骨髓间充质干细胞膜片和脐静脉内皮细胞共培养模型的构建及鉴定
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摘要
背景:细胞膜片技术是近年来热门的组织工程学技术,但是膜片中间部位的营养供给和移植后血供问题易导致失败,所以预血管化是提高组织工程材料移植后存活率的关键。目的:比较在不同氧含量下,人骨髓间充质干细胞和脐静脉内皮细胞共培养形成预血管化骨髓间充质干细胞膜片的生物学性能。方法:实验分2组:常氧共培养组即常氧(体积分数20%O2)条件下形成人骨髓间充质干细胞膜片,常氧条件下加入人脐静脉内皮细胞共培养;低氧共培养组,即低氧(体积分数2%O2)条件下形成人骨髓间充质干细胞膜片,常氧条件下加入人脐静脉内皮细胞共培养。采用免疫荧光染色技术观察并比较共培养7d两组的微血管生成情况,并通过ELISA法比较不同氧含量下骨髓间充质干细胞膜片内血管内皮生长因子的水平。结果与结论:①与常氧共培养组比较,低氧共培养组的血管更长、血管密度更高、管间交互连接更多(P <0.05);②低氧共培养组的血管内皮生长因子水平高于常氧共培养组(P <0.05);③结果证实,低氧共培养组较常氧共培养组膜片更利于血管生长,对于建立优化的预血管化膜片具有可行性。
BACKGROUND: Cell sheet technology is a recent popular technique for tissue engineering. However, a failure is prone to occur because of poor nutrient supply in the middle part of the membrane and post-transplantation blood supply, so pre-vascularization determines the survival rate of tissue engineering materials after transplantation.OBJECTIVE: To investigate the pre-vascularization of bone marrow mesenchymal stem cell sheets co-cultured with human umbilical vein endothelial cells under different oxygen tensions.METHODS: Bone marrow mesenchymal stem cell sheets were prepared under physiological hypoxia(2% O2) and normoxia(20% O2), and then co-cultured with human umbilical vein endothelial cells under normoxia. At 7 days of co-culture the angiogenesis was observed and compared by immunofluorescence staining. The level of vascular endothelial growth factor was compared under different oxygen tensions using ELISA.RESULTS AND CONCLUSION: Compared with the normoxia co-culture group, there were longer microvessels, higher vessel density and more microvessel networks in the hypoxia co-culture group(P < 0.05). The expression of vascular endothelial growth factor in the hypoxia co-culture group was higher than that in the normoxia co-culture group(P < 0.05). Therefore, the hypoxia co-culture is more beneficial to the growth of microvessels than the normoxia co-culture, and it is feasible to construct an optimized prevascularized cell sheets.
BACKGROUND: Cell sheet technology is a recent popular technique for tissue engineering. However, a failure is prone to occur because of poor nutrient supply in the middle part of the membrane and post-transplantation blood supply, so pre-vascularization determines the survival rate of tissue engineering materials after transplantation.OBJECTIVE: To investigate the pre-vascularization of bone marrow mesenchymal stem cell sheets co-cultured with human umbilical vein endothelial cells under different oxygen tensions.METHODS: Bone marrow mesenchymal stem cell sheets were prepared under physiological hypoxia(2% O2) and normoxia(20% O2), and then co-cultured with human umbilical vein endothelial cells under normoxia. At 7 days of co-culture the angiogenesis was observed and compared by immunofluorescence staining. The level of vascular endothelial growth factor was compared under different oxygen tensions using ELISA.RESULTS AND CONCLUSION: Compared with the normoxia co-culture group, there were longer microvessels, higher vessel density and more microvessel networks in the hypoxia co-culture group(P < 0.05). The expression of vascular endothelial growth factor in the hypoxia co-culture group was higher than that in the normoxia co-culture group(P < 0.05). Therefore, the hypoxia co-culture is more beneficial to the growth of microvessels than the normoxia co-culture, and it is feasible to construct an optimized prevascularized cell sheets.
引文
[1]李欣,金作林,吴琼,等.应用牙周膜干细胞-牙囊干细胞复合细胞膜片同种异体移植修复比格犬牙周组织缺损的研究[J].口腔疾病防治,2016,24(4):204-210.
[2] Iwata T, Yamato M, Tsuchioka H, et al. Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model. Biomaterials. 2009;30(14):2716-2723.
[3]文艺,杨鸿旭,刘倩,等.骨髓间充质干细胞联合血管内皮祖细胞修复骨质疏松性牙槽骨缺损[J].中国组织工程研究,2016,20(19):2748-2755.
[4] Yang J, Yamato M, Kohno C, et al. Cell sheet engineering:recreating tissues without biodegradable scaffolds.Biomaterials. 2005;26(33):6415-6422.
[5] Haraguchi Y, Shimizu T, Sasagawa T, et al. Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro. Nat Protoc. 2012;7(5):850-858.
[6] Asakawa N, Shimizu T, Tsuda Y, et al. Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering. Biomaterials. 2010;31(14):3903-3909.
[7] Novosel EC, Kleinhans C, Kluger PJ. Vascularization is the key challenge in tissue engineering. Adv Drug Deliv Rev.2011;63(4-5):300-311.
[8] Mascarenhas S, Avalos B, Ardoin SP. An update on stem cell transplantation in autoimmune rheumatologic disorders. Curr Allergy Asthma Rep. 2012;12(6):530-540.
[9] Yuan X, Tsai AC, Farrance I, et al. Aggregation of culture expanded human mesenchymal stem cells in microcarrier-based bioreactor. Biochem Eng J. 2018;131:39-46.
[10] Liu Y, Ming L, Luo H, et al. Integration of a calcined bovine bone and BMSC-sheet 3D scaffold and the promotion of bone regeneration in large defects. Biomaterials. 2013;34(38):9998-10006.
[11] Stoppato M, Stevens HY, Carletti E, et al. Influence of scaffold properties on the inter-relationship between human bone marrow derived stromal cells and endothelial cells in pro-osteogenic conditions. Acta Biomater. 2015;25:16-23.
[12] Pedersen TO, Blois AL, Xue Y, et al. Mesenchymal stem cells induce endothelial cell quiescence and promote capillary formation. Stem Cell Res Ther. 2014;5(1):23.
[13] Huang CC, Pan WY, Tseng MT, et al. Enhancement of cell adhesion, retention, and survival of HUVEC/cbMSC aggregates that are transplanted in ischemic tissues by concurrent delivery of an antioxidant for therapeutic angiogenesis. Biomaterials. 2016;74;53-63.
[14] Gurel PG, Torun KG, Hasirci V. Influence of co-culture on osteogenesis and angiogenesis of bone marrow mesenchymal stem cells and aortic endothelial cells.Microvasc Res. 2016;108:1-9.
[15]梁源,隋珂,尚冯青,等.血管内皮祖细胞/骨髓间充质干细胞复合细胞膜片的构建[J].中国组织工程研究,2014,18(41):6561-6566.
[16] Zhilai Z, Biling M, Sujun Q, et al. Preconditioning in lowered oxygen enhances the therapeutic potential of human umbilical mesenchymal stem cells in a rat model of spinal cord injury.Brain Res. 2016;1642;426-435.
[17] Kobayashi J, Kikuchi A, Aoyagi T, et al. Cell sheet tissue engineering:Cell sheet preparation, harvesting/manipulation,and transplantation. J Biomed Mater Res A. 2019;107(5):955-967.
[18]孟文霞,郭薇,李志强,等.血管生成相关因子在口腔扁平苔藓中的表达[J].口腔疾病防治,2017,25(11):712-717.
[19] Xu R, Sun Y, Chen Z, et al. Hypoxic preconditioning inhibits hypoxia-induced apoptosis of cardiac progenitor cells via the PI3K/Akt-DNMT1-p53 pathway. Sci Rep. 2016;6:30922.
[20] Mohyeldin A, Garzon-Muvdi T, Quinones-Hinojosa A. Oxygen in stem cell biology:a critical component of the stem cell niche. Cell Stem Cell. 2010;7(2):150-161.
[21] Grayson WL, Zhao F, Bunnell B, et al. Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells. Biochem Biophys Res Commun. 2007;358(3):948-953.
[22] Basciano L, Nemos C, Foliguet B, et al. Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent status. BMC Cell Biol. 2011;12:12.
[23] Teixeira FG, Panchalingam KM, Anjo SI, et al. Do hypoxia/normoxia culturing conditions change the neuroregulatory profile of Wharton Jelly mesenchymal stem cell secretome? Stem Cell Res Ther. 2015;6;133.
[24] Habib R, Haneef K, Naeem N, et al. Hypoxic stress and IL-7gene overexpression enhance the fusion potential of rat bone marrow mesenchymal stem cells with bovine renal epithelial cells. Mol Cell Biochem. 2015;403(1-2):125-137.
[25] Chen J, Zhang D, Li Q, et al. Effect of different cell sheet ECM microenvironment on the formation of vascular network.Tissue Cell. 2016;48(5):442-451.
[26] Amiri F, Jahanian-Najafabadi A, Roudkenar MH. In vitro augmentation of mesenchymal stem cells viability in stressful microenvironments:in vitro augmentation of mesenchymal stem cells viability. Cell Stress Chaperones. 2015;20(2):237-251.
[27] Qin HH, Filippi C, Sun S, et al. Hypoxic preconditioning potentiates the trophic effects of mesenchymal stem cells on co-cultured human primary hepatocytes. Stem Cell Res Ther.2015;6;237.
[28] Fan L, Zhang C, Yu Z, et al. Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and osteogenesis in rabbit femoral head osteonecrosis. Bone. 2015;81:544-553.
[29] Denu RA, Hematti P. Effects of oxidative stress on mesenchymal stem cell biology. Oxid Med Cell Longev. 2016;2016:2989076.
[2] Iwata T, Yamato M, Tsuchioka H, et al. Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model. Biomaterials. 2009;30(14):2716-2723.
[3]文艺,杨鸿旭,刘倩,等.骨髓间充质干细胞联合血管内皮祖细胞修复骨质疏松性牙槽骨缺损[J].中国组织工程研究,2016,20(19):2748-2755.
[4] Yang J, Yamato M, Kohno C, et al. Cell sheet engineering:recreating tissues without biodegradable scaffolds.Biomaterials. 2005;26(33):6415-6422.
[5] Haraguchi Y, Shimizu T, Sasagawa T, et al. Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro. Nat Protoc. 2012;7(5):850-858.
[6] Asakawa N, Shimizu T, Tsuda Y, et al. Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering. Biomaterials. 2010;31(14):3903-3909.
[7] Novosel EC, Kleinhans C, Kluger PJ. Vascularization is the key challenge in tissue engineering. Adv Drug Deliv Rev.2011;63(4-5):300-311.
[8] Mascarenhas S, Avalos B, Ardoin SP. An update on stem cell transplantation in autoimmune rheumatologic disorders. Curr Allergy Asthma Rep. 2012;12(6):530-540.
[9] Yuan X, Tsai AC, Farrance I, et al. Aggregation of culture expanded human mesenchymal stem cells in microcarrier-based bioreactor. Biochem Eng J. 2018;131:39-46.
[10] Liu Y, Ming L, Luo H, et al. Integration of a calcined bovine bone and BMSC-sheet 3D scaffold and the promotion of bone regeneration in large defects. Biomaterials. 2013;34(38):9998-10006.
[11] Stoppato M, Stevens HY, Carletti E, et al. Influence of scaffold properties on the inter-relationship between human bone marrow derived stromal cells and endothelial cells in pro-osteogenic conditions. Acta Biomater. 2015;25:16-23.
[12] Pedersen TO, Blois AL, Xue Y, et al. Mesenchymal stem cells induce endothelial cell quiescence and promote capillary formation. Stem Cell Res Ther. 2014;5(1):23.
[13] Huang CC, Pan WY, Tseng MT, et al. Enhancement of cell adhesion, retention, and survival of HUVEC/cbMSC aggregates that are transplanted in ischemic tissues by concurrent delivery of an antioxidant for therapeutic angiogenesis. Biomaterials. 2016;74;53-63.
[14] Gurel PG, Torun KG, Hasirci V. Influence of co-culture on osteogenesis and angiogenesis of bone marrow mesenchymal stem cells and aortic endothelial cells.Microvasc Res. 2016;108:1-9.
[15]梁源,隋珂,尚冯青,等.血管内皮祖细胞/骨髓间充质干细胞复合细胞膜片的构建[J].中国组织工程研究,2014,18(41):6561-6566.
[16] Zhilai Z, Biling M, Sujun Q, et al. Preconditioning in lowered oxygen enhances the therapeutic potential of human umbilical mesenchymal stem cells in a rat model of spinal cord injury.Brain Res. 2016;1642;426-435.
[17] Kobayashi J, Kikuchi A, Aoyagi T, et al. Cell sheet tissue engineering:Cell sheet preparation, harvesting/manipulation,and transplantation. J Biomed Mater Res A. 2019;107(5):955-967.
[18]孟文霞,郭薇,李志强,等.血管生成相关因子在口腔扁平苔藓中的表达[J].口腔疾病防治,2017,25(11):712-717.
[19] Xu R, Sun Y, Chen Z, et al. Hypoxic preconditioning inhibits hypoxia-induced apoptosis of cardiac progenitor cells via the PI3K/Akt-DNMT1-p53 pathway. Sci Rep. 2016;6:30922.
[20] Mohyeldin A, Garzon-Muvdi T, Quinones-Hinojosa A. Oxygen in stem cell biology:a critical component of the stem cell niche. Cell Stem Cell. 2010;7(2):150-161.
[21] Grayson WL, Zhao F, Bunnell B, et al. Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells. Biochem Biophys Res Commun. 2007;358(3):948-953.
[22] Basciano L, Nemos C, Foliguet B, et al. Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent status. BMC Cell Biol. 2011;12:12.
[23] Teixeira FG, Panchalingam KM, Anjo SI, et al. Do hypoxia/normoxia culturing conditions change the neuroregulatory profile of Wharton Jelly mesenchymal stem cell secretome? Stem Cell Res Ther. 2015;6;133.
[24] Habib R, Haneef K, Naeem N, et al. Hypoxic stress and IL-7gene overexpression enhance the fusion potential of rat bone marrow mesenchymal stem cells with bovine renal epithelial cells. Mol Cell Biochem. 2015;403(1-2):125-137.
[25] Chen J, Zhang D, Li Q, et al. Effect of different cell sheet ECM microenvironment on the formation of vascular network.Tissue Cell. 2016;48(5):442-451.
[26] Amiri F, Jahanian-Najafabadi A, Roudkenar MH. In vitro augmentation of mesenchymal stem cells viability in stressful microenvironments:in vitro augmentation of mesenchymal stem cells viability. Cell Stress Chaperones. 2015;20(2):237-251.
[27] Qin HH, Filippi C, Sun S, et al. Hypoxic preconditioning potentiates the trophic effects of mesenchymal stem cells on co-cultured human primary hepatocytes. Stem Cell Res Ther.2015;6;237.
[28] Fan L, Zhang C, Yu Z, et al. Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and osteogenesis in rabbit femoral head osteonecrosis. Bone. 2015;81:544-553.
[29] Denu RA, Hematti P. Effects of oxidative stress on mesenchymal stem cell biology. Oxid Med Cell Longev. 2016;2016:2989076.
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