139 research outputs found
Facile fabrication of microperforated membranes with re-useable SU-8 molds for organs-on-chips
Microperforated membranes are essential components of various organ-on-a-chip (OOC) barrier models devel- oped to study transport of molecular compounds and cells across cell layers in e.g. the intestine and blood-brain barrier. These OOC membranes have two functions: 1) to support growth of cells on one or both sides, and 2) to act as a filter-like barrier to separate adjacent compartments. Thin, microperforated poly(dimethylsiloxane) (PDMS) membranes can be fabricated by micromolding from silicon molds comprising arrays of micropillars for the formation of micropores. However, these molds are made by deep reactive ion etching (DRIE) and are expensive to fabricate. We describe the micromolding of thin PDMS membranes with easier-to-make, SU-8 epoxy photoresist molds. With a multilayer, SU-8, pillar microarray mold, massively parallel arrays of micropores can be formed in a thin layer of PDMS, resulting in a flexible barrier membrane that can be easily incorporated and sealed between other layers making up the OOC device. The membranes we describe here have a 30-μm thickness, with 12-μm-diameter circular pores arranged at a 100-μm pitch in a square array. We show application of these membranes in gut-on-a-chip devices, and expect that the reported fabrication strategy will also be suitable for other membrane dimension
Environmental and centrifugal factors influencing the visco-elastic properties of oral biofilms in vitro
Centrifugal compaction causes changes in the surface properties of bacterial cells. It has been shown previously that the surface properties of planktonic cells change with increasing centrifugal compaction. This study aimed to analyze the influences of centrifugal compaction and environmental conditions on the visco-elastic properties of oral biofilms. Biofilms were grown out of a layer of initially adhering streptococci, actinomyces or a combination of these. Different uni-axial deformations were induced on the biofilms and the load relaxations were measured over time. Linear-Regression-Analysis demonstrated that both the centrifugation coefficient for streptococci and induced deformation influenced the percentage relaxation. Centrifugal compaction significantly influenced relaxation only upon compression of the outermost 20% of the biofilm (p < 0.05), whereas biofilm composition became influential when 50% deformation was induced, invoking re-arrangement of the bacteria in deeper biofilm structures. In summary, the effects of centrifugal compaction of initially adhering, centrifuged bacteria extend to the visco-elastic properties of biofilms, indicating that the initial bacterial layer influences the structure of the entire biofilm
Role of adhesion forces in mechanosensitive channel gating inStaphylococcus aureusadhering to surfaces
Mechanosensitive channels in bacterial membranes open or close in response to environmental changes to allow transmembrane transport, including antibiotic uptake and solute efflux. In this paper, we hypothesize that gating of mechanosensitive channels is stimulated by forces through which bacteria adhere to surfaces. Hereto, channel gating is related with adhesion forces to different surfaces of a Staphylococcus aureus strain and its isogenic ΔmscL mutant, deficient in MscL (large) channel gating. Staphylococci becoming fluorescent due to uptake of calcein, increased with adhesion force and were higher in the parent strain (66% when adhering with an adhesion force above 4.0 nN) than in the ΔmscL mutant (40% above 1.2 nN). This suggests that MscL channels open at a higher critical adhesion force than at which physically different, MscS (small) channels open and contribute to transmembrane transport. Uptake of the antibiotic dihydrostreptomycin was monitored by staphylococcal killing. The parent strain exposed to dihydrostreptomycin yielded a CFU reduction of 2.3 log-units when adhering with an adhesion force above 3.5 nN, but CFU reduction remained low (1.0 log-unit) in the mutant, independent of adhesion force. This confirms that large channels open at a higher critical adhesion-force than small channels, as also concluded from calcein transmembrane transport. Collectively, these observations support our hypothesis that adhesion forces to surfaces play an important role, next to other established driving forces, in staphylococcal channel gating. This provides an interesting extension of our understanding of transmembrane antibiotic uptake and solute efflux in infectious staphylococcal biofilms in which bacteria experience adhesion forces from a wide variety of surfaces, like those of other bacteria, tissue cells, or implanted biomaterials
Two-Stage Interpretation of Changes in TEER of Intestinal Epithelial Layers Protected by Adhering Bifidobacteria During E. coli Challenges
Mechanisms of gastrointestinal protection by probiotic bacteria against infection involve amongst others, modulation of intestinal epithelial barrier function. Trans-epithelial electrical resistance (TEER) is widely used to evaluate cellular barrier functions. Here, we developed a two-stage interpretative model of the time-dependence of the TEER of epithelial layers grown in a transwell during Escherichia coli challenges in the absence or presence of adhering bifidobacteria. E. coli adhesion in absence or presence of adhering bifidobacteria was enumerated using selective plating. After 4-8 h, E. coli challenges increased TEER to a maximum due to bacterial adhesion and increased expression of a tight-junction protein [zonula occludens-1 (ZO-1)], concurrent with a less dense layer structure, that is indicative of mild epithelial layer damage. Before the occurrence of a TEER-maximum, decreases in electrical conductance (i.e., the reciprocal TEER) did not relate with para-cellular dextran-permeability, but after occurrence of a TEER-maximum, dextran-permeability and conductance increased linearly, indicative of more severe epithelial layer damage. Within 24 h after the occurrence of a TEER maximum, TEER decreased to below the level of unchallenged epithelial layers demonstrating microscopically observable holes and apoptosis. Under probiotic protection by adhering bifidobacteria, TEER-maxima were delayed or decreased in magnitude due to later transition from mild to severe damage, but similar linear relations between conductance and dextran permeability were observed as in absence of adhering bifidobacteria. Based on the time-dependence of the TEER and the relation between conductance and dextran-permeability, it is proposed that bacterial adhesion to epithelial layers first causes mild damage, followed by more severe damage after the occurrence of a TEER-maximum. The mild damage caused by E. coli prior to the occurrence of TEER maxima was reversible upon antibiotic treatment, but the severe damage after occurrence of TEER maxima could not be reverted by antibiotic treatment. Thus, single-time TEER is interpretable in two ways, depending whether increasing to or decreasing from its maximum. Adhering bifidobacteria elongate the time-window available for antibiotic treatment to repair initial pathogen damage to intestinal epithelial layers.</p
Antimicrobial loading of nanotubular titanium surfaces favoring surface coverage by mammalian cells over bacterial colonization
Titanium is frequently used for dental implants, percutaneous pins and screws or orthopedic joint prostheses. Implant surfaces can become peri-operatively contaminated by surgically introduced bacteria during implantation causing lack of surface coverage by mammalian cells and subsequent implant failure. Especially implants that have to function in a bacteria-laden environment such as dental implants or percutaneous pins, cannot be surgically implanted while being kept sterile. Accordingly, contaminating bacteria adhering to implant surfaces hamper successful surface coverage by mammalian cells required for long-term functioning. Here, nanotubular titanium surfaces were prepared and loaded with Ag nanoparticles or gentamicin with the aim of killing contaminating bacteria in order to favor surface coverage by mammalian cells. In mono-cultures, unloaded nanotubules did not cause bacterial killing, but loading of Ag nanoparticles or gentamicin reduced the number of adhering Staphylococcus aureus or Pseudomonas aeruginosa CFUs. A gentamicin-resistant Staphylococcus epidermidis was only killed upon loading with Ag nanoparticles. However, unlike low-level gentamicin loading, loading with Ag nanoparticles also caused tissue-cell death. In bi-cultures, low-level gentamicin-loading of nanotubular titanium surfaces effectively eradicated contaminating bacteria favoring surface coverage by mammalian cells. Thus, care must be taken in loading nanotubular titanium surfaces with Ag nanoparticles, while low-level gentamicin-loaded nanotubular titanium surfaces can be used as a local antibiotic delivery system to negate failure of titanium implants due to peri-operatively introduced, contaminating bacteria without hampering surface coverage by mammalian cells
Possibilities and impossibilities of magnetic nanoparticle use in the control of infectious biofilms
Targeting of chemotherapeutics towards a tumor site by magnetic nanocarriers is considered promising in tumor-control. Magnetic nanoparticles are also considered for use in infection-control as a new means to prevent antimicrobial resistance from becoming the number one cause of death by the year 2050. To this end, magnetic nanoparticles can either be loaded with an antimicrobial for use as a delivery vehicle or modified to acquire intrinsic antimicrobial properties. Magnetic nanoparticles can also be used for the local generation of heat to kill infectious microorganisms. Although appealing for tumor- and infectioncontrol, injection in the blood circulation may yield reticuloendothelial uptake and physical obstruction in organs that yield reduced targeting efficiency. This can be prevented with suitable surface modification. However, precise techniques to direct magnetic nanoparticles towards a target site are lacking. The problem of precise targeting is aggravated in infection-control due to the micrometer-size of infectious biofilms, as opposed to targeting of nanoparticles towards centimeter-sized tumors. This review aims to identify possibilities and impossibilities of magnetic targeting of nanoparticles for infection-control. We first review targeting techniques and the spatial resolution they can achieve as well as surface-chemical modifications of magnetic nanoparticles to enhance their targeting efficiency and antimicrobial efficacy. It is concluded that targeting problems encountered in tumor-control using magnetic nanoparticles, are neglected in most studies on their potential application in infection-control. Currently biofilm targeting by smart, self-adaptive and pH-responsive, antimicrobial nanocarriers for instance, seems easier to achieve than magnetic targeting. This leads to the conclusion that magnetic targeting of nanoparticles for the control of micrometer-sized infectious biofilms may be less promising than initially expected. However, using propulsion rather than precise targeting of magnetic nanoparticles in a magnetic field to traverse through infectious-biofilms can create artificial channels for enhanced antibiotic transport. This is identified as a more feasible, innovative application of magnetic nanoparticles in infection-control than precise targeting and distribution of magnetic nanoparticles over the depth of a biofilm. (C) 2021 Published by Elsevier Ltd on behalf of The editorial office of Journal of Materials Science & Technology
On-demand pulling-off of magnetic nanoparticles from biomaterial surfaces through implant-associated infectious biofilms for enhanced antibiotic efficacy
Biomaterial-associated infections can occur any time after surgical implantation of biomaterial implants and limit their success rates. On-demand, antimicrobial release coatings have been designed, but in vivo release triggers uniquely relating with infection do not exist, while inadvertent leakage of antimicrobials can cause exhaustion of a coating prior to need. Here, we attach magnetic-nanoparticles to a biomaterial surface, that can be pulled-off in a magnetic field through an adhering, infectious biofilm. Magnetic-nanoparticles remained stably attached to a surface upon exposure to PBS for at least 50 days, did not promote bacterial adhesion or negatively affect interaction with adhering tissue cells. Nanoparticles could be magnetically pulled-off from a surface through an adhering biofilm, creating artificial water channels in the biofilm. At a magnetic-nanoparticle coating concentration of 0.64 mg cm-2, these by-pass channels increased the penetrability of Staphylococcus aureus and Pseudomonas aeruginosa biofilms towards different antibiotics, yielding 10-fold more antibiotic killing of biofilm inhabitants than in absence of artificial channels. This innovative use of magnetic-nanoparticles for the eradication of biomaterial-associated infections requires no precise targeting of magnetic-nanoparticles and allows more effective use of existing antibiotics by breaking the penetration barrier of an infectious biofilm adhering to a biomaterial implant surface on-demand
Homogeneous Distribution of Magnetic, Antimicrobial-Carrying Nanoparticles through an Infectious Biofilm Enhances Biofilm-Killing Efficacy
Magnetic, antimicrobial-carrying nanoparticles provide a promising, new and direly needed antimicrobial strategy against infectious bacterial biofilms. Penetration and accumulation of antimicrobials over the thickness of a biofilm is a conditio sine qua non for effective killing of biofilm inhabitants. Simplified schematics on magnetic-targeting always picture homogeneous distribution of magnetic, antimicrobial-carrying nanoparticles over the thickness of biofilms, but this is not easy to achieve. Here, gentamicin-carrying magnetic nanoparticles (MNPs-G) were synthesized through gentamicin conjugation with iron-oxide nanoparticles and used to demonstrate the importance of their homogeneous distribution over the thickness of a biofilm. Diameters of MNPs-G were around 60 nm, well below the limit for reticuloendothelial rejection. MNPs-G killed most ESKAPE-panel pathogens, including Escherichia coli, equally as well as gentamicin in solution. MNPs-G distribution in a Staphylococcus aureus biofilm was dependent on magnetic-field exposure time and most homogeneous after 5 min magnetic-field exposure. Exposure of biofilms to MNPs-G with 5 min magnetic-field exposure yielded not only homogeneous distribution of MNPs-G, but concurrently better staphylococcal killing at all depths than that of MNPs, gentamicin in solution, and MNPs-G, or after other magnet-field exposure times. In summary, homogeneous distribution of gentamicin-carrying magnetic nanoparticles over the thickness of a staphylococcal biofilm was essential for killing biofilm inhabitants and required optimizing of the magnetic-field exposure time. This conclusion is important for further successful development of magnetic, antimicrobial carrying nanoparticles toward clinical application
Thermo-resistance of ESKAPE-panel pathogens, eradication and growth prevention of an infectious biofilm by photothermal, polydopamine-nanoparticles in vitro
Nanotechnology offers many novel infection-control strategies that may help prevent and treat antimicrobial-resistant bacterial infections. Here, we synthesized polydopamine, photothermal-nanoparticles (PDA-NPs) without further surface-functionalization to evaluate their potential with respect to biofilm-control. Most ESKAPE-panel pathogens in suspension with photothermal-nanoparticles showed three- to four-log-unit reductions upon Near-Infra-Red (NIR)-irradiation, but for enterococci only less than two-log unit reduction was observed. Exposure of existing Staphylococcus aureus biofilms to photothermal-nanoparticles followed by NIR-irradiation did not significantly kill biofilm-inhabitants. This indicates that the biofilm mode of growth poses a barrier to penetration of photothermal-nanoparticles, yielding dissipation of heat to the biofilm-surrounding rather than in its interior. Staphylococcal biofilm-growth in the presence of photothermal-nanoparticles could be significantly prevented after NIR-irradiation because PDA-NPs were incorporated in the biofilm and heat dissipated inside it. Thus, unmodified photothermal nanoparticles have potential for prophylactic infection-control, but data also constitute a warning for possible development of thermo-resistance in infectious pathogens.</p
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