Suppression of osteogenic differentiation and mitochondrial function change in human periodontal ligament stem cells by melatonin at physiological levels

Abstract

N-Acetyl-5-methoxytryptamine (melatonin, MT) at pharmacological concentrations promotes the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells; however, its role at physiological concentrations (1 pM–10 nM) remains unclear. We explored the effects of 1 pM–1 µM MT on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and its underlying mitochondrial dynamics-mediated mechanisms. T he PDLSC phenotype was detected by flow cytometry and evaluated for three-line differentiation. Alkaline phosphatase activity assay and Alizarin red staining were used to evaluate osteogenic differentiation. Osteogenesis-related gene and protein expression levels were measured by quantitative reverse transcription -polymerase chain reaction and western blotting. Mitochondrial function assays were performed using reactive oxygen species, ATP and NAD+/NADH kits and molecular mechanisms of mitochondrial dynamics-related proteins were assessed by western blotting. Our results have shown that physiological MT concentrations induced differentiation of hPDLSCs and down-regulated osteopontin (OPN) and osteocalcin (OCN) expression levels, which were restored or even up-regulated by 1 µM MT (lowest pharmacological concentration). Compared to the osteogenic induction alone, this treatment decreased the intracellular ATP content, whereas the intracellular reactive oxygen species level and NAD+/NADH ratio were increased. Mitochondrial function- and dynamics-related protein expression levels were consistent with those of osteogenic genes following osteogenic induction and MT treatment of hPDLSCs at various physiological concentrations. Physiological MT concentrations inhibited the osteogenic differentiation of hPDLSCs and simultaneously altered mitochondrial function. These findings provide insights into the stem cell tissue engineering and functions of MT.

Effect of Melatonin for Regulating Mesenchymal Stromal Cells and Derived Extracellular Vesicles

“…Nevertheless, some experiments have produced conflicting results. In contrast to previous findings, one study reported that physiological melatonin concentrations (1 pM–10 nM) inhibited the osteogenic differentiation of human periodontal ligament stromal cells, while 1 μM melatonin (the lowest pharmacological concentration) promoted it ( Zheng et al, 2020 ). The acute melatonin treatment was also more effective than chronic exposure, which may be due to the desensitization of the melatonin receptors ( Radio et al, 2006 ).…”

Combined Effects of Low‐Frequency Pulsed Electromagnetic Field and Melatonin on Ovariectomy‐Induced Bone Loss in Mice

“…We found that PEMF therapy had a positive effect on the expression of OPN, which is similar to the findings of Yin et al [2018]; however, MEL seemed to have no effect on the expression of this gene. Recently, Zheng et al [2020] demonstrated that the OPN expression level in human periodontal ligament stem cells (hPDLSCs) was downregulated by physiological MEL concentrations (1 pM-10 nM) and upregulated by the lowest pharmacological concentration (1 µM). Moreover, in a study on the mandibular distraction osteogenesis in rats, Acikan et al [2018] found that the OPN level was higher in the MEL groups with dosages of 25 and 50 mg/kg than in the control group, and the 50 mg/kg dose group had higher OPN levels than the 25 mg/kg dose group (P < 0.05).…”

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

“…According to the pre‐experiments, we observed that RNA and protein level reached the peak at the 8th day after osteogenic induction and melatonin treatment. Consequently, we conducted the measurement of osteogenesis and subsequent mitochondrial function tests, which is consistent with most previous literature on the osteogenesis of periodontal ligament cells 14,52,53 …”

“…Park et al also confirmed that melatonin promoted osteoblastic differentiation through the BMP/ERK/Wnt signaling pathways 50 . However, our previous research revealed that physiological concentrations of melatonin (10 nM, 100 pM, and 1 pM) had suppressed osteogenesis of hPDLSCs 14 . A possible explanation could be that regulating effect of melatonin is concentration‐dependent, Qiu et al 51 reported that melatonin inhibited the proliferation, differentiation, and apoptosis of osteoblasts in a concentration‐dependent manner.…”

“…Some randomized controlled clinical trials demonstrated that melatonin treatment was associated with reduction in periodontal inflammation and can significantly improve clinical periodontal parameters, such as probing depth and clinical attachment loss 11,12 . However, some studies found that melatonin could suppress both osteoblasts and osteoclasts, 13 and physiological levels of melatonin can suppress osteogenesis of osteogenic differentiation of hPDLSCs by inhibiting mitochondrial functions 14 . Therefore, the influence of melatonin on osteogenesis of human periodontal ligament cells is not thoroughly understood and still needs further exploration.…”