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Oxygen Gradients in Collagen-I Gels
Automated micromanipulator with fiber optic profiling oxygen microsensor as a method to evaluate oxygen gradients in collagen-I gels for tissue engineering
Laura Heimann1, Paul Wolff1, Elizabeth Rosado Balmayor1, Martijn van Griensven1, Gregor Liebsch2, Robert Meier2, Martin Gutbrod2
1Experimental Trauma Surgery, Technical University of Munich (TUM), Munich, Germany
2PreSens GmbH, Regensburg, Germany
Adipose-derived mesenchymal stem cell loaded collagen-I gels were evaluated with the automated profiling system by PreSens (Automated Micromanipulator AM and Profiling O2 microsensor PM) regarding oxygen gradients. The system can be used inside the incubator and offers high spatial resolution measurements down to 10 µm. In this study, control gels without cells were compared to gels loaded with two different cell densities. The results clearly show that oxygen values depend on cell content and cell amount. Unloaded gels remain at plateau oxygen levels in contrast to gels containing cells. In the latter, the more cells a gel contains the lower the inner oxygen tension drops, probably due to higher consumption.
In case of delayed healing or non-unions of long bones (10-40 % of all cases), a need exists for an approach that accelerates bone healing. One potentiality constitutes tissue engineering, consisting of a scaffold (matrix), cells and stimulating factors. Limiting aspect here is the short diffusion distance of oxygen of about 200 µm. Without oxygen, the whole construct will die, so it’s tremendously important to be able to observe oxygenation in situ.
PreSens developed several measurement systems based on a fluorescence quenching technique that does not consume oxygen itself and does not generate an electric potential at the measured spot. In the experiments described here, an automated profiling system was used to evaluate oxygen gradients developing in cell-seeded scaffolds.
The Automated Micromanipulator incorporates an optical oxygen microsensor based on a silica fiber (Fig. 1). It allows a very precise and vibration-free positioning with a high resolution up to 10 µm along the z-axis to generate exact oxygen gradient data in various scaffold materials.
Materials & Methods
Before starting the measurement process, calibration needs to be done with sodium sulfite solution (0% oxygen) and air saturated distilled water (100% air saturation). Collagen-I-gels were prepared with 10 x concentrated DMEM, distilled water and 3 different amount of cells (no cells, low and high cell density). Gelation was finished after 2 hours of incubation and media was added on top of the gels. The Microx 4 measurement unit was installed inside the incubator and controlled from outside with a laptop. The sensor was placed at the border between air and media and moved in 100µm steps towards the bottom of the well plate. The Software PreSens Profiling Studio enabled the measurement of variable step sizes, measuring velocities and wait times.
Adipose-derived Mesenchymal Stem Cells (AdMSCs) in Collagen-I Gels 5 weeks in vitro
Gels were cultivated for 5 weeks and evaluated every week. Figure 2 and 3 show that from day 0 on, an obvious oxygen gradient and difference between the 3 conditions could be observed. The control gel without cells stayed at plateau values of 19 % O2. Gels with lower cell density always showed higher oxygen levels than gels with more cells. Furthermore, oxygen levels decreased with every week, starting at about 18 % O2 immediately after gelation, 17 % O2 after 3 days in vitro, 16.5 % O2 after 7 - 28 days in vitro until reaching a plateau level of 16 % O2.
The Automated Profiling System is capable to detect smallest oxygen micro-gradients in collagen-I constructs. With this valuable information it is possible to determine which oxygen tension occurs in which depth of a hydrogel. In the context of Tissue Engineering, this could constitute a very useful tool to e.g. observe oxygenation of cell-loaded constructs, which are planned to be implanted into patients.