Melatonin promoted osteogenesis of human periodontal ligament cells by regulating mitochondrial functions through the translocase of the outer mitochondrial membrane 20

Background and objective: Melatonin plays an important role in various beneficial functions, including promoting differentiation. However, effects on osteogenic differentiation, especially in human periodontal cells (hPDLCs), still remain inconclusive. Mitochondria are highly dynamic organelles that play an important role in various biological processes in cells, including energy metabolism and oxidative stress reaction. Furthermore, the translocase of the outer mitochondrial membrane 20 (TOM20) is responsible for recognizing and transporting precursor proteins. Thus, the objective of this study was to evaluate the functionality of melatonin on osteogenesis in human periodontal cells and to explore the involved mechanism of mitochondria.

Methods: The hPDLCs were extracted and identified by flow cytometry and multilineage differentiation. We divided hPDLCs into control group, osteogenic induction group, and osteogenesis with melatonin treatment group (100, 10, and 1 μM). Then we used a specific siRNA to achieve interference of TOM20. Alizarin red and Alkaline phosphatase staining and activity assays were performed to evaluate osteogenic differentiation. Osteogenesis-related genes and proteins were measured by qPCR and western blot. Mitochondrial functions were tested using ATP, NAD+/NADH, JC-1, and Seahorse Mito Stress Test kits. Finally, TOM20 and mitochondrial dynamics-related molecules expression were also assessed by qPCR and western blot.

Results: Our results showed that melatonin-treated hPDLCs had higher calcification and ALP activity as well as upregulated OCN and Runx2 expression at mRNA and protein levels, which was the most obvious in 1 μM melatonin-treated group. Meanwhile, melatonin supplement elevated intracellular ATP production and mitochondrial membrane potential by increasing mitochondrial oxidative metabolism, hence causing a lower NAD⁺ /NADH ratio. In addition, we also found that melatonin treatment raised TOM20 level and osteogenesis and mitochondrial functions were both suppressed after knocking down TOM20.

Conclusion: We found that melatonin promoted osteogenesis of hPDLCs and 1 μM melatonin had the most remarkable effect. Melatonin treatment can reinforce mitochondrial functions by upregulating TOM20.