Diplomová práce se zabývá přípravou 3D porézních kolagenových skafoldů metodou lyofilizace a jejich modifikací bioaktivními látkami. K modifikaci byly použity přírodní polysacharidy – chitosan, vápenatá oxidovaná celulóza a chitin/chitosan-glukanový komplex. Mechanické vlastnosti skafoldů byly upraveny síťováním pomocí karbodiimidů. Růstové faktory byly dodány formou destičkového lyzátu. Byl zkoumán vliv biologicky aktivních aditiv, siťovacího činidla a obohacení růstovými faktory na vlastnosti připravených skaffoldů a jejich bioaktivitu v tkáních živých organismů. Konkrétně byly studovány morfologické vlastnosti, struktura, porozita, botnání, stabilita, chemické složení, teplota denaturace a biologické vlastnosti. K charakterizaci byly použity metody rastrovací elektronová mikroskopie, infračervená spektroskopie, diferenční kompenzační kalorimetrie a konfokální mikroskop. Připravené kolagenové substráty obohaceny bioaktivním aditivem a destičkovým lyzátem mohou být využity v biomedicíně jako skafoldy pro růst buněk v systémech s nízkou mechanickou zátěží.

V průběhu projektu byly vyvinuty unikátní kolagenní pěny z kolagenu získaného z kůže sladkovodní ryby (kapr obecný, Cyprinus carpio). Pomocí síťování karbodiimidem byl překonán problém s nestabilitou kolagenní matrix z kolagenu získávaného z chladnokrevných živočichů při tělesné teplotě savců. Následně byly pěny impregnovány antibiotiky (gentamicin a vankomycin) a opětovně lyofilizovány, což je postup, který zajišťuje požadovanou koncentraci antibiotika bez rizika následného vymytí při dalších technologických krocích. Uvedený produkt je, na rozdíl od přípravků z nesíťovanéhokolagenu, stabilní i při sterilizaci gamma zářením. Finální sterilizovaný produkt byl testován in vivo na potkaním modelu infikované rány. Byla prokázána efektivita v léčbě potenciálně letální infekce Pseudomonas aeruginosa a kmene Stafylococcus aureus rezistentní k meticilinu (MRSA). Vzhledem k vysoké potřebě profylaxe a terapie infekcí pooperačních a jiných ran právě výše uvedenými polyrezistentními původci se jedná o slibný prostředek k budoucímu klinickému využití. Zkušenosti, které jsme získali v průběhu uvolnování ATB z kolagenních pěn budou v dalším vyvoji použity pro impregnaci zevní kolagenní vrstvy cévní protézy, čímž bychom mohli eliminovat jednu z největších nevýhod a rizik spojených s použitím umělých materiálu a tím je...

Tissue engineering is an emerging interdisciplinary field developing new ways of treatment of patient's tissue defects using artificial substitutes. Skin tissue engineering is developing skin substitutes and wound dressings that would replace current treatment using autologous, allogeneic or xenogenic substitutes. There are high demands on materials which should serve as a scaffolds for dermal fibroblasts and keratinocytes. They must be non-cytotoxic and biodegradable with a rate proportional to formation of a new tissue. The materials should support adhesion and proliferation of the cells and even they could release growth factors and antimicrobial substance to enhance healing and new tissue formation. In this master thesis, the cell adhesion and proliferation were evaluated on sodium carboxymethyl cellulose (Hcel® NaT), poly-ε-caprolactone (PCL), poly-L-lactide-co-ε-caprolactone (PLA/PCL) and cellulose acetate (AC) nanofiber membranes. Primary human dermal fibroblasts and HaCaT cell line keratinocytes were selected for evaluation. The cell adhesion was observed by fluorescent microscopy, the proliferation was determined by metabolic assay (WST-1) and the material cytotoxicity was evaluated in xCELLigence® system. Materials did not show cytotoxic effects on the cells. However, the materials did...

Purpose: The aim of this work was to characterize the healthy human cornea and the cornea of patients suffering from posterior polymorphous corneal dystrophy (PPCD) using different antibodies. Despite the fact that PPCD is a very rare disorder, one of the largest groups of PPCD patients in the world comes from the Czech Republic. This offers us the opportunity to investigate the changes on the clinical, cellular and molecular levels. Material and Methods: A collection of 25 control corneas as well as 16 pathological corneas from PPCD patients were used. Epithelial (cytokeratins) and mesothelial markers (mesothelin, calbindin 2, HBME-1 protein) were detected in all layers of the healthy corneas using immunocyto- and immunohistochemistry. The expression of all markers was confirmed using molecular methods as well (RT-PCR and Western blot). Changes in the expression of cytokeratins and changes in the extracellular matrix structure (collagen IV and VIII) were studied in the PPCD corneas. Combined fluorescent immunohistochemistry with fluorescence in situ hybridization were used in order to characterize the origin of abnormal cells on the posterior graft surface, which cause the recurrence of the PPCD after penetrating keratoplasty surgery. Results: Changes in the cytokeratin expression (strong...

This master’s thesis contains a description of mesenchymal stem cells (MSC). The work includes knowledge about the process of migration and differentiation of MSCs. The theoretical part of this thesis also deals with the problem of cell cultivation in 2D and 3D environments. The most used materials for creating 3D scaffolds are described here. The practical part contains the description of the cell culture protocol for passaging and also describes the experiment in the cell laboratory. The results of the experiment are discussed using confocal microscope images. The proposed experiment was tested with a sufficient number of repetitions. The processing of microscopic images was performed in the MATLAB programming environment.

Author: Bc. Marie Zasadilová Title: Incidence of Joint Hypermobility Syndrome in Anxienty Patients Objectives: The aim of this study is to find out what ist he incidence of Joint hypermobility syndrome in the research group of probands with anxiety disorder, on the base of collected theoretical knowledge. Methods: The group of patients with diagnosis of anxiety disorder was examined on presence of joint hypermobility syndrome. For the examination was used standardised test scale Beighton score. The data was statistically processed, prevalence of hypermobility in the research group was compared with prevalece in common population. Results: Prevalence of joint hypermobility syndrome in the research group was 44,88%, that is about 31,88% more, than in common population. Hypermobility was found in 65% of female part oft he research group, that is about 25% more, than in common female population. Prevalence in male part of research group was 16,67%, about 5,17% more than in common male population. The hypotesis, that prevalence of joint hypermobility syndrome will be hihger in the research group than in common population, was affirmed. Average result of Beighton score in group of probands was 4,38 points, the most common result was 2 points. Skewness and krtosis of the histogram curve corresponds with...

Osteogenesis imperfecta is an inherited disorder caused mainly by collagen type I genes mutations, COL1A1 and COL1A2. These mutations affect especially connective tissue. Disease is characterized by fragile bones, deformations and increased frequency of fractures. It's worldwide extensive disorder regardless of age, sex, nationality or races. The incidence is 1: 16 - 20 000 births. Currently, we described nine clinically distinct forms of Osteogenesis imperfecta. Only the first four types OI, type I-IV, are caused by collagen type I genes mutations . In these nine types there are distinguished mild and severe forms. Type II and III are lethal forms, death occur offen during prenatal period or in the first days of the life affected individuals. Characteristic clinical features of collagen forms OI are an increased incidence of fractures, deformations of bones, blue sclera, hearing loss, Dentinogenesis imperfecta small or subnormal growth (Marini, 2010). This study alignment is mainly the description of the clinical forms, exploring the molecular basis of disease and determine the relationship between the type and position of the mutation and the resulting phenotype of affected individuals. We have analysed exons 31-40, including associated non-coding regions, of the COL1A1 gene (so-called MLBR =...

Purpose: The aim of this work was to characterize the healthy human cornea and the cornea of patients suffering from posterior polymorphous corneal dystrophy (PPCD) using different antibodies. Despite the fact that PPCD is a very rare disorder, one of the largest groups of PPCD patients in the world comes from the Czech Republic. This offers us the opportunity to investigate the changes on the clinical, cellular and molecular levels. Material and Methods: A collection of 25 control corneas as well as 16 pathological corneas from PPCD patients were used. Epithelial (cytokeratins) and mesothelial markers (mesothelin, calbindin 2, HBME-1 protein) were detected in all layers of the healthy corneas using immunocyto- and immunohistochemistry. The expression of all markers was confirmed using molecular methods as well (RT-PCR and Western blot). Changes in the expression of cytokeratins and changes in the extracellular matrix structure (collagen IV and VIII) were studied in the PPCD corneas. Combined fluorescent immunohistochemistry with fluorescence in situ hybridization were used in order to characterize the origin of abnormal cells on the posterior graft surface, which cause the recurrence of the PPCD after penetrating keratoplasty surgery. Results: Changes in the cytokeratin expression (strong...

The goal of this thesis was to invent and perform a testing protocol, witch can detect connective tissue mechanical and structural properties simultaneously in patients with clubfoot. Based on literature findings, it was presumed that connective tissue differ between medial and lateral side of foot. Because of low availability of specimen, it was important to draw and mention crucial alterations of testing protokol, for minimization of failed measurments. For reasons above, two specimens diveded into two samples was used for experiment. Described and discused methodics may enable of reader to insigt to drawbacks of this examination. For usability of this protocol, a setting and testing of few hypotheses was performed. One axis tensial testing with SHG microscopy examination was used in combination for experiment. One of the main result was finding, that structural differencies witch were expected, were not distinctive in samples in untensioned state. But, distingtive differencies may be drawed in tensioned samples. This differences for low number of specimen, cannot show any tendence. Results of tensial testing showed, that samples from medial side of foot can have higher toughness and higher fragility. For future acquisition of tendencies in specimen differencies, it is important to set particular...

This thesi s is eoneerned with the deteetion of posttranslational modifieation of proteins. These proteins are identified using liquid ehromatography and eapillary eleetrophoresis, their eoupling and eoupling to mass speetrometry. The first part of the theoretieal ehapter is devoted to the strueture, classifieation and metabolits ehanges of proteins in the human body. Major attention is foeused on eollagen, its strueture and individual eollagen types. The seeond part eonsiders the problems of nonenzymatie glyeation and its influenee on the body and engage in produets of posttranslational modifieation. The third part summarizes the individual separation proeedures and analysis of posttranslationaly modified proteins. The seeond ehapter deseribes experimental parameters, including ehemieals, instrumentation, materials and methods. The preparation of individual samples is also deseribed. All results and findings are summarized and diseussed in the last ehapter. This ehapter is divided in two parts. The first part foeuses on problems of in vivo experiments. The seeond part foeuses on problems of in vitro experiments. Powered by TCPDF (www.tcpdf.org)