Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.
- MeSH
- exozómy * chemie MeSH
- karcinom * metabolismus patologie MeSH
- lektiny analýza metabolismus MeSH
- lidé MeSH
- polysacharidy analýza metabolismus MeSH
- tekutá biopsie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The present study investigates the effect of an oxidized nanocrystalline diamond (O-NCD) coating functionalized with bone morphogenetic protein 7 (BMP-7) on human osteoblast maturation and extracellular matrix mineralization in vitro and on new bone formation in vivo. The chemical structure and the morphology of the NCD coating and the adhesion, thickness and morphology of the superimposed BMP-7 layer have also been assessed. The material analysis proved synthesis of a conformal diamond coating with a fine nanostructured morphology on the Ti6Al4V samples. The homogeneous nanostructured layer of BMP-7 on the NCD coating created by a physisorption method was confirmed by AFM. The osteogenic maturation of hFOB 1.19 cells in vitro was only slightly enhanced by the O-NCD coating alone without any increase in the mineralization of the matrix. Functionalization of the coating with BMP-7 resulted in more pronounced cell osteogenic maturation and increased extracellular matrix mineralization. Similar results were obtained in vivo from micro-CT and histological analyses of rabbit distal femurs with screws implanted for 4 or 12 weeks. While the O-NCD-coated implants alone promoted greater thickness of newly-formed bone in direct contact with the implant surface than the bare material, a further increase was induced by BMP-7. It can be therefore concluded that O-NCD coating functionalized with BMP-7 is a promising surface modification of metallic bone implants in order to improve their osseointegration.
- MeSH
- biokompatibilní potahované materiály chemie farmakologie MeSH
- diamant chemie MeSH
- extracelulární matrix MeSH
- kostní morfogenetický protein 7 * farmakologie MeSH
- králíci MeSH
- osteointegrace * MeSH
- slitiny MeSH
- titan MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Purpose: Nanomaterials for antimicrobial applications have gained interest in recent years due to the increasing bacteria resistance to conventional antibiotics. Wound sterilization, water treatment and surface decontamination all avail from multifunctional materials that also possess excellent antibacterial properties, eg zinc oxide (ZnO). Here, we assess and compare the effects of synthesized hedgehog-like ZnO structures and commercial ZnO particles with and without mixing on the inactivation of bacteria on surfaces and in liquid environments. Methods: Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria in microbial culture medium were added to reverse spin bioreactors that contained different concentrations of each ZnO type to enable dynamic mixing of the bacteria-ZnO suspensions. Optical density of the bacteria-ZnO suspensions was measured in real-time and the number of viable bacteria after 24 h exposure was determined using standard microbiological techniques. The concentration of zinc ion generated from ZnO dissolution in different liquid types was estimated from the dynamic interaction exposure. Static antibacterial tests without agitation in liquid media and on agar surface were performed for comparison. Results: A correlation between increasing ZnO particle concentration and reduction in viable bacteria was not monotonous. The lowest concentration tested (10 µg/mL) even stimulated bacteria growth. The hedgehog ZnO was significantly more antibacterial than commercial ZnO particles at higher concentrations (up to 1000 µg/mL tested), more against E. coli than S. aureus. Minimum inhibitory concentration in microwell plates was correlated with those results. No inhibition was detected for any ZnO type deposited on agar surface. Zinc ion release was greatly suppressed in cultivation media. Scanning electron microscopy images revealed that ZnO needles can pierce membrane of bacteria whereas the commercial ZnO nanoparticles rather agglomerate on the cell surface. Conclusion: The inhibition effects are thus mainly controlled by the interaction dynamics between bacteria and ZnO, where mixing greatly enhances antibacterial efficacy of all ZnO particles. The efficacy is modulated also by ZnO particle shapes, where hedgehog ZnO has superior effect, in particular at lower concentrations. However, at too low concentrations, ZnO can stimulate bacteria growth and must be thus used with caution.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- biomimetické materiály chemie farmakologie MeSH
- Escherichia coli účinky léků růst a vývoj MeSH
- ježkovití * MeSH
- mikrobiální testy citlivosti MeSH
- oxid zinečnatý chemie farmakologie MeSH
- Staphylococcus aureus účinky léků růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Due to high biocompatibility, miniaturization, optical transparency and low production cost together with high radiation hardness the diamond-based sensors are considered promising for radiation medicine and biomedicine in general. Here we present detection of fibroblast cell culture properties by nanocrystalline diamond solution-gated field-effect transistors (SG-FET), including effects of gamma irradiation. We show that blank nanocrystalline diamond field-effect biosensors are stable at least up to 300 Gy of γ irradiation. On the other hand, gate current of the diamond SG-FET biosensors with fibroblastic cells increases exponentially over an order of magnitude with increasing radiation dose. Extracellular matrix (ECM) formation is also detected and analyzed by correlation of electronic sensor data with optical, atomic force, fluorescence, and scanning electron microscopies.
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
In this interdisciplinary project, innovated diamond coatings will be prepared on bone and vascular implants in order to enhance their histocompatibility for biomedical applications. The implants will be based on metallic substrates used in medicine (Ti, Ti-6Al-4V, stainless steel). In addition, novel types of materials with embedded gold and diamond nanoparticles, polymers, self-standing diamond structures and composites will be developed for further enhanced implant functionality. We will optimize hierarchically organized micro- and nano-structured implants as well as their surface chemistry and wettability by lasers and plasma treatments. The implants will be further functionalized using BMP-7 for enhancing their osseointegration or heparin for promoting endothelial regeneration and preventing restenosis. The developed constructs will be tested and immunologically screened in cultures of bone and vascular cells. The most promising implants will be evaluated in vivo by implantation into the tibia or carotid arteries of rabbits, followed by histological analysis.
V tomto mezioborovém projektu budou připraveny inovované diamantové povlaky kostních a cévních implantátů pro zvýšení jejich histokompatibility při biomedicínských aplikacích. Implantáty budou založeny na kovech běžně užívaných v medicíně (Ti, Ti-6Al-4V, nerez ocel). Pro další zlepšení funkce budou navíc zkoumány nové materiály s nanočásticemi zlata a diamantu, polymery, samonosnými diamantovými strukturami a jejich kompozity. Hierarchicky organizovaná mikro- a nanostruktura implantátů, jejich povrchové chemické složení a smáčivost budou optimalizovány pomocí laseru a plazmatu. Implantáty budou dále funkcionalizovány kostním morfogenetickým proteinem-7 (BMP-7) pro zvýšení jejich osteointegrace nebo heparinem pro zvýšení regenerace endotelu a prevenci restenózy. Nově vyvinuté materiály budou testovány in vitro především z hlediska imunitní aktivace buněk. Vybrané materiály pak budou implantovány in vivo do tibie a krčních tepen králíka a následně bude histologicky sledována reakce přilehlých i vzdálených tkání.
- MeSH
- biokompatibilní materiály terapeutické užití MeSH
- experimenty na zvířatech MeSH
- intravitální mikroskopie MeSH
- kostní morfogenetický protein 7 MeSH
- kovové nanočástice terapeutické užití MeSH
- nanodiamanty terapeutické užití MeSH
- polymery terapeutické užití MeSH
- protézy a implantáty MeSH
- regenerativní lékařství MeSH
- techniky in vitro MeSH
- testování materiálů MeSH
- tkáňové inženýrství MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- biomedicínské inženýrství
- technika lékařská, zdravotnický materiál a protetika
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Cell fate modulation by adapting the surface of a biocompatible material is nowadays a challenge in implantology, tissue engineering as well as in construction of biosensors. Nanocrystalline diamond (NCD) thin films are considered promising in these fields due to their extraordinary physical and chemical properties and diverse ways in which they can be modified structurally and chemically. The initial cell distribution, the rate of cell adhesion, distance of cell migration and also the cell proliferation are influenced by the NCD surface termination. Here, we use real-time live-cell imaging to investigate the above-mentioned processes on oxidized NCD (NCD-O) and hydrogenated NCD (NCD-H) to elucidate cell preference to the NCD-O especially on surfaces with microscopic surface termination patterns. Cells adhere more slowly and migrate farther on NCD-H than on NCD-O. Cells seeded with a fetal bovine serum (FBS) supplement in the medium move across the surface prior to adhesion. In the absence of FBS, the cells adhere immediately, but still exhibit different migration and proliferation on NCD-O/H regions. We discuss the impact of these effects on the formation of cell arrays on micropatterned NCD. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1469-1478, 2017.
Cell migration plays an important role in many biological systems. A relatively simple stochastic model is developed and used to describe cell behavior on chemically patterned substrates. The model is based on three parameters: the speed of cell movement (own and external), the probability of cell adhesion, and the probability of cell division on the substrate. The model is calibrated and validated by experimental data obtained on hydrogen- and oxygen-terminated patterns on diamond. Thereby, the simulations reveal that: (1) the difference in the cell movement speed on these surfaces (about 1.5×) is the key factor behind the formation of cell arrays on the patterns, (2) this difference is provided by the presence of fetal bovine serum (validated by experiments), and (3) the directional cell flow promotes the array formation. The model also predicts that the array formation requires mean distance of cell travel at least 10% of intended stripe width. The model is generally applicable for biosensors using diverse cells, materials, and structures.
- MeSH
- biologické modely MeSH
- buněčná adheze * MeSH
- diamant chemie MeSH
- lidé MeSH
- lokomoce * MeSH
- nádorové buněčné linie MeSH
- osteoblasty fyziologie MeSH
- pohyb buněk * MeSH
- povrchové vlastnosti * MeSH
- proliferace buněk * MeSH
- statistické modely MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We show the influence of osteoblastic SAOS-2 cells on the transfer characteristics of nanocrystalline diamond solution-gated field-effect transistors (SGFET) prepared on glass substrates. Channels of these fully transparent SGFETs are realized by hydrogen termination of undoped diamond film. After cell cultivation, the transistors exhibit about 100× increased leakage currents (up to 10nA). During and after the cell delamination, the transistors return to original gate currents. We propose a mechanism where this triggering effect is attributed to ions released from adhered cells, which depends on the cell adhesion morphology, and could be used for cell culture monitoring.
- MeSH
- akční potenciály * MeSH
- biosenzitivní techniky přístrojové vybavení MeSH
- buněčná adheze * MeSH
- diamant chemie MeSH
- elektronické tranzistory * MeSH
- ionty MeSH
- lidé MeSH
- nádorové buňky kultivované MeSH
- nádory kostí * MeSH
- osteosarkom * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The authors show that nanocrystalline diamond (NCD) thin films prepared by microwave plasma enhanced chemical vapor deposition apparatus with a linear antenna delivery system are well compatible with epithelial cells (5637 human bladder carcinoma) and significantly improve the cell adhesion compared to reference glass substrates. This is attributed to better adhesion of adsorbed layers to diamond as observed by atomic force microscopy (AFM) beneath the cells. Moreover, the cell morphology can be adjusted by appropriate surface treatment of diamond by using hydrogen and oxygen plasma. Cell bodies, cytoplasmic rims, and filopodia were characterized by Peakforce AFM. Oxidized NCD films perform better than other substrates under all conditions (96% of cells adhered well). A thin adsorbed layer formed from culture medium and supplemented with fetal bovine serum (FBS) covered the diamond surface and played an important role in the cell adhesion. Nevertheless, 50-100 nm large aggregates formed from the RPMI medium without FBS facilitated cell adhesion also on hydrophobic hydrogenated NCD (increase from 23% to 61%). The authors discuss applicability for biomedical uses.
- MeSH
- buněčná adheze * MeSH
- diamant chemie MeSH
- epitelové buňky cytologie fyziologie ultrastruktura MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- nádorové buněčné linie MeSH
- nanostruktury chemie MeSH
- tvar buňky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
