Expression And Significance Of Notch Receptors And Ligands During Canine Mandible Nonvascular Transport Distraction Osteogenesis To Neovascularization

Objective:To study the function of Notch signal pathway and the mechanism of neovascularization during canine Mandible Nonvascular Transport Distraction Osteogenesis(NTDO) by comparatively investigating the expression of Notch receptors and ligands during the process of two different means of operation, NTDO and acutely lengthening.Methods:Twelve adult dogs were randomly divided into NTDO group and acutely lengthening group(control group) with six dogs in each group. A 10.0mm in length by 10.0mm in height segmental skeletal defect was created in the inferior half of right mandibular body by osteotomy on NTDO group dogs. Then a 15.0mm in length by 10.0mm in height free nonvascular bone fragment distal to the defect was made as the nonvascular transport, and a custom-made internal distractor was installed in place as designed on each dog. After 5-day latency period, the distraction was performed at a rate of 1 mm/day for 10 days. The same osteotomy were performed on dogs in acutely lengthening group, then the free nonvascular transport were directly moved from the distal of the defect to the mesial of the defect during surgery, and were fixed to the mandibular body with titanium plates. All subjects were sacrificed after 1 week of consolidation. Specimens were harvested in and around the distraction gap. And macrography and X-ray observation, flow cytometry detection, histological and immunohistochemical examinations of Specimens were carried out.Results:1) General oberservation:Samples from all subjects were successfully achieved according to the experimental design without halfway death, wound infection, distraction device and titanium nails falling off. The distraction region and transport disc were surrounded by periosteum.2) X-ray examination:The boundary of distraction gap was clear and a transparent area was observed in the center of the gap.No bone formation was found in both distraction gaps and lengthing gaps.3) flow cytometry examination:1.27% of CD34(+) and CD133(+) cells were found in tissue of NTDO group and 0.65% of CD34(+) and CD133(+) cells were found in tissue of control group. Most of the rest cells were CD34(-) and CD133(-) cells.4) TEM examination:Immature capillaries formed by a single endothelial cell were found in NTDO group. Mature capillaries formed by a plurality of endothelial cells were found in control group.5) HE staining micrograph examination:Compared with the control group, abundant small diameter capillaries and cells can be observed in distraction gap.6) Immunohistochemistry:Positive stain of CD105, Notch1, Notch2, D114 and Jag2 were found in both NTDO group and control group. The expression of CD 105, Notch1, Notch2 and D114 in NTDO group were higher than that in control group(P<0.05). The expression of Jag2 was no statistic difference between two groups (p>0.05)7) Western Blot:The expression of Notch1, Notch2 and D114 in NTDO group were higher than that in control group(P<0.05). The expression of Jag2 was no statistic difference between two groups (p>0.05)Conclusions:EPCs are involved in the angiogenesis process of mandibular NTDO; The mechanical stimulation may increase the expression of Notch1, Notch2 and D114 in distraction region; The mechanical stimulation caused by distraction may promote angiogenesis; The distraction stimulation may activate the D114/Notchl signaling pathway to promote the angiogenesis during mandibular NTDO.