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EN
Currently, studies on the formation of microbiological biofilm on biomaterial surfaces and medical devices have become an important aspect in medicine. In vivo studies of the biofilm development show a high correlation between the environmental factors and attachment of microorganisms to artificial surfaces. This paper is devoted to determining the impact of specific environmental stimuli and parameters of cultivation of Candida albicans yeasts on their adhesion to AISI-316L and Ti6Al4V substrates. Environmental parameters include variable glucose concentrations in YPG growth medium (0, 2, 4 and 10% wt.). Development of yeasts was also analyzed in terms of variable nutrients level i.e. transferring samples from medium with low nutrients level to other with higher glucose content. Another studied parameter was incubation time and how much fungal colonization varies during 24 and 48 h of incubation. For all samples, the number of both live and dead cells was taken into account. The results showed that with the increase in sugar content in culture media, biofilm development was observed, especially on AISI 316L surface. Also, significant changes in the number of adhered cells were observed with higher glucose concentration in medium after 24 h of incubation. Almost similar number of attached cells was observed for low glucose concentrations on both studied materials.
PL
Opracowanie przybliża problem bezpieczeństwa użytkowania produktów innowacyjnych technologii, odwołując się do przykładów produktów wprowadzonych do środowiska w przeszłości, które do dziś stanowią poważne zagrożenie dla zdrowia człowieka i jego środowiska. Ponadto przedstawiona jest hipoteza biologicznej identyfikacji produktów.
EN
The paper discusses the problem of safety of innovative technology products, giving examples of products introduced into the environment in the past and which still pose a serious threat to human health and to the environment. In addition, it presents the hypothesis of the biological identification of products.
PL
Postęp w naukach biologicznych i medycznych jest obecnie coraz silniej uzależniony od gromadzenia i poddawania analizie informacji o wzajemnych relacjach między komórkami żywych organizmów a powierzchniami materiałów abiotycznych. Szczególnie istotne jest to w dziedzinach związanych z wykorzystaniem biomateriałów, ale taka zależność jest też silnie widoczna w obszarze obejmującym przemysł przetwórczo-spożywczy. Oddziaływania materiał - komórka żywa, w znacznej mierze wykraczają poza dotychczas postrzegany zakres standardowego wykorzystania biomateriałów, a odkrycia na polu interakcji powierzchni materiałów z komórkami mają coraz większe znaczenie w prawidłowym użytkowaniu nowoczesnych systemów dystrybucji wody pitnej, a także przemysłowych systemów chłodzenia i obiegu cieczy. Wspomniany postęp stawia kolejne wyzwania przed inżynierią materiałową, która odpowiada za opracowywanie nowych technologii i materiałów mających spełniać coraz bardziej rygorystyczne oczekiwania użytkowników. Lepsze zrozumienie zależności w jakich pozostaje układ środowisko - organizmy żywe - materiał inżynierski prowadzą do wyeliminowania materiałów stwarzających potencjalnie zagrożenie, z jednoczesnym wprowadzeniem nowych materiałów, lepiej spełniających swoje funkcje. W procesie opracowania materiałów o wysokiej biozgodności uwagę naukowców w dużej mierze skupia grupa problemów związanych z zasiedlaniem powierzchni abiotycznych przez mikroorganizmy. Ograniczenie lub wyeliminowanie procesu kolonizacja powierzchni biomateriałów, zwykle prowadzącego do rozwinięcia się trudno zwalczanego biofilmu, stanowi istotne wyzwanie dla nowoopracowywanych biomateriałów. W literaturze fachowej dostrzegane są niekorzystne efekty, obserwowane przede wszystkim w praktyce klinicznej, do jakich doprowadza rozwiniecie się biofilmu na powierzchniach implantów. Opisywany jest także efekt wzrostu oporności takich kolonii bakteryjnych na działania układu immunologicznego oraz farmakoterapię. Jedną z możliwych modyfikacji powierzchni, pozwalających na ograniczenie zasiedlania ich przez mikroorganizmy, jest wprowadzenie elementów o działaniu bakterio- i grzybobójczym. W Instytucie Inżynierii Materiałowej opracowany został materiał, wytwarzany metodą hybrydową RF PACVD/MS, łączący bioinertny charakter warstw węglowych z bakteriobójczymi właściwościami jonów srebra. Powłoka tego typu pozwala na znaczące ograniczenie adhezji bakterii prowadzącej do rozwoju biofilmu na powierzchni materiału, przy jednoczesnej znikomej cytotoksyczności dla komórek organizmów wyższych. Wyniki eksperymentów prowadzonych z wykorzystaniem modelowych hodowli bakterii E.coli (DH5a) oraz komórek ludzkich osteoblastów (Saos 2) i komórek śródbłonka (EA.hy 926) w pełni potwierdziły zakładane właściwości nowego materiału.
EN
Progress in biological sciences and medicine is today increasingly dependent on collecting and examining information on the relationship between the cells of living organisms and abiotic surfaces of different materials. This is especially important in areas related to the use of biomaterials, and this dependence is particularly evident in certain areas such as the food industry. Interactions between material - living cells, has until recently been largely considered outside the scope of the standard use of biomaterials. The discovery of cell interactions with material surfaces are of growing importance in the proper use of modern drinking water distribution systems, industrial cooling systems and fluid circulation. This progress poses new challenges for materials engineering, which is responsible for the development of new technologies and materials that must meet increasingly stringent standards. With a better understanding of relationships present in such a system: environment - living cells - material engineering, it will be possible to eliminate any materials that poses a potential threat, and introduce new materials, that perform their functions more efficiently. In the process of developing materials with high a degree of biocompatibility, much of the research has focused on the problem of abiotic surface colonization by different microorganisms. One of the most significant challenges in developing new biomaterials is the reduction or elimination of biomaterial surface colonization, which usually leads to the development of a biofilm. In the literature the adverse effects of such interactions have been observed, primarily in clinical practice and the development of biofilms on the surface of implants. Increased resistance of these bacterial colonies to the human immune system and drug therapy has also been described. One possible material surface modification, which could reduce microorganism colonization, is the introduction of elements that exhibit bactericidal and fungicidal activity. The Institute of Materials Engineering has developed a material produced using a hybrid method RF PACVD/MS, which combines the bioinert nature of carbon layers and the bactericidal properties of silver ions. Such a coating can significantly reduce the degree bacterial adhesion, which results in the development of a biofilm on the surface materials, whilst having a negligible cytotoxic effect on the cells of higher organisms. Results of experiments conducted using the model bacterium E. coli culture (DH5a) together with human osteoblastic (Saos 2) and endothelial cells (EA.hy 926) have fully confirmed the assumed properties of this new material.
EN
Biomaterials used for medical implants or instruments production can cause numerous undesirable effects in human organism. They may affect cells being in a direct contact with them and can cause changes in genes expression, and as a consequence, also in protein profile of these cells. The aim of the present work was to examine an effect of medical steel 316L, poly-paraxylylene (Parylene) and nanocrystalline diamond (NCD) surfaces on protein expression in human endothelial cell line EA.hy 926.Cells were grown in Dulbecco’s MEM (DMEM) supplemented with antibiotics (penicillin and streptomycin), glucose, 10% heat inactivated fetal bovine serum and HAT-supplement. After 48h of incubation cells were washed with PBS and treated with lysis buffer (7M urea, 2M thiourea, 4% CHAPS, 2 % IPG buffer pH 4-7, 1% DTT). Proteins were purified from cell lysates with 2-D CleanUp Kit, and concentration was assessed with 2D Quant Kit. After overnight rehydration of IEF strips (pH 4-7, 11cm), in the presence of purified proteins, isoelectric focusing procedure was performed until 40kVh. Then, stripes were equilibrated, and focused proteins were separated in 12,5% polyacrylamide gels (SDS PAGE). Silver stained gels were recorded with ImageScanner and analyzed with ImageMaster 2D Platinium 6.0 (GE Healthcare) software. Numerous changes in protein expression were detected in endothelial cells exposed to artificial surfaces of tested materials (see TABLE I).
13
Content available Blood platelets apoptosis in hemodialyzed patients
EN
Blood platelet proteome of hemodialyzed uremic patients exhibits significant difference in comparison to the blood platelet proteome of healthy subjects. This alteration is manifested by the presence of high concentrations of low molecular peptides within the whole range of pI. Increased platelet apoptosis has been put forward as a possible cause of this phenomenon (1). The aim of the present research was to assess whether blood platelet populations from hemodialyzed uremic patients exhibit more binding sites for Annexin V (a marker of apoptosis) than control samples from healthy donors. Blood was obtained from uremic patients immediately before and after hemodialysis. At the same time samples from control healthy donors were also collected. Blood was anticoagulated with sodium citrate and was immediately exposed to propidium iodide, fluorescent labeled Annexin V and CD61 antibodies. The samples were incubated for 10 minutes in the dark and next the labeled samples were processed in a BectonDickinson FACScan flow cytofluorymeter. Our preliminary study was performed for 12 hemodialyzed patients, 13nondialyzed uremic patientsand 12 controls. It was found that the blood platelet population of hemodialyzed patients exhibited significantly higher level of fluorescence intensity attributed to Annexin V. Furthermore, this intensity was comparable before and after hemodialysis and was independent on patient age. The results support the hypothesis that blood platelet contact with artificial surfaces during the process of hemodialysys may be partially responsible for triggering blood platelet apoptosis.
EN
Investigations of the biofilm formation processes on biomaterials surfaces are focused on typical physiologic conditions - aerobic condition. In experimental works onto model strains, and also in the clinical reports there is a lack of the information related to the mechanisms engaged in biofilm grown under conditions of limited access to oxygen or in entirely anaerobic environment. Presented results show the colonization of medical steel 316L surface and creation of biofilm structures by E. coli bacteria’s, under oxygen deficit conditions or its total lack. In studied arrangement the change of the dynamics of bacterium growth was observed, which led to considerable slowdown of the biofilm development. Bacteria were observed with use of the combination of fluorescent dyes - bis-benzimide and propidium iodide – which makes it possible the distinction of live/death bacteria. In oxygen deficiency and in anaerobic conditions multiplication of death bacteria level in comparison to standard conditions was observed (suitably 34% and 41% dead cells, oxygenic conditions - 1%). The limitation of oxygen utilization possibility by the bacteria, which colonized investigated surfaces also resulted in decreased sizes reached by the cells.
15
Content available remote Interaction of parylene C with biological objects
EN
The aim of the present work was to examine the interactions of parylene C with such selected biological objects as: blood plasma proteins, platelets, endothelial cells, and bacterial biofilm produced by E. coli cells. The results obtained strongly support the thesis that parylene C is a material worth considering for biomedical use. Parylene C coating on polished medical steel significantly reduces platelet adhesion to this surface. On the other hand, in the case of the surface of machined medical steel coated with parylene C, the number of adhered platelets is significantly higher. This also means that surface texture of substrate material is very well reproduced by parylene C coating and is an important factor facilitating the platelet adhesion. Adsorption of plasma proteins at parylene C surface is very effective, and this finding confirms a notion that cell interaction with surfaces is mediated by the adsorbed proteins. In the light of the above, a high susceptibility of parylene C surface to bacterial colonization is easy to explain. The results showing reduced proliferation and changes in endothelial cell gene expression should also be seriously analysed when parylene C is considered for the use in contact with blood vessels.
EN
Microscopic methods usable for sample surface imaging and subsequent qualitative and quantitative evaluation of platelet adhesion to the surface of the biomaterial studied were compared. It was shown, making use of the samples of medical steel (AISI 316L), that such tools as surface imaging with scanning electron microscopy (SEM), glutaraldehyde induced fluorescence technique (GIFT) and metallurgical microscopy (MM) are equivalent in evaluating surface platelet adhesion. The importance of biological variability of blood samples for a proper result assessment and the necessity of using internal standards were also considered.
PL
Przeprowadzone przez nas badania wstępne dotyczyły sprawdzenia zdolności do wzrostu komórek śródbłonka ludzkiego na powierzchni wybranych biomateriałów, takich jak stal medyczna, stopy tytanu oraz materiałów modyfikowanych takich jak TiN i NCD (diament nanokrystaliczny). Komórki śródbłonka hodowano na powierzchni próbek, w kształcie dysków. Próbki wykonane były ze stali medycznej (AISI 316 L) w stanie dostawy, stali medycznej polerowanej, stali medycznej polerowanej i pokrytej warstwą NCD, stopu tytanu Ti6AI4V, tego samego stopu pokrytego warstwą NCD oraz stopu tytanu z warstwą TiN. Na podstawie hodowli komórek śródbłonka w ciągu 19 godzin wykazaliśmy, że były one zdolne do wzrostu na każdej z badanych powierzchni biomateriałów, jednak wykazywały różną zdolność do adhezji i przeżycia na każdej z tych powierzchni. Zgodnie z malejącą liczbą komórek znalezionych na badanych powierzchniach, można je uporządkować w następujący sposób: surowa stal medyczna, polerowana stal medyczna, polerowana stal medyczna pokryta warstwą NCD, stop tytanu pokryty warstwą NCD, stop tytanu, i stop tytanu pokryty warstwą TiN. Analiza lizatów komórkowych, z zastosowaniem elektroforezy dwukierunkowej, wykazała znaczne różnice w profilach białkowych komórek hodowanych w obecności różnych biomateriałów.
EN
Our preliminary study concerned the ability of human endothelial cells to grow on the surface of selected biomaterials, such as medical steel and titanium alloys, both unmodified and surface modified with TiN and NCD (nanocrystalline diamond). Cells were cultured on the surface of discoidal shape samples of the following materials: crude medical steel (AISI 316 L), polished medical steel, NCD coated polished medical steel, titanium alloy Ti6AI4V, NCD coated titanium alloy, and titanium alloy coated with TiN. Following 19 hours of culture growth of endothelial cells on the above biomaterials, no surface entirely free from the cells was found. It was found, however, that the cells revealed different ability do adhere and grow on each of these surfaces. The sequence of materials exhibiting an order of detected cells, decreasing from the highest number to the Iowest one, was as follows: crude medical steel, polished medical steel, NCD coated polished medical steel, NCD coated titanium alloy, titanium alloy and titanium alloy with TiN surface. A 2D electrophoresis of protein lysates of endothelial cells, grown on different biomaterial surfaces, revealed significant differences in protein profile of the cells cultured in the presence of different biomaterials.
PL
Adsorpcja i odkładanie się białek osoczowych na sztucznych powierzchniach implantów medycznych, wspomaga proces adhezji komórek. Zjawisko to stanowi poważny problem w implantologii. Użycie biosensorów SPR, pracujących w systemie BiaCore, umożliwia badania odkładania się białek na powierzchniach tytanu i na warstwach NCD (nanocrystalline diamond), w realnym czasie trwania procesu. Pokazano, że sensory pokryte bardzo cienkimi warstwami (poniżej 30 nm), zarówno tytanu jak i NCD, charakteryzują się czułością porównywalną do czułości sensora kontrolnego.
EN
Adsorption and deposition of plasma proteins at artificial surfaces of medical implants promote celi adhesion process. SPR-biosensors, working in the BiaCore system, enable studies in a real time of the phenomenon of protein deposition onto surfaces of titanium and NCD (nanocrystalline diamond). It was shown, that sensors covered with very thin layers (less then 30 nm) of both, titanium and NCD, exhibit similar sensitivity as a control gold-surface sensor.
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