Construction and preliminary study on biological characteristics of composite cell sheets of mesenchymal stem cells and endothelial progenitor cells derived from peripheral blood_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XING Fei , DUAN Xin , LIU Ming , CHEN Jialei , LONG Cheng , CHEN Ran , SUN Jiachen , WU Shuang , CHEN Li ,  XIANG Zhou

Department of Orthopedics, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China;

Corresponding author：

XIANG Zhou, Email: xiangzhou15@hotmail.com

Keywords：

Peripheral blood mesenchymal stem cells; endothelial progenitor cells; cell sheets; tissue engineering; tissue repair

DOI：

10.7507/1002-1892.201901087

Video：

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Objective To separate peripheral blood mesenchymal stem cells (PBMSC) and peripheral blood endothelial progenitor cells (PBEPC) from peripheral blood, and investigate the biological characteristics of composite cell sheets of PBMSC and PBEPC.Methods The peripheral blood of healthy adult New Zealand white rabbits was extracted and PBMSC and PBEPC were separated by density gradient centrifugation. Morphological observation and identification of PBMSC and PBEPC were performed. The 3rd generation of PBMSC and PBEPC were used to construct a composite cell sheet at a ratio of 1∶1, and the 3rd generation of PBMSC was used to construct a single cell sheet as control. The distributions of cells in two kinds of cell sheets were observed by HE staining. In addition, the expression of alkaline phosphatase (ALP), osteocalcin (OCN), and vascular endothelial growth factor (VEGF) in the supernatants of cell sheets were observed by ELISA at 1, 5, and 10 days after osteogenic induction.Results The morphology of PBMSC was spindle-shaped or polygonal, and PBMSC had good abilities of osteogenic and adipogenic differentiation. The morphology of PBEPC was paved stone-like, and the tube-forming test of PBEPC was positive. Two kinds of cell sheets were white translucent. The results of HE staining showed that the composite cell sheet had more cell layers and higher cell density than the single cell sheet. The expressions of ALP, OCN, and VEGF in the supernatant of the two groups of cell sheets increased with the time of induction. The expression of OCN in the group of composite cell sheet was significantly higher than that in the group of single cell sheet on the 5th and 10th day, ALP on the 10th day was significantly higher than that in the group of single cell sheet, VEGF expression on the 1st, 5th, and 10th day was significantly higher than that in the group of single cell sheet, all showing significant differences (P<0.05), and there was no significant difference between the two groups at other time points (P>0.05).Conclusion PBMSC have stable differentiation ability, and they have good application prospects because of their minimally invasive access. Composite cell membranes constructed by co-culture of two kinds of cells and induction of membrane formation provides a new idea and exploration for tissue defect repair.

Citation： XING Fei, DUAN Xin, LIU Ming, CHEN Jialei, LONG Cheng, CHEN Ran, SUN Jiachen, WU Shuang, CHEN Li, XIANG Zhou. Construction and preliminary study on biological characteristics of composite cell sheets of mesenchymal stem cells and endothelial progenitor cells derived from peripheral blood. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(1): 109-115. doi: 10.7507/1002-1892.201901087 Copy

1.	Maia FR, Carvalho MR, Oliveira JM, et al. Tissue engineering strategies for osteochondral repair. Adv Exp Med Biol, 2018, 1059: 353-371.
2.	Boyce ST, Lalley AL. Tissue engineering of skin and regenerative medicine for wound care. Burns Trauma, 2018, 6: 4.
3.	Shimizu T, Yamato M, Kikuchi A, et al. Cell sheet engineering for myocardial tissue reconstruction. Biomaterials, 2003, 24(13): 2309-2316.
4.	Wang C, Hei F, Ju Z, et al. Differentiation of urine-derived human induced pluripotent stem cells to alveolar type Ⅱ epithelial cells. Cell Reprogram, 2016, 18(1): 30-36.
5.	Gao H, Li B, Zhao L, et al. Influence of nanotopography on periodontal ligament stem cell functions and cell sheet based periodontal regeneration. Int J Nanomedicine, 2015, 10: 4009-4027.
6.	Gao LN, An Y, Lei M, et al. The effect of the coumarin-like derivative osthole on the osteogenic properties of human periodontal ligament and jaw bone marrow mesenchymal stem cell sheets. Biomaterials, 2013, 34(38): 9937-9951.
7.	Tsumanuma Y, Iwata T, Washio K, et al. Comparison of different tissue-derived stem cell sheets for periodontal regeneration in a canine 1-wall defect model. Biomaterials, 2011, 32(25): 5819-5825.
8.	Qi Y, Niu L, Zhao T, et al. Combining mesenchymal stem cell sheets with platelet-rich plasma gel/calcium phosphate particles: a novel strategy to promote bone regeneration. Stem Cell Res Ther, 2015, 6: 256.
9.	Shimizu T, Akahane M, Morita Y, et al. The regeneration and augmentation of bone with injectable osteogenic cell sheet in a rat critical fracture healing model. Injury, 2015, 46(8): 1457-1464.
10.	Yan J, Zhang C, Zhao Y, et al. Non-viral oligonucleotide antimiR-138 delivery to mesenchymal stem cell sheets and the effect on osteogenesis. Biomaterials, 2014, 35(27): 7734-7749.
11.	Fu WL, Xiang Z, Huang FG, et al. Coculture of peripheral blood-derived mesenchymal stem cells and endothelial progenitor cells on strontium-doped calcium polyphosphate scaffolds to generate vascularized engineered bone. Tissue Eng Part A, 2015, 21(5-6): 948-959.
12.	Valenzuela CD, Allori AC, Reformat DD, et al. Characterization of adipose-derived mesenchymal stem cell combinations for vascularized bone engineering. Tissue Eng Part A, 2013, 19(11-12): 1373-1385.
13.	Matsumoto T, Kuroda R, Mifune Y, et al. Circulating endothelial/skeletal progenitor cells for bone regeneration and healing. Bone, 2008, 43(3): 434-439.
14.	Fu WL, Xiang Z, Huang FG, et al. Combination of granulocyte colony-stimulating factor and CXCR4 antagonist AMD3100 for effective harvest of endothelial progenitor cells from peripheral blood and in vitro formation of primitive endothelial networks. Cell Tissue Bank, 2016, 17(1): 161-169.
15.	Ito A, Ino K, Kobayashi T, et al. The effect of RGD peptide-conjugated magnetite cationic liposomes on cell growth and cell sheet harvesting. Biomaterials, 2005, 26(31): 6185-6193.
16.	Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res, 2007, 100(9): 1249-1260.
17.	Li C, Zhang W, Jiang X, et al. Human-placenta-derived mesenchymal stem cells inhibit proliferation and function of allogeneic immune cells. Cell Tissue Res, 2007, 330(3): 437-446.
18.	Zvaifler NJ, Marinova-Mutafchieva L, Adams G, et al. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res, 2000, 2(6): 477-488.
19.	Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science, 1997, 275(5302): 964-967.
20.	Povsic TJ, Adams SD, Zavodni KL, et al. Aldehyde dehydrogenase activity allows reliable EPC enumeration in stored peripheral blood samples. J Thromb Thrombolysis, 2009, 28(3): 259-265.
21.	Kado M, Tanaka R, Arita K, et al. Human peripheral blood mononuclear cells enriched in endothelial progenitor cells via quality and quantity controlled culture accelerate vascularization and wound healing in a porcine wound model. Cell Transplant, 2018, 27(7): 1068-1079.
22.	Zhang Y, Su N, Luo F, et al. Deletion of Fgfr1 in osteoblasts enhances mobilization of EPCs into peripheral blood in a mouse endotoxemia model. Int J Biol Sci, 2014, 10(9): 1064-1071.
23.	Hasegawa M, Yamato M, Kikuchi A, et al. Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model. Tissue Eng, 2005, 11(3-4): 469-478.
24.	Okano T, Yamada N, Sakai H, et al. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly (N-isopropylacrylamide). J Biomed Mater Res, 1993, 27(10): 1243-1251.
25.	Wang J, Zhang R, Shen Y, et al. Recent advances in cell sheet technology for periodontal regeneration. Curr Stem Cell Res Ther, 2014, 9(3): 162-173.
26.	Yano F, Hojo H, Ohba S, et al. Cell-sheet technology combined with a thienoindazole derivative small compound TD-198946 for cartilage regeneration. Biomaterials, 2013, 34(22): 5581-5587.
27.	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.
28.	Zhou Y, Chen F, Ho ST, et al. Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts. Biomaterials, 2007, 28(5): 814-824.
29.	Ji AR, Ku SY, Cho MS, et al. Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage. Exp Mol Med, 2010, 42(3): 175-186.
30.	Wei F, Qu C, Song T, et al. Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity. J Cell Physiol, 2012, 227(9): 3216-3224.
31.	张楠, 刘娜, 孙楚, 等. 新型微弧氧化涂层镁-锌-钙合金支架/自体颗粒骨修复兔临界性骨缺损的研究. 中国修复重建外科杂志, 2018, 32(3): 298-305.
32.	殷建, 王斌, 朱超, 等. 局部注射促血管生成素 2 调控自噬促进体内组织工程人工骨早期血管化和骨缺损修复的研究. 中国修复重建外科杂志, 2018, 32(9): 1150-1156.
33.	Kaigler D, Pagni G, Park CH, et al. Stem cell therapy for craniofacial bone regeneration: a randomized, controlled feasibility trial. Cell Transplant, 2013, 22(5): 767-777.
34.	Khojasteh A, Fahimipour F, Jafarian M, et al. Bone engineering in dog mandible: Coculturing mesenchymal stem cells with endothelial progenitor cells in a composite scaffold containing vascular endothelial growth factor. J Biomed Mater Res B Appl Biomater, 2016, 105(7): 1767-1777.
35.	Seebach C, Henrich D, Wilhelm K, et al. Endothelial progenitor cells improve directly and indirectly early vascularization of mesenchymal stem cell-driven bone regeneration in a critical bone defect in rats. Cell Transplant, 2012, 21(8): 1667-1677.
36.	Zigdon-Giladi H, Bick T, Lewinson D, et al. Mesenchymal stem cells and endothelial progenitor cells stimulate bone regeneration and mineral density. J Periodontol, 2014, 85(7): 984-990.
37.	Liang Y, Wen L, Shang F, et al. Endothelial progenitors enhanced the osteogenic capacities of mesenchymal stem cells in vitro and in a rat alveolar bone defect model. Arch Oral Biol, 2016, 68: 123-130.

1. Maia FR, Carvalho MR, Oliveira JM, et al. Tissue engineering strategies for osteochondral repair. Adv Exp Med Biol, 2018, 1059: 353-371.
2. Boyce ST, Lalley AL. Tissue engineering of skin and regenerative medicine for wound care. Burns Trauma, 2018, 6: 4.
3. Shimizu T, Yamato M, Kikuchi A, et al. Cell sheet engineering for myocardial tissue reconstruction. Biomaterials, 2003, 24(13): 2309-2316.
4. Wang C, Hei F, Ju Z, et al. Differentiation of urine-derived human induced pluripotent stem cells to alveolar type Ⅱ epithelial cells. Cell Reprogram, 2016, 18(1): 30-36.
5. Gao H, Li B, Zhao L, et al. Influence of nanotopography on periodontal ligament stem cell functions and cell sheet based periodontal regeneration. Int J Nanomedicine, 2015, 10: 4009-4027.
6. Gao LN, An Y, Lei M, et al. The effect of the coumarin-like derivative osthole on the osteogenic properties of human periodontal ligament and jaw bone marrow mesenchymal stem cell sheets. Biomaterials, 2013, 34(38): 9937-9951.
7. Tsumanuma Y, Iwata T, Washio K, et al. Comparison of different tissue-derived stem cell sheets for periodontal regeneration in a canine 1-wall defect model. Biomaterials, 2011, 32(25): 5819-5825.
8. Qi Y, Niu L, Zhao T, et al. Combining mesenchymal stem cell sheets with platelet-rich plasma gel/calcium phosphate particles: a novel strategy to promote bone regeneration. Stem Cell Res Ther, 2015, 6: 256.
9. Shimizu T, Akahane M, Morita Y, et al. The regeneration and augmentation of bone with injectable osteogenic cell sheet in a rat critical fracture healing model. Injury, 2015, 46(8): 1457-1464.
10. Yan J, Zhang C, Zhao Y, et al. Non-viral oligonucleotide antimiR-138 delivery to mesenchymal stem cell sheets and the effect on osteogenesis. Biomaterials, 2014, 35(27): 7734-7749.
11. Fu WL, Xiang Z, Huang FG, et al. Coculture of peripheral blood-derived mesenchymal stem cells and endothelial progenitor cells on strontium-doped calcium polyphosphate scaffolds to generate vascularized engineered bone. Tissue Eng Part A, 2015, 21(5-6): 948-959.
12. Valenzuela CD, Allori AC, Reformat DD, et al. Characterization of adipose-derived mesenchymal stem cell combinations for vascularized bone engineering. Tissue Eng Part A, 2013, 19(11-12): 1373-1385.
13. Matsumoto T, Kuroda R, Mifune Y, et al. Circulating endothelial/skeletal progenitor cells for bone regeneration and healing. Bone, 2008, 43(3): 434-439.
14. Fu WL, Xiang Z, Huang FG, et al. Combination of granulocyte colony-stimulating factor and CXCR4 antagonist AMD3100 for effective harvest of endothelial progenitor cells from peripheral blood and in vitro formation of primitive endothelial networks. Cell Tissue Bank, 2016, 17(1): 161-169.
15. Ito A, Ino K, Kobayashi T, et al. The effect of RGD peptide-conjugated magnetite cationic liposomes on cell growth and cell sheet harvesting. Biomaterials, 2005, 26(31): 6185-6193.
16. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res, 2007, 100(9): 1249-1260.
17. Li C, Zhang W, Jiang X, et al. Human-placenta-derived mesenchymal stem cells inhibit proliferation and function of allogeneic immune cells. Cell Tissue Res, 2007, 330(3): 437-446.
18. Zvaifler NJ, Marinova-Mutafchieva L, Adams G, et al. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res, 2000, 2(6): 477-488.
19. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science, 1997, 275(5302): 964-967.
20. Povsic TJ, Adams SD, Zavodni KL, et al. Aldehyde dehydrogenase activity allows reliable EPC enumeration in stored peripheral blood samples. J Thromb Thrombolysis, 2009, 28(3): 259-265.
21. Kado M, Tanaka R, Arita K, et al. Human peripheral blood mononuclear cells enriched in endothelial progenitor cells via quality and quantity controlled culture accelerate vascularization and wound healing in a porcine wound model. Cell Transplant, 2018, 27(7): 1068-1079.
22. Zhang Y, Su N, Luo F, et al. Deletion of Fgfr1 in osteoblasts enhances mobilization of EPCs into peripheral blood in a mouse endotoxemia model. Int J Biol Sci, 2014, 10(9): 1064-1071.
23. Hasegawa M, Yamato M, Kikuchi A, et al. Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model. Tissue Eng, 2005, 11(3-4): 469-478.
24. Okano T, Yamada N, Sakai H, et al. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly (N-isopropylacrylamide). J Biomed Mater Res, 1993, 27(10): 1243-1251.
25. Wang J, Zhang R, Shen Y, et al. Recent advances in cell sheet technology for periodontal regeneration. Curr Stem Cell Res Ther, 2014, 9(3): 162-173.
26. Yano F, Hojo H, Ohba S, et al. Cell-sheet technology combined with a thienoindazole derivative small compound TD-198946 for cartilage regeneration. Biomaterials, 2013, 34(22): 5581-5587.
27. 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.
28. Zhou Y, Chen F, Ho ST, et al. Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts. Biomaterials, 2007, 28(5): 814-824.
29. Ji AR, Ku SY, Cho MS, et al. Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage. Exp Mol Med, 2010, 42(3): 175-186.
30. Wei F, Qu C, Song T, et al. Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity. J Cell Physiol, 2012, 227(9): 3216-3224.
31. 张楠, 刘娜, 孙楚, 等. 新型微弧氧化涂层镁-锌-钙合金支架/自体颗粒骨修复兔临界性骨缺损的研究. 中国修复重建外科杂志, 2018, 32(3): 298-305.
32. 殷建, 王斌, 朱超, 等. 局部注射促血管生成素 2 调控自噬促进体内组织工程人工骨早期血管化和骨缺损修复的研究. 中国修复重建外科杂志, 2018, 32(9): 1150-1156.
33. Kaigler D, Pagni G, Park CH, et al. Stem cell therapy for craniofacial bone regeneration: a randomized, controlled feasibility trial. Cell Transplant, 2013, 22(5): 767-777.
34. Khojasteh A, Fahimipour F, Jafarian M, et al. Bone engineering in dog mandible: Coculturing mesenchymal stem cells with endothelial progenitor cells in a composite scaffold containing vascular endothelial growth factor. J Biomed Mater Res B Appl Biomater, 2016, 105(7): 1767-1777.
35. Seebach C, Henrich D, Wilhelm K, et al. Endothelial progenitor cells improve directly and indirectly early vascularization of mesenchymal stem cell-driven bone regeneration in a critical bone defect in rats. Cell Transplant, 2012, 21(8): 1667-1677.
36. Zigdon-Giladi H, Bick T, Lewinson D, et al. Mesenchymal stem cells and endothelial progenitor cells stimulate bone regeneration and mineral density. J Periodontol, 2014, 85(7): 984-990.
37. Liang Y, Wen L, Shang F, et al. Endothelial progenitors enhanced the osteogenic capacities of mesenchymal stem cells in vitro and in a rat alveolar bone defect model. Arch Oral Biol, 2016, 68: 123-130.

Chinese Journal of Reparative and Reconstructive Surgery

Construction and preliminary study on biological characteristics of composite cell sheets of mesenchymal stem cells and endothelial progenitor cells derived from peripheral blood

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