Liu Jun, Hilderink Janneke, Groothuis Tom A M, Otto Cees, van Blitterswijk Clemens A, de Boer Jan
MIRA Research Institute, Department of Tissue Regeneration, University of Twente, Enschede, The Netherlands.
MIRA Research Institute, Department of Biophysical Engineering, University of Twente, Enschede, The Netherlands.
J Tissue Eng Regen Med. 2015 Aug;9(8):952-60. doi: 10.1002/term.1654. Epub 2013 Jan 24.
Limited nutrient diffusion in three-dimensional (3D) constructs is a major concern in tissue engineering. Therefore, monitoring nutrient availability and diffusion within a scaffold is an important asset. Since nutrients come in various forms, we have investigated the diffusion of the oxygen, luciferin and dextran molecules within tissue-engineered constructs using optical imaging technologies. First, oxygen availability and diffusion were investigated, using transgenic cell lines in which a hypoxia-responsive element drives expression of the green fluorescent protein gene. Using confocal imaging, we observed oxygen limitation, starting at around 200 µm from the periphery in the context of agarose gel with 1 million CHO cells. Diffusion of luciferin was monitored real-time in agarose gels using a cell line in which the luciferase gene was driven by a constitutively active CMV promoter. Gel concentration affected the diffusion rate of luciferin. Furthermore, we assessed the diffusion rates of fluorescent dextran molecules of different molecular weights in biomaterials by fluorescence recovery after photobleaching (FRAP) and observed that diffusion depended on both molecular size and gel concentration. In conclusion, we have validated a set of efficient tools to investigate molecular diffusion of a range of molecules and to optimize biomaterials design in order to improve nutrient delivery.
三维（3D）构建物中有限的营养物质扩散是组织工程中的一个主要问题。因此，监测支架内营养物质的可用性和扩散是一项重要的工作。由于营养物质有多种形式，我们使用光学成像技术研究了组织工程构建物中氧气、荧光素和葡聚糖分子的扩散。首先，利用缺氧反应元件驱动绿色荧光蛋白基因表达的转基因细胞系研究了氧气的可用性和扩散。使用共聚焦成像，我们观察到在含有100万个中国仓鼠卵巢（CHO）细胞的琼脂糖凝胶环境中，从周边约200 µm处开始出现氧气限制。利用荧光素酶基因由组成型活性巨细胞病毒（CMV）启动子驱动的细胞系，在琼脂糖凝胶中实时监测荧光素的扩散。凝胶浓度影响荧光素的扩散速率。此外，我们通过光漂白后荧光恢复（FRAP）评估了不同分子量的荧光葡聚糖分子在生物材料中的扩散速率，并观察到扩散取决于分子大小和凝胶浓度。总之，我们验证了一套有效的工具，用于研究一系列分子的分子扩散，并优化生物材料设计以改善营养物质输送。
