不同血管化策略构建组织工程骨的研究
发布时间：2018-09-10 07:33
【摘要】：目的： 组织工程的发展为骨缺损的修复治疗开辟了一条崭新的道路。然而对于大型哺乳动物大范围或受区血供不佳的骨缺损,由于组织工程骨植入体内后不能及时与机体建立有效血液循环,成骨效果不稳定。组织工程骨的血管化成为目前骨组织工程的研究重点。目前构建血管化组织工程骨的方法主要包括支架设计开发、应用细胞因子、体外联合内皮祖细胞和体内预血管化等。体外联合内皮祖细胞的方法在大动物体内研究较少；体内预血管化的方法探讨较多,但缺乏横向比较。为此,我们采用不同血管化策略在比格犬体内构建组织工程骨,明确体外联合内皮祖细胞的方法对组织工程骨成骨效果的影响,比较两种体内预血管化方法的成血管和促进成骨作用,为临床构建血管化组织工程骨提供参考。 研究方法： 1.应用密度梯度离心结合贴壁筛选法分离、培养、扩增比格犬BMSCs,并将BMSCs向骨、软骨和脂肪方向诱导分化鉴定；应用密度梯度离心法和差速贴壁法分离比格犬骨髓来源的内皮祖细胞(EPCs),并进行表面标志和细胞功能鉴定。 2.在体外构建EPCs/BMSCs/TCP、EPCs/TCP、BMSCs/TCP细胞支架复合物,植入裸鼠皮下,分别在术后6周和12周取材,Micro-CT和组织学检测各组的成骨能力。 3.在体外构建EPCs/BMSCs/TCP和BMSCs/TCP细胞支架复合物,植入比格犬下肢肌肉袋内,术后12周取材,Micro-CT、组织学和免疫组织化学检测各组的成血管及成骨能力。 4.通过显微外科的技术,吻合比格犬下肢隐动静脉,形成动静脉环路(AVLoop),构建AV Loop血管化组织工程骨模型；同时采用比格犬隐动静脉远端结扎后置入细胞支架复合物内,构建血管束置入(Vascular bundle,VB)血管化组织工程骨模型；在术后不同时间点通过CTA和B超进行模型验证。 5.组织工程骨体内构建6月后取材,采用灌注Microfil观察、Micro-CT扫描并重建分析、以及组织学染色比较VB组和AV Loop组的组织工程骨血管化的差异。 6.在不同时间点测量组织工程骨CT值；体内构建6月后取材,采用Micro-CT扫描分析,以及组织学染色比较VB组和AV Loop组的组织工程骨成骨效果的差异。 结果： 1.种子细胞的分离、培养和鉴定:BMSCs能够向骨、软骨和脂肪方向分化；EPCs能够摄取DiI-ac-LDL和结合FITC-UEA-1,在Matrigel上形成血管腔样结构并且VWF染色呈现阳性。 2.联合EPCs裸鼠体内构建组织工程骨：联合EPCs组的骨密度和成骨面积均高于单独BMSCs组(p0.05)。 3.联合EPCs比格犬体内构建组织工程骨：Micro-CT和组织学分析显示,联合EPCs和单独BMSCs的骨密度和成骨面积无统计学差异(p0.05)；VWF免疫组织化学分析表明,联合EPCs组的血管数量高于单独BMSCs组(p0.05);CT值分析提示,联合EPCs组相对CT值高于单独BMSCs组(p0.05)；组织学成骨面积分析提示,联合EPCs成骨面积大于单独BMSCs组(p0.05)。 4.不同体内预血管化方法的模型：CTA检测提示AV Loop组的血管环在第2W、4W、8W通畅,B超证实血管环在6月仍通畅；VB组血管束不显影。 5.不同体内预血管化方法构建组织工程骨的成血管比较：灌注Microfil并进行Micro-CT扫描分析,结果表明VB组和AV Loop组生成的血管总体积和表面积无明显差异(p0.05)；VWF免疫组织化学分析显示VB组和AV Loop组的血管数量无明显差异(p0.05)。 6.不同体内预血管化方法构建组织工程骨的成骨比较：CT、Micro-CT以及组织学分析显示,VB组和AV Loop组的组织工程骨骨密度和成骨面积均无明显差异(p0.05)。 结论 1.联合EPCs作为种子细胞在裸鼠体内能够促进BMSCs的成骨；联合EPCs作为种子细胞在比格犬体内能够促进组织工程骨的成骨和血管生成。 2.采用比格犬下肢隐动静脉可以成功构建体内AV Loop及VB血管化组织工程骨模型,为构建大体积血管化组织工程骨提供了参考方案。 3.采用VB及AV Loop体内血管化均能提高组织工程骨的血管化和成骨效果,并且两种方法促进组织工程骨血管化和成骨的效果无明显差异；而VB组具有手术操作简单和成功率高的优势。
[Abstract]:Objective:
The development of tissue engineering has opened up a new way for repairing bone defect. However, the vascularization of tissue engineered bone has become the current bone defect because tissue engineered bone can not establish effective blood circulation with the body in time after implantation. At present, the methods of constructing vascularized tissue-engineered bone mainly include the design and development of scaffolds, the application of cytokines, the combination of EPCs in vitro and in vivo pre-vascularization, etc. In this study, we used different vascularization strategies to construct tissue-engineered bone in Beagle dogs. We clarified the effect of combined endothelial progenitor cells in vitro on the osteogenesis of tissue-engineered bone, and compared the vascularization and osteogenesis of the two in vivo pre-vascularization methods to provide reference for clinical construction of vascularized tissue-engineered bone.
Research methods:
1. BMSCs from beagle dogs were isolated, cultured and amplified by density gradient centrifugation combined with adherence screening, and differentiated into bone, cartilage and fat. EPCs from Beagle dogs'bone marrow were isolated by density gradient centrifugation and differential adherence screening, and their surface markers and cell functions were identified.
2. EPCs/BMSCs/TCP, EPCs/TCP, BMSCs/TCP scaffold composites were constructed in vitro and implanted subcutaneously in nude mice. The osteogenic capacity of each group was examined by micro-CT and histology at 6 and 12 weeks after operation.
3. EPCs/BMSCs/TCP and BMSCs/TCP scaffold composites were constructed in vitro and implanted into the lower limb muscle pockets of Beagle dogs.
4. The saphenous arteries and veins of Beagle dogs were anastomosed by microsurgical technique to form AVLoop vascularized tissue engineered bone model. Vascular bundle (VB) vascularized tissue engineered bone model was constructed after distal ligation of saphenous arteries and veins of Beagle dogs. The model was verified by CTA and B ultrasound at different time points.
5. Tissue-engineered bone samples were taken 6 months after construction. The vascularization of tissue-engineered bone was compared between VB group and AV Loop group by perfusion microfil observation, micro-CT scanning and reconstruction analysis, and histological staining.
6. The CT value of tissue-engineered bone was measured at different time points; the tissue-engineered bone samples were taken 6 months after construction in vivo and analyzed by micro-CT scanning and histological staining.
Result:
1. Isolation, culture and identification of seed cells: BMSCs can differentiate into bone, cartilage and fat; EPCs can ingest DiI-ac-LDL and bind FITC-UEA-1, form vascular lumen-like structure on Matrigel and show positive VWF staining.
2. Tissue engineered bone was constructed in nude mice with EPCs: BMD and osteogenic area of combined EPCs group were higher than those of BMSCs group (p0.05).
3. Tissue-engineered bone in combination with EPCs in Beagle dogs: Micro-CT and histological analysis showed that there was no significant difference in BMD and osteogenic area between EPCs and BMSCs alone (p0.05); VWF immunohistochemical analysis showed that the number of blood vessels in combination with EPCs was higher than that in BMSCs alone (p0.05); CT value analysis indicated that the relative CT value of combined EPCs group was higher than that of BMSCs alone (p0.05). In BMSCs alone group (p0.05), histological analysis of osteogenic area showed that the combined EPCs osteogenic area was larger than BMSCs alone group (p0.05).
4. Models with different methods of pre-vascularization in vivo: CTA showed that the vascular ring of AV Loop group was unobstructed at 2W, 4W and 8W, and the vascular ring was still unobstructed in 6 months by B-ultrasound, while the vascular bundle of VB group was not developed.
5. Comparison of vascularization of tissue-engineered bone constructed by different in vivo pre-vascularization methods: Microfil perfusion and micro-CT scanning analysis showed that there was no significant difference in the total volume and surface area of blood vessels between VB group and AV Loop group (p0.05); VWF immunohistochemical analysis showed no significant difference in the number of blood vessels between VB group and AV Loop group (p0.05).
6. Comparison of osteogenesis of tissue-engineered bone constructed by different methods of in vivo pre-vascularization: CT, Micro-CT and histological analysis showed that there was no significant difference between VB group and AV Loop group in BMD and osteogenesis area (p0.05).
conclusion
1. The combination of EPCs as seed cells can promote the osteogenesis of BMSCs in nude mice and EPCs as seed cells can promote the osteogenesis and angiogenesis of tissue-engineered bone in Beagle dogs.
2. AV Loop and VB vascularized tissue-engineered bone models in vivo can be successfully constructed by using the saphenous arteries and veins of the lower extremities of Beagle dogs, providing a reference scheme for the construction of large-scale vascularized tissue-engineered bone.
3. Vascularization in vivo with VB and AV Loop can improve the vascularization and osteogenesis of tissue-engineered bone, and there is no significant difference between the two methods in promoting vascularization and osteogenesis of tissue-engineered bone.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08
本文编号：2233793
[Abstract]:Objective:
The development of tissue engineering has opened up a new way for repairing bone defect. However, the vascularization of tissue engineered bone has become the current bone defect because tissue engineered bone can not establish effective blood circulation with the body in time after implantation. At present, the methods of constructing vascularized tissue-engineered bone mainly include the design and development of scaffolds, the application of cytokines, the combination of EPCs in vitro and in vivo pre-vascularization, etc. In this study, we used different vascularization strategies to construct tissue-engineered bone in Beagle dogs. We clarified the effect of combined endothelial progenitor cells in vitro on the osteogenesis of tissue-engineered bone, and compared the vascularization and osteogenesis of the two in vivo pre-vascularization methods to provide reference for clinical construction of vascularized tissue-engineered bone.
Research methods:
1. BMSCs from beagle dogs were isolated, cultured and amplified by density gradient centrifugation combined with adherence screening, and differentiated into bone, cartilage and fat. EPCs from Beagle dogs'bone marrow were isolated by density gradient centrifugation and differential adherence screening, and their surface markers and cell functions were identified.
2. EPCs/BMSCs/TCP, EPCs/TCP, BMSCs/TCP scaffold composites were constructed in vitro and implanted subcutaneously in nude mice. The osteogenic capacity of each group was examined by micro-CT and histology at 6 and 12 weeks after operation.
3. EPCs/BMSCs/TCP and BMSCs/TCP scaffold composites were constructed in vitro and implanted into the lower limb muscle pockets of Beagle dogs.
4. The saphenous arteries and veins of Beagle dogs were anastomosed by microsurgical technique to form AVLoop vascularized tissue engineered bone model. Vascular bundle (VB) vascularized tissue engineered bone model was constructed after distal ligation of saphenous arteries and veins of Beagle dogs. The model was verified by CTA and B ultrasound at different time points.
5. Tissue-engineered bone samples were taken 6 months after construction. The vascularization of tissue-engineered bone was compared between VB group and AV Loop group by perfusion microfil observation, micro-CT scanning and reconstruction analysis, and histological staining.
6. The CT value of tissue-engineered bone was measured at different time points; the tissue-engineered bone samples were taken 6 months after construction in vivo and analyzed by micro-CT scanning and histological staining.
Result:
1. Isolation, culture and identification of seed cells: BMSCs can differentiate into bone, cartilage and fat; EPCs can ingest DiI-ac-LDL and bind FITC-UEA-1, form vascular lumen-like structure on Matrigel and show positive VWF staining.
2. Tissue engineered bone was constructed in nude mice with EPCs: BMD and osteogenic area of combined EPCs group were higher than those of BMSCs group (p0.05).
3. Tissue-engineered bone in combination with EPCs in Beagle dogs: Micro-CT and histological analysis showed that there was no significant difference in BMD and osteogenic area between EPCs and BMSCs alone (p0.05); VWF immunohistochemical analysis showed that the number of blood vessels in combination with EPCs was higher than that in BMSCs alone (p0.05); CT value analysis indicated that the relative CT value of combined EPCs group was higher than that of BMSCs alone (p0.05). In BMSCs alone group (p0.05), histological analysis of osteogenic area showed that the combined EPCs osteogenic area was larger than BMSCs alone group (p0.05).
4. Models with different methods of pre-vascularization in vivo: CTA showed that the vascular ring of AV Loop group was unobstructed at 2W, 4W and 8W, and the vascular ring was still unobstructed in 6 months by B-ultrasound, while the vascular bundle of VB group was not developed.
5. Comparison of vascularization of tissue-engineered bone constructed by different in vivo pre-vascularization methods: Microfil perfusion and micro-CT scanning analysis showed that there was no significant difference in the total volume and surface area of blood vessels between VB group and AV Loop group (p0.05); VWF immunohistochemical analysis showed no significant difference in the number of blood vessels between VB group and AV Loop group (p0.05).
6. Comparison of osteogenesis of tissue-engineered bone constructed by different methods of in vivo pre-vascularization: CT, Micro-CT and histological analysis showed that there was no significant difference between VB group and AV Loop group in BMD and osteogenesis area (p0.05).
conclusion
1. The combination of EPCs as seed cells can promote the osteogenesis of BMSCs in nude mice and EPCs as seed cells can promote the osteogenesis and angiogenesis of tissue-engineered bone in Beagle dogs.
2. AV Loop and VB vascularized tissue-engineered bone models in vivo can be successfully constructed by using the saphenous arteries and veins of the lower extremities of Beagle dogs, providing a reference scheme for the construction of large-scale vascularized tissue-engineered bone.
3. Vascularization in vivo with VB and AV Loop can improve the vascularization and osteogenesis of tissue-engineered bone, and there is no significant difference between the two methods in promoting vascularization and osteogenesis of tissue-engineered bone.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08
【参考文献】
相关期刊论文 前1条
1 吴雪晖;许建中;王序全;罗飞;曾玲;谭洪波;;血管内皮祖细胞对组织工程骨成骨能力影响的实验研究[J];重庆医学;2006年22期
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