同一供者及不同供者的人胎盤MSCs與臍靜脈內皮細胞復合培養的比較研究_《中國修復重建外科雜志》

作者：

楊婧 , 項舟 , 魏代清 , 黃瀟 , 高宇 , 高博 , 舒濤 , 段鑫 , 張興棟

四川大學華西醫院骨科（成都，610041）;

關鍵詞：

組織工程骨血管化人胎盤MSCs 人臍靜脈內皮細胞復合培養

DOI：

10.7507/1002-1892.20130202

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目的比較同一供者及不同供者的人胎盤MSCs（human placenta-derived MSCs，HPMSCs）與人臍靜脈內皮細胞（human umbilical vein endothelial cells，HUVECs）三維復合培養效果，為體外構建三維血管化組織工程骨提供實驗依據。方法取健康足月產婦自愿捐贈的胎盤及臍帶組織，采用Ⅳ型膠原酶消化和人淋巴細胞分層液密度梯度離心法分離培養HPMSCs，通過流式細胞學檢測細胞表型，取第2代細胞向成骨細胞誘導培養鑒定；以Ⅰ型膠原酶消化分離培養HUVECs，通過Ⅷ因子相關抗原因子免疫組織化學染色進行鑒定。實驗分為兩組，A組取不同供者的第3代HUVECs和成骨誘導培養3周的第3代HPMSCs以1∶1比例復合種植于膠原水凝膠，制備細胞-膠原水凝膠復合物；B組將同一供者的以上兩種細胞同法復合制備復合物。復合培養第1、3、5、7天取材，行CD31免疫熒光染色，于激光共聚焦顯微鏡下觀察其成血管傾向，測量復合物中的索道長度和節點數，并進行統計學分析。結果經鑒定，成功分離培養HPMSCs及HUVECs。CD31免疫熒光染色示，與A組相比，B組細胞-膠原水凝膠復合物中的HUVECs可在三維空間上較早、較好地伸展，在水凝膠內部相互交織能力較強，形成的索道更長，節點更多，網絡更密集。第7天時B組索道長度和節點數分別為（8.11 ± 0.62）mm/mm2及（21.30 ± 1.41）個/mm2，顯著優于A組的（6.68 ± 0.35） mm/mm2及（17.10 ± 1.10）個/mm2，差異均有統計學意義（t=0.894，P=0.000；t=0.732，P=0.000）。結論將同一供者的HPMSCs與HUVECs體外復合種植于膠原水凝膠支架上，比不同供者來源的細胞形成的網絡成血管傾向更明顯，交織連續性好，層次豐富。

引用本文： 楊婧,項舟,魏代清,黃瀟,高宇,高博,舒濤,段鑫,張興棟. 同一供者及不同供者的人胎盤MSCs與臍靜脈內皮細胞復合培養的比較研究. 中國修復重建外科雜志, 2013, 27(8): 916-922. doi: 10.7507/1002-1892.20130202 復制

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16.	Sumita Y, Honda MJ, Ohara T, et al. Performance of collagen sponge as a 3-D scaffold for tooth-tissue engineering. Biomaterials, 2006, 27(17): 3238-3248.
17.	Liu C, Xia Z, Czernuszka JT. Design and development of three-dimensional scaffolds for tissue engineering. Chemical Engineering Research and Design, 2007, 85(7): 1051-1064.
18.	Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell, 1982, 30(1): 215-224.
19.	Schindler OS. Current concepts of articular cartilage repair. Acta Orthop Belg, 2011, 77(6): 709-726.
20.	Lee SH, Shin H. Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage tissue engineering. Adv Drug Deliv Rev, 2007, 59(4-5): 339-359.
21.	Balakrishnan B, Banerjee R. Biopolymer-based hydrogels for cartilage tissue engineering. Chem Rev, 2011, 111(8): 4453-4474.
22.	Wenger A, Stahl A, Weber H, et al. Modulation of in vitro angiogenesis in a three-dimensional spheroidal coculture model for bone tissue engineering. Tissue Eng, 2004, 10(9-10): 1536-1547.
23.	Borges J, Tegtmeier FT, Padron NT, et al. Chorioallantoic membrane angiogenesis model for tissue engineering: a new twist on a classic model. Tissue Eng, 2003, 9(3): 441-450.
24.	Unger RE, Sartoris A, Peters K, et al. Tissue-like self-assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillary-like structures on three-dimensional porous biomaterials. Biomaterials, 2007, 28(27): 3965-3976.
25.	Lutolf MP. Biomaterials: Spotlight on hydrogels. Nat Mater, 2009, 8(6): 451-453.
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27.	Kanga Y, Kima S, Fahrenholtzb M, et al. Osteogenic and angiogenic potentials of monocultured and co-cultured human-bone-marrow-derived mesenchymal stem cells and human-umbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold. Acta Biomaterialia, 2013, 9(1): 4906-4915.
28.	Li H, Daculsi R, Grellier M, et al. The role of vascular actors in two dimensional dialogue of human bone marrow stromal cell and endothelial cell for inducing self-assembled network. PLoS One, 2011, 6(2): e16767.
29.	Tsigkou O, Pomerantseva I, Spencerc JA, et al. Engineered vascularized bone grafts. Proc Natl Acad Sci U S A, 2010, 107(8): 3311-3316.

1. Lovett M, Lee K, Edwards A, et al. Vascularization strategies for tissue engineering. Tissue Eng Part B Rev, 2009, 15(3): 353-370.
2. Dohle E, Fuchs S, Kolbe M, et al. Sonic hedgehog promotes angiogenesis and osteogenesis in a coculture system consisting of primary osteoblasts and outgrowth endothelial cells. Tissue Eng Part A, 2010, 16(4): 1235-1237.
3. Carmeliet P. Angiogenesis in life, disease and medicine. Nature, 2005, 438(7070): 932-936.
4. 參考文獻.
5. 楊志明. 組織工程基礎與臨床. 成都: 四川科學技術出版社, 2000: 1-11.
6. Kolbe M, Xiang Z, Dohle E, et al. Paracrine effects influenced by cell culture medium and consequences on microvessel-like structures in cocultures of mesenchymal stem cells and outgrowth endothelial cells. Tissue Eng Part A, 2011, 17(17-18): 2199-2212.
7. Orkin SH, Morrison SJ. Stem-cell competition. Nature, 2002, 418(6893): 25-27.
8. Fukuchi Y, Nakajima H, Sugiyama D, et al. Human placenta-derived cells have mesenchymal stem/progenitor cell potential. Stem Cells, 2004, 22(5): 649-658.
9. Longo UG, Loppini M, Berton A, et al. Stem cells from umbilical cord and placenta for musculoskeletal tissue engineering. Curr Stem Cell Res Ther, 2012, 7(4): 272-281.
10. Maruyama Y. The human endothelial cell in tissue culture. Z Zellforsch Mikrosk Anat, 1963, 60: 69-79.
11. Jaffe EA, Nachman RL, Becker CG, et al. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest, 1973, 52(11): 2745-2756.
12. Gimbrone MA Jr, Cotran RS, Folkman J. Human vascular endothelial cells in culture. Growth and DNA synthesis. J Cell Biol, 1974, 60(3): 673-684.
13. Herring M, Gardner A, Glover J. A single-staged technique for seeding vascular grafts with autogenous endothelium. Surgery, 1978, 84(4): 498-504.
14. Baudin B, Bruneel A, Bosselut N, et al. A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc, 2007, 2(3): 481-485.
15. Marin V, Kaplanski G, Grès S, et al. Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells. J Immunol Methods, 2001, 254(1-2): 183-190.
16. Sumita Y, Honda MJ, Ohara T, et al. Performance of collagen sponge as a 3-D scaffold for tooth-tissue engineering. Biomaterials, 2006, 27(17): 3238-3248.
17. Liu C, Xia Z, Czernuszka JT. Design and development of three-dimensional scaffolds for tissue engineering. Chemical Engineering Research and Design, 2007, 85(7): 1051-1064.
18. Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell, 1982, 30(1): 215-224.
19. Schindler OS. Current concepts of articular cartilage repair. Acta Orthop Belg, 2011, 77(6): 709-726.
20. Lee SH, Shin H. Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage tissue engineering. Adv Drug Deliv Rev, 2007, 59(4-5): 339-359.
21. Balakrishnan B, Banerjee R. Biopolymer-based hydrogels for cartilage tissue engineering. Chem Rev, 2011, 111(8): 4453-4474.
22. Wenger A, Stahl A, Weber H, et al. Modulation of in vitro angiogenesis in a three-dimensional spheroidal coculture model for bone tissue engineering. Tissue Eng, 2004, 10(9-10): 1536-1547.
23. Borges J, Tegtmeier FT, Padron NT, et al. Chorioallantoic membrane angiogenesis model for tissue engineering: a new twist on a classic model. Tissue Eng, 2003, 9(3): 441-450.
24. Unger RE, Sartoris A, Peters K, et al. Tissue-like self-assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillary-like structures on three-dimensional porous biomaterials. Biomaterials, 2007, 28(27): 3965-3976.
25. Lutolf MP. Biomaterials: Spotlight on hydrogels. Nat Mater, 2009, 8(6): 451-453.
26. Sharma MB, Limaye LS, Kale VP, et al. Mimicking the functional hematopoietic stem cell niche in vitro: recapitulation of marrow physiology by hydrogel-based three-dimensional cultures of mesenchymal stromal cells. Haematologica, 2012, 97(5): 651-660.
27. Kanga Y, Kima S, Fahrenholtzb M, et al. Osteogenic and angiogenic potentials of monocultured and co-cultured human-bone-marrow-derived mesenchymal stem cells and human-umbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold. Acta Biomaterialia, 2013, 9(1): 4906-4915.
28. Li H, Daculsi R, Grellier M, et al. The role of vascular actors in two dimensional dialogue of human bone marrow stromal cell and endothelial cell for inducing self-assembled network. PLoS One, 2011, 6(2): e16767.
29. Tsigkou O, Pomerantseva I, Spencerc JA, et al. Engineered vascularized bone grafts. Proc Natl Acad Sci U S A, 2010, 107(8): 3311-3316.

《中國修復重建外科雜志》

同一供者及不同供者的人胎盤MSCs與臍靜脈內皮細胞復合培養的比較研究

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《中國修復重建外科雜志》

同一供者及不同供者的人胎盤MSCs與臍靜脈內皮細胞復合培養的比較研究

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