Background: Our study focuses on the fabrication of appropriate scaffolds for skin wound healing. This research brings valuable insights into the molecular mechanisms of adhesion, proliferation, and control of cell behavior through the extracellular matrix represented by synthetic biodegradable nanofibrous membranes coated by biomolecules. Methods: Nanofibrous polylactic acid (PLA) membranes were prepared by a needle-less electrospinning technology. These membranes were coated with fibrin according to two preparation protocols, and additionally they were coated with fibronectin in order to increase the cell affinity for colonizing the PLA membranes. The adhesion, growth, and extracellular matrix protein production of neonatal human dermal fibroblasts were evaluated on the nanofibrous membranes. Results: Our results showed that fibrin-coated membranes improved the adhesion and proliferation of human dermal fibroblasts. The morphology of the fibrin nanocoating seems to be crucial for the adhesion of fibroblasts, and consequently for their phenotypic maturation. Fibrin either covered the individual fibers in the membrane (F1 nanocoating), or covered the individual fibers and also formed a fine homogeneous nanofibrous mesh on the surface of the membrane (F2 nanocoating), depending on the mode of fibrin preparation. The fibroblasts on the membranes with the F1 nanocoating remained in their typical spindle-like shape. However, the cells on the F2 nanocoating were spread mostly in a polygon-like shape, and their proliferation was significantly higher. Fibronectin formed an additional mesh attached to the surface of the fibrin mesh, and further enhanced the cell adhesion and growth. The relative gene expression and protein production of collagen I and fibronectin were higher on the F2 nanocoating than on the F1 nanocoating. Conclusion: A PLA membrane coated with a homogeneous fibrin mesh seems to be promising for the construction of temporary full-thickness skin tissue substitutes.
- MeSH
- buněčná adheze fyziologie MeSH
- buněčné kultury přístrojové vybavení metody MeSH
- extracelulární matrix metabolismus MeSH
- fibrin chemie farmakologie MeSH
- fibroblasty cytologie účinky léků MeSH
- fibronektiny metabolismus MeSH
- kolagen typu I metabolismus MeSH
- kultivované buňky MeSH
- kůže cytologie MeSH
- lidé MeSH
- membrány umělé MeSH
- nanostruktury chemie MeSH
- nanotechnologie metody MeSH
- polyestery chemie MeSH
- proliferace buněk fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články 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
Fibrin is a versatile biopolymer that has been extensively used in tissue engineering. In this paper fibrin nanostructures prepared using a technique based on the catalytic effect of fibrin-bound thrombin are presented. This technique enables surface-attached thin fibrin networks to form with precisely regulated morphology without the development of fibrin gel in bulk solution. Moreover, the influence of changing the polymerization time, along with the antithrombin III and heparin concentrations on the morphology of fibrin nanostructures was explored. The binding of bioactive molecules (fibronectin, laminin, collagen, VEGF, bFGF, and heparin) to fibrin nanostructures was confirmed. These nanostructures can be used for the surface modification of artificial biomaterials designed for different biomedical applications (e.g. artificial vessels, stents, heart valves, bone and cartilage constructs, skin grafts, etc.) in order to promote the therapeutic outcome.
Fibrin plays an important role during wound healing and skin regeneration. It is often applied in clinical practice for treatment of skin injuries or as a component of skin substitutes. We prepared electrospun nanofibrous membranes made from poly(l-lactide) modified with a thin fibrin nanocoating. Fibrin surrounded the individual fibers in the membrane and also formed a thin fibrous mesh on several places on the membrane surface. The cell-free fibrin nanocoating remained stable in the cell culture medium for 14 days and did not change its morphology. On membranes populated with human dermal fibroblasts, the rate of fibrin degradation correlated with the degree of cell proliferation. The cell spreading, mitochondrial activity, and cell population density were significantly higher on membranes coated with fibrin than on nonmodified membranes, and this cell performance was further improved by the addition of ascorbic acid in the cell culture medium. Similarly, fibrin stimulated the expression and synthesis of collagen I in human dermal fibroblasts, and this effect was further enhanced by ascorbic acid. The expression of beta1-integrins was also improved by fibrin, and on pure polylactide membranes, it was slightly enhanced by ascorbic acid. In addition, ascorbic acid promoted deposition of collagen I in the form of a fibrous extracellular matrix. Thus, the combination of nanofibrous membranes with a fibrin nanocoating and ascorbic acid seems to be particularly advantageous for skin tissue engineering.
- MeSH
- buněčná diferenciace MeSH
- elektrochemie metody MeSH
- extracelulární matrix metabolismus MeSH
- fibrin chemie metabolismus MeSH
- fibroblasty cytologie metabolismus MeSH
- fluorescenční protilátková technika MeSH
- imunoenzymatické techniky MeSH
- kolagen genetika metabolismus MeSH
- kultivované buňky MeSH
- kůže cytologie metabolismus MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- nanovlákna chemie MeSH
- polyestery chemie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proliferace buněk MeSH
- regenerace fyziologie MeSH
- tkáňové inženýrství metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Heart disease, including valve pathologies, is the leading cause of death worldwide. Despite the progress made thanks to improving transplantation techniques, a perfect valve substitute has not yet been developed: once a diseased valve is replaced with current technologies, the newly implanted valve still needs to be changed some time in the future. This situation is particularly dramatic in the case of children and young adults, because of the necessity of valve growth during the patient's life. Our review focuses on the current status of heart valve (HV) therapy and the challenges that must be solved in the development of new approaches based on tissue engineering. Scientists and physicians have proposed tissue-engineered heart valves (TEHVs) as the most promising solution for HV replacement, especially given that they can help to avoid thrombosis, structural deterioration and xenoinfections. Lastly, TEHVs might also serve as a model for studying human valve development and pathologies.
- MeSH
- bioprotézy * MeSH
- dítě MeSH
- endoteliální buňky cytologie fyziologie MeSH
- fetální krev cytologie fyziologie MeSH
- fibrin chemie MeSH
- indukované pluripotentní kmenové buňky cytologie fyziologie MeSH
- kolagen chemie MeSH
- kyselina hyaluronová chemie MeSH
- lidé MeSH
- ovce MeSH
- prasata MeSH
- srdeční chlopně umělé * MeSH
- srdeční chlopně patologie chirurgie MeSH
- tkáňové inženýrství metody MeSH
- tkáňové podpůrné struktury * MeSH
- zvířata MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy 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
The aim of the study was to evaluate the effect of a cell-free hyaluronate/type I collagen/fibrin composite scaffold containing polyvinyl alcohol (PVA) nanofibers enriched with liposomes, basic fibroblast growth factor (bFGF) and insulin on the regeneration of osteochondral defects. A novel drug delivery system was developed on the basis of the intake effect of liposomes encapsulated in PVA nanofibers. Time-controlled release of insulin and bFGF improved MSC viability in vitro. Nanofibers functionalized with liposomes also improved the mechanical characteristics of the composite gel scaffold. In addition, time-controlled release of insulin and bFGF stimulated MSC recruitment from bone marrow in vivo. Cell-free composite scaffolds containing PVA nanofibers enriched with liposomes, bFGF, and insulin were implanted into seven osteochondral defects of miniature pigs. Control defects were left untreated. After 12 weeks, the composite scaffold had enhanced osteochondral regeneration towards hyaline cartilage and/or fibrocartilage compared with untreated defects that were filled predominantly with fibrous tissue. The cell-free composite scaffold containing PVA nanofibers, liposomes and growth factors enhanced migration of the cells into the defect, and their differentiation into chondrocytes; the scaffold was able to enhance the regeneration of osteochondral defects in minipigs.
- MeSH
- buněčná diferenciace MeSH
- buňky kostní dřeně cytologie MeSH
- chondrocyty cytologie MeSH
- fibrin chemie MeSH
- fibroblastový růstový faktor 2 aplikace a dávkování MeSH
- inzulin aplikace a dávkování MeSH
- kolagen typu I chemie MeSH
- kyselina hyaluronová chemie MeSH
- liposomy MeSH
- mezenchymální kmenové buňky cytologie MeSH
- miniaturní prasata MeSH
- modul pružnosti MeSH
- nanovlákna aplikace a dávkování chemie MeSH
- polyvinylalkohol chemie MeSH
- prasata MeSH
- regenerace kostí * MeSH
- tkáňové podpůrné struktury MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
V Ústavu hematologie a krevní transfuze se problematikou vrozených poruch ve fibrinogenu zabýváme již více než 10 let a během této doby se nám podařilo nalézt mutaci v molekule fibrinogenu u více než 30 rodin. Tato práce přináší přehled 8 vybraných strukturálně nebo klinicky zajímavých případů dysfibrinogenemie, které se nám podařilo charakterizovat. Jednotlivé případy se liší především svými klinickými projevy. Studium vrozených poruch ve fibrinogenu přináší důležité informace o strukturálních a funkčních částech molekuly fibrinogenu, které se aktivně podílejí na nejrůznějších fyziologických procesech, především na hemostáze. U zhruba 1/3 pacientů s trombózami nebyla doposud nalezena příčina rozvoje trombotických komplikací, je možné, že některé jsou skryty za doposud neodhalenými mutacemi fibrinogenu. Mutace Bβ Arg237Ser, γ Tyr363Asn a Aα Asn106Asp se manifestovaly tromboticky. U nositelů těchto mutací byly zaznamenány jak hluboké žilní trombózy, tak i plicní embolie. V případě mutací Aα Gly13Glu, Aα Arg16Cys, γ Tyr262Cys a γ Arg275His byla zjištěna manifestace krvácivá, s různou intenzitou. Mutace Aα Arg16His se u 7letého pacienta zatím nemanifestovala klinickými příznaky, není však vyloučeno, protože se jedná o mutaci v místě odštěpování fibrinopeptidu A, že se mutace klinicky projeví v pozdějším věku. Výskyt posttranslačně modifikovaného fibrinogenu byl popsán ve spojitosti s řadou různých onemocnění. Ta bývají především spojena s oxidačním stresem, kdy dochází k nekontrolovatelnému nárůstu oxidantů. Oxidanty mění strukturu i funkci fibrinogenu. Námi zvolené modelové systémy reprezentovaly podmínky oxidačního stresu při různých onemocněních. K popisu strukturních změn jsme využili metodu stanovení karbonylů, SDS-PAGE s následnou imunodetekcí. K detekci vzniku tyrosylových radikálů jsme mimo SDA-PAGE s imunodetekcí použili i stanovení pomocí fluorescence. K určení míry oxidačního charakteru fibrinogenu jsme použili OFR (oxidative fibrinogen reactivity). Dále jsme sledovali vliv oxidativních změn na architekturu fibrinové sítě (skenovací elektronová mikroskopie), dále na interakci fibrinogenu s trombinem (odštěpování fibrinopeptidů, turbidimetrické sledování vzniku fibrinové sítě) a krevními destičkami (statická a dynamická adheze krevních destiček). Působením modifikačních látek za podmínek oxidačního stresu vznikají v molekule fibrinogenu nové karbonylové skupiny a tvoří se tyrosylové radikály. U většiny modifikací dochází jak k ovlivnění tvorby fibrinové sítě, tak i interakce s krevními destičkami. Celkové funkční vyznění vzniklých změn závisí na charakteru a intenzitě oxidačního činidla a, což je velmi důležité, pohybuje se od protitrombogenního až po výrazně trombogenní.
At the Institute of Hematology and Blood Transfusion, we have been studying hereditary dysfibrinogenemia for more than ten years. During this period we have described more than 30 families in the Czech Republic with inherited mutations in fibrinogen. This paper provides an overview of 8 interesting cases of dysfibrinogenemia which we have characterized. Individual cases differ mainly in their clinical manifestations. The study of congenital fibrinogen disorders provides scientists and clinicians important information on structural and functional aspects of the fibrinogen molecule during various physiological processes, especially hemostasis. For roughly one third of the patients with thrombosis the causes of their thrombotic complications have not been found yet. It is therefore possible that at least some of them might be the result of mutations in fibrinogen molecule, especially if these changes do not have to affect basic coagulation tests. Mutations Bβ Arg237Ser, γ Tyr363Asn, and Aα Asn106Asp have thrombotic manifestation. The carriers of these mutations reported both deep vein thrombosis and pulmonary embolism. Mutations Aα Gly13Glu, Aα Arg16Cys, γ Tyr262Cys, and γ Arg275His have bleeding manifestation with varying intensity. A seven year old carrier of the Aα Arg16His mutation has been asymptomatic; however clinical manifestation of the mutation in the future cannot be excluded because the mutation is situated in the site of fibrinopeptide A release. Posttranslational modified fibrinogen is linked with various diseases. These diseases are associated with oxidative stress which leads to uncontrolled production of oxidants. The oxidants modify structure as well as affect function of fibrinogen. We used several oxidative reagents mimicking various (patho)physiological states. We characterized the structural changes with quantification of carbonyls groups and SDS-PAGE followed by immunodetection. Tyrosyl radicals were also detected by SDS-PAGE with immunodetection and by fluorescent determinations. To determine the extent of oxidative nature of the fibrinogen we used OFR (Oxidative Fibrinogen Reactivity). We also studied the influence of these structural changes on the fibrin network architecture (scanning electron microscopy), the interaction of fibrinogen with thrombin (fibrinopeptides release, turbidimetric monitoring of fibrin network formation) and platelets (static and dynamic adhesion of platelets). New carbonyl groups and tyrosyl radicals were formed in fibrinogen. Most modifications occured both to influence fibrin network as well as its interaction with platelets. The overall effect of the functional changes depended on the nature and intensity of the oxidizing agent and what is very important, ranged from protitrombogennic to significantly thrombogenic.
- Klíčová slova
- dysfibrinogenemie,
- MeSH
- agregace trombocytů MeSH
- bodová mutace genetika MeSH
- dědičné koagulopatie genetika MeSH
- dítě MeSH
- dospělí MeSH
- fibrin chemie MeSH
- fibrinogen genetika MeSH
- fibrinogeny abnormální * fyziologie genetika MeSH
- fibrinolýza MeSH
- hemokoagulace * genetika MeSH
- hemoragické poruchy etiologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mikroskopie elektronová rastrovací MeSH
- mladý dospělý MeSH
- mutace genetika MeSH
- oxidační stres * fyziologie genetika MeSH
- techniky in vitro MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
The present study aimed to investigate the roles of fibrin deposition and protease activated receptor-1 (PAR-1) in renal cytokine/chemokine production and inflammatory cell infiltration in rats of different ages. Acute inflammation was induced by lipopolysaccharide (LPS) in rats which were then treated with tranexamic acid (TA), TA+urokinase (UK) or TA+low-molecular-weight heparin (HP). Fibrin deposition, inflammatory cells and expressions of PAR-1, monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) were detected. A reduction in fibrin deposition and PAR-1 expression in the LPS+TA+HP group was associated with decreased infiltration of inflammatory cells and down-regulated expressions of MCP-1 and ICAM-1. In the LPS+TA+UK group, the fibrin deposition, but not the PAR-1 expression, was reduced, However, the infiltration of inflammatory cells decreased and the expressions of MCP-1 and ICAM-1 down-regulated. There were significant differences in the fibrin deposition, infiltration of inflammatory cells and expression of PAR-1, MCP-1 and ICAM-1 between young and old rats undergoing the same treatment. These findings demonstrated that fibrin deposition plays more important roles than PAR-1 dose in cytokine/chemokine production and inflammatory cell infiltration in vivo, and ageing may deteriorate the fibrin deposition-induced production of cytokines/chemokines and infiltration of inflammatory cells.
- MeSH
- barvení a značení metody využití MeSH
- chemokiny chemie izolace a purifikace účinky léků MeSH
- cytokiny chemie účinky léků MeSH
- experimenty na zvířatech MeSH
- fibrin chemie účinky léků MeSH
- financování organizované MeSH
- hemokoagulace genetika imunologie MeSH
- ledviny cytologie enzymologie účinky léků MeSH
- northern blotting metody využití MeSH
- potkani Wistar MeSH
- receptor PAR-1 genetika imunologie účinky léků MeSH
- stárnutí MeSH
- statistika jako téma MeSH
- western blotting metody využití MeSH
- zánět MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
Hydrogels prepared from a mixture of fibrin and high-molecular weight (MW) hyaluronic acid (HA) were found to be suitable scaffolds for chondrocyte seeding and pig knee cartilage regeneration. Collagen in the hydrogels is not necessary for the formation of biomechanically stable tissue. Regenerated cartilage showed very good biomechanical and histological properties only 6 months after implantation. Notably, the quality of the healing process was dependent on the initial chondrocyte concentration of the scaffolds. These experiments were performed according to good laboratory practice (GLP).
- MeSH
- biokompatibilní materiály chemie MeSH
- biomechanika MeSH
- chondrocyty cytologie fyziologie MeSH
- chondrogeneze MeSH
- chrupavka fyziologie chirurgie MeSH
- fibrin chemie MeSH
- hydrogely MeSH
- kyselina hyaluronová chemie MeSH
- miniaturní prasata MeSH
- prasata MeSH
- protézy a implantáty MeSH
- regenerace MeSH
- testování materiálů MeSH
- tkáňové inženýrství MeSH
- tkáňové podpůrné struktury chemie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
