材料诱导成骨中成骨细胞调控破骨细胞分化、凋亡的作用机制的研究

The previous studies showed that scaffold microstructure may play a key role in osteoinduction.However,the exact mechanismis currently unknown.The reasons which affected bone formation and osteoblast differentiation or not 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 RAW264.7 cell line simulating osteoclast cells to establish a co-culture system between the two cell lines in vitro, and investigate osteoblast and osteoclast progenitor biological behavior in terms of cell adhesion,viability, proliferation, differentiation and bioresorption, aimed at proposing a complete and reliable picture of bone cell behavior, comprehensive of both the osteogenesis and the bone resorption processes when the cells seeded on scaffold with different sized micropores.To investigate the detailed mechanism involved in the regulatory role of osteoblast seeded on different sized-micropore scaffold upon osteoclast differentiation, the expressions of OPG, RANKL, and EphA2 in osteoblasts and the expressions of p-p65 and EphA2 in osteoclasts were detected by RT-PCR, Western blot and immunochemistry staining. To investigate the effect of osteoblast seeded on different sized-micropore scaffold upon osteoclast apoptosis and its mechanism, flow cytometry, Hoechst staining and the expressions of Fas, FasL, Casepase-8 and Caspase-3 in osteoclasts were determined. The aim of this project is to explore the effect of osteoblast on osteoclast and its mechanism while the microstructure inducing bone formation, and to provide the experimental basis for the application of the microstructure in the bone regeneration.

目前研究显示骨组织工程中支架材料微结构在启动骨诱导性中发挥着关键作用，但是微结构启动骨生成的内在机理尚不清楚，影响骨生成与否的原因亦有待进一步研究。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（HA）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，通过体内实验筛选能否体内异位诱导成骨的微孔结构；体外实验建立MG63成骨样细株胞和RAW264.7 破骨前体细胞株共同培养体系，并在该体系中采用定量Rt-PCR、Western-blotting、流式细胞计数等方法，研究在不同大小的支架微孔结构作用下成骨细胞对破骨细胞的生物学作用，特别是对破骨细胞分化与凋亡调控的影响，并进一步研究其分子生物学机制，探讨影响骨生成的因素。本研究可望从成骨细胞和破骨细胞相互作用的角度揭示影响材料微结构诱导成骨成功与否的机理，也为材料微结构诱导成骨在将来的临床应用提供实验依据。

目前研究显示在骨组织工程中支架材料微孔结构在启动骨诱导性中发挥着关键作用，但是其启动骨生成的内在机理尚不清楚，影响骨生成与否的原因亦有待进一步研究。在这一过程中，作为骨改建的两种主要细胞成骨细胞和破骨细胞如何相互作用相互影响，目前尚不清楚。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(HA)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，通过体内实验筛选能否体内异位诱导成骨的微孔结构；体外实验建立ME3T3-E1成骨样细株胞和RAW264.7 破骨前体细胞株共同培养体系，并在该体系中采用定量Rt-PCR、Western-blotting、流式细胞计数等方法，研究在不同大小的支架微孔结构作用下成骨细胞对破骨细胞的生物学作用，特别是对破骨细胞分化与凋亡调控的影响，并进一步研究其分子生物学机制，探讨影响骨生成的因素。结果显示：在不同的微孔结构的材料中，ME3T3-E1细胞对RAW264.7细胞的生物学行为影响（粘附率的检查、增殖活性的检查、破骨细胞骨向分化方面）有差异。共培养体系中成骨细胞与破骨细胞的活性鉴定显示：HA2支架材料共培养体系中成骨细胞与破骨细胞的活性最强，HA1支架材料共培养体系中成骨细胞与破骨细胞ALP表达与TRAP表达最高。并且：HA1支架材料中ME3T3-E1细胞和RAW264.7细胞的骨向分化指标最高，HA2支架材料最能促进ME3T3-E1细胞和RAW264.7细胞的增殖活性。本研究可望从成骨细胞和破骨细胞相互作用的角度揭示影响材料微结构诱导成骨成功与否的机理，也为材料微结构诱导成骨在将来的临床应用提供实验依据。