Melatonin Promotes Osteoblast Differentiation and Improves Fracture Healing in Mice by Activating TrkA

Background: Melatonin (MLT) is a hormone that regulates circadian rhythms and has various biological functions. Recent studies have indicated that MLT also plays a significant role in the skeletal system by modulating bone formation and resorption. The aim of this study is to investigate the impact of MLT on the in vitro osteogenic differentiation of MC3T3-E1 cells, a mouse preosteoblast cell line, and the in vivo healing of fractures in mice. Methods: The mice underwent bilateral fibular fractures for a duration of 2 weeks. MLT was given at 14 days post-fracture, after which time they were sacrificed. Staining with hematoxylin and eosin (H&E) was used to analyze the callus. MC3T3-E1 cells were exposed to MLT for 7 days. The extent of osteogenic differentiation was assessed using alizarin red staining. The expressions of alkaline phosphatase (ALP), collagen type 1 alpha 1 chain (COL1A1), osteocalcin (OCN), and runt-related transcription factor 2 (Runx2) were detected using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Additionally, the expressions of tropomyosin receptor kinase A (TrkA), metallothionein-1 (MT1) and metallothionein-2 (MT2) in animal tissues and cells were detected by Western blotting. Results: The expression of murine (mouse) calvaria 3T3-E1 (MC3T3-E1) cells treated with MLT was observed. The MLT group exhibited a higher proportion of osteogenic differentiation than the Control group, as evidenced by alizarin red staining and qRTPCR detection of osteogenic differentiation markers (p < 0.01). The Western blot analysis revealed a higher expression of MLT receptors in the MLT group than the control group (p < 0.01). TrkA expression was enhanced following MLT administration (p < 0.01). Additional studies revealed that the level of osteogenic differentiation in the overexpression tropomyosin receptor kinase A (OE-TrkA) group surpassed that of the MLT group (p < 0.001) but was decreased in the Si-TrkA group. MT1 and MT2 expressions were increased in the OE-TrkA group but were decreased in the Si-TrkA group (p < 0.001). Mechanical and histological tests in animal models demonstrated an augmentation in the level of osteoclast differentiation following MLT treatment (p < 0.01). The Western blots revealed an upregulation in TrkA expression following MLT treatment (p < 0.01). The utilization of TrkA activator and inhibitor revealed that MLT stimulated osteogenic differentiation and facilitated fracture healing through the activation of TrkA (p < 0.01). Conclusion: In summary, our study reveals that MLT stimulates the fracture healing in mice and the osteogenic differentiation of MC3T3-E1 cells through the activation of TrkA signaling pathway. Our findings suggest that MLT is a potential therapeutic agent for enhancing bone regeneration and repairing bone defects.