Léčba diabetických ran je problematická, časově i finančně náročná a omezující pro pacienty. Krevní destičky jsou fyziologicky přítomné v procesu hojení akutní rány, kde produkují růstové a angiogenní faktory, cytokiny, chemokiny, molekuly zprostředkující adhesi buněk a další bioaktivní látky napomáhající hojení. Lyzát připravený z plazmy obohacené o krevní destičky bude zabudován do degradovatelných i nedegradovatelných nanovlákenných nosičů připravených ze syntetických i přirozených polymerů dále modifikovaných fibrinem, a to s cílem postupného uvolňování růstových faktorů a dalších biologicky aktivních látek. Bioaktivita těchto nanovlákenných krytů bude hodnocena in vitro v kulturách lidských keratinocytů, dermálních fibroblastů, cévních endotelových buněk a kmenových buněk tukové tkáně. Nanovlákenné kryty s nejlepšími vlastnostmi budou aplikovány in vivo na rány u diabetických potkanů, a následně bude hodnocena jejich schopnost stimulace hojení ran.; Treatment of diabetic wounds is problematic, long-lasting, expensive and restrictive for patients. Platelets are physiologically present in the process of healing of acute wounds, where they secret growth and angiogenic factors, cytokines, chemokines, cell adhesion-mediating molecules and other bioactive substances that support healing. Lysate prepared from platelet-rich plasma will be incorporated into degradable and non-degradable nanofibrous dressings, prepared from synthetic and natural polymers further modified with fibrin, in order to assure controlled release of growth factors and other bioactive substances. Bioactivity of these dressings will be evaluated in vitro in cultures of human keratinocytes, dermal fibroblasts, vascular endothelial cells and adipose tissue-derived stem cells. Nanofibrous dressings with the best properties will be applied in vivo on wounds of diabetic rats, and their ability to stimulate wound healing will be evaluated.

• Klíčová slova
• řízené uvolňování, diabetické rány, lyzát z trombocytů, diabetický potkan, hojení ran, wound healing, diabetic wounds, platelet lysate, control release, diabetic rat, plasma obohacená destičkami, epidermální keratinocyty, dermální fibroblasty, kmenové buňky tukové tkáně, platelet-rich plasma, epidermal keratinocytes, dermal fibroblasts, adipose-tissue stem cells,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

An ideal decellularized allogenic or xenogeneic cardiovascular graft should be capable of preventing thrombus formation after implantation. The antithrombogenicity of the graft is ensured by a confluent endothelial cell layer formed on its surface. Later repopulation and remodeling of the scaffold by the patient's cells should result in the formation of living autologous tissue. In the work presented here, decellularized porcine pericardium scaffolds were modified by growing a fibrin mesh on the surface and inside the scaffolds, and by attaching heparin and human vascular endothelial growth factor (VEGF) to this mesh. Then the scaffolds were seeded with human adipose tissue-derived stem cells (ASCs). While the ASCs grew only on the surface of the decellularized pericardium, the fibrin-modified scaffolds were entirely repopulated in 28 d, and the scaffolds modified with fibrin, heparin and VEGF were already repopulated within 6 d. Label free mass spectrometry revealed fibronectin, collagens, and other extracellular matrix proteins produced by ASCs during recellularization. Thin layers of human umbilical endothelial cells were formed within 4 d after the cells were seeded on the surfaces of the scaffold, which had previously been seeded with ASCs. The results indicate that an artificial tissue prepared by in vitro recellularization and remodeling of decellularized non-autologous pericardium with autologous ASCs seems to be a promising candidate for cardiovascular grafts capable of accelerating in situ endothelialization. ASCs resemble the valve interstitial cells present in heart valves. An advantage of this approach is that ASCs can easily be collected from the patient by liposuction.

• MeSH
• bioprotézy MeSH
• decelularizovaná extracelulární matrix chemie   MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• endoteliální buňky cytologie   MeSH
• extracelulární matrix metabolismus   MeSH
• fibrinogen chemie   MeSH
• fibronektiny chemie   MeSH
• fluorescenční mikroskopie MeSH
• kmenové buňky MeSH
• kolagen chemie   MeSH
• lidé MeSH
• lipektomie MeSH
• perikard metabolismus   patologie   MeSH
• prasata MeSH
• proliferace buněk MeSH
• srdeční chlopně * MeSH
• techniky in vitro MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury * chemie   MeSH
• trombin chemie   MeSH
• tuková tkáň cytologie   MeSH
• vaskulární endoteliální růstový faktor A metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Autologous and allogenic human pericardia used as biomaterials for cardiovascular surgery are traditionally crosslinked with glutaraldehyde. In this work, we have evaluated the resistivity to collagenase digestion and the cytotoxicity of human pericardium crosslinked with various concentrations of glutaraldehyde in comparison with pericardium crosslinked by genipin, nordihydroguaiaretic acid, tannic acid, and in comparison with unmodified pericardium. Crosslinking retained the wavy-like morphology of native pericardium visualized by second harmonic generation microscopy. The collagenase digestion products were analyzed using SDS-PAGE, capillary electrophoresis, and a hydroxyproline assay. Glutaraldehyde and genipin crosslinking protected the native pericardium efficiently against digestion with collagenase III. Only low protection was provided by the other crosslinking agents. The cytotoxicity of crosslinked pericardium was evaluated using xCELLigence by monitoring the viability of porcine valve interstitial cells cultured in eluates from crosslinked pericardium. The highest cell index, reflecting both the number and the shape of the monitored cells was observed in eluates from genipin. Crosslinking pericardium grafts with genipin therefore seems to be a promising alternative procedure to the traditional crosslinking with glutaraldehyde, because it provides similarly high protection against degradation with collagenase, without cytotoxic effects.

• MeSH
• biokompatibilní materiály MeSH
• glutaraldehyd MeSH
• iridoidy MeSH
• kyselina nordihydroguaiaretová MeSH
• lidé MeSH
• perikard chemie   MeSH
• reagencia zkříženě vázaná * MeSH
• taniny MeSH
• transplantáty chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• srovnávací studie MeSH

V současné době tvoří 40% náhrad srdečních chlopní náhrady biologické, především z vepřové aortální chlopně a hovězího perikardu, které jsou síťované glutaraldehydem. Tyto xenogenní náhrady však často degenerují nebo kalcifikují, což omezuje jejich životnost. V tomto projektu chceme vyvinout novou biologickou náhradu srdeční aortální chlopně, s použitím lidského nebo vepřového perikardu, který bude osazen kmenovými buňkami z tukové tkáně a endotelovými buňkami. Takto připravená autologní chlopenní náhrada bude v budoucnu testována in vivo na miniprasatech. Perikard bude nejprve decelularizován, pokryt biomolekulárními strukturami na podporu recelularizace a endotelizace. Konstrukt osazený oběma buněčnými typy bude kultivován v dynamickém bioreaktoru. Mechanické zatěžování bude indukovat diferenciaci kmenových buněk na intersticiální buňky chlopně a na buňky hladkého svalu a bude podporovat produkci mezibuněčné hmoty a to povede k celkovému zlepšení mechanických vlastností perikardu.; Nowadays, biological heart valve prostheses are used in 40% of valve replacements. The predominant materials are porcine aortic valves and bovine pericardial valves preserved by glutaraldehyde. Xenogeneic prostheses, however, suffer from progressive calcific and noncalcific deterioration and limited durability. In the project, we will develop new biological aortic heart valve prostheses based on human or porcine pericardium that will be seeded with human adipose tissue-derived stem cells and endothelial cells intended as autologous for further in vivo experiments in minipigs. First of all, the pericardium will be decellularized, coated with biomolecular structures for supporting recellularization and endothelialization. The construct seeded with both cell types will be cultivated in a dynamic bioreactor. The mechanical loading of the tissue will induce the differentiation of stem cells into valve interstitial cells or smooth muscle cells, and extracellular matrix production, thus improving the mechanical properties of the pericardium.

• MeSH
• bioprotézy MeSH
• bioreaktory MeSH
• kmenové buňky MeSH
• lidé MeSH
• mechanický stres MeSH
• perikard MeSH
• prasata MeSH
• primární buněčná kultura MeSH
• srdeční chlopně umělé MeSH
• tuková tkáň MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH

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

Diamond-like carbon (DLC) thin films are promising for use in coating orthopaedic, dental and cardiovascular implants. The problem of DLC layers lies in their weak layer adhesion to metal implants. Chromium is used as a dopant for improving the adhesion of DLC films. Cr-DLC layers were prepared by a hybrid technology, using a combination of pulsed laser deposition (PLD) from a graphite target and magnetron sputtering. Depending on the deposition conditions, the concentration of Cr in the DLC layers moved from zero to 10.0 at.%. The effect of DLC layers with 0.0, 0.9, 1.8, 7.3, 7.7 and 10.0 at.% Cr content on the adhesion and osteogenic differentiation of human osteoblast-like Saos-2 cells was assessed in vitro. The DLC samples that contained 7.7 and 10.0 at.% of Cr supported cell spreading on day 1 after seeding. On day three after seeding, the most apparent vinculin-containing focal adhesion plaques were also found on samples with higher concentrations of chromium. On the other hand, the expression of type I collagen and alkaline phosphatase at the mRNA and protein level was the highest on Cr-DLC samples with a lower concentration of Cr (0-1.8 at.%). We can conclude that higher concentrations of chromium supported cell adhesion; however DLC and DLC doped with a lower concentration of chromium supported osteogenic cell differentiation.

• MeSH
• alkalická fosfatasa metabolismus   MeSH
• biokompatibilní potahované materiály MeSH
• buněčná adheze * MeSH
• buněčná diferenciace * MeSH
• buněčné linie MeSH
• chrom chemie   MeSH
• diamant chemie   MeSH
• fokální adheze MeSH
• kolagen typu I metabolismus   MeSH
• kovy chemie   MeSH
• lasery MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• osteoblasty cytologie   MeSH
• osteogeneze MeSH
• povrchové vlastnosti MeSH
• stanovení celkové genové exprese MeSH
• talin chemie   MeSH
• uhlík chemie   MeSH
• vinkulin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Ti-6Al-4V-based nanotubes were prepared on a Ti-6Al-4V surface by anodic oxidation on 10 V, 20 V, and 30 V samples. The 10 V, 20 V, and 30 V samples and a control smooth Ti-6Al-4V sample were evaluated in terms of their chemical composition, diameter distribution, and cellular response. The surfaces of the 10 V, 20 V, and 30 V samples consisted of nanotubes of a relatively wide range of diameters that increased with the voltage. Saos-2 cells had a similar initial adhesion on all nanotube samples to the control Ti-6Al-4V sample, but it was lower than on glass. On day 3, the highest concentrations of both vinculin and talin measured by enzyme-linked immunosorbent assay and intensity of immunofluorescence staining were on 30 V nanotubes. On the other hand, the highest concentrations of ALP, type I collagen, and osteopontin were found on 10 V and 20 V samples. The final cellular densities on 10 V, 20 V, and 30 V samples were higher than on glass. Therefore, the controlled anodization of Ti-6Al-4V seems to be a useful tool for preparing nanostructured materials with desirable biological properties.

• MeSH
• aktiny metabolismus   MeSH
• biologické markery metabolismus   MeSH
• buněčná adheze účinky léků   MeSH
• buněčná diferenciace účinky léků   MeSH
• fluorescenční protilátková technika MeSH
• fotoelektronová spektroskopie MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nanotrubičky chemie   MeSH
• osteoblasty cytologie   účinky léků   MeSH
• osteogeneze účinky léků   MeSH
• povrchové vlastnosti MeSH
• proliferace buněk účinky léků   MeSH
• titan farmakologie   MeSH
• velikost částic * MeSH
• viabilita buněk účinky léků   MeSH
• vinkulin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Currently used vascular prostheses are hydrophobic and do not allow endothelial cell (EC) adhesion and growth. The aim of this study was to prepare fibrin (Fb)-based two-dimensional (2D) and three-dimensional (3D) assemblies coated with extracellular matrix (ECM) proteins and to evaluate the EC adhesion, proliferation and differentiation on these assemblies in vitro. Coating of Fb with collagen, laminin (LM), and fibronectin (FN) was proved using the surface plasmon resonance technique. On all Fb assemblies, ECs reached higher cell densities than on polystyrene after 3 and 7 days of culture. Immunoflurescence staining showed better assembly of talin and vinculin into focal adhesion plaques, and also more apparent staining of vascular endothelial cadherin on surface-attached 3D Fb and protein-coated Fb assemblies. On these samples, ECs also contained a lower concentration of intercellular adhesion molecule-1, measured by enzyme-linked immunosorbent assay. Higher concentrations of CD31 (platelet-endothelial cell adhesion molecule-1) were found on 3D Fb coated with LM, and higher concentrations of von Willebrand factor were found on 3D Fb coated with type I collagen or LM in comparison to 2D Fb layers. The results indicate that ECM protein-coated 2D and 3D Fb assemblies can be used for versatile applications in various tissue replacements where endothelialization is desirable, for example, vascular prostheses and heart valves.

• MeSH
• buněčná adheze MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• endoteliální buňky cytologie   MeSH
• fibrin chemie   MeSH
• kolagen chemie   MeSH
• laminin chemie   MeSH
• skot MeSH
• tkáňové podpůrné struktury chemie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hydroxyapatite (HA) is considered to be a bioactive material that favorably influences the adhesion, growth, and osteogenic differentiation of osteoblasts. To optimize the cell response on the hydroxyapatite composite, it is desirable to assess the optimum concentration and also the optimum particle size. The aim of our study was to prepare composite materials made of polydimethylsiloxane, polyamide, and nano-sized (N) or micro-sized (M) HA, with an HA content of 0%, 2%, 5%, 10%, 15%, 20%, 25% (v/v) (referred to as N0-N25 or M0-M25), and to evaluate them in vitro in cultures with human osteoblast-like MG-63 cells. For clinical applications, fast osseointegration of the implant into the bone is essential. We observed the greatest initial cell adhesion on composites M10 and N5. Nano-sized HA supported cell growth, especially during the first 3 days of culture. On composites with micro-size HA (2%-15%), MG-63 cells reached the highest densities on day 7. Samples M20 and M25, however, were toxic for MG-63 cells, although these composites supported the production of osteocalcin in these cells. On N2, a higher concentration of osteopontin was found in MG-63 cells. For biomedical applications, the concentration range of 5%-15% (v/v) nano-size or micro-size HA seems to be optimum.

• MeSH
• cytoskeletální proteiny metabolismus   MeSH
• fyziologie buňky účinky léků   MeSH
• hydroxyapatit chemie   farmakologie   MeSH
• lidé MeSH
• mikrosféry MeSH
• nádorové buněčné linie MeSH
• nanočástice chemie   MeSH
• osteoblasty MeSH
• osteokalcin metabolismus   MeSH
• osteopontin metabolismus   MeSH
• rozpustnost MeSH
• spektrometrie rentgenová emisní MeSH
• velikost částic * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Autologous vein grafts are often used for treating damaged vessels, e.g. arteriovenous fistulas or arterial bypass conduits. Veins have a different histological structure from arteries, which often leads to intimal hyperplasia and graft restenosis. The aim of this study was to develop a perivascular sirolimus-delivery system that would release the antiproliferative drug sirolimus in a controlled manner. Polyester Mesh I was coated with purasorb, i.e. a copolymer of L-lactide and ɛ-caprolactone, with dissolved sirolimus; Mesh II was coated with two copolymer layers; the layer with dissolved sirolimus was overlaid with pure purasorb. This arrangement allowed sirolimus to be released for 6 and 4 weeks, for Mesh I and Mesh II, respectively. Mesh II released sirolimus more homogeneously, without the initial burst effect during the first week. However, the cumulative release curve was steeper at later time points than the curve for Mesh I. Both meshes inhibited proliferation of rat vascular smooth muscle cells during 14-day culture in vitro and preserved excellent cell viability. Newly developed sirolimus-releasing perivascular meshes are promising devices for preventing autologous graft restenosis.

• MeSH
• biokompatibilní potahované materiály MeSH
• farmaceutická chemie MeSH
• farmaceutická technologie metody   MeSH
• kardiovaskulární látky aplikace a dávkování   chemie   farmakologie   MeSH
• kinetika MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• léky s prodlouženým účinkem MeSH
• mikroskopie elektronová rastrovací MeSH
• molekulová hmotnost MeSH
• myocyty hladké svaloviny účinky léků   MeSH
• nosiče léků MeSH
• okluze cévního štěpu prevence a kontrola   MeSH
• polyestery chemie   MeSH
• potkani Wistar MeSH
• povrchové vlastnosti MeSH
• příprava léků MeSH
• proliferace buněk účinky léků   MeSH
• rozpustnost MeSH
• sirolimus aplikace a dávkování   chemie   farmakologie   MeSH
• stabilita léku MeSH
• svaly hladké cévní účinky léků   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Various techniques for coating synthetic surfaces with fibrin structures were tested for seeding bovine pulmonary artery endothelial cells (EC). Two-dimensional fibrin (Fb) structures (2D Fb) were obtained by successively repeating adsorption of fibrinogen (Fbg), incubating the surface with thrombin (Thr) solution, and inhibiting surface-attached Thr. Three-dimensional fibrin networks immobilized at the surface (3D Fb) were formed by catalytic action of surface-attached thrombin on an ambient Fbg solution. Ultra-thin 3D Fbs were obtained if thrombin inhibitors antithrombin III and heparin were added into an Fbg solution. The formation of surface fibrin structures was observed in situ using surface plasmon resonance. The morphology of the structures was studied by transmission and scanning electron microscopy. A polylactide fibrous scaffold was modified with a surface fibrin film without filling the inner pores with a bulk gel. The growth of EC seeded on a polystyrene surface coated with the Fb films was evaluated by the number and morphology of the adhering ECs and the concentration of beta-actin, vinculin, alpha(v)-intergrin, and von Willebrand factor (vWF). The best initial cell spreading after 1 day was observed on 2D Fb and ultra-thin 3D Fb. The highest concentration of vWF, a marker of EC differentiation, was observed after 3 days on thick 3D Fbs. The highest EC population densities after 7 days were observed on 2D Fb and thick 3D Fb.

• MeSH
• adsorpce MeSH
• aktiny metabolismus   MeSH
• antithrombin III metabolismus   MeSH
• biokompatibilní potahované materiály chemie   metabolismus   MeSH
• buněčné kultury MeSH
• endoteliální buňky cytologie   metabolismus   MeSH
• fibrin metabolismus   ultrastruktura   MeSH
• fibrinogen metabolismus   MeSH
• gely chemie   metabolismus   MeSH
• heparin metabolismus   MeSH
• integrin alfaV metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• polyestery chemie   MeSH
• povrchové vlastnosti MeSH
• skot MeSH
• testování materiálů MeSH
• tkáňové podpůrné struktury chemie   MeSH
• trombin metabolismus   MeSH
• vinkulin metabolismus   MeSH
• von Willebrandův faktor metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• hodnotící studie MeSH
• práce podpořená grantem MeSH