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Nanofibrous scaffolds in controlled delivery of autologous growth factors
Buzgo, Matej ; Amler, Evžen (advisor) ; Gášková, Dana (referee)
Platelet preparations are a source of various autologous growth factors and have numerous applications in tissues engineering. The aim of this work was to development electrospun nanofiber scaffolds with platelet preparations. Scaffolds based on the adhesion of platelets on nanofiber meshes were developed. The scaffolds were able to enhance chondrocyte proliferation in vitro. The main disadvantage of this system is the burst release of growth factors immediately after adhesion. To overcome this, we developed coaxially electrospun scaffolds with incorporated alpha granules. Alpha granules are novel platelet preparations with high amounts of growth factors. This system was able to stimulate chondrocyte proliferation and maintain TGF- 1 concentrations for 7 days. Additionally, a novel drug delivery system with coaxially incorporated liposomes was developed. Liposomes incorporated into nanofibers remain intact and can be used for the delivery of various molecules. The ability to maintain HRP activity was compared to systems based on coaxial electrospinning with liposomes, coaxial electrospinning without liposomes and blend electrospinning. When compared to other systems, coaxial electrospinning with liposomes preserves enzyme activity twice as long. These results clearly indicate the potential of...
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Application of nanofiber scaffolds for vesel regeneration
Bezděková, Dagmar ; Amler, Evžen (advisor) ; Holzerová, Kristýna (referee)
Although plenty of systems for vessel regeneration have been developed, no system is successful in small diameter (under 6 mm) vessel replacement yet. Synthetic materials, such as Dacron and ePTFE, have good results in large vessels replacement, but they cause thrombosis in small vessels. In addition, they are not degradable and do not allow a natural remodeling of the vessel system. Furthemore, endothelial cells, which are essential for creating natural antithrombogenic endothelium, do not adhere on these materials, as well as smooth muscle cells. Decellularized xenogenic material is the non-synthetic alternative for vessel regeneration. Appropriate detergent removes donor's cells and only extracellular matrix remains, which is able to host acceptor's cells. The main disadvantages of this system are difficulties with animal's nurture and structure violations after detergent is used. It appears that electrospun materials are the best alternative. The relatively simple process can be modified in many ways and provides then a scaffold, which mimics extracellular matrix. A big advantage of this process is the possibility to incorporate bioactive substances into a fiber. The substances serve there as an attractant for blood cells or as an anticoagulation factor. In combination with the progenitor cells seems...
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Preparation, characterization and testing of blood derivatives for applications in regenerative medicine
Sovková, Věra ; Amler, Evžen (advisor) ; Kolářová, Hana (referee) ; Vránová, Jana (referee)
Platelet products can be used, thanks to the broad range of bioactive molecules, either as a supplement for cell cultering in vitro alone or for development of cell- or cell-free scaffolds in diverse fields in regenerative medicine. The aim of this study was to prepare several types of platelet products. The concentration of selected molecules were observed. Subsequently, these products were tested with cell cultures in vitro alone or in combination with nanofibres scaffolds prepared by electrospinning or centrifugal spinning. It was found out, that platelets products contains chemokine RANTES and growth factor PDGF in the highest concentrations. It was further discovered the content of pro and antiinflammatory in terleukins and other growth factors. Platelet lysat in concentration 7% is sufficient to replace FBS in keratinocytes and fibroblasts cultures. In the other experiments, platelets in different concentrations were adhered to the scaffolds prepared by electrospinning and centrifugal spinning. Thus prepared scaffolds promote the proliferation and viability of all tested cell types in dose-dependent manner. In the last experiment, the individual components of platelet concentrate were separated and characterized. Their effect to the cell culture were tested. It was examinated the synergic...
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Biomechanical response of scaffold on mechanical loading
Anděrová, Jana ; Jelen, Karel (advisor) ; Amler, Evžen (referee)
The purpose of this work is to identify the parameters of scaffold's mechanical properties by observing/monitoring their response to defined external mechanical strain. The first part of the work is summarizing the knowledge about the required properties of scaffolds, their production and the factors influencing production. The practical part of the work concerns itself with measurement, analysis and evaluation of data based on proprietary methodology. Based on the results at this stage of the research, we can confirm, that scaffolds have viscoelastic, or viscoplastic character and its response depends on the magnitude of deformation, state of hydration, ratio of solutions and period of networking. Keywords: scaffod, tensile test, rheologic model
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Detection and visualization of native and artificial chondrogenous tissue using biophysical methods
Rampichová, Michala ; Amler, Evžen (advisor) ; Rosina, Jozef (referee) ; Kolářová, Hana (referee)
Tissue engineering is a rapidly developing field, based on using scaffolds made from natural or synthetic materials in combination with cells and stimulating factors for the replacement of damaged or lost tissue. It is very important to evaluate qualities of these scaffolds, which are necessary for cell proliferation and their mechanical support. The aim of this study was to develop a suitable scaffold for chondrocyte proliferation, scaffold functionalization and detection of cells and proteins of extracellular matrix (ECM) using methods of fluorescence and confocal microscopy. Another aim was to test an appropriate scaffold on a big animal model in vivo. Several scaffolds from natural and synthetic materials, in the form of microfibers, non-woven textiles, gels and foams were prepared for this study. Scaffolds were seeded with chondrocytes and cell adhesion, proliferation and synthesis of ECM proteins were detected. Methods of fluorescence microscopy, confocal microscopy and second harmonic generation (SHG) were used for visualization of cells and proteins. A hydrogel based on fibrin and hyaluronan was used as an scaffold for osteochondral defect regeneration in minipigs study. Prepared scaffolds showed high biocompatibility, good chondrocyte adhesion and ECM proteins synthesis. Moreover, microfibrous...
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Characterization of functionalized fibres for mesenchymal stem cells cultivation and differentiation
Greplová, Jarmila ; Amler, Evžen (advisor) ; Rosina, Jozef (referee)
Modification of nanofibers is an actual trend in tissue engineering. Polyvinylacohol (PVA) is nontoxic and biodegradable polymer suitable for preparation of submicron fibers by electrospinning. Main disadvantage of PVA fibers is rapid degradation in aqueous environment. On the other hand surface of fibers contains free hydroxyl group that could be chemically modified. In recent work, chemical modification of PVA nanofibers prepared by needleless electrospinning was investigated. Polyethylenglykol (PEG) linker was introduced to the fiber surface by acylation (PVA-PEG) and further modified by biotin (PVA-PEG-b) as a function agent. Process of chemical modification does not affected fibrous morphology of samples. Interestingly, linkage of PEG-b linker promoted stability of PVA in aqueous environment. PVA-PEG-b sample was stable for 41 days. Stability of samples was strongly dependent on amount of introduced PEG-b linker, thus proposed method of modification allows to prepare nanofibers of different solubility. Additionally, biocompatibility of chemically modified nanofibers with both mesenchymal stem cells (MSC) and chondrocytes was determined. Proliferation of both cell types was not sufficient and number of cells decreased in time, probably because of high hydrophility of modified PVA scaffold. To...
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