Although the proliferative effects at the wavelengths of the red and near-infrared region were proven in many studies [18–20], it is thought that there is a need for a design of a more efficient optical system to obtain consistent outcomes. In addition to this problem, the majority of the existing studies still evaluates the possible effect of the wavelengths in the range of 600 to 700 nm [21], but only the minor researches show the promise of other wavelengths in the visible spectrum, such as the wavelengths in the range of 400 to 600 nm [16, 17, 22].
Throughout the improvement of the Portable Multicolor LED-Based System for photobiomodulation therapy, we have paid attention to produce a system that is portable, cost-effective, small-sized, lightweight, durable, user-friendly, self-supporting, and easy to clean. We have designed this optical system with 9 LEDs (3-pieces of blue (460–475 nm), 3-pieces of green (515–535 nm), and 3-pieces of orange-LED light sources (585–595 nm)) and each of them corresponded to a single well of the 12-well plate to illuminate the single well homogenously with a single LED. The three wells of the culture plate were left blank without any LED to be used as the control group in the same experiment.
In this system, power LED light sources and 15° LED lenses were used together to provide a more homogenous illumination in the wells, due to the non-coherent structure of LED light and the location of LEDs which was the middle-top of each well. Here, LED lenses played a crucial role in reducing the spread of light to ensure homogenous illumination as in the work of Hwang et al [23]. Furthermore, black insulating materials that fit the border of a well were produced from the PLA filament to inhibit the transmission of the light beam from a well to the adjacent ones. To obtain maximum performance from the LEDs, transistors, LED drivers, and aluminum coolers were used and the LEDs were connected in series. With the help of the transistors, the LED drivers, and the aluminum cooler, the internal environment of the device was kept cooler during its performance and LEDs worked efficiently without losing their performance. Furthermore, a 12V 1.5A adaptor was used to activate 3-pieces metal-oxide-semiconductor field-effect transistors (MOSFETs) and LED Drivers, and to lead the 9-pieces of LEDs by Arduino Uno. Thus, the current and the voltages of the LED were kept constant without any loss during its operation and the output powers that were obtained from a single LED for each color were enough and suitable to induce photobiomodulation.
Until recently, it has been believed that the laser devices are more suitable light sources to induce photobiomodulation [9], because of their specific characteristics which are being collimated, coherent, and monochromatic [10]. The LEDs are energy-saving, affordable, and safe alternatives to lasers with having a smaller range of wavelengths. They have great promise in photobiomodulation, too. Generally, red-LEDs have been handled in clinics and scientific researches [22], but the information for the use of blue, green, and orange-LEDs are limited. Even so, it is known that low energy blue, green, and orange LEDs can promote several biological effects on the tissue and cellular levels [16, 17, 22].
Chabert et al. worked with an LED system that emitted the wavelengths of 590 nm yellow light, 630 nm red light, and a combination of them as an orange light at 4, 8, and 12 J/cm2 energy densities on the cell viability and the morphology of the normal human keratinocyte and fibroblast cells. They irradiated the cells twice with a 48 h interval. The best outcome regarding the cell morphology of the fibroblasts was obtained with 12 J/cm2 energy density. For both fibroblasts and keratinocytes, orange LED irradiation induced better improvements in the cell shape, cell viability, and protein expression such as Collagen 1. Thus, it was stated that orange light can be a promising wavelength to combat skin aging [17]. Similarly in our study, the highest proliferative effect on keratinocyte cells obtained with the triple orange-LED application at the energy densities of 3 J/cm2 and 5 J/cm2 with the rates of 150.73% and 214.53%, respectively.
Wang et al. examined the effects of 420 nm blue-LED array, 540 nm green filtered lamp, 660 nm red diode laser, and 810 nm NIR diode laser at the fluence of 3 J/cm2 for five times in every two days on the osteogenic differentiation of human adipose-derived stem cells (hASCs) for three weeks. Blue and green light irradiations at 3 J/cm2 provided an increment in the intracellular calcium concentration, and others did not induce dramatic increases. Thereupon, they stated that 420 nm blue and 540 nm green lights were more effective on the osteogenic differentiation with respect to 660 nm red and 810 nm NIR light [16]. In our study, we did not examine the cell differentiation, but we have evaluated the wound healing and cell proliferation issues on keratinocytes. We have observed that triple green light application had a great impact, similar to the study of Wang et al., on the cell viability of keratinocytes with a proliferation rate of 196%.
Zhu et al. examined the effect of blue light irradiation on the osteogenic differentiation and the cell proliferation of human gingival mesenchymal stem cells (hGMSCs) via blue-LEDs at 1, 2, 4, and 6 J/cm2 energy densities. They showed that there was a significant increase in osteogenic differentiation and it was proved with real-time PCR analysis of the specific genes, especially after the 2, 4, and 6 J/cm2 light applications. Besides, it was found that blue-light irradiation inhibited cell proliferation in the same study. It was thought that these stem cells took the path for cell differentiation. Thus, there was not any significant increase in cell viability [22]. On the contrary, triple blue light application in our study revealed successful outcomes in cell proliferation of keratinocytes at 5 J/cm2 energy density, with a rate of nearly 150%.
In the experimental part of this study with the portable multicolor LED-based photobiomodulation system, it was observed that the triple light treatment accelerated the wound healing process significantly and increased the cell viability of the keratinocytes with each wavelength at the different energy densities. Any of these applications created considerable differences when compared with the control group. The highest cell proliferation rates were achieved with the green-LED treatment at 1 J/cm2 and the orange-LED treatment at 5 J/cm2. At the beginning of this study, we hypothesized that the Portable Multicolor LED-Based System will be an important design for the examination of different wavelengths in the photobiomodulation process. Now, the obtained results confirmed this hypothesis with the positive outcomes of the green and orange-LED light applications on the accelerated wound healing of keratinocytes.