Research Of ECs Induced From MNCs Of Rabbit Marrow Improving Vascularization Of DBM

Tissue engineering provide a new approach to reconstruct large bone defects. Researchers have made great progress in many aspects,such as:cells,scaffold,cell factors and cell culture, but seeding cells can not get enough nutrition from blood,then vascularization of bone-tissue substitutes is necessary.Bone derived stem cells are potential stem cells,and bone marrow MNCs can be differentiated into cells with endothelial phenotype.Researchers find bone marrow derived ECs can improve neovascularization in vivo.DBM is a hopeful scaffold of bone tissue engineering for its construction and compatibility.Due to these analysis,we want to find a simple way to isolate and differentiate ECs from bone marrow marrow mononuclear cells(MSCs),gain enough induced ECs, find an effective way to seed derived cells into DBM,culture and observe derived cells on the scaffold,then implant these complex of derived ECs and scaffold into rabbit bone defect model to observe vascularization of DBM and healing of bone defect.Objective:1. To research the efficient way of isolating and proliferating of induced ECs from the rabbit marrow MNCs,to measure the number of induced ECs and to observe the main biological characteristic of induced ECs in vitro and gain enough induced ECs.2. To research the growth and proliferation of induced ECs in fibrin sealant in vitro;to culture and observe induced ECs in DBM seeded by fibrin sealant improving primary vasculogenesis in scaffold in vitro.3. To observe vascularization of complex of induced ECs and DBM after the complex was implanted in model of rabbit ulnar defect.Methods:1. MNCs in rabbit marrow were isolated by the gradient centrifugation and cultivated to isolate and induce ECs. Every passage of ECs were measured and analyzed. Gowth and proliferation and the changes of ECs morphology were observed during the passage cultivation. The immunohistochemical staining of CD31 and vWF and function of ECs were checked.2. 1×10~6 induced ECs were seeded into 1ml fibrin sealant, the growth and proliferation of induced ECs were observed under microscope ,and the growth curve were drawn by the way of MTT. 1×10~7 induced ECs were seeded into DBM by fibrin sealant, then vasculogenesis of induced ECs were confirmed under microscope, HE and electon microscope.3. The three experimential groups were :cultured complex of DBM and induced ECs ,DBM and vacant,each experimental group consisted of 6 animals per time points. Specimens were taken X ray ,infused India ink,stained with HE,stained immunehistochemically for CD31 and Brdu respectively after 2,4,8w ,then conuted MVD.Results:1. After cultivated 24h,the cluster of round cells sits on top of spindle-shape adherent cells ;after 4d the cluster of cells dispears. After cultivated for 12-14d ,number the second passage ECs derived from 1ml bone marrow was (3.1±0.76)×10~6;induced ECs were monolayer arrayed to form cobblelike shape. The cells of 2nd passage showed positive staining of CD31 and vWF, and uptake of DiI-LDL and binding of UEA.2. The second passage induced ECs grow in three-dimensional in fibrin sealant, become fusiform in 3th day and emerge tube-form after 6 days.The growth curve showed"S"form. The second passage induced ECs grow in DBM ,become fusiform in 3th day and emerge tube-form and endothelial-like structure after 6 days.3. X-rays,spicemens and HE of sections show that group of cultured complex of DBM and induced ECs have more bony callus in 8 weeks.The pictures of infusing India ink display more neovascularization in group of cultured complex of DBM and induced ECs than other groups.Counting MVD through pictures of immunehistochemical for CD31 reveal complex of DBM and induced ECs group (2,4,8w) have higher density of blood vessels than other group in neogenesis bone(P<0.01).Fluorescence and immunehistochemical of Brdu are positive in the complex of DBM and induced ECs group.Conclusion:1. Centrifugated rabbit bone marrow MNCs can be differentiated into cells with endothelial phenotype and proliferate well in vitro using EGM-2.2. The induced ECs grow and proliferate well in fibrin sealant in vitro. It is feasible that fibrin sealant can act as matrix in the process of seeding the induced ECs in bone tissue engineering.3. The induced ECs can emerge tube-form and endothelial-like structure in DBM in vitro, this results testify induced ECs in DBM seeded by fibrin sealant improve primary vasculogenesis in scaffold in vitro.4. The results reveal cultured complex of DBM and Rabbit bone marrow MNCs induced ECs enhance vascularization of DBM after implanting into model of rabbit nular defect.5. The induced ECs may act as seeding cells in neovascularization of bone tissue engineering in future.