不同血管化策略構(gòu)建組織工程骨的研究
發(fā)布時間：2018-09-10 07:33
【摘要】：目的： 組織工程的發(fā)展為骨缺損的修復(fù)治療開辟了一條嶄新的道路。然而對于大型哺乳動物大范圍或受區(qū)血供不佳的骨缺損,由于組織工程骨植入體內(nèi)后不能及時與機(jī)體建立有效血液循環(huán),成骨效果不穩(wěn)定。組織工程骨的血管化成為目前骨組織工程的研究重點。目前構(gòu)建血管化組織工程骨的方法主要包括支架設(shè)計開發(fā)、應(yīng)用細(xì)胞因子、體外聯(lián)合內(nèi)皮祖細(xì)胞和體內(nèi)預(yù)血管化等。體外聯(lián)合內(nèi)皮祖細(xì)胞的方法在大動物體內(nèi)研究較少；體內(nèi)預(yù)血管化的方法探討較多,但缺乏橫向比較。為此,我們采用不同血管化策略在比格犬體內(nèi)構(gòu)建組織工程骨,明確體外聯(lián)合內(nèi)皮祖細(xì)胞的方法對組織工程骨成骨效果的影響,比較兩種體內(nèi)預(yù)血管化方法的成血管和促進(jìn)成骨作用,為臨床構(gòu)建血管化組織工程骨提供參考。 研究方法： 1.應(yīng)用密度梯度離心結(jié)合貼壁篩選法分離、培養(yǎng)、擴(kuò)增比格犬BMSCs,并將BMSCs向骨、軟骨和脂肪方向誘導(dǎo)分化鑒定；應(yīng)用密度梯度離心法和差速貼壁法分離比格犬骨髓來源的內(nèi)皮祖細(xì)胞(EPCs),并進(jìn)行表面標(biāo)志和細(xì)胞功能鑒定。 2.在體外構(gòu)建EPCs/BMSCs/TCP、EPCs/TCP、BMSCs/TCP細(xì)胞支架復(fù)合物,植入裸鼠皮下,分別在術(shù)后6周和12周取材,Micro-CT和組織學(xué)檢測各組的成骨能力。 3.在體外構(gòu)建EPCs/BMSCs/TCP和BMSCs/TCP細(xì)胞支架復(fù)合物,植入比格犬下肢肌肉袋內(nèi),術(shù)后12周取材,Micro-CT、組織學(xué)和免疫組織化學(xué)檢測各組的成血管及成骨能力。 4.通過顯微外科的技術(shù),吻合比格犬下肢隱動靜脈,形成動靜脈環(huán)路(AVLoop),構(gòu)建AV Loop血管化組織工程骨模型；同時采用比格犬隱動靜脈遠(yuǎn)端結(jié)扎后置入細(xì)胞支架復(fù)合物內(nèi),構(gòu)建血管束置入(Vascular bundle,VB)血管化組織工程骨模型；在術(shù)后不同時間點通過CTA和B超進(jìn)行模型驗證。 5.組織工程骨體內(nèi)構(gòu)建6月后取材,采用灌注Microfil觀察、Micro-CT掃描并重建分析、以及組織學(xué)染色比較VB組和AV Loop組的組織工程骨血管化的差異。 6.在不同時間點測量組織工程骨CT值；體內(nèi)構(gòu)建6月后取材,采用Micro-CT掃描分析,以及組織學(xué)染色比較VB組和AV Loop組的組織工程骨成骨效果的差異。 結(jié)果： 1.種子細(xì)胞的分離、培養(yǎng)和鑒定:BMSCs能夠向骨、軟骨和脂肪方向分化；EPCs能夠攝取DiI-ac-LDL和結(jié)合FITC-UEA-1,在Matrigel上形成血管腔樣結(jié)構(gòu)并且VWF染色呈現(xiàn)陽性。 2.聯(lián)合EPCs裸鼠體內(nèi)構(gòu)建組織工程骨：聯(lián)合EPCs組的骨密度和成骨面積均高于單獨BMSCs組(p0.05)。 3.聯(lián)合EPCs比格犬體內(nèi)構(gòu)建組織工程骨：Micro-CT和組織學(xué)分析顯示,聯(lián)合EPCs和單獨BMSCs的骨密度和成骨面積無統(tǒng)計學(xué)差異(p0.05)；VWF免疫組織化學(xué)分析表明,聯(lián)合EPCs組的血管數(shù)量高于單獨BMSCs組(p0.05);CT值分析提示,聯(lián)合EPCs組相對CT值高于單獨BMSCs組(p0.05)；組織學(xué)成骨面積分析提示,聯(lián)合EPCs成骨面積大于單獨BMSCs組(p0.05)。 4.不同體內(nèi)預(yù)血管化方法的模型：CTA檢測提示AV Loop組的血管環(huán)在第2W、4W、8W通暢,B超證實血管環(huán)在6月仍通暢；VB組血管束不顯影。 5.不同體內(nèi)預(yù)血管化方法構(gòu)建組織工程骨的成血管比較：灌注Microfil并進(jìn)行Micro-CT掃描分析,結(jié)果表明VB組和AV Loop組生成的血管總體積和表面積無明顯差異(p0.05)；VWF免疫組織化學(xué)分析顯示VB組和AV Loop組的血管數(shù)量無明顯差異(p0.05)。 6.不同體內(nèi)預(yù)血管化方法構(gòu)建組織工程骨的成骨比較：CT、Micro-CT以及組織學(xué)分析顯示,VB組和AV Loop組的組織工程骨骨密度和成骨面積均無明顯差異(p0.05)。 結(jié)論 1.聯(lián)合EPCs作為種子細(xì)胞在裸鼠體內(nèi)能夠促進(jìn)BMSCs的成骨；聯(lián)合EPCs作為種子細(xì)胞在比格犬體內(nèi)能夠促進(jìn)組織工程骨的成骨和血管生成。 2.采用比格犬下肢隱動靜脈可以成功構(gòu)建體內(nèi)AV Loop及VB血管化組織工程骨模型,為構(gòu)建大體積血管化組織工程骨提供了參考方案。 3.采用VB及AV Loop體內(nèi)血管化均能提高組織工程骨的血管化和成骨效果,并且兩種方法促進(jìn)組織工程骨血管化和成骨的效果無明顯差異；而VB組具有手術(shù)操作簡單和成功率高的優(yōu)勢。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R318.08
本文編號：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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R318.08
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 吳雪暉;許建中;王序全;羅飛;曾玲;譚洪波;;血管內(nèi)皮祖細(xì)胞對組織工程骨成骨能力影響的實驗研究[J];重慶醫(yī)學(xué);2006年22期
，本文編號：2233793
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