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

Engineering artificial skin constructs is an ongoing challenge. An ideal material for hosting skin cells is still to be discovered. A promising candidate is low-cost cellulose, which is commonly fabricated in the form of a mesh and is applied as a wound dressing. Unfortunately, the structure and the topography of current cellulose meshes are not optimal for cell growth. To enhance the surface structure and the physicochemical properties of a commercially available mesh, we coated the mesh with wood-derived cellulose nanofibrils (CNFs). Three different types of mesh coatings are proposed in this study as a skin cell carrier: positively charged cationic cellulose nanofibrils (cCNFs), negatively charged anionic cellulose nanofibrils (aCNFs), and a combination of these two materials (c+aCNFs). These cell carriers were seeded with normal human dermal fibroblasts (NHDFs) or with human adipose-derived stem cells (ADSCs) to investigate cell adhesion, spreading, morphology, and proliferation. The negatively charged aCNF coating significantly improved the proliferation of both cell types. The positively charged cCNF coating significantly enhanced the adhesion of ADSCs only. The number of NHDFs was similar on the cCNF coatings and on the noncoated pristine cellulose mesh. However, the three-dimensional (3D) structure of the cCNF coating promoted cell survival. The c+aCNF construct proved to combine benefits from both types of CNFs, which means that the c+aCNF cell carrier is a promising candidate for further application in skin tissue engineering.

• MeSH
• celulosa * MeSH
• hydrogely MeSH
• kmenové buňky MeSH
• kůže * MeSH
• lidé MeSH
• tkáňové inženýrství MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• 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

Background: Repairs to deep skin wounds continue to be a difficult issue in clinical practice. A promising approach is to fabricate full-thickness skin substitutes with functions closely similar to those of the natural tissue. For many years, a three-dimensional (3D) collagen hydrogel has been considered to provide a physiological 3D environment for co-cultivation of skin fibroblasts and keratinocytes. This collagen hydrogel is frequently used for fabricating tissue-engineered skin analogues with fibroblasts embedded inside the hydrogel and keratinocytes cultivated on its surface. Despite its unique biological properties, the collagen hydrogel has insufficient stiffness, with a tendency to collapse under the traction forces generated by the embedded cells. Methods: The aim of our study was to develop a two-layer skin construct consisting of a collagen hydrogel reinforced by a nanofibrous poly-L-lactide (PLLA) membrane pre-seeded with fibroblasts. The attractiveness of the membrane for dermal fibroblasts was enhanced by coating it with a thin nanofibrous fibrin mesh. Results: The fibrin mesh promoted the adhesion, proliferation and migration of the fibroblasts upwards into the collagen hydrogel. Moreover, the fibroblasts spontaneously migrating into the collagen hydrogel showed a lower tendency to contract and shrink the hydrogel by their traction forces. The surface of the collagen was seeded with human dermal keratinocytes. The keratinocytes were able to form a basal layer of highly mitotically-active cells, and a suprabasal layer. Conclusion: The two-layer skin construct based on collagen hydrogel with spontaneously immigrated fibroblasts and reinforced by a fibrin-coated nanofibrous membrane seems to be promising for the construction of full-thickness skin substitute.

• MeSH
• fibrin farmakologie   MeSH
• fibroblasty cytologie   účinky léků   MeSH
• hydrogely farmakologie   MeSH
• keratinocyty cytologie   účinky léků   MeSH
• kolagen farmakologie   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• membrány umělé * MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• nanovlákna chemie   MeSH
• novorozenec MeSH
• pohyb buněk účinky léků   MeSH
• polyestery farmakologie   MeSH
• proliferace buněk účinky léků   MeSH
• škára cytologie   MeSH
• umělá kůže * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• novorozenec MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Idiopathic pes equinovarus (clubfoot) is a congenital deformity of the feet and lower legs. Clubfoot belongs to a group of fibro-proliferative disorders but its origin remains unknown. Our study aimed to achieve the first complex proteomic comparison of clubfoot contracted tissue of the foot (medial side; n = 16), with non-contracted tissue (lateral side; n = 13). We used label-free mass spectrometry quantification and immunohistochemistry. Seven proteins were observed to be significantly upregulated in the medial side (asporin, collagen type III, V, and VI, versican, tenascin-C, and transforming growth factor beta induced protein) and four in the lateral side (collagen types XII and XIV, fibromodulin, and cartilage intermediate layer protein 2) of the clubfoot. Comparison of control samples from cadavers brought only two different protein concentrations (collagen types I and VI). We also revealed pathological calcification and intracellular positivity of transforming growth factor beta only in the contracted tissue of clubfoot. Most of the 11 differently expressed proteins are strongly related to the extracellular matrix architecture and we assume that they may play specific roles in the pathogenesis of this deformity. These proteins seem to be promising targets for future investigations and treatment of this disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

• MeSH
• dítě MeSH
• extracelulární matrix - proteiny metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• kalcinóza MeSH
• lidé MeSH
• pes equinovarus etiologie   metabolismus   MeSH
• předškolní dítě MeSH
• prospektivní studie MeSH
• proteom MeSH
• transformující růstový faktor beta metabolismus   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Due to the internal structure of the knee joint, the ability to characterize and quantify the dynamic response of the meniscal tissue directly in vivo is highly problematic. The main purpose of this study was to investigate the behaviour of the meniscus under loading conditions. Four healthy young females were included. To obtain T2* values in the meniscus, the vTE sequence was used with 10 echoes ranging from 0.8 to 10.1 ms. Submilisecond first echo time is a great advantage of vTE sequence allowing for precise mapping of relatively short T2*. The two-parametric least squares fitting procedure was used to calculate T2* pixel-wise. A custom-made diamagnetic apparatus was developed to simulate stress conditions on the lower limb in a conventional MR scanner. vTE T2* was performed in five consecutive scans, 6:10 min apart. Three different compartments of the medial and lateral meniscus were segmented. The differences at the different time-points were calculated. A constant increase of T2* times after compression was statistically significant in the anterior horn of the medial meniscus. T2* mapping with variable echo time sequence might be a satisfactorily sensitive technique to detect the changes of meniscus physiology under loading conditions in vivo.

• MeSH
• dospělí MeSH
• kloubní chrupavka * diagnostické zobrazování   MeSH
• kolenní kloub MeSH
• lidé MeSH
• magnetická rezonanční tomografie metody   MeSH
• menisky tibiální * diagnostické zobrazování   fyziologie   MeSH
• zátěžový test * MeSH
• zobrazování trojrozměrné MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH
• práce podpořená grantem MeSH

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

Heat-treated polyacrylonitrile (HT-PAN), also referred to as black orlon (BO), is a promising carbon-based material used for applications in tissue engineering and regenerative medicine. To the best of our knowledge, no such complex bone morphology-mimicking three-dimensional (3D) BO structure has been reported to date. We report that BO can be easily made into 3D cryogel scaffolds with porous structures, using succinonitrile as a porogen. The cryogels possess a porous morphology, similar to bone tissue. The prepared scaffolds showed strong osteoconductive activity, providing excellent support for the adhesion, proliferation, and mitochondrial activity of human bone-derived cells. This effect was more apparent in scaffolds prepared from a matrix with a higher content of PAN (i.e., 10% rather than 5%). The scaffolds with 10% of PAN also showed enhanced mechanical properties, as revealed by higher compressive modulus and higher compressive strength. Therefore, these scaffolds have a robust potential for use in bone tissue engineering.

• MeSH
• akrylové pryskyřice chemie   MeSH
• kosti a kostní tkáň MeSH
• lidé MeSH
• pevnost v tlaku MeSH
• poréznost MeSH
• tkáňové inženýrství MeSH
• tkáňové podpůrné struktury MeSH
• vysoká teplota MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Fibrillar collagen in tendons and its natural development in rabbits are discussed in this paper. Achilles tendons from newborn (~7 days) to elderly (~38 months) rabbits were monitored in intact (n tendons=24) and microtome sectioned (n tendons=11) states with label-free second harmonic generation microscopy. After sectioning, the collagen fiber pattern was irregular for the younger animals and remained oriented parallel to the load axis of the tendon for the older animals. In contrast, the collagen fiber pattern in the intact samples followed the load axis for all the age groups. However, there was a significant difference in the tendon crimp pattern appearance between the age groups. The crimp amplitude (A) and wavelength (Λ) started at very low values (A=2.0±0.6 µm, Λ=19±4 µm) for the newborn animals. Both parameters increased for the sexually mature animals (>5 months old). When the animals were fully mature the amplitude decreased but the wavelength kept increasing. The results revealed that the microtome sectioning artifacts depend on the age of animals and that the collagen crimp pattern reflects the physical growth and development.

• MeSH
• Achillova šlacha cytologie   růst a vývoj   ultrastruktura   MeSH
• biomechanika fyziologie   MeSH
• extracelulární matrix fyziologie   MeSH
• fibrilární kolageny metabolismus   ultrastruktura   MeSH
• konfokální mikroskopie MeSH
• králíci MeSH
• mikroskopie elektronová rastrovací MeSH
• pevnost v tahu fyziologie   MeSH
• polarizační mikroskopie MeSH
• stárnutí fyziologie   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH