Raziskave interakcij med celicami in biopolimernimi materiali z naprednimi eksperimentalnimi metodami kot osnova za študij biokompatibilnosti polimerov

The last two decades have been determined by the development in the field of tissue engineering. Beside the constant progress in new biomaterials and scaffold fabrication methods, currently the main focus is to understand scaffolds biocompatibility. In our thesis, physical aspects of scaffold biocompatibility were studied by correlating molecular to macro scale physical properties of scaffolds with cell attachment and cell growth. In order to focus on scaffold physical properties, scaffolds were prepared by the same chemical composition of natural polymer gelatin excluding biochemical effects on the cell response. Scaffold with different physical properties were obtained by changing the temperature, pH and crosslinker degree during the cryogelation and populated by the fibroblast cells. Advanced experimental biophysical methods were applied to determine the polymer mobility via electron paramagnetic resonance (EPR) with spin labelling, the scaffold mechanical properties via rheometry, dynamic mechanical analysis (DMA) and nanoindentation using atomic force microscope (AFM) and the scaffold porosity via confocal fluorescence microscopy (CFM). The anisotropy of the molecular mobility of the side chains of polymers in the crosslinked gelatin structure was found to correlate with the initial cell growth (throughout the first week) the best of all the physical properties measured. About five times less efficient cell growth was measured on the scaffolds with highly mobile, spatially nonrestricted dynamics of the polymer side chains, in comparison with cell growth on the scaffolds with the restricted rotational motion of polymers. The result indicates that cells identify and respond to the degree of polymer mobility, where partially immobile phase is necessary for efficient cell attachment and efficient cell growth. So far, the molecular mobility of polymers constituting tissue engineering materials has never been studied thoroughly with respect to its influence on cell response, and therefore may represent a new experimental approach in understanding biocompatibility. To further understand cell-scaffold interaction, the study focused also on the first events during cell attachment - bond formation between the cell surface proteins and the specific binding sites on the material. In our thesis, cell adhesion dynamics was investigated in real-time on the surfaces of gelatin scaffolds with different physical properties using spatially-controlled cell manipulation by the optical tweezers and the confocal fluorescence microscopy detection. Our goal was to elucidate, if the adhesion dynamics can be correlated with cell growth and if it can be dependent on the scaffold polymer molecular mobility. Quantitative characterization of the optical tweezers force applied during cell-scaffold adhesion analysis was done by viscous drag force calibration and dynamic cell sequential trapping of individual cells. The maximal force on a trapped cell not causing the thermal damage was measured up to 200 pN, with nearly linearly increasing force profile across the cell towards the plasma membrane. By submicron spatial resolution of cell manipulation, we managed to quantify probability of cell adhesion, cell adhesion strength and mechanism of cell attachment, including the formation of the membrane tethers, which slow down the adhesion process. Adhesion strength was classified according to the displacement of the attached cell under the force of optical tweezers measured in the direction of the scaffold surface.Cell adhesion was shown to significantly correlate with cell growth in the first days of culture, while the adhesion itself seems to be dependent on the molecular mobility of surface polymers. The result indicates that the interactions during the first seconds may markedly direct further cell response. The developed methodology for cell adhesion analysis on the surfaces of 3D scaffolds serves as a good tool to forecast scaffold biocompatibility.Obdobje zadnjih dvajsetih let zaznamuje velik napredek na področju raziskav tkivnoinženirskih nosilcev za uporabo v regenerativni medicini. Poleg raziskav novih biomaterialov in načinov priprave nosilcev se daje velik pomen razumevanju biokompatibilnosti. V doktorski študiji smo proučevali vplive različnih fizikalnih lastnosti polimernih nosilcev iz želatine na molekularni kot tudi makroskopski skali na pritrjevanje in rast celic. Biokemijski vpliv na odziv celic smo izločili z uporabo enake kemijske sestave pri pripravi nosilcev. Porozne nosilce z različnimi fizikalnimi lastnostmi smo uspeli pripraviti s spreminjanjem temperature, pH in stopnje premreženja tekom sočasnega zamrzovanja in premreževanja. Za karakterizacijo fizikalnih lastnosti smo uporabili napredne biofizikalne eksperimentalne metode: elektronsko paramagnetno resonanco (EPR) s spinskim označevanjem molekul za analizo molekularne mobilnosti stranskih verig polimerov, reometrijo, dinamično mehansko analizo (DMA) in nanoindentacijo z mikroskopom na atomsko silo (AFM) za analizo mehanskih lastnosti nosilcev na makroskopski in mikroskopski skali ter konfokalno fluorescenčno mikroskopijo (CFM) za analizo poroznosti nosilcev na mikroskopski skali. Ugotovili smo, da med merjenimi lastnostmi anizotropija molekularne mobilnosti stranskih verig polimerov v premreženi želatinski strukturi najbolj korelira z rastjo celic v prvih dneh. Izmerili smo približno petkrat slabšo rast celic na nosilcih z izrazito, neomejeno prostorsko mobilnostjo stranskih verig polimerov v primerjavi z rastjo celic na nosilcih z omejeno mobilnostjo le teh. Rezultat nakazuje na to, da celice prepoznajo in se odzovejo na stopnjo molekularne mobilnosti polimerov, kjer je za uspešno pritrditev in uspešno rast le teh potrebna delno nemobilna faza polimerov v sami strukturi. Te fizikalne lastnosti v dosedanjih raziskavah vpliva lastnosti tkivno inženirskih materialov na odziv celic še niso natančno raziskovali in bi lahko predstavljala nov eksperimentalni pristop za boljše razumevanje biokompatibilnosti. Za nadaljnje razumevanje interakcije nosilec-celica, smo se v doktorski študiji posvetili analizi dinamike pritrjevanja celic ‒ nastanek prvih vezi med celičnimi transmembranskimi proteini in specifičnimi ligandi na površini materiala.V doktorski študiji smo proučevali dinamiko pritrjevanja posameznih celic v realnem času na površinah želatinskih nosilcev z različnimi fizikalnimi lastnostmi s prostorsko nadzorovano manipulacijo celic s pomočjo optične pincete in detekcije s konfokalno fluorescenčno mikroskopijo. Želeli smo pojasniti ali dinamika pritrjevanja korelira z rastjo celic in ali na dinamiko lahko vpliva molekularna mobilnost polimerov. Silo tekom analize pritrjevanja smo kvantitativno določili s predhodno umeritvijo z viskozno silo okoliške tekočine in umeritvijo s sekvenčno dinamično manipulacijo posameznih celic. Izmerjena največja sila optične pincete na celico pri kateri še nismo zaznali termične poškodbe je bila 200 pN, pri čemer sila pada v približku linearno iz lege pincete na robu celice radialno v smeri proti težišču, centru celice. S podmikronsko prostorsko ločljivostjo manipulacije celic na stiku s površino nosilca smo natančno določili verjetnost za pritrditev celic, moč pritrditve celic in mehanizme pritrditve celic, med katerimi je tudi takšen preko nastalih membranskih izrastkov, ki upočasni proces pritrjevanja. Moč pritrditve celic smo razvrstili glede na premik pritrjene celice v smeri tangencialno na površino nosilca pod vplivom sile optične pincete. Pokazali smo, da dinamika pritrjevanja celic res korelira z rastjo celic v prvih dneh, medtem ko kaže, da je pritrjevanje res odvisno od molekularne mobilnosti polimerov na površini nosilca. Interakcije v prvih sekundah stika tako lahko zelo vplivajo oz. usmerjajo nadaljnji odziv celic. Razvito metoda analize pritrjevanja celic na površine 3D nosilcev lahko služi kot dobro orodje za določitev biokompatibilnosti nosilcev

Images for: Characterization of blood coagulation dynamics and oxygenation in ex-vivo retinal vessels by fluorescence hyperspectral imaging

Images for the manuscript/paper titled Characterization of blood coagulation dynamics and oxygenation in ex-vivo retinal vessels by fluorescence hyperspectral imagin

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Videos are showing experimental results of the blood coagulation dynamics study in ex‐vivo retinal vessels

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