Figure 1
From: Atelocollagen promotes chondrogenic differentiation of human adipose-derived mesenchymal stem cells
Mixture materials and cell distribution, viability and proliferation. (A) Observation of mixed materials. Only Fibrin (a, * indication), thrombin (b, * indication), and the mixture of fibrin and thrombin (c, * indication). The fibrin, thrombin, and atelocollagen mixtures were observed with the substances expected of collagen under a microscope (d, ** indication). Scale bar; 200 µm. (B) Cell and materials distribution. Gel beads made with two different mixture ratios: 2 × 106 cells/0.8 mL hMSCs mixed with 0.2 mL thrombin in one syringe and 1 mL fibrin in the other syringe (a and b); and 2 × 106 cells/0.8 mL hMSCs mixed with 0.2 mL thrombin in one syringe and 0.2 mL atelocollagen mixed with 0.8 mL fibrin in the other syringe (c and d). hMSCs mixed well with materials and were well distributed (a–d, black arrow). Beads maintained their round shape well after mixture bead formation. Atelocollagen was observed (c and d, * black indication). Scale bar; 200 µm. (C) Biochemical staining of cell viability in mixture beads. Fluorescent dye staining after mixing for control I group (mixture of fibrin, hMSCs, and thrombin cultured in basal medium), control II group (mixture of fibrin, hMSCs, and thrombin cultured in chondrogenic differentiation medium), and the atelocollagen group (mixture of fibrin, atelocollagen, hMSCs, and thrombin cultured in chondrogenic differentiation medium). Viable cells were green, and dead cells were red when they were stained with calcein acetoxymethyl ester (calcein-AM) and ethidium homodimer-1 (EthD-1). These beads showed stable cell viability in all groups during culture. Especially, as cell morphology maintained a round shape in the atelocollagen group after 21 days of culture (l, white arrow), but not in other groups. Scale bar; 200 µm. (D) Cell proliferation and viability. Cells counts after all group bead digestion were measured using type I collagenase on days 0, 7, 14, and 21. After day 0 and 21 of culture period, the number of cells increased from 3.837 ± 0.283 × 104 to 3.142 ± 0.682 × 105 in control I group, 3.784 ± 0.207 × 104 to 3.12 ± 0.494 × 105 in control II group and 3.758 ± 0.217 × 104 to 3.002 ± 0.223 × 105 in the atelocollagen group (average ± SD) (Fig. 1D, blue color; **P < 0.01, red and green color; ***P < 0.001). Cell viability of all groups was stable on day 21 with 94.8 ± 2.326% in control I group, 94.83 ± 2.926% in control II group and 94.5 ± 2.152% in the atelocollagen group (average ± SD%). There was no significant difference in the comparison of all groups (#P > 0.05).
