Abstract: BACKGROUND: Slow bone repair and poor bone formation quality are still problems during masquelet technique in the treatment of large segment bone defects. H-type vessels can induce osteogenesis, enhance the local angiogenesis and osteogenesis coupling, and promote bone repair. However, there are few reports on the role of H-type blood vessels in the bone induced membrane. 
OBJECTIVE: To construct a large segment bone defect model of SD rat tibia, observe the expression characteristics of H-type blood vessels in the bone induced membrane, then to identify the expression peak point of H-type blood vessels in the bone induced membrane and determine the optimal period of bone grafting.
METHODS: Sixty SD rats were randomly divided into a control group (n=30) and a model group (n=30) by random number table method. The two groups were further divided into three subgroups at 4, 6, and 8 weeks after bone cement implantation, with 10 rats in each group. A 4 mm bone defect model of the right tibia was constructed in both the control and the model groups. Polymethyl methacrylate bone cement was implanted in the model group to induce bone biomembrane formation, while bone cement was not implanted in the control group. At 4, 6, and 8 weeks after bone cement implantation, 6 rats were randomly selected at each time point. The bone induction membrane tissue was cut from the model group, and the non-bone soft tissue of the corresponding part was cut from the control group. The dynamic expressions of H-type blood vessels in the bone induced membrane were identified by immunofluorescence. The morphological changes of the bone induced membrane were observed by hematoxylin-eosin staining. The formation of blood vessels in the bone induced membrane was observed by angiography. The expression levels of osteoblast-specific transcription factor in the bone induced membrane were detected by immunohistochemistry. Four rats remained at each time point. In the model group, the bone induced membrane was cut open and the bone cement was removed and autologous coccyx was implanted. In the control group, autologous coccyx was implanted in the bone defect area. Micro-CT evaluation of the tibial defect was performed 8 weeks after bone grafting. 
RESULTS AND CONCLUSION: (1) Immunofluorescence staining showed that the expression of H-type vessels in the model group was most obvious 6 weeks after bone cement implantation, and the expression of H-type vessels in the model group at each time point after bone cement implantation was higher than that in the control group (P < 0.05). (2) Hematoxylin-eosin staining and angiography showed that the number and volume of new blood vessels at each time point after bone cement implantation in the model group were greater than those in the control group (P < 0.05). The order of the number and volume of new blood vessels in the model group was: 8 weeks after bone cement implantation > 6 weeks after bone cement implantation > 4 weeks after bone cement implantation. (3) Immunohistochemical staining showed that the positive expression of osteoblast-specific transcription factors at each time point after bone cement implantation in the model group was higher than that in the control group (P < 0.05), and the positive expression of osteoblast-specific transcription factors in the model group was most obvious 6 weeks after bone cement implantation. (4) Micro-CT detection showed that the bone repair effect of the three subgroups in the model group was significantly better than that of the corresponding subgroups in the control group, and the bone repair effect of the subgroup in the model group 6 weeks after bone cement implantation was better than that of the subgroups 4 and 8 weeks after bone cement implantation. The results indicate that H-type blood vessels are dynamically expressed in the bone induced membrane and reached a peak 6 weeks after bone cement implantation. Good bone repair effects can be obtained by the bone induced membrane bone grafting 6 weeks after bone cement implantation. 

Key words: Masquelet technique, bone induced membrane, H-type vessels, dynamic expression, bone repair, massive bone defect, bone cement, engineered bone material