COMPARATIVE STUDY ON COMBINED CULTURE OF HUMAN PLACENTA-DERIVED MESENCHYMAL STEM CELLS AND HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS FROM SAME AND DIFFERENT INDIVIDUALS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YANG Jing , XIANG Zhou , WEI Daiqing , HUANG Xiao , GAO Yu , GAO Bo , SHU Tao , DUAN Xin , ZHANG Xingdong.

Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China. Corresponding author: XIANG Zhou, E-mail: xiangzhou15@hotmail.com;;
ZHANG Xingdong, E-mail: zhangxd@scu.edu.cn;

Corresponding author：

Keywords：

Tissue engineered bone; Vascularization; Human placenta-derived mesenchymal stem cells; Human umbilical vein endothelial cells; Combined culture

DOI：

10.7507/1002-1892.20130202

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Objective To investigate the protocols of combined culture of human placenta-derived mesenchymal stem cells (HPMSCs) and human umbilical vein endothelial cells (HUVECs) from the same and different individuals on collagen material, to provide the. Methods Under voluntary contributions, HPMSCs were isolated and purified from human full-term placenta using collagenase IV digestion and lymphocyte separation medium, and confirmed by morphology methods and flow cytometry, and then passage 2 cells were cultured under condition of osteogenic induction. HUVECs were isolated from fresh human umbilical vein by collagenase I digestion and subcultured to purification, and cells were confirmed by immunocytochemical staining of von Willebrand factor (vWF). There were 2 groups for experiment. Passage 3 osteoblastic induced HPMSCs were co-cultured with HUVECs (1 ∶ 1) from different individuals in group A and with HUVECs from the same individual in group B on collagen hydrogel. Confocal laser scanning microscope was used to observe the cellular behavior of the cell-collagen composites at 1, 3, 5, and 7 days after culturing. Results Flow cytometry showed that HPMSCs were bly positive for CD90 and CD29, but negative for CD31, CD45, and CD34. After induction, alizarin red, alkaline phosphatase, and collagenase I staining were positive. HUVECs displayed cobble-stone morphology and stained positively for endothelial cell marker vWF. The immunofluorescent staining of CD31 showed that HUVECs in the cell-collagen composite of group B had richer layers, adhered and extended faster and better in three-dimension space than that of group A. At 7 days, the class-like microvessel lengths and the network point numbers were (6.68 ± 0.35) mm/mm2 and (17.10 ± 1.10)/mm2 in group A, and were (8.11 ± 0.62) mm/mm2 and (21.30 ± 1.41)/mm2 in group B, showing significant differences between the 2 groups (t=0.894, P=0.000; t=0.732, P=0.000). Conclusion Composite implant HPMSCs and HUVECs from the same individual on collagen hydrogel is better than HPMSCs and HUVECs from different individuals in integrity and continuity of the network and angiogenesis.

Citation： YANG Jing,XIANG Zhou,WEI Daiqing,HUANG Xiao,GAO Yu,GAO Bo,SHU Tao,DUAN Xin,ZHANG Xingdong.. COMPARATIVE STUDY ON COMBINED CULTURE OF HUMAN PLACENTA-DERIVED MESENCHYMAL STEM CELLS AND HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS FROM SAME AND DIFFERENT INDIVIDUALS. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(8): 916-922. doi: 10.7507/1002-1892.20130202 Copy

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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.
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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.
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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. 参考文献.
5. 杨志明. 组织工程基础与临床. 成都: 四川科学技术出版社, 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.

Chinese Journal of Reparative and Reconstructive Surgery

COMPARATIVE STUDY ON COMBINED CULTURE OF HUMAN PLACENTA-DERIVED MESENCHYMAL STEM CELLS AND HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS FROM SAME AND DIFFERENT INDIVIDUALS

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