Does Phototherapy Promote Wound Healing? Limitations of Blue Light Irradiation

Introduction. Phototherapy is gaining increased attention in the research and treatment of various diseases. In particular, the use of blue light seems to bear promise, owing to its antimicrobial and immune-modulating properties; however, research focused on the effects of blue light on keratinocytes and reepithelization is rare. In addition, few studies to date have evaluated devices that are used in daily hospital routine. Objective. This study investigated the effects of phototherapy on keratinocytes with 2 established devices in vitro. Materials and Methods. Human adult low calcium high temperature keratinocytes were irradiated with 2 different devices, and the effects on scratch wound closure, proliferation, cell viability, and cytokine expression were evaluated. Results. Blue light irradiation reduced reepithelization at high doses in a scratch wound healing model (wound closure on day 1: control group, 25.57 percentage points [PP] ± 2.36 standard deviation vs Device A for 10 minutes, 1.33 PP ± 1.01) and mitochondrial activity measured with resazurin conversion (Device A for 10 minutes, 33.28% ± 12.34). Irradiated cells demonstrated a lower ratio of proliferating cell nuclear antigen-positive cells and, as a result, lower proliferation. Conclusions. Blue light reduces keratinocyte proliferation and migration at high doses and therefore could negatively affect wound healing. Available irradiation devices for possible use in wound therapy should be critically scrutinized and evaluated with in vitro methods prior to clinical use.

Introduction

In recent years, phototherapy has been an increasing focus of wound healing research.¹ Sufficient evidence supports the positive effects of light treatment of different wavelengths on wound healing for a range of tissues and wounds, such as venous and diabetic ulcers,^2,3ligaments,⁴ tendons,^5,6 bone,^7,8and cartilage.⁹

Blue light is part of the visible light spectrum and has a wavelength of approximately 380 nm to 450 nm. It appears to have immune-modulating/anti-inflammatory effects^10-12 and increases angiogenesis.¹³ It also has antimicrobial properties,¹⁴ as irradiation reduces bacteria such as Pseudomonas aeruginosa,^15,16 Propionibacterium acnes,¹⁷ Salmonella enterica,¹⁸ Staphylococcus aureus,¹⁶ and methicillin-resistant S aureus.^19-22 There is also investigation into the use of irradiation in the treatment of chronic inflammatory and hyperproliferative skin diseases such as psoriasis and atopic dermatitis.²³ The use of blue light improves hyperproliferative skin conditions by reducing proliferation and induction of differentiation.²⁴ This can result in improved pruritus, sleep, and quality of life for patients with severe atopic dermatitis as well as reduced frequency and intensity of disease exacerbations and reduced use of topical corticosteroids.²⁵ Irradiation with Device A (DermoDyne; DermoDyne HealthCare) has been shown to induce significant clinical improvement in atopic eczema of the hand and foot when used thrice-weekly for 30 minutes at a time for a duration of 4 weeks.¹² Treatment with Device B (EmoLED; Emoled Srl) has resulted in an improved healing process in superficial skin wounds after irradiation as well as reduced inflammatory response, higher collagen content, and better-recovered skin morphology.^26,27

These findings suggest that blue light irradiation may be a promising alternative wound healing therapy, especially for wound infections and chronic inflammatory skin diseases, with only minor side effects. Of note, short-term irradiation with visible blue light in dermatologic practice is considered to be safe.¹¹

Although several studies indicate positive effects of blue light irradiation in vivo, little data exist about the effects of blue light on keratinocytes and reepithelization., Additionally, only few studies have addressed the effects on keratinocytes with devices that are used in daily hospital or clinical practice. The present study aimed to investigate the effect of blue light irradiation on reepithelization, proliferation, cell viability, and cytokine expression after irradiation with devices that are already in use in daily clinical practice. Because the effects of in vivo wound healing are difficult to quantify as a result of various confounding factors (eg, age, perfusion, bacterial contamination), the authors wanted measure solely the effects of blue light irradiation on keratinocytes in vitro. To this end, the authors investigated the rate of wound healing in vitro using a scratch assay wound-healing model, and cell viability and cytokine release following irradiation with Device A and Device B were examined.

The aim of this study was to investigate the effects of blue light irradiation on keratinocytes in vitro with devices that are used in daily hospital routine.

Materials and Methods

Cell culture

Cell culture is the process by which cells are grown outside their natural environment to investigate the effect of an intervention under controlled conditions. Because the goal was to study wound healing effects, human adult low calcium high temperature keratinocyte (HaCaT) cells were used. This cell line has been used several times for this purpose. The HaCaT cells were grown in 175 cm² cell culture flasks in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 1% penicillin/streptomycin, and phenol red at 37°C and 5% CO2. When the cells became confluent, all medium was removed; cells were washed twice with phosphate-buffered saline (PBS) without Ca2+ and then detached using 0.25% trypsin in buffered ethylenediaminetetraacetic acid. Detachment was stopped with DMEM supplemented with 5% FBS, 1% penicillin/streptomycin, and phenol red. Following centrifugation and resuspension, cells were counted in a Neubauer chamber before plating.

Blue light irradiation

For irradiation therapy, Device A and Device B were used.

Device A is an ultraviolet-free irradiation device used to treat skin disorders such as atopic dermatitis, psoriasis, eczema, and alopecia areata.^12,25 It emits blue light impulses at a wavelength of 400 nm to 450 nm (28.9 J/cm²) at a glass surface on the top of the device, onto which cell culture plates are placed. Because the device also releases low heat, well plates are placed 2 cm from the light source. Treatment groups underwent blue light irradiation for 1 minute, 5 minutes, or 10 minutes.

Device B emits blue light at a wavelength of 400 nm to 450 nm.²⁸ The irradiation program lasts for 1 minute (120 mW/cm²), whereby the distance to the irradiation area of 4 cm is controlled by distance measurement. This device has been successfully used in previous studies.^26,27,29 According to the manufacturer, each Device B application has a duration of 60 seconds and is performed on a circular area with a diameter of 50 mm. After 60 seconds, the irradiation stops automatically. Longer treatment was not intended by the manufacturer, so longer irradiation applications were not performed in this study.

Nonirradiated HaCaT cells served as the control group.

Scratch assay

The scratch assay is a well-established and reproducible technique that is commonly used to investigate cell proliferation and migration.³⁰ For the purposes of the present study 150 000 cells were seeded in 24-well plates with 0.5 mL DMEM supplemented with 5% FBS, 1% penicillin/streptomycin, and phenol red. After 3 days of incubation, when cells had reached complete confluence, the cell monolayer was wounded using a sterile blue (100 µL–1000 µL) pipette tip, creating a straight scratch line in the cell monolayer. Afterward, cells were washed several times with PBS until all remaining cell debris was removed. Finally, 0.5 mL of new DMEM supplemented with 5% FBS, 1% penicillin/streptomycin, and phenol red was added, and the scratch was photographed at ×4 magnification (t0 photo). Cells then were irradiated as previously described. As there was no additional medium change after this step, cytokines and growth factors that may have been released by the irradiated cells were not removed with the supernatant. After 24 hours (t24 photo) and 48 hours (t48 photo), additional images were captured of the same view frame (Figure 1A). The size of the wound (percentage of cell-free/uncovered area in the image as a whole) in these images was determined by TScratch, an open-source computer program designed for automated analysis of monolayer wound healing assays.³¹ Wound closure was calculated per the Formula for each day following the start of the study.

The supernatant was collected for cytokine enzyme-linked immunosorbent assay (ELISA) analysis.

Resazurin conversion

The assay is based on the ability of living cells to convert a redox dye (resazurin) into an end product (resorufin) that both emits fluorescence and undergoes a colorimetric change. The assay is commonly used to evaluate cell viability based on this measurement of mitochondrial activity.

For resazurin conversion, 20 000 cells were seeded in 96-well plates with 100 µL DMEM supplemented with 5% FBS, 1% penicillin/streptomycin, and phenol red and were incubated for 3 days. Then the cell culture medium was changed, and blue light irradiation was performed as previously described. After 48 hours of incubation, 10 µL of a 0.025% (w/v) resazurin solution (in PBS) was added to the cells. After 30 minutes of incubation at 37°C, fluorescence intensity of the formed resorufin was measured (excitation/emission equals 540 nm/590 nm) and corrected to background. The control group was set as a reference.

Sulforhodamine B staining

The sulforhodamine B (SRB) assay is an established technique developed by Skehan et al³² as a cytotoxicity assay for anticancer drug screening. Because SRB binds to surface proteins under acidic conditions, it can be used to assess total protein content and, thus, cell viability.

For SRB staining, 20 000 cells were seeded in 96-well plates with 100 µL DMEM supplemented with 5% FBS, 1% penicillin/streptomycin, and phenol red and incubated for 3 days. The cell culture medium then was changed, and blue light irradiation was performed as previously described. The cells were incubated for 48 hours and then fixed with ethanol. All well plates were then stored at −20°C for further investigations. The ethanol was removed. The cells were washed once with tap water, and the plates were air-dried in a temperature-controlled room (room temperature, 21°C ± 1 standard deviation (SD); relative humidity, 36%–42%; no additional warming). Cells were covered with 50 µL SRB solution (4% SRB with acetic acid) and incubated while protected from light. After 30 minutes of incubation, SRB solution was removed. The remaining unbound SRB was washed 4 times with acetic acid solution until fully removed. The bound SRB was resolved with 100 µL 10 mM unbuffered tris(hydroxymethyl)aminomethane solution. Finally, absorbance was measured at wavelength (ƛ) of 565 nm (SRB) and ƛ of 690 nm (impurities), and optical density (OD) 690 nm was subtracted from OD 565 nm.

The control group was designated as a reference.

Proliferating cell nuclear antigen immunostaining

Proliferating cell nuclear antigen (PCNA) is a key component of the DNA replication system and is involved in the process of DNA elongation, recombination, methylation, and repair³³ and thus can be used to determine cells in S phase. The dye Hoechst 33342 instead binds to DNA, and this dye is commonly used to stain the nucleolus.³⁴

For PCNA immunostaining, 20 000 cells were seeded in 96-well plates with 100 µL DMEM supplemented with 5% FBS, 1% penicillin/streptomycin, and phenol red and incubated for 3 days. The cell culture medium then was changed, and blue light irradiation was performed as previously described. After 48 hours, cells were fixed with 100 µL 4% formaldehyde for 10 minutes and then washed with PBS and stored at 4°C for further investigation. The cells then were permeabilized with 100 µL 0.2% Triton X-100 for 20 minutes and washed again with PBS, after which 100 µL bovine serum albumin 5% was added to each well and left for 1 hour. After 1 hour of incubation, cells were washed again with PBS, after which 50 µL anti-PCNA antibody (PCNA-AB ab92522 [Abcam], 1:200 in PBS) was added and the cells incubated overnight. The next day, cells were again washed with PBS and 50 µL 1:500 second antibody (mouse anti-rabbit IgG-HRP sc-2357 [Santa Cruz Biotech], 1:500)/Hoechst 33342 (1:1000) was added and incubated for 2 hours. Next, cells were washed with PBS and cells were photographed with ×4 magnification in triplicates. Hoechst-positive and PCNA-positive cells were counted with ImageJ (National Institute of Health) and the ratio of PCNA positive cells was calculated (PCNApositive/Hoechstpositive) to determine the ratio of cells in S phase after irradiation.

Live/dead staining

Calcein acetoxymethyl ester (calcein-AM) is a cell-permeable, non-fluorescent dye that is converted into green fluorescent calcein by esterases in the cell cytoplasm. Ethidium homodimer-1 can enter only cells with damaged membranes. After having intercalated into DNA, it produces a bright red fluorescent signal. Thus, living cells are stained in green and dead cells in red.³⁵

For live/dead staining, 20 000 cells were seeded in 96-well plates with 100 µL DMEM supplemented with 5% FBS, 1% penicillin/streptomycin, and phenol red and incubated for 3 days. The cell culture medium then was changed, and blue light irradiation was performed as previously described. After 48 hours, the staining solution (1:1000 calcein-AM, 1:1000 Hoechst, and 1:1000 ethidium bromide in 100 µL PBS) was pipetted on the cells and incubated in the dark for 30 minutes. Next, cells were washed with PBS and images were obtained at ×20 magnification.

Cytokine ELISAs

Keratinocytes produce a variety of cytokines.³⁶ Therefore, the authors investigated cytokine concentration after irradiation in the remaining supernatants of the scratch assay. The cytokine Standard ABTS ELISA Development Kits (interferon [IFN]-γ, tumor necrosis factor α [TNF- α], interleukin [IL]-6) were purchased from PeproTech.

Statistics

All data are given as mean plus or minus SD. Data were analyzed using the Kruskal-Wallis test with the Dunn multiple comparison test to analyze differences between groups. All experiments were repeated four times (n = 4).

All analyses were performed using the Prism statistical software package (version 6; GraphPad Software).

Results

Scratch assay

Blue light irradiation reduced reepithelization (proliferation and migration) on avergae in all irradiated groups (Figure 1). The control group showed wound closure of 25.57 PP ± 2.36 on day 1 (Figure 1B). Irradiation with Device A reduced reepithelization in a time-dependent and dose-dependent manner (1 min, 20.51 PP ± 3.88; 5 minutes, 7.86 PP ± 5.95; 10 minutes, 1.33 PP ± 1.01), whereas values in the 5-minute and the 10-minute groups were significantly lower compared with the control group (P ≤ .05 and P ≤ .001, respectively). Reepithelization in the Device B group (20.61 PP ± 1.87) was comparable to the corresponding Device A group at an irradiation time of 1 minute.

Similar results were obtained on day 2 after irradiation (Figure 1C). Faster wound closure was achieved in the control group (10.67 PP ± 1.44) than in the Device A group (1 min, 10.59 PP ± 2.64; 5 minutes, 4.17 PP ± 2.23; 10 minutes, 1.42 PP ± 0.83) and in the Device B (11.16 PP ± 1.74) irradiated HaCaT cells. Once again, the group irradiated for 10 minutes using Device A exhibited significantly lower wound closure compared with the control group (P ≤ .05).

Cell viability

Blue light irradiation with Device A (Figure 2A) reduced resazurin conversion by mean compared with the control group (100% ± 4.76) in a dose-dependent manner (1 min, 72.24% ± 25.84; 5 minutes, 63.94% ± 10.37; 10 minutes, 33.28% ± 12.34), whereas the longest irradiation time of 10 minutes showed significantly lower resazurin conversion (P ≤ .01). Irradiation with Device B reduced resazurin conversion to 91.09% ± 26.91.

No statistically significant differences were observed with SRB staining (Figure 2B). All groups showed similar SRB content (range, 95.77% ± 6.70 to 99.43% ± 2.47) compared with the control group (100% ± 1.55).

PCNA immunostaining

Blue light irradiation reduced the ratio of PCNA-positive cells compared with the control group (Figure 3). There were fewer cells in irradiated groups than in the control group. Additionally, the nucleolus of irradiated cells seemed to be larger than the nucleolus in the control group (data not shown). Compared with the control group (0.80% ± 0.12), the ratio of PCNA-positive cells was lower in all treatment groups (Device A 1 minute, 0.36% ± 0.30; Device A 5 minutes, 0.69% ± 0.08; Device A 10 minutes, 0.34% ± 0.09; Device B, 0.46% ± 0.25). The ratio was significantly lower in the 10-minute Device A group compared with the control group (P ≤ .01).

Live/dead staining

To determine the toxicity of the irradiation procedure, cells were stained with calcein-AM (green equals cytoplasm of living cells), ethidium homodimer-1 (red equals DNA of damaged cells), and Hoechst 33342 (blue equals nucleolus). As depicted in Figure 4, irradiation resulted in considerably fewer cells, but the ratio of nonviable cells to viable cells seemed similar between the control group and the group treated with 5 minutes of irradiation.

Cytokine secretion

After irradiation, cytokine release was measured with ELISA in the supernatants (Figure 5). No IL-6 could be detected in the supernatants, and no significant differences could be measured between the supernatants concerning the release of IFN-γ after irradiation (control group, 2.16 ng/mL ± 0.45; Device A 1 minute, 1.78 ng/mL ± 0.30; Device A 5 minutes, 1.49 ng/mL ± 0.40; Device A 10 minutes, 2.45 ng/mL ± 1.23; Device B, 1.65 ng/mL ± 0.19), although there was a descending tendency in the Device A group at shorter irradiation time. Likewise, the concentration of TNF-α was not significantly different compared with the control group (control group, 2.79 ng/mL ± 3.54; Device A 1 minute, 1.71 ng/mL ± 2.49; Device A 5 minutes, 2.36 ng/mL ± 3.01; Device A 10 minutes, 2.39 ng/mL ± 2.31; Device B, 6.34 ng/mL ± 3.85).

Discussion

In the research and treatment of various diseases. The use of blue light seems to be particularly promising for wound healing and hyperproliferative skin diseases owing to its antimicrobial and immune-modulating properties. Although several studies indicate positive effects of blue light irradiation in vivo, little data exist about the effects of blue light on keratinocytes and reepithelization. Therefore, the present study investigated the effects of phototherapy on keratinocytes with 2 established devices in vitro.

The data from this study indicated that blue light irradiation can reduce keratinocyte proliferation, migration, and mitochondrial activity but that the irradiation has no effect on the release of IFN-γ and TNF-α.

Mamalis et al³⁷ showed that blue light (415 nm ± 15) reduces fibroblast proliferation in a dose-dependent manner, whereas viability as measured with trypan blue counting seems not to be affected by doses up to 80 J/cm². Slower in vitro wound closure was described by Masson-Meyers et al³⁵ when fibroblasts were irradiated with blue light (470 nm at fluences ranging from 3 J/cm² to 55 J/cm²); only high doses resulted in delayed wound closure. Interestingly, irradiation resulted in significantly lower IL-6 concentrations in the supernatants even at the same incubation time of 24 hours. Both of these studies^35,37 evaluated the effect of blue light irradiation on fibroblasts, but it is likely that blue light has the same effect on different cell types (eg, keratinocytes in the present study) and reduces cell proliferation in general. The present study did not detect any differences in SRB staining that support the data presented by Mamalis et al.⁴¹ Trypan blue cell counting and SRB staining are quantitative methods used to describe the amount of cell death or protein content; however, unlike resazurin conversion, neither of these methods enables qualitative assertions concerning cell metabolism.

Avola et al³⁸ showed that blue light (450 nm) irradiation decreased cell viability measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of keratinocytes and fibroblasts in a dose-dependent manner while hydroxytyrosol alleviated this effect. In contrast, those study authors³⁸ observed that PCNA was significantly increased after blue light LED treatment of 45 J/cm² and 15 J/cm². This is supported by the present findings, since MTT assay and resazurin conversion both are based on the capacity of mitochondrial metabolization of specific substances.

Godley et al³⁹ previously showed that light exposure (390 nm–550 nm) to human primary retinal epithelial cells significantly damaged mitochondrial DNA compared with dark-maintained controls, and irradiated cells continued to show an increasing loss of viability measured at MTT assay. The elevated levels of PCNA in the study by Avola et al³⁸ possibly could be explained by DNA damage caused by irradiation since PCNA also plays an important role in DNA repair processes.⁴⁰ Because a different wavelength (450 nm) was used, it is possible that wavelengths closer to the ultraviolet spectrum may induce more damage to DNA and thus should not be recommended for wound treatment.

Teuschl et al⁴¹ investigated scratch wound closure and viability of fibroblasts, myoblasts, and keratinocytes. Those authors detected decreased proliferation and augmented apoptosis in all 3 cell types after blue light (470 nm) irradiation, whereas red light (630 nm) irradiation was found to promote proliferation.⁴¹

Adamskaya et al⁴² showed that blue light irradiation (ƛ, 470 nm; intensity, 50 mW/cm²) for 10 minutes for 5 consecutive days improved reepithelization in an in vivo rat model. They suggested that illumination is an easily applicable, safe, and cost-effective method of treatment for surface wounds.⁴² Similar results were attained in an ex vivo model.⁴³ These findings seem to be contrary to the findings of the present in vitro study. However, blue light, due to interference with hemoglobin, can induce a temperature rise in a bleeding wound, close to the denaturation temperature of blood proteins,⁴⁴ which in consequence leads to faster wound healing in vivo²⁷ (whereby Device B was used as irradiation device in this study). In a study evaluating wound healing in 12 mice treated with Device B, Rossi et al²⁶ observed a faster healing process, reduced inflammatory response, higher collagen content, and better-recovered skin morphology in the treated tissue. It is possible that these positive effects on coagulation and, thus, wound healing in vivo predominate over the slower reepithelization caused by irradiation observed in vitro in the present study.

Bashir et al⁴⁵ showed that irradiation with ultraviolet B (290 nm–320 nm) induces the expression of TNF-α, whereas irradiation with ultraviolet A (320 nm–400 nm) does not. Shnitkind et al¹⁰ irradiated HaCaT cells with blue light (420 nm at 54 mJ/cm² and 134 mJ/cm²) and detected an inhibition of cytokine-induced production of IL-1α in the irradiation groups. They concluded that narrow-band blue light has anti-inflammatory effects on keratinocytes by decreasing the cytokine-induced production of IL-1α. Although no significant differences were detected in the present study, blue light irradiation seemed to decrease IFN-γ concentrations at lower doses. It could be that high-dose irradiation increased IFN-γ secretion, likely owing to high irradiation toxicity.

Limitations

Wound healing is a complex process involving a lot of different cells and physiological processes. The present study is limited due to its in vitro experimental setting, investigating only reepithelization in a keratinocyte cell line.

Conclusions

Several studies indicate the positive effects of blue-light phototherapy in the management of wounds and hyperproliferative skin diseases. Despite these positive effects, the present study indicates that irradiation with established devices may significantly reduce proliferation, migration, and cell metabolism of keratinocytes in vitro even after a short application time at high doses. Although this study is limited by its in vitro approach, the results indicate that blue light irradiation could also harm keratinocytes. Thus, use of phototherapy in wound therapy should be critically scrutinized and evaluated in vitro prior to clinical use.

Acknowledgements

Authors: Markus Denzinger, MD¹; Manuel Held, MD²; Sabrina Krauss, MD²; Christian Knorr, MD¹; Clemens Memmel, MD¹; Adrien Daigeler, MD²; and Wiebke Eisler, MD²

Affiliations: ¹Department of Pediatric Surgery, Klinik St. Hedwig, University Medical Center Regensburg, Regensburg, Germany; ²Clinic for Plastic, Reconstructive, Hand and Burn Surgery, BG Trauma Center, Eberhard-Karls-Universität, Tübingen, Germany

Correspondence: Markus Denzinger, MD, Department of Pediatric Surgery, Klinik St. Hedwig, University Medical Center Regensburg, Regensburg, Germany; m.denzinger@t-online.de

Disclosure: DermoDyne (Device A) was provided by DermoDyne HealthCare for the investigations. The authors disclose no financial or other conflicts of interest.

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