five minutes. The surface of the gels were washed with 2mL HBSS, and 2mL DMEM was added followed by incubation in a five-percent CO2 in air environment at 37?C.
Analysis of contraction. Contraction of collagen lattices was determined by averaging the longest and shortest diameter of each lattice measured by calipers. Calculation of the diameters represents the mean and standard deviation of 18 lattices (n = 18).
Cell viability. The viability of the fibroblasts was monitored using Trypan blue staining during the successive culture steps and the preparation of collagen gels. At the end of the experiment (96 hours), the cells were released from the collagen lattice by digestion of the collagen gel using 0.2-percent collagenase in cell culture medium for 10 minutes, resuspended in DMEM, and tested for viability. Viability was expressed as the percentage of viable cells remaining in the cell suspension.
Photomicroscopy. Photomicrographs were taken at 48, 72, and 96 hours after incubation in the collagen lattice using an Olympus CK2 inverted microscope with a photomicrograph attachment PM10-AK-2 and an Olympus C35 DA/2 camera. Kodak Ektachrome color slide film was used.
Statistical analysis. All of the results were expressed as the mean ± standard error (SEM). The statistical significance of the results was assessed using the Student’s t-test, and a P value of < 0.05 was considered to be significant.
Contraction morphology of NF cultured from different sites of the body. The cells were evenly dispersed throughout the collagen lattice, spherical in shape, and showed signs of spreading out after 24 hours. By 48 hours, the cells were beginning to elongate and acquire the typical morphology of fibroblasts. In general, at 72 hours the cells showed a marked change in morphology and assumed their typical bipolar spindle form with several long processes (Figure 2). It was evident that the cells cultured from the limb and abdomen were able to arrange themselves in a more organized pattern. The contracted gels at 96 hours revealed a regular arrangement of fibroblasts with more and longer processes (Figure 3).
In-situ morphological characteristics of GT and L929 cells in collagen lattices. Fibroblasts cultured from GT were spherical when seeded into the collagen lattice but within a few hours had rapidly elongated and become spindle shaped with long processes. In contrast, the L929 cells remained rounded for much longer (Figure 4) and did not show signs of elongating until 72 hours.
Collagen contraction mediated by normal fibroblasts. The fibroblasts from the limb, axilla, and abdomen started to contract sooner than the other cells, and at 24 hours, there is a significant difference between the limb and the other cell types (Figure 5). At 48 hours, however, cells from the other sites were contracting faster, and there was only a significant difference between the limb and the eyelid fibroblasts.
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