Photobiomodulation Effects of Single Near-Infrared (825 nm), Green (525 nm) and Combination of Wavelengths on Adipose-Derived Mesenchymal Stem Cell Physiology

Photobiomodulation (PBM) has been used as a bio stimulatory tool for adipose-derived mesenchymal stem cells (ADMSCs). The goal of this in vitro research was to examine the effects of combined and/or single applications of near infra-red (NIR) and green PBM using 5 J/cm 2 on ADMSCs. The results indicated that the viability of ADMSCs are not affected by single or combined wavelengths of 525 and 825 nm at 5 J/cm 2 . However, PBM signicantly stimulated cell metabolism seen by an increase in proliferation, it also upregulated intracellular ROS and MMP using 825 nm and 525 nm wavelengths. The combined wavelength irradiation mimicked results found for 825 nm during ATP measurement, cell concentration and migration rate, however signicant MMP stimulation and increased ROS production was achieved. In conclusion, results indicate that the combination wavelengths of 525 and 825 nm can be used in the expansion and differentiation of ADMSCs for regenerative purposes.


Introduction
The use of adipose-derived mesenchymal stem cells (ADMSCs) have become widespread as they can be easily harvested with less-invasive means, are abundantly available and can, furthermore be differentiated into many phenotypes [1][2][3] . Differentiation of ADMSCs can be triggered by the induction of biological growth factors or chemical inducers [4][5][6] . By this means, ADMSCs have a high differentiation potential in vitro into lineages that range from neuronal, osteogenic, chondrogenic, adipocyte, to myogenic 4 . For example, ADMSCs have been transdifferentiated into neuronal-like cells in media that contained inducers such as zinc chloride, broblast growth factor, nerve growth factor, forskolin, or 3isobutyl-1-methylxanthine [7][8][9][10] . Other studies have also shown that ADMSCs can be transdifferentiated to osteoblasts by introducing inducers such as ascorbate-2-phosphate, dexamethasone, and βglycerolphosphate 6 . Thus, ADMSCs can be applied to regenerative medicine in a vast number of ways, such as recovery from neurodegenerative diseases or osteoporosis 11,12 . Photobiomodulation (PBM) has become popular for its ability to either upregulate or downregulate biological processes at a molecular level depending on the laser parameters applied 13 . Parameters that are considered are wavelength, which can range from the visible to the near-infrared (NIR) spectrum of light at wavelengths between 400-1100 nm and uence or energy applied [14][15][16][17] . Where the effects of various laser uences are dose dependent. Fluences of 0.5-2 J/cm 2 has no signi cant effect on ADMSC proliferation 18 , 3-5 J/cm 2 has signi cant photobiomodulation 19 . Speci cally, photobiomodulation triggers chromophores in the mitochondria, stimulating the electron transport chain to generate more adenosine triphosphate (ATP) and increase mitochondrial membrane potential (MMP) 20,21 . Research showed that lower wavelengths, 451 and 540 nm, had no signi cant effects on cell proliferation, whereas higher wavelengths, 660 and 810 nm, upregulated proliferation of ADMSCs 14 . The use of green light between 450-580 nm has shown to stimulate differentiation of ADMSCs into osteoblasts 22,23 , where green light has been seen to stimulate various transcription factors involved in cell differentiation 24 .
Furthermore, the migration abilities of cells, important for homing, are also stimulated when exposed to NIR light 25 . However, the effect of combining upper and lower wavelengths remains to be explored. Therefore, the current study explored the effect of green and NIR laser light at 525 and 825 nm respectively, as well as the combination of the two wavelengths using 5 J/cm 2 . The effect on morphology, migration, proliferation, viability, cytotoxicity, intracellular ROS and MMP of the ADMSCs following photobiomodulation treatment was explored.

Biochemical Analysis
Morphology of ADMSCs treated with PBM Morphology results show a dense monolayer of ADMSCs that was relatively homogenous with a spindlelike shape and smooth cellular surface typically observed in healthy ADMSCs (Fig. 1). Experimental groups of ADMSCs that were exposed to laser irradiation at different wavelengths of 525 and 825 nm and uence of 5 J/cm 2 had no visible morphological modi cations in comparison to that of the control, they also showed a concentrated cell population. Of these experimental groups, the group treated with 825 nm NIR laser light was the densest 24 h, 48 h and 7 days post irradiation.

Cell viability and proliferation
To determine the effects of PBM on cell viability, we used the trypan blue dye exclusion assay. Viable cells inhibited penetration of the dye into the cells, indicative of their intact membrane, whereas cells with diminished membranes allowed for the dye to enter the cells. Results show ( Fig. 2A) that there were no signi cant effects on cell viability when using NIR, green and NIR-green irradiation at 5 J/cm 2 on ADMSCs. All cells maintained a high cell viability. However, a negligible decrease in viability is seen using green irradiation after 24 and 48h.
Along with establishing cellular viability the effects of various PBM wavelengths on cell proliferation kinetics was measured by means of cell concentration. Viable cell counts of treatment groups were compared to their respective controls. Results show (Fig. 2B) that 24 h after PBM treatment using NIR and the combination had a signi cant increase in cell number. All treatment groups were signi cantly higher than their control after 48 h. Combination treatment indicated a statistical signi cance after 7 days, whereas other treatments had no signi cance compared to the control.
To characterize the effects of different wavelengths of PBM on the proliferation of ADMSCs relative to its metabolism, we used the ATP Luminescence Assay. This test uses luciferase to generate a luminescent signal that is equal to the amount of ATP in the sample. The amount of ATP present was proportional to the proliferation rate of the cells, where an increase in ATP suggests an increase in mitochondrial stimulation and cell proliferation and vice versa. Cell proliferation was determined over a period post PBM treatment. Results show that NIR-PBM signi cantly increased the proliferation rate 24 and 48 h after irradiation (Fig. 2C). Green and combination wavelengths kept a slight upregulation in proliferation compared to the control cells after 24 h and a statistically signi cant increase is seen at 48 h and 7 days.
It was however seen that the cells had reached a stationary phase after 7 days post NIR treatment, due to cells reaching con uency in the growth environment 26 .

Reactive oxygen species (ROS) detection
Although physiologically up-regulated ROS production is essential for maintenance of stem cell activities, abnormally high levels of ROS can harm MSCs 27 . Therefore, the expression of ROS in the ADMSCs was determined in order to establish what effects the PBM at various wavelengths have on intracellular ROS production. It is seen that (Fig. 3) control cells receiving no treatment, representing normal healthy ADMSCs have slight ROS activity. The increase in ROS activity over time is due to the increase in cell number. Treatment groups compared to the control group show an increase in ROS levels when using NIR-irradiation, green-irradiation, and their combination. After 24 h NIR-irradiation increased ROS levels the most. At 48 h it was green-irradiation and after 7 days the combination wavelengths increased ROS levels the most. It should be mentioned that the increase in intracellular ROS did not have a detrimental effect on the cells where previous results show increased proliferation activity, and healthy viable cells.

Mitochondrial membrane potential ΔΨm
Light is primarily absorbed in the mitochondria in mammalian cells speci cally cytochrome c allowing for dissociation of nitric oxide from cytochrome c restoring electron transport and increasing mitochondrial membrane potential (MMP) 28 . In the following experiment, the effect of NIR 825 nm, Green 525 nm, and NIR-Green 825 + 525 nm laser light exposure on the MMP was measured by immuno uorescence microscopy 24 h, 48 h, and 7 days following irradiation (Fig. 4A). Results show that cells maintained their MMP over time after irradiation. With the cell mitochondria uorescing a bright red. Analysis (Fig. 4B) revealed that NIR-Green PBM signi cantly (P < 0.05) stimulated the MMP at 24 h, 48 h, and 7 days post irradiation when compared to the unirradiated control group. Although NIR and green PBM did upregulate MMP in comparison to the untreated control group, it was not signi cant.

Cell migration: 'central scratch test' method
Cell migration was calculated using the 'central scratch method', where a centralized scratch was created to examine cell motility through closing of the scratch. Cell migration was observed morphologically over time 0, 24, 48 h and 7 days as the cells close the central scratch (Fig. 5).
The ndings showed that all treatment groups had migrated signi cantly over time, compared to the untreated control cells, and that all treated samples travelled a similar distance (Fig. 6A). Irradiation using various wavelengths signi cantly promoted closure of the scratched area compared to cells not receiving irradiation treatment (Fig. 6B), where the combination treatment had the least amount of open area left.

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Various studies have explored the effects of PBM on ADMSCs. Studies included the use of single blue, green, red and NIR wavelengths. Results showed various outcomes depending on the laser wavelength and uences used, along with power output 14,25 and cell seeding density. The ndings from this study using combination wavelengths revealed that ADMSCs exposed to PBM formed a denser monolayer following incubation when compared to the control group that was not exposed to PBM. Speci cally, ADMSCs exposed to NIR 825 nm laser light displayed the densest monolayer formation 48 h and 7 days post irradiation. This is similar to Nurković et al that applied 808 nm laser light at 3 J/cm 2 and saw an increase in the number of cells when observing morphology 29 . Although cells treated with combination 525 + 825 nm wavelengths showed a visible increase in the monolayer, it was not as dense as that of cells exposed to NIR 825 nm laser light.
Cell viability was not affected using PBM of 5 J/cm 2 and wavelengths in the green, NIR and their combination wavelengths. All cells maintained a high viability rate, indicating that these PBM parameters do not have a negative effect on the cells in vitro. Nurković et al also found that NIR treatment did not signi cantly affect MSC viability by itself 29 . Similar ndings were observed in vivo where no signi cant increase/ decrease was noted after PBM treatment using 890 nm accounting for the increase in proliferation measured thereafter 30 . Similarly when using green irradiation, previous studies show that PBM at 470 nm 31 and at 405 nm 32 had no effect on cell viability. Therefore, the combination of 825 nm and 525 nm showed a persistent maintainable high viability insigni cant of the untreated controls.
We found that NIR-PBM has a positive effect on ADMSC metabolism showing a signi cant increase in cell proliferation when using an energy density of 5 J/cm 2 , corroborating results from previous studies using NIR-irradiation 14,29 . However the use of green light (525 nm) had shown maintainable proliferation after 24 h and a signi cant increase after 48 h, when using 5 J/cm 2 , this is similar to Anwer et al that saw an increase in proliferation following laser exposure of green PBM at 532 nm 33 . The signi cant increase in cell proliferation is due to the stimulated metabolic activity seen in the increase in ATP proliferation. This is in contrast to Wang et al that saw a dose dependent decrease in proliferation 14 and another study by Fekrazad et al that applied green light at 532 nm and saw an insigni cant decrease in proliferation 22 , these results can be attributed to the difference in laser power density and initial cell seeding density. The combination wavelengths used in this study however, showed a time dependent increase in cell proliferation with a signi cantly higher rate after 48 h and 7 days compared to their respective controls. Untreated ADMSCs maintained a small but steady upregulation in intracellular ROS over time, where ROS detection increases as cell numbers increase. However, treatment groups did show a slight increase compared to untreated controls in intracellular ROS at 24 h for NIR-PBM, at 48 h for green-PBM and at 7 days combination treatment. This indicates that PBM treatments maintained and upregulated ROS generation which can direct their stem cell fate and improve their therapeutic effect 27 . This is seen during various differentiation directions leading to various degrees of ROS production. Where adipogenic 34 , chondrogenic 35 , osteogenic 36 and neural 37 differentiation had all shown physiologically increased ROS levels.
It was observed that the MMP of the cells were maintained throughout the incubation period. Further investigation, through uorescent intensity measurement, combination wavelengths showed a signi cant increase in MMP 24 h, 48h, and 7 days post irradiation. NIR and green laser irradiation also showed an increase in uorescent intensity when compared to the control, albeit insigni cant. Interestingly, Wang et al found that NIR (810 nm) stimulated MMP, but green laser light (540 nm) inhibited the MMP 14  In conclusion the effects of using various single and combination PBM wavelengths for possible use in regenerative medicine when using ADMSCs as a cell source for in vitro proliferation and differentiation was evaluated. As discussed PBM using NIR irradiation showed signi cant bio stimulatory effects on the cells aiding in cell proliferation, viability and migration which can be applied prior to differentiation of the cells to achieve an abundant stem cell source. Furthermore, green light showed signi cant bio stimulation promoting cell proliferation, although not as signi cant as NIR-PBM. Notably the combination uses of NIR-and green light signi cantly promotes the induction of ADMSC differentiation through bio stimulation of intracellular ROS production and MMP. Concluding that using combination wavelengths of NIR and green PBM can aid in the proliferation, differentiation and possible maintenance of the cells differentiated state prior to terminal differentiation and con rmation of cell function. Furthermore, NIR light could be used to increase the cell yield of the terminally differentiated cells, where future recommendations should include the evaluation of the combination treatment in vivo in order to establish the effects on cell transplantation for regenerative purposes and cell homing.
The time exposure of each respective laser wavelength was calculated using the following formula:

Statistical Analysis
Biochemical assays were conducted in triplicate n = 3. Spectrophotometry studies were performed using a blank sample taken from the relevant data gathered. Statistical analyses were carried out on data sets, student's t-test was used to compare two groups and one-way ANOVA to compare all groups together with SigmaPlot program version 12. Data obtained from migration morphology is quantitatively analysed using ImageJ, a public Java domain image processing system (National Institute of Health Bethesda, MD USA). All data is expressed as a mean ± SE. The statistical signi cances were expressed by tables and graphs P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) and the standard error was de ned by dispersion bars.
Data availability and editing, AC, MJVR and HA; visualization, AC and MJVR; supervision, HA; project administration, AC, MJVR and HA; funding acquisition, AC, MJVR and HA. All authors have read and agreed to the nal version of the manuscript.