Effect of different culture conditions on biological characteristics of fibroblasts derived from human hypertrophic scar

Objective To investigate the effect of different culture conditions on cell proliferation, apoptosis, collagen synthesis and TGF-β1 expression of fibroblasts derived from human hypertrophic scar(HSF). Methods Fibroblasts were isolated from human hypertrophic scar and cultured in vitro. Cells of passage 4 to 6 were cultured with 10%O2+ 100% culture medium for 48 h, and then divided into four groups, 10%O2+ 100% culture medium group (control group), 7%O2+ 70% culture medium group (experimental group 1), 4%O2+ 40% culture medium group (experimental group 2) and 1%O2+ 10% culture medium group (experimental group 3) according to the different culture conditions. There are 4 samples in each group. MTT assay, Flow cytometer and the Annexin V-FITC/PI double-staining were performed to detect the cell proliferation, S phase cell ratio and cell apoptosis. Hydroxyproline assay kit was adopted to detect the collagen synthesis of the fibroblasts. Protein expression levels of transforming growth factor β1 (TGF -β1)were determined with Western-blotting. SPSS 13.0 software was used to analyze the data. Analysis of variance ( oneway ANOVA) and SNK test were used to evaluate significant differences among these groups. Results ①Compared with control group, the cell proliferation rate was higher in the experimental group 1(P<0.05), while it significantly decreased in experimental group 3 (P<0.05). There was a statistically significant difference among groups (F=163.32, P<0.01). ②The collagen synthesis was much higher in experimental group 1 compared with control group (0.56±0.01 vs 0.42±0.01, P<0.05), and was lower in experimental group 3 (0.48±0.01 , P<0.05). There was a statistically significant difference among groups (F=357.69, P<0.01). ③The S phase cell ratio in the four groups were(19.40±0.37)%, (22.83±0.28)%, (20.94±0.38)% and(14.54±0.21)% respectively. Compared with control group, the S phase cell ratio was significantly higher in experimental group 1 and experimental group 2 (P<0.05), while it was significantly lower in experimental group 3(P<0.05). There was a statistically significant difference among groups (F=357.69, P<0.01). ④ The expression of TGF-β1 increased significantly in experimental group 1 and 2(P<0.05)compared with control group. However, experimental group 3 showed less TGF-β1 expression. There was a statistically significant difference among groups (F=357.69, P<0.01). ⑤The ratio of living cells in the four groups was (96.21±0.29)%, (96.61±0. 24)%, (96.61±0.17)% and(95.36±0.15)% respectively. There was no significant difference between control group and experimental group 1 and 2, while it was significantly decreased in experimental group 3 (P<0.05). There was a statistically significant difference among groups(F=12.38, P<0.01). Conclusions Mild hypoxia and low concentration of culture medium promote cell proliferation, collagen synthesis, TGF-β1 expression and cell viability of fibroblasts derived from hypertrophic scar. However, severe hypoxia and low concentration of culture medium decrease cell biological characteristics and promote cell apoptosis. Key words: Hypertrophic scar; Fibroblast; Hypoxia; Culture conditions