心肌补片的制备与结构功能特点

doi:10.3969/j.issn.2095-4344.2015.38.026

摘要/Abstract

摘要：

背景：组织工程心肌补片可以通过体内移植，使种子细胞准确到达梗死心肌区域并进行再生修复，达到改善心功能的治疗效果。
目的：回顾制备组织工程心肌补片的构建方法和策略，分析不同构建方法制备心肌补片的结构功能特点。
方法：由第一作者检索2000至2014年Web of science核心数据库、PudMed数据库，检索主题词为“cardiac infarction；cell patch”。
结果与结论：制备组织工程心肌补片的方法和策略有：叠加单层细胞片获取复层细胞结构；将胶原凝胶等材料与细胞混合形成复层结构；在支架材料上种植细胞；使细胞齐向排列等。目前关于组织工程心肌补片的研究显示，将细胞片进行体内移植后，种子细胞在心肌梗死区域停留率、存活率与旁分泌作用等，在很大程度上影响细胞片对梗死心肌的再生修复作用。现有的细胞片构建方法中以温敏法构建技术最为成熟、研究使用最为广泛，与此同时，其他构建方法也可不同程度地提高种子细胞的增殖、分化及旁分泌能力，增加细胞片的力学强度与厚度以增加移植可操作性。研究人员常结合多种方法用于制备获取生物学及力学特性良好的组织工程心肌补片。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

关键词: 生物材料, 材料相容性, 心肌补片, 组织工程, 心肌梗死, 构建方法

Abstract:

BACKGROUND: In recent years, a lot of studies have reported the feasibility and efficacy of transplanting seed cells via tissue-engineered cardiac patches into specific infarcted myocardial area, helping regeneration of damaged heart tissue and improving heart functions.
OBJECTIVE: To review the methods and strategies of fabricating tissue-engineered cardiac patches and to analyze the structure and functional characteristics of cardiac patches prepared by different methods.
METHODS: The first author retrieved relevant articles about the fabrication methods of tissue engineered cardiac patches by searching PubMed and Web of science data bases. The key words were “cardiac infarction, cell death”.
RESULTS AND CONCLUSION: The fabrication methods and strategies of tissue-engineered cardiac patches are as follows: (1) obtaining multi-layer cell sheets by overlapping monolayer cell sheets; (2) combing gelatin with cells to construct multi-layer cell sheets; (3) seeding cells on scaffolds; (4) homogeneous arrangement of cells. Recent studies about tissue-engineered cardiac patches have demonstrated that the biological activities of seed cells, such as retention, survival and paracrine effects, greatly influence the effect of cardiac patches on the repair and regeneration of infarcted myocardial tissues. Temperature-sensitive method is the most mature and wide-spread used constructing technique; besides, other strategies also successfully improve the biological activities of seed cells to different extents, and they also increase the mechanical strength and thickness of cell

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: 生物材料, 材料相容性, 心肌补片, 组织工程, 心肌梗死, 构建方法

中图分类号:

R318

王颖薇，秦子夕，武征. 心肌补片的制备与结构功能特点[J]. 中国组织工程研究, 2015, 19(38): 6211-6216.

Wang Ying-wei, Qin Zi-xi, Wu Zheng. Fabrication and functional characteristics of cardiac patches [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(38): 6211-6216.

参考文献

[1] Sekiya N,Tobita K,Beckman S,et al. Muscle-derived Stem Cell Sheets Support Pump Function and Prevent Cardiac Arrhythmias in a Model of Chronic Myocardial Infarction. Mol Ther.2013;21(3):662-669.
[2] Narita T,Shintani Y,Ikebe C,et al.The Use of Scaffold-free Cell Sheet Technique to Refine Mesenchymal Stromal Cell-based Therapy for Heart Failure.Mol Ther.2013;21:860-867.
[3] Hamdi H,Planat-Benard V,Bel A,et al.Epicardial adipose stem cell sheets results in greater post-infarction survival than intramyocardial injections.Cardiovasc Res.2011;91:483-491.
[4] Hamdi H,Boitard SE,Planat-Benard V,et al.Efficacy of epicardially delivered adipose stroma cell sheets in dilated cardiomyopathy.Cardiovasc Res.2013;99(4):640-647.
[5] Narita T,Shintani Y,Ikebe C,et al.The use of cell-sheet technique eliminates arrhythmogenicity of skeletal myoblast-based therapy to the heart with enhanced therapeutic effects.Int J Cardiol.2013;168:261-269.
[6] Shimizu T,Yamato M,Kikuchi A,et al.Cell sheet engineering for myocardial tissue reconstruction. Biomaterials.2003;24: 2309-2316.
[7] Miyahara Y,Nagaya N,Kataoka M,et al.Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med.2006;12(4):459-465.
[8] Uchinaka A,Kawaguchi N,Hamada Y,et al.Transplantation of myoblast sheets that secrete the novel peptide SVVYGLR improves cardiac function in failing hearts. Cardiovasc Res. 2013;99:102-110.
[9] Uchinaka A,Kawaguchi N,Hamada Y,et al.Transplantation of elastin-secreting myoblast sheets improves cardiac function in infarcted rat heart. Mol Cell Biochem.2012;368(1-2): 203-214.
[10] Shimizu T,Sekine H,Isoi Y,et al.Long-term survival and growth of pulsatile myocardial tissue grafts engineered by the layering of cardiomyocyte sheets.Tissue Eng. 2006;12(3): 499-507.
[11] Okura H,Matsuyama A,Lee CM,et al.Cardiomyoblast-like cells differentiated from human adipose tissue-derived mesenchymal stem cells improve left ventricular dysfunction and survival in a rat myocardial infarction model.Tissue Eng Part C Methods. 2010;16(3):417-425.
[12] Matsuura K, Honda A, Nagai T,et al.Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice.J Clin Invest. 2009;119(8): 2204–2217.
[13] Masumoto H,Matsuo T,Yamamizu K,et al.Pluripotent Stem Cell-Engineered Cell Sheets Reassembled with Defined Cardiovascular Populations Ameliorate Reduction in Infarct Heart Function Through Cardiomyocyte-Mediated Neovascularization.Stem cells. 2012;30:1196-1205.
[14] Chang DH,Wen ZL,Wang YH,et al.Ultrastructural features of ischemic tissue following application of a bio-membrane based progenitor cardiomyocyte patch for myocardial infarction repair.PLoS One.2014;9(10):e107296.
[15] Alshammary S,Fukushima S,Miyagawa S,et al.Impact of cardiac stem cell sheet transplantation on myocardial infarction. SurgToday.2013;43(9):970-976.
[16] Shimizu T,Yamato M,Isoi Y,et al.Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces.Circ Res.2002;90:E40-E48.
[17] Kobayashi H,Shimizu T,Yamato M,et al.Fibroblast sheets co-cultured with endothelial progenitor cells improve cardiac function of infarcted hearts.J Artif Organs.2008; 11(3): 141-147.
[18] Matsuura K,Wada M,Shimizu T,et al.Creation of human cardiac cell sheets using pluripotent stem cells. Biochem Biophys Res Commun.2012;425(2):321-327.
[19] Matsuura K,Wada M,Konishi K,et al. Fabrication of mouse embryonic stem cell-derived layered cardiac cell sheets using a bioreactor culture system.PLoS One. 2012;7(12):e52176.
[20] Sasagawa T,Shimizu T,Sekiya S,et al.Comparison of angiogenic potential between prevascular and non-prevascular layered adipose-derived stem cell-sheets in early post-transplanted period.J Biomed Mater Res A.2014; 102(2):358-365.
[21] Ngo TX,Nagamori E,Kikuchi T,et al. Endothelial cell behavior inside myoblast sheets with different thickness. Biotechnol Lett. 2013;35(7):1001-1008.
[22] Tadakuma K,Tanaka N,Haraguchi Y,et al.A device for the rapid transfer/transplantation of living cell sheets with the absence of cell damage. Biomaterials.2013;34:9018-9025.
[23] Yeh TS,Fang YHD,Lu CH,et al.Baculovirus-transduced, VEGF-expressing adipose-derived stem cell sheet for the treatment of myocardium infarction. Biomaterials.2014;35: 174-184.
[24] Guo Y,Zhang XZ,Wei Y,et al.Culturing of ventricle cells at high density and construction of engineered cardiac cell sheets without scaffold.Int Heart J. 2009;50(5):653-662.
[25] Itabashi Y,Miyoshi S,Kawaguchi H,et al.A new method for manufacturing cardiac cell sheets using fibrin-coated dishes and its electrophysiological studies by optical mapping. Artificial Organs.2005;29(2):95-103.
[26] Suzuki R,Hattori F,Itabashi Y,et al.Omentopexy enhances graft function in myocardial cell sheet transplantation. Biochem Biophys Res Commun. 2009;387(2):353-359.
[27] Stevens KR, Pabon L, Muskheli V,et al.Scaffold-free human cardiac tissue patch created from embryonic stem cells. Tissue Eng Part A.2009;15(6):1211-1222.
[28] Stevens KR,Kreutziger KL,Dupras SK,et al.Physiological function and transplantation of scaffold-free and vascularized human cardiac muscle tissue. Proc Natl Acad Sci U S A. 2009; 106(39):16568-16573.
[29] Akiyama H,Ito A,Sato M, et al.Construction of cardiac tissue rings using a magnetic tissue fabrication technique.Int J Mol Sci.2010;11(8):2910-2920.
[30] Ishii M,Shibata R,Shimizu Y,et al.Multilayered adipose-derived regenerative cell sheets created by a novel magnetite tissue engineering method for myocardial infarction. Int J Cardiol.2014;175:545-553.
[31] Kito T,Shibata R,Ishii M,et al.iPS cell sheets created by a novel magnetite tissue engineering method for reparative angiogenesis. Sci Rep.2013;3:1418.
[32] Shin SR,Aghaei-Ghareh-Bolagh B,Gao XG,et al. Layer-by-Layer Assembly of 3D Tissue Constructs with Functionalized Graphene.Adv Funct Mater.2014;24(39): 6136-6144.
[33] Arana M,Pena E,Abizanda G,et al.Preparation and characterization of collagen-based ADSC-carrier sheets for cardiovascular application.Acta Biomater.2013;9:6075-6083.
[34] Arana M,Gavira JJ,Pena E,et al.Epicardial delivery of collagen patches with adipose-derived stem cells in rat and minipig models of chronic myocardial infarction. Biomaterials. 2014;35:143-151.
[35] Hamdi H,Planat-Benard V,Bel A,et al.Long-term functional benefits of epicardial patches as cell carriers.Cell Transplant. 2014;23:87-96.
[36] Yang MC,Wang SS,Chou NK,et al.The cardiomyogenic differentiation of rat mesenchymal stem cells on silk fibroin-polysaccharide cardiac patches in vitro.Biomaterials. 2009;30:3757-3765.
[37] Chi NH,Yang MC,Chung TW,et al.Cardiac repair achieved by bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid patches in a rat of myocardial infarction model. Biomaterials.2012;33:5541-5551.
[38] Piao H,Kwon JS,Piao S,et al. Effects of cardiac patches engineered with bone marrow-derived mononuclear cells and PGCL scaffolds in a rat myocardial infarction model. Biomaterials.2007;28:641-649.
[39] Siepe M,Giraud MN,Liljensten E,et al.Construction of skeletal myoblast-based polyurethane scaffolds for myocardial repair. Artif Organs.2007;31(6):425-433.
[40] Giraud MN,Flueckiger R,Cook S,et al.Long-term evaluation of myoblast seeded patches implanted on infarcted rat hearts. Artif Organs.2010;34:E184-192.
[41] Wickham AM,Islam MM,Mondal D,et al. Polycaprolactone- thiophene-conjugated carbon nanotube meshes as scaffolds for cardiac progenitor cells.J Biomed Mater Res B Appl Biomater.2014;102(7):1553-1561.
[42] Wei HJ,Chen CH,Lee WY,et al.Bioengineered cardiac patch constructed from multilayered mesenchymal stem cells for myocardial repair.Biomaterials. 2008;29:3547-3556.
[43] Chen CH,Wei HJ,Lin WW,et al.Porous tissue grafts sandwiched with multilayered mesenchymal stromal cell sheets induce tissue regeneration for cardiac repair. Cardiovasc Res.2008;80(1):88-95.
[44] Huang CC,Liao CK,Yang MJ,et al. A strategy for fabrication of a three-dimensional tissue construct containing uniformly distributed embryoid body-derived cells as a cardiac patch. Biomaterials.2010;31:6218-6227.
[45] Lin YD,Ko MC,Wu ST,et al.A nanopatterned cell-seeded cardiac patch prevents electro-uncoupling and improves the therapeutic efficacy of cardiac repair. Biomater Sci.2014;2: 567-580.
[46] Jiao A,Trosper NE,Yang HS,et al.Thermoresponsive Nanofabricated Substratum for the Engineering of Three-Dimensional Tissues with Layer-by-Layer Architectural Control. Acs Nano.2014;8:4430-4439.
[47] Fujita H,Shimizu K,Nagamori E.Application of a cell sheet-polymer film complex with temperature sensitivity for increased mechanical strength and cell alignment capability. Biotechnol Bioeng.2009;103(2):370-377.
[48] Liau B,Christoforou N, Leong KW, et al.Pluripotent stem cell-derived cardiac tissue patch with advanced structure and function. Biomaterials.2011;32:9180-9187.
[49] Bian WN,Liau B,Badie N,et al.Mesoscopic hydrogel molding to control the 3D geometry of bioartificial muscle tissues.Nat Protoc.2009;4:1522-1534.
[50] Sakaguchi K,Shimizu T,Horaguchi S,et al.In vitro engineering of vascularized tissue surrogates. Sci Rep.2013;3:1316.
[51] Kadota S,Minami I,Morone N,et al.Development of a reentrant arrhythmia model in human pluripotent stem cell-derived cardiac cell sheets. Eur Heart J.2013;34:1147-1156.

引言

材料方法

讨论

在无载体的心肌补片产品中，由于细胞片的成分与天然材料十分接近，这为种子细胞进行增殖分化等生命活动提供了较理想的微环境，同时细胞片的天然组成很大程度地避免了由非天然材料体内移植后引起的炎症反应、材料降解等问题。但目前无载体细胞片的构建仍然存在一些需要解决的问题：首先，细胞片结构形成有赖于细胞间连接，而这导致了体外构建周期长的问题；其次此类细胞片厚度一般较薄，需要通过后续的人工叠加操作才能得到厚层组织，在一定程度上增加了细胞片构建的操作难度，但可以开发相应的辅助工具解决一部分困难；另外，种子细胞的空间排列一般也较难控制。在有载体心肌补片中，一方面，支架材料的使用直接提高了细胞片整体的厚度，增加了细胞片移植应用的可操作性；另一方面，不同支架材料的性质也会影响种子细胞在其上贴附、增殖、分化及旁分泌等一系列的生物学活性。除此之外，可以对支架材料表面进行改性处理，使种子细胞在支架材料上以一定的方向齐性排列并建立有效连接，可以很大程度地提高信号在细胞间的传导效率，利于细胞间进行有效信息传递。然而，由于大部分细胞片载体使用的支架材料为胶原支架或高分子合成支架，此类非天然材料进行体内移植后可能会出现由材料降解等原因引起的宿主炎症反应，因此在选用或制备支架材料的过程中，除了有目的地设计支架材料的结构和功能外，还应充分考虑材料本身的生物相容性问题。在进行细胞片构建时，研究者应该根据目的细胞片的功能特点相应地选择构建方法与策略。考虑到种子细胞的生物学活性是其发挥修复作用的关键因素，在构建方案的设计上，除了需要关注细胞片的片层结构及力学结构外，还可以在构建过程中设计维持或提高种子细胞生物学活性的方案，如对种子细胞进行旁分泌因子基因转染^[23]，种植营养因子与细胞因子以提高种子细胞的活性^[35]，使用动态培养系统保证种子细胞的营养供应等^[50]。另外，还可以利用组织工程原理及技术优势，制备体外心肌功能检测的细胞片模型，如人多功能干细胞诱导的心肌细胞片可作为体外检测心率失常的模型^[51]。
中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程