Biological characteristics of osteoclast exosomes and their role in the osteogenic differentiation of somatic cells prior to osteogenesis

This study aimed to investigate the biological characteristics of osteoclast exocrine bodies and their role in the differentiation of somatic cells, so as to find out the key factors involved in osteoclast exosomatic growth and osteogenesis. RANKL (Receptor Activator for Nuclear Factor-κ B Ligand) induced factor was used to induce the osteoclast differentiation of Raw 264.7 cells, and TRAP (Tartrate resistant acid phosphatase) staining was employed to identify induced cells. Ultra-filtration centrifugation was used to separate OC-exosomes from osteoclast supernatant, while Western blot was employed to detect the expression characteristics of exosomal proteins CD9 and CD63. PKH67 labeled exosomes were observed to target kusao cells, which were divided into 3 groups, i.e., the complete medium group (group A), the osteoblast induced group (group B), and the osteogenesis induced liquid + OC-exosomes group (group C). The medium was changed on the next day and after 14-day culture. Using Western blot, alizarin red staining and Von Kossa silver staining, the role of OC-exosomes in the differentiation of kusao cells was clarified. Results showed that TRAP staining showed osteoclasts as irregular and TRAP positive giant cells with a red multicore and a large volume. Microcapsule membrane structures with a uniform size were detected in osteoclast supernatant, and the expression of CD9 and CD63 proteins was confirmed by Western blot. In addition, the Western blot results showed that the expression of RUNX2 (Runt-related transcription factor 2) protein in group B was 1.254 times of that in group A and 2.636 times of that in group C. Furthermore, alizarin red staining showed that the ratios of calcium salt deposition area to the total area in group A, group B and group C were 0.208%, 3.469%, and 20.724%, respectively. Von Kossa silver staining showed that the ratios of calcium salt deposition area to the total area in group A, group B and group C were 0.064%, 2.636%, and 20.872%, respectively. To sum up, OC-exosomes can promote the osteogenic differentiation of osteoblast cells (kusao cells).