FABRICATION OF PRE-VASCULARIZED CELL SHEETS BASED ON RABBIT BONE MARROW MESENCHYMAL STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

FENGYuxia ^1,2 , WANGYang ¹ , LIQin ¹ ,  MADongyang ² ,  RENLiling ¹

1. Department of Orthodontics, Stomatological Hospital, School of Stomatology, Lanzhou University, Lanzhou Gansu, 730000, P. R. China;
2. Department of Oral and Maxillofacial Surgery, Lanzhou General Hospital, Lanzhou Command of Chinese PLA, Lanzhou Gansu, 730000, P. R. China;

Corresponding author：

MADongyang, Email: doctormdy@hotmail.com; RENLiling, Email: renlil@lzu.edu.cn

Keywords：

Cell sheet; Pre-vascularization; Bone marrow mesenchymal stem cells; Endothelial like cells; Rabbit

DOI：

10.7507/1002-1892.20140277

Video：

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Objective To explore a new method of developing a pre-vascularized cell sheets. Methods Bone marrow mesenchymal stem cells (BMSCs) from 3-week-old Japanese white rabbits were harvested and cultured. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were added into the culture medium to differentiate into endothelial like cells (ECs) from BMSCs (experimental group), and non-induced cells served as the control group. The cell morphology was observed; and the von Willebrand factor (vWF) and CD31 immunofluorescent staining was used to identify the induced BMSCs. The 2nd generation BMSCs were seeded on a cell culture dish at a cell density of 9×10⁴cells/cm² and cultured for 14 days to form a thick cell sheet, and ECs from BMSCs were then seeded on the BMSCs sheet at a cell density of 5×10⁴ cells/cm² to develop pre-vascularized cell sheets and cultured for 3, 7, and 14 days (group A); non-induced BMSCs sheet and only ECs from BMSCs were used as group B and group C, respectively. The CD31 immunofluorescent staining and histological analysis were performed to evaluate the pre-vascularized cell sheet. Results BMSCs changed from long fusiform to cobblestone-like morphology after induced by VEGF and bFGF. The expressions of CD31 and vWF were positive in experimental group, but were negative in control group, which suggested that BMSCs have the ability to differentiate into ECs under this condition. After the ECs were seeded on the BMSCs sheet, the ECs migrated and rearranged; intracellular vacuoles and networks were observed. Furthermore, immunofluorescent staining for CD31 also revealed a developing process of tube formation after the ECs were seeded on the BMSCs sheet. The histological evaluations indicated the microvessel lumen formed. However, no similar change was observed in groups B and C. Conclusion BMSCs have the ability to differentiate into ECs after induced by VEGF and bFGF. ECs from BMSCs can develop into vascular network constructs when seeded on the BMSCs sheet, which provides a new method for engineering pre-vascularized tissue construction.

Citation： FENGYuxia, WANGYang, LIQin, MADongyang, RENLiling. FABRICATION OF PRE-VASCULARIZED CELL SHEETS BASED ON RABBIT BONE MARROW MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(10): 1278-1283. doi: 10.7507/1002-1892.20140277 Copy

1.	Jain RK, Au P, Tam J, et al.Engineering vascularized tissue.Nat Biotechnol, 2005, 23(7):821-823.
2.	Liu X, Zhang G, Hou C, et al.Vascularized bone tissue formation induced by fiber-reinforced scaffolds cultured with osteoblasts and endothelial cells.Biomed Res Int, 2013, 2013:854917.
3.	Novosel EC, Kleinhans C, Kluger PJ.Vascularization is the key challenge in tissue engineering.Adv Drug Deliv Rev, 2011, 63(4):300-311.
4.	徐红珍, 苏俭生.组织工程骨的血管化研究进展.中华临床医师杂志(电子版), 2010, 4(4):456-458.
5.	Ohashi K, Yokoyama T, Yamato M, et al.Engineering functional two-and three-dimensional liver systems in vivo using hepatic tissue sheets.Nat Med, 2007, 13(7):880-885.
6.	Nagamori E, Ngo TX, Takezawa Y, et al.Network formation through active migration of human vascular endothelial cells in a multilayered skeletal myoblast sheet.Biomaterials, 2013, 34(3):662-668.
7.	Okano T.Current progress of cell sheet tissue engineering and future perspective.TissueEng Part A, 2014, 2014:24417540.
8.	Elloumi-Hannachi I, Yamato M, Okano T.Cell sheet engineering:a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine.J Intern Med, 2010, 267(1):54-70.
9.	Tsigkou O, Pomerantseva I, Spencer JA, et al.Engineered vascularized bone grafts.Proc Natl Acad Sci, 2010, 107(8):3311-3316.
10.	Au P, Tam J, Fukumura D, et al.Bone marrow-derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature.Blood, 2008, 111(9):4551-4558.
11.	Staton CA, Reed MW, Brown NJ.A critical analysis of current in vitro and in vivo angiogenesis assays.Int J Exp Pathol, 2009, 90(3):195-221.
12.	Pankajakshan D, Kansal V, Agrawal DK.In vitro differentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells.J Tissue Eng Regen Med, 2013, 7(11):911-920.
13.	Santos MI, Unger RE, Sousa RA, et al.Crosstalk between osteoblasts and endothelial cells co-cultured on a polycaprolactone-starch scaffold and the in vitro development of vascularization.Biomaterials, 2009, 30(26):4407-4415.
14.	袁心刚, 范顺阳, 石磊, 等.人骨髓间充质干细胞向血管内皮细胞诱导的研究.中国实用医刊, 2010, 37(11):4-7.
15.	马东洋.基于骨髓基质干细胞构建无外支架组织工程骨的实验研究.西安:第四军医大学, 2010.
16.	林红.间充质干细胞和其诱导内皮细胞联合种植构筑血管化组织工程骨修复兔大段骨缺损.上海:复旦大学, 2007.
17.	Murohara T.Therapeutic vasculogenesis using human cord blood-derived endothelial progenitors.Trends Cardiovasc Med, 2001, 11(8):303-307.
18.	Ma D, Ren L, Liu Y, et al.Engineering scaffold-free bone tissue using bone marrow stromal cell sheets.J Orthop Res, 2010, 28(5):697-702.
19.	Zhang R, Gao Z, Geng W, et al.Engineering vascularized bone graft with osteogenic and angiogenic lineage differentiated bone marrow mesenchymal stem cells.Artificial Organs, 2012, 36(12):1036-1046.
20.	Ma D, Ren L, Yao H, et al.Locally injection of cell sheet fragments enhances new bone formation in mandibular distraction osteogenesis:a rabbit model.J Orthop Res, 2013, 31(7):1082-1088.

1. Jain RK, Au P, Tam J, et al.Engineering vascularized tissue.Nat Biotechnol, 2005, 23(7):821-823.
2. Liu X, Zhang G, Hou C, et al.Vascularized bone tissue formation induced by fiber-reinforced scaffolds cultured with osteoblasts and endothelial cells.Biomed Res Int, 2013, 2013:854917.
3. Novosel EC, Kleinhans C, Kluger PJ.Vascularization is the key challenge in tissue engineering.Adv Drug Deliv Rev, 2011, 63(4):300-311.
4. 徐红珍, 苏俭生.组织工程骨的血管化研究进展.中华临床医师杂志(电子版), 2010, 4(4):456-458.
5. Ohashi K, Yokoyama T, Yamato M, et al.Engineering functional two-and three-dimensional liver systems in vivo using hepatic tissue sheets.Nat Med, 2007, 13(7):880-885.
6. Nagamori E, Ngo TX, Takezawa Y, et al.Network formation through active migration of human vascular endothelial cells in a multilayered skeletal myoblast sheet.Biomaterials, 2013, 34(3):662-668.
7. Okano T.Current progress of cell sheet tissue engineering and future perspective.TissueEng Part A, 2014, 2014:24417540.
8. Elloumi-Hannachi I, Yamato M, Okano T.Cell sheet engineering:a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine.J Intern Med, 2010, 267(1):54-70.
9. Tsigkou O, Pomerantseva I, Spencer JA, et al.Engineered vascularized bone grafts.Proc Natl Acad Sci, 2010, 107(8):3311-3316.
10. Au P, Tam J, Fukumura D, et al.Bone marrow-derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature.Blood, 2008, 111(9):4551-4558.
11. Staton CA, Reed MW, Brown NJ.A critical analysis of current in vitro and in vivo angiogenesis assays.Int J Exp Pathol, 2009, 90(3):195-221.
12. Pankajakshan D, Kansal V, Agrawal DK.In vitro differentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells.J Tissue Eng Regen Med, 2013, 7(11):911-920.
13. Santos MI, Unger RE, Sousa RA, et al.Crosstalk between osteoblasts and endothelial cells co-cultured on a polycaprolactone-starch scaffold and the in vitro development of vascularization.Biomaterials, 2009, 30(26):4407-4415.
14. 袁心刚, 范顺阳, 石磊, 等.人骨髓间充质干细胞向血管内皮细胞诱导的研究.中国实用医刊, 2010, 37(11):4-7.
15. 马东洋.基于骨髓基质干细胞构建无外支架组织工程骨的实验研究.西安:第四军医大学, 2010.
16. 林红.间充质干细胞和其诱导内皮细胞联合种植构筑血管化组织工程骨修复兔大段骨缺损.上海:复旦大学, 2007.
17. Murohara T.Therapeutic vasculogenesis using human cord blood-derived endothelial progenitors.Trends Cardiovasc Med, 2001, 11(8):303-307.
18. Ma D, Ren L, Liu Y, et al.Engineering scaffold-free bone tissue using bone marrow stromal cell sheets.J Orthop Res, 2010, 28(5):697-702.
19. Zhang R, Gao Z, Geng W, et al.Engineering vascularized bone graft with osteogenic and angiogenic lineage differentiated bone marrow mesenchymal stem cells.Artificial Organs, 2012, 36(12):1036-1046.
20. Ma D, Ren L, Yao H, et al.Locally injection of cell sheet fragments enhances new bone formation in mandibular distraction osteogenesis:a rabbit model.J Orthop Res, 2013, 31(7):1082-1088.

Chinese Journal of Reparative and Reconstructive Surgery

FABRICATION OF PRE-VASCULARIZED CELL SHEETS BASED ON RABBIT BONE MARROW MESENCHYMAL STEM CELLS

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