ESPE Abstracts

Introduction: Human breast milk (HBM) contains various components with critical roles in supporting early human growth and development. HBM is highly enriched in miRNAs, short, non-coding RNAs of 18 to 25 nucleotides in length, involved in the development, differentiation, proliferation, metabolism, and death of cells and tissues. miRNAs have been linked to growth control, apoptosis, epigenetic modifications, developmental programming, stem cell differentiation, and increase growth by targeting genes involved in development. The growth plate shows growth activity only with breast milk products for the first six months, and continues to work in the following period in the interaction of other sources and hormones taken with nutrition. This study explores the functions of exosomes derived from HBM on growth plate cell lines in vitro.

Methods: Breast milk exosomes were isolated and characterized by positive CD63 and negative calnexin markers from western blot, nanoparticle tracking analysis and transmission electron microscopy. To assess cytotoxicity-cell viability using an MTS assay, cultured growth plate chondrocytes were incubated with HBM exosomes and the effective dose was determined. As a result of MTS analysis, 200 mg/dl was found to be optimum. in vitro scratch experiments were performed to analyze the effects of exosomes on the migratory capacity of chondrocytes. Cell cycle progression for different doses of exosomes in growth plate chondrocytes was assessed by flow cytometry. Expression of cartilage extracellular matrix components (Types I and II collagen and aggrecan) was assessed using reverse transcription coupled polymerase chain reaction (RT-PCR). As a result of PCR experiments, the amount of increase and decrease in gene expressions were analyzed.

Results: HBM exosomes can be endocytosed and effects of exosome application on growth plate cells were analyzed by MTS, Scratch assay, cell cycle and PCR techniques. After determining the optimum dose with MTS analysis, genes and expressions specific to chondrocytes and extracellular matrices such as aggrecan, type I and II collagen, Sox-9 were analyzed by PCR. HBM exosome treatment significantly attenuated the inhibitory effect of IL-1β on the proliferation and migration of chondrocytes. Exosomes activated signaling associated with extracellular matrix production of chondrocytes, a key signaling event in chondrocyte metabolic activities. It also increased expression of genes specific for growth, extracellular matrix secretory function and survival of cartilage cells. Finally, exosomes significantly increased chondrocyte proliferation and no toxic effects were observed.

Conclusion: In conclusion, our data identified HBM derived exosomes as a crucial regulator of chondrogenesis and may represent a potential strategy for the treatment of growth disorder.