Jun Liu, Janneke Hilderink, Tom A.M. Groothuis, Cees Otto, Clemens A. van Blitterswijk and Jan de Boer
Journal of Tissue Engineering and Regenerative Medicine 2013
Limited nutrient diffusion in three-dimensional (3D) constructs is amajor 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 mm 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.