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The co-culture of keratinocytes and fibroblasts on a multi-layered polyester nanofibrous membrane enriched with platelet lysate
Blanquer, Andreu ; Filová, Elena ; Jenčová, V. ; Brož, Antonín ; Kuželová Košťáková, E. ; Lisnenko, M. ; Procházková, R. ; Bačáková, Lucie
The prevalence of chronic wounds is increasing due to the population ageing and specific illnesses like diabetes mellitus and vascular diseases. Nanofibrous membranes fabricated using synthetic polymers are promising materials to enhance skin wound healing. PCL and PVA membranes are being studied to be used as scaffolds for skin tissue engineering and hydrogels for controlled drug delivery, respectively. The present study considers the development of a multi-layered membrane made of PCL and PVA loaded with platelet lysate (PL). PCL nanofibers allowed cell adhesion and growth, whereas PVA acted as a hydrogel that releases the bioactive compounds of platelet lysate. The cytocompatibility of the membranes containing PL and without it was demonstrated on two cell types involved in wound healing, i.e. keratinocytes and fibroblasts. Both cell types were able to adhere and proliferate on the membranes. In addition, the membrane containing PL enhanced the proliferation of fibroblasts. A co-culture study was also performed by seeding each cell type on one side of the membrane. The cells were co-cultured for 7 days and the results showed that PL increased the proliferation of cells achieving a monolayer of keratinocytes or fibroblasts on each side of the membrane. Thus, the beneficial effect of PCL-PVA+PL membranes on monocultures and co-cultures of skin cells was demonstrated, and these membranes can be considered potential scaffolds for treatment of chronic wounds.
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Interactions of skin and stem cells with polymer nanofibres for construction of skin substitutes
Tomšů, Júlia ; Bačáková, Lucie (advisor) ; Sedmera, David (referee) ; Jendelová, Pavla (referee)
The skin is the largest organ of a human body with a crucial role in the maintenance of homeostasis; therefore any extensive skin injury leads to severe complications. Since the application of auto-, allo- and xeno-grafts is accompanied by severe problems like the source limitation and the graft rejection, a bioengineered skin substitute seems to be one of the promising healing approach. This work is focused mainly on the construction of a pre- vascularized skin substitute consisting of a collagen hydrogel reinforced by a biodegradable nanofibrous membrane. Another strategy described in this work is the development of temporary cellulose-based wound dressings. For both research strategies, various cell types were utilized, i.e. normal human dermal fibroblasts (NHDFs), human keratinocytes (hKs), adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). In order to enhance the cell adhesion and growth, the synthetic nanofibrous membranes were improved by protein nanocoatings. It was found out that NHDFs and ADSCs preferred fibrin nanocoatings, mainly thin fibrin homogeneous mesh on the surface of the membrane. Keratinocytes rather adhered and stratified on collagen substrates. These observations further motivated the construction of the bi-layered construct, where...
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Differentiation of keratinocytes: molecular markers and potential of influencing them in vitro
Ondrúšková, Denisa ; Filová, Elena (advisor) ; Porubská, Bianka (referee)
Keratinocytes are the most abundant skin cells found in epidermis. They are divided into proliferative basal stem cells, which are in close contact with basement membrane and suprabasal differentiating cells. Basal keratinocytes express K5 and K14 keratins and give rise to differentiating layers via delamination or asymmetric division. The firstly formed layer is stratum spinosum that expresses keratins K1 and K10 and involucrin, and, subsequently, it passes into the stratum granulosum, in which cells express loricrin and profilagrin. The last layer of epidermis is the stratum corneum formed by corneocytes that finally desquamate. Keratinocytes participate in the process of skin regeneration and can be isolated and cultivated. Their cultivation can be affected by various factors, such as selection of suitable materiál (nanofibers/gels) and suitable culture media, which can be enriched with growth factors, platelet lysat, vitamins and other substances. When culturing them, it also depends on whether the cells are entirely immersed in medium or growing on liquid/air interface. To approximate in vivo conditions and to study interaction between cell populations, keratinocytes are often cultured in co-cultures with different cells such as fibroblasts, endothelial cells, monocytes and others....
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Encapsulation of active substances and possibilities of their application in anti-aging products
Horváthová, Iveta ; Němcová, Andrea (referee) ; Skoumalová, Petra (advisor)
The aims of this thesis were encapsulation of selected active substances and their possible application in cosmetic products with anti-aging effects. 6 kinds of active substances were studied – lipophilic vitamin E and vitamin A, hydrophilic vitamin B1 and vitamin B2, and phenolic compounds ferulic acid and chlorogenic acid. In theoretocal part, the general characteristics of active compounds and their use are described. It also includes the description of used methods. Practical part was focused on characterisation of active compounds from the perspective of antioxidant activity and encapsulation into liposomes from soy and sunflower lecithin. Encapsulation effectivity, size, stability and long-term stability of liposomes after four weeks were determined. The most suitable liposomes with active compounds were also tested for safety on human skin cells using the MTT test of cytotoxicity on keratinocytes HaCaT. Furthernore, 3 types of cosmetic products were prepared – day cream, night cream and skin serum. Each kind of cosmetic product was devided into 4 types – the product without any active compounds, the product containing free active compounds, the product with liposomes with active compouds and the product containing empty liposomes. As active compounds all our tested substances were used. Final products were tested 14 days on 15 female volunteers. Before and after products usage parametres of their skin were measured to analyze effect of products on skin rejuvenation. Volunteers were also rating sensory analysis of cosmetic products. The part of this thesis was also creating an online questionnaire about anti-aging cosmetics for publicity to find out the popularity of this kind of cosmetics.
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Preparation of nanoparticles and nanofibers with antimicrobial components
Kubišová, Veronika ; Slaninová, Eva (referee) ; Skoumalová, Petra (advisor)
This thesis addresses the problem of inadequate current wound therapy and presents a solution in the form of nanomaterial-based wound dressings (coverings). Specifically, it focuses on the development and characterization of various nanofibrous materials with integrated liposome particles that could serve as a source of therapeutic agents and be useful specifically in the field of a wound therapy. The review in the introductory part of the thesis first focused on the mentioned problem, which describes the shortcomings of existing conventional dressing materials. A description of human skin from an anatomical and functional point of view was not omitted, nor was the wound healing process itself. Different types of covering materials were also presented. However, a large part was focused on nanomaterials and their use in the field of the skin wound therapy. The nanomaterials mentioned were mainly liposome particles and nanofibres, as the experimental part of the work was focused on these structures. The description dealt with the characterization of these structures as well as the biopolymers used for their preparation. However, the aim was to prepare nanofibers with liposome content, so the method of forming such systems was described. Various therapeutic agents are also an integral part of the covering materials, especially those that suppress the development of infection and reduce wound pain; therefore, the search focused on the antibiotic ampicillin and the analgesic ibuprofen. The experimental part was devoted to the actual production of nanofibers with liposomes and also to the qualitative demonstration of the presence of liposomes in electrostatically prepared nanofibers. The selected polymeric components of these systems were polyhydroxybutyrate (PHB) and gelatine. However, the nanofibers and liposome particles (as well as combined liposomes with PHB) were first prepared in their own form and characterized mainly in terms of the gradual release of the drug substances. The results obtained were then compared with those of therapeutic drug delivery via combined nanofibrous structures with liposomes. For these combined structures, the aim was to achieve synergy in drug delivery between these systems. The aim of this work was to create a biomaterial covering with the controlled drug release. The drugs contained in these materials were the aforementioned ampicillin and ibuprofen. An important part of the work was then to determine the safety of the prepared materials which were tested for cytotoxicity, where the tests performed were MTT test and LDH test. And the actual wound healing ability of the nanofibers was then monitored in a scratch test or a "wound healing" test. At the end of the paper, recommendations for future work on this topic are given.
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Molecular mechanisms of activation and modulation of TRPV3 receptor
Chvojka, Štěpán ; Vlachová, Viktorie (advisor) ; Novotný, Jiří (referee)
Transient receptor potential vanilloid 3 receptor channel (TRPV3) is a thermosensitive ion channel expressed in skin keratinocytes. There, in a molecular complex with the epidermal growth factor receptor (EGFR) contributes to proliferation and terminal differentiation of keratinocytes, temperature detection, pain and pruritus. TRPV3 is activated by a number of exogenous compounds, such as carvacrol from oregano, thymol from thyme and eugenol from clove. Its unique feature is sensitization, TRPV3 channel activity successively increases upon repeated stimulation. The molecular basis of this process is not yet understood. One of the considered possibility is a direct phosphorylation of TRPV3 protein through signaling pathways involving EGFR and mitogen-activated protein kinase MAPK1 / MAPK3 (also called ERK2 / ERK1). In this thesis we investigated whether sensitization of TRPV3 which is expressed in a human cell line immortalized keratinocytes could be influenced by mutations on the predicted consensual phosphorylation sites for MAPK1 / MAPK3. We used electrophysiological patch-clamp technique and tested eight mutants, in which was threonine or serine replaced with aspartic acid mimicking phosphorylation. We identified six residues where the mutations influenced at least one of the functional...
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New possibilities in wound healing
Nováková, Laura ; Szotkowski, Martin (referee) ; Skoumalová, Petra (advisor)
The diploma thesis is focused on the study of fibrous wound dressings prepared by electrospinning method from natural biopolymers. Three active ingredients were added to the dressings: ampicillin, ibuprofen and collagenase, which are responsible for relieving pain, reducing the risk of infection and selectively removing necrotic tissue in the wound. The theoretical part describes the therapeutic dressings currently available on the market and the most common methods of nanofiber production. The experimental part evaluates the optimization of the preparation of gelatin, alginate and chitosan fibrous wound dressings, which were subsequently enriched with active substances and their gradual release into the model environment was determined spectrophotometrically. Antimicrobial effects against E.coli and S. epidermidis strains andantifungal activity against C. glabrata yeast were monitored. Finally, two cytotoxicity tests on the human keratinocyte cell line HaCaT confirmed the safety of the prepared products, which can serve as bioactive skin dressings in the future.
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Characterization of newly developed fluorescence probes in cellular systems
Kadlecová, Julie ; Hubálek Kalbáčová, Marie (advisor) ; Hendrych, Tomáš (referee)
Nanoparticles (NP) are currently a progressive area of scientific research. The possibility of synthesizing them according to the required parameters opens up possibilities for their wide use also in biomedicine. One example is a nanoparticle that can detect cellular processes, such as pH. We already know that the pH of healthy and cancer cells differs by the opposite gradient on the intracellular and extracellular side of the membrane. In this context, this work deals with the study of fluorescent silicon nanoparticles (SiNP) tested on a human keratinocyte cell line from a healthy donor (HaCaT) and from skin cancer donor (A431). Once found that even the highest concentrations of SiNP used are not cytotoxic, they can be further studied by fluorescence, confocal and super-resolution microscopy. In order to assess the pH detection properties of these SiNPs, a method for measuring intracellular pH with a fluorescent raciometric probe SNARF-1 using fluorescence spectroscopy and flow cytometry was introduced. Since the pH values of the intracellular environment are closely related to cellular metabolism, the metabolism of A431 and HaCaT cells was characterized and compared. To do this, methods for measuring analog glucose consumption (2-NBDG) and another new method for measuring real-time metabolism...
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Use of plasma jet for wound healing
Dvořáková, Eva ; Márová, Ivana (referee) ; Skoumalová, Petra (advisor)
This diploma thesis was focused on the possibility of using a plasma nozzle to accelerate the wound healing process. The benefits of using low-temperature plasma in medicine or biomedical applications are known from many studies, and low-temperature plasma is already used to sterilize medical devices, materials or surgical instruments. Some studies also report a high potential of usinh plasma nozzle in the treatment of skin wounds. In the experimental part of this work, an in vitro wound healing test was performed using two different low-temperature plasma sources. Source No. 1 was a surface wave microwave discharge and source No. 2 was a torch microwave discharge. An in vitro scratch healing test was performed on a monolayer of HaCaT keratinocytes and testing was performed using various parameters. The influence of the plasma treatment time was monitored, as well as the influence of the plasma discharge power and also the influence of the argon working gas flow. Especially when using a torch microwave discharge, faster wound healing was recorded at most of the parameters used compared to the control. Thus, it can be said that this source appears to be potentially suitable for faster wound healing. Furthermore, in the work using the MTT cytotoxicity test, the viability of skin cells after their plasmination was also monitored using the same conditions as in the in vitro wound healing test. When performed in the standard MTT assay, none of the settings or sources used showed any cytotoxic effects on keratinocytes. LDH cytotoxicity tests were also performed concurrently to verify the accuracy of the MTT assays. The results of both tests agreed and the use of low-temperature plasma in skin treatment can be considered as safe. Overall, the results show that the plasma nozzle can find use in medicine in the healing of skin wounds and chronic defects as a potentially fast, inexpensive and effective method.
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