Study Of Dynamic Pressure Promote The Angiogenesis Of Bone Matrix Objective

ObjectiveTo research the efficient way of isolating and proliferating of induced EPCs from the rat marrow MNCs, to observe the main biological characteristic of induced EPCs in vitro. Identify it by means of immunohistochemistry and immunofluorescence. PrePared DBM samples of pig in vitro, observe the morphology by Scanning electron microscope. To culture and observe induced EPCs in DBM. Put the complex of induced EPCs and DBM on the bioreactor, exert them appropriate pressure. We could observe the ability to form blood vessels.MethodsThe separation, culture, purifies and identification of EPCs Use the Ficoll density gradient centrifuge combined with difference-speed adherence screening method to separate MNCs from rat bone marrow. Identify the induced EPCs by means of immunohistochemistry and immunofluorescence. Through the organization of fixed, defatted, decalcified and other steps use of spine vertebral body, we prepared DBM samples of pig in vitro. Divided scaffolds into two groups A and groups B. Induced EPCs were seeded into DBM. The cell-seeded scaffolds of groups A were dynamically loaded in compression using a sine wave at 1 Hz,5% strain in the media-filled chamber for 4 h on days 5 of culture. We did not interfere the groups B.Both of two groups were cultured two weeks.Then we observed the ability of EPCs to form blood vessels. Primer design; Extract total RNA from cells with TrIzol; Reverse transcription reaction; PCR.ResultsAt the beginning of original generation cultured, the cells are small or big and circular majority. After 24h, difference-speed adherence. After 36h,a few cells adhere, some cells gradually extend to spindle-shaped. At 7th day, there are cell colonies, at 10-12d, the cells fuse to the monolayer like endothelial cell as the paving-stone shape.The cells paved surface of the scaffolds. We could see neighboring cells fused with each other. There were formation of blood vessels. It is obvious that A group more than B group. Test the mRNA expression of vWF and F1k-1 during the EPCs differentiationby RT-PCR. A group was significantly stronger than that of B group.ConclusionsThe method of Ficoll density gradient centrifuge combined with difference-speed adherence screening method may separate EPCs from rat bone marrow. Growth state stability, and proliferative capacity. We considerd that EPC is a kind of ideal seed cells. DBM is a good kind of carrier cage material,when it combines together with EPC,,it has become organization engineering bone,then we give press on it,the bone graft has been vascularized,so it has clinical application on the direction of repair bone defect.