Blue LED light (420 nm) has successfully been used to induce hemostasis through a photo-thermo-coagulation process: light absorption by hemoglobin triggers a local temperature increase, leading to a coagulation effect. Besides hemoglobin, there are other macromolecules, such as cytochromes, that are able to absorb blue light: after irradiation, these ubiquitous cellular components can trigger one or more intracellular pathway that modulates the healing process, in combination with the coagulation effect. The aim of this study is to investigate the molecular effects of 30s treatment with a Blue LED device in two different murine model wounds. In the first model we studied a superficial wound, and in particular the inflammatory response by an immunohistochemical and morphological analysis of the many cellular types involved in this phase of the healing process. The second model is a full-thickness wound: a customized ELISA kit enabled to study EGF, bFGF, VEGF, TNF-α, MMP-2 and PRO-MMP-9 at different postoperative time points (1, 3, 6, 9, 24 hours and 7 and 14 days after the treatment). A modulation of these parameters was evidenced in the early phase of the wound healing process, while at longer follow up times no differences are pointed out.
Keloids are an exuberant response to cutaneous wound healing, characterized by an exaggerated synthesis of collagen probably due to the increase of fibroblasts activity and their proliferation rate. Currently, there are not definitive treatments or pharmacological therapies able to prevent keloid formation and its recurrence. In the last years, physical treatments have been proposed and among them the photobiomodulation therapy. In this work, the effects of Blue LED light (410-430 nm wavelength, 0.69 W/cm2 power density, 5÷60s treatment time) were evaluated on seven boundary keloid tissues by using two different colorimetric assays. Micro-Raman spectroscopy was used to explore direct effects of the Blue LED light on the endogenous cellular redox system and in particular to probe any variation in the oxidation state of the photosensitive heme-protein Cytochrome C (Cyt C) upon irradiation. We also investigated the effects of Blue LED light on membrane currents correlated to cell cycle modulation by patch-clamp recordings. Twenty-four hours after irradiation, a significant reduction of cell metabolism and proliferation was observed. The decrease in cell metabolism was maintained up to 48 hours when we found also an increased reduction in cell proliferation. Electrophysiological recordings showed an enhancement of voltage-dependent outward currents elicited by a depolarizing ramp protocol after a 30s irradiation. Data indicates that Blue LED light irradiation directly affects human keloid fibroblasts: it possesses a long lasting inhibitory effect on cell metabolism and proliferation whereas acutely increases membrane currents. Similar responses were obtained in our recent works conducted on human keloid tissues. The proposed photomodulation treatment by using Blue LED light represents a non-invasive approach in the management of hypertrophic scars and keloids.
Keloids scars are an abnormal overgrowth of fibrotic tissue in response to an injury. The current treatments show several limits and do not represent a definitive solution or a prevention protocol. In a preliminary study, we irradiated two samples of human keloid fibroblasts with a Blue LED light, evidencing a possible modulation of their activity in vitro. In the current study, we use primary fibroblasts cultures from eight keloid tissues (from seven selected patients undergoing aesthetic surgery). The fibroblasts were irradiated with a Blue LED light and the treatment time was varied in the range 5÷60s. After irradiation, cell metabolism and cell proliferation were studied by the use of two colorimetric tests, CCK-8 and SRB (Sigma-Aldrich, Saint Louis, Missouri, USA). The analysis was performed 24 and 48h after the treatment. We thus evidenced that the Blue LED light induces a modulation of the fibroblasts metabolism; this effect is particularly evident at 30s irradiation time. We also evaluated the impact of Blue LED light on membrane currents by performing whole-cell patch-clamp recordings. We observed a significant increase of voltage dependent outward currents activated by a depolarizing ramp-protocol upon Blue LED light irradiation (@30s exposure). This effect was maintained in K+ free-solutions, thus ruling out the involvement of K+ channels. In conclusion, we demonstrated that the Blue LED light has a photobiomodulation effect in fibroblasts from human keloids. This effect can be proposed as a possible treatment of the wound site in human patients to prevent keloid scars occurrence.
After irradiation with a blue LED light photocoagulator, a faster healing process is observed in superficial skin wounds. This device has been used in order to induce a thermal effect and haemostasis in superficial abrasions. Our previous in vivo study in rat and mouse models focused on the inflammatory phase within the healing process, showed a light-induced modulation, which leads to a shortened healing time and to a better recovery of the dermal tissue. Here we describe a new series of experiments that have been conducted producing two superficial abrasions on the shaved-back of mice, treating the one wound with the blue light and leaving the other one healing without any treatment. The healthy skin was used as a control. The animals were observed during healing and sacrificed at different and selected time points. Wound tissue samples have been harvested both from the treated and untreated areas and examined by histopathological and immunofluorescence analysis, SHG imaging, and confocal microscopy. The results of the study point out the interaction among different cells type and the collagen morphology restoration as obtained in different pathological mice models treated with blue LED light.
Blue LED light irradiation is currently under investigation because of its effect in wound healing improvement. In this context, several mechanisms of action are likely to occur at the same time, consistently with the presence of different light absorbers within the skin. In our previous studies we observed the wound healing in superficial abrasions in an in vivo murine model. The results evidenced that both inflammatory infiltrate and myofibroblasts activity increase after irradiation. In this study we focused on evaluating the consequences of light absorption in fibroblasts from human cells culture: they play a key role in wound healing, both in physiological conditions and in pathological ones, such as keloid scarring. In particular we used keloids fibroblasts as a new target in order to investigate a possible metabolic or cellular mechanism correlation. Human keloid tissues were excised during standard surgery and immediately underwent primary cell culture extraction. Fibroblasts were allowed to grow in the appropriate conditions and then exposed to blue light. A metabolic colorimetric test (WST-8) was then performed. The tests evidenced an effect in mitochondrial activity, which could be modulated by the duration of the treatment. Electrophysiology pointed out a different behavior of irradiated fibroblasts. In conclusion, the Blue LED light affects the metabolic activity of fibroblasts and thus the cellular proliferation rate. No specific effect was found on keloid fibroblasts, thus indicating a very basic intracellular component, such as cytochromes, being the target of the treatment.