To tailor cell-surface interactions, precise and controlled attachment of cell-adhesive motifs is required, while any background non-specific cell and protein adhesion has to be blocked effectively. Herein, a versatile and highly reproducible antifouling surface modification based on "clickable" groups and hierarchically structured diblock copolymer brushes for the controlled attachment of cells is reported. The polymer brush architecture combines an antifouling bottom block of poly(2-hydroxyethyl methacrylate) poly(HEMA) and an ultrathin azide-bearing top block, which can participate in well-established "click" reactions including the highly selective copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction under mild conditions. This straightforward approach allows the rapid conjugation of a cell-adhesive, alkyne-bearing cyclic RGD peptide motif, enabling subsequent specific attachment of NIH 3T3 fibroblasts, their extensive proliferation and confluent cell sheet formation after 48 h of incubation. The generally applicable strategy presented in this report can be employed for surface functionalization with diverse alkyne-bearing biological moieties via CuAAC or copper-free alkyne-azide cycloaddition protocols, making it a versatile functionalization approach and a promising tool for tissue engineering, biomaterial implant design, and other applications that require surfaces supporting highly specific cell attachment.

• MeSH
• alkyny chemie   farmakologie   MeSH
• antiinfekční látky chemická syntéza   farmakologie   MeSH
• azidy chemie   farmakologie   MeSH
• biokompatibilní materiály chemická syntéza   farmakologie   MeSH
• buňky NIH 3T3 MeSH
• click chemie MeSH
• cykloadiční reakce MeSH
• katalýza MeSH
• myši MeSH
• oligopeptidy chemie   MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• proliferace buněk účinky léků   MeSH
• tkáňové inženýrství MeSH
• tkáňové podpůrné struktury * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

While many types of biomaterials have been evaluated in experimental spinal cord injury (SCI) research, little is known about the time-related dynamics of the tissue infiltration of these scaffolds. We analyzed the ingrowth of connective tissue, axons and blood vessels inside the superporous poly (2-hydroxyethyl methacrylate) hydrogel with oriented pores. The hydrogels, either plain or seeded with mesenchymal stem cells (MSCs), were implanted in spinal cord transection at the level of Th8. The animals were sacrificed at days 2, 7, 14, 28, 49 and 6 months after SCI and histologically evaluated. We found that within the first week, the hydrogels were already infiltrated with connective tissue and blood vessels, which remained stable for the next 6 weeks. Axons slowly and gradually infiltrated the hydrogel within the first month, after which the numbers became stable. Six months after SCI we observed rare axons crossing the hydrogel bridge and infiltrating the caudal stump. There was no difference in the tissue infiltration between the plain hydrogels and those seeded with MSCs. We conclude that while connective tissue and blood vessels quickly infiltrate the scaffold within the first week, axons show a rather gradual infiltration over the first month, and this is not facilitated by the presence of MSCs inside the hydrogel pores. Further research which is focused on the permissive micro-environment of the hydrogel scaffold is needed, to promote continuous and long-lasting tissue regeneration across the spinal cord lesion.

• MeSH
• axony patologie   MeSH
• biokompatibilní materiály chemie   MeSH
• časové faktory MeSH
• fyziologická neovaskularizace MeSH
• hydrogely MeSH
• krysa rodu rattus MeSH
• oligopeptidy chemie   MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• poranění míchy patologie   patofyziologie   terapie   MeSH
• poréznost MeSH
• potkani Wistar MeSH
• regenerace míchy fyziologie   MeSH
• testování materiálů MeSH
• tkáňové podpůrné struktury chemie   MeSH
• transplantace mezenchymálních kmenových buněk * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

The aim of the present study is to develop new magnetic polymer microspheres with functional groups available for easy protein and antibody binding. Monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA-COOH) microspheres ~4 µm in diameter and containing ∼1 mmol COOH/g were synthesized by multistep swelling polymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA), which was followed by MCMEMA hydrolysis. The microspheres were rendered magnetic by precipitation of iron oxide inside the pores, which made them easily separable in a magnetic field. Properties of the resulting magnetic poly(2-hydroxyethyl methacrylate) (mgt.PHEMA) particles with COOH functionality were examined by scanning and transmission electron microscopy (SEM and TEM), static volumetric adsorption of helium and nitrogen, mercury porosimetry, Fourier transform infrared (FTIR) and atomic absorption spectroscopy (AAS), and elemental analysis. Mgt.PHEMA microspheres were coupled with p46/Myo1C protein purified from blood serum of multiple sclerosis (MS) patients, which enabled easy isolation of monospecific anti-p46/Myo1C immunoglobulin G (IgG) antibodies from crude antibody preparations of mouse blood serum. High efficiency of this approach was confirmed by SDS/PAGE, Western blot, and dot blot analyses. The newly developed mgt.PHEMA microspheres conjugated with a potential disease biomarker, p46/Myo1C protein, are thus a promising tool for affinity purification of antibodies, which can improve diagnosis and treatment of MS patients.

• MeSH
• imobilizované proteiny chemie   imunologie   MeSH
• imunoglobulin G imunologie   izolace a purifikace   MeSH
• lidé MeSH
• magnetismus metody   MeSH
• magnety chemie   MeSH
• mikrosféry MeSH
• myosin typu I chemie   imunologie   MeSH
• myši MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• protein - isoformy chemie   imunologie   MeSH
• roztroušená skleróza diagnóza   imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Molecular diagnostics may provide tailored and cost efficient treatment for infectious disease and cancer. Rolling circle amplification (RCA) of padlock probes guarantees high specificity to identify nucleic acid targets down to single nucleotide resolution in a multiplex fashion. This makes the assay suitable for molecular analysis of various diseases, and interesting to integrate into automated devices for point-of-care analysis. A critical prerequisite for many molecular assays is (i) target-specific isolation from complex clinical samples and (ii) removal of reagents, inhibitors and contaminants between reaction steps. Efficient solid supports are therefore essential to enable multi-step, multi-analyte protocols. Superparamagnetic micro- and nanoparticles, with large surface area and rapid liquid-phase kinetics, are attractive for multi-step protocols. Recently, streptavidin-modified magnetic monodispersed poly(2-hydroxyethyl methacrylate) (STV-mag.PHEMA) microspheres were developed by multiple swelling polymerization. They are easily separated by a magnet and exhibit low non-specific protein sorption. In this study, the performance and the binding efficiency of STV-mag.PHEMA was addressed by circle-to-circle amplification (C2CA). A lower number of RCA products were detected as compared to the gold standard Dynabeads. Nevertheless, this study was the first to successfully adapt STV-mag.PHEMA microspheres as solid support in a DNA-based protocol, which is an important finding. The STV-mag.PHEMA microspheres were larger with about 16 times less surface area as compared to the Dynabeads, which might partly explain the lower rolling circle product (RCP) count obtained. Further research is currently ongoing comparing particles of similar sizes and optimizing reaction conditions to establish their full utility in the field. Ultimately, low cost and versatile particles are a great resource to facilitate future clinical molecular diagnostics.

• MeSH
• DNA chemie   metabolismus   MeSH
• imobilizované proteiny chemie   metabolismus   MeSH
• magnetismus MeSH
• mikrosféry * MeSH
• mikroskopie elektronová rastrovací MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• streptavidin chemie   metabolismus   MeSH
• techniky amplifikace nukleových kyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The architecture and mechanical properties of a scaffold for spinal cord injury treatment must provide tissue integration as well as effective axonal regeneration. Previous work has demonstrated the cell-adhesive and growth-promoting properties of the SIKVAV (Ser-Ile-Lys-Val-Ala-Val)-modified highly superporous poly(2-hydroxethyl methacrylate) (PHEMA) hydrogels. The aim of the current study was to optimize the porosity and mechanical properties of this type of hydrogel in order to develop a suitable scaffold for the repair of spinal cord tissue. Three types of highly superporous PHEMA hydrogels with oriented pores of ~60 µm diameter, porosities of 57-68% and equivalent stiffness characterized by elasticity moduli in the range 3-45 kPa were implanted into a spinal cord hemisection, and their integration into the host tissue, as well as the extent of axonal ingrowth into the scaffold pores, were histologically evaluated. The best tissue response was found with a SIKVAV-modified PHEMA hydrogel with 68% porosity and a moderate modulus of elasticity (27 kPa in the direction along the pores and 3.6 kPa in the perpendicular direction). When implanted into a spinal cord transection, the hydrogel promoted tissue bridging as well as aligned axonal ingrowth. In conclusion, a prospective oriented scaffold architecture of SIKVAV-modified PHEMA hydrogels has been developed for spinal cord injury repair; however, to develop an effective treatment for spinal cord injury, multiple therapeutic approaches are needed.

• MeSH
• axony patologie   MeSH
• biokompatibilní materiály chemie   MeSH
• hojení ran MeSH
• hydrogely chemie   MeSH
• krysa rodu rattus MeSH
• mechanický stres MeSH
• mezenchymální kmenové buňky cytologie   MeSH
• mikroskopie elektronová rastrovací MeSH
• oligopeptidy chemie   MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• poranění míchy rehabilitace   terapie   MeSH
• poréznost MeSH
• potkani Wistar MeSH
• pružnost MeSH
• regenerace nervu MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury chemie   MeSH
• tlak MeSH
• zelené fluorescenční proteiny chemie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The non-specific binding of non-target species to functionalized surfaces of biosensors continues to be challenge for biosensing in real-world media. Three different low-fouling and functionalizable surface platforms were employed to study the effect of functionalization on fouling resistance from several types of undiluted media including blood plasma and food media. The surface platforms investigated in this work included two polymer brushes: hydroxy-functional poly(2-hydroxyethyl methacrylate) (pHEMA) and carboxy-functional poly(carboxybetaine acrylamide) (pCBAA), and a standard OEG-based carboxy-functional alkanethiolate self-assembled monolayer (AT-SAM). The wet and dry polymer brushes were analyzed by AFM, ellipsometry, FT-IRRAS, and surface plasmon resonance (SPR). The surfaces were functionalized by the covalent attachment of antibodies, streptavidin, and oligonucleotides and the binding and biorecognition characteristics of the coatings were compared. We found that functionalization did not substantially affect the ultra-low fouling properties of pCBAA (plasma fouling of ~20 ng/cm(2)), a finding in contrast with pHEMA that completely lost its resistance to fouling after the activation of hydroxyl groups. Blocking a functionalized AT-SAM covalently with BSA decreased fouling down to the level comparable to unblocked pCBAA. However, the biorecognition capability of blocked functionalized AT-SAM was poor in comparison with functionalized pCBAA. Limits of detection of Escherichia coli O157:H7 in undiluted milk were determined to be 6×10(4), 8×10(5), and 6×10(5) cells/ml for pCBAA, pHEMA, and AT-SAM-blocked, respectively. Effect of analyte size on biorecognition activity of functionalized coatings was investigated and it was shown that the best performance in terms of overall fouling resistance and biorecognition capability is provided by pCBAA.

• MeSH
• adsorpce MeSH
• akrylamidy chemie   MeSH
• Escherichia coli izolace a purifikace   MeSH
• limita detekce MeSH
• mléko mikrobiologie   MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• polymery chemie   MeSH
• povrchová plasmonová rezonance metody   MeSH
• povrchové vlastnosti MeSH
• sulfhydrylové sloučeniny chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Superporous poly(2-hydroxyethyl methacrylate) is successfully used as a scaffold material for tissue engineering; however, it lacks functional groups that support cell adhesion. The objective of this study was to investigate the cell-adhesive properties of biomimetic ligands, such as laminin-derived Ac-CGGASIKVAVS-OH (SIKVAV) peptide and fibronectin subunits (Fn), as well as small molecules exemplified by 2-mercaptoethanol (ME) and cysteine (Cys), immobilized on a copolymer of 2-hydroxyethyl methacrylate (HEMA) with 2-aminoethyl methacrylate (AEMA) by a maleimide-thiol coupling reaction. The maleimide group was introduced to the P(HEMA-AEMA) hydrogels by the reaction of their amino groups with N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). Mesenchymal stem cells (MSCs) were used to investigate the cell adhesive properties of the modified hydrogels. A significantly larger area of cell growth as well as a higher cell density were found on Fn- and SIKVAV-modified hydrogels when compared to the ME- and Cys-modified supports or neat P(HEMA-AEMA). Moreover, Fn-modification strongly stimulated cell proliferation. The ability of MSCs to differentiate into adipocytes and osteoblasts was maintained on both Fn- and SIKVAV-modifications, but it was reduced on ME-modified hydrogels and neat P(HEMA-AEMA). The results show that the immobilization of SIKVAV and Fn-subunits onto superporous P(HEMA-AEMA) hydrogels via a GMBS coupling reaction improves cell adhesive properties. The high proliferative activity observed on Fn-modified hydrogels suggests that the immobilized Fn-subunits maintain their bioactivity and thus represent a promising tool for application in tissue engineering.

• MeSH
• buněčná diferenciace MeSH
• cystein chemie   MeSH
• fibronektiny chemie   MeSH
• hydrogely * MeSH
• krysa rodu rattus MeSH
• merkaptoethanol chemie   MeSH
• mezenchymální kmenové buňky cytologie   MeSH
• mikroskopie elektronová rastrovací MeSH
• peptidy chemie   MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• povrchové vlastnosti MeSH
• tkáňové inženýrství * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

To minimize non-specific protein adsorption on macroporous poly(glycidyl methacrylate) and poly(2-hydroxyethyl methacrylate) microspheres containing amino and/or carboxyl groups, the microspheres are coated with α,ω-bis-carboxy poly(ethylene glycol) and amino-terminated poly(ethylene glycol-co-propylene glycol) or α-methoxy-ω-amino poly(ethylene glycol). Adsorption of bovine serum albumin (BSA), γ-globulin, (125) I-BSA, pepsin, and chymotrypsin on neat and PEGylated microspheres is determined by UV-VIS spectroscopy of supernatants and eluates or by measurement of radioactivity in an ionization chamber. Neat and PEGylated microspheres adsorb 0.8-70% and 0.02-44% of protein, respectively.

• MeSH
• adsorpce MeSH
• biotechnologie metody   MeSH
• chymotrypsin chemie   MeSH
• gama-globuliny chemie   MeSH
• kyseliny polymethakrylové chemie   MeSH
• mikrosféry * MeSH
• molekulární struktura MeSH
• polyethylenglykoly chemie   MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• radioizotopy jodu chemie   MeSH
• sérový albumin hovězí chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The structural properties of microfiber meshes made from poly(2-hydroxyethyl methacrylate) (PHEMA) were found to significantly depend on the chemical composition and subsequent cross-linking and nebulization processes. PHEMA microfibres showed promise as scaffolds for chondrocyte seeding and proliferation. Moreover, the peak liposome adhesion to PHEMA microfiber scaffolds observed in our study resulted in the development of a simple drug anchoring system. Attached foetal bovine serum-loaded liposomes significantly improved both chondrocyte adhesion and proliferation. In conclusion, fibrous scaffolds from PHEMA are promising materials for tissue engineering and, in combination with liposomes, can serve as a simple drug delivery tool.

• MeSH
• biokompatibilní materiály chemie   MeSH
• buněčná adheze MeSH
• chondrocyty cytologie   MeSH
• fluorescenční mikroskopie metody   MeSH
• konfokální mikroskopie metody   MeSH
• lékové transportní systémy MeSH
• liposomy chemie   MeSH
• nosiče léků chemie   MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• polymery chemie   MeSH
• proliferace buněk MeSH
• racionální návrh léčiv MeSH
• reagencia zkříženě vázaná chemie   MeSH
• skot MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury chemie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Modifications of poly(2-hydroxyethyl methacrylate) (PHEMA) with cholesterol and the introduction of large pores have been developed to create highly superporous hydrogels that promote cell-surface interactions and that can serve as a permissive scaffold for spinal cord injury (SCI) treatment. Highly superporous cholesterol-modified PHEMA scaffolds have been prepared by the bulk radical copolymerization of 2-hydroxyethyl methacrylate (HEMA), cholesterol methacrylate (CHLMA), and ethylene dimethacrylate (EDMA) cross-linking agent in the presence of ammonium oxalate crystals to establish interconnected pores in the scaffold. Moreover, 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA) was incorporated in the polymerization recipe and hydrolyzed, thus introducing carboxyl groups in the hydrogel to control its swelling and softness. The hydrogels supported the in vitro adhesion and proliferation of rat mesenchymal stem cells. In an in vivo study of acute rat SCI, hydrogels were implanted to bridge a hemisection cavity. Histological evaluation was done 4 weeks after implantation and revealed the good incorporation of the implanted hydrogels into the surrounding tissue, the progressive infiltration of connective tissue and the ingrowth of neurofilaments, Schwann cells, and blood vessels into the hydrogel pores. The results show that highly superporous cholesterol-modified PHEMA hydrogels have bioadhesive properties and are able to bridge a spinal cord lesion.

• MeSH
• biokompatibilní materiály chemie   MeSH
• buněčná adheze MeSH
• cholesterol chemie   MeSH
• experimentální implantáty MeSH
• hydrogely chemie   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• mechanický stres MeSH
• methakryláty chemie   MeSH
• mezenchymální kmenové buňky cytologie   fyziologie   MeSH
• molekulární struktura MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• poranění míchy patologie   MeSH
• poréznost MeSH
• potkani Wistar MeSH
• proliferace buněk MeSH
• regenerace míchy MeSH
• regenerace nervu MeSH
• testování materiálů MeSH
• tkáňové podpůrné struktury chemie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH