Abstract
PURPOSE: Infrared low-level laser therapy (LLLT) has shown great promise in promoting cell proliferation and viability, making it a valuable tool in regenerative medicine. This study investigated how the interval between sessions shapes the response to 970 nm LLLT in murine osteoblast cultures by delivering three 10 J/cm² sessions separated by 24-48 h and measuring proliferation, reactive oxygen species (ROS), cytotoxicity, and apoptosis, with the goal of informing protocol design for bone regeneration. METHODS: Two osteoblast cultures were used, one control and the other LLL-treated group. The latter consisted of three irradiation sessions (10 J/cm(2) each) applied at 24, 48, and 96 h. RESULTS: The experimental results showed a significant increase in cell proliferation after two and three sessions (p < 0.05), while ROS levels progressively accumulated, peaking after the third session (p < 0.001). Cell viability remained above 90% in both groups during the first 48 h; however, a slight but significant reduction was observed in the LLLT group at 96 h. Apoptosis levels were lower in LLLT-treated cells during early phases (24-48 h), suggesting a transient cytoprotective effect that diminished after the third session. These findings indicate that infrared LLLT promotes cell proliferation without inducing cytotoxicity or programmed cell death. CONCLUSION: The results demonstrate that applying three infrared LLLT sessions of 10 J/cm² applied at 24, 48, and 96 h promotes osteoblastic proliferation and viability without inducing cytotoxicity or apoptosis. The proposed protocol, defined by energy dose and irradiation timing, provides a safe and effective strategy for bone tissue engineering.