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www.nature.com/scientificreports/ Figure 4. Effects of pulsed LED light on 3D cell proliferation and vasculogenesis in 1:0.01 co-cultures of HUVEC with ASC. (A,B) Representative images of LED stimulated fibrin clots containing GFP-HUVEC/ ASC co-cultures in a ratio of 1:0.01 after 4 days (A) and 7 days (B) of culture. (C) An increase of the area occupied by cells was determined in all light treated groups which reached significance with red and green light. (D) A trend to enhanced proliferation determined as occupied area was still observable after 7 days with red light being more potent. This effect, however, was not significant € The form factor of GFP-HUVEC was significantly reduced in all LED light stimulated groups, indicating increased cell stretching necessary to form cell-cell interactions. (F) After 7 days cell elongation was detectable in cells treated with green and red light. (G) Quantification of cellular junctions in 1:1 HUVEC/ASC co-cultures after one week showed a trend to enhanced network formation with red light treatment and a significant influence of green light. *P < 0.05, **P < 0.01. away from the beads, this number significantly increased in the green light group to 47.8 ± 19.9 cells (P = 0.047) and to 53.2 ± 7.1 cells in the red group (P = 0.0041). Blue light showed no effect and was in the same range as the control group. Effects of LLLT on endothelial cells embedded in 3D fibrin matrices. The effects of LED light of different wavelengths on endothelial cells placed on an extracellular matrix were tested using the Matrigel assay. Neither wavelength did have a significant effect on the formation of primitive endothelial networks (Supp. Figure 1). Since this assay is restricted to vasculogenic influences within 24 h we proceeded with our well-established 3D co-culture model of GFP-HUVEC and human ASC in a fibrin matrix (1:0.01 HUVEC/ASC seeding ratio). Figure 4 shows representative images of stimulated 3D co-cultures after 4 days (Fig. 4A) and on day 7 (Fig. 4B). At the 1:0.01 ratio no mature network formation was detected within 7 days. However, cells in the LED-stimulated groups showed increased cell elongation indicating enhanced activity to form cell-cell interac- tions. Quantification of the occupied area confirmed also in this co-culture model that stimulation with green and red light increased GFP-HUVEC proliferation (Fig. 4C). The area occupied by cells was significantly enhanced by red (~40%) and green (~45%) light treatment. The quantification in Fig. 4E represents form factor changes of SCientifiC REpORTS | 7: 10700 | DOI:10.1038/s41598-017-11061-y 4PDF Image | impact of wavelengths of LED light-therapy on endothelial cells
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