Med Laser 2022; 11(2): 104-109  https://doi.org/10.25289/ML.2022.11.2.104
Comparison of the 660 nm and 532 nm wavelengths for photobiomodulation therapy on dermal wounds in mice
Hyun Seok Ryu1,2, Seung Hoon Woo1,3
1Beckman Laser Institute Korea, Dankook University, Cheonan, Republic of Korea
2Interdisciplinary Program for Medical Laser, Dankook University, Cheonan, Republic of Korea
3Department of Otorhinolaryngology Head and Neck Surgery, Dankook University School of Medicine, Cheonan, Republic of Korea
Correspondence to: Seung Hoon Woo
E-mail: lesaby@hanmail.net
ORCID: https://orcid.org/0000-0001-7560-1140
Received: March 10, 2022; Accepted: March 15, 2022; Published online: June 30, 2022.
© Korean Society for Laser Medicine and Surgery. All rights reserved.

This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background and Objectives
The objective of this study was to compare the effects of photobiomodulation using the 660 nm diode and 532 nm diode-pumped solid-state (DPSS) lasers on dermal wound healing in mice.
Materials and Methods
The dermal wound was induced by a circular incision on the mice's back using a No. 15 surgical blade. The mice were treated with photobiomodulation at an energy density of 4 J/cm2 daily for 10 days before sacrifice. The macroscopic and histological observations of the wound were done on days 3, 7, and 10.
Results
The dermal wound area reduction rates in the diode laser, DPSS laser, and control groups were 34.2% ± 3.5%, 33.2% ± 2.4%, 24.0% ± 2.7% on the 3rd day, 72.2% ± 2.8%, 64.8% ± 3.5%, 42.8% ± 5.0% on the 7th day, and 87.2% ± 3.7%, 82.2% ± 7.9%, 71.4% ± 4.0% on the 10th day (p < 0.05), respectively. Histological analysis showed that the laser treatment improved granulation tissue formation, epithelialization, collagen deposition, and vascularization compared to the control.
Conclusion
Diode and DPSS lasers significantly improved dermal wound healing in mouse models. Further investigations are needed to confirm the mechanisms involved during photobiomodulation on dermal wound healing.
Keywords: Photobiomodulation therapy; Wound healing; Low-level light therapy
INTRODUCTION

The use of lasers to promote wound healing was reported in the 1970s. Since then, lasers of various wavelengths and irradiation methods have been reported for wound healing.1-3 Photobiomodulation using lasers can induce biochemical and physiological effects in various effect enzymes, cells, tissues, and institutions. It is known that photobiomodulation can promote cell differentiation,4,5 increase collagen synthesis,6,7 or increase the expression of growth factors in cells.8,9 A low-power laser is a laser that produces an output of less than 1 W (watt). When calculated in terms of energy including laser irradiation time, it generally can stimulate cells mainly in the range of 0.05 to 10 J.10

Due to the increasing prevalence of diabetes and ischemic diseases, surgical wounds could not be healed. Thus, more and more patients are in trouble. Cancer patients such as those with head and neck need to receive radiation therapy as preoperative guided treatment. Thus, the need for methods that can promote wound healing in the head and neck area is increasing.

In this study, a 660 nm diode laser and a 532 nm diode-pumped solid-state (DPSS) laser were treated in wound-induced mice to compare the photobiomodulation effects. To investigate the effect of photobiomodulation on wound healing, histological changes and changes in wound shape were measured. Results of this study could be used as basic data for developing an irradiation method with an optimum healing effect.

MATERIALS AND METHODS

Experimental animals and laser

Forty-five mice (Institute of Cancer Research) were randomly divided into three groups. Group 1, the control group (n = 15), was observed after wound induction. Group 2 (n = 15) was irradiated with a diode laser and group 3 (n = 15) was irradiated with a DPSS laser. As lasers, a Golden Light (GoldenLight, Daejeon, Republic of Korea) 660 nm photodynamic therapeutic diode laser and a 532 nm DPSS laser (Laser-Compact, Moscow, Russia) were used. The animal experiments were carried out according to the guidelines set by the Institutional Animal Care and Use Committee at Dankook University (DK-21-042).

Wound induction

After anesthesia by injecting 0.2 ml of a mixture of Ketamine 2 ml, Rompun 1 ml, and distilled water 9 ml into each mouse through the peritoneum, the hair behind the spine was removed with an animal root removal cream and a circular wound with a radius of 20 mm at a constant depth was made up to the fascia layer using a number 15 surgical blade.

Laser irradiation and biopsy

After wound induction, group 1 was performed a biopsy at the wound site on days 3, 7, and 10 for those that healed spontaneously. For groups 2 and 3, induced wounds were irradiated with a laser at the same power density (4 J/cm2). Group 2 was irradiated with a diode laser daily and then biopsied on days 3, 7, and 10. Group 3 was irradiated with a DPSS laser daily and biopsied on days 3, 7, and 10 (Fig. 1).

Figure 1. Photobiomodulation therapy images. (A) Control group; (B) 660 nm diode laser; and (C) 532 nm DPSS laser. DPSS, diode-pumped solid-state.

Macroscopic comparison of wound areas

After inducing a wound with a radius of 20 mm, cross-sectional areas of the wound on the 3rd, 7th, and 10th days were determined. Considering that circular wounds were not healed uniformly, the major axis of the wound and the minor axis of the vertical plane of the major axis were measured and compared.

Histopathological observation

Biopsy tissues were processed and observed under an optical microscope. Granulation tissue formation, epithelialization, collagen deposition, and vascularization were evaluated by a pathologist. These five items were scored to compare the degree of wound healing (Table 1).10 Statistical processing was performed using one-way ANOVA at a significance level of 0.05. All data were analyzed using SPSS ver. 10.0 (SPSS Inc., Chicago, IL, USA).

Table 1 . Criteria for scoring histologic sections

ScoreParameterCriteria
1-3EpithelializationNone to very minimal
Cellular contentNone to very minimal
Granulation tissueNone to sparse amount
Collagen depositionNone
VascularityNone
EpithelializationMinimal (less than half of diameter) to moderate (more than half of diameter)
Cellular contentPredominantly inflammatory cells
Collagen depositionNone to thin at wound center
Collagen depositionFew collagen fibers
VascularityFew capillaries
EpithelializationCompletely epithelialized
Cellular contentMore fibroblasts, still with inflammatory cells
7-9Granulation tissue7, sparse at wound center
8, thin layer at wound center, few collagen fibers
9, thicker layer, more collagen
Collagen depositionModerate collagen fibers
VascularityModerate neovascularity
EpithelializationThicker epithelial layer
Cellular contentPredominantly fibroblasts
10-12Granulation tissueUniformly thick
Collagen depositionModerate-to-extensive collagen deposited
VascularityExtensive neovascularity
EpithelializationThick epithelium
Cellular contentFewer number of fibroblasts in dermis
13-15Granulation tissueUniformly thick
Collagen depositionDense, organized, oriented collagen fibers
VascularityWell-defined capillary systems

RESULTS

Changes in wound area after treatment with photobiomodulation

In the control group (spontaneous healing group), healing rates (%) of the cross-sectional area measured on the 3rd, 7th, and 10th days were 24.0% ± 2.7%, 42.8% ± 5.0%, 71.4% ± 4.0%, respectively. In the diode laser group, healing rates were 34.2% ± 3.5%, 72.2% ± 2.8%, and 87.2% ± 3.7% respectively. In the DPSS laser group, these rates were 33.2% ± 2.4%, 64.8% ± 3.5%, and 82.2% ± 7.9%, respectively. From day 3, the diode laser group and the DPSS laser group both showed significantly decreased wound areas and increased healing rates compared to the control group. However, there was no significant difference in wound area or healing rate when the two laser groups were compared (Table 2, Fig. 2).

Table 2 . Wound closure in DPSS laser and diode laser

GroupWound closure (%)

Day 3Day 7Day 10
Control24.0 ± 2.742.8 ± 5.071.4 ± 4.0
660 nm diode34.2 ± 3.5*72.2 ± 2.8*87.2 ± 3.7*
532 nm DPSS33.2 ± 2.4*64.8 ± 3.5*82.2 ± 7.9*

The results of wound closure (%) were represented as mean ± standard deviation in each group.

DPSS, diode-pumped solid-state.

*p < 0.05 as compared to control.



Figure 2. Macroscopic observation of wound area at 7 days. (A) Control; (B) 660 nm diode laser; and (C) 532 nm DPSS laser. DPSS, diode-pumped solid-state.

Change in histological score after treatment with photobiomodulation

In the control group, histological scores measured on the 3rd, 7th, and 10th days of wound healing were 3.6 ± 1.1, 7.0 ± 1.0, and 9.0 ± 0.8, respectively. In the diode laser group, there were 4.4 ± 1.8, 9.4 ± 0.5, 11.2 ± 0.8, respectively, and in the DPSS laser group, there were 3.4 ± 0.9, 9.0 ± 0.7, and 10.8 ± 0.8, respectively. From day 7, histological scores showed no significant differences between the diode laser group and the DPSS laser group. However, their histological scores for wound healing were increased significantly compared to the control group (Table 3, Fig. 3).

Table 3 . Histologic score in 660 nm diode laser and 532 nm DPSS laser

GroupHistologic score

Day 3Day 7Day 10
Control3.6 ± 1.17.0 ± 1.09.0 ± 0.8
660 nm diode4.4 ± 1.89.4 ± 0.5*11.2 ± 0.8*
532 nm DPSS3.4 ± 0.99.0 ± 0.7*10.8 ± 0.8*

Evaluation of histological score was based on Table 1.

DPSS, diode-pumped solid-state.

*p < 0.05 as compared to control.



Figure 3. Histological analysis of H&E stain tissue sections (magnification ×100). (A) Control group was observed granulation tissue at the wound center and moderate epithelialization change (score = 6, based on Table 1). (B) Diode laser group was observed multiple extensive neovascularizations (score = 10, based on Table 1). (C) Diode-pumped solid-state laser group was observed uniformly thick granulation tissue at full layer and fibroblast-dominant cellular content (arrow) (score = 10, based on Table 1).
DISCUSSION

Wound healing is a complex series of biological reactions that go through inflammation, proliferation, synthesis, and eruption processes. The inflammatory phase involves hemostasis, activation of platelet degranulation, complement, and agglutination. These reactions are essential for macrophages and secretions of various cytokines. During the proliferative phase, endothelial cells and fibroblasts play important roles. Fibroblasts migrate in the surrounding normal tissue and change into the so-called wound fibro-blast from collagen. The formation of collagens is regulated by collagen-degrading enzymes after the differentiation phase begins, they are deposited in tissues. These deposited tissues occur over a period of about five weeks. They are important factors in determining the elasticity and appearance of the wound after recovery. It is the large winds of both an operator and the patient that induce these wound healings more quickly and improve the elasticity and appearance of the healed wounds. Promoting wound healing is becoming more important when there are many side effects of peripheral blood vessels and tissues due to diabetes and hypertension.11 The recovery of surgical wounds caused by radiation before and after surgery is a major problem.

Since Mester et al.12 reported that low-power lasers could stimulate wound healing, low-power lasers have been tried to treat various types of wounds and ulcers that have failed due to existing healing methods. It has been shown that low-power lasers can also improve recovery from fractures and radiation-induced osteonecrosis and that the range of applications for low-power lasers can be further expanded.13 However, the results of each research group are somewhat different due to the different experimental animals used, the tissue and the size of the wound, the methodological difference to confirm the wound healing result, and the variety of the laser type and energy intensity used. As a result, its commercialization is being delayed.

Various variables should be considered to maximize the effect of a low-power laser on wound healing. First, the laser used can influence the result. Differences in wavelength and laser output method can be variables. Results can vary depending on the laser irradiation time, that is, the amount of energy applied to the wound. The process of wound healing is also an important variable. In addition, when the laser is applied in the process of wound healing such as the inflammatory phase, the proliferative phase, and the remodeling phase could affect the result. Some studies have reported that the direction and angle at which the laser is applied to the wound might also make a difference in would healing.14

According to Karu15, the degree of increase of DNA synthesis rate from the living body differs depending on the wavelength of the laser. The 660 nm diode laser and 532 nm DPSS laser used in this study also increased wound healing compared to the control. However, no significant difference in wound healing was observed between the two laser groups. The DPSS (532 nm) laser technology used in this study is a technology that uses a high-power semiconductor laser as a pumping light source, unlike the existing laser that used a discharge tube in an industrial solid laser system such as the Nd:YAG laser system. Characteristics of the laser are improved through changes in the waveform, increasing life expectancy and energy density is increased. Because the exposure time is decreased, the effect of medical treatment is maintained compared to existing lasers and has various advantages such as less damage to surrounding tissues.

The amount of energy applied to the wound may be a variable in promoting wound healing. In general, when one irradiation energy is 10 J or more per unit area, it rather damages the tissue and suppresses biosynthesis. In this study, histological analysis was performed after irradiating at 4 J of energy per unit area. As a result, wound healing was promoted significantly from the 7th day. Before that, the amount of laser energy absorbed by the mouse tissue was low. The period during which the laser was involved was characterized at around 7 days in the process of each inflammatory phase, growth phase, and remodeling phase from the mouse wound. However, considering the apparent recovery rate of the wound area, there was an increase significantly from day 3 in the laser-irradiated group. It can be inferred that there is a time lag between the apparent wound and actual histological changes that occur.

The mechanism by which low-power laser promotes wound healing involves mitochondria activation, adenosine triphosphate synthesis, DNA and RNA synthesis promotion, protein synthesis promotion, enzyme reaction regulation, intracellular and extracellular pH regulation, and cell metabolism activation.16-19 In this study, it was confirmed that the activation process of cell metabolism at the microscopic level was further increased by the low-power laser. The cross-sectional area of the wound was also externally increased. It was observed that cells healed rapidly at significant levels after laser irradiation. It was presumed that the laser activated the photo acceptor of mitochondria and induced oxidation of the pre-respiratory nicotinamide adenine dinucleotide hydride pool, induced nucleic acid synthesis, cell alkalinization, and so on. Ultimately, such changes might have resulted in the activation of the secretion of transforming growth factor, fibroblast growth factor, platelet-derived growth factor, interleukin, and the like.20-22

In the future, we plan to use a low-power laser to heal wounds in artificially induced diabetes rats (diabetic mice) and study how the laser affects vascular necrosis and ischemic changes caused by diabetes. It is expected that the scope of application will increase even for humans. In addition, studies on the effect of low-power lasers on the healing of surgical wounds after radiation therapy should be conducted in parallel. Research on the optimum century and optimum irradiation time of lasers that can show the maximum effect of promoting wound healing without inducing side effects on the human body needs to be preceded using different lasers, enzyme-linked immunoassay, monoclonal antibody, and so on. When more specific studies on the mechanism of wound healing are combined with immunohistochemical analysis methods using different lasers, molecular-level analysis using electron microscopy, and tensiometers, the range of practical clinical use of output lasers can be further expanded. In conclusion, as a result of this study, after inducing a wound in a mouse model, lasers of 660 nm and 532 nm showed significant healing-enhancing effects compared to the untreated control group. Further research on the optimal laser environment is needed to confirm and maximize its effectiveness.

FUNDING

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2020R1I1A3072797).

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS

Concept and design: All authors. Analysis and interpretation: HSR. Data collection: HSR. Writing the article: HSR. Critical revision of the article: SHW. Final approval of the article: SHW. Statistical analysis: SHW. Obtained funding: SHW. Overall responsibility: SHW.

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