血管铸型方法检测组织工程骨修复羊胫骨缺损的远期血管化

Detection of long-term vascularization at the large defect of tibial diaphysis repaired with tissue engineered bone by vascular casting in goats

目的采用血管铸型方法对组织工程技术修复中国青山羊胫骨大段骨缺损的远期血管化情况进行检测。方法中国青山羊3只，制备单侧胫骨20mm的骨与骨膜缺损，缺损部位内植入组织工程骨-珊瑚羟基磷灰石／骨髓基质干细胞（coral hydroxyapatite／bone marrow stem cells，CHAP／BMSCs）。术后3年分别行双侧后肢股动脉甲基丙烯酸甲酯血管铸型灌注，灌注后采用大体解剖、未脱钙骨片、扫描电镜等方法检测组织工程骨的远期血管化，并与健侧相同部位正常骨对照，观察其血管化效果。结果血管灌注后大体解剖观察示组织工程骨血管来源于周围软组织、髓腔血管及两端正常骨皮质。灌注后未脱钙骨磨片显示组织工程骨内微血管沿哈弗管和伏克曼管分布交织形成网状。采用图像分析仪比较组织工程骨与正常骨横切面磨片上血管相对面积，分别为（763．89±47．38）U／10万U与（788．00±65．83）U／10万U，差异无统计学意义（t=0．390，P〉0．05）。结论采用CHAP／BMSCs修复山羊胫骨大段骨缺损远期观察结果表明，新生骨远期血管化效果与正常骨无异，为组织工程骨下一步的临床应用提供可靠的依据。

Objective To observe the long-term vascularization at the large defect of goat tibial shaft repaired with tissue engineered bone by using vascular casting. Methods Three Chinese goats were used in 2001 to create the models of lateral 20 mm bone and periosteum defect at the left hind tibia. The defects were reconstructed by filling coral hydroxyapatite （CHAP） plus induced bone marrow stem cells （BMSc）. Vascular casting and perfusion with methyl methacrylate was done 3 years postoperation for femoral arteries at bilateral hind limbs to compare the long-term vascularization at the experimental side with normal vascularization at the opposite side by gross anatomy, ground sections without decalcification and scanning electron microscope. Results Gross anatomy after vascular casting disclosed that the vessels of tissue-engineered bone grafts derived from anadesma coated outside, medullary cavity and normal cortical bone. Ground sections of vascular casting samples without decalcification revealed that the capillaries of engineered-bone grafts went along the Haversian and Volkmarm canals and interlaced into net. Transverse sections of tissueengineered bone grafts and opposite normal cortical bone presented no significant difference with respect to their vaso-area ratio detected by image-analyzer （P〉0.05）. The diaphyseal defects were substituted completely by new-born osseous tissues. Conclusion Long-term vascularization at the large bone defect repaired with tissue engineered bone can be the same as the normal one. The finding strengthens the evidence for clinical application of tissue engineered bone.