Monitoring nutrient transport in tissue-engineered grafts.
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.
MIRA Research Institute, Department of Tissue Regeneration, University of Twente, Enschede, The Netherlands.
Jun
J
Liu
MIRA Research Institute, Department of Tissue Regeneration, University of Twente, Enschede, The Netherlands.
Janneke
J
Hilderink
MIRA Research Institute, Department of Biophysical Engineering, University of Twente, Enschede, The Netherlands.
Tom A M
TA
Groothuis
MIRA Research Institute, Department of Biophysical Engineering, University of Twente, Enschede, The Netherlands.
Cees
C
Otto
MIRA Research Institute, Department of Tissue Regeneration, University of Twente, Enschede, The Netherlands.
Clemens A
CA
van Blitterswijk
MIRA Research Institute, Department of Tissue Regeneration, University of Twente, Enschede, The Netherlands.
Jan
J
de Boer
D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004058:Diffusion; D049454:Firefly Luciferin / Q000737:chemistry; D036681:Fluorescence Recovery After Photobleaching; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D000860:Hypoxia; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008156:Luciferases / Q000378:metabolism; D018613:Microscopy, Confocal; D055095:Optics and Photonics; D010100:Oxygen / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry; D023822:Tissue Engineering / Q000379:methods*
D001672:Biocompatible Materials; D049452:Green Fluorescent Proteins; D049454:Firefly Luciferin; D012685:Sepharose; D008156:Luciferases; D010100:Oxygen
3D scaffold; FRAP; bioluminescent imaging; hypoxia; nutrient supply; tissue engineering