2.1.

Cell Culture

U2OS human osteosarcoma cells were obtained from the American Type Tissue Culture Collection (ATCC, Rockville, Maryland). Cells were grown in T-75 flasks (Corning, Fisher, Pittsburgh, Pennsylvania) in a 37°C, 5% ${\mathrm{CO}}_2/95\%$ air incubator using Dulbecco’s modified Eagle’s medium and supplemented with 10% fetal bovine serum (Mediatach, Manassas, Virginia). Cells were trypsinized with TrypLE (Invitrogen Life Technologies, Carlsbad, California), seeded into 6-well or 96-well plates (Corning, Fisher) and grown to confluence.

2.2.

Light Irradiation Devices

The light sources and parameters of delivery were previously described.²⁰ Briefly two light sources were used, a laser (Biolitec AG, Jena, Germany) and a light-emitting diode (LED) array (Biophotas Inc., Tustin, California) with the following parameters: Laser 652 and 806 nm with a full bandwidth of 5 nm; LED 637 and 901 nm with full bandwidths of 17 and 69 nm, respectively. The duration of treatment time for both sources was 1800 s with a power density of 5.57 and $1.30\mathrm{mW}/{\mathrm{cm}}^2$ for the red and near infrared ranges, respectively, and with corresponding energy densities of 10.02 and 2.334. The spot diameter from the laser source was 12.5 cm. The laser and LED power were measured with a FieldMate laser power meter (Coherent, Santa Clara, California). All samples were kept under low level light or in the dark before and after irradiation.

2.3.

Heat Measurements

Cell culture temperature was measured using a RAYNGER MX4 (Raytek, Santa Cruz, California) infrared-detecting thermometer/laser temperature gun as previously described.²⁰ A fan was used to eliminate heat transfer from the LED circuitry. The cell culture temperature was measured before and after treatment.

2.4.

Pharmacological Treatments

Cells were treated with the following compounds: $50\text{-}\mu \mathrm{M}$ sodium azide, $100\text{-}\mu \mathrm{M}$ sodium cyanide (Sigma-Aldrich, St. Louis, Missouri), $5\text{-}\mu \mathrm{M}$ U1026 (Promega, Madison, Wisconsin) and $5\text{-}\mu \mathrm{M}$ NO-Cbi.

2.5.

Combined Pharmacological and Light Treatment

Cells were exposed to LLLT for 30 min and 1 h later received a single treatment of $5\text{-}\mu \mathrm{M}$ NO-Cbi.

2.6.

Preparation of NO-Cbi

Cobinamide was produced by base hydrolysis of hydroxocobalamin (Sigma-Aldrich) as described previously.²¹ The cobalt was reduced from a $+3$ to $+2$ valency state using a two-molar excess of ascorbic acid in a deoxygenated solution. NO gas (99.99% pure, Matheson Gas Co.) was bubbled through the reduced cobinamide solution, and excess NO was removed by bubbling argon through the solution.²² Concentrated stock solutions of NO-Cbi were diluted in deoxygenated sterile water, and the diluted NO-Cbi was added to the cells using a Hamilton syringe (Hamilton, Reno, Nevada).

2.7.

Scratch Wound Closure Assay

The scratch wound closure assay has been previously described.¹⁹ Briefly 600 to $700\text{-}\mu \mathrm{m}$-wide single-line scratches were mechanically generated in a cell monolayer by a $200\text{-}\mu \mathrm{l}$ plastic tip ($\sim 450$ to $600\mu \mathrm{m}$ at the tip) across the length of the well. Multiple images were acquired along the length of the scratch to ensure representative sampling areas for each gap. Cells from both edges of the wound migrated into the open area, which was measured at 24 h. The change in area was quantified using a custom MATLAB script, which blurred the image and subtracted the blurred image from the original image. Smooth regions (the scratch wound) have less of a difference between the blurred and original images, thus generating a clear wound boundary. Once the boundary was determined pseudocolor was added by the custom script, which mapped the gap of the scratch wound. Images with similar intensities can be quantified using the same threshold and blur-block parameters; however, these may be adjusted for images with varying intensities. This method allowed for very accurate measurement of wound areas. The wound closure rate was determined by plotting changes in the wound area as a function of time.

2.8.

MTS Cell Proliferation

U2OS cells were seeded at $2\times {10}^4$ per well in a 96-well plate format and incubated overnight to permit cell attachment. At 24-h post-treatment ($\mathrm{LLLT}\pm \mathrm{NO}\text{-}\mathrm{Cbi}$) cell proliferation was determined using an MTS [3(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] proliferation assay (CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay; Promega, Madison, Wisconsin) according to the manufacturer’s instructions. The MTS dye was added directly to the cell culture media and incubated with the cells for 2 h at 37°C. The dye absorbance at 490 nm was measured and plotted for all treatment conditions.

2.9.

Microscopy

Phase contrast images were captured through a $10\times$ magnification Ph1 na (numerical aperture) 0.25 objective (Zeiss, Jena, Germany). An inverted microscope (Axiovert 135, Zeiss) with a charge-coupled device (CCD) camera ($\mathrm{ORCA}\text{-}{\mathrm{R}}^2$ Hamamatsu, Bridgewater, New Jersey) was used. Images were acquired using custom Robolase II software.²³

2.10.

Cellular ROS Detection

About $2.0\times {10}^4$ cells were seeded into each well of a black 96-well plate and allowed to attach overnight. Cells were loaded with $25\text{-}\mu \mathrm{M}$ cell permeant reagent (2’,7’-dichlorofluorescein diacetate, DCFDA, Abcam, Cambridge, Massachusetts) for 45 min. This compound detects hydrogen peroxide, superoxide, and other ROS species.²⁴ Cells were then washed and treated with LLLT. ROS levels were measured immediately after LLLT treatment and detected using a plate reader with a 485-nm excitation and 535-nm emission filter set.

2.11.

ATP Measurement

Cells treated as described above were exposed to LLLT. Cells were extracted immediately after LLLT treatment, and the ATP concentration in the extracts was measured according to manufacturer’s instructions using the ATPLite kit (PerkinElmer Life Sciences, Boston, Massachusetts).

2.12.

Oxygen Consumption

A total of $1.0\times {10}^6$ intact live cells were loaded into the chamber of an Oxytherm System type Clark electrode (Hansatech Instruments, Pentney, England) as a suspension after trypsinzation. Oxygen consumption rates were allowed to stabilize for 5 min prior to data acquisition by Oxygraph Plus software (Hansatech Instruments, Pentney, England). LLLT was applied to the sample while in the chamber. No respiratory substrates were added to the sample mixture as oxygen levels were measured from live cells. Oxygen consumption was measured in live cells in real time for 5 min. The rate of oxygen consumption was determined using the software curve fitting function.

2.13.

Cyanide Analysis

A concentration of $100\text{-}\mu \mathrm{M}$ NaCN dissolved in 0.1-M NaOH was administered to cell cultures in a confluent 6-well dish followed by either immediate LLLT, or no treatment. After 30 min, the culture media was harvested by aspiration, and the cells were washed three times in PBS before being extracted in situ using 1 ml of RIPA Lysis and Extraction buffer (Thermo Scientific, Rockford, Illinois). A volume of $500\mu l$ of cell extract or media was added to the outer well of a Conway microdiffusion cell and $500\mu \mathrm{l}$ of 10% trichloroacetic acid (TCA) was added to the opposite side of the same well to prevent premature mixing. Next a volume of $250\mu \mathrm{l}$ of ice-cold $30\text{-}\mu \mathrm{M}$ cobinamide (Cbi) in 0.1-M NaOH was added to the inner well.²¹ Once the cells were capped, the TCA was mixed with the sample to convert ${\mathrm{CN}}^{-}$ to HCN, which is volatile. The Conway cells were incubated for 25 min at room temperature to allow for the cyanide to diffuse to the center well and bind to Cbi. The solution in the center well was collected, and absorbance at 366 nm was measured using a spectrophotometer. Cyanide concentrations of the samples were determined from a standard curve.

2.14.

Western Blots

Cells were serum starved overnight, lysed and harvested 8 min after treatment in Laemmli buffer heated to 100°C (BioRad, Hercules, California), and lysates were sonicated and resolved on SDS-PAGE gels. The proteins were transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh) and blocked for 1 h with Pierce Protein-Free Blocking buffer (Thermo Scientific). They were incubated overnight with antibodies for phospho-ERK1/2(Thr202/Tyr203) and total ERK1/2 at 1:10,000 in TBS/5% BSA (Cell Signaling Technology). Membranes were then incubated with an anti-rabbit-HRP antibody at $1:2000$ for 1 h and developed using a luminol-based chemiluminescent substrate. pERK1/2 and ERK intensity for each condition was quantified separately using ImageJ²⁵ and then normalized respectively to total ERK1/2 and the untreated control.

2.15.

Statistical Analysis

Data are presented as $\text{mean}\pm \text{standard error of the mean}(\mathrm{SEM})$. Student’s t-tests were used for experiments that contained only two variables, and one-way analysis of variance followed by Bonferroni’s post-test was used for experiments containing three or more variables. A $P$ value $<0.05$ was considered statistically significant. All experiments have been repeated at least three times with the technical replicate number indicated for each dataset. The presented data are an average of all replicates resulting from at least three separate experiments.

3.1.

Combination of NO-Cbi and LLLT Induces Wound Healing Much More Effectively Than Either Treatment Alone

As we showed previously, NO-Cbi and LLLT enhance wound healing in cellular models [Figs. 1(a) and 1(b)].^19,20 We now show that combining NO-Cbi and LLLT markedly enhanced wound healing compared to either treatment alone [Figs. 1(a) and 1(b)]. This effect was observed for all LLLT wavelengths tested and for light delivered by a laser or LEDs. No statistically significant difference in cell proliferation was observed for all conditions tested compared to the untreated control [Fig. 1(c)].

Fig. 1

Wound healing induced by low level light therapy (LLLT) and NO-Cbi. (a and b) U2OS cells received a mechanical scratch wound followed by treatment with LLLT, $5\mu \mathrm{M}$ NO-Cbi, or the combination of LLLT and NO-Cbi. Wound recovery was followed for a period of 24 h at which time the final wound area was determined and normalized to the initial wound size. The images being shown are at 24-h post-treatment. The normalized average wound area was determined by dividing the final wound area by the initial quantified wound area and averaging the replicates. The green area marks the wound. Bar, $100\mu \mathrm{m}$. (c) The MTS proliferation assay showed no statistically significant differences between the untreated control. Data represent the $\text{mean}\pm \text{standard error}$ of the mean (SEM) of at least three separate experiments with $n=12$ (a and b) or $n=6$ for (c) per experiment. ****$P<0.0001$, ***$P<0.001$, and **$P<0.01$ compared to untreated control or indicated comparison.

3.2.

C-ox and Electron Transport are Required for LLLT to Enhance Wound Healing

We performed several sets of experiments to determine if the effects of LLLT were mediated through the electron transport system and a C-ox-dependent mechanism. First, we found that sodium azide, a C-ox inhibitor, abolished LLLT-enhanced wound healing [Figs. 2(a) and 2(b)]. Second, we observed increased oxygen consumption occurred almost immediately following LLLT (Table 1). Third, LLLT decreased ROS and increased ATP concentrations in U2OS cells [Figs. 3(a) and 3(b)], indicating increased electron flow through the electron transport chain. And fourth, we took advantage of the tight binding of cyanide to C-ox, and treated cells with nontoxic concentrations of cyanide, i.e., cyanide concentrations that did not reduce rates of wound healing, and then measured the intracellular cyanide concentration. We found that the cyanide concentration was significantly lower in extracts of LLLT-treated cells than in control cells (Table 2). These data suggest that cyanide bound to C-ox within the cell was released when the cells were exposed to LLLT, and that the cyanide diffused into the culture media. The predicted increase in the cyanide concentration in the media was extremely small ($\sim 5\mathrm{pmol}$), which is below the detection limit of the assay, and, indeed, we found no change in the media cyanide concentration (Table 2). We provide a more in-depth explanation of this observation in the discussion section.

Fig. 2

Sodium azide inhibits wound healing after LLLT Treatment. (a and b). A mechanical scratch wound was generated in U2OS cells, and the cells were treated with $50\text{-}\mu \mathrm{M}$ sodium azide, a cytochrome C oxidase inhibitor. The wound closure rate was quantified after 24 h. The images being shown are at 24-h post-treatment. The green area marks the wound. Bar, $100\mu \mathrm{m}$. Data represent the $\text{mean}\pm \mathrm{SEM}$ of at least three separate experiments with $n=12$ per experiment. ****$P<0.0001$ and ***$P<0.001$ compared to untreated control.

Table 1

Low level light therapy (LLLT) increases oxygen consumption rates. Oxygen consumption rates were measured in intact U2OS cells for 5 min, and then for an additional 5 min immediately following LLLT treatment. Data represent the mean±SEM of at least three separate experiments with n=3 per experiment. ****P<0.0001 compared to untreated control.

Fig. 3

LLLT reduces reactive oxygen species (ROS) and increases ATP concentrations. (a) U2OS cells were incubated with DCFDA dye and cellular ROS was measured as described in Sec. 2. (b) A cell extract was prepared immediately following LLLT treatment and ATP was measured using a luciferase-based assay. Data represent the $\text{mean}\pm \mathrm{SEM}$ of at least three separate experiments with $n=4$ per experiment. ****$P<0.0001$, ***$P<0.001$, and **$P<0.01$ compared to untreated control.

Table 2

LLLT causes cyanide disassociation from C-ox. U2OS cells were treated with 100-μM sodium cyanide, and then some cells were immediately exposed to LLLT for 30 min. Media and cell extract samples were harvested and cyanide concentration was determined. Data represent the mean±SEM of at least three separate experiments with n=2 per experiment. **P<0.01 compared to untreated control.

3.3.

NO-Cbi-Induced and LLLT Induce ERK Activation

As shown previously, we found that NO-Cbi increased ERK1/2 activation [Fig. 4(a)]. LLLT also increased ERK1/2 activation at all wavelengths tested [Fig. 4(a)]. The combination of NO-Cbi and LLLT yielded varying degrees of ERK1/2 activation compared to individual treatments; however, differences between conditions were minimal. The scratch migration assay was conducted in combination with specific inhibitors for mitogen-activated protein kinase/extracellular-signal-regulated kinase (MEK/ERK) and C-ox using U0126 and sodium azide respectively [Fig. 4(b)]. The laser 652-nm wavelength was selected to investigate the interplay between light and NO-Cbi, because strong activation of the ERK1/2 pathway was observed. The U0126 inhibitor prevented both the NO-Cbi and laser 652-nm enhancement of wound healing. Sodium azide only prevented laser 652 nm while having no effect on NO-Cbi. The beneficial effect from the combined treatment of NO-Cbi and laser 652 nm was decreased when either inhibitor was used. When added alone, neither U0126 or sodium azide, had an effect on wound healing in the absence of NO-Cbi, laser 652 nm or both combined. These data indicate that both individual and combined treatments of NO-Cbi and laser 652 nm require MEK/ERK1/2.

Fig. 4

Western blot and migration analysis of phospho-ERK1/2. (a) U2OS cells were treated with NO-Cbi, LLLT, or a combination of LLLT and NO-Cbi. ERK1/2 phosphorylation and total ERK1/2 expression were determined by western blot analysis (left). The intensity of pERK1/2 expression was quantified and then normalized to the total ERK1/2 expression and the untreated control (right). (b) A mechanical scratch wound was generated in U2OS cells, and the cells were treated with $50\text{-}\mu \mathrm{M}$ sodium azide, a C-ox inhibitor and $5\text{-}\mu \mathrm{M}$ U0126, an MEK-specific inhibitor. The wound closure rate was quantified after 24 h. Data are representative of at least three separate experiments for (a) and data represent the $\text{mean}\pm \mathrm{SEM}$ of at least three separate experiments with $n=12$ per experiment for (b). ****$P<0.0001$ and *$P<0.05$ compared to untreated control.