Effects of sclerostin-single chain antibody fragment on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Antagonism of bone sclerosis protein can stimulate osteogenesis and increase bone synthesis and metabolism through the Wnt/β-catenin signaling pathway. OBJECTIVE: To investigate the effects of sclerostin-single chain antibody fragment (Scl-scFv) on the proliferation and osteogenic differentiation of C57BL/6 mouse bone marrow mesenchymal stem cells (BMSCs). METHODS: BMSCs were isolated from C57BL/6 mice using whole bone marrow adherence method. Alizarin red staining was performed at the 14thday of osteogenic induction, and oil red O staining performed at the 7thday of adipogenic induction. Passage 3 BMSCs were cultured with α-MEM complete medium with (experimental) or without (control) 50 μg/L Scl-scFv aScl-scFv. Real-time PCR was used to detect type 1 collagen, alkaline phosphatase, RUNX2, osteopontin, osteocalcin at the 7thday of culture and meanwhile, alkaline phosphatase staining was done; western blot assay was used to detect expression of type 1 collagen and osteopontin proteins, and ELISA was used to detect the level of osteocalcin in the cell supernatant at 4, 7, 10 days of culture. RESULTS AND CONCLUSION: Formation of calcium nodules and orangered oil droplets was obviously visible in the BMSCs after osteogenic and adipogenic induction, respectively. Over time, the absorbance value showed no difference between the experimental and control group. Compared with the control group, the experimental group showed significant increases in mRNA and protein expression of type 1 collagen and osteopontin as well as in protein expression of alkaline phosphatase, RUNX2 and osteocalcin (P < 0.05). Moreover, stronger alkaline phosphatase staining was found in the experimental group relative to the control group. These findings indicate that Scl-scFv has no effect on BMSCs proliferation,but can promote the osteogenic capacity of BMSCs in vitro.