Department of Biotechnology, Biomedical and Translational Sciences, University of Parma, Parma, Italy.
Clin Oral Implants Res. 2014 Feb;25(2):e133-9. doi: 10.1111/clr.12075. Epub 2012 Dec 5.
Phosphoserine-based functionalization has been proposed as a tool to improve integration of endosseous implants by promoting osteoblast adhesion and differentiation in vitro. The present work investigates whether phosphoserine-tethered poly(epsilon-lysine) dendrons, when applied as a film to titanium surfaces, enhance the differentiation of osteoblastic cells and the activation of Wnt/β-catenin signaling.
These films were tested in a murine model of calvaria-derived MC3T3 osteoblastic cells, primary bone marrow cells and mesenchymal, undifferentiated C2C12 cells. Gene expression was assayed by Real Time PCR, and activation of Wnt signaling pathway was measured with a reporter assay.
Dendrons increased expression of alkaline phosphatase and osteocalcin, two osteoblastic markers, in both murine osteoblastic MC3T3 cells and primary bone marrow cells. The expression of osteoprotegerin, a protein opposing osteoclastogenesis was also significantly higher in cells growing on dendron-coated substrates both at 3 and 6 days of culture. Similarly, the mRNA levels of Wisp-2 and of β-catenin, two Wnt target genes, were also markedly increased in this group at day 6. The activation of this signaling pathway in cells growing on the dendron-coated surfaces was confirmed by use of a TCF/β-catenin reporter system in the C2C12 cell line.
The findings of the present study show that phosphoserine-tethered poly(epsilon-lysine) dendron films act as stimuli for the activation of specific signal cascades and promote the differentiation of adhering progenitor cells into an osteoblastic phenotype.
磷酸丝氨酸基功能化已被提议作为一种工具，通过促进成骨细胞在体外的黏附和分化来提高骨内植入物的整合。本研究旨在探讨将磷酸丝氨酸键合的聚（ε-赖氨酸）树突应用于钛表面作为薄膜时，是否会增强成骨细胞的分化和 Wnt/β-连环蛋白信号的激活。
这些薄膜在鼠颅骨衍生的 MC3T3 成骨细胞、原代骨髓细胞和间充质、未分化的 C2C12 细胞的模型中进行了测试。通过实时 PCR 检测基因表达，并通过报告基因测定测量 Wnt 信号通路的激活。
树突增加了两种成骨细胞标志物，即碱性磷酸酶和骨钙素在鼠源性成骨细胞 MC3T3 细胞和原代骨髓细胞中的表达。在培养 3 和 6 天时，在树突涂层底物上生长的细胞中，骨保护素的表达也显著更高，骨保护素是一种对抗破骨细胞形成的蛋白。同样，在第 6 天，Wisp-2 和β-连环蛋白（两种 Wnt 靶基因）的 mRNA 水平也在该组中明显增加。在 C2C12 细胞系中使用 TCF/β-连环蛋白报告系统证实了该信号通路在树突涂层表面生长的细胞中的激活。
本研究的结果表明，磷酸丝氨酸键合的聚（ε-赖氨酸）树突薄膜作为刺激物，激活特定的信号级联，并促进附着的祖细胞分化为成骨细胞表型。
