Green LED and three‐dimensional (3D) scaffolds have recently received extensive attentions due to their impact on cell proliferation and differentiation. Melatonin, a circadian rhythm‐regulating hormone, is involved in some physiological phenomena including testosterone biosynthesis. Lower testosterone biosynthesis results in some disorders such as puberty retarding, andropause, and muscle weakness. Therefore, our aim was to investigate the proliferation of Leydig cells and their testosterone‐related Gene expression and secretion under the influence of 3D scaffold, green light and melatonin. The experimental groups of TM3 cells embedded in the 3D scaffold, were exposed to green light, melatonin, both and all three factors. Expression of cell cycle genes including PCNA, CYCLIND1, CDC2 and CDKN1B, and testosterone related genes; GATA4 and RORα were also examined. 3D scaffold enhanced Leydig cells proliferation, and testosterone‐related genes expression. While melatonin decreased cell proliferation and testosterone‐related genes expression. Green light did not significantly change the results but slightly decreased cell proliferation and testosterone synthesis. The combination of green light with melatonin significantly reduced the proliferation rate of TM3 cells and the expression of steroidogenic genes, while the combination of green light with scaffold improved the results. In general, the use of scaffolding enhances proliferation and testosterone‐related genes expression of TM3 Leydig cells. Also, application of green light and scaffolding reduces the deleterious effects of melatonin on these cells.

Different effects of fibrin scaffold, green LED and melatonin on cell proliferation and differentiation have received extensive attentions. Testosterone, the most important sex hormone is secreted by Leydig cells to regulate spermatogenesis and many other phenomena in the body organs. The aim of the present study was to evaluate the proliferation of Leydig cells and their testosterone secretion under the influence of three-dimensional scaffold culture, green light irradiation and melatonin. The experimental groups, were TM3 cells incorporated in 3D scaffold culture, exposed to green light and also exposed to melatonin individually, in pairs and three factors together. Cell proliferation and testosterone secretion were measured after 72 hr. Cell cycle genes including PCNA, CYCLIND1, CDC2 and CDKN1B and, testosterone related genes; GATA4 and RORα were also examined. In general, three-dimensional scaffold increased Leydig cells proliferation, testosterone secretion and the expression of the related genes. On the other hand, melatonin decreased cell proliferation and testosterone synthesis. Green light did not significantly change the results but slightly decreased cell proliferation and testosterone synthesis. Combination of green light with melatonin significantly reduced the outcome while, light with scaffold increased the results. Application of light + scaffold + melatonin covered the decreasing effect of melatonin.