Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro
Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro
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DOI：
10.1089/10763270360728107
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发表时间：
2003-12-01
影响因子：
--
通讯作者：
Guldberg, RE
中科院分区：
文献类型：
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作者：
Cartmell, SH;Porter, BD;Guldberg, RE
由于运输限制，静态培养中组织工程化构建体中心的细胞活性通常相对于构建体周边降低。我们设计了一种组织培养系统，其通过三维（3D）多孔细胞构建体灌注培养基以改善构建体内的营养输送和废物去除。本研究检测了培养基灌注速率对细胞接种的3D骨支架内细胞活力、增殖和基因表达的影响。将MC 3 T3-E1成骨细胞样细胞接种于人骨小梁支架，并以0.01、0.1、0.2和1.0 mL/min的流速灌注1周。培养1周后的共聚焦显微镜检查表明，1.0 mL/min的流速导致整个结构中大量细胞死亡，而降低流速导致活细胞比例增加，特别是在结构的中心。DNA分析显示，相对于0.2 mL/min和静态对照，0.01 mL/min流速下的细胞增殖增加。相反，Runx 2，骨钙素和碱性磷酸酶的mRNA表达上调0.2 mL/min与较低的流速相比，通过实时RT-PCR定量。这些数据表明，介质灌注可能有利于3-D组织在体外的发展，通过增强运输的营养物质和废物内的结构，并提供流动介导的机械刺激。
Cellular activity at the center of tissue-engineered constructs in static culture is typically decreased relative to the construct periphery because of transport limitations. We have designed a tissue culture system that perfuses culture medium through three-dimensional (3D) porous cellular constructs to improve nutrient delivery and waste removal within the constructs. This study examined the effects of medium perfusion rate on cell viability, proliferation, and gene expression within cell-seeded 3D bone scaffolds. Human trabecular bone scaffolds were seeded with MC3T3-E1 osteoblast-like cells and perfused for 1 week at flow rates of 0.01, 0.1, 0.2, and 1.0 mL/min. Confocal microscopy after 1 week of culture indicated that a flow rate of 1.0 mL/min resulted in substantial cell death throughout the constructs whereas lowering the flow rate led to an increasing proportion of viable cells, particularly at the center of the constructs. DNA analysis showed increases in cell proliferation at a flow rate of 0.01 mL/min relative to 0.2 mL/min and static controls. Conversely, mRNA expressions of Runx2, osteocalcin, and alkaline phosphatase were upregulated at 0.2 mL/min compared with lower flow rates as quantified by real-time RT-PCR. These data suggest that medium perfusion may benefit the development of 3-D tissues in vitro by enhancing transport of nutrients and waste within the constructs and providing flow-mediated mechanical stimuli.