RESUMO
OBJECTIVES: Biofilm studies have been mostly dedicated to the major human fungal pathogen Candida albicans, whereas much less is known about this virulence factor in Candida glabrata, certainly under in vivo conditions. This study provides a deeper understanding of the biofilm development of C. glabrata, its architecture and susceptibility profile to fluconazole and echinocandins. METHODS: In vitro and in vivo C. glabrata biofilms were developed inside serum-coated triple-lumen catheters placed in 24-well polystyrene plates or implanted subcutaneously in the back of a rat, respectively. Scanning electron microscopy and confocal scanning laser microscopy were used to visualize the biofilm architecture. Quantitative real-time PCR was used to demonstrate the expression profile of EPA1, EPA3, EPA6 and AWP1-AWP7 during in vivo biofilm formation. RESULTS: Mature biofilms were observed within the first 48 h and the amount of biofilm reached its maximum by 6 days. Architecturally, mature C. glabrata biofilms consisted of a thick network of yeast cells embedded in an extracellular matrix. Moreover, in vivo biofilms were susceptible to echinocandin drugs, whereas fluconazole remained ineffective. Gene expression profiling revealed that EPA3, EPA6, AWP2, AWP3 and AWP5 were up-regulated in in vivo biofilms compared with in vitro biofilms. CONCLUSIONS: C. glabrata is a unique microorganism, which, despite the lack of transition to the hyphal form, formed thick biofilms inside foreign bodies in vivo. To our knowledge, this is the first study that has described in vivo C. glabrata biofilm development and its architectural changes in detail and provides an insight into the susceptibility profile, as well as the gene expression machinery, of biofilm-associated infections.
Assuntos
Biofilmes/crescimento & desenvolvimento , Candida glabrata/fisiologia , Candidíase/microbiologia , Candidíase/patologia , Corpos Estranhos/complicações , Animais , Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida glabrata/efeitos dos fármacos , Candida glabrata/crescimento & desenvolvimento , Modelos Animais de Doenças , Equinocandinas/farmacologia , Feminino , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Microscopia Confocal , Microscopia Eletrônica de Varredura , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Resultado do TratamentoRESUMO
In this study, we demonstrated that in vitro Candida albicans biofilms grown in the presence of diclofenac showed increased susceptibility to caspofungin. These findings were further confirmed using a catheter-associated biofilm model in rats. C. albicans-inoculated catheters retrieved from rats that were treated with both diclofenac and caspofungin contained significantly fewer biofilm cells and showed no visible biofilms inside the catheter lumens, as documented by scanning electron microscopy, as compared to catheters retrieved from rats receiving only caspofungin or diclofenac. This report indicates that diclofenac could be useful in combination therapy with caspofungin to treat C. albicans biofilm-associated infections.
Assuntos
Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Diclofenaco/farmacologia , Equinocandinas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/farmacologia , Antifúngicos/administração & dosagem , Antifúngicos/farmacologia , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Caspofungina , Catéteres/microbiologia , Diclofenaco/administração & dosagem , Farmacorresistência Fúngica , Sinergismo Farmacológico , Equinocandinas/administração & dosagem , Lipopeptídeos , RatosRESUMO
Although Ti alloys are generally regarded to be highly corrosion resistant, inflammatory conditions following surgery can instigate breakdown of the TiO2 passivation layer leading to an increased metal ion release. Furthermore proteins present in the surrounding tissue will readily adsorb on a titanium surface after implantation. In this paper alternating current electrophoretic deposition (AC-EPD) of bovine serum albumin (BSA) on Ti6Al4V was investigated in order to increase the corrosion resistance and control the protein adsorption capability of the implant surface. The Ti6Al4V surface was characterized with SEM, XPS and ToF-SIMS after long-term immersion tests under physiological conditions and simulated inflammatory conditions either in Dulbecco's Modified Eagle Medium (DMEM) or DMEM supplemented with fetal calf serum (FCS). The analysis showed an increased adsorption of amino acids and proteins from the different immersion solutions. The BSA coating was shown to prevent selective dissolution of the vanadium (V) rich ß-phase, thus effectively limiting metal ion release to the environment. Electrochemical impedance spectroscopy measurements confirmed an increase of the corrosion resistance for BSA coated surfaces as a function of immersion time due to the time-dependent adsorption of the different amino acids (from DMEM) and proteins (from FCS) as observed by ToF-SIMS analysis.
Assuntos
Materiais Revestidos Biocompatíveis/farmacologia , Eletricidade , Eletroforese , Próteses e Implantes , Soroalbumina Bovina/farmacologia , Titânio/farmacologia , Ligas , Animais , Cálcio/análise , Bovinos , Corrosão , Espectroscopia Dielétrica , Nanopartículas/ultraestrutura , Fósforo/análise , Espectroscopia Fotoeletrônica , Espectrometria de Massa de Íon SecundárioRESUMO
Additive manufacturing techniques such as Selective Laser Melting (SLM) allow carefully controlled production of complex porous structures such as scaffolds. These advanced structures can offer many interesting advantages over conventionally produced products in terms of biological response and patient specific design. The surface finish of AM parts is often poor because of the layer wise nature of the process and adhering particles. Loosening of these particles after implantation should be avoided, as this could put the patient's health at risk. In this study the use of hydrochloric acid and hydrogen peroxide mixtures for surface treatment of cobalt-chromium F75 scaffolds produced by SLM is investigated. A 27% HCl and 8% H2O2 etchant proved effective in removing adhering particles while retaining the quasi-static and fatigue performance of the scaffolds.
Assuntos
Cromo , Cobalto , Próteses e Implantes , Alicerces Teciduais , Materiais Biocompatíveis , Humanos , Ácido Clorídrico , Peróxido de Hidrogênio , Lasers , Teste de Materiais , Porosidade , PósRESUMO
Additive manufacturing techniques such as Selective Laser Melting (SLM) allow carefully controlled production of complex porous structures such as scaffolds. These advanced structures can offer many interesting advantages over conventionally produced products in terms of biological response and patient specific design. The surface finish of AM parts is often poor because of the layer wise nature of the process and adhering particles. Loosening of these particles after implantation should be avoided, as this could put the patient's health at risk. In this study the use of hydrochloric acid and hydrogen peroxide mixtures for surface treatment of cobalt-chromium F75 scaffolds produced by SLM is investigated. A 27% HCl and 8% H2O2 etchant proved effective in removing adhering particles while retaining the quasi-static and fatigue performance of the scaffolds.
Assuntos
Cromo , Cobalto , Alicerces Teciduais , Humanos , Ácido Clorídrico , Peróxido de Hidrogênio , Lasers , Porosidade , PósRESUMO
One prominent cause of implant failure is infection; therefore, research is focusing on developing surface coatings that render the surface resistant to colonization by micro-organisms. Permanently attached coatings of antimicrobial molecules are of particular interest because of the reduced cytoxicity and lower risk of developing resistance compared to controlled release coatings. In this study, we focus on the chemical grafting of bioactive molecules on titanium. To concentrate the molecules at the metallic implant surface, we propose electrophoretic deposition (EPD) applying alternating current (AC) signals with an asymmetrical wave shape. We show that for the model molecule bovine serum albumin (BSA), as well as for the clinically relevant antifungal lipopeptide caspofungin (CASP), the deposition yield is drastically improved by superimposing a DC offset in the direction of the high-amplitude peak of the AC signal. Additionally, in order to produce immobilized CASP coatings, this experimental AC/DC-EPD method is combined with an established surface activation protocol. Principle component analysis (PCA) of time-of-flight secondary ion mass spectrometry (ToF-SIMS) data confirm the immobilization of CASP with higher yield as compared to a diffusion-controlled process, and higher purity than the clinical CASP starting suspensions. Scratch testing data indicate good coating adhesion. Importantly, the coatings remain active against the fungal pathogen C. albicans as shown by in vitro biofilm experiments. In summary, this paper delivers a proof-of-concept for the application of AC-EPD as a fast grafting tool for antimicrobial molecules without compromising their activities.
Assuntos
Titânio/química , Anti-Infecciosos , Materiais Revestidos Biocompatíveis , Eletricidade , Eletroforese , Próteses e ImplantesRESUMO
Bone implants with open porosity enable fast osseointegration, but also present an increased risk of biofilm-associated infections. We design a novel implant material consisting of a mesoporous SiO2 diffusion barrier (pore diameter: 6.4 nm) with controlled drug release functionality integrated in a macroporous Ti load-bearing structure (fully interconnected open porosity: 30%; pore window size: 0.5-2.0 µm). Using an in vitro tool consisting of Ti/SiO2 disks in an insert set-up, through which molecules can diffuse from feed side to release side, a continuous release without initial burst effect of the antibiofilm compound toremifene is sustained for at least 9 days, while release concentrations (up to 17 µM daily) increase with feed concentrations (up to 4mM). Toremifene diffusivity through the SiO2 phase into H2O is estimated around 10(-13)m(2)/s, suggesting configurational diffusion through mesopores. Candida albicans biofilm growth on the toremifene-release side is significantly inhibited, establishing a proof-of-concept for the drug delivery functionality of mesoporous SiO2 incorporated into a high-strength macroporous Ti carrier. Next-generation implants made of this composite material and equipped with an internal reservoir (feed side) can yield long-term controlled release of antibiofilm compounds, effectively treating infections on the implant surface (release side) over a prolonged time.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Liberação Controlada de Fármacos , Dióxido de Silício/química , Titânio/química , Toremifeno/química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Tamanho da Partícula , Porosidade , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
Electrophoretic deposition (EPD) is a colloidal production process developed in the early 20th century. Industrial scale EPD for the production of electronic components and phosphorescent screens and in the form of cataphoretic painting has known some success. Despite its limited practical applications, the inherent versatility of EPD has never ceased to fuel research into this technique. One of the major drives of this research was to render the method more environmentally friendly by enabling deposition from aqueous suspensions. One particular route, suggested to circumvent the problems caused by the use of water in EPD, is the use of alternating or pulsed fields. Recently, the use of alternating fields in EPD has been investigated for the deposition of biological matter in the form of cells and molecules. With this new avenue of research opening up and coinciding with a rise in biotechnological processes, one can expect a renewed interest in traditional EPD and fundamental research on the use of pulsed and alternating fields in this technique. Hence, this review attempts to summarize a century's worth of both fundamental and applied research for scientists venturing into the field of EPD.
RESUMO
The effect of short chained organic acids and bases on the surface energy and wetting properties of submicrometer alumina powder was assessed. The surface chemistry of treated powders was determined by means of Diffuse Reflectance Infrared Fourier Transform spectroscopy and compared to untreated powder. The wetting of powders was measured using a modified Washburn method, based on the use of precompacted powder samples. The geometric factor needed to calculate the contact angle was derived from measurements of the porous properties of the powder compacts. Contact angle measurements with several probe liquids before and after modification allowed a theoretical estimation of the surface energy based on the surface tension component theory. Trends in the surface energy components were linked to observations in infrared spectra. The results showed that the hydrophobic character of the precompacted powder depends on both the chain length and polar group of the modifying agent.