Effects of enoxaparin on osteogenic differentiation of bone marrow stem cells and their exosomes / 中华创伤骨科杂志

ABSTRACT

Objective:To study the effects of enoxaparin on osteogenic differentiation of bone marrow stem cells (BMSCs) and the exosomes derived from BMSCs.Methods:After the BMSCs from 4-week old male SD rats were cultured, their surface antigen and multilineage differentiation potentials were identified. Subsequently, the BMSCs were incubated with osteogenic differentiation medium containing 10 IU/mL enoxaparin for 14 days. After the exosomes derived from BMSCs (BMSC-Exos) were extracted by the kit method, their structure was observed by transmission electron microscopy and their surface antigen CD63 detected by Western blot. Alizarin red staining was used to analyze the osteogenic differentiation of BMSCs. Osteogenic proteins including OCN and BMP-2 in BMSCs and exosomes were detected.Results:The spindle-shaped BMSCs isolated and cultured showed a uniform spiral pattern. They expressed highly the surface markers CD29 and CD44 but lowly CD34 and CD45, indicating that the majority of the cells were BMSCs. BMSC-Exos, in an oval shape with a diameter of about 30 to 80 nm, expressed CD63. Alizarin red staining showed that the number of mineralized nodules in the enoxaparin treatment group was significantly lower than that in the control group. Western blot analysis indicated that enoxaparin inhibited the expression of osteocalcin (OCN) and BMP-2 in BMSCs. ELISA results showed that the protein levels of OCN(48.81 ng/mL ± 8.23 ng/mL) and BMP-2 (311.45 pg/mL ± 27.59 pg/mL) in the BMSCs treated with enoxaparin were significantly lower than those of OCN (80.43 ng/mL ± 10.74 ng/mL) and BMP-2 (399.23 pg/mL ± 32.25 pg/mL) in the control BMSCs ( P<0.05). The contents of OCN (1.45 ng/mL±0.15 ng/mL) and BMP-2 (18.47 pg/mL ± 0.54 pg/mL) in the exosomes from BMSCs treated with enoxaparin were significantly higher than those of OCN (1.00 ng/mL ± 0.12 ng/mL) and BMP-2 (9.07 ng/mL ± 0.36 pg/mL) in the exosomes from control BMSCs ( P<0.05). Conclusions:Enoxaparin may inhibit the osteogenic differentiation of BMSCs. The mechanism of this might be related to its direct inhibition against the expression of OCN and BMP-2 in BMSCs and its indirect reduction of OCN and BMP-2 in BMSCs through the exhaust of the above proteins in the form of exosomes.