人脂肪来源干细胞的可塑性及其在体外脂肪组织构建中的应用基础研究
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摘要
在组织工程的研究与应用中，种子细胞的来源、功能、规模化扩增、分化能力和老化等是制约组织工程发展的重要瓶颈问题之一。成体干细胞因其具有强大的自我更新、多系分化等特性，给组织工程种子细胞的选择带来了新希望。脂肪组织来源的间充质干细胞因其具有易于自体取材，大量获取、来源广泛的优势现已逐渐成为脂肪组织工程种子细胞的热点研究对象。我们对脂肪间充质干细胞的生物学特性和可塑性进行了初步的研究，并且利用旋转生物反应器和微载体培养技术对其进行了体外规模化扩增的探讨。此外，我们以脂肪间充质干细胞为种子细胞，以可生物降解的聚羟基乙酸／聚乳酸的共聚物(polylactic／glycolic acid polymer，PLGA)或胶原／藻酸盐为支架材料，对体外人工脂肪组织构建也进行了初步的探索。研究表明，脂肪间充质干细胞具有良好的自我增殖和多系分化潜力，不仅能够向脂肪、成骨等组织细胞分化，也能够向肝、内皮组织细胞分化。它能够在旋转生物反应器中规模化扩增并且保持着稳定的生物学特性。这种模拟微重力的旋转反应器有利于细胞聚集体的形成和体外脂肪组织的构建。本研究为脂肪间充质干细胞在以细胞治疗和组织工程为基础的软组织缺损修复中的应用奠定了一定的理论和技术基础。
For cell-based tissue engineering, expansion and maintenance of cell remains a major obstacle in the fields of tissue engineering. Together with the potential of multilineage differentiation and extensive proliferation of adult stem cell, these characteristics may make it a promising source for the tissue engineering. Recent studies showed that adipose-derived stem cells (ADSCs), although a relatively new addition to the growing list of adult stem cells sources, can differentiate into multiple lineages when placed in an appropriate environment. Human adipose tissue is plentiful, easily procured, and appropriate for autologous transplantation, so they could be an alternative and promising stem cell source for tissue engineering strategies in the future. We have investigated and characterized the biology and plasticity of hADSCs. As an important component of tissue engineering, the bioreactor system plays an important role in providing an optimized environment for cell growth, expansion and functional 3-D tissue development. To overcome the scarcity of cells expansion, we have explored the availability of large scale cultivation of hADSCs utilizing collagen-treated microbeads as carrier in a rotary cell culture system (RCCS), a commercially available bioreactor developed by NASA. Moreover, in present study, we examined the potential of polylactic/glycolic acid polymer (PLGA) non-woven meshes, poly (L-lactic acid) polymer (PLLA), and mixture of sodium alginate /gelatin as biodegradable scaffolds to develop a biohybrid construct as well as hADSCs for ex vivo adipose tissue engineering. It was showed that hADSCs possess high capacity for self-replication and multi-differentiate potential, and can be expanded in large scale in RCCS, in which hADSCs can maintain their phenotype and function. It also demonstrated that simulated microgravity culture of cell/scaffolds in RCCS may be beneficial to cell aggregation, formation of 3-dimensional architecture and construction of adipose tissue in vitro. This study would provide the proof of concept of adipose-derived stem cells-based therapy and tissue engineering for soft tissue repair or other clinical applications.
For cell-based tissue engineering, expansion and maintenance of cell remains a major obstacle in the fields of tissue engineering. Together with the potential of multilineage differentiation and extensive proliferation of adult stem cell, these characteristics may make it a promising source for the tissue engineering. Recent studies showed that adipose-derived stem cells (ADSCs), although a relatively new addition to the growing list of adult stem cells sources, can differentiate into multiple lineages when placed in an appropriate environment. Human adipose tissue is plentiful, easily procured, and appropriate for autologous transplantation, so they could be an alternative and promising stem cell source for tissue engineering strategies in the future. We have investigated and characterized the biology and plasticity of hADSCs. As an important component of tissue engineering, the bioreactor system plays an important role in providing an optimized environment for cell growth, expansion and functional 3-D tissue development. To overcome the scarcity of cells expansion, we have explored the availability of large scale cultivation of hADSCs utilizing collagen-treated microbeads as carrier in a rotary cell culture system (RCCS), a commercially available bioreactor developed by NASA. Moreover, in present study, we examined the potential of polylactic/glycolic acid polymer (PLGA) non-woven meshes, poly (L-lactic acid) polymer (PLLA), and mixture of sodium alginate /gelatin as biodegradable scaffolds to develop a biohybrid construct as well as hADSCs for ex vivo adipose tissue engineering. It was showed that hADSCs possess high capacity for self-replication and multi-differentiate potential, and can be expanded in large scale in RCCS, in which hADSCs can maintain their phenotype and function. It also demonstrated that simulated microgravity culture of cell/scaffolds in RCCS may be beneficial to cell aggregation, formation of 3-dimensional architecture and construction of adipose tissue in vitro. This study would provide the proof of concept of adipose-derived stem cells-based therapy and tissue engineering for soft tissue repair or other clinical applications.
引文
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2 Niechajev I, Sevcuk O. Long term results of fat transplantation: clinical and histologic studies. Plast Reconstr Surg, 1994, 94:496-506
3 Gerszten PC. A formal risk assessment of silicone breast implants. Biomaterials, 1999, 20:1063-1069
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5 SiggelkowW, Faridi A, Spiritus K, et al. Histological analysis of silicone breast implant capsules and correlation with capsular contracture. Biomaterials, 2003, 24:1101-1109
6 Shenaq SM, Yuksel E. New research in breast reconstruction: adipose tissue engineering. Clin Plast Surg, 2002, 29: 111-125
7 Patrick CW. Breast tissue engineering. Annu Rev Biomed Eng, 2004, 6:109-130
8 Kawaguchi N, Toriyama K, Nicodemou-Lena E, et al. De novo adipogenesis in mice at the site of injection of basement membrane and basic fibroblast growth factor. Proc Natl Acad Sci U.S.A, 1998,95: 1062-1066
9 Masuda T, Furue M, Matsuda T. Photocured, styrenated gelatin-based microspheres for de novo adipogenesis through corelease of basic fibroblast growth factor, insulin, and insulin-like growth factor I. Tissue Eng , 2004,10:523-535
10 Kimura Y, Ozeki M, Inamoto T, et al. Adipose tissue engineering based on human preadipocytes combined with gelatin microspheres containing basic fibroblast growth factor. Biomaterials, 2003,24: 2513-2521
11 Patrick CWJr, Zheng B, Johnston C, et al. Long-term implantation of preadipocyte-seeded PLGA scaffolds. Tissue Eng, 2002, 8: 283-293
12 Cho SW, Kim SS, Rhie JW, et al. Engineering of volume-stable adipose tissues. Biomaterials, 2005,26:3577-3585
13 Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 2002,13: 4279-4295
14 Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng, 2001, 7: 211-228
15 Morizono K, De Ugarte DA, Zhu M, et al. Multilineage cells from adipose tissue as gene
delivery vehicles. Hum Gene Ther, 2003, 14(l):59-66
16 Kanemura Y, Mori H, Kobayashi S, et al. Evaluation of in vitro proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity. J Neurosci Res, 2002, 69: 869-879
17 Langer R, Vacanti JP. Tissue engineering. Science, 1993,260(5110):920-926
18 Stock UA, Vacanti JP. Tissue engineering: current state and prospects. Annu Rev Med, 2001, 52:443-451
19 Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411):143-147
20 Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 2002,418(6893):41-49
21 Horwitz EM, Prockop DJ, Gordon PL, et al. Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood, 2001, 97(5):1227-1231
22 Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol, 2000, 18(2):307-316
23 Shahdadfar A, Fronsdal K, Haug T, et al. In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem Cells, 2005,23:1357-1366
24 De Ugarte DA, Alfonso Z, Zuk PA, et al. Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow. Immunol Lett, 2003, 89(2-3):267-270
25 Sen A, Lea2currieYR, Sujkowska D, et al. Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous. J Cell Biochem, 2001,81 :3122319
26 Aust L, Devlin B, Foster SJ, et al. Yield of human adipose derived adult stem cells from liposuction aspirates. Cytotherapy, 2004, 6(1): 7-14
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