Stichopus japonicus Polysaccharide Stimulates Osteoblast Differentiation through Activation of the Bone Morphogenetic Protein Pathway in MC3T3-E1 Cells

on osteogenic differentiation of MC3T3-E1 cells. Osteogenic differentiation was measured via histochemical staining of alkaline phosphatase (ALP) assay, alizarin red staining of mineralization assay, Western blotting, ELISA, and a qRT-PCR evaluation for the expression of BMP-2, runt-related transcription factor-2 (Runx-2), osteocalcin (OCN), osteopontin (OPN), and collagen type I (Col I) in MC3T3-E1 cells. Immunofluorescence assay was utilized to assess the BMP-2 localized on the cell surface. The results illustrated that SP-2 was able to increase ALP expression and accelerate the mineralization. Osteoblasts cultured on BMP-2/SP-2 substrate increased the expression levels of BMP-2, Runx-2, Col I, OCN, and OPN. SP-2 increased the binding efficiency involving a BMP-2 and its cell surface receptor. The dose of 5 μg/mL SP-2 used showed the best function of inducing osteoblast differentiation. These findings indicated that SP-2 is a more effective enhancer that cooperated with BMP-2 to induce osteoblastic differentiation by utilizing the BMP-2 signaling pathway.