IMPE Abstracts

Objective: The growth plate is the metabolically highly active cartilage portion of the bone where linear growth takes place. Chondrocytes in this region are affected by many hormones and local factors in the process of proliferation, maturation, hypertrophy, extracellular vesicle release and cell death. Melatonin, one of these factors, affects blastic activity, proliferation and mineralization in a circadian rhythm through its receptors in cartilage. In the growth plate model created as stated in the literature, effects of melatonin-stimulated exosomes (MSE) originating from adipose-derived mesenchymal stem cells were examined.

Methods: MSE was added to the cultured growth plate chondrocytes in various concentrations (25, 50, 100 μg/ ml), to assess cytotoxicity-cell viability at days 1, 2 and 3 using an MTS assay. In addition, in vitro scratch assays were performed to analyse the migration capacity of chondrocytes when treated with MSE. Cell cycle progression for different doses of MSE in growth plate chondrocytes was evaluated by flow cytometry. The expression of cartilage extracellular matrix components (Types I and II collagen and aggrecan) was evaluated using reverse transcription coupled polymerase chain reaction (RT-PCR).

Results: MTS assay showed that the cell viability was found to reduce upon MSE (25 and 50 μg/ml) treatment compared to the control group. The addition of 100 μg/ml was found to significantly increase the cell viability of chondrocytes treated with MSE. The results of the scratch assay test revealed MSE group with concentration 100 µg/ml significantly increased migration of chondrocytes compared to experimental groups at 24 and 48-hour time points. Flow cytometry analyses showed that proportion of chondrocytes in S phase increased significantly with subsequent reduction in G0/G1 phase of cell cycle on treatment with MSE (P< 0.05). Thus, MSE increased the rate of chondrocyte proliferation by promoting transition through G1/S phase of cell cycle. Real-time PCR demonstrated that MSE induced the expression of SOX9, COL2A1, and aggrecan, but at the same time simultaneously inhibited the expression of COL1A1.

Conclusion: We observed that in growth plate homeostasis, melatonin and exosome can be effective when they are together. These results support further investigation of melatonin in combination with exosome for potential use in growing children requiring treatment.

Exosome, melatonin, growth plate