Combination effect of low-level laser and orthokine for improving wound healing: In vitro study

Wound healing is a complex biological process that relies heavily on fibroblast viability, oxidative balance, and regulated cell proliferation. Low-level laser therapy (LLLT) and autologous conditioned serum (ACS), under the tradename of Orthokine, have each been reported to modulate inflammation and support tissue repair, yet their combined cellular effects remain insufficiently defined. This study aimed to evaluate the individual and combined effects of these compounds on the human dermal fibroblast (HDF) cell line. The HDF cell line was cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS). Following this, the cell line was treated with LLLT doses (2, 5, 7, and 10 J/cm²) and Orthokine concentrations (1, 2, 3, 5, and 10% v/v). Viability and proliferation were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Reactive Oxygen Species (ROS) measurement, apoptosis, and cell cycle phase analysis were performed by flow cytometry. Results showed that cell viability following Orthokine treatment increased in a dose- and time-dependent manner, and the highest effects were observed at 5% and 10%. LLLT treatment also enhanced viability. The combination of 5% Orthokine with 5 J/cm² LLLT produced the most notable effects, improving viability, decreasing apoptosis, and increasing S-phase frequency. LLLT and Orthokine positively influence fibroblast function for wound healing. Their combination improved cell viability, preventing apoptosis and promoting cell cycle progression. This approach may contribute to the development of more effective, non-invasive wound healing therapies.