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. 2023 Oct 11;21(2):e14335. doi: 10.1111/iwj.14335

Effect of a portable light emitting diode device on wound healing in a rat model

Han Gyu Cha 1, Joon Hur 2, Changsik John Pak 2, Joon Pio Hong 2, Hyunsuk Peter Suh 2,✉
PMCID: PMC10824621  PMID: 37822047

Abstract

Light‐emitting diode (LED) lights produce a variety of wavelengths that have demonstrable efficacy in therapeutic and aesthetic fields. However, a repetitive treatment regimen is required to produce treatment outcomes, which has created a need for portable LED devices. In this study, we aimed to develop a portable therapeutic LED device and investigate its healing effect on excisional wounds in a rat model. The 35 × 35 mm‐sized LED device was used on a total of 30 rats with full‐thickness wounds that were divided into two groups depending on radiation intensity (11.1 and 22.2 mW/cm2 group). LED irradiation was performed every 24 h for 30 min, over 14 days, in direct contact with the wound. Percentage wound closure was measured by photographic quantification and was assessed histologically using haematoxylin and eosin (H&E) and Masson's Trichrome staining, and immunohistochemistry for Vascular endothelial growth factor (VEGF) and CD31. Percentage wound closure was significantly higher in 22.2 mW/cm2 irradiated wounds than that in the control wounds on days 7 and 10. The area of collagen deposition was remarkably larger in 22.2 mW/cm2 irradiated wounds than that in the control, with more horizontally organized fibres. CD31 immunostaining confirmed a significant increase in the number of microvessels in 22.2 mW/cm2 irradiated wounds than that in the control wounds, although there was no difference in VEGF immunostaining. Our novel portable LED device accelerates wound healing in a rat model, raising the possibility that portable LED devices can combine convenience with accessibility to play an innovative role in wound dressing.

Keywords: LED, light emitting diode, portable LED, wound healing

1. INTRODUCTION

A light‐emitting diode (LED) is a complex semiconductor that converts electricity to noncoherent light with a narrow spectrum and a peak wavelength. Early LEDs produced low and unstable light with a broad wavelength until the National Aeronautics and Space Administration developed a quasimonochromatic LED that produces a very narrow spectrum of light and has a significant effect on wound healing. 1 , 2 At present, the evolution of LED technology has produced devices that emit light at wavelengths ranging from ultraviolet to near‐infrared (247–1300 nm). 3

The variety of wavelengths produced by LEDs has allowed for the expansion of its clinical applications and LEDs have been effective in both therapeutic and aesthetic fields. Blue LED light (400–470 nm), which penetrates up to 1 mm of skin, has been proven to treat skin conditions such as acne, actinic keratosis, and psoriasis. 4 , 5 , 6 Yellow LED light (570–590 nm), which penetrates into the papillary dermis, has been applied mainly to photoaged skin and post‐laser treatment. 7 , 8 Red LED light (630–670 nm) penetrates up to 2–3 mm and has been the most widely used in LED studies, where its efficacy has been demonstrated in scar treatment, skin rejuvenation, wound healing, and the treatment of premalignant lesions. 9 , 10 , 11 , 12 Infrared LED light (800–1200 nm) which penetrates the skin to a depth of 5–10 mm has been applied in the treatment of wounds and cellulite. 13 , 14 , 15

One of the very few disadvantages of LED light treatments is the need for a repetitive treatment regimen, which may require patients to visit a hospital daily, which is both tedious and costly. Therefore, there is a need for portable LED devices and various home‐use LED devices have been marketed recently, mainly for aesthetic and dermatologic purposes. 16 , 17 However, there are very few portable LED devices designed specifically for therapeutic applications such as pain management, wound healing, or the treatment of post‐procedural side effects. 18 , 19 In particular, there are very few studies that have demonstrated the effect of a portable LED device on wound treatment. 20 , 21 The necessity of daily dressing in addition to LED light therapy is likely to be the main obstacle limiting the development of a home‐use portable LED device for wound healing.

Therefore, the aim of this study was to develop a home‐use portable LED device and investigate its effect on excisional wound healing in a rat model.

2. MATERIALS AND METHODS

2.1. LED devices

The LED device (35 mm × 35 mm) used in this study was manufactured at the R&D Center of CG Bio Co., Ltd. (Seongnam, Korea). First, a circuit was formed on a flexible copper‐clad laminate (FCCL) dry film, and LED chips (19‐213/R6SC‐AU2V2B/3 T, EVERLIGHT Electronics Co., Ltd., New Taipei, Taiwan) were fixed to it. A via hole was formed to conduct electricity on the FCCL film, which was fabricated from a composite material of styrene and acrylate (Figure 1A). After the inner wall of the via hole was plated with copper, the film with via hole was completely compressed by applying high temperature (90 ± 10°C) and pressure (5 ± 1 kgf/cm2), with a specific laminating roller speed (1.5 ± 0.5 m/min). A circuit was created on the dry film under UV irradiation using the develop‐etch‐strip process. A polyimide cover was attached to the film to protect the formed circuit from high temperature (165 ± 15°C) and pressure (30 ± 10 kgf/cm2) conditions, and the polyimide surface was plated with gold to prevent oxidation and improve soldering performance. After the outside edge of the film was cut using a mould press, the LED chips and parts were mounted on the surface. We confirmed that the completed LED dressing was functional when the power was turned on, after connection to a 2 cm‐sized lithium manganese dioxide round cell battery (Figure 1B), and measured the wavelength, irradiance, and energy per area using a spectrometer. The peak wavelength of the completed LED dressing was 632 nm (range: 617.5–633.5 nm). Two types of LED dressings with light radiation values of 11.1 and 22.2 mW/cm2 were fabricated by varying the resistance of the driver that controls the current. When each of the two LED dressings was applied for 30 min, the amount of energy per area received by the wound surface was 20 and 40 J/cm2, respectively. The LED dressings were applied as close as possible to the wound surface to promote wound healing.

FIGURE 1.

FIGURE 1

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Home‐use portable light‐emitting diode (LED) device. (A) LED device structure. (B) A 35 × 35 mm‐sized LED device connected to a 2 cm‐sized lithium manganese dioxide round cell battery.

2.2. Animal models and LED treatment

Ten‐week‐old Wistar male rats (Orient Bio, Inc., Seongnam, Korea) weighing 220 g each were housed under a 12/12 h light/dark cycle, and standard laboratory water and food were provided ad libitum. Temperature (25 ± 2°C) and humidity (50 ± 5%) were strictly regulated. A total of 30 rats were divided into two groups depending on radiation intensity (11.1 and 22.2 mW/cm2 group) and 20 × 20 mm‐sized square full‐thickness wounds extending through the panniculus carnosus were made under sterile conditions using a No.10 blade, on both sides of the rat dorsum, symmetrically. LED irradiation was applied every 24 h for 30 min on the left‐side wound by direct contact, while the right‐side wound served as a non‐irradiated control. Skin temperature changes were monitored during the entire LED treatment using an infrared camera (Figure 2A,B). Occlusive dressing was performed daily after LED irradiation using a 5‐mm thickness foam fixed with Ioban™ (3 M, St. Paul, MIN) (Figure 2C). LED irradiation was performed for 14 days, and three rats in each group were euthanized on days 3, 7, 10, and 14 after skin sampling for histological analysis. Ethics approval for this study was obtained from the Institutional Animal Care and Use Committee of Asan Institute for Life Sciences (2019‐02‐091) and all animal procedures were conducted in accordance with institutional guidelines.

FIGURE 2.

FIGURE 2

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Experimental model with light‐emitting diode (LED) device and foam dressing. (A) LED irradiation was done every 24 h for 30 min. (B) No skin temperature change was observed during LED treatment. (C) Occlusive dressings were applied after LED treatment.

2.3. Measurement of wound closure

The size of the wound area in each group was measured on days 3, 7, 10, and 14 by analysing digital photographs of irradiated and control wounds using ImageJ software 1.53 (National Institute of Health, Bethesda, MD, USA). Percentage wound closure was calculated in comparison to the initial wound size and was presented as a mean percentage ± standard deviation (SD).

2.4. Histology and immunohistochemistry

For histological and immunohistochemical analyses, skin samples were obtained on days 3, 7, 10, and 14, and fixed in 4% paraformaldehyde overnight, then embedded in paraffin. Tissue sections (5 μm thick) were processed for haematoxylin and eosin (H&E) and Masson's trichrome staining, as well as immunostaining. Neoepidermal length was measured in H&E‐stained sections and collagen deposition was quantified by measuring relative colour intensity in Masson's trichrome‐stained sections using ImageJ software. Collagen fibre orientation was also calculated by scoring 1, 2, and 3 for vertical, mixed, and horizontal fibres, respectively. The inflammation score was calculated according to the infiltration of inflammatory cells, scored as absent, mild, moderate, and extensive as 1, 2, 3, and 4. For immunostaining, the samples were immunostained with primary antibodies against Vascular endothelial growth factor (VEGF) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and CD31 (Abcam, Cambridge, UK). Average optical density was calculated by quantifying the percentage of VEGF‐immunostained area using ImageJ software. Microvessel density was counted in 3 random fields of view in CD31‐immunostained sections at 200× magnification visualized with a light microscope. Vessels were identified as circular or ovoid‐shaped brown structures with a luminal space.

2.5. Statistical analysis

We compared the percentage wound closure of the control and irradiated wounds in each rat by analysing all data using Mann–Whitney and Kruskal‐Wallis tests in SPSS version 20.0 (IBM corp., Armonk, NY, USA). Statistical significance was defined as p < 0.05.

3. RESULTS

3.1. Wound closure after LED treatment

Percentage wound closure was significantly higher in wounds treated with 22.2 mW/cm2 (78.4 ± 4.5% and 94.2 ± 4.5%) than that in the control wounds (74.4 ± 6.9% and 89.7 ± 1.2%) on day 7 and 10, respectively (p < 0.05) (Figure 3).

FIGURE 3.

FIGURE 3

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Wound closure comparison. (A) Percentage wound closure in control and light‐emitting diode (LED)‐treated wounds on days 3, 7, and 10 (*p < 0.05). (B) Representative photographs of the full‐thickness wound during the healing process.

3.2. Histology

Neoepidermal length was relatively longer in 22.2 mW/cm2 irradiated wounds than in the control wounds throughout the wound‐healing process (Figure 4). The area of collagen deposition was larger in 22.2 mW/cm2 irradiated wounds compared to that in the control wounds, with a significant difference evident on day 3 (p < 0.001). The collagen fibre orientation score was also higher in 22.2 mW/cm2 irradiated wounds, which displayed more horizontally organized fibres than the control wounds (Figure 5). The inflammation score was lower in the irradiated wounds throughout the wound‐healing process, although this difference was not statistically significant (Figure 6).

FIGURE 4.

FIGURE 4

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Haematoxylin and eosin (H&E) staining of wound sections (×40) representing different neoepidermal lengths. (A) 22.2 mW/cm2 treated wound and (B) control wound on day 10. (C) Comparative graph showing the difference in neoepidermal lengths between two groups in the course of wound healing.

FIGURE 5.

FIGURE 5

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Masson's trichrome staining of wound sections (×100) on day 14. (A) Increased number of horizontal collagen fibres in 22.2 mW/cm2 treated wound compared with (B) control wound. (C) The area of collagen deposition and (D) the collagen fibre orientation was calculated using Image J (***p < 0.001).

FIGURE 6.

FIGURE 6

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Inflammation score calculated in haematoxylin and eosin (H&E) staining according to the infiltration of inflammatory cells scored as absent, mild, moderate, and extensive as 1, 2, 3, and 4.

3.3. Immunohistochemistry of CD31 and VEGF

Immunostaining of CD31 confirmed that there was a significant increase in the number of microvessels in the 22.2 mW/cm2 irradiated wounds compared with those in the control wounds on day 14 (Figure 7), although VEGF immunostaining showed no differences regardless of LED treatment (Figure 8).

FIGURE 7.

FIGURE 7

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Immunohistochemical staining for CD31 in wound samples (×200) on day 14. (A) Increased number of microvessels in 22.2 mW/cm2 treated wound compared with (B) control wound. (C) Microvessel density was calculated using Image J (*p < 0.05).

FIGURE 8.

FIGURE 8

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Average optical density calculated in Vascular endothelial growth factor (VEGF) immunostaining by quantifying the percentage of VEGF‐immunostained area.

4. DISCUSSION

The use of photobiomodulation (including LED devices) is expanding in various fields due to the non‐invasive and low‐risk nature of this therapy. Although it is mainly used in dermatologic rejuvenation, the therapeutic applications of LED treatment are garnering interest in the field of wound healing, based on the outcomes of LED exposure on skin, which include increased ATP, modulation of reactive oxygen species, alteration of collagen synthesis, and stimulation of angiogenesis. Red LEDs, in particular, have been proven to activate fibroblast growth factor (FGF), and increase the levels of type 1 procollagen and matrix metalloproteinase‐9. 3 , 22 , 23 However, in practice, the use of LEDs for wound healing requires a repetitive treatment regimen to maximize its effects and accelerate the healing process. Therefore, the development of a portable LED device is essential to provide patients with convenient, easy access to this valuable therapeutic intervention.

In this study, we have demonstrated the efficacy of our novel portable LED device, which is small and light enough to facilitate daily use by individuals. The light irradiation value of 22.2 mW/cm2 was selected based on the results from a pilot study (data not shown) in which we tested 11.1, 22.2, 33.3, and 44.4 mW/cm2 LED intensities to identify a radiation intensity that generates sufficient power without a temperature change that might induce burn injury. We found that radiation with 22.2 mW/cm2 for 30 min (40 J/cm2) produced the most efficient power without temperature change that was also capable of accelerating wound healing compared with higher radiation energies that had elevated temperature which might result in burn injury. Therefore, this study was designed to compare the effects of 11.1 and 22.2 mW/cm2 radiation intensities on 20 × 20 mm sized square full‐thickness wounds by applying red LEDs directly to the wound with a foam dressing cover. Wounds were incised on each side of the dorsum, with one side set as the control wound, to reduce individual differences.

Our analyses indicated that the percentage of wound closure was significantly higher in the 22.2 mW/cm2 wound compared with that of the control wound in the same individual from day 7 onwards. Wound closure was also faster in the 22.2 mW/cm2 group compared with the 11.1 mW/cm2 group from day 10 onwards. These results correlated with neoepidermal length in histological analyses, where neoepidermal length in the 22.2 mW/cm2 group was remarkably longer than that of the control wounds throughout the wound‐healing process. In addition, collagen deposition was increased in the 22.2 mW/cm2 irradiated wounds than that in the control wounds throughout the wound‐healing process, with abundant evidence of horizontally‐oriented collagen fibres. We believe that relatively abundant collagen is a marker of an upregulated proliferative phase of the wound‐healing cascade, which starts approximately 3 days after injury and lasts up to 3 weeks in a healing wound. As collagen synthesis is an integral requirement for granulation, we predict that treatment with 40 J/cm2 LED light will result in increased granulation tissue by upregulating collagen synthesis. In contrast, the inflammatory reaction that is known to be activated in the early phase of wound healing within a week was not affected by 40 J/cm2 red LED treatment. Consequently, we assume that the accelerated wound closure that was evident a week after the start of LED treatment was the result of upregulated proliferation rather than inflammation.

However, it is worth noting that the effect of our portable LED on vascularization and angiogenesis was not coincident, according to the results from VEGF and CD31 immunostaining. Although the CD31 immunostaining suggested that microvessel density had increased, there was no difference in VEGF immunostaining. These results may imply that our LED treatment stimulated angiogenesis by activating other growth factors, rather than VEGF. A previous in vitro study reported that red LED irradiation exhibits an inhibitory effect on VEGF expression, which correlates with our findings. 24 In addition, an in vivo study using BALB/c mice implanted with HeLa cells also demonstrated that red LED light attenuated VEGF expression. 25 Indeed, many studies have indicated that FGF is the primary growth factor stimulated by red LED irradiation. 26 , 27 One fundamental reason for these varying results may be the different energy densities employed in these studies and elucidation of the molecular mechanisms stimulated by different red LED energy densities is an ongoing research question, one which may be a limitation of the present study.

The energy density of 40 J/cm2 used in this study is higher than that used in previous studies focused on the application of red LEDs for wound healing in rat models; most of these studies used 1–20 J/cm2. 1 , 28 , 29 , 30 , 31 The positive effect of this higher dose in comparison to that in previous studies does not reflect a positive dosage response relationship. There are contradictory studies regarding positive and negative dosage response relationships in response to LED irradiation, and high energy densities of 50–100 J/cm2 displayed an inhibitory effect, resulting in delayed wound healing. 29 , 32 Therefore, the energy density of 40 J/cm2 in the current study may represent the maximum dosage that enables accelerated wound healing in a rat model.

Thus far, a number of different LED modalities have been developed in non‐contact forms where additional distance from the device and space are required for use. This is a drawback for patients with acute or chronic wounds and is one of the reasons that we sought to develop a portable LED device that could be used for direct contact in combination with dressing material. In this study, we confirmed that the efficacy of our contact LED device is not associated with thermal injury or other complications. In addition, as the coverage of the foam dressing over the LED device was efficient enough to absorb exudative fluid and moisturize the wound bed, we are planning to incorporate it as a new type of wound dressing material. To actualize a direct contact LED wound dressing, we would first need to diversify the LED device size to account for variable wound sizes, as well as minimize the size of the portable battery required to maintain consistent energy for wound healing.

5. CONCLUSIONS

In conclusion, our novel portable red LED device accelerates the wound‐healing process in a rat model. A portable LED device will provide convenience and accessibility to patients as it is light and small enough to carry and may lead to innovative roles in therapeutic wound dressing.

FUNDING INFORMATION

This work was financially supported by Young Medical Scientist Research Grant through the Daewoong Foundation.

CONFLICT OF INTEREST STATEMENT

The authors declare that there is no conflict of interest.

ACKNOWLEDGEMENTS

This study was supported by the Daewoong Foundation and CG Bio Co., Ltd.

Cha HG, Hur J, Pak CJ, Hong JP, Suh HP. Effect of a portable light emitting diode device on wound healing in a rat model. Int Wound J. 2024;21(2):e14335. doi: 10.1111/iwj.14335

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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