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1.
Raziskave interakcij med celicami in biopolimernimi materiali z naprednimi eksperimentalnimi metodami kot osnova za študij biokompatibilnosti polimerov
Rok Podlipec, 2015, doktorska disertacija

Opis: 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.
Ključne besede: polymer molecular mobility, mechanical response, morphology, scaffold biocompatibility, cell growth, single cell manipulation, cell adhesion dynamics, optical tweezers, electron paramagnetic resonance, dynamical mechanical analysis, nanoindentation, fluorescence microscopy and microspectroscopy
Objavljeno v DKUM: 06.10.2015; Ogledov: 2395; Prenosov: 170
.pdf Celotno besedilo (5,95 MB)

2.
EFFECT OF GELATINE SCAFFOLDS FABRICATION AS POLYPROPYLENE MESH COAT ON IMPLANT BIOCOMPATIBILITY
Selestina Gorgieva, 2014, doktorska disertacija

Opis: This work presents the methodological study, processing and optimization of novel, technologically acceptable procedure for in situ coating of polypropylene (PP) mesh (used for hernia treatment) with physico-chemically, mechanically and micro-structurally different gelatin (GEL) scaffolds to assess implant composite biocompatibility impact. In order to systematically follow the experimental work progress and respective achievements, whole research path is subdivided into three main sections. In the first section, the procedure for fabrication of gradiently micro-porous GELscaffolds on the cryo-unit’s cooling plate surface, using spatiotemporal and temperature- controlled gelation and freezing, followed by lyophylizaton was studied. Subsequently, cross-linking procedure using different molarities of reagents (EDC and NHS) and reaction media (100% PBS or 20/80% PBS/EtOH mixture) was performed for variable time extensions (1-24 h), rendering scaffolds physico-chemical properties. In this way, scaffolds with micro-structures having porosity gradient from 100 µm to 1000 µm and pores with rounded to ellipsoid morphology were formed, which, in combination with ethanol (EtOH) addition in cross-linking media modulates the swelling capacity towards twice lower percentages (~600%) comparing with scaffolds cross-linked in 100% PBS. Whilst the presence of EtOH reduce the cross-linking kinetic by retaining the scaffolds’ micro-structure formed during freezing, the 100% PBS and higher EDC molarity resulted in 40% cross-linking degree, being expressed as a thermal resistance up to 73 °C. The presented integral fabrication procedure was shown to allow tuning of both, the physical and micro-structural properties of scaffold, utilized in preparation of materials for specific biomedical applications. In the second part, the complex relation between surface and interface-related physico-chemical properties and gradient micro-structuring of 3D GELscaffolds, being fabricated by simultaneous temperature- controlled freeze-thawing cycles and in situ cross-linking using variable conditions (pH and molarity of carbodiimide reagent) and fibroblast cells viability (by tracking of their spreading and morphology) was established. Rarely- populated cells with rounded morphology and small elongations were observed on scaffolds with apparently negatively- charged surface with a lower cross-linking degree (CD) and consequently higher molecular mobility and availability of cell-recognition sequences, in comparison with the prominently- elongated and densely- populated cells on a scaffold’s with positively- charged surface, higher CD and lower mobility. Surface micro-structure effect was demonstrated by cell’s vacuolization and their pure inter-communication being present on scaffold’s bottom side with smaller pores (25±19 µm) and thinner pore walls (9±5 µm), over the air- exposed side with twice bigger pores (56±38 µm) and slightly thicker pore walls (12±6 µm). Strong correlation of preparation conditions (pH and reagents molarity) with CD (r2=0.96) and moderate correlation with local molecular mobility (r2 =-0.44), as well as micro-structure features being related to temperature gradient, imply on possibility to modulate scaffold’s properties in a direction to guide cell’s viability and most likely its genotype development. The third part presents an innovative strategy for the fabrication of bio-active PPmesh-GELscaffold composites with a potential for abdominal hernia treatment, where mesothelial cells in-growth have to be stimulated together with fibroblasts on-site proliferation, while formation of fibrin-developing, viscera-to-abdominal wall adhesions should be reduced, together with bacteria- related infections. In this respect, the plasma pre-activated PPmesh was coated with micro-structured GELscaffold, with pore size in 50 µm to 100 µm range at the upper-side and loosely- porous network at the composite bottom side, being modulated by sample thickness and freezing end- temperature applied. Simultaneously, the
Ključne besede: gelatin, targeted cross-linking, controlled freezing, gradiental micro-porosity, scaffold, surface and interface chemistry, physico-mechanical properties, polypropylene mesh, composite, biocompatibility.
Objavljeno v DKUM: 07.05.2014; Ogledov: 2295; Prenosov: 169
.pdf Celotno besedilo (4,98 MB)

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