In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.

• MeSH
• blastocysta účinky léků   metabolismus   MeSH
• fertilizace in vitro MeSH
• fibroblastový růstový faktor 2 * farmakologie   MeSH
• insulinu podobný růstový faktor I * farmakologie   MeSH
• kultivační média * chemie   farmakologie   MeSH
• leukemický inhibiční faktor * farmakologie   MeSH
• oocyty MeSH
• prasata embryologie   genetika   MeSH
• proteomika MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The purpose of dermal substitutes is to mimic the basic properties of the extracellular matrix of human skin. The application of dermal substitutes to the defect reduces the formation of hypertrophic scars and improves the scar quality. This study aims to develop an original dermal substitute enriched with stable fibroblast growth factor 2 (FGF2-STAB®) and test it in an animal model. METHODS: Dermal substitutes based on collagen/chitosan scaffolds or collagen/chitosan scaffolds with nanofibrous layer were prepared and enriched with FGF2-STAB® at concentrations of 0, 0.1, 1.0, and 10.0 μg ‧ cm-2. The performance of these dermal substitutes was tested in vivo on artificially formed skin defects in female swine. The outcomes were evaluated using cutometry at 3 and 6 months. In addition, visual appearance was assessed based on photos of the scars at 1-month, 3-month and 6-month follow-ups using Yeong scale and Visual Analog Scale. RESULTS: The dermal substitute was fully integrated into all defects and all wounds healed successfully. FGF2-STAB®-enriched matrices yielded better results in cutometry compared to scaffolds without FGF2. Visual evaluation at 1, 3, and 6 months follow-ups detected no significant differences among groups. The FGF2-STAB® effectiveness in improving the elasticity of scar tissues was confirmed in the swine model. This effect was independently observed in the scaffolds with nanofibres as well as in the scaffolds without nanofibres. CONCLUSION: The formation of scars with the best elasticity was exhibited by addition 1.0 μg ‧ cm-2of FGF2-STAB® into the scaffolds, although it had no significant effect on visual appearance at longer follow-ups. This study creates the basis for further translational studies of the developed product and its progression into the clinical phase of the research.

• MeSH
• chitosan * MeSH
• fibroblastový růstový faktor 2 * MeSH
• hojení ran účinky léků   MeSH
• jizva hypertrofická MeSH
• kolagen MeSH
• kůže MeSH
• modely nemocí na zvířatech MeSH
• nanovlákna terapeutické užití   MeSH
• popálení MeSH
• prasata MeSH
• pružnost * MeSH
• tkáňové podpůrné struktury MeSH
• umělá kůže * MeSH
• viskozita MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• fibroblastový růstový faktor 2 * MeSH
• fibroblasty MeSH
• hojení ran MeSH
• lidé MeSH
• popálení * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH

Several peripheral membrane proteins are known to form membrane pores through multimerization. In many cases, in biochemical reconstitution experiments, a complex distribution of oligomeric states has been observed that may, in part, be irrelevant to their physiological functions. This phenomenon makes it difficult to identify the functional oligomeric states of membrane lipid interacting proteins, for example, during the formation of transient membrane pores. Using fibroblast growth factor 2 (FGF2) as an example, we present a methodology applicable to giant lipid vesicles by which functional oligomers can be distinguished from nonspecifically aggregated proteins without functionality. Two distinct populations of fibroblast growth factor 2 were identified with (i) dimers to hexamers and (ii) a broad population of higher oligomeric states of membrane-associated FGF2 oligomers significantly distorting the original unfiltered histogram of all detectable oligomeric species of FGF2. The presented statistical approach is relevant for various techniques for characterizing membrane-dependent protein oligomerization.

• MeSH
• buněčná membrána metabolismus   MeSH
• fibroblastový růstový faktor 2 * metabolismus   MeSH
• lipidy MeSH
• membránové proteiny * metabolismus   MeSH
• membrány MeSH
• multimerizace proteinu MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

FGF2 is a cell survival factor involved in tumor-induced angiogenesis that is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here, we demonstrate that both PI(4,5)P2-dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further revealed cholesterol to enhance FGF2 binding to PI(4,5)P2-containing lipid bilayers. Based on extensive atomistic molecular dynamics (MD) simulations and membrane tension experiments, we proposed cholesterol to modulate FGF2 binding to PI(4,5)P2 by (i) increasing head group visibility of PI(4,5)P2 on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P2 molecules triggering FGF2 oligomerization, and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 toward the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2.

• MeSH
• buněčná membrána metabolismus   MeSH
• cholesterol * metabolismus   MeSH
• fibroblastový růstový faktor 2 * metabolismus   MeSH
• fosfatidylinositol-4,5-difosfát * metabolismus   MeSH
• lipidové dvojvrstvy * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Long-term delivery of growth factors and immunomodulatory agents is highly required to support the integrity of tissue in engineering constructs, e.g., formation of vasculature, and to minimize immune response in a recipient. However, for proteins with a net positive charge at the physiological pH, controlled delivery from negatively charged alginate (Alg) platforms is challenging due to electrostatic interactions that can hamper the protein release. In order to regulate such interactions between proteins and the Alg matrix, we propose to complex proteins of interest in this study - CXCL12, FGF-2, VEGF - with polyanionic heparin prior to their encapsulation into Alg microbeads of high content of α-L-guluronic acid units (high-G). This strategy effectively reduced protein interactions with Alg (as shown by model ITC and SPR experiments) and, depending on the protein type, afforded control over the protein release for at least one month. The released proteins retained their in vitro bioactivity: CXCL12 stimulated the migration of Jurkat cells, and FGF-2 and VEGF induced proliferation and maturation of HUVECs. The presence of heparin also intensified protein biological efficiency. The proposed approach for encapsulation of proteins with a positive net charge into high-G Alg hydrogels is promising for controlled long-term protein delivery under in vivo conditions.

• MeSH
• algináty chemie   MeSH
• chemokin CXCL12 chemie   MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• fibroblastový růstový faktor 2 chemie   MeSH
• heparin chemie   MeSH
• lidé MeSH
• mikrosféry MeSH
• nádorové buněčné linie MeSH
• tkáňové inženýrství MeSH
• vaskulární endoteliální růstový faktor A chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

Umělé kožní náhrady řídící proces hojení ran se používají v léčbě chronických kožních defektů a akutních ran. Závažným problémem je jejich vysoká cena a rizika pocházející z infekčních komplikací. Zatímco dvojvrstevná pěna Integra vyžaduje dvoukrokový chirurgický postup, jenovrstevný Matriderm použitelný v jednom kroku není schválen v ČR. Námi navrhovaná 3D nanostrukturní bio-náhrada se skládá z antibakteriální kolagen/chitosanové porézní pěny (dermální náhrada) s přísadou hyperstabilního fibroblastového růstového faktoru FGF2 a potažená elastickou nanovlákennou vrstvou (dermálně-epidermální spoj). Vrstva dermální náhrady umožňuje prorůstání nativních fibroblastů a cév, zatímco nanoporézní mukoadhezivní membrána simuluje "lepidlovou" mezivrstvu zajišťující dobrou přilnavost k epidermálnímu autograftu. V rámci tohoto projektu bude navržená bio-náhrada hodnocena fyzikálně-chemicky, biomechanicky, in-vitro pomocí fibroblastů, keratinocytů a makrofágů a následně použita na hojení defektu kůže v celé jeho tloušťce v preklinické studii na experimentálním modelu prasete.; The artificial skin substitutes are applied in the treatment of chronic skin defects and acute wounds to control wound healing process. However, a significant problem is their high price and risks originated from infectious complications. Whereas bilayer sponge Integra involves two step surgery procedure, the monolayer Matriderm applicable in one step is not approved in Czech. Our proposed 3D nanostructured bio-substitute is consisted of antibacterial collagen/chitosan porous sponge (dermal substitute) doped with hyperstable fibroblast growth factor FGF2 and covered with elastic nanofiber layer (dermal-epidermal junction). The dermal substitute layer facilitates ingrowth of native fibroblasts and blood vessels while nanoporous mucoadhesive membrane simulates the “glue-like” interlayer ensuring good adhesion to the epidermis autograft. In this project, proposed bio-substitute will be evaluated physico-chemically, biomechanically, in-vitro on fibroblasts, keratinocytes and macrophages followed by treatment of full thickness skin defect in preclinical study on experimental pig model.

• MeSH
• biokompatibilní materiály terapeutické užití   MeSH
• biopolymery terapeutické užití   MeSH
• fibroblastový růstový faktor 2 terapeutické užití   MeSH
• hojení ran MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• nanostruktury terapeutické užití   MeSH
• prasata MeSH
• protézy a implantáty MeSH
• tkáně MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• hodnotící studie MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• technika lékařská, zdravotnický materiál a protetika
• chirurgie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

In a biological system, nanoparticles (NPs) may interact with biomolecules. Specifically, the adsorption of proteins on the nanoparticle surface may influence both the nanoparticles' and proteins' overall bio-reactivity. Nevertheless, our knowledge of the biocompatibility and risk of exposure to nanomaterials is limited. Here, in vitro and ex ovo biocompatibility of naturally based crosslinked freeze-dried 3D porous collagen/chitosan scaffolds, modified with thermostable fibroblast growth factor 2 (FGF2-STAB®), to enhance healing and selenium nanoparticles (SeNPs) to provide antibacterial activity, were evaluated. Biocompatibility and cytotoxicity were tested in vitro using normal human dermal fibroblasts (NHDF) with scaffolds and SeNPs and FGF2-STAB® solutions. Metabolic activity assays indicated an antagonistic effect of SeNPs and FGF2-STAB® at high concentrations of SeNPs. The half-maximal inhibitory concentration (IC50) of SeNPs for NHDF was 18.9 µg/ml and IC80 was 5.6 µg/ml. The angiogenic properties of the scaffolds were monitored ex ovo using a chick chorioallantoic membrane (CAM) assay and the cytotoxicity of SeNPs over IC80 value was confirmed. Furthermore, the positive effect of FGF2-STAB® at very low concentrations (0.01 µg/ml) on NHDF metabolic activity was observed. Based on detailed in vitro testing, the optimal concentrations of additives in the scaffolds were determined, specifically 1 µg/ml of FGF2-STAB® and 1 µg/ml of SeNPs. The scaffolds were further subjected to antimicrobial tests, where an increase in selenium concentration in the collagen/chitosan scaffolds increased the antibacterial activity. This work highlights the antimicrobial ability and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® and SeNPs. Moreover, we suggest that these sponges could be used as scaffolds for growing cells in systems with low mechanical loading in tissue engineering, especially in dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration. Due to their antimicrobial properties, these scaffolds are also highly promising for tissue replacement requiring the prevention of infection.

• MeSH
• antibakteriální látky MeSH
• biokompatibilní materiály farmakologie   MeSH
• buněčné linie MeSH
• chitosan farmakologie   MeSH
• fibroblastový růstový faktor 2 farmakologie   MeSH
• fibroblasty účinky léků   MeSH
• hojení ran MeSH
• kolagen farmakologie   MeSH
• lidé MeSH
• nanočástice chemie   terapeutické užití   MeSH
• poréznost MeSH
• selen chemie   farmakologie   MeSH
• testování materiálů MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Vascular endothelial growth factor-A165 (VEGF-A165) and fibroblast growth factor-2 (FGF-2) are currently used for the functionalization of biomaterials designed for tissue engineering. We have developed a new simple method for heterologous expression and purification of VEGF-A165 and FGF-2 in the yeast expression system of Pichia pastoris. The biological activity of the growth factors was assessed in cultures of human and porcine adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). When added into the culture medium, VEGF-A165 stimulated proliferation only in HUVECs, while FGF-2 stimulated the proliferation of both cell types. A similar effect was achieved when the growth factors were pre-adsorbed to polystyrene wells. The effect of our recombinant growth factors was slightly lower than that of commercially available factors, which was attributed to the presence of some impurities. The stimulatory effect of the VEGF-A165 on cell adhesion was rather weak, especially in ADSCs. FGF-2 was a potent stimulator of the adhesion of ADSCs but had no to negative effect on the adhesion of HUVECs. In sum, FGF-2 and VEGF-A165 have diverse effects on the behavior of different cell types, which maybe utilized in tissue engineering.

• MeSH
• buněčná adheze účinky léků   MeSH
• endoteliální buňky pupečníkové žíly (lidské) cytologie   metabolismus   MeSH
• fibroblastový růstový faktor 2 chemie   genetika   farmakologie   MeSH
• kmenové buňky cytologie   metabolismus   MeSH
• lidé MeSH
• prasata MeSH
• proliferace buněk účinky léků   MeSH
• rekombinantní proteiny chemie   farmakologie   MeSH
• vaskulární endoteliální růstový faktor A chemie   genetika   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Ateroskleróza je charakterizovaná perzistujúcim zápalom cievnej steny a je považovaná za hlavnú príčinu napomáhajúcu rozvoju kardiovaskulárnych ochorení, ktoré sú celosvetovo vedúcou príčinou úmrtí. Z dôvodu rozšírenia aterosklerózy a jej komplikácií sa zvyšuje potreba včasnej a pokiaľ je to možné neinvazívnej diagnostiky, aby sa predišlo vzniku fatálnych alebo invalidizujúcich komplikácií aterosklerózy. Na detekciu aterosklerotických plátov sa využívajú zobrazovacie metódy aj klinické vyšetrenia, ktoré zachytávajú až pláty hemodynamicky významné. Lepšia prevencia aterosklerózy si žiada vyhľadávanie vysokorizikových jedincov vo včasných štádiách. Zápal sa prejavuje počas celého priebehu aterogenézy, teda aj v štádiu subklinickej aterosklerózy, kedy je možné stanoviť koncentráciu zápalových biomarkerov v krvi. Biomarkery sú predmetom záujmu pre jednoduchosť stanovenia v plazme či sére a možnosť ich využitia na diagnostické, prognostické aj terapeutické účely. Pozitívne korelácie s aterosklerózou a jej komplikáciami boli dokázané u biomarkerov asociovaných s metabolizmom kostí ako fibro­blastový rastový faktor 23, osteokalcín, osteoglycín, osteopontín či osteoprotegerín.

Atherosclerosis is characterized by persistent inflammation of the vascular wall and is considered to be a major cause contributing to the development of cardiovascular disease, which is the leading cause of death worldwide. Due to the prevalence of atherosclerosis and its complications, the need for early and, if possible, non-invasive dia­gnosis is increasing in order to prevent the development of fatal or disabling complications of atherosclerosis. Ima­ging methods as well as clinical examina­tions are used for the detection of atherosclerotic plaques, which capture up to hemodynamically significant plaques. Better prevention of atherosclerosis requires the search for high-risk indivi­duals in early stages. Inflammation manifests itself throughout the course of atherogenesis, i.e. also in the stage of subclinical atherosclerosis, when it is possible to determine the concentration of inflammatory biomarkers in the blood. Biomarkers are of interest for the simplicity of determination in plasma or serum and the possibility of their use for diagnostic, prognostic and therapeutic purposes. Positive correlations with atherosclerosis and its complications have been demonstrated in biomarkers associated with bone meta­bolism such as fibroblast growth factor 23, osteocalcin, osteoglycin, osteopontin or osteoprotegerin.

• Klíčová slova
• osteoglycin,
• MeSH
• ateroskleróza * diagnóza   patofyziologie   MeSH
• biologické markery MeSH
• fibroblastový růstový faktor 2 analýza   MeSH
• lidé MeSH
• osteokalcin analýza   MeSH
• osteopontin analýza   MeSH
• osteoporóza * diagnóza   patofyziologie   MeSH
• osteoprotegerin analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH