Impact of Melatonin on RAW264.7 Macrophages during Mechanical Strain.

The concentration of melatonin is elevated during the night when patients mainly wear removable orthodontic appliances. Next to periodontal ligament fibroblasts and osteoblasts, macrophages react to mechanical strain with an increased expression of inflammatory mediators. Here, we investigated the impact of melatonin on RAW264.7 macrophages exposed to tensile or compressive strain occurring during orthodontic tooth movement in the periodontal ligament. Before exposure to mechanical strain for 4 h, macrophages were pre-incubated with different melatonin concentrations for 24 h, to determine the dependence of melatonin concentration. Afterwards, we performed experiments with and without mechanical strain, the most effective melatonin concentration (25 µM), and the melatonin receptor 2 (MT2) specific antagonist 4P-PDOT. The expression of inflammatory genes and proteins was investigated by RT-qPCR, ELISAs, and immunoblot. Both tensile and compressive strain increased the expression of the investigated inflammatory factors interleukin-1-beta, interleukin-6, tumor necrosis factor alpha, and prostaglandin endoperoxide synthase-2. This effect was inhibited by the addition of melatonin. Incubation with 4P-PDOT blocked this anti-inflammatory effect of melatonin. Melatonin had an anti-inflammatory effect on macrophages exposed to mechanical strain, independent of the type of mechanical strain. As inhibition was possible with 4P-PDOT, the MT2 receptor might be involved in the regulation of the observed effects.

Impact of melatonin on periodontal ligament fibroblasts during mechanical strain.

BACKGROUND: The endogenous hormone melatonin regulates the circadian rhythm and impacts on bone metabolism. As patient compliance to wear removable orthodontic appliances is generally higher at night, when melatonin release is increased, a boosting effect on tooth movement would be favourable for therapy, whereas an inhibiting effect would indicate daytime wear to be more therapy-effective. We hypothesize that melatonin has either a stimulating or impeding effect on the expression profile of periodontal ligament fibroblasts (PDLF) during simulated orthodontic compressive and tensile strain, which would suggest either an accelerating or inhibiting impact on orthodontic tooth movement in vivo. METHODS: PDLF were preincubated with melatonin for 24 h and then subjected to tensile or compressive strain to mimic tension and pressure sides in PDL. In addition, the selective melatonin MTNR1B-receptor antagonist 4P-PDOT was used. We investigated melatonin effects on collagen synthesis, expression of inflammatory and bone-remodelling genes/proteins by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assays, and total collagen assays. PDLF-induced osteoclastogenesis was analysed in a coculture model by tartrate-resistant acid phosphatise (TRAP) staining. RESULTS: Expression of melatonin receptors in PDLF was not affected by compressive strain. Melatonin increased expression of inflammatory factors and elevated collagen synthesis during mechanical strain. Melatonin showed no effects on OPG or RANKL expression without mechanical strain, but increased RANKL gene expression during compression. CONCLUSIONS: Expression of melatonin receptors by PDLF enable them to detect fluctuating melatonin concentrations in the periodontal ligament. Melatonin increased collagen synthesis and expression of inflammatory mediators, but had no effect on genes involved in bone remodelling. Therefore, we suggest that melatonin has no accelerating effect on PDLF-induced osteoclastogenesis.