engleski

The study and experimental approaches of cell-scaffold contact

Efficient tissue regenerative medicine treatments of bone/cartilage lesions which depend mainly on biocompatible properties of implanted materials due to complexity of the tissue still remain a huge challenge (L. Kock, C. C. Donekelaar, K. Ito, Cell Tissue Res (2012) 347, 613-627). Modern tissue regeneration approaches often rely on tissue engineering based on scaffolding. Proliferation of cells through the scaffold, cell attachment onto the scaffold and ability of population growth on a day-time scale seem to be the straightforward criteria of good scaffold candidates. On the other hand, extensive testing is applied also to avoid immune responses and undesired too fast or too slow degradation. All of the above issues compose the so-called biocompatibility, which actually implies the emerging material-cell interactions and the consequential responses of both material and living cells. It is straightforward that biocompatibility is the property of the interacting pair, i.e. both the material and the cell. To qualify the material-cell pair as biocompatible the cell-material interactions should be predicted from the properties of both material and cells under various conditions. Regarding to the available knowledge this is still very difficult task. To contribute to the area, we tried to understand the scaffold-cell contact from molecular biophysical perspective. Firstly, local mobility of the gelatin polymers constructing the scaffold was studying through spin and fluorescent labeling at the same sites that were used to crosslink the gelatin network to construct the scaffold. With spin labeling and electron paramagnetic resonance spectroscopy (EPR) the local side-chain conformational motions have been characterized with the same methodology that was applied in protein structure determination (Štrancar et al. Eur. Biophys. J. 2010, vol.39, 499-511). The most interesting observation is that cells populate the scaffold very efficiently despite the fact that the gelatin side-chains and the backbone itself are very flexible with the scaffold macro structure (sort of gel) being conserved. Secondly, the interaction between the (L-929 fibroblast) cell membrane and the gelatin scaffold was characterized using optical tweezing (1064 nm NIR laser) by pulling scaffold and the cell membrane. Additional unspecific micro-beads were used to increase the interaction sensitivity. At the same time membrane architecture has been traced through fluorescent labeling by membrane-anchored environmentally-sensitive fluorophores based on NBD fluorophore with confocal fluorescence microspectroscopy (Arsov et al. Biomed. optics express 2011, vol. 2, 2083-2095). Correlation between membrane changes, cell attachment and forces to the material were searched for.

cell-scaffold contact; electron paramagnetic resonance spectroscopy; optical tweezer

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