Background Periosteum plays critical roles in de novo bone formation and fracture repair. Wnt16 has been regarded as a key regulator in periosteum bone formation. However, the role of Wnt16 in periosteum derived cells (PDCs) osteogenic differentiation remains unclear. The study goal is to uncover whether and how Wnt16 acts on the osteogenesis of PDCs. Methods We detected the variation of Wnt16 mRNA expression in PDCs, which were isolated from mouse femur and identified by flow cytometry, cultured in osteogenic medium for 14 days, then knocked down and over-expressed Wnt16 in PDCs to analysis its effects in osteogenesis. Further, we seeded PDCs (Wnt16 over-expressed/vector) in β-tricalcium phosphate cubes, and transplanted this complex into a critical size calvarial defect. Lastly, we used immunofluorescence, Topflash and NFAT luciferase reporter assay to study the possible downstream signaling pathway of Wnt16. Results Wnt16 mRNA expression showed an increasing trend in PDCs under osteogenic induction for 14 days. Wnt16 shRNA reduced mRNA expression of Runx2, collage type I (Col-1) and osteocalcin (OCN) after 7 days of osteogenic induction, as well as alizarin red staining intensity after 21days. Wnt16 also increased the mRNA expression of Runx2 and OCN and the protein production of Runx2 and Col-1 after 2 days of osteogenic stimulation. In the orthotopic transplantation assay, more bone volume, trabecula number and less trabecula space were found in Wnt16 over-expressed group. Besides, in the newly formed tissue Brdu positive area was smaller and Col-1 was larger in Wnt16 over-expressed group compared to the control group. Finally, Wnt16 upregulated CTNNB1/β-catenin expression and its nuclear translocation in PDCs, also increased Topflash reporter luciferase activity. By contrast, Wnt16 failed to increase NFAT reporter luciferase activity. Conclusion Together, Wnt16 plays a positive role in regulating PDCs osteogenesis, and Wnt16 may have a potential use in improving bone regeneration.

中文翻译：

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Wnt16信号在体外和体内促进骨膜衍生细胞的成骨细胞分化

背景骨膜在从头骨形成和骨折修复中起关键作用。Wnt16 被认为是骨膜骨形成的关键调节因子。然而，Wnt16 在骨膜衍生细胞 (PDC) 成骨分化中的作用仍不清楚。研究目标是揭示 Wnt16 是否以及如何作用于 PDC 的成骨。方法我们检测PDCs中Wnt16 mRNA表达的变化，从小鼠股骨中分离并通过流式细胞仪鉴定，在成骨培养基中培养14天，然后在PDCs中敲低并过表达Wnt16，以分析其在成骨中的作用。此外，我们将 PDC（Wnt16 过表达/载体）接种到 β-磷酸三钙立方体中，并将​​该复合物移植到临界大小的颅骨缺损中。最后，我们使用免疫荧光，Topflash 和 NFAT 荧光素酶报告基因检测，研究 Wnt16 可能的下游信号通路。结果Wnt16 mRNA表达在成骨诱导14天后PDCs中呈增加趋势。Wnt16 shRNA 在成骨诱导 7 天后降低了 Runx2、collage type I (Col-1) 和骨钙素 (OCN) 的 mRNA 表达，以及 21 天后的茜素红染色强度。Wnt16 还增加了成骨刺激 2 天后 Runx2 和 OCN 的 mRNA 表达以及 Runx2 和 Col-1 的蛋白质产生。在原位移植实验中，Wnt16过表达组的骨量、小梁数量较多，小梁间隙较少。此外，与对照组相比，Wnt16过表达组新形成的组织中Brdu阳性区域较小，Col-1较大。最后，Wnt16 上调 CTNNB1/β-catenin 表达及其在 PDC 中的核转位，也增加了 Topflash 报告荧光素酶活性。相比之下，Wnt16 未能增加 NFAT 报告荧光素酶活性。结论 Wnt16在调节PDCs成骨中发挥积极作用，Wnt16可能在改善骨再生方面具有潜在用途。