Photobiomodulation at 830 nm Stimulates Migration, Survival and Proliferation of Fibroblast Cells

Figures & data

Table 1 Laser Parameters

Variables	Current Study
Wavelength (nm)	830
Light source	Diode laser
Wave emission	Continuous wave
Spot size (cm^2)	9.1
Power output (mW)	105
Power density (mW/cm^2)	11.54
Irradiation time	7 min 10s
Energy density (J/cm^2)	5
Energy (J)	45.5

Table 2 Cell Migration Rate (%) in Normal Wounded (NW), Diabetic Wounded (DW) and Hypoxic Diabetic Wounded (HDW) Cells After Irradiation at 830 Nm with 5 J/cm² at 24, 48, and 72 h Compared with Their Corresponding Non‐irradiated (0 J/cm²) Wounded Models

Incubation Time After Irradiation
	24 h		48 h		72 h
Models	0 J/cm^2	5 J/cm^2	0 J/cm^2	5 J/cm^2	0 J/cm^2	5 J/cm^2
NW	26.8 ± 1	43.2 ± 3*	80.7 ± 2	84.1 ± 1	100	100
DW	19.0 ± 2	43.1 ± 3*	78.4 ± 1	100***	100	100
HDW	40.1 ± 4	44.2 ± 1	73.5 ± 2	100**	100	100

Note: *p < 0.05, **p < 0.01 and ***p < 0.001 (±SEM) represents statistical significance when compared to non-irradiated cells.

Figure 1 (A) Normal wounded (NW), (B) Diabetic wounded (DW) and (C) Hypoxic diabetic wounded (HDW) WS1 fibroblast cells at 0, 24, 48, and 72 h post‐irradiation at 830 nm with 5 J/cm² (magnification 200x, scale bar = 200 µm), non-irradiated (0 J/cm²) cells served as controls.

Figure 2 Cellular viability as evaluated by the CellTiter-Glo luminescent cell viability assay (ATP luminescence measured in relative light units, RLU).

Notes: Luminescence was recorded in irradiated (5 J/cm²) and non-irradiated (0 J/cm²) normal (N), normal wounded (NW), diabetic (D), diabetic wounded (DW) and hypoxic diabetic wounded (HDW) cells and analyzed 24, 48 and 72 h post-irradiation (830 nm). *p < 0.05 and **p < 0.01 (±SEM) represent statistical significance when compared to non-irradiated cells.

Figure 3 Cellular viability as assessed by the ApoTox-Glo triplex assay in WS1 cells.

Notes: Cell viability was measured in irradiated (5 J/cm²) and non-irradiated (0 J/cm²) normal (N), normal wounded (NW), diabetic (D), diabetic wounded (DW) and hypoxic diabetic wounded (HDW) cells and analyzed 24, 48 and 72 h post-irradiation (830 nm). *p < 0.05, **p < 0.01 and ***p < 0.001 (±SEM) represent statistical significance when compared to non-irradiated cells.

Figure 4 Cell cytotoxicity as assessed by the ApoTox-Glo triplex assay in WS1 cells.

Notes: Cytotoxicity was measured in irradiated (5 J/cm²) and non-irradiated (0 J/cm²) normal (N), normal wounded (NW), diabetic (D), diabetic wounded (DW) and hypoxic diabetic wounded (HDW) cells and analyzed 24, 48 and 72 h post-irradiation (830 nm). *p < 0.05, **p < 0.01 and ***p < 0.001 (±SEM) represent statistical significance when compared to non-irradiated cells.

Figure 5 Caspase 3/7 activation as assessed by the ApoTox-Glo triplex assay in WS1 cells (expressed in RLU).

Notes: Caspase 3/7 activation was measured in irradiated (5 J/cm²) and non-irradiated (0 J/cm²) normal (N), normal wounded (NW), diabetic (D), diabetic wounded (DW) and hypoxic diabetic wounded (HDW) cells and analyzed 24, 48 and 72 h post-irradiation (830 nm). *p < 0.05 and **p < 0.01 (±SEM) represent statistical significance when compared to non-irradiated cells.

Table 3 Cell Proliferation Was Determined by Flow Cytometry (% Cells in S, G0/G1 and G2/M Phase) in Non-Irradiated (0 J/cm²) and Irradiated (5 J/cm²) Normal (N), Normal Wounded (NW), Diabetic (D), Diabetic Wounded (DW) and Hypoxic Diabetic Wounded (HDW) Cells 24, 48 and 72 h Post-Irradiation at 830 Nm

	G0/G1 Phase		S Phase		G2/M Phase
24 h	0 J/cm^2	5 J/cm^2	0 J/cm^2	5 J/cm^2	0 J/cm^2	5 J/cm^2
N	70.3 ± 2	76.7 ± 5.2	7.9 ± 0.3	13.2 ± 1.9	5.7 ± 1.5	7.8 ± 1.5
NW	73.5 ± 5.6	74.4 ± 1.9	8.1 ± 0.3	9.7 ± 0.7*	11.4 ± 1.5	11.2 ± 2.2
D	72.4 ± 3.2	71.4 ± 3.2	10.7 ± 0.4	13.3 ± 0.5***	9.1 ± 0.2	8.0 ± 1.0
DW	68.6 ± 2.1	65.5 ± 2.1	13.1 ± 0.7	13.9 ± 0.9	9.8 ± 0.3	12.8 ± 0.4*
HDW	58.9 ± 1.7	61.5 ± 0.3	8.4 ± 0.6	10.8 ± 0.4	3.7 ± 1.1	3.4 ± 0.6
48 h
N	54.6 ± 4.7	46.5 ± 3.8*	16.0 ± 0.4	18.1 ± 1	4.8 ± 1.0	5.7 ± 1.1
NW	54.9 ± 3.6	54.3 ± 3.7	13.5 ± 0.5	16.7 ± 0.6**	4.5 ± 0.3	6.7 ± 1.2
D	50.8 ± 3.6	54.6 ± 4.5	10 ± 0.3	12.1 ± 0.6**	6.6 ± 1.2	4.9 ± 1.0**
DW	47.9 ± 4.4	50.4 ± 1.6	5.2 ± 0.4	7.6 ± 0.7**	6.9 ± 0.6	3.6 ± 1.0
HDW	50.8 ± 0.7	50.2 ± 1.7	4.2 ± 0.5	7.1 ± 0.9	4.6 ± 0.6	6.2 ± 0.5**
72 h
N	54.3 ± 2.3	61.6 ± 2.0**	20.4 ± 0.7	26.1 ± 1.7*	2.5 ± 0.5	9.3 ± 0.4**
NW	53.1 ± 1.5	53.7 ± 1.5	17.1 ± 0.3	18.1 ± 0.2	5.2 ± 0.5	6.7 ± 1.0
D	49.6 ± 3.7	53.2 ± 1.8	11.9 ± 1.0	15.5 ± 0.4	4.3 ± 0.5	7.5 ± 0.4*
DW	54.1± 3.1	52.0 ± 2.0	8.1 ± 0.2	9.3 ± 0.4	4 ± 0.6	2 ± 0.2
HDW	48.3 ± 0.8	53.7 ± 1.0	6.0 ± 0.1	7.8 ± 0.3**	1.7 ± 0.2	2.6 ± 0.5

Note: *p < 0.05, **p < 0.01 and ***p < 0.001 (±SEM) represents statistical significance when compared to non-irradiated cells.

Figure 6 Presence of TGF-β1 in culture media measured using ELISA in irradiated (5 J/cm²) and non-irradiated (0 J/cm²) normal (N), normal wounded (NW), diabetic (D), diabetic wounded (DW) and hypoxic diabetic wounded (HDW) cells and analyzed 24, 48 and 72 h post-irradiation (830 nm).

Note: *p < 0.05 and **p < 0.01 (±SEM) represent statistical significance when compared to non-irradiated cells.

Figure 7 Presence of pTGF-βR1 measured using ELISA in irradiated (5 J/cm²) and non-irradiated (0 J/cm²) normal (N), normal wounded (NW), diabetic (D), diabetic wounded (DW) and hypoxic diabetic wounded (HDW) cells and analyzed 24, 48 and 72 h post-irradiation (830 nm).

Notes: *p < 0.05, **p < 0.01 and ***p < 0.001 (±SEM) represent statistical significance when compared to non-irradiated cells.

Figure 8 Presence of p-Smad2/3 measured using ELISA in irradiated (5 J/cm²) and non-irradiated (0 J/cm²) normal (N), normal wounded (NW), diabetic (D), diabetic wounded (DW) and hypoxic diabetic wounded (HDW) cells and analyzed 24, 48 and 72 h post-irradiation (830 nm).

Note: *p < 0.05 and **p < 0.01 (±SEM) represent statistical significance when compared to non-irradiated cells.