微孔结构启动骨诱导性中Wnt经典信号通路的作用机制

The previous studies showed that scaffold microstructure may play a key role in osteoinduction.However,the exact mechanismis currently unknown.How canonical-Wnt signaling pathway which involved in bone formation and osteoblast differentiation acts in this process is still unclear and is worth of studying.Based on our previous work, hydroxyapatite micropores were produced by using different sized graphite powder as porogen excluding its effects on the scaffold's chemistrical composition and crystality. In this project, screening through in vivo experiments to decide which porous structure can induce ectopic bone formation in Beagle's. Then use human osteoblast-like MG63 cells simulating bone marrow mesenchymal cells and use mouse myoblast cell line C2C12 simulating myoblast cells, to observe the differences in different porous structure on the canonical-Wnt signaling pathway regulation in the cell osteogenic differentiation by in vitro experiments. To test this, MG63 cells and C2C12 cells were cultured respectively on microstructured hydroxyapatite plate or smooth hydroxyapatite plate. Expressions of ALP, RUNX2, OCN and collagen protein I by RT-PCR technique were used to determine the osteoblastic differentiation of the cells.Expression of Wnt pathway receptors, activators and inhibitors was measured by qPCR.To confirm that canonical signaling was involved, cells were cultured daily with exogenous Wnt3A (canonical pathway) or Wnt5A (non-canonical pathway). Alternatively, cells were cultured with antibodies to Wnt3A or Wnt5A to validate that Wnt proteins secreted by the cells were mediating cell responses to the surface.MG63 cells and C2C12 cells stably silenced for DKK1 or DKK2 are grown on hydroxyapatite surfaces DKK1 and DKK2 mRNA and protein were measured for all cell types. And to study the effect of hydroxyapatite micropore on the osteoblast differentiation of cultured cells. The aim of this project is to explore the mechanism of the microstructure inducing bone formation by cell signaling pathway, and to provide the experimental basis for the application of the microstructure in the bone regeneration.

目前研究显示材料微结构在启动骨诱导性中发挥着关键作用，但是微结构启动骨诱导性的内在机制尚不清楚，作为在成骨过程中发挥关键作用的Wnt细胞信号通路如何在该过程中发挥作用亦有待进一步研究。研究者在前期研究的基础上，利用碳粉作为造孔剂来形成羟基磷灰石支架的微孔结构、控制微孔的大小并排除对材料的化学构成的影响，通过体内实验筛选出能够体内异位成骨的微孔结构；并以MG63细胞模拟骨髓间充质细胞，以C2C12细胞模拟成肌细胞，通过体外实验观察不同微孔结构在细胞骨向分化过程中对Wnt经典信号通路调控影响的差异、不同微孔结构对细胞骨向分化的影响；并进行Wnt信号通路抑制剂DKK1、DKK2干预实验来研究Wnt信号抑制剂对细胞骨向分化的影响。课题旨在探讨Wnt经典信号通路在微孔结构启动骨诱导性中的机制，从而为微结构在骨再生中的应用提供实验依据。

目前研究显示材料微结构在启动骨诱导性中发挥着关键作用，但是微结构启动骨诱导性的内在机制尚不清楚，作为在成骨过程中发挥关键作用的wnt细胞信号通路如何在该过程中发挥作用亦有待进一步研究。研究者在前期研究的基础上，利用碳粉作为造孔剂来形成羟基磷灰石支架的微孔结构、控制微孔的大小并排除对材料的化学构成的影响，通过体内实验筛选出能够体内以为成骨的微孔结构；并以MG63细胞模拟骨髓间充质细胞，通过体外实验观察不同微孔结构在细胞骨向分化过程中对wnt经典信号通路调控影响的差异、不同微孔结构对细胞骨向分化的影响；并进行wnt信号通路抑制剂DKK1、DKK2干预实验来研究wnt信号抑制剂对细胞骨向分化的影响。结果显示：定性RT-PCR示MG63在材料表面培养8天后, RUNX2在不同材料的表面的表达并不相同,在HAP5上可有阳性表达，在HAP1表面有弱阳性表达，而在HAP0和对照组则无明显表达。ALP在HAP5上可有弱阳性表达，在HAP1， HAP0和对照组则无明显表达；定量RT-PCR示ALP在三种不同材料表面的表达并不相同P<0.05），在HAP5组的表达要高与其它组，RUNX2在不同材料的表面的表达有差异P<0.05），HAP5及HAP1组要高与HAP0组，HAP5组与HAP1组之间有差异。同样，MG63细胞在不同微孔大小的HA表面培养，在培养液中分别加入Wnt3a 或 Wnt5a干预，结果显示：Wnt3a能促进MG63细胞的骨向分化和BMP的生成。DKK1沉默能减少MG63细胞的增殖，但是会促进细胞骨向分化，而DKK2沉默能抑制细胞骨向分化。本课题结果提示：支架材料微孔结构在启动骨诱导中发挥着重要作用，在这一过程中经典信号通路可能在其中起重要作用。