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.

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.

• 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

INTRODUCTION: The formation of diabetic ulcers (DU) is a common complication for diabetic patients resulting in serious chronic wounds. There is therefore, an urgent need for complex treatment of this problem. This study examines a bioactive wound dressing of a biodegradable electrospun nanofibrous blend of poly(L-lactide-co-ε-caprolactone) and poly(ε-caprolactone) (PLCL/PCL) covered by a thin fibrin layer for sustained delivery of bioactive molecules. METHODS: Electrospun PLCL/PCL nanofibers were coated with fibrin-based coating prepared by a controlled technique and enriched with human platelet lysate (hPL), fibroblast growth factor 2 (FGF), and vascular endothelial growth factor (VEGF). The coating was characterized by scanning electron microscopy and fluorescent microscopy. Protein content and its release rate and the effect on human saphenous vein endothelial cells (HSVEC) were evaluated. RESULTS: The highest protein amount is achieved by the coating of PLCL/PCL with a fibrin mesh containing 20% v/v hPL (NF20). The fibrin coating serves as an excellent scaffold to accumulate bioactive molecules from hPL such as PDGF-BB, fibronectin (Fn), and α-2 antiplasmin. The NF20 coating shows both fast and a sustained release of the attached bioactive molecules (Fn, VEGF, FGF). The dressing significantly increases the viability of human saphenous vein endothelial cells (HSVECs) cultivated on a collagen-based wound model. The exogenous addition of FGF and VEGF during the coating procedure further increases the HSVECs viability. In addition, the presence of α-2 antiplasmin significantly stabilizes the fibrin mesh and prevents its cleavage by plasmin. DISCUSSION: The NF20 coating supplemented with FGF and VEGF provides a promising wound dressing for the complex treatment of DU. The incorporation of various bioactive molecules from hPL and growth factors has great potential to support the healing processes by providing appropriate stimuli in the chronic wound.

• MeSH
• alfa-2-antiplasmin MeSH
• endoteliální buňky MeSH
• hojení ran MeSH
• lidé MeSH
• nanovlákna * MeSH
• obvazy MeSH
• polyestery farmakologie   MeSH
• vaskulární endoteliální růstový faktor A * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

PURPOSE: Due to thromboembolic complications and in-stent-stenosis after flow diverter (FD) treatment, the long-term use of dual antiplatelet treatment (DAPT) is mandatory. The tested nano-coating has been shown to reduce material thrombogenicity and promote endothelial cell proliferation in vitro. We compared the biocompatibility of coated (Derivo Heal) and non-coated (Derivo bare) FDs with DAPT in an animal model. METHODS: Derivo® bare (n = 10) and Derivo® Heal (n = 10) FD were implanted in the common carotid arteries (CCAs) of New Zealand white rabbits. One additional FD, alternately a Derivo bare (n = 5) or Derivo Heal (n = 5), was implanted in the abdominal aorta (AA) for assessment of the patency of branch arteries. Histopathological examinations were performed after 28 days. Angiography was performed before and after FD implantation and at follow-up. RESULTS: Statistical analysis of the included specimens showed complete endothelialization of all FDs with no significant differences in neointima thickness between Derivo® bare and Derivo® Heal (CCA: p = 0.91; AA: p = 0.59). A significantly reduced number of macrophages in the vessel wall of the Derivo Heal was observed for the CCA (p = 0.02), and significantly reduced fibrin and platelet deposition on the surface of the Derivo Heal was observed for the AA. All branch arteries of the stented aorta remained patent. CONCLUSION: In this animal model, the novel fibrin-based coated FD showed a similar blood and tissue compatibility as the non-coated FD.

• MeSH
• arteria carotis communis MeSH
• biokompatibilní potahované materiály MeSH
• fibrin * MeSH
• králíci MeSH
• neointima MeSH
• stenty * MeSH
• trombocyty MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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
• thrombin chemie   MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury * 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

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.

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.

• 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

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body's circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 °C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (β-thromboglobulin [β-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

• MeSH
• beta-thromboglobulin metabolismus   MeSH
• biologické markery metabolismus   MeSH
• biologické modely * MeSH
• cévní protézy * MeSH
• heparin farmakologie   MeSH
• imunitní systém metabolismus   MeSH
• komplement metabolismus   MeSH
• krevní oběh účinky léků   fyziologie   MeSH
• krevní obraz MeSH
• krevní plazma MeSH
• lidé MeSH
• odběr vzorku krve MeSH
• pankreatická elastasa metabolismus   MeSH
• stenty MeSH
• testování materiálů metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH

Decellularized human pericardium is under study as an allogenic material for cardiovascular applications. The effects of crosslinking on the mechanical properties of decellularized pericardium were determined with a uniaxial tensile test, and the effects of crosslinking on the collagen structure of decellularized pericardium were determined by multiphoton microscopy. The viability of human umbilical vein endothelial cells seeded on decellularized human pericardium and on pericardium strongly and weakly crosslinked with glutaraldehyde and with genipin was evaluated by means of an MTS assay. The viability of the cells, measured by their metabolic activity, decreased considerably when the pericardium was crosslinked with glutaraldehyde. Conversely, the cell viability increased when the pericardium was crosslinked with genipin. Coating both non-modified pericardium and crosslinked pericardium with a fibrin mesh or with a mesh containing attached heparin and/or fibronectin led to a significant increase in cell viability. The highest degree of viability was attained for samples that were weakly crosslinked with genipin and modified by means of a fibrin and fibronectin coating. The results indicate a method by which in vivo endothelialization of human cardiac allografts or xenografts could potentially be encouraged.

• MeSH
• alografty MeSH
• biokompatibilní materiály * chemie   MeSH
• biomechanika MeSH
• endoteliální buňky pupečníkové žíly (lidské) cytologie   metabolismus   MeSH
• fibrin MeSH
• fibronektiny MeSH
• glutaraldehyd MeSH
• heterografty MeSH
• iridoidy MeSH
• kolagen chemie   ultrastruktura   MeSH
• lidé MeSH
• mikroskopie fluorescenční multifotonová MeSH
• perikard chemie   transplantace   ultrastruktura   MeSH
• pevnost v tahu MeSH
• povrchová plasmonová rezonance MeSH
• reagencia zkříženě vázaná MeSH
• testování materiálů MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Long-term performance of implanted cardiovascular grafts can be ensured if living endothelium overgrows their surface. Surface modifications to implants are therefore being sought that can encourage endothelialization while preventing thrombus formation until the natural endothelium is formed. In the present study, heparin was covalently attached to a fibrin mesh grown from a polyvinyl chloride (PVC) substrate surface by the catalytic action of surface immobilized thrombin on a fibrinogen solution. The coating prevented platelet activation, thrombin generation and clot formation, and reduced inflammatory reactions when exposed to fresh human whole blood circulating in a Chandler loop model. In addition, in vitro seeded human umbilical vein and human saphenous vein endothelial cells showed considerably enhanced attachment and proliferation on the coating. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2995-3005, 2017.

• MeSH
• aktivace trombocytů účinky léků   MeSH
• antikoagulancia chemie   farmakologie   MeSH
• biokompatibilní potahované materiály chemie   farmakologie   MeSH
• buněčná adheze účinky léků   MeSH
• cévní protézy škodlivé účinky   MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• endoteliální buňky cytologie   účinky léků   MeSH
• fibrin chemie   MeSH
• hematokrit MeSH
• hemokoagulace účinky léků   MeSH
• heparin chemie   farmakologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• trombóza krev   etiologie   prevence a kontrola   MeSH
• vena saphena cytologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Protein-coated resorbable synthetic polymeric nanofibrous membranes are promising for the fabrication of advanced skin substitutes. We fabricated electrospun polylactic acid and poly(lactide-co-glycolic acid) nanofibrous membranes and coated them with fibrin or collagen I. Fibronectin was attached to a fibrin or collagen nanocoating, in order further to enhance the cell adhesion and spreading. Fibrin regularly formed a coating around individual nanofibers in the membranes, and also formed a thin noncontinuous nanofibrous mesh on top of the membranes. Collagen also coated most of the fibers of the membrane and randomly created a soft gel on the membrane surface. Fibronectin predominantly adsorbed onto a thin fibrin mesh or a collagen gel, and formed a thin nanofibrous structure. Fibrin nanocoating greatly improved the attachment, spreading, and proliferation of human dermal fibroblasts, whereas collagen nanocoating had a positive influence on the behavior of human HaCaT keratinocytes. In addition, fibrin stimulated the fibroblasts to synthesize fibronectin and to deposit it as an extracellular matrix. Fibrin coating also showed a tendency to improve the ultimate tensile strength of the nanofibrous membranes. Fibronectin attached to fibrin or to a collagen coating further enhanced the adhesion, spreading, and proliferation of both cell types.

• MeSH
• buněčná adheze MeSH
• extracelulární matrix metabolismus   MeSH
• fibrin metabolismus   MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fibronektiny metabolismus   MeSH
• keratinocyty cytologie   metabolismus   MeSH
• kolagen metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• nanovlákna chemie   MeSH
• pevnost v tahu MeSH
• polymery chemie   MeSH
• proliferace buněk MeSH
• tkáňové inženýrství MeSH
• tkáňové podpůrné struktury chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH