The Mechanism Research On Notch、VEGF-A、And Ang-1 Signaling Pathways Regulating Neovascularization In Nonvascular Transport Distraction Osteogenesis

ObjectiveBone is rich in vascular tissue, new bone formation depends on good blood supply and nutrition support, the closely spatio-temporal relationship between vascular and bone cells maintains bone integrity. Therefore, angiogenesis plays a quite important role during the bone development and fracture repair. Also, traditional transport disc distraction osteogenesis requires that every bone segment should have sufficient blood supply to ensure exuberant metabolic activity, superior nutrient supply and the normal bone tissue metabolism in the process of distraction osteogenesis. This research is based on accidental clinical practice, in which we applied traditional transport disc distraction osteogenesis to repair a wide range of jaw bone defects, unexpectedly found that free bone transport disc completely stripped periosteum and soft tissue can still be successfully used to repaire jaw defects. After that, we put forward the concept of “nonvascular transport distraction osteogenesis, NTDO”.Then our team constructed successfully mandibular nonvascular transport distraction osteogenesis animal models and confirmed free bone disc stripped periosteum and lost blood supply can still be distracted osteogenesis. NTDO mode breaks the principles for traditional transport disc distraction osteogenesis that emphasizes bone transport disc must be retained periosteum and the surrounding soft tissue as far as possible to provide adequate blood supply, NTDO provides a new treatment for thoses patients, due to the large bone defects whose transport disc can not be kept periosteal and soft tissue attached or no enough blood supply for surrounding tissue of transport disc. However precise mechanism of angiogenesis in the process of NTDO and the space-time coupling of angiogenesis and new bone formation are still unknown, and to clarify the mechanism of angiogenesis in the process of NTDO. Exploring cellular and molecular mechanism of angiogenesis can provide theoretical basis for NTDO and preferably help to guide the clinical application of this technology.Endothelial progenitor cells(EPCs) were applied as seed cells in this study, by activating Notch signal transduction pathway on the EPCs, we studied Notch signal transduction pathway in the distraction zone of new blood vessels series molecular control events, further clarifed neovascularization mechanism in nonvascular transport distraction osteogenesis, which will provide theoretical foundation for NTDO. The NTDO technology will provide a new prospect with treatment in those cases that in reconstruction of postoperation tumors and cranial and maxillofacial plastic traditional transport disc can not be used to repair large bone defects.Methods1. The research on in vitro isolation, cultivation and identification of canine bone marrow endothelial progenitor cells(EPCs): Health hybrid dogs, aged 1~2 years, were punctured in the bilateral tibia and pumped tibia bone marrow 20 ml. Density gradient centrifugation combined with adherent culture method using special induction medium were used to isolate and cultivate canine bone marrow endothelial progenitor cells; By inverted phase contrast microscope to observe cell morphology; By flow cytometry to detect cell surface markers; By immunofluorescence double staining and VEGFR-2, v WF immunohistochemical to identify canine bone marrow endothelial progenitor cells; By Matrigel tubular formation assay to detect neovascularition ability for endothelial progenitor cells.2. The experimental study of integrating h Notch1.ICN into EPCs by lentivirus gene transfection: constructing h Notch1. ICN lentivirus vector, measuring virus drops and best MOI infected cells; after that h Notch1. ICN gene was transfected into the second or third generation of EPCs by using lentivirus as vector; after lentivirus gene transfection methods of QPCR and Western blot were used to detect the expression of h Notch1. ICN into EPCs.3. The research on overexpression of h Notch1. ICN impact on EPCs biological behaviors: CCK 8 method was used to test overexpresstion of h Notch1. ICN gene how to influence EPCs survivability; Annexin V/PI methods was used to detect overexpresstion of h Notch1.ICN how to influence EPCs apoptosis; Flow cytometry instrument was used to test overexpresstion of h Notch1. ICN how to influence EPCs cell cycle; Transwell was used to investigate through overexpression h Notch1. ICN EPCs migration across endothelial ability, and EPCs adhesive ability to activation of endothelial cells and angiogenesis ability; After lentivirus gene transfection the methods of QPCR and Western blot was used to detect Notch signaling pathway downstream signaling molecules Hes and Hey 1 m RNA and protein expression.4. The experimental research on the mechanism of Notch signaling pathway promoting the neovascularization in NTDO: 27 healthy mongrel dogs were randomly divided into 3 groups, nine dogs in each group. Group A: experimental group(injection autogenous EPCs modified with h Notch1. ICN gene); B: the control group(injection of autogenous EPCs); Group C: blank group(injection of normal saline). Constructing canine mandibular model of nonvascular transport distraction osteogenesis and fabricating nonvascular transport disc and then installing distractor. 5 dogs each group were euthanized after distraction1 week, 2 weeks, 4 weeks, new bone formation and reconstruction were examined by general observation, X-ray observation; The structure of new blood vessels in distracting gap was examined By H&E dyeing; The number of blood was examined through Chalkley method quantitative detection; Ang-1 VEGF-1 m RNA and protein expression in distracting gap were respectively detected by real time fluorescence quantitative polymerase chain reaction(q PCR) and western blot.Results1. The research on in vitro isolation, cultivation and identification of canine bone marrow endothelial progenitor cells(EPCs): Primitive EPCs were cultivated after 5d, by microscopy the volume of adherent cells began to increase, grew like colony type, formed a long strip, spindle or polygon. Parts of cells formed colony. The cell morphology is polygonal cells in center of cell and surrounding long spindle cells arranged like radial. After cultivation to 7~10 d, sub-culture cells proliferated fast, groups of cells showed like a typical "paving stone" or "pebbles" arrangement; Flow cytometry results showed that the expression of CD133 and CD34 were positive; Immunohistochemical results of both v WF and VEGFR-2 were positive; Immunofluorescence double staining results showed that EPCs can not only absorb Di I-ac LDL but also combine with FITC-UEA-1, had typical characteristics of endothelium, which indicated the positive cells were EPCs that were differentiating into endothelial cells. Matrigel tubular formation assay showed that EPCs can form new blood vessels.2. The research on integrating h Notch1.ICN into EPCs by lentivirus gene transfection: h Notch1. ICN lentivirus vector was successfully constructed. MOI value for canine endothelial progenitor cells(Epcs) was 10~20; Average virus drops was 1.02e+08 IU/ml; h Notch1.ICN m RNA expression be detected by QPCR after transfection; Lentivirus plasmid of h Notch l. ICN overexpression expressed in 293 T cells be dectected by Western Blot.3. The research on overexpression of h Notch 1.ICN impact on EPCs biological behaviors: Overexpression of h Notch 1.ICN gene in EPCs was not significantly influence survival vitality with CCK-8 method(p﹥0.05); Overexpression of h Notch 1.ICN gene in EPCs could strengthen antiapoptotic ability of EPCs with Annexin V/PI method(p<0.05); Overexpression of h Notch 1.ICN gene in EPCs increased the number of EPCs staying in the G2 phase with Flow cytometry detection(p<0.05); Overexpression of h Notch 1.ICN gene in EPCs promoted EPCs to adhere to activating endothelial cells, promoted EPCs to migrate across endothelial and finally promoted tube structure formation(p<0.05). The results about lentivirus gene transfected EPCs showed downstream signaling molecules on Notch signaling Hes 1 and Hey 1 m RNA and protein expression were increased with QPCR(p<0.05)and Western blot method.4. The experimental research on the mechanism of Notch signaling pathway promoting the neovascularization in NTDO: All animals were completed distraction, all animals lived to time of sampling with no infection and death. The weight of all experimental animals also increased to different extent. The distractor placed in mandibular was fixed well with no fracture and loose phenomenon. General pathology, HE staining and X-ray showed that new bone formation from high to low was A>B>C. The number of new blood vessels was detected by Chalkley method and decreased gradually between three groups with time. There were differences between groups after statistical analysis(p<0.05). Detection of Ang-1 with q PCR at each time point in A group was significantly higher than that of group B and C(p<0.01); Expression of VEGF-A was higher than that in group B(p<0.05) at 2 and 4 weeks, significantly higher than B at 1 week(p<0.01); Expression of VEGF-A was significantly higher than that of group C(p<0.01); Expression of VEGF-A and Ang-1 protein were detected with Western blot. The results were A>B>C, but their expression decreased gradually with time.Conclusions1. Overexpression of h Notch 1. ICN gene promotes downstream signaling molecules on Notch signaling Hes 1 and Hey 1 m RNA and protein expression; Sustained activation of Notch 1 signal does not affect EPCs survival vitality but makes cell cycle arrest in G2 and delays the progress of cell cycle, does not make EPCs enter into the next cell cycle, inhibits EPCs differentiation and strengthens EPCs antiapoptotic ability.2. Overexpression of h Notch 1.ICN gene promotes that EPCs adheres to activating endothelial cell, migrates across endothelium and forms tube cavity structure.3. The neovascularization promoting effects in NTDO of h Notch 1.ICN gene is medicated Notch signal increases expression of VEGF-A and Ang-1.